催化Aldol加成反应的新型枯草芽孢杆菌BS168酰基氨肽酶的表达和应用

Cloning and Application of a New Acylaminoacyl Peptidase from Bacillus subtilis 168 for Aldol Reaction

从枯草芽孢杆菌(Bacillus subtilis 168)基因组中的ORF32230出发,通过氨基酸序列分析推测其可能为酰基氨肽酶基因,并与典型的脯氨酸寡肽酶家族成员一致,含有2个独立的结构域,活性中心由催化三联体丝氨酸-天冬氨酸-组氨酸(Ser-Asp-His)组成.将BSU32230的基因片段与p ET-21a载体相连,转入BLP(DE3)表达菌中,在0.5 mmol/L异丙基-β-D-硫代半乳糖苷(IPTG)存在及20℃条件下诱导表达该蛋白.利用硫酸铵沉淀与Ni亲和层析对BSU32230蛋白进行纯化,并通过实验证明该蛋白同时具有酯酶和肽酶2种活性.该酶最佳反应温度为50℃,最佳p H值为8.0,40℃下半衰期约29 h,在p H=4~10范围内稳定.该酶能够在有机相中催化不对称Aldol加成反应,且反应产物的立体选择性较好(84.6%).

Acylaminoacyl peptidase has been named acyl-peptide releasing enzyme （AARE） , catalyses the removal of an N-acylated amino acid from N-c^-aeylpeptides. In this research, a novel acyl-peptide releasing enzyme gene（BSU32230） from Bacillus subtilis 168 was cloned and expressed in Escherichia eoli BLP DE3 codon plus to produce acyl-peptide releasing enzyme. The recombinant enzyme was purified in two steps： ammonium sulfate precipitation and Ni^2＋-column affinity chromatography. The optimum temperature and pH of enzyme were 50℃ and 8.0, respectively. The half-life of recombinant enzyme at 40℃ was 29 h, the enzyme was stable at pH range from 4 to 10. This report demonstrated the acylaminoacyl peptidase from Bacillus subtilis peptidase can catalyze aldol reaction and showed high enantioselectivity. The reaction provided optically active secondary alcohol with satisfying enantioselectivity（84.6% e.e. ）.