Shinsaku Hayashida

Characteristics of raw-starch-digesting glucoamylase from thermophilic Rhizomucor pusillus

Abstract A newly isolated thermophilic fungus, NH-139, identified as Rhizumucor pusillus (Lindt) Schipper produced only a single form of raw-starch-absorbable, raw-starch-digesting glucoamylase on solid wheat bran medium at 45°C. The electrophoretically homogenous preparation of glucoamylase, molecular weight 68,000, had its optimal temperature on gelatinized starch at 65°C and on raw corn starch at 50°C. However, this raw-starch-digesting glucoamylase, unlike other glucoamylases, could not completely hydrolyze glycogen but hydrolyzed it to the extent of 80% as glucose, and is classified as type B. The subtilisin-modified glucoamylase of this strain, molecular weight 60,000, still belonged to type B in the hydrolysis curve on glycogen and lost the ability to digest and adsorb onto raw starch.

Cellulases of Humicola insolens and Humicola grisea

Publisher Summary The enzymatic hydrolysis of native cellulose is a complex process requiring the participation of several enzymes. There are three major types of cellulolytic enzymes produced by fungi: cellobiohydrolase, endoglucanase, and β-glucosidase. Cellobiohydrolase is the enzyme with the highest affinity for cellulose and is capable of degrading crystalline cellulose (Avicel). Endoglucanase usually hydrolyzes only soluble substrates like CMC (carboxymethylcellulose). β-Glucosidase is the enzyme that hydrolyzes cellotriose and cellobiose to glucose. This chapter describes the thermostable cellobiohydrolase, endoglucanase, β-glucosidase from Humicola insolens and the Avicel-disintegrating endoglucanase from Humicola grisea. The chapter describes the assay method that is based on the release of reducing sugars or glucose following the incubation of Avicel, CMC, or cellobiose with the enzyme.

A Novel Raw-Starch-Digesting Yeast α-Amylase from Lipomyces starkeyi HN-606

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.

Temperature-sensitive mutants of a thermotolerant yeast, Hansenula polymorpha

Abstract Temperature-sensitive mutants (TS-1 and TS-7) of a thermotolerant yeast, Hansemula polymorpha CK-1, were isolated. The mutants were unable to grow at 50°C, the maximum growth temperature of the wild type. Mutants TS-1 and TS-7 grown at 20°C showed 33 and 50% viabilities after 6 h of incubation of 50°C, respectively. Mutant TS-1 showed little variation of the degree of fatty acid unsaturation (1.26–1.28/mol) and mutant TS-7 had an almost constant sterol/phospholipid molar ratio (0.31–0.34) at 20, 30 and 40°C, although the wild type had a decrease of the degree of fatty acid unsaturation from 1.56 at 20°C to 1.30 at 40°C and an increase of the sterol/phospholipid molar ratio from 0.26 at 20°C to 0.54 at 40°C.

Xylanase of Talaromyces byssochlamydoides

Publisher Summary The behavior of xylanase toward various substrates shows that it contains at least two enzyme activities; one is an endoxylanase that is responsible for the initial breakdown of the xylan chain and the formation of oligosaccharides and the other is β-xylosidase that causes a gradual fragmentation of the oligosaccharides leading finally to xylose as the end product. Many xylanases produced by various microorganisms have been investigated and some have been obtained in homogeneous states on disc electrophoresis. This chapter describes the thermostable xylanase of Talaromyces byssochlamydoides YH-50. The chapter discusses the assay method that is based on the release of xylose following the incubation of xylan with the enzyme. It describes the purification procedure and properties of the enzyme.

Saccharomyces uvarum inulyticus var. nov., a new high-concentration ethanol tolerant yeast from rice wine

SummaryA new alcohol tolerant yeast which had the ability to ferment glucose, galactose, maltose, trehalose, melizitose, α-methyl-d-glucoside, melibiose and raffinose (complete) was isolated from fermented rice wine. This new isolate which was morphologically, culturally and physiologically similar to type species Saccharomyces uvarum Beijerinck was considered as a variety of the species, hence, designated as Saccharomyces uvarum inulyticus var. nov., because of its extraordinary ability to ferment inulin into ethanol and hyper-tolerance to high concentration of ethanol at a wide range of temperature. The new variant produced 21.6%, 20%, 18.6%, and 9.8% v/v ethanol at 15, 20, 30, and 40° C, respectively, when fermentation was carried out by the stepwise addition of sucrose to the synthetic medium. The addition of fungal mycelia into the synthetic medium increased the fermentation rate and ethanol concentration up to 22.4%, 21.8%, 20.1%, and 11.6% v/v at 15, 20, 30, and 40° C, respectively. The new variant was found to grow well at 40° C and did not require any vitamin or growth factor, such as biotin, calcium pantothenate, inositol, niacin, p-aminobenzoic acid, pyridoxine hydrochloride, riboflavin and thiamine hydrochloride for normal growth.

Characterization of a mutant of thiostrepton-producing Streptomyces azureus ATCC 14921 and its plasmids

Abstract A mutant, strain PK10, of Streptomyces azureus ATCC 14921 and its two plasmids were characterized and compared with another mutant, PK 100, and its plasmid. One PK 10 plasmid of 8.8 kb was identical to a pock-forming plasmid, pSA1.1, of PK100. The other olasmid which was found only in PK10 nd named pSA1.2 (size, 7.6 kb), was a non-pock forming derivative of pSA1.1 with deletions in two different regions (about 1.2 kb and 30 b long). The pcok-forming ability of strain PK10 on a plasmid-free strain was lower than that of strain PK100 which contained only pSA1.1. Strain PK10 had fewer copies of pSA1.1 than strain PK100, and had normal spore formation and thiostrepton production, which were depressed in the strain PK100. The pSA1.1 from both PK10 and PK100 amplified to 20 to 30 copies in the transformants and inhibited theri spore formation and thiostrepton production. Thus, the function of pSA1.1 appeared to be depressed by pSA1.2.

Distribution of bph Genes Encoding Biphenyl and Polychlorinated Biphenyl Degrading Enzymes in Soil Bacteria

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.

Frequent occurrence of diploid a or α sporogenous mater cells and construction of ploidy series in the sake yeast kyokai no. 9

Abstract 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.

Isolation and growth characteristics of a nystatin-resistant mutant of a thermotolerant yeast, Hansenula polymorpha

Abstract 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.