Fusing a Carbohydrate-Binding Module into the Aspergillus usamii β-Mannanase to Improve Its Thermostability and Cellulose-Binding Capacity by In Silico Design

Abstract

The AuMan5A, an acidophilic glycoside hydrolase (GH) family 5 β-mannanase derived from Aspergillus usamii YL-01-78, consists of an only catalytic domain (CD). To perfect enzymatic properties of the AuMan5A, a family 1 carbohydrate-binding module (CBM) of the Trichoderma reesei cellobiohydrolase I (TrCBH I), having the lowest binding free energy with cellobiose, was selected by in silico design, and fused into its C-terminus forming a fusion β-mannanase, designated as AuMan5A-CBM. Then, its encoding gene, Auman5A-cbm, was constructed as it was designed theoretically, and expressed in Pichia pastoris GS115. SDS-PAGE analysis displayed that both recombinant AuMan5A-CBM (reAuMan5A-CBM) and AuMan5A (reAuMan5A) were secreted into the cultured media with apparent molecular masses of 57.3 and 49.8 kDa, respectively. The temperature optimum of the reAuMan5A-CBM was 75°C, being 5°C higher than that of the reAuMan5A. They were stable at temperatures of 68 and 60°C, respectively. Compared with reAuMan5A, the reAuMan5A-CBM showed an obvious decrease in K m and a slight alteration in V max. In addition, the fusion of a CBM of the TrCBH I into the AuMan5A contributed to its cellulose-binding capacity.

Controlled Aggregation and Increased Stability of β-Glucuronidase by Cellulose Binding Domain Fusion

“…Interestingly, GusA-CBD exhibited maximal activity at higher temperatures and acidic pH levels compared to free GusA. In a previous study, a family I carbohydrate-binding module of Trichoderma reesei cellobiohydrolase was fused with β-mannanase, resulting in an optimal temperature that was 5–8°C higher than that of free β-mannanase [13]. These results indicate that GusA-CBD is relatively stable at higher temperatures and more acidic pH values than free enzyme.…”

“…Additionally, constructing synthetic scaffolds using the CBD-based system may further improve the metabolite conversion rate by increasing local enzyme concentration and reducing intermediate loss caused by diffusion or side reactions [5]. For example, the fusion of various CBD molecules to the N- or C-termini of other enzymes may enhance enzyme catalytic efficiency by increasing their local concentrations around the polysaccharide substrates such as insoluble cellulose, and/or improve catalytic activity and thermostability [13,23]. …”

Enhancement of acetyl xylan esterase activity on cellulose acetate through fusion to a family 3 cellulose binding module

“…Binding of AnAXE and AnAXE-CtCBM3 to corncob xylan was examined by native affinity gel electrophoresis as described by Freelove et al [40]. Briefly, native polyacrylamide gels prepared for these analyses contained 7.5% (w/v) bis-acrylamide, 25 mM Tris/250 mM glycine buffer (pH 8.6) and 0.01% (w/v) of corncob xylan.…”

“…While published impacts of CBM addition on enzyme thermostability vary, earlier studies demonstrate the potential of CBMs from thermophilic organisms to increase the thermostability of appended catalytic domains [40,41]. Accordingly, to evaluate the effect of the CtCBM3 module on AnAXE stability, the half-life of AnAXE at 60 • C was used to as a baseline to study impacts of the CtCBM3 fusion.…”

Fusion of a family 20 carbohydrate‐binding module (CBM20) with cyclodextrin glycosyltransferase of Geobacillus sp. CHB1 improves catalytic efficiency

“…Several studies have shown that the fusion of CBMs into other enzymes can improve the catalytic activity or the substrate affinity. For example, fusion enzyme was constructed by fusing CBM to the C‐terminus region of β‐mannanase, resulting in an obvious decrease in Km and a slight alteration in Vmax . Chen et al fused the CBM from Alkalimonas amylolytica α‐amylase (CBM A my) to cyclodextrin glycosyltransferase from Paenibacillus macerans to improve 2‐O‐D‐glucopyranosyl‐L‐ascorbicacid (AA‐2G) productivity .…”