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1.
Ecotoxicol Environ Saf ; 191: 110184, 2020 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-31935556

RESUMO

Laccases play a significant role in remedying dye pollutants. Most of these enzymes are originated from terrestrial fungi and bacteria, thus they are not proper to be used in the environments with neutral/alkaline pH, or they may require laborious extraction/purification steps. These limitations can be solved using marine spore laccases through high stability and easy to use application. In the current study, laccase activity of the marine spore -forming Bacillus sp. KC2 was measured according to the guaiacol and syringaldazine oxidation. Abiotic stresses like pH of 6, temperature of 37 °C and 0.3 mM CuSO4 (in comparison with optimal sporulation conditions: pH of 8, temperature of 20 °C and 0.0 mM CuSO4) enhanced laccase formation in sporal coat. Maximum activity of enzyme was observed at 50 °C and pH 7, which did not change in the alkaline pH and temperature range of 20-70 °C. Results indicated ions, inhibitors and solvent stability of the enzyme and its activity were stimulated by Co2+, Mn2+, PMSF, acetone, acetonitrile, ethanol, and methanol. The spore laccase could decolorize synthetic dyes from various chemical groups including azo (acid orange, amaranth, trypan blue, congo red, and amido black), indigo (indigo carmine), thiazine (methylene blue, and toluidine blue), and triarylmethane (malachite green) with ABTS/syringaldazine mediators after 5 h. Degradation products were not toxic against Sorghum vulgare and Artemia salina model organisms. The enzyme mediator system showed high potentials for dye bioremediation over a wide range of harsh conditions.


Assuntos
Corantes/metabolismo , Lacase/metabolismo , Água do Mar/microbiologia , Esporos Bacterianos/enzimologia , Poluentes Químicos da Água/metabolismo , Bacillus/enzimologia , Biodegradação Ambiental , Concentração de Íons de Hidrogênio , Oxirredução , Temperatura
2.
Prep Biochem Biotechnol ; 50(1): 91-97, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31517567

RESUMO

Xylanases have gained increasing importance due to their diverse applications in the food, paper, and pharmaceutical industries, however, the production of these enzymes currently uses expensive substrates. It has already been estimated that more than 30% of the enzyme production cost originates from the substrate. The present study aimed to optimize the production of extracellular xylanases by the Bacillus sp. TC-DT 13 using solid-state fermentation with agro-industrial residues, with a view at reducing the production cost of these enzymes. All the agro-industrial residues were tested in submerged fermentation to select the best inductor to produce xylanase. Among these residues, wheat bran was selected as the best inducer of xylanase production with 1500 U/mL. Regarding solid-state fermentation, the use of wheat bran as the only fermentation substrate was used and a ratio of 1:4 moisture over a time of 144 hours induced higher amount of xylanase reaching 2943 U/g. The use of carbon and nitrogen sources did not result in the increase in production of xylanolitic enzymes. The use of agro-industrial residues in the solid-state fermentation, besides increasing the production of xylanase, reduces the cost of production and is an environmentally friendly alternative.


Assuntos
Bacillus/enzimologia , Fibras na Dieta/metabolismo , Endo-1,4-beta-Xilanases/metabolismo , Bacillus/metabolismo , Carbono/metabolismo , Fermentação , Microbiologia Industrial/economia , Microbiologia Industrial/métodos , Nitrogênio/metabolismo , Temperatura
3.
Recent Pat Biotechnol ; 14(1): 5-15, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31333132

RESUMO

BACKGROUND: Xylanases of thermophilic origin are more robust and stable and hence more suitable for industrial applications. The aim of the research was to develop a patent using a robust mutant exhibiting enhanced xylanase activity. The strain (Bacillus aestuarii SC-2014) subjected to mutagenesis is thermophilic in origin and hence it is envisioned that the enhancement of its catalytic potential will enhance its industrial applicability. OBJECTIVE: The main aim was to develop a stable and vigorous mutant having higher xylanase activity and improved thermostability. METHODS: The bacterial strain isolated from the Tattapani hot springs of Himachal Pradesh (India) was mutagenized by single separate exposure of Ethyl methane sulphonate (EMS) and N-methyl N-nitro N-nitrosoguanidine (MNNG). RESULTS: A mutant library was generated and extensive screening led to the identification of the most potent mutant strain selected and designated as Bacillus sp. SC-2014 EMS200 (MTCC number 25046) which displayed not only enhanced xylanase activity and thermo stability but also appreciable genetic stability. This strain displayed a 3-fold increase in enzyme activity and simultaneously, a significant reduction in fermentation time from 72 h to 48 h was also observed. The xylanase gene from wild and mutant strain was cloned, sequenced and subjected to molecular docking. Two mutations H121D and S123T were present inside the binding pocket. CONCLUSION: Mutation H121D made the binding pocket more acidic and charged, thus enhancing the xylanase activity for mutant protein. Mutations also resulted in charged amino acids (Y99K and H121D) which were identified as a probable cause for enhancing the thermostability of mutant protein.


Assuntos
Proteínas de Bactérias , Endo-1,4-beta-Xilanases , Engenharia de Proteínas/métodos , Bacillus/enzimologia , Bacillus/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação/genética , Endo-1,4-beta-Xilanases/química , Endo-1,4-beta-Xilanases/genética , Endo-1,4-beta-Xilanases/metabolismo , Estabilidade Enzimática , Fontes Termais/microbiologia , Temperatura Alta , Simulação de Acoplamento Molecular , Mutação
4.
J Agric Food Chem ; 68(1): 235-241, 2020 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-31822063

RESUMO

Enzymatic production of xylitol is a promising alternative to the chemical hydrogenation process. However, it encounters problems that are largely due to protein susceptibility to environmental factors. In this study, to develop a robust, practical enzymatic process for xylitol production, a coupled enzyme system consisting of formate dehydrogenase (FDH), glucose dehydrogenase (GDH), and xylose reductase (XR) was constructed, wherein the alkaline product produced by FDH and the acidic product produced by GDH could neutralize each other during cofactor regeneration. After optimization of conditions, a pH-neutralization, redox-balanced process was developed that could be carried out in pure water requiring no pH regulation. As a result, a xylitol production of 273.6 g/L that is much higher than those yet reported was obtained from 2 M xylose in 24 h, with a relatively high productivity of 11.4 g/(L h). The strategy demonstrated here can be adapted for the production of other NADH-consuming products.


Assuntos
Formiato Desidrogenases/química , Glucose 1-Desidrogenase/química , Água/química , Xilitol/química , Aldeído Redutase/química , Bacillus/enzimologia , Proteínas de Bactérias/química , Biocatálise , Candida tropicalis/enzimologia , Proteínas Fúngicas/química , Concentração de Íons de Hidrogênio , Oxirredução
5.
Arch Microbiol ; 202(1): 63-75, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31485713

RESUMO

Silver nanoparticles (AgNPs) were synthesized using cell-free filtrates of some mosquitocidal Bacilli. They showed the optical absorption peaks at 386-412 nm. They were polycrystalline spherical, hexagonal, cuboidal, rod and anisotropic shapes as detected by TEM. These nanoparticles were negatively charged with sizes ranging from 15 to 21 nm average diameter as detected by DLS. FTIR spectra showed that the main absorption bands of biomolecules capping AgNPs appeared at average wave numbers of 3435 cm-1 [ν(N-H) of amide A overlapped by ν(O-H)], 1631 cm-1 [(ν(C=O) of amide I], 1396 cm-1 [ν(C-N) of amide I], 2929 cm-1 (aliphatic C-H) and 1040 cm-1 (C-C-O). FTIR spectra confirmed the presence of protein biomolecules in the bacterial filtrate-formed coat covering AgNPs through free amide groups resulting in their stabilization in the aqueous medium. Nitrate reductase activity was found in all tested bacterial filtrates and ranged from 1.66 to 2.51 µmol/ml/min. These findings point to the probable role of nitrate reductase in reducing silver ions to silver nanoparticles and their stabilization. Tested AgNPs were multi-bioactive nanometals and showed mosquitocidal, bactericidal, fungicidal and virucidal activities. In addition, they exhibited highly synergistic mosquitocidal effect to spore toxin complex of mosquitocidal Bacilli at a very low concentration. AgNPs exhibited activities that were not or slightly cytotoxic to MA 104 cell line at tested concentrations. Therefore, they can be applied in the medical field. Finally, this study offered a simple, highly efficient, eco-friendly, economic method for biosynthesis of multi-bioactive AgNPs by some mosquitocidal Bacilli.


Assuntos
Bacillus/fisiologia , Nanopartículas Metálicas/química , Prata/química , Prata/farmacologia , Animais , Antibacterianos/química , Bacillus/enzimologia , Bacillus/metabolismo , Linhagem Celular , Chlorocebus aethiops , Nanopartículas Metálicas/ultraestrutura , Microscopia Eletrônica de Transmissão , Nitrato Redutase/metabolismo , Espectroscopia de Infravermelho com Transformada de Fourier
6.
Prep Biochem Biotechnol ; 50(3): 260-271, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31762381

RESUMO

Anti-leukemic enzyme L-asparaginase despite having significant applicability in medicine, holds side effects attributed to glutaminase activity and endotoxin content. Glutaminase activity proves to be toxic to non-tumor cells as glutamine is an essential amino acid. Endotoxin illicit the production of vasoactive amines and induce septic shock. Hence there is a need for glutaminase free L-asparaginase with minimum endotoxin level. The report aims at the development of a downstream process for purification of glutaminase free L-asparaginase and subsequent endotoxin removal. Producing bacteria were isolated from various soil samples and screened initially for asparaginase and glutaminase activity. The glutaminase free L-asparaginase producing bacteria were identified as Bacillus altitudinis. Production of L-asparaginase was optimized. The optimum medium comprised of comprising Lactose (1.5 g/L), NaCl (1.2 g/L), Yeast extract (5 g/L), L-asparagine (20 g/L) with pH 7.0 and incubation time of 18 h. Kinetic parameters Km and Vmax were computed to be 9.09x10-2M and 0.09 M/S. L-asparaginase Purification was achieved with a specific activity of 800 U/mg of enzyme. Molecular weight of the purified L-asparaginase was determined to be around 35 KDa using SDS-PAGE. The developed process also brought down the endotoxin content below the FDA recommended level. The endotoxin content of the purified enzyme was determined to be 0.015EU/mL.


Assuntos
Antineoplásicos , Asparaginase , Bacillus/enzimologia , Endotoxinas/análise , Microbiologia do Solo , Antineoplásicos/química , Antineoplásicos/isolamento & purificação , Asparaginase/química , Asparaginase/isolamento & purificação
7.
Enzyme Microb Technol ; 132: 109411, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31731971

RESUMO

Biocatalytic oxidations mediated by laccases are gaining importance due to their versatility and beneficial environmental effects. In this study, the oxidation of 1,4-dihydropyridines has been performed using three different types of bacterial laccase-based catalysts: purified laccase from Bacillus licheniformis ATCC 9945a (BliLacc), Escherichia coli whole cells expressing this laccase, and bacterial nanocellulose (BNC) supported BliLacc catalysts. The catalysts based on bacterial laccase were compared to the commercially available Trametes versicolor laccase (TvLacc). The oxidation product of 2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate was obtained within 7-24 h with good yields (70-99%) with all three biocatalysts. The substrate scope was examined with five additional 1,4-dihydropyridines, one of which was oxidized in high yield. Whole-cell biocatalyst was stable when stored for up to 1-month at 4 °C. In addition, evidence has been provided that multicopper oxidase CueO from the E. coli expression host contributed to the oxidation efficiency of the whole-cell biocatalyst. The immobilized whole-cell biocatalyst showed satisfactory activity and retained 37% of its original activity after three biotransformation cycles.


Assuntos
Bacillus/enzimologia , Biocatálise , Di-Hidropiridinas/metabolismo , Lacase/metabolismo , Enzimas Imobilizadas/metabolismo , Concentração de Íons de Hidrogênio , Oxirredução , Temperatura
8.
Enzyme Microb Technol ; 132: 109416, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31731975

RESUMO

A thermostable lipase from Bacillus thermocatenulatus was glycosylated by forming the consensus sequence (-NXS/T-) for N-linked glycosylation by site-directed mutagenesis. Among the eight BTL2 mutants including the consensus sequence, six BTL2 mutants, A277 N, A290 N, Y200 N, T236 N, T238 N, and P261 N, were glycosylated. Among the six mutants, glycosylated A277 N and T236 N showed higher stability in the presence of 25% (v/v) DMSO (74.3 and 72.8% of initial activity was remained after incubation at 45 °C for 20 h, respectively) than deglycosylated A277 N and T236 N (57.2 and 45.1% of initial activity was remained, respectively). These glycosylated mutants also showed higher remaining activity than wild-type BTL2 (56.0% of the initial activity were remained). Furthermore, the glycosylated mutant T236 N showed longer half-lives in the presence of 25% (v/v) ethylene glycol, DMSO, and DMF (161, 133, and 56.7 h at 45 °C, respectively) than deglycosylated mutant T236 N (107, 91.9, and 42.8 h, respectively). N-linked glycosylation may be a promising approach for preparing enzymes to retain their activity in the presence of organic solvents.


Assuntos
Bacillus/enzimologia , Proteínas de Bactérias/metabolismo , Lipase/metabolismo , Compostos Orgânicos/química , Solventes/química , Bacillus/genética , Proteínas de Bactérias/genética , Estabilidade Enzimática , Glicosilação , Lipase/genética , Mutagênese Sítio-Dirigida , Mutação
9.
Int J Mol Sci ; 20(24)2019 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-31835712

RESUMO

Mycobacteria produce two major lipoglycans, lipomannan (LM) and lipoarabinomannan (LAM), whose broad array of biological activities are tightly related to the fine details of their structure. However, the heterogeneity of these molecules in terms of internal and terminal covalent modifications and complex internal branching patterns represent significant obstacles to their structural characterization. Previously, an endo-α-(1→6)-D-mannanase from Bacillus circulans proved useful in cleaving the mannan backbone of LM and LAM, allowing the reducing end of these molecules to be identified as Manp-(1→6) [Manp-(1→2)]-Ino. Although first reported 45 years ago, no easily accessible form of this enzyme was available to the research community, a fact that may in part be explained by a lack of knowledge of its complete gene sequence. Here, we report on the successful cloning of the complete endo-α-(1→6)-D-mannanase gene from Bacillus circulans TN-31, herein referred to as emn. We further report on the successful production and purification of the glycosyl hydrolase domain of this enzyme and its use to gain further insight into its substrate specificity using synthetic mannoside acceptors as well as LM and phosphatidyl-myo-inositol mannoside precursors purified from mycobacteria.


Assuntos
Bacillus/enzimologia , Bacillus/genética , Clonagem Molecular , Genes Bacterianos , Manosiltransferases/genética , Lipopolissacarídeos/química , Lipopolissacarídeos/metabolismo , Manosídeos/metabolismo , Manosiltransferases/química , Manosiltransferases/isolamento & purificação , Mycobacterium smegmatis/metabolismo , Domínios Proteicos , Especificidade por Substrato
10.
Molecules ; 24(21)2019 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-31671673

RESUMO

Chitosanase plays an important role in the production of chitooligosaccharides (CHOS), which possess various biological activities. Herein, a glycoside hydrolase (GH) family 46 chitosanase-encoding gene, csnB, was cloned from marine bacterium Bacillus sp. BY01 and heterologously expressed in Escherichia coli. The recombinant chitosanase, CsnB, was optimally active at 35 °C and pH 5.0. It was also revealed to be a cold-adapted enzyme, maintaining 39.5% and 40.4% of its maximum activity at 0 and 10 °C, respectively. Meanwhile, CsnB showed wide pH-stability within the range of pH 3.0 to 7.0. Then, an improved reaction condition was built to enhance its thermostability with a final glycerol volume concentration of 20%. Moreover, CsnB was determined to be an endo-type chitosanase, yielding chitosan disaccharides and trisaccharides as the main products. Overall, CsnB provides a new choice for enzymatic CHOS production.


Assuntos
Adaptação Biológica , Bacillus/enzimologia , Temperatura Baixa , Glicosídeo Hidrolases/genética , Água do Mar/microbiologia , Sequência de Aminoácidos , Ácido Edético/farmacologia , Estabilidade Enzimática/efeitos dos fármacos , Glicerol/farmacologia , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/isolamento & purificação , Concentração de Íons de Hidrogênio , Hidrólise , Metais/farmacologia , Filogenia , Dodecilsulfato de Sódio/farmacologia
11.
J Agric Food Chem ; 67(43): 11948-11954, 2019 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-31577435

RESUMO

Corn gluten hydrolysate (CGH) was prepared by food-grade bacterial proteases, alcalase and neutral protease. Digestion of CGH with carboxypeptidase A and leucine aminopeptidase extensively changed the elution patterns of peptides as observed from reversed phase high performance liquid chromatography-mass spectrometry (LC-MS), whereas digestion with pepsin and trypsin hardly affected the elution patterns. Twenty-five major peptides in CGH were identified. After digestion with exopeptidases, only prolyl dipeptides and pyroglutamyl di- and tripeptides remained, whereas the other 17 peptides completely disappeared. On the other hand, all 25 peptides remained after digestion with pepsin and trypsin. These facts suggest that a majority of short-chain peptides in food protein hydrolysates are degraded by exopeptidases during digestion and absorption processes. Thus, susceptibility to exopeptidases should be considered for prediction of bioactive peptide upon ingestion, which has not been considered in most of previous studies on food-derived bioactive peptides.


Assuntos
Proteínas de Bactérias/química , Glutens/química , Peptídeo Hidrolases/química , Peptídeos/química , Zea mays/química , Bacillus/enzimologia , Biocatálise , Exopeptidases/química , Espectrometria de Massas , Hidrolisados de Proteína/química
12.
J Basic Microbiol ; 59(12): 1185-1194, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31617605

RESUMO

Pectinases are a group of enzymes, which catalyze the breakdown of pectin with numerous applications in various industries. Microbes are the predominant pectinase producers. In the present study, bacterial species were isolated from the soil of a vegetable and fruit dump yard area in a market. The species screened and isolated were identified as Bacillus tequilensis SALBT, and the media and culture conditions were optimized for enhanced production of total pectinases. Maximum pectinolytic activity was observed with 1.5% (w/v) pectin concentration with a combination of yeast extract as nitrogen source and MgSO4 as a metal ion source. Carbon/nitrogen in 2:1 ratio (w/v) yielded the maximum pectinase production with pH and temperature of the medium of 7.5°C and 40°C, respectively. Pectinase activity was determined by the dinitrosalicylic acid method. The pectinase production was relatively stable in the presence of various surfactants like Tween (20, 40, 60, and 80) and sodium dodecyl sulfate (SDS), whereas Triton X-100 showed an inhibitory effect. Mass production of the enzyme in optimized media and partial purification was performed by ammonium sulfate precipitation followed by dialysis. The approximate molecular weight of the partially purified pectinase was found to be 35 kDa by SDS-polyacrylamide gel electrophoresis. Application studies such as demucilaging coffee beans and juice clarification were also performed. The findings revealed that B. tequilensis SALBT with pectinase activity has the ability to remove the mucilage layer of pulped coffee seeds, and the partially purified pectinases found to be effective in clarifying juice.


Assuntos
Bacillus/enzimologia , Coffea/química , Microbiologia de Alimentos , Sucos de Frutas e Vegetais/análise , Poligalacturonase/metabolismo , Bacillus/classificação , Bacillus/genética , Meios de Cultura/química , DNA Bacteriano/genética , Concentração de Íons de Hidrogênio , Peso Molecular , Pectinas/metabolismo , Filogenia , Poligalacturonase/química , Poligalacturonase/isolamento & purificação , RNA Ribossômico 16S/genética , Sementes/química , Análise de Sequência de DNA , Microbiologia do Solo , Temperatura
13.
Enzyme Microb Technol ; 130: 109363, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31421720

RESUMO

GH11 xylanase XynJ from Bacillus sp. strain 41M-1 has a ß-jellyroll fold composed of eight ß strands with a deep active-site cleft. We hypothesized that the thermostability of XynJ will increase if the flexibility of the ß strands in the jellyroll structure is decreased without impairing activity. To verify this hypothesis, we introduced random mutations into Tyr13-Arg104 and Gly169-Tyr194, both of which are located in the ß-jellyroll fold of XynJ, to construct a site saturation mutagenesis library. By screening 576 clones followed by site saturation mutation analysis of Thr82, T82A was selected as the most thermostable variant. In the hydrolysis of beechwood xylan at pH 7.8, the temperatures required to reduce initial activity by 50% in 15 min were 61 °C for the wild-type XynJ (WT) and 65 °C for T82A. The optimum hydrolysis temperatures were 60 °C for WT and 65 °C for T82A. There was little difference in the kcat and Km values and the pH dependence of activity between WT and T82A. Crystallographic analysis of WT and T82A revealed that thermostabilization by the T82A mutation might result from the removal of unfavorable van der Waals interactions. Thus, a highly thermostable XynJ variant was generated without impairing activity using this mutation strategy.


Assuntos
Bacillus/enzimologia , Bacillus/genética , Endo-1,4-beta-Xilanases/genética , Temperatura Alta , Mutagênese , Endo-1,4-beta-Xilanases/metabolismo , Estabilidade Enzimática , Modelos Moleculares
14.
J Microbiol ; 57(10): 900-909, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31463786

RESUMO

In the present study, a laccase gene (BaLc) from a lignin degrading bacterium, Bacillus atrophaeus, has been cloned and expressed in Escherichia coli. The optimal catalytic activity of the protein was achieved at 5.5 pH and 35°C temperature, measured by oxidation of ABTS. The Km and Vmax values were determined as 1.42 mM and 4.16 µmole/min, respectively. To achieve the enzyme recovery, the biocatalyst (BaLc) was covalently attached onto the functionalized iron magnetic-nanoparticles. The nanoparticles were characterized by zeta-potential and FTIR analyses. The immobilized BaLc enzyme was physico-kinetically characterized, exhibiting retention of 60% of the residual activity after ten reaction cycles of ABTS oxidation. The immobilized biocatalyst system was tested for its biotechnological exploitability in plant juice processing, achieving 41-58% of phenol reduction, 41-58% decolorization, 50-59% turbidity reduction in the extracts of banana pseudo-stem and sweet sorghum stalk, and apple fruit juice. This is the first study to demonstrate the use of nanoparticle-laccase conjugate in juice clarification. The findings suggest that B. atrophaus laccase is a potential catalytic tool for plant juice bioprocessing activities.


Assuntos
Bacillus/enzimologia , Proteínas de Bactérias/química , Lacase/química , Bacillus/química , Biocatálise , Estabilidade Enzimática , Enzimas Imobilizadas/química , Manipulação de Alimentos , Sucos de Frutas e Vegetais/análise , Concentração de Íons de Hidrogênio , Cinética , Nanopartículas
15.
Int J Biol Macromol ; 140: 1064-1072, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31454643

RESUMO

The present study was aimed to evaluate the Methyltrioctylammonium Chloride (IL) and Sodium hydroxide effect on sugarcane bagasse (SB) structure and its subsequent utilization to produce cellulase from a thermophilic bacterium Bacillus aestuarii UE25. The strain was isolated from a crocodile pond of Manghopir, Karachi. Ten different factors affecting IL pretreatment of SB and cellulase production by UE25 were evaluated by Plackett-Burman design and three significant factors were optimized by employing Box Behnken design. Under optimum conditions, the strain produced 118.4 IU mL-1 of EG and 75.01 IU mL-1 of BGL that corroborated well with the predicted values by the model. Scanning electron microscopy, gravimetric analysis, Fourier transform infrared spectroscopy and NMR of SB revealed removal of lignin, decrease in cellulose content and structural changes in the SB after pretreatment and fermentation. The data provide prospects of utilizing this IL in comparison to imidazolium based IL for pretreatment of biomass.


Assuntos
Celulase/metabolismo , Celulose/química , Lignina/isolamento & purificação , Compostos de Amônio Quaternário/química , Saccharum/química , Temperatura , Bacillus/enzimologia , Celulose/metabolismo , Celulose/ultraestrutura , Estabilidade Enzimática , Espectroscopia de Ressonância Magnética , Filogenia , Saccharum/ultraestrutura , Espectroscopia de Infravermelho com Transformada de Fourier , Especificidade por Substrato , Termogravimetria
16.
Int J Biol Macromol ; 140: 129-139, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31408655

RESUMO

GH42 enzymes are potential candidates for bifunctional ß-galactosidase/α-L-arabinopyranosidase. A novel GH42 enzyme (BaBgal42A) from Bacillus was identified, the recombinant BaBgal42A hydrolyzed not only ß-D-galactopyranosidic bonds in pNP-ß-D-galactopyranoside, oNP-ß-D-galactopyranoside, lactose, galactan, and arabinan but also α-L-arabinopyranosidic linkages in pNP-α-L-arabinopyranoside, wheat arabinoxylan and galactan. The Km values of BaBgal42A for pNP-ß-D-galactopyranoside and pNP-α-L-arabinopyranoside were 2.76 and 16.23 mM, respectively. Investigation of cooperative activities of BaBgal42A with cognate enzymes revealed that BaBgal42A showed obvious synergy with an endo-ß-1,4-galactanase (BaGal53A) in the decomposition of galactan, supplementing BaBgal42A resulted in a 0.56-fold increase in the release of reducing sugars; BaBgal42A also exhibited a little synergy with its cognate endoxylanase (BaXynA)/α-L-arabinofuranosidase (BaAraA) in hydrolyzing wheat arabinoxylan/arabinan, addition of BaBgal42A released 12.7%/7.8% more reducing sugars than that produced by BaXynA/BaAraA alone. Moreover, BaBgal42A is a cold-adapted enzyme, exhibiting 28-46% of the maximal activity at the range of 5-20 °C and its activity was slightly stimulated by addition of Na+, K+, or Ca2+ at low concentrations. This study not only expands the diversity within GH42 family, but also provides new insights into the role of microbial GH42 enzymes, which would contribute to its potential application in polysaccharides degradation and milk lactose hydrolysis.


Assuntos
Bacillus/enzimologia , Proteínas de Bactérias/química , Glicosídeo Hidrolases/química , Polissacarídeos/química , Triticum/química , Xilanos/química , beta-Galactosidase/química , Bacillus/genética , Proteínas de Bactérias/genética , Glicosídeo Hidrolases/genética , beta-Galactosidase/genética
17.
J Appl Genet ; 60(3-4): 427-430, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31407219

RESUMO

Aminotransferases have attracted considerable attention due to their extraordinary potential for the biosynthesis of chiral amines. Research on transaminase genes can facilitate their application to various fields. Herein, 89 putative aminotransferase genes potentially encoding useful biocatalysts were identified in three Bacillus strains genomes by genome annotation. Enzymes encoded by genes ota3, ota8, otae6, otae21, otaf1, otaf8, and otaf26 belong to pyridoxine 5'-phosphate-dependent enzyme class IV. These seven ω-aminotransferase genes are highly conserved according to phylogenetic tree and bioinformatics analyses, as are the putative lysine catalytic residues in the corresponding enzymes (ω-BPTA 1-7). The enzymes may possess similar activity to ω-aminotransferases from Arthrobacter sp. KNK 168. The potential application of these novel enzymes for the synthesis of medicinal amino compounds will be explored in future genetic engineering studies.


Assuntos
Genoma Bacteriano/genética , Família Multigênica/genética , Filogenia , Transaminases/genética , Bacillus/enzimologia , Bacillus/genética , Bacillus amyloliquefaciens/enzimologia , Bacillus amyloliquefaciens/genética , Biologia Computacional , DNA Bacteriano/genética , Análise de Sequência de DNA , Transaminases/classificação
18.
Food Chem ; 301: 125266, 2019 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-31387037

RESUMO

ß-Xylosidase, of the glycoside hydrolase family 43 from Bacillus sp. HJ14, was expressed in Escherichia coli. Recombinant ß-xylosidase (rHJ14GH43) exhibited maximum activity at 25 °C, approximately 15, 45, and 88% of maximum activity at 0, 10, and 20 °C, respectively, and poor stability at temperatures over 20 °C. rHJ14GH43 showed moderate or high activity, but poor stability, in NaCl, KCl, NaNO3, KNO3, Na2SO4, and (NH4)2SO4 at concentrations from 3.0 to 30.0% (w/v). The crystal structure of rHJ14GH43 was resolved and showed higher structural flexibility due to fewer salt bridges and hydrogen bonds compared to mesophilic and thermophilic ß-xylosidases. High structural flexibility is presumed to be a key factor for catalytic adaptations to low temperatures and high salt concentrations. Approximately one-third of the surface of rHJ14GH43 is positively charged, which may be the primary factor responsible for poor stability in high neutral salt environments.


Assuntos
Bacillus/enzimologia , Xilosidases/metabolismo , Estabilidade Enzimática , Escherichia coli/genética , Concentração de Íons de Hidrogênio , Proteínas Recombinantes/metabolismo , Temperatura
19.
Int J Biol Macromol ; 140: 825-832, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31401271

RESUMO

An alkali-tolerant bacterial isolate CH7 was isolated from Chilika Lake sediments that produced α-amylase at high pH. It showed >15 mm zone diameter at pH 11.0 on agar plate containing 1% starch hence considered for study. It survived up to 10% NaCl concentration on nutrient agar plates. Taxonomically it was identified as Bacillus sp. strain SP-CH7 having GenBank Accession No. KU761266. Optimized alkaline α-amylase, AA7 showed 202.857 specific activity (IU/mg), 65.95 purification fold and 25.94 yield (%) respectively. Molecular weight was revealed by SDS-PAGE as 55 kDa. The Km value was 0.688 mg/mL and so was 2.342 IU/mL Vmax obtained for AA7 towards starch. This enzyme showed 92.79% and 91.14% stability at pH 11.0 and 13.0 respectively and up to 75 °C stability was also noted. Enzyme activity was stimulated by Co2+, Na+ and Ca2+. It was stable in presence of EDTA, SDS, all the powdered detergents as well as with liquid detergent "Ezee" which vouches for its potential application in Detergent industries.


Assuntos
Bacillus/enzimologia , Detergentes/química , Lagos/microbiologia , alfa-Amilases/química , Bacillus/genética , Bacillus/isolamento & purificação , Sequência de Bases , Fenômenos Químicos , Biologia Computacional/métodos , Sequência Consenso , Estabilidade Enzimática , Peso Molecular , Filogenia , RNA Ribossômico 16S/genética , Microbiologia da Água , alfa-Amilases/genética , alfa-Amilases/isolamento & purificação
20.
Biochim Biophys Acta Proteins Proteom ; 1867(11): 140261, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31401312

RESUMO

Bacillus lipases are industrially attractive enzymes due to their broad substrate specificity and optimum alkaline pH. However, narrow temperature range of action and low thermostability restrain their optimal use and thus, necessitate attention. Several laboratories are engaged in protein engineering of Bacillus lipases to generate variants with improved attributes for decades using techniques such as directed evolution or rational design. This review summarizes the effect of mutations on the conformational changes through in silico modeling and their manifestation with respect to various biochemical parameters. Various studies have been put together to develop a perspective on the molecular basis of biocatalysis of lipases holding industrial importance.


Assuntos
Substituição de Aminoácidos , Bacillus/enzimologia , Proteínas de Bactérias/química , Temperatura Alta , Lipase/química , Bacillus/genética , Proteínas de Bactérias/genética , Estabilidade Enzimática/genética , Lipase/genética , Mutação de Sentido Incorreto , Relação Estrutura-Atividade
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