Akira Obayashi

Improvement of ethanol production of themophilic Clostridium sp. by Mutation

Abstract Three thermophilic Clostridium strains were isolated from soil as cellulose-fermenting bacteria wich produced ethanol, lactic acid, and acetic acid from cellulose at 60°C. To increase ethanol productivity, strains no. 187 was mutated with N-methyl-N′-nitro-N-nitrosguanidine. The resultant mutant, no 187-102-27, was superior to the original strain in ethanol production, and produced less lactic and acetic acid. The activities of some enzymes involved in the biosynthesis of the lactic and acetic acid of mutant no. 187-102-27 were lower than those of the original strain. These results are highly consistent with the acid production of the mutant strain being low.

Evaluation of ethanol productivity from cellulose by clostridium thermocellum

Abstract Clostridium thermocellum , a thermophillic anaerobe, directly converts cellulose to ethanol. To estimate its ethanol production from cellulose, we used a new method based on material balance by which the efficiencies of the enzymes that convert cellulose to ethanol were calculated. Using this method, the maximum efficiency of ethanol production of two strains of C. thermocellum was estimated to be 0.05, with 0.67 as the theoretical maximum.

NADH production from NAD+ using malic enzyme of Pseudomonas diminuta IFO-13182

Abstract Permeable cells and a sonicate of Pseudomonas diminuta IFO-13182 produced NADH from NAD − by an NAD- and NADP-linked malic enzyme [ l -malate: NAD(P) + oxidoreductase (EC 1.1.1.39)] reaction in the presence of l -malic acid and divalent metal ions. Optimum conditions for NADH production were examined, including malate concentration, NAD + concentration, pH, and buffer type. Under suitable conditions, the conversion ratio of NADH from NAD + reached 100% after 3 h of incubation at 30°C and the amount of NADH accumulated was 14 μmol ml −1 of reaction mixture. The NADH produced in such enzyme reaction was purified in powder form. The purity of NADH produced was 96.6% from its coenzyme activity with alcohol dehydrogenase.

Use of Koji prepared with a high citric acid producing mutant of Aspergillus usamii as a raw material for Saké brewing

Abstract There is a possibility of developing a new kind of sake in which the refreshing sour taste of citric acid is introduced. In this study, we bred a new mutant of Aspergillus usamii mut. shiro-usamii that produced much citric acid. The koji prepared with the mutant contained about 20 mg of citric acid per gram of dry koji, twice that of the koji of the parental strain. The activities of a-amylase, glucoamylase, and acidic protease in the koji prepared with the mutant were 82%, 94%, and 95%, respectively, those of the parental strain. Using this koji with the mutant, sake was produced. The levels of citric acid and isoamyl acetate were 5.1 and 1.4 times, respectively, those of sake prepared with koji of A. oryzae. Sensory tests indicated that sake made with koji with the mutant was refreshingly sour, with a good aroma.

Isolation of Plasmids from Thermophilic Clostridia and Construction of Shuttle Vectors in Escherichia coli and Cellulolytic Clostridia

Abstract Four small plasmids (1.5–7.2 kb) were found in three strains of thermophilic clostridia. Two shuttle vectors, pCL1 and pCS1, used in Escherichia coli and some strains of Clostridium were constructed from plasmid pTA688L or pTA688S and the E. coli plasmid pBR328. The transformation system examined in strains of thermophilic Clostridium , which have cellulolytic activities, yielded about 1–7 × 10 2 transformants per microgram of pCS1 DNA. The DNA from these transformants was of the same size as that of pCS1. Thus, the host-vector system could be useful for the study of genes involved in the cellulolytic enzymes of species of Clostridium .

Screening for strains of Aspergillus oryzae that degrade carbamide for use in sake brewing

Abstract We screened 65 strains of Aspergillus oryzae for ones that degraded carbamide, Two strains, IFO 5238 and IFO 30113, were selected and used to prepare rice koji . Sake made with the rice koji prepared with these strains had less than half (less than 15 ppm) of the carbamide in sake made with rice koji prepared with commercial spores of A. oryzae .

Mirin-making at low alcohol concentration with Koji prepared with a new mutant of Aspergillus usamii

Abstract We examined the productivity of mirin-making and the quality if the mirin produced using koji prepared with a new mutant (mutant CA) that originated from Aspergillus usamii mut. shiro-usamii. The koji prepared with the mutant CA contained a large amount of citric acid. Therefore, the concentration of the brewing alcohol added to prevent bacterial contamination of the mash was decreased to 6.0% from the 12.5% needed when the mash was made with koji of the conventional Aspergillus oryzae. A mash containing this low concentration of alcohol was incubated with koji of the mutant CA and enzyme preparations such as α-amylase (6,000 DU/kg mash) from Bacillus subtilis and acidic protease (1,000 PU/kg mash) from Aspergillus niger. The starch and protein in this mash was sufficiently digested. The yield of mirin obtained from this mash was high (96% based on the mash weight), and the resulting mirin contained much citric acid, malic acid, succinic acid, nitrogen compounds, isomaltose, isomaltotriose, and oligosaccarides. The taste of the mirin was refreshingly sour and flavorsome.

Evaluation of koji prepared with various molds for mirin-making

Abstract Koji is one of the raw materials used for mirin -making, and it is traditionally prepared with Aspergillus oryzae . To improve productivity and to be available for a variety of mirin , various koji were prepared with A. niger, A. awamori, A. usamii mut. shiro-usamii , and Rhizopus oligosporus and examined for possible to use in mirin -making. The degrees of digestion of the starch and protein in various koji were at its highest with either koji prepared with A. oryzae at pH 7.0 or with A. usamii mut. shiro-usamii at pH 3.0. The degree of digestion of the protein in koji made with A. usamii mut. shiro-usamii was decreased in koji digestion at higher temperature (at over 50°C) compared to that in koji made with A. oryzae . We examined the ratio of koji to steamed glutinous rice to identify the optimum value for mirin production. The optimum ratios for digestion of starch in the mash with koji of A. oryzae and A. usamii mut. shiro-usamii were 0.10 and 0.15, respectively. These values were in agreement with the 0.10–0.25 established by experience. Therefore, of the five strains tested, A. usamii mut. shiro-usamii , among the tested molds was the most suitable strain for preparing koji in mirin -making without decreasing productivity, while lending new qualities to the mirin . The koji prepared with A. oryzae had a good smell but the koji prepared with A. usamii mut. shiro-usamii had a little mold smell. Then if this little mold smell could be improved, this koji can be used in mirin -making. The koji prepared with A. niger contained the most citric acid of all strains but the digestivities of starch and protein in the koji was less than those of koji prepared with A. oryzae and A. usamii mut. shiro-usamii . The digestivities of starch and protein in koji prepared with A. awamori were the lowest values. In 35% alcohol, its productivities in mirin -making seemed to be low. Because of the highest content of lactic acid, the koji prepared with R. oligosporus seemed to be suitable for mirin -making. Then if its productivity could be improved, this koji can be used in mirin -making.

Relationship between conditions for steaming rice and productivity in mirin-making

Abstract The relationship between the conditions used to prepare steamed rice and its enzymatic digestion for mirin-making was examined. The degree of gelatinization, G, of the starch in the steamed rice was directly related to the enzymatic digestion, and is represented by Eqs. 1 and 2 for regular and glutinous rice, respectively. G=79.88+(-18.13+0.306 Th) M G=93.08+(-2.69+0.149 Th) M where Th is the temperature of steaming and M is the water content of the soaked rice. Therefore, the degree of gelatinization of starch in steamed rice could be predicted. The water content of regular rice, which was low after soaking, can be estimated by Eq. 3, M=M 0 + exp (0.125 Tw-(10.51-0.00222 t)) where M0 is the water content of rice soaked for 24 h at 20°C and Tw and t are the temperatures and times of soaking, respectively. The optimum water content of soaked regular rice for mirin-making was calculated using Eq. 3. Thus, the design of a system for the steaming process made it possible to obtain steamed rice of the desired degree of gelatinization.

Relationship between enzyme efficiency and alcohol concentration in Mirin mash

Abstract In mirin-making, the influence of alcohol concentration on enzyme activities that originated from koji and from enzyme preparations were analyzed quantitatively in order to determine the optimum concentration for production. The influences were complex. The efficiency of the enzymes in the mash with alcohol could be described by the ratio of the maximum digestibility, Dm, of the components in the raw materials to a constant, tm, which is related to the velocity (Eqs. 1 and 2). D=D m (1- exp (- T 30 t m )) D m t m = lim T 30 →0 d d t (D m (1- exp (- T 30 t m where T30 is the incubation time at 30°C, D is the digestion after T30, and Dm/tm corresponds to the enzyme activity, usually represented as the initial velocities of the enzymes. Analysis of Dm and tm made it possible to achieve maximum productivity in the making of mirin.