Hiroshi Ooshima

Microwave treatment of cellulosic materials for their enzymatic hydrolysis

SummaryRice straw and bagasse with water content 84 or 94% were irradiated with microwave (2450MHz) in sealed glass vessels. This treatment enhanced markedly the accessibility of the cellulosic materials for the enzymatic hydrolysis: for example, 1.6 times in the rice straw by the microwave treatment at 170 °C for 5 min and 3.2 times in the bagasse by the treatment at 200 °C for 5 min, compared with the untreated.

Production of hydrogen by a hydrogenase-deficient mutant of Rhodobacter capsulatus

The characteristics of Rhodobacter capsulatus ST410, a mutant of the wild strain B100 lacking hydrogenase activity, were investigated from the viewpoint of hydrogen production. When 30 mM dl-malate and 7 mM l-glutamate were used as carbon and nitrogen sources, respectively, in an argon atmosphere, a specific hydrogen evolution rate of 0.14 ml/h/mg-dry cells was obtained at 6600 lx and 33°C. The evolution rate strongly depended on the light intensity: the higher the light intensity, the larger the evolution rate became up to at least 6600 lx. R. capsulatus ST410 converted 60 mM malate to hydrogen at a yield of 68%, calculated as a percentage of the stoichiometric maximum for the complete conversion of the carbon source to H2 and CO2. On the other hand, when the wild strain was used under the same conditions, the yield was only 25%. R. capsulatus ST410 converted not only malate but also glucose and cellobiose to hydrogen with good yields (60% for 30 mM glucose and 66% for 7.5 mM cellobiose). Ethanolamine was found to be a good nitrogen source, which permitted a large amount of hydrogen to be evolved and also depressed the cell growth to low levels.

Pretreatment of bagasse by nonionic surfactant for the enzymatic hydrolysis

Abstract Enzymatic hydrolysis of bagasse was accelerated by pretreatment with 3·33% wt nonionic surfactant (Tween 20; polyoxyethylene sorbitan monolaurate) at 170–190°C. The amount of the lignin remaining in the pretreated bagasse decreased by about 22–27% and the enzymatic hydrolysis rate increased, because of an increase in the surface area of cellulose accessible to enzyme, compared to those pretreated with water. This effect is based on the fact that surfactant makes hydrophobic degradation products extractable to water. The pretreatment effect varied with the HLB (hydrophile-lipophile balance) values of surfactant. The hydrophilic surfactant having high HLB values was useful for the extraction of hydrophobic degradation products from lignin and hemicellulose. In the pretreatment at 190°C, an addition of Tween 20 to the UCT-solvent (binary solvent with water, having upper critical-temperature in the manual solubility curve) increased the effect of UCT-solvent pretreatment. This is because the surfactant makes the separation of the degradation products easier.

Enzymatic activity of cellulase adsorbed on cellulose and its change during hydrolysis

Hydrolysis of pure cellulose Avicel has been carried out, using Meicelase fromTrichodertna viride, where the enzymatic activity of cellulase adsorbed on cellulose and its changes during the hydrolysis were investigated. A rapid drop of the hydrolysis rate during the reaction, that is always observed in enzymatic hydrolysis of cellulose, could be explained by a decline of specific activity of adsorbed enzyme, and it was implied that the decline results from a loss of synergistic action between endoglucanase and exoglucanase. An empirical equation expresses the change of hydrolysis rate during the reaction and also shows that the change of the hydrolysis rate is caused by the decline of the specific enzymatic activity of adsorbed enzyme.

Synthesis of aspartame precursor by solid thermolysin in organic solvent

SummaryThe enzymatic synthesis of a peptide compound was carried out successfully in homogeneous organic solvent.Solid Thermolysin was found to catalyze the synthetic reaction of N-benzyloxycarbonyl-L-aspartyl-L-phenylalanine methyl ester (Z-APM; a precursor of sweetner Aspartame) from N-benzyloxycarbonyl-L-aspartic acid (Z-L-Asp) and L-phenylalanine methyl ester (L-PheOMe) in a 98 percent organic medium (ethylacetate∶benzene∶methanol∶water=50∶29∶19∶2). The dissolution of enzyme was not observed. The optimal pH shifted to acidic side by 1.0 pH unit, compared with that in aqueous medium. The enzymatic activity of solid thermolysin with an average size of 3.4×9.5 μm was determined to be 0.18 μmoles-product/(mg-solid)·h under the initial concentrations of L-PheOMe of 0.1M and Z-L-Asp of 0.05M, and at pH 6.0 and 40°C.

n-acylation of β-amino alcohol by acyl migration following enzyme-catalyzed esterification

Abstract Lipase-catalyzed n -acylations of β-amino alcohols such as ethanolamine and l -serine were investigated. To prepare n -acyl derivatives by taking advantage of the acyl migration, we first carried out a screening of suitable enzymes for the desired reaction. As a result, we found a higher activity for n -acylation with Lipase Q L. This lipase had higher hydrolytic activity for the o-acyl compound but not the n -acyl compound. The observation shows that n -acylation results from the esterification and successive acyl migration into the amino group. Using Lipase Q L, we then investigated the n -acylation of ethanolamine or l -serine with fatty acids as acyl donors. The reaction parameters for the n -acylation were clarified.

Light intensity distribution in the externally illuminated cylindrical photo-bioreactor and its application to hydrogen production by Rhodobacter capsulatus

The light distribution in the externally illuminated cylindrical photo-bioreactor for production of hydrogen by a photosynthetic bacterium Rhodobacter capsulatus ST-410 was estimated. The estimation was performed on the basis of the Matsuura and Smiths diffuse model [1]. In the diffuse model, the incident light rays are assumed to proceed in every direction and the local intensity is calculated as the sum of the intensities of light. Since Lambert-Beers law, extensively used in photometry, was not useful for explaining the decrease in the intensity of light by the biomass, an empirical expression was used. The measurement of the intensities from every direction was conducted in an externally illuminated cylindrical photo-bioreactor having an inner diameter of 60mm and a working volume of 550ml. The obtained results confirmed our estimation. The light distribution was applied to estimate the hydrogen production by R. capsulatus ST-410 using the same photo-bioreactor. The overall hydrogen-production rate was successfully estimated.

Simple screening method for lipase for transesterification in organic solvent

We investigated lipase-catalyzed hydrolysis in water and dioxane—water with a simple colorimetric method. We screened 24 lipases for the ability to hydrolyze p-nitrophenyl esters as chromogenic substrates. Their hydrolytic activities were varied by adding dioxane. Most of the lipases showed high activity in hydrolysis in water, but some showed activity in 50% dioxane—water several tens times higher than those in water. Moreover, several lipases with hydrolytic abilities in 50% dioxane—water also catalyzed the transesterification of p-nitrophenol using fatty acid vinyl esters. We found it possible that a useful lipase for transesterification can be selected by measuring the hydrolysis activity of p-nitrophenyl ester in 50% dioxane—water.

The initial stage of crystallization of lysozyme: a differential scanning calorimetric (DSC) study

The initial stage of crystallization of hen-egg-white lysozyme was investigated. Lysozyme molecules formed a structure just after the supersaturated solution was prepared. The structure could be understood as changes in solution three-dimensional structure induced by attractive interactions or aggregates with an average size of 6.5 nm. The structure formation, however, did not immediately link to crystal growth. An induction period was needed before the start of crystal growth. DSC analysis of the supersaturated solution revealed that the structure thus formed at the beginning of crystallization is transformed just before the end of the induction period. DSC analysis also revealed that the driving force of the transformation is the formation of hydrophobic bonds between lysozyme molecules. Our understanding of the behavior of lysozyme molecules before crystal growth was furthered by measurement of the ζ-potential of the aggregates. Based on the present results, a possible model of the early stage of crystal growth of lysozyme was proposed.

Resistance of yeast and bacterial spores to high voltage electric pulses

Abstract Spores of a yeast, Saccharomyces cerevisiae , and a bacterium, Bacillus subtilis , were exposed to high voltage electric pulses. The viabilities of spores and vegetative cells of the yeast were significantly decreased after the electric pulse treatment, and some of the spores and almost all of the cells were stained red with an agent, phloxine B. On the other hand, (endo) spores of the bacterium were highly resistant to the electric pulses and little decrease in viability was observed, although the viability of vegetative cells was sharply lowered. The results revealed marked structural and/or biochemical differences between eukaryotic and prokaryotic spores.