底物亲和设计提高腈水解酶Nit6803活性

Substrate affinity design for the improvement of nitrilase Nit6803 activity

腈水解酶(Nitrilase,EC 3.5.5.1),是一类可以将腈类物质一步水解为羧酸的酶,是多种重要大宗化工品、药物中间体的理想生物催化剂。但是天然酶活性低、热稳定性差限制了其在工业上的应用。近年来通过蛋白质改造来解决酶活性与稳定性间的“trade-off”效应以提升催化性能的研究较多。该研究提出一种酶-底物亲和设计改造策略,以来源于Syechocystis sp.PCC6803的腈水解酶Nit6803作为改造对象,结合基于Rosetta的Cartesian_ddG方法和基于自由能微扰的酶-底物亲和力计算,对Nit6803的催化口袋进行单点突变及组合突变设计。基于此获得了活性显著提升的单点突变体F64Y、W170G,及组合突变体F64Y/W170G。其中,F64Y/W170G的比酶活力达到(22.48±0.64)U/mg,为野生型的4.56倍,且该突变体的热稳定性不低于野生型。通过分批补加3-氰基吡啶进行全细胞催化表明F64Y/W170G催化能力强于野生型,在达到相同转化率情况下极大的缩短了催化时间。结果表明,该研究提出的设计策略可以有效提升酶的活性而不影响其稳定性,为酶的理性设计改造提供了新的思路。

Nitrilase(EC 3.5.5.1)is an ideal biocatalyst for a variety of essential bulk chemicals and pharmaceutical intermediates,due to its capability to catalyze nitrile to carboxylic acid with high stereoselectivity under mild reaction conditions.However,the low activity and poor thermal stability of natural enzymes still limit its industrial application.In recent years,it has been a research hotspot to break through the bottleneck of‘trade-off’between activity and stability through protein engineering.This study proposes a novel enzyme-substrate affinity design strategy.The nitrilase Nit6803 derived from Syechocystis sp.PCC6803 was improved in activity through the rational design combining the Cartesian_ddG method in Rosetta suite and the enzyme-substrate affinity calculation based on free energy perturbation.The single-point mutants F64Y,W170G,and combination mutant F64Y/W170G with significantly improved activity were obtained.Among them,the specific enzyme activity of F64Y/W170G reached(22.48±0.64)U/mg,which was 4.56 times that of the wild type,and the thermal stability maintained.Whole-cell catalysis by adding 3-cyanopyridine in batches showed that F64Y/W170G had stronger catalytic ability than wild type,and greatly shortened the catalysis time when reaching the same conversion rate.The results demonstrated that the engineering strategy proposed in this study can effectively enhance the enzyme activity without decreasing its stability,which provides a new idea for the rational design of enzymes.