Engineering a family 27 carbohydrate-binding module into an Aspergillus usamii ß-mannanase to perfect its enzymatic properties.

A family 27 carbohydrate-binding module of a Thermotoga maritima ß-mannanase (TmCBM27) was chosen from the carbohydrate-active enzyme database by computer-aided design, possessing the lowest binding free energy with mannopentaose. To improve the enzymatic properties of a glycoside hydrolase family 5 ß-mannanase from Aspergillus usamii (AuMan5A), two fusion ß-mannanases, AuMan5A-F-M and AuMan5A-R-M, were designed by fusing a TmCBM27 into its C-terminus linked with a flexible peptide F (GGGGS)3 and rigid peptide R (EAAAK)3. Two fusion enzyme genes, Auman5A-F-m and Auman5A-R-m, were constructed as designed theoretically by overlapping PCR. Then, Auman5A and two fusion genes were expressed in Pichia pastoris GS115. Three recombinant ß-mannanases, reAuMan5A, reAuMan5A-F-M and reAuMan5A-R-M, were purified to homogeneity with specific activities of 230.6, 153.3 and 241.7 U/mg. The temperature optimum of reAuMan5A-R-M was 70°C, identical with that of reAuMan5A, while its thermostability and melting temperature (Tm) reached 68°C and 74.9°C, being 8.0°C and 8.4°C higher than those of the latter, respectively. Additionally, the Km values of reAuMan5A-R-M, towards locust bean gum, konjac gum and guar gum, significantly decreased to 0.9, 1.9 and 2.5 mg/mL from 1.7, 3.8 and 4.2 mg/mL of reAuMan5A, while its kcat/Km (catalytic efficiency) values increased to 287.8, 163.7 and 84.4 mL/mg⋅s from 171.2, 97.6 and 56.0 mL/mg⋅s of the latter, respectively. These results verified that the fusion of a TmCBM27 into the C-terminus of AuMan5A mediated by (EAAAK)3 linker contributed to its improved thermostability and catalytic efficiency.