Molecular cloning of the isoamyl alcohol oxidase-encoding gene (mreA) from Aspergillus oryzae.

Isoamyl alcohol oxidase (IAAOD) is a novel enzyme that catalyzes the formation of isovaleraldehyde, which is the main component of mureka that gives sake an off-flavor (Yamashita et al. Biosci. Biotechnol. Biochem., 63, 1216-1222, 1999). We cloned the genomic DNA sequence encoding IAAOD from a koji mold, Aspergillus oryzae, using a PCR-amplified DNA fragment corresponding to the partial amino acid sequences of the purified protein as a probe. The cloned gene comprises 1903 bp of an open reading frame with three putative introns and encodes 567 amino acids with a presumed signal peptide consisting of 24 amino acids at the N-terminus. Moreover, nine potential N-glycosylation sites were present. Homology search on amino acid sequence showed that IAAOD has a region significantly similar to those conserved in FAD-dependent oxidoreductases. Southern hybridization analysis revealed that the cloned gene exists as a single copy in the A. oryzae RIB 40 chromosome. The cloned gene was overexpressed under the control of the amyB promoter in A. oryzae. The isovaleraldehyde-producing activity in the culture supernatant of one transformant was over 800 times as high as that of transformant with the control vector. This result demonstrates that the cloned gene encodes IAAOD. We named this novel alcohol oxidase gene "mreA".

Effects of aldehyde dehydrogenase and acetyl-CoA synthetase on acetate formation in sake mash.

To reveal the mechanism of the production of acetate by sake yeast (Saccharomyces cerevisiae), the expression of genes encoding aldehyde dehydrogenase (ALD), acetyl-CoA synthetase (ACS) and acetyl-CoA hydrolase (ACH), which are related to acetate production, was investigated. Northern blot analysis using total RNA of sake yeast isolated from sake mash revealed that all of the tested genes, ACS1, ACS2, ALD2/3, ALD4, ALD6 and ACH1, were transcribed during sake fermentation. Transcription of ALD2/3 was detected only in the early stage of sake fermentation. A static culture of sake yeast in hyperosmotic media including 1 M sorbitol or 20% glucose resulted in high acetate production and increased transcription of ALD2/3. This is the same result as reported in an aerobic condition, and induction of ALD2/3 seemed to be one reason for high acetate production at high glucose concentration during fermentation. Overexpression of ACS2 resulted in low acetate production both during small-scale sake fermentation and in a static liquid culture. On the other hand, over-expression of ACS1 did not change acetate productivity significantly in a static culture. These results indicate that ALD2/3 and ACS2 play important roles for acetate production during sake fermentation.

Purification and Characterization of Isoamyl Alcohol Oxidase ("Mureka"-Forming Enzyme).

Isoamyl alcohol oxidase (IAAOD) was purified to apparent homogeneity on SDS-PAGE from ultrafiltration (UF) concentrated sake. IAAOD was a glycoprotein, a monomeric protein with an apparent molecular mass of 73 and 87 kDa, by SDS-PAGE and gel filtration on HPLC, respectively. IAAOD showed high substrate specificity toward C5 branched-chain alkyl alcohol (isoamyl alcohol), and no activity toward shorter (C1-C4) or longer (C7-C10) alkyl alcohols tested. IAAOD was stable between pH 3.0-6.0 at 25°C. The optimum pH was 4.5 at 35°C. Heavy metal ions, p-chloromercuribenzoate (PCMB), hydrazine, and hydroxylamine strongly inhibited the enzyme activity, and an anti-oxidant like L-ascorbate did also. Isovaleraldehyde was produced markedly in pasteurized sake by adding purified IAAOD, therefore, we concluded that it was the enzyme that causes formation of mureka, an off-flavor of sake, the main component of which is isovaleraldehyde.

Magnetic resonance imaging of acute cerebellar ataxia: report of a case with gadolinium enhancement and review of the literature.

A 10-year-old girl exhibited severe cerebellar ataxia following acute enterocolitis, and was diagnosed as having acute cerebellar ataxia (ACA). MRI of the brain in the acute stage revealed moderate swelling of the cerebellum and abnormal signal intensity enhanced with gadolinium in the cerebellar hemisphere. This is the first report of an ACA case with positive gadolinium enhancement. Cases of ACA with MRI abnormalities are reviewed and the clinical entity of ACA is discussed in association with autoimmune encephalitis.

Interaction of Protein Particles with Lipids in Soybean Milk.

The protein in soybean milk exists as 11S and 7S globulins, and the particles formed from them. The lipid content and composition in the protein fractions and effects of defatting on the form of the protein particles were investigated. The size-distribution of protein particles in both raw and heated soybean milk (soymilk) was not influenced by defatting with hexane, but the number of large particles were slightly increased. The protein particles from raw and heated soymilk samples contained 60% and 3% of the total lipid, respectively. Almost all neutral lipid in the particles of raw soymilk was moved to a floating fraction by heating, but a half of the phospholipids was retained in the particles. The protein components from the hexane-defatted meal were similar to those from whole meal, but those from the C-M-de-fatted meal contained remarkably little ß-conglycinin. C-M-de-fatting (Removal of polar lipids) caused a reduction in the particulate fraction, and the addition of phospholipids (lecithin) promoted the formation of protein particles.

Chromosomal transformation in Saccharomyces cerevisiae with DNA isolated by pulse field gel electrophoresis.

We isolated and purified yeast chromosome DNA molecules using pulse field gel electrophoresis (PFG). The isolated DNA had nearly the same size as the native chromosomal DNA on PFG. We could directly transform Saccharomyces cerevisiae yeasts with it, and obtain transformants that were selected by complementation of several markers. They had new chromosome DNA bands observed on PFG. The new chromosome was very stable during mitosis and mating processes, and each of the three homologous chromosomes in the derivative zygotes of transformants was separated equally in daughter cells.