Dataset Information

Significantly Enhanced Synthesis of Aromatic Esters of Arbutin Catalyzed by Immobilized Lipase in Co-solvent Systems.

ABSTRACT: Highly efficient and regioselective synthesis of pharmacologically interesting aromatic esters of arbutin catalyzed by immobilized lipase from Penicillium expansum in co-solvent systems was successfully carried out. As compared to tetrahydrofuran solvent, the initial rate and substrate conversion of arbutin vanilylation were markedly enhanced in tetrahydrofuran-isopropyl ether (20%, v/v). Moreover, the effects of three reaction parameters (enzyme amount, temperature and substrate molar ratio of vinyl vanillic acid to arbutin) on 6'-O-vanilloyl-arbutin synthesis were scrutinized and the key process parameters were optimized using response surface methodology (RSM). The experimental data were fitted well to a second order polynomial model by using multiple regression analysis. The best combination of variables was 50°C, 93 U/mL and 11 for the reaction temperature, the enzyme amount and mole ratio of arbutin to vinyl vanilic acid, respectively, and which the reaction rate, substrate conversion and regioselectivity were as high as 8.2 mM/h, 93 and 99%. It was worth noting that a variety of aromatic esters of arbutin were obtained with much higher conversion (93-99%) at these optimal conditions.

SUBMITTER: Yang R

PROVIDER: S-EPMC7180213 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Significantly Enhanced Synthesis of Aromatic Esters of Arbutin Catalyzed by Immobilized Lipase in Co-solvent Systems.

Yang Rongling R Nie Zekun Z Xu Ningning N Zhao Xiangjie X Wang Zhaoyu Z Luo Hongzhen H

Frontiers in bioengineering and biotechnology 20200417

Highly efficient and regioselective synthesis of pharmacologically interesting aromatic esters of arbutin catalyzed by immobilized lipase from <i>Penicillium expansum</i> in co-solvent systems was successfully carried out. As compared to tetrahydrofuran solvent, the initial rate and substrate conversion of arbutin vanilylation were markedly enhanced in tetrahydrofuran-isopropyl ether (20%, v/v). Moreover, the effects of three reaction parameters (enzyme amount, temperature and substrate molar ra ...[more]

PMID: 32363180

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Project description:An enhanced stability of enzymes in organic solvents is desirable under industrial conditions. The potential of lipases as biocatalysts is mainly limited by their denaturation in polar alcohols. In this study, we focused on selected solvent tunnels in lipase from Geobacillus stearothermophilus T6 to improve its stability in methanol during biodiesel synthesis. Using rational mutagenesis, bulky aromatic residues were incorporated to occupy solvent channels and induce aromatic interactions leading to a better inner core packing. The chemical and structural characteristics of each solvent tunnel were systematically analyzed. Selected residues were replaced with Phe, Tyr, or Trp. Overall, 16 mutants were generated and screened in 60% methanol, from which 3 variants showed an enhanced stability up to 81-fold compared with that of the wild type. All stabilizing mutations were found in the longest tunnel detected in the "closed-lid" X-ray structure. The combination of Phe substitutions in an A187F/L360F double mutant resulted in an increase in unfolding temperature (Tm ) of 7°C in methanol and a 3-fold increase in biodiesel synthesis yield from waste chicken oil. A kinetic analysis with p-nitrophenyl laurate revealed that all mutants displayed lower hydrolysis rates (k cat), though their stability properties mostly determined the transesterification capability. Seven crystal structures of different variants were solved, disclosing new ?-? or CH/? intramolecular interactions and emphasizing the significance of aromatic interactions for improved solvent stability. This rational approach could be implemented for the stabilization of other enzymes in organic solvents.IMPORTANCE Enzymatic synthesis in organic solvents holds increasing industrial opportunities in many fields; however, one major obstacle is the limited stability of biocatalysts in such a denaturing environment. Aromatic interactions play a major role in protein folding and stability, and we were inspired by this to redesign enzyme voids. The rational protein engineering of solvent tunnels of lipase from Geobacillus stearothermophilus is presented here, offering a promising approach to introduce new aromatic interactions within the enzyme core. We discovered that longer tunnels leading from the surface to the enzyme active site were more beneficial targets for mutagenesis for improving lipase stability in methanol during biodiesel biosynthesis. A structural analysis of the variants confirmed the generation of new interactions involving aromatic residues. This work provides insights into stability-driven enzyme design by targeting the solvent channel void.

Dataset Information

Significantly Enhanced Synthesis of Aromatic Esters of Arbutin Catalyzed by Immobilized Lipase in Co-solvent Systems.

Publications

Significantly Enhanced Synthesis of Aromatic Esters of Arbutin Catalyzed by Immobilized Lipase in Co-solvent Systems.

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