Enhancing the catalytic activity of a novel GH5 cellulase GtCel5 from Gloeophyllum trabeum CBS 900.73 by site-directed mutagenesis on loop 6

Abstract

BackgroundCellulases of glycosyl hydrolase (GH) family 5 share a (β/α)8 TIM-barrel fold structure with eight βα loops surrounding the catalytic pocket. These loops exposed on the surface play a vital role in protein functions, primarily due to the interactions of some key amino acids with solvent and ligand molecules. It has been reported that motions of these loops facilitate substrate access and product release, and loops 6 and 7 located at the substrate entrance of the binding pocket promote proton transfer reaction at the catalytic site motions. However, the role of these flexible loops in catalysis of GH5 cellulase remains to be explored.ResultsIn the present study, an acidic, mesophilic GH5 cellulase (with optimal activity at pH 4.0 and 70 °C), GtCel5, was identified in Gloeophyllum trabeum CBS 900.73. The specific activities of GtCel5 toward CMC-Na, barley β-glucan, and lichenan were 1117 ± 43, 6257 ± 26 and 5318 ± 54 U/mg, respectively. Multiple sequence alignment indicates that one amino acid residue at position 233 on the loop 6 shows semi-conservativeness and might contribute to the great catalytic performance. Saturation mutagenesis at position 233 was then conducted to reveal the vital roles of this position in enzyme properties. In comparison to the wild type, variants N233A and N233G showed decreased optimal temperature (− 10 °C) but increased activities (27 and 70%) and catalytic efficiencies (kcat/Km; 45 and 52%), respectively. The similar roles of position 233 in catalytic performance were also verified in the other two GH5 homologs, TeEgl5A and PoCel5, by reverse mutation. Further molecular dynamics simulations suggested that the substitution of asparagine with alanine or glycine may introduce more hydrogen bonds, increase the flexibility of loop 6, enhance the interactions between enzyme and substrate, and thus improve the substrate affinity and catalytic efficiency.ConclusionThis study proposed a novel cellulase with potentials for industrial application. A specific position was identified to play key roles in cellulase–substrate interactions and enzyme catalysis. It is of great importance for understanding the binding mechanism of GH5 cellulases, and provides an effective strategy to improve the catalytic performance of cellulases.Electronic supplementary materialThe online version of this article (10.1186/s13068-018-1080-5) contains supplementary material, which is available to authorized users.

Roles of Carbohydrate-Binding Module (CBM) of an Endo-��-1,4-glucanase (Cel5L) from Bacillus sp. KD1014 in Thermostability and Small-Substrate Hydrolyzing Activity

“…However, the two enzymes showed low but moderate hydrolytic activities, 35.0 and 32.7%, respectively, for lichenan (another type of β-1,3-1,4-glucan with different the ratio of β-1,3 to β-1,4 ratio). This result was similar to that of BlCel5B of B. licheniformis [37], but differed from those of BsCel5A of B. subtilis 168 and the GtCel5 of G. trabeum, which had higher lichenase activity A C C E P T E D than CMCase activity [2,11], and the Cel5B of T. fusca with undetectable lichenases activity [38]. Both Cel5L-p50 and Cel5L-p35 showed low activities for laminarin (β-1,3-1,6-glucan), however, the activity of Cel5L-p35 was 2.8 times higher than that of Cel5L-p50 (71.6% and 25.2%, respectively).…”

“…The relative activities of Cel5L-p50 and Cel5L-p35 for barley β-glucan (β-1,3-1,4-glucan) were 377.0 and 246.7%, respectively, as compared with CMC (β-1,4-glucan) ( Table 2). These higher activities for barley β-glucan than CMC resembled those of GH5-2 endoglucanases [29,36] and a fungal GH5-5 cellulase GtCel5 of Gloeophyllum trabeum [2]. However, the two enzymes showed low but moderate hydrolytic activities, 35.0 and 32.7%, respectively, for lichenan (another type of β-1,3-1,4-glucan with different the ratio of β-1,3 to β-1,4 ratio).…”

“…Complex polysaccharides can be hydrolyzed by cellulases such as endo-β-1,4-glucanase, exo-β-1,4-glucanase, and β-glucosidase. Among them, endo-β-1,4glucanases, which are grouped into 13 glycoside hydrolase (GH) families; 5-9, 12, 44, 45, 48, 51, 74, 124, and 131 [2], play important roles in hydrolysis as they cleave internal glycosidic bonds in the chains [3]. Most of the enzymes grouped in GH families 5-9, 12, 44, 45 and 48 are cellulases acting on cellulose [4].…”

Engineering Robust Cellulases for Tailored Lignocellulosic Degradation Cocktails

“…Another target area is the catalytic site, the targeting non-catalytic amino acids achieved a 1.9-fold improvement in the catalytic efficiency in an endoglucanase [79]. In addition, loops and residues that may interact with the substrate have been widely studied [80][81][82][83]. As a semi-rational approach also focused on protein areas that interact with the substrate, strategies employed a complete diversity for selected positions.…”

“…Regarding endoglucanases, the attention is different, some works have aimed toward the increase of activity [80], but most works have focused on producing a robust catalyst that can be employed in harsh conditions. Most studies have contemplated improving their thermostability, expanding their pH range, and increasing resistance in non-conventional media.…”