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Over-Expression of a Proline Specific Aminopeptidase from Aspergillus oryzae JN-412 and Its Application in Collagen Degradation

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Abstract

A strain that exhibited intracellular proline-specific aminopeptidase (PAP) activity was isolated from soy sauce koji and identified as Aspergillus oryzae JN-412. The gene coding PAP was cloned and efficiently expressed in Escherichia coli BL21 in a biologically active form. The highest specific activity reached 52.28 U mg−1 at optimum cultivation conditions. The recombinant enzyme was purified 3.3-fold to homogeneity with a recovery of 36.7 % from cell-free extract using Ni-affinity column chromatography. It appeared as a single protein band on SDS-PAGE with molecular mass of approximately 50 kDa. The purified enzyme exhibited the highest activity at 60 °C and pH 7.5. The enzyme activity was inhibited by PMSF and ions like Zn2+ and Cu2+. DTT, β-mercaptoethanol, EDTA, and ions like Co2+, Mg2+, Mn2+, and Ca2+ had no influence on enzyme activity, whereas Ni2+ enhanced the enzyme activity. By using collagen as a substrate, the purified recombinant prolyl aminopeptidase contributed to the hydrolysis of collagen when used in combination with neutral protease, and free amino acids in collagen hydrolysates was significantly increased.

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Acknowledgments

This work was supported by the National High Technology Research and Development Program of China (863 Program, 2011AA100905) and the Program for New Century Excellent Talents in University (NCET-12-0878).

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  1. The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122, China

    Guo-Wei Ding, Nan-Di Zhou & Ya-Ping Tian

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  1. Guo-Wei Ding
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Correspondence to Ya-Ping Tian.

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Ding, GW., Zhou, ND. & Tian, YP. Over-Expression of a Proline Specific Aminopeptidase from Aspergillus oryzae JN-412 and Its Application in Collagen Degradation. Appl Biochem Biotechnol 173, 1765–1777 (2014). https://doi.org/10.1007/s12010-014-0963-6

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