Molecular Evolution In Vitro To Improve Enzymatic Characteristics Of β-glucosidase

In this paper,we cloned the wild-type p-glucosidase gene from the genomic DNA of Paenibacillus polymyxa BN1 strain and then transferred it into Escherichia coli for further improvements,Firstly,we raised the heterologous expression level of the P-glucosidase gene by codon-optimization through site-directed mutagenesis,and then we enhanced the catalytic efficency of β-glucosidase through directed evolution and activity-driven screening method on the basis of the optimized high heterologous expressive gene.Moreover,to improve the thermal stability of β-glucosidase,we transferred the mutations reported in some literatures which could improve the thermal stability of P-glucosidase A to the mutant with the highest catalytic efficiency obtained at the last step and therefore we gained the thermostable mutant.To further probe into the oligomeric state of β-glucosidase and the impact of temperature to the enzymatic oligomeric state,we conducted the non-denaturing gel electrophoresis.Finally,we speculated the spatial relative position of β-glucosidase’s activated dimmer by comparing the weak interaction between monomeric P-glucosidase molecules.The main results were shown as follows:The P-glucosidase gene we obtained had a length of 1434 bp,which has four bases changes to the reported bglA gene from P.polymyxa and brings two amino acid substitutions,T385A and R422C.We used DEAE cellulose DE-52 anion exchange column and Ni-NTA agarose affinity chromatography to purify the recombinantβ-glucosidase,then analyzed it by means of SDS-PAGE.From the experimental data,we found the purified product was a single band with a molecular weight of around 52 kDa,as well as the optimal temperature and pH was 35 ℃ and 6.0,respectively,and the optimal substrate was o-nitrophenyl-β-D-glucopyranoside.However,the catalytic activity to substrate cellobiose was the highest,and the Km/Kcat value of 4-nitrophenyl-p-D-glucopyranoside is 45.1(mmol/L)-1s-1.Codon optimization results shown that the codons appeared in high frequency in the β-glucosidase gene while appeared in low frequency in E.coli genomic DNA had the greatest impact on the expression of the enzyme.When these codons were replaced by high-frequency codons of E.coli,the expression level of P-glucosidase was increased by 5 to 6 times.Through combining the beneficial mutations,a higher expression mutant WT-13 was obtained with an increase of relative expression index by 13 times.By screening with esculin medium plate and microplates from mutant library constructed through the method of error-prone PCR,two mutants with high OD405/OD600 value were obtained,m1 and m2.The catalytic efficiency of m1(Y349H)mutant was increased by three times than that of wild-type β-glucosidase,while the corresponding value was 1.7 times of m2(Q311R).The kinetic parameters elucidated that,Y349H mutation mainly improves the catalytic activity of the enzyme,while Q311R improves the affinity between the enzyme and substrates,but the latter has a negative effect on the catalytic activity.Structural analysis showed that Y349H leads to changes of the intramolecular hydrogen bonds,which results in the increase of the molecular energy.Whereas,Q311R,located on the surface of the molecule,can decrease the molecule energy without changes of hydrogen bonds.A thermal stability mutant m13(Y349H/E96K/A17S/N437K)was obtained by combining all beneficial mutations.The half-life time of the mutant was 75 min in 50℃,which was about 50 times to ml.The catalytic efficiency of m13 was 73.9(mmol/L)-1s-1,about 1.6 times to that of the wild-type β-glucosidase.The relative expression index was 13.6,about 15 times to that of wild-type gene.The results of Native-PAGE analysis clarified that β-glucosidase exists as several forms of monomer,dimer,tetramer and octamer in PB buffer.The catalytic activity would not be detected when the enzyme presented as the form of monomer,which was different from those of polymers.Since the β-glucosidase was relatively stable at low temperature,but when temperature was increased,the quaternary structure of the enzyme would be destroyed primarily,and then all the polymeric molecules were depolymerized to be monomers.At this very moment,the enzyme began losing its catalytic activity,if the incubation temperature raised,the monomer molecule was also affected,subsequently the enzyme loss the whole catalytic activity.By comparing the weak interactions between the monomeric β-glucosidase,we found that there was no perfect dimer from the octamer structure can be regarded as the functional dimer unit.Relatively,the dimer along the 4-fold axis meet the experimental fact best,so we hypothesized this dimer was the minimum active structural unit of β-glucosidase from P.polymyxa BN1 strain.