Engineering the enantioselectivity and thermostability of a (+)- γ-lactamase from Microbacterium hydrocarbonoxydans for the kinetic resolution of Vince lactam.

In this study, the enantioselectivity and thermostability of a (+)-γ-lactamase from Microbacterium hydrocarbonoxydans as the catalyst in the kinetic resolution of Vince lactam were improved. Enantiomerically pure (-)-Vince lactam is the key synthon in the synthesis of antiviral drugs such as carbovir and abacavir, which are used to fight against human HIV and hepatitis B viruses. The work was initialized by using the combinatorial active-site saturation test strategy to engineer the enantioselectivity of the enzyme. The approach resulted in two mutants, Val54Ser and Val54Leu, which catalyzed the hydrolysis of Vince lactam to give the (-)-Vince lactam with 99.2% (E >200) ee and 99.5% ee (E >200), respectively. To improve the thermostability of the enzyme, eleven residues with high B-factors calculated by B-FITTER or high root mean square fluctuation (RMSF) values from the molecular dynamics simulation were selected. Six mutants with increased thermostability were obtained. Finally, the mutants generated with improved enantioselectivity and mutants evolved for enhanced thermostability were combined. Several variants showing (+)-selectivity (E value >200) and improved thermostability were observed. These engineered enzymes are good candidates to serve as enantioselective catalysts for the preparation of enantiomerically pure Vince lactam.Importance Enzymatic kinetic resolution of the racemic Vince lactam using (+)-γ-lactamase is the most often utilized means of resolv...
Source: Applied and Environmental Microbiology - Category: Microbiology Authors: Tags: Appl Environ Microbiol Source Type: research