Search

The behaviour of halophilic and alkaliphilic lactic acid bacteria (HALAB) during maturation of salted and surface-ripened soft cheese remains unclear; thus, physiological properties of HALAB was investigated. Marinilactibacillus psychrotolerans was the predominant HALAB (106–1010 cfu g−1) in the cheese rind of Maroilles cheese. To elucidate HALAB behaviour during maturation, sterilised cheese was matured using M. psychrotolerans B7-9-5 as an adjunct culture. During the ripening period, concentrations of lactate and acetate decreased and increased, respectively. Strain B7-9-5 produced only acetate from lactate under aerobic conditions, and the relevant genes are present in its genome. Unlike in the marine strain, strain B7-9-5 contains gene clusters for lactose and citrate catabolism and utilised these carbohydrates for growth. Thus, when carbon sources are limited during ripening, this strain survives by utilising, if any, lactate, lactose and citrate, and it may contribute to flavour formation during ripening of salted and surface-ripened soft cheeses.

Latently existing non-starter lactic acid bacteria (NSLAB) were isolated from the French Brie de Meaux cheese using 12 isolation media with different NaCl concentrations and pH. Lactobacilli were frequently isolated using lactobacilli MRS agar, generally used for LAB isolation. Halophilic NSLAB (Tetragenococcus halophilus and Tetragenococcus koreensis) and slightly alkaliphilic NSLAB (Vagococcus lutrae), not detected using lactobacilli MRS agar or PCR-DGGE, were isolated using saline or alkaline media at log 6 to 8 cfu g−1 in the cheese rind. Their NaCl and pH optima were close to those of their isolation conditions. Phylogenetic independence of the T. halophilus isolate and genome characteristics and growth behaviour that allow T. koreensis and V. lutrae to survive in cheese rind via aerobic production of acetate from lactose were revealed. Thus, general laboratory isolation procedures should include various NaCl concentrations and pH conditions to capture a diversity of NSLAB in a living state from cheese.

The RpoH in Acetobacter pasteurianus NBRC3283 was characterized. It was revealed that the rpoH controls the expression of groEL, dnaKJ, grpE, and clpB to different extents. In addition, the rpoH disruption mutant became apt to be affected by heat, ethanol, and acetic acid, indicating its importance in acetic acid fermentation.

The grpE gene in Acetobacter pasteurianus NBRC 3283 was cloned and characterized, to elucidate the mechanism underlying the resistance of acetic acid bacteria to the stressors existing during acetic acid fermentation. This gene was found to be located in tandem with two related genes, appearing on the genome in the order grpE–dnaK–dnaJ. A σ32-type promoter sequence was found in the upstream region of grpE. The relative transcription levels of grpE, dnaK, and dnaJ mRNA were in the ratio of approximately 1:2:0.1, and the genes were transcribed as grpE–dnaK, dnaK, and dnaJ. The transcription level of grpE was elevated by heat shock and treatment with ethanol. Co-overexpression of GrpE with DnaK/J in cells resulted in improved growth compared to the single overexpression of DnaK/J in high temperature or ethanol-containing conditions, suggesting that GrpE acts cooperatively with DnaK/J for expressing resistance to those stressors considered to exist during acetic acid fermentation. Our findings indicate that GrpE is closely associated with adaptation to stressors in A. pasteurianus and may play an important role in acetic acid fermentation.

Amphibacillus is a genus for Gram-positive, spore-forming, rod-shaped, facultatively anaerobic bacteria with low-G+C content of DNA, established by Niimura et al. in 1990. Amphibacillus xylanus, the type species of the genus, grows well under both strictly anaerobic and aerobic conditions in spite of lacking any isoprenoid quinones, cytochromes, and catalase. Amphibacillus fermentum and Amphibacillus tropicus were later proposed by Zhilina et al. in 2001 for the isolates from a soda lake. In this paper, we revealed the latter two species also lacked isoprenoid quinones, cytochrome and catalase, and that they grew well under strictly anaerobic and aerobic conditions. The consistent growth of A. xylanus under both conditions is due to the presence of anaerobic and aerobic pathways for glucose metabolism in the organism. Although A. fermentum and A. tropicus are supposed to have a side enzymatic pyruvate pathway to produce lactate under both conditions, the two species have two major pyruvate metabolic pathways as observed in A. xylanus. Analysis data indicated that NADH formed both by the aerobic pyruvate pathway and by the glycolytic pathway was re-oxidized by the NADH oxidase in A. fermentum and A. tropicus as well as A. xylanus, and furthermore that the NADH oxidase-Prx (AhpC) system, i.e., NADH oxidase scavenging hydrogen peroxide with Prx, also functions in A. tropicus as observed with A. xylanus. Not only the taxonomical character of the genus Amphibacillus but also the growth characterization based on the two metabolic pathways and unique oxygen metabolism are distinctive in those traits from other facultative anaerobes.

The bacterium Acetobacter pasteurianus can ferment acetic acid, a process that proceeds at the risk of oxidative stress. To understand the stress response, we investigated catalase and OxyR in A. pasteurianus NBRC3283. This strain expresses only a KatE homolog as catalase, which is monofunctional and growth dependent. Disruption of the oxyR gene increased KatE activity, but both the katE and oxyR mutant strains showed greater sensitivity to hydrogen peroxide as compared to the parental strain. These mutant strains showed growth similar to the parental strain in the ethanol oxidizing phase, but their growth was delayed when cultured in the presence of acetic acid and of glycerol and during the acetic acid peroxidation phase. The results suggest that A. pasteurianus cells show different oxidative stress responses between the metabolism via the membrane oxidizing pathway and that via the general aerobic pathway during acetic acid fermentation.

各種ゴマ種子および脱脂粉末に，各種微生物を培養したものについて，抗血栓効果を調べるため，試料の50％エタノール抽出物について，ヒト血液を用いた血流改善，血小板凝集抑制，活性酸素消去の各試験を行った．（1）中米産ゴマ脱脂ゴマ粉末（セサミフラワー）は殆ど効果を示さないが，これにAsp. niger（NRIC 1222株）を7日間以上培養したものは，いずれの試験においても強い効果を示した．Asp. awamoriとAsp. oryzaeの場合は弱い効果がみられたが，B. nattoでは殆ど効果がみられなかった．（2）各試料の抽出物のリグナン含量の変化を調べた結果，このセサミフラワーには，セサミン，セサモリンは含有されているが，遊離セサミノールは全くない．Asp. nigerを培養した場合，セサミン，セサモリンは殆ど変化しないが，著量の遊離セサミノールが生成し，Asp. awamori, Asp. oryzaeの場合にもセサミノールは生成するが，比較的少なかった．（3）原料のフラワーを完全脱脂した試料に，Asp. niger培養，および加水分解酵素処理したものは，いずれの場合にも強い血液機能改善効果がみられ，セサミノールが著量に存在していた．またその脱脂フラワーを70％アルコールで抽出し，配糖体を除去後にAsp. nigerを培養した物では，改善効果はみられなかった．（4）タイ産黒ゴマ，パラグアイ産白ゴマ，高リグナンゴマのいずれの試料において，血流改善効果は種子において僅かにみられただけであったが，Asp. nigerの培養で増大した．発芽による影響はあまり見られなかった．血小板凝集抑制効果は種子でみられ，培養，発芽処理により効果はあまり変わらなかった．これらゴマ種子の場合，セサミン，セサモリンは高濃度に存在するが，セサミノールは遊離のものはなく，配糖体も殆どないと推定された．ただ発芽後微生物培養したものに少量の生成がみられた．（5）Asp. nigerまたは複数の酵素作用により発現する血流改善効果などの抗血栓機能には，それらの処理により，配糖体から生成したセサミノールが主に関与することを強く示唆するものである．（6）標品のセサミン，セサミノール，セサモールについて同様な血液試験をした結果，セサミノールにはいずれも有効で，とくに血流改善効果では非常に強い効果がみられた．セサミンは血小板凝集阻害のみに効果が見られ，セサモールには，いずれも弱い効果がみられるだけであった．（7）Asp. niger培養で著量（3～5％）に生成するクエン酸は，このような高濃度においては，培養抽出物の血栓予防効果に多少寄与しているものと思われた．（8）本研究により，セサミノール配糖体を多く含むゴマ試料に，その配糖体を有効に遊離セサミノールにする作用を持つ微生物の培養は，酵素作用を用いて加工することにより，強い抗血栓効果を持つ新しい機能性食品素材を作成する可能性が示された．

The dnaKJ operon of Acetobacter aceti was cloned and sequenced. The profile of the gene configuration was similar to that of other alpha-proteobacteria. In the DnaK and DnaJ proteins of A. aceti, the characteristic domains/motifs reported in other organisms were well conserved. This operon was transcribed in response to a temperature shift and exposure to ethanol/acetic acid. The overexpression of this operon in A. aceti resulted in improved growth compared to the control strain at high temperature or in the presence of ethanol, suggesting a correlation to resistance against stressors present during fermentation, although the overexpression did not increase the resistance to acetic acid.

ESR spectra were measured directly on a marked greening reaction mixture of Et-caffeate and a primary amino compound in alkali solution under aeration. A clear hyperfine structure was commonly detected early in the greening reaction with different amino compounds. Its hyperfine spectrum split into seven peaks was analyzed and found to be due to the oxidized free radical product of the Et-caffeate using an authentic sample system. Another type of hyperfine ESR spectrum was observed later in the reaction, and was altered with different amino compounds. The hyperfine structure for n-butylamine split into 12 lines. The latter type of free radical products were assumed to be a semiquinone type radical compounds of the trihydroxy benzacridine derivative, which was identified as the principal structure of the green and yellow pigments formed by this greening reaction system. A formation mechanism of the green pigment and related products involving these free radical products is proposed.

We isolated protease-productive bacteria from fish sauces collected in Vietnam and investigated their characteristics. Bacteria were detected from all the fish sauces on the low saline agar medium including 2.5% sodium chloride, and viable cell counts in the fish sauces were 1.0×102 to 1.4×106 cfu/ml. On the other hand, high halophilic bacteria which grow well on the high saline agar medium including 20% sodium chloride, were not found in all the fish sauces. Nine strains of protease-productive bacteria were picked out of the isolates of bacteria which grew on the low saline agar medium, and five of them were coccus and four were bacillus. All the strains of coccus producing protease were identified with Staphylococcus xylosus, and had a high halotolerant and did not have high protease productivity. All the strains of bacillus producing protease were identified with Bacillus, and their characteristics came closest to Bacillus firmus. Therefore, DNA homology of the strain of bacillus with B. firmus IFO 3330 was measured. Consequetly, it was confirmed that the strains of bacillus were not B. firmus. These strains had high halotolerant and high protease productivity, so it was suggested that they were involved in the maturing of the fish sauces.

The esterase encoding genes, est1 and est2 , were cloned from Acetobacter pasteurianus . Nucleotide sequence analysis of est1 revealed a gene of 954 bp, and est1 coded for an arylesterase with a molecular weight of 34863 Da consisting of 317 amino acids. The est2 gene contained an open reading frame composed of 1221 bp encoding an esterase with a molecular weight of 43389 Da consisting of 406 amino acids. The est1 gene showed some similarity, but the est2 gene showed no significant homology to other esterases reported in various microorganisms. Northern blot analysis of total RNA from A. pasteurianus revealed that transcription of the est1 gene was induced only when the cells were grown in a medium containing ethanol, and suggested that the est1 transcript is monocistronic. In contrast, transcription of the est2 gene was repressed in the presence of ethanol. In the absence of ethanol, expression of the est2 -mRNA, capable of encoding a multiple number of proteins, was revealed by Northern blot analysis. In addition, deletion analysis indicated that the 5′-region of the est2 gene contained a cis-acting domain for est2 transcriptional regulation. Analysis of the est1 promoter using the chloramphenicol acetyltransferase gene as a reporter gene showed that the promoter within the 305-bp fragment upstream of the ATG initiation codon was responsible for the transcription in cells grown in the presence of ethanol. Primer extension analysis of est1 -mRNA showed that the transcription initiation site was 49 bp upstream from the ATG initiation codon. The results of a gel mobility shift assay indicated that there is a regulatory protein related to est1 regulation, which may have some relation to the ethanol resistance of Acetobacter sp.

Acetobacter sp. produce ethylacetate during acetic acid fermentation. We found that high ethylacetate-producing strains contained more intracellular esterase than low ethylacetate-producing strains. Acetobacter pasteurianus N-23, a high ethylacetate-producing strain, formed two major intracellular esterases (esterase-1 and esterase-2) that were regulated by ethanol. These two esterases were purified to homogeneity by means of hydrophobic interaction, ion exchange, gel filtration, and hydroxyapatite chromatographies. The two esterases hydrolyzed various esters, but only esterase-1 showed ethylacetate synthesis activity at pH 3. The molecular weights of esterase-1 and esterase-2 were 72 kDa and 44 kDa, respectively, as determined by gel filtration, and 38 kDa and 42 kDa, respectively, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Esterase-1 was inhibited by cysteine enzyme inhibitors such as diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride. These esterases may play some role in the production of ethylacetate by Acetobacter sp. during vinegar fermentation.

1) 熱源に遠赤外線を用い,撹拌しつつ種子を均一に(±5℃の範囲で)一定条件で加熱することのできる実験用焙煎装置を作成した.2) この装置を用い,ゴマ種子を,170～175℃ (60, 90, 120, 160分),180～185℃ (10, 15, 30, 45, 60, 100分),190～195℃ (2, 5, 10, 15分),200～205℃ (2, 5分)の各条件で焙煎し,搾油した.その試料について褐変度(420nmの吸光度)と抗酸化性(重量法)を調べた.その結果,焙煎温度が170～175℃では長時間焙煎しても褐変度,抗酸化性ともに30分以降は平衡に達した.焙煎時間よりも温度の影響が大きく,特に,190℃以上で短時間でも急激に褐変度,抗酸化性とも上昇した.今回の条件では200-205℃, 5分の焙煎が褐変度,抗酸化性とも最も高い値を示した.3) ゴマ種子焙煎時にセサモリンの分解で生成するセサモールは,焙煎温度の上昇とともに増加し,200℃ 5分で13.02mg/100g生成した.トコフェロールは焙煎温度による影響はあまりなく,高温で若干分解される傾向であった.4) 焙煎により生成したセサモールを150℃, 30分の低温焙煎油に添加して抗酸化試験を行った結果,高温焙煎時に生成するセサモール量(16mg/100g)を添加した場合も,高温焙煎油の極めて高い抗酸化性は得られなかった.

壼酢の発酵醪より,乳酸菌,酵母,酢酸菌を分離して同定を行い,壺酢製造における微生物のフローラを調べた.(1) 壺酢発酵中の乳酸菌叢については,仕込み1, 5, 10, 20, 30日目の発酵醪より,計230株の乳酸菌を分離して同定試験を行った結果,仕込み5日目はL. plantarumが主勢菌株で,10～20日目ではL. casei subsp. caseiが増殖し,30日目ではL. coryniformis subsp. coryniformisが68%を占め,残りはL. sakeが20%, L. brevisが12%であった.仕込み1ヵ月間で乳酸菌は,3種類の菌種の交代があった.(2) 壺酢発酵中の酵母菌叢については,仕込み5, 10, 20, 30日目の発酵醪より,計110株の酵母を分離して同定試験を行った結果,全ての分離菌株はS. cerevisiaeであり,発酵初期のエタノール生成に大きく関与していることが分かった.(3) 壺酢発酵中の酢酸菌叢については,仕込み45, 60, 75, 90, 120日目の発酵醪より,計150株の酢酸菌を分離して同定試験を行った結果,各試料共A. pasteurianusが65～81%と主勢菌株で,A. acetiが18～35%の割合を占めていた.(4) 壺酢製造における微生物菌叢については,仕込み初期には同定された乳酸菌により,1%前後の乳酸が生成され,雑菌の増殖を防いでいる.乳酸の生成と同時に酵母のS. cerevisiaeが増殖し,アルコール発酵が行われ,アルコールが生成される.アルコール生成が終了すると,酢酸菌のA. pasteurianusが主勢で,A. acetiも増殖し酢酸発酵が行われ,酢酸が生成される.このように壺酢製造における微生物菌叢を明らかにした.

The angiotensin I-converting enzyme (ACE) inhibitory activity of fish sauce, the product with the strongest ACE inhibitory activity among liquid fermented foods, was fractionated into 2 fractions with ethanol treatment. The major part of the inhibitory substance was contained in the supernatant fraction (S-fraction) and its intraperitoneal administration had a hypotensive effect on spontaneously hypertensive rat (SHR). By octadecyl-silane column chromatography and two successive kinds of gel filtration, the inhibitory activity of the S-fraction was further separated into several fractions indicating that the strong inhibitory activity of the fish sauce is caused by a combined action of various kinds of inhibitory substances present. From the fraction with the lowest 50% inhibition concentration IC50 value, three kinds of dipeptides, glycyl-tryptophan, isoleucyl-tryptophan and valyl-tryptophan, were isolated as the ACE inhibitory compounds. Oral administration of these peptides to SHR showed a hypotensive effect.

Nine novel strains of halophilic and alkaliphilic lactic acid bacteria isolated from European soft and semi-hard cheeses by using a saline, alkaline medium (7 % NaCl, pH 9.5) were taxonomically characterized. The isolates were Gram-stain-positive, non-sporulating and non-motile. They lacked catalase and quinones. Under anaerobic cultivation conditions, lactate was produced from d-glucose with the production of formate, acetate and ethanol with a molar ratio of approximately 2 : 1 : 1. Under aerobic cultivation conditions, acetate and lactate were produced from d-glucose. The isolates were slightly halophilic, highly halotolerant and alkaliphilic. The optimum NaCl concentration for growth ranged between 2.0 % and 5.0 % (w/v), with a growth range of 0–1 % to 15–17.5 %. The optimum pH for growth ranged between 8.5 and 9.5, with a growth range of 7.0–7.5 to 9.5–10.0. Comparative sequence analysis of the 16S rRNA genes revealed that the isolates occupied a phylogenetic position within the genus Alkalibacterium , showing the highest sequence similarity (98.2 %) to Alkalibacterium kapii T22-1-2T. The isolates constituted a single genomic species with DNA–DNA hybridization values of 79–100 % among the isolates and Alkalibacterium , from which the isolates were different in motility and flagellation, growth responses to NaCl concentrations and pH, and profiles of sugar fermentation. The DNA G+C contents were between 36.0 and 37.6 mol%. The cell-wall peptidoglycan was type A4β, Orn-d-Asp. The major components of cellular fatty acids were C14 : 0, C16 : 0 and C16 : 1ω9c. Based on the phenotypic characteristics and genetic distinctness, the isolates are classified as a novel species within the genus Alkalibacterium , for which the name Alkalibacterium gilvum sp. nov. is proposed. The type strain is 3AD-1T ( = DSM 25751T = JCM 18271T).

Cassava cortex was successfully used in the induction of linamarase in Aspergillus oryzae in place of linamarin which is an expensive inducer. Induction by cassava cortex was attributed to its linamarin content. However, besides linamarin, some other components in the cortex may have enhanced induction or stabilized the enzyme induced thus resulting in increased linamarase production. Statistical evaluation showed the significant influence (p

Two novel strains of marine lactic acid bacteria, isolated from decaying marine algae collected from a subtropical area of Japan, are described. The isolates, designated O24-2Tand O25-2, were Gram-positive, non-sporulating and non-motile. They lacked catalase and quinones. Under anaerobic cultivation conditions, lactate was produced from glucose with the production of formate, acetate and ethanol in a molar ratio of approximately 2 : 1 : 1. Under aerobic cultivation conditions, acetate and lactate were produced from carbohydrates and related compounds. The isolates were slightly halophilic, highly halotolerant and alkaliphilic. They were able to grow in 0–17.0 % (w/v) NaCl, with optimum growth of strains O24-2Tand O25-2 at 1.0–3.0 and 1.0–2.0 % (w/v) NaCl, respectively. Growth of strain O24-2Twas observed at pH 7.5–9.5, with optimum growth at pH 8.0–8.5. Comparative 16S rRNA gene sequence analysis revealed that the isolates occupied a phylogenetic position within the genusAlkalibacterium, showing highest similarity (99.6 %) toAlkalibacterium putridalgicolaT129-2-1T. Although sequence similarity was high, the DNA–DNA relatedness value between strain O24-2TandA.putridalgicolaT129-2-1Twas 27 %, indicating that they are members of distinct species. The DNA G+C contents of O24-2Tand O25-2 were 43.7 and 44.4 mol%, respectively, and DNA–DNA relatedness between the isolates was 89 %. The cell-wall peptidoglycan was type A4β, Orn-d-Asp. The major cellular fatty acid components were C14 : 0, C16 : 0and C16 : 1ω9c. Based on phenotypic characteristics and genetic distinctiveness, the isolates were classified as representatives of a novel species within the genusAlkalibacterium, for which the nameAlkalibacterium subtropicumsp. nov. is proposed; the type strain is O24-2T( = DSM 23664T = NBRC 107172T).

We describe 10 new strains of marine lactic acid bacteria isolated from decaying marine algae, decaying seagrass, raw fish, salted fish and salted and fermented shrimp paste ('ka-pi') collected from a temperate area of Japan and Thailand. The isolates are Gram-positive and non-sporulating. They have motility with peritrichous flagella depending on the strains. They lack catalase and quinones. Under anaerobic conditions, lactate yields were 64-93 % of the glucose consumed; residual products were formate, acetate and ethanol with a molar ratio of approximately 2 : 1 : 1. The pH of the fermentation medium markedly affected the product composition; at higher pH, the yield of lactate decreased (15-48 % at pH 9.0) and yields of other products increased, retaining the molar ratio. Under aerobic conditions, acetate and lactate were produced from carbohydrates and related compounds. The isolates were slightly halophilic, highly halotolerant and alkaliphilic. The optimum NaCl concentration for growth ranged between 0.5 and 4.0 % (w/v), depending on the strain, with a growth range of between 0 and 17-21 % (11 % for one isolate). The optimum pH for growth ranged between 8.0 and 9.5, with a growth range of 6.0-11.0, depending on the strains. Comparative sequence analysis of the 16S rRNA genes revealed that the isolates occupied three phylogenetic positions within the genus Alkalibacterium, showing 97.1-99.8 % similarity to Alkalibacterium indicireducens. DNA-DNA hybridization values (

Angiotensin I converting enzyme (ACE, E.C. 3.4.15.1) inhibitory activity were measured with 11 kinds (31 items) of fermented foods. Strong inhibitory activity was detected in soy sauce, fish sauce, natto, nyufu, and cheese, but not in mirin, sake, or vinegar.

Phosphatidylcholine (PC) is a major component of membranes not only in eukaryotes, but also in several bacteria, including Acetobacter. To identify the PC biosynthetic pathway and its role in Acetobacter sp., we have studied Acetobacter aceti IFO3283, which is characterized by high ethanol oxidizing ability and high resistance to acetic acid. The pmt gene of A. aceti, encoding phosphatidylethanolamine N-methyltransferase (Pmt), which catalyzes methylation of phosphatidylethanolamine (PE) to PC, has been cloned and sequenced. One recombinant plasmid that complemented the PC biosynthesis was isolated from a gene library of the genomic DNA of A. aceti. The pmt gene encodes a polypeptide with molecular mass of either 25125, 26216, or 29052 for an about 27-kDa protein. The sequence of this gene showed significant similarity (44.3% identity in the similar sequence region) with the Rhodobacter sphaeroides pmtA gene which is involved in PE N-methylation. When the pmt gene was expressed in E. coli, which lacks PC, the Pmt activity and PC formation were clearly demonstrated. A. aceti strain harboring an interrupted pmt allele, pmt::Km, was constructed. The pmt disruption was confirmed by loss of Pmt and PC, and by Southern blot analyses. The null pmt mutant contained no PC, but tenfold more PE and twofold more phosphatidylglycerol (PG). The pmt disruptant did not show any dramatic effects on growth in basal medium supplemented with ethanol, but the disruption caused slow growth in basal medium supplemented with acetate. These results suggest that the lack of PC in the A. aceti membrane may be compensated by the increases of PE and PG by an unknown mechanism, and PC in A. aceti membrane is related to its acetic acid tolerance.

A marked greening observed in some foods such as sweet potato, burdock, and others during food processing was shown to be due to green pigment formation by the condensation reaction of two molecules of chlorogenic acid or caffeic acid ester with one molecule of a primary amino compound under aeration in alkaline solution. Reduction of the green pigment by ascorbic acid or NaBH4 gave a yellow product, which readily turn green and then blue in air. The reduced and acetylated product of the green pigment was identified to be a novel trihydroxy benzacridine derivative, and the yellowish ethanol solution of this product immediately turned green upon addition of butyl amine or diluted alkali. Therefore, the green pigment was assumed to be an oxidized quinone type product of trihydroxy benzacridine. This identification of the structure was supported by the correspondence of the measured absorption spectra with those calculated by the molecular orbital method. A possible charge transfer complex between products of different oxidation steps in green solution was proposed.

The regulation mechanism for expression of the ethanol inducible esterase gene, est1, was investigated in A. pasteurianus. Deletion analysis of the 5' non coding region of est1 showed that the FNR-binding consensus sequence is important in the induction of est1 by ethanol. Cells grown under oxygen starvation produced esterase-1 in not only the presence but also the absence of ethanol. These results suggest that the induction of est1-expression depends on the oxygen concentration, and the gene may be induced by a FNR-like factor activated by a decrease in the intracellular oxygen concentration.

Using a mutant defective in cysteine uptake, which is resistant to a toxic analog of cysteine, allylglycine, we searched for a gene that complements the defect in cysteine uptake in a yeast genomic library and found a DNA fragment causing the recovery of cysteine uptake and sensitivity to allylglycine. The gene in the fragment was identical to MUP1, the high affinity methionine permease gene. We conclude that Mup1 is a major permease in cysteine uptake.

Among Saccharomyces cerevisiae strains each defective in one of 11 amino acid permeases, a lysine permease disruptant (DK) exhibited about 2-fold reductions in maximum cell density and fermentation ability compared to the parent in a synthetic medium. These unusual properties of DK were found to result from the requirement of biotin for growth, in contrast to the parent whose growth was not dependent on external biotin. The rate of 14C-labeled biotin uptake and the intracellular free biotin content of DK were 2-2.5 fold lower than in the parent. We suggest that lysine permease in S. cerevisiae has the ability to transport both lysine and biotin.

Soybeans, tea leaves, and mushrooms were conspicuously rich in spermidine, while oranges contained a large amount of putrescine. Among the fermented foods, soy sauces were rich in putrescine and histamine, while Japanese sake contained plenty of agmatine. These polyamines are thought to be produced from amino acids during fermentation with amino acid decarboxylases formed by the micro-organisms.

Various conditions in the Bacillus brevis transformation by electroporation were investigated to increase the efficiency. Competent cell volume and presence of polyethylene glycol 6000 and single stranded DNA on pulsing were critical for the efficiency. As a result, we established the improved method by which 10(7)-10(8) transformants/microgram plasmid DNA could be obtained.

A crystalline product resulted from the reduction and acetylation of the green pigment formed from the reaction of methyl caffeate and butylamine. Its structure was determined to be that of a novel benzacridine, i.e., dimethyl-7-butyl-6, 9, 10-triacetoxybenz[k, l]- acridine-1, 2-dicarboxylate.