Catalysis Science & Technology
·
2023
·Vol. 13
·期 20
·5880-5891
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IF 5
Jinling Xu,Haisheng Zhou,Jiaqi Xu,Ziyuan Wang,Zhonglang Yu,Zhe Wang,Hongyu Zhang,Haoran Yu,Jianping Wu,Lirong Yang
Nitrile hydratase (NHase), an excellent biocatalyst, has been widely used for the production of amides, but the exothermic hydration reaction leads to its rapid inactivation, hindering its industrial applications, which requires the thermostability of NHase to be enhanced. In this study, employing NHase from Bordetella petrii DSM 12804 (NHAB) as the object, a computational strategy using synergetic energy and correlated configuration for redesigning enzymes (SECURE) was proposed to prune the reasonable mutant library and assemble effective single mutations, thus maximizing the thermostability of NHAB. Among the mutants, the best variant, A6M/B4M, combined six mutations in its α-subunit (S30T, A71D, A74D, A78R, S81T, and A133P) and four mutations in its β-subunit (L25F, G27Y, N59P, and A173N), showing an increase in Tm by 13.2 °C, an 866.0-fold prolonged half-life at 50 °C, and an 11.2% increase in activity. Then, the catalytic efficiency dramatically increased to 249.5 g L−1 acrylamide in 5 batches compared with that of 166.5 g L−1 by the wild type in 3 batches. Finally, the synergistic effect on the mutant represented by an overall change in structure and newly formed intermolecular interactions through dynamic simulations accounted for the enhanced thermostability. Thus, SECURE was demonstrated to be practical for the redesign of multimeric NHase, which also provided guidance for computational thermostability engineering of other industrial biocatalysts.
研究领域:
生物化学
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生物技术
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计算生物学
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酶学
✓ 已验证
产品关联
DOI
高影响因子
BIOPROCESS AND BIOSYSTEMS ENGINEERING
·
2023
·
IF 3.8
Jia Dong-Xu,Yu Hai,Wang Fan,Jin Li-Qun,Liu Zhi-Qiang,Zheng Yu-Guo
Cellobiose 2-epimerase (CE) is ideally suited to synthesize lactulose from lactose, but the poor thermostability and catalytic efficiency restrict enzymatic application. Herein, a non-characterized CE originating from Caldicellulosiruptor morganii (CmCE) was discovered in the NCBI database. Then, a smart mutation library was constructed based on FoldX ΔΔG calculation and modeling structure analysis, from which a positive mutant D226G located within the α 8 /α 9 loop exhibited longer half-lives at 65–75 °C as well as lower K m and higher k cat / K m values compared with CmCE. Molecular modeling demonstrated that the improvement of D226G was largely attributed to the rigidification of the flexible loop, the compactness of the catalysis pocket and the increment of substrate-binding capability. Finally, the yield of synthesizing lactulose catalyzed by D226G reached 45.5%, higher than the 35.9% achieved with CmCE. The disclosed effect of the flexible loop on enzymatic stability and catalysis provides insight to redesign efficient CEs to biosynthesize lactulose. Graphical abstract
研究领域:
生物技术
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酶学
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分子生物学
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化学工程
✓ 已验证
产品关联
DOI
PubMed
高影响因子
INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
·
2021
·
IF 6.953
Anni Li,Cheng Cheng,Weimin Qi,Xin Pan,Xuanping Xu,Xinyu Wang,Chunying Wu,Jianlin Chu,Bingfang He
Penicillin G acylase (PGA) was an important biocatalyst for enzymatic production of second-generation cephalosporin. PGA from Achromobacter xylosoxidans PX02 ( Ax PGA) showed relatively lower identity to Ec PGA (54.9% in α subunit and 51.7% in β subunit), which could synthesize cefamandole in the kinetically controlled N -acylation ( kcNa ). Semi-rational design of Ax PGA and “small and smart” mutant libraries were developed with minimal screening to improve cefamandole production. A triple mutant αR141A/αF142I/βF24G by combining the mutational sites (βF24, αR141, and αF142) from different subunits of Ax PGA showed better performance in cefamandole production, with 4.2-fold of improvement in the (k cat /K m ) AD value for activated acyl donor ( R )-Methyl mandelate. Meanwhile, the (k cat /K m ) Ps value for cefamandole by mutant αR141A/αF142I/βF24G was sharply dropped by 25.5 times, indicating its highly synthetic activity and extremely low hydrolysis of cefamandole. Strikingly, the triple mutant αR141A/αF142I/βF24G could form cefamandole with a yield of 85% at an economical substrate ratio (acyl donor/nucleophile) of 1.3:1 (82% at 1.1:1), which advanced the greener and more sustainable process of cefamandole production than the wild type. Furtherly, the improved synthetic ability and lower hydrolysis of cefamandole by mutant were rationalized using molecular docking.
研究领域:
生物化学
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生物技术
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酶学
✓ 已验证
DOI
PubMed
高影响因子
BIOORGANIC CHEMISTRY
·
2022
·
IF 5.307
Hua-Tao Liu,Chun-Yue Weng,Shen-Yuan Xu,Shu-Fang Li,Ya-Jun Wang,Yu-Guo Zheng
Traditional screening methods of enzyme engineering often require building large mutant libraries to screen for potentially beneficial sites, which are often time-consuming and labor-intensive with low mining efficiency. In this study, a novel enzyme engineering strategy was established to modify carbonyl reductase Ls CR for the synthesis of (1 S )-2-chloro-1-(3,4-difluorophenyl) ethanol (( S )-CFPL), which is a key intermediate of anticoagulant drug ticagrelor. The strategy was developed by combining HotSpot, FireProt and multiple sequence alignment, resulting in the construction of a “small and smart” mutant library including 10 mutations. Among them, 5 mutations were positive, resulting in a 50% mining accuracy of beneficial sites. Finally, a highly active mutant Ls CR M3 (N101D/A117G/F147L) was obtained by further screening through saturation mutation and iterative mutation. Compared with wild type (WT) Ls CR, the catalytic activity of Ls CR M3 was increased by 4.7 times, the catalytic efficiency k cat / K M value was increased by 2.9 times, and the half-life t 1/2 at 40 °C was increased by 1.3 times. Due to the low aqueous solubility of the substrate 2-chloro-1-(3,4-difluorophenyl) ethanone (CFPO), isopropanol was used as not only the co-substrate but also co-solvent. In the presence of 40% ( v / v ) isopropanol, Ls CR M3 completely reduced 400 g/L CFPO to enantiomerically pure CFPL (99.9%, e.e. ) in 11 h with a space–time yield (STY) as high as 809 g/L∙d.
研究领域:
酶工程
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生物催化
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化学生物学
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药物化学
✓ 已验证
DOI
PubMed
高影响因子
STAR Protocols
·
2024
·
IF 1.3
Shihao Song,Binbin Cui,Xiaohan Kong,Yinyue Deng
Summary Here, we present a protocol for identifying the components of a quorum sensing signaling system in bacteria. We describe the steps for characterizing the novel response regulator and receptor of the cis -2-dodecenoic acid (BDSF) quorum sensing signaling system in Burkholderia cenocepacia . The technical assays of this protocol include generating a random mutant library, chromatin immunoprecipitation sequencing (ChIP-seq), electrophoretic mobility transfer assay (EMSA), microscale thermophoresis (MST), and molecular simulation docking. For complete details on the use and execution of this protocol, please refer to Li et al. 1 and Yang et al. 2
研究领域:
微生物学
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分子生物学
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遗传学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
危险品化学品经营许可证（带存储）