Hemicellulolytic microorganisms play a significant role in nature by recycling hemicellulose, one of the main components of plant polysaccharides. Xylanases (EC 3.2.1.8) catalyze the hydrolysis of xylan, the major constituent of hemicellulose. The use of these enzymes could greatly improve the overall economics of processing lignocellulosic materials for the generation of liquid fuels and chemicals. Recently cellulase-free xylanases have received great attention in the development of environmentally friendly technologies in the paper and pulp industry. In microorganisms that produce xylanases low molecular mass fragments of xylan and their positional isomers play a key role in regulating its biosynthesis. Xylanase and cellulase production appear to be regulated separately, although the pleiotropy of mutations, which causes the elimination of both genes, suggests some linkage in the synthesis of the two enzymes. Xylanases are found in a cornucopia of organisms and the genes encoding them have been cloned in homologous and heterologous hosts with the objectives of overproducing the enzyme and altering its properties to suit commercial applications. Sequence analyses of xylanases have revealed distinct catalytic and cellulose binding domains, with a separate non-catalytic domain that has been reported to confer enhanced thermostability in some xylanases. Analyses of three-dimensional structures and the properties of mutants have revealed the involvement of specific tyrosine and tryptophan residues in the substrate binding site and of glutamate and aspartate residues in the catalytic mechanism. Many lines of evidence suggest that xylanases operate via a double displacement mechanism in which the anomeric configuration is retained, although some of the enzymes catalyze single displacement reactions with inversion of configuration. Based on a dendrogram obtained from amino acid sequence similarities the evolutionary relationship between xylanases is assessed. In addition the properties of xylanases from extremophilic organisms have been evaluated in terms of biotechnological applications.

Molecular and biotechnological aspects of xylanases

Multiplicity of beta-1,4-xylanase in microorganisms: functions and applications.

Thermophilic fungi are a small assemblage in mycota that have a minimum temperature of growth at or above 20°C and a maximum temperature of growth extending up to 60 to 62°C. As the only representatives of eukaryotic organisms that can grow at temperatures above 45°C, the thermophilic fungi are valuable experimental systems for investigations of mechanisms that allow growth at moderately high temperature yet limit their growth beyond 60 to 62°C. Although widespread in terrestrial habitats, they have remained underexplored compared to thermophilic species of eubacteria and archaea. However, thermophilic fungi are potential sources of enzymes with scientific and commercial interests. This review, for the first time, compiles information on the physiology and enzymes of thermophilic fungi. Thermophilic fungi can be grown in minimal media with metabolic rates and growth yields comparable to those of mesophilic fungi. Studies of their growth kinetics, respiration, mixed-substrate utilization, nutrient uptake, and protein breakdown rate have provided some basic information not only on thermophilic fungi but also on filamentous fungi in general. Some species have the ability to grow at ambient temperatures if cultures are initiated with germinated spores or mycelial inoculum or if a nutritionally rich medium is used. Thermophilic fungi have a powerful ability to degrade polysaccharide constituents of biomass. The properties of their enzymes show differences not only among species but also among strains of the same species. Their extracellular enzymes display temperature optima for activity that are close to or above the optimum temperature for the growth of organism and, in general, are more heat stable than those of the mesophilic fungi. Some extracellular enzymes from thermophilic fungi are being produced commercially, and a few others have commercial prospects. Genes of thermophilic fungi encoding lipase, protease, xylanase, and cellulase have been cloned and overexpressed in heterologous fungi, and pure crystalline proteins have been obtained for elucidation of the mechanisms of their intrinsic thermostability and catalysis. By contrast, the thermal stability of the few intracellular enzymes that have been purified is comparable to or, in some cases, lower than that of enzymes from the mesophilic fungi. Although rigorous data are lacking, it appears that eukaryotic thermophily involves several mechanisms of stabilization of enzymes or optimization of their activity, with different mechanisms operating for different enzymes.

Thermophilic Fungi: Their Physiology and Enzymes

Microbial 2,3-butanediol production: A state-of-the-art review

Production of fungal xylanases

Production of thermostable xylanase by a thermophilic fungus, Thermoascus aurantiacus

Several highly cellulolytic fungi were initially screened for their cellulase and β-glucosidase activities. Culture filtrates from Trichoderma E58 demonstrated the highest β-glucosidase activity, giving a value of 1.0 IU/mg protein. A variety of different cellulose and pretreated wood substrates were hydrolysed by various cellulase preparations. The importance of the pretreatment conditions and ensuing chemical extractions of the cellulosic substrates was demonstrated by the range of sugar and ethanol values obtained after saccharification and fermentation of the liberated sugars. The rate of hydrolysis of the cellulosic substrate by Trichoderma E58 culture filtrates, concentrated culture filtrates, and whole cell cultures was compared. An ethanol value of 2.2% (w/v) could be obtained after hydrolysis of 5% Solka floe by concentrated culture filtrates of Trichoderma E58 and fermentation of the liberated sugars by Zymomonas mobilis or Saccharomyces cerevisiae.

Ethanol fermentation of enzymatically hydrolysed pretreated wood fractions using Trichoderma cellulases, Zymomonas mobilis, and Saccharomyces cerevisiae

The reducing sugars, glucose, and ethanol produced during growth of the anaerobes Clostridium thermocellum and Acetivibrio cellulolyticus on cellulose were assayed. Zymomonas mobilis was grown under similar conditions and could ferment glucose to ethanol. The ethanol production by the cellulolytic bacteria alone and in co-culture with Zymomonas is described. Approximately 27% of a 1% cellulose substrate could be converted to ethanol by this co-culture.

A one step process for the conversion of cellulose to ethanol using anaerobic microorganisms in mono- and co-culture

A sequential co-culture approach was investigated for the conversion of lignocellulosic substrates to butanediol and ethanol. Growth of Clostridium thermocellum on solka floc and aspenwood xylan resulted in the release of extracellular endoglucanase and xylanase enzymes into the culture medium. Low levels of fermentation products were formed and unutilized sugars accumulated in the medium. Inoculation of Klebsiella pneumoniae as a sequential culture resulted in the rapid utilization of the accumulated sugars and the formation of additional fermentation products, including butanediol, ethanol, and acetoin. This approach was applicable to the use of mixed cellulose and hemicellulose substrates, including steam-exploded aspenwood. Further improvement in solvent production from steam-exploded substrates could be obtained by using a fed-batch approach to circumvent the problem of inhibitors associated with the natural substrates.

Maria K. H. Chan

Papers

Production of thermostable xylanase by a thermophilic fungus, Thermoascus aurantiacus

Ethanol fermentation of enzymatically hydrolysed pretreated wood fractions using Trichoderma cellulases, Zymomonas mobilis, and Saccharomyces cerevisiae

A one step process for the conversion of cellulose to ethanol using anaerobic microorganisms in mono- and co-culture

Butanediol production from lignocellulosic substrates by Klebsiella pneumoniae grown in sequential co-culture with Clostridium thermocellum