Studies on the mechanism of hydrolysis of cellulose and active site analysis of beta-1,4-Glucan Glucano-hydrolase from thermoascus auran

Abstract

Synopsis of the Thesis Entitled “Studies on the Mechanism of Hydrolysis of Cellulose and Active Site Analysis of Beta-1,4-Glucan Glucanohydrolase from Thermoascus aurantiacus” Submitted by M. S. Narasimha Swamy for the Ph.D. Degree at the Indian Institute of Science, Bangalore – 560 012, India

The present thesis deals with the physicochemical and functional characterization of cellulases from the thermophilic fungus Thermoascus aurantiacus, molecular adsorption of these enzymes to cellulose, and active site analysis of ?-1,4-glucan glucanohydrolase. Attempts were also made to clone the gene encoding ?-1,4-glucan glucanohydrolase in E. coli.

Cellulose is an unbranched linear polymer of ?-1,4-linked D-glucose units found abundantly in nature. The enzymatic hydrolysis of this renewable carbon source has evoked considerable interest in recent years because of its industrial importance. Success in this task requires a clear understanding of the mechanism of enzymatic hydrolysis of cellulose as well as the properties of cellulases employed. Cellulases of Thermoascus aurantiacus have been chosen as a model system in view of their potential to saccharify native cellulose.

?-1,4-glucan glucanohydrolase (endocellulase) and ?-1,4-glucan cellobiohydrolase (exocellulase) were purified from culture filtrates of Thermoascus aurantiacus using ion-exchange and molecular sieve chromatographic techniques (Khandke et al., 1989). Purity of the enzyme preparations was established by native PAGE, SDS-PAGE, and IEF.

Endocellulase (32 kDa): An acidic protein (pI 3.4), active over a wide pH range (2.0–8.5). Highly thermostable, retaining full activity at 70 °C for over 200 hours at pH 5.0–6.0. Hydrolyzed carboxymethyl cellulose (CMC) and phosphoric acid–swollen cellulose (PSC) efficiently. Optimum temperature for CMC hydrolysis was 78 °C (pH 3.5), while PSC hydrolysis showed broad optima (50–85 °C, pH 3.5–5.5). Major products: cellobiose, cellotriose, and cellotetraose.

Exocellulase (58 kDa): Acidic protein (pI 3.7), active over pH 3.0–7.5. Hydrolyzed microcrystalline cellulose (MCC) and PSC. Optimum activity at pH 3.0–4.5 and 65–70 °C. Products: mainly cellobiose with traces of glucose.

Adsorption Studies Cellulases must adsorb to cellulose before hydrolysis. Adsorption behavior of endocellulase and exocellulase was studied using antibodies and a standardized micro-ELISA method for selective quantification. Both enzymes bound more efficiently to swollen cellulose than crystalline cellulose.

Endocellulase adsorption followed Langmuir kinetics; exocellulase deviated.

Increasing temperature decreased adsorption of endocellulase but not exocellulase.

Increasing pH reduced endocellulase adsorption to PSC but had no effect on exocellulase.

Ionic strength had little effect.

Exocellulase enhanced adsorption of endocellulase, but the reverse was not true.

Active Site Analysis Chemical modification studies were carried out to identify functional groups involved in catalysis:

Trp residues: Modification with N-bromosuccinimide (NBS) inhibited activity in a concentration-dependent manner. Oxidation of one Trp caused 40% loss of PSC activity; two Trp oxidations caused 70% loss (PSC) and 30% loss (CMC); three Trp oxidations abolished activity. Substrates protected against inactivation, suggesting essential Trp residues near the active site.

Carboxyl groups: Modification with EEDQ and EDC inhibited activity. Kinetic analysis suggested roles in binding (CMC) and catalysis (PSC). Low EDC concentrations modified one essential carboxyl group (loss of PSC activity only), while higher concentrations modified two groups (loss of both PSC and CMC activity).

These results indicate distinct roles of Trp and carboxyl groups in maintaining activity toward PSC and CMC, suggesting possible allosites in the active site.

Gene Cloning Preliminary cloning studies were carried out: genomic DNA of T. aurantiacus was partially digested and cloned into the EcoRI site of pUC19. Transformants were screened using antibody probes. Two colonies expressed enzyme activity, with insert sizes of 1.3 kb and 0.3 kb.

Summary Physicochemical properties of endocellulase and exocellulase from Thermoascus aurantiacus were studied.

Adsorption behavior of cellulases onto cellulose was characterized.

Roles of Trp and carboxyl groups in catalysis and binding were elucidated.

Preliminary cloning of the endocellulase gene in E. coli was attempted.