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.
Highlights
Carboxypeptidases (CPs) catalyze the release of Cterminal amino acids from proteins and peptides (Sebastian Tanco et al, 2013)
The carboxypeptidase A (CPA) gene was successfully amplified from the genomic DNA of Bacillus pumilus ML413 strain by polymerase chain reaction (PCR)
A new carboxypeptidase A gene isolated from B. pumilus was successfully cloned into a Generally Recognized As Safe (GRAS) bacterium-B. subtilis 168
Summary
Carboxypeptidases (CPs) catalyze the release of Cterminal amino acids from proteins and peptides (Sebastian Tanco et al, 2013). Some non-digestive zinc carboxypeptidases are involved in hormone and neuropeptide processing, bioactive peptide activation or inactivation, or functional modulation of regulatory proteins (Joshi et al, 1999). They are used to remove ochratoxin A (Abrunhosa et al, 2007) and as a debitterizing reagent in the hydrolysis of soybean protein (Fang et al, 2005). Carboxypeptidases are classified into several types, carboxypeptidases that have a stronger preference for those amino acids containing aromatic or branched hydrocarbon chains are called carboxypeptidase A (A for aromatic/aliphatic), carboxypeptidases that cleave positively charged amino acids (arginine, lysine) are called carboxypeptidase B (B for basic), metallocarboxypeptidase that cleaves a C-terminal glutamate from the peptide N-acetyl-L-aspartyl-L-glutamate is called "glutamate carboxypeptidase ́
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