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

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Bacillus alcalophilus isolated 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 for Bacillus alcalophilus was developed. Additional copies of the apr4 gene were transferred into the strain Bacillus alcalophilus and 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) × 104 U/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.

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349-354

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January 2014

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© 2014 Trans Tech Publications Ltd. All Rights Reserved

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[1] R. Gupta, Q. Beg, P. Lorenz, Bacterial alkaline proteases: molecular approaches and industrial applications, Applied microbiology and biotechnology 59 (2002) 15-32.

DOI: 10.1007/s00253-002-0975-y

Google Scholar

[2] J. Singh, R. Vohra, D. Sahoo, Alkaline protease from a new obligate alkalophilic isolate of Bacillus sphaericus, Biotechnology letters 21 (1999) 921-924.

Google Scholar

[3] Q. Zhang, E. Smith, J. Shen, D. Bishop, An ethoxylated alkyl phosphate (anionic surfactant) for the promotion of activities of proteases and its potential use in the enzymatic processing of wool, Biotechnol. lett. 28 (2006) 717-723.

DOI: 10.1007/s10529-006-9049-z

Google Scholar

[4] C.G. Kumar, H. Takagi, Microbial alkaline proteases: from a bioindustrial viewpoint, Biotechnol. adv. 17 (1999) 561-594.

Google Scholar

[5] K. -H. Maurer, Detergent proteases, Curr. opin. Biotechnol. 15 (2004) 330-334.

Google Scholar

[6] K. Cheng, F. -P. Lu, M. Li, L. -L. Liu, X. -M. Liang, Purification and biochemical characterization of a serine alkaline protease TC4 from a new isolated Bacillus alcalophilus TCCC11004 in detergent formulations, Afr. J. Biotechnol . 9 (2010).

Google Scholar

[7] Y.A.V. Yomantas, E.G. Abalakina, L.I. Golubeva, L.Y. Gorbacheva, S.V. Mashko, Overproduction of Bacillus amyloliquefaciens extracellular glutamyl-endopeptidase as a result of ectopic multi-copy insertion of an efficiently-expressed mpr gene into the Bacillus subtilis chromosome, Microbial. Cell. Factories 10 (2011).

DOI: 10.1186/1475-2859-10-64

Google Scholar

[8] K.E. Tyo, P.K. Ajikumar, G. Stephanopoulos, Stabilized gene duplication enables long-term selection-free heterologous pathway expression, Nat. Biotechnol . 27 (2009) 760-765.

DOI: 10.1038/nbt.1555

Google Scholar

[9] S. -C. Wu, S. -L. Wong, Development of improved pUB110-based vectors for expression and secretion studies in< i> Bacillus subtilis</i>, J. Biotechnol. 72 (1999) 185-195.

DOI: 10.1016/s0168-1656(99)00101-7

Google Scholar

[10] L. Keay, P.W. Moser, B.S. Wildi, Proteases of the genus Bacillus. II. Alkaline proteases, Biotechnol bioengin. 12 (1970) 213-249.

DOI: 10.1002/bit.260120206

Google Scholar

[11] A.D. Holland, H.M. Rothfuss, M.E. Lidstrom, Development of a defined medium supporting rapid growth for Deinococcus radiodurans and analysis of metabolic capacities, Appl. Microbiol. Biotechnol. 72 (2006) 1074-1082.

DOI: 10.1007/s00253-006-0399-1

Google Scholar

[12] E. Amador, J.F. Martín, J.M. Castro, A Brevibacterium lactofermentum 16S rRNA gene used as target site for homologous recombination, FEMS microbiology letters 185 (2000) 199-204.

DOI: 10.1111/j.1574-6968.2000.tb09062.x

Google Scholar

[13] C. Condon, D. Liveris, C. Squires, I. Schwartz, C.L. Squires, rRNA operon multiplicity in Escherichia coli and the physiological implications of rrn inactivation, J. Bacteriol. 177 (1995) 4152-4156.

DOI: 10.1128/jb.177.14.4152-4156.1995

Google Scholar

[14] G. Rossolini, M. Riccio, E. Gallo, C. Galeotti, Kluyveromyces lactis rDNA as a target for multiple integration by homologous recombination, Gene. 119 (1992) 75-81.

DOI: 10.1016/0378-1119(92)90068-z

Google Scholar

[15] A. Pisabarro, A. Correia, J.F. Martín, Characterization of the rrnB operon of the plant pathogen Rhodococcus fascians and targeted integrations of exogenous genes at rrn loci, Appl. Environ. Microbiol . 64 (1998) 1276-1282.

DOI: 10.1128/aem.64.4.1276-1282.1998

Google Scholar