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

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• 作者：Hongyu Yang ; Qiang Zhu ; Nandi Zhou…
• 关键词：Prolyl aminopeptidase ; Pichia pastoris ; N ; glycosylation ; Thermostability
• 刊名：World Journal of Microbiology & Biotechnology
• 出版年：2016
• 出版时间：November 2016
• 年：2016
• 卷：32
• 期：11
• 全文大小：1,399 KB
• 刊物类别：Chemistry and Materials Science
• 刊物主题：Chemistry
  Applied Microbiology
  Biotechnology
  Biochemistry
  Environmental Biotechnology
  Microbiology
  
• 出版者：Springer Netherlands
• ISSN：1573-0972
• 卷排序：32

文摘

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.