NDLI: Optimized expression of prolyl aminopeptidase in Pichia pastoris and its characteristics after glycosylation

Content Provider	Springer Nature Link
Author	Yang, Hongyu Zhu, Qiang Zhou, Nandi Tian, Yaping
Copyright Year	2016
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$^{−1}$.
Starting Page	1
Ending Page	11
Page Count	11
File Format	PDF
ISSN	09593993
Journal	World Journal of Microbiology and Biotechnology
Volume Number	32
Issue Number	11
e-ISSN	15730972
Language	English
Publisher	Springer Netherlands
Publisher Date	2016-09-15
Publisher Place	Dordrecht
Access Restriction	One Nation One Subscription (ONOS)
Subject Keyword	Prolyl aminopeptidase Pichia pastoris N-glycosylation Thermostability Applied Microbiology Biotechnology Biochemistry Environmental Engineering/Biotechnology Microbiology
Content Type	Text
Resource Type	Article
Subject	Physiology Medicine Applied Microbiology and Biotechnology Biotechnology

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