scholarly journals Improving the Production of Salt-Tolerant Glutaminase by Integrating Multiple Copies of Mglu into the Protease and 16S rDNA Genes of Bacillus subtilis 168

Molecules ◽  
2019 ◽  
Vol 24 (3) ◽  
pp. 592 ◽  
Author(s):  
Xian Zhang ◽  
Zhaoyang Xu ◽  
Song Liu ◽  
Kai Qian ◽  
Meijuan Xu ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Bacillus Subtilis ◽  
16S Rdna ◽  
Micrococcus Luteus ◽  
Rdna Locus ◽  
Subtilis Strain ◽  
Enzyme Activity Assay ◽  
Bacillus Subtilis 168 ◽  
Fed Batch Fermentation ◽  
Multiple Copies

In this study, the Micrococcus luteus K-3 glutaminase was successfully over-expressed in the GRAS (Generally Recognized as Safe) Bacillus subtilis strain 168 by integration of the Mglu gene in the 16S rDNA locus. This was done in order to screen a strain producing high levels of recombinant glutaminase from the selected candidates. The transcription of the glutaminase genes in the B. subtilis 168 chromosome and the expression of glutaminase protein was further assessed by qPCR, SDS-PAGE analysis and an enzyme activity assay. To further increase the production of glutaminase, the nprB and nprE genes, which encode specific proteases, were disrupted by integration of the Mglu gene. After continuous cell culturing without the addition of antibiotics, the integrated recombinant strains showed excellent genetic stability, demonstrating favorable industrialization potential. After the fermentation temperature was optimized, a 5-L bioreactor was used for fed-batch fermentation of the recombinant glutaminase producing strain at 24 °C, and the highest enzyme activity achieved was approximately 357.6 U/mL.

Gene cloning and characterization of glycine oxidase from newly isolated Bacillus subtilis strain R5

Biologia ◽  
2011 ◽  
Vol 66 (1) ◽  
pp. 1-7 ◽  
Author(s):  
Farrukh Jamil ◽  
Naeem Rashid ◽  
Qurra-tul-Ann Gardner ◽  
Muhammad Akhtar
Keyword(s):  
Enzyme Activity ◽  
Bacillus Subtilis ◽  
Specific Activity ◽  
Subtilis Strain ◽  
Amino Acid Residues ◽  
Expression And Purification ◽  
Gene Encoding ◽  
Glycine Oxidase ◽  
Bacillus Subtilis Strain R5

AbstractThe gene encoding the glycine oxidase from Bacillus subtilis strain R5 (goxR) was cloned and expressed in Escherichia coli. The gene consisted of 1,110 nucleotides that encoded a protein (GoxR) of 369 amino acid residues with a molecular mass of 40,761 Da. The GoxR exhibited 98.6% identity with glycine oxidase from B. subtilis strain 168. Gene expression and purification of the recombinant GoxR were performed. The recombinant GoxR existed in a homotetramer form. The recombinant protein effectively catalyzed the oxidation of glycine and d-alanine. The specific activity of the purified recombinant GoxR was 0.96 U/mg when glycine was used as a substrate and 1.0 U/mg when d-alanine was substrate. The enzyme displayed its highest activity at pH 8.0 and at a temperature of 50°C. The activation energy of the reaction catalyzed by the enzyme was calculated to be 26 kJ/mol. The enzyme activity was significantly inhibited in the presence of organic solvents. No enhancement of enzyme activity was observed in the presence of metal cations. The experimental results presented in this study demonstrate that the enzyme was a bonafide glycine oxidase.

A new method for multiple gene inactivations in Bacillus subtilis 168, producing a strain free of selectable markers

2011 ◽  
Vol 57 (5) ◽  
pp. 427-436 ◽  
Author(s):  
Chong Zhang ◽  
Xiaohui Zhang ◽  
Zhengying Yao ◽  
Yaping Lu ◽  
Fengxia Lu ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Resistance Gene ◽  
Target Genes ◽  
Genome Structure ◽  
Subtilis Strain ◽  
Gene Target ◽  
Selectable Markers ◽  
Vector Integration ◽  
Bacillus Subtilis 168 ◽  
Counter Selection

This study describes a novel method for repeated gene inactivation in Bacillus subtilis 168. A B. subtilis strain (BS-PS) that is conditionally auxotrophic for lysine was obtained by replacing the PlysA promoter with the Pspac promoter. The homologous recombination integration vector PLC-T was constructed to contain lacI, which encodes a Pspac promoter repressor, and the chloromycetin resistance gene. Target genes were manipulated by generating an insertion sequence with two homologous arms and the target gene in PLC-T to create a specific integrating vector. Integration into the BS-PS chromosome occurred by a single crossover at either of the two homologous arms. The resulting transitional strain (BS-PS-PI) was chloromycetin resistant and lysine auxotrophic and had an unstable genome structure because of the duplication. Excision of lacI and chloromycetin resistance gene was achieved by a second single crossover at the duplication. Recovery of a lysine prototroph functioned as counter-selection and was identified by PCR. In this work, we inactivated nprE and aprE, two protease genes secreted by B. subtilis 168 free of selectable markers.

scholarly journals Bacterial Tolerance to Heavy Metal Contents Present in Contaminated and Uncontaminated Soils

2016 ◽  
Vol 29 (2) ◽  
pp. 56-61
Author(s):  
FZ Tanu ◽  
S Hoque
Keyword(s):  
Heavy Metal ◽  
Bacillus Subtilis ◽  
Bacillus Cereus ◽  
Metal Tolerance ◽  
Micrococcus Luteus ◽  
Heavy Metal Tolerance ◽  
Subtilis Strain ◽  
Rrna Gene ◽  
Bacterial Strains ◽  
Metal Tolerant Bacteria

Present study dealt with identification of some heavy metal tolerant bacteria from contaminated industrial soils of Dhaka Export Processing Zone (DEPZ) at Savar, tannery area at Hazaribagh and uncontaminated agricultural soils of Dhamrai and Kushtia in Bangladesh and determination of their tolerance to chromium (Cr6+) and cadmium (Cd2+). A total of 15 isolates from four soil samples were provisionally identified as different species of Bacillus, Micrococcus and Pseudomonas based on their morphological, physiological, and biochemical characteristics. Among them eight colonies were separated based on high level of heavy metal tolerance and identified at molecular level by PCR technique and 16S rRNA gene sequencing as Micrococcus luteus strain P43 (E4), Bacillus pocheonensis strain TR2-6 (T6), Bacillus megaterium strain H2 (T8), Bacillus amyloliquefaciens strain SCSAAB0007 (D10), Bacillus cereus isolate PGBw4 (D11), Bacillus cereus strain ES-4a1 (K12), Bacillus subtilis strain 1320, (K13), and Bacillus subtilis strain DP14 (K14). The Maximum Tolerable Concentration (MTC) of bacterial strains to Cr6+ and Cd2+ ranged between 250-1250 ?g/ml and 30-150 ?g/ml, respectively in nutrient broth medium. From the metal tolerance investigation Bacillus was found as the most heavy metal tolerant to both Cr6+ and Cd2+ among the three genera. The identified heavy metal tolerant bacteria could be useful for the bioremediation of heavy metal contaminated environment.Bangladesh J Microbiol, Volume 29, Number 2, Dec 2012, pp 56-61

Expression and Enzyme Activity Assay of pfu with Molecular Chaperone

2010 ◽  
Vol 31 (6) ◽  
pp. 499-503
Author(s):  
Hai-Jun ZHANG ◽  
Jun YANG ◽  
Xiao-Guang LIU ◽  
Xiang-Yang HU
Keyword(s):  
Enzyme Activity ◽  
Molecular Chaperone ◽  
Activity Assay ◽  
Enzyme Activity Assay

Antibacterial Flavonoids Against Oral Bacteria of Enterococcus Faecalis ATCC 29212 from Sarang Semut (Myrmecodia pendans) and Its Inhibitor Activity Against Enzyme MurA

2019 ◽  
Vol 16 (3) ◽  
pp. 290-296 ◽  
Author(s):  
Dikdik Kurnia ◽  
Eti Apriyanti ◽  
Cut Soraya ◽  
Mieke H. Satari
Keyword(s):  
Enzyme Activity ◽  
Enterococcus Faecalis ◽  
Ethyl Acetate Fraction ◽  
Antibacterial Activities ◽  
Oral Bacteria ◽  
Diffusion Method ◽  
Enzyme Activity Assay ◽  
Antibacterial Compounds ◽  
Ic50 Values ◽  
Positive Controls

Background: A significant number of antibiotics are known to inhibit peptidoglycan synthesis in the cross-linking stage, while the drug fosfomycin is the only one known to inhibit MurA. Escalated antibiotic resistance has had an impact on the efficacy of fosfomycin, thus demanding the discovery of suitable substitutes with improved potential for MurA inhibition. The aim of this work is to isolate antibacterial compounds from Sarang Semut (Myrmecodia pendans) and to evaluate their antibacterial activity against pathogenic oral bacteria of Enterococcus faecalis ATCC 29212 and inhibitory activity against MurA enzyme. Methods: The antibacterial compounds from Sarang Semut were isolated by a bioactivity-guided separation method with various solvents and combination of column chromatography on normal and reverse phases. The compounds with concentrations of 1000 and 5000 ppm were assessed against E. faecalis ATCC 29212 by agar well diffusion method, with chlorhexidine and fosfomycin being used as positive controls. Results: Two antibacterial compounds isolated from Sarang Semut were identified as two new flavonoids derivates of 1 (10 mg) and 2 (4 mg). Both compounds were tested for antibacterial activities against E. faecalis. MIC values of compounds 1 and 2 were 8.15 and 8.05 mm at 1000 ppm and 8.62 and 8.55 mm at 5000 ppm, respectively. MBC values were 156 and 625 ppm for 1 and 625 and 2500 ppm for 2, respectively. In an inhibitory murA enzyme activity assay, compounds 1 and 2 were shown to inhibit the enzyme activity by IC50 values of 21.7 and 151.3 ppm. Conclusion: The study demonstrated that ethyl acetate fraction of Sarang Semut contained antibacterial flavonoids as active constituents that showed activity against E. faecalis. These results showed the plant’s potential in herbal medicine and the development of new antibacterial agent for pathogenic dental caries.

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