BioMatNet Item: AIR2-CT93-1272 - Cellulases and Hemicellulases from the Thermophilic Fungus Thermoascus aurantiacus

AIR2-CT93-1272
Cellulases and Hemicellulases from the Thermophilic Fungus Thermoascus aurantiacus

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Summary Information

	Proposal No:	AIR2-CT93-1272
	Date Prepared:	July 1996, June 1997, April 1998
	Source:	Second Annual Progress Report Final Technical Report Summary Final Technical Report

Second Annual Progress Report

INTRODUCTION

Plant cell walls contain three main components: cellulose which is insoluble and forms crystalline, compact fibres which are highly resistant to degradation; hemicellulose which is amorphous and more easily degraded; and lignin. The thermophilic fungus Thermoascus aurantiacus produces active hemicellulases - in particular, xylanase. Below, the main aspects of the project are highlighted.

This item is based on the final report of project 0321, which was subtitled Investigation of the Structure and Mechanism of Individual Enzymes and Study of the Organization of the Cellulase Complex and the second annual report from project 1271. The objective of the proposed work was to improve the feasibility of enzymatic conversion of cellulosic materials into sugars by gaining a better understanding of the factors that effect the efficiency of conversion by the enzymes responsible. The released sugars can be used as a carbon source for fermentations generating chemicals, pharmaceuticals and fuels, as well as in modification of animal feed. Such plant cell wall degrading enzymes also find applications in fibre processing, vegetable oil extraction, flavour release, textiles, detergent and chemicals. The main problems with current commercial enzymes relate to heat-stability, activity and pH profile. In both projects reported here thermophilic (heat loving) organisms are used since these provide a source of more stable enzymes. However, the approaches differ, reflecting differences between the bacterial system (in which the enzymes are organised into a complex known as the cellulosome) and the fungal system in which a wide range of enzymes are released into the growth medium.

OBJECTIVE

The primary objective is to obtain high yields of stable and efficient enzymes from a thermophilic fungus, Thermoascus aurantiacus, which produces a complete cellulase and hemicellulase enzyme system, for use in valorization of agricultural wastes of agronomic importance from colder (wheat straw) and warmer (sorghum bagasse) regions of the Community. Changes in policy concerning wheat straw burning combined with an increasing interest in sweet sorghum as a non-food crop will result in millions of tonnes of such residues becoming available. Biological treatment will yield sugars, chemicals, environmentally friendly paper pulp, peat substitutes and organic fertilizers. In a wider context the cellulolytic enzymes have applications in depolymerization of wastes from the agro-food industries and in the food industry for example in preparation of instant coffee, natural flavours and colours, and cleaning of industrial filters. Previous work on bacterial and fungal systems has established mechanisms, and exposed the limitations of the commercially available enzymes. This is the first integrated project aimed at overcoming deficiencies in enzyme yield, stability and specific activity using a thermophilic fungus, in this case T. aurantiacus.

This work will produce enzymes to be used at or above 70 慢 in industrial processes. Although these enzymes are potentially suited for commercial use, their yield and efficiency require improvement.

This will be done by protein engineering and cloning into T. reesei, whilst strain improvement of T. aurantiacus will also be attempted using UV and chemical mutagenesis. Mutants resistant to catabolite repression will be selected. Individual enzymes will be purified and characterized using novel substrates and physical techniques including crystallography. Based on their structure-function relationships, research will focus on cloning and modifying these enzymes in order to increase their yield and catalytic efficiency in collaboration with an industrial partner. Cellulase and hemicellulase production by wild and mutant strains will be optimized at pilot scale and used in trials for pulping and saccharification.

RESULTS

To date, a number of proteins enzymes have been isolated, purified and screened for use in saccharification, textiles, stone-washing and animal feed. Crude filtrates fractionated using ion-exchange, gel filtration, chromato-focusing and affinity chromatographies yielded seven cellulases (two b-glucosidases, two endoglucanases and four exoglucanases), six hemicellulases (two endoxylanases, one xylosidase, two acetyl esterase and one arabinofuranosidase with xylosidase activity). The major b-glucosidase, the major endoglucanase and the major endoxylanase have been characterised and the N-terminal sequence of four cellulase (major b-glucosidasemajor endoglucanase and two exoglucanases) components investigated. In addition, antibodies have been raised for six cellulase components and the amino acid composition of all seven cellulase components have been determined. Enzyme purification and characterisation was aided by the synthesis of specific affinity gels, model cello-oligosaccharides, specific ligands and inhibitors. Scale up of production and evaluation of low-cost culture medium were investigated for both submerged culture and solid state fermentation. Mutagenesis and cloning to enable the more interesting proteins to be expressed in other systems and/or to provide information which could enable the thermal stability of existing systems to be improved, as well as to increase the yield of protein and decrease end product and catabolic inhibition, have both proved more difficult than anticipated and are receiving priority in the final year. Structural studies have been initiated with production of good crystals and subsequent X-ray data-sets for the major endoglucanase and endoxylanase, which are currently being resolved.

Final Technical Report

Activities

Three approaches were taken to improve the cost-effectiveness of protein production: optimisation of growth conditions; production of mutants and cloning. In addition the links between function, structure and enhanced thermal stability were investigated as were possible applications. A PC-based information system covering many aspects of the production, science and use of plant cell wall degrading enzymes was constructed.

Methods

A number of strains of T.a were characterised and distributed to the participants. Attempts were made to optimise culture conditions, using low cost media, so that T.a produced elevated levels of cellulase and hemicellulase, in both liquid and solid culture as well as to investigate and model the production and distribution of enzymes between the cells and the media. Bulk filtrates were produced for protein purification and characterisation, using conventional techniques of ion exchange, gel filtration and isoelectric focusing as well as novel affinity gels produced during the project. Fractionation and purification procedures were established for seven cellulase (two b-glucosidases, two endoglucanases and four exoglucanases) and six hemicellulase (two endoxylanases, one xylosidase, two acetyl esterase and one arabinofuranosidase with xylosidase activity) components which were purified and characterised.

Artificial substrates, specific ligands and inhibitors, including xyloside, xylobioside, glucoside, lactoside, cellobioside and cellotrioside of 4 methyl umbelliferone (MU) were prepared where necessary (RUG) and used to characterise proteins and label crystals for X-ray analysis in structural work. In addition 4-MU derivatives of xylobiose and b-D-glucopyranosyl-xylopyranose were prepared (used to differentiate xylanases belonging to family F and family G), as were acetate esters and esters derived from dihydroferulic acid and 4-hydroxyphenylpropionic acid (used to differentiate acetyl esterase and ferulic acid esterase activities). Antibodies were raised for six cellulase components and the amino acid composition of all seven cellulase components and xylanase determined. The major endoglucanase and the major xylanase were both crystallised for structural studies resulting in a final model for xylanase, comparison of which with other family 10 xylanases, family 5 cellulases and family 17 barley glucanases suggests that hydrophobic interactions, hydrogen bonds and helix dipole stabilisation contribute to the thermostability of T.a, and the ways in which substrate specificity is effected. Structure studies of the major endoglucanase from T.a progressed fairly well, resulting in a good initial map, which will provide detailed information on substrate binding once the structure is fully refined.

Enzymes were screened in various applications, including saccharification, fabric processing, animal feed and human food modification. The possibility of using some of the enzymes in catalytic synthesis was also evaluated. An integrated information system covering plant cell wall degrading enzymes was constructed, combining an `expert system' based on multimedia presentation software covering organisms, enzymes and applications with various databases and graphic presentation packages covering composition of lignocellulosic raw materials, structure of plant cell wall degrading enzymes, commercial suppliers of enzymes, glossary and reference sections, accessible through an integrated front end, which could be developed further as a CD-ROM or site on the World Wide Web.

Exploitation

Both industrial partners obtained results capable of further development to commercial products. Sufficient information concerning potential uses of the enzymes in a number of applications as well as initial results from cloning of genes into another fungus should enable development towards commercial applications. A series of affinity materials, substrates and ligands were also developed which will be available to other researchers investigating glycolases (polysaccharide degrading enzymes). The information software developed is being developed further . The possibility of extending the Information System as an educational tool is being investigated.

Summary Final Technical Report

1. Partnership
This project was co-ordinated by CPL Scientific Ltd (CPL - UK) and has three contractors: The Institute of Food Research (IFR, Reading - UK), the Biosystems Technology Laboratory of the National Technical University of Athens (NTUA - Greece) and CAYLA a French company. Additional contributions were made by the Laboratorium voor Biochimie, University of Gent (RUG - Belgium) and the Laboratoire de Genie de Procedes et de Technologie Alimentaire in Villeneuve d'Ascq (INRA - France) as sub-contractors to CPL.

2. Objectives
The primary objectives of this work was to obtain high yields of thermostable extracellular proteins (enzymes) from the fungus Thermoascus aurantiacus (T.a) as well as separate and fully characterise these in terms of their ability to degrade plant cell walls (cellulases and hemicellulases), in order to use them in commercial applications. Three approaches were taken to improve the cost-effectiveness of protein production: optimisation of growth (NTUA), production of mutants (INRA) and cloning (CAYLA). In addition the links between function, structure and enhanced thermal stability (IFR) were investigated as were possible applications (CAYLA). A PC-based information system covering many aspects of the production, science and use of plant cell wall degrading enzymes was constructed (CPL).

3. Project Work
A number of strains of T.a were characterised and distributed to the participants. Attempts were made to optimise culture conditions, using low cost media, so that T.a produced elevated levels of cellulase and hemicellulase, in both liquid and solid culture as well as to investigate and model the production and distribution of enzymes between the cells and the media. Bulk filtrates were produced (CAYLA) for protein purification and characterisation (IFR), using conventional techniques of ion-exchange, gel filtration and isoelectric focusing as well as novel affinity gels produced during the project (RUG). Fractionation and purification procedures were established for seven cellulase (two b -glucosidases, two endoglucanases and four exoglucanases) and six hemicellulase (two endoxylanases, one xylosidase, two acetyl esterase and one arabinofuranosidase with xylosidase activity) components which were purified to homogeneity in mg quantities and characterised (IFR).

Artificial substrates, specific ligands and inhibitors, including xyloside, xylobioside, glucoside, lactoside, cellobioside and cellotrioside of 4-methylumbelliferone (MU) were prepared where necessary (RUG) and used to characterise proteins and lable crystals for X-ray analysis in structural work. In addition 4-MU derivatives of xylobiose and b -D-glucopyranosyl-xylopyranose were prepared (used to differentiate xylanases belonging to family F and family G), as were acetate esters and esters derived from dihydroferulic acid and 4-hydroxyphenylpropionic acid (used to differentiate acetyl esterase and ferulic acid esterase activities).

Antibodies were raised for six cellulase components and the amino acid composition of all seven cellulase components and xylanase determined. The major endoglucanase and the major xylanase were both crystallised for structural studies (IFR) resulting in a final model for xylanase, comparison of which with other family 10 xylanases, family 5 cellulases and family 17 barley glucanases suggests that hydrophobic interactions, hydrogen bonds and helix dipole stabilisation contribute to the thermostability of T.a, and the ways in which substrate specificity is effected.

Structure studies of the major endoglucanase from T.a progressed fairly well, resulting in a good initial map, which will provide detailed information on substrate binding once the structure is fully refined.

Due to numerous technical problems in handling this organism, extracting DNA and obtaining results by PCR, sequencing and cloning work did not go as far as hoped (CAYLA), nor were mutants capable of over-expressing active cell wall degrading enzymes obtained (INRA). However, sufficient protein was obtained for screening in various applications, including saccharification, fabric processing, animal feed and human food modification (CAYLA). The possibility of using some of the enzymes in catalytic synthesis was evaluated (IFR).

An integrated information system covering plant cell wall degrading enzymes was constructed (CPL). This combined an 'expert system' based on multimedia presentation software covering organisms, enzymes and applications with various databases and graphic presentation packages covering composition of lignocellulosic raw materials, structure of plant cell wall degrading enzymes, commercial suppliers of enzymes, glossary and reference sections, accessible through an integrated front end, which could be developed further as a CD-ROM or site on the World Wide Web.

4. Exploitation Results
Both industrial partners (CAYLA and CPL) obtained results capable of further development to commercial products. Sufficient information concerning potential uses of the enzymes in a number of applications as well as initial results from cloning of genes into another fungus has encouraged CAYLA to continue development towards a patentable application. The software developed by CPL is being developed further, applied both to EU-funded information dissemination activites related to the non-food agro-industrial research carried out under AIR and FAIR and to similar products covering areas such as non-chemical inputs for agriculture and forestry (agrobiologicals), sweeteners and organic waste treatment. The possibility of extending the Information System as an educational tool is being investigated.

The non-commercial partners benefited through expansion of their basic research knowledge base, providing training resulting in submission of a number of PhD theses (IFR and NTUA) as well as contributing to the scientific literature. In particular NTUA increased their experience of solid state fermentation, coming up with a novel reactor; IFR gained further information contributing to general hypotheses concerning structural-functional relationships and the basis of thermal stability in plant cell wall degrading enzymes while RUG developed a series of afffinity materials, substrates and ligands which will be available to other researchers investigating glycolases (polysaccharide degrading enzymes). With further work the basic research carried out by these partners could lead to development of commercial solid state fermentation for enzyme production, use of some of the enzymes in chemical catalysis and sale of some of the chemicals as research reagents.