A novel polysaccharide hydrolase cDNA (celD) from Neocallimastix patriciarum encoding three multi-functional catalytic domains with high endoglucanase, cellobiohydrolase and xylanase activities
TL;DR: Substrate competition studies showed that carboxymethylcellulose and xylan appear to compete with methylumbelliferyl cellobioside for the same active site within each domain of celD.
Abstract: A plant polysaccharide hydrolase cDNA, designated celD, was isolated from a cDNA library of the rumen fungus Neocallimastix patriciarum. The enzyme encoded by celD had endoglucanase, cellobiohydrolase and xylanase activities. Deletion analysis revealed that celD cDNA can be truncated to code for three catalytically active domains. Each domain had the same substrate specificity as the enzyme produced by the untruncated celD and also possessed cellulose-binding capacity. Substrate competition studies showed that carboxymethylcellulose and xylan appear to compete with methylumbelliferyl cellobioside for the same active site within each domain. Expression of celD transcript in the rumen fungus was constitutive and was not affected by the presence of cellulose in the culture medium.
Citations
More filters
TL;DR: A concluding discussion identifies unresolved issues pertaining to microbial cellulose utilization, suggests approaches by which such issues might be resolved, and contrasts a microbially oriented cellulose hydrolysis paradigm to the more conventional enzymatically oriented paradigm in both fundamental and applied contexts.
Abstract: Fundamental features of microbial cellulose utilization are examined at successively higher levels of aggregation encompassing the structure and composition of cellulosic biomass, taxonomic diversity, cellulase enzyme systems, molecular biology of cellulase enzymes, physiology of cellulolytic microorganisms, ecological aspects of cellulase-degrading communities, and rate-limiting factors in nature. The methodological basis for studying microbial cellulose utilization is considered relative to quantification of cells and enzymes in the presence of solid substrates as well as apparatus and analysis for cellulose-grown continuous cultures. Quantitative description of cellulose hydrolysis is addressed with respect to adsorption of cellulase enzymes, rates of enzymatic hydrolysis, bioenergetics of microbial cellulose utilization, kinetics of microbial cellulose utilization, and contrasting features compared to soluble substrate kinetics. A biological perspective on processing cellulosic biomass is presented, including features of pretreated substrates and alternative process configurations. Organism development is considered for "consolidated bioprocessing" (CBP), in which the production of cellulolytic enzymes, hydrolysis of biomass, and fermentation of resulting sugars to desired products occur in one step. Two organism development strategies for CBP are examined: (i) improve product yield and tolerance in microorganisms able to utilize cellulose, or (ii) express a heterologous system for cellulose hydrolysis and utilization in microorganisms that exhibit high product yield and tolerance. A concluding discussion identifies unresolved issues pertaining to microbial cellulose utilization, suggests approaches by which such issues might be resolved, and contrasts a microbially oriented cellulose hydrolysis paradigm to the more conventional enzymatically oriented paradigm in both fundamental and applied contexts.
4,769 citations
TL;DR: 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.
Abstract: 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.
686 citations
TL;DR: In general, the systems produced by different microorganisms for the hydrolysis of a particular polysaccharide comprise similar enzymes from the same families, which are complex on two quite different levels.
Abstract: Microorganisms are efficient degraders of starch, chitin, and the polysaccharides in plant cell walls. Attempts to purify hydrolases led to the realization that a microorganism may produce a multiplicity of enzymes, referred to as a system, for the efficient utilization of a polysaccharide. In order to fully characterize a particular enzyme, it must be obtained free of the other components of a system. Quite often, this proves to be very difficult because of the complexity of a system. This realization led to the cloning of the genes encoding them as an approach to eliminating other components. More than 400 such genes have been cloned and sequenced, and the enzymes they encode have been grouped into more than 50 families of related amino acid sequences. The enzyme systems revealed in this manner are complex on two quite different levels. First, many of the individual enzymes are complex, as they are modular proteins comprising one or more catalytic domains linked to ancillary domains that often include one or more substrate-binding domains. Second, the systems are complex, comprising from a few to 20 or more enzymes, all of which hydrolyze a particular substrate. Systems for the hydrolysis of plant cell walls usually contain more components than systems for the hydrolysis of starch and chitin because the cell walls contain several polysaccharides. In general, the systems produced by different microorganisms for the hydrolysis of a particular polysaccharide comprise similar enzymes from the same families.
386 citations
TL;DR: The phylogenetic novelty of the hydrolases suggests that some of them may have potential for new applications in biocatalysis and the sequences and contexts of neighbouring genes suggested suggested tentative phylogenetic assignments of the rumen organisms producing the retrieved enzymes.
Abstract: A metagenome expression library of bulk DNA extracted from the rumen content of a dairy cow was established in a phage lambda vector and activity-based screening employed to explore the functional diversity of the microbial flora. Twenty-two clones specifying distinct hydrolytic activities (12 esterases, nine endo-beta-1,4-glucanases and one cyclodextrinase) were identified in the library and characterized. Sequence analysis of the retrieved enzymes revealed that eight (36%) were entirely new and formed deep-branched phylogenetic lineages with no close relatives among known ester- and glycosyl-hydrolases. Bioinformatic analyses of the hydrolase gene sequences, and the sequences and contexts of neighbouring genes, suggested tentative phylogenetic assignments of the rumen organisms producing the retrieved enzymes. The phylogenetic novelty of the hydrolases suggests that some of them may have potential for new applications in biocatalysis.
277 citations
TL;DR: This review describes the life-cycles and physiology of anaerobic fungi, details their interactions with other rumen micro-organisms and assesses their contribution to the digestion of plant material in herbivores.
228 citations
References
More filters
Book•
15 Jan 2001
TL;DR: Molecular Cloning has served as the foundation of technical expertise in labs worldwide for 30 years as mentioned in this paper and has been so popular, or so influential, that no other manual has been more widely used and influential.
Abstract: Molecular Cloning has served as the foundation of technical expertise in labs worldwide for 30 years. No other manual has been so popular, or so influential. Molecular Cloning, Fourth Edition, by the celebrated founding author Joe Sambrook and new co-author, the distinguished HHMI investigator Michael Green, preserves the highly praised detail and clarity of previous editions and includes specific chapters and protocols commissioned for the book from expert practitioners at Yale, U Mass, Rockefeller University, Texas Tech, Cold Spring Harbor Laboratory, Washington University, and other leading institutions. The theoretical and historical underpinnings of techniques are prominent features of the presentation throughout, information that does much to help trouble-shoot experimental problems. For the fourth edition of this classic work, the content has been entirely recast to include nucleic-acid based methods selected as the most widely used and valuable in molecular and cellular biology laboratories. Core chapters from the third edition have been revised to feature current strategies and approaches to the preparation and cloning of nucleic acids, gene transfer, and expression analysis. They are augmented by 12 new chapters which show how DNA, RNA, and proteins should be prepared, evaluated, and manipulated, and how data generation and analysis can be handled. The new content includes methods for studying interactions between cellular components, such as microarrays, next-generation sequencing technologies, RNA interference, and epigenetic analysis using DNA methylation techniques and chromatin immunoprecipitation. To make sense of the wealth of data produced by these techniques, a bioinformatics chapter describes the use of analytical tools for comparing sequences of genes and proteins and identifying common expression patterns among sets of genes. Building on thirty years of trust, reliability, and authority, the fourth edition of Mol
215,169 citations
TL;DR: A chemically modified phage T7 DNA polymerase has three properties that make it ideal for DNA sequencing by the chain-termination method; the enzyme is highly processive, catalyzing the polymerization of thousands of nucleotides without dissociating, and processive synthesis with dITP in place of dGTP eliminates band compressions.
Abstract: A chemically modified phage T7 DNA polymerase has three properties that make it ideal for DNA sequencing by the chain-termination method. The enzyme is highly processive, catalyzing the polymerization of thousands of nucleotides without dissociating. By virtue of the modification the 3' to 5' exonuclease activity is eliminated. The modified polymerase efficiently uses nucleotide analogs that increase the electrophoretic resolution of bands in gels. Consequently, dideoxynucleotide-terminated fragments have highly uniform radioactive intensity throughout the range of a few to thousands of nucleotides in length. There is virtually no background due to terminations at pause sites or secondary-structure impediments. Processive synthesis with dITP in place of dGTP eliminates band compressions, making possible the unambiguous determination of sequences from a single orientation.
2,128 citations
TL;DR: The interaction of the direct dye Congo red with intact beta-D-glucans provides the basis for a rapid and sensitive assay system for bacterial strains possessing beta-( 1 leads to 4),(1 leads to 3)-D- glucanohydrolase, and beta-(1 leading to 4)-D -glucanhydrolase activities.
Abstract: The interaction of the direct dye Congo red with intact beta-D-glucans provides the basis for a rapid and sensitive assay system for bacterial strains possessing beta-(1 leads to 4),(1 leads to 3)-D-glucanohydrolase, beta-(1 leads to 4)-D-glucanohydrolase, and beta-(1 leads to 3)-D-glucanohydrolase activities. A close correspondence was observed between cellulolytic activity and beta-(1 leads to 4)-D-glucanohydrolase and beta-(1 leads to 4),(1 leads to 3)-D-glucanohydrolase activities in isolates from the bovine rumen. Many of these isolates also possessed beta-(1 leads to 3)-D-glucanohydrolase activity, and this characteristic may have taxonomic significance.
1,783 citations
Book•
01 Jan 1969
TL;DR: The reviewer was left with the impression that more attention is paid to these tests in the author's departments than is usually done in Great Britain.
Abstract: Chapter I contains an adequate account of the chemistry of the plasma proteins. This section could, of course, have been expanded, but to have done so would have upset the balance of the book. Study of this chapter would be a good introduction to the more detailed original literature. The chemical methods of determining proteins are described in Chapter II. Chapter III-' Methods of Examination '-describes the procedures such as salting-in, salting-out, precipitation by various combinations of organic solvents at low concentration and electrolytes (cf. the studies of E. J. Cohn and his colleagues), electrophoresis (in free solution and on supporting media), immunoelectrophoresis and ultracentrifugation, which have provided so much of the present body of knowledge concerning the plasma proteins. Chapter IV, 'Clinical Chemical Methods ', and Chapter V, ' Clinical Significance of the Plasma Proteins ', deal with the practical details and the interpretation of the results of the various ' empirical' tests which depend upon changes in the composition and stability of the plasma proteins. The reviewer was left with the impression that more attention is paid to these tests in the author's departments than is usually done in Great Britain. Chapter VI, 'Clinical Significance of the Dysproteinaemias and Paraproteinaemias', reduplicates some of the material given in the earlier chapters but is, in the present writer's opinion, the best in the book.
1,408 citations
TL;DR: The beta-1, 4-glycanases appear to have arisen by the shuffling of a relatively small number of progenitor sequences, and some of the enzymes contain repeated sequences up to 150 amino acids in length.
836 citations
"A novel polysaccharide hydrolase cD..." refers background in this paper
...Structural studies have revealed that many cellulases consist of at least two distinct functional domains: a catalytic domain and a cellulose-binding domain (Gilkes et al., 1991 ; Goyal et aZ., 1991 ; BCguin, 1990)....
[...]
...For most other cellulases, the enzyme activity and cellulose-binding capacity are located in two discrete domains, which are usually separated by sequences rich in proline and hydroxyamino residues (Gilkes et al., 1991; Goyal et al., 1991; Bkguin, 1990)....
[...]