Kosaku Ishimi

Growth of organic solvent-tolerant Pseudomonas aeruginosa LST-03 in the presence of various organic solvents and production of lipolytic enzyme in the presence of cyclohexane.

Pseudomonas aeruginosa LST-03 grew in a liquid medium in the presence of organic solvents of which the log P value was equal to or greater than 2.4. Its growth in the presence of organic solvent was followed quantitatively by measuring the dry cell mass. Production of the lipolytic enzyme of P. aeruginosa LST-03 cultivated in the presence of cyclohexane was also investigated. Although this strain can grow in the presence of cyclohexane under conditions in which the supply of oxygen is limited, aeration is required for production of lipolytic enzyme. The lipolytic activity of the supernatant of the culture in the presence of cyclohexane was stabler than that in the absence of organic solvent.

Kinetics and mechanism of enzymatic hydrolysis of gelatin layers of X-ray film and release of silver particles

Abstract Used X-ray film contains a large number of silver particles in its gelatin layers. With the aim of developing an efficient enzymatic process for the recovery of silver and polyester film from used X-ray film, hydrolysis experiments were performed using a stirred-tank reactor in batch operation. On the bottom of the tank a sheet of X-ray film was fixed so that only one of the film surfaces would be exposed to a solution containing the alkaline protease B21-2 from alkalophilic Bacillus sp. The time courses of gelatin hydrolysis, measured by using unexposed film which was prepared by developing X-ray film without exposure to light and which did not contain any silver, showed that at any stirring speed there existed induction periods in which hydrolysis was very slow. When the stirring speed was higher, the induction period and the time required to complete the hydrolysis were shortened due to an increase in the transfer rate of the enzyme through the liquid film. The rate of gelatin hydrolysis of unexposed film increased with the enzyme concentration. The time courses measured using exposed film, which was prepared by developing X-ray film after it had been exposed homogeneously to light and which contained fine silver particles, showed that the induction periods for the release of silver particles were much longer than those for gelatin hydrolysis. The presence of silver particles did not significantly affect the gelatin hydrolysis. The above experimental results on gelatin hydrolysis were well explained by a proposed model which took into consideration a number of physical processes, such as diffusion of the enzyme and hydrolysis products through the liquid film on the surface of the gelatin layer, in addition to the chemical processes.

Hydrogen Production from Glucose by Anaerobes

Various anaerobes were cultivated in media containing glucose. When 100 mL of thioglycollate medium containing 2.0% (w/v) glucose was used, Clostridium butyricum ATCC 859, NBRC 3315, and NBRC 13949 evolved 227–243 mL of biogas containing about 180 mL of hydrogen in 1 day. Although some strains had some resistance against oxygen, C. butyricum ATCC 859 and 860 did not have it. C.butyricum NBRC 3315 and Enterobacter aerogenes NBRC 13534 produced hydrogen in the presence of glucose or pyruvic acid, and E. aerogenes NBRC 13534 produced hydrogen by not only glucose and pyruvic acid but also dextrin, sucrose, maltose, galactose, fructose, mannose, and mannitol. When a medium containing 0.5% (w/v) yeast extract and 2.0% (w/v) glucose was used, E. aerogenes NBRC 13534 evolved more biogas and hydrogen than C. butyricum NBRC 3315 in the absence of reducing agent.

Stabilities and Conformational Transitions of Various Proteases in the Presence of an Organic Solvent

The half‐life of the activity of the PST‐01 protease that was secreted by organic solvent‐tolerant Pseudomonas aeruginosa PST‐01 was very long in the presence of methanol as compared to that in the absence of methanol. The conformational transitions of the PST‐01 protease, α‐chymotrypsin, thermolysin, and subtilisin in the presence and absence of methanol were monitored by measuring the CD spectra. The conformational stabilities of the PST‐01 protease and subtilisin in the presence of methanol were higher than those in the absence of methanol. This resulted in high stability of these proteases in the presence of methanol. Furthermore, it was suggested that the organic solvent stabilities of enzymes were closely related to the secondary structure by monitoring the conformational transitions of polyamino acids, which form the particular conformations, in the presence and absence of methanol.

Cloning, expression and characterization of a lipase gene (lip3) from Pseudomonas aeruginosa LST-03

A lipase gene (lip3) was cloned from the Pseudomonas aeruginosa strain LST-03 (which tolerates organic solvents) and expressed in Escherichia coli. The cloned sequence includes an ORF consisting of 945 nucleotides, encoding a protein of 315 amino acids (Lip3 lipase, 34.8 kDa). The predicted Lip3 lipase belongs to the class of serine hydrolases; the catalytic triad consists of the residues Ser-137, Asp-258, and His-286. The gene cloned in the present study does not encode the LST-03 lipase, a previously isolated solvent-stable lipase secreted by P. aeruginosa LST-03, because the N-terminal amino acid sequence of the Lip3 lipase differs from that of the LST-03 lipase. Although the effects of pH on the activity and stability of the Lip3 lipase, and the temperature optimum of the enzyme, were similar to those of the LST-03 lipase, the relative activity of the Lip3 lipase at lower temperatures (0–35°C) was higher than that of the LST-03 lipase. In the absence of organic solvents, the half-life of the Lip3 lipase was similar to that of the LST-03 lipase. However, in the presence of most of the organic solvents tested in this study (the exceptions were ethylene glycol and glycerol), the stability of the Lip3 lipase was lower than that of the LST-03 lipase.

Cloning, Expression, and Characterization of a Lipolytic Enzyme Gene (lip8) from Pseudomonas aeruginosa LST-03

A lipolytic enzyme gene (lip8) was cloned from organic solvent-tolerant Pseudomonas aeruginosa LST-03 and sequenced. In the sequenced nucleotides, an open reading frame consisting of 1,173 nucleotides and encoding 391 amino acids was found. Lip8 is considered to belong to the family VIII of lipolytic enzymes whose serine in the consensus sequence of -Ser-Xaa-Xaa-Lys- acts as catalytic nucleophile. The gene was expressed in Escherichia coli and purified by a combination of ammonium sulfate fractionation and hydrophobic interaction and ion-exchange chromatographies to homogeneity on SDS-PAGE analysis. The optimum temperature and heat stability of Lip8 were not as high as those of Lip3 and LST-03 lipase, two other lipolytic enzymes from the same strain. Addition of glycerol to a solution containing Lip8 stabilized this enzyme. By measuring the activities against various triacylglycerols and fatty acid methyl esters having carbon chains of different lengths, Lip8 was categorized as an esterase which has higher activities against fatty acid methyl esters with short-chain fatty acids.

Peptide Synthesis of Aspartame Precursor Using Organic‐Solvent‐Stable PST‐01 Protease in Monophasic Aqueous‐Organic Solvent Systems

The PST‐01 protease is a metalloprotease that has zinc ion at the active center and is very stable in the presence of water‐soluble organic solvents. The reaction rates and the equilibrium yields of the aspartame precursor N‐carbobenzoxy‐l‐aspartyl‐l‐phenylalanine methyl ester (Cbz‐Asp‐Phe‐OMe) synthesis from N‐carbobenzoxy‐l‐aspartic acid (Cbz‐Asp) and l‐phenylalanine methyl ester (Phe‐OMe) in the presence of water‐soluble organic solvents were investigated under various conditions. Higher reaction rate and yield of Cbz‐Asp‐Phe‐OMe were attained by the PST‐01 protease when 30 mM Cbz‐Asp and 500 mM Phe‐OMe were used. The maximum reaction rate was obtained pH 8.0 and 37 °C. In the presence of dimethylsulfoxide (DMSO), glycerol, methanol, and ethylene glycol, higher reaction rates were obtained. The equilibrium yield was the highest in the presence of DMSO. The equilibrium yield of Cbz‐Asp‐Phe‐OMe using the PST‐01 protease attained 83% in the presence of 50% (v/v) DMSO.

Synthesis of New Polymer-Bound Adenine Nucleotides Using Starburst PAMAM Dendrimers

Two types of new polymer‐bound adenine nucleotides were synthesized by coupling adenine nucleotides (ATP and ADP) with starburst polyamidoamine (PAMAM) dendrimers. The first type was obtained by coupling native adenine nucleotides directly with a carboxy‐terminated PAMAM dendrimer. In the second type, the nucleotides were modified by introducing a spacer arm containing a carboxylic end group ( N6‐R‐ATP and N6‐R‐ADP) and coupled with an amine‐terminated PAMAM dendrimer. Both types of the dendrimers were coupled with native or the modified nucleotides using the well‐known carbodiimide activation technique. The optimum coupling pH and temperature were 4 and 30 °C, respectively, for preparing the carboxy‐terminated PAMAM‐bound ATP or ADP, and were 9 and 50 °C, respectively, for preparing the amine‐terminated PAMAM‐bound N6‐R‐ATP or N6‐R‐ADP. The ATP or ADP contents in the synthesized polymers were found to be 4 mol of ATP or of ADP/mol of carboxy‐terminated PAMAM‐bound ATP or ADP and 25 mol of ATP or of ADP/mol of amine‐terminated PAMAM‐bound N6‐R‐ATP or N6‐R‐ADP. The coenzymatic activities relative to the native ATP of the carboxy‐terminated PAMAM‐bound ATP against glucokinase and hexokinase were 16 and 7%, respectively, and those of the amine‐terminated PAMAM‐bound N6‐R‐ATP 2 and 1%, respectively. The coenzymatic activities relative to the native ADP of the carboxy‐terminated PAMAM‐bound ADP and the amine‐terminated PAMAM‐bound N6‐R‐ADP against acetate kinase were 24 and 3.5%, respectively.

Experimental Investigation of Fructose 1, 6-Diphosphate Production and Simultaneous ATP Regeneration by Conjugated Enzymes in an Ultrafiltration Hollow-Fiber Reactor

The enzymatic synthesis of fructose 1,6-diphosphate (FDP) from glucose and the enzymatic ATP regeneration were performed simultaneously in an ultrafiltration hollow-fiber reactor using both the purified enzymes and the crude cell extract of Bacillus stearothermophilus. The process consisted of the three-step synthetic reactions catalyzed by glucokinase, phosphoglucose isomerase and phosphofructokinase, and the ATP regeneration reaction catalyzed by acetate kinase. The experimental results of the yield and the recycle number agreed well with the theoretical predictions calculated using a computer program developed in our preceding study. This proved the validity of this computer program for predicting or analyzing the reactor performance.

Simulation of Particle Growth in the Dispersion Polymerization of Styrene: The Termination Rate Constant in Particles

A model is proposed for simulating the particle growth in the dispersion polymerization of styrene in ethanol. The model is based on the following assumption: (i) the termination reaction in an ethanol-phase and the chain-transfert reactions in the ethanol-phase and particles can be neglected, (ii) the mean volume of the radicals captured by particles is approximately equivalent to that of monomeric radical, and (iii) the termination rate constant in particles is β gel times that of the ethanol-phase. The experimental results of the conversion, the particle diameter and the particle number measured as the reaction time of 2 h were used to determine the initial conditions. When the termination rate constant in particles was taken to be about 1/130 of that of the ethanol phase, the calculation results of the conversion and the particle diameter were in good agreement with the experimental data.