Construction of a trifunctional cellulase and expression in Saccharomyces cerevisiae using a fusion protein

Abstract

BackgroundCellulose is the most important component of lignocellulose, and its degradation requires three different types of enzymes to act synergistically. There have been reports of single gene duality, but no gene has been described to have more than two functions. Cloning and expression of fusion cellulases containing more than two kinds of catalytic domains has not been reported thus far.ResultsWe synthesized three different cellulase genes and linked the three catalytic domains with a (G4S)3 flexible linker. The trifunctional cellulase gene (BCE) containing three types of cellulase functions was constructed and expressed in S. cerevisiae successfully. The β-glucosidase, the exoglucanase and the endoglucanase activity of the trifunctional cellulase BCE reached 16.80 IU/mg, 2.26 IU/mg and 20.67 IU/mg, which was 46.27, 6.73 and 46.20% higher than the activities of the β-glucosidase BG, the endoglucanase CBH and the endoglucanase EG. The filter paper enzyme activity of BCE was higher than those of BG, CBH and EG, reached 2.04 IU/mg.ConclusionsThe trifunctional cellulase BCE was designed based on β-glucosidase BG, endoglucanase EG and exoglucanase CBH, and it possessed β-glucosidase activity, endoglucanase activity and exoglucanase activity simultaneously. The BCE has better filter paper activity, it means the potential practical application.Electronic supplementary materialThe online version of this article (10.1186/s12896-018-0454-x) contains supplementary material, which is available to authorized users.

Formation of recombinant bifunctional fusion protein: A newer approach to combine the activities of two enzymes in a single protein

“…The size of the amplified DNA fragments was determined on agarose gel by comparing with the 100+500 bp DNA ladder and photographed under the Bio-Rad Gel documentation system. Three constructs, namely (i) protease, (ii) chitinase, and (iii) chitinase fused with protease, were constructed by using flexible glycine-serine peptide linker (GGGGS, (G4S)2) [43]. The amplified and purified fragments were inserted into the vector pUC18 mini-Tn7T purchased from add gene, USA.…”

“…The glycine-serine linker is flexible and helps in the proper folding of the enzymes. The fusion of three catalytic domains with a (G4S) 3 flexible linker showed trifunctional cellulase activity in Saccharomyces cerevisiae [43]. They found the higher activity of fusion protein as compared to individual clones.…”

Improved cellulase expression in diploid yeast strains enhanced consolidated bioprocessing of pretreated corn residues

“…Cultures were spot inoculated to screen for endoglucanase and β-glucosidase enzyme activity. The endoglucanase activity was monitored on 2% agar plates containing 1% (w/v) carboxymethylcellulose (Sigma-Aldrich) as the only carbohydrate source [35]. Plates were incubated for 72 h at 30 °C and zone formation was visualised by staining with 0.1% (w/v) Congo red (Sigma-Aldrich) for 15 min and destaining with 1 M NaCl for 30 min.…”

Mutants with Enhanced Cellobiose-Fermenting Ability from Thermotolerant Kluyveromyces marxianus DMKU 3-1042, Which Are Beneficial for Fermentation with Cellulosic Biomass

“…Consequently, the production cost of ethanol is high. In order to solve this problem, attempts have been made to breed enzyme-producing strains [41][42][43] or to produce cellulolytic enzymes in fermenting yeast [44,45]. The purpose of this study was to increase the expression of enzyme genes inherent in yeast, and, as a result, we succeeded in developing strains that are able to produce one of the three main enzymes required for cellulose degradation.…”