近平滑假丝酵母ATCC 7330羰基还原酶CpCR突变体酶的稳定性研究

Stability of CpCR mutants from Candida parapsilosis ATCC 7330 carbonyl reductase

通过蛋白质理性设计和定点突变技术,在近平滑假丝酵母ATCC 7330羰基还原酶wtCpCR的基础上,构建5个突变体酶A98N、S307N、G262N、S216N和S258N,考察了有机溶剂、温度、剪切力、氧气、辅酶NADPH浓度对突变体酶的稳定性影响。研究表明,A98 N在磷酸盐(potassium phosphate,PB)缓冲溶液中比酶活力提高10%,而S307 N没有酶活力,其他3个突变体比酶活力均有降低;A98 N和G262 N在PB/甲基叔丁基醚的双相体系中界面稳定性比原始酶wtCpCR分别提高1.7和1.4倍;4个突变体酶的耐热特性略有下降,T_(50)值介于31~36℃;在100、200 r/min条件下,4个突变体耐剪切力均增强,在300 r/min条件下G262 N突变酶的耐剪切稳定性比wtCpCR增强1.3倍;G262 N突变酶在有氧条件下的稳定性更好,比野生酶提高1.4倍,半衰期(t_(1/2))达到11.87 h;辅酶浓度为0~0.4 mmol/L时,G262 N突变酶更加稳定。该研究为该羰基还原酶wtCpCR的多点突变进一步改善酶稳定性提供重要的科学依据,同时可提高该酶在双相体系生物催化工艺的经济性。

Using the protein rational design and site-directed mutagenesis technology,five mutants with A98N,S307N,G262N,S216N and S258N substitution in carbonyl reductase(wtCpCR)from Candida parapsilosis ATCC 7330 were constructed.And their stability in organic solvents,temperature,shear resistance,oxygen resistance,coenzyme NADPH concentration were studied.The results showed that compared with the enzymatic activity of wtCpCR in potassium dihydrogen phosphate/dibasic potassium phosphate(PB)buffer,the enzymatic activity of the A98N mutant increased by 10%,but the mutant S307N had no enzymatic activity and the activity of other mutants slightly decreased.The interfacial stability of A98N and G262N mutant in the PB/methyl tert-butyl ether biphasic media increased 1.7 and 1.4-fold compared to than that of the wtCpCR,respectively.The thermal resistance properties of four mutants slightly reduced,with T_(50) values ranging from 31-36℃.Under the conditions of 100 and 200 r/min,the shear resistance of the four mutants was enhanced.When the speed was 300 r/min,the stability of the G262N mutant was 1.3 times higher than that of wtCpCR.The G262N mutant enzyme had better stability under aerobic conditions,with a t_(1/2) value of 11.87 h,which was 1.4 times higher than that of the wild-type enzyme.When the NADPH concentration was 0-0.4 mmol/L,the G262N mutant was much more stable.This study has provided very important scientific evidences for multiple mutations to improve stability modification of the carbonyl reductase from Candida parapsilosis(CpCR),which can improve its economy of biocatalysis process in biphasic reaction media.