Characterization of a Cellulomonas fimi exoglucanase/xylanase-endoglucanase gene fusion which improves microbial degradation of cellulosic biomass

Abstract

Effective degradation of cellulose requires multiple classes of enzyme working together. However, naturally occurring cellulases with multiple catalytic domains seem to be rather rare in known cellulose-degrading organisms. A fusion protein made from Cellulomonas fimi exo- and endo- glucanases, Cex and CenA which improves breakdown of cellulose is described. A homologous carbohydrate binding module (CBM-2) present in both glucanases was fused to give a fusion protein CxnA. CxnA or unfused constructs (Cex+CenA, Cex, or CenA) were expressed in Escherichia coli and Citrobacter freundii. The latter recombinant strains were cultured at the expense of cellulose filter paper. The expressed CxnA had both exo- and endo- glucanase activities. It was also exported to the supernatant as were the non-fused proteins. In addition, the hybrid CBM from the fusion could bind to microcrystalline cellulose. Growth of C. freundii expressing CxnA was superior to that of cells expressing the unfused proteins. Physical degradation of filter paper was also faster with the cells expressing fusion protein than the other constructs. Our results show that fusion proteins with multiple catalytic domains can improve the efficiency of cellulose degradation. Such fusion proteins could potentially substitute cloning of multiple enzymes as well as improving product yields.

Two-colour fluorescence fluorimetric analysis for direct quantification of bacteria and its application in monitoring bacterial growth in cellulose degradation systems

“…As the world's fossil fuel reserves deplete, there is a growing need to develop sustainable fuels (Creutzig et al, 2015, Fulton et al, 2015, Nuffield Council on Bioethics, 2011 (Duedu and French, 2016, French et al, 2015, Lakhundi et al, 2016 making it potentially easier to develop an ideal biofuel producing microorganism (IBPM) (French, 2009…”

Synergistic hydrolysis of filter paper by recombinant cellulase cocktails leveraging a key cellobiase, Cba2, of Cellulomonas biazotea

“…Despite working under unoptimized conditions, the filter paper activities obtained from the cooperation between Cba2 and Cba4 ( Figure 6 ) were found to be comparable to those achieved by some commercial cellulase preparations, ranging from 7.7 to 69.9 U ml −1 ( Yu et al, 2016 ). To date, although a large number of examples of cooperative action happening between Eng and Exg has been documented ( Ghose and Bisaria, 1979 ; Medve et al, 1998 ; Duedu and French, 2016 ), despite the presence of a wide collection of β-glucosidases characterized from a broad spectrum of microbial species, the ability of these enzymes to interact cooperatively among themselves has been rarely reported ( Mansfield et al, 1999 ). Leveraging the application of recombinant bacterial cellulases and the superb performance of Cba2 working either alone or in conjunction with Cba4, our work presented here provides evidence, for the first time, to demonstrate the occurrence of synergies among not only members of different types of cellulases, but also among the less commonly known cellobiases.…”

Molecular insight into cellulose degradation by the phototrophic green alga Scenedesmus

“…The 3D structure of this protein is available and there are also numerous works on the kinetic characterization of the enzyme and the identification of important amino acid residues for catalysis. 39 Among all the Scenedesmaceae genomes analyzed we found at least 11 enzymes, all of them putative GH10 bifunctional cellulase/ xylanase proteins (Figure 13A). These enzymes are monomeric proteins with a molecular mass ranging from 50 to 65 kDa, although there are also smaller variants of about 41 kDa in some fungi, such as Sclerotium rolfsii.…”