Characterization and gene cloning of a maltotriose-forming exo-amylase from Kitasatospora sp. MK-1785.

A maltotriose-forming amylase (G3Amy) from Kitasatospora sp. MK-1785 was successfully isolated from a soil sample by inhibiting typical extracellular α-amylases using a proteinaceous α-amylase inhibitor. G3Amy was purified from the MK-1785 culture supernatant and characterized. G3Amy produced maltotriose as the principal product from starch and was categorized as an exo-α-amylase. G3Amy could also transfer maltotriose to phenolic and alcoholic compounds. Therefore, G3Amy can be useful for not only maltotriose manufacture but also maltooligosaccharide-glycoside synthesis. Further, the G3Amy gene was cloned and expressed in Escherichia coli cells. Analysis of its deduced amino acid sequence revealed that G3Amy consisted of an N-terminal GH13 catalytic domain and two C-terminal repeat starch-binding domains belonging to CBM20. It is suggested that natural G3Amy was subjected to proteolysis at N-terminal region of the anterior CBM20 in the C-terminal region. As with natural G3Amy, recombinant G3Amy could produce and transfer maltotriose from starch.

Cloning and sequencing of the cDNA encoding beta-glucosidase 1 from Aspergillus aculeatus.

A cDNA was isolated from an Aspergillus aculeatus cDNA library using synthetic oligodeoxyribonucleotide mixtures that corresponded to the internal amino acid (aa) sequence of mature beta-glucosidase 1 (BGL1). Analysis of the nucleotide sequence of the cloned cDNA insert revealed a 2580-bp open reading frame (ORF) that encoded a 860-aa protein. The deduced aa sequence of the ORF shared sequence similarity with several BGL from other microorganisms.

Cloning and secretive expression of the gene encoding the proteinaceous alpha-amylase inhibitor paim from Streptomyces corchorusii.

A gene encoding the proteinaceous alpha-amylase inhibitor Paim was cloned and sequenced. Southern analysis and the amino acid sequence deduced from the cloned gene indicated that Paim isoforms were encoded in the same gene. When the gene was expressed in Escherichia coli and Streptomyces lividans, recombinant Paim inhibitors were produced in the periplasmic space and in the culture supernatant, respectively. The purified inhibitors had different N-terminal sequences from those of the authentic inhibitors.

Overexpression and purification of Aspergillus aculeatus beta-mannosidase and analysis of the integrated gene in Aspergillus oryzae.

An expression plasmid for the manB gene encoding Aspergillus aculeatus beta-d-mannosidase (MANB) was constructed by using an expression vector carrying an improved promoter. After transformation of A. oryzae by the plasmid, several transformants formed colonies emitting fluorescence on a plate containing 4-methylumbelliferyl beta-d-mannopyranoside (MU-Man) under UV-irradiation. The transformant that displayed the strongest fluorescence, named A. oryzae BMN1, produced about 270 mg MANB/l in liquid culture. Recombinant MANB overproduced in BMN1 was purified by two steps of column chromatography to a single protein band on SDS-polyacrylamide gel electrophoresis and had a molecular weight of 130,000. Analyses by Southern blotting and genomic PCR demonstrated that a single copy of the plasmid was integrated into the chromosome by recombination at the niaD locus.

The conserved tryptophan-arginine-tyrosine motif of a proteinaceous alpha-amylase inhibitor T-76 from Streptomyces nitrosporeus is important for inhibition of animal alpha-amylases but not for an alpha-amylase from Bacillus sp. no. 195.

Site-directed mutagenesis of Trp-16, Arg-17, and Tyr-18, which were thought to form a putative active site in proteinaceous alpha-amylase inhibitor T-76 from Streptomyces nitrosporeus for inhibition, was performed. The mutation at the site (W16A, R17A, and Y18A) resulted in a marked decrease in inhibitory activity against all animal alpha-amylases tested. Only the alpha-amylase from Bacillus sp. no. 195 (BAA) remained sensitive to all the constructed mutant inhibitors. A competition between T-76 mutants and the wild-type for porcine pancreatic alpha-amylase (PPA) suggest that the loss of inhibitory activity against PPA in mutant inhibitors was due to the decrease in their binding ability for PPA. T-76 formed a complex with BAA as well as PPA at the stoichiometric ratio of 1:1. A competition between BAA and the PPA/T-76 complex suggests that PPA and BAA might bind to the same region or regions close to each other on the T-76 molecule. These results indicate that the conserved Trp-Arg-Tyr motif of T-76 is involved in the interaction between T-76 and PPA while other amino acid residues seem to be important for the T-76/BAA interaction. Since the BAA-type alpha-amylase is the actual target of the inhibitors from microbes in comparison with animal alpha-amylases, BAA might be a better material than PPA to elucidate the "true" function of proteinaceous alpha-amylase inhibitors.

Development of an efficient production method for beta-mannosidase by the creation of an overexpression system in Aspergillus aculeatus.

AIM: To develop an overexpression system in Aspergillus aculeatus in order to establish an efficient overproduction method of beta-mannosidase (MANB). METHODS AND RESULTS: An overexpression plasmid for the manB gene, encoding A. aculeatus MANB, was constructed and introduced into A. aculeatus cells. The gene was overexpressed under an improved promoter containing 12 copies of Region III cis-elements of Aspergillus oryzae in the transformant, and it secreted 2.56 mg MANB ml(-1) in liquid culture, which obtained a 9.4-fold higher productivity than that achieved in an overexpression system in A. oryzae. Most of the secreted protein in the cultured medium of the transformed A. aculeatus was the overproduced enzyme. CONCLUSIONS: Aspergillus aculeatus with the introduced overexpression plasmid produced 2.56 mg MANB ml(-1) in cultured medium. The improved promoter with A. oryzae Region III functioned in A. aculeatus; thus the strain is an expectant host for recombinant protein productions. SIGNIFICANCE AND IMPACT OF THE STUDY: The overexpression system with the improved promoter in A. aculeatus brought the highest productivity of MANB reported to date. The expression system would be a strong bioindustrial tool for protein production.

New type of starch-binding domain: the direct repeat motif in the C-terminal region of Bacillus sp. no. 195 alpha-amylase contributes to starch binding and raw starch degrading.

The alpha-amylase from Bacillus sp. no. 195 (BAA) consists of two domains: one is the catalytic domain similar to alpha-amylases from animals and Streptomyces in the N-terminal region; the other is the functionally unknown domain composed of an approx. 90-residue direct repeat in the C-terminal region. The gene coding for BAA was expressed in Streptomyces lividans TK24. Three active forms of the gene products were found. The pH and thermal profiles of BAAs, and their catalytic activities for p-nitrophenyl maltopentaoside and soluble starch, showed almost the same behaviours. The largest, 69 kDa, form (BAA-alpha) was of the same molecular mass as that of the mature protein estimated from the nucleotide sequence, and had raw-starch-binding and -degrading abilities. The second largest, 60 kDa, form (BAA-beta), whose molecular mass was the same as that of the natural enzyme from Bacillus sp. no. 195, was generated by proteolytic processing between the two repeat sequences in the C-terminal region, and had lower activities for raw starch binding and degrading than those of BAA-alpha. The smallest, 50 kDa, form (BAA-gamma) contained only the N-terminal catalytic domain as a result of removal of the C-terminal repeat sequence, which led to loss of binding and degradation of insoluble starches. Thus the starch adsorption capacity and raw-starch-degrading activity of BAAs depends on the existence of the repeat sequence in the C-terminal region. BAA-alpha was specifically adsorbed on starch or dextran (alpha-1,4 or alpha-1,6 glucan), and specifically desorbed with maltose or beta-cyclodextrin. These observations indicated that the repeat sequence of the enzyme was functional in the starch-binding domain (SBD). We propose the designation of the homologues to the SBD of glucoamylase from Aspergillus niger as family I SBDs, the homologues to that of glucoamylase from Rhizopus oryzae as family II, and the homologues of this repeat sequence of BAA as family III.

Expression of Aspergillus aculeatus No. F-50 cellobiohydrolase I (cbhI) and beta-glucosidase 1 (bgl1) genes by Saccharomyces cerevisiae.

A cellobiohydrolase I (cbhI) and a beta-glucosidase 1 (bgl1) gene of Aspergillus aculeatus were expressed in Saccharomyces cerevisiae. The transformed cells secreted the enzymes efficiently in an active form. The recombinant CBHI gave two bands of different molecular mass (110 and 90 kDa) and the recombinant BGL1 gave one band (180 kDa) by SDS-PAGE. The recombinant CBHI and BGL1 had the same enzymatical properties as the native enzyme except for the specific activity toward cellulosic substrates. By the combination of three different types of cellulases, FI-CMCase, CBHI, and BGL1, we could hydrolyze Avicel up to 59% under our experimental conditions.

Cloning and sequencing of an endoglucanase gene from Scopulariopsis brevicaulis TOF-1212, and its expression in Saccharomyces cerevisiae.

The egI gene, encoding a major endoglucanase (EGI) of Scopulariopsis brevicaulis TOF-1212, was cloned and sequenced. The eglgene consisted of 868 bp with one intron and encoded a protein of 229 amino acids with a calculated molecular mass of 22,392 daltons. The EGI was assigned to a family 45 of glycosyl hydrolases and showed high similarity with other fungal endoglucanases, especially with those of Humicola grisea and Fusarium oxysporum, on the basis of hydrophobic cluster analysis. The egI gene was expressed under the promoter of the phosphoglycerate kinase gene (PGK) in Saccharomyces cerevisiae. The transformed cells were able to secrete the enzyme efficiently in an active form.

Molecular cloning and expression of proteinaceous alpha-amylase inhibitor gene from Streptomyces nitrosporeus in Escherichia coli.

The proteinaceous alpha-amylase inhibitor, T-76, gene was cloned by screening a Streptomyces nitrosporeus genomic library using a deoxyinosine-containing probe corresponding to the amino acid sequence of the inhibitor. The nucleotide sequence of the insert of a positive clone had an open reading frame of 330 bp that encoded a polypeptide of 110 amino acid residues with a calculated molecular mass of 11,306 daltons. The polypeptide begins with proximal basic amino acids and a region rich in hydrophobic amino acids that possibly act as a signal peptide for secretion, which is followed by a sequence consistent with the amino-terminal amino acid sequence of the T-76 inhibitor. Escherichia coli cells harboring the plasmid derivatives for expression produced the inhibitor in their periplasmic space. The amino-terminal sequence of the inhibitor produced by an E. coli transformant was identical to that of the T-76 inhibitor secreted by S. nitrosporeus. The amino acid sequence of the inhibitor deduced from nucleotide sequence showed significant homology to other proteinaceous alpha-amylase inhibitors.

Cloning and sequencing of beta-mannosidase gene from Aspergillus aculeatus no. F-50.

The manB gene, coding for a unique beta-mannosidase (MANB) of Aspergillus aculeatus, was cloned from genomic and cDNA libraries, and sequenced. The gene consists of 2,811 bp encoding a polypeptide of 937 amino acid residues with a molecular mass of 104,214 Da. The A. aculeatus MANB shared amino acid sequence identity with MANB of human (24%), goat (24%), bovine (24%), and Caenorhabditis elegans (22%). When the A. aculeatus MANB was compared with other related enzymes, a Glu residue corresponding to the active site identified by the Escherichia coli beta-galactosidase and the human beta-guclonidase was conserved. This is the first fungal gene that encodes MANB.