Purification and characterization of a O-methyltransferase capable of methylating 2-hydroxy-3-alkylpyrazine from Vitis vinifera L. (cv. Cabernet Sauvignon).

An S-adenosyl-L-methionine-dependent O-methyltransferase capable of methylating 2-hydroxy-3-alkylpyrazine (HP) was purified 7,300-fold to apparent homogeneity with an 8.2% overall recovery from Vitis vinifera L. (cv.

AFLP analysis of type strains and laboratory and industrial strains of Saccharomyces sensu stricto and its application to phenetic clustering.

Using nine primer pairs, amplified fragment length polymorphism (AFLP) analysis was conducted to characterize industrial, laboratory and type strains of Saccharomyces sensu stricto. S. cerevisiae, S.

S-Adenosyl-L-methionine-dependent O-methylation of 2-hydroxy-3-alkylpyrazine in wine grapes: a putative final step of methoxypyrazine biosynthesis.

The final step of 2-methoxy-3-alkylpyrazine (MP) biosynthesis has been presumed to involve O-methylation of 2-hydroxy-3-alkylpyarzine (HP), although this reaction has never been demonstrated in organisms. We detected 2-hydroxy-3-isobutylpyrazine (IBHP) and 2-hydroxy-3-isopropylpyrazine (IPHP) in unripe grapes, and S-adenosyl-L-methionine-dependent O-methyltransferase (OMT) activity toward HP in crude extracts from young shoots and unripe grapes that accumulate MP at different levels. The levels of HP in the berries and stems were estimated by using 2-hydroxy-3-sec-butylpyrazine as an internal standard.

Transport of pyruvate in Saccharomyces cerevisiae and cloning of the gene encoded pyruvate permease.

Pyruvate uptake in Saccharomyces cerevisiae was not observed at 0 degrees C and was prevented by the uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP). The initial uptake rate of S. cerevisiae kyokai No.

Molecular cloning and characterization of a transcriptional activator gene, amyR, involved in the amylolytic gene expression in Aspergillus oryzae.

A gene, designated amyR, coding for a transcriptional activator involved in amylolytic gene expression has been cloned from Aspergillus oryzae by screening for a clone that enabled to reverse the reduced expression of the alpha-amylase gene (amyB) promoter. amyR encodes 604 amino acid residues of a putative DNA-binding protein carrying a zinc binuclear cluster motif (Zn(II)2Cys6) belonging to the GAL4 family of transcription factors. The amyR gene disruptants showed a significant restricted growth on starch medium and produced little of the amylolytic enzymes including alpha-amylase and glucoamylase compared with a non-disruptant, indicating that amyR is a transcriptional activator gene involved in starch/maltose-induced efficient expression of the amylolytic genes in A.

The bglA gene of Aspergillus kawachii encodes both extracellular and cell wall-bound beta-glucosidases.

We cloned the genomic DNA and cDNA of bglA, which encodes beta-glucosidase in Aspergillus kawachii, based on a partial amino acid sequence of purified cell wall-bound beta-glucosidase CB-1. The nucleotide sequence of the cloned bglA gene revealed a 2,933-bp open reading frame with six introns that encodes an 860-amino-acid protein. Based on the deduced amino acid sequence, we concluded that the bglA gene encodes cell wall-bound beta-glucosidase CB-1.

Structure of the glucan-binding sugar chain of Tip1p, a cell wall protein of Saccharomyces cerevisiae.

Tip1p is one of the major cell wall mannoproteins of Saccharomyces cerevisiae and is presumed to be synthesized as a glycosylphosphatidylinositol (GPI)-anchored form. We purified Tip1p from a glucanase extract of yeast cell walls and analyzed the sugar chain involved in the cell wall linkage. One mol of glucanase-extracted Tip1p contained 7.

Purification and characterization of a feruloylesterase from Aspergillus awamori.

A feruloylesterase was purified from the extracellular broth of Aspergillus awamori grown on wheat bran culture. The purified enzyme gave a single band on SDS-polyacrylamide gel electrophoresis and isoelectric focusing, with an apparent M(r) of 35,000 and a pI of 3.8, respectively.

Purification and characterization of extracellular and cell wall bound beta-glucosidases from Aspergillus kawachii.

We isolated two extracellular beta-glucosidases (EX-1: 145 kDa, EX-2: 130 kDa) and one cell wall bound beta-glucosidase (CB-1: 120 kDa) from Aspergillus kawachii and characterized their physical and kinetic properties. From the results of N-terminal amino acid sequence, enzymatic parameters and deglycosylation of the three purified enzymes, we strongly suggest that these three enzymes were products of the same gene, modified by different degree of glycosylation. All three purified beta-glucosidases adsorbed to the purified cell wall fraction of this strain.

Sed1p is a major cell wall protein of Saccharomyces cerevisiae in the stationary phase and is involved in lytic enzyme resistance.

A 260-kDa structural cell wall protein was purified from sodium dodecyl sulfate-treated cell walls of Saccharomyces cerevisiae by incubation with Rarobacter faecitabidus protease I, which is a yeast-lytic enzyme. Amino acid sequence analysis revealed that this protein is the product of the SED1 gene. SED1 was formerly identified as a multicopy suppressor of erd2, which encodes a protein involved in retrieval of luminal endoplasmic reticulum proteins from the secretory pathway.

Transformation system for a wastewater treatment yeast, Hansenula fabianii J640: isolation of the orotidine-5'-phosphate decarboxylase gene (URA3) and uracil auxotrophic mutants.

A transformation system for Hansenula fabianii J640, a commonly used wastewater treatment yeast, was constructed. As a host cell, a uracil auxotrophic mutant designated as H. fabianii J640 u-1, which was confirmed to have a mutation at the locus of the gene for orotidine-5'-phosphate (OMP) decarboxylase (URA3), was obtained by positive selection using 5-fluoroorotic acid.

Identification and analysis of a static culture-specific cell wall protein, Tir1p/Srp1p in Saccharomyces cerevisiae.

A 100-kDa protein was found to be a major cell wall protein in Saccharomyces cerevisiae cells cultured without shaking, but was not present in cells cultured with shaking. The amino acid sequence of this protein was identical to the sequence of Tir1p/Srp1p. TIR1/SRP1 has previously been identified as a gene induced by glucose, cold shock or anaerobiosis and was believed to be a cell membrane protein but not a cell wall protein.

An Aspergillus awamori acetylesterase: purification of the enzyme, and cloning and sequencing of the gene.

An inducible acetylesterase was purified from the culture medium of Aspergillus awamori strain IFO4033 growing on wheat-bran culture by ion-exchange, gel-filtration and hydrophobic-interaction chromatographies. The purified enzyme had an Mr of 31000 and contained Asn-linked oligosaccharides. The enzyme liberated acetic acid from wheat bran, hydrolysed only alpha-naphthyl acetate and propionate when aromatic esters were used for the substrate, and was tentatively classified as a carboxylic esterase (EC 3.

Raw-starch-digesting and thermostable alpha-amylase from the yeast Cryptococcus sp. S-2: purification, characterization, cloning and sequencing.

A starch-degrading enzyme produced by the yeast Cryptococcus sp. S-2 was purified in only one step by using an alpha-cyclodextrin-Sepharose 6B column, and was characterized as an alpha-amylase (EC 3.2.

Acid xylanase from yeast Cryptococcus sp. S-2: purification, characterization, cloning, and sequencing.

A xylan-degrading enzyme produced by yeast Cryptococcus sp. S-2 was isolated and purified, and characterized as an endoxylanase (1,4-beta-D-xylan xylanohydrolase [EC 3.2.

Methoxypyrazine Content of Japanese Red Wines.

Three 2-methoxy-3-alkylpyrazines (MPs) in Japanese red wine and grape samples were determined by GC-EIMS, using 2-methyl-3-n-propylpyrazine as an internal standard. MPs in the Cabernet Sauvignon red wines were derived not only from the pulp but also from other parts of the grape berries. All of the Cabernet Sauvignon red wines made annually from 1975 to 1994 contained 2-methoxy-3-isobutylpyrazine (isobutylMP), although those made from well-ripened grapes had a low isobutylMP level.

Cloning, characterization and overproduction of nuclease S1 gene (nucS) from Aspergillus oryzae.

The nuclease S1 gene (nucS) from Aspergillus oryzae was isolated using a polymerase-chain-reaction-amplified DNA fragment as a probe, and a 2.6-kb SalI-EcoRI fragment containing the nucS gene was sequenced. It was deduced that the nucS gene had two short introns, 49 and 50 nucleotides in length.

Cloning and nucleotide sequence of the ribonuclease T1 gene (rntA) from Aspergillus oryzae and its expression in Saccharomyces cerevisiae and Aspergillus oryzae.

A genomic DNA encoding ribonuclease (RNase) T1 from Aspergillus oryzae was cloned using a synthetic oligonucleotide probe. The cloned gene (designated rntA) encoded functional RNase T1, since an A. oryzae transformant with multiple copies of the rntA gene showed higher RNase T1 activity (over 200 times) than a transformant with a vector.

Molecular cloning of CWP1: a gene encoding a Saccharomyces cerevisiae cell wall protein solubilized with Rarobacter faecitabidus protease I.

A yeast cell wall glycoprotein with a molecular weight of 40,000, named gp40, was solubilized from SDS-extracted cell wall of Saccharomyces cerevisiae by incubation with Rarobacter faecitabidus protease I, which is a yeast-lytic enzyme. Based on its amino acid sequence, we cloned and sequenced the gene encoding the precursor of gp40, named CWP1; cell wall protein gene. The DNA sequence of the CWP1 gene was identical to YKL443, an open reading frame identified in a genome sequencing program for yeast chromosome XI.

Electrophoretic karyotype and gene assignment to chromosomes of Aspergillus oryzae.

An electrophoretic karyotype of Aspergillus oryzae was obtained by transverse altering-field electrophoresis. Seven chromosomal bands were found. With Schizosaccharomyces pombe chromosomes as size standards, we estimated the sizes of the chromosomes to be 7.

Cloning and nucleotide sequence of the acid protease-encoding gene (pepA) from Aspergillus oryzae.

We have cloned a genomic DNA sequence encoding the acid protease (PEPA) from Aspergillus oryzae using a 0.6-kb fragment as a probe. This fragment was amplified by the polymerase chain reaction (PCR) using oligonucleotide primers designed from the partial amino acid sequences of peptide fragments of the purified protein.

Molecular structure of Rarobacter faecitabidus protease I. A yeast-lytic serine protease having mannose-binding activity.

Rarobacter faecitabidus protease I (RPI) is a serine protease exhibiting lytic activity toward living yeast cells. RPI is similar to elastase in its substrate specificity and has a lectin-like affinity for mannose. The gene encoding RPI was cloned to elucidate its structure and function.

Cloning and sequencing of the xynA gene encoding xylanase A of Aspergillus kawachii.

We have cloned the xynA gene coding for xylanase A, a major component of the xylanase family, from Aspergillus kawachii. The cDNA was isolated from an A. kawachii cDNA library by immunoscreening using antibody raised against the purified xylanase A protein.