title: Optimized expression of prolyl aminopeptidase in Pichia pastoris and its characteristics after glycosylation.
abstract: Prolyl aminopeptidases are specific exopeptidases that catalyze the hydrolysis of the N-terminus proline residue of peptides and proteins. In the present study, the prolyl aminopeptidase gene (pap) from Aspergillus oryzae JN-412 was optimized through the codon usage of Pichia pastoris. Both the native and optimized pap genes were inserted into the expression vector pPIC9 K and were successfully expressed in P. pastoris. Additionally, the activity of the intracellular enzyme expressed by the recombinant optimized pap gene reached 61.26 U mL(-1), an activity that is 2.1-fold higher than that of the native gene. The recombinant enzyme was purified by one-step elution through Ni-affinity chromatography. The optimal temperature and pH of the purified PAP were 60 °C and 7.5, respectively. Additionally, the recombinant PAP was recovered at a yield greater than 65 % at an extremely broad range of pH values from 6 to 10 after treatment at 50 °C for 6 h. The molecular weight of the recombinant PAP decreased from 50 kDa to 48 kDa after treatment with a deglycosylation enzyme, indicating that the recombinant PAP was completely glycosylated. The glycosylated PAP exhibited high thermo-stability. Half of the activity remained after incubation at 50 °C for 50 h, whereas the remaining activity of PAP expressed in E. coli was only 10 % after incubation at 50 °C for 1 h. PAP could be activated by the appropriate salt concentration and exhibited salt tolerance against NaCl at a concentration up to 5 mol L(-1).
authors.affiliation: The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122, China.
authors.forename: Hongyu
authors.initials: H
authors.lastname: Yang
authors.affiliation: The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122, China.
authors.forename: Qiang
authors.initials: Q
authors.lastname: Zhu
authors.affiliation: The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122, China.
authors.forename: Nandi
authors.initials: N
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: Yaping
authors.initials: Y
authors.lastname: Tian
mesh_terms: D000626:Aminopeptidases / Q000235:genetics* / Q000378:metabolism*; D001236:Aspergillus oryzae / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D010843:Pichia / Q000235:genetics* / Q000254:growth & development; D011994:Recombinant Proteins; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D005656:Fungal Proteins; D011994:Recombinant Proteins; D000626:Aminopeptidases; C022450:prolyl aminopeptidase
keywords: N-glycosylation; Pichia pastoris; Prolyl aminopeptidase; Thermostability