Efficient production of (: R)-3-TBDMSO glutaric acid methyl monoester 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 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.