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Improving activity and enantioselectivity of lipase via immobilization on macroporous resin for resolution of racemic 1- phenylethanol in non-aqueous medium.

ABSTRACT: BACKGROUND: Burkholderia cepacia lipase (BCL) has been proved to be capable of resolution reactions. However, its free form usually exhibits low stability, bad resistance and no reusability, which restrict its further industrial applications. Therefore, it is of great importance to improve the catalytic performance of free lipase in non-aqueous medium. RESULTS: In this work, macroporous resin NKA (MPR-NKA) was utilized as support for lipase immobilization. Racemic transesterification of 1-phenylethanol with vinyl acetate was chosen as model reaction. Compared with its free form, the enzyme activity and enantioselectivity (ees) of the immobilized lipase have been significantly enhanced. The immobilized BCL exhibited a satisfactory thermostability over a wide range of temperature (from 10 to 65°C) and an excellent catalytic efficiency. After being used for more than 30 successive batches, the immobilized lipase still kept most of its activity. In comparison with other immobilized lipases, the immobilized BCL also exhibits better catalytic efficiency, which indicates a significant potential in industrial applications. CONCLUSION: The results of this study have proved that MPR-NKA was an excellent support for immobilization of lipase via the methods of N2 adsorption-desorption, scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS) and Fourier transform-infrared spectroscopy (FT-IR). The improvement of enzyme activity and ees for the immobilized lipase was closely correlated with the alteration of its secondary structure. This information may contribute to a better understanding of the mechanism of immobilization and enzymatic biotransformation in non-aqueous medium.

SUBMITTER: Li X

PROVIDER: S-EPMC4228463 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Improving activity and enantioselectivity of lipase via immobilization on macroporous resin for resolution of racemic 1- phenylethanol in non-aqueous medium.

Li Xiang X Huang Shuangshuang S Xu Li L Yan Yunjun Y

BMC biotechnology 20131029

<h4>Background</h4>Burkholderia cepacia lipase (BCL) has been proved to be capable of resolution reactions. However, its free form usually exhibits low stability, bad resistance and no reusability, which restrict its further industrial applications. Therefore, it is of great importance to improve the catalytic performance of free lipase in non-aqueous medium.<h4>Results</h4>In this work, macroporous resin NKA (MPR-NKA) was utilized as support for lipase immobilization. Racemic transesterificatio ...[more]

PMID: 24168516

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Project description:The NADPH-dependent (S)-carbonyl reductaseII from Candida parapsilosis catalyzes acetophenone to chiral phenylethanol in a very low yield of 3.2%. Site-directed mutagenesis was used to design two mutants Ala220Asp and Glu228Ser, inside or adjacent to the substrate-binding pocket. Both mutations caused a significant enantioselectivity shift toward (R)-phenylethanol in the reduction of acetophenone. The variant E228S produced (R)-phenylethanol with an optical purity above 99%, in 80.2% yield. The E228S mutation resulted in a 4.6-fold decrease in the K M value, but nearly 5-fold and 21-fold increases in the k cat and k cat/K M values with respect to the wild type. For NADPH regeneration, Bacillus sp. YX-1 glucose dehydrogenase was introduced into the (R)-phenylethanol pathway. A coexpression system containing E228S and glucose dehydrogenase was constructed. The system was optimized by altering the coding gene order on the plasmid and using the Shine-Dalgarno sequence and the aligned spacing sequence as a linker between them. The presence of glucose dehydrogenase increased the NADPH concentration slightly and decreased NADP(+) pool 2- to 4-fold; the NADPH/NADP(+) ratio was improved 2- to 5-fold. The recombinant Escherichia coli/pET-MS-SD-AS-G, with E228S located upstream and glucose dehydrogenase downstream, showed excellent performance, giving (R)-phenylethanol of an optical purity of 99.5 % in 92.2% yield in 12 h in the absence of an external cofactor. When 0.06 mM NADP(+) was added at the beginning of the reaction, the reaction duration was reduced to 1 h. Optimization of the coexpression system stimulated an over 30-fold increase in the yield of (R)-phenylethanol, and simultaneously reduced the reaction time 48-fold compared with the wild-type enzyme. This report describes possible mechanisms for alteration of the enantiopreferences of carbonyl reductases by site mutation, and cofactor rebalancing pathways for efficient chiral alcohols production.

Dataset Information

Improving activity and enantioselectivity of lipase via immobilization on macroporous resin for resolution of racemic 1- phenylethanol in non-aqueous medium.

Publications

Improving activity and enantioselectivity of lipase via immobilization on macroporous resin for resolution of racemic 1- phenylethanol in non-aqueous medium.

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