Jia Hao-Yu, Yang Zi-Yue, Chen Qi, Zong Min-Hua, Li Ning
School of Food Science and Engineering, South China University of Technology, Guangzhou, China.
State Key Laboratory of Bioreactor Engineering, Shanghai Collaborative Innovation Center for Biomanufacturing, East China University of Science and Technology, Shanghai, China.
Front Chem. 2021 May 13;9:610091. doi: 10.3389/fchem.2021.610091. eCollection 2021.
Catalytic promiscuity is a promising starting point for improving the existing enzymes and even creating novel enzymes. In this work, site-directed mutagenesis was performed to improve promiscuous alcohol dehydrogenase activity of reductive aminase from (RedAm). RedAm showed the cofactor preference toward NADPH in reductive aminations, while it favored NADH in the reduction reactions. Some key amino acid residues such as N93, I118, M119, and D169 were identified for mutagenesis by molecular docking. Variant N93A showed the optimal pH and temperature of 8 and 30°C, respectively, in the reduction of 5-hydroxymethylfurfural (HMF). The thermostability was enhanced upon mutation of N93 to alanine. The catalytic efficiency of variant N93A ( / , 23.6 mM s) was approximately 2-fold higher compared to that of the wild-type (WT) enzyme (13.1 mM s). The improved catalytic efficiency of this variant may be attributed to the reduced steric hindrance that stems from the smaller side chain of alanine in the substrate-binding pocket. Both the WT enzyme and variant N93A had broad substrate specificity. () cells harboring plain vector enabled selective reduction of biobased furans to target alcohols, with the conversions of 35-95% and the selectivities of >93%. The introduction of variant N93A to resulted in improved substrate conversions (>98%) and selectivities (>99%).
催化多效性是改进现有酶甚至创造新酶的一个有前景的起点。在这项工作中，进行了定点诱变以提高来自[具体来源未提及]的还原氨基酶（RedAm）的混杂乙醇脱氢酶活性。RedAm在还原胺化反应中对NADPH表现出辅因子偏好，而在还原反应中则偏好NADH。通过分子对接确定了一些关键氨基酸残基，如N93、I118、M119和D169用于诱变。变体N93A在还原5-羟甲基糠醛（HMF）时，最佳pH和温度分别为8和30°C。将N93突变为丙氨酸后热稳定性增强。变体N93A的催化效率（/，23.6 mM s）比野生型（WT）酶（13.1 mM s）高出约2倍。该变体催化效率的提高可能归因于底物结合口袋中丙氨酸较小侧链导致的空间位阻减小。WT酶和变体N93A都具有广泛的底物特异性。携带空载体的[具体细胞未提及]细胞能够将生物基呋喃选择性还原为目标醇，转化率为35 - 95%，选择性>93%。将变体N93A引入[具体细胞未提及]导致底物转化率提高（>98%）和选择性提高（>99%）。
