新颖微生物低温酯酶EstP8的酶学性质研究与在手性催化中的应用

从假单胞菌Pseudonocardia antitumoralis HUP007基因组中克隆了一个1 041bp的酯酶基因EstP8，编码的蛋白具有377个氨基酸残基。在E.coli BL21（DE3）中实现酯酶EstP8的高效异源表达和纯化。EstP8为脂肪酶家族Ⅳ中的一员，具有HGGG保守序列。EstP8最适底物为对硝基苯酚乙酸酯（p-NPO），最适温度和pH分别为50℃和8.0。EstP8催化p-NPO水解反应的活性、V_max和K_m分别达到105.19U/mg、89.4μM/min、1.144mM。EstP8在pH7.0~8.0范围内具有良好的pH稳定性；在4℃时，酯酶相对活力为41.78%，在10~40℃内具有很好温度稳定性。EstP8对大部分金属离子有很好的耐受性，低浓度的Cu²⁺、Mn²⁺、Zn²⁺对该酶的活性有激活作用。辛烷、庚烷、甲苯、丙酮、DMF等有机溶剂对EstP8的活性同样具有激活作用。酯酶EstP8还可以通过水解拆分高效地制备手性（R）-1-苯基乙醇；添加有机溶剂可以很好地促进该酯酶的光学选择性和产率，在共溶剂甲苯的存在下，所制备的（R）-1-苯基乙醇的e.e.和产率可达91%和18%；在共溶剂DMSO的存在下，所制备的（S）-乙酸苏合香酯的e.e.和产率可达98%和60%。酯酶EstP8在手性生物催化等诸多工业领域有很好的应用潜力。

Functional Characterization of a Novel Microbial Psychrophilic Lipase and Its Utilization in Stereo-Selective Biocatalysis

From the genome of Pseudomonas antitumoralis HUP007, an esterase gene harboring 1041 bp and encoding a protein EstP8 of 377 amino acid was cloned. Esterase EstP8 was efficiently expressed in E. coli BL21(DE3) and further purified. Esterase EstP8 belongs to the IV family of lipases and contains conserved HGGG motif. The optimal substrate of esterase EstP8 was p-NPO, the optimal working temperature of esterase EstP8 was 50℃ and the optimal working pH of esterase EstP8 was 8.0. The hydrolysis activity of esterase EstP8 toward p-NPO was 105.19 U/mg, with V_max and K_m being 89.4 μmol/min and 1.144 mM, respectively. Esterase EstP8 remained high activity at pH7.0~8.0. The relative activity of esterase EstP8 was 41.78% at 4℃ and esterase EstP8 behaved very good thermo-stability at 10~40℃. EstP8 exhibited very good resistance to most metal ions tested. The addition of Cu²⁺, Mn²⁺ or Zn²⁺ of low concentrations could even stimulate the activity of EstP8. Organic solvents such as isooctane, methylbenzene, acetone and DMF could stimulate the activity of EstP8. Esterase EstP8 could generate chiral (R)-1-phenylethanol through kinetic resolution. The addition of organic solvents could well stimulate the stereo-selectivity and conversion during kinetic resolution. The e.e. and conversion of generated (R)-1-phenylethanol could reach 91% and 18%, respectively, in the presence of methylbenzene. The e.e. and conversion of generated (S)-styrallyl acetate could reach 98% and 60%, respectively, in the presence of DMSO. Psychrophilic esterase EstP8 possesses good potential in diverse industries such as stereo-selective biocatalysis.