AimsWe aimed to explore Yarrowia lipolytica carbonyl reductases as effective biocatalysts and to develop efficient asymmetric reduction systems for chiral alcohol synthesis.Methods and ResultsYarrowia lipolytica carbonyl reductase genes were obtained via homologous sequence amplification strategy. Two carbonyl reductases, YaCRI and YaCRII, were identified and characterized, and used to catalyse the conversion of 2‐hydroxyacetophenone (2‐HAP) to optically pure (S)‐1‐phenyl‐1,2‐ethanediol. Enzymatic assays revealed that YaCRI and YaCRII exhibited specific activities of 6·96 U mg−1 (99·8% e.e.) and 7·85 U mg−1 (99·9% e.e.), respectively, and showed moderate heat resistance at 40–50°C and acid tolerance at pH 5·0–6·0. An efficient whole‐cell two‐phase system was established using reductase‐expressing recombinant Escherichia coli. The conversion of 2‐HAP (20·0 g l−1) conversion with the solvent of dibutyl phthalate was approximately 70‐fold higher than in water. Furthermore, the two recombinant E. coli displayed biocatalyst activity and enantioselectivity towards several different carbonyl compounds, and E. coli BL21 (DE3)/pET‐28a‐yacrII showed a broad substrate spectrum.ConclusionsA new whole‐cell recombinant E. coli‐based bioreduction system for enantiopure alcohol synthesis with high enantioselectivity at high substrate concentrations was developed.Significance and Impact of the StudyWe proposed a promising approach for the efficient preparation of enantiopure chiral alcohols.
To improve the reusability and organic solvent tolerance of microbial lipase and expand the application of lipase (hydrolysis, esterification, and transesterification), we immobilized marine microbial lipase using different methods and determined the properties of immobilized lipases. Considering the activity and cost of immobilized lipase, the concentration of lipase was fixed at 2 mg/mL. The optimal temperature of immobilized lipases was 40°C and 5°C higher than free lipase. The activities of immobilized lipases were much higher than free lipase at alkaline pH (more than 50% at pH 12). The free lipase lost most activity (35.3%) and immobilized lipases retained more than 46.4% of their initial activity after 3 h heat treatment at 70°C. At alkaline pH, immobilized lipases were more stable than free lipase (more than 60% residue activity at pH 11 for 3 h). Immobilized lipases retained 80% of their activity after 5 cycles and increased enzyme activity (more than 108.7%) after 3 h treatment in tert-butanol. Immobilization of lipase which improved reusability of lipase and provided a chance to expand the application of marine microbial lipase in organic system expanded the application range of lipase to catalyze hydrolysis and esterification in harsh condition.
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