A cDNA coding for glucoamylase P of Hormoconis resinae was cloned using a synthetic oligonucleotide probe coding for a peptide fragment of the purified enzyme and polyclonal anti-glucoamylase antibodies. Nucleotide-sequence analysis revealed an open reading frame of 1848 base pairs coding for a protein of 616 amino-acid residues. Comparison with other fungal glucoamylase amino-acid sequences showed homologies of 37-48%. The glucoamylase cDNA, when introduced into Saccharomyces cerevisiae under the control of the yeast ADC1 promoter, directed the secretion of active glucoamylase P into the growth medium.

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...

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The effect of the upstream sequences of the yeast ADH1 promoter on the expression of Hormoconis resinae glucoamylase P by Saccharomyces cerevisiae was studied. Sequence analysis of the 5'-terminal region of the promoter revealed sequence patterns resembling a transcription start point and the binding site for the regulatory protein ADR1. A short promoter was constructed by deleting all the promoter sequences upstream of nucleotide -409, including the upstream activating sequence UASRPG. A medium-length promoter was constructed by deleting a fragment of 558 bp containing the putative upstream transcription start point but not the UAS. The short promoter increased the glucoamylase expression level 1.6-fold compared with the long promoter, but the beginning of secretion was delayed by about 10 h probably because of the absence of the UAS. The medium-length promoter directed expression of the glucoamylase without an initial delay, with the enzyme activity lying between the activities produced under the long and short promoters. Northern blot analysis confirmed the secretion patterns of the strains with different promoters but failed to reveal any transcripts starting at the putative upstream start point.

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