Construction Of Activity-based Probes On Hydrolytic Enzyme For Application In Separation And Identification Of Microbial Stereoselectivity Lipase

With the continuous development of enzyme engineering,gene engineering and proteomics,the application of lipases on the synthesis of chiral drug intermediates has become more and more widespread.It has been a critical problem that how to extract stereoselective lipases in vivo quickly and efficiently.Nowadays,activity-based probes,a kind of small molecules that can mark particular proteins,have been reported to separate and purify important proteins of medical and biological value in organisms.A lipase from Aspergillus tamari ZJUT ZQ013 had already been screened before.Herein,the corresponding activity-based probes,which are capable to separate and purify the target lipase from the fungus by chromatography were designed and synthesized.Then the structure of lipase was analyzed and identified.Subsequently,the gene of lipase was cloned and expressed through genetic engineering approach.At last,the enzymatic properties and kinetic parameters were studied.In this paper,three activity-based probes?Rac-amio-probe,D-amio-probe,and L-amio-probe?with the corresponding substrates of three configurations of N-Boc-2-methylaminobutyric were designed and synthesized.The report groups of the probes are biotin,which provide convenience for enrichment and purification of the target enzyme.Also,the identify groups are connected to the report groups by“click”.The probes were then used to co-incubate with the crude extract of Aspergillus tamarii ZJUT ZQ013,followed by affinity chromatography with streptomycin to rapidly extract the target lipase from microbial cells.The result of SDS-PAGE showed the weight of enzyme was 38kDa,and mass spectrometry and N-terminal sequences were identified.In an attempt to verify the specificity of the probe,the experiment with commodity enzyme as control was taken.It turned out that the probe can specifically identify and separate the target enzyme.The steroselectivity of the target enzyme with three corresponding probes?Rac-amio-probe,D-amio-probe,and L-amio-probe?were also studied,with a result suggesting that the lipase could hydrolysis Rac-and L-aminobutyric acid ester,giving the corresponding?S?-aminobutyric acid.Meanwhile,it also proved that the target lipase was?S?-configuration lipase.Total RNA from Aspergillus tamari ZJUT ZQ013 were extracted to obtain cDNA by reverse transcription,and the gene fragment by PCR was finally received.After this,the gene fragment was connected to the vector pEASY-E2 and expressed in E.coli BL21?DE3?.The activity of the recombinant enzyme was 1528.5U/L,which was ten times of the wild fungus enzyme.The gene fragment was found to be 921 bp,encoding 306amino acids.A database search using NCBI BLAST showed a best matching score with acylgylcerol lipase.And then its secondary structure and tertiary structure were simulated for further study of its catalysis mechanism.After that,the recombinant enzyme was purified by a Ni-NTA column affinity chromatography.Finally,the enzymatic properties and kinetic parameters were investigated.The results showed that the enzyme preferred to hydrolysis short chain of triolein.The enzyme was quite stable in a wide pH range with an optimum pH of 8.5,and it had good thermal stability under 50?.When adding metal ions into the reaction system,Fe²⁺showed the promoting effect on enzyme activity,while Cu²⁺and Mn2+had a great inhibition.Among the organic solvents,glycerol,DMSO,acetone,n-Hexane and toluene improved the enzyme activity,while THF inhibited the activity.In surfactants,Tween 20,Tween 80 and Triton X-100 activated the enzyme,while SDS remarkably suppressed the enzyme.As to inhibitors,EDTA,PMSF and DEPC obviously inhibit the activity,but DTT could enhance the enzyme activity.Using p NPA as a substrate,the K_m and V_maxax of the enzyme were 5.2 mM and 30.12 U/mg,respectively,suggesting that the enzyme had a good affinity to the substrate.In this work,active-based probes were utilized to quickly extract the stereoselectivity lipase from microbial cells for the first time,and a preliminary research on the properties of this enzyme was conducted.The identify group of the active-based probes used in this experiment was the enzyme substrate itself,which provided a new idea for the design of active-based probes.Meanwhile,the identification group was flexible,so changes could be made for separation and purification of different enzymes in the future.