Kiyohisa Imada

Purification and properties ofβ-fructofuranosidase fromAureobasidium sp. ATCC 20524

SummaryPurification and properties of two β-fructofuranosidases, which produce 1-kestose (1F-β-fructofuranosyl-sucrose) from sucrose, fromAureobasidium sp. ATCC 20524 are reported. The enzymes were purified to homogeneity by fractionations involving ethanol, calcium acetate and ammonium sulfate and DEAE-Cellulofine and Sephadex G-200 chromatography. Molecular weights of the enzymes were estimated to be about 318000 (P-1) and 346000 (P-2) daltons by gel filtration. The enzymes were glycoproteins that contained about 30% (w/v) (P-1) and 53% (w/v) (P-2) carbohydrate. The optimum pH for the enzymatic reactions were 4.5–5.5 (P-1) and 4.5–6 (P-2). The enzymes were stable over a wide pH range (4–9). The optimum reaction temperatures for both enzymes were 50–55°C and they retained more than 94% (P-1) and 98% (P-2) activities at 50°C after 15 min. TheKm values for sucrose were 0.47 M (P-1) and 0.65 M (P-2). The enzymes were inhibited by mercury, copper and lead ions as well asp-chloromercuribenzoate.

Production of a fructosyl-transferring enzyme byAureobasidium sp. ATCC 20524

SummaryProduction of a fructose-transferring enzyme byAureobasidium sp. ATCC 20524 and reaction conditions for the production of fructooligosaccharide, isokestose, were studied. The maximum total enzymatic activity of culture broth was 103.2 U/ml. The optimum reation pH and temperature of intracellular enzyme was 5–6 and 50°C, respectively.

Immobilization of a fructosyl-transferring enzyme from aureobasidium sp. on shirasu porous glass

Abstract A fructosyl-transferring enzyme from Aureobasidium sp. ATCC 20524 which produces 1-kestose from sucrose was immobilized onto a shirasu porous glass (1142 U g −1 support). The enzymatic profiles of the preparation were almost identical to those of the native one except that its stability was improved. The column packed with it was stable for more than 30 d during a continuous operation.

Continous production of 1-kestose by β-fructofuranosidase immobilized onshirasu porous glass

Summary1-Kestose was produced continously and selectively from 40% (w/v) sucrose solution at fast flow rate by a column packed with an immobilized β-fructofuranosidase onshirasu porous glass.

Observation of the chemical structure of fructooligosaccharide produced by an enzyme fromAureobasidium sp. ATCC 20524

The chemical structure of the oligosaccharide produced from sucrose by an enzyme extracted fromAureobasidium sp. ATCC 20524 was observed. The GC-MS analysis by methylation indicated that this oligosaccharide is composed of 2-linked, 1,2-linked fructose and 1-linked glucose. The [13C]-NMR spectrum indicated that the 1,2-linked glycosidic linkages of fructose of this oligosaccharide areβ, and the 1-linked glycosidic linkages of fructose are α. This investigation suggested that this oligosaccharide is isokestose.

Long-term continuous reaction of immobilized β-fructofuranosidase

Summaryβ-Fructofuranosidase was immobilized by alginate gel at high efficiency (92 %). The extreme long-term continuous reaction (half-life, 275 days) was achieved by the immobilized enzyme using sucrose at high concentration (500 mg ml−1) to produce fructo-olicosaccharides, such as 1-kestose (Fruβ2→1Fruβ2→1aGlc) and nystose (Fruβ2→1Fruβ2→1Fruβ2→1aGlc).

Immobilization ofβ-fructofuranosidase fromAureobasidium sp. ATCC 20524 on porous silica

Summaryβ-Fructofuranosidase P-1 fromAureobasidium sp. ATCC 20524, which produces a fructo-oligosaccharide (1-kestose) from sucrose, was immobilized covalently onto alkylamine porous silica with glutaraldehyde at high efficiency (44.4%). Optimum pore diameter of porous silica for immobilization of the enzyme was 91.7 nm. The enzymatic profiles of immobilized enzyme were almost identical to the native one except its stabilities to temperature and metal ions were improved. 1-Kestose was produced continuously and selectively from 40% (w/v) sucrose at fast flow rates by a column packed with the immobilized enzyme for up to 26 days, and the effluent concentration of 1-kestose remained in the range 113–135 mg ml−1.

Purification and properties of glucosyltransferase fromAureobasidium

SummaryPurification and properties of glucosyltransferase, which produces panose (Glcα1→6Glcα1→4Glc) and isomaltose (Glcα1→6Glc) from maltose (Glcα1→4Glc), are reported. The enzyme, fromAureobasidium, was purified to homogeneity by fractionations involving ammonium sulfate and DEAE-Cellulofine, S-Sepharose Fast Flow and Sephadex G-200 chromatography. Molecular mass of the enzyme was estimated to be 395 kDa by gel filtration. The enzyme was identified as a glycoprotein which contains 32% (w/w) carbohydrate. The optimum pH for the enzymatic reaction was 4.5–5.5 and the enzyme was stable over a pH range of 4–6. The optimum reaction temperature for the enzyme was 65°C and the enzyme retained more than 96% activity at 60°C after 15 min. The enzyme produced panose from maltose by means of a high efficiency (45.5%) glucosyl-transfer reaction. The enzyme was inhibited by metal ions, such as those of mercury, silver and aluminum, and also by organic inhibitors, especially nitrilotriacetic acid.

Utilisation of soybean residue for the production of β-fructofuranosidase

Abstract An industrial waste, soybean residue, was used for the production of a useful microbial enzyme, β-fructofuranosidase, by fermentation. The enzyme was produced by Aspergillus japonicus using soybean residue (3–15%, w/v) as the only medium component other than sucrose. Sucrose at 15% (w/v) was the best carbon source for enzyme production. The maximum enzymatic activity reached approximately 9 × 10 3 U/50 ml medium after 48 h. The cell growth reached at least 0·6 g dry cells/50 ml medium after 120 h. The fermentation by Asp. japonicus to produce β-fructofuranosidase was suggested as a method to recycle soybean residue.

Effect of deglycosylation on the properties of β-fructofuranosidaseP-1 fromAureobasidium sp. ATCC 20524

SummaryMost of the carbohydrate moiety of β-fructofuranosidaseP-1 fromAureobasidium sp. ATCC 20524 was removed by endo-β-N-acetylglucosaminidase F. A subunit of 94000 Da was observed in SDS-PAGE after deglycosylation. TheKm value for sucrose was not changed by deglycosylation but the stability at pH 4–5 and 50°C was decreased. The deglycosylated enzyme was more sensitive to proteases such as pronase E and subtilisin than the native enzyme. It is considered that the carbohydrate moiety of β-fructofuranosidaseP-1 contributes to the stability of the enzyme but is not essential in its catalytic function.