Replacing a piece of loop-structure in the substrate-binding groove of Aspergillus usamii β-mannanase, AuMan5A, to improve its enzymatic properties by rational design.

Abstract

To perfect the enzymatic properties of AuMan5A, a mesophilic glycoside hydrolase (GH) family 5 β-mannanase from Aspergillus usamii, its loop-structure substitution was carried out by rational design and followed by megaprimer PCR. Based on the structural analysis and enzymatic property comparison of various β-mannanases, a piece of loop-structure with seven amino acids between two β-strands (βD and βE) in the substrate-binding groove, named "Loop DE," was speculated to be correlative to the thermostability and catalytic efficiency of GH family 5 β-mannanases. Therefore, three AuMan5A's mutants, AuMan5A-Af, AuMan5A-An, and AuMan5A-Th, were designed by substituting a Loop DE sequence ((316)KSPDGGN(322)) of AuMan5A with the corresponding sequences of other three family 5 β-mannanases, respectively. Then, the mutant-encoding genes, Auman5A-Af, Auman5A-An, and Auman5A-Th, were constructed as designed theoretically and then expressed in Pichia pastoris GS115. The expressed recombinant AuMan5A-Af (re-AuMan5A-Af) displayed the temperature optimum (T opt) of 75°C, T m value of 76.6°C and half-life (t 1/2) of 480min at 70°C, which were 10 and 12.1°C higher and 48-fold longer than those of re-AuMan5A, respectively. Its catalytic efficiency (k cat/K m) was 12.7-fold that of re-AuMan5A. What is more, the site-directed mutagenesis of D320G in AuMan5A-Af was performed. The T opt and t 1/2 of expressed re-AuMan5A-Af(D320G) decreased to 70°C and 40min, respectively, while its k cat/K m was only 35% of that of re-AuMan5A-Af. These results demonstrated that the mutation of G320 (in AuMan5A) into D320 (in AuMan5A-Af) through Loop DE substitution was mainly responsible for the thermostability and catalytic efficiency improvement of AuMan5A-Af.