高等学校化学学报 ›› 2020, Vol. 41 ›› Issue (5): 1018.doi: 10.7503/cjcu20190621
收稿日期:
2019-12-02
出版日期:
2020-05-10
发布日期:
2020-02-07
通讯作者:
穆晓清
E-mail:xqmu@jiangnan.edu.cn
基金资助:
WU Tao1,2,MU Xiaoqing1,2,*(),NIE Yao1,XU Yan1
Received:
2019-12-02
Online:
2020-05-10
Published:
2020-02-07
Contact:
Xiaoqing MU
E-mail:xqmu@jiangnan.edu.cn
Supported by:
摘要:
基于同源建模建立了Bacillus cereus胺脱氢酶(BcAmDH)的三维结构, 采用半理性设计方法, 对底物结合口袋附近的8个氨基酸残基(L42, G43, M67, A115, E116, T136, V293和V296)分别进行单点饱和突变, 通过显色法筛选出3个正向突变位点(116, 136和293). 进一步采用迭代饱和突变策略对这3个正向位点进行组合突变, 获得最优突变株V293A/E116V/T136S, 其对苯乙酮还原反应的催化效率达到2.54 L·min-1·mmol-1, 比BcAmDH提高了719%; 与BcAmDH相比, 最优突变株在催化苯乙酮的不对称还原反应时, 底物浓度由100 mmol/L提高至300 mmol/L, 转化率由42.1%提高至80.2%. 分子对接结果表明, 突变株底物结合口袋的位阻减小和底物进出通道的扩大是提高催化效率的主要原因.
中图分类号:
TrendMD:
吴涛,穆晓清,聂尧,徐岩. 迭代饱和突变提高Bacillus cereus胺脱氢酶对苯乙酮还原的催化效率. 高等学校化学学报, 2020, 41(5): 1018.
WU Tao,MU Xiaoqing,NIE Yao,XU Yan. Improving Catalytic Efficiency of Bacillus Cereus Amine Dehydrogenase for Acetophenone Reduction by Iterative Saturation Mutagenesis . Chem. J. Chinese Universities, 2020, 41(5): 1018.
Fig.1 3D structure of GcLeuDH and BcAmDH(A) and distribution of selected amino acid(B) 3D structure of GcLeuDH and BcAmDH are shown as cyan and green cartoon, respectively. The eight selected amino acid residues(L42, G43, M67, A115, E116, T136, V293, V296) and the two residues initially mutated to generate AmDH activity(S70, L263) are shown as green and yellow sticks, respectively.
Fig.2 High-throughput screening results of site-directed saturation mutagenesis library(A) and color-rendering results of 116-site saturation mutagenesis library(B) Color-rendering results of BcAmDH and positive mutants are shown in black rectangle and red circle, respectively.
Fig.3 SDS-PAGE analysis(A) and relative activity(B) of positive mutants obtained by site-directed saturation mutagenesis (A) M: low molecular protein marker; lane 1—7 represent crude enzyme for BcAmDH, V293A, T136G, T136M, E116L, E116C and E116V, respectively. (B) 1. BcAmDH; 2. V293A; 3. T136G; 4. T136M; 5. E116L; 6. E116C; 7. E116V.
Fig.4 Relative activity of positive mutants obtained by iterative saturation mutagenesis a. BcAmDH; b. V293A; c. V293A/E116V; d. V293A/E116C; e. V293A/E116K; f. V293A/E116V/T136S; g. V293A/E116V/T136C.
Mutant | Acetophenone | NADH | ||||
---|---|---|---|---|---|---|
Km/ (mmol·L-1) | kcat/ min-1 | (kcat/Km)/ (L·min-1·mmol-1) | Km/ (mmol·L-1) | kcat/ min-1 | (kcat/Km)/ (L·min-1·mmol-1) | |
BcAmDH | 31.11±1.74 | 9.58±0.51 | 0.31 | 0.026±0.0025 | 4.13±0.32 | 158.85 |
V293A | 36.18±1.65 | 26.02±1.48 | 0.72 | 0.031±0.0023 | 8.77±0.76 | 282.90 |
V293A/E116V | 31.52±1.92 | 44.89±3.37 | 1.42 | 0.028±0.0034 | 16.09±0.97 | 574.64 |
V293A/E116V/T136S | 28.87±1.22 | 73.25±6.21 | 2.54 | 0.027±0.0031 | 21.14±1.31 | 782.96 |
Table 1 Kinetic parameters of BcAmDH and three mutants
Mutant | Acetophenone | NADH | ||||
---|---|---|---|---|---|---|
Km/ (mmol·L-1) | kcat/ min-1 | (kcat/Km)/ (L·min-1·mmol-1) | Km/ (mmol·L-1) | kcat/ min-1 | (kcat/Km)/ (L·min-1·mmol-1) | |
BcAmDH | 31.11±1.74 | 9.58±0.51 | 0.31 | 0.026±0.0025 | 4.13±0.32 | 158.85 |
V293A | 36.18±1.65 | 26.02±1.48 | 0.72 | 0.031±0.0023 | 8.77±0.76 | 282.90 |
V293A/E116V | 31.52±1.92 | 44.89±3.37 | 1.42 | 0.028±0.0034 | 16.09±0.97 | 574.64 |
V293A/E116V/T136S | 28.87±1.22 | 73.25±6.21 | 2.54 | 0.027±0.0031 | 21.14±1.31 | 782.96 |
Fig.5 Molecular docking results of BcAmDH and mutant V293A/E116V/T136S with acetophenone (A) Molecular docking results of BcAmDH and mutant V293A/E116V/T136S with acetophenone, 3D structures of BcAmDH and mutant V293A/E116V/T136S are shown as green and white surface, 116, 136 and 293 are the numbering of amino acid residues; (B) molecular docking results of BcAmDH and mutant V293A/E116V/T136S with acetophenone, 3D structures of BcAmDHand mutant V293A/E116V/T136S are shown as green and white cartoon, E116V, T136S and V293A refer to mutation results; molecular docking results of BcAmDH(C) and mutant V293A/E116V/T136S with acetophenon(D), showing the effect of V293A mutation near the substrate side chain phenyl group on the shape of the substrate binding pocket; molecular docking results of BcAmDH(E) and mutant V293A/E116V/T136S with acetophenon(F), showing the effect of E116V and T136S mutations at the substrate entry channel on the shape of the substrate binding pocket. The substrate acetophenone is shown as green sticks with BcAmDH and white sticks with mutant V293A/E116V/T136S.
Fig.6 Time course of asymmetric reduction of acetophenone with different concentrations by BcAmDH and mutant V293A/E116V/T136S a. 100 mmol/L Acetophenone with BcAmDH; b. 100 mmol/L acetophenone with V293A/E116V/T136S; c. 200 mmol/L acetophenone with V293A/E116V/T136S; d. 300 mmol/L acetophenone with V293A/E116V/T136S.
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