Molecular Catalysis
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2022
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IF 5.089
Jiacheng Zhang,Jieyu Zhou,Guochao Xu,Ye Ni
Kp ADH is a promising biocatalyst for the synthesis of chiral bulky-bulky alcohols. Substrate specificity toward diaryl ketone substrates with different substituents at phenyl group was investigated. The results reveal that the para -position of diaryl ketones plays important role in manipulating the stereoselectivity of Kp ADH. Two vital residues, E214 and T215, were identified through single-point mutagenesis, and saturation mutagenesis was performed to evaluate their contributions to the stereoselectivity. Furthermore, stereodivergent evolution of Kp ADH was achieved through combinatorial mutagenesis. Among them, E214I/T215S/S237A (ISA) and F161V/S196G/E214G (VGG) exhibited e.e. values of >99% ( S ) and 99% ( R ) toward [4-(trifluoromethyl)phenyl]-2-pyridinylmethanone ( 5a ), and e.e. values of >99% ( S ) and 98% ( R ) toward (4-methylphenyl)-2-pyridinylmethanone ( 6a ), respectively. Kinetic characterization and interaction analysis further prove that the eliminated side-chain collision and enhanced electrostatic interactions are mainly responsible for the increased catalytic efficiency and stereoselectivity. This study provides beneficial mutants with complementary stereoselectivity toward diaryl ketones with para -substituents and also provides guidance for the engineering of homologous alcohol dehydrogenases toward bulky-bulky ketones.
研究领域:
生物化学
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酶工程
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高影响因子
ACS Catalysis
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2018
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IF 11.384
Guo-Chao Xu,Yue Wang,Ming-Hui Tang,Jie-Yu Zhou,Jing Zhao,Rui-Zhi Han,Ye Ni
A hydroclassified combinatorial saturation mutagenesis (HCSM) strategy was proposed for reshaping the substrate binding pocket by dividing 20 amino acids into four groups based on their hydrophobicity and size. These smart HCSM libraries could significantly reduce screening effort especially for the simultaneous mutagenesis of three or more residues and lacking high throughput screening methods. Employing HCSM strategy, the stereoselectivity of KpADH, an alcohol dehydrogenase from Kluyveromyces polysporus, was efficiently improved to 99.4% ee. (4-Chlorophenyl)(pyridin-2-yl)methanone (CPMK), generally regarded as a “hard-to-reduce” ketone, was used as a model substrate, and its corresponding chiral alcohol products could be utilized as antihistamine precursors. The best variant 50C10 displayed higher binding affinity and catalytic efficiency toward CPMK with KM/kcat of 59.3 s–1·mM–1, 3.51-fold that of KpADH. Based on MD simulations, increased difference between two binding pockets, enhanced hydrophobicity, and π–π and halogen–alkyl interactions were proposed to favor the enantioselective recognition and substrate binding in 50C10. Substrate spectrum analysis revealed that 50C10 exhibited improved enantioselectivity toward diaryl ketones especially with halo- or other electron-withdrawing groups. As much as 500 mM CPMK could be asymmetrically reduced into chiral diaryl alcohols with ee of 99.4% and a space–time yield of 194 g·L–1·d–1 without addition of external NADP+. This study provides an effective mutagenesis strategy for the protein engineering of substrate specificity and enantioselectivity.
研究领域:
蛋白质工程
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生物催化
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酶催化
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手性合成
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药物前体
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分子动力学模拟
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生物化学
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高影响因子
Processes
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2025
·Vol. 14
·期 1
·55
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IF 2.8
Bao Fan,Jiale Xiong,Cuiluan Ma,Yu-Cai He
Gastrodin is a bioactive component of traditional Chinese medicine, exhibiting anti-cancer, anti-inflammatory, antioxidant and neuroprotective properties. It has broad application prospects in health foods, pharmaceuticals and cosmetics. In recent years, the conversion of biomass-derived aldehydes into high-value-added chemicals has garnered widespread attention. In this study, gastrodin was biosynthesized via a dual-enzyme coupling system consisting of UGTBL1-Δ60 and KpADH. Specifically, lignin-derivedp-hydroxybenzaldehyde was used as the substrate. First, the glycosylation ofp-hydroxybenzaldehyde by UGTBL1-Δ60 yieldedp-hydroxybenzaldehydeβ-glucoside, generating the glycosylation reaction solution. Subsequently, bioreduction of the glycosylation product by KpADH produced gastrodin. Under the optimal reaction conditions (0.05 g/mL KpADH whole cells, 50 mM glucose, pH 7.5 and 30 °C) a gastrodin yield of 82.8% was achieved within 12 h. Moreover, both UGTBL1-Δ60 and KpADH retained high catalytic activity after multiple reaction cycles. This study establishes a green and efficient biocatalytic approach for gastrodin synthesis, and also provides new insights into the high-value utilization of lignin.
研究领域:
生物催化
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合成生物学
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生物化工
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可再生资源
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天然产物化学
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高影响因子
Molecular Catalysis
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2025
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IF 4.9
Bo Fan,Run Lin,Xinyu Gu,Ruibing Cui,Ruiqing Yang,Yu-Cai He
Alcohols can be extensively used in the production of fine chemicals, such as fuel additives, food supplements and pharmaceutical intermediates, which can be acquired by the reduction of biomass-derived aldehyde. In this study, the recombinant Escherichia coli KPADH was genetically constructed through the co-expression of alcohol dehydrogenase from Kluyveromyces polysporus and glucose dehydrogenase from Bacillus subtilis . Biomass-derived aldehydes were biologically converted into alcohols in a benign system. In deep eutectic solvent (DES) choline chloride:glycerol-H 2 O (choline chloride:glycerol, 10 wt %) containing E. coli KPADH cells (0.1 g/mL), HMF (50 mM), glucose (2 equivalents), and NAD + (0.1 μM/mM HMF), bioreduction of HMF into 2,5-bis(hydroxymethyl)furan was implemented, acquiring a high yield of 98.1 % within 8 h. Other biobased compounds, such as isovanillin, vanillin, p -hydroxybenzaldehyde, furfural, and benzaldehyde were also converted into their corresponding alcohols. According to the molecular docking results, HMF had the lowest CDOCKER energy value of -27.422 kcal/mol. It was found that HMF displayed strong binding affinity with E. coli KPADH, resulting in enhanced binding stability and superior catalytic reduction activity. This study demonstrated a green and highly effective biocatalytic method for the synthesis of biobased alcohols.
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高影响因子
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