半理性设计进化土曲霉来源的ω-转氨酶AtTA热稳定性  

Semi-rational evolution ofω-transaminase from Aspergillus terreus for enhancing the thermostability

作　　者：蔡婷婷 曹佳仁 邱帅 吕常江 樊芳芳 胡升[2] 赵伟睿 梅乐和[2,3,4] 黄俊 CAI Tingting;CAO Jiaren;QIU Shuai;LYU Changjiang;FAN Fangfang;HU Sheng;ZHAO Weirui;MEI Lehe;HUANG Jun(School of Biological and Chemical Engineering,Zhejiang University of Science and Technology,Hangzhou 310023,Zhejiang,China;School of Biological and Chemical Engineering,Ningbo Tech University,Ningbo 315100,Zhejiang,China;College of Chemical and Biological Engineering,Zhejiang University,Hangzhou 310027,Zhejiang,China;Jinhua Advanced Research Institute,Jinhua 321019,Zhejiang,China)

机构地区：[1]浙江科技学院生物与化学工程学院,浙江杭州310023 [2]浙大宁波理工学院生物与化学工程学院,浙江宁波315100 [3]浙江大学化学工程与生物工程学院,浙江杭州310027 [4]金华高等研究院,浙江金华321019

出　　处：《生物工程学报》2023年第6期2126-2140,共15页Chinese Journal of Biotechnology

基　　金：国家自然科学基金(32071268);宁波市“科技创新2025”重大专项(2020Z080)。

摘　　要：转氨酶(ω-transaminase,ω-TA)作为一种天然的生物催化剂,在手性胺类化合物的合成中具有较好的应用前景。但ω-TA在催化非天然底物的反应过程中存在稳定性差、活性低的缺陷,大大限制了ω-TA的应用。为改善此缺陷,针对来源于土曲霉(Aspergillus terreus)的(R)-ω-TA(At TA),采用基于分子动力学模拟的计算机辅助设计与随机突变、组合突变相结合的策略进行酶的热稳定性改造,获得了热稳定性与活性同步提高的最佳突变酶At TA-E104D/A246V/R266Q(M3)。与At TA野生酶(wild-type,WT)相比,M3的半衰期t1/2(35℃)由17.8 min提升至102.7 min,提升了4.8倍,半失活温度T5010比WT(38.1℃)提高2.2℃。最佳突变酶M3对丙酮酸和1-(R)-苯乙胺的催化效率分别是野生酶的1.59倍和1.56倍。分子动力学模拟与分子对接结果表明,分子内氢键与疏水相互作用的增加所导致α-螺旋的加固稳定是酶热稳定性提升的主要原因;底物分子与结合口袋氨基酸之间氢键相互作用的增加以及底物结合口袋体积的增大是导致M3催化效率提升的主要原因。底物谱测定结果表明,相较于WT,M3对11种芳香酮类化合物的催化性能均有所提升,进一步说明M3对手性胺的合成具有更高的应用价值。ω-transaminase(ω-TA)is a natural biocatalyst that has good application potential in the synthesis of chiral amines.However,the poor stability and low activity ofω-TA in the process of catalyzing unnatural substrates greatly hampers its application.To overcome these shortcomings,the thermostability of(R)-ω-TA(AtTA)from Aspergillus terreus was engineered by combining molecular dynamics simulation assisted computer-aided design with random and combinatorial mutation.An optimal mutant AtTA-E104D/A246V/R266Q(M3)with synchronously enhanced thermostability and activity was obtained.Compared with the wildtype(WT)enzyme,the half-life t1/2(35℃)of M3 was prolonged by 4.8-time(from 17.8 min to 102.7 min),and the half deactivation temperature(1050 T)was increased from 38.1℃to 40.3℃.The catalytic efficiencies toward pyruvate and 1-(R)-phenylethylamine of M3 were 1.59-and 1.56-fold that of WT.Molecular dynamics simulation and molecular docking showed that the reinforced stability ofα-helix caused by the increase of hydrogen bond and hydrophobic interaction in molecules was the main reason for the improvement of enzyme thermostability.The enhanced hydrogen bond of substrate with surrounding amino acid residues and the enlarged substrate binding pocket contributed to the increased catalytic efficiency of M3.Substrate spectrum analysis revealed that the catalytic performance of M3 on 11 aromatic ketones were higher than that of WT,which further showed the application potential of M3 in the synthesis of chiral amines.

关 键 词：手性胺 ω-转氨酶 热稳定性 半理性设计 分子动力学模拟 Α-螺旋 

分 类 号：Q55[生物学—生物化学]