Study On The Expression And Stability Transformation Of The Hydroxynitrile Lyases Gene From Arabidopsis Thaliana

Objective To Construct the expression vectors of Hydroxynitrile lyases gene from Arabidopsis thaliana and express it in Escherichia coli BL21(DE3).The production technology of heterologous HNL were optimized to enhance the intracellular expression level and viability in Escherichia coli BL21(DE3).Heterologous HNL was purified and investigated,then the functional properties of the enzyme were explored immediately.Based on the technology of protein engineering,the stability of heterologous HNL was enhanced after molecular modification.Methods 1.Based on the bias of codon,we optimized the codon of hydroxynitrile lyases gene from Arabidopsis thaliana.The fragment of HNL gene from Arabidopsis thaliana was amplified by PCR.Using pET-28a(+),pET-30a(+),pET-32a(+)as vectors and cloning HNL gene into them through gene recombination technology.After the identification by two restriction enzymes digestion and DNA sequencing,they were transformed into Escherichia coli BL21(DE3)by thermal stimulation method.We used single factor method to optimize enzyme-producing condition with different expression vectors,induction strength,induction temperature,induction time and induction opportu-nity,respectively.To compare dry weight of cells after induction,the activity of AtHNL was expressed in Escherichia coli BL21(DE3)were analyzed by UV,and analysis proteinexpression by SDS-PAGE.2.Induced expression was accroading to the technology of production which was optimized,fusion protein was purified after cell collection,suspension,ultrasonic disruption and Ni-chelating column.SDS-PAGE analysis showed their molecular weight and expression.Investigated the activity of AtHNL by UV,and analyzed its enzymatic properties,like the appropriate pH and temperature,thermal stability,Km value,Vmax value,simultaneously.3.Cloning the gene of linker into its upstream primer and amplified AtHNL-linker gene by PCR.Helix structure which acted as a stabilising role was lead into N-terminu of target protein,the helix gene(1USE,1Y66,1YBK)from different sources was cloned into the vector pET-28a(+),then constructed recombinant plasmids pET-28a(+)-1USE,pET-28a(+)-1Y66 and pET-28a(+)-1YBK.Subsequently,AtHNL-linker gene was cloned into those vectors.This was confirmed by restriction enzyme digestion and DNA sequencing,then transformed into Escherichia coli BL21(DE3)by method of thermal stimulation.The expression of recombinant HNL was induced and purified by Ni-chelating column,and its molecular weight and expression were analyzed by SDS-PAGE.Investigated the activity of 1Y66-AtHNL by UV,the appropriate pH and thermal stability were characterized,comrpared with pET-28a(+)-HNL-At.Results 1.pET-28a(+)-HNL-At,pET-30a(+)-HNL-At,pET-32a(+)-HNL-At were correctly constructed,the gene of hydroxynitrile lyase from was expressed successfully in Escherichia coli after induced by IPTG.The completed fermentation condition optimization was induction strength of 0.504 mmol·L-1,induction temperature of 30℃,induction time of 6 h,induction opportunity of OD600=0.9.And its maximum activity reached 29.3 U·mL-1 in the fermentation broth,achieved by 114.78%compared with unoptimization.2.pET-28a(+)-HNL-At was purified by Ni-chelating column,a single band with relative molecular mass of about 30 kDa were obtained by SDS-PAGE.Through the analysis of enzymatic properties,it was showed that the optimal activity of the target protein pET-28a(+)-HNL-At was determined at pH 5.0,the temperature of 30℃,the Km of 0.476 mmol-1,the Vmax of 0.245 μmol·mL-1·min-1 and the specific activity was 213.9 U·mg-1,respectively.3.1USE-AtHNL,1Y66-AtHNL,1YBK-AtHNL were successfully constructed.At the same time,the mainly expression products of 1USE-AtHNL and 1YBK-AtHNL were inclusion bodies,and their enzyme activities were 10.06 U·L-1 and 10.51 U·mL-1,respectively.The expression products of 1Y66-AtHNL was soluble proteins and the enzyme activity was 10.51 U·mL-1,a single band with relative molecular mass of about 38.5 kDa by SDS-PAGE.Then,the pH stability of 1 Y66-AtHNL was increased by 100%at pH 3.5-4.5.In the optimal reaction pH of 5,60℃ for 2.5 h,enzyme activity had remained 50%.Conclusions 1.The study has successfully constructed 3 recombinant plasmids,they are stable expression of engineering bacteria was obtained,after transformed into Escherichia coli BL21(DE3).And the high efficiently heterologous expression of the recombinant HNL was achieved in Escherichia coli.2.pET-28a(+)-HNL-At was purified by Ni-chelating column,the method was conveniently and efficiently,and the characterization of the enzyme was correct,provide reliable guarantee for further study.3.The transformation stabitily of pET-28a(+)-HNL-At was successfully achieved in Escherichia coli.We found the inclusion bodies from 1USE-AtHNL and 1YBK-AtHNL showed enzyme activity.The pH stability and thermal stability of 1Y66-AtHNL was better than pET-28a(+)-HNL-At.This result lays the foundation for further research.