title: Over-expression of a proline specific aminopeptidase from Aspergillus oryzae JN-412 and its application in collagen degradation.
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.
authors.affiliation: The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122, China.
authors.forename: Guo-Wei
authors.initials: GW
authors.lastname: Ding
authors.forename: Nan-Di
authors.initials: ND
authors.lastname: Zhou
authors.forename: Ya-Ping
authors.initials: YP
authors.lastname: Tian
mesh_terms: D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D001236:Aspergillus oryzae / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D003094:Collagen / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005818:Genetic Engineering / Q000379:methods*; D006868:Hydrolysis; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011392:Proline / Q000378:metabolism*; D017433:Protein Structure, Secondary; D059748:Proteolysis; D013379:Substrate Specificity
chemical_list: D003094:Collagen; D011392:Proline; D000626:Aminopeptidases; C022450:prolyl aminopeptidase