Exoproteome analysis of Clostridium cellulovorans in natural soft-biomass degradation

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Abstract

Clostridium cellulovorans is an anaerobic, cellulolytic bacterium, capable of effectively degrading various types of soft biomass. Its excellent capacity for degradation results from optimization of the composition of the protein complex (cellulosome) and production of non-cellulosomal proteins according to the type of substrates. In this study, we performed a quantitative proteome analysis to determine changes in the extracellular proteins produced by C. cellulovorans for degradation of several types of natural soft biomass. C. cellulovorans was cultured in media containing bagasse, corn germ, rice straw (natural soft biomass), or cellobiose (control). Using an isobaric tag method and a liquid chromatograph equipped with a long monolithic silica capillary column/mass spectrometer, we identified 372 proteins in the culture supernatant. Of these, we focused on 77 saccharification-related proteins of both cellulosomal and non-cellulosomal origins. Statistical analysis showed that 18 of the proteins were specifically produced during degradation of types of natural soft biomass. Interestingly, the protein Clocel_3197 was found and commonly involved in the degradation of every natural soft biomass studied. This protein may perform functions, in addition to its known metabolic functions, that contribute to effective degradation of natural soft biomass.

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The cellulosomes: multienzyme machines for degradation of plant cell wall polysaccharides.

Edward A. Bayer, Jean-Pierre Belaich, Yuval Shoham … (2004)

The discrete multicomponent, multienzyme cellulosome complex of anaerobic cellulolytic bacteria provides enhanced synergistic activity among the different resident enzymes to efficiently hydrolyze intractable cellulosic and hemicellulosic substrates of the plant cell wall. A pivotal noncatalytic subunit called scaffoldin secures the various enzymatic subunits into the complex via the cohesin-dockerin interaction. The specificity characteristics and tenacious binding between the scaffoldin-based cohesin modules and the enzyme-borne dockerin domains dictate the supramolecular architecture of the cellulosome. The diversity in cellulosome architecture among the known cellulosome-producing bacteria is manifest in the arrangement of their genes in either multiple-scaffoldin or enzyme-linked clusters on the genome. The recently described three-dimensional crystal structure of the cohesin-dockerin heterodimer sheds light on the critical amino acids that contribute to this high-affinity protein-protein interaction. In addition, new information regarding the regulation of cellulosome-related genes, budding genetic tools, and emerging genomics of cellulosome-producing bacteria promises new insight into the assembly and consequences of the multienzyme complex.

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Evolution, substrate specificity and subfamily classification of glycoside hydrolase family 5 (GH5)

Henrik Aspeborg, Pedro M Coutinho, Yang Wang … (2012)

Background The large Glycoside Hydrolase family 5 (GH5) groups together a wide range of enzymes acting on β-linked oligo- and polysaccharides, and glycoconjugates from a large spectrum of organisms. The long and complex evolution of this family of enzymes and its broad sequence diversity limits functional prediction. With the objective of improving the differentiation of enzyme specificities in a knowledge-based context, and to obtain new evolutionary insights, we present here a new, robust subfamily classification of family GH5. Results About 80% of the current sequences were assigned into 51 subfamilies in a global analysis of all publicly available GH5 sequences and associated biochemical data. Examination of subfamilies with catalytically-active members revealed that one third are monospecific (containing a single enzyme activity), although new functions may be discovered with biochemical characterization in the future. Furthermore, twenty subfamilies presently have no characterization whatsoever and many others have only limited structural and biochemical data. Mapping of functional knowledge onto the GH5 phylogenetic tree revealed that the sequence space of this historical and industrially important family is far from well dispersed, highlighting targets in need of further study. The analysis also uncovered a number of GH5 proteins which have lost their catalytic machinery, indicating evolution towards novel functions. Conclusion Overall, the subfamily division of GH5 provides an actively curated resource for large-scale protein sequence annotation for glycogenomics; the subfamily assignments are openly accessible via the Carbohydrate-Active Enzyme database at http://www.cazy.org/GH5.html.

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Substrate and Enzyme Characteristics that Limit Cellulose Hydrolysis.

Mansfield, Mooney, Saddler (1999)

The ability and, consequently, the limitations of various microbial enzyme systems to completely hydrolyze the structural polysaccharides of plant cell walls has been the focus of an enormous amount of research over the years. As more and more of these extracellular enzymatic systems are being identified and characterized, clear similarities and differences are being elucidated. Although much has been learned concerning the structures, kinetics, catalytic action, and interactions of enzymes and their substrates, no single mechanism of total lignocellulosic saccharification has been established. The heterogeneous nature of the supramolecular structures of naturally occurring lignocellulosic matrices make it difficult to fully understand the interactions that occur between enzyme complexes and these substrates. However, it is apparent that the efficacy of enzymatic complexes to hydrolyze these substrates is inextricably linked to the innate structural characteristics of the substrate and/or the modifications that occur as saccharification proceeds. This present review is not intended to conclusively answer what factors control polysaccharide biodegradation, but to serve as an overview illustrating some of the potential enzymatic and structural limitations that invariably influence the complete hydrolysis of lignocellulosic polysaccharides.

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Author and article information

Contributors

Kohei Esaka: esaka.kohei.42x@st.kyoto-u.ac.jp

Shunsuke Aburaya: aburaya.shunsuke.37r@st.kyoto-u.ac.jp

Hironobu Morisaka: morisaka@kais.kyoto-u.ac.jp

Kouichi Kuroda: k_kuro@kais.kyoto-u.ac.jp

Mitsuyoshi Ueda: miueda@kais.kyoto-u.ac.jp

Journal

Journal ID (nlm-ta): AMB Express

Journal ID (iso-abbrev): AMB Express

Title: AMB Express

Publisher: Springer Berlin Heidelberg (Berlin/Heidelberg )

ISSN (Electronic): 2191-0855

Publication date (Electronic): 24 January 2015

Publication date PMC-release: 24 January 2015

Publication date Collection: December 2015

Volume: 5

Issue: 1

Electronic Location Identifier: 2

Affiliations

[ ]Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto Japan

[ ]Kyoto Integrated Science and Technology Bio-Analysis Center, Shimogyo-ku, Kyoto Japan

Article

Publisher ID: 89

DOI: 10.1186/s13568-014-0089-9

PMC ID: 4305082

SO-VID: bbfb431b-c351-4a7b-a73b-96dbe009f245

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This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited.

History

Date received : 21 November 2014

Date accepted : 23 December 2014

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ScienceOpen disciplines: Biotechnology

Keywords: clostridium cellulovorans,cellulosome,soft-biomass degradation,proteome analysis,monolithic column

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ScienceOpen disciplines: Biotechnology

Keywords: clostridium cellulovorans, cellulosome, soft-biomass degradation, proteome analysis, monolithic column

Exoproteome analysis of Clostridium cellulovorans in natural soft-biomass degradation

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Most cited references 30

The cellulosomes: multienzyme machines for degradation of plant cell wall polysaccharides.

Evolution, substrate specificity and subfamily classification of glycoside hydrolase family 5 (GH5)

Substrate and Enzyme Characteristics that Limit Cellulose Hydrolysis.

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