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Issue 61, 2017
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Efficient production of (R)-3-TBDMSO glutaric acid methyl monoester by manipulating the substrate pocket of Pseudozyma antarctica lipase B

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Abstract

Optically pure (R)-3-substituted glutaric acid methyl monoesters are multifunctional chiral building blocks used in the pharmaceutical industry. In the current study, a combined in silico/mutagenesis approach was used to improve the performance of Pseudozyma antarctica lipase B (CALB) as a biocatalyst in the asymmetric synthesis of (R)-3-t-butyl-dimethyl-silyloxy (TBDMSO) glutaric acid methyl monoester (R-J6). Candidate amino acids that likely affected the enantioselectivity of CALB were identified by substrate structure analysis. Mutant variants were screened in silico; CALB enantioselectivity was reversed and enhanced based on molecular docking analyses, followed by reshaping of the substrate pocket. EF5 CALB variant, generated by semi-rational design and selected by high-throughput screening, exhibited high R-selectivity with an eeR (enantiomeric excess) value of 85%, while the wild-type (WT) CALB showed S-selectivity; the kcat/KM of EF5 towards R-J6 increased 14-fold, from 0.59 to 8.29 mM−1 s−1. Compared with WT CALB, the affinity of EF5 for 3-TBDMSO glutaric anhydride increased 2.31-fold. By optimizing the fermentation conditions for the yeast host for protein production and enzyme immobilization conditions, the hydrolytic activity of EF5 was increased to 2401.5 ± 5.3 U mL−1 and 2706.7 ± 11.4 U g−1, respectively. The yield of R-J6 generated by the EF5 variant in non-aqueous media increased to 55 ± 1.6 g L−1, with an eeR value of 98.5%. Semi-rational design was hence successfully employed to generate gram quantities of (R)-3-substituted glutaric acid monoesters with enormous potential and high ee.

Graphical abstract: Efficient production of (R)-3-TBDMSO glutaric acid methyl monoester by manipulating the substrate pocket of Pseudozyma antarctica lipase B

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Publication details

The article was received on 29 May 2017, accepted on 25 Jul 2017 and first published on 03 Aug 2017


Article type: Paper
DOI: 10.1039/C7RA06016E
RSC Adv., 2017,7, 38264-38272
  • Open access: Creative Commons BY license
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    Efficient production of (R)-3-TBDMSO glutaric acid methyl monoester by manipulating the substrate pocket of Pseudozyma antarctica lipase B

    J. Wu, H. Wang, B. Yang, W. Song, C. Liang and L. Liu, RSC Adv., 2017, 7, 38264
    DOI: 10.1039/C7RA06016E

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