Comparison of two glucoamylases from Hormoconis resinae

Abstract

Two extracellular glucoamylases (EC 3.2.1.3), glucoamylase P and glucoamylase S, were purified to homogeneity from the culture medium of Hormoconis resinae (ATCC 20495; formerly Cl&sprium resinae) by a new method. Their apparent molecular masses (71 kDa glucoamylase P; 78 kDa glucoamylase S) and catalytic properties agreed well with those previously reported in the literature. Heat inactivation studies suggested that the high debranching (1,6-glycosidic) activity of glucoamylase P preparations (measured with pullulan) may reside in the same protein molecule as its l,&glycosidic activity (measured with soluble starch). Although glucoamylase S had virtually no debranching activity, it cross-reacted with polyclonal antibodies raised against glucoamylase P, and the two enzymes had very similar amino acid compositions. However, peptide mapping and amino-terminal sequencing studies of the peptides showed that the two enzymes have different sequences and must be encoded by different genes. IntroductionEnzymes that degrade poly-and oligosaccharides are commercially important for the food and fermentation industries. Glucoamylases (1,4-glucan glucohydrolases ; EC 3.2.1 .3) release glucose units sequentially from the nonreducing end of polymeric carbohydrates. The ability of glucoamylases to hydrolyse 1,6-glycosidic bonds is called debranching activity and is of great importance in industrial processes requiring complete degradation of starch to glucose.Glucoamylases are produced by a wide variety of micro-organisms (Manjunath et al., 1983). Most of the known glucoamylases have very low activity towards 1,6-glycosidic bonds. The analytical measurement of debranching activity is not straightforward, since the activity of glucoamylases on substrates containing only 1,6-glycosidic bonds is usually quite low. To avoid this difficulty, substrates like pullulan (every third bond 1,6-glycosidic) are often used to estimate 1,6-hydrolysing activity. Many fungal glucoamylases have been reported to exist in multiple forms (Lineback & Bauman, 1970;Tsuboi et al., 1974;Yamasaki et al., 1977;Takahashi et al., 1978Takahashi et al., , 1981. In order to clone the appropriate gene it is essential to know if these multiple forms are coded by different genes, or if they are results of post-translational al. (1984) these two forms of the enzyme are products of two differently spliced mRNA molecules coded by the same gene, whereas Svensson et al. (1986) have pointed out that proteolytic modification also affects the multiplicity of this enzyme.The fungus Hormoconis resinae (ATCC 20495; formerly Cladosporium resinae) has been reported to produce two forms of glucoamylase which exhibit different substrate specificities and have different molecular masses and PI values (McCleary & Anderson, 1980). The smaller glucoamylase P has a very high debranching activity, while the larger glucoamylase S has virtually no debranching activity. In the present work, we examined whether the 1,4-and 1,6-glycosidic activities of glucoamylase P are both functions...

Purification and biochemical characterization of a thermostable extracellular glucoamylase produced by the thermotolerant fungus Paecilomyces variotii

“…Similar results were reported for other fungal glucoamylases [25,27,34], and might be due to an increased stability of the enzyme-substrate complex [35]. Another alternative explication, suggested by Fagerstrom et al [30], is that the high concentration of nonreducing ends of starch will act to protect the enzyme against thermal denaturation. In respect of pH stability, the enzyme was stable over the period of 1 h in the pH range of 2.5-8.0 at 4°C (Fig.…”

“…5a). Glucoamylases have in general pH optima between 3.5 and 6, and they are thermostable only at temperatures below 60°C [30]. These results show that the enzyme presented a high level of stability when compared with some glucoamylases of A. niger [31][32][33].…”

Application of artificial neural networks to modelling of starch hydrolysis by glucoamylase

“…Starch consists of a mixture of branched amylopectin based on chains of a-1,4-linked a-D-glucose units with a-1,6-glucosidic bonds at the branch points and almost linear amylose composed of mainly a-1,4-linked glucose residues. GA attacks mainly the a-1,4-glucosidic bonds from the nonreducing ends of the polysaccharide chains, releasing successively D-glucose molecules in the b-con®guration, whereas the activity towards the a-1,6 bonds at the chain ends is very low [5,18]. Although GA is capable of hydrolysing all the glucosidic linkages in starch, D-glucose yields higher than 95% of the theoretical ones are not achieved because: Furthermore, during starch hydrolysis by GA an``overshoot'' of the equilibrium concentration (86% for a corn starch solution) occurs as the a-1,4-bonds scission predominates [24].…”

Effect of upstream sequences of the ADH1 promoter on the expression of Hormoconis resinae glucoamylase P by Saccharomyces cerevisiae

“…Glucoamylase activity was determined as described previously [1,16], using soluble starch as substrate. One unit was defined as the amount of enzyme releasing 1 /zmol glucose per min at 30°C.…”

“…a Activity in the culture supernatant. b Calculated assuming a specific activity of 14.4 U mg-1 [16]. c Living cells counted on YPD plates.…”