ACS Synthetic Biology
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2024
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IF 3.7
Jialing Wang,Jie Chen,Kemin Lv,Zhen Gao,Jiahuang Li,Bin Wu,Bingfang He,Gerhard Schenk
Processive endoglucanases have generated significant interest due to their bifunctionality in the degradation of cellulose and low product inhibition. However, enhancing their catalytic efficiency through engineering remains a formidable challenge. To address this bottleneck, our engineering efforts targeted loop regions located in the substrate channel of processive endoglucanase EG5C-1. Guided by a comparative analysis of characteristic structural features of the substrate channels in cellobiohydrolase, endoglucanase, and processive endoglucanase, a highly active triple mutant CM6 (N105H/T205S/D233L) was generated that had a 5.1- and 4.7-fold increase in catalytic efficiency toward soluble substrate carboxymethyl cellulose-Na and insoluble substrate phosphoric acid-swollen cellulose (PASC), compared with wild-type EG5C-1. Furthermore, this mutant exhibited greater processivity compared to EG5C-1. Molecular dynamics simulations unveiled that the mutations in the loop regions reshaped the substrate channel, leading to a deeper cleft, resembling the closed channel configuration of cellobiohydrolases. The increased compactness of the substrate channel induced changes in the substrate binding mode and substrate deformation, thereby enhancing both binding affinity and catalytic efficiency. Moreover, metadynamics simulations demonstrated that the processive velocity of cellulose chain through the binding channel in mutant CM6 surpassed that observed in EG5C-1.
研究领域:
生物化学
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酶工程
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PubMed
高影响因子
NEW JOURNAL OF CHEMISTRY
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2023
·Vol. 47
·期 28
·13279-13285
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IF 3.3
Junling Guo,Zhongyi Cheng,Lukasz Peplowski,Zhemin Zhou
Isonicotinamide has important roles and application prospects in pharmaceutical, medical, and materials sciences. The biosynthesis of isonicotinamide has great advantages over chemical methods, but the biosynthesis of isonicotinamide has been reported for three decades and has not been industrialized yet. In this study, a thermophilic NHase from Carbonactinospora thermoautotrophicus was selected and then its activity was substantially improved by a “toolbox” screening strategy. The specific activity of the βL48D mutant towards 4-cyanopyridine was 307.02 ± 3.50 U mg−1, which was 13.9 times higher than that of the WT C.t NHase (22.01 ± 1.30 U mg−1). The structural analysis demonstrated that introduction of Asp into the βLeu48 site resulted in a larger catalytic cavity and better substrate orientations. Finally, a recombinant strain E. coli BL21 of the βL48D mutant was cultured at high cell density in a 5 L fermenter for high-level isonicotinamide production, developing a powerful process for isonicotinamide bio-production. The yield of isonicotinamide reached 2.89 M (352 g L−1), the substrate (4-cyanopyridine) was exhausted and the conversion was 100%. These results prove that C.t NHase is a promising competitor for future industrial production of biosynthetic isonicotinamide.
研究领域:
生物化学
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工业微生物学
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药物化学
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DOI
高影响因子
Scientific Reports
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2015
·Vol. 5
·期 1
·1-12
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IF 5.578
Yang Yang,Zhang Xinxin,Yin Qiang,Fang Wei,Fang Zemin,Wang Xiaotang,Zhang Xuecheng,Xiao Yazhong
β-Glucosidases are enzymes that hydrolyze β-glycosidic bonds to release non-reducing terminal glucosyl residues from glycosides and oligosaccharides and thus have significant application potential in industries. However, most β-glucosidases are feedback inhibited by the glucose product, which restricts their application. Remarkably, some β-glucosidases of the glycoside hydrolase (GH) 1 family are tolerant to or even stimulated by glucose. Elucidation of the mechanisms of glucose tolerance and stimulation of the GH1 β-glucosidases will be crucial to improve their application through enzyme engineering. In this study, by comparing the primary and tertiary structures of two GH1 β-glucosidases with distinct glucose dependence, some putative glucose-dependence relevant sites were mutated to investigate their exact roles. Both biochemical and structural characterization of the mutants suggested that some sites at the entrance and middle of the substrate channel regulate the effects of glucose and the relative binding affinity/preference of these sites to glucose modulates the glucose dependence. A mechanism was therefore proposed to interpret the glucose dependence of GH1 β-glucosidases. This research provides fresh insight into our current understanding of the properties and mechanisms of GH1 β-glycosidases and related enzymes that modulate their activity via feedback control mechanism.
研究领域:
生物化学
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酶学
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生物技术
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高影响因子
INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
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2022
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IF 8.025
Junling Guo,Julia Berdychowska,Qianpeng Lai,Yiwei Meng,Zhongyi Cheng,Lukasz Peplowski,Zhemin Zhou
Nitrile hydratase (NHase; EC 4.2.1.84) is widely used to synthesize the corresponding amides from nitriles, which is the most successful green biocatalyst. However, the limited acceptability of substrates and instability under harsh reaction conditions have hindered its widespread industrial application. Here, a gene encoding an extremophilic NHase from Streptomyces thermoautotrophicus ( S.t NHase) was successfully overexpressed in Escherichia coli . The enzyme exhibited excellent thermostability, retaining >50 % of residual activity after heat treatment at 65 °C for 252 min. To further improve the catalytic performance of S.t NHase, semi-rational engineering of its substrate access tunnel was performed. A mutant βL48D showed a specific activity of 566.18 ± 18.86 U/mg towards 3-cyanopyridine, which was 7.7 times higher than its parent enzyme (73.80 ± 5.76 U/mg). Molecular dynamics simulation showed that the introduction of aspartic acid into βLeu48 resulted in a larger and more frequent opening of the substrate access tunnel entrance. On this basis, a “toolbox” containing various mutants on the substrate access tunnel was further established, whose catalytic activity towards various nitrile substrates was extensively improved, showing great potential for efficient synthesis of multiple high-value amides.
研究领域:
生物催化
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酶工程
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工业生物技术
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蛋白质工程
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PubMed
高影响因子
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
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2024
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IF 2.5
He Liu,Qian Gao,Kaiyue Zhang,Meng Xu,Hualei Wang,Dongzhi Wei
( R )-selective transaminases have the potential to act as efficient biocatalysts for the synthesis of important pharmaceutical intermediates. However, their low catalytic efficiency and unfavorable equilibrium limit their industrial application. Seven ( R )-selective transaminases were identified using homologous sequence mining. Beginning with the optimal candidate from Mycolicibacterium hippocampi , virtual mutagenesis and substrate tunnel engineering were performed to improve catalytic efficiency. The obtained variant, T282S/Q137E, exhibited 3.68-fold greater catalytic efficiency ( k cat / K m ) than the wild-type enzyme . Using substrate fed-batch and air sweeping processes, effective conversion of 100 mM 4-hydroxy-2-butanone was achieved with a conversion rate of 93 % and an ee value > 99.9 %. This study provides a basis for mutation of ( R )-selective transaminases and offers an efficient biocatalytic process for the asymmetric synthesis of ( R )-3-aminobutanol.
研究领域:
生物化学
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酶工程
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合成生物学
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PubMed
高影响因子
CHINESE JOURNAL OF CATALYSIS
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2024
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IF 15.7
Zhenhua Wu,Jiafu Shi,Boyu Zhang,Yushuai Jiao,Xiangxuan Meng,Ziyi Chu,Yu Chen,Yiran Cheng,Zhongyi Jiang
ABSTRACT Enhancing catalytic activity of multi-enzyme in vitro through substrate channeling effect is promising yet challenging. Herein, conjugated microporous polymers (CMPs)-scaffolded integrated enzyme cascade systems (I-ECSs) are constructed through co-entrapping glucose oxidase (GOx) and horseradish peroxidase (HRP), in which hydrogen peroxide (H 2 O 2 ) is the intermediate product. The interplay of low-resistance mass transfer pathway and appropriate pore wall-H 2 O 2 interactions facilitates the directed transfer of H 2 O 2 , resulting in 2.4-fold and 5.0-fold elevation in catalytic activity compared to free ECSs and separated ECSs, respectively. The substrate channeling effect could be regulated by altering the mass ratio of GOx to HRP. Besides, I-ECSs demonstrate excellent stabilities in harsh environments and multiple recycling.
研究领域:
生物催化
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材料科学
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酶工程
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DOI
高影响因子
JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
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2024
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IF 5.7
Weijun Leng,Ying Li,Li Yuan,Xiuting Li,Ruichang Gao
Protein glutaminases (PG; EC = 3.5.1.44) are enzymes known for enhancing protein functionality. In this study, we cloned and expressed the gene chryb3 encoding protein glutaminase PG3, exhibiting 39.4 U/mg specific activity. Mature-PG3 featured a substrate channel surrounded by aromatic and hydrophobic amino acids at positions 38–45 and 78–84, with Val81 playing a pivotal role in substrate affinity. The dynamic opening and closing motions between Gly65, Thr66, and Cys164 at the catalytic cleft greatly influence substrate binding and product release. Redesigning catalytic pocket and cocatalytic region produced combinatorial mutant MT6 showing a 2.69-fold increase in specific activity and a 2.99-fold increase at t65 °C1/2. Furthermore, MT6 boosted fish myofibrillar protein (MP) solubility without NaCl. Key residues such as Thr3, Asn54, Val81, Tyr82, Asn107, and Ser108 were vital for PG3-myosin interaction, particularly Asn54 and Asn107. This study sheds light on the catalytic mechanism of PG3 and guided its rational engineering and utilization in low-salt fish MP product production.
研究领域:
生物化学
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酶学
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蛋白质工程
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食品科学
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DOI
PubMed
高影响因子
iScience
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2024
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IF 4.6
Jiawei Wang,Xingyu Ouyang,Shiyu Meng,Jiayi Li,Liangxu Liu,Chaofeng Li,Hengrun Li,Haotian Zheng,Chao Liao,Yi-Lei Zhao,Jun Ni
Lignin valorization is crucial for achieving economic and sustainable biorefinery processes. However, the enzyme substrate preferences involved in lignin degradation remain poorly understood, and low activity toward specific substrates presents a significant challenge to the efficient utilization of lignin. In this study, we investigated the substrate promiscuity of Th Ado, a key enzyme involved in lignin valorization. Pre-reaction state analysis revealed that a hydrogen bond network is critical in determining substrate selectivity. By performing targeted saturation mutagenesis on residues surrounding the substrate tunnels, we identified the Y205W and Y205Q mutants, which demonstrated 0.73-fold and 0.72-fold enhancements in activity, respectively. Structural analysis indicated that the redirection of the original substrate tunnel may be responsible for the improved activity. Our study provides essential insights into the substrate preference mechanisms of lignin degrading enzymes and suggests that this tunnel-redirection strategy can be extended to other promiscuous enzymes.
研究领域:
生物化学
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生物技术
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酶学
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可持续化学
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PubMed
高影响因子
ACS Applied Materials & Interfaces
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2021
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IF 9.229
Zhenfu Wang,Yang Liu,Xiaoyan Dong,Yan Sun
Chemoenzymatic catalysis combining the traits of chemical and enzymatic catalysis provides tremendous possibilities for the design of biosynthetic pathways utilizing inorganic catalysts and enzymes. However, the efficiency of chemoenzymatic catalysis is usually governed by the synergy and compatibility of the two catalysts. Here, we report for the first time the catalase-like activity of cobalt phosphate nanocrystals (CoPs). By a one-pot biomimetic mineralization with CoPs and l-amino acid oxidase (LAAO) under a mild condition, we have fabricated a hybrid nanobiocatalyst, LAAO@CoPs, for the chemoenzymatic synthesis of α-keto acid. The as-fabricated nanobiocatalyst with directly contacted catalytic sites of the enzyme and nanozyme maximizes the substrate channeling effects for in situ chemical decomposition of the oxidative intermediate, H2O2, during the enzymatic oxidation of l-tryptophan (l-Trp), thus minimizing the H2O2 accumulation and byproduct generation. Benefiting from the superiority of LAAO@CoPs, complete conversion (100.0%) of l-Trp to indole pyruvic acid is achieved, over two times higher than the yield of the free LAAO system (47.6%). Meanwhile, LAAO@CoPs show high stabilities against heat and proteolytic treatments. This work offers a new design approach for constructing a high-performance nanobiocatalyst for cascade reactions, especially for those systems with toxic or reactive intermediates.
研究领域:
纳米技术
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生物催化
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化学生物学
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酶学
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产品关联
DOI
PubMed
高影响因子
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