Gene Cloning,Expression,Characteristics And Application Of Carboxylesterase From Bacillus Velezensis

Carboxylesterase is important enzyme in biocatalysis research which generally exists in different living organisms.It can catalyze the hydrolysis of carboxylic acid esters effectively,and plays an important role in clinical,pharmaceutical,environmental restoration and so on.In this paper,the mining,cloning,expression,enzymatic properties of carboxylesterase from Bacillus velezensis SYBC H47,and its application in degradation of four phthalate ester?PAEs?were studied.The main research results were showed as follows:?1?Sixty-six esterase genes were screened from the konwn genome of B.velezensis SYBC H47 by functional annotation.After the further classification,four carboxylesterase genes were obtained,namely:baces01,baces02,baces03,and baces04.?2?The analysis of nucleic acid and amino acid sequence of baces01-04 revealed that the ORF of the four genes was 603,1449,741,and 870 bp,respectively.The protein molecular weight was predicted as 22.4,52.8,28.3,and 31.9 kDa,respectively.The amino acid sequences of BaCEs01,BaCEs02,BaCEs03,and BaCEs04 all contain the conserved motif of Gly-Xxx-Ser-Xxx-Gly and the serine hydrolase catalytic triad Ser-Asp-His.BaCEs01-BaCEs04 belongs to the esterase families VI,VII,VIII,and VI,respectively.?3?Four carboxylesterase genes from Bacillus velezensis SYBC H47 were successfully cloned and expressed in E.coli BL21?DE3?.The results of enzyme activity measurement indicated that BaCEs01-04 all have carboxylesterase activity.?4?The study of enzyme properties showed that the optimal pH of BaCEs01-04 was 6.5,6.5,7.0,and 7.5,respectively.The optimal temperature of BaCEs01-04 was 40,45,45,and65?,respectively.The effects of metal ions,inhibitors,surfactant,and organic solvents on the activity were studied.The result showed that Cu²⁺inhibited the activity of recombination enzyme,while NH₄⁺slightly enhanced the activity of BaCEs01,BaCEs02,and BaCEs4.The inhibitor PMSF obviously inhibited the activity of recombination enzyme,and the organic solvents isopropanol,acetone,and n-hexane enhanced the activity of BaCEs04.The hydrolytic activity of BaCEs01-04 on pNP-esters with different carbon chain lengths?C2-C16?was studied.Recombinases showed higher activity on substrates with shorter carbon chain lengths.Using different concentrations pNPC₂,pNPC₄,and pNPC₆ as substrate to analysis kinetic parameters.For BaCEs01,the K_m values were 1.28,0.95,1.36 mmol L^-1,respectively,the k_cat/K_m values were 14.16,30.53,10.51 L·mmol^-1·s^-1,respectively.For BaCEs02,the K_m values were 0.34,0.67,0.70 mmol L^-1,respectively,the k_cat/K_m values were 341.47,130.24,112.94L·mmol^-1·s^-1,respectively.For BaCEs03,the K_m value were 2.48,1.93,2.84 mmol L^-1,respectively,the k_cat/K_m values were 7.14,10.73,3.90 L·mmol^-1·s^-1,respectively.For BaCEs04,the K_m values were 0.68,0.73,0.55 mmol L^-1,respectively,the k_cat/K_m values were 27.81,67.70,24.60 L·mmol^-1·s^-1,respectively.?5?The qualitative and quantitative analysis of the enzymatic degradation of dimethyl phthalate?DMP?,diethyl phthalate?DEP?,dipropyl phthalate?DPRP?,and dibutyl phthalate?DBP?were used thin layer chromatography?TLC?and liquid chromatography-mass spectrometry?LC-MS?analysis.After 5 h treatment,the degradation ratio of DMP and DEP exceeded 25%and 50%,respectively.The degradation ratio of DPRP was 60.9,94.8,55.9,and77.9%,respectively.The degradation ratio of DBP was 70.8,88.4,66.8,and 86.9%,respectively.The residual substrate and the corresponding monoalkyl phthalate?MNP?in the degradation products were detected by MS analysis.Further experiment showed that MNP cannot be degraded by BaCEs01-04,which indicated that four recombinases could hydrolyzed one ester bond of PAEs,and generated corresponding MNP.