A Dual‐Focus Workflow for Simultaneously Engineering High Activity and Thermal Stability in Methyl Parathion Hydrolase

Angewandte Chemie, Journal Year: 2024, Volume and Issue: 136(46)

Published: Aug. 10, 2024

Abstract Industrial fermentation applications typically require enzymes that exhibit high stability and activity at temperatures. However, efforts to simultaneously improve these properties are usually limited by a trade‐off between activity. This report describes computational strategy enhance both thermal of the mesophilic organophosphate‐degrading enzyme, methyl parathion hydrolase (MPH). To predict hotspot mutation sites, we assembled library features associated with target for each residue then prioritized candidate sites hierarchical clustering. Subsequent in silico screening multiple algorithms simulate selective pressures yielded subset 23 mutations. Iterative parallel mutations improved yielded, MPHase‐m5b, which exhibited 13.3 °C higher T m 4.2 times catalytic than wild‐type (WT) MPH over wide temperature range. Systematic analysis crystal structures, molecular dynamics (MD) simulations, quantum mechanics/molecular mechanics (QM/MM) calculations revealed wider entrance active site increased substrate access an extensive network interactions outside reinforced αβ/βα sandwich architecture stability. study thus provides advanced, rational design framework efficiency engineering highly active, thermostable biocatalysts industrial applications.

Language: Английский

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Biosynthesis of Methyl (2S,3R)-2-[(Benzoylamino)methyl]-3-hydroxybutanoate in High Space-Time Yield with Immobilized Engineered Carbonyl Reductase

ACS Sustainable Chemistry & Engineering, Journal Year: 2025, Volume and Issue: unknown

Published: Jan. 9, 2025

4-Acetoxy-azacyclobutanone (4AA) is a highly demanded chemical compound used in the production of Penem and Carbapenem antibiotics. However, its synthesis constrained by preparation methyl (2S,3R)-2-[(benzoylamino)methyl]-3-hydroxybutanoate [(2S,3R)-BHME]. In light stringent environmental regulations, there an urgent need to develop effective enzymatic method using 2-benzoylaminomethyl-3-oxy-butyrate ester (BOME) as substrate. This study mined carbonyl reductase AxSDR from Algoriella xinjiangensis, which asymmetrically reduces BOME (2S,3R)-BHME isopropanol (IPA) cosubstrate. The mechanisms underlying high stereoselectivity, substrate selectivity, limited activity toward were analyzed computer-aided technology. Based on these analyses, was rationally designed, leading identification triple-point variant, G94T/H145Y/Y188L (Mu3), exhibited 2-fold increase catalytic efficiency. After condition optimization, Mu3 cells able convert 300 mM BOME, achieving space-time yield 15.1 g/L/h. sustainability biosynthesis further enhanced immobilizing IPA-tolerant amino resin. immobilized enzyme Mu3-imm increased 75.3 g/(L·h) maintained at 50.2 after 100 uses. These results demonstrate significant industrial application potential reducing costs risks associated with downstream products such 4-AA, Penem,

Language: Английский

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Advances in aldo-keto reductases immobilization for biocatalytic synthesis of chiral alcohol

International Journal of Biological Macromolecules, Journal Year: 2024, Volume and Issue: 274, P. 133264 - 133264

Published: June 18, 2024

Language: Английский

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A Dual‐Focus Workflow for Simultaneously Engineering High Activity and Thermal Stability in Methyl Parathion Hydrolase

Angewandte Chemie International Edition, Journal Year: 2024, Volume and Issue: 63(46)

Published: Aug. 10, 2024

Industrial fermentation applications typically require enzymes that exhibit high stability and activity at temperatures. However, efforts to simultaneously improve these properties are usually limited by a trade-off between activity. This report describes computational strategy enhance both thermal of the mesophilic organophosphate-degrading enzyme, methyl parathion hydrolase (MPH). To predict hotspot mutation sites, we assembled library features associated with target for each residue then prioritized candidate sites hierarchical clustering. Subsequent in silico screening multiple algorithms simulate selective pressures yielded subset 23 mutations. Iterative parallel mutations improved yielded, MPHase-m5b, which exhibited 13.3 °C higher T

Language: Английский

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Directly mining and identifying a cellobiose 2-epimerase with isomerization activity by phylogenetic analysis

Food Bioscience, Journal Year: 2024, Volume and Issue: 62, P. 105232 - 105232

Published: Oct. 2, 2024

Language: Английский

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Reversing the Enantioselectivity of Enzymatic Dynamic Kinetic Asymmetric Transformations in the Synthesis of Fused Lactones

ACS Catalysis, Journal Year: 2024, Volume and Issue: unknown, P. 17480 - 17488

Published: Nov. 12, 2024

The rational design of one ketoreductase into stereocomplementary variants for controlling the stereoselectivity bulky chiral molecules bearing contiguous stereocenters is highly desirable and challenging. Herein, we report protein engineering from Chryseobacterium sp. CA49 (ChKRED20) through targeted mutagenesis only two key residues (Y188 H145) located in enzyme pocket, achieving precise stereocontrol over synthesis tricyclic fused lactones (highest reversing enantioselectivity >99:1 e.r. to <1:99 e.r.). Notably, both kinetic resolution asymmetric reduction (KR-AR) dynamic transformation (DyKAT) were observed this system. In KR-AR process, ChKRED20 exclusively convert (R)- or (S)-keto esters corresponding enantio- diastereoenriched (R,S)- (S,R)-cis-lactones deliver leftover (S)- (R)-keto esters. On contrary, DyKAT unreactive configurations substrates undergo efficient equilibration via an enolization protonation–deprotonation enzymes. Computational studies are also conducted get insight origin enantioselectivity.

Language: Английский

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From discovery to application: Enabling technology-based optimizing carbonyl reductases biocatalysis for active pharmaceutical ingredient synthesis

Biotechnology Advances, Journal Year: 2024, Volume and Issue: unknown, P. 108496 - 108496

Published: Dec. 1, 2024

Language: Английский

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Hot off the Press

Natural Product Reports, Journal Year: 2024, Volume and Issue: 41(6), P. 868 - 872

Published: Jan. 1, 2024

A personal selection of 32 recent papers is presented covering various aspects current developments in bioorganic chemistry and novel natural products, such as penihemeroterpenoid from Penicillium herquei .

Language: Английский

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Rational mutagenesis of an epoxide hydrolase and its structural mechanism for the enantioselectivity improvement toward chiral ortho-fluorostyrene oxide

International Journal of Biological Macromolecules, Journal Year: 2024, Volume and Issue: unknown, P. 136864 - 136864

Published: Oct. 1, 2024

Language: Английский

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