Molecular Engineering of Fungal GH5 and GH26 Beta-(1,4)-Mannanases toward Improvement of Enzyme Activity

Abstract

Microbial mannanases are biotechnologically important enzymes since they target the hydrolysis of hemicellulosic polysaccharides of softwood biomass into simple molecules like manno-oligosaccharides and mannose. In this study, we have implemented a strategy of molecular engineering in the yeast Yarrowia lipolytica to improve the specific activity of two fungal endo-mannanases, PaMan5A and PaMan26A, which belong to the glycoside hydrolase (GH) families GH5 and GH26, respectively. Following random mutagenesis and two steps of high-throughput enzymatic screening, we identified several PaMan5A and PaMan26A mutants that displayed improved kinetic constants for the hydrolysis of galactomannan. Examination of the three-dimensional structures of PaMan5A and PaMan26A revealed which of the mutated residues are potentially important for enzyme function. Among them, the PaMan5A-G311S single mutant, which displayed an impressive 8.2-fold increase in kcat/KM due to a significant decrease of KM, is located within the core of the enzyme. The PaMan5A-K139R/Y223H double mutant revealed modification of hydrolysis products probably in relation to an amino-acid substitution located nearby one of the positive subsites. The PaMan26A-P140L/D416G double mutant yielded a 30% increase in kcat/KM compared to the parental enzyme. It displayed a mutation in the linker region (P140L) that may confer more flexibility to the linker and another mutation (D416G) located at the entrance of the catalytic cleft that may promote the entrance of the substrate into the active site. Taken together, these results show that the directed evolution strategy implemented in this study was very pertinent since a straightforward round of random mutagenesis yielded significantly improved variants, in terms of catalytic efiiciency (kcat/KM).

Isolation and characterization of novel multifunctional recombinant family 26 glycoside hydrolase from Mehsani buffalo rumen metagenome

“…The rapid, sensitive, and substantial cost reduction in next‐generation sequencing has dramatically speeded up the development in sequence‐based metagenomics. Several mannanases, xylanases, cellulases, and esterases from fungi and bacteria , were isolated. GHs with bi‐ or multifunctional activities enhance the degradation of cellulose and hemicellulose biomass in bioethanol production .…”

Reassembly and co-crystallization of a family 9 processive endoglucanase from its component parts: Structural and functional significance of the intermodular linker

“…Studies in cellulolytic fungi revealed that linkers undergo modifications such as glycoslation and have also been shown to directly bind to the cellulose substrate (Beckham et al 2012;Payne et al 2013;Sammond et al 2012;Srisodsuk et al 1993). Point mutations in different fungal GH-CBM linkers have also been shown to significantly affect the activity of 506 the enzymes and their stability (Couturier et al 2013;Lu et al 2014).…”

Plant biomass degrading ability of the coprophilic ascomycete fungus Podospora anserina

“…Hence, a directed evolution strategy was carried out to improve the catalytic efficiencies of PaMan5A and PaMan26A. It allowed the identification of hot-spots within the active site cleft of both mannanases (Couturier et al, 2013b). In particular, a mutant of PaMan26A with an increased activity exhibited two mutations, P140L and D416G.…”

“…The former was located in the linker between the catalytic domain and the CBM, the latter at the entrance of the catalytic cleft. It was hypothesized that the substitution of a proline by a leucine residue increased the flexibility, and thus resulted in an improved enzymatic activity, while the loss of the D416 side chain could facilitate the entrance of the substrate into the active site (Couturier et al, 2013b).…”