Molecular Modification And Functional Characterization For Improving Thermostability Of Pullulanase Based On Computational Design

Pullulanase(EC 3.2.1.41),a sort of enzyme debranching starch,is capable of uniquely hydrolyzing?-1,6-glucosidic linkages of amylopectin,limit dextrin,pullulan and other branched polysaccharides.At present,the pullulanase of acid and high temperature resistance is widely utilized in starch sugar industry,and it has become a key enzyme in the production of high quality syrup.However,the pullulanase are not only utilized for production of syrup with high quality,but also versatile for synthesizes of high value-added products to meet the advanced starch processing industry.Pullulanase from Bacillus thermoleovorans US105(BtPul)is a neutral and thermophile pullulanase,with enzyme activity 219.2 U/mg at optimal reaction conditions of pH 6.0 and 70°C,underlying that BtPul has potencial in the fields of high-value utilization of raw materials of starch.However,the thermostability of BtPul need progressing for the industrial application of starch processing.This research focused on improving the thermostability of BtPul based on the sequence and structure information of BtPul,as well as computation-aided engineering.In addition,the activity and thermostability of BtPul were further improve by addition of chemical reagents.Moreover,the extracellular secretion efficiency of BtPul in E.coli was improved by signal peptide fusion into the N-terminus of BtPul.The main results are listed as follows:(1)Based on the traditional site-directed mutagenesis,we combined multiple thermostability computational tools(FoldX,dDFIRE,and I-Mutant 3.0)to improve thermostability of BtPul.17 potential stable mutants were selected by the three thermostability computational tools and were constructed for further experiments.The T_m of S25E,D138F,P207W,C691R,G692M and T694F were increased by from 1.1?up to 3.8?,with half-lives 178 min,173 min,187 min,202 min,265 min and 210 min,which were 1.39 times,1.35 times,1.46 times,1.58 times,2.07 times and 1.64 times that of WT,respectively.G692M was the optimum mutant,and its specific activity and other enzymatic properties were almost not affected by the mutation.(2)Based on non-traditional site-directed mutagenesis,we introduced a"staple"consisted of noncanonical amino acid(O-2-bromoethyl-tyrosine,O2be Y)into the N/C-terminal domain of BtPul to increase its thermostability.8 mutants were selected according to the prediction through Rosetta Match,FoldX,and Rosetta ddg?monomer for potential“stapling”sites screening.The half-life of single mutant T73(O2be Y)-171C was 330 min,which was 2.57 times that of WT,and its T_m was increased by 7.0?.The half-lives of combined mutants,T73(O2be Y)-171C/T126F and T73(O2be Y)-171C/T126F/A72R,were 425 min and 398 min,respectively,which were 3.32 times and 3.11 times that of WT.In addition,the specific activities of the two combination mutants also increased to 282.9 U/mg and 316.6 U/mg,respectively.The results of measurement of kinetic parameters of the two combination mutants showed lower K_m(6.47±0.2 mg/mL and 4.9±0.2 mg/mL),as well as higher k_cat/K_m(88.2±4.9 mL/mg/s and 85.5±5.3 mL/mg/s).(3)Further improvment of the activity and thermostability of BtPul was conducted via the addition of chemical reagents.We explored effects of mutiple common chemical reagents on the activity and thermostability of BtPul.At a certain concentration,Mn²⁺,Mg²⁺and Triton X-100 showed remarkable activation on the activity of BtPul,by which the BtPul enzyme activities were increased by 105.1%,95.7%and 78.0%,respectively.Mg²⁺,maltose,and gelatin showed greate promotion on the thermostability of BtPul,by which the residual enzyme activities were increased by 12.0%,38.0%and 49.0%at360 min,respectively.The residual enzyme activity of BtPul was increased to 49.5%by addition of Cu²⁺,while relative activity declined to 14.2%.(4)We finally improved secretion efficiency of BtPul in recombinant E.coli.Through fousion of signal peptide with N/C-terminus of BtPul,the extracellular enzyme activity of Pe IB-G692M increased to 4.8 U/mL,which was 2.4 times that of G692M,and its proportion of extracellular BtPul was 9.7 times that of G692M.The extracellular enzyme activity of Opt _PelB-G692M,whose codon of PelB was further optimized,was increased to 29.8 U/mL,which was 6.2 times that of Pe IB-G692M and14.9 times that of G692M.The total enzyme activity of Opt _PelB-G692M increased from50.0 U/mL up to 160.0 U/mL,and its proportion of extracellular BtPul also increased from 9.6%to 18.6%.