脯氨酸氨肽酶枯草芽孢杆菌分泌表达及特性表征

Secretory Expression and Characterization of Prolyl Aminopeptidase inBacillus subtilis

DOI：10.3969/j.issn.1673-1689.2018.11.003

中文关键词: 脯氨酸氨肽酶 枯草芽孢杆菌 分泌表达 特性表征

英文关键词: prolyl aminopeptidase,Bacillus subtilis,secretory expression,characterization

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中文摘要:

构建重组枯草芽孢杆菌大量分泌米曲霉脯氨酸氨肽酶，纯化后对其基本酶学性质进行表征。将脯氨酸氨肽酶cDNA序列从载体pMD19-pap上克隆后连接到载体pMA5上得到重组质粒，转化枯草芽孢杆菌WB600后进行分泌表达。通过在培养基中加入5% D-山梨醇和2 mmol/L CaCl2，胞外酶活由7.5 U/mL提高到36 U/mL。通过硫酸铵盐析、Hitrap Q和SuperdexTM 75对重组脯氨酸氨肽酶进行了纯化，纯化后的比酶活为247.3 U/mg，纯化倍数达到8.8倍。该酶最适温度为50 ℃，最适pH为7.5，米氏常数Km和最大反应速率Vmax分别为0.171 mmol/L和55.99 μmol/（L·min）。

英文摘要:

Construct recombinantBacillus subtilisto over-expressionAspergillus oryzaeprolyl minopeptidase，then purified and characterized its basic enzyme properties. The pap gene was cloned from pMD19-pap vector and ligated into pMA5 to generate recombinant pMA5-pap expression vector. Then the vector was transformed intoBacillus subtilisWB600 competent cells and secretory expressed. By adding 5% D-sorbitol and 2 mmol/L CaCl2 in TB medium，the enzyme activity in fermented supernatant increased from 7.5 to 36.0 U/mL. The recombinant prolyl aminopeptidase was purified by ammonium sulfate，Hitrap Q anion exchange chromatography and SuperdexTM 75 gel chromatography，the purification factor was 8.8 and the specific activity of the purified enzyme was 247.3 U/mg. The purified enzyme has the highest activity at pH 7.5 and 50 ℃，theKm andVmax was estimated to be 0.171 mmol/L and 55.99 μmol/（L·min） respectively.

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