Yasuyuki Isono

Degradation of aliphatic polyester films by commercially available lipases with special reference to rapid and complete degradation of poly(L-lactide) film by lipase PL derived from Alcaligenes sp.

Commercial lipases were examined for their degradation efficiency of aliphatic polyester films. In 100 days immersion of polyester films in lipase solutions at37 °C at pH 7.0,Lipase Asahi derived from Chromobacterium viscosum degraded polybutylene succinate-co-adipate (PBSA), poly (ε-caprolactone) (PCL) and polybutylene succinate (PBS), and Lipase F derived from Rhizopus niveus degraded PBSA and PCL during 4–17 days. Lipase F-AP15 derived fromRhizopus orizae could degrade PBSA in 22 days. In these cases, PBS and PBSA were mainly degraded to dimers, whereas PCL was mainly degraded to monomers. Only poly(3-hydroxybutyrate-co-3-hydroxyvalerate)(PHB/V) and poly (L-lactide) (PLA) were not degraded in the experiments. However, PLA degraded completely at 55 °C, pH 8.5 with Lipase PL during 20 days. This result could be explained with the sequential reactions of the chemical hydrolysis of the polymer to oligomers at higher pH and temperature, and the succeeding enzymatic hydrolysis of oligomers to the monomers.

Immobilization of saccharomyces cerevisiae for ethanol fermentation on γ-alumina particles using a spray-dryer

Saccharomyces cerevisiae was immobilized on γ-alumina particles with binder polymer by a spray-drying process. Batch fermentations of sucrose to ethanol were performed using the immobilized cells. The optimum pH was 4, and this helped to minimize microbial contamination and facilitate electrostatic attraction between the γ-alumina particles and the yeast cells. Addition of yeast extract resulted in high ethanol conversion. The reasons for differences between cellulose acetate phthalate (CAP) and styrene-maleic acid co-polymer (SMC) as binders on ethanol conversion were not apparent. The release of binder with the SMC from the γ-alumina was less than that from the CAP. Presoaking of γ-alumina particles in resin for binder solution before the immobilization using a spray-dryer was effective in achieving high ethanol conversion.

Solvent-Free Esterification of Oleic Acid and Oleyl Alcohol Using Membrane Reactor and Lipase-Surfactant Complex

Oleyl alcohol and oleic acid were esterified using lipase-surfactant complex (LSC) in a solvent-free reaction system. LSC showed a higher reaction rate than the crude enzyme in the reaction system. Optimum LSC activity was obtained at 180 mg/g-solution water addition. A preliminary study of the membrane reactor system using a microfiltration membrane and a solvent-free reaction was performed, and the feasibility of the reactor system was confirmed.

Wax ester synthesis in a membrane reactor with lipase-surfactant complex in hexane

A lipase-surfactant complex (LSC) was prepared by mixing an aqueous solution of lipase (E.C. 3.1.1.3) derived fromPseudomonas sp. with an alcoholic solution of sorbitan monostearate. This LSC was applied to the catalysis of esterification of cetyl alcohol and palmitic acid to synthesize was ester in hexane. A membrane reactor system was investigated for the repeated batch synthesis of wax ester with LSC. High conversions were maintained during repeated experiments; however, the flux decreased during the run. Sponge washing of the membrane restored the flux to 70% of its original value.

Enzymic peptide synthesis using a microaqueous highly concentrated amino acid mixture

Abstract A concentrated microaqueous solution of the amino acids N-(benzyloxycarbonyl)- l -aspartic acid (ZA) and l -phenylalanine methyl ester (PM) was used at a suitable pH for the enzymic synthesis of ZAPM. Dehydrochlorination of PM–HCl was first carried out by aqueous/chloroform biphasic extraction, then the chloroform phase which extracted PM was concentrated with a rotary evaporator. In this operation, concentrated dehydrochlorinated PM solution at a concentration higher than 3.5 M could be prepared. The pH value of the reaction media was regulated by setting the ratio of PM/ZA. High enzymic activity of ZAPM synthesis was observed at a PM/ZA ratio of 3.3 which gives the suitable pH value for the enzyme. Water addition of 100 μl in the reaction media consisted of 1.0 ml of PM solution (3.77 M) and 300 mg of ZA (1.13 mmol) was effective in increasing enzymic activity.

Lipase-surfactant complex as catalyst of interesterification and esterification in organic media

A lipase-surfactant complex (LSC) was prepared by mixing an aqueous solution of lipase MF-30 derived from Pseudomonas sp. and an alcoholic solution of sorbitan monostearate. The LSC efficiently catalyzed the interesterification of triglyceride and fatty acid in hexane, as well as that of palm oil and fatty acid. The LSC catalyzed the esterification of fatty acids with mono- and diglyceride, methanol and cetyl alcohol in hexane. Catalytic activities of the LSC were higher than those of a crude lipase in esterification.

Hollow-fibre enzyme reactor integrated with solvent extraction for synthesis of aspartame precursor

Abstract A new process for the simultaneous enzymic synthesis and purification of N-(benzyloxycarbonyl)- L -aspartyl- L -phenylalanine methyl ester (ZAPM), a precursor of aspartame, has been developed. The enzymic reaction between N-(benzyloxycarbonyl)- L -aspartic acid (ZA) and L -phenylalanine methyl ester (PM) was carried out in a biphasic hollow-fibre rector with an aqueous phase an a butyl acetate phase. The reaction took place in the aqueous phase and by maintaining the pH at 5, the product (ZAPM) was extracted into the organic phase. Product purity was greater than 90% and reasonable productivity could be achieved with this system.

New selective perstraction system with charged membrane

Abstract A new perstraction system using a charged membrane was developed in order to obtain high selectivity by liquid-liquid equilibrium and electrostatic rejection effects. A mixture of N- beenzyloxycarbonyl)- l -aspartic acid (ZA), l -phenylalanine methyl ester (PM) and N- (benzyloxycarbonyl)- l -phenylalanine methyl ester (ZAPM) was used as the model solution. The aqueous phase was adjusted to pH 6 and using a positively charged membrane, only ZAPM was extracted into the organic phase. The separations between ZA and ZAPM, and between PM and ZAPM were performed by bi-phasic extraction and electrostatic rejection, respectively. The utility of this hybrid separation system, combining perstraction with charged membrane separation is demonstrated.

Performance of Perstractive Enzyme Reactor for Synthesis of Aspartame Precursor

A perstractive enzyme reactor was used for the synthesis of N-(benzyloxycarbonyl)-L-aspartyl-L-phenylalanine methyl ester (ZAPM), the precursor of the artificial sweetener, aspartame. The synthesis of ZAPM in the reactor proceeded by an enzymatic reaction between N-(benzyloxycarbonyl)-L-aspartic acid (ZA) and L-phenylalanine methyl ester (PM) in the aqueous phase. The synthesized ZAPM in the aqueous phase was mainly extracted into the organic phase, therefore, the concentration of ZAPM in the aqueous phase could be kept low. As a result, high conversion of ZAPM was obtained with this system. The partition coefficients of substances in the aqueous/butyl acetate biphasic system, the mass transfer coefficients of substances through the membrane and the enzymatic kinetics of ZAPM synthesis were determined experimentally. The reaction model which was based on the material mass balance equations was discussed to estimate the performance of the perstractive enzyme reactor system. The calculation values using the model and the experimental data showed good agreement with the concentration changes of the substances in the system.

Preparation of lipase-surfactant complex for the catalysis of triglyceride hydrolysis in heterogeneous reaction systems

To facilitate lipase (E.C.3.1.1.3) catalyzed hydrolysis reaction of triglycerides in heterogeneous reaction systems, lipase-surfactant complex (LSC) was prepared by mixing an aqueous solution of lipase and surfactant in ethanol. The LSC prepared from lipase MF-30 derived from Pseudomonas sp. and Nonion SP-60R (sorbitan mono-stearate) showed a high LSC yield and activity recovery. About 30% of the protein in the crude lipase was recovered in the LSC precipitate, and further addition of surfactant to the supernatant was ineffective. Our preparation method for the selective separation of the catalytic active protein from a crude enzyme was suggested.