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SUMMARY 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.

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An extracellular β-glucosidase from Thermoascus aurantiacus was purified to homogeneity by DEAE-Sepharose, Ultrogel AcA 44 and Mono-P column chromatography. The enzyme was a homotrimer, with a monomer molecular mass of 120kDa; only the trimer was optimally active at 80°C and at pH 4.5. At 90°C, the enzyme showed 70% of its optimal activity. It was stable at pH 5.2 and at temperatures up to 70°C for 48h, but stability decreased above 70°C and at pH values above and below 5.0. The enzyme hydrolysed aryl and alkyl β-d-glucosides and cello-oligosaccharides, and was specific for substrates with a β-glycosidic linkage. The hydroxy groups at positions 2, 4 and 6 of a glucose residue at the non-reducing end of a disaccharide appeared to be essential for catalysis. The enzyme had the lowest Km towards p-nitrophenyl β-d-glucoside (0.1137mM) and the highest kcat towards cellobiose and β,β-trehalose (17052min-1). It released one glucose unit at a time from the non-reducing end of cello-oligosaccharides, and the rate of hydrolysis decreased with an increase in chain length. Glucose and d-δ-gluconolactone inhibited the β-glucosidase competitively, with Ki values of 0.29mM and 8.3nM respectively, while methanol, ethanol and propan-2-ol activated the enzyme. The enzyme catalysed the synthesis of methyl, ethyl and propyl β-d-glucosides in the presence of methanol, ethanol and propan-2-ol respectively with either glucose or cellobiose, although cellobiose was preferred. An acidic pH favoured hydrolysis and transglycosylation, but high concentrations of alcohols favoured the latter reaction. The stereochemistry of cellobiose hydrolysis revealed that β-glucosidase from T. aurantiacus is a retaining glycosidase, while N-terminal amino acid sequence alignment indicated that it is a member of glycoside hydrolase family 3.

This review considers the structure of cellulose and hemicellulose, enzymology of cellulases and xylanases (sources, enzyme assays, substrate specificity, kinetics and subsite mapping, inhibition, transferase activity, mode of action, synergism, catalytic mechanism), other characteristics of cellulases and xylanases (induction and regulation, physicochemical properties, their multiplicity, adsorption, architecture and classification, structure/function relationships) and applications.

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An extracellular P-glucosidase from Fusarium oxysporum was purified to homogeneity by gelfiltration and ion-exchange chromatographies. The enzyme, a monomeric protein of 11 0 kDa, was maximally active at pH 5.0-6.0 and at 60°C. It hydrolysed l-+4-linked aryl-p-glucosides and 1-4-linked, 1-3-linked and 1+6-linked P-glucosides. The apparent K, and k,, values for p-nitrophenyl ,8-D-glucopyranoside (4-NpGlcp) and cellobiose were 0.093 (K,,,), 1.07 mM (kcat) and 1802 (Km), 461.5 min-' (k,,,), respectively. Glucose and gluconolactone inhibited the enzyme competitively with K, values of 2.05 mM and 3.03 pM, respectively. Alcohols activated the enzyme; butanol showed maximum effect (2.2-fold at 0.5 M) while methanol increased the activity by 1.4-fold at 1 M. The enzyme catalysed the synthesis of methylglucosides, ethylglucoside and propylglucosides, as well as trisaccharides in the presence of different alcohols and disaccharides, respectively. In addition, the enzyme hydrolysed the unsubstituted and methylumbelliferyl cello-oligosaccharides [MeUmb(Glc),] but the rate of hydrolysis decreased with increasing chain length. Analysis of products released from MeUmb(Glc), as a function of time revealed that the enzyme attacked these substrates in a stepwise manner and from both ends. Thus, P-glucosidase from E oxysporum, with the above interesting properties, could be of commercial interest.Many fungi, including Fusarium oxysporum, when grown on cellulose, produce endoglucanase [endo-(l+4)-& D-glucanase], exoglucanase [exo-(I -4)-P-~-glucanase) and P-glucosidase (cellobiase or P-D-glucohydrolase) [ 1 -61. Of these, endoglucanases and exoglucanases act synergistically to effect extensive solubilization of crystalline cellulose [7, 81. This suggested that P-glucosidase has no direct role in cellulolysis. Based on in vitro saccharification, Sternberg [9] reported that the addition of P-glucosidase from Aspergillus niger to the cellulase preparation of Trichoderma reesei stimulated the rate and extent of cellulose hydrolysis by relieving