Showing papers by "Akio Sugihara published in 2001"

Enzymatic conversion of waste edible oil to biodiesel fuel in a fixed-bed bioreactor

Abstract: The conversion of waste edible oil to biodiesel fuel in a fixed-bed bioreactor was investigated. Three-step methanolysis of waste oil was conducted using three columns packed with 3 g of immobilized Candida antarctica lipase. A mixture of waste oil and 1/3 molar equivalent of methanol against total fatty acids in the oil was used as substrate for the first-step reaction, and mixtures of the first- and second-step eluates and 1/3 molar equivalent of methanol were used for the second- and third-step reactions, respectively. Ninety percent of waste oil was converted to the corresponding methyl esters (ME) by feeding substrate mixtures into the first, second, and third reactors at flow rates of 6, 6 and 4 mL/h, respectively. We also attempted one-step methanolysis of waste oil. When a mixture of waste oil and 90% ME-containing eluate (1∶3, wt/wt) and an equimolar amount of methanol against total fatty acids in the waste oil was fed into a reactor packed with 3 g of immobilized C. antarctica lipase at a flow rate of 4 mL/h, the ME content in the eluate reached 90%. The immobilized biocatalyst could be used for 100 d in the two reaction systems without significant decrease in its activity. Waste oil contained 1980 ppm water and 2.5% free fatty acids, but these contaminants had little influence on enzymatic production of biodiesel fuel.

Enzymatic synthesis of high-purity structured lipids with caprylic acid at 1,3-positions and polyunsaturated fatty acid at 2-position

Abstract: We attempted to synthesize high-purity structured triacylglycerols (TAG) with caprylic acid (CA) at the 1,3-positions and a polyunsaturated fatty acid (PUFA) at the 2-position by a two-step enzymatic method. The first step was synthesis of TAG of PUFA (TriP), and the second step was acidolysis of TriP with CA. Candida antarctica lipase was effective for the first reaction. When a reaction medium of PUFA/glycerol (3∶1, mol/mol) and 5% immobilized Candida lipase was mixed for 24 h at 40°C and 15 mm Hg, syntheses of TAG of γ-linolenic, arachidonic, eicosapentaenoic, and docosahexaenoic acids reached 89, 89, 88, and 83%, respectively. In these reactions, the lipase could be used for at least 10 cycles without significant loss of activity. In the second step, the resulting trieicosapentaenoin was acidolyzed at 30°C for 48h with 15 mol parts CA using 7% of immobilized Rhizopus delemar lipase. The CA content in the acylglycerol fraction reached 40 mol%. To increase the content further, the acylglycerols were extracted from the reaction mixture with n-hexane and were allowed to react again with CA under conditions similar to those of the first acidolysis. After three successive acidolysis reactions, the CA content reached 66 mol%. The content of dicapryloyl-eicosapentaenoyl-glycerol reached 86 wt% of acylglycerols, and the ratio of 1,3-dicapryloyl-2-eicosapentaenoyl-glycerol to 1(3),2-dicapryloyl-3(1)-eicosapentaenoyl-glycerol was 98∶2 (w/w). In this reaction, the lipase could be used for at least 20 cycles without significant loss of activity. Repeated acidolysis of the other TriP with CA under similar conditions synthesized 1,3-dicapryloyl-2-γ-linolenoyl-glycerol, 1,3-dicapryloyl-2-arachidonoyl-glycerol, and 1,3-dicapryloyl-2-docosahexaenoyl-glycerol in yields of 58, 87, and 19 wt%, respectively.

Use of thermostable Fusarium heterosporum lipase for production of structured lipid containing oleic and palmitic acids in organic solvent-free system

Abstract: A newly developed 1,3-positionally specific thermostable lipase from Fusarium heterosporum (named R275A lipase) was immobilized on Dowex WBA for the production of structured lipid by acidolysis of tripalmitin (PPP) with oleic acid (OA). The immobilized catalyst was fully activated by pretreatment at 50°C in a PPP/OA mixture containing 2% water. The pretreatment caused concomitant hydrolysis, but the hydrolysis was repressed using a substrate without water in the subsequent reactions. The optimal reaction conditions were determined as follows: A mixture of PPP/OA (1∶2, w/w) and 8% immobilized lipase catalyst was incubated at 50°C for 24 h with shaking at 130 oscillations/min. The acidolysis reached 50% under these conditions, and the contents of triolein, 1,3-dioleoyl-2-palmitoyl-glycerol, 1(3),2-dioleoyl-3(1)-palmitoyl-glycerol, 1(3),2-palmitoyl-3(1)-oleoyl-glycerol, 1,3-dipalmitoyl-2-oleoyl-glycerol, and PPP in the reaction mixture were 8, 36, 4, 28, 1, and 6 mol%, respectively. The stabilities of immobilized R275A lipase catalyst and two immobilized catalysts containing Rhizopus delemar or Rhizomucor miehei lipases were compared under the conditions mentioned above, with the catalysts being transferred to fresh substrate every 24 h. The half-life of the R275A lipase catalyst was 370 d, which was significantly longer than those of Rhizopus and Rhizomucor lipase catalysts.

A Novel α-Amino-Acid Esterase from Bacillus mycoides Capable of Forming Peptides of DD- and DL-Configurations

Abstract: A novel alpha-amino-acid esterase possessing some properties favorable for the synthesis of D-amino acid-containing peptides has been purified from the culture broth of Bacillus mycoides. The enzyme consisted of 4 subunits of 39 kDa, had an isoelectric point of 7.0, and showed its maximum activity at around 47 degrees C and pH 7.6. The enzyme activity was strongly depressed by phenylmethanesulfonyl fluoride, but not by penicillin G or ampicillin, suggesting that the protein is a serine enzyme lacking penicillin-binding ability. The enzyme hydrolyzed a variety of D- and L-amino acid methyl esters with concomitant formation of homooligomers from D-Phe, D-Trp, D-Tyr, and D-Asp(OCH(3)) methyl esters, but it did not act on the D- or L-amino acid amides tested. Incubation of a mixture of Ac-D-Phe-OMe and D-/L-Leu-NH(2) with the enzyme yielded Ac-D-Phe-D-/L-Leu-NH(2) together with Ac-D-Phe-OH, the hydrolysate of the carboxyl component. To its credit, the enzyme failed to hydrolyze casein as well as peptides including diastereomers of diphenylalanine and dialanine, indicating that the enzyme would not cause secondary hydrolysis of once-formed peptides. These observations indicate the potential utility of the newly isolated enzyme for the synthesis of D-amino acid-containing peptides.

New method for producing xanthophyll educt and method for purifying the same

Abstract: PROBLEM TO BE SOLVED: To provide a method for producing a xanthophyll educt from a xanthophyll ester or a composition containing the same, simply and in a good efficiency. SOLUTION: This method for producing the xanthophyll educt is constituted by allowing a lipase to act on a suspension consisting of the xanthophyll ester or the composition containing the same, water and an alcohol for converting the xanthophylls ester to the xanthophyll educt.

Method for concentrating ceramide glycoside

Abstract: (57) [Summary] (Modifications required) [Problem] To provide high-concentration ceramide glycosides from plant materials Provided is a concentration method which is industrially mass-produced. SOLUTION: A step of hydrolyzing a plant material to recover an oil, a step of subjecting the oil to distillation to obtain a distillation residue, a step of adding an organic solvent such as ethanol or acetone to the distillation residue, and separating the solvent, The ceramide glycoside is concentrated by combining a step of adding a second organic solvent and separating the solvent after the step of separating.

Method for purifying crude xanthophyll

Abstract: PROBLEM TO BE SOLVED: To provide a new method for the purification of crude xanthophylls. SOLUTION: Crude xanthophyll obtained by the solvent extraction of algae is purified by treating the xanthophyll with lipase in the presence of water under acidic condition at 4-90 deg.C without adjusting the pH to effect the selective hydrolysis of neutral lipids (mono, di or triglyceride) existing as foreign materials, separating the lipase-treated liquid into oil and water and separating free fatty acids from the obtained oil layer.

Method for producing fatty acid lower alcohol ester

Abstract: PROBLEM TO BE SOLVED: To produce various alcohol esters of unsaturated fatty acids having physiological activity and useful for pharmaceuticals, etc., under mild condition in high yield without causing the problems such as the isomerization risk, the discoloration and the generation of degradation odor. SOLUTION: A fatty acid lower alcohol ester is produced by etherifying a fatty acid with a lower alcohol in a reaction system free from organic solvent except for the fatty acid and the lower alcohol and having a water content of <=10% in the presence of a lipase free from fatty acid selectivity.