Abstract
Chiral Vince lactam (γ-lactam) is an important precursor of many carbocyclic nucleoside analogues and pharmaceuticals. Here, a (+)-γ-lactamase encoding gene delm from Delftia sp. CGMCC 5755 was identified through genome hunting. To achieve its soluble and functional expression, Escherichia coli and Bacillus subtilis expression systems were introduced. Compared with E. coli system, recombinant (+)-γ-lactamase showed improved protein solubility and catalytic activity in B. subtilis 168. Reaction conditions for enantioselective resolution of γ-lactam were optimized to be at 30 °C, pH 9.0, and 300 rpm when employing the recombinant B. subtilis 168/pMA5-delm whole cells. Kinetic analysis showed that the apparent V max and K m were 0.595 mmol/(min · gDCW) and 378 mmol/L, respectively. No obvious substrate inhibition was observed. In a 500-mL reaction system, enantioselective resolution of 100 g/L γ-lactam was achieved with 10 g/L dry cells, resulting in 55.2 % conversion and 99 % ee of (−)-γ-lactam. All above suggested that recombinant B. subtilis 168/pMA5-delm could potentially be applied in the preparation of optically pure (−)-γ-lactam.
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Acknowledgments
We are grateful to the National High Technology Research and Development Program of China (973 Program, 2011CB710800), National Natural Science Foundation of China (21276112, 31401634), the Fundamental Research Funds for the Central Universities (JUSRP51409B), the Program of Introducing Talents of Discipline to Universities (111-2-06), and a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions for the financial support of this research.
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All the authors certify that this manuscript is original and has not been published and will not be submitted elsewhere for publication while being considered by Applied Microbiology and Biotechnology. And, the study is not split up into several parts to increase the quantity of submissions and submitted to various journals or to one journal over time. No data have been fabricated or manipulated (including images) to support your conclusions. No data, text, or theories by others are presented as if they were our own. The submission has been received explicitly from all co-authors. And, authors whose names appear on the submission have contributed sufficiently to the scientific work and therefore share collective responsibility and accountability for the results. The authors declare that they have no conflict of interest. This article does not contain any studies with human participants or animals performed by any of the authors. Informed consent was obtained from all individual participants included in the study.
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Xue, TY., Xu, GC., Han, RZ. et al. Soluble Expression of (+)-γ-Lactamase in Bacillus subtilis for the Enantioselective Preparation of Abacavir Precursor. Appl Biochem Biotechnol 176, 1687–1699 (2015). https://doi.org/10.1007/s12010-015-1670-7
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DOI: https://doi.org/10.1007/s12010-015-1670-7