Modulation of the thermostability and substrate specificity of Candida rugosa lipase1 by altering the acyl-binding residue Gly414 at the α-helix-connecting bend

Zhang, Xiaofei; Zhang, Yong; Yang, Guangyu; Xie, Yuan; Xu, Lishi; An, Jiao; Cui, Li; Feng, Yan

doi:10.1016/j.enzmictec.2015.08.006

Cited by 14 publications

(12 citation statements)

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“…Lip1 is a mesophilic enzyme with an optimum temperature of 40 C, starts inactivation at 45 C, has a half-life of less than 1 hour at 50 C, and loses nearly all catalytic activity at 60 C. 7,8 Poor thermal stability prevents Lip1 from being applied in industrial processes where relatively high temperatures are required. 9 Literature surveys reveal that enhancing thermal stability of CRL via protein engineering is rarely reported. Recent strategies to stabilize Lip1 have focused on residues at the active center.…”

Section: Introductionmentioning

confidence: 99%

Identification of a hot-spot to enhanceCandida rugosalipase thermostability by rational design methods

Chen

Fang

et al. 2018

RSC Adv.

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Section: Introductionmentioning

confidence: 99%

Identification of a hot-spot to enhanceCandida rugosalipase thermostability by rational design methods

Chen

Fang

et al. 2018

RSC Adv.

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“…LIP1 is supposed to present in an equilibrium between its open and closed states in solution 21 22 . The optimal temperature of LIP1 is 45 °C, and it loses nearly all activity after 60 °C incubation for 10 min 23 24 . Those residues with high B-factor ranking located within 10 Å of the catalytic residue Ser209 were chosen as candidates for site-saturated mutagenesis and ordered recombination mutagenesis (ORM).…”

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confidence: 99%

A general and efficient strategy for generating the stable enzymes

Zhang

Yang

Zhang

et al. 2016

Sci Rep

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The local flexibility of an enzyme’s active center plays pivotal roles in catalysis, however, little is known about how the flexibility of these flexible residues affects stability. In this study, we proposed an active center stabilization (ACS) strategy to improve the kinetic thermostability of Candida rugosa lipase1. Based on the B-factor ranking at the region ~10 Å within the catalytic Ser209, 18 residues were selected for site-saturation mutagenesis. Based on three-tier high-throughput screening and ordered recombination mutagenesis, the mutant VarB3 (F344I/F434Y/F133Y/F121Y) was shown to be the most stable, with a 40-fold longer in half-life at 60 °C and a 12.7 °C higher Tm value than that of the wild type, without a decrease in catalytic activity. Further analysis of enzymes with different structural complexities revealed that focusing mutations on the flexible residues within around 10 Å of the catalytic residue might increase the success rate for enzyme stabilization. In summary, this study identifies a panel of flexible residues within the active center that affect enzyme stability. This finding not only provides clues regarding the molecular evolution of enzyme stability but also indicates that ACS is a general and efficient strategy for exploring the functional robustness of enzymes for industrial applications.

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“…Engineering proteins for substrate specificity is a particularly exciting and challenging field for industrial or academic research. 7 The X-ray structure of free Asp189Ser rat trypsin revealed that this single amino acid mutation at the bottom of the substrate-binding pocket resulted in extensive structural changes around the mutated site. 22 In the present study, the proteolysis activity of the enzyme was routinely determined using BQRR as the substrate.…”

Section: Discussionmentioning

confidence: 99%

“…For instance, the substrate specificity of a β‐glycosidase mutant was altered due to its electrostatic effect and extending the side chains, which affected substrate binding during catalysis . Similarly, G414W displayed a shift in substrate preference compared to Candida rugosa lipase 1 because of strong steric hindrance that blocked the entrance to the binding site . Such successful transformations of enzyme properties have laid the foundation for our experiment, as it is feasible that the substrate specificity of MBSP can be changed by gene modification.…”

Section: Introductionmentioning

confidence: 99%

Altering the substrate specificity of a myofibril‐bound serine proteinase from Crucian carp by site‐directed mutagenesis

Mei

et al. 2018

J of Chemical Tech & Biotech

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BACKGROUND Myofibril‐bound serine proteinase (MBSP) comprises a class of trypsin‐type serine proteinases that can specifically cleave the carboxyl side of peptide bonds of arginine or lysine residues. To obtain higher specificity MBSPs, mutants were designed based on the substrate‐binding pocket and constructed through site‐directed mutagenesis. RESULTS The wild‐type (WT) and mutants were expressed in Pichia pastoris, and its enzymatic properties indicated that the mutants D170S, D170T, D170K and D170T/G203A were not biologically active. However, S171A specifically cleaved arginine residues and G203A preferably hydrolyzed lysine residues. The secondary structures of mutants were approximately similar to that of the WT according to the results of circular dichroism spectroscopy. Additionally, molecular docking showed that substrates were able to combine with S171A within the critical areas through hydrophobic interaction, hydrogen bonds and van der Waals electrostatic force. CONCLUSION The structural stability of MBSP was consistent using amino acid substitution. Due to the interaction pattern of substrates and mutant enzyme changes, only S171A was selective in cutting Arg residues, which illustrates how the catalytic triad and substrate‐binding pocket of MBSP plays an important role during enzymatic hydrolysis of substrates. © 2018 Society of Chemical Industry

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Modulation of the thermostability and substrate specificity of Candida rugosa lipase1 by altering the acyl-binding residue Gly414 at the α-helix-connecting bend

Cited by 14 publications

References 50 publications

Identification of a hot-spot to enhanceCandida rugosalipase thermostability by rational design methods

Identification of a hot-spot to enhanceCandida rugosalipase thermostability by rational design methods

A general and efficient strategy for generating the stable enzymes

Altering the substrate specificity of a myofibril‐bound serine proteinase from Crucian carp by site‐directed mutagenesis

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