African Journal of
Biotechnology

  • Abbreviation: Afr. J. Biotechnol.
  • Language: English
  • ISSN: 1684-5315
  • DOI: 10.5897/AJB
  • Start Year: 2002
  • Published Articles: 12240

Full Length Research Paper

Cloning, over-expression, and characterization of a new carboxypeptidase A gene of Bacillus pumilus ML413 in Bacillus subtilis 168

TRAORE Tahirou
  • TRAORE Tahirou
  • Laboratory of Applied Microbiology and Metabolic Engineering, School of Biotechnology, Jiangnan University, Wuxi, Jiangsu Province 214122, China.
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Junping zhou
  • Junping zhou
  • Laboratory of Applied Microbiology and Metabolic Engineering, School of Biotechnology, Jiangnan University, Wuxi, Jiangsu Province 214122, China.
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Zhiming Rao
  • Zhiming Rao
  • Laboratory of Applied Microbiology and Metabolic Engineering, School of Biotechnology, Jiangnan University, Wuxi, Jiangsu Province 214122, China.
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Genchu Huang
  • Genchu Huang
  • Laboratory of Applied Microbiology and Metabolic Engineering, School of Biotechnology, Jiangnan University, Wuxi, Jiangsu Province 214122, China.
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Taowei Yang
  • Taowei Yang
  • Laboratory of Applied Microbiology and Metabolic Engineering, School of Biotechnology, Jiangnan University, Wuxi, Jiangsu Province 214122, China.
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Xian Zhang
  • Xian Zhang
  • Laboratory of Applied Microbiology and Metabolic Engineering, School of Biotechnology, Jiangnan University, Wuxi, Jiangsu Province 214122, China.
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Meijuan Xu
  • Meijuan Xu
  • Laboratory of Applied Microbiology and Metabolic Engineering, School of Biotechnology, Jiangnan University, Wuxi, Jiangsu Province 214122, China.
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  •  Received: 28 January 2016
  •  Accepted: 08 April 2016
  •  Published: 27 April 2016

Abstract

Carboxypeptidase A (CPAs) are a well-studied group of zinc-containing exopeptidases that facilitate the breakdown of proteins and peptides during metabolism. Carboxypeptidase A is typically produced in mammalian pancreatic, brain and other tissues. A new gene encoding carboxypeptidase A in the prokaryote Bacillus pumilus was amplified by polymerase chain reaction (PCR), ligated into the shuttle vector pMA5, and cloned in a GRAS bacteria-Bacillus subtilis 168 host. This gene sequence contained a 1621 bp open reading frame that encodes a protein of 540 amino acids. The optimum pH and temperature for enzyme activity were 7.5 and 50°C, respectively. The enzyme was quite stable at neutral pH and maintained about 65% activity following a 24 h incubation at 40°C. The Km of this CPA was 0.1 mM, much higher than in mammalian species. Glycerol, ammonium sulfate, and sodium citrate improved enzyme activity under optimal culture condition. The carboxypeptidase activity in recombinant B. subtilis 168 reached a maximum of 179 U ml-1 in a 5 L fermentator when cultured on improved medium. The over expression of carboxypeptidase A in Bacillus subtilis has commercial applications.

Key words: Bacillus pumilus, Bacillus subtilis 168, over-expression, orthogonal arrays, carboxypeptidase A, metallocarboxypeptidase.