Using Molecular Simulation to Guide Protein Engineering for Biocatalysis in Organic Solvents

Cui, Haiyang; Vedder, Markus; Schwaneberg, Ulrich; Davari, Mehdi D.

doi:10.1007/978-1-0716-1826-4_10

Cited by 8 publications

(7 citation statements)

References 103 publications

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“…Integrating the experimental results of enzymatic activity profiles and the comprehensive computational studies, we found the reduced specific activity of all three enzymes (BSLA, CBHI, and EG) mainly resulted from three factors: (i) increased structural compactness, (ii) overall water stripping, and (iii) competitive inhibition by glycerol in the substrate binding site. Previous studies also confirmed such factors played essential roles in modifying the solvent resistance and enzyme activity in nonconventional media. ,, …”

Section: Resultsmentioning

confidence: 59%

“…Previous studies also confirmed such factors played essential roles in modifying the solvent resistance and enzyme activity in nonconventional media. 43,58,77 As shown in Figure 1, the phenomena of decrease (i.e., BSLA), activation (i.e., CBHI), and neutralization (i.e., EG) were all detected by the experimental stability performance studies as incubation time changes. Our MD simulation studies also demonstrated that learning the role of glycerol in modifying the stability of enzymes is a pretty complicated task.…”

Section: The Overall Structural Change Of Enzymes In Various Concentr...mentioning

confidence: 90%

“…Although these studies have made significant progress in the mechanistic understanding of protein–protectant systems, the inconsequential statements and reasoning indicated that further studies were still required. Also, more and more studies proved the importance of quantitative experimental evidence to compare with theoretical and molecular dynamics (MD) simulation results. − …”

Section: Introductionmentioning

confidence: 99%

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The Role of Glycerol in Preserving Proteins Needs to Be Reconsidered

Wang

Sheng

Cui

et al. 2022

ACS Sustainable Chem. Eng.

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The safe and efficient stabilization and preservation of enzymes, therapeutic proteins, and biomaterials are increasingly important in biology, medicine, and pharmaceutics. Various protectants have been explored, but their protective actions on protein structures remain unclear. Herein, we present an all-around protectant–protein interaction landscape by investigating the behaviors of Bacillus subtilis lipase A (BSLA), cellobiohydrolase I from Trichoderma reesei (CBHI), and endoglucanase from Penicillium Verruculosum (EG) in various concentrations of glycerol. Surprisingly, decreased, neutralization, and activation effects were observed for three industrial enzymes during the long-term storage examination, respectively, demonstrating that a universal condition for protein preservation might not exist. Alignment of the experimental catalytic activity profiles and computational molecular dynamics simulation reveals that (1) the overall structure of enzymes in glycerol remains stable; (2) specific activity reduction mainly results from three factors: (a) increased structural compactness, (b) overall water stripping, and (c) competitive inhibition by glycerol in the substrate binding site; (3) H-bond interactions are the main driving force that governs the structural dynamics, water stripping, and glycerol accumulation in glycerol cosolvents. Also, these gained insights are most likely to be transferred to other polyol additive systems for rationally stabilizing and preserving biomaterials in structural biology, biocatalysis, and biotransformation fields.

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Section: Resultsmentioning

confidence: 59%

Section: The Overall Structural Change Of Enzymes In Various Concentr...mentioning

confidence: 90%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

The Role of Glycerol in Preserving Proteins Needs to Be Reconsidered

Wang

Sheng

Cui

et al. 2022

ACS Sustainable Chem. Eng.

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“…Enhancing enzyme thermal stability employs varied methods such as media engineering, chemical modification, protein engineering, and immobilization . Protein engineering, especially computer-aided rational design, garners attention .…”

Section: Introductionmentioning

confidence: 99%

Enhancing the Thermal Stability and Enzyme Activity of Ketopantoate Hydroxymethyltransferase through Interface Modification Engineering

Cai,

Shi,

Wang

et al. 2024

J. Agric. Food Chem.

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Ketopantoate hydroxymethyltransferase (KPHMT) plays a pivotal role in D-pantothenic acid biosynthesis. Most KPHMTs are homodecamers with low thermal stability, posing challenges for protein engineering and limiting output enhancement. Previously, a high-enzyme activity KPHMT mutant (K25A/E189S) from Corynebacterium glutamicum was screened as mother strain (M0). Building upon this strain, our study focused on interface engineering modifications, employing a multifaceted approach including integrating folding-free energy calculation, B-factor analysis, and conserved site analysis. Preliminary screening led to the selection of five mutants in the interface�E106S, E98T, E98N, S247I, and S247D�showing improved thermal stability, culminating in the double-site mutant M8 (M0-E98N/S247D). M8 exhibited a T 1/2 value of 288.79 min at 50 °C, showing a 3.29fold increase compared to M0. Meanwhile, the T m value of M8 was elevated from 53.2 to 59.6 °C. Investigations of structural and molecular dynamics simulations revealed alterations in surface electrostatic charge distribution and the formation of increased hydrogen bonds between subunits, contributing to enhanced thermal stability. This investigation corroborates the efficacy of interface engineering modifications in bolstering KPHMT stability while showing its potential for positively impacting industrial Dpantothenic acid synthesis.

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“…subtilis Lipase A (also known as Lipase A and LipA) is one of the smallest known lipases, consisting of just 181 amino acid residues . LipA, with an α–β hydrolase fold (Figure A), has been a subject of many experimental and computational studies with an aim to enhance its thermostability, − catalytic activity, − and solvent resistance. − The catalytic triad consists of Ser77, Asp133, and His156. It is characterized as a lidless lipase because of the lack of interfacial activation (for the triacetyl glycerol substrate) and the absence of any conventional lid structure (usually a loop or α-helix covering the active site) in the crystal structures (CS). , However, exchanging the loop Ala39-Gly52 with an existing lid sequence has been reported to reduce the catalytic activity of LipA in an aqueous solution, which raised the question if this particular loop can be considered as a lid for LipA .…”

Section: Introductionmentioning

confidence: 99%