Showing papers by "Akio Sugihara published in 1994"
TL;DR: Geotrichum candidum lipase was found to be suitable for their concentration in glycerides as discussed by the authors, and was used to extract polyunsaturated fatty acids in good yield.
Abstract: Three lipases, isolated previously in our laboratory, each with different fatty acid and positional specificities, and a known lipase fromCandida cylindracea were screened for concentrating docosahexaenoic (DHA) and eicosapentaenoic (EPA) acids in glycerides.Geotrichum candidum lipase was found to be suitable for their concentration in glycerides. Tuna oil was treated at 30°C with this lipase for 16 h, and 33.5% hydrolysis resulted in the production of glycerides containing 48.7% of DHA and EPA. The hydrolysis was not increased despite adding further lipase, so the glycerides were extracted, and the reaction was repeated. The second hydrolysis produced glycerides containing 57.5% of DHA and EPA in a 54.5% yield, with recovery of 81.5% of initial DHA and EPA. Of the total glycerides, 85.5% were triglycerides. These results showed thatG. candidum lipase was effective in producing glycerides that contained a high concentration of polyunsaturated fatty acids in good yield.
115 citations
TL;DR: A 1.3-kbp lipase cDNA was isolated from the cDNA library by colony hybridization with an oligonucleotide probe corresponding to the N-terminal amino acid sequence, and it was suggested that the catalytic triad was composed of Ser144, Asp198, and His256.
Abstract: Fusarium heterosporum produces a solvent-tolerant lipase. A 1.3-kbp lipase cDNA was isolated from the cDNA library by colony hybridization with an oligonucleotide probe corresponding to the N-terminal amino acid sequence. Nucleotide sequence analysis showed an open reading frame of 999 bp, and the deduced amino acid sequence contained the N-terminal sequence determined by Edman degradation and the consensus pentapeptide (-Gly-X-Ser-X-Gly-), which is conserved in lipase, esterase, and serine protease. The mature lipase consisted of 301 amino acid residues with a molecular mass of 32.7 kDa, preceded by the putative signal peptide or preprosequence. The enzyme was homologous to lipases from Humicola lanuginosa (39% homology), Rhizomucor miehei (32%), Rhizopus delemar (32%), and Rhizopus niveus (32%), and to mono- and diacylglycerol lipase from Penicillium camembertii (38%). Comparison of this lipase with these homologous enzymes suggested that the catalytic triad was composed of Ser144, Asp198, and His256, and that the oxyanion hole was formed with Ser 82.
52 citations
TL;DR: Geotrichum candidum ATCC34614 produces a major and three minor forms of lipase with similar molecular masses, but different positional and fatty acid specificities; the major and one of the minor forms were confirmed to cleave both the inside and outside ester bonds of triolein indiscriminately.
Abstract: Geotrichum candidum ATCC34614 produces a major (I) and three minor (II, III and IV) forms of lipase with similar molecular masses, but different positional and fatty acid specificities. The major and one of the minor (II) forms were confirmed to cleave both the inside and outside ester bonds of triolein indiscriminately. The non-positional specificity was also retained on 1,3-dipalmitoyl-2-oleoyl-glycerol (POP) hydrolysis. In contrast, the remaining two forms (III and IV) showed unusual positional specificity; they cleaved the inside (2-position) ester bond of triolein at nearly twice the rate of cleavage at the 1(3)-position. The preference for the inside ester bond was increased upon POP hydrolysis.
34 citations
TL;DR: An intracellular carboxylesterase from Pseudomonas sp.
Abstract: An intracellular carboxylesterase from Pseudomonas sp. was overproduced in E. coli, and purified to homogeneity by a combination of hydrogen bond chromatography, gel filtration, and hydrophobic interaction chromatography. Gel filtration and SDS-PAGE suggested that the purified enzyme consisted of two subunits of molecular mass of 28 kDa. Its isoelectric point was 5.9. The enzyme was thermolabile, and showed its maximum activity at 22 degrees C (pH 7.5). Methyl propionate was hydrolyzed at the highest rate among the fatty acid methyl esters tested. PMSF, DFP, PCMB, and HgCl2 inhibited the enzyme markedly, suggesting that serine and/or cysteine is in or near the active site.
14 citations
TL;DR: Moreau et al. as discussed by the authors described the gene cloning, structures, and production mechanisms of microbial lipases and their production by recombinant microbes and extracted the total RNA was extracted with 4 M guanidine thiocyanate solution from log-phase cells showing the maximum rate of specific lipase production.
Abstract: In this chapter, the authors describe the gene cloning, structures, and production mechanisms of microbial lipases and their production by recombinant microbes. The total RNA was extracted with 4 M guanidine thiocyanate solution from log-phase cells showing the maximum rate of specific lipase production. The primary structures of bacterial lipases were deduced from the nucleotide sequences of the cloned genes. Moreau et al. carried out the following chemical modification to investigate the role of serine in the common sequence: Ser-152 of porcine pancreatic lipase in the common sequence was specifically modified with diethyl p-nitrophenyl phosphate. Iizumi et al. attempted the lipase overproduction by a self-cloning system of KWI-56. Rhizomucor miehei lipase is synthesized as a precursor, with a signal sequence, a propeptide, and a mature enzyme. The analysis of the NH2-terminal amino acid sequence indicated that the precursor containing the propeptide was processed in and secreted from a recombinant A.
7 citations
Patent•
22 Feb 1994
TL;DR: In this article, a new DNA fragment was obtained by preparing a cDNA library of Fusarium heterosporum 100 (FERM P-12810) coding an amino acid sequence of the formula, screening this library by a colony hybridization method, etc., selecting a recombinant plasmid containing a gene coding a lipase having high resistance to organic solvents, and forming the fragment into a small fragment.
Abstract: PURPOSE:To obtain a new DNA fragment useful for obtaining a protein having a lipase activity having resistance to organic solvents in high productivity and high purity. CONSTITUTION:This DNA fragment has a generic information of a protein having a lipase activity being high in resistance to organic solvents. The fragment is obtained by preparing e.g. cDNA library of Fusarium heterosporum 100 (FERM P-12810) coding an amino acid sequence of the formula, screening this library by a colony hybridization method, etc., selecting a recombinant plasmid containing a gene coding a lipase having high resistance to organic solvents and forming the plasmid into a small fragment.
3 citations
Patent•
25 Jan 1994
TL;DR: In this article, a microorganism in Geotrichum is used to produce lipase from fish oil containing high unsaturated fatty acids and free acids and glycerol are removed from the reaction mixture.
Abstract: PURPOSE: To concentrate the glyceride of the invention containing an abundant amount of EPA and DHA by hydrolyzing lipid containing high unsaturated fatty acids and removing the free fatty acids and glycerol as hydrolysates. CONSTITUTION: Lipid such as fish oil containing high unsaturated fatty acids is hydrolyzed with lipase which is produced by a microorganism in Geotrichum, free acids and glycerol are removed from the reaction mixture to concentrate the high unsaturated fatty acids in the glycerides. High unsaturated fatty acid glycerides concentrated more efficiently than in the case where the lipase from the yeast in Candida is used. COPYRIGHT: (C)1995,JPO
1 citations