Enhanced catalytic site thermal stability of cold-adapted esterase EstK by a W208Y mutation

Boyineni, Jerusha; Kim, Junyoung; Kang, Beom Sik; Lee, ChangWoo; Jang, Sei‐Heon

doi:10.1016/j.bbapap.2014.03.009

Cited by 14 publications

(27 citation statements)

References 18 publications

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“…However, there are still some barriers to the use of esterases in industrial applications, such as low production yield, limited pH and thermal stability, and poor performance in organic solvents ( 5 , 6 ). For certain industrial applications, functional modification of the enzymes based on rational protein design is necessary to enhance their catalytic efficiency, substrate specificity, enantioselectivity, and thermostability ( 7 – 10 ). Better knowledge of their structure and function will facilitate rational design.…”

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confidence: 99%

Structural insights into the substrate specificity of two esterases from the thermophilic Rhizomucor miehei

Yang

Qin

Duan

et al. 2015

Journal of Lipid Research

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Two hormone-sensitive lipase (HSL) family esterases (RmEstA and RmEstB) from the thermophilic fungus Rhizomucor miehei, exhibiting distinct substrate specificity, have been recently reported to show great potential in industrial applications. In this study, the crystal structures of RmEstA and RmEstB were determined at 2.15 Å and 2.43 Å resolutions, respectively. The structures of RmEstA and RmEstB showed two distinctive domains, a catalytic domain and a cap domain, with the classical α/β-hydrolase fold. Catalytic triads consisting of residues Ser161, Asp262, and His292 in RmEstA, and Ser164, Asp261, and His291 in RmEstB were found in the respective canonical positions. Structural comparison of RmEstA and RmEstB revealed that their distinct substrate specificity might be attributed to their different substrate-binding pockets. The aromatic amino acids Phe222 and Trp92, located in the center of the substrate-binding pocket of RmEstB, blocked this pocket, thus narrowing its catalytic range for substrates (C2–C8). Two mutants (F222A and W92F in RmEstB) showing higher catalytic activity toward long-chain substrates further confirmed the hypothesized interference. This is the first report of HSL family esterase structures from filamentous fungi.jlr The information on structure-function relationships could open important avenues of exploration for further industrial applications of esterases.

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confidence: 99%

Structural insights into the substrate specificity of two esterases from the thermophilic Rhizomucor miehei

Yang

Qin

Duan

et al. 2015

Journal of Lipid Research

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“…These characteristics were similar to those of other reported cold-adapted esterases. For instance, a cold-adapted EstK cloned from Pseudomonas mandelii [ 18 – 20 ]. Phthalate ester hydrolase gene was identified from biofilms of a wastewater treatment plant shows activity at low temperatures [ 21 ].…”

Section: Discussionmentioning

confidence: 99%

A novel cold-adapted esterase from Enterobacter cloacae: Characterization and improvement of its activity and thermostability via the site of Tyr193Cys

Gao

Bandikari

et al. 2018

Microb Cell Fact

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BackgroundIn industries lipolytic reactions occur in insensitive conditions such as high temperature thus novel stout esterases with unique properties are attracts to the industrial application. Protein engineering is the tool to obtain desirable characters of enzymes. A novel esterase gene was isolated from South China Sea and subjected to a random mutagenesis and site directed mutagenesis for higher activity and thermo-stability compared to wild type.ResultsA novel esterase showed the highest hydrolytic activity against p-nitrophenyl acetate (pNPA, C2) and the optimal activity at 40 °C and pH 8.5. It was a cold-adapted enzyme and retained approximately 40% of its maximum activity at 0 °C. A mutant, with higher activity and thermo-stability was obtained by random mutagenesis. Kinetic analysis indicated that the mutant Val29Ala/Tyr193Cys shown 43.5% decrease in Km, 2.6-fold increase in Kcat, and 4.7-fold increase in Kcat/Km relative to the wild type. Single mutants V29A and Y193C were constructed and their kinetic parameters were measured. The results showed that the values of Km, Kcat, and Kcat/Km of V29A were similar to those of the wild type while Y193C showed 52.7% decrease in Km, 2.7-fold increase in Kcat, and 5.6-fold increase in Kcat/Km compared with the wild type. The 3-D structure and docking analysis revealed that the replacement of Tyr by Cys could enlarge the binding pocket. Moreover Y193C also showed a better thermo-stability for the reason its higher hydrophobicity and retained 67% relative activity after incubation for 3 h at 50 °C.ConclusionsThe superior quality of modified esterase suggested it has great potential application in extreme conditions and the mutational work recommended that important information for the study of esterase structure and function.Electronic supplementary materialThe online version of this article (10.1186/s12934-018-0885-z) contains supplementary material, which is available to authorized users.

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“…Multiple sequence alignment of cold-adapted enzymes with homologous mesophilic and thermophilic enzymes provides valuable insight into the rational design of mutations in cold-adapted enzymes, whereby the corresponding amino acids of thermophilic enzymes are considered for mutagenesis [46]. Mutation of tryptophan 208 in the active site wall of cold-adapted Pseudomonas mandelii esterase EstK to tyrosine, in which tyrosine is highly conserved in the corresponding position of hyperthermophilic esterases, conferred catalytic site thermal stability in EstK via a strengthened hydrogen bond [47]. However, the prediction of mutation sites and proper amino acid substitutions remains a challenge [46].…”

Section: Protein Engineering To Improve the Stability Of Cold-adaptedmentioning

confidence: 99%

Improving the Stability of Cold-Adapted Enzymes by Immobilization

2017

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Cold-adapted enzymes have gained considerable attention as biocatalysts that show high catalytic activity at low temperatures. However, the use of cold-adapted enzymes at ambient temperatures has been hindered by their low thermal stabilities caused by their inherent structural flexibilities. Accordingly, protein engineering and immobilization have been employed to improve the thermal stability of cold-adapted enzymes. Immobilization has been shown to increase the thermal stability of cold-adapted enzymes at the critical temperatures at which denaturation begins. This review summarizes progress in immobilization of cold-adapted enzymes as a strategy to improve their thermal and organic solvent stabilities.

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Enhanced catalytic site thermal stability of cold-adapted esterase EstK by a W208Y mutation

Cited by 14 publications

References 18 publications

Structural insights into the substrate specificity of two esterases from the thermophilic Rhizomucor miehei

Structural insights into the substrate specificity of two esterases from the thermophilic Rhizomucor miehei

A novel cold-adapted esterase from Enterobacter cloacae: Characterization and improvement of its activity and thermostability via the site of Tyr193Cys

Improving the Stability of Cold-Adapted Enzymes by Immobilization

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