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

• Record Type: Journal Article
• Authors: Wu, Jing; Wang, Hongjiang; Yang, Bin; Song, Wei; Liang, Chenchen; Liu, Liming
• Is Part Of: RSC advances. Volume 7:Issue 61(2017)
• Page Start: 38264
• Page End: 38272
• Publication Date: 2017-08-03
• Subjects: Chemistry -- Periodicals; 540.5
• DOI: 10.1039/c7ra06016e
• Languages: English
• ISSNs: 2046-2069

Abstract

Abstract : Efficient production of optically pure ( R )-3-substituted glutaric acid methyl monoesters, the multifunctional chiral building blocks used in the pharmaceutical industry, by manipulating the substrate pocket of Pseudozyma antarctica lipase B. 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 ee R (enantiomeric excess) value of 85%, while the wild-type (WT) CALB showed S -selectivity; the k cat / K M 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 ee R 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.