Discovery,Engineering And Application Of Highly Substrate-Tolerant(-)-γ-Lactamase For Enzymatic Preparation Of(+)-vince Lactam

Compared with the traditional chemical synthesis,biocatalysis has the characteristics of high efficiency,good selectivity,mild reaction conditions,environmental friendliness,etc.α/βhydrolases are a class of enzymes,which are widely used in pharamcetical,food,washing and paper making industries.This paper mainly studied the screening of α/β hydrolase and their application in the chiral pharmaceutical intermediates and natural flavors.In the main part of this thesis,a novel(-)-y-lactamase SvGL was identified by a genome data-mining approach,which could enantioselectively hydrolyze(-)-y-lactam to obtain(+)-y-lactam In the appendix part,a novel esterase AbMBH was identified from the genomic library of Acinetobacter sp.ECU2040,which could enantioselectively hydrolyze l-nenthyl benzoate to obtain/-menthol.Firstly,11 enzymes were successfully overexpressed and then assayed using Vince lactam as substrate.The(-)-y-lactamase SvGL from Streptomyces viridochromogenes DSM 40736 exhibited excellent performance in the aspects of activity,thermostability,enantioselectivity and substrate concentration tolerance.Consequently,SvGL was chosen for the further study.SvGL belongs to the α/β hydrolase fold family and shares a similar catalytic mechanism with esterases.Multiple sequences alignment of SvGL indicated that the conserved motif GFSMG locates between residues 97-101,and the catalytic triad is constituted by Ser99,Asp229 and His258,while the oxyanion hole is composed of the backbone N atoms of Phe33 and Met100.SvGL exhibited excellent stability over a pH range of 6.0 to 9.0 for 24 h,and the optimal pH was 8.0.SvGL displayed maximal activity at 40℃.The T505 and half-life were 76.6℃ and 13.0 h at 70℃,respectively.Kinetic constants of KM and kcat were 87.4 mM and 104 s-1,respectively,and the specificity constant(kcat/KM)is 1.22 mM-1 s-1.The specific activity of SvGL towards Vince lactam was 189 U mg-1.Additionally,SvGL also displayed the p-nitrophenyl ester esterase activity of 9.77 U mg-1 towards pNPA,the perhydrolase activity of 10.4 U mg-1 towards H2O2,and lactonase activity of 7.67 U mg-1 towards 3,4-dihydroiso co umar iaIn the third part,two mutants SvGLL130Y and VvGLL1301 with reduced values were obtained.The specific activity of purified SvGLL130Y and SvGLL1301 towards 10 mM of Vince lactam showed 2.43-and 1.68-fold greater than SvGL,respectively.Compared to SvGL,the kcat/KM of SvGLL130Y increased from 1.22 to 2.41 mM-1 s-1,while that of SvGLL-1301 increased from 1.22 to 1.88 mM-1 s-1.Structure analysis indicated that the space orientation of side chain of Ile and Tyr were changed and the substrate binding pocket of two mutants were also apparently expanded.The binding of(-)-y-lactam with enzyme of wild type and mutants by molecular docking were analyzed,respectively.For mutant SvGLL130Y,the r(C-O)and affinity increased slightly,however,a in the r(N-N)was reduced significantly from 3.7 to 2.9 A.For mutant SvGLL1301,no obvious change was observed.SvGLL130Y and SvGLL1301 also displayed 2.55-and 1.62-fold higher esterase activities towards pNPA,11.9-and 2.38-fold lower promiscuous oxidase activities towards H2O2.For lactonase activity,there was no obvious improved.Compared with SvGL,the kcat/KM values of mutants SvGLL130Y and SvGLL1301 towards y-lactam increased by 1.98-and 1.5-fold,respectively,however,obvious substrate inhibition was found in both mutants under high substrate concentration.Mutant SvGLL130y exhibited serious substrate inhibition,the conversion rate was only 1%under 3.0 M Vince lactam for 8 h The activity of mutant SvGLL1301 was severely suppressed under 4.0 M Vince lactam,the conversion rate was 21.8%for 7.5 h and 22.8%for 10 h.However,the conversion rate of SvGL has reached 48.4%for 8 h.Finally,using SvGL as a biocatalyst,an efficient and environmentally benign process for the production of(+)-γ-lactam was developed.The process allwed enantioselective resolution of 436.5 g L-1 racemic γ-lactam with only 0.2 g L-1 of lyophilized cell-free extract,affording an extremely high substrate/catalyst ratio of 2183(g/g),a space-time yield of 458 g L-1 d-1,and a very low E fector(environmental factor)of 5.7(kg waste per kg product)even when the process water is included.These results demonstrate that this bioprocess is a promising route for the green manufacture of(+)-y-lactam.