Using 16S rDNA as Target Site for Homologous Recombination to Improve the Alkaline Protease Production of Bacillus alcalophilus

2014 ◽  
Vol 886 ◽  
pp. 349-354
Author(s):  
Qing Shan Mo ◽  
Yao Tian ◽  
Hui Tu Zhang ◽  
Ling Jun Bu ◽  
Fu Ping Lu
Keyword(s):  
16S Rdna ◽  
Alkaline Protease ◽  
Genetic Manipulation ◽  
Protease Production ◽  
Protease Gene ◽  
Wild Type ◽  
Recombinant Strains ◽  
Manipulation System ◽  
Rdna Sites ◽  
Alkaline Protease Gene

Bacillus alcalophilusisolated was used for the production of alkaline protease. The enzyme encoded by alkaline protease gene (apr4) gene. To further improve the production of the strain for industrial requirement, a genetic manipulation system forBacillus alcalophiluswas developed. Additional copies of theapr4 gene were transferred into the strainBacillus alcalophilusand integrated into the 16S rDNA sites, yielding a series of recombinant strains. One of these recombinant strains, designated K23, exhibited superior properties for production of alkaline protease. the protease activity of K23 achieved by (6.19 ± 0.34) × 104U/ml, which is approximately 111.3% higher than that of the wild-type ones for 50-h fermentation. In addition, the new strain was genetically stable for more than 100 generations. These superior characteristics make it to be more suitable than the wild-type strain for alkaline protease production.

scholarly journals Involvement of Stringent Factor RelA in Expression of the Alkaline Protease Gene aprE in Bacillus subtilis

2001 ◽  
Vol 183 (15) ◽  
pp. 4648-4651 ◽  
Author(s):  
Michihiro Hata ◽  
Mitsuo Ogura ◽  
Teruo Tanaka
Keyword(s):  
Bacillus Subtilis ◽  
Alkaline Protease ◽  
Double Mutant ◽  
Regulatory System ◽  
Protease Gene ◽  
Wild Type ◽  
Double Mutation ◽  
Two Component ◽  
In The Wild ◽  
Alkaline Protease Gene

ABSTRACT Expression of Bacillus subtilis aprE, encoding an extracellular alkaline protease, is positively regulated by phosphorylated DegU, the regulator of a two-component regulatory system, DegS-DegU. We found that the expression of anaprE′-′lacZ fusion was greatly reduced in a disruption mutant with a mutation of relA, which encodes the stringent factor RelA. The level of DegU in the relA mutant was similar to that in the wild-type cell. A relA degU double mutation did not result in a further decrease of theaprE′-′lacZ level found in a degU single mutant. The expression of the aprE′-′lacZ fusion in therelA mutant was stimulated by multicopy degR or the degU32(Hy) and degS200(Hy) mutations that cause the stabilization of phosphorylated DegU. Furthermore, the expression of sacB′-′lacZ, which is also dependent on phosphorylated DegU, was stimulated by the relA mutation, and this stimulation was not seen in the relA degU double mutant. These results show that RelA (or its product guanosine-3′,5′-bisdiphosphate [pp Gpp]) does not affect the phosphorylation of DegU and suggest that it participates in the expression of aprE and sacB through the regulation of DegU-dependent transcription.

scholarly journals Characterization and Optimization of Alkaline Protease Production from Bacillus licheniformis HSW-16 Isolated from Sambhar Salt Lake

2015 ◽  
Vol 3 (2) ◽  
pp. 347-351
Author(s):  
Rajnish Prakash Singh ◽  
Prabhat Nath Jha
Keyword(s):  
Bacillus Licheniformis ◽  
Alkaline Protease ◽  
Salt Lake ◽  
Industrial Applications ◽  
Protease Production ◽  
Protease Gene ◽  
Potential Source ◽  
Alkaline Protease Gene ◽  
Bacterium Bacillus ◽  
Ph Range

Halophilic microorganisms are recognized as potential source of secondary metabolites including enzymes and drugs with wide agricultural and industrial applications. In the present study protease producing halotolerant bacterium Bacillus licheniformis HSW-16 was isolated from hypersaline Sambhar lake, Rajasthan India. Protease production was performed by using azocasein as substrate. Confirmation of protease production was also done by amplification of alkaline protease gene and sequencing. The various nutritional factors such as carbon and nitrogen source and other physiological parameters like pH, temperature, incubation time and agitation speed were optimized for optimum protease production. The enzyme was active in pH range 7-10, temperature 25 °C-40 °C and salt concentration of 1.5M. The characteristics demonstrated by this isolate showed that it could be used as a potential source of enzyme.Int J Appl Sci Biotechnol, Vol 3(2): 347-351 DOI: http://dx.doi.org/10.3126/ijasbt.v3i2.12757 

scholarly journals Isolation and Molecular Identification of Auxotrophic Mutants to Develop a Genetic Manipulation System for the HaloarchaeonNatrinemasp. J7-2

Archaea ◽  
2015 ◽  
Vol 2015 ◽  
pp. 1-16 ◽  
Author(s):  
Jie Lv ◽  
Shuai Wang ◽  
Yuchen Wang ◽  
Yuping Huang ◽  
Xiangdong Chen
Keyword(s):  
Genomic Dna ◽  
Genetic Manipulation ◽  
Gene Mutations ◽  
Transformation Method ◽  
Wild Type ◽  
Auxotrophic Mutants ◽  
Archaeal Species ◽  
Manipulation System ◽  
Exogenous Genes ◽  
Mutant Phenotypes

Our understanding of the genusNatrinemais presently limited due to the lack of available genetic tools. Auxotrophic markers have been widely used to construct genetic systems in bacteria and eukaryotes and in some archaeal species. Here, we isolated four auxotrophic mutants ofNatrinemasp. J7-2, via 1-methyl-3-nitro-1-nitroso-guanidin mutagenesis, and designated them as J7-2-1, J7-2-22, J7-2-26, and J7-2-52, respectively. The mutant phenotypes were determined to be auxotrophic for leucine (J7-2-1), arginine (J7-2-22 and J7-2-52), and lysine (J7-2-26). The complete genome and the biosynthetic pathways of amino acids in J7-2 identified that the auxotrophic phenotype of three mutants was due to gene mutations inleuB(J7-2-1),dapD(J7-2-26), andargC(J7-2-52). These auxotrophic phenotypes were employed as selectable makers to establish a transformation method. The transformation efficiencies were determined to be approximately 103transformants perµg DNA. And strains J7-2-1 and J7-2-26 were transformed into prototrophic strains with the wild type genomic DNA, amplified fragments of the corresponding genes, or the integrative plasmids carrying the corresponding genes. Additionally, exogenous genes,bgaHoramyHgene, were expressed successfully in J7-2-1. Thus, we have developed a genetic manipulation system for theNatrinemagenus based on the isolated auxotrophic mutants ofNatrinemasp. J7-2.

scholarly journals Cloning, characterization, and multiple chromosomal integration of a Bacillus alkaline protease gene.

1991 ◽  
Vol 57 (4) ◽  
pp. 901-909 ◽  
Author(s):  
J C van der Laan ◽  
G Gerritse ◽  
L J Mulleners ◽  
R A van der Hoek ◽  
W J Quax
Keyword(s):  
Alkaline Protease ◽  
Protease Gene ◽  
Chromosomal Integration ◽  
Alkaline Protease Gene
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