Mining Of Thermostable(+)-?-Lactamases And Application In Resolution Of Vince Lactam

Optically pure Vince lactam is an important pharmaceutical intermediate,such as(–)-vince lactam can be used in the synthesis of antiviral drugs such as abacavir and palamivir,while(+)-Vince lactam can be used in the synthesis of drug targets such as megliptin and MK-0812.Asymmetric resolution of racemic Vince lactam is an important method to obtain optically pure Vince lactam.Compared with chemical resolution and physical recrystallization,asymmetric hydrolysis of racemic Vince lactam by stereoselective?-lactamase to synthesize chiral Vince lactam has the advantages of mild conditions,high optical purity and environmental friendliness,exibiting industrial application potential.In this study,through genome mining,we obtained two putative(+)-?-lactamases based on the activity and stereoselectivity evaluation.They are(+)-?-lactamase FoLM from Fusarium oxysporum and TmLM from thermaerbactaster marianensis.Based on multiple sequence alignment and phylogenetic tree analysis,FoLM and TmLM are the type II(+)-?-lactamases,belonging to the family of formamide/acetamide.The enzymatic properties of FoLM and TmLM were characterized.The optimal p H and temperature of FoLM and TmLM were all at p H 7.0 and 60°C.Under the optimum conditions,the specific activity of FoLM and TmLM was 2.7 U·mg^–1and 13.7 U·mg^–1 respectively.Kinetic parameters analysis showed that K_m and V_max of FoLM and TmLM toward Vince lactam were 37.6 mmol·L^-1 and 4.4?mol·min^-1·mg^-1,32.2 mmol·L^-1 and 16.2?mol·min^-1·mg^-1 respectively.Both TmLM and FoLM are stable at higher temperature,with half-lives of 32 h and 33 h at 50°C,17 h and 20 h at 70°C,and could retain 50%of intial activity at 90°C for 4 h.Both FoLM and TmLM can catalyze the hydrolysis of?-caprolactam,although the activity is very low.However,it is difficult for FoLM and TmLM to catalyze monocyclic amide compounds such as?-heptyl lactam,?-lauryl lactam and caprolactam,indicating FoLM and TmLM have high substrate specificity.Application of FoLM and TmLM in the asymmetric resoltuon of Vince lactam.Whole cells of recombinant E.coli p ET28a-FoLM and E.coli p ET28a-TmLM were adopted and the reactions were performed at 60°C and p H 7.0,respectively.It was found that E.coli p ET28a-TmLM had better substrate tolerance.When the substrate concentration was 1.0 M,the asymmetric resolution could be completed within 10 h,with conversion ratio of 51.1%,and ee value of 99.9%.When the substrate concentration was further increased to 1.5 M,the final conversion was only 38.4%,even if the reaction time was prolonged.Considering the substrate inhibition,substrate was added in fed-batch to alleviate the substrate inhibition.Finally,2 M(218 g·L^–1)racemic Vince lactam could be enantioselectively resolved by 10g·L^–1 lyophilized cells in a 250 m L reaction mixture.After 24 h reaction,the conversion rate reached 50.2%,the ee value was 99.6%,and the substrate to catalyst ratio(S/C)was 21.8g·g^–1.After extraction and rotary evaporation under vaccum,16.5 g product was obtained with a recovery of 60.5%and the product was confirmed to be(–)Vince lactam by NMR.In this study,two thermostable type II(+)-?-lactamases,FoLM and TmLM,were obtained,which can enantioselectively hydrolyze Vince lactam.The reaction system for the synthesis of chiral Vince lactam was established using E.coli p ET28a-TmLM.As much as 2.0M Vince lactam could be enantioselectively resolved.In summary,this study provides efficient biocatalyst for the synthesis of chiral?-lactams.