Acetone-dried Cells Research Articles

Stereoselective reduction of ethyl 4-chloro-3-oxobutanoate by fungi.

The enantioselectivity of ECAA to ECHB by eight fungi of four genus was evaluated. All strains showed (S)-selectivity, and Cylindrocarpon sclerotigenum IFO 31855 gave the highest yield and good optical purity (e.e.; >99%). Cell-free extract and acetone-dried cells of C. sclerotigenum IFO 31855 reduced ECAA to (S)-ECHB in the presence of NADPH (e.e.; >99%) and the e.e. was not decreased by heat treatment of the cell-free extract or the acetone-dried cells. The active fractions shown by two peaks on a DEAE-Toyopearl 650 M column gave preferentially (S)-ECHB (e.e.; >99%).

One-step synthesis of n-octyl β-D-xylotrioside, xylobioside and xyloside from xylan and n-octanol using acetone powder of Aureobasidium pullulans in supercritical fluids

n-Octyl β-d-xylotrioside, xylobioside and xyloside, which are effective next-generation surfactants, can be prepared by the one-step reaction of xylan and n-octanol using the acetone powder (acetone-dried cells) of Aureobasidium pullulansas the enzyme source of xylanase in supercritical carbon dioxide (CO2) and fluoroform (CHF3) fluids. Yields of the n-octyl glycosides were significantly increased using supercritical CHF3, and the total yields of the n-octyl β-d-glycosides exceeded 250 mg g−1 xylan. n-Octyl β-d-xylotrioside was produced only by using supercritical fluids.

Effect of Additives on Transesterification Activity of Rhizopus chinensis Lipase.

The transesterification activity of powder lipase prepared from the purified lipase of Rhizopus chinensis cells by freeze-drying was quite low compared with that of acetone-dried cells. Additives which could enhance the transesterification activity of the freeze-dried powder lipase were screened. The freeze-dried lipases prepared with certain fatty acid methylesters or certain types of surfactants exhibited high transesterification activity. It was shown that not only the solubility of the freeze-dried lipase in n-hexane but also the organic solvent-stability was enhanced when methyl stearate was added to the lipase solution at the freeze-drying step.

Effect of additives on transesterification activity of Rhizopus chinensis lipase

The transesterification activity of powder lipase prepared from the purified lipase of Rhizopus chinensis cells by freeze-drying was quite low compared with that of acetone-dried cells. Additives which could enhance the transesterification activity of the freeze-dried powder lipase were screened. The freeze-dried lipases prepared with certain fatty acid methylesters or certain types of surfactants exhibited high transesterification activity. It was shown that not only the solubility of the freeze-dried lipase in n-hexane but also the organic solvent-stability was enhanced when methyl stearate was added to the lipase solution at the freeze-drying step.

Biocatalytic One-step Synthesis of n-Octyl β-d-Xylotrioside and Xylobioside from Xylan and n-Octanol in Supercritical Carbon Dioxide

Abstract n-Octyl β-d-xylotrioside and xylobioside, which are effective next generation surfactants, can be prepared by the one-step reaction of xylan and n-octanol using acetone powder (acetone-dried cells) of Aureobasidium pullulans KK415 (ATCC 201145) as the enzyme source of xylanase in supercritical carbon dioxide (scCO2). Yields of the octyl glycosides were significantly increased using scCO2. Furthermore, without scCO2, n-octyl xylotrioside was hardly produced.

Synthesis of optically active ethyl 4-chloro-3-hydroxybutanoate by microbial reduction.

A total of 400 yeast strains were examined for the ability to reduce ethyl 4-chloroacetoacetate (COBE) to ethyl 4-chloro-3-hydroxybutyrate (CHBE) by using acetone-dried cells in the presence of a coenzyme-recycling system in water/n-butyl acetate. We discovered some yeast strains that reduced COBE to (S)-CHBE. Heating of acetone-dried cells of the selected yeast strains increased the optical purity of the product. There may be several enzymes that can reduce COBE stereoselectively in the same yeast cells. The cultured broth of Candida magnoliae accumulated 90 g/l (S)-CHBE (96.6% enantiomeric excess, e.e.) in the presence of glucose, NADP and glucose dehydrogenase in n-butyl acetate. When these cells were heated, the stereoselectivity of the reduction increased to 99% e.e. (S)-CHBE is one of the useful chiral building blocks applicable to the synthesis of some pharmaceuticals. We expect that the cheap and industrial production of this important chiral compound will follow the discovery of this yeast strain.

One-pot synthesis of alkyl β-D-xylobioside from xylan and alcohol by acetone powder of Aureobasidium pullulans

Alkyl β-D-xylobioside was prepared by the one-pot reaction of xylan and a fatty alcohol using acetone powder (acetone-dried cells) of Aureobasidium pullulans KK415 (ATCC 201145). Both the yields and reaction speed were significantly increased using the acetone powder compared to those of the cell-free culture filtrate. Using 0.066% acetone powder, 2-ethylhexyl, 1-octyl and 2-octyl b-D-xylobiosides were produced in the yields of 118, 80 and 74 mg per g xylan, respectively, when alcohol and xylan were incubated at 65°C for 16 h. On the other hand, C8 alkyl xylo-bioside was hardly produced by the resting cells of A. pullulans.

Removal and bioconversion of phenol in wastewater by a thermostable β-tyrosinase

This study explores an enzymatic method for removing phenol from the wastewater created during the manufacture of phenolic resin. The enzyme used was a thermostable β-tyrosinase catalyzing the synthesis of l-tyrosine from phenol, pyruvate, and ammonia. As the reaction proceeds, l-tyrosine precipitates as insoluble aggregates because l-tyrosine is barely soluble in water. The enzymatic removal of phenol was effective at pH values ranging from 6.5–9.0 and temperatures below 70°C. The optimal concentration of each substrate was determined as 60 m m phenol, 0.1 m pyruvate, and 0.4 m ammonia. When the enzyme was used in an intact cell or acetone-dried cell state instead of the cell-free extract, the optimal concentration of phenol was increased up to 120 m m. By treating wastewater containing 100 m m phenol with acetone-dried cells at 37°C, we could reduce the concentration of phenol to 8 m m within 24 h.

Intracellular lipase production by Rhizopus chinensis using biomass support particles in a circulating bed fermentor

To utilize Rhizopus chinensis cells directly as a catalyst for industrial interesterification of oils and fats, the mass production of acetone-dried cells immobilized in Biomass Support Particles (BSPs) which show high intracellular lipase activity was examined using a circulating bed fermentor (CBF). First, the effects of aeration rate and number of BSPs were investigated in batch culture. The results revealed that the aeration rate had a significant influence on the formation of intracellular lipase. Therefore, a comparatively low aeration rate (4 vvm) capable of attaining good circulation of BSPs in the CBF was adequate for the lipase production. The effects of meat extract (main component of medium) feeding on intracellular lipase production was also examined by constant-feed rate fed-batch cultures. The results indicated that the specific feed rate of meat extract q s controlled in the range of 0.04–0.08 (g-meat extract)(g-cell) −1(h) −1 might enhance intracellular lipase activity, whereas the cellular growth in BSPs was independent of the meat extract feed rate. Therefore, the fed-batch culture in which the specific feed rate of meat extract q s was controlled at 0.05 by varying the meat extract feed rate stepwise using a microcomputer was carried out, and the intracellular lipase activity of immobilized cells could be enhanced 1.3–1.5 times that obtained in batch cultivation. The specific feed rate of meat extract q s is an effective index for the production of intracellular lipase of Rh. chinensis.

Amino acid racemase with broad substrate specificity, its properties and use in phenylalanine racemization

Broad substrate specificity amino acid racemase (EC 5.1.1.10) was purified from a crude extract of Pseudomonas putida SCRC-744 to near homogeneity. The enzyme has an isoelectric point of 7.6 and a molecular weight of 62,000–65,000. The enzyme showed a broad substrate specificity toward amino acids, utilizing d-glutamine as the best substrate. d-Phenylalanine acted as a substrate to 1% the velocity for d-glutamine. Maximal reaction velocities were observed at 50°–60°C and around pH 8. The apparent Km values for d-glutamine and d-phenylalanine were 7.8 mM and 25.7 mM, respectively. Both enantiomers of phenylalanine were efficiently racemized by acetone-dried cells of P. putida SCRC-744.

Culture conditions for intracellular lipase production by Rhizopus chinensis and its immobilization within biomass support particles

Acetone-dried cells of Rhizopus chinensis (with a 1,3-positional specificity lipase) were investigated for the interestierification reaction of olive oil and methyl stearate. First, the culture conditions for intracellular lipase production were examined, and then the activities of dried cells obtained from immobilization in Biomass Support Particles (BSPs) were compared with those of freely suspended cells. It was clear from cultivation of freely suspended cells that intracellular lipase activity for the interesterification reaction was enhanced sifnificantly by the presence of oleic acid, oil, and tea oil, but that the presence of glucose reduced the activity. The specific activity of dried cells within BSPs increased 7-fold compared with that obrained from freely suspended cells. The process presented here, using immobilization within BSPs, can provide cells directly as a catalyst with high activity, where cells become immobilized simply during batch operation, and no special preparation of cells is necessary. Therefore, the reaction system using dried cells immobilized within BSPs is a promising interesterifcation process for industrial applications.

Interesterification of fats and oils by immobilized fungus at constant water concentration

The kinetics of enzymatic interesterification of oils and fats, using acetone-dried cells of Rhizopus chinensis immobilized on biomass support particles as a lipase catalyst, were investigated in batch operations at several constant water concentrations. Even under microaqueous ( i.e., low-water-content) conditions, not only interesterification but also hydrolysis occured, and the water content in the reaction system decreased. The reaction rates of interesterification and hydrolysis at constant water concentrations were determined. For the reactions between olive oil and methyl stearate at several water concentrations, the parameters involved in the reaction model were determined by a trial-and-error method so as to make the calculated results correlate with the experimental data. The relationship between the parameters obtained and water concentration were examined. The rate constants involved in the reaction model of both interesterification and hydrolysis increased or decreased monotonically with the increasing water content, while the apparent activity of the lipase catalyst for interesterification had a maximum value at a water concentration of about 50 ppm. This suggests that when the water content is excessive the hydrolysis activity of lipase is accelerated more than its interesterification activity, and that when the water content is too little lipase activity can not be activated for either hydrolysis or interesterification.

Polyagglutinating and non-typable strains of Pseudomonas aeruginosa in cystic fibrosis

Serologically polyagglutinating (PA) and non-typable (NT) strains of Pseudomonas aeruginosa are frequently isolated from cystic fibrosis (CF) patients, but are uncommon in other patients. From serologically typical parent strains, we isolated two variants (one PA, the other NT) which differed from the parent in bacteriophage susceptibility or in sensitivity to the bactericidal action of normal human serum. The PA and NT variants (strains 7/1 and 18S respectively) reacted with antiserum to the parent strains 7 and 18R but did not absorb homologous specific O antibody from antiserum to the parent strains. In contrast the parent strains absorbed anti-PA and anti-NT antibodies from antisera to the variant strains. The yield of lipopolysaccharide (LPS) from acetone-dried cells of the parent strain 7 was similar to that of the PA derivative; but the NT strain 18S yielded only half the LPS of its parent strain. LPS of the variant 7/1 gave a banding profile by SDS-PAGE similar to that of the parent LPS 7, but lacked high-molecular-weight components. LPS of the variant 18S appeared to be grossly different in profile from LPS 18R. Of 533 isolates of P. aeruginosa that were tested with O antisera and with antisera to the two variants, 15% were O-typable and 22% were O-non-typable; 26% reacted with anti-PA serum alone, 10% with anti-NT serum alone, and 27% were agglutinated by both sera. There was a statistically significant correlation between serum sensitivity of CF isolates and their reaction with the PA or NT antisera.

Product inhibition of the enzymatic hydrolysis of lactose

Kinetic experiments have been conducted with acetone-dried cells of Kluyveromyces fragilis to study product inhibition of the enzymatic hydrolysis of lactose. Both hydrolytic products, d-glucose and d-galactose, showed efficient inhibition effect on enzyme activity. The fact that d-glucose and d-galactose are mutually exclusive for the inhibition was verified by Dixon plots. The kinetic constants were also estimated using the experimental data. The rate equation was derived based on a multiple inhibition model of competitive inhibition of d-galactose and non-competitive inhibition of d-glucose. The good agreement between experiment and prediction indicated the validity of the established model .

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).

Synthesis of Fatty Acid Esters byCorynebacteriumsp. S-401

Resting cells and acetone-dried cells of Corynebacterium sp. S-401 catalyzed the fatty acid ester synthesis of various alcohols and fatty acids. These reactions were carried out in phosphate buffer and/or organic solvents. In some cases synthetic reactions of esters in nonpolar solvents, such as n-hexane and benzene, gave better results compared with those obtained in phosphate buffer.

Synthesis of fatty acid esters by Corynebacterium sp. S-401.

Resting cells and acetone-dried cells of Corynebacterium sp. S-401 catalyzed the fatty acid ester synthesis of various alcohols and fatty acids. These reactions were carried out in phosphate buffer and/or organic solvents. In some cases synthetic reactions of esters in nonpolar solvents, such as n-hexane and benzene, gave better results compared with those obtained in phosphate buffer.

Application of an immobilized Escherichia coli cell tube in analysis of L-threonine.

Acetone-dried cells of Escherichia coli with an enhanced level of biodegradative L-threonine deaminase (EC 4.2.1.16) were easily and tightly immobilized to the inner surface of glass tubes by use of an emulsion-type of photo-crosslinkable resin prepolymer. A continuous flow system equipped with the microbial cell tube thus prepared was proved to be applicable for assay of L-threonine or L-serine in fermentation broth with rapidity (each assay, less than 9min), excellent substrate specificity (specific for L-threonine and L-serine) and high stability over repeated assays (at least 60 assays).

Assay of cephalosporin C with an immobilized microbial cell tube system.

Acetone-dried cells of Citrobacter freundii having cephalosporinase were easily and tightly immobilized to the inner surface of glass tubes by use of an emulsion-type of photo-crosslinkable resin prepolymer. The microbial cell tubes thus prepared were used in a continuous flow system to measure cephalosporin C by potentiometry. A good response was observed in the concentration range from 0.1 mM to 4mM of cephalosporin C. This system was active on cephalosporin C, deacetylcephalosporin C and deacetoxycephalosporin C, indicating the possibility for assaying the total amount of cephalosporins in fermentation broth as cephalosporin C values. The enzyme in the microbial cell tubes was stable after repeated use in at least 200 assays.

Application of urethane prepolymers to immobilization of biocatalysts: .DELTA.1-dehydrogenation of hydrocortisone by Arthrobacter simplex cells entrapped with urethane prepolymers.

Acetone-dried cells of Arthrobacter simplex having appreciable steroid Δ1-dehydrogenase activity were immobilized by mixing the cell suspension with water-miscible urethane prepolymers synthesized from toluene diisocyanate and polyether diols. The entrapped cell activity in the transformation of hydrocortisone to prednisolone was affected by the properties of urethane prepolymers, such as the isocyanate group content in prepolymers, the molecular weight of polyether diols and the ethylene oxide content in diols. The addition of 10% of organic solvents, such as methanol and glycols, to the aqueous reaction mixture enhanced the solubility of the substrate greatly and the reaction rate of the immobilized cells. The activity of immobilized cells remained high even in the system containing 30% of methanol, which drastically inhibited the activity of free cells. The presence of an electron acceptor, phenazine methosulfate or 2,6-dichlorophenolindophenol, significantly stimulated the steroid conversion with entr...