Synergistic effect of Aspergillus niger and Trichoderma reesei enzyme sets on the saccharification of wheat straw and sugarcane bagasse.

PubMed

van den Brink, Joost; Maitan-Alfenas, Gabriela Piccolo; Zou, Gen; Wang, Chengshu; Zhou, Zhihua; Guimarães, Valéria Monteze; de Vries, Ronald P

2014-10-01

Plant-degrading enzymes can be produced by fungi on abundantly available low-cost plant biomass. However, enzymes sets after growth on complex substrates need to be better understood, especially with emphasis on differences between fungal species and the influence of inhibitory compounds in plant substrates, such as monosaccharides. In this study, Aspergillus niger and Trichoderma reesei were evaluated for the production of enzyme sets after growth on two "second generation" substrates: wheat straw (WS) and sugarcane bagasse (SCB). A. niger and T. reesei produced different sets of (hemi-)cellulolytic enzymes after growth on WS and SCB. This was reflected in an overall strong synergistic effect in releasing sugars during saccharification using A. niger and T. reesei enzyme sets. T. reesei produced less hydrolytic enzymes after growth on non-washed SCB. The sensitivity to non-washed plant substrates was not reduced by using CreA/Cre1 mutants of T. reesei and A. niger with a defective carbon catabolite repression. The importance of removing monosaccharides for producing enzymes was further underlined by the decrease in hydrolytic activities with increased glucose concentrations in WS media. This study showed the importance of removing monosaccharides from the enzyme production media and combining T. reesei and A. niger enzyme sets to improve plant biomass saccharification. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Enhanced bioprocessing of lignocellulose: Wood-rot fungal saccharification and fermentation of corn fiber to ethanol

NASA Astrophysics Data System (ADS)

Shrestha, Prachand

This research aims at developing a biorefinery platform to convert corn-ethanol coproduct, corn fiber, into fermentable sugars at a lower temperature with minimal use of chemicals. White-rot (Phanerochaete chrysosporium), brown-rot (Gloeophyllum trabeum) and soft-rot (Trichoderma reesei) fungi were used in this research to biologically break down cellulosic and hemicellulosic components of corn fiber into fermentable sugars. Laboratory-scale simultaneous saccharification and fermentation (SSF) process proceeded by in-situ cellulolytic enzyme induction enhanced overall enzymatic hydrolysis of hemi/cellulose from corn fiber into simple sugars (mono-, di-, tri-saccharides). The yeast fermentation of hydrolyzate yielded 7.1, 8.6 and 4.1 g ethanol per 100 g corn fiber when saccharified with the white-, brown-, and soft-rot fungi, respectively. The highest corn-to-ethanol yield (8.6 g ethanol/100 g corn fiber) was equivalent to 42 % of the theoretical ethanol yield from starch and cellulose in corn fiber. Cellulase, xylanase and amylase activities of these fungi were also investigated over a week long solid-substrate fermentation of corn fiber. G. trabeum had the highest activities for starch (160 mg glucose/mg protein.min) and on day three of solid-substrate fermentation. P. chrysosporium had the highest activity for xylan (119 mg xylose/mg protein.min) on day five and carboxymethyl cellulose (35 mg glucose/mg protein.min) on day three of solid-substrate fermentation. T. reesei showed the highest activity for Sigma cell 20 (54.8 mg glucose/mg protein.min) on day 5 of solid-substrate fermentation. The effect of different pretreatments on SSF of corn fiber by fungal processes was examined. Corn fiber was treated at 30 °C for 2 h with alkali [2% NaOH (w/w)], alkaline peroxide [2% NaOH (w/w) and 1% H2O 2 (w/w)], and by steaming at 100 °C for 2 h. Mild pretreatment resulted in improved ethanol yields for brown- and soft-rot SSF, while white-rot and Spezyme CP SSFs showed

Simultaneous saccharification and viscosity reduction of cassava pulp using a multi-component starch- and cell-wall degrading enzyme for bioethanol production.

PubMed

Poonsrisawat, Aphisit; Paemanee, Atchara; Wanlapatit, Sittichoke; Piyachomkwan, Kuakoon; Eurwilaichitr, Lily; Champreda, Verawat

2017-10-01

In this study, an efficient ethanol production process using simultaneous saccharification and viscosity reduction of raw cassava pulp with no prior high temperature pre-gelatinization/liquefaction step was developed using a crude starch- and cell wall-degrading enzyme preparation from Aspergillus aculeatus BCC17849. Proteomic analysis revealed that the enzyme comprised a complex mixture of endo- and exo-acting amylases, cellulases, xylanases, and pectina ses belonging to various glycosyl hydrolase families. Enzymatic hydrolysis efficiency was dependent on the initial solid loading in the reaction. Reduction in mixture viscosity was observed with a rapid decrease in complex viscosity from 3785 to 0.45 Pa s with the enzyme dosage of 2.19 mg/g on a dried weight basis within the first 2 h, which resulted from partial destruction of the plant cell wall fiber and degradation of the released starch granules by the enzymes as shown by scanning electron microscopy. Saccharification of cassava pulp at an initial solid of 16% (w/v) in a bench-scale bioreactor resulted in 736.4 mg glucose/g, which is equivalent to 82.92% glucose yield based on the total starch and glucan in the substrate, after 96 h at 40 °C. Simultaneous saccharification and fermentation of cassava pulp by Saccharomyces cerevisiae with the uncooked enzymatic process led to a final ethanol concentration of 6.98% w/v, equivalent to 96.7% theoretical yield based on the total starch and cellulose content. The results demonstrated potential of the enzyme for low-energy processing of cassava pulp in biofuel industry.

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.

Enhanced Ethanol Production from De-Ashed Paper Sludge by Simultaneous Saccharification and Fermentation and Simultaneous Saccharification and Co-Fermentation

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

Kang, L.; Wang, W.; Pallapolu, V. R.

2011-11-01

A previous study demonstrated that paper sludges with high ash contents can be converted to ethanol by simultaneous saccharification and fermentation (SSF) or simultaneous saccharification and co-fermentation (SSCF). High ash content in the sludge, however, limited solid loading in the bioreactor, causing low product concentration. To overcome this problem, sludges were de-ashed before SSF and SSCF. Low ash content in sludges also increased the ethanol yield to the extent that the enzyme dosage required to achieve 70% yield in the fermentation process was reduced by 30%. High solid loading in SSF and SSCF decreased the ethanol yield. High agitation andmore » de-ashing of the sludges were able to restore the part of the yield loss caused by high solid loading. Substitution of the laboratory fermentation medium (peptone and yeast extract) with corn steep liquor did not bring about any adverse effects in the fermentation. Fed-batch operation of the SSCF and SSF using low-ash content sludges was effective in raising the ethanol concentration, achieving 47.8 g/L and 60.0 g/L, respectively.« less

Simultaneous pretreatment and saccharification: green technology for enhanced sugar yields from biomass using a fungal consortium.

PubMed

Dhiman, Saurabh Sudha; Haw, Jung-Rim; Kalyani, Dayanand; Kalia, Vipin C; Kang, Yun Chan; Lee, Jung-Kul

2015-03-01

Two different biomasses were subjected to simultaneous pretreatment and saccharification (SPS) using a cocktail of hydrolytic and oxidizing enzymes. Application of a novel laccase as a detoxifying agent caused the removal of 49.8% and 32.6% of phenolic contents from the soaked rice straw and willow, respectively. Hydrolysis of soaked substrates using a newly developed fungal consortium resulted in saccharification yield of up to 74.2% and 63.6% for rice straw and willow, respectively. A high saccharification yield was obtained with soaked rice straw and willow without using any hazardous chemicals. The efficiency of each step related to SPS was confirmed by atomic force microscopy. The suitability of the developed SPS process was further confirmed by converting the hydrolysate from the process into bioethanol with 72.4% sugar conversion efficiency. To the best of our knowledge, this is the first report on the development of a less tedious, single-pot, and eco-friendly SPS methodology. Copyright © 2014 Elsevier Ltd. All rights reserved.

Peracetic acid-ionic liquid pretreatment to enhance enzymatic saccharification of lignocellulosic biomass.

PubMed

Uju; Abe, Kojiro; Uemura, Nobuyuki; Oshima, Toyoji; Goto, Masahiro; Kamiya, Noriho

2013-06-01

To enhance enzymatic saccharification of pine biomass, the pretreatment reagents peracetic acid (PAA) and ionic liquid (IL) were validated in single reagent pretreatments or combination pretreatments with different sequences. In a 1h saccharification, 5-25% cellulose conversion was obtained from the single pretreatment of PAA or IL. In contrast, a marked enhancement in conversion rates was achieved by PAA-IL combination pretreatments (45-70%). The PAA followed by IL (PAA+IL) pretreatment sequence was the most effective for preparing an enzymatic digestible regenerated biomass with 250-fold higher glucose formation rates than untreated biomass and 2- to 12-fold higher than single pretreatments with PAA or IL alone. Structural analysis confirmed that this pretreatment resulted in biomass with highly porous structural fibers associated with the reduction of lignin content and acetyl groups. Using the PAA+IL sequence, biomass loading in the pretreatment step can be increased from 5% to 15% without significant decrease in cellulose conversion. Copyright © 2013 Elsevier Ltd. All rights reserved.

Saccharification of sunflower stalks using lignocellulases from a fungal consortium comprising Pholiota adiposa and Armillaria gemina.

PubMed

Ramachandran, Priyadharshini; Kim, Tae-Su; Dhiman, Saurabh Sudha; Li, Jinglin; Park, Ji-Hyun; Choi, Joon-Ho; Kim, Jae Young; Kim, Dongwook; Lee, Jung-Kul

2015-09-01

Lignocellulases from Armillaria gemina and Pholiota adiposa are efficient in hydrolyzing aspen and poplar biomass, respectively. In the present study, lignocellulosic enzymes obtained from a fungal consortium comprising P. adiposa and A. gemina were used for the saccharification of sunflower stalks. Sunflower stalks were thermochemically pretreated using 2 % NaOH at 50 °C for 24 h. The saccharification process parameters including substrate concentration, enzyme loading, pH, and temperature were optimized using response surface methodology to improve the saccharification yield. The highest enzymatic hydrolysis (84.3 %) was obtained using the following conditions: enzyme loading 10 FPU/g-substrate, substrate 5.5 %, temperature 50 °C, and pH 4.5. The hydrolysis yield obtained using the enzymes from the fungal consortium was equivalent to that obtained using a mixture of commercial enzymes Celluclast and Novozyme β-glucosidase. Addition of up to 500 ppm of heavy metal ions (As, Cu, Fe, Mn, Ni, Pb, and Zn) during saccharification did not significantly affect the saccharification yield. Thus, the biomass grown for phytoremediation of heavy metals can be used for the production of reducing sugars followed by ethanol fermentation.

Optimization of liquid fermentation of microbial consortium WSD-5 followed by saccharification and acidification of wheat straw.

PubMed

Wen, Boting; Yuan, Xufeng; Cao, Yanzhuan; Liu, Yan; Wang, Xiaofen; Cui, Zongjun

2012-08-01

The microbial consortium WSD-5 is composed of bacteria and fungi, and the cooperation and symbiosis of the contained microbes enhance the degradation ability of WSD-5. Experiment results showed that the highest cellulase and hemicellulase were obtained when ventilation volume was 4 L/min, stirring rate was 0 rpm, and substrate loading rate was 3%. After 6 days of cultivation, a 67.60% loss in wheat straw dry weight was observed. The crude enzyme secreted from WSD-5 after optimization was evaluated by experiments of saccharification and acidification. The maximum concentration of reducing sugars was 3254 mg/L after 48 h saccharification. The concentration of sCOD peaked on day 2 with a value of 4345 mg/L during acidification, and the biogas yield and methane yield were 22.3% and 32.3% higher than un-acidified samples. This study is the first attempt to explore both the saccharification and the acidification ability of crude enzymes secreted by microbial consortium. Copyright © 2012 Elsevier Ltd. All rights reserved.

Correlating physical changes and enhanced enzymatic saccharification of pine flour pretreated by Ν-Methylmorpholine-Ν-oxide

Treesearch

Ye Liu; Qixin Zhong; Siqun Wang; Zhiyong Cai

2011-01-01

Pretreatment of lignocellulosic biomass by Ν-methylmorpholine-Ν-oxide (NMMO), a solvent used in the textile industry to dissolve cellulose for production of regenerated cellulose fibers, was observed to enhance significantly enzymatic saccharification and fermentation. The enhancement was speculated to have been caused by reduced cellulose crystallinity...

Simultaneously saccharification and fermentation approach as a tool for enhanced fossil fuels biodesulfurization.

PubMed

Paixão, Susana M; Arez, Bruno F; Roseiro, José C; Alves, Luís

2016-11-01

Biodesulfurization can be a complementary technology to the hydrodesulfurization, the commonly physical-chemical process used for sulfur removal from crude oil. The desulfurizing bacterium Gordonia alkanivorans strain 1B as a fructophilic microorganism requires fructose as C-source. In this context, the main goal of this work was the optimization of a simultaneous saccharification and fermentation (SSF) approach using the Zygosaccharomyces bailii strain Talf1 crude enzymes with invertase activity and sucrose as a cheaper fructose-rich commercial C-source (50% fructose) towards dibenzothiophene (DBT) desulfurization by strain 1B. The determination of optimal conditions, for both sucrose hydrolysis and DBT desulfurization was carried out through two sequential experimental uniform designs according to the Doehlert distribution for two factors: pH (5.5-7.5) and temperature (28-38 °C), with the enzyme load of 1.16 U/g/L; and enzyme load (0-4 U/g/L) and temperature (28-38 °C), with pH at 7.5. Based on 2-hydroxybiphenyl production, the analysis of the response surfaces obtained pointed out for pH 7.5, 32 °C and 1.8 U/g/L as optimal conditions. Further optimized SSF of sucrose during the DBT desulfurization process permitted to attain a 4-fold enhanced biodesulfurization. This study opens a new focus of research through the exploitation of sustainable low cost sucrose-rich feedstocks towards a more economical viable bioprocess scale-up. Copyright © 2016 Elsevier Ltd. All rights reserved.

Enhancement of enzymatic saccharification of Eucalyptus globulus: steam explosion versus steam treatment.

PubMed

Martin-Sampedro, Raquel; Revilla, Esteban; Villar, Juan C; Eugenio, Maria E

2014-09-01

Steam explosion and steam pre-treatment have proved capable of enhancing enzymatic saccharification of lignocellulosic materials. However, until now, these methods had not been compared under the same operational conditions and using the same raw material. Both pre-treatments lead to increased yields in the saccharification of Eucalyptus globulus; but results have been better with steam pre-treatments, despite the more accessible surface of exploded samples. The reason for this finding could be enzymatic inhibition: steam explosion causes a more extensive extraction of hemicelluloses and releases a greater amount of degradation products which can inhibit enzymatic action. Enzymatic inhibition is also dependent on the amount and chemical structure of lignin, which was also a contributing factor to the lower enzymatic yields obtained with the most severe pre-treatment. Thus, the highest yields (46.7% glucose and 73.4% xylose yields) were obtained after two cycle of steam treatment, of 5 and 3 min, at 183°C. Copyright © 2014 Elsevier Ltd. All rights reserved.

On-site cellulase production and efficient saccharification of corn stover employing cbh2 overexpressing Trichoderma reesei with novel induction system.

PubMed

Li, Yonghao; Zhang, Xiaoyue; Xiong, Liang; Mehmood, Muhammad Aamer; Zhao, Xinqing; Bai, Fengwu

2017-08-01

Although on-site cellulase production offers cost-effective saccharification of lignocellulosic biomass, low enzyme titer is still a barrier for achieving robustness. In the present study, a strain of T. reesei was developed for enhanced production of cellulase via overexpression of Cellobiohydrolase II. Furthermore, optimum enzyme production was achieved using a novel inducer mixture containing synthesized glucose-sophorose (MGD) and alkali pre-treated corn stover (APCS). Within 60h, a remarkably higher cellulase productivity and activity were achieved in the fed-batch fermentation using the optimized ratio of MGD and APCS in the inducer mixture, compared to those reported using cellulosic biomass as the sole inducer. After the enzyme production, APCS was added directly into the fermentation broth at 20% solid loading, which produced 122.5g/L glucose and 40.21g/L xylose, leading to the highest yield reported so far. The improved enzyme titers during on-site cellulase production would benefit cost-competitive saccharification of lignocellulosic biomass. Copyright © 2017 Elsevier Ltd. All rights reserved.

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.

Enhanced Inulin Saccharification by Self-Produced Inulinase from a Newly Isolated Penicillium sp. and its Application in D-Lactic Acid Production.

PubMed

Zheng, Zhaojuan; Xu, Qianqian; Liu, Peng; Zhou, Fan; Ouyang, Jia

2018-03-10

In order to find an alternative for commercial inulinase, a strain XL01 identified as Penicillium sp. was screened for inulinase production. The broth after cultivated was centrifuged, filtered, and used as crude enzyme for the following saccharification. At pH 5.0 and 50 °C, the crude enzyme released 84.9 g/L fructose and 20.7 g/L glucose from 120 g/L inulin in 72 h. In addition, simultaneous saccharification and fermentation of chicory flour for D-lactic acid production was carried out using the self-produced crude inulinase and Lactobacillus bulgaricus CGMCC 1.6970. A high D-lactic acid titer and productivity of 122.0 g/L and 1.69 g/(L h) was achieved from 120 g/L chicory flour in 72 h. The simplicity for inulinase production and the high efficiency for D-lactic acid fermentation provide a perspective and profitable industrial biotechnology for utilization of the inulin-rich biomass.

Short time ionic liquids pretreatment on lignocellulosic biomass to enhance enzymatic saccharification.

PubMed

Uju; Shoda, Yasuhiro; Nakamoto, Aya; Goto, Masahiro; Tokuhara, Wataru; Noritake, Yoshiyuki; Katahira, Satoshi; Ishida, Nobuhiro; Nakashima, Kazunori; Ogino, Chiaki; Kamiya, Noriho

2012-01-01

The potential of 1-buthyl-3-methylpyridinium chloride, [Bmpy][Cl], as a pretreatment solvent for lignocellulosic biomasses, Bagasse and Eucalyptus, was investigated. The yields of regenerated biomasses ranged between 35% and 96%, and varied according to the pretreatment time, type of ionic liquid (IL) and biomass. The pretreatment of the biomass with [Bmpy][Cl] resulted in up to 8-fold increase in the cellulose conversion when compared with the untreated biomass. For a short pretreatment period (i.e., 10 min), [Bmpy][Cl] showed better performance than 1-ethyl-3-methylimidazolium acetate ([Emim][OAc]) with respect to the initial enzymatic saccharification rates. The increase in the reaction rates with [Emim][OAc] treatment was because of a reduction in the cellulose crystallinity. In contrast, a decrease in the crystallinity index was not clearly observed for the biomass pretreated with [Bmpy][Cl], and the enhancement of the enzymatic saccharification rates using this IL is presumably due to a reduction in the degree of polymerization of cellulose in the biomass. Copyright © 2011. Published by Elsevier Ltd.

Enhanced enzymatic hydrolysis of waste paper for ethanol production using separate saccharification and fermentation.

PubMed

Guerfali, Mohamed; Saidi, Adel; Gargouri, Ali; Belghith, Hafedh

2015-01-01

Ethanol produced from lignocellulosic biomass is a renewable alternative to diminishing petroleum-based liquid fuels. In this study, the feasibility of ethanol production from waste paper using the separate hydrolysis and fermentation (SHF) was investigated. Two types of waste paper materials, newspaper and office paper, were evaluated for their potential to be used as a renewable feedstock for the production of fermentable sugars via enzymatic hydrolysis of their cellulose fractions. Hydrolysis step was conducted with a mixture of cellulolytic enzymes produced locally by Trichoderma reesei Rut-C30 (cellulase-overproducing mutant) and Aspergillus niger F38 cultures. Surfactant pretreatment effect on waste paper enzymatic digestibility was studied and Triton X-100 at 0.5 % (w w(-1)) has improved the digestibility of newspaper about 45 %. The effects of three factors (dry matter quantity, phosphoric acid pretreatment and hydrolysis time) on the extent of saccharification were also assessed and quantified by using a methodical approach based on response surface methodology. Under optimal hydrolysis conditions, maximum degrees of saccharification of newspaper and office paper were 67 and 92 %, respectively. Sugars released from waste paper were subsequently converted into ethanol (0.38 g ethanol g(-1) sugar) with Saccharomyces cerevisiae CTM-30101.

Increased saccharification yields from aspen biomass upon treatment with enzymatically generated peracetic acid.

PubMed

Duncan, Shona; Jing, Qing; Katona, Adrian; Kazlauskas, Romas J; Schilling, Jonathan; Tschirner, Ulrike; Aldajani, Waleed Wafa

2010-03-01

The recalcitrance of lignocellulosic biomass to enzymatic release of sugars (saccharification) currently limits its use as feedstock for biofuels. Enzymatic hydrolysis of untreated aspen wood releases only 21.8% of the available sugars due primarily to the lignin barrier. Nature uses oxidative enzymes to selectively degrade lignin in lignocellulosic biomass, but thus far, natural enzymes have been too slow for industrial use. In this study, oxidative pretreatment with commercial peracetic acid (470 mM) removed 40% of the lignin (from 19.9 to 12.0 wt.% lignin) from aspen and enhanced the sugar yields in subsequent enzymatic hydrolysis to about 90%. Increasing the amount of lignin removed correlated with increasing yields of sugar release. Unfortunately, peracetic acid is expensive, and concentrated forms can be hazardous. To reduce costs and hazards associated with using commercial peracetic acid, we used a hydrolase to catalyze the perhydrolysis of ethyl acetate generating 60-70 mM peracetic acid in situ as a pretreatment to remove lignin from aspen wood. A single pretreatment was insufficient, but multiple cycles (up to eight) removed up to 61.7% of the lignin enabling release of >90% of the sugars during saccharification. This value corresponds to a predicted 581 g of fermentable sugars from 1 kg of aspen wood. Improvements in the enzyme stability are needed before the enzymatically generated peracetic acid is a commercially viable alternative.

Lignin triggers irreversible cellulase loss during pretreated lignocellulosic biomass saccharification.

PubMed

Gao, Dahai; Haarmeyer, Carolyn; Balan, Venkatesh; Whitehead, Timothy A; Dale, Bruce E; Chundawat, Shishir Ps

2014-01-01

Non-productive binding of enzymes to lignin is thought to impede the saccharification efficiency of pretreated lignocellulosic biomass to fermentable sugars. Due to a lack of suitable analytical techniques that track binding of individual enzymes within complex protein mixtures and the difficulty in distinguishing the contribution of productive (binding to specific glycans) versus non-productive (binding to lignin) binding of cellulases to lignocellulose, there is currently a poor understanding of individual enzyme adsorption to lignin during the time course of pretreated biomass saccharification. In this study, we have utilized an FPLC (fast protein liquid chromatography)-based methodology to quantify free Trichoderma reesei cellulases (namely CBH I, CBH II, and EG I) concentration within a complex hydrolyzate mixture during the varying time course of biomass saccharification. Three pretreated corn stover (CS) samples were included in this study: Ammonia Fiber Expansion(a) (AFEX™-CS), dilute acid (DA-CS), and ionic liquid (IL-CS) pretreatments. The relative fraction of bound individual cellulases varied depending not only on the pretreated biomass type (and lignin abundance) but also on the type of cellulase. Acid pretreated biomass had the highest levels of non-recoverable cellulases, while ionic liquid pretreated biomass had the highest overall cellulase recovery. CBH II has the lowest thermal stability among the three T. reesei cellulases tested. By preparing recombinant family 1 carbohydrate binding module (CBM) fusion proteins, we have shown that family 1 CBMs are highly implicated in the non-productive binding of full-length T. reesei cellulases to lignin. Our findings aid in further understanding the complex mechanisms of non-productive binding of cellulases to pretreated lignocellulosic biomass. Developing optimized pretreatment processes with reduced or modified lignin content to minimize non-productive enzyme binding or engineering pretreatment

Effect of harvesting date on the composition and saccharification of Miscanthus x giganteus.

PubMed

Le Ngoc Huyen, T; Rémond, C; Dheilly, R M; Chabbert, B

2010-11-01

The chemical composition of the whole aerial biomass and isolated organs of Miscanthus x giganteus was examined for saccharification into fermentable sugars at early and late harvesting dates. Delayed harvest was mainly related to increased amounts of cell wall and ester-linked phenolic acids. Addition of an enzyme cocktail (cellulases, beta-glucosidase and xylanase) resulted in similar enzyme digestibilities at the two harvesting dates, ranging from 11-13% and 8-9% of the cellulose and arabinoxylan, respectively. However, the internodes, leaves and sheaths varied in cell wall content and composition and gave rise to different saccharification yields with internodes being the most recalcitrant organs. Non-cell wall fraction was estimated as the amount of material extracted by neutral detergent solution, and accounted for 23% of the whole aerial biomass harvested at an early date. However, saccharification yields from the miscanthus biomass did not change after soluble fraction removal. An ammonia pretreatment improved enzyme efficiency on early-harvested miscanthus, to a greater extent than on late-harvested biomass. This trend was confirmed for two different years of harvesting. Copyright 2010 Elsevier Ltd. All rights reserved.

Addition of alkali to the hydrothermal-mechanochemical treatment of Eucalyptus enhances its enzymatic saccharification.

PubMed

Ishiguro, Maki; Endo, Takashi

2014-02-01

The effects of alkali on hydrothermal-mechanochemical treatment (hydrothermal treatment combined with wet-milling) were examined with the aim of improving pretreatment of lignocellulosic biomass before enzymatic saccharification. After enzymatic saccharification, the highest glucose yield was obtained by autoclaving at 170°C in the presence of 20% NaOH per substrate weight. The wood fiber was unraveled into finer nanofibers by hydrothermal-mechanochemical treatment, thus increasing the specific surface area of the substrate from 11 to 132m(2)/g. Adding 20% NaOH to the treatment further increased the specific surface area of the already fibrillated substrate by 76% (232m(2)/g) due to lignin removal and ester bond cleavage between lignin and hemicellulose. This increase in specific surface area was closely related to the increase in enzymatic digestibility; therefore, NaOH addition may have enhanced the effect of hydrothermal-mechanochemical treatment. Copyright © 2013 Elsevier Ltd. All rights reserved.

Enzymatic saccharification of brown seaweed for production of fermentable sugars.

PubMed

Sharma, Sandeep; Horn, Svein Jarle

2016-08-01

This study shows that high drying temperatures negatively affect the enzymatic saccharification yield of the brown seaweed Saccharina latissima. The optimal drying temperature of the seaweed in terms of enzymatic sugar release was found to be 30°C. The enzymatic saccharification process was optimized by investigating factors such as kinetics of sugar release, enzyme dose, solid loading and different blend ratios of cellulases and an alginate lyase. It was found that the seaweed biomass could be efficiently hydrolysed to fermentable sugars using a commercial cellulase cocktail. The inclusion of a mono-component alginate lyase was shown to improve the performance of the enzyme blend, in particular at high solid loadings. At 25% dry matter loading a combined glucose and mannitol concentration of 74g/L was achieved. Copyright © 2016 Elsevier Ltd. All rights reserved.

Enzymatic saccharification and fermentation of cellulosic date palm wastes to glucose and lactic acid

PubMed Central

Alrumman, Sulaiman A.

2016-01-01

The bioconversion of cellulosic wastes into high-value bio-products by saccharification and fermentation processes is an important step that can reduce the environmental pollution caused by agricultural wastes. In this study, enzymatic saccharification of treated and untreated date palm cellulosic wastes by the cellulases from Geobacillus stearothermophilus was optimized. The alkaline pre-treatment of the date palm wastes was found to be effective in increasing the saccharification percentage. The maximum rate of saccharification was found at a substrate concentration of 4% and enzyme concentration of 30 FPU/g of substrate. The optimum pH and temperature for the bioconversions were 5.0 and 50 °C, respectively, after 24 h of incubation, with a yield of 31.56 mg/mL of glucose at a saccharification degree of 71.03%. The saccharification was increased to 94.88% by removal of the hydrolysate after 24 h by using a two-step hydrolysis. Significant lactic acid production (27.8 mg/mL) was obtained by separate saccharification and fermentation after 72 h of incubation. The results indicate that production of fermentable sugar and lactic acid is feasible and may reduce environmental pollution by using date palm wastes as a cheap substrate. PMID:26887233

Enzymatic saccharification and fermentation of cellulosic date palm wastes to glucose and lactic acid.

PubMed

Alrumman, Sulaiman A

2016-01-01

The bioconversion of cellulosic wastes into high-value bio-products by saccharification and fermentation processes is an important step that can reduce the environmental pollution caused by agricultural wastes. In this study, enzymatic saccharification of treated and untreated date palm cellulosic wastes by the cellulases from Geobacillus stearothermophilus was optimized. The alkaline pre-treatment of the date palm wastes was found to be effective in increasing the saccharification percentage. The maximum rate of saccharification was found at a substrate concentration of 4% and enzyme concentration of 30 FPU/g of substrate. The optimum pH and temperature for the bioconversions were 5.0 and 50°C, respectively, after 24h of incubation, with a yield of 31.56mg/mL of glucose at a saccharification degree of 71.03%. The saccharification was increased to 94.88% by removal of the hydrolysate after 24h by using a two-step hydrolysis. Significant lactic acid production (27.8mg/mL) was obtained by separate saccharification and fermentation after 72h of incubation. The results indicate that production of fermentable sugar and lactic acid is feasible and may reduce environmental pollution by using date palm wastes as a cheap substrate. Copyright © 2015 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

Engineering the cell wall by reducing de-methyl-esterified homogalacturonan improves saccharification of plant tissues for bioconversion.

PubMed

Lionetti, Vincenzo; Francocci, Fedra; Ferrari, Simone; Volpi, Chiara; Bellincampi, Daniela; Galletti, Roberta; D'Ovidio, Renato; De Lorenzo, Giulia; Cervone, Felice

2010-01-12

Plant cell walls represent an abundant, renewable source of biofuel and other useful products. The major bottleneck for the industrial scale-up of their conversion to simple sugars (saccharification), to be subsequently converted by microorganisms into ethanol or other products, is their recalcitrance to enzymatic saccharification. We investigated whether the structure of pectin that embeds the cellulose-hemicellulose network affects the exposure of cellulose to enzymes and consequently the process of saccharification. Reduction of de-methyl-esterified homogalacturonan (HGA) in Arabidopsis plants through the expression of a fungal polygalacturonase (PG) or an inhibitor of pectin methylesterase (PMEI) increased the efficiency of enzymatic saccharification. The improved enzymatic saccharification efficiency observed in transformed plants could also reduce the need for acid pretreatment. Similar results were obtained in PG-expressing tobacco plants and in PMEI-expressing wheat plants, indicating that reduction of de-methyl-esterified HGA may be used in crop species to facilitate the process of biomass saccharification.

Corn fiber, cobs and stover: enzyme-aided saccharification and co-fermentation after dilute acid pretreatment.

PubMed

Van Eylen, David; van Dongen, Femke; Kabel, Mirjam; de Bont, Jan

2011-05-01

Three corn feedstocks (fibers, cobs and stover) available for sustainable second generation bioethanol production were subjected to pretreatments with the aim of preventing formation of yeast-inhibiting sugar-degradation products. After pretreatment, monosaccharides, soluble oligosaccharides and residual sugars were quantified. The size of the soluble xylans was estimated by size exclusion chromatography. The pretreatments resulted in relatively low monosaccharide release, but conditions were reached to obtain most of the xylan-structures in the soluble part. A state of the art commercial enzyme preparation, Cellic CTec2, was tested in hydrolyzing these dilute acid-pretreated feedstocks. The xylose and glucose liberated were fermented by a recombinant Saccharomyces cerevisiae strain. In the simultaneous enzymatic saccharification and fermentation system employed, a concentration of more than 5% (v/v) (0.2g per g of dry matter) of ethanol was reached. Copyright © 2011 Elsevier Ltd. All rights reserved.

pH-Induced Lignin Surface Modification to Reduce Nonspecific Cellulase Binding and Enhance Enzymatic Saccharification of Lignocelluloses

Treesearch

Hongming Lou; J.Y. Zhu; Tian Qing Lan; Huranran Lai; Xueqing Qiu

2013-01-01

We studied the mechanism of the significant enhancement in the enzymatic saccharification of lignocelluloses at an elevated pH of 5.5–6.0. Four lignin residues with different sulfonic acid contents were isolated from enzymatic hydrolysis of lodgepole pine pretreated by either dilute acid (DA) or sulfite pretreatment to overcome recalcitrance of lignocelluloses (SPORL...

Saccharification of woody biomass using glycoside hydrolases from Stereum hirsutum.

PubMed

Jeya, Marimuthu; Kalyani, Dayanand; Dhiman, Saurabh Sudha; Kim, Hoon; Woo, Seongmin; Kim, Dongwook; Lee, Jung-Kul

2012-08-01

Enzymatic saccharification of woody biomasses was performed using glycoside hydrolases from Stereum hirsutum, a newly isolated fungal strain found to secrete efficient glycoside hydrolases. The strain showed the highest β-glucosidase, cellobiohydrolase, endoglucanase, endoxylanase, laccase, and filter paper activity of 10.3, 1.7, 10.3, 29.9, 0.12, and 0.58 U/ml, respectively. Among the various biomasses tested for saccharification, pine biomass produced maximum reducing sugar. Response surface methodology was used to optimize the hydrolysis of pine biomass to achieve the highest level of sugars. The parameters including enzyme, substrate concentration, temperature and pH were found to be critical for the conversion of pine biomass into sugars. Maximum saccharification of 49.7% (435 mg/g-substrate) was obtained after 96 h of hydrolysis. A close agreement between the experimental results and the model predictions was achieved. S. hirsutum could be a good choice for the production of reducing sugars from cellulosic biomasses. Copyright © 2012 Elsevier Ltd. All rights reserved.

Enzymatic saccharification of liquid hot water and dilute sulfuric acid pretreated oil palm empty fruit bunch and sugarcane bagasse

NASA Astrophysics Data System (ADS)

Risanto, L.; Fitria; Fajriutami, T.; Hermiati, E.

2018-03-01

Oil palm empty fruit bunch (OPEFB) and sugarcane bagasse (SB) are potential feedstocks for the production of bioethanol. In this study OPEFB and SB were pretreated by liquid hot water and dilute sulfuric acid (3% H2SO4), and continued with enzymatic saccharification. Heating treatment for both methods was conducted in an autoclave at 121 °C for 1 hr. The saccharification was performed up to 72 hours with cellulase enzyme loading of 10, 20, and 30 FPU per g biomass. Results showed that OPEFB and SB pretreated with H2SO4 produced higher reducing sugars than those pretreated by liquid hot water. Higher enzyme loading also resulted in higher reducing sugars. Reducing sugars obtained from enzymatic saccharification of OPEFB were higher than those obtained from SB. The highest total reducing sugars (50.48 g/100 g biomass) was obtained from OPEFB pretreated with 3% H2SO4 at enzyme loading of 30 FPU per g biomass.

Methods of saccharification of polysaccharides in plants

DOEpatents

Howard, John; Fake, Gina

2014-04-29

Saccharification of polysaccharides of plants is provided, where release of fermentable sugars from cellulose is obtained by adding plant tissue composition. Production of glucose is obtained without the need to add additional .beta.-glucosidase. Adding plant tissue composition to a process using a cellulose degrading composition to degrade cellulose results in an increase in the production of fermentable sugars compared to a process in which plant tissue composition is not added. Using plant tissue composition in a process using a cellulose degrading enzyme composition to degrade cellulose results in decrease in the amount of cellulose degrading enzyme composition or exogenously applied cellulase required to produce fermentable sugars.

Enhanced enzymatic saccharification of corn stover by in situ modification of lignin with poly (ethylene glycol) ether during low temperature alkali pretreatment.

PubMed

Lai, Chenhuan; Tang, Shuo; Yang, Bo; Gao, Ziqi; Li, Xin; Yong, Qiang

2017-11-01

A novel pretreatment process of corn stover was established in this study by in situ modification of lignin with poly (ethylene glycol) diglycidyl ether (PEGDE) during low temperature alkali pretreatment. The addition of PEGDE obviously improved the enzymatic hydrolysis by covalently modifying the residual lignins in substrates. Under the optimized conditions (pretreated with 10% (w/w) NaOH and 10% (w/w) PEGDE at 70°C for 2.5h), the total fermentable sugar yield was increased by 46.4%, from 23.7g to 34.7g per 100g raw materials. Additionally, the remaining activities of exo-glucanase and β-glucosidase in supernatant were increased by 58.6% and 40.6% respectively, demonstrating that the enhancement of enzymatic hydrolysis was mainly due to the alleviation of enzyme non-productive binding. Although the isolated lignin modified with PEGDE enhanced the enzymatic hydrolysis of substrates as well, this in situ lignin modification provided an efficient but simple way to improve enzymatic saccharification. Copyright © 2017. Published by Elsevier Ltd.

Process for whole cell saccharification of lignocelluloses to sugars using a dual bioreactor system

DOEpatents

Lu, Jue [Okemos, MI; Okeke, Benedict [Montgomery, AL

2012-03-27

The present invention describes a process for saccharification of lignocelluloses to sugars using whole microbial cells, which are enriched from cultures inoculated with paper mill waste water, wood processing waste and soil. A three-member bacterial consortium is selected as a potent microbial inocula and immobilized on inedible plant fibers for biomass saccharification. The present invention further relates the design of a dual bioreactor system, with various biocarriers for enzyme immobilization and repeated use. Sugars are continuously removed eliminating end-product inhibition and consumption by cell.

Production by Tobacco Transplastomic Plants of Recombinant Fungal and Bacterial Cell-Wall Degrading Enzymes to Be Used for Cellulosic Biomass Saccharification.

PubMed

Longoni, Paolo; Leelavathi, Sadhu; Doria, Enrico; Reddy, Vanga Siva; Cella, Rino

2015-01-01

Biofuels from renewable plant biomass are gaining momentum due to climate change related to atmospheric CO2 increase. However, the production cost of enzymes required for cellulosic biomass saccharification is a major limiting step in this process. Low-cost production of large amounts of recombinant enzymes by transgenic plants was proposed as an alternative to the conventional microbial based fermentation. A number of studies have shown that chloroplast-based gene expression offers several advantages over nuclear transformation due to efficient transcription and translation systems and high copy number of the transgene. In this study, we expressed in tobacco chloroplasts microbial genes encoding five cellulases and a polygalacturonase. Leaf extracts containing the recombinant enzymes showed the ability to degrade various cell-wall components under different conditions, singly and in combinations. In addition, our group also tested a previously described thermostable xylanase in combination with a cellulase and a polygalacturonase to study the cumulative effect on the depolymerization of a complex plant substrate. Our results demonstrate the feasibility of using transplastomic tobacco leaf extracts to convert cell-wall polysaccharides into reducing sugars, fulfilling a major prerequisite of large scale availability of a variety of cell-wall degrading enzymes for biofuel industry.

A Weibull statistics-based lignocellulose saccharification model and a built-in parameter accurately predict lignocellulose hydrolysis performance.

PubMed

Wang, Mingyu; Han, Lijuan; Liu, Shasha; Zhao, Xuebing; Yang, Jinghua; Loh, Soh Kheang; Sun, Xiaomin; Zhang, Chenxi; Fang, Xu

2015-09-01

Renewable energy from lignocellulosic biomass has been deemed an alternative to depleting fossil fuels. In order to improve this technology, we aim to develop robust mathematical models for the enzymatic lignocellulose degradation process. By analyzing 96 groups of previously published and newly obtained lignocellulose saccharification results and fitting them to Weibull distribution, we discovered Weibull statistics can accurately predict lignocellulose saccharification data, regardless of the type of substrates, enzymes and saccharification conditions. A mathematical model for enzymatic lignocellulose degradation was subsequently constructed based on Weibull statistics. Further analysis of the mathematical structure of the model and experimental saccharification data showed the significance of the two parameters in this model. In particular, the λ value, defined the characteristic time, represents the overall performance of the saccharification system. This suggestion was further supported by statistical analysis of experimental saccharification data and analysis of the glucose production levels when λ and n values change. In conclusion, the constructed Weibull statistics-based model can accurately predict lignocellulose hydrolysis behavior and we can use the λ parameter to assess the overall performance of enzymatic lignocellulose degradation. Advantages and potential applications of the model and the λ value in saccharification performance assessment were discussed. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Thermal assisted alkaline pretreatment of rice husk for enhanced biomass deconstruction and enzymatic saccharification: Physico-chemical and structural characterization.

PubMed

Shahabazuddin, Md; Sarat Chandra, T; Meena, S; Sukumaran, R K; Shetty, N P; Mudliar, S N

2018-04-21

Thermal assisted alkaline pretreatment (TAAP) of rice husk (RH) was investigated to facilitate enzymatic saccharification by enhancing the enzyme accessibility to cellulosic components. Statistically guided experiments based on the Box-Behnken design involving four factors viz. biomass loading, particle size, NaOH loading and reaction time was considered for optimization. The maximum sugar yield of 371 mg g -1 biomass was obtained at optimized pretreatment condition [biomass loading (10% w/w), particle size (0.25-0.625 mm), NaOH loading (2% w/w), and reaction time (40 min)]. The TAAP of RH resulted in the efficient removal of lignin (14.9-54% (w/w)) with low hemicellulose solubilization [10.7-33.1% (w/w)] and with a simultaneous increase in cellulose concentration [32.65-51.65% (w/w)]. The SEM analysis indicated increased porosity and biomass disruption during TAAP. The FTIR analysis showed progressive removal of noncellulosic constituents, and XRD analysis revealed an increase in cellulose crystallinity post-TAAP indicating the effectiveness of pretreatment. Copyright © 2018 Elsevier Ltd. All rights reserved.

Epigallocatechin gallate incorporation into lignin enhances the alkaline delignification and enzymatic saccharification of cell walls

PubMed Central

2012-01-01

Background Lignin is an integral component of the plant cell wall matrix but impedes the conversion of biomass into biofuels. The plasticity of lignin biosynthesis should permit the inclusion of new compatible phenolic monomers such as flavonoids into cell wall lignins that are consequently less recalcitrant to biomass processing. In the present study, epigallocatechin gallate (EGCG) was evaluated as a potential lignin bioengineering target for rendering biomass more amenable to processing for biofuel production. Results In vitro peroxidase-catalyzed polymerization experiments revealed that both gallate and pyrogallyl (B-ring) moieties in EGCG underwent radical cross-coupling with monolignols mainly by β–O–4-type cross-coupling, producing benzodioxane units following rearomatization reactions. Biomimetic lignification of maize cell walls with a 3:1 molar ratio of monolignols and EGCG permitted extensive alkaline delignification of cell walls (72 to 92%) that far exceeded that for lignified controls (44 to 62%). Alkali-insoluble residues from EGCG-lignified walls yielded up to 34% more glucose and total sugars following enzymatic saccharification than lignified controls. Conclusions It was found that EGCG readily copolymerized with monolignols to become integrally cross-coupled into cell wall lignins, where it greatly enhanced alkaline delignification and subsequent enzymatic saccharification. Improved delignification may be attributed to internal trapping of quinone-methide intermediates to prevent benzyl ether cross-linking of lignin to structural polysaccharides during lignification, and to the cleavage of ester intra-unit linkages within EGCG during pretreatment. Overall, our results suggest that apoplastic deposition of EGCG for incorporation into lignin would be a promising plant genetic engineering target for improving the delignification and saccharification of biomass crops. PMID:22889353

Ultrasonic pretreatment for enhanced saccharification and fermentation of ethanol production from corn

NASA Astrophysics Data System (ADS)

Montalbo-Lomboy, Melissa T.

The 21st Century human lifestyle has become heavily dependent on hydrocarbon inputs. Energy demand and the global warming effects due to the burning of fossil fuels have continued to increase. Rising awareness of the negative environmental and economic impacts of hydrocarbon dependence has led to a resurgence of interest in renewable energy sources such as ethanol. Fuel ethanol is known to be a cleaner and renewable source of energy relative to gasoline. Many studies have agreed that fuel ethanol has reduced greenhouse gas (GHG) emissions and has larger overall energy benefits compared to gasoline. Currently, the majority of the fuel ethanol in the United States is produced from corn using dry-grind milling process. The typical dry-grind ethanol plant incorporates jet cooking using steam to cook the corn slurry as pretreatment for saccharification; an energy intensive step. In aiming to reduce energy usage, this study evaluated the use of ultrasonics as an alternative to jet cooking. Ultrasonic batch experiments were conducted using a Branson 2000 Series bench-scale ultrasonic unit operating at a frequency of 20 kHz and a maximum output of 2.2 kW. Corn slurry was sonicated at varying amplitudes from 192 to 320 mumpeak-to-peak(p-p) for 0-40 seconds. Enzyme stability was investigated by adding enzyme (STARGEN(TM)001) before and after sonication. Scanning electron micrograph (SEM) images and particle size distribution analysis showed a nearly 20-fold size reduction by disintegration of corn particles due to ultrasonication. The results also showed a 30% improvement in sugar release of sonicated samples relative to the control group (untreated). The efficiency exceeded 100% in terms of relative energy gain from the additional sugar released due to ultrasonication compared to the ultrasonic energy applied. Interestingly, enzymatic activity was enhanced when sonicated at low and medium power. This result suggested that ultrasonic energy did not denature the enzymes

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.

The simultaneous saccharification and fermentation of pretreated woody crops to ethanol

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

Spindler, D.D.; Wyman, C.E.; Grohmann, K.

1991-12-31

Four promising woody crops (Populus maximowiczii x nigra (NE388), P. trichocarpa x deltoides (N11), P. tremuloides, and Sweetgum Liquidambar styraciflua) were pretreated by dilute sulfuric acid and evaluated in the simultaneous saccharification and fermentation (SSF) process for ethanol production. The yeast Saccharomyces cerevisiae was used in the fermentations alone, and in mixed cultures with {beta}-glucosidase producing Brettanomyces clausenii. Commercial Genencor 150L cellulose enyme was either employed alone or supplemented with {beta}-glucosidase. All SSFs were run at 37{degrees}C for 8 d and compared to saccharifications at 45{degrees}C under the same enzyme loadings. S. cerevisiae alone achieved the highest ethanol yields andmore » rates of hydrolysis at the higher enzyme loadings, whereas the mixed culture performed better at the lower enzyme loadings without {beta}-glucosidase supplementation. The best overall rates of fermentation (3 d) and final theoretical ethanol yields (86-90%) were achieved with P. maximowiczii x nigra (NE388) and Sweetgum Liquidambar styraciflua, followed by P. tremuloides and P. trichocarpa x deltoides (N11) with slightly slower rates and lower yields. Although there were some differences in SSF performance, all these pretreated woody crops show promise as substrates for ethanol production.« less

Comparative study on the conventional and non thermal simultaneous saccharification and fermentation of Manihot glaziovii root starch

NASA Astrophysics Data System (ADS)

Hargono, Kumoro, Andri Cahyo; Jos, Bakti

2015-12-01

Inconventional ethanol production process, starch is converted into dextrins via liquefaction using α-amylase enzyme at high temperature (90-120°C). Then, dextrins are saccharified by glucoamylase to obtain to monomeric sugars (glucose). Recently, a granular starch hydrolyzing enzymes (GSHE), Stargen 002, was developed to convert starch into dextrins at low temperature (< 32°C) and hydrolyzes dextrins into glucose. The subject of this research was to compare ethanol production using a granular starch hydrolyzing enzymes and conventional enzymatic liquefaction and saccharification in cassava starch processing. Starch slurry concentrations were 20% w/v, and dosage of enzymes 0.50, 1.0 and 2%, respectively, were studied. After 48 hr process the final ethanol concentration for the respective enzyme concentration for conventional process were 34.90, 36.16 and 42.10 g/L, whereas for the non-thermal treatment, final ethanol concentration were 46.4, 57.62 and 59.65 g/L, respectively. By implementation of this non thermal process, the use of energy can be saved by carrying out saccharification step at lower temperature (30°C) could be realized.

Enhanced bioethanol production by fed-batch simultaneous saccharification and co-fermentation at high solid loading of Fenton reaction and sodium hydroxide sequentially pretreated sugarcane bagasse.

PubMed

Zhang, Teng; Zhu, Ming-Jun

2017-04-01

A study on the fed-batch simultaneous saccharification and co-fermentation (SSCF) of Fenton reaction combined with NaOH pretreated sugarcane bagasse (SCB) at a high solid loading of 10-30% (w/v) was investigated. Enzyme feeding mode, substrate feeding mode and combination of both were compared with the batch mode under respective solid loadings. Ethanol concentrations of above 80g/L were obtained in batch and enzyme feeding modes at a solid loading of 30% (w/v). Enzyme feeding mode was found to increase ethanol productivity and reduce enzyme loading to a value of 1.23g/L/h and 9FPU/g substrate, respectively. The present study provides an economically feasible process for high concentration bioethanol production. Copyright © 2017 Elsevier Ltd. All rights reserved.

Steam-exploded biomass saccharification is predominately affected by lignocellulose porosity and largely enhanced by Tween-80 in Miscanthus.

PubMed

Sun, Dan; Alam, Aftab; Tu, Yuanyuan; Zhou, Shiguang; Wang, Yanting; Xia, Tao; Huang, Jiangfeng; Li, Ying; Zahoor; Wei, Xiaoyang; Hao, Bo; Peng, Liangcai

2017-09-01

In this study, total ten Miscanthus accessions exhibited diverse cell wall compositions, leading to largely varied hexoses yields at 17%-40% (% cellulose) released from direct enzymatic hydrolysis of steam-exploded (SE) residues. Further supplied with 2% Tween-80 into the enzymatic digestion, the Mis7 accession showed the higher hexose yield by 14.8-fold than that of raw material, whereas the Mis10 had the highest hexoses yield at 77% among ten Miscanthus accessions. Significantly, this study identified four wall polymer features that negatively affect biomass saccharification as p<0.05 or 0.01 in the SE residues, including cellulose DP, Xyl and Ara of hemicellulose, and S-monomer of lignin. Based on Simons' stain, the SE porosity (defined by DY/DB) was examined to be the unique positive factor on biomass enzymatic digestion. Hence, this study provides the potential strategy to enhance biomass saccharification using optimal biomass process technology and related genetic breeding in Miscanthus and beyond. Copyright © 2017 Elsevier Ltd. All rights reserved.

Optimization of a novel sequential alkalic and metal salt pretreatment for enhanced delignification and enzymatic saccharification of corn cobs.

PubMed

Sewsynker-Sukai, Yeshona; Gueguim Kana, E B

2017-11-01

This study presents a sequential sodium phosphate dodecahydrate (Na 3 PO 4 ·12H 2 O) and zinc chloride (ZnCl 2 ) pretreatment to enhance delignification and enzymatic saccharification of corn cobs. The effects of process parameters of Na 3 PO 4 ·12H 2 O concentration (5-15%), ZnCl 2 concentration (1-5%) and solid to liquid ratio (5-15%) on reducing sugar yield from corn cobs were investigated. The sequential pretreatment model was developed and optimized with a high coefficient of determination value (0.94). Maximum reducing sugar yield of 1.10±0.01g/g was obtained with 14.02% Na 3 PO 4 ·12H 2 O, 3.65% ZnCl 2 and 5% solid to liquid ratio. Scanning electron microscopy (SEM) and Fourier Transform Infrared analysis (FTIR) showed major lignocellulosic structural changes after the optimized sequential pretreatment with 63.61% delignification. In addition, a 10-fold increase in the sugar yield was observed compared to previous reports on the same substrate. This sequential pretreatment strategy was efficient for enhancing enzymatic saccharification of corn cobs. Copyright © 2017 Elsevier Ltd. All rights reserved.

Biomass saccharification is largely enhanced by altering wall polymer features and reducing silicon accumulation in rice cultivars harvested from nitrogen fertilizer supply.

PubMed

Zahoor; Sun, Dan; Li, Ying; Wang, Jing; Tu, Yuanyuan; Wang, Yanting; Hu, Zhen; Zhou, Shiguang; Wang, Lingqiang; Xie, Guosheng; Huang, Jianliang; Alam, Aftab; Peng, Liangcai

2017-11-01

In this study, two rice cultivars were collected from experimental fields with seven nitrogen fertilizer treatments. All biomass samples contained significantly increased cellulose contents and reduced silica levels, with variable amounts of hemicellulose and lignin from different nitrogen treatments. Under chemical (NaOH, CaO, H 2 SO 4 ) and physical (hot water) pretreatments, biomass samples exhibited much enhanced hexoses yields from enzymatic hydrolysis, with high bioethanol production from yeast fermentation. Notably, both degree of polymerization (DP) of cellulose and xylose/arabinose (Xyl/Ara) ratio of hemicellulose were reduced in biomass residues, whereas other wall polymer features (cellulose crystallinity and monolignol proportion) were variable. Integrative analysis indicated that cellulose DP, hemicellulosic Xyl/Ara and silica are the major factors that significantly affect cellulose crystallinity and biomass saccharification. Hence, this study has demonstrated that nitrogen fertilizer supply could largely enhance biomass saccharification in rice cultivars, mainly by reducing cellulose DP, hemicellulosic Xyl/Ara and silica in cell walls. Copyright © 2017 Elsevier Ltd. All rights reserved.

Steam explosion distinctively enhances biomass enzymatic saccharification of cotton stalks by largely reducing cellulose polymerization degree in G. barbadense and G. hirsutum.

PubMed

Huang, Yu; Wei, Xiaoyang; Zhou, Shiguang; Liu, Mingyong; Tu, Yuanyuan; Li, Ao; Chen, Peng; Wang, Yanting; Zhang, Xuewen; Tai, Hongzhong; Peng, Liangcai; Xia, Tao

2015-04-01

In this study, steam explosion pretreatment was performed in cotton stalks, leading to 5-6 folds enhancements on biomass enzymatic saccharification distinctive in Gossypium barbadense and Gossypium hirsutum species. Sequential 1% H2SO4 pretreatment could further increase biomass digestibility of the steam-exploded stalks, and also cause the highest sugar-ethanol conversion rates probably by releasing less inhibitor to yeast fermentation. By comparison, extremely high concentration alkali (16% NaOH) pretreatment with raw stalks resulted in the highest hexoses yields, but it had the lowest sugar-ethanol conversion rates. Characterization of wall polymer features indicated that biomass saccharification was enhanced with steam explosion by largely reducing cellulose DP and extracting hemicelluloses. It also showed that cellulose crystallinity and arabinose substitution degree of xylans were the major factors on biomass digestibility in cotton stalks. Hence, this study has provided the insights into cell wall modification and biomass process technology in cotton stalks and beyond. Copyright © 2015 Elsevier Ltd. All rights reserved.

Simultaneous Saccharification and Fermentation of Sugar Beet Pulp for Efficient Bioethanol Production

PubMed Central

Berłowska, Joanna; Balcerek, Maria; Dziekońska-Kubczak, Urszula; Patelski, Piotr; Dziugan, Piotr

2016-01-01

Sugar beet pulp, a byproduct of sugar beet processing, can be used as a feedstock in second-generation ethanol production. The objective of this study was to investigate the effects of pretreatment, of the dosage of cellulase and hemicellulase enzyme preparations used, and of aeration on the release of fermentable sugars and ethanol yield during simultaneous saccharification and fermentation (SSF) of sugar beet pulp-based worts. Pressure-thermal pretreatment was applied to sugar beet pulp suspended in 2% w/w sulphuric acid solution at a ratio providing 12% dry matter. Enzymatic hydrolysis was conducted using Viscozyme and Ultraflo Max (Novozymes) enzyme preparations (0.015–0.02 mL/g dry matter). Two yeast strains were used for fermentation: Ethanol Red (S. cerevisiae) (1 g/L) and Pichia stipitis (0.5 g/L), applied sequentially. The results show that efficient simultaneous saccharification and fermentation of sugar beet pulp was achieved. A 6 h interval for enzymatic activation between the application of enzyme preparations and inoculation with Ethanol Red further improved the fermentation performance, with the highest ethanol concentration reaching 26.9 ± 1.2 g/L and 86.5 ± 2.1% fermentation efficiency relative to the theoretical yield. PMID:27722169

Simultaneous Saccharification and Fermentation of Sugar Beet Pulp for Efficient Bioethanol Production.

PubMed

Berłowska, Joanna; Pielech-Przybylska, Katarzyna; Balcerek, Maria; Dziekońska-Kubczak, Urszula; Patelski, Piotr; Dziugan, Piotr; Kręgiel, Dorota

2016-01-01

Sugar beet pulp, a byproduct of sugar beet processing, can be used as a feedstock in second-generation ethanol production. The objective of this study was to investigate the effects of pretreatment, of the dosage of cellulase and hemicellulase enzyme preparations used, and of aeration on the release of fermentable sugars and ethanol yield during simultaneous saccharification and fermentation (SSF) of sugar beet pulp-based worts. Pressure-thermal pretreatment was applied to sugar beet pulp suspended in 2% w/w sulphuric acid solution at a ratio providing 12% dry matter. Enzymatic hydrolysis was conducted using Viscozyme and Ultraflo Max (Novozymes) enzyme preparations (0.015-0.02 mL/g dry matter). Two yeast strains were used for fermentation: Ethanol Red ( S. cerevisiae ) (1 g/L) and Pichia stipitis (0.5 g/L), applied sequentially. The results show that efficient simultaneous saccharification and fermentation of sugar beet pulp was achieved. A 6 h interval for enzymatic activation between the application of enzyme preparations and inoculation with Ethanol Red further improved the fermentation performance, with the highest ethanol concentration reaching 26.9 ± 1.2 g/L and 86.5 ± 2.1% fermentation efficiency relative to the theoretical yield.

Saccharification of citrus wastes by immobilized polygalacturonase in an improved alginate matrix.

PubMed

Ramírez-Tapias, Yuly A; Lapasset Laumann, Aldana S; Britos, Claudia N; Rivero, Cintia W; Trelles, Jorge A

2017-12-01

Enzyme immobilization using hydrogels is a low-cost and effective system for the degradation of bulk pectin derived from orange industry residues. Polygalacturonases obtained from four different bacterial strains of Streptomyces genus were immobilized in alginate gel and assayed for pectin hydrolysis. The enzyme from Streptomyces halstedii ATCC 10897 proved to be superior and more stable within the alginate matrix. Furthermore, a new strategy to improve alginate bead stability using a mixture of calcium and strontium is reported; this technique allowed enhancing the mechanical properties by combining different amounts of these cations for ionotropic gelation. The developed biocatalyst showed maximum hydrolysis at 2 h, generating 1.54 mg/mL of reducing sugars and decreasing the viscosity of polygalacturonic acid by 98.9%. Reusability up to 29 successive reactions (58 h) demonstrated a very stable performance. The heterogeneous biocatalyst was used in the enzymatic saccharification of orange peel albedo (2.23 mg/mL) for adding value to this agro-waste by industrial exploitation.

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.

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.

Effects of different cellulases on the release of phenolic acids from rice straw during saccharification.

PubMed

Xue, Yiyun; Wang, Xiahui; Chen, Xingxuan; Hu, Jiajun; Gao, Min-Tian; Li, Jixiang

2017-06-01

Effects of different cellulases on the release of phenolic acids from rice straw during saccharification were investigated in this study. All cellulases tested increased the contents of phenolic acids during saccharification. However, few free phenolic acids were detected, as they were present in conjugated form after saccharification when the cellulases from Trichoderma reesei, Trichoderma viride and Aspergillus niger were used. On the other hand, phenolic acids were present in free form when the Acremonium cellulolyticus cellulase was used. Assays of enzyme activity showed that, besides high cellulase activity, the A. cellulolyticus cellulase exhibited high feruloyl esterase (FAE) activity. A synergistic interaction between FAE and cellulase led to the increase in free phenolic acids, and thus an increase in antioxidative and antiradical activities of the phenolic acids. Moreover, a cost estimation demonstrated the feasibility of phenolic acids as value-added products to reduce the total production cost of ethanol. Copyright © 2017 Elsevier Ltd. All rights reserved.

Evaluation of nanoparticle-immobilized cellulase for improved ethanol yield in simultaneous saccharification and fermentation reactions

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

Lupoi, Jason; Smith, Emily

2011-12-01

Ethanol yields were 2.1 (P = 0.06) to 2.3 (P = 0.01) times higher in simultaneous saccharification and fermentation (SSF) reactions of microcrystalline cellulose when cellulase was physisorbed on silica nanoparticles compared to enzyme in solution. In SSF reactions, cellulose is hydrolyzed to glucose by cellulase while yeast simultaneously ferments glucose to ethanol. The 35 C temperature and the presence of ethanol in SSF reactions are not optimal conditions for cellulase. Immobilization onto solid supports can stabilize the enzyme and promote activity at non-optimum reaction conditions. Mock SSF reactions that did not contain yeast were used to measure saccharification productsmore » and identify the mechanism for the improved ethanol yield using immobilized cellulase. Cellulase adsorbed to 40 nm silica nanoparticles produced 1.6 times (P = 0.01) more glucose than cellulase in solution in 96 h at pH 4.8 and 35 C. There was no significant accumulation (<250 {mu}g) of soluble cellooligomers in either the solution or immobilized enzyme reactions. This suggests that the mechanism for the immobilized enzyme's improved glucose yield compared to solution enzyme is the increased conversion of insoluble cellulose hydrolysis products to soluble cellooligomers at 35 C and in the presence of ethanol. The results show that silica-immobilized cellulase can be used to produce increased ethanol yields in the conversion of lignocellulosic materials by SSF.« less

Co-solvent pretreatment reduces costly enzyme requirements for high sugar and ethanol yields from lignocellulosic biomass.

PubMed

Nguyen, Thanh Yen; Cai, Charles M; Kumar, Rajeev; Wyman, Charles E

2015-05-22

We introduce a new pretreatment called co-solvent-enhanced lignocellulosic fractionation (CELF) to reduce enzyme costs dramatically for high sugar yields from hemicellulose and cellulose, which is essential for the low-cost conversion of biomass to fuels. CELF employs THF miscible with aqueous dilute acid to obtain up to 95 % theoretical yield of glucose, xylose, and arabinose from corn stover even if coupled with enzymatic hydrolysis at only 2 mgenzyme  gglucan (-1) . The unusually high saccharification with such low enzyme loadings can be attributed to a very high lignin removal, which is supported by compositional analysis, fractal kinetic modeling, and SEM imaging. Subsequently, nearly pure lignin product can be precipitated by the evaporation of volatile THF for recovery and recycling. Simultaneous saccharification and fermentation of CELF-pretreated solids with low enzyme loadings and Saccharomyces cerevisiae produced twice as much ethanol as that from dilute-acid-pretreated solids if both were optimized for corn stover. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Process for concentrated biomass saccharification

DOEpatents

Hennessey, Susan M.; Seapan, Mayis; Elander, Richard T.; Tucker, Melvin P.

2010-10-05

Processes for saccharification of pretreated biomass to obtain high concentrations of fermentable sugars are provided. Specifically, a process was developed that uses a fed batch approach with particle size reduction to provide a high dry weight of biomass content enzymatic saccharification reaction, which produces a high sugars concentration hydrolysate, using a low cost reactor system.

Understanding the cellulolytic system of Trichoderma harzianum P49P11 and enhancing saccharification of pretreated sugarcane bagasse by supplementation with pectinase and α-L-arabinofuranosidase.

PubMed

Delabona, Priscila da Silva; Cota, Júnio; Hoffmam, Zaira Bruna; Paixão, Douglas Antonio Alvaredo; Farinas, Cristiane Sanchez; Cairo, João Paulo Lourenço Franco; Lima, Deise Juliana; Squina, Fábio Marcio; Ruller, Roberto; Pradella, José Geraldo da Cruz

2013-03-01

Supplementation of cellulase cocktails with accessory enzymes can contribute to a higher hydrolytic capacity in releasing fermentable sugars from plant biomass. This study investigated which enzymes were complementary to the enzyme set of Trichoderma harzianum in the degradation of sugarcane bagasse. Specific activities of T. harzianum extract on different substrates were compared with the extracts of Penicillium echinulatum and Trichoderma reesei, and two commercial cellulase preparations. Complementary analysis of the secretome of T. harzianum was also used to identify which enzymes were produced during growth on pretreated sugarcane bagasse. These analyses enabled the selection of the enzymes pectinase and α-L-arabinofuranosidase (AF) to be further investigated as supplements to the T. harzianum extract. The effect of enzyme supplementation on the efficiency of sugarcane bagasse saccharification was evaluated using response surface methodology. The supplementation of T. harzianum enzymatic extract with pectinase and AF increased the efficiency of hydrolysis by up to 116%. Copyright © 2012 Elsevier Ltd. All rights reserved.

Chemical characteristics and enzymatic saccharification of lignocellulosic biomass treated using high-temperature saturated steam: comparison of softwood and hardwood.

PubMed

Asada, Chikako; Sasaki, Chizuru; Hirano, Takeshi; Nakamura, Yoshitoshi

2015-04-01

This study investigated the effect of high-temperature saturated steam treatments on the chemical characteristics and enzymatic saccharification of softwood and hardwood. The weight loss and chemical modification of cedar and beech wood pieces treated at 25, 35, and 45 atm for 5 min were determined. Fourier transform infrared and X-ray diffraction analyses indicated that solubilization and removal of hemicellulose and lignin occurred by the steam treatment. The milling treatment of steam-treated wood enhanced its enzymatic saccharification. Maximum enzymatic saccharification (i.e., 94% saccharification rate of cellulose) was obtained using steam-treated beech at 35 atm for 5 min followed by milling treatment for 1 min. However, the necessity of the milling treatment for efficient enzymatic saccharification is dependent on the wood species. Copyright © 2015 Elsevier Ltd. All rights reserved.

Modeling and experimental studies on intermittent starch feeding and citrate addition in simultaneous saccharification and fermentation of starch to flavor compounds.

PubMed

Chavan, Abhijit R; Raghunathan, Anuradha; Venkatesh, K V

2009-04-01

Simultaneous saccharification and fermentation (SSF) is a combined process of saccharification of a renewable bioresource and fermentation process to produce products, such as lactic acid and ethanol. Recently, SSF has been extensively used to convert various sources of cellulose and starch into fermentative products. Here, we present a study on production of buttery flavors, namely diacetyl and acetoin, by growing Lactobacillus rhamnosus on a starch medium containing the enzyme glucoamylase. We further develop a structured kinetics for the SSF process, which includes enzyme and growth kinetics. The model was used to simulate the effect of pH and temperature on the SSF process so as to obtain optimum operating conditions. The model was experimentally verified by conducting SSF using an initial starch concentration of 100 g/L. The study demonstrated that the developed kinetic was able to suggest strategies for improved productivities. The developed model was able to accurately predict the enhanced productivity of flavors in a three stage process with intermittent addition of starch. Experimental and simulations demonstrated that citrate addition can also lead to enhanced productivity of flavors. The developed optimal model for SSF was able to capture the dynamics of SSF in batch mode as well as in a three stage process. The structured kinetics was also able to quantify the effect of multiple substrates present in the medium. The study demonstrated that structured kinetic models can be used in the future for design and optimization of SSF as a batch or a fed-batch process.

Rapid saccharification for production of cellulosic biofuels.

PubMed

Lee, Dae-Seok; Wi, Seung Gon; Lee, Soo Jung; Lee, Yoon-Gyo; Kim, Yeong-Suk; Bae, Hyeun-Jong

2014-04-01

The economical production of biofuels is hindered by the recalcitrance of lignocellulose to processing, causing high consumption of processing enzymes and impeding hydrolysis of pretreated lignocellulosic biomass. We determined the major rate-limiting factor in the hydrolysis of popping pre-treated rice straw (PPRS) by examining cellulase adsorption to lignin and cellulose, amorphogenesis of PPRS, and re-hydrolysis. Based on the results, equivalence between enzyme loading and the open structural area of cellulose was required to significantly increase productive adsorption of cellulase and to accelerate enzymatic saccharification of PPRS. Amorphogenesis of PPRS by phosphoric acid treatment to expand open structural area of the cellulose fibers resulted in twofold higher cellulase adsorption and increased the yield of the first re-hydrolysis step from 13% to 46%. The total yield from PPRS was increased to 84% after 3h. These results provide evidence that cellulose structure is one of major effects on the enzymatic hydrolysis. Copyright © 2014 Elsevier Ltd. All rights reserved.

Liquid nitrogen pretreatment of eucalyptus sawdust and rice hull for enhanced enzymatic saccharification.

PubMed

Castoldi, Rafael; Correa, Vanesa G; de Morais, Gutierrez Rodrigues; de Souza, Cristina G M; Bracht, Adelar; Peralta, Rosely A; Peralta-Muniz Moreira, Regina F; Peralta, Rosane M

2017-01-01

In this work, liquid nitrogen was used for the first time in the pretreatment of plant biomasses for purposes of enzymatic saccharification. After treatment (cryocrushing), the initial rates of the enzymatic hydrolysis of eucalyptus sawdust and rice hull were increased more than ten-fold. Cryocrushing did not modify significantly the contents of cellulose, hemicellulose and lignin in both eucalyptus sawdust and rice hulls. However, substantial disorganization of the lignocellulosic materials in consequence of the pretreatment could be observed by electron microscopy. Cryocrushing was highly efficient in improving the saccharification of the holocellulose component of the plant biomasses (from 4.3% to 54.1% for eucalyptus sawdust and from 3.9% to 40.6% for rice hull). It is important to emphasize that it consists in a simple operation with low requirements of water and chemicals, no corrosion, no release of products such as soluble phenolics, furfural and hydroxymethylfurfural and no waste generation. Copyright © 2016 Elsevier Ltd. All rights reserved.

Saccharification of bamboo carbohydrates for the production of ethanol

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

De Menezes, T.J.B.; Azzini, A.; Dos Santos, C.L.M.

1983-04-01

Bamboo carbohydrates were hydrolyzed with commercial amylases and a mixture of fungal culture broths containing cellulolytic and hemicellulolytic enzymes. The effects of cooking temperature and the size of fiber particles were also investigated. It was found that the higher the cooking temperature, the higher the rate of sugar formation and the lower the viscosity of the slurry. Additions of cellulose and hemicellulose digesting enzymes increased the sugar yield and decreased the viscosity of both the cooked and noncooked slurries. A smaller size of particle appeared to favor the average saccharification rate. Although glucose, xylose, and cellobiose were present in themore » hydrolysates, only 50% of the total carbohydrate was digested, and 78.9% of this was converted to reducing sugars. The alcohol efficiency for the fermentation of cooked and noncooked mashes by Saccharomyces was about 85%.« less

Saccharification and liquefaction of cassava starch: an alternative source for the production of bioethanol using amylolytic enzymes by double fermentation process

PubMed Central

2014-01-01

Background Cassava starch is considered as a potential source for the commercial production of bioethanol because of its availability and low market price. It can be used as a basic source to support large-scale biological production of bioethanol using microbial amylases. With the progression and advancement in enzymology, starch liquefying and saccharifying enzymes are preferred for the conversion of complex starch polymer into various valuable metabolites. These hydrolytic enzymes can selectively cleave the internal linkages of starch molecule to produce free glucose which can be utilized to produce bioethanol by microbial fermentation. Results In the present study, several filamentous fungi were screened for production of amylases and among them Aspergillus fumigatus KIBGE-IB33 was selected based on maximum enzyme yield. Maximum α-amylase, amyloglucosidase and glucose formation was achieved after 03 days of fermentation using cassava starch. After salt precipitation, fold purification of α-amylase and amyloglucosidase increased up to 4.1 and 4.2 times with specific activity of 9.2 kUmg-1 and 393 kUmg-1, respectively. Concentrated amylolytic enzyme mixture was incorporated in cassava starch slurry to give maximum glucose formation (40.0 gL-1), which was further fermented using Saccharomyces cerevisiae into bioethanol with 84.0% yield. The distillate originated after recovery of bioethanol gave 53.0% yield. Conclusion An improved and effective dual enzymatic starch degradation method is designed for the production of bioethanol using cassava starch. The technique developed is more profitable due to its fast liquefaction and saccharification approach that was employed for the formation of glucose and ultimately resulted in higher yields of alcohol production. PMID:24885587

Ethanol production via simultaneous saccharification and fermentation of sodium hydroxide treated corn stover using Phanerochaete chrysosporium and Gloeophyllum trabeum.

PubMed

Vincent, Micky; Pometto, Anthony L; van Leeuwen, J Hans

2014-04-01

Ethanol was produced via the simultaneous saccharification and fermentation (SSF) of dilute sodium hydroxide treated corn stover. Saccharification was achieved by cultivating either Phanerochaete chrysosporium or Gloeophyllum trabeum on the treated stover, and fermentation was then performed by using either Saccharomyces cerevisiae or Escherichia coli K011. Ethanol production was highest on day 3 for the combination of G. trabeum and E. coli K011 at 6.68 g/100g stover, followed by the combination of P. chrysosporium and E. coli K011 at 5.00 g/100g stover. SSF with S. cerevisiae had lower ethanol yields, ranging between 2.88 g/100g stover at day 3 (P. chrysosporium treated stover) and 3.09 g/100g stover at day 4 (G. trabeum treated stover). The results indicated that mild alkaline pretreatment coupled with fungal saccharification offers a promising bioprocess for ethanol production from corn stover without the addition of commercial enzymes. Published by Elsevier Ltd.

Combined substrate, enzyme and yeast feed in simultaneous saccharification and fermentation allow bioethanol production from pretreated spruce biomass at high solids loadings

PubMed Central

2014-01-01

Background Economically feasible cellulosic ethanol production requires that the process can be operated at high solid loadings, which currently imparts technical challenges including inefficient mixing leading to heat and mass transfer limitations and high concentrations of inhibitory compounds hindering microbial activity during simultaneous saccharification and fermentation (SSF) process. Consequently, there is a need to develop cost effective processes overcoming the challenges when working at high solid loadings. Results In this study we have modified the yeast cultivation procedure and designed a SSF process to address some of the challenges at high water insoluble solids (WIS) content. The slurry of non-detoxified pretreated spruce when used in a batch SSF at 19% (w/w) WIS was found to be inhibitory to Saccharomyces cerevisiae Thermosacc that produced 2 g l-1 of ethanol. In order to reduce the inhibitory effect, the non-washed solid fraction containing reduced amount of inhibitors compared to the slurry was used in the SSF. Further, the cells were cultivated in the liquid fraction of pretreated spruce in a continuous culture wherein the outflow of cell suspension was used as cell feed to the SSF reactor in order to maintain the metabolic state of the cell. Enhanced cell viability was observed with cell, enzyme and substrate feed in a SSF producing 40 g l-1 ethanol after 96 h corresponding to 53% of theoretical yield based on available hexose sugars compared to 28 g l-1 ethanol in SSF with enzyme and substrate feed but no cell feed resulting in 37% of theoretical yield at a high solids loading of 20% (w/w) WIS content. The fed-batch SSF also significantly eased the mixing, which is usually challenging in batch SSF at high solids loading. Conclusions A simple modification of the cell cultivation procedure together with a combination of yeast, enzyme and substrate feed in a fed-batch SSF process, made it possible to operate at high solids loadings in a

Combined substrate, enzyme and yeast feed in simultaneous saccharification and fermentation allow bioethanol production from pretreated spruce biomass at high solids loadings.

PubMed

Koppram, Rakesh; Olsson, Lisbeth

2014-04-08

Economically feasible cellulosic ethanol production requires that the process can be operated at high solid loadings, which currently imparts technical challenges including inefficient mixing leading to heat and mass transfer limitations and high concentrations of inhibitory compounds hindering microbial activity during simultaneous saccharification and fermentation (SSF) process. Consequently, there is a need to develop cost effective processes overcoming the challenges when working at high solid loadings. In this study we have modified the yeast cultivation procedure and designed a SSF process to address some of the challenges at high water insoluble solids (WIS) content. The slurry of non-detoxified pretreated spruce when used in a batch SSF at 19% (w/w) WIS was found to be inhibitory to Saccharomyces cerevisiae Thermosacc that produced 2 g l-1 of ethanol. In order to reduce the inhibitory effect, the non-washed solid fraction containing reduced amount of inhibitors compared to the slurry was used in the SSF. Further, the cells were cultivated in the liquid fraction of pretreated spruce in a continuous culture wherein the outflow of cell suspension was used as cell feed to the SSF reactor in order to maintain the metabolic state of the cell. Enhanced cell viability was observed with cell, enzyme and substrate feed in a SSF producing 40 g l-1 ethanol after 96 h corresponding to 53% of theoretical yield based on available hexose sugars compared to 28 g l-1 ethanol in SSF with enzyme and substrate feed but no cell feed resulting in 37% of theoretical yield at a high solids loading of 20% (w/w) WIS content. The fed-batch SSF also significantly eased the mixing, which is usually challenging in batch SSF at high solids loading. A simple modification of the cell cultivation procedure together with a combination of yeast, enzyme and substrate feed in a fed-batch SSF process, made it possible to operate at high solids loadings in a conventional bioreactor. The

Automated saccharification assay for determination of digestibility in plant materials.

PubMed

Gomez, Leonardo D; Whitehead, Caragh; Barakate, Abdellah; Halpin, Claire; McQueen-Mason, Simon J

2010-10-27

Cell wall resistance represents the main barrier for the production of second generation biofuels. The deconstruction of lignocellulose can provide sugars for the production of fuels or other industrial products through fermentation. Understanding the biochemical basis of the recalcitrance of cell walls to digestion will allow development of more effective and cost efficient ways to produce sugars from biomass. One approach is to identify plant genes that play a role in biomass recalcitrance, using association genetics. Such an approach requires a robust and reliable high throughput (HT) assay for biomass digestibility, which can be used to screen the large numbers of samples involved in such studies. We developed a HT saccharification assay based on a robotic platform that can carry out in a 96-well plate format the enzymatic digestion and quantification of the released sugars. The handling of the biomass powder for weighing and formatting into 96 wells is performed by a robotic station, where the plant material is ground, delivered to the desired well in the plates and weighed with a precision of 0.1 mg. Once the plates are loaded, an automated liquid handling platform delivers an optional mild pretreatment (< 100°C) followed by enzymatic hydrolysis of the biomass. Aliquots from the hydrolysis are then analyzed for the release of reducing sugar equivalents. The same platform can be used for the comparative evaluation of different enzymes and enzyme cocktails. The sensitivity and reliability of the platform was evaluated by measuring the saccharification of stems from lignin modified tobacco plants, and the results of automated and manual analyses compared. The automated assay systems are sensitive, robust and reliable. The system can reliably detect differences in the saccharification of plant tissues, and is able to process large number of samples with a minimum amount of human intervention. The automated system uncovered significant increases in the

Understanding the effects of lignosulfonate on enzymatic saccharification of pure cellulose

Treesearch

Hongming Lou; Haifeng Zhou; Xiuli Li; Mengxia Wang; J.Y. Zhu; Xueqing Qiu

2014-01-01

The effects of lignosulfonate (LS) on enzymatic saccharification of pure cellulose were studied. Four fractions of LS with different molecular weight (MW) prepared by ultrafiltration of a commercial LS were applied at different loadings to enzymatic hydrolysis of Whatman paper under different pH. Using LS fractions with low MW and high degree of sulfonation can enhance...

Ethanosolv Pretreatment of Bamboo with Dilute Acid for Efficient Enzymatic Saccharification

Treesearch

Zhiqiang Li; Zehui Jiang; Benhua Fei; Zhiyong Cai; Xuejun Pan

2012-01-01

Bamboo is a potential lignocellulosic biomass for the production of bioethanol because of its high cellulose and hemicelluloses content. In this research, ethanosolv pretreatment catalyzed by sulfuric acid was studied in order to enhance enzymatic saccharification of moso bamboo. The addition of 2% (w/w on bamboo) sulfuric acid in water or 75% (v/v) ethanol was...

Ethanol production from ensiled rice straw and whole-crop silage by the simultaneous enzymatic saccharification and fermentation process.

PubMed

Shinozaki, Yukiko; Kitamoto, Hiroko K

2011-03-01

Silage production from rice straw and whole-plant forage paddy rice is increasing in Japan because of decrease in rice consumption. One potential use for this silage is bioethanol production. In this study, we analyzed the effectiveness of three different commercially available cellulases at saccharification of sun-dried rice straw, ensiled rice straw, and rice whole-crop silage (WCS). Furthermore, the ethanol productivity of the simultaneous saccharification and ethanol fermentation process (SSF) from the same plant substrates was analyzed. Among the three kinds of cellulases tested (Novozymes NS50013, Genencor GC220, and Acremonium cellulase), Acremonium cellulase showed the highest ethanol production for the three plant substrates, and the WCS produced the highest ethanol level. Analysis of the enzymatic degradation activity of the cellulases revealed that Acremonium cellulase contained remarkably high glucoamylase and pectinase side activities relative to the other cellulase preparations. The addition of glucoamylase and pectinase to the other two cellulases significantly increased ethanol productivity to levels observed for the Acremonium cellulase preparation, which showed little enhanced performance with the addition of the same enzymes. Finally, we tested whether milling and sterilization had an effect on ethanol production and found that sterilized silage produced higher ethanol levels but that the milling process had no significant effect. These results show that (i) silage made from whole-plant rice can be used for bioethanol production and (ii) the proper selection and combination of commercially available enzymes can make SSF more cost efficient by removing the need for a pre-treatment step. Copyright © 2010 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Lignosulfonate To Enhance Enzymatic Saccharification of Lignocelluloses: Role of Molecular Weight and Substrate Lignin

Treesearch

Haifeng Zhou; Hongming Lou; Dongjie Yang; J.Y. Zhu; Xueqing Qiu

2013-01-01

This study conducted an investigation of the effect of lignosulfonate (LS) on enzymatic saccharification of lignocelluloses. Two commercial LSs and one laboratory sulfonated kraft lignin were applied to Whatman paper, dilute acid and SPORL (sulfite pretreatment to overcome recalcitrance of lignocelluloses) pretreated aspen, and kraft alkaline and SPORL pretreated...

Characterization of truncated endo-β-1,4-glucanases from a compost metagenomic library and their saccharification potentials.

PubMed

Lee, Jae Pil; Lee, Hyun Woo; Na, Han Beur; Lee, Jun-Hee; Hong, Yeo-Jin; Jeon, Jeong-Min; Kwon, Eun Ju; Kim, Sung Kyum; Kim, Hoon

2018-04-23

A gene encoding an endo-β-1,4-glucanase (Cel6H-f481) was cloned from a compost metagenomic library. The gene, cel6H-f481, was composed of 1446 bp to encode a fused protein of 481 amino acid residues (50,429 Da), i.e., 445 residues (Cel6H-445) from the metagenome, and 36 residues from the pUC19 vector at N-terminus. Cel6H-445 belonged to glycosyl hydrolase (GH) family 6 and showed 71% identity with Actinotalea fermentans endoglucanase with low coverage. Several active bands of truncated forms were observed by activity staining of the crude extract. Major truncated enzymes of 35 (Cel6H-p35) and 23 kDa (Cel6H-p23) were separated by HiTrap Q chromatography. The two enzymes had the same optimum temperature (50 °C) and pH (5.5), but Cel6H-p35 was more thermostable than Cel6H-p23 and other GH6 endoglucanases reported. Both enzymes efficiently hydrolyzed carboxymethyl-cellulose (CMC) and barley β-glucan, but hardly hydrolyzed other substrates tested. The V max of Cel6H-p35 for CMC was 1.4 times greater than that of Cel6H-p23. The addition of the crude enzymes to a commercial enzyme set increased the saccharification of pretreated rice straw powder by up to 30.9%. These results suggest the N-terminal region of Cel6H-p35 contributes to thermostability and specific activity, and that the enzymes might be a useful additive for saccharification. Copyright © 2018 Elsevier B.V. All rights reserved.

Characterization of Cellulase Enzyme Inhibitors Formed During the Chemical Pretreatments of Rice Straw

NASA Astrophysics Data System (ADS)

Rajan, Kalavathy

Production of fuels and chemicals from a renewable and inexpensive resource such as lignocellulosic biomass is a lucrative and sustainable option for the advanced biofuel and bio-based chemical platform. Agricultural residues constitute the bulk of potential feedstock available for cellulosic fuel production. On a global scale, rice straw is the largest source of agricultural residues and is therefore an ideal crop model for biomass deconstruction studies. Lignocellulosic biofuel production involves the processes of biomass conditioning, enzymatic saccharification, microbial fermentation and ethanol distillation, and one of the major factors affecting its techno-economic feasibility is the biomass recalcitrance to enzymatic saccharification. Preconditioning of lignocellulosic biomass, using chemical, physico-chemical, mechanical and biological pretreatments, is often practiced such that biomass becomes available to downstream processing. Pretreatments, such as dilute acid and hot water, are effective means of biomass conversion. However, despite their processing importance, preconditioning biomass also results in the production of carbohydrate and lignin degradation products that are inhibitory to downstream saccharification enzymes. The saccharification enzyme cocktail is made up of endo-cellulase, exo-cellulase and beta-glucosidase enzymes, whose role is to cleave cellulose polymers into glucose monomers. Specifically, endo-cellulase and exo-cellulase enzymes cleave cellulose chains in the middle and at the end, resulting in cellobiose molecules, which are hydrolyzed into glucose by beta-glucosidase. Unfortunately, degradation compounds generated during pretreatment inhibit the saccharification enzyme cocktail. Various research groups have identified specific classes of inhibitors formed during biomass pretreatment and have studied their inhibitory effect on the saccharification cocktail. These various research groups prepared surrogate solutions in an attempt to

Substrate-Related Factors Affecting Enzymatic Saccharification of Lignocelluloses: Our Recent Understanding

Treesearch

Shao-Yuan Leu; J.Y. Zhu

2013-01-01

Enzymatic saccharification of cellulose is a key step in conversion of plant biomass to advanced biofuel and chemicals. Many substrate-related factors affect saccharification. Rather than examining the role of each individual factor on overall saccharification efficiency, this study examined how each factor affects the three basic processes of a heterogeneous...

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.

The effect of nonenzymatic protein on lignocellulose enzymatic hydrolysis and simultaneous saccharification and fermentation.

PubMed

Wang, Hui; Kobayashi, Shinichi; Hiraide, Hatsue; Cui, Zongjun; Mochidzuki, Kazuhiro

2015-01-01

Nonenzymatic protein was added to cellulase hydrolysis and simultaneous saccharification and fermentation (SSF) of different biomass materials. Adding bovine serum albumin (BSA) and corn steep before cellulase enhanced enzyme activity in solution and increased cellulose and xylose conversion rates. The cellulose conversion rate of filter paper hydrolysis was increased by 32.5 % with BSA treatment. When BSA was added before cellulase, the remaining activity in the solution was higher than that in a control without BSA pretreatment. During SSF with pretreated rice straw as the substrate, adding 1.0 mg/mL BSA increased the ethanol yield by 13.6 % and final xylose yield by 42.6 %. The results indicated that lignin interaction is not the only mechanism responsible for the positive BSA effect. BSA had a stabilizing effect on cellulase and relieved cumulative sugar inhibition of enzymatic hydrolysis of biomass materials. Thus, nonenzymatic protein addition represents a promising strategy in the biorefining of lignocellulose materials.

Immobilization of indigenous holocellulase on iron oxide (Fe2O3) nanoparticles enhanced hydrolysis of alkali pretreated paddy straw.

PubMed

Kumar, Ajay; Singh, Surender; Tiwari, Rameshwar; Goel, Renu; Nain, Lata

2017-03-01

The holocellulase from Aspergillus niger SH3 was characterized and found to contain 125 proteins including cellulases (26), hemicellulases (21), chitinases (10), esterases (6), amylases (4) and hypothetical protein (32). The crude enzyme was immobilized on five different nanoparticles (NPs) via physical adsorption and covalent coupling methods. The enzyme-nanoparticle complexes (ENC) were screened for protein binding, enzymatic activities and immobilization efficiency. Magnetic enzyme-nanoparticle complexes (MENC) showed higher immobilization efficiency (60-80%) for most of the enzymes. MENC also showed better catalytic efficiencies in term of higher V max and lower K m than free enzyme. Saccharification yields from alkali treated paddy straw were higher (375.39mg/gds) for covalently immobilized MENC than free enzyme (339.99mg/gds). The immobilized enzyme was used for two cycles of saccharification with 55% enzyme recovery. Hence, this study for the first time demonstrated the immobilization of indigenous enzyme and its utilization for saccharification of paddy straw. Copyright © 2016 Elsevier B.V. All rights reserved.

Lignin as a facilitator, not a barrier, during saccharification by brown rot fungi

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

Schilling, Jonathan S.; Tschirner, Ulrike; Blanchette, Robert A

2012-11-28

This research focused on the biology of a group of wood-degrading fungi that cause brown rot in wood, with particular attention to the potential to mimic this biological approach ex situ for bioprocessing lignocellulosic biomass. Supported by the long-standing theory that these fungi use a two-step oxidative/enzymatic approach during brown rot, our team’s objectives were as follows: 1) to determine the discrete timing of lignin modifications, 2) to correlate these alterations with biocatalyst efficiency and ingress into plant cell walls, and 3) to reproduce modifications prior to saccharification for efficient bioprocessing. The core findings of our research were that 1)more » lignin modifications occur nearly coincident with enzyme secretion during brown rot and 2) there is no specificity to the benefit that a brown rot pretreatment has on the efficacy of cellulases – it is a general enhancement best predicted by chemical changes to lignin and side-chain hemicellulose sugars. In our work, this meant we could attain and predict broad improvements in saccharification using commercial cellulase cocktails, in some cases more than three-fold of that in untreated biomass. This project was completed with minimal variance from the original project management plan (PMP), resulting in fourteen presentations and posters, four peer-reviewed publications, and one additional publication now in review. The publications have been valuable to other scientists working toward similar goals and have been cited in thirteen peer-reviewed publications written by others since 2010. We are working with ADM to advance application options for industry, building on the lessons learned during this DOE award period.« less

Ethanol production from sunflower meal biomass by simultaneous saccharification and fermentation (SSF) with Kluyveromyces marxianus ATCC 36907.

PubMed

Camargo, Danielle; Gomes, Simone D; Sene, Luciane

2014-11-01

The lignocellulosic materials are considered promising renewable resources for ethanol production, but improvements in the processes should be studied to reduce operating costs. Thus, the appropriate enzyme loading for cellulose saccharification is critical for process economics. This study aimed at evaluating the concentration of cellulase and β-glucosidase in the production of bioethanol by simultaneous saccharification and fermentation (SSF) of sunflower meal biomass. The sunflower biomass was pretreated with 6% H2SO4 (w/v), at 121 °C, for 20 min, for hemicellulose removal and delignificated with 1% NaOH. SSF was performed with Kluyveromyces marxianus ATCC 36907, at 38 °C, 150 rpm, for 72 h, with different enzyme concentrations (Cellulase Complex NS22086-10, 15 and 20 FPU/gsubstrate and β-Glucosidase NS22118, with a cellulase to β-glucosidase ratio of 1.5:1; 2:1 and 3:1). The best condition for ethanol production was cellulase 20 FPU/gsubstrate and β-glucosidase 13.3 CBU/gsubstrate, resulting in 27.88 g/L ethanol, yield of 0.47 g/g and productivity of 0.38 g/L h. Under this condition the highest enzymatic conversion of cellulose to glucose was attained (87.06%).

Kinetic modeling of multi-feed simultaneous saccharification and co-fermentation of pretreated birch to ethanol.

PubMed

Wang, Ruifei; Koppram, Rakesh; Olsson, Lisbeth; Franzén, Carl Johan

2014-11-01

Fed-batch simultaneous saccharification and fermentation (SSF) is a feasible option for bioethanol production from lignocellulosic raw materials at high substrate concentrations. In this work, a segregated kinetic model was developed for simulation of fed-batch simultaneous saccharification and co-fermentation (SSCF) of steam-pretreated birch, using substrate, enzymes and cell feeds. The model takes into account the dynamics of the cellulase-cellulose system and the cell population during SSCF, and the effects of pre-cultivation of yeast cells on fermentation performance. The model was cross-validated against experiments using different feed schemes. It could predict fermentation performance and explain observed differences between measured total yeast cells and dividing cells very well. The reproducibility of the experiments and the cell viability were significantly better in fed-batch than in batch SSCF at 15% and 20% total WIS contents. The model can be used for simulation of fed-batch SSCF and optimization of feed profiles. Copyright © 2014 Elsevier Ltd. All rights reserved.

Enhanced production of bioethanol from waste of beer fermentation broth at high temperature through consecutive batch strategy by simultaneous saccharification and fermentation.

PubMed

Khattak, Waleed Ahmad; Khan, Taous; Ha, Jung Hwan; Ul-Islam, Mazhar; Kang, Min-Kyung; Park, Joong Kon

2013-10-10

Malt hydrolyzing enzymes and yeast glycolytic and fermentation enzymes in the waste from beer fermentation broth (WBFB) were identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). A new 'one-pot consecutive batch strategy' was developed for efficient bio-ethanol production by simultaneous saccharification and fermentation (SSF) using WBFB without additional enzymes, microbial cells, or carbohydrates. Bio-ethanol production was conducted in batches using WBFB supernatant in the first phase at 25-67°C and 50rpm, followed by the addition of 3% WBFB solid residue to the existing culture broth in the second phase at 67°C. The ethanol production increased from 50 to 102.5g/L when bare supernatant was used in the first phase, and then to 219g ethanol/L in the second phase. The amount of ethanol obtained using this strategy was almost equal to that obtained using the original WBFB containing 25% solid residue at 33°C, and more than double that obtained when bare supernatant was used. Microscopic and gel electrophoresis studies revealed yeast cell wall degradation and secretion of cellular material into the surrounding medium. Scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR) supported the existence of enzymes in WBFB involved in bioethanol production at elevated temperatures. The results of this study will provide insight for the development of new strategies for biofuel production. Copyright © 2013 Elsevier Inc. All rights reserved.

Thermoascus aurantiacus is a promising source of enzymes for biomass deconstruction under thermophilic conditions.

PubMed

McClendon, Shara D; Batth, Tanveer; Petzold, Christopher J; Adams, Paul D; Simmons, Blake A; Singer, Steven W

2012-07-28

Thermophilic fungi have attracted increased interest for their ability to secrete enzymes that deconstruct biomass at high temperatures. However, development of thermophilic fungi as enzyme producers for biomass deconstruction has not been thoroughly investigated. Comparing the enzymatic activities of thermophilic fungal strains that grow on targeted biomass feedstocks has the potential to identify promising candidates for strain development. Thielavia terrestris and Thermoascus aurantiacus were chosen for characterization based on literature precedents. Thermoascus aurantiacus and Thielavia terrestris were cultivated on various biomass substrates and culture supernatants assayed for glycoside hydrolase activities. Supernatants from both cultures possessed comparable glycoside hydrolase activities when incubated with artificial biomass substrates. In contrast, saccharifications of ionic liquid pretreated switchgrass (Panicum virgatum) revealed that T. aurantiacus enzymes released more glucose than T. terrestris enzymes over a range of protein mass loadings and temperatures. Temperature-dependent saccharifications demonstrated that the T. aurantiacus proteins retained higher levels of activity compared to a commercial enzyme mixture sold by Novozymes, Cellic CTec2, at elevated temperatures. Enzymes secreted by T. aurantiacus released glucose at similar protein loadings to CTec2 on dilute acid, ammonia fiber expansion, or ionic liquid pretreated switchgrass. Proteomic analysis of the T. aurantiacus culture supernatant revealed dominant glycoside hydrolases from families 5, 7, 10, and 61, proteins that are key enzymes in commercial cocktails. T. aurantiacus produces a complement of secreted proteins capable of higher levels of saccharification of pretreated switchgrass than T. terrestris enzymes. The T. aurantiacus enzymatic cocktail performs at the same level as commercially available enzymatic cocktail for biomass deconstruction, without strain development or

Thermoascus aurantiacus is a promising source of enzymes for biomass deconstruction under thermophilic conditions

PubMed Central

2012-01-01

Background Thermophilic fungi have attracted increased interest for their ability to secrete enzymes that deconstruct biomass at high temperatures. However, development of thermophilic fungi as enzyme producers for biomass deconstruction has not been thoroughly investigated. Comparing the enzymatic activities of thermophilic fungal strains that grow on targeted biomass feedstocks has the potential to identify promising candidates for strain development. Thielavia terrestris and Thermoascus aurantiacus were chosen for characterization based on literature precedents. Results Thermoascus aurantiacus and Thielavia terrestris were cultivated on various biomass substrates and culture supernatants assayed for glycoside hydrolase activities. Supernatants from both cultures possessed comparable glycoside hydrolase activities when incubated with artificial biomass substrates. In contrast, saccharifications of ionic liquid pretreated switchgrass (Panicum virgatum) revealed that T. aurantiacus enzymes released more glucose than T. terrestris enzymes over a range of protein mass loadings and temperatures. Temperature-dependent saccharifications demonstrated that the T. aurantiacus proteins retained higher levels of activity compared to a commercial enzyme mixture sold by Novozymes, Cellic CTec2, at elevated temperatures. Enzymes secreted by T. aurantiacus released glucose at similar protein loadings to CTec2 on dilute acid, ammonia fiber expansion, or ionic liquid pretreated switchgrass. Proteomic analysis of the T. aurantiacus culture supernatant revealed dominant glycoside hydrolases from families 5, 7, 10, and 61, proteins that are key enzymes in commercial cocktails. Conclusions T. aurantiacus produces a complement of secreted proteins capable of higher levels of saccharification of pretreated switchgrass than T. terrestris enzymes. The T. aurantiacus enzymatic cocktail performs at the same level as commercially available enzymatic cocktail for biomass deconstruction

High-throughput Saccharification assay for lignocellulosic materials.

PubMed

Gomez, Leonardo D; Whitehead, Caragh; Roberts, Philip; McQueen-Mason, Simon J

2011-07-03

Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be used in establishing a biorefinery. These raw materials would constitute a new industrial platform, which is both sustainable and carbon neutral, to replace the current dependency on fossil fuel. The recalcitrance to deconstruction observed in lignocellulosic materials is produced by several intrinsic properties of plant cell walls. Crystalline cellulose is embedded in matrix polysaccharides such as xylans and arabinoxylans, and the whole structure is encased by the phenolic polymer lignin, that is also difficult to digest (1). In order to improve the digestibility of plant materials we need to discover the main bottlenecks for the saccharification of cell walls and also screen mutant and breeding populations to evaluate the variability in saccharification (2). These tasks require a high throughput approach and here we present an analytical platform that can perform saccharification analysis in a 96-well plate format. This platform has been developed to allow the screening of lignocellulose digestibility of large populations from varied plant species. We have scaled down the reaction volumes for gentle pretreatment, partial enzymatic hydrolysis and sugar determination, to allow large numbers to be assessed rapidly in an automated system. This automated platform works with milligram amounts of biomass, performing ball milling under controlled conditions to reduce the plant materials to a standardised particle size in a reproducible manner. Once the samples are ground, the automated formatting robot dispenses specified and recorded amounts of material into the corresponding wells of 96 deep well plate (Figure 1). Normally, we dispense the same material into 4 wells to have 4 replicates for analysis. Once the plates are filled with the plant material in the desired layout, they are manually moved to a liquid handling station (Figure 2

Rapid and Complete Enzyme Hydrolysis of Lignocellulosic Nanofibrils

Treesearch

Raquel Martin-Sampedro; Ilari Filpponen; Ingrid C. Hoeger; J.Y. Zhu; Janne Laine; Orlando J. Rojas

2012-01-01

Rapid enzymatic saccharification of lignocellulosic nanofibrils (LCNF) was investigated by monitoring nanoscale changes in mass via quartz crystal microgravimetry and also by measuring reducing sugar yields. In only a few minutes LCNF thin films were completely hydrolyzed upon incubation in multicomponent enzyme systems. Conversion to sugars and oligosaccharides of...

Acetone-butanol-ethanol production from Kraft paper mill sludge by simultaneous saccharification and fermentation.

PubMed

Guan, Wenjian; Shi, Suan; Tu, Maobing; Lee, Yoon Y

2016-01-01

Paper mill sludge (PS), a solid waste from pulp and paper industry, was investigated as a feedstock for acetone-butanol-ethanol (ABE) production by simultaneous saccharification and fermentation (SSF). ABE fermentation of paper sludge by Clostridium acetobutylicum required partial removal of ash in PS to enhance its enzymatic digestibility. Enzymatic hydrolysis was found to be a rate-limiting step in the SSF. A total of 16.4-18.0g/L of ABE solvents were produced in the SSF of de-ashed PS with solid loading of 6.3-7.4% and enzyme loading of 10-15FPU/g-glucan, and the final solvent yield reached 0.27g/g sugars. No pretreatment and pH control were needed in ABE fermentation of paper sludge, which makes it an attractive feedstock for butanol production. The results suggested utilization of paper sludge should not only consider the benefits of buffering effect of CaCO3 in fermentation, but also take into account its inhibitory effect on enzymatic hydrolysis. Published by Elsevier Ltd.

Characterization of cellulolytic extract from Pycnoporus sanguineus PF-2 and its application in biomass saccharification.

PubMed

Falkoski, Daniel Luciano; Guimarães, Valéria Monteze; de Almeida, Maíra Nicolau; Alfenas, Acelino Couto; Colodette, Jorge Luiz; de Rezende, Sebastião Tavares

2012-03-01

The aim of this work was to evaluate the biochemical features of the white-rot fungi Pycnoporus sanguineus cellulolytic complex and its utilization to sugarcane bagasse hydrolysis. When cultivated under submerged fermentation using corn cobs as carbon source, P. sanguineus produced high FPase, endoglucanase, β-glucosidase, xylanase, mannanase, α-galactosidase, α-arabinofuranosidase, and polygalacturonase activities. Cellulase activities were characterized in relation to pH and temperature. β-Glucosidase and FPase activities were higher at 55 °C, pH 4.5, and endoglucanase activity was higher at 60 °C, in a pH range of 3.5-4.0. All cellulase activities were highly stable at 40 and 50 °C through 48 h of pre-incubation. Crude enzymatic extract from P. sanguineus was applied in a saccharification experiment using acid-treated and alkali-treated sugarcane bagasse as substrate, and the hydrolysis yields were compared to that obtained by a commercial cellulase preparation. Reducing sugar yields of 60.4% and 64.0% were reached when alkali-treated bagasse was hydrolyzed by P. sanguineus extract and commercial cellulase, respectively. Considering the glucose production, it was observed that P. sanguineus extract and commercial cellulase ensured yields of 22.6% and 36.5%, respectively. The saccharification of acid-treated bagasse was lower than that of alkali-treated bagasse regardless of the cellulolytic extract. The present work showed that P. sanguineus has a great potential as an enzyme producer for biomass saccharification.

Impact of Pretreatment Technologies on Saccharification and Isopentenol Fermentation of Mixed Lignocellulosic Feedstocks

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

Shi, Jian; George, Kevin W.; Sun, Ning

2015-02-28

In order to enable the large-scale production of biofuels or chemicals from lignocellulosic biomass, a consistent and affordable year-round supply of lignocellulosic feedstocks is essential. Feedstock blending and/or densification offers one promising solution to overcome current challenges on biomass supply, i.e., low energy and bulk densities and significant compositional variations. Therefore, it is imperative to develop conversion technologies that can process mixed pelleted biomass feedstocks with minimal negative impact in terms of overall performance of the relevant biorefinery unit operations: pretreatment, fermentable sugar production, and fuel titers. We processed the mixture of four feedstocks—corn stover, switchgrass, lodgepole pine, and eucalyptusmore » (1:1:1:1 on dry weight basis)—in flour and pellet form using ionic liquid (IL) 1-ethyl-3-methylimidazolium acetate, dilute sulfuric acid (DA), and soaking in aqueous ammonia (SAA) pretreatments. Commercial enzyme mixtures, including cellulases and hemicellulases, were then applied to these pretreated feedstocks at low to moderate enzyme loadings to determine hydrolysis efficiency. Results show significant variations on the chemical composition, crystallinity, and enzymatic digestibility of the pretreated feedstocks across the different pretreatment technologies studied. The advanced biofuel isopentenol was produced during simultaneous saccharification and fermentation (SSF) of pretreated feedstocks using an engineered Escherichia coli strain. Results show that IL pretreatment liberates the most sugar during enzymatic saccharification, and in turn led to the highest isopentenol titer as compared to DA and SAA pretreatments. This study provides insights on developing biorefinery technologies that produce advanced biofuels based on mixed feedstock streams.« less

Associating cooking additives with sodium hydroxide to pretreat bamboo residues for improving the enzymatic saccharification and monosaccharides production.

PubMed

Huang, Caoxing; He, Juan; Wang, Yan; Min, Douyong; Yong, Qiang

2015-10-01

Cooking additive pulping technique is used in kraft mill to increase delignification degree and pulp yield. In this work, cooking additives were firstly applied in the sodium hydroxide pretreatment for improving the bioconversion of bamboo residues to monosaccharides. Meanwhile, steam explosion and sulfuric acid pretreatments were also carried out on the sample to compare their impacts on monosaccharides production. Results indicated that associating anthraquinone with sodium hydroxide pretreatment showed the best performance in improving the original carbohydrates recovery, delignification, enzymatic saccharification, and monosaccharides production. After consecutive pretreatment and enzymatic saccharification process, 347.49 g, 307.48 g, 142.93 g, and 87.15 g of monosaccharides were released from 1000 g dry bamboo residues pretreated by sodium hydroxide associating with anthraquinone, sodium hydroxide, steam explosion and sulfuric acid, respectively. The results suggested that associating cooking additive with sodium hydroxide is an effective pretreatment for bamboo residues to enhance enzymatic saccharification for monosaccharides production. Copyright © 2015 Elsevier Ltd. All rights reserved.

High titer ethanol production from simultaneous enzymatic saccharification and fermentation of aspen at high solids : a comparison between SPORL and dilute acid pretreatments

Treesearch

J.Y. Zhu; R. Gleisner; C.T. Scott; X.L. Luo; S. Tian

2011-01-01

Native aspen (Populus tremuloides) was pretreated using sulfuric acid and sodium bisulfite (SPORL) and dilute sulfuric acid alone (DA). Simultaneous enzymatic saccharification and fermentation (SSF) was conducted at 18% solids using commercial enzymes with cellulase loadings ranging from 6 to 15 FPU/g glucan and Saccharomyces cerevisiae Y5. Compared with DA...

Fungal cellulases as an aid for the saccharification of cassava

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

De Menezes, T.J.B.; Arakaki, T.; DeLamo, P.R.

1978-01-01

Culture broths of cellulolytic fungi were used together with commercial amylases to enhance the saccharification of cassava starch slurry. The addition of appropriate concentration of cellulase from Trichoderma viride and from a basidiomycete from soil increased both the rate of sugar formation and the degree of solubilization and decreased the viscosity of the hydrolyzates. Owing to the improvement of the rhetorical properties of the must and the additional sugar produced, an increased ethanol yield would be expected from the alcohol fermentation of this hydrolyzate.

Assessment of integrated process based on hydrothermal and alkaline treatments for enzymatic saccharification of sweet sorghum stems.

PubMed

Sun, Shao-Long; Sun, Shao-Ni; Wen, Jia-Long; Zhang, Xue-Ming; Peng, Feng; Sun, Run-Cang

2015-01-01

In this study, sweet sorghum stem was subjected to hydrothermal pretreatment (HTP) and alkaline post-treatment to enhance its saccharification ratio by reducing its recalcitrance. The results showed that the HTP (110-210°C, 0.5-2.0h) significantly degraded hemicelluloses, and the pretreatment at the temperature higher than 190°C led to the partial degradation of the cellulose. As compared to the sole HTP, the integrated process removed most of lignin and hemicelluloses, which incurred a higher cellulose saccharification ratio. Under an optimum condition evaluated (HTP at 170°C for 0.5h and subsequent 2% NaOH treatment), 77.5% saccharification ratio was achieved, which was 1.8, 2.0 and 5.5 times as compared to the only HTP pretreated substrates, alkaline treated substrates alone and the raw material without pretreatment, respectively. Clearly, the integrated process can be considered as a promising approach to achieve an efficient conversion of lignocellulose to fermentable glucose. Copyright © 2014 Elsevier Ltd. All rights reserved.

Controlled expression of pectic enzymes in Arabidopsis thaliana enhances biomass conversion without adverse effects on growth.

PubMed

Tomassetti, Susanna; Pontiggia, Daniela; Verrascina, Ilaria; Reca, Ida Barbara; Francocci, Fedra; Salvi, Gianni; Cervone, Felice; Ferrari, Simone

2015-04-01

Lignocellulosic biomass from agriculture wastes is a potential source of biofuel, but its use is currently limited by the recalcitrance of the plant cell wall to enzymatic digestion. Modification of the wall structural components can be a viable strategy to overcome this bottleneck. We have previously shown that the expression of a fungal polygalacturonase (pga2 from Aspergillus niger) in Arabidopsis and tobacco plants reduces the levels of de-esterified homogalacturonan in the cell wall and significantly increases saccharification efficiency. However, plants expressing pga2 show stunted growth and reduced biomass production, likely as a consequence of an extensive loss of pectin integrity during the whole plant life cycle. We report here that the expression in Arabidopsis of another pectic enzyme, the pectate lyase 1 (PL1) of Pectobacterium carotovorum, under the control of a chemically inducible promoter, results, after induction of the transgene, in a saccharification efficiency similar to that of plants expressing pga2. However, lines with high levels of transgene induction show reduced growth even in the absence of the inducer. To overcome the problem of plant fitness, we have generated Arabidopsis plants that express pga2 under the control of the promoter of SAG12, a gene expressed only during senescence. These plants expressed pga2 only at late stages of development, and their growth was comparable to that of WT plants. Notably, leaves and stems of transgenic plants were more easily digested by cellulase, compared to WT plants, only during senescence. Expression of cell wall-degrading enzymes at the end of the plant life cycle may be therefore a useful strategy to engineer crops unimpaired in biomass yield but improved for bioconversion. Copyright © 2014 Elsevier Ltd. All rights reserved.

OsCESA9 conserved-site mutation leads to largely enhanced plant lodging resistance and biomass enzymatic saccharification by reducing cellulose DP and crystallinity in rice

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

Li, Fengcheng; Xie, Guosheng; Huang, Jiangfeng

Genetic modification of plant cell walls has been posed to reduce lignocellulose recalcitrance for enhancing biomass saccharification. Several dozen CESA mutants have been reported since cellulose synthase (CESA) gene was first identified, but almost all mutants exhibit the defective phenotypes in plant growth and development. Here, the rice (Oryza sativa) Osfc16 mutant with substitutions (W481C, P482S) at P-CR conserved site in CESA9 shows a slightly affected plant growth and higher biomass yield by 25%–41% compared with wild type (Nipponbare, a japonica variety). Chemical and ultrastructural analyses indicate that Osfc16 has a significantly reduced cellulose crystallinity (CrI) and thinner secondary cellmore » walls compared with wild type. CESA co-IP detection, together with implementations of a proteasome inhibitor (MG132) and two distinct cellulose inhibitors (Calcofluor, CGA), shows that CESA9 mutation could affect integrity of CESA4/7/9 complexes, which may lead to rapid CESA proteasome degradation for low-DP cellulose biosynthesis. These may reduce cellulose CrI, which improves plant lodging resistance, a major and integrated agronomic trait on plant growth and grain production, and enhances biomass enzymatic saccharification by up to 2.3-fold and ethanol productivity by 34%–42%. Our study has for the first time reported a direct modification for the low-DP cellulose production that has broad applications in biomass industries.« less

OsCESA9 conserved-site mutation leads to largely enhanced plant lodging resistance and biomass enzymatic saccharification by reducing cellulose DP and crystallinity in rice

DOE PAGES

Li, Fengcheng; Xie, Guosheng; Huang, Jiangfeng; ...

2017-03-15

Genetic modification of plant cell walls has been posed to reduce lignocellulose recalcitrance for enhancing biomass saccharification. Several dozen CESA mutants have been reported since cellulose synthase (CESA) gene was first identified, but almost all mutants exhibit the defective phenotypes in plant growth and development. Here, the rice (Oryza sativa) Osfc16 mutant with substitutions (W481C, P482S) at P-CR conserved site in CESA9 shows a slightly affected plant growth and higher biomass yield by 25%–41% compared with wild type (Nipponbare, a japonica variety). Chemical and ultrastructural analyses indicate that Osfc16 has a significantly reduced cellulose crystallinity (CrI) and thinner secondary cellmore » walls compared with wild type. CESA co-IP detection, together with implementations of a proteasome inhibitor (MG132) and two distinct cellulose inhibitors (Calcofluor, CGA), shows that CESA9 mutation could affect integrity of CESA4/7/9 complexes, which may lead to rapid CESA proteasome degradation for low-DP cellulose biosynthesis. These may reduce cellulose CrI, which improves plant lodging resistance, a major and integrated agronomic trait on plant growth and grain production, and enhances biomass enzymatic saccharification by up to 2.3-fold and ethanol productivity by 34%–42%. Our study has for the first time reported a direct modification for the low-DP cellulose production that has broad applications in biomass industries.« less

Simultaneous Saccharification and Fermentation and Partial Saccharification and Co-Fermentation of Lignocellulosic Biomass for Ethanol Production

NASA Astrophysics Data System (ADS)

Doran-Peterson, Joy; Jangid, Amruta; Brandon, Sarah K.; Decrescenzo-Henriksen, Emily; Dien, Bruce; Ingram, Lonnie O.

Ethanol production by fermentation of lignocellulosic biomass-derived sugars involves a fairly ancient art and an ever-evolving science. Production of ethanol from lignocellulosic biomass is not avant-garde, and wood ethanol plants have been in existence since at least 1915. Most current ethanol production relies on starch- and sugar-based crops as the substrate; however, limitations of these materials and competing value for human and animal feeds is renewing interest in lignocellulose conversion. Herein, we describe methods for both simultaneous saccharification and fermentation (SSF) and a similar but separate process for partial saccharification and cofermentation (PSCF) of lignocellulosic biomass for ethanol production using yeasts or pentose-fermenting engineered bacteria. These methods are applicable for small-scale preliminary evaluations of ethanol production from a variety of biomass sources.

Lignin biosynthesis perturbations affect secondary cell wall composition and saccharification yield in Arabidopsis thaliana

PubMed Central

2013-01-01

Background Second-generation biofuels are generally produced from the polysaccharides in the lignocellulosic plant biomass, mainly cellulose. However, because cellulose is embedded in a matrix of other polysaccharides and lignin, its hydrolysis into the fermentable glucose is hampered. The senesced inflorescence stems of a set of 20 Arabidopsis thaliana mutants in 10 different genes of the lignin biosynthetic pathway were analyzed for cell wall composition and saccharification yield. Saccharification models were built to elucidate which cell wall parameters played a role in cell wall recalcitrance. Results Although lignin is a key polymer providing the strength necessary for the plant’s ability to grow upward, a reduction in lignin content down to 64% of the wild-type level in Arabidopsis was tolerated without any obvious growth penalty. In contrast to common perception, we found that a reduction in lignin was not compensated for by an increase in cellulose, but rather by an increase in matrix polysaccharides. In most lignin mutants, the saccharification yield was improved by up to 88% cellulose conversion for the cinnamoyl-coenzyme A reductase1 mutants under pretreatment conditions, whereas the wild-type cellulose conversion only reached 18%. The saccharification models and Pearson correlation matrix revealed that the lignin content was the main factor determining the saccharification yield. However, also lignin composition, matrix polysaccharide content and composition, and, especially, the xylose, galactose, and arabinose contents influenced the saccharification yield. Strikingly, cellulose content did not significantly affect saccharification yield. Conclusions Although the lignin content had the main effect on saccharification, also other cell wall factors could be engineered to potentially increase the cell wall processability, such as the galactose content. Our results contribute to a better understanding of the effect of lignin perturbations on plant cell

Production and purification of amylolytic enzymes for saccharification of microalgal biomass.

PubMed

Rodrigues, Éllen Francine; Ficanha, Aline Matuella Moreira; Dallago, Rogério Marcos; Treichel, Helen; Reinehr, Christian Oliveira; Machado, Tainara Paula; Nunes, Greice Borges; Colla, Luciane Maria

2017-02-01

The aim of this study was the production of amylolytic enzymes by solid state or submerged fermentations (SSF or SF, respectively), followed by purification using chemical process or microfiltration and immobilization of purified enzymes in a polyurethane support. The free and immobilized enzymes obtained were used to evaluate enzymatic hydrolysis of the polysaccharides of Spirulina. Microfiltration of the crude extracts resulted in an increase in their specific activity and thermal stability at 40°C and 50°C for 24h, as compared to extracts obtained by SSF and SF. Immobilization of polyurethane purified enzyme produced yields of 332% and 205% for the enzymes obtained by SF and SSF, respectively. Free or immobilized enzymes favor the generation of fermentable sugar, being the application of the purified and immobilized enzymes in the hydrolysis of microalgal polysaccharides considered a promising alternative towards development of the bioethanol production process from microalgal biomass. Copyright © 2016 Elsevier Ltd. All rights reserved.

Linkage Mapping of Stem Saccharification Digestibility in Rice

PubMed Central

Hua, Cangmei; Sun, Lili; Ali, Imran; Huang, Linli; Yu, Chunyan; Simister, Rachael; Steele-King, Clare; Gan, Yinbo; McQueen-Mason, Simon J.

2016-01-01

Rice is the staple food of almost half of the world population, and in excess 90% of it is grown and consumed in Asia, but the disposal of rice straw poses a problem for farmers, who often burn it in the fields, causing health and environmental problems. However, with increased focus on the development of sustainable biofuel production, rice straw has been recognized as a potential feedstock for non-food derived biofuel production. Currently, the commercial realization of rice as a biofuel feedstock is constrained by the high cost of industrial saccharification processes needed to release sugar for fermentation. This study is focused on the alteration of lignin content, and cell wall chemotypes and structures, and their effects on the saccharification potential of rice lignocellulosic biomass. A recombinant inbred lines (RILs) population derived from a cross between the lowland rice variety IR1552 and the upland rice variety Azucena with 271 molecular markers for quantitative trait SNP (QTS) analyses was used. After association analysis of 271 markers for saccharification potential, 1 locus and 4 pairs of epistatic loci were found to contribute to the enzymatic digestibility phenotype, and an inverse relationship between reducing sugar and lignin content in these recombinant inbred lines was identified. As a result of QTS analyses, several cell-wall associated candidate genes are proposed that may be useful for marker-assisted breeding and may aid breeders to produce potential high saccharification rice varieties. PMID:27415441

Simultaneous saccharification and fermentation of fungal pretreated cornstalk for hydrogen production using Thermoanaerobacterium thermosaccharolyticum W16.

PubMed

Zhao, Lei; Cao, Guang-Li; Wang, Ai-Jie; Guo, Wan-Qian; Ren, Hong-Yu; Ren, Nan-Qi

2013-10-01

In this research, environmentally friendly fungal pretreatment was first adopted for deconstruction of cornstalk. Then the fungal-pretreated cornstalk was employed to produce hydrogen in simultaneous saccharification and fermentation (SSF) using crude enzyme from Trichoderma viride and Thermoanaerobacterium thermosaccharolyticum W16. The influence of various factors including substrate concentration, initial pH, and enzyme loading on hydrogen production were evaluated. The highest hydrogen yield of 89.3 ml/g-cornstalk was obtained with an initial pH 6.5, 0.75% substrate concentration, and 34 FPU/g cellulose. Compared the result with SSF of physical or chemical pretreated lignocellulosic materials, this research suggested an economic and efficient way for hydrogen production from lignocellulosic biomass. Copyright © 2013 Elsevier Ltd. All rights reserved.

Mechanistic insight into ultrasound induced enhancement of simultaneous saccharification and fermentation of Parthenium hysterophorus for ethanol production.

PubMed

Singh, Shuchi; Agarwal, Mayank; Sarma, Shyamali; Goyal, Arun; Moholkar, Vijayanand S

2015-09-01

This paper presents investigations into mechanism of ultrasound assisted bioethanol synthesis using Parthenium hysterophorus biomass through simultaneous saccharification and fermentation (SSF) mode. Approach of coupling experimental results to mathematical model for SSF using Genetic Algorithm based optimization has been adopted. Comparison of model parameters for experiments with mechanical shaking and sonication (10% duty cycle) give an interesting mechanistic account of influence of ultrasound on SSF system. A 4-fold rise in ethanol and cell mass productivity is seen with ultrasound. The analysis reveals following facets of influence of ultrasound on SSF: increase in Monod constant for glucose for cell growth, maximal specific growth rate and inhibition constant of cell growth by glucose and reduction in specific cell death rate. Values of inhibition constant of cell growth by ethanol (K3E), and constants for growth associated (a) and non-growth associated (b) ethanol production remained unaltered with sonication. Beneficial effects of ultrasound are attributed to enhanced cellulose hydrolysis, enhanced trans-membrane transport of substrate and products as well as dilution of the toxic substances due to micro-convection induced by ultrasound. Intrinsic physiological functioning of cells remained unaffected by ultrasound as indicated by unaltered values of K3E, a and b. Copyright © 2015 Elsevier B.V. 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.

Microwave-assisted inorganic salt pretreatment of sugarcane leaf waste: Effect on physiochemical structure and enzymatic saccharification.

PubMed

Moodley, Preshanthan; Kana, E B Gueguim

2017-07-01

This paper presents a method to pretreat sugarcane leaf waste using microwave-assisted (MA) inorganic salt to enhance enzymatic saccharification. The effects of process parameters of salt concentration, microwave power intensity and pretreatment time on reducing sugar yield from sugarcane leaf waste were investigated. Pretreatment models based on MA-NaCl, MA-ZnCl 2 and MA-FeCl 3 were developed with high coefficients of determination (R 2 >0.8) and optimized. Maximum reducing sugar yield of 0.406g/g was obtained with 2M FeCl 3 at 700W for 3.5min. Scanning electron microscopy (SEM), Fourier Transform Infrared analysis (FTIR) and X-ray diffraction (XRD) showed major changes in lignocellulosic structure after MA-FeCl 3 pretreatment with 71.5% hemicellulose solubilization. This regime was further assessed on sorghum leaves and Napier grass under optimal MA-FeCl 3 conditions. A 2-fold and 3.1-fold increase in sugar yield respectively were observed compared to previous reports. This pretreatment was highly effective for enhancing enzymatic saccharification of lignocellulosic biomass. Copyright © 2017. Published by Elsevier Ltd.

Incorporation of flavonoid derivatives or pentagalloyl glucose into lignin enhances cell wall saccharification following mild alkaline or acidic pretreatments

USDA-ARS?s Scientific Manuscript database

Partial substitution of normal monolignols with phenolic precursors from other metabolic pathways may improve the susceptibility of lignified biomass to chemical pretreatment and enzymatic saccharification for biofuel production. Flavonoids and gallate esters readily undergo oxidative coupling react...

Pipeline transport and simultaneous saccharification of corn stover.

PubMed

Kumar, Amit; Cameron, Jay B; Flynn, Peter C

2005-05-01

Pipeline transport of corn stover delivered by truck from the field is evaluated against a range of truck transport costs. Corn stover transported by pipeline at 20% solids concentration (wet basis) or higher could directly enter an ethanol fermentation plant, and hence the investment in the pipeline inlet end processing facilities displaces comparable investment in the plant. At 20% solids, pipeline transport of corn stover costs less than trucking at capacities in excess of 1.4 M drytonnes/yr when compared to a mid range of truck transport cost (excluding any credit for economies of scale achieved in the ethanol fermentation plant from larger scale due to multiple pipelines). Pipelining of corn stover gives the opportunity to conduct simultaneous transport and saccharification (STS). If current enzymes are used, this would require elevated temperature. Heating of the slurry for STS, which in a fermentation plant is achieved from waste heat, is a significant cost element (more than 5 cents/l of ethanol) if done at the pipeline inlet unless waste heat is available, for example from an electric power plant located adjacent to the pipeline inlet. Heat loss in a 1.26 m pipeline carrying 2 M drytonnes/yr is about 5 degrees C at a distance of 400 km in typical prairie clay soils, and would not likely require insulation; smaller pipelines or different soil conditions might require insulation for STS. Saccharification in the pipeline would reduce the need for investment in the fermentation plant, saving about 0.2 cents/l of ethanol. Transport of corn stover in multiple pipelines offers the opportunity to develop a large ethanol fermentation plant, avoiding some of the diseconomies of scale that arise from smaller plants whose capacities are limited by issues of truck congestion.

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.

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.

How cell wall complexity influences saccharification efficiency in Miscanthus sinensis

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

De Souza, Amanda P.; Kamei, Claire L. Alvim; Torres, Andres F.

The production of bioenergy from grasses has been developing quickly during the last decade, with Miscanthus being among the most important choices for production of bioethanol. However, one of the key barriers to producing bioethanol is the lack of information about cell wall structure. Cell walls are thought to display compositional differences that lead to emergence of a very high level of complexity, resulting in great diversity in cell wall architectures. In this work, a set of different techniques was used to access the complexity of cell walls of different genotypes of Miscanthus sinensis in order to understand how theymore » interfere with saccharification efficiency. Three genotypes of M. sinensis displaying different patterns of correlation between lignin content and saccharification efficiency were subjected to cell wall analysis by quantitative/qualitative analytical techniques such as monosaccharide composition, oligosaccharide profiling, and glycome profiling. When saccharification efficiency was correlated negatively with lignin, the structural features of arabinoxylan and xyloglucan were found to contribute positively to hydrolysis. In the absence of such correlation, different types of pectins, and some mannans contributed to saccharification efficiency. In conclusion, different genotypes of M. sinensis were shown to display distinct interactions among their cell wall components, which seem to influence cell wall hydrolysis.« less

How cell wall complexity influences saccharification efficiency in Miscanthus sinensis

DOE PAGES

De Souza, Amanda P.; Kamei, Claire L. Alvim; Torres, Andres F.; ...

2015-04-23

The production of bioenergy from grasses has been developing quickly during the last decade, with Miscanthus being among the most important choices for production of bioethanol. However, one of the key barriers to producing bioethanol is the lack of information about cell wall structure. Cell walls are thought to display compositional differences that lead to emergence of a very high level of complexity, resulting in great diversity in cell wall architectures. In this work, a set of different techniques was used to access the complexity of cell walls of different genotypes of Miscanthus sinensis in order to understand how theymore » interfere with saccharification efficiency. Three genotypes of M. sinensis displaying different patterns of correlation between lignin content and saccharification efficiency were subjected to cell wall analysis by quantitative/qualitative analytical techniques such as monosaccharide composition, oligosaccharide profiling, and glycome profiling. When saccharification efficiency was correlated negatively with lignin, the structural features of arabinoxylan and xyloglucan were found to contribute positively to hydrolysis. In the absence of such correlation, different types of pectins, and some mannans contributed to saccharification efficiency. In conclusion, different genotypes of M. sinensis were shown to display distinct interactions among their cell wall components, which seem to influence cell wall hydrolysis.« less

Kinetic modeling of simultaneous saccharification and fermentation of corn starch for ethanol production.

PubMed

Białas, Wojciech; Czerniak, Adrian; Szymanowska-Powałowska, Daria

2014-01-01

Fuel ethanol production, using a simultaneous saccharification and fermentation process (SSF) of native starch from corn flour, has been performed using Saccharomyces cerevisiae and a granular starch hydrolyzing enzyme. The quantitative effects of mash concentration, enzyme dose and pH were investigated with the use of a Box-Wilson central composite design protocol. Proceeding from results obtained in optimal fermentation conditions, a kinetics model relating the utilization rates of starch and glucose as well as the production rates of ethanol and biomass was tested. Moreover, scanning electron microscopy (SEM) was applied to investigate corn starch granule surface after the SFF process. A maximum ethanol concentration of 110.36 g/l was obtained for native corn starch using a mash concentration of 25%, which resulted in ethanol yield of 85.71%. The optimal conditions for the above yield were found with an enzyme dose of 2.05 ml/kg and pH of 5.0. These results indicate that by using a central composite design, it is possible to determine optimal values of the fermentation parameters for maximum ethanol production. The investigated kinetics model can be used to describe SSF process conducted with granular starch hydrolyzing enzymes. The SEM micrographs reveal randomly distributed holes on the surface of granules.

Statistical optimization of recycled-paper enzymatic hydrolysis for simultaneous saccharification and fermentation via central composite design.

PubMed

Liu, Qing; Cheng, Ke-ke; Zhang, Jian-an; Li, Jin-ping; Wang, Ge-hua

2010-01-01

A central composite design of the response surface methodology (RSM) was employed to study the effects of temperature, enzyme concentration, and stirring rate on recycled-paper enzymatic hydrolysis. Among the three variables, temperature and enzyme concentration significantly affected the conversion efficiency of substrate, whereas stirring rate was not effective. A quadratic polynomial equation was obtained for enzymatic hydrolysis by multiple regression analysis using RSM. The results of validation experiments were coincident with the predicted model. The optimum conditions for enzymatic hydrolysis were temperature, enzyme concentration, and stirring rate of 43.1 degrees C, 20 FPU g(-1) substrate, and 145 rpm, respectively. In the subsequent simultaneous saccharification and fermentation (SSF) experiment under the optimum conditions, the highest 28.7 g ethanol l(-1) was reached in the fed-batch SSF when 5% (w/v) substrate concentration was used initially, and another 5% added after 12 h fermentation. This ethanol output corresponded to 77.7% of the theoretical yield based on the glucose content in the raw material.

Saccharification of rice straw by cellulase from a local Trichoderma harzianum SNRS3 for biobutanol production.

PubMed

Rahnama, Nooshin; Foo, Hooi Ling; Abdul Rahman, Nor Aini; Ariff, Arbakariya; Md Shah, Umi Kalsom

2014-12-12

Rice straw has shown to be a promising agricultural by-product in the bioconversion of biomass to value-added products. Hydrolysis of cellulose, a main constituent of lignocellulosic biomass, is a requirement for fermentable sugar production and its subsequent bioconversion to biofuels such as biobutanol. The high cost of commercial enzymes is a major impediment to the industrial application of cellulases. Therefore, the use of local microbial enzymes has been suggested. Trichoderma harzianum strains are potential CMCase and β-glucosidase producers. However, few researches have been reported on cellulase production by T. harzianum and the subsequent use of the crude cellulase for cellulose enzymatic hydrolysis. For cellulose hydrolysis to be efficiently performed, the presence of the whole set of cellulase components including exoglucanase, endoglucanase, and β-glucosidase at a considerable concentration is required. Biomass recalcitrance is also a bottleneck in the bioconversion of agricultural residues to value-added products. An effective pretreatment could be of central significance in the bioconversion of biomass to biofuels. Rice straw pretreated using various concentrations of NaOH was subjected to enzymatic hydrolysis. The saccharification of rice straw pretreated with 2% (w/v) NaOH using crude cellulase from local T. harzianum SNRS3 resulted in the production of 29.87 g/L reducing sugar and a yield of 0.6 g/g substrate. The use of rice straw hydrolysate as carbon source for biobutanol fermentation by Clostridium acetobutylicum ATCC 824 resulted in an ABE yield, ABE productivity, and biobutanol yield of 0.27 g/g glucose, 0.04 g/L/h and 0.16 g/g glucose, respectively. As a potential β-glucosidase producer, T. harzianum SNRS3 used in this study was able to produce β-glucosidase at the activity of 173.71 U/g substrate. However, for cellulose hydrolysis to be efficient, Filter Paper Activity at a considerable concentration is also required to initiate the

Redox-initiated hydrogel system for detection and real-time imaging of cellulolytic enzyme activity.

PubMed

Malinowska, Klara H; Verdorfer, Tobias; Meinhold, Aylin; Milles, Lukas F; Funk, Victor; Gaub, Hermann E; Nash, Michael A

2014-10-01

Understanding the process of biomass degradation by cellulolytic enzymes is of urgent importance for biofuel and chemical production. Optimizing pretreatment conditions and improving enzyme formulations both require assays to quantify saccharification products on solid substrates. Typically, such assays are performed using freely diffusing fluorophores or dyes that measure reducing polysaccharide chain ends. These methods have thus far not allowed spatial localization of hydrolysis activity to specific substrate locations with identifiable morphological features. Here we describe a hydrogel reagent signaling (HyReS) system that amplifies saccharification products and initiates crosslinking of a hydrogel that localizes to locations of cellulose hydrolysis, allowing for imaging of the degradation process in real time. Optical detection of the gel in a rapid parallel format on synthetic and natural pretreated solid substrates was used to quantify activity of T. emersonii and T. reesei enzyme cocktails. When combined with total internal reflection fluorescence microscopy and AFM imaging, the reagent system provided a means to visualize enzyme activity in real-time with high spatial resolution (<2 μm). These results demonstrate the versatility of the HyReS system in detecting cellulolytic enzyme activity and suggest new opportunities in real-time chemical imaging of biomass depolymerization. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Nanocaged enzymes with enhanced catalytic activity and increased stability against protease digestion

NASA Astrophysics Data System (ADS)

Zhao, Zhao; Fu, Jinglin; Dhakal, Soma; Johnson-Buck, Alexander; Liu, Minghui; Zhang, Ting; Woodbury, Neal W.; Liu, Yan; Walter, Nils G.; Yan, Hao

2016-02-01

Cells routinely compartmentalize enzymes for enhanced efficiency of their metabolic pathways. Here we report a general approach to construct DNA nanocaged enzymes for enhancing catalytic activity and stability. Nanocaged enzymes are realized by self-assembly into DNA nanocages with well-controlled stoichiometry and architecture that enabled a systematic study of the impact of both encapsulation and proximal polyanionic surfaces on a set of common metabolic enzymes. Activity assays at both bulk and single-molecule levels demonstrate increased substrate turnover numbers for DNA nanocage-encapsulated enzymes. Unexpectedly, we observe a significant inverse correlation between the size of a protein and its activity enhancement. This effect is consistent with a model wherein distal polyanionic surfaces of the nanocage enhance the stability of active enzyme conformations through the action of a strongly bound hydration layer. We further show that DNA nanocages protect encapsulated enzymes against proteases, demonstrating their practical utility in functional biomaterials and biotechnology.

Nanocaged enzymes with enhanced catalytic activity and increased stability against protease digestion

PubMed Central

Zhao, Zhao; Fu, Jinglin; Dhakal, Soma; Johnson-Buck, Alexander; Liu, Minghui; Zhang, Ting; Woodbury, Neal W.; Liu, Yan; Walter, Nils G.; Yan, Hao

2016-01-01

Cells routinely compartmentalize enzymes for enhanced efficiency of their metabolic pathways. Here we report a general approach to construct DNA nanocaged enzymes for enhancing catalytic activity and stability. Nanocaged enzymes are realized by self-assembly into DNA nanocages with well-controlled stoichiometry and architecture that enabled a systematic study of the impact of both encapsulation and proximal polyanionic surfaces on a set of common metabolic enzymes. Activity assays at both bulk and single-molecule levels demonstrate increased substrate turnover numbers for DNA nanocage-encapsulated enzymes. Unexpectedly, we observe a significant inverse correlation between the size of a protein and its activity enhancement. This effect is consistent with a model wherein distal polyanionic surfaces of the nanocage enhance the stability of active enzyme conformations through the action of a strongly bound hydration layer. We further show that DNA nanocages protect encapsulated enzymes against proteases, demonstrating their practical utility in functional biomaterials and biotechnology. PMID:26861509

Nanocaged enzymes with enhanced catalytic activity and increased stability against protease digestion.

PubMed

Zhao, Zhao; Fu, Jinglin; Dhakal, Soma; Johnson-Buck, Alexander; Liu, Minghui; Zhang, Ting; Woodbury, Neal W; Liu, Yan; Walter, Nils G; Yan, Hao

2016-02-10

Cells routinely compartmentalize enzymes for enhanced efficiency of their metabolic pathways. Here we report a general approach to construct DNA nanocaged enzymes for enhancing catalytic activity and stability. Nanocaged enzymes are realized by self-assembly into DNA nanocages with well-controlled stoichiometry and architecture that enabled a systematic study of the impact of both encapsulation and proximal polyanionic surfaces on a set of common metabolic enzymes. Activity assays at both bulk and single-molecule levels demonstrate increased substrate turnover numbers for DNA nanocage-encapsulated enzymes. Unexpectedly, we observe a significant inverse correlation between the size of a protein and its activity enhancement. This effect is consistent with a model wherein distal polyanionic surfaces of the nanocage enhance the stability of active enzyme conformations through the action of a strongly bound hydration layer. We further show that DNA nanocages protect encapsulated enzymes against proteases, demonstrating their practical utility in functional biomaterials and biotechnology.

Thermo-chemical pretreatment and enzymatic hydrolysis for enhancing saccharification of catalpa sawdust.

PubMed

Jin, Shuguang; Zhang, Guangming; Zhang, Panyue; Li, Fan; Fan, Shiyang; Li, Juan

2016-04-01

To improve the reducing sugar production from catalpa sawdust, thermo-chemical pretreatments were examined and the chemicals used including NaOH, Ca(OH)2, H2SO4, and HCl. The hemicellulose solubilization and cellulose crystallinity index (CrI) were significantly increased after thermo-alkaline pretreatments, and the thermo-Ca(OH)2 pretreatment showed the best improvement for reducing sugar production comparing to other three pretreatments. The conditions of thermo-Ca(OH)2 pretreatment and enzymatic hydrolysis were systematically optimized. Under the optimal conditions, the reducing sugar yield increased by 1185.7% comparing to the control. This study indicates that the thermo-Ca(OH)2 pretreatment is ideal for the saccharification of catalpa sawdust and that catalpa sawdust is a promising raw material for biofuel. Copyright © 2016 Elsevier Ltd. All rights reserved.

Mild chemical pretreatments are sufficient for complete saccharification of steam-exploded residues and high ethanol production in desirable wheat accessions.

PubMed

Zahoor; Tu, Yuanyuan; Wang, Lingqiang; Xia, Tao; Sun, Dan; Zhou, Shiguang; Wang, Yanting; Li, Ying; Zhang, Heping; Zhang, Tong; Madadi, Meysam; Peng, Liangcai

2017-11-01

In this study, a combined pretreatment was performed in four wheat accessions using steam explosion followed with different concentrations of H 2 SO 4 or NaOH, leading to increased hexoses yields by 3-6 folds from enzymatic hydrolysis. Further co-supplied with 1% Tween-80, Talq90 and Talq16 accessions exhibited an almost complete enzymatic saccharification of steam-exploded (SE) residues after 0.5% H 2 SO 4 or 1% NaOH pretreatment, with the highest bioethanol yields at 18.5%-19.4%, compared with previous reports about wheat bioethanol yields at 11%-17% obtained under relatively strong pretreatment conditions. Furthermore, chemical analysis indicated that much enhanced saccharification in Talq90 and Talq16 may be partially due to their relatively low cellulose CrI and DP values and high hemicellulose Ara and H-monomer levels in raw materials and SE residues. Hence, this study has not only demonstrated a mild pretreatment technology for a complete saccharification, but it has also obtained the high ethanol production in desirable wheat accessions. Copyright © 2017 Elsevier Ltd. All rights reserved.

Host cell capable of producing enzymes useful for degradation of lignocellulosic material

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

Los, Alrik Pieter; Sagt, Cornelis Maria Jacobus; Schoonneveld-Bergmans, Margot Elisabeth Francoise

The invention relates to a host cell comprising at least four different heterologous polynucleotides chosen from the group of polynucleotides encoding cellulases, hemicellulases and pectinases, wherein the host cell is capable of producing the at least four different enzymes chosen from the group of cellulases, hemicellulases and pectinases, wherein the host cell is a filamentous fungus and is capable of secretion of the at least four different enzymes. This host cell can suitably be used for the production of an enzyme composition that can be used in a process for the saccharification of cellulosic material.

Enhanced enzymatic saccharification of pretreated biomass using glycerol thermal processing (GTP).

PubMed

Zhang, Wei; Sathitsuksanoh, Noppadon; Barone, Justin R; Renneckar, Scott

2016-01-01

Biomass was heated (200-240°C) in the presence of glycerol, for 4-12 min, under shear to disrupt the native cell wall architecture. The impact of this method, named glycerol thermal processing (GTP), on saccharification efficiency of the hardwood Liquidambar styraciflua, and a control cellulose sample was studied as a function of treatment severity. Furthermore, the enzymatic conversion of samples with varying compositions was studied after extraction of the structural polymers. Interestingly, the sweet gum processed materials crystallinity index increased by 10% of the initial value. The experiments revealed that the residual lignin was not a barrier to limiting the digestibility of cellulose after pretreatment yielding up to 70% glucose based on the starting wood material. Further xylan removal greatly improved the cellulose hydrolysis rate, converting nearly 70% of the cellulose into glucose within 24h, and reaching 78% of ultimate glucan digestibility after 72 h. Copyright © 2015 Elsevier Ltd. All rights reserved.

Organic acids associated with saccharification of cellulosic wastes during solid-state fermentation.

PubMed

El-Naggar, Noura El-Ahmady; El-Hersh, Mohammed Saad

2011-02-01

Saccharification of five cellulosic wastes, i.e. rice husks, wheat bran, corn cobs, wheat straw and rice straw by three cellulytic fungi, i.e. Aspergillus glaums MN1, Aspergillus oryzae MN2 and Penicillium purpurogenum MN3, during solid-state fermentation (SSF) was laboratory studied. Rice husks, wheat bran, and corn cobs were selected as inducers of glucose production in the tested fungi. An incubation interval of 10 days was optimal for glucose production. Maximal activities of the cellulases FP-ase, CMC-ase, and p-glucosidase were detected during SSF of rice husks by P. purpurogenum; however, a-amylase activity (7.2 U/g) was comparatively reduced. Meanwhile, the productivities of FP-ase, CMC-ase, and β-glucosidase were high during SSF of rice husks by A glaucus; however, they decreased during SSF of corn cobs by P. purpurogenum. Addition of rock phosphate (RP) (75 mg P(2)O(5)) decreased the pH of SSF media. (NH(4))(2)SO(4) was found to be less inducer of cellulytic enzymes, during SSF of rice husks by A. glaucus or A. oryzae; it also induced phytase production and solubilization of RP. The organic acids associated with saccharification of the wastes studied have also been investigated. The highest concentration of levulinic acid was detected (46.15 mg/g) during SSF of corn cobs by P. purpurogenum. Likewise, oxalic acid concentration was 43.20 mg/g during SSF of rice husks by P. purpurogenum.

Utilization of radiation technique on the saccharification and fermentation of biomass

NASA Astrophysics Data System (ADS)

Kaetsu, I.; Kumakura, M.; Fujimura, T.; Yoshii, F.; Kojima, T.; Tamada, M.

The application of irradiation technique to the process of saccharification and subsequent fermentation of cellulosic wastes such as chaff and rice straw to obtain ethanol, was investigated. It was found that when waste raw materials were irradiated by ?-ray or electron beam, they became accessible to the subsequent enzymatic saccharification reaction. Irradiation of 10 7-10 8 Rad was enough for this effect. Some kind of additives reduced necessary dosage for this pretreatment. Cellulase, Trichoderma reesei which produce cellulase, and yeast were immobilized as biocatalysts for biomass conversion by radiation-induced polymerization of glass-forming monomer at low temperature. The immobilized cellulase showed almost same activity of glucose production as the native cellulase. Continuous saccharification reaction was carried out by using the immobilized cellulase. The immobilized Trichoderma reesei and the immobilized yeast showed almost same activity as the intact biocatalysts. It was concluded that the continuous saccharification and subsequent fermentation could be carried out effectively by using the immobilized biocatalysts. Spinach chloroplasts were immobilized by the same method as the first step for the conversion of water into hydrogen gas using solar energy. The immobilized chloroplasts kept the O 2 evolution activity in storage more than 30 days at 4°C. Thermostatility of chloroplasts was also improved greatly by the immobilization.

Partial purification of saccharifying and cell wall-hydrolyzing enzymes from malt in waste from beer fermentation broth.

PubMed

Khattak, Waleed Ahmad; Kang, Minkyung; Ul-Islam, Mazhar; Park, Joong Kon

2013-06-01

A number of hydrolyzing enzymes that are secreted from malt during brewing, including cell wall-hydrolyzing, saccharide-hydrolyzing, protein-degrading, lipid-hydrolyzing, and polyphenol and thiol-hydrolyzing enzymes, are expected to exist in an active form in waste from beer fermentation broth (WBFB). In this study, the existence of these enzymes was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, after which enzyme extract was partially purified through a series of purification steps. The hydrolyzing enzyme activity was then measured under various conditions at each purification step using carboxymethyl cellulose as a substrate. The best hydrolyzing activities of partially purified enzymes were found at pH 4.5 and 50 °C in a citrate buffer system. The enzymes showed highest thermal stability at 30 °C when exposed for prolonged time. As the temperature increased gradually from 25 to 70 °C, yeast cells in the chemically defined medium with enzyme extract lost their cell wall and viability earlier than those without enzyme extract. Cell wall degradation and the release of cell matrix into the culture media at elevated temperature (45-70 °C) in the presence of enzyme extract were monitored through microscopic pictures. Saccharification enzymes from malt were relatively more active in the original WBFB than supernatant and diluted sediments. The presence of hydrolyzing enzymes from malt in WBFB is expected to play a role in bioethanol production using simultaneous saccharification and fermentation without the need for additional enzymes, nutrients, or microbial cells via a cell-free enzyme system.

Simultaneous saccharification and fermentation of acid-pretreated rapeseed meal for succinic acid production using Actinobacillus succinogenes.

PubMed

Chen, Kequan; Zhang, Han; Miao, Yelian; Wei, Ping; Chen, Jieyu

2011-04-07

Rapeseed meal was evaluated for succinic acid production by simultaneous saccharification and fermentation using Actinobacillus succinogenes ATCC 55618. Diluted sulfuric acid pretreatment and subsequent hydrolysis with pectinase was used to release sugars from rapeseed meal. The effects of culture pH, pectinase loading and yeast extract concentration on succinic acid production were investigated. When simultaneous saccharification and fermentation of diluted acid pretreated rapeseed meal with a dry matter content of 12.5% (w/v) was performed at pH 6.4 and a pectinase loading of 2% (w/w, on dry matter) without supplementation of yeast extract, a succinic acid concentration of 15.5 g/L was obtained at a yield of 12.4 g/100g dry matter. Fed-batch simultaneous saccharification and fermentation was carried out with supplementation of concentrated pretreated rapeseed meal and pectinase at 18 and 28 h to yield a final dry matter content of 20.5% and pectinase loading of 2%, with the succinic acid concentration enhanced to 23.4 g/L at a yield of 11.5 g/100g dry matter and a productivity of 0.33 g/(Lh). This study suggests that rapeseed meal may be an alternative substrate for the efficient production of succinic acid by A. succinogenes without requiring nitrogen source supplementation. Copyright © 2011 Elsevier Inc. All rights reserved.

Ultrasound enhanced glucose release from corn in ethanol plants.

PubMed

Khanal, Samir Kumar; Montalbo, Melissa; van Leeuwen, J; Srinivasan, Gowrishankar; Grewell, David

2007-12-01

This work evaluated the use of high power ultrasonic energy to treat corn slurry in dry corn milling ethanol plants to enhance liquefaction and saccharification for ethanol production. Corn slurry samples obtained before and after jet cooking were subjected to ultrasonic pretreatment for 20 and 40 s at amplitudes of vibration ranging from 180 to 299 microm(pp) (peak to peak amplitude in microm). The resulting samples were then exposed to enzymes (alpha-amylase and glucoamylase) to convert cornstarch into glucose. A comparison of scanning electron micrographs of raw and sonicated samples showed the development of micropores and the disruption of cell walls in corn mash. The corn particle size declined nearly 20-fold following ultrasonic treatment at high power settings. The glucose release rate from sonicated samples increased as much as threefold compared to the control group. The efficiency of ultrasound exceeded 100% in terms of energy gain from the sugar released over the ultrasonic energy supplied. Enzymatic activity was enhanced when the corn slurry was sonicated with simultaneous addition of enzymes. This finding suggests that the ultrasonic energy did not degrade or denature the enzymes during the pretreatment.

Enhanced enzyme stability through site-directed covalent immobilization.

PubMed

Wu, Jeffrey Chun Yu; Hutchings, Christopher Hayden; Lindsay, Mark Jeffrey; Werner, Christopher James; Bundy, Bradley Charles

2015-01-10

Breakthroughs in enzyme immobilization have enabled increased enzyme recovery and reusability, leading to significant decreases in the cost of enzyme use and fueling biocatalysis growth. However, current enzyme immobilization techniques suffer from leaching, enzyme stability, and recoverability and reusability issues. Moreover, these techniques lack the ability to control the orientation of the immobilized enzymes. To determine the impact of orientation on covalently immobilized enzyme activity and stability, we apply our PRECISE (Protein Residue-Explicit Covalent Immobilization for Stability Enhancement) system to a model enzyme, T4 lysozyme. The PRECISE system uses non-canonical amino acid incorporation and the Huisgen 1,3-dipolar cycloaddition "click" reaction to enable directed enzyme immobilization at rationally chosen residues throughout an enzyme. Unlike previous site-specific systems, the PRECISE system is a truly covalent immobilization method. Utilizing this system, enzymes immobilized at proximate and distant locations from the active site were tested for activity and stability under denaturing conditions. Our results demonstrate that orientation control of covalently immobilized enzymes can provide activity and stability benefits exceeding that of traditional random covalent immobilization techniques. PRECISE immobilized enzymes were 50 and 73% more active than randomly immobilized enzymes after harsh freeze-thaw and chemical denaturant treatments. Copyright © 2014 Elsevier B.V. All rights reserved.

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

Enzymatic Hydrolysis and Simultaneous Saccharification and Fermentation of Soybean Processing Intermediates for the Production of Ethanol and Concentration of Protein and Lipids

PubMed Central

Long, Craig C.; Gibbons, William

2012-01-01

Carbohydrates in soybeans are generally undesirable due to their low digestibility and because they “dilute” more valuable components (proteins, lipids). To remove these carbohydrates and raise the titer of more valuable components, ethanol production was investigated. Commercial enzymes (Novozyme cellulase, β-glucosidase, and pectinase) were added to ground soybeans (SB), soybean meal (SBM), soybean hulls (SH), and soybean white flakes (WF) at a 10% solids loading rate to quantify hydrolyzed glucan. Saccharification resulted in glucan reductions of 28%, 45%, 76%, and 80% (SBM, SB, SH, WF, resp.). Simultaneous saccharification and fermentation (SSF) trials were conducted at 5%, 10%, 15%, and 20% solids loading with Saccharomyces cerevisiae NRRL Y-2034 and Scheffersomyces stipitis NRRL Y-7124, with protein, fiber, and lipids analyzed at SSF 10% solids and saccharification trials. S. cerevisiae and S. stipitis produced ~3–12.5 g/L ethanol and ~2.5–8.6 g/L ethanol, respectively, on SB, SBM, and WF over all solid loading rates. SH resulted in higher ethanol titers for both S. cerevisiae (~9–23 g/L) and S. stipitis (~9.5–14.5 g/L). Protein concentrations decreased by 2.5–10% for the SB, SBM, and WF, but increased by 53%–55% in SH. Oil concentrations increased by ~50% for SB; by ~500%–1300% for the others. PMID:23762751

Enhancement of In Situ Enzymatic Saccharification of Corn Stover by a Stepwise Sodium Hydroxide and Organic Acid Pretreatment.

PubMed

Qing, Qing; Guo, Qi; Zhou, Linlin; He, Yucai; Wang, Liqun; Zhang, Yue

2017-01-01

A stepwise pretreatment method that combines sodium hydroxide and organic acid pretreatments was proposed and investigated to maximize the recovery of main constituents of lignocellulose. The sodium hydroxide pretreatment was firstly optimized by a designed orthogonal experiment with the optimum pretreatment conditions determined as 1 wt% NaOH at 70 °C for 1 h, and 60.42 % of lignin was successfully removed during this stage. In the second stage, 0.5 % acetic acid was selected to pretreat the first-stage solid residue at 80 °C for 40 min in order to decompose hemicelluloses to soluble oligomers or monomers. Then, the whole slurry was subjected to in situ enzymatic saccharification by cellullase with a supplementation of xylanase to further degrade the xylooligosaccharides generated during the acetic acid pretreatment. The maximum reducing sugar and glucose yields achieved were 20.74 and 12.03 g/L, respectively. Furthermore, rapid ethanol fermentation and a yield of 80.3 % also testified this pretreatment method, and the in situ saccharification did not bring any negative impact on ethanol fermentation and has a broad application prospect.

Ethanol and anaerobic conditions reversibly inhibit commercial cellulase activity in thermophilic simultaneous saccharification and fermentation (tSSF)

PubMed Central

2012-01-01

Background A previously developed mathematical model of low solids thermophilic simultaneous saccharification and fermentation (tSSF) with Avicel was unable to predict performance at high solids using a commercial cellulase preparation (Spezyme CP) and the high ethanol yield Thermoanaerobacterium saccharolyticum strain ALK2. The observed hydrolysis proceeded more slowly than predicted at solids concentrations greater than 50 g/L Avicel. Factors responsible for this inaccuracy were investigated in this study. Results Ethanol dramatically reduced cellulase activity in tSSF. At an Avicel concentration of 20 g/L, the addition of ethanol decreased conversion at 96 hours, from 75% in the absence of added ethanol down to 32% with the addition of 34 g/L initial ethanol. This decrease is much greater than expected based on hydrolysis inhibition results in the absence of a fermenting organism. The enhanced effects of ethanol were attributed to the reduced, anaerobic conditions of tSSF, which were shown to inhibit cellulase activity relative to hydrolysis under aerobic conditions. Cellulose hydrolysis in anaerobic conditions was roughly 30% slower than in the presence of air. However, this anaerobic inhibition was reversed by exposing the cellulase enzymes to air. Conclusion This work demonstrates a previously unrecognized incompatibility of enzymes secreted by an aerobic fungus with the fermentation conditions of an anaerobic bacterium and suggests that enzymes better suited to industrially relevant fermentation conditions would be valuable. The effects observed may be due to inactivation or starvation of oxygen dependent GH61 activity, and manipulation or replacement of this activity may provide an opportunity to improve biomass to fuel process efficiency. PMID:22703989

Simultaneous or separated; comparison approach for saccharification and fermentation process in producing bio-ethanol from EFB

NASA Astrophysics Data System (ADS)

Bardant, Teuku Beuna; Dahnum, Deliana; Amaliyah, Nur

2017-11-01

Simultaneous Saccharification Fermentation (SSF) of palm oil (Elaeis guineensis) empty fruit bunch (EFB) pulp were investigated as a part of ethanol production process. SSF was investigated by observing the effect of substrate loading variation in range 10-20%w, cellulase loading 5-30 FPU/gr substrate and yeast addition 1-2%v to the ethanol yield. Mathematical model for describing the effects of these three variables to the ethanol yield were developed using Response Surface Methodology-Cheminformatics (RSM-CI). The model gave acceptable accuracy in predicting ethanol yield for Simultaneous Saccharification and Fermentation (SSF) with coefficient of determination (R2) 0.8899. Model validation based on data from previous study gave (R2) 0.7942 which was acceptable for using this model for trend prediction analysis. Trend prediction analysis based on model prediction yield showed that SSF gave trend for higher yield when the process was operated in high enzyme concentration and low substrate concentration. On the other hand, even SHF model showed better yield will be obtained if operated in lower substrate concentration, it still possible to operate in higher substrate concentration with slightly lower yield. Opportunity provided by SHF to operate in high loading substrate make it preferable option for application in commercial scale.

Fusion of agarase and neoagarobiose hydrolase for mono-sugar production from agar.

PubMed

Alkotaini, Bassam; Han, Nam Soo; Kim, Beom Soo

2017-02-01

In enzymatic saccharification of agar, endo- and exo-agarases together with neoagarobiose hydrolase (NABH) are important key enzymes for the sequential hydrolysis reactions. In this study, a bifunctional endo/exo-agarase was fused with NABH for production of mono-sugars (D-galactose and 3,6-anhydro-L-galactose) from agar using only one fusion enzyme. Two fusion enzymes with either bifunctional agarase (Sco3476) or NABH (Zg4663) at the N-terminus, Sco3476-Zg4663 (SZ) and Zg4663-Sco3476 (ZS), were constructed. Both fusion enzymes exhibited their optimal agarase and NABH activities at 40 and 35 °C, respectively. Fusions SZ and ZS enhanced the thermostability of the NABH activity, while only fusion SZ showed a slight enhancement in the NABH catalytic efficiency (K cat /K M ) from 14.8 (mg/mL) -1  s -1 to 15.8 (mg/mL) -1  s -1 . Saccharification of agar using fusion SZ resulted in 2-fold higher mono-sugar production and 3-fold lower neoagarobiose accumulation when compared to the physical mixture of Sco3476 and Zg4663. Therefore, this fusion has the potential to reduce enzyme production cost, decrease intermediate accumulation, and increase mono-sugar yield in agar saccharification.

Elucidation of Factors Effecting Enzymatic Saccharification using Transgenic Hardwoods

NASA Astrophysics Data System (ADS)

Min, Douyong

Three groups of transgenic wood samples were used as starting materials to elucidate the recalcitrance of enzymatic saccharification with/without pretreatments. The first group of transgenic wood samples is low lignin P. trichocarpa. The second group is low xylan P. trichocarpa. The third one is 12 hybrid poplars which have different levels of S/V ratio and lignin content. Four pretreatments were carried out in this research including dilute sulfuric acid, green liquor, auto hydrolysis and ozone delignification. The behavior among pretreatments as a function of removal of lignin appears to be different. Lignin is the major factor of recalcitrance of the lignocellulosic material to ethanol conversion process. Xylan also plays key role in this process. In addition, the crude milled wood lignin was isolated from these three groups of transgenic samples. Lignin carbohydrate complexes was characterized by 1H-13C HMQC and 13C NMR. Thus the effect of LCCs on enzymatic saccharification was elucidated. High S/V ratio propels the lignin removal during pretreatments however; high S/V ratio retards the enzymatic saccharification on the lignocellulosic material without pretreatments. The level of LCCs linkages accounts for additional recalcitrance of the lignocellulosic material to ethanol conversion process. The amount of LCCs linkages is affected by xylan content, lignin content and S/V ratio.

Starch saccharification and fermentation of uncooked sweet potato roots for fuel ethanol production.

PubMed

Zhang, Peng; Chen, Caifa; Shen, Yanhu; Ding, Tielin; Ma, Daifu; Hua, Zichun; Sun, Dongxu

2013-01-01

An energy-saving ethanol fermentation technology was developed using uncooked fresh sweet potato as raw material. A mutant strain of Aspergillus niger isolated from mildewed sweet potato was used to produce abundant raw starch saccharification enzymes for treating uncooked sweet potato storage roots. The viscosity of the fermentation paste of uncooked sweet potato roots was lower than that of the cooked roots. The ethanol fermentation was carried out by Zymomonas mobilis, and 14.4 g of ethanol (87.2% of the theoretical yield) was produced from 100g of fresh sweet potato storage roots. Based on this method, an energy-saving, high efficient and environment-friendly technology can be developed for large-scale production of fuel ethanol from sweet potato roots. Copyright © 2012 Elsevier Ltd. All rights reserved.

Enzymatic Saccharification of Lignocelluloses Should be Conducted at Elevated pH 5.2-6.2

Treesearch

T.Q. Lan; Hongming Lou; J.Y. Zhu

2013-01-01

This study revealed that cellulose enzymatic saccharification response curves of lignocellulosic substrates were very different from those of pure cellulosic substrates in terms of optimal pH and pH operating window. The maximal enzymatic cellulose saccharification of lignocellulosic substrates occurs at substrate suspension

Malbranchea cinnamomea: A thermophilic fungal source of catalytically efficient lignocellulolytic glycosyl hydrolases and metal dependent enzymes.

PubMed

Mahajan, Chhavi; Basotra, Neha; Singh, Surender; Di Falco, Marcos; Tsang, Adrian; Chadha, B S

2016-01-01

This study reports thermophilic fungus Malbranchea cinnamomea as an important source of lignocellulolytic enzymes. The secretome analysis using LC-MS/MS orbitrap showed that fungus produced a spectrum of glycosyl hydrolases (cellulase/hemicellulase), polysaccharide lyases (PL) and carbohydrate esterases (CE) in addition to cellobiose dehydrogenase (CDH) indicating the presence of functional classical and oxidative cellulolytic mechanisms. The protein fractions in the secretome resolved by ion exchange chromatography were analyzed for ability to hydrolyze alkali treated carrot grass (ATCG) in the presence of Mn(2+)/Cu(2+). This strategy in tandem with peptide mass fingerprinting led to identification of metal dependent protein hydrolases with no apparent hydrolytic activity, however, showed 5.7 folds higher saccharification in presence of Mn(2+). Furthermore, adding different protein fractions to commercial cellulase (Novozymes: Cellic CTec2) resulted in enhanced hydrolysis of ATCG ranging between 1.57 and 3.43 folds indicating the enzymes from M. cinnamomea as catalytically efficient. Copyright © 2015 Elsevier Ltd. All rights reserved.

Process analysis and optimization of simultaneous saccharification and co-fermentation of ethylenediamine-pretreated corn stover for ethanol production.

PubMed

Qin, Lei; Zhao, Xiong; Li, Wen-Chao; Zhu, Jia-Qing; Liu, Li; Li, Bing-Zhi; Yuan, Ying-Jin

2018-01-01

Improving ethanol concentration and reducing enzyme dosage are main challenges in bioethanol refinery from lignocellulosic biomass. Ethylenediamine (EDA) pretreatment is a novel method to improve enzymatic digestibility of lignocellulose. In this study, simultaneous saccharification and co-fermentation (SSCF) process using EDA-pretreated corn stover was analyzed and optimized to verify the constraint factors on ethanol production. Highest ethanol concentration was achieved with the following optimized SSCF conditions at 6% glucan loading: 12-h pre-hydrolysis, 34 °C, pH 5.4, and inoculum size of 5 g dry cell/L. As glucan loading increased from 6 to 9%, ethanol concentration increased from 33.8 to 48.0 g/L, while ethanol yield reduced by 7%. Mass balance of SSCF showed that the reduction of ethanol yield with the increasing solid loading was mainly due to the decrease of glucan enzymatic conversion and xylose metabolism of the strain. Tween 20 and BSA increased ethanol concentration through enhancing enzymatic efficiency. The solid-recycled SSCF process reduced enzyme dosage by 40% (from 20 to 12 mg protein/g glucan) to achieve the similar ethanol concentration (~ 40 g/L) comparing to conventional SSCF. Here, we established an efficient SSCF procedure using EDA-pretreated biomass. Glucose enzymatic yield and yeast viability were regarded as the key factors affecting ethanol production at high solid loading. The extensive analysis of SSCF would be constructive to overcome the bottlenecks and improve ethanol production in cellulosic ethanol refinery.

One-step process of hydrothermal and alkaline treatment of wheat straw for improving the enzymatic saccharification.

PubMed

Sun, Shaolong; Zhang, Lidan; Liu, Fang; Fan, Xiaolin; Sun, Run-Cang

2018-01-01

To increase the production of bioethanol, a two-step process based on hydrothermal and dilute alkaline treatment was applied to reduce the natural resistance of biomass. However, the process required a large amount of water and a long operation time due to the solid/liquid separation before the alkaline treatment, which led to decrease the pure economic profit for production of bioethanol. Therefore, four one-step processes based on order of hydrothermal and alkaline treatment have been developed to enhance concentration of glucose of wheat straw by enzymatic saccharification. The aim of the present study was to systematically evaluated effect for different one-step processes by analyzing the physicochemical properties (composition, structural change, crystallinity, surface morphology, and BET surface area) and enzymatic saccharification of the treated substrates. In this study, hemicelluloses and lignins were removed from wheat straw and the morphologic structures were destroyed to various extents during the four one-step processes, which were favorable for cellulase absorption on cellulose. A positive correlation was also observed between the crystallinity and enzymatic saccharification rate of the substrate under the conditions given. The surface area of the substrate was positively related to the concentration of glucose in this study. As compared to the control (3.0 g/L) and treated substrates (11.2-14.6 g/L) obtained by the other three one-step processes, the substrate treated by one-step process based on successively hydrothermal and alkaline treatment had a maximum glucose concentration of 18.6 g/L, which was due to the high cellulose concentration and surface area for the substrate, accompanying with removal of large amounts of lignins and hemicelluloses. The present study demonstrated that the order of hydrothermal and alkaline treatment had significant effects on the physicochemical properties and enzymatic saccharification of wheat straw. The one

Alkaline organosolv pretreatment of corn stover for enhancing the enzymatic digestibility.

PubMed

Yuan, Wei; Gong, Zhiwei; Wang, Guanghui; Zhou, Wenting; Liu, Yi; Wang, Xuemin; Zhao, Mi

2018-06-14

In the present study, a sodium hydroxide-methanol solution (SMs) pretreatment of corn stover was described to overcome biomass recalcitrance for the first time. Effects of sodium hydroxide loading, solid-to-liquid ratio, processing time and temperature on enzymatic saccharification were studied in detail. The SMs pretreatment could significantly enhance the enzyme accessibility of corn stover, minimize the degradation of sugar polymers, and decrease the energy consumption. 97.5% glucan and 83.5% xylan were preserved in the regenerated corn stover under the optimal condition. Subsequent enzymatic digestibilities of glucan and xylan reached 97.2% and 80.3%, respectively. The enzyme susceptibility of the regenerated samples was explained by their physical and chemical characteristics. This strategy provides a promising alternative for better techno-economic of the lignocelluloses-to-sugars routes. Copyright © 2018 Elsevier Ltd. All rights reserved.

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.

Simultaneous saccharification of inulin and starch using commercial glucoamylase and the subsequent bioconversion to high titer sorbitol and gluconic acid.

PubMed

An, Kehong; Hu, Fengxian; Bao, Jie

2013-12-01

A new bioprocess for production of sorbitol and gluconic acid from two low-cost feedstocks, inulin and cassava starch, using a commercially available enzyme was proposed in this study. The commercial glucoamylase GA-L NEW from Genencor was found to demonstrate a high inulinase activity for hydrolysis of inulin into fructose and glucose. The glucoamylase was used to replace the expensive and not commercially available inulinase enzyme for simultaneous saccharification of inulin and starch into high titer glucose and fructose hydrolysate. The glucose and fructose in the hydrolysate were converted into sorbitol and gluconic acid using immobilized whole cells of the recombinant Zymomonas mobilis strain. The high gluconic acid concentration of 193 g/L and sorbitol concentration of 180 g/L with the overall yield of 97.3 % were obtained in the batch operations. The present study provided a practical production method of sorbitol and gluconic acid from low cost feedstocks and enzymes.

The relation between xyr1 overexpression in Trichoderma harzianum and sugarcane bagasse saccharification performance.

PubMed

da Silva Delabona, Priscila; Rodrigues, Gisele Nunes; Zubieta, Mariane Paludetti; Ramoni, Jonas; Codima, Carla Aloia; Lima, Deise Juliana; Farinas, Cristiane Sanchez; da Cruz Pradella, José Geraldo; Seiboth, Bernhard

2017-03-20

This work investigates the influence of the positive regulator XYR1 of Trichoderma harzianum on the production of cellulolytic enzymes, using sugarcane bagasse as carbon source. Constitutive expression of xyr1 was achieved under the control of the strong Trichoderma reesei pki1 promoter. Five clones with xyr1 overexpression achieved higher xyr1 expression and greater enzymatic productivity when cultivated under submerged fermentation, hence validating the genetic construction for T. harzianum. Clone 5 presented a relative expression of xyr1 26-fold higher than the parent strain and exhibited 66, 37, and 36% higher values for filter paper activity, xylanase activity, and β-glucosidase activity, respectively, during cultivation in a stirred-tank bioreactor. The overexpression of xyr1 in T. harzianum resulted in an enzymatic complex with significantly improved performance in sugarcane bagasse saccharification, with an enhancement of 25% in the first 24h. Our results also show that constitutive overexpression of xyr1 leads to the induction of several important players in biomass degradation at early (24h) and also late (48h) timepoints of inoculation. However, we also observed that the carbon catabolite repressor CRE1 was upregulated in xyr1 overexpression mutants. These findings demonstrate the feasibility of improving cellulase production by modifying regulator expression and suggest an attractive approach for increasing total cellulase productivity in T. harzianum. Copyright © 2017 Elsevier B.V. All rights reserved.

A study of overproduction and enhanced secretion of enzymes. Quarterly report

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

Dashek, W.V.

1993-09-01

Wood decay within forests, a significant renewable photosynthetic energy resource, is caused primarily by Basidiomycetous fungi, e.g., white rot fungi. These organisms possess the ability to degrade lignin, cellulose and hemicellulose, the main organic polymers of wood. In the case of the white rot fungi, e.g., Coriolus versicolor, the capacity results from the fungus` ability to elaborate extracellular cellulolytic and ligninolytic enzymes. With regard to the latter, at least one of the enzymes, polyphenol oxidase (PPO) appears within a defined growth medium. This proposal focuses on the over-production and enhanced secretion of PPO, cellulase and lignin peroxidase. There are twomore » major sections to the proposal: (1) overproduction of lignocellulolytic enzymes by genetic engineering methodologies and hyper-production and enhanced secretion of these enzymes by biochemical/electro microscopical techniques and (2) the biochemical/electron microscopical method involves substrate induction and the time-dependent addition of respiration and PPO enzymes.« less

Scaffoldless engineered enzyme assembly for enhanced methanol utilization

DOE PAGES

Price, J. Vincent; Chen, Long; Whitaker, W. Brian; ...

2016-10-24

Methanol is an important feedstock derived from natural gas and can be chemically converted into commodity and specialty chemicals at high pressure and temperature. Although biological conversion of methanol can proceed at ambient conditions, there is a dearth of engineered microorganisms that use methanol to produce metabolites. In nature, methanol dehydrogenase (Mdh), which converts methanol to formaldehyde, highly favors the reverse reaction. Thus, efficient coupling with the irreversible sequestration of formaldehyde by 3-hexulose-6-phosphate synthase (Hps) and 6-phospho-3-hexuloseisomerase (Phi) serves as the key driving force to pull the pathway equilibrium toward central metabolism. An emerging strategy to promote efficient substrate channelingmore » is to spatially organize pathway enzymes in an engineered assembly to provide kinetic driving forces that promote carbon flux in a desirable direction. Here, we report a scaffoldless, self-assembly strategy to organize Mdh, Hps, and Phi into an engineered supramolecular enzyme complex using an SH3–ligand interaction pair, which enhances methanol conversion to fructose-6-phosphate (F6P). To increase methanol consumption, an “NADH Sink” was created using Escherichia coli lactate dehydrogenase as an NADH scavenger, thereby preventing reversible formaldehyde reduction. Combination of the two strategies improved in vitro F6P production by 97-fold compared with unassembled enzymes. The beneficial effect of supramolecular enzyme assembly was also realized in vivo as the engineered enzyme assembly improved whole-cell methanol consumption rate by ninefold. This approach will ultimately allow direct coupling of enhanced F6P synthesis with other metabolic engineering strategies for the production of many desired metabolites from methanol.« less

Scaffoldless engineered enzyme assembly for enhanced methanol utilization

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

Price, J. Vincent; Chen, Long; Whitaker, W. Brian

Methanol is an important feedstock derived from natural gas and can be chemically converted into commodity and specialty chemicals at high pressure and temperature. Although biological conversion of methanol can proceed at ambient conditions, there is a dearth of engineered microorganisms that use methanol to produce metabolites. In nature, methanol dehydrogenase (Mdh), which converts methanol to formaldehyde, highly favors the reverse reaction. Thus, efficient coupling with the irreversible sequestration of formaldehyde by 3-hexulose-6-phosphate synthase (Hps) and 6-phospho-3-hexuloseisomerase (Phi) serves as the key driving force to pull the pathway equilibrium toward central metabolism. An emerging strategy to promote efficient substrate channelingmore » is to spatially organize pathway enzymes in an engineered assembly to provide kinetic driving forces that promote carbon flux in a desirable direction. Here, we report a scaffoldless, self-assembly strategy to organize Mdh, Hps, and Phi into an engineered supramolecular enzyme complex using an SH3–ligand interaction pair, which enhances methanol conversion to fructose-6-phosphate (F6P). To increase methanol consumption, an “NADH Sink” was created using Escherichia coli lactate dehydrogenase as an NADH scavenger, thereby preventing reversible formaldehyde reduction. Combination of the two strategies improved in vitro F6P production by 97-fold compared with unassembled enzymes. The beneficial effect of supramolecular enzyme assembly was also realized in vivo as the engineered enzyme assembly improved whole-cell methanol consumption rate by ninefold. This approach will ultimately allow direct coupling of enhanced F6P synthesis with other metabolic engineering strategies for the production of many desired metabolites from methanol.« less

Scaffoldless engineered enzyme assembly for enhanced methanol utilization

PubMed Central

Price, J. Vincent; Chen, Long; Whitaker, W. Brian; Papoutsakis, Eleftherios; Chen, Wilfred

2016-01-01

Methanol is an important feedstock derived from natural gas and can be chemically converted into commodity and specialty chemicals at high pressure and temperature. Although biological conversion of methanol can proceed at ambient conditions, there is a dearth of engineered microorganisms that use methanol to produce metabolites. In nature, methanol dehydrogenase (Mdh), which converts methanol to formaldehyde, highly favors the reverse reaction. Thus, efficient coupling with the irreversible sequestration of formaldehyde by 3-hexulose-6-phosphate synthase (Hps) and 6-phospho-3-hexuloseisomerase (Phi) serves as the key driving force to pull the pathway equilibrium toward central metabolism. An emerging strategy to promote efficient substrate channeling is to spatially organize pathway enzymes in an engineered assembly to provide kinetic driving forces that promote carbon flux in a desirable direction. Here, we report a scaffoldless, self-assembly strategy to organize Mdh, Hps, and Phi into an engineered supramolecular enzyme complex using an SH3–ligand interaction pair, which enhances methanol conversion to fructose-6-phosphate (F6P). To increase methanol consumption, an “NADH Sink” was created using Escherichia coli lactate dehydrogenase as an NADH scavenger, thereby preventing reversible formaldehyde reduction. Combination of the two strategies improved in vitro F6P production by 97-fold compared with unassembled enzymes. The beneficial effect of supramolecular enzyme assembly was also realized in vivo as the engineered enzyme assembly improved whole-cell methanol consumption rate by ninefold. This approach will ultimately allow direct coupling of enhanced F6P synthesis with other metabolic engineering strategies for the production of many desired metabolites from methanol. PMID:27791059

The identification of and relief from Fe3+ inhibition for both cellulose and cellulase in cellulose saccharification catalyzed by cellulases from Penicillium decumbens.

PubMed

Wang, Mingyu; Mu, Ziming; Wang, Junli; Hou, Shaoli; Han, Lijuan; Dong, Yanmei; Xiao, Lin; Xia, Ruirui; Fang, Xu

2013-04-01

Lignocellulosic biomass is an underutilized, renewable resource that can be converted to biofuels. The key step in this conversion is cellulose saccharification catalyzed by cellulase. In this work, the effect of metal ions on cellulose hydrolysis by cellulases from Penicillium decumbens was reported for the first time. Fe(3+) and Cu(2+) were shown to be inhibitory. Further studies on Fe(3+) inhibition showed the inhibition takes place on both enzyme and substrate levels. Fe(3+) treatment damages cellulases' capability to degrade cellulose and inhibits all major cellulase activities. Fe(3+) treatment also reduces the digestibility of cellulose, due to its oxidation. Treatment of Fe(3+)-treated cellulose with DTT and supplementation of EDTA to saccharification systems partially relieved Fe(3+) inhibition. It was concluded that Fe(3+) inhibition in cellulose degradation is a complicated process in which multiple inhibition events occur, and that relief from Fe(3+) inhibition can be achieved by the supplementation of reducing or chelating agents. Copyright © 2013 Elsevier Ltd. All rights reserved.

Saccharification of newspaper waste after ammonia fiber expansion or extractive ammonia

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

Montella, Salvatore; Balan, Venkatesh; da Costa Sousa, Leonardo

Here, the lignocellulosic fractions of municipal solid waste (MSW) can be used as renewable resources due to the widespread availability, predictable and low pricing and suitability for most conversion technologies. In particular, after the typical paper recycling loop, the newspaper waste (NW) could be further valorized as feedstock in biorefinering industry since it still contains up to 70 % polysaccharides. In this study, two different physicochemical methods— ammonia fiber expansion (AFEX) and extractive ammonia (EA) were tested for the pretraetment of NW. Furthermore, based on the previously demonstrated ability of the recombinant enzymes endocellulase rCelStrep, α-larabinofuranosidase rPoAbf and its evolvedmore » variant rPoAbf F435Y/Y446F to improve the saccharification of different lignocellulosic pretreated biomasses (such as corn stover and Arundo donax), in this study these enzymes were tested for the hydrolysis of pretreated NW, with the aim of valorizing the lignocellulosic fractions of the MSW. In particular, a mixture of purified enzymes containing cellulases, xylanases and accessory hemicellulases, was chosen as reference mix and rCelStrep and rPoAbf or its variant were replaced to EGI and Larb. The results showed that these enzymatic mixes are not suitable for the hydrolysis of NW after AFEX or EA pretreatment. On the other hand, when the enzymes rCelStrep, rPoAbf and rPoAbf F435Y/Y446F were tested for their effect in hydrolysis of pretreated NW by addition to a commercial enzyme mixture, it was shown that the total polysaccharides conversion yield reached 37.32 % for AFEX pretreated NW by adding rPoAbf to the mix whilst the maximum sugars conversion yield for EA pretreated NW was achieved 40.80 % by adding rCelStrep. The maximum glucan conversion yield obtained (45.61 % for EA pretreated NW by adding rCelStrep to the commercial mix) is higher than or comparable to those reported in recent manuscripts adopting hydrolysis conditions similar to

Saccharification of newspaper waste after ammonia fiber expansion or extractive ammonia

DOE PAGES

Montella, Salvatore; Balan, Venkatesh; da Costa Sousa, Leonardo; ...

2016-03-02

Here, the lignocellulosic fractions of municipal solid waste (MSW) can be used as renewable resources due to the widespread availability, predictable and low pricing and suitability for most conversion technologies. In particular, after the typical paper recycling loop, the newspaper waste (NW) could be further valorized as feedstock in biorefinering industry since it still contains up to 70 % polysaccharides. In this study, two different physicochemical methods— ammonia fiber expansion (AFEX) and extractive ammonia (EA) were tested for the pretraetment of NW. Furthermore, based on the previously demonstrated ability of the recombinant enzymes endocellulase rCelStrep, α-larabinofuranosidase rPoAbf and its evolvedmore » variant rPoAbf F435Y/Y446F to improve the saccharification of different lignocellulosic pretreated biomasses (such as corn stover and Arundo donax), in this study these enzymes were tested for the hydrolysis of pretreated NW, with the aim of valorizing the lignocellulosic fractions of the MSW. In particular, a mixture of purified enzymes containing cellulases, xylanases and accessory hemicellulases, was chosen as reference mix and rCelStrep and rPoAbf or its variant were replaced to EGI and Larb. The results showed that these enzymatic mixes are not suitable for the hydrolysis of NW after AFEX or EA pretreatment. On the other hand, when the enzymes rCelStrep, rPoAbf and rPoAbf F435Y/Y446F were tested for their effect in hydrolysis of pretreated NW by addition to a commercial enzyme mixture, it was shown that the total polysaccharides conversion yield reached 37.32 % for AFEX pretreated NW by adding rPoAbf to the mix whilst the maximum sugars conversion yield for EA pretreated NW was achieved 40.80 % by adding rCelStrep. The maximum glucan conversion yield obtained (45.61 % for EA pretreated NW by adding rCelStrep to the commercial mix) is higher than or comparable to those reported in recent manuscripts adopting hydrolysis conditions similar to

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.

Simultaneous saccharification and extractive fermentation of lignocellulosic materials into lactic acid in a two-zone fermentor-extractor system.

PubMed

Iyer, P V; Lee, Y Y

1999-01-01

Simultaneous saccharification and extractive fermentation of lignocellulosic materials into lactic acid was investigated using a two-zone bioreactor. The system is composed of an immobilized cell reactor, a separate column reactor containing the lignocellulosic substrate and a hollow-fiber membrane. It is operated by recirculating the cell free enzyme (cellulase) solution from the immobilized cell reactor to the column reactor through the membrane. The enzyme and microbial reactions thus occur at separate locations, yet simultaneously. This design provides flexibility in reactor operation as it allows easy separation of the solid substrate from the microorganism, in situ removal of the product and, if desired, different temperatures in the two reactor sections. This reactor system was tested using pretreated switchgrass as the substrate. It was operated under a fed-batch mode with continuous removal of lactic acid by solvent extraction. The overall lactic acid yield obtainable from this bioreactor system is 77% of the theoretical.

Valorization of kitchen biowaste for ethanol production via simultaneous saccharification and fermentation using co-cultures of the yeasts Saccharomyces cerevisiae and Pichia stipitis.

PubMed

Ntaikou, Ioanna; Menis, Nikolaos; Alexandropoulou, Maria; Antonopoulou, Georgia; Lyberatos, Gerasimos

2018-04-30

The biotransformation of the pre-dried and shredded organic fraction of kitchen waste to ethanol was investigated, via co-cultures of the yeasts Saccharomyces cerevisiae and Pichia stipitis (Scheffersomyces stipitis). Preliminary experiments with synthetic media were performed, in order to investigate the effect of different operational parameters on the ethanol production efficiency of the co-culture. The control of the pH and the supplementation with organic nitrogen were shown to be key factors for the optimization of the process. Subsequently, the ethanol production efficiency from the waste was assessed via simultaneous saccharification and fermentation experiments. Different loadings of cellulolytic enzymes and mixtures of cellulolytic with amylolytic enzymatic blends were tested in order to enhance the substrate conversion efficiency. It was further shown that for solids loading up to 40% waste on dry mass basis, corresponding to 170 g.L -1 initial concentration of carbohydrates, no substrate inhibition occurred, and ethanol concentration up to 45 g.L -1 was achieved. Copyright © 2018 Elsevier Ltd. All rights reserved.

Production and Partial Characterization of α-Amylase Enzyme from Bacillus sp. BCC 01-50 and Potential Applications

PubMed Central

Qureshi, Abdul Sattar; Khushk, Imrana; Ali, Chaudhry Haider; Lashari, Safia; Bhutto, Muhammad Aqeel; Mangrio, Ghulam Sughra; Lu, Changrui

2017-01-01

Amylase is an industrially important enzyme and applied in many industrial processes such as saccharification of starchy materials, food, pharmaceutical, detergent, and textile industries. This research work deals with the optimization of fermentation conditions for α-amylase production from thermophilic bacterial strain Bacillus sp. BCC 01-50 and characterization of crude amylase. The time profile of bacterial growth and amylase production was investigated in synthetic medium and maximum enzyme titer was observed after 60 h. In addition, effects of different carbon sources were tested as a substrate for amylase production and molasses was found to be the best. Various organic and inorganic compounds, potassium nitrate, ammonium chloride, sodium nitrate, urea, yeast extract, tryptone, beef extract, and peptone, were used and beef extract was found to be the best among the nitrogen sources used. Temperature, pH, agitation speed, and size of inoculum were also optimized. Highest enzyme activity was obtained when the strain was cultured in molasses medium for 60 h in shaking incubator (150 rpm) at 50°C and pH 8. Crude amylase showed maximal activity at pH 9 and 65°C. Enzyme remained stable in alkaline pH range 9-10 and 60–70°C. Crude amylase showed great potential for its application in detergent industry and saccharification of starchy materials. PMID:28168200

Production and Partial Characterization of α-Amylase Enzyme from Bacillus sp. BCC 01-50 and Potential Applications.

PubMed

Simair, Altaf Ahmed; Qureshi, Abdul Sattar; Khushk, Imrana; Ali, Chaudhry Haider; Lashari, Safia; Bhutto, Muhammad Aqeel; Mangrio, Ghulam Sughra; Lu, Changrui

2017-01-01

Amylase is an industrially important enzyme and applied in many industrial processes such as saccharification of starchy materials, food, pharmaceutical, detergent, and textile industries. This research work deals with the optimization of fermentation conditions for α -amylase production from thermophilic bacterial strain Bacillus sp. BCC 01-50 and characterization of crude amylase. The time profile of bacterial growth and amylase production was investigated in synthetic medium and maximum enzyme titer was observed after 60 h. In addition, effects of different carbon sources were tested as a substrate for amylase production and molasses was found to be the best. Various organic and inorganic compounds, potassium nitrate, ammonium chloride, sodium nitrate, urea, yeast extract, tryptone, beef extract, and peptone, were used and beef extract was found to be the best among the nitrogen sources used. Temperature, pH, agitation speed, and size of inoculum were also optimized. Highest enzyme activity was obtained when the strain was cultured in molasses medium for 60 h in shaking incubator (150 rpm) at 50°C and pH 8. Crude amylase showed maximal activity at pH 9 and 65°C. Enzyme remained stable in alkaline pH range 9-10 and 60-70°C. Crude amylase showed great potential for its application in detergent industry and saccharification of starchy materials.

Deficiency of cellulase activity measurements for enzyme evaluation.

PubMed

Pryor, Scott W; Nahar, Nurun

2010-11-01

Switchgrass was used as a model feedstock to determine the influence of pretreatment conditions and biomass quality on enzymatic hydrolysis using different enzyme products. Dilute sulfuric acid and soaking in aqueous ammonia pretreatments were used to produce biomass with varied levels of hemicellulose and lignin sheathing. Pretreated switchgrass solids were tested with simple enzymatic hydrolysis and simultaneous saccharification and fermentation (SSF) with three commercial enzyme products: Accellerase 1000 (Genencor), Spezyme CP (Genencor)/Novozyme 188 (Novozymes), and Celluclast/Novozyme 188 (Novozymes). Enzymes were loaded on a common activity basis (FPU/g cellulose and CBU/g cellulose). Despite identical enzyme loadings, glucose yields were significantly different for both acid and alkaline pretreatments but differences diminished as hydrolysis progressed for acid-pretreated biomass. Cellobiose concentrations in Accellerase treatments indicated an initial beta-glucosidase limitation that became less significant over time. SSF experiments showed that differences in glucose and ethanol yields could not be attributed to enzyme product inhibition. Yield discrepancies of glucose or ethanol in acid pretreatment, alkaline pretreatment, and acid pretreatment/SSF were as much as 15%, 19%, and 5%. These results indicate that standardized protocols for measuring enzyme activity may not be adequate for assessing activity using pretreated biomass substrates.

Simultaneous saccharification and fermentation of Agave tequilana fructans by Kluyveromyces marxianus yeasts for bioethanol and tequila production.

PubMed

Flores, Jose-Axel; Gschaedler, Anne; Amaya-Delgado, Lorena; Herrera-López, Enrique J; Arellano, Melchor; Arrizon, Javier

2013-10-01

Agave tequilana fructans (ATF) constitute a substrate for bioethanol and tequila industries. As Kluyveromyces marxianus produces specific fructanases for ATF hydrolysis, as well as ethanol, it can perform simultaneous saccharification and fermentation. In this work, fifteen K. marxianus yeasts were evaluated to develop inoculums with fructanase activity on ATF. These inoculums were added to an ATF medium for simultaneous saccharification and fermentation. All the yeasts, showed exo-fructanhydrolase activity with different substrate specificities. The yeast with highest fructanase activity in the inoculums showed the lowest ethanol production level (20 g/l). Five K. marxianus strains were the most suitable for the simultaneous saccharification and fermentation of ATF. The volatile compounds composition was evaluated at the end of fermentation, and a high diversity was observed between yeasts, nevertheless all of them produced high levels of isobutyl alcohol. The simultaneous saccharification and fermentation of ATF with K. marxianus strains has potential for industrial application. Copyright © 2013 Elsevier Ltd. All rights reserved.

Carbon nanotube-lipase hybrid nanoflowers with enhanced enzyme activity and enantioselectivity.

PubMed

Li, Kai; Wang, Jianhua; He, Yaojia; Abdulrazaq, Miaad Adnan; Yan, Yunjun

2018-06-19

Various nanoflowers are synthesized as supports for different methods of enzyme immobilization; however, the activities of these immobilized enzymes are limited because of their confinement in the nanoflowers. In order to increase the performance of nanoflowers, in this study, different protein-phosphate hybrid nanostructures were successfully synthesized and further enhanced by carbon nanotubes (CNTs) under the same conditions. Only Cu 3 (PO 4 ) 2 complex nanostructures exhibited flower-like structures and showed excellent results after enhancement with CNTs in this framework. An esterification reaction between lauric acid and 1-dodecanol was used to test enzyme activity during immobilization, revealing that the Cu 3 (PO 4 ) 2 /CNT/protein complex exhibited 68-fold higher activity relative to free lipase and 51-fold higher than that of Cu 3 (PO 4 ) 2 /Burkholderia cepacia lipase hybrid nanoflowers in the absence of CNTs. All three hybrid nanostructures showed good performance and exhibited excellent reusability in resolution reactions between 1-phenylethanol and vinyl acetate. Additionally, the substrate enantiomeric excess (ee s ) reached 98% in only 10 min, and the corresponding Cu 3 (PO 4 ) 2 /CNT/protein complex could be recycled eight times without obvious loss of activity. This approach involving nanoflowers enhanced with CNTs will be highly beneficial for decreasing mass-transfer resistance and providing enhanced enzyme loading along with promising potential for industrial application. Copyright © 2018 Elsevier B.V. All rights reserved.

Saccharification of Miscanthus x giganteus, incorporation of lignocellulosic by-product in cementitious matrix.

PubMed

Le Ngoc Huyen, Tran; Queneudec T'kint, Michèle; Remond, Caroline; Chabbert, Brigitte; Dheilly, Rose-Marie

2011-11-01

Given the non competition of miscanthus with food and animal feed, this lignocellulosic species has attracted attention as a possible biofuel resource. However, sustainability of ethanol production from lignocelluloses biomass would imply reduction in the consumption of chemicals and/or energetic means, but also valorization of the lignocellulosic by-product remaining from enzymatic saccharification. Introduction of these by-products into a cementitious matrix could be used in manufacturing a lightweight composite. Miscanthus biomass was submitted to chemical pretreatments followed by saccharification using an enzymatic cocktail. Residues from saccharification were then mixed with a cementitious matrix. Given their mechanical properties and a good adherence between cement and by-product, the hardened materials could be used. However, the delay in the beginning of setting time is too long, which prevents the direct use of by-product into cementitious matrix. Preliminary experiments using a setting accelerator in the cementitious matrix permitted significant reduction in the setting time delay. Copyright © 2011 Académie des sciences. Published by Elsevier SAS. All rights reserved.

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.

Aspen pectate lyase PtxtPL1-27 mobilizes matrix polysaccharides from woody tissues and improves saccharification yield

PubMed Central

2014-01-01

Background Wood cell walls are rich in cellulose, hemicellulose and lignin. Hence, they are important sources of renewable biomass for producing energy and green chemicals. However, extracting desired constituents from wood efficiently poses significant challenges because these polymers are highly cross-linked in cell walls and are not easily accessible to enzymes and chemicals. Results We show that aspen pectate lyase PL1-27, which degrades homogalacturonan and is expressed at the onset of secondary wall formation, can increase the solubility of wood matrix polysaccharides. Overexpression of this enzyme in aspen increased solubility of not only pectins but also xylans and other hemicelluloses, indicating that homogalacturonan limits the solubility of major wood cell wall components. Enzymatic saccharification of wood obtained from PL1-27-overexpressing trees gave higher yields of pentoses and hexoses than similar treatment of wood from wild-type trees, even after acid pretreatment. Conclusions Thus, the modification of pectins may constitute an important biotechnological target for improved wood processing despite their low abundance in woody biomass. PMID:24450583

Lignocellulosic Fermentation of Wild Grass Employing Recombinant Hydrolytic Enzymes and Fermentative Microbes with Effective Bioethanol Recovery

PubMed Central

Das, Saprativ P.; Ghosh, Arabinda; Gupta, Ashutosh; Das, Debasish

2013-01-01

Simultaneous saccharification and fermentation (SSF) studies of steam exploded and alkali pretreated different leafy biomass were accomplished by recombinant Clostridium thermocellum hydrolytic enzymes and fermentative microbes for bioethanol production. The recombinant C. thermocellum GH5 cellulase and GH43 hemicellulase genes expressed in Escherichia coli cells were grown in repetitive batch mode, with the aim of enhancing the cell biomass production and enzyme activity. In batch mode, the cell biomass (A 600 nm) of E. coli cells and enzyme activities of GH5 cellulase and GH43 hemicellulase were 1.4 and 1.6 with 2.8 and 2.2 U·mg−1, which were augmented to 2.8 and 2.9 with 5.6 and 3.8 U·mg−1 in repetitive batch mode, respectively. Steam exploded wild grass (Achnatherum hymenoides) provided the best ethanol titres as compared to other biomasses. Mixed enzyme (GH5 cellulase, GH43 hemicellulase) mixed culture (Saccharomyces cerevisiae, Candida shehatae) system gave 2-fold higher ethanol titre than single enzyme (GH5 cellulase) single culture (Saccharomyces cerevisiae) system employing 1% (w/v) pretreated substrate. 5% (w/v) substrate gave 11.2 g·L−1 of ethanol at shake flask level which on scaling up to 2 L bioreactor resulted in 23 g·L−1 ethanol. 91.6% (v/v) ethanol was recovered by rotary evaporator with 21.2% purification efficiency. PMID:24089676

Open-mouthed hybrid microcapsules with elevated enzyme loading and enhanced catalytic activity.

PubMed

Shi, Jiafu; Zhang, Shaohua; Wang, Xiaoli; Jiang, Zhongyi

2014-10-25

Open-mouthed hybrid microcapsules (HMCs) are synthesized through a hard-templating method. When utilized for enzyme immobilization and enzymatic catalysis, the open-mouthed HMCs show high enzyme loading capability, enhanced catalytic activity and desirable recycling stability, due to their fully exposed outer and inner surfaces.

Simultaneous detoxification, saccharification, and ethanol fermentation of weak-acid hydrolyzates

USDA-ARS?s Scientific Manuscript database

Lignocellulosic feedstocks can be prepared for ethanol fermentation by pre-treatment with a dilute mineral acid catalyst that hydrolyzes the hemicellulose and opens up the plant cell wall fibers for subsequent enzymatic saccharification. The acid catalyzed reaction scheme is sequential whereby rele...

Cognitive enhancers (nootropics). Part 2: drugs interacting with enzymes. Update 2014.

PubMed

Froestl, Wolfgang; Muhs, Andreas; Pfeifer, Andrea

2014-01-01

Scientists working in the field of Alzheimer's disease and, in particular, cognitive enhancers are very productive. The review on Drugs interacting with Enzymes was accepted in August 2012. However, this field is very dynamic. New potential targets for the treatment of Alzheimer's disease were identified. This update describes drugs interacting with 60 enzymes versus 43 enzymes in the first paper. Some compounds progressed in their development, while many others were discontinued. The present review covers the evolution of research in this field through April 2014.

Batch bioethanol production via the biological and chemical saccharification of some Egyptian marine macroalgae.

PubMed

Soliman, Ramadan M; Younis, Sherif A; El-Gendy, Nour Sh; Mostafa, Soha S M; El-Temtamy, Seham A; Hashim, Ahmed I

2018-04-19

Marine seaweeds (macroalgae) cause eutrophication problem and affects the touristic activities. The success of the production of the third generation bioethanol from marine macroalgae depends mainly on the development of an ecofriendly and eco-feasible pretreatment (i.e. hydrolysis) technique, a highly effective saccharification step and finally an efficient bioethanol fermentation step. Therefore, this study aimed to investigate the potentiality of different marine macroalgal strains, collected from Egyptian coasts, for bioethanol production via different saccharification processes. Different marine macroalgal strains; red Jania rubens, green Ulva lactuca. and brown Sargassum latifolium, have been collected from Egyptian Mediterranean and Red Sea shores. Different hydrolysis processes were evaluated to maximize the extraction of fermentable sugars; thermo-chemical hydrolysis with diluted acids (HCl and H 2 SO 4 ) and base (NaOH), hydrothermal hydrolysis followed by saccharification with different fungal strains and finally, thermo-chemical hydrolysis with diluted HCl, followed by fungal saccharification. The hydrothermal hydrolysis of Sargassum latifolium followed by biological saccharification using Trichoderma asperellum RM1 produced maximum total sugars of 510 mg g -1 macroalgal biomass. The integration of the hydrothermal and fungal hydrolyses of the macroalgal biomass with a separate batch fermentation of the produced sugars using two Saccharomyces cerevisiae strains, produced approximately 0.29 g bioethanol g -1 total reducing sugars. A simulated regression modeling for the batch bioethanol fermentation was also performed. This study, supported the possibility of using seaweeds as a renewable source of bioethanol, throughout a suggested integration of macroalgal biomass hydrothermal- and fungal- hydrolysis with a separate batch bioethanol fermentation process of the produced sugars. The usage of marine macroalgae (i.e. seaweeds) as feedstock for bioethanol

Ethanol production from rice winery waste-rice wine cake by simultaneous saccharification and fermentation without cooking.

PubMed

Vu, Van Hanh; Kim, Keun

2009-10-01

Ethanol production by simultaneous saccharification and fermentation (SSF) of low-value rice wine cake (RWC) without cooking was investigated. RWC is the filtered solid waste of fermented rice wine mash and contains 53% of raw starch. RWC slurry was mixed with raw-starch-digesting enzyme of Rhizopus sp. and yeast for SSF. The yeast strain used was selected from 300 strains for RWC fermentation and identified as Saccharomyces cerevisiae KV25. High efficiency (94%) of ethanol production was achieved at optimal condition of uncooked RWC slurry containing 23.03% of starch. The optimal SSF condition determined was 1.125 unit of raw-starch-digesting enzyme per one gram of RWC, 30 degrees C of fermentation temperature, 4.5 of pH slurry, 36 h-age of seeding culture, initial yeast cell 2 x 10(7) per ml slurry, 17 mM urea as nitrogen additive, 0.25 mM Cu(2+) as metal ion additives, 90 h of fermentation time. In this optimal condition, ethanol production by SSF of uncooked RWC slurry was improved to 16.8% (v/v) from 15.1% (v/v) of pre-optimization.

Precision breeding for RNAi suppression of a major 4-coumarate:coenzyme A ligase gene improves cell wall saccharification from field grown sugarcane.

PubMed

Jung, Je Hyeong; Kannan, Baskaran; Dermawan, Hugo; Moxley, Geoffrey W; Altpeter, Fredy

2016-11-01

Sugarcane (Saccharum spp. hybrids) is a major feedstock for commercial bioethanol production. The recent integration of conversion technologies that utilize lignocellulosic sugarcane residues as well as sucrose from stem internodes has elevated bioethanol yields. RNAi suppression of lignin biosynthetic enzymes is a successful strategy to improve the saccharification of lignocellulosic biomass. 4-coumarate:coenzyme A ligase (4CL) is a key enzyme in the biosynthesis of phenylpropanoid metabolites, such as lignin and flavonoids. Identifying a major 4CL involved in lignin biosynthesis among multiple isoforms with functional divergence is key to manipulate lignin biosynthesis. In this study, two full length 4CL genes (Sh4CL1 and Sh4CL2) were isolated and characterized in sugarcane. Phylogenetic, expression and RNA interference (RNAi) analysis confirmed that Sh4CL1 is a major lignin biosynthetic gene. An intragenic precision breeding strategy may facilitate the regulatory approval of the genetically improved events and was used for RNAi suppression of Sh4CL1. Both, the RNAi inducing cassette and the expression cassette for the mutated ALS selection marker consisted entirely of DNA sequences from sugarcane or the sexually compatible species Sorghum bicolor. Field grown sugarcane with intragenic RNAi suppression of Sh4CL1 resulted in reduction of the total lignin content by up to 16.5 % along with altered monolignol ratios without reduction in biomass yield. Mature, field grown, intragenic sugarcane events displayed 52-76 % improved saccharification efficiency of lignocellulosic biomass compared to wild type (WT) controls. This demonstrates for the first time that an intragenic approach can add significant value to lignocellulosic feedstocks for biofuel and biochemical production.

Improved in situ saccharification of cellulose pretreated by dimethyl sulfoxide/ionic liquid using cellulase from a newly isolated Paenibacillus sp. LLZ1.

PubMed

Hu, Dongxue; Ju, Xin; Li, Liangzhi; Hu, Cuiying; Yan, Lishi; Wu, Tianyun; Fu, Jiaolong; Qin, Ming

2016-02-01

A cellulase producing strain was newly isolated from soil samples and identified as Paenibacillus sp. LLZ1. A novel aqueous-dimethyl sulfoxide (DMSO)/1-ethyl-3-methylimidazolium diethyl phosphate ([Emin]DEP)-cellulase system was designed and optimized. In the pretreatment, DMSO was found to be a low-cost substitute of up to 70% ionic liquid to enhance the cellulose dissolution. In the enzymatic saccharification, the optimum pH and temperature of the Paenibacillus sp. LLZ1 cellulase were identified as 6.0 and 40°C, respectively. Under the optimized reaction condition, the conversion of microcrystalline cellulose and bagasse cellulose increased by 39.3% and 37.6%, compared with unpretreated cellulose. Compared to current methods of saccharification, this new approach has several advantages including lower operating temperature, milder pH, and less usage of ionic liquid, indicating a marked progress in environmental friendly hydrolysis of biomass-based materials. Copyright © 2015 Elsevier Ltd. All rights reserved.

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.

Methods and compositions for simultaneous saccharification and fermentation

DOEpatents

Ingram, Lonnie O'Neal; Zhou, Shengde

2006-04-11

The invention provides compositions and methods for the synergistic degradation of oligosaccharides by endoglucanases. The invention further provides recombinant host cells containing one or more genes encoding endoglucanses which are capable of the synergistic degradation of oligosaccharides. Preferred host cells of the invention are ethanologenic and capable of carrying out simultaneous saccharification and fermentation resulting in the production of ethanol from complex cellulose substrates.

Whole slurry saccharification and fermentation of maleic acid-pretreated rice straw for ethanol production.

PubMed

Jung, Young Hoon; Park, Hyun Min; Kim, Kyoung Heon

2015-09-01

We evaluated the feasibility of whole slurry (pretreated lignocellulose) saccharification and fermentation for producing ethanol from maleic acid-pretreated rice straw. The optimized conditions for pretreatment were to treat rice straw at a high temperature (190 °C) with 1 % (w/v) maleic acid for a short duration (3 min ramping to 190 °C and 3 min holding at 190 °C). Enzymatic digestibility (based on theoretical glucose yield) of cellulose in the pretreated rice straw was 91.5 %. Whole slurry saccharification and fermentation of pretreated rice straw resulted in 83.2 % final yield of ethanol based on the initial quantity of glucan in untreated rice straw. These findings indicate that maleic acid pretreatment results in a high yield of ethanol from fermentation of whole slurry even without conditioning or detoxification of the slurry. Additionally, the separation of solids and liquid is not required; therefore, the economics of cellulosic ethanol fuel production are significantly improved. We also demonstrated whole slurry saccharification and fermentation of pretreated lignocellulose, which has rarely been reported.

Enhanced enzyme kinetic stability by increasing rigidity within the active site.

PubMed

Xie, Yuan; An, Jiao; Yang, Guangyu; Wu, Geng; Zhang, Yong; Cui, Li; Feng, Yan

2014-03-14

Enzyme stability is an important issue for protein engineers. Understanding how rigidity in the active site affects protein kinetic stability will provide new insight into enzyme stabilization. In this study, we demonstrated enhanced kinetic stability of Candida antarctica lipase B (CalB) by mutating the structurally flexible residues within the active site. Six residues within 10 Å of the catalytic Ser(105) residue with a high B factor were selected for iterative saturation mutagenesis. After screening 2200 colonies, we obtained the D223G/L278M mutant, which exhibited a 13-fold increase in half-life at 48 °C and a 12 °C higher T50(15), the temperature at which enzyme activity is reduced to 50% after a 15-min heat treatment. Further characterization showed that global unfolding resistance against both thermal and chemical denaturation also improved. Analysis of the crystal structures of wild-type CalB and the D223G/L278M mutant revealed that the latter formed an extra main chain hydrogen bond network with seven structurally coupled residues within the flexible α10 helix that are primarily involved in forming the active site. Further investigation of the relative B factor profile and molecular dynamics simulation confirmed that the enhanced rigidity decreased fluctuation of the active site residues at high temperature. These results indicate that enhancing the rigidity of the flexible segment within the active site may provide an efficient method for improving enzyme kinetic stability.

Krebs cycle metabolon formation: metabolite concentration gradient enhanced compartmentation of sequential enzymes.

PubMed

Wu, Fei; Pelster, Lindsey N; Minteer, Shelley D

2015-01-25

Dynamics of metabolon formation in mitochondria was probed by studying diffusional motion of two sequential Krebs cycle enzymes in a microfluidic channel. Enhanced directional co-diffusion of both enzymes against a substrate concentration gradient was observed in the presence of intermediate generation. This reveals a metabolite directed compartmentation of metabolic pathways.

Cellulase retention and sugar removal by membrane ultrafiltration during lignocellulosic biomass hydrolysis.

PubMed

Knutsen, Jeffrey S; Davis, Robert H

2004-01-01

Technologies suitable for the separation and reuse of cellulase enzymes during the enzymatic saccharification of pretreated corn stover are investigated to examine the economic and technical viability of processes that promote cellulase reuse while removing inhibitory reaction products such as glucose and cellobiose. The simplest and most suitable separation is a filter with relatively large pores on the order of 20-25 mm that retains residual corn stover solids while passing reaction products such as glucose and cellobiose to form a sugar stream for a variety of end uses. Such a simple separation is effective because cellulase remains bound to the residual solids. Ultrafiltration using 50-kDa polyethersulfone membranes to recover cellulase enzymes in solution was shown not to enhance further the saccharification rate or overall conversion. Instead, it appears that the necessary cellulase enzymes, including beta-glucosidase, are tightly bound to the substrate; when fresh corn stover is contacted with highly washed residual solids, without the addition of fresh enzymes, glucose is generated at a high rate. When filtration was applied multiple times, the concentration of inhibitory reaction products such as glucose and cellobiose was reduced from 70 to 10 g/L. However, an enhanced saccharification performance was not observed, most likely because the concentration of the inhibitory products remained too high. Further reduction in the product concentration was not investigated, because it would make the reaction unnecessarily complex and result in a product stream that is much too dilute to be useful. Finally, an economic analysis shows that reuse of cellulase can reduce glucose production costs, especially when the enzyme price is high. The most economic performance is shown to occur when the cellulase enzyme is reused and a small amount of fresh enzyme is added after each separation step to replace lost or deactivated enzyme.

Analysis, pretreatment and enzymatic saccharification of different fractions of Scots pine

PubMed Central

2014-01-01

Background Forestry residues consisting of softwood are a major lignocellulosic resource for production of liquid biofuels. Scots pine, a commercially important forest tree, was fractionated into seven fractions of chips: juvenile heartwood, mature heartwood, juvenile sapwood, mature sapwood, bark, top parts, and knotwood. The different fractions were characterized analytically with regard to chemical composition and susceptibility to dilute-acid pretreatment and enzymatic saccharification. Results All fractions were characterized by a high glucan content (38-43%) and a high content of other carbohydrates (11-14% mannan, 2-4% galactan) that generate easily convertible hexose sugars, and by a low content of inorganic material (0.2-0.9% ash). The lignin content was relatively uniform (27-32%) and the syringyl-guaiacyl ratio of the different fractions were within the range 0.021-0.025. The knotwood had a high content of extractives (9%) compared to the other fractions. The effects of pretreatment and enzymatic saccharification were relatively similar, but without pretreatment the bark fraction was considerably more susceptible to enzymatic saccharification. Conclusions Since sawn timber is a main product from softwood species such as Scots pine, it is an important issue whether different parts of the tree are equally suitable for bioconversion processes. The investigation shows that bioconversion of Scots pine is facilitated by that most of the different fractions exhibit relatively similar properties with regard to chemical composition and susceptibility to techniques used for bioconversion of woody biomass. PMID:24641769

Analysis, pretreatment and enzymatic saccharification of different fractions of Scots pine.

PubMed

Normark, Monica; Winestrand, Sandra; Lestander, Torbjörn A; Jönsson, Leif J

2014-03-19

Forestry residues consisting of softwood are a major lignocellulosic resource for production of liquid biofuels. Scots pine, a commercially important forest tree, was fractionated into seven fractions of chips: juvenile heartwood, mature heartwood, juvenile sapwood, mature sapwood, bark, top parts, and knotwood. The different fractions were characterized analytically with regard to chemical composition and susceptibility to dilute-acid pretreatment and enzymatic saccharification. All fractions were characterized by a high glucan content (38-43%) and a high content of other carbohydrates (11-14% mannan, 2-4% galactan) that generate easily convertible hexose sugars, and by a low content of inorganic material (0.2-0.9% ash). The lignin content was relatively uniform (27-32%) and the syringyl-guaiacyl ratio of the different fractions were within the range 0.021-0.025. The knotwood had a high content of extractives (9%) compared to the other fractions. The effects of pretreatment and enzymatic saccharification were relatively similar, but without pretreatment the bark fraction was considerably more susceptible to enzymatic saccharification. Since sawn timber is a main product from softwood species such as Scots pine, it is an important issue whether different parts of the tree are equally suitable for bioconversion processes. The investigation shows that bioconversion of Scots pine is facilitated by that most of the different fractions exhibit relatively similar properties with regard to chemical composition and susceptibility to techniques used for bioconversion of woody biomass.

On-Site Production of Cellulolytic Enzymes by the Sequential Cultivation Method.

PubMed

Farinas, Cristiane S; Florencio, Camila; Badino, Alberto C

2018-01-01

The conversion of renewable lignocellulosic biomass into fuels, chemicals, and high-value materials using the biochemical platform has been considered the most sustainable alternative for the implementation of future biorefineries. However, the high cost of the cellulolytic enzymatic cocktails used in the saccharification step significantly affects the economics of industrial large-scale conversion processes. The on-site production of enzymes, integrated to the biorefinery plant, is being considered as a potential strategy that could be used to reduce costs. In such approach, the microbial production of enzymes can be carried out using the same lignocellulosic biomass as feedstock for fungal development and biofuels production. Most of the microbial cultivation processes for the production of industrial enzymes have been developed using the conventional submerged fermentation. Recently, a sequential solid-state followed by submerged fermentation has been described as a potential alternative cultivation method for cellulolytic enzymes production. This chapter presents the detailed procedure of the sequential cultivation method, which could be employed for the on-site production of the cellulolytic enzymes required to convert lignocellulosic biomass into simple sugars.

Thermophilic Enzyme or Mesophilic Enzyme with Enhanced Thermostability: Can We Draw a Line?

PubMed

Jing, Xiaomin; Evangelista Falcon, Wilfredo; Baudry, Jerome; Serpersu, Engin H

2017-07-27

Aminoglycoside nucleotidyltransferase 4' (ANT) is a homodimeric enzyme that modifies the C4'-OH site of aminoglycoside antibiotics by nucleotidylation. A few single- and double-residue mutants of this enzyme (T130K, D80Y, and D80Y/T130K) from Bacillus stearothermophilus show increased thermostability. This article investigates how such residue replacements, which are distant from the active site and monomer-monomer interface, result in various changes of the thermostability of the enzyme. In this work, we show that the thermodynamic properties of enzyme-ligand complexes and protein dynamics may be indicators of a thermophilic behavior. Our data suggests that one of the single-site mutants of ANT, D80Y, may be a thermophilic protein and the other thermostable mutant, T130K, is actually a more heat-stable variant of the mesophilic wild type (WT) with a higher T m . Our data also suggest that T130K and D80Y adopt different global dynamics strategies to achieve different levels of thermostability enhancement and that the differences between the properties of the species can be described in terms of global dynamics rather than in terms of specific structural features. Thermophilicity of the D80Y comes at the cost of less favorable thermodynamic parameters for ligand binding relative to WT. On the other hand, the T130K species exhibits the same affinity to ligands and the same thermodynamic parameters of complex formation as the WT enzyme. These observations suggest that a quantitative characterization of ligand binding and protein dynamics can be used to differentiate thermophilic proteins from their simply more heat-stable mesophilic counterparts.

Enhancement of cellulosome-mediated deconstruction of cellulose by improving enzyme thermostability.

PubMed

Moraïs, Sarah; Stern, Johanna; Kahn, Amaranta; Galanopoulou, Anastasia P; Yoav, Shahar; Shamshoum, Melina; Smith, Matthew A; Hatzinikolaou, Dimitris G; Arnold, Frances H; Bayer, Edward A

2016-01-01

The concerted action of three complementary cellulases from Clostridium thermocellum, engineered to be stable at elevated temperatures, was examined on a cellulosic substrate and compared to that of the wild-type enzymes. Exoglucanase Cel48S and endoglucanase Cel8A, both key elements of the natural cellulosome from this bacterium, were engineered previously for increased thermostability, either by SCHEMA, a structure-guided, site-directed protein recombination method, or by consensus-guided mutagenesis combined with random mutagenesis using error-prone PCR, respectively. A thermostable β-glucosidase BglA mutant was also selected from a library generated by error-prone PCR that will assist the two cellulases in their methodic deconstruction of crystalline cellulose. The effects of a thermostable scaffoldin versus those of a largely mesophilic scaffoldin were also examined. By improving the stability of the enzyme subunits and the structural component, we aimed to improve cellulosome-mediated deconstruction of cellulosic substrates. The results demonstrate that the combination of thermostable enzymes as free enzymes and a thermostable scaffoldin was more active on the cellulosic substrate than the wild-type enzymes. Significantly, "thermostable" designer cellulosomes exhibited a 1.7-fold enhancement in cellulose degradation compared to the action of conventional designer cellulosomes that contain the respective wild-type enzymes. For designer cellulosome formats, the use of the thermostabilized scaffoldin proved critical for enhanced enzymatic performance under conditions of high temperatures. Simple improvement in the activity of a given enzyme does not guarantee its suitability for use in an enzyme cocktail or as a designer cellulosome component. The true merit of improvement resides in its ultimate contribution to synergistic action, which can only be determined experimentally. The relevance of the mutated thermostable enzymes employed in this study as components

Final Technical Report - Consolidating Biomass Pretreatment with Saccharification by Resolving the Spatial Control Mechanisms of Fungi

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

Schilling, Jonathan

Consolidated bioprocessing (CBP) of lignocellulose combines enzymatic sugar release (saccharification) with fermentation, but pretreatments remain separate and costly. In nature, lignocellulose-degrading brown rot fungi consolidate pretreatment and saccharification, likely using spatial gradients to partition these incompatible reactions. With the field of biocatalysis maturing, reaction partitioning is increasingly reproducible for commercial use. Therefore, my goal was to resolve the reaction partitioning mechanisms of brown rot fungi so that they can be applied to bioconversion of lignocellulosic feedstocks. Brown rot fungi consolidate oxidative pretreatments with saccharification and are a focus for biomass refining because 1) they attain >99% sugar yield without destroyingmore » lignin, 2) they use a simplified cellulase suite that lacks exoglucanase, and 3) their non-enzymatic pretreatment is facilitative and may be accelerated. Specifically, I hypothesized that during brown rot, oxidative pretreatments occur ahead of enzymatic saccharification, spatially, and the fungus partitions these reactions using gradients in pH, lignin reactivity, and plant cell wall porosity. In fact, we found three key results during these experiments for this work: 1) Brown rot fungi have an inducible cellulase system, unlike previous descriptions of a constitutive mechanism. 2) The induction of cellulases is delayed until there is repression of oxidatively-linked genes, allowing the brown rot fungi to coordinate two incompatible reactions (oxidative pretreatment with enzymatic saccharification, to release wood sugars) in the same pieces of wood. 3) This transition is mediated by the same wood sugar, cellobiose, released by the oxidative pretreatment step. Collectively, these findings have been published in excellent journal outlets and have been presented at conferences around the United States, and they offer clear targets for gene discovery en route to making biofuels and

Impact of enzyme replacement therapy on cardiac morphology and function and late enhancement in Fabry's cardiomyopathy.

PubMed

Beer, Meinrad; Weidemann, Frank; Breunig, Frank; Knoll, Anita; Koeppe, Sabrina; Machann, Wolfram; Hahn, Dietbert; Wanner, Christoph; Strotmann, Jörg; Sandstede, Jörn

2006-05-15

The present study evaluated the evolution of cardiac morphology, function, and late enhancement as a noninvasive marker of myocardial fibrosis, and their inter-relation during enzyme replacement therapy in patients with Fabry's disease using magnetic resonance imaging and color Doppler myocardial imaging. Late enhancement, which was present in up to 50% of patients, was associated with increased left ventricular mass, the failure of a significant regression of hypertrophy during enzyme replacement therapy, and worse segmental myocardial function. Late enhancement may predict the effect of enzyme replacement therapy on left ventricular mass and cardiac function.

Kinetic modeling of cellulosic biomass to ethanol via simultaneous saccharification and fermentation: Part I. Accommodation of intermittent feeding and analysis of staged reactors.

PubMed

Shao, Xiongjun; Lynd, Lee; Wyman, Charles; Bakker, André

2009-01-01

The model of South et al. [South et al. (1995) Enzyme Microb Technol 17(9): 797-803] for simultaneous saccharification of fermentation of cellulosic biomass is extended and modified to accommodate intermittent feeding of substrate and enzyme, cascade reactor configurations, and to be more computationally efficient. A dynamic enzyme adsorption model is found to be much more computationally efficient than the equilibrium model used previously, thus increasing the feasibility of incorporating the kinetic model in a computational fluid dynamic framework in the future. For continuous or discretely fed reactors, it is necessary to use particle conversion in conversion-dependent hydrolysis rate laws rather than reactor conversion. Whereas reactor conversion decreases due to both reaction and exit of particles from the reactor, particle conversion decreases due to reaction only. Using the modified models, it is predicted that cellulose conversion increases with decreasing feeding frequency (feedings per residence time, f). A computationally efficient strategy for modeling cascade reactors involving a modified rate constant is shown to give equivalent results relative to an exhaustive approach considering the distribution of particles in each successive fermenter.

Enhancement of Cellulose Degradation by Cattle Saliva

PubMed Central

Seki, Yasutaka; Kikuchi, Yukiko; Kimura, Yoshihiro; Yoshimoto, Ryo; Takahashi, Masatoshi; Aburai, Kenichi; Kanai, Yoshihiro; Ruike, Tatsushi; Iwabata, Kazuki; Sugawara, Fumio; Sakai, Hideki; Abe, Masahiko; Sakaguchi, Kengo

2015-01-01

Saccharification of cellulose is a promising technique for producing alternative source of energy. However, the efficiency of conversion of cellulose into soluble sugar using any currently available methodology is too low for industrial application. Many additives, such as surfactants, have been shown to enhance the efficiency of cellulose-to-sugar conversion. In this study, we have examined first whether cattle saliva, as an additive, would enhance the cellulase-catalyzed hydrolysis of cellulose, and subsequently elucidated the mechanism by which cattle saliva enhanced this conversion. Although cattle saliva, by itself, did not degrade cellulose, it enhanced the cellulase-catalyzed degradation of cellulose. Thus, the amount of reducing sugar produced increased approximately 2.9-fold by the addition of cattle saliva. We also found that non-enzymatic proteins, which were present in cattle saliva, were responsible for causing the enhancement effect. Third, the mechanism of cattle saliva mediated enhancement of cellulase activity was probably similar to that of the canonical surfactants. Cattle saliva is available in large amounts easily and cheaply, and it can be used without further purification. Thus, cattle saliva could be a promising additive for efficient saccharification of cellulose on an industrial scale. PMID:26402242

Pretreating wheat straw by phosphoric acid plus hydrogen peroxide for enzymatic saccharification and ethanol production at high solid loading.

PubMed

Qiu, Jingwen; Ma, Lunjie; Shen, Fei; Yang, Gang; Zhang, Yanzong; Deng, Shihuai; Zhang, Jing; Zeng, Yongmei; Hu, Yaodong

2017-08-01

Wheat straw was pretreated by phosphoric acid plus hydrogen peroxide (PHP) for enzymatic hydrolysis and ethanol fermentation at high solid loadings. Results indicated solid loading could reach 20% with 77.4% cellulose-glucose conversion and glucose concentration of 164.9g/L in hydrolysate, it even was promoted to 25% with only 3.4% decrease on cellulose-glucose conversion as the pretreated-wheat straw was dewatered by air-drying. 72.9% cellulose-glucose conversion still was achieved as the minimized enzyme input of 20mg protein/g cellulose was employed for hydrolysis at 20% solid loading. In the corresponding conditions, 100g wheat straw can yield 11.2g ethanol with concentration of 71.2g/L by simultaneous saccharification and fermentation. Thus, PHP-pretreatment benefitted the glucose or ethanol yield at high solid loadings with lower enzyme input. Additionally, decreases on the maximal cellulase adsorption and the direct-orange/direct-blue indicated drying the PHP-pretreated substrates negatively affected the hydrolysis due to the shrinkage of cellulase-size-accommodable pores. Copyright © 2017 Elsevier Ltd. All rights reserved.

Improvement of enzymatic saccharification yield in Arabidopsis thaliana by ectopic expression of the rice SUB1A-1 transcription factor

PubMed Central

Núñez-López, Lizeth; Aguirre-Cruz, Andrés

2015-01-01

Saccharification of polysaccharides releases monosaccharides that can be used by ethanol-producing microorganisms in biofuel production. To improve plant biomass as a raw material for saccharification, factors controlling the accumulation and structure of carbohydrates must be identified. Rice SUB1A-1 is a transcription factor that represses the turnover of starch and postpones energy-consuming growth processes under submergence stress. Arabidopsis was employed to test if heterologous expression of SUB1A-1 or SUB1C-1 (a related gene) can be used to improve saccharification. Cellulolytic and amylolytic enzymatic treatments confirmed that SUB1A-1 transgenics had better saccharification yield than wild-type (Col-0), mainly from accumulated starch. This improved saccharification yield was developmentally controlled; when compared to Col-0, young transgenic vegetative plants yielded 200–300% more glucose, adult vegetative plants yielded 40–90% more glucose and plants in reproductive stage had no difference in yield. We measured photosynthetic parameters, starch granule microstructure, and transcript abundance of genes involved in starch degradation (SEX4, GWD1), juvenile transition (SPL3-5) and meristematic identity (FUL, SOC1) but found no differences to Col-0, indicating that starch accumulation may be controlled by down-regulation of CONSTANS and FLOWERING LOCUS T by SUB1A-1 as previously reported. SUB1A-1 transgenics also offered less resistance to deformation than wild-type concomitant to up-regulation of AtEXP2 expansin and BGL2 glucan-1,3,-beta-glucosidase. We conclude that heterologous SUB1A-1 expression can improve saccharification yield and softness, two traits needed in bioethanol production. PMID:25780769

Mechanisms of Enhanced Catalysis in Enzyme-DNA Nanostructures Revealed through Molecular Simulations and Experimental Analysis.

PubMed

Gao, Yingning; Roberts, Christopher C; Toop, Aaron; Chang, Chia-En A; Wheeldon, Ian

2016-08-03

Understanding and controlling the molecular interactions between enzyme substrates and DNA nanostructures has important implications in the advancement of enzyme-DNA technologies as solutions in biocatalysis. Such hybrid nanostructures can be used to create enzyme systems with enhanced catalysis by controlling the local chemical and physical environments and the spatial organization of enzymes. Here we have used molecular simulations with corresponding experiments to describe a mechanism of enhanced catalysis due to locally increased substrate concentrations. With a series of DNA nanostructures conjugated to horseradish peroxidase, we show that binding interactions between substrates and the DNA structures can increase local substrate concentrations. Increased local substrate concentrations in HRP(DNA) nanostructures resulted in 2.9- and 2.4-fold decreases in the apparent Michaelis constants of tetramethylbenzidine and 4-aminophenol, substrates of HRP with tunable binding interactions to DNA nanostructures with dissociation constants in the micromolar range. Molecular simulations and kinetic analysis also revealed that increased local substrate concentrations enhanced the rates of substrate association. Identification of the mechanism of increased local concentration of substrates in close proximity to enzymes and their active sites adds to our understanding of nanostructured biocatalysis from which we can develop guidelines for enhancing catalysis in rationally designed systems. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Metal accumulation by sunflower (Helianthus annuus L.) and the efficacy of its biomass in enzymatic saccharification

PubMed Central

Dhiman, Saurabh Sudha; Zhao, Xin; Li, Jinglin; Kim, Dongwook; Kalia, Vipin C.; Kim, In-Won; Kim, Jae Young; Lee, Jung-Kul

2017-01-01

Accumulation of metal contaminants in soil as a result of various industrial and anthropogenic activities has reduced soil fertility significantly. Phytoextraction of metal contaminants can improve soil fertility and provide inexpensive feedstock for biorefineries. We investigated the hyperaccumulation capacity of sunflower (Helianthus annuus) biomass by cultivating these plants in various concentrations of metal contaminants. Sunflowers were grown in soils contaminated with various levels of heavy metals (10–2,000 mg/kg dry soil). The degree of metal uptake by different parts of the biomass and the residual concentration in the soil were estimated through inductively coupled plasma mass spectrometry. An almost 2.5-fold hyperaccumulation of Zn2+ was observed in the leaf and flower biomass compared with the concentration in the soil. For the subsequent saccharification of biomass with hyperaccumulated contaminants, a fungal lignocellulosic consortium was used. The fungal consortium cocktail retained more than 95% filter paper activity with 100 mM Ni2+ ions even after 36 h. The highest saccharification yield (SY, 87.4%) was observed with Ni2+ as the contaminant (10 mg/kg dry wt), whereas Pb2+ (251.9 mg/kg dry wt) was the strongest inhibitor of biomass hydrolysis, resulting in only a 30% SY. Importantly, the enzyme cocktail produced by the fungal consortium resulted in almost the same SY (%) as that obtained from a combination of commercial cellulase and β-glucosidase. Significant sugar conversion (61.7%) from H. annuus biomass hydrolysate occurred, resulting in the production of 11.4 g/L of bioethanol. This is the first study to assess the suitability of phytoremediated sunflower biomass for bioethanol production. PMID:28437478

Metal accumulation by sunflower (Helianthus annuus L.) and the efficacy of its biomass in enzymatic saccharification.

PubMed

Dhiman, Saurabh Sudha; Zhao, Xin; Li, Jinglin; Kim, Dongwook; Kalia, Vipin C; Kim, In-Won; Kim, Jae Young; Lee, Jung-Kul

2017-01-01

Accumulation of metal contaminants in soil as a result of various industrial and anthropogenic activities has reduced soil fertility significantly. Phytoextraction of metal contaminants can improve soil fertility and provide inexpensive feedstock for biorefineries. We investigated the hyperaccumulation capacity of sunflower (Helianthus annuus) biomass by cultivating these plants in various concentrations of metal contaminants. Sunflowers were grown in soils contaminated with various levels of heavy metals (10-2,000 mg/kg dry soil). The degree of metal uptake by different parts of the biomass and the residual concentration in the soil were estimated through inductively coupled plasma mass spectrometry. An almost 2.5-fold hyperaccumulation of Zn2+ was observed in the leaf and flower biomass compared with the concentration in the soil. For the subsequent saccharification of biomass with hyperaccumulated contaminants, a fungal lignocellulosic consortium was used. The fungal consortium cocktail retained more than 95% filter paper activity with 100 mM Ni2+ ions even after 36 h. The highest saccharification yield (SY, 87.4%) was observed with Ni2+ as the contaminant (10 mg/kg dry wt), whereas Pb2+ (251.9 mg/kg dry wt) was the strongest inhibitor of biomass hydrolysis, resulting in only a 30% SY. Importantly, the enzyme cocktail produced by the fungal consortium resulted in almost the same SY (%) as that obtained from a combination of commercial cellulase and β-glucosidase. Significant sugar conversion (61.7%) from H. annuus biomass hydrolysate occurred, resulting in the production of 11.4 g/L of bioethanol. This is the first study to assess the suitability of phytoremediated sunflower biomass for bioethanol production.

Enhancing polyphenol extraction from unripe apples by carbohydrate-hydrolyzing enzymes.

PubMed

Zheng, Hu-zhe; Hwang, In-Wook; Chung, Shin-Kyo

2009-12-01

The effects of process variables such as enzyme types, enzyme ratio, reaction temperature, pH, time, and ethanol concentration on the extraction of unripe apple polyphenol were investigated. The results indicated that Viscozyme L had the strongest effect on polyphenols extraction and was selected to study the polyphenol composition. The ratio of enzyme (Viscozyme L) to substrate (2 fungal beta-glucanase units (FBG)) at 0.02, reaction at pH 3.7, 50 degrees C for 12 h, and ethanol concentration of 70% were chosen as the most favorable extraction condition. Total phenolic content (TPC), reducing sugar content (RSC), and extraction yield increased by about 3, 1.5, and 2 times, respectively, compared with control. The contents of p-coumaric acid, ferulic acid, and caffeic acid increased to 8, 4, and 32 times, respectively. The enzyme-aided polyphenol extraction process from unripe apples might be applied to food industry for enhancing bioactive compound production.

Enhancing polyphenol extraction from unripe apples by carbohydrate-hydrolyzing enzymes*

PubMed Central

Zheng, Hu-zhe; Hwang, In-Wook; Chung, Shin-Kyo

2009-01-01

The effects of process variables such as enzyme types, enzyme ratio, reaction temperature, pH, time, and ethanol concentration on the extraction of unripe apple polyphenol were investigated. The results indicated that Viscozyme L had the strongest effect on polyphenols extraction and was selected to study the polyphenol composition. The ratio of enzyme (Viscozyme L) to substrate (2 fungal beta-glucanase units (FBG)) at 0.02, reaction at pH 3.7, 50 °C for 12 h, and ethanol concentration of 70% were chosen as the most favorable extraction condition. Total phenolic content (TPC), reducing sugar content (RSC), and extraction yield increased by about 3, 1.5, and 2 times, respectively, compared with control. The contents of p-coumaric acid, ferulic acid, and caffeic acid increased to 8, 4, and 32 times, respectively. The enzyme-aided polyphenol extraction process from unripe apples might be applied to food industry for enhancing bioactive compound production. PMID:19946955

Pretreatment of woody and herbaceous biomass for enzymatic saccharification using sulfuric acid-free ethanol cooking.

PubMed

Teramoto, Yoshikuni; Lee, Seung-Hwan; Endo, Takashi

2008-12-01

A sulfuric acid-free ethanol cooking (SFEC) treatment was developed to achieve complete saccharification of the cellulosic component of eucalyptus and baggase flour, thereby avoiding the problems associated with the use of strong acid catalysts. Cutter-milled flours were exposed to an ethanol (EtOH)/water/acetic acid mixture in an autoclave. Enzymatic hydrolysis experiments of the pretreated samples demonstrated that almost complete conversion of the cellulosic components to glucose was achieved under optimal conditions. A large-scale trial revealed that there was little consumption of in-feed EtOH during SFEC; therefore, it is considered that most part EtOH used can be essentially recovered and reused. Field emission scanning electron microscopy showed that SFEC induced the formation of pores ranging in size from approximately 10 to several 100nm. It can be assumed that the porous surface was due to the partial removals of lignin and hemicellulose, which improved the accessibility of the enzyme onto the substrate.

A new beta-glucosidase producing yeast for lower-cost cellulosic ethanol production from xylose-extracted corncob residues by simultaneous saccharification and fermentation

USDA-ARS?s Scientific Manuscript database

Conventional cellulose-to-ethanol conversion by simultaneous saccharification and fermentation (SSF)requires enzymatic saccharification using both cellulase and ß-glucosidase allowing cellulose utilization by common ethanologenic yeast. Here we report a new yeast strain of Clavispora NRRL Y-50464 th...

NanoCluster Beacons as reporter probes in rolling circle enhanced enzyme activity detection

NASA Astrophysics Data System (ADS)

Juul, Sissel; Obliosca, Judy M.; Liu, Cong; Liu, Yen-Liang; Chen, Yu-An; Imphean, Darren M.; Knudsen, Birgitta R.; Ho, Yi-Ping; Leong, Kam W.; Yeh, Hsin-Chih

2015-04-01

As a newly developed assay for the detection of endogenous enzyme activity at the single-catalytic-event level, Rolling Circle Enhanced Enzyme Activity Detection (REEAD) has been used to measure enzyme activity in both single human cells and malaria-causing parasites, Plasmodium sp. Current REEAD assays rely on organic dye-tagged linear DNA probes to report the rolling circle amplification products (RCPs), the cost of which may hinder the widespread use of REEAD. Here we show that a new class of activatable probes, NanoCluster Beacons (NCBs), can simplify the REEAD assays. Easily prepared without any need for purification and capable of large fluorescence enhancement upon hybridization, NCBs are cost-effective and sensitive. Compared to conventional fluorescent probes, NCBs are also more photostable. As demonstrated in reporting the human topoisomerases I (hTopI) cleavage-ligation reaction, the proposed NCBs suggest a read-out format attractive for future REEAD-based diagnostics.As a newly developed assay for the detection of endogenous enzyme activity at the single-catalytic-event level, Rolling Circle Enhanced Enzyme Activity Detection (REEAD) has been used to measure enzyme activity in both single human cells and malaria-causing parasites, Plasmodium sp. Current REEAD assays rely on organic dye-tagged linear DNA probes to report the rolling circle amplification products (RCPs), the cost of which may hinder the widespread use of REEAD. Here we show that a new class of activatable probes, NanoCluster Beacons (NCBs), can simplify the REEAD assays. Easily prepared without any need for purification and capable of large fluorescence enhancement upon hybridization, NCBs are cost-effective and sensitive. Compared to conventional fluorescent probes, NCBs are also more photostable. As demonstrated in reporting the human topoisomerases I (hTopI) cleavage-ligation reaction, the proposed NCBs suggest a read-out format attractive for future REEAD-based diagnostics. Electronic

Recombinant hosts suitable for simultaneous saccharification and fermentation

DOEpatents

Ingram, Lonnie O'Neal; Zhou, Shengde

2007-06-05

The invention provides recombinant host cells containing at least one heterologous polynucleotide encoding a polysaccharase under the transcriptional control of a surrogate promoter capable of increasing the expression of the polysaccharase. In addition, the invention further provides such hosts with genes encoding secretory protein/s to facilitate the secretion of the expressed polysaccharase. Preferred hosts of the invention are ethanologenic and capable of carrying out simultaneous saccharification fermentation resulting in the production of ethanol from complex cellulose substrates.

Microwave assisted alkaline pretreatment to enhance enzymatic saccharification of catalpa sawdust.

PubMed

Jin, Shuguang; Zhang, Guangming; Zhang, Panyue; Li, Fan; Wang, Siqi; Fan, Shiyang; Zhou, Shuqiong

2016-12-01

Catalpa sawdust, a promising biofuel production biomass, was pretreated by microwave-water, -NaOH, and -Ca(OH) 2 to enhance enzymatic digestibility. After 48h enzymatic hydrolysis, microwave-Ca(OH) 2 pretreated sample showed the highest reducing sugar yield. The content of hemicellulose and lignin in catalpa sawdust decreased after microwave-alkali pretreatment. SEM observation showed that the catalpa sawdust surface with microwave-Ca(OH) 2 pretreatment suffered the most serious erosion. Crystallinity index of catalpa sawdust increased after all three kinds of pretreatment. The optimum conditions of microwave-Ca(OH) 2 pretreatment were particle size of 40mesh, Ca(OH) 2 dosage of 2.25% (w/v), microwave power of 400W, pretreatment time of 6min, enzyme loading of 175FPU/g, and hydrolysis time of 96h, and the reducing sugar yield of microwave-Ca(OH) 2 pretreated catalpa sawdust reached 402.73mg/g, which increased by 682.15% compared with that of raw catalpa sawdust. The catalpa sawdust with microwave-Ca(OH) 2 pretreatment is promising for biofuel production with great potential. Copyright © 2016 Elsevier Ltd. All rights reserved.

Evaluation of Ethanol Production Activity by Engineered Saccharomyces cerevisiae Fermenting Cellobiose through the Phosphorolytic Pathway in Simultaneous Saccharification and Fermentation of Cellulose.

PubMed

Lee, Won-Heong; Jin, Yong-Su

2017-09-28

In simultaneous saccharification and fermentation (SSF) for production of cellulosic biofuels, engineered Saccharomyces cerevisiae capable of fermenting cellobiose has provided several benefits, such as lower enzyme costs and faster fermentation rate compared with wild-type S. cerevisiae fermenting glucose. In this study, the effects of an alternative intracellular cellobiose utilization pathway-a phosphorolytic pathway based on a mutant cellodextrin transporter (CDT-1 (F213L)) and cellobiose phosphorylase (SdCBP)-was investigated by comparing with a hydrolytic pathway based on the same transporter and an intracellular β-glucosidase (GH1-1) for their SSF performances under various conditions. Whereas the phosphorolytic and hydrolytic cellobiose-fermenting S. cerevisiae strains performed similarly under the anoxic SSF conditions, the hydrolytic S. cerevisiae performed slightly better than the phosphorolytic S. cerevisiae under the microaerobic SSF conditions. Nonetheless, the phosphorolytic S. cerevisiae expressing the mutant CDT-1 showed better ethanol production than the glucose-fermenting S. cerevisiae with an extracellular β-glucosidase, regardless of SSF conditions. These results clearly prove that introduction of the intracellular cellobiose metabolic pathway into yeast can be effective on cellulosic ethanol production in SSF. They also demonstrate that enhancement of cellobiose transport activity in engineered yeast is the most important factor affecting the efficiency of SSF of cellulose.

Cotton Stalk Pretreatment Using Daedalea flavida, Phlebia radiata, and Flavodon flavus: Lignin Degradation, Cellulose Recovery, and Enzymatic Saccharification.

PubMed

Meehnian, Harmanpreet; Jana, Asim K

2017-04-01

Lignocellulolytic enzyme activities of selective fungi Daedalea flavida MTCC 145 (DF-2), Phlebia radiata MTCC 2791 (PR), and non-selective fungus Flavodon flavus MTCC 168 (FF) were studied for pretreatment of cotton stalks. Simultaneous productions of high LiP and laccase activities by DF-2 during early phase of growth were effective for lignin degradation 27.83 ± 1.25 % (w/w of lignin) in 20-day pretreatment. Production of high MnP activity without laccase in the early growth phase of PR was ineffective and delayed lignin degradation 24.93 ± 1.53 % in 25 days due to laccase production at later phase. With no LiP activity, low activities of MnP and laccase by FF yielded poor lignin degradation 15.09 ± 0.6 % in 20 days. Xylanase was predominant cellulolytic enzyme produced by DF-2, resulting hemicellulose as main carbon and energy source with 83 % of cellulose recovery after 40 days of pretreatment. The glucose yield improved more than two fold from 20-day DF-2 pretreated cotton stalks after enzymatic saccharification.

Bioethanol Production From Banana Stem By Using Simultaneous Saccharification and Fermentation (SSF)

NASA Astrophysics Data System (ADS)

Kusmiyati; Mustofa, A.; Jumarmi

2018-05-01

The rapid growth and development of industries in the world result in a greater energy needs. Some studies show that ethanol can be used as an alternative energy. However, bioethanol production from food raw materials such as sugar and starch has drawback that cause the food crisis. This aim of this study was to convert banana stem into bioethanol. Banana stem contained of 44.6% cellulose, 36.0% hemicellulose and 19.4% lignin. After banana stems were pretreated with acid (H2SO4) and alkaline (NaOH) at a concentration of 2% w/v at 121 °C for 30 minutes, then subsequently the simultaneous saccharification and fermentation (SSF) were carried out by using mixed cultures of Aspergillus niger, Trichoderma reesei and Zymomonas mobilis at various enzymes ratios of (1:1:1), (1:2:1), (1:2:2), (1:1:2) and various pH (4, 5 and 6) with SSF time for 144 hours and temperature of 30°C. The results show that acid pretreatment showed better results than the alkali pretreatment. After acid pretreatment and alkali pretreatment, lignin content of pretreted banana stem reduced to 15.92% and 16.34%, respectively, cellulose increased to 52.11% and 50.6% respectively, hemicellulose reduced to 28.45% and 28.83%, respectively The SSF showed that pH 5 gave the highest bioethanol. The highest concentration of bioethanol (8.51 g/L) was achieved at the SSF process at pH 5 with a ratio Aspergillus niger, Trichoderma reesei and Zymomonas mobilis enzymes of (1:1:2).

Characterization of a Multimodular Endo-β-1,4-Glucanase (Cel9K) from Paenibacillus sp. X4 with a Potential Additive for Saccharification.

PubMed

Lee, Jae Pil; Kim, Yoon A; Kim, Sung Kyum; Kim, Hoon

2018-04-28

An endo-β-1,4-glucanase gene, cel 9K, was cloned using the shot-gun method from Paenibacillus sp. X4, which was isolated from alpine soil. The gene was 2,994 bp in length, encoding a protein of 997 amino acid residues with a predicted signal peptide composed of 32 amino acid residues. Cel9K was a multimodular enzyme, and the molecular mass and theoretical pI of the mature Cel9K were 103.5 kDa and 4.81, respectively. Cel9K contains the GGxxDAGD, PHHR, GAxxGG, YxDDI, and EVxxDYN motifs found in most glycoside hydrolase family 9 (GH9). The protein sequence showed the highest similarity (88%) with the cellulase of Bacillus sp. BP23 in comparison to the enzymes with reported properties. The enzyme was purified by chromatography using HiTrap Q, CHT-II, and HiTrap Butyl HP. Using SDS-PAGE/activity staining, the molecular mass of Cel9K was estimated to be 93 kDa, which is a truncated form produced by the proteolytic cleavage of its C-terminus. Cel9K was optimally active at pH 5.5 and 50°C and showed a half-life of 59.2 min at 50°C. The CMCase activity was increased to more than 150% in the presence of 2 mM Na⁺, K⁺, and Ba²⁺, but decreased significantly to less than 50% by Mn²⁺ and Co²⁺. The addition of Cel9K to a commercial enzyme set (Celluclast 1.5L + Novozym 188) increased the saccharification of the pretreated reed and rice straw powders by 30.4 and 15.9%, respectively. The results suggest that Cel9K can be used to enhance the enzymatic conversion of lignocellulosic biomass to reducing sugars as an additive.

Optimization of enzymatic hydrolysis for ethanol production by simultaneous saccharification and fermentation of wastepaper.

PubMed

Sangkharak, Kanokphorn

2011-11-01

The present study investigated the development of high sugar production by optimization of an enzymatic hydrolysis process using both conventional and statistical methods, as well as the production of ethanol by the selected wastepaper source. Among four sources of pretreated wastepaper including office paper, newspaper, handbills and cardboard, office paper gave the highest values of cellulose (87.12%) and holocelluloses (89.07%). The effects of the amount of wastepaper, the pretreatment method and the type of enzyme on reducing sugar production from office paper were studied using conventional methods. The highest reducing sugar production (1851.28 µg L(-1); 37.03% conversion of glucose) was obtained from the optimal condition containing 40 mg of office paper, pretreated with stream explosion and hydrolysed with the combination of cellulase from Aspergillus niger and Trichoderma viride at the fixed loading rate of 20 FPU g(-1) sample. The effects of interaction of wastepaper amount and enzyme concentration as well as incubation time were studied by a statistical method using central composite design. The optimal medium composition consisted of 43.97 µg L(-1), 28.14 FPU g(-1) sample and 53.73 h of wastepaper, enzyme concentration and incubation time, respectively, and gave the highest amount of sugar production (2184.22 µg L(-1)) and percentage conversion of glucose (43.68%). The ethanol production from pretreated office paper using Saccharomyces cerevisiae in a simultaneous saccharification and fermentation process was 21.02 g L(-1) after 36 h of cultivation, corresponding to an ethanol volumetric production rate of 0.58 g ethanol L(-1) h(-1).

Production of l(+)-lactic acid from acid pretreated sugarcane bagasse using Bacillus coagulans DSM2314 in a simultaneous saccharification and fermentation strategy.

PubMed

van der Pol, Edwin C; Eggink, Gerrit; Weusthuis, Ruud A

2016-01-01

Sugars derived from lignocellulose-rich sugarcane bagasse can be used as feedstock for production of l(+)-lactic acid, a precursor for renewable bioplastics. In our research, acid-pretreated bagasse was hydrolysed with the enzyme cocktail GC220 and fermented by the moderate thermophilic bacterium Bacillus coagulans DSM2314. Saccharification and fermentation were performed simultaneously (SSF), adding acid-pretreated bagasse either in one batch or in two stages. SSF was performed at low enzyme dosages of 10.5-15.8 FPU/g DW bagasse. The first batch SSF resulted in an average productivity of 0.78 g/l/h, which is not sufficient to compete with lactic acid production processes using high-grade sugars. Addition of 1 g/l furfural to precultures can increase B. coagulans resistance towards by-products present in pretreated lignocellulose. Using furfural-containing precultures, productivity increased to 0.92 g/l/h, with a total lactic acid production of 91.7 g in a 1-l reactor containing 20% W/W DW bagasse. To increase sugar concentrations, bagasse was solubilized with a liquid fraction, obtained directly after acid pretreatment. Solubilizing the bagasse fibres with water increased the average productivity to 1.14 g/l/h, with a total lactic acid production of 84.2 g in a 1-l reactor. Addition of bagasse in two stages reduced viscosity during SSF, resulting in an average productivity in the first 23 h of 2.54 g/l/h, similar to productivities obtained in fermentations using high-grade sugars. Due to fast accumulation of lactic acid, enzyme activity was repressed during two-stage SSF, resulting in a decrease in productivity and a slightly lower total lactic acid production of 75.6 g. In this study, it is shown that an adequate production of lactic acid from lignocellulose was successfully accomplished by a two-stage SSF process, which combines acid-pretreated bagasse, B. coagulans precultivated in the presence of furfural as microorganism, and GC220 as enzyme

Potential of the waste from beer fermentation broth for bio-ethanol production without any additional enzyme, microbial cells and carbohydrates.

PubMed

Ha, Jung Hwan; Shah, Nasrullah; Ul-Islam, Mazhar; Park, Joong Kon

2011-08-10

The potential of the waste from beer fermentation broth (WBFB) for the production of bio-ethanol using a simultaneous saccharification and fermentation process without any extra additions of saccharification enzymes, microbial cells or carbohydrate was tested. The major microbial cells in WBFB were isolated and identified. The variations in compositions of WBFB with stock time were investigated. There was residual activity of starch hydrolyzing enzymes in WBFB. The effects of reaction modes e.g. static and shaking on bio-ethanol production were studied. After 7 days of cultivation using the supernatant of WBFB at 30 °C the ethanol concentration reached 103.8 g/L in shaking culture and 91.5 g/L in static culture. Agitation experiments conducted at a temperature-profile process in which temperature was increased from 25 to 67 °C shortened the simultaneous process time. The original WBFB was more useful than the supernatant of WBFB in getting the higher concentration of ethanol and reducing the fermentation time. From this whole study it was found that WBFB is a cheap and suitable source for bio-ethanol production. Copyright © 2011 Elsevier Inc. All rights reserved.

High solid simultaneous saccharification and fermentation of wet oxidized corn stover to ethanol.

PubMed

Varga, Enikõ; Klinke, Helene B; Réczey, Kati; Thomsen, Anne Belinda

2004-12-05

In this study ethanol was produced from corn stover pretreated by alkaline and acidic wet oxidation (WO) (195 degrees C, 15 min, 12 bar oxygen) followed by nonisothermal simultaneous saccharification and fermentation (SSF). In the first step of the SSF, small amounts of cellulases were added at 50 degrees C, the optimal temperature of enzymes, in order to obtain better mixing condition due to some liquefaction. In the second step more cellulases were added in combination with dried baker's yeast (Saccharomyces cerevisiae) at 30 degrees C. The phenols (0.4-0.5 g/L) and carboxylic acids (4.6-5.9 g/L) were present in the hemicellulose rich hydrolyzate at subinhibitory levels, thus no detoxification was needed prior to SSF of the whole slurry. Based on the cellulose available in the WO corn stover 83% of the theoretical ethanol yield was obtained under optimized SSF conditions. This was achieved with a substrate concentration of 12% dry matter (DM) acidic WO corn stover at 30 FPU/g DM (43.5 FPU/g cellulose) enzyme loading. Even with 20 and 15 FPU/g DM (corresponding to 29 and 22 FPU/g cellulose) enzyme loading, ethanol yields of 76 and 73%, respectively, were obtained. After 120 h of SSF the highest ethanol concentration of 52 g/L (6 vol.%) was achieved, which exceeds the technical and economical limit of the industrial-scale alcohol distillation. The SSF results showed that the cellulose in pretreated corn stover can be efficiently fermented to ethanol with up to 15% DM concentration. A further increase of substrate concentration reduced the ethanol yield significant as a result of insufficient mass transfer. It was also shown that the fermentation could be followed with an easy monitoring system based on the weight loss of the produced CO2.

Organic acid pretreatment of oil palm trunk: effect on enzymatic saccharification and ethanol production.

PubMed

Rattanaporn, Kittipong; Tantayotai, Prapakorn; Phusantisampan, Theerawut; Pornwongthong, Peerapong; Sriariyanun, Malinee

2018-04-01

Effective lignocellulosic biomass saccharification is one of the crucial requirements of biofuel production via fermentation process. Organic acid pretreatments have been gained much interests as one of the high potential methods for promoting enzymatic saccharification of lignocellulosic materials due to their lower hazardous properties and lower production of inhibitory by-products of fermentation than typical chemical pretreatment methods. In this study, three organic acids, including acetic acid, oxalic acid, and citric acid, were examined for improvement of enzymatic saccharification and bioethanol production from oil palm trunk biomass. Based on response surface methodology, oxalic acid pretreated biomass released the maximum reducing sugar of 144 mg/g-pretreated biomass at the optimum condition, which was higher than untreated samples for 2.30 times. The released sugar yield of oil palm trunk also corresponded to the results of FT-IR analysis, which revealed the physical modification of cellulose and hemicellulose surface structures of pretreated biomass. Nevertheless, citric acid pretreatment is the most efficient pretreatment method to improve bioethanol fermentation of Saccharomyces cerevisiae TISTR 5606 at 1.94 times higher than untreated biomass. These results highlighted the selection of organic acid pretreatment as a potential method for biofuel production from oil palm trunk feedstocks.

Compositions for enhancing hydroysis of cellulosic material by cellulolytic enzyme compositions

DOEpatents

Quinlan, Jason; Xu, Feng; Sweeney, Matthew; Johansen, Katja Salomon

2014-09-30

The present invention relates to compositions comprising a GH61 polypeptide having cellulolytic enhancing activity and an organic compound comprising a carboxylic acid moiety, a lactone moiety, a phenolic moiety, a flavonoid moiety, or a combination thereof, wherein the combination of the GH61 polypeptide having cellulolytic enhancing activity and the organic compound enhances hydrolysis of a cellulosic material by a cellulolytic enzyme compared to the GH61 polypeptide alone or the organic compound alone. The present invention also relates to methods of using the compositions.

Defying the activity-stability trade-off in enzymes: taking advantage of entropy to enhance activity and thermostability.

PubMed

Siddiqui, Khawar Sohail

2017-05-01

The biotechnological applications of enzymes are limited due to the activity-stability trade-off, which implies that an increase in activity is accompanied by a concomitant decrease in protein stability. This premise is based on thermally adapted homologous enzymes where cold-adapted enzymes show high intrinsic activity linked to enhanced thermolability. In contrast, thermophilic enzymes show low activity around ambient temperatures. Nevertheless, genetically and chemically modified enzymes are beginning to show that the activity-stability trade-off can be overcome. In this review, the origin of the activity-stability trade-off, the thermodynamic basis for enhanced activity and stability, and various approaches for escaping the activity-stability trade-off are discussed. The role of entropy in enhancing both the activity and the stability of enzymes is highlighted with a special emphasis placed on the involvement of solvent water molecules. This review is concluded with suggestions for further research, which underscores the implications of these findings in the context of productivity curves, the Daniel-Danson equilibrium model, catalytic antibodies, and life on cold planets.

Guanidinylated Neomycin Conjugation Enhances Intranasal Enzyme Replacement in the Brain.

PubMed

Tong, Wenyong; Dwyer, Chrissa A; Thacker, Bryan E; Glass, Charles A; Brown, Jillian R; Hamill, Kristina; Moremen, Kelley W; Sarrazin, Stéphane; Gordts, Philip L S M; Dozier, Lara E; Patrick, Gentry N; Tor, Yitzhak; Esko, Jeffrey D

2017-12-06

Iduronidase (IDUA)-deficient mice accumulate glycosaminoglycans in cells and tissues and exhibit many of the same neuropathological symptoms of patients suffering from Mucopolysaccharidosis I. Intravenous enzyme-replacement therapy for Mucopolysaccharidosis I ameliorates glycosaminoglycan storage and many of the somatic aspects of the disease but fails to treat neurological symptoms due to poor transport across the blood-brain barrier. In this study, we examined the delivery of IDUA conjugated to guanidinoneomycin (GNeo), a molecular transporter. GNeo-IDUA and IDUA injected intravenously resulted in reduced hepatic glycosaminoglycan accumulation but had no effect in the brain due to fast clearance from the circulation. In contrast, intranasally administered GNeo-IDUA entered the brain rapidly. Repetitive intranasal treatment with GNeo-IDUA reduced glycosaminoglycan storage, lysosome size and number, and neurodegenerative astrogliosis in the olfactory bulb and primary somatosensory cortex, whereas IDUA was less effective. The enhanced efficacy of GNeo-IDUA was not the result of increased nose-to-brain delivery or enzyme stability, but rather due to more efficient uptake into neurons and astrocytes. GNeo conjugation also enhanced glycosaminoglycan clearance by intranasally delivered sulfamidase to the brain of sulfamidase-deficient mice, a model of Mucopolysaccharidosis IIIA. These findings suggest the general utility of the guanidinoglycoside-based delivery system for restoring missing lysosomal enzymes in the brain. Copyright © 2017 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.

Enhancing the functional properties of thermophilic enzymes by chemical modification and immobilization.

PubMed

Cowan, Don A; Fernandez-Lafuente, Roberto

2011-09-10

The immobilization of proteins (mostly typically enzymes) onto solid supports is mature technology and has been used successfully to enhance biocatalytic processes in a wide range of industrial applications. However, continued developments in immobilization technology have led to more sophisticated and specialized applications of the process. A combination of targeted chemistries, for both the support and the protein, sometimes in combination with additional chemical and/or genetic engineering, has led to the development of methods for the modification of protein functional properties, for enhancing protein stability and for the recovery of specific proteins from complex mixtures. In particular, the development of effective methods for immobilizing large multi-subunit proteins with multiple covalent linkages (multi-point immobilization) has been effective in stabilizing proteins where subunit dissociation is the initial step in enzyme inactivation. In some instances, multiple benefits are achievable in a single process. Here we comprehensively review the literature pertaining to immobilization and chemical modification of different enzyme classes from thermophiles, with emphasis on the chemistries involved and their implications for modification of the enzyme functional properties. We also highlight the potential for synergies in the combined use of immobilization and other chemical modifications. Copyright © 2011 Elsevier Inc. All rights reserved.

Microbial β-glucosidases from cow rumen metagenome enhance the saccharification of lignocellulose in combination with commercial cellulase cocktail

PubMed Central

2012-01-01

Background A complete saccharification of plant polymers is the critical step in the efficient production of bio-alcohols. Beta-glucosidases acting in the degradation of intermediate gluco-oligosaccharides produced by cellulases limit the yield of the final product. Results In the present work, we have identified and then successfully cloned, expressed, purified and characterised 4 highly active beta-glucosidases from fibre-adherent microbial community from the cow rumen. The enzymes were most active at temperatures 45–55°C and pH 4.0-7.0 and exhibited high affinity and activity towards synthetic substrates such as p-nitrophenyl-beta-D-glucopyranoside (pNPbetaG) and pNP-beta-cellobiose, as well as to natural cello-oligosaccharides ranging from cellobiose to cellopentaose. The apparent capability of the most active beta-glucosidase, herein named LAB25g2, was tested for its ability to improve, at low dosage (31.25 units g-1 dry biomass, using pNPbetaG as substrate), the hydrolysis of pre-treated corn stover (dry matter content of 20%; 350 g glucan kg-1 dry biomass) in combination with a beta-glucosidase-deficient commercial Trichoderma reseei cellulase cocktail (5 units g-1 dry biomass in the basis of pNPbetaG). LAB25g2 increased the final hydrolysis yield by a factor of 20% (44.5 ± 1.7% vs. 34.5 ± 1.5% in control conditions) after 96–120 h as compared to control reactions in its absence or in the presence of other commercial beta-glucosidase preparations. The high stability (half-life higher than 5 days at 50°C and pH 5.2) and 2–38000 fold higher (as compared with reported beta-glucosidases) activity towards cello-oligosaccharides may account for its performance in supplementation assays. Conclusions The results suggest that beta-glucosidases from yet uncultured bacteria from animal digestomes may be of a potential interest for biotechnological processes related to the effective bio-ethanol production in combination with low dosage of commercial cellulases

Distinct Geographical Distribution of the Miscanthus Accessions with Varied Biomass Enzymatic Saccharification

PubMed Central

Li, Xukai; Liao, Haofeng; Fan, Chunfen; Hu, Huizhen; Li, Ying; Li, Jing; Yi, Zili; Cai, Xiwen; Peng, Liangcai; Tu, Yuanyuan

2016-01-01

Miscanthus is a leading bioenergy candidate for biofuels, and it thus becomes essential to characterize the desire natural Miscanthus germplasm accessions with high biomass saccharification. In this study, total 171 natural Miscanthus accessions were geographically mapped using public database. According to the equation [P(H/L| East) = P(H/L∩East)/P(East)], the probability (P) parameters were calculated on relationships between geographical distributions of Miscanthus accessions in the East of China, and related factors with high(H) or low(L) values including biomass saccahrification under 1% NaOH and 1% H2SO4 pretreatments, lignocellulose features and climate conditions. Based on the maximum P value, a golden cutting line was generated from 42°25’ N, 108°22’ E to 22°58’ N, 116°28’ E on the original locations of Miscanthus accessions with high P(H|East) values (0.800–0.813), indicating that more than 90% Miscanthus accessions were originally located in the East with high biomass saccharification. Furthermore, the averaged insolation showed high P (H|East) and P(East|H) values at 0.782 and 0.754, whereas other climate factors had low P(East|H) values, suggesting that the averaged insolation is unique factor on Miscanthus distributions for biomass saccharification. In terms of cell wall compositions and wall polymer features, both hemicelluloses level and cellulose crystallinity (CrI) of Miscanthus accessions exhibited relative high P values, suggesting that they should be the major factors accounting for geographic distributions of Miscanthus accessions with high biomass digestibility. PMID:27532636

An Electrochemical Impedance Spectroscopy System for Monitoring Pineapple Waste Saccharification.

PubMed

Conesa, Claudia; Ibáñez Civera, Javier; Seguí, Lucía; Fito, Pedro; Laguarda-Miró, Nicolás

2016-02-04

Electrochemical impedance spectroscopy (EIS) has been used for monitoring the enzymatic pineapple waste hydrolysis process. The system employed consists of a device called Advanced Voltammetry, Impedance Spectroscopy & Potentiometry Analyzer (AVISPA) equipped with a specific software application and a stainless steel double needle electrode. EIS measurements were conducted at different saccharification time intervals: 0, 0.75, 1.5, 6, 12 and 24 h. Partial least squares (PLS) were used to model the relationship between the EIS measurements and the sugar determination by HPAEC-PAD. On the other hand, artificial neural networks: (multilayer feed forward architecture with quick propagation training algorithm and logistic-type transfer functions) gave the best results as predictive models for glucose, fructose, sucrose and total sugars. Coefficients of determination (R²) and root mean square errors of prediction (RMSEP) were determined as R² > 0.944 and RMSEP < 1.782 for PLS and R² > 0.973 and RMSEP < 0.486 for artificial neural networks (ANNs), respectively. Therefore, a combination of both an EIS-based technique and ANN models is suggested as a promising alternative to the traditional laboratory techniques for monitoring the pineapple waste saccharification step.

An Electrochemical Impedance Spectroscopy System for Monitoring Pineapple Waste Saccharification

PubMed Central

Conesa, Claudia; Ibáñez Civera, Javier; Seguí, Lucía; Fito, Pedro; Laguarda-Miró, Nicolás

2016-01-01

Electrochemical impedance spectroscopy (EIS) has been used for monitoring the enzymatic pineapple waste hydrolysis process. The system employed consists of a device called Advanced Voltammetry, Impedance Spectroscopy & Potentiometry Analyzer (AVISPA) equipped with a specific software application and a stainless steel double needle electrode. EIS measurements were conducted at different saccharification time intervals: 0, 0.75, 1.5, 6, 12 and 24 h. Partial least squares (PLS) were used to model the relationship between the EIS measurements and the sugar determination by HPAEC-PAD. On the other hand, artificial neural networks: (multilayer feed forward architecture with quick propagation training algorithm and logistic-type transfer functions) gave the best results as predictive models for glucose, fructose, sucrose and total sugars. Coefficients of determination (R2) and root mean square errors of prediction (RMSEP) were determined as R2 > 0.944 and RMSEP < 1.782 for PLS and R2 > 0.973 and RMSEP < 0.486 for artificial neural networks (ANNs), respectively. Therefore, a combination of both an EIS-based technique and ANN models is suggested as a promising alternative to the traditional laboratory techniques for monitoring the pineapple waste saccharification step. PMID:26861317

Importance of cellulase cocktails favoring hydrolysis of cellulose.

PubMed

Victoria, Juliet; Odaneth, Annamma; Lali, Arvind

2017-07-03

Depolymerization of lignocellulosic biomass is catalyzed by groups of enzymes whose action is influenced by substrate features and the composition of cellulase preparation. Cellulases contain a mixture of variety of enzymes, whose proportions dictate the saccharification of biomass. In the current study, four cellulase preparation varying in their composition were used to hydrolyze two types of alkali-treated biomass (aqueous ammonia-treated rice straw and sodium hydroxide-treated rice straw) to study the effect on catalytic rate, saccharification yields, and sugar release profile. We found that substrate features affected the extent of saccharification but had minimal effect on the sugar release pattern. In addition, complete hydrolysis to glucose was observed with enzyme preparation having at least a cellobiase units (CBU)/carboxymethyl cellulose (CMC) ratio (>0.15), while a modified enzyme ratio can be used for oligosaccharide synthesis. Thus, cellulase preparation with defined ratios of the three main enzymes can improve the saccharification which is of utmost importance in defining the success of lignocellulose-based economies.

Effect of pretreatment on simultaneous saccharification and fermentation of hardwood into acetone/butanol

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

Shah, M.M.; Song, S.K.; Lee, Y.Y.

1991-12-31

The effectiveness of pretreatments on hardwood substrate was investigated in connection with its subsequent conversion by simultaneous saccharification and fermentation (SSF), using Clostridium acetobutylicum. The main objectives of the pretreatment were to achieve efficient separation of lignin from carbohydrates, and to obtain maximum sugar yield on enzymatic hydrolysis of pretreated wood. Two methods have given promising results: (1) supercritical CO{sub 2}-SO{sub 2} treatment, and (2) monoethanolamine (MEA) treatment. The MEA pretreatment removed above 90% of hardwood lignin while retaining 83% of carbohydrates. With CO{sub 2}-SO{sub 2} pretreatment, the degree of lignin separation was lower. Under the scheme of SSF, themore » pretreated hardwood was converted to acetone, butanol, and ethanol (ABE) via single stage processing by cellulose enzyme system and C. acetobutylicum cells. The product yield in the process was such that 15 g of ABE/100 g of dry aspen wood was produced. In the overall process of SSF, the enzymatic hydrolysis was found to be the rate-limiting step. The ability of C. acetobutylicum to metabolize various 6-carbon and 5-carbon sugars resulted in efficient utilization of all available sugars from hardwood.« less

Valorisation of food and beverage waste via saccharification for sugars recovery.

PubMed

Kwan, Tsz Him; Ong, Khai Lun; Haque, Md Ariful; Kwan, Wing Hei; Kulkarni, Sandeep; Lin, Carol Sze Ki

2018-05-01

Valorisation of mixed food and beverage (F&B) waste was studied for the recovery of sugars via saccharification. Glucoamylase and sucrase were employed to hydrolyse the starch and sucrose present in the mixed F&B waste because of the high cost-effectiveness for such recovery. The Michaelis-Menten kinetics model suggests that preservatives and additives in beverages did not inhibit glucoamylase and sucrase during saccharification. High levels of glucose (228.1 g L -1 ) and fructose (55.7 g L -1 ) were efficiently produced within 12 h at a solid-to-liquid ratio of 37.5% (w/v) in 2.5 L bioreactors. An overall conversion yield of 0.17 g sugars per g of mixed F&B waste was obtained in mass balance analysis. Lastly, possible industrial applications of the sugar-rich hydrolysate and by-products are discussed. This study is believed to cast insights into F&B waste recycling via biotechnology to produce high-value added products to promote the establishment of a circular bio-economy. Copyright © 2018 Elsevier Ltd. All rights reserved.

Enzymatic saccharification and bioethanol production from Cynara cardunculus pretreated by steam explosion.

PubMed

Fernandes, Maria C; Ferro, Miguel D; Paulino, Ana F C; Mendes, Joana A S; Gravitis, Janis; Evtuguin, Dmitry V; Xavier, Ana M R B

2015-06-01

The correct choice of the specific lignocellulosic biomass pretreatment allows obtaining high biomass conversions for biorefinery implementations and cellulosic bioethanol production from renewable resources. Cynara cardunculus (cardoon) pretreated by steam explosion (SE) was involved in second-generation bioethanol production using separate hydrolysis and fermentation (SHF) or simultaneous saccharification and fermentation (SSF) processes. Steam explosion pretreatment led to partial solubilisation of hemicelluloses and increased the accessibility of residual polysaccharides towards enzymatic hydrolysis revealing 64% of sugars yield against 11% from untreated plant material. Alkaline extraction after SE pretreatment of cardoon (CSEOH) promoted partial removal of degraded lignin, tannins, extractives and hemicelluloses thus allowing to double glucose concentration upon saccharification step. Bioethanol fermentation in SSF mode was faster than SHF process providing the best results: ethanol concentration 18.7 g L(-1), fermentation efficiency of 66.6% and a yield of 26.6g ethanol/100 g CSEOH or 10.1 g ethanol/100 g untreated cardoon. Copyright © 2015 Elsevier Ltd. All rights reserved.

Effects of drying-induced fiber hornification on enzymatic saccharification of lignocelluloses

Treesearch

Xiaolin Luo; Junyong Zhu

2011-01-01

This study investigated the effect of fiber hornification during drying on lignocellulosic substrate enzymatic saccharification. Two chemically pretreated wood substrates and one commercial bleached kraft hardwood pulp were used. Heat drying at 105 and 150°C and air drying at 50% RH and 23.8°C for different durations were applied to produce substrate...

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.

Enzyme-assisted extraction enhancing the umami taste amino acids recovery from several cultivated mushrooms.

PubMed

Poojary, Mahesha M; Orlien, Vibeke; Passamonti, Paolo; Olsen, Karsten

2017-11-01

In this study, enzyme-assisted extraction was performed to extract umami taste and total free amino acids (FAAs) from the six different mushrooms including shiitake (Lentinus edodes), oyster (Pleurotus ostreatus), tea tree (Agrocybe aegerita) and, white, brown and portobello champignons (Agaricus bisporus). β-Glucanase and Flavourzyme® were used as the enzymes for cell wall and proteins hydrolysis, respectively. It was found that β-glucanase treatment alone did not enhance the extraction efficiency, however in combination, β-glucanase and Flavourzyme® enhanced the extraction efficiency significantly up to 20-fold compared to conventional HCl mediated extraction, depending on the mushroom species. The optimal conditions for the enzyme treatment were: water as extraction solvent (initial pH = 7), enzyme concentration of 5% v/w each of β-glucanase and Flavourzyme®, temperature 50°C and an incubation time of 1h. White and brown champignons were found to be the richest source of umami taste FAAs (26.75±1.07 and 25.6±0.9mg/g DM, respectively). Copyright © 2017 Elsevier Ltd. All rights reserved.

Enhancement of catalytic activity and thermostability of a thermostable cellobiohydrolase from Chaetomium thermophilum by site-directed mutagenesis.

PubMed

Han, Chao; Li, Weiguang; Hua, Chengyao; Sun, Fengqing; Bi, Pengsheng; Wang, Qunqing

2018-05-20

Enzymatic saccharification of lignocellulosic biomass is increasingly applied in agricultural and industrial applications. Nevertheless, low performance in the extreme environment severely prevents the utilization of commercial enzyme preparations. To obtain cellobiohydrolases with improved catalytic activity and thermostability, structure-based rational design was performed based on a thermostable cellobiohydrolase CtCel6 from Chaetomium thermophilum. In the present study, four conserved and noncatalytic residue substitutions were generated via site-directed mutagenesis. Mutations were heterologously expressed in yeast Pichia pastoris, purified, and ultimately assayed for enzymatic characteristics. The mutant Y119F increased the catalytic activity 1.82-, 1.65- and 1.43-fold against β-d-glucan, phosphoric acid swollen cellulose (PASC) and carboxymethylcellulose sodium (CMC-Na), respectively. In addition, S131 W effectively enhanced the enzyme's heat resistance to elevated temperatures. The half-life (t 1/2 ) of this mutant enzyme was increased 1.42- and 2.40-fold at 80 °C and 90 °C, respectively, compared to the wild-type. This study offers initial insight into the biological function of the conserved and noncatalytic residues of thermostable cellobiohydrolases and provides a valid approach to the improvement of enzyme redesign proposal. Copyright © 2018 Elsevier B.V. All rights reserved.

Effects of wet-pressing-induced fiber hornification on enzymatic saccharification of lignocelluloses

Treesearch

X.L. Luo; Junyong Zhu; Roland Gleisner; H.Y. Zhan

2011-01-01

This article reports the effect of wet-pressing-induced fiber hornification on enzymatic saccharification of lignocelluloses. A wet cellulosic substrate of bleached kraft eucalyptus pulp and two wet sulfite-pretreated lignocellulosic substrates of aspen and lodgepole pine were pressed to various moisture (solids) contents by variation of pressing pressure and pressing...

Alkaline peroxide pretreatment of corn stover for enzymatic saccharification and ethanol production

USDA-ARS?s Scientific Manuscript database

Alkaline hydrogen peroxide (AHP) pretreatment and enzymatic saccharification were evaluated for conversion of corn stover cellulose and hemicellulose to fermentable sugars. Corn stover used in this study contained 37.0±0.2% cellulose, 26.8±0.2% hemicellulose and 18.0±0.1% lignin on dry basis. Unde...

Evaluation of a recombinant insect-derived amylase performance in simultaneous saccharification and fermentation process with industrial yeasts.

PubMed

Celińska, Ewelina; Borkowska, Monika; Białas, Wojciech

2016-03-01

Starch is the dominant feedstock consumed for the bioethanol production, accounting for 60 % of its global production. Considering the significant contribution of bioethanol to the global fuel market, any improvement in its major operating technologies is economically very attractive. It was estimated that up to 40 % of the final ethanol unit price is derived from the energy input required for the substrate pre-treatment. Application of raw starch hydrolyzing enzymes (RSHE), combined with operation of the process according to a simultaneous saccharification and fermentation (SSF) strategy, constitutes the most promising solutions to the current technologies limitations. In this study, we expressed the novel RSHE derived from an insect in Saccharomyces cerevisiae strain dedicated for the protein overexpression. Afterwards, the enzyme performance was assessed in SSF process conducted by industrial ethanologenic or thermotolerant yeast species. Comparison of the insect-derived RSHE preparation with commercially available amylolytic RSH preparation was conducted. Our results demonstrate that the recombinant alpha-amylase from rice weevil can be efficiently expressed and secreted with its native signal peptide in S. cerevisiae INVSc-pYES2-Amy1 expression system (accounting for nearly 72 % of the strain's secretome). Application of the recombinant enzyme-based preparation in SSF process secured sufficient amylolytic activity for the yeast cell propagation and ethanol formation from raw starch. (Oligo)saccharide profiles generated by the compared preparations differed with respect to homogeneity of the sugar mixtures. Concomitantly, as demonstrated by a kinetic model developed in this study, the kinetic parameters describing activity of the compared preparations were different.

[Effect of N-terminal truncation of Bacillus acidopullulyticus pullulanase on enzyme properties and functions].

PubMed

Chen, A'na; Liu, Xiuxia; Dai, Xiaofeng; Zhan, Jinling; Peng, Feng; Li, Lu; Wang, Fen; Li, Song; Yang, Yankun; Bai, Zhonghu

2016-03-01

We constructed different N-terminal truncated variants based on Bacillus acidopullulyticus pullulanase 3D structure (PDB code 2WAN), and studied the effects of truncated mutation on soluble expression, enzymatic properties, and application in saccharification. Upon expression, the variants of X45 domain deletion existed as inclusion bodies, whereas deletion of CBM41 domain had an effective effect on soluble expression level. The variants that lack of CBM41 (M1), lack of X25 (M3), and lack both of CBM41 and X25 (M5) had the same optimal pH (5.0) and optimal temperature (60 degrees C) with the wild-type pullulanase (WT). The K(m) of M1 and M5 were 1.42 mg/mL and 1.85 mg/mL, respectively, 2.4- and 3.1-fold higher than that of the WT. k(cat)/K(m) value of M5 was 40% lower than that of the WT. Substrate specificity results show that the enzymes exhibited greater activity with the low-molecular-weight dextrin than with high-molecular-weight soluble starch. When pullulanases were added to the saccharification reaction system, the dextrose equivalent of the WT, M1, M3, and M5 were 93.6%, 94.7%, 94.5%, and93.1%, respectively. These results indicate that the deletion of CBM41 domain and/or X25 domain did not affect the practical application in starch saccharification process. Furthermore, low-molecular-weight variants facilitate the heterologous expression. Truncated variants may be more suitable for industrial production than the WT.

Parameter Estimation for Simultaneous Saccharification and Fermentation of Food Waste Into Ethanol Using Matlab Simulink

NASA Astrophysics Data System (ADS)

Davis, Rebecca Anne

The increase in waste disposal and energy costs has provided an incentive to convert carbohydrate-rich food waste streams into fuel. For example, dining halls and restaurants discard foods that require tipping fees for removal. An effective use of food waste may be the enzymatic hydrolysis of the waste to simple sugars and fermentation of the sugars to ethanol. As these wastes have complex compositions which may change day-to-day, experiments were carried out to test fermentability of two different types of food waste at 27° C using Saccharomyces cerevisiae yeast (ATCC4124) and Genencor's STARGEN™ enzyme in batch simultaneous saccharification and fermentation (SSF) experiments. A mathematical model of SSF based on experimentally matched rate equations for enzyme hydrolysis and yeast fermentation was developed in Matlab Simulink®. Using Simulink® parameter estimation 1.1.3, parameters for hydrolysis and fermentation were estimated through modified Michaelis-Menten and Monod-type equations with the aim of predicting changes in the levels of ethanol and glycerol from different initial concentrations of glucose, fructose, maltose, and starch. The model predictions and experimental observations agree reasonably well for the two food waste streams and a third validation dataset. The approach of using Simulink® as a dynamic visual model for SSF represents a simple method which can be applied to a variety of biological pathways and may be very useful for systems approaches in metabolic engineering in the future.

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...

Simultaneous saccharification and fermentation of industrial sweetpotatoes for ethanol production and anthocyanins extraction

USDA-ARS?s Scientific Manuscript database

A simultaneous saccharification fermentation (SSF) system was studied for ethanol production in flour industrial sweetpotato (ISP) feedstocks (lines: white DM02-180 and purple NC-413) as an integrated cost saving process, and to examine the feasibility of extracting anthocyanins from flour purple IS...

Barrier height enhancement of metal/semiconductor contact by an enzyme biofilm interlayer

NASA Astrophysics Data System (ADS)

Ocak, Yusuf Selim; Gul Guven, Reyhan; Tombak, Ahmet; Kilicoglu, Tahsin; Guven, Kemal; Dogru, Mehmet

2013-06-01

A metal/interlayer/semiconductor (Al/enzyme/p-Si) MIS device was fabricated using α-amylase enzyme as a thin biofilm interlayer. It was observed that the device showed an excellent rectifying behavior and the barrier height value of 0.78 eV for Al/α-amylase/p-Si was meaningfully larger than the one of 0.58 eV for conventional Al/p-Si metal/semiconductor (MS) contact. Enhancement of the interfacial potential barrier of Al/p-Si MS diode was realized using enzyme interlayer by influencing the space charge region of Si semiconductor. The electrical properties of the structure were executed by the help of current-voltage and capacitance-voltage measurements. The photovoltaic properties of the structure were executed under a solar simulator with AM1.5 global filter between 40 and 100 mW/cm2 illumination conditions. It was also reported that the α-amylase enzyme produced from Bacillus licheniformis had a 3.65 eV band gap value obtained from optical method.

Thermophilic Bacillus coagulans requires less cellulases for simultaneous saccharification and fermentation of cellulose to products than mesophilic microbial biocatalysts.

PubMed

Ou, Mark S; Mohammed, Nazimuddin; Ingram, L O; Shanmugam, K T

2009-05-01

Ethanol production from lignocellulosic biomass depends on simultaneous saccharification of cellulose to glucose by fungal cellulases and fermentation of glucose to ethanol by microbial biocatalysts (SSF). The cost of cellulase enzymes represents a significant challenge for the commercial conversion of lignocellulosic biomass into renewable chemicals such as ethanol and monomers for plastics. The cellulase concentration for optimum SSF of crystalline cellulose with fungal enzymes and a moderate thermophile, Bacillus coagulans, was determined to be about 7.5 FPU g(-1) cellulose. This is about three times lower than the amount of cellulase required for SSF with Saccharomyces cerevisiae, Zymomonas mobilis, or Lactococcus lactis subsp. lactis whose growth and fermentation temperature optimum is significantly lower than that of the fungal cellulase activity. In addition, B. coagulans also converted about 80% of the theoretical yield of products from 40 g/L of crystalline cellulose in about 48 h of SSF with 10 FPU g(-1) cellulose while yeast, during the same period, only produced about 50% of the highest yield produced at end of 7 days of SSF. These results show that a match in the temperature optima for cellulase activity and fermentation is essential for decreasing the cost of cellulase in cellulosic ethanol production.

The heat released during catalytic turnover enhances the diffusion of an enzyme

PubMed Central

Riedel, Clement; Gabizon, Ronen; Wilson, Christian A. M.; Hamadani, Kambiz; Tsekouras, Konstantinos; Marqusee, Susan; Pressé, Steve; Bustamante, Carlos

2015-01-01

Recent studies have shown that the diffusivity of enzymes increases in a substrate-dependent manner during catalysis1,2. Although this observation has been reported and characterized for several different systems3–10, the precise origin of this phenomenon is unknown. Calorimetric methods are often used to determine enthalpies from enzyme-catalysed reactions and can therefore provide important insight into their reaction mechanisms11,12. The ensemble averages involved in traditional bulk calorimetry cannot probe the transient effects that the energy exchanged in a reaction may have on the catalyst. Here we obtain single-molecule fluorescence correlation spectroscopy data and analyse them within the framework of a stochastic theory to demonstrate a mechanistic link between the enhanced diffusion of a single enzyme molecule and the heat released in the reaction. We propose that the heat released during catalysis generates an asymmetric pressure wave that results in a differential stress at the protein–solvent interface that transiently displaces the centre-of-mass of the enzyme (chemoacoustic effect). This novel perspective on how enzymes respond to the energy released during catalysis suggests a possible effect of the heat of reaction on the structural integrity and internal degrees of freedom of the enzyme. PMID:25487146

The heat released during catalytic turnover enhances the diffusion of an enzyme

DOE PAGES

Riedel, Clement; Gabizon, Ronen; Wilson, Christian A. M.; ...

2014-12-10

Recent studies have shown that the diffusivity of enzymes increases in a substrate-dependent manner during catalysis. Although this observation has been reported and characterized for several different systems, the precise origin of this phenomenon is unknown. Calorimetric methods are often used to determine enthalpies from enzyme-catalysed reactions and can therefore provide important insight into their reaction mechanisms. The ensemble averages involved in traditional bulk calorimetry cannot probe the transient effects that the energy exchanged in a reaction may have on the catalyst. Here we obtain single-molecule fluorescence correlation spectroscopy data and analyse them within the framework of a stochastic theorymore » to demonstrate a mechanistic link between the enhanced diffusion of a single enzyme molecule and the heat released in the reaction. We propose that the heat released during catalysis generates an asymmetric pressure wave that results in a differential stress at the protein-solvent interface that transiently displaces the centre-of-mass of the enzyme (chemoacoustic effect). We find this novel perspective on how enzymes respond to the energy released during catalysis suggests a possible effect of the heat of reaction on the structural integrity and internal degrees of freedom of the enzyme.« less

Development of a Commerical Enzyme System for Lignocellulosic Biomass Saccharification

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

Kumar, Manoj

2011-02-14

Lignocellulosic biomass is the most abundant, least expensive renewable natural biological resource for the production of biobased products and bioenergy is important for the sustainable development of human civilization in 21st century. For making the fermentable sugars from lignocellulosic biomass, a reduction in cellulase production cost, an improvement in cellulase performance, and an increase in sugar yields are all vital to reduce the processing costs of biorefineries. Improvements in specific cellulase activities for non-complexed cellulase mixtures can be implemented through cellulase engineering based on rational design or directed evolution for each cellulase component enzyme, as well as on the reconstitutionmore » of cellulase components. In this paper, we will provide DSM's efforts in cellulase research and developments and focus on limitations. Cellulase improvement strategies based on directed evolution using screening on relevant substrates, screening for higher thermal tolerance based on activity screening approaches such as continuous culture using insoluble cellulosic substrates as a powerful selection tool for enriching beneficial cellulase mutants from the large library. We will illustrate why and how thermostable cellulases are vital for economic delivery of bioproducts from cellulosic biomass using biochemical conversion approach.« less

Bacterial cellulose production from cotton-based waste textiles: enzymatic saccharification enhanced by ionic liquid pretreatment.

PubMed

Hong, Feng; Guo, Xiang; Zhang, Shuo; Han, Shi-fen; Yang, Guang; Jönsson, Leif J

2012-01-01

Cotton-based waste textiles were explored as alternative feedstock for production of bacterial cellulose (BC) by Gluconacetobacter xylinus. The cellulosic fabrics were treated with the ionic liquid (IL) 1-allyl-3-methylimidazolium chloride ([AMIM]Cl). [AMIM]Cl caused 25% inactivation of cellulase activity at a concentration as low as of 0.02 g/mL and decreased BC production during fermentation when present in concentrations higher than 0.0005 g/mL. Therefore, removal of residual IL by washing with hot water was highly beneficial to enzymatic saccharification as well as BC production. IL-treated fabrics exhibited a 5-7-fold higher enzymatic hydrolysis rate and gave a seven times larger yield of fermentable sugars than untreated fabrics. BC from cotton cloth hydrolysate was obtained at an yield of 10.8 g/L which was 83% higher than that from the culture grown on glucose-based medium. The BC from G. xylinus grown on IL-treated fabric hydrolysate had a 79% higher tensile strength than BC from glucose-based culture medium which suggests that waste cotton pretreated with [AMIM]Cl has potential to serve as a high-quality carbon source for BC production. Copyright © 2011 Elsevier Ltd. All rights reserved.

Coniferyl ferulate incorporation into lignin enhances the alkaline delignification and enzymatic degradation of cell walls.

PubMed

Grabber, John H; Hatfield, Ronald D; Lu, Fachuang; Ralph, John

2008-09-01

Incorporating ester interunit linkages into lignin could facilitate fiber delignification and utilization. In model studies with maize cell walls, we examined how partial substitution of coniferyl alcohol (a normal monolignol) with coniferyl ferulate (an ester conjugate from lignan biosynthesis) alters the formation and alkaline extractability of lignin and the enzymatic hydrolysis of structural polysaccharides. Coniferyl ferulate moderately reduced lignification and cell-wall ferulate copolymerization with monolignols. Incorporation of coniferyl ferulate increased lignin extractability by up to 2-fold in aqueous NaOH, providing an avenue for producing fiber with less noncellulosic and lignin contamination or of delignifying at lower temperatures. Cell walls lignified with coniferyl ferulate were more readily hydrolyzed with fibrolytic enzymes, both with and without alkaline pretreatment. Based on our results, bioengineering of plants to incorporate coniferyl ferulate into lignin should enhance lignocellulosic biomass saccharification and particularly pulping for paper production.

Application of thermophilic enzymes and water jet system to cassava pulp.

PubMed

Chaikaew, Siriporn; Maeno, Yuka; Visessanguan, Wonnop; Ogura, Kota; Sugino, Gaku; Lee, Seung-Hwan; Ishikawa, Kazuhiko

2012-12-01

Co-production of fermentable sugars and nanofibrillated cellulose from cassava pulp was achieved by the combination of thermophilic enzymes (endoglucanase, β-glucosidase, and α-amylase) and a new atomization system (Star Burst System; SBS), which employs opposing water jets. The SBS represents a key technology for providing cellulose nanofibers and improving the enzymatic saccharification of cassava pulp. Depending on the enzymes used, the production of glucose from cassava pulp treated with the SBS was 1.2- to 2.5-fold higher than that from pulp not treated with the SBS. Nanofibrillated cellulose with the gel-like property in suspension was produced (yield was over 90%) by α-amylase treatment, which completely released trapped starch granules from the fibrous cell wall structure of cassava pulp pretreated with the SBS. The SBS provides an environmentally low-impact pretreatment system for processing biomass material into value-added products. Copyright © 2012 Elsevier Ltd. All rights reserved.

High bioethanol titre from Manihot glaziovii through fed-batch simultaneous saccharification and fermentation in Automatic Gas Potential Test System.

PubMed

Moshi, Anselm P; Crespo, Carla F; Badshah, Malik; Hosea, Kenneth M M; Mshandete, Anthony Manoni; Mattiasson, Bo

2014-03-01

A process for the production of high bioethanol titre was established through fed-batch and simultaneous saccharification and fermentation (FB-SSF) of wild, non-edible cassava Manihot glaziovii. FB-SSF allowed fermentation of up to 390g/L of starch-derived glucose achieving high bioethanol concentration of up to 190g/L (24% v/v) with yields of around 94% of the theoretical value. The wild cassava M. glaziovii starch is hydrolysable with a low dosage of amylolytic enzymes (0.1-0.15% v/w, Termamyl® and AMG®). The Automatic Gas Potential Test System (AMPTS) was adapted to yeast ethanol fermentation and demonstrated to be an accurate, reliable and flexible device for studying the kinetics of yeast in SSF and FB-SSF. The bioethanol derived stoichiometrically from the CO2 registered in the AMPTS software correlated positively with samples analysed by HPLC (R(2)=0.99). Copyright © 2013 Elsevier Ltd. All rights reserved.

Designing Improved Enzymes of industrial application from marine microorganisms using Protein Engineering

NASA Astrophysics Data System (ADS)

Prajapati, A. S.; Panchal, K.; Subramanian, R. B.; Patel, D. H.; Sudhir, A. P.; Dave, B. R.

2015-12-01

Global demand for energy has grown with the development of new industries, requiring constant improvement and search for new sources of energy. One of the challenges today is releasing the energy of glucose that nature has cleverly locked into lignocellulosic biomass. Potent and efficient enzyme preparations need to be developed for the enzymatic saccharification process to be more economical. Approaches like enzyme engineering, reconstitution of enzyme mixtures and bioprospecting for superior enzymes are gaining importance. The ocean is considered to be a great reservoir of biodiversity. Because enzymes have unequalled advantages, many industries are keenly interested in adapting enzymatic methods for their processes. Microbial communities in marine environments are ecologically relevant as intermediaries of energy and play an important role in nutrient regeneration cycles as decomposers of dead and decaying organic matter. The exploitation of marine bacteria in the search for improved enzymes or strategies provides a means to upgrade feasibility for lignocellulosic biomass conversion, ultimately providing means to a 'greener' technology. Several industrial enzymes are derived from terrestrial sources, whereas, marine environment which encompasses about 71 percent of the earth's surface and a vast resources for useful enzymes, remain unexplored. Marine microorganisms take active part in the mineralization of complex organic matter through degradative pathways of their metabolism. Bacteria from marine environments secrete different enzymes based on their habitat and their ecological functions. Therefore marine microbial enzymes have become the focal point of interest. Even though many of these enzymes are being isolated, the efficiency of hydrolysis is very poor. This could be overcome by altering the substrate specificity of lignocellulases. Protein engineering could prove to be useful to improve the catalytic function these enzymes.

Production of L- and D-lactic acid from waste Curcuma longa biomass through simultaneous saccharification and cofermentation.

PubMed

Nguyen, Cuong Mai; Kim, Jin-Seog; Nguyen, Thanh Ngoc; Kim, Seul Ki; Choi, Gyung Ja; Choi, Yong Ho; Jang, Kyoung Soo; Kim, Jin-Cheol

2013-10-01

Simultaneous saccharification and cofermentation (SSCF) of Curcuma longa waste biomass obtained after turmeric extraction to L- and D-lactic acid by Lactobacillus coryniformis and Lactobacillus paracasei, respectively, was investigated. This is a rich, starchy, agro-industrial waste with potential for use in industrial applications. After optimizing the fermentation of the biomass by adjusting nitrogen sources, enzyme compositions, nitrogen concentrations, and raw material concentrations, the SSCF process was conducted in a 7-l jar fermentor at 140 g dried material/L. The maximum lactic acid concentration, average productivity, reducing sugar conversion and lactic acid yield were 97.13 g/L, 2.7 g/L/h, 95.99% and 69.38 g/100 g dried material for L-lactic acid production, respectively and 91.61 g/L, 2.08 g/L/h, 90.53% and 65.43 g/100 g dried material for D-lactic acid production, respectively. The simple and efficient process described in this study could be utilized by C. longa residue-based lactic acid industries without requiring the alteration of plant equipment. Copyright © 2013 Elsevier Ltd. All rights reserved.

Improved lignocellulose conversion to biofuels with thermophilic bacteria and thermostable enzymes.

PubMed

Bhalla, Aditya; Bansal, Namita; Kumar, Sudhir; Bischoff, Kenneth M; Sani, Rajesh K

2013-01-01

Second-generation feedstock, especially nonfood lignocellulosic biomass is a potential source for biofuel production. Cost-intensive physical, chemical, biological pretreatment operations and slow enzymatic hydrolysis make the overall process of lignocellulosic conversion into biofuels less economical than available fossil fuels. Lignocellulose conversions carried out at ≤ 50 °C have several limitations. Therefore, this review focuses on the importance of thermophilic bacteria and thermostable enzymes to overcome the limitations of existing lignocellulosic biomass conversion processes. The influence of high temperatures on various existing lignocellulose conversion processes and those that are under development, including separate hydrolysis and fermentation, simultaneous saccharification and fermentation, and extremophilic consolidated bioprocess are also discussed. Copyright © 2012 Elsevier Ltd. All rights reserved.

Microwave-Assisted Alkali Pre-Treatment, Densification and Enzymatic Saccharification of Canola Straw and Oat Hull

PubMed Central

Agu, Obiora S.; Tabil, Lope G.; Dumonceaux, Tim

2017-01-01

The effects of microwave-assisted alkali pre-treatment on pellets’ characteristics and enzymatic saccharification for bioethanol production using lignocellulosic biomass of canola straw and oat hull were investigated. The ground canola straw and oat hull were immersed in distilled water, sodium hydroxide and potassium hydroxide solutions at two concentrations (0.75% and 1.5% w/v) and exposed to microwave radiation at power level 713 W and three residence times (6, 12 and 18 min). Bulk and particle densities of ground biomass samples were determined. Alkaline-microwave pre-treated and untreated samples were subjected to single pelleting test in an Instron universal machine, pre-set to a load of 4000 N. The measured parameters, pellet density, tensile strength and dimensional stability were evaluated and the results showed that the microwave-assisted alkali pre-treated pellets had a significantly higher density and tensile strength compared to samples that were untreated or pre-treated by microwave alone. The chemical composition analysis showed that microwave-assisted alkali pre-treatment was able to disrupt and break down the lignocellulosic structure of the samples, creating an area of cellulose accessible to cellulase reactivity. The best enzymatic saccharification results gave a high glucose yield of 110.05 mg/g dry sample for canola straw ground in a 1.6 mm screen hammer mill and pre-treated with 1.5% NaOH for 18 min, and a 99.10 mg/g dry sample for oat hull ground in a 1.6 mm screen hammer mill and pre-treated with 0.75% NaOH for 18 min microwave-assisted alkali pre-treatments. The effects of pre-treatment results were supported by SEM analysis. Overall, it was found that microwave-assisted alkali pre-treatment of canola straw and oat hull at a short residence time enhanced glucose yield. PMID:28952504

Microwave-Assisted Alkali Pre-Treatment, Densification and Enzymatic Saccharification of Canola Straw and Oat Hull.

PubMed

Agu, Obiora S; Tabil, Lope G; Dumonceaux, Tim

2017-03-26

The effects of microwave-assisted alkali pre-treatment on pellets' characteristics and enzymatic saccharification for bioethanol production using lignocellulosic biomass of canola straw and oat hull were investigated. The ground canola straw and oat hull were immersed in distilled water, sodium hydroxide and potassium hydroxide solutions at two concentrations (0.75% and 1.5% w/v) and exposed to microwave radiation at power level 713 W and three residence times (6, 12 and 18 min). Bulk and particle densities of ground biomass samples were determined. Alkaline-microwave pre-treated and untreated samples were subjected to single pelleting test in an Instron universal machine, pre-set to a load of 4000 N. The measured parameters, pellet density, tensile strength and dimensional stability were evaluated and the results showed that the microwave-assisted alkali pre-treated pellets had a significantly higher density and tensile strength compared to samples that were untreated or pre-treated by microwave alone. The chemical composition analysis showed that microwave-assisted alkali pre-treatment was able to disrupt and break down the lignocellulosic structure of the samples, creating an area of cellulose accessible to cellulase reactivity. The best enzymatic saccharification results gave a high glucose yield of 110.05 mg/g dry sample for canola straw ground in a 1.6 mm screen hammer mill and pre-treated with 1.5% NaOH for 18 min, and a 99.10 mg/g dry sample for oat hull ground in a 1.6 mm screen hammer mill and pre-treated with 0.75% NaOH for 18 min microwave-assisted alkali pre-treatments. The effects of pre-treatment results were supported by SEM analysis. Overall, it was found that microwave-assisted alkali pre-treatment of canola straw and oat hull at a short residence time enhanced glucose yield.

Production of D-lactic acid from defatted rice bran by simultaneous saccharification and fermentation.

PubMed

Tanaka, Takaaki; Hoshina, Masahiro; Tanabe, Suguru; Sakai, Kenji; Ohtsubo, Sadami; Taniguchi, Masayuki

2006-01-01

Production of d-lactic acid from rice bran, one of the most abundant agricultural by-products in Japan, is studied. Lactobacillus delbrueckii subsp. delbrueckii IFO 3202 and defatted rice bran powder after squeezing rice oil were used for the production. Since the rice bran contains polysaccharides as starch and cellulose, we coupled saccharification with amylase and cellulase to lactic acid fermentation. The indigenous bacteria in the rice bran produced racemic lactic acid in the saccharification at pH 6.0-6.8. Thus the pH was controlled at 5.0 to suppress the growth of the indigenous bacteria. L. delbrueckii IFO 3202 produced 28 kgm(-3) lactic acid from 100 kgm(-3) rice bran after 36 h at 37 degrees C. The yield based on the amount of sugars soluble after 36-h hydrolysis of the bran by amylase and cellulase (36 kgm(-3) from 100 kgm(-3) of the bran) was 78%. The optical purity of produced d-lactic acid was 95% e.e.

A study of over production and enhanced secretion of enzymes. Quarterly report 1

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

Dashek, W.V.

1992-12-28

The current project is concerned with the over-production and enhanced secretion of PPO, cellulase and lignin peroxidase. The project is divided into two segments: over-production of lignocellulolytic enzymes by genetic engineering methodologies and hyper-production and enhanced secretion of these enzymes by biochemical/electron microscopical techniques. The former approach employs recombinant DNA procedures, ligation of appropriate nuclease generated DNA fragments into a vector and the subsequent transformation of Escherichia coli to yield E. coli harboring a C. versicolor DNA insert. The biochemistry/electron microscopical method involves substrate induction and the time-dependent addition of respiration and PPO inhibitors to elevate C.versicolor`s ability to synthesizemore » and secrete lignocellulosic enzymes. In this connection, cell fractionation/kinetic analysis, TEM immunoelectron microscopic localization and TEM substrate localization of PPO are being employed to assess the route of secretion. Both approaches will culminate in the batch culture of either E. coli or C. versicolor, in a fermentor with the subsequent development of rapid isolation and purification procedures to yield elevated quantities of pure lignocellulosic enzymes. During the past year, research effort were directed toward determining the route of polyphenol oxidase (PPO) secretion by the wood-decay fungus, Coriolus versicolor. In addition, research activities were continued to over-produce and to purify PPO as well as define the time-dependent intra- and extra-cellular appearances of C. versicolor ligninases and cellulases.« less

The ultrasound-enhanced bioscouring performance of four polygalacturonase enzymes obtained from rhizopus oryzae

USDA-ARS?s Scientific Manuscript database

An analytical and statistical method has been developed to measure the ultrasound-enhanced bioscouring performance of milligram quantities of endo- and exo-polygalacturonase enzymes obtained from Rhizopus oryzae fungi. UV-Vis spectrophotometric data and a general linear mixed models procedure indic...

Bioethanol production from ball milled bagasse using an on-site produced fungal enzyme cocktail and xylose-fermenting Pichia stipitis.

PubMed

Buaban, Benchaporn; Inoue, Hiroyuki; Yano, Shinichi; Tanapongpipat, Sutipa; Ruanglek, Vasimon; Champreda, Verawat; Pichyangkura, Rath; Rengpipat, Sirirat; Eurwilaichitr, Lily

2010-07-01

Sugarcane bagasse is one of the most promising agricultural by-products for conversion to biofuels. Here, ethanol fermentation from bagasse has been achieved using an integrated process combining mechanical pretreatment by ball milling, with enzymatic hydrolysis and fermentation. Ball milling for 2 h was sufficient for nearly complete cellulose structural transformation to an accessible amorphous form. The pretreated cellulosic residues were hydrolyzed by a crude enzyme preparation from Penicillium chrysogenum BCC4504 containing cellulase activity combined with Aspergillus flavus BCC7179 preparation containing complementary beta-glucosidase activity. Saccharification yields of 84.0% and 70.4% for glucose and xylose, respectively, were obtained after hydrolysis at 45 degrees C, pH 5 for 72 h, which were slightly higher than those obtained with a commercial enzyme mixture containing Acremonium cellulase and Optimash BG. A high conversion yield of undetoxified pretreated bagasse (5%, w/v) hydrolysate to ethanol was attained by separate hydrolysis and fermentation processes using Pichia stipitis BCC15191, at pH 5.5, 30 degrees C for 24 h resulting in an ethanol concentration of 8.4 g/l, corresponding to a conversion yield of 0.29 g ethanol/g available fermentable sugars. Comparable ethanol conversion efficiency was obtained by a simultaneous saccharification and fermentation process which led to production of 8.0 g/l ethanol after 72 h fermentation under the same conditions. This study thus demonstrated the potential use of a simple integrated process with minimal environmental impact with the use of promising alternative on-site enzymes and yeast for the production of ethanol from this potent lignocellulosic biomass. 2009. Published by Elsevier B.V.

Current knowledge on agarolytic enzymes and the industrial potential of agar-derived sugars.

PubMed

Yun, Eun Ju; Yu, Sora; Kim, Kyoung Heon

2017-07-01

Agar is a major cell wall carbohydrate of red macroalgae (Rhodophyta). Sugars derived from agar, such as agarooligosaccharides (AOSs), neoagarooligosaccharides (NAOSs), neoagarobiose (NAB), and 3,6-anhydro-L-galactose (L-AHG), possess various physiological activities. These agar-derived sugars can be produced by hydrolysis using chemicals or agarolytic enzymes. Despite the industrial potential of agar-derived sugars, their application has been hampered mainly due to the absence of efficient processes for the liquefaction and saccharification of agar. In this review, we have focused on strategies for producing high value-added sugars from agarose via chemical or enzymatic liquefaction and enzymatic saccharification. The liquefaction of agarose is a key step for preventing gelling and increasing the solubility of agarose in water by prehydrolyzing agarose into AOSs or NAOSs. For the industrial use of agar-derived sugars, AOS, NAOS, NAB, and L-AHG can be used as functional biomaterials owing to their physiological activities such as antiinflammation, skin whitening, and moisturizing. Recently, it was reported that AHG could be considered as a new anticariogenic sugar to replace xylitol. This review provides a comprehensive overview of processes for the hydrolysis of agar or agarose to produce high value-added sugars and the industrial application of these sugars.

Fundamental Challenges in Mechanistic Enzymology: Progress toward Understanding the Rate Enhancements of Enzymes

PubMed Central

Herschlag, Daniel; Natarajan, Aditya

2013-01-01

Enzymes are remarkable catalysts that lie at the heart of biology, accelerating chemical reactions to an astounding extent with extraordinary specificity. Enormous progress in understanding the chemical basis of enzymatic transformations and the basic mechanisms underlying rate enhancements over the past decades is apparent. Nevertheless, it has been difficult to achieve a quantitative understanding of how the underlying mechanisms account for the energetics of catalysis, because of the complexity of enzyme systems and the absence of underlying energetic additivity. We review case studies from our own work that illustrate the power of precisely defined and clearly articulated questions when dealing with such complex and multi-faceted systems, and we also use this approach to evaluate our current ability to design enzymes. We close by highlighting a series of questions that help frame some of what remains to be understood, and we encourage the reader to define additional questions and directions that will deepen and broaden our understanding of enzymes and their catalysis. PMID:23488725

Fundamental challenges in mechanistic enzymology: progress toward understanding the rate enhancements of enzymes.

PubMed

Herschlag, Daniel; Natarajan, Aditya

2013-03-26

Enzymes are remarkable catalysts that lie at the heart of biology, accelerating chemical reactions to an astounding extent with extraordinary specificity. Enormous progress in understanding the chemical basis of enzymatic transformations and the basic mechanisms underlying rate enhancements over the past decades is apparent. Nevertheless, it has been difficult to achieve a quantitative understanding of how the underlying mechanisms account for the energetics of catalysis, because of the complexity of enzyme systems and the absence of underlying energetic additivity. We review case studies from our own work that illustrate the power of precisely defined and clearly articulated questions when dealing with such complex and multifaceted systems, and we also use this approach to evaluate our current ability to design enzymes. We close by highlighting a series of questions that help frame some of what remains to be understood, and we encourage the reader to define additional questions and directions that will deepen and broaden our understanding of enzymes and their catalysis.

Enhancement of fruit shelf life by suppressing N-glycan processing enzymes.

PubMed

Meli, Vijaykumar S; Ghosh, Sumit; Prabha, T N; Chakraborty, Niranjan; Chakraborty, Subhra; Datta, Asis

2010-02-09

In a globalized economy, the control of fruit ripening is of strategic importance because excessive softening limits shelf life. Efforts have been made to reduce fruit softening in transgenic tomato through the suppression of genes encoding cell wall-degrading proteins. However, these have met with very limited success. N-glycans are reported to play an important role during fruit ripening, although the role of any particular enzyme is yet unknown. We have identified and targeted two ripening-specific N-glycoprotein modifying enzymes, alpha-mannosidase (alpha-Man) and beta-D-N-acetylhexosaminidase (beta-Hex). We show that their suppression enhances fruit shelf life, owing to the reduced rate of softening. Analysis of transgenic tomatoes revealed approximately 2.5- and approximately 2-fold firmer fruits in the alpha-Man and beta-Hex RNAi lines, respectively, and approximately 30 days of enhanced shelf life. Overexpression of alpha-Man or beta-Hex resulted in excessive fruit softening. Expression of alpha-Man and beta-Hex is induced by the ripening hormone ethylene and is modulated by a regulator of ripening, rin (ripening inhibitor). Furthermore, transcriptomic comparative studies demonstrate the down-regulation of cell wall degradation- and ripening-related genes in RNAi fruits. It is evident from these results that N-glycan processing is involved in ripening-associated fruit softening. Genetic manipulation of N-glycan processing can be of strategic importance to enhance fruit shelf life, without any negative effect on phenotype, including yield.

SpyRings Declassified: A Blueprint for Using Isopeptide-Mediated Cyclization to Enhance Enzyme Thermal Resilience.

PubMed

Schoene, C; Bennett, S P; Howarth, M

2016-01-01

Enzymes often have marginal stability, with unfolding typically leading to irreversible denaturation. This sensitivity is a major barrier, both for de novo enzyme development and for expanding enzyme impact beyond the laboratory. Seeking an approach to enhance resilience to denaturation that could be applied to a range of different enzymes, we developed SpyRing cyclization. SpyRings contain genetically encoded SpyTag (13 amino acids) on the N-terminus and SpyCatcher (12kDa) on the C-terminus of the enzyme, so that the Spy partners spontaneously react together through an irreversible isopeptide bond. SpyRing cyclization gave major increases in thermal resilience, including on a model for enzyme evolution, β-lactamase, and an industrially important enzyme in agriculture and nutrition, phytase. We outline the SpyRing rationale, including comparison of SpyRing cyclization to other cyclization strategies. The cloning strategy is presented for the simple insertion of enzyme genes for recombinant expression. We discuss structure-based approaches to select suitable enzyme cyclization targets. Approaches to evaluate the cyclization reaction and its effect on enzyme resilience are described. We also highlight the use of differential scanning calorimetry to understand how SpyRing cyclization promotes enzyme refolding. Efficiently searching sequence space will continue to be important for enzyme improvement, but the SpyRing platform may be a valuable rational adjunct for conferring resilience. © 2016 Elsevier Inc. All rights reserved.

Enhancing a Pathway-Genome Database (PGDB) to capture subcellular localization of metabolites and enzymes: the nucleotide-sugar biosynthetic pathways of Populus trichocarpa.

PubMed

Nag, Ambarish; Karpinets, Tatiana V; Chang, Christopher H; Bar-Peled, Maor

2012-01-01

Understanding how cellular metabolism works and is regulated requires that the underlying biochemical pathways be adequately represented and integrated with large metabolomic data sets to establish a robust network model. Genetically engineering energy crops to be less recalcitrant to saccharification requires detailed knowledge of plant polysaccharide structures and a thorough understanding of the metabolic pathways involved in forming and regulating cell-wall synthesis. Nucleotide-sugars are building blocks for synthesis of cell wall polysaccharides. The biosynthesis of nucleotide-sugars is catalyzed by a multitude of enzymes that reside in different subcellular organelles, and precise representation of these pathways requires accurate capture of this biological compartmentalization. The lack of simple localization cues in genomic sequence data and annotations however leads to missing compartmentalization information for eukaryotes in automatically generated databases, such as the Pathway-Genome Databases (PGDBs) of the SRI Pathway Tools software that drives much biochemical knowledge representation on the internet. In this report, we provide an informal mechanism using the existing Pathway Tools framework to integrate protein and metabolite sub-cellular localization data with the existing representation of the nucleotide-sugar metabolic pathways in a prototype PGDB for Populus trichocarpa. The enhanced pathway representations have been successfully used to map SNP abundance data to individual nucleotide-sugar biosynthetic genes in the PGDB. The manually curated pathway representations are more conducive to the construction of a computational platform that will allow the simulation of natural and engineered nucleotide-sugar precursor fluxes into specific recalcitrant polysaccharide(s). Database URL: The curated Populus PGDB is available in the BESC public portal at http://cricket.ornl.gov/cgi-bin/beocyc_home.cgi and the nucleotide-sugar biosynthetic pathways can

Enhancing a Pathway-Genome Database (PGDB) to capture subcellular localization of metabolites and enzymes: the nucleotide-sugar biosynthetic pathways of Populus trichocarpa

PubMed Central

Nag, Ambarish; Karpinets, Tatiana V.; Chang, Christopher H.; Bar-Peled, Maor

2012-01-01

Understanding how cellular metabolism works and is regulated requires that the underlying biochemical pathways be adequately represented and integrated with large metabolomic data sets to establish a robust network model. Genetically engineering energy crops to be less recalcitrant to saccharification requires detailed knowledge of plant polysaccharide structures and a thorough understanding of the metabolic pathways involved in forming and regulating cell-wall synthesis. Nucleotide-sugars are building blocks for synthesis of cell wall polysaccharides. The biosynthesis of nucleotide-sugars is catalyzed by a multitude of enzymes that reside in different subcellular organelles, and precise representation of these pathways requires accurate capture of this biological compartmentalization. The lack of simple localization cues in genomic sequence data and annotations however leads to missing compartmentalization information for eukaryotes in automatically generated databases, such as the Pathway-Genome Databases (PGDBs) of the SRI Pathway Tools software that drives much biochemical knowledge representation on the internet. In this report, we provide an informal mechanism using the existing Pathway Tools framework to integrate protein and metabolite sub-cellular localization data with the existing representation of the nucleotide-sugar metabolic pathways in a prototype PGDB for Populus trichocarpa. The enhanced pathway representations have been successfully used to map SNP abundance data to individual nucleotide-sugar biosynthetic genes in the PGDB. The manually curated pathway representations are more conducive to the construction of a computational platform that will allow the simulation of natural and engineered nucleotide-sugar precursor fluxes into specific recalcitrant polysaccharide(s). Database URL: The curated Populus PGDB is available in the BESC public portal at http://cricket.ornl.gov/cgi-bin/beocyc_home.cgi and the nucleotide-sugar biosynthetic pathways can

Restructuring the crystalline cellulose hydrogen bond network enhances its depolymerization rate

Treesearch

Shishir P.S. Chundawat; Giovanni Bellesia; Nirmal Uppugundla; Leonardo da Costa Sousa; Dahai Gao; Albert M. Cheh; Umesh P. Agarwal; Christopher M. Bianchetti; George N. Phillips; Paul Langan; Venkatesh Balan; S. Gnanakaran; Bruce E. Dale

2011-01-01

Conversion of lignocellulose to biofuels is partly inefficient due to the deleterious impact of cellulose crystallinity on enzymatic saccharification. We demonstrate how the synergistic activity of cellulases was enhanced by altering the hydrogen bond network within crystalline cellulose fibrils. We provide a molecular-scale explanation of these phenomena through...

Experiment of Enzyme Kinetics Using Guided Inquiry Model for Enhancing Generic Science Skills

NASA Astrophysics Data System (ADS)

Amida, N.; Supriyanti, F. M. T.; Liliasari

2017-02-01

This study aims to enhance generic science skills of students using guided inquiry model through experiments of enzyme kinetics. This study used quasi-experimental methods, with pretest-posttestnonequivalent control group design. Subjects of this study were chemistry students enrolled in biochemistry lab course, consisted of 18 students in experimental class and 19 students in control class. Instrument in this study were essay test that involves 5 indicators of generic science skills (i.e. direct observation, causality, symbolic language, mathematical modeling, and concepts formation) and also student worksheets. The results showed that the experiments of kinetics enzyme using guided inquiry model have been enhance generic science skills in high category with a value of average of 0.77. Four indicators classified in the high category are direct observation, causality, symbolic language, and mathematical modeling with the value of 0,73 0,70; 0,96; dan 0,85. Meanwhile, indicator of concepts formation in the medium category with a value of 0.62

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.

Development of a semi-continuous two-stage simultaneous saccharification and fermentation process for enhanced 2,3-butanediol production by Klebsiella oxytoca.

PubMed

Moon, S-K; Kim, D-K; Park, J M; Min, J; Song, H

2018-04-01

Klebsiella oxytoca naturally produces a large amount of 2,3-butanediol (2,3-BD), a promising chemical with wide industrial applications, along with various by-products. Previously, we have developed a metabolically engineered K. oxytoca ΔldhA ΔpflB strain to reduce the formation of by-products. To improve 2,3-BD productivity and examine the stability of K. oxytoca ΔldhA ΔpflB strain for industrial application, a semi-continuous two-stage simultaneous saccharification and fermentation (STSSF) process was developed. The STSSF with the K. oxytoca ΔldhA ΔpflB mutant using cassava as a carbon source could produce 108 ± 3·73 g (2,3- BD )  l -1 with a yield of 0·45 g (2,3- BD )  g (glucose) -1 and a productivity of 3·00 g (2,3- BD ) l -1  h -1 . No apparent changes in the final titre, yield and productivity of 2,3-BD were observed for up to 20 cycles of STSSF. Also, microbial contamination and spontaneous mutation of the host strain with potential detrimental effects on fermentation efficiency did not occur during the whole fermentation period. These results strongly underpin that the K. oxytoca ΔldhA ΔpflB mutant is stable and that the STSSF process is commercially exploitable. There is growing interest in the production of 2,3-butanediol (2,3-BD) from renewable resources by microbial fermentation because of its wide applications to specialty and commodity chemical industries. Klebsiella oxytoca usually produces 2,3-BD as a major end product during the fermentation of carbohydrates. This is the first study to provide a high-efficiency simultaneous saccharification and 2,3-BD fermentation process. Also, this study proves the stability of a metabolically engineered 2,3-BD overproducing K. oxytoca strain for industrial application. © 2018 The Society for Applied Microbiology.

Comparison of Dilute Acid and Ionic Liquid Pretreatment of Switchgrass: Biomass Recalcitrance, Delignification and Enzymatic Saccharification

USDA-ARS?s Scientific Manuscript database

The efficiency of two biomass pretreatment technologies, dilute acid hydrolysis and dissolution in an ionic liquid, are compared in terms of delignification, saccharification efficiency and saccharide yields with switchgrass serving as a model bioenergy crop. When subject to ionic liquid pretreatme...

Effects of tea saponin on glucan conversion and bonding behaviour of cellulolytic enzymes during enzymatic hydrolysis of corncob residue with high lignin content

PubMed Central

2013-01-01

Background Recently, interest in the utilization of corncob residue (CCR, with high lignin of 45.1%) as a feedstock for bioethanol has been growing. Surfactants have been one of the most popular additives intended to prevent the inhibitory effect of lignin on cellulolytic enzymes, thereby improving hydrolysis. In this study, the effects of biosurfactant tea saponin (TS) on the enzymatic hydrolysis of CCR and the bonding behavior of cellulolytic enzymes to the substrate were investigated. The surface tension in the supernatant was also detected to obtain information about the characteristics and stability of TS. Results The glucose concentration was 17.15 mg/mL at 120 hours of hydrolysis with the low loading of cellulolytic enzymes (7.0 FPU/g cellulose and 10.5 BGU/g cellulose) and 5% CCR. The optimal dosage of TS was its critical micelle concentration (cmc, 1.80 mg/mL). The glucose yield was enhanced from 34.29 to 46.28 g/100 g dry matter by TS. The results indicate that TS can promote the adsorption of cellulolytic enzymes on the substrate and mediate the release of adsorbed enzymes. Meanwhile, TS improves the recovery of the cellulolytic enzymes after a hydrolysis cycle and prevents deactivation of the enzymes during the intense shaking process. The surface tension in supernatants of digested CCR with TS remained at 50.00 mN/m during the course of hydrolysis. It is interesting to note that biosurfactant TS can maintain the surface tension in supernatants, despite its digestibility by cellulolytic enzymes. Conclusions Serving as an accelerant of lignocellulose hydrolysis, TS can also be degraded by the cellulolytic enzymes and release glucose while retaining stability, which reduces the cost of both the cellulolytic enzymes and the additive. As the glucose from the TS could be utilized by yeast, further efforts will investigate the mechanism of function and the application of TS in the production of ethanol by simultaneous saccharification and fermentation

Self-assembly of amphiphilic janus particles into monolayer capsules for enhanced enzyme catalysis in organic media.

PubMed

Cao, Wei; Huang, Renliang; Qi, Wei; Su, Rongxin; He, Zhimin

2015-01-14

Encapsulation of enzymes during the creation of an emulsion is a simple and efficient route for enhancing enzyme catalysis in organic media. Herein, we report a capsule with a shell comprising a monolayer of silica Janus particles (JPs) (referred to as a monolayer capsule) and a Pickering emulsion for the encapsulation of enzyme molecules for catalysis purposes in organic media using amphiphilic silica JPs as building blocks. We demonstrate that the JP capsules had a monolayer shell consisting of closely packed silica JPs (270 nm). The capsules were on average 5-50 μm in diameter. The stability of the JP capsules (Pickering emulsion) was investigated with the use of homogeneous silica nanoparticles as a control. The results show that the emulsion stabilized via amphiphilic silica JPs presented no obvious changes in physical appearance after 15 days, indicating the high stability of the emulsions and JP capsules. Furthermore, the lipase from Candida sp. was chosen as a model enzyme for encapsulation within the JP capsules during their formation. The catalytic performance of lipase was evaluated according to the esterification of 1-hexanol with hexanoic acid. It was found that the specific activity of the encapsulated enzymes (28.7 U mL(-1)) was more than 5.6 times higher than that of free enzymes in a biphasic system (5.1 U mL(-1)). The enzyme activity was further increased by varying the volume ratio of water to oil and the JPs loadings. The enzyme-loaded capsule also exhibited high stability during the reaction process and good recyclability. In particular, the jellification of agarose in the JP capsules further enhanced their operating stability. We believe that the monolayer structure of the JP capsules, together with their high stability, rendered the capsules to be ideal enzyme carriers and microreactors for enzyme catalysis in organic media because they created a large interfacial area and had low mass transfer resistance through the monolayer shell.

Interlinked population balance and cybernetic models for the simultaneous saccharification and fermentation of natural polymers.

PubMed

Ho, Yong Kuen; Doshi, Pankaj; Yeoh, Hak Koon; Ngoh, Gek Cheng

2015-10-01

Simultaneous Saccharification and Fermentation (SSF) is a process where microbes have to first excrete extracellular enzymes to break polymeric substrates such as starch or cellulose into edible nutrients, followed by in situ conversion of those nutrients into more valuable metabolites via fermentation. As such, SSF is very attractive as a one-pot synthesis method of biological products. However, due to the co-existence of multiple biochemical steps, modeling SSF faces two major challenges. The first is to capture the successive chain-end and/or random scission of the polymeric substrates over time, which determines the rate of generation of various fermentable substrates. The second is to incorporate the response of microbes, including their preferential substrate utilization, to such a complex broth. Each of the above-mentioned challenges has manifested itself in many related areas, and has been competently but separately attacked with two diametrically different tools, i.e., the Population Balance Modeling (PBM) and the Cybernetic Modeling (CM), respectively. To date, they have yet to be applied in unison on SSF resulting in a general inadequacy or haphazard approaches to examine the dynamics and interactions of depolymerization and fermentation. To overcome this unsatisfactory state of affairs, here, the general linkage between PBM and CM is established to model SSF. A notable feature is the flexible linkage, which allows the individual PBM and CM models to be independently modified to the desired levels of detail. A more general treatment of the secretion of extracellular enzyme is also proposed in the CM model. Through a case study on the growth of a recombinant Saccharomyces cerevisiae capable of excreting a chain-end scission enzyme (glucoamylase) on starch, the interlinked model calibrated using data from the literature (Nakamura et al., Biotechnol. Bioeng. 53:21-25, 1997), captured features not attainable by existing approaches. In particular, the effect

Enzymes and other agents that enhance cell wall extensibility

NASA Technical Reports Server (NTRS)

Cosgrove, D. J.

1999-01-01

Polysaccharides and proteins are secreted to the inner surface of the growing cell wall, where they assemble into a network that is mechanically strong, yet remains extensible until the cells cease growth. This review focuses on the agents that directly or indirectly enhance the extensibility properties of growing walls. The properties of expansins, endoglucanases, and xyloglucan transglycosylases are reviewed and their postulated roles in modulating wall extensibility are evaluated. A summary model for wall extension is presented, in which expansin is a primary agent of wall extension, whereas endoglucanases, xyloglucan endotransglycosylase, and other enzymes that alter wall structure act secondarily to modulate expansin action.

Silencing CAFFEOYL SHIKIMATE ESTERASE Affects Lignification and Improves Saccharification in Poplar.

PubMed

Saleme, Marina de Lyra Soriano; Cesarino, Igor; Vargas, Lívia; Kim, Hoon; Vanholme, Ruben; Goeminne, Geert; Van Acker, Rebecca; Fonseca, Fernando Campos de Assis; Pallidis, Andreas; Voorend, Wannes; Junior, José Nicomedes; Padmakshan, Dharshana; Van Doorsselaere, Jan; Ralph, John; Boerjan, Wout

2017-11-01

Caffeoyl shikimate esterase (CSE) was recently shown to play an essential role in lignin biosynthesis in Arabidopsis ( Arabidopsis thaliana ) and later in Medicago truncatula However, the general function of this enzyme was recently questioned by the apparent lack of CSE activity in lignifying tissues of different plant species. Here, we show that down-regulation of CSE in hybrid poplar ( Populus tremula × Populus alba ) resulted in up to 25% reduced lignin deposition, increased levels of p -hydroxyphenyl units in the lignin polymer, and a relatively higher cellulose content. The transgenic trees were morphologically indistinguishable from the wild type. Ultra-high-performance liquid chromatography-mass spectrometry-based phenolic profiling revealed a reduced abundance of several oligolignols containing guaiacyl and syringyl units and their corresponding hydroxycinnamaldehyde units, in agreement with the reduced flux toward coniferyl and sinapyl alcohol. These trees accumulated the CSE substrate caffeoyl shikimate along with other compounds belonging to the metabolic classes of benzenoids and hydroxycinnamates. Furthermore, the reduced lignin amount combined with the relative increase in cellulose content in the CSE down-regulated lines resulted in up to 62% more glucose released per plant upon limited saccharification when no pretreatment was applied and by up to 86% and 91% when acid and alkaline pretreatments were used. Our results show that CSE is not only important for the lignification process in poplar but is also a promising target for the development of improved lignocellulosic biomass crops for sugar platform biorefineries. © 2017 American Society of Plant Biologists. All Rights Reserved.

Multimodal analysis of pretreated biomass species highlights generic markers of lignocellulose recalcitrance.

PubMed

Herbaut, Mickaël; Zoghlami, Aya; Habrant, Anouck; Falourd, Xavier; Foucat, Loïc; Chabbert, Brigitte; Paës, Gabriel

2018-01-01

Biomass recalcitrance to enzymatic hydrolysis has been assigned to several structural and chemical factors. However, their relative importance remains challenging to evaluate. Three representative biomass species (wheat straw, poplar and miscanthus) were submitted to four standard pretreatments (dilute acid, hot water, ionic liquid and sodium chlorite) in order to generate a set of contrasted samples. A large array of techniques, including wet chemistry analysis, porosity measurements using NMR spectroscopy, electron and fluorescence microscopy, were used in order to determine possible generic factors of biomass recalcitrance. The pretreatment conditions selected allowed obtaining samples displaying different susceptibility to enzymatic hydrolysis (from 3 up to 98% of the initial glucose content released after 96 h of saccharification). Generic correlation coefficients were calculated between the measured chemical and structural features and the final saccharification rates. Increases in porosity displayed overall strong positive correlations with saccharification efficiency, but different porosity ranges were concerned depending on the considered biomass. Lignin-related factors displayed highly negative coefficients for all biomasses. Lignin content, which is likely involved in the correlations observed for porosity, was less detrimental to enzymatic hydrolysis than lignin composition. Lignin influence was highlighted by the strong negative correlation with fluorescence intensity which mainly originates from monolignols in mature tissues. Our results provide a better understanding of the factors responsible for biomass recalcitrance that can reasonably be considered as generic. The correlations with specific porosity ranges are biomass species-dependent, meaning that enzymes cocktails with fitted enzyme size are likely to be needed to optimise saccharification depending on the biomass origin. Lignin composition, which probably influences its structure, is the

Bioethanol production from the nutrient stress-induced microalga Chlorella vulgaris by enzymatic hydrolysis and immobilized yeast fermentation.

PubMed

Kim, Kyoung Hyoun; Choi, In Seong; Kim, Ho Myeong; Wi, Seung Gon; Bae, Hyeun-Jong

2014-02-01

The microalga Chlorella vulgaris is a potential feedstock for bioenergy due to its rapid growth, carbon dioxide fixation efficiency, and high accumulation of lipids and carbohydrates. In particular, the carbohydrates in microalgae make them a candidate for bioethanol feedstock. In this study, nutrient stress cultivation was employed to enhance the carbohydrate content of C. vulgaris. Nitrogen limitation increased the carbohydrate content to 22.4% from the normal content of 16.0% on dry weight basis. In addition, several pretreatment methods and enzymes were investigated to increase saccharification yields. Bead-beating pretreatment increased hydrolysis by 25% compared with the processes lacking pretreatment. In the enzymatic hydrolysis process, the pectinase enzyme group was superior for releasing fermentable sugars from carbohydrates in microalgae. In particular, pectinase from Aspergillus aculeatus displayed a 79% saccharification yield after 72h at 50°C. Using continuous immobilized yeast fermentation, microalgal hydrolysate was converted into ethanol at a yield of 89%. Copyright © 2013 Elsevier Ltd. 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.

Paradoxical enhancement of chemoreceptor detection sensitivity by a sensory adaptation enzyme

PubMed Central

Han, Xue-Sheng; Dahlquist, Frederick W.; Parkinson, John S.

2017-01-01

A sensory adaptation system that tunes chemoreceptor sensitivity enables motile Escherichia coli cells to track chemical gradients with high sensitivity over a wide dynamic range. Sensory adaptation involves feedback control of covalent receptor modifications by two enzymes: CheR, a methyltransferase, and CheB, a methylesterase. This study describes a CheR function that opposes the signaling consequences of its catalytic activity. In the presence of CheR, a variety of mutant serine chemoreceptors displayed up to 40-fold enhanced detection sensitivity to chemoeffector stimuli. This response enhancement effect did not require the known catalytic activity of CheR, but did involve a binding interaction between CheR and receptor molecules. Response enhancement was maximal at low CheR:receptor stoichiometry and quantitative analyses argued against a reversible binding interaction that simply shifts the ON–OFF equilibrium of receptor signaling complexes. Rather, a short-lived CheR binding interaction appears to promote a long-lasting change in receptor molecules, either a covalent modification or conformation that enhances their response to attractant ligands. PMID:28827352

Identification and Characterization of a 25 kDa Protein That Is Indispensable for the Efficient Saccharification of Eisenia bicyclis in the Digestive Fluid of Aplysia kurodai

PubMed Central

Tsuji, Akihiko; Kuwamura, Shuji; Shirai, Akihiro; Yuasa, Keizo

2017-01-01

The digestive fluid of the sea hare Aplysia kurodai can liberate approximately 2.5 mg of glucose from 10 mg of dried Eisenia bicyclis powder. Although laminaran, a major storage polysaccharide in E. bicyclis, is easily digested to glucose by the synergistic action of the 110 and 210 kDa A. kurodai β-glucosidases (BGLs), glucose is not liberated from E. bicyclis by direct incubation with these BGLs. To clarify this discrepancy, we searched for an Eisenia hydrolysis enhancing protein (EHEP) in the digestive fluid of A. kurodai. A novel 25 kDa protein that enhances E. bicyclis saccharification by β-glucosidases was purified to a homogeneous state from the digestive fluid of A. kurodai, and its cDNA was cloned from total cDNAs reverse-transcribed from hepatopancreas total RNA. The E. bicyclis extract strongly inhibited BGLs, suggesting some compound within this brown alga functioned as a feeding deterrent. However, when E. bicyclis was incubated with BGLs in the presence of EHEP, glucose production was markedly increased. As E. bicyclis is rich in phlorotannin, which are only found in brown algae, our study suggested that these compounds are the main BGL inhibitors in E. bicyclis extract. EHEP protects BGLs from phlorotannin inhibition by binding to phlorotannins and forming an insoluble complex with phloroglucinol and phlorotannins. These findings indicated that EHEP plays a key role in the saccharification of brown seaweeds containing phlorotannins in the digestive fluid of A. kurodai. This is the first report of EHEP as a phlorotannin-binding protein that protects BGLs from inhibition. PMID:28129373

Production of d-lactic acid from hardwood pulp by mechanical milling followed by simultaneous saccharification and fermentation using metabolically engineered Lactobacillus plantarum.

PubMed

Hama, Shinji; Mizuno, Shino; Kihara, Maki; Tanaka, Tsutomu; Ogino, Chiaki; Noda, Hideo; Kondo, Akihiko

2015-01-01

This study focused on the process development for the d-lactic acid production from cellulosic feedstocks using the Lactobacillus plantarum mutant, genetically modified to produce optically pure d-lactic acid from both glucose and xylose. The simultaneous saccharification and fermentation (SSF) using delignified hardwood pulp (5-15% loads) resulted in the lactic acid titers of 55.2-84.6g/L after 72h and increased productivities of 1.77-2.61g/L/h. To facilitate the enzymatic saccharification of high-load pulp at a fermentation temperature, short-term (⩽10min) pulverization of pulp was conducted, leading to a significantly improved saccharification with the suppressed formation of formic acid by-product. The short-term milling followed by SSF resulted in a lactic acid titer of 102.3g/L, an optical purity of 99.2%, and a yield of 0.879g/g-sugars without fed-batch process control. Therefore, the process presented here shows promise for the production of high-titer d-lactic acid using the L. plantarum mutant. Copyright © 2015 Elsevier Ltd. All rights reserved.

Simultaneous saccharification and ethanol fermentation of oxalic acid pretreated corncob assessed with response surface methodology

Treesearch

Jae-Won Lee; Rita C.L.B. Rodrigues; Thomas W. Jeffries

2009-01-01

Response surface methodology was used to evaluate optimal time, temperature and oxalic acid concentration for simultaneous saccharification and fermentation (SSF) of corncob particles by Pichia stipitis CBS 6054. Fifteen different conditions for pretreatment were examined in a 23 full factorial design with six axial points. Temperatures ranged from 132 to 180º...

Ionic liquid-tolerant cellulase enzymes

DOEpatents

Gladden, John; Park, Joshua; Singer, Steven; Simmons, Blake; Sale, Ken

2017-10-31

The present invention provides ionic liquid-tolerant cellulases and method of producing and using such cellulases. The cellulases of the invention are useful in saccharification reactions using ionic liquid treated biomass.

A Novel simultaneous-Saccharification-Fermentation Strategy for Efficient Co-fermentation of C5 and C6 Sugars Using Native, Non-GMO Yeasts

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

Varanasi, Sasidhar; Relue, Patricia

Economic bioethanol production is critically dependent upon the ability to convert both the hexose (C6) and pentose (C5) sugars resulting from cellulose and hemicellulose. C5 sugars are not readily fermentable by native Saccharomyces cerevisiae. Genetically Modified Organisms (GMOs) are designed to ferment xylose, but their stability, ethanol yield, environmental impact, and survival under conditions of industrial fermentation are unproven. In this project, we developed a novel approach for efficient fermentation of both C5 and C6 sugars using native S. Cerevisiae by exploiting its ability to produce ethanol from xylulose - the keto-isomer of xylose. While the isomerization of xylose tomore » xylulose can be accomplished via commercially (and cheaply) available Xylose Isomerase (XI) (Sweetzyme™), this conversion has an extremely unfavorable equilibrium (xylose:xylose is about 5:1). To address this, we developed two alternate strategies. In the first, the two enzymes XI and urease are coimmobilized on solid support particles to enable complete isomerization of xylose to xylulose under pH conditions suitable for fermentation, in a simultaneous-isomerization-fermentation (SIF) mode. The ability of our technology to conduct isomerization of xylose under pH conditions suitable for both saccharification and fermentation opens the possibility of SSF with native yeasts for the first time. Herein, we performed specific research tasks for implementation of our technology in several modes of operation, including simultaneous-isomerization-and-fermentation (SIF), simultaneous-saccharification-and-isomerization (SSI) followed by fermentation, and SSF mode with the biomass feedstock poplar. The projected economics of our process are very favorable in comparison to the costs associated with engineering, licensing and propagating GMOs. This novel fermentation technology is readily accessible to rural farming economies for implementation in cellulosic ethanol production

Cognitive enhancers (nootropics). Part 2: drugs interacting with enzymes.

PubMed

Froestl, Wolfgang; Muhs, Andreas; Pfeifer, Andrea

2013-01-01

Cognitive enhancers (nootropics) are drugs to treat cognition deficits in patients suffering from Alzheimer's disease, schizophrenia, stroke, attention deficit hyperactivity disorder, or aging. Cognition refers to a capacity for information processing, applying knowledge, and changing preferences. It involves memory, attention, executive functions, perception, language, and psychomotor functions. The term nootropics was coined in 1972 when memory enhancing properties of piracetam were observed in clinical trials. In the meantime, hundreds of drugs have been evaluated in clinical trials or in preclinical experiments. To classify the compounds, a concept is proposed assigning drugs to 19 categories according to their mechanism(s) of action, in particular drugs interacting with receptors, enzymes, ion channels, nerve growth factors, re-uptake transporters, antioxidants, metal chelators, and disease modifying drugs meaning small molecules, vaccines, and monoclonal antibodies interacting with amyloid-β and tau. For drugs whose mechanism of action is not known, they are either classified according to structure, e.g., peptides, or their origin, e.g., natural products. This review covers the evolution of research in this field over the last 25 years.

Steam pretreatment of agricultural residues facilitates hemicellulose recovery while enhancing enzyme accessibility to cellulose.

PubMed

Chandra, Richard P; Arantes, Valdeir; Saddler, Jack

2015-06-01

The origins of lignocellulosic biomass and the pretreatment used to enhance enzyme accessibility to the cellulosic component are known to be strongly influenced by various substrate characteristics. To assess the impact that fibre properties might have on enzymatic hydrolysis, seven agricultural residues were characterised before and after steam pretreatment using a single pretreatment condition (190°C, 5min, 3% SO2) previously shown to enhance fractionation and hydrolysis of the cellulosic component of corn stover. When the fibre length, width and coarseness, viscosity, water retention value and cellulose crystallinity were monitored, no clear correlation was observed between any single substrate characteristic and the substrate's ease of enzymatic hydrolysis. However, the amount of hemicellulose that was solubilised during pretreatment correlated (r(2)=0.98) with the effectiveness of enzyme hydrolysis of each pretreated substrate. Simons's staining, to measure the cellulose accessibility, showed good correlation (r(2)=0.83) with hemicellulose removal and the extent of enzymatic hydrolysis. Copyright © 2015 Elsevier Ltd. All rights reserved.

Sexual crossing of thermophilic fungus Myceliophthora heterothallica improved enzymatic degradation of sugar beet pulp.

PubMed

Aguilar-Pontes, Maria Victoria; Zhou, Miaomiao; van der Horst, Sjors; Theelen, Bart; de Vries, Ronald P; van den Brink, Joost

2016-01-01

Enzymatic degradation of plant biomass requires a complex mixture of many different enzymes. Like most fungi, thermophilic Myceliophthora species therefore have a large set of enzymes targeting different linkages in plant polysaccharides. The majority of these enzymes have not been functionally characterized, and their role in plant biomass degradation is unknown. The biotechnological challenge is to select the right set of enzymes to efficiently degrade a particular biomass. This study describes a strategy using sexual crossing and screening with the thermophilic fungus Myceliophthora heterothallica to identify specific enzymes associated with improved sugar beet pulp saccharification. Two genetically diverse M. heterothallica strains CBS 203.75 and CBS 663.74 were used to generate progenies with improved growth on sugar beet pulp. One progeny, named SBP.F1.2.11, had a different genetic pattern from the parental strains and had improved saccharification activity after the growth on 3 % sugar beet pulp. The improved SBP saccharification was not explained by altered activities of the major (hemi-)cellulases. Exo-proteome analysis of progeny and parental strains after 7-day growth on sugar beet pulp showed that only 17 of the 133 secreted CAZy enzymes were more abundant in progeny SBP.F1.2.11. Particularly one enzyme belonging to the carbohydrate esterase family 5 (CE5) was more abundant in SBP.F1.2.11. This CE5-CBM1 enzyme, named as Axe1, was phylogenetically related to acetyl xylan esterases. Biochemical characterization of Axe1 confirmed de-acetylation activity with optimal activities at 75-85 °C and pH 5.5-6.0. Supplementing Axe1 to CBS 203.75 enzyme set improved release of xylose and glucose from sugar beet pulp. This study identified beneficial enzymes for sugar beet pulp saccharification by selecting progeny with improved growth on this particular substrate. Saccharification of sugar beet pulp was improved by supplementing enzyme mixtures with a previously

Sonocatalytic-Fenton reaction for enhanced OH radical generation and its application to lignin degradation.

PubMed

Ninomiya, Kazuaki; Takamatsu, Hiromi; Onishi, Ayaka; Takahashi, Kenji; Shimizu, Nobuaki

2013-07-01

The present study demonstrated that the combined use of the sonocatalytic reaction (using ultrasound and titanium dioxide) and the Fenton reaction exhibited synergistically enhanced hydroxyl (OH) radical generation. Dihydroxybenzoic acid (DHBA) concentration as index of OH radical generation was 13 and 115 μM at 10 min in the sonocatalytic reaction and Fenton reaction, respectively. On the other hand, the DHBA concentration was 378 μM at 10 min in the sonocatalytic-Fenton reaction. The sonocatalytic-Fenton reaction was used for degradation of lignin. The lignin degradation ratio was 1.8%, 49.9%, and 60.0% at 180 min in the sonocatalytic reaction, Fenton reaction, and sonocatalytic-Fenton reaction, respectively. Moreover, the sonocatalytic-Fenton reaction was applied to pretreatment of lignocellulosic biomass to enhance subsequent enzymatic saccharification. The cellulose saccharification ratio was 11%, 14%, 16% and 25% at 360 min of pretreatment by control reaction, the sonocatalytic reaction, Fenton reaction, and sonocatalytic-Fenton reaction, respectively. Copyright © 2013 Elsevier B.V. All rights reserved.

In vitro bioconversion of chitin to pyruvate with thermophilic enzymes.

PubMed

Honda, Kohsuke; Kimura, Keisuke; Ninh, Pham Huynh; Taniguchi, Hironori; Okano, Kenji; Ohtake, Hisao

2017-09-01

Chitin is the second most abundant organic compound on the planet and thus has been regarded as an alternative resource to petroleum feedstocks. One of the key challenges in the biological conversion of biomass-derived polysaccharides, such as cellulose and chitin, is to close the gap between optimum temperatures for enzymatic saccharification and microbial fermentation and to implement them in a single bioreactor. To address this issue, in the present study, we aimed to perform an in vitro, one-pot bioconversion of chitin to pyruvate, which is a precursor of a wide range of useful metabolites. Twelve thermophilic enzymes, including that for NAD + regeneration, were heterologously produced in Escherichia coli and semi-purified by heat treatment of the crude extract of recombinant cells. When the experimentally decided concentrations of enzymes were incubated with 0.5 mg mL -1 colloidal chitin (equivalent to 2.5 mM N-acetylglucosamine unit) and an adequate set of cofactors at 70°C, 0.62 mM pyruvate was produced in 5 h. Despite the use of a cofactor-balanced pathway, determination of the pool sizes of cofactors showed a rapid decrease in ATP concentration, most probably due to the thermally stable ATP-degrading enzyme(s) derived from the host cell. Integration of an additional enzyme set of thermophilic adenylate kinase and polyphosphate kinase led to the deceleration of ATP degradation, and the final product titer was improved to 2.1 mM. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Simultaneous saccharification and bioethanol production from corn cobs: Process optimization and kinetic studies.

PubMed

Sewsynker-Sukai, Yeshona; Gueguim Kana, E B

2018-08-01

This study investigates the simultaneous saccharification and fermentation (SSF) process for bioethanol production from corn cobs with prehydrolysis (PSSF) and without prehydrolysis (OSSF). Two response surface models were developed with high coefficients of determination (>0.90). Process optimization gave high bioethanol concentrations and bioethanol conversions for the PSSF (36.92 ± 1.34 g/L and 62.36 ± 2.27%) and OSSF (35.04 ± 0.170 g/L and 58.13 ± 0.283%) models respectively. Additionally, the logistic and modified Gompertz models were used to study the kinetics of microbial cell growth and ethanol formation under microaerophilic and anaerobic conditions. Cell growth in the OSSF microaerophilic process gave the highest maximum specific growth rate (µ max ) of 0.274 h -1 . The PSSF microaerophilic bioprocess gave the highest potential maximum bioethanol concentration (P m ) (42.24 g/L). This study demonstrated that microaerophilic rather than anaerobic culture conditions enhanced cell growth and bioethanol production, and that additional prehydrolysis steps do not significantly impact on the bioethanol concentration and conversion in SSF process. Copyright © 2018 Elsevier Ltd. All rights reserved.

Two-step size reduction and post-washing of steam exploded corn stover improving simultaneous saccharification and fermentation for ethanol production.

PubMed

Liu, Zhi-Hua; Chen, Hong-Zhang

2017-01-01

The simultaneous saccharification and fermentation (SSF) of corn stover biomass for ethanol production was performed by integrating steam explosion (SE) pretreatment, hydrolysis and fermentation. Higher SE pretreatment severity and two-step size reduction increased the specific surface area, swollen volume and water holding capacity of steam exploded corn stover (SECS) and hence facilitated the efficiency of hydrolysis and fermentation. The ethanol production and yield in SSF increased with the decrease of particle size and post-washing of SECS prior to fermentation to remove the inhibitors. Under the SE conditions of 1.5MPa and 9min using 2.0cm particle size, glucan recovery and conversion to glucose by enzymes were 86.2% and 87.2%, respectively. The ethanol concentration and yield were 45.0g/L and 85.6%, respectively. With this two-step size reduction and post-washing strategy, the water utilization efficiency, sugar recovery and conversion, and ethanol concentration and yield by the SSF process were improved. Copyright © 2016 Elsevier Ltd. All rights reserved.

Development of a highly sensitive chemiluminescence enzyme immunoassay using enhanced luminol as substrate.

PubMed

Tao, Xiaoqi; Wang, Wenjun; Wang, Zhanhui; Cao, Xingyuan; Zhu, Jinghui; Niu, Lanlan; Wu, Xiaoping; Jiang, Haiyang; Shen, Jianzhong

2014-06-01

In this study, a high sensitivity chemiluminescence enzyme immunoassay (CLEIA) based on novel enhancers was developed. Under optimal conditions, we developed an enhanced chemiluminescence reaction (ECR) catalyzed by horseradish peroxidase (HRP-C) in the presence of 3-(10'-phenothiazinyl) propane-1-sulfonate (SPTZ) and 4-morpholinopyridine (MORP) as enhancers. The limit of detection of the newly prepared chemiluminescent cocktail for HRP was 0.33 pg/well, which is lower than that of commercial Super Signal substrate. The results showed that this novel chemiluminescent cocktail can significantly increase the light output of HRP-catalyzed ECR, which can be translated into a corresponding improvement in sensitivity. Similar improvements were observed in CLEIA for the determination of chloramphenicol in milk. In addition, the ECR of N-azoles as secondary enhancer was also presented. Copyright © 2013 John Wiley & Sons, Ltd.

Silencing CAFFEOYL SHIKIMATE ESTERASE Affects Lignification and Improves Saccharification in Poplar1[OPEN

PubMed Central

2017-01-01

Caffeoyl shikimate esterase (CSE) was recently shown to play an essential role in lignin biosynthesis in Arabidopsis (Arabidopsis thaliana) and later in Medicago truncatula. However, the general function of this enzyme was recently questioned by the apparent lack of CSE activity in lignifying tissues of different plant species. Here, we show that down-regulation of CSE in hybrid poplar (Populus tremula × Populus alba) resulted in up to 25% reduced lignin deposition, increased levels of p-hydroxyphenyl units in the lignin polymer, and a relatively higher cellulose content. The transgenic trees were morphologically indistinguishable from the wild type. Ultra-high-performance liquid chromatography-mass spectrometry-based phenolic profiling revealed a reduced abundance of several oligolignols containing guaiacyl and syringyl units and their corresponding hydroxycinnamaldehyde units, in agreement with the reduced flux toward coniferyl and sinapyl alcohol. These trees accumulated the CSE substrate caffeoyl shikimate along with other compounds belonging to the metabolic classes of benzenoids and hydroxycinnamates. Furthermore, the reduced lignin amount combined with the relative increase in cellulose content in the CSE down-regulated lines resulted in up to 62% more glucose released per plant upon limited saccharification when no pretreatment was applied and by up to 86% and 91% when acid and alkaline pretreatments were used. Our results show that CSE is not only important for the lignification process in poplar but is also a promising target for the development of improved lignocellulosic biomass crops for sugar platform biorefineries. PMID:28878037

Treatment of biomass to obtain fermentable sugars

DOEpatents

Dunson, Jr., James B.; Tucker, Melvin [Lakewood, CO; Elander, Richard [Evergreen, CO; Hennessey, Susan M [Avondale, PA

2011-04-26

Biomass is pretreated using a low concentration of aqueous ammonia at high biomass concentration. Pretreated biomass is further hydrolyzed with a saccharification enzyme consortium. Fermentable sugars released by saccharification may be utilized for the production of target chemicals by fermentation.

Farm Deployable Microbial Bioreactor for Fuel Ethanol Production

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

Okeke, Benedict

Research was conducted to develop a farm and field deployable microbial bioreactor for bioethanol production from biomass. Experiments were conducted to select the most efficient microorganisms for conversion of plant fiber to sugars for fermentation to ethanol. Mixtures of biomass and surface soil samples were collected from selected sites in Alabama black belt counties (Macon, Sumter, Choctaw, Dallas, Montgomery, Lowndes) and other areas within the state of Alabama. Experiments were conducted to determine the effects of culture parameters on key biomass saccharifying enzymes (cellulase, beta-glucosidase, xylanase and beta-xylosidase). A wide-scale sampling of locally-grown fruits in Central Alabama was embarked tomore » isolate potential xylose fermenting microorganisms. Yeast isolates were evaluated for xylose fermentation. Selected microorganisms were characterized by DNA based methods. Factors affecting enzyme production and biomass saccharification were examined and optimized in the laboratory. Methods of biomass pretreatment were compared. Co-production of amylolytic enzymes with celluloytic-xylanolytic enzymes was evaluated; and co-saccharification of a combination of biomass, and starch-rich materials was examined. Simultaneous saccharification and fermentation with and without pre-saccharifcation was studied. Whole culture broth and filtered culture broth simultaneous saccahrifcation and fermentation were compared. A bioreactor system was designed and constructed to employ laboratory results for scale up of biomass saccharification.« less

High temperature pre-digestion of corn stover biomass for improved product yields

DOE PAGES

Brunecky, Roman; Hobdey, Sarah E.; Taylor, Larry E.; ...

2014-12-03

Introduction: The efficient conversion of lignocellulosic feedstocks remains a key step in the commercialization of biofuels. One of the barriers to cost-effective conversion of lignocellulosic biomass to sugars remains the enzymatic saccharification process step. Here, we describe a novel hybrid processing approach comprising enzymatic pre-digestion with newly characterized hyperthermophilic enzyme cocktails followed by conventional saccharification with commercial enzyme preparations. Dilute acid pretreated corn stover was subjected to this new procedure to test its efficacy. Thermal tolerant enzymes from Acidothermus cellulolyticus and Caldicellulosiruptor bescii were used to pre-digest pretreated biomass at elevated temperatures prior to saccharification by the commercial cellulase formulation.more » Results: We report that pre-digestion of biomass with these enzymes at elevated temperatures prior to addition of the commercial cellulase formulation increased conversion rates and yields when compared to commercial cellulase formulation alone under low solids conditions. In conclusion, Our results demonstrating improvements in rates and yields of conversion point the way forward for hybrid biomass conversion schemes utilizing catalytic amounts of hyperthermophilic enzymes.« less

White-rot fungal pretreatment of wheat straw with Phanerochaete chrysosporium for biohydrogen production: simultaneous saccharification and fermentation.

PubMed

Zhi, Zelun; Wang, Hui

2014-07-01

This paper demonstrates biohydrogen production was enhanced by white-rot fungal pretreatment of wheat straw (WS) through simultaneous saccharification and fermentation (SSF). Wheat straw was pretreated by Phanerochaete chrysosporium at 30 °C under solid state fermentation for 12 days, and lignin was removed about 28.5 ± 1.3 %. Microscopic structure observation combined thermal gravity and differential thermal gravity analysis further showed that the lignocellulose structure obviously disrupted after fungal pretreatment. Subsequently, the pretreated WS and crude cellulases prepared from Trichoderma atroviride were applied in SSF for hydrogen production using Clostridium perfringens. The maximum hydrogen yield was obtained to be 78.5 ± 3.4 ml g(-1)-pretreated WS, which was about 1.8-fold than the unpretreated group. Furthermore, the modified Gompertz model was applied study the progress of cumulative H(2) production. This work developed a novel bio-approach to improve fermentative H(2) yield from lignocellulosic biomass.

Simultaneous saccharification and fermentation of steam exploded duckweed: Improvement of the ethanol yield by increasing yeast titre

PubMed Central

Zhao, X.; Moates, G.K.; Elliston, A.; Wilson, D.R.; Coleman, M.J.; Waldron, K.W.

2015-01-01

This study investigated the conversion of Lemna minor biomass to bioethanol. The biomass was pre-treated by steam explosion (SE, 210 °C, 10 min) and then subjected to simultaneous saccharification and fermentation (SSF) using Cellic® CTec 2 (20 U or 0.87 FPU g−1 substrate) cellulase plus β-glucosidase (2 U g−1 substrate) and a yeast inoculum of 10% (v/v or 8.0 × 107 cells mL−1). At a substrate concentration of 1% (w/v) an ethanol yield of 80% (w/w, theoretical) was achieved. However at a substrate concentration of 20% (w/v), the ethanol yield was lowered to 18.8% (w/w, theoretical). Yields were considerably improved by increasing the yeast titre in the inoculum or preconditioning the yeast on steam exploded liquor. These approaches enhanced the ethanol yield up to 70% (w/w, theoretical) at a substrate concentration of 20% (w/v) by metabolising fermentation inhibitors. PMID:26210138

Steam explosion treatment for ethanol production from branches pruned from pear trees by simultaneous saccharification and fermentation.

PubMed

Sasaki, Chizuru; Okumura, Ryosuke; Asada, Chikako; Nakamura, Yoshitoshi

2014-01-01

This study investigated the production of ethanol from unutilized branches pruned from pear trees by steam explosion pretreatment. Steam pressures of 25, 35, and 45 atm were applied for 5 min, followed by enzymatic saccharification of the extracted residues with cellulase (Cellic CTec2). High glucose recoveries, of 93.3, 99.7, and 87.1%, of the total sugar derived from the cellulose were obtained from water- and methanol-extracted residues after steam explosion at 25, 35, and 45 tm, respectively. These values corresponded to 34.9, 34.3, and 27.1 g of glucose per 100 g of dry steam-exploded branches. Simultaneous saccharification and fermentation experiments were done on water-extracted residues and water- and methanol-extracted residues by Kluyveromyces marxianus NBRC 1777. An overall highest theoretical ethanol yield of 76% of the total sugar derived from cellulose was achieved when 100 g/L of water- and methanol-washed residues from 35 atm-exploded pear branches was used as substrate.

Conversion of Aqueous Ammonia-Treated Corn Stover to Lactic Acid by Simultaneous Saccharification and Cofermentation

NASA Astrophysics Data System (ADS)

Zhu, Yongming; Lee, Y. Y.; Elander, Richard T.

Treatment of corn stover with aqueous ammonia removes most of the structural lignin, whereas retaining the majority of the carbohydrates in the solids. After treatment, both the cellulose and hemicellulose in corn stover become highly susceptible to enzymatic digestion. In this study, corn stover treated by aqueous ammonia was investigated as the substrate for lactic acid production by simultaneous saccharification and cofermentation (SSCF). A commercial cellulase (Spezyme-CP) and Lactobacillus pentosus American Type Culture Collection (ATCC) 8041 (Spanish Type Culture Collection [CECT]-4023) were used for hydrolysis and fermentation, respectively. In batch SSCF operation, the carbohydrates in the treated corn stover were converted to lactic acid with high yields, the maximum lactic acid yield reaching 92% of the stoichiometric maximum based on total fermentable carbohydrates (glucose, xylose, and arabinose). A small amount of acetic acid was also produced from pentoses through the phosphoketolase pathway. Among the major process variables for batch SSCF, enzyme loading and the amount of yeast extract were found to be the key factors affecting lactic acid production. Further tests on nutrients indicated that corn steep liquor could be substituted for yeast extract as a nitrogen source to achieve the same lactic acid yield. Fed-batch operation of the SSCF was beneficial in raising the concentration of lactic acid to a maximum value of 75.0 g/L.

On energy consumption for size-reduction and yields from subsequent enzymatic saccharification of pretreated lodgepole pine

Treesearch

W. Zhu; Junyong Zhu; Roland Gleisner; X.J. Pan

2010-01-01

This study investigated the effects of chemical pretreatment and disk-milling conditions on energy consumption for size-reduction and the efficiency of enzymatic cellulose saccharification of a softwood. Lodgepole pine wood chips produced from thinnings of a 100-year-old unmanaged forest were pretreated by hot-water, dilute-acid, and two SPORL processes (Sulfite...

Expression of a mutated SPT15 gene in Saccharomyces cerevisiae enhances both cell growth and ethanol production in microaerobic batch, fed-batch, and simultaneous saccharification and fermentations.

PubMed

Seong, Yeong-Je; Park, Haeseong; Yang, Jungwoo; Kim, Soo-Jung; Choi, Wonja; Kim, Kyoung Heon; Park, Yong-Cheol

2017-05-01

The SPT15 gene encodes a Saccharomyces cerevisiae TATA-binding protein, which is able to globally control the transcription levels of various metabolic and regulatory genes. In this study, a SPT15 gene mutant (S42N, S78R, S163P, and I212N) was expressed in S. cerevisiae BY4741 (BSPT15-M3), of which effects on fermentative yeast properties were evaluated in a series of culture types. By applying different nitrogen sources and air supply conditions in batch culture, organic nitrogen sources and microaerobic condition were decided to be more favorable for both cell growth and ethanol production of the BSPT15-M3 strain than the control S. cerevisiae BY4741 strain expressing the SPT15 gene (BSPT15wt). Microaerobic fed-batch cultures of BSPT15-M3 with glucose shock in the presence of high ethanol content resulted in a 9.5-13.4% higher glucose consumption rate and ethanol productivity than those for the BSPT15wt strain. In addition, BSPT15-M3 showed 4.5 and 3.9% increases in ethanol productivity from cassava hydrolysates and corn starch in simultaneous saccharification and fermentation processes, respectively. It was concluded that overexpression of the mutated SPT15 gene would be a potent strategy to develop robust S. cerevisiae strains with enhanced cell growth and ethanol production abilities.

Systematic optimization of fed-batch simultaneous saccharification and fermentation at high-solid loading based on enzymatic hydrolysis and dynamic metabolic modeling of Saccharomyces cerevisiae.

PubMed

Unrean, Pornkamol; Khajeeram, Sutamat; Laoteng, Kobkul

2016-03-01

An integrative simultaneous saccharification and fermentation (SSF) modeling is a useful guiding tool for rapid process optimization to meet the techno-economic requirement of industrial-scale lignocellulosic ethanol production. In this work, we have developed the SSF model composing of a metabolic network of a Saccharomyces cerevisiae cell associated with fermentation kinetics and enzyme hydrolysis model to quantitatively capture dynamic responses of yeast cell growth and fermentation during SSF. By using model-based design of feeding profiles for substrate and yeast cell in the fed-batch SSF process, an efficient ethanol production with high titer of up to 65 g/L and high yield of 85 % of theoretical yield was accomplished. The ethanol titer and productivity was increased by 47 and 41 %, correspondingly, in optimized fed-batch SSF as compared to batch process. The developed integrative SSF model is, therefore, considered as a promising approach for systematic design of economical and sustainable SSF bioprocessing of lignocellulose.

Batch fermentation options for high titer bioethanol production from a SPORL pretreated Douglas-Fir forest residue without detoxification

Treesearch

Mingyan Yang; Hairui Ji; Junyong Zhu

2016-01-01

This study evaluated batch fermentation modes, namely, separate hydrolysis and fermentation (SHF), quasi-simultaneous saccharification and fermentation (Q-SSF), and simultaneous saccharification and fermentation (SSF), and fermentation conditions, i.e., enzyme and yeast loadings, nutrient supplementation and sterilization, on high titer bioethanol production from SPORL...

Enhancement of photoassimilate utilization by manipulation of starch regulatory enzymes

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

Okita, Thomas W.

maturation to a starch granule. Although Pho1 catalyzes a reversible reaction, our DoE supported studies clearly demonstrated that the kinetic properties of this enzyme strongly favor synthesis of starch and that these catalytic properties are independent of the L80 peptide, a structural domain that is absent in phosphorylases from other organisms. Interesting expression of a Pho1 lacking the L80 peptide enhanced plant growth and seed yields, suggesting that Pho1 has a second function in controlling growth. Overall, results from these biochemical and physiological studies have increased our fundamental understanding on how these important starch regulatory enzymes operate at the molecular level and in planta, which will collectively aid in efforts to increase the utilization of higher plants as a renewable source of energy.« less

Enzyme-MOF (metal-organic framework) composites.

PubMed

Lian, Xizhen; Fang, Yu; Joseph, Elizabeth; Wang, Qi; Li, Jialuo; Banerjee, Sayan; Lollar, Christina; Wang, Xuan; Zhou, Hong-Cai

2017-06-06

The ex vivo application of enzymes in various processes, especially via enzyme immobilization techniques, has been extensively studied in recent years in order to enhance the recyclability of enzymes, to minimize enzyme contamination in the product, and to explore novel horizons for enzymes in biomedical applications. Possessing remarkable amenability in structural design of the frameworks as well as almost unparalelled surface tunability, Metal-Organic Frameworks (MOFs) have been gaining popularity as candidates for enzyme immobilization platforms. Many MOF-enzyme composites have achieved unprecedented results, far outperforming free enzymes in many aspects. This review summarizes recent developments of MOF-enzyme composites with special emphasis on preparative techniques and the synergistic effects of enzymes and MOFs. The applications of MOF-enzyme composites, primarily in transferation, catalysis and sensing, are presented as well. The enhancement of enzymatic activity of the composites over free enzymes in biologically incompatible conditions is emphasized in many cases.

Synergistic enhancement of cellulase pairs linked by consensus ankyrin repeats: determination of the roles of spacing, orientation and enzyme identity

PubMed Central

Cunha, Eva S.; Hatem, Christine L.; Barrick, Doug

2017-01-01

Biomass deconstruction to small simple sugars is a potential approach to biofuels production, however the highly recalcitrant nature of biomass limits the economic viability of this approach. Thus, research on efficient biomass degradation is necessary to achieve large-scale production of biofuels. Enhancement of cellulolytic activity by increasing synergism between cellulase enzymes holds promise in achieving high-yield biofuels production. Here we have inserted cellulase pairs from extremophiles into hyper-stable α-helical consensus ankyrin repeat domain scaffolds. Such chimeric constructs allowed us to optimize arrays of enzyme pairs against a variety of cellulolytic substrates. We found that endocellulolytic domains CelA (CA) and Cel12A (C12A) act synergistically in the context of ankyrin repeats, with both three and four repeat spacing. The extent of synergy differs for different substrates. Also, having C12A N-terminal to CA provides greater synergy than the reverse construct, especially against filter paper. In contrast, we do not see synergy for these enzymes in tandem with CelK (CK) catalytic domain, a larger exocellulase, demonstrating the importance of enzyme identity in synergistic enhancement. Furthermore, we found endocellulases CelD and CA with three repeat spacing to act synergistically against filter paper. Importantly, connecting CA and C12A with a disordered linker of similar contour length, shows no synergistic enhancement, indicating that synergism results from connecting these domains with folded ankyrin repeats. These results show that ankyrin arrays can be used to vary spacing and orientation between enzymes, helping to design and optimize artificial cellulosomes, providing a novel architecture for synergistic enhancement of enzymatic cellulose degradation. PMID:27071357

Cognitive enhancing effect of angiotensin-converting enzyme inhibitors and angiotensin receptor blockers on learning and memory

PubMed Central

Nade, V. S.; Kawale, L. A.; Valte, K. D.; Shendye, N. V.

2015-01-01

Objective: The present study was designed to investigate cognitive enhancing property of angiotensin-converting enzymes inhibitors (ACEI) and angiotensin receptor blockers (ARBs) in rats. Materials and Methods: The elevated plus maze (EPM), passive avoidance test (PAT), and water maze test (WMT) were used to assess cognitive enhancing activity in young and aged rats. Ramipril (10 mg/kg, p.o.), perindopril (10 mg/kg, i.p), losartan (20 mg/kg, i.p), and valsartan (20 mg/kg, p.o) were administered to assess their effect on learning and memory. Scopolamine (1 mg/kg, i.p) was used to impair cognitive function. Piracetam (200 mg/kg, i.p) was used as reference drug. Results: All the treatments significantly attenuated amnesia induced by aging and scopolamine. In EPM, aged and scopolamine-treated rats showed an increase in transfer latency (TL) whereas, ACEI and ARBs showed a significant decrease in TL. Treatment with ACEI and ARBs significantly increased step down latencies and decreased latency to reach the platform in target quadrant in young, aged and scopolamine-treated animals in PAT and WMT, respectively. The treatments inhibited acetylcholinesterase (AChE) enzyme in the brain. Similarly, all the treatments attenuated scopolamine-induced lipid peroxidation and normalize antioxidant enzymes. Conclusion: The results suggest that the cognitive enhancing effect of ACEI and ARBs may be due to inhibition of AChE or by regulation of antioxidant system or increase in formation of angiotensin IV. PMID:26069362

Synergistic Enhancement of Enzyme Performance and Resilience via Orthogonal Peptide-Protein Chemistry Enabled Multilayer Construction.

PubMed

Zhang, Xue-Jian; Wang, Xiao-Wei; Sun, Jiaxing; Su, Chao; Yang, Shuguang; Zhang, Wen-Bin

2018-05-16

Protein immobilization is critical to utilize their unique functions in diverse applications. Herein, we report that orthogonal peptide-protein chemistry enabled multilayer construction can facilitate the incorporation of various folded structural domains, including calmodulin in different states, affibody and dihydrofolate reductase (DHFR). An extended conformation is found to be the most advantageous for steady film growth. The resulting protein thin films exhibit sensitive and selective responsive behaviors to bio-signals (Ca2+, TFP, NADPH, etc.) and fully maintain the catalytic activity of DHFR. The approach is applicable to different substrates such as hydrophobic gold and hydrophilic silica microparticles. The DHFR enzyme can be immobilized onto silica microparticles with tunable amounts. The multi-layer set-up exhibits a synergistic enhancement of DHFR activity with increasing number of bilayers and also makes the embedded DHFR more resilient to lyophilization. Therefore, this is a convenient and versatile method for protein immobilization with potential benefits of synergistic enhancement in enzyme performance and resilience.

Enzyme Engineering for In Situ Immobilization.

PubMed

Rehm, Fabian B H; Chen, Shuxiong; Rehm, Bernd H A

2016-10-14

Enzymes are used as biocatalysts in a vast range of industrial applications. Immobilization of enzymes to solid supports or their self-assembly into insoluble particles enhances their applicability by strongly improving properties such as stability in changing environments, re-usability and applicability in continuous biocatalytic processes. The possibility of co-immobilizing various functionally related enzymes involved in multistep synthesis, conversion or degradation reactions enables the design of multifunctional biocatalyst with enhanced performance compared to their soluble counterparts. This review provides a brief overview of up-to-date in vitro immobilization strategies while focusing on recent advances in enzyme engineering towards in situ self-assembly into insoluble particles. In situ self-assembly approaches include the bioengineering of bacteria to abundantly form enzymatically active inclusion bodies such as enzyme inclusions or enzyme-coated polyhydroxyalkanoate granules. These one-step production strategies for immobilized enzymes avoid prefabrication of the carrier as well as chemical cross-linking or attachment to a support material while the controlled oriented display strongly enhances the fraction of accessible catalytic sites and hence functional enzymes.

Enhanced bioethanol production from pretreated corn stover via multi-positive effect of casein micelles.

PubMed

Eckard, Anahita Dehkhoda; Muthukumarappan, Kasiviswanathan; Gibbons, William

2013-05-01

Casein polypeptides containing substructures of αs1-casein, β-casein, k-casein, αs2-casein were used as a lignin-blocker at above critical micelles concentration to improve the bioethanol production of dilute acid, lime, alkali, extrusion and AFEX pretreated corn stover (CS). Application of 0.5 g/g glucan of casein was found to effectively increase the glucose yield of CS pretreated with dilute acid, lime, alkali, extrusion and AFEX by 31.9%, 17.0%, 22.7%, 29.5%, and 17.4%, respectively with no positive impact on Avicel. Consequently 96 h simultaneous saccharification and fermentation (SSF) of these hydrolysates reduced the fermentation period by up to 48 h and increased the theoretical yield of ethanol by 8.48-33.7% compared to control. Application of casein during saccharification reduced the enzyme utilization by 33.0%. Recycling of hydrolysate from casein-treated CS for a 2nd round hydrolysis resulted in average glucose yield of 36.4% compared to 29.0% control. Copyright © 2012 Elsevier Ltd. All rights reserved.

Downregulation of RWA genes in hybrid aspen affects xylan acetylation and wood saccharification.

PubMed

Pawar, Prashant Mohan-Anupama; Ratke, Christine; Balasubramanian, Vimal K; Chong, Sun-Li; Gandla, Madhavi Latha; Adriasola, Mathilda; Sparrman, Tobias; Hedenström, Mattias; Szwaj, Klaudia; Derba-Maceluch, Marta; Gaertner, Cyril; Mouille, Gregory; Ezcurra, Ines; Tenkanen, Maija; Jönsson, Leif J; Mellerowicz, Ewa J

2017-06-01

High acetylation of angiosperm wood hinders its conversion to sugars by glycoside hydrolases, subsequent ethanol fermentation and (hence) its use for biofuel production. We studied the REDUCED WALL ACETYLATION (RWA) gene family of the hardwood model Populus to evaluate its potential for improving saccharification. The family has two clades, AB and CD, containing two genes each. All four genes are expressed in developing wood but only RWA-A and -B are activated by master switches of the secondary cell wall PtNST1 and PtMYB21. Histochemical analysis of promoter::GUS lines in hybrid aspen (Populus tremula × tremuloides) showed activation of RWA-A and -B promoters in the secondary wall formation zone, while RWA-C and -D promoter activity was diffuse. Ectopic downregulation of either clade reduced wood xylan and xyloglucan acetylation. Suppressing both clades simultaneously using the wood-specific promoter reduced wood acetylation by 25% and decreased acetylation at position 2 of Xylp in the dimethyl sulfoxide-extracted xylan. This did not affect plant growth but decreased xylose and increased glucose contents in the noncellulosic monosaccharide fraction, and increased glucose and xylose yields of wood enzymatic hydrolysis without pretreatment. Both RWA clades regulate wood xylan acetylation in aspen and are promising targets to improve wood saccharification. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

Enhanced cellulase recovery without β-glucosidase supplementation for cellulosic ethanol production using an engineered strain and surfactant.

PubMed

Huang, Renliang; Guo, Hong; Su, Rongxin; Qi, Wei; He, Zhimin

2017-03-01

Recycling cellulases by substrate adsorption is a promising strategy for reducing the enzyme cost of cellulosic ethanol production. However, β-glucosidase has no carbohydrate-binding module (CBM). Thus, additional enzymes are required in each cycle to achieve a high ethanol yield. In this study, we report a new method of recycling cellulases without β-glucosidase supplementation using lignocellulosic substrate, an engineered strain expressing β-glucosidase and Tween 80. The cellulases and Tween 80 were added to an aqueous suspension of diluted sulfuric acid/ammonia-treated corncobs in a simultaneous saccharification and fermentation (SSF) process for ethanol production. Subsequently, the addition of fresh pretreated corncobs to the fermentation liquor and remaining solid residue provided substrates with absorbed cellulases for the next SSF cycle. This method provided excellent ethanol production in three successive SSF cycles without requiring the addition of new cellulases. For a 10% (w/v) solid loading, a cellulase dosage of 30 filter paper units (FPU)/g cellulose, 0.5% Tween 80, and 2 g/L of the engineered strain, approximately 90% of the initial ethanol concentration from the first SSF process was obtained in the next two SSF processes, with a total ethanol production of 306.27 g/kg corncobs and an enzyme productivity of 0.044 g/FPU. Tween 80 played an important role in enhancing cellulase recovery. This new enzyme recycling method is more efficient and practical than other reported methods. Biotechnol. Bioeng. 2017;114: 543-551. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Effect of enzyme secreting bacterial pretreatment on enhancement of aerobic digestion potential of waste activated sludge interceded through EDTA.

PubMed

Kavitha, S; Adish Kumar, S; Yogalakshmi, K N; Kaliappan, S; Rajesh Banu, J

2013-12-01

In this study, the effect of Ethylene diamine tetra acetic acid (EDTA) on Extracellular polymeric substance (EPS) removal tailed with bacterial enzymatic pretreatment on aerobic digestion of activated sludge was studied. In order to enhance the accessibility of sludge to the enzyme secreting bacteria; the extracellular polymeric substances were removed using EDTA. EDTA efficiently removed the EPS with limited cell lysis and enhanced the sludge enzyme activity at its lower concentration of 0.2 g/g SS. The sludge was then subjected to bacterial pretreatment to enhance the aerobic digestion. In aerobic digestion the best results in terms of Suspended solids (SS) reduction (48.5%) and COD (Chemical oxygen demand) solubilization (47.3%) was obtained in experimental reactor than in control. These results imply that aerobic digestion can be enhanced efficiently through bacterial pretreatment of EPS removed sludge. Copyright © 2013 Elsevier Ltd. All rights reserved.

Alkali-explosion pretreatment of straw and bagasse for enzymic hydrolysis.

PubMed

Puri, V P; Pearce, G R

1986-04-01

Sugarcane bagasse and wheat straw were subjected to alkali treatment at 200 degrees C for 5 min and at 3.45 MPa gas pressure (steam and nitrogen), followed by an explosive discharge through a defibrating nozzle, in an attempt to improve the rate and extent of digestibility. The treatment resulted in the solubilization of 40-45% of the components and in the production of a pulp that gave saccharification yields of 80 and 65% in 8 h for bagasse and wheat straw, respectively. By comparison, alkali steaming at 200 degrees C (1.72 MPa) for 5 min gave saccharification yields of only 58 and 52% in 48 h. The increase in temperature from 140 to 200 degrees C resulted in a gradual increase in in vitro organic matter digestibility (IVOMD) for both the substrates. Also, the extent of alkalinity during pretreatment appears to effect the reactivity of the final product towards enzymes. Pretreatment times ranging from 5 to 60 caused a progressive decline in the IVOMD of bagasse and wheat straw by the alkali explosion method and this was accompanied by a progressive decrease in pH values after explosion. In the alkali-steaming method, pretreatment time had no apparent effect with either substrate. An analysis of the alkali-exploded products showed that substantial amounts of hemicellulose and a small proportion of the lignin were solubilized. The percentage crystallinity of the cellulose did not alter in either substrate but there was a substantial reduction in the degree of polymerization. The superiority of the alkali-explosion pretreatment is attributed to the efficacy of fiber separation and disintegration; this increases the surface area and reduces the degree of polymerization.

Phenotypic selection of a wild Saccharomyces cerevisiae strain for simultaneous saccharification and co-fermentation of AFEX pretreated corn stover

Treesearch

Mingie Jin; Cory Sarks; Christa Gunawan; Benjamin D. Bice; Shane P. Simonett; Ragothaman Avanasi Narasimhan; Laura B. Willis; Bruce E. Dale; Venkatesh Balan; Trey K. Sato

2013-01-01

Simultaneous saccharification and co-fermentation (SSCF) process involves enzymatic hydrolysis of pretreated lignocellulosic biomass and fermentation of glucose and xylose in one bioreactor. The optimal temperatures for enzymatic hydrolysis are higher than the standard fermentation temperature of ethanologenic Saccharomyces cerevisiae. Moreover,...

Hydroxycinnamate Conjugates as Potential Monolignol Replacements: In vitro Lignification and Cell Wall Studies with Rosmarinic Acid

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

Yuki, Tobimatsu; Sasikumar, Elumalai; Grabber, John H.

2012-04-01

The plasticity of lignin biosynthesis should permit the inclusion of new compatible phenolic monomers, such as rosmarinic acid (RA) and analogous catechol derivatives, into cell-wall lignins that are consequently less recalcitrant to biomass processing. In vitro lignin polymerization experiments revealed that RA readily underwent peroxidase-catalyzed copolymerization with monolignols and lignin oligomers to form polymers with new benzodioxane inter-unit linkages. Incorporation of RA permitted extensive depolymerization of synthetic lignins by mild alkaline hydrolysis, presumably by cleavage of ester intra-unit linkages within RA. Copolymerization of RA with monolignols into maize cell walls by in situ peroxidases significantly enhanced alkaline lignin extractability andmore » promoted subsequent cell wall saccharification by fungal enzymes. Incorporating RA also improved cell wall saccharification by fungal enzymes and by rumen microflora even without alkaline pretreatments, possibly by modulating lignin hydrophobicity and/or limiting cell wall cross-linking. Consequently, we anticipate that bioengineering approaches for partial monolignol substitution with RA and analogous plant hydroxycinnamates would permit more efficient utilization of plant fiber for biofuels or livestock production.« less

Production of L-lactic Acid from Biomass Wastes Using Scallop Crude Enzymes and Novel Lactic Acid Bacterium

NASA Astrophysics Data System (ADS)

Yanagisawa, Mitsunori; Nakamura, Kanami; Nakasaki, Kiyohiko

In the present study, biomass waste raw materials including paper mill sludge, bamboo, sea lettuce, and shochu residue (from a distiller) and crude enzymes derived from inedible and discarded scallop parts were used to produce L-lactic acid for the raw material of biodegradable plastic poly-lactic acid. The activities of cellulase and amylase in the crude enzymes were 22 and 170units/L, respectively, and L-lactic acid was produced from every of the above mentioned biomass wastes, by the method of liquid-state simultaneous saccharification and fermentation (SSF) . The L-lactic acid concentrations produced from sea lettuce and shochu residue, which contain high concentration of starch were 3.6 and 9.3g/L, respectively, and corresponded to greater than 25% of the conversion of glucans contained in these biomass wastes. Furthermore, using the solid state SSF method, concentrations as high as 13g/L of L-lactic acid were obtained from sea lettuce and 26g/L were obtained from shochu residue.

Comparative evaluation of chemical and enzymatic saccharification of mixotrophically grown de-oiled microalgal biomass for reducing sugar production.

PubMed

Pancha, Imran; Chokshi, Kaumeel; Maurya, Rahulkumar; Bhattacharya, Sourish; Bachani, Pooja; Mishra, Sandhya

2016-03-01

For the commercialization of microalgal based biofuels, utilization of de-oiled carbohydrate rich biomass is important. In the present study, chemo-enzymatic hydrolysis of mixotrophically grown Scenedesmus sp. CCNM 1077 de-oiled biomass is evaluated. Among the chemical hydrolysis, use of 0.5M HCl for 45 min at 121°C resulted in highest saccharification yield of 37.87% w/w of de-oiled biomass. However, enzymatic hydrolysis using Viscozyme L at loading rate of 20 FBGU/g of de-oiled biomass, pH 5.5 and temperature 45°C for 72 h resulted in saccharification yield of 43.44% w/w of de-oiled biomass. Further, 78% ethanol production efficiency was achieved with enzymatically hydrolyzed de-oiled biomass using yeast Saccharomyces cerevisiae ATCC 6793. These findings of the present study show application of mixotrophically grown de-oiled biomass of Scenedesmus sp. CCNM 1077 as promising feedstock for bioethanol production. Copyright © 2016 Elsevier Ltd. All rights reserved.

Applying functional metagenomics to search for novel lignocellulosic enzymes in a microbial consortium derived from a thermophilic composting phase of sugarcane bagasse and cow manure.

PubMed

Colombo, Lívia Tavares; de Oliveira, Marcelo Nagem Valério; Carneiro, Deisy Guimarães; de Souza, Robson Assis; Alvim, Mariana Caroline Tocantins; Dos Santos, Josenilda Carlos; da Silva, Cynthia Canêdo; Vidigal, Pedro Marcus Pereira; da Silveira, Wendel Batista; Passos, Flávia Maria Lopes

2016-09-01

Environments where lignocellulosic biomass is naturally decomposed are sources for discovery of new hydrolytic enzymes that can reduce the high cost of enzymatic cocktails for second-generation ethanol production. Metagenomic analysis was applied to discover genes coding carbohydrate-depleting enzymes from a microbial laboratory subculture using a mix of sugarcane bagasse and cow manure in the thermophilic composting phase. From a fosmid library, 182 clones had the ability to hydrolyse carbohydrate. Sequencing of 30 fosmids resulted in 12 contigs encoding 34 putative carbohydrate-active enzymes belonging to 17 glycosyl hydrolase (GH) families. One third of the putative proteins belong to the GH3 family, which includes β-glucosidase enzymes known to be important in the cellulose-deconstruction process but present with low activity in commercial enzyme preparations. Phylogenetic analysis of the amino acid sequences of seven selected proteins, including three β-glucosidases, showed low relatedness with protein sequences deposited in databases. These findings highlight microbial consortia obtained from a mixture of decomposing biomass residues, such as sugar cane bagasse and cow manure, as a rich resource of novel enzymes potentially useful in biotechnology for saccharification of lignocellulosic substrate.

Synergistic enhancement of cellulase pairs linked by consensus ankyrin repeats: Determination of the roles of spacing, orientation, and enzyme identity.

PubMed

Cunha, Eva S; Hatem, Christine L; Barrick, Doug

2016-08-01

Biomass deconstruction to small simple sugars is a potential approach to biofuels production; however, the highly recalcitrant nature of biomass limits the economic viability of this approach. Thus, research on efficient biomass degradation is necessary to achieve large-scale production of biofuels. Enhancement of cellulolytic activity by increasing synergism between cellulase enzymes holds promise in achieving high-yield biofuels production. Here we have inserted cellulase pairs from extremophiles into hyperstable α-helical consensus ankyrin repeat domain scaffolds. Such chimeric constructs allowed us to optimize arrays of enzyme pairs against a variety of cellulolytic substrates. We found that endocellulolytic domains CelA (CA) and Cel12A (C12A) act synergistically in the context of ankyrin repeats, with both three and four repeat spacing. The extent of synergy differs for different substrates. Also, having C12A N-terminal to CA provides greater synergy than the reverse construct, especially against filter paper. In contrast, we do not see synergy for these enzymes in tandem with CelK (CK) catalytic domain, a larger exocellulase, demonstrating the importance of enzyme identity in synergistic enhancement. Furthermore, we found endocellulases CelD and CA with three repeat spacing to act synergistically against filter paper. Importantly, connecting CA and C12A with a disordered linker of similar contour length shows no synergistic enhancement, indicating that synergism results from connecting these domains with folded ankyrin repeats. These results show that ankyrin arrays can be used to vary spacing and orientation between enzymes, helping to design and optimize artificial cellulosomes, providing a novel architecture for synergistic enhancement of enzymatic cellulose degradation. Proteins 2016; 84:1043-1054. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Heterogeneous Expression and Functional Characterization of Cellulose-Degrading Enzymes from Aspergillus niger for Enzymatic Hydrolysis of Alkali Pretreated Bamboo Biomass.

PubMed

Ali, Nasir; Ting, Zhang; Li, Hailong; Xue, Yong; Gan, Lihui; Liu, Jian; Long, Minnan

2015-09-01

Enzymatic hydrolysis of cellulosic biomass has caught much attention because of modest reaction conditions and environment friendly conditions. To reduce the cost and to achieve good quantity of cellulases, a heterologous expression system is highly favored. In this study, cellulose-degrading enzymes, GH3 family β-glucosidase (BGL), GH7 family-related cellobiohydrolases (CBHs), and endoglucanase (EG) from a newly isolated Aspergillus niger BE-2 are highly expressed in Pichia pastoris GS115. The strain produced EG, CBHs, and BGL enzymatic concentration of 0.56, 0.11, and 22 IU/mL, respectively. Mode of actions of the recombinant enzymes for substrate specificity and end product analysis are verified and found specific for cellulose degradation. Bamboo biomass saccharification with A. niger cellulase released a high level of fermentable sugars. Hydrolysis parameters are optimized to obtain reducing sugars level of 3.18 g/L. To obtain reducing sugars from a cellulosic biomass, A. niger could be a good candidate for enzymes resource of cellulase to produce reducing sugars from a cellulosic biomass. This study also facilitates the development of highly efficient enzyme cocktails for the bioconversion of lignocellulosic biomass into monosaccharides and oligosaccharides.

Optimization of the simultaneous saccharification and fermentation process using thermotolerant yeasts.

PubMed

Ballesteros, I; Oliva, J M; Ballesteros, M; Carrasco, J

1993-01-01

Different treatments to improve the thermotolerance of fermenting yeasts for simultaneous ethanol saccharification and fermentation process of cellulosic materials have been examined. Yeasts of the genera Saccharomyces and Kluyveromyces were tested for growth and fermentation at progressively higher temperatures in the range of 42-47 degrees C. The best results were obtained with K. marxianus LG, which was then submitted to different treatments in order to achieve thermotolerant clones. A total of 35 new clones were obtained that dramatically improved the SSF of 10% Solka-floc substrate at 45 degrees C when compared to the original strain, some with ethanol concentrations as high as 33 g/L.

Model-based optimization and scale-up of multi-feed simultaneous saccharification and co-fermentation of steam pre-treated lignocellulose enables high gravity ethanol production.

PubMed

Wang, Ruifei; Unrean, Pornkamol; Franzén, Carl Johan

2016-01-01

High content of water-insoluble solids (WIS) is required for simultaneous saccharification and co-fermentation (SSCF) operations to reach the high ethanol concentrations that meet the techno-economic requirements of industrial-scale production. The fundamental challenges of such processes are related to the high viscosity and inhibitor contents of the medium. Poor mass transfer and inhibition of the yeast lead to decreased ethanol yield, titre and productivity. In the present work, high-solid SSCF of pre-treated wheat straw was carried out by multi-feed SSCF which is a fed-batch process with additions of substrate, enzymes and cells, integrated with yeast propagation and adaptation on the pre-treatment liquor. The combined feeding strategies were systematically compared and optimized using experiments and simulations. For high-solid SSCF process of SO2-catalyzed steam pre-treated wheat straw, the boosted solubilisation of WIS achieved by having all enzyme loaded at the beginning of the process is crucial for increased rates of both enzymatic hydrolysis and SSCF. A kinetic model was adapted to simulate the release of sugars during separate hydrolysis as well as during SSCF. Feeding of solid substrate to reach the instantaneous WIS content of 13 % (w/w) was carried out when 60 % of the cellulose was hydrolysed, according to simulation results. With this approach, accumulated WIS additions reached more than 20 % (w/w) without encountering mixing problems in a standard bioreactor. Feeding fresh cells to the SSCF reactor maintained the fermentation activity, which otherwise ceased when the ethanol concentration reached 40-45 g L(-1). In lab scale, the optimized multi-feed SSCF produced 57 g L(-1) ethanol in 72 h. The process was reproducible and resulted in 52 g L(-1) ethanol in 10 m(3) scale at the SP Biorefinery Demo Plant. SSCF of WIS content up to 22 % (w/w) is reproducible and scalable with the multi-feed SSCF configuration and model-aided process

Enhanced saccharification of sugarcane bagasse using soluble cellulase supplemented with immobilized β-glucosidase.

PubMed

Borges, Diogo Gontijo; Baraldo, Anderson; Farinas, Cristiane Sanchez; Giordano, Raquel de Lima Camargo; Tardioli, Paulo Waldir

2014-09-01

The β-glucosidase (BG) enzyme plays a vital role in the hydrolysis of lignocellulosic biomass. Supplementation of the hydrolysis reaction medium with BG can reduce inhibitory effects, leading to greater conversion. In addition, the inclusion of immobilized BG can be a useful way of increasing enzyme stability and recyclability. BG was adsorbed on polyacrylic resin activated by carboxyl groups (BG-PC) and covalently attached to glyoxyl-agarose (BG-GA). BG-PC exhibited similar behavior to soluble BG in the hydrolysis of cellobiose, while BG-GA hydrolyzed the same substrate at a lower rate. However, the thermal stability of BG-GA was higher than that of free BG. Hydrolysis of pretreated sugarcane bagasse catalyzed by soluble cellulase supplemented with immobilized BG improved the conversion by up to 40% after 96 h of reaction. Both derivatives remained stable up to the third cycle and losses of activity were less than 50% after five cycles. Copyright © 2014 Elsevier Ltd. All rights reserved.

A Multispecies Fungal Biofilm Approach to Enhance the Celluloyltic Efficiency of Membrane Reactors for Consolidated Bioprocessing of Plant Biomass

PubMed Central

Xiros, Charilaos; Studer, Michael H.

2017-01-01

The constraints and advantages in cellulolytic enzymes production by fungal biofilms for a consolidated bioconversion process were investigated during this study. The biofilm cultivations were carried out in reactors designed for consolidated bioprocessing Multispecies Biofilm Membrane reactors, (MBM) where an aerobic fungal biofilm produces the lignocellulolytic enzymes while a fermenting microorganism forms the fermentation product at anaerobic conditions. It was shown that although mycelial growth was limited in the MBM reactors compared to submerged cultivations, the secretion of cellulolytic enzymes per cell dry weight was higher. When Trichoderma reesei was used as the sole enzyme producer, cellobiose accumulated in the liquid medium as the result of the deficiency of β-glucosidase in the fungal secretome. To enhance β-glucosidase activity, T. reesei was co-cultivated with A. phoenicis which is a β-glucosidase overproducer. The two fungi formed a multispecies biofilm which produced a balanced cellulolytic cocktail for the saccharification of plant biomass. The mixed biofilm reached a 2.5 fold increase in β-glucosidase production, compared to the single T. reesei biofilm. The enzymatic systems of single and mixed biofilms were evaluated regarding their efficiency on cellulosic substrates degradation. Washed solids from steam pretreated beechwood, as well as microcrystalline cellulose were used as the substrates. The enzymatic system of the multispecies biofilm released four times more glucose than the enzymatic system of T. reesei alone from both substrates and hydrolyzed 78 and 60% of the cellulose content of washed solids from beechwood and microcrystalline cellulose, respectively. PMID:29067006

Ectomycorrhizal fungi enhance nitrogen and phosphorus nutrition of Nothofagus dombeyi under drought conditions by regulating assimilative enzyme activities.

PubMed

Alvarez, Maricel; Huygens, Dries; Olivares, Erick; Saavedra, Isabel; Alberdi, Miren; Valenzuela, Eduardo

2009-08-01

Drought stress conditions (DC) reduce plant growth and nutrition, restraining the sustainable reestablishment of Nothofagus dombeyi in temperate south Chilean forest ecosystems. Ectomycorrhizal symbioses have been documented to enhance plant nitrogen (N) and phosphorus (P) uptake under drought, but the regulation of involved assimilative enzymes remains unclear. We studied 1-year-old N. dombeyi (Mirb.) Oerst. plants in association with the ectomycorrhizal fungi Pisolithus tinctorius (Pers.) Coker & Couch. and Descolea antartica Sing. In greenhouse experiments, shoot and root dry weights, mycorrhizal colonization, foliar N and P concentrations, and root enzyme activities [glutamate synthase (glutamine oxoglutarate aminotransferase (GOGAT), EC 1.4.1.13-14), glutamine synthetase (GS, EC 6.3.1.2), glutamate dehydrogenase (GDH, EC 1.4.1.2-4), nitrate reductase (NR, EC 1.6.6.1), and acid phosphomonoesterase (PME, EC 3.1.3.1-2)] were determined as a function of soil-water content. Inoculation of N. dombeyi with P. tinctorius and D. antartica significantly stimulated plant growth and increased plant foliar N and P concentrations, especially under DC. Ectomycorrhizal inoculation increased the activity of all studied enzymes relative to non-mycorrhizal plants under drought. We speculate that GDH is a key enzyme involved in the enhancement of ectomycorrhizal carbon (C) availability by fuelling the tricarboxylic acid (TCA) cycle under conditions of drought-induced carbon deficit. All studied assimilative enzymes of the ectomycorrhizal associations, involved in C, N, and P transfers, are closely interlinked and interdependent. The up-regulation of assimilative enzyme activities by ectomycorrhizal fungal root colonizers acts as a functional mechanism to increase seedling endurance to drought. We insist upon incorporating ectomycorrhizal inoculation in existing Chilean afforestation programs.

Paper sludge (PS) to bioethanol: Evaluation of virgin and recycle mill sludge for low enzyme, high-solids fermentation.

PubMed

Boshoff, Sonja; Gottumukkala, Lalitha Devi; van Rensburg, Eugéne; Görgens, Johann

2016-03-01

Paper sludge (PS) from the paper and pulp industry consists primarily of cellulose and ash and has significant potential for ethanol production. Thirty-seven PS samples from 11 South African paper and pulp mills exhibited large variation in chemical composition and resulting ethanol production. Simultaneous saccharification and fermentation (SSF) of PS in fed-batch culture was investigated at high solid loadings and low enzyme dosages. Water holding capacity and viscosity of the PS influenced ethanol production at elevated solid loadings of PS. High viscosity of PS from virgin pulp mills restricted the solid loading to 18% (w/w) at an enzyme dosage of 20 FPU/gram dry PS (gdPS), whereas an optimal solid loading of 27% (w/w) was achieved with corrugated recycle mill PS at 11 FPU/gdPS. Ethanol concentration and yield of virgin pulp and corrugated recycle PS were 34.2g/L at 66.9% and 45.5 g/L at 78.2%, respectively. Copyright © 2015 Elsevier Ltd. All rights reserved.

Enzyme-enhanced fluorescence detection of DNA on etched optical fibers.

PubMed

Niu, Shu-yan; Li, Quan-yi; Ren, Rui; Zhang, Shu-sheng

2009-05-15

A novel DNA biosensor based on enzyme-enhanced fluorescence detection on etched optical fibers was developed. The hybridization complex of DNA probe and biotinylated target was formed on the etched optical fiber, and was then bound with streptavidin labeled horseradish peroxidase (streptavidin-HRP). The target DNA was quantified through the fluorescent detection of bi-p,p'-4-hydroxyphenylacetic acid (DBDA) generated from the substrate 4-hydroxyphenylacetic acid (p-HPA) under the catalysis of HRP, with a detection limit of 1 pM and a linear range from 1.69 pM to 169 pM. It is facile to regenerate this sensor through surface treatment with concentrated urea solution. It was discovered that the sensor can retain 70% of its original activity after three detection-regeneration cycles.

Model-based complete enzymatic production of 3,6-anhydro-L-galactose from red algal biomass.

PubMed

Pathiraja, Duleepa; Lee, Saeyoung; Choi, In-Geol

2018-06-13

3,6-Anhydro-L-galactose (L-AHG) is a bioactive constituent of agar polysaccharides. To be used as a cosmetic or pharmaceutical ingredient, L-AHG is more favorably prepared by enzymatic saccharification of agar using a combination of agarolytic enzymes. Determining the optimum enzyme combination from natural repertoire is a bottleneck for designing an efficient enzymatic hydrolysis process. We consider all theoretical enzymatic saccharification routes in the natural agarolytic pathway of a marine bacterium, Saccharophagus degradans 2-40. Among these routes, three representative routes were determined by removing redundant enzymatic reactions. We simulated each L-AHG production route by simple kinetic models and validated the reaction feasibility by experimental procedure. The optimal enzyme mixture (with 67.3% maximum saccharification yield) was composed of endo-type β-agarase, exo-type β-agarase, agarooligosaccharolytic β-galactosidase and α-neoagarobiose hydrolase. This approach will reduce time and effort for developing a coherent enzymatic process to produce L-AHG on mass scale.

Biomimetic cell wall model studies to identify new lignin bioengineering targets for improving biomass susceptibility to pretreatment and enzymatic saccharification

USDA-ARS?s Scientific Manuscript database

Increasingly, bioengineering of lignin to contain atypical building blocks from other metabolic pathways is being pursued to custom-design lignin that is easier to remove by chemical pretreatments and less inhibitory toward polysaccharide saccharification. Because plants produce such a diverse array...

Angiotensin converting enzyme over expression in myelocytes enhances the immune response

PubMed Central

Bernstein, Kenneth E.; Gonzalez-Villalobos, Romer A.; Giani, Jorge F.; Shah, Kandarp; Bernstein, Ellen; Janjulia, Tea; Koronyo, Yosef; Shi, Peng D.; Koronyo-Hamaoui, Maya; Fuchs, Sebastien; Shen, Xiao Z.

2015-01-01

Angiotensin converting enzyme (ACE) plays an important role in blood pressure control. ACE also has effects on renal function, reproduction, hematopoiesis and several aspects of the immune response. ACE 10/10 mice over express ACE in monocytic cells; macrophages from ACE 10/10 mice demonstrate increased polarization towards a proinflammatory phenotype. As a result, ACE 10/10 mice have a highly effective immune response following challenge with either melanoma, bacterial infection or Alzheimer’s disease. The ACE 10/10 mice suggest that enhanced monocytic function greatly contributes to the ability of the immune response to defend against a wide variety of antigenic and non-antigenic challenges. PMID:24633750

Early lignin pathway enzymes and routes to chlorogenic acid in switchgrass (Panicum virgatum L.).

PubMed

Escamilla-Treviño, Luis L; Shen, Hui; Hernandez, Timothy; Yin, Yanbin; Xu, Ying; Dixon, Richard A

2014-03-01

Studying lignin biosynthesis in Panicum virgatum (switchgrass) has provided a basis for generating plants with reduced lignin content and increased saccharification efficiency. Chlorogenic acid (CGA, caffeoyl quinate) is the major soluble phenolic compound in switchgrass, and the lignin and CGA biosynthetic pathways potentially share intermediates and enzymes. The enzyme hydroxycinnamoyl-CoA: quinate hydroxycinnamoyltransferase (HQT) is responsible for CGA biosynthesis in tobacco, tomato and globe artichoke, but there are no close orthologs of HQT in switchgrass or in other monocotyledonous plants with complete genome sequences. We examined available transcriptomic databases for genes encoding enzymes potentially involved in CGA biosynthesis in switchgrass. The protein products of two hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyltransferase (HCT) genes (PvHCT1a and PvHCT2a), closely related to lignin pathway HCTs from other species, were characterized biochemically and exhibited the expected HCT activity, preferring shikimic acid as acyl acceptor. We also characterized two switchgrass coumaroyl shikimate 3'-hydroxylase (C3'H) enzymes (PvC3'H1 and PvC3'H2); both of these cytochrome P450s had the capacity to hydroxylate 4-coumaroyl shikimate or 4-coumaroyl quinate to generate caffeoyl shikimate or CGA. Another switchgrass hydroxycinnamoyl transferase, PvHCT-Like1, is phylogenetically distant from HCTs or HQTs, but exhibits HQT activity, preferring quinic acid as acyl acceptor, and could therefore function in CGA biosynthesis. The biochemical features of the recombinant enzymes, the presence of the corresponding activities in plant protein extracts, and the expression patterns of the corresponding genes, suggest preferred routes to CGA in switchgrass.

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.

Highly thermostable and pH-stable cellulases from Aspergillus niger NS-2: properties and application for cellulose hydrolysis.

PubMed

Bansal, Namita; Janveja, Chetna; Tewari, Rupinder; Soni, Raman; Soni, Sanjeev Kumar

2014-01-01

Optimization of cultural conditions for enhanced cellulase production by Aspergillus niger NS-2 were studied under solid-state fermentation. Significant increase in yields (CMCase 463.9 ± 20.1 U/g, FPase 101.1 ± 3.5 U/g and β-glucosidase 99 ± 4.0 U/g) were obtained under optimized conditions. Effect of different nutritional parameters was studied to induce the maximum production of cellulase complex. Scale-up studies for enzyme production process were carried out. Characterization studies showed that enzymes produced by A. niger NS-2 were highly temperature- and pH stable. At 50 °C, the half life for CMCase, FPase, β-glucosidase were approximately 240 h. Cellulases from A. niger NS-2 were stable at 35 °C for 24 h over a broader pH range of 3.0-9.0. We examined the feasibility of using steam pretreatment to increase the saccharification yields from various lignocellulosic residues for sugar release which can potentially be used in bioethanol production. Saccharification of pretreated dry potato peels, carrot peels, composite waste mixture, orange peels, onion peels, banana peels, pineapple peels by crude enzyme extract from A. niger NS-2, resulted in very high cellulose conversion efficiencies of 92-98 %.

Effect of steam explosion on waste copier paper alone and in a mixed lignocellulosic substrate on saccharification and fermentation

PubMed Central

Elliston, Adam; Wilson, David R.; Wellner, Nikolaus; Collins, Samuel R.A.; Roberts, Ian N.; Waldron, Keith W.

2015-01-01

This study evaluated steam (SE) explosion on the saccharification and simultaneous saccharification and fermentation (SSF) of waste copier paper. SE resulted in a colouration, a reduction in fibre thickness and increased water absorption. Changes in chemical composition were evident at severities greater than 4.24 resulting in a loss of xylose and the production of breakdown products known to inhibit fermentation (particularly formic acid and acetic acid). SE did not improve final yields of glucose or ethanol, and at severities 4.53 and 4.83 reduced yields probably due to the effect of breakdown products and fermentation inhibitors. However, at moderate severities of 3.6 and 3.9 there was an increase in initial rates of hydrolysis which may provide a basis for reducing processing times. Co-steam explosion of waste copier paper and wheat straw attenuated the production of breakdown products, and may also provide a basis for improving SSF of lignocellulose. PMID:25846183

Cellulase stability, adsorption/desorption profiles and recycling during successive cycles of hydrolysis and fermentation of wheat straw.

PubMed

Rodrigues, Ana Cristina; Felby, Claus; Gama, Miguel

2014-03-01

The potential of enzymes recycling after hydrolysis and fermentation of wheat straw under a variety of conditions was investigated, monitoring the activity of the enzymes in the solid and liquid fractions, using low molecular weight substrates. A significant amount of active enzymes could be recovered by recycling the liquid phase. In the early stage of the process, enzyme adsorb to the substrate, then gradually returning to the solution as the saccharification proceeds. At 50°C, normally regarded as an acceptable operational temperature for saccharification, the enzymes (Celluclast) significantly undergo thermal deactivation. The hydrolysis yield and enzyme recycling efficiency in consecutive recycling rounds can be increased by using high enzyme loadings and moderate temperatures. Indeed, the amount of enzymes in the liquid phase increased with its thermostability and hydrolytic efficiency. This study contributes towards developing effective enzymes recycling strategies and helping to reduce the enzyme costs on bioethanol production. Copyright © 2014 Elsevier Ltd. All rights reserved.

Simultaneous saccharification and fermentation of steam exploded duckweed: Improvement of the ethanol yield by increasing yeast titre.

PubMed

Zhao, X; Moates, G K; Elliston, A; Wilson, D R; Coleman, M J; Waldron, K W

2015-10-01

This study investigated the conversion of Lemna minor biomass to bioethanol. The biomass was pre-treated by steam explosion (SE, 210°C, 10 min) and then subjected to simultaneous saccharification and fermentation (SSF) using Cellic® CTec 2 (20 U or 0.87 FPU g(-1) substrate) cellulase plus β-glucosidase (2 U g(-1) substrate) and a yeast inoculum of 10% (v/v or 8.0×10(7) cells mL(-1)). At a substrate concentration of 1% (w/v) an ethanol yield of 80% (w/w, theoretical) was achieved. However at a substrate concentration of 20% (w/v), the ethanol yield was lowered to 18.8% (w/w, theoretical). Yields were considerably improved by increasing the yeast titre in the inoculum or preconditioning the yeast on steam exploded liquor. These approaches enhanced the ethanol yield up to 70% (w/w, theoretical) at a substrate concentration of 20% (w/v) by metabolising fermentation inhibitors. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

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).

Suitable technological conditions for enzymatic hydrolysis of waste paper by Novozymes® enzymes NS50013 and NS50010.

PubMed

Brummer, Vladimir; Skryja, Pavel; Jurena, Tomas; Hlavacek, Viliam; Stehlik, Petr

2014-10-01

Waste paper belongs to a group of quantitatively the most produced waste types. Enzymatic hydrolysis is becoming a suitable way to treat this type of waste and at the same time, to produce a valuable liquid biofuel, because reducing sugars solutions that are formed during the process of saccharification can be a precursor for following or simultaneous fermentation. If it will be possible to make the enzymatic hydrolysis of the waste paper economically viable, it could serve as one of the new ways to lower the dependence of the transport sector on oil in the future. Only several studies comparing the enzymatic hydrolysis of different waste papers were performed in the past; they are summarized in this manuscript. In our experimental trials, suitable technological conditions for waste paper enzymatic hydrolysis using enzymes from Novozymes® biomass kit: enzymes NS50013 and NS50010 were investigated. The following enzymatic hydrolysis parameters in laboratory scale trials were verified on high cellulose content substrates-filter paper and cellulose pulp: type of buffer, pH, temperature, concentration of the substrate, loading of the enzyme and rate of stirring.

Simultaneous saccharification and co-fermentation of paper sludge to ethanol by Saccharomyces cerevisiae RWB222--Part I: kinetic modeling and parameters.

PubMed

Zhang, Jiayi; Shao, Xiongjun; Townsend, Oliver V; Lynd, Lee R

2009-12-01

A kinetic model was developed to predict batch simultaneous saccharification and co-fermentation (SSCF) of paper sludge by the xylose-utilizing yeast Saccharomyces cerevisiae RWB222 and the commercial cellulase preparation Spezyme CP. The model accounts for cellulose and xylan enzymatic hydrolysis and competitive uptake of glucose and xylose. Experimental results show that glucan and xylan enzymatic hydrolysis are highly correlated, and that the low concentrations of xylose encountered during SSCF do not have a significant inhibitory effect on enzymatic hydrolysis. Ethanol is found to not only inhibit the specific growth rate, but also to accelerate cell death. Glucose and xylose uptake rates were found to be competitively inhibitory, but this did not have a large impact during SSCF because the sugar concentrations are low. The model was used to evaluate which constants had the greatest impact on ethanol titer for a fixed substrate loading, enzyme loading, and fermentation time. The cellulose adsorption capacity and cellulose hydrolysis rate constants were found to have the greatest impact among enzymatic hydrolysis related constants, and ethanol yield and maximum ethanol tolerance had the greatest impact among fermentation related constants.

Improved enzymatic saccharification of steam exploded cotton stalk using alkaline extraction and fermentation of cellulosic sugars into ethanol.

PubMed

Keshav, Praveen K; Naseeruddin, Shaik; Rao, L Venkateswar

2016-08-01

Cotton stalk, a widely available and cheap agricultural residue lacking economic alternatives, was subjected to steam explosion in the range 170-200°C for 5min. Steam explosion at 200°C and 5min led to significant hemicellulose solubilization (71.90±0.10%). Alkaline extraction of steam exploded cotton stalk (SECOH) using 3% NaOH at room temperature for 6h led to 85.07±1.43% lignin removal with complete hemicellulose solubilization. Besides, this combined pretreatment allowed a high recovery of the cellulosic fraction from the biomass. Enzymatic saccharification was studied between steam exploded cotton stalk (SECS) and SECOH using different cellulase loadings. SECOH gave a maximum of 785.30±8.28mg/g reducing sugars with saccharification efficiency of 82.13±0.72%. Subsequently, fermentation of SECOH hydrolysate containing sugars (68.20±1.16g/L) with Saccharomyces cerevisiae produced 23.17±0.84g/L ethanol with 0.44g/g yield. Copyright © 2016 Elsevier Ltd. All rights reserved.

Engineering Cellulase Enzymes for Bioenergy

NASA Astrophysics Data System (ADS)

Atreya, Meera Elizabeth

methods. Protein engineering targets to improve cellulases include reducing enzyme inhibition, improving inter-enzyme synergy, and increasing enzyme thermotolerance. Ameliorating enzyme inhibition could improve catalytic activity and thus the speed of conversion from biomass to fermentable sugars. Improved enzyme synergy could reduce the enzyme loading required to achieve equivalent biomass conversion. Finally, thermostable enzymes could enable more biomass to be processed at a time, due to high temperatures decreasing the viscosity of biomass slurries. A high-temperature enzyme saccharification reaction could also decrease the risk of contamination in the resulting concentrated sugar solution. Throughout my PhD, I have explored research projects broadly across all of these topics, with the most success in addressing the issue of enzyme inhibition. Cellulase enzyme Cel7A is the most abundant cellulase employed by natural systems for cellulose hydrolysis. Cellobiohydrolase enzymes like Cel7A break down cellulose into cellobiose (two glucose molecules). Unfortunately, upon cleavage, this product molecule interferes with continued hydrolysis activity of Cel7A; the strong binding of cellobiose in the active site can obstruct the enzyme from processing down the cellulase chain. This phenomenon, known as product inhibition, is a bottleneck to efficient biomass breakdown. Using insights from computational protein modeling studies, I experimentally generated and tested mutant Cel7A enzymes for improved tolerance to cellobiose. Indeed, this strategy yielded Cel7A enzymes exhibiting reduced product inhibition, including some mutants completely impervious to cellobiose. The improvements in tolerance to cellobiose, however, resulted in an overall reduction of enzyme activity for the mutants tested. Nevertheless, my findings substantiated computational reports with experimental evidence and pinpointed an amino acid residue in the Cel7A product binding site that is of interest for

Synthetic CO2-fixation enzyme cascades immobilized on self-assembled nanostructures that enhance CO2/O2 selectivity of RubisCO.

PubMed

Satagopan, Sriram; Sun, Yuan; Parquette, Jon R; Tabita, F Robert

2017-01-01

With increasing concerns over global warming and depletion of fossil-fuel reserves, it is attractive to develop innovative strategies to assimilate CO 2 , a greenhouse gas, into usable organic carbon. Cell-free systems can be designed to operate as catalytic platforms with enzymes that offer exceptional selectivity and efficiency, without the need to support ancillary reactions of metabolic pathways operating in intact cells. Such systems are yet to be exploited for applications involving CO 2 utilization and subsequent conversion to valuable products, including biofuels. The Calvin-Benson-Bassham (CBB) cycle and the enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) play a pivotal role in global CO 2 fixation. We hereby demonstrate the co-assembly of two RubisCO-associated multienzyme cascades with self-assembled synthetic amphiphilic peptide nanostructures. The immobilized enzyme cascades sequentially convert either ribose-5-phosphate (R-5-P) or glucose, a simpler substrate, to ribulose 1,5-bisphosphate (RuBP), the acceptor for incoming CO 2 in the carboxylation reaction catalyzed by RubisCO. Protection from proteolytic degradation was observed in nanostructures associated with the small dimeric form of RubisCO and ancillary enzymes. Furthermore, nanostructures associated with a larger variant of RubisCO resulted in a significant enhancement of the enzyme's selectivity towards CO 2 , without adversely affecting the catalytic activity. The ability to assemble a cascade of enzymes for CO 2 capture using self-assembling nanostructure scaffolds with functional enhancements show promise for potentially engineering entire pathways (with RubisCO or other CO 2 -fixing enzymes) to redirect carbon from industrial effluents into useful bioproducts.

Modified kinetics of enzymes interacting with nanoparticles

NASA Astrophysics Data System (ADS)

Díaz, Sebastián. A.; Breger, Joyce C.; Malanoski, Anthony; Claussen, Jonathan C.; Walper, Scott A.; Ancona, Mario G.; Brown, Carl W.; Stewart, Michael H.; Oh, Eunkeu; Susumu, Kimihiro; Medintz, Igor L.

2015-08-01

Enzymes are important players in multiple applications, be it bioremediation, biosynthesis, or as reporters. The business of catalysis and inhibition of enzymes is a multibillion dollar industry and understanding the kinetics of commercial enzymes can have a large impact on how these systems are optimized. Recent advances in nanotechnology have opened up the field of nanoparticle (NP) and enzyme conjugates and two principal architectures for NP conjugate systems have been developed. In the first example the enzyme is bound to the NP in a persistent manner, here we find that key factors such as directed enzyme conjugation allow for enhanced kinetics. Through controlled comparative experiments we begin to tease out specific mechanisms that may account for the enhancement. The second system is based on dynamic interactions of the enzymes with the NP. The enzyme substrate is bound to the NP and the enzyme is free in solution. Here again we find that there are many variables , such as substrate positioning and NP selection, that modify the kinetics.

Comparison of bio-hydrogen production yield capacity between asynchronous and simultaneous saccharification and fermentation processes from agricultural residue by mixed anaerobic cultures.

PubMed

Li, Yameng; Zhang, Zhiping; Zhu, Shengnan; Zhang, Huan; Zhang, Yang; Zhang, Tian; Zhang, Quanguo

2018-01-01

Taken common agricultural residues as substrate, dark fermentation bio-hydrogen yield capacity from asynchronous saccharification and fermentation (ASF) and simultaneous saccharification and fermentation (SSF) was investigated. The highest hydrogen yield of 472.75mL was achieved with corncob using ASF. Hydrogen yield from corn straw, rice straw, corncob and sorghum stalk by SSF were 20.54%,10.31%,13.99% and 5.92% higher than ASF, respectively. The experimental data fitted well to the modified Gompertz model. SSF offered a distinct advantage over ASF with respect to reducing overall process time (60h of SSF, 108h of ASF). Meanwhile, SSF performed better than SSF with respect to shortening the lag-stage. The major metabolites of anaerobic fermentation hydrogen production by ASF and SSF were butyric acid and acetic acid. Copyright © 2017 Elsevier Ltd. All rights reserved.

Alkali-based pretreatments distinctively extract lignin and pectin for enhancing biomass saccharification by altering cellulose features in sugar-rich Jerusalem artichoke stem.

PubMed

Li, Meng; Wang, Jun; Yang, Yuezhou; Xie, Guanghui

2016-05-01

Jerusalem artichoke (JA) has been known as a potential nonfood feedstock for biofuels. Based on systems analysis of total 59 accessions, both soluble sugar and ash could positively affect biomass digestibility after dilute sodium hydroxide pretreatment (A). In this study, one representative accession (HEN-3) was used to illustrate its enzymatic digestibility with pretreatments of ultrasonic-assisted dilute sodium hydroxide (B), alkaline peroxide (C), and ultrasonic-assisted alkaline peroxide (D). Pretreatment D exhibited the highest hexose release rate (79.4%) and total sugar yield (10.4 g/L), which were 2.4 and 2.6 times higher, respectively, than those of the control. The analysis of cellulose crystalline index (CrI), cellulose degree of polymerization (DP), thermal behavior and SEM suggested that alkali-based pretreatments could distinctively extract lignin and pectin polymers, leading to significant alterations of cellulose CrI and DP for high biomass saccharification. Additionally, hydrogen peroxide (H2O2) could significant reduce the generation of fermentation inhibitors during alkali-based pretreatments. Copyright © 2016 Elsevier Ltd. All rights reserved.

Process integration for simultaneous saccharification, fermentation, and recovery (SSFR): Production of butanol from corn stover using Clostridium beijerinckii P260

USDA-ARS?s Scientific Manuscript database

A simultaneous saccharification, fermentation, and recovery (SSFR) process was developed for production of acetone butanol ethanol (AB or ABE), of which butanol is the main product, from corn stover employing Clostridium beijerinckii P260. Of the 86 gL^-1^ corn stover, over 97% of the sugars were r...

A novel film-pore-surface diffusion model to explain the enhanced enzyme adsorption of corn stover pretreated by ultrafine grinding.

PubMed

Zhang, Haiyan; Chen, Longjian; Lu, Minsheng; Li, Junbao; Han, Lujia

2016-01-01

Ultrafine grinding is an environmentally friendly pretreatment that can alter the degree of polymerization, the porosity and the specific surface area of lignocellulosic biomass and can, thus, enhance cellulose hydrolysis. Enzyme adsorption onto the substrate is a prerequisite for the enzymatic hydrolysis process. Therefore, it is necessary to investigate the enzyme adsorption properties of corn stover pretreated by ultrafine grinding. The ultrafine grinding pretreatment was executed on corn stover. The results showed that ultrafine grinding pretreatment can significantly decrease particle size [from 218.50 μm of sieve-based grinding corn stover (SGCS) to 17.45 μm of ultrafine grinding corn stover (UGCS)] and increase the specific surface area (SSA), pore volume (PV) and surface composition (SSA: from 1.71 m(2)/g of SGCS to 2.63 m(2)/g of UGCS, PV: from 0.009 cm(3)/g of SGCS to 0.024 m(3)/g of UGCS, cellulose surface area: from 168.69 m(2)/g of SGCS to 290.76 m(2)/g of UGCS, lignin surface area: from 91.46 m(2)/g of SGCS to 106.70 m(2)/g of UGCS). The structure and surface composition changes induced by ultrafine grinding increase the enzyme adsorption capacity from 2.83 mg/g substrate of SGCS to 5.61 mg/g substrate of UGCS. A film-pore-surface diffusion model was developed to simultaneously predict the enzyme adsorption kinetics of both the SGCS and UGCS. Satisfactory predictions could be made with the model based on high R (2) and low RMSE values (R (2) = 0.95 and RMSE = 0.16 mg/g for the UGCS, R (2) = 0.93 and RMSE = 0.09 mg/g for the SGCS). The model was further employed to analyze the rate-limiting steps in the enzyme adsorption process. Although both the external-film and internal-pore mass transfer are important for enzyme adsorption on the SGCS and UGCS, the UGCS has a lower internal-pore resistance compared to the SGCS. Ultrafine grinding pretreatment can enhance the enzyme adsorption onto corn stover by altering structure and

Enhanced biomass delignification and enzymatic saccharification of canola straw by steam-explosion pretreatment.

PubMed

Garmakhany, Amir Daraei; Kashaninejad, Mahdi; Aalami, Mehran; Maghsoudlou, Yahya; Khomieri, Mortza; Tabil, Lope G

2014-06-01

In recent decades, bioconversion of lignocellulosic biomass to biofuel (ethanol and biodiesel) has been extensively investigated. The three main chemical constituents of biomass are cellulose, hemicellulose and lignin. Cellulose and hemicellulose are polysaccharides of primarily fermentable sugars, glucose and xylose respectively. Hemicellulose also includes small fermentable fractions of arabinose, galactose and mannose. The main issue in converting lignocellulosic biomass to fuel ethanol is the accessibility of the polysaccharides for enzymatic breakdown into monosaccharides. This study focused on the use of steam explosion as the pretreatment method for canola straw as lignocellulosic biomass. Result showed that steam explosion treatment of biomass increased cellulose accessibility and it hydrolysis by enzyme hydrolysis. Following 72 h of enzyme hydrolysis, a maximum cellulose conversion to glucose yield of 29.40% was obtained for the steam-exploded sample while the control showed 11.60% glucose yields. Steam explosion pretreatment increased glucose production and glucose yield by 200% and 153.22%, respectively, compared to the control sample. The crystalline index increased from 57.48% in untreated canola straw to 64.72% in steam-exploded samples. Steam explosion pretreatment of biomass increased cellulose accessibility, and enzymatic hydrolysis increased glucose production and glucose yield of canola straw. © 2013 Society of Chemical Industry.

Simultaneous saccharification and cofermentation of peracetic acid-pretreated biomass.

PubMed

Teixeira, L C; Linden, J C; Schroeder, H A

2000-01-01

Previous work in our laboratories has demonstrated the effectiveness of peracetic acid for improving enzymatic digestibility of lignocellulosic materials. The use of dilute alkali solutions as a pre-pretreatment prior to peracetic acid lignin oxidation increased carbohydrate hydrolysis yields in a synergistic as opposed to additive manner. Deacetylation of xylan is easily achieved using dilute alkali solutions under mild conditions. In this article, we evaluate the effectiveness of peracetic acid combined with an alkaline pre-pretreatment through simultaneous saccharification and cofermentation (SSCF) of pretreated hybrid poplar wood and sugar cane bagasse. Respective ethanol yields of 92.8 and 91.9% of theoretical are achieved using 6% NaOH/15% peracetic acid-pretreated substrates and recombinant Zymomonas mobilis CP4/pZB5. Reduction of acetyl groups of the lignocellulosic materials is demonstrated following alkaline pre-pretreatments. Such processing may be helpful in reducing peracetic acid requirements. The influence of deacetylation is more significant in combined pretreatments using lower peracetic acid loadings.

Celluclast and Cellic® CTec2: Saccharification/fermentation of wheat straw, solid-liquid partition and potential of enzyme recycling by alkaline washing.

PubMed

Rodrigues, Ana Cristina; Haven, Mai Østergaard; Lindedam, Jane; Felby, Claus; Gama, Miguel

2015-11-01

The hydrolysis/fermentation of wheat straw and the adsorption/desorption/deactivation of cellulases were studied using Cellic(®) CTec2 (Cellic) and Celluclast mixed with Novozyme 188. The distribution of enzymes - cellobiohydrolase I (Cel7A), endoglucanase I (Cel7B) and β-glucosidase - of the two formulations between the residual substrate and supernatant during the course of enzymatic hydrolysis and fermentation was investigated. The potential of recyclability using alkaline wash was also studied. The efficiency of hydrolysis with an enzyme load of 10 FPU/g cellulose reached >98% using Cellic(®) CTec2, while for Celluclast a conversion of 52% and 81%, was observed without and with β-glucosidase supplementation, respectively. The decrease of Cellic(®) CTec2 activity observed along the process was related to deactivation of Cel7A rather than of Cel7B and β-glucosidase. The adsorption/desorption profiles during hydrolysis/fermentation revealed that a large fraction of active enzymes remained adsorbed to the solid residue throughout the process. Surprisingly, this was the case of Cel7A and β-glucosidase from Cellic, which remained adsorbed to the solid fraction along the entire process. Alkaline washing was used to recover the enzymes from the solid residue. This method allowed efficient recovery of Celluclast enzymes; however, this may be achieved only when minor amounts of cellulose remain present. Regarding the Cellic formulation, neither the presence of cellulose nor lignin restricted an efficient desorption of the enzymes at alkaline pH. This work shows that the recycling strategy must be customized for each particular formulation, since the enzymes found e.g. in Cellic and Celluclast bear quite different behaviour regarding the solid-liquid distribution, stability and cellulose and lignin affinity. Copyright © 2015 Elsevier Inc. All rights reserved.

Crosslinked Enzyme Aggregates in Hierarchically-Ordered Mesoporous Silica: A Simple and Effective Method for Enzyme Stabilization

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

Kim, Moon Il; Kim, Jungbae; Lee, Jinwoo

2007-02-01

alpha-chymotrypsin (CT) and lipase (LP) were immobilized in hierarchically-ordered mesocellular mesoporous silica (HMMS) in a simple but effective way for the enzyme stabilization, which was achieved by the enzyme adsorption followed by glutaraldehyde (GA) crosslinking. This resulted in the formation of nanometer scale crosslinked enzyme aggregates (CLEAs) entrapped in the mesocellular pores of HMMS (37 nm), which did not leach out of HMMS through narrow mesoporous channels (13 nm). CLEA of alpha-chymotrypsin (CLEA-CT) in HMMS showed a high enzyme loading capacity and significantly increased enzyme stability. No activity decrease of CLEA-CT was observed for two weeks under even rigorously shakingmore » condition, while adsorbed CT in HMMS and free CT showed a rapid inactivation due to the enzyme leaching and presumably autolysis, respectively. With the CLEA-CT in HMMS, however, there was no tryptic digestion observed suggesting that the CLEA-CT is not susceptible to autolysis. Moreover, CLEA of lipase (CLEA-LP) in HMMS retained 30% specific activity of free lipase with greatly enhanced stability. This work demonstrates that HMMS can be efficiently employed as host materials for enzyme immobilization leading to highly enhanced stability of the immobilized enzymes with high enzyme loading and activity.« less

Immobilized enzymes: understanding enzyme - surface interactions at the molecular level.

PubMed

Hoarau, Marie; Badieyan, Somayesadat; Marsh, E Neil G

2017-11-22

Enzymes immobilized on solid supports have important and industrial and medical applications. However, their uses are limited by the significant reductions in activity and stability that often accompany the immobilization process. Here we review recent advances in our understanding of the molecular level interactions between proteins and supporting surfaces that contribute to changes in stability and activity. This understanding has been facilitated by the application of various surface-sensitive spectroscopic techniques that allow the structure and orientation of enzymes at the solid/liquid interface to be probed, often with monolayer sensitivity. An appreciation of the molecular interactions between enzyme and surface support has allowed the surface chemistry and method of enzyme attachement to be fine-tuned such that activity and stability can be greatly enhanced. These advances suggest that a much wider variety of enzymes may eventually be amenable to immobilization as green catalysts.

Two new β-glucosidases from ethanol-fermenting fungus Mucor circinelloides NBRC 4572: enzyme purification, functional characterization, and molecular cloning of the gene.

PubMed

Kato, Yasuo; Nomura, Taiji; Ogita, Shinjiro; Takano, Maki; Hoshino, Kazuhiro

2013-12-01

Two β-glucosidases (BGLs 1 and 2) were purified to homogeneity from the extracellular enzyme preparations of the ethanol-fermenting Mucor circinelloides NBRC 4572 statically grown on rice straw. BGLs 1 and 2 are monomeric glycoproteins whose apparent molecular masses (Ms) are around 78 kDa, which decreased by approximately 10 kDa upon enzymatic deglycosylation. Both BGLs showed similar enzyme characteristics in optimal temperature and pH, stability, and inhibitors. They were active against a wide range of aryl-β-glucosides and β-linked glucose oligosaccharides. Their amino acid sequences shared 81% identity and exhibited less than 60% identity with the known family-3 BGLs. Considering properties such as reduced inhibition by ethanol, glucose, and cellobiose, low transglucosylation activity, wider substrate range, less binding affinity to lignocellulosic materials, and abundant expression, BGL1 is likely to be more suitable for bioethanol production than BGL2 via simultaneous saccharification and fermentation of rice straw with M. circinelloides.

Enhancement in ionic liquid tolerance of cellulase immobilized on PEGylated graphene oxide nanosheets: Application in saccharification of lignocellulose.

PubMed

Xu, Jiaxing; Sheng, Zhenhuan; Wang, Xinfeng; Liu, Xiaoyan; Xia, Jun; Xiong, Peng; He, Bingfang

2016-01-01

The objective of the present work was to improve ionic liquid (IL) tolerance of cellulase based on the exploration of functional nanoscale carriers for potential application in lignocellulosic biorefinery. PEGylated graphene oxide (GO) composite was successfully fabricated by chemical binding of 4-arm-PEG-NH2 and GO and applied to the immobilization of cellulase. The PEGylated GO-Cellulase retained 61% of the initial activity in 25% (w/v) 1-butyl-3-methylimidazolium chloride ([Bmim][Cl]) while free cellulase only retained 2%. The IL stability was enhanced more than 30 times. The relatively minor change in Km value (from 2.7 to 3.2mgmL(-1)) after the immobilization suggested that PEGylated GO-Cellulase was capable of closely mimicking the performance of free enzyme. After treating rice straw with [Bmim][Cl] and dilution to a final IL concentration of 15% (w/v), the slurry was directly hydrolyzed using PEGylated GO-Cellulase without IL removing and a high hydrolysis rate of 87% was achieved. Copyright © 2015 Elsevier Ltd. All rights reserved.

Macrophage mediated PCI enhanced gene-directed enzyme prodrug therapy

NASA Astrophysics Data System (ADS)

Christie, Catherine E.; Zamora, Genesis; Kwon, Young J.; Berg, Kristian; Madsen, Steen J.; Hirschberg, Henry

2015-03-01

Photochemical internalization (PCI) is a photodynamic therapy-based approach for improving the delivery of macromolecules and genes into the cell cytosol. Prodrug activating gene therapy (suicide gene therapy) employing the transduction of the E. coli cytosine deaminase (CD) gene into tumor cells, is a promising method. Expression of this gene within the target cell produces an enzyme that converts the nontoxic prodrug, 5-FC, to the toxic metabolite, 5-fluorouracil (5-FU). 5-FC may be particularly suitable for brain tumors, because it can readily cross the bloodbrain barrier (BBB). In addition the bystander effect, where activated drug is exported from the transfected cancer cells into the tumor microenvironment, plays an important role by inhibiting growth of adjacent tumor cells. Tumor-associated macrophages (TAMs) are frequently found in and around glioblastomas. Monocytes or macrophages (Ma) loaded with drugs, nanoparticles or photosensitizers could therefore be used to target tumors by local synthesis of chemo attractive factors. The basic concept is to combine PCI, to enhance the ex vivo transfection of a suicide gene into Ma, employing specially designed core/shell NP as gene carrier.

An Improved Ultrasensitive Enzyme-Linked Immunosorbent Assay Using Hydrangea-Like Antibody-Enzyme-Inorganic Three-in-One Nanocomposites.

PubMed

Wei, Tianxiang; Du, Dan; Zhu, Mei-Jun; Lin, Yuehe; Dai, Zhihui

2016-03-01

Protein-inorganic nanoflowers, composed of protein and copper(II) phosphate (Cu3(PO4)2), have recently grabbed people's attention. Because the synthetic method requires no organic solvent and because of the distinct hierarchical nanostructure, protein-inorganic nanoflowers display enhanced catalytic activity and stability and would be a promising tool in biocatalytical processes and biological and biomedical fields. In this work, we first coimmobilized the enzyme, antibody, and Cu3(PO4)2 into a three-in-one hybrid protein-inorganic nanoflower to enable it to possess dual functions: (1) the antibody portion retains the ability to specifically capture the corresponding antigen; (2) the nanoflower has enhanced enzymatic activity and stability to produce an amplified signal. The prepared antibody-enzyme-inorganic nanoflower was first applied in an enzyme-linked immunosorbent assay to serve as a novel enzyme-labeled antibody for Escherichia coli O157:H7 (E. coli O157:H7) determination. The detection limit is 60 CFU L(-1), which is far superior to commercial ELISA systems. The three-in-one antibody (anti-E. coli O157:H7 antibody)-enzyme (horseradish peroxidase)-inorganic (Cu3(PO4)2) nanoflower has some advantages over commercial enzyme-antibody conjugates. First, it is much easier to prepare and does not need any complex covalent modification. Second, it has fairly high capture capability and catalytic activity because it is presented as aggregates of abundant antibodies and enzymes. Third, it has enhanced enzymatic stability compared to the free form of enzyme due to the unique hierarchical nanostructure.

Enhancement in the sensitivity of microfluidic enzyme-linked immunosorbent assays through analyte preconcentration.

PubMed

Yanagisawa, Naoki; Dutta, Debashis

2012-08-21

In this Article, we describe a microfluidic enzyme-linked immunosorbent assay (ELISA) method whose sensitivity can be substantially enhanced through preconcentration of the target analyte around a semipermeable membrane. The reported preconcentration has been accomplished in our current work via electrokinetic means allowing a significant increase in the amount of captured analyte relative to nonspecific binding in the trapping/detection zone. Upon introduction of an enzyme substrate into this region, the rate of generation of the ELISA reaction product (resorufin) was observed to increase by over a factor of 200 for the sample and 2 for the corresponding blank compared to similar assays without analyte trapping. Interestingly, in spite of nonuniformities in the amount of captured analyte along the surface of our analysis channel, the measured fluorescence signal in the preconcentration zone increased linearly with time over an enzyme reaction period of 30 min and at a rate that was proportional to the analyte concentration in the bulk sample. In our current study, the reported technique has been shown to reduce the smallest detectable concentration of the tumor marker CA 19-9 and Blue Tongue Viral antibody by over 2 orders of magnitude compared to immunoassays without analyte preconcentration. When compared to microwell based ELISAs, the reported microfluidic approach not only yielded a similar improvement in the smallest detectable analyte concentration but also reduced the sample consumption in the assay by a factor of 20 (5 μL versus 100 μL).

BSA treatment to enhance enzymatic hydrolysis of cellulose in lignin containing substrates.

PubMed

Yang, Bin; Wyman, Charles E

2006-07-05

Cellulase and bovine serum albumin (BSA) were added to Avicel cellulose and solids containing 56% cellulose and 28% lignin from dilute sulfuric acid pretreatment of corn stover. Little BSA was adsorbed on Avicel cellulose, while pretreated corn stover solids adsorbed considerable amounts of this protein. On the other hand, cellulase was highly adsorbed on both substrates. Adding a 1% concentration of BSA to dilute acid pretreated corn stover prior to enzyme addition at 15 FPU/g cellulose enhanced filter paper activity in solution by about a factor of 2 and beta-glucosidase activity in solution by about a factor of 14. Overall, these results suggested that BSA treatment reduced adsorption of cellulase and particularly beta-glucosidase on lignin. Of particular note, BSA treatment of pretreated corn stover solids prior to enzymatic hydrolysis increased 72 h glucose yields from about 82% to about 92% at a cellulase loading of 15 FPU/g cellulose or achieved about the same yield at a loading of 7.5 FPU/g cellulose. Similar improvements were also observed for enzymatic hydrolysis of ammonia fiber explosion (AFEX) pretreated corn stover and Douglas fir treated by SO(2) steam explosion and for simultaneous saccharification and fermentation (SSF) of BSA pretreated corn stover. In addition, BSA treatment prior to hydrolysis reduced the need for beta-glucosidase supplementation of SSF. The results are consistent with non-specific competitive, irreversible adsorption of BSA on lignin and identify promising strategies to reduce enzyme requirements for cellulose hydrolysis. (c) 2006 Wiley Periodicals, Inc.

Evaluation of Relationships between Growth Rate, Tree Size, Lignocellulose Composition, and Enzymatic Saccharification in Interspecific Corymbia Hybrids and Parental Taxa.

PubMed

Healey, Adam L; Lee, David J; Lupoi, Jason S; Papa, Gabriella; Guenther, Joel M; Corno, Luca; Adani, Fabrizio; Singh, Seema; Simmons, Blake A; Henry, Robert J

2016-01-01

In order for a lignocellulosic bioenergy feedstock to be considered sustainable, it must possess a high rate of growth to supply biomass for conversion. Despite the desirability of a fast growth rate for industrial application, it is unclear what effect growth rate has on biomass composition or saccharification. We characterized Klason lignin, glucan, and xylan content with response to growth in Corymbia interspecific F1 hybrid families (HF) and parental species Corymbia torelliana and C. citriodora subspecies variegata and measured the effects on enzymatic hydrolysis from hydrothermally pretreated biomass. Analysis of biomass composition within Corymbia populations found similar amounts of Klason lignin content (19.7-21.3%) among parental and hybrid populations, whereas glucan content was clearly distinguished within C. citriodora subspecies variegata (52%) and HF148 (60%) as compared to other populations (28-38%). Multiple linear regression indicates that biomass composition is significantly impacted by tree size measured at the same age, with Klason lignin content increasing with diameter breast height (DBH) (+0.12% per cm DBH increase), and glucan and xylan typically decreasing per DBH cm increase (-0.7 and -0.3%, respectively). Polysaccharide content within C. citriodora subspecies variegata and HF-148 were not significantly affected by tree size. High-throughput enzymatic saccharification of hydrothermally pretreated biomass found significant differences among Corymbia populations for total glucose production from biomass, with parental Corymbia torelliana and hybrids HF-148 and HF-51 generating the highest amounts of glucose (~180 mg/g biomass, respectively), with HF-51 undergoing the most efficient glucan-to-glucose conversion (74%). Based on growth rate, biomass composition, and further optimization of enzymatic saccharification yield, high production Corymbia hybrid trees are potentially suitable for fast-rotation bioenergy or biomaterial production.

Evaluation of Relationships between Growth Rate, Tree Size, Lignocellulose Composition, and Enzymatic Saccharification in Interspecific Corymbia Hybrids and Parental Taxa

PubMed Central

Healey, Adam L.; Lee, David J.; Lupoi, Jason S.; Papa, Gabriella; Guenther, Joel M.; Corno, Luca; Adani, Fabrizio; Singh, Seema; Simmons, Blake A.; Henry, Robert J.

2016-01-01

In order for a lignocellulosic bioenergy feedstock to be considered sustainable, it must possess a high rate of growth to supply biomass for conversion. Despite the desirability of a fast growth rate for industrial application, it is unclear what effect growth rate has on biomass composition or saccharification. We characterized Klason lignin, glucan, and xylan content with response to growth in Corymbia interspecific F1 hybrid families (HF) and parental species Corymbia torelliana and C. citriodora subspecies variegata and measured the effects on enzymatic hydrolysis from hydrothermally pretreated biomass. Analysis of biomass composition within Corymbia populations found similar amounts of Klason lignin content (19.7–21.3%) among parental and hybrid populations, whereas glucan content was clearly distinguished within C. citriodora subspecies variegata (52%) and HF148 (60%) as compared to other populations (28–38%). Multiple linear regression indicates that biomass composition is significantly impacted by tree size measured at the same age, with Klason lignin content increasing with diameter breast height (DBH) (+0.12% per cm DBH increase), and glucan and xylan typically decreasing per DBH cm increase (-0.7 and -0.3%, respectively). Polysaccharide content within C. citriodora subspecies variegata and HF-148 were not significantly affected by tree size. High-throughput enzymatic saccharification of hydrothermally pretreated biomass found significant differences among Corymbia populations for total glucose production from biomass, with parental Corymbia torelliana and hybrids HF-148 and HF-51 generating the highest amounts of glucose (~180 mg/g biomass, respectively), with HF-51 undergoing the most efficient glucan-to-glucose conversion (74%). Based on growth rate, biomass composition, and further optimization of enzymatic saccharification yield, high production Corymbia hybrid trees are potentially suitable for fast-rotation bioenergy or biomaterial production

Evaluation of Relationships between Growth Rate, Tree Size, Lignocellulose Composition, and Enzymatic Saccharification in Interspecific Corymbia Hybrids and Parental Taxa

DOE PAGES

Healey, Adam L.; Lee, David J.; Lupoi, Jason S.; ...

2016-11-18

In order for a lignocellulosic bioenergy feedstock to be considered sustainable,it must possess a high rate of growth to supply biomass for conversion. Despite the desirability of a fast growth rate for industrial application,it is unclear what effect growth rate has on biomass composition or saccharification. We characterized Klason lignin,glucan,and xylan content with response to growth in Corymbia interspecific F1 hybrid families (HF) and parental species Corymbia torelliana and C. citriodora subspecies variegata and measured the effects on enzymatic hydrolysis from hydrothermally pretreated biomass. Analysis of biomass composition within Corymbia populations found similar amounts of Klason lignin content (19.7–21.3%) amongmore » parental and hybrid populations,whereas glucan content was clearly distinguished within C. citriodora subspecies variegata (52%) and HF148 (60%) as compared to other populations (28–38%). Multiple linear regression indicates that biomass composition is significantly impacted by tree size measured at the same age,with Klason lignin content increasing with diameter breast height (DBH) (+0.12% per cm DBH increase),and glucan and xylan typically decreasing per DBH cm increase (-0.7 and -0.3%,respectively). Polysaccharide content within C. citriodora subspecies variegata and HF-148 were not significantly affected by tree size. High-throughput enzymatic saccharification of hydrothermally pretreated biomass found significant differences among Corymbia populations for total glucose production from biomass,with parental Corymbia torelliana and hybrids HF-148 and HF-51 generating the highest amounts of glucose (~180 mg/g biomass,respectively),with HF-51 undergoing the most efficient glucan-to-glucose conversion (74%). Based on growth rate,biomass composition,and further optimization of enzymatic saccharification yield,high production Corymbia hybrid trees are potentially suitable for fast-rotation bioenergy or biomaterial production.« less

Evaluation of Relationships between Growth Rate, Tree Size, Lignocellulose Composition, and Enzymatic Saccharification in Interspecific Corymbia Hybrids and Parental Taxa

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

Healey, Adam L.; Lee, David J.; Lupoi, Jason S.

In order for a lignocellulosic bioenergy feedstock to be considered sustainable,it must possess a high rate of growth to supply biomass for conversion. Despite the desirability of a fast growth rate for industrial application,it is unclear what effect growth rate has on biomass composition or saccharification. We characterized Klason lignin,glucan,and xylan content with response to growth in Corymbia interspecific F1 hybrid families (HF) and parental species Corymbia torelliana and C. citriodora subspecies variegata and measured the effects on enzymatic hydrolysis from hydrothermally pretreated biomass. Analysis of biomass composition within Corymbia populations found similar amounts of Klason lignin content (19.7–21.3%) amongmore » parental and hybrid populations,whereas glucan content was clearly distinguished within C. citriodora subspecies variegata (52%) and HF148 (60%) as compared to other populations (28–38%). Multiple linear regression indicates that biomass composition is significantly impacted by tree size measured at the same age,with Klason lignin content increasing with diameter breast height (DBH) (+0.12% per cm DBH increase),and glucan and xylan typically decreasing per DBH cm increase (-0.7 and -0.3%,respectively). Polysaccharide content within C. citriodora subspecies variegata and HF-148 were not significantly affected by tree size. High-throughput enzymatic saccharification of hydrothermally pretreated biomass found significant differences among Corymbia populations for total glucose production from biomass,with parental Corymbia torelliana and hybrids HF-148 and HF-51 generating the highest amounts of glucose (~180 mg/g biomass,respectively),with HF-51 undergoing the most efficient glucan-to-glucose conversion (74%). Based on growth rate,biomass composition,and further optimization of enzymatic saccharification yield,high production Corymbia hybrid trees are potentially suitable for fast-rotation bioenergy or biomaterial production.« less

Efficient chemical and enzymatic saccharification of the lignocellulosic residue from Agave tequilana bagasse to produce ethanol by Pichia caribbica.

PubMed

Saucedo-Luna, Jaime; Castro-Montoya, Agustin Jaime; Martinez-Pacheco, Mauro Manuel; Sosa-Aguirre, Carlos Ruben; Campos-Garcia, Jesus

2011-06-01

Bagasse of Agave tequilana (BAT) is the residual lignocellulosic waste that remains from tequila production. In this study we characterized the chemical composition of BAT, which was further saccharified and fermented to produce ethanol. BAT was constituted by cellulose (42%), hemicellulose (20%), lignin (15%), and other (23%). Saccharification of BAT was carried out at 147 °C with 2% sulfuric acid for 15 min, yielding 25.8 g/l of fermentable sugars, corresponding to 36.1% of saccharificable material (cellulose and hemicellulose contents, w/w). The remaining lignocellulosic material was further hydrolyzed by commercial enzymes, ~8.2% of BAT load was incubated for 72 h at 40 °C rendering 41 g/l of fermentable sugars corresponding to 73.6% of the saccharificable material (w/w). Mathematic surface response analysis of the acid and enzymatic BAT hydrolysis was used for process optimization. The results showed a satisfactory correlation (R (2) = 0.90) between the obtained and predicted responses. The native yeast Pichia caribbica UM-5 was used to ferment sugar liquors from both acid and enzymatic hydrolysis to ethanol yielding 50 and 87%, respectively. The final optimized process generated 8.99 g ethanol/50 g of BAT, corresponding to an overall 56.75% of theoretical ethanol (w/w). Thus, BAT may be employed as a lignocellulosic raw material for bioethanol production and can contribute to BAT residue elimination from environment.

A novel hybrid organosolv: steam explosion method for the efficient fractionation and pretreatment of birch biomass.

PubMed

Matsakas, Leonidas; Nitsos, Christos; Raghavendran, Vijayendran; Yakimenko, Olga; Persson, Gustav; Olsson, Eva; Rova, Ulrika; Olsson, Lisbeth; Christakopoulos, Paul

2018-01-01

The main role of pretreatment is to reduce the natural biomass recalcitrance and thus enhance saccharification yield. A further prerequisite for efficient utilization of all biomass components is their efficient fractionation into well-defined process streams. Currently available pretreatment methods only partially fulfill these criteria. Steam explosion, for example, excels as a pretreatment method but has limited potential for fractionation, whereas organosolv is excellent for delignification but offers poor biomass deconstruction. In this article, a hybrid method combining the cooking and fractionation of conventional organosolv pretreatment with the implementation of an explosive discharge of the cooking mixture at the end of pretreatment was developed. The effects of various pretreatment parameters (ethanol content, duration, and addition of sulfuric acid) were evaluated. Pretreatment of birch at 200 °C with 60% v/v ethanol and 1% w/w biomass H 2 SO 4 was proven to be the most efficient pretreatment condition yielding pretreated solids with 77.9% w/w cellulose, 8.9% w/w hemicellulose, and 7.0 w/w lignin content. Under these conditions, high delignification of 86.2% was demonstrated. The recovered lignin was of high purity, with cellulose and hemicellulose contents not exceeding 0.31 and 3.25% w/w, respectively, and ash to be < 0.17% w/w in all cases, making it suitable for various applications. The pretreated solids presented high saccharification yields, reaching 68% at low enzyme load (6 FPU/g) and complete saccharification at high enzyme load (22.5 FPU/g). Finally, simultaneous saccharification and fermentation (SSF) at 20% w/w solids yielded an ethanol titer of 80 g/L after 192 h, corresponding to 90% of the theoretical maximum. The novel hybrid method developed in this study allowed for the efficient fractionation of birch biomass and production of pretreated solids with high cellulose and low lignin contents. Moreover, the explosive

Comparison of ultrasonic and CO₂laser pretreatment methods on enzyme digestibility of corn stover.

PubMed

Tian, Shuang-Qi; Wang, Zhen-Yu; Fan, Zi-Luan; Zuo, Li-Li

2012-01-01

To decrease the cost of bioethanol production, biomass recalcitrance needs to be overcome so that the conversion of biomass to bioethanol becomes more efficient. CO(2) laser irradiation can disrupt the lignocellulosic physical structure and reduce the average size of fiber. Analyses with Fourier transform infrared spectroscopy, specific surface area, and the microstructure of corn stover were used to elucidate the enhancement mechanism of the pretreatment process by CO(2) laser irradiation. The present work demonstrated that the CO(2) laser had potential to enhance the bioconversion efficiency of lignocellulosic waste to renewable bioethanol. The saccharification rate of the CO(2) laser pretreatment was significantly higher than ultrasonic pretreatment, and reached 27.75% which was 1.34-fold of that of ultrasonic pretreatment. The results showed the impact of CO(2) laser pretreatment on corn stover to be more effective than ultrasonic pretreatment.

Pretreatment of bamboo residues with Coriolus versicolor for enzymatic hydrolysis.

PubMed

Zhang, Xiaoyu; Xu, Chunyan; Wang, Hongxun

2007-08-01

Pretreatment by a white-rot fungus Coriolus versicolor B1 under different conditions and saccharification of bamboo were investigated. The saccharification rate was significantly enhanced and a maximum saccharification rate of 37.0% was achieved after pretreatment. Reducing sugars yield was 223.2 mg/g of bamboo residues, which was 2.34 times that of the raw material. It was feasible to treat bamboo residues with B1 for the saccharification of bamboo.

Enhanced simultaneous saccharification and fermentation of pretreated beech wood by in situ treatment with the white rot fungus Irpex lacteus in a membrane aerated biofilm reactor.

PubMed

Brethauer, Simone; Robert Lawrence, Shahab; Michael Hans-Peter, Studer

2017-08-01

The aim of the present study was to investigate the combination of steam pretreatment and biological treatment with lignin degrading fungal strains in order to enable efficient bioprocessing of beech wood to ethanol. In a sequential process of steam and fungal pretreatment followed by enzymatic hydrolysis, Irpex lacteus almost doubled the glucose yield for mildly pretreated beech wood, but could not improve yields for more severely pretreated substrates. However, when simultaneous saccharification and fermentation is combined with in situ I. lacteus treatment, which is enabled by the application of a membrane aerated biofilm reactor, ethanol yields of optimally steam pretreated beech could be improved from 65 to 80%. Generally, in situ fungal treatment during bioprocessing of lignocellulose is an interesting method to harness the versatile abilities of white rot fungi. Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.

Cytokinin oxidase from Phaseolus vulgaris callus tissues. Enhanced in vitro activity of the enzyme in the presence of copper-imidazole complexes

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

Chatfield, J.M.; Armstrong, D.J.

1987-07-01

The effects of metal ions on cytokinin oxidase activity extracted from callus tissues of Phaseolus vulgaris L. cv Great Northern have been examined using an assay based on the oxidation of N/sup 6/-(..delta../sup 2/-isopentenyl)-adenine-2,8-/sup 3/H (i/sup 6/ Ade) to adenine (Ade). The addition of cupric ions to reaction mixtures containing imidazole buffer markedly enhanced cytokinin oxidase activity. In the presence of optimal concentrations of copper and imidazole, cytokinin oxidase activity was stimulated more than 20-fold. The effect was enzyme dependent, specific for copper, and observed only in the presence of imidazole. The substrate specificity of the copper-imidazole enhanced reaction, asmore » judged by substrate competition tests, was the same as that observed in the absence of copper and imidazole. Similarly, in tests involving DEAE-cellulose chromatography, elution profiles of cytokinin oxidase activity determined using a copper-imidazole enhanced assay were identical to those obtained using an assay without copper and imidazole. On the basis of these results, the addition of copper and imidazole to reaction mixtures used to assay for cytokinin oxidase activity is judged to provide a reliable and specific assay of greatly enhanced sensitivity for the enzyme. The mechanism by which copper and imidazole enhance cytokinin oxidase activity is not certain, but the reaction catalyzed by the enzyme was not inhibited by anaerobic conditions when these reagents were present. This observation suggests that copper-imidazole complexes are substituting for oxygen in the reaction mechanism by which cytokinin oxidase effects cleavage of the N/sup 6/-side chain of i/sup 6/ Ade.« less

Production of ethanol from a mixture of waste paper and kitchen waste via a process of successive liquefaction, presaccharification, and simultaneous saccharification and fermentation.

PubMed

Nishimura, Hiroto; Tan, Li; Kira, Noriko; Tomiyama, Shigeo; Yamada, Kazuo; Sun, Zhao-Yong; Tang, Yue-Qin; Morimura, Shigeru; Kida, Kenji

2017-09-01

Efficient ethanol production from waste paper requires the addition of expensive nutrients. To reduce the production cost of ethanol from waste paper, a study on how to produce ethanol efficiently by adding kitchen waste (potentially as a carbon source, nutrient source, and acidity regulator) to waste paper was performed and a process of successive liquefaction, presaccharification, and simultaneous saccharification and fermentation (L+PSSF) was developed. The individual saccharification performances of waste paper and kitchen waste were not influenced by their mixture. Liquefaction of kitchen waste at 90°C prior to presaccharification and simultaneous saccharification and fermentation (PSSF) was essential for efficient ethanol fermentation. Ethanol at concentrations of 46.6 or 43.6g/l was obtained at the laboratory scale after fermentation for 96h, even without pH adjustment and/or the addition of extra nutrients. Similarly, ethanol at a concentration of 45.5g/l was obtained at the pilot scale after fermentation for 48h. The ethanol concentration of L+PSSF of the mixture of waste paper and kitchen waste was comparable to that of PSSF of waste paper with added nutrients (yeast extract and peptone) and pH adjustment using H 2 SO 4 , indicating that kitchen waste is not only a carbon source but also an excellent nutrient source and acidity regulator for fermentation of the mixture of waste paper and kitchen waste. Copyright © 2017. Published by Elsevier Ltd.

Biomimicry enhances sequential reactions of tethered glycolytic enzymes, TPI and GAPDHS.

PubMed

Mukai, Chinatsu; Gao, Lizeng; Bergkvist, Magnus; Nelson, Jacquelyn L; Hinchman, Meleana M; Travis, Alexander J

2013-01-01

Maintaining activity of enzymes tethered to solid interfaces remains a major challenge in developing hybrid organic-inorganic devices. In nature, mammalian spermatozoa have overcome this design challenge by having glycolytic enzymes with specialized targeting domains that enable them to function while tethered to a cytoskeletal element. As a step toward designing a hybrid organic-inorganic ATP-generating system, we implemented a biomimetic site-specific immobilization strategy to tether two glycolytic enzymes representing different functional enzyme families: triose phosphoisomerase (TPI; an isomerase) and glyceraldehyde 3-phosphate dehydrogenase (GAPDHS; an oxidoreductase). We then evaluated the activities of these enzymes in comparison to when they were tethered via classical carboxyl-amine crosslinking. Both enzymes show similar surface binding regardless of immobilization method. Remarkably, specific activities for both enzymes were significantly higher when tethered using the biomimetic, site-specific immobilization approach. Using this biomimetic approach, we tethered both enzymes to a single surface and demonstrated their function in series in both forward and reverse directions. Again, the activities in series were significantly higher in both directions when the enzymes were coupled using this biomimetic approach versus carboxyl-amine binding. Our results suggest that biomimetic, site-specific immobilization can provide important functional advantages over chemically specific, but non-oriented attachment, an important strategic insight given the growing interest in recapitulating entire biological pathways on hybrid organic-inorganic devices.

Biomimicry Enhances Sequential Reactions of Tethered Glycolytic Enzymes, TPI and GAPDHS

PubMed Central

Mukai, Chinatsu; Gao, Lizeng; Bergkvist, Magnus; Nelson, Jacquelyn L.; Hinchman, Meleana M.; Travis, Alexander J.

2013-01-01

Maintaining activity of enzymes tethered to solid interfaces remains a major challenge in developing hybrid organic-inorganic devices. In nature, mammalian spermatozoa have overcome this design challenge by having glycolytic enzymes with specialized targeting domains that enable them to function while tethered to a cytoskeletal element. As a step toward designing a hybrid organic-inorganic ATP-generating system, we implemented a biomimetic site-specific immobilization strategy to tether two glycolytic enzymes representing different functional enzyme families: triose phosphoisomerase (TPI; an isomerase) and glyceraldehyde 3-phosphate dehydrogenase (GAPDHS; an oxidoreductase). We then evaluated the activities of these enzymes in comparison to when they were tethered via classical carboxyl-amine crosslinking. Both enzymes show similar surface binding regardless of immobilization method. Remarkably, specific activities for both enzymes were significantly higher when tethered using the biomimetic, site-specific immobilization approach. Using this biomimetic approach, we tethered both enzymes to a single surface and demonstrated their function in series in both forward and reverse directions. Again, the activities in series were significantly higher in both directions when the enzymes were coupled using this biomimetic approach versus carboxyl-amine binding. Our results suggest that biomimetic, site-specific immobilization can provide important functional advantages over chemically specific, but non-oriented attachment, an important strategic insight given the growing interest in recapitulating entire biological pathways on hybrid organic-inorganic devices. PMID:23626684

Optimization of fermentation parameters for production of ethanol from kinnow waste and banana peels by simultaneous saccharification and fermentation.

PubMed

Sharma, Naresh; Kalra, K L; Oberoi, Harinder Singh; Bansal, Sunil

2007-12-01

A study was taken up to evaluate the role of some fermentation parameters like inoculum concentration, temperature, incubation period and agitation time on ethanol production from kinnow waste and banana peels by simultaneous saccharification and fermentation using cellulase and co-culture of Saccharomyces cerevisiae G and Pachysolen tannophilus MTCC 1077. Steam pretreated kinnow waste and banana peels were used as substrate for ethanol production in the ratio 4:6 (kinnow waste: banana peels). Temperature of 30°C, inoculum size of S. cerevisiae G 6% and (v/v) Pachysolen tannophilus MTCC 1077 4% (v/v), incubation period of 48 h and agitation for the first 24 h were found to be best for ethanol production using the combination of two wastes. The pretreated steam exploded biomass after enzymatic saccharification containing 63 gL(-1) reducing sugars was fermented with both hexose and pentose fermenting yeast strains under optimized conditions resulting in ethanol production, yield and fermentation efficiency of 26.84 gL(-1), 0.426 gg (-1) and 83.52 % respectively. This study could establish the effective utilization of kinnow waste and banana peels for bioethanol production using optimized fermentation parameters.

Restricting lignin and enhancing sugar deposition in secondary cell walls enhances monomeric sugar release after low temperature ionic liquid pretreatment

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

Scullin, Chessa; Cruz, Alejandro G.; Chuang, Yi -De

Lignocellulosic biomass has the potential to be a major source of renewable sugar for biofuel production. Before enzymatic hydrolysis, biomass must first undergo a pretreatment step in order to be more susceptible to saccharification and generate high yields of fermentable sugars. Lignin, a complex, interlinked, phenolic polymer, associates with secondary cell wall polysaccharides, rendering them less accessible to enzymatic hydrolysis. Herein, we describe the analysis of engineered Arabidopsis lines where lignin biosynthesis was repressed in fiber tissues but retained in the vessels, and polysaccharide deposition was enhanced in fiber cells with little to no apparent negative impact on growth phenotype.

Restricting lignin and enhancing sugar deposition in secondary cell walls enhances monomeric sugar release after low temperature ionic liquid pretreatment

DOE PAGES

Scullin, Chessa; Cruz, Alejandro G.; Chuang, Yi -De; ...

2015-07-04

Lignocellulosic biomass has the potential to be a major source of renewable sugar for biofuel production. Before enzymatic hydrolysis, biomass must first undergo a pretreatment step in order to be more susceptible to saccharification and generate high yields of fermentable sugars. Lignin, a complex, interlinked, phenolic polymer, associates with secondary cell wall polysaccharides, rendering them less accessible to enzymatic hydrolysis. Herein, we describe the analysis of engineered Arabidopsis lines where lignin biosynthesis was repressed in fiber tissues but retained in the vessels, and polysaccharide deposition was enhanced in fiber cells with little to no apparent negative impact on growth phenotype.

Lignosulfonate-mediated cellulase adsorption: enhanced enzymatic saccharification of lignocellulose through weakening nonproductive binding to lignin

Treesearch

Zhaojiang Wang; JY Zhu; Yingjuan Fu; Menghua Qin; Zhiyong Shao; Jungang Jiang; Fang Yang

2013-01-01

Thermochemical pretreatment of lignocellulose is crucial to bioconversion in the fields of biorefinery and biofuels. However, the enzyme inhibitors in pretreatment hydrolysate make solid substrate washing and hydrolysate detoxification indispensable prior to enzymatic hydrolysis. Sulfite pretreatment to overcome recalcitrance of lignocelluloses (SPORL) is a relatively...

Hematopoietic Stem Cell Regeneration Enhanced by Ectopic Expression of ROS-detoxifying Enzymes in Transplant Mice

PubMed Central

Miao, Weimin; XuFeng, Richard; Park, Moo-Rim; Gu, Haihui; Hu, Linping; Kang, Jin Wook; Ma, Shihui; Liang, Paulina H; Li, Yanxin; Cheng, Haizi; Yu, Hui; Epperly, Michael; Greenberger, Joel; Cheng, Tao

2013-01-01

High levels of reactive oxygen species (ROS) can exhaust hematopoietic stem cells (HSCs). Thus, maintaining a low state of redox in HSCs by modulating ROS-detoxifying enzymes may augment the regeneration potential of HSCs. Our results show that basal expression of manganese superoxide dismutase (MnSOD) and catalase were at low levels in long-term and short-term repopulating HSCs, and administration of a MnSOD plasmid and lipofectin complex (MnSOD-PL) conferred radiation protection on irradiated recipient mice. To assess the intrinsic role of elevated MnSOD or catalase in HSCs and hematopoietic progenitor cells, the MnSOD or catalase gene was overexpressed in mouse hematopoietic cells via retroviral transduction. The impact of MnSOD and catalase on hematopoietic progenitor cells was mild, as measured by colony-forming units (CFUs). However, overexpressed catalase had a significant beneficial effect on long-term engraftment of transplanted HSCs, and this effect was further enhanced after an insult of low-dose γ-irradiation in the transplant mice. In contrast, overexpressed MnSOD exhibited an insignificant effect on long-term engraftment of transplanted HSCs, but had a significant beneficial effect after an insult of sublethal irradiation. Taken together, these results demonstrate that HSC function can be enhanced by ectopic expression of ROS-detoxifying enzymes, especially after radiation exposure in vivo. PMID:23295952

Sugarcane bagasse pretreatment using three imidazolium-based ionic liquids; mass balances and enzyme kinetics

PubMed Central

2012-01-01

Background Effective pretreatment is key to achieving high enzymatic saccharification efficiency in processing lignocellulosic biomass to fermentable sugars, biofuels and value-added products. Ionic liquids (ILs), still relatively new class of solvents, are attractive for biomass pretreatment because some demonstrate the rare ability to dissolve all components of lignocellulosic biomass including highly ordered (crystalline) cellulose. In the present study, three ILs, 1-butyl-3-methylimidazolium chloride ([C4mim]Cl), 1-ethyl-3-methylimidazolium chloride ([C2mim]Cl), 1-ethyl-3-methylimidazolium acetate ([C2mim]OAc) are used to dissolve/pretreat and fractionate sugarcane bagasse. In these IL-based pretreatments the biomass is completely or partially dissolved in ILs at temperatures greater than 130°C and then precipitated by the addition of an antisolvent to the IL biomass mixture. For the first time mass balances of IL-based pretreatments are reported. Such mass balances, along with kinetics data, can be used in process modelling and design. Results Lignin removals of 10% mass of lignin in bagasse with [C4mim]Cl, 50% mass with [C2mim]Cl and 60% mass with [C2mim]OAc, are achieved by limiting the amount of water added as antisolvent to 0.5 water:IL mass ratio thus minimising lignin precipitation. Enzyme saccharification (24 h, 15FPU) yields (% cellulose mass in starting bagasse) from the recovered solids rank as: [C2mim]OAc(83%) > >[C2mim]Cl(53%) = [C4mim]Cl(53%). Composition of [C2mim]OAc-treated solids such as low lignin, low acetyl group content and preservation of arabinosyl groups are characteristic of aqueous alkali pretreatments while those of chloride IL-treated solids resemble aqueous acid pretreatments. All ILs are fully recovered after use (100% mass as determined by ion chromatography). Conclusions In all three ILs regulated addition of water as an antisolvent effected a polysaccharide enriched precipitate since some of the lignin remained dissolved

Effect of pretreatment severity in continuous steam explosion on enzymatic conversion of wheat straw: Evidence from kinetic analysis of hydrolysis time courses.

PubMed

Monschein, Mareike; Nidetzky, Bernd

2016-01-01

Focusing on continuous steam explosion, the influence of pretreatment severity due to varied acid loading on hydrolysis of wheat straw by Trichoderma reesei cellulases was investigated based on kinetic evaluation of the saccharification of each pretreated substrate. Using semi-empirical descriptors of the hydrolysis time course, key characteristics of saccharification efficiency were captured in a quantifiable fashion. Not only hydrolysis rates per se, but also the transition point of their bi-phasic decline was crucial for high saccharification degree. After 48h the highest saccharification was achieved for substrate pretreated at relatively low severity (1.2% acid). Higher severity increased enzyme binding to wheat straw, but reduced the specific hydrolysis rates. Higher affinity of the lignocellulosic material for cellulases does not necessarily result in increased saccharification, probably because of lignin modifications occurring at high pretreatment severities. At comparable severity, continuous pretreatment produced a substrate more susceptible to enzymatic hydrolysis than the batch process. Copyright © 2015 Elsevier Ltd. All rights reserved.

Cognitive enhancers (Nootropics). Part 3: drugs interacting with targets other than receptors or enzymes. Disease-modifying drugs. Update 2014.

PubMed

Froestl, Wolfgang; Pfeifer, Andrea; Muhs, Andreas

2014-01-01

Scientists working in the field of Alzheimer's disease and, in particular, cognitive enhancers, are very productive. The review "Drugs interacting with Targets other than Receptors or Enzymes. Disease-modifying Drugs" was accepted in October 2012. In the last 20 months, new targets for the potential treatment of Alzheimer's disease were identified. Enormous progress was realized in the pharmacological characterization of natural products with cognitive enhancing properties. This review covers the evolution of research in this field through May 2014.

Optimization of High Solids Dilute Acid Hydrolysis of Spent Coffee Ground at Mild Temperature for Enzymatic Saccharification and Microbial Oil Fermentation.

PubMed

Wang, Hui-Min David; Cheng, Yu-Shen; Huang, Chi-Hao; Huang, Chia-Wei

2016-10-01

Soluble coffee, being one of the world's most popular consuming drinks, produces a considerable amount of spent coffee ground (SCG) along with its production. The SCG could function as a potential lignocellulosic feedstock for production of bioproducts. The objective of this study is to investigate the possible optimal condition of dilute acid hydrolysis (DAH) at high solids and mild temperature condition to release the reducing sugars from SCG. The optimal condition was found to be 5.3 % (w/w) sulfuric acid concentration and 118 min reaction time. Under the optimal condition, the mean yield of reducing sugars from enzymatic saccharification of defatted SCG acid hydrolysate was 563 mg/g. The SCG hydrolysate was then successfully applied to culture Lipomyces starkeyi for microbial oil fermentation without showing any inhibition. The results suggested that dilute acid hydrolysis followed by enzymatic saccharification has the great potential to convert SCG carbohydrates to reducing sugars. This study is useful for the further developing of biorefinery using SCG as feedstock at a large scale.

Dynamic modeling and analyses of simultaneous saccharification and fermentation process to produce bio-ethanol from rice straw.

PubMed

Ko, Jordon; Su, Wen-Jun; Chien, I-Lung; Chang, Der-Ming; Chou, Sheng-Hsin; Zhan, Rui-Yu

2010-02-01

The rice straw, an agricultural waste from Asians' main provision, was collected as feedstock to convert cellulose into ethanol through the enzymatic hydrolysis and followed by the fermentation process. When the two process steps are performed sequentially, it is referred to as separate hydrolysis and fermentation (SHF). The steps can also be performed simultaneously, i.e., simultaneous saccharification and fermentation (SSF). In this research, the kinetic model parameters of the cellulose saccharification process step using the rice straw as feedstock is obtained from real experimental data of cellulase hydrolysis. Furthermore, this model can be combined with a fermentation model at high glucose and ethanol concentrations to form a SSF model. The fermentation model is based on cybernetic approach from a paper in the literature with an extension of including both the glucose and ethanol inhibition terms to approach more to the actual plants. Dynamic effects of the operating variables in the enzymatic hydrolysis and the fermentation models will be analyzed. The operation of the SSF process will be compared to the SHF process. It is shown that the SSF process is better in reducing the processing time when the product (ethanol) concentration is high. The means to improve the productivity of the overall SSF process, by properly using aeration during the batch operation will also be discussed.

Green coconut mesocarp pretreated by an alkaline process as raw material for bioethanol production.

PubMed

Soares, Jimmy; Demeke, Mekonnen M; Foulquié-Moreno, Maria R; Van de Velde, Miet; Verplaetse, Alex; Fernandes, Antonio Alberto Ribeiro; Thevelein, Johan M; Fernandes, Patricia Machado Bueno

2016-09-01

Cocos nucifera L., coconut, is a palm of high importance in the food industry, but a considerable part of the biomass is inedible. In this study, the pretreatment and saccharification parameters NaOH solution, pretreatment duration and enzyme load were evaluated for the production of hydrolysates from green coconut mesocarp using 18% (w/v) total solids (TS). Hydrolysates were not detoxified in order to preserve sugars solubilized during the pretreatment. Reduction of enzyme load from 15 to 7.5 filter paper cellulase unit (FPU)/g of biomass has little effect on the final ethanol titer. With optimized pretreatment and saccharification, hydrolysates with more than 7% (w/v) sugars were produced in 48h. Fermentation of the hydrolysate using industrial Saccharomyces cerevisiae strains produced 3.73% (v/v) ethanol. Our results showed a simple pretreatment condition with a high-solid load of biomass followed by saccharification and fermentation of undetoxified coconut mesocarp hydrolysates to produce ethanol with high titer. Copyright © 2016 Elsevier Ltd. All rights reserved.

An aldonolactonase AltA from Penicillium oxalicum mitigates the inhibition of β-glucosidase during lignocellulose biodegradation.

PubMed

Peng, Shengjuan; Cao, Qing; Qin, Yuqi; Li, Xuezhi; Liu, Guodong; Qu, Yinbo

2017-05-01

Efficient deconstruction of lignocellulose is achieved by the synergistic action of various hydrolytic and oxidative enzymes. However, the aldonolactones generated by oxidative enzymes have inhibitory effects on some cellulolytic enzymes. In this work, D-glucono-1,5-lactone was shown to have a much stronger inhibitory effect than D-glucose and D-gluconate on β-glucosidase, a vital enzyme during cellulose degradation. AltA, a secreted enzyme from Penicillium oxalicum, was identified as an aldonolactonase which can catalyze the hydrolysis of D-glucono-1,5-lactone to D-gluconic acid. In the course of lignocellulose saccharification conducted by cellulases from P. oxalicum or Trichoderma reesei, supplementation of AltA was able to relieve the decrease of β-glucosidase activity obviously with a stimulation of glucose yield. This boosting effect disappeared when sodium azide and ethylenediaminetetraacetic acid (EDTA) were added to the saccharification system to inhibit the activities of oxidative enzymes. In summary, we describe the first heterologous expression of a fungal secreted aldonolactonase and its application as an efficient supplement of cellulolytic enzyme system for lignocellulose biodegradation.

Enzyme-assisted supercritical carbon dioxide extraction of black pepper oleoresin for enhanced yield of piperine-rich extract.

PubMed

Dutta, Sayantani; Bhattacharjee, Paramita

2015-07-01

Black pepper (Piper nigrum L.), the King of Spices is the most popular spice globally and its active ingredient, piperine, is reportedly known for its therapeutic potency. In this work, enzyme-assisted supercritical carbon dioxide (SC-CO2) extraction of black pepper oleoresin was investigated using α-amylase (from Bacillus licheniformis) for enhanced yield of piperine-rich extract possessing good combination of phytochemical properties. Optimization of the extraction parameters (without enzyme), mainly temperature and pressure, was conducted in both batch and continuous modes and the optimized conditions that provided the maximum yield of piperine was in the batch mode, with a sample size of 20 g of black pepper powder (particle diameter 0.42 ± 0.02 mm) at 60 °C and 300 bar at 2 L/min of CO2 flow. Studies on activity of α-amylase were conducted under these optimized conditions in both batch and continuous modes, with varying amounts of lyophilized enzyme (2 mg, 5 mg and 10 mg) and time of exposure of the enzyme to SC-CO2 (2.25 h and 4.25 h). The specific activity of the enzyme increased by 2.13 times when treated in the continuous mode than in the batch mode (1.25 times increase). The structural changes of the treated enzymes were studied by (1)H NMR analyses. In case of α-amylase assisted extractions of black pepper, both batch and continuous modes significantly increased the yields and phytochemical properties of piperine-rich extracts; with higher increase in batch mode than in continuous. Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Characterization of a novel swollenin from Penicillium oxalicum in facilitating enzymatic saccharification of cellulose

PubMed Central

2013-01-01

Background Plant expansins and fungal swollenin that can disrupt crystalline cellulose have great potential for applications in conversion of biomass. Recent studies have been mainly focused on Trichoderma reesei swollenin that show relatively low activity in the promotion of cellulosic hydrolysis. Our aim was to isolate a novel swollenin with greater disruptive activity, to establish an efficient way of producing recombinant swollenin, and to optimize the procedure using swollenin in facilitation of cellulosic hydrolysis. Results A novel gene encoding a swollenin-like protein, POSWOI, was isolated from the filamentous fungus Penicillium oxalicum by Thermal Asymmetric Interlaced PCR (TAIL-PCR). It consisted of a family 1 carbohydrate-binding module (CBM1) followed by a linker connected to a family 45 endoglucanase-like domain. Using the cellobiohydrolase I promoter, recombinant POSWOI was efficiently produced in T. reesei with a yield of 105 mg/L, and showed significant disruptive activity on crystalline cellulose. Simultaneous reaction with both POSWOI and cellulases enhanced the hydrolysis of crystalline cellulose Avicel by approximately 50%. Using a POSWOI-pretreatment procedure, cellulases can produce nearly twice as many reducing sugars as without pretreatment. The mechanism by which POSWOI facilitates the saccharification of cellulose was also studied using a cellulase binding assay. Conclusion We present a novel fungal swollenin with considerable disruptive activity on crystalline cellulose, and develop a better procedure for using swollenin in facilitating cellulosic hydrolysis. We thus provide a new approach for the effective bioconversion of cellulosic biomass. PMID:23688024

Enhancement in multiple lignolytic enzymes production for optimized lignin degradation and selectivity in fungal pretreatment of sweet sorghum bagasse.

PubMed

Mishra, Vartika; Jana, Asim K; Jana, Mithu Maiti; Gupta, Antriksh

2017-07-01

The objective of this work was to study the increase in multiple lignolytic enzyme productions through the use of supplements in combination in pretreatment of sweet sorghum bagasse (SSB) by Coriolus versicolor such that enzymes act synergistically to maximize the lignin degradation and selectivity. Enzyme activities were enhanced by metallic salts and phenolic compound supplements in SSF. Supplement of syringic acid increased the activities of LiP, AAO and laccase; gallic acid increased MnP; CuSO 4 increased laccase and PPO to improve the lignin degradations and selectivity individually, higher than control. Combination of supplements optimized by RSM increased the production of laccase, LiP, MnP, PPO and AAO by 17.2, 45.5, 3.5, 2.4 and 3.6 folds respectively for synergistic action leading to highest lignin degradation (2.3 folds) and selectivity (7.1 folds). Enzymatic hydrolysis of pretreated SSB yielded ∼2.43 times fermentable sugar. This technique could be widely applied for pretreatment and enzyme productions. Copyright © 2017 Elsevier Ltd. All rights reserved.

Cocoa-enriched diet enhances antioxidant enzyme activity and modulates lymphocyte composition in thymus from young rats.

PubMed

Ramiro-Puig, Emma; Urpí-Sardà, Mireia; Pérez-Cano, Francisco J; Franch, Angels; Castellote, Cristina; Andrés-Lacueva, Cristina; Izquierdo-Pulido, Maria; Castell, Margarida

2007-08-08

Cocoa is a rich source of flavonoids, mainly (-)-epicatechin, (+)-catechin, and procyanidins. This article reports the effect of continuous cocoa intake on antioxidant capacity in plasma and tissues, including lymphoid organs and liver, from young rats. Weaned Wistar rats received natural cocoa (4% or 10% food intake) for three weeks, corresponding to their infancy. Flavonoid absorption was confirmed through the quantification of epicatechin metabolites in urine. Total antioxidant capacity (TAC) and the activity of antioxidant enzymes, superoxide dismutase (SOD) and catalase, were examined. Cocoa intake enhanced TAC in all tissues especially in thymus. Moreover, thymus SOD and catalase activities were also dose-dependently increased by cocoa. It was also analyzed whether the enhanced antioxidant system in thymus could influence its cellular composition. An increase in the percentage of thymocytes in advanced development stage was found. In summary, cocoa diet enhances thymus antioxidant defenses and influences thymocyte differentiation.

Enhancing solubility of deoxyxylulose phosphate pathway enzymes for microbial isoprenoid production

PubMed Central

2012-01-01

Background Recombinant proteins are routinely overexpressed in metabolic engineering. It is well known that some over-expressed heterologous recombinant enzymes are insoluble with little or no enzymatic activity. This study examined the solubility of over-expressed homologous enzymes of the deoxyxylulose phosphate pathway (DXP) and the impact of inclusion body formation on metabolic engineering of microbes. Results Four enzymes of this pathway (DXS, ISPG, ISPH and ISPA), but not all, were highly insoluble, regardless of the expression systems used. Insoluble dxs (the committed enzyme of DXP pathway) was found to be inactive. Expressions of fusion tags did not significantly improve the solubility of dxs. However, hypertonic media containing sorbitol, an osmolyte, successfully doubled the solubility of dxs, with the concomitant improvement in microbial production of the metabolite, DXP. Similarly, sorbitol significantly improved the production of soluble and functional ERG12, the committed enzyme in the mevalonate pathway. Conclusion This study demonstrated the unanticipated findings that some over-expressed homologous enzymes of the DXP pathway were highly insoluble, forming inclusion bodies, which affected metabolite formation. Sorbitol was found to increase both the solubility and function of some of these over-expressed enzymes, a strategy to increase the production of secondary metabolites. PMID:23148661

High level expression of Acidothermus cellulolyticus β-1, 4-endoglucanase in transgenic rice enhances the hydrolysis of its straw by cultured cow gastric fluid

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

Chou, Hong L.; Dai, Ziyu; Hsieh, Chia W.

Large-scale production of effective cellulose hydrolytic enzymes is the key to the bioconversion of agricultural residues to ethanol. The goal of this study was to develop a rice plant as a bioreactor for the large-scale production of cellulose hydrolytic enzymes via genetic transformation, and to simultaneously improve rice straw as an efficient biomass feedstock for conversion of cellulose to glucose. In this study, the cellulose hydrolytic enzyme {beta}-1, 4-endoglucanase (E1) from the thermophilic bacterium Acidothermus cellulolyticus was overexpressed in rice through Agrobacterium-mediated transformation. The expression of the bacterial gene in rice was driven by the constitutive Mac promoter, a hybridmore » promoter of Ti plasmid mannopine synthetase promoter and cauliflower mosaic virus 35S promoter enhancer with the signal peptide of tobacco pathogenesis-related protein for targeting the protein to the apoplastic compartment for storage. A total of 52 transgenic rice plants from six independent lines expressing the bacterial enzyme were obtained, which expressed the gene at high levels with a normal phenotype. The specific activities of E1 in the leaves of the highest expressing transgenic rice lines were about 20 fold higher than those of various transgenic plants obtained in previous studies and the protein amounts accounted for up to 6.1% of the total leaf soluble protein. Zymogram and temperature-dependent activity analyses demonstrated the thermostability of the enzyme and its substrate specificity against cellulose, and a simple heat treatment can be used to purify the protein. In addition, hydrolysis of transgenic rice straw with cultured cow gastric fluid yielded almost twice more reducing sugars than wild type straw. Taken together, these data suggest that transgenic rice can effectively serve as a bioreactor for large-scale production of active, thermostable cellulose hydrolytic enzymes. As a feedstock, direct expression of large amount of

Heterologous expression of Talaromyces emersonii cellobiohydrolase Cel7A in Trichoderma reesei increases the efficiency of corncob residues saccharification.

PubMed

Sun, Ningning; Qian, Yuanchao; Wang, Weiwei; Zhong, Yaohua; Dai, Meixue

2018-07-01

Improve the hydrolysis efficiency of the Trichoderma reesei cellulase system by heterologously expressing cellobiohydrolase Cel7A (Te-Cel7A) from the thermophilic fungus Talaromyces emersonii. Te-Cel7A was expressed in T. reesei under control of the cdna1 promoter and the generated transformant QTC14 could successfully secrete Te-Cel7A into the supernatant using glucose as carbon source. The recombinant Te-Cel7A had a temperature optimum at 65 °C and an optimal pH of 5, which were similar to those from the native host. The culture supernatant of QTC14 exhibited a 28.8% enhancement in cellobiohydrolase activity and a 65.2% increase in filter paper activity relative to that of the parental strain QP4. Moreover, the QTC14 cellulase system showed higher thermal stability than that of the parental strain QP4. In the saccharification of delignified corncob residue, the cellulose conversion of QTC14 showed 13.9% higher than that of QP4 at the end of reaction. The thermophilic fungus-derived cellulases could be efficiently expressed by T. reesei and the recombinant cellulases had potential applications for biomass conversion.

Simultaneous Saccharification and Fermentation of Sugar Beet Pulp with Mixed Bacterial Cultures for Lactic Acid and Propylene Glycol Production.

PubMed

Berlowska, Joanna; Cieciura, Weronika; Borowski, Sebastian; Dudkiewicz, Marta; Binczarski, Michal; Witonska, Izabela; Otlewska, Anna; Kregiel, Dorota

2016-10-17

Research into fermentative production of lactic acid from agricultural by-products has recently concentrated on the direct conversion of biomass, whereby pure sugars are replaced with inexpensive feedstock in the process of lactic acid production. In our studies, for the first time, the source of carbon used is sugar beet pulp, generated as a by-product of industrial sugar production. In this paper, we focus on the simultaneous saccharification of lignocellulosic biomass and fermentation of lactic acid, using mixed cultures with complementary assimilation profiles. Lactic acid is one of the primary platform chemicals, and can be used to synthesize a wide variety of useful products, including green propylene glycol. A series of controlled batch fermentations was conducted under various conditions, including pretreatment with enzymatic hydrolysis. Inoculation was performed in two sequential stages, to avoid carbon catabolite repression. Biologically-synthesized lactic acid was catalytically reduced to propylene glycol over 5% Ru/C. The highest lactic acid yield was obtained with mixed cultures. The yield of propylene glycol from the biological lactic acid was similar to that obtained with a water solution of pure lactic acid. Our results show that simultaneous saccharification and fermentation enables generation of lactic acid, suitable for further chemical transformations, from agricultural residues.

Process development of starch hydrolysis using mixing characteristics of Taylor vortices.

PubMed

Masuda, Hayato; Horie, Takafumi; Hubacz, Robert; Ohmura, Naoto; Shimoyamada, Makoto

2017-04-01

In food industries, enzymatic starch hydrolysis is an important process that consists of two steps: gelatinization and saccharification. One of the major difficulties in designing the starch hydrolysis process is the sharp change in its rheological properties. In this study, Taylor-Couette flow reactor was applied to continuous starch hydrolysis process. The concentration of reducing sugar produced via enzymatic hydrolysis was evaluated by varying operational variables: rotational speed of the inner cylinder, axial velocity (reaction time), amount of enzyme, and initial starch content in the slurry. When Taylor vortices were formed in the annular space, efficient hydrolysis occurred because Taylor vortices improved the mixing of gelatinized starch with enzyme. Furthermore, a modified inner cylinder was proposed, and its mixing performance was numerically investigated. The modified inner cylinder showed higher potential for enhanced mixing of gelatinized starch and the enzyme than the conventional cylinder.

Simultaneous saccharification and fermentation of ground corn stover for the production of fuel ethanol using Phanerochaete chrysosporium, Gloeophyllum trabeum, Saccharomyces cerevisiae, and Escherichia coli K011.

PubMed

Vincent, Micky; Pometto, Anthony L; van Leeuwen, J Hans

2011-07-01

Enzymatic saccharification of corn stover using Phanerochaete chrysosporium and Gloeophyllum trabeum and subsequent fermentation of the saccharification products to ethanol by Saccharomyces cerevisiae and Escherichia coli K011 were achieved. Prior to simultaneous saccharification and fermentation (SSF) for ethanol production, solid-state fermentation was performed for four days on ground corn stover using either P. chrysosporium or G. trabeum to induce in situ cellulase production. During SSF with S. cerevisiae or E. coli, ethanol production was the highest on day 4 for all samples. For corn stover treated with P. chrysosporium, the conversion to ethanol was 2.29 g/100 g corn stover with S. cerevisiae as the fermenting organism, whereas for the sample inoculated with E. coli K011, the ethanol production was 4.14 g/100 g corn stover. Corn stover treated with G. trabeum showed a conversion 1.90 and 4.79 g/100 g corn stover with S. cerevisiae and E. coli K011 as the fermenting organisms, respectively. Other fermentation co-products, such as acetic acid and lactic acid, were also monitored. Acetic acid production ranged between 0.45 and 0.78 g/100 g corn stover, while no lactic acid production was detected throughout the 5 days of SSF. The results of our experiment suggest that it is possible to perform SSF of corn stover using P. chrysosporium, G. trabeum, S. cerevisiae and E. coli K011 for the production of fuel ethanol.

Enhancement of proteolytic enzyme activity excreted from Bacillus stearothermophilus for a thermophilic aerobic digestion process.

PubMed

Kim, Young-Kee; Bae, Jin-Hye; Oh, Byung-Keun; Lee, Won Hong; Choi, Jeong-Woo

2002-04-01

Proteolysis is one of the main enzymatic reactions involved in waste activated sludge (WAS) digestion. In this study, proteases excreted from Bacillus stearothermophilus (ATCC 31197) were classified, and an enhancement of protease activity was achieved using economical chemical additives for WAS digestion. Proteases excreted from B. stearothermophilus were classified into two families: serine and metallo-proteases. Various metal ions were investigated as additives which could potentially enhance protease activity. It was observed that Ca2+ and Fe2+ could markedly activate these enzymes. These results were applied to thermophilic aerobic digestion (TAD) of industrial WAS using B. stearothermophilus. The addition of these divalent ions enhanced the degradation performance of the TAD process in terms of reducing the total suspended solids (TSSs), the dissolved organic carbon (DOC) content, and the intracellular and extracellular protein concentrations. The best result, with respect to protein reduction in a digestion experiment, was obtained by the addition of 2 mM Ca2+. Therefore, a proposed TAD process activated by calcium addition can be successfully used for industrial and municipal WAS digestion to the upgrading of TAD process performance.

The role of carbon starvation in the induction of enzymes that degrade plant-derived carbohydrates in Aspergillus niger

PubMed Central

van Munster, Jolanda M.; Daly, Paul; Delmas, Stéphane; Pullan, Steven T.; Blythe, Martin J.; Malla, Sunir; Kokolski, Matthew; Noltorp, Emelie C.M.; Wennberg, Kristin; Fetherston, Richard; Beniston, Richard; Yu, Xiaolan; Dupree, Paul; Archer, David B.

2014-01-01

Fungi are an important source of enzymes for saccharification of plant polysaccharides and production of biofuels. Understanding of the regulation and induction of expression of genes encoding these enzymes is still incomplete. To explore the induction mechanism, we analysed the response of the industrially important fungus Aspergillus niger to wheat straw, with a focus on events occurring shortly after exposure to the substrate. RNA sequencing showed that the transcriptional response after 6 h of exposure to wheat straw was very different from the response at 24 h of exposure to the same substrate. For example, less than half of the genes encoding carbohydrate active enzymes that were induced after 24 h of exposure to wheat straw, were also induced after 6 h exposure. Importantly, over a third of the genes induced after 6 h of exposure to wheat straw were also induced during 6 h of carbon starvation, indicating that carbon starvation is probably an important factor in the early response to wheat straw. The up-regulation of the expression of a high number of genes encoding CAZymes that are active on plant-derived carbohydrates during early carbon starvation suggests that these enzymes could be involved in a scouting role during starvation, releasing inducing sugars from complex plant polysaccharides. We show, using proteomics, that carbon-starved cultures indeed release CAZymes with predicted activity on plant polysaccharides. Analysis of the enzymatic activity and the reaction products, indicates that these proteins are enzymes that can degrade various plant polysaccharides to generate both known, as well as potentially new, inducers of CAZymes. PMID:24792495

The role of carbon starvation in the induction of enzymes that degrade plant-derived carbohydrates in Aspergillus niger.

PubMed

van Munster, Jolanda M; Daly, Paul; Delmas, Stéphane; Pullan, Steven T; Blythe, Martin J; Malla, Sunir; Kokolski, Matthew; Noltorp, Emelie C M; Wennberg, Kristin; Fetherston, Richard; Beniston, Richard; Yu, Xiaolan; Dupree, Paul; Archer, David B

2014-11-01

Fungi are an important source of enzymes for saccharification of plant polysaccharides and production of biofuels. Understanding of the regulation and induction of expression of genes encoding these enzymes is still incomplete. To explore the induction mechanism, we analysed the response of the industrially important fungus Aspergillus niger to wheat straw, with a focus on events occurring shortly after exposure to the substrate. RNA sequencing showed that the transcriptional response after 6h of exposure to wheat straw was very different from the response at 24h of exposure to the same substrate. For example, less than half of the genes encoding carbohydrate active enzymes that were induced after 24h of exposure to wheat straw, were also induced after 6h exposure. Importantly, over a third of the genes induced after 6h of exposure to wheat straw were also induced during 6h of carbon starvation, indicating that carbon starvation is probably an important factor in the early response to wheat straw. The up-regulation of the expression of a high number of genes encoding CAZymes that are active on plant-derived carbohydrates during early carbon starvation suggests that these enzymes could be involved in a scouting role during starvation, releasing inducing sugars from complex plant polysaccharides. We show, using proteomics, that carbon-starved cultures indeed release CAZymes with predicted activity on plant polysaccharides. Analysis of the enzymatic activity and the reaction products, indicates that these proteins are enzymes that can degrade various plant polysaccharides to generate both known, as well as potentially new, inducers of CAZymes. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Adding tetrahydrofuran to dilute acid pretreatment provides new insights into substrate changes that greatly enhance biomass deconstruction by Clostridium thermocellum and fungal enzymes

DOE PAGES

Thomas, Vanessa A.; Donohoe, Bryon S.; Li, Mi; ...

2017-11-30

Consolidated bioprocessing (CBP) by anaerobes, such as Clostridium thermocellum, which combine enzyme production, hydrolysis, and fermentation are promising alternatives to historical economic challenges of using fungal enzymes for biological conversion of lignocellulosic biomass. However, limited research has integrated CBP with real pretreated biomass, and understanding how pretreatment impacts subsequent deconstruction by CBP vs. fungal enzymes can provide valuable insights into CBP and suggest other novel biomass deconstruction strategies. This study focused on determining the effect of pretreatment by dilute sulfuric acid alone (DA) and with tetrahydrofuran (THF) addition via co-solvent-enhanced lignocellulosic fractionation (CELF) on deconstruction of corn stover and Populusmore » with much different recalcitrance by C. thermocellum vs. fungal enzymes and changes in pretreated biomass related to these differences.« less

Adding tetrahydrofuran to dilute acid pretreatment provides new insights into substrate changes that greatly enhance biomass deconstruction by Clostridium thermocellum and fungal enzymes

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

Thomas, Vanessa A.; Donohoe, Bryon S.; Li, Mi

Consolidated bioprocessing (CBP) by anaerobes, such as Clostridium thermocellum, which combine enzyme production, hydrolysis, and fermentation are promising alternatives to historical economic challenges of using fungal enzymes for biological conversion of lignocellulosic biomass. However, limited research has integrated CBP with real pretreated biomass, and understanding how pretreatment impacts subsequent deconstruction by CBP vs. fungal enzymes can provide valuable insights into CBP and suggest other novel biomass deconstruction strategies. This study focused on determining the effect of pretreatment by dilute sulfuric acid alone (DA) and with tetrahydrofuran (THF) addition via co-solvent-enhanced lignocellulosic fractionation (CELF) on deconstruction of corn stover and Populusmore » with much different recalcitrance by C. thermocellum vs. fungal enzymes and changes in pretreated biomass related to these differences.« less

Comparison of Ultrasonic and CO2 Laser Pretreatment Methods on Enzyme Digestibility of Corn Stover

PubMed Central

Tian, Shuang-Qi; Wang, Zhen-Yu; Fan, Zi-Luan; Zuo, Li-Li

2012-01-01

To decrease the cost of bioethanol production, biomass recalcitrance needs to be overcome so that the conversion of biomass to bioethanol becomes more efficient. CO2 laser irradiation can disrupt the lignocellulosic physical structure and reduce the average size of fiber. Analyses with Fourier transform infrared spectroscopy, specific surface area, and the microstructure of corn stover were used to elucidate the enhancement mechanism of the pretreatment process by CO2 laser irradiation. The present work demonstrated that the CO2 laser had potential to enhance the bioconversion efficiency of lignocellulosic waste to renewable bioethanol. The saccharification rate of the CO2 laser pretreatment was significantly higher than ultrasonic pretreatment, and reached 27.75% which was 1.34-fold of that of ultrasonic pretreatment. The results showed the impact of CO2 laser pretreatment on corn stover to be more effective than ultrasonic pretreatment. PMID:22605970

Expression of a bacterial 3-dehydroshikimate dehydratase reduces lignin content and improves biomass saccharification efficiency.

PubMed

Eudes, Aymerick; Sathitsuksanoh, Noppadon; Baidoo, Edward E K; George, Anthe; Liang, Yan; Yang, Fan; Singh, Seema; Keasling, Jay D; Simmons, Blake A; Loqué, Dominique

2015-12-01

Lignin confers recalcitrance to plant biomass used as feedstocks in agro-processing industries or as source of renewable sugars for the production of bioproducts. The metabolic steps for the synthesis of lignin building blocks belong to the shikimate and phenylpropanoid pathways. Genetic engineering efforts to reduce lignin content typically employ gene knockout or gene silencing techniques to constitutively repress one of these metabolic pathways. Recently, new strategies have emerged offering better spatiotemporal control of lignin deposition, including the expression of enzymes that interfere with the normal process for cell wall lignification. In this study, we report that expression of a 3-dehydroshikimate dehydratase (QsuB from Corynebacterium glutamicum) reduces lignin deposition in Arabidopsis cell walls. QsuB was targeted to the plastids to convert 3-dehydroshikimate - an intermediate of the shikimate pathway - into protocatechuate. Compared to wild-type plants, lines expressing QsuB contain higher amounts of protocatechuate, p-coumarate, p-coumaraldehyde and p-coumaryl alcohol, and lower amounts of coniferaldehyde, coniferyl alcohol, sinapaldehyde and sinapyl alcohol. 2D-NMR spectroscopy and pyrolysis-gas chromatography/mass spectrometry (pyro-GC/MS) reveal an increase of p-hydroxyphenyl units and a reduction of guaiacyl units in the lignin of QsuB lines. Size-exclusion chromatography indicates a lower degree of lignin polymerization in the transgenic lines. Therefore, our data show that the expression of QsuB primarily affects the lignin biosynthetic pathway. Finally, biomass from these lines exhibits more than a twofold improvement in saccharification efficiency. We conclude that the expression of QsuB in plants, in combination with specific promoters, is a promising gain-of-function strategy for spatiotemporal reduction of lignin in plant biomass. © 2015 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The

Expression of a bacterial 3-dehydroshikimate dehydratase reduces lignin content and improves biomass saccharification efficiency

DOE PAGES

Eudes, Aymerick; Sathitsuksanoh, Noppadon; Baidoo, Edward E. K.; ...

2015-01-13

Lignin confers recalcitrance to plant biomass used as feedstocks in agro-processing industries or as source of renewable sugars for the production of bioproducts. The metabolic steps for the synthesis of lignin building blocks belong to the shikimate and phenylpropanoid pathways. Genetic engineering efforts to reduce lignin content typically employ gene knockout or gene silencing techniques to constitutively repress one of these metabolic pathways. Recently, new strategies have emerged offering better spatiotemporal control of lignin deposition, including the expression of enzymes that interfere with the normal process for cell wall lignification. In this study, we report that expression of a 3-dehydroshikimatemore » dehydratase (QsuB from Corynebacterium glutamicum) reduces lignin deposition in Arabidopsis cell walls. QsuB was targeted to the plastids to convert 3-dehydroshikimate – an intermediate of the shikimate pathway – into protocatechuate. Compared to wild-type plants, lines expressing QsuB contain higher amounts of protocatechuate, p-coumarate, p-coumaraldehyde and p-coumaryl alcohol, and lower amounts of coniferaldehyde, coniferyl alcohol, sinapaldehyde and sinapyl alcohol. 2D-NMR spectroscopy and pyrolysis-gas chromatography/mass spectrometry (pyro-GC/MS) reveal an increase of p-hydroxyphenyl units and a reduction of guaiacyl units in the lignin of QsuB lines. Size-exclusion chromatography indicates a lower degree of lignin polymerization in the transgenic lines. Therefore, our data show that the expression of QsuB primarily affects the lignin biosynthetic pathway. Finally, biomass from these lines exhibits more than a twofold improvement in saccharification efficiency. We conclude that the expression of QsuB in plants, in combination with specific promoters, is a promising gain-of-function strategy for spatiotemporal reduction of lignin in plant biomass.« less

Spontaneous interfacial reaction between metallic copper and PBS to form cupric phosphate nanoflower and its enzyme hybrid with enhanced activity.

PubMed

He, Guangli; Hu, Weihua; Li, Chang Ming

2015-11-01

We herein report the spontaneous interfacial reaction between copper foil with 0.01 M phosphate buffered saline (PBS) to form free-standing cupric phosphate (Cu3(PO4)2) nanoflowers at ambient temperature. The underlying chemistry was thoroughly investigated and it is found that the formation of nanoflower is synergistically caused by dissolved oxygen, chlorine ions and phosphate ions. Enzyme-Cu3(PO4)2 hybrid nanoflower was further prepared successfully by using an enzyme-dissolving PBS solution and the enzymes in the hybrid exhibit enhanced biological activity. This work provides a facile route for large-scale synthesis of hierarchical inorganic and functional protein-inorganic hybrid architectures via a simple one-step solution-immersion reaction without using either template or surfactant, thus offering great potential for biosensing application among others. Copyright © 2015 Elsevier B.V. All rights reserved.

Lignin Films from Spruce, Eucalyptus, and Wheat Straw Studied with Electroacoustic and Optical Sensors: Effect of Composition and Electrostatic Screening on Enzyme Binding.

PubMed

Pereira, Antonio; Hoeger, Ingrid C; Ferrer, Ana; Rencoret, Jorge; Del Rio, José C; Kruus, Kristiina; Rahikainen, Jenni; Kellock, Miriam; Gutiérrez, Ana; Rojas, Orlando J

2017-04-10

Lignins were isolated from spruce, wheat straw, and eucalyptus by using the milled wood lignin (MWL) method. Functional groups and compositional analyses were assessed via 2D NMR and 31 P NMR to realize their effect on enzyme binding. Films of the lignins were fabricated and ellipsometry, atomic force microscopy, and water contact angle measurements were used for their characterization and to reveal the changes upon enzyme adsorption. Moreover, lignin thin films were deposited on quartz crystal microgravimetry (QCM) and surface plasmon (SPR) resonance sensors and used to gain further insights into the lignin-cellulase interactions. For this purpose, a commercial multicomponent enzyme system and a monocomponent Trichoderma reesei exoglucanase (CBH-I) were considered. Strong enzyme adsorption was observed on the various lignins but compared to the multicomponent cellulases, CBH-I displayed lower surface affinity and higher binding reversibility. This resolved prevalent questions related to the affinity of this enzyme with lignin. Remarkably, a strong correlation between enzyme binding and the syringyl/guaiacyl (S/G) ratio was found for the lignins, which presented a similar hydroxyl group content ( 31 P NMR): higher protein affinity was determined on isolated spruce lignin (99% G units), while the lowest adsorption occurred on isolated eucalyptus lignin (70% S units). The effect of electrostatic interactions in enzyme adsorption was investigated by SPR, which clearly indicated that the screening of charges allowed more extensive protein adsorption. Overall, this work furthers our understanding of lignin-cellulase interactions relevant to biomass that has been subjected to no or little pretreatment and highlights the widely contrasting effects of the nature of lignin, which gives guidance to improve lignocellulosic saccharification and related processes.

Enhanced Colorimetric Immunoassay Accompanying with Enzyme Cascade Amplification Strategy for Ultrasensitive Detection of Low-Abundance Protein

PubMed Central

Gao, Zhuangqiang; Hou, Li; Xu, Mingdi; Tang, Dianping

2014-01-01

Methods based on enzyme labels have been developed for colorimetric immunoassays, but most involve poor sensitivity and are unsuitable for routine use. Herein, we design an enhanced colorimetric immunoassay for prostate-specific antigen (PSA) coupling with an enzyme-cascade-amplification strategy (ECAS-CIA). In the presence of target PSA, the labeled alkaline phosphatase on secondary antibody catalyzes the formation of palladium nanostructures, which catalyze 3,3′,5,5′-tetramethylbenzidine-H2O2 system to produce the colored products, thus resulting in the signal cascade amplification. Results indicated that the ECAS-CIA presents good responses toward PSA, and allows detection of PSA at a concentration as low as 0.05 ng mL−1. Intra- and inter-assay coefficients of variation are below 9.5% and 10.7%, respectively. Additionally, the methodology is validated for analysis of clinical serum specimens with consistent results obtained by PSA ELISA kit. Importantly, the ECAS-CIA opens a new horizon for protein diagnostics and biosecurity. PMID:24509941

Improved pretreatment of lignocellulosic biomass using enzymatically-generated peracetic acid.

PubMed

Yin, DeLu Tyler; Jing, Qing; AlDajani, Waleed Wafa; Duncan, Shona; Tschirner, Ulrike; Schilling, Jonathan; Kazlauskas, Romas J

2011-04-01

Release of sugars from lignocellulosic biomass is inefficient because lignin, an aromatic polymer, blocks access of enzymes to the sugar polymers. Pretreatments remove lignin and disrupt its structure, thereby enhancing sugar release. In previous work, enzymatically generated peracetic acid was used to pretreat aspen wood. This pretreatment removed 45% of the lignin and the subsequent saccharification released 97% of the sugars remaining after pretreatment. In this paper, the amount of enzyme needed is reduced tenfold using first, an improved enzyme variant that makes twice as much peracetic acid and second, a two-phase reaction to generate the peracetic acid, which allows enzyme reuse. In addition, the eight pretreatment cycles are reduced to only one by increasing the volume of peracetic acid solution and increasing the temperature to 60 °C and the reaction time to 6h. For the pretreatment step, the weight ratio of peracetic acid to wood determines the amount of lignin removed. Copyright © 2011 Elsevier Ltd. All rights reserved.

Evaluation of microwave-assisted pretreatment of lignocellulosic biomass immersed in alkaline glycerol for fermentable sugars production.

PubMed

Diaz, Ana Belen; Moretti, Marcia Maria de Souza; Bezerra-Bussoli, Carolina; Carreira Nunes, Christiane da Costa; Blandino, Ana; da Silva, Roberto; Gomes, Eleni

2015-06-01

A pretreatment with microwave irradiation was applied to enhance enzyme hydrolysis of corn straw and rice husk immersed in water, aqueous glycerol or alkaline glycerol. Native and pretreated solids underwent enzyme hydrolysis using the extract obtained from the fermentation of Myceliophthora heterothallica, comparing its efficiency with that of the commercial cellulose cocktail Celluclast®. The highest saccharification yields, for both corn straw and rice husk, were attained when biomass was pretreated in alkaline glycerol, method that has not been previously reported in literature. Moreover, FTIR, TG and SEM analysis revealed a more significant modification in the structure of corn straw subjected to this pretreatment. Highest global yields were attained with the crude enzyme extract, which might be the result of its content in a great variety of hydrolytic enzymes, as revealed zymogram analysis. Moreover, its hydrolysis efficiency can be improved by its supplementation with commercial β-glucosidase. Copyright © 2015 Elsevier Ltd. All rights reserved.

Characteristics of Corn Stover Pretreated with Liquid Hot Water and Fed-Batch Semi-Simultaneous Saccharification and Fermentation for Bioethanol Production

PubMed Central

Li, Xuezhi; Lu, Jie; Zhao, Jian; Qu, Yinbo

2014-01-01

Corn stover is a promising feedstock for bioethanol production because of its abundant availability in China. To obtain higher ethanol concentration and higher ethanol yield, liquid hot water (LHW) pretreatment and fed-batch semi-simultaneous saccharification and fermentation (S-SSF) were used to enhance the enzymatic digestibility of corn stover and improve bioconversion of cellulose to ethanol. The results show that solid residues from LHW pretreatment of corn stover can be effectively converted into ethanol at severity factors ranging from 3.95 to 4.54, and the highest amount of xylan removed was approximately 89%. The ethanol concentrations of 38.4 g/L and 39.4 g/L as well as ethanol yields of 78.6% and 79.7% at severity factors of 3.95 and 4.54, respectively, were obtained by fed-batch S-SSF in an optimum conditions (initial substrate consistency of 10%, and 6.1% solid residues added into system at the prehydrolysis time of 6 h). The changes in surface morphological structure, specific surface area, pore volume and diameter of corn stover subjected to LHW process were also analyzed for interpreting the possible improvement mechanism. PMID:24763192

Biomass enzymatic saccharification is determined by the non-KOH-extractable wall polymer features that predominately affect cellulose crystallinity in corn.

PubMed

Jia, Jun; Yu, Bin; Wu, Leiming; Wang, Hongwu; Wu, Zhiliang; Li, Ming; Huang, Pengyan; Feng, Shengqiu; Chen, Peng; Zheng, Yonglian; Peng, Liangcai

2014-01-01

Corn is a major food crop with enormous biomass residues for biofuel production. Due to cell wall recalcitrance, it becomes essential to identify the key factors of lignocellulose on biomass saccharification. In this study, we examined total 40 corn accessions that displayed a diverse cell wall composition. Correlation analysis showed that cellulose and lignin levels negatively affected biomass digestibility after NaOH pretreatments at p<0.05 & 0.01, but hemicelluloses did not show any significant impact on hexoses yields. Comparative analysis of five standard pairs of corn samples indicated that cellulose and lignin should not be the major factors on biomass saccharification after pretreatments with NaOH and H2SO4 at three concentrations. Notably, despite that the non-KOH-extractable residues covered 12%-23% hemicelluloses and lignin of total biomass, their wall polymer features exhibited the predominant effects on biomass enzymatic hydrolysis including Ara substitution degree of xylan (reverse Xyl/Ara) and S/G ratio of lignin. Furthermore, the non-KOH-extractable polymer features could significantly affect lignocellulose crystallinity at p<0.05, leading to a high biomass digestibility. Hence, this study could suggest an optimal approach for genetic modification of plant cell walls in bioenergy corn.

Biomass Enzymatic Saccharification Is Determined by the Non-KOH-Extractable Wall Polymer Features That Predominately Affect Cellulose Crystallinity in Corn

PubMed Central

Wu, Leiming; Wang, Hongwu; Wu, Zhiliang; Li, Ming; Huang, Pengyan; Feng, Shengqiu; Chen, Peng; Zheng, Yonglian; Peng, Liangcai

2014-01-01

Corn is a major food crop with enormous biomass residues for biofuel production. Due to cell wall recalcitrance, it becomes essential to identify the key factors of lignocellulose on biomass saccharification. In this study, we examined total 40 corn accessions that displayed a diverse cell wall composition. Correlation analysis showed that cellulose and lignin levels negatively affected biomass digestibility after NaOH pretreatments at p<0.05 & 0.01, but hemicelluloses did not show any significant impact on hexoses yields. Comparative analysis of five standard pairs of corn samples indicated that cellulose and lignin should not be the major factors on biomass saccharification after pretreatments with NaOH and H2SO4 at three concentrations. Notably, despite that the non-KOH-extractable residues covered 12%–23% hemicelluloses and lignin of total biomass, their wall polymer features exhibited the predominant effects on biomass enzymatic hydrolysis including Ara substitution degree of xylan (reverse Xyl/Ara) and S/G ratio of lignin. Furthermore, the non-KOH-extractable polymer features could significantly affect lignocellulose crystallinity at p<0.05, leading to a high biomass digestibility. Hence, this study could suggest an optimal approach for genetic modification of plant cell walls in bioenergy corn. PMID:25251456

An overview of technologies for immobilization of enzymes and surface analysis techniques for immobilized enzymes

PubMed Central

Mohamad, Nur Royhaila; Marzuki, Nur Haziqah Che; Buang, Nor Aziah; Huyop, Fahrul; Wahab, Roswanira Abdul

2015-01-01

The current demands of sustainable green methodologies have increased the use of enzymatic technology in industrial processes. Employment of enzyme as biocatalysts offers the benefits of mild reaction conditions, biodegradability and catalytic efficiency. The harsh conditions of industrial processes, however, increase propensity of enzyme destabilization, shortening their industrial lifespan. Consequently, the technology of enzyme immobilization provides an effective means to circumvent these concerns by enhancing enzyme catalytic properties and also simplify downstream processing and improve operational stability. There are several techniques used to immobilize the enzymes onto supports which range from reversible physical adsorption and ionic linkages, to the irreversible stable covalent bonds. Such techniques produce immobilized enzymes of varying stability due to changes in the surface microenvironment and degree of multipoint attachment. Hence, it is mandatory to obtain information about the structure of the enzyme protein following interaction with the support surface as well as interactions of the enzymes with other proteins. Characterization technologies at the nanoscale level to study enzymes immobilized on surfaces are crucial to obtain valuable qualitative and quantitative information, including morphological visualization of the immobilized enzymes. These technologies are pertinent to assess efficacy of an immobilization technique and development of future enzyme immobilization strategies. PMID:26019635

Enzyme Kinetics in Microgravity

NASA Astrophysics Data System (ADS)

Liu, C. C.; Licata, V. J.

2010-04-01

The kinetics of some enzymes have been found to be enhanced by the microgravity environment. This is a relatively small effect, but is sufficient to have physiological effects and to impact pharmaceutical therapy in microgravity.

Enzymes in Fish and Seafood Processing

PubMed Central

Fernandes, Pedro

2016-01-01

Enzymes have been used for the production and processing of fish and seafood for several centuries in an empirical manner. In recent decades, a growing trend toward a rational and controlled application of enzymes for such goals has emerged. Underlying such pattern are, among others, the increasingly wider array of enzyme activities and enzyme sources, improved enzyme formulations, and enhanced requirements for cost-effective and environmentally friendly processes. The better use of enzyme action in fish- and seafood-related application has had a significant impact on fish-related industry. Thus, new products have surfaced, product quality has improved, more sustainable processes have been developed, and innovative and reliable analytical techniques have been implemented. Recent development in these fields are presented and discussed, and prospective developments are suggested. PMID:27458583

21 CFR 864.9400 - Stabilized enzyme solution.

Code of Federal Regulations, 2013 CFR

2013-04-01

...) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Products Used In Establishments That Manufacture Blood and Blood Products § 864.9400 Stabilized enzyme solution. (a) Identification. A stabilized enzyme solution is a reagent intended for medical purposes that is used to enhance the reactivity of red blood...

21 CFR 864.9400 - Stabilized enzyme solution.

Code of Federal Regulations, 2012 CFR

2012-04-01

...) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Products Used In Establishments That Manufacture Blood and Blood Products § 864.9400 Stabilized enzyme solution. (a) Identification. A stabilized enzyme solution is a reagent intended for medical purposes that is used to enhance the reactivity of red blood...

21 CFR 864.9400 - Stabilized enzyme solution.

Code of Federal Regulations, 2014 CFR

2014-04-01

...) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Products Used In Establishments That Manufacture Blood and Blood Products § 864.9400 Stabilized enzyme solution. (a) Identification. A stabilized enzyme solution is a reagent intended for medical purposes that is used to enhance the reactivity of red blood...

Production of raw cassava starch-degrading enzyme by Penicillium and its use in conversion of raw cassava flour to ethanol.

PubMed

Lin, Hai-Juan; Xian, Liang; Zhang, Qiu-Jiang; Luo, Xue-Mei; Xu, Qiang-Sheng; Yang, Qi; Duan, Cheng-Jie; Liu, Jun-Liang; Tang, Ji-Liang; Feng, Jia-Xun

2011-06-01

A newly isolated strain Penicillium sp. GXU20 produced a raw starch-degrading enzyme which showed optimum activity towards raw cassava starch at pH 4.5 and 50 °C. Maximum raw cassava starch-degrading enzyme (RCSDE) activity of 20 U/ml was achieved when GXU20 was cultivated under optimized conditions using wheat bran (3.0% w/v) and soybean meal (2.5% w/v) as carbon and nitrogen sources at pH 5.0 and 28 °C. This represented about a sixfold increment as compared with the activity obtained under basal conditions. Starch hydrolysis degree of 95% of raw cassava flour (150 g/l) was achieved after 72 h of digestion by crude RCSDE (30 U/g flour). Ethanol yield reached 53.3 g/l with fermentation efficiency of 92% after 48 h of simultaneous saccharification and fermentation of raw cassava flour at 150 g/l using the RCSDE (30 U/g flour), carried out at pH 4.0 and 40 °C. This strain and its RCSDE have potential applications in processing of raw cassava starch to ethanol.

Restoration of Traditional Korean Nuruk and Analysis of the Brewing Characteristics.

PubMed

Lee, Jang-Eun; Lee, Ae Ran; Kim, HyeRyun; Lee, Eunjung; Kim, Tae Wan; Shin, Woo Chang; Kim, Jae Ho

2017-05-28

In this study, a total of 58 different kinds of nuruk (a traditional Korean fermentation starter) were prepared, including 46 kinds of restored nuruk from ancient documents. Each nuruk was evaluated by analysis of its saccharification power, and the enzyme activities of glucoamylase, α-amylase, β-amylase, protease, and β-glucanase. The range of saccharification power (sp) of the restored nuruk ranged between 85 and 565 sp. The diastatic enzymes, α-amylase, β-amylase, and glucoamylase, were significantly correlated to the saccharification power value; conversely, β-glucanase and protease did not have a correlative relationship with saccarification power. In addition, their brewing properties on chemical and organoleptic aspects of traditional alcoholic beverage production were compared. Each raw and supplementary material contained in nuruk showed its own unique characteristics on Korean alcoholic beverage brewing. For the first time, in this study, the traditional Korean nuruk types mentioned in ancient documents were restored using modernized production methods, and also characterized based on their brewing properties. Our results could be utilized as a basis for further study of traditional alcoholic beverages and their valuable microorganisms.

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.

Extracellular Enzyme Activity Profile in a Chemically Enhanced Water Accommodated Fraction of Surrogate Oil: Toward Understanding Microbial Activities After the Deepwater Horizon Oil Spill

PubMed Central

Kamalanathan, Manoj; Xu, Chen; Schwehr, Kathy; Bretherton, Laura; Beaver, Morgan; Doyle, Shawn M.; Genzer, Jennifer; Hillhouse, Jessica; Sylvan, Jason B.; Santschi, Peter; Quigg, Antonietta

2018-01-01

Extracellular enzymes and extracellular polymeric substances (EPS) play a key role in overall microbial activity, growth and survival in the ocean. EPS, being amphiphilic in nature, can act as biological surfactant in an oil spill situation. Extracellular enzymes help microbes to digest and utilize fractions of organic matter, including EPS, which can stimulate growth and enhance microbial activity. These natural processes might have been altered during the 2010 Deepwater Horizon oil spill due to the presence of hydrocarbon and dispersant. This study aims to investigate the role of bacterial extracellular enzymes during exposure to hydrocarbons and dispersant. Mesocosm studies were conducted using a water accommodated fraction of oil mixed with the chemical dispersant, Corexit (CEWAF) in seawater collected from two different locations in the Gulf of Mexico and corresponding controls (no additions). Activities of five extracellular enzymes typically found in the EPS secreted by the microbial community – α- and β-glucosidase, lipase, alkaline phosphatase, leucine amino-peptidase – were measured using fluorogenic substrates in three different layers of the mesocosm tanks (surface, water column and bottom). Enhanced EPS production and extracellular enzyme activities were observed in the CEWAF treatment compared to the Control. Higher bacterial and micro-aggregate counts were also observed in the CEWAF treatment compared to Controls. Bacterial genera in the order Alteromonadaceae were the most abundant bacterial 16S rRNA amplicons recovered. Genomes of Alteromonadaceae commonly have alkaline phosphatase and leucine aminopeptidase, therefore they may contribute significantly to the measured enzyme activities. Only Alteromonadaceae and Pseudomonadaceae among bacteria detected here have higher percentage of genes for lipase. Piscirickettsiaceae was abundant; genomes from this order commonly have genes for leucine aminopeptidase. Overall, this study provides insights

Miscanthus as cellulosic biomass for bioethanol production.

PubMed

Lee, Wen-Chien; Kuan, Wei-Chih

2015-06-01

The members of the genus Miscanthus are potential feedstocks for biofuels because of the promising high yields of biomass per unit of planted area. This review addresses species, cultivation, and lignocellulose composition of Miscanthus, as well as pretreatment and enzyme saccharification of Miscanthus biomass for ethanol fermentation. The average cellulose contents in dried biomass of Miscanthus floridulus, Miscanthus sinensis, Miscanthus sacchariflorus, and Miscanthus × giganteus (M × G) are 37.2, 37.6, 38.9, and 41.1% wt/wt, respectively. A number of pretreatment methods have been applied in order to enhance digestibility of Miscanthus biomass for enzymatic saccharification. Pretreatment of Miscanthus using liquid hot water or alkaline results in a significant release of glucose; while glucose yields can be 90% or higher if a pretreatment like AFEX that combines both chemical and physical processes is used. As ethanol is produced by yeast fermentation of the hydrolysate from enzymatic hydrolysis of residual solids (pulp) after pretreatment, theoretical ethanol yields are 0.211-0.233 g/g-raw biomass if only cellulose is taken into account. Simultaneous saccharification and fermentation of pretreated M × G and M. lutarioriparius results in experimental ethanol yields of 0.13 and 0.15 g/g-raw biomass, respectively. Co-production of value-added products can reduce the overall production cost of bioethanol. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Selection of thermotolerant yeasts for simultaneous saccharification and fermentation (SSF) of cellulose to ethanol.

PubMed

Ballesteros, I; Ballesteros, M; Cabañas, A; Carrasco, J; Martín, C; Negro, M J; Saez, F; Saez, R

1991-01-01

A total of 27 yeast strains belonging to the groups Candida, Saccharomyces, and Kluyveromyces were screened for their ability to grow and ferment glucose at temperatures ranging 32-45 degrees C. K. marxianus and K. fragilis were found to be the best ethanol producing organisms at the higher temperature tested and, so, were selected for subsequent simultaneous saccharification and fermentation (SSF) studies. SSF experiments were performed at 42 and 45 degrees C, utilizing Solkafloc (10%) as cellulose substrate and a cellulase loading of 15 FPU/g substrate. Best results were achieved at 42 degrees C with K. marxianus L. G. and K. fragilis L. G., both of which produced close to 38 g/L ethanol and 0.5 ethanol yield, in 78 h.

Enhanced Transformation of TNT by Arabidopsis Plants Expressing an Old Yellow Enzyme

PubMed Central

Zhu, Bo; Peng, Ri-He; Fu, Xiao-Yan; Jin, Xiao-Fen; Zhao, Wei; Xu, Jing; Han, Hong-Juan; Gao, Jian-Jie; Xu, Zhi-Sheng; Bian, Lin; Yao, Quan-Hong

2012-01-01

2,4,6-Trinitrotoluene (TNT) is released in nature from manufacturing or demilitarization facilities, as well as after the firing or detonation of munitions or leakage from explosive remnants of war. Environmental contamination by TNT is associated with human health risks, necessitating the development of cost-effective remediation techniques. The lack of affordable and effective cleanup technologies for explosives contamination requires the development of better processes. In this study, we present a system for TNT phytoremediation by overexpressing the old yellow enzyme (OYE3) gene from Saccharomyces cerevisiae. The resulting transgenic Arabidopsis plants demonstrated significantly enhanced TNT tolerances and a strikingly higher capacity to remove TNT from their media. The current work indicates that S. cerevisiae OYE3 overexpression in Arabidopsis is an efficient method for the phytoremoval and degradation of TNT. Our findings have the potential to provide a suitable remediation strategy for sites contaminated by TNT. PMID:22808068

Rational enhancement of enzyme performance in organic solvents. Final technical report, 1992--1996

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

Klibanov, A.M.

1996-12-31

This research focused on the following: the dependence of enzymatic activity of several model hydrolases in nonaqueous solvents; control of substrate selectivity of the protease subtilisin Carlsberg by the solvent; control of catalytic activity and enantioselectivity of this enzyme in organic solvents by immobilization support; lipase-catalyzed acylation of sugars in anhydrous hydrophobic media; the possibility of accelerating enzymatic processes in organic solvents by certain cosolvents; whether lipase catalysis in organic solvents can be enhanced by introducing interfaces in the in the reaction medium; the structure of proteins suspended in organic solvents; improving enzymatic enantioselectivity in organic solvents; analyzing the plungemore » in enzymatic activity upon replacing water with organic solvents; and the structural basis for the phenomenon of molecular memory of imprinted proteins in organic solvents.« less

Formulation of enzyme blends to maximize the hydrolysis of alkaline peroxide pretreated alfalfa hay and barley straw by rumen enzymes and commercial cellulases

PubMed Central

2014-01-01

Background Efficient conversion of lignocellulosic biomass to fermentable sugars requires the synergistic action of multiple enzymes; consequently enzyme mixtures must be properly formulated for effective hydrolysis. The nature of an optimal enzyme blends depends on the type of pretreatment employed as well the characteristics of the substrate. In this study, statistical experimental design was used to develop mixtures of recombinant glycosyl hydrolases from thermophilic and anaerobic fungi that enhanced the digestion of alkaline peroxide treated alfalfa hay and barley straw by mixed rumen enzymes as well as commercial cellulases (Accelerase 1500, A1500; Accelerase XC, AXC). Results Combinations of feruloyl and acetyl xylan esterases (FAE1a; AXE16A_ASPNG), endoglucanase GH7 (EGL7A_THITE) and polygalacturonase (PGA28A_ASPNG) with rumen enzymes improved straw digestion. Inclusion of pectinase (PGA28A_ASPNG), endoxylanase (XYN11A_THITE), feruloyl esterase (FAE1a) and β-glucosidase (E-BGLUC) with A1500 or endoglucanase GH7 (EGL7A_THITE) and β-xylosidase (E-BXSRB) with AXC increased glucose release from alfalfa hay. Glucose yield from straw was improved when FAE1a and endoglucanase GH7 (EGL7A_THITE) were added to A1500, while FAE1a and AXE16A_ASPNG enhanced the activity of AXC on straw. Xylose release from alfalfa hay was augmented by supplementing A1500 with E-BGLUC, or AXC with EGL7A_THITE and XYN11A_THITE. Adding arabinofuranosidase (ABF54B_ASPNG) and esterases (AXE16A_ASPNG; AXE16B_ASPNG) to A1500, or FAE1a and AXE16A_ASPNG to AXC enhanced xylose release from barley straw, a response confirmed in a scaled up assay. Conclusion The efficacy of commercial enzyme mixtures as well as mixed enzymes from the rumen was improved through formulation with synergetic recombinant enzymes. This approach reliably identified supplemental enzymes that enhanced sugar release from alkaline pretreated alfalfa hay and barley straw. PMID:24766728

Formulation of enzyme blends to maximize the hydrolysis of alkaline peroxide pretreated alfalfa hay and barley straw by rumen enzymes and commercial cellulases.

PubMed

Badhan, Ajay; Wang, Yuxi; Gruninger, Robert; Patton, Donald; Powlowski, Justin; Tsang, Adrian; McAllister, Tim

2014-04-26

Efficient conversion of lignocellulosic biomass to fermentable sugars requires the synergistic action of multiple enzymes; consequently enzyme mixtures must be properly formulated for effective hydrolysis. The nature of an optimal enzyme blends depends on the type of pretreatment employed as well the characteristics of the substrate. In this study, statistical experimental design was used to develop mixtures of recombinant glycosyl hydrolases from thermophilic and anaerobic fungi that enhanced the digestion of alkaline peroxide treated alfalfa hay and barley straw by mixed rumen enzymes as well as commercial cellulases (Accelerase 1500, A1500; Accelerase XC, AXC). Combinations of feruloyl and acetyl xylan esterases (FAE1a; AXE16A_ASPNG), endoglucanase GH7 (EGL7A_THITE) and polygalacturonase (PGA28A_ASPNG) with rumen enzymes improved straw digestion. Inclusion of pectinase (PGA28A_ASPNG), endoxylanase (XYN11A_THITE), feruloyl esterase (FAE1a) and β-glucosidase (E-BGLUC) with A1500 or endoglucanase GH7 (EGL7A_THITE) and β-xylosidase (E-BXSRB) with AXC increased glucose release from alfalfa hay. Glucose yield from straw was improved when FAE1a and endoglucanase GH7 (EGL7A_THITE) were added to A1500, while FAE1a and AXE16A_ASPNG enhanced the activity of AXC on straw. Xylose release from alfalfa hay was augmented by supplementing A1500 with E-BGLUC, or AXC with EGL7A_THITE and XYN11A_THITE. Adding arabinofuranosidase (ABF54B_ASPNG) and esterases (AXE16A_ASPNG; AXE16B_ASPNG) to A1500, or FAE1a and AXE16A_ASPNG to AXC enhanced xylose release from barley straw, a response confirmed in a scaled up assay. The efficacy of commercial enzyme mixtures as well as mixed enzymes from the rumen was improved through formulation with synergetic recombinant enzymes. This approach reliably identified supplemental enzymes that enhanced sugar release from alkaline pretreated alfalfa hay and barley straw.

ZnO-Based Amperometric Enzyme Biosensors

PubMed Central

Zhao, Zhiwei; Lei, Wei; Zhang, Xiaobing; Wang, Baoping; Jiang, Helong

2010-01-01

Nanostructured ZnO with its unique properties could provide a suitable microenvironment for immobilization of enzymes while retaining their biological activity, and thus lead to an expanded use of this nanomaterial for the construction of electrochemical biosensors with enhanced analytical performance. ZnO-based enzyme electrochemical biosensors are summarized in several tables for an easy overview according to the target biosensing analyte (glucose, hydrogen peroxide, phenol and cholesterol), respectively. Moreover, recent developments in enzyme electrochemical biosensors based on ZnO nanomaterials are reviewed with an emphasis on the fabrications and features of ZnO, approaches for biosensor construction (e.g., modified electrodes and enzyme immobilization) and biosensor performances. PMID:22205864

Vertical Integration of Biomass Saccharification of Enzymes for Sustainable Cellulosic Biofuel Production in a Biorefinery

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

Kumar, Manoj

2011-05-09

These are a set of slides from this conference. Lignocellulosic biomass is the most abundant, least expensive renewable natural biological resource for the production of biobased products and bioenergy is important for the sustainable development of human civilization in 21st century. For making the fermentable sugars from lignocellulosic biomass, a reduction in cellulase production cost, an improvement in cellulase performance, and an increase in sugar yields are all vital to reduce the processing costs of biorefineries. Improvements in specific cellulase activities for non-complexed cellulase mixtures can be implemented through cellulase engineering based on rational design or directed evolution for eachmore » cellulase component enzyme, as well as on the reconstitution of cellulase components. In this paper, we will provide DSM's efforts in cellulase research and developments and focus on limitations. Cellulase improvement strategies based on directed evolution using screening on relevant substrates, screening for higher thermal tolerance based on activity screening approaches such as continuous culture using insoluble cellulosic substrates as a powerful selection tool for enriching beneficial cellulase mutants from the large library. We will illustrate why and how thermostable cellulases are vital for economic delivery of bioproducts from cellulosic biomass using biochemical conversion approach.« less

Release of enzymes from lysosomes by irradiation and the relation of lipid peroxide formation to enzyme release

PubMed Central

Wills, E. D.; Wilkinson, A. E.

1966-01-01

1. Acid phosphatase, cathepsin and β-glucuronidase are released from rat-liver lysosomes by irradiation in vitro. Enzyme release is detectable after a dose of 1krad and increases with dose up to 100krads. 2. Maximum radiation effects were observed when the lysosomes were kept for 20hr. at 4° or 20° after irradiation. 3. An atmosphere of nitrogen considerably decreases enzyme release from lysosomes. 4. Enzyme release is enhanced by ascorbic acid and decreased by vitamin E. 5. Irradiation causes formation of lipid peroxides in lysosomes, and enzyme release increases with lipid peroxide formation. 6. It is suggested that lipid peroxide formation leads to rupture of the lysosome membrane and allows release of the contained hydrolytic enzymes. PMID:5964962

Enhancement of hepatic detoxification enzyme activity by dietary mercuric acetate

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

Wagstaff, D.J.

1973-01-01

This report deals with stimulation of liver microsomal enzymes by dietary mercuric acetate (HgAc) and interactions of HgAc with phenobarbital sodium (PB). There is a diphasic response of microsomal enzymes in rats exposed to mercurials. Detoxication activity increased as the dietary dose of HgAc was increased. Liver weight was unaffected by ingestion of HgAc . Toxicity of HgAc increased with dosage. There were no deaths among animals fed diets of 2000 ppM HgAc or less but all five animals fed the diet of 5000 ppM died after five but before ten days on the experiment. The mercury-phenobarbital interactions support speculationmore » that mercury in combination with other chemicals in the environment may have enzyme stimulatory capacity at low exposure levels. 25 references, 1 figure, 1 table.« less

Salt-responsive lytic polysaccharide monooxygenases from the mangrove fungus Pestalotiopsis sp. NCi6.

PubMed

Patel, Ilabahen; Kracher, Daniel; Ma, Su; Garajova, Sona; Haon, Mireille; Faulds, Craig B; Berrin, Jean-Guy; Ludwig, Roland; Record, Eric

2016-01-01

Lytic polysaccharide monooxygenases (LPMOs) belong to the "auxiliary activities (AA)" enzyme class of the CAZy database. They are known to strongly improve the saccharification process and boost soluble sugar yields from lignocellulosic biomass, which is a key step in the efficient production of sustainable economic biofuels. To date, most LPMOs have been characterized from terrestrial fungi, but novel fungal LPMOs isolated from more extreme environments such as an estuary mangrove ecosystem could offer enzymes with unique properties in terms of salt tolerance and higher stability under harsh condition. Two LPMOs secreted by the mangrove-associated fungus Pestalotiopsis sp. NCi6 (PsLPMOA and PsLPMOB) were expressed in the yeast Pichia pastoris and produced in a bioreactor with >85 mg L(-1) for PsLPMOA and >260 mg L(-1) for PsLPMOB. Structure-guided homology modeling of the PsLPMOs showed a high abundance of negative surface charges, enabling enhanced protein stability and activity in the presence of sea salt. Both PsLPMOs were activated by a cellobiose dehydrogenase (CDH) from Neurospora crassa, with an apparent optimum of interaction at pH 5.5. Investigation into their regioselective mode of action revealed that PsLPMOA released C1- and C4-oxidized cello-oligosaccharide products, while PsLPMOB released only C4-oxidized products. PsLPMOA was found to cleave polymeric cellulose in the presence of up to 6 % sea salt, which emphasizes the use of sea water in the industrial saccharification process with improved ecological footprints. Two new LPMOs from the mangrove fungus Pestalotiopsis sp. NCi6 were found to be fully reactive against cellulose. The combined hydrolytic activities of these salt-responsive LPMOs could therefore facilitate the saccharification process using sea water as a reaction medium for large-scale biorefineries.

[Interaction between CYP450 enzymes and metabolism of traditional Chinese medicine as well as enzyme activity assay].

PubMed

Lu, Tu-lin; Su, Lian-lin; Ji, De; Gu, Wei; Mao, Chun-qin

2015-09-01

Drugs are exogenous compounds for human bodies, and will be metabolized by many enzymes after administration. CYP450 enzyme, as a major metabolic enzyme, is an important phase I drug metabolizing enzyme. In human bodies, about 75% of drug metabolism is conducted by CYP450 enzymes, and CYP450 enzymes is the key factor for drug interactions between traditional Chinese medicine( TCM) -TCM, TCM-medicine and other drug combination. In order to make clear the interaction between metabolic enzymes and TCM metabolism, we generally chose the enzymatic activity as an evaluation index. That is to say, the enhancement or reduction of CYP450 enzyme activity was used to infer the inducing or inhibitory effect of active ingredients and extracts of traditional Chinese medicine on enzymes. At present, the common method for measuring metabolic enzyme activity is Cocktail probe drugs, and it is the key to select the suitable probe substrates. This is of great significance for study drug's absorption, distribution, metabolism and excretion (ADME) process in organisms. The study focuses on the interaction between TCMs, active ingredients, herbal extracts, cocktail probe substrates as well as CYP450 enzymes, in order to guide future studies.

Aβ-degrading enzymes: potential for treatment of Alzheimer disease.

PubMed

Miners, James Scott; Barua, Neil; Kehoe, Patrick Gavin; Gill, Steven; Love, Seth

2011-11-01

There is increasing evidence that deficient clearance of β-amyloid (Aβ) contributes to its accumulation in late-onset Alzheimer disease (AD). Several Aβ-degrading enzymes, including neprilysin (NEP), insulin-degrading enzyme, and endothelin-converting enzyme reduce Aβ levels and protect against cognitive impairment in mouse models of AD. The activity of several Aβ-degrading enzymes rises with age and increases still further in AD, perhaps as a physiological response to minimize the buildup of Aβ. The age- and disease-related changes in expression of more recently recognized Aβ-degrading enzymes (e.g. NEP-2 and cathepsin B) remain to be investigated, and there is strong evidence that reduced NEP activity contributes to the development of cerebral amyloid angiopathy. Regardless of the role of Aβ-degrading enzymes in the development of AD, experimental data indicate that increasing the activity of these enzymes (NEP in particular) has therapeutic potential in AD, although targeting their delivery to the brain remains a major challenge. The most promising current approaches include the peripheral administration of agents that enhance the activity of Aβ-degrading enzymes and the direct intracerebral delivery of NEP by convection-enhanced delivery. In the longer term, genetic approaches to increasing the intracerebral expression of NEP or other Aβ-degrading enzymes may offer advantages.

Effects of moisture enhancement, enzyme treatment, and blade tenderization on the processing characteristics and tenderness of beef semimembranosus steaks.

PubMed

Pietrasik, Z; Shand, P J

2011-05-01

The individual and combined effects of moisture enhancement with a salt/phosphate solution (ME), blade tenderization (BT), and enzyme injection with proteinases derived from Aspergillus oryzae or Bacillus subtilis on cooking properties, Warner-Bratzler shear force (WBSF), and sensory characteristics of beef semimembranosus were investigated. ME significantly (P < 0.01) reduced WBSF and increased (P < 0.05) sensory scores for juiciness and tenderness. BT increased (P < 0.05) initial and overall tenderness scores and made connective tissue less perceptible. BT combined with ME resulted in the highest initial and overall tenderness scores, however, combining ME with either proteinase was as effective for reducing WBSF and increasing tenderness, particularly at 20 (vs. 10) ppm enzyme inclusion. Tenderness of enzyme-injected steaks was increased without compromising other palatability attributes. All treatments increased the frequency of steaks rated slightly tender or higher, with the ME+BT combination, or ME with inclusion of 20 ppm of either proteinase, being most effective. Copyright © 2010 The American Meat Science Association. Published by Elsevier Ltd. All rights reserved.

Bioprospecting thermophiles for cellulase production: a review.

PubMed

Acharya, Somen; Chaudhary, Anita

2012-07-01

Most of the potential bioprospecting is currently related to the study of the extremophiles and their potential use in industrial processes. Recently microbial cellulases find applications in various industries and constitute a major group of industrial enzymes. Considerable amount of work has been done on microbial cellulases, especially with resurgence of interest in biomass ethanol production employing cellulases and use of cellulases in textile and paper industry. Most efficient method of lignocellulosic biomass hydrolysis is through enzymatic saccharification using cellulases. Significant information has also been gained about the physiology of thermophilic cellulases producers and process development for enzyme production and biomass saccharification. The review discusses the current knowledge on cellulase producing thermophilic microorganisms, their physiological adaptations and control of cellulase gene expression. It discusses the industrial applications of thermophilic cellulases, their cost of production and challenges in cellulase research especially in the area of improving process economics of enzyme production.

Deciphering the molecular mechanisms behind cellulase production in Trichoderma reesei, the hyper-cellulolytic filamentous fungus.

PubMed

Shida, Yosuke; Furukawa, Takanori; Ogasawara, Wataru

2016-09-01

The filamentous fungus Trichoderma reesei is a potent cellulase producer and the best-studied cellulolytic fungus. A lot of investigations not only on glycoside hydrolases produced by T. reesei, but also on the machinery controlling gene expression of these enzyme have made this fungus a model organism for cellulolytic fungi. We have investigated the T. reesei strain including mutants developed in Japan in detail to understand the molecular mechanisms that control the cellulase gene expression, the biochemical and morphological aspects that could favor this phenotype, and have attempted to generate novel strains that may be appropriate for industrial use. Subsequently, we developed recombinant strains by combination of these insights and the heterologous-efficient saccharifing enzymes. Resulting enzyme preparations were highly effective for saccharification of various biomass. In this review, we present some of the salient findings from the recent biochemical, morphological, and molecular analyses of this remarkable cellulase hyper-producing fungus.

Molecular dynamics investigation of the ionic liquid/enzyme interface: application to engineering enzyme surface charge.

PubMed

Burney, Patrick R; Nordwald, Erik M; Hickman, Katie; Kaar, Joel L; Pfaendtner, Jim

2015-04-01

Molecular simulations of the enzymes Candida rugosa lipase and Bos taurus α-chymotrypsin in aqueous ionic liquids 1-butyl-3-methylimidazolium chloride and 1-ethyl-3-methylimidazolium ethyl sulfate were used to study the change in enzyme-solvent interactions induced by modification of the enzyme surface charge. The enzymes were altered by randomly mutating lysine surface residues to glutamate, effectively decreasing the net surface charge by two for each mutation. These mutations resemble succinylation of the enzyme by chemical modification, which has been shown to enhance the stability of both enzymes in ILs. After establishing that the enzymes were stable on the simulated time scales, we focused the analysis on the organization of the ionic liquid substituents about the enzyme surface. Calculated solvent charge densities show that for both enzymes and in both solvents that changing positively charged residues to negative charge does indeed increase the charge density of the solvent near the enzyme surface. The radial distribution of IL constituents with respect to the enzyme reveals decreased interactions with the anion are prevalent in the modified systems when compared to the wild type, which is largely accompanied by an increase in cation contact. Additionally, the radial dependence of the charge density and ion distribution indicates that the effect of altering enzyme charge is confined to short range (≤1 nm) ordering of the IL. Ultimately, these results, which are consistent with that from prior experiments, provide molecular insight into the effect of enzyme surface charge on enzyme stability in ILs. © 2015 Wiley Periodicals, Inc.

Immobilization of microbial cell and yeast cell and its application to biomass conversion using radiation techniques

NASA Astrophysics Data System (ADS)

Kaetsu, Isao; Kumakura, Minoru; Fujimura, Takashi; Kasai, Noboru; Tamada, Masao

The recent results of immobilization of cellulase-producing cells and ethanol-fermentation yeast by radiation were reported. The enzyme of cellulase produced by immobilized cells was used for saccharification of lignocellulosic wastes and immobilized yeast cells were used for fermentation reaction from glucose to ethanol. The wastes such as chaff and bagasse were treated by γ-ray or electron-beam irradiation in the presence of alkali and subsequent mechanical crushing, to form a fine powder less than 50 μm in diameter. On the other hand, Trichoderma reesei as a cellulase-producing microbial cell was immobilized on a fibrous carrier having a specific porous structure and cultured to produce cellulase. The enzymatic saccharification of the pretreated waste was carried out using the produced cellulase. The enhanced fermentation process to produce ethanol from glucose with the immobilized yeast by radiation was also studied. The ethanol productivity of immobilized growing yeast cells thus obtained was thirteen times that of free yeast cells in a 1:1 volume of liquid medium to immobilized yeast cells.

Effects of enzyme loading, densification, and storage on AFEX-pretreated biomass for ethanol production.

PubMed

Biersbach, Gwen; Rijal, Binod; Pryor, Scott W; Gibbons, William R

2015-12-01

Corn stover, switchgrass, and prairie cordgrass were treated with an ammonia fiber expansion (AFEX) process and a novel densification method (ComPAKco). Separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) were used to evaluate impacts of densification. ComPAKco densification is characterized by low-temperature and low-energy requirements, resulting in compacted biomass briquettes (CBB) 1-2 cm square, with a bulk density of 380-460 kg/m(3). Feedstocks were evaluated before and following AFEX pretreatment, after densification, and after storage. Two enzyme doses were tested. The low rate used 5 filter paper units (FPU) of Spezyme CP (cellulase) and 21.3 cellobiase units (CBU) of Novozyme 188 (aka NS50010 [β-glucosidase]) per gram of glucan. The high dosage rate was three times higher and resulted in 40-56 % and 33-82 % higher ethanol yields with SHF and SSF, respectively. Trials revealed no adverse effect on ethanol yield following densification or 6-month storage of densified, AFEX-pretreated feedstocks.

Enhanced hydrolysis of cellulose hydrogels by morphological modification.

PubMed

Alfassi, Gilad; Rein, Dmitry M; Cohen, Yachin

2017-11-01

Cellulose is one of the most abundant bio-renewable materials on earth, yet the potential of cellulosic bio-fuels is not fully exploited, primarily due to the high costs of conversion. Hydrogel particles of regenerated cellulose constitute a useful substrate for enzymatic hydrolysis, due to their porous and amorphous structure. This article describes the influence of several structural aspects of the cellulose hydrogel on its hydrolysis. The hydrogel density was shown to be directly proportional to the cellulose concentration in the initial solution, thus affecting its hydrolysis rate. Using high-resolution scanning electron microscopy, we show that the hydrogel particles in aqueous suspension exhibit a dense external surface layer and a more porous internal network. Elimination of the external surface layer accelerated the hydrolysis rate by up to sixfold and rendered the process nearly independent of cellulose concentration. These findings may be of practical relevance to saccharification processing costs, by reducing required solvent quantities and enzyme load.

Enzyme leaps fuel antichemotaxis

PubMed Central

Jee, Ah-Young; Dutta, Sandipan; Cho, Yoon-Kyoung

2018-01-01

There is mounting evidence that enzyme diffusivity is enhanced when the enzyme is catalytically active. Here, using superresolution microscopy [stimulated emission-depletion fluorescence correlation spectroscopy (STED-FCS)], we show that active enzymes migrate spontaneously in the direction of lower substrate concentration (“antichemotaxis”) by a process analogous to the run-and-tumble foraging strategy of swimming microorganisms and our theory quantifies the mechanism. The two enzymes studied, urease and acetylcholinesterase, display two families of transit times through subdiffraction-sized focus spots, a diffusive mode and a ballistic mode, and the latter transit time is close to the inverse rate of catalytic turnover. This biochemical information-processing algorithm may be useful to design synthetic self-propelled swimmers and nanoparticles relevant to active materials. Executed by molecules lacking the decision-making circuitry of microorganisms, antichemotaxis by this run-and-tumble process offers the biological function to homogenize product concentration, which could be significant in situations when the reactant concentration varies from spot to spot. PMID:29255047

Antitumor Synergism and Enhanced Survival with a Tumor Vasculature-Targeted Enzyme Prodrug System, Rapamycin, and Cyclophosphamide.

PubMed

Krais, John J; Virani, Needa; McKernan, Patrick H; Nguyen, Quang; Fung, Kar-Ming; Sikavitsas, Vassilios I; Kurkjian, Carla; Harrison, Roger G

2017-09-01

Mutant cystathionine gamma-lyase was targeted to phosphatidylserine exposed on tumor vasculature through fusion with Annexin A1 or Annexin A5. Cystathionine gamma-lyase E58N, R118L, and E338N mutations impart nonnative methionine gamma-lyase activity, resulting in tumor-localized generation of highly toxic methylselenol upon systemic administration of nontoxic selenomethionine. The described therapeutic system circumvents systemic toxicity issues using a novel drug delivery/generation approach and avoids the administration of nonnative proteins and/or DNA required with other enzyme prodrug systems. The enzyme fusion exhibits strong and stable in vitro binding with dissociation constants in the nanomolar range for both human and mouse breast cancer cells and in a cell model of tumor vascular endothelium. Daily administration of the therapy suppressed growth of highly aggressive triple-negative murine 4T1 mammary tumors in immunocompetent BALB/cJ mice and MDA-MB-231 tumors in SCID mice. Treatment did not result in the occurrence of negative side effects or the elicitation of neutralizing antibodies. On the basis of the vasculature-targeted nature of the therapy, combinations with rapamycin and cyclophosphamide were evaluated. Rapamycin, an mTOR inhibitor, reduces the prosurvival signaling of cells in a hypoxic environment potentially exacerbated by a vasculature-targeted therapy. IHC revealed, unsurprisingly, a significant hypoxic response (increase in hypoxia-inducible factor 1 α subunit, HIF1A) in the enzyme prodrug-treated tumors and a dramatic reduction of HIF1A upon rapamycin treatment. Cyclophosphamide, an immunomodulator at low doses, was combined with the enzyme prodrug therapy and rapamycin; this combination synergistically reduced tumor volumes, inhibited metastatic progression, and enhanced survival. Mol Cancer Ther; 16(9); 1855-65. ©2017 AACR . ©2017 American Association for Cancer Research.

Optimization of parameters for enhanced oil recovery from enzyme treated wild apricot kernels.

PubMed

Rajaram, Mahatre R; Kumbhar, Baburao K; Singh, Anupama; Lohani, Umesh Chandra; Shahi, Navin C

2012-08-01

Present investigation was undertaken with the overall objective of optimizing the enzymatic parameters i.e. moisture content during hydrolysis, enzyme concentration, enzyme ratio and incubation period on wild apricot kernel processing for better oil extractability and increased oil recovery. Response surface methodology was adopted in the experimental design. A central composite rotatable design of four variables at five levels was chosen. The parameters and their range for the experiments were moisture content during hydrolysis (20-32%, w.b.), enzyme concentration (12-16% v/w of sample), combination of pectolytic and cellulolytic enzyme i.e. enzyme ratio (30:70-70:30) and incubation period (12-16 h). Aspergillus foetidus and Trichoderma viride was used for production of crude enzyme i.e. pectolytic and cellulolytic enzyme respectively. A complete second order model for increased oil recovery as the function of enzymatic parameters fitted the data well. The best fit model for oil recovery was also developed. The effect of various parameters on increased oil recovery was determined at linear, quadric and interaction level. The increased oil recovery ranged from 0.14 to 2.53%. The corresponding conditions for maximum oil recovery were 23% (w.b.), 15 v/w of the sample, 60:40 (pectolytic:cellulolytic), 13 h. Results of the study indicated that incubation period during enzymatic hydrolysis is the most important factor affecting oil yield followed by enzyme ratio, moisture content and enzyme concentration in the decreasing order. Enzyme ratio, incubation period and moisture content had insignificant effect on oil recovery. Second order model for increased oil recovery as a function of enzymatic hydrolysis parameters predicted the data adequately.

Lignocellulosic butanol production from Napier grass using semi-simultaneous saccharification fermentation.

PubMed

He, Chi-Ruei; Kuo, Yu-Yuan; Li, Si-Yu

2017-05-01

Napier grass is a potential feedstock for biofuel production because of its strong adaptability and wide availability. Compositional analysis has been done on Napier grass which was collected from a local area of Taiwan. By comparing acid- and alkali-pretreatment, it was found that the alkali-pretreatment process is favorable for Napier grass. An overall glucose yield of 0.82g/g-glucose total can be obtained with the combination of alkali-pretreatment (2.5wt% NaOH, 8wt% sample loading, 121°C, and a reaction time of 40min) and enzymatic hydrolysis (40FPU/g-substrate). Semi-simultaneous saccharification fermentation (sSSF) was carried out, where enzymatic hydrolysis and ABE fermentation were operated in the same batch. It was found that after 24-h hydrolysis, followed by 96-h fermentation, the butanol and acetone concentrations reached 9.45 and 4.85g/L, respectively. The butanol yield reached 0.22g/g-sugar glucose+xylose . Finally, the efficiency of butanol production from Napier grass was calculated at 31%. Copyright © 2017 Elsevier Ltd. All rights reserved.

Nanotechnology Enabled Enhancement of Enzyme Activity and Thermostability: Study on Impaired Pectate Lyase from Attenuated Macrophomina phaseolina in Presence of Hydroxyapatite Nanoparticle

PubMed Central

Dutta, Nalok; Mukhopadhyay, Arka; Dasgupta, Anjan Kr.; Chakrabarti, Krishanu

2013-01-01

In this paper we show that hydroxyapatite nanoparticles (NP) can not only act as a chaperon (by imparting thermostability) but can serve as a synthetic enhancer of activity of an isolated extracellular pectate lyase (APL) with low native state activity. The purified enzyme (an attenuated strain of Macrophomina phaseolina) showed feeble activity at 50°C and pH 5.6. However, on addition of 10.5 µg/ml of hydroxyapatite nanoparticles (NP), APL activity increased 27.7 fold with a 51 fold increase in half-life at a temperature of 90°C as compared to untreated APL. The chaperon like activity of NP was evident from entropy–enthalpy compensation profile of APL. The upper critical temperature for such compensation was elevated from 50°C to 90°C in presence of NP. This dual role of NP in enhancing activity and conferring thermostability to a functionally impaired enzyme is reported for the first time. PMID:23691068

Remote Ischemic Preconditioning Enhances the Expression of Genes Encoding Antioxidant Enzymes and Endoplasmic Reticulum Stress-Related Proteins in Rat Skeletal Muscle.

PubMed

Park, Ui Jun; Kim, Hyoung Tae; Cho, Won Hyun; Park, Jae Hyoung; Jung, Hye Ra; Kim, Min Young

2016-12-01

Ischemic preconditioning (IPC), including remote IPC (rIPC) and direct IPC (dIPC), is a promising method to decrease ischemia-reperfusion (IR) injury. This study tested the effect of both rIPC and dIPC on the genes for antioxidant enzymes and endoplasmic reticulum (ER) stress-related proteins. Twenty rats were randomly divided into the control and study groups. In the control group (n=10), the right hind limb was sham-operated. The left hind limb (IscR) of the control group underwent IR injury without IPC. In the study group (n=10), the right hind limb received IR injury after 3 cycles of rIPC. The IscR received IR injury after 3 cycles of dIPC. Gene expression was analyzed by Quantitative real-time polymerase chain reaction from the anterior tibialis muscle. The expression of the antioxidant enzyme genes including glutathione peroxidase (GPx), superoxide dismutase (SOD) 1 and catalase (CAT) were significantly reduced in IscR compared with sham treatment. In comparison with IscR, rIPC enhanced the expression of GPx, SOD2, and CAT genes. dIPC enhanced the expression of SOD2 and CAT genes. The expression of SOD2 genes was consistently higher in rIPC than in dIPC, but the difference was only significant for SOD2. The expression of genes for ER stress-related proteins tended to be reduced in IscR in comparison with sham treatment. However, the difference was only significant for C/EBP homologous protein (CHOP). In comparison with IscR, rIPC significantly up-regulated activating transcription factor 4 and CHOP, whereas dIPC up-regulated CHOP. Both rIPC and dIPC enhanced expression of genes for antioxidant enzymes and ER stress-related proteins.

Cold-Adapted Enzymes

NASA Astrophysics Data System (ADS)

Georlette, D.; Bentahir, M.; Claverie, P.; Collins, T.; D'amico, S.; Delille, D.; Feller, G.; Gratia, E.; Hoyoux, A.; Lonhienne, T.; Meuwis, M.-a.; Zecchinon, L.; Gerday, Ch.

In the last few years, increased attention has been focused on enzymes produced by cold-adapted micro-organisms. It has emerged that psychrophilic enzymes represent an extremely powerful tool in both protein folding investigations and for biotechnological purposes. Such enzymes are characterised by an increased thermosensitivity and, most of them, by a higher catalytic efficiency at low and moderate temperatures, when compared to their mesophilic counterparts. The high thermosensitivity probably originates from an increased flexibility of either a selected area of the molecular edifice or the overall protein structure, providing enhanced abilities to undergo conformational changes during catalysis at low temperatures. Structure modelling and recent crystallographic data have allowed to elucidate the structural parameters that could be involved in this higher resilience. It was demonstrated that each psychrophilic enzyme adopts its own adaptive strategy. It appears, moreover, that there is a continuum in the strategy of protein adaptation to temperature, as the previously mentioned structural parameters are implicated in the stability of thermophilic proteins. Additional 3D crystal structures, site-directed and random mutagenesis experiments should now be undertaken to further investigate the stability-flexibility-activity relationship.

Hydrolysis of lignocelluloses by penicillium funiculosum cellulase

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

Mishra, C.; Rao, M.; Seeta, R.

1984-04-01

Enzymatic hydrolysis of cellulose is a promising method for the conversion of waste cellulose to glucose. During the past few years, the development of this technology has proceeded rapidly, with significant advances made in enzyme production, pretreatment, and hydrolysis. A variety of fungi are reported to produce cellulases but among these Trichoderma reesei and its mutants are powerful producers of cellulases. However, the search for new and possibly better sources of cellulase is continued due to the low levels of beta-glucosidase of T. reesei. Penicillium funiculosum produces a complete cellulase having endo-beta-1,4-glucanase (15-20 U/mL), exo-beta-1,4-glucanase (1.5-2.0 U/mL), and high beta-glucosidasemore » (8-10 U/mL). The saccharification of alkali-treated cotton and bagasse by P. funiculosum enzyme was 70 and 63%, respectively. It was possible to obtain glucose concentration as high as 30% using 50% bagasse. It is of interest that the percent saccharification of cellulosic substrates with the Penicillium enzyme is comparable to that of T. reesei cellulase when the same amount of filter paper activity is used, although the endo-glucanase activity of the latter is two to three times higher. This communication reports the studies on saccharification of lignocelluloses by P. funiculosum cellulase and certain studies on the kinetic aspects. (Refs. 15).« less

An enhanced in vivo stable isotope labeling by amino acids in cell culture (SILAC) model for quantification of drug metabolism enzymes.

PubMed

MacLeod, A Kenneth; Fallon, Padraic G; Sharp, Sheila; Henderson, Colin J; Wolf, C Roland; Huang, Jeffrey T-J

2015-03-01

Many of the enzymes involved in xenobiotic metabolism are maintained at a low basal level and are only synthesized in response to activation of upstream sensor/effector proteins. This induction can have implications in a variety of contexts, particularly during the study of the pharmacokinetics, pharmacodynamics, and drug-drug interaction profile of a candidate therapeutic compound. Previously, we combined in vivo SILAC material with a targeted high resolution single ion monitoring (tHR/SIM) LC-MS/MS approach for quantification of 197 peptide pairs, representing 51 drug metabolism enzymes (DME), in mouse liver. However, as important enzymes (for example, cytochromes P450 (Cyp) of the 1a and 2b subfamilies) are maintained at low or undetectable levels in the liver of unstimulated metabolically labeled mice, quantification of these proteins was unreliable. In the present study, we induced DME expression in labeled mice through synchronous ligand-mediated activation of multiple upstream nuclear receptors, thereby enhancing signals for proteins including Cyps 1a, 2a, 2b, 2c, and 3a. With this enhancement, 115 unique, lysine-containing, Cyp-derived peptides were detected in the liver of a single animal, as opposed to 56 in a pooled sample from three uninduced animals. A total of 386 peptide pairs were quantified by tHR/SIM, representing 68 Phase I, 30 Phase II, and eight control proteins. This method was employed to quantify changes in DME expression in the hepatic cytochrome P450 reductase null (HRN) mouse. We observed compensatory induction of several enzymes, including Cyps 2b10, 2c29, 2c37, 2c54, 2c55, 2e1, 3a11, and 3a13, carboxylesterase (Ces) 2a, and glutathione S-transferases (Gst) m2 and m3, along with down-regulation of hydroxysteroid dehydrogenases (Hsd) 11b1 and 17b6. Using DME-enhanced in vivo SILAC material with tHR/SIM, therefore, permits the robust analysis of multiple DME of importance to xenobiotic metabolism, with improved utility for the study of

Targeting Heparan Sulfate Proteoglycans and their Modifying Enzymes to Enhance Anticancer Chemotherapy Efficacy and Overcome Drug Resistance.

PubMed

Lanzi, Cinzia; Zaffaroni, Nadia; Cassinelli, Giuliana

2017-01-01

Targeting heparan sulfate proteoglycans (HSPGs) and enzymes involved in heparan sulfate (HS) chain editing is emerging as a new anticancer strategy. The involvement of HSPGs in tumor cell signaling, inflammation, angiogenesis and metastasis indicates that agents able to inhibit aberrant HSPG functions can potentially act as multitarget drugs affecting both tumor cell growth and the supportive boost provided by the microenvironment. Moreover, accumulating evidence supports that an altered expression or function of HSPGs, or of the complex enzyme system regulating their activities, can also depress the tumor response to anticancer treatments in several tumor types. Thereby, targeting HSPGs or HSPG modifying enzymes appears an appealing approach to enhance chemotherapy efficacy. A great deal of effort from academia and industry has led to the development of agents mimicking HS, and/or inhibiting HSPG modifying enzymes. Inhibitors of Sulf-2, an endosulfatase that edits the HS sulfation pattern, and inhibitors of heparanase, the endoglycosidase that produces functional HS fragments, appear particularly promising. In fact, a Sulf-2 inhibitor (OKN-007), and two heparanase inhibitors/HS mimics (roneparstat, PG545) are currently under early clinical investigation. In this review, we summarized preclinical studies in experimental tumor models of the main chemical classes of Sulf-2 and heparanase inhibitors. We described examples of different mechanisms through which heparanase and HSPGs, often in cooperation, may impact tumor sensitivity to various antitumor agents. Finally, we reported a few preclinical studies showing increased antitumor efficacy obtained with the use of candidate clinical HS mimics in combination regimens. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Production of lactic acid from the mixture of softwood pre-hydrolysate and paper mill sludge by simultaneous saccharification and fermentation.

PubMed

Shi, Suan; Kang, Li; Lee, Y Y

2015-03-01

Paper mill sludge is a solid waste material composed of pulp residues and ash generated from pulping and paper making process. The carbohydrate portion of the sludges from Kraft/Recycle paper mill has chemical and physical characteristics similar to those of commercial wood pulp. Because of its high carbohydrate content and well-dispersed structure, the sludge can be biologically converted to value-added products without pretreatment. In bioconversion of solid feedstock such as paper mill sludge, a certain amount of water must be present to attain fluidity. In this study, hemicellulose pre-hydrolysate, in place of water, was added to the sludge to increase the concentration of the final product. Pre-hydrolysate was obtained by hot-water treatment of pine wood in which the total sugar concentration reached 4 wt.%. The mixture was processed by simultaneous saccharification and fermentation (SSF) using enzymes (cellulase and pectinase) and Lactobacillus rhamnosus (ATCC-10863). Pectinase was added to hydrolyze mannose oligomers in the pre-hydrolysate to monomers. During the SSF of the mixture, calcium carbonate in the paper sludge acted as a buffer, yielding calcium lactate as the final product. External pH control was unnecessary due to the buffer action of calcium carbonate that maintained the pH near optimum for the SSF. The lactic acid yield in the range of 80-90 % of the theoretical maximum was obtained. Use of the mixed feed of pre-hydrolysate and pulp mill sludges in the SSF raised the product concentration to 60 g of lactate/L.

Enhancement of catalytic activity of enzymes by heating in anhydrous organic solvents: 3D structure of a modified serine proteinase at high resolution.

PubMed

Sharma, S; Tyagi, R; Gupta, M N; Singh, T P

2001-01-01

For the first time, it is demonstrated that exposure of an enzyme to anhydrous organic solvents at optimized high temperature enhances its catalytic power through local changes at the binding region. Six enzymes, namely, proteinase K, wheat germ acid phosphatase, alpha-amylase, beta-glucosidase, chymotrypsin and trypsin were exposed to acetonitrile at 70 degrees C for three hr. The activities of these enzymes were found to be considerably enhanced. In order to understand the basis of this change in the activity of these enzymes, proteinase K was analyzed in detail using X-ray diffraction method. The overall structure of the enzyme was found to be similar to the native structure in aqueous environment. The hydrogen bonding system of the catalytic triad remained intact after the treatment. However, the water structure in the substrate binding site underwent some rearrangement as some of the water molecules were either displaced or completely absent. The most striking observation concerning the water structure was the complete deletion of the water molecule which occupied the position at the so-called oxyanion hole in the active site of the native enzyme. Three acetonitrile molecules were found in the present structure. All the acetonitrile molecules were located in the recognition site. Interlinked through water molecules, the sites occupied by acetonitrile molecules were independent of water molecules. The acetonitrile molecules are involved in extensive interactions with the protein atoms. The methyl group of one of the acetonitrile molecules (CCN1) interacts simultaneously with the hydrophobic side chains of Leu 96, Ile 107 and Leu 133. The development of such a hydrophobic environment at the recognition site introduced a striking conformation change in Ile 107 by rotating its side chain about C alpha-C beta bond by 180 degrees to bring about the delta-methyl group within the range of attractive van der Waals interactions with the methyl group of CCN1. A similar

The effectiveness of dietary sunflower meal and exogenous enzyme on growth, digestive enzymes, carcass traits, and blood chemistry of broilers.

PubMed

Alagawany, Mahmoud; Attia, Adel I; Ibrahim, Zenat A; Mahmoud, Reda A; El-Sayed, Sabry A

2017-05-01

High costs of conventional protein feed sources including soybean meal (SBM) generated the need for finding other alternatives. Thus, the present study was designed to evaluate the impact of graded replacements of SBM by sunflower seed meal (SFM) with or without enzyme supplementation on growth performance, digestive enzymes, carcass traits, and blood profile of broiler chickens. A total of 240 unsexed 1-week-old broiler chicks (Hubbard) were randomly divided into eight treatment groups of 30 chicks each in five replicates each of six chicks in a factorial design (4 × 2) arrangement, including four levels of SFM (0, 25, 50, and 75% replacing SBM) and two levels of enzyme (0- or 0.1-g/kg diet) supplementation. Performance traits including feed conversion ratio, body weight, and weight gain were significantly (P < 0.01) improved with increasing SFM up to 50% substitution for SBM or with enzyme supplementation in broiler diet during the experiment. However, feed intake of broiler chicks was decreased with enzyme supplementation (P < 0.05). The activities of digestive enzymes (protease and amylase) were significantly (P < 0.05) influenced and enhanced by SFM and enzyme inclusion in diets, respectively. The activities of protease and amylase were improved with SFM diet supplemented with 0.1 g/kg enzyme in comparison with those with the un-supplemented diet. The evaluated carcass traits were not statistically (P > 0.05) influenced by feeding SFM meal or enzyme addition. Biochemical blood parameters were significantly (P < 0.01) affected by SFM, enzyme, or their interaction in broiler diets, except for globulin that was not affected by dietary enzyme. It is concluded that increasing SFM level in the diet up to 50% replacing SBM with the supplementation of enzyme improved the growth performance and enhanced positively carcass traits as well as the activity of digestive enzymes in broiler chickens.

Enhanced efficiency of biological excess sludge hydrolysis under anaerobic digestion by additional enzymes.

PubMed

Yang, Qi; Luo, Kun; Li, Xiao-ming; Wang, Dong-bo; Zheng, Wei; Zeng, Guang-ming; Liu, Jing-jin

2010-05-01

In this investigation, the effects of commercial enzyme preparation containing alpha amylase and neutral protease on hydrolysis of excess sludge and the kinetic analysis of hydrolysis process were evaluated. The results indicated that amylase treatment displayed higher hydrolysis efficiency than that of protease. VSS reduction greatly increased to 39.70% for protease and 54.24% for amylase at the enzyme dosage of 6% (w/w), respectively. The hydrolysis rate of sludge improved with temperature increasing from 40 to 50 degrees Celsius, which could be well described by the amended Arrhenius equation. Mixed-enzyme had great impact on sludge solubilisation than single enzyme. The mixture of two enzymes (protease:amylase=1:3) resulted in optimum hydrolysis efficiency, the efficiency of solids hydrolysis increased from 10% (control test) to 68.43% at the temperature of 50 degrees Celsius. Correspondingly, the concentration of reducing sugar and NH(4)(+)-N improved about 377% and 201%, respectively. According to the kinetic analysis of enzymatic hydrolysis process, VSS solubilisation process within prior 4 h followed first-order kinetics. Compared with control test, the hydrolysis rate improved significantly at 50 degrees Celsius when either single enzyme or mixed-enzyme was added. Copyright 2009. Published by Elsevier Ltd.

Traffic jams reduce hydrolytic efficiency of cellulase on cellulose surface.

PubMed

Igarashi, Kiyohiko; Uchihashi, Takayuki; Koivula, Anu; Wada, Masahisa; Kimura, Satoshi; Okamoto, Tetsuaki; Penttilä, Merja; Ando, Toshio; Samejima, Masahiro

2011-09-02

A deeper mechanistic understanding of the saccharification of cellulosic biomass could enhance the efficiency of biofuels development. We report here the real-time visualization of crystalline cellulose degradation by individual cellulase enzymes through use of an advanced version of high-speed atomic force microscopy. Trichoderma reesei cellobiohydrolase I (TrCel7A) molecules were observed to slide unidirectionally along the crystalline cellulose surface but at one point exhibited collective halting analogous to a traffic jam. Changing the crystalline polymorphic form of cellulose by means of an ammonia treatment increased the apparent number of accessible lanes on the crystalline surface and consequently the number of moving cellulase molecules. Treatment of this bulky crystalline cellulose simultaneously or separately with T. reesei cellobiohydrolase II (TrCel6A) resulted in a remarkable increase in the proportion of mobile enzyme molecules on the surface. Cellulose was completely degraded by the synergistic action between the two enzymes.

Bioprospecting thermophiles for cellulase production: a review

PubMed Central

Acharya, Somen; Chaudhary, Anita

2012-01-01

Most of the potential bioprospecting is currently related to the study of the extremophiles and their potential use in industrial processes. Recently microbial cellulases find applications in various industries and constitute a major group of industrial enzymes. Considerable amount of work has been done on microbial cellulases, especially with resurgence of interest in biomass ethanol production employing cellulases and use of cellulases in textile and paper industry. Most efficient method of lignocellulosic biomass hydrolysis is through enzymatic saccharification using cellulases. Significant information has also been gained about the physiology of thermophilic cellulases producers and process development for enzyme production and biomass saccharification. The review discusses the current knowledge on cellulase producing thermophilic microorganisms, their physiological adaptations and control of cellulase gene expression. It discusses the industrial applications of thermophilic cellulases, their cost of production and challenges in cellulase research especially in the area of improving process economics of enzyme production. PMID:24031898

Protective effect of bioflavonoid myricetin enhances carbohydrate metabolic enzymes and insulin signaling molecules in streptozotocin–cadmium induced diabetic nephrotoxic rats

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

Kandasamy, Neelamegam; Ashokkumar, Natarajan, E-mail: npashokkumar1@gmail.com

Diabetic nephropathy is the kidney disease that occurs as a result of diabetes. The present study was aimed to evaluate the therapeutic potential of myricetin by assaying the activities of key enzymes of carbohydrate metabolism, insulin signaling molecules and renal function markers in streptozotocin (STZ)–cadmium (Cd) induced diabetic nephrotoxic rats. After myricetin treatment schedule, blood and tissue samples were collected to determine plasma glucose, insulin, hemoglobin, glycosylated hemoglobin and renal function markers, carbohydrate metabolic enzymes in the liver and insulin signaling molecules in the pancreas and skeletal muscle. A significant increase of plasma glucose, glycosylated hemoglobin, urea, uric acid, creatinine,more » blood urea nitrogen (BUN), urinary albumin, glycogen phosphorylase, glucose-6-phosphatase, and fructose-1,6-bisphosphatase and a significant decrease of plasma insulin, hemoglobin, hexokinase, glucose-6-phosphate dehydrogenase, glycogen and glycogen synthase with insulin signaling molecule expression were found in the STZ–Cd induced diabetic nephrotoxic rats. The administration of myricetin significantly normalizes the carbohydrate metabolic products like glucose, glycated hemoglobin, glycogen phosphorylase and gluconeogenic enzymes and renal function markers with increase insulin, glycogen, glycogen synthase and insulin signaling molecule expression like glucose transporter-2 (GLUT-2), glucose transporter-4 (GLUT-4), insulin receptor-1 (IRS-1), insulin receptor-2 (IRS-2) and protein kinase B (PKB). Based on the data, the protective effect of myricetin was confirmed by its histological annotation of the pancreas, liver and kidney tissues. These findings suggest that myricetin improved carbohydrate metabolism which subsequently enhances glucose utilization and renal function in STZ–Cd induced diabetic nephrotoxic rats. - Highlights: • Diabetic rats are more susceptible to cadmium nephrotoxicity. • Cadmium plays as a

Pullulanase: Role in Starch Hydrolysis and Potential Industrial Applications

PubMed Central

Hii, Siew Ling; Tan, Joo Shun; Ling, Tau Chuan; Ariff, Arbakariya Bin

2012-01-01

The use of pullulanase (EC 3.2.1.41) has recently been the subject of increased applications in starch-based industries especially those aimed for glucose production. Pullulanase, an important debranching enzyme, has been widely utilised to hydrolyse the α-1,6 glucosidic linkages in starch, amylopectin, pullulan, and related oligosaccharides, which enables a complete and efficient conversion of the branched polysaccharides into small fermentable sugars during saccharification process. The industrial manufacturing of glucose involves two successive enzymatic steps: liquefaction, carried out after gelatinisation by the action of α-amylase; saccharification, which results in further transformation of maltodextrins into glucose. During saccharification process, pullulanase has been used to increase the final glucose concentration with reduced amount of glucoamylase. Therefore, the reversion reaction that involves resynthesis of saccharides from glucose molecules is prevented. To date, five groups of pullulanase enzymes have been reported, that is, (i) pullulanase type I, (ii) amylopullulanase, (iii) neopullulanase, (iv) isopullulanase, and (v) pullulan hydrolase type III. The current paper extensively reviews each category of pullulanase, properties of pullulanase, merits of applying pullulanase during starch bioprocessing, current genetic engineering works related to pullulanase genes, and possible industrial applications of pullulanase. PMID:22991654

Enhancement of synthetic Trichoderma-based enzyme mixtures for biomass conversion with an alternative family 5 glycosyl hydrolase from Sporotrichum thermophile.

PubMed

Ye, Zhuoliang; Zheng, Yun; Li, Bingyao; Borrusch, Melissa S; Storms, Reginald; Walton, Jonathan D

2014-01-01

Enzymatic conversion of lignocellulosic materials to fermentable sugars is a limiting step in the production of biofuels from biomass. We show here that combining enzymes from different microbial sources is one way to identify superior enzymes. Extracts of the thermophilic fungus Sporotrichum thermophile (synonym Myceliophthora thermophila) gave synergistic release of glucose (Glc) and xylose (Xyl) from pretreated corn stover when combined with an 8-component synthetic cocktail of enzymes from Trichoderma reesei. The S. thermophile extracts were fractionated and an enhancing factor identified as endo-β1,4-glucanase (StCel5A or EG2) of subfamily 5 of Glycosyl Hydrolase family 5 (GH5_5). In multi-component optimization experiments using a standard set of enzymes and either StCel5A or the ortholog from T. reesei (TrCel5A), reactions containing StCel5A yielded more Glc and Xyl. In a five-component optimization experiment (i.e., varying four core enzymes and the source of Cel5A), the optimal proportions for TrCel5A vs. StCel5A were similar for Glc yields, but markedly different for Xyl yields. Both enzymes were active on lichenan, glucomannan, and oat β-glucan; however, StCel5A but not TrCel5A was also active on β1,4-mannan, two types of galactomannan, and β1,4-xylan. Phylogenetically, fungal enzymes in GH5_5 sorted into two clades, with StCel5A and TrCel5A belonging to different clades. Structural differences with the potential to account for the differences in performance were deduced based on the known structure of TrCel5A and a homology-based model of StCel5A, including a loop near the active site of TrCel5A and the presence of four additional Trp residues in the active cleft of StCel5A. The results indicate that superior biomass-degrading enzymes can be identified by exploring taxonomic diversity combined with assays in the context of realistic enzyme combinations and realistic substrates. Substrate range may be a key factor contributing to superior performance

Enhancement of Synthetic Trichoderma-Based Enzyme Mixtures for Biomass Conversion with an Alternative Family 5 Glycosyl Hydrolase from Sporotrichum thermophile

PubMed Central

Ye, Zhuoliang; Zheng, Yun; Li, Bingyao; Borrusch, Melissa S.; Storms, Reginald; Walton, Jonathan D.

2014-01-01

Enzymatic conversion of lignocellulosic materials to fermentable sugars is a limiting step in the production of biofuels from biomass. We show here that combining enzymes from different microbial sources is one way to identify superior enzymes. Extracts of the thermophilic fungus Sporotrichum thermophile (synonym Myceliophthora thermophila) gave synergistic release of glucose (Glc) and xylose (Xyl) from pretreated corn stover when combined with an 8-component synthetic cocktail of enzymes from Trichoderma reesei. The S. thermophile extracts were fractionated and an enhancing factor identified as endo-β1,4- glucanase (StCel5A or EG2) of subfamily 5 of Glycosyl Hydrolase family 5 (GH5_5). In multi-component optimization experiments using a standard set of enzymes and either StCel5A or the ortholog from T. reesei (TrCel5A), reactions containing StCel5A yielded more Glc and Xyl. In a five-component optimization experiment (i.e., varying four core enzymes and the source of Cel5A), the optimal proportions for TrCel5A vs. StCel5A were similar for Glc yields, but markedly different for Xyl yields. Both enzymes were active on lichenan, glucomannan, and oat β-glucan; however, StCel5A but not TrCel5A was also active on β1,4-mannan, two types of galactomannan, and β1,4-xylan. Phylogenetically, fungal enzymes in GH5_5 sorted into two clades, with StCel5A and TrCel5A belonging to different clades. Structural differences with the potential to account for the differences in performance were deduced based on the known structure of TrCel5A and a homology-based model of StCel5A, including a loop near the active site of TrCel5A and the presence of four additional Trp residues in the active cleft of StCel5A. The results indicate that superior biomass-degrading enzymes can be identified by exploring taxonomic diversity combined with assays in the context of realistic enzyme combinations and realistic substrates. Substrate range may be a key factor contributing to superior

Activation of immobilized enzymes by acoustic wave resonance oscillation.

PubMed

Nishiyama, Hiroshi; Watanabe, Tomoya; Inoue, Yasunobu