| Pullulanase,a well-known starch-debranching enzyme,has been widely used to catalyze the hydrolysis of α-1,6-glycosidic linkages at branching points in pullulan,amylopectin,and related saccharides,with great application potentials in food,brewing,bioenergy,and pharmaceutical industries.Although extensive studies have been done to discover,express and engineer pullulanases,only few pullulanases displaying excellent characteristics have hitherto been reported,especially cold-adapted pullulanases keeping high catalytic activity at ambient temperatures with application potential in cold starch hydrolysis.With the development of starch hydrolysis technology and the expansion of starch deep processing,screening pullulanase with desirable characteristics to reduce the production cost,simplify the process,and enhance the utilization of starch resources is an important development direction.In this study,a novel cold-adapted pullulanase(PulPB1)from Bacillus methanolicus PB1 strain was successfully expressed,and characterized.Based on the sequence information and structure model,we modified the N-terminal domain of PulPB1,and explored its effect on the catalytic properties.Furthermore,the thermostability of PulPB1 was improved through rational design.The main research results are listed as follows:1.A novel pullulanase(PulPB1)from Bacillus methanolicus PB1 was successfully expressed in Escherichia coli BL21(DE3)with recombinant plamid p ET-28a-PulPB1.Based on the optimization of induction conditions,E.coli BL21(DE3)was cultured in LB medium at 37℃ until the absorbance at 600 nm reached 0.8.Expression was then induced by adding 0.2 m M IPTG and culturing at 20℃ for 16 h.After purifing with Ni-affinity chromatography,the activity of PulPB1 toward pullulan was determined as 292 U/mg.2.PulPB1 was determined to be a cold-adapted type I pullulanase with maximum activity at 50℃ and p H 5.5.The recombinant PulPB1 showed great stability,its half-life at 50℃ was 137 h.PulPB1 can efficiently hydrolyze pullulan and amylopectin,with activities of 292 and 184 U/mg at 50℃ and p H 5.5,respectively.Moreover,PulPB1 is promising for application in cold amylopectin hydrolysis,due to the increased catalytic efficiency when combining PulPB1 with amyloglucosidase for amylopectin hydrolysis at 40℃.3.The N-terminal domain of PulPB1 was characterized as CBM68 domain and it significantly affect the enzymatic performance.Following truncation of the N-terminal domain,the solubility of truncated variant was significantly reduced,its activity towards pullulan decreased markedly from 292 to 141 U/mg and the half-life at 50℃ decreased from 137 to 10 h.On the other hand,the catalytic activity of N-domain chimeric variant was 27% higher than that of PulPB1,and the stability was slightly reduced.Therefore,CBM68 is involved in the formation and stability of the tertiary structure of PulPB1,and has effects on the substrate affinity and catalytic efficiency.4.To improve the stability of PulPB1,a library containing 26 mutants was constructed based on structural information and sequence alignment.Mutants M-(S243L)and M-(K253P)with improved stability were selected.A combined mutant M-(S243L-K253P)was then constructed to further enhance the thermostability.Its half-life at 60℃ reaches 107.8 min,which is 3.7-fold that of the wild-type PulPB1.Structure analysis revealed the molecular basis for the improved stability of PulPB1 mutants.The introductions of Leu and Pro enhanced the local hydrophobic interaction and the rigidity of the loop,resulting in the increased enzyme stability. |
PDF Full Text Request