A newly isolated strain, MU-2, which produces very high β-fructofuranosidase activity, was identified asAspergillus japonicus. For enzyme production by the strain, sucrose at 20% (w/v) was the best carbon source and yeast extract at 1.5 to 3% (w/v) the best nitrogen source. Total enzymatic activity and cell growth were at maximum after 48 h, at 1.57×10(4) U/flask and 0.81 g dry cells/flask, respectively. The optimum pH value of the enzymatic reaction was between 5.0 and 5.5 and the optimum temperature 60 to 65°C. The enzyme produced 1-kestose (O-β-D-fructofuranosyl-(2→1)-β-D-fructofuranosyl α-D-glucopyranoside) and nystose (O-β-D-fructofuranosyl-(2→1)-β-D-fructofuranosyl-(2→1)-β-D-fructofuranosyl α-D-glucopyranoside) from sucrose by fructosyl-transferring activity. The strain was found to be very useful for industrial production of β-fructofuranosidase.

An NADPH-dependent aldehyde reductase (ALR) isolated from a red yeast, Sporobolomyces salmonicolor, catalyzes the reduction of a variety of carbonyl compounds. To investigate its primary structure, we cloned and sequenced the cDNA coding for ALR. The aldehyde reductase gene (ALR) comprises 969 bp and encodes a polypeptide of 35,232 Da. The deduced amino acid sequence showed a high degree of similarity to other members of the aldo-keto reductase superfamily. Analysis of the genomic DNA sequence indicated that the ALR gene was interrupted by six introns (two in the 5 noncoding region and four in the coding region). Southern hybridization analysis of the genomic DNA from S. salmonicolor indicated that there was one copy of the gene. The ALR gene was expressed in Escherichia coli under the control of the tac promoter. The enzyme expressed in E. coli was purified to homogeneity and showed the same catalytic properties as did the enzyme from S. salmonicolor. Aldehyde reductase (ALR) (EC 1.1.1.2), aldose reductase (EC 1.1.1.21), and carbonyl reductase (EC 1.1.1.184) catalyze the NADPH-dependent reduction of a variety of carbonyl compounds and are widely distributed in mammalian and plant tissues. The amino acid sequences of aldose reductases and ALRs show significant similarity, but that of carbonyl reductase does not show similarity to the other sequences. These enzymes are members of the aldo-keto reductase superfamily (3), which includes prostaglandin F synthase (29),-crystalline (8), ␦-3-ketosteroid 5-␤-reductase (17), a soybean reductase (30), and chlordecone reductase (34). The yeast GCY gene product (16), the p100/11E gene product of Leishmania major (21), and mouse androgen-dependent protein (19), whose functions are not known, also show a high degree of sequence similarity with members of the aldo-keto reductase superfamily. The physiological roles of the aldo-keto reductases have not been established. It is suggested that under physiological conditions aldose reductase participates in osmoregulation, but under hyperglycaemic conditions it contributes to the onset and development of severe complications in diabetes. We isolated an NADPH-dependent ALR from the red yeast Sporobolomyces salmonicolor, which in addition to catalyzing common substrates of ALRs (35) can catalyze the asymmetric reduction of ethyl 4-chloro-3-oxobutanoate to ethyl (R)-4chloro-3-hydroxybutanoate, a promising chiral building block for chemical synthesis of L-carnitine. L-Carnitine deficiency is a common secondary problem associated with many metabolic diseases, and oral administration of the compound produces a

β-Fructofuranosidase fromAspergillus japonicus, which produces 1-kestose (O-β-D-fructofuranosyl-(2→1)-β-D-fructofuranosyl α-D-glucopyranoside) and nystose (O-β-D-fructofuranosyl-(2→1)-β-D-fructofuranosyl-(2→1)-β-D-fructofuranosyl α-D-glucopyranoside) from sucrose, was purified to homogeneity by fractionation with calcium acetate and ammonium sulphate and chromatography with DEAE-Cellulofine and Sephadex G-200. Its molecular size was estimated to be about 304,000 Da by gel filtration. The enzyme was a glycoprotein which contained about 20% (w/w) carbohydrate. Optimum pH for the enzymatic reaction was 5.5 to 6. The enzyme was stable over a wide pH range, from pH 4 to 9. Optimum reaction temperature for the enzyme was 60 to 65°C and it was stable below 60°C. The Km value for sucrose was 0.21M. The enzyme was inhibited by metal ions, such as those of silver, lead and iron, and also byp-chloromercuribenzoate.

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