The permeabilization of yeast cells with methanol, ethanol, and isopropyl alcohol under various conditions was studied to develop the preparation method of high activity whole cell biocatalysts. Recombinant Saccharomyces cerevisiae, which intracellularly overexpresses glyoxalase I and catalyzes the conversion of methylglyoxal to S-lactoylglutathione in the presence of glutathione, was used as the model system. The permeabilization treatments with alcohols significantly enhanced the activities of yeast cells. Especially, the initial S-lactoylglutathione production rates of cells permeabilized with 40% ethanol and isopropyl alcohol solutions for 10 min at 4 degrees C were high and were 364 and 582 times larger than those of untreated cells, respectively. These permeabilized yeast cells retained high activities during repeated batch reactions. Even in third batch reaction, they showed approximately 70-80% of the activity in the first batch. The plasma membrane of S. cerevisiae cells was damaged by the treatment with alcohol solutions in such a way that leakage of glyoxalase I from the cells is rather small and that both substrate and product show very high permeability. The initial S-lactoylglutathione production rates of these permeabilized cells were 1.5-2.5 times larger than those of glyoxalase I in cell extracts prepared by ethyl acetate method from the same amount of cells. These results demonstrate that the recombinant S. cerevisiae cells permeabilized with alcohol solutions under the optimum condition are very effective whole cell biocatalysts. Copyright 1999 John Wiley & Sons, Inc.

Methylglyoxal is an endogenous cytotoxic compound formed as a byproduct of glycolysis. We systematically analyzed the metabolic fate of methylglyoxal in various microorganisms and found that glyoxalase I is a ubiquitous and critical enzyme for its detoxification. We found that glyoxalase I consists of five segments (regions I-V) which are conserved among the glyoxalase Is of various species. We hypothesize that yeast glyoxalase I evolved by gene duplication and here show supporting evidence. We also found that expression of the structural gene for the glyoxalase I of S. cerevisiae is induced by osmotic stress through the HOG-MAG kinase signaling pathway. We identified the physiological significance of this glycolytic-methylglyoxal pathway in S, cerevisiae.

The EST2 gene, encoding an isoamyl acetate-hydrolyzing esterase, was disrupted in a diploid strain of Saccharomyces cerevisiae UT-1 (MATa/MATα ura3/ura3 trp1/trp1 EST2/EST2), which is derived from the industrial sake yeast Kyokai no. 701 (strain K-701), by using two disruption plasmids (pDest2U, est2::URA3; and pDest2T, est2::TRP1) sequentially. Genomic Southern blot analysis revealed that both loci of the EST2 gene on the chromosome of strain UT-1 were disrupted. The resultant mutants were named UTUT-1 and UTUT-2 (a/MATα ura3/ura3 trp1/trp1 est2::URA3/est2::TRP1). Deficiency in Est2p esterase was also confirmed by activity staining of the gel after native-polyacrylamide gel electrophoresis of cell extracts of the two mutant strains. Small scale sake brewing was carried out using these sake yeasts and the strains they were derived from, and their brewing properties were compared. The fermentation profiles of the four strains (strains K-701, UT-1, UTUT-1, and UTUT-2) were largely similar. The components of the resulting sake were also similar except for the acetate ester concentration, although strains UTUT-1 and UTUT-2 produced approximately 2-times more isoamyl acetate than the wild type K-701. These resuts strongly suggest that the EST2 gene product is likely to play a crucial role in the hydrolysis of isoamyl acetate in the sake mash. Strains UTUT-1 and UTUT-2, deficient in Est2p esterase, are suitable for sake brewing.

In continuation to our search of fullerenes in usual soots, sixteen additional samples of Chinese ink stick have been analyzed by HPLC. When combined with our previous results, we recognize that C60-content in Chinese ink sticks decreases with time following the first order rate law. This novel observation was interpreted in terms of diffusion-dissipation of C60 molecules through the solid stick.

A mutant deficient carboxylesterase which hydrolyzes isoamyl acetate (est2) was isolated from a yeast, Saccharomyces cerevisiae. The EST2 gene was cloned and its nucleotide sequence determined. The EST2 gene has an open reading frame of 714 bp (238 amino acids), and the molecular weight of the gene product calculated from the predicted amino acid sequence was 27,304.66. The EST2 gene product lacks the consensus sequence (Gly-Xaa-Ser-Xaa-Gly) that is observed in the serine type esterase or lipase, although a quite similar sequence (Ala-Cys-Ser-Ala-Gly) was found. Genomic Southern analysis revealed that the EST2 gene was present as a single copy on the chromosomal DNA of laboratory strains of S. cerevisiae as well as the sake brewing yeasts. The est2 mutant accumulated approximately 19 times higher amounts of isoamyl acetate compared with the parent strain in laboratory scale sake brewing. Therefore, the EST2 gene product is likely to play a crucial role in the hydrolysis of isoamyl acetate in sake mash.

Hansenula yeasts are known to be producers of esters. Among the Hansenula genera, H. mrakii IFO 0895 was highly resistant to ethyl acetate. The yeast could use ethyl acetate as a sole source of carbon. Iso-amyl acetate is one of the fruit flavor esters of sake (Japanese rice wine) and the production of the ester by H. mrakii was investigated using a small scale-sake brewing system. H. mrakii produced comparable amount of iso-amyl acetate with an industrially used sake brewing yeast, Saccharomyces cerevisiae Kyokai No. 7. Esterases are believed to be involved in the production of esters by H. mrakii.

NAD(P)H dehydrogenase was purified approximately 480-fold from Saccharomyces cerevisiae with 6.5% activity yield. The enzyme was homogeneous on polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be 40,000–44,000 by gel filtration on Sephadex G-150 column chromatography and SDS-polyacrylamide gel electrophoresis. The Km values for NADPH and NADH were 7.3 μM and 0.1 mM, respectively. The activity of the enzyme increased approximately 4-fold with Cu2+. FAD, FMN and cytochrome c were not effective as electron acceptors, although Fe(CN)63− was slightly effective. NADH generated by the reaction of lactaldehyde dehydrogenase in the glycolytic methylglyoxal pathway will be reoxidized by NAD(P)H dehydrogenase. NAD(P)H dehydrogenase thus may contribute to the reduction/oxidation system in the glycolytic methylglyoxal pathway to maintain the flux of methylglyoxal to lactic acid via lactaldehyde.

When two types of alginate lyases [poly-mannuronate (poly M)-specific and poly-guluronate (poly G)-specific lyases] were incubated on agar plates containing alginate and the surfaces of the plates covered with CaCl2 solution, poly M-specific and poly G-specific lyases yielded white-halos and white-rings, respectively. The substrate specificity of alginate lyase was easily determined by discriminating between the types of gellation (i.e. halo or ring formation) caused by the interaction between calcium ions and depolymerized alginates.

Conditions for the production of bacterial alginate-specific lyase (A1-III) by Bacillus subtilis transformed with an inducible secretion vector pISA412 harboring the A1-III gene from a bacterium (Flavobacterium sp.) of strain A1 were studied. Galactose at around 3% was found as the most efficient carbon source in the pH region between 7.0∼8.5. A higher amount of potassium phosphate was required to repress degradation of A1-III produced in medium. Under the most preferable culture conditions determined, production of the lyase reached approximately 0.3 mg/ml. The properties of A1-III purified from the medium were comparable with those of A1-III present in strain A1 in terms of molecular size, substrate specificity and in N-terminal amino acid sequence.

Klebsiella sp. LF 1202, previously isolated from soil assimilates adenosine as a sole source of carbon and nitrogen. The bacterium inducibly formed both purine nucleoside phosphorylase (PNPase) and uridine phosphorylase (UPase), simultaneously, when cultured in adenosine-containing medium or uridine-containing medium. PNPase and UPase were purified to homogeneity on SDS-PAGE, and some properties were examined. Enzymic production of 1-β-d-ribofuranosyl-1, 2, 4,-triazole-3-carboxamide (Virazole), an anti-DNA virus pharmaceutical drug, was demonstrated using the combined reaction of both enzymes.

Sporulation of the yeast Saccharomyces cerevisiae 4011 and other strains of S. cerevisiae was induced by d- or l-lysine during growth in a nutrient-rich medium. Potassium acetate was not required for the sporulation. The effects of other d- or l-amino acids on sporulation, if any, were negligible or very slight. Although the sporulation efficiency varied depending on the yeast strain, lysine concentration, pH of the medium and incubation period, the results obtained indicated that nutritional starvation is not a prerequisite for the sporulation of yeast cells, and that lysine or a certain cellular molecule involved in the metabolism of the amino acid has the ability to cause meiosis and sporulation in yeast cells.

A bacterium (strain Al) isolated from a ditch produces three kinds of intracellular alginate lyases [Al-I (molecular weight: M.W. 60,000), Al-II-1 (M.W. 60,000) and Al-II-2]; the former two lyases have been purified and characterized (Yonemoto et al., J. Ferment. Bioeng., 72, 152–157, 1991). As part of a series of studies, Al-II-2 lyase was purified from cell-free extract of the bacterium. The lyase, with a M.W. of 25,000, depolymerized sodium-, potassium- and propyleneglycol alginates most efficiently at pH 8.0, 70°C, but it was inactive toward bacterial alginates with O-acetyl groups.

Journal Article Effects of Electric Shock on Escherichia coli K-12: Purification of a 23-kDa Protein from Electric Shock-induced Fluid of E. coli Get access Yoshiharu Inoue, Yoshiharu Inoue Research Institute for Food Science, Kyoto University, Uji, Kyoto 611, Japan Search for other works by this author on: Oxford Academic Google Scholar Akira Kimura Akira Kimura Research Institute for Food Science, Kyoto University, Uji, Kyoto 611, Japan Search for other works by this author on: Oxford Academic Google Scholar Bioscience, Biotechnology, and Biochemistry, Volume 57, Issue 5, 1 January 1993, Pages 867–869, https://doi.org/10.1271/bbb.57.867 Published: 01 January 1993 Article history Published: 01 January 1993 Received: 27 January 1993

A high expression system of the γ-glutamylcysteine synthetase gene (gshl) of Escherichia coli B was constructed, and rapid purification of GSH-I was performed. The active site of GSH-I was analysed by chemical modification, and Lys, Arg and His residues seemed to be involved in the active site of the enzyme. Among them, His residues were substituted to Ala by site-directed mutagenesis, and His-150 was found to be essential for the activity of GSH-I.

A bacterium (strain A1) isolated from a ditch synthesized three types of intracellular alginate lyases: A1-I (molecular weight [M.W.] 60,000), A1-II-2 (M.W. 25,000) and A1-III (M.W. 38,000). The nucleotide sequence of the gene for A1-I lyase, which has been cloned in Escherichia coli DH1 was determined. The open reading frame of the gene encoded 622 amino acids with a calculated M.W. of 69,153. The N-terminal amino acid sequence of A1-I lyase purified from strain A1 or E. coli DH1 cells transformed with the A1-I lyase gene was consistent with the deduced sequence from 55His to 74Ala, indicating that the A1-I lyase was synthesized as a precursor with a M.W. of 69,153 and then processed to a mature form with a M.W. of 63,681. The N-terminal sequence of the first twenty amino acids of A1-III lyase was found to match that of A1-I lyase. The N-terminal sequence of the first twenty amino acids of A1-II-2 lyase was consistent with the deduced amino acid sequence from 414Ala to 433Val in the nucleotide sequence of the A1-I lyase gene. These results indicated that the A1-I lyase was further processed to generate A1-II-2 and A1-III lyase species.

Wild type cells of Hansenula mrakii IFO 0895 were highly resistant to the oxidative stress caused by lipid hydroperoxide. The resistance was due to a glutathione peroxidase (GSHPx) which was induced when the yeast was cultured in a medium containing lipid hydroperoxide (Inoue, Y. et al. , Agric. Biol. Chem., 54, 3289–3293, 1990). In order to investigate the role of GSHPx, two mutants sensitive to lipid hydroperoxide were isolated. The phenotypes of the mutants were temperature-dependent; i.e. , the mutants could grow at 28°C, but not at 35°C in the presence of lipid hydroperoxide. The mutants failed to induce the GSHPx at 35°C. However, the enzyme induced at 28°C and prepared from both mutants was stable after incubation at 37°C for 1 h.

A DNA fragment enhancing resistance against oxidative stress caused by several peroxides was cloned from the yeast Saccharomyces cerevisiae, and the corresponding gene was designated OSR; oxidative stress-resistant gene. By amplification of the OSR gene in the yeast cell, the transformant became resistant to tert-butyl hydroperoxide, linoleic acid hydroperoxide and hydrogen peroxide, whereas it became hypersensitive to cumene hydroperoxide. Thus, the dosage of the OSR gene in the yeast cell seemed to affect the behaviour of the transformant against peroxides. The resistance of the transformant was suppressed by the addition of buthionine sulfoximine, a potent inhibitor of glutathione biosynthesis, into a medium containing lipid hydroperoxide; thus a glutathione-dependent resistance mechanism was suggested to be concerned with the phenotype of the transformant.

As the result of the development of recDNA techniques, it has become possible to manipulate genes and breed useful microorganisms. Among various transformants bred in our laboratory to produce large amounts of useful compounds 2 topics will be described in this article. One is the breeding of a transformant of E. coli which accumulates tuna growth hormone. The other is the breeding of a jumbo yeast and production of S-lactoylglutathione.