R. A. J. Warren

Cellulose Hydrolysis by Bacteria and Fungi

Publisher Summary The chapter focuses on the recent advances in understanding the structural and functional organization of individual cellulases, their regulation, and the ways in which the multiple enzyme components of cellulolytic systems cooperate. It overviews the cellulose structures because cellulose is more than a homopolymer of β-1 ,4 linked glucose units. An appreciation of its complex physical organization and its interactions with other plant cell wall components is central for understanding of the mechanisms of cellulase action. Cellulose nearly always occurs in close association with plant cell wall matrix polysaccharides so that enzymes such as xylanases are intimately involved in the attack of cellulose in vivo. Cellulases effect important changes to their substrate before releasing soluble products and the key to understanding cellulase action rest in the examination of these events. Progress in this area is limited by the availability of appropriate analytical tools although new techniques, such as atomic force microscopy are promising. The properties of cellulases are profoundly altered by the presence of trace enzyme contaminants. Future studies in vitro are proposed to be restricted to enzymes from recombinant sources. The reasons for the individual cellulolytic bacteria and fungi requiring many related cellulases with specificities that overlap is still not clear, but perhaps this is because the complexity of the substrates and of the task these microorganisms face is underestimated.

A scheme for designating enzymes that hydrolyse the polysaccharides in the cell walls of plants

A scheme is proposed for designating enzymes that hydrolyse the polysaccharides in the cell walls of plants. These enzymes are predominantly β‐1,4‐glycanases. The scheme is based on the classification of the catalytic domains of glycoside hydrolases into families of related amino acid sequences. The new designation for an enzyme indicates its family and, because all members of a family have these characteristics in common, its three‐dimensional fold and stereospecificity of hydrolysis. The scheme is intended to simplify comparison of the systems of enzymes produced by different microorganisms for the hydrolysis of plant cell walls.

Structure of the gene encoding the exoglucanase of Cellulomonas fimi

In Cellulomonas fimi the cex gene encodes an exoglucanase (Exg) involved in the degradation of cellulose. The gene now has been sequenced as part of a 2.58-kb fragment of C. fimi DNA. The cex coding region of 1452 bp (484 codons) was identified by comparison of the DNA sequence to the N-terminal amino acid (aa) sequence of the Exg purified from C. fimi. The Exg sequence is preceded by a putative signal peptide of 41 aa, a translational initiation codon, and a sequence resembling a ribosome-binding site five nucleotides (nt) before the initiation codon. The nt sequence immediately following the translational stop codon contains four inverted repeats, two of which overlap, and which can be arranged in stable secondary structures. The codon usage in C. fimi appears to be quite different from that of Escherichia coli. A dramatic (98.5%) bias occurs for G or C in the third position for the 35 codons utilized in the cex gene.

Glycosylation of bacterial cellulases prevents proteolytic cleavage between functional domains

Glycosylated cellulases from Cellulomonas fimi were compared with their non‐glycosylated counterparts synthesized in Escherichia coli from recombinant DNA. Glycosylation of the enzymes does not significantly affect their kinetic properties, or their stabilities towards heat and pH. However, the glycosylated enzymes are protected from attack by a C. fimi protease when bound to cellulose, while the non‐glycosylated enzymes yield active, truncated products with greatly reduced affinity for cellulose.

THE TOPOLOGY OF THE SUBSTRATE BINDING CLEFTS OF GLYCOSYL HYDROLASE FAMILY 10 XYLANASES ARE NOT CONSERVED

The crystal structures of family 10 xylanases indicate that the distal regions of their active sites are quite different, suggesting that the topology of the substrate binding clefts of these enzymes may vary. To test this hypothesis, we have investigated the rate and pattern of xylooligosaccharide cleavage by the family 10 enzymes, Pseudomonas fluorescens subsp.cellulosa xylanase A (XYLA) and Cellulomonas fimi exoglucanase, Cex. The data showed that Cex contained three glycone and two aglycone binding sites, while XYLA had three glycone and four aglycone binding sites, supporting the view that the topologies of substrate binding clefts in family 10 glycanases are not highly conserved. The importance of residues in the substrate binding cleft of XYLA in catalysis and ligand binding were evaluated using site-directed mutagenesis. In addition to providing insight into the function of residues in the glycone region of the active site, the data showed that the aromatic residues Phe-181, Tyr-255, and Tyr-220 play important roles in binding xylose moieties, via hydrophobic interactions, at subsites +1, +3, and +4, respectively. Interestingly, the F181A mutation caused a much larger reduction in the activity of the enzyme against xylooligosaccharides compared with xylan. These data, in conjunction with a previous study (Charnock, S. J., Lakey, J. H., Virden, R., Hughes, N., Sinnott, M. L., Hazlewood, G. P., Pickersgill, R., and Gilbert, H. J. (1997)J. Biol. Chem. 272, 2942–2951), suggest that the binding of xylooligosaccharides at the −2 and +1 subsites ensures that the substrates occupy the −1 and +1 subsites and thus preferentially form productive complexes with the enzyme. Loss of ligand binding at either subsite results in small substrates forming nonproductive complexes with XYLA by binding to distal regions of the substrate binding cleft.

MOLECULAR CLONING OF A CELLULOMONAS FIMI CELLULOSE GENG IN ESCHERICIA COLI

A sensitive and simple immunoassay was developed to screen Escherichia coli transformed with recombinant DNA plasmids carrying a cellulase gene. The assay was used to identify a recombinant DNA plasmid carrying at least one cellulase gene from Cellulomonas fimi. The enzyme present in extracts of E. coli carrying the plasmid was active in catalysing the hydrolysis of carboxymethylcellulose as indicated by the production of reducing sugars.Abstract A sensitive and simple immunoassay was developed to screen Escherichia coli transformed with recombinant DNA plasmids carrying a cellulase gene. The assay was used to identify a recombinant DNA plasmid carrying at least one cellulase gene from Cellulomonas fimi . The enzyme present in extracts of E. coli carrying the plasmid was active in catalysing the hydrolysis of carboxymethylcellulose as indicated by the production of reducing sugars.

Directed evolution of new glycosynthases from Agrobacterium β-glucosidase: a general screen to detect enzymes for oligosaccharide synthesis

BACKGROUND Oligosaccharide synthesis is becoming increasingly important to industry as diverse therapeutic roles for these molecules are discovered. The chemical synthesis of oligosaccharides on an industrial scale is often prohibitively complex and costly. An alternative, that of enzymatic synthesis, is limited by the difficulty of obtaining an appropriate enzyme. A general screen for enzymes that catalyze the synthesis of the glycosidic bond would enable the identification and engineering of new or improved enzymes. RESULTS Glycosynthases are nucleophile mutants of retaining glycosidases that efficiently catalyze the synthesis of the glycosidic linkage by condensing an activated glycosyl fluoride donor with a suitable acceptor sugar. A novel agar plate-based coupled-enzyme screen was developed (using a two-plasmid system) and used to select an improved glycosynthase from a library of mutants. CONCLUSIONS Plate-based coupled-enzyme screens of this type are extremely valuable for identification of functional synthetic enzymes and can be applied to the evolution of a range of glycosyl transferases.

The Cellulase System of Cellulomonas fimi

Supernatants from cultures of Cellulomonas fimi contained up to 10 components with CM-cellulase activity as determined by non-denaturing PAGE. Some of the active components were glycosylated. The activity profiles of the supernatants, as determined by PAGE, varied with the cellulosic substrate used for the growth of a culture, with culture age, and with storage of the supernatants. These variations were a consequence of proteolysis and a reduction in the glycosylation of some of the components. Proteinase activity in the supernatants was induced by growth of C. fimi on cellulosic substrates. Proteolysis and a reduction in glycosylation resulted in the conversion of slow-moving into fast-moving components on non-denaturing PAGE. The fast-moving components had a reduced ability to bind to an insoluble cellulosic substrate such as Avicel. Several of the CM-cellulase activities in culture supernatants were immunologically related. In contrast to the large number of CM-cellulases found in the supernatant, substrate-bound activity comprised only three slow-moving components, at least some of which were glycosylated. It was concluded that the cellulase system of C. fimi was composed of only three enzymes, and that these enzymes had a great affinity for, and were stabilized by binding to, an insoluble cellulosic substrate. Enzymes which accumulated free in the culture medium were subject to limited proteolysis and de-glycosylation which generated a variety of products, some of which retained enzymic activity.

Isolation and characterization of Escherichia coli clones expressing cellulase genes from Cellulomonas fimi

A sensitive immunoassay developed previously was used to isolate a series of Escherichia coli clones expressing cellulase genes from Cellulomonas fimi. The clones fell into three groups. Clones in the first group contained plasmids with a 6.6 kb insert of C. fimi DNA, were strongly antigenic, and contained low levels of CM-cellulase activity. Those in the second group contained plasmids with a 5.0 kb insert, were weakly antigenic, and contained high levels of CM-cellulase activity. Those in the third group contained plasmids with a 5.6 kb insert, were weakly antigenic, and contained low levels of CM-cellulase activity. Restriction analysis showed that the three groups carried different cellulase genes. All of these CM-cellulase activities were exported to the periplasm in E. coli. but with different efficiencies. These activities represented important components of the C. fimi cellulase complex. Their properties indicated that they were different from each other and that they probably had complementary actions.

Visualization of the adsorption of a bacterial endo-β-1,4-glucanase and its isolated cellulose-binding domain to crystalline cellulose

Endo-beta-1,4-glucanase A (CenA), a cellulase from the bacterium Cellulomonas fimi, is composed of two domains: a catalytic domain and a cellulose-binding domain. Adsorption of CenA and its isolated cellulose-binding domain (CBD.PTCenA) to Valonia cellulose microcrystals was examined by transmission electron microscopy using an antibody sandwich technique (CenA/CBD.PTCenA-alpha CenA IgG-protein A-gold conjugate). Adsorption of both CenA and CBD.PTCenA occurred along the lengths of the microcrystals, with an apparent preference for certain crystal faces or edges. CenA or CBD.PTCenA, but not the isolated catalytic domain, were shown to prevent the flocculation of microcrystalline bacterial cellulose. The cellulose-binding domain may assist crystalline cellulose hydrolysis in vitro by promoting substrate dispersion.