Studies on B-glucosidase of the thermophilic fungus sporotrictum thermophilic

Abstract

An interesting behaviour of glucosidase concerns its entrapment and release into the extracellular medium depending on the carbon source used for cultivation of the fungus. Berg and Pettersson (1977) and Vaheri et al. (1979) found that cellobiose favours the exclusive retention of the enzyme in T. reesei. In Sporotrichum pulverulentum, the enzyme was extracellular when the fungus was grown on cellulose and cell bound when it was grown with cellobiose (Deshpande et al., 1978). These observations have generated considerable interest in glucosidase as a model enzyme system to study the mechanism of enzyme release from fungal mycelia. Kubicek (1981) found that glucosidase could be effectively released from cell wall preparations of T. reesei when treated with laminarinase and chitinase. Since glucan and chitin form the rigid structure of fungal cell walls, he proposed that glucosidase is located at the chitin– glucan polymers of the cell wall. Kubicek (1982) noted that glucosidase excretion in T. pseudokoningii was correlated with cell wall bound 1,3 glucanase activity in lytic cultures. 1,3 Glucanase has been specifically localized by immunofluorescence microscopy in the tips, new septa, and branching areas of the hyphae (Kritzman et al., 1978), and is believed to have a role in cell wall loosening during hyphal morphogenesis. Kubicek (1982) proposed that glucosidase is bound to glucan in the hyphal wall and that its excretion into the culture fluid reflects cell wall bound lytic enzyme action on glucan. Understanding the precise location of glucosidase is very important for understanding the mechanism of cellulase induction. Glucosidases have transferase activity. It has been suggested that a plasma membrane glucosidase may be responsible in vivo for the formation of sophorose (2 O glucopyranosyl D glucose) from cellulose hydrolysis products, and that sophorose may be the true inducer of cellulase in T. reesei (Umile and Kubicek, 1986; Kubicek, 1987). In Sporotrichum pulverulentum, however, cellobiose is a better inducer of cellulase than sophorose (Eriksson and Hamp, 1978).

Properties of Glucosidase The glucosidase of Botryodiplodia theobromae, which has been particularly well studied by Umezurike (1975a), shows interesting structural and catalytic properties. The enzyme is a high molecular weight protein (350,000–380,000), which upon storage dissociates into lower molecular weight species (170,000–180,000; 83,000–87,000; and 45,000–47,000). Electron microscopy revealed that the native molecule is an octamer. The enzyme, when dissociated by carboxyamidomethylation of the reduced form, showed one enzymatically inactive protein band on electrophoresis with a molecular weight of 10,000–12,000. These results suggested a slow association–dissociation of type: 8(n) 2(4n) 4(2n) 8(n) where n is a monomer consisting of about four catalytically inactive subunits. The average molecular weights of the octamer (8n), tetramer (4n), dimer (2n), and monomer (n) were approximately 320,000; 160,000; 80,000; and 40,000, respectively. Since reported molecular weights of glucosidases from various sources fall within these ranges, Umezurike (1975a) postulated that enzymes from different organisms may represent a homologous series of macromolecules. Kinetic studies on hydrolysis of p nitrophenyl D glucoside by B. theobromae glucosidase in the presence of various substances (maltose, glucose) indicated the presence of two binding sites: a donor site and an acceptor site (Umezurike, 1971). The acceptor site exhibited broad specificity, accepting fructose, sucrose, glycerol and methanol as glucosyl acceptors (Umezurike, 1975b). Very low concentrations (<0.1 mM) of cellobiose activated, but higher concentrations strongly inhibited, glucosidase activity with arylglucoside as the glycosyl donor (Umezurike, 1971). It was suggested that at low concentrations cellobiose binds to the acceptor site, whereas at higher concentrations it binds competitively to the substrate binding site, which includes both the glucose binding (glycone) and aglycone binding subsites. Chirico and Brown (1987) showed that the active site of extracellular glucosidase of T. reesei is composed of three subsites. Michaelis constants for cello oligosaccharide hydrolysis by glucosidases have been measured to evaluate the enzyme’s role in cellulose hydrolysis. For glucosidases from A. niger (King and Smibert, 1963) and S. rolfsii (Shewale and Sadana, 1981), the Km values decreased 4 to 20 fold as chain length increased. The Km values for T. reesei glucosidase decreased from cellobiose to cellotetraose and then remained constant from cellotetraose to cellohexaose. In contrast, Km values for two glucosidases purified from T. koningii increased with chain length (Wood and McCrae, 1982). Shewale and Sadana (1981) interpreted the chain length dependence of Km values as indicating that the function of glucosidase in the cellulase system must be considered alongside the roles of other cellulolytic components, and not solely from kinetic parameters of one enzyme.

Postulated Functions for Glucosidase Cellulose Saccharification Sternberg et al. (1977) studied the influence of glucosidase on the saccharification of cellulose. Crystalline cellulose (15%) was treated with crude T. viride cellulase (filter paper cellulase activity = 3.0 U/ml) in the absence and presence of varying concentrations of A. niger glucosidase. Saccharification was monitored over 50 hours by assaying supernatants for total sugars and glucose. They observed that the time required to reach any given sugar concentration with cellulase alone was reduced by 50% upon addition of glucosidase (3.0 U/ml). In other words, cellulose hydrolysis rate increased significantly upon glucosidase addition. This stimulatory effect was explained by glucosidase reducing cellobiose levels in the hydrolysate, thereby relieving cellobiose mediated product inhibition of cellulase.