The Fusion Of β-mannanase With The Family 27 CBM And Optimization Of The Peptide Linker

Mannan endo β-1,4-mannosidase(endo-β-1,4-D-mannanase, EC 3.2.1.78) can randomly hydrolyze the β-1,4 linkages in the main chain of linear mannan polymer or its derivatives to low molecular weight, water soluble oligosacharrides. Research shows that it can be widely used in food, feed, fiber, liquid fuel, textile, pulp bleaching and other industrial fields. AuMan5 A, a glycoside hydrolase family 5 β-mannanase is derived from Aspergillus usamii. Although it displayed high optimum temperature and strong pH tolerance, the applicability of AuMan5 A was still limited by its poor stability and weak substrate affinity. To perfect its enzymatic properties, a family 27 carbohydrate-binding module(CBM) was fused into AuMan5 A linked separately with different linkers. Finally, the β-mannanase with excellent properties was successfully obtained.Several family 27 CBM amino acid sequences were obtained from the CAZy database and then the homology alignment between or among the sequences was accomplished. Subsequently, the phylogenetic tree of CBM27 was constructed. The 3-D structures of the candidate CBM27 were homologically modeled, respectively. The interactions between CBM27 and mannopentaose were predicted by molecular docking simulation. The binding free energies of the candidate CBM27 with mannopentaose were calculated separately. Finally, TmCBM27 from T. maritima MSB8 β-mannanase, possessing the lowest binding free energy of-209.99 kcal/mol, was selected.The Auman5A-cbm27 or cbm27-Auman5 A were constructed by fusing TmCBM27 into the C terminus or N terminus of AuMan5 A by overlapping PCR, respectively. Then, the recombinant expression vectors, pPIC9K-Auman5A-cbm27 and pPIC9K-cbm27-Auman5 A, were constructed. The resulting recombinant vectors and pPIC9K-Auman5 A were separately transformed into P. pastoris GS115 by electroporation. Recombinant β-mannanases reAuMan5A、reAuMan5A-CBM27 and reCBM27-AuMan5 A showed the specific activities of 230.6 U/mg、280.3 U/mg and 281.1 U/mg, after expressed by menthanol induction and purified, respectively. The enzymatic properties of three recombinant β-mannanases were analyzed. The Topt of them were 70°C, 70°C and 60°C, respectively. reAuMan5A-CBM27 was thermostable at or below 68°C, which was 8°C or 10°C higher than that(60°C or 58°C) of reAuMan5 A or reCBM27-AuMan5 A. The Tm values of reAuMan5 A, reAuMan5A-CBM27 and reCBM27-AuMan5 A were 66.5°C, 74.2°C and 61.3°C, respectively, which were consistent with their temperature stability levels. The Km values of three recombinant β-mannanases for locust bean gum were 1.71 mg/mL, 0.94 mg/m L and 1.26 mg/mL, respectively. These results demonstrated that the fusion of a TmCBM27 into the C-terminus of AuMan5 A contributed to its improved thermostability and substrate affinity, because TmCBM27 provided thermo-protection to the catalytic domain and it possessed the mannan-binding specificity.A flexible peptide linker(F,(GGGGS)3) or rigid peptide linker(R,(EAAAK)3) was chosen to optimize the linker of reAuMan5A-CBM27. Two fusion β-mannanase genes, Auman5A-F-cbm27 and Auman5A-R-cbm27, were constructed using overlapping PCR. Then, they were expressed in P. pastoris GS115, respectively. The specific activities of the recombinant fusions, reAuMan5A-F-C and reAuMan5A-R-C, were 217.2 U/mg and 341.7 U/mg, respectively. The enzymatic properties of reAuMan5A-F-C and reAuMan5A-R-C were characterized. Their temperature optima were 65°C and 70°C, respectively, and they were highly stable at temperatures of 60°C and 70°C, respectively. The Tm values of the two recombinant β-mannanases, reAuMan5A-F-C and reAuMan5A-R-C, were 68.4°C and 74.9°C. The data showed that the α-helical linker could improve the stability of reAuMan5A-R-C. The Km values of the two fusions for locust bean gum were 1.95 mg/mL and 0.73 mg/mL, respectively. The fusion enzyme reAuMan5A-R-C was obtained with strong substrate affinity, which would make it a good candidate for industrial application.We chose reAuMan5A-R-C to hydrolyze locust bean gum. Under the optimized reaction conditions of 30 mg/m L locust bean gum concentration, enzyme amount 60 U/g locust bean gum at 60°C for 6 h,the hydrolysis rate of locust bean gum could reach to 55.5%. To our knowledge, this is the first report on the improvement of the enzymatic properties of AuMan5 A by computer-aided design and gene fusion technology.