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Two saccharifying α-amylases with different molecular masses were purified from Bacillus subtilis IFO 3108. The higher molecular mass α-amylase 1 (RBSA-1, MM 67 kDa) was able to adsorb to α-cyclodextrin (α-CD) sepharose CL-6B and hydrolyze raw starch. RBSA-1 showed weak adsorption to raw corn starch over the wide pH range of 5.0–9.0. At low pH (5.0–6.0), RBSA-1 exhibited high adsorption to raw potato starch. The lower molecular mass α-amylase 2 (BSA-2, MM 45 kDa) exhibited enzymatic properties similar to RBSA-1, however, was unable to adsorb to α-CD sepharose CL-6B and failed to hydrolyze raw starch. On the other hand, a liquefying type α-amylase (RBLA), purified from Bacillus sp., exhibited remarkable adsorption to raw starches tested over a wide pH range (5.0–9.0). This pH range corresponded to that suitable for the digestion of raw starches. The adsorption of RBSA-1 and RBLA to α-CD sepharose CL-6B was pH-independent, however, the extent of adsorption of RBSA-1 (70–85%) was much greater than that of RBLA (30–45%). The hydrolysis of raw corn starch was specifically inhibited by α-CD for both enzymes. The Ki value (0.44 mM) for RBSA-1 was much lower than that for RBLA (3.44 mM).

The digestion of raw starch by a glucoamylase (GA MU-H) from a mutant strain of Aspergillus awamori var. kawachi was closely correlated with mannoside chains O-linked to the Gp-I region (A470-V514), but not sugar chains N-linked to catalytic GAI' domain of GA MU-H. The partial replacement of mannose residues by glucose residues led to a significant decrease raw starch digestion. By the substitution of D2O for H2O in the reaction mixture, the raw starch digestion of GA MU-H decreased to 80% of that at 30 degrees C, although the rate of hydrolysis of soluble starch by and the ability to bind beta-cyclodextrin of GA MU-H were unchanged. Glycerol, known as an antichaotropic reagent, decreased the raw starch digestion of GA MU-H significantly. However, it did not have any effect on the enzymatic activity for soluble starch when soluble starch was the substrate. The efficient digestion of raw starch with raw starch-digesting glucoamylase needed the mannoside chains O-linked to the Gp-I region, which were suggested to contribute to digestion of raw starch through the interaction with water.

The extradiol ring-cleavage dioxygenases derived from seven different Pseudomonas strains were expressed in Escherichia coli and the substrate specificities were investigated for a variety of catecholic compounds. The substrate range of four 2,3-dihydroxybiphenyl dioxygenases from biphenyl-utilizing bacteria, 3-methylcatechol dioxygenase from toluene utilizing Pseudomonas putida F1, 1,2-dihydroxynaphthalene dioxygenase from a NAH7 plasmid, and catechol 2,3-dioxygenase from a TOL plasmid pWW0 were compared. Among the dioxygenases, that from Pseudomonas pseudoalcaligenes KF707 showed a very narrow substrate range. Contrary to this, the dioxygenase from pWW0 showed a relaxed substrate range. The seven extradiol dioxygenases from the various Pseudomonas strains are highly diversified in terms of substrate specificity.

Endomitotic diploidization occurred as a result of heat treatment of spores during germination of the industrial yeast S. cerevisiae. Polyploid cells were obtained in both heterothallic and homothallic strains. In the heterothallic strains, the resultant diploid cells, with the ability to mate, were used to construct tetraploids. In the homothallic strains, tetraploid cells were obtained directly after a mating-type switch. In each strain, the highest frequency (approximately 10% of viable cells) of diploidization was obtained when spores were cultured for 30 min in liquid medium to initiate germination followed by subsequent heat treatment at 55°C for 10min.

An amylolytic yeast strain, isolated from Thai Loogpang Lauw and identified as Lipomyces starkeyi HN-606, was found to produce a novel yeast α -amylase characteristically showing raw-starch digestibility almost to the same extent as the commercial amylolytic Aspergillus , but with a lower culture temperature of 15°C rather than 25°C as optimal for growth. The yeast α -amylase (MW 56,000) was adsorbed onto raw corn-starch and could digest it to form glucose, maltose and maltotriose. α -Cyclodextrin at less than 10 mM completely inhibited the raw-starch adsorption and digestion with a K l value of 0.38 mM, but could hardly inhibit gelatinized-starch hydrolysis.

A protease- and glycosidase-negative mutant F-2035, of Aspergillus awamori var. kawachi has been isolated that produces a glucoamylase that contains a large carbohydrate moiety and has enhanced ability to digest raw starch (GA MU-H; MW, 110,000). This enzyme digested raw corn starch 2.5 times faster than did the parental glucoamylase I (GA I; MW, 90,000). When grown from an enriched seed culture, this mutant also produced a glucoamylase with less ability to digests raw starch (GA MU-L; MW, 110,000). Its activity was 25% of that of GA MU-H with raw starch. Both GA MU-H and GA MU-L proved to be identical to GA I in terms of adsorption to raw starch, molar activity against gelatinized starch, amino acid composition, and terminal amino acid sequence. The carbohydrate contents of GA I, GA MU-H, and GA MU-L were 17%, 33%, and 33% by weight, respectively. The carbohydrates of GA I and GA MU-H were mostly mannose, but that of GA MU-L was composed of mannose (71%) and glucose (26%). Partial removal of the carbohyd...

A single form of exo-type cellulase (Exo I; MW, 65,000), purified from a Trichoderma viride protease-depressed mutant, HK-75, digested Avicel to cellobiose exowise, and hydrolyzed cellotriose, cellotetraose, and cellopentaose in the strict manner of splitting off by cellobiose units. Exo I, however, hydrolyzed cellohexaose by both cellobiose and cellotriose units. Exo I was proteolyzed by papain into two fragments; GPExo (MW, 9,000) and Exo I' (MW, 56,000). The GPExo intensively adsorbed onto Avicel but did not hydrolyze it. Exo I' had nearly identical activity to that of intact Exo I toward cellooligosaccharides but was almost inert to Avicel in digestion and adsorption. Sequence analysis of N-terminal and C-terminal amino acids showed that GPExo was between Gly435 and Leu496 and Exo I' between Glu1 and Gly434 in Exo I. Exo I therefore consists of two domains, one for adsorption to Avicel, as demonstrated by the Avicel-affinity site, GPExo and the other for the cleavage of glycosidic linkages as demonstrated in Exo I'.

Field studies on the control of potato common scab were done with non-antibiotic Actinomycete biofertilizer, which was manufactured from swine feces with coprophilous actinomycetes and acidified to pH 5.0 with sulfuric acid. The field soil at pH 5.0 was mixed with 0.56 kg of the acidified biofertilizer per subplot (1.0 x 1.2 m) to a depth of the 15 cm. The scab severity, in terms of the percent coverage of the total surface area of potato tubers, was 10.7%, in the control, while the lesions were slight in the biofertilizer subplot, scab severity being only 2.30/0, and the potato production was increased 3 times with the biofertilizer. The viable counts of Streptomyces scabies in the biofertilizer subplot decreased to 1 × 103 per gram soil from 5 × 105 by 12 weeks, while the inhabitant fluorescent pseudomonads increased to 2.4 × 107 per gram soil from 3 × 104, and the increased fluorescent pseudomonads were antagonistic to the pathogenic strains.

Three bacterial strains, A41, B47, and MM4, isolated from sea sediment and aeration tanks of a sewage plant, identified as Pseudomonas stutzeri, showed lethal activity against the red tide phytoplankton Chattonella antiqua. Upon the addition of the culture broth of bacteria, C. antiqua lost its mobility and lysed. Their lethal activity reached a maximum in the nutrient broth containing 3% NaCl at 25°C (the lowest lethal concentration; 0.5%) for 2 to 6 days of cultivation. Addition of the A41 culture broth to a coculture of C. antiqua and killifish (Oryzias latipes) showed the selective toxicity on C. antiqua.

A degenerated strain of Clostridium saccharoperbutylacetonicum N1-4 ATCC13564 was isolated after 30 subcultures and was designated as DGN32. The product yields of DGN32 drastically decreased to below one-third of those of N1-4, but its acetone productivity remained the same as that of N1-4 when the culture pH was at 5.1. DGN32 with or without pH control evolved almost equimolar amounts of CO2 and H2 throughout the fermentation. The amount of NADH consumed for the acid and solvent production of DGN32 corresponded to that of NADH generated in the EMP pathway. These results suggested that the electron flow coupled with pyruvate metabolism to acetyl-CoA in DGN32 would be kept for H2 evolution, but not for NADH generation. However DGN32 cultivated with a redox dye (benzyl viologen) preferentially produced butanol though the butanol production rate was comparatively low. In DGN32 there was suppression of the NADH generation system coupled with pyruvate metabolism to acetyl-CoA, which could be essential for the ...

A mutant, AS2-1, was isolated by UV irradiation from Clostridium saccharoperbutylacetonicum Nl-4 ATCC 13564 producing hyper butanol at the molar ratio of acetone–butanol–ethanol (3.3:9.5:1). The mutant proved to be deficient in the production of acetone, butanol, and acetic acid, but enhanced the ethanol (17.7 times increase) and newly produced isovaleric acid at a molar ratio of 1:1. The maximum production of ethanol and isovaleric acid reached 10.6 g/l (0.23 M) and 19.8 g/l (0.20 m) from glucose at 77.7 g/l (0.43 m) in TYA medium. As the equimolar ratio of ethanol and isovaleric acid was also produced in an amino acid-free minimal medium containing ammonium acetate as a nitrogen source, isovaleric acid was not produced from amino acids, but de novo synthesized from the ammonium and glucose.

Mutant AS2-1, derived from Clostridium saccharoperbutylacetonicum N1-4 ATCC 13564, did not produce acetone, butanol, and acetic acid, but had enhanced ethanol production and newly produced isovaleric acid at the molar ratio of 1:1. The mutant was defective in thiolase (acetyl-CoA acetyltransferase) activity, while the parent N1-4 showed high thiolase activity (103.5 units/g protein). Addition of allyl alcohol into the mutant culture significantly decreased the production of both ethanol and isovaleric acid. AS2-1 mutant, therefore, had NAD+ — and NAD(P)+-dependent aldehyde dehydrogenase on the pathway to ethanol and isovaleric acid, respectively. It was suggested that an excess of electrons accumulated as the result of the deficiency in thiolase was consumed for the reductive formation of isovalerate from pyruvate. The equation of the novel ethanol–isovaleric acid fermentation was concluded as 1.5 mol glucose = 1 mol ethanol + 1 mol isovaleric acid + 2 mol CO2 + 2 mol H2O +1 mol H2.

Diploid cells with ability to mate, hereafter referred to as diploid mater cells, were obtained at significant frequencies by the heat treatment of haploid spores at the early germination stage in Saccharomyces cerevisiae heterothallic strain CG5M (a/alpha diploid cells heterozygous for five auxotrophic markers). The highest frequency (ca. 11%) of diploidization was obtained from viable cells after heat treatment at 55 degrees C for 10 min when spores were precultivated for 30 min in liquid medium to initiate the germination. The diploid mater cells obtained were homozygous for mating type and for the auxotrophic markers. The diploidization of a spore is thus concluded to be due to endomitotic events in germinating heat-treated spores.

Glucoamylase I (GAI) from Aspergillus awamori var. kawachi hydrolyzes raw starch efficiently and is composed of three functional domains: the amino-terminal catalytic GAI' domain (A-1 to V-469), the threonine- and serine-rich O-glycosylated Gp-I domain (A-470 to V-514), and the carboxy-terminal raw starch-binding Cp domain (A-515 to R-615). In order to investigate the role of the Gp-I domain, an additional repeat of Gp-I and internal deletions of the entire Gp-I sequence or parts of the Gp-I sequence were introduced within Gp-I. All mutant genes as well as the wild-type gene were inserted into a yeast-secretion vector, YEUp3H alpha, and expressed in Saccharomyces cerevisiae. Wild-type GAI expressed in yeast cells (GAY), GAGpI, having an extra Gp-I, and GA delta 470-493, lacking the A-470-to-T-493 sequences of Gp-I, were successfully secreted into the culture medium. On the other hand, GA delta 470-507, lacking A-470 to S-507, and GA delta GpI, lacking the entire Gp-I (A-470-to-V-514) sequence, failed to be secreted and remained in the yeast cells. The carbohydrate content of GAGpI was 1.2 times higher than that of GAY and 2.4 times higher than that of the original GAI. The raw starch digestibility of GAGpI was almost the same as that of GAY but was 1.5 times faster than that of GAI.(ABSTRACT TRUNCATED AT 250 WORDS)

Sixteen biphenyl and polychlorinated biphenyl (PCB)-degrading strains were isolated from soil at various locations and characterized. All the isolates were gram negative bacteria that include eleven species of Pseudomonas, one Achromobacter, one belonging to the CDC Group VE, Biotype I, while three strains were unidentified. Most of these strains grew on basal salts media supplemented with a wide variety of aromatic compounds, which include substituted biphenyls and biphenyl related compounds. Southern blot analysis using two previously cloned bph genes as probes revealed that four (three Pseudomonas, one unidentified) out of the sixteen strains showed significant hybridization with the well studied bph genes of Pseudomonas pseudoalcaligenes KF707. The immunological cross reactivity of the 2,3dihydroxybiphenyl dioxygenases and hydrolases from the four strains corresponded well to the DNA homology. The existence of almost identical bph genes in different soil bacteria indicates that certain chromosomal bph operons have a mechanism of transfer within bacterial populations in the environment. However, the twelve other biphenyl utilizing isolates did not show any significant hybridization with the KF707 bph probe, while all of the sixteen strains did not hybridize with the Moraxella sp. KF704 bph probe. These results suggest the diversity of PCB-degrading genotypes.

Carboxy-terminal deletions were introduced into the raw starch-binding domain (A-515 to R-615) encoded by the gene for glucoamylase I (GAI) from Aspergillus awamori var. kawachi. Genes coding for proteins designated GA596 (A-1 to E-596), GA570 (A-1 to A-570), and GA559 (A-1 to N-559) were constructed and resulted in truncated proteins. All of the mutant genes were expressed heterologously in Saccharomyces cerevisiae. GA596 adsorbed to raw starch and digested it. GA570 and GA559 did not adsorb to raw starch or to an alpha-cyclodextrin-Sepharose CL-4B gel under our experimental conditions. However, GA570 was able to digest raw starch, and the digestion of raw starch by GA570 was inhibited by beta-cyclodextrin. Residue Trp-562 of GAI, which was suggested previously to contribute to formation of an inclusion complex with raw starch, was replaced by Leu (GAW562L), Phe (GAW562F), and Gly (GAW562G). GAW562L and GAW562F adsorbed to raw starch and an alpha-cyclodextrin gel, but GAW562G did not. Although GAW562L digested raw starch to the same extent as wild-type GAI (designated GAY), GAW562F and GAW562G exhibited less ability to digest raw starch. On the basis of our results, it appears that the sequence around Trp-562, PL(W-562)YVTVTLPA, is the minimal sequence necessary for digestion of raw starch and that hydrophobic residue Trp-562 contributes to formation of an inclusion complex. The sequence near Trp-589, which has abundant hydrogen bond-forming residues and the charged amino acid residues needed for stable adsorption to raw starch, probably assists in the formation of the inclusion complex.

Engineering of hybrid gene clusters between the toluene metabolic tod operon and the biphenyl metabolic bph operon greatly enhanced the rate of biodegradation of trichloroethylene. Escherichia coli cells carrying a hybrid gene cluster composed of todC1 (the gene encoding the large subunit of toluene terminal dioxygenase in Pseudomonas putida F1), bphA2 (the gene encoding the small subunit of biphenyl terminal dioxygenase in Pseudomonas pseudoalcaligenes KF707), bphA3 (the gene encoding ferredoxin in KF707), and bphA4 (the gene encoding ferredoxin reductase in KF707) degraded trichloroethylene much faster than E. coli cells carrying the original toluene dioxygenase genes (todC1C2BA) or the original biphenyl dioxygenase genes (bphA1A2A3A4).

alpha-, beta-, and gamma-cyclodextrins (CDs) completely inhibited raw starch digestion by glucoamylase I (GA I, MW 90,000) from Aspergillus awamori var. kawachi, and inhibited by 85% the raw starch adsorption of GA I at the CD concentrations of 1-5 mM. CDs at 1-5 mM did not inhibit gelatinized starch hydrolysis by GA I, but at the concentration of 50 mM, they inhibited such hydrolysis slightly. GA I was specifically adsorbed onto CD-Sepharose 6B, but glucoamylase I' (GA I', MW 73,000), which does not adsorb onto or digest raw starch, from the same strain was not adsorbed onto that gel. The adsorption of the glucoamylases onto raw starch and CD-Sepharose 6B was correlated to their digestion of raw starch. The hydrophobic adsorption of GA I onto CDs and raw starch occurred competitively at the Cp region, which is on the C-terminal side of Gp-I in the site for raw starch affinity of GA I, and inclusion complexes were formed.

The enzymatically inactive but raw-starch-adsorbable peptide fragments designated as Gp-pan P and Gp-pan I were obtained from a tryptic digest of heat-inactivated hog pancreatic alpha-amylase. These two glycopeptide fragments were purified with Sephadex G-75, DEAE-Sephadex A-50, and HPLC and were found to be homogeneous on disc gel electrophoresis. Gp-pan P and I had molecular weights of 20,000 and 30,000 with SDS-PAGE, carbohydrate contents of 10% and 7%, N-terminal amino acids Gly-Trp and Ala-Val, and C-terminal amino acids Gly-Arg and Ile-Lys. Gp-pan P had promotive but Gp-pan I inhibitory effects on raw starch digestion by Aspergillus awamori var. kawachi glucoamylase I and Bacillus subtilis 65 alpha-amylase.

A high copy number plasmid pCS86 (3.0 kb) was isolated from Clostridium acetobutylicum strain No. 86. For developing the vector plasmid for gene cloning of saccharolytic clostridia, two hybrid plasmids were constructed by joining pCS86 to the E. coli promoter probe vector pKK232-8 carrying the promoterless gene of chloramphenicol acetyltransferase (CAT). One of the hybrid plasmids designated as pTY10 replicated and expressed the CAT gene in E. coli, and transformed a plasmid-free strain, C. acetobutylicum strain No. 220, as a shuttle vector. This is the first shuttle vector constructed from C. acetobutylicum and E. coli plasmids. Reciprocal transformation was possible between E. coli and C. acetobutylicum, though deletion occurred in the sequence originated from pKK232-8. A deleted plasmid pTY101 only replicated in Clostridium, though it showed a high copy number.

A purine auxotroph with pale purple spores and a derivative with yellowish orange spores were obtained from the wild-type strain of Streptomyces azureus ATCC 14921, which has bluish green spores. The changed color or pigmentation in the mutants was limited to the spores. They accumulated AIR (5′-phosphoribosyl-5-aminoimidazole) due to the lack of AIR carboxylase activity.

Diploid a or α sporogenous mater cells were frequently obtained in the sake yeast Kyokai no. 9 by heat treatment followed by random spore plating. Polyploid cells were constructed between the opposite mater cells by the mass mating technique, followed by the isolation of the zygotes using a micromanipulator.

Aspergillus awamori var. kawachi produced selectively one of the three types of glucoamylases under the following different conditions in the submerged cultures at the controlled pH of 6.5 to 7.0: (1) the raw-starch digestive glucoamylase, type I, by a cultivation in the synthetic medium containing ammonium citrate as a nitrogen source, deficient in zinc and sufficient in phosphate, for a shorter period of 30 hr at 35°C; (2) one of the raw-starch indigestive glucoamylases, type I, showing the same hydrolysis curves for gelatinized starch as the type I, by a cultivation in the synthetic medium containing ammonium acetate as a nitrogen source, sufficient in zinc, for 50 hr at 30°C; (3) the other of the raw-starch indigestive glucoamylases, type II, that was less susceptible for α-1,6 glucosidic linkages of gelatinized starch, by a cultivation in the synthetic medium containing glucose, casein-peptone and sufficient amount of inorganic elements, especially calcium and zinc, for 96 hr at 25°C.

A thermophilic fungus, strain YH-78, identified as Humicola grisea var. thermoidea Cooney et Emerson, was newly isolated from compost heaps. This strain exhibited high CMCase activity in cultures on wheat bran medium (50°C, 4 days), but Avicelase activity in the culture filtrate was very low. The culture filtrate significantly disintegrated filter paper and hydrolyzed carboxymethyl cellulose to 45% as glucose at 50°C. The activity of CMCase in the culture filtrate was partially adsorbed onto Avicel. The CMCase component adsorbed onto Avicel was purified to homogeneity by disc electrophoresis. The purified CMCase component was optimally active at 50°C and pH 5.0, and completely adsorbed onto Avicel at pH 5.0 at 4°C for 10 min. The enzyme showed high activity toward CMC and no activity toward Avicel, but showed a high synergistic action together with the cellulase from Humicola insolens YH-8 on hydrolysis of Avicel.

Glucoamylase I (MW 90,000; N-terminal alanine) was selectively produced by a submerged culture of Aspergillus awamori var. kawachi as an enzyme having the ability to digest even raw starches, and was converted into a modified active glucoamylase decreasing in molecular weight during the digestion with the acid protease of the same mold strain. The modified glucoamylase (MW 83,000; N-terminal alanine plus isoleucine) was resembled to an intermediate type of glucoamylase I′, and exhibited hydrolysis curves for gelatinized potato starch and maltose in the same manner as that of glucoamylase I; but it did not digest raw starches and was not adsorbed onto cornstarch, and showed the lower hydrolysis limit of 80 percent for glycogen. The carbohydrate contents of glucoamylase I decreased 59 percent during the digestion with fungal α-mannosidase. The carbohydrate-split glucoamylase (MW 86,000; N-terminal alanine) was unstable at pH 9 or at 65°C in the same extent as glucoamylase I′, but was identical with glucoamy...

The glucoamylase I which was purified from a culture of Aspergillus awamori var. kawachi as an enzyme having an ability to digest raw starch, was converted to a modified enzyme with decrease in molecular weight and carbohydrate content by incubation with subtilisin in 0.01 m phosphate buffer, pH 6.8, at 30°C. The modified glucoamylase, isolated by the DEAE-Sephadex A–50 column chromatography, showed a 77% specific activity, and the same manner of the hydrolysis curve for gelatinized potato starch and maltose as the native enzyme, but showed no digestibility for raw starches nor adsorbability onto raw corn-starch, and a lower hydrolysis limit of 80 per cent for glycogen. Stabilities also decreased at pH 9 and at 65°C, respectively. Protease was proved to change the properties of glucoamylase.

The relative amounts of metals in the cell wall of Clostridium saccharoperbutylacetonicum ATCC 13564 were found to be as follows: Fe>Al_??_Mg>K>Na>Ca_??_Zn>Cu>Hg_??_Mn>Co. Of particular note here is the very high Fe content and the specific Fe-binding properties of cell wall. The iron physiology in cell wall was somewhat different from those of Mg and Cu, which were used for comparison. The in vivo accumulation of Fe into cell wall increased vigorously after 6hr of cultivation (early to middle log phase) and continued until the late stage of the log phase (about 15hr). The maximum content was ca. 1100μg/g of dry weight of cell wall, when Fe2+ was supplemented to the medium at 3.6×10-5M. In vivo accumulation of Mg increased in the first 6hr of cultivation, and then decreased. Its maximum content was ca. 360μg/g, when Mg2+ was supplemented at 1.2×10-3M. The accumulation of Fe and Mg was closely related to the cell growth. On the other hand, toxic Cu rapidly bound to cell wall for initial short periods regardless of the growth phase. Its maximum content was ca. 60μg/g when no Cu2+ was added; when Cu2+ was intentionally supplemented at 2×10-5M, its maximum content was ca. 200μg/g. The relative metal binding in cell walls in vitro was in the order of Cu2+>Fe2+>Mg2+. Fe had a high affinity with the peptidoglycan moiety of the cell wall. Mg had some affinity to the polysaccharide moiety of the cell wall. Cu seemed to have a high affinity with some unidentified constituents of the cell wall.

Sucrose-induced autolysis of Clostridium saccharoperbutylacetonicum (ATCC 13564) was inhibited by various chemical inhibitors, including known enzyme inhibitors (such as heavy metal ions and p-chloromercuric benzoate) and fixative agents (such as formalin and glutaraldehyde). Fradiomycin (neomycin) also inhibited the lysis at the concentration of above 50pg/ml. The organisms whose growth had been inhibited by the antibiotics sucn as ch loramphenicol , were res i s tan t to sucroseinduced autolysis. The lysate produced by sucrose treatment had lytic activity, autolysin activity, on isolated cell wall. During the lysis, autolysin was released from the cells concomitantly with the degradation products of cell wall labelled wi th I%-D-alanine. In sp i te of these re lease of ce l l wal l -degradat ion products and autolysin, the cell number and cellular content of macromolecules (DNA, RNA and protein) were hardly decreased. We suggest that sucrose-induced autolysis is due to the ac t ion of au to lys in and progresses wi thout des t ruc t ion of pro toplasmic membrane .

An exogenous ternary complex composed of Tween 80, ergosterol, and albumin increased the final ethanol concentration of fermentation by sake yeasts from 17.2 to 19.0% (vol/vol) and reduced the fermentation time from 30 to 25 days. Likewise, a complex of monoolein, albumin, and either ergosterol or ergosteryl oleate increased the final ethanol concentration of fermentation to 19.7 or 19.8% (vol/vol), respectively, and reduced the fermentation time to 25 days. Both Tween 80 and monoolein promoted the fermentative activity (Q CO 2 ) of cells, and the effect was enhanced by the presence of ergosterol.

A biofertilizer, showing antagonistic activity against potato common scab in pot tests, was produced from swine feces with a newly isolated strain, CH-33, identified as Streptomyces albidoflavus. This strain characteristically grew on fresh swine feces at 20~35°C without sterili-zation or any additives, and produced an antibiotic substance against Streptomyces scabies, the common scab-pathogen, during composting. The addition of the biofertilizer at from 0.1 g to 1.6 g total nitrogen (N) per 600 g humic volcanic ash soil in a pot did not inhibit the growth of Brassica rapa var. perviridis but increased it, even at the highest nitrogen content tested. Common scab was completely inhibited when the biofertilizer was added at 0.1 g to 1.6g as nitrogen (N) per 4 kg of scab-infected soil in a pot. Thus a biofertilizer suppressing plant pathogenic microorganisms was developed.

The raw-starch-digesting glucoamylase I (GAI) of Aspergillus awamori var. kawachi contains a glycopeptide I (Gp-I) region as the raw-starch-affinity site essential for its raw-starch-adsorption and raw-starch-digestion. Molecular cloning of the GAI gene and analysis of its nucleotide sequence revealed some nucleotide replacements, in comparison with the Aspergillus niger and Aspergillus awamori glucoamylase genes. The deduced total amino acid sequence of GAI contained 35 replacements and one deletion, of amino acid residues, and therefore a total of 615 residues, in comparison with the A. niger and A. awamori glucoamylases. The raw-starch-adsorbable Gp-I region in GAI was located between Ala470 and Val514 near the C-terminal, consisting of ATGG- TTTTATTTGSGGVTSTSKTTTTASKTSTTTSSTSCTTPTAV.

TitleTransfection and Transformation Systems for Pock-Forming andThiostrepton-Producing Streptomyces azureusAuthor(s) Ogata, Seiya; Koyama-Miyoshi, Yuko; Hayashida, ShinsakuCitation九州大学農学部紀要 ¦¦ 29(4) ¦¦ p179-188; Journal of the Faculty ofAgriculture, Kyushu University ¦¦ 29(4) ¦¦ p179-188Issue Date 1985-06URL http://hdl.handle.net/2324/23804Right

A nystatin-resistant mutant (NR-21) of a thermotolerant yeast, Hansenula polymorpha CK-1, was isolated by mutagenesis with ethyl methanesulfonate, followed by selection for resistance to nystatin (50 units/ml). The mutant was defective in ergosterol biosynthesis. Specific growth rates (h −1 of the mutant were reduced to 0.35 at 40°C and 0.16 at 50°C as compared with the wild type (0.53 at 40°C and 0.28 at 50°C). The mutant grown with ergosterol-phosphatidylcholine emulsion at 50°C incorporated ergosterol and its specific growth rate was increased to 0.41, which was comparable to that of the wild type grown under the same conditions.