scholarly journals Cloning and Expression of theγ-Polyglutamic Acid Synthetase GenepgsBCA inBacillus subtilisWB600

2016 ◽  
Vol 2016 ◽  
pp. 1-7 ◽  
Author(s):  
Biaosheng Lin ◽  
Zhijuan Li ◽  
Huixia Zhang ◽  
Jiangwen Wu ◽  
Maochun Luo
Keyword(s):  
Bacillus Subtilis ◽  
Fermentation Broth ◽  
Recombinant Expression ◽  
Product Yield ◽  
Industrial Applications ◽  
Cloning And Expression ◽  
Polyglutamic Acid ◽  
Recombinant Bacteria ◽  
Expression Product ◽  
Clone And Express

To clone and express theγ-polyglutamic acid (γ-PGA) synthetase genepgsBCA inBacillus subtilis, a pWB980 plasmid was used to construct and transfect the recombinant expression vector pWB980-pgsBCA intoBacillus subtilisWB600.PgsBCAwas expressed under the action of a P43 promoter in the pWB980 plasmid. Our results showed that the recombinant bacteria had the capacity to synthesizeγ-PGA. The expression product was secreted extracellularly into the fermentation broth, with a product yield of 1.74 g/L or higher.γ-PGA samples from the fermentation broth were purified and characterized. Hydrolysates ofγ-PGA presented in single form, constituting simple glutamic acid only, which matched the characteristics of the infrared spectra of theγ-PGA standard, and presented as multimolecular aggregates with a molecular weight within the range of 500–600 kDa. Expressing theγ-PGA synthetase genepgsBCAinB. subtilissystem has potential industrial applications.

scholarly journals Cloning, Expression, and Characterization of a Novel Thermophilic Monofunctional Catalase fromGeobacillussp. CHB1

2016 ◽  
Vol 2016 ◽  
pp. 1-8 ◽  
Author(s):  
Xianbo Jia ◽  
Jichen Chen ◽  
Chenqiang Lin ◽  
Xinjian Lin
Keyword(s):  
Amino Acid ◽  
Specific Activity ◽  
Fermentation Broth ◽  
Residual Activity ◽  
Industrial Applications ◽  
Activity Level ◽  
High Yield ◽  
E Coli ◽  
Wide Range ◽  
Clone And Express

Catalases are widely used in many scientific areas. A catalase gene (Kat) fromGeobacillussp. CHB1 encoding a monofunctional catalase was cloned and recombinant expressed inEscherichia coli(E. coli), which was the first time to clone and express this type of catalase ofgenus Geobacillusstrains as far as we know. ThisKatgene was 1,467 bp in length and encoded a catalase with 488 amino acid residuals, which is only 81% similar to the previously studiedBacillussp. catalase in terms of amino acid sequence. Recombinant catalase was highly soluble inE. coliand made up 30% of the totalE. coliprotein. Fermentation broth of the recombinantE. colishowed a high catalase activity level up to 35,831 U/mL which was only lower than recombinantBacillussp. WSHDZ-01 among the reported catalase production strains. The purified recombinant catalase had a specific activity of 40,526 U/mg andKmof 51.1 mM. The optimal reaction temperature of this recombinant enzyme was 60°C to 70°C, and it exhibited high activity over a wide range of reaction temperatures, ranging from 10°C to 90°C. The enzyme retained 94.7% of its residual activity after incubation at 60°C for 1 hour. High yield and excellent thermophilic properties are valuable features for this catalase in industrial applications.

scholarly journals Expression and Characterization ofGeobacillus stearothermophilusSR74 Recombinantα-Amylase inPichia pastoris

2015 ◽  
Vol 2015 ◽  
pp. 1-9 ◽  
Author(s):  
Sivasangkary Gandhi ◽  
Abu Bakar Salleh ◽  
Raja Noor Zaliha Raja Abd Rahman ◽  
Thean Chor Leow ◽  
Siti Nurbaya Oslan
Keyword(s):  
Specific Activity ◽  
Thermophilic Bacteria ◽  
Recombinant Expression ◽  
Expression System ◽  
Alcohol Oxidase ◽  
Product Yield ◽  
Inducible Promoter ◽  
Industrial Applications ◽  
Maximum Activity ◽  
Gene Encoding

Geobacillus stearothermophilusSR74 is a locally isolated thermophilic bacteria producing thermostable and thermoactiveα-amylase. Increased production and commercialization of thermostableα-amylase strongly warrant the need of a suitable expression system. In this study, the gene encoding the thermostableα-amylase inG. stearothermophilusSR74 was amplified, sequenced, and subcloned intoP. pastorisGS115 strain under the control of a methanol inducible promoter, alcohol oxidase (AOX). Methanol induced recombinant expression and secretion of the protein resulted in high levels of extracellular amylase production. YPTM medium supplemented with methanol (1% v/v) was the best medium and once optimized, the maximum recombinantα-amylase SR74 achieved in shake flask was 28.6 U mL−1at 120 h after induction. The recombinant 59 kDaα-amylase SR74 was purified 1.9-fold using affinity chromatography with a product yield of 52.6% and a specific activity of 151.8 U mg−1. The optimum pH ofα-amylase SR74 was 7.0 and the enzyme was stable between pH 6.0–8.0. The purified enzyme was thermostable and thermoactive, exhibiting maximum activity at 65°C with a half-life (t1/2) of 88 min at 60°C. In conclusion, thermostableα-amylase SR74 fromG. stearothermophilusSR74 would be beneficial for industrial applications, especially in liquefying saccrification.

scholarly journals A highly thermotolerant laccase produced by Cerrena unicolor strain CGMCC 5.1011 for complete and stable malachite green decolorization

AMB Express ◽  
2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Yanhua Yao ◽  
Guimei Zhou ◽  
Yonghui Lin ◽  
Xinqi Xu ◽  
Jie Yang
Keyword(s):  
Malachite Green ◽  
Fermentation Broth ◽  
Nitrogen Sources ◽  
Life Time ◽  
Industrial Applications ◽  
Dynamics Simulation ◽  
Carbon And Nitrogen ◽  
Cdna Sequences ◽  
Cerrena Unicolor ◽  
Future Work

Abstract Laccases are a class of multi-copper oxidases with important industrial values. A thermotolerant laccase produced by a basidiomycete fungal strain Cerrena unicolor CGMCC 5.1011 was studied. With glycerin and peptone as the carbon and nitrogen sources, respectively, a maximal laccase activity of 121.7 U/mL was attained after cultivation in the shaking flask for 15 days. Transcriptomics analysis revealed an expressed laccase gene family of 12 members in C. unicolor strain CGMCC 5.1011, and the gene and cDNA sequences were cloned. A glycosylated laccase was purified from the fermentation broth of Cerrena unicolor CGMCC 5.1011 and corresponded to Lac2 based on MALDI-TOF MS/MS identification. Lac2 was stable at pH 5.0 and above, and was resistant to organic solvents. Lac2 displayed remarkable thermostability, with half-life time of 1.67 h at 70 ºC. Consistently, Lac2 was able to completely decolorize malachite green (MG) at high temperatures, whereas Lac7 from Cerrena sp. HYB07 resulted in accumulation of colored MG transformation intermediates. Molecular dynamics simulation of Lac2 was conducted, and possible mechanisms underlying Lac2 thermostability were discussed. The robustness of C. unicolor CGMCC 5.1011 laccase would not only be useful for industrial applications, but also provide a template for future work to develop thermostable laccases.

scholarly journals Laccase and Its Mutant Displayed on the Bacillus subtilis Spore Coat for Oxidation of Phenolic Compounds in Organic Solvents

Catalysts ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 606
Author(s):  
Silu Sheng ◽  
Edgardo T. Farinas
Keyword(s):  
Bacillus Subtilis ◽  
Organic Solvents ◽  
Environmental Changes ◽  
Sinapic Acid ◽  
Product Yield ◽  
Spore Coat ◽  
Subtilis Spore ◽  
Bacillus Subtilis Spore ◽  
Cota Laccase ◽  
Inert Surface

Enzymes displayed on the Bacillus subtilis spore coat have several features that are useful for biocatalysis. The enzyme is preimmobilized on an inert surface of the spore coat, which is due to the natural sporulation process. As a result, protein stability can be increased, and they are resistant to environmental changes. Next, they would not lyse under extreme conditions, such as in organic solvents. Furthermore, they can be easily removed from the reaction solution and reused. The laboratory evolved CotA laccase variant T480A-CotA was used to oxidize the following phenolic substrates: (+)-catechin, (−)-epicatechin, and sinapic acid. The kinetic parameters were determined and T480A-CotA had a greater Vmax/Km than wt-CotA for all substrates. The Vmax/Km for T480A-CotA was 4.1, 5.6, and 1.4-fold greater than wt-CotA for (+)-catechin, (−)-epicatechin, and sinapic acid, respectively. The activity of wt-CotA and T480A-CotA was measured at different concentrations from 0–70% in organic solvents (dimethyl sulfoxide, ethanol, methanol, and acetonitrile). The Vmax for T480A-CotA was observed to be greater than the wt-CotA in all organic solvents. Finally, the T480A-CotA was recycled 7 times over a 23-h period and up to 60% activity for (+)-catechin remained. The product yield was up to 3.1-fold greater than the wild-type.

scholarly journals The multifunctionality of expression systems in Bacillus subtilis: Emerging devices for the production of recombinant proteins

2021 ◽  
pp. 153537022110301
Author(s):  
Caio Coutinho de Souza ◽  
Jander Matos Guimarães ◽  
Soraya dos Santos Pereira ◽  
Luis André Morais Mariúba
Keyword(s):  
Bacillus Subtilis ◽  
Recombinant Proteins ◽  
Large Scale ◽  
Recombinant Expression ◽  
Academic Research ◽  
Future Research ◽  
Expression Systems ◽  
Bioactive Substances ◽  
Exogenous Dna ◽  
Chemical Inducers

Bacillus subtilis is a successful host for producing recombinant proteins. Its GRAS (generally recognized as safe) status and its remarkable innate ability to absorb and incorporate exogenous DNA into its genome make this organism an ideal platform for the heterologous expression of bioactive substances. The factors that corroborate its value can be attributed to the scientific knowledge obtained from decades of study regarding its biology that has fostered the development of several genetic engineering strategies, such as the use of different plasmids, engineering of constitutive or double promoters, chemical inducers, systems of self-inducing expression with or without a secretion system that uses a signal peptide, and so on. Tools that enrich the technological arsenal of this expression platform improve the efficiency and reduce the costs of production of proteins of biotechnological importance. Therefore, this review aims to highlight the major advances involving recombinant expression systems developed in B. subtilis, thus sustaining the generation of knowledge and its application in future research. It was verified that this bacterium is a model in constant demand and studies of the expression of recombinant proteins on a large scale are increasing in number. As such, it represents a powerful bacterial host for academic research and industrial purposes.

scholarly journals Enhancement of L-amino Acid Oxidase Production by Bacillus Subtilis H-8 With Oxygen-vector and Asymmetric Degradation of DL Arginine to D-arginine

2020 ◽  
Author(s):  
peng xu ◽  
Changpei Pan ◽  
Gongcheng Cui ◽  
ChunYan Wei ◽  
Lijuan Wang ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Oleic Acid ◽  
Amino Acid ◽  
Fermentation Broth ◽  
Cation Exchange Resin ◽  
Optical Purity ◽  
Phenyl Isothiocyanate ◽  
Acid Oxidase ◽  
Amino Acid Oxidase ◽  
Oxygen Vector

Abstract The Bacillus subtilis H-8 independently screened by our laboratory can produce L-amino acid oxidase (L-AAO), and DL-arginine can be degraded asymmetrically by suspending the wet bacteria in the degradation liquid. By adding oxygen-vectors to the fermentation medium, the collected amount of wet bacteria can be increased. Taking n-dodecane, n-hexadecane, oleic acid, paraffin, and n-hexane as oxygen-vectors, the optimal oxygen-vector oleic acid was 1.2% (v/v). The weight of wet cells increased by 66.83% compared with before, and the activity of L-AAO in fermentation broth increased by 38.88% compared with before. The standard sample DL-arginine was derivatized by phenyl isothiocyanate, and then subjected to high performance liquid chromatography(HPLC), and the obtained peak area and arginine content were used as standard curves to measure the DL-arginine. The content of D-arginine and L-arginine in the initial degradation solution was 50% each, and the bacterial cells are added to the initial degradation solution of DL-arginine. After 21 hours of reaction, L-arginine was completely Degraded, remaining 47% of D-arginine.D-alanine was easily extracted from the reaction solution using cation-exchange resin,after centrifugation, decolorization, concentration and vacuum drying, and the chemical and optical purity of the extracted d-alanine was 92.68 and 97.46%, respectively.

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