Studies on starch hydrolysis using native and immobilized glucoamylases purification, characterization.......

Abstract

Glucoamylase (alpha-1,4-glucan glucohydrolase, EC 3.2.1.3) has been a subject of considerable importance in recent years due to its ability to hydrolyze starch directly to glucose. Although extensive studies were carried out on the purification and characterization of glucoamylase from several mesophilic fungi, the information available on this enzyme from thermophilic sources is very meager. The present investigation was directed towards studying the detailed characteristics of glucoamylase from thermophilic fungi and to develop a thermostable immobilized glucoamylase that may eventually be employed for the commercial production of glucose from starch.

Of the thermophilic fungi tested, Thermomyces lanuginosus ML-I elaborated higher yields of a thermostable glucoamylase. This fungus was therefore selected for detailed investigations.

Studies on the effect of various carbohydrates on the production of glucoamylase indicated that glucoamylase from T. lanuginosus is an inducible enzyme. Glucoamylase was induced maximally when the fungus was grown in the presence of starch, dextrin, dextran, maltose, or glycerol. Growth kinetics of T. lanuginosus revealed a differential pattern of appearance of glucoamylase at different stages of growth. The enzyme exists only in the mycelium in the initial stages of growth and appears in the culture medium at a later stage. Studies on this aspect indicated that the enzyme is synthesized as a precursor and is released into the extracellular medium by a specific processing mechanism.

Glucoamylase from the culture filtrates of T. lanuginosus was purified by two procedures. The purified enzyme was established to be homogeneous by several criteria. The enzyme is a glycoprotein with a molecular weight of 57,000 daltons. It was optimally active at a pH of 4.8–5.2 and at a temperature of 70°C. The enzyme is an exoglucanase catalyzing the hydrolysis of successive glucose residues from the non-reducing ends of the starch molecule. The enzyme is specific for alpha-1,4-glucosidic linkages and exhibited an increase in activity and a decrease in Km value with increasing chain length of the substrate molecule. The enzyme was inhibited by D-glucono-?-lactone in a non-competitive manner.

The catalytic and stability characteristics of glucoamylase from T. lanuginosus were compared with those of the enzyme purified from a mesophilic fungus, Rhizopus sp. These comparative studies indicated that the thermophilic glucoamylase is catalytically more efficient than the mesophilic enzyme and that the thermophilic enzyme exhibited remarkable stability towards heat and denaturing agents.

Glucoamylase from T. lanuginosus was immobilized by adsorption onto DEAE-cellulose and by entrapment in polyacrylamide gels, whereas the enzyme from Rhizopus sp. was immobilized by covalent attachment to AE-cellulose and CK-Sephadex. The catalytic behavior of the immobilized enzymes was investigated in relation to their native counterparts. These studies indicated that the immobilized enzymes exhibited altered chemical and kinetic properties. This behavior is explained on the basis of microenvironmental effects arising as a result of electrostatic interactions and/or diffusional effects. Studies on the operational stability of the immobilized enzymes suggested that these derivatives are susceptible to thermal denaturation at higher temperatures.

Entrapment in concentrated polyacrylamide or polyacrylate gels was used as an alternate approach to prepare thermostable immobilized enzyme derivatives. The thermal stability of the model enzymes, such as glucoamylase from T. lanuginosus and Rhizopus sp., and that of glucose oxidase from Aspergillus niger was enhanced several-fold by their entrapment in polyacrylamide gels. The enzymes entrapped in polyacrylate gels, on the other hand, exhibited a different behavior, probably owing to microenvironmental effects arising due to the charged nature of the gel matrix. These results indicated the possible application of this system to the continuous production of glucose from starch.