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 480 min 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 40 min, 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.

A multi‐functional genetic manipulation system and its use in high‐level expression of a β‐mannanase mutant with high specific activity in Pichia pastoris

“…However, substituting S199 by the most flexible amino acid residue G could facilitate the flexibility of 187–198 loop. Improving the flexibility of the catalytic core is a widely adopted strategy for enhancing the catalytic activity of the enzyme (Huang et al ., 2014; Dong et al ., 2016; Zheng et al., 2018). P191 M, P194E and S199G could directly or indirectly increase the flexibility of the catalytic core of enzyme, thus presumably facilitating the reaction at the catalytic residue E190.…”

Improving the temperature characteristics and catalytic efficiency of a mesophilic xylanase from Aspergillus oryzae, AoXyn11A, by iterative mutagenesis based on in silico design

“…The protein/enzyme with high T m meant it has a high thermostability ( Jang et al 2010). The T m of xylanase here was measured by protein thermal shift (PTS) method (Dong et al 2016), using a PTS Kit (Applied Biosystems, Carlsbad, CA, USA) and a LightCycler 480II 96 Real-Time PCR system (Roche, Basel, Switzerland). Three replicates were conducted independently.…”

Applications of Microbial β-Mannanases

“…Numerous studies have shown that the alteration of the loop structures in proximity to the active centers of the glycoside hydrolyzes may enhance their catalytic efficiency and/or thermostability. Dong et al (2016) have sought to improve the characteristics of an Aspergillus usamii derived β-mannanase (AuMan5A) by substitution of its loop structure. Based on enzymatic characteristics and structural analysis of various GH 5 β-mannanases, three mutants (AuMan5A, AuMAN5A-An, AuMAN5A-AF) are designed by substituting a piece of loop structure with seven amino acids in the substrate binding groove of AuMan5A with the corresponding sequences of three other family 5 β-mannanases, respectively.…”