title: Secretory Expression, Purification, Characterization, and Application of an Aspergillus oryzae Prolyl Aminopeptidase in Bacillus subtilis.
abstract: cDNA coding a prolyl aminopeptidase (PAP) was cloned from Aspergillus oryzae and over expressed in Bacillus subtilis with a 6×His tag in N-terminus. The recombinant prolyl aminopeptidase was secreted to extracellular by adding 2 mM CaCl2and 5% D-sorbitol in TB medium; the enzyme activity in fermented supernatant increased from 7.2 to 41.5 U mL-1. It has been purified 4.3-fold through Ni-chelating affinity chromatography with a recovery of 47.3%. The purified enzyme is stable below 50 °C and within pH 6-11, and with the highest activity at pH 7.5 and 50 °C. Several kinds of salt can activate enzyme activity in a certain concentration and the relative activity was 127.02% even when the concentration of NaCl reached 4.36 M. It cleaved N-terminal Pro residues from many peptides but shown different hydrolysis rates for various Pro-X dipeptides or peptides which are of different lengths. It combined with alkaline protease and leucine aminopeptidase to hydrolyze casein, many free amino acid especially proline and small peptide of hydrolysate increased significantly.
authors.affiliation: The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122, China.
authors.forename: Kai-Dao
authors.initials: KD
authors.lastname: Wang
authors.affiliation: The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122, China.
authors.forename: Ke-Hong
authors.initials: KH
authors.lastname: Wang
authors.affiliation: The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122, China.
authors.forename: Nan-Di
authors.initials: ND
authors.lastname: Zhou
authors.affiliation: The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122, China. biochem@jiangnan.edu.cn.
authors.forename: Ya-Ping
authors.initials: YP
authors.lastname: Tian
mesh_terms: D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D001236:Aspergillus oryzae / Q000201:enzymology* / Q000235:genetics; D001412:Bacillus subtilis / Q000166:cytology / Q000235:genetics*; D002364:Caseins / Q000378:metabolism; D004274:DNA, Recombinant / Q000235:genetics; D005110:Extracellular Space / Q000187:drug effects / Q000235:genetics; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D015202:Protein Engineering / Q000379:methods*; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D002364:Caseins; D004274:DNA, Recombinant; D000626:Aminopeptidases; C022450:prolyl aminopeptidase
keywords: Application; Bacillus subtilis; Characterization; Prolyl aminopeptidase; Secretion