Michael P. Coughlan

Kinetic parameters and mode of action of the cellobiohydrolases produced by Talaromyces emersonii.

Three forms of cellobiohydrolase (EC 3.2.1.91), CBH IA, CBH IB and CBH II, were isolated to apparent homogeneity from culture filtrates of the aerobic fungus Talaromyces emersonii. The three enzymes are single sub-unit glycoproteins, and unlike most other fungal cellobiohydrolases are characterised by noteworthy thermostability. The kinetic properties and mode of action of each enzyme against polymeric and small soluble oligomeric substrates were investigated in detail. CBH IA, CBH IB and CBH II catalyse the hydrolysis of microcrystalline cellulose, albeit to varying extents. Hydrolysis of a soluble cellulose derivative (CMC) and barley 1,3;1,4-beta-D-glucan was not observed. Cellobiose (G2) is the main reaction product released by CBH IA, CBH IB, and CBH II from microcrystalline cellulose. All three CBHs are competitively inhibited by G2; inhibition constant values (K(i)) of 2.5 and 0.18 mM were obtained for CBH IA and CBH IB, respectively (4-nitrophenyl-beta-cellobioside as substrate), while a K(i) of 0.16 mM was determined for CBH II (2-chloro-4-nitrophenyl-beta-cellotrioside as substrate). Bond cleavage patterns were determined for each CBH on 4-methylumbelliferyl derivatives of beta-cellobioside and beta-cellotrioside (MeUmbG(n)). While the Tal. emersonii CBHs share certain properties with their counterparts from Trichoderma reesei, Humicola insolens and other fungal sources, distinct differences were noted.

Enzymic hydrolysis of cellulose: an overview

Abstract An overview is presented of the factors militating against the saccharification of lignocellulosic materials, pretreatments that may be used to improve on the extent of saccharification, recent advances in our understanding of the structure of cellulose, and the production, properties and mechanisms of action of the cellulolytic enzyme systems.

Aldehyde Oxidase, Xanthine Oxidase and Xanthine Dehydrogenase; Hydroxylases containing Molybdenum, Iron-Sulphur and Flavin

Publisher Summary The group of enzymes collectively and descriptively known as the molybdenum iron-sulphur flavin hydroxylases includes aldehyde oxidase, xanthine oxidase, and xanthine dehydrogenase. As a group and as individuals, they are among the most unique of proteins. The promiscuity of these enzymes is reflected in their behavior with respect to substrates. Distinctions between aldehyde and xanthine oxidases and between xanthine oxidase and dehydrogenase have not always been made. Aldehyde oxidase, xanthine oxidase, and xanthine dehydrogenase catalyze, though at widely different rates, the hydroxylation of a wide variety of purines, pteridines, pyrimidines, other heterocyclic nitrogenous compounds, and aldehydes, whether aliphatic, aromatic, or heteroaromatic. These enzymes differ not only in substrate specificity but also with respect to the position on the substrate that is hydroxylated. This is most evident with substrates, such as purines and pteridines, having more than one site available for hydroxylation.

The cellulolytic system of Talaromyces emersonii: Purification and characterization of the extracellular and intracellular β-glucosidases

Abstract The thermophyllic fungus Talaromyces emersonii produces three extracellular and one intracellular enzymes exhibiting β-glucosidase (1,4-(1,3;1,4)-β- d -glucan 4-glucanohydrolase, EC 3.2.1.21) activity. Two of the extracellular forms β-glucosidase I and β-glucosidase III have been purified as has the intracellular, β-glucosidase IV. The pH and temperature, optima, stability, kinetic parameters and substrate specificity of each has been determined. We conclude that β-glucosidase I and β-glucosidase IV are true cellobiases while β-glucosidase III is an exo-β-1,4-glucose hydrolase.

Cellulose Degradation by Fungi

Cellulose is the most abundant organic macromolecule on earth. It has been estimated that total biomass (fossil fuels excepted) amounts to about 1·8 × 1012 tonnes, 1 × 1011 tonnes being replenished each year by photosynthesis (Bassham, 1975; Stephens & Heichel, 1975). Since 40% of this biomass consists of cellulose (Brown, 1983), one may calculate that 7 × 1011 tonnes of this material exist, mainly in higher plants, and that annual productivity is about 4 × 1010 tonnes. The magnitude of these figures can be appreciated by noting that the rate of cellulose synthesis is equivalent to 70 kg per person per day (Lutzen et al., 1983) or to 50 000 barrels of oil per second in energy terms (Sienko & Plane, 1976). Indeed, current terrestrial biomass, at 640 billion tonnes of oil equivalent, is equal to total proven fossil fuel reserves (De Montelambert, 1983). But, unlike the latter it is constantly being renewed. Lignocellulosic wastes or residues of forest, agriculture, industrial or domestic origin, are generated in great quantities especially in the more developed countries. Many reports have suggested that much of the demand for fuels and chemical feedstocks, currently met by oil, could be met by appropriate exploitation of such wastes (Avgerinos & Wang, 1980; Chartier, 1981; Eveleigh, 1982; Brown, 1983; Grohman & Villet, 1983; Hall, 1983; Sheppard & Lipinsky, 1983; Sinskey, 1983; Soltes, 1983; Eveleigh, 1984; Lloyd, 1984).

Synergistic hydrolysis of cellulose by components of the extracellular cellulase system of Talaromyces emersonii

Forecasts of shortages in food and fossil fuel have stimulated an increased interest in cellulose as a potential alternative [l--S]. To this end the enzymic degradation of cellulase is being investigated. Many organisms possess enzymes that can hydrolyze soluble celluloses. By contrast, few species produce the ‘complete’ cellulase complex capable of catalying the extensive degradation of the crystalline substrates found in nature [ 6-81. Talaromyces emersonii, a thermophyllic fungus, when grown on cellulose-containing media, produces an extracellular enzyme system that catalyzes the degradation of various insoluble crystalline celluloses [9,10]. The observed extent of hydrolysis of these substrates implied that the cellulase system produced by this organism is a ‘complete’ complex. Here we provide direct evidence that such is indeed the case.

Production of thermostable xylan-degrading enzymes by Talaromyces emersonii☆

Abstract Talaromyces emersonii strains CBS814.70 and UCG208 produce multiple forms of xylan-degrading enzymes when grown by liquid- or solid-state cultivation methods on glucose, lactose, cellulose, xylan and a variety of straws and pulps. Activity in crude extracts is optimal at 80°C and the enzymes produced by liquid cultivation have half-life values ranging from 50 to 250 min at 80°C, pH 5.

The cellulolytic system of talaromyces emersonii identification of the various components produced during growth on cellulosic media

Talaromyces emersonii, a thermophyllic fungus was grown on cellulose/corn steep liquor/NH4NO3 medium. The kinetics of growth and extracellular cellulase production were measured. The enzyme system was found to be comprised of four to five forms of exo-β-1,4-glucanase (cellobiohydrolase; 1,4-β-d-glucan cellobiohydrolase, EC 3.2.1.91), at least two forms of endo-1,4-β-glucanase (1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4) and three enzymes exhibiting β-glucosidase (cellobiase; β-d-glucoside glucohydrolase, EC 3.2.1.21) activity. One of the latter, termed β-glucosidase III, is induced concurrently with the cellulases but disappears from the medium because of the low pH that develops during growth. The cellulases by contrast are more acid-stable. Later in the growth cycle a second from of β-glucosidase, termed β-glucosidase I, accumulates. An intracellular β-glucosidase, β-glucosidase IV, was also detected. The possible functions of these enzymes are discussed.

Biochemical characteristics of two endo-β-1,4-xylanases produced byPenicillium capsulatum

SummaryTwo endo-β-1,4-xylan xylanohydrolases (EC 3.2.1.8), XynA and XynB, from solid-state cultures ofPenicillium capsulatum, were purified to apparent homogeneity as judged by electrophoresis and isoelectric focusing. Each is a single subunit glycoprotein. XynA containing 97 mol carbohydrate·mol−1 protein, while XynB contains 63 mol·mol−1.Mr and pI values are 28 500, 5.0–5.2 (XynA) and 29 500, 5.0–5.2 (XynB), respectively. Both enzymes are most active at pH 4 and 47–48°C, and have half-lives of 32 min (XynA) and 13 min (XynB) at pH 4, 60°C. Each form catalyzed the hydrolysis of a variety of xylans, albeit with different degrees of efficiency. In addition, XynB catalyzed extensive degradation of barley β-glucan, CM-cellulose and, to a lesser extent, lichenan, but kinetic parameters indicate that it is primarily a xylanase. The products of hydrolysis of various xylans and xylopentaose differed for each enzyme and ranged from xylose to xyloheptaose depending on the substrate used. Each enzyme is endo-acting and has transferase as well as direct hydrolase activity. Inactivation byN-bromosuccinimide indicated the possible involvement of tryptophan in binding and/or catalysis.

Isolation of mutants of Talaromyces emersonii CBS 814.70 with enhanced cellulase activity

Abstract By a combination of genetic mutation and modification of growth medium the cellulase [see 1,4-(1,3;1,4)-β- d -glucan 4-glucanohydrolase, EC 3.2.1.4 etc.] activity of culture filtrates of Talaromyces emersonii CBS 814.70 has been increased four-fold to approximately 2 U ml −1 and a productivity of 20–25 Ul −1 h −1 . At 50°C this system was completely stable for at least 24 h. At 60°C in static reaction mixtures 19% of the original activity was lost compared with 21% when mixtures were shaken. At 70°C the loss of activity after 4 h was 64% without shaking and 70% when shaken. The cellulase system from Trichoderma reesei was decidedly less stable than that of Talaromyces emersonii under each of the above conditions . The ability of each enzyme system, separately and together, to digest beet pulp was investigated.