Photo-crosslinkable Resin Research Articles

Recent Advances in the use of Immobilized Lipases Directed Toward the Asymmetric Syntheses of Complex Molecules

Water-insoluble compounds can be substrates for enzymatic reactions when lipases are immobilized properly and suitable organic solvents are used. In this review, three type of lipase immobilization method and their application to the asymmetric syntheses of complex molecules are described. Lipases immobilized with Celite or synthetic prepolymers such as urethane prepolymer and photo-crosslinkable resin prepolymer have been applied for the kinetic resolution of many kinds of water-insoluble substrate.Phospholipid-lipase aggregates with ether linkages are novel and have been found to function effectively as immobilized lipases in asymmetric hydrolysis or esterification reactions in water-saturated organic solvent. The phospholipid-lipase aggregates are considered to have a stacked bilayer based on X-ray diffraction analysis structure of the lipid in the crystalline phase.

Immobilization of trichloroethylene-degrading bacterium, Methylocystis sp. strain M in different matrices

The trichloroethylene (TCE) degradation activities of a TCE-degrading methanotroph, Methylocystis sp. strain M, which was immobilized in different matrices have been investigated to identify a suitable matrix. Cells immobilized in Ca-alginate, κ-carrageenan, and agarose showed higher or almost the same degradation activity in comparison with that of free cells, while low activity was observed in the cells immobilized in photo-crosslinkable resin, polyurethane, and polyelectrolyte complex. In repeated use, only the agarose-immobilized cells were not damaged and retained about 40% of the initial TCE degradation activity among the cells immobilized in the three agents. The fundamental degradation characteristics of the immobilized cells were also investigated using agarose-immobilized cells. The maximum degradation rate ( V max) of TCE was 3.1 μg of TCE/mg of dry cells·h, while the saturation constant ( K s) was 56.5 μM. The optimum pH was found to be 7.0 and the optimum temperature 35°C.

Immobilization of Thiobacillus ferrooxidans using various polymers as matrix.

The cells of acidophilic iron-oxidizing bacteria (Thiobacillus ferrooxidans) were immobilized using five kinds of polymers (photo-crosslinkable resin, agar, calcium alginate, k-carrageenan, and gelrite) as gel matrix, and the characteristics of cell immobilization were revealed. Among these gels, gelrite showed promise for application in iron oxidation under strongly acidic conditions. In continuous cultures of iron-oxidizing bacteria immobilized in gelrite beads, a steady state of strong iron oxidation was maintained for a month. The thick layer of iron-oxide precipitates was formed around reddish-brown "activated beads" that harbored a large number of iron-oxidizing bacteria and were the sources of cells living in the liquid phase.

Immobilization of animal cells using photo-crosslinkable resin

A photo-crosslinkable resin, BIX12, was selected from among various photo-crosslinkable resins for the immobilization of animal cells. BIX12 had no cytotoxic effect on the growth of hybridoma cells and the production of monoclonal antibody, although other photo-crosslinkable resins had significant inhibitory effects. Using BIX12-alginate hybrid gel particles, hybridoma cells could grow in the resins and produce monoclonal antibody. For the continuous production of monoclonal antibody, perfusion culture using a fluidized-bed bioreactor with direct air bubbling was carried out. By this cultivation, monoclonal antibody could be produced stably for more than 50 d. A high viable cell density of more than 10 7 cells/ml-gel was attained, and the antibody productivity was improved 8.5-fold compared with conventional suspension culture using a spinner flask. Anchorage-dependent cells were also immobilized in the resin particles by three immobilization procedures. Among these procedures, porous BIX12 formed by adding gelatin powder provided good support strength and allowed the cells to grow on the surface inside of the support.

Characteristics of Zymomonas mobilis immobilized by photo-crosslinkable resin in ethanol fermentation

Zymomonas mobilis, an ethanol-producing bacterium, was immobilized in hydrophilic photo-crosslinked resin gels to form a biocatalyst. The molecular structure of the photo-crosslinkable resin could be modulated so as to minimize a disadvantage of this bacterium—poor-tolerance to salts in molasses. Characteristics of Z. mobilis immobilized by photo-crosslinkable resin gel, such as fermentability, cell growth in gel, the potential of gel materials, diffusion of materials, and salt distribution are discussed. ENTG-3800 photo-crosslinkable resin was selected as the most suitable entrapping material for Z. mobilis, especially in using molasses.

Continuous ethanol fermentation in molasses medium using Zymomonas mobilis immobilized in photo-crosslinkable resin gels

Zymomonas mobilis B-69 147, an ethanol-producing bacterium, was immobilized in photo-crosslinkable resin gels to form a biocatalyst system. Continuous ethanol fermentation with this immobilized Zymomonas was carried out in molasses and compared to that with immobilized yeast. As a result of operating this process for two weeks, a productivity of 60 g/ l·h based on immobilized gel was obtained with improvement in the poor tolerance to salts of Zymomonas. The productivity of immobilized Z. mobilis was superior to that of immobilized yeast.

Kinetic properties of cassava ?-glucosidase immobilized in photo-crosslinkable resins

Photo-crosslinkable resins (ENT-1000, ENT-2000 and ENT-4000) were used. Immobilized β-glucosidase showed lower substrate affinity, better thermal stability, optimum pH range shifted towards the alkaline region and higher temperature optimum. β-Glucosidase entrapped in ENT-4000 exhibited properties closest to those of the free enzyme.

光硬化性樹脂プレポリマーを用いる脱窒菌の固定化に関する研究

The denitrifiers isolated from the activated sludge of night soil treatment plant were immobilized using a photo-crosslinkable resin prepolymer.Denitrifying activity was strongly inhibited during the first period but the activity was increased in the medium containing nitrate and recovered after 1 day. Acetate was an effective hydrogen source and soap was also effective. Methanol could not serve as a hydrogen source. The optimum pH for denitrification by the immobilized bacteria was 7.0 for nitrite and 7.5 for nitrate, and the optimum temperature ranged 30°C to 40°C. The lowest CH3COONa/NOx-N ratio nessary for getting the highest activity was 4 for nitrite (BOD/NO2-N=2) and 5 for nitrate (BOD/NO3-N=2.5).Photo-crosslinkable resin prepolymer gels exhibited stable denitrification activity to the secondarily treated sewage contained 70mg/l of soap.

光硬化性樹脂による生体触媒の固定

光硬化性樹脂による生体触媒の固定

Conversion of d-xylose into xylitol by immobilized cells of Candida pelliculosa and Methanobacterium sp. HU

The enzymatic conversion of d-xylose into xylitol by the immobilized cells of Candida pelliculosa (NADP + dependent xylose reductase) coupled with the immobilized cells of Methanobacterium sp. HU (hydrogenase and F 120-NADP + oxidoreductase) was done using hydrogen as an electron donor of NADP +. Benzene treatment of the co-immobilized cells with a photo-crosslinkable resin prepolymer, ENT 4000, increased the permeability of NADP (H) into the cells. Besides, treatments with glutaraldehyde and hexamethylenediamine to the immubilized cells were done to enhance the stability of immobilized-cell activity. Thus, the continuous production of xylitol in a column reactor packed with the co-immobilized cells could operate stably for 2 weeks.

Formula omitted] methyl ester production by immobilized Nocardia opaca: Thermolysin under hydrogen high pressure in a water-organic solvent system

N-( Benzyloxycarbonyl)- l-aspartyl- l-phenylalanine methyl ester (Z-APM) has been produced from phenylpyruvate methyl ester (PPyOMe) and N-(benzyloxycarbonyl)-aspartic acid (Z-Asp) under hydrogen high pressure in a water-ethyl acetate system. Z-APM was produced in a two step reaction using immobilized Nocardia opaca and thermolysin. Phenylalanine methyl ester (PheOMe) was produced from PPyOMe, NH 4Cl and H 2 by immobilized N. opaca. The PheOMe production rate increased linearly with increasing hydrogen pressure. The PheOMe production rate was 0.41 μmol/min/g dry cells under 100 atm of hydrogen. Z-APM was produced from PheOMe and Z-Asp by immobilized thermolysin. The highest activity (22.2 μmol/min/g thermolysin) was obtained by coupling N. opaca immobilized in calcium alginate and thermolysin immobilized in photo-crosslinkable resin prepolymer ENTP-4000. When repeated use was carried out without reactivation of the immobilized N. opaca, Z-APM production was not observed after triple use. Therefore, immobilized N. opaca was separated from immobilized thermolysin and reactivated periodically in nutrient broth for 20 h. As a result, the immobilized system retained 72% of the initial activity after being used 5 times for over 100 h.

A study of some photocrosslinkable resins using i.r. spectroscopy

The kinetics of photoinduced crosslinking of a number of polymers containing chalcone units have been studied. The concentration of the chalcone groups and the nature of the backbone were varied in a controlled manner. The rates of photocrosslinking cannot be analysed by the simple application of solution kinetics. Factors which affect the rate of cure (e.g. concentration of chalcone units, thickness of films and temperature) were examined and reasons are given for the failure of the simple kinetic model.

Oxygen diffusivity of synthetic gels derived from prepolymers

The oxygen-diffusivity (D m ) of 16 different gels formed with synthetic prepolymers (photo-crosslinkable resins, urethane resins and photosensitive resins), and that of calcium alginate (for comparison) was determined, using an oxygen electrode covered by the gel membranes with stepwise enzymatic removal of O2 from the buffer solution. The water content of the gels was found to be decisive for the O2-diffusivity of the gels: gels with the highest water content showed also the highest D m . From these findings, the suitability of different polymeric gels for substrate conversion and biosensor systems could be predicted.

Hydrolysis of triglycerides by immobilized thermostable lipase from Humicola lanuginosa.

Thermostable lipase from Humicola lanuginosa No. 3 was immobilized by entrapment in photo-crosslinkable resin prepolymers, urethane prepolymers or Ca-alginate. Adsorption to different types of resins or gels, and covalent binding methods were also employed. From all the methods examined, adsorption on Amberlite XAD-7 followed by crosslinking with glutaraldehyde was superior, as to several points. Firstly, the adsorption efficiency for lipase was relatively high, being about 70%. Secondly, the optimal temperature was about 10°C higher than that for free lipase. The resin immobilized lipase was not affected by pH changes, and finally it also exhibited excellent stability as to repeated batch hydrolysis of triglycerides, both in a shaking flask system and in a recycling column reactor for more than 2 and 3 weeks, respectively.

Oriented immobilization of bacteriorhodopsin in synthetic polymer membranes by use of electric static field

Application of an electric field during the process of resin formations induced oriented immobilization of bacteriorhodopsin (BRp) in the form of purple membrane, isolated from Halobacterium halobium, within a membrane prepared from a photo-crosslinkable resin prepolymer through entrapment. The observed photoelectric current upon illumination of the membrane was highly enhanced by this oriented immobilization.

Application of entrapped growing yeast cells to peptide secretion system

Efficient production of α-mating factor with immobilized growing Saccharomyces cerevisiae cells was examined in order to establish a secretion system for useful peptides using the α-mating factor secretion system. The free yeast cells produced α-mating factor into the cultured medium, but α-mating factor was gradually degraded in situ. Based on the criteria of α-mating factor productivity, cell growth in gels, cell leakage from gels, and mechanical strength of gels, a mixture of a hydrophilic neutral photo-crosslinkable resin prepolymer (ENT-2000) and an anionic prepolymer (ENTA-2) (8:2 by weight) was employed as gel material, among various kinds of natural and synthetic supports, to entrap the yeast cells. Anionic photo-crosslinkable resins selectively entrapped α-mating factor-degrading enzymes which were produced by the cells and degraded α-mating factor formed. The immobilized growing yeast cells could be used successfully for continuous production of α-mating factor in a packed column reactor for at least 30 days.

Novel Leu-Lys-specific peptidase (leulysin) produced by gel-entrapped yeast cells

Saccharomyces cerevisiae cells entrapped with a neutral hydrophilic photo-crosslinkable resin prepolymers specifically excreted into a cultured medium a new type of a peptidase, which cleaved Leu-Lys bond of α-mating factor. The enzyme was purified by membrane filtration followed by ion exchange chromatography and gel filtration chromatography. The purified enzyme showed a strict substrate specificity on internal Leu-Lys bond. Leu-Lys bond near terminus of a molecule and Leu-X and X-Lys bonds examined so far were not hydrolyzed by the enzyme.

Immobilized bromoperoxidase of Corallina pilulifera as a multifunctional halogenating biocatalyst

The partially purified bromoperoxidase of Corallina pilulifera (Corallinaceae, Rhodophyta) was immobilized on the following matrices: Cellulofine (covalent binding), DEAE-Cellulofine (ionic binding), or alkylsilane treated Controlled-Pore Glass (physical adsorption), and entrapped in the soft gels using photo-crosslinkable resin prepolymer (ENT-2000), polyurethane prepolymer (PU-6), or κ-carrageenan. These different forms of immobilized bromoperoxidase were tested for the bromination reactions of monochlorodimedone and uracil. The immobilization techniques using DEAE-Cellulofine and ENT-2000 were found to be suitable for the bromoperoxidase reaction. The immobilized enzyme on DEAE-Cellulofine showed the highest activity and a half-life of 45 days when it was used for the conversion of uracil to 5-bromouracil.

Control of enzyme-distribution in enzyme-membrane by electric field.

By dint of electric field given during the polymerization process, alkaline phosphatase from porcine intestine was immobilized asymmetrically in photocrosslinkable resin prepolymer membrane through entrapment. Catalytic activities of two sides of the membrane and the transport-catalysis behavior were measured.It was found that the conversion of the substrate, present in one chamber, to the product and the transport of the latter to the other side of the membrane were more efficiently carried out when the enzyme distribution in the matrix was inclined to the side of the membrane facing to the substrate-free chamber.The same procedure was successfully applied to coimmobilize alcohol dehydrogenase from equine liver and formaldehyde dehydrogenase from yeast in one membrane with different (complementary) distributions of the enzymes with respect to the membrane thickness.

Sequential conversion of cortexolone to prednisolone by immobilized mycelia of Curvularia lunata and immobilized cells of Arthrobacter simplex

Two-step bioconversion of cortexolone (Reichstein's Compound S) to its Δ1-dehaydro-11β-hydroxy derivative, prednisolone, was successfully performed by the combined use of immobilized Curvularia lunata mycelia and immobilized Arthrobacter simplex cells. Immobilized living mycelia of C. lunata having a high 11β-hydroxylation activity were prepared by in situ germination of spores entrapped in photo-crosslinked resin gels of a suitable net-work structure. Acetone-dried cells of A. simplex having an induced steroid Δ1-dehydrogenase activity were also entrapped with photo-crosslinkable resin prepolymers and used for Δ- dehydrogenation of hydrocortisone to prednisolone. For the production of prednisolone from cortexolone, the combination of sequential steps, 11β-hydroxylation and subsequent Δ1-dehydrogenation, was found to be suitable. Each immobilized microbial cell system was stable and could be used for the sequential reactions repeatedly (operational period, 25 days).