Amylases From A Thermophilic Fungus Thermomyces Lanuginosus Iisc 91 :Their Purification And Properties

Abstract

A knowledge of molecular properties and structure of heat-stable enzymes is important for the understanding of basic principles governing thermo stability of proteins and evolution of life at high temperatures. Information on functional characteristics of thermo stable enzymes is necessary also for improving existing biotechnologies and developing new ones. Because of these reasons enzymes from thermophilic organisms are being exploited. In this context, amylolytic enzymes represent a useful choice for investigation from both basic and applied points of view. a-Amylase and glucoamylase hydrolyse starch into oligosaccharides and glucose, respectively. In the present study a thermophilic fungus, Thermomyces lanuginosus, was selected as a source of thermostable starch-degrading enzymes. The main objectives of this research were to understand the physicochemical properties, mechanism of starch utilization by T. lanuginosus and effect of heat, on amylo1ytic enzymes.

Purification of amylolytic enzymes A strain of T, lanuginosus, IlSc 91, isolated from a manure heap in our laboratory was found to produce higher levels of extra cellular amylolytic enzymes than strains obtained from culture collection in U.S.A, and Europe. This strain produced 4 units of glucoamylase and 40 units of a-amylase per ml of culture filtrate when grown on 2% starch at 50 C. Culture filtrate was used as the starting material for purification of these enzymes. Glucoamylase and a-amylase were purified by ultrafiltration and a combination of ion exchange and gel-filtration chromatography 93- and 112-fold with 30 and 41% recovery, respectively; Homogeneity of purified enzymes was established by the criteria of native- PAGE, SDS-PAGE, gel-filtration on HPLC and N-terminal amino acid analysis. Some of the physicochernical properties of these enzymes were studied. Physicochemical characteristics Glucoernylase is a monomeric glycoprotien (carbohydrate content 11 %, w/w) and has a molecular weight, of 45 kDa. It produces only glucose from starch. Km and Vmax, for soluble potato starch are 0.04 mg ml-1 and 666 p mole glucose min-1 mg protein-1, respectively. The enzyme is optimally active at 70 C at pH 6.0. Its activation energy is 14.0 kCal mole-1. It has melting temperature of 73 C. Molar extinction coefficient of glucoamylase is 5.5 x 104 mole-1 cm-l. It is stable at 60°C for > 7h. The enzyme is rich in alanine, serine and aspartate/ asparagine. Glucoamylase contains alanine as the N-terminal amino acid. It does not contain cysteine. Purified a-amylase is a homodimeric protein of 40 kDa and contains 5% (w/w) carbohydrate. It liberates oligosaccharides from starch with maltose being the principle product of hydrolysis. The Km for soluble starch is 2.5 mg ml-1. A high Vmax, of 8000 mg starch min-1 mg protein-1 was found. The enzyme is optimally active at 65°C at pH 5.6. The activation energy is 10.9 kCal mole-1. At 50DC, which is the optimal temperature of growth of T. lanuginosus, purified a-amylase is completely stable for over 6 h. Ca2+ increases the melting temperature of a-amylase from 66°C to 73°C. a-Amylase requires Ca2+ for its activity and structural stabi1it.y The molar extinction coefficient of the enzyme is 4.7 x 10' mole-1 cm-1 a- Amylase is rich in aspartate / asparagine, glutamatme /glutamine, alanine, glycine and leucine. It does not contain cysteine. a- Amylase contains alanine as the N-t.ermina1 amino acid. Hydrolysis of starch by a-amylase and glucoamylase Experiments were done to understand the role of a-amylase and glucoamylase in the utilization of starch by T. lanuginosus. Crude and purified amylase preparations hydrolyse raw potato starch slightly more efficiently than soluble potato starch. The extent of starch hydrolysis by a mixture of a-amylase and glucoamylase is equal to that by culture filtrate containing the same amount of enzyme activities, Electrophoresis of crude culture filtrate proteins on native-PAGE and activity staining on gel showed the presence of one species each of a-amylase and glucoamylase. This suggests that in T. lanuginosus hydrolysis of starch is mediated by one species each of extracellular a-amylase and glucoamylase. The hydrolysis of starch by a mixture of a-amylase and glucoamylase is equal to the arithmetic sum of hydrolysis by individual enzyme showing that the enzymes do not act synergistically. a-Amylase is the main starch depolymerizing enzyme. Conversion of starch into glucose by glucoamylase does not require the presence of a-amylase. Starch is hydralysed to a maximum of 72 and 97% by glucoamylase and a-amylase, respectively. Effect of heat on a-amylase The effect, of heat, on a-amylase and glucoamylase was studied with the view to obtain information on the thermal inactivation of these proteins. Five-min heat treatment of the native a-amylase (40 kDa) results in the specific conversion of all protein molecules into partially active (approximately 50% residual activity) and SDS-undissociable dimer of 45 kDa. a-Amylase (45 kDa) after 5-min heat treatment. is partially active and can he rendered completely active by incubation at 37°C for 3 h. This altered form of a-amylase is not due to the formation of disulfide linkage in protein because the enzyme does not contain cysteine and b mercaptoethanol does not prevent heat-induced structural change. Heat, treatment, for 20 min or more results in further structural changes which result in the irreversible inactivation of the enzyme. Prolonged heating (>40 min) probably causes the degradation of protein. Reactivation of 20-min heat-inactivated a-amylase occurs specifically at 37°C or 50°C within 3 h but not at lower temperatures (0°C or 4°C). Native-PAGE analysis of the native and 20-min heated-reactivated a-amylase shows that the reactivated sample is comprised of two protein species of different charge and/or mass. Activity staining shows that only one of these protein band is active and it has electrophoretic mobility identical to that of the native enzyme. Native and the active fraction of 20-min heated-reactivated a-amylase possess similar specific activity. This suggests that it is cat8alytmically and perhaps structurally similar to the native enzyme. The native and the reactivated a-amylase are resist ant to trypsin digestion. However, heat- inactivated a-amylase is degraded into low molecular weight, peptides. These observation suggest that heat-inactivated a-amylase is partially unfolded, Unlike the native, the heat-treated (94"C, 5 min) a-amylase can not be stained with AgN03 while both forms can be stained with Coomassie brilliant blue R and by Schiff's base. On the basis of these observations a tentative model was proposed for the effect of heat on a-amylase (Fig.) Staining by + + Staining by AgNO3, + + Staining by Schiff 's base + + Sensitivity to trypsin + +

Figure : Schematic represent ation of heat-induced changes in a-amylase