The sequence of a 1409 base pair restriction fragment containing the B. subtilis gene for (1-3), (1-4)-beta-D-glucan endoglucanase is reported. The gene is encoded in a 726 base pair segment. The deduced amino acid sequence of the protein has a hydrophobic signal peptide at the NH2-terminus similar to those found in five other secreted proteins from Bacillus. The gene is preceded by a sequence resembling promoters for the vegetative B. subtilis RNA polymerase. This is followed by a sequence resembling a B. subtilis ribosome binding site nine nucleotides before the first codon of the gene. Two sequences, one before and one after the gene, can be arranged in secondary structures similar to transcriptional terminators. There is also a short open reading frame coding for a hydrophobic protein on the minus strand just upstream from the beta-glucanase gene. A possible role for this gene in the control of expression of beta-glucanase is suggested.

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The nucleotide sequence of a 4.39‐kb DNA fragment encoding the α‐glucosidase gene of Candida tsukubaensis is reported. The cloned gene contains a major open reading frame (ORF 1) which encodes the α‐glucosidase as a single precursor polypeptide of 1070 amino acids with a predicted molecular mass of 119 kDa. N‐terminal amino acid sequence analysis of the individual subunits of the purified enzyme, expressed in the recombinant host Saccharomyces cerevisiae, confirmed that the α‐glucosidase precursor is proteolytically processed by removal of an N‐terminal signal peptide to yield the two peptide subunits 1 and 2, of molecular masses 63–65 kDa and 50–52 kDa, respectively. Both subunits are secreted by the heterologous host S. cerevisiae in a glycosylated form. Coincident with its efficient expresion in the heterologus host, the C. tsukubaensisα‐glucosidase gene contains many of the canonical features of highly expressed S. cerevisiae genes. There is considerable sequence similarity between C. tsukubaensisα‐glucosidase, the rabbit sucrase‐isomaltase complex (proSI) and human lysosomal acid α‐glucosidase. The cloned DNA fragment from C. tsukubaensis contains a second open reading frame (ORF 2) which has the capacity to encode a polypeptide of 170 amino acids. The function and identity of the polypeptide encoded by ORF 2 is not known.

The molecular cloning of an alpha-glucosidase gene isolated from a Candida tsukubaensis (CBS 6389) genomic library in Saccharomyces cervisiae is reported. The cloned gene is contained within a 6.2 kb Sau3A DNA fragment and directs the synthesis and secretion of an amylolytic enzyme into the extracellular medium of the recombinant host, S. cerevisiae. The cloned enzyme was found to have an unusually broad substrate specificity and is capable of hydrolysing alpha-1,2, alpha-1,3, alpha-1,4 and alpha-1,6 linked, as well as aryl and alkyl, D-glucosides. On the basis of its substrate specificity profile, the cloned enzyme was classified as an alpha-glucosidase (E.C. 3.2.1.20). It has a pH optimum in the range 4.2-4.6, a temperature optimum of 58 degrees C and is readily inactivated at pasteurization temperature (60 degrees C). Southern blot analysis failed to reveal any homology between the cloned gene and genomic DNA isolated from other well characterized amylolytic yeasts. A rapid plate-assay, based on the utilization of a chromogenic substrate X-alpha-D-glucoside to detect the expression of the cloned alpha-glucosidase in S. cerevisiae transformants, was developed.

The endo-beta-1,3-1,4-glucanase gene from B. subtilis was placed under yeast promoter control in a number of different yeast expression vectors. The hybrid plasmids were transformed into S. cerevisiae where they directed the synthesis of varying amounts of active enzyme. The presence of B. subtilis DNA sequences 5' to the initiation codon for the B. subtilis beta-glucanase gene reduced expression of the gene in yeast. A 1,000-fold increase in the yield of beta-glucanase was obtained using the ADH1 promoter compared with the CYC1 promoter.

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The endo-beta-1,3-1,4-glucanase enzyme of Bacillus subtilis C120, when synthesized in Escherichia coli, is located mainly in the cytoplasm, but enzyme activity is also detected in the periplasmic space and in the extracellular medium. The proportion recovered in the extracellular medium is not altered by changes in the levels of synthesis of the enzyme. Lysis of E. coli cells is ruled out as the cause of the secretion by the normal localization of beta-galactosidase, an intracellular protein. However, beta-lactamase, which is normally found in the periplasmic space, is detected in the extracellular medium of E. coli transformants containing beta-glucanase plasmids, suggesting that the presence of beta-glucanase in the cell alters the permeability of the outer membrane. The beta-glucanase proteins found in the extracellular medium, the periplasmic space and the cytoplasm have the same electrophoretic mobilities as the secreted enzyme of B. subtilis. Amino-terminal sequencing has shown that the beta-glucanase enzyme in the intracellular fraction of E. coli is processed at a site two amino acids distant from the processing site used in B. subtilis.

The alpha-glucosidase gene of Candida tsukubaensis is contained within a 3.47 kb BamH1-Mlul fragment which, when introduced into Saccharomyces cerevisiae AH22 on a yeast-Escherichia coli shuttle vector, allows the transformants to utilize maltose as sole carbon source. Thus, the cloned gene confers a dominant selectable phenotype on transformed strains of S. cerevisiae which are otherwise unable to grow in nutrient media containing maltose, dextrin or other alpha-1.4-linked alpha-D-glucopyranosides, specifically hydrolysed by the alpha-glucosidase. The cloned enzyme expressed in yeast is secreted into the extracellular medium in a glycosylated form which accounts for up to 60% of the secreted protein and has a molecular size of 70-80 kilodalton (kDa). Deglycosylation of the alpha-glucosidase showed that the enzyme is composed of two distinct polypeptides with subunit molecular weights of 63-65 kDa (peptide 1) and 50-52 kDa (peptide 2). An increase in the level of expression of the alpha-glucosidase by yeast transformants in selective minimal medium was obtained by using a vector with increased copy number containing the leu2-d gene as selectable marker. The alpha-glucosidase gene promoter functions more effectively than the Gall-10 promoter in directing alpha-glucosidase expression in S. cerevisiae. It also directs the expression of high levels of beta-galactosidase activity in yeast when fused to a promoterless E. coli lacZ gene. Expression of the alpha-glucosidase gene under the control of its own promoter is constitutive, orientation dependent and not subject to catabolite repression.