Thermostable microbial xylanases for pulp and paper industries: trends, applications and further perspectives.

PubMed

Kumar, Vishal; Marín-Navarro, Julia; Shukla, Pratyoosh

2016-02-01

Xylanases are enzymes with biotechnological relevance in a number of fields, including food, feed, biofuel, and textile industries. Their most significant application is in the paper and pulp industry, where they are used as a biobleaching agent, showing clear economic and environmental advantages over chemical alternatives. Since this process requires high temperatures and alkali media, the identification of thermostable and alkali stable xylanases represents a major biotechnological goal in this field. Moreover, thermostability is a desirable property for many other applications of xylanases. The review makes an overview of xylanase producing microorganisms and their current implementation in paper biobleaching. Future perspectives are analyzed focusing in the efforts carried out to generate thermostable enzymes by means of modern biotechnological tools, including metagenomic analysis, enzyme molecular engineering and nanotechnology. Furthermore, structural and mutagenesis studies have revealed critical sites for stability of xylanases from glycoside hydrolase families GH10 and GH11, which constitute the main classes of these enzymes. The overall conclusions of these works are summarized here and provide relevant information about putative weak spots within xylanase structures to be targeted in future protein engineering approaches.

Structure-Specificity Relationships of an Intracellular Xylanase from Geobacillus stearothermophilus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Solomon,V.; Teplitsky, A.; Shulami, S.

2007-01-01

Geobacillus stearothermophilus T-6 is a thermophilic Gram-positive bacterium that produces two selective family 10 xylanases which both take part in the complete degradation and utilization of the xylan polymer. The two xylanases exhibit significantly different substrate specificities. While the extracellular xylanase (XT6; MW 43.8 kDa) hydrolyzes the long and branched native xylan polymer, the intracellular xylanase (IXT6; MW 38.6 kDa) preferentially hydrolyzes only short xylo-oligosaccharides. In this study, the detailed three-dimensional structure of IXT6 is reported, as determined by X-ray crystallography. It was initially solved by molecular replacement and then refined at 1.45 {angstrom} resolution to a final R factormore » of 15.0% and an R{sub free} of 19.0%. As expected, the structure forms the classical ({alpha}/{beta}){sub 8} fold, in which the two catalytic residues (Glu134 and Glu241) are located on the inner surface of the central cavity. The structure of IXT6 was compared with the highly homologous extracellular xylanase XT6, revealing a number of structural differences between the active sites of the two enzymes. In particular, structural differences derived from the unique subdomain in the carboxy-terminal region of XT6, which is completely absent in IXT6. These structural modifications may account for the significant differences in the substrate specificities of these otherwise very similar enzymes.« less

Maize pollen coat xylanase facilitates pollen tube penetration into silk during sexual reproduction.

PubMed

Suen, Der Fen; Huang, Anthony H C

2007-01-05

Cell wall hydrolases are well documented to be present on pollen, but their roles on the stigma during sexual reproduction have not been previously demonstrated. We explored the function of the tapetum-synthesized xylanase, ZmXYN1, on maize (Zea mays L.) pollen. Transgenic lines (xyl-less) containing little or no xylanase in the pollen coat were generated with use of an antisense construct of the xylanase gene-coding region driven by the XYN1 gene promoter. Xyl-less and wild-type plants had similar vegetative growth. Electron microscopy revealed no appreciable morphological difference in anther cells and pollen between xyl-less lines and the wild type, whereas immunofluorescence microscopy and biochemical analyses indicated an absence of xylanase on xyl-less pollen. Xyl-less pollen germinated as efficiently as wild-type pollen in vitro in a liquid medium but less so on gel media of increasing solidity or on silk, which is indicative of partial impaired water uptake. Once germinated in vitro or on silk, the xyl-less and wild-type pollen tubes elongated at comparable rates. Tubes of germinated xyl-less pollen on silk did not penetrate into the silk as efficiently as tubes of wild-type pollen, and this lower efficiency could be overcome by the addition of xylanase to the silk. For wild-type pollen, coat xylanase activity on oat spelled xylan in vitro and tube penetration into silk were inhibited by xylose but not glucose. The overall findings indicate that maize pollen coat xylanase facilitates pollen tube penetration into silk via enzymatic xylan hydrolysis.

Functional characterization of Penicillium occitanis Pol6 and Penicillium funiculosum GH11 xylanases.

PubMed

Driss, Dorra; Berrin, Jean Guy; Juge, Nathalie; Bhiri, Fatma; Ghorbel, Raoudha; Chaabouni, Semia Ellouz

2013-08-01

Xylanases are hemicellulolytic enzymes, which are responsible for the degradation of heteroxylans constituting the lignocellulosic plant cell wall. Xylanases from the GH11 family are considered as true xylanases because of their high substrate specificity. In order to study in depth a crucial difference in the thumb region between two closely related xylanases from Penicillium in terms of kinetic parameters and inhibition sensitivity, the GH11 xylanases from Penicillium occitanis Pol6 (PoXyn3) and from Penicillium funiculosum (PfXynC) were heterologously expressed in Pichia pastoris. The PoXyn3 and PfXynC cDNAs encoding mature xylanases were cloned into pGAPZαA vectors and integrated into the genome of P. pastoris X-33 under the control of the glyceraldehyde 3-phosphate dehydrogenase constitutive promoter. PfXynC was expressed as a His-tagged recombinant protein and purified from the supernatant homogeneity by a one-step purification protocol using immobilized metal affinity chromatography. The recombinant PoXyn3 was purified using a single anion-exchange chromatography. The purified recombinant enzymes were optimally active at 45°C and pH 4.0 for PoXyn3 and 40°C and pH 3.0 for PfXynC. The measured kinetic parameters (k(cat) and Vmax) showed that PfXynC was five times more active than PoXyn3 irrespective of the substrate whereas the apparent affinity (K(m)) was similar. The recombinant enzymes showed distinct sensitivity to the Triticum aestivum xylanase inhibitor TAXI-I. Copyright © 2013 Elsevier Inc. All rights reserved.

A Simple Method for the Determination of Xylanase Activity on Insoluble Substrates

USDA-ARS?s Scientific Manuscript database

The propensity for a xylanase to convert insoluble (arabino)xylan into soluble oligosaccharides is an important parameter in the baking, pulp and paper, prebiotics, and biofuel industries. Current methods for determining xylanase activity on insoluble substrates are labor intensive, non-specific, or...

Xylanase production with xylan rich lignocellulosic wastes by a local soil isolate of Trichoderma viride

PubMed Central

Goyal, Meenakshi; Kalra, K.L.; Sareen, V.K.; Soni, G.

2008-01-01

In the present study, cultural and nutritional conditions for enhanced production of xylanase by a local soil isolate of Trichoderma viride, using various lignocellulosic substrates in submerged culture fermentation have been optimized. Of the lignocellulosics used, maize straw was the best inducer followed by jowar straw for xylanase production. The highest activity achieved was between 14 to 17 days of fermentation. A continuous increase in xylanase production was observed with increasing level of lignocellulosics in the medium and highest activity was observed with maize straw at 5% level. Xylanase production with higher levels of lignocellulosics (3 to 5%) of maize, jowar and barseem was found to be higher as compared to that with commercial xylan as carbon source. Sodium nitrate was the best nitrogen source among the six sources used. Maximum xylanase production was achieved with initial medium pH of 3.5–4.0 and incubation temperature of 25ºC.The enzyme preparation was effective in bringing about saccharification of different lignocellulosics. The xylanase production could be further improved by using alkali treated straw as carbon source. PMID:24031262

A novel cellulase free alkaliphilic xylanase from alkali tolerant Penicillium citrinum: production, purification and characterization.

PubMed

Dutta, T; Sengupta, R; Sahoo, R; Sinha Ray, S; Bhattacharjee, A; Ghosh, S

2007-02-01

The enzymatic hydrolysis of xylan has potential economic and environment-friendly applications. Therefore, attention is focused here on the discovery of new extremophilic xylanase in order to meet the requirements of industry. An extracellular xylanase was purified from the culture filtrate of P. citrinum grown on wheat bran bed in solid substrate fermentation. Single step purification was achieved using hydrophobic interaction chromatography. The purified enzyme showed a single band on SDS-PAGE with an apparent molecular weight of c. 25 kDa and pI of 3.6. Stimulation of the activity by beta mercaptoethanol, dithiotheritol (DTT) and cysteine was observed. Moderately thermostable xylanase showed optimum activity at 50 degrees C at pH 8.5. Xylanase purified from P. citrinum was alkaliphilic and moderately thermostable in nature. The present work reports for the first time the purification and characterization of a novel endoglucanase free alkaliphilic xylanase from the alkali tolerant fungus Penicillium citrinum. The alkaliphilicity and moderate thermostability of this xylanase may have potential implications in paper and pulp industries.

Benefits from additives and xylanase during enzymatic hydrolysis of bamboo shoot and mature bamboo.

PubMed

Li, Kena; Wang, Xiao; Wang, Jingfeng; Zhang, Junhua

2015-09-01

Effects of additives (BSA, PEG 6000, and Tween 80) on enzymatic hydrolysis of bamboo shoot and mature bamboo fractions (bamboo green, bamboo timber, bamboo yellow, bamboo node, and bamboo branches) by cellulases and/or xylanase were evaluated. The addition of additives was comparable to the increase of cellulase loadings in the conversion of cellulose and xylan in bamboo fractions. Supplementation of xylanase (1 mg/g DM) with cellulases (10 FPU/g DM) in the hydrolysis of bamboo fractions was more efficient than addition of additives in the production of glucose and xylose. Moreover, addition of additives could further increase the glucose release from different bamboo fractions by cellulases and xylanase. Bamboo green exhibited the lowest hydrolyzability. Almost all of the polysaccharides in pretreated bamboo shoot fractions were hydrolyzed by cellulases with the addition of additives or xylanase. Additives and xylanase showed great potential for reducing cellulase requirement in the hydrolysis of bamboo. Copyright © 2015 Elsevier Ltd. All rights reserved.

Structural Insight into and Mutational Analysis of Family 11 Xylanases: Implications for Mechanisms of Higher pH Catalytic Adaptation.

PubMed

Bai, Wenqin; Zhou, Cheng; Zhao, Yueju; Wang, Qinhong; Ma, Yanhe

2015-01-01

To understand the molecular basis of higher pH catalytic adaptation of family 11 xylanases, we compared the structures of alkaline, neutral, and acidic active xylanases and analyzed mutants of xylanase Xyn11A-LC from alkalophilic Bacillus sp. SN5. It was revealed that alkaline active xylanases have increased charged residue content, an increased ratio of negatively to positively charged residues, and decreased Ser, Thr, and Tyr residue content relative to non-alkaline active counterparts. Between strands β6 and β7, alkaline xylanases substitute an α-helix for a coil or turn found in their non-alkaline counterparts. Compared with non-alkaline xylanases, alkaline active enzymes have an inserted stretch of seven amino acids rich in charged residues, which may be beneficial for xylanase function in alkaline conditions. Positively charged residues on the molecular surface and ionic bonds may play important roles in higher pH catalytic adaptation of family 11 xylanases. By structure comparison, sequence alignment and mutational analysis, six amino acids (Glu16, Trp18, Asn44, Leu46, Arg48, and Ser187, numbering based on Xyn11A-LC) adjacent to the acid/base catalyst were found to be responsible for xylanase function in higher pH conditions. Our results will contribute to understanding the molecular mechanisms of higher pH catalytic adaptation in family 11 xylanases and engineering xylanases to suit industrial applications.

A multipurpose immobilized biocatalyst with pectinase, xylanase and cellulase activities

PubMed Central

Dalal, Sohel; Sharma, Aparna; Gupta, Munishwar Nath

2007-01-01

Background The use of immobilized enzymes for catalyzing various biotransformations is now a widely used approach. In recent years, cross-linked enzyme aggregates (CLEAs) have emerged as a novel and versatile biocatalyst design. The present work deals with the preparation of a CLEA from a commercial preparation, Pectinex™ Ultra SP-L, which contains pectinase, xylanase and cellulase activities. The CLEA obtained could be used for any of the enzyme activities. The CLEA was characterized in terms of kinetic parameters, thermal stability and reusability in the context of all the three enzyme activities. Results Complete precipitation of the three enzyme activities was obtained with n-propanol. When resulting precipitates were subjected to cross-linking with 5 mM glutaraldehyde, the three activities initially present (pectinase, xylanase and cellulase) were completely retained after cross-linking. The Vmax/Km values were increased from 11, 75 and 16 to 14, 80 and 19 in case of pectinase, xylanase and cellulase activities respectively. The thermal stability was studied at 50°C, 60°C and 70°C for pectinase, xylanase and cellulase respectively. Half-lives were improved from 17, 22 and 32 minutes to 180, 82 and 91 minutes for pectinase, xylanase and cellulase respectively. All three of the enzymes in CLEA could be reused three times without any loss of activity. Conclusion A single multipurpose biocatalyst has been designed which can be used for carrying out three different and independent reactions; 1) hydrolysis of pectin, 2) hydrolysis of xylan and 3) hydrolysis of cellulose. The preparation is more stable at higher temperatures as compared to the free enzymes. PMID:17880745

A multipurpose immobilized biocatalyst with pectinase, xylanase and cellulase activities.

PubMed

Dalal, Sohel; Sharma, Aparna; Gupta, Munishwar Nath

2007-06-08

The use of immobilized enzymes for catalyzing various biotransformations is now a widely used approach. In recent years, cross-linked enzyme aggregates (CLEAs) have emerged as a novel and versatile biocatalyst design. The present work deals with the preparation of a CLEA from a commercial preparation, Pectinex Ultra SP-L, which contains pectinase, xylanase and cellulase activities. The CLEA obtained could be used for any of the enzyme activities. The CLEA was characterized in terms of kinetic parameters, thermal stability and reusability in the context of all the three enzyme activities. Complete precipitation of the three enzyme activities was obtained with n-propanol. When resulting precipitates were subjected to cross-linking with 5 mM glutaraldehyde, the three activities initially present (pectinase, xylanase and cellulase) were completely retained after cross-linking. The V(max)/K(m) values were increased from 11, 75 and 16 to 14, 80 and 19 in case of pectinase, xylanase and cellulase activities respectively. The thermal stability was studied at 50 degrees C, 60 degrees C and 70 degrees C for pectinase, xylanase and cellulase respectively. Half-lives were improved from 17, 22 and 32 minutes to 180, 82 and 91 minutes for pectinase, xylanase and cellulase respectively. All three of the enzymes in CLEA could be reused three times without any loss of activity. A single multipurpose biocatalyst has been designed which can be used for carrying out three different and independent reactions; 1) hydrolysis of pectin, 2) hydrolysis of xylan and 3) hydrolysis of cellulose. The preparation is more stable at higher temperatures as compared to the free enzymes.

The Use of Xylanases from Different Microbial Origin in Bread Baking and Their Effects on Bread Qualities

NASA Astrophysics Data System (ADS)

Al-Widyan, Omar; Khataibeh, Moayad H.; Abu-Alruz, Khaled

Effects of xylanases on bread quality were examined. Enzymes used were endo-xylanase (EC 3.2.1.8) from different sources of microorganisms. Baked loaves were assessed for Loaves volume, colour and staling rate. Xylanases produced from rumen microorganisms M6 had clearly positive effects on loaf volume of bread as well as anti-firming potential. M3 (produced from Trichoderma longibrachiatum) improved crumb softness. The use of xylanase for breadmaking lowered firmness of bread crumb effectively compared with control loaf. It can be summarized that xylanases had significant positive effects on bread characteristics. In particular, they had advantage in retarding the staling rate of bread. It is recommended that the optimum dosage of enzymes, method of application in industrial scale especially with xylanase should be studied further in order to gain the great advantages of enzyme addition in breadmaking.

Structural Insight into and Mutational Analysis of Family 11 Xylanases: Implications for Mechanisms of Higher pH Catalytic Adaptation

PubMed Central

Bai, Wenqin; Zhou, Cheng; Zhao, Yueju; Wang, Qinhong; Ma, Yanhe

2015-01-01

To understand the molecular basis of higher pH catalytic adaptation of family 11 xylanases, we compared the structures of alkaline, neutral, and acidic active xylanases and analyzed mutants of xylanase Xyn11A-LC from alkalophilic Bacillus sp. SN5. It was revealed that alkaline active xylanases have increased charged residue content, an increased ratio of negatively to positively charged residues, and decreased Ser, Thr, and Tyr residue content relative to non-alkaline active counterparts. Between strands β6 and β7, alkaline xylanases substitute an α-helix for a coil or turn found in their non-alkaline counterparts. Compared with non-alkaline xylanases, alkaline active enzymes have an inserted stretch of seven amino acids rich in charged residues, which may be beneficial for xylanase function in alkaline conditions. Positively charged residues on the molecular surface and ionic bonds may play important roles in higher pH catalytic adaptation of family 11 xylanases. By structure comparison, sequence alignment and mutational analysis, six amino acids (Glu16, Trp18, Asn44, Leu46, Arg48, and Ser187, numbering based on Xyn11A-LC) adjacent to the acid/base catalyst were found to be responsible for xylanase function in higher pH conditions. Our results will contribute to understanding the molecular mechanisms of higher pH catalytic adaptation in family 11 xylanases and engineering xylanases to suit industrial applications. PMID:26161643

Purification and Characterization of Haloalkaline, Organic Solvent Stable Xylanase from Newly Isolated Halophilic Bacterium-OKH

PubMed Central

Sanghvi, Gaurav; Jivrajani, Mehul; Patel, Nirav; Jivrajani, Heta; Bhaskara, Govinal Badiger; Patel, Shivani

2014-01-01

A novel, alkali-tolerant halophilic bacterium-OKH with an ability to produce extracellular halophilic, alkali-tolerant, organic solvent stable, and moderately thermostable xylanase was isolated from salt salterns of Mithapur region, Gujarat, India. Identification of the bacterium was done based upon biochemical tests and 16S rRNA sequence. Maximum xylanase production was achieved at pH 9.0 and 37°C temperature in the medium containing 15% NaCl and 1% (w/v) corn cobs. Sugarcane bagasse and wheat straw also induce xylanase production when used as carbon source. The enzyme was active over a range of 0–25% sodium chloride examined in culture broth. The optimum xylanase activity was observed at 5% sodium chloride. Xylanase was purified with 25.81%-fold purification and 17.1% yield. Kinetic properties such as Km and Vmax were 4.2 mg/mL and 0.31 μmol/min/mL, respectively. The enzyme was stable at pH 6.0 and 50°C with 60% activity after 8 hours of incubation. Enzyme activity was enhanced by Ca2+, Mn2+, and Mg2+ but strongly inhibited by heavy metals such as Hg2+, Fe3+, Ni2+, and Zn2+. Xylanase was found to be stable in organic solvents like glutaraldehyde and isopropanol. The purified enzyme hydrolysed lignocellulosic substrates. Xylanase, purified from the halophilic bacterium-OKH, has potential biotechnological applications. PMID:27350996

Enhancement of thermoalkaliphilic xylanase production by Pichia pastoris through novel fed-batch strategy in high cell-density fermentation.

PubMed

Shang, Tingting; Si, Dayong; Zhang, Dongyan; Liu, Xuhui; Zhao, Longmei; Hu, Cong; Fu, Yu; Zhang, Rijun

2017-06-21

Xylanase degrades xylan into monomers of various sizes by catalyzing the endohydrolysis of the 1,4-β-D-xylosidic linkage randomly, possessing potential in wide industrial applications. Most of xylanases are susceptible to be inactive when suffering high temperature and high alkaline process. Therefore, it is necessary to develop a high amount of effective thermoalkaliphilic xylanases. This study aims to enhance thermoalkaliphilic xylanase production in Pichia pastoris through fermentation parameters optimization and novel efficient fed-batch strategy in high cell-density fermentation. Recombinant xylanase activity increased 12.2%, 7.4%, 12.0% and 9.9% by supplementing the Pichia pastoris culture with 20 g/L wheat bran, 5 mg/L L-histidine, 10 mg/L L-tryptophan and 10 mg/L L-methionine in shake flasks, respectively. Investigation of nutritional fermentation parameters, non-nutritional fermentation parameters and feeding strategies in 1 L bioreactor and 1 L shake flask revealed that glycerol and methanol feeding strategies were the critical factors for high cell density and xylanase activity. In 50 L bioreactor, a novel glycerol feeding strategy and a four-stage methanol feeding strategy with a stepwise increase in feeding rate were developed to enhance recombinant xylanase production. In the initial 72 h of methanol induction, the linear dependence of xylanase activity on methanol intake was observed (R 2  = 0.9726). The maximum xylanase activity was predicted to be 591.2 U/mL, while the actual maximum xylanase activity was 560.7 U/mL, which was 7.05 times of that in shake flask. Recombinant xylanase retained 82.5% of its initial activity after pre-incubation at 80 °C for 50 min (pH 8.0), and it exhibited excellent stability in the broad temperature (60-80 °C) and pH (pH 8.0-11.0) ranges. Efficient glycerol and methanol fed-batch strategies resulting in desired cell density and xylanase activity should be applied in other P. pastoris

Cellulase and Xylanase Production from Three Isolates of Indigenous Endophytic Fungi

NASA Astrophysics Data System (ADS)

Yopi; Tasia, W.; Melliawati, R.

2017-12-01

Cellulases and hemicellulases have good potential to be used in energy production, in pulp, paper, textile industries, as well as in animal feed industries. Moreover, its utilization in food industries also cannot be ignored, among others, cellulase and xylanase roles in bakery, wine, and fruit and vegetables juice production. One of the potential enzyme source is endophytic fungi. Object of this study is to explore the potency of endophytic fungi isolated from medicinal plants as source of cellulolytic and xylanolytic enzymes. HL.47F.216 is endophytic fungi isolated from traditional medicinal plants ironwood tree was determined as xylanase producer. HL.51F.235 from pin-flower tree is cellulase producer, while CBN.6F.29 which produces both xylanase and cellulase is originated from Madagascar periwinkle. HL.47F.216 showed 2.5 cm in clear zone diameter and its xylanase activity was 0.262 U/mL with optimum condition pH 7 at 50°C. HL.51F.235 showed 2.4 cm clear zone diameter and 0.239 U/mL of cellulase activity at pH 5 and 70°C. CBN.6F.29 showed 2.8 cm and 0.394 U/mL (pH 5, 40°C) for its cellulase activity, while 2.3 cm and 0.439 U/mL (pH 8, 70°C) for its xylanase activity. Xylanase from HL.47F.216 and CBN.6F.29 showed low molecular masses of 20 kDa and 37-50 kDa, respectively. Molecular masses for cellulases from HL.51F.235 and CBN.6F.29 were 25 and 50 kDa for HL.51F.235 and 100 kDa for CBN.6F.29. Based on macroscopic and microscopic identification, fungal isolate CBN.6F.29 is a member of Class Coelomycetes, while HL.47F.216 was Acremonium sp. and HL.51F.235 was Aspergillus nigri.

Utilization of deoiled Jatropha curcas seed cake for production of xylanase from thermophilic Scytalidium thermophilum.

PubMed

Joshi, Chetna; Khare, S K

2011-01-01

Jatropha curcas is a major biodiesel crop. Large amount of deoiled cake is generated as by-product during biodiesel production from its seeds. Deoiled J. curcas seed cake was assessed as substrate for the production of xylanase from thermophilic fungus Scytalidium thermophilum by solid-state fermentation. The seed cake was efficiently utilized by S. thermophilum for its growth during which it produced good amount of heat stable extracellular xylanase. The solid-state fermentation conditions were optimized for maximum xylanase production. Under the optimized conditions viz. deoiled seed cake supplemented with 1% oat-spelt xylan, adjusted to pH 9.0, moisture content 1:3 w/v, inoculated with 1×10(6) spores per 5 g cake and incubated at 45 °C, 1455 U xylanase/g deoiled seed cake was obtained. The xylanase was useful in biobleaching of paper pulp. Solid-state fermentation of deoiled cake appears a potentially viable approach for its effective utilization. Copyright © 2010 Elsevier Ltd. All rights reserved.

Purification and characterization of a thermostable hypothetical xylanase from Aspergillus oryzae HML366.

PubMed

He, Haiyan; Qin, Yongling; Li, Nan; Chen, Guiguang; Liang, Zhiqun

2015-03-01

In the current study, fermentation broth of Aspergillus oryzae HML366 in sugar cane bagasse was subjected to ultrafiltration and ion exchange chromatography, and two xylanases, XynH1 and XynH2, were purified. Time-of-flight mass spectrometry coupled with SDS-PAGE analysis revealed that XynH1 is identical to the hypothetical A. oryzae RIB40 protein XP_001826985.1, with a molecular weight of 33.671 kDa. Likewise, XynH2 was identified as xylanase XynF1 with a molecular weight of 35.402 kDa. Sequence analysis indicated that XynH1 belongs to glycosyl hydrolases family 10. The specific activity of XynH1 was measured at 476.9 U/mg. Optimal xylanase activity was observed at pH 6.0, and enzyme remained active within pH 4.0-10.0 and at a temperature below 70 °C. Mg(2+), Mn(2+), Ca(2+), and K(+) enhanced the XynH1 xylanase activity to 146, 122, 114, and 108%, respectively. XynH1 hydrolyzed Birchwood xylan and Larchwood xylan effectively. The K m and V max of XynH1 values determined were 1.16 mM and 336 μmol/min/mg with Birchwood xylan as the substrate. A. oryzae HML366 xylanase XynH1 showed superior heat and pH tolerance, therefore may have significant applications in paper and biofuel industries. These studies constitute the first investigation of the xylanase activities of the hypothetical protein XP_001826985.1 form A. oryzae.

Xylanase supplementation on enzymatic saccharification of dilute acid pretreated poplars at different severities

Treesearch

Chao Zhang; Xinshu Zhuang; Zhao Jiang Wang; Fred Matt; Franz St. John; J.Y. Zhu

2013-01-01

Three pairs of solid substrates from dilute acid pretreatment of two poplar wood samples were enzymatically hydrolyzed by cellulase preparations supplemented with xylanase. Supplementation of xylanase improved cellulose saccharification perhaps due to improved cellulose accessibility by xylan hydrolysis. Total xylan removal directly affected enzymatic cellulose...

Production, Purification, and Characterization of a Major Penicillium glabrum Xylanase Using Brewer's Spent Grain as Substrate

PubMed Central

Beitel, Susan Michelz; Fortkamp, Diana; Terrasan, César Rafael Fanchini; de Almeida, Alex Fernando

2013-01-01

In recent decades, xylanases have been used in many processing industries. This study describes the xylanase production by Penicillium glabrum using brewer's spent grain as substrate. Additionally, this is the first work that reports the purification and characterization of a xylanase using this agroindustrial waste. Optimal production was obtained when P. glabrum was grown in liquid medium in pH 5.5, at 25 °C, under stationary condition for six days. The xylanase from P. glabrum was purified to homogeneity by a rapid and inexpensive procedure, using ammonium sulfate fractionation and molecular exclusion chromatography. SDS-PAGE analysis revealed one band with estimated molecular mass of 18.36 kDa. The optimum activity was observed at 60 °C, in pH 3.0. The enzyme was very stable at 50 °C, and high pH stability was verified from pH 2.5 to 5.0. The ion Mn2+ and the reducing agents β-mercaptoethanol and DTT enhanced xylanase activity, while the ions Hg2+, Zn2+, and Cu2+ as well as the detergent SDS were strong inhibitors of the enzyme. The use of brewer's spent grain as substrate for xylanase production cannot only add value and decrease the amount of this waste but also reduce the xylanase production cost. PMID:23762855

Effects of structure and xylanase treatment of brewers' spent grain on performance and nutrient availability in broiler chickens.

PubMed

Denstadli, V; Westereng, B; Biniyam, H G; Ballance, S; Knutsen, S H; Svihus, B

2010-06-01

1. A factorial (2 x 3) feeding trial was set up to investigate the effects of coarse or finely ground brewers' spent grain (BSG) and xylanase treatment, either with no xylanase, top-dressed with xylanase or pre-treated with xylanase. 2. The experimental diets shared the same basal formulation and were fed to male broiler chickens (Ross 308) housed in individual cages from 12 to 29 d of age. 3. Xylanase pre-treatment reduced the dietary concentration of arabinoxylan by 15-30%. Pellet durability increased when BSG was ground. 4. Feed utilisation was significantly higher (6%) when the birds were given coarse BSG rather than ground BSG, whereas there was no significant effect of enzyme treatment. Apparent metabolisable energy was unaffected by the dietary treatments. 5. The overall starch digestibility was high (99%), with no dietary differences, whereas ileal protein digestibility was low (57%). Xylanase top-dressing tended to improve ileal protein digestibility but, in general, xylanase treatment had no major effect on overall performance in male broilers given diets with BSG.

Effect of exogenous xylanase on rumen in vitro gas production and degradability of wheat straw.

PubMed

Togtokhbayar, Norovsambuu; Cerrillo, María A; Rodríguez, Germán Buendía; Elghandour, Mona M M Y; Salem, Abdelfattah Z M; Urankhaich, Chuluunbaatar; Jigjidpurev, Sukhbaatar; Odongo, Nicholas E; Kholif, Ahmed E

2015-08-01

The objective of this study was to determine effects of xylanase on in vitro gas production (GP) and in sacco degradability of wheat straw. Rumen fluid was obtained from three Mongolian native goats fitted with permanent rumen cannulas. The trial consisted of five doses (0, 0.5, 1.0, 1.5, 2.0 μL/g of substrate) of a commercial xylanase (Dyadic® xylanase PLUS, Dyadic International, Inc., Jupiter, FL, USA). For the in sacco degradability, different levels of xylanase enzyme were added directly onto 2 g of wheat straw in nylon bags and incubated in the rumen for 3, 6, 12, 24 and 48 h to estimate degradability of wheat straw. Total GP increased (P < 0.001) at all times of incubation at intermediate levels of xylanase. Methane production had a similar pattern at 3 and 12 h of incubation; increased linearly at 24 h of incubation, and was unaffected at 6 and 48 h of incubation. Rumen NH3 -N concentration increased linearly at 3 h and the highest values were observed with intermediate enzyme levels. All ruminal volatile fatty acids increased linearly with intermediate levels of the fibrolytic enzyme. The in sacco rate of dry matter degradation decreased linearly (P = 0.020) with increasing enzymes. Intermediate levels of xylanase improved rumen kinetic fermentation and degradability. The outcome of this research indicated that the application of xylanase enzyme could improve in vitro GP fermentation of wheat straw. © 2015 Japanese Society of Animal Science.

Xylanase production from marine derived Trichoderma pleuroticola 08ÇK001 strain isolated from Mediterranean coastal sediments.

PubMed

Korkmaz, Melih N; Ozdemir, Sennur C; Uzel, Ataç

2017-10-01

Xylanases constitutes one the most important enzymes with diverse applications in different industries such as bioethanol production, animal feedstock production, production of xylo-oligosaccharides, baking industry, paper and pulp industry, xylitol production, fruit juice, and beer finishing, degumming, and agriculture. Currently, industrial xylanases are mainly produced by Aspergillus and Trichoderma members. Since the marine environments are less studied compared to terrestrial environments and harbors great microbial diversity we aimed to investigate the xylanase production of 88 marine-derived filamentous fungal strains. These strains are semi-quantitatively screened for their extracellular xylanase production and Trichoderma pleuroticola 08ÇK001 xylanase activity was further characterized. Optimum pH and temperature was determined as 5.0 and 50 °C, respectively. The enzyme preparation retained 53% of its activity at pH 5.0 after 1 h and have found resistant against several ions and compounds such as K + , Ba 2+ , Na + , β-mercaptoethanol, Triton X-100 and toluene. This study demonstrates that marine-derived fungal strains are prolific sources for xylanase production and presents the first report about the production and characterization of xylanase from a marine derived T. pleuroticola strain. The characteristics of T. pleuroticola 08ÇK001 xylanase activity indicate possible employment in some industrial processes such as animal feed, juice and wine industries or paper pulping applications. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Gene Expression and Molecular Characterization of a Xylanase from Chicken Cecum Metagenome

PubMed Central

AL-Darkazali, Hind; Meevootisom, Vithaya

2017-01-01

A xylanase gene xynAMG1 with a 1,116-bp open reading frame, encoding an endo-β-1,4-xylanase, was cloned from a chicken cecum metagenome. The translated XynAMG1 protein consisted of 372 amino acids including a putative signal peptide of 23 amino acids. The calculated molecular mass of the mature XynAMG1 was 40,013 Da, with a theoretical pI value of 5.76. The amino acid sequence of XynAMG1 showed 59% identity to endo-β-1,4-xylanase from Prevotella bryantii and Prevotella ruminicola and 58% identity to that from Prevotella copri. XynAMG1 has two conserved motifs, DVVNE and TEXD, containing two active site glutamates and an invariant asparagine, characteristic of GH10 family xylanase. The xynAMG1 gene without signal peptide sequence was cloned and fused with thioredoxin protein (Trx.Tag) in pET-32a plasmid and overexpressed in Escherichia coli Tuner™(DE3)pLysS. The purified mature XynAMG1 was highly salt-tolerant and stable and displayed higher than 96% of its catalytic activity in the reaction containing 1 to 4 M NaCl. It was only slightly affected by common organic solvents added in aqueous solution to up to 5 M. This chicken cecum metagenome-derived xylanase has potential applications in animal feed additives and industrial enzymatic processes requiring exposure to high concentrations of salt and organic solvents. PMID:28751915

Developing a xylanase XYNZG from Plectosphaerella cucumerina for baking by heterologously expressed in Kluyveromyces lactis.

PubMed

Zhan, Fei Xiang; Wang, Qin Hong; Jiang, Si Jing; Zhou, Yu Ling; Zhang, Gui Min; Ma, Yan He

2014-12-16

Xylanase can replace chemical additives to improve the volume and sensory properties of bread in the baking. Suitable baking xylanase with improved yield will promote the application of xylanase in baking industry. The xylanase XYNZG from the Plectosphaerella cucumerina has been previously characterized by heterologous expression in Pichia pastoris. However, P. pastoris is not a suitable host for xylanase to be used in the baking process since P. pastoris does not have GRAS (Generally Regarded As Safe) status and requires large methanol supplement during the fermentation in most conditions, which is not allowed to be used in the food industry. Kluyveromyces lactis, as another yeast expression host, has a GRAS status, which has been successfully used in food and feed applications. No previous work has been reported concerning the heterologous expression of xylanase gene xynZG in K. lactis with an aim for application in baking. The xylanase gene xynZG from the P. cucumerina was heterologously expressed in K. lactis. The recombinant protein XYNZG in K. lactis presented an approximately 19 kDa band on SDS-PAGE and zymograms analysis. Transformant with the highest halo on the plate containing the RBB-xylan (Remazol Brilliant Blue-xylan) was selected for the flask fermentation in different media. The results indicated that the highest activity of 115 U/ml at 72 h was obtained with the YLPU medium. The mass spectrometry analysis suggested that the hydrolytic products of xylan by XYNZG were mainly xylobiose and xylotriose. The results of baking trials indicated that the addition of XYNZG could reduce the kneading time of dough, increase the volume of bread, improve the texture, and have more positive effects on the sensory properties of bread. Xylanase XYNZG is successfully expressed in K. lactis, which exhibits the highest activity among the published reports of the xylanase expression in K. lactis. The recombinant XYNZG can be used to improve the volume and sensory

Xylanases, nucleic acids encoding them and methods for making and using them

DOEpatents

Gray, Kevin A; Dirmeier, Reinhard

2013-07-16

The invention relates to enzymes having xylanase, mannanase and/or glucanase activity, e.g., catalyzing hydrolysis of internal .beta.-1,4-xylosidic linkages or endo-.beta.-1,4-glucanase linkages; and/or degrading a linear polysaccharide beta-1,4-xylan into xylose. Thus, the invention provides methods and processes for breaking down hemicellulose, which is a major component of the cell wall of plants, including methods and processes for hydrolyzing hemicelluloses in any plant or wood or wood product, wood waste, paper pulp, paper product or paper waste or byproduct. In addition, methods of designing new xylanases, mannanases and/or glucanases and methods of use thereof are also provided. The xylanases, mannanases and/or glucanases have increased activity and stability at increased pH and temperature.

Aureobasidium pullulans xylanase, gene and signal sequence

DOEpatents

Xin-Liang, Li; Ljungdahl, Lars G.

1997-01-01

A xylanase from Aureobasidium pullulans having a high specific activity is provided as well as a signal protein for controlling excretion into cell culture medium of proteins to which it is attached. DNA encoding these proteins is also provided.

Aureobasidium pullulans xylanase, gene and signal sequence

DOEpatents

Li Xinliang; Ljungdahl, L.G.

1997-01-07

A xylanase from Aureobasidium pullulans having a high specific activity is provided, as well as a signal protein for controlling excretion into cell culture medium of proteins to which it is attached. DNA encoding these proteins is also provided. 4 figs.

Response of broiler chickens fed wheat-based diets to xylanase supplementation.

PubMed

Gonzalez-Ortiz, G; Sola-Oriol, D; Martinez-Mora, M; Perez, J F; Bedford, M R

2017-08-01

The objective of this study was to evaluate the effect of xylanase supplementation on performance, footpad score (FPS), nutrient digestibility, and intestinal morphometry in broiler chickens. Two-hundred-eighty-eight Ross 308 broiler chicks (one d old) were placed in one of 3 experimental treatments: positive control (PC), negative control (NC) (-150 kcal/kg), and XYL (NC supplemented with xylanase). Each treatment had 8 replicates with 12 animals each. Starter (zero to 21 d) and grower (21 to 42 d) diets, based on wheat and soy-bean meal, were available ad libitum. Body weight gain (BWG) and feed intake were measured, and mortality corrected feed conversion ratio (FCR) was calculated. The relative weights of the empty gastrointestinal tract compartments and FPS were recorded on d 21 and d 42. On d 42, ileal contents were collected for nutrient digestibility determination. Statistical comparisons were performed using one-way ANOVA (JMP Pro 12). The reduction of energy resulted in lighter birds at the end of the study (PC: 2,710 vs. NC: 2,546 g; P = 0.030) whereas xylanase supplementation numerically increased BWG by 84 g (P = 0.229) and improved FCR by 12 points (P = 0.145) compared with the NC. Significant differences in FPS were observed on d 21. Score 0 (no lessions) was predominant in PC and XYL treatments, while score 1 (mild lesions) had a higher frequency in NC birds. Xylanase supplementation numerically increased organic matter (5.9%) and energy (4.7%) utilization with values above those observed with the NC. No treatment effects were observed in any of the morphometric measurements, with the exception of the gizzard (P = 0.036) and the ileum (P = 0.088) on d 42. Xylanase resulted in higher relative gizzard weights compared to NC (P = 0.102). Supplementation of broiler diets with xylanase tended to influence performance, which may be due to a better utilization of nutrients. The increase in gizzard and ileum weights in birds 42 d but not 21 d of age

Immobilization of Aspergillus niger xylanase on magnetic latex beads.

PubMed

Tyagi, R; Gupta, M N

1995-04-01

Xylanase from Pectinex 3XL was purified 70-fold by precipitation with an enteric polymer, Eudragit S-100. The purified xylanase was immobilized on magnetic latex beads via carbodi-imide coupling. The immobilized preparation showed 80% of the total activity bound to the beads. The pH optimum remained unchanged at 6.0 and the Km increased from 0.25 g/100 ml (free enzyme) to 0.39 g/100 ml on immobilization. Immobilization resulted in significant thermal stability at 60 degrees C. The time course of hydrolysis of xylan at 60 degrees C by free enzyme as well as immobilized enzyme was also studied.

Effect of additives on adsorption and desorption behavior of xylanase on acid-insoluble lignin from corn stover and wheat straw.

PubMed

Li, Yanfei; Ge, Xiaoyan; Sun, Zongping; Zhang, Junhua

2015-06-01

The competitive adsorption between cellulases and additives on lignin in the hydrolysis of lignocelluloses has been confirmed, whereas the effect of additives on the interaction between xylanase and lignin is not clear. In this work, the effects of additives, poly(ethylene glycol) 2000, poly(ethylene glycol) 6000, Tween 20, and Tween 80, on the xylanase adsorption/desorption onto/from acid-insoluble lignin from corn stover (CS-lignin) and wheat straw (WS-lignin) were investigated. The results indicated that the additives could adsorb onto isolated lignin and reduce the xylanase adsorption onto lignin. Compared to CS-lignin, more additives could adsorb onto WS-lignin, making less xylanase adsorbed onto WS-lignin. In addition, the additives could enhance desorption of xylanase from lignin, which might be due to the competitive adsorption between xylanase and additives on lignin. The released xylanase from lignin still exhibited hydrolytic capacity in the hydrolysis of isolated xylan and xylan in corn stover. Copyright © 2015 Elsevier Ltd. All rights reserved.

Xylanases, nucleic acids encoding them and methods for making and using them

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gray, Kevin A.; Dirmeier, Richard

The invention relates to enzymes having xylanase, mannanase and/or glucanase activity, e.g., catalyzing hydrolysis of internal .beta.-1,4-xylosidic linkages or endo-.beta.-1,4-glucanase linkages; and/or degrading a linear polysaccharide beta-1,4-xylan into xylose. Thus, the invention provides methods and processes for breaking down hemicellulose, which is a major component of the cell wall of plants, including methods and processes for hydrolyzing hemicelluloses in any plant or wood or wood product, wood waste, paper pulp, paper product or paper waste or byproduct. In addition, methods of designing new xylanases, mannanases and/or glucanases and methods of use thereof are also provided. The xylanases, mannanases and/or glucanasesmore » have increased activity and stability at increased pH and temperature.« less

Effects of dietary xylanase supplementation on performance and functional digestive parameters in broilers fed wheat-based diets.

PubMed

Liu, Wen-Chao; Kim, In-Ho

2017-03-01

This experiment was conducted to evaluate dietary xylanase supplementation in broilers wheat-based diets on performance and functional digestive parameters including ileal digesta viscosity, apparent ileal digestibility, intestinal morphology and microflora, digestive enzyme activities, and excreta odor content. A total of 600 1-day-old Ross 308 male broilers with an initial average BW of 45 ± 0.6 g were randomly allotted into 4 treatments with 10 replicate pens per treatment and 15 broilers in each pen for 35 d. The 4 dietary treatments were wheat-based diets and supplemented with 0, 1,875, 3,750, and 5,625 XU/kg xylanase. Xylanase supplementation improved (linear, P < 0.05) the body weight gain and decreased (linear, P < 0.05) the feed conversion ratio during d 1 to 18 and for the duration of the experiment. Dietary supplementation of xylanase led to a decrease (linear, P < 0.01) in ileal digesta viscosity. The apparent ileal digestibility of dry matter (DM), crude protein (CP), gross energy, and most amino acids (with the exception of Ile, Phe, Asp, Glu, and Pro) were increased (linear, P < 0.05) by xylanase supplementation. Increasing the dietary xylanase levels improved (linear, P < 0.05) the villus height and the ratio of villus height to crypt depth of the duodenum, jejunum, and ileum. In addition, inclusion of xylanase increased (linear, P < 0.05) the Lactobacillus numbers in the ileum and cecum, while decreased the ileal E. coli counts (linear, P < 0.01) and cecal E. coli populations (linear, P < 0.01; quadratic, P < 0.05). There were no significant differences (P > 0.05) in trypsin, amylase, and protease activities of small intestine among dietary treatments. Furthermore, xylanase supplementation reduced excreta NH3 (linear, P < 0.05; quadratic, P < 0.05) and total mercaptan (R.SH) (linear, P < 0.01) concentration. Taken together, dietary xylanase supplementation in broilers wheat-based diets had beneficial effects on growth

Solid-state Fermentation of Xylanase from Penicillium canescens 10-10c in a Multi-layer-packed Bed Reactor

NASA Astrophysics Data System (ADS)

Assamoi, Antoine A.; Destain, Jacqueline; Delvigne, Frank; Lognay, Georges; Thonart, Philippe

Xylanase is produced by Penicillium canescens 10-10c from soya oil cake in static conditions using solid-state fermentation. The impact of several parameters such as the nature and the size of inoculum, bed-loading, and aeration is evaluated during the fermentation process. Mycelial inoculum gives more production than conidial inoculum. Increasing the quantity of inoculum enhances slightly xylanase production. Forced aeration induces more sporulation of strain and reduces xylanase production. However, forced moistened air improves the production compared to production obtained with forced dry air. In addition, increasing bed-loading reduces the specific xylanase production likely due to the incapacity of the Penicillium strain to grow deeply in the fermented soya oil cake mass. Thus, the best cultivation conditions involve mycelial inoculum form, a bed loading of 1-cm height and passive aeration. The maximum xylanase activity is obtained after 7 days of fermentation and attains 10,200 U/g of soya oil cake. These levels are higher than those presented in the literature and, therefore, show all the potentialities of this stock and this technique for the production of xylanase.

Temperature influence on fluorescence intensity and enzyme activity of the fusion protein of GFP and hyperthermophilic xylanase.

PubMed

Zhang, Chong; Liu, Min-Sheng; Xing, Xin-Hui

2009-09-01

By constructing the expression system for fusion protein of GFPmut1 (a green fluorescent protein mutant) with the hyperthermophilic xylanase obtained from Dictyoglomus thermophilum Rt46B.1, the effects of temperature on the fluorescence of GFP and its relationship with the activities of GFP-fused xylanase have been studied. The fluorescence intensities of both GFP and GFP-xylanase have proved to be thermally sensitive, with the thermal sensitivity of the fluorescence intensity of GFP-xylanase being 15% higher than that of GFP. The lost fluorescence intensity of GFP inactivated at high temperature of below 60 degrees C in either single or fusion form can be completely recovered by treatment at 0 degrees C. By the fluorescence recovery of GFP domain at low temperature, the ratios of fluorescence intensity to xylanase activity (Rgfp/Axyl) at 15 degrees C and 37 degrees C have been compared. Even though the numbers of molecules of GFP and xylanase are equivalent, the Rgfp/Axyl ratio at 15 degrees C is ten times of that at 37 degrees C. This is mainly due to the fact that lower temperature is more conducive to the correct folding of GFP than the hyperthermophilic xylanase during the expression. This study has indicated that the ratio of GFP fluorescence to the thermophilic enzyme activity for the fusion proteins expressed at different temperatures could be helpful in understanding the folding properties of the two fusion partners and in design of the fusion proteins.

Cloning and heterologous expression of cellulose free thermostable xylanase from Bacillus brevis.

PubMed

Goswami, Girish K; Krishnamohan, Medichtrla; Nain, Vikrant; Aggarwal, Chetana; Ramesh, Bandarupalli

2014-01-01

Xylanase gene isolated from Bacillus brevis was expressed in E. coli BL21. Sequencing of the gene (Gen Bank accession number: HQ179986) showed that it belongs to family 11 xylanases. The recombinant xylanase was predominantly secreted to culture medium and showed mesophilic nature (optimum activity at 55°C and pH 7.0). The cell free culture medium exhibited 30 IU/ml xylanse activity. The enzyme did not show any cellulose activity and was active under wide range of temperature (40°C to 80°C) and pH (4 to 9). The enzyme showed considerable thermo stability and regained over 90% of activity, when returned to 55°C after boiling for 5 min. These physiochemical properties of B. brevis xylanse show high potential of its applications in paper and pulp industry.

Thermoresistant xylanases from Trichoderma stromaticum: Application in bread making and manufacturing xylo-oligosaccharides.

PubMed

Carvalho, Elck Almeida; Dos Santos Góes, Laís Mendes; Uetanabaro, Ana Paula T; da Silva, Erik Galvão Paranhos; Rodrigues, Luciano Brito; Pirovani, Carlos Priminho; da Costa, Andréa Miura

2017-04-15

The enzymes Xyl1 and Xyl2 from T. stromaticum were purified and identified by mass spectrometry (MALDI-TOF/MS). Xyl1 contained three proteins with similarity to xylanase family 10, 62 and anarabinofuranosidase of the Trichoderma genus and Xyl2 contained a protein with similarity to endo-1,4-β-xylanase. High xylanase activity was found at 50°C for Xyl1 and 60°C for Xyl2 and pH 5.0 for both, retaining more than 80% of activities for one hour at 60°C and pH 5-8. Ag 2+ and β-mercaptoethanol increased while SDS and EDTA inhibited the xylanase activity of both Xyl1 and Xyl2 extracts. The Km and V max values for purified Xyl2 were 9.6mg/mL and 28.57μmol/min/mg, respectively. In application tests, both Xyl1 and Xyl2 were effective in degrading beechwood xylan to produce xylo-oligosaccharides. In baking, adding Xyl1 increased the softness and volume of wheat bread and whole grain bread, qualities increasingly desired by consumers in this segment. Copyright © 2016 Elsevier Ltd. All rights reserved.

Alkalistable endo-β-1,4-xylanase production from a newly isolated alkalitolerant Penicillium sp. SS1 using agro-residues.

PubMed

Bajaj, Bijender Kumar; Sharma, Mukul; Sharma, Sunny

2011-09-01

Thermostable and alkalitolerant xylanases have got intense research focus due to their vast applications in various industries including pulp and paper, food, feed, textile, biofuel, etc. In the present investigation, a Penicillum sp. SS1 isolated from degrading woody material was found to produce moderately thermoactive and alkalistable endo-β-1,4-xylanase (xylanase). Maximum xylanase production was observed after fourth day of fermentation (43.84 IU/ml). The organism produced substantial quantities of xylanase using agricultural residues like wheat bran (20.6 IU/ml), rice bran (21.8 IU/ml) and sawdust (10.7 IU/ml) as carbon sources. The enzyme preparation was totally free of filter paper activity (FPase) and possessed negligible carboxymethyl cellulase (CMCase) activity; this could be an important feature of enzyme if the intended application of enzyme is in pulp and paper industries. Among nitrogen sources examined, yeast extract supported maximum xylanase production (45.74 IU/ml), and was followed by soybean meal (22.2 IU/ml) and ammonium sulphate (20 IU/ml). Maximum xylanase production was observed at initial medium pH 9 (25.6 IU/ml); however, at pH 8 and 10 also significantly high enzyme titre was observed (24 and 21.2 IU/ml, respectively). Thus, Penicillium sp. SS1 displayed capability of growing and producing xylanase at high alkaline pH (8-10). Maximum xylanase activity was reported at 50 °C, however, significantly high activity was observed at 60 °C (65.4%), however, at 70-80 °C activity was lost considerably. At 50-60 °C the enzyme retained very high activity up to 30-60 min (91-100%), however, prolonged incubation (90 min) caused considerable activity reduction (residual activity 63-68%).

Effects of exogenous xylanase on performance, nutrient digestibility and caecal thermal profiles of broilers given wheat-based diets.

PubMed

Cowieson, A J; Masey O'Neill, H V

2013-06-01

1. Five dietary treatments were used in a 49 d broiler trial to assess the effect of xylanase on performance, nutrient digestibility and thermal profiles of the caeca and head. Treatments included an industry-standard control diet and four further diets where xylanase was introduced with or without a metabolisable energy density dilution either from day one or the introduction was delayed until d 28. 2. The addition of xylanase with no associated energy dilution from day one resulted in the most consistent beneficial effects on performance, with significant improvements in weight gain compared with the industry-standard to d 28 and at d 49. Addition of xylanase from d 28 (with no energy dilution) was the second most successful strategy and resulted in a significant improvement in feed conversion ratio (FCR) from d 29 to 49 and overall. 3. Addition of xylanase improved ileal digestible energy values at d 28 by around 0.35 MJ/kg and ileal nitrogen digestibility coefficients by around 3%. On d 49 xylanase improved ileal digestible energy values by around 0.9 MJ/kg and ileal nitrogen digestibility coefficients by around 4.6%. 4. Thermal imaging of the head and caeca of three birds per replicate on d 49 revealed a significant increase in caecal surface temperature following xylanase addition with no effect on head temperature profile. These increases were particularly large (around 1.4ºC, or 3.9%) when xylanase was added from day one with no corresponding energy dilution in feed formulation. 5. It can be concluded that supplemental xylanase is effective in improving performance and nutrient digestibility in broilers given wheat-based diets. The correlation between the magnitude of this effect and the increased temperature in the caeca presents additional evidence that the hind-gut microflora may play an important, if yet unquantified, role in the outworking of these mechanisms.

Polypeptides having xylanase activity and polynucleotides encoding same

DOE Office of Scientific and Technical Information (OSTI.GOV)

Spodsberg, Nikolaj

The present invention relates to isolated polypeptides having xylanase activity and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having xylanase activity and polynucleotides encoding same

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lopez de Leon, Alfredo; Rey, Michael

The present invention relates to isolated polypeptides having xylanase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having xylanase activity and polynucleotides encoding same

DOEpatents

Lopez de Leon, Alfredo; Rey, Michael

2010-12-14

The present invention relates to isolated polypeptides having xylanase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having xylanase activity and polynucleotides encoding same

DOEpatents

Lopez de Leon, Alfredo; Rey, Michael

2016-05-31

The present invention relates to isolated polypeptides having xylanase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having xylanase activity and polynucleotides encoding same

DOEpatents

Tang, Lan; Liu, Ye; Duan, Junxin; Ding, Hanshu

2013-04-30

The present invention relates to isolated polypeptides having xylanase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having xylanase activity and polynucleotides encoding same

DOEpatents

Tang, Lan; Liu, Ye; Duan, Junxin; Hanshu, Ding

2012-10-30

The present invention relates to isolated polypeptides having xylanase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having xylanase activity and polynucleotides encoding same

DOEpatents

Lopez de Leon, Alfredo; Rey, Michael

2015-01-27

The present invention relates to isolated polypeptides having xylanase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having xylanase activity and polynucleotides encoding same

DOEpatents

Spodsberg, Nikolaj

2014-10-21

The present invention relates to isolated polypeptides having xylanase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having xylanase activity and polynucleotides encoding same

DOEpatents

Spodsberg, Nikolaj

2017-05-02

The present invention relates to isolated polypeptides having xylanase activity and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having xylanase activity and polynucleotides encoding same

DOEpatents

Spodsberg, Nikolaj

2014-10-14

The present invention relates to isolated polypeptides having xylanase activity and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Sequential and simultaneous strategies for biorefining of wheat straw using room temperature ionic liquids, xylanases and cellulases.

PubMed

Husson, Eric; Auxenfans, Thomas; Herbaut, Mickael; Baralle, Manon; Lambertyn, Virginie; Rakotoarivonina, Harivoni; Rémond, Caroline; Sarazin, Catherine

2018-03-01

Sequential and simultaneous strategies for fractioning wheat straw were developed in combining 1-ethyl-3-methyl imidazolium acetate [C2mim][OAc], endo-xylanases from Thermobacillus xylanilyticus and commercial cellulases. After [C2mim][OAc]-pretreatment, hydrolysis catalyzed by endo-xylanases of wheat straw led to efficient xylose production with very competitive yield (97.6 ± 1.3%). Subsequent enzymatic saccharification allowed achieving a total degradation of cellulosic fraction (>99%). These high performances revealed an interesting complementarity of [C2mim][OAc]- and xylanase-pretreatments for increasing enzymatic digestibility of cellulosic fraction in agreement with the structural and morphological changes of wheat straw induced by each of these pretreatment steps. In addition a higher tolerance of endo-xylanases from T. xylaniliticus to [C2mim][AcO] until 30% v/v than cellulases from T. reesei was observed. Based on this property, a simultaneous strategy combining [C2mim][OAc]- and endo-xylanases as pretreatment in a one-batch produced xylose with similar yield than those obtained by the sequential strategy. Copyright © 2017 Elsevier Ltd. All rights reserved.

Production of xylanase from an alkali tolerant Streptomyces sp. 7b under solid-state fermentation, its purification, and characterization.

PubMed

Bajaj, Bijender Kumar; Singh, Narendera Pratap

2010-11-01

Streptomyces sp. 7b showed highest xylanase activity among 41 bacterial isolates screened under submerged fermentation. The organism grew over broad pH (5-11) and temperatures range (25-55 degrees C) and displayed maximum xylanase production on wheat bran (1230 U/g) under solid-state fermentation. Xylanase production was enhanced substantially (76%-77%) by inclusion of trypton (2180 U/g) or beef extract (2170 U/g) and moderately (36%-46%) by yeast extract (1800 U/g) or soybean meal (1670 U/g). Inclusion of readily utilizable sugars such as glucose, maltose, fructose, lactose or xylose in the substrate repressed the xylanase production. The optimum initial pH of the medium for maximum enzyme production was 7 to 8; however, appreciable level of activity was obtained at pH 6 (1,680 U/g) and 9 (1,900 U/g). Most appropriate solid to liquid ratio for maximum xylanase production in solid-state fermentation was found to be 1:2.5. The organism produced a single xylanase of molecular weight of approximately 30 kDa as analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis after purification with ammonium sulfate precipitation, and carboxy methyl sephadex chromatography. The enzyme was purified to the extent of 5.68-fold by salt precipitation and ion-exchange chromatography. Optimum temperature and pH for maximum xylanase activity were 50 degrees C and 6, respectively.

Production of xylanases by mangrove fungi from the Philippines and their application in enzymatic pretreatment of recycled paper pulps.

PubMed

Torres, Jeremy Martin O; Dela Cruz, Thomas Edison E

2013-04-01

Mangrove fungi are vastly unexplored for enzymes with industrial application. This study aimed to assess the biocatalytic activity of mangrove fungal xylanases on recycled paper pulp. Forty-four mangrove fungal (MF) isolates were initially screened for xylanolytic activity in minimal medium with corn cob xylan as the sole carbon source. Eight MF were further cultivated under submerged fermentation for the production of crude xylanases. These crude enzymes were then characterized and tested for the pretreatment of recycled paper pulps. Results showed that 93 % of the tested MF isolates exhibited xylanolytic activity in solid medium. In submerged fermentation, salinity improved the growth of the fungal isolates but did not influence xylanase production. The crude xylanases were mostly optimally active at 50 °C and pH 7. Changes in pH had a greater effect on xylanase stability than temperature. More than half of the activity was lost at pH 9 for majority of the crude enzymes. However, two thermophilic xylanases from Fusarium sp. KAWIT-A and Aureobasidium sp. 2LIPA-M and one alkaliphilic xylanase from Phomopsis sp. MACA-J were also produced. All crude enzymes exhibited cellulase activities ranging from 4 to 21 U/ml. Enzymatic pretreatment of recycled paper pulps with 5 % consistency produced 70-650 mg of reducing sugars per gram of pulp at 50 °C after 60 min. The release of high amounts of reducing sugars showed the potential of mangrove fungal crude xylanases in the local paper and pulp industry. The diverse properties shown by the tested crude enzymes also indicate its potential applications to other enzyme-requiring industries.

A novel method for simultaneous purification and immobilization of a xylanase-lichenase chimera via SpyTag/SpyCatcher spontaneous reaction.

PubMed

Lin, Yuanqing; Jin, Wenhui; Wang, Jindan; Cai, Zhengwen; Wu, Shuyu; Zhang, Guangya

2018-08-01

We generated a bifunctional enzyme chimera containing the xylanase and lichenase coupled with SpyTag between them. Meanwhile, we generated another chimera containing SpyCatcher and elastin-like polypeptides (ELPs). As ELPs could bond to the xylanase-lichenase chimera through SpyTag/SpyCatcher spontaneous reaction in mild condition, which would lead to the formation of a 3-arm star multifunctional chimera. We purified the xylanase-lichenase by the non-chromatographic purification tag of ELPs. Interestingly, 57.5% of the xylanase and 47.2% of the lichenase in chimera self-assembled into insoluble active particles during the process of purification, which could serve as immobilized bifunctional enzymes. Notably, the immobilized chimera xylanase-lichenase showed a remarkable stability even after 10 reaction cycles, which retained around 56% (lichenase) and 44% (xylanase) of their initial activities, respectively. Moreover, the enhanced thermostability of the immobilized enzymes was also achieved. After incubating at 60 °C for 60 min, the residual activity of the immobilized lichenase was 35%, while the free one was only 24%. Unexpectedly, the free xylanase almost lost its activity when incubated at 55 °C for 60 min, whereas the immobilized xylanase retained 10% of its activity. However, the catalytic efficiency (k cat /K m ) of the free xylanase was 1.7-fold higher than the immobilized one, while the free lichenase was 1.1-fold higher than the immobilized one. This is among the first known reports that two enzymes are purified and immobilized in one-step. This novel strategy is easy to scale up and may meet the demands of biofuel industry. It would have great potentials in other biotechnological fields, such as the multifunctional biomaterials systems. Copyright © 2018 Elsevier Inc. All rights reserved.

Synergism of cellulase, xylanase, and pectinase on hydrolyzing sugarcane bagasse resulting from different pretreatment technologies.

PubMed

Li, Jingbo; Zhou, Pengfei; Liu, Hongmei; Xiong, Chunjiang; Lin, Jianghai; Xiao, Wenjuan; Gong, Yingxue; Liu, Zehuan

2014-03-01

Sugarcane bagasse (SCB) resulting from different pretreatments was hydrolyzed by enzyme cocktails based on replacement of cellulase (Celluclast 1.5 L:Novozym 188=1FPU:4pNPGU) by xylanase or pectinase at different proportions. Lignin content of NaOH pretreated SCB and hemicellulose content of H2SO4 pretreated SCB were the lowest. NaOH pretreatment showed the best for monosaccharide production among the four pretreatments. Synergism was apparently observed between cellulase and xylanase for monosaccharide production from steam exploded SCB (SESB), NaOH, and H2O2 pretreated SCB. No synergism was observed between cellulase and pectinase for producing glucose. Additionally, no synergism was present when H2SO4 pretreated SCB was used. Replacement of 20% of the cellulase by xylanase enhanced the glucose yield by 6.6%, 8.8%, and 9.5% from SESB, NaOH, and H2O2 pretreated SCB, respectively. Degree of synergism between cellulase and xylanase had positive relationship with xylan content and was affected by hydrolysis time. Copyright © 2014 Elsevier Ltd. All rights reserved.

Combination of Xylanase and Debranching Enzymes Specific to Wheat Arabinoxylan Improve the Growth Performance and Gut Health of Broilers.

PubMed

Lei, Zhao; Shao, Yuxin; Yin, Xiaonan; Yin, Dafei; Guo, Yuming; Yuan, Jianmin

2016-06-22

Arabinoxylan (AX) is the major antinutritional factor of wheat. This study evaluated the synergistic effects of xylanase and debranching enzymes (arabinofuranosidase [ABF] and feruloyl esterase [FAE]) on AX. During in vitro tests, the addition of ABF or FAE accelerated the hydrolysis of water-soluble AX (WE-AX) and water-insoluble AX (WU-AX) and produced more xylan oligosaccharides (XOS) than xylanase alone. XOS obtained from WE-AX stimulated greater proliferation of Lactobacillus brevis and Bacillus subtilis than did fructo-oligosaccharides (FOS) and glucose. During in vivo trials, xylanase increased the average daily growth (ADG), decreased the feed-conversion ratio (FCR), and reduced the digesta viscosity of jejunum and intestinal lesions of broilers fed a wheat-based diet on day 36. ABF or FAE additions further improved these effects. Broilers fed a combination of xylanase, ABF, and FAE exhibited the best growth. In conclusion, the synergistic effects among xylanase, ABF, and FAE increased AX degradation, which improve the growth performance and gut health of broilers.

Interactions between xylanase and glucanase in maize-soy-based diets for broilers.

PubMed

Cowieson, A J; Bedford, M R; Ravindran, V

2010-04-01

1. The effect of various doses of xylanase and glucanase on the performance and ileal nutrient digestibility of broiler chickens fed on maize/soy-based diets was evaluated. 2. A total of 960 male broilers were used in separate growth and digestibility trials with each involving 10 treatments and 6 replicates. The 10 treatments included a positive control reference diet, a negative control diet with a lower energy density and 8 further diets where xylanase and glucanase were added to the negative control, individually and in combination. 3. Birds which received the negative control diet returned poorer (6 points; P < 0.05) feed conversion ratios compared with those fed on the positive control, confirming the lower energy density of the negative control diet. Ileal digestibility of energy determined at 21 and 42 d was also significantly lower for the negative control compared with the positive control. At d 21 birds that received the negative control diet returned lower ileal amino acid digestibility (for most amino acids) compared with their counterparts fed on the positive control whereas at d 42 this effect was not apparent. 4. Supplementation of the negative control with both glucanase and xylanase improved feed conversion ratio (FCR) and ileal nutrient digestibility. When both enzymes were added simultaneously a sub-additive effect (i.e. simultaneous use resulted in benefits greater than either enzyme independently but less than the sum of the individual effects) was observed, with the best performance achieved with the combination of xylanase (16 000 BXU/kg) and glucanase (30 000 BU/kg). 5. It can be concluded that the removal of oil to accommodate the anticipated energy digestibility improvement with enzymes can have deleterious effects on FCR and ileal amino acid digestibility in young broilers which enzymes may not adequately mitigate. Thus, in order to maximise the response to non-starch polysaccharide degrading enzymes in maize/soy-based broiler diets, it

Production of xylanase and β-xylosidase from autohydrolysis liquor of corncob using two fungal strains.

PubMed

Michelin, Michele; Polizeli, Maria de Lourdes T M; Ruzene, Denise S; Silva, Daniel P; Ruiz, Héctor A; Vicente, António A; Jorge, João A; Terenzi, Héctor F; Teixeira, José A

2012-09-01

Agroindustrial residues are materials often rich in cellulose and hemicellulose. The use of these substrates for the microbial production of enzymes of industrial interest is mainly due to their high availability associated with their low cost. In this work, corncob (CCs) particles decomposed to soluble compounds (liquor) were incorporated in the microbial growth medium through autohydrolysis, as a strategy to increase and undervalue xylanase and β-xylosidase production by Aspergillus terricola and Aspergillus ochraceus. The CCs autohydrolysis liquor produced at 200 °C for 5, 15, 30 or 50 min was used as the sole carbon source or associated with untreated CC. The best condition for enzyme synthesis was observed with CCs submitted to 30 min of autohydrolysis. The enzymatic production with untreated CCs plus CC liquor was higher than with birchwood xylan for both microorganisms. A. terricola produced 750 total U of xylanase (144 h cultivation) and 30 total U of β-xylosidase (96-168 h) with 0.75% untreated CCs and 6% CCs liquor, against 650 total U of xylanase and 2 total U of β-xylosidase in xylan; A. ochraceus produced 605 total U of xylanase and 56 total U of β-xylosidase (168 h cultivation) with 1% untreated CCs and 10% CCs liquor against 400 total U of xylanase and 38 total U of β-xylosidase in xylan. These results indicate that the treatment of agroindustrial wastes through autohydrolysis can be a viable strategy in the production of high levels of xylanolytic enzymes.

Development of a bifunctional xylanase-cellulase chimera with enhanced activity on rice and barley straws using a modular xylanase and an endoglucanase procured from camel rumen metagenome.

PubMed

Khalili Ghadikolaei, Kamran; Akbari Noghabi, Kambiz; Shahbani Zahiri, Hossein

2017-09-01

The camel rumen metagenome is an untapped source of glycoside hydrolases. In this study, novel genes encoding for a modular xylanase (XylC) and a cellulase (CelC) were isolated from a camel rumen metagenome and expressed in Escherichia coli BL21 (DE3). XylC with xylanase (Xyn), CBM, and carbohydrate esterase (CE) domains was characterized as a β-1,4-endoxylanase with remarkable catalytic activity on oat-spelt xylan (K cat  = 2919 ± 57 s -1 ). The implication of XylC's modular structure in its high catalytic activity was analyzed by truncation and fusion construction with CelC. The resulting fusions including Cel-CBM, Cel-CBM-CE, and Xyn-CBM-Cel showed remarkable enhancement in CMCase activity with K cat values of 742 ± 12, 1289 ± 34.5, and 2799 ± 51 s -1 compared to CelC with a K cat of 422 ± 3.5 s -1 . It was also shown that the bifunctional Xyn-CBM-Cel with synergistic xylanase/cellulase activities was more efficient than XylC and CelC in hydrolysis of rice and barley straws.

Growth performance, nutrient utilization, and digesta characteristics in broiler chickens fed corn or wheat diets without or with supplemental xylanase.

PubMed

Kiarie, E; Romero, L F; Ravindran, V

2014-05-01

Efficacy of supplemental xylanase on growth performance, nutrient utilization, and digesta characteristics in broiler chickens fed corn- or wheat-based diets was investigated. In experiment 1, 192 male broilers (8 birds/pen; n = 6) were fed 4 diets (corn or wheat without or with 1,250 xylanase units/kg) in 2 phases (starter, d 0-21 and grower, d 22-42). There was no interaction (P > 0.05) between diet and xylanase on performance (d 0-42). Wheat diets resulted (P < 0.01) in better performance than corn diets, whereas xylanase-fed birds had improved (P < 0.01) BW gain (2,457 vs. 2,275 g) and feed per gain (1.677 vs. 1.762) relative to birds not fed xylanase. In experiment 2, TiO2 (0.3%) was added in starter diets used in experiment 1, allocated to 13-d-old broiler chicks (n = 6) housed in cages (6 birds/cage) and fed up to d 21. Excreta samples were obtained from d 17 to 20 and birds were euthanized on d 21 for digesta. Corn diets had a greater concentration (10.7 vs. 9.8%) of insoluble nonstarch polysaccharides (NSP) than wheat diets, which in turn had more than twice the concentration of soluble NSP. There was an interaction (P < 0.03) between diet type and xylanase on jejunal digesta viscosity but not (P > 0.10) on apparent ileal digestibilities of nutrients, cecal volatile fatty acids, and AMEn. In this context, diet type influenced (P < 0.05) cecal volatile fatty acids and retention of nutrients and fiber but did not affect (P = 0.45) AMEn. In contrast, xylanase-fed birds showed higher (P < 0.05) ceca digesta acetic acid, apparent ileal digestibilities of nutrients, and retention of components. As a result, birds fed xylanase had higher AMEn (3,059 vs. 2,995 kcal/kg; P < 0.01) compared with birds not fed xylanase. Although wheat diets had superior growth performance, the AMEn was similar in both diets. Xylanase improved growth performance and AMEn independent of diet type, suggesting hydrolysis of both soluble and insoluble NSP.

Production of beta-xylanase by a Thermomyces lanuginosus MC 134 mutant on corn cobs and its application in biobleaching of bagasse pulp.

PubMed

Kumar, Kuttanpillai Santhosh; Manimaran, Ayyachamy; Permaul, Kugen; Singh, Suren

2009-05-01

The production of hemicellulases by Thermomyces lanuginosus SK using oatspelts xylan was examined during submerged cultivation. A high level of extracellular xylanase (346+/-10 U ml(-1)) production was observed on the fifth day; however, accessory enzyme levels were low. T. lanuginosus SK was further subjected to UV and N-methyl-N-nitro-N-nitrosoguanidine mutagenesis. The T. lanuginosus MC 134 mutant showed a 1.5 fold increase in xylanase production on oatspelts xylan, compared to the wild type strain. Xylanase production was further enhanced to 3299+/-95 U ml(-1) by using corn cobs under optimized growth conditions. A reduction in xylanase production was observed in a 5 L fermenter. Also, the biobleaching efficiency of crude xylanase was evaluated on bagasse pulp, and a brightness of 46.07+/-0.05% was observed with the use of 50 U of crude xylanase per gram of pulp. This brightness was 3.6 points higher than that of the untreated samples. Reducing sugars (25.78+/-0.14 mg g(-1)) and UV-absorbing lignin-derived compound values were considerably higher in xylanase-treated samples. T. lanuginosus MC 134 has a potential application in the pulp and paper industries.

Production of a cellulase-free alkaline xylanase from Bacillus pumilus MTCC 5015 by submerged fermentation and its application in biobleaching.

PubMed

Thomas, Leya; Sindhu, Raveendran; Binod, Parameswaran; Pandey, Ashok

2015-06-01

Here, we described the production of a cellulase-free alkaline xylanase from Bacillus pumilus MTCC 5015 by submerged fermentation and its application in biobleaching. Various process parameters affecting xylanase production by B. pumilus were optimized by adopting a Plackett-Burman design (PBD) as well as Response surface methodology (RSM). These statistical methods aid in improving the enzyme yield by analysing the individual crucial components of the medium. Maximum production was obtained with 4% yeast extract, 0.08% magnesium sulphate, 30 h of inoculum age, incubation temperature of 33.5 degrees C and pH 9.0. Under optimized conditions, the xylanase activity was 372 IU/ml. Media engineering improved a 5-fold increase in the enzyme production. Scanning electron microscopy (SEM) showed significant changes on the surface of xylanase treated pulps as a result of xylan hydrolysis. Increased roughness of paper carton fibres was apparent in scanning electron micrograph due to opening of the micro fibrils present on the surface by xylanase action. The untreated pulp did not show any such change. These results demonstrated that the B. pumilus MTCC 5015 xylanase was effective in bio-bleaching of paper carton.

Cloning, Expression and Characteristics of a Novel Alkalistable and Thermostable Xylanase Encoding Gene (Mxyl) Retrieved from Compost-Soil Metagenome

PubMed Central

Verma, Digvijay; Kawarabayasi, Yutaka; Miyazaki, Kentaro; Satyanarayana, Tulasi

2013-01-01

Background The alkalistable and thermostable xylanases are in high demand for pulp bleaching in paper industry and generating xylooligosaccharides by hydrolyzing xylan component of agro-residues. The compost-soil samples, one of the hot environments, are expected to be a rich source of microbes with thermostable enzymes. Methodology/Principal Findings Metagenomic DNA from hot environmental samples could be a rich source of novel biocatalysts. While screening metagenomic library constructed from DNA extracted from the compost-soil in the p18GFP vector, a clone (TSDV-MX1) was detected that exhibited clear zone of xylan hydrolysis on RBB xylan plate. The sequencing of 6.321 kb DNA insert and its BLAST analysis detected the presence of xylanase gene that comprised 1077 bp. The deduced protein sequence (358 amino acids) displayed homology with glycosyl hydrolase (GH) family 11 xylanases. The gene was subcloned into pET28a vector and expressed in E. coli BL21 (DE3). The recombinant xylanase (rMxyl) exhibited activity over a broad range of pH and temperature with optima at pH 9.0 and 80°C. The recombinant xylanase is highly thermostable having T1/2 of 2 h at 80°C and 15 min at 90°C. Conclusion/Significance This is the first report on the retrieval of xylanase gene through metagenomic approach that encodes an enzyme with alkalistability and thermostability. The recombinant xylanase has a potential application in paper and pulp industry in pulp bleaching and generating xylooligosaccharides from the abundantly available agro-residues. PMID:23382818

Screening of Thermophilic Bacteria Produce Xylanase from Sapan Sungai Aro Hot Spring South Solok

NASA Astrophysics Data System (ADS)

Irdawati, I.; Syamsuardi, S.; Agustien, A.; Rilda, Y.

2018-04-01

xylanase is one of the enzymes with great prospects as hemicellulose hydrolyzing enzyme. Global annual market demand for this enzyme reach US 200 million. This enzyme catalyzes the xylan (hemicellulose) reactions breaking into xilooligosakarida and xylose. Xylanase can be applied to various industrial sectors such as bread, sugar xylose, biofuels, especially in bleaching paper (bleaching) pulp. Xylanase Isable to replace conventional chemical bleaching using chlorine that is not friendly for the environment. Currently xylanase production is extracted from the thermophilic bacteria for enzyme stability at high temperatures that are suitable for industrial applications. Thermophilic bacteria can be isolated from a hot spring, one of the which is a source of Sapan Sungai Aro Hot Spring, located in the district South Solok. The aim of this study was to select and identification of thermophilic bacteria can produce xylanase.This roomates is a descriptive study, which was Carried out in the Laboratory of Microbiology, Mathematic and Science Faculty of Padang State University, and Laboratory of Bacteriology, BasoVeterinary Research Center. The research procedure consisted of the preparation and sterilization of materials and tools, medium manufacturing, regeneration, selection and identification. Selection is performed by using a semiquantitative screening plate that contains xylan substrate. Identification is based on microscopic and biochemical characteristics until the genus level.Selection results Showed 12 out of 16 isolates had xilanolitik activity, with the highest activity is SSA2 with xilanolitik index of 0.74. The top five index producehigestxilanolitik isolates that are SSA2, SSA3 and SSA4 identified as Bacillus sp. 1., and SSAS6 and SSA7 is Bacillus sp. 2.

Production, partial purification and characterization of xylanase using Nicotiana tabacum leaf dust as substrate.

PubMed

Acharya, Komal P; Shilpkar, Prateek

2016-03-01

Isolated Bacillus sp. was used in the present study for production of xylanase from Nicotiana tabacum leaf dust. The strain was able to give a maximum of 1.77 Uml⁻¹ xylanase activity under optimized fermentation conditions which was further increased upto 2.77 Uml⁻¹ after extraction and partial purification of enzyme. After partial purification, the enzyme was characterized and it gave the highest xylanase activity at pH 7.0, when 0.2 ml enzyme was incubated with 2.0% substrate (Nicotiana tabacum leaf dust) for 60 min at 60°C. Saccharification study of Nicotiana tabacum leaf dust with partially purified enzyme revealed that 18.4% reducing sugar was released in 20 hrs incubation, and TLC and HPTLC analysis showed that xylose and glucose sugars were obtained after hydrolysis of substrate. FTIR analysis confirmed decomposition of substrate.

Polypeptides having xylanase activity and polynucleotides encoding the same

DOEpatents

Spodsberg, Nikolaj [Bagsvaed, DK

2014-01-07

The present invention relates to isolated polypeptides having xylanase activity and isolated polynucleotides encoding the polypeptides. The inventino also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Oligomerization triggered by foldon: a simple method to enhance the catalytic efficiency of lichenase and xylanase.

PubMed

Wang, Xinzhe; Ge, Huihua; Zhang, Dandan; Wu, Shuyu; Zhang, Guangya

2017-07-03

Effective and simple methods that lead to higher enzymatic efficiencies are highly sough. Here we proposed a foldon-triggered trimerization of the target enzymes with significantly improved catalytic performances by fusing a foldon domain at the C-terminus of the enzymes via elastin-like polypeptides (ELPs). The foldon domain comprises 27 residues and can forms trimers with high stability. Lichenase and xylanase can hydrolyze lichenan and xylan to produce value added products and biofuels, and they have great potentials as biotechnological tools in various industrial applications. We took them as the examples and compared the kinetic parameters of the engineered trimeric enzymes to those of the monomeric and wild type ones. When compared with the monomeric ones, the catalytic efficiency (k cat /K m ) of the trimeric lichenase and xylanase increased 4.2- and 3.9- fold. The catalytic constant (k cat ) of the trimeric lichenase and xylanase increased 1.8- fold and 5.0- fold than their corresponding wild-type counterparts. Also, the specific activities of trimeric lichenase and xylanase increased by 149% and 94% than those of the monomeric ones. Besides, the recovery of the lichenase and xylanase activities increased by 12.4% and 6.1% during the purification process using ELPs as the non-chromatographic tag. The possible reason is the foldon domain can reduce the transition temperature of the ELPs. The trimeric lichenase and xylanase induced by foldon have advantages in the catalytic performances. Besides, they were easier to purify with increased purification fold and decreased the loss of activities compared to their corresponding monomeric ones. Trimerizing of the target enzymes triggered by the foldon domain could improve their activities and facilitate the purification, which represents a simple and effective enzyme-engineering tool. It should have exciting potentials both in industrial and laboratory scales.

Enhanced production of xylanase from locally isolated fungal strain using agro-industrial residues under solid-state fermentation.

PubMed

Abdullah, Roheena; Nisar, Kinza; Aslam, Aafia; Iqtedar, Mehwish; Naz, Shagufta

2015-01-01

This study is related to the isolation of fungal strain for xylanase production using agro-industrial residues. Forty fungal strains with xylanolytic potential were isolated by using xylan agar plates and quantitatively screened in solid-state fermentation. Of all the tested isolates, the strain showing highest ability to produce xylanase was assigned the code Aspergillus niger LCBT-14. For the enhanced production of the enzyme, five different fermentation media were evaluated. Out of all media, M4 containing wheat bran gave maximum enzyme production. Effect of different variables including incubation time, temperature, pH, carbon and nitrogen sources has been investigated. The optimum enzyme production was obtained after 72 h at 30°C and pH 4. Glucose as a carbon source while ammonium sulphate and yeast extract as nitrogen sources gave maximum xylanase production (946 U/mL/min). This study was successful in producing xylanase by A. niger LCBT-14 economically by utilising cheap indigenous substrate.

A xylose-stimulated xylanase-xylose binding protein chimera created by random nonhomologous recombination.

PubMed

Ribeiro, Lucas Ferreira; Tullman, Jennifer; Nicholes, Nathan; Silva, Sérgio Ruschi Bergamachi; Vieira, Davi Serradella; Ostermeier, Marc; Ward, Richard John

2016-01-01

Saccharification of lignocellulosic material by xylanases and other glycoside hydrolases is generally conducted at high concentrations of the final reaction products, which frequently inhibit the enzymes used in the saccharification process. Using a random nonhomologous recombination strategy, we have fused the GH11 xylanase from Bacillus subtilis (XynA) with the xylose binding protein from Escherichia coli (XBP) to produce an enzyme that is allosterically stimulated by xylose. The pT7T3GFP_XBP plasmid containing the XBP coding sequence was randomly linearized with DNase I, and ligated with the XynA coding sequence to create a random XynA-XBP insertion library, which was used to transform E. coli strain JW3538-1 lacking the XBP gene. Screening for active XBP was based on the expression of GFP from the pT7T3GFP_XBP plasmid under the control of a xylose inducible promoter. In the presence of xylose, cells harboring a functional XBP domain in the fusion protein (XBP+) showed increased GFP fluorescence and were selected using FACS. The XBP+ cells were further screened for xylanase activity by halo formation around xylanase producing colonies (XynA+) on LB-agar-xylan media after staining with Congo red. The xylanase activity ratio with xylose/without xylose in supernatants from the XBP+/XynA+ clones was measured against remazol brilliant blue xylan. A clone showing an activity ratio higher than 1.3 was selected where the XynA was inserted after the asparagine 271 in the XBP, and this chimera was denominated as XynA-XBP271. The XynA-XBP271 was more stable than XynA at 55 °C, and in the presence of xylose the catalytic efficiency was ~3-fold greater than the parental xylanase. Molecular dynamics simulations predicted the formation of an extended protein-protein interface with coupled movements between the XynA and XBP domains. In the XynA-XBP271 with xylose bound to the XBP domain, the mobility of a β-loop in the XynA domain results in an increased access to the

Energy and nutrient utilization of broiler chickens fed corn-soybean meal and corn-based diets supplemented with xylanase.

PubMed

Stefanello, C; Vieira, S L; Carvalho, P S; Sorbara, J O B; Cowieson, A J

2016-08-01

A study was conducted to evaluate the effects of increased levels of a β-xylanase on energy and nutrient utilization of broiler chickens fed corn-soy diets. A total of 480 slow feathering Cobb × Cobb 500 male broilers were randomly distributed to 10 treatments having 8 replicates of 6 birds each. Birds were fed a common starter diet to d 14 post hatch (3,050 kcal/kg AMEn, 21.7% CP, 1.05% Ca, and 0.53% nPP). The experimental diets were provided afterwards until 25 d. Two experimental diets, a conventional corn/soy-based basal diet (CS) and the basal diet in which 40% of the diet was displaced by corn (CN), were fed as-is or supplemented with 50, 100, 150, or 200 fungal β-xylanase units (FXU)/kg. Dietary treatments were distributed factorially as a 2 × 5 arrangement. Samples of feed, excreta, and ileal digesta were analyzed for determination of ileal digestible energy (IDE), metabolizable energy, and total tract retention of protein and lipid. No interactions between diet and xylanase were observed. The CS diets had higher (P < 0.05) energy utilization and nutrient digestibility when compared to the CN diets. AMEn and IDE were improved (P < 0.05) by 192 and 145 kcal/kg, respectively, when diets were supplemented with 100 FXU/kg xylanase. The xylanase added to the CN diet led to quadratic increases (P < 0.05) in IDE (Y = - 0.014x(2) + 2.570x + 3,155; r(2) = 0.60) and in AMEn (Y = - 0.016x(2) + 3.982x + 3,155; r(2) = 0.68). Crude protein digestibility and AMEn were linearly increased (P < 0.05) when xylanase was added to the CN diet. In conclusion, energy utilization and digestibility of crude protein and dry matter increased with xylanase supplementation in corn/soy-based diets. When xylanase was tested in the CS diet, 92 and 124 FXU/kg maximized the energy release effect; however, the maximum energy response in the CN diet or corn was not achieved until 200 FXU/kg. © 2016 Poultry Science Association Inc.

Production of Sporotrichum thermophile xylanase by solid state fermentation utilizing deoiled Jatropha curcas seed cake and its application in xylooligosachharide synthesis.

PubMed

Sadaf, Ayesha; Khare, S K

2014-02-01

De-oiled Jatropha curcas seed cake, a plentiful by-product of biodiesel industry was used as substrate for the production of a useful xylanase from Sporotrichum thermophile in solid state fermentation. Under the optimized conditions, 1025U xylanase/g (deoiled seed cake) was produced. The xylanase exhibited half life of 4h at 45°C and 71.44min at 50°C respectively. It was stable in a broad pH range of 7.0-11.0. Km and Vmax were 12.54mg/ml and 454.5U/ml/min respectively. S. thermophile xylanase is an endoxylanase free of exoxylanase activity, hence advantageous for xylan hydrolysis to produce xylooligosachharides. Hydrolysis of oat spelt xylan by S. thermophile xylanase yielded 73% xylotetraose, 15.4% xylotriose and 10% xylobiose. The S. thermophile endoxylanase thus seem potentially useful in the food industries. Copyright © 2013 Elsevier Ltd. All rights reserved.

Polypeptides having xylanase activity and polynucleotides encoding same

DOE Office of Scientific and Technical Information (OSTI.GOV)

Spodsberg, Nikolaj; Shaghasi, Tarana

The present invention relates to polypeptides having xylanase activity, catalytic domains, and carbohydrate binding domains, and polynucleotides encoding the polypeptides, catalytic domains, and carbohydrate binding domains. The present invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides, catalytic domains, and carbohydrate binding domains.

Molecular Dynamics Approach in Designing Thermostable Aspergillus niger Xylanase

NASA Astrophysics Data System (ADS)

Malau, N. D.; Sianturi, M.

2017-03-01

Molecular dynamics methods we have applied as a tool in designing thermostable Aspergillus niger Xylanase, by examining Root Mean Square Deviation (RMSD) and The Stability of the Secondary Structure of enzymes structure at its optimum temperature and compare with its high temperature behavior. As RMSD represents structural fluctuation at a particular temperature, a better understanding of this factor will suggest approaches to bioengineer these enzymes to enhance their thermostability. In this work molecular dynamic simulations of Aspergillus niger xylanase (ANX) have been carried at 400K (optimum catalytic temperature) for 2.5 ns and 500K (ANX reported inactive temperature) for 2.5 ns. Analysis have shown that the Root Mean Square Deviation (RMSD) significant increase at higher temperatures compared at optimum temperature and some of the secondary structures of ANX that have been damaged at high temperature. Structural analysis revealed that the fluctuations of the α-helix and β-sheet regions are larger at higher temperatures compared to the fluctuations at optimum temperature.

Enzymatic saccharification of seaweeds into fermentable sugars by xylanase from marine Bacillus sp. strain BT21.

PubMed

Parab, Pankaj; Khandeparker, Rakhee; Amberkar, Ujwala; Khodse, Vishwas

2017-10-01

Enzymatic hydrolysis of seaweed biomass was studied using xylanase produced from marine bacteria Bacillus sp. strain BT21 through solid-state fermentation of wheat bran. Three types of seaweeds, Ahnfeltia plicata , Padina tetrastromatica and Ulva lactuca , were selected as representatives of red, brown, and green seaweeds, respectively. Seaweed biomass was pretreated with hot water. The efficiency of pretreated biomass to release reducing sugar by the action of xylanase as well as the type of monosaccharide released during enzyme saccharification of seaweed biomass was studied. It was seen that pretreated biomass of seaweed A. plicata, U. lactuca , and P. tetrastroma , at 121 °C for 45 min, followed by incubation with 50 IU xylanase released reducing sugars of 233 ± 5.3, 100 ± 6.1 and 73.3 ± 4.1 µg/mg of seaweed biomass, respectively. Gas chromatography analysis illustrated the release of xylose, glucose, and mannose during the treatment process. Hot water pre-treatment process enhanced enzymatic conversion of biomass into sugars. This study revealed the important role of xylanase in saccharification of seaweed, a promising feedstock for third-generation bioethanol production.

Cloning and in-silico analysis of beta-1,3-xylanase from psychrophilic yeast, Glaciozyma antarctica PI12

NASA Astrophysics Data System (ADS)

Nor, Nooraisyah Mohamad; Bakar, Farah Diba Abu; Mahadi, Nor Muhammad; Murad, Abdul Munir Abdul

2015-09-01

A beta-1,3-xylanase (EC 3.2.1.32) gene from psychrophilic yeast, Glaciozyma antarctica has been identified via genome data mining. The enzyme was grouped into GH26 family based on Carbohydrate Active Enzyme (CaZY) database. The molecular weight of this protein was predicted to be 42 kDa and is expected to be soluble for expression. The presence of signal peptide suggested that this enzyme may be released extracellularly into the marine environment of the host's habitat. This supports the theory that such enzymatic activity is required for degradation of nutrients of polysaccharide origins into simpler carbohydrates outside the environment before it could be taken up inside the cell. The sequence for this protein showed very little conservation (< 30%) with other beta-1,3-xylanases from available databases. Based on the phylogenetic analysis, this protein also showed distant relationship to other xylanases from eukaryotic origin. The protein may have undergone major substitution in its gene sequence order to adapt to the cold climate. This is the first report of beta-1,3-xylanase gene isolated from a psychrophilic yeast.

Bleach boosting effect of xylanase A from Bacillus halodurans C-125 in ECF bleaching of wheat straw pulp.

PubMed

Lin, Xiao-qiong; Han, Shuang-yan; Zhang, Na; Hu, Hui; Zheng, Sui-ping; Ye, Yan-rui; Lin, Ying

2013-02-05

Past studies have revealed major difficulties in applications of xylanase in the pulp and paper industry as enzymes isolated from many different species could not tolerate high temperatures or highly alkaline conditions. The thermostable xylanase A from Bacillus halodurans C-125 (C-125 xylanase A) was successfully cloned and expressed in Pichia pastoris with a yield as high as 3361 U/mL in a 2 L reactor. Its thermophilic and basophilic properties (optimal activity at 70 °C and pH 9.0), together with the fact it is cellulase-free, render this enzyme attractive for compatible applications in the pulp and paper industry. The pretreatment of wheat straw pulp with C-125 xylanase A at pH 9.0 and 70 °C for 90 min induced the release of both chromophores (Ab(237), Ab(254), Ab(280)) and hydrophobic compounds (Ab(465)) into the filtrate as well as sugar degradation. Moreover, the addition of 10 U xylanase to 1 g wheat straw pulp (dry weight) as pretreatment improved brightness by 5.2% ISO and decreased the kappa number by 5.0% when followed by hydrogen peroxide bleaching. In addition, compared with two commercial enzymes, Pulpzyme HC and AU-PE89, which are normally incorporated in ECF bleaching of wheat straw pulp, C-125 xylanase A proved to be more effective in enhancing brightness as well as preserving paper strength properties. When evaluating the physical properties of pulp samples, such as tensile index, tearing index, bursting index, and post-color (PC) number, the enzymes involved in pretreating pulps exhibited better or the same performances as chemical treatment. Compared with chemical bleaching, chlorine consumption can be significantly reduced by 10% for xylanase-pretreated wheat straw pulp while maintaining the brightness together with the kappa number at the same level. Scanning electron microscopy revealed significant surface modification of enzyme-pretreated pulp fibers with no marked fiber disruptions. Copyright © 2012 Elsevier Inc. All rights reserved.

[Correlation between thermostability of the xylanase EvXyn11(TS) and its N-terminal disulfide bridge].

PubMed

Min, Rou; Li, Jianfang; Gao, Shujuan; Zhang, Huimin; Wu, Jing; Wu, Minchen

2013-04-04

To reveal the correlation between thermostability of xylanase EvXyn11(TS) and its N-terminal disulfide bridge, an EvXyn11(TS)-encoding gene (Syxyn11) was synthesized and subjected to site-directed mutagenesis. Multiple homology alignment of protein primary structures between the EvXyn11(TS) and several GH family 11 xylanases displayed that, in their N-termini, only EvXyn11(TS) contained a disulfide bridge (Cys5-Cys32), whose effect on the xylanase thermostability was predicted by molecular dynamics simulation. We constructed a gene Syxyn11(M), encoding the mutated xylanase (EvXyn11(M)) without N-terminal disulfide bridge. Then, Syxyn11 and Syxyn11(M) were expressed in Pichia pastoris GS115, and temperature and pH properties of the expressed enzymes were analyzed. The analytical results displayed that the temperature optimum of EvXyn11(M) was 70 degrees C, which was 15 degrees C lower than that of EvXyn11(TS). The half-life (t1/2(90)) of EvXyn11(TS) at 90 degrees C was 32 min, while the t1/2(70) of EvXyn11(M) at 70 degrees C was only 8.0 min. The important role of the N-terminal disulfide bridge on the thermostability of EvXyn11(TS) was first predicted by molecular dynamics simulation, and confirmed by site-directed mutagenesis. This work provided a novel strategy to improve thermostabilities of the mesophilic family 11 xylanases with high specific activities.

Optimization of moistening solution concentration on xylanase activity in solid state fermentation from oil palm empty fruit bunches

NASA Astrophysics Data System (ADS)

Mardawati, Efri; Parlan; Rialita, Tita; Nurhadi, Bambang

2018-03-01

Xylanase is an enzyme used in the industrial world, including food industry. Xylanase can be utilized as a 1,4-β-xylosidic endo-hydrolysis catalyst of xylanase, a hemicellulose component for obtaining a xylose monomer. This study aims to determine the optimum concentration of the fermentation medium using Response Surface Method (RSM) in the production of xylanase enzyme from oil palm empty fruit bunches (OPEFB) through solid state fermentation process. The variables varied in this study used factor A (ammonium sulphate concentration 1.0-2.0 g/L), B (concentration of potassium dihydrogen phosphate 1.5-2.5 g/L) and C (urea concentration 0.2 – 0.5 g/L). The data was analysed by using Design Expert version 10.0.1.0 especially CCD with total 17 running including 3 times replicated of canter point. Trichoderma viride was used for the process production of xylanase enzyme. The ratio between substrate and moistening solution used was 0.63 g / mL with temperature of 32.80C, 60 h incubation time. The analysis of enzyme activity was done by DNS method with 1% xylan as substrate. Analysis of protein content in enzyme was done by Bradford method. The optimum of moistening solution concentration in this fermentation was obtained. They are, the ammonium sulphate concentration of 1.5 g/L, potassium dihydrogen phosphate 2.0 g/L and urea 0.35 g/L with activity of 684.70 U/mL, specific activity enzyme xylanase 6261.58 U/mg, protein content 0.1093 U/mg, the model was validated using experiment design with perfect reliability value 0.96.

Engineering improved thermostability of the GH11 xylanase from Neocallimastix patriciarum via computational library design.

PubMed

Bu, Yifan; Cui, Yinglu; Peng, Ying; Hu, Meirong; Tian, Yu'e; Tao, Yong; Wu, Bian

2018-04-01

Xylanases, which cleave the β-1,4-glycosidic bond between xylose residues to release xylooligosaccharides (XOS), are widely used as food additives, animal feeds, and pulp bleaching agents. However, the thermally unstable nature of xylanases would hamper their industrial application. In this study, we used in silico design in a glycoside hydrolase family (GH) 11 xylanase to stabilize the enzyme. A combination of the best mutations increased the apparent melting temperature by 14 °C and significantly enhanced thermostability and thermoactivation. The variant also showed an upward-shifted optimal temperature for catalysis without compromising its activity at low temperatures. Moreover, a 10-fold higher XOS production yield was obtained at 70 °C, which compensated the low yield obtained with the wild-type enzyme. Collectively, the variant constructed by the computational strategy can be used as an efficient biocatalyst for XOS production at industrially viable conditions.

High copy and stable expression of the xylanase XynHB in Saccharomyces cerevisiae by rDNA-mediated integration.

PubMed

Fang, Cheng; Wang, Qinhong; Selvaraj, Jonathan Nimal; Zhou, Yuling; Ma, Lixin; Zhang, Guimin; Ma, Yanhe

2017-08-18

Xylanase is a widely-used additive in baking industry for enhancing dough and bread quality. Several xylanases used in baking industry were expressed in different systems, but their expression in antibiotic free vector system is highly essential and safe. In the present study, an alternative rDNA-mediated technology was developed to increase the copy number of target gene by integrating it into Saccharomyces cerevisiae genome. A xylanase-encoding gene xynHB from Bacillus sp. was cloned into pHBM367H and integrated into S. cerevisiae genome through rDNA-mediated recombination. Exogenous XynHB expressed by recombinant S. cerevisiae strain A13 exhibited higher degradation activity towards xylan than other transformants. The real-time PCR analysis on A13 genome revealed the presence of 13.64 copies of xynHB gene. Though no antibiotics have been used, the genetic stability and the xylanase activity of xynHB remained stable up to 1,011 generations of cultivation. S. cerevisiae strain A13 expressing xylanase reduced the required kneading time and increased the height and diameter of the dough size, which would be safe and effective in baking industry as no antibiotics-resistance risk. The new effective rDNA-mediated technology without using antibiotics here provides a way to clone other food related industrial enzymes for applications.

Comparison of several ethanol productions using xylanase, inorganic salts, surfactant

NASA Astrophysics Data System (ADS)

Wu, Yan; Lu, Jie; Yang, Rui-feng; Song, Wen-jing; Li, Hai-ming; Wang, Hai-song; Zhou, Jing-hui

2017-03-01

Liquid hot water (LHW) pretreatment is an effective and environmentally friendly method to produce bioethanol with lignocellulosic materials. Corn stover was pretreated with liquid hot water (LHW) and then subjected to semi-simultaneous saccharification and fermentation (S-SSF) to obtain high ethanol concentration and yield. The present study aimed to confirm the effect of several additives on the fermentation digestibility of unwashed WIS of corn stover pretreated with LHW. So we also investigated the process, such as enzyme addition, inorganic salts, surfactant and different loading Triton. Results show that high ethanol concentration is necessary to add xylanase in the stage of saccharification. The ethanol concentration increased mainly with magnesium ion on fermentation. Comparing with Tween 80, Span 80 and Polyethylene glycol, Triton is the best surfactant. In contrast to using xylanase and Triton respectively, optimization can make up the lack of stamina and improve effect of single inorganic salts.

Improvement of the optimum pH of Aspergillus niger xylanase towards an alkaline pH by site-directed mutagenesis.

PubMed

Li, Fei; Xie, Jingcong; Zhang, Xuesong; Zhao, Linguo

2015-01-01

In an attempt to shift the optimal pH of the xylanase B (XynB) from Aspergillus niger towards alkalinity, target mutation sites were selected by alignment between Aspergillus niger xylanase B and other xylanases that have alkalophilic pH optima that highlight charged residues in the eight-residues-longer loop in the alkalophilic xylanase. Multiple engineered XynB mutants were created by site-directed mutagenesis with substitutions Q164K and Q164K+D117N. The variant XynB-117 had the highest optimum pH (at 5.5), which corresponded to a basic 0.5 pH unit shift when compared with the wild-type enzyme. However, the optimal pH of the XynB- 164 mutation was not changed, similar to the wild type. These results suggest that the residues at positions 164 and 117 in the eight-residues-longer loop and the cleft's edge are important in determining the pH optima of XynB from Aspergillus niger.

Optimization of Xylanase Production from Penicillium sp.WX-Z1 by a Two-Step Statistical Strategy: Plackett-Burman and Box-Behnken Experimental Design

PubMed Central

Cui, Fengjie; Zhao, Liming

2012-01-01

The objective of the study was to optimize the nutrition sources in a culture medium for the production of xylanase from Penicillium sp.WX-Z1 using Plackett-Burman design and Box-Behnken design. The Plackett-Burman multifactorial design was first employed to screen the important nutrient sources in the medium for xylanase production by Penicillium sp.WX-Z1 and subsequent use of the response surface methodology (RSM) was further optimized for xylanase production by Box-Behnken design. The important nutrient sources in the culture medium, identified by the initial screening method of Placket-Burman, were wheat bran, yeast extract, NaNO3, MgSO4, and CaCl2. The optimal amounts (in g/L) for maximum production of xylanase were: wheat bran, 32.8; yeast extract, 1.02; NaNO3, 12.71; MgSO4, 0.96; and CaCl2, 1.04. Using this statistical experimental design, the xylanase production under optimal condition reached 46.50 U/mL and an increase in xylanase activity of 1.34-fold was obtained compared with the original medium for fermentation carried out in a 30-L bioreactor. PMID:22949884

Optimization of Xylanase production from Penicillium sp.WX-Z1 by a two-step statistical strategy: Plackett-Burman and Box-Behnken experimental design.

PubMed

Cui, Fengjie; Zhao, Liming

2012-01-01

The objective of the study was to optimize the nutrition sources in a culture medium for the production of xylanase from Penicillium sp.WX-Z1 using Plackett-Burman design and Box-Behnken design. The Plackett-Burman multifactorial design was first employed to screen the important nutrient sources in the medium for xylanase production by Penicillium sp.WX-Z1 and subsequent use of the response surface methodology (RSM) was further optimized for xylanase production by Box-Behnken design. The important nutrient sources in the culture medium, identified by the initial screening method of Placket-Burman, were wheat bran, yeast extract, NaNO(3), MgSO(4), and CaCl(2). The optimal amounts (in g/L) for maximum production of xylanase were: wheat bran, 32.8; yeast extract, 1.02; NaNO(3), 12.71; MgSO(4), 0.96; and CaCl(2), 1.04. Using this statistical experimental design, the xylanase production under optimal condition reached 46.50 U/mL and an increase in xylanase activity of 1.34-fold was obtained compared with the original medium for fermentation carried out in a 30-L bioreactor.

Ultrasounds pretreatment of olive pomace to improve xylanase and cellulase production by solid-state fermentation.

PubMed

Leite, Paulina; Salgado, José Manuel; Venâncio, Armando; Domínguez, José Manuel; Belo, Isabel

2016-08-01

Olive mills generate a large amount of waste that can be revaluated. This work aim to improve the production lignocellulolytic enzymes by solid-state fermentation using ultrasounds pretreated olive mill wastes. The composition of olive mill wastes (crude and exhausted olive pomace) was compared and several physicochemical characteristics were significantly different. The use of both wastes in SSF was evaluated and a screening of fungi for xylanase and cellulase production was carried out. After screening, the use of exhausted olive pomace and Aspergillus niger led to the highest enzyme activities, so that they were used in the study of ultrasounds pre-treatment. The results showed that the sonication led to a 3-fold increase of xylanase activity and a decrease of cellulase activity. Moreover, the liquid fraction obtained from ultrasounds treatment was used to adjust the moisture of solid and a positive effect on xylanase (3.6-fold increase) and cellulase (1.2-fold increase) production was obtained. Copyright © 2016 Elsevier Ltd. All rights reserved.

Improvement for enhanced xylanase production by Cellulosimicrobium cellulans CKMX1 using central composite design of response surface methodology.

PubMed

Walia, Abhishek; Mehta, Preeti; Guleria, Shiwani; Shirkot, Chand Karan

2015-12-01

The effects of yeast extract (X 1 ), NH 4 NO 3 (X 2 ), peptone (X 3 ), urea (X 4 ), CMC (X 5 ), Tween 20 (X 6 ), MgSO 4 (X 7 ), and CaCO 3 (X 8 ) on production of xylanase from Cellulosimicrobium cellulans CKMX1 were optimized by statistical analysis using response surface methodology (RSM). The RSM was used to optimize xylanase production by implementing the Central composite design. Statistical analysis of the results showed that the linear, interaction and quadric terms of these variables had significant effects. However, only the linear effect of X 4 , X 5 , interaction effect of X 1 X 7 , X 1 X 8 , X 2 X 3 , X 2 X 8 , X 3 X 6 , X 3 X 8 , X 4 X 6 , X 4 X 7 , X 5 X 7 , X 5 X 8 and quadratic effect of X 3 2 , X 5 2 and X 7 2 found to be insignificant terms in the quadratic model and had no response at significant level. The minimum and maximum xylanase production obtained was 331.50 U/g DBP and 1027.65 U/g DBP, respectively. The highest xylanase activity was obtained from Run No. 30, which consisted of yeast extract (X 1 ), 1.00 g (%); NH 4 NO 3 (X 2 ), 0.20 g (%); peptone (X 3 ), 1.00 g (%); urea (X 4 ), 10 mg (%); CMC (X 5 ), 1.00 g (%); Tween 20 (X 6 ), 0.02 mL (%); CaCO 3 (X 7 ), 0.50 g (%) and MgSO 4 (X 8 ), 9.0 g (%). The optimization resulted in 3.1-fold increase of xylanase production, compared with the lowest xylanase production of 331.50 U/g DBP after 72 h of incubation in stationary flask experiment. Application of cellulase-free xylanase in pulp biobleaching from C. cellulans CKMX1 under C-E P -D sequence has been shown to bring about a 12.5 % reduction of chlorine, decrease of 0.8 kappa points (40 %), and gain in brightness was 1.42 % ISO points in 0.5 % enzyme treated pulp as compared to control.

Influence of method of whole wheat inclusion and xylanase supplementation on the performance, apparent metabolisable energy, digestive tract measurements and gut morphology of broilers.

PubMed

Wu, Y B; Ravindran, V; Thomas, D G; Birtles, M J; Hendriks, W H

2004-06-01

1. The aim of the present study was to examine the influence of whole wheat inclusion and xylanase supplementation on the performance, apparent metabolisable energy (AME), digesta viscosity, and digestive tract measurements of broilers fed on wheat-based diets. The influence of the method of whole wheat inclusion (pre- or post-pelleting) was also compared. A 3 x 2 factorial arrangement of treatments was used with three diet forms (648 g/kg ground wheat [GW], GW replaced by 200 g/kg of whole wheat before [WW1] or after cold-pelleting [WW2]) and two xylanase levels (0 and 1000 XU/kg diet). 2. Birds given diets containing whole wheat had improved weight gains, feed efficiency and AME compared to those fed on diets containing ground wheat. The relative gizzard weight of birds fed WW2 diets was higher than in those fed GW and WW1 diets. Pre-pelleting inclusion of whole wheat had no effect on relative gizzard weight. Post-pelleting inclusion of whole wheat resulted in greater improvements in feed efficiency and AME than the pre-pelleting treatment. 3. Xylanase supplementation significantly improved weight gain, feed efficiency and AME, irrespective of the wheat form used. Viscosity of the digesta in the duodenum, jejunum and ileum were reduced by xylanase addition. Xylanase supplementation reduced the relative weight of the pancreas. 4. Neither xylanase supplementation nor whole wheat inclusion influenced the relative weight and length of the small intestine. 5. Xylanase supplementation increased ileal villus height. A significant interaction between diet form and xylanase was observed for ileal crypt depth. Xylanase supplementation had no effect on crypt depth in birds fed on diets containing GW, but increased the crypt depth in WW2 diets. No significant effects of diet form and xylanase supplementation were observed for the thickness of the tunica muscularis layer of gizzard or villus height, crypt depth, goblet cell numbers or epithelial thickness in the ileum. 6

Determination of the modes of action and synergies of xylanases by analysis of xylooligosaccharide profiles over time using fluorescence-assisted carbohydrate electrophoresis.

PubMed

Gong, Weili; Zhang, Huaiqiang; Tian, Li; Liu, Shijia; Wu, Xiuyun; Li, Fuli; Wang, Lushan

2016-07-01

The structure of xylan, which has a 1,4-linked β-xylose backbone with various substituents, is much more heterogeneous and complex than that of cellulose. Because of this, complete degradation of xylan needs a large number of enzymes that includes GH10, GH11, and GH3 family xylanases together with auxiliary enzymes. Fluorescence-assisted carbohydrate electrophoresis (FACE) is able to accurately differentiate unsubstituted and substituted xylooligosaccharides (XOS) in the heterogeneous products generated by different xylanases and allows changes in concentrations of specific XOS to be analyzed quantitatively. Based on a quantitative analysis of XOS profiles over time using FACE, we have demonstrated that GH10 and GH11 family xylanases immediately degrade xylan into sizeable XOS, which are converted into smaller XOS in a much lower speed. The shortest substituted XOS produced by hydrolysis of the substituted xylan backbone by GH10 and GH11 family xylanases were MeGlcA(2) Xyl3 and MeGlcA(2) Xyl4 , respectively. The unsubstituted xylan backbone was degraded into xylose, xylobiose, and xylotriose by both GH10 and GH11 family xylanases; the product profiles are not family-specific but, instead, depend on different subsite binding affinities in the active sites of individual enzymes. Synergystic action between xylanases and β-xylosidase degraded MeGlcA(2) Xyl4 into xylose and MeGlcA(2) Xyl3 but further degradation of MeGlcA(2) Xyl3 required additional enzymes. Synergy between xylanases and β-xylosidase was also found to significantly accelerate the conversion of XOS into xylose. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Effect of Aspergillus niger xylanase on dough characteristics and bread quality attributes.

PubMed

Ahmad, Zulfiqar; Butt, Masood Sadiq; Ahmed, Anwaar; Riaz, Muhammad; Sabir, Syed Mubashar; Farooq, Umar; Rehman, Fazal Ur

2014-10-01

The present study was conducted to investigate the impact of various treatments of xylanase produced by Aspergillus niger applied in bread making processes like during tempering of wheat kernels and dough mixing on the dough quality characteristics i.e. dryness, stiffness, elasticity, extensibility, coherency and bread quality parameters i.e. volume, specific volume, density, moisture retention and sensory attributes. Different doses (200, 400, 600, 800 and 1,000 IU) of purified enzyme were applied to 1 kg of wheat grains during tempering and 1 kg of flour (straight grade flour) during mixing of dough in parallel. The samples of wheat kernels were agitated at different intervals for uniformity in tempering. After milling and dough making of both types of flour (having enzyme treatment during tempering and flour mixing) showed improved dough characteristics but the improvement was more prominent in the samples receiving enzyme treatment during tempering. Moreover, xylanase decreased dryness and stiffness of the dough whereas, resulted in increased elasticity, extensibility and coherency and increase in volume & decrease in bread density. Xylanase treatments also resulted in higher moisture retention and improvement of sensory attributes of bread. From the results, it is concluded that dough characteristics and bread quality improved significantly in response to enzyme treatments during tempering as compared to application during mixing.

Computational design of an endo-1,4-[beta]-xylanase ligand binding site

DOE Office of Scientific and Technical Information (OSTI.GOV)

Morin, Andrew; Kaufmann, Kristian W.; Fortenberry, Carie

2012-09-05

The field of computational protein design has experienced important recent success. However, the de novo computational design of high-affinity protein-ligand interfaces is still largely an open challenge. Using the Rosetta program, we attempted the in silico design of a high-affinity protein interface to a small peptide ligand. We chose the thermophilic endo-1,4-{beta}-xylanase from Nonomuraea flexuosa as the protein scaffold on which to perform our designs. Over the course of the study, 12 proteins derived from this scaffold were produced and assayed for binding to the target ligand. Unfortunately, none of the designed proteins displayed evidence of high-affinity binding. Structural characterizationmore » of four designed proteins revealed that although the predicted structure of the protein model was highly accurate, this structural accuracy did not translate into accurate prediction of binding affinity. Crystallographic analyses indicate that the lack of binding affinity is possibly due to unaccounted for protein dynamics in the 'thumb' region of our design scaffold intrinsic to the family 11 {beta}-xylanase fold. Further computational analysis revealed two specific, single amino acid substitutions responsible for an observed change in backbone conformation, and decreased dynamic stability of the catalytic cleft. These findings offer new insight into the dynamic and structural determinants of the {beta}-xylanase proteins.« less

Catalytic performance of corn stover hydrolysis by a new isolate Penicillium sp. ECU0913 producing both cellulase and xylanase.

PubMed

Shi, Qian-Qian; Sun, Jie; Yu, Hui-Lei; Li, Chun-Xiu; Bao, Jie; Xu, Jian-He

2011-07-01

A fungal strain, marked as ECU0913, producing high activities of both cellulase and xylanase was newly isolated from soil sample collected near decaying straw and identified as Penicillium sp. based on internal transcribed spacer sequence homology. The cultivation of this fungus produced both cellulase (2.40 FPU/ml) and xylanase (241 IU/ml) on a stepwisely optimized medium at 30 °C for 144 h. The cellulase and xylanase from Penicillium sp. ECU0913 was stable at an ambient temperature with half-lives of 28 and 12 days, respectively. Addition of 3 M sorbitol greatly improved the thermostability of the two enzymes, with half-lives increased by 2.3 and 188-folds, respectively. Catalytic performance of the Penicillium cellulase and xylanase was evaluated by the hydrolysis of corn stover pretreated by steam explosion. With an enzyme dosage of 50 FPU/g dry substrate, the conversions of cellulose and hemicellulose reached 77.2% and 47.5%, respectively, without adding any accessory enzyme.

One-step zymogram method for the simultaneous detection of cellulase/xylanase activity and molecular weight estimation of the enzyme.

PubMed

Cano-Ramírez, Claudia; Santiago-Hernández, Alejandro; Rivera-Orduña, Flor Nohemí; Pineda-Mendoza, Rosa María; Zúñiga, Gerardo; Hidalgo-Lara, María Eugenia

2017-02-01

Here, we describe a zymographic method for the simultaneous detection of enzymatic activity and molecular weight (MW) estimation, following a single electrophoresis step. This involved separating cellulase and xylanase activities from bacteria and fungi, obtained from different sources, such as commercial extracts, crude extract and purified proteins, under denaturing conditions, by 10% polyacrylamide gel electrophoresis, using polyacrylamide gels copolymerized with 1% (w/v) carboxymethylcellulose or beechwood xylan as substrates. Then, enzymes were refolded by treatment with 2.5% Triton X-100 in an appropriate buffer for each enzymatic activity, and visualized by Coomassie blue staining for MW estimation. Finally, Congo red staining revealed bio-active cellulase and xylanase bands after electrophoretic separation of the proteins in the preparations. This method may provide a useful additional tool for screening of particular cellulase and xylanase producers, identification and MW estimation of polypeptides that manifest these activities, and for monitoring and control of fungal and bacterial cellulase and xylanase production. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Constitutive expression of the xylanase inhibitor TAXI-III delays Fusarium head blight symptoms in durum wheat transgenic plants.

PubMed

Moscetti, Ilaria; Tundo, Silvio; Janni, Michela; Sella, Luca; Gazzetti, Katia; Tauzin, Alexandra; Giardina, Thierry; Masci, Stefania; Favaron, Francesco; D'Ovidio, Renato

2013-12-01

Cereals contain xylanase inhibitor (XI) proteins which inhibit microbial xylanases and are considered part of the defense mechanisms to counteract microbial pathogens. Nevertheless, in planta evidence for this role has not been reported yet. Therefore, we produced a number of transgenic plants constitutively overexpressing TAXI-III, a member of the TAXI type XI that is induced by pathogen infection. Results showed that TAXI-III endows the transgenic wheat with new inhibition capacities. We also showed that TAXI-III is correctly secreted into the apoplast and possesses the expected inhibition parameters against microbial xylanases. The new inhibition properties of the transgenic plants correlate with a significant delay of Fusarium head blight disease symptoms caused by Fusarium graminearum but do not significantly influence leaf spot symptoms caused by Bipolaris sorokiniana. We showed that this contrasting result can be due to the different capacity of TAXI-III to inhibit the xylanase activity of these two fungal pathogens. These results provide, for the first time, clear evidence in planta that XI are involved in plant defense against fungal pathogens and show the potential to manipulate TAXI-III accumulation to improve wheat resistance against F. graminearum.

Mechanisms for kappa reduction and color removal by xylanases

Treesearch

Thomas W. Jeffries; Mark Davis; Brian Rosin; Larry L. Landucci

1998-01-01

Xylanases reduce kappa and release UV- and visibly absorptive materials from kraft pulps. The extents of these actions depend on the origin and processing of the pulp, access of enzymes to the substrate, and the natures of the enzymes. Hexeneuronic acid (HexA) is a component of kraft pulp xylans that accounts for a fraction of the kappa content. It absorbs strongly in...

Use of xylanase in the TCF bleaching of eucalyptus kraft pulp

DOE Office of Scientific and Technical Information (OSTI.GOV)

Roncero, B.; Vidal, T.; Torres, A.L.

1996-10-01

Environmental pressures are forcing the pulp and paper industry to develop new technologies that reduce or eliminate the presence of various contaminants in bleaching plant effluents. Oxygen delignification techniques, replacement of elemental chlorine with chlorine dioxide, ozone, hydrogen peroxide and new agents as well as the use of xylanase enzymes for biobleaching, reduce o eliminate the production of chlorinated organic substances. This paper compares the sequence XOZP with OZP in the bleaching of Eucalyptus globulus kraft pulps. It has been studied the influence of enzymatic treatment on the consumption of bleaching agents: ozone and hydrogen peroxide. Chemical, physical, optical andmore » refining properties of pulps, as well as COD and colour of effluent are also studied. The xylanase treatment is positive and it is possible to manufacture fully bleached pulps at high brightness and viscosity without using chlorine compounds at a low ozone and hydrogen peroxide consumption.« less

Xylanase production by Burkholderia sp. DMAX strain under solid state fermentation using distillery spent wash.

PubMed

Mohana, Sarayu; Shah, Amita; Divecha, Jyoti; Madamwar, Datta

2008-11-01

Xylanase production by a newly isolated strain of Burkholderia sp. was studied under solid state fermentation using anaerobically treated distillery spent wash. Response surface methodology (RSM) involving Box-Behnken design was employed for optimizing xylanase production. The interactions between distillery effluent concentration, initial pH, moisture ratio and inoculum size were investigated and modeled. Under optimized conditions, xylanase production was found to be in the range of 5200-5600 U/g. The partially purified enzyme recovered after ammonium sulphate fractionation showed maximum activity at 50 degrees C and pH 8.6. Kinetic parameters like Km and Vmax for xylan were found to be 12.75 mg/ml and 165 micromol/mg/min. In the presence of metal ions such as Ca2+, Co2+, Mn2+, Ba2+, Mg2+ and protein disulphide reducing agents such as beta-mercaptoethanol and dithiotheritol (DTT) the activity of enzyme increased, where as strong inhibition of enzyme activity was observed in the presence of Cu2+, Ag+, Fe2+ and SDS. The crude enzyme hydrolysed lignocellulosic substrate, wheat bran as well as industrial pulp.

The influence of sorbitol on the production of cellulases and xylanases in an airlift bioreactor.

PubMed

Ritter, Carla Eliana Todero; Fontana, Roselei Claudete; Camassola, Marli; da Silveira, Maurício Moura; Dillon, Aldo José Pinheiro

2013-11-01

The production of cellulases and xylanases by Penicillium echinulatum in an airlift bioreactor was evaluated. In batch production, we tested media with isolated or associated cellulose and sorbitol. In fed-batch production, we tested cellulose addition at two different times, 30 h and 48 h. Higher liquid circulation velocities in the downcomer were observed in sorbitol 10 g L(-1) medium. In batch production, higher FPA (filter paper activity) and endoglucanase activities were obtained with cellulose (7.5 g L(-1)) and sorbitol (2.5 g L(-1)), 1.0 U mL(-1) (120 h) and 6.4 U m L(-1) (100 h), respectively. For xylanases, the best production condition was cellulose 10 g L(-1), which achieved 5.5 U mL(-1) in 64 h. The fed-batch process was favorable for obtaining xylanases, but not for FPA and endoglucanases, suggesting that in the case of cellulases, the inducer must be added early in the process. Copyright © 2013 Elsevier Ltd. All rights reserved.

Enhancement of Cellulase and Xylanase Production Using pH-Shift and Dissolved Oxygen Control Strategy with Streptomyces griseorubens JSD-1.

PubMed

Zhang, Dan; Luo, Yanqing; Chu, Shaohua; Zhi, Yuee; Wang, Bin; Zhou, Pei

2016-01-01

In this study, the production of cellulase and xylanase by Streptomyces griseorubens JSD-1 was improved by integrating the pH-shift and dissolved oxygen (DO)-constant control strategies. The pH-shift control strategy was carried out by analyzing the specific cell growth rate (μ) and specific enzyme formation rate (Q p) of S. griseorubens JSD-1. The pH was controlled at 8.0 during the first 48 h to maintain high cell growth, which then shifted to 7.5 after 48 h to improve the production of cellulase and xylanase. Using this method, the maximum activities of cellulase, xylanase, and filter paper enzyme (FPase) increased by 47.9, 29.5, and 113.6 %, respectively, compared to that obtained without pH control. On the basis of pH-shift control, the influence of DO concentrations on biomass and enzyme production was further investigated. The maximum production of cellulase, xylanase, and FPase reached 114.38 ± 0.96 U mL(-1), 330.57 ± 2.54 U mL(-1), and 40.11 ± 0.38 U mL(-1), which were about 1.6-fold, 0.6-fold, and 3.2-fold higher than that of neutral pH without DO control conditions. These results supplied a functional approach for improving cellulase and xylanase production.

Effect of combined xylanase and phytase on growth performance, apparent total tract digestibility, and carcass characteristics in growing pigs fed corn-based diets containing high-fiber coproducts.

PubMed

Jang, Y D; Wilcock, P; Boyd, R D; Lindemann, M D

2017-09-01

Phytate has been shown to be an antinutrient, and the feeding of high levels of phytase can break down phytate to improve nutrient utilization and pig performance. Dietary xylanase targets arabinoxylan breakdown, thereby improving energy utilization in pigs. However, the effects of simultaneous supplementation have not been clearly determined. Crossbred pigs ( = 45; mean initial weight, 26.4 ± 0.2 kg) were allotted to 1 of 9 treatments to evaluate the effects of both xylanase (endo-1,4-β xylanase [EC 3.2.1.8]) and phytase (6-phytase [EC 3.1.3.26]) supplementation as follows: 1) positive control (PC), a corn-soybean meal-based diet with 15% corn distillers dried grains with solubles, 15% wheat middlings, and 13% corn germ meal; 2) negative control (NC), ME was reduced by 103 kcal/kg from the PC diet by replacement of fat with corn starch; 3) NC + phytase (500 phytase units (FTU)/kg diet); 4) NC + phytase (1,000 FTU/kg diet); 5) NC + phytase (2,000 FTU/kg diet); 6) NC + xylanase (24,000 xylanase units [BXU]/kg diet); 7) NC + phytase (500 FTU/kg diet) + xylanase (24,000 BXU/kg diet); 8) NC + phytase (1,000 FTU/kg diet) + xylanase (24,000 BXU/kg diet); and 9) NC + phytase (2,000 FTU/kg diet) + xylanase (24,000 BXU/kg diet). All diets were formulated to meet nutrient requirements before phytase and xylanase addition to the diets. There were no significant interactions between xylanase and phytase supplementation on growth performance, carcass characteristics, and apparent total tract digestibility (ATTD). The ADG ( < 0.01, quadratic) and G:F ( < 0.05, linear) for the overall period increased as phytase level increased. The ATTD of P increased as phytase supplementation level increased ( < 0.05, linear and quadratic). The ATTD of DM, NDF, ether extract ( < 0.05), and hemicellulose ( = 0.05) increased quadratically as phytase level increased. Estimated carcass lean percentage and lean gain increased ( < 0.05, linear) as phytase level increased. Xylanase supplementation

Characterization of a novel xylanase gene from rumen content of Hu sheep.

PubMed

Wang, Qian; Luo, Yang; He, Bo; Jiang, Lin-Shu; Liu, Jian-Xin; Wang, Jia-Kun

2015-12-01

A novel xylanase gene, xyn-lxy, was cloned from a metagenomic fosmid library, which was previously constructed from the rumen contents of Hu sheep and was functionally characterized in Escherichia coli. The open reading frame was composed of 1923 bp and encoded for 640 amino acids, including a catalytic domain of glycosyl hydrolase family 10 and carbohydrate-binding module 9. The gene showed 97 % identity with uncultured bacterium Contig1552 but low similarity with xylanases from known cellulolytic-degrading microorganisms in the rumen. The recombinant XYN-LXY showed a specific activity of 664.7 U mg(-1). The optimal temperature and pH of the enzyme were 50 °C and 6.0, respectively. Specifically, XYN-LXY was exclusively activated by Mn(2+) among all of the cations and reducing agents tested in this study. An enzymatic hydrolysis assay revealed that XYN-LXY degraded birchwood xylan into xylooligosaccharide with a low degree of polymerization. After incubation for 4 h, the concentration of the dominant product, xylobiose, was 2.297 ± 0.175 mg ml(-1) (74.07 % of total product) followed by xylose with a concentration of 0.656 ± 0.010 mg ml(-1) (21.14 % of total product). The XYN-LXY exhibited deep degradation effects on the xylan substrate, which were rarely observed with endo-xylanase, making it a promising candidate for industrial application, especially in biofuel production.

Enhanced sugar production from pretreated barley straw by additive xylanase and surfactants in enzymatic hydrolysis for acetone-butanol-ethanol fermentation.

PubMed

Yang, Ming; Zhang, Junhua; Kuittinen, Suvi; Vepsäläinen, Jouko; Soininen, Pasi; Keinänen, Markku; Pappinen, Ari

2015-01-01

This study aims to improve enzymatic sugar production from dilute sulfuric acid-pretreated barley straw for acetone-butanol-ethanol (ABE) fermentation. The effects of additive xylanase and surfactants (polyethylene glycol [PEG] and Tween) in an enzymatic reaction system on straw hydrolysis yields were investigated. By combined application of 2g/100g dry-matter (DM) xylanase and PEG 4000, the glucose yield was increased from 53.2% to 86.9% and the xylose yield was increased from 36.2% to 70.2%, which were considerably higher than results obtained with xylanase or surfactant alone. The ABE fermentation of enzymatic hydrolysate produced 10.8 g/L ABE, in which 7.9 g/L was butanol. The enhanced sugar production increased the ABE yield from 93.8 to 135.0 g/kg pretreated straw. The combined application of xylanase and surfactants has a large potential to improve sugar production from barley straw pretreated with a mild acid and that the hydrolysate showed good fermentability in ABE production. Copyright © 2015 Elsevier Ltd. All rights reserved.

Obtaining cellulose binding and hydrolyzing activity of a family 11 hybrid xylanase by fusion with xylan binding domain.

PubMed

Liu, Ming-Qi; Dai, Xian-Jun; Liu, Guang-Fu; Wang, Qian

2013-03-01

The xylan binding domain (XBD) and linker sequences (LS) from thermostable and thermophilic Thermomonospora fusca xylanase A (TfxA) was fused to the carboxyl-terminus of a family 11 hybrid xylanase ATx. The constructed chimera (ATxX) was successfully expressed in Pichia pastoris, partially purified to homogeneity, and then characterized in detail. After 96-h 0.25% methanol induction, the xylanase and cellulose activity of ATxX from pPATxX1 transformant culture medium supernatant were 452.1 U/mg and 19.3 U/mg, respectively. SDS-PAGE analysis revealed that the molecular mass of ATxX was about 33.01 kDa. 3.7% ATxX was bound after incubation with 1% microcrystal cellulose at 25 °C for 3 h, while the ATx did not show cellulose binding-hydrolyzing ability. These results suggested that ATx obtained cellulose binding and hydrolyzing ability by fusing with XBD and LS. Enzymatic studies showed that the temperature and pH optimum of the ATxX xylanase activity were 60 °C and pH 5.0, respectively, which were the same as that of ATx. The temperature and pH optimum of the ATxX cellulase activity were 60 °C and pH 6.0, respectively. The major hydrolytic products released by ATxX from birchwood xylan were xylotriose and xylohexaose. Xylooligosaccharides from xylobiose to xylohexaose could be hydrolyzed by ATxX. Mode of action analysis showed that the chimeric ATxX was an endo-acting enzyme. The XBD and LS plays an important role in the binding and hydrolyzing of xylanase to insoluble substrates. Copyright © 2012 Elsevier Inc. All rights reserved.

A Comparison of Polysaccharide Substrates and Reducing Sugar Methods for the Measurement of endo-1,4-β-Xylanase.

PubMed

McCleary, Barry V; McGeough, Paraic

2015-11-01

The most commonly used method for the measurement of the level of endo-xylanase in commercial enzyme preparations is the 3,5-dinitrosalicylic acid (DNS) reducing sugar method with birchwood xylan as substrate. It is well known that with the DNS method, much higher enzyme activity values are obtained than with the Nelson-Somogyi (NS) reducing sugar method. In this paper, we have compared the DNS and NS reducing sugar assays using a range of xylan-type substrates and accurately compared the molar response factors for xylose and a range of xylo-oligosaccharides. Purified beechwood xylan or wheat arabinoxylan is shown to be a suitable replacement for birchwood xylan which is no longer commercially available, and it is clearly demonstrated that the DNS method grossly overestimates endo-xylanase activity. Unlike the DNS assay, the NS assay gave the equivalent colour response with equimolar amounts of xylose, xylobiose, xylotriose and xylotetraose demonstrating that it accurately measures the quantity of glycosidic bonds cleaved by the endo-xylanase. The authors strongly recommend cessation of the use of the DNS assay for measurement of endo-xylanase due to the fact that the values obtained are grossly overestimated due to secondary reactions in colour development.

The Botrytis cinerea xylanase Xyn11A contributes to virulence with its necrotizing activity, not with its catalytic activity

PubMed Central

2010-01-01

Background The Botrytis cinerea xylanase Xyn11A has been previously shown to be required for full virulence of this organism despite its poor contribution to the secreted xylanase activity and the low xylan content of B. cinerea hosts. Intriguingly, xylanases from other fungi have been shown to have the property, independent of the xylan degrading activity, to induce necrosis when applied to plant tissues, so we decided to test the hypothesis that secreted Xyn11A contributes to virulence by promoting the necrosis of the plant tissue surrounding the infection, therefore facilitating the growth of this necrotroph. Results We show here that Xyn11A has necrotizing activity on plants and that this capacity is conserved in site-directed mutants of the protein lacking the catalytic activity. Besides, Xyn11A contributes to the infection process with the necrotizing and not with the xylan hydrolyzing activity, as the catalytically-impaired Xyn11A variants were able to complement the lower virulence of the xyn11A mutant. The necrotizing activity was mapped to a 30-amino acids peptide in the protein surface, and this region was also shown to mediate binding to tobacco spheroplasts by itself. Conclusions The main contribution of the xylanase Xyn11A to the infection process of B. cinerea is to induce necrosis of the infected plant tissue. A conserved 30-amino acids region on the enzyme surface, away from the xylanase active site, is responsible for this effect and mediates binding to plant cells. PMID:20184750

Phylogenetic classification of Aureobasidium pullulans strains for production of pullulan and xylanase

USDA-ARS?s Scientific Manuscript database

This study tests the hypothesis that phylogenetic classification can predict whether A. pullulans strains will produce useful levels of the commercial polysaccharide, pullulan, or the valuable enzyme, xylanase. To test this hypothesis, 19 strains of A. pullulans with previously described phenotypes...

Effects of microbial xylanase on digestibility of dry matter, organic matter, neutral detergent fiber, and energy and the concentrations of digestible and metabolizable energy in rice coproducts fed to weanling pigs.

PubMed

Casas, G A; Stein, H H

2016-05-01

The objective of this experiment was to test the hypothesis that the apparent total tract digestibility (ATTD) of DM, OM, fiber, and GE by weanling pigs and the concentration of DE and ME in full-fat rice bran (FFRB), defatted rice bran (DFRB), brown rice, and broken rice is improved if microbial xylanase is added to the diet. Eighty pigs (13.6 ± 0.8 kg initial BW) were allotted to 10 diets with 8 replicate pigs per diet in a randomized complete block design with 2 blocks of 40 pigs. A basal diet based on corn and soybean meal and 4 diets containing corn, soybean meal, and each of the 4 rice coproducts were formulated. The rice coproducts and corn and soybean meal were the only sources of energy in the diets. Five additional diets that were similar to the initial 5 diets with the exception that they also contained 16,000 units of xylanase (Econase XT-25; AB Vista, Marlborough, UK) were also formulated. All diets also contained 1,500 units of microbial phytase (Quantum Blue 5G; AB Vista). The DE and ME and the ATTD of DM, OM, fiber, and GE in diets and ingredients were calculated using the direct method and the difference method, respectively. Results indicated that the concentrations of DE and ME (DM basis) in FFRB and DFRB increased ( < 0.05) if xylanase was used. Broken rice had a greater ( < 0.05) concentration of DE and ME than FFRB and DFRB if no xylanase was added to the diets, but if xylanase was used, no differences in ME among FFRB, brown rice, and broken rice were observed. The ATTD of DM was greater ( < 0.05) in ingredients with xylanase than in ingredients without xylanase and there was a tendency ( = 0.067) for the ATTD of OM to be greater if xylanase was used. The ATTD of NDF in FFRB was greater ( < 0.05) when xylanase was added than if no xylanase was used, whereas the ATTD of NDF in DFRB was not affected by the addition of xylanase. In conclusion, if no xylanase was used, broken rice and brown rice have greater concentrations of DE and ME than FFRB

Influence of phytase and xylanase, individually or in combination, on performance, apparent metabolisable energy, digestive tract measurements and gut morphology in broilers fed wheat-based diets containing adequate level of phosphorus.

PubMed

Wu, Y B; Ravindran, V; Thomas, D G; Birtles, M J; Hendriks, W H

2004-02-01

1. The aim of the present study was to examine the influence of microbial phytase and xylanase, individually or in combination, on performance, apparent metabolisable energy, digesta viscosity, digestive tract measurements and gut morphology in broilers fed on wheat-soy diets containing adequate phosphorus (P). The wheat-soy basal diet was formulated to contain 4.5 g/kg non-phytate P and the experimental diets were formulated by supplementing the basal diet with xylanase (1000 xylanase units/kg diet), phytase (500 phytase units/kg diet) or a combination of phytase and xylanase. 2. Supplemental phytase improved the weight gains and feed efficiency by 17.5 and 2.9%, respectively. Corresponding improvements due to the addition of xylanase were 16.5 and 4.9%, respectively. The combination of phytase and xylanase caused no further improvements in broiler performance. 3. Individual additions of xylanase or phytase resulted in numerical improvements in apparent metabolisable energy (AME), but the differences were not significant. The combination of the two enzymes significantly increased AME. Addition of xylanase and the combination of the two enzymes reduced the viscosity of digesta in all sections of the intestine. Phytase supplementation reduced digesta viscosity in the duodenum and ileum, but not in the jejunum. 4. Enzyme supplementation lowered the relative weight and length of the small intestine. Additions of xylanase and phytase reduced the relative weight of the small intestine by 15.5 and 11.4%, respectively, while the corresponding reductions in the relative length of the small intestine were 16.5 and 14.1%, respectively. The combination of phytase and xylanase had no further effects on the relative weight and length of the small intestine compared with the xylanase group. 5. The addition of phytase increased villus height in the duodenum and decreased the number of goblet cells in the jejunum compared with those on the unsupplemented basal diet. Xylanase

Structural Insights into the Thermophilic Adaption Mechanism of Endo-1,4-β-Xylanase from Caldicellulosiruptor owensensis.

PubMed

Liu, Xin; Liu, Tengfei; Zhang, Yuebin; Xin, Fengjiao; Mi, Shuofu; Wen, Boting; Gu, Tianyi; Shi, Xinyuan; Wang, Fengzhong; Sun, Lichao

2018-01-10

Xylanases (EC 3.2.1.8) are a kind of enzymes degrading xylan to xylooligosaccharides (XOS) and have been widely used in a variety of industrial applications. Among them, xylanases from thermophilic microorganisms have distinct advantages in industries that require high temperature conditions. The CoXynA gene, encoding a glycoside hydrolase (GH) family 10 xylanase, was identified from thermophilic Caldicellulosiruptor owensensis and was overexpressed in Escherichia coli. Recombinant CoXynA showed optimal activity at 90 °C with a half-life of about 1 h at 80 °C and exhibited highest activity at pH 7.0. The activity of CoXynA activity was affected by a variety of cations. CoXynA showed distinct substrate specificities for beechwood xylan and birchwood xylan. The crystal structure of CoXynA was solved and a molecular dynamics simulation of CoXynA was performed. The relatively high thermostability of CoXynA was proposed to be due to the increased overall protein rigidity resulting from the reduced length and fluctuation of Loop 7.

Effect of xylanase supplementation of cellulase on digestion of corn stover solids prepared by leading pretreatment technologies.

PubMed

Kumar, Rajeev; Wyman, Charles E

2009-09-01

Solids resulting from pretreatment of corn stover by ammonia fiber expansion (AFEX), ammonia recycled percolation (ARP), controlled pH, dilute acid, lime, and sulfur dioxide (SO(2)) technologies were hydrolyzed by enzyme cocktails based on cellulase supplemented with beta-glucosidase at an activity ratio of 1:2, respectively, and augmented with up to 11.0 g xylanase protein/g cellulase protein for combined cellulase and beta-glucosidase mass loadings of 14.5 and 29.0 mg protein (about 7.5 and 15 FPU, respectively)/g of original potential glucose. It was found that glucose release increased nearly linearly with residual xylose removal by enzymes for all pretreatments despite substantial differences in their relative yields. The ratio of the fraction of glucan removed by enzymes to that for xylose was defined as leverage and correlated statistically at two combined cellulase and beta-glucosidase mass loadings with pretreatment type. However, no direct relationship was found between leverage and solid features following different pretreatments such as residual xylan or acetyl content. However, acetyl content not only affected how xylanase impacted cellulase action but also enhanced accessibility of cellulose and/or cellulase effectiveness, as determined by hydrolysis with purified CBHI (Cel7A). Statistical modeling showed that cellulose crystallinity, among the main substrate features, played a vital role in cellulase-xylanase interactions, and a mechanism is suggested to explain the incremental increase in glucose release with xylanase supplementation.

Structural analysis of xylanase inhibitor protein I (XIP-I), a proteinaceous xylanase inhibitor from wheat (Triticum aestivum, var. Soisson).

PubMed Central

Payan, Françoise; Flatman, Ruth; Porciero, Sophie; Williamson, Gary; Juge, Nathalie; Roussel, Alain

2003-01-01

A novel class of proteinaceous inhibitors exhibiting specificity towards microbial xylanases has recently been discovered in cereals. The three-dimensional structure of xylanase inhibitor protein I (XIP-I) from wheat (Triticum aestivum, var. Soisson) was determined by X-ray crystallography at 1.8 A (1 A=0.1 nm) resolution. The inhibitor possesses a (beta/alpha)(8) barrel fold and has structural features typical of glycoside hydrolase family 18, namely two consensus regions, approximately corresponding to the third and fourth barrel strands, and two non-proline cis -peptide bonds, Ser(36)-Phe and Trp(256)-Asp (in XIP-I numbering). However, detailed structural analysis of XIP-I revealed several differences in the region homologous with the active site of chitinases. The catalytic glutamic acid residue of family 18 chitinases [Glu(127) in hevamine, a chitinase/lysozyme from the rubber tree (Hevea brasiliensis)] is conserved in the structure of the inhibitor (Glu(128)), but its side chain is fully engaged in salt bridges with two neighbouring arginine residues. Gly(81), located in subsite -1 of hevamine, where the reaction intermediate is formed, is replaced by Tyr(80) in XIP-I. The tyrosine side chain fills the subsite area and makes a strong hydrogen bond with the side chain of Glu(190) located at the opposite side of the cleft, preventing access of the substrate to the catalytic glutamic acid. The structural differences in the inhibitor cleft structure probably account for the lack of activity of XIP-I towards chitin. PMID:12617724

An extremely thermophilic anaerobic bacterium Caldicellulosiruptor sp. F32 exhibits distinctive properties in growth and xylanases during xylan hydrolysis.

PubMed

Ying, Yu; Meng, Dongdong; Chen, Xiaohua; Li, Fuli

2013-08-15

An anaerobic, extremely thermophilic, and cellulose- and xylan-degrading bacterium F32 was isolated from biocompost. Sequence analysis of the 16S rRNA gene of this strain showed that it was closely related to Caldicellulosiruptor saccharolyticus DSM 8903 (99.0% identity). Physiological and biochemical data also supported that identification of strain F32 as a Caldicellulosiruptor species. The proteins secreted by Caldicellulosiruptor sp. F32 grown on xylan showed a xylanase activity of 7.74U/mg, which was 2.5 times higher than that of C. saccharolyticus DSM 8903. Based on the genomic sequencing data, 2 xylanase genes, JX030400 and JX030401, were identified in Caldicellulosiruptor sp. F32. The xylanase encoded by JX030401 shared 97% identity with Csac_0696 of C. saccharolyticus DSM 8903, while that encoded by JX030400 shared 94% identity with Athe_0089 of C. bescii DSM 6725, which was not found in the genome of strain DSM 8903. Xylanse encoded by JX030400 had 9-fold higher specific activity than JX030401. Our results indicated that although the 2 strains shared high identity, the xylanase system in Caldicellulosiruptor sp. F32 was more efficient than that in C. saccharolyticus DSM 8903. Copyright © 2013 Elsevier Inc. All rights reserved.

A highly thermostable alkaline cellulase-free xylanase from thermoalkalophilic Bacillus sp. JB 99 suitable for paper and pulp industry: purification and characterization.

PubMed

Shrinivas, Dengeti; Savitha, Gunashekaran; Raviranjan, Kumar; Naik, Gajanan Ramchandra

2010-11-01

A highly thermostable alkaline xylanase was purified to homogeneity from culture supernatant of Bacillus sp. JB 99 using DEAE-Sepharose and Sephadex G-100 gel filtration with 25.7-fold increase in activity and 43.5% recovery. The molecular weight of the purified xylanase was found to be 20 kDA by SDS-PAGE and zymogram analysis. The enzyme was optimally active at 70 °C, pH 8.0 and stable over pH range of 6.0-10.0.The relative activity at 9.0 and 10.0 were 90% and 85% of that of pH 8.0, respectively. The enzyme showed high thermal stability at 60 °C with 95% of its activity after 5 h. The K (m) and V (max) of enzyme for oat spelt xylan were 4.8 mg/ml and 218.6 µM min(-1) mg(-1), respectively. Analysis of N-terminal amino acid sequence revealed that the xylanase belongs to glycosyl hydrolase family 11 from thermoalkalophilic Bacillus sp. with basic pI. Substrate specificity showed a high activity on xylan-containing substrate and cellulase-free nature. The hydrolyzed product pattern of oat spelt xylan on thin-layer chromatography suggested xylanase as an endoxylanase. Due to these properties, xylanase from Bacillus sp. JB 99 was found to be highly compatible for paper and pulp industry.

Heterologous expression of xylanase enzymes in lipogenic yeast Yarrowia lipolytica

DOE PAGES

Wang, Wei; Wei, Hui; Alahuhta, Markus; ...

2014-12-02

In order to develop a direct microbial sugar conversion platform for the production of lipids, drop-in fuels and chemicals from cellulosic biomass substrate, we chose Yarrowia lipolytica as a viable demonstration strain. Y. lipolytica is known to accumulate lipids intracellularly and is capable of metabolizing sugars to produce lipids; however, it lacks the lignocellulose-degrading enzymes needed to break down biomass directly. While research is continuing on the development of a Y. lipolytica strain able to degrade cellulose, in this study, we present successful expression of several xylanases in Y. lipolytica. The XynII and XlnD expressing Yarrowia strains exhibited an abilitymore » to grow on xylan mineral plates. This was shown by Congo Red staining of halo zones on xylan mineral plates. Enzymatic activity tests further demonstrated active expression of XynII and XlnD in Y. lipolytica. Furthermore, synergistic action in converting xylan to xylose was observed when XlnD acted in concert with XynII. Finally, the successful expression of these xylanases in Yarrowia further advances us toward our goal to develop a direct microbial conversion process using this organism.« less

Xylanase 30 A from Clostridium thermocellum functions as a glucuronoxylan xylanohydrolase

Treesearch

Franz J. St John; Casey Crooks; Diane Dietrich; Jason Hurlbert

2017-01-01

Endoxylanases classified into glycoside hydrolase family 30 subfamily 8 (GH30-8) have been shown to hydrolyze glucuronoxylan with dependence upon the glucuronic acid (GlcA) appendage. In a recent report, the GH30-8 xylanase from Clostridium thermocellum (CtXyn30A) was shown to hydrolyze arabinoxylan which contains no GlcA. Protein structure...

A thermostable Gloeophyllum trabeum xylanase with potential for the brewing industry.

PubMed

Wang, Xiaoyu; Luo, Huiying; Yu, Wangning; Ma, Rui; You, Shuai; Liu, Weina; Hou, Lingyu; Zheng, Fei; Xie, Xiangming; Yao, Bin

2016-05-15

A xylanase gene of glycoside hydrolase family 10, GtXyn10, was cloned from Gloeophyllum trabeum CBS 900.73 and expressed in Pichia pastoris GS115. Purified recombinant GtXyn10 exhibited significant activities to xylan (100.0%), lichenan (11.2%), glucan (15.2%) and p-nitrophenol-β-cellobiose (18.6%), demonstrated the maximum xylanase and glucanase activities at pH 4.5-5.0 and 75°C, retained stability over the pH range of 2.0-7.5 and at 70°C, and was resistant to pepsin and trypsin, most metal ions and SDS. Multiple sequence alignment and modeled-structure analysis identified a unique Gly48 in GtXyn10, and site-directed mutagenesis of Gly48 to Lys improved the temperature optimum up to 80°C. Under simulated mashing conditions, GtXyn10 (80U) reduced the mash viscosity by 12.8% and improved the filtration rate by 31.3%. All these properties above make GtXyn10 attractive for potential applications in the feed and brewing industries. Copyright © 2015 Elsevier Ltd. All rights reserved.

Novel structural features of xylanase A1 from Paenibacillus sp. JDR-2

Treesearch

Franz J. St John; James F. Preston; Edwin Pozharski

2012-01-01

The Gram-positive bacterium Paenibacillus sp. JDR-2 (PbJDR2) has been shown to have novel properties in the utilization of the abundant but chemically complex hemicellulosic sugar glucuronoxylan. Xylanase A1 of PbJDR2 (PbXynA1) has been implicated in an efficient process in which extracellular...

Xylanase supplementation of a wheat-based diet improved nutrient digestion and mRNA expression of intestinal nutrient transporters in broiler chickens infected with Clostridium perfringens.

PubMed

Guo, Shuangshuang; Liu, Dan; Zhao, Xu; Li, Changwu; Guo, Yuming

2014-01-01

Necrotic enteritis caused by Clostridium perfringens has become prevalent in the European Union due to the withdrawal of antibiotics in poultry feed. In an experiment with a 2 × 2 factorial arrangement, 336 one-day-old male broiler chicks (Ross 308) were assigned to 4 groups with or without C. perfringens challenge and fed wheat-based diets supplemented with or without xylanase at 5,500 U/kg of diet. The study aimed to investigate effects of xylanase addition on growth performance as well as nutrient digestion and absorption of C. perfringens-infected broilers. Before challenge (d 0-14), xylanase-supplemented birds had greater ADG and lower feed conversion ratio (FCR; P < 0.05). During infection (d 14-21), challenge tended to decrease ADG (P = 0.063) and significantly increased FCR (P < 0.05), whereas xylanase addition greatly reduced FCR (P < 0.05). Clostridium perfringens infection decreased AME values and apparent ileal digestibility of DM of diets (P < 0.05). Xylanase supplementation increased AME values regardless of infection and apparent ileal digestibility of CP in challenged birds (P < 0.05). Activities of duodenal α-amylase and chymotrypsin and pancreatic trypsin were decreased by C. perfringens infection (P < 0.05). Xylanase supplementation elevated pancreatic chymotrypsin activity and reduced duodenal α-amylase and trypsin activities (P < 0.05). It also decreased jejunal α-amylase activity and increased pancreatic α-amylase as well as jejunal sucrase activities in uninfected birds (P < 0.05). The duodenal mRNA expression of sodium glucose cotransporter 1 (SGLT1), H(+)-dependent peptide transporter 1 (PepT1), and liver fatty acid-binding protein (L-FABP) were downregulated (P < 0.05), but ileal SGLT1 gene expression was increased by infection (P < 0.05). Xylanase addition upregulated expression of jejunal SGLT1, PepT1, and L-FABP genes as well as ileal PepT1 and L-FABP genes in challenged broilers (P < 0.05). In conclusion, xylanase supplementation

Co-cultivation of mutant Penicillium oxalicum SAU(E)-3.510 and Pleurotus ostreatus for simultaneous biosynthesis of xylanase and laccase under solid-state fermentation.

PubMed

Dwivedi, Pallavi; Vivekanand, V; Pareek, Nidhi; Sharma, Amit; Singh, Rajesh P

2011-10-01

Co-cultivation of mutant Penicillium oxalicum SAU(E)-3.510 and Pleurotus ostreatus MTCC 1804 was evaluated for the production of xylanase-laccase mixture under solid-state fermentation (SSF) condition. Growth compatibility between mutant P. oxalicum SAU(E)-3.510 and white rot fungi (P. ostreatus MTCC 1804, Trametes hirsuta MTCC 136 and Pycnoporus sp. MTCC 137) was analyzed by growing them on potato dextrose agar plate. Extracellular enzyme activities were determined spectrophotometrically. Under derived conditions, paired culturing of mutant P. oxalicum SAU(E)-3.510 and P. ostreatus MTCC 1804 resulted in 58% and 33% higher levels of xylanase and laccase production, respectively. A combination of sugarcane bagasse and black gram husk in a ratio of 3:1 was found to be the most ideal solid substrate and support for fungal colonization and enzyme production during co-cultivation. Maximum levels of xylanase (8205.31 ± 168.31 IU g(-1)) and laccase (375.53 ± 34.17 IU g(-1)) during SSF were obtained by using 4 g of solid support with 80% of moisture content. Furthermore, expressions of both xylanase and laccase were characterized during mixed culture by zymogram analysis. Improved levels of xylanase and laccase biosynthesis were achieved by co-culturing the mutant P. oxalicum SAU(E)-3.510 and P. ostreatus MTCC 1804. This may be because of efficient substrate utilization as compared to their respective monocultures in the presence of lignin degradation compounds because of synergistic action of xylanase and laccase. Understanding and developing the process of co-cultivation appears productive for the development of mixed enzyme preparation with tremendous potential for biobleaching. Copyright © 2011 Elsevier B.V. All rights reserved.

Improvement of oxygen transfer coefficient during Penicillium canescens culture. Influence of turbine design, agitation speed, and air flow rate on xylanase production.

PubMed

Gaspar, A; Strodiot, L; Thonart, P

1998-01-01

To improve xylanase productivity from Penicillium canescens 10-10c culture, an optimization of oxygen supply is required. Because the strain is sensitive to shear forces, leading to lower xylanase productivity as to morphological alteration, vigorous mixing is not desired. The influence of turbine design, agitation speed, and air flow rate on K1a (global mass transfer coefficient, h(-1)) and enzyme production is discussed. K1a values increased with agitation speed and air flow rate, whatever the impeller, in our assay conditions. Agitation had more influence on K1a values than air flow, when a disk-mounted blade's impeller (DT) is used; an opposite result was obtained with a hub-mounted pitched blade's impeller (PBT). Xylanase production appeared as a function of specific power (W/m3), and an optimum was found in 20 and 100 L STRs fitted with DT impellers. On the other hand, the use of a hub-mounted pitched blade impeller (PBT8), instead of a disk-mounted blade impeller (DT4), reduced the lag time of hemicellulase production and increased xylanase productivity 1.3-fold.

Effects of exogenous phytase and xylanase, individually or in combination, and pelleting on nutrient digestibility, available energy content of wheat and performance of growing pigs fed wheat-based diets.

PubMed

Yang, Y Y; Fan, Y F; Cao, Y H; Guo, P P; Dong, B; Ma, Y X

2017-01-01

Two experiments were conducted to determine the effects of adding exogenous phytase and xylanase, individually or in combination, as well as pelleting on nutrient digestibility, available energy content of wheat and the performance of growing pigs fed wheat-based diets. In Experiment 1, forty-eight barrows with an initial body weight of 35.9±0.6 kg were randomly assigned to a 2×4 factorial experiment with the main effects being feed form (pellet vs meal) and enzyme supplementation (none, 10,000 U/kg phytase, 4,000 U/kg xylanase or 10,000 U/kg phytase plus 4,000 U/kg xylanase). The basal diet contained 97.8% wheat. Pigs were placed in metabolic cages for a 7-d adaptation period followed by a 5-d total collection of feces and urine. Nutrient digestibility and available energy content were determined. Experiment 2 was conducted to evaluate the effects of pelleting and enzymes on performance of wheat for growing pigs. In this experiment, 180 growing pigs (35.2±9.0 kg BW) were allocated to 1 of 6 treatments according to a 2×3 factorial treatment arrangement with the main effects being feed form (meal vs pellet) and enzyme supplementation (0, 2,500 or 5,000 U/kg xylanase). In Experiment 1, there were no interactions between feed form and enzyme supplementation. Pelleting reduced the digestibility of acid detergent fiber (ADF) by 6.4 percentage units (p<0.01), increased the digestibility of energy by 0.6 percentage units (p<0.05), and tended to improve the digestibility of crude protein by 0.5 percentage units (p = 0.07) compared with diets in mash form. The addition of phytase improved the digestibility of phosphorus (p<0.01) and calcium (p<0.01) by 6.9 and 7.6 percentage units respectively compared with control group. Adding xylanase tended to increase the digestibility of crude protein by 1.0 percentage units (p = 0.09) and increased the digestibility of neutral detergent fiber (NDF) (p<0.01) compared with control group. Supplementation of the xylanase

Studies on carboxymethyl cellulase and xylanase activities of anaerobic fungal isolate CR4 from the bovine rumen.

PubMed

Matsui, Hiroki; Ban-Tokuda, Tomomi

2008-12-01

An anaerobic fungal isolate, CR4, was isolated from the bovine rumen. The DNA sequence of internal transcribed spacer region 1 showed that CR4 belonged to the genus Caecocmyces. The dry matter digestibility of timothy hay by anaerobic fungal isolate CR4 was determined. The effects of carbohydrate growth substrates on carboxymethyl cellulase (CMCase) and xylanase activities also were examined. The extent of dry matter digestibility of timothy hay was 31% at 6 days' incubation. The highest specific activity of CMCase in the culture supernatant (SN) fraction was observed in xylose culture. The activity of CMCase was not detected in the SN fraction of cellobiose and xylan or in the cell-bound fraction of all growth substrates. The highest specific activity of xylanase in the SN fraction was observed in glucose culture. These results suggest that fiber-degrading enzyme activities were affected by growth substrates and that CR4 is xylanolytic. Zymogram analysis showed that CR4 produces three CMCases of molecular mass (95, 89, and 64 kDa) and three xylanases of molecular mass (82, 73, and 66 kDa). This is the first demonstration showing the molecular mass of fiber-degrading enzymes of Caecomyces.

Xylanase supplementation improves the nutritive value of diets containing high levels of sorghum distillers' dried grains with solubles for broiler chickens.

PubMed

Barekatain, Mohammad Reza; Choct, Mingan; Iji, Paul A

2013-05-01

An experiment was conducted to investigate the effect of sorghum distillers' dried grains with solubles (sDDGS) and xylanase supplementation in broiler chicken diets. A total of 432 Cobb-500 day-old male broiler chicks were used in a 4 × 2 factorial design (0, 100, 200 or 300 g kg(-1) sDDGS with or without xylanase supplementation). Each treatment was replicated six times with nine birds per replicate in a 35 day study. Feed intake was increased (P < 0.001) throughout the study with the inclusion of dietary sDDGS. Body weight gain (BWG) was unaffected except for the last 2 weeks of study, when birds that received 200 and 300 g kg(-1) sDDGS had higher (P < 0.001) BWG. Feed conversion ratio (FCR) increased (P < 0.05) as sDDGS in the diet rose to 100 g kg(-1) during the starter phase and the whole period of study. Over the starter period, xylanase supplementation improved (P < 0.05) FCR, particularly for the highest inclusion of sDDGS. Protein digestibility deteriorated (P < 0.001) as sDDGS in the diet rose to 300 g kg(-1) . Xylanase reduced (P < 0.001) the concentration of xylose in the ileum of birds. The activities of sucrase and maltase in the jejunal mucosa were reduced when birds were offered 200 and 300 g kg(-1) sDDGS. Incorporation of sDDGS increased (P < 0.01) the total short-chain fatty acid concentration in the caeca. The results showed that diets containing large amounts of sDDGS will benefit from xylanase supplementation, particularly in terms of FCR. © 2012 Society of Chemical Industry.

Xylanase from the extremely thermophilic bacterium Caldocellum saccharolyticum: Overexpression of the gene in Escherichia coli and characterization of the gene product

DOE Office of Scientific and Technical Information (OSTI.GOV)

Luethi, E.; Jasmat, N.B.; Bergquist, P.L.

A xylanase encoded by the xynA gene of the extreme thermophile Caldocellum saccharolyticum was overexpressed in Escherichia coli by cloning the gene downstream from the temperature-inducible {lambda} P{sub R} and P{sub L} promoters of the expression vector pJLA602. Induction of up to 55 times was obtained by growing the cells at 42{degrees}C, and the xylanase made up of 20% of the whole-cell protein content. The enzyme was located in the cytoplasmic fraction in E.coli. The temperature and pH optima were determined to be 70{degrees}C and pH 5.5 to 6, respectively. The xylanase was stable for at least 72 h ifmore » incubated at 60{degrees}C, with half-lives of 8 to 9 h at 70{degrees}C and 2 to 3 min at 80{degrees}C. The enzyme had high activity on xylan and ortho-nitrophenyl {beta}-D-xylopyranoside and some activity on carboxymethyl cellulose and para-nitrophenyl {beta}-D-cellobioside. The gene was probably expressed from its own promoter in E. coli. Translation of the xylanase overproduced in E. coli seemed to initiate at a GTG codon and not at an ATG codon as previously determined.« less

Effects of rhamnolipid on the cellulase and xylanase in hydrolysis of wheat straw.

PubMed

Wang, Hong-Yuan; Fan, Bing-Quan; Li, Chun-Hua; Liu, Shuang; Li, Min

2011-06-01

The effects of biosurfactant rhamnolipid (RL) and chemical surfactant Triton X-100 on the production of cellulases and xylanase from Penicillium expansum (P. expansum) in untreated, acid- and alkali-pretreated wheat straw submerged fermentations were studied, and the influences on the activity and stability of Cellulase R-10 were also investigated. The results showed that RL and Triton X-100 enhanced the activities of cellulases and xylanase to different extents and the stimulatory effects of RL were superior to those of Triton X-100. During the peak enzyme production phase, RL (60 RE mg/l) increased cellulases activities by 25.5-102.9%, in which the raise of the same enzyme in acid-pretreated straw broths was the most. It was found that the reducing sugars by hydrolyzing wheat straw with Cellulase R-100 were not visibly increased after adding RL. However, it distinctly protected Cellulase R-10 from degradation or inactivation, keeping the reducing sugars yield at about 17%. Copyright © 2011. Published by Elsevier Ltd.

Alteration of white-rot basidiomycetes cellulase and xylanase activities in the submerged co-cultivation and optimization of enzyme production by Irpex lacteus and Schizophyllum commune.

PubMed

Metreveli, Eka; Kachlishvili, Eva; Singer, Steven W; Elisashvili, Vladimir

2017-10-01

Mono and dual cultures of four white-rot basidiomycete species were evaluated for cellulase and xylanase activity under submerged fermentation conditions. Co-cultivation of Pycnoporus coccineus or Trametes hirsuta with Schizophyllum commune displayed antagonistic interactions resulting in the decrease of endoglucanase and total cellulase activities. In contrast, increases in cellulase and xylanase activity were revealed through the compatible interactions of Irpex lacteus with S. commune. Co-cultivation conditions were optimized for maximum enzyme production by I. lacteus and S. commune, the best producers of cellulase/xylanase and β-glucosidase, respectively. An optimized medium for the target enzyme production by the mixed culture was established in a laboratory fermenter yielding 7U/mL total cellulase, 142U/mL endoglucanase, 104U/mL xylanase, and 5.2U/mL β-glucosidase. The dual culture approach resulted in an enzymatic mixture with 11% improved lignocellulose saccharification potential compared to enzymes from a monoculture of I. lacteus. Copyright © 2017 Elsevier Ltd. All rights reserved.

Isolation, purification and characterisation of low molecular weight xylanase from Bacillus pumilus SSP-34.

PubMed

Subramaniyan, S

2012-04-01

Low molecular weight endo-xylanase from Bacillus pumilus SSP-34 was purified to homogeneity using ion exchange and size exclusion chromatographies. Xylanases were isolated by novel purification protocol which includes the use of anion exchange matrix such as DEAE Sepharose CL 6B with less affinity towards enzyme protein. The purified B. pumilus SSP-34 have a molecular weight of 20 kDa, with optimum pH and temperature at 6.0 and 50 °C, respectively. The enzyme was stable at 50 °C for 30 min. It showed remarkable stability at pH values ranging from 4.5 to 9 when the reaction was carried out at 50 °C. K (m) and V (max) values, determined with oats spelts xylan were 6.5 mg ml⁻¹ and 1,233 μmol min⁻¹ mg⁻¹ protein, respectively, and the specific activity was 1,723 U mg⁻¹.

Nutrient and fiber utilization responses of supplemental xylanase in broiler chickens fed wheat based diets are independent of the adaptation period to test diets.

PubMed

Kiarie, E; Walsh, M C; Romero, L F; Arent, S; Ravindran, V

2017-09-01

The effects of adaptation (AD) to xylanase-supplemented diets on nutrient and fiber utilization in 21-d-old broilers were investigated. Six treatments, arranged in two levels of AD (starting at d 0 or d 14 of age) and three levels of xylanase (0 or 2,500 or 5,000 xylanase units/kg feed) were used. All diets had 500 phytase U/kg and 0.3% TiO2 as indigestible marker. A total of 384 d old male broiler (Ross 308) chicks were divided into two groups. The first group was assigned on weight basis to 24 cages (8 chicks per cage) and randomly allocated to the diets from d 0. Birds in the second group were reared on a commercial starter diet in the same room for 13 d. On d 14, the birds were individually weighed, assigned on weight basis to 24 cages (8 chicks per cage), and randomly allocated to the diets. Birds had free access to experimental diets and water. Excreta samples were collected from d 18 to 21. On d 21, all birds were euthanized to access ileal digesta. There was no interaction (P > 0.05) between AD and xylanase on the apparent ileal digestibility (AID) and apparent retention (AR) of components. The main effect of AD was such that the birds exposed to diets for 7 d (d 14 to 21) had higher (P < 0.01) AID of energy than those exposed for 21 d (d 0 to 21). In contrast, birds exposed to diets for 21 d had higher (P < 0.05) AMEn and AR of neutral detergent fiber. Xylanase improvements (P < 0.01) in the AID of energy and AMEn were dose dependent and coincided with linear improvements (P < 0.05) in the AID of nitrogen, fat, and starch. In conclusion, xylanase improvements on retention of fiber and nutrients were independent of AD (7 or 21 d) suggesting that the xylanase effects are not transitional. Greater retention of fiber with longer AD is suggestive of possible microbial adaptation. © 2017 Poultry Science Association Inc.

Effects of exogenous phytase and xylanase, individually or in combination, and pelleting on nutrient digestibility, available energy content of wheat and performance of growing pigs fed wheat-based diets

PubMed Central

Yang, Y. Y.; Fan, Y. F.; Cao, Y. H.; Guo, P. P.; Dong, B.; Ma, Y. X.

2017-01-01

Objective Two experiments were conducted to determine the effects of adding exogenous phytase and xylanase, individually or in combination, as well as pelleting on nutrient digestibility, available energy content of wheat and the performance of growing pigs fed wheat-based diets. Methods In Experiment 1, forty-eight barrows with an initial body weight of 35.9±0.6 kg were randomly assigned to a 2×4 factorial experiment with the main effects being feed form (pellet vs meal) and enzyme supplementation (none, 10,000 U/kg phytase, 4,000 U/kg xylanase or 10,000 U/kg phytase plus 4,000 U/kg xylanase). The basal diet contained 97.8% wheat. Pigs were placed in metabolic cages for a 7-d adaptation period followed by a 5-d total collection of feces and urine. Nutrient digestibility and available energy content were determined. Experiment 2 was conducted to evaluate the effects of pelleting and enzymes on performance of wheat for growing pigs. In this experiment, 180 growing pigs (35.2±9.0 kg BW) were allocated to 1 of 6 treatments according to a 2×3 factorial treatment arrangement with the main effects being feed form (meal vs pellet) and enzyme supplementation (0, 2,500 or 5,000 U/kg xylanase). Results In Experiment 1, there were no interactions between feed form and enzyme supplementation. Pelleting reduced the digestibility of acid detergent fiber (ADF) by 6.4 percentage units (p<0.01), increased the digestibility of energy by 0.6 percentage units (p<0.05), and tended to improve the digestibility of crude protein by 0.5 percentage units (p = 0.07) compared with diets in mash form. The addition of phytase improved the digestibility of phosphorus (p<0.01) and calcium (p<0.01) by 6.9 and 7.6 percentage units respectively compared with control group. Adding xylanase tended to increase the digestibility of crude protein by 1.0 percentage units (p = 0.09) and increased the digestibility of neutral detergent fiber (NDF) (p<0.01) compared with control group. Supplementation of

Effects of Xylanase Supplementation on Growth Performance, Nutrient Digestibility and Non-starch Polysaccharide Degradation in Different Sections of the Gastrointestinal Tract of Broilers Fed Wheat-based Diets

PubMed Central

Zhang, L.; Xu, J.; Lei, L.; Jiang, Y.; Gao, F.; Zhou, G. H.

2014-01-01

This experiment was performed to investigate the effects of exogenous xylanase supplementation on performance, nutrient digestibility and the degradation of non-starch polysaccharides (NSP) in different sections of the gastrointestinal tract (GIT) of broilers fed wheat-based diets. A total of 120 7-day-old Arbor Acres broiler chicks were randomly allotted to two wheat-based experimental diets supplemented with 0 or 1.0 g/kg xylanase. Each treatment was composed of 6 replicates with 10 birds each. Diets were given to the birds from 7 to 21 days of age. The results showed that xylanase supplementation did not affect feed intake, but increased body weight gain of broiler at 21 day of age by 5.8% (p<0.05) and improved feed-to-gain ratio by 5.0% (p<0.05). Xylanase significantly increased (p<0.05) ileal digestibilities of crude protein (CP) by 3.5%, starch by 9.3%, soluble NSP by 43.9% and insoluble NSP by 42.2% relative to the control group, respectively. Also, compared with the control treatment, xylanase addition increased (p<0.05) total tract digestibilities of dry matter by 5.7%, CP by 4.1%, starch by 6.3%, soluble NSP by 50.8%, and had a tendency to increase (p = 0.093) insoluble NSP by 19.9%, respectively. The addition of xylanase increased the concentrations of arabinose and xylose in the digesta of gizzard, duodenum, jejunum, and ileum (p<0.05), and the order of their concentration was ileum>jejunum>duodenum>>gizzard> caecum. The supplementation of xylanse increased ileal isomaltriose concentration (p<0.05), but did not affect the concentrations of isomaltose, panose and 1-kestose in the digesta of all GIT sections. These results suggest that supplementation of xylanase to wheat-based diets cuts the arabinoxylan backbone into small fragments (mainly arabinose and xylose) in the ileum, jejunum and duodenum, and enhances digestibilites of nutrients by decreasing digesta viscosity. The release of arabinose and xylose in the small intestine may also be the important

The complete conformational free energy landscape of β-xylose reveals a two-fold catalytic itinerary for β-xylanases.

PubMed

Iglesias-Fernández, Javier; Raich, Lluís; Ardèvol, Albert; Rovira, Carme

2015-02-01

Unraveling the conformational catalytic itinerary of glycoside hydrolases (GHs) is a growing topic of interest in glycobiology, with major impact in the design of GH inhibitors. β-xylanases are responsible for the hydrolysis of glycosidic bonds in β-xylans, a group of hemicelluloses of high biotechnological interest that are found in plant cell walls. The precise conformations followed by the substrate during catalysis in β-xylanases have not been unambiguously resolved, with three different pathways being proposed from structural analyses. In this work, we compute the conformational free energy landscape (FEL) of β-xylose to predict the most likely catalytic itineraries followed by β-xylanases. The calculations are performed by means of ab initio metadynamics, using the Cremer-Pople puckering coordinates as collective variables. The computed FEL supports only two of the previously proposed itineraries, 2 S O → [ 2,5 B] ǂ → 5 S 1 and 1 S 3 → [ 4 H 3 ] ǂ → 4 C 1 , which clearly appear in low energy regions of the FEL. Consistently, 2 S O and 1 S 3 are conformations preactivated for catalysis in terms of free energy/anomeric charge and bond distances. The results however exclude the O E → [ O S 2 ] ǂ → B 2,5 itinerary that has been recently proposed for a family 11 xylanase. Classical and ab initio QM/MM molecular dynamics simulations reveal that, in this case, the observed O E conformation has been enforced by enzyme mutation. These results add a word of caution on using modified enzymes to inform on catalytic conformational itineraries of glycoside hydrolases.

Identification and use of the putative Bacteroides ovatus xylanase promoter for the inducible production of recombinant human proteins.

PubMed

Hamady, Zaed Z R; Farrar, Mark D; Whitehead, Terence R; Holland, Keith T; Lodge, J Peter A; Carding, Simon R

2008-10-01

The use of genetically modified bacteria to deliver biologically active molecules directly to the gut has become an increasingly attractive area of investigation. The challenge of regulation of production of the therapeutic molecule and colonization of the bowel led us to investigate Bacteroides ovatus for the production of these molecules, due to its ability to colonize the colon and xylan utilization properties. Here we have identified the putative xylanase promoter. The 5' region of the corresponding mRNA was determined by 5'RACE analysis and the transcription initiation site was identified 216 bp upstream of the ATG start codon. The putative xylanase promoter was regulated by xylan in a dose- and time-dependent manner, and repressed by glucose. This promoter was subsequently used to direct the controlled expression of a gene encoding the human intestinal trefoil factor (TFF-3) after integration as a single copy into the chromosome of B. ovatus. The resulting strain produced biologically active TFF-3 in the presence of xylan. These findings identify the B. ovatus xylanase operon promoter and show that it can be utilized to direct xylan-inducible expression of heterologous eukaryotic genes in B. ovatus.

Xylanase and β-xylosidase production by Aspergillus ochraceus: new perspectives for the application of wheat straw autohydrolysis liquor.

PubMed

Michelin, Michele; Polizeli, Maria de Lourdes T M; Ruzene, Denise S; Silva, Daniel P; Vicente, António A; Jorge, João A; Terenzi, Héctor F; Teixeira, José A

2012-01-01

The xylanase biosynthesis is induced by its substrate-xylan. The high xylan content in some wastes such as wheat residues (wheat bran and wheat straw) makes them accessible and cheap sources of inducers to be mainly applied in great volumes of fermentation, such as those of industrial bioreactors. Thus, in this work, the main proposal was incorporated in the nutrient medium wheat straw particles decomposed to soluble compounds (liquor) through treatment of lignocellulosic materials in autohydrolysis process, as a strategy to increase and undervalue xylanase production by Aspergillus ochraceus. The wheat straw autohydrolysis liquor produced in several conditions was used as a sole carbon source or with wheat bran. The best conditions for xylanase and β-xylosidase production were observed when A. ochraceus was cultivated with 1% wheat bran added of 10% wheat straw liquor (produced after 15 min of hydrothermal treatment) as carbon source. This substrate was more favorable when compared with xylan, wheat bran, and wheat straw autohydrolysis liquor used separately. The application of this substrate mixture in a stirred tank bioreactor indicated the possibility of scaling up the process to commercial production.

Bi-functional fusion enzyme EG-M-Xyn displaying endoglucanase and xylanase activities and its utility in improving lignocellulose degradation.

PubMed

Chen, Chin-Chung; Gao, Guo-Jhan; Kao, Ai-Ling; Tsai, Zheng-Chia

2018-05-01

In this study, the gene fusion of endoglucanase (EG, one of cellulases) from Teleogryllus emma and xylanase (Xyn, one of hemicellulases) from Thermomyces lanuginosus was constructed to generate a fusion enzyme (EG-M-Xyn). Through the expression and purification by ultrafiltration and size-exclusion chromatography, the purified EG-M-Xyn had a molecular weight of 75.5 kDa and exhibited the specific activity of CMCase and xylanase as 306.8 U/mg and 1227.3 U/mg, respectively. The K m values (CMC and beechwood xylan) were 6.8 and 60.6 mg mL -1 while catalytic efficiency (k cat /K m ) values of CMCase and xylanase were 3280 and 38,797 min -1  mg -1  mL, respectively. EG-M-Xyn exerted great properties for its great potential in improving the enzymatic hydrolysis of lignocellulosics to produce fermentable sugars. First, EG-M-Xyn showed mild reaction pH and temperature of 5.5 and 50 °C, respectively. Secondly, EG-M-Xyn exhibited great heat tolerance of T 1/2 values of 173 (CMCase) and 693 min (xylanase). Lastly and most importantly, application of EG-M-Xyn in combination with Ctec2 (commercial enzyme) in the saccharification led to a 10-20% net increase in fermentable sugars liberated from pretreated rice straw in comparison to the Ctec2 alone group. In conclusion, EG-M-Xyn had great potential in generating fermentable sugars from renewable agro-residues for biofuel and fine chemical industry. Copyright © 2017 Elsevier B.V. All rights reserved.

A new chitinase-like xylanase inhibitor protein (XIP) from coffee (Coffea arabica) affects Soybean Asian rust (Phakopsora pachyrhizi) spore germination

PubMed Central

2011-01-01

Background Asian rust (Phakopsora pachyrhizi) is a common disease in Brazilian soybean fields and it is difficult to control. To identify a biochemical candidate with potential to combat this disease, a new chitinase-like xylanase inhibitor protein (XIP) from coffee (Coffea arabica) (CaclXIP) leaves was cloned into the pGAPZα-B vector for expression in Pichia pastoris. Results A cDNA encoding a chitinase-like xylanase inhibitor protein (XIP) from coffee (Coffea arabica) (CaclXIP), was isolated from leaves. The amino acid sequence predicts a (β/α)8 topology common to Class III Chitinases (glycoside hydrolase family 18 proteins; GH18), and shares similarity with other GH18 members, although it lacks the glutamic acid residue essential for catalysis, which is replaced by glutamine. CaclXIP was expressed as a recombinant protein in Pichia pastoris. Enzymatic assay showed that purified recombinant CaclXIP had only residual chitinolytic activity. However, it inhibited xylanases from Acrophialophora nainiana by approx. 60% when present at 12:1 (w/w) enzyme:inhibitor ratio. Additionally, CaclXIP at 1.5 μg/μL inhibited the germination of spores of Phakopsora pachyrhizi by 45%. Conclusions Our data suggests that CaclXIP belongs to a class of naturally inactive chitinases that have evolved to act in plant cell defence as xylanase inhibitors. Its role on inhibiting germination of fungal spores makes it an eligible candidate gene for the control of Asian rust. PMID:21299880

A new chitinase-like xylanase inhibitor protein (XIP) from coffee (Coffea arabica) affects Soybean Asian rust (Phakopsora pachyrhizi) spore germination.

PubMed

Vasconcelos, Erico A R; Santana, Celso G; Godoy, Claudia V; Seixas, Claudine D S; Silva, Marilia S; Moreira, Leonora R S; Oliveira-Neto, Osmundo B; Price, Daniel; Fitches, Elaine; Filho, Edivaldo X F; Mehta, Angela; Gatehouse, John A; Grossi-De-Sa, Maria F

2011-02-07

Asian rust (Phakopsora pachyrhizi) is a common disease in Brazilian soybean fields and it is difficult to control. To identify a biochemical candidate with potential to combat this disease, a new chitinase-like xylanase inhibitor protein (XIP) from coffee (Coffea arabica) (CaclXIP) leaves was cloned into the pGAPZα-B vector for expression in Pichia pastoris. A cDNA encoding a chitinase-like xylanase inhibitor protein (XIP) from coffee (Coffea arabica) (CaclXIP), was isolated from leaves. The amino acid sequence predicts a (β/α)8 topology common to Class III Chitinases (glycoside hydrolase family 18 proteins; GH18), and shares similarity with other GH18 members, although it lacks the glutamic acid residue essential for catalysis, which is replaced by glutamine. CaclXIP was expressed as a recombinant protein in Pichia pastoris. Enzymatic assay showed that purified recombinant CaclXIP had only residual chitinolytic activity. However, it inhibited xylanases from Acrophialophora nainiana by approx. 60% when present at 12:1 (w/w) enzyme:inhibitor ratio. Additionally, CaclXIP at 1.5 μg/μL inhibited the germination of spores of Phakopsora pachyrhizi by 45%. Our data suggests that CaclXIP belongs to a class of naturally inactive chitinases that have evolved to act in plant cell defence as xylanase inhibitors. Its role on inhibiting germination of fungal spores makes it an eligible candidate gene for the control of Asian rust.

Rapid 2,2'-bicinchoninic-based xylanase assay compatible with high throughput screening

Treesearch

William R. Kenealy; Thomas W. Jeffries

2003-01-01

High-throughput screening requires simple assays that give reliable quantitative results. A microplate assay was developed for reducing sugar analysis that uses a 2,2'-bicinchoninic-based protein reagent. Endo-1,4-â-D-xylanase activity against oat spelt xylan was detected at activities of 0.002 to 0.011 IU ml−1. The assay is linear for sugar...

Phylogenetic analysis of β-xylanase SRXL1 of Sporisorium reilianum and its relationship with families (GH10 and GH11) of Ascomycetes and Basidiomycetes

PubMed Central

Álvarez-Cervantes, Jorge; Díaz-Godínez, Gerardo; Mercado-Flores, Yuridia; Gupta, Vijai Kumar; Anducho-Reyes, Miguel Angel

2016-01-01

In this paper, the amino acid sequence of the β-xylanase SRXL1 of Sporisorium reilianum, which is a pathogenic fungus of maize was used as a model protein to find its phylogenetic relationship with other xylanases of Ascomycetes and Basidiomycetes and the information obtained allowed to establish a hypothesis of monophyly and of biological role. 84 amino acid sequences of β-xylanase obtained from the GenBank database was used. Groupings analysis of higher-level in the Pfam database allowed to determine that the proteins under study were classified into the GH10 and GH11 families, based on the regions of highly conserved amino acids, 233–318 and 180–193 respectively, where glutamate residues are responsible for the catalysis. PMID:27040368

Molecular cloning and expression in Saccharomyces cerevisiae of two Aspergillus nidulans xylanase genes.

PubMed Central

Pérez-Gonzalez, J A; De Graaff, L H; Visser, J; Ramón, D

1996-01-01

Two Aspergillus nidulans genes, xlnA and xlnB, encoding the X22 and X24 xylanases from this fungus, respectively, have been cloned and sequenced. Their cDNAs have been expressed in a laboratory Saccharomyces cerevisiae strain under the control of a constitutive yeast promoter, resulting in the construction of recombinant xylanolytic yeast strains. PMID:8787417

Secretome analysis of the thermophilic xylanase hyper-producer Thermomyces lanuginosus SSBP cultivated on corn cobs.

PubMed

Winger, A M; Heazlewood, J L; Chan, L J G; Petzold, C J; Permaul, K; Singh, S

2014-11-01

Thermomyces lanuginosus is a thermophilic fungus known for its ability to produce industrially important enzymes including large amounts of xylanase, the key enzyme in hemicellulose hydrolysis. The secretome of T. lanuginosus SSBP was profiled by shotgun proteomics to elucidate important enzymes involved in hemicellulose saccharification and to characterise the presence of other industrially interesting enzymes. This study reproducibly identified a total of 74 proteins in the supernatant following growth on corn cobs. An analysis of proteins revealed nine glycoside hydrolase (GH) enzymes including xylanase GH11, β-xylosidase GH43, β-glucosidase GH3, α-galactosidase GH36 and trehalose hydrolase GH65. Two commercially produced Thermomyces enzymes, lipase and amylase, were also identified. In addition, other industrially relevant enzymes not currently explored in Thermomyces were identified including glutaminase, fructose-bisphosphate aldolase and cyanate hydratase. Overall, these data provide insight into the novel ability of a cellulase-free fungus to utilise lignocellulosic material, ultimately producing a number of enzymes important to various industrial processes.

Gene cloning, overexpression, and characterization of a xylanase from Penicillium sp. CGMCC 1669.

PubMed

Liu, Wanli; Shi, Pengjun; Chen, Qiang; Yang, Peilong; Wang, Guozeng; Wang, Yaru; Luo, Huiying; Yao, Bin

2010-09-01

A xylanase-encoding gene, xyn11F63, was isolated from Penicillium sp. F63 CGMCC1669 using degenerated polymerase chain reaction (PCR) and thermal asymmetric interlaced (TAIL)-PCR techniques. The full-length chromosomal gene consists of 724 bp, including a 73-bp intron, and encodes a 217 amino acid polypeptide. The deduced amino acid sequence of xyn11F63 shows the highest identity of 70% to the xylanase from Penicillium sp. strain 40, which belongs to glycosyl hydrolases family 11. The gene was overexpressed in Pichia pastoris, and its activity in the culture medium reached 516 U ml(-1). After purification to electrophoretic homogeneity, the enzyme showed maximal activity at pH 4.5 and 40 degrees C, was stable at acidic buffers of pH 4.5-9.0, and was resistant to proteases (proteinase K, trypsin, subtilisin A, and alpha-chymotrypsin). The specific activity, K (m), and V (max) for oat spelt xylan substrate was 7,988 U mg(-1), 22.2 mg ml(-1), and 15,105.7 micromol min(-1) mg(-1), respectively. These properties make XYN11F63 a potential economical candidate for use in feed and food industrial applications.

Insight of endo-1,4-xylanase II from Trichoderma reesei: conserved water-mediated H-bond and ion pairs interactions.

PubMed

Vijayakumar, Balakrishnan; Velmurugan, Devadasan

2013-12-01

Endo-1,4-Xylanase II is an enzyme which degrades the linear polysaccharide beta-1,4-xylan into xylose. This enzyme shows highest enzyme activity around 55 °C, even without being stabilized by the disulphide bridges. A set of nine high resolution crystal structures of Xylanase II (1.11-1.80 Å) from Trichoderma reesei were selected and analyzed in order to identify the invariant water molecules, ion pairs and water-mediated ionic interactions. The crystal structure (PDB-id: 2DFB) solved at highest resolution (1.11 Å) was chosen as the reference and the remaining structures were treated as mobile molecules. These structures were then superimposed with the reference molecule to observe the invariant water molecules using 3-dimensional structural superposition server. A total of 37 water molecules were identified to be invariant molecules in all the crystal structures, of which 26 invariant molecules have hydrogen bond interactions with the back bone of residues and 21 invariant water molecules have interactions with side chain residues. The structural and functional roles of these water molecules and ion pairs have been discussed. The results show that the invariant water molecules and ion pairs may be involved in maintaining the structural architecture, dynamics and function of the Endo-1,4-Xylanase II.

The disruption of two salt bridges of the cold-active xylanase XynGR40 results in an increase in activity, but a decrease in thermostability.

PubMed

Wang, Guozeng; Wu, Jingjing; Lin, Juan; Ye, Xiuyun; Yao, Bin

2016-12-02

Cold-active xylanases are of great interest due to their large potential for application in the food industry. In this study, salt bridges of the eight glycoside hydrolase (GH) family 10 cold-active xylanases reported to date were predicted and the salt bridges specific to the cold-active xylanase XynGR40 were identified. Seven mutants were constructed to disrupt salt bridges specific to XynGR40. The results suggested that five mutants lost their xylanase activity, while the other two mutants, D30N and D83N, displayed different properties when compared with the wild-type XynGR40. First, both mutations showed an obvious decrease in thermostability, with the T 1/2 of D30N and D83N at 50 °C being about one half and one sixth of the wild-type, respectively. Second, both D30N and D83N had a higher specific activity than the wild-type, with activities about 13 and 163% higher, respectively. Third, both D30N and D83N had high k cat and K m values, which resulted in a higher catalytic efficiency of the mutant D83N, but a lower catalytic efficiency of the mutant D30N compared to the wild-type. Our results suggested that salt bridges play important roles in both the activity and thermostability of the cold-active xylanase XynGR40. The mutant D83N had a higher k cat and higher relative activity at low temperatures than the wild-type, and is a good candidate for application in the food industry. Copyright © 2016 Elsevier Inc. All rights reserved.

Engineering a thermostable fungal GH10 xylanase, importance of N-terminal amino acids.

PubMed

Song, Letian; Tsang, Adrian; Sylvestre, Michel

2015-06-01

Xylanases are used in many industrial processes including pulp bleaching, baking, detergent, and the hydrolysis of plant cell wall in biofuels production. In this work we have evolved a single domain GH10 xylanase, Xyn10A_ASPNG, from Aspergillus niger to improve its thermostability. We introduced a rational approach involving as the first step a computational analysis to guide the design of a mutagenesis library in targeted regions which identified thermal important residues that were subsequently randomly mutagenized through rounds of iterative saturation mutagenesis (ISM). Focusing on five residues, four rounds of ISM had generated a quintuple mutant 4S1 (R25W/V29A/I31L/L43F/T58I) which exhibited thermal inactivation half-life (t1/2 ) at 60°C that was prolonged by 30 folds in comparison with wild-type enzyme. Whereas the wild-type enzyme retained 0.2% of its initial activity after a heat treatment of 10 min at 60°C and was completely inactivated after 2 min at 65°C, 4S1 mutant retained 30% of its initial activity after 15 min heating at 65°C. Furthermore, the mutant melting temperature (Tm ) increased by 17.4°C compared to the wild type. Each of the five mutations in 4S1 was found to contribute to thermoresistance, but the dramatic improvement of enzyme thermoresistance of 4S1 was attributed to the synergistic effects of the five mutations. Comparison of biochemical data and model structure between 4S1 and the wild-type enzyme suggested that the N-terminal coil of the enzyme is important in stabilizing GH10 xylanase structure. Based on model structure analyses, we propose that enforced hydrophobic interactions within N-terminal elements and between N- and C-terminal ends are responsible for the improved thermostability of Xyn10A_ASPNG. © 2015 Wiley Periodicals, Inc.

Genomic characterization of plant cell wall degrading enzymes and in silico analysis of xylanases and polygalacturonases of Fusarium virguliforme.

PubMed

Chang, Hao-Xun; Yendrek, Craig R; Caetano-Anolles, Gustavo; Hartman, Glen L

2016-07-12

Plant cell wall degrading enzymes (PCWDEs) are a subset of carbohydrate-active enzymes (CAZy) produced by plant pathogens to degrade plant cell walls. To counteract PCWDEs, plants release PCWDEs inhibitor proteins (PIPs) to reduce their impact. Several transgenic plants expressing exogenous PIPs that interact with fungal glycoside hydrolase (GH)11-type xylanases or GH28-type polygalacturonase (PG) have been shown to enhance disease resistance. However, many plant pathogenic Fusarium species were reported to escape PIPs inhibition. Fusarium virguliforme is a soilborne pathogen that causes soybean sudden death syndrome (SDS). Although the genome of F. virguliforme was sequenced, there were limited studies focused on the PCWDEs of F. virguliforme. Our goal was to understand the genomic CAZy structure of F. viguliforme, and determine if exogenous PIPs could be theoretically used in soybean to enhance resistance against F. virguliforme. F. virguliforme produces diverse CAZy to degrade cellulose and pectin, similar to other necrotorphic and hemibiotrophic plant pathogenic fungi. However, some common CAZy of plant pathogenic fungi that catalyze hemicellulose, such as GH29, GH30, GH44, GH54, GH62, and GH67, were deficient in F. virguliforme. While the absence of these CAZy families might be complemented by other hemicellulases, F. virguliforme contained unique families including GH131, polysaccharide lyase (PL) 9, PL20, and PL22 that were not reported in other plant pathogenic fungi or oomycetes. Sequence analysis revealed two GH11 xylanases of F. virguliforme, FvXyn11A and FvXyn11B, have conserved residues that allow xylanase inhibitor protein I (XIP-I) binding. Structural modeling suggested that FvXyn11A and FvXyn11B could be blocked by XIP-I that serves as good candidate for developing transgenic soybeans. In contrast, one GH28 PG, FvPG2, contains an amino acid substitution that is potentially incompatible with the bean polygalacturonase-inhibitor protein II (PvPGIP2

Fast automated online xylanase activity assay using HPAEC-PAD.

PubMed

Cürten, Christin; Anders, Nico; Juchem, Niels; Ihling, Nina; Volkenborn, Kristina; Knapp, Andreas; Jaeger, Karl-Erich; Büchs, Jochen; Spiess, Antje C

2018-01-01

In contrast to biochemical reactions, which are often carried out under automatic control and maintained overnight, the automation of chemical analysis is usually neglected. Samples are either analyzed in a rudimentary fashion using in situ techniques, or aliquots are withdrawn and stored to facilitate more precise offline measurements, which can result in sampling and storage errors. Therefore, in this study, we implemented automated reaction control, sampling, and analysis. As an example, the activities of xylanases on xylotetraose and soluble xylan were examined using high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). The reaction was performed in HPLC vials inside a temperature-controlled Dionex™ AS-AP autosampler. It was started automatically when the autosampler pipetted substrate and enzyme solution into the reaction vial. Afterwards, samples from the reaction vial were injected repeatedly for 60 min onto a CarboPac™ PA100 column for analysis. Due to the rapidity of the reaction, the analytical method and the gradient elution of 200 mM sodium hydroxide solution and 100 mM sodium hydroxide with 500 mM sodium acetate were adapted to allow for an overall separation time of 13 min and a detection limit of 0.35-1.83 mg/L (depending on the xylooligomer). This analytical method was applied to measure the soluble short-chain products (xylose, xylobiose, xylotriose, xylotetraose, xylopentaose, and longer xylooligomers) that arise during enzymatic hydrolysis. Based on that, the activities of three endoxylanases (EX) were determined as 294 U/mg for EX from Aspergillus niger, 1.69 U/mg for EX from Bacillus stearothermophilus, and 0.36 U/mg for EX from Bacillus subtilis. Graphical abstract Xylanase activity assay automation.

Production of prebiotic-xylooligosaccharides from alkali pretreated mahogany and mango wood sawdust by using purified xylanase of Clostridium strain BOH3.

PubMed

Rajagopalan, Gobinath; Shanmugavelu, Kavitha; Yang, Kun-Lin

2017-07-01

Xylooligosaccharides (XOS) are emerging prebiotics which can be produced from lignocellulosic biomass including agro-residues and hardwood. In this study, we report the production of XOS from thermal-alkali pretreated hardwood such as mahogany and mango by using a purified xylanase from Clostridium strain BOH3. In the first approach, pure xylan is extracted from mahogany and mango hardwood and then the pure xylan is hydrolyzed by using the xylanase. In this case, 572 and 504mg XOS/g pure xylan were obtained from mahogany and mango woods, respectively. In the second approach, the same xylanase is employed to hydrolyze sawdust of hardwood after different types of pretreatments. After a thermal (121°C for 15min) pretreatment under a mild alkaline (0.05N NaOH) condition, the pretreated mahogany and mango sawdust can be utilized directly to produce 89.5 and 67.6mg XOS/g pretreated sawdust, respectively. XOS produced from the pretreated sawdust show strong prebiotic effects on Bifidobacteria and Lactobacilli. This report shows the possibility of producing XOS from pretreated woody wastes without using pure xylan as a substrate. Copyright © 2017 Elsevier Ltd. All rights reserved.

On the specificity and mode of action of a xylanase from Trametes hirsuta (Wulf.) Pilát.

PubMed

Kubacková, M; Karácsonyi, S; Bilisics, L; Toman, R

1979-11-01

The mode of action of the extracellular endo-(1 leads to 4)-beta-D-xylanase produced by Trametes hirsuta on a (4-0-methyl-D-glucurono)-D-xylan and a modified, essentially neutral D-xylan from white willow (Salix alba L.) has been studied. Xylotetraose and xylohexaose, together with aldotetraouronic and aldohexaouronic acids, were the main products. The acidic oligosaccharides had a 4-O-methyl-D-glucopyranosyluronic acid group attached to the non-reducing D-xylosyl end-group. The action pattern of the xylanase corresponds to that of a typical endo-enzyme that acts more readily in the middle of chain, and the specific region of its action appears to involve five D-xylosyl residues. The products of the enzymic treatment of the D-xylan have revealed a regular distribution of the 4-O-methyl-D-glucopyranosyluronic acid groups attached to the D-xylan backbone.

Cellulose and hemicellulose-degrading enzymes in Fusarium commune transcriptome and functional characterization of three identified xylanases.

PubMed

Huang, Yuhong; Busk, Peter Kamp; Lange, Lene

2015-06-01

Specific enzymes from plant-pathogenic microbes demonstrate high effectiveness for natural lignocellulosic biomass degradation and utilization. The secreted lignocellulolytic enzymes of Fusarium species have not been investigated comprehensively, however. In this study we compared cellulose and hemicellulose-degrading enzymes of classical fungal enzyme producers with those of Fusarium species. The results indicated that Fusarium species are robust cellulose and hemicellulose degraders. Wheat bran, carboxymethylcellulose and xylan-based growth media induced a broad spectrum of lignocellulolytic enzymes in Fusarium commune. Prediction of the cellulose and hemicellulose-degrading enzymes in the F. commune transcriptome using peptide pattern recognition revealed 147 genes encoding glycoside hydrolases and six genes encoding lytic polysaccharide monooxygenases (AA9 and AA11), including all relevant cellulose decomposing enzymes (GH3, GH5, GH6, GH7, GH9, GH45 and AA9), and abundant hemicellulases. We further applied peptide pattern recognition to reveal nine and seven subfamilies of GH10 and GH11 family enzymes, respectively. The uncharacterized XYL10A, XYL10B and XYL11 enzymes of F. commune were classified, respectively, into GH10 subfamily 1, subfamily 3 and GH11 subfamily 1. These xylanases were successfully expressed in the PichiaPink™ system with the following properties: the purified recombinant XYL10A had interesting high specific activity; XYL10B was active at alkaline conditions with both endo-1,4-β-d-xylanase and β-xylosidase activities; and XYL11 was a true xylanase characterized by high substrate specificity. These results indicate that F. commune with genetic modification is a promising source of enzymes for the decomposition of lignocellulosic biomass. Copyright © 2015 Elsevier Inc. All rights reserved.

Partial Optimization of Endo-1, 4-Β-Xylanase Production by Aureobasidium pullulans Using Agro-Industrial Residues

PubMed Central

Nasr, Shaghayegh; Soudi, Mohammad Reza; Hatef Salmanian, Ali; Ghadam, Parinaz

2013-01-01

Objective(s) : Although bacteria and molds are the pioneering microorganisms for production of many enzymes, yet yeasts provide safe and reliable sources of enzymes with applications in food and feed. Materials and Methods: Single xylanase producer yeast was isolated from plant residues based on formation of transparent halo zones on xylan agar plates. The isolate showed much greater endo-1, 4-β-xylanase activity of 2.73 IU/ml after optimization of the initial extrinsic conditions. It was shown that the strain was also able to produce β-xylosidase (0.179 IU/ml) and α-arabinofuranosidase (0.063 IU/ml). Identification of the isolate was carried out and the endo-1, 4-β-xylanaseproduction by feeding the yeast cells on agro-industrial residues was optimized using one factor at a time approach. Results: The enzyme producer strain was identified as Aureobasidiumpullulans. Based on the optimization approach, an incubation time of 48 hr at 27°C, inoculum size of 2% (v/v), initial pH value of 4 and agitation rate of 90 rpm were found to be the optimal conditions for achieving maximum yield of the enzyme. Xylan, containing agricultural residues, was evaluated as low-cost alternative carbon source for production of xylanolytic enzymes. The production of xylanase enzyme in media containing wheat bran as the sole carbon source was very similar to that of the medium containing pure beechwoodxylan. Conclusion:This finding indicates the feasibility of growing of A. pullulans strain SN090 on wheat bran as an alternate economical substrate in order for reducing the costs of enzyme production and using this fortified agro-industrial byproduct in formulation of animal feed. PMID:24570830

Single-step purification and characterization of an extreme halophilic, ethanol tolerant and acidophilic xylanase from Aureobasidium pullulans NRRL Y-2311-1 with application potential in the food industry.

PubMed

Yegin, Sirma

2017-04-15

An extracellular xylanase from Aureobasidium pullulans NRRL Y-2311-1 produced on wheat bran was purified by a single-step chromatographic procedure. The enzyme had a molecular weight of 21.6kDa. The optimum pH and temperature for xylanase activity were 4.0 and 30-50°C, respectively. The enzyme was stable in the pH range of 3.0-8.0. The inactivation energy of the enzyme was calculated as 218kJmol -1 . The xylanase was ethanol tolerant and kept complete activity in the presence of 10% ethanol. Likewise, it retained almost complete activity at a concentration range of 0-20% NaCl. In general, the enzyme was resistant to several metal ions and reagents. Mg 2+ , Zn 2+ , Cu 2+ , K 1+ , EDTA and β-mercaptoethanol resulted in enhanced xylanase activity. The K m and V max values on beechwood xylan were determined to be 19.43mgml -1 and 848.4Uml -1 , respectively. The enzyme exhibits excellent characteristics and could, therefore, be a promising candidate for application in food and bio-industries. Copyright © 2016 Elsevier Ltd. All rights reserved.

Role in pathogenesis of two endo-beta-1,4-xylanase genes from the vascular wilt fungus Fusarium oxysporum.

PubMed

Gómez-Gómez, E; Ruíz-Roldán, M C; Di Pietro, A; Roncero, M I G; Hera, C

2002-04-01

A gene, xyl4, whose predicted amino acid sequence shows significant homology with family 11 xylanases, was identified from the tomato vascular wilt fungus Fusarium oxysporum f. sp. lycopersici. Expression of xyl4 is induced on oat spelt xylan as the carbon source, subject to carbon catabolite repression and preferentially expressed at alkaline ambient pH. Transcript levels of xyl4 on an inducing carbon source are differentially regulated by the nature and concentration of the nitrogen source. As shown by RT-PCR, xyl4 is expressed by F. oxysporum during the entire cycle of infection on tomato plants. Targeted inactivation of xyl4 and of xyl3, a previously identified gene of F. oxysporum f. sp. lycopersici encoding a family 10 xylanase, had no detectable effect on virulence on tomato plants, demonstrating that both genes are not essential for pathogenicity.

Engineering increased thermostability in the GH-10 endo-1,4-ß-xylanase from Thermoascus aurantiacus CBMAI 756

USDA-ARS?s Scientific Manuscript database

The GH10 endo-xylanase from Thermoascus aurantiacus CBMAI 756 (XynA) is industrially attractive due to its considerable thermostability and high specific activity. Considering the possibility of a further improvement in thermostability, eleven mutants were created in the present study via site-direc...

Structure determination of the extracellular xylanase from Geobacillus stearothermophilus by selenomethionyl MAD phasing.

PubMed

Teplitsky, A; Mechaly, A; Stojanoff, V; Sainz, G; Golan, G; Feinberg, H; Gilboa, R; Reiland, V; Zolotnitsky, G; Shallom, D; Thompson, A; Shoham, Y; Shoham, G

2004-05-01

Xylanases are hemicellulases that hydrolyze the internal beta-1,4-glycoside bonds of xylan. The extracellular thermostable endo-1,4-beta-xylanase (EC 3.2.1.8; XT6) produced by the thermophilic bacterium Geobacillus stearothermophilus T-6 was shown to bleach pulp optimally at pH 9 and 338 K and was successfully used in a large-scale biobleaching mill trial. The xylanase gene was cloned and sequenced. The mature enzyme consists of 379 amino acids, with a calculated molecular weight of 43 808 Da and a pI of 9.0. Crystallographic studies of XT6 were performed in order to study the mechanism of catalysis and to provide a structural basis for the rational introduction of enhanced thermostability by site-specific mutagenesis. XT6 was crystallized in the primitive trigonal space group P3(2)21, with unit-cell parameters a = b = 112.9, c = 122.7 A. A full diffraction data set for wild-type XT6 has been measured to 2.4 A resolution on flash-frozen crystals using synchrotron radiation. A fully exchanged selenomethionyl XT6 derivative (containing eight Se atoms per XT6 molecule) was also prepared and crystallized in an isomorphous crystal form, providing full selenium MAD data at three wavelengths and enabling phase solution and structure determination. The structure of wild-type XT6 was refined at 2.4 A resolution to a final R factor of 15.6% and an R(free) of 18.6%. The structure demonstrates that XT6 is made up of an eightfold TIM-barrel containing a deep active-site groove, consistent with its 'endo' mode of action. The two essential catalytic carboxylic residues (Glu159 and Glu265) are located at the active site within 5.5 A of each other, as expected for 'retaining' glycoside hydrolases. A unique subdomain was identified in the carboxy-terminal part of the enzyme and was suggested to have a role in xylan binding. The three-dimensional structure of XT6 is of great interest since it provides a favourable starting point for the rational improvement of its already high

Characterization of the arabinoxylan-degrading machinery of the thermophilic bacterium Herbinix hemicellulosilytica-Six new xylanases, three arabinofuranosidases and one xylosidase.

PubMed

Mechelke, M; Koeck, D E; Broeker, J; Roessler, B; Krabichler, F; Schwarz, W H; Zverlov, V V; Liebl, W

2017-09-10

Herbinix hemicellulosilytica is a newly isolated, gram-positive, anaerobic bacterium with extensive hemicellulose-degrading capabilities obtained from a thermophilic biogas reactor. In order to exploit its potential as a source for new industrial arabinoxylan-degrading enzymes, six new thermophilic xylanases, four from glycoside hydrolase family 10 (GH10) and two from GH11, three arabinofuranosidases (1x GH43, 2x GH51) and one β-xylosidase (GH43) were selected. The recombinantly produced enzymes were purified and characterized. All enzymes were active on different xylan-based polysaccharides and most of them showed temperature-vs-activity profiles with maxima around 55-65°C. HPAEC-PAD analysis of the hydrolysates of wheat arabinoxylan and of various purified xylooligosaccharides (XOS) and arabinoxylooligosaccharides (AXOS) was used to investigate their substrate and product specificities: among the GH10 xylanases, XynB showed a different product pattern when hydrolysing AXOS compared to XynA, XynC, and XynD. None of the GH11 xylanases was able to degrade any of the tested AXOS. All three arabinofuranosidases, ArfA, ArfB and ArfC, were classified as type AXH-m,d enzymes. None of the arabinofuranosidases was able to degrade the double-arabinosylated xylooligosaccharides XA 2+3 XX. β-Xylosidase XylA (GH43) was able to degrade unsubstituted XOS, but showed limited activity to degrade AXOS. Copyright © 2017. Published by Elsevier B.V.

Xylanase and feruloyl esterase from actinomycetes cultures could enhance sugarcane bagasse hydrolysis in the production of fermentable sugars.

PubMed

Rahmani, Nanik; Kahar, Prihardi; Lisdiyanti, Puspita; Hermiati, Euis; Lee, Jaemin; Yopi; Prasetya, Bambang; Ogino, Chiaki; Kondo, Akihiko

2018-02-23

The addition of enzymes that are capable of degrading hemicellulose has a potential to reduce the need for commercial enzymes during biomass hydrolysis in the production of fermentable sugars. In this study, a high xylanase producing actinomycete strain (Kitasatospora sp. ID06-480) and the first ethyl ferulate producing actinomycete strain (Nonomuraea sp. ID06-094) were selected from 797 rare actinomycetes, respectively, which were isolated in Indonesia. The addition (30%, v/v) of a crude enzyme supernatant from the selected strains in sugarcane bagasse hydrolysis with low-level loading (1 FPU/g-biomass) of Cellic® CTec2 enhanced both the released amount of glucose and reducing sugars. When the reaction with Ctec2 was combined with crude enzymes containing either xylanase or feruloyl esterase, high conversion yield of glucose from cellulose at 60.5% could be achieved after 72 h-saccharification.

Engineering increased thermostability in the thermostable GH-11 xylanase from Thermobacillus xylanilyticus.

PubMed

Paës, Gabriel; O'Donohue, Michael J

2006-09-18

Enzymatic hydrolysis constitutes an attractive strategy for biorefining of abundant, low-cost agricultural by-products such as wheat bran and straw. However, to adopt such an approach, efficient enzymes are required, in particular xylanases. To promote heat-induced disorganization of the complex cell wall network in wheat bran and thus increase enzymatic hydrolysis, we have attempted to improve the thermoresistance of a GH-11 xylanase that is already moderately thermostable. Using a previously described engineering strategy that involves the introduction of disulphide bridges, a mutant (Tx-xyl-SS3) displaying enhanced thermostability and thermoactivity was obtained. The half life at 70 degrees C (180 min) of Tx-xyl-SS3 is 10-fold greater than that of the wild type enzyme and its specific activity is almost doubled (3500 IU mg(-1)). Despite these improvements, Tx-xyl-SS3 was unsuitable for use at significantly higher reaction temperatures (i.e. 85-95 degrees C) and thus the initial objective of this study remained unaccomplished. However, unexpectedly even at the normal hydrolytic temperature (60 degrees C), Tx-xyl-SS3 was able to solubilize 50% of the wheat bran arabinoxylans, 10 points more than the wild type enzyme in parallel reactions. The data presented here show that this improvement is not directly linked to the increase in thermostability and/or thermoactivity, but rather to other unidentified changes to physico-chemical properties that may allow Tx-xyl-SS3 to better penetrate the cell wall network in wheat bran.

Purification and biochemical properties of multiple xylanases from Aspergillus ochraceus tolerant to Hg2+ ion and a wide range of pH.

PubMed

Michelin, Michele; Silva, Tony M; Jorge, João A; Polizeli, Maria de Lourdes T M

2014-09-01

Production of multiple xylanases, in which each enzyme has a specific characteristic, can be one strategy to achieve the effective hydrolysis of xylan. Three xylanases (xyl 1, xyl 2, and xyl 3) from Aspergillus ochraceus were purified by chromatography using diethylaminoethyl (DEAE) cellulose, Biogel P-60, and Sephadex G-100 columns. These enzymes are glycoproteins of low molecular weight with an optimum temperature at 60 °C. The glycosylation presented is apparently not related to thermostability, since xyl 3 (20 % carbohydrate) was more thermostable than xyl 2 (67 % carbohydrate). Xyl 3 was able to retain most of its activity in a wide range of pH (3.5-8.0), while xyl 1 and xyl 2 presented optimum pH of 6.0. Xyl 1 and xyl 2 were activated by 5 and 10 mM MnCl2 and CoCl2, while xyl 3 was activated by 1 mM of the same compounds. Interestingly, xyl 2 presented high tolerance toward mercury ion. Xylanases from A. ochraceus hydrolyzed xylans of different origins, such as birchwood, oat spelt, larchwood, and eucalyptus (around 90 % or more), except xyl 2 and xyl 3 that hydrolyzed with lesser efficiency eucalyptus (66.7 %) and oat spelt (44.8 %) xylans.

Efficient production of lignocellulolytic enzymes xylanase, β-xylosidase, ferulic acid esterase and β-glucosidase by the mutant strain Aspergillus awamori 2B.361 U2/1

PubMed Central

Gottschalk, Leda Maria Fortes; de Sousa Paredes, Raquel; Teixeira, Ricardo Sposina Sobral; da Silva, Ayla Sant’Ana; da Silva Bon, Elba Pinto

2013-01-01

The production of xylanase, β-xylosidase, ferulic acid esterase and β-glucosidase by Aspergillus awamori 2B.361 U2/1, a hyper producer of glucoamylase and pectinase, was evaluated using selected conditions regarding nitrogen nutrition. Submerged cultivations were carried out at 30 °C and 200 rpm in growth media containing 30 g wheat bran/L as main carbon source and either yeast extract, ammonium sulfate, sodium nitrate or urea, as nitrogen sources; in all cases it was used a fixed molar carbon to molar nitrogen concentration of 10.3. The use of poor nitrogen sources favored the accumulation of xylanase, β-xylosidase and ferulic acid esterase to a peak concentrations of 44,880; 640 and 118 U/L, respectively, for sodium nitrate and of 34,580, 685 and 170 U/L, respectively, for urea. However, the highest β-glucosidase accumulation of 10,470 U/L was observed when the rich organic nitrogen source yeast extract was used. The maxima accumulation of filter paper activity, xylanase, β-xylosidase, ferulic acid esterase and β-glucosidase by A. awamori 2B.361 U2/1 was compared to that produced by Trichoderma reesei Rut-C30. The level of β-glucosidase was over 17-fold higher for the Aspergillus strain, whereas the levels of xylanase and β-xylosidase were over 2-fold higher. This strain also produced ferulic acid esterase (170 U/L), which was not detected in the T. reesei culture. PMID:24294256

Effects of beta-glucanase and xylanase supplementation on gastrointestinal digestive enzyme activities of weaned piglets fed a barley-based diet.

PubMed

Fan, C L; Han, X Y; Xu, Z R; Wang, L J; Shi, L R

2009-04-01

The effects of supplementing a barley-based diet for weaned piglets withexogenous beta-glucanase and xylanase on gastrointestinal digestiveenzyme activities were investigated. Thirty-six cross-bred weaned pigletswere randomly assigned to two groups with three pens based on sexand mass. Each group was fed on the diet based on barley with or withoutadded beta-glucanase and xylanase (0.15%) for a 4-week period. Theresults showed that enzyme supplementation improved growth performanceof piglets significantly (p < 0.05), but had no effect (p = 0.091)on average daily feed intake. The results also showed that supplementationof beta-glucanase and xylanase had no effect on pepsin activity in gastriccontents but slightly decreased (p = 0.092) the pepsin activity ingastric mucosa. Meanwhile, no effect of enzyme supplementation ontrypsin activity in duodenal contents was observed. However, the activitiesof amylase and lipase in duodenal contents were significantly(p < 0.05) decreased, whereas the activities of maltase, sucrase andgamma-glutamyl transpeptidase (gamma-GT) in jejunal and ileal mucosa wereenhanced significantly (p < 0.05). The improvement of disaccharidaseand gamma-GT activity may be attributed to the positive impacts of exogenousenzymes on digestion and absorption of the nutrients. In conclusion,the current results indicated that supplementation with enzymes in barley-based diets could improve the growth performance of piglets,decrease the activities of amylase and lipase in duodenal contents andincrease the activities of disaccharidase and gamma-GT in jejunal and ilealmucosa.

Trichoderma reesei xylanase 5 is defective in the reference strain QM6a but functional alleles are present in other wild-type strains.

PubMed

Ramoni, Jonas; Marchetti-Deschmann, Martina; Seidl-Seiboth, Verena; Seiboth, Bernhard

2017-05-01

Trichoderma reesei is a paradigm for the regulation and industrial production of plant cell wall-degrading enzymes. Among these, five xylanases, including the glycoside hydrolase (GH) family 11 XYN1 and XYN2, the GH10 XYN3, and the GH30 XYN4 and XYN6, were described. By genome mining and transcriptome analysis, a further putative xylanase, encoded by xyn5, was identified. Analysis of xyn5 from the genome-sequenced reference strain T. reesei QM6a shows that it encodes a non-functional, truncated form of XYN5. However, non-truncated orthologues are present in other genome sequenced Trichoderma spp., and sequencing of xyn5 in other T. reesei wild-type isolates shows that they harbor a putative functional xyn5 allele. In silico analysis and 3D modeling revealed that the encoded XYN5 has significant structural similarities to xylanases of the GH11 family, including a GH-typical substrate binding groove and a carboxylate pair in the active site. The xyn5 of wild-type strain TUCIM1282 was recombinantly expressed in a T. reesei strain with a (hemi)cellulase-free background and the corresponding protein purified to apparent homogeneity. The pH and temperature optima and the kinetic parameters of the purified XYN5 were pH 4, 50 °C, and V max  = 2646 nkat/mg with a K m of 9.68 mg/ml. This functional xyn5 allele was used to replace the mutated version which led to an overall increase of the xylanolytic activity. These findings are of particular importance as GH11 xylanases are of high biotechnological relevance, and T. reesei is one of the main industrial producers of such lignocellulose-degrading enzymes.

Assembly of Xylanases into Designer Cellulosomes Promotes Efficient Hydrolysis of the Xylan Component of a Natural Recalcitrant Cellulosic Substrate

PubMed Central

Moraïs, Sarah; Barak, Yoav; Hadar, Yitzhak; Wilson, David B.; Shoham, Yuval; Lamed, Raphael; Bayer, Edward A.

2011-01-01

ABSTRACT In nature, the complex composition and structure of the plant cell wall pose a barrier to enzymatic degradation. Nevertheless, some anaerobic bacteria have evolved for this purpose an intriguing, highly efficient multienzyme complex, the cellulosome, which contains numerous cellulases and hemicellulases. The rod-like cellulose component of the plant cell wall is embedded in a colloidal blend of hemicelluloses, a major component of which is xylan. In order to enhance enzymatic degradation of the xylan component of a natural complex substrate (wheat straw) and to study the synergistic action among different xylanases, we have employed a variation of the designer cellulosome approach by fabricating a tetravalent complex that includes the three endoxylanases of Thermobifida fusca (Xyn10A, Xyn10B, and Xyn11A) and an Xyl43A β-xylosidase from the same bacterium. Here, we describe the conversion of Xyn10A and Xyl43A to the cellulosomal mode. The incorporation of the Xyl43A enzyme together with the three endoxylanases into a common designer cellulosome served to enhance the level of reducing sugars produced during wheat straw degradation. The enhanced synergistic action of the four xylanases reflected their immediate juxtaposition in the complex, and these tetravalent xylanolytic designer cellulosomes succeeded in degrading significant (~25%) levels of the total xylan component of the wheat straw substrate. The results suggest that the incorporation of xylanases into cellulosome complexes is advantageous for efficient decomposition of recalcitrant cellulosic substrates—a distinction previously reserved for cellulose-degrading enzymes. PMID:22086489

Finding stable cellulase and xylanase: evaluation of the synergistic effect of pH and temperature.

PubMed

Farinas, Cristiane S; Loyo, Marcel Moitas; Baraldo, Anderson; Tardioli, Paulo W; Neto, Victor Bertucci; Couri, Sonia

2010-12-31

Ethanol from lignocellulosic biomass has been recognized as one of the most promising alternatives for the production of renewable and sustainable energy. However, one of the major bottlenecks holding back its commercialization is the high costs of the enzymes needed for biomass conversion. In this work, we studied the enzymes produced from a selected strain of Aspergillus niger under solid state fermentation. The cellulase and xylanase enzymatic cocktail was characterized in terms of pH and temperature by using response surface methodology. Thermostability and kinetic parameters were also determined. The statistical analysis of pH and temperature effects on enzymatic activity showed a synergistic interaction of these two variables, thus enabling to find a pH and temperature range in which the enzymes have a higher activity. The results obtained allowed the construction of mathematical models used to predict endoglucanase, β-glucosidase and xylanase activities under different pH and temperature conditions. Optimum temperature values for all three enzymes were found to be in the range between 35°C and 60°C, and the optimum pH range was found between 4 and 5.5. The methodology employed here was very effective in estimating enzyme behavior under different process conditions. Copyright © 2010 Elsevier B.V. All rights reserved.

Supplementation with xylanase and β-xylosidase to reduce xylo-oligomer and xylan inhibition of enzymatic hydrolysis of cellulose and pretreated corn stover

PubMed Central

2011-01-01

Background Hemicellulose is often credited with being one of the important physical barriers to enzymatic hydrolysis of cellulose, and acts by blocking enzyme access to the cellulose surface. In addition, our recent research has suggested that hemicelluloses, particularly in the form of xylan and its oligomers, can more strongly inhibit cellulase activity than do glucose and cellobiose. Removal of hemicelluloses or elimination of their negative effects can therefore become especially pivotal to achieving higher cellulose conversion with lower enzyme doses. Results In this study, cellulase was supplemented with xylanase and β-xylosidase to boost conversion of both cellulose and hemicellulose in pretreated biomass through conversion of xylan and xylo-oligomers to the less inhibitory xylose. Although addition of xylanase and β-xylosidase did not necessarily enhance Avicel hydrolysis, glucan conversions increased by 27% and 8% for corn stover pretreated with ammonia fiber expansion (AFEX) and dilute acid, respectively. In addition, adding hemicellulase several hours before adding cellulase was more beneficial than later addition, possibly as a result of a higher adsorption affinity of cellulase and xylanase to xylan than glucan. Conclusions This key finding elucidates a possible mechanism for cellulase inhibition by xylan and xylo-oligomers and emphasizes the need to optimize the enzyme formulation for each pretreated substrate. More research is needed to identify advanced enzyme systems designed to hydrolyze different substrates with maximum overall enzyme efficacy. PMID:21702938

Isolation, screening and characterization of a novel extracellular xylanase from Aspergillus niger (KP874102.1) and its application in orange peel hydrolysis.

PubMed

Uday, Uma Shankar Prasad; Majumdar, Ria; Tiwari, Onkar Nath; Mishra, Umesh; Mondal, Abhijit; Bandyopadhyay, Tarun Kanti; Bhunia, Biswanath

2017-12-01

In the present work, a potent xylanase producing fungal strain Aspergillus niger (KP874102.1) was isolated through cultural and morphological observations from soil sample of Baramura forest, Tripura west, India. 28S rDNA technique was applied for genomic identification of this fungal strain. The isolated strain was found to be phylogenetically closely related to Aspergillus niger. Kinetic constants such as K m and V max for extracellular xylanase were determined using various substrate such as beech wood xylan, oat spelt xylan and CM cellulose through Lineweaver-Burk plot. K m , V max and K cat for beech wood xylan are found to be 2.89mg/ml, 2442U and 426178Umlmg -1 respectively. Crude enzyme did not show also CM cellulose activity. The relative efficiency of oat spelt xylan was found to be 0.819 with respect to beech wood xylan. After acid hydrolysis, enzyme was able to produce reducing sugar with 17.7, 35.5, 50.8 and 65% (w/w) from orange peel after 15, 30, 45 and 60min incubation with cellulase free xylanase and maximum reducing sugar formation rate was found to be 55.96μg/ml/min. Therefore, the Aspergillus niger (KP874102.1) is considered as a potential candidate for enzymatic hydrolysis of orange peel. Copyright © 2017 Elsevier B.V. All rights reserved.

Influence of a direct-fed microbial and xylanase enzyme on the dietary energy uptake efficiency and performance of broiler chickens.

PubMed

Murugesan, Ganapathi Raj; Persia, Michael E

2015-09-01

Efficacy of a multi-strain direct-fed microbial product (PoultryStar(®) ME; PS) and a xylanase enzyme product on the dietary energy utilization efficiency and resulting performance in broiler chickens was evaluated. Apart from performance parameters, cecal and serum metabolites and activities of hepatic enzymes involved in energy metabolism were also determined. Ross 308 chicks were fed one of four experimental diets [control (CON), CON + PS, CON + xylanase and CON + PS + xylanase] using a 2 × 2 factorial arrangement from 1-21 days of age. Cecal proportions of propionate and butyrate, as well as total short-chain fatty acid concentration were increased (P <0.01) by PS suggesting increased fermentation of dietary fiber. Both additives reduced (P <0.01) serum non-esterified free fatty acids, while PS reduced (P <0.01) serum triglyceride. Hepatic glycogen concentration was increased (P <0.01) by both additives. Changes in these serum metabolites and hepatic glycogen indicate the influence of additives in swiftly transitioning the birds from fasting to feeding metabolism. The activity of hepatic glucose-6-phosphate dehydrogenase (G6PDH) was increased (P <0.01) by PS. Elevated hepatic glycogen and G6PDH activity indicate increased glucose-sparing potential. Feed conversion ratio (FCR) was lowered by both additives, while the magnitude of reduction was higher with the combination. The combination worked synergistically, compared to their individual effects, to increase dietary energy uptake and hepatic energy retention. The combination additively increased the FCR, suggesting involvement of synergistic modes of actions. © 2014 Society of Chemical Industry.

A xylanase gene directly cloned from the genomic DNA of alkaline wastewater sludge showing application potential in the paper industry.

PubMed

Zhao, Yanyu; Luo, Huiying; Meng, Kun; Shi, Pengjun; Wang, Guozeng; Yang, Peilong; Yuan, Tiezheng; Yao, Bin

2011-09-01

A xylanase gene, aws-2x, was directly cloned from the genomic DNA of the alkaline wastewater sludge using degenerated PCR and modified TAIL-PCR. The deduced amino acid sequence of AWS-2x shared the highest identity (60%) with the xylanase from Chryseobacterium gleum belonging to the glycosyl hydrolase GH family 10. Recombinant AWS-2x was expressed in Escherichia coli BL21 (DE3) and purified to electrophoretic homogeneity. The enzyme showed maximal activity at pH 7.5 and 55 °C, maintained more than 50% of maximal activity when assayed at pH 9.0, and was stable over a wide pH range from 4.0 to 11.0. The specific activity of AWS-2x towards hardwood xylan (beechwood and birchwood xylan) was significantly higher than that to cereal xylan (oat spelt xylan and wheat arabinoxylan). These properties make AWS-2x a potential candidate for application in the pulp and paper industry.

Designing cross-linked xylanase aggregates for bioconversion of agroindustrial waste biomass towards potential production of nutraceuticals.

PubMed

Hero, J S; Romero, C M; Pisa, J H; Perotti, N I; Olivaro, C; Martinez, M A

2018-05-01

Immobilized biocatalysts design has the potential to efficiently produce valuable bioproducts from lignocellulosic biomass. Among them, the carrier-free immobilization through the cross-linked enzyme aggregates technology is a simple and low-cost alternative. A two steps statistical approach was utilized to evaluate the synthesis of a cross-linked enzyme aggregate from a xylanolytic preparation, which was produced by Cohnella sp. AR92 grown in a peptone-based culture medium. The resulting immobilized biocatalyst, Xyl-CLEA, was significate more stable (25 to 45%) towards temperatures up to 50°C with respect to the free enzyme, and retained over 50% of its initial activity after 5 consecutive cycles of reuse. By means of infrared spectroscopy and electron microscopy, the Xyl-CLEA showed architectural features described as signature of type I and type II of protein aggregates. These, were the result of the simultaneous aggregation of a multiplicity of proteins from the crude enzymatic extract. The enzymatic activity was assessed using alkali pretreated sugar cane bagasse as substrate. Whereas the free enzymatic preparation released xylose as the main product, the immobilized xylanase produced xylooligosaccharides, thus showing that the immobilization procedure modified the potential application of the extracellular xylanase from Conhella sp. AR92. Copyright © 2018 Elsevier B.V. All rights reserved.

Contribution of ethanol-tolerant xylanase G2 from Aspergillus oryzae on Japanese sake brewing.

PubMed

Sato, Yuichiro; Fukuda, Hisashi; Zhou, Yan; Mikami, Shigeaki

2010-12-01

We purified three xylanase isozymes (XynF1, XynF3 and XynG2) from a solid-state Aspergillus oryzae RIB128 culture using chromatography. The results of our sake-brewing experiment, in which we used exogenously supplemented enzymes, revealed that only XynG2 improved the alcohol yield and the material utilization. The alcohol yield of the XynG2 batch displayed an increase of 4.4% in comparison to the control, and the amount of sake cake decreased by 4.6%. The contribution of XynG2 was further confirmed through our brewing experiment in which we used the yeast heterogeneously expressing fungal xylanase isozymes. Interestingly XynG1, an enzyme with a XynG2-like sequence that is more vulnerable to ethanol, did not improve the sake-mash fermentation. The stability of XynG2 in ethanol was prominent, and it retained most of its original activity after we exposed it to 80% ethanol for 30min, whereas the stability of the other isozymes in ethanol, including XynG1, was much lower (20-25% ethanol). We concluded, therefore, that the improvement of material utilization achieved with XynG2 is primarily attributable to its characteristically high stability in ethanol, thereby, effectively degrading rice endosperm cell walls under high-alcohol conditions such as a sake-mash environment. Copyright © 2010 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

An isolated Amycolatopsis sp. GDS for cellulase and xylanase production using agricultural waste biomass.

PubMed

Kshirsagar, S D; Saratale, G D; Saratale, R G; Govindwar, S P; Oh, M K

2016-01-01

The aim of this study was to evaluate an isolate of Amycolatopsis sp. GDS for cellulase and xylanase production, their characterization, and its application to the preparation of biomass feedstock for ethanol production. A novel potent cellulolytic bacterial strain was isolated and identified as Amycolatopsis sp. GDS. The strain secreted high levels of cellulase and xylanase in the presence of agricultural waste biomass. The enzymes were thermostable and active up to 70°C. Interestingly, the enzymes were expressed well at higher NaCl (up to 2·5 mol l(-1) ) and ionic liquid (10%) concentrations, so that they could be used during the pretreatment of biomass. Enzyme stability in the presence of organic solvents, surfactants and oxidizing agents was also noted. Crude enzymes from Amycolatopsis sp. GDS resulted in comparable saccharification (60%) of wheat straw to commercial enzymes (64%). The cellulolytic enzymes from Amycolatopsis sp. GDS were stable, expressed well under conditions with various chemicals, and yielded significant amounts of hydrolysates from the biomass. The high bioethanol production using yeast co-cultures with enzymatic hydrolysates highlights the significance of selecting the strain and substrate for biofuel production. This study demonstrates the importance of the isolate Amycolatopsis sp. GDS that secretes high levels of cellulase and hemicellulase by utilizing agricultural waste biomass and its application in the preparation of biomass feedstock and sequential ethanol fermentation. © 2015 The Society for Applied Microbiology.

C-Terminal carbohydrate-binding module 9_2 fused to the N-terminus of GH11 xylanase from Aspergillus niger.

PubMed

Xu, Wenxuan; Liu, Yajuan; Ye, Yanxin; Liu, Meng; Han, Laichuang; Song, Andong; Liu, Liangwei

2016-10-01

The 9_2 carbohydrate-binding module (C2) locates natively at the C-terminus of the GH10 thermophilic xylanase from Thermotoga marimita. When fused to the C-terminus, C2 improved thermostability of a GH11 xylanase (Xyn) from Aspergillus niger. However, a question is whether the C-terminal C2 would have a thermostabilizing effect when fused to the N-terminus of a catalytic module. A chimeric enzyme, C2-Xyn, was created by step-extension PCR, cloned in pET21a(+), and expressed in E. coli BL21(DE3). The C2-Xyn exhibited a 2 °C higher optimal temperature, a 2.8-fold longer thermostability, and a 4.5-fold higher catalytic efficiency on beechwood xylan than the Xyn. The C2-Xyn exhibited a similar affinity for binding to beechwood xylan and a higher affinity for oat-spelt xylan than Xyn. C2 is a thermostabilizing carbohydrate-binding module and provides a model of fusion at an enzymatic terminus inconsistent with the modular natural terminal location.

An improved TCF sequence for biobleaching kenaf pulp: influence of the hexenuronic acid content and the use of xylanase.

PubMed

Andreu, Glòria; Vidal, Teresa

2014-01-01

Enzymatic delignification with laccase from Trametes villosa used in combination with chemical mediators (acetosyringone, acetovanillone and 1-hydroxybenzotriazole) to improve the totally chlorine-free (TCF) bleaching of kenaf pulp was studied. The best final pulp properties were obtained by using an LHBTQPo sequence developed by incorporating a laccase-mediator stage into an industrial bleaching sequence involving chelation and peroxide stages. The new sequence resulted in increased kenaf pulp delignification (90.4%) and brightness (77.2%ISO) relative to a conventional TCF chemical sequence (74.5% delignification and 74.5% brightness). Also, the sequence provided bleached kenaf fibers with high cellulose content (pulp viscosity of 890 g·mL(-1) vs 660 g·mL(-1)). Scanning electron micrographs revealed that xylanase altered fiber surfaces and facilitated reagent access as a result. However, the LHBTX (xylanase) stage removed 21% of hexenuronic acids in kenaf pulp. These recalcitrant compounds spent additional bleaching reagents and affected pulp properties after peroxide stage. Copyright © 2013 Elsevier Ltd. All rights reserved.

Solid-state fermentation of oil palm frond petiole for lignin peroxidase and xylanase-rich cocktail production.

PubMed

Mohamad Ikubar, Mohamed Roslan; Abdul Manan, Musaalbakri; Md Salleh, Madihah; Yahya, Adibah

2018-05-01

In current practice, oil palm frond leaflets and stems are re-used for soil nutrient recycling, while the petioles are typically burned. Frond petioles have high commercialization value, attributed to high lignocellulose fiber content and abundant of juice containing free reducing sugars. Pressed petiole fiber is the subject of interest in this study for the production of lignocellulolytic enzyme. The initial characterization showed the combination of 0.125 mm frond particle size and 60% moisture content provided a surface area of 42.3 m 2 /g, porosity of 12.8%, and density of 1.2 g/cm 3 , which facilitated fungal solid-state fermentation. Among the several species of Aspergillus and Trichoderma tested, Aspergillus awamori MMS4 yielded the highest xylanase (109 IU/g) and cellulase (12 IU/g), while Trichoderma virens UKM1 yielded the highest lignin peroxidase (222 IU/g). Crude enzyme cocktail also contained various sugar residues, mainly glucose and xylose (0.1-0.4 g/L), from the hydrolysis of cellulose and hemicellulose. FT-IR analysis of the fermented petioles observed reduction in cellulose crystallinity ( I 900/1098 ), cellulose-lignin ( I 900/1511 ), and lignin-hemicellulose ( I 1511/1738 ) linkages. The study demonstrated successful bioconversion of chemically untreated frond petioles into lignin peroxidase and xylanase-rich enzyme cocktail under SSF condition.

The effect of reduced calorie diets, with and without fat, and the use of xylanase on performance characteristics of broilers between 0 and 42 days.

PubMed

O'Neill, H V Masey; Mathis, G; Lumpkins, B S; Bedford, M R

2012-06-01

When decreasing the energy value of broiler diets, nonstarch polysaccharide degrading enzymes, such as xylanase, are often used. In doing so, they are assigned an energy value and considered to contribute energy to the diet. The aim of this study was to determine the effect of decreasing energy in a broiler diet by 100 kCal/kg on performance and whether the expected drop in performance could be recovered with the use of xylanase. Two formulations were used to provide decreased energy diets, both with and without supplementary fat. Six hundred 1-d-old male Cobb broilers were placed in a 2 × 3 full factorial design in 5 randomized complete blocks. The treatments were enzyme dose at 0 or 16,000 U/kg, and the 3 different diets [positive control (PC), negative control 1, without fat (NC1), and negative control 2 with fat (NC2)]. At no point were there any interactions between diet type and enzyme inclusion; where there was an effect of xylanase, it was consistent regardless of the diet type. There was a significant effect of diet type on feed intake between d 0 to 35; NC1 and NC2 had significantly increased feed intake compared with the PC (P = 0.006). The feed conversion ratio was significantly increased in birds fed the negative control diets during 0 to 35 d and 0 to 42 d (P = 0.003 and P = 0.002, respectively). However, feed conversion ratio was significantly improved by the addition of enzyme during periods 0 to 35 d and 0 to 42 d. There were no significant main effects between d 0 and 21 (all responses P > 0.1). Liveability was not affected by any of the treatments (P > 0.1). Decreasing energy in broiler diets results in worsened performance. The use of xylanase may improve feed conversion ratio. The use of some fat may help, so the whole diet composition should be considered in conjunction with enzyme dose to achieve the best advantage.

Preparation, Purification, and Secondary Structure Determination of Bacillus Circulans Xylanase. A Molecular Laboratory Incorporating Aspects of Molecular Biology, Biochemistry, and Biophysical Chemistry

ERIC Educational Resources Information Center

Russo, Sal; Gentile, Lisa

2006-01-01

A project module designed for biochemistry or cellular and molecular biology student which involves determining the secondary structure of Bacillus circulans xylanase (BCX) by circular dichroism (CD) spectroscopy under conditions that compromise its stabilizing intramolecular forces is described. The lab model enhanced students knowledge of the…

Antioxidant activity of xylooligosaccharides produced from glucuronoxylan by Xyn10A and Xyn30D xylanases and eucalyptus autohydrolysates.

PubMed

Valls, Cristina; Pastor, F I Javier; Vidal, Teresa; Roncero, M Blanca; Díaz, Pilar; Martínez, Josefina; Valenzuela, Susana V

2018-08-15

Antioxidant activity of xylooligosaccharides (XOS) released from beechwood and birchwood glucuronoxylans by two different xylanases, one from family GH10 (Xyn10A) and another from family GH30 (Xyn30D) was examined. The ABTS (2, 2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) method was used, since it resulted more accurate for the antioxidant activity determination of XOS. Thin layer chromatography and MALDI-TOF MS analysis showed that Xyn10A produced a mixture of neutral and acidic XOS whereas the XOS produced by Xyn30D were all acidic, containing a methylglucuronic acid (MeGlcA) ramification. These acidic XOS, MeGlcA substituted, showed a strongly higher antioxidant activity than the XOS produced by Xyn10A (80% vs. 10% respectively, at 200 μg mL -1 ). Moreover, the antioxidant activity increased with the degree of polymerization of XOS, and depended on the xylan substrate used. The antioxidant capacity of eucalyptus autohydrolysates after xylanase treatment was also analysed, showing a decrease of their antioxidant activity simultaneous with the decrease in XOS length. Copyright © 2018 Elsevier Ltd. All rights reserved.

Effect of cellulase, xylanase and α-amylase combinations on the rheological properties of Chinese steamed bread dough enriched in wheat bran.

PubMed

Liu, Wenjun; Brennan, Margaret Anne; Serventi, Luca; Brennan, Charles Stephen

2017-11-01

The present study investigates the effects of α-amylase (6 and 10ppm), xylanase (70 and 120ppm) and cellulase (35 and 60ppm) on the rheological properties of bread dough. The mixing property of dough was measured by using a DoughLAB. The extension and stickiness of dough were analysed using the Texture Analyzer. The results illustrate that the addition of single enzyme and enzyme combinations can increase the extensibility, softening, mixing tolerance index (MTI) and stickiness, whereas decrease the resistance to extension. For water absorption, the addition of single enzyme had no significant effect, while the combination enzyme significantly (p<0.05) decreased the values from 63.9 to 59.6% (wheat flour dough) and 71.4-67.1% (dough incorporated with 15% wheat bran). Compared to the single enzyme with the value of 34.1mm, enzyme combination (6, 120 and 60ppm) increased the extensibility of wheat flour dough by up to 42%. Additionally, combination of α-amylase, xylanase and cellulase had a synergetic effect on the dough rheology. Copyright © 2017 Elsevier Ltd. All rights reserved.

Enzymatic hydrolysis of pretreated Alfa fibers (Stipa tenacissima) using β-d-glucosidase and xylanase of Talaromyces thermophilus from solid-state fermentation.

PubMed

Mallek-Fakhfakh, Hanen; Fakhfakh, Jawhar; Walha, Kamel; Hassairi, Hajer; Gargouri, Ali; Belghith, Hafedh

2017-10-01

This work aims at realizing an optimal hydrolysis of pretreated Alfa fibers (Stipa tenacissima) through the use of enzymes produced from Talaromyces thermophilus AX4, namely β-d-glucosidase and xylanase, by a solid state fermentation process of an agro-industrial waste (wheat bran supplemented with lactose). The carbon source was firstly selected and the optimal values of three other parameters were determined: substrate loading (10g), moisture content (85%) and production time (10days); which led to an optimized enzymatic juice. The outcome was then supplemented with cellulases of T. reesei and used to optimize the enzymatic saccharification of alkali-pretreated Alfa fibers (PAF). The maximum saccharification yield of 83.23% was achieved under optimized conditions (substrate concentration 3.7% (w/v), time 144h and enzyme loading of 0.8 FPU, 15U CMCase, 60U β-d-glucosidase and 125U xylanase).The structural modification of PAF due to enzymatic saccharification was supported by the changes of morphologic and chemical composition observed through macroscopic representation, FTIR and X-Ray analysis. Copyright © 2017 Elsevier B.V. All rights reserved.

Enhancing cellulase production by overexpression of xylanase regulator protein gene, xlnR, in Talaromyces cellulolyticus cellulase hyperproducing mutant strain.

PubMed

Okuda, Naoyuki; Fujii, Tatsuya; Inoue, Hiroyuki; Ishikawa, Kazuhiko; Hoshino, Tamotsu

2016-10-01

We obtained strains with the xylanase regulator gene, xlnR, overexpressed (HXlnR) and disrupted (DXlnR) derived from Talaromyces cellulolyticus strain C-1, which is a cellulase hyperproducing mutant. Filter paper degrading enzyme activity and cellobiohydrolase I gene expression was the highest in HXlnR, followed by C-1 and DXlnR. These results indicate that the enhancement of cellulase productivity was succeeded by xlnR overexpression.

Prediction of ingredient quality and the effect of a combination of xylanase, amylase, protease and phytase in the diets of broiler chicks. 2. Energy and nutrient utilisation.

PubMed

Cowieson, A J; Singh, D N; Adeola, O

2006-08-01

1. In order to investigate the effects of xylanase, amylase, protease and phytase in the diets of broiler chickens containing graded concentrations of metabolisable energy (ME), two 42-d experiments were conducted using a total of 2208 broiler chicks (8 treatments with 12 replicate pens in each experiment). 2. Four diets including one positive and three negative control diets were used. Three maize/soybean meal-based negative control (NC) diets were formulated to be identical in available phosphorus (P), calcium (Ca) and amino acids but NC1 contained approximately 0.17 MJ/kg less ME than NC2 and approximately 0.34 MJ/kg less ME than NC3. A positive control (PC) was fed for comparison and was formulated to be adequate in all nutrients, providing approximately 0.63 MJ/kg ME, 0.13% available P, 0.12% Ca and 1 to 2% amino acids more than NC1. 3. The reduction in nutrient density between NC1 and PC was determined using ingredient quality models Avichecktrade mark Corn and Phychecktrade mark that can predict the response to exogenous enzymes in maize/soybean meal-based broiler diets. Supplementation of each diet with or without a cocktail of xylanase, amylase, protease and phytase gave a total of 8 dietary treatments in a 4 x 2 factorial arrangement. The same treatments and diet designs were used in both experiments but conducted in different locations using different batches of maize, soybean meal and minor ingredients. 4. In both experiments, digestibility was improved by the addition of exogenous enzymes, particularly those for P, Ca and certain amino acids. In addition, the supplementation of the PC with enzymes elicited a positive response indicating that over-the-top addition of xylanase, amylase, protease and phytase may offer a nutritionally and economically viable alternative to feed cost reduction. 5. It can be concluded that the digestibility of nutrients by broilers fed on maize/soybean meal-based diets can be improved by the use of a combination of xylanase

Heterologous expression of family 10 xylanases from Acidothermus cellulolyticus enhances the exoproteome of Caldicellulosiruptor bescii and growth on xylan substrates

DOE PAGES

Kim, Sun -Ki; Chung, Daehwan; Himmel, Michael E.; ...

2016-08-22

The ability to deconstruct plant biomass without conventional pretreatment has made members of the genus Caldicellulosiruptor the target of investigation for the consolidated processing of lignocellulosic biomass to biofuels and bioproducts. These Gram-positive bacteria are hyperthermophilic anaerobes and the most thermophilic cellulolytic organisms so far described. They use both C5 and C6 sugars simultaneously and have the ability to grow well on xylan, a major component of plant cell walls. This is an important advantage for their use to efficiently convert biomass at yields sufficient for an industrial process. For commodity chemicals, yield from substrate is perhaps the most importantmore » economic factor. In an attempt to improve even further the ability of C. bescii to use xylan, we introduced two xylanases from Acidothermus cellulolyticus. Acel_0180 includes tandem carbohydrate-binding modules (CBM2 and CBM3) located at the C-terminus, one of which, CBM2, is not present in C. bescii. Also, the sequences of Xyn10A and Acel_0180 have very little homology with the GH10 domains present in C. bescii. For these reasons, we selected these xylanases as potential candidates for synergistic interaction with those in the C. bescii exoproteome. As a result, heterologous expression of two xylanases from Acidothermus cellulolyticus in Caldicellulosiruptor bescii resulted in a modest, but significant increase in the activity of the exoproteome of C. bescii on xylan substrates. Even though the increase in extracellular activity was modest, the ability of C. bescii to grow on these substrates was dramatically improved suggesting that the xylan substrate/microbe interaction substantially increased deconstruction over the secreted free enzymes alone. In conclusion, we anticipate that the ability to efficiently use xylan, a major component of plant cell walls for conversion of plant biomass to products of interest, will allow the conversion of renewable, sustainable, and

GH10 XynA is the main xylanase identified in the crude enzymatic extract of Paenibacillus sp. A59 when grown on xylan or lignocellulosic biomass.

PubMed

Ghio, Silvina; Insani, Ester M; Piccinni, Florencia E; Talia, Paola M; Grasso, Daniel H; Campos, Eleonora

2016-01-01

A novel bacterial isolate with polysaccharides degrading activity was identified as Paenibacillus sp., and named Paenibacillus sp. A59. Even though it is a strict mesophile, optimal xylanase activity of the crude enzymatic extract was achieved between 50°C and 70°C and more than 60% of the activity was retained after incubation for 48h at 50°C, indicating thermotolerance of the enzymes involved. The extract was also active on pre-treated sugarcane residue (SCR) and wheat straw, releasing xylobiose and xylose as the main products, therefore confirming its predominantly xylanolytic activity. By zymograms and mass spectrometry of crude enzymatic extracts of xylan or SCR cultures, a 32kDa GH10 beta- 1,4- endoxylanase with xylanase and no CMCase activity was identified. We named this enzyme XynA and it was the only xylanase identified under both conditions assayed, suggesting that it is a good candidate for recombinant expression and evaluation in hemicelluloses deconstruction applications. Also, a protein with two S-layer homology domains (SLH) and a large uncharacterized C-terminal domain as well as an ABC substrate binding protein were identified in crude extracts of SCR cultures. We propose that Paenibacillus sp. A59 uses a system similar to anaerobic and other Gram positive bacteria, with SLH-domain proteins anchoring polysaccharide-degrading enzymes close to the membrane and the substrate binding protein assisting translocation of simple sugars to the cell interior. Copyright © 2016 Elsevier GmbH. All rights reserved.

Site-directed mutagenesis of GH10 xylanase A from Penicillium canescens for determining factors affecting the enzyme thermostability.

PubMed

Denisenko, Yury A; Gusakov, Alexander V; Rozhkova, Aleksandra M; Osipov, Dmitry O; Zorov, Ivan N; Matys, Veronika Yu; Uporov, Igor V; Sinitsyn, Arkady P

2017-11-01

In order to investigate factors affecting the thermostability of GH10 xylanase A from Penicillium canescens (PcXylA) and to obtain its more stable variant, the wild-type (wt) enzyme and its mutant forms, carrying single amino acid substitutions, were cloned and expressed in Penicillium verruculosum B1-537 (niaD-) auxotrophic strain under the control of the cbh1 gene promoter. The recombinant PcXylA-wt and I6V, I6L, L18F, N77D, Y125R, H191R, S246P, A293P mutants were successfully expressed and purified for characterization. The mutations did not affect the enzyme specific activity against xylan from wheat as well as its pH-optimum of activity. One mutant (L18F) displayed a higher thermostability relative to the wild-type enzyme; its half-life time at 50-60°C was 2-2.5-fold longer than that for the PcXylA-wt, and the melting temperature was 60.0 and 56.1°C, respectively. Most of other mutations led to decrease in the enzyme thermostability. This study, together with data of other researchers, suggests that multiple mutations should be introduced into GH10 xylanases in order to dramatically improve their stability. Copyright © 2017 Elsevier B.V. All rights reserved.

Concomitant production of xylanases and cellulases from Trichoderma longibrachiatum MDU-6 selected for the deinking of paper waste.

PubMed

Chutani, Preeti; Sharma, Krishna Kant

2016-05-01

Sixty fungal cultures were isolated from agricultural soil, industrial soil, forest canopy soil having decomposed leaf litter and compost samples collected from different regions of India. Fifteen fungal cultures were selected qualitatively for the production of xylanase and cellulases and were identified employing ITS, NS and MNS primers. The enzyme cocktail consisting of 3811 IU g(-1) of xylanase and 9.9 IU g(-1) of cellulase from Trichoderma longibrachiatum MDU-6 was selected quantitatively for the deinking of diverse paper wastes. The enzyme production increased two fold when produced at tray level in comparison with flasks. The enzyme cocktail was effective in the deinking of old newspaper samples with significant removal of chromophores, phenolics and hydrophobic compounds and less sugar loss. While in case of examination papers and laser printed papers, ink removal was not very significant. Moreover, the sugar loss was significantly high in case of examination papers. The deinking results were further confirmed with FTIR analysis. Deinked newspaper pulp sample shows brightness of 52%, which was 9.6% high than its control sample. The ERIC value for deinked newspaper pulp was found to be 655.9 ppm. Thereafter, the deinked newspaper pulp was examined under light microscope after differential staining with safranin and malachite green and also examined under scanning and transmission electron microscope, which revealed fibrillation and perforation.

The effects of xylanase supplementation on growth, digestion, circulating hormone and metabolite levels, immunity and gut microflora in cockerels fed on wheat-based diets.

PubMed

Gao, F; Jiang, Y; Zhou, G H; Han, Z K

2007-08-01

1. The xylanase product used in this study was derived from a genetically modified isolate of Aspergillus niger. Two trials were conducted to investigate the effects of xylanase supplementation on growth, digestion, circulating hormone and metabolite levels, immune parameters and composition of the gut microflora in cockerels fed on wheat-based diets. 2. The experimental diets consisted of a wheat-based control diet supplemented with 0 or 0.1% enzyme preparation. The diets were fed between 7 and 21 d of age. 3. Enzyme supplementation improved growth and feed conversion efficiency. The addition of enzyme to wheat-based diet increased the apparent total digestibility of dry matter (DM), crude protein and fat. 4. Enzyme supplementation reduced the relative weight of digestive organs to a certain extent, but there was no significant difference. Enzyme supplementation reduced digesta viscosity in the jejunum. There was no significant difference between the two experimental groups in counts of lactobacillus and coliform bacteria in the caeca. 5. Enzyme supplementation increased the concentration of blood thyroxine (T(4)), insulin-like growth factor I (IGF-I) and insulin, reduced the concentrations of blood uric acid, but had no significant effect on the concentrations of blood glucose and triiodothyronine (T(3)). 6. Enzyme supplementation increased the relative weight of spleen of cockerels, serum antibody titres to Newcastle disease virus (NDV), lymphocyte proliferation in response to phytohaemagglutinin (PHA) and the natural killer (NK) cell activity. 7. It is concluded that supplementation with an enzyme preparation (xylanase), which hydrolyses non-starch polysaccharides can improve growth in cockerels fed on wheat-based diets. This improvement is achieved through enzyme effects on digestion, absorption, metabolism and immunity of cockerels.

Production of spent mushroom substrate hydrolysates useful for cultivation of Lactococcus lactis by dilute sulfuric acid, cellulase and xylanase treatment.

PubMed

Qiao, Jian-Jun; Zhang, Yan-Fei; Sun, Li-Fan; Liu, Wei-Wei; Zhu, Hong-Ji; Zhang, Zhijun

2011-09-01

Spent mushroom substrate (SMS) was treated with dilute sulfuric acid followed by cellulase and xylanase treatment to produce hydrolysates that could be used as the basis for media for the production of value added products. A L9 (3(4)) orthogonal experiment was performed to optimize the acid treatment process. Pretreatment with 6% (w/w) dilute sulfuric acid at 120°C for 120 min provided the highest reducing sugar yield of 267.57 g/kg SMS. No furfural was detected in the hydrolysates. Exposure to 20PFU of cellulase and 200 XU of xylanase per gram of pretreated SMS at 40°C resulted in the release of 79.85 g/kg or reducing sugars per kg acid pretreated SMS. The dilute sulfuric acid could be recycled to process fresh SMS four times. SMS hydrolysates neutralized with ammonium hydroxide, sodium hydroxide, or calcium hydroxide could be used as the carbon source for cultivation of Lactococcus lactis subsp. lactis W28 and a cell density of 2.9×10(11)CFU/mL could be obtained. The results provide a foundation for the development of value-added products based on SMS. Copyright © 2011 Elsevier Ltd. All rights reserved.

Prediction of ingredient quality and the effect of a combination of xylanase, amylase, protease and phytase in the diets of broiler chicks. 1. Growth performance and digestible nutrient intake.

PubMed

Cowieson, A J; Singh, D N; Adeola, O

2006-08-01

1. A total of 2208 broiler chicks were used in two growth experiments (8 treatments and 12 replicate pens in each experiment) to assess the effects of xylanase, amylase, protease and phytase in maize-based diets. 2. A positive control diet was formulated containing adequate nutrient concentrations. A negative control diet was formulated to contain approximately 628 kJ/kg, 0.13%, 0.12% and 1 to 2% less metabolisable energy (ME), phosphorus (P), calcium (Ca) and amino acids, respectively, than the positive control. In addition, two further negative control diets that contained 167 or 334 kJ/kg more ME, respectively, than negative control 1 were formulated. 3. A further 4 dietary treatments were made by supplementing each of the 4 negative control diets with a combination of xylanase, amylase, protease and phytase, resulting in 8 dietary treatments in a 4 by 2 factorial arrangement. 4. The scale of the removal of energy, P, Ca and amino acids from the positive control diet was determined using least square models based on in vivo data for both the xylanase/amylase/protease cocktail and for phytase and it was predicted that performance of birds fed on negative control 1 would be returned by supplemental enzymes to that of those fed on the positive control. 5. In both experiments there was a significantly poorer performance in birds fed on the negative control 1 than in those fed on the positive control. The poorer weight gain and feed conversion ratio could be attributed in part to a reduced intake of digestible energy, P, nitrogen (N) and amino acids associated with birds fed on the negative control diet. 6. Supplementation of the negative control diets with the enzyme combination returned performance to that of the positive control in both experiments. 7. These data indicate that exogenous xylanase, amylase, protease and phytase can be used successfully in a strategically formulated low nutrient density diet to maintain performance to that of birds fed on a

Generation of transgenic wheat (Triticum aestivum L.) accumulating heterologous endo-xylanase or ferulic acid esterase in the endosperm.

PubMed

Harholt, Jesper; Bach, Inga C; Lind-Bouquin, Solveig; Nunan, Kylie J; Madrid, Susan M; Brinch-Pedersen, Henrik; Holm, Preben B; Scheller, Henrik V

2010-04-01

Endo-xylanase (from Bacillus subtilis) or ferulic acid esterase (from Aspergillus niger) were expressed in wheat under the control of the endosperm-specific 1DX5 glutenin promoter. Constructs both with and without the endoplasmic reticulum retention signal (Lys-Asp-Glu-Leu) KDEL were used. Transgenic plants were recovered in all four cases but no qualitative differences could be observed whether KDEL was added or not. Endo-xylanase activity in transgenic grains was increased between two and threefold relative to wild type. The grains were shrivelled and had a 25%-33% decrease in mass. Extensive analysis of the cell walls showed a 10%-15% increase in arabinose to xylose ratio, a 50% increase in the proportion of water-extractable arabinoxylan, and a shift in the MW of the water-extractable arabinoxylan from being mainly larger than 85 kD to being between 2 and 85 kD. Ferulic acid esterase-expressing grains were also shrivelled, and the seed weight was decreased by 20%-50%. No ferulic acid esterase activity could be detected in wild-type grains whereas ferulic acid esterase activity was detected in transgenic lines. The grain cell walls had 15%-40% increase in water-unextractable arabinoxylan and a decrease in monomeric ferulic acid between 13% and 34%. In all the plants, the observed changes are consistent with a plant response that serves to minimize the effect of the heterologously expressed enzymes by increasing arabinoxylan biosynthesis and cross-linking.

Generation of transgenic wheat (Triticum aestivum L.) accumulating heterologous endo-xylanase or ferulic acid esterase in the endosperm

DOE Office of Scientific and Technical Information (OSTI.GOV)

Harholt, Jesper; Bach, Inga C; Lind-Bouquin, Solveig

2009-12-08

Endo-xylanase (from Bacillus subtilis) or ferulic acid esterase (from Aspergillus niger) were expressed in wheat under the control of the endosperm specific 1DX5 glutenin promoter. Constructs both with and without the endoplasmic reticulum retention signal KDEL were used. Transgenic plants were recovered in all four cases but no qualitative differences could be observed whether KDEL was added or not. Endo-xylanase activity in transgenic grains was increased between two and three fold relative to wild type. The grains were shriveled and had a 25-33% decrease in mass. Extensive analysis of the cell walls showed a 10-15% increase in arabinose to xylosemore » ratio, a 50% increase in the proportion of water extractable arabinoxylan, and a shift in the MW of the water extractable arabinoxylan from being mainly larger than 85 kD to being between 2 kD and 85 kD. Ferulic acid esterase expressing grains were also shriveled and the seed weight was decreased by 20-50%. No ferulic acid esterase activity could be detected in wild type grains whereas ferulic acid esterase activity was detected in transgenic lines. The grain cell walls had 15-40% increase in water unextractable arabinoxylan and a decrease in monomeric ferulic acid between 13 and 34%. In all the plants the observed changes are consistent with a plant response that serves to minimize the effect of the heterologously expressed enzymes by increasing arabinoxylan biosynthesis and cross-linking.« less

Thermophilic fungi as new sources for production of cellulases and xylanases with potential use in sugarcane bagasse saccharification.

PubMed

de Cassia Pereira, J; Paganini Marques, N; Rodrigues, A; Brito de Oliveira, T; Boscolo, M; da Silva, R; Gomes, E; Bocchini Martins, D A

2015-04-01

To obtain new cellulases and xylanases from thermophilic fungi; evaluate their potential for sugarcane bagasse saccharification. Thirty-two heat-tolerant fungi were isolated from the environment, identified (morphological/molecular tools) and the production of the enzymes was evaluated by solid state fermentation using lignocellulosic materials as substrates. Myceliophthora thermophila JCP 1-4 was the best producer of endoglucanase (357·51 U g(-1) ), β-glucosidase (45·42 U g(-1) ), xylanase (931·11 U g(-1) ) and avicelase (3·58 U g(-1) ). These enzymes were most active at 55-70°C and stable at 30-60°C. Using crude enzymatic extract from M. thermophila JCP 1-4 to saccharify sugarcane bagasse pretreated with microwaves and glycerol, glucose and xylose yields obtained were 15·6 and 35·13% (2·2 and 1·95 g l(-1) ), respectively. All isolated fungi have potential to produce the enzymes; M. thermophila JCP 1-4 enzymatic extract have potential to be better explored in saccharification experiments. Pretreatment improved enzymatic saccharification, as sugar yields were much higher than those obtained from in natura bagasse. Myceliophthora thermophila JCP 1-4 produces avicelase (not commonly found among fungi; important to hydrolyse crystalline cellulose) and a β-glucosidase resistant to glucose inhibition, interesting characteristics for saccharification experiments. © 2015 The Society for Applied Microbiology.

Energy utilization and growth performance of chickens fed novel wheat inbred lines selected for different pentosan levels with and without xylanase supplementation

PubMed Central

Pirgozliev, V.; Rose, S. P.; Pellny, T.; Amerah, A. M.; Wickramasinghe, M.; Ulker, M.; Rakszegi, M.; Bedo, Z.; Shewry, P. R.; Lovegrove, A.

2015-01-01

Different F5 recombinant inbred lines from the cross Yumai 34 × Ukrainka were grown in replicated trials on a single site in one harvest year at Rothamsted Research. A total of 10 samples from those lines were harvested and used in a broiler experiment. Twenty nutritionally complete meal-form diets that had 630 g/kg of wheat with different amounts of pentosan, with and without exogenous xylanase supplementation, were used to compare broiler growth performance and determine apparent metabolizable energy corrected for N retention (AMEn). We examined the relationship between the nutritive value of the wheat samples and their chemical compositions and results of quality tests. The amounts of total and water soluble pentosans in wheat samples ranged from 36.7 to 48.0 g/kg DM, and 6.7 to 11.6 g/kg DM, respectively. The mean crude oil and protein contents of the wheat samples were 10.5 and 143.9 g/kg DM, respectively. The average determined value for the kinematic viscosity was 0.0018 mPa.s, and 2.1 mPa.s for the dynamic viscosity. The AMEn of the wheat-based diets had a maximum range of 0.47 MJ/kg DM within the ten wheat samples that were tested. Xylanase supplementation improved (P < 0.05) dietary AMEn, dry matter, and fat digestibility coefficients. There was a positive (P < 0.05) relationship between in vitro kinematic viscosity of the wheat samples and the total pentosan content. There was a negative relationship between the total pentosan content in the wheat and broiler growth performance. An increase by 10 g of pentosan per kg of wheat reduced (P < 0.001) daily feed intake and weight gain by 2.9 g and 3.5 g, respectively. The study shows that the feeding quality of wheat samples can be predicted by their total pentosan content. Supplementary xylanase improved energy and nutrient availability of all wheat samples that was independent of differences in pentosan content. PMID:25595480

Concomitant production of cellulase and xylanase by thermophilic mould Sporotrichum thermophile in solid state fermentation and their applicability in bread making.

PubMed

Bala, Anju; Singh, Bijender

2017-06-01

Sporotrichum thermophile BJAMDU5 secreted high titres of xylanolytic and cellulolytic enzymes in solid state fermentation using mixture of wheat straw and cotton oil cake (ratio 1:1) at 45 °C, pH 5.0 after 72 h inoculated with 2.9 × 10 7  CFU/mL conidiospores. Supplementation of solid medium with lactose and ammonium sulphate further enhanced the production of hydrolytic enzymes. Among different surfactants studied, Tween 80 enhanced the production of all enzymes [3455 U/g DMR (dry mouldy residue), 879.26 U/g DMR, 976.28 U/g DMR and 35.10 U/g DMR for xylanase, CMCase (Carboxymethylcellulase), FPase (Filter paper activity) and β-glucosidase, respectively] as compared to other surfactants. Recycling of solid substrate reduced the production of all these enzymes after second cycle. End products analysis by TLC showed the ability of hydrolytic enzymes of S. thermophile to liberate monomeric (xylose and glucose) as well as oligomeric (xylobiose, cellobiose and higher ones) sugars. Supplementation of enzyme resulted in improved nutritional properties of the bread. Formation of oligomeric sugars by xylanase enzyme of S. thermophile BJAMDU5 make it a good candidate in food industry.

Biochemical and biophysical characterization of novel GH10 xylanase prospected from a sugar cane bagasse compost-derived microbial consortia.

PubMed

Evangelista, Danilo Elton; Kadowaki, Marco Antonio Seiki; Mello, Bruno Luan; Polikarpov, Igor

2018-04-01

Environmental issues are promoting the development of innovative technologies for the production of renewable energy and "green products" from plant biomass residues. These technologies rely on the conversion of the plant cell wall (PCW) polysaccharides into simple sugars, which involve synergistic activities of different PCW degrading enzymes, including xylanases; these are widely applied in food and feed sectors, paper and textile industries, among others. We cloned, expressed and biochemically characterized a novel xylanase (Xyn10) from the GH10 identified in a metatranscriptome of compost-derived microbial consortia and determined its low-resolution SAXS molecular envelope in solution. Our results reveal that Xyn10 is a monomeric flexible globular enzyme, with high stability with a broad pH range from 4 to 10 and optimal activity conditions at pH 7 and 40 °C. Only 10% of activity loss was observed after the enzyme was incubated for 30 h at 40 °C with a pH ranging from 5 to 10. Moreover, Xyn10 maintained 100% of its initial activity after incubation for 120 h at 40 °C and 51% after incubation for 24 h at 50 °C (pH = 7.0). Xyn10 shows endocatalytic activity towards xylan and arabinoxylan, liberating xylose, xylobiose, 1,2-α-d-methylglucuronic acid decorated xylotriose, and 1,3-α-l-arabinofuranose decorated xylobiose and xylotriose oligosaccharides. Copyright © 2017 Elsevier B.V. All rights reserved.

Production of xylanase and protease by Penicillium janthinellum CRC 87M-115 from different agricultural wastes.

PubMed

Oliveira, Luciana A; Porto, Ana L F; Tambourgi, Elias B

2006-04-01

Five agricultural wastes were evaluated in submerged fermentation for xylanolytic enzymes production by Penicillium janthinellum. The wastes were hydrolyzed in acid medium and the liquid fraction was used for cultivation. Corn cob (55.3 U/mL) and oat husk (54.8 U/mL) were the best inducers of xylanase. Sugar cane bagasse (23.0 U/mL) and corn husk (23.8 U/mL) were moderately good, while cassava peel was negligible. Protease production was very low in all agro-industrial residues. The maximum biomass yields were 1.30 and 1.17 g/L for cassava peel and corn husk after 180 h, respectively. Xylanolytic activity showed a cell growth associated profile.

Bioflocculants' production from a cellulase-free xylanase-producing Pseudomonas boreopolis G22 by degrading biomass and its application in cost-effective harvest of microalgae.

PubMed

Guo, Haipeng; Hong, Chuntao; Zhang, Cheng; Zheng, Bingsong; Jiang, Dean; Qin, Wensheng

2018-05-01

The major problem for industrial application of bioflocculants is its high production cost. Here, a novel bacterium Pseudomonas boreopolis G22, which can secret a cellulase-free xylanase and simultaneously produce bioflocculants (MBF-G22) through directly converting untreated biomass, was isolated. The bioflocculants' production of G22 was closely related to its xylanase activity, hydrolysis ability of biomass and the hemicellulose loss caused by G22. The optimal fermentation conditions with the highest bioflocculants' yield (3.75 mg g -1 dry biomass) were obtained at the fermentation time of 96 h, incubation temperature of 30 °C, inoculum concentration of 1.0% and biomass concentration of 1.0% in an initial pH value of 7.0. MBF-G22 mainly consisted of polysaccharides (63.3%) with a molecular weight of 3.982 × 10 6  Da and showed the highest flocculating efficiency of 97.1% at a dosage of 3.5 mg L -1 . In addition, MBF-G22 showed high flocculating efficiency of microalgae (95.7%) at a dosage of 80 mg L -1 . Copyright © 2018 Elsevier Ltd. All rights reserved.

Impact of a multicarbohydrase containing α-galactosidase and xylanase on ileal digestible energy, crude protein digestibility, and ileal amino acid digestibility in broiler chickens.

PubMed

Jasek, A; Latham, R E; Mañón, A; Llamas-Moya, S; Adhikari, R; Poureslami, R; Lee, J T

2018-06-08

Exogenous enzymatic supplementation of poultry feeds, including α-galactosidase and xylanase, has been shown to increase metabolically available energy, although little information has been published on the impact on amino acid digestibility. An experiment was conducted to investigate a multicarbohydrase containing α-galactosidase and xylanase on amino acid digestibility, ileal digestible energy (IDE), and CP in male broiler chicks. The experiment was a 2 × 2 (diet × enzyme) factorial arrangement with 15 replicates of 8 male broilers per replicate raised for 21 d in a battery setting. The 2 dietary treatments included a positive control (PC) and a negative control (NC) diet formulated to contain 2.5% less calculated AME and digestible amino acids. Each of these diets was fed with and without enzyme. Broilers were fed a starter diet from 0-14 d (crumble) and a grower from 14-21 d (pellet). Birds were sampled on day 21 to determine ileal amino acid digestibility, IDE, and CP digestibility. Titanium dioxide (TiO2) was used as an indigestible marker for the determination of digestibility coefficients. Total ileal amino acid digestibility was increased (P = 0.008) by 3.80% with the inclusion of enzyme. Methionine and lysine digestibility was improved (P < 0.05) with the inclusion of enzyme by 3.37% and 2.61%, respectively. Enzyme inclusion increased (P = 0.001) cysteine digestibility by 9.3%. Diet-influenced ileal amino acid digestibility with tryptophan, threonine, isoleucine, and valine digestibility being increased (P < 0.05) in the PC when compared to the NC. IDE was decreased (P = 0.037) in broilers fed the NC diet by 100 kcal/kg feed when compared to broilers fed the PC diet. Enzyme inclusion increased (P = 0.047) IDE value by 90 kcal/kg. Crude protein digestibility was not influenced by diet; however, similar improvements in CP digestibility with enzyme inclusion were observed as with energy. These data support the benefits of a multicarbohydrase containing �

Response surface methodology optimization of partitioning of xylanase form Aspergillus Niger by metal affinity polymer-salt aqueous two-phase systems.

PubMed

Fakhari, Mohamad Ali; Rahimpour, Farshad; Taran, Mojtaba

2017-09-15

Aqueous two phase affinity partitioning system using metal ligands was applied for partitioning and purification of xylanase produced by Aspergillus Niger. To minimization the number of experiments for the design parameters and develop predictive models for optimization of the purification process, response surface methodology (RSM) with a face-centered central composite design (CCF) has been used. Polyethylene glycol (PEG) 6000 was activated using epichlorohydrin, covalently linked to iminodiacetic acid (IDA), and the specific metal ligand Cu was attached to the polyethylene glycol-iminodiacetic acid (PEG-IDA). The influence of some experimental variables such as PEG (10-18%w/w), sodium sulfate (8-12%), PEG-IDA-Cu 2+ concentration (0-50% w/w of total PEG), pH of system (4-8) and crude enzyme loading (6-18%w/w) on xylanase and total protein partitioning coefficient, enzyme yield and enzyme specific activity were systematically evaluated. Two optimal point with high enzyme partitioning factor 10.97 and yield 79.95 (including 10% PEG, 12% Na 2 SO 4 , 50% ligand, pH 8 and 6% crude enzyme loading) and high specific activity in top phase 42.21 (including 14.73% PEG, 8.02% Na 2 SO 4 , 28.43% ligand, pH 7.7 and 6.08% crude enzyme loading) were attained. The adequacy of the RSM models was verified by a good agreement between experimental and predicted results. Copyright © 2017 Elsevier B.V. All rights reserved.

Crystal structures of native and xylosaccharide-bound alkali thermostable xylanase from an alkalophilic Bacillus sp. NG-27: Structural insights into alkalophilicity and implications for adaptation to polyextreme conditions

PubMed Central

Manikandan, Karuppasamy; Bhardwaj, Amit; Gupta, Naveen; Lokanath, Neratur K.; Ghosh, Amit; Reddy, Vanga Siva; Ramakumar, Suryanarayanarao

2006-01-01

Crystal structures are known for several glycosyl hydrolase family 10 (GH10) xylanases. However, none of them is from an alkalophilic organism that can grow in alkaline conditions. We have determined the crystal structures at 2.2 Å of a GH10 extracellular endoxylanase (BSX) from an alkalophilic Bacillus sp. NG-27, for the native and the complex enzyme with xylosaccharides. The industrially important enzyme is optimally active and stable at 343 K and at a pH of 8.4. Comparison of the structure of BSX with those of other thermostable GH10 xylanases optimally active at acidic or close to neutral pH showed that the solvent-exposed acidic amino acids, Asp and Glu, are markedly enhanced in BSX, while solvent-exposed Asn was noticeably depleted. The BSX crystal structure when compared with putative three-dimensional homology models of other extracellular alkalophilic GH10 xylanases from alkalophilic organisms suggests that a protein surface rich in acidic residues may be an important feature common to these alkali thermostable enzymes. A comparison of the surface features of BSX and of halophilic proteins allowed us to predict the activity of BSX at high salt concentrations, which we verified through experiments. This offered us important lessons in the polyextremophilicity of proteins, where understanding the structural features of a protein stable in one set of extreme conditions provided clues about the activity of the protein in other extreme conditions. The work brings to the fore the role of the nature and composition of solvent-exposed residues in the adaptation of enzymes to polyextreme conditions, as in BSX. PMID:16823036

Potential application of waste from castor bean (Ricinus communis L.) for production for xylanase of interest in the industry.

PubMed

Herculano, Polyanna Nunes; Moreira, Keila Aparecida; Bezerra, Raquel Pedrosa; Porto, Tatiana Souza; de Souza-Motta, Cristina Maria; Porto, Ana Lúcia Figueiredo

2016-12-01

Xylanases activity (XY) from Aspergillus japonicus URM5620 produced by Solid-State Fermentation (SSF) of castor press cake (Ricinus communis) on different conditions of production and extraction by PEG/citrate aqueous two-phase system (ATPS) were investigated. XY production was influenced by substrate amount (5-10 g), initial moisture (15-35 %), pH (4.0-6.0) and temperature (25-35 °C), obtaining the maximum activity of 29,085 ± 1808 U g ds -1 using 5.0 g of substrate with initial moisture of 15 % at 25 °C and pH 6.0, after 120 h of fermentation. The influence of PEG molar mass (1000-8000 g mol -1 ), phase concentrations (PEG 20.0-24.0 % w/w and sodium citrate 15-20 % w/w) and pH (6.0-8.0) on partition coefficient, purification factor, yield and selectivity of XY were determinate. Enzyme partitioning into the PEG rich phase was favored by M PEG 8000 (g mol -1 ), C PEG 24 % (w/w), C C 20 % (w/w) and pH 8.0, resulting in partition coefficient of 50.78, activity yield of 268 %, 7.20-fold purification factor and selectivity of 293. A. japonicus URM5620 has a potential role in the development of a bioprocess for the XY production using low-cost media. In addition, the present study proved it is feasible to extract xylanase from SSF by adopting the one step ATPS consisting of PEG/citrate.

Biochemical and thermodynamic characteristics of thermo-alkali-stable xylanase from a novel polyextremophilic Bacillus halodurans TSEV1.

PubMed

Kumar, Vikash; Satyanarayana, T

2013-09-01

The purified extracellular xylanase of polyextremophilic Bacillus halodurans TSEV1 has been visualized as a single band on SDS-PAGE and eluted as single peak by gel filtration, with a molecular mass of 40 kDa. The peptide finger print and cloned xylanase gene sequence analyses indicate that this enzyme belongs to GH family 10. The active site carboxyl residues are mainly involved in catalysis, while tryptophan residues are involved in substrate binding. The enzyme is optimally active at 80 °C and pH 9.0, and stable in the pH range of 7.0-12.0 with T 1/2 of 35 min at 80 °C (pH 9.0). Activation energy for birch wood xylan hydrolysis is 30.51 kJ mol(-1). The K m, V max and k cat (birchwood xylan) are 2.05 mg ml(-1), 333.33 μmol mg(-1 )min(-1) and 3.33 × 10(4) min(-1), respectively. The pKa1 and pKa2 of ionizable groups of the active site that influence V max are 8.51 and 11.0. The analysis of thermodynamic parameters for xylan hydrolysis suggests this as a spontaneous process. The enzyme is resistant to chemical denaturants like urea and guanidinium-HCl. The site-directed mutagenesis of catalytic glutamic acid residues (E196 and E301) resulted in a complete loss of activity. The birch wood xylan hydrolyzate contained xylobiose and xylotriose as the main products without any trace of xylose, and the enzyme hydrolyzes xylotetraose and xylopentaose rapidly to xylobiose. Thermo-alkali-stability, resistance to various chemical denaturants and mode of action make it a useful biocatalyst for generating xylo-oligosaccharides from agro-residues and bleaching of pulp in paper industries.

High-level soluble expression of a thermostable xylanase from thermophilic fungus Thermomyces lanuginosus in Escherichia coli via fusion with OsmY protein.

PubMed

Le, Yilin; Wang, Huilei

2014-07-01

A thermostable xylanase is encoded by xynA from fungus Thermomyces lanuginosus. The problem emerged from overexpression of xynA in Escherichia coli has been the formation of inclusion bodies. Here we describe the xynA was fused with the hyperosmotically inducible periplasmic protein of E. coli, OsmY. The fusion protein OsmY-xynA was expressed as almost all soluble form. The soluble expression level of fusion protein reached 98±6U/ml when cells containing pET-OsmY-xynA were expressed without IPTG induction at 37°C. The induction is probably due to auto-induction due to lactose in the medium (Studier (2005) [21]). The cells harboring pET-OsmY-xynA expressed an activity level about 24 times higher than that expressed from pET-20b-xynA. Xylanase activity was observed in the extracellular (36±1.3U/ml) and the periplasmic (42±4U/ml) when cells containing pET-OsmY-xynA were induced without IPTG addition. After the cold osmotic shock procedure followed by nickel affinity chromatography, the purified fusion protein showed a single band on SDS-PAGE gel with a molecular mass of 44kDa. The purified fusion enzyme exhibited the highest activity at 65°C and pH 6.0. Copyright © 2014 Elsevier Inc. All rights reserved.

A novel GH10 xylanase from Penicillium sp. accelerates saccharification of alkaline-pretreated bagasse by an enzyme from recombinant Trichoderma reesei expressing Aspergillus β-glucosidase.

PubMed

Shibata, Nozomu; Suetsugu, Mari; Kakeshita, Hiroshi; Igarashi, Kazuaki; Hagihara, Hiroshi; Takimura, Yasushi

2017-01-01

Trichoderma reesei is considered a candidate fungal enzyme producer for the economic saccharification of cellulosic biomass. However, performance of the saccharifying enzymes produced by T. reesei is insufficient. Therefore, many attempts have been made to improve its performance by heterologous protein expression. In this study, to increase the conversion efficiency of alkaline-pretreated bagasse to sugars, we conducted screening of biomass-degrading enzymes that showed synergistic effects with enzyme preparations produced by recombinant T. reesei . Penicillium sp. strain KSM-F532 produced the most effective enzyme to promote the saccharification of alkaline-pretreated bagasse. Biomass-degrading enzymes from strain KSM-F532 were fractionated and analyzed, and a xylanase, named PspXyn10, was identified. The amino acid sequence of PspXyn10 was determined by cDNA analysis: the enzyme shows a modular structure consisting of glycoside hydrolase family 10 (GH10) and carbohydrate-binding module family 1 (CBM1) domains. Purified PspXyn10 was prepared from the supernatant of a recombinant T. reesei strain. The molecular weight of PspXyn10 was estimated to be 55 kDa, and its optimal temperature and pH for xylanase activity were 75 °C and pH 4.5, respectively. More than 80% of the xylanase activity was maintained at 65 °C for 10 min. With beechwood xylan as the substrate, the enzyme had a K m of 2.2 mg/mL and a V max of 332 μmol/min/mg. PspXyn10ΔCBM, which lacked the CBM1 domain, was prepared by limited proteolysis. PspXyn10ΔCBM showed increased activity against soluble xylan, but decreased saccharification efficiency of alkaline-pretreated bagasse. This result indicated that the CBM1 domain of PspXyn10 contributes to the enhancement of the saccharification efficiency of alkaline-pretreated bagasse. A recombinant T. reesei strain, named X2PX10, was constructed from strain X3AB1. X3AB1 is an Aspergillus aculeatus β-glucosidase-expressing T. reesei PC-3-7. X2PX10

Biological pretreatment of rice straw with Streptomyces griseorubens JSD-1 and its optimized production of cellulase and xylanase for improved enzymatic saccharification efficiency.

PubMed

Zhang, Dan; Luo, Yanqing; Chu, Shaohua; Zhi, Yuee; Wang, Bin; Zhou, Pei

2016-08-17

Biological pretreatment of rice straw and production of reducing sugars by hydrolysis of bio-pretreated material with Streptomyces griseorubens JSD-1 was investigated. After 10 days of incubation, various chemical compositions of inoculated rice straw were degraded and used for further enzymatic hydrolysis studies. The production of cellulolytic enzyme by S. griseorubens JSD-1 favored the conversion of cellulose to reducing sugars. The culture medium for cellulolytic enzyme production by using agro-industrial wastes was optimized through response surface methodology. According to the response surface analysis, the concentrations of 11.13, 20.34, 4.61, and 2.85 g L(-1) for rice straw, wheat bran, peptone, and CaCO3, respectively, were found to be optimum for cellulase and xylanase production. Then the hydrolyzed spent Streptomyces cells were used as a nitrogen source and the maximum filter paper cellulase, carboxymethylcellulase, and xylanase activities of 25.79, 78.91, and 269.53 U mL(-1) were achieved. The crude cellulase produced by S. griseorubens JSD-1 was subsequently used for the hydrolysis of bio-pretreated rice straw, and the optimum saccharification efficiency of 88.13% was obtained, indicating that the crude enzyme might be used instead of commercial cellulase during a saccharification process. These results give a basis for further study of bioethanol production from agricultural cellulosic waste.

Comparison of a xylanase and a complex of non starch polysaccharide-degrading enzymes with regard to performance and bacterial metabolism in weaned piglets.

PubMed

Vahjen, Wilfried; Osswald, Tanja; Schäfer, Klaus; Simon, Ortwin

2007-04-01

Weaned piglets were fed a wheat based diet either non-supplemented or supplemented with a multi-enzyme preparation or a xylanase mono-enzyme preparation, respectively. Both enzyme preparations increased live weight gain nonsignificantly, but only animals of the xylanase group showed a trend (p = 0.076) for an improved feed conversion. Only precaecal digestibility of total amino acids was improved significantly when the mono-enzyme preparation was added. Improvements of digestibility of crude fat, crude protein and starch did not reach the significance level. Both enzyme preparations reduced jejunal viscosity, however viscosity in the colon was only reduced by the mono-enzyme preparation. Both enzymes significantly reduced Lactobacillus spp. cell numbers as well as bacterial metabolites in the stomach and showed similar nonsignificant modifications in jejunum contents except for acetate in the mono-enzyme group. Total jejunal bile acids were unchanged. Compared to the control, the ratio of the main conjugated to the main deconjugated bile acid was significantly higher in the mono-enzyme group. This study has shown that the mono- and multi-enzyme preparation can lead to improved performance in wheat based diets for piglets. Like in poultry, the main mode of action seems to be the reduction of small intestinal viscosity. However, the generation of fermentable carbohydrates by the multi-enzyme preparation may mask beneficial effects on performance due to the development of an active bile acid deconjugating microbiota in the small intestine.

The thermophilic biomass-degrading fungus Thielavia terrestris Co3Bag1 produces a hyperthermophilic and thermostable β-1,4-xylanase with exo- and endo-activity.

PubMed

García-Huante, Yolanda; Cayetano-Cruz, Maribel; Santiago-Hernández, Alejandro; Cano-Ramírez, Claudia; Marsch-Moreno, Rodolfo; Campos, Jorge E; Aguilar-Osorio, Guillermo; Benitez-Cardoza, Claudia G; Trejo-Estrada, Sergio; Hidalgo-Lara, María Eugenia

2017-01-01

A hyperthermophilic and thermostable xylanase of 82 kDa (TtXynA) was purified from the culture supernatant of T. terrestris Co3Bag1, grown on carboxymethyl cellulose (CMC), and characterized biochemically. TtXynA showed optimal xylanolytic activity at pH 5.5 and at 85 °C, and retained more than 90% of its activity at a broad pH range (4.5-10). The enzyme is highly thermostable with a half-life of 23.1 days at 65 °C, and active in the presence of several metal ions. Circular dichroism spectra strongly suggest the enzyme gains secondary structures when temperature increases. TtXynA displayed higher substrate affinity and higher catalytic efficiency towards beechwood xylan than towards birchwood xylan, oat-spelt xylan, and CMC. According to its final hydrolysis products, TtXynA displays endo-/exo-activity, yielded xylobiose, an unknown oligosaccharide containing about five residues of xylose and a small amount of xylose on beechwood xylan. Finally, this report represents the description of the first fungal hyperthermophilic xylanase which is produced by T. terrestris Co3Bag1. Since TtXynA displays relevant biochemical properties, it may be a suitable candidate for biotechnological applications carried out at high temperatures, like the enzymatic pretreatment of plant biomass for the production of bioethanol.

Rationalising pKa shifts in Bacillus circulans xylanase with computational studies.

PubMed

Xiao, Kela; Yu, Haibo

2016-11-09

Bacillus circulans xylanase (BcX), a family 11 glycoside hydrolase, catalyses the hydrolysis of xylose polymers with a net retention of stereochemistry. Glu172 in BcX is believed to act as a general acid by protonating the aglycone during glycosylation, and then as a general base to facilitate the deglycosylation step. The key to the dual role of this general acid/base lies in its protonation states, which depend on its intrinsic pK a value and the specific environment which it resides within. To fully understand the detailed molecular features in BcX to establish the dual role of Glu172, we present a combined study based on both atomistic simulations and empirical models to calculate pK a shifts for the general acid/base Glu172 in BcX at different functional states. Its pK a values and those of nearby residues, obtained based on QM/MM free energy calculations, MCCE and PROPKA, show a good agreement with available experimental data. Additionally, our study provides additional insights into the effects of structural and electrostatic perturbations caused by mutations and chemical modifications, suggesting that the local solvation environment and mutagenesis of the residues adjacent to Glu172 establish its dual role during hydrolysis. The strengths and limitations of various methods for calculating pK a s and pK a shifts have also been discussed.

Continuous xylanase production with Aspergillus nidulans under pyridoxine limitation using a trickle bed reactor.

PubMed

Müller, Michael; Prade, Rolf A; Segato, Fernando; Atiyeh, Hasan K; Wilkins, Mark R

2015-01-01

A trickle bed reactor (TBR) with recycle was designed and tested using Aspergillus nidulans with a pyridoxine marker and over-expressing/secreting recombinant client xylanase B (XynB). The pyridoxine marker prevented the fungus from synthesizing its own pyridoxine and fungus was unable to grow when no pyridoxine was present in the medium; however, enzyme production was unaffected. Uncontrolled mycelia growth that led to clogging of the TBR was observed when fungus without a pyridoxine marker was used for XynB production. Using the fungus with pyridoxine marker, the TBR was operated continuously for 18 days and achieved a XynB output of 41 U/ml with an influent and effluent flow rate of 0.5 ml/min and a recycle flow rate of 56 ml/min. Production yields in the TBR were 1.4 times greater than a static tray culture and between 1.1 and 67 times greater than yields for SSF enzyme production stated in the literature. Copyright © 2015 Elsevier Ltd. All rights reserved.

Structure features of GH10 xylanase from Caldicellulosiruptor bescii: implication for its thermophilic adaption and substrate binding preference.

PubMed

Zhang, Yong; An, Jiao; Yang, Guangyu; Zhang, Xiaofei; Xie, Yuan; Chen, Liuqing; Feng, Yan

2016-10-01

Caldicellulosiruptor bescii is the most thermophilic cellulolytic species of organisms known to date. In our previous study, GH10 xylanase CbXyn10B from C. bescii displayed outstanding hydrolytic activity toward various xylans at high temperatures. To understand the structural basis for this protein's catalysis and thermostability, we solved the crystal structures of CbXyn10B and its complexes with xylooligosaccharides. These structural models were used to guide comparison with its mesophilic counterpart PbXyn10B. A distinctive structural feature is that thermophilic CbXyn10B presents a relatively stable interaction between the extended loops L7 and L8 in the catalytic cleft by an extensive hydrogen bonding network, which is mediated by Lys 306 , Arg 314 and three well-ordered water molecules. Moreover, a unique aromatic cluster consisting of Try 17 , Phe 20 , Phe 21 , and Phe 337 may enhance the interaction between the N- and C- terminus. Targeted mutagenesis demonstrated that these interactions substantially contribute to enzyme stabilization, as indicated by a considerable decrease in the melting temperature (T m ) of CbXyn10B by substituting critical residues with Ala. Therefore, it was shown that not only the aromatic interaction connecting protein termini but also the extensive hydrogen bonding network formed between surface loops could restrict the local structural flexibility and contribute significantly to the overall stability of enzymes. Furthermore, the xylooligosaccharides were found to tightly bind to the glycone subsites of xylanase, indicating higher affinities at these subsites and reflecting its substrate binding preference. Our results suggest that CbXyn10B is stabilized with distinct rigidity at the catalytic cleft as well as the terminal regions, which provides insights into the evolutionary strategy for accommodating the functional needs of GH10 enzymes to high temperature. © The Author 2016. Published by Oxford University Press on behalf of

Endoglucanase and xylanase production by Bacillus sp. AR03 in co-culture.

PubMed

Hero, Johan S; Pisa, José H; Perotti, Nora I; Romero, Cintia M; Martínez, María A

2017-07-03

The behavior of three isolates retrieved from different cellulolytic consortia, Bacillus sp. AR03, Paenibacillus sp. AR247 and Achromobacter sp. AR476-2, were examined individually and as co-cultures in order to evaluate their ability to produce extracellular cellulases and xylanases. Utilizing a peptone-based medium supplemented with carboxymethyl cellulose (CMC), an increase estimation of 1.30 and 1.50 times was obtained by the co-culture containing the strains AR03 and AR247, with respect to enzyme titles registered by their individual cultivation. On the contrary, the extracellular enzymatic production decreased during the co-cultivation of strain AR03 with the non-cellulolytic Achromobacter sp. AR476-2. The synergistic behavior observed through the combined cultivation of the strains AR03 and AR247 might be a consequence of the consumption by Paenibacillus sp. AR247 of the products of the CMC hydrolysis (i.e., cellobiose and/or cello-oligosaccharides), which were mostly generated by the cellulase producer Bacillus sp. AR03. The effect observed could be driven by the requirement to fulfill the nutritional supply from both strains on the substrate evaluated. These results would contribute to a better description of the degradation of the cellulose fraction of the plant cell walls in nature, expected to an efficient utilization of renewable sources.

Applicability of thermo-alkali-stable and cellulase-free xylanase from a novel thermo-halo-alkaliphilic Bacillus halodurans in producing xylooligosaccharides.

PubMed

Kumar, Vikash; Satyanarayana, T

2011-11-01

An alkaliphilic, moderately thermophilic and halophilic bacterial isolate capable of producing a high titer of extracellular thermo-alkali-stable, cellulase-free endoxylanase was isolated from the paper mill effluents. It was identified as Bacillus halodurans. The purified xylanase was active from pH 7 to 12 and 30 to 100°C with optimal activity at pH 9.0 and 80°C. It had T(1/2) values of 40 and 15 min at 70 and 80°C, respectively. Activity was stimulated by dithiothreitol but strongly inhibited by N-bromosuccinimide. Its action on birchwood xylan and agro-residues liberated xylooligosaccharides of 2-7 degree of polymerization, and thus, the mode of action is similar to endoxylanases of the family 10 glucoside hydrolases.

The effects of transportation stress on Japanese quail (Coturnix Coturnix japonica) fed corn-based diet in comparison with wheat-based diet supplemented with xylanase and phytase.

PubMed

Mehraei Hamzekolaei, M H; Zamani Moghaddam, A K; Tohidifar, S S; Dehghani Samani, A; Heydari, A

2016-08-01

Harvesting, handling and transporting quails to the slaughterhouses, other farms and laboratories might covertly reduce their welfare. The aim of this study was to evaluate the effects of two major sources of energy in poultry nutrition on reducing transportation stress in Japanese quail (Coturnix Coturnix japonica). Male quails (n = 60) were divided into two groups. The first group was fed corn-based diet, and the second was fed wheat-based diet supplemented with xylanase and phytase. At the end of the experiment (day 35), quails were subjected to 80 km of transportation. Immediately on arrival and after 24 h, heterophil counts, lymphocyte counts and H:L ratios were measured. On arrival, H counts were lower, L counts were higher, and H:L ratios were lower for corn-fed group. After 24 h, wheat-fed group showed lower increment of H counts, greater increment of L counts and also decrement of H:L ratios rather than corn-fed group which showed increment of H:L ratios. However, these ratios were still lower in corn-fed group. Results indicate that corn-based diets can help Japanese quail to better resist transportation stress, although it seems that feeding wheat-based diets supplemented with xylanase and phytase could have positive effects for coping better with stress after journeys. Journal of Animal Physiology and Animal Nutrition © 2015 Blackwell Verlag GmbH.

Improvement of bread making quality by supplementation with a recombinant xylanase produced by Pichia pastoris.

PubMed

de Queiroz Brito Cunha, Carolina Cândida; Gama, Aline Rodrigues; Cintra, Lorena Cardoso; Bataus, Luiz Artur Mendes; Ulhoa, Cirano José

2018-01-01

Xylanases (EC 3.2.1.8) are hydrolytic enzymes, which randomly cleave the β-1,4-linked xylose residues from xylan. The synthetic gene xynBS27 from Streptomyces sp. S27 was successfully cloned and expressed in Pichia pastoris. The full-length gene consists of 729 bp and encodes 243 amino acids including 51 residues of a putative signal peptide. This enzyme was purified in two steps and was shown to have a molecular weight of 20 kDa. The purified r-XynBS27 was active against beechwood xylan and oat spelt xylan as expected for GH 11 family. The optimum pH and temperature values for the enzyme were 6.0 and 75 °C, respectively. The Km and Vmax were 12.38 mg/mL and 13.68 μmol min/mg, respectively. The r-XynBS27 showed high xylose tolerance and was inhibited by some metal ions and by SDS. r-XynBS27 was employed as an additive in the bread making process. A decrease in firmness, stiffness and consistency, and improvements in specific volume and reducing sugar content were recorded.

Thermal and chemical denaturation of Bacillus circulans xylanase: A biophysical chemistry laboratory module.

PubMed

Raabe, Richard; Gentile, Lisa

2008-11-01

A number of institutions have been, or are in the process of, modifying their biochemistry major to include some emphasis on the quantitative physical chemistry of biomolecules. Sometimes this is done as a replacement for part for the entire physical chemistry requirement, while at other institutions this is incorporated as a component into the traditional two-semester biochemistry series. The latter is the model used for biochemistry and molecular biology majors at the University of Richmond, whose second semester of biochemistry is a course entitled Proteins: Structure, Function, and Biophysics. What is described herein is a protein thermodynamics laboratory module, using the protein Bacillus circulans xylanase, which reinforces many lecture concepts, including: (i) the denatured (D) state ensemble of a protein can be different, depending on how it was populated; (ii) intermediate states may be detected by some spectroscopic techniques but not by others; (iii) the use and assumptions of the van't Hoff approach to calculate ΔH(o) , ΔS(o) , and ΔG(o) (T) for thermal protein unfolding transitions; and (iv) the use and assumptions of an approach that allows determination of the Gibb's free energy of a protein unfolding transition based on the linear dependence of ΔG(o) on the concentration of denaturant used. This module also requires students to design their own experimental protocols and spend time in the primary literature, both important parts of an upper division lab. Copyright © 2008 International Union of Biochemistry and Molecular Biology, Inc.

Thumb-loops up for catalysis: a structure/function investigation of a functional loop movement in a GH11 xylanase

PubMed Central

Paës, Gabriel; Cortés, Juan; Siméon, Thierry; O'Donohue, Michael J.; Tran, Vinh

2012-01-01

Dynamics is a key feature of enzyme catalysis. Unfortunately, current experimental and computational techniques do not yet provide a comprehensive understanding and description of functional macromolecular motions. In this work, we have extended a novel computational technique, which combines molecular modeling methods and robotics algorithms, to investigate functional motions of protein loops. This new approach has been applied to study the functional importance of the so-called thumb-loop in the glycoside hydrolase family 11 xylanase from Thermobacillus xylanilyticus (Tx-xyl). The results obtained provide new insight into the role of the loop in the glycosylation/deglycosylation catalytic cycle, and underline the key importance of the nature of the residue located at the tip of the thumb-loop. The effect of mutations predicted in silico has been validated by in vitro site-directed mutagenesis experiments. Overall, we propose a comprehensive model of Tx-xyl catalysis in terms of substrate and product dynamics by identifying the action of the thumb-loop motion during catalysis. PMID:24688637

Ethanol/water pulps from sugar cane straw and their biobleaching with xylanase from Bacillus pumilus.

PubMed

Moriya, Regina Y; Gonçalves, Adilson R; Duarte, Marta C T

2007-04-01

The influence of independent variables (temperature and time) on the cooking of sugar cane straw with ethanol/water mixtures was studied to determine operating conditions that obtain pulp with high cellulose contents and a low lignin content. An experimental 2(2) design was applied for temperatures of 185 and 215 degrees C, and time of 1 and 2.5 h with the ethanol/water mixture concentration and constant straw-to-solvent ratio. The system was scaled-up at 200 degrees C cooking temperature for 2 h with 50% ethanol-water concentration, and 1:10 (w/v) straw-to-solvent ratio to obtain a pulp with 3.14 cP viscosity, 58.09 kappa-number, and the chemical composition of the pulps were 3.2% pentosan and 31.5% lignin. Xylanase from Bacillus pumilus was then applied at a loading of 5-150 IU/g dry pulp in the sugar cane straw ethanol/water pulp at 50 degrees C for 2 and 20 h. To ethanol/water pulps, the best enzyme dosage was found to be 20 IU/g dry pulp at 20 h, and a high enzyme dosage of 150 IU/g dry pulp did not decrease the kappa-number of the pulp.

Raw sugarcane bagasse as carbon source for xylanase production by Paenibacillus species: a potential degrader of agricultural wastes.

PubMed

Di Marco, Enzo; Soraire, Pablo M; Romero, Cintia M; Villegas, Liliana B; Martínez, María Alejandra

2017-08-01

Paenibacillus species isolated from a variety of natural sources have shown to be important glycoside hydrolases producers. These enzymes play a key role in bio-refining applications, as they are central biocatalysts for the processing of different types of polymers from vegetal biomass. Xylanase production by three native isolates belonging to the genus Paenibacillus was approached by utilizing mineral-based medium and agricultural by-products as a convenient source to produce biocatalysts suitable for their degradation. While varieties of alkali pretreated sugarcane bagasse were useful substrates for the strains from Paenibacillus genus evaluated, raw sugarcane bagasse was the most effective substrate for endoxylanase production by Paenibacillus sp. AR247. This strain was then selected to further improvement of its enzyme production by means of a two-step statistical approach. It was determined that the carbon source, provided as an inexpensive agro-waste, as well as phosphate and magnesium were the culture media components that most influenced the enzyme production, which was improved three times compared to the screening results.

Substituting Both the N-Terminal and "Cord" Regions of a Xylanase from Aspergillus oryzae to Improve Its Temperature Characteristics.

PubMed

Li, Chuang; Li, Jianfang; Wang, Rui; Li, Xueqing; Li, Jinping; Deng, Chao; Wu, Minchen

2018-02-06

To improve the temperature characteristics of AoXyn11A, a mesophilic glycoside hydrolase family (GHF) 11 xylanase from Aspergillus oryzae CICC40186, its N-terminal and "cord" regions were selected to be substituted by means of the computer-aided analysis and calculation. In brief, one mutant, named ATX11A 41 , possessing the lowest root-mean-square deviation (RMSD) value was designed based on the molecular dynamics (MD) simulation by substituting the N-terminal 41 amino acids of AoXyn11A with the corresponding 42 ones of pXYL11, a thermophilic GHF11 xylanase from Thermobifida fusca. On the basis of the primary structure alignment of pXYL11 with ATX11A 41 (or AoXyn11A), another mutant, named ATX11A 41/cord , was designed by substituting the cord region ( 93 GTYNPGSGG 101 ) of ATX11A 41 with the corresponding one ( 93 GTYRPTG 99 ) of pXYL11. Both mutant-encoding genes, ATx11A 41 and ATx11A 41/cord , were constructed as designed theoretically by a megaprimer PCR technique and were expressed in Pichia pastoris GS115. The specific activities of recombinant (re) AoXyn11A, ATX11A 41 , and ATX11A 41/cord were 2916.7, 2667.6, and 2457.0 U/mg, respectively. The analysis of temperature characteristics displayed that the temperature optimum (T opt ) of reATX11A 41 or reATX11A 41/cord was 65 °C, which was 15 °C higher than that of reAoXyn11A. The thermal inactivation half-life (t 1/2 ) values of reATX11A 41 and reATX11A 41/cord at 60 °C were 55 and 83 min, respectively, whereas that of reAoXyn11A was only 18 min at 50 °C. The melting temperature (T m ) values of reAoXyn11A, reATX11A 41 , and reATX11A 41/cord were 54.2, 66.7, and 71.9 °C, respectively. In conclusion, the above findings indicated that the substitution of both the N-terminal and cord regions of a mesophilic AoXyn11A greatly contributed to its improved temperature characteristics.

Solid state production of polygalacturonase and xylanase by Trichoderma species using cantaloupe and watermelon rinds.

PubMed

Mohamed, Saleh A; Al-Malki, Abdulrahman L; Khan, Jalaluddin A; Kabli, Saleh A; Al-Garni, Saleh M

2013-10-01

Different solid state fermentation (SSF) sources were tested such as cantaloupe and watermelon rinds, orange and banana peels, for the production of polygalacturonase (PG) and xylanase (Xyl) by Trichoderma harzianum and Trichoderma virens. The maximum production of both PG and Xyl were obtained by T. harzianum and T. virnes grown on cantaloupe and watermelon rinds, respectively. Time course, moisture content, temperature, pH, supplementation with carbon and nitrogen sources were optimized to achieve the maximum production of both PG and Xyl of T. harzianum and T. virens using cantaloupe and watermelon rinds, respectively. The maximum production of PG and Xyl of T. harzianum and T. virens was recorded at 4-5 days of incubation, 50-66% moisture, temperature 28-35°C and pH 6-7. The influence of supplementary carbon and nitrogen sources was studied. For T. harzianum, lactose enhanced PG activity from 87 to 120 units/g solid, where starch and maltose enhanced Xyl activity from 40 to 55-60 units/g solid for T. virnes. Among the nitrogen sources, ammonium sulphate, ammonium nitrate, yeast extract and urea increased PG activity from 90 to 110-113 units/g solid for T. harzianum. Similarly, ammonium chloride, ammonium sulphate and yeast extract increased Xyl activity from 45 to 55-70 units/g solid for T. virens.

A high performance Trichoderma reesei strain that reveals the importance of xylanase III in cellulosic biomass conversion.

PubMed

Nakazawa, Hikaru; Kawai, Tetsushi; Ida, Noriko; Shida, Yosuke; Shioya, Kouki; Kobayashi, Yoshinori; Okada, Hirofumi; Tani, Shuji; Sumitani, Jun-Ichi; Kawaguchi, Takashi; Morikawa, Yasushi; Ogasawara, Wataru

2016-01-01

The ability of the Trichoderma reesei X3AB1strain enzyme preparations to convert cellulosic biomass into fermentable sugars is enhanced by the replacement of xyn3 by Aspergillus aculeatus β-glucosidase 1 gene (aabg1), as shown in our previous study. However, subsequent experiments using T. reesei extracts supplemented with the glycoside hydrolase (GH) family 10 xylanase III (XYN III) and GH Family 11 XYN II showed increased conversion of alkaline treated cellulosic biomass, which is rich in xylan, underscoring the importance of XYN III. To attain optimal saccharifying potential in T. reesei, we constructed two new strains, C1AB1 and E1AB1, in which aabg1 was expressed heterologously by means of the cbh1 or egl1 promoters, respectively, so that the endogenous XYN III synthesis remained intact. Due to the presence of wild-type xyn3 in T. reesei E1AB1, enzymes prepared from this strain were 20-30% more effective in the saccharification of alkaline-pretreated rice straw than enzyme extracts from X3AB1, and also outperformed recent commercial cellulase preparations. Our results demonstrate the importance of XYN III in the conversion of alkaline-pretreated cellulosic biomass by T. reesei. Copyright © 2015 Elsevier Inc. All rights reserved.

D-Xylose fermentation, xylitol production and xylanase activities by seven new species of Sugiyamaella.

PubMed

Sena, Letícia M F; Morais, Camila G; Lopes, Mariana R; Santos, Renata O; Uetanabaro, Ana P T; Morais, Paula B; Vital, Marcos J S; de Morais, Marcos A; Lachance, Marc-André; Rosa, Carlos A

2017-01-01

Sixteen yeast isolates identified as belonging to the genus Sugiyamaella were studied in relation to D-xylose fermentation, xylitol production, and xylanase activities. The yeasts were recovered from rotting wood and sugarcane bagasse samples in different Brazilian regions. Sequence analyses of the internal transcribed spacer (ITS) region and the D1/D2 domains of large subunit rRNA gene showed that these isolates belong to seven new species. The species are described here as Sugiyamaella ayubii f.a., sp. nov. (UFMG-CM-Y607 T  = CBS 14108 T ), Sugiyamaella bahiana f.a., sp. nov. (UFMG-CM-Y304 T  = CBS 13474 T ), Sugiyamaella bonitensis f.a., sp. nov. (UFMG-CM-Y608 T  = CBS 14270 T ), Sugiyamaella carassensis f.a., sp. nov. (UFMG-CM-Y606 T  = CBS 14107 T ), Sugiyamaella ligni f.a., sp. nov. (UFMG-CM-Y295 T  = CBS 13482 T ), Sugiyamaella valenteae f.a., sp. nov. (UFMG-CM-Y609 T  = CBS 14109 T ) and Sugiyamaella xylolytica f.a., sp. nov. (UFMG-CM-Y348 T  = CBS 13493 T ). Strains of the described species S. boreocaroliniensis, S. lignohabitans, S. novakii and S. xylanicola, isolated from rotting wood of Brazilian ecosystems, were also compared for traits relevant to xylose metabolism. S. valenteae sp. nov., S. xylolytica sp. nov., S. bahiana sp. nov., S. bonitensis sp. nov., S. boreocarolinensis, S. lignohabitans and S. xylanicola were able to ferment D-xylose to ethanol. Xylitol production was observed for all Sugiyamaella species studied, except for S. ayubii sp. nov. All species studied showed xylanolytic activity, with S. xylanicola, S. lignohabitans and S. valenteae sp. nov. having the highest values. Our results suggest these Sugiyamaella species have good potential for biotechnological applications.

Thermostability improvement of a streptomyces xylanase by introducing proline and glutamic acid residues.

PubMed

Wang, Kun; Luo, Huiying; Tian, Jian; Turunen, Ossi; Huang, Huoqing; Shi, Pengjun; Hua, Huifang; Wang, Caihong; Wang, Shuanghe; Yao, Bin

2014-04-01

Protein engineering is commonly used to improve the robustness of enzymes for activity and stability at high temperatures. In this study, we identified four residues expected to affect the thermostability of Streptomyces sp. strain S9 xylanase XynAS9 through multiple-sequence analysis (MSA) and molecular dynamic simulations (MDS). Site-directed mutagenesis was employed to construct five mutants by replacing these residues with proline or glutamic acid (V81P, G82E, V81P/G82E, D185P/S186E, and V81P/G82E/D185P/S186E), and the mutant and wild-type enzymes were expressed in Pichia pastoris. Compared to the wild-type XynAS9, all five mutant enzymes showed improved thermal properties. The activity and stability assays, including circular dichroism and differential scanning calorimetry, showed that the mutations at positions 81 and 82 increased the thermal performance more than the mutations at positions 185 and 186. The mutants with combined substitutions (V81P/G82E and V81P/G82E/D185P/S186E) showed the most pronounced shifts in temperature optima, about 17°C upward, and their half-lives for thermal inactivation at 70°C and melting temperatures were increased by >9 times and approximately 7.0°C, respectively. The mutation combination of V81P and G82E in adjacent positions more than doubled the effect of single mutations. Both mutation regions were at the end of long secondary-structure elements and probably rigidified the local structure. MDS indicated that a long loop region after positions 81 and 82 located at the end of the inner β-barrel was prone to unfold. The rigidified main chain and filling of a groove by the mutations on the bottom of the active site canyon may stabilize the mutants and thus improve their thermostability.

A family 30 glucurono-xylanase from Bacillus subtilis LC9: Expression, characterization and its application in Chinese bread making.

PubMed

Guo, Yalan; Gao, Zhen; Xu, Jiaxing; Chang, Siyuan; Wu, Bin; He, Bingfang

2018-05-22

A GH30-8 endoxylanase was identified from an environmental Bacillus subtilis isolate following growth selection on aspen wood glucuronoxylan. The putative endoxylanase was cloned for protein expression and characterization in the Gram-positive protease deficient protein expression host B. subtilis WB800. The extracellular activity obtained was 55 U/mL, which was 14.5-fold higher than that obtained with the native species. The apparent molecular mass of BsXyn30 was estimated as 43 kDa by SDS-PAGE. BsXyn30 showed an optimal activity at pH 7.0 and 60 °C. Recombinant BsXyn30 displayed maximum activity against aspen wood xylan, followed by beechwood xylan but showed no catalytic activity on arabinose-substituted xylans. Analysis of hydrolyzed products of beechwood xylan by thin-layer chromatography and mass spectroscopy revealed the presence of xylooligosaccharides with a single methyl-glucuronic acid residue. BsXyn30 exhibited very low activity for hydrolysis xylotetraose and xylopentaose, but had no detectable activity against xylobiose and xylotriose. Using BsXyn30 as an additive in breadmaking, a decrease in water-holding capacity, an increase in dough expansion as well as improvements in volume and specific volume of the bread were recorded. Thus, the present study provided the basis for the application of GH30 xylanase in breadmaking. Copyright © 2018 Elsevier B.V. All rights reserved.

Detection and characterization of serine and threonine hydroxyl protons in Bacillus circulans xylanase by NMR spectroscopy.

PubMed

Brockerman, Jacob A; Okon, Mark; McIntosh, Lawrence P

2014-01-01

Hydroxyl protons on serine and threonine residues are not well characterized in protein structures determined by both NMR spectroscopy and X-ray crystallography. In the case of NMR spectroscopy, this is in large part because hydroxyl proton signals are usually hidden under crowded regions of (1)H-NMR spectra and remain undetected by conventional heteronuclear correlation approaches that rely on strong one-bond (1)H-(15)N or (1)H-(13)C couplings. However, by filtering against protons directly bonded to (13)C or (15)N nuclei, signals from slowly-exchanging hydroxyls can be observed in the (1)H-NMR spectrum of a uniformly (13)C/(15)N-labeled protein. Here we demonstrate the use of a simple selective labeling scheme in combination with long-range heteronuclear scalar correlation experiments as an easy and relatively inexpensive way to detect and assign these hydroxyl proton signals. Using auxtrophic Escherichia coli strains, we produced Bacillus circulans xylanase (BcX) labeled with (13)C/(15)N-serine or (13)C/(15)N-threonine. Signals from two serine and three threonine hydroxyls in these protein samples were readily observed via (3)JC-OH couplings in long-range (13)C-HSQC spectra. These scalar couplings (~5-7 Hz) were measured in a sample of uniformly (13)C/(15)N-labeled BcX using a quantitative (13)C/(15)N-filtered spin-echo difference experiment. In a similar approach, the threonine and serine hydroxyl hydrogen exchange kinetics were measured using a (13)C/(15)N-filtered CLEANEX-PM pulse sequence. Collectively, these experiments provide insights into the structural and dynamic properties of several serine and threonine hydroxyls within this model protein.

Effect of pretreatments and endo-1,4-β-xylanase hydrolysis of canola meal and mustard bran for production of oligosaccharides.

PubMed

Yuan, Lin; Scanlon, Martin G; Eskin, N A Michael; Thiyam-Hollander, Usha; Aachary, Ayyappan A

2015-01-01

Alkali/acid-pretreated canola meal and mustard bran were subjected to endo-1,4-β-xylanase (T. longibrachiatum) hydrolysis for oligosaccharide production. Pretreatments significantly (α = 0.05) increased the relative content of pentose sugars, especially in alkali-pretreated canola meal (∼44 %) and mustard bran (∼72 %). The amounts of pentosan (g/100 g) in acid- and alkali-pretreated canola meal were 7.50 and 8.21 and in corresponding mustard bran were 8.67 and 10.39, respectively. These pretreated substrates produced a pentose content (g/100 g) of 2.10 ± 0.14 (18 h) and 2.95 ± 0.10 (24 h), respectively, during hydrolysis. As per UPLC-MS data, the main oligosaccharides in the hydrolyzates of alkali-pretreated substrates are xylo-glucuronic acid and xylobiose. The release of total phenolics of the hydrolyzates increased until 18 h irrespective of the type of substrate or pretreatment. Hydrolyzates of acid-pretreated substrates indicated more total antioxidant activity than alkali-pretreated substrates, attributed to its high phenolic content. The study suggests the potential of canola meal and mustard bran for the production of oligosaccharides, wherein the use of various combinations of cell-wall-degrading enzymes and its optimization may result in a better yield, with simultaneous production of endogenous phenolics.

Ethanol production by Escherichia coli from Arundo donax biomass under SSF, SHF or CBP process configurations and in situ production of a multifunctional glucanase and xylanase.

PubMed

Loaces, Inés; Schein, Sima; Noya, Francisco

2017-01-01

Diluted acid or liquid hot water (LHW) pretreated Arundo donax biomass was converted into ethanol under separated hydrolysis and fermentation (SHF) or simultaneous saccharification and fermentation (SSF) using Escherichia coli as the fermentative organism. Up to 0.26gL -1 h -1 and 25.0gL -1 of ethanol were obtained with diluted acid pretreated biomass under SSF compared to 0.17gL -1 h -1 and 24gL -1 under SHF. LHW pretreated biomass elicited 25% lower yields on average. Saccharification was carried out with Cellic CTec2 cocktail. Alternatively, under a consolidated bioprocess (CBP) where the ethanologenic bacteria was complemented with a novel multifunctional glucanase and xylanase, ethanol concentration was 7.6gL -1 and 7.2gL -1 after 96h for dilute acid or LHW pretreated biomass, respectively, without any prior saccharification step. According to these results, a bacterial fermentative host combined with in situ enzyme expression can improve ethanol production from A. donax biomass. Copyright © 2016 Elsevier Ltd. All rights reserved.

A xylanase with broad pH and temperature adaptability from Streptomyces megasporus DSM 41476, and its potential application in brewing industry.

PubMed

Qiu, Zhenhua; Shi, Pengjun; Luo, Huiying; Bai, Yingguo; Yuan, Tiezheng; Yang, Peilong; Liu, Suchun; Yao, Bin

2010-05-05

A xylanase gene, xynAM6, was isolated from the genomic DNA library of Streptomyces megasporus DSM 41476 using colony PCR screening method. The 1440-bp full-length gene encodes a 479-amino acid peptide consisting of a putative signal peptide of 36 residues, a family 10 glycoside hydrolase domain and a family 2 carbohydrate-binding module. The mature peptide of xynAM6 was successfully expressed in Pichia pastoris GS115. The optimal pH and temperature were pH 5.5 and 70°C, respectively. The enzyme showed broad temperature adaptability (>60% of the maximum activity at 50-80°C), had good thermostability at 60°C and 70°C, remained stable at pH 4.0-11.0, and was resistant to most proteases. The Km and Vmax values for oat spelt xylan were 1.68mgml(-1) and 436.76μmolmin(-1)mg(-1), respectively, and 2.33mgml(-1) and 406.93μmolmin(-1)mg(-1) for birchwood xylan, respectively. The hydrolysis products of XYNAM6 were mainly xylose and xylobiose. Addition of XYNAM6 (80U) to the brewery mash significantly reduced the filtration rate and viscosity by 36.33% and 35.51%, respectively. These favorable properties probably make XYNAM6 a good candidate for application in brewing industry. Copyright © 2010 Elsevier Inc. All rights reserved.

Production of fibrolytic enzymes by Aspergillus japonicus C03 using agro-industrial residues with potential application as additives in animal feed.

PubMed

Facchini, Fernanda Dell Antonio; Vici, Ana Claudia; Reis, Victor Ricardo Amin; Jorge, João Atilio; Terenzi, Héctor Francisco; Reis, Ricardo Andrade; Polizeli, Maria de Lourdes Teixeira de Moraes

2011-03-01

Solid-state fermentation obtained from different and low-cost carbon sources was evaluated to endocellulases and endoxylanases production by Aspergillus japonicus C03. Regarding the enzymatic production the highest levels were observed at 30 °C, using soy bran added to crushed corncob or wheat bran added to sugarcane bagasse, humidified with salt solutions, and incubated for 3 days (xylanase) or 6 days (cellulase) with 70% relative humidity. Peptone improved the xylanase and cellulase activities in 12 and 29%, respectively. The optimum temperature corresponded to 60 °C and 50-55 °C for xylanase and cellulase, respectively, both having 4.0 as optimum pH. Xylanase was fully stable up to 40 °C, which is close to the rumen temperature. The enzymes were stable in pH 4.0-7.0. Cu++ and Mn++ increased xylanase and cellulase activities by 10 and 64%, respectively. A. japonicus C03 xylanase was greatly stable in goat rumen fluid for 4 h during in vivo and in vitro experiments.

Structure-based protein engineering for thermostable and alkaliphilic enhancement of endo-β-1,4-xylanase for applications in pulp bleaching.

PubMed

Boonyapakron, Katewadee; Jaruwat, Aritsara; Liwnaree, Benjamas; Nimchua, Thidarat; Champreda, Verawat; Chitnumsub, Penchit

2017-10-10

In the pulp bleaching industry, enzymes with robust activity at high pH and temperatures are desirable for facilitating the pre-bleaching process with simplified processing and minimal use of chlorinated compounds. To engineer an enzyme for this purpose, we determined the crystal structure of the Xyn12.2 xylanase, a xylan-hydrolyzing enzyme derived from the termite gut symbiont metagenome, as the basis for structure-based protein engineering to improve Xyn12.2 stability in high heat and alkaline conditions. Engineered cysteine pairs that generated exterior disulfide bonds increased the k cat of Xyn12.2 variants and melting temperature at all tested conditions. These improvements led to up to 4.2-fold increases in catalytic efficiency at pH 9.0, 50°C for 1h and up to 3-fold increases at 60°C. The most effective variants, XynTT and XynTTTE, exhibited 2-3-fold increases in bagasse hydrolysis at pH 9.0 and 60°C compared to the wild-type enzyme. Overall, engineering arginines and phenylalanines for increased pK a and hydrogen bonding improved enzyme catalytic efficiency at high stringency conditions. These modifications were the keys to enhancing thermostability and alkaliphilicity in our enzyme variants, with XynTT and XynTTTE being especially promising for their application to the pulp and paper industry. Copyright © 2017 Elsevier B.V. All rights reserved.

Generation of xylooligosaccharides from microwave irradiated agroresidues using recombinant thermo-alkali-stable endoxylanase of the polyextremophilic bacterium Bacillus halodurans expressed in Pichia pastoris.

PubMed

Kumar, Vikash; Satyanarayana, T

2015-03-01

The recombinant Pichia pastoris harboring the endoxylanase gene (TSEV1xyl) of Bacillus halodurans TSEV1 yielded a high titer of extracellular xylanase (502±23 U ml(-1)) on induction with methanol. The purified recombinant xylanase (TSEV1xyl) displayed optimal activity at 80°C and pH 9.0. The glycosylated recombinant xylanase exhibited higher thermostability (T1/2 of 45 min at 80°C) than the native enzyme (T1/2 of 35 min at 80°C). The agroresidues subjected to pretreatment (soaking in alkali followed by microwave irradiation) liberated xylooligosaccharides (XOS) upon hydrolysis with the recombinant xylanase. The removal of unhydrolyzed agroresidues, xylanase and xylose from the hydrolysate by two-step ultrafiltration led to the purification of XOS as confirmed by TLC as well as HPLC analysis. Copyright © 2014 Elsevier Ltd. All rights reserved.

Thermomyces lanuginosus: properties of strains and their hemicellulases.

PubMed

Singh, Suren; Madlala, Andreas M; Prior, Bernard A

2003-04-01

The non-cellulolytic Thermomyces lanuginosus is a widespread and frequently isolated thermophilic fungus. Several strains of this fungus have been reported to produce high levels of cellulase-free beta-xylanase both in shake-flask and bioreactor cultivations but intraspecies variability in terms of beta-xylanase production is apparent. Furthermore all strains produce low extracellular levels of other hemicellulases involved in hemicellulose hydrolysis. Crude and purified hemicellulases from this fungus are stable at high temperatures in the range of 50-80 degrees C and over a broad pH range (3-12). Various strains are reported to produce a single xylanase with molecular masses varying between 23 and 29 kDa and pI values between 3.7 and 4.1. The gene encoding the T. lanuginosus xylanase has been cloned and sequenced and is shown to be a member of family 11 glycosyl hydrolases. The crystal structure of the xylanase indicates that the enzyme consists of two beta-sheets and one alpha-helix and forms a rigid complex with the three central sugars of xyloheptaose whereas the peripheral sugars might assume different configurations thereby allowing branched xylan chains to be accepted. The presence of an extra disulfide bridge between the beta-strand and the alpha-helix, as well as to an increase in the density of charged residues throughout the xylanase might contribute to the thermostability. The ability of T. lanuginosus to produce high levels of cellulase-free thermostable xylanase has made the fungus an attractive source of thermostable xylanase with potential as a bleach-boosting agent in the pulp and paper industry and as an additive in the baking industry.

One-step combined focused epPCR and saturation mutagenesis for thermostability evolution of a new cold-active xylanase.

PubMed

Acevedo, Juan Pablo; Reetz, Manfred T; Asenjo, Juan A; Parra, Loreto P

2017-05-01

Enzymes active at low temperature are of great interest for industrial bioprocesses due to their high efficiency at a low energy cost. One of the particularities of naturally evolved cold-active enzymes is their increased enzymatic activity at low temperature, however the low thermostability presented in this type of enzymes is still a major drawback for their application in biocatalysis. Directed evolution of cold-adapted enzymes to a more thermostable version, appears as an attractive strategy to fulfill the stability and activity requirements for the industry. This paper describes the recombinant expression and characterization of a new and highly active cold-adapted xylanase from the GH-family 10 (Xyl-L), and the use of a novel one step combined directed evolution technique that comprises saturation mutagenesis and focused epPCR as a feasible semi-rational strategy to improve the thermostability. The Xyl-L enzyme was cloned from a marine-Antarctic bacterium, Psychrobacter sp. strain 2-17, recombinantly expressed in E. coli strain BL21(DE3) and characterized enzymatically. Molecular dynamic simulations using a homology model of the catalytic domain of Xyl-L were performed to detect flexible regions and residues, which are considered to be the possible structural elements that define the thermolability of this enzyme. Mutagenic libraries were designed in order to stabilize the protein introducing mutations in some of the flexible regions and residues identified. Twelve positive mutant clones were found to improve the T 50 15 value of the enzyme, in some cases without affecting the activity at 25°C. The best mutant showed a 4.3°C increase in its T 50 15 . The efficiency of the directed evolution approach can also be expected to work in the protein engineering of stereoselectivity. Copyright © 2017 Elsevier Inc. All rights reserved.

Effect of exogenous xylanase, amylase, and protease as single or combined activities on nutrient digestibility and growth performance of broilers fed corn/soy diets

PubMed Central

Amerah, A. M.; Romero, L. F.; Awati, A.; Ravindran, V.

2017-01-01

Abstract Two trials (a 42-d performance and a 21-d cohort digestibility) were conducted to evaluate the performance and nutrient digestibility of broilers fed corn diets supplemented with exogenous xylanase, amylase, and protease as single or combined activities. A nutritionally adequate, positive control (PC) diet was formulated. The negative control (NC) diet was formulated to be lower in metabolizable energy (∼86 kcal/kg diet) and digestible amino acids (1 to 2%) compared to PC. The other 4 treatments were based on the NC and they were either supplemented with xylanase (X), amylase (A), protease (P), or a combination of X, A, and P (XAP; to provide 2,000 U of X, 200 U of A, and 4,000 U of P/kg diet). All diets were marginal in AvP and Ca and contained a background of phytase (1,000 FTU/kg). In each trial, male broiler (Ross 308) chicks were allocated to the 5 treatments (10 replicates of 20 birds/pen and 9 replicates of 8 birds/cage for the performance and digestibility trials, respectively). In the digestibility trial, ileal digesta was collected on d21 for the determination of nutrient utilization. Data were subjected to one-way ANOVA and means were separated by Tukey's HSD test. Only the XAP improved (P < 0.05) AMEn compared to NC. X, A or XAP improved (P < 0.05) N digestibility and apparent ileal digestible energy (AIDE). Both P and XAP improved N retention. The relative improvement in energy digestibility due to enzyme supplementation was greater at the ileal level than that measured in the excreta. The measured changes on AIDE due to supplemental enzymes were much higher than the sum of calculated contributions from starch, fat, and protein. Supplementation of all enzymes reduced (P < 0.05) ileal flow of soluble rhamnose and mannose relative to NC. In the performance trial, both X and XAP improved (P < 0.05) weight gain (WG) and only XAP improved (P < 0.05) FCR compared to NC during the starter phase (1-21d). Over the entire period (1–42d), WG

Improving the temperature characteristics and catalytic efficiency of a mesophilic xylanase from Aspergillus oryzae, AoXyn11A, by iterative mutagenesis based on in silico design.

PubMed

Li, Xue-Qing; Wu, Qin; Hu, Die; Wang, Rui; Liu, Yan; Wu, Min-Chen; Li, Jian-Fang

2017-12-01

To improve the temperature characteristics and catalytic efficiency of a glycoside hydrolase family (GHF) 11 xylanase from Aspergillus oryzae (AoXyn11A), its variants were predicted based on in silico design. Firstly, Gly 21 with the maximum B-factor value, which was confirmed by molecular dynamics (MD) simulation on the three-dimensional structure of AoXyn11A, was subjected to site-saturation mutagenesis. Thus, one variant with the highest thermostability, AoXyn11A G21I , was selected from the mutagenesis library, E. coli/Aoxyn11A G21X (X: any one of 20 amino acids). Secondly, based on the primary structure multiple alignment of AoXyn11A with seven thermophilic GHF11 xylanases, AoXyn11A Y13F or AoXyn11A G21I-Y13F , was designed by replacing Tyr 13 in AoXyn11A or AoXyn11A G21I with Phe. Finally, three variant-encoding genes, Aoxyn11A G21I , Aoxyn11A Y13F and Aoxyn11A G21I-Y13F , were constructed by two-stage whole-plasmid PCR method, and expressed in Pichia pastoris GS115, respectively. The temperature optimum (T opt ) of recombinant (re) AoXyn11A G21I-Y13F was 60 °C, being 5 °C higher than that of reAoXyn11A G21I or reAoXyn11A Y13F , and 10 °C higher than that of reAoXyn11A. The thermal inactivation half-life (t 1/2 ) of reAoXyn11A G21I-Y13F at 50 °C was 240 min, being 40-, 3.4- and 2.5-fold longer than those of reAoXyn11A, reAoXyn11A G21I and reAoXyn11A Y13F . The melting temperature (T m ) values of reAoXyn11A, reAoXyn11A G21I , reAoXyn11A Y13F and reAoXyn11A G21I-Y13F were 52.3, 56.5, 58.6 and 61.3 °C, respectively. These findings indicated that the iterative mutagenesis of both Gly21Ile and Tyr13Phe improved the temperature characteristics of AoXyn11A in a synergistic mode. Besides those, the catalytic efficiency (k cat /K m ) of reAoXyn11A G21I-Y13F was 473.1 mL mg -1 s -1 , which was 1.65-fold higher than that of reAoXyn11A.

Highly thermo-halo-alkali-stable β-1,4-endoxylanase from a novel polyextremophilic strain of Bacillus halodurans.

PubMed

Kumar, Vikash; Syal, Poonam; Satyanarayana, T

2013-05-01

A novel bacterial isolate, capable of producing extracellular highly thermostable, halo-alkali-stable and cellulase-free xylanase, was isolated from soil and identified as Bacillus halodurans TSPV1 by polyphasic approach. The Plackett-Burman design identified wheat bran, lactose, tryptone and NaCl as the factors that significantly affect xylanase production, and thus, these were optimized by response surface methodology. The data analysis suggested that optimum levels of wheat bran (15-20 g L(-1)), lactose (1.0-1.5 g L(-1)), tryptone (2-2.5 g L(-1)) and NaCl (7.0-8.0 g L(-1)) support 6.75-fold higher xylanase production than that in the un-optimized medium. The xylanase is optimally active at 90 °C and pH 10, and stable for 4 h at 90 °C (T 1/2 60 h) over a broad range of NaCl concentrations (0-29 %). This is the first report on the isolation of polyextremophilic B. halodurans strain that produces thermo-halo-alkali-stable xylanase in submerged fermentation. This enzyme efficiently saccharifies agro residues like wheat bran and corncobs. Fifty-six percent of hemicellulose of wheat bran could be hydrolyzed by xylanase (100 U g(-1) substrate) along with cellulase (22 U FPase and 50 U CMCase g(-1)). The xylanase, being thermo-alkali stable and cellulase free, can find applications in pre-bleaching of paper pulps and hydrolysis of xylan in agricultural residues.

Production of xylanolytic enzymes by Aspergillus terricola in stirred tank and airlift tower loop bioreactors.

PubMed

Michelin, Michele; Polizeli, Maria de Lourdes Teixeira de Moraes; Silva, Daniel Pereira da; Ruzene, Denise Santos; Vicente, António Augusto; Jorge, João Atílio; Terenzi, Héctor Francisco; Teixeira, José António

2011-12-01

Fungi producing high xylanase levels have attracted considerable attention because of their potential industrial applications. Batch cultivations of Aspergillus terricola fungus were evaluated in stirred tank and airlift bioreactors, by using wheat bran particles suspended in the cultivation medium as substrate for xylanase and β-xylosidase production. In the stirred tank bioreactor, in physical conditions of 30°C, 300 rpm, and aeration of 1 vvm (1 l min⁻¹), with direct inoculation of fungal spores, 7,475 U l⁻¹ xylanase was obtained after 36 h of operation, remaining constant after 24 h. In the absence of air injection in the stirred tank reactor, limited xylanase production was observed (final concentration 740 U l⁻¹). When the fermentation process was realized in the airlift bioreactor, xylanase production was higher than that observed in the stirred tank bioreactor, being 9,265 U l⁻¹ at 0.07 vvm (0.4 l min⁻¹) and 12,845 U l⁻¹ at 0.17 vvm (1 l min⁻¹) aeration rate.

Nucleo-cytoplasmic shuttling dynamics of the transcriptional regulators XYR1 and CRE1 under conditions of cellulase and xylanase gene expression in Trichoderma reesei

PubMed Central

Lichius, Alexander; Seidl-Seiboth, Verena; Seiboth, Bernhard; Kubicek, Christian P

2014-01-01

Trichoderma reesei is a model for investigating the regulation of (hemi-)cellulase gene expression. Cellulases are formed adaptively, and the transcriptional activator XYR1 and the carbon catabolite repressor CRE1 are main regulators of their expression. We quantified the nucleo-cytoplasmic shuttling dynamics of GFP-fusion proteins of both transcription factors under cellulase and xylanase inducing conditions, and correlated their nuclear presence/absence with transcriptional changes. We also compared their subcellular localization in conidial germlings and mature hyphae. We show that cellulase gene expression requires de novo biosynthesis of XYR1 and its simultaneous nuclear import, whereas carbon catabolite repression is regulated through preformed CRE1 imported from the cytoplasmic pool. Termination of induction immediately stopped cellulase gene transcription and was accompanied by rapid nuclear degradation of XYR1. In contrast, nuclear CRE1 rapidly decreased upon glucose depletion, and became recycled into the cytoplasm. In mature hyphae, nuclei containing activated XYR1 were concentrated in the colony center, indicating that this is the main region of XYR1 synthesis and cellulase transcription. CRE1 was found to be evenly distributed throughout the entire mycelium. Taken together, our data revealed novel aspects of the dynamic shuttling and spatial bias of the major regulator of (hemi-)cellulase gene expression, XYR1, in T. reesei. PMID:25302561

Isolation, screening, and identification of potential cellulolytic and xylanolytic producers for biodegradation of untreated oil palm trunk and its application in saccharification of lemongrass leaves.

PubMed

Ang, S K; Yahya, Adibah; Abd Aziz, Suraini; Md Salleh, Madihah

2015-01-01

This study presents the isolation and screening of fungi with excellent ability to degrade untreated oil palm trunk (OPT) in a solid-state fermentation system (SSF). Qualitative assay of cellulases and xylanase indicates notable secretion of both enzymes by 12 fungal strains from a laboratory collection and 5 strains isolated from a contaminated wooden board. High production of these enzymes was subsequently quantified in OPT in SSF. Aspergillus fumigates SK1 isolated from cow dung gives the highest xylanolytic activity (648.448 U g(-1)), generally high cellulolytic activities (CMCase: 48.006, FPase: 6.860, beta-glucosidase: 16.328 U g(-1)) and moderate lignin peroxidase activity (4.820 U/g), and highest xylanolytic activity. The xylanase encoding gene of Aspergillus fumigates SK1 was screened using polymerase chain reaction by a pair of degenerate primers. Through multiple alignment of the SK1 strain's xylanase nucleotide sequences with other published xylanases, it was confirmed that the gene belonged to the xylanase glycoside hydrolase family 11 (GH11) with a protein size of 24.49 kD. Saccharification of lemongrass leaves using crude cellulases and xylanase gives the maximum reducing sugars production of 6.84 g/L with glucose as the major end product and traces of phenylpropanic compounds (vanillic acid, p-coumaric acid, and ferulic acid).

Characterization and identification of the xylanolytic enzymes from Aspergillus fumigatus Z5.

PubMed

Miao, Youzhi; Li, Juan; Xiao, Zhizhuang; Shen, Qirong; Zhang, Ruifu

2015-06-23

Plant biomass, the most abundant natural material on earth, represents a vast source of food and energy in nature. As the main component of plant biomass, xylan is a complex polysaccharide comprising a linear β(1,4)-linked backbone of xylosyl residues substituted by acetyl, arabinosyl, glucuronysyl and 4-O-methylglucuronycyl residues. Aspergillus fumigatus Z5 is an efficient plant biomass depolymerization fungus. In this study, its crude xylanolytic enzymes were characterized and identified by two-dimensional gel electrophoresis (2-DE). The optimal temperature for the crude xylanases was close to 60 °C, the highest xylanase activity was achieved at pH ranged from 3 to 6, and the crude xylanases also showed a very broad region of pH (3-11) stability. The maximal xylanase activity of 21.45 U · ml(-1) was observed in the fourth day of cultivation at 50 °C and 150 rpm with 2 % xylan as the sole carbon source. Zymogram analysis indicated that there were more than seven secreted proteins with xylanase activity. In the crude enzyme, two major endoxylanases, five cellulases and several associated enzymes were identified to be involved in the hydrolysis of polysaccharides. Of the total 13 xylanase genes in the Z5 genome, 11 were observed using q-PCR to be induced by xylan, one of which, An endo-1,4-β-xylanase with a low secretion level, was also expressed and characterized. The final hydrolysis products of xylan by crude enzyme mainly consisted of xylobiose. This study provides a comprehensive understanding of the depolymerization of xylan by Z5 and will help to design enzymatic strategies for plant biomass utilization.

Phenolic acid esterases, coding sequences and methods

DOEpatents

Blum, David L.; Kataeva, Irina; Li, Xin-Liang; Ljungdahl, Lars G.

2002-01-01

Described herein are four phenolic acid esterases, three of which correspond to domains of previously unknown function within bacterial xylanases, from XynY and XynZ of Clostridium thermocellum and from a xylanase of Ruminococcus. The fourth specifically exemplified xylanase is a protein encoded within the genome of Orpinomyces PC-2. The amino acids of these polypeptides and nucleotide sequences encoding them are provided. Recombinant host cells, expression vectors and methods for the recombinant production of phenolic acid esterases are also provided.

Screening and characterizing of xylanolytic and xylose-fermenting yeasts isolated from the wood-feeding termite, Reticulitermes chinensis

PubMed Central

Xie, Rongrong; Zhou, Feng; Huang, Miao

2017-01-01

The effective fermentation of xylose remains an intractable challenge in bioethanol industry. The relevant xylanase enzyme is also in a high demand from industry for several biotechnological applications that inevitably in recent times led to many efforts for screening some novel microorganisms for better xylanase production and fermentation performance. Recently, it seems that wood-feeding termites can truly be considered as highly efficient natural bioreactors. The highly specialized gut systems of such insects are not yet fully realized, particularly, in xylose fermentation and xylanase production to advance industrial bioethanol technology as well as industrial applications of xylanases. A total of 92 strains from 18 yeast species were successfully isolated and identified from the gut of wood-feeding termite, Reticulitermes chinensis. Of these yeasts and strains, seven were identified for new species: Candida gotoi, Candida pseudorhagii, Hamamotoa lignophila, Meyerozyma guilliermondii, Sugiyamaella sp.1, Sugiyamaella sp. 2, and Sugiyamaella sp.3. Based on the phylogenetic and phenotypic characterization, the type strain of C. pseudorhagii sp. nov., which was originally designated strain SSA-1542T, was the most frequently occurred yeast from termite gut samples, showed the highly xylanolytic activity as well as D-xylose fermentation. The highest xylanase activity was recorded as 1.73 and 0.98 U/mL with xylan or D-xylose substrate, respectively, from SSA-1542T. Among xylanase-producing yeasts, four novel species were identified as D-xylose-fermenting yeasts, where the yeast, C. pseudorhagii SSA-1542T, showed the highest ethanol yield (0.31 g/g), ethanol productivity (0.31 g/L·h), and its fermentation efficiency (60.7%) in 48 h. Clearly, the symbiotic yeasts isolated from termite guts have demonstrated a competitive capability to produce xylanase and ferment xylose, suggesting that the wood-feeding termite gut is a promising reservoir for novel xylanases

Effect of exogenous xylanase, amylase, and protease as single or combined activities on nutrient digestibility and growth performance of broilers fed corn/soy diets.

PubMed

Amerah, A M; Romero, L F; Awati, A; Ravindran, V

2017-04-01

Two trials (a 42-d performance and a 21-d cohort digestibility) were conducted to evaluate the performance and nutrient digestibility of broilers fed corn diets supplemented with exogenous xylanase, amylase, and protease as single or combined activities. A nutritionally adequate, positive control (PC) diet was formulated. The negative control (NC) diet was formulated to be lower in metabolizable energy (∼86 kcal/kg diet) and digestible amino acids (1 to 2%) compared to PC. The other 4 treatments were based on the NC and they were either supplemented with xylanase (X), amylase (A), protease (P), or a combination of X, A, and P (XAP; to provide 2,000 U of X, 200 U of A, and 4,000 U of P/kg diet). All diets were marginal in AvP and Ca and contained a background of phytase (1,000 FTU/kg). In each trial, male broiler (Ross 308) chicks were allocated to the 5 treatments (10 replicates of 20 birds/pen and 9 replicates of 8 birds/cage for the performance and digestibility trials, respectively). In the digestibility trial, ileal digesta was collected on d21 for the determination of nutrient utilization. Data were subjected to one-way ANOVA and means were separated by Tukey's HSD test. Only the XAP improved (P < 0.05) AMEn compared to NC. X, A or XAP improved (P < 0.05) N digestibility and apparent ileal digestible energy (AIDE). Both P and XAP improved N retention. The relative improvement in energy digestibility due to enzyme supplementation was greater at the ileal level than that measured in the excreta. The measured changes on AIDE due to supplemental enzymes were much higher than the sum of calculated contributions from starch, fat, and protein. Supplementation of all enzymes reduced (P < 0.05) ileal flow of soluble rhamnose and mannose relative to NC. In the performance trial, both X and XAP improved (P < 0.05) weight gain (WG) and only XAP improved (P < 0.05) FCR compared to NC during the starter phase (1-21d). Over the entire period (1-42d), WG and FI

Heat and pH stability of alkali-extractable corn arabinoxylan and its xylanase-hydrolyzate and their viscosity behavior.

PubMed

Rumpagaporn, Pinthip; Kaur, Amandeep; Campanella, Osvaldo H; Patterson, John A; Hamaker, Bruce R

2012-01-01

In in vitro batch fermentations, both alkali-extractable corn arabinoxylan (CAX) and its xylanase-hydrolyzate (CH) were utilized by human fecal microbiota and produced similar short chain fatty acid (SCFA) contents and desirable long fermentation profiles with low initial gas production. Fortification of these arabinoxylans into processed foods would contribute desirable dietary fiber benefits to humans. Heat and pH stability, as well as viscosity behavior of CAX and CH were investigated. Size exclusion chromatography was used to analyze the molecular size distribution after treatment at different pH's and heating temperatures for different time periods. Treated under boiling and pressure cooking conditions at pH 3, CAX was degraded to a smaller molecular size, whereas the molecular size of the CH showed only a minor decrease. CAX and CH were mostly stable at neutral pH, except when CAX was treated under pressure for 60 min that slightly lowered molecular size. At 37 °C, neither CAX nor CH was adversely affected by treatment at low or neutral pH. The viscosities of solutions containing 5% and 10% of CAX were 48.7 and 637.0 mPa.s, respectively that were higher than those of solutions containing 5% and 10% of its hydrolyzate at shear rate 1 s⁻¹. The CAX solutions showed Newtonian flow behavior, whereas shear-thinning behavior was observed in CH solutions. In conclusion, the hydrolyzate of CAX has potential to be used in high fiber drinks due to its favorable fermentation properties, higher pH and heat stability, lower and shear-thinning viscosity, and lighter color than the native CAX. Arabinoxylan extracted by an alkali from corn bran is a soluble fiber with a desirable low initial and extended fermentation property. Corn arabinoxylan hydrolyzate using an endoxylanase was much more stable at different levels of acidity and heat than the native arabinoxylan, and showed lower solution viscosity and shear-thinning property that indicates its potential as an

Influence of partial replacement of ground wheat with whole wheat and exogenous enzyme supplementation on growth performance, nutrient digestibility and energy utilization in young broilers.

PubMed

Abdollahi, M R; Ravindran, V; Amerah, A M

2016-10-01

This study investigated the influence of pre-pelleting inclusion of whole wheat (WW) and exogenous enzyme supplementation on growth performance, coefficient of apparent ileal nutrient digestibility (CAID) and apparent metabolizable energy (AME) in broilers fed wheat-based pelleted diets. A 2 × 3 factorial arrangement of treatments was used with two methods of wheat inclusion [622 g/kg ground wheat (GW) and 250 g/kg WW replaced GW (wt/wt) pre-pelleting (PWW)] and three enzymes (xylanase, phytase and xylanase plus phytase). A total of 288, one-day-old male broilers (Ross 308) were individually weighed and allocated to 36 cages (8 broilers/cage), and the cages were randomly assigned to the six dietary treatments. Birds fed PWW diets gained more (p < 0.05) weight than those fed GW diets. There was no effect (p > 0.05) of WW inclusion on feed intake (FI). Phytase alone increased (p < 0.05) FI compared to xylanase or the combination. Whole wheat inclusion increased (p < 0.05) the gain-to-feed ratio (G:F). Feeding xylanase plus phytase and phytase-added diets resulted in the greatest and lowest G:F, respectively, with xylanase supplemented diets being intermediate. Birds fed PWW diets had greater (p < 0.05) relative gizzard weights than those fed GW diets. There was no effect (p > 0.05) of WW inclusion on the CAID of nitrogen (N), starch and fat. Combination of xylanase and phytase resulted in greater (p < 0.05) digestibility of N, starch and fat than that of individual additions. Feeding PWW diets resulted in greater (p < 0.05) AME values than GW diets. Combination of xylanase and phytase increased (p < 0.05) the AME compared to the diets with individual additions of xylanase or phytase. The current results suggest that the influence of pre-pelleting WW inclusion and exogenous enzymes on nutrient digestibility and broiler performance is not additive. Journal of Animal Physiology and Animal Nutrition © 2016 Blackwell Verlag GmbH.

Mode of action of family 10 and 11 endoxylanases on water-unextractable arabinoxylan.

PubMed

Vardakou, Maria; Katapodis, Petros; Samiotaki, Martina; Kekos, Dimitris; Panayotou, George; Christakopoulos, Paul

2003-11-01

Microbial endo-beta-1,4-xylanases (EXs, EC 3.2.1.8) belonging to glycanase families 10 and 11 differ in their action on water-unextractable arabinoxylan (WU-AX). WU-AX was incubated with different levels of a Thermoascus aurantiacus family 10 and a Sporotrichum thermophile family 11 endoxylanases. At 10 g l(-1) arabinoxylan, enzyme concentrations (KE values) needed to obtain half-maximal hydrolysis rates (V(max) values) were 4.4 nM for the xylanase from T. aurantiacus and 7.1 nM for the xylanase from S. thermophile. Determination of Vmax/KE revealed that the family 10 enzyme hydrolysed two times more efficiently WU-AX than the family 11 enzyme. Molecular weights of the products formed were assessed and separation of feruloyl-oligosaccharides was achieved by anion-exchange and size-exclusion chromatography (SEC). The main difference between the feruloylated products by xylanases of family 10 and 11 concerned the length of the products containing feruloyl-arabinosyl substitution. The xylanase from T. aurantiacus liberated from WU-AX a feruloyl arabinoxylodisaccharide (FAX2) as the shortest feruloylated fragment in contrast with the enzyme from S. thermophile, which liberated a feruloyl arabinoxylotrisaccharide (FAX3). These results indicated that different factors govern WU-AX breakdown by the two endoxylanases.

Impact of orientation of carbohydrate binding modules family 22 and 6 on the catalytic activity of Thermotoga maritima xylanase XynB.

PubMed

Tajwar, Razia; Shahid, Saher; Zafar, Rehan; Akhtar, Muhammad Waheed

2017-11-01

Xylanase XynB of the hyperthermophile Thermotoga maritima, which belongs to glycoside hydrolase family 10 (GH10), does not have an associated carbohydrate binding module (CBM) in the native state. CBM6 and CBM22 from a thermophile Clostridium thermocellum were fused to the catalytic domain of XynB (XynB-C) to determine the effects on activity and other properties. XynB-B22C and XynB-CB22, produced by fusing CBM22 to the N- and C-terminal of XynB-C, showed 1.7- and 3.24-fold increase in activity against the insoluble birchwood xylan, respectively. Similarly, CBM6 when attached to the C-terminal of XynB-C resulted in 2.0-fold increase in activity, whereas its attachment to the N-terminal did not show any increase of activity. XynB-B22C and XynB-CB22 retained all the activity, whereas XynB-B6C and XynB-CB6 lost 17 and 11% of activity, respectively, at 60°C for 4h. Thermostability data and the secondary structure contents obtained by molecular modelling are in agreement with the data from circular dichroism analysis. Molecular modelling analysis showed that the active site residues of the catalytic domain and the binding residues of CBM6 and CBM22 were located on the surface of molecule, except XynB-B6C, where the binding residues were found somewhat buried. In the case of XynB-CB22, the catalytic and the binding residues seem to be located favorably adjacent to each other, thus showing higher increase in activity. This study shows that the active site residues of the catalytic domain and the binding residues of the CBM are arranged in a unique fashion, not reported before. Copyright © 2017 Elsevier Inc. All rights reserved.

Investigation of enzyme formulation on pretreated switchgrass.

PubMed

Falls, Matthew; Shi, Jian; Ebrik, Mirvat A; Redmond, Tim; Yang, Bin; Wyman, Charles E; Garlock, Rebecca; Balan, Venkatesh; Dale, Bruce E; Pallapolu, V Ramesh; Lee, Y Y; Kim, Youngmi; Mosier, Nathan S; Ladisch, Michael R; Hames, Bonnie; Thomas, Steve; Donohoe, Bryon S; Vinzant, Todd B; Elander, Richard T; Warner, Ryan E; Sierra-Ramirez, Rocio; Holtzapple, Mark T

2011-12-01

This work studied the benefits of adding different enzyme cocktails (cellulase, xylanase, β-glucosidase) to pretreated switchgrass. Pretreatment methods included ammonia fiber expansion (AFEX), dilute-acid (DA), liquid hot water (LHW), lime, lime+ball-milling, soaking in aqueous ammonia (SAA), and sulfur dioxide (SO(2)). The compositions of the pretreated materials were analyzed and showed a strong correlation between initial xylan composition and the benefits of xylanase addition. Adding xylanase dramatically improved xylan yields for SAA (+8.4%) and AFEX (+6.3%), and showed negligible improvement (0-2%) for the pretreatments with low xylan content (dilute-acid, SO(2)). Xylanase addition also improved overall yields with lime+ball-milling and SO(2) achieving the highest overall yields from pretreated biomass (98.3% and 93.2%, respectively). Lime+ball-milling obtained an enzymatic yield of 92.3kg of sugar digested/kg of protein loaded. Copyright © 2011 Elsevier Ltd. All rights reserved.

An ascomycota coculture in batch bioreactor is better than polycultures for cellulase production.

PubMed

Hernández, Christian; Milagres, Adriane M F; Vázquez-Marrufo, Gerardo; Muñoz-Páez, Karla María; García-Pérez, José Antonio; Alarcón, Enrique

2018-07-01

Efficient hydrolysis of holocellulose depends on a proper balance between cellulase (endoglucanase, exoglucanase, β-glucosidase) and xylanase activities. The present study aimed to induce the production of cellulases and xylanases using liquid cultures (one, two, three, and four fungal strains on the same bioreactor) of wild strains of Trichoderma harzianum, Aspergillus niger, and Fusarium oxysporum. The strains were identified by amplification and analysis of the ITS rDNA region and the obtained sequences were deposited in Genbank. Enzymes (endoglucanase, exoglucansae, β-glucosidase, and xylanase activities) and the profile of extracellular protein isoforms (SDS-PAGE) produced by different fungal combinations (N = 14) were analyzed by Pearson's correlation matrix and principal component analysis (PCA). According to our results, induction of endoglucanase (19.02%) and β-glucosidase (6.35%) were obtained after 4 days when A. niger and F. oxysporum were cocultured. The combination of A. niger-T. harzianum produced higher endoglucanase in a shorter time than monocultures. On the contrary, when more than two strains were cultured in the same reactor, the relationships of competition were established, trending to diminish the amount of enzymes and the extracellular protein isoforms produced. The xylanase production was sensible to stress produced by mixed cultures, decreasing their activity. This is important when the aim is to produce cellulase-free xylanase. In addition, exoglucanase activity did not change in the combinations tested.

Feeding value of field beans (Vicia faba L. var. minor) with and without enzyme containing tannase, pectinase and xylanase activities for broilers.

PubMed

Abdulla, Jalil Mahmwd; Rose, Stephen Paul; Mackenzie, Alexander Mackay; Pirgozliev, Vasil Radoslavov

2017-04-01

Effects of field beans with various tannin content and exogenous enzyme mixture containing tannase, pectinase and xylanase activities on N-corrected dietary apparent metabolisable energy (AMEn), coefficients of dry matter (DMR) and nitrogen retention (NR), fat digestibility, gastrointestinal tract (GIT) development, jejunal villus morphometry, ileal digesta viscosity and sialic acid were examined. Birds' growth performance and energy conversion ratio (ECR) were also measured. Birds were fed one of eight mash diets. The Control diet contained as major ingredients wheat (400 g/kg) and soybean meal (SBM) (127 g/kg and 221 g crude protein/kg and 12.83 MJ AMEn/kg. To reduce nutrient density, the Control diet also contained washed sand at 119 g/kg. Another three diets containing 300 g/kg of each of three experimental field bean cultivar samples in replacement for SBM and sand were also mixed. Each diet was fed to nine pens with two male Ross 308 broilers. Diets high in tannin had low AMEn, ECR, DMR and NR (p < 0.001). Feeding field beans increased (p < 0.001) the weights of the pancreas and the proventriculus and gizzard (PG) of the birds. Supplementing diets with the enzyme mixture improved (p < 0.001) feed conversion efficiency, AMEn and all nutrient utilisation coefficients despite the tannins in diets. The enzyme mixture reduced ileal digesta viscosity (p < 0.001) and the weight of pancreas, total GIT and PG (p < 0.05) of the birds. It can be concluded that the feeding value of field beans with different tannin contents may vary when fed to broilers. The supplementation of the enzyme mixture improved the feeding value of diets for broilers. The beneficial effect of the addition of the enzyme mixture seems to be mediated through reduced ileal digesta viscosity and improved nutrient availability.

An additional aromatic interaction improves the thermostability and thermophilicity of a mesophilic family 11 xylanase: structural basis and molecular study.

PubMed Central

Georis, J.; de Lemos Esteves, F.; Lamotte-Brasseur, J.; Bougnet, V.; Devreese, B.; Giannotta, F.; Granier, B.; Frère, J. M.

2000-01-01

In a general approach to the understanding of protein adaptation to high temperature, molecular models of the closely related mesophilic Streptomyces sp. S38 Xyl1 and thermophilic Thermomonospora fusca TfxA family 11 xylanases were built and compared with the three-dimensional (3D) structures of homologous enzymes. Some of the structural features identified as potential contributors to the higher thermostability of TfxA were introduced in Xyl1 by site-directed mutagenesis in an attempt to improve its thermostability and thermophilicity. A new Y11-Y16 aromatic interaction, similar to that present in TfxA and created in Xyl1 by the T11Y mutation, improved both the thermophilicity and thermostability. Indeed, the optimum activity temperature (70 vs. 60 degrees C) and the apparent Tm were increased by about 9 degrees C, and the mutant was sixfold more stable at 57 degrees C. The combined mutations A82R/F168H/N169D/delta170 potentially creating a R82-D169 salt bridge homologous to that present in TfxA improved the thermostability but not the thermophilicity. Mutations R82/D170 and S33P seemed to be slightly destabilizing and devoid of influence on the optimal activity temperature of Xyl1. Structural analysis revealed that residues Y11 and Y16 were located on beta-strands B1 and B2, respectively. This interaction should increase the stability of the N-terminal part of Xyl1. Moreover, Y11 and Y16 seem to form an aromatic continuum with five other residues forming putative subsites involved in the binding of xylan (+3, +2, +1, -1, -2). Y11 and Y16 might represent two additional binding subsites (-3, -4) and the T11Y mutation could thus improve substrate binding to the enzyme at higher temperature and thus the thermophilicity of Xyl1. PMID:10752608

Enhancement of Penicillium echinulatum glycoside hydrolase enzyme complex.

PubMed

dos Santos Costa, Patrícia; Büchli, Fernanda; Robl, Diogo; Delabona, Priscila da Silva; Rabelo, Sarita Candida; Pradella, José Geraldo da Cruz

2016-05-01

The enhancement of enzyme complex produced by Penicillium echinulatum grown in several culture media components (bagasse sugarcane pretreated by various methods, soybean meal, wheat bran, sucrose, and yeast extract) was studied to increment FPase, xylanase, pectinase, and β-glucosidase enzyme activities. The present results indicated that culture media composed with 10 g/L of the various bagasse pretreatment methods did not have any substantial influence with respect to the FPase, xylanase, and β-glucosidase attained maximum values of, respectively, 2.68 FPU/mL, 2.04, and 115.4 IU/mL. On the other hand, proposed culture media to enhance β-glucosidase production composed of 10 g/L steam-exploded bagasse supplemented with soybean flour 5.0 g/L, yeast extract 1.0 g/L, and sucrose 10.0 g/L attained, respectively, 3.19 FPU/mL and 3.06 IU/mL while xylanase was maintained at the same level. The proteomes obtained from the optimized culture media for enhanced FPase, xylanase, pectinase, and β-glucosidase production were analyzed using mass spectrometry and a panel of GH enzyme activities against 16 different substrates. Culture medium designed to enhance β-glucosidase activity achieved higher enzymatic activities values (13 measured activities), compared to the culture media for FPase/pectinase (9 measured activities) and xylanase (7 measured activities), when tested against the 16 substrates. Mass spectrometry analyses of secretome showed a consistent result and the greatest number of spectral counts of Cazy family enzymes was found in designed β-glucosidase culture medium, followed by FPase/pectinase and xylanase. Most of the Cazy identified protein was cellobiohydrolase (GH6 and GH7), endoglucanase (GH5), and endo-1,4-β-xylanase (GH10). Enzymatic hydrolysis of hydrothermally pretreated sugarcane bagasse performed with β-glucosidase enhanced cocktail achieved 51.4 % glucose yield with 10 % w/v insoluble solids at enzyme load of 15 FPU/g material. Collectively the

Starch digestibility, energy utilization, and growth performance of broilers fed corn-soybean basal diets supplemented with enzymes.

PubMed

Stefanello, C; Vieira, S L; Santiago, G O; Kindlein, L; Sorbara, J O B; Cowieson, A J

2015-10-01

A study was conducted to evaluate the effects of dietary α-amylase and β-xylanase supplementation of corn-soy diets, formulated with or without supplemental phytase, on growth performance, energy utilization, and starch digestibility in broiler chickens. A total of 336 slow-feathering, Cobb × Cobb 500 male broilers were randomly distributed to 6 treatments having 8 replicates of 7 birds each. Birds were fed a common starter diet to d 14 post-hatch (3,050 kcal/kg AMEn, 21.7% CP, 1.05% Ca, and 0.53% nPP). The experimental diets were provided afterwards until d 25. A 2 × 3 factorial arrangement of 2 control diets (basal = corn-soy diet without added phytase or PHY = corn-soy diet formulated with 1,000 phytase units/kg) and 3 carbohydrase supplementations (0, 80 kilo-Novo α-amylase units/kg, or 80 kilo-Novo α-amylase units/kg + 100 fungal β-xylanase units/kg) was used from d 14 to 25. Excreta were collected from 21 to 24 d and all birds were euthanized at 25 d for jejunum and ileum content collection. Samples of feed, excreta, and jejunal and ileal digesta were analyzed for determination of total tract retention and ileal apparent digestibility. No interactions between diet and carbohydrase were observed. Broilers fed diets formulated with phytase or supplemented with amylase + xylanase had higher BW gain (BWG) and lower FCR (P < 0.05) when compared with birds fed diets without carbohydrases. Relative to the basal diet, AMEn was increased (P < 0.01) by 70 kcal/kg and 99 kcal/kg when birds were fed the diet supplemented with amylase and amylase + xylanase, respectively. Starch digestibility in the jejunum and ileum was increased (P < 0.05) by 3.5% and 2.4%, respectively, when birds were fed the diet supplemented with amylase + xylanase. Results from this experiment show that corn-soy diets having phytase and supplemented with amylase and xylanase led to increased growth performance, AMEn, and starch digestibility in broilers. Furthermore, the efficacy of

Next-generation non-starch polysaccharide-degrading, multi-carbohydrase complex rich in xylanase and arabinofuranosidase to enhance broiler feed digestibility.

PubMed

Cozannet, Pierre; Kidd, Michael T; Montanhini Neto, Roberto; Geraert, Pierre-André

2017-08-01

This study was carried out to evaluate the effect of a multi-carbohydrase complex (MCC) rich in xylanase (Xyl) and arabinofuranosidase (Abf) on overall broiler feed digestibility in broilers. Energy utilization and digestibility of dry matter (DM), organic matter (OM), protein, starch, fat, and insoluble and soluble fibers were measured using the mass-balance method. The experiment was carried out on 120 broilers (3-week-old chickens). Broilers were distributed over 8 treatments to evaluate the effect of the dietary arabinoxylan content and nutrient density with and without MCC (Rovabio® Advance). The graded content of arabinoxylan (AX) was obtained using different raw materials (wheat, rye, barley, and dried distillers' wheat). Diet-energy density was modified with added fat. Measurements indicated that nutrient density and AX content had a significant effect on most digestibility parameters. Apparent metabolizable energy (AME) was significantly increased (265 kcal kg-1) by MCC. The addition of MCC also resulted in significant improvement in the digestibility of all evaluated nutrients, with average improvements of 3.0, 3.3, 3.2, 3.0, 6.2, 2.9, 5.8, and 3.8% units for DM, OM, protein, starch, fat, insoluble and soluble fibers, and energy utilization, respectively. The interaction between MCC and diet composition was significant for the digestibility of OM, fat, protein, and energy. Nutrient digestibility and diet AME were negatively correlated with AX content (P < 0.001). However, the addition of MCC resulted in a reduction of this negative effect (P < 0.001). The AME of diets with and without the addition of MCC were successfully predicted by the diet digestible nutrient (i.e., starch, protein, fat, insoluble and soluble fibers) content with and without MCC (R2 = 0.87; RSD = 78 kcal kg-1). This study confirms that the presence of AX in wheat-based diets and wheat-based diets with other cereals and cereal by-products reduces nutrient digestibility in broiler

Effects of enzymes to improve sensory quality of frozen dough bread and analysis on its mechanism.

PubMed

Wang, Xuan; Pei, Dudu; Teng, Yuefei; Liang, Jianfen

2018-01-01

Baking quality of frozen dough is negatively affected by dough weakening and by a reduction in both yeast viability and activity during freezing and frozen storage. The objective of this study was to investigate effects of different enzymes, such as α-amylase, xylanase, celluase, glucose oxidase, and lipase on the texture and sensory quality of bread after frozen storage, as well as on dough properties, in terms of fermentation characteristics, freezable water contents and microstructure. Except for α-amylase, other enzymes improved the bread sensory quality and got higher overall acceptability, especially xylanase. Dough fermentative behavior showed that the maximum heights of frozen dough were increased by 33.2, 19.7 and 7.4%, respectively with xylanase, cellulase and lipase. Cellulase lowered gas holding ability of dough. Thermodynamic properties indicated that addition of enzyme decreased the freezable water contents in frozen dough. Scanning electronic microscopy revealed that freezing and frozen storage disrupted dough gluten network causing separation of starch granules from the gluten matrix. Inclusion of cellulase, xylanase and lipase made the frozen dough having a more continuous gluten network and smoother surface, and glucose oxidase increased the stability of the gluten work.

Bioprocessing of wheat bran for the production of lignocellulolytic enzyme cocktail by Cotylidia pannosa under submerged conditions.

PubMed

Sharma, Deepika; Garlapat, Vijay Kumar; Goel, Gunjan

2016-04-02

Characterization and production of efficient lignocellulytic enzyme cocktails for biomass conversion is the need for biofuel industry. The present investigation reports the modeling and optimization studies of lignocellulolytic enzyme cocktail production by Cotylidia pannosa under submerged conditions. The predominant enzyme activities of cellulase, xylanase and laccase were produced in the cocktail through submerged conditions using wheat bran as a substrate. A central composite design approach was utilized to model the production process using temperature, pH, incubation time and agitation as input variables with the goal of optimizing the output variables namely cellulase, xylanase and laccase activities. The effect of individual, square and interaction terms on cellulase, xylanase and laccase activities were depicted through the non-linear regression equations with significant R(2) and P-values. An optimized value of 20 U/ml, 17 U/ml and 13 U/ml of cellulase, xylanase and laccase activities, respectively, were obtained with a media pH of 5.0 in 77 h at 31C, 140 rpm using wheatbran as a substrate. Overall, the present study introduces a fungal strain, capable of producing lignocellulolytic enzyme cocktail for subsequent applications in biofuel industry.

Bioprocessing of wheat bran for the production of lignocellulolytic enzyme cocktail by Cotylidia pannosa under submerged conditions

PubMed Central

Sharma, Deepika; Garlapat, Vijay Kumar; Goel, Gunjan

2016-01-01

ABSTRACT Characterization and production of efficient lignocellulytic enzyme cocktails for biomass conversion is the need for biofuel industry. The present investigation reports the modeling and optimization studies of lignocellulolytic enzyme cocktail production by Cotylidia pannosa under submerged conditions. The predominant enzyme activities of cellulase, xylanase and laccase were produced in the cocktail through submerged conditions using wheat bran as a substrate. A central composite design approach was utilized to model the production process using temperature, pH, incubation time and agitation as input variables with the goal of optimizing the output variables namely cellulase, xylanase and laccase activities. The effect of individual, square and interaction terms on cellulase, xylanase and laccase activities were depicted through the non-linear regression equations with significant R2 and P-values. An optimized value of 20 U/ml, 17 U/ml and 13 U/ml of cellulase, xylanase and laccase activities, respectively, were obtained with a media pH of 5.0 in 77 h at 31C, 140 rpm using wheatbran as a substrate. Overall, the present study introduces a fungal strain, capable of producing lignocellulolytic enzyme cocktail for subsequent applications in biofuel industry. PMID:26941214

Molecular Mechanisms Associated with Xylan Degradation by Xanthomonas Plant Pathogens*

PubMed Central

Santos, Camila Ramos; Hoffmam, Zaira Bruna; de Matos Martins, Vanesa Peixoto; Zanphorlin, Leticia Maria; de Paula Assis, Leandro Henrique; Honorato, Rodrigo Vargas; Lopes de Oliveira, Paulo Sérgio; Ruller, Roberto; Murakami, Mario Tyago

2014-01-01

Xanthomonas pathogens attack a variety of economically relevant plants, and their xylan CUT system (carbohydrate utilization with TonB-dependent outer membrane transporter system) contains two major xylanase-related genes, xynA and xynB, which influence biofilm formation and virulence by molecular mechanisms that are still elusive. Herein, we demonstrated that XynA is a rare reducing end xylose-releasing exo-oligoxylanase and not an endo-β-1,4-xylanase as predicted. Structural analysis revealed that an insertion in the β7-α7 loop induces dimerization and promotes a physical barrier at the +2 subsite conferring this unique mode of action within the GH10 family. A single mutation that impaired dimerization became XynA active against xylan, and high endolytic activity was achieved when this loop was tailored to match a canonical sequence of endo-β-1,4-xylanases, supporting our mechanistic model. On the other hand, the divergent XynB proved to be a classical endo-β-1,4-xylanase, despite the low sequence similarity to characterized GH10 xylanases. Interestingly, this enzyme contains a calcium ion bound nearby to the glycone-binding region, which is required for catalytic activity and structural stability. These results shed light on the molecular basis for xylan degradation by Xanthomonas and suggest how these enzymes synergistically assist infection and pathogenesis. Our findings indicate that XynB contributes to breach the plant cell wall barrier, providing nutrients and facilitating the translocation of effector molecules, whereas the exo-oligoxylanase XynA possibly participates in the suppression of oligosaccharide-induced immune responses. PMID:25266726

Prospect for Developing a Consolidated Bioprocessing (CBP) Strain Using Xylan as the Substrate: the Case Study of Yarrowia lipolytica

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Wei; Wei, Hui; Alahuhta, Markus

2016-07-08

To achieve the goal of developing a direct microbial sugar conversion platform for the production of lipids and drop-in fuels from cellulosic biomass substrate, Yarrowia lipolytica was used to investigate its potential for being developed as CBP strain by expressing cellulase and xylanase enzymes. Y. lipolytica is known to accumulate lipids intracellularly and is capable of metabolizing glucose and xylose to produce lipids; however, due to the lack of the biomass degrading enzymes, it cannot directly utilize lignocellulosic substrates as carbon sources. While research is continuing on the development of a Y. lipolytica strain able to degrade cellulose, in thismore » study, we present successful expression of several xylanases in Y. lipolytica. To the best of our knowledge, this is the first study introducing heterologous hemicellulose genes into the genome of Y. lipolytica. SDS-PAGE and western blotting analysis showed that the endo-xylanase gene XynII and exo-xylosidase gene XlnD were successfully expressed and secreted, and the expressed xylanases were likely either not or sparsely glycosylated, which is advantageous for expression of heterologous proteins from any species. Enzymatic activity tests further demonstrated active expression of XynII and XlnD in Y. lipolytica. Furthermore, synergistic action on converting xylan to xylose was observed when XlnD worked in concert with XynII. XlnD was able to work on the xylo-oligomers generated by XynII, enhancing the xylan conversion to monomeric xylose. The successful expression of these xylanases in Yarrowia further advances us towards our goal to develop a direct microbial conversion process using this organism. and xylose to produce lipids; however, due to the lack of the biomass degrading enzymes, it cannot directly utilize lignocellulosic substrates as carbon sources. While research is continuing on the development of a Y. lipolytica strain able to degrade cellulose, in this study, we present successful

Effects of exogenous xylanase on performance, nutrient digestibility, volatile fatty acid production and digestive tract thermal profiles of broilers fed on wheat- or maize-based diet.

PubMed

Masey-O'Neill, H V; Singh, M; Cowieson, A J

2014-01-01

1. A previous experiment reported that caecal temperature was negatively correlated with d 49 feed conversion ratio (FCR). This increased temperature in the caeca may indicate a prebiotic effect. An experiment was designed to investigate whether caecal temperature was affected in diets based on maize and whether other portions of the tract were affected. 2. A total of 25 Ross 308-d-old male broilers were allocated to each of 8 replicate pens per treatment. Treatments followed a 2 × 3 factorial design: two diets based on wheat or maize and three levels of enzyme addition, 0, 16 000 or 32 000 BXU/kg. Growth performance was assessed between d 1 and 49. Digestibility measurements were taken at d 28 and 49. On d 49, the excised small and large intestine of each bird was thermally imaged, weighed and volatile fatty acids (VFA) measured. 3. On d 28 and d 49, birds on the maize diets had higher feed intake and weight gain than those offered wheat diets. Additionally, on d 28, birds that received the maize diet had lower FCR than those offered the wheat diet. Enzyme improved FCR at d 49, independently of cereal. On d 28, enzyme improved the coefficient of apparent ileal DM digestibility and the coefficient of apparent ileal nitrogen digestibility. Enzyme only improved apparent ileal digestible energy in wheat-based diets (interactive term). On d 49, all digestibility parameters were improved by enzyme. Enzyme increased gizzard weight in maize-fed birds and the caeca of those fed wheat were heavier. The higher enzyme dose decreased duodenal temperature. In summary of VFA data, wheat-based diets produced more total VFAs and the total amount also increased with enzyme. 4. It appears from this study that there is equal potential in both wheat and maize diets for xylanase to improve performance of broilers probably through different mechanisms.

Investigation of the indigenous fungal community populating barley grains: Secretomes and xylanolytic potential.

PubMed

Sultan, Abida; Frisvad, Jens C; Andersen, Birgit; Svensson, Birte; Finnie, Christine

2017-10-03

The indigenous fungal species populating cereal grains produce numerous plant cell wall-degrading enzymes including xylanases, which could play important role in plant-pathogen interactions and in adaptation of the fungi to varying carbon sources. To gain more insight into the grain surface-associated enzyme activity, members of the populating fungal community were isolated, and their secretomes and xylanolytic activities assessed. Twenty-seven different fungal species were isolated from grains of six barley cultivars over different harvest years and growing sites. The isolated fungi were grown on medium containing barley flour or wheat arabinoxylan as sole carbon source. Their secretomes and xylanase activities were analyzed using SDS-PAGE and enzyme assays and were found to vary according to species and carbon source. Secretomes were dominated by cell wall degrading enzymes with xylanases and xylanolytic enzymes being the most abundant. A 2-DE-based secretome analysis of Aspergillus niger and the less-studied pathogenic fungus Fusarium poae grown on barley flour and wheat arabinoxylan resulted in identification of 82 A. niger and 31 F. poae proteins many of which were hydrolytic enzymes, including xylanases. The microorganisms that inhabit the surface of cereal grains are specialized in production of enzymes such as xylanases, which depolymerize plant cell walls. Integration of gel-based proteomics approach with activity assays is a powerful tool for analysis and characterization of fungal secretomes and xylanolytic activities which can lead to identification of new enzymes with interesting properties, as well as provide insight into plant-fungal interactions, fungal pathogenicity and adaptation. Understanding the fungal response to host niche is of importance to uncover novel targets for potential symbionts, anti-fungal agents and biotechnical applications. Copyright © 2017 Elsevier B.V. All rights reserved.

Extracellular Xylanopectinolytic Enzymes by Bacillus subtilis ADI1 from EFB's Compost

PubMed Central

Nawawi, Muhammad Hariadi; Mohamad, Rosfarizan; Tahir, Paridah Md.

2017-01-01

Microbial xylanase and pectinase are two extremely valuable enzymes, which have captivated much attention. This can be seen from the increased demand for these enzymes by many industrial sectors. This study investigates the isolation and screening of extracellular xylanopectinolytic enzymes-producing bacteria in a submerged fermentation (SmF). Samples are collected from the compost of empty fruit bunch (EFB) at Biocompost Pilot Plant, located at Biorefinery Plant, Universiti Putra Malaysia. From the experiment, out of 20 isolates, 11 isolates show xylanase or/and pectinase activity, and only one isolate (EFB-11) shows the concurrent activities of xylanase and pectinase. These activities are selected for enzyme production under submerged fermentation (quantitative screening). At the 72nd hour of incubation, xylanase and pectinase show the highest production, which ranges about 42.33 U/mL and 62.17 U/mL (with low amount of cellulase present), supplemented with 2% (w/v) of rice bran as carbon source at incubation temperature level, which is 30°C. Meanwhile, the pH of media is shifted to 8.42, which indicates that EFB-11 isolate is alkalotolerant bacteria and identified as Bacillus subtilis ADI1. This strain proves to have potential in agroindustrial bioconversion and has a promising ability to scale up to an industrial scale. PMID:28523288

Comparative evaluation of fermented and non-fermented de-oiled rice bran with or without exogenous enzymes supplementation in the diet of Labeo rohita (Hamilton, 1822).

PubMed

Ranjan, Amit; Sahu, Narottam Prasad; Deo, Ashutosh Dharmendra; Kumar, H Sanath; Kumar, Sarvendra; Jain, Kamal Kant

2018-03-29

A 60-day feeding trial was conducted to study the effect of exogenous enzymes (xylanase and phytase) supplementation in the non-fermented and fermented de-oiled rice bran (DORB)-based diet of Labeo rohita. Four test diets (T1-DORB-based diet, T2-fermented DORB-based diet, T3-phytase and xylanase supplemented DORB-based diet, and T4-phytase and xylanase supplemented fermented DORB-based diet) were formulated and fed to the respective groups. Test diets T3 and T4 were supplemented with 0.01% xylanase (16,000 U kg -1 ) and 0.01% phytase (500 U kg -1 ) enzymes. One hundred twenty juveniles of L. rohita, with an average weight 5.01 ± 0.02 g, were stocked in 12 uniform size plastic rectangular tanks in triplicate with 10 fishes per tank following a completely randomized design (CRD). Exogenous enzyme supplementation to the T3 group significantly improved the growth performance of L. rohita (p < 0.05). Fermented DORB fed groups registered significantly lower growth irrespective of the supplementation of exogenous enzymes. The carcass composition (except CP %), enzyme activities (except amylase activity), globulin, and A/G ratio did not vary significantly (p > 0.05). Based on the results of the present study, it is concluded that exogenous enzyme supplementation significantly increases the growth of fish fed with DORB-based diet.

Effect of supplementing a fibrous diet with a xylanase and β-glucanase blend on growth performance, intestinal glucose uptake, and transport-associated gene expression in growing pigs.

PubMed

Agyekum, A K; Sands, J S; Regassa, A; Kiarie, E; Weihrauch, D; Kim, W K; Nyachoti, C M

2015-07-01

The present study evaluated supplemental carbohydrase effect on performance, intestinal nutrient uptake, and transporter mRNA expressions in growing pigs offered a high-fiber diet manufactured with distillers dried grains with solubles (DDGS). Twenty-four pigs (22.4 ± 0.7 kg BW) were randomly assigned to 1of 3 nutritionally adequate diets (8 pigs per diet) based on corn and soybean meal (SBM) with either 0 (control) or 30% DDGS (high fiber [HF]). The third diet was supplemented with a xylanase and β-glucanase blend (XB) in addition to the 30% DDGS (HF+XB). Parameters determined were ADFI, ADG, G:F, plasma glucose and plasma urea nitrogen (PUN) concentrations, jejunal tissue electrophysiological properties, and mRNA expressions of the sodium-dependent glucose transport 1 (SGLT1) and cationic AA transporter, bo,+AT, in the jejunal and ileal tissues. In addition, mRNA expressions of the short-chain fatty acid transporters, monocarboxylate transporter 1 (MCT1) and sodium-coupled monocarboxylate transporter, and mucin genes were quantified in the ileum. Feed intake, plasma glucose, and jejunal tissue electrophysiological properties were not affected (P > 0.05) by diet. However, control-fed pigs had superior growth rate and feed efficiency and higher PUN (P < 0.05) than HF- and HF+XB-fed pigs. The HF diet increased (P < 0.05) SGLT1 mRNA expression in the jejunum and decreased (P < 0.05) bo,+ mRNA expression in the ileum. The XB supplementation also increased bo,+ mRNA expression in the ileum relative to HF-fed pigs. Additionally, MCT1 mRNA expression was greater (P < 0.05) in the ileum of the HF- and HF+XB-fed pigs. In the present study, XB supplementation influenced nutrient transporter mRNA expression, although it was not accompanied by improved pig performance.

Hydrolyzabilities of different corn stover fractions after aqueous ammonia pretreatment.

PubMed

Sun, Zongping; Ge, Xiaoyan; Xin, Donglin; Zhang, Junhua

2014-02-01

The effect of aqueous ammonia pretreatment on the hydrolysis of different corn stover fractions (rind, husk, leaf, and pith) by xylanase (XYL) with cellulases (CELs) was evaluated. The aqueous ammonia pretreatment had excellent delignification ability (above 66%) for different corn stover fractions. The corn rind exhibited the lowest susceptibility to aqueous ammonia pretreatment. The pretreated rind showed the lowest hydrolyzability by CEL and XYL, which was supported by a high content of crystalline cellulose in the hydrolyzed residues of rind, as confirmed by X-ray diffraction (XRD). With the addition of 1 mg XYL/g dry matter, a high glucose yield (above 90%) could be obtained from the pretreated rind by CEL. The results revealed that a high hydrolyzate yield of corn rind after aqueous ammonia pretreatment could be obtained with 1 mg xylanase/g dry matter, showing that aqueous ammonia pretreatment and xylanase addition to cellulases have great potential for the efficient hydrolysis of corn stover without previous fractionation.

Direct production of feruloyl oligosaccharides and hemicellulase inducement and distribution in a newly isolated Aureobasidium pullulans strain.

PubMed

Yu, Xiao-hong; Gu, Zhen-xin

2014-02-01

Studies were carried out to screen and identify strains that are able to directly produce ferulic oligosaccharides (FOs) from wheat bran (WB). The inducement and distribution of hemicellulases from strain 2012, which was identified as a non-melanin secreting strain of Aureobasidium pullulans (A. pullulans), were also determined. In a 60 g/L WB solution, A. pullulans could produce 545 nmol/L FOs, 64.12 IU/mL xylanase and 0.14 IU/mL ferulic acid esterase (FAE). A. pullulans was cultivated in media with WB, glucose, xylose, sucrose, lactose or xylan as the carbon source, and hemicellulases were mainly induced by xylan and WB and inhibited by glucose and sucrose. Xylanase and FAE were mainly present in the culture filtrate, xylosidase in the hyphal filaments and arabinofuranosidase was a membrane-bound enzyme. The yield of FOs was positively correlated to the hemicellulases activity, and significantly positively (P < 0.05) correlated to the xylanase activity (r = 0.992).

The ACEII recombinant Trichoderma reesei QM9414 strains with enhanced xylanase production and its applications in production of xylitol from tree barks.

PubMed

Xiong, Lili; Kameshwar, Ayyappa Kumar Sista; Chen, Xi; Guo, Zhiyun; Mao, Canquan; Chen, Sanfeng; Qin, Wensheng

2016-12-28

ACEII transcription factor plays a significant role in regulating the expression of cellulase and hemicellulase encoding genes. Apart from ACEII, transcription factors such as XYR1, CRE1, HAP2/3/5 complex and ACEI function in a coordinated pattern for regulating the gene expression of cellulases and hemicellulases. Studies have demonstrated that ACEII gene deletion results in decreased total cellulase and xylanase activities with reduced transcript levels of lignocellulolytic enzymes. In this study, we have successfully transformed the ACEII transcription factor encoding gene in Trichoderma reesei to significantly improve its degrading abilities. Transformation experiments on parental strain T. reesei QM9414 has resulted in five genetically engineered strains T/Ace2-2, T/Ace2-5, T/Ace2-8, T/Ace5-4 and T/Ace10-1. Among which, T/Ace2-2 has exhibited significant increase in enzyme activity by twofolds, when compared to parental strain. The T/Ace2-2 was cultured on growth substrates containing 2% bark supplemented with (a) sugar free + MA medium (b) glucose + MA medium and (c) xylose + MA medium. The bark degradation efficiency of genetically modified T/Ace2-2 strain was assessed by analyzing the xylitol production yield using HPAEC. By 6th day, about 10.52 g/l of xylitol was produced through enzymatic conversion of bark (2% bark + MA + xylose) by the T/Ace2-2 strain and by 7th day the conversion rate was found to be 0.21 g/g. Obtained results confirmed that bark growth medium supplemented with D-xylose has profoundly increased the conversion rate of bark by T/Ace2-2 strain when compared to sugar free and glucose supplemented growth media. Results obtained from scanning electron microscopy has endorsed our current results. Bark samples inoculated with T/Ace2-2 strain has showed large number of degraded cells with clearly visible cavities and fractures, by exposing the microfibrillar interwoven complex. We propose a cost effective and ecofriendly method for

The efficacy of dietary xylanase and phytase in broiler chickens fed expeller-extracted camelina meal.

PubMed

Pekel, A Y; Horn, N L; Adeola, O

2017-01-01

A study was conducted to investigate the effects of dietary phytase (PHY) and xylanase (XYL) on growth performance, nutrient utilization, and intestinal characteristics in broilers fed corn-soybean meal-based diets with added expeller-extracted camelina meal (CM). The corn-soybean meal-based diets without or with CM was formulated to contain 2.9 or 2.7 g/kg non phytate phosphorus, respectively. A total of 384 male Ross 708 broilers were allocated to 8 dietary treatments in a randomized complete block design with 6 replicates per treatment, from 7 to 21 d post hatching. The experiment consisted of a 2 × 2 × 2 factorial arrangement of treatments with 2 dietary CM levels (zero or 100 g/kg), 2 dietary levels of XYL (zero or 800 unit/kg), and 2 dietary levels of PHY (zero or 4,000 unit/kg). Chromic oxide was included in the diets as an indigestible marker. Growth was measured throughout the experiment and excreta were collected on d 18 to 21 post hatching for measurement of nutrient and energy retention. On d 21 post hatching, broilers were euthanized by CO 2 asphyxiation, ileal digesta was collected for nutrient and energy digestibility measurements, and the left tibia was removed for bone ash measurement. Furthermore, duodenal digesta was collected and a segment of the mid jejunum was excised for viscosity and morphology measurement, respectively. Broilers fed 100 g/kg CM diets showed lower (P < 0.001) BW gain and G:F than those fed zero g/kg CM diets for 7 to 14 d and 7 to 21 d post hatching periods, respectively. Phytase supplementation improved (P < 0.05) all the growth parameters measured regardless of CM addition. Phytase supplementation resulted in an increase (P < 0.05) in jejunum villus height but there was no impact of CM or XYL. Additionally, duodenal digesta viscosity increased (P < 0.001) with added CM. Tibia ash and weight increased (P < 0.001) with PHY supplementation. There was a CM × PHY interaction (P < 0.05) on percentage tibia ash

Optimization of enzyme-assisted improvement of polyphenols and free radical scavenging activity in red rice bran: A statistical and neural network-based approach.

PubMed

Prabhu, Ashish A; Jayadeep, A

2017-04-21

The current study is focused on optimizing the parameters involved in enzymatic processing of red rice bran for maximizing total polyphenol (TP) and free radical scavenging activity (FRSA). The sequential optimization strategies using central composite design (CCD) and artificial neural network (ANN) modeling linked with genetic algorithm (GA) was performed to study the effect of incubation time (60-90 min), xylanase concentration (5-10 mg/g), cellulase concentration (5-10 mg/g) on the response, i.e., total polyphenol and FRSA. The result showed that incubation time has a negative effect on the response, while the square effect of xylanase and cellulase showed positive effect on the response. A maximum TP of 2,761 mg ferulic acid Eq/100 g bran and FRSA of 778.4 mg Catechin Eq/100 g bran was achieved with incubation time (min) = 60.491; xylanase (mg/g) = 5.4633; cellulase (mg/g) = 11.5825. Furthermore, ANN-GA-based optimization showed better predicting capabilities as compared to CCD.

Enhancing enzymatic hydrolysis of xylan by adding sodium lignosulfonate and long-chain fatty alcohols.

PubMed

Lou, Hongming; Yuan, Long; Qiu, Xueqing; Qiu, Kexian; Fu, Jinguo; Pang, Yuxia; Huang, Jinhao

2016-01-01

Sodium lignosulfonate (SXSL) and long-chain fatty alcohols (LFAs) could enhance the enzymatic hydrolysis of xylan, and the compound of SXSL and LFAs have synergies on the enzymatic hydrolysis. SXSL shows a strong enhancement in buffer pH range from 4.0 to 6.0. The enhancement increased with the SXSL dosage and the xylanase loading. The cellulose and lignin in corncob substrate could not only adsorb xylanase nonproductively, but also seriously reduce the accessibility of xylanase on xylan to impede the enzymatic hydrolysis of xylan. Cellulase could break the plant cell wall structure of corncob and make additives work better. The xylose yield of corncob at 72h increased from 59.4% to 73.7% by adding the compound of 5g/L SXSL and 0.01% (v/v) n-decanol, which was higher than that without cellulase and additives by 30.7%. Meanwhile, the glucose yield at 72h of corncob increased from 45.8% to 62.3%. Copyright © 2015 Elsevier Ltd. All rights reserved.

The complete conformational free energy landscape of β-xylose reveals a two-fold catalytic itinerary for β-xylanases† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c4sc02240h Click here for additional data file.

PubMed Central

Iglesias-Fernández, Javier; Raich, Lluís; Ardèvol, Albert

2015-01-01

Unraveling the conformational catalytic itinerary of glycoside hydrolases (GHs) is a growing topic of interest in glycobiology, with major impact in the design of GH inhibitors. β-xylanases are responsible for the hydrolysis of glycosidic bonds in β-xylans, a group of hemicelluloses of high biotechnological interest that are found in plant cell walls. The precise conformations followed by the substrate during catalysis in β-xylanases have not been unambiguously resolved, with three different pathways being proposed from structural analyses. In this work, we compute the conformational free energy landscape (FEL) of β-xylose to predict the most likely catalytic itineraries followed by β-xylanases. The calculations are performed by means of ab initio metadynamics, using the Cremer–Pople puckering coordinates as collective variables. The computed FEL supports only two of the previously proposed itineraries, 2SO → [2,5B]ǂ → 5S1 and 1S3 → [4H3]ǂ → 4C1, which clearly appear in low energy regions of the FEL. Consistently, 2SO and 1S3 are conformations preactivated for catalysis in terms of free energy/anomeric charge and bond distances. The results however exclude the OE → [OS2]ǂ → B2,5 itinerary that has been recently proposed for a family 11 xylanase. Classical and ab initio QM/MM molecular dynamics simulations reveal that, in this case, the observed OE conformation has been enforced by enzyme mutation. These results add a word of caution on using modified enzymes to inform on catalytic conformational itineraries of glycoside hydrolases. PMID:29560204

Identification and characterisation of xylanolytic yeasts isolated from decaying wood and sugarcane bagasse in Brazil.

PubMed

Lara, Carla A; Santos, Renata O; Cadete, Raquel M; Ferreira, Carla; Marques, Susana; Gírio, Francisco; Oliveira, Evelyn S; Rosa, Carlos A; Fonseca, César

2014-06-01

In this study, yeasts associated with lignocellulosic materials in Brazil, including decaying wood and sugarcane bagasse, were isolated, and their ability to produce xylanolytic enzymes was investigated. A total of 358 yeast isolates were obtained, with 198 strains isolated from decaying wood and 160 strains isolated from decaying sugarcane bagasse samples. Seventy-five isolates possessed xylanase activity in solid medium and were identified as belonging to nine species: Candida intermedia, C. tropicalis, Meyerozyma guilliermondii, Scheffersomyces shehatae, Sugiyamaella smithiae, Cryptococcus diffluens, Cr. heveanensis, Cr. laurentii and Trichosporon mycotoxinivorans. Twenty-one isolates were further screened for total xylanase activity in liquid medium with xylan, and five xylanolytic yeasts were selected for further characterization, which included quantitative analysis of growth in xylan and xylose and xylanase and β-D-xylosidase activities. The yeasts showing the highest growth rate and cell density in xylan, Cr. laurentii UFMG-HB-48, Su. smithiae UFMG-HM-80.1 and Sc. shehatae UFMG-HM-9.1a, were, simultaneously, those exhibiting higher xylanase activity. Xylan induced the highest level of (extracellular) xylanase activity in Cr. laurentii UFMG-HB-48 and the highest level of (intracellular, extracellular and membrane-associated) β-D-xylosidase activity in Su. smithiae UFMG-HM-80.1. Also, significant β-D-xylosidase levels were detected in xylan-induced cultures of Cr. laurentii UFMG-HB-48 and Sc. shehatae UFMG-HM-9.1a, mainly in extracellular and intracellular spaces, respectively. Under xylose induction, Cr. laurentii UFMG-HB-48 showed the highest intracellular β-D-xylosidase activity among all the yeast tested. C. tropicalis UFMG-HB 93a showed its higher (intracellular) β-D-xylosidase activity under xylose induction and higher at 30 °C than at 50 °C. This study revealed different xylanolytic abilities and strategies in yeasts to metabolise xylan and

High-level expression of thermostable cellulolytic enzymes in tobacco transplastomic plants and their use in hydrolysis of an industrially pretreated Arundo donax L. biomass.

PubMed

Castiglia, Daniela; Sannino, Lorenza; Marcolongo, Loredana; Ionata, Elena; Tamburino, Rachele; De Stradis, Angelo; Cobucci-Ponzano, Beatrice; Moracci, Marco; La Cara, Francesco; Scotti, Nunzia

2016-01-01

Biofuels production from plant biomasses is a complex multi-step process with important economic burdens. Several biotechnological approaches have been pursued to reduce biofuels production costs. The aim of the present study was to explore the production in tobacco plastome of three genes encoding (hemi)cellulolytic enzymes from thermophilic and hyperthermophilic bacterium and Archaea, respectively, and test their application in the bioconversion of an important industrially pretreated biomass feedstock (A. donax) for production of second-generation biofuels. The selected enzymes, endoglucanase, endo-β-1,4-xylanase and β-glucosidase, were expressed in tobacco plastome with a protein yield range from 2 % to more than 75 % of total soluble proteins (TSP). The accumulation of endoglucanase (up to 2 % TSP) gave altered plant phenotypes whose severity was directly linked to the enzyme yield. The most severe seedling-lethal phenotype was due to the impairment of plastid development associated to the binding of endoglucanase protein to thylakoids. Endo-β-1,4-xylanase and β-glucosidase, produced at very high level without detrimental effects on plant development, were enriched (fourfold) by heat treatment (105.4 and 255.4 U/mg, respectively). Both plastid-derived biocatalysts retained the main features of the native or recombinantly expressed enzymes with interesting differences. Plastid-derived xylanase and β-glucosidase resulted more thermophilic than the E. coli recombinant and native counterpart, respectively. Bioconversion experiments, carried out at 50 and 60 °C, demonstrated that plastid-derived enzymes were able to hydrolyse an industrially pretreated giant reed biomass. In particular, the replacement of commercial enzyme with plastid-derived xylanase, at 60 °C, produced an increase of both xylose recovery and hydrolysis rate; whereas the replacement of both xylanase and β-glucosidase produced glucose levels similar to those observed with the

Characterization of lignocellulolytic activities from fungi isolated from the deep-sea sponge Stelletta normani.

PubMed

Batista-García, Ramón Alberto; Sutton, Thomas; Jackson, Stephen A; Tovar-Herrera, Omar Eduardo; Balcázar-López, Edgar; Sánchez-Carbente, María Del Rayo; Sánchez-Reyes, Ayixon; Dobson, Alan D W; Folch-Mallol, Jorge Luis

2017-01-01

Extreme habitats have usually been regarded as a source of microorganisms that possess robust proteins that help enable them to survive in such harsh conditions. The deep sea can be considered an extreme habitat due to low temperatures (<5°C) and high pressure, however marine sponges survive in these habitats. While bacteria derived from deep-sea marine sponges have been studied, much less information is available on fungal biodiversity associated with these sponges. Following screening of fourteen fungi isolated from the deep-sea sponge Stelletta normani sampled at a depth of 751 metres, three halotolerant strains (TS2, TS11 and TS12) were identified which displayed high CMCase and xylanase activities. Molecular based taxonomic approaches identified these strains as Cadophora sp. TS2, Emericellopsis sp. TS11 and Pseudogymnoascus sp. TS 12. These three fungi displayed psychrotolerance and halotolerant growth on CMC and xylan as sole carbon sources, with optimal growth rates at 20°C. They produced CMCase and xylanase activities, which displayed optimal temperature and pH values of between 50-70°C and pH 5-8 respectively, together with good thermostability and halotolerance. In solid-state fermentations TS2, TS11 and TS12 produced CMCases, xylanases and peroxidase/phenol oxidases when grown on corn stover and wheat straw. This is the first time that CMCase, xylanase and peroxidase/phenol oxidase activities have been reported in these three fungal genera isolated from a marine sponge. Given the biochemical characteristics of these ligninolytic enzymes it is likely that they may prove useful in future biomass conversion strategies involving lignocellulosic materials.

Characterization of lignocellulolytic activities from fungi isolated from the deep-sea sponge Stelletta normani

PubMed Central

Batista-García, Ramón Alberto; Sutton, Thomas; Jackson, Stephen A.; Tovar-Herrera, Omar Eduardo; Balcázar-López, Edgar; Sánchez-Carbente, María del Rayo; Sánchez-Reyes, Ayixon; Dobson, Alan D. W.

2017-01-01

Extreme habitats have usually been regarded as a source of microorganisms that possess robust proteins that help enable them to survive in such harsh conditions. The deep sea can be considered an extreme habitat due to low temperatures (<5°C) and high pressure, however marine sponges survive in these habitats. While bacteria derived from deep-sea marine sponges have been studied, much less information is available on fungal biodiversity associated with these sponges. Following screening of fourteen fungi isolated from the deep-sea sponge Stelletta normani sampled at a depth of 751 metres, three halotolerant strains (TS2, TS11 and TS12) were identified which displayed high CMCase and xylanase activities. Molecular based taxonomic approaches identified these strains as Cadophora sp. TS2, Emericellopsis sp. TS11 and Pseudogymnoascus sp. TS 12. These three fungi displayed psychrotolerance and halotolerant growth on CMC and xylan as sole carbon sources, with optimal growth rates at 20°C. They produced CMCase and xylanase activities, which displayed optimal temperature and pH values of between 50–70°C and pH 5–8 respectively, together with good thermostability and halotolerance. In solid-state fermentations TS2, TS11 and TS12 produced CMCases, xylanases and peroxidase/phenol oxidases when grown on corn stover and wheat straw. This is the first time that CMCase, xylanase and peroxidase/phenol oxidase activities have been reported in these three fungal genera isolated from a marine sponge. Given the biochemical characteristics of these ligninolytic enzymes it is likely that they may prove useful in future biomass conversion strategies involving lignocellulosic materials. PMID:28339473

Enhanced enzymatic hydrolysis of lignocellulose by optimizing enzyme complexes.

PubMed

Zhang, Mingjia; Su, Rongxin; Qi, Wei; He, Zhimin

2010-03-01

To enhance the conversion of the cellulose and hemicellulose, the corncob pretreated by aqueous ammonia soaking was hydrolyzed by enzyme complexes. The saturation limit for cellulase (Spezyme CP) was determined as 15 mg protein/g glucan (50 filter paper unit (FPU)/g glucan). The accessory enzymes (beta-glucosidase, xylanase, and pectinase) were supplemented to hydrolyze cellobiose (cellulase-inhibiting product), hemicellulose, and pectin (the component covering the fiber surfaces), respectively. It was found that beta-glucosidase (Novozyme 188) loading of 1.45 mg protein/g glucan [30 cellobiase units (CBU)/g glucan] was enough to eliminate the cellobiose inhibitor, and 2.9 mg protein/g glucan (60 CBU/g glucan) was the saturation limit. The supplementation of xylanase and pectinase can increase the conversion of cellulose and hemicellulose significantly. The yields of glucose and xylose enhanced with the increasing enzyme loading, but the increasing trend became low at high loading. Compared with xylanase, pectinase was more effective to promote the hydrolysis of cellulose and hemicellulose. The supplementation of pectinase with 0.12 mg protein/g glucan could increase the yields of glucose and xylose by 7.5% and 29.3%, respectively.

Hydrolysis of lignocellulosic feedstock by novel cellulases originating from Pseudomonas sp. CL3 for fermentative hydrogen production.

PubMed

Cheng, Chieh-Lun; Chang, Jo-Shu

2011-09-01

A newly isolated indigenous bacterium Pseudomonas sp. CL3 was able to produce novel cellulases consisting of endo-β-1,4-d-glucanase (80 and 100 kDa), exo-β-1,4-d-glucanase (55 kDa) and β-1,4-d-glucosidase (65 kDa) characterized by enzyme assay and zymography analysis. In addition, the CL3 strain also produced xylanase with a molecular weight of 20 kDa. The optimal temperature for enzyme activity was 50, 45, 45 and 55 °C for endo-β-1,4-d-glucanase, exo-β-1,4-d-glucanase, β-1,4-d-glucosidase and xylanase, respectively. All the enzymes displayed optimal activity at pH 6.0. The cellulases/xylanase could hydrolyze cellulosic materials very effectively and were thus used to hydrolyze natural agricultural waste (i.e., bagasse) for clean energy (H2) production by Clostridium pasteurianum CH4 using separate hydrolysis and fermentation process. The maximum hydrogen production rate and cumulative hydrogen production were 35 ml/L/h and 1420 ml/L, respectively, with a hydrogen yield of around 0.96 mol H2/mol glucose. Copyright © 2011 Elsevier Ltd. All rights reserved.

Mathematical model-based optimization of physico-enzymatic hydrolysis of Pinus roxburghii needles for the production of reducing sugars.

PubMed

Vats, Siddharth; Maurya, Devendra Prasad; Jain, Ayushi; Mall, Varija; Negi, Sangeeta

2013-11-01

The objective of this study was to optimize the physico-enzymatic pretreatment of P. roxburghii fallen foliage (needles) to produce reducing sugars through response surface methodology (RSM) with central composite face centered design (CCD). Under this, five parameters, i.e., concentration of laccase, cellulose and xylanase, steam explosion pressure and incubation period, at three levels with twenty six runs were taken into account. Cellulase, xylanase and laccase enzymes with activity 4.563, 38.32 and 0.05 IU/mL, respectively, were produced from locally isolated microbial strains. The analysis of variance (ANOVA) was applied for the validation of the predicted model at 95% of confidence level. This model predicted 334 mg/g release of reducing sugars on treating P. roxburghii fallen foliage with 1.18 mL of cellulose, 0.31 mL of xylanase and 0.01 mL of laccase, 14.39 psi steam explosion pressure and 24 h of incubation time. The experimental results obtained were in good agreement to predicted values, making it a reliable optimized model for five factors in combination to predict reducing sugar yield for ethanol production for bio-fuel industry.

Combined enzymatic and physical deinking methodology for efficient eco-friendly recycling of old newsprint.

PubMed

Virk, Antar Puneet; Puri, Minakshi; Gupta, Vijaya; Capalash, Neena; Sharma, Prince

2013-01-01

The development in the deinking process has made recycled fiber a major part of the raw material for pulp and paper industry. Enzymes have revolutionized the deinking process obtaining brightness levels surpassing conventional deinking processes. This study explores the deinking efficiencies of bacterial alkalophilic laccase (L) and xylanase (X) enzymes along with physical deinking methods of microwaving (MW) and sonication (S) for recycling of old newsprint (ONP). The operational parameters viz. enzyme dose, pH and treatment time for X and L deinking were optimized statistically using Response Surface Methodology. Laccase did not require any mediator supplementation for deinking. Deinking of ONP pulp with a combination of xylanase and laccase enzymes was investigated, and fiber surface composition and morphological changes were studied using X-ray diffraction, fourier transform infrared spectroscopy and scanning electron microscopy. Compared to the pulp deinked with xylanase (47.9%) or laccase (62.2%) individually, the percentage reduction of effective residual ink concentration (ERIC) was higher for the combined xylanase/laccase-deinked pulp (65.8%). An increase in brightness (21.6%), breaking length (16.5%), burst factor (4.2%) tear factor (6.9%), viscosity (13%) and cellulose crystallinity (10.3%) along with decrease in kappa number (22%) and chemical consumption (50%) were also observed. Surface appeared more fibrillar along with changes in surface functional groups. A combination of physical and enzymatic processes (S-MW-XL) for deinking further improved brightness (28.8%) and decreased ERIC (73.9%) substantially. This is the first report on deinking of ONP with laccase without any mediator supplementation. XL pretreatment resulted in marked improvement in paper quality and a new sequence being reported for deinking (S-MW-XL) will contribute further in decreasing chemical consumption and making the process commercially feasible.

Combined Enzymatic and Physical Deinking Methodology for Efficient Eco-Friendly Recycling of Old Newsprint

PubMed Central

Virk, Antar Puneet; Puri, Minakshi; Gupta, Vijaya; Capalash, Neena; Sharma, Prince

2013-01-01

Background The development in the deinking process has made recycled fiber a major part of the raw material for pulp and paper industry. Enzymes have revolutionized the deinking process obtaining brightness levels surpassing conventional deinking processes. This study explores the deinking efficiencies of bacterial alkalophilic laccase (L) and xylanase (X) enzymes along with physical deinking methods of microwaving (MW) and sonication (S) for recycling of old newsprint (ONP). Methods and Results The operational parameters viz. enzyme dose, pH and treatment time for X and L deinking were optimized statistically using Response Surface Methodology. Laccase did not require any mediator supplementation for deinking. Deinking of ONP pulp with a combination of xylanase and laccase enzymes was investigated, and fiber surface composition and morphological changes were studied using X-ray diffraction, fourier transform infrared spectroscopy and scanning electron microscopy. Compared to the pulp deinked with xylanase (47.9%) or laccase (62.2%) individually, the percentage reduction of effective residual ink concentration (ERIC) was higher for the combined xylanase/laccase-deinked pulp (65.8%). An increase in brightness (21.6%), breaking length (16.5%), burst factor (4.2%) tear factor (6.9%), viscosity (13%) and cellulose crystallinity (10.3%) along with decrease in kappa number (22%) and chemical consumption (50%) were also observed. Surface appeared more fibrillar along with changes in surface functional groups. A combination of physical and enzymatic processes (S-MW-XL) for deinking further improved brightness (28.8%) and decreased ERIC (73.9%) substantially. Conclusion This is the first report on deinking of ONP with laccase without any mediator supplementation. XL pretreatment resulted in marked improvement in paper quality and a new sequence being reported for deinking (S-MW-XL) will contribute further in decreasing chemical consumption and making the process

Enzymes in Commercial Cellulase Preparations Bind Differently to Dioxane Extracted Lignins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yarbrough, John M.; Mittal, Ashutosh; Katahira, Rui

Commercial fungal cellulases used in biomass-to-biofuels processes can be grouped into three general classes: native, augmented, and engineered. To evaluate lignin binding affinities of different enzyme activities in various commercial cellulase formulations in order to determine if enzyme losses due to lignin binding can be modulated by using different enzymes of the same activity We used water:dioxane (1:9) to extract lignin from pretreated corn stover. Commercial cellulases were incubated with lignin and the unbound supernatants were evaluated for individual enzyme loss by SDS=PAGE and these were correlated with activity loss using various pNP-sugar substrates. Colorimetric assays for general glycosyl hydrolasemore » activities showed distinct differences in enzyme binding to lignin for each enzyme activity. Native systems demonstrated low binding of endo- and exo-cellulases, high binding of xylanase, and moderate ..beta..-glucosidase binding. Engineered cellulase mixtures exhibited low binding of exo-cellulases, very strong binding of endocellulases and ..beta..- glucosidase, and mixed binding of xylanase activity. The augmented cellulase had low binding of exocellulase, high binding of endocellulase and xylanase, and moderate binding of ..beta..-glucosidase activities. Bound and unbound activities were correlated with general molecular weight ranges of proteins as measured by loss of proteins bands in bound fractions on SDS-PAGE gels. Lignin-bound high molecular weight bands correlated with binding of ..beta..-glucosidase activity. While ..beta..-glucosidases demonstrated high binding in many cases, they have been shown to remain active. Bound low molecular weight bands correlated with xylanase activity binding. Contrary to other literature, exocellulase activity did not show strong lignin binding. The variation in enzyme activity binding between the three classes of cellulases preparations indicate that it is certainly possible to alter the binding of

Production of Cellulolytic and Hemicellulolytic Enzymes From Aureobasidium pulluans on Solid State Fermentation

NASA Astrophysics Data System (ADS)

Leite, Rodrigo Simões Ribeiro; Bocchini, Daniela Alonso; da Silva Martins, Eduardo; Silva, Dênis; Gomes, Eleni; da Silva, Roberto

This article investigates a strain of the yeast Aureobasidium pullulans for cellulase and hemicellulase production in solid state fermentation. Among the substrates analyzed, the wheat bran culture presented the highest enzymatic production (1.05 U/mL endoglucanase, 1.3 U/mL β-glucosidase, and 5.0 U/mL xylanase). Avicelase activity was not detected. The optimum pH and temperature for xylanase, endoglucanase and β-glucosidase were 5.0 and 50, 4.5 and 60, 4.0 and 75°C, respectively. These enzymes remained stable between a wide range of pH. The β-glucosidase was the most thermostable enzyme remaining 100% active when incubated at 75°C for 1 h.

Bioconversion of Xylan to triglycerides by oil-rich yeasts. [Cryptococcus albidus; Cryptococcus terricoluus; Trichosporon

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fall, R.; Phelps, P.; Spindler, D.

A series of lipid-accumulating yeasts was examined for their potential to saccharify xylan and accumulate triglyceride. Of the genera tested, including Candida, Cryptococcus, Lipomyces, Rhodosporidium, Rhodotorula, and Trichosporon, only Crytococcus and Trichosporon isolates saccharified xylan. All of the strains could assimilate xylose and accumuate triglyceride under nitrogen-limiting conditions. Strains of Cryptococcus albidus were found to be especially useful for a one-step saccharification of xylan coupled to triglyceride synthesis. Crytococcus terricolus, a strain constitutive for lipid accumulation, lacked extracellular xylanase, but did assimilate xylose and xylobiose and was able to continuously convert xylan to triglyceride if the culture medium was supplementedmore » with xylanase. 22 references.« less

Mixed submerged fermentation with two filamentous fungi for cellulolytic and xylanolytic enzyme production.

PubMed

Garcia-Kirchner, O; Muñoz-Aguilar, M; Pérez-Villalva, R; Huitrón-Vargas, C

2002-01-01

The efficient saccharification of lignocellulosic materials requires the cooperative actions of different cellulase enzyme activities: exoglucanase, endoglucanase, beta-glucosidase, and xylanase. Previous studies with the fungi strains Aureobasidium sp. CHTE-18, Penicillium sp. CH-TE-001, and Aspergillus terreus CH-TE-013, selected mainly because of their different cellulolytic and xylanolytic activities, have demonstrated the capacity of culture filtrates of cross-synergistic action in the saccharification of native sugarcane bagasse pith. In an attempt to improve the enzymatic hydrolysis of different cellulosic materials, we investigated a coculture fermentation with two of these strains to enhance the production of cellulases and xylanases. The 48-h batch experimental results showed that the mixed culture of Penicillium sp. CH-TE-001 and A. terreus CH-TE-013 produced culture filtrates with high protein content, cellulase (mainly beta-glucosidase), and xylanase activities compared with the individual culture of each strain. The same culture conditions were used in a simple medium with mineral salts, corn syrup liquor, and sugarcane bagasse pith as the sole carbon source with moderate shaking at 29 degrees C. Finally, we compared the effect of the cell-free culture filtrates obtained from the mixed and single fermentations on the saccharification of different kinds of cellulosic materials.

Contribution of protein, starch, and fat to the apparent ileal digestible energy of corn- and wheat-based broiler diets in response to exogenous xylanase and amylase without or with protease.

PubMed

Romero, L F; Sands, J S; Indrakumar, S E; Plumstead, P W; Dalsgaard, S; Ravindran, V

2014-10-01

The ileal energy contribution of protein, starch, and fat in response to 2 exogenous enzyme combinations was studied in 2 digestibility assays with 21- (experiment 1; 432 birds) and 42-d-old (experiment 2; 288 birds) Ross 308 broiler chickens. A 2 × 2 × 3 factorial arrangement of treatments with 2 base grains (corn or wheat), without or with high fiber ingredients (corn distillers dried grains with solubles and canola meal), and 3 enzyme treatments was implemented. Enzyme treatments, fed from 12 to 21 d or 32 to 42 d, were 1) without enzymes, 2) with xylanase from Trichoderma ressei (2,000 U/kg) and amylase from Bacillus licheniformis (200 U/kg; XA), or 3) with XA plus protease from Bacillus subtilis (4,000 U/kg; XAP). All diets contained Escherichia coli phytase (500 FTU/kg). Apparent ileal digestibility (AID) of protein, starch, and fat, as well as the apparent ileal digestible energy, were determined using titanium dioxide as inert marker. A generalized mixed model was used to test main effects and 2-way interactions at P < 0.05. An enzyme × grain interaction was detected for AID of starch at 21 and 42 d, and AID of fat at 21 d, with greater effects of enzymes in wheat-based compared with corn-based diets, but significant increments due to enzymes compared with controls in both diet types. Apparent ileal digestibility of fat at 42 d increased with enzyme supplementation compared with the control treatments. The XA and XAP treatments gradually (P < 0.05) increased AID of protein at 21 d, but only XAP increased AID of protein compared with the control at 42 d. Compared with the controls, XA increased AID energy by 52 or 87 kcal, and XAP by 104 or 152 kcal/kg of DM at 21 or 42 d, respectively. The caloric contribution of starch, fat, and protein were affected differentially by base grain and the presence of fibrous ingredients at 21 and 42 d of age. ©2014 Poultry Science Association Inc.

Contribution of protein, starch, and fat to the apparent ileal digestible energy of corn- and wheat-based broiler diets in response to exogenous xylanase and amylase without or with protease1

PubMed Central

Romero, L. F.; Sands, J. S.; Indrakumar, S. E.; Plumstead, P. W.; Dalsgaard, S.; Ravindran, V.

2014-01-01

The ileal energy contribution of protein, starch, and fat in response to 2 exogenous enzyme combinations was studied in 2 digestibility assays with 21- (experiment 1; 432 birds) and 42-d-old (experiment 2; 288 birds) Ross 308 broiler chickens. A 2 × 2 × 3 factorial arrangement of treatments with 2 base grains (corn or wheat), without or with high fiber ingredients (corn distillers dried grains with solubles and canola meal), and 3 enzyme treatments was implemented. Enzyme treatments, fed from 12 to 21 d or 32 to 42 d, were 1) without enzymes, 2) with xylanase from Trichoderma ressei (2,000 U/kg) and amylase from Bacillus licheniformis (200 U/kg; XA), or 3) with XA plus protease from Bacillus subtilis (4,000 U/kg; XAP). All diets contained Escherichia coli phytase (500 FTU/kg). Apparent ileal digestibility (AID) of protein, starch, and fat, as well as the apparent ileal digestible energy, were determined using titanium dioxide as inert marker. A generalized mixed model was used to test main effects and 2-way interactions at P < 0.05. An enzyme × grain interaction was detected for AID of starch at 21 and 42 d, and AID of fat at 21 d, with greater effects of enzymes in wheat-based compared with corn-based diets, but significant increments due to enzymes compared with controls in both diet types. Apparent ileal digestibility of fat at 42 d increased with enzyme supplementation compared with the control treatments. The XA and XAP treatments gradually (P < 0.05) increased AID of protein at 21 d, but only XAP increased AID of protein compared with the control at 42 d. Compared with the controls, XA increased AID energy by 52 or 87 kcal, and XAP by 104 or 152 kcal/kg of DM at 21 or 42 d, respectively. The caloric contribution of starch, fat, and protein were affected differentially by base grain and the presence of fibrous ingredients at 21 and 42 d of age. PMID:25071229

Enzymatic activities produced by mixed Saccharomyces and non-Saccharomyces cultures: relationship with wine volatile composition.

PubMed

Maturano, Yolanda Paola; Assof, Mariela; Fabani, María Paula; Nally, María Cristina; Jofré, Viviana; Rodríguez Assaf, Leticia Anahí; Toro, María Eugenia; Castellanos de Figueroa, Lucía Inés; Vazquez, Fabio

2015-11-01

During certain wine fermentation processes, yeasts, and mainly non-Saccharomyces strains, produce and secrete enzymes such as β-glucosidases, proteases, pectinases, xylanases and amylases. The effects of enzyme activity on the aromatic quality of wines during grape juice fermentation, using different co-inoculation strategies of non-Saccharomyces and Saccharomyces cerevisiae yeasts, were assessed in the current study. Three strains with appropriate enological performance and high enzymatic activities, BSc562 (S. cerevisiae), BDv566 (Debaryomyces vanrijiae) and BCs403 (Candida sake), were assayed in pure and mixed Saccharomyces/non-Saccharomyces cultures. β-Glucosidase, pectinase, protease, xylanase and amylase activities were quantified during fermentations. The aromatic profile of pure and mixed cultures was determined at the end of each fermentation. In mixed cultures, non-Saccharomyces species were detected until day 4-5 of the fermentation process, and highest populations were observed in MSD2 (10% S. cerevisiae/90% D. vanrijiae) and MSC1 (1% S. cerevisiae/99% C. sake). According to correlation and multivariate analysis, MSD2 presented the highest concentrations of terpenes and higher alcohols which were associated with pectinase, amylase and xylanase activities. On the other hand, MSC1 high levels of β-glucosidase, proteolytic and xylanolytic activities were correlated to esters and fatty acids. Our study contributes to a better understanding of the effect of enzymatic activities by yeasts on compound transformations that occur during wine fermentation.

InXy and SeXy, compact heterologous reporter proteins for mammalian cells.

PubMed

Fluri, David A; Kelm, Jens M; Lesage, Guillaume; Baba, Marie Daoud-El; Fussenegger, Martin

2007-10-15

Mammalian reporter proteins are essential for gene-function analysis, drugscreening initiatives and as model product proteins for biopharmaceutical manufacturing. Bacillus subtilis can maintain its metabolism by secreting Xylanase A (XynA), which converts xylan into shorter xylose oligosaccharides. XynA is a family 11 xylanase monospecific for D-xylose containing substrates. Mammalian cells transgenic for constitutive expression of wild-type xynA showed substantial secretion of this prokaryotic enzyme. Deletion analysis confirmed that a prokaryotic signal sequence encoded within the first 81 nucleotides was compatible with the secretory pathway of mammalian cells. Codon optimization combined with elimination of the prokaryotic signal sequence resulted in an exclusively intracellular mammalian Xylanase A variant (InXy) while replacement by an immunoglobulin-derived secretion signal created an optimal secreted Xylanase A derivative (SeXy). A variety of chromogenic and fluorescence-based assays adapted for use with mammalian cells detected InXy and SeXy with high sensitivity and showed that both reporter proteins resisted repeated freeze/thaw cycles, remained active over wide temperature and pH ranges, were extremely stable in human serum stored at room temperature and could independently be quantified in samples also containing other prominent reporter proteins such as the human placental alkaline phosphatase (SEAP) and the Bacillus stearothermophilus-derived secreted alpha-amylase (SAMY). Glycoprofiling revealed that SeXy produced in mammalian cells was N- glycosylated at four different sites, mutation of which resulted in impaired secretion. SeXy was successfully expressed in a variety of mammalian cell lines and primary cells following transient transfection and transduction with adeno-associated virus particles (AAV) engineered for constitutive SeXy expression. Intramuscular injection of transgenic AAVs into mice showed significant SeXy levels in the bloodstream

A novel member of glycoside hydrolase family 30 subfamily 8 with altered substrate specificity

PubMed Central

St John, Franz J.; Dietrich, Diane; Crooks, Casey; Pozharski, Edwin; González, Javier M.; Bales, Elizabeth; Smith, Kennon; Hurlbert, Jason C.

2014-01-01

Endoxylanases classified into glycoside hydrolase family 30 subfamily 8 (GH30-8) are known to hydrolyze the hemicellulosic polysaccharide glucuronoxylan (GX) but not arabinoxylan or neutral xylooligosaccharides. This is owing to the specificity of these enzymes for the α-1,2-linked glucuronate (GA) appendage of GX. Limit hydrolysis of this substrate produces a series of aldouronates each containing a single GA substituted on the xylose penultimate to the reducing terminus. In this work, the structural and biochemical characterization of xylanase 30A from Clostridium papyro­solvens (CpXyn30A) is presented. This xylanase possesses a high degree of amino-acid identity to the canonical GH30-8 enzymes, but lacks the hallmark β8–α8 loop region which in part defines the function of this GH30 subfamily and its role in GA recognition. CpXyn30A is shown to have a similarly low activity on all xylan substrates, while hydrolysis of xylohexaose revealed a competing transglycosylation reaction. These findings are directly compared with the model GH30-8 enzyme from Bacillus subtilis, XynC. Despite its high sequence identity to the GH30-8 enzymes, CpXyn30A does not have any apparent specificity for the GA appendage. These findings confirm that the typically conserved β8–α8 loop region of these enzymes influences xylan substrate specificity but not necessarily β-1,4-xylanase function. PMID:25372685

Phenylacetic and Phenylpropionic Acids Do Not Affect Xylan Degradation by Ruminococcus albus

PubMed Central

Reveneau, Carine; Adams, Sarah E.; Cotta, M. A.; Morrison, M.

2003-01-01

Since the addition of either ruminal fluid or a combination of phenylacetic and phenylpropionic acids (PAA/PPA) has previously been shown to dramatically improve cellulose degradation and growth of Ruminococcus albus, it was of interest to determine the effects of these additives on xylan-grown cultures. Although cell-bound xylanase activity increased when either PAA/PPA or ruminal fluid was added to the growth medium, total xylanase did not change, and neither of these supplements affected the growth or xylan-degrading capacity of R. albus 8. Similarly, neither PAA/PPA nor ruminal fluid affected xylan degradation by multiple strains of R. albus when xylan prepared from oat spelts was used as a carbohydrate source. These results show that the xylanolytic potential of R. albus is not conditional on the availability of PAA/PPA or other components of ruminal fluid. PMID:14602663

Enzymatic hydrolysis of lignocellulosic biomass by Kitasatospora sp. to produce xylo-oligosaccharides (XOS)

NASA Astrophysics Data System (ADS)

Rahmani, Nanik; Jannah, Alifah Mafatikhul; Lisdiyanti, Puspita; Prasetya, Bambang; Yopi

2017-11-01

The optimizations of enzymatic hydrolysis to produce of xylo-oligosaccharides (XOs) from three different lignocellulosic biomasses were investigated. Sugarcane bagasse, oil palm empty fruit bunch, and rice straw contain rich hemicelluloses especially hetero-xylan which can be hydrolyzes by endo-xylanase enzyme. Enzymatic hydrolysis of sugarcane bagasse by endo-xylanase from Kitasatospora sp. was optimum at temperature hydrolysis 30 °C using 16 U of enzyme concentrations and 4 % substrate concentrations, while oil palm empty fruit bunchwas optimum at temperature hydrolysis 30 °C using 16 U of enzyme concentrations and 5 % substrate concentrations, and rice straw was optimum at 40 °C temperature hydrolysis using 16 U of enzyme concentrations and 4 % substrate concentrations. The hydrolysis products were analyzed by TLC and HPLC. The main product hydrolysis for sugarcane bagasse, oil palm empty fruit bunch and rice straw are xylobiose.

Effects of exogenous proteases without or with carbohydrases on nutrient digestibility and disappearance of non-starch polysaccharides in broiler chickens

PubMed Central

Olukosi, O. A.; Beeson, L. A.; Englyst, K.; Romero, L. F.

2015-01-01

The objective of the current study was to evaluate the effect of a subtilisin protease, without or with inclusion of carbohydrases, on digestibility and retention of energy and protein, as well as the solubilization and disappearance of non-starch polysaccharides (NSP) from corn-soybean meal based diets fed to broiler chickens. Two hundred eighty-eight Ross 308 male broiler chickens were used for the experiment. On d 14, the birds were weighed and allocated to 6 treatments and 8 replicates per treatment with 6 birds per replicate. Treatments were: 1) corn-soybean meal based control diet; 2) control diet plus supplemental protease at 5,000 (P5000) protease units (PU)/kg); 3) control plus 10,000 PU/kg protease (P10000); or control plus an enzyme combination containing xylanase, amylase, and protease (XAP) added to achieve protease activity of: 4) 2,500 PU/kg (XAP2500); 5) 5,000 PU/kg (XAP5000); or 6) 10,000 PU/kg (XAP10000). The enzymes in XAP were combined at fixed ratios of 10:1:25 of xylanase:amylase:protease. Data were analyzed by ANOVA and specific orthogonal contrasts between treatments were performed. Addition of xylanase and amylase increased (P < 0.05) the ileal digestibility of protein by 4.2% and 2.1% at XAP5000 and XAP10000, respectively (relative to P5000 and P10000, respectively), exhibiting a plateau after the XAP5000 dose. Increment (P < 0.05) in AME due to protease was evident, particularly in P10000. At the ileal level, XAP reduced (P < 0.05) the flow of insoluble xylose and arabinose, which indicates an increase in the solubilization of arabinoxylan polymers in the small intestine. Protease on its own reduced (P < 0.05) the flow of insoluble arabinose but did not affect the flow of insoluble xylose. XAP reduced (P < 0.05) the pre-cecal flow of insoluble and total glucose and galactose. It was concluded that whereas protease by itself improved nutrient utilization and increased solubilization of NSP components, at the lower dose, a

The variation in chemical composition of barley feed with or without enzyme supplementation influences nutrient digestibility and subsequently affects performance in piglets.

PubMed

Clarke, L C; Sweeney, T; Curley, E; Duffy, S K; Rajauria, G; O'Doherty, J V

2018-06-01

This study investigates the effect of dietary supplementation of a β-glucanase and β-xylanase enzyme mix to barley based diets, at two different chemical compositions achieved through different agronomical conditions on growth performance, coefficient of apparent total tract digestibility (CATTD) of nutrients, selected faecal microbial populations and faecal scores in piglets. Sixty-four piglets (11.7 kg (SD 0.96)) housed in pens of two were assigned to one of four dietary treatments (n = 8). The dietary treatments were as follows: (T1) low quality barley diet, (T2) low quality barley diet containing a β-glucanase and β-xylanase enzyme supplement, (T3) high quality barley diet and (T4) high quality barley diet containing a β-glucanase and β-xylanase enzyme supplement. Piglets offered the low quality barley-based diet had a higher (p < .05) average daily gain (ADG) (0.73 vs. 0.69 kg, SEM 0.001), gain:feed (G:F) ratio (0.61 vs. 0.58 kg, SEM 0.011) and a higher CATTD (p < .001) of dry matter (DM), organic matter (OM), nitrogen (N), ash, gross energy (GE) and neutral detergent fibre (NDF) compared with piglets offered the high quality barley diet. Piglets offered the high quality barley-based diet had reduced faecal scores compared to piglets offered the low quality barley-based diet (2.44 vs. 2.57, SEM 0.036) (p < .05). There was a higher population of Lactobacillus spp. (11.6 vs. 10.5 log gene copy number/g faeces, SEM 0.177) (p < .001) and total volatile fatty acid (VFA) concentration (185 vs. 165 mmol/g faeces, SEM 5.658) (p < .001) in the faeces of piglets offered the high quality barley-based diet compared to piglets offered the low quality barley-based diet. The inclusion of a β-glucanase and β-xylanase enzyme complex had no effect on any variable measured. In conclusion, the higher quality barley-based diet showed beneficial effects on the faecal Lactobacillus spp. population and faecal scores of the piglets; however, the higher level of

Phylogenetic classification of Aureobasidium pullulans strains for production of feruloyl esterase

USDA-ARS?s Scientific Manuscript database

The objective was to phylogenetically classify diverse strains of A. pullulans and determine their production of feruloyl esterase. Seventeen strains from the A. pullulans literature were phylogenetically classified. Phenotypic traits of color variation and endo-ß-1,4-xylanase overproduction were as...

Diversity in production of xyaln-degrading enzymes among species belonging to the Trichoderma section Longibrachiatum

USDA-ARS?s Scientific Manuscript database

Xylan is an important part of plant biomass and represents a renewable raw material for biorefineries. Contrary to cellulose, the structure of hemicellulose is quite complex. Therefore, the biodegradation of xylan needs the cooperation of many enzymes. For industrial production of xylanase multienzy...

Comparison of aqueous ammonia and dilute acid pretreatment of bamboo fractions: Structure properties and enzymatic hydrolysis.

PubMed

Xin, Donglin; Yang, Zhong; Liu, Feng; Xu, Xueru; Zhang, Junhua

2015-01-01

The effect of two pretreatments methods, aqueous ammonia (SAA) and dilute acid (DA), on the chemical compositions, cellulose crystallinity, morphologic change, and enzymatic hydrolysis of bamboo fractions (bamboo yellow, timber, green, and knot) was compared. Bamboo fractions with SAA pretreatment had better hydrolysability than those with DA pretreatment. High crystallinity index resulted in low hydrolysis yield in the conversion of SAA pretreated bamboo fractions, not DA pretreated fractions. The increase of cellulase loading had modestly positive effect in the hydrolysis of both SAA and DA pretreated bamboo fractions, while supplement of xylanase significantly increased the hydrolysis of the pretreated bamboo fractions, especially after SAA pretreatment. The results indicated that SAA pretreatment was more effective than DA pretreatment in conversion of bamboo fractions, and supplementation of xylanase was necessary in effective conversion of the SAA pretreated fractions into fermentable sugars. Copyright © 2014 Elsevier Ltd. All rights reserved.

Release of feruloylated oligosaccharides from wheat bran through submerged fermentation by edible mushrooms.

PubMed

Xie, Chunyan; Wu, Zhiyan; Guo, Hongzhen; Gu, Zhenxin

2014-07-01

Wheat bran, a by-product of the flour industry, is believed to be a raw material for the production of feruloylated oligosaccharides (FOs) because of its high content of conjiont ferulic acid (FA). Studies were carried out to identify edible mushrooms that are able to release FOs from wheat bran. All the six tested mushrooms (Pleurotus ostreatus, Hericium erinaceum, Auricularia auricula, Cordyceps militaris, Agrocybe chaxingu, and Ganoderma lucium) were found to release FOs, and Agrocybe chaxingu had the highest yield, reaching 35.4 µM in wheat bran broth. Enzymes detection showed that these species secreted extracellular enzymes during fermentation, including cellulase and xylanase. Agrocybe chaxingu secreted the significant amount of xylanase (180 mU ml(-1) ), which was responsible for the release of FOs from wheat bran, while Hericium erinaceum secreted FA esterase which could disassemble FOs. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

S-protected thiolated hydroxyethyl cellulose (HEC): Novel mucoadhesive excipient with improved stability.

PubMed

Leonaviciute, Gintare; Bonengel, Sonja; Mahmood, Arshad; Ahmad Idrees, Muneeb; Bernkop-Schnürch, Andreas

2016-06-25

The aim of this study was the design of novel S-protected thiolated hydroxyethyl cellulose (HEC) and the assessment of its mucoadhesive properties and biodegradability compared to the corresponding unmodified polymer. Thiolated HEC was S-protected via disulfide bond formation between 6-mercaptonicotinamide (6-MNA) and the thiol substructures of the polymer. In vitro screening of mucoadhesive properties was accomplished using two different methods: rotating cylinder studies and viscosity measurements. Moreover, biodegradability of these polymers by cellulase, xylanase and lysozyme was evaluated. MTT and LDH assays were performed on Caco-2 cells to determine the cytotoxicity of S-protected thiolated HEC. Thiolated HEC displayed 280.09±1.70μmol of free thiol groups per gram polymer. S-protected thiolated HEC exhibiting 270.8±21.11μmol immobilized 6-MNA ligands per gram of polymer was shown being 2.4-fold more mucoadhesive compared to thiolated HEC. No mucoadhesion was observed in case of unmodified HEC. Results were in a good agreement with rheological studies. The presence of free thiol moieties likely caused lower degree of hydrolysis by xylanase, whereas the degradation by both enzymes cellulase and xylanase was more hampered when 6-MNA was introduced as ligand for thiol group's protection. Findings in cell viability revealed that all three conjugates were non-toxic. S-protection of thiolated hydroxyethyl cellulose improved mucoadhesive properties and provided pronounced stability towards enzymatic attack, that makes this excipient superior for non-invasive drug administration over thiolated and unmodified forms. Copyright © 2016 Elsevier Ltd. All rights reserved.

Production and characterization of multi-polysaccharide degrading enzymes from Aspergillus aculeatus BCC199 for saccharification of agricultural residues.

PubMed

Suwannarangsee, Surisa; Arnthong, Jantima; Eurwilaichitr, Lily; Champreda, Verawat

2014-10-01

Enzymatic hydrolysis of lignocellulosic biomass into fermentable sugars is a key step in the conversion of agricultural by-products to biofuels and value-added chemicals. Utilization of a robust microorganism for on-site production of biomass-degrading enzymes has gained increasing interest as an economical approach for supplying enzymes to biorefinery processes. In this study, production of multi-polysaccharide-degrading enzymes from Aspergillus aculeatus BCC199 by solid-state fermentation was improved through the statistical design approach. Among the operational parameters, yeast extract and soybean meal as well as the nonionic surfactant Tween 20 and initial pH were found as key parameters for maximizing production of cellulolytic and hemicellulolytic enzymes. Under the optimized condition, the production of FPase, endoglucanase, β-glucosidase, xylanase, and β-xylosidase was achieved at 23, 663, 88, 1,633, and 90 units/g of dry substrate, respectively. The multi-enzyme extract was highly efficient in the saccharification of alkaline-pretreated rice straw, corn cob, and corn stover. In comparison with commercial cellulase preparations, the BCC199 enzyme mixture was able to produce remarkable yields of glucose and xylose, as it contained higher relative activities of β-glucosidase and core hemicellulases (xylanase and β-xylosidase). These results suggested that the crude enzyme extract from A. aculeatus BCC199 possesses balanced cellulolytic and xylanolytic activities required for the efficient saccharification of lignocellulosic biomass feedstocks, and supplementation of external β-glucosidase or xylanase was dispensable. The work thus demonstrates the high potential of A. aculeatus BCC199 as a promising producer of lignocellulose-degrading enzymes for the biomass conversion industry.

Microplate-based active/inactive 1 screen for biomass degrading enzyme library purification and gene discovery

USDA-ARS?s Scientific Manuscript database

We present here a whole-cell and permeabilized E. coli cell 1' active/inactive microplate screen for ß-D-xylosidase, xylanase, endocellulase, and ferulic acid esterase enzyme activities which are critical for the enzymatic deconstruction of biomass for fuels and chemicals. Transformants from genomic...

Structural and Functional Analyses of a Glycoside Hydrolase Family 5 Enzyme with an Unexpected [beta]-Fucosidase Activity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yoshida, Shosuke; Park, David S.; Bae, Brian

2012-02-15

We present characterization of PbFucA, a family 5 glycoside hydrolase (GH5) from Prevotella bryantii B{sub 1}4. While GH5 members typically are xylanases, PbFucA shows no activity toward xylan polysaccharides. A screen against a panel of p-nitrophenol coupled sugars identifies PbFucA as a {beta}-D-fucosidase. We also present the 2.2 {angstrom} resolution structure of PbFucA and use structure-based mutational analysis to confirm the role of catalytically essential residues. A comparison of the active sites of PbFucA with those of family 5 and 51 glycosidases reveals that while the essential catalytic framework is identical between these enzymes, the steric contours of the respectivemore » active site clefts are distinct and likely account for substrate discrimination. Our results show that members of this cluster of orthologous group (COG) 5520 have {beta}-D-fucosidase activities, despite showing an overall sequence and structural similarity to GH-5 xylanases.« less

Optimization of enzymatic extraction of pectin from Opuntia ficus indica cladodes after mucilage removal.

PubMed

Bayar, Nadia; Friji, Marwa; Kammoun, Radhouane

2018-02-15

In this study, pectin was isolated from Opuntia ficus indica (OFI) cladodes after removing mucilage using the xylanase and cellulase. The process variables were optimized by the Box Behnken design with three factors at three levels. The optimal extraction condition obtained was: liquid to solid (LS), cellulase to xylanase and enzymes to matter ratios of 22ml/g, 2:1U/U and 4U/g, respectively. The simulated extraction yield of 17.91% was validated by the experimental result (16.67±0.30). The enzyme-extracted pectin from OFI cladodes (EAEPC) was low methylated, with a high uronic acid content, a water and oil holding capacity of 5.42g/g and 1.23g/g, respectively, a good foam and emulsion stability and important DPPH radical scavenging activity. Both the OFI cladodes and enzymatic process present promising alternatives to traditional sources and extraction processes of pectin, respectively. EAEPC thus represents a promising additive in food industries. Copyright © 2017. Published by Elsevier Ltd.

The global regulator LaeA controls production of citric acid and endoglucanases in Aspergillus carbonarius.

PubMed

Linde, Tore; Zoglowek, Marta; Lübeck, Mette; Frisvad, Jens Christian; Lübeck, Peter Stephensen

2016-08-01

The global regulatory protein LaeA is known for regulating the production of many kinds of secondary metabolites in Aspergillus species, as well as sexual and asexual reproduction, and morphology. In Aspergillus carbonarius, it has been shown that LaeA regulates production of ochratoxin. We have investigated the regulatory effect of LaeA on production of citric acid and cellulolytic enzymes in A. carbonarius. Two types of A. carbonarius strains, having laeA knocked out or overexpressed, were constructed and tested in fermentation. The knockout of laeA significantly decreased the production of citric acid and endoglucanases, but did not reduce the production of beta-glucosidases or xylanases. The citric acid accumulation was reduced with 74-96 % compared to the wild type. The endoglucanase activity was reduced with 51-78 %. Overexpression of LaeA seemed not to have an effect on citric acid production or on cellulose or xylanase activity.

Evaluation of gastrointestinal bacterial population for the production of holocellulose enzymes for biomass deconstruction

PubMed Central

Asem, Dhaneshwaree; Leo, Vincent Vineeth; Passari, Ajit Kumar; Tonsing, Mary Vanlalhruaii; Joshi, J. Beslin; Uthandi, Sivakumar; Hashem, Abeer; Abd_Allah, Elsayed Fathi

2017-01-01

The gastrointestinal (GI) habitat of ruminant and non-ruminant animals sustains a vast ensemble of microbes that are capable of utilizing lignocellulosic plant biomass. In this study, an indigenous swine (Zovawk) and a domesticated goat (Black Bengal) were investigated to isolate bacteria having plant biomass degrading enzymes. After screening and enzymatic quantification of eighty-one obtained bacterial isolates, Serratia rubidaea strain DBT4 and Aneurinibacillus aneurinilyticus strain DBT87 were revealed as the most potent strains, showing both cellulase and xylanase production. A biomass utilization study showed that submerged fermentation (SmF) of D2 (alkaline pretreated pulpy biomass) using strain DBT4 resulted in the most efficient biomass deconstruction with maximum xylanase (11.98 U/mL) and FPase (0.5 U/mL) activities (55°C, pH 8). The present study demonstrated that bacterial strains residing in the gastrointestinal region of non-ruminant swine are a promising source for lignocellulose degrading microorganisms that could be used for biomass conversion. PMID:29023528

Evaluation of gastrointestinal bacterial population for the production of holocellulose enzymes for biomass deconstruction.

PubMed

Asem, Dhaneshwaree; Leo, Vincent Vineeth; Passari, Ajit Kumar; Tonsing, Mary Vanlalhruaii; Joshi, J Beslin; Uthandi, Sivakumar; Hashem, Abeer; Abd Allah, Elsayed Fathi; Singh, Bhim Pratap

2017-01-01

The gastrointestinal (GI) habitat of ruminant and non-ruminant animals sustains a vast ensemble of microbes that are capable of utilizing lignocellulosic plant biomass. In this study, an indigenous swine (Zovawk) and a domesticated goat (Black Bengal) were investigated to isolate bacteria having plant biomass degrading enzymes. After screening and enzymatic quantification of eighty-one obtained bacterial isolates, Serratia rubidaea strain DBT4 and Aneurinibacillus aneurinilyticus strain DBT87 were revealed as the most potent strains, showing both cellulase and xylanase production. A biomass utilization study showed that submerged fermentation (SmF) of D2 (alkaline pretreated pulpy biomass) using strain DBT4 resulted in the most efficient biomass deconstruction with maximum xylanase (11.98 U/mL) and FPase (0.5 U/mL) activities (55°C, pH 8). The present study demonstrated that bacterial strains residing in the gastrointestinal region of non-ruminant swine are a promising source for lignocellulose degrading microorganisms that could be used for biomass conversion.

Enzymatic saccharification of biologically pre-treated wheat straw with white-rot fungi.

PubMed

Dias, Albino A; Freitas, Gil S; Marques, Guilhermina S M; Sampaio, Ana; Fraga, Irene S; Rodrigues, Miguel A M; Evtuguin, Dmitry V; Bezerra, Rui M F

2010-08-01

Wheat straw was submitted to a pre-treatment by the basidiomycetous fungi Euc-1 and Irpex lacteus, aiming to improve the accessibility of cellulose towards enzymatic hydrolysis via previous selective bio-delignification. This allowed the increase of substrate saccharification nearly four and three times while applying the basidiomycetes Euc-1 and I. lacteus, respectively. The cellulose/lignin ratio increased from 2.7 in the untreated wheat straw to 5.9 and 4.6 after the bio-treatment by the basidiomycetes Euc-1 and I. lacteus, respectively, thus evidencing the highly selective lignin biodegradation. The enzymatic profile of both fungi upon bio-treatment of wheat straw have been assessed including laccase, manganese-dependent peroxidase, lignin peroxidase, carboxymethylcellulase, xylanase, avicelase and feruloyl esterase activities. The difference in efficiency and selectivity of delignification within the two fungi treatments was interpreted in terms of specific lignolytic enzyme profiles and moderate xylanase and cellulolytic activities. (c) 2010 Elsevier Ltd. All rights reserved.

Cloning and characterization of alpha-glucuronidase enzyme

USDA-ARS?s Scientific Manuscript database

Hemicellulose is the second largest source of biomass on Earth. Xylan, a polymer of beta-1,4-linked xylose residues, is a common component of hemicellulose. The enzymes xylanase and beta-xylosidase hydrolyze the xylan into xylose which can then be fermented into value-added products. However, the...

Enzyme processes for pulp and paper : a review of recent developments

Treesearch

William R. Kenealy; Thomas W. Jeffries

2003-01-01

The pulp and paper industry is applying new, ecologically sound technology in its manufacturing processes. Many interesting enzymatic applications have been proposed in the literature. Implemented technologies tend to change the existing industrial process as little as possible. Commercial applications include xylanases in prebleaching kraft pulps and various enzymes...

Antagonists' impact on enzymatic response in wilt infected cotton plants

USDA-ARS?s Scientific Manuscript database

A number of PR-proteins possess enzymatic activity. As such, these proteins maybe indicators of defensive response of plants. Thus, we have conducted a comparative analysis of beta-1,3-glucanase, peroxidase and xylanase activity in cotton plants to determine how these enzymes are affected by the pat...

Bifidobacterium longum D2 enhances microbial degradation of long-chain arabinoxylans in an in vitro model of the proximal colon.

PubMed

Truchado, P; Van den Abbeele, P; Rivière, A; Possemiers, S; De Vuyst, L; Van de Wiele, T

2015-01-01

Long-chain arabinoxylans (LC-AX) are degraded in the colon by intestinal bacteria possessing AX-degrading enzymes, such as bifidobacteria. Enzymatic activity of intestinal bacterial might vary depending on the composition of the gut microbiota. To compare the enzymatic activities of the bacterial gut communities of two healthy individuals (donors D1 and D2), these bacterial communities were inoculated into in vitro model M-SHIME(®). Differences in xylanase activities and denaturing gradient gel electrophoresis profiles, in particular a DNA-band corresponding with Bifidobacterium longum, were found in the proximal colon vessel. 16S rRNA gene sequencing analysis demonstrated the presence of two different B. longum species in these bacterial communities, showing 99% gene sequence similarity with B. longum NCC2705 and B. longum. subsp. longum KACC 91563, respectively, further referred to as B. longum D1 and B. longum D2. When grown on LC-AX as the sole added energy source, B. longum D2 displayed significantly higher activities of β-xylanase (5.3-fold), β-xylosidase (2.9-fold), and α-arabinofuranosidase (1.5-fold), respectively, compared to B. longum D1. When B. longum D2 was inoculated in the M-SHIME, inoculated with the bacterial gut communities of the individual with low AX-degrading enzyme activities, the β-xylanase activity increased (1.5-fold) in the proximal vessel. We demonstrated the presence of differences in LC-AX degrading enzyme activities of the bacterial gut communities of two individuals in the in vitro M-SHIME model, which could be linked to the presence of a potent AX-degrading B. longum (D2) strain.

Droplet-based microfluidic high-throughput screening of heterologous enzymes secreted by the yeast Yarrowia lipolytica.

PubMed

Beneyton, Thomas; Thomas, Stéphane; Griffiths, Andrew D; Nicaud, Jean-Marc; Drevelle, Antoine; Rossignol, Tristan

2017-01-31

Droplet-based microfluidics is becoming an increasingly attractive alternative to microtiter plate techniques for enzymatic high-throughput screening (HTS), especially for exploring large diversities with lower time and cost footprint. In this case, the assayed enzyme has to be accessible to the substrate within the water-in-oil droplet by being ideally extracellular or displayed at the cell surface. However, most of the enzymes screened to date are expressed within the cytoplasm of Escherichia coli cells, which means that a lysis step must take place inside the droplets for enzyme activity to be assayed. Here, we take advantage of the excellent secretion abilities of the yeast Yarrowia lipolytica to describe a highly efficient expression system particularly suitable for the droplet-based microfluidic HTS. Five hydrolytic genes from Aspergillus niger genome were chosen and the corresponding five Yarrowia lipolytica producing strains were constructed. Each enzyme (endo-β-1,4-xylanase B and C; 1,4-β-cellobiohydrolase A; endoglucanase A; aspartic protease) was successfully overexpressed and secreted in an active form in the crude supernatant. A droplet-based microfluidic HTS system was developed to (a) encapsulate single yeast cells; (b) grow yeast in droplets; (c) inject the relevant enzymatic substrate; (d) incubate droplets on chip; (e) detect enzymatic activity; and (f) sort droplets based on enzymatic activity. Combining this integrated microfluidic platform with gene expression in Y. lipolytica results in remarkably low variability in the enzymatic activity at the single cell level within a given monoclonal population (<5%). Xylanase, cellobiohydrolase and protease activities were successfully assayed using this system. We then used the system to screen for thermostable variants of endo-β-1,4-xylanase C in error-prone PCR libraries. Variants displaying higher thermostable xylanase activities compared to the wild-type were isolated (up to 4.7-fold improvement

Ligand bound structures of a glycosyl hydrolase family 30 glucuronoxylan xylanohydrolase

Treesearch

Franz St. Johns; Jason C. Hurlbert; John D. Rice; James F. Preston; Edwin Pozharski

2011-01-01

Xylanases of glycosyl hydrolase family 30 (GH30) have been shown to cleave β-1,4 linkages of 4-O-methylglucuronoxylan (MeGXn) as directed by the position along the xylan chain of an α-1,2-linked 4-O-methylglucuronate (MeGA) moiety. Complete hydrolysis of MeGXn by...

Genome structure of bacillus cereus tsu1 and genes involved in cellulose degradation and poly-3-hydroxybutyrate synthesis

USDA-ARS?s Scientific Manuscript database

In previous work, we reported on the isolation and genome sequence analysis of Bacillus cereus strain tsu1 NCBI accession number JPYN00000000. The 36 scaffolds in the assembled tsu1 genome were all aligned with B. cereus B4264 genome with variations. Genes encoding for xylanase and cellulase and the...

Identification of Pectin Degrading Enzymes Secreted by Xanthomonas oryzae pv. oryzae and Determination of Their Role in Virulence on Rice

PubMed Central

Tayi, Lavanya; Maku, Roshan V.; Patel, Hitendra Kumar; Sonti, Ramesh V.

2016-01-01

Xanthomonas oryzae pv.oryzae (Xoo) causes the serious bacterial blight disease of rice. Xoo secretes a repertoire of plant cell wall degrading enzymes (CWDEs) like cellulases, xylanases, esterases etc., which act on various components of the rice cell wall. The major cellulases and xylanases secreted by Xoo have been identified and their role in virulence has been determined. In this study, we have identified some of the pectin degrading enzymes of Xoo and assessed their role in virulence. Bioinformatics analysis indicated the presence of four pectin homogalacturonan (HG) degrading genes in the genome of Xoo. The four HG degrading genes include one polygalacturonase (pglA), one pectin methyl esterase (pmt) and two pectate lyases (pel and pelL). There was no difference in the expression of pglA, pmt and pel genes by laboratory wild type Xoo strain (BXO43) grown in either nutrient rich PS medium or in plant mimic XOM2 medium whereas the expression of pelL gene was induced in XOM2 medium as indicated by qRT-PCR experiments. Gene disruption mutations were generated in each of these four genes. The polygalacturonase mutant pglA- was completely deficient in degrading the substrate Na-polygalacturonicacid (PGA). Strains carrying mutations in the pmt, pel and pelL genes were as efficient as wild type Xoo (BXO43) in cleaving PGA. These observations clearly indicate that PglA is the major pectin degrading enzyme produced by Xoo. The pectin methyl esterase, Pmt, is the pectin de-esterifying enzyme secreted by Xoo as evident from the enzymatic activity assay performed using pectin as the substrate. Mutations in the pglA, pmt, pel and pelL genes have minimal effects on virulence. This suggests that, as compared to cellulases and xylanases, the HG degrading enzymes may not have a major role in the pathogenicity of Xoo. PMID:27907079

The Glucuronic Acid Utilization Gene Cluster from Bacillus stearothermophilus T-6

PubMed Central

Shulami, Smadar; Gat, Orit; Sonenshein, Abraham L.; Shoham, Yuval

1999-01-01

A λ-EMBL3 genomic library of Bacillus stearothermophilus T-6 was screened for hemicellulolytic activities, and five independent clones exhibiting β-xylosidase activity were isolated. The clones overlap each other and together represent a 23.5-kb chromosomal segment. The segment contains a cluster of xylan utilization genes, which are organized in at least three transcriptional units. These include the gene for the extracellular xylanase, xylanase T-6; part of an operon coding for an intracellular xylanase and a β-xylosidase; and a putative 15.5-kb-long transcriptional unit, consisting of 12 genes involved in the utilization of α-d-glucuronic acid (GlcUA). The first four genes in the potential GlcUA operon (orf1, -2, -3, and -4) code for a putative sugar transport system with characteristic components of the binding-protein-dependent transport systems. The most likely natural substrate for this transport system is aldotetraouronic acid [2-O-α-(4-O-methyl-α-d-glucuronosyl)-xylotriose] (MeGlcUAXyl3). The following two genes code for an intracellular α-glucuronidase (aguA) and a β-xylosidase (xynB). Five more genes (kdgK, kdgA, uxaC, uxuA, and uxuB) encode proteins that are homologous to enzymes involved in galacturonate and glucuronate catabolism. The gene cluster also includes a potential regulatory gene, uxuR, the product of which resembles repressors of the GntR family. The apparent transcriptional start point of the cluster was determined by primer extension analysis and is located 349 bp from the initial ATG codon. The potential operator site is a perfect 12-bp inverted repeat located downstream from the promoter between nucleotides +170 and +181. Gel retardation assays indicated that UxuR binds specifically to this sequence and that this binding is efficiently prevented in vitro by MeGlcUAXyl3, the most likely molecular inducer. PMID:10368143

New perspective on glycoside hydrolase binding to lignin from pretreated corn stover

DOE PAGES

Yarbrough, John M.; Mittal, Ashutosh; Mansfield, Elisabeth; ...

2015-12-18

Background: Non-specific binding of cellulases to lignin has been implicated as a major factor in the loss of cellulase activity during biomass conversion to sugars. It is believed that this binding may strongly impact process economics through loss of enzyme activities during hydrolysis and enzyme recycling scenarios. The current model suggests glycoside hydrolase activities are lost though non-specific/non-productive binding of carbohydrate-binding domains to lignin, limiting catalytic site access to the carbohydrate components of the cell wall. Results: In this study, we compared component enzyme affinities of a commercial Trichoderma reesei cellulase formulation, Cellic CTec2, towards extracted corn stover lignin usingmore » sodium dodecyl sulfate-polyacrylamide gel electrophoresis and p-nitrophenyl substrate activities to monitor component binding, activity loss, and total protein binding. Protein binding was strongly affected by pH and ionic strength. β-D-glucosidases and xylanases, which do not have carbohydrate-binding modules (CBMs) and are basic proteins, demonstrated the strongest binding at low ionic strength, suggesting that CBMs are not the dominant factor in enzyme adsorption to lignin. Despite strong adsorption to insoluble lignin, β-D-glucosidase and xylanase activities remained high, with process yields decreasing only 4–15 % depending on lignin concentration. Conclusion: We propose that specific enzyme adsorption to lignin from a mixture of biomass-hydrolyzing enzymes is a competitive affinity where β-D-glucosidases and xylanases can displace CBM interactions with lignin. Process parameters, such as temperature, pH, and salt concentration influence the individual enzymes’ affinity for lignin, and both hydrophobic and electrostatic interactions are responsible for this binding phenomenon. Moreover, our results suggest that concern regarding loss of critical cell wall degrading enzymes to lignin adsorption may be unwarranted when complex

Identification of Pectin Degrading Enzymes Secreted by Xanthomonas oryzae pv. oryzae and Determination of Their Role in Virulence on Rice.

PubMed

Tayi, Lavanya; Maku, Roshan V; Patel, Hitendra Kumar; Sonti, Ramesh V

2016-01-01

Xanthomonas oryzae pv.oryzae (Xoo) causes the serious bacterial blight disease of rice. Xoo secretes a repertoire of plant cell wall degrading enzymes (CWDEs) like cellulases, xylanases, esterases etc., which act on various components of the rice cell wall. The major cellulases and xylanases secreted by Xoo have been identified and their role in virulence has been determined. In this study, we have identified some of the pectin degrading enzymes of Xoo and assessed their role in virulence. Bioinformatics analysis indicated the presence of four pectin homogalacturonan (HG) degrading genes in the genome of Xoo. The four HG degrading genes include one polygalacturonase (pglA), one pectin methyl esterase (pmt) and two pectate lyases (pel and pelL). There was no difference in the expression of pglA, pmt and pel genes by laboratory wild type Xoo strain (BXO43) grown in either nutrient rich PS medium or in plant mimic XOM2 medium whereas the expression of pelL gene was induced in XOM2 medium as indicated by qRT-PCR experiments. Gene disruption mutations were generated in each of these four genes. The polygalacturonase mutant pglA- was completely deficient in degrading the substrate Na-polygalacturonicacid (PGA). Strains carrying mutations in the pmt, pel and pelL genes were as efficient as wild type Xoo (BXO43) in cleaving PGA. These observations clearly indicate that PglA is the major pectin degrading enzyme produced by Xoo. The pectin methyl esterase, Pmt, is the pectin de-esterifying enzyme secreted by Xoo as evident from the enzymatic activity assay performed using pectin as the substrate. Mutations in the pglA, pmt, pel and pelL genes have minimal effects on virulence. This suggests that, as compared to cellulases and xylanases, the HG degrading enzymes may not have a major role in the pathogenicity of Xoo.

Comprehensive analysis of the cellulolytic system reveals its potential for deconstruction of lignocellulosic biomass in a novel Streptomyces sp.

PubMed

Pinheiro, Guilherme L; de Azevedo-Martins, Allan C; Albano, Rodolpho M; de Souza, Wanderley; Frases, Susana

2017-01-01

The giant snail Achatina fulica is considered an invasive species in most territories in which it was introduced, due to its ability to process a large amount of lignocellulose as a consequence of the presence of a cellulolytic-associated microflora. Streptomyces are well known as crucial agents in the decomposition of complex polymers in soil environments and also as cellulolytic symbionts commonly associated with herbivore insects. Here, we employed a combination of genomic and biochemical tools for a detailed evaluation of the cellulolytic potential of Streptomyces sp. I1.2, an aerobic bacterium isolated from the intestinal lumen of A. fulica in a screening for cellulolytic bacteria. Genomic analysis revealed that the ratio and diversity of CAZy domains and GH families coded by Streptomyces sp. I1.2 are comparable to those present in other highly cellulolytic bacteria. After growth on crystalline cellulose or sugarcane bagasse as sole carbon sources, the functionality of several genes encoding endoglucanases, cellobiohydrolases, xylanases, CBMs, and one β-glucosidase were confirmed by the combination of enzymatic activity measurements, zymography, TLC, and cellulose-binding assays. The endoglucanases secreted by this isolate were stable at 50 °C and exhibited activity over a broad pH range between 4.0 and 8.0. The endoglucanases and cellobiohydrolases secreted by Streptomyces sp. I1.2 exhibited specific activities that were similar to the levels present in a commercial cellulase preparation from Trichoderma reesei, while I1.2 xylanase levels were even 350 % higher. The results presented here show that Streptomyces sp. I1.2 is promising for future biotechnological applications, since it is able to produce endoglucanases, cellobiohydrolases, and xylanases in appreciable amounts when grown on a low-cost residue such as sugarcane bagasse.

New perspective on glycoside hydrolase binding to lignin from pretreated corn stover

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yarbrough, John M.; Mittal, Ashutosh; Mansfield, Elisabeth

Background: Non-specific binding of cellulases to lignin has been implicated as a major factor in the loss of cellulase activity during biomass conversion to sugars. It is believed that this binding may strongly impact process economics through loss of enzyme activities during hydrolysis and enzyme recycling scenarios. The current model suggests glycoside hydrolase activities are lost though non-specific/non-productive binding of carbohydrate-binding domains to lignin, limiting catalytic site access to the carbohydrate components of the cell wall. Results: In this study, we compared component enzyme affinities of a commercial Trichoderma reesei cellulase formulation, Cellic CTec2, towards extracted corn stover lignin usingmore » sodium dodecyl sulfate-polyacrylamide gel electrophoresis and p-nitrophenyl substrate activities to monitor component binding, activity loss, and total protein binding. Protein binding was strongly affected by pH and ionic strength. β-D-glucosidases and xylanases, which do not have carbohydrate-binding modules (CBMs) and are basic proteins, demonstrated the strongest binding at low ionic strength, suggesting that CBMs are not the dominant factor in enzyme adsorption to lignin. Despite strong adsorption to insoluble lignin, β-D-glucosidase and xylanase activities remained high, with process yields decreasing only 4–15 % depending on lignin concentration. Conclusion: We propose that specific enzyme adsorption to lignin from a mixture of biomass-hydrolyzing enzymes is a competitive affinity where β-D-glucosidases and xylanases can displace CBM interactions with lignin. Process parameters, such as temperature, pH, and salt concentration influence the individual enzymes’ affinity for lignin, and both hydrophobic and electrostatic interactions are responsible for this binding phenomenon. Moreover, our results suggest that concern regarding loss of critical cell wall degrading enzymes to lignin adsorption may be unwarranted when complex

Cellulolytic enzymes, nucleic acids encoding them and methods for making and using them

DOEpatents

Gray, Kevin A [San Diego, CA; Zhao, Lishan [Emeryville, CA; Cayouette, Michelle H [San Diego, CA

2012-01-24

The invention provides polypeptides having any cellulolytic activity, e.g., a cellulase activity, a endoglucanase, a cellobiohydrolase, a beta-glucosidase, a xylanase, a mannanse, a .beta.-xylosidase, an arabinofuranosidase, and/or an oligomerase activity, polynucleotides encoding these polypeptides, and methods of making and using these polynucleotides and polypeptides. In one aspect, the invention is directed to polypeptides having any cellulolytic activity, e.g., a cellulase activity, e.g., endoglucanase, cellobiohydrolase, beta-glucosidase, xylanase, mannanse, .beta.-xylosidase, arabinofuranosidase, and/or oligomerase activity, including thermostable and thermotolerant activity, and polynucleotides encoding these enzymes, and making and using these polynucleotides and polypeptides. In one aspect, the invention provides polypeptides having an oligomerase activity, e.g., enzymes that convert recalcitrant soluble oligomers to fermentable sugars in the saccharification of biomass. The polypeptides of the invention can be used in a variety of pharmaceutical, agricultural, food and feed processing and industrial contexts. The invention also provides compositions or products of manufacture comprising mixtures of enzymes comprising at least one enzyme of this invention.

Xylose and cellulose fractionation from corncob with three different strategies and separate fermentation of them to bioethanol.

PubMed

Chen, Yefu; Dong, Boyu; Qin, Weijun; Xiao, Dongguang

2010-09-01

To the aim of efficient utilization of both of xylose and cellulose, a laboratory xylose/cellulose fractionation and separate fermentation (XCFSF) bioethanol process was performed. Three xylose/cellulose fractionation strategies: (A) dilute sulfur acid hydrolysis and detoxification, (B) lime pretreatment and xylanase hydrolysis, (C) bio-treatment with Phanerochaete chrysosporium and xylanase hydrolysis were applied to corn cobs. As a result, the maximum xylose yields obtained from A, B and C fractionation methods were 78.47%, 57.84% and 42.54%, respectively, and 96.81%, 92.14% and 80.34% of cellulose were preserved in the corresponding solid residues. The xylose dissolved in acid and enzymatic hydrolysates was fermented to ethanol by Candida shahatae and the cellulose remaining in solid residues was converted to ethanol by simultaneous saccharification and fermentation (SSF) with Saccharomyces cerevisiae. Finally, for A, B, C fractionation methods, 70.40%, 52.87%, 39.22% of hemicellulose and 89.77%, 84.30%, 71.90% of cellulose in corn cobs was converted to ethanol, respectively. Copyright 2010 Elsevier Ltd. All rights reserved.

Facilitating the enzymatic saccharification of pulped bamboo residues by degrading the remained xylan and lignin-carbohydrates complexes.

PubMed

Huang, Caoxing; He, Juan; Li, Xin; Min, Douyong; Yong, Qiang

2015-09-01

Kraft pulping was performed on bamboo residues and its impact on the chemical compositions and the enzymatic digestibility of the samples were investigated. To improve the digestibility of sample by degrading the xylan and lignin-carbohydrates complexes (LCCs), xylanase and α-L-arabinofuranosidase (AF) were supplemented with cellulase. The results showed more carbohydrates were remained in the samples pulped with low effective alkali (EA) charge, compared to conventional kraft pulping. When 120 IU/g xylanase and 15 IU/g AF were supplemented with 20 FPU/g cellulase, the xylan degradation yield of the sample pulped with 12% EA charge increased from 68.20% to 88.35%, resulting in an increased enzymatic saccharification efficiency from 58.98% to 83.23%. The amount of LCCs in this sample decreased from 8.63/100C9 to 2.99/100C9 after saccharification with these enzymes. The results indicated that degrading the remained xylan and LCCs in the pulp could improve its enzymatic digestibility. Copyright © 2015 Elsevier Ltd. All rights reserved.

Purification, crystallization and preliminary X-ray analysis of a thermostable glycoside hydrolase family 43 β-xylosidase from Geobacillus thermoleovorans IT-08

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rohman, Ali; Oosterwijk, Niels van; Kralj, Slavko

2007-11-01

The β-xylosidase was crystallized using PEG 6000 as precipitant. 5% PEG 6000 yielded bipyramid-shaped tetragonal crystals diffracting to 1.55 Å resolution, and 13% PEG 6000 gave rectangular monoclinic crystals diffracting to 1.80 Å resolution. The main enzymes involved in xylan-backbone hydrolysis are endo-1,4-β-xylanase and β-xylosidase. β-Xylosidase converts the xylo-oligosaccharides produced by endo-1,4-β-xylanase into xylose monomers. The β-xylosidase from the thermophilic Geobacillus thermoleovorans IT-08, a member of glycoside hydrolase family 43, was crystallized at room temperature using the hanging-drop vapour-diffusion method. Two crystal forms were observed. Bipyramid-shaped crystals belonging to space group P4{sub 3}2{sub 1}2, with unit-cell parameters a = bmore » = 62.53, c = 277.4 Å diffracted to 1.55 Å resolution. The rectangular crystals belonged to space group P2{sub 1}, with unit-cell parameters a = 57.94, b = 142.1, c = 153.9 Å, β = 90.5°, and diffracted to 1.80 Å resolution.« less

Use of a new Trichoderma harzianum strain isolated from the Amazon rainforest with pretreated sugar cane bagasse for on-site cellulase production.

PubMed

Delabona, Priscila da Silva; Farinas, Cristiane Sanchez; da Silva, Mateus Ribeiro; Azzoni, Sindelia Freitas; Pradella, José Geraldo da Cruz

2012-03-01

The on-site production of cellulases is an important strategy for the development of sustainable second-generation ethanol production processes. This study concerns the use of a specific cellulolytic enzyme complex for hydrolysis of pretreated sugar cane bagasse. Glycosyl hydrolases (FPase, xylanase, and β-glucosidase) were produced using a new strain of Trichoderma harzianum, isolated from the Amazon rainforest and cultivated under different conditions. The influence of the carbon source was first investigated using shake-flask cultures. Selected carbon sources were then further studied under different pH conditions using a stirred tank bioreactor. Enzymatic activities up to 121 FPU/g, 8000 IU/g, and 1730 IU/g of delignified steam-exploded bagasse+sucrose were achieved for cellulase, xylanase and β-glucosidase, respectively. This enzymatic complex was used to hydrolyze pretreated sugar cane bagasse. A comparative evaluation, using an enzymatic extract from Trichoderma reesei RUTC30, indicated similar performance of the T. harzianum enzyme complex, being a potential candidate for on-site production of enzymes. Copyright © 2011 Elsevier Ltd. All rights reserved.

Tow steps biohydrogen production: biomass pretreatment and fermentation

NASA Astrophysics Data System (ADS)

Ma, C.; Yang, H. H.; Guo, L. J.

2010-03-01

This paper investigated the pretreatment of cornstalk and integrated dark-photo fermentation for hydrogen production. Five parameters of the pretreatment experiments, including NaOH concentration, temperature, residence time, and dosage of cellulase and xylanase, were optimized through the L25 (5≙5) orthogonal test. The optimal NaOH concentration, temperature, residence time, and dosage of cellulase and xylanase were 0.5wt%, 115 °C, 3 h, 0.08g/g cornstalk, 0.08g/g cornstalk, respectively. Under the optimal conditions, 0.31g glucose/g cornstalk was obtained. The two-step fermentation consisted of dark fermentation and photo fermentation. The pretreated cornstalk was used as the substrate for dark fermentation, with cow dung as the inoculum. Then the effluents of dark fermentation were employed as the substrate for photo fermentation by photosynthetic bacteria. H2 yield of dark fermentation was 116.7 mL/g cornstalk, with H2 concentration of 41%. After photo fermentation, the total H2 yield increased to 294 mL/g cornstalk.

Characterization of cellulolytic enzymes and bioH2 production from anaerobic thermophilic Clostridium sp. TCW1.

PubMed

Lo, Yung-Chung; Huang, Chi-Yu; Cheng, Chieh-Lun; Lin, Chiu-Yue; Chang, Jo-Shu

2011-09-01

A thermophilic anaerobic bacterium Clostridium sp. TCW1 was isolated from dairy cow dung and was used to produce hydrogen from cellulosic feedstock. Extracellular cellulolytic enzymes produced from TCW1 strain were identified as endoglucanases (45, 53 and 70 kDa), exoglucanase (70 kDa), xylanases (53 and 60 kDa), and β-glucosidase (45 kDa). The endoglucanase and xylanase were more abundant. The optimal conditions for H2 production and enzyme production of the TCW1 strain were the same (60 °C, initial pH 7, agitation rate of 200 rpm). Ten cellulosic feedstock, including pure or natural cellulosic materials, were used as feedstock for hydrogen production by Clostridium strain TCW1 under optimal culture conditions. Using filter paper at 5.0 g/L resulted in the most effective hydrogen production performance, achieving a H2 production rate and yield of 57.7 ml/h/L and 2.03 mol H2/mol hexose, respectively. Production of cellulolytic enzyme activities was positively correlated with the efficiency of dark-H2 fermentation. Copyright © 2011 Elsevier Ltd. All rights reserved.

[Biodiversity and enzymes of culturable facultative-alkaliphilic actinobacteria in saline-alkaline soil in Fukang, Xinjiang].

PubMed

Zhang, Yongguang; Liu, Qing; Wang, Hongfei; Zhang, Daofeng; Chen, Jiyue; Zhang, Yuanming; Li, Wenjun

2014-02-04

In order to analyze the biodiversity of cultivable facultative-alkaliphilic actinobacteria and the enzymes they produced. Total 10 soil samples were collected from saline-alkaline environments of Fukang, Xinjiang province. Facultative-alkaliphilic actinobacteria strains were isolated and identified by 16S rRNA gene sequence analysis. Enzymes including amylase, proteinase, xylanase, and cellulase were detected. Total 116 facultative-alkaliphilic actinobacterial strains and 4 alkali-tolerant actinobacterial strains were isolated from the samples, and those strains were distributed within 22 genera in 13 families and 8 orders of actinobacteria based on their 16S rRNA gene sequence analysis. The ratio of non-predominant Streptomyces and Nocardiopsis strains were 53.3%. The positive rates of amylase, proteinase, xylanase and cellulase were 35.8, 37.6, 28.3 and 17.5%, respectively. Diverse facultative-alkaliphilic actinobacteria were discovered from saline-alkaline environments of Fukang. Facultative-alkaliphilic actinobacteria are a potential source for enzymes. The study would facilitate the knowledge of the diversity of facultative-alkaliphilic actinobacteria, and provide the technical basis for exploration of facultative-alkaliphilic actinobacteria resources.

Enhanced cellulase producing mutants developed from heterokaryotic Aspergillus strain.

PubMed

Kaur, Baljit; Oberoi, H S; Chadha, B S

2014-03-01

A heterokaryon 28, derived through protoplast fusion between Aspergillus nidulans and Aspergillus tubingensis (Dal8), was subjected cyclic mutagenesis followed by selection on increasing levels of 2-deoxy glucose (2-DG) as selection marker. The derived deregulated cellulase hyper producing mutant '64', when compared to fusant 28, produced 9.83, 7.8, 3.2, 4.2 and 19.74 folds higher endoglucanase, β-glucosidase, cellobiohydrolase, FPase and xylanase, respectively, under shake cultures. The sequence analysis of PCR amplified β-glucosidase gene from wild and mutant showed nucleotide deletion/substitution. The mutants showed highly catalytic efficient β-glucosidase as evident from low Km and high Vmax values. The expression profiling through zymogram analysis also indicated towards over-expression of cellulases. The up/down regulated expressed proteins observed through SDS-PAGE were identified by Peptide mass fingerprinting The cellulase produced by mutants in conjunction with cellulase free xylanase derived from Thermomyces lanuginosus was used for efficient utilization of alkali treated rice straw for obtaining xylo-oligosaccharides and ethanol. Copyright © 2014 Elsevier Ltd. All rights reserved.

Comparative evaluation of lignocellulolytic activities of filamentous cultures of monocentric and polycentric anaerobic fungi.

PubMed

Dagar, Sumit Singh; Kumar, Sanjay; Mudgil, Priti; Puniya, Anil Kumar

2018-04-01

Sixteen strains of monocentric and polycentric anaerobic fungi were evaluated for cellulase, xylanase and esterase activities. Though strain level variations were observed among all genera, Neocallimastix and Orpinomyces strains exhibited the highest lignocellulolytic activities. The esterase activities of monocentric group of anaerobic fungi were better than the polycentric group. Copyright © 2018 Elsevier Ltd. All rights reserved.

Draft Genome Sequence of Bacillus stratosphericus LAMA 585, Isolated from the Atlantic Deep Sea

PubMed Central

Cabral, Alencar; Andreote, Fernando Dini; Cavalett, Angélica; Pessatti, Marcos Luiz; Dini-Andreote, Francisco; da Silva, Marcus Adonai Castro

2013-01-01

Bacillus stratosphericus LAMA 585 was isolated from the Mid-Atlantic-Ridge seafloor (5,500-m depth). This bacterium presents the capacity for cellulase, xylanase, and lipase production when growing aerobically in marine-broth media. Genes involved in the tolerance of oligotrophic and extreme conditions and prospection of biotechnological products were annotated in the draft genome (3.7 Mb). PMID:23640380

Enzyme Production by Industrially Relevant Fungi Cultured on Coproduct From Corn Dry Grind Ethanol Plants

NASA Astrophysics Data System (ADS)

Ximenes, Eduardo A.; Dien, Bruce S.; Ladisch, Michael R.; Mosier, Nathan; Cotta, Michael A.; Li, Xin-Liang

Distillers dried grain with solubles (DDGS) is the major coproduct produced at a dry grind ethanol facility. Currently, it is sold primarily as a ruminant animal feed. DDGS is low cost and relatively high in protein and fiber contents. In this study, DDGS was investigated as carbon source for extracellular hydrolytic enzyme production. Two filamentous fungi, noted for their high cellulolytic and hemicellulolytic enzyme titers, were grown on DDGS: Trichoderma reesei Rut C-30 and Aspergillus niger NRRL 2001. DDGS was either used as delivered from the plant (untreated) or after being pretreated with hot water. Both microorganisms secreted a broad range of enzymes when grown on DDGS. Higher xylanase titers were obtained when cultured on hot water DDGS compared with growth on untreated DDGS. Maximum xylanase titers were produced in 4 d for A. niger and 8 d for T. reesei in shake flask cultures. Larger amounts of enzymes were produced in bioreactors (5L) either equipped with Rushton (for T. reesei) or updraft marine impellers (A. niger). Initial production titers were lower for bioreactor than for flask cultures, especially for T. reesei cultures. Improvement of enzyme titers were obtained using fed-batch feeding schemes.

Characterization of Lignocellulolytic Activities from a Moderate Halophile Strain of Aspergillus caesiellus Isolated from a Sugarcane Bagasse Fermentation

PubMed Central

Miranda-Miranda, Estefan; Sánchez-Reyes, Ayixón; Cuervo-Soto, Laura; Aceves-Zamudio, Denise; Atriztán-Hernández, Karina; Morales-Herrera, Catalina; Rodríguez-Hernández, Rocío; Folch-Mallol, Jorge

2014-01-01

A moderate halophile and thermotolerant fungal strain was isolated from a sugarcane bagasse fermentation in the presence of 2 M NaCl that was set in the laboratory. This strain was identified by polyphasic criteria as Aspergillus caesiellus. The fungus showed an optimal growth rate in media containing 1 M NaCl at 28°C and could grow in media added with up to 2 M NaCl. This strain was able to grow at 37 and 42°C, with or without NaCl. A. caesiellus H1 produced cellulases, xylanases, manganese peroxidase (MnP) and esterases. No laccase activity was detected in the conditions we tested. The cellulase activity was thermostable, halostable, and no differential expression of cellulases was observed in media with different salt concentrations. However, differential band patterns for cellulase and xylanase activities were detected in zymograms when the fungus was grown in different lignocellulosic substrates such as wheat straw, maize stover, agave fibres, sugarcane bagasse and sawdust. Optimal temperature and pH were similar to other cellulases previously described. These results support the potential of this fungus to degrade lignocellulosic materials and its possible use in biotechnological applications. PMID:25162614

Enzymatic pulp upgrade for producing high-value cellulose out of a Kraft paper pulp.

PubMed

Hutterer, Christian; Kliba, Gerhard; Punz, Manuel; Fackler, Karin; Potthast, Antje

2017-07-01

The high-yield separation of polymeric parts from wood-derived lignocellulosic material is indispensable in biorefinery concepts. For the separation of cellulose and xylan from hardwood paper pulps to obtain pulps of high cellulose contents, simple alkaline extractions were found to be the most suitable technology, although having certain limitations. These are embodied by residual alkali resistant xylan incorporated in the pulp matrix. Further purification in order to produce pure cellulose with a low uniformity could be achieved selectively degrading residual xylan and depolymerizing the cellulose macromolecules by xylanase and cellulase. The latter help to adjust cellulose chain lengths for certain dissolving pulp grades while reducing the demand for ozone in subsequent TCF bleaching. Experiments applying different commercially available enzyme preparations revealed the dependency of xylanase performance on the residual xylan content in pulps being stimulated by additional cellulase usage. The action of the latter strongly depends on the cellulose allomorphy confirming the impact of the pulp morphology. Hence, the combined application of both types of enzymes offers a high potential for upgrading pulps in order to produce a pure and high-value cellulose product. Copyright © 2017 Elsevier Inc. All rights reserved.

Functional display of family 11 endoxylanases on the surface of phage M13.

PubMed

Beliën, T; Hertveldt, K; Van den Brande, K; Robben, J; Van Campenhout, S; Volckaert, G

2005-02-09

Two family 11 endoxylanases (EC 3.2.1.8) were functionally displayed on the surface of bacteriophage M13. The genes encoding endo-1,4-xylanase I from Aspergillus niger (ExlA) and endo-1,4-xylanase A from Bacillus subtilis (XynA) were fused to the gene encoding the minor coat protein g3p in phagemid vector pHOS31. Phage rescue resulted in functional monovalent display of the enzymes as was demonstrated by three independent tests. Firstly, purified recombinant phage particles showed a clear hydrolytic activity in an activity assay based on insoluble, chromagenic arabinoxylan substrate. Secondly, specific binding of endoxylanase displaying phages to immobilized endoxylanase inhibitors was demonstrated by interaction ELISA. Finally, two rounds of selection and amplification in a biopanning procedure against immobilized endoxylanase inhibitor were performed. Phages displaying endoxylanases were strongly enriched from background phages displaying unrelated proteins. These results open perspectives to use phage display for analysing protein-protein interactions at the interface between endoxylanases and their inhibitors. In addition, this technology should enable engineering of endoxylanases into novel variants with altered binding properties towards endoxylanase inhibitors.

Impact of cell wall-degrading enzymes on water-holding capacity and solubility of dietary fibre in rye and wheat bran.

PubMed

Petersson, Karin; Nordlund, Emilia; Tornberg, Eva; Eliasson, Ann-Charlotte; Buchert, Johanna

2013-03-15

Rye and wheat bran were treated with several xylanases and endoglucanases, and the effects on physicochemical properties such as solubility, viscosity, water-holding capacity and particle size as well as the chemical composition of the soluble and insoluble fractions of the bran were studied. A large number of enzymes with well-defined activities were used. This enabled a comparison between enzymes of different origins and with different activities as well as a comparison between the effects of the enzymes on rye and wheat bran. The xylanases derived from Bacillus subtilis were the most effective in solubilising dietary fibre from wheat and rye bran. There was a tendency for a higher degree of degradation of the soluble or solubilised dietary fibre in rye bran than in wheat bran when treated with most of the enzymes. None of the enzymes increased the water-holding capacity of the bran or the viscosity of the aqueous phase. The content of insoluble material decreased as the dietary fibre was solubilised by the enzymes. The amount of material that may form a network to retain water in the system was thereby decreased. © 2012 Society of Chemical Industry.

Cellulose- and xylan-degrading thermophilic anaerobic bacteria from biocompost.

PubMed

Sizova, M V; Izquierdo, J A; Panikov, N S; Lynd, L R

2011-04-01

Nine thermophilic cellulolytic clostridial isolates and four other noncellulolytic bacterial isolates were isolated from self-heated biocompost via preliminary enrichment culture on microcrystalline cellulose. All cellulolytic isolates grew vigorously on cellulose, with the formation of either ethanol and acetate or acetate and formate as principal fermentation products as well as lactate and glycerol as minor products. In addition, two out of nine cellulolytic strains were able to utilize xylan and pretreated wood with roughly the same efficiency as for cellulose. The major products of xylan fermentation were acetate and formate, with minor contributions of lactate and ethanol. Phylogenetic analyses of 16S rRNA and glycosyl hydrolase family 48 (GH48) gene sequences revealed that two xylan-utilizing isolates were related to a Clostridium clariflavum strain and represent a distinct novel branch within the GH48 family. Both isolates possessed high cellulase and xylanase activity induced independently by either cellulose or xylan. Enzymatic activity decayed after growth cessation, with more-rapid disappearance of cellulase activity than of xylanase activity. A mixture of xylan and cellulose was utilized simultaneously, with a significant synergistic effect observed as a reduction of lag phase in cellulose degradation.

Screening and characterization of thermo-active enzymes of biotechnological interest produced by thermophilic Bacillus isolated from hot springs in Tunisia.

PubMed

Thebti, Wajdi; Riahi, Yosra; Gharsalli, Rawand; Belhadj, Omrane

2016-01-01

As part of the contribution to the global efforts in research of thermostable enzymes being of industrial interest, we focus on the isolation of thermophilic bacteria from Tunisian hot springs. Among the collection of 161 strains of thermophilic Bacillus isolated from different samples of thermal water in Tunisia, 20% are capable of growing at 100°C and the rest grow at 70°C or above. Preliminary activity tests on media supplemented with enzyme-substrates confirmed that 35 strains produced amylases, 37 - proteases, 43 - cellulases, 31 - xylanases and 37 - mannanases. The study of the effect of temperature on enzyme activity led to determination of the optimal temperatures of activities that vary between 60 and 100°C. Several enzymes were active at high temperatures (80, 90 and 100°C) and kept their activity even at 110°C. Several isolated strains producing enzymes with high optimal temperatures of activity were described for the first time in this study. Both strains B62 and B120 are producers of amylase, protease, cellulase, xylanase, and mannanase. The sequencing of 16S DNA identified isolated strains as Geobacillus kaustophillus, Aeribacillus pallidus, Geobacillus galactosidasus and Geobacillus toebii.

Production of multi-fiber modifying enzyme from Mamillisphaeria sp. for refining of recycled paper pulp.

PubMed

Laothanachareon, Thanaporn; Khonzue, Parichart; Rattanaphan, Nakul; Tinnasulanon, Phungjai; Apawasin, Saowanee; Paemanee, Atchara; Ruanglek, Vasimon; Tanapongpipat, Sutipa; Champreda, Verawat; Eurwilaichitr, Lily

2011-01-01

Enzymatic modification of pulp is receiving increasing interest for energy reduction at the refining step of the paper-making process. In this study, the production of a multi-fiber modifying enzyme from Mamillisphaeria sp. BCC8893 was optimized in submerged fermentation using a response-surface methodology. Maximal production was obtained in a complex medium comprising wheat bran, soybean, and rice bran supplemented with yeast extract at pH 6.0 and a harvest time of 7 d, resulting in 9.2 IU/mL of carboxymethyl cellulase (CMCase), 14.9 IU/mL of filter paper activity (FPase), and 242.7 IU/mL of xylanase. Treatment of old corrugated container pulp at 0.2-0.3 IU of CMCase/g of pulp led to reductions in refining energy of 8.5-14.8%. The major physical properties were retained, including tensile and compression strength. Proteomic analysis showed that the enzyme was a complex composite of endo-glucanases, cellobiohydrolases, beta-1,4-xylanases, and beta-glucanases belonging to various glycosyl hydrolase families, suggestive of cooperative enzyme action in fiber modification, providing the basis for refining efficiency.

Agricultural waste from the tequila industry as substrate for the production of commercially important enzymes.

PubMed

Huitron, C; Perez, R; Sanchez, A E; Lappe, P; Rocha Zavaleta, L

2008-01-01

Approximately 1 million tons of Agave tequilana plants are processed annually by the Mexican Tequila industry generating vast amounts of agricultural waste. The aim of this study was to investigate the potential use of Agave tequilana waste as substrate for the production of commercially important enzymes. Two strains of Aspergillus niger (CH-A-2010 and CH-A-2016), isolated from agave fields, were found to grow and propagate in submerged cultures using Agave tequilana waste as substrate. Isolates showed simultaneous extracellular inulinase, xylanase, pectinase, and cellulase activities. Aspergillus CH-A-2010 showed the highest production of inulinase activity (1.48 U/ml), whereas Aspergillus niger CH-A-2016 produced the highest xylanase (1.52 U/ml) and endo-pectinase (2.7U/ml) activities. In both cases production of enzyme activities was significantly higher on Agave tequilana waste than that observed on lemon peel and specific polymeric carbohydrates. Enzymatic hydrolysis of raw A. tequilana stems and leaves, by enzymes secreted by the isolates yielded maximum concentrations of reducing sugars of 28.2 g/l, and 9.9 g/l respectively. In conclusion, Agave tequilana waste can be utilized as substrate for the production of important biotechnological enzymes.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Wei; Wei, Hui; Alahuhta, Markus

In order to develop a direct microbial sugar conversion platform for the production of lipids, drop-in fuels and chemicals from cellulosic biomass substrate, we chose Yarrowia lipolytica as a viable demonstration strain. Y. lipolytica is known to accumulate lipids intracellularly and is capable of metabolizing sugars to produce lipids; however, it lacks the lignocellulose-degrading enzymes needed to break down biomass directly. While research is continuing on the development of a Y. lipolytica strain able to degrade cellulose, in this study, we present successful expression of several xylanases in Y. lipolytica. The XynII and XlnD expressing Yarrowia strains exhibited an abilitymore » to grow on xylan mineral plates. This was shown by Congo Red staining of halo zones on xylan mineral plates. Enzymatic activity tests further demonstrated active expression of XynII and XlnD in Y. lipolytica. Furthermore, synergistic action in converting xylan to xylose was observed when XlnD acted in concert with XynII. Finally, the successful expression of these xylanases in Yarrowia further advances us toward our goal to develop a direct microbial conversion process using this organism.« less

USSR and Eastern Europe Scienific Abstracts. Biomedical and Behavioral Sciences, Number 60

DTIC Science & Technology

1976-12-27

DECOMPOSITION OF CELLULOSE-CONTAINING WASTES BY THE HEAT-TOLERANT FUNGUS ASPERGILLUS TERREUS 17 p Moscow MIKROBIOL. PROM-ST«. REF. SB. [Microbiological...heat-tolerant fungus Aspergillus terreus 17 p grows and forms cellulolytic enzymes and xylanase in such agricultural wastes as barley and wheat chaff...cellulose subtrate. Chaetomium globosum activity produced the C^ cellulase enzyme but little protease. A flavus, A. niger and Penicillium purpurogenum

Strategies for improved isopropanol-butanol production by a Clostridium strain from glucose and hemicellulose through consolidated bioprocessing.

PubMed

Xin, Fengxue; Chen, Tianpeng; Jiang, Yujiang; Dong, Weiliang; Zhang, Wenming; Zhang, Min; Wu, Hao; Ma, Jiangfeng; Jiang, Min

2017-01-01

High cost of traditional substrates and formation of by-products (such as acetone and ethanol) in acetone-butanol-ethanol (ABE) fermentation hindered the large-scale production of biobutanol. Here, we comprehensively characterized a newly isolated solventogenic and xylanolytic Clostridium species, which could produce butanol at a high ratio with elimination of ethanol and conversion of acetone to more value-added product, isopropanol. Ultimately, direct butanol production from hemicellulose was achieved with efficient expression of indigenous xylanase by the novel strain via consolidated bioprocessing. A novel wild-type Clostridium sp. strain NJP7 was isolated and characterized in this study, which was capable of fermenting monosaccharides, e.g., glucose into butanol via a fermentative acetone-isopropanol-butanol pathway. With enhancement of buffering capacity and alcohol dehydrogenase activities, butanol and isopropanol titer by Clostridium sp. strain NJP7 was improved to 12.21 and 1.92 g/L, respectively, and solvent productivity could be enhanced to 0.44 g/L/h. Furthermore, with in situ extraction with biodiesel, the amount of butanol and isopropanol was finally improved to 25.58 and 5.25 g/L in the fed-batch mode. Meanwhile, Clostridium sp. strain NJP7 shows capability of direct isopropanol-butanol production from hemicelluloses with expression of indigenous xylanase. 2.06 g/L of butanol and 0.54 g/L of isopropanol were finally achieved through the temperature-shift simultaneous saccharification and fermentation, representing the highest butanol production directly from hemicellulose. The co-production of isopropanol with butanol by the newly isolated Clostridium sp. strain NJP7 would add on the economical values for butanol fermentation. Furthermore, the high isopropanol-butanol production with in situ extraction would also greatly enhance the economic feasibility for fermentative production of butanol-isopropanol in large scale. Meanwhile, its direct

Visualization of enzyme activities inside earthworm biopores by in situ soil zymography

NASA Astrophysics Data System (ADS)

Thu Duyen Hoang, Thi; Razavi, Bahar. S.; Blagodatskaya, Evgenia; Kuzyakov, Yakov

2015-04-01

Earthworms can strongly activate microorganisms, increase microbial and enzyme activities and consequently the turnover of native soil organic matter. In extremely dynamic microhabitats and hotspots as biopores made by earthworms, the in situ enzyme activities are a footprint of complex biotic interactions. The effect of earthworms on the alteration of enzyme activities inside biopores and the difference between bio-pores and earthworm-free soil was visualized by in situ soil zymography (Spohn and Kuzyakov, 2014). For the first time, we prepared quantitative imaging of enzyme activities in biopores. Furthermore, we developed the zymography technique by direct application of a substrate saturated membrane to the soil to obtain better spatial resolution. Lumbricus terrestris L. was placed into transparent box (15×20×15cm). Simultaneously, maize seed was sown in the soil. Control soil box with maize and without earthworm was prepared in the same way. After two weeks when bio-pore systems were formed by earthworm, we visualized in situ enzyme activities of five hydrolytic enzymes (β-glucosidase, cellobiohydrolase, chitinase, xylanase, leucine aminopeptidase) and phosphatase. Followed by non-destructive zymography, biopore samples and control soil were destructively collected to assay enzyme kinetics by fluorogenically labeled substrates method. Zymography showed higher activity of β-glucosidase, chitinase, xylanase and phosphatase in biopores comparing to bulk soil. These differences were further confirmed by fluorimetric microplate enzyme assay detected significant difference of Vmax in four above mentioned enzymes. Vmax of β-glucosidase, chitinase, xylanase and phosphatase in biopores is 68%, 108%, 50% and 49% higher than that of control soil. However, no difference in cellobiohydrolase and leucine aminopeptidase kinetics between biopores and control soil were detected. This indicated little effect of earthworms on protein and cellulose transformation in soil

A culture-based study of the bacterial communities within the guts of nine longicorn beetle species and their exo-enzyme producing properties for degrading xylan and pectin.

PubMed

Park, Doo-Sang; Oh, Hyun-Woo; Jeong, Won-Jin; Kim, Hyangmi; Park, Ho-Yong; Bae, Kyung Sook

2007-10-01

In this study, bacterial communities within the guts of several longicorn beetles were investigated by a culture-dependent method. A total of 142 bacterial strains were isolated from nine species of longicorn beetle, including adults and larvae. A comparison of their partial 16S rRNA gene sequences showed that most of the bacteria constituting the gut communities can typically be found in soil, plants and the intestines of animals, and approximately 10% were proposed as unreported. Phylogenetic analysis demonstrated that the bacterial species comprised 7 phyla, and approximately half were Gammaproteobacteria. Actinobacteria were the second most populous group (19%), followed by Firmicutes (13%) and Alphaproteobacteria (11%). Betaproteobacteria, Flavobacteria, and Acidobacteria were minor constituents. The taxonomic compositions of the isolates were variable according to the species of longicorn beetle. Particularly, an abundance of Actinobacteria existed in Moechotypa diphysis and Mesosa hirsute, which eat broadleaf trees; however, no Actinobacteria were isolated from Corymbia rubra and Monochamus alternatus, which are needle-leaf eaters. Considerable proportions of xylanase and pectinase producing bacteria in the guts of the longicorn beetles implied that the bacteria may play an important role in the digestion of woody diets. Actinobacteria and Gammaproteobacteria were the dominant xylanase producers in the guts of the beetles.

Nitrogen amendment of green waste impacts microbial community, enzyme secretion and potential for lignocellulose decomposition

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yu, Chaowei; Harrold, Duff R.; Claypool, Joshua T.

Microorganisms involved in biomass deconstruction are an important resource for organic waste recycling and enzymes for lignocellulose bioconversion. The goals of this paper were to examine the impact of nitrogen amendment on microbial community restructuring, secretion of xylanases and endoglucanases, and potential for biomass deconstruction. Communities were cultivated aerobically at 55 °C on green waste (GW) amended with varying levels of NH 4Cl. Bacterial and fungal communities were determined using 16S rRNA and ITS region gene sequencing and PICRUSt (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States) was applied to predict relative abundance of genes involved in lignocellulose hydrolysis.more » Nitrogen amendment significantly increased secretion of xylanases and endoglucanases, and microbial activity; enzyme activities and cumulative respiration were greatest when nitrogen level in GW was between 4.13–4.56 wt% (g/g), but decreased with higher nitrogen levels. The microbial community shifted to one with increasing potential to decompose complex polymers as nitrogen increased with peak potential occurring between 3.79–4.45 wt% (g/g) nitrogen amendment. Finally, the results will aid in informing the management of nitrogen level to foster microbial communities capable of secreting enzymes that hydrolyze recalcitrant polymers in lignocellulose and yield rapid decomposition of green waste.« less

Pretreatment of Corn Stover by Low Moisture Anhydrous Ammonia (LMAA) in a Pilot-Scale Reactor and Bioconversion to Fuel Ethanol and Industrial Chemicals.

PubMed

Nghiem, Nhuan P; Senske, Gerard E; Kim, Tae Hyun

2016-04-01

Corn stover (CS) adjusted to 50, 66, and 70 % moisture was pretreated by the low moisture anhydrous ammonia (LMAA) process in a pilot-scale ammoniation reactor. After ammoniation, the 70 % moisture CS was treated at 90 and 100 °C whereas the others were treated at 90 °C only. The 70 % moisture pretreated CS then was subjected to a storage study under non-sterile conditions for 3 months. It was found that storage time did not have significant effects on the compositions of the pretreated materials and their hydrolysis by commercial enzymes. The 70 % moisture CS treated at 90 °C was used for preparation of a mix sugar hydrolysate (MSH) using combination of cellulase and xylanase. The MSH was used to prepare a corn mash at 9.5 wt% solid then subjected to ethanol fermentation by Escherichia coli KO11. The 66 % moisture CS treated at 90 °C was hydrolyzed with xylanase to make a xylose-rich hydrolysate (XRH), which was subsequently used for butyric acid fermentation by Clostridium tyrobutyricum. The resultant cellulose-enriched residue was hydrolyzed with cellulase to make a glucose-rich hydrolysate (GRH), which was subsequently used for succinic acid fermentation by E. coli AFP184.

Richness of endophytic fungi isolated from Opuntia ficus-indica Mill. (Cactaceae) and preliminary screening for enzyme production.

PubMed

Bezerra, J D P; Santos, M G S; Svedese, V M; Lima, D M M; Fernandes, M J S; Paiva, L M; Souza-Motta, C M

2012-05-01

Opuntia ficus-indica Mill. (forage cactus) is farmed with relative success in the semi-arid region of the Brazilian northeast for commercial purposes, particularly as forage and food. Endophytic microorganisms are those that can be isolated inside plant tissues and can be a new source to production of enzymes with different potentialities. The objective of this study was to describe the richness of endophytic fungi from O. ficus-indica and to detect the capacity of these species to produce extracellular hydrolytic enzymes. Forty-four endophytic fungi species were isolated. Among them, the most commonly found were Cladosporium cladosporioides (20.43%) and C. sphaerospermum (15.99%). Acremonium terricola, Monodictys castaneae, Penicillium glandicola, Phoma tropica and Tetraploa aristata are being reported for the first time as endophytic fungi for Brazil. The majority of isolated fungi exhibited enzymatic potential. Aspergillus japonicus and P. glandicola presented pectinolytic activity. Xylaria sp. was the most important among the other 14 species with positive cellulase activity. All 24 isolates analysed were xylanase-positive. Protease was best produced by isolate PF103. The results indicate that there is a significant richness of endophytic fungi in O. ficus-indica, and that these isolates indicate promising potential for deployment in biotechnological processes involving production of pectinases, cellulases, xylanases and proteases.

Streptomyces sp. TEM 33 possesses high lipolytic activity in solid-state fermentation in comparison with submerged fermentation.

PubMed

Cadirci, Bilge Hilal; Yasa, Ihsan; Kocyigit, Ali

2016-01-01

Solid-state fermentation (SSF) is a bioprocess that doesn't need an excess of free water, and it offers potential benefits for microbial cultivation for bioprocesses and product development. In comparing the antibiotic production, few detailed reports could be found with lipolytic enzyme production by Streptomycetes in SSF. Taking this knowledge into consideration, we prefer to purify Actinomycetes species as a new source for lipase production. The lipase-producing strain Streptomyces sp. TEM 33 was isolated from soil and lipase production was managed by solid-state fermentation (SSF) in comparison with submerged fermentation (SmF). Bioprocess-affecting factors like initial moisture content, incubation time, and various carbon and nitrogen additives and the other enzymes secreted into the media were optimized. Lipase activity was measured as 1.74 ± 0.0005 U/g dry substrate (gds) by the p-nitrophenylpalmitate (pNPP) method on day 6 of fermentation with 71.43% final substrate moisture content. In order to understand the metabolic priority in SSF, cellulase and xylanase activity of Streptomyces sp. TEM33 was also measured. The microorganism degrades the wheat bran to its usable form by excreting cellulases and xylanases; then it secretes the lipase that is necessary for degrading the oil in the medium.

Insights into the plant polysaccharide degradation potential of the xylanolytic yeast Pseudozyma brasiliensis.

PubMed

Kaupert Neto, Antonio Adalberto; Borin, Gustavo Pagotto; Goldman, Gustavo Henrique; Damásio, André Ricardo de Lima; Oliveira, Juliana Velasco de Castro

2016-03-01

In second-generation (2G) bioethanol production, plant cell-wall polysaccharides are broken down to release fermentable sugars. The enzymes of this process are classified as carbohydrate-active enzymes (CAZymes) and contribute substantially to the cost of biofuel production. A novel basidiomycete yeast species, Pseudozyma brasiliensis, was recently discovered. It produces an endo-β-1,4-xylanase with a higher specific activity than other xylanases. This enzyme is essential for the hydrolysis of biomass-derived xylan and has an important role in 2G bioethanol production. In spite of the P. brasiliensis biotechnological potential, there is no information about how it breaks down polysaccharides. For the first time, we characterized the secretome of P. brasiliensis grown on different carbon sources (xylose, xylan, cellobiose and glucose) and also under starvation conditions. The growth and consumption of each carbohydrate and the activity of the CAZymes of culture supernatants were analyzed. The CAZymes found in its secretomes, validated by enzymatic assays, have the potential to hydrolyze xylan, mannan, cellobiose and other polysaccharides. The data show that this yeast is a potential source of hydrolases, which can be used for biomass saccharification. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Modelling in situ enzyme potential of soils: a tool to predict soil respiration from agricultural fields

NASA Astrophysics Data System (ADS)

Shahbaz Ali, Rana; Poll, Christian; Demyan, Scott; Nkwain Funkuin, Yvonne; Ingwersen, Joachim; Wizemann, Hans-Dieter; Kandeler, Ellen

2014-05-01

The fate of soil organic carbon (SOC) is one of the largest uncertainties in predicting future climate and terrestrial ecosystem functions. Extra-cellular enzymes, produced by microorganisms, perform the very first step in SOC degradation and serve as key components in global carbon cycling. Very little information is available about the seasonal variation in the temperature sensitivity of soil enzymes. Here we aim to model in situ enzyme potentials involved in the degradation of either labile or recalcitrant organic compounds to understand the temporal variability of degradation processes. To identify the similarities in seasonal patterns of soil respiration and in situ enzyme potentials, we compared the modelled in situ enzyme activities with weekly measured soil CO2 emissions. Arable soil samples from two different treatments (4 years fallow and currently vegetated plots; treatments represent range of carbon input into soil) were collected every month from April, 2012 to April, 2013, from two different study regions (Kraichgau and Swabian Alb) in Southwest Germany. The vegetation plots were under crop rotation in both study areas. We measured activities of three enzymes including β-glucosidase, xylanase and phenoloxidase at five different temperatures. We also measured soil microbial biomass in form of microbial carbon (Cmic). Land-use and area had significant effects (P < 0.001) on the microbial biomass; fallow plots having less Cmic than vegetation plots. Potential activities of β-glucosidase (P < 0.001) and xylanase (P < 0.01) were significantly higher in the vegetation plots of the Swabian Alb region than in the Kraichgau region. In both study areas, enzyme activities were higher during vegetation period and lower during winter which points to the importance of carbon input and/or temperature and soil moisture. We calculated the temperature sensitivity (Q10) of enzyme activities based on laboratory measurements of enzyme activities at a range of incubation

Bioabatement with xylanase supplementation to reduce enzymatic hydrolysis inhibitors

USDA-ARS?s Scientific Manuscript database

Bioabatement, using the fungus Coniochaeta ligniaria NRRL30616 can effectively eliminate enzyme inhibitors from pretreated biomass hydrolysis. However, our recent research suggested that bioabatement had no beneficial effect on removing xylo-oligomers which were identified as strong inhibitors to ce...

The effect of Fusarium culmorum infection and deoxynivalenol (DON) application on proteome response in barley cultivars Chevron and Pedant.

PubMed

Kosová, Klára; Chrpová, Jana; Šantrůček, Jiří; Hynek, Radovan; Štěrbová, Lenka; Vítámvás, Pavel; Bradová, Jana; Prášil, Ilja Tom

2017-10-03

Fusarium head blight (FHB) disease adversely affects grain quality and final yield in small-grain cereals including barley. In the present study, the effect of an artificial infection with Fusarium culmorum and an application of deoxynivalenol (DON) on barley spikes of cultivars Chevron and Pedant during flowering was investigated at grain mid-dough stage (BBCH 73) 10days after pathogen inoculation (10 dai). Proteomic analysis using a two-dimensional differential gel electrophoresis (2D-DIGE) technique coupled with LC-MS/MS investigated 98 protein spots revealing quantitative or qualitative differences between the experimental variants. Protein functional annotation of 93 identified protein spots revealed that most affected functional groups represent storage proteins (globulins, hordeins), followed by proteins involved in carbohydrate metabolism (α-amylase inhibitor, β-amylase, glycolytic enzymes), amino acid metabolism (aminotransferases), defence response (chitinase, xylanase inhibitor, serpins, SGT1, universal stress protein USP), protein folding (chaperones, chaperonins), redox metabolism (ascorbate-glutathione cycle), and proteasome-dependent protein degradation. The obtained results indicate adverse effects of infection on plant proteome as well as an active plant response to pathogen as shown by enhanced levels of several inhibitors of pathogen-produced degradation enzymes (α-amylase inhibitor, xylanase inhibitor, serpins), chaperones, and other stress-related proteins (SGT1, USP). Genotypic differences were found in hordein abundance between Chevron and Pedant. Copyright © 2017 Elsevier B.V. All rights reserved.

A Highly Efficient Xylan-Utilization System in Aspergillus niger An76: A Functional-Proteomics Study.

PubMed

Gong, Weili; Dai, Lin; Zhang, Huaiqiang; Zhang, Lili; Wang, Lushan

2018-01-01

Xylan constituted with β-1,4-D-xylose linked backbone and diverse substituted side-chains is the most abundant hemicellulose component of biomass, which can be completely and rapidly degraded into fermentable sugars by Aspergillus niger . This is of great value for obtaining renewable biofuels and biochemicals. To clarify the underlying mechanisms associated with highly efficient xylan degradation, assimilation, and metabolism by A. niger , we utilized functional proteomics to analyze the secreted proteins, sugar transporters, and intracellular proteins of A. niger An76 grown on xylan-based substrates. Results demonstrated that the complete xylanolytic enzyme system required for xylan degradation and composed of diverse isozymes was secreted in a sequential order. Xylan-backbone-degrading enzymes were preferentially induced by xylose or other soluble sugars, which efficiently produced large amounts of xylooligosaccharides (XOS) and xylose; however, XOS was more efficient than xylose in triggering the expression of the key transcription activator XlnR, resulting in higher xylanase activity and shortening xylanase-production time. Moreover, the substituted XOS was responsible for improving the abundance of side-chain-degrading enzymes, specific transporters, and key reductases and dehydrogenases in the pentose catabolic pathway. Our findings indicated that industries might be able to improve the species and concentrations of xylan-degrading enzymes and shorten fermentation time by adding abundant intermediate products of natural xylan (XOS) to cultures of filamentous fungi.

Increased production of biomass-degrading enzymes by double deletion of creA and creB genes involved in carbon catabolite repression in Aspergillus oryzae.

PubMed

Ichinose, Sakurako; Tanaka, Mizuki; Shintani, Takahiro; Gomi, Katsuya

2018-02-01

In a previous study, we reported that a double gene deletion mutant for CreA and CreB, which constitute the regulatory machinery involved in carbon catabolite repression, exhibited improved production of α-amylase compared with the wild-type strain and single creA or creB deletion mutants in Aspergillus oryzae. Because A. oryzae can also produce biomass-degrading enzymes, such as xylolytic and cellulolytic enzymes, we examined the production levels of those enzymes in deletion mutants in this study. Xylanase and β-glucosidase activities in the wild-type were hardly detected in submerged culture containing xylose as the carbon source, whereas those enzyme activities were significantly increased in the single creA deletion (ΔcreA) and double creA and creB deletion (ΔcreAΔcreB) mutants. In particular, the ΔcreAΔcreB mutant exhibited >100-fold higher xylanase and β-glucosidase activities than the wild-type. Moreover, in solid-state culture, the β-glucosidase activity of the double deletion mutant was >7-fold higher than in the wild-type. These results suggested that deletion of both creA and creB genes could also efficiently improve the production levels of biomass-degrading enzymes in A. oryzae. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Step enzymatic hydrolysis of sodium hydroxide-pretreated Chinese liquor distillers' grains for ethanol production.

PubMed

Liu, Yue-Hong; Wu, Zheng-Yun; Yang, Jian; Yuan, Yu-Ju; Zhang, Wen-Xue

2014-01-01

Distillers' grains are a co-product of ethanol production. In China, only a small portion of distillers' grains have been used to feed the livestock because the amount was so huge. Nowadays, it has been reported that the distillers' grains have the potential for fuel ethanol production because they are composed of lignocelluloses and residual starch. In order to effectively convert distillers' grains to fuel ethanol and other valuable production, sodium hydroxide pretreatment, step-by-step enzymatic hydrolysis, and simultaneous saccharification and fermentation (SSF) were investigated. The residual starch was first recycled from wet distillers' grains (WDG) with glucoamylase to obtain glucose-rich liquid. The total sugar concentration was 21.3 g/L, and 111.9% theoretical starch was hydrolyzed. Then the removed-starch dry distillers' grains (RDDG) were pretreated with NaOH under optimal conditions and the pretreated dry distillers' grains (PDDG) were used for xylanase hydrolysis. The xylose concentration was 19.4 g/L and 68.6% theoretical xylose was hydrolyzed. The cellulose-enriched dry distillers' grains (CDDG) obtained from xylanase hydrolysis were used in SSF for ethanol production. The ethanol concentration was 42.1 g/L and the ethanol productivity was 28.7 g/100 g CDDG. After the experiment, approximately 80.6% of the fermentable sugars in WDG was converted to ethanol.

Optimization of sodium hydroxide pretreatment and enzyme loading for efficient hydrolysis of rice straw to improve succinate production by metabolically engineered Escherichia coli KJ122 under simultaneous saccharification and fermentation.

PubMed

Sawisit, Apichai; Jampatesh, Surawee; Jantama, Sirima Suvarnakuta; Jantama, Kaemwich

2018-07-01

Rice straw was pretreated with sodium hydroxide (NaOH) before subsequent use for succinate production by Escherichia coli KJ122 under simultaneous saccharification and fermentation (SSF). The NaOH pretreated rice straw was significantly enhanced lignin removal up to 95%. With the optimized enzyme loading of 4% cellulase complex + 0.5% xylanase (endo-glucanase 67 CMC-U/g, β-glucosidase 26 pNG-U/g and xylanase 18 CMC-U/g dry biomass), total sugar conversion reached 91.7 ± 0.8% (w/w). The physicochemical analysis of NaOH pretreated rice straw indicated dramatical changes in its structure, thereby favoring enzymatic saccharification. In batch SSF, succinate production of 69.8 ± 0.3 g/L with yield and productivity of 0.84 g/g pretreated rice straw and 0.76 ± 0.02 g/L/h, respectively, was obtained. Fed-batch SSF significantly improved succinate concentration and productivity to 103.1 ± 0.4 g/L and 1.37 ± 0.07 g/L/h with a comparable yield. The results demonstrated a feasibility of sequential saccharification and fermentation of rice straw as a promising process for succinate production in industrial scale. Copyright © 2018 Elsevier Ltd. All rights reserved.

A Highly Efficient Xylan-Utilization System in Aspergillus niger An76: A Functional-Proteomics Study

PubMed Central

Gong, Weili; Dai, Lin; Zhang, Huaiqiang; Zhang, Lili; Wang, Lushan

2018-01-01

Xylan constituted with β-1,4-D-xylose linked backbone and diverse substituted side-chains is the most abundant hemicellulose component of biomass, which can be completely and rapidly degraded into fermentable sugars by Aspergillus niger. This is of great value for obtaining renewable biofuels and biochemicals. To clarify the underlying mechanisms associated with highly efficient xylan degradation, assimilation, and metabolism by A. niger, we utilized functional proteomics to analyze the secreted proteins, sugar transporters, and intracellular proteins of A. niger An76 grown on xylan-based substrates. Results demonstrated that the complete xylanolytic enzyme system required for xylan degradation and composed of diverse isozymes was secreted in a sequential order. Xylan-backbone-degrading enzymes were preferentially induced by xylose or other soluble sugars, which efficiently produced large amounts of xylooligosaccharides (XOS) and xylose; however, XOS was more efficient than xylose in triggering the expression of the key transcription activator XlnR, resulting in higher xylanase activity and shortening xylanase-production time. Moreover, the substituted XOS was responsible for improving the abundance of side-chain-degrading enzymes, specific transporters, and key reductases and dehydrogenases in the pentose catabolic pathway. Our findings indicated that industries might be able to improve the species and concentrations of xylan-degrading enzymes and shorten fermentation time by adding abundant intermediate products of natural xylan (XOS) to cultures of filamentous fungi. PMID:29623069

Combined subcritical water and enzymatic hydrolysis for reducing sugar production from coconut husk

NASA Astrophysics Data System (ADS)

Muharja, Maktum; Junianti, Fitri; Nurtono, Tantular; Widjaja, Arief

2017-05-01

Coconut husk wastes are abundantly available in Indonesia. It has a potential to be used into alternative renewable energy sources such as hydrogen using enzymatic hydrolysis followed by a fermentation process. Unfortunately, enzymatic hydrolysis is hampered by the complex structure of lignocellulose, so the cellulose component is hard to degrade. In this study, Combined Subcritical Water (SCW) and enzymatic hydrolysis are applied to enhance fermentable, thereby reducing production of sugar from coconut husk. There were two steps in this study, the first step was coconut husk pretreated by SCW in batch reactor at 80 bar and 150-200°C for 60 minutes reaction time. Secondly, solid fraction from the results of SCW was hydrolyzed using the mixture of pure cellulose and xylanase enzymes. Analysis was conducted on untreated and SCW-treated by gravimetric assay, liquid fraction after SCW and solid fraction after enzymatic hydrolysis using DNS assay. The maximum yield of reducing sugar (including xylose, arabinose glucose, galactose, mannose) was 1.254 gr per 6 gr raw material, representing 53.95% of total sugar in coconut husk biomass which was obtained at 150°C 80 bar for 60 minutes reaction time of SCW-treated and 6 hour of enzymatic hydrolysis using mixture of pure cellulose and xylanase enzymes (18.6 U /gram of coconut husk).

Suppression of xylan endotransglycosylase PtxtXyn10A affects cellulose microfibril angle in secondary wall in aspen wood.

PubMed

Derba-Maceluch, Marta; Awano, Tatsuya; Takahashi, Junko; Lucenius, Jessica; Ratke, Christine; Kontro, Inkeri; Busse-Wicher, Marta; Kosik, Ondrej; Tanaka, Ryo; Winzéll, Anders; Kallas, Åsa; Leśniewska, Joanna; Berthold, Fredrik; Immerzeel, Peter; Teeri, Tuula T; Ezcurra, Ines; Dupree, Paul; Serimaa, Ritva; Mellerowicz, Ewa J

2015-01-01

Certain xylanases from family GH10 are highly expressed during secondary wall deposition, but their function is unknown. We carried out functional analyses of the secondary-wall specific PtxtXyn10A in hybrid aspen (Populus tremula × tremuloides). PtxtXyn10A function was analysed by expression studies, overexpression in Arabidopsis protoplasts and by downregulation in aspen. PtxtXyn10A overexpression in Arabidopsis protoplasts resulted in increased xylan endotransglycosylation rather than hydrolysis. In aspen, the enzyme was found to be proteolytically processed to a 68 kDa peptide and residing in cell walls. Its downregulation resulted in a corresponding decrease in xylan endotransglycosylase activity and no change in xylanase activity. This did not alter xylan molecular weight or its branching pattern but affected the cellulose-microfibril angle in wood fibres, increased primary growth (stem elongation, leaf formation and enlargement) and reduced the tendency to form tension wood. Transcriptomes of transgenic plants showed downregulation of tension wood related genes and changes in stress-responsive genes. The data indicate that PtxtXyn10A acts as a xylan endotransglycosylase and its main function is to release tensional stresses arising during secondary wall deposition. Furthermore, they suggest that regulation of stresses in secondary walls plays a vital role in plant development. © 2014 The Authors. New Phytologist © 2014 New Phytologist Trust.

Evaluation of certain food additives and contaminants.

PubMed

2004-01-01

This report represents the conclusions of a Joint FAO/WHO Expert Committee convened to evaluate the safety of various food additives, with a view to recommending acceptable daily intakes (ADIs) and to prepare specifications for the identity and purity of food additives. The first part of the report contains a general discussion of the principles governing the toxicological evaluation of food additives (including flavouring agents) and contaminants, assessments of intake, and the establishment and revision of specifications for food additives. A summary follows of the Committee's evaluations of toxicological and intake data on various specific food additives (alpha-amylase from Bacillus lichenformis containing a genetically engineered alpha-amylase gene from B. licheniformis, annatto extracts, curcumin, diacetyl and fatty acid esters of glycerol, D-tagatose, laccase from Myceliophthora thermophila expressed in Aspergillus oryzae, mixed xylanase, beta-glucanase enzyme preparation produced by a strain of Humicola insolens, neotame, polyvinyl alcohol, quillaia extracts and xylanase from Thermomyces lanuginosus expressed in Fusarium venenatum), flavouring agents, a nutritional source of iron (ferrous glycinate, processed with citric acid), a disinfectant for drinking-water (sodium dichloroisocyanurate) and contaminants (cadmium and methylmercury). Annexed to the report are tables summarizing the Committee's recommendations for ADIs of the food additives, recommendations on the flavouring agents considered, and tolerable intakes of the contaminants considered, changes in the status of specifications and further information requested or desired.

Production of Biomass-Degrading Multienzyme Complexes under Solid-State Fermentation of Soybean Meal Using a Bioreactor

PubMed Central

Vitcosque, Gabriela L.; Fonseca, Rafael F.; Rodríguez-Zúñiga, Ursula Fabiola; Bertucci Neto, Victor; Couri, Sonia; Farinas, Cristiane S.

2012-01-01

Biomass-degrading enzymes are one of the most costly inputs affecting the economic viability of the biochemical route for biomass conversion into biofuels. This work evaluates the effects of operational conditions on biomass-degrading multienzyme production by a selected strain of Aspergillus niger. The fungus was cultivated under solid-state fermentation (SSF) of soybean meal, using an instrumented lab-scale bioreactor equipped with an on-line automated monitoring and control system. The effects of air flow rate, inlet air relative humidity, and initial substrate moisture content on multienzyme (FPase, endoglucanase, and xylanase) production were evaluated using a statistical design methodology. Highest production of FPase (0.55 IU/g), endoglucanase (35.1 IU/g), and xylanase (47.7 IU/g) was achieved using an initial substrate moisture content of 84%, an inlet air humidity of 70%, and a flow rate of 24 mL/min. The enzymatic complex was then used to hydrolyze a lignocellulosic biomass, releasing 4.4 g/L of glucose after 36 hours of saccharification of 50 g/L pretreated sugar cane bagasse. These results demonstrate the potential application of enzymes produced under SSF, thus contributing to generate the necessary technological advances to increase the efficiency of the use of biomass as a renewable energy source. PMID:23365723

Morphological and enzymatic response of the thermotolerant fungus Fomes sp. EUM1 in solid state fermentation under thermal stress.

PubMed

Ordaz-Hernández, Armando; Ortega-Sánchez, Eric; Montesinos-Matías, Roberto; Hernández-Martínez, Ricardo; Torres-Martínez, Daniel; Loera, Octavio

2016-08-01

Thermotolerance of the fungus Fomes sp. EUM1 was evaluated in solid state fermentation (SSF). This thermotolerant strain improved both hyphal invasiveness (38%) and length (17%) in adverse thermal conditions exceeding 30°C and to a maximum of 40°C. In contrast, hyphal branching decreased by 46% at 45°C. The production of cellulases over corn stover increased 1.6-fold in 30°C culture conditions, xylanases increased 2.8-fold at 40°C, while laccase production improved 2.7-fold at 35°C. Maximum production of lignocellulolytic enzymes was obtained at elevated temperatures in shorter fermentation times (8-6 days), although the proteases appeared as a thermal stress response associated with a drop in lignocellulolytic activities. Novel and multiple isoenzymes of xylanase (four bands) and cellulase (six bands) were secreted in the range of 20-150 kDa during growth in adverse temperature conditions. However, only a single laccase isoenzyme (46 kDa) was detected. This is the first report describing the advantages of a thermotolerant white-rot fungus in SSF. These results have important implications for large-scale SSF, where effects of metabolic heat are detrimental to growth and enzyme production, which are severely affected by the formation of high temperature gradients. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

An Alignment-Free Algorithm in Comparing the Similarity of Protein Sequences Based on Pseudo-Markov Transition Probabilities among Amino Acids

PubMed Central

Li, Yushuang; Yang, Jiasheng; Zhang, Yi

2016-01-01

In this paper, we have proposed a novel alignment-free method for comparing the similarity of protein sequences. We first encode a protein sequence into a 440 dimensional feature vector consisting of a 400 dimensional Pseudo-Markov transition probability vector among the 20 amino acids, a 20 dimensional content ratio vector, and a 20 dimensional position ratio vector of the amino acids in the sequence. By evaluating the Euclidean distances among the representing vectors, we compare the similarity of protein sequences. We then apply this method into the ND5 dataset consisting of the ND5 protein sequences of 9 species, and the F10 and G11 datasets representing two of the xylanases containing glycoside hydrolase families, i.e., families 10 and 11. As a result, our method achieves a correlation coefficient of 0.962 with the canonical protein sequence aligner ClustalW in the ND5 dataset, much higher than those of other 5 popular alignment-free methods. In addition, we successfully separate the xylanases sequences in the F10 family and the G11 family and illustrate that the F10 family is more heat stable than the G11 family, consistent with a few previous studies. Moreover, we prove mathematically an identity equation involving the Pseudo-Markov transition probability vector and the amino acids content ratio vector. PMID:27918587

Hydrolysis of various thai agricultural biomasses using the crude enzyme from Aspergillus aculeatus iizuka FR60 isolated from soil

PubMed Central

Boonmee, Atcha

2012-01-01

In this study, forty-two fungi from soil were isolated and tested for their carboxymethyl cellulase (CMCase) and xylanase activities. From all isolates, the fungal isolate FR60, which was identified as Aspergillus aculeatus Iizuka, showed high activities in both CMCase and xylanase with 517 mU/mg protein and 550 mU/mg protein, respectively. The crude enzyme from A. aculeatus Iizuka FR60 could hydrolyze several agricultural residues such as corncob, and sweet sorghum leaf and stalk at comparable rates with respect to the tested commercial enzymes and with a maximum rate in rice hull hydrolysis (29 μg sugar g-1 dry weight substrate mg-1 enzyme hr-1). The highest amount of glucose was obtained from corncob by using the crude enzyme from A. aculeatus Iizuka FR60 (10.1 g/100 g dry substrate). From overall enzymatic treatment results, the lowest sugar yield was from rice hulls treatment (1.6 g/100 g dry weight) and the highest amount of reducing sugar was obtained from rice straw treatment (15.3 g/100 g dry weight). Among tested agricultural wastes, rice hull could not be effectively hydrolyzed by enzymes, whereas sugarcane leaf and stalk, and peanut shell could be effectively hydrolyzed (30-31% total sugar comparing with total sugar yield from acid treatment). PMID:24031852

Knocking out Bcsas1 in Botrytis cinerea impacts growth, development, and secretion of extracellular proteins, which decreases virulence.

PubMed

Zhang, Zhanquan; Qin, Guozheng; Li, Boqiang; Tian, Shiping

2014-06-01

Pathogenic fungi usually secrete a series of virulence factors to the extracellular environment to facilitate infection. Rab GTPases play a central role in the secretory pathway. To explore the function of Rab/GTPase in filamentous fungi, we knocked out a Rab/GTPase family gene, Bcsas1, in Botrytis cinerea, an aggressive fungal pathogen that infects more than 200 plant species. A detailed analysis was conducted on the virulence and the secretory capability of the mutants. The results indicated that knockout of Bcsas1 inhibited hyphal development and reduced sporulation of B. cinerea on potato dextrose agar plates resulting in reduced virulence on various fruit hosts. Knocking out the Bcsas1 gene led to an accumulation of transport vesicles at the hyphal tip, significantly reduced extracellular protein content, and lowered the activity of polygalacturonase and xylanase in the extracellular medium. However, mutation of Bcsas1 did not affect the expression of genes encoding polygalacturonase and xylanase, suggesting the secretion of these two family enzymes was suppressed in the mutant. Moreover, a comparative analysis of the secretome provided further evidence that the disruption of Bcsas1 in mutant strains significantly depressed the secretion of polysaccharide hydrolases and proteases. The results indicate that Bcsas1, the Rab8/SEC4-like gene, plays a crucial role in development, protein secretion, and virulence of B. cinerea.

Evaluation of certain food additives and contaminants. Eightieth report of the Joint FAO/WHO Expert Committee on Food Additives.

PubMed

2016-01-01

This report represents the conclusions of a Joint FAO/WHO Expert Committee convened to evaluate the safety of various food additives and contaminants and to prepare specifications for identity and purity. The first part of the report contains a brief description of general considerations addressed at the meeting, including updates on matters of interest to the work of the Committee. A summary follows of the Committee's evaluations of technical, toxicological and/or dietary exposure data for seven food additives (benzoates; lipase from Fusarium heterosporum expressed in Ogataea polymorpha; magnesium stearate; maltotetraohydrolase from Pseudomonas stutzeri expressed in Bacillus licheniformis; mixed β-glucanase, cellulase and xylanase from Rasamsonia emersonii; mixed β-glucanase and xylanase from Disporotrichum dimorphosporum; polyvinyl alcohol (PVA)- polyethylene glycol (PEG) graft copolymer) and two groups of contaminants (non-dioxin-like polychlorinated biphenyls and pyrrolizidine alkaloids). Specifications for the following food additives were revised or withdrawn: advantame; annatto extracts (solavnt extracted bixin, ad solvent-extracted norbixin); food additives containing aluminium and/or silicon (aluminium silicate; calcium aluminium silicate; calcium silicate; silicon dioxide, amorphous; sodium aluminium silicate); and glycerol ester of gum rosin. Annexed to the report are tables or text summarizing the toxicological and dietary exposure information and information on specifications as well as the Committees recommendations on the food additives and contaminants considered at this meeting.

Digestive enzymes from workers and soldiers of termite Nasutitermes corniger.

PubMed

Lima, Thâmarah de Albuquerque; Pontual, Emmanuel Viana; Dornelles, Leonardo Prezzi; Amorim, Poliana Karla; Sá, Roberto Araújo; Coelho, Luana Cassandra Breitenbach Barroso; Napoleão, Thiago Henrique; Paiva, Patrícia Maria Guedes

2014-10-01

The digestive apparatus of termites may have several biotechnological applications, as well as being a target for pest control. This report discusses the detection of cellulases (endoglucanase, exoglucanase, and β-glucosidase), hemicellulases (β-xylosidase, α-l-arabinofuranosidase, and β-d-xylanase), α-amylase, and proteases (trypsin-like, chymotrypsin-like, and keratinase-type) in gut extracts from Nasutitermes corniger workers and soldiers. Additionally, the effects of pH (3.0-11.0) and temperature (30-100°C) on enzyme activities were evaluated. All enzymes investigated were detected in the gut extracts of worker and soldier termites. Endoglucanase and β-xylanase were the main cellulase and hemicellulase, respectively. Zymography for proteases of worker extracts revealed polypeptides of 22, 30, and 43kDa that hydrolyzed casein, and assays using protease inhibitors showed that serine proteases were the main proteases in worker and soldier guts. The determined enzyme activities and their response to different pH and temperature values revealed that workers and soldiers contained a distinct digestive apparatus. The ability of these termites to efficiently digest the main components of lignocellulosic materials stimulates the purification of gut enzymes. Further investigation into their biotechnological potential as well as whether the enzymes detected are produced by the termites or by their symbionts is needed. Copyright © 2014 Elsevier Inc. All rights reserved.

A reusable multipurpose magnetic nanobiocatalyst for industrial applications.

PubMed

Perwez, Mohammad; Ahmad, Razi; Sardar, Meryam

2017-10-01

A multipurpose magnetic nanobiocatalyst is developed by conjugating Pectinex 3XL (a commercial enzyme containing pectinase, xylanase and cellulase activities) on 3-aminopropyl triethoxysilane activated magnetic nanoparticles. The nanobiocatalyst retained 87% of pectinase, 69% of xylanase and 58% of cellulase activity after conjugation on modified nanoparticles as compared to their soluble counterparts. Thermal stability data at 70°C showed increase in enzyme stability after conjugation to nanoparticles and the kinetic parameters (K m and V max ) remain unaltered after immobilization. The immobilized enzyme system can be successfully used upto 5th cycle after that slight decrease in enzyme activities was observed. The nanobiocatalyst retained high pectinase activities in organic solvents and chemical reagents as compared to free enzymes. DLS data shows that the nanoparticles size increases from 63nm to 86nm after immobilization. Atomic Force Microscopy data confirms the deposition of enzymes on the nanoparticles. The nanobiocatalyst was used for the clarification of pine apple and orange juice and was also used for the production of bioethanol. Hydrolysis of pretreated wheat straw produced 1.39g/l and 1.59g/l after treatment with free Pectinex 3xL and nanobiocatalyst respectively. The concentration of bioethanol also increases by 1.4 fold as compared to the free enzyme. Copyright © 2017 Elsevier B.V. All rights reserved.

Production and immobilization of enzymes by solid-state fermentation of agroindustrial waste.

PubMed

Romo Sánchez, Sheila; Gil Sánchez, Irene; Arévalo-Villena, María; Briones Pérez, Ana

2015-03-01

The recovery of by-products from agri-food industry is currently one of the major challenges of biotechnology. Castilla-La Mancha produces around three million tons of waste coming from olive oil and wine industries, both of which have a pivotal role in the economy of this region. For this reason, this study reports on the exploitation of grape skins and olive pomaces for the production of lignocellulosic enzymes, which are able to deconstruct the agroindustrial waste and, therefore, reuse them in future industrial processes. To this end, solid-state fermentation was carried out using two local fungal strains (Aspergillus niger-113 N and Aspergillus fumigatus-3). In some trials, a wheat supplementation with a 1:1 ratio was used to improve the growth conditions, and the particle size of the substrates was altered through milling. Separate fermentations were run and collected after 2, 4, 6, 8, 10 and 15 days to monitor enzymatic activity (xylanase, cellulase, β-glucosidase, pectinase). The highest values were recorded after 10 and 15 days of fermentation. The use of A. niger on unmilled grape skin yielded the best outcomes (47.05 U xylanase/g by-product). The multi-enzymatic extracts obtained were purified, freeze dried, and immobilized on chitosan by adsorption to assess the possible advantages provided by the different techniques.

Microplate-Based Evaluation of the Sugar Yield from Giant Reed, Giant Miscanthus and Switchgrass after Mild Chemical Pre-Treatments and Hydrolysis with Tailored Trichoderma Enzymatic Blends.

PubMed

Cianchetta, Stefano; Bregoli, Luca; Galletti, Stefania

2017-11-01

Giant reed, miscanthus, and switchgrass are considered prominent lignocellulosic feedstocks to obtain fermentable sugars for biofuel production. The bioconversion into sugars requires a delignifying pre-treatment step followed by hydrolysis with cellulase and other accessory enzymes like xylanase, especially in the case of alkali pre-treatments, which retain the hemicellulose fraction. Blends richer in accessory enzymes than commercial mix can be obtained growing fungi on feedstock-based substrates, thus ten selected Trichoderma isolates, including the hypercellulolytic strain Trichoderma reesei Rut-C30, were grown on giant reed, miscanthus, or switchgrass-based substrates. The produced enzymes were used to saccharify the corresponding feedstocks, compared to a commercial enzymatic mix (6 FPU/g). Feedstocks were acid (H 2 SO 4 0.2-2%, w/v) or alkali (NaOH 0.02-0.2%, w/v) pre-treated. A microplate-based approach was chosen for most of the experimental steps due to the large number of samples. The highest bioconversion was generally obtained with Trichoderma harzianum Or4/99 enzymes (78, 89, and 94% final sugar yields at 48 h for giant reed, miscanthus, and switchgrass, respectively), with significant increases compared to the commercial mix, especially with alkaline pre-treatments. The differences in bioconversion yields were only partially caused by xylanases (maximum R 2  = 0.5), indicating a role for other accessory enzymes.

Cellulolytic enzyme expression and simultaneous conversion of lignocellulosic sugars into ethanol and xylitol by a new Candida tropicalis strain.

PubMed

Mattam, Anu Jose; Kuila, Arindam; Suralikerimath, Niranjan; Choudary, Nettem; Rao, Peddy V C; Velankar, Harshad Ravindra

2016-01-01

Lignocellulosic ethanol production involves major steps such as thermochemical pretreatment of biomass, enzymatic hydrolysis of pre-treated biomass and the fermentation of released sugars into ethanol. At least two different organisms are conventionally utilized for producing cellulolytic enzymes and for ethanol production through fermentation, whereas in the present study a single yeast isolate with the capacity to simultaneously produce cellulases and xylanases and ferment the released sugars into ethanol and xylitol has been described. A yeast strain isolated from soil samples and identified as Candida tropicalis MTCC 25057 expressed cellulases and xylanases over a wide range of temperatures (32 and 42 °C) and in the presence of different cellulosic substrates [carboxymethylcellulose and wheat straw (WS)]. The studies indicated that the cultivation of yeast at 42 °C in pre-treated hydrolysate containing 0.5 % WS resulted in proportional expression of cellulases (exoglucanases and endoglucanases) at concentrations of 114.1 and 97.8 U g(-1) ds, respectively. A high xylanase activity (689.3 U g(-1) ds) was also exhibited by the yeast under similar growth conditions. Maximum expression of cellulolytic enzymes by the yeast occurred within 24 h of incubation. Of the sugars released from biomass after pretreatment, 49 g L(-1) xylose was aerobically converted into 15.8 g L(-1) of xylitol. In addition, 25.4 g L(-1) glucose released after the enzymatic hydrolysis of biomass was fermented by the same yeast to obtain an ethanol titer of 7.3 g L(-1). During the present study, a new strain of C. tropicalis was isolated and found to have potential for consolidated bioprocessing (CBP) applications. The strain could grow in a wide range of process conditions (temperature, pH) and in the presence of lignocellulosic inhibitors such as furfural, HMF and acetic acid. The new yeast produced cellulolytic enzymes over a wide temperature range and in the presence of

Silencing of the tomato phosphatidylinositol-phospholipase C2 (SlPLC2) reduces plant susceptibility to Botrytis cinerea.

PubMed

Gonorazky, Gabriela; Guzzo, María Carla; Abd-El-Haliem, Ahmed M; Joosten, Matthieu H A J; Laxalt, Ana María

2016-12-01

The tomato [Solanum lycopersicum (Sl)] phosphatidylinositol-phospholipase C (PI-PLC) gene family is composed of six members, named SlPLC1 to SlPLC6, differentially regulated on pathogen attack. We have previously shown that the fungal elicitor xylanase induces a raise of SlPLC2 and SlPLC5 transcripts and that SlPLC2, but not SlPLC5, is required for xylanase-induced expression of defense-related genes. In this work we studied the role of SlPLC2 in the interaction between tomato and the necrotrophic fungus Botrytis cinerea. Inoculation of tomato leaves with B. cinerea increases SlPLC2 transcript levels. We knocked-down the expression of SlPLC2 by virus-induced gene silencing and plant defense responses were analyzed upon B. cinerea inoculation. SlPLC2 silenced plants developed smaller necrotic lesions concomitantly with less proliferation of the fungus. Silencing of SlPLC2 resulted as well in a reduced production of reactive oxygen species. Upon B. cinerea inoculation, transcript levels of the salicylic acid (SA)-defense pathway marker gene SlPR1a were diminished in SlPLC2 silenced plants compared to non-silenced infected plants, while transcripts of the jasmonic acid (JA)-defense gene markers Proteinase Inhibitor I and II (SlPI-I and SlPI-II) were increased. This implies that SlPLC2 participates in plant susceptibility to B. cinerea. © 2016 BSPP and John Wiley & Sons Ltd.

Development of eco-friendly process for the production of bioethanol from banana peel using inhouse developed cocktail of thermo-alkali-stable depolymerizing enzymes.

PubMed

Prakash, Heena; Chauhan, Prakram Singh; General, Thiyam; Sharma, A K

2018-07-01

Conversion of agro-industrial wastes to energy is an innovative approach for waste valorization and management which also mitigates environmental pollution. In this view, present study investigated the feasibility of producing bioethanol from banana peels using cocktail of depolymerizing enzyme/s. We isolated Geobacillus stearothermophilus HPA19 from natural resource which produces cocktail of thermo-alkali-stable xylano-pectino-cellulolytic enzyme/s using wheat bran within 24 h. The optimal temperature and pH for xylanase, filter paper cellulase and pectinase were 80, 70 and 80 °C, and 9.0, 8.0 and 9.0, respectively. Cocktail enzymes showed stability at high temperature (80 °C) and pH (10.0). Ni 2+ and Zn 2+ promoted the relative activity of xylanase and FPase, whereas Na + , Ca 2+ and K + promoted pectinase activity. Cocktail was assessed in saccharification of banana peel. Reducing sugar obtained (37.06 mg ml -1 ) after one variable at a time (OVAT) method is greatly influenced by enzyme dose. Further, response surface methodology was used to optimize saccharification leading to twofold increase in reducing sugar. Maximum ethanol production (21.1 gl -1 ) was achieved through fermentation giving the efficiency of 76.5% within 30 h. Hence utilization of waste biomass for production of value-added products through biotechnological intervention not only helps to combat environmental pollution but also contributes significantly to the economy.

Effect of growth substrate, method of fermentation, and nitrogen source on lignocellulose-degrading enzymes production by white-rot basidiomycetes.

PubMed

Elisashvili, Vladimir; Kachlishvili, Eva; Penninckx, Michel

2008-11-01

The exploration of seven physiologically different white rot fungi potential to produce cellulase, xylanase, laccase, and manganese peroxidase (MnP) showed that the enzyme yield and their ratio in enzyme preparations significantly depends on the fungus species, lignocellulosic growth substrate, and cultivation method. The fruit residues were appropriate growth substrates for the production of hydrolytic enzymes and laccase. The highest endoglucanase (111 U ml(-1)) and xylanase (135 U ml(-1)) activities were revealed in submerged fermentation (SF) of banana peels by Pycnoporus coccineus. In the same cultivation conditions Cerrena maxima accumulated the highest level of laccase activity (7,620 U l(-1)). The lignified materials (wheat straw and tree leaves) appeared to be appropriate for the MnP secretion by majority basidiomycetes. With few exceptions, SF favored to hydrolases and laccase production by fungi tested whereas SSF was appropriate for the MnP accumulation. Thus, the Coriolopsis polyzona hydrolases activity increased more than threefold, while laccase yield increased 15-fold when tree leaves were undergone to SF instead SSF. The supplementation of nitrogen to the control medium seemed to have a negative effect on all enzyme production in SSF of wheat straw and tree leaves by Pleurotus ostreatus. In SF peptone and ammonium containing salts significantly increased C. polyzona and Trametes versicolor hydrolases and laccase yields. However, in most cases the supplementation of media with additional nitrogen lowered the fungi specific enzyme activities. Especially strong repression of T. versicolor MnP production was revealed.

Three feruloyl esterases in Cellulosilyticum ruminicola H1 act synergistically to hydrolyze esterified polysaccharides.

PubMed

Li, Jiabao; Cai, Shichun; Luo, Yuanming; Dong, Xiuzhu

2011-09-01

Feruloyl esterases (Faes) constitute a subclass of carboxyl esterases that specifically hydrolyze the ester linkages between ferulate and polysaccharides in plant cell walls. Until now, the described microbial Faes were mainly from fungi. In this study, we report that Cellulosilyticum ruminicola H1, a previously described fibrolytic rumen bacterium, possesses three different active feruloyl esterases, FaeI, FaeII, and FaeIII. Phylogenetic analysis classified the described bacterial Faes into two types, FaeI and FaeII in type I and FaeIII in type II. Substrate specificity assays indicated that FaeI is more active against the ester bonds in natural hemicelluloses and FaeIII preferentially attacks the ferulate esters with a small moiety, such as methyl groups, while FaeII is active on both types of substrates. Among the three feruloyl esterase genes, faeI was the only one induced significantly by xylose and xylan, while pectin appeared to moderately induce the three genes during the late log phase to stationary phase. Western blot analysis determined that FaeI and FaeIII were secreted and cytoplasmic proteins, respectively, whereas FaeII seemed to be cell associated. The addition of FaeI and FaeII but not FaeIII enhanced the activity of a xylanase on maize cob, suggesting a synergy of the former two with xylanase. Hence, we propose that the three feruloyl esterases work in concert to hydrolyze ferulate esters in natural hemicelluloses.

Feasibility test of utilizing Saccharophagus degradans 2-40(T) as the source of crude enzyme for the saccharification of lignocellulose.

PubMed

Jung, Young Hoon; Kim, Hyun Kyung; Song, Du-Sup; Choi, In-Geol; Yang, Taek Ho; Lee, Hee Jong; Seung, Doyoung; Kim, Kyoung Heon

2014-04-01

In the conversion of lignocellulose into high-value products, including fuels and chemicals, the production of cellulase and the enzymatic hydrolysis for producing fermentable sugar are the largest contributors to the cost of production of the final products. The marine bacterium Saccharophagus degradans 2-40(T) can degrade more than ten different complex polysaccharides found in the ocean, including cellulose and xylan. Accordingly, S. degradans has been actively considered as a practical source of crude enzymes needed for the saccharification of lignocellulose to produce ethanol by others including a leading commercial company. However, the overall enzyme system of S. degradans for hydrolyzing cellulose and hemicellulose has not been quantitatively evaluated yet in comparison with commercial enzymes. In this study, the inductions and activities of cellulase and xylanase of cell-free lysate of S. degradans were investigated. The growth of S. degradans cells and the activities of cellulase and xylanase were promoted by adding 2 % of cellulose and xylan mixture (cellulose:xylan = 4:3 in mass ratio) to the aquarium salt medium supplemented with 0.2 % glucose. The specific cellulase activity of the cell-free lysate of S. degradans, as determined by the filter paper activity assay, was approximately 70 times lower than those of commercial cellulases, including Celluclast 1.5 L and Accellerase 1000. These results imply that significant improvement in the cellulase activity of S. degradans is needed for the industrial uses of S. degradans as the enzyme source.

Cellulolytic and Xylanolytic Potential of High β-Glucosidase-Producing Trichoderma from Decaying Biomass

DOE PAGES

Okeke, Benedict C.

2014-08-17

Availability, cost and efficiency of microbial enzymes for lignocellulose bioconversion are central to sustainable biomass ethanol technology. Fungi enriched from decaying biomass and surface soil mixture displayed an array of strong cellulolytic and xylanolytic activities. Strains SG2 and SG4 produced a promising array of cellulolytic and xylanolytic enzymes including β-glucosidase, usually low in cultures of Trichoderma species. Nucleotide sequence analysis of internal transcribed spacer 2 (ITS2) region of rRNA gene revealed that strains SG2 and SG4 are closely related to Trichoderma inhamatum, Trichoderma piluliferum and Trichoderma aureoviride. Trichoderma sp. SG2 correspondingly displayed as much as 9.84±1.12, 48.02±2.53 and 30.10±1.11 unitsmore » mL-1 of cellulase, xylanase and β-glucosidase. Ten times dilution of culture supernatant of strain SG2 revealed that activities were about 5.34, 8.45, and 2.05 orders of magnitude higher than observed in crude culture filtrate for cellulase, xylanase, and β-glucosidase respectively, indicating that more enzymes are present to contact with substrates in biomass sacharification. In parallel experiments Trichoderma species SG2 and SG4 produced more β-glucosidase than the industrial strain Trichoderma reesei RUT-C30. Results indicate that strains SG2 and SG4 have potential for low cost in-house production of primary lignocellulose-hydrolyzing enzymes for production of biomass saccharides and biofuel in the field.« less

Strong cellulase inhibition by Mannan polysaccharides in cellulose conversion to sugars.

PubMed

Kumar, Rajeev; Wyman, Charles E

2014-07-01

Cellulase enzymes contribute a major fraction of the total cost for biological conversion of lignocellulosic biomass to fuels and chemicals. Although a several fold reduction in cellulase production costs and enhancement of cellulase activity and stability have been reported in recent years, sugar yields are still lower at low enzyme doses than desired commercially. We recently reported that hemicellulose xylan and its oligomers strongly inhibit cellulase and that supplementation of cellulase with xylanase and β-xylosidase would significantly reduce such inhibition. In this study, mannan polysaccharides and their enzymatically prepared hydrolyzates were discovered to be strongly inhibitory to fungal cellulase in cellulose conversion (>50% drop in % relative conversion), even at a small concentration of 0.1 g/L, and inhibition was much greater than experienced by other known inhibitors such as cellobiose, xylooligomers, and furfural. Furthermore, cellulase inhibition dramatically increased with heteromannan loading and mannan substitution with galactose side units. In general, enzymatically prepared hydrolyzates were less inhibitory than their respective mannan polysaccharides except highly substituted ones. Supplementation of cellulase with commercial accessory enzymes such as xylanase, pectinase, and β-glucosidase was effective in greatly relieving inhibition but only for less substituted heteromannans. However, cellulase supplementation with purified heteromannan specific enzymes relieved inhibition by these more substituted heteromannans as well, suggesting that commercial preparations need to have higher amounts of such activities to realize high sugar yields at the low enzyme protein loadings needed for low cost fuels production. © 2014 Wiley Periodicals, Inc.

Cloning, expression and characterization of β-xylosidase from Aspergillus niger ASKU28.

PubMed

Choengpanya, Khuanjarat; Arthornthurasuk, Siriphan; Wattana-amorn, Pakorn; Huang, Wan-Ting; Plengmuankhae, Wandee; Li, Yaw-Kuen; Kongsaeree, Prachumporn T

2015-11-01

β-Xylosidases catalyze the breakdown of β-1,4-xylooligosaccharides, which are produced from degradation of xylan by xylanases, to fermentable xylose. Due to their important role in xylan degradation, there is an interest in using these enzymes in biofuel production from lignocellulosic biomass. In this study, the coding sequence of a glycoside hydrolase family 3 β-xylosidase from Aspergillus niger ASKU28 (AnBX) was cloned and expressed in Pichia pastoris as an N-terminal fusion protein with the α-mating factor signal sequence (α-MF) and a poly-histidine tag. The expression level was increased to 5.7 g/l in a fermenter system as a result of optimization of only five codons near the 5' end of the α-MF sequence. The recombinant AnBX was purified to homogeneity through a single-step Phenyl Sepharose chromatography. The enzyme exhibited an optimal activity at 70°C and at pH 4.0-4.5, and a very high kinetic efficiency toward a xyloside substrate. AnBX demonstrated an exo-type activity with retention of the β-configuration, and a synergistic action with xylanase in hydrolysis of beechwood xylan. This study provides comprehensive data on characterization of a glycoside hydrolase family 3 β-xylosidase that have not been determined in any prior investigations. Our results suggested that AnBX may be useful for degradation of lignocellulosic biomass in bioethanol production, pulp bleaching process and beverage industry. Copyright © 2015 Elsevier Inc. All rights reserved.

Celluloytic enzymes, nucleic acids encoding them and methods for making and using them

DOEpatents

Gray, Kevin A; Zhao, Lishan; Cayouette, Michelle H

2015-11-04

The invention is directed to polypeptides having any cellulolytic activity, e.g., a cellulase activity, e.g., endoglucanase, cellobiohydrolase, beta-glucosidase, xylanase, mannanse, .beta.-xylosidase, arabinofuranosidase, and/or oligomerase activity, including thermostable and thermotolerant activity, and polynucleotides encoding these enzymes, and making and using these polynucleotides and polypeptides. The polypeptides of the invention can be used in a variety of pharmaceutical, agricultural, food and feed processing and industrial contexts. The invention also provides compositions or products of manufacture comprising mixtures of enzymes comprising at least one enzyme of this invention.

Celluloytic enzymes, nucleic acids encoding them and methods for making and using them

DOEpatents

Gray, Kevin A.; Zhao, Lishan; Cayouette, Michelle H.

2015-09-08

The invention is directed to polypeptides having any cellulolytic activity, e.g., a cellulase activity, e.g., endoglucanase, cellobiohydrolase, beta-glucosidase, xylanase, mannanse, .beta.-xylosidase, arabinofuranosidase, and/or oligomerase activity, including thermostable and thermotolerant activity, and polynucleotides encoding these enzymes, and making and using these polynucleotides and polypeptides. The polypeptides of the invention can be used in a variety of pharmaceutical, agricultural, food and feed processing and industrial contexts. The invention also provides compositions or products of manufacture comprising mixtures of enzymes comprising at least one enzyme of this invention.

[Comparative characteristics of microbial proteases by the level of hydrolysis of protein substrates].

PubMed

Rimareva, L V; Overchenko, M B; Serba, E M; Trifonova, V V

1997-01-01

Screening of enzyme preparations displaying a maximum proteolytic activity at pH 4.0-5.5 and effecting deep proteolysis of plant proteins was performed. Amyloprotooryzin prepared from Aspergillus oryzae 387 containing a complex of proteolytic enzymes was the most effective. The amino acid composition of the hydrolysates obtained was studied. Amyloprotooryzin increased the contents of amino acids by 108-227%, depending on the substrate used. The enzymatic complex of amyloprotooryzin was studied; in addition, proteases, alpha-amylase, exo-beta-glucanase, and xylanase were detected in the complex.

Delayed ripening of banana fruit by salicylic acid.

PubMed

Srivastava; Dwivedi

2000-09-08

Salicylic acid treatment has been found to delay the ripening of banana fruits (Musa acuminata). Fruit softening, pulp:peel ratio, reducing sugar content, invertase and respiration rate have been found to decrease in salicylic acid treated fruits as compared with control ones. The activities of major cell wall degrading enzymes, viz. cellulase, polygalacturonase and xylanase were found to be decreased in presence of salicylic acid. The major enzymatic antioxidants namely, catalase and peroxidase, were also found to be decreased in presence of salicylic acid during banana fruit ripening.

Enzyme activity screening of thermophilic bacteria isolated from Dusun Tua Hot Spring, Malaysia

NASA Astrophysics Data System (ADS)

Msarah, Marwan; Ibrahim, Izyanti; Aqma, Wan Syaidatul

2018-04-01

Thermophilic bacteria have biotechnological importance due to the availability of unique enzymes which are stable in extreme circumstances. The aim of this study includes to isolate thermophilic bacteria from hot spring and screen for important enzyme activities. Water samples from the Dusun Tua Hot Spring were collected and the physiochemical characterisation of water was measured. Eight thermophilic bacteria were isolated and determined to have at least three strong enzyme activity including protease, lipase, amylase, cellulase, pectinase and xylanase. The results showed that HuluC2 displayed all the enzyme activities and can be further studied.

Regulation of Microbe-Associated Molecular Pattern-Induced Hypersensitive Cell Death, Phytoalexin Production, and Defense Gene Expression by Calcineurin B-Like Protein-Interacting Protein Kinases, OsCIPK14/15, in Rice Cultured Cells1[W][OA

PubMed Central

Kurusu, Takamitsu; Hamada, Jumpei; Nokajima, Hiroshi; Kitagawa, Youichiro; Kiyoduka, Masahiro; Takahashi, Akira; Hanamata, Shigeru; Ohno, Ryoko; Hayashi, Teruyuki; Okada, Kazunori; Koga, Jinichiro; Hirochika, Hirohiko; Yamane, Hisakazu; Kuchitsu, Kazuyuki

2010-01-01

Although cytosolic free Ca2+ mobilization induced by microbe/pathogen-associated molecular patterns is postulated to play a pivotal role in innate immunity in plants, the molecular links between Ca2+ and downstream defense responses still remain largely unknown. Calcineurin B-like proteins (CBLs) act as Ca2+ sensors to activate specific protein kinases, CBL-interacting protein kinases (CIPKs). We here identified two CIPKs, OsCIPK14 and OsCIPK15, rapidly induced by microbe-associated molecular patterns, including chitooligosaccharides and xylanase (Trichoderma viride/ethylene-inducing xylanase [TvX/EIX]), in rice (Oryza sativa). Although they are located on different chromosomes, they have over 95% nucleotide sequence identity, including the surrounding genomic region, suggesting that they are duplicated genes. OsCIPK14/15 interacted with several OsCBLs through the FISL/NAF motif in yeast cells and showed the strongest interaction with OsCBL4. The recombinant OsCIPK14/15 proteins showed Mn2+-dependent protein kinase activity, which was enhanced both by deletion of their FISL/NAF motifs and by combination with OsCBL4. OsCIPK14/15-RNAi transgenic cell lines showed reduced sensitivity to TvX/EIX for the induction of a wide range of defense responses, including hypersensitive cell death, mitochondrial dysfunction, phytoalexin biosynthesis, and pathogenesis-related gene expression. On the other hand, TvX/EIX-induced cell death was enhanced in OsCIPK15-overexpressing lines. Our results suggest that OsCIPK14/15 play a crucial role in the microbe-associated molecular pattern-induced defense signaling pathway in rice cultured cells. PMID:20357140

Isolation of amylolytic, xylanolytic, and cellulolytic microorganisms extracted from the gut of the termite Reticulitermes santonensis by means of a micro-aerobic atmosphere.

PubMed

Tarayre, Cédric; Brognaux, Alison; Bauwens, Julien; Brasseur, Catherine; Mattéotti, Christel; Millet, Catherine; Destain, Jacqueline; Vandenbol, Micheline; Portetelle, Daniel; De Pauw, Edwin; Eric, Haubruge; Francis, Frédéric; Thonart, Philippe

2014-05-01

The aim of this work was to isolate enzyme-producing microorganisms from the tract of the termite Reticulitermes santonensis. The microorganisms were extracted from the guts and anaerobic (CO₂ or CO₂/H₂) and micro-aerobic atmospheres were used to stimulate growth. Three different strategies were tried out. First, the sample was spread on Petri dishes containing solid media with carboxymethylcellulose, microcrystalline cellulose or cellobiose. This technique allowed us to isolate two bacteria: Streptomyces sp. strain ABGxAviA1 and Pseudomonas sp. strain ABGxCellA. The second strategy consisted in inoculating a specific liquid medium containing carboxymethylcellulose, microcrystalline cellulose, or cellobiose. The samples were then spread on Petri dishes with the same specific medium containing carboxymethylcellulose, microcrystalline cellulose, or cellobiose. This led to the isolation of the mold Aspergillus sp. strain ABGxAviA2. Finally, the third strategy consisted in heating the first culture and spreading samples on agar plates containing rich medium. This led to the isolation of the bacterium Bacillus subtilis strain ABGx. All those steps were achieved in controlled atmospheres. The four enzyme-producing strains which were isolated were obtained by using a micro-aerobic atmosphere. Later, enzymatic assays were performed on the four strains. Streptomyces sp. strain ABGxAviA1 was found to produce only amylase, while Pseudomonas sp. strain ABGxCellA was found to produce β-glucosidase as well. Aspergillus sp. strain ABGxAviA2 showed β-glucosidase, amylase, cellulase, and xylanase activities. Finally, B. subtilis strain ABGx produced xylanase and amylase.

A novel bZIP transcription factor ClrC positively regulates multiple stress responses, conidiation and cellulase expression in Penicillium oxalicum.

PubMed

Lei, Yunfeng; Liu, Guodong; Yao, Guangshan; Li, Zhonghai; Qin, Yuqi; Qu, Yinbo

2016-06-01

Cellulase production in filamentous fungi is largely regulated at the transcriptional level, and several transcription factors have been reported to be involved in this process. In this study, we identified ClrC, a novel transcription factor in cellulase production in Penicillium oxalicum. ClrC and its orthologs have a highly conserved basic leucine zipper (bZIP) DNA binding domain, and their biological functions have not been explored. Deletion of clrC resulted in pleiotropic effects, including altered growth, reduced conidiation and increased sensitivity to oxidative and cell wall stresses. In particular, the clrC deletion mutant ΔclrC showed 46.1% ± 8.1% and 58.0% ± 8.7% decreases in production of filter paper enzyme and xylanase activities in cellulose medium, respectively. In contrast, 57.4% ± 10.0% and 70.9% ± 19.4% increased production of filter paper enzyme, and xylanase was observed in the clrC overexpressing strain, respectively. The transcription levels of major cellulase genes, as well as two cellulase transcriptional activator genes, clrB and xlnR, were significantly downregulated in ΔclrC, but substantially upregulated in clrC overexpressing strains. Furthermore, we observed that the absence of ClrC reduced full induction of cellulase expression even in the clrB overexpressing strain. These results indicated that ClrC is a novel and efficient engineering target for improving cellulolytic enzyme production in filamentous fungi. Copyright © 2016 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Cellulolytic and xylanolytic potential of high β-glucosidase-producing Trichoderma from decaying biomass.

PubMed

Okeke, Benedict C

2014-10-01

Availability, cost, and efficiency of microbial enzymes for lignocellulose bioconversion are central to sustainable biomass ethanol technology. Fungi enriched from decaying biomass and surface soil mixture displayed an array of strong cellulolytic and xylanolytic activities. Strains SG2 and SG4 produced a promising array of cellulolytic and xylanolytic enzymes including β-glucosidase, usually low in cultures of Trichoderma species. Nucleotide sequence analysis of internal transcribed spacer 2 (ITS2) region of rRNA gene revealed that strains SG2 and SG4 are closely related to Trichoderma inhamatum, Trichoderma piluliferum, and Trichoderma aureoviride. Trichoderma sp. SG2 crude culture supernatant correspondingly displayed as much as 9.84 ± 1.12, 48.02 ± 2.53, and 30.10 ± 1.11 units mL(-1) of cellulase, xylanase, and β-glucosidase in 30 min assay. Ten times dilution of culture supernatant of strain SG2 revealed that total activities were about 5.34, 8.45, and 2.05 orders of magnitude higher than observed in crude culture filtrate for cellulase, xylanase, and β-glucosidase, respectively, indicating that more enzymes are present to contact with substrates in biomass saccharification. In parallel experiments, Trichoderma species SG2 and SG4 produced more β-glucosidase than the industrial strain Trichoderma reesei RUT-C30. Results indicate that strains SG2 and SG4 have potential for low cost in-house production of primary lignocellulose-hydrolyzing enzymes for production of biomass saccharides and biofuel in the field.

Shiitake Medicinal Mushroom, Lentinus edodes (Higher Basidiomycetes) Productivity and Lignocellulolytic Enzyme Profiles during Wheat Straw and Tree Leaf Bioconversion.

PubMed

Elisashvili, Vladimir; Kachlishvili, Eva; Asatiani, Mikheil D

2015-01-01

Two commercial strains of Lentinus edodes have been comparatively evaluated for their productivity and lignocellulolytic enzyme profiles in mushroom cultivation using wheat straw or tree leaves as the growth substrates. Both substrates are profitable for recycling into shiitake fruit bodies. L. edodes 3715 gave the lowest yield of mushroom during tree leaves bioconversion with the biological efficiency (BE) 74.8% while the L. edodes 3721 BE achieved 83.4%. Cultivation of shiitake on wheat straw, especially in the presence of additional nitrogen source, increased the L. edodes 3721 BE to 92-95.3% owing to the high hydrolases activity and favorable conditions. Despite the quantitative variations, each strain of L. edodes had a similar pattern for secreting enzymes into the wheat straw and tree leaves. The mushrooms laccase and MnP activities were high during substrate colonization and declined rapidly during primordia appearance and fruit body development. While oxidase activity decreased, during the same period cellulases and xylanase activity raised sharply. Both cellulase and xylanase activity peaked at the mature fruit body stage. When mushrooms again shifted to the vegetative growth, oxidase activity gradually increased, whereas the hydrolases activity dropped rapidly. The MnP, CMCase, and FP activities of L. edodes 3721 during cultivation on wheat straw were higher than those during mushroom growth on tree leaves whereas the laccase activity was rather higher in fermentation of tree leaves. Enrichment of wheat straw with an additional nitrogen source rather favored to laccase, MnP, and FPA secretion during the vegetative stage of the L. edodes 3721 growth.

Cellulolytic and xylanolytic enzymes from thermophilic Aspergillus terreus RWY.

PubMed

Sharma, Reetika; Kocher, Gurvinder Singh; Bhogal, Ravinder Singh; Oberoi, Harinder Singh

2014-12-01

Thermophilic Aspergillus terreus RWY produced cellulases and xylanases in optimal concentrations at 45 °C in solid state fermentation process, though enzyme production was also observed at 50 and 55 °C. Filter paper cellulase (FP), endoglucanase (EG), β-glucosidase (BGL), cellobiohydrolase (CBH), xylanase, β-xylosidase, α-L-arabinofuranosidase and xylan esterase activities for A. terreus RWY at 45 °C in 72 h were 11.3 ± 0.65, 103 ± 6.4, 122.5 ± 8.7, 10.3 ± 0.66, 872 ± 22.5, 22.1 ± 0.75, 126.4 ± 8.4 and 907 ± 15.5 U (g-ds)(-1) , respectively. Enzyme was optimally active at temperatures and pH ranging between 50-60 °C and 4.0-6.0, respectively. The half life (T1/2 ) of 270 and 240 min at 70 and 75 °C, respectively for the enzyme indicates its stability at higher temperatures. The addition of MnCl2 , CoCl2 , and FeCl3 significantly enhanced cellulase activity. Enzyme demonstrated multiplicity by having seven, one and three isoform(s) for EG, CBH and BGL, respectively. Significant production of functionally active consortium of cellulolytic and xylanolytic enzymes from A. terreus RWY makes it a potential candidate in bioprocessing applications. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Towards complete hydrolysis of soy flour carbohydrates by enzyme mixtures for protein enrichment: A modeling approach.

PubMed

Loman, Abdullah Al; Ju, Lu-Kwang

2016-05-01

Soy protein is a well-known nutritional supplement in proteinaceous food and animal feed. However, soybeans contain complex carbohydrate. Selective carbohydrate removal by enzymes could increase the protein content and remove the indigestibility of soy products for inclusion in animal feed. Complete hydrolysis of soy flour carbohydrates is challenging due to the presence of proteins and different types of non-structural polysaccharides. This study is designed to guide complex enzyme mixture required for hydrolysis of all types of soy flour carbohydrates. Enzyme broths from Aspergillus niger, Aspergillus aculeatus and Trichoderma reesei fermentations were evaluated in this study for soy carbohydrate hydrolysis. The resultant hydrolysate was measured for solubilized carbohydrate by both total carbohydrate and reducing sugar analyses. Conversion data attained after 48h hydrolysis were first fitted with models to determine the maximum fractions of carbohydrate hydrolyzable by each enzyme group, i.e., cellulase, xylanase, pectinase and α-galactosidase. Kinetic models were then developed to describe the increasing conversions over time under different enzyme activities and process conditions. The models showed high fidelity in predicting soy carbohydrate hydrolysis over broad ranges of soy flour loading (5-25%) and enzyme activities: per g soy flour, cellulase, 0.04-30 FPU; xylanase, 3.5-618U; pectinase, 0.03-120U; and α-galactosidase, 0.01-60U. The models are valuable in guiding the development and production of optimal enzyme mixtures toward hydrolysis of all types of carbohydrates present in soy flour and in optimizing the design and operation of hydrolysis reactor and process. Copyright © 2016 Elsevier Inc. All rights reserved.

Isolation and partial characterization of a mutant of Penicillium funiculosum for the saccharification of straw

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hoffman, R.M.; Wood, T.M.

1985-01-01

Clearing of agar plates containing ball-milled, delignified straw has been used for screening mutants of Penicillium funiculosum IMI 87160 III. The effects of glycerol and a number of sugars on the clearing were investigated for selecting derepressed mutants. The ..beta..-glucosidase synthesis by one such mutant, C22c, in shake flasks containing straw was not repressed by 5% glycerol, whereas activities on filter paper, CM-cellulose, and p-nitrophenyl-..beta..-xylosidase were only partially derepressed; xylanase was extensively derepressed. The evidence for separate control of the enzymes involved in the solubilization of straw is discussed. 23 references.

Pectinase from Trichoderma reesei QM 9414

DOE Office of Scientific and Technical Information (OSTI.GOV)

Haltmeier, T.; Leisola, M.; Ulmer, D.

1983-06-01

The present study was undertaken to determine whether T. reesei produces pectinolytic enzymes and, if so, to determine their action pattern and specificity. The aim was also to find out the practical importance of pectinolytic activity in the hydrolysis of cellulosic materials. It was found that the presence of pectinase does not appear to facilitate the hydrolysis of cellulose in plant material through any cell-separating mechanism. It is concluded that cellulase and xylanase activities are the important activities in the practical hydrolysis of cellulosic materials and that lignin is the real limiting factor. (Refs. 12).

Enzymatic activity in the surface microlayer and subsurface water in the harbour channel

NASA Astrophysics Data System (ADS)

Perliński, Piotr; Mudryk, Zbigniew J.; Antonowicz, Józef

2017-09-01

Hydrolytic activity of eight extracellular enzymes was determined spectrofluorimetric method in the surface microlayer and subsurface water in the harbour channel in Ustka. The ranking order of the potential enzyme activity rates in the studied water layers was as follows: lipase > phosphatase > aminopeptidase > β-glucosidase > α-glucosidase > xylanase > cellulase > chitinase. The level of activity of all studied hydrolases was higher in the surface microlayer than subsurface water. No clear gradients in the level of enzymatic activity were determined along the horizontal profile of the studied channel. Activity of extracellular enzymes was strongly influenced by the season.

Effect of exogenous enzymes in maize-based diets varying in nutrient density for young broilers: growth performance and digestibility of energy, minerals and amino acids.

PubMed

Cowieson, A J; Ravindran, V

2008-01-01

1. A total of 192 male broilers (Cobb 500) were used in a growth and digestibility assay, involving a 2 x 2 factorial arrangement of treatments, to assess the effects of an enzyme cocktail of xylanase, amylase and protease in maize-based diets. 2. The following two diets were formulated: a positive control diet containing adequate nutrient concentrations for broiler starters as per breeder recommendations and a negative control diet to contain approximately 0.63 MJ/kg apparent metabolisable energy (AME) and 3% amino acids less than the positive control. 3. A further two dietary treatments were developed by supplementing each control diet with an enzyme product containing xylanase, amylase and protease. 4. Birds fed on the negative control diet had poorer weight gain and feed efficiency than those given the positive control diet. There was no effect of diet or enzyme on feed intake. The digestibility of nitrogen, calcium, phosphorus and most amino acids were unaffected by dietary nutrient density. 5. Supplementation of both the positive and negative control diets with the enzyme improved weight gain and feed efficiency compared with the unsupplemented diets. In the case of the negative control, supplemental enzyme improved performance to that of the unsupplemented positive control diet. There was no interaction between diet and enzyme for either weight gain or FCR, suggesting similar beneficial responses regardless of the nutrient density of the diet. 6. In both diets, enzyme supplementation improved AME by an average of 3% (0.35 MJ/kg DM) and nitrogen retention by an average of 11.7% (26 g/kg DM intake vs 29 g/kg DM intake). There was no interaction between diet and enzyme for AME or nitrogen retention. 7. Ileal digestibilities of calcium and phosphorus were not influenced by supplemental enzyme, whereas the digestibility of nitrogen and most amino acids was improved by enzyme addition compared with the unsupplemented control diets. There was no interaction between

Lignocellulose-Adapted Endo-Cellulase Producing Streptomyces Strains for Bioconversion of Cellulose-Based Materials.

PubMed

Ventorino, Valeria; Ionata, Elena; Birolo, Leila; Montella, Salvatore; Marcolongo, Loredana; de Chiaro, Addolorata; Espresso, Francesco; Faraco, Vincenza; Pepe, Olimpia

2016-01-01

Twenty-four Actinobacteria strains, isolated from Arundo donax, Eucalyptus camaldulensis and Populus nigra biomass during natural biodegradation and with potential enzymatic activities specific for the degradation of lignocellulosic materials, were identified by a polyphasic approach. All strains belonged to the genus Streptomyces ( S .) and in particular, the most highly represented species was Streptomyces argenteolus representing 50% of strains, while 8 strains were identified as Streptomyces flavogriseus (synonym S. flavovirens ) and Streptomyces fimicarius (synonyms Streptomyces acrimycini, Streptomyces baarnensis, Streptomyces caviscabies , and Streptomyces flavofuscus ), and the other four strains belonged to the species Streptomyces drozdowiczii, Streptomyces rubrogriseus, Streptomyces albolongus , and Streptomyces ambofaciens . Moreover, all Streptomyces strains, tested for endo and exo-cellulase, cellobiase, xylanase, pectinase, ligninase, peroxidase, and laccase activities using qualitative and semi-quantitative methods on solid growth medium, exhibited multiple enzymatic activities (from three to six). The 24 strains were further screened for endo-cellulase activity in liquid growth medium and the four best endo-cellulase producers ( S. argenteolus AE58P, S. argenteolus AE710A, S. argenteolus AE82P, and S. argenteolus AP51A) were subjected to partial characterization and their enzymatic crude extracts adopted to perform saccharification experiments on A. donax pretreated biomass. The degree of cellulose and xylan hydrolysis was evaluated by determining the kinetics of glucose and xylose release during 72 h incubation at 50°C from the pretreated biomass in the presence of cellulose degrading enzymes (cellulase and β-glucosidase) and xylan related activities (xylanase and β-xylosidase). The experiments were carried out utilizing the endo-cellulase activities from the selected S. argenteolus strains supplemented with commercial β-gucosidase and

Enzymatic processing of pigmented and non pigmented rice bran on changes in oryzanol, polyphenols and antioxidant activity.

PubMed

Prabhu, Ashish A; Jayadeep, A

2015-10-01

Bran from different rice varieties is a treasure of nutrients and nutraceuticals, and its use is limited due to the poor sensory and functional properties. Application of enzymes can alter the functional and phytochemical properties. So the effect of endo-xylanase, cellulase and their combination on microstructural, nutraceutical and antioxidant properties of pigmented (Jyothi) and non-pigmented (IR64) rice bran were investigated. Scanning electron micrograph revealed micro structural changes in fibre structures on processing. All the enzymatic processing methods resulted in an increase in the content of oryzanol, soluble, bound and total polyphenols, flavonoid and tannin. It also showed an increase in the bioactivity with respect to free radical scavenging activity and total antioxidant activity. However, extent of the increase in bio-actives varied with the type of bran and enzyme application method. Endo-xylanase showed higher percentage difference compared to controls of Jyothi and IR64 bran extracts respectively in the content of the bound (10 & 19 %) and total (20 & 14 %) polyphenols. Combination of both the enzymes resulted in higher percentage increase of bioactive components and properties. It resulted in greater percentage difference compared to controls of Jyothi and IR64 extracts respectively in the content of soluble (58 & 17 %) and total (21 & 14 %) polyphenols, flavonoids (12 & 38 %), γ-oryzanol (10 & 12 %), free radical scavenging activity (64 & 30 %) and total antioxidant activity (82 & 136 %). It may be concluded that enzymatic bio-processing of bran with cellulose and hemicellulose degrading enzymes can improve its nutraceutical properties, and it may be used for development of functional foods.

Novel Endoxylanases of the Moderately Thermophilic Polysaccharide-Degrading Bacterium Melioribacter roseus.

PubMed

Rakitin, Andrey L; Ermakova, Alexandra Y; Ravin, Nikolai V

2015-09-01

Three endoxylanase-encoding genes from the moderately themophilic chemoorganotrophic bacterium Melioribacter roseus were cloned and expressed in Escherichia coli. Genes xyl2091 (Mros_2091) and xyl2495 (Mros_2495) encode GH10 family hydrolases, whereas xyl2090 (Mros_2090) represents the GH30 family. In addition to catalytic domains, Xyl2090 and Xyl2091 contain carbohydrate-binding modules that could facilitate their binding to xylans and Por sorting domains associated with the sorting of proteins from the periplasm to the outer membrane, where they are covalently attached. Recombinant endoxylanase Xyl2495 exhibited a high specific activity of 1,920 U/mg on birchwood xylan at 40°C. It is active at low temperatures, exhibiting more than 30% of the maximal activity even at 0°C. Endoxylanases Xyl2090 and Xyl2091 have lower specific activities but higher temperature optima at 80°C and 65°C, respectively. Analysis of xylan hydrolysis products revealed that Xyl2090 generates xylo-oligosaccharides longer than xylopentaose. Xylose and xylobiose are the major products of xylan hydrolysis by the recombinant Xyl2091 and Xyl2495. No activity against cellulose was observed for all enzymes. The presence of three xylanases ensures efficient xylan hydrolysis by M. roseus. The highly processive "free" endoxylanase Xyl2495 could hydrolyze xylan under moderate temperatures. Xylan hydrolysis at elevated temperatures could be accomplished by concerted action of two cell-bound xylanases; Xyl2090 that probably degrades xylans to long xylo-oligosaccharides, and Xyl2091 hydrolyzing them to xylose and xylobiose. The new endoxylanases could be useful for saccharification of lignocellulosic biomass in biofuels production, bleaching of paper pulp, and obtaining low molecular weight xylooligosaccharides.

Manipulation of dietary protein and nonstarch polysaccharide to control swine manure emissions.

PubMed

Clark, O Grant; Moehn, Soenke; Edeogu, Ike; Price, Jason; Leonard, Jeremy

2005-01-01

Odor and greenhouse gas (GHG) emissions from stored pig (Sus scrofa) manure were monitored for response to changes in the crude protein level (168 or 139 g kg(-1), as-fed basis) and nonstarch polysaccharide (NSP) content [i.e., control, or modified with beet pulp (Beta vulgaris L.), cornstarch, or xylanase] of diets fed to pigs in a production setting. Each diet was fed to one of eight pens of pigs according to a 2 x 4, full-factorial design, replicated over three time blocks with different groups of animals and random assignment of diets. Manure from each treatment was characterized and stored in a separate, ventilated, 200-L vessel. Repeated measurements of odor, carbon dioxide (CO2), methane (CH4), and nitrous oxide (N2O) emissions from the vessels were taken every two weeks for eight weeks. Manure from high-protein diets had higher sulfur concentration and pH (P < or = 0.05). High-NSP (beet pulp) diets resulted in lower manure nitrogen and ammonia concentrations and pH (P < or = 0.05). Odor level and hedonic tone of exhaust air from the storage vessel headspaces were unaffected by the dietary treatments. Mean CO2 and CH4 emissions (1400 and 42 g d(-1) m(-3) manure, respectively) increased with lower dietary protein (P < or = 0.05). The addition of xylanase to high-protein diets caused a decrease in manure CO2 emissions, but an increase when added to low-protein diets (P < or = 0.05). Nitrous oxide emissions were negligible. Contrary to other studies, these results do not support the use of dietary protein reduction to reduce emissions from stored swine manure.

Enzymatic production of ferulic acid from defatted rice bran by using a combination of bacterial enzymes.

PubMed

Uraji, Misugi; Kimura, Masayo; Inoue, Yosikazu; Kawakami, Kayoko; Kumagai, Yuya; Harazono, Koichi; Hatanaka, Tadashi

2013-11-01

Ferulic acid (FA), which is present in the cell walls of some plants, is best known for its antioxidant property. By combining a commercial enzyme that shows FA esterase activity with several Streptomyces carbohydrate-hydrolyzing enzymes, we succeeded in enhancing the enzymatic production of FA from defatted rice bran. In particular, the combination of three xylanases, an α-L-arabinofuranosidase, and an acetyl xylan esterase from Streptomyces spp. produced the highest increase in the amount of released FAs among all the enzymes in the Streptomyces enzymes library. This enzyme combination also had an effect on FA production from other biomasses, such as raw rice bran, wheat bran, and corncob.

Purification of the major endoglucanase from Aspergillus fumigatus Fresenius.

PubMed

Parry, J B; Stewart, J C; Heptinstall, J

1983-08-01

Aspergillus fumigatus (Fresenius), IMI 246651, A.T.C.C. 46324, produces two beta-glucosidase enzymes, cotton-solubilizing activity, xylanase and endoglucanase enzymes which can be separated by gel-filtration chromatography. The major endoglucanase does not bind to concanavalin A-Sepharose and does not stain with periodic acid/Schiff reagent. It is homogeneous on polyacrylamide isoelectric focusing (pI = 7.1) and has a mol.wt. of 12500 by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The endoglucanase produces glucose and a mixture of oligosaccharides from cellulose; the purified enzyme has a small dextranase activity. It is stable at 50 degrees C and pH 6.

Effect of Flavonoid-Rich Extract of Glycyrrhiza glabra on Gut-Friendly Microorganisms, Commercial Probiotic Preparations, and Digestive Enzymes.

PubMed

Asha, Mannanthendil Kumaran; Debraj, Debnath; Dethe, Shekhar; Bhaskar, Anirban; Muruganantham, Nithyanantham; Deepak, Mundkinajeddu

2017-05-04

Flavonoid-rich extract prepared from Glycyrrhiza glabra has been found to be beneficial in patients with functional dyspepsia and was reported to possess some gut health-promoting properties such as antioxidant, anti-inflammatory and anti-Helicobacter pylori activities. In the present study, the flavonoid-rich extract of Glycyrrhiza glabra was evaluated for its compatibility with probiotic strains (Lactobacillus casei, Lactobacillus fermentum, Lactobacillus plantarum, and Streptococcus thermophilus), commercial probiotic drinks, and digestive enzymes (pancreatic α-amylase, α-glucosidase, phytase, xylanase, and pancreatic lipase). Results of this study indicated that the flavonoid-rich extract of Glycyrrhiza glabra is compatible with the tested probiotic strains, probiotic drinks and digestive enzymes.

Establishment of ruminal enzyme activities and fermentation capacity in dairy calves from birth through weaning.

PubMed

Rey, M; Enjalbert, F; Monteils, V

2012-03-01

The objectives of this study were to characterize the establishment of ruminal fermentation and enzymatic activities in dairy calves from birth to weaning (d 83). Six Holstein calves, immediately separated from their mother at birth, were fed colostrum for 3 d after birth, and thereafter milk replacer, starter pelleted concentrate, and hay until d 83 of age. Ruminal samples were collected from each calf every day for the first 10 d, and additionally at d 12, 15, 19, 22, 26, 29, 33, 36, 40, 43, 47, 50, 55, 62, 69, and 83. Ruminal samples were collected 1h after milk feeding with a stomach tube. The pH and redox potential (E(h)) were immediately measured. Samples were kept for further determination of ammonia nitrogen (NH(3)-N) and volatile fatty acid (VFA) concentrations, and xylanase, amylase, urease, and protease activities. Ruminal pH averaged 6.69, 5.82, and 6.34, from d 1 to 9, d 10 to 40, and d 43 to 83 of age, respectively. At first day of life, the ruminal E(h) value was positive (+224 mV). From d 2 to 9, d 10 to 40, and d 43 to 83 of age, ruminal E(h) averaged -164, -115, and -141 mV, respectively. From d 1 to 3, d 4 to 22, and d 26 to 83 of age, NH(3)-N concentration averaged 60.1, 179.8, and 58.2 mg/L, respectively. No VFA were detected in ruminal samples collected on d 1 of life of calves. From d 2 to 10 and d 12 to 83 of age, ruminal total VFA concentration averaged 19.5 and 84.4mM, respectively. Neither ruminal xylanase or amylase activities were observed at d 1 of age. From d 5 to 15 and d 19 to 83 of age, the xylanase activity averaged 182.2 and 62.4 μmol of sugar released per hour per gram of ruminal content dry matter (DM), respectively. From d 5 to 83 of age, the amylase activity reached 35.4 μmol of sugar released per hour per gram of ruminal content DM. The ruminal ureolytic activity was observed with an average value of 6.9 μg of NH(3)-N released per minute per gram of ruminal content DM over the 83-d experimental period. From d 1 to 4 and d

Rice bacterial blight pathogen Xanthomonas oryzae pv. oryzae produces multiple DSF-family signals in regulation of virulence factor production

PubMed Central

2010-01-01

Background Xanthomonas oryzae pv. oryzae (Xoo) is the causal agent of rice bacterial blight disease. Xoo produces a range of virulence factors, including EPS, extracellular enzyme, iron-chelating siderophores, and type III-secretion dependent effectors, which are collectively essential for virulence. Genetic and genomics evidence suggest that Xoo might use the diffusible signal factor (DSF) type quorum sensing (QS) system to regulate the virulence factor production. However, little is known about the chemical structure of the DSF-like signal(s) produced by Xoo and the factors influencing the signal production. Results Xoo genome harbours an rpf cluster comprising rpfB, rpfF, rpfC and rpfG. The proteins encoded by these genes are highly homologous to their counterparts in X. campestris pv. campestris (Xcc), suggesting that Xcc and Xoo might use similar mechanisms for DSF biosynthesis and autoregulation. Consistent with in silico analysis, the rpfF mutant was DSF-deficient and the rpfC mutant produced about 25 times higher DSF-like activity than the wild type Xoo strain KACC10331. From the supernatants of rpfC mutant, we purified three compounds showing strong DSF-like activity. Mass spectrometry and NMR analysis revealed that two of them were the previously characterized DSF and BDSF; the third one was a novel unsaturated fatty acid with 2 double bonds and was designated as CDSF in this study. Further analysis showed that all the three DSF-family signals were synthesized via the enzyme RpfF encoded by Xoo2868. DSF and BDSF at a final concentration of 3 μM to the rpfF mutant could fully restore its extracellular xylanase activity and EPS production to the wild type level, but CDSF was less active than DSF and BDSF in induction of EPS and xylanase. DSF and CDSF shared a similar cell density-dependent production time course with the maximum production being detected at 42 h after inoculation, whereas the maximum production of BDSF was observed at 36 h after

Responses of non-starch polysaccharide-degrading enzymes on digestibility and performance of growing pigs fed a diet based on corn, soya bean meal and Chinese double-low rapeseed meal.

PubMed

Fang, Z F; Peng, J; Liu, Z L; Liu, Y G

2007-08-01

This study was conducted to investigate the effect of two distinct enzyme preparations on nutrients' digestibility and growth performance of growing pigs fed diets based on corn, soya bean meal and Chinese double-low rapeseed meal (DLRM). The two enzyme preparations were Enzyme R, a preparation extracted from fermentation of a non-GMO fungus Penicillum funiculosum, developed for multi-grain and multi-animal species; and Enzyme P, a xylanase preparation from Trichoderma longibrachiatum, for pigs fed corn-based diets only. Both enzymes were tested at 0, 0.25 and 0.50 g/kg feed using 70 crossbred male pigs (Large Yorkshire x Landrace) in five dietary treatments and seven replicates in each treatment, for growth period from 27 to 68 kg live weight in 49 days. Results showed that the supplementation of both enzymes (1) increased total-tract digestibility of dietary energy from 77.5% (control) to 81.4% (Enzyme R, p < 0.05) and 81.9% (Enzyme P, p < 0.05); of neutral detergent fibre from 41.0% (control) to 57.8% (Enzyme R, p < 0.05) and 60.0% (Enzyme P, p < 0.05); (2) improved average daily gain from 786 g (control) to 829 g (Enzyme R, p < 0.05) and 846 g (Enzyme P, p < 0.05); and numerical increases in feed intake from 1.96 kg/pig/day (control) to 2.01 (Enzyme R) and 2.00 (p > 0.05) and feed conversion ratio from 2.50 (control) to 2.42 (Enzyme R) and 2.36 (Enzyme P, p < 0.05); (3) there was a dose response but no significant differences were observed in enzyme efficacy between the two enzyme preparations. The present study demonstrated beneficial effects of applying xylanase-based enzymes to improve feeding values of pig diets based on corn, soya bean meal and DLRM.

Proteomic analysis of extracellular proteins from Aspergillus oryzae grown under submerged and solid-state culture conditions.

PubMed

Oda, Ken; Kakizono, Dararat; Yamada, Osamu; Iefuji, Haruyuki; Akita, Osamu; Iwashita, Kazuhiro

2006-05-01

Filamentous fungi are widely used for the production of homologous and heterologous proteins. Recently, there has been increasing interest in Aspergillus oryzae because of its ability to produce heterologous proteins in solid-state culture. To provide an overview of protein secretion by A. oryzae in solid-state culture, we carried out a comparative proteome analysis of extracellular proteins in solid-state and submerged (liquid) cultures. Extracellular proteins prepared from both cultures sequentially from 0 to 40 h were subjected to two-dimensional electrophoresis, and protein spots at 40 h were identified by peptide mass fingerprinting using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry. We also attempted to identify cell wall-bound proteins of the submerged culture. We analyzed 85 spots from the solid-state culture and 110 spots from the submerged culture. We identified a total of 29 proteins, which were classified into 4 groups. Group 1 consisted of extracellular proteins specifically produced in the solid-state growth condition, such as glucoamylase B and alanyl dipeptidyl peptidase. Group 2 consisted of extracellular proteins specifically produced in the submerged condition, such as glucoamylase A (GlaA) and xylanase G2 (XynG2). Group 3 consisted of proteins produced in both conditions, such as xylanase G1. Group 4 consisted of proteins that were secreted to the medium in the solid-state growth condition but trapped in the cell wall in the submerged condition, such as alpha-amylase (TAA) and beta-glucosidase (Bgl). A Northern analysis of seven genes from the four groups suggested that the secretion of TAA and Bgl was regulated by trapping these proteins in the cell wall in submerged culture and that secretion of GlaA and XynG2 was regulated at the posttranscriptional level in the solid-state culture.

A biodegradation study of forest biomass by Aspergillus niger F7: correlation between enzymatic activity, hydrolytic percentage and biodegradation index

PubMed Central

Sharma, Nivedita; Kaushal, Richa; Gupta, Rakesh; Kumar, Sanjeev

2012-01-01

Aspergillus niger F7 isolated from soil was found to be the potent producer of cellulase and xylanase. The residue of forest species Toona ciliata, Celtris australis, Cedrus deodara and Pinus roxburghii was selected as substrate for biodegradation study due to its easy availability and wide use in industry. It was subjected to alkali (sodium hydroxide) treatment for enhancing its degradation. Biodegradation of forest waste by hydrolytic enzymes (cellulase and xylanase) secreted by A. niger under solid state fermentation (SSF) was explored. SSF of pretreated forest biomass was found to be superior over untreated forest biomass. Highest extracellular enzyme activity of 2201±23.91 U/g by A. niger was shown in pretreated C. australis wood resulting in 6.72±0.20 percent hydrolysis and 6.99±0.23 biodegradation index (BI). The lowest BI of 1.40±0.08 was observed in untreated saw dust of C. deodara having the least enzyme activity of 238±1.36 U/g of dry matter. Biodegradation of forest biomass under SSF was increased many folds when moistening agent i.e. tap water had been replaced with modified basal salt media (BSM). In BSM mediated degradation of forest waste with A. niger, extracellular enzyme activity was increased up to 4089±67.11 U/g of dry matter in turn resulting in higher BI of 15.4±0.41 and percent hydrolysis of 19.38±0.81 in pretreated C. australis wood. A. niger exhibited higher enzyme activity on pretreated biomass when moistened with modified BSM in this study. Statistically a positive correlation has been drawn between these three factors i.e. enzyme activity, BI and percent hydrolysis of forest biomass thus proving their direct relationship with each other. PMID:24031853

Whole Proteome Analyses on Ruminiclostridium cellulolyticum Show a Modulation of the Cellulolysis Machinery in Response to Cellulosic Materials with Subtle Differences in Chemical and Structural Properties

PubMed Central

Badalato, Nelly; Guillot, Alain; Sabarly, Victor; Dubois, Marc; Pourette, Nina; Pontoire, Bruno; Robert, Paul; Bridier, Arnaud; Monnet, Véronique; Sousa, Diana Z.; Durand, Sylvie; Mazéas, Laurent; Buléon, Alain; Bouchez, Théodore; Mortha, Gérard

2017-01-01

Lignocellulosic materials from municipal solid waste emerge as attractive resources for anaerobic digestion biorefinery. To increase the knowledge required for establishing efficient bioprocesses, dynamics of batch fermentation by the cellulolytic bacterium Ruminiclostridium cellulolyticum were compared using three cellulosic materials, paper handkerchief, cotton discs and Whatman filter paper. Fermentation of paper handkerchief occurred the fastest and resulted in a specific metabolic profile: it resulted in the lowest acetate-to-lactate and acetate-to-ethanol ratios. By shotgun proteomic analyses of paper handkerchief and Whatman paper incubations, 151 proteins with significantly different levels were detected, including 20 of the 65 cellulosomal components, 8 non-cellulosomal CAZymes and 44 distinct extracytoplasmic proteins. Consistent with the specific metabolic profile observed, many enzymes from the central carbon catabolic pathways had higher levels in paper handkerchief incubations. Among the quantified CAZymes and cellulosomal components, 10 endoglucanases mainly from the GH9 families and 7 other cellulosomal subunits had lower levels in paper handkerchief incubations. An in-depth characterization of the materials used showed that the lower levels of endoglucanases in paper handkerchief incubations could hypothetically result from its lower crystallinity index (50%) and degree of polymerization (970). By contrast, the higher hemicellulose rate in paper handkerchief (13.87%) did not result in the enhanced expression of enzyme with xylanase as primary activity, including enzymes from the “xyl-doc” cluster. It suggests the absence, in this material, of molecular structures that specifically lead to xylanase induction. The integrated approach developed in this work shows that subtle differences among cellulosic materials regarding chemical and structural characteristics have significant effects on expressed bacterial functions, in particular the

Novel thermophilic hemicellulases for the conversion of lignocellulose for second generation biorefineries.

PubMed

Cobucci-Ponzano, Beatrice; Strazzulli, Andrea; Iacono, Roberta; Masturzo, Giuseppe; Giglio, Rosa; Rossi, Mosè; Moracci, Marco

2015-10-01

The biotransformation of lignocellulose biomasses into fermentable sugars is a very complex procedure including, as one of the most critical steps, the (hemi) cellulose hydrolysis by specific enzymatic cocktails. We explored here, the potential of stable glycoside hydrolases from thermophilic organisms, so far not used in commercial enzymatic preparations, for the conversion of glucuronoxylan, the major hemicellulose of several energy crops. Searches in the genomes of thermophilic bacteria led to the identification, efficient production, and detailed characterization of novel xylanase and α-glucuronidase from Alicyclobacillus acidocaldarius (GH10-XA and GH67-GA, respectively) and a α-glucuronidase from Caldicellulosiruptor saccharolyticus (GH67-GC). Remarkably, GH10-XA, if compared to other thermophilic xylanases from this family, coupled good specificity on beechwood xylan and the best stability at 65 °C (3.5 days). In addition, GH67-GC was the most stable α-glucuronidases from this family and the first able to hydrolyse both aldouronic acid and aryl-α-glucuronic acid substrates. These enzymes, led to the very efficient hydrolysis of beechwood xylan by using 7- to 9-fold less protein (concentrations <0.3 μM) and in much less reaction time (2h vs 12h) if compared to other known biotransformations catalyzed by thermophilic enzymes. In addition, remarkably, together with a thermophilic β-xylosidase, they catalyzed the production of xylose from the smart cooking pre-treated biomass of one of the most promising energy crops for second generation biorefineries. We demonstrated that search by the CAZy Data Bank of currently available genomes and detailed enzymatic characterization of recombinant enzymes allow the identification of glycoside hydrolases with novel and interesting properties and applications. Copyright © 2015 Elsevier Inc. All rights reserved.

Proteomic Analysis of Extracellular Proteins from Aspergillus oryzae Grown under Submerged and Solid-State Culture Conditions

PubMed Central

Oda, Ken; Kakizono, Dararat; Yamada, Osamu; Iefuji, Haruyuki; Akita, Osamu; Iwashita, Kazuhiro

2006-01-01

Filamentous fungi are widely used for the production of homologous and heterologous proteins. Recently, there has been increasing interest in Aspergillus oryzae because of its ability to produce heterologous proteins in solid-state culture. To provide an overview of protein secretion by A. oryzae in solid-state culture, we carried out a comparative proteome analysis of extracellular proteins in solid-state and submerged (liquid) cultures. Extracellular proteins prepared from both cultures sequentially from 0 to 40 h were subjected to two-dimensional electrophoresis, and protein spots at 40 h were identified by peptide mass fingerprinting using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry. We also attempted to identify cell wall-bound proteins of the submerged culture. We analyzed 85 spots from the solid-state culture and 110 spots from the submerged culture. We identified a total of 29 proteins, which were classified into 4 groups. Group 1 consisted of extracellular proteins specifically produced in the solid-state growth condition, such as glucoamylase B and alanyl dipeptidyl peptidase. Group 2 consisted of extracellular proteins specifically produced in the submerged condition, such as glucoamylase A (GlaA) and xylanase G2 (XynG2). Group 3 consisted of proteins produced in both conditions, such as xylanase G1. Group 4 consisted of proteins that were secreted to the medium in the solid-state growth condition but trapped in the cell wall in the submerged condition, such as α-amylase (TAA) and β-glucosidase (Bgl). A Northern analysis of seven genes from the four groups suggested that the secretion of TAA and Bgl was regulated by trapping these proteins in the cell wall in submerged culture and that secretion of GlaA and XynG2 was regulated at the posttranscriptional level in the solid-state culture. PMID:16672490

Identification of thermophilic bacterial strains producing thermotolerant hydrolytic enzymes from manure compost.

PubMed

Charbonneau, David M; Meddeb-Mouelhi, Fatma; Boissinot, Maurice; Sirois, Marc; Beauregard, Marc

2012-03-01

Ten thermophilic bacterial strains were isolated from manure compost. Phylogenetic analysis based on 16S rRNA genes and biochemical characterization allowed identification of four different species belonging to four genera: Geobacillus thermodenitrificans, Bacillus smithii, Ureibacillus suwonensis and Aneurinibacillus thermoaerophilus. PCR-RFLP profiles of the 16S-ITS-23S rRNA region allowed us to distinguish two subgroups among the G. thermodenitrificans isolates. Isolates were screened for thermotolerant hydrolytic activities (60-65°C). Thermotolerant lipolytic activities were detected for G. thermodenitrificans, A. thermoaerophilus and B. smithii. Thermotolerant protease, α-amylase and xylanase activities were also observed in the G. thermodenitrificans group. These species represent a source of potential novel thermostable enzymes for industrial applications.

Experimental mixture design as a tool to enhance glycosyl hydrolases production by a new Trichoderma harzianum P49P11 strain cultivated under controlled bioreactor submerged fermentation.

PubMed

Delabona, Priscila da Silva; Farinas, Cristiane Sanchez; Lima, Deise Juliana da Silva; Pradella, José Geraldo da Cruz

2013-03-01

This work investigates the glycosyl hydrolase (GH) profile of a new Trichoderma harzianum strain cultivated under controlled bioreactor submerged fermentation. The influence of different medium components (delignified steam-exploded sugarcane bagasse, sucrose, and soybean flour) on GH biosynthesis was assessed using experimental mixture design (EMD). Additionally, the effect of increased component concentrations in culture media selected from the EMD was studied. It was found that that a mixed culture medium could significantly maximize GH biosynthesis rate, especially for xylanase enzymes which achieved a 2-fold increment. Overall, it was demonstrated that T. harzianumP49P11 enzymes have a great potential to be used in the deconstruction of biomass. Copyright © 2012 Elsevier Ltd. All rights reserved.

Thermal and Chemical Denaturation of "Bacillus circulans" Xylanase: A Biophysical Chemistry Laboratory Module

ERIC Educational Resources Information Center

Raabe, Richard; Gentile, Lisa

2008-01-01

A number of institutions have been, or are in the process of, modifying their biochemistry major to include some emphasis on the quantitative physical chemistry of biomolecules. Sometimes this is done as a replacement for part for the entire physical chemistry requirement, while at other institutions this is incorporated as a component into the…

Improving biogas production from microalgae by enzymatic pretreatment.

PubMed

Passos, Fabiana; Hom-Diaz, Andrea; Blanquez, Paqui; Vicent, Teresa; Ferrer, Ivet

2016-01-01

In this study, enzymatic pretreatment of microalgal biomass was investigated under different conditions and evaluated using biochemical methane potential (BMP) tests. Cellulase, glucohydrolase and an enzyme mix composed of cellulase, glucohydrolase and xylanase were selected based on the microalgae cell wall composition (cellulose, hemicellulose, pectin and glycoprotein). All of them increased organic matter solubilisation, obtaining high values already after 6h of pretreatment with an enzyme dose of 1% for cellulase and the enzyme mix. BMP tests with pretreated microalgae showed a methane yield increase of 8 and 15% for cellulase and the enzyme mix, respectively. Prospective research should evaluate enzymatic pretreatments in continuous anaerobic reactors so as to estimate the energy balance and economic cost of the process. Copyright © 2015 Elsevier Ltd. All rights reserved.

Lignocellulose-degrading enzymes from termites and their symbiotic microbiota.

PubMed

Ni, Jinfeng; Tokuda, Gaku

2013-11-01

Lignocellulose-the dry matter of plants, or "plant biomass"-digestion is of increasing interest in organismal metabolism research, specifically the conversion of biomass into biofuels. Termites efficiently decompose lignocelluloses, and studies on lignocellulolytic systems may elucidate mechanisms of efficient lignocellulose degradation in termites as well as offer novel enzyme sources, findings which have significant potential industrial applications. Recent progress in metagenomic and metatranscriptomic research has illuminated the diversity of lignocellulolytic enzymes within the termite gut. Here, we review state-of-the-art research on lignocellulose-degrading systems in termites, specifically cellulases, xylanases, and lignin modification enzymes produced by termites and their symbiotic microbiota. We also discuss recent investigations into heterologous overexpression of lignocellulolytic enzymes from termites and their symbionts. Copyright © 2013 Elsevier Inc. All rights reserved.

Cellulosic bioethanol production from Jerusalem artichoke (Helianthus tuberosus L.) using hydrogen peroxide-acetic acid (HPAC) pretreatment.

PubMed

Song, Younho; Wi, Seung Gon; Kim, Ho Myeong; Bae, Hyeun-Jong

2016-08-01

Jerusalem artichoke (JA) is recognized as a suitable candidate biomass crop for bioethanol production because it has a rapid growth rate and high biomass productivity. In this study, hydrogen peroxide-acetic acid (HPAC) pretreatment was used to enhance the enzymatic hydrolysis and to effectively remove the lignin of JA. With optimized enzyme doses, synergy was observed from the combination of three different enzymes (RUT-C30, pectinase, and xylanase) which provided a conversion rate was approximately 30% higher than the rate with from treatment with RUT-C30 alone. Fermentation of the JA hydrolyzates by Saccharomyces cerevisiae produced a fermentation yield of approximately 84%. Therefore, Jerusalem artichoke has potential as a bioenergy crop for bioethanol production. Copyright © 2016 Elsevier Ltd. All rights reserved.

Ingestion, enzymatic digestion and absorption of particles derived from different vegetal sources by the cockle Cerastoderma edule

NASA Astrophysics Data System (ADS)

Arambalza, U.; Urrutia, M. B.; Navarro, E.; Ibarrola, I.

2010-10-01

Ingestion, enzymatic digestion and absorption of particulate detrital matter derived from six different vegetal sources by the common cockle Cerastoderma edule was analyzed in a series of seasonal experiments performed in March, May and October 2005. Two green macroalgae: Ulva lactuca and Enteromorpha sp; two vascular plants: Spartina maritima and Juncus maritimus, the red macroalgae Gracilaria gracilis; and the microalgae Isochrysis galbana were used in experiments. Detrital matter was elaborated by freeze-drying, grinding and sieving (< 63 μm) vegetal tissues. Mono-specific detrital diets of similar organic content (≈ 60-70%) were elaborated by mixing detritus with ashed silt. We measured i) the biochemical composition of different detritus, ii) physiological components of the absorptive balance (i.e. clearance, ingestion, rejection and absorption rate and absorption efficiency), iii) the capability of the digestive gland to hydrolyze carbohydrates from different detritus (digestibility), as well as iv) glandular cellulase and xylanase activities. Detritus type, season and the interaction detritus-season exerted significant effects upon all the physiological components of absorptive balance. Effects were light at the pre-absorptive level, however, huge variations associated to absorption efficiency promoted large significant differences in absorption rates (AR) of different kind of detritus: irrespective of season, highest values corresponded to cockles fed the green macroalgae ( Ulva and Enteromorpha) and lowest to those fed the vascular plant Juncus maritimus. Recorded significant differences in enzymatic digestibility among detritus were found to explain ≈ 40% of differences recorded in AR, and the following regression could be fitted: AR = 0.232 (± 0.032) * Digestibility + 0,072 (± 0.015); r 2 = 0.415; F = 51.036; p < 0.001. Digestibility of Ulva and Enteromorpha was found to be significantly correlated with cellulase activity in the digestive gland

Secretome analysis of Trichoderma reesei and Aspergillus niger cultivated by submerged and sequential fermentation processes: Enzyme production for sugarcane bagasse hydrolysis.

PubMed

Florencio, Camila; Cunha, Fernanda M; Badino, Alberto C; Farinas, Cristiane S; Ximenes, Eduardo; Ladisch, Michael R

2016-08-01

Cellulases and hemicellulases from Trichoderma reesei and Aspergillus niger have been shown to be powerful enzymes for biomass conversion to sugars, but the production costs are still relatively high for commercial application. The choice of an effective microbial cultivation process employed for enzyme production is important, since it may affect titers and the profile of protein secretion. We used proteomic analysis to characterize the secretome of T. reesei and A. niger cultivated in submerged and sequential fermentation processes. The information gained was key to understand differences in hydrolysis of steam exploded sugarcane bagasse for enzyme cocktails obtained from two different cultivation processes. The sequential process for cultivating A. niger gave xylanase and β-glucosidase activities 3- and 8-fold higher, respectively, than corresponding activities from the submerged process. A greater protein diversity of critical cellulolytic and hemicellulolytic enzymes were also observed through secretome analyses. These results helped to explain the 3-fold higher yield for hydrolysis of non-washed pretreated bagasse when combined T. reesei and A. niger enzyme extracts from sequential fermentation were used in place of enzymes obtained from submerged fermentation. An enzyme loading of 0.7 FPU cellulase activity/g glucan was surprisingly effective when compared to the 5-15 times more enzyme loadings commonly reported for other cellulose hydrolysis studies. Analyses showed that more than 80% consisted of proteins other than cellulases whose role is important to the hydrolysis of a lignocellulose substrate. Our work combined proteomic analyses and enzymology studies to show that sequential and submerged cultivation methods differently influence both titers and secretion profile of key enzymes required for the hydrolysis of sugarcane bagasse. The higher diversity of feruloyl esterases, xylanases and other auxiliary hemicellulolytic enzymes observed in the enzyme

Identification of a novel fungus, Leptosphaerulina chartarum SJTU59 and characterization of its xylanolytic enzymes.

PubMed

Wu, Qiong; Li, Yaqian; Li, Yingying; Gao, Shigang; Wang, Meng; Zhang, Tailong; Chen, Jie

2013-01-01

Xylanolytic enzymes are widely used in processing industries, e.g., pulp and paper, food, livestock feeds, and textile. Furthermore, certain xylanotic enzymes have demonstrated the capability to improve the resistance and immunity of plants. Screening of high-yield microbial xylanolytic enzyme producers is significant for improving large-scale cost-effective xylanolytic enzyme production. This study provided new evidence of high-level xylanolytic enzyme production by a novel fungus, designated Leptosphaerulina chartarum SJTU59. Under laboratory conditions, L. chartarum SJTU59 produced xylanolytic enzymes of up to 17.566 U/mL (i.e., 878.307 U/g substrate). The enzyme solution was relatively stable over a wide range of pH (pH 3.0 to pH 9.0) and temperature (40°C to 65°C) while showing high resistance to the majority of metal ions tested. Composition analysis of the hydrolytic products of xylan showed sufficient degradation by xylanolytic enzymes from L. chartarum SJTU59, mainly the monosaccharide xylose, and a small amount of xylobiose were enzymatically produced; whereas in the presence of sufficient xylan substrates, mainly xylooligosaccharides, an emerging prebiotic used in food industry, were produced. In addition, the xylanolytic enzyme preparation from L. chartarum SJTU59 could initiate tissue necrosis and oxidative burst in tobacco leaves, which may be related to enhanced plant defense to adversity and disease. L. chartarum SJTU59 possessed a complex xylanolytic enzyme system, from which two novel endo-β-1,4-xylanases of the glycoside hydrolase (GH) family 10, one novel endo-β-1,4-xylanase of the GH family 11, and one novel β-xylosidase of the GH family 43 were obtained via rapid amplification of complementary DNA ends. Given the high yield and stable properties of xylanolytic enzymes produced by L. chartarum SJTU59, future studies will be conducted to characterize the properties of individual xylanolytic enzymes from L. chartarum SJTU59. xylanolytic

The effects of high dose of two manganese supplements (organic and inorganic) on the rumen microbial ecosystem.

PubMed

Kišidayová, Svetlana; Pristaš, Peter; Zimovčáková, Michaela; Blanár Wencelová, Monika; Homol'ová, Lucia; Mihaliková, Katarína; Čobanová, Klaudia; Grešáková, Ľubomíra; Váradyová, Zora

2018-01-01

Little is known about the effects of the high dose and types of manganese supplements on rumen environment at manganese intake level close above the limit of 150 mg/kg of dry feed matter. The effects of high dose of two manganese supplements (organic and inorganic) on rumen microbial ecosystem after four months of treatment of 18 lambs divided into three treatment groups were studied. We examined the enzyme activities (α-amylase, xylanase, and carboxymethyl cellulase), total and differential microscopic counts of rumen ciliates, total microscopic counts of bacteria, and fingerprinting pattern of the eubacterial and ciliates population analyzed by PCR-DGGE. Lambs were fed a basal diet with a basal Mn content (34.3 mg/kg dry matter; control) and supplemented either with inorganic manganous sulfate or organic Mn-chelate hydrate (daily 182.7, 184 mg/kg dry matter of feed, respectively). Basal diet, offered twice daily, consisted of ground barley and hay (268 and 732 g/kg dry matter per animal and day). The rumens of the lambs harbored ciliates of the genera of Entodinium, Epidinium, Diplodinium, Eudiplodinium, Dasytricha, and Isotricha. No significant differences between treatment groups were observed in the total ciliate number, the number of ciliates at the genus level, as well as the total number of bacteria. Organic Mn did decrease the species richness and diversity of the eubacterial population examined by PCR-DGGE. No effects of type of Mn supplement on the enzyme activities were observed. In comparison to the control, α-amylase specific activities were decreased and carboxymethyl-cellulase specific activities were increased by the Mn supplements. Xylanase activities were not influenced. In conclusion, our results suggested that the intake of tested inorganic and organic manganese supplements in excess may affect the specific groups of eubacteria. More studies on intake of Mn supplements at a level close to the limit can reveal if the changes in microbial

The effects of high dose of two manganese supplements (organic and inorganic) on the rumen microbial ecosystem

PubMed Central

Pristaš, Peter; Zimovčáková, Michaela; Blanár Wencelová, Monika; Homol'ová, Lucia; Mihaliková, Katarína; Čobanová, Klaudia; Grešáková, Ľubomíra; Váradyová, Zora

2018-01-01

Little is known about the effects of the high dose and types of manganese supplements on rumen environment at manganese intake level close above the limit of 150 mg/kg of dry feed matter. The effects of high dose of two manganese supplements (organic and inorganic) on rumen microbial ecosystem after four months of treatment of 18 lambs divided into three treatment groups were studied. We examined the enzyme activities (α-amylase, xylanase, and carboxymethyl cellulase), total and differential microscopic counts of rumen ciliates, total microscopic counts of bacteria, and fingerprinting pattern of the eubacterial and ciliates population analyzed by PCR-DGGE. Lambs were fed a basal diet with a basal Mn content (34.3 mg/kg dry matter; control) and supplemented either with inorganic manganous sulfate or organic Mn-chelate hydrate (daily 182.7, 184 mg/kg dry matter of feed, respectively). Basal diet, offered twice daily, consisted of ground barley and hay (268 and 732 g/kg dry matter per animal and day). The rumens of the lambs harbored ciliates of the genera of Entodinium, Epidinium, Diplodinium, Eudiplodinium, Dasytricha, and Isotricha. No significant differences between treatment groups were observed in the total ciliate number, the number of ciliates at the genus level, as well as the total number of bacteria. Organic Mn did decrease the species richness and diversity of the eubacterial population examined by PCR-DGGE. No effects of type of Mn supplement on the enzyme activities were observed. In comparison to the control, α-amylase specific activities were decreased and carboxymethyl-cellulase specific activities were increased by the Mn supplements. Xylanase activities were not influenced. In conclusion, our results suggested that the intake of tested inorganic and organic manganese supplements in excess may affect the specific groups of eubacteria. More studies on intake of Mn supplements at a level close to the limit can reveal if the changes in microbial

Potential uses of spent mushroom substrate and its associated lignocellulosic enzymes.

PubMed

Phan, Chia-Wei; Sabaratnam, Vikineswary

2012-11-01

Mushroom industries generate a virtually in-exhaustible supply of a co-product called spent mushroom substrate (SMS). This is the unutilised substrate and the mushroom mycelium left after harvesting of mushrooms. As the mushroom industry is steadily growing, the volume of SMS generated annually is increasing. In recent years, the mushroom industry has faced challenges in storing and disposing the SMS. The obvious solution is to explore new applications of SMS. There has been considerable discussion recently about the potentials of using SMS for production of value-added products. One of them is production of lignocellulosic enzymes such as laccase, xylanase, lignin peroxidase, cellulase and hemicellulase. This paper reviews scientific research and practical applications of SMS as a readily available and cheap source of enzymes for bioremediation, animal feed and energy feedstock.

Probiotic activity of lignocellulosic enzyme as bioactivator for rice husk degradation

NASA Astrophysics Data System (ADS)

Lamid, Mirni; Al-Arif, Anam; Warsito, Sunaryo Hadi

2017-02-01

The utilization of lignocellulosic enzyme will increase nutritional value of rice husk. Cellulase consists of C1 (β-1, 4-glucan cellobiohydrolase or exo-β-1,4glucanase), Cc (endo-β-1,4-glucanase) and component and cellobiose (β-glucocidase). Hemicellulase enzyme consists of endo-β-1,4-xilanase, β-xilosidase, α-L arabinofuranosidase, α-D-glukuronidaseand asetil xilan esterase. This research aimed to study the activity of lignocellulosic enzyme, produced by cows in their rumen, which can be used as a bioactivator in rice husk degradation. This research resulted G6 and G7 bacteria, producing xylanase and cellulase with the activity of 0.004 U mL-1 and 0.021 U mL-1; 0.003 ( U mL-1) and 0.026 (U mL-1) respectively.

On the assessments of arabinoxylan localization and enzymatic modifications for enhanced protein networking and its structural impact on rye dough and bread.

PubMed

Döring, Clemens; Hussein, Mohamed A; Jekle, Mario; Becker, Thomas

2017-08-15

For rye dough structure, it is hypothesised that the presence of arabinoxylan hinders the proteins from forming a coherent network. This hypothesis was investigated using fluorescent-stained antibodies that bind to the arabinoxylan chains. Image analysis proves that the arabinoxylan surrounds the proteins, negatively affecting protein networking. Further, it is hypothesised that the dosing of xylanase and transglutaminase has a positive impact on rye dough and bread characteristics; the findings in this study evidenced that this increases the protein network by up to 38% accompanied by a higher volume rise of 10.67%, compared to standard rye dough. These outcomes combine a product-oriented and physiochemical design of a recipe, targeting structural and functional relationships, and demonstrate a successful methodology for enhancing rye bread quality. Copyright © 2017 Elsevier Ltd. All rights reserved.

Ground Based Program for the Physical Analysis of Macromolecular Crystal Growth

NASA Technical Reports Server (NTRS)

Malkin, Alexander J.

1999-01-01

In a reported period in situ atomic force microscopy was utilized in our laboratory to study mechanisms of growth and kinetics of crystallization of ten protein and virus crystals. These included canavalin, thaumatin, apoferritin, lipase, catalase, t-RNA, lysozyme, xylanase, turnip yellow mosaic virus (TYMV) and satellite tobacco mosaic virus (STMV). We have also designed and constructed in our laboratory both in situ conventional two-beam Michelson and phase shift Mach-Zenhder interferometers. Computer software for the processing of the interferometric images was developed as well. Interferometric techniques were applied for studies of growth kinetics and transport phenomena in crystallization of several macromolecular crystals. As a result of this work we have published 21 papers and have given many presentations at international and national meetings. A list of these publications and conference presentations is attached.

Extremozymes: A Potential Source for Industrial Applications.

PubMed

Dumorné, Kelly; Córdova, David Camacho; Astorga-Eló, Marcia; Renganathan, Prabhaharan

2017-04-28

Extremophilic microorganisms have established a diversity of molecular strategies in order to survive in extreme conditions. Biocatalysts isolated by these organisms are termed extremozymes, and possess extraordinary properties of salt allowance, thermostability, and cold adaptivity. Extremozymes are very resistant to extreme conditions owing to their great solidity, and they pose new opportunities for biocatalysis and biotransformations, as well as for the development of the economy and new line of research, through their application. Thermophilic proteins, piezophilic proteins, acidophilic proteins, and halophilic proteins have been studied during the last few years. Amylases, proteases, lipases, pullulanases, cellulases, chitinases, xylanases, pectinases, isomerases, esterases, and dehydrogenases have great potential application for biotechnology, such as in agricultural, chemical, biomedical, and biotechnological processes. The study of extremozymes and their main applications have emerged during recent years.

Utilization of xylan by yeasts and its conversion to ethanol by Pichia stipitis strains. [Cryptococcus; Pichia stipitis; Candida shehatae

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, H.; Biely, P.; Latta, R.K.

Yeasts able to grow on D-xylose were screened for the ability to hydrolyze xylan. Xylanase activity was found to be rare; a total of only 19 of more than 250 strains yielded a positive test result. The activity was localized largely in the genus Cryptococcus and in Pichia stipitis and its anamorph Candida shehatae. The ability to hydrolyze xylan was generally uncoupled from that to hydrolyze cellulose; only three of the xylan-positive strains also yielded a positive test for cellulolytic activity. Of the 19 xylanolytic strains. 2. P. stipitis CBS 5773 and CBS 5775, converted xylan into ethanol, with aboutmore » 60% of a theoretical yield computed on the basis of the amount of D-xylose present originally that could be released by acid hydrolysis.« less

Effect of polysaccharide admixtures on expression of multiple polysaccharide-degrading enzymes in Microbulbifer strain CMC-5.

PubMed

Jonnadula, RaviChand; Imran, Md; Poduval, Preethi B; Ghadi, Sanjeev C

2018-03-01

Microbulbifer strain CMC-5 produces agarase, alginate lyase, xylanase, carboxymethyl cellulase and carrageenase. The extracellular production of the above carbohydrases was investigated by growing Microbulbifer strain CMC-5 in a sea water based medium containing homologous/heterologous polysaccharides as a single substrate or as a combination of mixed assorted substrate. Presence of singular homologous polysaccharides in the growth medium induces respective carbohydrase at high levels. Any two polysaccharides in various combinations produced high level of homologous carbohydrase and low level of other heterologous carbohydrase. All five carbohydrases were consistently produced by strain CMC-5, when carboxymethyl cellulose was included as one of the substrate in dual substrate combination, or in presence of mix blends of all five polysaccharides. Interestingly, thalli of Gracilaria sp. that contain agar and cellulose predominantly in their cell wall induces only agarase expression in strain CMC-5.

Lignases and aldo-keto reductases for conversion of lignin-containing materials to fermentable products

DOEpatents

Scharf, Michael; Sethi, Amit

2016-09-13

Termites have specialized digestive systems that overcome the lignin barrier in wood to release fermentable simple sugars. Using the termite Reticulitermes flavipes and its gut symbionts, high-throughput titanium pyrosequencing and proteomics approaches experimentally compared the effects of lignin-containing diets on host-symbiont digestome composition. Proteomic investigations and functional digestive studies with recombinant lignocellulases conducted in parallel provided strong evidence of congruence at the transcription and translational levels and provide enzymatic strategies for overcoming recalcitrant lignin barriers in biofuel feedstocks. Briefly described, therefore, the disclosure provides a system for generating a fermentable product from a lignified plant material, the system comprising a cooperating series of at least two catalytically active polypeptides, where said catalytically active polypeptides are selected from the group consisting of: cellulase Cell-1, .beta.-glu cellulase, an aldo-keto-reductase, a catalase, a laccase, and an endo-xylanase.

Enzymes and chelating agent in cotton pretreatment.

PubMed

Csiszár, E; Losonczi, A; Szakács, G; Rusznák, I; Bezúr, L; Reicher, J

2001-08-23

Desized cotton fabric and cotton seed-coat fragments (impurities) have been treated with commercial cellulase (Celluclast 1.5 L), hemicellulase-pectinase (Viscozyme 120 L) and xylanase (Pulpzyme HC) enzymes. Seed-coat fragments hydrolyzed much faster than the cotton fabric itself. This relative difference in hydrolysis rates makes possible a direct enzymatic removal of seed-coat fragments from desized cotton fabric. Addition of chelating agents such as ethylenediamine-tetra-acetic acid (EDTA) markedly enhanced the directed enzyme action. Pretreatments carried out in acidic solution at pH 5 increased the lightness of seed-coat fragments, contrary to the samples treated in neutral medium at pH 7. Alkaline scouring resulted in darker seed-coat fragments except for the samples pretreated with Pulpzyme HC plus EDTA. This effect is similar to that observed in the biobleaching process in pulp and paper industry.

Compositional Changes and Baking Performance of Rye Dough As Affected by Microbial Transglutaminase and Xylanase.

PubMed

Grossmann, Isabel; Döring, Clemens; Jekle, Mario; Becker, Thomas; Koehler, Peter

2016-07-20

Doughs supplemented with endoxylanase (XYL) and varying amounts of microbial transglutaminase (TG) were analyzed by sequential protein extraction, quantitation of protein fractions and protein types, and determination of water-extractable arabinoxylans. With increasing TG activity, the concentration of prolamins and glutelins decreased and increased, respectively, and the prolamin-to-glutelin ratio strongly declined. The overall amount of extractable protein decreased with increasing TG level showing that cross-linking by TG provided high-molecular-weight protein aggregates. The decrease of the high-molecular-weight arabinoxylan fraction and the concurrent increase of the medium-molecular-weight fraction confirmed the degradation of arabinoxylans by XYL. However, XYL addition did not lead to significant improved cross-linking of rye proteins by TG. Volume and crumb hardness measurements of bread showed increased protein connectivity induced by XYL and TG. Significant positive effects on the final bread quality were especially obtained by XYL addition.

Fiber degradation potential of natural co-cultures of Neocallimastix frontalis and Methanobrevibacter ruminantium isolated from yaks (Bos grunniens) grazing on the Qinghai Tibetan Plateau.

PubMed

Wei, Ya-Qin; Long, Rui-Jun; Yang, Hui; Yang, Hong-Jian; Shen, Xi-Hui; Shi, Rui-Fang; Wang, Zhi-Ye; Du, Jun-Guo; Qi, Xiao-Jin; Ye, Qian-Hong

2016-06-01

Several natural anaerobic fungus-methanogen co-cultures have been isolated from rumen and feces source of herbivores with strong fiber degrading ability. In this study, we isolated 7 Neocallimastix with methanogen co-cultures from the rumen of yaks grazing on the Qinghai Tibetan Plateau. Based on morphological characteristics and internal transcribed spacer 1 sequences (ITS1), all the fungi were identified as Neocallimastix frontalis. The co-cultures were confirmed as the one fungus - one methanogen pattern by the PCR-denatured gradient gel electrophoresis (DGGE) assay. All the methanogens were identified as Methanobrevibacter ruminantium by 16s rRNA gene sequencing. We investigated the biodegrading capacity of the co-culture (N. frontalis + M. ruminantium) Yaktz1 on wheat straw, corn stalk and rice straw in a 7 days-incubation. The in vitro dry matter digestibility (IVDMD), acid detergent fiber digestibility (ADFD) and neural detergent fiber digestibility (NDFD) values of the substrates in the co-culture were significantly higher than those in the mono-culture N. frontalis Yaktz1. The co-culture exhibited high polysaccharide hydrolase (xylanase and FPase) and esterase activities. The xylanase in the co-culture reached the highest activity of 12500 mU/ml on wheat straw at the day 3 of the incubation. At the end of the incubation, 3.00 mmol-3.29 mmol/g dry matter of methane were produced by the co-culture. The co-culture also produced high level of acetate (40.00 mM-45.98 mM) as the end-product during the biodegradation. Interestingly, the N. frontalis Yaktz1 mono-culture produced large amount of lactate (8.27 mM-11.60 mM) and ethanol (163.11 mM-242.14 mM), many times more than those recorded in the previously reported anaerobic fungi. Our data suggests that the (N. frontalis + M. ruminantium) Yaktz1 co-culture and the N. frontalis Yaktz1 mono-culture both have great potentials for different industrial use. Copyright © 2016. Published by

Synthetic enzyme mixtures for biomass deconstruction: production and optimization of a core set.

PubMed

Banerjee, Goutami; Car, Suzana; Scott-Craig, John S; Borrusch, Melissa S; Aslam, Nighat; Walton, Jonathan D

2010-08-01

The high cost of enzymes is a major bottleneck preventing the development of an economically viable lignocellulosic ethanol industry. Commercial enzyme cocktails for the conversion of plant biomass to fermentable sugars are complex mixtures containing more than 80 proteins of suboptimal activities and relative proportions. As a step toward the development of a more efficient enzyme cocktail for biomass conversion, we have developed a platform, called GENPLAT, that uses robotic liquid handling and statistically valid experimental design to analyze synthetic enzyme mixtures. Commercial enzymes (Accellerase 1000 +/- Multifect Xylanase, and Spezyme CP +/- Novozyme 188) were used to test the system and serve as comparative benchmarks. Using ammonia-fiber expansion (AFEX) pretreated corn stover ground to 0.5 mm and a glucan loading of 0.2%, an enzyme loading of 15 mg protein/g glucan, and 48 h digestion at 50 degrees C, commercial enzymes released 53% and 41% of the available glucose and xylose, respectively. Mixtures of three, five, and six pure enzymes of Trichoderma species, expressed in Pichia pastoris, were systematically optimized. Statistical models were developed for the optimization of glucose alone, xylose alone, and the average of glucose + xylose for two digestion durations, 24 and 48 h. The resulting models were statistically significant (P < 0.0001) and indicated an optimum composition for glucose release (values for optimized xylose release are in parentheses) of 29% (5%) cellobiohydrolase 1, 5% (14%) cellobiohydrolase 2, 25% (25%) endo-beta1,4-glucanase 1, 14% (5%) beta-glucosidase, 22% (34%) endo-beta1,4-xylanase 3, and 5% (17%) beta-xylosidase in 48 h at a protein loading of 15 mg/g glucan. Comparison of two AFEX-treated corn stover preparations ground to different particle sizes indicated that particle size (100 vs. 500 microm) makes a large difference in total digestibility. The assay platform and the optimized "core" set together provide a starting

The GH51 α-l-arabinofuranosidase from Paenibacillus sp. THS1 is multifunctional, hydrolyzing main-chain and side-chain glycosidic bonds in heteroxylans.

PubMed

Bouraoui, Hanen; Desrousseaux, Marie-Laure; Ioannou, Eleni; Alvira, Pablo; Manaï, Mohamed; Rémond, Caroline; Dumon, Claire; Fernandez-Fuentes, Narcis; O'Donohue, Michael J

2016-01-01

Conceptually, multi-functional enzymes are attractive because in the case of complex polymer hydrolysis having two or more activities defined by a single enzyme offers the possibility of synergy and reduced enzyme cocktail complexity. Nevertheless, multi-functional enzymes are quite rare and are generally multi-domain assemblies with each activity being defined by a separate protein module. However, a recent report described a GH51 arabinofuranosidase from Alicyclobacillus sp. A4 that displays both α-l-arabinofuranosidase and β-d-xylanase activities, which are defined by a single active site. Following on from this, we describe in detail another multi-functional GH51 arabinofuranosidase and discuss the molecular basis of multifunctionality. THSAbf is a GH51 α-l-arabinofuranosidase. Characterization revealed that THSAbf is active up to 75 °C, stable at 60 °C and active over a broad pH range (4-7). THSAbf preferentially releases para-nitrophenyl from the l-arabinofuranoside (k cat/K M = 1050 s(-1) mM(-1)) and to some extent from d-galactofuranoside and d-xyloside. THSAbf is active on 4-O-methylglucuronoxylans from birch and beechwood (10.8 and 14.4 U mg(-1), respectively) and on sugar beet branched and linear arabinans (1.1 ± 0.24 and 1.8 ± 0.1 U mg(-1)). Further investigation revealed that like the Alicyclobacillus sp. A4 α-l-arabinofuranosidase, THSAbf also displays endo-xylanase activity, cleaving β-1,4 bonds in heteroxylans. The optimum pH for THASAbf activity is substrate dependent, but ablation of the catalytic nucleophile caused a general loss of activity, indicating the involvement of a single active center. Combining the α-l-arabinofuranosidase with a GH11 endoxylanase did not procure synergy. The molecular modeling of THSAbf revealed a wide active site cleft and clues to explain multi-functionality. The discovery of single active site, multifunctional enzymes such as THSAbf opens up exciting avenues for enzyme engineering and the

Enzyme effects on extruded diets for dogs with soybean meal as a substitute for poultry by-product meal.

PubMed

Tortola, L; Souza, N G; Zaine, L; Gomes, M O S; Matheus, L F O; Vasconcellos, R S; Pereira, G T; Carciofi, A C

2013-05-01

The effects of exogenous enzymes supplementation on kibble diets for dogs formulated with soybean meal (SBM) as a substitute for poultry by-product meal (PM) was investigated on nutrient digestibility, fermentation products formation, post-prandial urea response and selected faecal bacteria counts. Two kibble diets with similar compositions were used in two trials: PM-based diet (28.9% of PM; soybean hulls as a fibre source) and SBM-based diet (29.9% of SBM). In experiment 1, the SBM diet was divided into three diets: SBM-0, without enzyme addition; SBM-1, covered after extrusion with 7500 U protease/kg and 45 U cellulase/kg; and SBM-2, covered with 15,000 U protease/kg and 90 U cellulase/kg. In experiment 2, the SBM diet was divided into three diets: SBM-0; SBM-1, covered with 140 U protease/kg; 8 U cellulase/kg, 800 U pectinase/kg, 60 U phytase/kg, 40 U betaglucanase/kg and 20 U xylanase/kg; and SMB-2, covered with 700 U protease/kg, 40 U cellulase/kg, 4000 U pectinase/kg, 300 U phytase/kg, 200 U betaglucanase/kg and 100 U xylanase/kg. Each experiment followed a block design with six dogs per diet. Data were submitted to analysis of variance and means compared by orthogonal and polynomial contrasts (p < 0.05). In both experiments, nutrients and energy digestibility did not differ between diets (p > 0.05). SBM consumption resulted in increased faecal moisture and production (p < 0.05), without effect on faecal score. Higher concentration of propionate, acetate and lactate, and lower ammonia and pH were found in the faeces of dogs fed SBM (p < 0.05). Higher post-prandial urea was verified in dogs fed SBM (p < 0.05). In experiment 2, the addition of enzymes increased faecal concentration of propionate, acetate and total short-chain fatty acid (p < 0.05) and tended to reduce post-prandial urea concentration (p = 0.06). Although with similar digestibility, SBM shows a worse utilization of absorbed amino acids than the PM. Soybean oligosaccharides can beneficially

Characterization of salt-adapted secreted lignocellulolytic enzymes from the mangrove fungus Pestalotiopsis sp.

PubMed

Arfi, Yonathan; Chevret, Didier; Henrissat, Bernard; Berrin, Jean-Guy; Levasseur, Anthony; Record, Eric

2013-01-01

Fungi are important for biomass degradation processes in mangrove forests. Given the presence of sea water in these ecosystems, mangrove fungi are adapted to high salinity. Here we isolate Pestalotiopsis sp. NCi6, a halotolerant and lignocellulolytic mangrove fungus of the order Xylariales. We study its lignocellulolytic enzymes and analyse the effects of salinity on its secretomes. De novo transcriptome sequencing and assembly indicate that this fungus possesses of over 400 putative lignocellulolytic enzymes, including a large fraction involved in lignin degradation. Proteomic analyses of the secretomes suggest that the presence of salt modifies lignocellulolytic enzyme composition, with an increase in the secretion of xylanases and cellulases and a decrease in the production of oxidases. As a result, cellulose and hemicellulose hydrolysis is enhanced but lignin breakdown is reduced. This study highlights the adaptation to salt of mangrove fungi and their potential for biotechnological applications.

Engineering fungal morphology for enhanced production of hydrolytic enzymes by Aspergillus oryzae SBS50 using microparticles.

PubMed

Singh, Bijender

2018-06-01

Effect of microparticles and silver nanoparticles was studied on the production of hydrolytic enzymes by a potent phytase-producing mould, Aspergillus oryzae SBS50. Addition of microparticles, viz. talc powder and aluminum oxide enhanced phytase production from 2894 to 3903 and 2847 to 4204 U/L, cellulase from 2529 to 4931 and 2455 to 3444 U/L, xylanase from 9067 to 9642 and 9994 to 14,783 U/L, amylase from 5880 to 11,000 and 6130 to 13,145 U/L, respectively. Fungal morphology was also engineered by the use of microparticles. Fungal pellet size was significantly reduced (~ 90%) by the addition of microparticles. Fermentation time was reduced from 4 to 3 days after the addition of microparticles, thus increasing the productivity of the enzymes significantly. These results confirmed the importance of microparticles in engineering fungal morphology for enhanced production of hydrolytic enzymes.

Isolation and characterization of fatty acid methyl ester (FAME)-producing Streptomyces sp. S161 from sheep (Ovis aries) faeces.

PubMed

Lu, Y; Wang, J; Deng, Z; Wu, H; Deng, Q; Tan, H; Cao, L

2013-09-01

An actinomycete producing oil-like mixtures was isolated and characterized. The strain was isolated from sheep faeces and identified as Streptomyces sp. S161 based on 16S rRNA gene sequence analysis. The strain showed cellulase and xylanase activities. The (1) H nuclear magnetic resonance (NMR) spectra of the mixtures showed that the mixtures were composed of fatty acid methyl esters (52·5), triglycerides (13·7) and monoglycerides (9·1) (mol.%). Based on the gas chromatography-mass spectrometry (GC-MS) analysis, the fatty acid methyl esters were mainly composed of C14-C16 long-chain fatty acids. The results indicated that Streptomyces sp. S161 could produce fatty acid methyl esters (FAME) directly from starch. To our knowledge, this is the first isolated strain that can produce biodiesel (FAME) directly from starch. © 2013 The Society for Applied Microbiology.

Influence of fluid dynamic conditions on enzymatic hydrolysis of lignocellulosic biomass: Effect of mass transfer rate.

PubMed

Wojtusik, Mateusz; Zurita, Mauricio; Villar, Juan C; Ladero, Miguel; Garcia-Ochoa, Felix

2016-09-01

The effect of fluid dynamic conditions on enzymatic hydrolysis of acid pretreated corn stover (PCS) has been assessed. Runs were performed in stirred tanks at several stirrer speed values, under typical conditions of temperature (50°C), pH (4.8) and solid charge (20% w/w). A complex mixture of cellulases, xylanases and mannanases was employed for PCS saccharification. At low stirring speeds (<150rpm), estimated mass transfer coefficients and rates, when compared to chemical hydrolysis rates, lead to results that clearly show low mass transfer rates, being this phenomenon the controlling step of the overall process rate. However, for stirrer speed from 300rpm upwards, the overall process rate is controlled by hydrolysis reactions. The ratio between mass transfer and overall chemical reaction rates changes with time depending on the conditions of each run. Copyright © 2016 Elsevier Ltd. All rights reserved.

Xylella fastidiosa requires polygalacturonase for colonization and pathogenicity in Vitis vinifera grapevines.

PubMed

Roper, M Caroline; Greve, L Carl; Warren, Jeremy G; Labavitch, John M; Kirkpatrick, Bruce C

2007-04-01

Xylella fastidiosa is the causal agent of Pierce's disease of grape, an economically significant disease for the grape industry. X. fastidiosa systemically colonizes the xylem elements of grapevines and is able to breach the pit pore membranes separating xylem vessels by unknown mechanisms. We hypothesized that X. fastidiosa utilizes cell wall degrading enzymes to break down pit membranes, based on the presence of genes involved in plant cell wall degradation in the X. fastidiosa genome. These genes include several beta-1,4 endoglucanases, several xylanases, several xylosidases, and one polygalacturonase (PG). In this study, we demonstrated that the pglA gene encodes a functional PG. A mutant in pglA lost pathogenicity and was compromised in its ability to systemically colonize Vitis vinifera grapevines. The results indicate that PG is required for X. fastidiosa to successfully infect grapevines and is a critical virulence factor for X. fastidiosa pathogenesis in grapevine.

Application of metagenomic techniques in mining enzymes from microbial communities for biofuel synthesis.

PubMed

Xing, Mei-Ning; Zhang, Xue-Zhu; Huang, He

2012-01-01

Feedstock for biofuel synthesis is transitioning to lignocelluosic biomass to address criticism over competition between first generation biofuels and food production. As microbial catalysis is increasingly applied for the conversion of biomass to biofuels, increased import has been placed on the development of novel enzymes. With revolutionary advances in sequencer technology and metagenomic sequencing, mining enzymes from microbial communities for biofuel synthesis is becoming more and more practical. The present article highlights the latest research progress on the special characteristics of metagenomic sequencing, which has been a powerful tool for new enzyme discovery and gene functional analysis in the biomass energy field. Critical enzymes recently developed for the pretreatment and conversion of lignocellulosic materials are evaluated with respect to their activity and stability, with additional explorations into xylanase, laccase, amylase, chitinase, and lipolytic biocatalysts for other biomass feedstocks. Copyright © 2012 Elsevier Inc. All rights reserved.

Bound Phenolics of Quinoa Seeds Released by Acid, Alkaline, and Enzymatic Treatments and Their Antioxidant and α-Glucosidase and Pancreatic Lipase Inhibitory Effects.

PubMed

Tang, Yao; Zhang, Bing; Li, Xihong; Chen, Peter X; Zhang, Hua; Liu, Ronghua; Tsao, Rong

2016-03-02

Unextractable phenolics from plant foods and their role in health benefits have become increasingly important. Meal residues of three quinoa seeds free of fat and extractable phenolics were subjected to acid, alkaline, and enzymatic hydrolyses. The total and individual phenolic compounds released were analyzed, and 19 phenolics, predominantly phenolic acids and several flavonoids, were identified. The concentration of bound phenolics was highest in black quinoa followed by red and white, regardless of the hydrolysis method. Higher phenolic contents also showed stronger antioxidant activities and inhibition of α-glucosidase and pancreatic lipase activities. Carbohydrases, that is, pectinase, xylanase and feruloyl esterase, which effectively liberated bound phenolics are known to be secreted by colonic bacteria, suggesting potential antioxidant and anti-inflammatory effects by these compounds in the large intestine during colonic fermentation. These results can also be applied to treat foods high in bound phenolics to enhance bioaccessibility.

Mathematical modeling of enzyme production using Trichoderma harzianum P49P11 and sugarcane bagasse as carbon source.

PubMed

Gelain, Lucas; da Cruz Pradella, José Geraldo; da Costa, Aline Carvalho

2015-12-01

A mathematical model to describe the kinetics of enzyme production by the filamentous fungus Trichoderma harzianum P49P11 was developed using a low cost substrate as main carbon source (pretreated sugarcane bagasse). The model describes the cell growth, variation of substrate concentration and production of three kinds of enzymes (cellulases, beta-glucosidase and xylanase) in different sugarcane bagasse concentrations (5; 10; 20; 30; 40 gL(-1)). The 10 gL(-1) concentration was used to validate the model and the other to parameter estimation. The model for enzyme production has terms implicitly representing induction and repression. Substrate variation was represented by a simple degradation rate. The models seem to represent well the kinetics with a good fit for the majority of the assays. Validation results indicate that the models are adequate to represent the kinetics for a biotechnological process. Copyright © 2015 Elsevier Ltd. All rights reserved.

Evaluation of Mucor indicus and Saccharomyces cerevisiae capability to ferment hydrolysates of rape straw and Miscanthus giganteus as affected by the pretreatment method.

PubMed

Lewandowska, Małgorzata; Szymańska, Karolina; Kordala, Natalia; Dąbrowska, Aneta; Bednarski, Włodzimierz; Juszczuk, Andrzej

2016-07-01

Rape straw and Miscanthus giganteus was pretreated chemically with oxalic acid or sodium hydroxide. The pretreated substrates were hydrolyzed with enzymatic preparations of cellulase, xylanase and cellobiase. The highest concentration of reducing sugars was achieved after hydrolysis of M. giganteus pretreated with NaOH (51.53gdm(-3)). In turn, the highest yield of enzymatic hydrolysis determined based on polysaccharides content in the pretreated substrates was obtained in the experiments with M. giganteus and oxalic acid (99.3%). Rape straw and M. giganteus hydrolysates were fermented using yeast Saccharomyces cerevisiae 7, NRRL 978 or filamentous fungus Mucor rouxii (Mucor indicus) DSM 1191. The highest ethanol concentration was determined after fermentation of M. giganteus hydrolysate pretreated with NaOH using S. cerevisiae (1.92% v/v). Considering cellulose content in the pretreated solid, the highest degree of its conversion to ethanol (86.2%) was achieved after fermentation of the hydrolysate of acid-treated M. giganteus using S. cerevisiae. Copyright © 2016 Elsevier Ltd. All rights reserved.

Economic Analysis of the Production of Amylases and Other Hydrolases by Aspergillus awamori in Solid-State Fermentation of Babassu Cake

PubMed Central

de Castro, Aline Machado; Carvalho, Daniele Fernandes; Freire, Denise Maria Guimarães; Castilho, Leda dos Reis

2010-01-01

Amylases are one of the most important industrial enzymes produced worldwide, with their major application being in ethanol manufacturing. This work investigated the production of amylases by solid-state fermentation of babassu cake, using the filamentous fungus Aspergillus awamori IOC-3914. Lab-scale experiments were carried out to generate input data for simulations of an industrial plant for amylase production. Additionally to the target enzymes, other hydrolases (cellulases, xylanases, and proteases) were also produced, enriching the final product. The most suitable fermentation time was 144 hours, when exoamylase and endoamylase activities of 40.5 and 42.7 U g−1 were achieved, respectively. A first evaluation showed a large impact of the inoculum propagation medium on production costs. Therefore, five propagation media were compared, and PDA medium presented the best cost-benefit ratio. The credits obtained from sales of fermented cake as a coproduct enabled a significant decrease in the production cost of the enzyme product, down to 10.40 USD kg−1. PMID:21048867

Mobilization of hydrophobic contaminants from soils by enzymatic depolymerization of soil organic matter.

PubMed

Wicke, Daniel; Reemtsma, Thorsten

2010-02-01

The effect of hydrolytic exoenzymes on the release of hydrophobic organic contaminants (HOC) from two different surface soils was studied in laboratory batch experiments. Incubation of the soils with cellulase with an activity fivefold above the inherent soil activity enhanced the release of hydrophobic contaminants (polycyclic aromatic hydrocarbons (PAH), polychlorinated biphenyls (PCB) and hydroxylated PCB) by 40-200%. Xylanase and invertase did not show measurable effects at comparable relative activity levels. This suggests that cellulose substructures are important for the retention of HOC in soil organic matter (SOM). Hydrolytic exoenzymes, and the microorganisms that release them, contribute to the mobilization of HOC from soil, by shifting the sorption equilibrium in the course of SOM transformation into dissolved organic matter or by facilitating HOC diffusion as a consequence of reduced rigidity of SOM. We conclude that not only biodegradation but also sorption and desorption of HOC in soil can be influenced by (micro-) biology and the factors that determine its activity.

Application of simultaneous saccharification and fermentation (SSF) from viscosity reducing of raw sweet potato for bioethanol production at laboratory, pilot and industrial scales.

PubMed

Zhang, Liang; Zhao, Hai; Gan, Mingzhe; Jin, Yanlin; Gao, Xiaofeng; Chen, Qian; Guan, Jiafa; Wang, Zhongyan

2011-03-01

The aim of this work was to research a bioprocess for bioethanol production from raw sweet potato by Saccharomyces cerevisiae at laboratory, pilot and industrial scales. The fermentation mode, inoculum size and pressure from different gases were determined in laboratory. The maximum ethanol concentration, average ethanol productivity rate and yield of ethanol after fermentation in laboratory scale (128.51 g/L, 4.76 g/L/h and 91.4%) were satisfactory with small decrease at pilot scale (109.06 g/L, 4.89 g/L/h and 91.24%) and industrial scale (97.94 g/L, 4.19 g/L/h and 91.27%). When scaled up, the viscosity caused resistance to fermentation parameters, 1.56 AUG/g (sweet potato mash) of xylanase decreased the viscosity from approximately 30000 to 500 cp. Overall, sweet potato is a attractive feedstock for be bioethanol production from both the economic standpoints and environmentally friendly. Copyright © 2011 Elsevier Ltd. All rights reserved.

Vine Trimming Shoots as Substrate for Ferulic Acid Esterases Production.

PubMed

Pérez-Rodríguez, N; Outeiriño, D; Torrado Agrasar, A; Domínguez, J M

2017-02-01

Ferulic acid esterases (FAE) possess a large variety of biotechnological applications mainly based on their ability to release ferulic acid from lignocellulosic matrixes. The use of vine trimming shoots (VTS), an agricultural waste, as substrate for the generation of this kind of esterases represents an attractive alternative to change the consideration of VTS from residue to resource. Furthermore, xylanase, cellobiase, and cellulase activities were quantified. Six microorganisms were screened for FAE production by solid-state fermentation, and the effects of the additional supplementation and substrate size were also tested. Finally, the process was scaled-up to a horizontal bioreactor where the influence of aeration in enzymatic activities was evaluated. Thus, the optimal FAE activity (0.44 U/g dry VTS) was attained by Aspergillus terreus CECT 2808, in non-additional supplementation media, using the larger particles size of substrate (≤ 5 mm) and at a flow rate of 0.7 L/min.

Lignocellulose degradation patterns, structural changes, and enzyme secretion by Inonotus obliquus on straw biomass under submerged fermentation.

PubMed

Xu, Xiangqun; Xu, Zhiqi; Shi, Song; Lin, Mengmeng

2017-10-01

This study examined the white rot fungus I. obliquus on the degradation of three types of straw biomass and the production of extracellular lignocellulolytic enzymes under submerged fermentation. The fungus process resulted in a highest lignin loss of 72%, 39%, and 47% in wheat straw, rice straw, and corn stover within 12days, respectively. In merely two days, the fungus selectively degraded wheat straw lignin by 37%, with only limited cellulose degradation (13%). Fourier transform infrared spectroscopy revealed that the fungus most effectively degraded the wheat straw lignin and rice straw crystalline cellulose. Scanning electronic microscopy showed the most pronounced structural changes in wheat straw. High activities of manganese peroxidase (159.0U/mL) and lignin peroxidase (123.4U/mL) were observed in wheat straw culture on Day 2 and 4, respectively. Rice straw was the best substrate to induce the production of cellulase and xylanase. Copyright © 2017 Elsevier Ltd. All rights reserved.

Application of solid waste from anaerobic digestion of poultry litter in Agrocybe aegerita cultivation: mushroom production, lignocellulolytic enzymes activity and substrate utilization.

PubMed

Isikhuemhen, Omoanghe S; Mikiashvili, Nona A; Kelkar, Vinaya

2009-06-01

The degradation and utilization of solid waste (SW) from anaerobic digestion of poultry litter by Agrocybe aegerita was evaluated through mushroom production, loss of organic matter (LOM), lignocellulolytic enzymes activity, lignocellulose degradation and mushroom nutrients content. Among the substrate combinations (SCs) tested, substrates composed of 10-20% SW, 70-80% wheat straw and 10% millet was found to produce the highest mushroom yield (770.5 and 642.9 g per 1.5 kg of substrate). LOM in all SCs tested varied between 8.8 and 48.2%. A. aegerita appears to degrade macromolecule components (0.6-21.8% lignin, 33.1-55.2% cellulose and 14-53.9% hemicellulose) during cultivation on the different SCs. Among the seven extracellular enzymes monitored, laccase, peroxidase and CMCase activities were higher before fruiting; while xylanase showed higher activities after fruiting. A source of carbohydrates (e.g., millet) in the substrate is needed in order to obtain yield and biological efficiency comparable to other commercially cultivated exotic mushrooms.

Investigation of mircroorganisms colonising activated zeolites during anaerobic biogas production from grass silage.

PubMed

Weiss, S; Zankel, A; Lebuhn, M; Petrak, S; Somitsch, W; Guebitz, G M

2011-03-01

The colonisation of activated zeolites (i.e. clinoptilolites) as carriers for microorganisms involved in the biogas process was investigated. Zeolite particle sizes of 1.0-2.5mm were introduced to anaerobic laboratory batch-cultures and to continuously operated bioreactors during biogas production from grass silage. Incubation over 5-84 days led to the colonisation of zeolite surfaces in small batch-cultures (500 ml) and even in larger scaled and flow-through disturbed bioreactors (28 l). Morphological insights were obtained by using scanning electron microscopy (SEM). Single strand conformation polymorphism (SSCP) analysis based on amplification of bacterial and archaeal 16S rRNA fragments demonstrated structurally distinct populations preferring zeolite as operational environment. via sequence analysis conspicuous bands from SSCP patterns were identified. Populations immobilised on zeolite (e.g. Ruminofilibacter xylanolyticum) showed pronounced hydrolytic enzyme activity (xylanase) shortly after re-incubation in sterilised sludge on model substrate. In addition, the presence of methanogenic archaea on zeolite particles was demonstrated. Copyright © 2010 Elsevier Ltd. All rights reserved.

Constitutive cellulase production from glucose using the recombinant Trichoderma reesei strain overexpressing an artificial transcription activator.

PubMed

Zhang, Xiaoyue; Li, Yonghao; Zhao, Xinqing; Bai, Fengwu

2017-01-01

The high cost of cellulase production presents biggest challenge in biomass deconstruction. Cellulase production by Trichoderma reesei using low cost carbon source is of great interest. In this study, an artificial transcription activator containing the Cre1 binding domain linked to the Xyr1 effector and binding domains was designed and constitutively overexpressed in T. reesei RUT C30. The recombinant strain T. reesei zxy-2 displayed constitutive cellulase production using glucose as a sole carbon source, and the production titer was 12.75-fold of that observed with T. reesei RUT C30 in shake flask culture. Moreover, FPase and xylanase titers of 2.63 and 108.72IU/mL, respectively, were achieved using glucose as sole carbon source within 48h in a 7-L fermenter by batch fermentation using T. reesei zxy-2. The crude enzyme obtained was used to hydrolyze alkali pretreated corn stover, and a high glucose yield of 99.18% was achieved. Copyright © 2016. Published by Elsevier Ltd.

Fungal solid state fermentation on agro-industrial wastes for acid wastewater decolorization in a continuous flow packed-bed bioreactor.

PubMed

Iandolo, Donata; Amore, Antonella; Birolo, Leila; Leo, Gabriella; Olivieri, Giuseppe; Faraco, Vincenza

2011-08-01

This study was aimed at developing a process of solid state fermentation (SSF) with the fungi Pleurotus ostreatus and Trametes versicolor on apple processing residues for wastewater decolorization. Both fungi were able to colonize apple residues without any addition of nutrients, material support or water. P. ostreatus produced the highest levels of laccases (up to 9U g(-1) of dry matter) and xylanases (up to 80U g(-1) of dry matter). A repeated batch decolorization experiment was set up with apple residues colonized by P. ostreatus, achieving 50% decolorization and 100% detoxification after 24h, and, adding fresh wastewater every 24h, a constant decolorization of 50% was measured for at least 1 month. A continuous decolorization experiment was set up by a packed-bed reactor based on colonized apple residues achieving a performance of 100mg dye L(-1)day(-1) at a retention time of 50h. Copyright © 2011 Elsevier Ltd. All rights reserved.

Effects of rhamnolipid and initial compost particle size on the two-stage composting of green waste.

PubMed

Zhang, Lu; Sun, Xiangyang

2014-07-01

Composting is a potential alternative to green waste incineration or deposition in landfills. The effects of the biosurfactant rhamnolipid (RL) (at 0.0%, 0.15%, and 0.30%) and initial compost particle size (IPS) (10, 15, and 25 mm) on a new, two-stage method for composting green waste was investigated. A combination of RL addition and IPS adjustment improved the quality of the finished compost in terms of its physical characteristics, pH, C/N ratio, nutrient content, cellulose and hemicellulose contents, water-soluble carbon (WSC) content, xylanase and CMCase activities, numbers of culturable microorganisms (bacteria, actinomycetes, and fungi), and toxicity to germinating seeds. The production of a stable and mature compost required only 24 days with the optimized two-stage composting method described here rather than the 90-270 days required with traditional composting. The best quality compost was obtained with 0.15% RL and an IPS of 15 mm. Copyright © 2014 Elsevier Ltd. All rights reserved.

Novel transgenic pigs with enhanced growth and reduced environmental impact

PubMed Central

Yang, Huaqiang; Liu, Dewu; Cai, Gengyuan; Li, Guoling; Mo, Jianxin; Wang, Dehua; Zhong, Cuili; Wang, Haoqiang; Sun, Yue; Shi, Junsong; Zheng, Enqin; Meng, Fanming; Zhang, Mao; He, Xiaoyan; Zhou, Rong; Zhang, Jian; Huang, Miaorong; Zhang, Ran; Li, Ning; Fan, Mingzhe; Yang, Jinzeng

2018-01-01

In pig production, inefficient feed digestion causes excessive nutrients such as phosphorus and nitrogen to be released to the environment. To address the issue of environmental emissions, we established transgenic pigs harboring a single-copy quad-cistronic transgene and simultaneously expressing three microbial enzymes, β-glucanase, xylanase, and phytase in the salivary glands. All the transgenic enzymes were successfully expressed, and the digestion of non-starch polysaccharides (NSPs) and phytate in the feedstuff was enhanced. Fecal nitrogen and phosphorus outputs in the transgenic pigs were reduced by 23.2–45.8%, and growth rate improved by 23.0% (gilts) and 24.4% (boars) compared with that of age-matched wild-type littermates under the same dietary treatment. The transgenic pigs showed an 11.5–14.5% improvement in feed conversion rate compared with the wild-type pigs. These findings indicate that the transgenic pigs are promising resources for improving feed efficiency and reducing environmental impact. PMID:29784082

Exploring surface characterization and electrostatic property of Hybrid Pennisetum during alkaline sulfite pretreatment for enhanced enzymatic hydrolysability.

PubMed

Yang, Ming; Wang, Jingfeng; Hou, Xincun; Wu, Juying; Fan, Xifeng; Jiang, Fan; Tao, Pan; Wang, Fan; Peng, Pai; Yang, Fangxia; Zhang, Junhua

2017-11-01

The surface characterization and electrostatic property of Hybrid Pennisetum (HP) after alkaline sulfite pretreatment were explored for enhanced enzymatic hydrolysability. The O/C ratio in HP increased from 0.34 to 0.60, and C1 concentration decreased from 62.5% to 31.6%, indicating that alkaline sulfite pretreatment caused poorer lignin but richer carbohydrate on HP surface. Zeta potential and sulfur element analysis indicated that more enzymes would preferably adsorb on the carbohydrate surface of alkaline sulfite pretreated HP because the lignin was sulfonated, which facilitated the decrease of non-productive adsorption. Glucose yield of alkaline sulfite pretreated HP reached to 100% by synergistic action of cellulase and xylanase in the hydrolysis, which was significantly higher than that of NaOH pretreated, and the concentration of glucose released was 1.52times higher. The results suggested that alkaline sulfite pretreatment had potential for improving the HP hydrolysability, and the surface characterization and electrostatic property facilitated the enzymatic digestibility. Copyright © 2017 Elsevier Ltd. All rights reserved.

A novel marine bacterium Isoptericola sp. JS-C42 with the ability to saccharifying the plant biomasses for the aid in cellulosic ethanol production.

PubMed

Santhi, Velayudhan Satheeja; Gupta, Ashutosh; Saranya, Somasundaram; Jebakumar, Solomon Robinson David

2014-06-01

The ever growing demands for food products such as starch and sugar produces; there is a need to find the sources for saccharification for cellulosic bioethanol production. This study provides the first evidence of the lignocellulolytic and saccharifying ability of a marine bacterium namely Isoptericola sp. JS-C42, a Gram positive actinobacterium with the cocci cells embedded on mycelia isolated from the Arabian Sea, India. It exhibited highest filter paper unit effect, endoglucanase, exoglucanase, cellobiohydrolase, β-glucosidase, xylanase and ligninase effect. The hydrolytic potential of the enzymes displayed the efficient saccharification capability of steam pretreated biomass. It was also found to degrade the paddy, sorghum, Acacia mangium and Ficus religiosa into simple reducing sugars by its efficient lignocellulose enzyme complex with limited consumption of sugars. Production of ethanol was also achieved with the Saccharomyces cerevisiae . Overall, it offers a great potential for the cellulosic ethanol production in an economically reliable and eco-friendly point-of-care.

[Bacterium Arthrobacter agilis UMCV2 and diverse amines inhibit in vitro growth of wood-decay fungi].

PubMed

Orozco-Mosqueda, M Del Carmen; Valencia-Cantero, Eduardo; López-Albarrán, Pablo; Martínez-Pacheco, Mauro; Velázquez-Becerra, Crisanto

2015-01-01

The kingdom Fungi is represented by a large number of organisms, including pathogens that deteriorate the main structural components of wood, such as cellulose, hemicellulose and lignin. The aim of our work was to characterize the antifungal activity in Arthrobacter agilis UMCV2 and diverse amines against wood-decaying fungi. Four fungal organisms (designated as UMTM) were isolated from decaying wood samples obtained from a forest in Cuanajo-Michoacán, México. Two of them showed a clear enzymatic activity of cellulases, xylanases and oxido-reducing enzymes and were identified as Hypocrea (UMTM3 isolate) and Fusarium (UMTM13 isolate). In vitro, the amines showed inhibitory effect against UMTM growth and one of the amines, dimethylhexadecylamine (DMA16), exhibited strong potential as wood preventive treatment, against the attack of decaying fungi. Copyright © 2015 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.

Hydrolytic enzyme activities in shiitake mushroom (Lentinula edodes) strains cultivated on coffee pulp.

PubMed

Mata, Gerardo; Salmones, Dulce; Pérez-Merlo, Rosalía

Hydrolytic enzyme production (cellulases, laminarinases and xylanases) was studied in cultures of Lentinula edodes on sterilized coffee pulp. Samples of substrate colonized by mycelia were taken after 7, 14, 21, 28 and 35 days of incubation at 25°C (W1 to W5) and during the fruiting period at different stages: formation of primordia (PF), first harvest (H) and one week after the first harvest (PH). The enzymatic activity was lower during the early mycelial growth and showed higher levels during the formation and development of fruiting bodies. During the reproductive stage of the fungus, the samples were subjected to a soaking treatment; however, it was not possible to relate this soaking treatment to the increase in enzyme production. The levels of enzymatic activity suggest that secretion of the studied enzymes does not influence the adaptability of the strains to the substrate. Copyright © 2016 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.

Onion skin waste as a valorization resource for the by-products quercetin and biosugar.

PubMed

Choi, In Seong; Cho, Eun Jin; Moon, Jae-Hak; Bae, Hyeun-Jong

2015-12-01

Onion skin waste (OSW), which is produced from processed onions, is a major industrial waste. We evaluated the use of OSW for biosugar and quercetin production. The carbohydrate content of OSW was analyzed, and the optimal conversion conditions were evaluated by varying enzyme mixtures and loading volumes for biosugar production and quercetin extraction. The enzymatic conversion rate of OSW to biosugar was 98.5% at 0.72 mg of cellulase, 0.16 mg of pectinase, and 1.0mg of xylanase per gram of dry OSW. Quercetin extraction also increased by 1.61-fold after complete enzymatic hydrolysis. In addition, the newly developed nano-matrix (terpyridine-immobilized silica-coated magnetic nanoparticles-zinc (TSMNP-Zn matrix) was utilized to separate quercetin from OSW extracts. The nano-matrix facilitated easy separation and purification of quercetin. Using the TSMNP-Zn matrix the quercetin was approximately 90% absorbed. In addition, the recovery yield of quercetin was approximately 75% after treatment with ethylenediaminetetraacetic acid. Copyright © 2015 Elsevier Ltd. All rights reserved.

Modelling of different enzyme productions by solid-state fermentation on several agro-industrial residues.

PubMed

Diaz, Ana Belen; Blandino, Ana; Webb, Colin; Caro, Ildefonso

2016-11-01

A simple kinetic model, with only three fitting parameters, for several enzyme productions in Petri dishes by solid-state fermentation is proposed in this paper, which may be a valuable tool for simulation of this type of processes. Basically, the model is able to predict temporal fungal enzyme production by solid-state fermentation on complex substrates, maximum enzyme activity expected and time at which these maxima are reached. In this work, several fermentations in solid state were performed in Petri dishes, using four filamentous fungi grown on different agro-industrial residues, measuring xylanase, exo-polygalacturonase, cellulose and laccase activities over time. Regression coefficients after fitting experimental data to the proposed model turned out to be quite high in all cases. In fact, these results are very interesting considering, on the one hand, the simplicity of the model and, on the other hand, that enzyme activities correspond to different enzymes, produced by different fungi on different substrates.

Myceliophthora thermophila syn. Sporotrichum thermophile: a thermophilic mould of biotechnological potential.

PubMed

Singh, Bijender

2016-01-01

Myceliophthora thermophila syn. Sporotrichum thermophile is a ubiquitous thermophilic mould with a strong ability to degrade organic matter during optimal growth at 45 °C. Both genome analysis and experimental data have suggested that the mould is capable of hydrolyzing all major polysaccharides found in biomass. The mould is able to secrete a large number of hydrolytic enzymes (cellulases, laccases, xylanases, pectinases, lipases, phytases and some other miscellaneous enzymes) employed in various biotechnological applications. Characterization of the biomass-hydrolyzing activity of wild and recombinant enzymes suggests that this mould is highly efficient in biomass decomposition at both moderate and high temperatures. The native enzymes produced by the mould are more efficient in activity than their mesophilic counterparts beside their low enzyme titers. The mould is able to synthesize various biomolecules, which are used in multifarious applications. Genome sequence data of M. thermophila also supported the physiological data. This review describes the biotechnological potential of thermophilic mould, M. thermophila supported by genomic and experimental evidences.

Some Properties of Extracellular Acetylxylan Esterase Produced by the Yeast Rhodotorula mucilaginosa†

PubMed Central

Lee, Hung; To, Rebecca J. B.; Latta, Roger K.; Biely, Peter; Schneider, Henry

1987-01-01

The red yeast Rhodotorula mucilaginosa produced an esterase that accumulated in the culture supernatant on induction with triacetin. The enzyme was specific for substrates bearing an O-acetyl group, but was relatively nonspecific for the rest of the molecule, which could consist of a phenol, a monosaccharide, a polysaccharide, or an aliphatic alcohol. The esterase was more active against acetylxylan and glucose β-d-pentaacetate than were a number of esterases from plant and animal sources, when activities on 4-nitrophenyl acetate were compared. The enzyme exhibited Michaelis-Menten kinetics and was active over a broad pH range (5.5 to 9.2), with an optimum between pH 8 and 10. In addition, the enzyme retained its activity for 2 h at 55°C. The yeast that produced the enzyme did not produce xylanase and, hence, is of interest for the production of acetylxylan esterase that is free of xylanolytic activity. PMID:16347498

Evaluation of thermophilic fungal consortium for paddy straw composting.

PubMed

Kumar, Adesh; Gaind, Sunita; Nain, Lata

2008-06-01

Out of 10 thermophilic fungi isolated from wheat straw, farm yard manure, and soil, only three showed highest cellobiase, carboxymethyl cellulase, xylanase, and FPase activities. They were identified as Aspergillus nidulans (Th(4)), Scytalidium thermophilum (Th(5)), and Humicola sp. (Th(10)). A fungal consortium of these three fungi was used to compost a mixture (1:1) of silica rich paddy straw and lignin rich soybean trash. The composting of paddy straw for 3 months, during summer period in North India, resulted in a product with C:N ratio 9.5:1, available phosphorus 0.042% and fungal biomass 6.512 mg of N-acetyl glucosamine/100 mg of compost. However, a C:N ratio of 10.2:1 and highest humus content of 3.3% was achieved with 1:1 mixture of paddy straw and soybean trash. The fungal consortium was effective in converting high silica paddy straw into nutritionally rich compost thereby leading to economical and environment friendly disposal of this crop residue.

Compost Grown Agaricus bisporus Lacks the Ability to Degrade and Consume Highly Substituted Xylan Fragments

PubMed Central

de Vries, Ronald P.; Gruppen, Harry; Kabel, Mirjam A.

2015-01-01

The fungus Agaricus bisporus is commercially grown for the production of edible mushrooms. This cultivation occurs on compost, but not all of this substrate is consumed by the fungus. To determine why certain fractions remain unused, carbohydrate degrading enzymes, water-extracted from mushroom-grown compost at different stages of mycelium growth and fruiting body formation, were analyzed for their ability to degrade a range of polysaccharides. Mainly endo-xylanase, endo-glucanase, β-xylosidase and β-glucanase activities were determined in the compost extracts obtained during mushroom growth. Interestingly, arabinofuranosidase activity able to remove arabinosyl residues from doubly substituted xylose residues and α-glucuronidase activity were not detected in the compost enzyme extracts. This correlates with the observed accumulation of arabinosyl and glucuronic acid substituents on the xylan backbone in the compost towards the end of the cultivation. Hence, it was concluded that compost grown A. bisporus lacks the ability to degrade and consume highly substituted xylan fragments. PMID:26237450

Enrichment, isolation and characterization of fungi tolerant to 1-ethyl-3-methylimidazolium acetate

DOE Office of Scientific and Technical Information (OSTI.GOV)

Singer, S.W.; Reddy, A. P.; Gladden, J. M.

2010-12-15

This work aims to characterize microbial tolerance to 1-ethyl-3-methylimidazolium acetate ([C2mim][OAc]), ionic liquid that has emerged as a novel biomass pretreatment for lignocellulosic biomass. Enrichment experiments performed using inocula treated with [C2mim][OAc] under solid and liquid cultivation yielded fungal populationsdominated by Aspergilli. Ionic liquid-tolerant Aspergillus isolates from these enrichments were capable of growing in a radial plate growth assay in the presence of 10% [C2mim][OAc]. When a [C2mim][OAc]-tolerant Aspergillus fumigatus strain was grown in the presence of switchgrass, endoglucanases and xylanases were secreted that retained residual enzymatic activity in the presence of 20% [C2mim][OAc]. The results of the study suggestmore » tolerance to ionic liquids is a general property of Aspergilli. Tolerance to an industrially important ionic liquid was discovered in a fungal genera that is widely used in biotechnology, including biomass deconstruction.« less

Xylan degradation by the human gut Bacteroides xylanisolvens XB1A(T) involves two distinct gene clusters that are linked at the transcriptional level.

PubMed

Despres, Jordane; Forano, Evelyne; Lepercq, Pascale; Comtet-Marre, Sophie; Jubelin, Gregory; Chambon, Christophe; Yeoman, Carl J; Berg Miller, Margaret E; Fields, Christopher J; Martens, Eric; Terrapon, Nicolas; Henrissat, Bernard; White, Bryan A; Mosoni, Pascale

2016-05-04

Plant cell wall (PCW) polysaccharides and especially xylans constitute an important part of human diet. Xylans are not degraded by human digestive enzymes in the upper digestive tract and therefore reach the colon where they are subjected to extensive degradation by some members of the symbiotic microbiota. Xylanolytic bacteria are the first degraders of these complex polysaccharides and they release breakdown products that can have beneficial effects on human health. In order to understand better how these bacteria metabolize xylans in the colon, this study was undertaken to investigate xylan breakdown by the prominent human gut symbiont Bacteroides xylanisolvens XB1A(T). Transcriptomic analyses of B. xylanisolvens XB1A(T) grown on insoluble oat-spelt xylan (OSX) at mid- and late-log phases highlighted genes in a polysaccharide utilization locus (PUL), hereafter called PUL 43, and genes in a fragmentary remnant of another PUL, hereafter referred to as rPUL 70, which were highly overexpressed on OSX relative to glucose. Proteomic analyses supported the up-regulation of several genes belonging to PUL 43 and showed the important over-production of a CBM4-containing GH10 endo-xylanase. We also show that PUL 43 is organized in two operons and that the knockout of the PUL 43 sensor/regulator HTCS gene blocked the growth of the mutant on insoluble OSX and soluble wheat arabinoxylan (WAX). The mutation not only repressed gene expression in the PUL 43 operons but also repressed gene expression in rPUL 70. This study shows that xylan degradation by B. xylanisolvens XB1A(T) is orchestrated by one PUL and one PUL remnant that are linked at the transcriptional level. Coupled to studies on other xylanolytic Bacteroides species, our data emphasize the importance of one peculiar CBM4-containing GH10 endo-xylanase in xylan breakdown and that this modular enzyme may be used as a functional marker of xylan degradation in the human gut. Our results also suggest that B. xylanisolvens

MICROBIAL FERMENTATION OF ABUNDANT BIOPOLYMERS: CELLULOSE AND CHITIN

DOE Office of Scientific and Technical Information (OSTI.GOV)

Leschine, Susan

Our research has dealt with seven major areas of investigation: i) characterization of cellulolytic members of microbial consortia, with special attention recently given to Clostridium phytofermentans, a bacterium that decomposes cellulose and produces uncommonly large amounts of ethanol, ii) investigations of the chitinase system of Cellulomonas uda; including the purification and characterization of ChiA, the major component of this enzyme system, iii) molecular cloning, sequence and structural analysis of the gene that encodes ChiA in C. uda, iv) biofilm formation by C. uda on nutritive surfaces, v) investigations of the effects of humic substances on cellulose degradation by anaerobic cellulolyticmore » microbes, vi) studies of nitrogen metabolism in cellulolytic anaerobes, and vii) understanding the molecular architecture of the multicomplex cellulase-xylanase system of Clostridium papyrosolvens. Also, progress toward completing the research of more recent projects is briefly summarized. Major accomplishments include: 1. Characterization of Clostridium phytofermentans, a cellulose-fermenting, ethanol-producing bacterium from forest soil. The characterization of a new cellulolytic species isolated from a cellulose-decomposing microbial consortium from forest soil was completed. This bacterium is remarkable for the high concentrations of ethanol produced during cellulose fermentation, typically more than twice the concentration produced by other species of cellulolytic clostridia. 2. Examination of the use of chitin as a source of carbon and nitrogen by cellulolytic microbes. We discovered that many cellulolytic anaerobes and facultative aerobes are able to use chitin as a source of both carbon and nitrogen. This major discovery expands our understanding of the biology of cellulose-fermenting bacteria and may lead to new applications for these microbes. 3. Comparative studies of the cellulase and chitinase systems of Cellulomonas uda. Results of these studies

Characterization of a Cellulomonas fimi exoglucanase/xylanase-endoglucanase gene fusion which improves microbial degradation of cellulosic biomass.

PubMed

Duedu, Kwabena O; French, Christopher E

2016-11-01

Effective degradation of cellulose requires multiple classes of enzyme working together. However, naturally occurring cellulases with multiple catalytic domains seem to be rather rare in known cellulose-degrading organisms. A fusion protein made from Cellulomonas fimi exo- and endo- glucanases, Cex and CenA which improves breakdown of cellulose is described. A homologous carbohydrate binding module (CBM-2) present in both glucanases was fused to give a fusion protein CxnA. CxnA or unfused constructs (Cex+CenA, Cex, or CenA) were expressed in Escherichia coli and Citrobacter freundii. The latter recombinant strains were cultured at the expense of cellulose filter paper. The expressed CxnA had both exo- and endo- glucanase activities. It was also exported to the supernatant as were the non-fused proteins. In addition, the hybrid CBM from the fusion could bind to microcrystalline cellulose. Growth of C. freundii expressing CxnA was superior to that of cells expressing the unfused proteins. Physical degradation of filter paper was also faster with the cells expressing fusion protein than the other constructs. Our results show that fusion proteins with multiple catalytic domains can improve the efficiency of cellulose degradation. Such fusion proteins could potentially substitute cloning of multiple enzymes as well as improving product yields. Copyright © 2016 Elsevier Inc. All rights reserved.

Relative contribution of phytates, fibers, and tannins to low iron and zinc in vitro solubility in pearl millet (Pennisetum glaucum) flour and grain fractions.

PubMed

Lestienne, Isabelle; Caporiccio, Bertrand; Besançon, Pierre; Rochette, Isabelle; Trèche, Serge

2005-10-19

In vitro digestions were performed on pearl millet flours with decreased phytate contents and on two dephytinized or nondephytinized pearl millet grain fractions, a decorticated fraction, and a bran fraction with low and high fiber and tannin contents, respectively. Insoluble residues of these digestions were then incubated with buffer or enzymatic solutions (xylanases and/or phytases), and the quantities of indigestible iron and zinc released by these different treatments were determined. In decorticated pearl millet grain, iron was chelated by phytates and by insoluble fibers, whereas zinc was almost exclusively chelated by phytates. In the bran of pearl millet grain, a high proportion of iron was chelated by iron-binding phenolic compounds, while the rest of iron as well as the majority of zinc were chelated in complexes between phytates and fibers. The low effect of phytase action on iron and zinc solubility of bran of pearl millet grain shows that, in the case of high fiber and tannin contents, the chelating effect of these compounds was higher than that of phytates.

An ammonium sulfate sensitive endoxylanase produced by Streptomyces.

PubMed

Simkhada, Jaya Ram; Yoo, Hah Young; Park, Don Hee; Choi, Yun Hee; Lee, Hyo Jeong; Kim, Seung Wook; Yoo, Jin Cheol

2013-06-01

Streptomyces sp. CSWu2 was newly isolated and identified from Korean soil. In culture medium, the strain produced a highly active endoxylanase (Xynwu2), which was purified to homogeneity by a single-step chromatography on Poros-HQ. The xylanase was ~38 kDa and its activity was maximal at 65 °C and pH 11.0. It was stable up to 60 °C and from pH 8.0 to 12.0, and its activity was slightly enhanced by nonionic detergents, but inhibited by EDTA, EGTA, and divalent metal ions. Intriguingly, Xynwu2 was highly sensitive to ammonium sulfate, but its completely suppressed activity was recovered by desalting out. Xynwu2 produced xylose and xylobiose as principal end products from xylan, suggesting an endoxylanase nature. Importantly, scanning electron microscopy showed Xynwu2 efficiently degraded corncobs, an agro-industrial waste material. We believe that Xynwu2 is a potential candidate for converting lignocellulosic waste material into simple sugars which could be used to produce bioethanol and other value-added products.

High-performance of Agaricus blazei fungus for the biological pretreatment of elephant grass.

PubMed

Dal Picolli, Thais; Regalin Aver, Kaliane; Claudete Fontana, Roselei; Camassola, Marli

2018-01-01

Biological pre-treatment seems to be promising being an eco-friendly process, with no inhibitor generated during the process. The potential for elephant grass pre-treatment with white degradation fungi Pleurotus ostreatus, Agaricus blazei, Lentinula edodes, Pleurotus citrinopileatus, and Pleurotus djamor, in isolated or mixed cultures of these strains, was evaluated. The highest activities of enzymes involved in the degradation of lignocellulosic biomass (laccases, endoglucanases, xylanases, and β-glucosidases) were observed for A. blazei, L. edodes and the combination of P. ostreatus and A. blazei. In the enzymatic hydrolysis, there was greater release of reducing sugars in the pre-treated elephant grass samples by A. blazei during 10 days (338.91 ± 7.39 mg g -1 of biomass). For this sample, higher lignin reductions, 24.81 and 57.45%, after 15 and 35 days of incubation, respectively, were also verified. These data indicate the potential of macromycetes such as A. blazei to perform biological pre-treatments. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:42-50, 2018. © 2017 American Institute of Chemical Engineers.

Polyphenols content of spent coffee grounds subjected to physico-chemical pretreatments influences lignocellulolytic enzymes production by Bacillus sp. R2.

PubMed

Khelil, Omar; Choubane, Slimane; Cheba, Ben Amar

2016-07-01

The objective of this study was to investigate the impact of polyphenols content changes issued after physico-chemical treatments of spent coffee grounds on lignocellulolytic enzymes production by Bacillus sp. R2. Total polyphenols of the collected substrates were extracted with water under autoclaving conditions. Results showed that polyphenols content of spent coffee grounds decreased with continued treatments. Untreated spent coffee grounds were the best substrate for cellulase and pectinase (1.33±0.06μ/ml and 0.32±0.02μ/ml respectively). A strong positive correlation was noticed between polyphenols content and cellulase and pectinase activities. However, xylanase and peroxidase correlated moderately with polyphenols content and their highest activities were registered with spent coffee grounds treated with boiling water and 1% EDTA (0.31±0.002μ/ml and 15.56±0.56μ/ml respectively). The obtained results indicate that polyphenols content of the pretreated substrates influences the production of lignocellulolytic enzymes by Bacillus sp. R2. Copyright © 2016. Published by Elsevier Ltd.

Evaluation of secretome of highly efficient lignocellulolytic Penicillium sp. Dal 5 isolated from rhizosphere of conifers.

PubMed

Rai, Rohit; Kaur, Baljit; Singh, Surender; Di Falco, Macros; Tsang, Adrian; Chadha, B S

2016-09-01

Penicillium sp. (Dal 5) isolated from rhizosphere of conifers from Dalhousie (Himachal Pradesh, India) was found to be an efficient cellulolytic strain. The culture under shake flask on CWR (cellulose, wheat bran and rice straw) medium produced appreciably higher levels of endoglucanase (35.69U/ml), β-glucosidase (4.20U/ml), cellobiohydrolase (2.86U/ml), FPase (1.2U/ml) and xylanase (115U/ml) compared to other Penicillium strains reported in literature. The mass spectroscopy analysis of Penicillium sp. Dal 5 secretome identified 108 proteins constituting an array of CAZymes including glycosyl hydrolases (GH) belonging to 24 different families, polysaccharide lyases (PL), carbohydrate esterases (CE), lytic polysaccharide mono-oxygenases (LPMO) in addition to swollenin and a variety of carbohydrate binding modules (CBM) indicating an elaborate genetic potential of this strain for hydrolysis of lignocellulosics. Further, the culture extract was evaluated for hydrolysis of alkali treated rice straw, wheat straw, bagasse and corn cob at 10% substrate loading rate. Copyright © 2016 Elsevier Ltd. All rights reserved.

Effects of thermo-chemical pretreatment plus microbial fermentation and enzymatic hydrolysis on saccharification and lignocellulose degradation of corn straw.

PubMed

Wang, Ping; Chang, Juan; Yin, Qingqiang; Wang, Erzhu; Zhu, Qun; Song, Andong; Lu, Fushan

2015-10-01

In order to increase corn straw degradation, the straw was kept in the combined solution of 15% (w/w) lime supernatant and 2% (w/w) sodium hydroxide with liquid-to-solid ratio of 13:1 (mL/g) at 83.92°C for 6h; and then added with 3% (v/v) H2O2 for reaction at 50°C for 2h; finally cellulase (32.3 FPU/g dry matter) and xylanase (550 U/g dry matter) was added to keep at 50°C for 48 h. The maximal reducing sugars yield (348.77 mg/g) was increased by 126.42% (P<0.05), and the degradation rates of cellulose, hemicellulose and lignin in pretreated corn straw with enzymatic hydrolysis were increased by 40.08%, 45.71% and 52.01%, compared with the native corn straw with enzymatic hydrolysis (P<0.05). The following study indicated that the combined microbial fermentation and enzymatic hydrolysis could further increase straw degradation and reducing sugar yield (442.85 mg/g, P<0.05). Copyright © 2015 Elsevier Ltd. All rights reserved.

Laccase produced by a thermotolerant strain of Trametes trogii LK13

PubMed Central

Yan, Jinping; Chen, Yuhui; Niu, Jiezhen; Chen, Daidi; Chagan, Irbis

2015-01-01

Thermophilic and thermotolerant micro-organisms strains have served as the natural source of industrially relevant and thermostable enzymes. Although some strains of the Trametes genus are thermotolerant, few Trametes strains were studied at the temperature above 30 °C until now. In this paper, the laccase activity and the mycelial growth rate for Trametes trogii LK13 are superior at 37 °C. Thermostability and organic cosolvent tolerance assays of the laccase produced at 37 °C indicated that the enzyme possessed fair thermostability with 50% of its initial activity at 80 °C for 5 min, and could remain 50% enzyme activity treated with organic cosolvent at the concentration range of 25%–50% (v/v). Furthermore, the test on production of laccase and lignocellulolytic enzymes showed the crude enzymes possessed high laccase level (1000 U g −1 ) along with low cellulose (2 U g −1 ) and xylanase (140 U g −1 ) activity. Thus, T. trogii LK13 is a potential strain to be applied in many biotechnological processes. PMID:26221089

Exploring the effects of treatments with carbohydrases to obtain a high-cellulose content pulp from a non-wood alkaline pulp.

PubMed

Beltramino, Facundo; Valls, Cristina; Vidal, Teresa; Roncero, M Blanca

2015-11-20

In this work, treatments with a xylanase (X) and carbohydrases mixture (Cx) were applied on a TCF bleached sisal pulp in order to obtain high-cellulose content fibers applicable on a wide range of uses. A limit of ≈12% w/w final content in hemicelluloses was found regardless of the enzymatic treatment assessed. An extraction with 4% and 9% w/v NaOH was performed for further hemicelluloses removal. We found that NaOH dose could be strongly reduced if combined with Cx or Cx+X treatments. Also, if necessary, a stronger reduction could be obtained with 9% w/v NaOH, which was found to be boosted in a 14% if performed after a treatment with Cx. An end-product with a low content in xylans (≈2.9% w/w) and in HexA (5.8μmol/odp) was obtained. Pulp Fock solubility was also increased (≈30%) by enzymatic treatments. HPLC analysis of effluents provided useful information of enzymatic catalytic mechanisms. Copyright © 2015 Elsevier Ltd. All rights reserved.

Production of anti-streptococcal liamocins from agricultural biomass by Aureobasidium pullulans.

PubMed

Leathers, Timothy D; Price, Neil P J; Manitchotpisit, Pennapa; Bischoff, Kenneth M

2016-12-01

Liamocins are unique heavier-than-water "oils" produced by certain strains of the fungus Aureobasidium pullulans. Liamocins have antibacterial activity with specificity for Streptococcus sp. Previous studies reported that liamocin yields were highest from strains of A. pullulans belonging to phylogenetic clades 8, 9, and 11, cultured on medium containing sucrose. In this study, 27 strains from these clades were examined for the first time for production of liamocins from agricultural biomass substrates. Liamocin yields were highest from strains in phylogenetic clade 11, and yields were higher from cultures grown on sucrose than from those grown on pretreated wheat straw. However, when supplementary enzymes (cellulase, β-glucosidase, and xylanase) were added, liamocin production on pretreated wheat straw was equivalent to that on sucrose. Liamocins produced from wheat straw were free of the melanin contamination common in sucrose-grown cultures. Furthermore, MALDI-TOF MS analysis showed that liamocins produced from wheat straw were under-acetylated, resulting in higher proportions of the mannitol A1 and B1 species of liamocin, the latter of which has the highest biological activity against Streptococcus sp.

Direct and efficient xylitol production from xylan by Saccharomyces cerevisiae through transcriptional level and fermentation processing optimizations.

PubMed

Li, Zhe; Qu, Hongnan; Li, Chun; Zhou, Xiaohong

2013-12-01

In this study, four engineered Saccharomyces cerevisiae carrying xylanase, β-xylosidase and xylose reductase genes by different transcriptional regulations were constructed to directly convert xylan to xylitol. According to the results, the high-copy number plasmid required a rigid selection for promoter characteristics, on the contrast, the selection of promoters could be more flexible for low-copy number plasmid. For cell growth and xylitol production, glucose and galactose were found more efficient than other sugars. The semi-aerobic condition and feeding of co-substrates were taken to improve the yield of xylitol. It was found that the strain containing high-copy number plasmid had the highest xylitol yield, but it was sensitive to the change of fermentation. However, the strain carrying low-copy number plasmid was more adaptable to different processes. By optimization of the transcriptional regulation and fermentation processes, the xylitol concentration could be increased of 1.7 folds and the yield was 0.71 g xylitol/g xylan. Copyright © 2013 Elsevier Ltd. All rights reserved.

Adsorption of monocomponent enzymes in enzyme mixture analyzed quantitatively during hydrolysis of lignocellulose substrates.

PubMed

Várnai, Anikó; Viikari, Liisa; Marjamaa, Kaisa; Siika-aho, Matti

2011-01-01

The adsorption of purified Trichoderma reesei cellulases (TrCel7A, TrCel6A and TrCel5A) and xylanase TrXyn11 and Aspergillus niger β-glucosidase AnCel3A was studied in enzyme mixture during hydrolysis of two pretreated lignocellulosic materials, steam pretreated and catalytically delignified spruce, along with microcrystalline cellulose (Avicel). The enzyme mixture was compiled to resemble the composition of commercial cellulase preparations. The hydrolysis was carried out at 35 °C to mimic the temperature of the simultaneous saccharification and fermentation (SSF). Enzyme adsorption was followed by analyzing the activity and the protein amount of the individual free enzymes in the hydrolysis supernatant. Most enzymes adsorbed quickly at early stages of the hydrolysis and remained bound throughout the hydrolysis, although the conversion reached was fairly high. Only with the catalytically oxidized spruce samples, the bound enzymes started to be released as the hydrolysis degree reached 80%. The results based on enzyme activities and protein assay were in good accordance. Copyright © 2010 Elsevier Ltd. All rights reserved.

Magnetic cross-linked enzyme aggregates (CLEAs): a novel concept towards carrier free immobilization of lignocellulolytic enzymes.

PubMed

Bhattacharya, Abhishek; Pletschke, Brett I

2014-01-01

The enzymatic conversion of lignocellulosic biomass into biofuels has been identified as an excellent strategy to generate clean energy. However, the current process is cost-intensive as an effective immobilization approach to reuse the enzyme(s) has been a major challenge. The present study introduces the concept and application of novel magnetic cross-linked enzyme aggregates (mag-CLEAs). Both mag-CLEAs and calcium-mag-CLEAs (Ca-mag-CLEAs) exhibited a 1.35 fold higher xylanase activity compared to the free enzyme and retained more than 80.0% and 90.0% activity, respectively, after 136h of incubation at 50°C, compared to 50% activity retained by CLEAs. A 7.4 and 9.0 fold higher sugar release from lime-pretreated and NH4OH pre-treated sugar bagasse, respectively, was achieved with Ca-mag-CLEAs compared to the free enzymes. The present study promotes the successful application of mag-CLEAs and Ca-mag-CLEAs as carrier free immobilized enzymes for the effective hydrolysis of lignocellulolytic biomass and associated biofuel feedstocks. Copyright © 2014 Elsevier Inc. All rights reserved.

Cost-effective simultaneous saccharification and fermentation of l-lactic acid from bagasse sulfite pulp by Bacillus coagulans CC17.

PubMed

Zhou, Jie; Ouyang, Jia; Xu, Qianqian; Zheng, Zhaojuan

2016-12-01

The main barriers to cost-effective lactic acid production from lignocellulose are the high cost of enzymes and the ineffective utilization of the xylose within the hydrolysate. In the present study, the thermophilic Bacillus coagulans strain CC17 was used for the simultaneous saccharification and fermentation (SSF) of bagasse sulfite pulp (BSP) to produce l-lactic acid. Unexpectedly, SSF by CC17 required approximately 33.33% less fungal cellulase than did separate hydrolysis and fermentation (SHF). More interestingly, CC17 can co-ferment cellobiose and xylose without any exogenous β-glucosidase in SSF. Moreover, adding xylanase could increase the concentration of lactic acid produced via SSF. Up to 110g/L of l-lactic acid was obtained using fed-batch SSF, resulting in a lactic acid yield of 0.72g/g cellulose. These results suggest that SSF using CC17 has a remarkable advantage over SHF and that a potentially low-cost and highly-efficient fermentation process can be established using this protocol. Copyright © 2016 Elsevier Ltd. All rights reserved.

On-site hydrolytic enzymes production from fungal co-cultivation of Bermuda grass and corn cob.

PubMed

Amaro-Reyes, Aldo; Gracida, Jorge; Huizache-Peña, Nelson; Elizondo-García, Norberto; Salazar-Martínez, José; García Almendárez, Blanca E; Regalado, Carlos

2016-07-01

Solid state fermentation (SSF) is used to produce industrial enzymes. The objective of this study was to use a co-culture of Aspergillus niger GS1 and Trichoderma reesei, grown on a mixture of Bermuda grass and corn cob to obtain fermented forage (FF) rich in hydrolytic enzymes, as a value added ingredient for animal feed. FPase, amylase and xylanase productivities (dry matter, DM) were 8.8, 181.4, and 42.1Ug(-1)h(-1), respectively (1U=reducing sugars released min(-1)), after 12-16h of SSF with C/N=60. Cellulose, hemicellulose and lignin decreased 1.6-, 2.7- and 1.9-fold (DM), respectively. In vitro ruminal and true digestibility of DM was improved 2.4- and 1.4-fold. Ruminal digestion of FF reduced 1.32-fold the acetate:propionate ratio, which may reduce the environmental impact of ruminants feeding. On-site hydrolytic enzymes productivity using SSF without enzymes extraction could be of economic potential for digestibility improvement in animal feed. Copyright © 2016 Elsevier Ltd. All rights reserved.

Protein enrichment of corn cob heteroxylan waste slurry by thermophilic aerobic digestion using Bacillus stearothermophilus.

PubMed

Ugwuanyi, J Obeta; Harvey, Linda M; McNeil, Brian

2008-10-01

Thermophilic aerobic digestion (TAD) of heteroxylan waste was implemented at waste load of 30gL(-1) with mineral nitrogen supplementation to study effect of the process on waste degradation, protein accretion and quality. Digestions were carried out at 45 50, 55, 60 and 65 degrees C using Bacillusstearothermophilus in a CSTR under batch conditions at 1.0vvm aeration rate, pH 7.0 for a maximum of 120h. Amylase and xylanase activities appeared rapidly in the digest, while basal protease activity appeared early in the digestion and increased towards end of the processes. Highest degradation of volatile suspended solid, hemicellulose and fibre occurred at 55 degrees C while highest degradation of total suspended solid occurred at 60 degrees C. Highest protein accretion (258.8%) and assimilation of mineral nitrogen and soluble protein occurred at 55 degrees C. The % content of amino acids of digest crude protein increased relative to raw waste and with digestion temperature. Quality of digest protein was comparable to the FAO standard for feed use. TAD has potentials for use in the protein enrichment of waste.