Two Strategies for Microbial Production of an Industrial Enzyme-Alpha-Amylase

NASA Technical Reports Server (NTRS)

Bernhardsdotter, Eva C. M. J.; Garriott, Owen; Pusey, Marc L.; Ng, Joseph D.

2003-01-01

Extremophiles are microorganisms that thrive in, from an anthropocentric view, extreme environments including hot springs, soda lakes and arctic water. This ability of survival at extreme conditions has rendered extremophiles to be of interest in astrobiology, evolutionary biology as well as in industrial applications. Of particular interest to the biotechnology industry are the biological catalysts of the extremophiles, the extremozymes, whose unique stabilities at extreme conditions make them potential sources of novel enzymes in industrial applications. There are two major approaches to microbial enzyme production. This entails enzyme isolation directly from the natural host or creating a recombinant expression system whereby the targeted enzyme can be overexpressed in a mesophilic host. We are employing both methods in the effort to produce alpha-amylases from a hyperthermophilic archaeon (Thermococcus) isolated from a hydrothermal vent in the Atlantic Ocean, as well as from alkaliphilic bacteria (Bacillus) isolated from a soda lake in Tanzania. Alpha-amylases catalyze the hydrolysis of internal alpha-1,4-glycosidic linkages in starch to produce smaller sugars. Thermostable alpha-amylases are used in the liquefaction of starch for production of fructose and glucose syrups, whereas alpha-amylases stable at high pH have potential as detergent additives. The alpha-amylase encoding gene from Thermococcus was PCR amplified using carefully designed primers and analyzed using bioinformatics tools such as BLAST and Multiple Sequence Alignment for cloning and expression in E.coli. Four strains of Bacillus were grown in alkaline starch-enriched medium of which the culture supernatant was used as enzyme source. Amylolytic activity was detected using the starch-iodine method.

Sequence similarities and evolutionary relationships of microbial, plant and animal alpha-amylases.

PubMed

Janecek, S

1994-09-01

Amino acid sequence comparison of 37 alpha-amylases from microbial, plant and animal sources was performed to identify their mutual sequence similarities in addition to the five already described conserved regions. These sequence regions were examined from structure/function and evolutionary perspectives. An unrooted evolutionary tree of alpha-amylases was constructed on a subset of 55 residues from the alignment of sequence similarities along with conserved regions. The most important new information extracted from the tree was as follows: (a) the close evolutionary relationship of Alteromonas haloplanctis alpha-amylase (thermolabile enzyme from an antarctic psychrotroph) with the already known group of homologous alpha-amylases from streptomycetes, Thermomonospora curvata, insects and mammals, and (b) the remarkable 40.1% identity between starch-saccharifying Bacillus subtilis alpha-amylase and the enzyme from the ruminal bacterium Butyrivibrio fibrisolvens, an alpha-amylase with an unusually large polypeptide chain (943 residues in the mature enzyme). Due to a very high degree of similarity, the whole amino acid sequences of three groups of alpha-amylases, namely (a) fungi and yeasts, (b) plants, and (c) A. haloplanctis, streptomycetes, T. curvata, insects and mammals, were aligned independently and their unrooted distance trees were calculated using these alignments. Possible rooting of the trees was also discussed. Based on the knowledge of the location of the five disulfide bonds in the structure of pig pancreatic alpha-amylase, the possible disulfide bridges were established for each of these groups of homologous alpha-amylases.

A novel strategy to improve protein secretion via overexpression of the SppA signal peptide peptidase in Bacillus licheniformis.

PubMed

Cai, Dongbo; Wang, Hao; He, Penghui; Zhu, Chengjun; Wang, Qin; Wei, Xuetuan; Nomura, Christopher T; Chen, Shouwen

2017-04-24

Signal peptide peptidases play an important role in the removal of remnant signal peptides in the cell membrane, a critical step for extracellular protein production. Although these proteins are likely a central component for extracellular protein production, there has been a lack of research on whether protein secretion could be enhanced via overexpression of signal peptide peptidases. In this study, both nattokinase and α-amylase were employed as prototypical secreted target proteins to evaluate the function of putative signal peptide peptidases (SppA and TepA) in Bacillus licheniformis. We observed dramatic decreases in the concentrations of both target proteins (45 and 49%, respectively) in a sppA deficient strain, while the extracellular protein yields of nattokinase and α-amylase were increased by 30 and 67% respectively in a strain overexpressing SppA. In addition, biomass, specific enzyme activities and the relative gene transcriptional levels were also enhanced due to the overexpression of sppA, while altering the expression levels of tepA had no effect on the concentrations of the secreted target proteins. Our results confirm that SppA, but not TepA, plays an important functional role for protein secretion in B. licheniformis. Our results indicate that the sppA overexpression strain, B. licheniformis BL10GS, could be used as a promising host strain for the industrial production of heterologous secreted proteins.

Construction of promoter-probe shuttle vectors for Escherichia coli and corynebacteria on the basis of promoterless alpha-amylase gene.

PubMed

Ugorcáková, J; Bukovská, G; Timko, J

2000-01-01

We constructed new promoter-probe vectors for E. coli and corynebacteria based on the promoterless alpha-amylase gene originating from Bacillus subtilis. Vectors pJUPAE1 and pJUPAE2 are suitable for isolation of transcriptionally active fragments from plasmids, phages or genomic DNA. alpha-Amylase activity can be easily visually detected on agar plates containing a chromogenic substrate, or by direct measurement of alpha-amylase activity.

Central carbon metabolism influences cellulase production in Bacillus licheniformis.

PubMed

Wang, J; Liu, S; Li, Y; Wang, H; Xiao, S; Li, C; Liu, B

2018-01-01

Bacillus licheniformis that can produce cellulase including endo glucanase and glucosidase is an important industrial microbe for cellulose degradation. The purpose of this research was to assess the effect of endo glucanase gene bglC and glucosidase gene bglH on the central metabolic flux in B. licheniformis. bglC and bglH were knocked out using homologous recombination method, respectively, and the corresponding knockout strains were obtained for 13 C metabolic flux analysis. A significant change was observed in metabolic fluxes after 13 C metabolic flux ratio analysis. In both of the knockout strains, the increased fluxes of the pentose phosphate pathway and malic enzyme reaction enabled an elevated supply of NADPH which provided enough reducing power for the in vivo synthesis reactions. The fluxes through tricarboxylic acid cycle and anaplerotic reactions increased fast in the two knockout strains, which meant more energy generated. The changed fluxes in central carbon metabolism provided a holistic view of the physiological status in B. licheniformis and possible targets for further strain engineering. Cellulase is very important in the field of agriculture and bioenergy because of its degrading effect on cellulosic biomass. This study presented the effect of central carbon metabolism on cellulase production in Bacillus licheniformis. The study also provided a holistic view of the physiological status in B. licheniformis. The shifted metabolism provided a quantitative evaluation of the biosynthesis of cellulase and a priority ranked target list for further strain engineering. © 2017 The Society for Applied Microbiology.

Characterization of the native form and the carboxy-terminally truncated halotolerant form of α-amylases from Bacillus subtilis strain FP-133.

PubMed

Takenaka, Shinji; Miyatake, Ayaka; Tanaka, Kosei; Kuntiya, Ampin; Techapun, Charin; Leksawasdi, Noppol; Seesuriyachan, Phisit; Chaiyaso, Thanongsak; Watanabe, Masanori; Yoshida, Ken-ichi

2015-06-01

Two amylases, amylase I and amylase II from Bacillus subtilis strain FP-133, were purified to homogeneity and characterized. Their stabilities toward temperature, pH, and organic solvents, and their substrate specificities toward polysaccharides and oligosaccharides were similar. Under moderately high salt conditions, both amylases were more stable than commercial B. licheniformis amylase, and amylase I retained higher amylase activity than amylase II. The N-terminal amino acid sequence, genomic southern blot analysis, and MALDI-TOFF-MS analysis indicated that the halotolerant amylase I was produced by limited carboxy-terminal truncation of the amylase II peptide. The deduced amino acid sequence of amylase II was >95% identical to that of previously reported B. subtilis α-amylases, but their carboxy-terminal truncation points differed. Three recombinant amylases--full-length amylase corresponding to amylase II, an artificially truncated amylase corresponding to amylase I, and an amylase with a larger artificial C-terminal truncation--were expressed in B. subtilis. The artificially truncated recombinant amylases had the same high amylase activity as amylase I under moderately high salt conditions. Sequence comparisons indicated that an increased ratio of Asp/Glu residues in the enzyme may be one factor responsible for increasing halotolerance. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Toxigenic Strains of Bacillus licheniformis Related to Food Poisoning

PubMed Central

Salkinoja-Salonen, M. S.; Vuorio, R.; Andersson, M. A.; Kämpfer, P.; Andersson, M. C.; Honkanen-Buzalski, T.; Scoging, A. C.

1999-01-01

Toxin-producing isolates of Bacillus licheniformis were obtained from foods involved in food poisoning incidents, from raw milk, and from industrially produced baby food. The toxin detection method, based on the inhibition of boar spermatozoan motility, has been shown previously to be a sensitive assay for the emetic toxin of Bacillus cereus, cereulide. Cell extracts of the toxigenic B. licheniformis isolates inhibited sperm motility, damaged cell membrane integrity, depleted cellular ATP, and swelled the acrosome, but no mitochondrial damage was observed. The responsible agent from the B. licheniformis isolates was partially purified. It showed physicochemical properties similar to those of cereulide, despite having very different biological activity. The toxic agent was nonproteinaceous; soluble in 50 and 100% methanol; and insensitive to heat, protease, and acid or alkali and of a molecular mass smaller than 10,000 g mol−1. The toxic B. licheniformis isolates inhibited growth of Corynebacterium renale DSM 20688T, but not all inhibitory isolates were sperm toxic. The food poisoning-related isolates were beta-hemolytic, grew anaerobically and at 55°C but not at 10°C, and were nondistinguishable from the type strain of B. licheniformis, DSM 13T, by a broad spectrum of biochemical tests. Ribotyping revealed more diversity; the toxin producers were divided among four ribotypes when cut with PvuII and among six when cut with EcoRI, but many of the ribotypes also contained nontoxigenic isolates. When ribotyped with PvuII, most toxin-producing isolates shared bands at 2.8 ± 0.2, 4.9 ± 0.3, and 11.7 ± 0.5 or 13.1 ± 0.8 kb. PMID:10508100

Molten Globule-Like Partially Folded State of Bacillus licheniformis α-Amylase at Low pH Induced by 1,1,1,3,3,3-Hexafluoroisopropanol

PubMed Central

Abd Halim, Adyani Azizah; Zaroog, Mohammed Suleiman; Abdul Kadir, Habsah; Tayyab, Saad

2014-01-01

Effect of 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) on acid-denatured Bacillus licheniformis α-amylase (BLA) at pH 2.0 was investigated by far-UV CD, intrinsic fluorescence, and ANS fluorescence measurements. Addition of increasing HFIP concentrations led to an increase in the mean residue ellipticity at 222 nm (MRE222 nm) up to 1.5 M HFIP concentration beyond which it sloped off. A small increase in the intrinsic fluorescence and a marked increase in the ANS fluorescence were also observed up to 0.4 M HFIP concentration, both of which decreased thereafter. Far- and near-UV CD spectra of the HFIP-induced state observed at 0.4 M HFIP showed significant retention of the secondary structures closer to native BLA but a disordered tertiary structure. Increase in the ANS fluorescence intensity was also observed with the HFIP-induced state, suggesting exposure of the hydrophobic clusters to the solvent. Furthermore, thermal denaturation of HFIP-induced state showed a non-cooperative transition. Taken together, all these results suggested that HFIP-induced state of BLA represented a molten globule-like state at pH 2.0. PMID:24977228

Bacillus licheniformis affects the microbial community and metabolic profile in the spontaneous fermentation of Daqu starter for Chinese liquor making.

PubMed

Wang, Peng; Wu, Qun; Jiang, Xuejian; Wang, Zhiqiang; Tang, Jingli; Xu, Yan

2017-06-05

Chinese liquor is produced from spontaneous fermentation starter (Daqu) that provides the microbes, enzymes and flavors for liquor fermentation. To improve the flavor character of Daqu, we inoculated Bacillus licheniformis and studied the effect of this strain on the community structure and metabolic profile in Daqu fermentation. The microbial relative abundance changed after the inoculation, including the increase in Bacillus, Clavispora and Aspergillus, and the decrease in Pichia, Saccharomycopsis and some other genera. This variation was also confirmed by pure culture and coculture experiments. Seventy-three metabolites were identified during Daqu fermentation process. After inoculation, the average content of aromatic compounds were significantly enriched from 0.37mg/kg to 0.90mg/kg, and the average content of pyrazines significantly increased from 0.35mg/kg to 5.71mg/kg. The increase in pyrazines was positively associated with the metabolism of the inoculated Bacillus and the native genus Clavispora, because they produced much more pyrazines in their cocultures. Whereas the increase in aromatic compounds might be related to the change of in situ metabolic activity of several native genera, in particular, Aspergillus produced more aromatic compounds in cocultures with B. licheniformis. It indicated that the inoculation of B. licheniformis altered the flavor character of Daqu by both its own metabolic activity and the variation of in situ metabolic activity. Moreover, B. licheniformis inoculation influenced the enzyme activity of Daqu, including the significant increase in amylase activity (from 1.3gstarch/g/h to 1.7gstarch/g/h), and the significant decrease in glucoamylase activity (from 627.6mgglucose/g/h to 445.6mgglucose/g/h) and esterase activity (from 28.1mgethylcaproate/g/100h to 17.2mgethylcaproate/g/100h). These effects of inoculation were important factors for regulating the metabolism of microbial communities, hence for improving the flavor profile

Inhibition of Bacillus licheniformis LMG 19409 from ropy cider by enterocin AS-48.

PubMed

Grande, M J; Lucas, R; Abriouel, H; Valdivia, E; Ben Omar, N; Maqueda, M; Martínez-Cañamero, M; Gálvez, A

2006-08-01

To determine the activity of enterocin AS-48 against ropy-forming Bacillus licheniformis from cider. Enterocin AS-48 was tested on B. licheniformis LMG 19409 from ropy cider in MRS-G broth, fresh-made apple juice and in two commercial apple ciders (A and B). Bacillus licheniformis was rapidly inactivated in MRS-G by 0.5 microg ml(-1)AS-48 and in fresh-made apple juice by 3 microg ml(-1). Concentration-dependent inactivation of this bacterium in two commercial apple ciders (A and B) stored at 4, 15 and 30 degrees C for 15 days was also demonstrated. Counts from heat-activated endospores in cider A plus AS-48 decreased very slowly. Application of combined treatments of heat (95 degrees C) and enterocin AS-48 reduced the time required to achieved complete inactivation of intact spores in cider A to 4 min for 6 microg ml(-1) and to 1 min for 12 microg ml(-1). D and z values also decreased as the bacteriocin concentration increased. Enterocin AS-48 can inhibit ropy-forming B. licheniformis in apple cider and increase the heat sensitivity of spores. Results from this study support the potential use of enterocin AS-48 to control B. licheniformis in apple cider.

Bacillus licheniformis in geogenic dust induces inflammation in respiratory epithelium.

PubMed

Pickering, Janessa; Teo, Teck Hui; Thornton, Ruth B; Kirkham, Lea-Ann; Zosky, Graeme R; Clifford, Holly D

2018-07-01

Exposure to environmental geogenic (or earth-derived) dust can lead to more frequent and severe infections in the human airway. Particulate matter < 10 µm (PM 10 ) is the component of air pollution that is commonly associated with the exacerbation of respiratory diseases. We have previously demonstrated that mice exposed to geogenic dust PM 10 experienced an exacerbation of inflammatory responses to influenza A virus. Whether geogenic dust PM 10 also exacerbates respiratory bacterial infection is not yet known, nor are the components of the dust that drive these responses. We treated airway bronchial epithelial cells (NuLi-1) with UV-irradiated geogenic dust PM 10 from six remote Western Australian towns. High levels of IL-6 and IL-8 production were observed, as well as persistent microbial growth. 16 S rRNA sequencing of the growth identified the microbe as Bacillus licheniformis, a spore-forming, environmentally abundant bacterium. We next investigated the interaction of B. licheniformis with respiratory epithelium in vitro to determine whether this exacerbated infection with a bacterial respiratory pathogen (non-typeable Haemophilus influenzae, NTHi). Heat treatment (100 °C) of all PM 10 samples eliminated B. licheniformis contamination and reduced epithelial inflammatory responses, suggesting that heat-labile and/or microbial factors were involved in the host response to geogenic dust PM 10 . We then exposed NuLi-1 epithelium to increasing doses of the isolated Bacillus licheniformis (multiplicity of infection of 10:1, 1:1 or 0.1:1 bacteria: cells) for 1, 3, and 24 h. B. licheniformis and NTHi infection (association and invasion) was assessed using a standard gentamicin survival assay, and epithelial release of IL-6 and IL-8 was measured using a bead based immunoassay. B. licheniformis was cytotoxic to NuLi-1 cells at 24 h. At 3 h post-challenge, B. licheniformis elicited high IL-6 and IL-8 inflammatory responses from NuLi-1 cells compared with

Biodegradation of food waste using microbial cultures producing thermostable α-amylase and cellulase under different pH and temperature.

PubMed

Awasthi, Mukesh Kumar; Wong, Jonathan W C; Kumar, Sunil; Awasthi, Sanjeev Kumar; Wang, Quan; Wang, Meijing; Ren, Xiuna; Zhao, Junchao; Chen, Hongyu; Zhang, Zengqiang

2018-01-01

The aim of this work was to study the biodegradation of food waste employing thermostable α-amylase and cellulase enzymes producing bacteria. Four potential isolates were identified which were capable of producing maximum amylase and cellulase and belong to the amylolytic strains, Brevibacillus borstelensis and Bacillus licheniformis; cellulolytic strains, Bacillus thuringiensis and Bacillus licheniformis, respectively. These strains were selected based on its higher cell density, enzymatic activities and stability at a wide range of pH and temperature compared to other strains. The results indicated that 1:1 ratio of pre and post consumed food wastes (FWs) were helpful to facilitate the degradation employing bacterial consortium. In addition, organic matter decomposition and chemical parameters of the end product quality also indicated that bacterial consortium was very effective for 1:1 ratio of FWs degradation as compared to the other treatments. Copyright © 2017 Elsevier Ltd. All rights reserved.

21 CFR 184.1012 - α-Amylase enzyme preparation from Bacillus stearothermophilus.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 3 2013-04-01 2013-04-01 false α-Amylase enzyme preparation from Bacillus... GENERALLY RECOGNIZED AS SAFE Listing of Specific Substances Affirmed as GRAS § 184.1012 α-Amylase enzyme preparation from Bacillus stearothermophilus. (a) α-Amylase enzyme preparation is obtained from the culture...

21 CFR 184.1012 - α-Amylase enzyme preparation from Bacillus stearothermophilus.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 3 2012-04-01 2012-04-01 false α-Amylase enzyme preparation from Bacillus... GENERALLY RECOGNIZED AS SAFE Listing of Specific Substances Affirmed as GRAS § 184.1012 α-Amylase enzyme preparation from Bacillus stearothermophilus. (a) α-Amylase enzyme preparation is obtained from the culture...

21 CFR 184.1012 - α-Amylase enzyme preparation from Bacillus stearothermophilus.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 3 2014-04-01 2014-04-01 false α-Amylase enzyme preparation from Bacillus... Specific Substances Affirmed as GRAS § 184.1012 α-Amylase enzyme preparation from Bacillus stearothermophilus. (a) α-Amylase enzyme preparation is obtained from the culture filtrate that results from a pure...

21 CFR 184.1012 - α-Amylase enzyme preparation from Bacillus stearothermophilus.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 3 2010-04-01 2009-04-01 true α-Amylase enzyme preparation from Bacillus... GENERALLY RECOGNIZED AS SAFE Listing of Specific Substances Affirmed as GRAS § 184.1012 α-Amylase enzyme preparation from Bacillus stearothermophilus. (a) α-Amylase enzyme preparation is obtained from the culture...

21 CFR 184.1012 - α-Amylase enzyme preparation from Bacillus stearothermophilus.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 3 2011-04-01 2011-04-01 false α-Amylase enzyme preparation from Bacillus... GENERALLY RECOGNIZED AS SAFE Listing of Specific Substances Affirmed as GRAS § 184.1012 α-Amylase enzyme preparation from Bacillus stearothermophilus. (a) α-Amylase enzyme preparation is obtained from the culture...

Fluorescent CdSe QDs containing Bacillus licheniformis bioprobes for Copper (II) detection in water.

PubMed

Yan, Zheng-Yu; Du, Qing-Qing; Wan, Dong-Yu; Lv, Hang; Cao, Zhi-Ran; Wu, Sheng-Mei

2017-12-01

Quantum dots (QDs) are semiconductor nanoparticles (NPs) that offer valuable functionality for cellular labeling, drug delivery, solar cells and quantum computation. In this study, we reported that CdSe QDs could be bio-synthesized in Bacillus licheniformis. After optimization, the obtained CdSe QDs exhibited a uniform particle size of 3.71±0.04nm with a maximum fluorescence emission wavelength at 550nm and the synthetical positive ratio can reach up to 87%. Spectral properties, constitution, particle sizes and crystalline phases of the CdSe QDs were systematically and integrally investigated. The CdSe QD-containing Bacillus licheniformis cells were further used as whole fluorescent bio-probes to detect copper (II) (Cu 2+ ) in water, which demonstrated a low limit of detection (0.91μM). The assay also showed a good selectivity for Cu 2+ over other ions including Al 3+ , Cd 2+ , Mg 2+ , K + , Na + , NH 4 + , Zn 2+ , CH 3 COO + , Pb 2+ and I - . Our study suggests the fluorescent CdSe QDs-containing Bacillus licheniformis bio-probes as a promising approach for detection of Cu 2+ in complex solution environment. Copyright © 2017. Published by Elsevier Inc.

Effects of alpha-amylase reaction mechanisms on analysis of resistant-starch contents.

PubMed

Moore, Samuel A; Ai, Yongfeng; Chang, Fengdan; Jane, Jay-lin

2015-01-22

This study aimed to understand differences in the resistant starch (RS) contents of native and modified starches obtained using two standard methods of RS content analysis: AOAC Method 991.43 and 2002.02. The largest differences were observed in native potato starch, cross-linked wheat distarch phosphate, and high-amylose corn starch stearic-acid complex (RS5) between using AOAC Method 991.43 with Bacillus licheniformis α-amylase (BL) and AOAC Method 2002.02 with porcine pancreatic α-amylase (PPA). To determine possible reasons for these differences, we hydrolyzed raw-starch granules with BL and PPA with equal activity at pH 6.9 and 37°C for up to 84 h and observed the starch granules displayed distinct morphological differences after the hydrolysis. Starches hydrolyzed by BL showed erosion on the surface of the granules; those hydrolyzed by PPA showed pitting on granule surfaces. These results suggested that enzyme reaction mechanisms, including the sizes of the binding sites and the reaction patterns of the two enzymes, contributed to the differences in the RS contents obtained using different methods of RS analysis. Copyright © 2014 Elsevier Ltd. All rights reserved.

Efficient expression of nattokinase in Bacillus licheniformis: host strain construction and signal peptide optimization.

PubMed

Wei, Xuetuan; Zhou, Yinhua; Chen, Jingbang; Cai, Dongbo; Wang, Dan; Qi, Gaofu; Chen, Shouwen

2015-02-01

Nattokinase (NK) possesses the potential for prevention and treatment of thrombus-related diseases. In this study, high-level expression of nattokinase was achieved in Bacillus licheniformis WX-02 via host strain construction and signal peptides optimization. First, ten genes (mpr, vpr, aprX, epr, bpr, wprA, aprE, bprA, hag, amyl) encoding for eight extracellular proteases, a flagellin and an amylase were deleted to obtain B. licheniformis BL10, which showed no extracellular proteases activity in gelatin zymography. Second, the gene fragments of P43 promoter, Svpr, nattokinase and TamyL were combined into pHY300PLK to form the expression vector pP43SNT. In BL10 (pP43SNT), the fermentation activity and product activity per unit of biomass of nattokinase reached 14.33 FU/mL and 2,187.71 FU/g respectively, which increased by 39 and 156 % compared to WX-02 (pP43SNT). Last, Svpr was replaced with SsacC and SbprA, and the maximum fermentation activity (33.83 FU/mL) was achieved using SsacC, which was 229 % higher than that of WX-02 (pP43SNT). The maximum NK fermentation activity in this study reaches the commercial production level of solid state fermentation, and this study provides a promising engineered strain for industrial production of nattokinase, as well as a potential platform host for expression of other target proteins.

Proteomics study of extracellular fibrinolytic proteases from Bacillus licheniformis RO3 and Bacillus pumilus 2.g isolated from Indonesian fermented food

NASA Astrophysics Data System (ADS)

Nur Afifah, Diana; Rustanti, Ninik; Anjani, Gemala; Syah, Dahrul; Yanti; Suhartono, Maggy T.

2017-02-01

This paper presents the proteomics study which includes separation, identification and characterization of proteins. The experiment on Indonesian fermented food such as extracellular fibrinolytic protease from Bacillus licheniformis RO3 and Bacillus pumilus 2.g isolated from red oncom and tempeh gembus was conducted. The experimental works comprise the following steps: (1) a combination of one- and two-dimensional electrophoresis analysis, (2) mass spectrometry analysis using MALDI-TOF-MS and (3) investigation using protein database. The result suggested that there were new two protein fractions of B. licheniformis RO3 and three protein fractions of B. pumilus 2.g. These result has not been previously reported.

[Maxillary sinus infection by Bacillus licheniformis: a case report from Djibouti].

PubMed

Garcia Hejl, C; Sanmartin, N; Samson, T; Soler, C; Koeck, J-L

2015-01-01

Aerobic, spore-forming gram-positive Bacillus spp infections are rare and reported mainly in immunocompromised hosts. We report a case of acute unilateral maxillary sinusitis, caused by Bacillus licheniformis, in a 35-year-old French soldier stationed in Djibouti. It was easily identifiable due to its typical culture and resistance profile. This case is interesting for two reasons: first, it is, to our knowledge, the first case of sinusitis attributed to this microbe, and second, it has rarely been described in immunocompetent patients without altered skin or mucous membranes.

Mode of alpha-amylase production by the shochu koji mold Aspergillus kawachii.

PubMed

Nagamine, Kazuki; Murashima, Kenji; Kato, Taku; Shimoi, Hitoshi; Ito, Kiyoshi

2003-10-01

Aspergillus kawachii produces two kinds of alpha-amylase, one is an acid-unstable alpha-amylase and the other is an acid-stable alpha-amylase. Because the quality of the shochu depends strongly on the activities of the alpha-amylases, the culture conditions under which these alpha-amylases are produced were examined. In liquid culture, acid-unstable alpha-amylase was produced abundantly, but, acid-stable alpha-amylase was not produced. The acid-unstable alpha-amylase was produced significantly when glycerol or glucose was used as a carbon source, similarly to the use of inducers such as starch or maltose. In liquid culture, A. kawachii assimilated starch at pH 3.0, but no alpha-amylase activity was recognized in the medium. Instead, the alpha-amylase was found to be trapped in the cell wall. The trapped form was identified as acid-unstable alpha-amylase. Usually, acid-unstable alpha-amylase is unstable at pH 3.0, so its stability appeared to be due to its immobilization in the cell wall. In solid-state culture, both kinds of alpha-amylase were produced. The production of acid-stable alpha-amylase seems to be solid-state culture-specific and was affected by the moisture content in the solid medium.

Isolation and Characterization of Thermophilic Bacteria from Jordanian Hot Springs: Bacillus licheniformis and Thermomonas hydrothermalis Isolates as Potential Producers of Thermostable Enzymes.

PubMed

Mohammad, Balsam T; Al Daghistani, Hala I; Jaouani, Atef; Abdel-Latif, Saleh; Kennes, Christian

2017-01-01

The aim of this study was the isolation and characterization of thermophilic bacteria from hot springs in Jordan. Ten isolates were characterized by morphological, microscopic, biochemical, molecular, and physiological characteristics. Sequencing of the 16S rDNA of the isolates followed by BLAST search revealed that nine strains could be identified as Bacillus licheniformis and one isolate as Thermomonas hydrothermalis . This is the first report on the isolation of Thermomonas species from Jordanian hot springs. The isolates showed an ability to produce some thermostable enzymes such as amylase, protease, cellulose, gelatins, and lecithin. Moreover, the UPGMA dendrogram of the enzymatic characteristics of the ten isolates was constructed; results indicated a high phenotypic diversity, which encourages future studies to explore further industrial and environmental applications.

Properties of Thermosensitive Extracellular α-Amylases of Bacillus subtilis

PubMed Central

Yamane, Kunio; Maruo, Bunji

1974-01-01

Enzymological properties of four thermosensitive α-amylases (M3, M9, M18, and M20) brought by different mutation sites in α-amylase structural gene of Bacillus subtilis were compared with those of the parental α-amylase NA64. Two thermosensitive α-amylases (M9 and M20) were altered not only in their thermosensitivity but also in their immunological properties, catalytic properties, molecular weights determined by the gel filtration on a Bio-Gel P-100 column, and others. The other two thermosensitive α-amylases (M3 and M18) were altered only in their thermosensitivity. Images PMID:4218234

alpha-Amylase gene of Streptomyces limosus: nucleotide sequence, expression motifs, and amino acid sequence homology to mammalian and invertebrate alpha-amylases.

PubMed Central

Long, C M; Virolle, M J; Chang, S Y; Chang, S; Bibb, M J

1987-01-01

The nucleotide sequence of the coding and regulatory regions of the alpha-amylase gene (aml) of Streptomyces limosus was determined. High-resolution S1 mapping was used to locate the 5' end of the transcript and demonstrated that the gene is transcribed from a unique promoter. The predicted amino acid sequence has considerable identity to mammalian and invertebrate alpha-amylases, but not to those of plant, fungal, or eubacterial origin. Consistent with this is the susceptibility of the enzyme to an inhibitor of mammalian alpha-amylases. The amino-terminal sequence of the extracellular enzyme was determined, revealing the presence of a typical signal peptide preceding the mature form of the alpha-amylase. Images PMID:3500166

Stress Responses of the Industrial Workhorse Bacillus licheniformis to Osmotic Challenges

PubMed Central

Schroeter, Rebecca; Hoffmann, Tamara; Voigt, Birgit; Meyer, Hanna; Bleisteiner, Monika; Muntel, Jan; Jürgen, Britta; Albrecht, Dirk; Becher, Dörte; Lalk, Michael; Evers, Stefan; Bongaerts, Johannes; Maurer, Karl-Heinz; Putzer, Harald; Hecker, Michael; Schweder, Thomas; Bremer, Erhard

2013-01-01

The Gram-positive endospore-forming bacterium Bacillus licheniformis can be found widely in nature and it is exploited in industrial processes for the manufacturing of antibiotics, specialty chemicals, and enzymes. Both in its varied natural habitats and in industrial settings, B. licheniformis cells will be exposed to increases in the external osmolarity, conditions that trigger water efflux, impair turgor, cause the cessation of growth, and negatively affect the productivity of cell factories in biotechnological processes. We have taken here both systems-wide and targeted physiological approaches to unravel the core of the osmostress responses of B. licheniformis. Cells were suddenly subjected to an osmotic upshift of considerable magnitude (with 1 M NaCl), and their transcriptional profile was then recorded in a time-resolved fashion on a genome-wide scale. A bioinformatics cluster analysis was used to group the osmotically up-regulated genes into categories that are functionally associated with the synthesis and import of osmostress-relieving compounds (compatible solutes), the SigB-controlled general stress response, and genes whose functional annotation suggests that salt stress triggers secondary oxidative stress responses in B. licheniformis. The data set focusing on the transcriptional profile of B. licheniformis was enriched by proteomics aimed at identifying those proteins that were accumulated by the cells through increased biosynthesis in response to osmotic stress. Furthermore, these global approaches were augmented by a set of experiments that addressed the synthesis of the compatible solutes proline and glycine betaine and assessed the growth-enhancing effects of various osmoprotectants. Combined, our data provide a blueprint of the cellular adjustment processes of B. licheniformis to both sudden and sustained osmotic stress. PMID:24348917

Liver alpha-amylase gene expression as an early obesity biomarker.

PubMed

Mojbafan, Marzieh; Afsartala, Zohreh; Amoli, Mahsa M; Mahmoudi, Mahdi; Yaghmaei, Parichehreh; Larijani, Bagher; Ebrahim-Habibi, Azadeh

2017-04-01

Obesity is a major health problem worldwide, for which preventive and therapeutic means are still needed. Alpha-amylase is a digestive enzyme whose inhibition has been targeted as a potential anti-obesity strategy. However, alpha-amylase gene expression has not been particularly attended to, and in contrast with pancreatic and salivary amylases, fewer studies have focused on liver alpha-amylase. The present study aimed at investigating the expression of alpha-amylase gene in obese and normal mice at RNA and protein level as well as acarbose effect on this gene expression in hepatocyte cell culture. Control and case groups were fed by normal mouse pellet and high-fat diet respectively, during 8 weeks. After this period, serum biochemical parameters including glucose, cholesterol, triglycerides, AST, ALT and alpha-amylase were assayed. Liver alpha-amylase gene was analyzed by real time PCR, and liver enzyme was assayed with Bernfeld and ELISA methods Hepatocyte cell culture derived from both group were also treated by acarbose and alpha-amylase activity and gene expression was analyzed by above mentioned methods. All biochemical factors showed an increase in obese mice, but the increase in ALT and AST were not statistically significant. Alpha-amylase levels were also increased in obese mice, both at RNA and protein level, while a decrease was seen in obese mice derived hepatocytes after acarbose treatment. Elevated liver alpha-amylase levels may be indicative of initial stages of obesity and the use of acarbose could be considered as a treatment of obesity which could be potentially effective at multiple levels. Copyright © 2016. Published by Elsevier Urban & Partner Sp. z o.o.

Development of an Efficient Genome Editing Tool in Bacillus licheniformis Using CRISPR-Cas9 Nickase.

PubMed

Li, Kaifeng; Cai, Dongbo; Wang, Zhangqian; He, Zhili; Chen, Shouwen

2018-03-15

Bacillus strains are important industrial bacteria that can produce various biochemical products. However, low transformation efficiencies and a lack of effective genome editing tools have hindered its widespread application. Recently, clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 techniques have been utilized in many organisms as genome editing tools because of their high efficiency and easy manipulation. In this study, an efficient genome editing method was developed for Bacillus licheniformis using a CRISPR-Cas9 nickase integrated into the genome of B. licheniformis DW2 with overexpression driven by the P43 promoter. The yvmC gene was deleted using the CRISPR-Cas9n technique with homology arms of 1.0 kb as a representative example, and an efficiency of 100% was achieved. In addition, two genes were simultaneously disrupted with an efficiency of 11.6%, and the large DNA fragment bacABC (42.7 kb) was deleted with an efficiency of 79.0%. Furthermore, the heterologous reporter gene aprN , which codes for nattokinase in Bacillus subtilis , was inserted into the chromosome of B. licheniformis with an efficiency of 76.5%. The activity of nattokinase in the DWc9nΔ7/pP43SNT-S sacC strain reached 59.7 fibrinolytic units (FU)/ml, which was 25.7% higher than that of DWc9n/pP43SNT-S sacC Finally, the engineered strain DWc9nΔ7 (Δ epr Δ wprA Δ mpr Δ aprE Δ vpr Δ bprA Δ bacABC ), with multiple disrupted genes, was constructed using the CRISPR-Cas9n technique. Taken together, we have developed an efficient genome editing tool based on CRISPR-Cas9n in B. licheniformis This tool could be applied to strain improvement for future research. IMPORTANCE As important industrial bacteria, Bacillus strains have attracted significant attention due to their production of biological products. However, genetic manipulation of these bacteria is difficult. The CRISPR-Cas9 system has been applied to genome editing in some bacteria, and CRISPR-Cas9n was proven to

Influence of media composition on the production of alkaline α-amylase from Bacillus subtilis CB-18.

PubMed

Ogbonnaya, Nwokoro; Odiase, Anthonia

2012-01-01

Starch, a homopolysaccharide is an important and an abundant food reserve and energy source. Starches are processed to yield different products which find many industrial applications. Alpha-amylases hydrolyze starch by cleaving α-1,4-glucosidic bonds and have been used in food, textile and pharmaceutical industries [Sun et al. 2010]. Enzymatic conversion of starch with amylase presents an economically superior alternative to the conventional method of starch gelatinization. Alkaline α-amylase has an important position in the global enzyme market as a constituent of detergent. In this paper, we screened soil bacteria and an isolate, alkalophilic Bacillus subtilis CB-18 was found to produce an alkaline α-amylase in different media. MATERIAL AND METHODS. Screening of the isolates for amylolytic activity was carried out by growing bacteria isolated from the soil in starch agar plates and subsequently staining the plates with iodine solution to reveal zones of hydrolysis of starch. The selected isolate, Bacillus subtlis CB-18 was grown in different media at alkaline pH to evaluate the influence of media composition on alkaline α-amylase production. Enzyme assay was carried out by growing the culture in a broth medium and obtaining cell - free culture supernatant after centrifugation at 2515 × g for 15 minutes Amylase activity was determined by incubating 0.5 ml of crude enzyme solution in 0.1M Tris/HCl buffer (pH 8.5) with 0.5 ml of 1% soluble starch solution. The reaction was terminated by the addition of DNS reagent and reducing sugar produced from the amylolytic reaction was determined. Bacillus subtilis CB-18 used for this work was selected because it produced 7 mm zone diameter on starch agar plate. This organism was cultured in different alkaline broth media containing 2% soluble starch as inducer carbohydrate for α-amylase production. Among the carbon sources used for enzyme production, sorbitol was the best to stimulate enzyme production with α-amylase

Effects of High Pressure on Bacillus licheniformis Spore Germination and Inactivation

PubMed Central

Borch-Pedersen, Kristina; Mellegård, Hilde; Reineke, Kai; Boysen, Preben; Sevenich, Robert; Lindbäck, Toril

2017-01-01

ABSTRACT Bacillus and Clostridium species form spores, which pose a challenge to the food industry due to their ubiquitous nature and extreme resistance. Pressurization at <300 MPa triggers spore germination by activating germination receptors (GRs), while pressurization at >300 MPa likely triggers germination by opening dipicolinic acid (DPA) channels present in the inner membrane of the spores. In this work, we expose spores of Bacillus licheniformis, a species associated with food spoilage and occasionally with food poisoning, to high pressure (HP) for holding times of up to 2 h. By using mutant spores lacking one or several GRs, we dissect the roles of the GerA, Ynd, and GerK GRs in moderately HP (mHP; 150 MPa)-induced spore germination. We show that Ynd alone is sufficient for efficient mHP-induced spore germination. GerK also triggers germination with mHP, although at a reduced germination rate compared to that of Ynd. GerA stimulates mHP-induced germination but only in the presence of either the intact GerK or Ynd GR. These results suggests that the effectiveness of the individual GRs in mHP-induced germination differs from their effectiveness in nutrient-induced germination, where GerA plays an essential role. In contrast to Bacillus subtilis spores, treatment with very HP (vHP) of 550 MPa at 37°C did not promote effective germination of B. licheniformis spores. However, treatment with vHP in combination with elevated temperatures (60°C) gave a synergistic effect on spore germination and inactivation. Together, these results provide novel insights into how HP affects B. licheniformis spore germination and inactivation and the role of individual GRs in this process. IMPORTANCE Bacterial spores are inherently resistant to food-processing regimes, such as high-temperature short-time pasteurization, and may therefore compromise food durability and safety. The induction of spore germination facilitates subsequent inactivation by gentler processing conditions

RNA-Seq of Bacillus licheniformis: active regulatory RNA features expressed within a productive fermentation.

PubMed

Wiegand, Sandra; Dietrich, Sascha; Hertel, Robert; Bongaerts, Johannes; Evers, Stefan; Volland, Sonja; Daniel, Rolf; Liesegang, Heiko

2013-10-01

The production of enzymes by an industrial strain requires a complex adaption of the bacterial metabolism to the conditions within the fermenter. Regulatory events within the process result in a dynamic change of the transcriptional activity of the genome. This complex network of genes is orchestrated by proteins as well as regulatory RNA elements. Here we present an RNA-Seq based study considering selected phases of an industry-oriented fermentation of Bacillus licheniformis. A detailed analysis of 20 strand-specific RNA-Seq datasets revealed a multitude of transcriptionally active genomic regions. 3314 RNA features encoded by such active loci have been identified and sorted into ten functional classes. The identified sequences include the expected RNA features like housekeeping sRNAs, metabolic riboswitches and RNA switches well known from studies on Bacillus subtilis as well as a multitude of completely new candidates for regulatory RNAs. An unexpectedly high number of 855 RNA features are encoded antisense to annotated protein and RNA genes, in addition to 461 independently transcribed small RNAs. These antisense transcripts contain molecules with a remarkable size range variation from 38 to 6348 base pairs in length. The genome of the type strain B. licheniformis DSM13 was completely reannotated using data obtained from RNA-Seq analyses and from public databases. The hereby generated data-sets represent a solid amount of knowledge on the dynamic transcriptional activities during the investigated fermentation stages. The identified regulatory elements enable research on the understanding and the optimization of crucial metabolic activities during a productive fermentation of Bacillus licheniformis strains.

Method for using a yeast alpha-amylase promoter

DOEpatents

Gao, Johnway; Skeen, Rodney S.; Hooker, Brian S.; Anderson, Daniel B.

2003-04-22

The present invention provides the promoter clone discovery of an alpha-amylase gene of a starch utilizing yeast strain Schwanniomyces castellii. The isolated alpha-amylase promoter is an inducible promoter, which can regulate strong gene expression in starch culture medium.

Models for the action of barley alpha-amylase isozymes on linear substrates.

PubMed

MacGregor, E A; MacGregor, A W; Macri, L J; Morgan, J E

1994-05-05

The formation of maltodextrins, G1 to G12, during the hydrolysis of amylose by alpha-amylases 1 and 2 from barley malt was followed by HPLC. Similar, but not identical, distributions of products were obtained with the two alpha-amylase components. Maltose, G6, and G7 were major products, but G7 was degraded as hydrolysis proceeded. alpha-Amylase 1 produced more G1 and G3 than did alpha-amylase 2 at all stages of hydrolysis. Products formed during the hydrolysis of G9, G10, G11, and G12 by the two alpha-amylases were also determined. A different spectrum of products was observed with each substrate and small differences were observed in the action pattern of the two alpha-amylases, e.g., G3 and G7 were the major products formed during the hydrolysis of G10 by alpha-amylase 1, whereas G2 and G8 were the major products formed by alpha-amylase 2 on the same substrate. These results were used to develop a model of the active site of barley malt alpha-amylases. This site contains ten contiguous subsites with the catalytic site situated between subsites 7 and 8. The model can be used to predict hydrolysis patterns of amylose and maltodextrins by cereal alpha-amylases.

Heterologous expression, biochemical characterization, and overproduction of alkaline α-amylase from Bacillus alcalophilus in Bacillus subtilis

PubMed Central

2011-01-01

Background Alkaline α-amylases have potential applications for hydrolyzing starch under high pH conditions in the starch and textile industries and as ingredients in detergents for automatic dishwashers and laundries. While the alkaline α-amylase gains increased industrial interest, the yield of alkaline α-amylases from wild-type microbes is low, and the combination of genetic engineering and process optimization is necessary to achieve the overproduction of alkaline α-amylase. Results The alkaline α-amylase gene from Bacillus alcalophilus JN21 (CCTCC NO. M 2011229) was cloned and expressed in Bacillus subtilis strain WB600 with vector pMA5. The recombinant alkaline α-amylase was stable at pH from 7.0 to 11.0 and temperature below 40°C. The optimum pH and temperature of alkaline α-amylase was 9.0 and 50°C, respectively. Using soluble starch as the substrate, the Km and Vmax of alkaline α-amylase were 9.64 g/L and 0.80 g/(L·min), respectively. The effects of medium compositions (starch, peptone, and soybean meal) and temperature on the recombinant production of alkaline α-amylase in B. subtilis were investigated. Under the optimal conditions (starch concentration 0.6% (w/v), peptone concentration 1.45% (w/v), soybean meal concentration 1.3% (w/v), and temperature 37°C), the highest yield of alkaline α-amylase reached 415 U/mL. The yield of alkaline α-amylase in a 3-L fermentor reached 441 U/mL, which was 79 times that of native alkaline α-amylase from B. alcalophilus JN21. Conclusions This is the first report concerning the heterologous expression of alkaline α-amylase in B. subtilis, and the obtained results make it feasible to achieve the industrial production of alkaline α-amylase with the recombinant B. subtilis. PMID:21978209

Screening alpha-glucosidase and alpha-amylase inhibitors from natural compounds by molecular docking in silico.

PubMed

Jhong, Chien-Hung; Riyaphan, Jirawat; Lin, Shih-Hung; Chia, Yi-Chen; Weng, Ching-Feng

2015-01-01

The alpha-glucosidase inhibitor is a common oral anti-diabetic drug used for controlling carbohydrates normally converted into simple sugars and absorbed by the intestines. However, some adverse clinical effects have been observed. The present study seeks an alternative drug that can regulate the hyperglycemia by down-regulating alpha-glucosidase and alpha-amylase activity by molecular docking approach to screen the hyperglycemia antagonist against alpha-glucosidase and alpha-amylase activities from the 47 natural compounds. The docking data showed that Curcumin, 16-hydroxy-cleroda-3,13-dine-16,15-olide (16-H), Docosanol, Tetracosanol, Antroquinonol, Berberine, Catechin, Quercetin, Actinodaphnine, and Rutin from 47 natural compounds had binding ability towards alpha-amylase and alpha-glucosidase as well. Curcumin had a better biding ability of alpha-amylase than the other natural compounds. Analyzed alpha-glucosidase activity reveals natural compound inhibitors (below 0.5 mM) are Curcumin, Actinodaphnine, 16-H, Quercetin, Berberine, and Catechin when compared to the commercial drug Acarbose (3 mM). A natural compound with alpha-amylase inhibitors (below 0.5 mM) includes Curcumin, Berberine, Docosanol, 16-H, Actinodaphnine/Tetracosanol, Catechin, and Quercetin when compared to Acarbose (1 mM). When taken together, the implication is that molecular docking is a fast and effective way to screen alpha-glucosidase and alpha-amylase inhibitors as lead compounds of natural sources isolated from medicinal plants. © 2015 International Union of Biochemistry and Molecular Biology.

Characterization of salivary alpha-amylase binding to Streptococcus sanguis

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

Scannapieco, F.A.; Bergey, E.J.; Reddy, M.S.

1989-09-01

The purpose of this study was to identify the major salivary components which interact with oral bacteria and to determine the mechanism(s) responsible for their binding to the bacterial surface. Strains of Streptococcus sanguis, Streptococcus mitis, Streptococcus mutans, and Actinomyces viscosus were incubated for 2 h in freshly collected human submandibular-sublingual saliva (HSMSL) or parotid saliva (HPS), and bound salivary components were eluted with 2% sodium dodecyl sulfate. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western transfer, alpha-amylase was the prominent salivary component eluted from S. sanguis. Studies with {sup 125}I-labeled HSMSL or {sup 125}I-labeled HPS also demonstrated a componentmore » with an electrophoretic mobility identical to that of alpha-amylase which bound to S. sanguis. Purified alpha-amylase from human parotid saliva was radiolabeled and found to bind to strains of S. sanguis genotypes 1 and 3 and S. mitis genotype 2, but not to strains of other species of oral bacteria. Binding of ({sup 125}I)alpha-amylase to streptococci was saturable, calcium independent, and inhibitable by excess unlabeled alpha-amylases from a variety of sources, but not by secretory immunoglobulin A and the proline-rich glycoprotein from HPS. Reduced and alkylated alpha-amylase lost enzymatic and bacterial binding activities. Binding was inhibited by incubation with maltotriose, maltooligosaccharides, limit dextrins, and starch.« less

Mixed-mode resins: taking shortcut in downstream processing of raw-starch digesting α-amylases

PubMed Central

Lončar, Nikola; Slavić, Marinela Šokarda; Vujčić, Zoran; Božić, Nataša

2015-01-01

Bacillus licheniformis 9945a α-amylase is known as a potent enzyme for raw starch hydrolysis. In this paper, a mixed mode Nuvia cPrime™ resin is examined with the aim to improve the downstream processing of raw starch digesting amylases and exploit the hydrophobic patches on their surface. This resin combines hydrophobic interactions with cation exchange groups and as such the presence of salt facilitates hydrophobic interactions while the ion-exchange groups enable proper selectivity. α-Amylase was produced using an optimized fed-batch approach in a defined media and significant overexpression of 1.2 g L−1 was achieved. This single step procedure enables simultaneous concentration, pigment removal as well as purification of amylase with yields of 96% directly from the fermentation broth. PMID:26492875

High-Level Heat Resistance of Spores of Bacillus amyloliquefaciens and Bacillus licheniformis Results from the Presence of a spoVA Operon in a Tn1546 Transposon

PubMed Central

Berendsen, Erwin M.; Koning, Rosella A.; Boekhorst, Jos; de Jong, Anne; Kuipers, Oscar P.; Wells-Bennik, Marjon H. J.

2016-01-01

Bacterial endospore formers can produce spores that are resistant to many food processing conditions, including heat. Some spores may survive heating processes aimed at production of commercially sterile foods. Recently, it was shown that a spoVA operon, designated spoVA2mob, present on a Tn1546 transposon in Bacillus subtilis, leads to profoundly increased wet heat resistance of B. subtilis spores. Such Tn1546 transposon elements including the spoVA2mob operon were also found in several strains of Bacillus amyloliquefaciens and Bacillus licheniformis, and these strains were shown to produce spores with significantly higher resistances to wet heat than their counterparts lacking this transposon. In this study, the locations and compositions of Tn1546 transposons encompassing the spoVA2mob operons in B. amyloliquefaciens and B. licheniformis were analyzed. Introduction of these spoVA2mob operons into B. subtilis 168 (producing spores that are not highly heat resistant) rendered mutant 168 strains that produced high-level heat resistant spores, demonstrating that these elements in B. amyloliquefaciens and B. licheniformis are responsible for high level heat resistance of spores. Assessment of growth of the nine strains of each species between 5.2°C and 57.7°C showed some differences between strains, especially at lower temperatures, but all strains were able to grow at 57.7°C. Strains of B. amyloliquefaciens and B. licheniformis that contain the Tn1546 elements (and produce high-level heat resistant spores) grew at temperatures similar to those of their Tn1546-negative counterparts that produce low-level heat resistant spores. The findings presented in this study allow for detection of B. amyloliquefaciens and B. licheniformis strains that produce highly heat resistant spores in the food chain. PMID:27994575

Use of Nonspecific, Glutamic Acid-Free, Media and High Glycerol or High Amylase as Inducing Parameters for Screening Bacillus Isolates Having High Yield of Polyglutamic Acid

PubMed Central

Baxi, Nandita N.

2014-01-01

Out of fifty-five Bacillus isolates obtained from ten different regional locations and sources, seven showed the ability to consistently produce specific extracellular polymeric substance (EPS) on rich as well as synthetic but nonspecific media which did not contain glutamic acid. The isolates were identified as either Bacillus licheniformis or Bacillus subtilis. The EPS from all isolates was resistant to alpha protease, proteinase K, and was thus of high molecular weight. Further it was detected after SDS-PAGE by methylene blue but not by coomassie blue R staining as in case of proteins with high proportion of acidic amino acids. Cell-free EPS, after acid hydrolysis, showed absence of carbohydrates and presence of only glutamic acid. Thus the native the EPS from all seven isolates was confirmed to be gamma polyglutamic acid (PGA) and not exopolysaccharide. The Bacillus isolate T which produced maximum polymer on all media tested had higher amylase: protease activity as compared to other strains. If inoculum was developed in rich medium as compared to synthetic medium, the PGA produced increased by twofold in the subsequent synthetic production medium. Similarly, use of inoculum consisting of young and vegetative cells also increased the PGA production by twofold though amount of inoculum did not affect yield of PGA. Though PGA was produced in even in the absence of glutamic acid supplementation in the production medium by all isolates, the yield of PGA increased by fourfold in the presence glutamic acid and the maximum yield was 30 g/l for isolate K. The supplementation of glutamine instead of glutamic acid into the medium caused an increase in the viscosity of the non-Newtonian solution of PGA. PMID:27379328

Use of Nonspecific, Glutamic Acid-Free, Media and High Glycerol or High Amylase as Inducing Parameters for Screening Bacillus Isolates Having High Yield of Polyglutamic Acid.

PubMed

Baxi, Nandita N

2014-01-01

Out of fifty-five Bacillus isolates obtained from ten different regional locations and sources, seven showed the ability to consistently produce specific extracellular polymeric substance (EPS) on rich as well as synthetic but nonspecific media which did not contain glutamic acid. The isolates were identified as either Bacillus licheniformis or Bacillus subtilis. The EPS from all isolates was resistant to alpha protease, proteinase K, and was thus of high molecular weight. Further it was detected after SDS-PAGE by methylene blue but not by coomassie blue R staining as in case of proteins with high proportion of acidic amino acids. Cell-free EPS, after acid hydrolysis, showed absence of carbohydrates and presence of only glutamic acid. Thus the native the EPS from all seven isolates was confirmed to be gamma polyglutamic acid (PGA) and not exopolysaccharide. The Bacillus isolate T which produced maximum polymer on all media tested had higher amylase: protease activity as compared to other strains. If inoculum was developed in rich medium as compared to synthetic medium, the PGA produced increased by twofold in the subsequent synthetic production medium. Similarly, use of inoculum consisting of young and vegetative cells also increased the PGA production by twofold though amount of inoculum did not affect yield of PGA. Though PGA was produced in even in the absence of glutamic acid supplementation in the production medium by all isolates, the yield of PGA increased by fourfold in the presence glutamic acid and the maximum yield was 30 g/l for isolate K. The supplementation of glutamine instead of glutamic acid into the medium caused an increase in the viscosity of the non-Newtonian solution of PGA.

Parallel beta/alpha-barrels of alpha-amylase, cyclodextrin glycosyltransferase and oligo-1,6-glucosidase versus the barrel of beta-amylase: evolutionary distance is a reflection of unrelated sequences.

PubMed

Janecek, S

1994-10-17

The structures of functionally related beta/alpha-barrel starch hydrolases, alpha-amylase, beta-amylase, cyclodextrin glycosyltransferase and oligo-1,6-glucosidase, are discussed, their mutual sequence similarities being emphasized. Since these enzymes (except for beta-amylase) along with the predicted set of more than ten beta/alpha-barrels from the alpha-amylase enzyme superfamily fulfil the criteria characteristic of the products of divergent evolution, their unrooted distance tree is presented.

High-level expression of nattokinase in Bacillus licheniformis by manipulating signal peptide and signal peptidase.

PubMed

Cai, D; Wei, X; Qiu, Y; Chen, Y; Chen, J; Wen, Z; Chen, S

2016-09-01

Nattokinase is an enzyme produced by Bacillus licheniformis and has potential to be used as a drug for treating cardiovascular disease due to its beneficial effects of preventing fibrin clots etc. However, the low activity and titre of this protein produced by B. licheniformis often hinders its application of commercial production. The aim of this work is to improve the nattokinase production by manipulating signal peptides and signal peptidases in B. licheniformis. The P43 promoter, amyL terminator and AprN target gene were used to form the nattokinase expression vector, pHY-SP-NK, which was transformed into B. licheniformis and nattokinase was expressed successfully. A library containing 81 predicted signal peptides was constructed for nattokinase expression in B. licheniformis, with the maximum activity being obtained under the signal peptide of AprE. Among four type I signal peptidases genes (sipS, sipT, sipV, sipW) in B. licheniformis, the deletion of sipV resulted in a highest decrease in nattokinase activity. Overexpression of sipV in B. licheniformis led to a nattokinase activity of 35·60 FU ml(-1) , a 4·68-fold improvement over activity produced by the initial strain. This work demonstrates the potential of B. licheniformis for industrial production of nattokinase through manipulation of signal peptides and signal peptidases expression. This study has screened the signal peptides of extracellular proteins of B. licheniformis for nattokinase production. Four kinds of Type I signal peptidases genes have been detected respectively in B. licheniformis to identify which one played the vital role for nattokinase production. This study provided a promising strain for industry production of nattokinase. © 2016 The Society for Applied Microbiology.

Correlation between salivary alpha-amylase and stress-related anxiety.

PubMed

Rashkova, Maya R; Ribagin, Lora S; Toneva, Nina G

2012-01-01

Salivary alpha-amylase is a useful biomarker that can be used in assessing human psychobiological and social behavioural processes. Studying it opens up possibilities for the creation of novel concepts concerning the interaction of biological and social processes and their impact on health and behaviour. The levels of salivary alpha-amylase and situation anxiety self-assessment using Spielberger test were measured twice in 30 individuals aged 21.37 +/- 0.96 yrs (18 females and 12 males): once during stressful situation (prior to examination) and, again a month later, in stress-free environment (during a training session). Salivary alpha-amylase was measured using kinetic reaction kit Salimetrics LLC--USA. The mean level of salivary alpha-amylase measured during the first measurement 156.0 +/- 93.33 U/ml. During the second measurement in the absence of intense stress, the levels were two times lower - 74.03 +/- 58.06 U/ml and the differences were statistically significant (P < 0.001). We found a statistically significant correlation between the levels of salivary alpha-amylase in both measurements (P < 0.01). The correlation coefficient was r = 0.472 (P < 0.01). The adapted version of the State-Trait Anxiety Inventory score (STAI) created by Spielberger is appropriate for assessment of stress-related anxiety in young individuals. Salivary alpha-amylase may be used as a biomarker for objective evaluation of the psychosomatic state of individuals in a stressful environment. The combination of psychological test and objective indicator such as salivary alpha-amylase is an excellent tool for objective evaluation of individual's state in stressful environment. Similar tests may be used in assessment of patients' behaviours at dental treatment that may be considered a stressor in most patients.

Nitrous oxide emission by the non-denitrifying, nitrate ammonifier Bacillus licheniformis.

PubMed

Sun, Yihua; De Vos, Paul; Heylen, Kim

2016-01-19

Firmicutes have the capacity to remove excess nitrate from the environment via either denitrification, dissimilatory nitrate reduction to ammonium or both. The recent renewed interest in their nitrogen metabolism has revealed many interesting features, the most striking being their wide variety of dissimilatory nitrate reduction pathways. In the present study, nitrous oxide production from Bacillus licheniformis, a ubiquitous Gram-positive, spore-forming species with many industrial applications, is investigated. B. licheniformis has long been considered a denitrifier but physiological experiments on three different strains demonstrated that nitrous oxide is not produced from nitrate in stoichiometric amounts, rather ammonium is the most important end-product, produced during fermentation. Significant strain dependency in end-product ratios, attributed to nitrite and ammonium, and medium dependency in nitrous oxide production were also observed. Genome analyses confirmed the lack of a nitrite reductase to nitric oxide, the key enzyme of denitrification. Based on the gene inventory and building on knowledge from other non-denitrifying nitrous oxide emitters, hypothetical pathways for nitrous oxide production, involving NarG, NirB, qNor and Hmp, are proposed. In addition, all publically available genomes of B. licheniformis demonstrated similar gene inventories, with specific duplications of the nar operon, narK and hmp genes as well as NarG phylogeny supporting the evolutionary separation of previously described distinct BALI1 and BALI2 lineages. Using physiological and genomic data we have demonstrated that the common soil bacterium B. licheniformis does not denitrify but is capable of fermentative dissimilatory nitrate/nitrite reduction to ammonium (DNRA) with concomitant production of N2O. Considering its ubiquitous nature and non-fastidious growth in the lab, B. licheniformis is a suitable candidate for further exploration of the actual mechanism of N2O

Genetic diversity of Bacillus sp producers of amylase isolated from the soil.

PubMed

Xavier, A R E O; Lima, E R; Oliveira, A M E; Cardoso, L; Santos, J; Cangussu, C H C; Leite, L N; Quirino, M C L; Júnior, I G C; Oliveira, D A; Xavier, M A S

2017-09-27

The microorganisms are the best source of extracellular enzymes since they allow an economical technology with low-resource consumption compared to animals and plants. The amylases are among the most important enzymes being the genus Bacillus one of the most investigated due to its ability to produce this enzyme. The objective of this study was to isolate and analyze the genetic diversity among bacteria of the genus Bacillus sp producer of amylase originated from the soil. To this end, soil samples were collected and submitted to the condition of extreme temperature. The serial dilution procedure followed by seeding on solid medium containing starch was used for isolation of strains that produce amylase. The microorganisms isolated were subjected to standard morphological methods for presumptive identification of the genus Bacillus. The PCR assay with the universal genetic marker 16S rDNA was used for confirmation of bacterial strain. All the 10 isolates presumptively identified as bacteria amplified a fragment of 370 bp corresponding to the 16S rDNA gene. The enzymatic activity was expressed as an enzymatic index (EI), after 24 h of incubation. All isolate producers of amylase exhibited EI ≥ 2.0. The determination of the genetic profile and the clonal relationship among the isolates were performed by the method of ERIC-PCR polymorphism. The isolates of Bacillus spp were divided into 2 groups (I and II). Through this method, the discriminatory capacity of this analysis of polymorphisms was verified in differing producer strains from those not producing amylase.

Effect of certain plant extracts on alpha-amylase activity.

PubMed

Prashanth, D; Padmaja, R; Samiulla, D S

2001-02-01

Ethanolic extracts of Punica granatum, Mangifera indica, Boerhaavia diffusa, Embelia ribes, Phyllanthus maderaspatensis, and Withania somnifera, were tested for their effect on alpha-amylase activity (in vitro). P. granatum and M. indica were found to exhibit interesting alpha-amylase inhibitory activity.

Purification and characterization of gamma poly glutamic acid from newly Bacillus licheniformis NRC20.

PubMed

Tork, Sanaa E; Aly, Magda M; Alakilli, Saleha Y; Al-Seeni, Madeha N

2015-03-01

γ-poly glutamic acid (γ-PGA) has received considerable attention for pharmaceutical and biomedical applications. γ-PGA from the newly isolate Bacillus licheniformis NRC20 was purified and characterized using diffusion distance agar plate, mass spectrometry and thin layer chromatography. All analysis indicated that γ-PGA is a homopolymer composed of glutamic acid. Its molecular weight was determined to be 1266 kDa. It was composed of L- and D-glutamic acid residues. An amplicon of 3050 represents the γ-PGA-coding genes was obtained, sequenced and submitted in genbank database. Its amino acid sequence showed high similarity with that obtained from B. licheniformis strains. The bacterium NRC 20 was independent of L-glutamic acid but the polymer production enhanced when cultivated in medium containing L-glutamic acid as the sole nitrogen source. Finally we can conclude that γ-PGA production from B. licheniformis NRC20 has many promised applications in medicine, industry and nanotechnology. Copyright © 2014 Elsevier B.V. All rights reserved.

Transglycosylation of naringin by Bacillus stearothermophilusMaltogenic amylase to give glycosylated naringin.

PubMed

Lee, S J; Kim, J C; Kim, M J; Kitaoka, M; Park, C S; Lee, S Y; Ra, M J; Moon, T W; Robyt, J F; Park, K H

1999-09-01

Naringin, a bitter compound in citrus fruits, was transglycosylated by Bacillus stearothermophilus maltogenic amylase reaction with maltotriose to give a series of mono-, di-, and triglycosylnaringins. Glycosylation products of naringin were observed by TLC and HPLC. The major glycosylation product was purified by using a Sephadex LH-20 column. The sturcture was determined by using MALDI-TOF MS, methylation analysis, and (1)H and (13)C NMR. The major transglycosylation product was maltosylnaringin, in which the maltose unit was attached by an alpha-1-->6 glycosidic linkage to the D-glucose moiety of naringin. This product was 250 times more soluble in water and 10 times less bitter than naringin.

Salivary Alpha-Amylase Correlates with Subjective Heat Pain Perception.

PubMed

Wittwer, Amrei; Krummenacher, Peter; La Marca, Roberto; Ehlert, Ulrike; Folkers, Gerd

2016-06-01

Self-reports of pain are important for an adequate therapy. This is a problem with patients and infants who are restricted in providing an accurate verbal estimation of their pain. Reliable, real-time, economical, and non-invasive physiological correlates might contribute to a more comprehensive description of pain. Salivary alpha-amylase constitutes one candidate biomarker, which reflects predominantly sympathetic nervous system alterations under stressful conditions and can be measured non-invasively. The current study investigated the effects of acute heat pain on salivary alpha-amylase activity. Heat pain tolerance was measured on the non-dominant forearm. Participants completed visual analog scales on pain intensity and unpleasantness. Saliva samples were collected directly after pain induction. Twenty-seven healthy volunteers were recruited for this study. While salivary alpha-amylase levels correlated positively with intensity and unpleasantness ratings in response to acute heat pain stimuli, there was no corresponding association with pain tolerance. Salivary alpha-amylase is suggested to be an indirect physiologic correlate of subjective heat pain perception. Future studies should address the role of salivary alpha-amylase depending on the origin of pain, the concerned tissue, and other pain assessment methods. © 2016 American Academy of Pain Medicine. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Zinc oxide nanoparticles as novel alpha-amylase inhibitors

NASA Astrophysics Data System (ADS)

Dhobale, Sandip; Thite, Trupti; Laware, S. L.; Rode, C. V.; Koppikar, Soumya J.; Ghanekar, Ruchika-Kaul; Kale, S. N.

2008-11-01

Amylase inhibitors, also known as starch blockers, contain substances that prevent dietary starches from being absorbed by the body via inhibiting breakdown of complex sugars to simpler ones. In this sense, these materials are projected as having potential applications in diabetes control. In this context, we report on zinc oxide nanoparticles as possible alpha-amylase inhibitors. Zinc oxide nanoparticles have been synthesized using soft-chemistry approach and 1-thioglycerol was used as a surfactant to yield polycrystalline nanoparticles of size ˜18 nm, stabilized in wurtzite structure. Conjugation study and structural characterization have been done using x-ray diffraction technique, Fourier transform infrared spectroscopy, UV-visible spectroscopy, and transmission electron microscopy. Cytotoxicity studies on human fibrosarcoma (HT-1080) and skin carcinoma (A-431) cell lines as well as mouse primary fibroblast cells demonstrate that up to a dose of 20 μg/ml, ZnO nanoparticles are nontoxic to the cells. We report for the first time the alpha-amylase inhibitory activity of ZnO nanoparticles wherein an optimum dose of 20 μg/ml was sufficient to exhibit 49% glucose inhibition at neutral pH and 35 °C temperature. This inhibitory activity was similar to that obtained with acarbose (a standard alpha-amylase inhibitor), thereby projecting ZnO nanoparticles as novel alpha-amylase inhibitors.

Alpha-amylase from the Hyperthermophilic Archaeon Thermococcus thioreducens

NASA Technical Reports Server (NTRS)

Bernhardsdotter, E. C. M. J.; Pusey, M. L.; Ng, M. L.; Garriott, O. K.

2003-01-01

Extremophiles are microorganisms that thrive in, from an anthropocentric view, extreme environments such as hot springs. The ability of survival at extreme conditions has rendered enzymes from extremophiles to be of interest in industrial applications. One approach to producing these extremozymes entails the expression of the enzyme-encoding gene in a mesophilic host such as E.coli. This method has been employed in the effort to produce an alpha-amylase from a hyperthermophile (an organism that displays optimal growth above 80 C) isolated from a hydrothermal vent at the Rainbow vent site in the Atlantic Ocean. alpha-amylases catalyze the hydrolysis of starch to produce smaller sugars and constitute a class of industrial enzymes having approximately 25% of the enzyme market. One application for thermostable alpha-amylases is the starch liquefaction process in which starch is converted into fructose and glucose syrups. The a-amylase encoding gene from the hyperthermophile Thermococcus thioreducens was cloned and sequenced, revealing high similarity with other archaeal hyperthermophilic a-amylases. The gene encoding the mature protein was expressed in E.coli. Initial characterization of this enzyme has revealed an optimal amylolytic activity between 85-90 C and around pH 5.3-6.0.

Posttranscriptional regulation of alpha-amylase II-4 expression by gibberellin in germinating rice seeds.

PubMed

Nanjo, Yohei; Asatsuma, Satoru; Itoh, Kimiko; Hori, Hidetaka; Mitsui, Toshiaki; Fujisawa, Yukiko

2004-06-01

Hormonal regulation of expression of alpha-amylase II-4 that lacks the gibberellin-response cis-element (GARE) in the promoter region of the gene was studied in germinating rice (Oryza sativa L.) seeds. Temporal and spatial expression of alpha-amylase II-4 in the aleurone layer were essentially identical to those of alpha-amylase I-1 whose gene contains GARE, although these were distinguishable in the embryo tissues at the early stage of germination. The gibberellin-responsible expression of alpha-amylase II-4 was also similar to that of alpha-amylase I-1. However, the level of alpha-amylase II-4 mRNA was not increased by gibberellin, indicating that the transcriptional enhancement of alpha-amylase II-4 expression did not occur in the aleurone. Gibberellin stimulated the accumulation of 45Ca2+ into the intracellular secretory membrane system. In addition, several inhibitors for Ca2+ signaling, such as EGTA, neomycin, ruthenium red (RuR), and W-7 prevented the gibberellin-induced expression of alpha-amylase II-4 effectively. While the gibberellin-induced expression of alpha-amylase II-4 occurred normally in the aleurone layer of a rice dwarf mutant d1 which is defective in the alpha subunit of the heterotrimeric G protein. Based on these results, it was concluded that the posttranscriptional regulation of alpha-amylase II-4 expression by gibberellin operates in the aleurone layer of germinating rice seed, which is mediated by Ca2+ but not the G protein.

Separation and determination of peptide metabolite of Bacillus licheniformis in a microbial fuel cell by high-speed capillary micellar electrokinetic chromatography.

PubMed

Wang, Wei; Bai, Ruiguang; Cai, Xiaoyu; Lin, Ping; Ma, Lihong

2017-11-01

A method using high-speed capillary micellar electrokinetic chromatography and a microbial fuel cell was applied to determine the metabolite of the peptides released by Bacillus licheniformis. Two peptides, l-carnosine and l-alanyl-l-glutamine were used as the substrate to feed Bacillus licheniformis in a microbial fuel cell. The metabolism process of the bacterium was monitored by analyzing the voltage outputs of the microbial fuel cell. A home-made spontaneous injection device was applied to perform high-speed capillary micellar electrokinetic chromatography. Under the optimized conditions, tryptophan, glycine, valine, tyrosine and the two peptides could be rapidly separated within 2.5 min with micellar electrokinetic chromatography mode. Then the method was applied to analyze the solutions sampled from the microbial fuel cell. After 92 h running, valine, as the metabolite, was successfully detected with concentration 3.90 × 10 -5 M. The results demonstrated that Bacillus licheniformis could convert l-carnosine and l-alanyl-l-glutamine into valine. The method employed in this work was proved to have great potential in analysis of metabolites, such as amino acids, for microorganisms. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Psychological stress-induced changes in salivary alpha-amylase and adrenergic activity.

PubMed

Kang, Younhee

2010-12-01

The aim of the study was to examine the relationships among salivary alpha-amylase, plasma catecholamines, blood pressure, and heart rate during psychological stress. This study used a pretest-post-test experimental design with a control group, using repeated measures. A total of 33 participants was divided into the experimental group (n = 16) that underwent a college academic final test as the psychological stress and the control group (n = 17) that did not undergo the test. The levels of salivary alpha-amylase and plasma catecholamines, blood pressure, and heart rate were measured seven times and stress and anxiety were measured once and twice, respectively, as subjective stress markers. Significant changes in the level of salivary alpha-amylase were found in response to psychological stress. However, the correlations of salivary alpha-amylase with the plasma catecholamines, blood pressure, and heart rate were only partially found to be statistically significant. In conclusion, it was shown that salivary alpha-amylase was sensitive to stress throughout this study. Thus, salivary alpha-amylase may be used to measure stress uninvasively in both clinical settings and nursing research where the effects of stress might be scrutinized. Furthermore, the mechanisms of illnesses that are induced by stress could be explored. © 2010 Blackwell Publishing Asia Pty Ltd.

Improvement of heterologous protein production in Aspergillus oryzae by RNA interference with alpha-amylase genes.

PubMed

Nemoto, Takashi; Maruyama, Jun-ichi; Kitamoto, Katsuhiko

2009-11-01

Aspergillus oryzae RIB40 has three alpha-amylase genes (amyA, amyB, and amyC), and secretes alpha-amylase abundantly. However, large amounts of endogenous secretory proteins such as alpha-amylase can compete with heterologous protein in the secretory pathway and decrease its production yields. In this study, we examined the effects of suppression of alpha-amylase on heterologous protein production in A. oryzae, using the bovine chymosin (CHY) as a reporter heterologous protein. The three alpha-amylase genes in A. oryzae have nearly identical DNA sequences from those promoters to the coding regions. Hence we performed silencing of alpha-amylase genes by RNA interference (RNAi) in the A. oryzae CHY producing strain. The silenced strains exhibited a reduction in alpha-amylase activity and an increase in CHY production in the culture medium. This result suggests that suppression of alpha-amylase is effective in heterologous protein production in A. oryzae.

Bottleneck in secretion of α-amylase in Bacillus subtilis.

PubMed

Yan, Shaomin; Wu, Guang

2017-07-19

Amylase plays an important role in biotechnology industries, and Gram-positive bacterium Bacillus subtilis is a major host to produce heterogeneous α-amylases. However, the secretion stress limits the high yield of α-amylase in B. subtilis although huge efforts have been made to address this secretion bottleneck. In this question-oriented review, every effort is made to answer the following questions, which look simple but are long-standing, through reviewing of literature: (1) Does α-amylase need a specific and dedicated chaperone? (2) What signal sequence does CsaA recognize? (3) Does CsaA require ATP for its operation? (4) Does an unfolded α-amylase is less soluble than a folded one? (5) Does α-amylase aggregate before transporting through Sec secretion system? (6) Is α-amylase sufficient stable to prevent itself from misfolding? (7) Does α-amylase need more disulfide bonds to be stabilized? (8) Which secretion system does PrsA pass through? (9) Is PrsA ATP-dependent? (10) Is PrsA reused after folding of α-amylase? (11) What is the fate of PrsA? (12) Is trigger factor (TF) ATP-dependent? The literature review suggests that not only the most of those questions are still open to answers but also it is necessary to calculate ATP budget in order to better understand how B. subtilis uses its energy for production and secretion.

Enhancement of L-valine production in Bacillus licheniformis by blocking three branched pathways.

PubMed

Liang, Chengwen; Huo, Yanli; Qi, Gaofu; Wei, Xuetuan; Wang, Qin; Chen, Shouwen

2015-06-01

Bacillus licheniformis WX-02 is used for the production of many valuable chemicals. Here, we have sought to improve L-valine production by blocking the metabolic pathways related to branched-chain amino acids. The synthesis genes of L-leucine (leuA) and L-isoleucine (ilvA) were deleted to obtain mutant strains. L-Valine yields of WX-02ΔleuA and WX-02ΔilvA reached 33.2 and 21.1 mmol/l, respectively, which are 22 and 14 times higher than the wild-type WX-02 (1.53 mmol/l). After further deletion of L-lactate dehydrogenase gene (ldh) from WX-02ΔleuA, the productivity reached 0.47 mmol/l h, an increase of 19 %. We provide a possibility to over-produce L-valine using genetically-modified B. licheniformis using remodeling of the biosynthetic pathway to L-valine.

Production and deactivation of biosurfactant by Bacillus licheniformis JF-2

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

Lin, Sungchyr; Sharma, M.M.; Georgiou, G.

Bacillus licheniformis JF-2 produces a lipopeptide surfactant with excellent interfacial properties (Lin et al., 1990, 1992). An HPLC assay was developed to monitor the concentration of the lipopeptide in the fermentation broth and was employed to determine the effect of the composition of the growth medium on biosurfactant production. A maximum concentration of 110 mg/L lipopeptide was obtained in optimized media with 1.0% (w/v) glucose as the carbon source. The maximum amount of surfactant was obtained in early stationary-phase cultures, but subsequently decreased rapidly and disappeared completely from the fermentation broth within 8 h. It was shown that the surfactantmore » is chemically stable in the culture supernatant but becomes internalized by stationary-phase cells. The apparent rate of surfactant internalization was not inhibited by carbonyl cyanide (m-chlorophenyl)hydrazone (CCCP), an uncoupler of oxidative phosphorylation, suggesting that it is not dependent on the availability of ATP and/or a charged membrane. A variety of physical and chemical treatments failed to release the surfactant from the cells. In minimal media the rate of surfactant internalization could be reduced by optimizing the concentration of phosphate and by increasing the amount of magnesium, whereas the nitrogen source, calcium, and trace salts had no effect. Since a related lipopeptide has been shown to be responsible for DNA transformation competence in certain Bacillus subtilis strains, it is possible that the internalization of the B. licheniformis JF-2 surfactant may be a developmentally important process related to the ability of the cells to take up extraneous DNA. 21 refs., 8 figs.« less

Polygalacturonase: production of pectin depolymerising enzyme from Bacillus licheniformis KIBGE IB-21.

PubMed

Rehman, Haneef Ur; Qader, Shah Ali Ul; Aman, Afsheen

2012-09-01

Polygalacturonase is an enzyme that hydrolyzes external and internal α (1-4) glycosidic bonds of pectin to decrease the viscosity of fruits juices and vegetable purees. Several bacterial strains were isolated from soil and rotten vegetables and screened for polygalacturonase production. The strain which produced maximum polygalacturonase was identified Bacillus licheniformis on the basis of taxonomic studies and 16S rDNA analysis. The isolated bacterial strain produced maximum polygalacturonase at 37 °C after 48 h of fermentation. Among various carbon sources apple pectin (1.0%) showed maximum enzyme production. Different agro industrial wastes were also used as substrate in batch fermentation and it was found that wheat bran is capable of producing high yield of enzyme. Maximum polygalacturonase production was obtained by using yeast extract (0.3%) as a nitrogen source. It was observed that B. licheniformis KIBGE IB-21 is capable of producing 1015 U/mg of polygalacturonase at neutral pH. Copyright © 2012 Elsevier Ltd. All rights reserved.

Molecular cloning and characterization of an alpha-amylase from Pichia burtonii 15-1.

PubMed

Kato, Saemi; Shimizu-Ibuka, Akiko; Mura, Kiyoshi; Takeuchi, Akiko; Tokue, Chiyoko; Arai, Soichi

2007-12-01

An alpha-amylase secreted by Pichia burtonii 15-1 isolated from a traditional starter murcha of Nepal, named Pichia burtonii alpha-amylase (PBA), was studied. The gene was cloned and its nucleotide sequence was determined. PBA was deduced to consist of 494 amino acid residues. It shared certain degrees of amino acid sequence identity with other homologous proteins: 60% with Schwanniomyces occidentalis alpha-amylase, 58% with Saccharomycopsis sp. alpha-amylase, and 47% with Taka-amylase A from Aspergillus oryzae. A three-dimensional structural model of PBA generated using the known three-dimensional structure of Taka-amylase A as a template suggested high structural similarity between them. Kinetic analysis revealed that the K(m) values of PBA were lower than those of Taka-amylase A for the oligosaccharides. Although the k(cat) values of PBA were lower than those of Taka-amylase A for the oligosaccharide substrates, the k(cat)/K(m) values of PBA were higher.

Long-term stability of diurnal salivary cortisol and alpha-amylase secretion patterns.

PubMed

Skoluda, Nadine; La Marca, Roberto; Gollwitzer, Mario; Müller, Andreas; Limm, Heribert; Marten-Mittag, Birgitt; Gündel, Harald; Angerer, Peter; Nater, Urs M

2017-06-01

This study aimed to investigate long-term stability and variability of diurnal cortisol and alpha-amylase patterns. Diurnal cortisol and alpha-amylase secretion patterns were assessed on a single workday with three waves of measurement across a total time period of 24months in 189 participants. Separate hierarchical linear models were analyzed, with and without a number of potential predictor variables (age, BMI, smoking, chronic stress, stress reactivity). While low long-term stability was found in diurnal cortisol, the stability of diurnal alpha-amylase was moderate across the time period of 24months. Several predictor variables had a positive impact on diurnal cortisol and alpha-amylase secretion patterns averaged across waves. Our findings underpin the notion that long-term stability is not necessarily warranted in longitudinal studies. It is important to choose an appropriate study design when attempting to disentangle clinically and biologically relevant changes from naturally occurring variations in diurnal cortisol and alpha-amylase. Copyright © 2017 Elsevier Inc. All rights reserved.

In vitro Ca(2+)-dependent maturation of milk-clotting recombinant Epr: minor extracellular protease: from Bacillus licheniformis.

PubMed

Ageitos, José Manuel; Vallejo, Juan Andrés; Serrat, Manuel; Sánchez-Pérez, Angeles; Villa, Tomás G

2013-06-01

The minor extracellular protease (Epr) is secreted into the culture medium during Bacillus licheniformis, strain USC13, stationary phase of growth. Whereas, B. subtilis Epr has been reported to be involved in swarming; the B. licheniformis protease is also involved in milk-clotting as shown by the curd forming ability of culture broths expressing this protein. The objectives of this study are the characterization of recombinant B. licheniformis Epr (minor extracellular protease) and the determination of its calcium-dependent activation process. In this work, we have cloned and expressed B. licheniformis Epr in Escherichia coli. We were also able to construct a tridimensional model for Epr based on its homology to Thermococcus kodakarensis pro-tk-subtilisin 2e1p, fervidolysin from Fervidobacterium pennivorans 1rv6, and B. lentus 1GCI subtilisin. Recombinant Epr was accumulated into inclusion bodies; after protein renaturation, Epr undergoes an in vitro calcium-dependent activation, similar to that described for tk protease. The recombinant Epr is capable of producing milk curds with the same clotting activity previously described for the native B. licheniformis Epr enzyme although further rheological and industrial studies should be carried out to confirm its real applicability. This work represents for the first time that Epr may be successfully expressed in a non-bacilli microorganism.

Development of a solid-state fermentation process for production of an alpha amylase with potentially interesting properties.

PubMed

Hashemi, Maryam; Razavi, Seyed Hadi; Shojaosadati, Seyed Abbas; Mousavi, Seyyed Mohammad; Khajeh, Khosro; Safari, Mohammad

2010-09-01

Ca-independency with potential activity and stability at low pH are among the most interesting characteristics of alpha-amylase in starch industry. In this attempt the synergetic effect of low pH on activity of crude Ca-independent alpha-amylase isolated from a native Bacillus sp. KR-8104 in solid-state fermentation (SSF) was studied using wheat bran (WB) as a substrate. The effects of different parameters including moisturizing agents, solid substrate to moisture ratio, particle size, incubation temperature and period, inoculum (v/w) and supplementation with 1% (w/w) different carbon and nitrogen sources on enzyme production were investigated. Maximum enzyme production of 140U/g dry fermented substrate was obtained from wheat bran moistened with tap water at a ratio of 1:1.5 and supplemented with 1% (w/w) NH(4)NO(3) and 1% (w/w) lactose after 48h incubation at 37 degrees C. Even though the production of alpha-amylase was lower at 40 and 45 degrees C, the viable cell count was higher. In addition response surface methodology (RSM) was applied to find optimum conditions of temperature and pH on crude amylase activity. Using central composite design (CCD) a quadratic mathematical model equation was derived for the prediction of enzyme activity. The results showed that the model was in good agreement with experimental results, with R(2)=0.90 (p<0.0001) and the low pH has a synergetic effect on enzyme activity at higher temperature. Copyright 2010 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Preparation and characterization of a dextran-amylase conjugate.

PubMed

Marshall, J J

1976-07-01

Bacillus amyloliquefaciens alpha-amylase was attached to dextran after activation of the polysaccharide by using a modification of the cyanogen bromide method. The soluble dextran-amylase conjugate was purified by molecular-sieve chromatography. The conjugated enzyme has greater stability than the unmodified enzyme at low pH values, during heat treatment, and on removal of calcium ions with a chelating agent. Attachment of dextran to alpha-amylase did not alter the Michaelis constant of the enzyme acting on starch. The polysaccharide-enzyme conjugate probably consists of a cross-linked aggregate of many dextran and many enzyme molecules, in which a proportion of the enzyme molecules, although not inactivated, are unable to express their activity, except after dextranase treatment.

The bean. alpha. -amylase inhibitor is encoded by a lectin gene

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

Moreno, J.; Altabella, T.; Chrispeels, M.J.

The common bean, Phaseolus vulgaris, contains an inhibitor of insect and mammalian {alpha}-amylases that does not inhibit plant {alpha}-amylase. This inhibitor functions as an anti-feedant or seed-defense protein. We purified this inhibitor by affinity chromatography and found that it consists of a series of glycoforms of two polypeptides (Mr 14,000-19,000). Partial amino acid sequencing was carried out, and the sequences obtained are identical with portions of the derived amino acid sequence of a lectin-like gene. This lectin gene encodes a polypeptide of MW 28,000, and the primary in vitro translation product identified by antibodies to the {alpha}-amylase inhibitor has themore » same size. Co- and posttranslational processing of this polypeptide results in glycosylated polypeptides of 14-19 kDa. Our interpretation of these results is that the bean lectins constitute a gene family that encodes diverse plant defense proteins, including phytohemagglutinin, arcelin and {alpha}-amylase inhibitor.« less

Cloning of a yeast alpha-amylase promoter and its regulated heterologous expression

DOEpatents

Gao, Johnway [Richland, WA; Skeen, Rodney S [Pendleton, OR; Hooker, Brian S [Kennewick, WA; Anderson, Daniel B [Pasco, WA

2003-04-01

The present invention provides the promoter clone discovery of an alpha-amylase gene of a starch utilizing yeast strain Schwanniomyces castellii. The isolated alpha-amylase promoter is an inducible promoter, which can regulate strong gene expression in starch culture medium.

Production of raw-starch-digesting α-amylase isoform from Bacillus sp. under solid-state fermentation and biochemical characterization.

PubMed

Božić, Nataša; Slavić, Marinela Šokarda; Gavrilović, Anja; Vujčić, Zoran

2014-07-01

α-Amylase production by solid-state fermentation of different Bacillus sp. was studied previously on different fermentation media. However, no study has been reported on the influence of selected media on expression of desired amylase isoforms such as raw-starch-digesting amylase (RSDA). In this paper, the influence of different inexpensive and available agro-resources as solid media (corn, wheat and triticale) on α-amylase isoform induction from three wild-type Bacillus sp., selected among one hundred strains tested, namely 9B, 12B and 24A was investigated. For all three strains, tested amylases were detected in the multiple forms; however, number and intensity of each form differed depending on the solid media used for growth. To determine which isoform from Bacillus sp. 12B was RSDA, the suspected isoform was purified. The optimum pH for the purified α-amylase isoform was 6.0-8.0, while the optimum temperature was 60-90 °C. Isoform was considerably thermostable and Ca(2+)-independent, and actually the only α-amylase active towards raw starch. Purification and characterization of RSDA showed that not all of the solid media tested induced RSDA. From an economic point of view, it might be significant to obtain pure isoenzyme for potential use in the raw-starch hydrolysis, since it was 5 times more efficient in raw corn starch hydrolysis than the crude amylase preparation.

Chitosanase purified from bacterial isolate Bacillus licheniformis of ruined vegetables displays broad spectrum biofilm inhibition.

PubMed

Muslim, Sahira Nsayef; Al-Kadmy, Israa M S; Hussein, Nadheema Hammood; Mohammed Ali, Alaa Naseer; Taha, Buthainah Mohammed; Aziz, Sarah Naji; Kheraif, Abdulaziz Abdullah Al; Divakar, Darshan Devang; Ramakrishnaiah, Ravikumar

2016-11-01

A number of bacterial species produces chitosanases which has variety of applications because of its high biodegradability, non-toxicity and antimicrobial assets. In the present study chitosanase is purified from new bacterial species Bacillus licheniformis from spoiled vegetable. This novel strain of Bacillus licheniformis isolated from spoilt cucumber and pepper samples has the ability to produce the chitosanase enzyme when grown on chitosan substrate. Study also examined its antibiofilm properties against diverse bacterial species with biofilm forming ability. The purified chitosanase inhibited the biofilm formation ability for all Gram-negative and Gram-positive biofilm-forming bacteria [biofilm producers] tested in this study in congo red agar and microtiter plate's methods. Highly antibiofilm activity of chitosanase was recorded against Pseudomonas aeruginosa followed by Klebsiella pneumoniae with reduction of biofilm formation upto 22 and 29%, respectively compared with [100] % of control. Biofilm formation has multiple role including ability to enhance resistance and self-protection from external stress. This chitosanase has promising benefit as antibiofilm agent against biofilm forming pathogenic bacteria and has promising application as alternative antibiofilm agents to combat the growing number of multidrug resistant pathogen-associated infections, especially in situation where biofilms are involved. Copyright © 2016 Elsevier Ltd. All rights reserved.

Structural characterization of Bacillus licheniformis Dahb1 exopolysaccharide-antimicrobial potential and larvicidal activity on malaria and Zika virus mosquito vectors.

PubMed

Abinaya, Muthukumar; Vaseeharan, Baskaralingam; Divya, Mani; Vijayakumar, Sekar; Govindarajan, Marimuthu; Alharbi, Naiyf S; Khaled, Jamal M; Al-Anbr, Mohammed N; Benelli, Giovanni

2018-04-27

Microbial polysaccharides produced by marine species play a key role in food and cosmetic industry, as they are nontoxic and biodegradable polymers. This investigation reports the isolation of exopolysaccharide from Bacillus licheniformis Dahb1 and its biomedical applications. Bacillus licheniformis Dahb1 exopolysaccharide (Bl-EPS) was extracted using the ethanol precipitation method and structurally characterized. FTIR and 1 H-NMR pointed out the presence of various functional groups and primary aromatic compounds, respectively. Bl-EPS exhibited strong antioxidant potential confirmed via DPPH radical, reducing power and superoxide anion scavenging assays. Microscopic analysis revealed that the antibiofilm activity of Bl-EPS (75 μg/ml) was higher against Gram-negative (Pseudomonas aeruginosa and Proteus vulgaris) bacteria over Gram-positive species (Bacillus subtilis and Bacillus pumilus). Bl-EPS led to biofilm inhibition against Candida albicans when tested at 75 μg/ml. The hemolytic assay showed low cytotoxicity of Bl-EPS at 5 mg/ml. Besides, Bl-EPS achieved LC 50 values < 80 μg/ml against larvae of mosquito vectors Anopheles stephensi and Aedes aegypti. Overall, our findings pointed out the multipurpose bioactivity of Bl-EPS, which deserves further consideration for pharmaceutical, environmental and entomological applications.

The difference in in vivo sensitivity between Bacillus licheniformis PerR and Bacillus subtilis PerR is due to the different cellular environments.

PubMed

Kim, Jung-Hoon; Won, Young-Bin; Ji, Chang-Jun; Yang, Yoon-Mo; Ryu, Su-Hyun; Ju, Shin-Yeong; Kwon, Yumi; Lee, Yeh-Eun; Lee, Jin-Won

2017-02-26

PerR, a member of Fur family of metal-dependent regulators, is a major peroxide sensor in many Gram positive bacteria, and controls the expression of genes involved in peroxide resistance. Bacillus licheniformis, a close relative to the well-studied model organism Bacillus subtilis, contains three PerR-like proteins (PerR BL , PerR2 and PerR3) in addition to Fur and Zur. In the present study, we characterized the role of PerR BL in B. licheniformis. In vitro and in vivo studies indicate that PerR BL , like PerR BS , uses either Fe 2+ or Mn 2+ as a corepressor and only the Fe 2+ -bound form of PerR BL senses low levels of H 2 O 2 by iron-mediated histidine oxidation. Interestingly, regardless of the difference in H 2 O 2 sensitivity, if any, between PerR BL and PerR BS , B. licheniformis expressing PerR BL or PerR BS could sense lower levels of H 2 O 2 and was more sensitive to H 2 O 2 than B. subtilis expressing PerR BL or PerR BS . This result suggests that the differences in cellular milieu between B. subtilis and B. licheniformis, rather than the intrinsic differences in PerR BS and PerR BL per se, affect the H 2 O 2 sensing ability of PerR inside the cell and the H 2 O 2 resistance of cell. In contrast, B. licheniformis and B. subtilis expressing Staphylococcus aureus PerR (PerR SA ), which is more sensitive to H 2 O 2 than PerR BL and PerR BS , were more resistant to H 2 O 2 than those expressing either PerR BL or PerR BS . This result indicates that the sufficient difference in H 2 O 2 susceptibility of PerR proteins can override the difference in cellular environment and affect the resistance of cell to H 2 O 2 . Copyright © 2017 Elsevier Inc. All rights reserved.

Patterns of cortisol and alpha-amylase reactivity to psychosocial stress in maltreated women

PubMed Central

Mielock, Alyssa S.; Morris, Matthew C.; Rao, Uma

2016-01-01

Background Childhood maltreatment can trigger enduring changes in major stress response systems, particularly in the context of major depressive disorder (MDD). However, the relative impact of maltreatment versus MDD on hypothalamic-pituitary-adrenal axis and sympathetic-adrenal-medullary system stress reactivity is not well understood. Method This study examined salivary cortisol and alpha-amylase responses to the Trier Social Stress Test (TSST) in 26 maltreated (15 with current MDD) and 26 non-maltreated (17 with current MDD) women. Results Maltreated women showed greater anticipatory cortisol reactivity during the TSST protocol compared to non-maltreated women. Maltreated women also showed rapid deceleration in cortisol levels. Whereas non-maltreated women showed initial declines in alpha-amylase levels but rapidly increasing alpha-amylase levels during the TSST protocol, maltreated women did not exhibit changes in alpha-amylase levels during the TSST protocol. Contrary to expectation, MDD did not impact cortisol or alpha-amylase responses. Limitations The present study is limited by retrospective report of childhood maltreatment, cross-sectional design, and modest sample sizes. Conclusions These findings suggest that childhood maltreatment plays a greater role driving alterations in cortisol and alpha-amylase stress reactivity than MDD. Understanding the biological embedding of maltreatment is critical for elucidating mechanisms linking these experiences to risk for negative mental and physical health outcomes. PMID:27875756

Patterns of cortisol and alpha-amylase reactivity to psychosocial stress in maltreated women.

PubMed

Mielock, Alyssa S; Morris, Matthew C; Rao, Uma

2017-02-01

Childhood maltreatment can trigger enduring changes in major stress response systems, particularly in the context of major depressive disorder (MDD). However, the relative impact of maltreatment versus MDD on hypothalamic-pituitary-adrenal axis and sympathetic-adrenal-medullary system stress reactivity is not well understood. This study examined salivary cortisol and alpha-amylase responses to the Trier Social Stress Test (TSST) in 26 maltreated (15 with current MDD) and 26 non-maltreated (17 with current MDD) women. Maltreated women showed greater anticipatory cortisol reactivity during the TSST protocol compared to non-maltreated women. Maltreated women also showed rapid deceleration in cortisol levels. Whereas non-maltreated women showed initial declines in alpha-amylase levels but rapidly increasing alpha-amylase levels during the TSST protocol, maltreated women did not exhibit changes in alpha-amylase levels during the TSST protocol. Contrary to expectation, MDD did not impact cortisol or alpha-amylase responses. The present study is limited by retrospective report of childhood maltreatment, cross-sectional design, and modest sample sizes. These findings suggest that childhood maltreatment plays a greater role driving alterations in cortisol and alpha-amylase stress reactivity than MDD. Understanding the biological embedding of maltreatment is critical for elucidating mechanisms linking these experiences to risk for negative mental and physical health outcomes. Copyright © 2016 Elsevier B.V. All rights reserved.

Biochemical Characterization of An Arginine-Specific Alkaline Trypsin from Bacillus licheniformis.

PubMed

Gong, Jin-Song; Li, Wei; Zhang, Dan-Dan; Xie, Min-Feng; Yang, Biao; Zhang, Rong-Xian; Li, Heng; Lu, Zhen-Ming; Xu, Zheng-Hong; Shi, Jin-Song

2015-12-17

In the present study, we isolated a trypsin-producing strain DMN6 from the leather waste and identified it as Bacillus licheniformis through a two-step screening strategy. The trypsin activity was increased up to 140 from 20 U/mL through culture optimization. The enzyme was purified to electrophoretic homogeneity with a molecular mass of 44 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the specific activity of purified enzyme is 350 U/mg with Nα-Benzoyl-L-arginine ethylester as the substrate. The optimum temperature and pH for the trypsin are 65 °C and pH 9.0, respectively. Also, the enzyme can be significantly activated by Ba(2+). This enzyme is relatively stable in alkaline environment and displays excellent activity at low temperatures. It could retain over 95% of enzyme activity after 180 min of incubation at 45 °C. The distinguished activity under low temperature and prominent stability enhance its catalytic potential. In the current work, the open reading frame was obtained with a length of 1371 nucleotides that encoded a protein of 456 amino acids. These data would warrant the B. licheniformis trypsin as a promising candidate for catalytic application in collagen preparation and leather bating through further protein engineering.

Biochemical characterization of the alpha-amylase inhibitor in mungbeans and its application in inhibiting the growth of Callosobruchus maculatus.

PubMed

Wisessing, Anussorn; Engkagul, Arunee; Wongpiyasatid, Arunee; Choowongkomon, Kiattawee

2010-02-24

The insect Callosobruchus maculatus causes considerable damage to harvested mungbean seeds every year, which leads to commercial losses. However, recent studies have revealed that mungbean seeds contain alpha-amylase inhibitors that can inhibit the protein C. maculatus, preventing growth and development of the insect larvae in the seed, thus preventing further damage. For this reason, the use of alpha-amylase inhibitors to interfere with the pest's digestion process has become an interesting alternative biocontrolling agent. In this study, we have isolated and purified the alpha-amylase inhibitor from mungbean seeds (KPS1) using ammonium sulfate precipitation, gel filtration chromatography and reversed phase HPLC. We found that the alpha-amylase inhibitor, isolated as a monomer, had a molecular weight of 27 kDa. The alpha-amylase inhibitor was purified 750-fold with a final yield of 0.4 mg of protein per 30 g of mungbean seeds. Its specific activity was determined at 14.5 U (mg of protein)(-1). Interestingly, we found that the isolated alpha-amylase inhibitor inhibits C. maculatus alpha-amylase but not human salivary alpha-amylase. After preincubation of the enzyme with the inhibitor, the mungbean alpha-amylase inhibitor inhibited C. maculatus alpha-amylase activity by decreasing V(max) while increasing the K(m) constant, indicating that the mungbean alpha-amylase is a mix noncompetitive inhibitor. The in vivo effect of alpha-amylase inhibitor on the mortality of C. maculatus shows that the alpha-amylase inhibitor acts on C. maculatus during the development stage, by reducing carbohydrate digestion necessary for growth and development, rather than during the end laying/hatching stage. Our results suggest that mungbean alpha-amylase inhibitor could be a useful future biocontrolling agent.

Effect of amino acids on tannase biosynthesis by Bacillus licheniformis KBR6.

PubMed

Mohapatra, Pradeep K Das; Pati, Bikas R; Mondal, Keshab C

2009-04-01

Microbial tannase (tannin acyl hydrolase, EC 3.1.1.20), a hydrolysable tannin-degrading enzyme, has gained importance in various industrial processes, and is used extensively in the manufacture of instant tea, beer, wine, and gallic acid. Tannase is an inducible enzyme, and hydrolysable tannin, especially tannic acid, is the sole inducer. This study is of the effect of various amino acids and their analogues on tannase biosynthesis by Bacillus licheniformis KBR6 to ascertain the mode of action of these growth factors on tannase biosynthesis from microbial origin. Enzyme production was carried out in enriched tannic acid medium through submerged fermentation for 20 h at 35 degrees C. Different amino acids at a concentration of 0.05 g% (w/v) were added to the culture medium immediately after sterilization. Culture supernatant was used as the source of the enzyme and the quantity of tannase was estimated by the colorimetric assay method. Growth of the organism was estimated according to biomass dry weight. Maximum tannase (2.87-fold that of the control) was synthesized by B. licheniformis KBR6 when alanine was added to the culture medium. Other amino acids, such as DL-serine, L-cystine, glycine, L-ornithine, aspartic acid, L-glutamic acid, DL-valine, L-leucine and L-lysine, also induced tannase synthesis. L-Cysteine monohydrochloride and DL-threonine were the most potent inhibitors. Regulation of tannase biosynthesis by B. licheniformis in the presence of various amino acids is shown. This information will be helpful for formulating an enriched culture medium for industrial-scale tannase production.

Draft Genome Sequence of Bacillus licheniformis Strain YNP1-TSU Isolated from Whiterock Springs in Yellowstone National Park

PubMed Central

O'Hair, Joshua A.; Li, Hui; Thapa, Santosh; Scholz, Matthew B.

2017-01-01

ABSTRACT Novel cellulolytic microorganisms can potentially influence second-generation biofuel production. This paper reports the draft genome sequence of Bacillus licheniformis strain YNP1-TSU, isolated from hydrothermal-vegetative microbiomes inside Yellowstone National Park. The assembled sequence contigs predicted 4,230 coding genes, 66 tRNAs, and 10 rRNAs through automated annotation. PMID:28254968

Alpha-amylase inhibitor-1 gene from Phaseolus vulgaris expressed in Coffea arabica plants inhibits alpha-amylases from the coffee berry borer pest.

PubMed

Barbosa, Aulus E A D; Albuquerque, Erika V S; Silva, Maria C M; Souza, Djair S L; Oliveira-Neto, Osmundo B; Valencia, Arnubio; Rocha, Thales L; Grossi-de-Sa, Maria F

2010-06-17

Coffee is an important crop and is crucial to the economy of many developing countries, generating around US$70 billion per year. There are 115 species in the Coffea genus, but only two, C. arabica and C. canephora, are commercially cultivated. Coffee plants are attacked by many pathogens and insect-pests, which affect not only the production of coffee but also its grain quality, reducing the commercial value of the product. The main insect-pest, the coffee berry borer (Hypotheneumus hampei), is responsible for worldwide annual losses of around US$500 million. The coffee berry borer exclusively damages the coffee berries, and it is mainly controlled by organochlorine insecticides that are both toxic and carcinogenic. Unfortunately, natural resistance in the genus Coffea to H. hampei has not been documented. To overcome these problems, biotechnological strategies can be used to introduce an alpha-amylase inhibitor gene (alpha-AI1), which confers resistance against the coffee berry borer insect-pest, into C. arabica plants. We transformed C. arabica with the alpha-amylase inhibitor-1 gene (alpha-AI1) from the common bean, Phaseolus vulgaris, under control of the seed-specific phytohemagglutinin promoter (PHA-L). The presence of the alpha-AI1 gene in six regenerated transgenic T1 coffee plants was identified by PCR and Southern blotting. Immunoblotting and ELISA experiments using antibodies against alpha-AI1 inhibitor showed a maximum alpha-AI1 concentration of 0.29% in crude seed extracts. Inhibitory in vitro assays of the alpha-AI1 protein against H. hampei alpha-amylases in transgenic seed extracts showed up to 88% inhibition of enzyme activity. This is the first report showing the production of transgenic coffee plants with the biotechnological potential to control the coffee berry borer, the most important insect-pest of crop coffee.

Application of Oxygen-Enriched Aeration in the Production of Bacitracin by Bacillus licheniformis

PubMed Central

Flickinger, M. C.; Perlman, D.

1979-01-01

The physiological effects of controlling the dissolved oxygen tension at 0.01, 0.02, and 0.05 atm by the use of oxygen-enriched aeration were investigated during growth and bacitracin production by Bacillus licheniformis ATCC 10716. Up to a 2.35-fold increase in the final antibiotic yield and a 4-fold increase in the rate of bacitracin synthesis were observed in response to O2-enriched aeration. The increase in antibiotic production was accompanied by increased respiratory activity and an increase in the specific productivity of the culture from 1.3 to 3.6 g of antibiotic per g of cell mass produced. Oxygen enrichment of the aeration decreased medium carbohydrate uptake and the maximum specific growth rate of B. licheniformis from 0.6 h−1 to as low as 0.15 h−1, depending upon the level of enrichment and the conditions of oxygen transfer rate (impeller speed). The response of this culture to O2 enrichment suggests that this method of controlling the dissolved oxygen tension for antibiotic-producing cultures may simulate conditions that would occur if the carbon source were fed slowly, as is often employed to optimize antibiotic production. Analysis of the biologically active bacitracins produced by B. licheniformis ATCC 10716 suggested that the ratio of biologically active peptides was not changed by O2 enrichment, nor were any new biologically active compounds formed. Images PMID:34361

Draft Genome Sequence of Bacillus licheniformis Strain GB2, a Hydrocarbon-Degrading and Plant Growth-Promoting Soil Bacterium.

PubMed

Gkorezis, Panagiotis; Van Hamme, Jonathan; Bottos, Eric; Thijs, Sofie; Balseiro-Romero, Maria; Monterroso, Carmela; Kidd, Petra Suzan; Rineau, Francois; Weyens, Nele; Sillen, Wouter; Vangronsveld, Jaco

2016-06-23

We report the 4.39 Mb draft genome of Bacillus licheniformis GB2, a hydrocarbonoclastic Gram-positive bacterium of the family Bacillaceae, isolated from diesel-contaminated soil at the Ford Motor Company site in Genk, Belgium. Strain GB2 is an effective plant-growth promoter useful for diesel fuel remediation applications based on plant-bacterium associations. Copyright © 2016 Gkorezis et al.

Tobacco plants transformed with the bean. alpha. ai gene express an inhibitor of insect. alpha. -amylase in their seeds. [Nicotiana tabacum; Tenebrio molitor

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

Altabella, T.; Chrispeels, M.J.

Bean (Phaseolus vulgaris L.) seeds contain a putative plant defense protein that inhibits insect and mammalian but not plant {alpha}-amylases. We recently presented strong circumstantial evidence that this {alpha}-amylase inhibitor ({alpha}Al) is encoded by an already-identified lectin gene whose product is referred to as lectin-like-protein (LLP). We have now made a chimeric gene consisting of the coding sequence of the lectin gene that encodes LLP and the 5{prime} and 3{prime} flanking sequences of the lectin gene that encodes phytohemagglutinin-L. When this chimeric gene was expressed in transgenic tobacco (Nicotiana tabacum), we observed in the seeds a series of polypeptides (M{submore » r} 10,000-18,000) that cross-react with antibodies to the bean {alpha}-amylase inhibitor. Most of these polypeptides bind to a pig pancreas {alpha}-amylase affinity column. An extract of the seeds of the transformed tobacco plants inhibits pig pancreas {alpha}-amylase activity as well as the {alpha}-amylase present in the midgut of Tenebrio molitor. We suggest that introduction of this lectin gene (to be called {alpha}ai) into other leguminous plants may be a strategy to protect the seeds from the seed-eating larvae of Coleoptera.« less

Metabolome analysis reveals the effect of carbon catabolite control on the poly(γ-glutamic acid) biosynthesis of Bacillus licheniformis ATCC 9945.

PubMed

Mitsunaga, Hitoshi; Meissner, Lena; Palmen, Thomas; Bamba, Takeshi; Büchs, Jochen; Fukusaki, Eiichiro

2016-04-01

Poly(γ-glutamic acid) (PGA) is a polymer composed of L- and/or D-glutamic acids that is produced by Bacillus sp. Because the polymer has various features as water soluble, edible, non-toxic and so on, it has attracted attention as a candidate for many applications such as foods, cosmetics and so on. However, although it is well known that the intracellular metabolism of Bacillus sp. is mainly regulated by catabolite control, the effect of the catabolite control on the PGA producing Bacillus sp. is largely unknown. This study is the first report of metabolome analysis on the PGA producing Bacillus sp. that reveals the effect of carbon catabolite control on the metabolism of PGA producing Bacillus licheniformis ATCC 9945. Results showed that the cells cultivated in glycerol-containing medium showed higher PGA production than the cells in glucose-containing medium. Furthermore, metabolome analysis revealed that the activators of CcpA and CodY, global regulatory proteins of the intracellular metabolism, accumulated in the cells cultivated in glycerol-containing and glucose-containing medium, respectively, with CodY apparently inhibiting PGA production. Moreover, the cells seemed to produce glutamate from citrate and ammonium using glutamine synthetase/glutamate synthase. Pulsed addition of di-ammonium hydrogen citrate, as suggested by the metabolome result, was able to achieve the highest value so far for PGA production in B. licheniformis. Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Modeling heat resistance of Bacillus weihenstephanensis and Bacillus licheniformis spores as function of sporulation temperature and pH.

PubMed

Baril, Eugénie; Coroller, Louis; Couvert, Olivier; Leguérinel, Ivan; Postollec, Florence; Boulais, Christophe; Carlin, Frédéric; Mafart, Pierre

2012-05-01

Although sporulation environmental factors are known to impact on Bacillus spore heat resistance, they are not integrated into predictive models used to calculate the efficiency of heating processes. This work reports the influence of temperature and pH encountered during sporulation on heat resistance of Bacillus weihenstephanensis KBAB4 and Bacillus licheniformis AD978 spores. A decrease in heat resistance (δ) was observed for spores produced either at low temperature, at high temperature or at acidic pH. Sporulation temperature and pH maximizing the spore heat resistance were identified. Heat sensitivity (z) was not modified whatever the sporulation environmental factors were. A resistance secondary model inspired by the Rosso model was proposed. Sporulation temperatures and pHs minimizing or maximizing the spore heat resistance (T(min(R)), T(opt(R)), T(max(R)), pH(min(R)) and pH(opt(R))) were estimated. The goodness of the model fit was assessed for both studied strains and literature data. The estimation of the sporulation temperature and pH maximizing the spore heat resistance is of great interest to produce spores assessing the spore inactivation in the heating processes applied by the food industry. Copyright © 2011 Elsevier Ltd. All rights reserved.

Physical and catalytic properties of alpha-amylase from Tenebrio molitor L. larvae.

PubMed Central

Buonocore, V; Poerio, E; Silano, V; Tomasi, M

1976-01-01

The amylase from Tenebrio molitor L. larvae (yellow mealworm) was characterized according to a number of its molecular and catalytic properties. The insect amylase is a single polypeptide chain with mol.wt. 68000, an isoelectric point of 4.0 and a very low content of sulphur-containing amino acids. The enzyme is a Ca2+-protein and behaves as an alpha-amylase. Removal of Ca2+ by exhaustive dialysis against water causes the irreversible inactivation of the enzyme. Moreover, the enzyme is activated by the presence in the assay mixture of Cl-, or some other inorganic anions that are less effective than Cl-, and is inhibited by F-. Optimal conditions of pH and temperature for the enzymic activity are 5.8 and 37 degrees C. The insect amylase exhibits an identical kinetic behaviour toward starch, amylose and amylopectin; the enzyme hydrolyses glycogen with a higher affinity constant. Compared with the non-insect alpha-amylases described in the literature, Tenebrio molitor amylase has a lower affinity for starch. PMID:942374

Effects of acetic acid and arginine on pH elevation and growth of Bacillus licheniformis in an acidified cucumber juice medium

USDA-ARS?s Scientific Manuscript database

Bacillus licheniformis has been shown to cause pH elevation in tomato products having an initial pH below 4.6 and metabiotic effects that can lead to the growth of pathogenic bacteria. Because of this, the organism poses a potential risk to acidified vegetable products; however, little is known abou...

A pathway closely related to the (D)-tagatose pathway of gram-negative enterobacteria identified in the gram-positive bacterium Bacillus licheniformis.

PubMed

Van der Heiden, Edwige; Delmarcelle, Michaël; Lebrun, Sarah; Freichels, Régine; Brans, Alain; Vastenavond, Christian M; Galleni, Moreno; Joris, Bernard

2013-06-01

We report the first identification of a gene cluster involved in d-tagatose catabolism in Bacillus licheniformis. The pathway is closely related to the d-tagatose pathway of the Gram-negative bacterium Klebsiella oxytoca, in contrast to the d-tagatose 6-phosphate pathway described in the Gram-positive bacterium Staphylococcus aureus.

Developing a new production host from a blueprint: Bacillus pumilus as an industrial enzyme producer.

PubMed

Küppers, Tobias; Steffen, Victoria; Hellmuth, Hendrik; O'Connell, Timothy; Bongaerts, Johannes; Maurer, Karl-Heinz; Wiechert, Wolfgang

2014-03-24

Since volatile and rising cost factors such as energy, raw materials and market competitiveness have a significant impact on the economic efficiency of biotechnological bulk productions, industrial processes need to be steadily improved and optimized. Thereby the current production hosts can undergo various limitations. To overcome those limitations and in addition increase the diversity of available production hosts for future applications, we suggest a Production Strain Blueprinting (PSB) strategy to develop new production systems in a reduced time lapse in contrast to a development from scratch.To demonstrate this approach, Bacillus pumilus has been developed as an alternative expression platform for the production of alkaline enzymes in reference to the established industrial production host Bacillus licheniformis. To develop the selected B. pumilus as an alternative production host the suggested PSB strategy was applied proceeding in the following steps (dedicated product titers are scaled to the protease titer of Henkel's industrial production strain B. licheniformis at lab scale): Introduction of a protease production plasmid, adaptation of a protease production process (44%), process optimization (92%) and expression optimization (114%). To further evaluate the production capability of the developed B. pumilus platform, the target protease was substituted by an α-amylase. The expression performance was tested under the previously optimized protease process conditions and under subsequently adapted process conditions resulting in a maximum product titer of 65% in reference to B. licheniformis protease titer. In this contribution the applied PSB strategy performed very well for the development of B. pumilus as an alternative production strain. Thereby the engineered B. pumilus expression platform even exceeded the protease titer of the industrial production host B. licheniformis by 14%. This result exhibits a remarkable potential of B. pumilus to be the

A Pathway Closely Related to the d-Tagatose Pathway of Gram-Negative Enterobacteria Identified in the Gram-Positive Bacterium Bacillus licheniformis

PubMed Central

Van der Heiden, Edwige; Lebrun, Sarah; Freichels, Régine; Brans, Alain; Vastenavond, Christian M.; Galleni, Moreno; Joris, Bernard

2013-01-01

We report the first identification of a gene cluster involved in d-tagatose catabolism in Bacillus licheniformis. The pathway is closely related to the d-tagatose pathway of the Gram-negative bacterium Klebsiella oxytoca, in contrast to the d-tagatose 6-phosphate pathway described in the Gram-positive bacterium Staphylococcus aureus. PMID:23524682

Enzymatic Properties of an Alkaline and Chelator Resistant alpha-amylase from the Alkaliphilic Bacillus sp. Isolate L1711

NASA Technical Reports Server (NTRS)

Bernhardsdotter, Eva C. M. J.; Pusey, Marc L.; Ng, Joseph D.; Garriott, Owen K.

2004-01-01

An alkaliphilic amylase producing bacterium, Bacillus sp. strain L 711, was selected among 13 soda lakes isolates. When grown at pH 10.5 and 37 C, strain L711 produced multiple forms of amylases in the culture broth. One of these, BAA, was purified from the culture supernatant by QAE column chromatography and preparative native gel electrophoresis. The molecular weight of BAA was determined to be 51 kDa by denaturing gel electrophoresis. The pH optima for activity below and above 40 C were 9.5 - 10.0 and 7.0 - 7.5 respectively. BAA was stable in the pH range 6-11 and was completely inactivated at 55 C. The thermostability was not increased in the presence of Ca(2+). The enzyme was strongly inhibited by Ca(2+), Zn(2+), Mg(2+), Mn(2+), Ba(2+) and Cu(2+), whereas the presence of Na(+), Co(2+) and EDTA (10 mM) enhanced enzymatic activity. The K(sub m), and specific activity of BAA on soluble starch were 1.9 mg/ml and 18.5 U/mg respectively. The main end products of hydrolysis were maltotetraose, maltose and glucose.

Effect of Bacillus licheniformis and Bacillus subtilis supplementation of ewe's feed on sheep milk production and young lamb mortality.

PubMed

Kritas, S K; Govaris, A; Christodoulopoulos, G; Burriel, A R

2006-05-01

The purpose of this pilot study was to evaluate under field conditions the effect of a probiotic containing Bacillus licheniformis and Bacillus subtilis on young lamb mortality and sheep milk production when administered in the late pregnancy and lactation feed of ewes. In a sheep farm, two groups of milking ewes with identical genetic material, management, nutrition, health status and similar production characteristics were formed. One group (46 ewes) served as control, while the other one (48 ewes) served as a probiotic-treated group. Both groups of ewes received a similar feeding regiment, but the ewes of the second group were additionally offered a probiotic product containing B. licheniformis and B. subtilis (BioPlus 2B, Chr. Hansen, Denmark) at the approximate dose of 2.56 x 10(9) viable spores per ewe per day. Lamb mortality during the 1.5 months suckling period, and milk yield during the 2 months of milk collection for commercial purposes have been recorded. In the non-treated control group, 13.1% mortality was observed versus 7.8% in the probiotic-treated group (P = 0.33), with mortality being mainly due to diarrhoea. Microbiological examination of diarrhoeic faeces from some of the dead lambs in both groups revealed the presence of Escherichia coli. The average daily milk yield per ewe was significantly lower in the control group (0.80 l) than that in the probiotic-treated group (0.93 l) (P < 0.05). Fat and protein content of milk in ewes that received probiotics was significantly (P < 0.05) increased compared with untreated ewes. It was concluded that supplementing ewe's feed with probiotics may have beneficial effect on subsequent milk yields, fat and protein content.

Draft Genome Sequences of Three Cellulolytic Bacillus licheniformis Strains Isolated from Imperial Geyser, Amphitheater Springs, and Whiterock Springs inside Yellowstone National Park

PubMed Central

O' Hair, Joshua A.; Li, Hui; Thapa, Santosh; Scholz, Matthew

2017-01-01

ABSTRACT Novel cellulolytic microorganisms are becoming more important for rapidly growing biofuel industries. This paper reports the draft genome sequences of Bacillus licheniformis strains YNP2-TSU, YNP3-TSU, and YNP5-TSU. These cellulolytic isolates were collected from several hydrothermal features inside Yellowstone National Park. PMID:28360181

Exposure to inhalable dust, wheat flour and alpha-amylase allergens in industrial and traditional bakeries.

PubMed

Bulat, Petar; Myny, Katrien; Braeckman, Lutgart; van Sprundel, Marc; Kusters, Edouard; Doekes, Gert; Pössel, Kerstin; Droste, Jos; Vanhoorne, Michel

2004-01-01

This study was designed to characterize exposure to inhalable dust, wheat flour and alpha-amylase allergens in industrial and traditional bakeries. The study included 70 bakeries from the northern part of Belgium. Based on the degree of automation and a clear division of individual job tasks, four bakeries were identified as industrial and the remaining 66 were identified as traditional ones. Personal, as well as stationary, samples of inhalable dust were collected during full shift periods, usually 5-7 h. The portable pumps aspirated 2 l/min through Teflon personal dust samplers (Millipore, pore size 1.0 microm) mounted in PAS-6 sampling heads. In the collected samples the inhalable dust, wheat flour and alpha-amylase allergens were determined. Wheat flour allergens were measured using enzyme-linked immunosorbent assay inhibition and an antiwheat IgG4 serum pool. The alpha-amylase allergens were measured using a sandwich enzyme immunoassay with affinity-purified polyclonal rabbit IgG antibodies. In total, 440 samples (300 personal and 140 stationary) were processed. The highest inhalable dust exposure was observed in traditional bakeries among bread [geometric mean (GM) 2.10 mg/m3] and bread and pastry workers (GM 1.80 mg/m3). In industrial bakeries the highest dust exposure was measured in bread-producing workers (GM 1.06 mg/m3). Similar relations were observed for wheat flour and alpha-amylase allergens. Bread baking workers in traditional bakeries had the highest exposure to both allergens (wheat flour GM 22.33 microg/m(3), alpha-amylase GM 0.61 ng/m3). The exposure to wheat flour and alpha-amylase allergens in industrial bakeries was higher in bread baking workers (wheat flour GM 6.15 microg/m3, alpha-amylase GM 0.47 ng/m3) than in bread packing workers (wheat flour GM 2.79 microg/m3, alpha-amylase GM 0.15 ng/m3). The data presented suggest that, on average, exposure in the Belgium bakeries studied-industrial as well as traditional-is lower than or similar to

Encapsulation of alpha-amylase into starch-based biomaterials: an enzymatic approach to tailor their degradation rate.

PubMed

Azevedo, Helena S; Reis, Rui L

2009-10-01

This paper reports the effect of alpha-amylase encapsulation on the degradation rate of a starch-based biomaterial. The encapsulation method consisted in mixing a thermostable alpha-amylase with a blend of corn starch and polycaprolactone (SPCL), which were processed by compression moulding to produce circular disks. The presence of water was avoided to keep the water activity low and consequently to minimize the enzyme activity during the encapsulation process. No degradation of the starch matrix occurred during processing and storage (the encapsulated enzyme remained inactive due to the absence of water), since no significant amount of reducing sugars was detected in solution. After the encapsulation process, the released enzyme activity from the SPCL disks after 28days was found to be 40% comparatively to the free enzyme (unprocessed). Degradation studies on SPCL disks, with alpha-amylase encapsulated or free in solution, showed no significant differences on the degradation behaviour between both conditions. This indicates that alpha-amylase enzyme was successfully encapsulated with almost full retention of its enzymatic activity and the encapsulation of alpha-amylase clearly accelerates the degradation rate of the SPCL disks, when compared with the enzyme-free disks. The results obtained in this work show that degradation kinetics of the starch polymer can be controlled by the amount of encapsulated alpha-amylase into the matrix.

A kinetic model to explain the maximum in alpha-amylase activity measurements in the presence of small carbohydrates.

PubMed

Baks, Tim; Janssen, Anja E M; Boom, Remko M

2006-06-20

The effect of the presence of several small carbohydrates on the measurement of the alpha-amylase activity was determined over a broad concentration range. At low carbohydrate concentrations, a distinct maximum in the alpha-amylase activity versus concentration curves was observed in several cases. At higher concentrations, all carbohydrates show a decreasing alpha-amylase activity at increasing carbohydrate concentrations. A general kinetic model has been developed that can be used to describe and explain these phenomena. This model is based on the formation of a carbohydrate-enzyme complex that remains active. It is assumed that this complex is formed when a carbohydrate binds to alpha-amylase without blocking the catalytic site and its surrounding subsites. Furthermore, the kinetic model incorporates substrate inhibition and substrate competition. Depending on the carbohydrate type and concentration, the measured alpha-amylase activity can be 75% lower than the actual alpha-amylase activity. The model that has been developed can be used to correct for these effects in order to obtain the actual amount of active enzyme. 2006 Wiley Periodicals, Inc.

The inability of Bacillus licheniformis perR mutant to grow is mainly due to the lack of PerR-mediated fur repression.

PubMed

Kim, Jung-Hoon; Yang, Yoon-Mo; Ji, Chang-Jun; Ryu, Su-Hyun; Won, Young-Bin; Ju, Shin-Yeong; Kwon, Yumi; Lee, Yeh-Eun; Youn, Hwan; Lee, Jin-Won

2017-06-01

PerR, a member of Fur family protein, is a metal-dependent H 2 O 2 sensing transcription factor that regulates genes involved in peroxide stress response. Industrially important bacterium Bacillus licheniformis contains three PerR-like proteins (PerR BL , PerR2, and PerR3) compared to its close relative Bacillus subtilis. Interestingly, unlike other bacteria including B. subtilis, no authentic perR BL null mutant could be established for B. licheniformis. Thus, we constructed a conditional perR BL mutant using a xylose-inducible promoter, and investigated the genes under the control of PerR BL . PerR BL regulon genes include katA, mrgA, ahpC, pfeT, hemA, fur, and perR as observed for PerR BS . However, there is some variation in the expression levels of fur and hemA genes between B. subtilis and B. licheniformis in the derepressed state. Furthermore, katA, mrgA, and ahpC are strongly induced, whereas the others are only weakly or not induced by H 2 O 2 treatment. In contrast to the B. subtilis perR null mutant which frequently gives rise to large colony phenotype mainly due to the loss of katA, the suppressors of B. licheniformis perR mutant, which can form colonies on LB agar, were all catalase-positive. Instead, many of the suppressors showed increased levels of siderophore production, suggesting that the suppressor mutation is linked to the fur gene. Consistent with this, perR fur double mutant could grow on LB agar without Fe supplementation, whereas perR katA double mutant could only grow on LB agar with Fe supplementation. Taken together, our data suggest that in B. licheniformis, despite the similarity in PerR BL and PerR BS regulon genes, perR is an essential gene required for growth and that the inability of perR null mutant to grow is mainly due to elevated expression of Fur.

Structural properties of hydrolyzed high-amylose rice starch by α-amylase from Bacillus licheniformis.

PubMed

Qin, Fengling; Man, Jianmin; Xu, Bin; Hu, Maozhi; Gu, Minghong; Liu, Qiaoquan; Wei, Cunxu

2011-12-14

High-amylose cereal starch has a great benefit on human health through its resistant starch (RS) content. Enzyme hydrolysis of native starch is very helpful in understanding the structure of starch granules and utilizing them. In this paper, native starch granules were isolated from a transgenic rice line (TRS) enriched with amylose and RS and hydrolyzed by α-amylase. Structural properties of hydrolyzed TRS starches were studied by X-ray powder diffraction, Fourier transform infrared, and differential scanning calorimetry. The A-type polymorph of TRS C-type starch was hydrolyzed faster than the B-type polymorph, but the crystallinity did not significantly change during enzyme hydrolysis. The degree of order in the external region of starch granule increased with increasing enzyme hydrolysis time. The amylose content decreased at first and then went back up during enzyme hydrolysis. The hydrolyzed starches exhibited increased onset and peak gelatinization temperatures and decreased gelatinization enthalpy on hydrolysis. These results suggested that the B-type polymorph and high amylose that formed the double helices and amylose-lipid complex increased the resistance to BAA hydrolysis. Furthermore, the spectrum results of RS from TRS native starch digested by pancreatic α-amylase and amyloglucosidase also supported the above conclusion.

One-Step Production of Immobilized α-Amylase in Recombinant Escherichia coli▿ †

PubMed Central

Rasiah, Indira A.; Rehm, Bernd H. A.

2009-01-01

Industrial enzymes are often immobilized via chemical cross-linking onto solid supports to enhance stability and facilitate repeated use in bioreactors. For starch-degrading enzymes, immobilization usually places constraints on enzymatic conversion due to the limited diffusion of the macromolecular substrate through available supports. This study describes the one-step immobilization of a highly thermostable α-amylase (BLA) from Bacillus licheniformis and its functional display on the surface of polyester beads inside engineered Escherichia coli. An optimized BLA variant (Termamyl) was N-terminally fused to the polyester granule-forming enzyme PhaC of Cupriavidus necator. The fusion protein lacking the signal sequence mediated formation of stable polyester beads exhibiting α-amylase activity. The α-amylase beads were assessed with respect to α-amylase activity, which was demonstrated qualitatively and quantitatively. The immobilized α-amylase showed Michaelis-Menten enzyme kinetics exerting a Vmax of about 506 mU/mg of bead protein with a Km of about 5 μM, consistent with that of free α-amylase. The stability of the enzyme at 85°C and the capacity for repeated usage in a starch liquefaction process were also demonstrated. In addition, structural integrity and functionality of the beads at extremes of pH and temperature, demonstrating their suitability for industrial use, were confirmed by electron microscopy and protein/enzyme analysis. This study proposes a novel, cost-effective method for the production of immobilized α-amylase in a single step by using the polyester granules forming protein PhaC as a fusion partner in engineered E. coli. PMID:19201981

Crystal Structure of Bacillus subtilis α-Amylase in Complex with Acarbose

PubMed Central

Kagawa, Masayuki; Fujimoto, Zui; Momma, Mitsuru; Takase, Kenji; Mizuno, Hiroshi

2003-01-01

The crystal structure of Bacillus subtilis α-amylase, in complex with the pseudotetrasaccharide inhibitor acarbose, revealed an hexasaccharide in the active site as a result of transglycosylation. After comparison with the known structure of the catalytic-site mutant complexed with the native substrate maltopentaose, it is suggested that the present structure represents a mimic intermediate in the initial stage of the catalytic process. PMID:14617662

LaaA, a novel high-active alkalophilic alpha-amylase from deep-sea bacterium Luteimonas abyssi XH031(T).

PubMed

Song, Qinghao; Wang, Yan; Yin, Chong; Zhang, Xiao-Hua

2016-08-01

Alpha-amylase is a kind of broadly used industrial enzymes, most of which have been exploited from terrestrial organism. Comparatively, alpha-amylase from marine environment was largely undeveloped. In this study, a novel alkalophilic alpha-amylase with high activity, Luteimonas abyssi alpha-amylase (LaaA), was cloned from deep-sea bacterium L. abyssi XH031(T) and expressed in Escherichia coli BL21. The gene has a length of 1428bp and encodes 475 amino acids with a 35-residue signal peptide. The specific activity of LaaA reached 8881U/mg at the optimum pH 9.0, which is obvious higher than other reported alpha-amylase. This enzyme can remain active at pH levels ranging from 6.0 to 11.0 and temperatures below 45°C, retaining high activity even at low temperatures (almost 38% residual activity at 10°C). In addition, 1mM Na(+), K(+), and Mn(2+) enhanced the activity of LaaA. To investigate the function of potential active sites, R227G, D229K, E256Q/H, H327V and D328V mutants were generated, and the results suggested that Arg227, Asp229, Glu256 and Asp328 were total conserved and essential for the activity of alpha-amylase LaaA. This study shows that the alpha-amylase LaaA is an alkali-tolerant and high-active amylase with strong potential for use in detergent industry. Copyright © 2016 Elsevier Inc. All rights reserved.

Validation of an assay for quantification of alpha-amylase in saliva of sheep

PubMed Central

Fuentes-Rubio, Maria; Fuentes, Francisco; Otal, Julio; Quiles, Alberto; Hevia, María Luisa

2016-01-01

The objective of this study was to develop a time-resolved immunofluorometric assay (TR-IFMA) for quantification of salivary alpha-amylase in sheep. For that purpose, after the design of the assay, an analytical and a clinical validation were carried out. The analytical validation of the assay showed intra- and inter-assay coefficients of variation (CVs) of 6.1% and 10.57%, respectively and an analytical limit of detection of 0.09 ng/mL. The assay also demonstrated a high level of accuracy, as determined by linearity under dilution. For clinical validation, a model of acute stress testing was conducted to determine whether expected significant changes in alpha-amylase were picked up in the newly developed assay. In that model, 11 sheep were immobilized and confronted with a sheepdog to induce stress. Saliva samples were obtained before stress induction and 15, 30, and 60 min afterwards. Salivary cortisol was measured as a reference of stress level. The results of TR-IFMA showed a significant increase (P < 0.01) in the concentration of alpha-amylase in saliva after stress induction. The assay developed in this study could be used to measure salivary alpha-amylase in the saliva of sheep and this enzyme could be a possible noninvasive biomarker of stress in sheep. PMID:27408332

Production of tannase by the immobilized cells of Bacillus licheniformis KBR6 in Ca-alginate beads.

PubMed

Mohapatra, P K D; Mondal, K C; Pati, B R

2007-06-01

The present study was aimed at finding the optimal conditions for immobilization of Bacillus licheniformis KBR6 cells in calcium-alginate (Ca-alginate) beads and determining the operational stability during the production of tannin-acyl-hydrolase (tannase) under semicontinous cultivation. The active cells of B. licheniformis KBR6 were immobilized in Ca-alginate and used for the production of tannase. The influence of alginate concentration (5, 10, 20 and 30 g l(-1)) and initial cell loading on enzyme production were studied. The production of tannase increased significantly with increasing alginate concentration and reached a maximum enzyme yield of 0.56 +/- 0.03 U ml(-1) at 20 g l(-1). This was about 1.70-fold higher than that obtained by free cells. The immobilized cells produced tannase consistently over 13 repeated cycles and reached a maximum level at the third cycle. Scanning electron microscope study indicated that the cells in Ca-alginate beads remain in normal shape. The Ca-alginate entrapment is a promising immobilization method of B. licheniformis KBR6 for repeated tannase production. Tannase production by immobilized cells is superior to that of free cells because it leads to higher volumetric activities within the same period of fermentation. This is the first report of tannase production from immobilized bacterial cells. The bacterium under study can produce higher amounts of tannase with respect to other fungal strains within a short cultivation period.

Biotransformation of benzo[a]pyrene by the thermophilic bacterium Bacillus licheniformis M2-7.

PubMed

Guevara-Luna, Joseph; Alvarez-Fitz, Patricia; Ríos-Leal, Elvira; Acevedo-Quiroz, Macdiel; Encarnación-Guevara, Sergio; Moreno-Godinez, Ma Elena; Castellanos-Escamilla, Mildred; Toribio-Jiménez, Jeiry; Romero-Ramírez, Yanet

2018-06-09

Benzo[a]pyrene (BaP) is recognized as a potentially carcinogenic and mutagenic hydrocarbon, and thus, its removal from the environment is a priority. The use of thermophilic bacteria capable of biodegrading or biotransforming this compound to less toxic forms has been explored in recent decades, since it provides advantages compared to mesophilic organisms. This study assessed the biotransformation of BaP by the thermophilic bacterium Bacillus licheniformis M2-7. Our analysis of the biotransformation process mediated by strain M2-7 on BaP shows that it begins during the first 3 h of culture. The gas chromatogram of the compound produced shows a peak with a retention time of 17.38 min, and the mass spectra shows an approximate molecular ion of m/z 167, which coincides with the molecular weight of the chemical formula C 6 H 4 (COOH) 2 , confirming a chemical structure corresponding to phthalic acid. Catechol 2,3-dioxygenase (C23O) enzyme activity was detected in minimal saline medium supplemented with BaP (0.33 U mg -1 of protein). This finding suggests that B. licheniformis M2-7 uses the meta pathway for biodegrading BaP using the enzyme C23O, thereby generating phthalic acid as an intermediate.

In vitro antagonistic activity and the protective effect of probiotic Bacillus licheniformis Dahb1 in zebrafish challenged with GFP tagged Vibrio parahaemolyticus Dahv2.

PubMed

Girija, Vairavan; Malaikozhundan, Balasubramanian; Vaseeharan, Baskaralingam; Vijayakumar, Sekar; Gobi, Narayanan; Del Valle Herrera, Marian; Chen, Jiann-Chu; Santhanam, Perumal

2018-01-01

In vitro antagonistic activity and the protective effect of probiotic Bacillus licheniformis Dahb1 in zebrafish (Danio rerio) challenged with GFP tagged Vibrio parahaemolyticus Dahv2 was studied. The cell free extract of probiotic B. licheniformis Dahb1 at 100 μg mL -1 showed growth inhibition of V. parahaemolyticus Dahv2 in vitro. B. licheniformis Dahb1 also inhibited the biofilm growth of GFP tagged V. parahaemolyticus Dahv2 at 100 μg mL -1 in vitro. The growth and survival of zebrafish was tested using probiotic B. licheniformis Dahb1. Weight (1.28 g) of zebrafish that received the cell free extract was much higher than in control (1.04 g). The mortality of zebrafish infected with GFP tagged V. parahaemolyticus Dahv2 at 10 7 Cfu mL -1 (Group IV) was 100%, whereas a complete survival of zebrafish that received the cell free extract of B. licheniformis Dahb1 at 10 7 Cfu mL -1 (Group VII) was observed after 30 days. The number of GFP tagged V. parahaemolyticus Dahv2 colonies in the intestine and gills significantly reduced after treatment with the cell free extract of B. licheniformis Dahb1. Furthermore, a significant decrease in the fluorescent colonies of GFP tagged V. parahaemolyticus Dahv2 was observed after treatment with the cell free extract of B. licheniformis Dahb1 under confocal laser scanning microscopy (CLSM). In conclusion, the cell free extract of B. licheniformis Dahb1 could prevent Vibrio infection by enhancing the growth and survival of zebrafish. Copyright © 2017 Elsevier Ltd. All rights reserved.

Effects of a dietary Aspergillus oryzae extract containing alpha-amylase activity on performance and carcass characteristics of finishing beef cattle.

PubMed

Tricarico, J M; Abney, M D; Galyean, M L; Rivera, J D; Hanson, K C; McLeod, K R; Harmon, D L

2007-03-01

Three experiments were conducted to examine the effects of an Aspergillus oryzae extract containing alpha-amylase activity on performance and carcass characteristics of finishing beef cattle. In Exp. 1, 120 crossbred steers were used in a randomized complete block design to evaluate the effects of roughage source (alfalfa hay vs. cottonseed hulls) and supplemental alpha-amylase at 950 dextrinizing units (DU)/kg of DM. Significant roughage source x alpha-amylase interactions (P < 0.05) were observed for performance. In steers fed cottonseed hulls, supplemental alpha-amylase increased ADG through d 28 and 112 and tended (P < 0.15) to increase ADG in all other periods. The increases in ADG were related to increased DMI and efficiency of gain during the initial 28-d period but were primarily related to increased DMI as the feeding period progressed. Supplemental alpha-amylase increased (P = 0.02) the LM area across both roughage sources. In Exp. 2, 96 crossbred heifers were used in a randomized complete block design with a 2 x 3 factorial arrangement of treatments to evaluate the effects of corn processing (dry cracked vs. high moisture) and supplemental alpha-amylase concentration (0, 580, or 1,160 DU/kg of DM). Alpha-amylase supplementation increased DMI (P = 0.05) and ADG (P = 0.03) during the initial 28 d on feed and carcass-adjusted ADG (P = 0.04) across corn processing methods. Longissimus muscle area was greatest (quadratic effect, P = 0.04), and yield grade was least (quadratic effect, P = 0.02) in heifers fed 580 DU of alpha-amylase/kg of DM across corn processing methods. In Exp. 3, 56 crossbred steers were used in a randomized complete block design to evaluate the effects of supplemental alpha-amylase (930 DU/kg of DM) on performance when DMI was restricted to yield a programmed ADG. Alpha-amylase supplementation did not affect performance when DMI was restricted. We conclude that dietary alpha-amylase supplementation of finishing beef diets may result in

Glutamate dehydrogenase (RocG) in Bacillus licheniformis WX-02: Enzymatic properties and specific functions in glutamic acid synthesis for poly-γ-glutamic acid production.

PubMed

Tian, Guangming; Wang, Qin; Wei, Xuetuan; Ma, Xin; Chen, Shouwen

2017-04-01

Poly-γ-glutamic acid (γ-PGA), a natural biopolymer, is widely used in cosmetics, medicine, food, water treatment, and agriculture owing to its features of moisture sequestration, cation chelation, non-toxicity and biodegradability. Intracellular glutamic acid, the substrate of γ-PGA, is a limiting factor for high yield in γ-PGA production. Bacillus subtilis and Bacillus licheniformis are both important γ-PGA producing strains, and B. subtilis synthesizes glutamic acid in vivo using the unique GOGAT/GS pathway. However, little is known about the glutamate synthesis pathway in B. licheniformis. The aim of this work was to characterize the glutamate dehydrogenase (RocG) in glutamic acid synthesis from B. licheniformis with both in vivo and in vitro experiments. By re-directing the carbon flux distribution, the rocG gene deletion mutant WX-02ΔrocG produced intracellular glutamic acid with a concentration of 90ng/log(CFU), which was only 23.7% that of the wild-type WX-02 (380ng/log(CFU)). Furthermore, the γ-PGA yield of mutant WX-02ΔrocG was 5.37g/L, a decrease of 45.3% compared to the wild type (9.82g/L). In vitro enzymatic assays of RocG showed that RocG has higher affinity for 2-oxoglutarate than glutamate, and the glutamate synthesis rate was far above degradation. This is probably the first study to reveal the glutamic acid synthesis pathway and the specific functions of RocG in B. licheniformis. The results indicate that γ-PGA production can be enhanced through improving intracellular glutamic acid synthesis. Copyright © 2017 Elsevier Inc. All rights reserved.

Close evolutionary relatedness among functionally distantly related members of the (alpha/beta)8-barrel glycosyl hydrolases suggested by the similarity of their fifth conserved sequence region.

PubMed

Janecek, S

1995-12-11

A short conserved sequence equivalent to the fifth conserved sequence region of alpha-amylases (173_LPDLD, Aspergillus oryzae alpha-amylase) comprising the calcium-ligand aspartate, Asp-175, was identified in the amino acid sequences of several members of the family of (alpha/beta)8-barrel glycosyl hydrolases. Despite the fact that the aspartate is not invariantly conserved, the stretch can be easily recognised in all sequences to be positioned 26-28 amino acid residues in front of the well-known catalytic aspartate (Asp-206, A. oryzae alpha-amylase) located in the beta 4-strand of the barrel. The identification of this region revealed remarkable similarities between some alpha-amylases (those from Bacillus megaterium, Bacillus subtilis and Dictyoglomus thermophilum) on the one hand and several different enzyme specificities (such as oligo-1,6-glucosidase, amylomaltase and neopullulanase, respectively) on the other hand. The most interesting example was offered by B. subtilis alpha-amylase and potato amylomaltase with the regions LYDWN and LYDWK, respectively. These observations support the idea that all members of the family of glycosyl hydrolases adopting the structure of the alpha-amylase-type (alpha/beta)8-barrel are mutually closely related and the strict evolutionary borders separating the individual enzyme specificities can be hardly defined.

Alpha-amylase Inhibition and Antioxidant Activity of Marine Green Algae and its Possible Role in Diabetes Management.

PubMed

Unnikrishnan, P S; Suthindhiran, K; Jayasri, M A

2015-10-01

In the continuing search for safe and efficient antidiabetic drug, marine algae become important source which provide several compounds of immense therapeutic potential. Alpha-amylase, alpha-glucosidase inhibitors, and antioxidant compounds are known to manage diabetes and have received much attention recently. In the present study, four green algae (Chaetomorpha aerea, Enteromorpha intestinalis, Chlorodesmis, and Cladophora rupestris) were chosen to evaluate alpha-amylase, alpha-glucosidase inhibitory, and antioxidant activity in vitro. The phytochemical constituents of all the extracts were qualitatively determined. Antidiabetic activity was evaluated by inhibitory potential of extracts against alpha-amylase and alpha-glucosidase by spectrophotometric assays. Antioxidant activity was determined by 2,2-diphenyl-1-picrylhydrazyl, hydrogen peroxide (H2O2), and nitric oxide scavenging assay. Gas chromatography-mass spectrometry (GC-MS) analysis was carried out to determine the major compound responsible for its antidiabetic action. Among the various extracts screened, chloroform extract of C. aerea (IC50 - 408.9 μg/ml) and methanol extract of Chlorodesmis (IC50 - 147.6 μg/ml) showed effective inhibition against alpha-amylase. The extracts were also evaluated for alpha-glucosidase inhibition, and no observed activity was found. Methanol extract of C. rupestris showed notable free radical scavenging activity (IC50 - 666.3 μg/ml), followed by H2O2 (34%) and nitric oxide (49%). Further, chemical profiling by GC-MS revealed the presence of major bioactive compounds. Phenol, 2,4-bis (1,1-dimethylethyl) and z, z-6,28-heptatriactontadien-2-one were predominantly found in the methanol extract of C. rupestris and chloroform extract of C. aerea. Our results demonstrate that the selected algae exhibit notable alpha-amylase inhibition and antioxidant activity. Therefore, characterization of active compounds and its in vivo assays will be noteworthy. Four green algae were

Alpha-amylase Inhibition and Antioxidant Activity of Marine Green Algae and its Possible Role in Diabetes Management

PubMed Central

Unnikrishnan, P. S.; Suthindhiran, K.; Jayasri, M. A.

2015-01-01

Aim: In the continuing search for safe and efficient antidiabetic drug, marine algae become important source which provide several compounds of immense therapeutic potential. Alpha-amylase, alpha-glucosidase inhibitors, and antioxidant compounds are known to manage diabetes and have received much attention recently. In the present study, four green algae (Chaetomorpha aerea, Enteromorpha intestinalis, Chlorodesmis, and Cladophora rupestris) were chosen to evaluate alpha-amylase, alpha-glucosidase inhibitory, and antioxidant activity in vitro. Materials and Methods: The phytochemical constituents of all the extracts were qualitatively determined. Antidiabetic activity was evaluated by inhibitory potential of extracts against alpha-amylase and alpha-glucosidase by spectrophotometric assays. Antioxidant activity was determined by 2,2-diphenyl-1-picrylhydrazyl, hydrogen peroxide (H2O2), and nitric oxide scavenging assay. Gas chromatography-mass spectrometry (GC-MS) analysis was carried out to determine the major compound responsible for its antidiabetic action. Results: Among the various extracts screened, chloroform extract of C. aerea (IC50 − 408.9 μg/ml) and methanol extract of Chlorodesmis (IC50 − 147.6 μg/ml) showed effective inhibition against alpha-amylase. The extracts were also evaluated for alpha-glucosidase inhibition, and no observed activity was found. Methanol extract of C. rupestris showed notable free radical scavenging activity (IC50 – 666.3 μg/ml), followed by H2O2 (34%) and nitric oxide (49%). Further, chemical profiling by GC-MS revealed the presence of major bioactive compounds. Phenol, 2,4-bis (1,1-dimethylethyl) and z, z-6,28-heptatriactontadien-2-one were predominantly found in the methanol extract of C. rupestris and chloroform extract of C. aerea. Conclusion: Our results demonstrate that the selected algae exhibit notable alpha-amylase inhibition and antioxidant activity. Therefore, characterization of active compounds and its in vivo

Optimization of novel and greener approach for the coproduction of uricase and alkaline protease in Bacillus licheniformis by Box-Behnken model.

PubMed

Pawar, Shweta V; Rathod, Virendra K

2018-01-02

This study explores a novel concept of coproduction of uricase and alkaline protease by Bacillus licheniformis using single substrate in single step. Seven local bacterial strains were screened for uricase production, amongst which B. licheniformis is found to produce highest uricase along with alkaline protease. Optimization of various factors influencing maximum enzyme coproduction by B. licheniformis is performed. Maximum enzyme productivity of 0.386 U/mL uricase and 0.507 U/mL alkaline protease is obtained at 8 hr of incubation period, 1% (v/v) inoculum, and at 0.2% (w/v) uric acid when the organism is cultivated at 25°C, 180 rpm, in a media containing xylose as a carbon source, urea as a nitrogen source, and initial pH of 9.5. The statistical experimental design method of Box-Behnken was further applied to obtain optimal concentration of significant parameters such as pH (9.5), uric acid concentration (0.1%), and urea concentration (0.05%). The maximum uricase and alkaline protease production by B. licheniformis using Box-Behnken design was 0.616 and 0.582 U/mL, respectively, with 1.6- and 1.13-fold increase as compared to one factor at a time optimized media. This study will be useful to develop an economic, commercially viable, and scalable process for simultaneous production of uricase and protease enzymes.

Enzymatic Properties of an Alkaline and Chelator Resistant Proportional to alpha-Amylase from the Alkaliphilic Bacillus sp. Isolate L1711

NASA Technical Reports Server (NTRS)

Bernhardsdotter, Eva C. M. J.; Pusey, Marc L.; Ng, Joseph D.; Garriott, Owen K.

2004-01-01

An alkaliphilic amylase producing bacterium, Bacillus sp. strain L1711, was selected among 13 soda lakes isolates. When grown at pH 10.5 and 370 C, strain L1711 produced multiple forms of amylases in the culture broth. One of these, BAA, was purified from the culture supernatant by QAE column chromatography and preparative native gel electrophoresis. The molecular weight of BAA was determined to be 51 kDa by denaturing gel electrophoresis. The pH optima for activity below and above 40 C were 9.5-10.0 and 7.0-7.5 respectively. BAA was stable in the pH range 6-11 and was completely inactivated at 55?C. The thermostability was not increased in the presence of Ca(2+). The enzyme was strongly inhibited by Ca(2+), Zn(2+), Mg(2+), Mn(2+), Ba(2+) and Cu(2+), whereas the presence of Na(+), Co2+ and EDTA (10 mM) enhanced enzymatic activity. The K(sub m) and specific activity of BAA on soluble starch were 1.9 mg/ml and 18.5 U/mg respectively. The main end products of hydrolysis were maltotetraose, maltose and glucose .

Progress of pancreatitis disease biomarker alpha amylase enzyme by new nano optical sensor.

PubMed

Attia, M S; Al-Radadi, Najlaa S

2016-12-15

A new nano optical sensor binuclear Pd-(2-aminothiazole) (urea), Pd(atz,ur) complex was prepared and characterized for the assessment of the activity of alpha amylase enzyme in urine and serum samples for early diagnosis of Pancreatitis disease. The assessment of alpha amylase activity is carried out by the quenching of the luminescence intensity of the nano optical sensor binuclear Pd(atz,ur) complex at 457nm by the 2-chloro-4-nitrophenol (2-CNP) which produced from the reaction of the enzyme with 2-chloro-4-nitrophenyl-α-d-maltotrioside (CNPG3) substrate. The remarkable quenching of the luminescence intensity at 457nm of nano Pd(atz,ur) doped in sol-gel matrix by various concentrations of the 2-CNP was successfully used as an optical sensor for the assessment of α-amylase activity. The calibration plot was achieved over the concentration range 8.5×10(-6) to 1.9×10(-9)molL(-1) 2-CNP with a correlation coefficient of (0.999) and a detection limit of (7.4×10(-10)molL(-1)). The method was used satisfactorily for the assessment of the α-amylase activity over activity range (3-321U/L) in different urine and serum samples of pancreatitis patients. The assessment of the alpha amylase biomarker by the proposed method increases its sensitivity (96.88%) and specificity (94.41%) for early diagnosis of pancreatitis diseases. Copyright © 2016 Elsevier B.V. All rights reserved.

Hyperproduction of γ-glutamyl transpeptidase from Bacillus licheniformis ER15 in the presence of high salt concentration.

PubMed

Bindal, Shruti; Gupta, Rani

2017-02-07

Microbial γ-glutamyl transpeptidases (GGTs) have been exploited in biotechnological, pharmaceutical, and food sectors for the synthesis of various γ-glutamyl compounds. But, till date, no bacterial GGTs are commercially available in the market because of lower levels of production from various sources. In the current study, production of GGT from Bacillus licheniformis ER15 was investigated to achieve high GGT titers. Hyperproduction of GGT from B. licheniformis ER15 was achieved with 6.4-fold enhancement (7921.2 ± 198.7 U/L) by optimization of culture medium following one-variable-at-a-time strategy and statistical approaches. Medium consisting of Na 2 HPO 4 : 0.32% (w/v); KH 2 PO 4 : 0.15% (w/v); starch: 0.1% (w/v); soybean meal: 0.5% (w/v); NaCl: 4.0% (w/v), and MgCl 2 : 5 mM was found to be optimal for maximum GGT titers. Maximum GGT titers were obtained, in the optimized medium at 37°C and 200 rpm, after 40 h. It was noteworthy that GGT production was a linear function of sodium chloride concentration, as observed during response surface methodology. While investigating the role of NaCl on GGT production, it was found that NaCl drastically decreased subtilisin concentration and indirectly increasing GGT recovery. B. licheniformis ER15 is proved to be a potential candidate for large-scale production of GGT enzyme and its commercialization.

Expression of Bacillus protease (Protease BYA) from Bacillus sp. Y in Bacillus subtilis and enhancement of its specific activity by site-directed mutagenesis-improvement in productivity of detergent enzyme-.

PubMed

Tobe, Seiichi; Shimogaki, Hisao; Ohdera, Motoyasu; Asai, Yoshio; Oba, Kenkichi; Iwama, Masanori; Irie, Masachika

2006-01-01

An attempt was made to express protease BYA produced by an alkalophilic Bacillus sp. Y in Bacillus subtilis by gene engineering methods. The gene encoding protease BYA was cloned from Bacillus sp. Y, and expression vector pTA71 was constructed from the amylase promoter of Bacillus licheniformis, DNA fragments encoding the open reading frame of protease BYA, and pUB110. Protease BYA was secreted at an activity level of 5100 APU/ml in the common industrial culture medium of Bacillus subtilis transformed with pTA71. We then attempted to increase the specific activity of protease BYA by site-directed mutagenesis. Amino acid residue Ala29 next to catalytic Asp30 was replaced by one of three uncharged amino acid residues (Val29, Leu29, Ile29), and each mutant enzyme was expressed and isolated from the culture medium. Val29 mutant enzyme was secreted at an activity level of greater than 7000 APU/ml in culture medium, and its specific activity was 1.5-fold higher than that of the wild-type enzyme. Other mutant enzymes had specific activity similar to that of the original one and were less stabile than the wild-type enzyme. It can be thought that the substitution at amino acid residue 29 affects the level of activity and stability of protease BYA.

Aerobic Fitness Level Affects Cardiovascular and Salivary Alpha Amylase Responses to Acute Psychosocial Stress.

PubMed

Wyss, Thomas; Boesch, Maria; Roos, Lilian; Tschopp, Céline; Frei, Klaus M; Annen, Hubert; La Marca, Roberto

2016-12-01

Good physical fitness seems to help the individual to buffer the potential harmful impact of psychosocial stress on somatic and mental health. The aim of the present study is to investigate the role of physical fitness levels on the autonomic nervous system (ANS; i.e. heart rate and salivary alpha amylase) responses to acute psychosocial stress, while controlling for established factors influencing individual stress reactions. The Trier Social Stress Test for Groups (TSST-G) was executed with 302 male recruits during their first week of Swiss Army basic training. Heart rate was measured continuously, and salivary alpha amylase was measured twice, before and after the stress intervention. In the same week, all volunteers participated in a physical fitness test and they responded to questionnaires on lifestyle factors and personal traits. A multiple linear regression analysis was conducted to determine ANS responses to acute psychosocial stress from physical fitness test performances, controlling for personal traits, behavioural factors, and socioeconomic data. Multiple linear regression revealed three variables predicting 15 % of the variance in heart rate response (area under the individual heart rate response curve during TSST-G) and four variables predicting 12 % of the variance in salivary alpha amylase response (salivary alpha amylase level immediately after the TSST-G) to acute psychosocial stress. A strong performance at the progressive endurance run (high maximal oxygen consumption) was a significant predictor of ANS response in both models: low area under the heart rate response curve during TSST-G as well as low salivary alpha amylase level after TSST-G. Further, high muscle power, non-smoking, high extraversion, and low agreeableness were predictors of a favourable ANS response in either one of the two dependent variables. Good physical fitness, especially good aerobic endurance capacity, is an important protective factor against health

Human Parotid Gland Alpha-Amylase Secretion as a Function of Chronic Hyperbaric Exposure

DTIC Science & Technology

1979-01-01

parotid ...Pullman, WA 99163 Gilman, S. C, G. J. Fischer, R. J. Biersner, R. D. Thornton, and D. A. Miller. 1979. Human parotid gland alpha-amylase secretion...as a function of chronic hyperbaric exposure. Undersea Biomed. Res. 6(3):303-307.—Secretion of a-amylase by the human parotid gland increased

Higher levels of salivary alpha-amylase predict failure of cessation efforts in male smokers.

PubMed

Dušková, M; Simůnková, K; Hill, M; Hruškovičová, H; Hoskovcová, P; Králíková, E; Stárka, L

2010-01-01

The ability to predict the success or failure of smoking cessation efforts will be useful for clinical practice. Stress response is regulated by two primary neuroendocrine systems. Salivary cortisol has been used as a marker for the hypothalamus-pituitary-adrenocortical axis and salivary alpha-amylase as a marker for the sympathetic adrenomedullary system. We studied 62 chronic smokers (34 women and 28 men with an average age of 45.2+/-12.9 years). The levels of salivary cortisol and salivary alpha-amylase were measured during the period of active smoking, and 6 weeks and 24 weeks after quitting. We analyzed the men separately from the women. The men who were unsuccessful in cessation showed significantly higher levels of salivary alpha-amylase over the entire course of the cessation attempt. Before stopping smoking, salivary cortisol levels were higher among the men who were unsuccessful in smoking cessation. After quitting, there were no differences between this group and the men who were successful in cessation. In women we found no differences between groups of successful and unsuccessful ex-smokers during cessation. In conclusions, increased levels of salivary alpha-amylase before and during smoking cessation may predict failure to quit in men. On the other hand, no advantage was found in predicting the failure to quit in women. The results of our study support previously described gender differences in smoking cessation.

Activation of bean (Phaseolus vulgaris) [alpha]-amylase inhibitor requires proteolytic processing of the proprotein

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

Pueyo, J.J.; Hunt, D.C.; Chrispeels, M.J.

Seeds of the common bean (Phaseolus vulgaris) contain a plant defense protein that inhibits the [alpha]-amylases of mammals and insects. This [alpha]-amylase inhibitor ([alpha]Al) is synthesized as a proprotein on the endoplasmic reticulum and is proteolytically processed after arrival in the protein storage vacuoles to polypeptides of relative molecular weight (M[sub r]) 15,000 to 18,000. The authors report two types of evidence that proteolytic processing is linked to activation of the inhibitory activity. First, by surveying seed extracts of wild accessions of P. vulgaris and other species in the genus Phaseolus, they found that antibodies to [alpha]Al recognize large (M[submore » r] 30,000-35,000) polypeptides as well as typical [alpha]Al processing products (M[sub r] 15,000-18,000). [alpha]Al activity was found in all extracts that had the typical [alpha]Al processed polypeptides, but was absent from seed extracts that lacked such polypeptides. Second, they made a mutant [alpha]Al in which asparagine-77 is changed to aspartic acid-77. This mutation slows down the proteolytic processing of pro-[alpha]Al when the gene is expressed in tobacco. When pro-[alpha]Al was separated from mature [alpha]Al by gel filtration, pro-[alpha]Al was found not to have [alpha]-amylase inhibitory activity. The authors interpret these results to mean that formation of the active inhibitor is causally related to proteolytic processing of the proprotein. They suggest that the polypeptide cleavage removes a conformation constraint on the precursor to produce the biochemically active molecule. 43 refs., 5 figs., 1 tab.« less

Effect of neohesperidin dihydrochalcone on the activity and stability of alpha-amylase: a comparative study on bacterial, fungal, and mammalian enzymes.

PubMed

Kashani-Amin, Elaheh; Ebrahim-Habibi, Azadeh; Larijani, Bagher; Moosavi-Movahedi, Ali Akbar

2015-10-01

Neohesperidin dihydrochalcone (NHDC) was recently introduced as an activator of mammalian alpha-amylase. In the current study, the effect of NHDC has been investigated on bacterial and fungal alpha-amylases. Enzyme assays and kinetic analysis demonstrated the capability of NHDC to significantly activate both tested alpha-amylases. The ligand activation pattern was found to be more similar between the fungal and mammalian enzyme in comparison with the bacterial one. Further, thermostability experiments indicated a stability increase in the presence of NHDC for the bacterial enzyme. In silico (docking) test locates a putative binding site for NHDC on alpha-amylase surface in domain B. This domain shows differences in various alpha-amylase types, and the different behavior of the ligand toward the studied enzymes may be attributed to this fact. Copyright © 2015 John Wiley & Sons, Ltd.

GFP tagged Vibrio parahaemolyticus Dahv2 infection and the protective effects of the probiotic Bacillus licheniformis Dahb1 on the growth, immune and antioxidant responses in Pangasius hypophthalmus.

PubMed

Gobi, Narayanan; Malaikozhundan, Balasubramanian; Sekar, Vijayakumar; Shanthi, Sathappan; Vaseeharan, Baskaralingam; Jayakumar, Rengarajan; Khudus Nazar, Abdul

2016-05-01

In this study, the pathogenicity of GFP tagged Vibrio parahaemolyticus Dahv2 and the protective effect of the probiotic strain, Bacillus licheniformis Dahb1 was studied on the Asian catfish, Pangasius hypophthalmus. The experiment was carried out for 24 days with three groups and one group served as the control (without treatment). In the first group, P. hypophthalmus was orally infected with 1 mL of GFP tagged V. parahaemolyticus Dahv2 at two different doses (10(5) and 10(7) cfu mL(-1)). In the second group, P. hypophthalmus was orally administrated with 1 ml of the probiotic B. licheniformis Dahb1 at two different doses (10(5) and 10(7) cfu mL(-1)). In the third group, P. hypophthalmus was orally infected first with 1 mL of GFP tagged V. parahaemolyticus Dahv2 followed by the administration of 1 mL of B. licheniformis Dahb1 (combined treatment) at two different doses (10(5) and 10(7) cfu mL(-1)). The growth, immune (myeloperoxidase, respiratory burst, natural complement haemolytic and lysozyme activity) and antioxidant (glutathione-S-transferase, reduced glutathione and total glutathione) responses of P. hypophthalmus were reduced after post infection of GFP tagged V. parahaemolyticus Dahv2 compared to control. However, after administration with the probiotic B. licheniformis Dahb1 at 10(5) cfu mL(-1), P. hypophthalmus showed significant increase in the growth, immune and antioxidant responses compared to 10(7) cfu mL(-1). On the otherhand, the growth, immune and antioxidant responses of P. hypophthalmus infected and administrated with combined GFP tagged Vibrio + Bacillus at 10(5) cfu mL(-1) were relatively higher than that of GFP tagged V. parahaemolyticus Dahv2 and control groups but lower than that of probiotic B. licheniformis Dahb1 groups. The results of the present study conclude that the probiotic B. licheniformis Dahb1 at 10(5) cfu mL(-1) has the potential to protect the P. hypophthalmus against V. parahaemolyticus Dahv2 infection by enhancing the growth

Production of alpha-amylase from Aspergillus oryzae for several industrial applications in a single step.

PubMed

Porfirif, María C; Milatich, Esteban J; Farruggia, Beatriz M; Romanini, Diana

2016-06-01

A one-step method as a strategy of alpha-amylase concentration and purification was developed in this work. This methodology requires the use of a very low concentration of biodegradable polyelectrolyte (Eudragit(®) E-PO) and represents a low cost, fast, easy to scale up and non-polluting technology. Besides, this methodology allows recycling the polymer after precipitation. The formation of reversible soluble/insoluble complexes between alpha-amylase and the polymer Eudragit(®) E-PO was studied, and their precipitation in selected conditions was applied with bioseparation purposes. Turbidimetric assays allowed to determine the pH range where the complexes are insoluble (4.50-7.00); pH 5.50 yielded the highest turbidity of the system. The presence of NaCl (0.05M) in the medium totally dissociates the protein-polymer complexes. When the adequate concentration of polymer was added under these conditions to a liquid culture of Aspergillus oryzae, purification factors of alpha-amylase up to 7.43 and recoveries of 88% were obtained in a simple step without previous clarification. These results demonstrate that this methodology is suitable for the concentration and production of alpha-amylase from this source and could be applied at the beginning of downstream processing. Copyright © 2016 Elsevier B.V. All rights reserved.

An organic solvent-, detergent-, and thermo-stable alkaline protease from the mesophilic, organic solvent-tolerant Bacillus licheniformis 3C5.

PubMed

Rachadech, W; Navacharoen, A; Ruangsit, W; Pongtharangkul, T; Vangnai, A S

2010-01-01

Bacillus licheniformis 3C5, isolated as mesophilic bacterium, exhibited tolerance towards a wide range of non-polar and polar organic solvents at 45 degrees C. It produced an extracellular organic solvent-stable protease with an apparent molecular mass of approximately 32 kDa. The inhibitory effect of PMSF and EDTA suggested it is likely to be an alkaline serine protease. The protease was active over abroad range of temperatures (45-70 degrees C) and pH (8-10) range with an optimum activity at pH 10 and 65 degrees C. It was comparatively stable in the presence ofa relatively high concentration (35% (v/v)) of organic solvents and various types of detergents even at a relatively high temperature (45 degrees C). The protease production by B. licheniformis 3C5 was growth-dependent. The optimization of carbon and nitrogen sources for cell growth and protease production revealed that yeast extract was an important medium component to support both cell growth and the protease production. The overall properties of the protease produced by B. licheniformis 3C5 suggested that this thermo-stable, solvent-stable, detergent-stable alkaline protease is a promising potential biocatalyst for industrial and environmental applications.

Molecular cloning of alpha-amylases from cotton boll weevil, Anthonomus grandis and structural relations to plant inhibitors: an approach to insect resistance.

PubMed

Oliveira-Neto, Osmundo B; Batista, João A N; Rigden, Daniel J; Franco, Octávio L; Falcão, Rosana; Fragoso, Rodrigo R; Mello, Luciane V; dos Santos, Roseane C; Grossi-de-Sá, Maria F

2003-01-01

Anthonomus grandis, the cotton boll weevil, causes severe cotton crop losses in North and South America. Here we demonstrate the presence of starch in the cotton pollen grains and young ovules that are the main A. grandis food source. We further demonstrate the presence of alpha-amylase activity, an essential enzyme of carbohydrate metabolism for many crop pests, in A. grandis midgut. Two alpha-amylase cDNAs from A. grandis larvae were isolated using RT-PCR followed by 5' and 3' RACE techniques. These encode proteins with predicted molecular masses of 50.8 and 52.7kDa, respectively, which share 58% amino acid identity. Expression of both genes is induced upon feeding and concentrated in the midgut of adult insects. Several alpha-amylase inhibitors from plants were assayed against A. grandis alpha-amylases but, unexpectedly, only the BIII inhibitor from rye kernels proved highly effective, with inhibitors generally active against other insect amylases lacking effect. Structural modeling of Amylag1 and Amylag2 showed that different factors seem to be responsible for the lack of effect of 0.19 and alpha-AI1 inhibitors on A. grandis alpha-amylase activity. This work suggests that genetic engineering of cotton to express alpha-amylase inhibitors may offer a novel route to A. grandis resistance.

Enzymatic laundry for old clothes: immobilized alpha-amylase from Bacillus sp. for the biocleaning of an ancient Coptic tunic.

PubMed

Ferrari, Martina; Mazzoli, Roberto; Morales, Simona; Fedi, Mariaelena; Liccioli, Lucia; Piccirillo, Anna; Cavaleri, Tiziana; Oliva, Cinzia; Gallo, Paolo; Borla, Matilde; Cardinali, Michela; Pessione, Enrica

2017-09-01

The classification and conservation of ancient artworks (belonging to collections) is of important cultural, historical, and economic concern. However, ancient textiles often display structural damage that renders them fragile and unsuitable for exhibition. One of the most common types of damage is linked to erroneous restoration treatments, among which the application of glues to consolidate cuts. Harsh strategies, such as mechanical or chemical treatments, are not suitable since they can cause further impairment of the fabric, whereas mild approaches, like wet cleaning, are often ineffective, as also demonstrated by the present study. Here, we have explored the possibility of using gellan-immobilized enzymes of bacterial origin (Bacillus alpha-amylase) to obtain a satisfactory starch removal from a damaged archaeological tunic-shroud from the Turin Egyptian Museum (Italy), without altering the original yarns or textile fibers. This method, already applied to clean casein-damaged wall paintings, as well as cotton, silk, and linen fabrics, has proved to be optimal for the treatment of a wool burial shroud and to be able to definitively solve fragile textile restoration problems. Moreover, efforts have been made to obtain insights into the artwork: a multidisciplinary approach has allowed to obtain a correct chronological attribution (radiocarbon dating) and fabric fiber characterization (SEM-EDX) as well as shed light on the colored parts and dark stains (FORS+IRFC and XRF). Finally, the evaluation of the type of glue, by Fourier transform infrared spectroscopy, has suggested the best enzyme for glue removal. These results have demonstrated that a mild bio-based approach is a successful tool for the treatment of archaeological textiles in critical conditions.

Bacillus licheniformis Contains Two More PerR-Like Proteins in Addition to PerR, Fur, and Zur Orthologues

PubMed Central

Ju, Shin-Yeong; Yang, Yoon-Mo; Ryu, Su-Hyun; Kwon, Yumi; Won, Young-Bin; Lee, Yeh-Eun; Youn, Hwan; Lee, Jin-Won

2016-01-01

The ferric uptake regulator (Fur) family proteins include sensors of Fe (Fur), Zn (Zur), and peroxide (PerR). Among Fur family proteins, Fur and Zur are ubiquitous in most prokaryotic organisms, whereas PerR exists mainly in Gram positive bacteria as a functional homologue of OxyR. Gram positive bacteria such as Bacillus subtilis, Listeria monocytogenes and Staphylococcus aureus encode three Fur family proteins: Fur, Zur, and PerR. In this study, we identified five Fur family proteins from B. licheniformis: two novel PerR-like proteins (BL00690 and BL00950) in addition to Fur (BL05249), Zur (BL03703), and PerR (BL00075) homologues. Our data indicate that all of the five B. licheniformis Fur homologues contain a structural Zn2+ site composed of four cysteine residues like many other Fur family proteins. Furthermore, we provide evidence that the PerR-like proteins (BL00690 and BL00950) as well as PerRBL (BL00075), but not FurBL (BL05249) and ZurBL (BL03703), can sense H2O2 by histidine oxidation with different sensitivity. We also show that PerR2 (BL00690) has a PerR-like repressor activity for PerR-regulated genes in vivo. Taken together, our results suggest that B. licheniformis contains three PerR subfamily proteins which can sense H2O2 by histidine oxidation not by cysteine oxidation, in addition to Fur and Zur. PMID:27176811

Enhanced Production and Characterization of a Solvent Stable Amylase from Solvent Tolerant Bacillus tequilensis RG-01: Thermostable and Surfactant Resistant

PubMed Central

Tiwari, Soni; Shukla, Neha; Mishra, Pooja; Gaur, Rajeeva

2014-01-01

Ten bacterial strains isolated from the soil samples in the presence of cyclohexane were screened for amylase production. Among them, culture RG-01 was adjudged as the best amylase producer and was identified as Bacillus tequilensis from MTCC, Chandigarh. The isolate showed maximum amylase production (8100 U/mL) in the presence of starch, peptone, and Ca2+ ions at 55°C pH 7.0 within 24 h of incubation. The enzyme was stable in the presence of n-dodecane, isooctane, n-decane, xylene, toluene, n-hexane, n-butanol, and cyclohexane, respectively. The presence of benzene, methanol, and ethanol marginally reduced the amylase stability, respectively. The enzyme was showed it 100% activity at 55°C and pH 7.0 with 119% and 127% stability at 55°C and pH 7.0, respectively. The enzyme was also stable in the presence of SDS, Tween-40, Tween-60, and Tween-80 (1%) and was found stimulatory effect, respectively. Only Triton-X-100 showed a moderate inhibitory effect (5%) on amylase activity. This isolate (Bacillus tequilensis RG-01) may be useful in several industrial applications owing to its thermotolerant and organic solvents and surfactants resistance characteristics. PMID:25401163

Poly-γ-glutamic acid produced from Bacillus licheniformis CGMCC 2876 as a potential substitute for polyacrylamide in the sugarcane industry.

PubMed

Yan, Shan; Yao, Haosheng; Chen, Zhen; Zeng, Shengquan; Xi, Xi; Wang, Yuanpeng; He, Ning; Li, Qingbiao

2015-01-01

As an environmentally friendly and industrially useful biopolymer, poly-γ-glutamic acid (γ-PGA) from Bacillus licheniformis CGMCC 2876 was characterized by the high-resolution mass spectrometry and (1)H NMR. A flocculating activity of 11,474.47 U mL(-1) obtained with γ-PGA, and the effects of carbon sources, ions, and chemical properties (D-/L-composition and molecular weight) on the production and flocculating activity of γ-PGA were discussed. Being a bioflocculant in the sugar refinery process, the color and turbidity of the sugarcane juice was IU 1,877.36 and IU 341.41 with 0.8 ppm of γ-PGA, respectively, which was as good as the most widely used chemically synthesized flocculant in the sugarcane industry--polyacrylamide with 1 ppm. The γ-PGA produced from B. licheniformis CGMCC 2876 could be a promising alternate of chemically synthesized flocculants in the sugarcane industry. © 2015 American Institute of Chemical Engineers.

General Subject 1. Report to ICUMSA on the determination of commercial alpha-amylase activity by a spectrophotometric method

USDA-ARS?s Scientific Manuscript database

A report is given on a new industrial method for the determination of the activity or strength of commercial alpha-amylase at a sugarcane factory or refinery, as well as a recommendation. At the present time, the activities or strengths of commercial alpha-amylases cannot be directly compared becau...

The effect of alpha amylase enzyme on quality of sweet sorghum juice for chrystal sugar

NASA Astrophysics Data System (ADS)

Marwati, T.; Cahyaningrum, N.; Widodo, S.; Astiati, U. T.; Budiyanto, A.; Wahyudiono; Arif, A. B.; Richana, N.

2018-01-01

Sweet sorghum juice (Sorghum bicolor L. Moench) has characteristics similar to sugar cane juice and potentially used for sugar substitutes that can support food security. Nevertheless the sweet sorghum juicecontain starch which impede sorghum sugar crystallization. Therefore, research on the enzymatic process is needed to convert starch into reducing sugar. The experimental design used was the Factorial Randomized Design with the first factor was alpha amylase enzyme concentration (0, 20, 40, 60, 80, 100, 120 μL/100 mL) and second factor was incubation time (0, 30, 60, 90 minute) at temperature 100°C. The experiment was conducted on fresh sweet sorghum. The results showed that the addition of the alpha amylase enzyme increased the content of reducing sugar and decreased levels of starch. Elevating concentration of alpha amylase enzyme will increase the reducing sugar content in sweet sorghum juice. The optimum alpha amylase enzyme concentration to produce the highest total sugar was 80 μL/100 mL of sweet sorghum juice with the optimum incubation time was 90 minutes. The results of this study are expected to create a new sweetener for sugar substitution. From the economic prospective aspect, sorghum is a potential crop and can be relied upon to support the success of the food diversification program which further leads to the world food security

Molecular Identification of a Newly Isolated Bacillus subtilis BI19 and Optimization of Production Conditions for Enhanced Production of Extracellular Amylase

PubMed Central

Dash, Biplab Kumar; Rahman, M. Mizanur; Sarker, Palash Kumar

2015-01-01

A study was carried out with a newly isolated bacterial strain yielding extracellular amylase. The phylogenetic tree constructed on the basis of 16S rDNA gene sequences revealed this strain as clustered with the closest members of Bacillus sp. and identified as Bacillus subtilis BI19. The effect of various fermentation conditions on amylase production through shake-flask culture was investigated. Rice flour (1.25%) as a cheap natural carbon source was found to induce amylase production mostly. A combination of peptone and tryptone as organic and ammonium sulfate as inorganic nitrogen sources gave highest yield. Maximum production was obtained after 24 h of incubation at 37°C with an initial medium pH 8.0. Addition of surfactants like Tween 80 (0.25 g/L) and sodium lauryl sulfate (0.2 g/L) resulted in 28% and 15% increase in enzyme production, respectively. Amylase production was 3.06 times higher when optimized production conditions were used. Optimum reaction temperature and pH for crude amylase activity were 50°C and 6.0, respectively. The crude enzyme showed activity and stability over a fair range of temperature and pH. These results suggest that B. subtilis BI19 could be exploited for production of amylase at relatively low cost and time. PMID:26180814

Salivary Alpha-Amylase Reactivity in Breast Cancer Survivors

PubMed Central

Wan, Cynthia; Couture-Lalande, Marie-Ève; Narain, Tasha A.; Lebel, Sophie; Bielajew, Catherine

2016-01-01

The two main components of the stress system are the hypothalamic-pituitary-adrenal (HPA) and sympathetic-adrenal-medullary (SAM) axes. While cortisol has been commonly used as a biomarker of HPA functioning, much less attention has been paid to the role of the SAM in this context. Studies have shown that long-term breast cancer survivors display abnormal reactive cortisol patterns, suggesting a dysregulation of their HPA axis. To fully understand the integrity of the stress response in this population, this paper explored the diurnal and acute alpha-amylase profiles of 22 breast cancer survivors and 26 women with no history of cancer. Results revealed that breast cancer survivors displayed identical but elevated patterns of alpha-amylase concentrations in both diurnal and acute profiles relative to that of healthy women, F (1, 39) = 17.95, p < 0.001 and F (1, 37) = 7.29, p = 0.010, respectively. The average area under the curve for the diurnal and reactive profiles was 631.54 ± 66.94 SEM and 1238.78 ± 111.84 SEM, respectively. This is in sharp contrast to their cortisol results, which showed normal diurnal and blunted acute patterns. The complexity of the stress system necessitates further investigation to understand the synergistic relationship of the HPA and SAM axes. PMID:27023572

Salivary Alpha-Amylase Reactivity in Breast Cancer Survivors.

PubMed

Wan, Cynthia; Couture-Lalande, Marie-Ève; Narain, Tasha A; Lebel, Sophie; Bielajew, Catherine

2016-03-23

The two main components of the stress system are the hypothalamic-pituitary-adrenal (HPA) and sympathetic-adrenal-medullary (SAM) axes. While cortisol has been commonly used as a biomarker of HPA functioning, much less attention has been paid to the role of the SAM in this context. Studies have shown that long-term breast cancer survivors display abnormal reactive cortisol patterns, suggesting a dysregulation of their HPA axis. To fully understand the integrity of the stress response in this population, this paper explored the diurnal and acute alpha-amylase profiles of 22 breast cancer survivors and 26 women with no history of cancer. Results revealed that breast cancer survivors displayed identical but elevated patterns of alpha-amylase concentrations in both diurnal and acute profiles relative to that of healthy women, F (1, 39) = 17.95, p < 0.001 and F (1, 37) = 7.29, p = 0.010, respectively. The average area under the curve for the diurnal and reactive profiles was 631.54 ± 66.94 SEM and 1238.78 ± 111.84 SEM, respectively. This is in sharp contrast to their cortisol results, which showed normal diurnal and blunted acute patterns. The complexity of the stress system necessitates further investigation to understand the synergistic relationship of the HPA and SAM axes.

Cell surface engineering of Bacillus subtilis improves production yields of heterologously expressed α-amylases.

PubMed

Cao, Haojie; van Heel, Auke J; Ahmed, Hifza; Mols, Maarten; Kuipers, Oscar P

2017-04-04

Bacillus subtilis is widely used as a cell factory for numerous heterologous proteins of commercial value and medical interest. To explore the possibility of further enhancing the secretion potential of this model bacterium, a library of engineered strains with modified cell surface components was constructed, and the corresponding influences on protein secretion were investigated by analyzing the secretion of α-amylase variants with either low-, neutral- or high- isoelectric points (pI). Relative to the wild-type strain, the presence of overall anionic membrane phospholipids (phosphatidylglycerol and cardiolipin) increased dramatically in the PssA-, ClsA- and double KO mutants, which resulted in an up to 47% higher secretion of α-amylase. Additionally, we demonstrated that the appropriate net charge of secreted targets (AmyTS-23, AmyBs and AmyBm) was beneficial for secretion efficiency as well. In B. subtilis, the characteristics of cell membrane phospholipid bilayer and the pIs of heterologous α-amylases appear to be important for their secretion efficiency. These two factors can be engineered to reduce the electrostatic interaction between each other during the secretion process, which finally leads to a better secretion yield of α-amylases.

Characterization of lipopeptides produced by Bacillus licheniformis using liquid chromatography with accurate tandem mass spectrometry.

PubMed

Favaro, Gabriella; Bogialli, Sara; Di Gangi, Iole Maria; Nigris, Sebastiano; Baldan, Enrico; Squartini, Andrea; Pastore, Paolo; Baldan, Barbara

2016-10-30

The plant endophyte Bacillus licheniformis, isolated from leaves of Vitis vinifera, was studied to individuate and characterize the presence of bioactive lipopeptides having amino acidic structures. Crude extracts of liquid cultures were analyzed by ultra-high-performance liquid chromatography (UHPLC) coupled to a quadrupole time-of-flight (QTOF) mass analyzer. Chromatographic conditions were optimized in order to obtain an efficient separation of the different isobaric lipopeptides, avoiding merged fragmentations of co-eluted isomeric compounds and reducing possible cross-talk phenomena. Composition of the amino acids was outlined through the interpretation of the fragmentation behavior in tandem high-resolution mass spectrometry (HRMS/MS) mode, which showed both common-class and peculiar fragment ions. Both [M + H](+) and [M + Na](+) precursor ions were fragmented in order to differentiate some isobaric amino acids, i.e. Leu/Ile. Neutral losses characteristic of the iso acyl chain were also evidenced. More than 90 compounds belonging to the classes of surfactins and lichenysins, known as biosurfactant molecules, were detected. Sequential LC/HRMS/MS analysis was used to identify linear and cyclic lipopeptides, and to single out the presence of a large number of isomers not previously reported. Some critical issues related to the simultaneous selection of different compounds by the quadrupole filter were highlighted and partially solved, leading to tentative assignments of several structures. Linear lichenysins are described here for the first time. The approach was proved to be useful for the characterization of non-target lipopeptides, and proposes a rationale MS experimental scheme aimed to investigate the difference in amino acid sequence and/or in the acyl chain of the various congeners, when standards are not available. Results expanded the knowledge about production of linear and cyclic bioactive compounds from Bacillus licheniformis, clarifying the

The green tea polyphenol (-)-epigallocatechin gallate precipitates salivary proteins including alpha-amylase: biochemical implications for oral health.

PubMed

Hara, Kumiko; Ohara, Masaru; Hayashi, Ikue; Hino, Takamune; Nishimura, Rumi; Iwasaki, Yoriko; Ogawa, Tetsuji; Ohyama, Yoshihiko; Sugiyama, Masaru; Amano, Hideaki

2012-04-01

Green tea is a popular drink throughout the world, and it contains various components, including the green tea polyphenol (-)-epigallocatechin gallate (EGCG). Tea interacts with saliva upon entering the mouth, so the interaction between saliva and EGCG interested us, especially with respect to EGCG-protein binding. SDS-PAGE revealed that several salivary proteins were precipitated after adding EGCG to saliva. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) peptide mass fingerprinting indicated that the major proteins precipitated by EGCG were alpha-amylase, S100, and cystatins. Surface plasmon resonance revealed that EGCG bound to alpha-amylase at dissociation constant (K(d)) = 2.74 × 10(-6) M, suggesting that EGCG interacts with salivary proteins with a relatively strong affinity. In addition, EGCG inhibited the activity of alpha-amylase by non-competitive inhibition, indicating that EGCG is effective at inhibiting the formation of fermentable carbohydrates involved in caries formation. Interestingly, alpha-amylase reduced the antimicrobial activity of EGCG against the periodontal bacterium Aggregatibacter actinomycetemcomitans. Therefore, we considered that EGCG-salivary protein interactions might have both protective and detrimental effects with respect to oral health. © 2012 Eur J Oral Sci.

Purification and properties of an alpha-amylase protein-inhibitor from Arachis hypogaea seeds.

PubMed

Irshad, M; Sharma, C B

1981-06-15

A protein showing highly specific inhibitory activity towards hog pancreatic and human salivary alpha-amylases (1,4-alpha-D-glucan glucanohydrolase, EC 3.2.1.1), but not towards plant and bacterial alpha-amylases, has been purified 197-fold from an aqueous extract of peanut cotyledons using heat treatment, (NH4)2SO4 precipitation and ion-exchange chromatography on DEAE- and CM-cellulose. The purified inhibitor was homogeneous by polyacrylamide gel electrophoresis. Its molecular weight, as determined by Sephadex G-100 gel-filtration, and its electrophoretic mobility at pH 8 relative to bromophenol blue, were 25 000 and 0.14, respectively. The inhibitory activity was relatively resistant to thermal treatment and markedly increased when the inhibitor was preincubated with the enzyme before the addition of starch. Further, the inhibition was found to be pH-dependent and non-competitive in nature.

Investigating the potential of Bacillus subtilis alpha-amylase as a pressure-temperature-time indicator for high hydrostatic pressure pasteurization processes.

PubMed

Grauwet, Tara; Van der Plancken, Iesel; Vervoort, Liesbeth; Hendrickx, Marc E; Van Loey, Ann

2009-01-01

The potential of Bacillus subtilis alpha-amylase (BSA) as a pressure-temperature-time indicator (pTTI) for high pressure pasteurization processing (400-600 MPa; T(i) 10-40 degrees C; 1-15 min) was investigated. A stepwise approach was followed for the development of an enzyme-based, extrinsic, isolated pTTI. First, based on literature data on the pressure stability, BSA was selected as a candidate indicator. Next to the accuracy and ease of the measurement of the indicator's response (residual activity) to the pressure treatment, the storage and handling stability of BSA at atmospheric pressure was verified. Second, the stability of BSA at a constant temperature (T) and time in function of pressure (p) was investigated. Solvent engineering was used to shift the inactivation window of BSA in the processing range of interest. Third, the enzyme (1 g/L BSA-MES 0.05 M pH 5.0) was kinetically calibrated under isobaric-isothermal conditions. Time dependent changes in activity could be modeled best by a first-order model. Except for low pressures and high temperatures, a synergistic effect between pressure and temperature could be observed. Based on the model selected to describe the combined p,T-dependency of the inactivation rate constant, an elliptically shaped isorate contour plot could be constructed, illustrating the processing range where BSA can be used to demonstrate temperature gradients. Fourth, the validity of the kinetic model was tested successfully under dynamic conditions similar to those used in food industry. Finally, the indicator was found suitable to demonstrate nonuniformity in two-sectional planes of a vertical, single vessel system. (c) 2009 American Institute of Chemical Engineers. Biotechnol. Prog., 2009.

Relationship Between Meditation Depth and Waking Salivary Alpha-Amylase Secretion Among Long-Term MBSR Instructors.

PubMed

Haslam, Alyson; Wirth, Michael D; Robb, Sara Wagner

2017-08-01

The purpose of this study was to characterize sympathetic activity by using waking salivary alpha-amylase (sAA) concentrations in a group of long-term meditation instructors and to examine the association between meditation (depth, dose and duration) and the waking alpha-amylase response. Salivary alpha-amylase samples were collected (immediately upon waking and at 15-min, 30-min and 45-min intervals after waking) from mindfulness-based stress reduction instructors to determine both the area under the curve and the awakening slope (difference in alpha-amylase concentrations between waking and 30-min post-waking). It was determined through general linear models that neither years of meditation nor meditation dose were associated with the awakening sAA slope, but higher scores for meditation depth (greater depth) was associated with a more negative (or steeper) awakening slope [Quartile (Q)1: -7 versus Q4: -21 U/mL; p = 0.06], in fully adjusted models. Older age (p = 0.04) and a later time of waking (p < 0.01) also were associated with less negative awakening slope values. Smoking was associated with lower area under the curve values (smokers: 1716 U/mL versus nonsmokers: 2107 U/mL; p = 0.05) in fully adjusted models. The results suggest a 'healthy' sAA waking slope among individuals who meditate more deeply. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Crystal structures of the psychrophilic alpha-amylase from Alteromonas haloplanctis in its native form and complexed with an inhibitor.

PubMed Central

Aghajari, N.; Feller, G.; Gerday, C.; Haser, R.

1998-01-01

Alteromonas haloplanctis is a bacterium that flourishes in Antarctic sea-water and it is considered as an extreme psychrophile. We have determined the crystal structures of the alpha-amylase (AHA) secreted by this bacterium, in its native state to 2.0 angstroms resolution as well as in complex with Tris to 1.85 angstroms resolution. The structure of AHA, which is the first experimentally determined three-dimensional structure of a psychrophilic enzyme, resembles those of other known alpha-amylases of various origins with a surprisingly greatest similarity to mammalian alpha-amylases. AHA contains a chloride ion which activates the hydrolytic cleavage of substrate alpha-1,4-glycosidic bonds. The chloride binding site is situated approximately 5 angstroms from the active site which is characterized by a triad of acid residues (Asp 174, Glu 200, Asp 264). These are all involved in firm binding of the Tris moiety. A reaction mechanism for substrate hydrolysis is proposed on the basis of the Tris inhibitor binding and the chloride activation. A trio of residues (Ser 303, His 337, Glu 19) having a striking spatial resemblance with serine-protease like catalytic triads was found approximately 22 angstroms from the active site. We found that this triad is equally present in other chloride dependent alpha-amylases, and suggest that it could be responsible for autoproteolytic events observed in solution for this cold adapted alpha-amylase. PMID:9541387

Natural products as alpha-amylase and alpha-glucosidase inhibitors and their hypoglycaemic potential in the treatment of diabetes: an update.

PubMed

Tundis, R; Loizzo, M R; Menichini, F

2010-04-01

The inhibition of alpha-glucosidase and alpha-amylase, enzymes involved in the digestion of carbohydrates, can significantly reduce the post-prandial increase of blood glucose and therefore can be an important strategy in the management of blood glucose level in type 2 diabetic and borderline patients. Currently, there is renewed interest in plant-based medicines and functional foods modulating physiological effects in the prevention and cure of diabetes and obesity. The plant kingdom is a wide field to search for natural effective oral hypoglycaemic agents that have slight or no side effects. More than ca. 1200 plant species have been recorded to be used empirically worldwide for their alleged hypoglycaemic activity. Therefore, natural alpha-glucosidase and alpha-amylase inhibitors from plant sources offer an attractive strategy for the control of hyperglycaemia. This article reviews recent data on plant extracts and isolated natural compounds that are being tested for their hypglycaemic activity, highlights ongoing research and considers the future persepctives.

Screening of traditional antidiabetic medicinal plants of Mauritius for possible alpha-amylase inhibitory effects in vitro.

PubMed

Kotowaroo, M I; Mahomoodally, M F; Gurib-Fakim, A; Subratty, A H

2006-03-01

In this study, seven exotic/indigenous medicinal plants of Mauritius, namely Coix lacryma-jobi (Poaceae), Aegle marmelos (Rutaceae), Artocarpus heterophyllus (Moraceae), Vangueria madagascariensis (Rubiaceae), Azadirachta indica (Meliaceae), Eriobotrya japonica (Rosaceae) and Syzigium cumini (Myrtaceae) were studied for possible effects on starch breakdown by alpha-amylase in vitro. The results showed that only Artocarpus heterophyllus significantly (p < 0.05) inhibited alpha-amylase activity in vitro. To confirm the observed effects, a further biochemical assay was undertaken to investigate the effects of Artocarpus heterophyllus on alpha-amylase activity using rat plasma in vitro. It was found that the aqueous leaf extract significantly (p < 0.05) inhibited alpha-amylase activity in rat plasma. The highest inhibitory activity (27.20 +/- 5.00%) was observed at a concentration of 1000 microg/mL. However, in both cases dose dependency was not observed. Enzyme kinetic studies using the Michaelis-Menten and Lineweaver-Burk equations were performed to establish the type of inhibition involved. In the presence of the plant extract the maximal velocity (Vmax) remained constant (1/150 g / L/s) whereas the Michaelis-Menten constant (Km) increased by 5.79 g / L, indicating that the aqueous leaf extract of Artocarpus heterophyllus behaved as a competitive inhibitor. Results from the present study tend to indicate that Artocarpus heterophyllus could act as a 'starch blocker' thereby reducing post-prandial glucose peaks. Copyright 2006 John Wiley & Sons, Ltd.

Two forms of alpha-amylase in mantle tissue of Mytilus galloprovincialis: purification and molecular properties of form II.

PubMed

Lombraña, M; Suárez, P; San Juan, F

2005-09-01

alpha-Amylase activity has been shown for the first time in a non-digestive tissue from Mytilus galloprovincialis. alpha-amylase from mussel mantle tissue has been purified by affinity chromatography on insoluble starch, followed by gel-filtration chromatography on Superdex-200. The chromatographic and electrophoretic behaviour of M. galloprovincialis alpha-amylase and stability characteristics suggest two forms of this enzyme: one form forming stable aggregates (form I) and a monomeric form (form II) that is more abundant, active and unstable. Both forms show an inverse quantitative variation. Purified form II was highly unstable and the molecular mass was estimated to be 66 kDa by sodium dodecyl sulphate (SDS)-gel electrophoresis. Maximum activity was noted at pH 6.5 and 35 degrees C.

Reflection on design and testing of pancreatic alpha-amylase inhibitors: an in silico comparison between rat and rabbit enzyme models

PubMed Central

2012-01-01

Background Inhibitors of pancreatic alpha-amylase are potential drugs to treat diabetes and obesity. In order to find compounds that would be effective amylase inhibitors, in vitro and in vivo models are usually used. The accuracy of models is limited, but these tools are nonetheless valuable. In vitro models could be used in large screenings involving thousands of chemicals that are tested to find potential lead compounds. In vivo models are still used as preliminary mean of testing compounds behavior in the whole organism. In the case of alpha-amylase inhibitors, both rats and rabbits could be chosen as in vivo models. The question was which animal could present more accuracy with regard to its pancreatic alpha-amylase. Results As there is no crystal structure of these enzymes, a molecular modeling study was done in order to compare the rabbit and rat enzymes with the human one. The overall result is that rabbit enzyme could probably be a better choice in this regard, but in the case of large ligands, which could make putative interactions with the −4 subsite of pancreatic alpha-amylase, interpretation of results should be made cautiously. Conclusion Molecular modeling tools could be used to choose the most suitable model enzyme that would help to identify new enzyme inhibitors. In the case of alpha-amylase, three-dimensional structures of animal enzymes show differences with the human one which should be taken into account when testing potential new drugs. PMID:23352052

Identification and properties of an alpha-amylase from a strain of Eubacterium sp. isolated from the rat intestinal tract.

PubMed

Delahaye, E P; Foglietti, M J; Andrieux, C; Chardon-Loriaux, I; Szylit, O; Raibaud, P

1991-01-01

1. A bacterial amylase was isolated from the intestinal content of monoxenic rats inoculated with Eubacterium sp. B86. 2. Affinity chromatography on cross-linked starch allowed its separation from rat endogenous amylases. 3. The bacterial enzyme was characterized by its pI, molecular weight and action pattern. It behaves as a typical endo-amylase (alpha-amylase).

Isolation and characterisation of a novel alpha-amylase from the extreme haloarchaeon Haloterrigena turkmenica.

PubMed

Santorelli, Marco; Maurelli, Luisa; Pocsfalvi, Gabriella; Fiume, Immacolata; Squillaci, Giuseppe; La Cara, Francesco; Del Monaco, Giovanni; Morana, Alessandra

2016-11-01

An extracellular halophilic alpha-amylase (AmyA) was produced by the haloarchaeon Haloterrigena turkmenica grown in medium enriched with 0.2% (w/v) starch. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and size exclusion chromatography (SEC) analyses showed a major band at 66.0kDa and a peak of 54.0kDa, respectively. Analysis of tryptic fragments of the protein present in the major SDS-PAGE band by nano-LC-ESI-MS/MS led to identification of the alpha-amylase catalytic region, encoded by the htur2110 gene, as the protein possessing the described activity. Optimal values for activity were 55°C, pH 8.5 and 2M NaCl, and high thermostability was showed at 55°C and 3M NaCl. AmyA activity was enhanced by Triton X-100 and was not influenced by n-hexane and chloroform. Starch hydrolysis produced different oligomers with maltose as the smallest end-product. The efficiency of AmyA in degrading starch contained in agronomic residues was tested in grape cane chosen as model substrate. Preliminary results showed that starch was degraded making the enzyme a potential candidate for utilization of agro-industrial waste in fuel and chemicals production. AmyA is one of the few investigated amylases produced by haloarchaea, and the first alpha-amylase described among microorganisms belonging to the genus Haloterrigena. Copyright © 2016 Elsevier B.V. All rights reserved.

Disruption in the cecal microbiota of chickens challenged with Clostridium perfringens and other factors was alleviated by Bacillus licheniformis supplementation.

PubMed

Lin, Yicen; Xu, Shuai; Zeng, Dong; Ni, Xueqin; Zhou, Mengjia; Zeng, Yan; Wang, Hesong; Zhou, Yi; Zhu, Hui; Pan, Kangcheng; Li, Guangyao

2017-01-01

Clostridium perfringens can induce necrotic enteritis of chickens, which causes large economic losses every year. Bacillus licheniformis, a probiotic, can inhibit the growth of pathogenic bacteria such as Clostridium perfringens, thereby improving the health status of chickens. However, from a microbial ecology perspective, the mechanisms by which alterations to the gut microbiota improve health remain unknown. In this study, we used Illumina MiSeq sequencing to investigate the cecal microbiota of a negative control group (NC), a C. perfringens and Eimeria challenge group with fishmeal supplementation (PC), a group supplemented with fishmeal and infected with coccidia (FC), and group PC with B. licheniformis supplementation (BL). We found that the health status of C. perfringens-challenged chickens was compromised, and that B. licheniformis improved the growth of the chickens challenged with pathogens. Microbial diversity analysis and taxonomic profiling of groups NC, PC, and FC revealed a disturbed cecal microflora of the birds with C. perfringens. We also characterized the microbiota of the chickens in the BL group using several methods. Principal coordinate analysis demonstrated that, compared with group PC, the bacterial community structure of group BL was more similar to that of group NC. Linear discriminant analysis with effect size revealed less differentially represented bacterial taxa between groups BL and NC than between groups PC and NC. In addition, groups BL and NC appeared to have similar overrepresented microbial taxa (such as Bacteroides, Helicobacter, Megamonas, and Akkermansia) compared with group PC. Finally, a phylogenetic investigation of communities by reconstruction of unobserved states analysis indicated that large differences existed between group PC and groups NC and BL. In conclusion, pre-treatment with B. licheniformis reduced the disturbance of the cecal microbiome induced by challenge with C. perfringens and other factors in broiler

Disruption in the cecal microbiota of chickens challenged with Clostridium perfringens and other factors was alleviated by Bacillus licheniformis supplementation

PubMed Central

Ni, Xueqin; Zhou, Mengjia; Zeng, Yan; Wang, Hesong; Zhou, Yi; Zhu, Hui; Pan, Kangcheng; Li, Guangyao

2017-01-01

Clostridium perfringens can induce necrotic enteritis of chickens, which causes large economic losses every year. Bacillus licheniformis, a probiotic, can inhibit the growth of pathogenic bacteria such as Clostridium perfringens, thereby improving the health status of chickens. However, from a microbial ecology perspective, the mechanisms by which alterations to the gut microbiota improve health remain unknown. In this study, we used Illumina MiSeq sequencing to investigate the cecal microbiota of a negative control group (NC), a C. perfringens and Eimeria challenge group with fishmeal supplementation (PC), a group supplemented with fishmeal and infected with coccidia (FC), and group PC with B. licheniformis supplementation (BL). We found that the health status of C. perfringens-challenged chickens was compromised, and that B. licheniformis improved the growth of the chickens challenged with pathogens. Microbial diversity analysis and taxonomic profiling of groups NC, PC, and FC revealed a disturbed cecal microflora of the birds with C. perfringens. We also characterized the microbiota of the chickens in the BL group using several methods. Principal coordinate analysis demonstrated that, compared with group PC, the bacterial community structure of group BL was more similar to that of group NC. Linear discriminant analysis with effect size revealed less differentially represented bacterial taxa between groups BL and NC than between groups PC and NC. In addition, groups BL and NC appeared to have similar overrepresented microbial taxa (such as Bacteroides, Helicobacter, Megamonas, and Akkermansia) compared with group PC. Finally, a phylogenetic investigation of communities by reconstruction of unobserved states analysis indicated that large differences existed between group PC and groups NC and BL. In conclusion, pre-treatment with B. licheniformis reduced the disturbance of the cecal microbiome induced by challenge with C. perfringens and other factors in broiler

In vitro antimicrobial effect of Satureja wiedemanniana against Bacillus species isolated from raw meat samples.

PubMed

Yucel, Nihal; Aslim, Belma; Ozdoğan, Hakan

2009-08-01

In this study a total of 30 raw meat samples obtained from Ankara, Turkey were screened for the presence of Bacillus species. Among the meat samples analyzed, the predominant species isolated was Bacillus circulans; other Bacillus species were identified as Bacillus firmus, Bacillus lentus, Bacillus megaterium, Bacillus licheniformis, Bacillus mycoides, Bacillus sphaericus, and Bacillus cereus. Minced meat samples were more contaminated with Bacillus species than sliced beef sample. From these samples, 242 Bacillus species isolates were obtained, which were investigated for proteolytic and lipolytic activity, associated with meat spoilage. Interestingly, some Bacillus strains produced the highest values of proteolytic/lipolytic activities. Nineteen Bacillus strains were selected among the 242 isolates according to their proteolytic/lipolytic activity with a clear zone diameter of > or =6 mm. The essential oil of Satureja wiedemanniana (Lalem) Velen was also tested against these 19 Bacillus species that had proteolytic and lipolytic activity. The essential oil yield obtained from the aerial parts of the plant was 0.35% (vol/wt). The inhibition zones of the essential oil obtained against all the Bacillus species were in the range of 5.0-12.0 mm. The oil showed high antimicrobial activities against B. licheniformis M 6(26), M 11(16), and M 12(1) strains. B. licheniformis 12(1) showed high lipolytic activity (18.0 mm). Also, B. licheniformis M 6(26) and M 11(16) showed high proteolytic activity (16.0 and 14.0 mm). These results may suggest that an essential oil of S. wiedemanniana can be used as a natural preservative in meat against spoilage bacteria.

Sociodemographic Risk, Parenting, and Effortful Control: Relations to Salivary Alpha-amylase and Cortisol in Early Childhood

PubMed Central

Taylor, Zoe E.; Spinrad, Tracy L.; VanSchyndel, Sarah K.; Eisenberg, Nancy; Huynh, Jacqueline; Sulik, Michael J.; Granger, Douglas A.

2012-01-01

Early sociodemographic risk, parenting, and temperament were examined as predictors of the activity of children’s (N = 148; 81 boys, 67 girls) hypothalamic-pituitary-adrenal axis and autonomic nervous system. Demographic risk was assessed at 18 months (T1), intrusive-overcontrolling parenting and effortful control were assessed at 30 months (T2), and salivary cortisol and alpha-amylase were collected at 72 (T3) months of age. Demographic risk at T1 predicted lower levels of children’s effortful control and higher levels of mothers’ intrusive-overcontrolling parenting at T2. Intrusive-overcontrolling parenting at T2 predicted higher levels of children’s cortisol and alpha-amylase at T3, but effortful control did not uniquely predict children’s cortisol or alpha-amylase. Findings support the open nature of stress responsive physiological systems to influence by features of the early caregiving environment and underscore the utility of including measures of these systems in prevention trials designed to influence child outcomes by modifying parenting behavior. PMID:22949301

Permissive role of the acidification caused by wheat aleurone layers upon. alpha. -amylase induction by GA sub 3

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

Rodriguez-Campos, E.; Bernal-Lugo, I.; Hamabata, A.

1989-04-01

Wheat aleurone has the capacity of acidifying the incubation medium in 1 to 2 pH units. The {alpha}-amylase induction by GA{sub 3} in isolated wheat aleurone layers is strongly dependent on acidic pH of the medium (pH < 5). To examine possible mechanisms {sup 35}-Met incorporation into proteins and {alpha}-amylase, in the presence of GA{sub 3} and Ca{sup 2+} at pH, 4, 5 and 6 was studied. Although {sup 35}-Met uptake decreased markedly ({approx} 90%) at pH 4 in thepresence of GA{sub 3}, incorporation into total protein did not change significantly from other conditions. Auto-radiography of SDS-PAGE showed that mostmore » of the amino acid was in the {alpha}-amylase band, meaning that the effect of acidic pH is specific for GA{sub 3} actions on aleurone tissue. On the other hand, an increase of protonated GA{sub 3} diffusion could be ruled out. Also, there was not {alpha}-amylase inactivation at pH 6. These findings point out to the important physiological role of the acidification caused by the aleurone.« less

Analysis of the extreme diversity of salivary alpha-amylase isoforms generated by physiological proteolysis using liquid chromatography-tandem mass spectrometry.

PubMed

Bailey, Ulla-Maja; Punyadeera, Chamindie; Cooper-White, Justin J; Schulz, Benjamin L

2012-12-12

Saliva is a crucial biofluid for oral health and is also of increasing importance as a non-invasive source of disease biomarkers. Salivary alpha-amylase is an abundant protein in saliva, and changes in amylase expression have been previously associated with a variety of diseases and conditions. Salivary alpha-amylase is subject to a high diversity of post-translational modifications, including physiological proteolysis in the oral cavity. Here we developed methodology for rapid sample preparation and non-targeted LC-ESI-MS/MS analysis of saliva from healthy subjects and observed an extreme diversity of alpha-amylase proteolytic isoforms. Our results emphasize the importance of consideration of post-translational events such as proteolysis in proteomic studies, biomarker discovery and validation, particularly in saliva. Copyright © 2012 Elsevier B.V. All rights reserved.

Cloning, molecular characterization and heterologous expression of AMY1, an alpha-amylase gene from Cryptococcus flavus.

PubMed

Galdino, Alexsandro S; Ulhoa, Cirano J; Moraes, Lídia Maria P; Prates, Maura V; Bloch, Carlos; Torres, Fernando A G

2008-03-01

A Cryptococcus flavus gene (AMY1) encoding an extracellular alpha-amylase has been cloned. The nucleotide sequence of the cDNA revealed an ORF of 1896 bp encoding for a 631 amino acid polypeptide with high sequence identity with a homologous protein isolated from Cryptococcus sp. S-2. The presence of four conserved signature regions, (I) (144)DVVVNH(149), (II) (235)GLRIDSLQQ(243), (III) (263)GEVFN(267), (IV) (327)FLENQD(332), placed the enzyme in the GH13 alpha-amylase family. Furthermore, sequence comparison suggests that the C. flavusalpha-amylase has a C-terminal starch-binding domain characteristic of the CBM20 family. AMY1 was successfully expressed in Saccharomyces cerevisiae. The time course of amylase secretion in S. cerevisiae resulted in a maximal extracellular amylolytic activity (3.93 U mL(-1)) at 60 h of incubation. The recombinant protein had an apparent molecular mass similar to the native enzyme (c. 67 kDa), part of which was due to N-glycosylation.

Highly thermostable, thermophilic, alkaline, SDS and chelator resistant amylase from a thermophilic Bacillus sp. isolate A3-15.

PubMed

Arikan, Burhan

2008-05-01

A thermostable alkaline alpha-amylase producing Bacillus sp. A3-15 was isolated from compost samples. There was a slight variation in amylase synthesis within the pH range 6.0 and 12.0 with an optimum pH of 8.5 (8mm zone diameter in agar medium) on starch agar medium. Analyses of the enzyme for molecular mass and amylolytic activity were carried out by starch SDS-PAGE electrophoresis, which revealed two independent bands (86,000 and 60,500 Da). Enzyme synthesis occurred at temperatures between 25 and 65 degrees C with an optimum of 60 degrees C on petri dishes. The partial purification enzyme showed optimum activity at pH 11.0 and 70 degrees C. The enzyme was highly active (95%) in alkaline range of pH (10.0-11.5), and it was almost completely active up to 100 degrees C with 96% of the original activity remaining after heat treatment at 100 degrees C for 30 min. Enzyme activity was enhanced in the presence of 5mM CaCl2 (130%) and inhibition with 5mM by ZnCl2, NaCl, Na-sulphide, EDTA, PMSF (3mM), Urea (8M) and SDS (1%) was obtained 18%, 20%, 36%, 5%, 10%, 80% and 18%, respectively. The enzyme was stable approximately 70% at pH 10.0-11.0 and 60 degrees C for 24h. So our result showed that the enzyme was both, highly thermostable-alkaline, thermophile and chelator resistant. The A3-15 amylase enzyme may be suitable in liquefaction of starch in high temperature, in detergent and textile industries and in other industrial applications.

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.

Determinants of wheat antigen and fungal alpha-amylase exposure in bakeries.

PubMed

Burstyn, I; Teschke, K; Bartlett, K; Kennedy, S M

1998-05-01

The study's objectives were to measure flour antigen exposure in bakeries and define the determinants of exposure. Ninety-six bakery workers, employed in seven different bakeries, participated in the study. Two side-by-side full-shift inhalable dust samples were obtained from each study participant on a single occasion. The flour antigen exposure was measured as wheat antigen and fungal alpha-amylase content of the water-soluble fraction of inhalable dust, assayed via enzyme-linked immunosorbent assays. During the entire sampling period bakers were observed and information on 14 different tasks was recorded at 15-minute intervals. Other production characteristics were also recorded for each sampling day and used in statistical modeling to identify significant predictors of exposure. The mean alpha-amylase antigen exposure was 22.0 ng/m3 (ranging from below the limit of detection of 0.1 ng/m3 to 307.1 ng/m3) and the mean wheat antigen exposure was 109 micrograms/m3 (ranging from below the limit of detection of 1 microgram/m3 to 1018 micrograms/m3). Regression models that explained 74% of variability in wheat antigen and alpha-amylase antigen exposures were constructed. The models indicated that tasks such as weighing, pouring, and operating dough-brakers increased flour antigen exposure, while packing and decorating resulted in lower exposures. Croissant, puff-pastry, and bread/bun production lines were associated with increased exposure, while cake production and substitution of dusting with the use of divider oil were associated with decreased exposure. Exposure levels can be reduced by the automation of forming tasks, alteration of tasks requiring pouring of flour, and changes to the types of products manufactured.

Induction of Osmoadaptive Mechanisms and Modulation of Cellular Physiology Help Bacillus licheniformis Strain SSA 61 Adapt to Salt Stress

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

Paul, Sangeeta; Aggarwal, Chetana; Thakur, Jyoti Kumar

Bacillus licheniformis strain SSA 61, originally isolated from Sambhar salt lake, was observed to grow even in the presence of 25 % salt stress. Osmoadaptive mechanisms of this halotolerant B. licheniformis strain SSA 61, for long-term survival and growth under salt stress, were determined. Proline was the preferentially accumulated compatible osmolyte. There was also increased accumulation of antioxidants ascorbic acid and glutathione. Among the different antioxidative enzymes assayed, superoxide dismutase played the most crucial role in defense against salt-induced stress in the organism. Adaptation to stress by the organism involved modulation of cellular physiology at various levels. There was enhancedmore » expression of known proteins playing essential roles in stress adaptation, such as chaperones DnaK and GroEL, and general stress protein YfkM and polynucleotide phosphorylase/polyadenylase. Proteins involved in amino acid biosynthetic pathway, ribosome structure, and peptide elongation were also overexpressed. Salt stress-induced modulation of expression of enzymes involved in carbon metabolism was observed. There was up-regulation of a number of enzymes involved in generation of NADH and NADPH, indicating increased cellular demand for both energy and reducing power.« less

Taxonomy of the Streptomyces strain ZG0656 that produces acarviostatin alpha-amylase inhibitors and analysis of their effects on blood glucose levels in mammalian systems.

PubMed

Geng, P; Bai, G; Shi, Q; Zhang, L; Gao, Z; Zhang, Q

2009-02-01

To clarify the taxonomic status of strain ZG0656 and analyse the effects of its acarviostatin products on blood glucose levels in mammalian systems. Our program to screen for new alpha-amylase inhibitors led to the isolation of strain ZG0656. The polyphasic taxonomic study revealed that strain ZG0656 represents a novel variation of Streptomyces coelicoflavus, for which we propose the name S. coelicoflavus var. nankaiensis. Four chemically distinct alpha-amylase inhibitors, acarviostatins I03, II03, III03 and IV03, were isolated from strain ZG0656. Acarviostatins III03 and IV03 are both novel oligomers. All four acarviostatins are mixed noncompetitive porcine pancreas alpha-amylase inhibitors. Acarviostatin III03 is the most potent alpha-amylase inhibitor known to date. Moreover, in the in vitro and in vivo experiments, acarviostatins III03 showed significant inhibition of starch hydrolysis and glucose transfer to blood. Strain ZG0656 is a novel variation of S. coelicoflavus, whose products are novel effective alpha-amylase inhibitors. Among the products, acarviostatins III03 could significantly depress blood glucose levels in mammalian systems and be developed towards a possible therapeutic agent for diabetes. Acarviostatin III03 is the most potent alpha-amylase inhibitor known to date. The oligomer will benefit the research on the relationship between alpha-amylase and various inhibitors and will offer more choices in diabetes treatments.

Quantitative digital image analysis of chromogenic assays for high throughput screening of alpha-amylase mutant libraries.

PubMed

Shankar, Manoharan; Priyadharshini, Ramachandran; Gunasekaran, Paramasamy

2009-08-01

An image analysis-based method for high throughput screening of an alpha-amylase mutant library using chromogenic assays was developed. Assays were performed in microplates and high resolution images of the assay plates were read using the Virtual Microplate Reader (VMR) script to quantify the concentration of the chromogen. This method is fast and sensitive in quantifying 0.025-0.3 mg starch/ml as well as 0.05-0.75 mg glucose/ml. It was also an effective screening method for improved alpha-amylase activity with a coefficient of variance of 18%.

Structure, function, and fate of the BlaR signal transducer involved in induction of beta-lactamase in Bacillus licheniformis.

PubMed Central

Zhu, Y; Englebert, S; Joris, B; Ghuysen, J M; Kobayashi, T; Lampen, J O

1992-01-01

The membrane-spanning protein BlaR is essential for the induction of beta-lactamase in Bacillus licheniformis. Its nature and location were confirmed by the use of an antiserum specific for its carboxy-terminal penicillin sensor, its function was studied by genetic dissection, and the structure of the penicillin sensor was derived from hydrophobic cluster analysis of the amino acid sequence by using, as a reference, the class A beta-lactamases with known three-dimensional structures. During the first 2 h after the addition of the beta-lactam inducer, full-size BlaR, bound to the plasma membrane, is produced, and then beta-lactamase is produced. By 2 h after induction, BlaR is present in various (membrane-bound and cytosolic) forms, and there is a gradual decrease in beta-lactamase production. The penicillin sensors of BlaR and the class D beta-lactamases show strong similarities in primary structures. They appear to have the same basic spatial disposition of secondary structures as that of the class A beta-lactamases, except that they lack several alpha helices and, therefore, have a partially uncovered five-stranded beta sheet and a more readily accessible active site. Alterations of BlaR affecting conserved secondary structures of the penicillin sensor and specific sites of the transducer annihilate beta-lactamase inducibility. Images PMID:1400165

Alpha-Amylase Inhibition and Antioxidative Capacity of Some Antidiabetic Plants Used by the Traditional Healers in Southeastern Nigeria

PubMed Central

Oyedemi, Blessing O.; Ijeh, Ifeoma I.; Ohanyerem, Princemartins E.; Aiyegoro, Olayinka A.

2017-01-01

Oxidative stress plays a significant role in the pathogenesis of metabolic syndrome including diabetes mellitus (DM). The inhibition of alpha-amylase is an important therapeutic target in the regulation of postprandial increase of blood glucose in diabetic patients. The present study investigated the alpha-amylase inhibitory and antioxidant potential of selected herbal drugs used in the treatment of DM by the traditional healers in Isiala Mbano and Ikwuano regions of southeastern Nigeria. Antioxidant activity was evaluated in terms of free radical scavenging, reducing power, and total phenolic (TPC) and flavonoid content (TFC) in consonance with the TLC profiling. The results showed that methanol crude extracts from Anacardium occidentale (AO) and Ceiba pentandra (CP) recorded higher TPC and TFC, potent free radical scavenging, and efficient reducing power (RP) as compared with other plant samples. All the plant extracts exhibited a relative alpha-amylase inhibition apart from Strophanthus hispidus (SH) extract with a negative effect. We discovered a mild to weak correlation between alpha-amylase inhibition or antioxidative capacity and the total phenol or flavonoid content. At least in part, the results obtained in this work support the traditional use of certain plant species in the treatment of patients with DM. PMID:28367491

Characterization of Lipopeptide Biosurfactants Produced by Bacillus licheniformis MB01 from Marine Sediments

PubMed Central

Chen, Yulin; Liu, Shiliang A.; Mou, Haijin; Ma, Yunxiao; Li, Meng; Hu, Xiaoke

2017-01-01

Antibiotic resistance has become one of the world’s most severe problems because of the overuse of antibiotics. Antibiotic-resistant bacteria are more difficult to kill and more expensive to treat. Researchers have been studied on antibiotic alternatives such as antimicrobial peptides and lipopeptides. A functional bacteria MB01 producing lipopeptides which can be used as bacteriostat was isolated from the Bohai Sea sediments, which had been identified as Bacillus licheniformis by the morphological, physiological, and biochemical identification and 16s rDNA sequence. The lipopeptides produced by MB01 were determined to be cyclic surfactin homologs by LC-ESI-MS structural identification after crude extraction and LH-20 chromatography. [M+H]+ m/z 994, 1008, 1022, and 1036 were all the characteristic molecular weight of surfactin homologs. CID analysis revealed that the molecular structure of the lipopeptides was Rn-Glu1-Leu/Ile2-Leu3-Val4-Asp5-Leu6-Leu/Ile7. The lipopeptides showed well resistance to UV light and the change of pH and temperature. PMID:28559889

Enhanced Production of Poly-γ-glutamic Acid by Bacillus licheniformis TISTR 1010 with Environmental Controls.

PubMed

Kongklom, Nuttawut; Shi, Zhongping; Chisti, Yusuf; Sirisansaneeyakul, Sarote

2017-07-01

Bacillus licheniformis TISTR 1010 was used for glutamic acid-independent production of poly-γ-glutamic acid (γ-PGA). A fed-batch production strategy was developed involving feedings of glucose, citric acid, and ammonium chloride at specified stages of the fermentation. With the dissolved oxygen concentration controlled at ≥50% of air saturation and the pH controlled at ~7.4, the fed-batch operation at 37 °C afforded a peak γ-PGA concentration of 39.9 ± 0.3 g L -1 with a productivity of 0.926 ± 0.006 g L -1  h -1 . The observed productivity was nearly threefold greater than previously reported for glutamic acid-independent production using the strain TISTR 1010. The molecular weight of γ-PGA was in the approximate range of 60 to 135 kDa.

A hyper-thermostable α-amylase from Pyrococcus furiosus accumulates in Nicotiana tabacum as functional aggregates.

PubMed

Zhu, Hong; Reynolds, L Bruce; Menassa, Rima

2017-06-19

Alpha amylase hydrolyzes α-bonds of polysaccharides such as starch and produces malto-oligosaccharides. Its starch saccharification applications make it an essential enzyme in the textile, food and brewing industries. Commercially available α-amylase is mostly produced from Bacillus or Aspergillus. A hyper-thermostable and Ca 2++ independent α-amylase from Pyrococcus furiosus (PFA) expressed in E.coli forms insoluble inclusion bodies and thus is not feasible for industrial applications. We expressed PFA in Nicotiana tabacum and found that plant-produced PFA forms functional aggregates with an accumulation level up to 3.4 g/kg FW (fresh weight) in field conditions. The aggregates are functional without requiring refolding and therefore have potential to be applied as homogenized plant tissue without extraction or purification. PFA can also be extracted from plant tissue upon dissolution in a mild reducing buffer containing SDS. Like the enzyme produced in P. furiosus and in E. coli, plant produced PFA preserves hyper-thermophilicity and hyper-thermostability and has a long shelf life when stored in lyophilized leaf tissue. With tobacco's large biomass and high yield, hyper-thermostable α-amylase was produced at a scale of 42 kg per hectare. Tobacco may be a suitable bioreactor for industrial production of active hyperthermostable alpha amylase.

Branched chain amino acids maintain the molecular weight of poly(γ-glutamic acid) of Bacillus licheniformis ATCC 9945 during the fermentation.

PubMed

Mitsunaga, Hitoshi; Meissner, Lena; Büchs, Jochen; Fukusaki, Eiichiro

2016-10-01

Poly(γ-glutamic acid) mainly produced by Bacillus spp. is an industrially important compound due to several useful features. Among them, molecular weight is an important characteristic affecting on the physical properties such as viscosities and negative charge densities. However, it is difficult to control the molecular size of PGA since it decreases during fermentation. Previous study reported that PGA produced in the media containing different carbon sources such as glucose and glycerol showed differences in molecular weight. Therefore in this study, the effect of carbon source on the PGA molecular weight was examined; with the aim of developing a strategy to maintain the high molecular weight of PGA during fermentation. Our result showed that the weight average molecular weight (Mw) of PGA of Bacillus licheniformis ATCC 9945 cultivated in the media containing PTS-sugars were higher than the medium containing glycerol (non-PTS). The result of metabolome analysis indicated the possibility of CodY (a global regulator protein) activation in the cells cultivated in the media containing PTS-sugars. To mimic this effect, branched-chain amino acids (BCAAs), which are activators of CodY, were added to a medium containing glycerol. As the result, the Mw of PGA in the BCAAs-supplemented media were maintained and high during the early production phase compared to the non BCAAs-supplemented medium. These results indicate that BCAAs can repress the PGA molecular weight reduction during fermentation in B. licheniformis ATCC 9945. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Influence of nitrogen source and pH value on undesired poly(γ-glutamic acid) formation of a protease producing Bacillus licheniformis strain.

PubMed

Meissner, Lena; Kauffmann, Kira; Wengeler, Timo; Mitsunaga, Hitoshi; Fukusaki, Eiichiro; Büchs, Jochen

2015-09-01

Bacillus spp. are used for the production of industrial enzymes but are also known to be capable of producing biopolymers such as poly(γ-glutamic acid). Biopolymers increase the viscosity of the fermentation broth, thereby impairing mixing, gas/liquid mass and heat transfer in any bioreactor system. Undesired biopolymer formation has a significant impact on the fermentation and downstream processing performance. This study shows how undesirable poly(γ-glutamic acid) formation of an industrial protease producing Bacillus licheniformis strain was prevented by switching the nitrogen source from ammonium to nitrate. The viscosity was reduced from 32 to 2.5 mPa s. A constant or changing pH value did not influence the poly(γ-glutamic acid) production. Protease production was not affected: protease activities of 38 and 46 U mL(-1) were obtained for ammonium and nitrate, respectively. With the presented results, protease production with industrial Bacillus strains is now possible without the negative impact on fermentation and downstream processing by undesired poly(γ-glutamic acid) formation.

[Activity of alpha-amylase and concentration of protein in saliva of pregnant women].

PubMed

Ciejak, Magdalena; Olszewska, Maria; Jakubowska, Katarzyna; Zebiełowicz, Dariusz; Safranow, Krzysztof; Chlubek, Dariusz

2007-01-01

One of the hypothetical reasons of the increased incidence of caries in women during the pregnancy may be the increased activity of alpha-amylase, which can be found in their saliva. The enzyme takes part in the process of decomposition of simple sugars, which make basic substrate for caries-causing bacteria. The aim of the paper was the evaluation of the influence of pregnancy and gestational age on the activity of alpha-amylase and the concentration of protein in women's saliva. The examined group consisted of 64 pregnant women at age 17-39, between 21st and 40th week of pregnancy. The control group consisted of 44 healthy women at age 20-35, who were not pregnant. In saliva, which was taken before morning meal, without stimulation, protein concentration was determined by Bradford method and the activity of amylase was determined by kinetic method. The activity of amylase correlated strongly and positively with protein concentration in saliva of both the pregnant (RS = +0.65; p < 0.00001) and the control group (RS = +0.74; p < 0.00001) women. There were no significant differences between examined parameters in the examined and the control group. It has been observed in the examined group, that there is the significant negative correlation between protein concentration in saliva and the week of pregnancy (RS = -0.35; p <0.01). It has been observed, in conducted researches, that there is no relation between the activity of amylase and the pregnancy and gestational age, which proves against the essential role of this enzyme in the increased caries incidence of pregnant women. However, the observed changes of total protein concentration in saliva during pregnancy, suggest that the exact cognition of proteins in pregnant women's saliva may reveal new mechanisms, which lead to an increase of caries risk.

Subsite mapping of enzymes. Application of the depolymerase computer model to two alpha-amylases.

PubMed Central

Allen, J D; Thoma, J A

1976-01-01

In the preceding paper (Allen and Thoma, 1976) we developed a depolymerase computer model, which uses a minimization routine to establish a subsite map for a depolymerase. In the present paper we show how the model is applied to experimental data for two alpha-amylases. Michaelis parameters and bond-cleavage frequencies for substrates of chain lengths up to twelve glucosyl units have been reported for Bacillus amyloliquefaciens, and a subsite map has been proposed for this enzyme [Thoma et al. (1971) J. Biol. Chem. 246, 5621-5635]. By applying the computer model to the experimental data, we have arrived at a ten-subsite map. We find that a significant improvement in this map is achieved by allowing the hydrolytic rate coefficient to vary as a function of the number of occupied subsites comprising the enzyme-binding region. The bond-cleavage frequencies, the enzyme is found to have eight subsites. A partial subsite map is arrived at, but the entire binding region cannot be mapped because Michaelis parameters are complicated by transglycosylation reactions. The hydrolytic rate coefficients for this enzyme are not constant. PMID:999630

Efficient recombinant expression and secretion of a thermostable GH26 mannan endo-1,4-beta-mannosidase from Bacillus licheniformis in Escherichia coli.

PubMed

Songsiriritthigul, Chomphunuch; Buranabanyat, Bancha; Haltrich, Dietmar; Yamabhai, Montarop

2010-04-11

Mannans are one of the key polymers in hemicellulose, a major component of lignocellulose. The Mannan endo-1,4-beta-mannosidase or 1,4-beta-D-mannanase (EC 3.2.1.78), commonly named beta-mannanase, is an enzyme that can catalyze random hydrolysis of beta-1,4-mannosidic linkages in the main chain of mannans, glucomannans and galactomannans. The enzyme has found a number of applications in different industries, including food, feed, pharmaceutical, pulp/paper industries, as well as gas well stimulation and pretreatment of lignocellulosic biomass for the production of second generation biofuel. Bacillus licheniformis is a Gram-positive endospore-forming microorganism that is generally non-pathogenic and has been used extensively for large-scale industrial production of various enzymes; however, there has been no previous report on the cloning and expression of mannan endo-1,4-beta-mannosidase gene (manB) from B. licheniformis. The mannan endo-1,4-beta-mannosidase gene (manB), commonly known as beta-mannanase, from Bacillus licheniformis strain DSM13 was cloned and overexpressed in Escherichia coli. The enzyme can be harvested from the cell lysate, periplasmic extract, or culture supernatant when using the pFLAG expression system. A total activity of approximately 50,000 units could be obtained from 1-l shake flask cultures. The recombinant enzyme was 6 x His-tagged at its C-terminus, and could be purified by one-step immobilized metal affinity chromatography (IMAC) to apparent homogeneity. The specific activity of the purified enzyme when using locust bean gum as substrate was 1672 +/- 96 units/mg. The optimal pH of the enzyme was between pH 6.0 - 7.0; whereas the optimal temperature was at 50 - 60 degrees C. The recombinant beta-mannanase was stable within pH 5 - 12 after incubation for 30 min at 50 degrees C, and within pH 6 - 9 after incubation at 50 degrees C for 24 h. The enzyme was stable at temperatures up to 50 degrees C with a half-life time of activity (tau1

Cortisol, salivary alpha-amylase and children's perceptions of their social networks.

PubMed

Ponzi, Davide; Muehlenbein, Michael P; Geary, David C; Flinn, Mark V

2016-01-01

In recent years there has been a growing interest in the use of social network analysis in biobehavioral research. Despite the well-established importance of social relationships in influencing human behavior and health, little is known about how children's perception of their immediate social relationships correlates with biological parameters of stress. In this study we explore the association between two measures of children's personal social networks, perceived network size and perceived network density, with two biomarkers of stress, cortisol and salivary alpha-amylase. Forty children (mean age = 8.30, min age = 5, and max age = 12) were interviewed to collect information about their friendships and three samples of saliva were collected. Our results show that children characterized by a lower pre-interview cortisol concentration and a lower salivary alpha-amylase reactivity to the interview reported the highest density of friendships. We discuss this result in light of the multisystem approach to the study of children's behavioral outcomes, emphasizing that future work of this kind is needed in order to understand the cognitive and biological mechanisms underlying children's and adolescents' social perceptual biases.

In situ analysis of Bacillus licheniformis biofilms: amyloid-like polymers and eDNA are involved in the adherence and aggregation of the extracellular matrix.

PubMed

Randrianjatovo-Gbalou, I; Rouquette, P; Lefebvre, D; Girbal-Neuhauser, E; Marcato-Romain, C-E

2017-05-01

This study attempts to determine which of the exopolymeric substances are involved in the adherence and aggregation of a Bacillus licheniformis biofilm. The involvement of extracellular proteins and eDNA were particularly investigated using DNase and proteinase K treatment. The permeability of the biofilms increased fivefold after DNase I treatment. The quantification of the matrix components showed that, irrespective to the enzyme tested, eDNA and amyloid-like polymers were removed simultaneously. Size-exclusion chromatography analyses supported these observations and revealed the presence of associated nucleic acid and protein complexes in the biofilm lysates. These data suggest that some extracellular DNA and amyloid-like proteins were closely interlaced within the matrix. Finally, confocal laser scanning microscopy imaging gave supplementary clues about the 3D organization of the biofilms, confirming that eDNA and exoproteins were essentially layered under and around the bacterial cells, whereas the amyloid-like fractions were homogeneously distributed within the matrix. These results confirm that some DNA-amyloid complexes play a key role in the modulation of the mechanical resistance of B. licheniformis biofilms. The study highlights the need to consider the whole structure of biofilms and to target the interactions between matrix components. A better understanding of B. licheniformis biofilm physiology and the structural organization of the matrix will strengthen strategies of biofilm control. © 2017 The Society for Applied Microbiology.

Potential of the bean alpha-amylase inhibitor alpha-AI-1 to inhibit alpha-amylase activity in true bugs(Hemiptera)

USDA-ARS?s Scientific Manuscript database

True bugs (Hemiptera) are an important pest complex not controlled by Bt crops. An alternative source of resistance includes inhibitors of digestive enzymes. aAI-1, an a-amylase inhibitor from the common bean, has been shown to inhibit a-amylases of bruchid pests of grain legumes. Here we quantify t...

Amelioration in secretion of hyperthermostable and Ca2+ -independent alpha-amylase of Geobacillus thermoleovorans by some polyamines and their biosynthesis inhibitor methylglyoxal-bis-guanylhydrazone.

PubMed

Uma Maheswar Rao, J L; Satyanarayana, T

2004-01-01

Effect of polyamines and their biosynthesis inhibitors on the production of hyperthermostable and Ca2+ -independent alpha-amylase by Geobacillus thermoleovorans MTCC 4220. The alpha-amylase was produced in starch-yeast extract-tryptone (SYT) broth with different polyamines (PA) and polyamine biosynthesis inhibitors, methylglyoxal-bis-guanylhydrazone (MGBG) and cyclohexylammonium sulphate (CHA) at 70 degrees C. The bacterial pellets were obtained after growing G. thermoleovorans at different temperatures, and used in determining total PA. The cell-free culture filtrates were used in alpha-amylase assays. During growth, total polyamines in biomass increased till 2 h, and thereafter, decreased gradually. The total polyamine content was very high in the biomass cultivated at 55 degrees C when compared with that of higher temperatures. Enzyme titre enhanced up to 70 degrees C, and thereafter declined. Extracellular enzyme and protein levels declined in the presence of exogenously added PA. The intracellular enzyme titres, however, were higher in putrescine (put) and spermidine (spd) than in spermine (spm). Polyamine biosynthesis inhibitor, MGBG enhanced secretion of alpha-amylase in a laboratory fermentor as well as shake flasks, although CHA did not affect it. The intracellular accumulation of put in the presence of MGBG appeared to enhance synthesis and secretion of alpha-amylase. Extracellular enzyme and protein levels were low in the presence of exogenously added PA, but their intracellular levels, however, were higher in put and spd than in spm. A substantial increase in the synthesis and secretion of alpha-amylase was attained in G. thermoleovorans in the presence of polyamine biosynthesis inhibitor MGBG.

Amylase production potentials of bacterial isolates obtained from the gut of Oryctes rhinoceros larvae

NASA Astrophysics Data System (ADS)

Aryati, P. C.; Pangastuti, A.; Sari, S. L. A.

2017-04-01

Amylase is one of the main enzymes used in industry, such as food, detergent, textile, and pharmaceutical industry. Amylase can be produced by plants, animals, and microorganisms. However, bacterial and fungal amylases have dominated application in industries. This research was aimed to determine amylolytic activity of bacteria isolated from the gut of Oryctes rhinoceros larvae. Based on clear zone formation, 9 from 11 isolates showed amylolytic activity. Isolates with the widest clear zone, i.e Bacillus subtilis GOR1, Bacillus cereus GOR3, and Bacillus pumilus GOR2, were screened for amylolytic activity based on reduction sugar production. The result showed that Bacillus subtilis GOR1 was the most potential as amylase producer, showed by the widest clear zone 5.224 cm2 and highest reduction sugar production 0.0235 mg/ml. Highest amylase specific activity (0.1447 U/mg protein) was obtained at 60°C and pH 7. Amylase activity was stable for 3 hours at 60°C with residual activity respectively was 59.7%.

The spectrum of low molecular weight alpha-amylase/protease inhibitor genes expressed in the US bread wheat cultivar Butte 86

PubMed Central

2011-01-01

Background Wheat grains accumulate a variety of low molecular weight proteins that are inhibitors of alpha-amylases and proteases and play an important protective role in the grain. These proteins have more balanced amino acid compositions than the major wheat gluten proteins and contribute important reserves for both seedling growth and human nutrition. The alpha-amylase/protease inhibitors also are of interest because they cause IgE-mediated occupational and food allergies and thereby impact human health. Results The complement of genes encoding alpha-amylase/protease inhibitors expressed in the US bread wheat Butte 86 was characterized by analysis of expressed sequence tags (ESTs). Coding sequences for 19 distinct proteins were identified. These included two monomeric (WMAI), four dimeric (WDAI), and six tetrameric (WTAI) inhibitors of exogenous alpha-amylases, two inhibitors of endogenous alpha-amylases (WASI), four putative trypsin inhibitors (CMx and WTI), and one putative chymotrypsin inhibitor (WCI). A number of the encoded proteins were identical or very similar to proteins in the NCBI database. Sequences not reported previously included variants of WTAI-CM3, three CMx inhibitors and WTI. Within the WDAI group, two different genes encoded the same mature protein. Based on numbers of ESTs, transcripts for WTAI-CM3 Bu-1, WMAI Bu-1 and WTAI-CM16 Bu-1 were most abundant in Butte 86 developing grain. Coding sequences for 16 of the inhibitors were unequivocally associated with specific proteins identified by tandem mass spectrometry (MS/MS) in a previous proteomic analysis of milled white flour from Butte 86. Proteins corresponding to WDAI Bu-1/Bu-2, WMAI Bu-1 and the WTAI subunits CM2 Bu-1, CM3 Bu-1 and CM16 Bu-1 were accumulated to the highest levels in flour. Conclusions Information on the spectrum of alpha-amylase/protease inhibitor genes and proteins expressed in a single wheat cultivar is central to understanding the importance of these proteins in both

Engineering Bacillus licheniformis as a thermophilic platform for the production of l-lactic acid from lignocellulose-derived sugars.

PubMed

Li, Chao; Gai, Zhongchao; Wang, Kai; Jin, Liping

2017-01-01

Bacillus licheniformis MW3 as a GRAS and thermophilic strain is a promising microorganism for chemical and biofuel production. However, its capacity to co-utilize glucose and xylose, the major sugars found in lignocellulosic biomass, is severely impaired by glucose-mediated carbon catabolite repression (CCR). In this study, a "dual-channel" process was implemented to engineer strain MW3 for simultaneous utilization of glucose and xylose, using l-lactic acid as a target product. A non-phosphotransferase system (PTS) glucose uptake route was activated via deletion of the glucose transporter gene ptsG and introduction of the galactose permease gene galP . After replacing the promoter of glucokinase gene glck with the strong promoter P als , the engineered strain recovered glucose consumption and utilized glucose and xylose simultaneously. Meanwhile, to improve the consumption rate of xylose in this strain, several measures were undertaken, such as relieving the regulation of the xylose repressor XylR, reducing the catabolite-responsive element, and optimizing the rate-limiting step. Knockout of ethanol and acetic acid pathway genes further increased lactic acid yield by 6.2%. The resultant strain, RH15, was capable of producing 121.9 g/L l-lactic acid at high yield (95.3%) after 40 h of fermentation from a mixture of glucose and xylose. When a lignocellulosic hydrolysate was used as the substrate, 99.3 g/L l-lactic acid was produced within 40 h, with a specific productivity of 2.48 g/[L h] and a yield of 94.6%. Our engineered strain B. licheniformis RH15 could thermophilically produced l-lactic acid from lignocellulosic hydrolysate with relatively high concentration and productivity at levels that were competitive with most reported cases of l-lactic acid-producers. Thus, the engineered strain might be used as a platform for the production of other chemicals. In addition to engineering the B. licheniformis strain, the "dual-channel" process might serve as an

Evaluation of various parameters of calcium-alginate immobilization method for enhanced alkaline protease production by Bacillus licheniformis NCIM-2042 using statistical methods.

PubMed

Potumarthi, Ravichandra; Subhakar, Ch; Pavani, A; Jetty, Annapurna

2008-04-01

Calcium-alginate immobilization method for the production of alkaline protease by Bacillus licheniformis NCIM-2042 was optimized statistically. Four variables, such as sodium-alginate concentration, calcium chloride concentration, inoculum size and agitation speed were optimized by 2(4) full factorial central composite design and subsequent analysis and model validation by a second-order regression equation. Eleven carbon, 11 organic nitrogen and seven inorganic nitrogen sources were screened by two-level Plackett-Burman design for maximum alkaline protease production by using optimized immobilized conditions. The levels of four variables, such as Na-alginate 2.78%; CaCl(2), 2.15%; inoculum size, 8.10% and agitation, 139 rpm were found to be optimum for maximal production of protease. Glucose, soybean meal and ammonium sulfate were resulted in maximum protease production at 644 U/ml, 720 U/ml, and 806 U/ml when screened for carbon, organic nitrogen and inorganic nitrogen sources, respectively, using optimized immobilization conditions. Repeated fed batch mode of operation, using optimized immobilized conditions, resulted in continuous operation for 12 cycles without disintegration of beads. Cross-sectional scanning electron microscope images have shown the growth pattern of B. licheniformis in Ca-alginate immobilized beads.

A chimeric α-amylase engineered from Bacillus acidicola and Geobacillus thermoleovorans with improved thermostability and catalytic efficiency.

PubMed

Parashar, Deepak; Satyanarayana, T

2016-04-01

The α-amylase (Ba-amy) of Bacillus acidicola was fused with DNA fragments encoding partial N- and C-terminal region of thermostable α-amylase gene of Geobacillus thermoleovorans (Gt-amy). The chimeric enzyme (Ba-Gt-amy) expressed in Escherichia coli displays marked increase in catalytic efficiency [K cat: 4 × 10(4) s(-1) and K cat/K m: 5 × 10(4) mL(-1) mg(-1) s(-1)] and higher thermostability than Ba-amy. The melting temperature (T m) of Ba-Gt-amy (73.8 °C) is also higher than Ba-amy (62 °C), and the CD spectrum analysis revealed the stability of the former, despite minor alteration in secondary structure. Langmuir-Hinshelwood kinetic analysis suggests that the adsorption of Ba-Gt-amy onto raw starch is more favourable than Ba-amy. Ba-Gt-amy is thus a suitable biocatalyst for raw starch saccharification at sub-gelatinization temperatures because of its acid stability, thermostability and Ca(2+) independence, and better than the other known bacterial acidic α-amylases.

Increasing the bioflocculant production and identifying the effect of overexpressing epsB on the synthesis of polysaccharide and γ-PGA in Bacillus licheniformis.

PubMed

Liu, Peize; Chen, Zhen; Yang, Lijie; Li, Qingbiao; He, Ning

2017-09-26

Polysaccharides and poly-γ-glutamic acid (γ-PGA) are biomacromolecules that have been reported as bioflocculants, and they exhibit high flocculating activity in many industrial applications. Bacillus licheniformis CGMCC 2876 can produce polysaccharide and γ-PGA bioflocculants under different culture conditions. Several key genes are involved in the metabolic pathway of polysaccharides in B. licheniformis, but the impacts of the regulation of these genes on the production of polysaccharide bioflocculants have not been illustrated completely. To increase the bioflocculant production and identify the correlation between the synthesis of polysaccharides and γ-PGA in B. licheniformis, a few key genes were investigated to explore their influence on the synthesis of the bioflocculants. Overexpressing epsB from the eps gene cluster not only improved the bioflocculant crude yield by 13.98% but also enhanced the flocculating activity by 117.92%. The composition of the bioflocculant from the epsB recombinant strain was 28.95% total sugar, 3.464% protein and 44.03% γ-PGA, while in the original strain, these components represented 53.67%, 3.246% and 34.13%, respectively. In combination with an analysis of the transcriptional levels of several key genes involved in γ-PGA synthesis in B. licheniformis, we inferred that epsB played a key role in the synthesis of both polysaccharide and γ-PGA. The bioflocculant production of the epsB recombinant strain was further evaluated during batch fermentation in a 2 L fermenter; the flocculating activity reached 9612.75 U/mL, and the bioflocculant yield reached 10.26 g/L after 72 h, representing increases of 224% and 36.62%, respectively, compared with the original strain. Moreover, we found that the tandem expression of phosphoglucomutase (pgcA) and UTP-glucose-1-phosphate uridylyltransferase (gtaB1) could enhance the crude yield of the bioflocculant by 20.77% and that the overexpression of epsA could enhance the bioflocculant

Rubusuaviins A-F, monomeric and oligomeric ellagitannins from Chinese sweet tea and their alpha-amylase inhibitory activity.

PubMed

Li, Haizhou; Tanaka, Takashi; Zhang, Ying-Jun; Yang, Chong-Ren; Kouno, Isao

2007-09-01

Six new ellagitannins herein, rubusuaviins A-F, were isolated from the aqueous acetone extract of Chinese sweet tea (Tien-cha, dried leaves of Rubus suavissimus S. LEE) together with seven known tannins. Rubusuaviin A was characterized as 1-O-galloyl-2,3-O-(S)-HHDP-4,6-O-(S)-sanguisorboyl-beta-D-glucopyranose. Rubusuaviins B, C, and E are dimeric, trimeric, and tetrameric ellagitannins, respectively, in which the sanguisorboyl groups were connected ellagitannin units. Rubusuaviins D and F were desgalloyl derivatives of rubusuaviins C and E, respectively. The inhibition of alpha-amylase activity by rubusuaviins and related ellagitannins was compared. Ellagitannins with beta-galloyl groups at the glucose C-1 positions showed stronger inhibition compared with the alpha-galloyl and desgalloyl compounds. The molecular weight of these compounds was not important for the inhibition of alpha-amylase activity.

Ultrasound assisted process intensification of uricase and alkaline protease enzyme co-production in Bacillus licheniformis.

PubMed

Pawar, Shweta V; Rathod, Virendra K

2018-07-01

Low energy ultrasound irradiation was used to enhance co-production of enzymes uricase and alkaline protease using Bacillus licheniformis NRRL 14209. Production of uricase and alkaline protease was evaluated for different ultrasound parameters such as ultrasound power, time of irradiation, duty cycle and growth stage of organisms at which irradiation is carried out. Maximum uricase production of 0.825 U/mL and alkaline protease of 0.646 U/mL have been obtained when fermentation broth was irradiated at 6 h of growth stage with 60 W power for 15 min of duration having 40% of duty cycle. The enzyme yield was found to be enhanced by a factor of 1.9-3.8 and 1.2-2.2 for uricase and alkaline protease respectively. Nevertheless, intracellular uricase was also observed in a fermentation broth after ultrasonic process intensification. The results indicate the effectiveness of low frequency ultrasound in improving enzyme yields with a vision of commercial applicability of the process. Copyright © 2018 Elsevier B.V. All rights reserved.

[The primary structure of the alpha-amylase inhibitor Hoe 467A from Streptomyces tendae 4158. A new class of inhibitors].

PubMed

Aschauer, H; Vértesy, L; Nesemann, G; Braunitzer, G

1983-10-01

The native or modified alpha-amylase inhibitor Hoe 467A - isolated from the culture medium of Streptomyces tendae 4158 - and overlapping peptides were degraded by the automatic Edman technique. The oxidized or aminoethylated or oxidized and maleoylated inhibitor was digested with trypsin and the native inhibitor with pepsin. Further digestion with Staphylococcus aureus proteinase was also carried out. After peptic digestion two cystin peptides were isolated, which allowed the establishment of the disulfide bonds. The alpha-amylase inhibitor is a polypeptid consisting of 74 amino-acid residues with a molecular mass of 7958 Da. The inhibitor is composed of all naturally occurring amino acids except methionine and phenylalanine and shows no sequence homology to known inhibitors. The clinical and pharmacological importance in respect to the inhibitors ability for inactivation of human salivary and pancreatic alpha-amylase is discussed. Especially the proteinase resistance of the inhibitor enables a clinical application in human (e.g. Diabetes mellitus) per os.

Efficient production of L-asparaginase from Bacillus licheniformis with low-glutaminase activity: optimization, scale up and acrylamide degradation studies.

PubMed

Mahajan, Richi V; Saran, Saurabh; Kameswaran, Karthikeya; Kumar, Vinod; Saxena, R K

2012-12-01

L-Asparaginase has potential as an anti-cancer drug and for prevention of acrylamide formation in fried and baked foods. Production of the enzyme by Bacillus licheniformis (RAM-8) was optimized by process engineering using a statistical modeling approach and a maximum yield of 32.26 IU/ml was achieved. The L-asparaginase exhibited glutaminase activity of only 0.8 IU/ml and would therefore be less prone to cause the side effects associated with asparaginase therapy compared to enzyme preparations with higher glutaminase activities. When production was carried out in a 30-L bioreactor, enzyme production reached 29.94 IU/ml in 15 h. The enzyme inhibited poly-acrylamide formation in 10% acrylamide solution and reduced acrylamide formation in fried potatoes by 80%. Copyright © 2012 Elsevier Ltd. All rights reserved.

Characterization of Biosurfactant Produced by Bacillus licheniformis TT42 Having Potential for Enhanced Oil Recovery.

PubMed

Suthar, Harish; Nerurkar, Anuradha

2016-09-01

Bacillus licheniformis TT42 produced a low-molecular weight anionic biosurfactant that reduced the surface tension of water from 72 to 27 mN/m and the interfacial tension from 12 to 0.05 mN/m against crude oil. We have earlier reported significant enhancement in oil recovery in laboratory sand pack columns and core flood studies, by biosurfactant-TT42 compared to standard strain, Bacillus mojavensis JF2. In the context of this application of the biosurfactant-TT42, its characterization was deemed important. In the preliminary studies, the biosurfactant-TT42 was found to be functionally stable at under conditions of temperature, pH, and salinity generally prevalent in oil reservoirs. Furthermore, the purified biosurfactant-TT42 was found to have a CMC of 22 mg/l. A newly developed activity staining TLC method was used for the purification of biosurfactant-TT42. Structural characterization of biosurfactant-TT42 using TLC, Fourier transform infrared spectroscopy (FTIR), GC-MS, and matrix-assisted laser desorption ionization time of flight (MALDI-TOF)/TOF suggested that it was a mixture of lipopeptide species, all having a common hydrophilic cyclic heptapeptide head with the sequence, Gln-Leu/Ileu-Leu/Ileu-Val-Asp-Leu/Ileu-Leu/Ileu linked to hydrophobic tails of different lengths of 3β-OH-fatty acids bearing 1043, 1057 and 1071 Da molecular weight, where 3β-OH-C19 fatty acid was predominant. This is the longest chain length of fatty acids reported in a lipopeptide.

Daily Social Interactions and the Biological Stress Response: Are There Age Differences in Links Between Social Interactions and Alpha-Amylase?

PubMed

Birditt, Kira S; Tighe, Lauren A; Nevitt, Michael R; Zarit, Steven H

2017-12-12

According to the strength and vulnerability integration (SAVI) model, older people are better able to avoid negative social interactions than younger people, but when they do experience negative interactions, they are equally or more emotionally and physiologically reactive than younger people. Less is known about the links between daily negative and positive social encounters and the sympathetic adrenal medullary system (a key stress pathway) and whether there are age differences in these links. This study considers whether negative and positive social interactions are associated with diurnal alpha-amylase (a measure of the sympathetic adrenal medullary system) and whether there are differences in these links by age. Participants were from the Daily Health, Stress, and Relationship Study, which includes a random sample of 89 individuals (aged 40-95) who completed 14 days of daily diary interviews and provided saliva samples four times a day (wake, 30 min after wake, lunch, and bedtime) for four of those days that were assayed for alpha-amylase. Days in which people reported more negative interactions were associated with flatter morning declines in alpha-amylase, indicating greater stress. Links between positive interactions and diurnal alpha-amylase varied by age group. Findings are consistent with the SAVI model indicating that older adults respond differently to social stimuli than younger people. © The Author(s) 2017. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Homogeneity and heterogeneity in amylase production by Bacillus subtilis under different growth conditions.

PubMed

Ploss, Tina N; Reilman, Ewoud; Monteferrante, Carmine G; Denham, Emma L; Piersma, Sjouke; Lingner, Anja; Vehmaanperä, Jari; Lorenz, Patrick; van Dijl, Jan Maarten

2016-03-29

Bacillus subtilis is an important cell factory for the biotechnological industry due to its ability to secrete commercially relevant proteins in large amounts directly into the growth medium. However, hyper-secretion of proteins, such as α-amylases, leads to induction of the secretion stress-responsive CssR-CssS regulatory system, resulting in up-regulation of the HtrA and HtrB proteases. These proteases degrade misfolded proteins secreted via the Sec pathway, resulting in a loss of product. The aim of this study was to investigate the secretion stress response in B. subtilis 168 cells overproducing the industrially relevant α-amylase AmyM from Geobacillus stearothermophilus, which was expressed from the strong promoter P(amyQ)-M. Here we show that activity of the htrB promoter as induced by overproduction of AmyM was "noisy", which is indicative for heterogeneous activation of the secretion stress pathway. Plasmids were constructed to allow real-time analysis of P(amyQ)-M promoter activity and AmyM production by, respectively, transcriptional and out-of-frame translationally coupled fusions with gfpmut3. Our results show the emergence of distinct sub-populations of high- and low-level AmyM-producing cells, reflecting heterogeneity in the activity of P(amyQ)-M. This most likely explains the heterogeneous secretion stress response. Importantly, more homogenous cell populations with regard to P(amyQ)-M activity were observed for the B. subtilis mutant strain 168degUhy32, and the wild-type strain 168 under optimized growth conditions. Expression heterogeneity of secretory proteins in B. subtilis can be suppressed by degU mutation and optimized growth conditions. Further, the out-of-frame translational fusion of a gene for a secreted target protein and gfp represents a versatile tool for real-time monitoring of protein production and opens novel avenues for Bacillus production strain improvement.

Utilization of Industrial Waste for the Production of Bio-Preservative from Bacillus licheniformis Me1 and Its Application in Milk and Milk-Based Food Products.

PubMed

Nithya, Vadakedath; Prakash, Maya; Halami, Prakash M

2018-06-01

The bio-preservative efficacy of a partially purified antibacterial peptide (ppABP) produced by Bacillus licheniformis Me1 in an economical medium developed using agro-industry waste was evaluated by direct application in milk and milk-based food products. The addition of ppABP in milk samples stored at 4 ± 2 °C and 28 ± 2 °C resulted in the growth inhibition of pathogens Listeria monocytogenes Scott A, Micrococcus luteus ATCC 9341, and Staphylococcus aureus FRI 722. The shelf life of milk samples with added ppABP increased to 4 days at 28 ± 2 °C, whereas curdling and off-odor were noticed in samples without ppABP. Furthermore, the milk samples with ppABP were sensorily acceptable. Antilisterial effect was also observed in cheese and paneer samples treated with ppABP. These results clearly indicate that the ppABP of B. licheniformis Me1 can be utilized as a bio-preservative to control the growth of spoilage and pathogenic bacteria, thereby reducing the risk of food-borne diseases.

Immobilization of pectin degrading enzyme from Bacillus licheniformis KIBGE IB-21 using agar-agar as a support.

PubMed

Rehman, Haneef Ur; Aman, Afsheen; Zohra, Raheela Rahmat; Qader, Shah Ali Ul

2014-02-15

Pectinase from Bacillus licheniformis KIBGE IB-21 was immobilized in agar-agar matrix using entrapment technique. Effect of different concentrations of agar-agar on pectinase immobilization was investigated and it was found that maximum immobilization was achieved at 3.0% agar-agar with 80% enzyme activity. After immobilization, the optimum temperature of enzyme increased from 45 to 50 °C and reaction time from 5 to 10 minutes as compared to free enzyme. Due to the limited diffusion of high molecular weight substrate, K(m) of immobilized enzyme slightly increased from 1.017 to 1.055 mg ml(-1), while Vmax decreased from 23,800 to 19,392 μM min(-1) as compared to free enzyme. After 120 h entrapped pectinase retained their activity up to 82% and 71% at 30 °C and 40 °C, respectively. The entrapped pectinase showed activity until 10th cycle and maintain 69.21% activity even after third cycle. Copyright © 2013 Elsevier Ltd. All rights reserved.

Purification and characterization of an alpha-amylase of Pichia burtonii isolated from the traditional starter "murcha" in Nepal.

PubMed

Takeuchi, Akiko; Shimizu-Ibuka, Akiko; Nishiyama, Yoshitaka; Mura, Kiyoshi; Okada, Sanae; Tokue, Chiyoko; Arai, Soichi

2006-12-01

Among more than 20 yeast strains isolated from the traditional starter "murcha" in Nepal, we characterized a yeast that might be involved in saccharification. This strain, identified as Pichia burtonii, produced an extracellular amylolytic enzyme when cultured in the presence of starch in the medium. Since no amylase secreted by P. burtonii has yet been reported, we purified the enzyme and determined its N-terminal amino acid sequence. Together with the results of a hydrolyzing activity assay toward various substrates, it was found to be an alpha-amylase. The purified enzyme, named Pichia burtonii alpha-amylase (PBA), was a glycoprotein with an apparent molecular mass of 51 kDa. Enzyme activity was optimal at pH 5.0 at 40 degrees C. The enzyme retained 80% of its original activity after incubation under the optimal pH condition at 50 degrees C for 30 min. The activity was inhibited by metal ions such as Cd(2+), Cu(2+), Hg(2+), Al(3+), and Zn(2+).

Efficient Expression of Maltohexaose-Forming α-Amylase from Bacillus stearothermophilus in Brevibacillus choshinensis SP3 and Its Use in Maltose Production

PubMed Central

Duan, Xuguo

2017-01-01

The maltohexaose-forming, Ca2+-independent α-amylase gene from Bacillus stearothermophilus (AmyMH) was efficiently expressed in Brevibacillus choshinensis SP3. To improve the production of AmyMH in B. choshinensis SP3, the temperature and initial pH of culture medium were optimized. In addition, single-factor and response surface methodologies were pursued to optimize culture medium. Addition of proline to the culture medium significantly improved the production of recombinant α-amylase in B. choshinensis SP3. This improvement may result from improved cellular integrity of recombinant B. choshinensis SP3 in existence of proline. Culture medium optimization resulted in an 8-fold improvement in α-amylase yield, which reached 1.72 × 104 U·mL−1. The recombinant α-amylase was applied to the production of maltose on a laboratory scale. A maltose content of 90.72%, which could be classified as an extremely high maltose syrup, could be achieved using 15% (m/v) corn starch as the substrate. This study demonstrated that the B. choshinensis SP3 expression system was able to produce substantial quantities of recombinant α-amylase that has potential application in the starch industry. PMID:29250543

Utilization of α-amylase enzyme from Bacillus stearothermophilus RSAII1B for maltodextrin production from sago starch

NASA Astrophysics Data System (ADS)

Arfah, R. A.; Ahmad, A.; Dali, S.; Djide, M. N.; Mahdalia; Arif, A. R.

2018-03-01

The dried sago flour derived from Palopo contains 28.80% amylose and 91.23% total carbohydrate. Based on the data, sago starch has the potential to become an alternative raw material for themaltodextrin production. Maltodextrin is one of the starch derivative products produced by hydrolysis process using the α-amylase enzyme with amaximum DE (dextrose equivalent) value of 20. The use of maltodextrin for food and pharmaceutical industries is increasing because of maltodextrin is widely used as thickener filler, surfactant and sugar substitute in milk powder. The aims of this study are to optimize the addition of enzyme concentration and hydrolysis time of α -amylase enzyme to obtain high quality ofmaltodextrin This study also aimed to characterization the obtained maltodextrin. The first step was isolation and purification α-amylase from the isolate of Bacillus stearothermophilus RSAII1B, followed by determination of the α-amylase concentration (0.05%, 0.07% and 0.09%) in 2.0% starch substrate, and the hydrolysis time ofα-amilase (60, 90, 120, 240 minutes). Maltodextrin characters observed were dextrose equivalent (DE), reducing sugar, moisture content, pH changes, color, solubility, viscosity, and total plate count (TPC). The results showed that the value of DE was 12.31, reducing sugar was 11.4%; water content was 10.92%; pH was 4.85; The color of maltodextrin powder was white bone color; solubility was 153.2 g/L; Viscositywas 210-240 cps, TPCwas 380 cfu/g. Maltodextrins produced from sago starch using the α-amylase enzyme from B.stearothermophillus RSAIIm met the quality requirements of SNI 7599: 2010.

Production and characterization of a thermostable alpha-amylase from Nocardiopsis sp. endophyte of yam bean.

PubMed

Stamford, T L; Stamford, N P; Coelho, L C; Araújo, J M

2001-01-01

Thermostable amylolytic enzymes have been currently investigated to improve industrial processes of starch degradation. Studies on production of alpha-amylase by Nocardiopsis sp., an endophytic actinomycete isolated from yam bean (Pachyrhizus erosus L. Urban), showed that higher enzyme levels were obtained at the end of the logarithmic growth phase after incubation for 72 h at pH 8.6. Maximum activity of alpha-amylase was obtained at pH 5.0 and 70 degrees C. The isolated enzyme exhibited thermostable properties as indicated by retention of 100% of residual activity at 70 degrees C, and 50% of residual activity at 90 degrees C for 10 min. Extracellular enzyme from Nocardiopsis sp. was purified by fractional precipitation with ammonium sulphate. After 60% saturation produced 1130 U mg-1 protein and yield was 28% with purification 2.7-fold. The enzyme produced by Nocardiopsis sp. has potential for industrial applications.

Peer Victimization and Aggression: Moderation by Individual Differences in Salivary Cortisol and Alpha-Amylase

ERIC Educational Resources Information Center

Rudolph, Karen D.; Troop-Gordon, Wendy; Granger, Douglas A.

2010-01-01

This research examined whether variations in salivary measures of the hypothalamic-pituitary-adrenal axis (cortisol) and autonomic nervous system (alpha amylase [sAA]) contribute to individual differences in the association between peer victimization and aggression. Children (N = 132; M age = 9.46 years, SD = 0.33) completed a measure of peer…

Bacillus licheniformis SA03 Confers Increased Saline-Alkaline Tolerance in Chrysanthemum Plants by Induction of Abscisic Acid Accumulation.

PubMed

Zhou, Cheng; Zhu, Lin; Xie, Yue; Li, Feiyue; Xiao, Xin; Ma, Zhongyou; Wang, Jianfei

2017-01-01

Soil saline-alkalization is a major abiotic stress that leads to low iron (Fe) availability and high toxicity of sodium ions (Na + ) for plants. It has recently been shown that plant growth promoting rhizobacteria (PGPR) can enhance the ability of plants to tolerate multiple abiotic stresses such as drought, salinity, and nutrient deficiency. However, the possible involvement of PGPR in improving saline-alkaline tolerance of plants and the underlying mechanisms remain largely unknown. In this study, we investigated the effects of Bacillus licheniformis (strain SA03) on the growth of Chrysanthemum plants under saline-alkaline conditions. Our results revealed that inoculation with SA03 alleviated saline-alkaline stress in plants with increased survival rates, photosynthesis and biomass. The inoculated plants accumulated more Fe and lower Na + concentrations under saline-alkaline stress compared with the non-inoculated plants. RNA-Sequencing analyses further revealed that SA03 significantly activated abiotic stress- and Fe acquisition-related pathways in the stress-treated plants. However, SA03 failed to increase saline-alkaline tolerance in plants when cellular abscisic acid (ABA) and nitric oxide (NO) synthesis were inhibited by treatment with fluridone (FLU) and 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO), respectively. Importantly, we also found that NO acted downstream of SA03-induced ABA to activate a series of adaptive responses in host plants under saline-alkaline stress. These findings demonstrated the potential roles of B. licheniformis SA03 in enhancing saline-alkaline tolerance of plants and highlighted the intricate integration of microbial signaling in regulating cellular Fe and Na + accumulation.

Isolation and characterization of a cold-active, alkaline, detergent stable α-amylase from a novel bacterium Bacillus subtilis N8.

PubMed

Arabacı, Nihan; Arıkan, Burhan

2018-05-28

A cold-active alkaline amylase producer Bacillus subtilis N8 was isolated from soil samples. Amylase synthesis optimally occurred at 15°C and pH 10.0 on agar plates containing starch. The molecular weight of the enzyme was found to be 205 kDa by performing SDS-PAGE. While the enzyme exhibited the highest activity at 25°C and pH 8.0, it was highly stable in alkaline media (pH 8.0-12.0) and retained 96% of its original activity at low temperatures (10-40°C) for 24 hr. While the amylase activity increased in the presence of β-mercaptoethanol (103%); Ba 2+ , Ca 2+ , Na + , Zn 2+ , Mn 2+ , H 2 O 2 , and Triton X-100 slightly inhibited the activity. The enzyme showed resistance to some denaturants: such as SDS, EDTA, and urea (52, 65, and 42%, respectively). N8 α-amylase displayed the maximum remaining activity of 56% with 3% NaCl. The major final products of starch were glucose, maltose, and maltose-derived oligosaccharides. This novel cold-active α-amylase has the potential to be used in the industries of detergent and food, bioremediation process and production of prebiotics.

Application of enzymatic apple pomace hydrolysate to production of 2,3-butanediol by alkaliphilic Bacillus licheniformis NCIMB 8059.

PubMed

Białkowska, Aneta M; Gromek, Ewa; Krysiak, Joanna; Sikora, Barbara; Kalinowska, Halina; Jędrzejczak-Krzepkowska, Marzena; Kubik, Celina; Lang, Siegmund; Schütt, Fokko; Turkiewicz, Marianna

2015-12-01

2,3-Butanediol (2,3-BD) synthesis by a nonpathogenic bacterium Bacillus licheniformis NCIMB 8059 from enzymatic hydrolysate of depectinized apple pomace and its blend with glucose was studied. In shake flasks, the maximum diol concentration in fed-batch fermentations was 113 g/L (in 163 h, from the hydrolysate, feedings with glucose) while in batch processes it was around 27 g/L (in 32 h, from the hydrolysate and glucose blend). Fed-batch fermentations in the 0.75 and 30 L fermenters yielded 87.71 g/L 2,3-BD in 160 h, and 72.39 g/L 2,3-BD in 94 h, respectively (from the hydrolysate and glucose blend, feedings with glucose). The hydrolysate of apple pomace, which was for the first time used for microbial 2,3-BD production is not only a source of sugars but also essential minerals.

Study of HMG-CoA Reductase Inhibition Activity of the Hydrolyzed Product of Snakehead Fish (Channa striata) Skin Collagen with 50 kDa Collagenase from Bacillus licheniformis F11.4

PubMed Central

Virginia, Agnes; Rachmawati, Heni; Riani, Catur; Retnoningrum, Debbie S.

2016-01-01

Bioactive peptides produced from enzymatic hydrolysis fibrous protein have been proven to have several biological activities. Previous study showed that the hydrolysis product of snakehead fish skin collagen with 26 kDa collagenase from Bacillus licheniformis F11.4 showed HMG-CoA (HMGR) inhibition activity. The aim of this research was to determine the ability of the hydrolysis product produced from snakehead fish skin collagen hydrolysed by 50 kDa collagenase from B. licheniformis F11.4 in inhibiting HMGR activity. Snakehead fish skin collagen was extracted using an acid method and collagenase was produced from B. licheniformis F11.4 using half-strength Luria Bertani (LB) medium containing 5% collagen. Crude collagenase was concentrated and fractionated using the DEAE Sephadex A-25 column eluted with increasing gradient concentrations of NaCl. Collagen, collagenase, and fractions were analyzed using SDS-PAGE and collagenolytic activity was analyzed by the zymography method. Collagenase with 50 kDa molecular weight presented in fraction one was used to hydrolyze the collagen. The reaction was done in 18 hours at 50°C. The hydrolysis product using 3.51 μg collagen and 9 ng collagenase showed 25.8% inhibition activity against pravastatin. This work shows for the first time that the hydrolysis product of snakehead fish skin collagen and 50 kDa collagenase from B. licheniformis F11.4 has potential as an anticholesterol agent. PMID:27110500

Study of HMG-CoA Reductase Inhibition Activity of the Hydrolyzed Product of Snakehead Fish (Channa striata) Skin Collagen with 50 kDa Collagenase from Bacillus licheniformis F11.4.

PubMed

Virginia, Agnes; Rachmawati, Heni; Riani, Catur; Retnoningrum, Debbie S

2016-01-01

Bioactive peptides produced from enzymatic hydrolysis fibrous protein have been proven to have several biological activities. Previous study showed that the hydrolysis product of snakehead fish skin collagen with 26 kDa collagenase from Bacillus licheniformis F11.4 showed HMG-CoA (HMGR) inhibition activity. The aim of this research was to determine the ability of the hydrolysis product produced from snakehead fish skin collagen hydrolysed by 50 kDa collagenase from B. licheniformis F11.4 in inhibiting HMGR activity. Snakehead fish skin collagen was extracted using an acid method and collagenase was produced from B. licheniformis F11.4 using half-strength Luria Bertani (LB) medium containing 5% collagen. Crude collagenase was concentrated and fractionated using the DEAE Sephadex A-25 column eluted with increasing gradient concentrations of NaCl. Collagen, collagenase, and fractions were analyzed using SDS-PAGE and collagenolytic activity was analyzed by the zymography method. Collagenase with 50 kDa molecular weight presented in fraction one was used to hydrolyze the collagen. The reaction was done in 18 hours at 50°C. The hydrolysis product using 3.51 μg collagen and 9 ng collagenase showed 25.8% inhibition activity against pravastatin. This work shows for the first time that the hydrolysis product of snakehead fish skin collagen and 50 kDa collagenase from B. licheniformis F11.4 has potential as an anticholesterol agent.

Evaluation of the alpha-amylase activity as an indicator of pasteurization efficiency and microbiological quality of liquid whole eggs.

PubMed

Silva, Guilherme Resende da; Menezes, Liliane Denize Miranda; Lanza, Isabela Pereira; Oliveira, Daniela Duarte de; Silva, Carla Aparecida; Klein, Roger Wilker Tavares; Assis, Débora Cristina Sampaio de; Cançado, Silvana de Vasconcelos

2017-09-01

In order to evaluate the efficiency of the pasteurization process in liquid whole eggs, an UV/visible spectrophotometric method was developed and validated for the assessment of alpha-amylase activity. Samples were collected from 30 lots of raw eggs (n = 30) and divided into three groups: one was reserved for analysis of the raw eggs, the second group was pasteurized at 61.1°C for 3.5 minutes (n = 30), and the third group was pasteurized at 64.4°C for 2.5 minutes (n = 30). In addition to assessing alpha-amylase activity, the microbiological quality of the samples was also evaluated by counting total and thermotolerant coliforms, mesophilic aerobic microorganisms, Staphylococcus spp., and Salmonella spp. The validated spectrophotometric method demonstrated linearity, with a coefficient of determination (R2) greater than 0.99, limits of detection (LOD) and quantification (LOQ) of 0.48 mg kg-1 and 1.16 mg kg-1, respectively, and acceptable precision and accuracy with relative standard deviation (RSD) values of less than 10% and recovery rates between 98.81% and 105.40%. The results for alpha-amylase activity in the raw egg samples showed high enzyme activity due to near-complete hydrolysis of the starch, while in the eggs pasteurized at 61.1°C, partial inactivation of the enzyme was observed. In the samples of whole eggs pasteurized at 64.4°C, starch hydrolysis did not occur due to enzyme inactivation. The results of the microbiological analyses showed a decrease (P < 0.0001) in the counts for all the studied microorganisms and in the frequency of Salmonella spp. in the pasteurized egg samples according to the two binomials under investigation, compared to the raw egg samples, which showed high rates of contamination (P < 0.0001). After pasteurization, only one sample (3.33%) was positive for Salmonella spp., indicating failure in the pasteurization process, which was confirmed by the alpha-amylase test. It was concluded that the validated methodology for

Symptoms of prenatal depression are associated with raised salivary alpha-amylase levels.

PubMed

Braithwaite, Elizabeth C; Ramchandani, Paul G; Lane, Tracy A; Murphy, Susannah E

2015-10-01

Prenatal depression increases risk for a number of adverse offspring outcomes, however the biological mechanisms underlying this association remain unclear. It has been suggested that maternal glucocorticoids may mediate this link, though supporting evidence has been mixed. An alternative mechanism of effect may be via depression-induced changes in maternal sympathetic nervous system (SNS) function. We examined this hypothesis by determining the relationship between symptoms of maternal prenatal depression and diurnal salivary alpha-amylase (sAA) levels. 76 pregnant women were recruited during either the second or third trimester of pregnancy. Participants self-reported depressive symptoms using the Edinburgh postnatal depression scale. Saliva samples, to be assayed for alpha-amylase activity, were collected at home over two working days. Participants with depressive symptoms in later pregnancy had elevated awakening sAA levels compared with non-depressed controls (t(73) = -2.737, p = 0.008), and continued to have raised sAA throughout the day (F(1) = 10.924, p = 0.002). Our findings highlight that symptoms of depression during late pregnancy are associated with increased maternal SNS activity. Thus, changes in maternal SNS function, which may include increased vasoconstriction and reduced foetal blood flow, could, in part, mediate associations between prenatal depression and adverse offspring outcomes. Copyright © 2015 Elsevier Ltd. All rights reserved.

[Mechanism of metabolic and ionic germination of "Bacillus licheniformis" spores treated with hydrogen peroxide (author's transl)].

PubMed

Cerf, O

1977-01-01

Spores of Bacillus licheniformis 109-2A0 lost their refractility and absorbancy at 640 nm in the presence of metabolizable molecules (L-alanine). The same occurred with spores treated with 4.4 mol/1 hydrogen peroxide, pH 2.0, at 65 degrees C, even after 5 min of treatment. In addition, these transformations could be promoted after 2 min of treatment by inorganic ions (KI). This possibility occurs following a kinetics of activation. Thermodynamic parameters showed this activation to be combined with a molecular re-organization. Loss of refractility or absorbancy, induced by L-ala or KI, was inhibited by inhibitors of membrane functions or of L-alanine dehydrogenase, enzyme of which a noticeable activity was demonstrated in treated spores. Only 10% of spore calcium leaked during the treatment. Therefore loss of refractility or absorbancy caused by molecules metabolizable or not seemed to correspond to a physiological germination. The first even of the metabolic, as well as or the ionic germination could well be a modification of the spore membrane proton-motive force.

Assessing Agreement Between Salivary Alpha Amylase Levels Collected by Passive Drool and Eluted Filter Paper in Adolescents With Cancer

PubMed Central

Ameringer, Suzanne; Munro, Cindy; Elswick, R.K.

2014-01-01

Purpose/Objectives To assess the validity of filter paper (FP) against the gold standard of passive drool (PD) for collecting salivary alpha amylase as a surrogate biomarker of psychological stress in adolescents with cancer. Design Part of a longitudinal, descriptive study of symptoms in adolescents with cancer during chemotherapy. Setting A pediatric hematology/oncology treatment center. Sample 33 saliva sample pairs from nine adolescents with cancer, aged 13–18 years. Methods Salivary alpha amylase was collected by PD and FP at four time points during a cycle of chemotherapy: days 1 (time 1) and 2 (time 2) of chemotherapy, day 7–10 (time 3), and day 1 of the next cycle (time 4). A random effects regression was used to assess the correlation between PD and FP values, and a Bland Altman analysis was conducted to assess agreement between the values. Main Research Variables Salivary alpha amylase. Findings The estimated correlation between PD and FP values was r = 0.91, p < 0.001. Regression results were also used to rescale FP values to the levels of the PD values because the FP values were on a different scale than the PD values. The Bland Altman analysis revealed that the agreement between the rescaled FP values and PD values was not satisfactory. Conclusions Eluted FP may not be a valid method for collecting salivary alpha amylase in adolescents with cancer. Implications for Nursing Psychological stress in adolescents with cancer may be linked to negative outcomes, such as greater symptom severity and post-traumatic stress disorder. Nurses need valid, efficient, biobehavioral measures to assess psychological stress in the clinical setting. PMID:22750901

Rope-producing strains of Bacillus spp. from wheat bread and strategy for their control by lactic acid bacteria.

PubMed

Pepe, Olimpia; Blaiotta, Giuseppe; Moschetti, Giancarlo; Greco, Teresa; Villani, Francesco

2003-04-01

Two types of white wheat bread (high- and low-type loaves) were investigated for rope spoilage. Thirty of the 56 breads tested developed rope spoilage within 5 days; the high-type loaves were affected by rope spoilage more than the low-type loaves. Sixty-one Bacillus strains were isolated from ropy breads and were characterized on the basis of their phenotypic and genotypic traits. All of the isolates were identified as Bacillus subtilis by biochemical tests, but molecular assays (randomly amplified polymorphic DNA PCR assay, denaturing gradient gel electrophoresis analysis, and sequencing of the V3 region of 16S ribosomal DNA) revealed greater Bacillus species variety in ropy breads. In fact, besides strains of B. subtilis, Bacillus licheniformis, Bacillus cereus, and isolates of Bacillus clausii and Bacillus firmus were also identified. All of the ropy Bacillus isolates exhibited amylase activity, whereas only 32.4% of these isolates were able to produce ropiness in bread slices after treatment at 96 degrees C for 10 min. Strains of lactic acid bacteria previously isolated from sourdough were first selected for antirope activity on bread slices and then used as starters for bread-making experiments. Prevention of growth of approximately 10(4) rope-producing B. subtilis G1 spores per cm(2) on bread slices for more than 15 days was observed when heat-treated cultures of Lactobacillus plantarum E5 and Leuconostoc mesenteroides A27 were added. Growth of B. subtilis G1 occurred after 7 days in breads started with Saccharomyces cerevisiae T22, L. plantarum E5, and L. mesenteroides A27.

The impact of single nucleotide polymorphism in monomeric alpha-amylase inhibitor genes from wild emmer wheat, primarily from Israel and Golan

PubMed Central

2010-01-01

Background Various enzyme inhibitors act on key insect gut digestive hydrolases, including alpha-amylases and proteinases. Alpha-amylase inhibitors have been widely investigated for their possible use in strengthening a plant's defense against insects that are highly dependent on starch as an energy source. We attempted to unravel the diversity of monomeric alpha-amylase inhibitor genes of Israeli and Golan Heights' wild emmer wheat with different ecological factors (e.g., geography, water, and temperature). Population methods that analyze the nature and frequency of allele diversity within a species and the codon analysis method (comparing patterns of synonymous and non-synonymous changes in protein coding sequences) were used to detect natural selection. Results Three hundred and forty-eight sequences encoding monomeric alpha-amylase inhibitors (WMAI) were obtained from 14 populations of wild emmer wheat. The frequency of SNPs in WMAI genes was 1 out of 16.3 bases, where 28 SNPs were detected in the coding sequence. The results of purifying and the positive selection hypothesis (p < 0.05) showed that the sequences of WMAI were contributed by both natural selection and co-evolution, which ensured conservation of protein function and inhibition against diverse insect amylases. The majority of amino acid substitutions occurred at the C-terminal (positive selection domain), which ensured the stability of WMAI. SNPs in this gene could be classified into several categories associated with water, temperature, and geographic factors, respectively. Conclusions Great diversity at the WMAI locus, both between and within populations, was detected in the populations of wild emmer wheat. It was revealed that WMAI were naturally selected for across populations by a ratio of dN/dS as expected. Ecological factors, singly or in combination, explained a significant proportion of the variations in the SNPs. A sharp genetic divergence over very short geographic distances compared to

Comparing Dental Stress in New Child Patients and Returning Patients Using Salivary Cortisol, Immunoglobulin-A and Alpha- Amylase.

PubMed

Alaki, Sumer M; Safi, Ayman; Ouda, Soliman; Nadhreen, Alaa

this study was aimed at comparing dental stress in children having their first dental visit to those returning for dental treatment using salivary biomarkers of stress including salivary cortisol (s-cortisol), Immunoglobulin-A (s-IgA) and alpha-amylase (s-α-amylase). Additionally, the study was aimed at monitoring the change in stress in new patients as they progressed from the waiting to the clinical areas. salivary samples were collected from 40 children who had not been to a dentist before and similar samples were collected from 40 children who were returning for completion of dental treatment. Salivary cortisol, s-IgA and s-α-amylase concentrations were obtained by Enzyme-linked Immunosorbent Assay (ELISA). salivary cortisol levels were higher for new patients at the waiting area compared to that at the dental chair (p=0.05). Salivary alpha-amylase significantly increased in new patients while being seated in the dental chair. Returning patients had higher s-α-amylase (p=0.001) and s-IgA (p=0.016) compared to new patients. Returning patients had the lowest level of s-cortisol when providers were faculty pediatric dentists than with students and interns (p=0.035). children coming in for their first dental visit may experience dental stress at the waiting area before being seated for dental examination. Returning children may experience higher levels of stress compared to new child patients possibly due to previous dental exposure.

Immunochemical Studies on α-Amylase III. Immunochemical Relationships Among Amylases from Various Microorganisms1

PubMed Central

Sirisinha, Stitaya; Allen, Peter Z.

1965-01-01

Sirishinha, Stitaya (University of Rochester School of Medicine and Dentistry, Rochester, N.Y.), and Peter Z. Allen. Immunochemical studies on α-amylase. III. Immunochemical relationships among amylases from various microorganisms. J. Bacteriol. 90:1120–1128. 1965.—Immunochemical relationships among amylases obtained from a selected group of microorganisms were examined, and a cross-reaction was detected between the α-amylases of Bacillus stearothermophilus and B. subtilis. Immunodiffusion and quantitative precipitin studies, as well as cross-neutralization tests, indicate that B. stearothermophilus α-amylase reacts with a portion of antibody present in antisera to crystalline B. subtilis α-amylase. Amylases from these two species thus have some aspects of structure in common. Limited data obtained by immunodiffusion suggest that groupings which confer cross-reactivity to the B. stearothermophilus enzyme are lost after exposure to mercaptoethanol in the presence of ethylenediamine-tetraacetate, followed by treatment with iodoacetamide. With the antisera employed and within the concentration range examined, no immunochemical cross-reaction was observed among amylases from Aspergillus oryzae, B. subtilis, B. polymyxa, B. macerans, Pseudomonas saccharophila, and Euglena sanguinis. Immunoelectrophoresis of partially purified B. stearothermophilus α-amylase by use of antiserum to the crude enzyme, together with localization of amylase activity in immunoelectrophoretic plates, suggests that B. stearothermophilus α-amylase is antigenic in the rabbit. Images PMID:5847799

Serum trypsin, alpha-amylase and lipase during bombesin stimulation in normal subjects and patients with pancreatic insufficiency.

PubMed

Hafkenscheid, J C; Hessels, M; Jansen, J B; Lamers, C B

1984-01-31

The effect of infusion of bombesin (60 pmol/kg 20 min) on pancreatic enzymes in serum was studied in 13 normal subjects and 12 patients with pancreatic insufficiency. In normal subjects administration of bombesin induced large increases in serum trypsin (p less than 0.01), while serum total alpha-amylase and pancreatic alpha-amylase did not change and serum lipase showed only a modest rise (0.01 less than p less than 0.05). Patients with pancreatic insufficiency had significantly lower serum concentrations of all enzymes studied (p less than 0.01) and in such patients bombesin did not change the concentrations of pancreatic enzymes in serum. It is concluded that determination of the serum trypsin response to bombesin may be of help in the diagnosis of pancreatic insufficiency.

Production, Characterization, and Application of Bacillus licheniformis W16 Biosurfactant in Enhancing Oil Recovery.

PubMed

Joshi, Sanket J; Al-Wahaibi, Yahya M; Al-Bahry, Saif N; Elshafie, Abdulkadir E; Al-Bemani, Ali S; Al-Bahri, Asma; Al-Mandhari, Musallam S

2016-01-01

The biosurfactant production by Bacillus licheniformis W16 and evaluation of biosurfactant based enhanced oil recovery (EOR) using core-flood under reservoir conditions were investigated. Previously reported nine different production media were screened for biosurfactant production, and two were further optimized with different carbon sources (glucose, sucrose, starch, cane molasses, or date molasses), as well as the strain was screened for biosurfactant production during the growth in different media. The biosurfactant reduced the surface tension and interfacial tension to 24.33 ± 0.57 mN m -1 and 2.47 ± 0.32 mN m -1 respectively within 72 h, at 40°C, and also altered the wettability of a hydrophobic surface by changing the contact angle from 55.67 ± 1.6 to 19.54°± 0.96°. The critical micelle dilution values of 4X were observed. The biosurfactants were characterized by different analytical techniques and identified as lipopeptide, similar to lichenysin-A. The biosurfactant was stable over wide range of extreme environmental conditions. The core flood experiments showed that the biosurfactant was able to enhance the oil recovery by 24-26% over residual oil saturation (S or ). The results highlight the potential application of lipopeptide biosurfactant in wettability alteration and microbial EOR processes.

The 2.0-A resolution structure of soybean beta-amylase complexed with alpha-cyclodextrin.

PubMed

Mikami, B; Hehre, E J; Sato, M; Katsube, Y; Hirose, M; Morita, Y; Sacchettini, J C

1993-07-13

New crystallographic findings are presented which offer a deeper understanding of the structure and functioning of beta-amylase, the first known exo-type starch-hydrolyzing enzyme. A refined three-dimensional structure of soybean beta-amylase, complexed with the inhibitor alpha-cyclodextrin, has been determined at 2.0-A resolution with a conventional R-value of 17.5%. The model contains 491 amino acid residues, 319 water molecules, 1 sulfate ion, and 1 alpha-cyclodextrin molecule. The protein consists of a core with an (alpha/beta)8 supersecondary structure, plus a smaller globular region formed by long loops (L3, L4, and L5) extending from beta-strands beta 3, beta 4, and beta 5. Between the two regions is a cleft that opens into a pocket whose floor contains the postulated catalytic center near the carboxyl group of Glu 186. The annular alpha-cyclodextrin binds in (and partly projects from) the cleft with its glucosyl O-2/O-3 face abutting the (alpha/beta)8 side and with its alpha-D(1 --> 4) glucosidic linkage progression running clockwise as viewed from that side. The ligand does not bind deeply enough to interact with the carboxyl group of Glu 186. Rather, it occupies most of the cleft entrance, strongly suggesting that alpha-cyclodextrin inhibits catalysis by blocking substrate access to the more deeply located reaction center. Of the various alpha-cyclodextrin interactions with protein residues in loops L4, L5, L6, and L7, most notable is the shallow inclusion complex formed with Leu 383 (in L7, on the core side of the cleft) through contacts of its methyl groups with the C-3 atoms of four of the ligand's D-glucopyranosyl residues. All six residues of the bound alpha-cyclodextrin are of 4C1 conformation and are joined by alpha-1,4 linkages with similar torsional angles to form a nearly symmetrical torus as reported for crystalline inclusion complexes with alpha-cyclodextrin. We envision a significant role for the methyl groups of Leu 383 at the cleft entrance

The Potential Source of B. licheniformis Contamination During Whey Protein Concentrate 80 Manufacture.

PubMed

Md Zain, Siti Norbaizura; Bennett, Rod; Flint, Steve

2017-03-01

The objective of this study was to determine the possible source of predominant Bacillus licheniformis contamination in a whey protein concentrate (WPC) 80 manufacturing plant. Traditionally, microbial contaminants of WPC were believed to grow on the membrane surfaces of the ultrafiltration plant as this represents the largest surface area in the plant. Changes from hot to cold ultrafiltration have reduced the growth potential for bacteria on the membrane surfaces. Our recent studies of WPCs have shown the predominant microflora B. licheniformis would not grow in the membrane plant because of the low temperature (10 °C) and must be growing elsewhere. Contamination of dairy products is mostly due to bacteria being released from biofilm in the processing plant rather from the farm itself. Three different reconstituted WPC media at 1%, 5%, and 20% were used for biofilm growth and our results showed that B. licheniformis formed the best biofilm at 1% (low solids). Further investigations were done using 3 different media; tryptic soy broth, 1% reconstituted WPC80, and 1% reconstituted WPC80 enriched with lactose and minerals to examine biofilm growth of B. licheniformis on stainless steel. Thirty-three B. licheniformis isolates varied in their ability to form biofilm on stainless steel with stronger biofilm in the presence of minerals. The source of biofilms of thermo-resistant bacteria such as B. licheniformis is believed to be before the ultrafiltration zone represented by the 1% WPC with lactose and minerals where the whey protein concentration is about 0.6%. © 2017 Institute of Food Technologists®.

Stilbene Glucoside, a Putative Sleep Promoting Constituent from Polygonum multiflorum Affects Sleep Homeostasis by Affecting the Activities of Lactate Dehydrogenase and Salivary Alpha Amylase.

PubMed

Wei, Qian; Ta, Guang; He, Wenjing; Wang, Wei; Wu, Qiucheng

2017-01-01

Chinese herbal medicine (CHM) has been used for treating insomnia for centuries. The most used CHM for insomnia was Polygonum multiflorum. However, the molecular mechanism for CHM preventing insomnia is unknown. Stilbene glucoside (THSG), an important active component of P. multiflorum, may play an important role for treating insomnia. To test the hypothesis, Kunming mice were treated with different dosages of THSG. To examine the sleep duration, a computer-controlled sleep-wake detection system was implemented. Electroencephalogram (EEG) and electromyogram (EMG) electrodes were implanted to determine sleep-wake state. RT-PCR and Western blot was used to measure the levels of lactate dehydrogenase (LDH) and saliva alpha amylase. Spearman's rank correlation coefficient was used to identify the strength of correlation between the variables. The results showed that THSG significantly prolonged the sleep time of the mice (p<0.01). THSG changed sleep profile by reducing wake and rapid eye movement (REM) period, and increasing non-REM period. RT-PCR and Western blot analysis showed that THSG could down-regulate the levels of LDH and saliva alpha amylase (p<0.05). The level of lactate and glucose was positively related with the activity of LDH and saliva alpha amylase (p<0.05), respectively. On the other hand, the activities of LDH and amylase were negatively associated with sleep duration (p<0.05). The levels of lactate and glucose affect sleep homeostasis. Thus, THSG may prevent insomnia by regulating sleep duration via LDH and salivary alpha amylase.

Studies on the production of alkaline α-amylase from Bacillus subtilis CB-18.

PubMed

Nwokoro, Ogbonnaya; Anthonia, Odiase

2015-01-01

various temperatures (25, 30, 35, 40, 45, 50, 55 and 60°C) in a thermo static water bath. The reactions were stopped by adding DNS reagent. The enzyme activity was therefore determined. Thermal stability was studied by incubating 0.5 ml of enzyme solution in 0.1 M Tris/HCl buffer (pH 8.5) and 0.5 ml of 1% (w/v) soluble starch (Merck) in 0.1 M Tris/HCl buffer (pH 8.5) for 3 h at various temperatures (20, 30, 40, 50, 60 and 70°C) for 60 min. The enzyme displayed optimal activity at pH 8.0 at which it produced maximum specific activity of 34.3 units/mg protein. Maximum stability was at pH 8.0 to 9.0. Maximum activity was observed at temperature of 50°C while thermo stability of the enzyme was observed at 40-50°C. The enzyme displayed a wide range of activities on starch and caused the release of 5.86, 4.75, 5.98, 3.44, 3.96, 8.84 mg/mL reducing sugar from cassava, potato, cocoyam, corn, rice and soluble starch respectively. This investigation reports some biochemical characterization of alkaline α-amylase from Bacillus subtilis CB-18. The substrate specificities of this enzyme on various starches suggested that the alkaline α-amylase enzyme had combined activities on raw and soluble starch.

Draft genome sequence of a thermostable, alkaliphilic α-amylase and protease producing Bacillus amyloliquefaciens strain KCP2.

PubMed

Prajapati, Vimalkumar S; Ray, Sanket; Narayan, Jitendra; Joshi, Chaitanya C; Patel, Kamlesh C; Trivedi, Ujjval B; Patel, R M

2017-12-01

Bacillus amyloliquefaciens strain KCP2 was isolated from municipal food waste samples collected in Vallabh Vidyanagar, Gujarat, India. Strain KCP2 is noteworthy due to its ability to produce a thermostable, alkaliphilic α-amylase and a protease. These enzymes have importance in several industrial processes including bread making, brewing, starch processing, pharmacy, and textile industries. Whole genome sequencing of strain KCP2 showed that the estimated genome size was 3.9 Mb, the G + C content was 46%, and it coded for 4113 genes.

Longitudinal and Immediate Effect of Kundalini Yoga on Salivary Levels of Cortisol and Activity of Alpha-Amylase and Its Effect on Perceived Stress.

PubMed

García-Sesnich, Jocelyn N; Flores, Mauricio Garrido; Ríos, Marcela Hernández; Aravena, Jorge Gamonal

2017-01-01

Stress is defined as an alteration of an organism's balance in response to a demand perceived from the environment. Diverse methods exist to evaluate physiological response. A noninvasive method is salivary measurement of cortisol and alpha-amylase. A growing body of evidence suggests that the regular practice of Yoga would be an effective treatment for stress. To determine the Kundalini Yoga (KY) effect, immediate and after 3 months of regular practice, on the perception of psychological stress and the salivary levels of cortisol and alpha-amylase activity. To determine the psychological perceived stress, levels of cortisol and alpha-amylase activity in saliva, and compare between the participants to KY classes performed for 3 months and a group that does not practice any type of yoga. The total sample consisted of 26 people between 18 and 45-year-old; 13 taking part in KY classes given at the Faculty of Dentistry, University of Chile and 13 controls. Salivary samples were collected, enzyme-linked immunosorbent assay was performed to quantify cortisol and kinetic reaction test was made to determine alpha-amylase activity. Perceived Stress Scale was applied at the beginning and at the end of the intervention. Statistical analysis was applied using Stata v11.1 software. Shapiro-Wilk test was used to determine data distribution. The paired analysis was fulfilled by t -test or Wilcoxon signed-rank test. T -test or Mann-Whitney's test was applied to compare longitudinal data. A statistical significance was considered when P < 0.05. KY practice had an immediate effect on salivary cortisol. The activity of alpha-amylase did not show significant changes. A significant decrease of perceived stress in the study group was found. KY practice shows an immediate effect on salivary cortisol levels and on perceived stress after 3 months of practice.

Bacillus licheniformis SA03 Confers Increased Saline–Alkaline Tolerance in Chrysanthemum Plants by Induction of Abscisic Acid Accumulation

PubMed Central

Zhou, Cheng; Zhu, Lin; Xie, Yue; Li, Feiyue; Xiao, Xin; Ma, Zhongyou; Wang, Jianfei

2017-01-01

Soil saline-alkalization is a major abiotic stress that leads to low iron (Fe) availability and high toxicity of sodium ions (Na+) for plants. It has recently been shown that plant growth promoting rhizobacteria (PGPR) can enhance the ability of plants to tolerate multiple abiotic stresses such as drought, salinity, and nutrient deficiency. However, the possible involvement of PGPR in improving saline–alkaline tolerance of plants and the underlying mechanisms remain largely unknown. In this study, we investigated the effects of Bacillus licheniformis (strain SA03) on the growth of Chrysanthemum plants under saline–alkaline conditions. Our results revealed that inoculation with SA03 alleviated saline–alkaline stress in plants with increased survival rates, photosynthesis and biomass. The inoculated plants accumulated more Fe and lower Na+ concentrations under saline–alkaline stress compared with the non-inoculated plants. RNA-Sequencing analyses further revealed that SA03 significantly activated abiotic stress- and Fe acquisition-related pathways in the stress-treated plants. However, SA03 failed to increase saline–alkaline tolerance in plants when cellular abscisic acid (ABA) and nitric oxide (NO) synthesis were inhibited by treatment with fluridone (FLU) and 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO), respectively. Importantly, we also found that NO acted downstream of SA03-induced ABA to activate a series of adaptive responses in host plants under saline–alkaline stress. These findings demonstrated the potential roles of B. licheniformis SA03 in enhancing saline–alkaline tolerance of plants and highlighted the intricate integration of microbial signaling in regulating cellular Fe and Na+ accumulation. PMID:28706529

Residues Phe103 and Phe149 are critical for the co-chaperone activity of Bacillus licheniformis GrpE.

PubMed

Lin, Min-Guan; Chi, Meng-Chun; Chen, Bo-En; Wang, Tzu-Fan; Lo, Huei-Fen; Lin, Long-Liu

2015-01-01

A tryptophan-free Bacillus licheniformis nucleotide exchange factor (BlGrpE) and its Trp mutants (F70W, F103W, F149W, F70/103W, F70/149W, F103/149W and F70/103/149W) were over-expressed and purified to near homogeneity. Simultaneous addition of B. licheniformis DnaJ, NR-peptide and individual variants synergistically stimulated the ATPase activity of a recombinant DnaK (BlDnaK) from the same bacterium by 3.1-14.7-fold, which are significantly lower than the synergistic stimulation (18.9-fold) of BlGrpE. Protein-protein interaction analysis revealed that Trp mutants relevant to amino acid positions 103 and 149 lost the ability to bind BlDnaK. Circular dichroism measurements indicate that F70W displayed a comparable level of secondary structure to that of BlGrpE, and the wild-type protein and the Trp mutants as well all experienced a reversible behavior of thermal denaturation. Guanidine hydrochloride (GdnHCl)-induced unfolding transition for BlGrpE was calculated to be 1.25 M corresponding to ΔG(N-U) of 4.29 kcal/mol, whereas the unfolding transitions of mutant proteins were in the range of 0.77-1.31 M equivalent to ΔG(N-U) of 2.41-4.14 kcal/mol. Taken together, the introduction of tryptophan residue, especially at positions 103 and 149, into the primary structure of BlGrpE has been proven to be detrimental to structural integrity and proper function of the protein. Copyright © 2014 Elsevier B.V. All rights reserved.

Cortisol and alpha-amylase as stress response indicators during pre-hospital emergency medicine training with repetitive high-fidelity simulation and scenarios with standardized patients.

PubMed

Valentin, Bernd; Grottke, Oliver; Skorning, Max; Bergrath, Sebastian; Fischermann, Harold; Rörtgen, Daniel; Mennig, Marie-Therese; Fitzner, Christina; Müller, Michael P; Kirschbaum, Clemens; Rossaint, Rolf; Beckers, Stefan K

2015-04-08

In emergency medicine, the benefits of high-fidelity simulation (SIM) are widely accepted and standardized patients (SP) are known to mimic real patients accurately. However, only limited data are available concerning physicians' stress markers within these training environments. The aim of this pilot study was to investigate repetitive stress among healthcare professionals in simulated pre-hospital emergency scenarios using either SIM or SPs. Teams with one emergency medical services (EMS) physician and two paramedics completed three SIM scenarios and two SP scenarios consecutively. To evaluate stress, salivary cortisol and alpha-amylase were measured in saliva samples taken before, during and after the scenarios. A total of 14 EMS physicians (29% female; mean age: 36.8 ± 5.0 years; mean duration of EMS-experience: 9.1 ± 5.8 years) and 27 paramedics (11% female; age: 30.9 ± 6.9 years; EMS experience: 8.1 ± 6.0 years) completed the study. Alpha-amylase and cortisol levels did not differ significantly between the two professions. Cortisol values showed a gradual and statistically significant reduction over time but little change was observed in response to each scenario. In contrast, alpha-amylase activity increased significantly in response to every SIM and SP scenario, but there was no clear trend towards an overall increase or decrease over time. Increases in salivary alpha-amylase activity suggest that both SIM and SP training produce stress among emergency healthcare professionals. Corresponding increases in salivary cortisol levels were not observed. Among physicians in the emergency setting, it appears that alpha-amylase provides a more sensitive measure of stress levels than cortisol.

Cognitive performance and morning levels of salivary cortisol and alpha-amylase in children reporting high vs. low daily stress perception.

PubMed

Maldonado, Enrique F; Fernandez, Francisco J; Trianes, M Victoria; Wesnes, Keith; Petrini, Orlando; Zangara, Andrea; Enguix, Alfredo; Ambrosetti, Lara

2008-05-01

The aim of the present study was to assess the effects of daily stress perception on cognitive performance and morning basal salivary cortisol and alpha-amylase levels in healthy children aged 9-12. Participants were classified by whether they had low daily perceived stress (LPS, n = 27) or a high daily perceived stress (HPS, n = 26) using the Children Daily Stress Inventory (CDSI). Salivary cortisol and alpha-amylase were measured at awakening and 30 minutes later. Cognitive performance was assessed using the Cognitive Drug Research assessment system. The HPS group exhibited significantly poorer scores on speed of memory (p < .05) and continuity of attention (p < .05) relative to the LPS group. The HPS group also showed significantly lower morning cortisol levels at awakening and at +30 minutes measures in comparison with the LPS group (p < .05), and mean morning cortisol levels were negatively correlated with speed of memory (p < .05) in the 53 participants. No significant differences were observed between both groups in alpha-amylase levels. These findings suggest that daily perceived stress in children may impoverish cognitive performance via its modulating effects on the HPA axis activity.

Adolescents' increasing stress response to social evaluation: pubertal effects on cortisol and alpha-amylase during public speaking.

PubMed

van den Bos, Esther; de Rooij, Mark; Miers, Anne C; Bokhorst, Caroline L; Westenberg, P Michiel

2014-01-01

Stress responses to social evaluation are thought to increase during adolescence, which may be due to pubertal maturation. However, empirical evidence is scarce. This study is the first to investigate the relation between pubertal development and biological responses to a social-evaluative stressor longitudinally. Participants performed the Leiden Public Speaking Task twice, with a 2-year interval (N = 217; age at Time 1: 8-17 years). The results support an increase in sensitivity to social evaluation during adolescence. The overall cortisol and alpha-amylase responses increased-both between and within participants-and were more strongly related to self-reported pubertal development than to age. The cortisol response shifted from speech delivery toward anticipation. The alpha-amylase response increased in both phases. © 2013 The Authors. Child Development © 2013 Society for Research in Child Development, Inc.

Bean alpha-amylase inhibitors in transgenic peas inhibit development of pea weevil larvae.

PubMed

de Sousa-Majer, Maria José; Hardie, Darryl C; Turner, Neil C; Higgins, Thomas J V

2007-08-01

This glasshouse study used an improved larval measurement procedure to evaluate the impact of transgenic pea, Pisum sativum L., seeds expressing a-amylase inhibitor (AI)-1 or -2 proteins on pea weevil, Bruchus pisorum L. Seeds of transgenic 'Laura' and 'Greenfeast' peas expressing alpha-(AI)-1 reduced pea weevil survival by 93-98%. Larval mortality occurred at an early instar. Conversely, in nontransgenic cultivars, approximately 98-99% of the pea weevils emerged as adults. By measuring the head capsule size, we determined that larvae died at the first to early third instar in alpha-(AI)-1 transgenic peas, indicating that this inhibitor is highly effective in controlling this insect. By contrast, transgenic Laura and 'Dundale' expressing alpha-(AI)-2 did not affect pea weevil survival, but they did delay larval development. After 77 d of development, the head capsule size indicated that the larvae were still at the third instar stage in transgenic alpha-(AI)-2 peas, whereas adult bruchids had developed in the nontransgenic peas.

The effects of autonomy support on salivary alpha-amylase: The role of individual differences.

PubMed

Sieber, Vanda; Schüler, Julia; Wegner, Mirko

2016-12-01

The empirical evidence for the relationship between autonomy-supportive environments and physiological stress is inconsistent. Whereas some studies report a decrease in stress in autonomy-supportive environments, other studies show a negative effect of autonomy on physiological stress. As previous research has not considered individual differences within this relationship, the present research aims to close this empirical gap by proposing that an implicit autonomy disposition, which is defined as a dispositional preference for self-determination, serves as a moderator. In an experiment, we tested whether the autonomy disposition moderates the effect of different teaching styles (controlling, autonomy-supportive, and neutral) on the acute physiological stress response (salivary alpha-amylase) in adolescents (N=69). The study revealed that participants with a high implicit autonomy disposition displayed lower salivary alpha-amylase responses when exposed to autonomy-supportive vignettes compared to when they were exposed to controlling or neutral teaching styles. The opposite pattern was found in students with a low implicit autonomy disposition. The results illustrate that experimentally induced variations in autonomy support lead to different physiological stress responses, depending on individual differences in the implicit autonomy disposition. Copyright © 2016 Elsevier Ltd. All rights reserved.

Combined Expression of Aspergillus nidulans Endoxylanase X24 and Aspergillus oryzae (alpha)-Amylase in Industrial Baker's Yeasts and Their Use in Bread Making.

PubMed

Monfort, A; Blasco, A; Prieto, J A; Sanz, P

1996-10-01

The Aspergillus nidulans endoxylanase X24 and the Aspergillus oryzae (alpha)-amylase cDNAs were placed under the control of the Saccharomyces cerevisiae actin promoter (pACT1) and introduced into baker's yeast. Bread made with transformants expressing both enzymes (YEpACT-AMY-ACT-X24) showed a 30% increase in volume and reduced firmness in comparison with that produced with a commercial strain. Endoxylanase X24 and (alpha)-amylase seem to act synergistically to improve the quality of bread in terms of volume and density.

Dynamic rheological properties of dough as affected by amylases from various sources.

PubMed

Doğan, Ismail S

2002-12-01

The effect of alpha-amylases from cereal, fungal, and bacterial sources on dough dynamic rheological properties was investigated. Dynamic rheological study of flour-and-water doughs during resting period showed significant changes in dough rheological properties as a function of alpha-amylases. Addition of alpha-amylases caused a time-dependent decrease in G', storage modulus. The enzyme action on starch during baking increased viscous flow properties. These changes were temperature-dependent. The thermal inactivation temperature of alpha-amylase plays an important role in modification of starch. Rheological changes in dough will alter the machinability of the dough and the quality of end products.

Combined Expression of Aspergillus nidulans Endoxylanase X24 and Aspergillus oryzae (alpha)-Amylase in Industrial Baker's Yeasts and Their Use in Bread Making

PubMed Central

Monfort, A.; Blasco, A.; Prieto, J. A.; Sanz, P.

1996-01-01

The Aspergillus nidulans endoxylanase X24 and the Aspergillus oryzae (alpha)-amylase cDNAs were placed under the control of the Saccharomyces cerevisiae actin promoter (pACT1) and introduced into baker's yeast. Bread made with transformants expressing both enzymes (YEpACT-AMY-ACT-X24) showed a 30% increase in volume and reduced firmness in comparison with that produced with a commercial strain. Endoxylanase X24 and (alpha)-amylase seem to act synergistically to improve the quality of bread in terms of volume and density. PMID:16535419

Effect of carbon and nitrogen sources on simultaneous production of α-amylase and green food packaging polymer by Bacillus sp. CFR 67.

PubMed

Sreekanth, M S; Vijayendra, S V N; Joshi, G J; Shamala, T R

2013-04-01

In this paper, effect of different carbon and nitrogen sources, including hydrolysates of rice bran and wheat bran, on simultaneous production of α-amylase (for hydrolysis of starch in food systems) and polyhydroxyalkanoates (PHA, a green biopolymer, which can be used as a packing material for foods) by Bacillus sp. CFR 67 was studied by submerged fermentation. Amongst various carbon sources tested, glucose and sucrose supported production of significantly (P < 0.05) higher amount of α-amylase (66 U/ml) and PHA (444 mg/l), respectively. Of the nitrogen sources tested, ammonium acetate and beef extract led to the production of maximum amount of amylase (36 U/ml) and PHA (592 mg/l), respectively. Supplementation of the production medium with wheat bran hydrolysate (50 ml/l) produced significantly higher amounts of amylase (73 U/ml) and PHA (524 mg/l). Thus this study indicated the potential of agro-residues for the production of value added biomolecules, which can reduce the cost of production of these molecules and enables to reduce the pollution mainly caused by the use of non biodegradable plastics.

Application of a statistical design to the optimization of parameters and culture medium for alpha-amylase production by Aspergillus oryzae CBS 819.72 grown on gruel (wheat grinding by-product).

PubMed

Kammoun, Radhouane; Naili, Belgacem; Bejar, Samir

2008-09-01

The production optimization of alpha-amylase (E.C.3.2.1.1) from Aspergillus oryzae CBS 819.72 fungus, using a by-product of wheat grinding (gruel) as sole carbon source, was performed with statistical methodology based on three experimental designs. The optimisation of temperature, agitation and inoculum size was attempted using a Box-Behnken design under the response surface methodology. The screening of nineteen nutrients for their influence on alpha-amylase production was achieved using a Plackett-Burman design. KH(2)PO(4), urea, glycerol, (NH(4))(2)SO(4), CoCl(2), casein hydrolysate, soybean meal hydrolysate, MgSO(4) were selected based on their positive influence on enzyme formation. The optimized nutrients concentration was obtained using a Taguchi experimental design and the analysis of the data predicts a theoretical increase in the alpha-amylase expression of 73.2% (from 40.1 to 151.1 U/ml). These conditions were validated experimentally and revealed an enhanced alpha-amylase yield of 72.7%.

Potentized Mercuric chloride and Mercuric iodide enhance alpha-amylase activity in vitro.

PubMed

Sukul, N C; De, A; Sukul, A; Sinhababu, S P

2002-10-01

Mercuric chloride 30c and Mercuric iodide 30c were prepared by successive dilution in 30 steps of 1:100 followed by sonication at 20KHz for 30s at each step. Both were prepared in two media: 90% ethanol and distilled water. Three preparations of Mercuric chloride 30 in water were used: 12-month old, 1-month old and 4-day old. The controls for the water and ethanol-water preparations were pure water 30c and 90% ethanol 30c, respectively. For the three water preparations there were three matched controls of water 30c of the same ages. Each potentized substance or its control was mixed with distilled water 1:100 before testing. Hydrolysis of starch by alpha-amylase was measured by the standard procedure after incubation for 15 min at 27 degrees C. Mercuric chloride 30c and Mercuric iodide 30c in both water and aqueous ethanol media, enhanced enzyme activity significantly, compared to their respective controls. Mercuric chloride 30c, prepared in water 12 months previously, produced no significant change in the enzyme activity compared to its control. We hypothesize that the structure of the active molecule imprinted on water polymers during the process of dynamization. The specifically structured water interacts with the active sites of alpha-amylase, modifying its activity. Ethanol molecules have large non-polar part stabilizing the water structure and thus retaining activity for a longer time.

Longitudinal and Immediate Effect of Kundalini Yoga on Salivary Levels of Cortisol and Activity of Alpha-Amylase and Its Effect on Perceived Stress

PubMed Central

García-Sesnich, Jocelyn N; Flores, Mauricio Garrido; Ríos, Marcela Hernández; Aravena, Jorge Gamonal

2017-01-01

Context: Stress is defined as an alteration of an organism's balance in response to a demand perceived from the environment. Diverse methods exist to evaluate physiological response. A noninvasive method is salivary measurement of cortisol and alpha-amylase. A growing body of evidence suggests that the regular practice of Yoga would be an effective treatment for stress. Aims: To determine the Kundalini Yoga (KY) effect, immediate and after 3 months of regular practice, on the perception of psychological stress and the salivary levels of cortisol and alpha-amylase activity. Settings and Design: To determine the psychological perceived stress, levels of cortisol and alpha-amylase activity in saliva, and compare between the participants to KY classes performed for 3 months and a group that does not practice any type of yoga. Subjects and Methods: The total sample consisted of 26 people between 18 and 45-year-old; 13 taking part in KY classes given at the Faculty of Dentistry, University of Chile and 13 controls. Salivary samples were collected, enzyme-linked immunosorbent assay was performed to quantify cortisol and kinetic reaction test was made to determine alpha-amylase activity. Perceived Stress Scale was applied at the beginning and at the end of the intervention. Statistical Analysis Used: Statistical analysis was applied using Stata v11.1 software. Shapiro–Wilk test was used to determine data distribution. The paired analysis was fulfilled by t-test or Wilcoxon signed-rank test. T-test or Mann–Whitney's test was applied to compare longitudinal data. A statistical significance was considered when P < 0.05. Results: KY practice had an immediate effect on salivary cortisol. The activity of alpha-amylase did not show significant changes. A significant decrease of perceived stress in the study group was found. Conclusions: KY practice shows an immediate effect on salivary cortisol levels and on perceived stress after 3 months of practice. PMID:28546677

Three camelid VHH domains in complex with porcine pancreatic alpha-amylase. Inhibition and versatility of binding topology.

PubMed

Desmyter, Aline; Spinelli, Silvia; Payan, Francoise; Lauwereys, Marc; Wyns, Lode; Muyldermans, Serge; Cambillau, Christian

2002-06-28

Camelids produce functional antibodies devoid of light chains and CH1 domains. The antigen-binding fragment of such heavy chain antibodies is therefore comprised in one single domain, the camelid heavy chain antibody VH (VHH). Here we report on the structures of three dromedary VHH domains in complex with porcine pancreatic alpha-amylase. Two VHHs bound outside the catalytic site and did not inhibit or inhibited only partially the amylase activity. The third one, AMD9, interacted with the active site crevice and was a strong amylase inhibitor (K(i) = 10 nm). In contrast with complexes of other proteinaceous amylase inhibitors, amylase kept its native structure. The water-accessible surface areas of VHHs covered by amylase ranged between 850 and 1150 A(2), values similar to or even larger than those observed in the complexes between proteins and classical antibodies. These values could certainly be reached because a surprisingly high extent of framework residues are involved in the interactions of VHHs with amylase. The framework residues that participate in the antigen recognition represented 25-40% of the buried surface. The inhibitory interaction of AMD9 involved mainly its complementarity-determining region (CDR) 2 loop, whereas the CDR3 loop was small and certainly did not protrude as it does in cAb-Lys3, a VHH-inhibiting lysozyme. AMD9 inhibited amylase, although it was outside the direct reach of the catalytic residues; therefore it is to be expected that inhibiting VHHs might also be elicited against proteases. These results illustrate the versatility and efficiency of VHH domains as protein binders and enzyme inhibitors and are arguments in favor of their use as drugs against diabetes.

Cloning and Characterization of an alpha-amylase Gene from the Hyperthermophilic Archaeon Thermococcus Thioreducens

NASA Technical Reports Server (NTRS)

Bernhardsdotter, Eva C. M. J.; Pusey, Mark L.; Ng, Joseph D.; Garriott, Owen K.

2004-01-01

The gene encoding an extracellular alpha-amylase, TTA, from the hyperthermophilic archaeon Thermococcus thioreducens was cloned and expressed in Escherichia coli. Primary structural analysis revealed high similarity with other a-amylases from the Thermococcus and Pyrococcus genera, as well as the four highly conserved regions typical for a-amylases. The 1374 bp gene encodes a protein of 457 amino acids, of which 435 constitute the mature protein preceded by a 22 amino acid signal peptide. The molecular weight of the purified recombinant enzyme was estimated to be 43 kDa by denaturing gel electrophoresis. Maximal enzymatic activity of recombinant TTA was observed at 90 C and pH 5.5 in the absence of exogenous Ca(2+), and the enzyme was considerably stable even after incubation at 90 C for 2 hours. The thermostability at 90 and 102 C was enhanced in the presence of 5 mM Ca(2+). The extraordinarily high specific activity (about 7.4 x 10(exp 3) U/mg protein at 90 C, pH 5.5 with soluble starch as substrate) together with its low pH optimum makes this enzyme an interesting candidate for starch processing applications.

Distinct differentiation of closely related species of Bacillus subtilis group with industrial importance.

PubMed

Jeyaram, Kumaraswamy; Romi, Wahengbam; Singh, Thangjam Anand; Adewumi, Gbenga Adedeji; Basanti, Khundrakpam; Oguntoyinbo, Folarin Anthony

2011-11-01

PCR amplification of 16S rRNA gene by universal primers followed by restriction fragment length polymorphism analysis using RsaI, CfoI and HinfI endonucleases, distinctly differentiated closely related Bacillus amyloliquefaciens, Bacillus licheniformis and Bacillus pumilus from Bacillus subtilis sensu stricto. This simple, economical, rapid and reliable protocol could be an alternative to misleading phenotype-based grouping of these closely related species. Copyright © 2011 Elsevier B.V. All rights reserved.

Norepinephrine transporter blocker atomoxetine increases salivary alpha amylase.

PubMed

Warren, Christopher M; van den Brink, Ruud L; Nieuwenhuis, Sander; Bosch, Jos A

2017-04-01

It has been suggested that central norepinephrine (NE) activity may be inferred from increases in salivary alpha-amylase (SAA), but data in favor of this proposition are limited. We administered 40mg of atomoxetine, a selective NE transporter blocker that increases central NE levels, to 24 healthy adult participants in a double-blind, placebo-controlled cross-over design. Atomoxetine administration significantly increased SAA secretion and concentrations at 75-180min after treatment (more than doubling baseline levels). Consistent with evidence that elevation in central NE is a co-determinant of hypothalamic-pituitary-adrenal axis activity, salivary cortisol also approximately doubled at the same time points. Moreover, changes in salivary cortisol positively correlated with SAA (0.44

Diurnal patterns of salivary alpha-amylase and cortisol secretion in female adolescent tennis players after 16 weeks of training.

PubMed

Filaire, Edith; Ferreira, Jose Pedro; Oliveira, Miguel; Massart, Alain

2013-07-01

We examined the effects of 16 weeks of training on diurnal pattern of salivary alpha-amylase (sAA), cortisol, and the ratio of sAA over cortisol (AOC) in 12 national adolescent female tennis players. Stress and recovery were also evaluated using the Recovery-Stress-Questionnaire for Athletes-RESTQ-Sport. Data were collected after a 2-week rest (January, W0), and 4 months after W0 (W16). Subjects collected five saliva samples throughout a day. While all participants displayed the previously shown decrease after awakening in adolescents at W0, they showed a rise in the alpha-amylase awakening response and a higher alpha-amylase activity output (p<0.01) at W16 compared to W0. For the daily rhythm of cortisol we found subjects having a low overall output of salivary cortisol (p<0.01) and a blunted response to awakening at W16. Furthermore, an increase in the ratio AOC at W16, and a negative correlation between this ratio and Sport-specific recovery score. Our findings offer support for the hypothesis that increase of training load during the study period induced asymmetry activation between the two stress systems, in relation to psychological alterations and performance decrease. These results provide encouragement to continue exploring the impact of training program using a psychobiological approach among young athletes in order to prevent fatigue and preserve the health of these athletes. Copyright © 2012 Elsevier Ltd. All rights reserved.

SusG: A Unique Cell-Membrane-Associated [alpha]-Amylase from a Prominent Human Gut Symbiont Targets Complex Starch Molecules

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

Koropatkin, Nicole M.; Smith, Thomas J.

SusG is an {alpha}-amylase and part of a large protein complex on the outer surface of the bacterial cell and plays a major role in carbohydrate acquisition by the animal gut microbiota. Presented here, the atomic structure of SusG has an unusual extended, bilobed structure composed of amylase at one end and an unprecedented internal carbohydrate-binding motif at the other. Structural studies further demonstrate that the carbohydrate-binding motif binds maltooligosaccharide distal to, and on the opposite side of, the amylase catalytic site. SusG has an additional starch-binding site on the amylase domain immediately adjacent to the active cleft. Mutagenesis analysismore » demonstrates that these two additional starch-binding sites appear to play a role in catabolism of insoluble starch. However, elimination of these sites has only a limited effect, suggesting that they may have a more important role in product exchange with other Sus components.« less

Interaction between wheat alpha-amylase/trypsin bi-functional inhibitor and mammalian digestive enzymes: Kinetic, equilibrium and structural characterization of binding.

PubMed

Cuccioloni, Massimiliano; Mozzicafreddo, Matteo; Ali, Ishtiaq; Bonfili, Laura; Cecarini, Valentina; Eleuteri, Anna Maria; Angeletti, Mauro

2016-12-15

Alpha-amylase/trypsin bi-functional inhibitors (ATIs) are non-gluten protein components of wheat and other cereals that can hypersensitise the human gastrointestinal tract, eventually causing enteropathies in predisposed individuals. These inhibitory proteins can act both directly by targeting specific pro-inflammatory receptors, and indirectly by impairing the activity of digestive enzymes, the latter event causing the accumulation of undigested peptides with potential immunogenic properties. Herein, according to a concerted approach based on in vitro and in silico methods we characterized kinetics, equilibrium parameters and modes of binding of the complexes formed between wheat ATI and two representative mammalian digestive enzymes, namely trypsin and alpha-amylase. Interestingly, we demonstrated ATI to target both enzymes with independent binding sites and with moderately high affinity. Copyright © 2016 Elsevier Ltd. All rights reserved.

Assessment of salivary amylase as a stress biomarker in pregnant patients.

PubMed

Guglielminotti, J; Dehoux, M; Mentré, F; Bedairia, E; Montravers, P; Desmonts, J-M; Longrois, D

2012-01-01

Chronic stress during pregnancy has been associated with worsened maternal and fetal outcomes. Acute stress immediately before spinal anaesthesia for caesarean section may contribute to hypotension. Therefore objective measures of acute stress may help identify women at risk of adverse outcomes. Salivary alpha-amylase is a stress biomarker that has so far been poorly investigated during pregnancy. The reference change value is the difference between two sequential results that must be exceeded for a change to be considered clinically relevant. Our first aim was to determine if salivary alpha-amylase increased in pregnant patients when subjected to the stress of transfer to the operating room. Our second aim was to determine if changes in salivary alpha-amylase were likely to be clinically significant by measuring reference change value in healthy volunteers. In 15 pregnant patients undergoing planned caesarean section under spinal anaesthesia, salivary alpha-amylase, systolic blood pressure, heart rate, and immediate anxiety were measured on the morning of surgery on the ward and again in the operating room. The reference change value was calculated from 18 healthy volunteers. A median 220% increase in salivary alpha-amylase activity (P=0.0015) and a 17% increase in systolic blood pressure (P=0.0006) were observed between the ward and operating room. No changes of immediate anxiety or heart rate were observed. Reference change value was ±76% in volunteers and 13 of the 15 pregnant patients had a salivary alpha-amylase increase greater than the reference change value. When pregnant women are taken to the operating room, a clinically and statistically significant increase in salivary alpha-amylase was observed. Further studies are required to define its clinical usefulness. Copyright © 2011 Elsevier Ltd. All rights reserved.

Study of starch fermentation at low pH by Lactobacillus fermentum Ogi E1 reveals uncoupling between growth and alpha-amylase production at pH 4.0.

PubMed

Calderon Santoyo, M; Loiseau, G; Rodriguez Sanoja, R; Guyot, J P

2003-01-15

Lactobacillus fermentum Ogi E1 is an amylolytic heterofermentative lactic acid bacterium previously isolated from ogi, a Benin maize sourdough. In the present study, the effect of different pH between 3.5 and 6.0 on starch fermentation products and alpha-amylase production was investigated. Whereas a pH of 5.0 was optimum for specific growth rate and lactic acid production, growth was only slightly affected at suboptimal pH of 4.0 and 6.0. Over a pH range of 6.0 to 3.5, yields of product formation from substrate and of biomass relative to ATP were constant. These results showed that L. fermentum Ogi E1 was particularly acid tolerant, and well adapted to the acid conditions that develop during natural fermentation of cereal doughs. This acid tolerance may partly explain the dominance of L. fermentum in various traditional African sourdoughs. Surprisingly, alpha-amylase production, unlike growth, dropped dramatically when the strain was cultivated at pH 4.0 with starch. With maltose as substrate, the yield of alpha-amylase relative to biomass remained unchanged at pH 4.0 and 5.0, unlike that observed with starch. Based on the distribution of enzyme activity between extra- and intracellular fractions and fermentation kinetics, it appears that starch was first hydrolyzed into dextrins by alpha-amylase activity, and maltose was produced from dextrins by extracellular enzyme activity, transferred into the cell and then hydrolyzed into glucose by intracellular alpha-glucosidase.

A comparative ecotoxicity analysis of α- and γ-phase aluminium oxide nanoparticles towards a freshwater bacterial isolate Bacillus licheniformis.

PubMed

Pakrashi, Sunandan; Kumar, Deepak; Iswarya, V; Bhuvaneshwari, M; Chandrasekaran, N; Mukherjee, Amitava

2014-12-01

Crystalline structure of nanoparticles may influence their physicochemical behaviour as well as their toxicological impact on biota. The differences in orientation of the atoms result in the variations in chemical stability. Thus, toxicological impacts of different crystalline phases of aluminium oxide nanoparticles are expected to vary. The present study brings out a comparative toxicity analysis of γ-phase and α-phase aluminium oxide nanoparticles of comparable hydrodynamic size range towards a freshwater bacterial isolate Bacillus licheniformis at low exposure concentrations (5, 1, 0.5 and 0.05 µg/mL). Upon 2-h exposure, the α-aluminium oxide particles showed lower toxicity than the γ-phase aluminium oxide. The lower level of oxidative stress generation and cell membrane damage in case of the α-phase aluminium oxide nanoparticles substantiated the toxicity results. The involvement of protein, lipopolysaccharides in nanoparticle-cell surface interaction, was noted in both the cases. To conclude, the crystallinity of aluminium oxide nanoparticles played an important role in the interaction and the toxicity response.

Norepinephrine infusion with and without alpha-adrenergic blockade by phentolamine increases salivary alpha amylase in healthy men.

PubMed

Kuebler, Ulrike; von Känel, Roland; Heimgartner, Nadja; Zuccarella-Hackl, Claudia; Stirnimann, Guido; Ehlert, Ulrike; Wirtz, Petra H

2014-11-01

Mental stress reliably induces increases in salivary alpha amylase (sAA), a suggested surrogate marker for sympathetic nervous system (SNS) reactivity. While stress-induced sAA increases correlate with norepinephrine (NE) secretion, a potential mediating role of noradrenergic mechanisms remains unclear. In this study, we investigated for the first time in humans whether a NE-stress-reactivity mimicking NE-infusion with and without alpha-adrenergic blockade by phentolamine would induce changes in sAA. In a single-blind placebo-controlled within-subjects design, 21 healthy men (29-66 years) took part in three different experimental trials varying in terms of substance infusion with a 1-min first infusion followed by a 15-min second infusion: saline-infusion (trial-1), NE-infusion (5 μg/min) without alpha-adrenergic blockade (trial-2), and with phentolamine-induced non-selective blockade of alpha1- and alpha2-adrenergic receptors (trial-3). Saliva samples were collected immediately before, during, and several times after substance infusion in addition to blood pressure and heart rate readings. Experimental trials significantly differed in sAA reactivity to substance-infusion (p=.001) with higher sAA reactivity following NE-infusion with (trial-3; p=.001) and without alpha-adrenergic-blockade (trial-2; p=.004) as compared to placebo-infusion (trial-1); sAA infusion reactivity did not differ between trial-2 and trial-3 (p=.29). Effective phentolamine application was verified by blood pressure and heart rate infusion reactivity. Salivary cortisol was not affected by NE, either with or without alpha-adrenergic-blockade. We found that NE-infusion stimulates sAA secretion, regardless of co-administered non-selective alpha-adrenergic blockade by phentolamine, suggesting that the mechanism underlying stress-induced sAA increases may involve NE. Copyright © 2014 Elsevier Ltd. All rights reserved.

Production and characterization of alpha-amylase from mango kernel by Fusarium solani NAIMCC-F-02956 using submerged fermentation.

PubMed

Kumar, Devendra; Yadav, Kaushlesh K; Muthukumar, M; Garg, Neelima

2013-11-01

Microbial production of enzymes using low valued agro industrial wastes is gaining importance globally. Mango is one of the major fruit processed into a variety of products. During processing 40-50% of solid waste is generated in form of peel and stones. After decortications of mango stone, kernel is obtained which is a rich source of starch (upto 60%). It was utilized as a substrate for alpha-amylase production using Fusarium soloni. Maximum alpha-amylase production (0.889 U g(-1)) was recorded using a substrate concentration of 5% (w/v), pH-4 and temperature 30 degrees C on 9th day of incubation. Supplementation of production medium with micronutrients viz., Ca2+, Fe2+ or Mg2+ improved the enzyme production while, Zn2+, B3+ or Mn2+ ions exhibited inhibitory effect. The extracellular protein was precipitated by ammonium sulphate up to 70% saturation, dialyzed and purified (27.84 fold) by gel-exclusion (Sephadex G-75) chromatography. Protein profiling on 12% SDS-PAGE revealed three bands corresponding to 26, 27 and 30 kDa molecular sizes. The optimum amylase activity was achieved at pH 5.0 at 40 degrees C. The Michaelis constant (KM), Vmax and activation energy (-Ea) were found to be 3.7 mg ml(-1), 0.24 U mg(-1) and 42.39 kJ mole(-1), respectively.

Enterotoxins and emetic toxins production by Bacillus cereus and other species of Bacillus isolated from Soumbala and Bikalga, African alkaline fermented food condiments.

PubMed

Ouoba, Labia Irene I; Thorsen, Line; Varnam, Alan H

2008-06-10

The ability of various species of Bacillus from fermented seeds of Parkia biglobosa known as African locust bean (Soumbala) and fermented seeds of Hibiscus sabdariffa (Bikalga) was investigated. The study included screening of the isolates by haemolysis on blood agar, detection of toxins in broth and during the fermentation of African locust bean using the Bacillus cereus Enterotoxin Reverse Passive Latex Agglutination test kit (BCET-RPLA) and the Bacillus Diarrhoeal Enterotoxin Visual Immunoassay (BDEVIA). Detection of genes encoding cytotoxin K (CytK), haemolysin BL (Hbl A, Hbl C, Hbl D), non-hemolytic enterotoxin (NheA, NheB, NheC) and EM1 specific of emetic toxin producers was also investigated using PCR with single pair and multiplex primers. Of 41 isolates, 29 Bacillus belonging to the species of B. cereus, Bacillus subtilis, Bacillus licheniformis and Bacillus pumilus showed haemolysis on blood agar. Using RPLA, enterotoxin production was detected for three isolates of B. cereus in broth and all B. cereus (9) in fermented seeds. Using BDEVIA, enterotoxin production was detected in broth as well as in fermented seeds for all B. cereus isolates. None of the isolates belonging to the other Bacillus species was able to produce enterotoxins either by RPLA or BDEVIA. Nhe genes were detected in all B. cereus while Hbl and CytK genes were detected respectively in five and six B. cereus strains. A weak presence of Hbl (A, D) and CytK genes was detected in two isolates of B. subtilis and one of B. licheniformis but results were inconsistent, especially for Hbl genes. The emetic specific gene fragment EM1 was not detected in any of the isolates studied.

Salivary Alpha Amylase as a Noninvasive Biomarker for Dental Fear and Its Correlation with Behavior of Children during Dental Treatment

PubMed Central

Noorani, Hina; Shivaprakash, PK

2014-01-01

ABSTrACT Objective: Objectives of our studies were to predict dental fear in a child patient depending on salivary alpha amylase (sAA) level before and after dental treatment and to evaluate correla­tion of later with behavior of child patient during dental treatment. Materials and methods: Seventy-seven children between age of 5 and 12 years were divided in three groups. Group 1 consisted of 25 school children who did not undergo any dental treatment. Groups 2 and 3 underwent dental treatment without and with local anesthesia respectively. Groups 2 and 3 were administered child fear survey schedule-dental subscale (CFSS-DS) questionnaire before treatment. Salivary samples were collected for sAA estimation in groups 2 and 3 children before and after completion of dental treatment and behavior during treatment was noted using Frankel behavior rating scale. Group 1 acted as control in which salivary sample was collected in absence of dental stress. Results: When groups 2 and 3 were combined, pretreatment sAA level had a statistically significant (p = 0.0094) correlation with CFSS-DS scores. Conclusion: Alpha amylase can be used as a screening tool to predict level of dental fear in a child patient. How to cite this article: Noorani H, Joshi HV, Shivaprakash PK. Salivary Alpha Amylase as a Noninvasive Biomarker for Dental Fear and Its Correlation with Behavior of Children during Dental Treatment. Int J Clin Pediatr Dent 2014;7(1):19-23. PMID:25206232

Purification of an alpha amylase from Aspergillus flavus NSH9 and molecular characterization of its nucleotide gene sequence.

PubMed

Karim, Kazi Muhammad Rezaul; Husaini, Ahmad; Sing, Ngieng Ngui; Sinang, Fazia Mohd; Roslan, Hairul Azman; Hussain, Hasnain

2018-04-01

In this study, an alpha-amylase enzyme from a locally isolated Aspergillus flavus NSH9 was purified and characterized. The extracellular α-amylase was purified by ammonium sulfate precipitation and anion-exchange chromatography at a final yield of 2.55-fold and recovery of 11.73%. The molecular mass of the purified α-amylase was estimated to be 54 kDa using SDS-PAGE and the enzyme exhibited optimal catalytic activity at pH 5.0 and temperature of 50 °C. The enzyme was also thermally stable at 50 °C, with 87% residual activity after 60 min. As a metalloenzymes containing calcium, the purified α-amylase showed significantly increased enzyme activity in the presence of Ca 2+ ions. Further gene isolation and characterization shows that the α-amylase gene of A. flavus NSH9 contained eight introns and an open reading frame that encodes for 499 amino acids with the first 21 amino acids presumed to be a signal peptide. Analysis of the deduced peptide sequence showed the presence of three conserved catalytic residues of α-amylase, two Ca 2+ -binding sites, seven conserved peptide sequences, and several other properties that indicates the protein belongs to glycosyl hydrolase family 13 capable of acting on α-1,4-bonds only. Based on sequence similarity, the deduced peptide sequence of A. flavus NSH9 α-amylase was also found to carry two potential surface/secondary-binding site (SBS) residues (Trp 237 and Tyr 409) that might be playing crucial roles in both the enzyme activity and also the binding of starch granules.

[Are amylases in bakery products and flour potential food allergens?].

PubMed

Baur, X; Sander, I; Jansen, A; Czuppon, A B

1994-05-21

The enzyme alpha-amylase from the mould Aspergillus oryzae (Asp o II) routinely used for the production of bread, cakes and pastries has in recent years been identified as an inhalative allergen for occupational diseases (bakers' asthma). It is doubtful whether this amylase in the final product, i.e. after the baking procedure, can still be regarded as an allergen. To clarify this question, detailed case histories on 138 subjects were recorded (98 allergics, 20 patients suffering form chronic intestinal diseases, 20 healthy controls). The clinical examinations included prick skin test and IgE antibody determination using one of the customary enzyme preparations. EAST showed a few of these 138 bread consumers to be weakly sensitized to the enzyme. One of the subjects displayed a significant reaction to alpha-amylase heated to 200 degrees C. As expected, eleven bakers sensitized to alpha-amylase by inhaling it in the workplace (positive prick test, positive case history) predominantly exhibited specific IgE antibodies to the native enzyme. Apart from one weakly positive finding, heated alpha-amylase yielded negative results in this collective. Baking conditions vary widely, especially with regard to single components, temperature and duration. Thus, further investigations as to residual allergenicity or the feasible occurrence of new antigenic determinants during the production of bread, cake and pastries are required. 27% of bakers examined and 9% of atopics showed antibodies to a flour inherent enzyme, a beta-amylase. On the whole, the selected conditions hinted at a weakly sensitizing potential inherent in baking flour and in added amylase.

Exploring potential applications of a novel extracellular polymeric substance synthesizing bacterium (Bacillus licheniformis) isolated from gut contents of earthworm (Metaphire posthuma) in environmental remediation.

PubMed

Biswas, Jayanta Kumar; Banerjee, Anurupa; Rai, Mahendra Kumar; Rinklebe, Jörg; Shaheen, Sabry M; Sarkar, Santosh Kumar; Dash, Madhab Chandra; Kaviraj, Anilava; Langer, Uwe; Song, Hocheol; Vithanage, Meththika; Mondal, Monojit; Niazi, Nabeel Khan

2018-05-22

The aim was to isolate, characterize, and explore potentials of gut bacteria from the earthworm (Metaphire posthuma) and imply these bacteria for remediation of Cu(II) and Zn(II). An extracellular polymeric substance (EPS) producing gut bacteria (Bacillus licheniformis strain KX657843) was isolated and identified based on 16S rRNA sequencing and phylogenetic analysis. The strain showed maximum tolerance of 8 and 6 mM for Cu(II) and Zn(II) respectively. It removed 34.5% of Cu(II) and 54.4% of Zn(II) at 25 mg L -1 after 72 and 96 h incubation respectively. The bacteria possessed a great potential to produce indole acetic acid (38.49 μg mL -1 ) at 5 mg mL -1 L-tryptophan following 12 days incubation. The sterilized seeds of mung beans (Vigna radiata) displayed greater germination and growth under bacterium enriched condition. We observed that the bacterial strain phosphate solubilization ability with a maximum of 204.2 mg L -1 in absence of Cu(II) and Zn(II). Endowed with biosurfactant property the bacterium exhibited 24% emulsification index. The bacterium offered significant potential of plant growth promotion, Cu(II) and Zn(II) removal, and as such this study is the first report on EPS producing B. licheniformis KX657843 from earthworm which can be applied as powerful tool in remediation programs of Cu(II) and Zn(II) contaminated sites.

Daytime Secretion of Salivary Cortisol and Alpha-Amylase in Preschool-Aged Children with Autism and Typically Developing Children

ERIC Educational Resources Information Center

Kidd, Sharon A.; Corbett, Blythe A.; Granger, Douglas A.; Boyce, W. Thomas; Anders, Thomas F.; Tager, Ira B.

2012-01-01

We examined daytime salivary cortisol and salivary alpha-amylase (sAA) secretion levels and variability in preschool-aged children with autism (AUT) and typically developing children (TYP). Fifty-two subjects (26 AUT and 26 TYP) were enrolled. Salivary samples were obtained at waking, midday, and bedtime on two consecutive days at three phases…

Maltose effects on barley malt diastatic power enzyme activity and thermostability at high isothermal mashing temperature: II. Alpha-amylase

USDA-ARS?s Scientific Manuscript database

Maltose, the primary product of starch degradation during mashing, has the potential as a compatible solute to affect the activity of and increase the thermostability of barley malt alpha-amylase activity at high temperatures used in mashing and temperatures above those normally used in mashing. To ...

Effects of microbial enzymes on starch and hemicellulose degradation in total mixed ration silages.

PubMed

Ning, Tingting; Wang, Huili; Zheng, Mingli; Niu, Dongze; Zuo, Sasa; Xu, Chuncheng

2017-02-01

This study investigated the association of enzyme-producing microbes and their enzymes with starch and hemicellulose degradation during fermentation of total mixed ration (TMR) silage. The TMRs were prepared with soybean curd residue, alfalfa hay (ATMR) or Leymus chinensis hay (LTMR), corn meal, soybean meal, vitamin-mineral supplements, and salt at a ratio of 25:40:30:4:0.5:0.5 on a dry matter basis. Laboratory-scale bag silos were randomly opened after 1, 3, 7, 14, 28, and 56 days of ensiling and subjected to analyses of fermentation quality, carbohydrates loss, microbial amylase and hemicellulase activities, succession of dominant amylolytic or hemicellulolytic microbes, and their microbial and enzymatic properties. Both ATMR and LTMR silages were well preserved, with low pH and high lactic acid concentrations. In addition to the substantial loss of water soluble carbohydrates, loss of starch and hemicellulose was also observed in both TMR silages with prolonged ensiling. The microbial amylase activity remained detectable throughout the ensiling in both TMR silages, whereas the microbial hemicellulase activity progressively decreased until it was inactive at day 14 post-ensiling in both TMR silages. During the early stage of fermentation, the main amylase-producing microbes were Bacillus amyloliquefaciens ( B. amyloliquefaciens ), B. cereus , B. licheniformis , and B. subtilis in ATMR silage and B. flexus , B. licheniformis , and Paenibacillus xylanexedens ( P. xylanexedens ) in LTMR silage, whereas Enterococcus faecium was closely associated with starch hydrolysis at the later stage of fermentation in both TMR silages. B. amyloliquefaciens , B. licheniformis , and B. subtilis and B. licheniformis , B. pumilus , and P. xylanexedens were the main source of microbial hemicellulase during the early stage of fermentation in ATMR and LTMR silages, respectively. The microbial amylase contributes to starch hydrolysis during the ensiling process in both TMR silages

Stability of thermostable alkaline protease from Bacillus licheniformis RP1 in commercial solid laundry detergent formulations.

PubMed

Sellami-Kamoun, Alya; Haddar, Anissa; Ali, Nedra El-Hadj; Ghorbel-Frikha, Basma; Kanoun, Safia; Nasri, Moncef

2008-01-01

The stability of crude extracellular protease produced by Bacillus licheniformis RP1, isolated from polluted water, in various solid laundry detergents was investigated. The enzyme had an optimum pH and temperature at pH 10.0-11.0 and 65-70 degrees C. Enzyme activity was inhibited by PMSF, suggesting that the preparation contains a serine-protease. The alkaline protease showed extreme stability towards non-ionic (5% Tween 20% and 5% Triton X-100) and anionic (0.5% SDS) surfactants, which retained 100% and above 73%, respectively, of its initial activity after preincubation 60 min at 40 degrees C. The RP1 protease showed excellent stability and compatibility with a wide range of commercial solid detergents at temperatures from 40 to 50 degrees C, suggesting its further application in detergent industry. The enzyme retained 95% of its initial activity with Ariel followed by Axion (94%) then Dixan (93.5%) after preincubation 60 min at 40 degrees C in the presence of 7 mg/ml of detergents. In the presence of Nadhif and New Det, the enzyme retained about 83.5% of the original activity. The effects of additives such as maltodextrin, sucrose and PEG 4000 on the stability of the enzyme during spray-drying and during subsequent storage in New Det detergent were also examined. All additives tested enhanced stability of the enzyme.

21 CFR 173.115 - Alpha-acetolactate decarboxylase (α-ALDC) enzyme preparation derived from a recombinant Bacillus...

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Alpha-acetolactate decarboxylase (α-ALDC) enzyme... FOOD FOR HUMAN CONSUMPTION Enzyme Preparations and Microorganisms § 173.115 Alpha-acetolactate decarboxylase (α-ALDC) enzyme preparation derived from a recombinant Bacillus subtilis. The food additive alpha...

Accuracy of alpha amylase in diagnosing microaspiration in intubated critically-ill patients.

PubMed

Dewavrin, Florent; Zerimech, Farid; Boyer, Alexandre; Maboudou, Patrice; Balduyck, Malika; Duhamel, Alain; Nseir, Saad

2014-01-01

Amylase concentration in respiratory secretions was reported to be a potentially useful marker for aspiration and pneumonia. The aim of this study was to determine accuracy of α-amylase in diagnosing microaspiration in critically ill patients. Retrospective analysis of prospectively collected data collected in a medical ICU. All patients requiring mechanical ventilation for at least 48 h, and included in a previous randomized controlled trial were eligible for this study, provided that at least one tracheal aspirate was available for α-amylase measurement. As part of the initial trial, pepsin was quantitatively measured in all tracheal aspirates during a 48-h period. All tracheal aspirates were frozen, allowing subsequent measurement of α-amylase for the purpose of the current study. Microaspiration was defined as the presence of at least one positive tracheal aspirate for pepsin (>200 ng.mL-1). Abundant microaspiration was defined as the presence of pepsin at significant level in >74% of tracheal aspirates. Amylase was measured in 1055 tracheal aspirates, collected from 109 patients. Using mean α-amylase level per patient, accuracy of α-amylase in diagnosing microaspiration was moderate (area under the receiver operator curve 0.72±0.05 [95%CI 0.61-0.83], for an α-amylase value of 1685 UI.L-1). However, when α-amylase levels, coming from all samples, were taken into account, area under the receiver operator curve was 0.56±0.05 [0.53-0.60]. Mean α-amylase level, and percentage of tracheal aspirates positive for α-amylase were significantly higher in patients with microaspiration, and in patients with abundant microaspiration compared with those with no microaspiration; and similar in patients with microaspiration compared with those with abundant microaspiration. α-amylase and pepsin were significantly correlated (r2 = 0.305, p = 0.001). Accuracy of mean α-amylase in diagnosing microaspiration is moderate. Further, when all α-amylase levels

Blunted cortisol response to psychosocial stress in atopic patients is associated with decrease in salivary alpha-amylase and aldosterone: Focus on sex and menstrual cycle phase.

PubMed

Hlavacova, N; Solarikova, P; Marko, M; Brezina, I; Jezova, D

2017-04-01

A decreased responsiveness of the hypothalamic-pituitary-adrenocortical axis to stress stimuli in patients with atopy is well documented. The aim of this study was to investigate personality traits, salivary alpha-amylase activity and the aldosterone response to psychosocial stress procedure based on public speech in atopic patients with respect to sex and the menstrual cycle (MC) phase. The study was performed in 106 subjects of both sexes, 53 atopic patients suffering from allergic rhinitis, allergic asthma or atopic dermatitis and 53 age-, sex-, the MC phase- and BMI- matched healthy controls. Substantially attenuated activity of alpha-amylase and reduced secretion of aldosterone during the psychosocial stress were observed in the whole sample of patients with atopy. Higher activity of alpha-amylase observed in the follicular compared to the luteal phase in healthy women was not present in atopic patients. In both males and females, atopy was associated with blunted cortisol response but no changes in the heart rate. Psychological characterization revealed a significantly higher trait anxiety and higher preference for avoidance-oriented coping strategy in female but not male atopic patients. These findings provide evidence that patients with atopy exhibit insufficient alpha-amylase and aldosterone responsiveness to psychosocial stress, thus suggesting decreased sympathetic activity. Potential disturbances in sex hormone status during the MC in female patients with atopy have to be considered in future research. Changes in personality traits were demonstrated in female atopic patients, but not in male patients. Copyright © 2017. Published by Elsevier Ltd.

Immunohistochemistry of active gibberellins and gibberellin-inducible alpha-amylase in developing seeds of morning glory.

PubMed

Nakayama, Akira; Park, Seijin; Zheng-Jun, Xu; Nakajima, Masatoshi; Yamaguchi, Isomaro

2002-07-01

Gibberellins (GAs) in developing seeds of morning glory (Pharbitis nil) were quantified and localized by immunostaining. The starch grains began to be digested after the GA contents had increased and reached a plateau. Immunohistochemical staining with the antigibberellin A(1)-methyl ester-antiserum, which has high affinity to biologically active GAs, showed that GA(1) and/or GA(3) were localized around starch grains in the integument of developing young seeds, suggesting the participation of GA-inducible alpha-amylase in this digestion. We isolated an alpha-amylase cDNA (PnAmy1) that was expressed in the immature seeds, and using an antibody raised against recombinant protein, it was shown that PnAmy1 was expressed in the immature seeds. GA responsiveness of PnAmy1 was shown by treating the young fruits 9 d after anthesis with GA(3). RNA-blot and immunoblot analyses showed that PnAmy1 emerged soon after the rapid increase of GA(1/3). An immunohistochemical analysis of PnAmy1 showed that it, like the seed GA(1/3), was also localized around starch grains in the integument of developing young seeds. The localization of GA(1/3) in the integument coincident with the expression of PnAmy1 suggests that both function as part of a process to release sugars for translocation or for the further development of the seeds.

The Characterisation of an Alkali-Stable Maltogenic Amylase from Bacillus lehensis G1 and Improved Malto-Oligosaccharide Production by Hydrolysis Suppression

PubMed Central

Abdul Manas, Nor Hasmaliana; Pachelles, Samson; Mahadi, Nor Muhammad; Illias, Rosli Md.

2014-01-01

A maltogenic amylase (MAG1) from alkaliphilic Bacillus lehensis G1 was cloned, expressed in Escherichia coli, purified and characterised for its hydrolysis and transglycosylation properties. The enzyme exhibited high stability at pH values from 7.0 to 10.0. The hydrolysis of β-cyclodextrin (β-CD) produced malto-oligosaccharides of various lengths. In addition to hydrolysis, MAG1 also demonstrated transglycosylation activity for the synthesis of longer malto-oligosaccharides. The thermodynamic equilibrium of the multiple reactions was shifted towards synthesis when the reaction conditions were optimised and the water activity was suppressed, which resulted in a yield of 38% transglycosylation products consisting of malto-oligosaccharides of various lengths. Thin layer chromatography and high-performance liquid chromatography analyses revealed the presence of malto-oligosaccharides with a higher degree of polymerisation than maltoheptaose, which has never been reported for other maltogenic amylases. The addition of organic solvents into the reaction further suppressed the water activity. The increase in the transglycosylation-to-hydrolysis ratio from 1.29 to 2.15 and the increased specificity toward maltopentaose production demonstrated the enhanced synthetic property of the enzyme. The high transglycosylation activity of maltogenic amylase offers a great advantage for synthesising malto-oligosaccharides and rare carbohydrates. PMID:25221964

Agro-industrial residues and starch for growth and co-production of polyhydroxyalkanoate copolymer and α-amylase by Bacillus sp. CFR-67.

PubMed

Shamala, T R; Vijayendra, S V N; Joshi, G J

2012-07-01

Polyhydroxyalkanoates (PHA) and α-amylase (α-1,4 glucan-4-glucanohydrolase, E.C. 3.2.1.1) were co-produced by Bacillus sp. CFR-67 using unhydrolysed corn starch as a substrate. Bacterial growth and polymer production were enhanced with the supplementation of hydrolysates of wheat bran (WBH) or rice bran (RBH) individually or in combination (5-20 g L(-1), based on weight of soluble substrates-SS). In batch cultivation, a mixture of WBH and RBH (1:1, 10 g L(-1) of SS) along with ammonium acetate (1.75 g L(-1)) and corn starch (30 g L(-1)) produced maximum quantity of biomass (10 g L(-1)) and PHA (5.9 g L(-1)). The polymer thus produced was a copolymer of polyhydroxybutyrate-co-hydroxyvalerate of 95:5 to 90:10 mol%. Presence of WBH and corn starch (10-50 g L(-1)) in the medium enhanced fermentative yield of α-amylase (2-40 U mL(-1) min(-1)). The enzyme was active in a wide range of pH (4-9) and temperature (40-60°C). This is the first report on simultaneous production of copolymer of bacterial PHA and α-amylase from unhydrolysed corn starch and agro-industrial residues as substrates.

A thermophilic alkalophilic α-amylase from Bacillus sp. AAH-31 shows a novel domain organization among glycoside hydrolase family 13 enzymes.

PubMed

Saburi, Wataru; Morimoto, Naoki; Mukai, Atsushi; Kim, Dae Hoon; Takehana, Toshihiko; Koike, Seiji; Matsui, Hirokazu; Mori, Haruhide

2013-01-01

α-Amylases (EC 3.2.1.1) hydrolyze internal α-1,4-glucosidic linkages of starch and related glucans. Bacillus sp. AAH-31 produces an alkalophilic thermophilic α-amylase (AmyL) of higher molecular mass, 91 kDa, than typical bacterial α-amylases. In this study, the AmyL gene was cloned to determine its primary structure, and the recombinant enzyme, produced in Escherichia coli, was characterized. AmyL shows no hydrolytic activity towards pullulan, but the central region of AmyL (Gly395-Asp684) was similar to neopullulanase-like α-amylases. In contrast to known neopullulanase-like α-amylases, the N-terminal region (Gln29-Phe102) of AmyL was similar to carbohydrate-binding module family 20 (CBM20), which is involved in the binding of enzymes to starch granules. Recombinant AmyL showed more than 95% of its maximum activity in a pH range of 8.2-10.5, and was stable below 65 °C and from pH 6.4 to 11.9. The kcat values for soluble starch, γ-cyclodextrin, and maltotriose were 103 s(-1), 67.6 s(-1), and 5.33 s(-1), respectively, and the Km values were 0.100 mg/mL, 0.348 mM, and 2.06 mM, respectively. Recombinant AmyL did not bind to starch granules. But the substitution of Trp45 and Trp84, conserved in site 1 of CBM20, with Ala reduced affinity to soluble starch, while the mutations did not affect affinity for oligosaccharides. Substitution of Trp61, conserved in site 2 of CBM20, with Ala enhanced hydrolytic activity towards soluble starch, indicating that site 2 of AmyL does not contribute to binding to soluble long-chain substrates.

THESIS-ABSTRACT Supplementation levels of exogenous alpha-amylase in broilers diets.

PubMed

Oliveira, H B; Silva, M I A; Mesquita, F R

2017-08-17

This study aimed to evaluate the supplementation levels of an exogenous alpha-amylase in broilers diets and compare two indicators in determining the diets energy. The experiment was divided into two parallel evaluations, being one of performance and the other of metabolism. In performance assay, 1,700 one-day-old Cobb-500 male chicks were used. The animals were distributed in 50 experimental plots and evaluated five treatments with ten replicates in a completely randomized design (CRD). The treatments were: a positive control (PC), a negative control (NC) and three alpha-amylase supplementation levels 200, 400 and 600 g/t, and the NC was formulated with 50 and 90 kcal of energy reduction in relation to the PC to the phases from 1 to 21 days and from 22 to 42 days, respectively. In the metabolism assay were used 240 animals, 150 birds for stage from 14 to 21 days and 90 birds to stage from 35 to 42 days of age and the treatments were the same as the performance assay, with six replicates per treatment in CRD. All diets of metabolism test contained the digestibility indicators Lipe ® (eucalyptus purified lignin) and chromic oxide (Cr 2 O 3 ), in concentrations of 0.05 and 1.0%, respectively. In the period from 1 to 21 days old, no significant differences were observed in weight gain (WG) (P > 0.05), however, feed intake (FI) was found higher by using 200 ppm of enzyme (P < 0.05) and better feed conversion (FC) with the PC (P < 0.05). From 22 to 42 days, no significant differences were observed on the WG (P > 0.05), but were observed lower FI and better FC to PC treatment (P < 0.05). In the period from 1 to 42 days of age, significant differences were also not observed on the WG (P > 0.05), but there was lower FI and better FC for the PC treatment (P < 0.05). The AMEn (apparent metabolizable energy corrected for nitrogen balance), determined using the total collection, reaffirmed the values ​​calculated for the PC and NC with intermediate data obtained from the

Effect of oxygen mass transfer rate on the production of 2,3-butanediol from glucose and agro-industrial byproducts by Bacillus licheniformis ATCC9789.

PubMed

Rebecchi, Stefano; Pinelli, Davide; Zanaroli, Giulio; Fava, Fabio; Frascari, Dario

2018-01-01

2,3-Butanediol (BD) is a largely used fossil-based platform chemical. The yield and productivity of bio-based BD fermentative production must be increased and cheaper substrates need to be identified, to make bio-based BD production more competitive. As BD bioproduction occurs under microaerobic conditions, a fine tuning and control of the oxygen transfer rate (OTR) is crucial to maximize BD yield and productivity. Very few studies on BD bioproduction focused on the use of non-pathogenic microorganisms and of byproducts as substrate. The goal of this work was to optimize BD bioproduction by the non-pathogenic strain Bacillus licheniformis ATCC9789 by (i) identifying the ranges of volumetric and biomass-specific OTR that maximize BD yield and productivity using standard sugar and protein sources, and (ii) performing a preliminary evaluation of the variation in process performances and cost resulting from the replacement of glucose with molasses, and beef extract/peptone with chicken meat and bone meal, a byproduct of the meat production industry. OTR optimization with an expensive, standard medium containing glucose, beef extract and peptone revealed that OTRs in the 7-15 mmol/L/h range lead to an optimal BD yield (0.43 ± 0.03 g/g) and productivity (0.91 ± 0.05 g/L/h). The corresponding optimal range of biomass-specific OTR was equal to 1.4-7.9 [Formula: see text], whereas the respiratory quotient ranged from 1.8 to 2.5. The switch to an agro-industrial byproduct-based medium containing chicken meat and bone meal and molasses led to a 50% decrease in both BD yield and productivity. A preliminary economic analysis indicated that the use of the byproduct-based medium can reduce by about 45% the BD production cost. A procedure for OTR optimization was developed and implemented, leading to the identification of a range of biomass-specific OTR and respiratory quotient to be used for the scale-up and control of BD bioproduction by Bacillus licheniformis

RNA interference-based silencing of the alpha-amylase (amy1) gene in Aspergillus flavus decreases fungal growth and aflatoxin production in maize kernels.

PubMed

Gilbert, Matthew K; Majumdar, Rajtilak; Rajasekaran, Kanniah; Chen, Zhi-Yuan; Wei, Qijian; Sickler, Christine M; Lebar, Matthew D; Cary, Jeffrey W; Frame, Bronwyn R; Wang, Kan

2018-06-01

Expressing an RNAi construct in maize kernels that targets the gene for alpha-amylase in Aspergillus flavus resulted in suppression of alpha-amylase (amy1) gene expression and decreased fungal growth during in situ infection resulting in decreased aflatoxin production. Aspergillus flavus is a saprophytic fungus and pathogen to several important food and feed crops, including maize. Once the fungus colonizes lipid-rich seed tissues, it has the potential to produce toxic secondary metabolites, the most dangerous of which is aflatoxin. The pre-harvest control of A. flavus contamination and aflatoxin production is an area of intense research, which includes breeding strategies, biological control, and the use of genetically-modified crops. Host-induced gene silencing, whereby the host crop produces siRNA molecules targeting crucial genes in the invading fungus and targeting the gene for degradation, has shown to be promising in its ability to inhibit fungal growth and decrease aflatoxin contamination. Here, we demonstrate that maize inbred B104 expressing an RNAi construct targeting the A. flavus alpha-amylase gene amy1 effectively reduces amy1 gene expression resulting in decreased fungal colonization and aflatoxin accumulation in kernels. This work contributes to the development of a promising technology for reducing the negative economic and health impacts of A. flavus growth and aflatoxin contamination in food and feed crops.

OPTIMIZATION OF ALKALINE Α-AMYLASE PRODUCTION BY THERMOPHILIC BACILL US SUBTILIS.

PubMed

Al-Johani, Nuha Bakeet; Al-Seeni, Madeha N; Ahmed, Youssri Mohamed

2017-01-01

Starch-degrading amylase enzyme is important in biotechnological applications as food, fermentation, textile, paper and pharmaceutical purposes. The aim of current study to isolate alkaline thermostable α-amylase bacteria and then study the composition of medium and culture conditions to optimize cells growth and a-amylase production. Thermophilic amylase producing bacterium was isolated from local hot water-springs in Gazan city Saudi Arabia. Phylogenetic analysis of 16 S rRNA sequence for the strain revealed that the strain have the same sequence of Bacillus subtilis . Maximum amylase production was observed, when B. subtilis cultured in medium containing starch at concentration 0.5%, and 10 g/L peptones as nitrogen source at pH 8.5 in when it was incubated for 48 h at 45°C. An amylase-producing bacterium were isolated from hot-spring water and was identified as B. subtilis . Amylase produced from B.subtilis had optimum temperature 45°C and pH 8.5 in shaking media.

Extracellular facile biosynthesis, characterization and stability of gold nanoparticles by Bacillus licheniformis.

PubMed

Singh, Sneha; Vidyarthi, Ambarish Sharan; Nigam, Vinod Kumar; Dev, Abhimanyu

2014-02-01

The development of a reliable, eco-friendly process for synthesis of gold nanoparticles (AuNPs) has gained impetus in recent years to counter the drawbacks of chemical and physical methods. This study illustrates simple, green synthesis of AuNPs in vitro using cell lysate supernatant (CLS) of non-pathogenic bacteria and to investigate its potential antimicrobial activity. Gold nanoparticles were synthesized by the reduction of precursor AuCl4- ions using the CLS of Bacillus licheniformis at 37°C upon 24 h of incubation. The nanoparticles were characterized for their morphology, particle size, optical absorption, zeta potential, and stability. Further the antimicrobial activity was assayed using cup-plate method. The process of biosynthesis was extracellular and the gold ions were reduced to stable nanogold of average size 38 nm. However, upon storage of AuNPs for longer duration at room temperature stability was influenced in terms of increase in particle size and decrease in zeta potential with respect to as synthesized nanoparticles. SEM micrographs revealed the spherical shape of AuNPs and EDX analysis confirmed the presence of gold in the sample. Also clear zone of inhibition was observed against Bacilllus subtilis MTCC 8364, Pseudomonas aeruginosa MTCC 7925, and Escherichia coli MTCC 1698 confirming the antimicrobial activity of AuNPs. The bioprocess under study was simple and less time consuming as compared to other methods as the need for harvesting AuNPs from within the microbial cells via downstream process will be eliminated. Nanoparticles exhibited good stability even in absence of external stabilizing agents. AuNPs showed good antimicrobial activity against several Gram-negative and Gram-positive pathogenic bacteria. The extracellular biosynthesis from CLS may serve as a suitable alternative for large scale synthesis of gold nanoparticles in vitro. The synthesis from lysed bacterial cell strongly suggests that exposure of microbial whole cells to the

Characterization of thermostable cellulase produced by Bacillus strains isolated from solid waste of carrageenan

NASA Astrophysics Data System (ADS)

Listyaningrum, N. P.; Sutrisno, A.; Wardani, A. K.

2018-03-01

Cellulase-producing bacteria was isolated from solid waste of carrageenan and identified as Bacillus licheniformis C55 by 16S rRNA sequencing. The optimum condition for cellulase production was obtained at pH and temperature of 8.0 and 50°C, respectively in a medium containing glucose as carbon source and 1.0% carboxymethyl cellulose (CMC) to stimulate the cellulase production. Most remarkably, the enzyme retained its relative activity over 50% after incubation at 50°C for 90 minutes. Substrate specificity suggested that the enzyme is an endoglucanase. The molecular mass of Bacillus licheniformis C55 crude cellulase was found about 18 kDa by SDS-PAGE analysis. This thermostable enzyme would facilitate development of more efficient and cost-effective forms of the process to convert lignocellulosic biomass into high-value products.

Alternative method for quantification of alfa-amylase activity.

PubMed

Farias, D F; Carvalho, A F U; Oliveira, C C; Sousa, N M; Rocha-Bezerrra, L C B; Ferreira, P M P; Lima, G P G; Hissa, D C

2010-05-01

A modification of the sensitive agar diffusion method was developed for macro-scale determination of alfa-amylase. The proposed modifications lower costs with the utilisation of starch as substrate and agar as supporting medium. Thus, a standard curve was built using alfa-amylase solution from Aspergillus oryzae, with concentrations ranging from 2.4 to 7,500 U.mL-1. Clear radial diffusion zones were measured after 4 hours of incubation at 20 A degrees C. A linear relationship between the logarithm of enzyme activities and the area of clear zones was obtained. The method was validated by testing alpha-amylase from barley at the concentrations of 2.4; 60; 300 and 1,500 U.mL-1. The proposed method turned out to be simpler, faster, less expensive and able to determine on a macro-scale alpha-amylase over a wide range (2.4 to 7,500 U.mL-1) in scientific investigation as well as in teaching laboratory activities.

The role of two isoenzymes of alpha-amylase of Araucaria araucana (Araucariaceae) on the digestion of starch granules during germination.

PubMed

Waghorn, Juana J; del Pozo, Talía; Acevedo, Elba A; Cardemil, Liliana A

2003-03-01

Starch is the principal reserve of Araucaria araucana seeds, and it is hydrolysed during germination mainly by alpha-amylase. There are several alpha-amylase isoenzymes whose patterns change in the embryo and in the megagametophyte from the one observed in quiescent seeds (T(0)) to a different one observed 90 h after imbibition (T(90)). The objective of this research was to study the roles of two purified alpha-amylase isoenzymes by in vitro digestion of starch granules extracted from the tissues at two times of imbibition: one is abundant in quiescent seeds and the other is abundant after 90 h of imbibition. The isoenzymes digested the starch granules of their own stage of germination better, since the isoenzyme T(0) digested starch granules mainly from quiescent seeds, while the isoenzyme T(90) digested starch mainly at 90 h of imbibition. The sizes of the starch granule and the tissue from which these granules originated make a difference to digestion by the isoenzymes. Embryonic isoenzyme T(0) digested large embryonic starch granules better than small and medium-sized granules, and better than those isolated from megagametophytes. Similarly isoenzyme T(90) digested small embryonic starch granules better than medium-sized and large granules, and better than those isolated from megagametophytes. However, a mixture of partially purified megagametophytic isoenzymes T(0) and T(90) digested the megagametophytic granules better than those isolated from embryos. Studies of in vitro sequential digestion of starch granules with these isoenzymes corroborated their specificity. The isoenzyme T(90) digested starch granules previously digested by the isoenzyme T(0). This suggests that in vivo these two isoenzymes may act sequentially in starch granule digestion.

A proprietary alpha-amylase inhibitor from white bean (Phaseolus vulgaris): A review of clinical studies on weight loss and glycemic control

PubMed Central

2011-01-01

Obesity, and resultant health hazards which include diabetes, cardiovascular disease and metabolic syndrome, are worldwide medical problems. Control of diet and exercise are cornerstones of the management of excess weight. Foods with a low glycemic index may reduce the risk of diabetes and heart disease as well as their complications. As an alternative to a low glycemic index diet, there is a growing body of research into products that slow the absorption of carbohydrates through the inhibition of enzymes responsible for their digestion. These products include alpha-amylase and glucosidase inhibitors. The common white bean (Phaseolus vulgaris) produces an alpha-amylase inhibitor, which has been characterized and tested in numerous clinical studies. A specific and proprietary product named Phase 2® Carb Controller (Pharmachem Laboratories, Kearny, NJ) has demonstrated the ability to cause weight loss with doses of 500 to 3000 mg per day, in either a single dose or in divided doses. Clinical studies also show that Phase 2 has the ability to reduce the post-prandial spike in blood glucose levels. Experiments conducted incorporating Phase 2 into food and beverage products have found that it can be integrated into various products without losing activity or altering the appearance, texture or taste of the food. There have been no serious side effects reported following consumption of Phase 2. Gastro-intestinal side effects are rare and diminish upon extended use of the product. In summary, Phase 2 has the potential to induce weight loss and reduce spikes in blood sugar caused by carbohydrates through its alpha-amylase inhibiting activity. PMID:21414227

A proprietary alpha-amylase inhibitor from white bean (Phaseolus vulgaris): a review of clinical studies on weight loss and glycemic control.

PubMed

Barrett, Marilyn L; Udani, Jay K

2011-03-17

Obesity, and resultant health hazards which include diabetes, cardiovascular disease and metabolic syndrome, are worldwide medical problems. Control of diet and exercise are cornerstones of the management of excess weight. Foods with a low glycemic index may reduce the risk of diabetes and heart disease as well as their complications. As an alternative to a low glycemic index diet, there is a growing body of research into products that slow the absorption of carbohydrates through the inhibition of enzymes responsible for their digestion. These products include alpha-amylase and glucosidase inhibitors. The common white bean (Phaseolus vulgaris) produces an alpha-amylase inhibitor, which has been characterized and tested in numerous clinical studies. A specific and proprietary product named Phase 2® Carb Controller (Pharmachem Laboratories, Kearny, NJ) has demonstrated the ability to cause weight loss with doses of 500 to 3000 mg per day, in either a single dose or in divided doses. Clinical studies also show that Phase 2 has the ability to reduce the post-prandial spike in blood glucose levels. Experiments conducted incorporating Phase 2 into food and beverage products have found that it can be integrated into various products without losing activity or altering the appearance, texture or taste of the food. There have been no serious side effects reported following consumption of Phase 2. Gastro-intestinal side effects are rare and diminish upon extended use of the product. In summary, Phase 2 has the potential to induce weight loss and reduce spikes in blood sugar caused by carbohydrates through its alpha-amylase inhibiting activity.

Development and application of active films for food packaging using antibacterial peptide of Bacillus licheniformis Me1.

PubMed

Nithya, V; Murthy, P S K; Halami, P M

2013-08-01

An attempt was made to evaluate the effectiveness of partially purified antibacterial peptide (ppABP) produced by Bacillus licheniformis Me1 for food preservation by means of active packaging. The active packaging films containing ppABP were developed using two different packing materials [low-density polyethylene (LDPE) and cellulose films] by two different methods: soaking and spread coating. The activated films showed antibacterial activity against pathogens. The release study of ppABP from coated film showed that the LDPE films liberated ppABP as soon as it comes in contact with water, while gradual release of coated ppABP was observed in case of cellulose films. The activated films showed residual activity in different simulating conditions, such as pH of food and storage temperatures. The activated films demonstrated its biopreservative efficacy in controlling the growth of pathogens in cheese and paneer. The ppABP-activated films were found to be effective for biopreservation. The ppABP from active films got diffused into the food matrix and reduced the growth rate and maximum growth population of the target micro-organism. Both types of ppABP-activated films can be used as a packaging material to control spoilage and pathogenic organisms in food, thereby extending the shelf life of foods. © 2013 The Society for Applied Microbiology.

Discovering an Accessible Enzyme: Salivary [alpha]-Amylase--"Prima Digestio Fit in Ore"--A Didactic Approach for High School Students

ERIC Educational Resources Information Center

Marini, Isabella

2005-01-01

Human salivary [alpha]-amylase is used in this experimental approach to introduce biology high school students to the concept of enzyme activity in a dynamic way. Through a series of five easy, rapid, and inexpensive laboratory experiments students learn what the activity of an enzyme consists of: first in a qualitative then in a semi-quantitative…

Chestnut astringent skin extract, an alpha-amylase inhibitor, retards carbohydrate absorption in rats and humans.

PubMed

Tsujita, Takahiro; Takaku, Takeshi; Suzuki, Tsuneo

2008-02-01

Inhibitors of carbohydrate-hydrolyzing enzyme play an important role to control postprandial blood glucose levels. In this paper, we investigated the effect of an ethanol extract from chestnut astringent skin (CAS) on alpha-amylase. Chestnut astringent skin extract strongly inhibited human and porcine pancreatic alpha-amylase. We also investigated the effect of CAS extract on carbohydrate absorption in rats and humans. Oral administration of CAS extract to normal rats fed corn starch (2 g/kg body weight), significantly suppressed the increase of blood glucose levels after starch loading in a dose-dependent manner. The effective dose of CAS extract required to achieve 20 and 40% suppression of the rise in blood glucose level was estimated to be 40 and 155 mg/kg body weight, respectively. Chestnut astringent skin extract also suppressed the rise in plasma insulin level and the fall in plasma non-esterified fatty acid level. In the type 2 diabetic rat model, CAS extract significantly suppressed the rise in blood glucose level after starch loading in a dose-dependent manner. Chestnut astringent skin extract also suppressed the rise in plasma glucose level after boiled rice loading in a dose-dependent manner in humans. The amount of CAS extract required to achieve 11 and 23% suppression in the rise in plasma glucose level was 300 and 600 mg/person, respectively. These results suggest that CAS extract retards absorption of carbohydrate and reduces post-prandial hyperglycemia.

Optimization of medium components and cultural variables for enhanced production of acidic high maltose-forming and Ca2+-independent α-amylase by Bacillus acidicola.

PubMed

Sharma, Archana; Satyanarayana, Tulasi

2011-05-01

The production of acidic α-amylase by a novel acidophilic bacterium Bacillus acidicola TSAS1 was optimized in submerged fermentation using statistical approaches. The process parameters that significantly affected α-amylase production (starch, K(2)HPO(4), inoculum size and temperature) were identified by Plackett and Burman design. The optimum levels of the significant variables as determined using central composite design of response surface methodology are starch (2.75%), K(2)HPO(4) (0.01%), inoculum size [2% (v/v) containing 1.9×10(8) CFU ml(-1)], and temperature (33°C). An overall 2.4 and 2.9-fold increase in enzyme production has been attained in batch and fed-batch fermentations in the laboratory fermentor, respectively. Copyright © 2011 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Characterisation of three starch degrading enzymes: thermostable β-amylase, maltotetraogenic and maltogenic α-amylases.

PubMed

Derde, L J; Gomand, S V; Courtin, C M; Delcour, J A

2012-11-15

Maltogenic α-amylase from Bacillus stearothermophilus (BStA) is widely used as bread crumb anti-firming enzyme. A maltotetraose-forming α-amylase from Pseudomonas saccharophila (PSA) was recently proposed as alternative, hence the need to compare both exo-acting enzymes with some endo-action component. A purely exo-acting thermostable β-amylase from Clostridium thermosulfurogenes (CTB) was included for reference purposes. Under the experimental conditions used, temperature optima of the enzymes are rather similar (60-65 °C), but temperature stability decreased in the order BStA, PSA and CTB. The action of the enzymes on different substrates and their impact on the rheological behaviour of maize starch suspensions demonstrated that, while CTB acts exclusively through an exo-action mechanism, BStA displayed limited endo-action which became more pronounced at higher temperatures. PSA has more substantial endo-action than BStA, which is rather temperature independent. This is important for their impact in processes such as breadmaking, where temperature is gradually increased. Copyright © 2012 Elsevier Ltd. All rights reserved.

Identification of Bacillus spp. from Bikalga, fermented seeds of Hibiscus sabdariffa: phenotypic and genotypic characterization.

PubMed

Ouoba, L I I; Parkouda, C; Diawara, B; Scotti, C; Varnam, A H

2008-01-01

To identify Bacillus spp. responsible of the fermentation of Hibiscus sabdariffa for production of Bikalga, an alkaline fermented food used as a condiment in Burkina Faso. Seventy bacteria were isolated from Bikalga produced in different regions of Burkina Faso and identified by phenotyping and genotyping using PCR amplification of the 16S-23S rDNA intergenic transcribed spacer (ITS-PCR), repetitive sequence-based PCR (rep-PCR) and DNA sequencing. The isolates were characterized as motile, rod-shaped, endospore forming, catalase positive, Gram-positive bacteria. ITS-PCR allowed typing mainly at species level. Rep-PCR was more discriminative and allowed a typing at ssp. level. The DNA sequencing combined with the Blast search program and fermentation profiles using API 50CHB system allowed an identification of the bacteria as Bacillus subtilis, B. licheniformis, B. cereus, B. pumilus, B. badius, Brevibacillus bortelensis, B. sphaericus and B. fusiformis. B. subtilis were the predominant bacterium (42) followed by B. licheniformis (16). Various species and ssp. of Bacillus are involved in fermentation of H. sabdariffa for production of Bikalga. Selection of starter cultures of Bacillus for controlled production of Bikalga, selection of probiotic bacteria.

Evaluation of certain food additives and contaminants.

PubMed

2004-01-01

This report represents the conclusions of a Joint FAO/WHO Expert Committee convened to evaluate the safety of various food additives, with a view to recommending acceptable daily intakes (ADIs) and to prepare specifications for the identity and purity of food additives. The first part of the report contains a general discussion of the principles governing the toxicological evaluation of food additives (including flavouring agents) and contaminants, assessments of intake, and the establishment and revision of specifications for food additives. A summary follows of the Committee's evaluations of toxicological and intake data on various specific food additives (alpha-amylase from Bacillus lichenformis containing a genetically engineered alpha-amylase gene from B. licheniformis, annatto extracts, curcumin, diacetyl and fatty acid esters of glycerol, D-tagatose, laccase from Myceliophthora thermophila expressed in Aspergillus oryzae, mixed xylanase, beta-glucanase enzyme preparation produced by a strain of Humicola insolens, neotame, polyvinyl alcohol, quillaia extracts and xylanase from Thermomyces lanuginosus expressed in Fusarium venenatum), flavouring agents, a nutritional source of iron (ferrous glycinate, processed with citric acid), a disinfectant for drinking-water (sodium dichloroisocyanurate) and contaminants (cadmium and methylmercury). Annexed to the report are tables summarizing the Committee's recommendations for ADIs of the food additives, recommendations on the flavouring agents considered, and tolerable intakes of the contaminants considered, changes in the status of specifications and further information requested or desired.

Salivary alpha amylase in on-call from home fire and emergency service personnel.

PubMed

Hall, Sarah J; Aisbett, Brad; Robertson, Samuel J; Ferguson, Sally A; Turner, Anne I

2017-11-01

The effect of working on-call from home on the sympatho-adrenal medullary system activity is currently unknown. This study had two aims, Aim 1: examine salivary alpha amylase awakening response (AAR) and diurnal salivary alpha amylase (sAA) profile in fire and emergency service workers who operate on-call from home following a night on-call with a call (NIGHT-CALL), a night on-call without a call (NO-CALL) and an off-call night (OFF-CALL), and Aim 2: explore whether there was an anticipatory effect of working on-call from home (ON) compared to when there was an off-call (OFF) on the diurnal sAA profile. Participants wore activity monitors, completed sleep and work diaries and collected seven saliva samples a day for one week. AAR area under the curve with respect to ground (AUC G ), AAR area under the curve with respect to increase (AUC I ), AAR reactivity, diurnal sAA slope, diurnal sAA AUC G and mean 12-h sAA concentrations were calculated. Separate generalised estimating equation models were constructed for each variable of interest for each aim. For Aim 1, there were no differences between NIGHT-CALL or NO-CALL and OFF-CALL for any response variable. For Aim 2, there was no difference between any response variable of interest when ON the following night compared to when OFF the following night ( n  = 14). These findings suggest that there is no effect of working on-call from home on sAA, but should be interpreted with caution, as overnight data were not collected. Future research, using overnight heart rate monitoring, could help confirm these findings. © 2017 The authors.

Evaluation of alpha- amylase inhibition by Urtica dioica and Juglans regia extracts.

PubMed

Rahimzadeh, Mahsa; Jahanshahi, Samaneh; Moein, Soheila; Moein, Mahmood Reza

2014-06-01

One strategy for the treatment of diabetes is inhibition of pancreatic α- amylase. Plants contains different chemical constituents with potential for inhibition of α-amylase and hence maybe used as therapeutic. Urtica dioica and Juglans regia Linn were tested for α-amylase inhibition. Different concentrations of leaf aqueous extracts were incubated with enzyme substrate solution and the activity of enzyme was measured. For determination of the type of inhibition, Dixon plot was depicted. Acarbose was used as the standard inhibitor. Both plant extracts showed time and concentration dependent inhibition of α-amylase. 60% inhibition was seen with 2 mg/ml of U. dioica and 0.4 mg/ml of J. regia aqueous extract. Dixon plots revealed the type of α-amylase inhibition by these two extracts as competitive inhibition. Determination of the type of α-amylase inhibition by these plant extracts could provide by successful use of plant chemicals as drug targets.

Evaluation of alpha- amylase inhibition by Urtica dioica and Juglans regia extracts

PubMed Central

Rahimzadeh, Mahsa; Jahanshahi, Samaneh; Moein, Soheila; Moein, Mahmood Reza

2014-01-01

Objective(s): One strategy for the treatment of diabetes is inhibition of pancreatic α- amylase. Plants contains different chemical constituents with potential for inhibition of α-amylase and hence maybe used as therapeutic. Materials and Methods: Urtica dioica and Juglans regia Linn were tested for α-amylase inhibition. Different concentrations of leaf aqueous extracts were incubated with enzyme substrate solution and the activity of enzyme was measured. For determination of the type of inhibition, Dixon plot was depicted. Acarbose was used as the standard inhibitor. Results: Both plant extracts showed time and concentration dependent inhibition of α-amylase. 60% inhibition was seen with 2 mg/ml of U. dioica and 0.4 mg/ml of J. regia aqueous extract. Dixon plots revealed the type of α-amylase inhibition by these two extracts as competitive inhibition. Conclusion: Determination of the type of α-amylase inhibition by these plant extracts could provide by successful use of plant chemicals as drug targets. PMID:25140210

Impact of amylases on biopolymer dynamics during storage of straight-dough wheat bread.

PubMed

Bosmans, Geertrui M; Lagrain, Bert; Fierens, Ellen; Delcour, Jan A

2013-07-03

When Bacillus stearothermophilus α-amylase (BStA), Pseudomonas saccharophila α-amylase (PSA), or Bacillus subtilis α-amylase (BSuA) was added to a bread recipe to impact bread firming, amylose crystal formation was facilitated, leading to lower initial crumb resilience. Bread loaves that best retained their quality were those obtained when BStA was used. The enzyme hindered formation of an extended starch network, resulting in less water immobilization and smaller changes in crumb firmness and resilience. BSuA led to extensive degradation of the starch network during bread storage with release of immobilized water, eventually resulting in partial structure collapse and poor crumb resilience. The most important effect of PSA was an increased bread volume, resulting in smaller changes in crumb firmness and resilience. A negative linear relation was found between NMR proton mobilities of water and biopolymers in the crumb and crumb firmness. The slope of that relation gave an indication of the strength of the starch network.

Degradation of complex carbohydrate: immobilization of pectinase from Bacillus licheniformis KIBGE-IB21 using calcium alginate as a support.

PubMed

Rehman, Haneef Ur; Aman, Afsheen; Silipo, Alba; Qader, Shah Ali Ul; Molinaro, Antonio; Ansari, Asma

2013-08-15

Pectinases are heterogeneous group of enzymes that catalyse the hydrolysis of pectin substances which is responsible for the turbidity and undesirable cloudiness in fruits juices. In current study, partially purified pectinase from Bacillus licheniformis KIBGE-IB21 was immobilized in calcium alginate beads. The effect of sodium alginate and calcium chloride concentration on immobilization was studied and it was found that the optimal sodium alginate and calcium chloride concentration was 3.0% and 0.2 M, respectively. It was found that immobilization increases the optimal reaction time for pectin degradation from 5 to 10 min and temperature from 45 to 55°C, whereas, the optimal pH remained same with reference to free enzyme. Thermal stability of enzyme increased after immobilization and immobilized pectinase retained more than 80% of its initial activity after 5 days at 30°C as compared with free enzyme which showed only 30% of residual activity. The immobilized enzyme also exhibited good operational stability and 65% of its initial activity was observed during third cycle. In term of pectinase immobilization efficiency and stability, this calcium alginate beads approach seemed to permit good results and can be used to make a bioreactor for various applications in food industries. Copyright © 2013 Elsevier Ltd. All rights reserved.

Alpha-amylase inhibitor, CS-1036 binds to serum amylase in a concentration-dependent and saturable manner.

PubMed

Honda, Tomohiro; Kaneno-Urasaki, Yoko; Ito, Takashi; Kimura, Takako; Matsushima, Nobuko; Okabe, Hiromi; Yamasaki, Atsushi; Izumi, Takashi

2014-03-01

(2R,3R,4R)-4-hydroxy-2-(hydroxymethyl)pyrrolidin-3-yl 4-O-(6-deoxy-β-D-glucopyranosyl)-α-D-glucopyranoside (CS-1036), which is an α-amylase inhibitor, exhibited biphasic and sustained elimination with a long t1/2 (18.4-30.0 hours) in rats and monkeys, but exhibited a short t1/2 (3.7-7.9 hours) in humans. To clarify the species differences in the t1/2, the plasma protein binding of CS-1036 was evaluated by ultrafiltration. A concentration-dependent and saturable plasma protein binding of CS-1036 was observed in rats and monkeys with the dissociation rate constant (KD) of 8.95 and 27.2 nM, and maximal binding capacity (Bmax) of 52.8 and 22.1 nM, respectively. By the assessments of the recombinant amylase and immunoprecipitation, the major binding protein of CS-1036 in rats was identified as salivary amylase (KD 5.64 nM). CS-1036 also showed concentration-dependent and saturable binding to human salivary and pancreatic amylase, with similar binding affinity in rats. However, the protein binding of CS-1036 was constant in human plasma (≤10.2%) due to the lower serum amylase level compared with rats and monkeys. From the calculation of the unbound fraction (fu) in plasma based on in vitro KD and Bmax, the dose-dependent increase in fu after oral administration is speculated to lead to a dose-dependent increase in total body clearance and a high area under the curve/dose at lower doses, such as 0.3 mg/kg in rats.

Identification and Molecular Characterization of Genes Coding Pharmaceutically Important Enzymes from Halo-Thermo Tolerant Bacillus

PubMed Central

Safary, Azam; Moniri, Rezvan; Hamzeh-Mivehroud, Maryam; Dastmalchi, Siavoush

2016-01-01

Purpose: Robust pharmaceutical and industrial enzymes from extremophile microorganisms are main source of enzymes with tremendous stability under harsh conditions which make them potential tools for commercial and biotechnological applications. Methods: The genome of a Gram-positive halo-thermotolerant Bacillus sp. SL1, new isolate from Saline Lake, was investigated for the presence of genes coding for potentially pharmaceutical enzymes. We determined gene sequences for the enzymes laccase (CotA), l-asparaginase (ansA3, ansA1), glutamate-specific endopeptidase (blaSE), l-arabinose isomerase (araA2), endo-1,4-β mannosidase (gmuG), glutaminase (glsA), pectate lyase (pelA), cellulase (bglC1), aldehyde dehydrogenase (ycbD) and allantoinases (pucH) in the genome of Bacillus sp. SL1. Results: Based on the DNA sequence alignment results, six of the studied enzymes of Bacillus sp. SL-1 showed 100% similarity at the nucleotide level to the same genes of B. licheniformis 14580 demonstrating extensive organizational relationship between these two strains. Despite high similarities between the B. licheniformis and Bacillus sp. SL-1 genomes, there are minor differences in the sequences of some enzyme. Approximately 30% of the enzyme sequences revealed more than 99% identity with some variations in nucleotides leading to amino acid substitution in protein sequences. Conclusion: Molecular characterization of this new isolate provides useful information regarding evolutionary relationship between B. subtilis and B. licheniformis species. Since, the most industrial processes are often performed in harsh conditions, enzymes from such halo-thermotolerant bacteria may provide economically and industrially appealing biocatalysts to be used under specific physicochemical situations in medical, pharmaceutical, chemical and other industries. PMID:28101462

Identification and Molecular Characterization of Genes Coding Pharmaceutically Important Enzymes from Halo-Thermo Tolerant Bacillus.

PubMed

Safary, Azam; Moniri, Rezvan; Hamzeh-Mivehroud, Maryam; Dastmalchi, Siavoush

2016-12-01

Purpose: Robust pharmaceutical and industrial enzymes from extremophile microorganisms are main source of enzymes with tremendous stability under harsh conditions which make them potential tools for commercial and biotechnological applications. Methods: The genome of a Gram-positive halo-thermotolerant Bacillus sp. SL1, new isolate from Saline Lake, was investigated for the presence of genes coding for potentially pharmaceutical enzymes. We determined gene sequences for the enzymes laccase (CotA), l-asparaginase (ansA3, ansA1), glutamate-specific endopeptidase (blaSE), l-arabinose isomerase (araA2), endo-1,4-β mannosidase (gmuG), glutaminase (glsA), pectate lyase (pelA), cellulase (bglC1), aldehyde dehydrogenase (ycbD) and allantoinases (pucH) in the genome of Bacillus sp. SL1. Results: Based on the DNA sequence alignment results, six of the studied enzymes of Bacillus sp. SL-1 showed 100% similarity at the nucleotide level to the same genes of B. licheniformis 14580 demonstrating extensive organizational relationship between these two strains. Despite high similarities between the B. licheniformis and Bacillus sp. SL-1 genomes, there are minor differences in the sequences of some enzyme. Approximately 30% of the enzyme sequences revealed more than 99% identity with some variations in nucleotides leading to amino acid substitution in protein sequences. Conclusion: Molecular characterization of this new isolate provides useful information regarding evolutionary relationship between B. subtilis and B. licheniformis species. Since, the most industrial processes are often performed in harsh conditions, enzymes from such halo-thermotolerant bacteria may provide economically and industrially appealing biocatalysts to be used under specific physicochemical situations in medical, pharmaceutical, chemical and other industries.

Role of Bacillus licheniformis VS16-Derived Biosurfactant in Mediating Immune Responses in Carp Rohu and its Application to the Food Industry

PubMed Central

Giri, Sib Sankar; Sen, Shib Sankar; Jun, Jin Woo; Sukumaran, V.; Park, Se Chang

2017-01-01

Multifarious applications of Bacillus licheniformis VS16-derived biosurfactant were explored. Labeo rohita fingerlings were injected intraperitoneally with 0.1 mL of phosphate-buffered saline (PBS) containing purified biosurfactant at 0 (control), 55 (S55), 110 (S110), 220 (S220), or 330 (S330) μg mL-1 concentrations. Various immunological parameters and the expression of immune-related genes were measured at 7, 14, and 21 days post-administration (dpa). At 21 dpa, fish were challenged with Aeromonas hydrophila and mortality was recorded for 14 days. Immune parameters such as lysozyme levels (39.29 ± 2.14 U mL-1), alternative complement pathway (61.21 ± 2.38 U mL-1), and phagocytic activities (33.37 ± 1.2%) were maximum (P < 0.05) in the S220 group at 14 dpa; but immunoglobulin levels (11.07 ± 0.83 mg mL-1) were highest in the S220 group at 7 dpa, compared to that in controls. Activities of digestive enzymes (amylase, protease, and lipase) were higher (P < 0.05) in the S220 and S330 groups than in the control group. Regarding cytokine gene expression, pro-inflammatory cytokines (TNF-α and IL-1β) were down-regulated (P < 0.05) in the S220 and S330 groups. Expression of IL-10, TGF-β, and IKB-α were up-regulated in the S220 and S330 groups at 14 dpa, with the highest levels in the S220 group. The expression of NF-κB p65 and IKK-β were down-regulated in treatment groups, and were lowest (P < 0.05) in the S220 group. The highest post-challenge survival rate (72.7%) was recorded in S220 group. Further, the potential of this substance to inhibit biofilm formation, and heavy metal removal from vegetables were also evaluated. Biosurfactant was effective in inhibiting biofilm formation up to 54.71 ± 1.27%. Moreover, it efficiently removed cadmium (Cd) from tested vegetables such as carrot, radish, ginger, and potato, with the highest removal efficiency (60.98 ± 1.29%) recorded in ginger contaminated with Cd. Collectively, these results suggest that isolated

Synthesis and Physicochemical Characterization of D-Tagatose-1-Phosphate: The Substrate of the Tagatose-1-Phosphate Kinase in the Phosphotransferase System-Mediated D-Tagatose Catabolic Pathway of Bacillus licheniformis.

PubMed

Van der Heiden, Edwige; Delmarcelle, Michaël; Simon, Patricia; Counson, Melody; Galleni, Moreno; Freedberg, Darón I; Thompson, John; Joris, Bernard; Battistel, Marcos D

2015-01-01

We report the first enzymatic synthesis of D-tagatose-1-phosphate (Tag-1P) by the multicomponent phosphoenolpyruvate:sugar phosphotransferase system (PEP-PTS) present in tagatose-grown cells of Klebsiella pneumoniae. Physicochemical characterization by (31)P and (1)H nuclear magnetic resonance spectroscopy reveals that, in solution, this derivative is primarily in the pyranose form. Tag-1P was used to characterize the putative tagatose-1-phosphate kinase (TagK) of the Bacillus licheniformis PTS-mediated D-tagatose catabolic pathway (Bli-TagP). For this purpose, a soluble protein fusion was obtained with the 6 His-tagged trigger factor (TF(His6)) of Escherichia coli. The active fusion enzyme was named TagK-TF(His6). Tag-1P and D-fructose-1-phosphate are substrates for the TagK-TF(His6) enzyme, whereas the isomeric derivatives D-tagatose-6-phosphate and D-fructose-6-phosphate are inhibitors. Studies of catalytic efficiency (kcat/Km) reveal that the enzyme specificity is markedly in favor of Tag-1P as the substrate. Importantly, we show in vivo that the transfer of the phosphate moiety from PEP to the B. licheniformis tagatose-specific Enzyme II in E. coli is inefficient. The capability of the PTS general cytoplasmic components of B. subtilis, HPr and Enzyme I to restore the phosphate transfer is demonstrated. © 2015 S. Karger AG, Basel.

Cloning, expression, purification and characterization of lipase from Bacillus licheniformis, isolated from hot spring of Himachal Pradesh, India.

PubMed

Kaur, Gagandeep; Singh, Amninder; Sharma, Rohit; Sharma, Vinay; Verma, Swati; Sharma, Pushpender K

2016-06-01

In the present investigation, a gene encoding extracellular lipase was cloned from a Bacillus licheniformis. The recombinant protein containing His-tag was expressed as inclusion bodies in Esherichia coli BL21DE3 cells, using pET-23a as expression vector. Expressed protein purified from the inclusion bodies demonstrated ~22 kDa protein band on 12 % SDS-PAGE. It exhibited specific activity of 0.49 U mg -1 and % yield of 8.58. Interestingly, the lipase displayed activity at wide range of pH and temperature, i.e., 9.0-14.0 pH and 30-80 °C, respectively. It further demonstrated ~100 % enzyme activity in presence of various organic solvents. Enzyme activity was strongly inhibited in the presence of β-ME. Additionally, the serine and histidine modifiers also inhibited the enzyme activities strongly at all concentrations that suggest their role in the catalytic center. Enzyme could retain its activity in presence of various detergents (Triton X-100, Tween 20, Tween 40, SDS). Sequence and structural analysis employing in silico tools revealed that the lipase contained two highly conserved sequences consisting of ITITGCGNDL and NLYNP, arranged as parallel β-sheet in the core of the 3D structure. The function of these conserve sequences have not fully understood.

Some structural features of the teichuronic acid of Bacillus licheniformis N.C.T.C. 6346 cell walls

PubMed Central

Hughes, R. C.; Thurman, P. F.

1970-01-01

A teichuronic acid, containing glucuronic acid and N-acetylgalactosamine, was purified from acid extracts of Bacillus licheniformis 6346 cell walls as described by Janczura, Perkins & Rogers (1961). After reduction of the carboxyl function of glucuronic acid residues in the polysaccharide the reduced polymer contains equimolar amounts of N-acetylgalactosamine and glucose. Methylation of the reduced polysaccharide by the Hakamori (1964) technique showed the glucose residues to be substituted on C-4. A disaccharide, 3-O-glucuronosylgalactosamine, was isolated from partial acid hydrolysates of teichuronic acid. After N-acetylation the disaccharide produces chromogen readily on heating at pH7, in agreement with C-3 substitution of the reducing N-acetylamino sugar. Teichuronic acid also produces chromogen under the same conditions, with concurrent elimination of a modified polysaccharide from C-3 of reducing terminal N-acetylgalactosamine residues of the teichuronic acid chains. The number-average chain lengths of several preparations of teichuronic acid were estimated from the amounts of chromogen produced in comparison with the N-acetylated disaccharide. The values obtained are in good agreement with the weight-average molecular weight determined by ultracentrifugal analysis. The reducing terminals of teichuronic acid are shown to be exclusively N-acetylgalactosamine by reduction with sodium boro[3H]hydride. The number-average chain lengths of the teichuronic acid preparations were estimated by the extent of in corporation of tritium and are in agreement with values obtained by the other methods. PMID:5419741

Immunoelectron microscopic double labeling of alkaline phosphatase and penicillinase with colloidal gold in frozen thin sections of Bacillus licheniformis 749/C.

PubMed Central

Guan, T; Ghosh, A; Ghosh, B K

1985-01-01

The subcellular distribution of alkaline phosphatase and penicillinase was determined by double labeling frozen thin sections of Bacillus licheniformis 749/C with colloidal gold-immunoglobulin G (IgG). Antipenicillinase and anti-alkaline phosphatase antibodies were used to prepare complexes with 5- and 15-nm colloidal gold particles, respectively. The character of the labeling of membrane-bound alkaline phosphatase and penicillinase was different: the immunolabels for alkaline phosphatase (15-nm particles) were bound to a few sites at the inner surface of the plasma membrane, and the gold particles formed clusters of various sizes at the binding sites; the immunolabels for penicillinase (5-nm particles), on the other hand, were bound to the plasma membrane in a dispersed and random fashion. In the cytoplasm, immunolabels for both proteins were distributed randomly, and the character of their binding was similar. The labeling was specific: pretreating the frozen thin sections with different concentrations of anti-alkaline phosphatase or penicillinase blocked the binding of the immunolabel prepared with the same antibody. Binding could be fully blocked by pretreatment with 800 micrograms of either antibody per ml. Images PMID:3876329

Positive dermal hypersensitivity and specific antibodies in workers exposed to bio-engineered enzymes

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

Biagini, R.E.; Henningsen, G.M.; Driscoll, R.

1991-03-15

Thirty-six employees who produced industrial enzymes from bio-engineered strains of bacteria and fungi were evaluated by skin prick testing and enzyme linked immunosorbent assays for specific IgE and IgG antibodies. The workers complained of asthma- and flu-like' symptoms which generally lessened away from work. The enzymes evaluated were {alpha}-amylase from A. niger (ind-AAN), B. licheniformis (ind-AAL) and B. subtilis (ind-AAS); purified {alpha}-amylase from B. subtilis (AAS) and A. niger (AAN); alkaline protease from B. licheniformis (ind-APL) and purified alkaline protease (APL); amylase glucosidase from A. niger (ind-AGN) and purified amylase glucosidase (AGN). Significantly positive skin tests were found for APL,more » AGN and ind-AAN. Significantly elevated specific IgE results were observed for AAN, AGN, and ind-AAN; elevated specific IgGs were observed for AAN, ind-AAN, ind-AAS, ind-AAL and ind-AGN. Radioimmunoassays of air filter samples (using sera with high Ab titers) for 4 of the ind-enzymes showed only ind-AAN at extremely high environmental levels. These results indicate that occupational exposure to some ind-enzymes causes immediate onset dermal hypersensitivity reactions. The results are equivocal as to whether these reactions are IgE mediated, as IgE titers were low. Contrary to this, IgG titers were extremely high and suggest that these biomarkers can be used as indicators of both individual exposure and environmental analyses.« less

Effects of sorghum (Sorghum bicolor (L.) Moench) tannins on alpha-amylase activity and in vitro digestibility of starch in raw and processed flours

USDA-ARS?s Scientific Manuscript database

The effect of condensed tannins (CT) on in vitro starch digestibility in cooked, wholegrain sorghum flours and on corn starch was investigated. CT extracts were also tested for their inhibitory effect on alpha-amylases. Rapidly digestible starch, slowly digestible starch, and resistant starch were n...

Antimicrobial Susceptibility of Bacillus Strains Isolated from Primary Starters for African Traditional Bread Production and Characterization of the Bacitracin Operon and Bacitracin Biosynthesis

PubMed Central

Sørensen, Kim I.; Thorsen, Line; Stuer-Lauridsen, Birgitte; Abdelgadir, Warda S.; Nielsen, Dennis S.; Derkx, Patrick M. F.; Jespersen, Lene

2012-01-01

Bacillus spp. are widely used as feed additives and probiotics. However, there is limited information on their resistance to various antibiotics, and there is a growing concern over the transfer of antibiotic resistance genes. The MIC for 8 antibiotics was determined for 85 Bacillus species strains, Bacillus subtilis subsp. subtilis (n = 29), Bacillus licheniformis (n = 38), and Bacillus sonorensis (n = 18), all of which were isolated from starters for Sudanese bread production. All the strains were sensitive to tetracycline (8.0 mg/liter), vancomycin (4.0 mg/liter), and gentamicin (4.0 mg/liter) but resistant to streptomycin. Sensitivity to clindamycin, chloramphenicol, and kanamycin was species specific. The erythromycin resistance genes ermD and ermK were detected by PCR in all of the erythromycin-resistant (MIC, ≥16.0 mg/liter) B. licheniformis strains and one erythromycin-sensitive (MIC, 4.0 mg/liter) B. licheniformis strain. Several amino acid changes were present in the translated ermD and ermK nucleotide sequences of the erythromycin-sensitive strain, which could indicate ErmD and ErmK protein functionalities different from those of the resistance strains. The ermD and ermK genes were localized on an 11.4-kbp plasmid. All of the B. sonorensis strains harbored the bacitracin synthetase gene, bacA, and the transporter gene bcrA, which correlated with their observed resistance to bacitracin. Bacitracin was produced by all the investigated species strains (28%), as determined by ultra-high-definition quadrupole time-of-flight liquid chromatography-mass spectrometry (UHD-QTOF LC/MS). The present study has revealed species-specific variations in the antimicrobial susceptibilities of Bacillus spp. and provides new information on MIC values, as well as the occurrence of resistance genes in Bacillus spp., including the newly described species B. sonorensis. PMID:22941078

Antimicrobial susceptibility of Bacillus strains isolated from primary starters for African traditional bread production and characterization of the bacitracin operon and bacitracin biosynthesis.

PubMed

Adimpong, David B; Sørensen, Kim I; Thorsen, Line; Stuer-Lauridsen, Birgitte; Abdelgadir, Warda S; Nielsen, Dennis S; Derkx, Patrick M F; Jespersen, Lene

2012-11-01

Bacillus spp. are widely used as feed additives and probiotics. However, there is limited information on their resistance to various antibiotics, and there is a growing concern over the transfer of antibiotic resistance genes. The MIC for 8 antibiotics was determined for 85 Bacillus species strains, Bacillus subtilis subsp. subtilis (n = 29), Bacillus licheniformis (n = 38), and Bacillus sonorensis (n = 18), all of which were isolated from starters for Sudanese bread production. All the strains were sensitive to tetracycline (8.0 mg/liter), vancomycin (4.0 mg/liter), and gentamicin (4.0 mg/liter) but resistant to streptomycin. Sensitivity to clindamycin, chloramphenicol, and kanamycin was species specific. The erythromycin resistance genes ermD and ermK were detected by PCR in all of the erythromycin-resistant (MIC, ≥16.0 mg/liter) B. licheniformis strains and one erythromycin-sensitive (MIC, 4.0 mg/liter) B. licheniformis strain. Several amino acid changes were present in the translated ermD and ermK nucleotide sequences of the erythromycin-sensitive strain, which could indicate ErmD and ErmK protein functionalities different from those of the resistance strains. The ermD and ermK genes were localized on an 11.4-kbp plasmid. All of the B. sonorensis strains harbored the bacitracin synthetase gene, bacA, and the transporter gene bcrA, which correlated with their observed resistance to bacitracin. Bacitracin was produced by all the investigated species strains (28%), as determined by ultra-high-definition quadrupole time-of-flight liquid chromatography-mass spectrometry (UHD-QTOF LC/MS). The present study has revealed species-specific variations in the antimicrobial susceptibilities of Bacillus spp. and provides new information on MIC values, as well as the occurrence of resistance genes in Bacillus spp., including the newly described species B. sonorensis.

Screening, Gene Cloning, and Characterizations of an Acid-Stable α-Amylase.

PubMed

Liu, Xinyu; Jia, Wei; An, Yi; Cheng, Kun; Wang, Mingdao; Yang, Sen; Chen, Hongge

2015-06-01

Based on its α-amylase activity at pH 5.0 and optimal pH of the crude enzyme, a strain (named B-5) with acid α-amylase production was screened. The B-5 strain was identified as Bacillus amyloliquefaciens through morphological, physiological, and biochemical characteristics analysis, as well as 16S rDNA phylogenetic analysis. Its α-amylase gene of GenBank Accession No. GU318401 was cloned and expressed in Escherichia coli. The purified recombinant α-amylase AMY-Ba showed the optimal pH of 5.0, and was stable at a pH range of 4.0-6.0. When hydrolyzing soluble starch, amylose, and amylopectin, AMY-Ba released glucose and maltose as major end products. The α-amylase AMY-Ba in this work was a different type from the well-investigated J01542 (GenBank Accession No.)-type α-amylase from the same species. AMY-Ba exhibited notable adsorption and hydrolysis ability towards various raw starches. Structure analysis of AMY-Ba suggested the presence of a new starch-binding domain at its C-terminal region.

Classification of microbial α-amylases for food manufacturing using proteinase digestion.

PubMed

Akiyama, Takumi; Yamazaki, Takeshi; Tada, Atsuko; Ito, Yusai; Otsuki, Noriko; Akiyama, Hiroshi

2014-09-01

Enzymes produced by microorganisms and plants are used as food additives to aid the processing of foods. Identification of the origin of these enzyme products is important for their proper use. Proteinase digestion of α-amylase products, followed by high performance liquid chromatography (HPLC) analysis, was applied to α-amylase from the mold Aspergillus species, the bacteria Bacillus species, and the actinomycetes Saccharomonospora species. Eighteen commercial products of α-amylase were digested with trypsin and endoproteinase Lys-C and HPLC analyzed. For some proteinase/sample combinations, the area of the intact α-amylase peak decreased and new peaks were detected after digestion. The presence and retention times of the novel peaks were used to group the products. The results from this method, called the proteinase digestion-HPLC method, allowed the classification of the α-amylase products into 10 groups, whereas the results from sodium dodecyl sulfate polyacrylamide gel electrophoresis allowed their classification into seven groups.

Synthesis and Physicochemical Characterization of D-Tagatose-1-phosphate: The Substrate of the Tagatose-1-Phosphate Kinase TagK in the PTS-mediated D-Tagatose Catabolic Pathway of Bacillus licheniformis

PubMed Central

Van der Heiden, Edwige; Delmarcelle, Michaël; Simon, Patricia; Counson, Melody; Galleni, Moreno; Freedberg, Darón I.; Thompson, John; Joris, Bernard; Battistel, Marcos D.

2015-01-01

We report the first enzymatic synthesis of D-tagatose-1-phosphate (Tag-1P) by the multi-component PEP-dependent:tag-PTS present in tagatose-grown cells of Klebsiella pneumoniae. Physicochemical characterization by 31P and 1H NMR spectroscopy reveals that, in solution, this derivative is primarily in the pyranose form. Tag-1P was used to characterize the putative tagatose-1-phosphate kinase (TagK) of the Bacillus licheniformis PTS-mediated D-Tagatose catabolic Pathway (Bli-TagP). For this purpose, a soluble protein fusion was obtained with the 6 His-tagged trigger factor (TFHis6) of Escherichia coli. The active fusion enzyme was named TagK-TFHis6. Tag-1P and D-fructose-1-phosphate (Fru-1P) are substrates for the TagK-TFHis6 enzyme, whereas the isomeric derivatives D-tagatose-6-phosphate (Tag-6P) and D-fructose-6-phosphate (Fru-6P) are inhibitors. Studies of catalytic efficiency (kcat/Km) reveal that the enzyme specificity is markedly in favor of Tag-1P as substrate. Importantly, we show in vivo that the transfer of the phosphate moiety from PEP to the B. licheniformis tagatose-specific enzyme II (EIITag) in E.coli is inefficient. The capability of the PTS general cytoplasmic components of B. subtilis, HPr and EI, to restore the phosphate transfer is demonstrated. PMID:26159072

Influence of the coating formulation on enzymatic digestibility and drug release from 5-aminosalicylic acid pellets coated with mixtures of high-amylose starch and Surelease intended for colon-specific drug delivery.

PubMed

Freire, Cristina; Podczeck, Fridrun; Veiga, Francisco; Sousa, João

2010-02-01

Colon-specific delivery of drugs can be achieved with dosage forms coated with biopolymers that are metabolized selectively by the colonic microflora and yet resistant to enzymatic digestion in the small intestine. The aim of this study was to study the influence of formulation factors on the performance of mixed films from high-amylose starches and Surelease((R)), applied using a spray-coating process, as potential colon-specific delivery devices. 5-Aminosalicylic acid-loaded pellets were prepared by an extrusion-spheronization process and film coated with mixtures of the starches and Surelease((R)). Optimization of the coating formulation, that is, starch-to-Surelease((R)) ratio, film-coating thickness, and type of starch, was undertaken first in enzyme-free media resembling the conditions in the stomach and small intestine. The effect of curing of the film coating on the drug release profile upon storage was also evaluated. Optimized coating formulations were further assessed for enzymatic digestibility using artificial gastric and intestinal juices containing commercially available pepsin and pancreatin or alpha-amylase from hog pancreas, respectively. Finally, drug release was assessed in fluid-simulating conditions in the colon (SCF) containing Bacillus licheniformis alpha-amylase. Film coatings comprising high-amylose starches and Surelease((R)) in a ratio of 1:2 (w/w) and film thickness of approximately 45 microm were able to withstand the chemical and enzymatic environment of the upper gastrointestinal tract, in particular, resisted degradation by the pancreatic alpha-amylases. Stability of the coatings during storage was achieved with additional curing. In SCF, these coatings were susceptible to enzymatic degradation. This study showed that high amylose starch-mixed films can be successfully used as colon-specific delivery devices. The preparation of the coating dispersions described is simple and rapid, without the need to extract the amylose component

Further Experiments on Gibberellin-Stimulated Amylase Production in Cereal Grains

ERIC Educational Resources Information Center

Coppage, Jo; Hill, T. A.

1973-01-01

Experiments conducted on wheat and barley grains to analyze activities of alpha- and beta-amylase enzymes. Gibberellins were used exogenously. Techniques are described in detail. Results on different cultivars revealed that beta-amylase was not an invariable result of imbibition. Techniques employed can be used by school students. (PS)

Induced calcium carbonate precipitation using Bacillus species.

PubMed

Seifan, Mostafa; Samani, Ali Khajeh; Berenjian, Aydin

2016-12-01

Microbially induced calcium carbonate precipitation is an emerging process for the production of self-healing concrete. This study was aimed to investigate the effects and optimum conditions on calcium carbonate biosynthesis. Bacillus licheniformis, Bacillus sphaericus, yeast extract, urea, calcium chloride and aeration were found to be the most significant factors affecting the biomineralization of calcium carbonate. It was noticed that the morphology of microbial calcium carbonate was mainly affected by the genera of bacteria (cell surface properties), the viscosity of the media and the type of electron acceptors (Ca 2+ ). The maximum calcium carbonate concentration of 33.78 g/L was achieved at the optimum conditions This value is the highest concentration reported in the literature.

Inhibitory effects of rosmarinic acid extracts on porcine pancreatic amylase in vitro.

PubMed

McCue, Patrick P; Shetty, Kalidas

2004-01-01

Porcine pancreatic alpha-amylase (PPA) was allowed to react with herbal extracts containing rosmarinic acid (RA) and purified RA. The derivatized enzyme-phytochemical mixtures obtained were characterized for residual amylase activity. These in vitro experiments showed that the amylase activity was inhibited in the presence of these phytochemicals. The extent of amylase inhibition correlated with increased concentration of RA. RA-containing oregano extracts yielded higher than expected amylase inhibition than similar amount of purified RA, suggesting that other phenolic compounds or phenolic synergies may contribute to additional amylase inhibitory activity. The significance of food-grade, plant-based amylase inhibitors for modulation of diabetes mellitus and other oxidation-linked diseases is hypothesized and discussed.

Cloning, enhanced expression and characterization of an α-amylase gene from a wild strain in B. subtilis WB800.

PubMed

Chen, Jing; Chen, Xianghua; Dai, Jun; Xie, Guangrong; Yan, Luying; Lu, Lina; Chen, Jianhua

2015-09-01

A Bacillus strain with high productivity of α-amylase isolated from a starch farm was identified as Bacillus amyloliquefaciens. The α-amylase encoding gene amy1 was cloned into pMD18-T vector and amplified in E. coli DH5α. Shuttle vector pP43MNX was reconstructed to obtain vector pP43X for heterologous expression of the α-amylase in B. subtilis WB800. Recombinant enzyme was sufficiently purified by precipitation, gel filtration and anion exchange with a specific activity of 5566 U/mg. The α-amylase sequence contains an open reading frame of 1545 bp, which encodes a protein of 514 amino acid residues with a predicted molecular mass of 58.4 kDa. The enzyme exhibited maximal activity at pH 6.0 and 60 °C. Catalytic efficiency of the recombinant α-amylase was inhibited by Hg(2+), Pb(2+) and Cu(2+), but stimulated by Li(+), Mn(2+) and Ca(2+). The purified enzyme showed decreased activity toward detergents (SDS, Tween 20 and Triton X-100). Compared with production by the wild strain, there was a 1.48-fold increase in the productivity of α-amylase in recombinant B. subtilis WB800. Copyright © 2015 Elsevier B.V. All rights reserved.

[POLYMORPHISM OF ALFA-AMYLASE AND CONJUGATION IN COMMON WHEAT ENZYME TYPES WITH QUANTITATIVE TRAITS OF PLANTS].

PubMed

Netsvetaev, V P; Bondarenko, L S; Motorina, I P

2015-01-01

Using polymorphism of alpha-amylase in the winter common wheat studied inheritance isoenzymes and its conjugation enzyme types with germinating grain on the "vine", grain productivity, plant height and time of ear formation. It is shown that the polymorphism isoenzyme of alpha-amylase wheat is limited by the presence of different loci whose products are similar in electrophoretic parameters. In this regard, one component of the enzyme can be controlling at one or two or three genes. Identification of a locus controlling alpha-amylase isoenzyme in the fast moving part of the electrophoretogram, designated as α-Amy-B7. Determine the distance of the locus to factor α-Amy-B6.

Inactivation of Bacillus spores inoculated in milk by Ultra High Pressure Homogenization.

PubMed

Amador Espejo, Genaro Gustavo; Hernández-Herrero, M M; Juan, B; Trujillo, A J

2014-12-01

Ultra High-Pressure Homogenization treatments at 300 MPa with inlet temperatures (Ti) of 55, 65, 75 and 85 °C were applied to commercial Ultra High Temperature treated whole milk inoculated with Bacillus cereus, Bacillus licheniformis, Bacillus sporothermodurans, Bacillus coagulans, Geobacillus stearothermophilus and Bacillus subtilis spores in order to evaluate the inactivation level achieved. Ultra High-Pressure Homogenization conditions at 300 MPa with Ti = 75 and 85 °C were capable of a spore inactivation of ∼5 log CFU/mL. Furthermore, under these processing conditions, commercial sterility (evaluated as the complete inactivation of the inoculated spores) was obtained in milk, with the exception of G. stearothermophilus and B. subtilis treated at 300 MPa with Ti = 75 °C. The results showed that G. stearothermophilus and B. subtilis have higher resistance to the Ultra High-Pressure Homogenization treatments applied than the other microorganisms inoculated and that a treatment performed at 300 MPa with Ti = 85 °C was necessary to completely inactivate these microorganisms at the spore level inoculated (∼1 × 10(6) CFU/mL). Besides, a change in the resistance of B. licheniformis, B. sporothermodurans, G. stearothermophilus and B. subtilis spores was observed as the inactivation obtained increased remarkably in treatments performed with Ti between 65 and 75 °C. This study provides important evidence of the suitability of UHPH technology for the inactivation of spores in high numbers, leading to the possibility of obtaining commercially sterile milk. Copyright © 2014 Elsevier Ltd. All rights reserved.

Kinetic studies of amylase and biomass production by Calvatia gigantea

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

Kekos, D.; Macris, B.J.

1987-01-01

Production of alpha-amylase (alpha-4, glucan 4-glucanohydrolase, EC 3.2.1.1) by microorganisms has been practiced for many years in small and large scale operations and the literature on this enzyme is voluminous. Aspergillus niger and Aspergillus oryzae have been reported as the main fungal species used for commercial production of the enzyme. On the other hand, large volumes of low-cost agricultural products such as acorn (the perisperm-free dry seed contains approximately 60% starch) are wasted in many countries and provide a challenge to biotechnology to efficiently utilize these rich sources of starch for the production of high added value products like enzymes.more » C. gigantea is an edible puffball excreting high levels of alpha-amylase when cultivated on different sources of starch containing elevated quantities of toxic tannic compounds. This fungus has been employed for the production of microbial protein from wastes and acorns containing high levels of toxic tannic compounds. The same fungus was also reported to grow on both hydrolyzable and condensed tannins as sole carbon sources. The present work was undertaken to investigate certain kinetic characteristics of alpha-amylase and biomass production by C. gigantea grown on soluble and acorn starch in a lab fermenter. (Refs. 18).« less

Distribution and identification of proteolytic Bacillus spp. in paddy field soil under rice cultivation.

PubMed

Watanabe, K; Hayano, K

1993-07-01

Proteolytic bacteria in paddy field soils under rice cultivation were characterized and enumerated using azocoll agar plates. Bacillus spp. were the proteolytic bacteria that were most frequently present, comprising 59% of the isolates. They were always the numerically dominant proteolytic bacteria isolated from three kinds of fertilizer treatments (yearly application of rice-straw compost and chemical fertilizer, yearly application of chemical fertilizer, and no fertilizer application) and at three different stages of rice development (vegetative growth stage, maximal tillering stage, and harvest stage). Of the 411 proteolytic bacteria isolated, 124 isolates had stronger proteolytic activity than others on the basis of gelatin liquefaction tests and most of them were Bacillus spp. (100% in 1989 and 92.4% in 1991). Bacillus subtilis and Bacillus cereus were the main bacteria of this group and Bacillus mycoides, Bacillus licheniformis, and Bacillus megaterium were also present. We conclude that these Bacillus spp. are the primary source of soil protease in these paddy fields.

Salivary Alpha-Amylase Activity Levels in Catatonic Schizophrenia Decrease after Electroconvulsive Therapy.

PubMed

Kanayama, Misako; Miyaoka, Tsuyoshi; Araki, Tomoko; Hayashida, Maiko; Hashioka, Sadayuki; Horiguchi, Jun

2018-01-01

Dysfunction of the autonomic nervous system (ANS) in schizophrenia has been detected by electrophysiological methods, but the underlying mechanisms remain unknown. Several studies have suggested that measuring salivary alpha-amylase activity levels is useful for evaluating the ANS activity and that sAA levels increase in schizophrenia and correlate with Brief Psychiatric Rating Scale (BPRS) scores. However, no study has examined the relationship between sAA activity levels and symptoms of schizophrenia with catatonic state. We present the case of a 59-year-old female with persistent catatonic schizophrenia treated by electroconvulsive therapy. We evaluated the ANS activity by measuring sAA activity levels before and after ECT, and we evaluated her symptoms using the BPRS and Bush-Francis Catatonia Rating Scale (BFCRS). ECT was highly effective and BPRS and BFCRS scores substantially decreased. sAA activity levels decreased from 125 kU/l to 33 kU/l. sAA activity levels could be a potential biomarker of schizophrenia with catatonic state.

Effects of early life adversity on cortisol/salivary alpha-amylase symmetry in free-ranging juvenile rhesus macaques.

PubMed

Petrullo, Lauren A; Mandalaywala, Tara M; Parker, Karen J; Maestripieri, Dario; Higham, James P

2016-11-01

Early life adversity (ELA) affects physiological and behavioral development. One key component is the relationship between the developing Hypothalamic-Pituitary-Adrenal (HPA) axis and the Sympathetic Nervous System (SNS). Recent studies suggest a relationship between early life adversity and asymmetry in cortisol (a measure of HPA activation) and salivary alpha-amylase (sAA: a correlate of SNS activation) responses to stress among human children, but to our knowledge there have been no comparable studies in nonhumans. Here, we investigate the responses of these two analytes in "low stress" and "high stress" situations in free-ranging juvenile rhesus macaques (Macaca mulatta) on Cayo Santiago, Puerto Rico. Behavioral data on maternal maltreatment were collected during the first 3months of life to determine individual rates of ELA, and saliva samples were collected from subjects noninvasively during juvenility. Irrespective of ELA, salivary alpha-amylase levels were lower in low stress situations and higher in high stress situations. For cortisol however, high ELA subjects exhibited higher low stress concentrations and blunted acute responses during high stress situations compared to moderate and low ELA subjects. Cortisol and sAA values were positively correlated among low ELA subjects, suggesting symmetry, but were uncorrelated or negatively correlated among moderate and high ELA subjects, suggesting asymmetry in these individuals. These findings indicate dysregulation of the stress response among juveniles maltreated during infancy: specifically, attenuated cortisol reactivity coupled with typical sAA reactivity characterize the stress response profiles of juveniles exposed to higher rates of ELA during the first 3months of life. Copyright © 2016 Elsevier Inc. All rights reserved.

General Subject 2. Report to ICUMSA on the determination of carry-over alpha-amylase activity in white and refined sugars by a spectrophotometric method

USDA-ARS?s Scientific Manuscript database

A report is given on a new industrial method for the determination of carry-over alpha-amylase activity in raw and refined sugars, as well as a recommendation. In recent years, there has been increased concern over carry-over activity of mostly high temperature (HT) and very high temperature (VHT) s...

Characterization of the Activity and Stability of Amylase from Saliva and Detergent: Laboratory Practicals for Studying the Activity and Stability of Amylase from Saliva and Various Commercial Detergents

ERIC Educational Resources Information Center

Valls, Cristina; Rojas, Cristina; Pujadas, Gerard; Garcia-Vallve, Santi; Mulero, Miquel

2012-01-01

This article presents two integrated laboratory exercises intended to show students the role of [alpha]-amylases (AAMYs) in saliva and detergents. These laboratory practicals are based on the determination of the enzymatic activity of amylase from saliva and different detergents using the Phadebas test (quantitative) and the Lugol test…

A glycoprotein α-amylase inhibitor from Withania somnifera differentially inhibits various α-amylases and affects the growth and development of Tribolium castaneum.

PubMed

Kasar, Sainath S; Marathe, Kiran R; Bhide, Amey J; Herwade, Abhijeet P; Giri, Ashok P; Maheshwari, Vijay L; Pawar, Pankaj K

2017-07-01

Identification and characterisation of plant defensive molecules enrich our resources to design crop protection strategies. In particular, plant-derived proteinaceous inhibitor(s) of insect digestive enzymes appear to be a safe, sustainable and attractive option. A glycoprotein having non-competitive α-amylase inhibitory activity with a molecular weight of 8.3 kDa was isolated and purified from seeds of Withania somnifera α-amylase inhibitor (WSAI). Its mass spectrometry analysis revealed 59% sequence coverage with Wrightide II-type α-amylase inhibitor from Wrightia religiosa. A dose-dependent inhibition of α-amylases from Aspergillus oryzae, Bacillus subtilis, Helicoverpa armigera and Tribolium castaneum was recorded. Interestingly, WSAI did not inhibit human salivary α-amylase significantly. When adults of T. castaneum were fed with WSAI (1.6 mg g -1 ), decrease in consumption, growth and efficiency of conversion of ingested food was evident, along with over fourfold increases in feeding deterrence index. A decline in larval residual α-amylase activity after feeding of WSAI resulted in a reduction in longevity of T. castaneum. The study reflects the significance of WSAI in affecting the overall growth and development of T. castaneum. Pre- and post-harvest pest resistive capability makes WSAI a potential candidate for insect pest management. Further, the effectiveness of this inhibitor could be explored either in formulations or through a transgenic approach. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

Heavy Metal Detoxification by Different Bacillus Species Isolated from Solar Salterns

PubMed Central

Syed, Shameer; Chinthala, Paramageetham

2015-01-01

The biosorption mechanism is an alternative for chemical precipitation and ultrafiltration which have been employed to treat heavy metal contamination with a limited success. In the present study, three species of Bacillus which were isolated from solar salterns were screened for their detoxification potential of the heavy metals, lead, chromium, and copper, by biosorption. Biosorption potential of each isolate was determined by Atomic Absorption Spectroscopy (AAS), Inductively Coupled Plasma-Optical Emission Spectroscopy (ICP-OES), and Energy Dispersive Spectroscopy (EDS) as the amount of metal present in the medium after the treatment with the isolates. Bacterial isolates, Bacillus licheniformis NSPA5, Bacillus cereus NSPA8, and Bacillus subtilis NSPA13, showed significant level of lead biosorption with maximum of 87–90% by Bacillus cereus NSPA8. The biosorption of copper and chromium was relatively low in comparison with lead. With the obtained results, we have concluded that the bacterial isolates are potential agents to treat metal contamination in more efficient and ecofriendly manner. PMID:26525498

Screening the thermophilic and hyperthermophilic bacterial population of three Iranian hot-springs to detect the thermostable α-amylase producing strain

PubMed Central

Fooladi, J; Sajjadian, A

2010-01-01

Background Screening is a routine procedure for isolation of microorganisms which are able to produce special metabolites. Purified thermostable α-amylase from bacterial sources is widely used in different industries. In this study we analyzed samples collected from three different hot springs in Iran to detect any strains capable of producing thermostable α-amylase. Materials and Methods Hot water samples from Larijan (67°C, pH 6.5), Mahallat (46°C, pH 7), and Meshkinshahr (82°C, pH 6), were cultivated in screening starch agar plates and incubated at 65°C for 24 hours. Thereafter, the plates were stained with Gram's iodine solution. Results and Discussion The bacterial colonies from the Meshkinshahr hot-spring produced the largest haloforming zone. Based on the phenotypic tests, the strain was identified as Bacillus sp. The culture condition was optimized for biosynthesis of α-amylase. The enzyme was produced at maximum level when it was incubated at 70°C in the presence of soluble starch (1%) at pH 6. The addition of calcium (10 mM) and peptone (1%) to the mineral medium, shortened the lag period and improved the growth and α-amylase synthesis. The addition of glucose (1%) to the culture greatly diminished the syntheses of α -amylase. Importantly, the enzyme extract retained 100% activity when incubated for 45 minutes at 100°C. Conclusion The Meshkinshahr hot-spring is rich in the Bacillus spp thermostable α-amylase producing strain of the thermophilic bacterial population. Iranian hot-springs like Meshkinshahr, have large microbial storages and can be used as sources of different biological products like enzymes. The enzyme which was produced with Bacillus sp. could hydrolyse polymers like starch and was used at laboratory scale successfully. PMID:22347550

Evening salivary cortisol and alpha-amylase at 14months and neurodevelopment at 4years: Sex differences.

PubMed

Andiarena, Ainara; Balluerka, Nekane; Murcia, Mario; Ibarluzea, Jesús; Glover, Vivette; Vegas, Oscar

2017-08-01

Stress system activity in early life can have long-term effects on neurodevelopment. The main aim of this study was to assess the association of child evening salivary cortisol and alpha-amylase basal levels at 14months of age with longer-term neuropsychological development at 4years in a low-risk population-based birth cohort derived from the INMA (Environment and Childhood) project in Spain. We included 186 parent-children pairs with information on both stress system activity and neurodevelopment. Both stress markers at 14months of age showed an association with neuropsychological development at 4years. Salivary cortisol showed a sex-specific pattern of association. In girls, cortisol levels at 14months were negatively associated with cognitive development [long-term declarative memory (β=-17.8, p=0.028; 95% CI=-33.2 to -2.5); executive function (β=-9.8, p=0.08; 95% CI=-21 to 1)] and gross motor development (β=-13; p=0.022; 95% CI=-24 to -2), whereas in boys cortisol levels were negatively associated with socioemotional development [autistic-like behaviours: Incidence Rate Ratio (IRR)=1.6, p=0.039; 95% CI=1.01 to 2.41]. Salivary alpha-amylase was positively associated with socioemotional development in boys only [social competence (β=2.11, p=0.013; 95% CI=0.47 to 3.72), autistic-like behaviours (IRR=0.93, p=0.042; 95% CI=0.87 to 0.99) and hyperactivity symptoms (IRR=0.81, p=0.021; 95% CI=0.69 to 0.97)]. These results suggest that stress system activity in early life is associated with longer-term neurodevelopment and that sex is an important factor in this relationship. Copyright © 2017 Elsevier Inc. All rights reserved.

alpha-Galactosidase from Bacillus megaterium VHM1 and its application in removal of flatulence-causing factors from soymilk.

PubMed

Patil, Aravind Goud G; K, Praveen Kumar S; Mulimani, Veerappa H; Veeranagouda, Yaligara; Lee, Kyoung

2010-11-01

A bacterial strain capable of producing extracellular alpha-galactosidase was isolated from sugar cane industrial waste soil sample. Microbiological, physiological, and biochemical studies revealed that isolate belonged to Bacillus sp,. Furthermore, 16S rDNA sequence analysis of new isolates was identified as Bacillus megaterium VHM1. The production of alpha-galactosidase was optimized by various physical culture conditions. Guar gum and yeast extract acted as the best carbon and nitrogen source, respectively for the production of alpha-galactosidase. The enzyme showed an optimum pH at 7.5 and was stable over a pH between 5 and 9. The enzyme was optimally active in 55degreesC and the enzyme was thermostable with half life of 120 minutes at 55 degrees C and lost their 90%, residual activity in 120 minutes at 60 degrees C. alpha-Galactosidase was strongly inhibited by Ag2, Cu2, and Hg2+ at 1mM concentration. The metal ions Fe2, Mn2+, and Mg2+ had no effect on alpha-galactosidase activity, Zn2+,Ni2+, and Ca2+ reduced the enzyme activity slightly. The B megaterium VHM1 enzyme treatment completely hydrolyzed flatulence-causing sugars of soymilk within one and half hour.

Salivary Cortisol, Salivary Alpha Amylase, and the Dental Anxiety Scale

PubMed Central

Sadi, Hana; Finkelman, Matthew; Rosenberg, Morton

2013-01-01

The aim of this study was to investigate the correlation between dental anxiety, salivary cortisol, and salivary alpha amylase (sAA) levels. Furthermore, the aim was to look into individual differences such as age, race, gender, any existing pain, or traumatic dental experience and their effect on dental anxiety. This study followed a cross-sectional design and included a convenience sample of 46. Every patient was asked to complete the Dental Anxiety Scale (DAS) and a basic demographic/dental history questionnaire. A saliva sample, utilizing the method of passive drooling, was then collected in 2-mL cryovials. Samples were analyzed for salivary cortisol and sAA levels by Salimetrics. Significant associations were observed between DAS scores and presence of pain and history of traumatic dental experience. However, no significant correlations were observed between DAS, cortisol, and sAA levels. Our study reconfirms that dental anxiety is associated with presence of pain and a history of traumatic dental experience. On the other hand, our study was the first to our knowledge to test the correlation between the DAS and sAA; nevertheless, our results failed to show any significant correlation between dental anxiety, cortisol, and sAA levels. PMID:23763559

Biosynthesis of silver nanoparticles using a probiotic Bacillus licheniformis Dahb1 and their antibiofilm activity and toxicity effects in Ceriodaphnia cornuta.

PubMed

Shanthi, Sathappan; Jayaseelan, Barbanas David; Velusamy, Palaniyandi; Vijayakumar, Sekar; Chih, Cheng Ta; Vaseeharan, Baskaralingam

2016-04-01

In the present study, we synthesized and characterized a probiotic Bacillus licheniformis cell free extract (BLCFE) coated silver nanoparticles (BLCFE-AgNPs). These BLCFE-AgNPs were characterized by UV-visible spectrophotometer, XRD, EDX, FTIR, TEM and AFM. A strong surface plasmon resonance centered at 422 nm in UV-visible spectrum indicates the formation of AgNPs. The XRD spectrum of silver nanoparticles exhibited 2θ values corresponding to the silver nanocrystal. TEM and AFM showed the AgNPs were spherical in shape within the range of 18.69-63.42 nm and the presence of silver was confirmed by EDX analysis. Light and Confocal Laser Scanning Microscope (CLSM) images showed a weak adherence and disintegrated biofilm formation of Vibrio parahaemolyticus Dav1 treated with BLCFE-AgNPs compared to control. This result suggests that BLCFE-AgNps may be used for the control of biofilm forming bacterial populations in the biomedical field. In addition, acute toxicity results concluded that BLCFE-AgNPs were less toxic to the fresh water crustacean Ceriodaphnia cornuta (50 μg/ml) when compared to AgNO3 (22 μg/ml). This study also reports a short term analysis (24 h) of uptake and depuration of BLCFE-AgNPs in C. cornuta. Copyright © 2016 Elsevier Ltd. All rights reserved.

Self-compassionate young adults show lower salivary alpha-amylase responses to repeated psychosocial stress.

PubMed

Breines, Juliana G; McInnis, Christine M; Kuras, Yuliya I; Thoma, Myriam V; Gianferante, Danielle; Hanlin, Luke; Chen, Xuejie; Rohleder, Nicolas

2015-10-01

In this study we tested the hypothesis that participants higher in dispositional self-compassion would show lower stress-induced reactivity of salivary alpha-amylase (sAA), a marker of sympathetic nervous system activation. Thirty-three healthy participants (18-34 years old) were exposed to a standardized laboratory stressor on two consecutive days. Self-compassion, self-esteem, and demographic factors were assessed by questionnaire and sAA was assessed at baseline and at 1, 10, 30, and 60 minutes following each stressor. Self-compassion was a significant negative predictor of sAA responses on both days. This relationship remained significant when controlling for self-esteem, subjective distress, age, gender, ethnicity, and Body Mass Index (BMI). These results suggest that self-compassion may serve as a protective factor against stress-induced physiological changes that have implications for health.

Self-compassionate young adults show lower salivary alpha-amylase responses to repeated psychosocial stress

PubMed Central

Breines, Juliana G.; McInnis, Christine M.; Kuras, Yuliya I.; Thoma, Myriam V.; Gianferante, Danielle; Hanlin, Luke; Chen, Xuejie; Rohleder, Nicolas

2015-01-01

In this study we tested the hypothesis that participants higher in dispositional self-compassion would show lower stress-induced reactivity of salivary alpha-amylase (sAA), a marker of sympathetic nervous system activation. Thirty-three healthy participants (18–34 years old) were exposed to a standardized laboratory stressor on two consecutive days. Self-compassion, self-esteem, and demographic factors were assessed by questionnaire and sAA was assessed at baseline and at 1, 10, 30, and 60 minutes following each stressor. Self-compassion was a significant negative predictor of sAA responses on both days. This relationship remained significant when controlling for self-esteem, subjective distress, age, gender, ethnicity, and Body Mass Index (BMI). These results suggest that self-compassion may serve as a protective factor against stress-induced physiological changes that have implications for health. PMID:26005394

Introduction of a unique tryptophan residue into various positions of Bacillus licheniformis DnaK.

PubMed

Chen, Bo-En; Lin, Min-Guan; Lo, Huei-Fen; Wang, Tzu-Fan; Chi, Meng-Chun; Lin, Long-Liu

2013-01-01

Site-directed mutagenesis together with biochemical and biophysical techniques were used to probe effects of single-tryptophan-incorporated mutations on a bacterial molecular chaperone, Bacillus licheniformis DnaK (BlDnaK). Specifically, five phenylalanine residues (Phe(120), Phe(174), Phe(186), Phe(378) and Phe(396)) of BlDnaK were individually replaced by single tryptophans, thus creating site-specific probes for the fluorescence analysis of the protein. The steady-state ATPase activity for BlDnaK, F120W, F174W, F186W, F378W, and F396W was determined to be 76.01, 52.82, 25.32, 53.31, 58.84, and 47.53 nmol Pi/min/mg, respectively. Complementation test revealed that the single mutation at codons 120, 186, and 378 of the dnaK gene still allowed an Escherichia coli dnaK756-Ts strain to grow at a stringent temperature of 44°C. Simultaneous addition of co-chaperones and NR-peptide did not synergistically stimulate the ATPase activity of F174W and F396W, and these two proteins were unable to assist the refolding of GdnHCl-denatured luciferase. The heat-induced denaturation of all variants could be fitted adequately to a three-state model, in agreement with the observation for the wild-type protein. By CD spectral analysis, GdnHCl-induced unfolding transition for BlDnaK was 1.51 M corresponding to ΔG(N-U) of 1.69 kcal/mol; however, the transitions for mutant proteins were 1.07-1.55 M equivalent to ΔG(N-U) of 0.94-2.93 kcal/mol. The emission maximum of single-tryptophan-incorporated variants was in the range of 333.2-335.8 nm. Acrylamide quenching analysis showed that the mutant proteins had a dynamic quenching constant of 3.0-4.2 M(-1). Taken together, these results suggest that the molecular properties of BlDnaK have been significantly changed upon the individual replacement of selected phenylalanine residues by tryptophan. Copyright © 2012 Elsevier B.V. All rights reserved.

Gallic acid-based alkyl esters synthesis in a water-free system by celite-bound lipase of Bacillus licheniformis SCD11501.

PubMed

Sharma, Shivika; Kanwar, Shamsher S; Dogra, Priyanka; Chauhan, Ghanshyam S

2015-01-01

Gallic acid (3, 4, 5- trihydroxybenzoic acid) is an important antioxidant, anti-inflammatory, and radical scavenging agent. In the present study, a purified thermo-tolerant extra-cellular lipase of Bacillus licheniformis SCD11501 was successfully immobilized by adsorption on Celite 545 gel matrix followed by treatment with a cross-linking agent, glutaraldehyde. The celite-bound lipase treated with glutaraldehyde showed 94.8% binding/retention of enzyme activity (36 U/g; specific activity 16.8 U/g matrix; relative increase in enzyme activity 64.7%) while untreated matrix resulted in 88.1% binding/retention (28.0 U/g matrix; specific activity 8.5 U/g matrix) of lipase. The celite-bound lipase was successfully used to synthesis methyl gallate (58.2%), ethyl gallate (66.9%), n-propyl gallate (72.1%), and n-butyl gallate (63.8%) at 55(o) C in 10 h under shaking (150 g) in a water-free system by sequentially optimizing various reaction parameters. The low conversion of more polar alcohols such as methanol and ethanol into their respective gallate esters might be due to the ability of these alcohols to severely remove water from the protein hydration shell, leading to enzyme inactivation. Molecular sieves added to the reaction mixture resulted in enhanced yield of the alkyl ester(s). The characterization of synthesised esters was done through fourier transform infrared (FTIR) spectroscopy and (1) H NMR spectrum analysis. © 2015 American Institute of Chemical Engineers.

Alpha-amylase serum levels in professional soccer players are not related with physical fitness.

PubMed

Sanchis-Gomar, Fabian; Alis, Rafael; Rampinini, Ermanno; Bosio, Andrea; Romagnoli, Marco; Lombardi, Giovanni; Lippi, Giuseppe

2017-03-01

Recent evidence has showed that serum or salivary values of α-amylase predict endurance running performance. In this study we investigate whether serum α-amylase concentration may be associated with training status during a competitive season and after a detraining period in professional soccer players. The study population consisted in 15 male professional soccer players from an Italian major league team (age [mean±SD] 27±5 years, weight 76.9±4.1 kg, height 1.82±0.05 m). Serum α-amylase levels were measured 3 times during the last part of a competitive season (January, March and May) and just before preseason training (July). Metabolic and cardiovascular fitness of soccer players was improved during the last part of the season. The levels of α-amylase did not change significantly throughout the study period (χ2=7.331, P=0.062), nor they were found to be associated with variation of physical fitness and training status. The α-amylase fluctuations throughout a competitive season and after vacation time were meaningless in professional soccer players. No significant associations with physical fitness variations could be observed. These results suggest that α-amylase concentration may be a useful parameter for identifying individual inclination to endurance exercise, but not for predicting actual training status.

Optimization of Levan Production by Cold-Active Bacillus licheniformis ANT 179 and Fructooligosaccharide Synthesis by Its Levansucrase.

PubMed

Xavier, Janifer Raj; Ramana, Karna Venkata

2017-03-01

Fructooligosaccharides (FOS) and levan attract much attention due to a wide range of applications in food technology and pharmaceutical and cosmetic industry. Bacillus licheniformis ANT 179, isolated from Antarctica soil, produced levansucrase and levan in a medium containing sucrose as carbon substrate. In this study, characterization of levansucrase and production of short-chain FOS and levan were investigated. Temperature and pH optimum of the enzyme were found to be 60 °C and pH 6.0, respectively. The optimization of fermentation conditions for levan production using sugarcane juice by response surface methodology (RSM) was carried out. Central composite rotatable design was used to study the main and the interactive effects of medium components: sugarcane juice and casein peptone concentration on levan production by the bacterium. The optimized medium with sugarcane juice at 20 % (v/v) and casein peptone at 2 % (w/v) was found to be optimal at an initial pH of 7.0 and incubation temperature of 35 °C for 48 h. Under these conditions, the maximum levan concentration was 50.25 g/L on wet weight basis and 16.35 g/L on dry weight basis. The produced inulin type FOS (kestose and neokestose) and levan were characterized by Fourier transform infrared spectroscopy (FT-IR) and nuclear magnetic resonance (NMR) analysis. The study revealed that the levansucrase could form FOS from sucrose. The locally available low-cost substrate such as sugarcane juice in the form of a renewable substrate is proposed to be suitable even for scale-up production of enzyme and FOS for industrial applications. The levan and FOS synthesized by the bacterium are suitable for food applications and biomedical uses as the bacterium has GRAS status and devoid of endotoxin as compared to other Gram-negative bacteria.

Pitting corrosion inhibition of aluminum 2024 by Bacillus biofilms secreting polyaspartate or gamma-polyglutamate.

PubMed

Ornek, D; Jayaraman, A; Syrett, B C; Hsu, C-H; Mansfeld, F B; Wood, T K

2002-04-01

Pitting corrosion of aluminum 2024 in Luria Bertani medium was reduced by the secretion of anionic peptides by engineered and natural Bacillus biofilms and was studied in continuous reactors using electrochemical impedance spectroscopy. Compared to sterile controls, pitting was reduced dramatically by the presence of the biofilms. The secretion of a 20 amino acid polyaspartate peptide by an engineered Bacillus subtilis WB600/pBE92-Asp biofilm slightly reduced the corrosion rate of the passive aluminum alloy at pH 6.5; however, the secretion of gamma-polyglutamate by a Bacillus licheniformis biofilm reduced the corrosion rate by 90% (compared to the B. subtilis WB600/pBE92 biofilm which did not secrete polyaspartate or gamma-polyglutamate). The corrosion potential ( E(corr)) of aluminum 2024 was increased by about 0.15-0.44 V due to the formation of B. subtilis and B. licheniformis biofilms as compared to sterile controls. The increase of E(corr) and the observed prevention of pitting indicate that the pitting potential ( E(pit)) had increased. This result and the further decrease of corrosion rates for the passive aluminum alloy suggest that the rate of the anodic metal dissolution reaction was reduced by an inhibitor produced by the biofilms. Purified gamma-polyglutamate also decreased the corrosion rates of aluminum 2024.

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.

Boosting isoprene production via heterologous expression of the Kudzu isoprene synthase gene (kIspS) into Bacillus spp. cell factory.

PubMed

Gomaa, Lamis; Loscar, Michael E; Zein, Haggag S; Abdel-Ghaffar, Nahed; Abdelhadi, Abdelhadi A; Abdelaal, Ali S; Abdallah, Naglaa A

2017-08-08

Isoprene represents a key building block for the production of valuable materials such as latex, synthetic rubber or pharmaceutical precursors and serves as basis for advanced biofuel production. To enhance the production of the volatile natural hydrocarbon isoprene, released by plants, animals and bacteria, the Kudzu isoprene synthase (kIspS) gene has been heterologously expressed in Bacillus subtilis DSM 402 and Bacillus licheniformis DSM 13 using the pHT01 vector. As control, the heterologous expression of KIspS in E. coli BL21 (DE3) with the pET28b vector was used. Isoprene production was analyzed using Gas Chromatography Flame Ionization Detector. The highest isoprene production was observed by recombinant B. subtilis harboring the pHT01-kIspS plasmid which produced 1434.3 μg/L (1275 µg/L/OD) isoprene. This is threefold higher than the wild type which produced 388 μg/L (370 μg/L/OD) isoprene, when both incubated at 30 °C for 48 h and induced with 0.1 mM IPTG. Additionally, recombinant B. subtilis produced fivefold higher than the recombinant B. licheniformis, which produced 437.2 μg/L (249 μg/L/OD) isoprene when incubated at 37 °C for 48 h induced with 0.1 mM IPTG. This is the first report of optimized isoprene production in B. licheniformis. However, recombinant B. licheniformis showed less isoprene production. Therefore, recombinant B. subtilis is considered as a versatile host for heterologous production of isoprene.

Isolation and Evaluation of Bacillus Strains for Industrial Production of 2,3-Butanediol.

PubMed

Song, Chan Woo; Rathnasingh, Chelladurai; Park, Jong Myoung; Lee, Julia; Song, Hyohak

2018-03-28

Biologically produced 2,3-butanediol (2,3-BDO) has diverse industrial applications. In this study, schematic isolation and screening procedures were designed to obtain generally regarded as safe (GRAS) and efficient 2,3-BDO producers. Over 4,000 candidate strains were isolated by pretreatment and enrichment, and the isolated Bacillus strains were further screened by morphological, biochemical, and genomic analyses. The screened strains were then used to test the utilization of the most common carbon (glucose, xylose, fructose, sucrose) and nitrogen (yeast extract, corn steep liquor) sources for the economical production of 2,3-BDO. Two-stage fed-batch fermentation was finally carried out to enhance 2,3-BDO production. In consequence, a newly isolated Bacillus licheniformis GSC3102 strain produced 92.0 g/l of total 2,3-BDO with an overall productivity and yield of 1.40 g/l/h and 0.423 g/g glucose, respectively, using a cheap and abundant nitrogen source. These results strongly suggest that B. licheniformis , which is found widely in nature, can be used as a host strain for the industrial fermentative production of 2,3-BDO.

Purification and DNA binding properties of the blaI gene product, repressor for the beta-lactamase gene, blaP, of Bacillus licheniformis.

PubMed Central

Grossman, M J; Lampen, J O

1987-01-01

The location of the repressor gene, blaI, for the beta-lactamase gene blaP of Bacillus licheniformis 749, on the 5' side of blaP, was confirmed by sequencing the bla region of the constitutive mutant 749/C. An amber stop codon, likely to result in a nonfunctional truncated repressor, was found at codon 32 of the 128 codon blaI open reading frame (ORF) located 5' to blaP. In order to study the DNA binding activity of the repressor, the structural gene for blaI, from strain 749, with its ribosome binding site was expressed using a two plasmid T7 RNA polymerase/promotor system (S. Tabor and C. C. Richardson. Proc. Natl. Acad. Sci. 82, 1074-1078 (1985). Heat induction of this system in Escherichia coli K38 resulted in the production of BlaI as 5-10% of the soluble cell protein. Repressor protein was then purified by ammonium sulfate fractionation and cation exchange chromatography. The sequence of the N-terminal 28 amino acid residues was determined and was as predicted from the DNA. Binding of BlaI to DNA was detected by the slower migration of protein DNA complexes during polyacrylamide gel electrophoresis. BlaI was shown to selectively bind DNA fragments carrying the promoter regions of blaI and blaP. Images PMID:3498148

Calcium carbonate precipitation by strain Bacillus licheniformis AK01, newly isolated from loamy soil: a promising alternative for sealing cement-based materials.

PubMed

Vahabi, Ali; Ramezanianpour, Ali Akbar; Sharafi, Hakimeh; Zahiri, Hossein Shahbani; Vali, Hojatollah; Noghabi, Kambiz Akbari

2015-01-01

The relevant experiments were designed to determine the ability of indigenous bacterial strains isolated from limestone caves, mineral springs, and loamy soils to induce calcium carbonate precipitation. Among all isolates examined in this study, an efficient carbonate-precipitating soil bacterium was selected from among the isolates and identified by 16S rRNA gene sequences as Bacillus licheniformis AK01. The ureolytic isolate was able to grow well on alkaline carbonate-precipitation medium and precipitate calcium carbonate more than 1 g L(-1). Fourier transform infrared (FTIR) spectroscopy, X-ray diffraction (XRD) analyses, and scanning electron microscopy (SEM)/energy-dispersive X-ray spectroscopy (EDX) examinations were performed in order to confirm the presence of calcium carbonate in the precipitate and to determine which polymorphs were present. The selected isolate was determined to be an appropriate candidate for application in a surface treatment of cement-based material to improve the properties of the mortar. Biodeposition of a layer of calcite on the surface of cement specimens resulted in filling in pore spaces. This could be an alternative method to improve the durability of the mortar. The kind of bacterial culture and medium composition had a profound impact on the resultant CaCO(3) crystal morphology. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Effects of exposure to GSM mobile phone base station signals on salivary cortisol, alpha-amylase, and immunoglobulin A.

PubMed

Augner, Christoph; Hacker, Gerhard W; Oberfeld, Gerd; Florian, Matthias; Hitzl, Wolfgang; Hutter, Jörg; Pauser, Gernot

2010-06-01

The present study aimed to test whether exposure to radiofrequency electromagnetic fields (RF-EMF) emitted by mobile phone base stations may have effects on salivary alpha-amylase, immunoglobulin A (IgA), and cortisol levels. Fifty seven participants were randomly allocated to one of three different experimental scenarios (22 participants to scenario 1, 26 to scenario 2, and 9 to scenario 3). Each participant went through five 50-minute exposure sessions. The main RF-EMF source was a GSM-900-MHz antenna located at the outer wall of the building. In scenarios 1 and 2, the first, third, and fifth sessions were "low" (median power flux density 5.2 microW/m(2)) exposure. The second session was "high" (2126.8 microW/m(2)), and the fourth session was "medium" (153.6 microW/m(2)) in scenario 1, and vice versa in scenario 2. Scenario 3 had four "low" exposure conditions, followed by a "high" exposure condition. Biomedical parameters were collected by saliva samples three times a session. Exposure levels were created by shielding curtains. In scenario 3 from session 4 to session 5 (from "low" to "high" exposure), an increase of cortisol was detected, while in scenarios 1 and 2, a higher concentration of alpha-amylase related to the baseline was identified as compared to that in scenario 3. IgA concentration was not significantly related to the exposure. RF-EMF in considerably lower field densities than ICNIRP-guidelines may influence certain psychobiological stress markers. Copyright © 2010 The Editorial Board of Biomedical and Environmental Sciences. Published by Elsevier B.V. All rights reserved.

Preparation and characterization of novel bioactive peptides responsible for angiotensin I-converting enzyme inhibition from wheat germ.

PubMed

Matsui, T; Li, C H; Osajima, Y

1999-07-01

Reported is the preparation of wheat germ (WG) hydrolyzate with potent angiotensin I-converting enzyme (ACE) inhibitory activity, and the characterization of peptides responsible for ACE inhibition. Successful hydrolyzate with the most potent ACE inhibitory activity was obtained by 0.5 wt.%-8 h Bacillus licheniformis alkaline protease hydrolysis after 3.0 wt.%-3 h alpha-amylase treatment of defatted WG (IC50; 0.37 mg protein ml(-1)). The activity of WG hydrolyzate was markedly increased by ODS and subsequent AG50W purifications (IC50; 0.018 mg protein ml(-1)). As a result of isolations by high performance liquid chromatographies, 16 peptides with the IC50 value of less than 20 microM, composed of 2-7 amino acid residues were identified from the WG hydrolyzate. Judging from the high content (260 mg in 100 g of AG50W fraction) and powerful ACE inhibitory activity (IC50; 0.48 microM), Ile-Val-Tyr was identified as a main contributor to the ACE inhibition of the hydrolyzate.

Detoxification and anti-nutrients reduction of Jatropha curcas seed cake by Bacillus fermentation.

PubMed

Phengnuam, Thanyarat; Suntornsuk, Worapot

2013-02-01

Jatropha curcas seed cake is a by-product generated from oil extraction of J. curcas seed. Although it contains a high amount of protein, it has phorbol esters and anti-nutritional factors such as phytate, trypsin inhibitor, lectin and saponin. It cannot be applied directly in the food or animal feed industries. This investigation was aimed at detoxifying the toxic and anti-nutritional compounds in J. curcas seed cake by fermentation with Bacillus spp. Two GRAS (generally recognized as safe) Bacillus strains used in the study were Bacillus subtilis and Bacillus licheniformis with solid-state and submerged fermentations. Solid-state fermentation was done on 10 g of seed cake with a moisture content of 70% for 7 days, while submerged fermentation was carried out on 10 g of seed cake in 100 ml distilled water for 5 days. The fermentations were incubated at the optimum condition of each strain. After fermentation, bacterial growth, pH, toxic and anti-nutritional compounds were determined. Results showed that B. licheniformis with submerged fermentation were the most effective method to degrade toxic and anti-nutritional compounds in the seed cake. After fermentation, phorbol esters, phytate and trypsin inhibitor were reduced by 62%, 42% and 75%, respectively, while lectin could not be eliminated. The reduction of phorbol esters, phytate and trypsin inhibitor was related to esterase, phytase and protease activities, respectively. J. curcas seed cake could be mainly detoxified by bacterial fermentation and the high-protein fermented seed cake could be potentially applied to animal feed. Copyright © 2012 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Diurnal Salivary Alpha-amylase Dynamics among Dementia Family Caregivers

PubMed Central

Liu, Yin; Granger, Douglas A.; Kim, Kyungmin; Klein, Laura C.; Almeida, David M.; Zarit, Steven H.

2016-01-01

Objective The study examined diurnal regulation of salivary alpha-amylase (sAA) in association with daily stressors, adult day services (ADS) use, and other caregiving characteristics. Methods A sample of 165 family caregivers of individuals with dementia (IWD) completed an 8-day diary study. Caregivers provided 5 saliva samples across the 8 days. On some days, caregivers provided all or most of the care. On other days, their relative attended ADS for part of the day. A 3-level unconditional linear spline model was fit to describe the typical sAA diurnal rhythms. Predictors were then added to the unconditional model to test the hypotheses on ADS use and daily stressors. Results Daily ADS use did not have an effect on diurnal sAA regulation. However, controlling for daily ADS use, greater ADS use over the 8 days was associated with a more prominent rise between 30 minutes after wake-up and before lunch, and a more prominent decline between before lunch and late afternoon. Fewer ADS days were associated with a more flattened sAA diurnal rhythm. Additionally, greater daily care-related stressor exposures had a within-person association with lower sAA levels in the late afternoon. Care-related stressor exposures had significant within- and between-person associations with sAA diurnal slopes. Furthermore, daily positive experiences had a significant between-person association with sAA diurnal slopes. Conclusions Caring for a disabled family member may heighten the vulnerability to potential physiological conditions. Respite from care stressors from ADS use may have some biobehavioral benefits on sAA regulations. PMID:27786517

Salivary Alpha Amylase and Cortisol Levels in Children with Global Developmental Delay and Their Relation with the Expectation of Dental Care and Behavior during the Intervention

ERIC Educational Resources Information Center

dos Santos, Marcio Jose Possari; Bernabe, Daniel Galera; Nakamune, Ana Claudia de Melo Stevanato; Perri, Silvia Helena Venturoli; de Aguiar, Sandra Maria Herondina Coelho Avila; de Oliveira, Sandra Helena Penha

2012-01-01

The purpose of this study was to analyze the alpha-amylase (sAA) and cortisol levels in children with Global developmental delay (GDD) before and after dental treatment and its association with the children's behavior during treatment. The morning salivary cortisol levels and activity of sAA of 33 children with GDD were evaluated before and after…

Production of itaconic acid in Escherichia coli expressing recombinant α-amylase using starch as substrate.

PubMed

Okamoto, Shusuke; Chin, Taejun; Nagata, Keisuke; Takahashi, Tetsuya; Ohara, Hitomi; Aso, Yuji

2015-05-01

Several studies on fermentative production of a vinyl monomer itaconic acid from hydrolyzed starch using Aspergillus terreus have been reported. Herein, we report itaconic acid production by Escherichia coli expressing recombinant α-amylase, using soluble starch as its sole carbon source. To express α-amylase in E. coli, we first constructed recombinant plasmids expressing α-amylases by using cell surface display technology derived from two amylolytic bacteria, Bacillus amyloliquefaciens NBRC 15535(T) and Streptococcus bovis NRIC 1535. The recombinant α-amylase from S. bovis (SBA) showed activity at 28°C, which is the optimal temperature for production of itaconic acid, while α-amylase from B. amyloliquefaciens displayed no noticeable activity. E. coli cells expressing SBA produced 0.15 g/L itaconic acid after 69 h cultivation under pH-stat conditions, using 1% starch as the sole carbon source. In fact, E. coli cells expressing SBA had similar growth rates when grown in the presence of 1% glucose or starch, thereby highlighting the expression of an active α-amylase that enabled utilization of starch to produce itaconic acid in E. coli. Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Some distinguishable properties between acid-stable and neutral types of alpha-amylases from acid-producing koji.

PubMed

Suganuma, Toshihiko; Fujita, Kiyotaka; Kitahara, Kanefumi

2007-11-01

The highly humid climate of Japan facilitates the growth of various molds. Among these molds, Aspergillus oryzae is the most important and popular in Japan, and has been used as yellow-koji in producing many traditional fermented beverages and foods, such as Japanese sake, and soy sauce. Taka-amylase A (TAA), a major enzyme produced by the mold, is well known worldwide to be a leading enzyme for industrial utilization and academic study, since many extensive studies have been carried out with TAA. In southern Kyushu, the other koji's of citric acid-producing molds have often been used, such as in the production of a traditional distilled liquor of shochu. The koji molds black-koji and white-koji produce two types of alpha-amylase, namely, acid-stable (AA) and common neutral (NA). The latter enzyme is enzymatically and genetically similar to TAA. In this review, we investigate AA from three molds, Aspergillus niger, A. kawachii and A. awamori, and the yeast Cryptococcus sp. regarding the distinguishable properties between AA and NA. (i) The N-terminus amino acid sequences of AA determined by molecular cloning started with the sequence of L-S-A-, whereas those of NA started with A-T-P-. (ii) Most of the full sequences of AA were composed of, besides a core catalytic domain, an extra domain of a hinge region and a carbohydrate binding domain, which could be responsible for raw-starch-digestibility. The AA from A. niger has no exceptionally extra domain, similarly to NA. (iii) Simple methods for distinguishing AA from NA using CNP-alpha-G3 and G5 as substrates were developed by our group. (iv) The number of subsite in AA on the basis of its cleavage pattern of maltooligosaccharides was estimated to be five, which differs from that of TAA, 7-9. AA has many advantages in industrial applications, such as its acid-stability, thermostability, and raw-starch digesting properties.

Isolation of potential probiotic Bacillus spp. and assessment of their subcellular components to induce immune responses in Labeo rohita against Aeromonas hydrophila.

PubMed

Ramesh, Dharmaraj; Vinothkanna, Annadurai; Rai, Amit Kumar; Vignesh, Venkada Subramanian

2015-08-01

Bacillus species isolated from the gut of healthy Labeo rohita (Hamilton) were screened for antibacterial activity against selected fish pathogens. Among the isolates, KADR5 and KADR6 showed antibacterial activity, tolerated low pH and high bile concentrations and were susceptibility to various antibiotics. Based on morphological and biochemical tests and 16S rRNA gene analysis the probiotic strains KADR5 and KADR6 were identified as Bacillus licheniformis and Bacillus pumilus, respectively. The immune stimulatory effect of subcellular components of probiotic Bacillus licheniformis KADR5 and Bacillus pumilus KADR6 in L. rohita against Aeromonas hydrophila infection was studied. Fish were immunized intraperitoneally in case of subcellular components [cell wall proteins (CWPs), extracellular proteins (ECPs), whole cell proteins (WCPs)] and orally in case of live cells (10(8) CFU/g of feed). After 14th day of administration, fishes from each group were challenged intraperitoneally with 0.1 ml of A. hydrophila cell suspension in PBS (10(5) cells ml(-1)). Groups immunized with subcellular components and live cells had significantly lower mortalities of 20-40% and 23-33%, respectively in comparison to control (80% mortality). The non specific immune factors in the cellular components and viable cells of the probiotics increased the expression of lysozyme and respiratory burst. Use of WCPs and CWPs resulted in better protection against A. hydrophila in L. rohita. Our results clearly reflect the potential of cellular components of the probiotics Bacillus species for the protection of fish against A. hydrophila infection by enhancing the immune response. Copyright © 2015 Elsevier Ltd. All rights reserved.

Isolation and characterization of a novel analyte from Bacillus subtilis SC-8 antagonistic to Bacillus cereus.

PubMed

Lee, Nam Keun; Yeo, In-Cheol; Park, Joung Whan; Kang, Byung-Sun; Hahm, Young Tae

2010-09-01

In this study, an effective substance was isolated from Bacillus subtilis SC-8, which was obtained from traditionally fermented soybean paste, cheonggukjang. The substance was purified by HPLC, and its properties were analyzed. It had an adequate antagonistic effect on Bacilluscereus, and its spectrum of activity was narrow. When tested on several gram-negative and gram-positive foodborne pathogenic bacteria such as Salmonella enterica, Salmonella enteritidis, Staphylococcus aureus, and Listeria monocytogenes, no antagonistic effect was observed. Applying the derivative from B. subtilis SC-8 within the same genus did not inhibit the growth of major soybean-fermenting bacteria such as Bacillus subtilis, Bacillus licheniformis, and Bacillus amyloquefaciens. The range of pH stability of the purified antagonistic substance was wide (from 4.0 to >10.0), and the substance was thermally stable up to 60 degrees C. In the various enzyme treatments, the antagonistic activity of the purified substance was reduced with proteinase K, protease, and lipase; its activity was partially destroyed with esterase. Spores of B. cereus did not grow at all in the presence of 5mug/mL of the purified antagonistic substance. The isolated antagonistic substance was thought to be an antibiotic-like lipopeptidal compound and was tentatively named BSAP-254 because it absorbed to UV radiation at 254nm. Copyright 2010 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Characterizing Novel Thermophilic Amylase Producing Bacteria From Taptapani Hot Spring, Odisha, India

PubMed Central

Sen, Sudip Kumar; Raut, Sangeeta; Satpathy, Soumya; Rout, Prangya Ranjan; Bandyopadhyay, Bidyut; Das Mohapatra, Pradeep Kumar

2014-01-01

Background: Amylases play a vital role in biotechnological studies and rank an important position in the world enzyme market (25% to 33%). Bioprocess method of amylase production is more effective than the other sources, since the technique is easy, cost effective, fast, and the enzymes of required properties can be procured. Objectives: The current study aimed to report the characteristics of novel amylase producing bacterial strains isolated from Taptapani hot spring, Odisha, India. Materials and Methods: Bacterial strains were isolated by dilution plating method from the water samples collected from Taptapani Hot Spring, Odisha and screened for amylase production through starch hydrolysis. The bacterial isolates were identified morphologically, biochemically, and finally by 16S rDNA profiling. Results: Based on the morphological, physiological, biochemical characteristics and the molecular characterization, the isolates SS1, SS2, and SS3 were identified as Bacillus barbaricus, Aeromonas veroni, and Stenotrophomonas maltophilia, respectively. The approximate molecular weight of enzymes from SS1, SS2, and SS3 strains were 19 kDa, 56 kDa and 49 kDa, respectively. Conclusions: The current report isolates, characterizes, and demonstrates the novel heat-adapted amylase-producing bacteria SS1, SS2 and SS3 from Taptapani hot spring, indicating its potentiality and stability under acidic conditions. PMID:25741425

The 53-kDa proteolytic product of precursor starch-hydrolyzing enzyme of Aspergillus niger has Taka-amylase-like activity.

PubMed

Ravi-Kumar, K; Venkatesh, K S; Umesh-Kumar, S

2007-04-01

The 53-kDa amylase secreted by Aspergillus niger due to proteolytic processing of the precursor starch-hydrolyzing enzyme was resistant to acarbose, a potent alpha-glucosidase inhibitor. The enzyme production was induced when A. niger was grown in starch medium containing the inhibitor. Antibodies against the precursor enzyme cross-reacted with the 54-kDa Taka-amylase protein of A. oryzae. It resembled Taka-amylase in most of its properties and also hydrolyzed starch to maltose of alpha-anomeric configuration. However, it did not degrade maltotriose formed during the reaction and was not inhibited by zinc ions.

Harsh discipline and behavior problems: the moderating effects of cortisol and alpha-amylase.

PubMed

Chen, Frances R; Raine, Adrian; Rudo-Hutt, Anna S; Glenn, Andrea L; Soyfer, Liana; Granger, Douglas A

2015-01-01

Numerous studies link harsh discipline to adjustment problems in youth, yet not all individuals exposed to harsh discipline develop behavior problems. Contemporary theory suggests that this relationship could be moderated by individual differences in environmentally sensitive biological systems. This study investigated whether the interaction between hypothalamic-pituitary-adrenal (HPA) activity and autonomic nervous system (ANS) arousal moderated the link between harsh discipline and behavior problems. Three saliva samples were collected on a single day from 425 inner city youth (50% male, age 11-12 years, 80% African American) and were later assayed for cortisol (HPA) and alpha-amylase (ANS). Problem behavior was assessed by self- and parent-report using the Child Behavior Checklist. Youth also reported the level of harsh discipline that they experienced. Harsh discipline was positively associated with externalizing and internalizing problems only when there were asymmetrical profiles of HPA activity and ANS arousal. This pattern was evident for boys but not girls. Findings are discussed in relation to prevailing theories suggesting that biological susceptibility translates adversity into risk for behavior problems. Copyright © 2014 Elsevier B.V. All rights reserved.

Purification and characterization of a thermostable α-amylase produced by the fungus Paecilomyces variotii.

PubMed

Michelin, Michele; Silva, Tony M; Benassi, Vivian M; Peixoto-Nogueira, Simone C; Moraes, Luiz Alberto B; Leão, Juliana M; Jorge, João A; Terenzi, Héctor F; Polizeli, Maria de Lourdes T M

2010-11-02

An α-amylase produced by Paecilomyces variotii was purified by DEAE-cellulose ion exchange chromatography, followed by Sephadex G-100 gel filtration and electroelution. The α-amylase showed a molecular mass of 75 kDa (SDS-PAGE) and pI value of 4.5. Temperature and pH optima were 60°C and 4.0, respectively. The enzyme was stable for 1 h at 55°C, showing a t₅₀ of 53 min at 60°C. Starch protected the enzyme against thermal inactivation. The α-amylase was more stable in alkaline pH. It was activated mainly by calcium and cobalt, and it presented as a glycoprotein with 23% carbohydrate content. The enzyme preferentially hydrolyzed starch and, to a lower extent, amylose and amylopectin. The K(m) of α-amylase on Reagen® and Sigma® starches were 4.3 and 6.2 mg/mL, respectively. The products of starch hydrolysis analyzed by TLC were oligosaccharides such as maltose and maltotriose. The partial amino acid sequence of the enzyme presented similarity to α-amylases from Bacillus sp. These results confirmed that the studied enzyme was an α-amylase ((1→4)-α-glucan glucanohydrolase). Copyright © 2010 Elsevier Ltd. All rights reserved.

Role of mechanical vs. chemical action in the removal of adherent Bacillus spores during CIP procedures.

PubMed

Faille, C; Bénézech, T; Blel, W; Ronse, A; Ronse, G; Clarisse, M; Slomianny, C

2013-04-01

This study was designed to evaluate the respective roles of mechanical and chemical effects on the removal of Bacillus spores during cleaning-in-place. This analysis was performed on 12 strains belonging to the Bacillus cereus group (B. cereus, Bacillus anthracis, Bacillus thuringiensis) or to less related Bacillus species (Bacillus pumilus, Bacillus licheniformis, Bacillus sporothermodurans, Bacillus subtilis). Adherent spores were subjected to rinsing-in-place (mechanical action) and cleaning-in-place (mechanical and chemical actions) procedures, the latter involving NaOH 0.5% at 60°C. Results revealed that mechanical action alone only removed between 53 and 89% of the attached spores at a shear stress of 500 Pa. This resistance to shear was not related to spore surface properties. Conversely, in the presence of NaOH at a shear stress of 4 Pa, spores were readily detached, with between 80 and 99% of the adherent spores detached during CIP and the chemical action greatly depended on the strain. This finding suggests that chemical action plays the major role during CIP, whose efficacy is significantly governed by the spore surface chemistry. Copyright © 2012 Elsevier Ltd. All rights reserved.

Unintended changes in protein expression revealed by proteomic analysis of seeds from transgenic pea expressing a bean alpha-amylase inhibitor gene.

PubMed

Chen, Hancai; Bodulovic, Greg; Hall, Prudence J; Moore, Andy; Higgins, Thomas J V; Djordjevic, Michael A; Rolfe, Barry G

2009-09-01

Seeds of genetically modified (GM) peas (Pisum sativum L.) expressing the gene for alpha-amylase inhibitor-1 (alphaAI1) from the common bean (Phaseolus vulgaris L. cv. Tendergreen) exhibit resistance to the pea weevil (Bruchus pisorum). A proteomic analysis was carried out to compare seeds from GM pea lines expressing the bean alphaAI1 protein and the corresponding alphaAI1-free segregating lines and non-GM parental line to identify unintended alterations to the proteome of GM peas due to the introduction of the gene for alphaAI1. Proteomic analysis showed that in addition to the presence of alphaAI1, 33 other proteins were differentially accumulated in the alphaAI1-expressing GM lines compared with their non-GM parental line and these were grouped into five expression classes. Among these 33 proteins, only three were found to be associated with the expression of alphaAI1 in the GM pea lines. The accumulation of the remaining 30 proteins appears to be associated with Agrobacterium-mediated transformation events. Sixteen proteins were identified after MALDI-TOF-TOF analysis. About 56% of the identified proteins with altered accumulation in the GM pea were storage proteins including legumin, vicilin or convicilin, phaseolin, cupin and valosin-containing protein. Two proteins were uniquely expressed in the alphaAI1-expressing GM lines and one new protein was present in both the alphaAI1-expressing GM lines and their alphaAI1-free segregating lines, suggesting that both transgenesis and transformation events led to demonstrable changes in the proteomes of the GM lines tested.

Different small, acid-soluble proteins of the alpha/beta type have interchangeable roles in the heat and UV radiation resistance of Bacillus subtilis spores.

PubMed Central

Mason, J M; Setlow, P

1987-01-01

Spores of Bacillus subtilis strains which carry deletion mutations in one gene (sspA) or two genes (sspA and sspB) which code for major alpha/beta-type small, acid-soluble spore proteins (SASP) are known to be much more sensitive to heat and UV radiation than wild-type spores. This heat- and UV-sensitive phenotype was cured completely or in part by introduction into these mutant strains of one or more copies of the sspA or sspB genes themselves; multiple copies of the B. subtilis sspD gene, which codes for a minor alpha/beta-type SASP; or multiple copies of the SASP-C gene, which codes for a major alpha/beta-type SASP of Bacillus megaterium. These findings suggest that alpha/beta-type SASP play interchangeable roles in the heat and UV radiation resistance of bacterial spores. Images PMID:3112127

Diurnal salivary alpha-amylase dynamics among dementia family caregivers.

PubMed

Liu, Yin; Granger, Douglas A; Kim, Kyungmin; Klein, Laura C; Almeida, David M; Zarit, Steven H

2017-02-01

The study examined diurnal regulation of salivary alpha-amylase (sAA) in association with daily stressors, adult day services (ADS) use, and other caregiving characteristics. A sample of 165 family caregivers of individuals with dementia (IWD) completed an 8-day diary study. Caregivers provided 5 saliva samples across the 8 days. On some days, caregivers provided all or most of the care. On other days, their relative attended ADS for part of the day. A 3-level unconditional linear spline model was fit to describe the typical sAA diurnal rhythms. Predictors were then added to the unconditional model to test the hypotheses on ADS use and daily stressors. Daily ADS use did not have an effect on diurnal sAA regulation. However, controlling for daily ADS use, greater ADS use over the 8 days was associated with a more prominent rise between 30 min after wake-up and before lunch, and a more prominent decline between before lunch and late afternoon. Fewer ADS days were associated with a more flattened sAA diurnal rhythm. Additionally, greater daily care-related stressor exposures had a within-person association with lower sAA levels in the late afternoon. Care-related stressor exposures had significant within- and between-person associations with sAA diurnal slopes. Furthermore, daily positive experiences had a significant between-person association with sAA diurnal slopes. Caring for a disabled family member may heighten the vulnerability to potential physiological conditions. Respite from care stressors from ADS use may have some biobehavioral benefits on sAA regulations. (PsycINFO Database Record (c) 2017 APA, all rights reserved).

In silico exploration of Red Sea Bacillus genomes for natural product biosynthetic gene clusters.

PubMed

Othoum, Ghofran; Bougouffa, Salim; Razali, Rozaimi; Bokhari, Ameerah; Alamoudi, Soha; Antunes, André; Gao, Xin; Hoehndorf, Robert; Arold, Stefan T; Gojobori, Takashi; Hirt, Heribert; Mijakovic, Ivan; Bajic, Vladimir B; Lafi, Feras F; Essack, Magbubah

2018-05-22

The increasing spectrum of multidrug-resistant bacteria is a major global public health concern, necessitating discovery of novel antimicrobial agents. Here, members of the genus Bacillus are investigated as a potentially attractive source of novel antibiotics due to their broad spectrum of antimicrobial activities. We specifically focus on a computational analysis of the distinctive biosynthetic potential of Bacillus paralicheniformis strains isolated from the Red Sea, an ecosystem exposed to adverse, highly saline and hot conditions. We report the complete circular and annotated genomes of two Red Sea strains, B. paralicheniformis Bac48 isolated from mangrove mud and B. paralicheniformis Bac84 isolated from microbial mat collected from Rabigh Harbor Lagoon in Saudi Arabia. Comparing the genomes of B. paralicheniformis Bac48 and B. paralicheniformis Bac84 with nine publicly available complete genomes of B. licheniformis and three genomes of B. paralicheniformis, revealed that all of the B. paralicheniformis strains in this study are more enriched in nonribosomal peptides (NRPs). We further report the first computationally identified trans-acyltransferase (trans-AT) nonribosomal peptide synthetase/polyketide synthase (PKS/ NRPS) cluster in strains of this species. B. paralicheniformis species have more genes associated with biosynthesis of antimicrobial bioactive compounds than other previously characterized species of B. licheniformis, which suggests that these species are better potential sources for novel antibiotics. Moreover, the genome of the Red Sea strain B. paralicheniformis Bac48 is more enriched in modular PKS genes compared to B. licheniformis strains and other B. paralicheniformis strains. This may be linked to adaptations that strains surviving in the Red Sea underwent to survive in the relatively hot and saline ecosystems.

Activity and cellular localization of amylases of rabbit cecal bacteria.

PubMed

Sirotek, K; Marounek, M; Suchorská, O

2006-01-01

Five 11-week-old rabbits, fed a commercial granulated feed, were slaughtered and cecal starch-degrading bacteria enumerated; total concentration of cultivable bacteria utilizing starch averaged 5.5 x 10(10) CFU/g. The activity and cellular localization of amylases was determined in 9 bacteria identified as Actinomyces israeli (strains AA2 and AD4), Bacteroides spp. (strain AA3), Dichelobacter nodosus (strain AA4), Mitsuokella multiacidus (strain AA6), Eubacterium spp. (strains AA7 and AB2), Clostridium spp. (strains AD1 and AA5). Four strains (AA3, AA4, AA5, AD4) produced extracellular amylases with an activity of 26-35 micromol of reducing sugars per h per mg of protein; in five strains (AA2, AA6, AA7, AB2, AD1) amylases were membrane-bound with an activity of 14-18 micromol of reducing sugars per h per mg of protein. All strains exhibited a low intracellular amylolytic activity. The pH optimum of amylases was 6.8-7.0. In strains producing extracellular amylases a substantial loss of viscosity was observed during incubations of cultivation supernatant with starch, similar to viscosity reduction in starch solutions treated with alpha-amylase; this indicates an endo-type (random cleavage) of extracellular amylase reaction in the bacteria under study. No strain possessed glucoamylase activity.

A new group of glycoside hydrolase family 13 α-amylases with an aberrant catalytic triad

PubMed Central

Sarian, Fean D.; Janeček, Štefan; Pijning, Tjaard; Ihsanawati; Nurachman, Zeily; Radjasa, Ocky K.; Dijkhuizen, Lubbert; Natalia, Dessy; van der Maarel, Marc J. E. C.

2017-01-01

α-Amylases are glycoside hydrolase enzymes that act on the α(1→4) glycosidic linkages in glycogen, starch, and related α-glucans, and are ubiquitously present in Nature. Most α-amylases have been classified in glycoside hydrolase family 13 with a typical (β/α)8-barrel containing two aspartic acid and one glutamic acid residue that play an essential role in catalysis. An atypical α-amylase (BmaN1) with only two of the three invariant catalytic residues present was isolated from Bacillus megaterium strain NL3, a bacterial isolate from a sea anemone of Kakaban landlocked marine lake, Derawan Island, Indonesia. In BmaN1 the third residue, the aspartic acid that acts as the transition state stabilizer, was replaced by a histidine. Three-dimensional structure modeling of the BmaN1 amino acid sequence confirmed the aberrant catalytic triad. Glucose and maltose were found as products of the action of the novel α-amylase on soluble starch, demonstrating that it is active in spite of the peculiar catalytic triad. This novel BmaN1 α-amylase is part of a group of α-amylases that all have this atypical catalytic triad, consisting of aspartic acid, glutamic acid and histidine. Phylogenetic analysis showed that this group of α-amylases comprises a new subfamily of the glycoside hydrolase family 13. PMID:28287181

Hypoglycemic activity of Pyrus biossieriana Buhse leaf extract and arbutin: Inhibitory effects on alpha amylase and alpha glucosidase

PubMed Central

Yousefi, Fatemeh; Mahjoub, Soleiman; Pouramir, Mahdi; Khadir, Fatemeh

2013-01-01

Background: The mechanism of hypoglycemic and hypolipidemic activities of Pyrus biossieriana Buhse leaf extract (PbBLE) and its phytochemical component arbutin, have not been well determined. The present study was performed to understand the hypoglycemic activity mechanisms of pbBLE and arbutin more clearly. Methods: In vitro enzymatic carbohydrate digestion with PbBLE and arbutin was assessed using α-amylase and α-glucosidase powders. The enzyme solutions were premixed with PbBLE and arbutin at different concentrations (0.1, 1, 10 and 100 mg/ml). Substrate solutions and colorimetric reagents were added to the reaction. The release of glucose was determined by spectrophotometric method. Acarbose was used as the positive control. Results: The extract (10, 100 mg/ ml) completely inhibit α- amylase and α- glucosidase activities. The extract produced higher reduction of α-amylase and α-glucosidase activity than arbutin. Inhibition at various concentrations (0.1, 1, 10, 100 mg/ml) were significantly different (p<0.05). Conclusion: Our results exhibited that both the extract and arbutin were able to suppress the enzymes strongly. PMID:24294470

Cloning and expression of an alpha-1,3-glucanase gene from Bacillus circulans KA-304: the enzyme participates in protoplast formation of Schizophyllum commune.

PubMed

Yano, Shigekazu; Wakayama, Mamoru; Tachiki, Takashi

2006-07-01

A culture filtrate of Bacillus circulans KA-304 grown on a cell-wall preparation of Schizophyllum commune has an activity to form protoplasts from S. commune mycelia, and a combination of alpha-1,3-glucanase and chitinase I, which were isolated from the filtrate, brings about the protoplast-forming activity. The gene of alpha-1,3-glucanase was cloned from B. circulans KA-304. It consists of 3,879 nucleotides, which encodes 1,293 amino acids including a putative signal peptide (31 amino acid residues), and the molecular weight of alpha-1,3-glucanase without the putative signal peptide was calculated to be 132,184. The deduced amino acid sequence of alpha-1,3-glucanase of B. circulans KA-304 showed approximately 80% similarity to that of mutanase (alpha-1,3-glucanase) of Bacillus sp. RM1, but no significant similarity to those of fungal mutanases. The recombinant alpha-1,3-glucanase was expressed in Escherichia coli Rosetta-gami B (DE 3), and significant alpha-1,3-glucanase activity was detected in the cell-free extract of the organism treated with isopropyl-beta-D-thiogalactopyranoside. The recombinant alpha-1,3-glucanase showed protoplast-forming activity when the enzyme was combined with chitinase I.

Effect of domestic cooking on the starch digestibility, predicted glycemic indices, polyphenol contents and alpha amylase inhibitory properties of beans (Phaseolis vulgaris) and breadfruit (Treculia africana).

PubMed

Chinedum, E; Sanni, S; Theressa, N; Ebere, A

2018-01-01

The effect of processing on starch digestibility, predicted glycemic indices (pGI), polyphenol contents and alpha amylase inhibitory properties of beans (Phaseolis vulgaris) and breadfruit (Treculia africana) was studied. Total starch ranged from 4.3 to 68.3g/100g, digestible starch ranged from 4.3 to 59.2 to 65.7g/100g for the raw and processed legumes; Resistance starch was not detected in most of the legumes except in fried breadfruit and the starches in both the raw and processed breadfruit were more rapidly digested than those from raw and cooked beans. Raw and processed breadfruit had higher hydrolysis curves than raw and processed beans with the amylolysis level in raw breadfruit close to that of white bread. Raw beans had a low glycemic index (GI); boiled beans and breadfruit had intermediate glycemic indices respectively while raw and fried breadfruit had high glycemic indices. Aqueous extracts of the food samples had weak α-amylase inhibition compared to acarbose. The raw and processed legumes contained considerable amounts of dietary phenols and flavonoids. The significant correlation (r=0.626) between α-amylase inhibitory actions of the legumes versus their total phenolic contents suggests the contribution of the phenolic compounds in these legumes to their α-amylase inhibitory properties. Copyright © 2017 Elsevier B.V. All rights reserved.

In vitro susceptibility of Bacillus spp. to selected antimicrobial agents.

PubMed Central

Weber, D J; Saviteer, S M; Rutala, W A; Thomann, C A

1988-01-01

Although often dismissed as contaminants when isolated from blood cultures, Bacillus spp. are increasingly recognized as capable of causing serious systemic infections. As part of a clinical-microbiological study, 89 strains of Bacillus spp. isolated from clinical blood cultures between 1981 and 1985 had their species determined and were tested for antimicrobial agent susceptibility to 18 antibiotics. Species of isolates were determined by the API 50CH and API 20E systems. Bacillus cereus (54 strains) was the most common species isolated, followed by B. megaterium (13 strains), B. polymyxa (5 strains), B. pumilus (4 strains), B. subtilis (4 strains), B. circulans (3 strains), B. amyloliquefaciens (2 strains), B. licheniformis (1 strain), and Bacillus spp. (3 strains). Microdilution MIC susceptibility tests revealed all B. cereus strains to be susceptible to imipenem, vancomycin, chloramphenicol, gentamicin, and ciprofloxacin. Non-B. cereus strains were most susceptible to imipenem, vancomycin, LY146032, and ciprofloxacin. Disk susceptibility testing suggested that B. cereus was rarely susceptible to penicillins, semisynthetic penicillins, or cephalosporins with the exception of mezlocillin. In contrast, many non-B. cereus strains were susceptible to penicillins, semisynthetic penicillins, and cephalosporins, but marked variability was noted among species. PMID:3395100

Sorbitol counteracts temperature- and chemical-induced denaturation of a recombinant α-amylase from alkaliphilic Bacillus sp. TS-23.

PubMed

Chi, Meng-Chun; Wu, Tai-Jung; Chen, Hsing-Ling; Lo, Huei-Fen; Lin, Long-Liu

2012-12-01

Enzymes are highly complex systems with a substantial degree of structural variability in their folded state. In the presence of cosolvents, fluctuations among vast numbers of folded and unfolded conformations occur via many different pathways; alternatively, certain conformations can be stabilized or destabilized. To understand the contribution of osmolytes to the stabilization of structural changes and enzymatic activity of a truncated Bacillus sp. TS-23 α-amylase (BACΔNC), we monitored amylolytic activity, circular dichroism, and fluorescence as a function of osmolytes. In the presence of trimethylamine N-oxide (TMAO) and sorbitol, BACΔNC activity was retained significantly at elevated temperatures. As compared to the control, the secondary structures of this enzyme were essentially conserved upon the addition of these two kinds of osmolytes. Fluorescence results revealed that the temperature-induced conformational change of BACΔNC was prevented by TMAO and sorbitol. However, glycerol did not provide profound protection against thermal denaturation of the enzyme. Sorbitol was further found to counteract guanidine hydrochloride- and SDS-induced denaturation of BACΔNC. Thus, some well-known naturally occurring osmolytes make a dominant contribution to the stabilization of BACΔNC.

Stress exacerbates pain in the everyday lives of women with fibromyalgia syndrome--The role of cortisol and alpha-amylase.

PubMed

Fischer, Susanne; Doerr, Johanna M; Strahler, Jana; Mewes, Ricarda; Thieme, Kati; Nater, Urs M

2016-01-01

Although fibromyalgia syndrome (FMS) is a chronic condition, its cardinal symptom pain is known to fluctuate over the day. Stress has often been claimed to exacerbate pain; however, there is barely any evidence on whether or not this is true on a day-to-day basis (and, alternatively, on whether pain leads to increased stress levels). Using an ecologically valid measurement design, we tested whether and how stress and pain are intertwined in participants with FMS. We additionally examined the role of the two major stress-responsive systems, the hypothalamic-pituitary-adrenal axis and the autonomic nervous system, as potential mediators of this relationship. An ambulatory assessment study was conducted over the course of 14 days. On each day, 32 females with FMS provided six diary entries on momentary stress and pain levels. Saliva samples were collected at the same time points to determine cortisol and alpha-amylase as indicators of stress-responsive systems. Higher stress at a given measurement time point was associated with higher reported pain levels at the subsequent time point (UC=1.47, p<0.001), but not vice versa (UC<0.01, p=0.179). The stress-pain relationship was neither mediated by momentary cortisol nor by alpha-amylase; however, momentary cortisol was independently associated with momentary pain (UC=0.27, p=0.009). Stress seems to be a powerful exacerbating factor for pain as experienced by patients with FMS in their everyday lives. Cortisol may be involved in the diurnal fluctuation of pain levels in patients with FMS. Future studies should identify relevant daily stressors in persons with FMS and scrutinize the mechanisms underlying the cortisol-pain relationship. Copyright © 2015 Elsevier Ltd. All rights reserved.

Structure of waxy maize starch hydrolyzed by maltogenic alpha-amylase in relation to its retrogradation

USDA-ARS?s Scientific Manuscript database

Maltogenic a-amylase is widely used as an antistaling agent in bakery foods. The objective of this study was to determine the degree of hydrolysis (DH) and starch structure after maltogenic amylase treatments in relation to its retrogradation. Waxy maize starch was cooked and hydrolyzed to different...

Daytime Secretion of Salivary Cortisol and Alpha-Amylase in Preschool-Aged Children with Autism and Typically Developing Children

PubMed Central

Corbett, Blythe A.; Granger, Douglas A.; Boyce, W. Thomas; Anders, Thomas F.; Tager, Ira B.

2013-01-01

We examined daytime salivary cortisol and salivary alpha-amylase (sAA) secretion levels and variability in preschool-aged children with autism (AUT) and typically developing children (TYP). Fifty-two subjects (26 AUT and 26 TYP) were enrolled. Salivary samples were obtained at waking, midday, and bedtime on two consecutive days at three phases (baseline, 3 months later, 6 months later). There were modest increases in waking cortisol and sAA levels in AUT relative to TYP, but the increases were not statistically significant. Important differences were observed in cortisol and sAA variability between AUT and TYP. There was also a graded response among AUT by functional status—cortisol and sAA secretion levels were higher when IQ was lower. PMID:22477468

Alpha-amylase reactivity in relation to psychopathic traits in adults.

PubMed

Glenn, Andrea L; Remmel, Rheanna J; Raine, Adrian; Schug, Robert A; Gao, Yu; Granger, Douglas A

2015-04-01

Recent investigations of the psychobiology of stress in antisocial youth have benefited from a multi-system measurement model. The inclusion of salivary alpha-amylase (sAA), a surrogate marker of autonomic/sympathetic nervous system (ANS) activity, in addition to salivary cortisol, a biomarker of the hypothalamic-pituitary-adrenal (HPA) axis functioning, has helped define a more complete picture of individual differences and potential dysfunction in the stress response system of these individuals. To the authors' knowledge, no studies have examined sAA in relation to antisocial behavior in adults or in relation to psychopathic traits specifically. In the present study, we examined sAA, in addition to salivary cortisol, in a relatively large sample (n=158) of adult males (M age=36.81, range=22-67 years; 44% African-American, 34% Caucasian, 16% Hispanic) recruited from temporary employment agencies with varying levels of psychopathic traits. Males scoring highest in psychopathy were found to have attenuated sAA reactivity to social stress compared to those scoring lower in psychopathy. No differential relationships with the different factors of psychopathy were observed. In contrast to studies of antisocial youth, there were no interactions between sAA and cortisol levels in relation to psychopathy, but there was a significant interaction between pre-stressor levels of sAA and cortisol. Findings reveal potential regulatory deficits in the fast-acting, 'fight or flight', component of the stress response in adult males with psychopathic traits, as well as abnormalities in how this system may interact with the HPA axis. Copyright © 2015 Elsevier Ltd. All rights reserved.

Cortisol and Alpha-amylase changes during an Ultra-Running Event.

PubMed

Deneen, Whitney P; Jones, Alexis B

2017-01-01

Elevated stress hormone concentrations can positively affect an athlete's overall performance during a competition, and in many cases, are necessary to be able to perform exercise. During extreme exercise, the body's ability to utilize energy efficiently can affect an athlete's performance. Elevated hormonal concentrations can have many benefits in regards to an athlete's overall performance during a competition. The purpose of this study was to examine the effects of long distance running, such as seen during an ultra-running event (distances beyond 26.2 miles), on the activity of the hypothalamic-pituitary-adrenocortical (HPA) axis production of cortisol (CORT) as compared to autonomic nervous system production of salivary alpha-amylase (AA). Despite the well-known effects of exercise on CORT and AA response, it is unclear what effect running beyond the marathon distance has on these levels. This study investigates what effect long duration cardio exercise, such as running up to 100K (kilometers) distance, has on the neuroendocrine system, by means of saliva samples provided by participants signed up for an ultra-marathon event. The findings of this study show that the autonomic nervous system may present a response signal during physical stress that is independent of the HPA axis response. At distances beyond the marathon length, the production of CORT and AA was found to be suppressed for athletes, which could help them in their continued performance. Furthermore, this study recognizes a difference in the overall male and female response to stress in regards to CORT and AA production.

Alpha-Amylase Reactivity in Relation to Psychopathic Traits in Adults

PubMed Central

Glenn, Andrea L.; Remmel, Rheanna J.; Raine, Adrian; Schug, Robert A.; Gao, Yu; Granger, Douglas A.

2015-01-01

Recent investigations of the psychobiology of stress in antisocial youth have benefited from a multi-system measurement model. The inclusion of salivary alpha-amylase (sAA), a surrogate marker of autonomic/sympathetic nervous system (ANS) activity, in addition to salivary cortisol, a biomarker of the hypothalamic-pituitary-adrenal (HPA) axis functioning, has helped define a more complete picture of individual differences and potential dysfunction in the stress response system of these individuals. To the authors' knowledge, no studies have examined sAA in relation to antisocial behavior in adults or in relation to psychopathic traits specifically. In the present study, we examined sAA, in addition to salivary cortisol, in a relatively large sample (n = 158) of adult males (M age = 36.81, range = 22-67 years; 44% African-American, 34% Caucasian, 16% Hispanic) recruited from temporary employment agencies with varying levels of psychopathic traits. Males scoring highest in psychopathy were found to have attenuated sAA reactivity to social stress compared to those scoring lower in psychopathy. No differential relationships with the different factors of psychopathy were observed. In contrast to studies of antisocial youth, there were no interactions between sAA and cortisol levels in relation to psychopathy, but there was a significant interaction between pre-stressor levels of sAA and cortisol. Findings reveal potential regulatory deficits in the fast-acting, ‘fight or flight’, component of the stress response in adult males with psychopathic traits, as well as abnormalities in how this system may interact with the HPA axis. PMID:25662339

Gibberellin induces alpha-amylase gene in seed coat of Ipomoea nil immature seeds.

PubMed

Nakajima, Masatoshi; Nakayama, Akira; Xu, Zheng-Jun; Yamaguchi, Isomaro

2004-03-01

Two full-length cDNAs encoding gibberellin 3-oxidases, InGA3ox1 and InGA3ox2, were cloned from developing seeds of morning glory (Ipomoea nil (Pharbitis nil) Choisy cv. Violet) with degenerate-PCR and RACEs. The RNA-blot analysis for these clones revealed that the InGA3ox2 gene was organ-specifically expressed in the developing seeds at 6-18 days after anthesis. In situ hybridization showed the signals of InGA3ox2 mRNA in the seed coat, suggesting that active gibberellins (GAs) were synthesized in the tissue, although no active GA was detected there by immunohistochemistry. In situ hybridization analysis for InAmy1 (former PnAmy1) mRNA showed that InAmy1 was also synthesized in the seed coat. Both InGA3ox2 and InAmy1 genes were expressed spatially overlapped without a clear time lag, suggesting that both active GAs and InAmy1 were synthesized almost simultaneously in seed coat and secreted to the integument. These observations support the idea that GAs play an important role in seed development by inducing alpha-amylase.

Alpha-amylase inhibitory activity and sterol composition of the marine algae, Sargassum glaucescens

PubMed Central

Payghami, Nasrin; Jamili, Shahla; Rustaiyan, Abdolhossein; Saeidnia, Soodabeh; Nikan, Marjan; Gohari, Ahmad Reza

2015-01-01

Background: Sargassum species (phaeophyceae) are economically important brown algae in southern parts of Iran. Sargassum is mainly harvested as a row material in alginate production industries and is a source of plant foods or plant bio-stimulants even a component of animal foods. Objective: In this study, Sargassum glaucescens, collected from the seashore of Chabahar, was employed for phytochemical and biological evaluations. Materials and Methods: For that purpose, the dried algae was extracted by methanol and subjected to different chromatographic separation methods. Results: Six sterols, fucosterol (1), 24(S)-hydroxy-24-vinylcholesterol (2), 24(R)-hydroxy-24-vinylcholesterol (3), stigmasterol (4), β-sitosterol (5) and cholesterol (6) were identified by spectroscopic methods including 1H-NMR, 13C-NMR and mass spectroscopy. In vitro alpha-amylase inhibitory test was performed on the methanolic extract and the results revealed a potent inhibition (IC50 = 8.9 ± 2.4 mg/mL) of the enzyme compared to acarbose as a positive control. Conclusion: Various biological activities and distribution of sterols in Sargassum genus have been critically reviewed here. The results concluded that these algae are a good candidate for further anti-diabetic investigations in animals and human. PMID:26692744

Co-Metabolic Degradation of β-Cypermethrin and 3-Phenoxybenzoic Acid by Co-Culture of Bacillus licheniformis B-1 and Aspergillus oryzae M-4.

PubMed

Zhao, Jiayuan; Chi, Yuanlong; Xu, Yingchao; Jia, Dongying; Yao, Kai

2016-01-01

The degradation efficiency of organic contaminants and their associated metabolites by co-culture of microbes is mainly limited by toxic intermediates from co-metabolic degradation. In this study, we investigated the degradation of β-cypermethrin (β-CY) and 3-phenoxybenzoic acid (3-PBA) by co-culture of Bacillus licheniformis B-1 and Aspergillus oryzae M-4, as well as the influences of β-CY and 3-PBA metabolites on their degradation and the growth of strains B-1 and M-4. Our results indicated that 100 mg/L β-CY was degraded by 78.85%, and 3-PBA concentration was 0.05 mg/L after 72 h. Compared with using only strain B-1, the half-life (t1/2) of β-CY by using the two strains together was shortened from 84.53 h to 38.54 h, and the yield coefficient of 3-PBA was decreased from 0.846 to 0.001. At 100 mg/L of 3-PBA and gallic acid, β-CY and 3-PBA degradation were only 17.68% and 40.45%, respectively. As the toxic intermediate derived from co-metabolic degradation of β-CY by strain B-1, 3-PBA was efficiently degraded by strain M-4, and gallic acid, as the toxic intermediate from co-metabolic degradation of 3-PBA by strain M-4, was efficiently degraded by strain B-1. These results provided a promising approach for efficient biodegradation of β-CY and 3-PBA.

Structure of Bacillus halmapalus α-amylase crystallized with and without the substrate analogue acarbose and maltose

PubMed Central

Lyhne-Iversen, Louise; Hobley, Timothy J.; Kaasgaard, Svend G.; Harris, Pernille

2006-01-01

Recombinant Bacillus halmapalus α-amylase (BHA) was studied in two different crystal forms. The first crystal form was obtained by crystallization of BHA at room temperature in the presence of acarbose and maltose; data were collected at cryogenic temperature to a resolution of 1.9 Å. It was found that the crystal belonged to space group P212121, with unit-cell parameters a = 47.0, b = 73.5, c = 151.1 Å. A maltose molecule was observed and found to bind to BHA and previous reports of the binding of a nonasaccharide were confirmed. The second crystal form was obtained by pH-induced crystallization of BHA in a MES–HEPES–boric acid buffer (MHB buffer) at 303 K; the solubility of BHA in MHB has a retrograde temperature dependency and crystallization of BHA was only possible by raising the temperature to at least 298 K. Data were collected at cryogenic temperature to a resolution of 2.0 Å. The crystal belonged to space group P212121, with unit-cell parameters a = 38.6, b = 59.0, c = 209.8 Å. The structure was solved using molecular replacement. The maltose-binding site is described and the two structures are compared. No significant changes were seen in the structure upon binding of the substrates. PMID:16946462

Amylase - urine

MedlinePlus

... page: //medlineplus.gov/ency/article/003607.htm Amylase - urine To use the sharing features on this page, ... test that measures the amount of amylase in urine. Amylase is an enzyme that helps digest carbohydrates. ...

Synthesis and in vitro study of benzofuran hydrazone derivatives as novel alpha-amylase inhibitor.

PubMed

Taha, Muhammad; Shah, Syed Adnan Ali; Imran, Syahrul; Afifi, Muhammad; Chigurupati, Sridevi; Selvaraj, Manikandan; Rahim, Fazal; Ullah, Hayat; Zaman, Khalid; Vijayabalan, Shantini

2017-12-01

The α-amylase acts as attractive target to treat type-2 diabetes mellitus. Therefore in discovering a small molecule as α-amylase inhibitor, we have synthesized benzofuran carbohydrazide analogs (1-25), characterized through different spectroscopic techniques such as 1 HNMR and EI-MS. All screened analog shows good α-amylase inhibitory potentials with IC 50 value ranging between 1.078±0.19 and 2.926±0.05µM when compared with acarbose having IC 50 =0.62±0.22µM. Only nine analogs among the series such as analogs 3, 5, 7, 8, 10, 12, 21, 23 and 24 exhibit good inhibitory potential with IC 50 values 1.644±0.128, 1.078±0.19, 1.245±0.25, 1.843±0.19, 1.350±0.24, 1.629±0.015, 1.353±0.232, 1.359±0.119 and 1.488±0.07µM when compare with standard drug acarbose. All other analogs showed good to moderate α-amylase inhibitory potentials. The SAR study was conducted on the basis of substituent difference at the phenyl ring. The binding interaction between analogs and active site of enzyme was confirmed by docking studies. Copyright © 2017 Elsevier Inc. All rights reserved.

Potency of Amylase-producing Bacteria and Optimization Amylase Activities

NASA Astrophysics Data System (ADS)

Indriati, G.; Megahati, R. R. P.; Rosba, E.

2018-04-01

Enzymes are capable to act as biocatalyst for a wide variety of chemical reactions. Amylase have potential biotechnological applications in a wide range of industrial processes and account for nearly 30% of the world’s enzyme market. Amylase are extracellular enzymes that catalyze the hydrolysis of internal α-1,4-glycosidic linkages in starch to dextrin, and other small carbohydrate molecules constituted of glucose units. Although enzymes are produced from animal and plant sources, the microbial sources are generally the most suitable for commercial applications. Bacteria from hot springs is widely used as a source of various enzymes, such as amylase. But the amount of amylase-producing bacteria is still very limited. Therefore it is necessary to search sources of amylase-producing bacteria new, such as from hot springs Pariangan. The purpose of this study was to isolation of amylase-producing bacteria from Pariangan hot spring, West Sumatera and amylase activity optimization. The results were obtained 12 isolates of thermophilic bacteria and 5 isolates of amyalse-producing bacteria with the largest amylolytic index of 3.38 mm. The highest amylase activity was obtained at 50°C and pH 7.5.

Effect of boiling and roasting on the fermentation of soybeans into dawadawa (soy-dawadawa).

PubMed

Dakwa, Sarah; Sakyi-Dawson, Esther; Diako, Charles; Annan, Nana Takyiwa; Amoa-Awua, Wisdom Kofi

2005-09-25

Soybeans which had initially been dehulled by either boiling (boiled/dehulled) or roasting (roasted/dehulled) before peeling, were cooked and fermented into dawadawa, a traditional food condiment. The micropopulation, enzymatic activities, proximate composition, amino acid, and aroma profiles of the two types of soybean dawadawa were evaluated during fermentation. Only minor differences were found in the microbial profiles of the two types of soy-dawadawa. Although boiled/dehulled soy-dawadawa initially had lower microbial counts, it recorded higher counts at the advanced stages of fermentation. Proteolytic and amylolytic Bacillus species including Bacillus subtilis, Bacillus pumilus, Bacillus licheniformis, Bacillus cereus, and Bacillus firmus dominated the micropopulation of the two types of soy-dawadawa with Bacillus subtilis accounting for about 50% of the Bacillus species in all samples. Lactic acid bacteria and yeasts occurred in low numbers in the two types of soy-dawadawa. The proximate composition of the two types of soy-dawadawa were similar, and their contents of moisture and protein increased whilst fat and ash decreased during fermentation. Both types of fermenting soy-dawadawa recorded similar levels of alpha-amylase activity, but boiled/dehulled soy-dawadawa showed slightly higher protease activity. The levels of isoleucine, leucine, lysine, phenylalanine, arginine and proline increased significantly with fermentation time in both types of soy-dawadawa. With respect to differences in their aroma profiles, hexanodecanol, octadecyl acetate, 1,2-dimethyl benzene, tetradecene, (E)-5-eicosene, cyclohexadecane, and hexacosane were found only in the roasted/dehulled samples, whilst 1,2-ethanediol, ethyl acetate, dimethyl disulfide, cyclotetradecane, decene, indole , 2 butyl-octenal, acetophenone, and toluene were found only in the boiled/dehulled samples. A market focus group showed preference for roasted/dehulled soy-dawadawa over boiled/dehulled soy

Enzymatic synthesis of γ-L-glutamyl-S-allyl-L-cysteine, a naturally occurring organosulfur compound from garlic, by Bacillus licheniformis γ-glutamyltranspeptidase.

PubMed

Chen, Yi-Yu; Lo, Huei-Fen; Wang, Tzu-Fan; Lin, Min-Guan; Lin, Long-Liu; Chi, Meng-Chun

2015-01-01

In the practical application of Bacillus licheniformis γ-glutamyltranspeptidase (BlGGT), we describe a straightforward enzymatic synthesis of γ-L-glutamyl-S-allyl-L-cysteine (GSAC), a naturally occurring organosulfur compound found in garlic, based on a transpeptidation reaction involving glutamine as the γ-glutamyl donor and S-allyl-L-cysteine as the acceptor. With the help of thin layer chromatography technique and computer-assisted image analysis, we performed the quantitative determination of GSAC. The optimum conditions for a biocatalyzed synthesis of GSAC were 200 mM glutamine, 200 mM S-allyl-L-cysteine, 50 mM Tris-HCl buffer (pH 9.0), and BlGGT at a final concentration of 1.0 U/mL. After a 15-h incubation of the reaction mixture at 60 °C, the GSAC yield for the free and immobilized enzymes was 19.3% and 18.3%, respectively. The enzymatic synthesis of GSAC was repeated under optimal conditions at 1-mmol preparative level. The reaction products together with the commercially available GSAC were further subjected to an ESI-MS/MS analysis. A significant signal with m/z of 291.1 and the protonated fragments at m/z of 73.0, 130.1, 145.0, and 162.1 were observed in the positive ESI-MS/MS spectrum, which is consistent with those of the standard compound. These results confirm the successful synthesis of GSAC from glutamine and S-allyl-L-cysteine by BlGGT. Copyright © 2015 Elsevier Inc. All rights reserved.

Thermostable Fe/Mn superoxide dismutase from Bacillus licheniformis SPB-13 from thermal springs of Himalayan region: Purification, characterization and antioxidative potential.

PubMed

Thakur, Abhishek; Kumar, Pradeep; Lata, Jeevan; Devi, Neena; Chand, Duni

2018-05-01

Superoxide dismutase (SOD; EC 1.15.1.1) is an enzyme that scavenges free radicals and increases the longevity. In this study, a thermostable superoxide dismutase (SOD) from Bacillus licheniformis SPB-13, from Himalayan region was purified to homogeneity using ion exchange chromatography (DEAE-Sepharose). The SDS and native PAGE analysis showed that SOD is composed of two subunits of 32 kDa each and total molecular mass of the enzyme was estimated as 68 kDa. The specific activity of enzyme was 3965.51 U/mg and was purified to 16.17 folds. The SOD showed maximum activity with 60 mM Tris-HCl buffer at pH 8.0 for 2 min of incubation. Enzyme along with FeCl 3 as metal ion remained active till 70 °C. After reaction variables optimization, enzyme activity increased from 3965.51 to 4015.72 U/mg. Kinetic analysis of SOD showed k m of 1.4 mM of NADH and V max of 10000 U/mg of protein. Turnover number (k cat ) and catalytic efficiency (k cat /K m ) were found to be 11,333 s -1 and 7092.2 s -1 ·mM -1 NADH. The activation energy (E a ) was calculated as 2.67 kJ·mol -1 . After typing, it was found to be a member of Fe/Mn SOD family with IC 50 value of 25 μg/ml, prevented the cell death at a concentration of 30 μg/ml and it increased the cell viability by 30%. Copyright © 2018 Elsevier B.V. All rights reserved.

Psychosocial determinants of diurnal alpha-amylase among healthy Quebec workers.

PubMed

Marchand, Alain; Juster, Robert-Paul; Lupien, Sonia J; Durand, Pierre

2016-04-01

Salivary alpha-amylase (sAA) is a stress-sensitive biomarker the shows promise as an indirect proxy of sympathetic-adrenal-medullary axis activities that are otherwise difficult to discern non-invasively. This comprehensive study investigated diurnal sAA in association with numerous psychosocial characteristics related to mental health, work stress, and non-work stress. Participants included 395 workers (56.1% women, age: M=41.3, SD=10.81) from across 34 distinct workplaces. Diurnal sAA was sampled over two non-consecutive work days at awakening, 30 min after awakening, 14h00, 16h00, and bedtime. Well-validated psychometrics and survey items were used to measure mental health (psychological distress, depression, burnout, work characteristics) (task design, demands, social relations, gratifications), and non-work characteristics (marital/parental status, economic statuses, marital and parental stress, work-family conflicts). Preliminary results revealed that men showed occasionally higher sAA concentrations than women. Multilevel regressions were used to analyze sAA concentrations nested according to levels (i) for each time-point, (ii) between workers, and (iii) across workplaces while covarying for time of awakening, sex, age, cigarette smoking, alcohol consumption, regular physical activity, psychotropic drug use, and body mass index. Main results revealed that psychological demands, support from colleagues, interpersonal conflicts, job recognition and job insecurity appear to be associated with diurnal sAA, while non-work factors did not. Our findings showing a distinct diurnal profile for sAA replicate and expand those of Nater et al. (2007, Psychoneuroendocrinology 32, 392-401), providing further evidence that sAA is associated to subjective psychosocial factors. Copyright © 2016 Elsevier Ltd. All rights reserved.

Four rice seed cDNA clones belonging to the alpha-amylase/trypsin inhibitor gene family encode potential rice allergens.

PubMed

Alvarez, A M; Fukuhara, E; Nakase, M; Adachi, T; Aoki, N; Nakamura, R; Matsuda, T

1995-07-01

Four rice seed proteins encoded by cDNAs belonging to the alpha-amylase/trypsin inhibitor gene family were overexpressed as TrpE-fusion proteins in E. coli. The expressed rice proteins were detected by SDS-PAGE as major proteins in bacterial cell lysates. Western blot analyses showed that all the recombinant proteins were immunologically reactive to rabbit polyclonal antibodies and to a mouse monoclonal antibody (25B9) specific for a previously isolated rice allergen of 16 kDa. Some truncated proteins from deletion mutants of the cDNAs retained their reactivity to the specific antibodies. These results suggest that the cDNAs encode potential rice allergens and that some epitopes of the recombinant proteins are still immunoreactive when they are expressed as their fragments.

Salivary flow and alpha-amylase: collection technique, duration, and oral fluid type.

PubMed

Beltzer, Emilie K; Fortunato, Christine K; Guaderrama, Melissa M; Peckins, Melissa K; Garramone, Bianca M; Granger, Douglas A

2010-09-01

There has been renewed interest in salivary alpha-amylase (sAA), a surrogate marker of autonomic/sympathetic activity, in biosocial research on stress vulnerability, reactivity, and recovery. This study explored the impact of saliva flow rate on sAA measurement by examining the influence of (1) the technique used to collect oral fluid-synthetic swab, cotton pledget, hydrocellulose microsponge, or passive drool; (2) collection point duration--the length of time the technique is employed (1-5min); and (3) oral fluid type--whole unstimulated saliva (not absorbed by any material) or oral fluid sampled from areas near the parotid, submandibular, or sublingual salivary glands. sAA activity (U/mL) was the highest in oral fluid collected from the parotid and submandibular gland areas. The volume (mL) of oral fluid collected increased, and the activity of sAA (U/mL) decreased, as collection point duration lengthened. The magnitude of these effects varied according to collection technique and oral fluid type. Across all conditions, there were positive correlations (range .70-.88) between sAA activity (U/mL) and sAA output (U/min). Management of these potential sources of measurement error will be essential to ensuring the success of future research on the correlates and concomitants of sAA activity, stress-related reactivity and recovery, and diurnal variation. Copyright 2010 Elsevier Inc. All rights reserved.

Mechanism of removal of undesirable residual amylase, insoluble starch, and select colorants from refinery streams by powdered activated carbons

USDA-ARS?s Scientific Manuscript database

There is a need in the world-wide sugar industry to find a practical and economical solution to remove or inactivate residual alpha-amylases that are high temperature stable from factory or refinery streams. A survey of refineries that used amylase and had activated carbon systems for decolorization...

Butyric acid released during milk lipolysis triggers biofilm formation of Bacillus species.

PubMed

Pasvolsky, Ronit; Zakin, Varda; Ostrova, Ievgeniia; Shemesh, Moshe

2014-07-02

Bacillus species form biofilms within milking pipelines and on surfaces of equipment in the dairy industry which represent a continuous hygiene problem and can lead to serious economic losses due to food spoilage and equipment impairment. Although much is known about the mechanism by which the model organism Bacillus subtilis forms biofilms in laboratory mediums in vitro, little is known of how these biofilms are formed in natural environments such as milk. Besides, little is known of the signaling pathways leading to biofilm formation in other Bacillus species, such as Bacillus cereus and Bacillus licheniformis, both of which are known to contaminate milk. In this study, we report that milk triggers the formation of biofilm-related structures, termed bundles. We show this to be a conserved phenomenon among all Bacillus members tested. Moreover, we demonstrate that the tasA gene, which encodes a major portion of the matrix which holds the biofilm together, is vital for this process. Furthermore, we show that the free fatty acid (FFA) - butyric acid (BA), which is released during lipolysis of milk fat and demonstrates antimicrobial activity, is the potent trigger for biofilm bundle formation. We finally show that BA-triggered biofilm bundle formation is mediated by the histidine kinase, KinD. Taken together, these observations indicate that BA, which is a major FFA within milk triggers biofilm formation in a conserved mechanism among members of the Bacillus genus. Copyright © 2014 Elsevier B.V. All rights reserved.

Salivary cortisol, alpha-amylase and immunoglobulin a responses to a morning session of basketball or volleyball training in boys aged 14-18 years.

PubMed

Bruzda-Zwiech, A; Konieczka, M; Hilt, A; Daszkowska, M; Grzegorczyk, J; Szczepańska, J

2017-01-01

This study investigates whether a single session of routine morning basketball or volleyball training affects saliva levels of cortisol, alpha-amylase (sAA) and secretory immunoglobulin A (sIgA) in boys aged 14–18 years. Twenty-nine boys who participate in basketball or volleyball training, recruited from the Marcin Gortat’s Athletic Championship School in Lodz, were enrolled in the study. The 90-minute routine exercise program included 15 minutes of warm-up followed by basketball or volleyball practice. Unstimulated saliva samples were collected prior to and immediately after the exercise, and were analysed using ELISA. One training session resulted in a significant increase of sAA concentration in all participants, as well as in the volleyball and basketball subgroups (p=0.00022; p=0.0029; p=0.0011; respectively). Post-exercise cortisol levels were significantly lower than pre-exercise levels (p=0.00002) throughout the group, as well as in the volleyball and basketball subgroups (p=0.0048; p=0.0019; p=0.0048; respectively). The exercise protocol did not significantly affect sIgA level, either in the whole examined group or the volleyball subgroup, however a weak significant increase of sIgA was observed in the basketball subgroup (p=0.046). The routine morning training session comprising a warm-up followed by basketball or volleyball practice seems to activate the sympatho-adrenal-medullary system, with a subsequent increase of alpha-amylase, but does not affect oral immunity in 14-18-year-old boys.

Phylogenetic distribution of phenotypic traits in bacillus thuringiensis analyzed by multilocus sequence typing

USDA-ARS?s Scientific Manuscript database

Strains from a collection of 3,639 diverse Bacillus thuringiensis isolates were classified based on phenotypic profiles resulting from six biochemical tests, including production of amylase (T), lecithinase (L), urease (U), acid from sucrose (S) and salicin (A), and the hydrolysis of esculin (E). St...

Self-compassion training modulates alpha-amylase, heart rate variability, and subjective responses to social evaluative threat in women

PubMed Central

Arch, Joanna J.; Brown, Kirk Warren; Dean, Derek J.; Landy, Lauren N.; Brown, Kimberley; Laudenslager, Mark L.

2014-01-01

A growing body of research has revealed that social evaluative stressors trigger biological and psychological responses that in chronic forms have been linked to aging and disease. Recent research suggests that self-compassion may protect the self from typical defensive responses to evaluation. We investigated whether brief training in self-compassion moderated biopsychological responses to the Trier Social Stress Test (TSST) in women. Compared to attention (placebo) and no-training control conditions, brief self-compassion training diminished sympathetic (salivary alpha-amylase), cardiac parasympathetic, and subjective anxiety responses, though not HPA-axis (salivary cortisol) responses to the TSST. Self-compassion training also led to greater self-compassion under threat relative to the control groups. In that social stress pervades modern life, self-compassion represents a promising approach to diminishing its potentially negative psychological and biological effects. PMID:24636501

Salivary cortisol and alpha-amylase levels during an assessment procedure correlate differently with risk-taking measures in male and female police recruits

PubMed Central

van den Bos, Ruud; Taris, Ruben; Scheppink, Bianca; de Haan, Lydia; Verster, Joris C.

2013-01-01

Recent laboratory studies have shown that men display more risk-taking behavior in decision-making tasks following stress, whilst women are more risk-aversive or become more task-focused. In addition, these studies have shown that sex differences are related to levels of the stress hormone cortisol (indicative of activation of the hypothalamus-pituitary-adrenocortical-axis): the higher the levels of cortisol the more risk-taking behavior is shown by men, whereas women generally display more risk-aversive or task-focused behavior following higher levels of cortisol. Here, we assessed whether such relationships hold outside the laboratory, correlating levels of cortisol obtained during a job-related assessment procedure with decision-making parameters in the Cambridge Gambling Task (CGT) in male and female police recruits. The CGT allows for discriminating different aspects of reward-based decision-making. In addition, we correlated levels of alpha-amylase [indicative of activation of the sympatho-adrenomedullary-axis (SAM)] and decision-making parameters. In line with earlier studies men and women only differed in risk-adjustment in the CGT. Salivary cortisol levels correlated positively and strongly with risk-taking measures in men, which was significantly different from the weak negative correlation in women. In contrast, and less strongly so, salivary alpha-amylase levels correlated positively with risk-taking in women, which was significantly different from the weak negative correlation with risk-taking in men. Collectively, these data support and extend data of earlier studies indicating that risky decision-making in men and women is differently affected by stress hormones. The data are briefly discussed in relation to the effects of stress on gambling. PMID:24474909

Enhanced catalysis of L-asparaginase from Bacillus licheniformis by a rational redesign.

PubMed

Sudhir, Ankit P; Agarwaal, Viplove V; Dave, Bhaumik R; Patel, Darshan H; Subramanian, R B

2016-05-01

L-Asparaginase (3.5.1.1) being antineoplastic in nature are used in the treatment of acute lymphoblastic leukemia (ALL). However glutaminase activity is the cause of various side effects when used as a drug against acute lymphoblastic leukemia (ALL). Therefore, there is a need of a novel L-asparaginase (L-ASNase) with low or no glutaminase activity. Such a property has been observed with L-ASNase from B. licheniformis (BliA). The enzyme being glutaminase free in nature paved the way for its improvement to achieve properties similar to or near to the commercially available L-ASNases. Rational enzyme engineering approach resulted in four mutants: G238N, E232A, D103V and Q112H. Among these the mutant enzyme, D103V, had a specific activity of 597.7IU/mg, which is higher than native (rBliA) (407.65IU/mg). Moreover, when the optimum temperature and in vitro half life were studied and compared with native BliA, D103V mutant BliA was better, showing tolerance to higher temperatures and a 3 fold higher half life. Kinetic studies revealed that the mutant D103V L-ASNase has increased substrate affinity, with Km value of 0.42mM and Vmax of 2778.9μmolmin(-1). Copyright © 2016 Elsevier Inc. All rights reserved.

Sporulation boundaries and spore formation kinetics of Bacillus spp. as a function of temperature, pH and a(w).

PubMed

Baril, Eugénie; Coroller, Louis; Couvert, Olivier; El Jabri, Mohammed; Leguerinel, Ivan; Postollec, Florence; Boulais, Christophe; Carlin, Frédéric; Mafart, Pierre

2012-10-01

Sporulation niches in the food chain are considered as a source of hazard and are not clearly identified. Determining the sporulation environmental boundaries could contribute to identify potential sporulation niches. Spore formation was determined in a Sporulation Mineral Buffer. The effect of incubation temperature, pH and water activity on time to one spore per mL, maximum sporulation rate and final spore concentration was investigated for a Bacillus weihenstephanensis and a Bacillus licheniformis strain. Sporulation boundaries of B. weihenstephanensis and of B. licheniformis were similar to, or included within, the range of temperatures, pH and water activities supporting growth. For instance, sporulation boundaries of B. weihenstephanensis were evaluated at 5°C, 35°C, pH 5.2 and a(w) 0.960 while growth boundaries were observed at 5°C, 37°C, pH 4.9 and a(w) 0.950. Optimum spore formation was determined at 30°C pH 7.2 for B. weihenstephanensis and at 45°C pH 7.2 for B. licheniformis. Lower temperatures and pH delayed the sporulation process. For instance, the time to one spore per mL was tenfold longer when sporulation occurred at 10°C and 20°C, for each strain respectively, than at optimum sporulation temperature. The relative effect of temperature and pH on sporulation rates and on growth rates is similar. This work suggests that the influence of environmental factors on the quantitative changes in sporulation boundaries and rates was similar to their influence on changes in growth rate. Copyright © 2012 Elsevier Ltd. All rights reserved.

Identification and Pathogenic Potential of Clinical Bacillus and Paenibacillus Isolates

PubMed Central

Celandroni, Francesco; Salvetti, Sara; Gueye, Sokhna Aissatou; Mazzantini, Diletta; Lupetti, Antonella; Senesi, Sonia; Ghelardi, Emilia

2016-01-01

The soil-related Bacillus and Paenibacillus species have increasingly been implicated in various human diseases. Nevertheless, their identification still poses problems in the clinical microbiology laboratory and, with the exception of Bacillus anthracis and Bacillus cereus, little is known on their pathogenicity for humans. In this study, we evaluated the use of matrix-assisted laser desorption—ionization time of flight mass spectrometry (MALDI-TOF MS) in the identification of clinical isolates of these genera and conducted genotypic and phenotypic analyses to highlight specific virulence properties. Seventy-five clinical isolates were subjected to biochemical and MALDI-TOF MS identification. 16S rDNA sequencing and supplemental tests were used to solve any discrepancies or failures in the identification results. MALDI-TOF MS significantly outperformed classical biochemical testing for correct species identification and no misidentification was obtained. One third of the collected strains belonged to the B. cereus species, but also Bacillus pumilus and Bacillus subtilis were isolated at high rate. Antimicrobial susceptibility testing showed that all the B. cereus, B. licheniformis, B. simplex, B. mycoides, Paenibacillus glucanolyticus and Paenibacillus lautus isolates are resistant to penicillin. The evaluation of toxin/enzyme secretion, toxin-encoding genes, motility, and biofilm formation revealed that B. cereus displays the highest virulence potential. However, although generally considered nonpathogenic, most of the other species were shown to swim, swarm, produce biofilms, and secrete proteases that can have a role in bacterial virulence. In conclusion, MALDI-TOF MS appears useful for fast and accurate identification of Bacillus and Paenibacillus strains whose virulence properties make them of increasing clinical relevance. PMID:27031639

Effect of thymol in heating and recovery media on the isothermal and non-isothermal heat resistance of Bacillus spores.

PubMed

Esteban, Maria-Dolores; Conesa, Raquel; Huertas, Juan-Pablo; Palop, Alfredo

2015-06-01

Members of the genus Bacillus include important food-borne pathogen and spoilage microorganisms for food industry. Essential oils are natural products extracted from herbs and spices, which can be used as natural preservatives in many foods because of their antibacterial, antifungal, antioxidant and anti-carcinogenic properties. The aim of this research was to explore the effect of the addition of different concentrations of thymol to the heating and recovery media on the thermal resistance of spores of Bacillus cereus, Bacillus licheniformis and Bacillus subtilis at different temperatures. While the heat resistance was hardly reduced when thymol was present in the heating medium, the effect in the recovery medium was greater, reducing the D100 °C values down to one third for B. subtilis and B. cereus when 0.5 mM thymol was added. This effect was dose dependent and was also observed at other heating temperatures. Copyright © 2014 Elsevier Ltd. All rights reserved.

Linamarase activities in Bacillus spp. responsible for thermophilic aerobic digestion of agricultural wastes for animal nutrition.

PubMed

Ugwuanyi, J Obeta; Harvey, L M; McNeil, B

2007-01-01

Thermophilic Bacillus spp. isolated from thermophilic aerobic digestion (TAD) of model agricultural slurry were screened for ability to secret linamarase activity and degrade linamarin, a cyanogenic glycoside toxin abundant in cassava. Screening was performed by both linamarin - picrate assay and by p-nitrophenyl beta-D-glucoside (PNPG) degradation, and results of both assays were related. Linamarase positive isolates were identified as Bacillus coagulans, Bacillus licheniformis and Bacillus stearothermophilus. Enzyme production was growth related and peak production was reached in 48 h in B. coagulans and 36 h in B. stearothermophilus. B. coagulans produced over 40 times greater activity than B. stearothermophilus. Enzyme productivity in shake flask was not strictly related to screening assay result. Crude enzyme of B. coagulans was optimally active at 75 degrees C while that of B. stearothermophilus was optimally active at 80 degrees C and both had optimum activity at pH 8.0. The thermophilic and neutrophilic- to marginally alkaline activity of the crude enzymes could be very useful in the detoxification and reprocessing of cyanogens containing cassava wastes by TAD for use in animal nutrition.

Gender differences in automatic thoughts and cortisol and alpha-amylase responses to acute psychosocial stress in patients with obsessive-compulsive personality disorder.

PubMed

Kanehisa, Masayuki; Kawashima, Chiwa; Nakanishi, Mari; Okamoto, Kana; Oshita, Harumi; Masuda, Koji; Takita, Fuku; Izumi, Toshihiko; Inoue, Ayako; Ishitobi, Yoshinobu; Higuma, Haruka; Ninomiya, Taiga; Akiyoshi, Jotaro

2017-08-01

Obsessive-compulsive personality disorder (OCPD) has a pervasive pattern of preoccupation with orderliness, perfection, and mental and interpersonal control at the expense of flexibility, openness, and efficiency. The aims of the present study were to explore the relationship between OCPD and psychological stress and psychological tests. We evaluated 63 OCPD patients and 107 healthy controls (HCs). We collected saliva samples from patients and controls before and after a social stress procedure, the Trier Social Stress Test (TSST), to measure the concentrations of salivary alpha-amylase (sAA) and salivary cortisol. The Childhood Trauma Questionnaire (CTQ), Profile of Mood State (POMS), State-Trait Anxiety Inventory (STAI), Beck Depression Inventory (BDI), Social Adaptation Self-Evaluation Scale (SASS), and Depression and Anxiety Cognition Scale (DACS) were administered to patients and HCs. Following TSST exposure, the salivary amylase and cortisol levels were significantly decreased in male patients compared with controls. Additionally, OCPD patients had higher CTQ, POMS, STAI, and BDI scores than HCs and exhibited significantly higher anxiety and depressive states. OCPD patients scored higher on future denial and threat prediction as per the DACS tool. According to a stepwise regression analysis, STAI, POMS, and salivary cortisol responses were independent predictors of OCPD. Our results suggested that attenuated sympathetic and parasympathetic reactivity in male OCPD patients occurs along with attenuated salivary amylase and cortisol responses to the TSST. In addition, there was a significant difference between OCPD patients and HCs in child trauma, mood, anxiety, and cognition. The finding support the modeling role of cortisol (20min) on the relationships between STAI trait and depression among OCPD. Copyright © 2017 Elsevier B.V. All rights reserved.

Cortisol and alpha amylase reactivity and timing of puberty: Vulnerabilities for antisocial behaviour in young adolescents

PubMed Central

Susman, Elizabeth J.; Granger, Douglas A; Blades, Keeva T.; Randazzo, William; Heaton, Jodi A.; Dorn, Lorah D.

2009-01-01

The theoretical framework proposed that cortisol and saliva alpha amylase (sAA) reactivitiy are vulnerabilities for antisocial behaviour. These indices of hypothalamic-pituitary-adrenal (HPA) and sympathetic-adrenal-medulary (SAM) components of the stress system, respectively, were considered vulnerabilities that also interact with the putative stressful transition of timing of puberty to predispose adolescents toward antisocial behaviour. The sample consisted of 8- to-13-year-old boys and girls (N=135) and a parent. For boys, timing of puberty moderated the association between cortisol and sAA reactivity and antisocial behaviour. Higher cortisol reactivity in later timing boys was related to a composite index of antisocial behaviour and rule-breaking behaviour problems. In contrast, lower sAA reactivity and earlier timing of puberty in boys was related to rule breaking and conduct disorder symptoms. The interaction between timing of puberty and HPA or SAM regulation and timing of puberty in boys suggests that reproductive, neuroendocrine mechanisms may be involved in the extensively documented adverse consequences of off-time pubertal development. PMID:19819639

Cortisol and alpha amylase reactivity and timing of puberty: vulnerabilities for antisocial behaviour in young adolescents.

PubMed

Susman, Elizabeth J; Dockray, Samantha; Granger, Douglas A; Blades, Keeva T; Randazzo, William; Heaton, Jodi A; Dorn, Lorah D

2010-05-01

The theoretical framework proposed that cortisol and saliva alpha amylase (sAA) reactivitiy are vulnerabilities for antisocial behaviour. These indices of hypothalamic-pituitary-adrenal (HPA) and sympathetic-adrenal-medulary (SAM) components of the stress system, respectively, were considered vulnerabilities that also interact with the putative stressful transition of timing of puberty to predispose adolescents toward antisocial behaviour. The sample consisted of 8- to-13-year-old boys and girls (N=135) and a parent. For boys, timing of puberty moderated the association between cortisol and sAA reactivity and antisocial behaviour. Higher cortisol reactivity in later timing boys was related to a composite index of antisocial behaviour and rule-breaking behaviour problems. In contrast, lower sAA reactivity and earlier timing of puberty in boys was related to rule breaking and conduct disorder symptoms. The interaction between timing of puberty and HPA or SAM regulation and timing of puberty in boys suggests that reproductive, neuroendocrine mechanisms may be involved in the extensively documented adverse consequences of off-time pubertal development. Copyright 2009 Elsevier Ltd. All rights reserved.

FT-Raman spectroscopic analysis of enhanced activity of supercritical carbon dioxide treated bacterial alpha-amylase.

PubMed

Paul, Kaninika; Dutta, Sayantani; Bhattacharjee, Paramita

2017-09-01

Our previous investigation on high pressure supercritical carbon dioxide treatment of a bacterial α-amylase had revealed enhanced activity of the same. 1 H NMR analysis of the activity enhanced enzyme led the authors to hypothesize that the enhancement was possibly owing to alterations in the active site of the enzyme. In the present study, the changes in the active site of the treated enzyme was analysed by Fourier-transform Raman (FT-Raman) spectroscopy. The spectra obtained revealed shifting of bands in the active site of α-amylase indicating a nudging effect of the bonds in this region consequent to high pressure treatment. Also, shifts in bands in the OH stretching vibration of water were observed in the enzyme spectra. These variations in the spectra confirmed changes in the active site as well as in the water associated with the same that perhaps had a concerted effect on the increased activity of α-amylase. Copyright © 2017 Elsevier Inc. All rights reserved.

Detergent-compatible bacterial amylases.

PubMed

Niyonzima, Francois N; More, Sunil S

2014-10-01

Proteases, lipases, amylases, and cellulases are enzymes used in detergent formulation to improve the detergency. The amylases are specifically supplemented to the detergent to digest starchy stains. Most of the solid and liquid detergents that are currently manufactured contain alkaline enzymes. The advantages of using alkaline enzymes in the detergent formulation are that they aid in removing tough stains and the process is environmentally friendly since they reduce the use of toxic detergent ingredients. Amylases active at low temperature are preferred as the energy consumption gets reduced, and the whole process becomes cost-effective. Most microbial alkaline amylases are used as detergent ingredients. Various reviews report on the production, purification, characterization, and application of amylases in different industry sectors, but there is no specific review on bacterial or fungal alkaline amylases or detergent-compatible amylases. In this mini-review, an overview on the production and property studies of the detergent bacterial amylases is given, and the stability and compatibility of the alkaline bacterial amylases in the presence of the detergents and the detergent components are highlighted.

Influence of heterologous MreB proteins on cell morphology of Bacillus subtilis.

PubMed

Schirner, Kathrin; Errington, Jeff

2009-11-01

The prokaryotic cytoskeletal protein MreB is thought to govern cell shape by positioning the cell wall synthetic apparatus at growth sites in the cell. In rod-shaped bacteria it forms helical filaments that run around the periphery of the rod during elongation. Gram-positive bacteria often contain more than one mreB gene. Bacillus subtilis has three mreB-like genes, mreB, mbl and mreBH, the first two of which have been shown to be essential under normal growth conditions. Expression of an mreB homologue from the closely related organism Bacillus licheniformis did not have any effect on cell growth or morphology. In contrast, expression of mreB from the phylogenetically more distant bacterium Clostridium perfringens produced shape defects and ultimately cell death, due to disruption of the endogenous MreB cytoskeleton. However, expression of either mreB(B. licheniformis) (mreB(Bl)) or mreB(C. perfringens) (mreB(Cp)) was sufficient to confer a rod shape to B. subtilis deleted for the three mreB isologues, supporting the idea that the three proteins have largely redundant functions in cell morphogenesis. Expression of mreBCD(Bl) could fully compensate for the loss of mreBCD in B. subtilis and led to the formation of rod-shaped cells. In contrast, expression of mreBCD(Cp) was not sufficient to confer a rod shape to B. subtilis Delta mreBCD, indicating that a complex of these three cell shape determinants is not enough for cell morphogenesis of B. subtilis.

Cloning and Characterization of an Alpha-amylase Gene from the Hyperthermophilic Archaeon Thermococcus Thioreducens

NASA Technical Reports Server (NTRS)

Bernhardsdotter, Eva C. M. J.; Pusey, Marc L.; Ng, Joseph D.; Garriott, Owen K.

2004-01-01

The gene encoding an extracellular a-amylase, TTA, from the hyperthermophilic archaeon Thermococcus thioreducens was cloned and expressed in Escherichia coli. Primary structural analysis revealed high similarity with other a-amylases from the Thermococcus and Pyrococcus genera, as well as the four highly conserved regions typical for a-amylases. The 1374 bp gene encodes a protein of 457 amino acids, of which 435 constitute the mature protein preceded by a 22 amino acid signal peptide. The molecular weight of the purified recombinant enzyme was estimated to be 43 kDa by denaturing gel electrophoresis. Maximal enzymatic activity of recombinant TTA was observed at 90 C and pH 5.5 in the absence of exogenous Ca(2+), and the enzyme was considerably stable even after incubation at 90 C for 2 hours. The thermostability at 90 and 102 C was enhanced in the presence of 5 mM Ca(2+). The extraordinarily high specific activity (about 7.4 x 10(exp 3) U/mg protein at 90 C, pH 5.5 with soluble starch as substrate) together with its low pH optimum makes this enzyme an interesting candidate for starch processing applications.

Individual Differences in Preschoolers' Salivary Cortisol and Alpha-Amylase Reactivity: Relations to Temperament and Maladjustment

PubMed Central

Spinrad, Tracy L.; Eisenberg, Nancy; Granger, Douglas A.; Eggum, Natalie D.; Sallquist, Julie; Haugen, RG; Kupfer, Anne; Hofer, Claire

2009-01-01

We examined the relations of 84 preschoolers' (43 boys; mean age = 54 months) situational stress reactivity to their observed emotions and mothers' reports of temperament and adjustment. Salivary cortisol and salivary alpha-amylase (sAA) were collected prior to, and following, a frustrating task. Children's anger, sadness, and positive affect were measured, and mothers reported on preschoolers' dispositional emotionality, regulation, impulsivity, and problem behaviors. Forty-seven percent of children had an increase in sAA and 52% had an increase in cortisol following the challenging task. On average, sAA levels showed the predicted pattern of rise following the frustrating task, followed by return to baseline. For cortisol, there was a mean increase from pre-task to 40 minutes post-test. sAA reactivity was associated with relatively low levels of dispositional anger and impulsivity and relatively high regulation, particularly for girls. sAA reactivity also was related to low externalizing problems for girls, but not boys. Although cortisol reactivity was unrelated to children's emotions and maladjustment, it was positively related to mothers' reports of regulation. The findings suggest that sAA reactivity in response to a frustrating social task may reflect girls' constrained behavior. PMID:19348808

Individual differences in preschoolers' salivary cortisol and alpha-amylase reactivity: relations to temperament and maladjustment.

PubMed

Spinrad, Tracy L; Eisenberg, Nancy; Granger, Douglas A; Eggum, Natalie D; Sallquist, Julie; Haugen, R G; Kupfer, Anne; Hofer, Claire

2009-06-01

We examined the relations of 84 preschoolers' (43 boys; mean age=54 months) situational stress reactivity to their observed emotions and mothers' reports of temperament and adjustment. Salivary cortisol and salivary alpha-amylase (sAA) were collected prior to, and following, a frustrating task. Children's anger, sadness, and positive affect were measured, and mothers reported on preschoolers' dispositional emotionality, regulation, impulsivity, and problem behaviors. Forty-seven percent of children had an increase in sAA and 52% had an increase in cortisol following the challenging task. On average, sAA levels showed the predicted pattern of rise following the frustrating task, followed by return to baseline. For cortisol, there was a mean increase from pre-task to 40 min post-test. sAA reactivity was associated with relatively low levels of dispositional anger and impulsivity and relatively high regulation, particularly for girls. sAA reactivity also was related to low externalizing problems for girls, but not boys. Although cortisol reactivity was unrelated to children's emotions and maladjustment, it was positively related to mothers' reports of regulation. The findings suggest that sAA reactivity in response to a frustrating social task may reflect girls' constrained behavior.

Directed evolution improves the fibrinolytic activity of nattokinase from Bacillus natto.

PubMed

Yongjun, Cai; Wei, Bao; Shujun, Jiang; Meizhi, Weng; Yan, Jia; Yan, Yin; Zhongliang, Zheng; Goulin, Zou

2011-12-01

Nattokinase (subtilisin NAT, NK) is a relatively effective microbial fibrinolytic enzyme that has been identified and characterized from Bacillus natto. In the current report, DNA family shuffling was used to improve the fibrinolytic activity of nattokinase. Three homologous genes from B. natto AS 1.107, Bacillus amyloliquefaciens CICC 20164 and Bacillus licheniformis CICC 10092 were shuffled to generate a mutant library. A plate-based method was used to screen the mutant libraries for improved activity. After three rounds of DNA shuffling, one desirable mutant with 16 amino acid substitutions was obtained. The mutant enzyme was purified and characterized. The kinetic measurements showed that the catalytic efficiency of the mutant NK was approximately 2.3 times higher than that of the wild-type nattokinase. In addition, the molecular modeling analysis suggested that the mutations affect the enzymatic function by changing the surface conformation of the substrate-binding pocket. The current study shows that the evolution of nattokinase with improved fibrinolytic activity by DNA family shuffling is feasible and provides useful references to facilitate the application of nattokinase in thrombolytic therapy. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Significantly enhancing recombinant alkaline amylase production in Bacillus subtilis by integration of a novel mutagenesis-screening strategy with systems-level fermentation optimization.

PubMed

Ma, Yingfang; Shen, Wei; Chen, Xianzhong; Liu, Long; Zhou, Zhemin; Xu, Fei; Yang, Haiquan

2016-01-01

Alkaline amylase has significant potential for applications in the textile, paper and detergent industries, however, low yield of which cannot meet the requirement of industrial application. In this work, a novel ARTP mutagenesis-screening method and fermentation optimization strategies were used to significantly improve the expression level of recombinant alkaline amylase in B. subtilis 168. The activity of alkaline amylase in mutant B. subtilis 168 mut-16# strain was 1.34-fold greater than that in the wild-type, and the highest specific production rate was improved from 1.31 U/(mg·h) in the wild-type strain to 1.57 U/(mg·h) in the mutant strain. Meanwhile, the growth of B. subtilis was significantly enhanced by ARTP mutagenesis. When the agitation speed was 550 rpm, the highest activity of recombinant alkaline amylase was 1.16- and 1.25-fold of the activities at 450 and 650 rpm, respectively. When the concentration of soluble starch and soy peptone in the initial fermentation medium was doubled, alkaline amylase activity was increased 1.29-fold. Feeding hydrolyzed starch and soy peptone mixture or glucose significantly improved cell growth, but inhibited the alkaline amylase production in B. subtilis 168 mut-16#. The highest alkaline amylase activity by feeding hydrolyzed starch reached 591.4 U/mL, which was 1.51-fold the activity by feeding hydrolyzed starch and soy peptone mixture. Single pulse feeding-based batch feeding at 10 h favored the production of alkaline amylase in B. subtilis 168 mut-16#. The results indicated that this novel ARTP mutagenesis-screening method could significantly improve the yield of recombinant proteins in B. subtilis . Meanwhile, fermentation optimization strategies efficiently promoted expression of recombinant alkaline amylase in B. subtilis 168 mut-16#. These findings have great potential for facilitating the industrial-scale production of alkaline amylase and other enzymes, using B. subtilis cultures as microbial cell

Invariant glycines and prolines flanking in loops the strand beta 2 of various (alpha/beta)8-barrel enzymes: a hidden homology?

PubMed Central

Janecek, S.

1996-01-01

The question of parallel (alpha/beta)8-barrel fold evolution remains unclear, owing mainly to the lack of sequence homology throughout the amino acid sequences of (alpha/beta)8-barrel enzymes. The "classical" approaches used in the search for homologies among (alpha/beta)8-barrels (e.g., production of structurally based alignments) have yielded alignments perfect from the structural point of view, but the approaches have been unable to reveal the homologies. These are proposed to be "hidden" in (alpha/beta)8-barrel enzymes. The term "hidden homology" means that the alignment of sequence stretches proposed to be homologous need not be structurally fully satisfactory. This is due to the very long evolutionary history of all (alpha/beta)8-barrels. This work identifies so-called hidden homology around the strand beta 2 that is flanked by loops containing invariant glycines and prolines in 17 different (alpha/beta)8-barrel enzymes, i.e., roughly in half of all currently known (alpha/beta)8-barrel proteins. The search was based on the idea that a conserved sequence region of an (alpha/beta)8-barrel enzyme should be more or less conserved also in the equivalent part of the structure of the other enzymes with this folding motif, given their mutual evolutionary relatedness. For this purpose, the sequence region around the well-conserved second beta-strand of alpha-amylase flanked by the invariant glycine and proline (56_GFTAIWITP, Aspergillus oryzae alpha-amylase numbering), was used as the sequence-structural template. The proposal that the second beta-strand of (alpha/beta)8-barrel fold is important from the evolutionary point of view is strongly supported by the increasing trend of the observed beta 2-strand structural similarity for the pairs of (alpha/beta)8-barrel enzymes: alpha-amylase and the alpha-subunit of tryptophan synthase, alpha-amylase and mandelate racemase, and alpha-amylase and cyclodextrin glycosyltransferase. This trend is also in agreement with the

Invariant glycines and prolines flanking in loops the strand beta 2 of various (alpha/beta)8-barrel enzymes: a hidden homology?

PubMed

Janecek, S

1996-06-01

The question of parallel (alpha/beta)8-barrel fold evolution remains unclear, owing mainly to the lack of sequence homology throughout the amino acid sequences of (alpha/beta)8-barrel enzymes. The "classical" approaches used in the search for homologies among (alpha/beta)8-barrels (e.g., production of structurally based alignments) have yielded alignments perfect from the structural point of view, but the approaches have been unable to reveal the homologies. These are proposed to be "hidden" in (alpha/beta)8-barrel enzymes. The term "hidden homology" means that the alignment of sequence stretches proposed to be homologous need not be structurally fully satisfactory. This is due to the very long evolutionary history of all (alpha/beta)8-barrels. This work identifies so-called hidden homology around the strand beta 2 that is flanked by loops containing invariant glycines and prolines in 17 different (alpha/beta)8-barrel enzymes, i.e., roughly in half of all currently known (alpha/beta)8-barrel proteins. The search was based on the idea that a conserved sequence region of an (alpha/beta)8-barrel enzyme should be more or less conserved also in the equivalent part of the structure of the other enzymes with this folding motif, given their mutual evolutionary relatedness. For this purpose, the sequence region around the well-conserved second beta-strand of alpha-amylase flanked by the invariant glycine and proline (56_GFTAIWITP, Aspergillus oryzae alpha-amylase numbering), was used as the sequence-structural template. The proposal that the second beta-strand of (alpha/beta)8-barrel fold is important from the evolutionary point of view is strongly supported by the increasing trend of the observed beta 2-strand structural similarity for the pairs of (alpha/beta)8-barrel enzymes: alpha-amylase and the alpha-subunit of tryptophan synthase, alpha-amylase and mandelate racemase, and alpha-amylase and cyclodextrin glycosyltransferase. This trend is also in agreement with the

Heart rate, salivary α-amylase activity, and cooperative behavior in previously naïve children receiving dental local anesthesia.

PubMed

Arhakis, Aristidis; Menexes, George; Coolidge, Trilby; Kalfas, Sotirios

2012-01-01

Psychosomatic indicators, such as heart rate (HR), salivary alpha amylase (sAA) activity, and behavior, can be used to determine stress. This study's aim was to assess the pattern of changes of salivary alpha amylase, heart rate, and cooperative behavior in previously naïve children receiving dental treatment under local anesthesia. Included were 30 children with no prior dental experience who needed 4 or more sessions of dental treatment involving local anesthesia. In each session, sAA, HR, and behavior were assessed before and during the application of local anesthesia and at the end of the treatment. The highest sAA value was always observed at the end of each session; overall, the value was lower in the fourth session. HR always increased during the local anesthesia, and did not vary across sessions. No significant relationship was found between child cooperation and either sAA or HR. In this sample, child cooperation may not be an accurate indicator of stress. Based on salivary alpha amylase activity changes, dental treatment involving local anesthesia in naïve children appeared to be less stressful after 3 sessions.

Characterization of Bacillus subtilis strains in Thua nao, a traditional fermented soybean food in northern Thailand.

PubMed

Inatsu, Y; Nakamura, N; Yuriko, Y; Fushimi, T; Watanasiritum, L; Kawamoto, S

2006-09-01

To clarify the diversity of Bacillus subtilis strains in Thua nao that produce high concentrations of products useful in food manufacturing and in health-promoting compounds. Production of amylase, protease, subtilisin NAT (nattokinase), and gamma-polyglutamic acid (PGA) by the Bacillus subtilis strains in Thua nao was measured. Productivity of protease NAT by these strains tended to be higher than by Japanese commercial natto-producing strains. Molecular diversity of isolated strains was analysed via randomly amplified polymorphic DNA-PCR fingerprinting. The strains were divided into 19 types, including a type with the same pattern as a Japanese natto-producing strain. B. subtilis strains that could be a resource for effective production of protease, amylase, subtilisin NAT, or PGA were evident in Thua nao produced in various regions in northern Thailand. This study clearly demonstrated the value of Thua nao as a potential resource of food-processing enzymes and health-promoting compounds.

Positions of Trp Codons in the Leader Peptide-Coding Region of the at Operon Influence Anti-Trap Synthesis and trp Operon Expression in Bacillus licheniformis▿

PubMed Central

Levitin, Anastasia; Yanofsky, Charles

2010-01-01

Tryptophan, phenylalanine, tyrosine, and several other metabolites are all synthesized from a common precursor, chorismic acid. Since tryptophan is a product of an energetically expensive biosynthetic pathway, bacteria have developed sensing mechanisms to downregulate synthesis of the enzymes of tryptophan formation when synthesis of the amino acid is not needed. In Bacillus subtilis and some other Gram-positive bacteria, trp operon expression is regulated by two proteins, TRAP (the tryptophan-activated RNA binding protein) and AT (the anti-TRAP protein). TRAP is activated by bound tryptophan, and AT synthesis is increased upon accumulation of uncharged tRNATrp. Tryptophan-activated TRAP binds to trp operon leader RNA, generating a terminator structure that promotes transcription termination. AT binds to tryptophan-activated TRAP, inhibiting its RNA binding ability. In B. subtilis, AT synthesis is upregulated both transcriptionally and translationally in response to the accumulation of uncharged tRNATrp. In this paper, we focus on explaining the differences in organization and regulatory functions of the at operon's leader peptide-coding region, rtpLP, of B. subtilis and Bacillus licheniformis. Our objective was to correlate the greater growth sensitivity of B. licheniformis to tryptophan starvation with the spacing of the three Trp codons in its at operon leader peptide-coding region. Our findings suggest that the Trp codon location in rtpLP of B. licheniformis is designed to allow a mild charged-tRNATrp deficiency to expose the Shine-Dalgarno sequence and start codon for the AT protein, leading to increased AT synthesis. PMID:20061467

Similarity of different beta-strands flanked in loops by glycines and prolines from distinct (alpha/beta)8-barrel enzymes: chance or a homology?

PubMed Central

Janecek, S.

1995-01-01

Many (alpha/beta)8-barrel enzymes contain their conserved sequence regions at or around the beta-strand segments that are often preceded and succeeded by glycines and prolines, respectively. alpha-Amylase is one of these enzymes. Its sequences exhibit a very low degree of similarity, but strong conservation is seen around its beta-strands. These conserved regions were used in the search for similarities with beta-strands of other (alpha/beta)8-barrel enzymes. The analysis revealed an interesting similarity between the segment around the beta 2-strand of alpha-amylase and the one around the beta 4-strand of glycolate oxidase that are flanked in loops by glycines and prolines. The similarity can be further extended on other members of the alpha-amylase and glycolate oxidase subfamilies, i.e., cyclodextrin glycosyltransferase and oligo-1,6-glucosidase, and flavocytochrome b2, respectively. Moreover, the alpha-subunit of tryptophan synthase, the (alpha/beta)8-barrel enzyme belonging to the other subfamily of (alpha/beta)8-barrels, has both investigated strands, beta 2 and beta 4, similar to beta 2 of alpha-amylase and beta 4 of glycolate oxidase. The possibilities of whether this similarity exists only by chance or is a consequence of some processes during the evolution of (alpha/beta)8-barrel proteins are briefly discussed. PMID:7549888

Similarity of different beta-strands flanked in loops by glycines and prolines from distinct (alpha/beta)8-barrel enzymes: chance or a homology?

PubMed

Janecek, S

1995-06-01

Many (alpha/beta)8-barrel enzymes contain their conserved sequence regions at or around the beta-strand segments that are often preceded and succeeded by glycines and prolines, respectively. alpha-Amylase is one of these enzymes. Its sequences exhibit a very low degree of similarity, but strong conservation is seen around its beta-strands. These conserved regions were used in the search for similarities with beta-strands of other (alpha/beta)8-barrel enzymes. The analysis revealed an interesting similarity between the segment around the beta 2-strand of alpha-amylase and the one around the beta 4-strand of glycolate oxidase that are flanked in loops by glycines and prolines. The similarity can be further extended on other members of the alpha-amylase and glycolate oxidase subfamilies, i.e., cyclodextrin glycosyltransferase and oligo-1,6-glucosidase, and flavocytochrome b2, respectively. Moreover, the alpha-subunit of tryptophan synthase, the (alpha/beta)8-barrel enzyme belonging to the other subfamily of (alpha/beta)8-barrels, has both investigated strands, beta 2 and beta 4, similar to beta 2 of alpha-amylase and beta 4 of glycolate oxidase. The possibilities of whether this similarity exists only by chance or is a consequence of some processes during the evolution of (alpha/beta)8-barrel proteins are briefly discussed.

Endocrine and emotional response to exclusion among women and men; cortisol, salivary alpha amylase, and mood.

PubMed

Helpman, Liat; Penso, Julia; Zagoory-Sharon, Orna; Feldman, Ruth; Gilboa-Schechtman, Eva

2017-05-01

Social exclusion is ubiquitous and painful. Evolutionary models indicate sex differences in coping with social stress. Recent empirical data suggest different sex patterns in hypothalamic-pituitary-adrenal (HPA) and sympathetic-adrenal-medullary (SAM) reactivity. The present study sought to test this hypothesis. We examined differences in endocrine and emotional response to exclusion by using a virtual ball tossing paradigm (Cyberball). Saliva samples and mood ratings were collected to reflect levels before, and repeatedly following, exclusion. The sample included 21 women and 23 men. Cortisol and salivary alpha amylase (sAA), biomarkers of the HPA and SAM systems, respectively, were used as indices of two arms of stress response. Following exclusion, all participants experienced mood worsening followed by mood improvement, with men reporting less distress than women. Women evinced decline in cortisol following the Cyberball task, whereas men's cortisol levels showed a non-significant rise, and then decline, following exclusion. Our results concur with previous findings showing SAM reactivity to be gender-neutral and HPA reactivity to be gender-divergent. Additional studies are needed to examine sex-specific response to social exclusion. Implications for individual differences in recovery from stress are discussed.

Smallpox and pan-Orthodox Virus Detection by Real-Time 3’-Minor Groove Binder TaqMan Assays Oil the Roche LightCycler and the Cepheid Smart Cycler Platforms

DTIC Science & Technology

2003-11-08

Bacillus anthracis BA0068 Ames Sterne SPS 97.13.213 Bacillus cereus Bacillus coagulans Bacillus licheniformis Bacillus macerans Bacillus ...megaterium Bacillus polymyxa Bacillus sphaericus Bacillus stearothermophilus Bacillus subtilis subsp. niger Bacillus thuringiensis Bacillus popilliae...varicella- zoster virus, and Bacillus anthracis DNA by LightCycler polymerase chain reaction after autoclaving:

Children’s cortisol and salivary alpha-amylase interact to predict attention bias to threatening stimuli

PubMed Central

Ursache, Alexandra; Blair, Clancy

2017-01-01

Physiological responses to threat occur through both the autonomic nervous system (ANS) and the hypothalamic pituitary adrenal (HPA) axis. Activity in these systems can be measured through salivary alpha-amylase (sAA) and salivary cortisol, respectively. Theoretical work and empirical studies have suggested the importance of examining the coordination of these systems in relation to cognitive functioning and behavior problems. Less is known, however, about whether these systems interactively predict more automatic aspects of attention processing such as attention toward emotionally salient threatening stimuli. We used a dot probe task to assess attention bias toward threatening stimuli in 347 kindergarten children. Cortisol and sAA were assayed from saliva samples collected prior to children’s participation in assessments on a subsequent day. Using regression analyses, we examined relations of sAA and cortisol to attention bias. Results indicate that cortisol and sAA interact in predicting attention bias. Higher levels of cortisol predicted greater bias toward threat for children who had high levels of sAA, but predicted greater bias away from threat for children who had low levels of sAA. These results suggest that greater symmetry in HPA and ANS functioning is associated with greater reliance on automatic attention processes in the face of threat. PMID:25455863

Significantly improving the yield of recombinant proteins in Bacillus subtilis by a novel powerful mutagenesis tool (ARTP): Alkaline α-amylase as a case study.

PubMed

Ma, Yingfang; Yang, Haiquan; Chen, Xianzhong; Sun, Bo; Du, Guocheng; Zhou, Zhemin; Song, Jiangning; Fan, You; Shen, Wei

2015-10-01

In this study, atmospheric and room temperature plasma (ARTP), a promising mutation breeding technique, was successfully applied to generate Bacillus subtilis mutants that yielded large quantities of recombinant protein. The high throughput screening platform was implemented to select those mutants with the highest yield of recombinant alkaline α-amylase (AMY), including the preferred mutant B. subtilis WB600 mut-12#. The yield and productivity of recombinant AMY in B. subtilis WB600 mut-12# increased 35.0% and 8.8%, respectively, the extracellular protein concentration of which increased 37.9%. B. subtilis WB600 mut-12# exhibited good genetic stability. Cells from B. subtilis WB600 mut-12# became shorter and wider than those from the wild-type. This study is the first to report a novel powerful mutagenesis tool (ARTP) that significantly improves the yield of recombinant proteins in B. subtilis and may therefore play an important role in the high expression level of proteins in recombinant microbial hosts. Copyright © 2015 Elsevier Inc. All rights reserved.

Effect of the lectins wheat germ agglutinin (WGA) and Ulex europaeus agglutinin (UEA-I) on the alpha-amylase secretion of rat pancreas in vitro and in vivo.

PubMed

Mikkat, U; Damm, I; Schröder, G; Schmidt, K; Wirth, C; Weber, H; Jonas, L

1998-05-01

Lectins are able to bind to cholecystokinin (CCK) receptors and other glycosylated membrane proteins. The lectins wheat germ agglutinin (WGA) and Ulex europaeus agglutinin (UEA-I) are used for affinity chromatography to isolate the highly glycosylated CCK-A receptor of pancreatic acinar cells. According to the working hypothesis that lectin binding to the CCK receptor should alter the ligand-receptor interaction, the effect of WGA and UEA-I on CCK-8-induced enzyme secretion was studied on isolated rat pancreatic acini in vitro. In vitro both lectins showed a dosage-dependent inhibition of CCK-8-induced alpha-amylase secretion of acini over 60 min. WGA showed a strong inhibitory effect on amylase secretion, approximately 40%, in vitro. UEA-I caused a smaller, but significant decrease, approximately 20%, in enzyme secretion of isolated acini. Additionally, both lectins inhibited cerulein/secretin- or cerulein-induced pancreatic secretion of rats in vivo, but not after secretin alone. The results are discussed with respect to a possible influence of both lectins on the interaction of CCK or cerulein with the CCK-A receptor.

Influences of AMY1 gene copy number and protein expression on salivary alpha-amylase activity before and after citric acid stimulation in splenic asthenia children.

PubMed

Yang, Zemin; Lin, Jing; Chen, Longhui; Zhang, Min; Yang, Xiaorong; Chen, Weiwen

2015-06-01

To compare the correlations between salivary alpha-amylase (sAA) activity and amylase, alpha 1 (salivary) gene (AMYl) copy number or its gene expression between splenic asthenia and healthy children, and investigate the reasons of attenuated sAA activity ratio before and after citric acid stimulation in splenic asthenia children. Saliva samples from 20 splenic asthenia children and 29 healthy children were collected before and after citric acid stimulation. AMYl copy number, sAA activity, and total sAA and glycosylated sAA contents were determined, and their correlations were analyzed. Although splenic asthenia and healthy children had no differences in AMY1 copy number, splenic asthenia children had positive correlations between AMY1 copy number and sAA activity before or after citric acid stimulation. Splenic asthenia children had a higher sAA glycosylated proportion ratio and glycosylated sAA content ratio, while their total sAA content ratio and sAA activity ratio were lower compared with healthy children. The glycosylated sAA content ratio was higher than the total sAA content ratio in both groups. Splenic asthenia and healthy children had positive correlations between total sAA or glycosylated sAA content and sAA activity. However, the role played by glycosylated sAA content in sAA activity in healthy children increased after citric acid stimulation, while it decreased in splenic asthenia children. Genetic factors like AMY1 copy number variations, and more importantly, sAA glycosylation abnormalities leading to attenuated sAA activity after citric acid stimulation, which were the main reasons of the attenuated sAA activity ratio in splenic asthenia children compared with healthy children.

Use of Probiotic Bacillus spp. in Rotifer (Brachionus plicatilis) and Artemia (Artemia urmiana) Enrichment: Effects on Growth and Survival of Pacific White Shrimp, Litopenaeus vannamei, Larvae.

PubMed

Jamali, Hadi; Imani, Ahmad; Abdollahi, Daruosh; Roozbehfar, Reza; Isari, Amin

2015-06-01

This study was to evaluate the effect of a preparation of Bacillus probiotic (Bacillus licheniformis and B. subtilis, 1:1) on growth and survival rate of Pacific white shrimp, Litopenaeus vannamei larvae. The larvae were fed on Artemia urmiana nauplii and Brachionus plicatilis enriched with the probiotic preparation at 1 × 10(6) CFU mL(-1) rate. The experimental setup was completely randomized design comprised of six treatments, namely solo Artemia nauplii (A) or rotifer (R), Artemia nauplii and rotifer without any enrichment (A + R), Artemia nauplii enrichment with probiotic bacilli (Bacillus licheniformis and B. subtilis) (A + B), rotifer enrichment with probiotic bacilli (R + B) and enriched Artemia nauplii and rotifer (A + R + B). All treatments were performed in triplicate. Chemical parameters of rearing water viz. pH, salinity and temperature were 7.5-8, 30-31 ppt and 31-32 °C, respectively. Photoperiod was 16L:8D. Shrimp larvae were fed Artemia nauplii and rotifers at 5-20 and 10-40 individuals per shrimp larvae four times a day, respectively. Growth and survival rate of larvae were determined at MII, MIII, PL1, PL4, PL7 and PL10 stages. Larvae in A + R + B treatment showed the highest total length (10.89 ± 0.51 mm), weight (674 ± 73 μg) and survival rate (65% ± 3.5). Lowest total length, weight and survival rate (7.96 ± 0.63 mm, 493 ± 52 μg and 24.5 ± 2.4%, respectively) were recorded in treatment B larvae. We concluded that Bacillus probiotic can improve growth and survival rate of Pacific white shrimp larvae without conceivably undesirable effects.

Enzymatic activities in different strains isolated from healthy and brittle leaf disease affected date palm leaves: study of amylase production conditions.

PubMed

Mouna, Jrad; Imen, Fendri; Choba Ines, Ben; Nourredine, Drira; Adel, Kadri; Néji, Gharsallah

2015-02-01

The present study aimed to investigate and compare the enzymatic production of endophytic bacteria isolated from healthy and brittle leaf disease affected date palm leaves (pectinase, cellulase, lipase, and amylase). The findings revealed that the enzymatic products from the bacterial isolates of healthy date palm leaves were primarily 33% amylolytic enzyme, 33 % cellulase, 25 % pectinase, and 25 % lipase. The isolates from brittle leaf disease date palm leaves, on the other hand, were noted to produce 16 % amylolytic enzyme, 20 % cellulose, 50 % pectinase, and 50 % lipase. The effects of temperature and pH on amylase, pectinase, and cellulose activities were investigated. The Bacillus subtilis JN934392 strain isolated from healthy date palm leaves produced higher levels of amylase activity at pH 7. A Box Behnken Design (BBD) was employed to optimize amylase extraction. Maximal activity was observed at pH and temperature ranges of pH 6-6.5 and 37-39 °C, respectively. Under those conditions, amylase activity was noted to be attained 9.37 U/ml. The results showed that the enzyme was able to maintain more than 50 % of its activity over a temperature range of 50-80 °C, with an optimum at 70 °C. This bacterial amylase showed high activity compared to other bacteria, which provides support for its promising candidacy for future industrial application.

Effect of tin oxide nanoparticle binding on the structure and activity of α-amylase from Bacillus amyloliquefaciens

NASA Astrophysics Data System (ADS)

Jahir Khan, Mohammad; Qayyum, Shariq; Alam, Fahad; Husain, Qayyum

2011-11-01

Proteins adsorbed on nanoparticles (NPs) are being used in biotechnology, biosensors and drug delivery. However, understanding the effect of NPs on the structure of proteins is still in a nascent state. In the present paper tin oxide (SnO2) NPs were synthesized by the reaction of SnCl4·5H2O in methanol via the sol-gel method and characterized by x-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR) and transmission electron microscopy (TEM). The binding of these SnO2-NPs with α-amylase was investigated by using UV-vis, fluorescence and circular dichroism (CD) spectroscopic techniques. A strong quenching of tryptophan fluorescence intensity in α-amylase was observed due to formation of a ground state complex with SnO2-NPs. Far-UV CD spectra showed that the secondary structure of α-amylase was changed in the presence of NPs. The Michaelis-Menten constant (Km), was found to be 26.96 and 28.45 mg ml - 1, while Vmax was 4.173 and 3.116 mg ml - 1 min - 1 for free and NP-bound enzyme, respectively.

Two novel, putatively cell wall-associated and glycosylphosphatidylinositol-anchored alpha-glucanotransferase enzymes of Aspergillus niger.

PubMed

van der Kaaij, R M; Yuan, X-L; Franken, A; Ram, A F J; Punt, P J; van der Maarel, M J E C; Dijkhuizen, L

2007-07-01

In the genome sequence of Aspergillus niger CBS 513.88, three genes were identified with high similarity to fungal alpha-amylases. The protein sequences derived from these genes were different in two ways from all described fungal alpha-amylases: they were predicted to be glycosylphosphatidylinositol anchored, and some highly conserved amino acids of enzymes in the alpha-amylase family were absent. We expressed two of these enzymes in a suitable A. niger strain and characterized the purified proteins. Both enzymes showed transglycosylation activity on donor substrates with alpha-(1,4)-glycosidic bonds and at least five anhydroglucose units. The enzymes, designated AgtA and AgtB, produced new alpha-(1,4)-glycosidic bonds and therefore belong to the group of the 4-alpha-glucanotransferases (EC 2.4.1.25). Their reaction products reached a degree of polymerization of at least 30. Maltose and larger maltooligosaccharides were the most efficient acceptor substrates, although AgtA also used small nigerooligosaccharides containing alpha-(1,3)-glycosidic bonds as acceptor substrate. An agtA knockout of A. niger showed an increased susceptibility towards the cell wall-disrupting compound calcofluor white, indicating a cell wall integrity defect in this strain. Homologues of AgtA and AgtB are present in other fungal species with alpha-glucans in their cell walls, but not in yeast species lacking cell wall alpha-glucan. Possible roles for these enzymes in the synthesis and/or maintenance of the fungal cell wall are discussed.

Biological indicators for steam sterilization: characterization of a rapid biological indicator utilizing Bacillus stearothermophilus spore-associated alpha-glucosidase enzyme.

PubMed

Albert, H; Davies, D J; Woodson, L P; Soper, C J

1998-11-01

The alpha-glucosidase enzyme was isolated from vegetative cells and spores of Bacillus stearothermophilus, ATCC 7953. Spore-associated enzyme had a molecular weight of approximately 92,700, a temperature optimum of 60 degrees C, and a pH optimum of 7.0-7.5. The enzyme in crude aqueous spore extract was stable for 30 min up to a temperature of 65 degrees C, above which the enzyme was rapidly denatured. The optimal pH for stability of the enzyme was approximately 7.2. The alpha-glucosidase in crude vegetative cell extract had similar characteristics to the spore-associated enzyme but its molecular weight was 86,700. The vegetative cell and spore-associated enzymes were cross-reactive. The enzymes are postulated to derive from a single gene product, which undergoes modification to produce the spore-associated form. The location of alpha-glucosidase in the spore coats (outside the spore protoplast) is consistent with the location of most enzymes involved in activation, germination and outgrowth.

Molecular, Biochemical, and Dietary Regulation Features of α-Amylase in a Carnivorous Crustacean, the Spiny Lobster Panulirus argus.

PubMed

Rodríguez-Viera, Leandro; Perera, Erick; Martos-Sitcha, Juan Antonio; Perdomo-Morales, Rolando; Casuso, Antonio; Montero-Alejo, Vivian; García-Galano, Tsai; Martínez-Rodríguez, Gonzalo; Mancera, Juan Miguel

2016-01-01

Alpha-amylases are ubiquitously distributed throughout microbials, plants and animals. It is widely accepted that omnivorous crustaceans have higher α-amylase activity and number of isoforms than carnivorous, but contradictory results have been obtained in some species, and carnivorous crustaceans have been less studied. In addition, the physiological meaning of α-amylase polymorphism in crustaceans is not well understood. In this work we studied α-amylase in a carnivorous lobster at the gene, transcript, and protein levels. It was showed that α-amylase isoenzyme composition (i.e., phenotype) in lobster determines carbohydrate digestion efficiency. Most frequent α-amylase phenotype has the lowest digestion efficiency, suggesting this is a favoured trait. We revealed that gene and intron loss have occurred in lobster α-amylase, thus lobsters express a single 1830 bp cDNA encoding a highly conserved protein with 513 amino acids. This protein gives rise to two isoenzymes in some individuals by glycosylation but not by limited proteolysis. Only the glycosylated isoenzyme could be purified by chromatography, with biochemical features similar to other animal amylases. High carbohydrate content in diet down-regulates α-amylase gene expression in lobster. However, high α-amylase activity occurs in lobster gastric juice irrespective of diet and was proposed to function as an early sensor of the carbohydrate content of diet to regulate further gene expression. We concluded that gene/isoenzyme simplicity, post-translational modifications and low Km, coupled with a tight regulation of gene expression, have arose during evolution of α-amylase in the carnivorous lobster to control excessive carbohydrate digestion in the presence of an active α-amylase.

Molecular, Biochemical, and Dietary Regulation Features of α-Amylase in a Carnivorous Crustacean, the Spiny Lobster Panulirus argus

PubMed Central

Martos-Sitcha, Juan Antonio; Perdomo-Morales, Rolando; Casuso, Antonio; Montero-Alejo, Vivian; García-Galano, Tsai; Martínez-Rodríguez, Gonzalo; Mancera, Juan Miguel

2016-01-01

Alpha-amylases are ubiquitously distributed throughout microbials, plants and animals. It is widely accepted that omnivorous crustaceans have higher α-amylase activity and number of isoforms than carnivorous, but contradictory results have been obtained in some species, and carnivorous crustaceans have been less studied. In addition, the physiological meaning of α-amylase polymorphism in crustaceans is not well understood. In this work we studied α-amylase in a carnivorous lobster at the gene, transcript, and protein levels. It was showed that α-amylase isoenzyme composition (i.e., phenotype) in lobster determines carbohydrate digestion efficiency. Most frequent α-amylase phenotype has the lowest digestion efficiency, suggesting this is a favoured trait. We revealed that gene and intron loss have occurred in lobster α-amylase, thus lobsters express a single 1830 bp cDNA encoding a highly conserved protein with 513 amino acids. This protein gives rise to two isoenzymes in some individuals by glycosylation but not by limited proteolysis. Only the glycosylated isoenzyme could be purified by chromatography, with biochemical features similar to other animal amylases. High carbohydrate content in diet down-regulates α-amylase gene expression in lobster. However, high α-amylase activity occurs in lobster gastric juice irrespective of diet and was proposed to function as an early sensor of the carbohydrate content of diet to regulate further gene expression. We concluded that gene/isoenzyme simplicity, post-translational modifications and low Km, coupled with a tight regulation of gene expression, have arose during evolution of α-amylase in the carnivorous lobster to control excessive carbohydrate digestion in the presence of an active α-amylase. PMID:27391425

Specialization of Bacillus in the Geochemically Challenged Environment of Death Valley

NASA Astrophysics Data System (ADS)

Kopac, S.

2014-04-01

Death Valley is the hottest, driest place in North America, a desert with soils containing toxic elements such as boron and lead. While most organisms are unable to survive under these conditions, a diverse community of bacteria survives here. What has enabled bacteria to adapt and thrive in a plethora of extreme and stressful environments where other organisms are unable to grow? The unique environmental adaptations that distinguish ecologically distinct bacterial groups (ecotypes) remain a mystery, in contrast to many animal species (perhaps most notably Darwin's ecologically distinct finch species). We resolve the ecological factors associated with recently diverged ecotypes of the soil bacteria Bacillus subtilis and Bacillus licheniformis, isolated from the dry, geochemically challenging soils of Death Valley, CA. To investigate speciation associated with challenging environmental parameters, we sampled soil transects along a 400m stretch that parallels a decrease in salinity adjacent to a salt flat; transects also encompass gradients in soil B, Cu, Fe, NO3, and P, all of which were quantified in our soil samples. We demarcated strains using Ecotype Simulation, a sequence-based algorithm. Each ecotype's habitat associations were determined with respect to salinity, B, Cu, Fe, NO3, and P. In addition, our sample strains were tested for tolerance of copper, boron and salinity (all known to inhibit growth at high concentrations) by comparing their growth over a 20 hour period. Ecotypes differed in their habitat associations with salinity, boron, copper, iron, and other ecological factors; these environmental dimensions are likely causing speciation of B. subtilis-licheniformis ecotypes at our sample site. Strains also differed in tolerance of boron and copper, providing evidence that our sequence-based demarcations reflect real differences in metabolism. By better understanding the relationship between bacterial speciation and the environment, we can begin to

Gedunin and Azadiradione: Human Pancreatic Alpha-Amylase Inhibiting Limonoids from Neem (Azadirachta indica) as Anti-Diabetic Agents.

PubMed

Ponnusamy, Sudha; Haldar, Saikat; Mulani, Fayaj; Zinjarde, Smita; Thulasiram, Hirekodathakallu; RaviKumar, Ameeta

2015-01-01

Human pancreatic α-amylase (HPA) inhibitors offer an effective strategy to lower postprandial hyperglycemia via control of starch breakdown. Limonoids from Azadirachta indica known for their therapeutic potential were screened for pancreatic α-amylase inhibition, a known anti-diabetic target. Studies were carried out to reveal their mode of action so as to justify their hypoglycemic potential. Of the nine limonoids isolated/semi-synthesized from A.indica and screened for α-amylase inhibition, azadiradione and exhibited potential inhibition with an IC50 value of 74.17 and 68.38 μM, respectively against HPA under in vitro conditions. Further screening on AR42J α-amylase secretory cell line for cytotoxicity and bioactivity revealed that azadiradione and gedunin exhibited cytotoxicity with IC50 of 11.1 and 13.4μM. Maximal secreted α-amylase inhibition of 41.8% and 53.4% was seen at 3.5 and 3.3μM, respectively. Michaelis-Menten kinetics suggested a mixed mode of inhibition with maltopentaose (Ki 42.2, 18.6 μM) and starch (Ki' 75.8, 37.4 μM) as substrate with a stiochiometry of 1:1 for both azadiradione and gedunin, respectively. The molecular docking simulation indicated plausible π-alkyl and alkyl-alkyl interactions between the aromatic amino acids and inhibitors. Fluorescence and CD confirmed the involvement of tryptophan and tyrosine in ligand binding to HPA. Thermodynamic parameters suggested that binding is enthalpically and entropically driven with ΔG° of -21.25 kJ mol-1 and -21.16 kJ mol-1 for azadiradione and gedunin, respectively. Thus, the limonoids azadiradione and gedunin could bind and inactivate HPA (anti-diabetic target) and may prove to be lead drug candidates to reduce/control post-prandial hyperglycemia.

Effect of starch and amylase on the expression of amylase-binding protein A in Streptococcus gordonii

PubMed Central

Nikitkova, A.E.; Haase, E.M.; Scannapieco, F.A.

2012-01-01

SUMMARY Streptococcus gordonii is a common oral commensal bacterial species in tooth biofilm (dental plaque) and specifically binds to salivary amylase through the surface exposed amylase-binding protein A (AbpA). When S. gordonii cells are pretreated with amylase, amylase bound to AbpA facilitates growth with starch as a primary nutrition source. The goal of this study was to explore possible regulatory effects of starch, starch metabolites and amylase on the expression of S. gordonii AbpA. An amylase ligand-binding assay was used to assess the expression of AbpA in culture supernatants and on bacterial cells from S. gordonii grown in defined medium supplemented with 1% starch, 0.5 mg ml−1 amylase, with starch and amylase together, or with various linear malto-oligosaccharides. Transcription of abpA was determined by reverse transcription quantitative polymerase chain reaction. AbpA was not detectable in culture supernatants containing either starch alone or amylase alone. In contrast, the amount of AbpA was notably increased when starch and amylase were both present in the medium. The expression of abpA was significantly increased (P < 0.05) following 40 min of incubation in defined medium supplemented with starch and amylase. Similar results were obtained in the presence of maltose and other short-chain malto-oligosacchrides. These results suggest that the products of starch hydrolysis produced from the action of salivary α-amylase, particularly maltose and maltotriose, regulate AbpA expression in S. gordonii. PMID:22759313

The diurnal course of salivary alpha-amylase in nurses: an investigation of potential confounders and associations with stress.

PubMed

Wingenfeld, Katja; Schulz, Michael; Damkroeger, Annika; Philippsen, Christine; Rose, Matthias; Driessen, Martin

2010-09-01

In psychoneuroendocrinology research, salivary measures have become increasingly important. While several studies focus on determinants of salivary cortisol such as age, gender, and gynaecological variables, less research has focused on confounding variables of salivary alpha-amylase (sAA). In a large sample of nurses (N=215) we analyzed the impact of age, gender, intake of oral contraceptives, smoking, coffee consumption as well as psychological parameters, such as work stress and burnout, on basal diurnal sAA release. Saliva was collected at 07:00 h, 11:30 h, 17:30 h, and 20:00 h on a working day during early shift. Only gender could be identified to have an impact on sAA, with females having a more pronounced sAA increase over the course of the day. Whereas depression, anxiety, work stress and burnout were not associated with sAA, a small negative correlation between social difficulties, measured with the Chronic Stress Screening Scale, and sAA could be identified. Copyright (c) 2010 Elsevier B.V. All rights reserved.

An Apple Fruit Fermentation (AFF) Treatment Improves the Composition of the Rhizosphere Microbial Community and Growth of Strawberry (Fragaria × ananassa Duch 'Benihoppe') Seedlings.

PubMed

Zhang, Jie; Pang, Hui; Ma, Mengxia; Bu, Yufen; Shao, Wei; Huang, Weijing; Ji, Qianlong; Yao, Yuncong

2016-01-01

Plant growth can be promoted by the application of apple fruit fermentation (AFF), despite unclear of the underlying mechanisms, the effects involved in AFF on rhizosphere microorganisms have been hypothesized. We investigated the consequences of applying AFF alone or in combination with Bacillus licheniformis to strawberry tissue culture seedlings in vitro, the analyses of Denaturing Gradient Gel Electrophoresis (DGGE) and 16S rDNA were performed to determine AFF effects on rhizosphere. Moreover, the growth index and antioxidant enzyme activities were determined 30 days after treatments. We identified five dominant bacteria in AFF: Coprinus atramentarius, Bacillus megaterium, Bacillus licheniformis, Weissella and B. subtilis. The greatest number of bacterial species were observed in the rhizosphere of control matrix (water treated), and the lowest diversity appeared in the rhizosphere soil treated with 108 cfu/mL B. licheniformis alone. Combining AFF plus B. licheniformis in one treatment resulted in the largest leaf area, plant height, root length, plant weight, and the markedly higher activities of antioxidant enzymes. We conclude that a combination of AFF plus B. licheniformis treatment to matrix can increase antioxidant enzymes activities in strawberry seedlings, optimize the status of rhizosphere microbial, and promote plant growth.

An Apple Fruit Fermentation (AFF) Treatment Improves the Composition of the Rhizosphere Microbial Community and Growth of Strawberry (Fragaria × ananassa Duch ‘Benihoppe’) Seedlings

PubMed Central

Bu, Yufen; Shao, Wei; Huang, Weijing; Ji, Qianlong; Yao, Yuncong

2016-01-01

Plant growth can be promoted by the application of apple fruit fermentation (AFF), despite unclear of the underlying mechanisms, the effects involved in AFF on rhizosphere microorganisms have been hypothesized. We investigated the consequences of applying AFF alone or in combination with Bacillus licheniformis to strawberry tissue culture seedlings in vitro, the analyses of Denaturing Gradient Gel Electrophoresis (DGGE) and 16S rDNA were performed to determine AFF effects on rhizosphere. Moreover, the growth index and antioxidant enzyme activities were determined 30 days after treatments. We identified five dominant bacteria in AFF: Coprinus atramentarius, Bacillus megaterium, Bacillus licheniformis, Weissella and B. subtilis. The greatest number of bacterial species were observed in the rhizosphere of control matrix (water treated), and the lowest diversity appeared in the rhizosphere soil treated with 108 cfu/mL B. licheniformis alone. Combining AFF plus B. licheniformis in one treatment resulted in the largest leaf area, plant height, root length, plant weight, and the markedly higher activities of antioxidant enzymes. We conclude that a combination of AFF plus B. licheniformis treatment to matrix can increase antioxidant enzymes activities in strawberry seedlings, optimize the status of rhizosphere microbial, and promote plant growth. PMID:27755580

Purification and Characterization of a Novel and Robust L-Asparaginase Having Low-Glutaminase Activity from Bacillus licheniformis: In Vitro Evaluation of Anti-Cancerous Properties

PubMed Central

Mahajan, Richi V.; Kumar, Vinod; Rajendran, Vinoth; Saran, Saurabh; Ghosh, Prahlad C.; Saxena, Rajendra Kumar

2014-01-01

L-asparaginase having low glutaminase has been a key therapeutic agent in the treatment of acute lymphpoblastic leukemia (A.L.L). In the present study, an extracellular L-asparaginase with low glutaminase activity, produced by Bacillus licheniformis was purified to homogeneity. Protein was found to be a homotetramer of 134.8 KDa with monomeric size of 33.7 KDa and very specific for its natural substrate i.e. L-asparagine. The activity of purified L-asparaginase enhanced in presence of cations including Na+ and K+, whereas it was moderately inhibited in the presence of divalent cations and thiol group blocking reagents. The purified enzyme was maximally active over the range of pH 6.0 to 10.0 and temperature of 40°C and enzyme was stable maximum at pH 9.0 and −20°C. CD spectra of L-asparaginase predicted the enzyme to consist of 63.05% α- helix and 3.29% β-sheets in its native form with T222 of 58°C. Fluorescent spectroscopy showed the protein to be stable even in the presence of more than 3 M GdHCl. Kinetic parameters Km, Vmax and kcat of purified enzyme were found as 1.4×10−5 M, 4.03 IU and 2.68×103 s−1, respectively. The purified L-asparaginase had cytotoxic activity against various cancerous cell lines viz. Jurkat clone E6-1, MCF-7 and K-562 with IC50 of 0.22 IU, 0.78 IU and 0.153 IU respectively. However the enzyme had no toxic effect on human erythrocytes and CHO cell lines hence should be considered potential candidate for further pharmaceutical use as an anticancer drug. PMID:24905227

Purification and characterization of a novel and robust L-asparaginase having low-glutaminase activity from Bacillus licheniformis: in vitro evaluation of anti-cancerous properties.

PubMed

Mahajan, Richi V; Kumar, Vinod; Rajendran, Vinoth; Saran, Saurabh; Ghosh, Prahlad C; Saxena, Rajendra Kumar

2014-01-01

L-asparaginase having low glutaminase has been a key therapeutic agent in the treatment of acute lymphpoblastic leukemia (A.L.L). In the present study, an extracellular L-asparaginase with low glutaminase activity, produced by Bacillus licheniformis was purified to homogeneity. Protein was found to be a homotetramer of 134.8 KDa with monomeric size of 33.7 KDa and very specific for its natural substrate i.e. L-asparagine. The activity of purified L-asparaginase enhanced in presence of cations including Na+ and K+, whereas it was moderately inhibited in the presence of divalent cations and thiol group blocking reagents. The purified enzyme was maximally active over the range of pH 6.0 to 10.0 and temperature of 40°C and enzyme was stable maximum at pH 9.0 and -20°C. CD spectra of L-asparaginase predicted the enzyme to consist of 63.05% α-helix and 3.29% β-sheets in its native form with T222 of 58°C. Fluorescent spectroscopy showed the protein to be stable even in the presence of more than 3 M GdHCl. Kinetic parameters Km, Vmax and kcat of purified enzyme were found as 1.4×10(-5) M, 4.03 IU and 2.68×10(3) s(-1), respectively. The purified L-asparaginase had cytotoxic activity against various cancerous cell lines viz. Jurkat clone E6-1, MCF-7 and K-562 with IC50 of 0.22 IU, 0.78 IU and 0.153 IU respectively. However the enzyme had no toxic effect on human erythrocytes and CHO cell lines hence should be considered potential candidate for further pharmaceutical use as an anticancer drug.

Concomitant production of detergent compatible enzymes by Bacillus flexus XJU-1.

PubMed

Niyonzima, Francois N; More, Sunil S

2014-01-01

A soil screened Bacillus flexus XJU-1 was induced to simultaneously produce alkaline amylase, alkaline lipase and alkaline protease at their optimum levels on a common medium under submerged fermentation. The basal cultivation medium consisted of 0.5% casein, 0.5% starch and 0.5% cottonseed oil as an inducer for protease, amylase, and lipase, respectively. The casein also served as nitrogen source for all 3 enzymes. The starch was also found to act as carbon source additive for both lipase and protease. Maximum enzyme production occurred on fermentation medium with 1.5% casein, 1.5% soluble starch, 2% cottonseed oil, 2% inoculum size, initial pH of 11.0, incubation temperature of 37 °C and 1% soybean meal as a nitrogen source supplement. The analysis of time course study showed that 24 h was optimum incubation time for amylase whereas 48 h was the best time for both lipase and protease. After optimization, a 3.36-, 18.64-, and 27.33-fold increase in protease, amylase and lipase, respectively was recorded. The lipase was produced in higher amounts (37.72 U/mL) than amylase and protease about 1.27 and 5.85 times, respectively. As the 3 enzymes are used in detergent formulations, the bacterium can be commercially exploited to secrete the alkaline enzymes for use in detergent industry. This is the first report for concomitant production of 3 alkaline enzymes by a bacterium.

Applications of thermal-gradients method for the optimization of α-amylase crystallization conditions based on dynamic and static light scattering data

NASA Astrophysics Data System (ADS)

Delboni, L. F.; Iulek, J.; Burger, R.; da Silva, A. C. R.; Moreno, A.

2002-02-01

The expression, purification, crystallization, and characterization by X-ray diffraction of α-amylase are described here. Dynamic and static light scattering methods with a temperature controller was used to optimize the crystallization conditions of α-amylase from Bacillus stearothermophilus an important enzyme in many fields of industrial activity. After applying thermal gradients for growing crystals, X-ray cryo-crystallographic methods were employed for the data collection. Crystals grown by these thermal-gradients diffracted up to a maximum resolution of 3.8 Å, which allowed the determination of the unit cell constants as follows: a=61.7 Å, b=86.7 Å, c=92.2 Å and space group C222 (or C222 1).

Aspergillus oryzae S2 alpha-amylase production under solid state fermentation: optimization of culture conditions.

PubMed

Sahnoun, Mouna; Kriaa, Mouna; Elgharbi, Fatma; Ayadi, Dorra-Zouari; Bejar, Samir; Kammoun, Radhouane

2015-04-01

Aspergillus oryzae S2 was assayed for alpha-amylase production under solid state fermentation (SSF). In addition to AmyA and AmyB already produced in monitored submerged culture, the strain was noted to produce new AmyB oligomeric forms, in particular a dominant tetrameric form named AmyC. The latter was purified to homogeneity through fractional acetone precipitation and size exclusion chromatography. SDS-PAGE and native PAGE analyses revealed that, purified AmyC was an approximately 172 kDa tetramer of four 42 kDa subunits. AmyC was also noted to display the same NH2-terminal amino acid sequence residues and approximately the same physico-chemical properties of AmyA and AmyB, to exhibit maximum activity at pH 5.6 and 60 °C, and to produce maltose and maltotriose as major starch hydrolysis end-products. Soyabean meal was the best substitute to yeast extract compared to fish powder waste and wheat gluten waste. AmyC production was optimized under SSF using statistical design methodology. Moisture content of 76.25%, C/N substrate ratio of 0.62, and inoculum size of 10(6.87) spores allowed maximum activity of 22118.34 U/g of dried substrate, which was 33 times higher than the one obtained before the application of the central composite design (CCD). Copyright © 2015 Elsevier B.V. All rights reserved.

Effect of temperature and pH on polygalacturonase production by pectinolytic bacteria Bacillus licheniformis strain GD2a in submerged medium from Raja Nangka (Musa paradisiaca var. formatypica) banana peel waste

NASA Astrophysics Data System (ADS)

Widowati, E.; Utami, R.; Mahadjoeno, E.; Saputro, G. P.

2017-04-01

The aim of this research were to determine the effect of temperature (45°C, 55°C, 65°C) and pH (5.0; 6.0; 7.0) on the increase of total cell count and polygalacturonase enzyme activity produced from raja nangka banana (Musa paradisiaca var. formatypica) peel waste by pectinolytic bacterial Bacillus licheniformis strain GD2a. This research applied two sample repetition and one analysis repetition. The result showed temperature and pH affect total cell count. The total cell count on 45°C and pH 7 recorded the highest number at 9.469 log cell/ml. Temperature and pH also affected pectin concentration at the end of fermentation. The lowest pectin concentration recorded at 45°C and pH 7 was 0.425 %. The highest enzyme activity recorded at 65°C and pH 7 was 0.204 U/ml. The highest enzyme protein concentration was recorded at 65°C and resulted as 0.310 mg/ml on pH 6. The highest specific activity was 19.527 U/mg at 65°C and pH 7. By this result, could be concluded that optimum condition process on polygalacturonase production was at 65°C and pH 7 because it gave highest enzyme activity result (0,204 U/ml).

Effect of the glass transition temperature on alpha-amylase activity in a starch matrix.

PubMed

Chaudhary, Vinita; Panyoyai, Naksit; Small, Darryl M; Shanks, Robert A; Kasapis, Stefan

2017-02-10

This study optimises a protocol for the estimation of α-amylase activity in a condensed starch matrix in the vicinity of the glass transition region. Enzymatic activity on the vitrified starch system was compared with that of a reference substrate, maltodextrin. The activity was assayed as the rate of release of reducing sugar using a dinitrosalicylic acid procedure. The condensed carbohydrate matrices served the dual purpose of acting as a substrate as well as producing a pronounced effect on the ability to enzymatic hydrolysis. Activation energies were estimated throughout the glass transition region of condensed carbohydrate preparations based on the concept of the spectroscopic shift factor. Results were used to demonstrate a considerable moderation by the mechanical glass transition temperature, beyond the expected linear effect of the temperature dependence, on the reaction rate of starch hydrolysis by α-amylase in comparison with the low-molecular weight chain of maltodextrin. Copyright © 2016. Published by Elsevier Ltd.

Selection and evaluation of Malaysian Bacillus spp. strains as potential probiotics in cultured tiger grouper (Epinephelus fuscoguttatus).

PubMed

Yasin, Ina-salwany Md; Razak, Nabilah Fatin; Natrah, F M I; Harmin, Sharr Azni

2016-07-01

A total of 58 Gram-positive bacteria strains were isolated from the marine environment and screened for potential probiotics for disease prevention and improving the productivity of tiger grouper Epinephelus fuscoguttatus larvae and juveniles. The bacteria were identified as Bacillus licheniformis, B. subtilis, B. circulans, B. sphaericus, B. cereus, Brevibacillus brevis, Corynebacterium propinquum, Leifsonia aquatica and Paenibacillus macerans. Only 24 strains showed antagonistic activities against four pathogenic strains; Vibrio alginolyticus, V. harveyi, V. parahaemolyticus and Aeromonas hydrophila, where two of the Bacillus strains, B12 and B45 demonstrated intermediate to highest level of inhibitory activity against these pathogenic strains, respectively. Further assessment by co-culture assay showed that Bacillus strain B12 exhibited a total inhibition of V. alginolyticus, while B45 strain displayed no inhibitory activity. Mixed culture of Bacillus B12 and B45 strains to outcompete V. alginolyticus was observed at a cell density of 10(7) CFU ml(-1). Molecular identification and phylogenetic tree analysis have categorized Bacillus strain B12 to the reference strains GQ340480 and JX290193 of? B. amyloliquafaciens, and Bacillus strain B45 with a reference strain JF496522 of B. subtilis. Safety tests of probionts by intraperitoneal administration of B12 and B45 strains at cell densities of 103, 105 and 10(7) CFU ml(-1) revealed no abnormalities and cent percent survival for healthy Epinephelus fuscoguttatus juveniles within 15 days of experimental period. Overall, the study revealed that Bacillus B12 strain possesses tremendous probiotic potential that could be used as a feed supplement in tiger grouper diets. ?

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

PubMed

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

2014-10-01

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

Isolation and characterization of Bacillus subtilis CH16 strain from chicken gastrointestinal tracts for use as a feed supplement to promote weight gain in broilers.

PubMed

Nguyen, A T V; Nguyen, D V; Tran, M T; Nguyen, L T; Nguyen, A H; Phan, T-N

2015-06-01

Spore-forming bacterial strains were isolated from chicken gastrointestinal tracts to develop a heat-stable feed supplement that promotes weight gain in broilers. Seven Bacillus strains having more than 90% sporulation were screened from the isolates and identified to be closely related with Bacillus subtilis and Bacillus licheniformis. Of the seven strains, B. subtilis CH16 was selected to develop a feed supplement for broilers, because it formed 100% heat-stable spores, grew rapidly at 42°C and quickly formed a biofilm. In large-scale trials in broilers (n ≥ 1150 per group), the group fed CH16 (3 × 10(6) CFU g(-1) pellet) showed higher average daily gain (ADG = 61·16) and lower food conversion ratio (FCR = 1·696) than did the group fed B. licheniformis CH22 (ADG = 57·10 and FCR = 1·792), the group fed B. subtilis HU58 (ADG = 51·90 and FCR = 1·868), BioPlus group (ADG = 59·32 and FCR = 1·807) and the control group (ADG = 56·02 and FCR = 1·880). In conclusion, CH16 spores significantly increased ADG by 9·17% and reduced FCR by 9·79% in broilers. The result supports the use of B. subtilis CH16 of chicken intestinal origin as a feed supplement that promote weight gain in broilers. Significance and impact of the study: This study reports screening of Bacillus strains isolated from chicken gastrointestinal tracts for development of a feed supplement that promote weight gain in broilers. Of the seven Bacillus isolates with high sporulation efficiency (≥90%), Bacillus subtilis CH16 strain showed the best growth and biofilm formation at body temperature of broilers (42°C). In large-scale trials in broilers (n ≥ 1150 per group), CH16 spores induced a 9·17% increase in daily weight gain (ADG) and a 9·79% reduction in FCR while the commercial BioPlus(®) YC induced only a 5·89% increase in ADG and a 3·88% reduction in FCR. © 2015 The Society for Applied Microbiology.

Mind your thoughts: associations between self-generated thoughts and stress-induced and baseline levels of cortisol and alpha-amylase.

PubMed

Engert, Veronika; Smallwood, Jonathan; Singer, Tania

2014-12-01

Stress is a major health burden in today's society. Research shows that negative cognitive styles are associated with increased stress reactivity, low mood and accelerated cellular aging. Our study sought to unravel the relationship between the content of self-generated thoughts and psychosocial stress measured in terms of hypothalamic-pituitary-adrenal axis and sympathetic activity. Features of self-generated thoughts were assessed using thought sampling while participants performed cognitive tasks following a stress induction or in a baseline condition. More negatively toned emotional thoughts and more social temporal thoughts with a past focus were associated with increased cortisol and alpha-amylase levels, both after stress and at baseline. More social temporal thoughts with a future focus, on the other hand, had an overall attenuating effect on the levels of both stress markers. Our results indicate a fundamental link between the thoughts and stress levels we experience. Understanding the mechanisms governing this mind-body association may have important implications for understanding and counteracting the high incidence of stress-related disorders in today's society. Copyright © 2014 Elsevier B.V. All rights reserved.

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

PubMed Central

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

2014-01-01

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

Biosynthesis of titanium dioxide nanoparticles using Bacillus amyloliquefaciens culture and enhancement of its photocatalytic activity for the degradation of a sulfonated textile dye Reactive Red 31.

PubMed

Khan, Razia; Fulekar, M H

2016-08-01

The present study aims at exploiting Bacillus amyloliquefaciens for the biosynthesis of titanium dioxide nanoparticles and also investigates role of bacterial enzymes in the biosynthesis of titanium dioxide nanoparticles. Bacterial synthesized as well as metal doped titanium dioxide nanoparticles were characterized by X-ray diffractometer (XRD), Fourier transform infrared spectroscopy (FTIR), Transmission electron microscopy (TEM), Energy dispersive X-ray spectroscopy (EDAX). Amylase activity (43.37IU) in culture supernatant evinced a potential involvement of extracellular enzyme in TiO2 nanoparticle biosynthesis. Crystallite size of bio-synthesized nanoparticles was found to be in the range of 15.23-87.6nm. FTIR spectroscopy and native-PAGE (Polyacrylamide Gel Electrophoresis) clearly indicated involvement of alpha amylase in biosynthesis of TiO2 nanoparticles and in their stabilization. TEM micrographs of the synthesized titanium dioxide nanoparticles revealed the formation of spherical nanoparticles with a size range of 22.11-97.28nm. Photocatalytic degradation of Reactive Red 31 (RR31) dye was carried out using bio-synthesized TiO2 nanoparticles under UV radiation. Photocatalytic activity of synthesized nanoparticles was enhanced by Ag, La, Zn and Pt doping. Platinum doped TiO2 showed highest potential (90.98%) in RR31 degradation as compared to undoped (75.83%). Copyright © 2016 Elsevier Inc. All rights reserved.

Amylase Test: MedlinePlus Lab Test Information

MedlinePlus

... page: https://medlineplus.gov/labtests/amylasetest.html Amylase Test To use the sharing features on this page, please enable JavaScript. What is an Amylase Test? An amylase test measures the amount of amylase ...

Autonomic markers associated with generalized social phobia symptoms: heart rate variability and salivary alpha-amylase.

PubMed

García-Rubio, María J; Espín, Laura; Hidalgo, Vanesa; Salvador, Alicia; Gómez-Amor, Jesús

2017-01-01

The study of autonomic nervous system changes associated with generalized social phobia (GSP) disorder has increased in recent years, showing contradictory results. The present study aimed to evaluate how young people with GSP reacted before, during, and after exposure to the Trier Stress Social Test (TSST), focusing on their autonomic changes (heart rate variability (HRV) and salivary alpha-amylase (sAA)) compared to a control group (non-GSP). Some psychological variables were also considered. Sex was specifically studied as a possible modulator of autonomic fluctuations and psychological state. Eighty young people were randomly distributed into two counterbalanced situations: stress condition (N = 18 and 21 for GSP and non-GSP, respectively) and control condition (N = 21 and 20 for GSP and non-GSP, respectively), where cardiovascular variables were continuously recorded. Psychological questionnaires about mood and perceived stress were filled out, and five saliva samples were collected to analyze sAA. GSP participants showed higher values on low- and high-frequency ratios (HR domains), compared to non-GSP people, during exposure to the TSST, but no differences were observed after the stressor. Furthermore, the two groups did not differ in sAA. Importantly, positive affect in GSP participants was modulated by sex. The present study suggests that the balance between high- and low-frequency domains of HRV is a key cardiovascular marker reflecting the stress response of GSP people, as well the importance of sex in positive affect when facing a stressful situation.

Crystal structure of Anoxybacillus α-amylase provides insights into maltose binding of a new glycosyl hydrolase subclass.

PubMed

Chai, Kian Piaw; Othman, Noor Farhan Binti; Teh, Aik-Hong; Ho, Kok Lian; Chan, Kok-Gan; Shamsir, Mohd Shahir; Goh, Kian Mau; Ng, Chyan Leong

2016-03-15

A new subfamily of glycosyl hydrolase family GH13 was recently proposed for α-amylases from Anoxybacillus species (ASKA and ADTA), Geobacillus thermoleovorans (GTA, Pizzo, and GtamyII), Bacillus aquimaris (BaqA), and 95 other putative protein homologues. To understand this new GH13 subfamily, we report crystal structures of truncated ASKA (TASKA). ASKA is a thermostable enzyme capable of producing high levels of maltose. Unlike GTA, biochemical analysis showed that Ca(2+) ion supplementation enhances the catalytic activities of ASKA and TASKA. The crystal structures reveal the presence of four Ca(2+) ion binding sites, with three of these binding sites are highly conserved among Anoxybacillus α-amylases. This work provides structural insights into this new GH13 subfamily both in the apo form and in complex with maltose. Furthermore, structural comparison of TASKA and GTA provides an overview of the conformational changes accompanying maltose binding at each subsite.

Unexpected high digestion rate of cooked starch by the Ct-Maltase-Glucoamylase small intestine mucosal alpha-glucosidase subunit

USDA-ARS?s Scientific Manuscript database

For starch digestion to glucose, two luminal alpha-amylases and four gut mucosal alpha-glucosidase subunits are employed. The aim of this research was to investigate, for the first time, direct digestion capability of individual mucosal alpha-glucosidases on cooked (gelatinized) starch. Gelatinized ...

Susceptibility to corrosion of laser welding composite arch wire in artificial saliva of salivary amylase and pancreatic amylase.

PubMed

Zhang, Chao; Liu, Jiming; Yu, Wenwen; Sun, Daqian; Sun, Xinhua

2015-10-01

In this study, laser-welded composite arch wire (CAW) with a copper interlayer was exposed to artificial saliva containing salivary amylase or pancreatic amylase, and the resultant corrosion behavior was studied. The purpose was to determine the mechanisms by which salivary amylase and pancreatic amylase contribute to corrosion. The effects of amylase on the electrochemical resistance of CAW were tested by potentiodynamic polarization measurements. The dissolved corrosion products were determined by ICP-OES, and the surfaces were analyzed by SEM, AFM and EDS. The results showed that both exposure to salivary amylase and pancreatic amylase significantly improved the corrosion resistance of CAW. Even isozyme could have different influences on the alloy surface. When performing in vitro research of materials to be used in oral cavity, the effect of α-amylase should be taken into account since a simple saline solution does not entirely simulate the physiological situation. Copyright © 2015 Elsevier B.V. All rights reserved.

Astilbe thunbergii reduces postprandial hyperglycemia in a type 2 diabetes rat model via pancreatic alpha-amylase inhibition by highly condensed procyanidins.

PubMed

Kato, Eisuke; Kushibiki, Natsuka; Inagaki, Yosuke; Kurokawa, Mihoko; Kawabata, Jun

2017-09-01

Type 2 diabetes mellitus (T2DM) is a common global health problem. Prevention of this disease is an important task, and functional food supplements are considered an effective method. We found potent pancreatic α-amylase inhibition in Astilbe thunbergii root extract (AT) and confirmed that AT treatment in a T2DM rat model reduces post-starch administration blood glucose levels. Activity-guided isolation revealed procyanidin (AT-P) as the α-amylase inhibitory component with IC 50  = 1.7 μg/mL against porcine pancreatic α-amylase. Structure analysis of AT-P revealed it is a B-type procyanidin comprised of four types of flavan-3-ols, some with a galloyl group, and catechin attached as the terminal unit. The abundant AT-P content and its comparable α-amylase inhibition to acarbose, the anti-diabetic medicine, suggest that AT is a promising food supplement for diabetes prevention.

Addition of Bacillus sp. inoculums in bedding for swine on a pilot scale: effect on microbial population and bedding temperature.

PubMed

Corrêa, E K; Ulguim, R R; Corrêa, L B; Castilhos, D D; Bianchi, I; Gil-Turnes, C; Lucia, T

2012-10-01

Thermal and microbiological characteristics of beddings for swine were compared according to their depth and of addition of inoculums. Bedding was added to boxes at 0.25 (25D) and 0.50 m (50D), with three treatments: control (no inoculums); T1, with 250 g of Bacillus cereus var. toyoii at 8.4 × 10(7) CFU; and T2, with 250 g of a pool of B. subtilis, Bacillus licheniformis and Bacillus polymyxa at 8.4 × 10(7) CFU (250 g for 25D and 500 g for 50D). Mean temperatures were 28.5 ± 3.9 at the surface and 35.2 ± 8.9 inside the beddings. The most probable number (MPN) of thermophilic bacteria was higher for T1 and T2 than for the control (P<0.05). The MPN of thermophilic bacteria and fungi was greater for D50 than for D25 (P<0.05). The use of 25D without inoculums is recommended due to the reduction of thermophilic microbiota. Copyright © 2012 Elsevier Ltd. All rights reserved.

Structural features, substrate specificity, kinetic properties of insect α-amylase and specificity of plant α-amylase inhibitors.

PubMed

Kaur, Rimaljeet; Kaur, Narinder; Gupta, Anil Kumar

2014-11-01

α-Amylase is an important digestive enzyme required for the optimal growth and development of insects. Several insect α-amylases had been purified and their physical and chemical properties were characterized. Insect α-amylases of different orders display variability in structure, properties and substrate specificity. Such diverse properties of amylases could be due to different feeding habits and gut environment of insects. In this review, structural features and properties of several insect α-amylases were compared. This could be helpful in exploring the diversity in characteristics of α-amylase between the members of the same class (insecta). Properties like pH optima are reflected in enzyme structural features. In plants, α-amylase inhibitors (α-AIs) occur as part of natural defense mechanisms against pests by interfering in their digestion process and thus could also provide access to new pest management strategies. AIs are quite specific in their action; therefore, these could be employed according to their effectiveness against target amylases. Potential of transgenics with α-AIs has also been discussed for insect resistance and controlling infestation. The differences in structural features of insect α-amylases provided reasons for their efficient functioning at different pH and the specificity towards various substrates. Various proteinaceous and non-proteinaceous inhibitors discussed could be helpful in controlling pest infestation. In depth detailed studies are required on proteinaceous α-AI-α-amylase interaction at different pH's as well as the insect proteinase action on these inhibitors before selecting the α-AI for making transgenics resistant to particular insect. Copyright © 2014 Elsevier Inc. All rights reserved.

Putative Virulence Factor Expression by Clinical and Food Isolates of Bacillus spp. after Growth in Reconstituted Infant Milk Formulae

PubMed Central

Rowan, Neil J.; Deans, Karen; Anderson, John G.; Gemmell, Curtis G.; Hunter, Iain S.; Chaithong, Thararat

2001-01-01

Forty-seven strains representing 14 different Bacillus species isolated from clinical and food samples were grown in reconstituted infant milk formulae (IMF) and subsequently assessed for adherence to, invasion of, and cytotoxicity toward HEp-2 and Caco-2 cells. Cell-free supernatant fluids from 38 strains (81%) were shown to be cytotoxic, 43 strains (91%) adhered to the test cell lines, and 23 strains (49%) demonstrated various levels of invasion. Of the 21 Bacillus cereus strains examined, 5 (24%) were invasive. A larger percentage of clinically derived Bacillus species (20%) than of similar species tested from the food environment were invasive. Increased invasion occurred after growth of selected Bacillus species in reconstituted IMF containing glucose. While PCR primer studies revealed that many different Bacillus species contained DNA sequences encoding the hemolysin BL (HBL) enterotoxin complex and B. cereus enterotoxin T, not all of these isolates expressed these diarrheagenic genes after growth in reconstituted IMF. Of the 47 Bacillus isolates examined, 3 isolates of B. cereus and 1 isolate of B. subtilis produced the HBL enterotoxin after 18 h of growth in brain heart infusion broth. However, eight isolates belonging to the species B. cereus, B. licheniformis, B. circulans, and B. megaterium were found to produce this enterotoxin after growth in reconstituted IMF when assessed with the B. cereus enterotoxin (diarrheal type) reversed passive latex agglutination (RPLA) kit. It is concluded that several Bacillus species occurring occasionally in clinical specimens and food samples are of potential medical significance due to the expression of putative virulence factors. PMID:11525980

Purification and Characterization of a Highly Efficient Calcium-Independent α-Amylase from Talaromyces pinophilus 1-95

PubMed Central

Xian, Liang; Wang, Fei; Luo, Xiang; Feng, Yu-Liang; Feng, Jia-Xun

2015-01-01

Alpha-amylase is a very important enzyme in the starch conversion process. Most of the α-amylases are calcium-dependent and exhibit poor performance in the simultaneous saccharification and fermentation process of industrial bioethanol production that uses starch as feedstock. In this study, an extracellular amylolytic enzyme was purified from the culture broth of newly isolated Talaromyces pinophilus strain 1-95. The purified amylolytic enzyme, with an apparent molecular weight of 58 kDa on SDS-PAGE, hydrolyzed maltopentaose, maltohexaose, and maltoheptaose into mainly maltose and maltotriose and minor amount of glucose, confirming the endo-acting mode of the enzyme, and hence, was named Talaromyces pinophilus α-amylase (TpAA). TpAA was most active at pH 4.0–5.0 (with the temperature held at 37°C) and 55°C (at pH 5.0), and stable within the pH range of 5.0–9.5 (at 4°C) and below 45°C (at pH 5.0). Interestingly, the Ca2+ did not improve its enzymatic activity, optimal temperature, or thermostability of the enzyme, indicating that the TpAA was Ca2+-independent. TpAA displayed higher enzyme activity toward malto-oligosaccharides and dextrin than other previously reported α-amylases. This highly active Ca2+-independent α-amylase may have potential applications in starch-to-ethanol conversion process. PMID:25811759