title: Soluble Expression of (+)-γ-Lactamase in Bacillus subtilis for the Enantioselective Preparation of Abacavir Precursor.
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.
authors.affiliation: The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122, Jiangsu, China.
authors.forename: Tian-Yun
authors.initials: TY
authors.lastname: Xue
authors.forename: Guo-Chao
authors.initials: GC
authors.lastname: Xu
authors.forename: Rui-Zhi
authors.initials: RZ
authors.lastname: Han
authors.forename: Ye
authors.initials: Y
authors.lastname: Ni
mesh_terms: D000581:Amidohydrolases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D001412:Bacillus subtilis / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D042742:Delftia / Q000201:enzymology / Q000235:genetics; D015224:Dideoxynucleosides / Q000737:chemistry*; D015870:Gene Expression; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D000099227:Abacavir
chemical_list: D000581:Amidohydrolases; D001426:Bacterial Proteins; D015224:Dideoxynucleosides; D011994:Recombinant Proteins; D000099227:Abacavir; C401943:gamma-lactamase