Recombinant Expression And Molecular Modification Of Thermotoga Maritima MSB8 Pullulanase

Pullulanase is a starch debranched enzyme that can specifically hydrolyze theα-1,6-glycosidic bond of amylopectin,often cooperating with amylase to completely hydrolyze starch.It plays an important role in industrial production such as resistant starch,food alcohol and fuel ethanol.Since the saccharification conditions are characterized by high temperature（55°C-60°C）and weak acid（pH 4.5-5.5）,the thermostability and acid resistance of pullulanase are strictly required.In this paper,the gene PulA derived from Thermotoga maritima MSB8 was elected from the database.After sequence optimization,it was expressed in E.coli BL21,and the enzymatic properties of the recombinant protein were analyzed.Based on the analysis of the amino acid sequence and domain of PulA,a series of mutants with different domains and site mutations were constructed to study the effects of domain and site directed on the catalytic activity and enzymatic properties of pullulanase.The main results are as follows:（1）Bioinformatics analysis of PulA from Thermotoga maritima MSB8 showed that the total length of the sequence was 2472 bp,encoding 824 amino acids and the molecular weight was about 94 kDa.PulA has four typical conserved domains（RegionⅠ-Ⅳ）and the unique YNWGYDP conserved sequence of type Ⅰ pullulanase.It can be determined that the pullulanase was type Ⅰ pullulanase and belonged to the glycoside hydrolase GH13 family.（2）The recombinant strain BL21-pET22b（+）-PulA was constructed and the target protein was successfully isolated by nickel-column affinity chromatography.The specific activity,K_mvalue and V_maxvalue of pullulanase were 7.81±0.09 U/mg,1.76±0.21 mg/mL and 0.41±0.09 U/mg.The enzymatic properties showed that the optimum pH of PulA was 5.5 and the optimum temperature was 95℃.After incubation at 40-90℃for 60 min,more than 80%of the residual activity remained.The above results showed that PulA was thermostability and acid resistance,which met the requirements of the saccharification process.（3）Four truncated mutants were obtained by excising the N-terminal and C-terminal domains of PulA:mutant PulA1 excised from CBM41 domain,mutant PulA2 excised from CBM41 and X domains,mutant PulA3 excised from CBM41-X-CBM4148 domains and mutant PulA4 excised from C-terminal domain.PulA3 and PulA4 lost activity,indicating that the C-terminal domain was a necessary domain to maintain pullulanase activity.The optimum reaction temperatures of and PulA2 were 85℃and 65℃respectively,which were 10℃and 5℃lower than that of PulA.The specific activity decreased to 54.5%and 26.4%of PulA.The K_mwas3.47 and 4.51 times that of PulA.The above results showed that N-terminal domains,such as CBM41,X and CBM48 domains,played an important role in the expression,structure maintenance and substrate binding of pullulanase.（4）Six mutants were obtained by site directed mutation.The results showed that the mutation did not change the optimal pH of PulA,but affected the thermal stability.The T_mof M1-M6 was 3.88,4.90,13.11,15.68,15.39 and 13.95℃lower than PulA respectively.The loss of hydrogen bond after site directed mutation may reduce the thermal stability of the mutant.