Kenzo Yokozeki

Application of immobilized lipase to regio-specific interesterification of triglyceride in organic solvent

SummaryLipase from Rhizopus delemar was immobilized by entrapment with photo-crosslinkable resin prepolymers or urethane prepolymers or by binding to various types of porous silica beads. The immobilized lipase preparations thus obtained were examined for their activity in converting olive oil to an interesterified fat (cacao butter-like fat), whose oleic acid moieties at 1- and 3-positions were replaced with stearic acid moieties, in the reaction solvent n-hexane. Although all of the immobilized preparations exhibited some activity, lipase adsorbed on Celite and then entrapped with a hydrophobic photo-crosslinkable resin prepolymer showed the highest activity, about 75% of that of lipase simply adsorbed onto Celite. Entrapment markedly enhanced the operational stability of lipase.

Polyunsaturated fatty acid saturation by gut lactic acid bacteria affecting host lipid composition

Significance Microorganisms in the gastrointestinal tract interact with their host in many ways. Lipid metabolism by gastrointestinal microbes generates multiple fatty acid species that can affect host health. In the representative gut bacterium Lactobacillus plantarum, we revealed a fatty acid metabolism, saturation metabolism of polyunsaturated fatty acid, that generates hydroxy fatty acids, oxo fatty acids, conjugated fatty acids, and partially saturated trans-fatty acids as intermediates. Furthermore, fatty acid analysis in mice suggests that the fatty acid metabolism by gastrointestinal microbes modifies fatty acid composition of the host. Therefore, functional investigations of lipid metabolisms of gastrointestinal microbes may provide new methods for improving our health by altering lipid metabolism related to the onset of metabolic syndrome. In the representative gut bacterium Lactobacillus plantarum, we identified genes encoding the enzymes involved in a saturation metabolism of polyunsaturated fatty acids and revealed in detail the metabolic pathway that generates hydroxy fatty acids, oxo fatty acids, conjugated fatty acids, and partially saturated trans-fatty acids as intermediates. Furthermore, we observed these intermediates, especially hydroxy fatty acids, in host organs. Levels of hydroxy fatty acids were much higher in specific pathogen-free mice than in germ-free mice, indicating that these fatty acids are generated through polyunsaturated fatty acids metabolism of gastrointestinal microorganisms. These findings suggested that lipid metabolism by gastrointestinal microbes affects the health of the host by modifying fatty acid composition.

Novel Enzymatic Method for the Production of Xylitol from D-Arabitol by Gluconobacter oxydans

Microorganisms capable of producing xylitol from D-arabitol were screened for. Of the 420 strains tested, three bacteria, belonging to the genera Acetobacter and Gluconobacter, produced xylitol from D-arabitol when intact cells were used as the enzyme source. Among them, Gluconobacter oxydans ATCC 621 produced 29.2 g/l xylitol from 52.4 g/l D-arabitol after incubation for 27 h. The production of xylitol was increased by the addition of 5% (v/v) ethanol and 5 g/l D-glucose to the reaction mixture. Under these conditions, 51.4 g/l xylitol was obtained from 52.4 g/l D-arabitol, a yield of 98%, after incubation for 27 h. This conversion consisted of two successive reactions, conversion of D-arabitol to D-xylulose by a membrane-bound D-arabitol dehydrogenase, and conversion of D-xylulose to xylitol by a soluble NAD-dependent xylitol dehydrogenase. Use of disruptants of the membrane-bound alcohol dehydrogenase genes suggested that NADH was generated via NAD-dependent soluble alcohol dehydrogenase.

Purification and Characterization of Novel Transglutaminase from Bacillus subtilis Spores

Transglutaminase activity was detected in suspensions of purified spores prepared from lysozyme-treated sporulating cells of Bacillus subtilis AJ 1307. The enzyme was easily solubilized from the spores upon incubation at pH 10.5 at 37°C. The transglutaminase activity was separated into two fractions upon purification by hydrophobic interaction chromatography (TG1 and TG2). Each enzyme was purified to electrophoretic homogeneity (about 1,000-fold). Both enzymes had the same molecular weight of 29,000 as estimated by SDS-PAGE, had the same N-terminal 30 amino acid sequence, and also showed the same optimal temperature (60°C) and pH (8.2). The purified enzyme catalyzed formation of cross-linked ε-(γ-glutamyl)lysine isopeptides, resulting in the gel-formation of protein solutions such as αs-casein and BSA.

A novel enzymatic method for the production of purine-2′-deoxyribonucleosides

Abstract The microbial production of purine-2′-deoxyribonucleosides from pyrimidine-2′-deoxyribonucleosides and purine bases was examined by the application of nucleoside phosphorylase using Enterobacter aerogenes AJ-11125 as the enzyme source. In this system, 2′-deoxyadenosine (dAR) was efficiently produced from 2′-deoxyuridine (dUR) and adenine. In contrast, 2′-deoxyguanosine (dGR) was scarcely produced from dUR and guanine, because of the low solubility of guanine. Under the conditions using guanosine (GR) with higher solubility than guanine as a guanine source, higher productivity of dGR was obtained, but the maximal molar yield obtained was less than 20%. To improve its productivity, we newly constructed a following enzymatic method via 2,6-diaminopurine-2′-deoxyriboside (dDAPR) as follows: production of dDAPR from dUR and 2,6-diaminopurine (DAP) by E. aerogenes AJ-11125, followed by the conversion of dDAPR to dGR by adenosine deaminase. Through the successive reactions, dGR was efficiently produced with high yield.

A novel l-isoleucine hydroxylating enzyme, l-isoleucine dioxygenase from Bacillus thuringiensis, produces (2S,3R,4S)-4-hydroxyisoleucine

The unique function of 4-hydroxyisoleucine (4-HIL) is to stimulate glucose-induced insulin secretion in a glucose-dependent manner. 4-HIL is distributed only in certain kinds of plants and mushrooms, but the biosynthetic mechanism of 4-HIL has not been elucidated. Moreover, 4-HIL-producing microorganisms have not been reported. l-isoleucine (l-Ile) hydroxylating activity producing 4-HIL was detected in a cell lysate of Bacillus thuringiensis strain 2e2 AKU 0251 obtained from the mid-late exponential phase of growth. Properties of the purified hydroxylase demonstrated that it is a alpha-ketoglutaric acid (alpha-KG) dependent l-Ile dioxygenase (IDO) and requires alpha-KG, ferric ion, and ascorbic acid for its maximum activity. IDO showed high stereoselectivity in l-Ile hydroxylation producing only (2S,3R,4S)-4-HIL. The N-terminal 22 amino acids sequence revealed high homology to a hypothetical protein (GenBank ID: RBTH_06809) in B. thuringiensis serovar israelensis ATCC 35646. The histidine motif, which is conserved in alpha-KG dependent dioxygenases, is found in RBTH_06809.

Metabolic diversity in biohydrogenation of polyunsaturated fatty acids by lactic acid bacteria involving conjugated fatty acid production

Lactobacillus plantarum AKU 1009a effectively transforms linoleic acid to conjugated linoleic acids of cis-9,trans-11-octadecadienoic acid (18:2) and trans-9,trans-11–18:2. The transformation of various polyunsaturated fatty acids by washed cells of L. plantarum AKU 1009a was investigated. Besides linoleic acid, α-linolenic acid [cis-9,cis-12,cis-15-octadecatrienoic acid (18:3)], γ-linolenic acid (cis-6,cis-9,cis-12–18:3), columbinic acid (trans-5,cis-9,cis-12–18:3), and stearidonic acid [cis-6,cis-9,cis-12,cis-15-octadecatetraenoic acid (18:4)] were found to be transformed. The fatty acids transformed by the strain had the common structure of a C18 fatty acid with the cis-9,cis-12 diene system. Three major fatty acids were produced from α-linolenic acid, which were identified as cis-9,trans-11,cis-15–18:3, trans-9,trans-11,cis-15–18:3, and trans-10,cis-15–18:2. Four major fatty acids were produced from γ-linolenic acid, which were identified as cis-6,cis-9,trans-11–18:3, cis-6,trans-9,trans-11–18:3, cis-6,trans-10–18:2, and trans-10-octadecenoic acid. The strain transformed the cis-9,cis-12 diene system of C18 fatty acids into conjugated diene systems of cis-9,trans-11 and trans-9,trans-11. These conjugated dienes were further saturated into the trans-10 monoene system by the strain. The results provide valuable information for understanding the pathway of biohydrogenation by anaerobic bacteria and for establishing microbial processes for the practical production of conjugated fatty acids, especially those produced from α-linolenic acid and γ-linolenic acid.

Linoleic Acid Isomerase in Lactobacillus plantarum AKU1009a Proved to Be a Multi-Component Enzyme System Requiring Oxidoreduction Cofactors

Linoleic acid isomerase in Lactobacillus plantarum was found to be a novel multi-component enzyme system widespread in membrane and soluble fractions. The isomerization reaction involved a hydration step, 10-hydroxy-12-octadecenoic acid production from linoleic acid, as part of the reaction, and the hydration reaction was catalyzed by the membrane fraction. Both membrane and soluble fractions were required for the whole isomerization reaction, i.e., conjugated linoleic acid (CLA) production from linoleic acid, and for CLA production from 10-hydroxy-12-octadecenoic acid, a reaction intermediate. The multi-component enzyme system was inhibited by o-phenanthroline, and divalent metal ions such as Ni2+ and Co2+ restored activity. Metal oxides such as VO4 3+, MoO4 2+, and MnO4 2+ enhanced activity. The multi-component enzyme systems required oxidoreduction cofactors such as NADH together with FAD or NADPH for total activity.

Novel multi-component enzyme machinery in lactic acid bacteria catalyzing C C double bond migration useful for conjugated fatty acid synthesis

Linoleic acid isomerase was identified as a multi-component enzyme system that consists of three enzymes that exist in both the membrane and soluble fractions of Lactobacillus plantarum. One enzyme (CLA-HY) is present in the membrane fraction, while two enzymes (CLA-DH and CLA-DC) exist in the soluble fraction. Three Escherichia coli transformants expressing CLA-HY, CLA-DH, and CLA-DC were constructed. Conjugated linoleic acid (CLA) and 10-hydroxy-12-octadecenoic acid were generated from linoleic acid only when all these three E. coli transformants were used as catalysts simultaneously. CLA-HY catalyzed the hydration reaction, a part of linoleic acid isomerization, to produce 10-hydroxy-12-octadecenoic acid. This multi-component enzyme system required oxidoreduction cofactors such as NADH and FAD. This is the first report to reveal enzymes genes and the elaborate machinery that synthesizes CLA, especially an important isomer of cis-9, trans-11-CLA, in lactic acid bacteria.

Characterization of Bacillus thuringiensis l-Isoleucine Dioxygenase for Production of Useful Amino Acids

ABSTRACT We determined the enzymatic characteristics of an industrially important biocatalyst, α-ketoglutarate-dependent l-isoleucine dioxygenase (IDO), which was found to be the enzyme responsible for the generation of (2S,3R,4S)-4-hydroxyisoleucine in Bacillus thuringiensis 2e2. Depending on the amino acid used as the substrate, IDO catalyzed three different types of oxidation reactions: hydroxylation, dehydrogenation, and sulfoxidation. IDO stereoselectively hydroxylated several hydrophobic aliphatic l-amino acids, as well as l-isoleucine, and produced (S)-3-hydroxy-l-allo-isoleucine, 4-hydroxy-l-leucine, (S)-4-hydroxy-l-norvaline, 4-hydroxy-l-norleucine, and 5-hydroxy-l-norleucine. The IDO reaction product of l-isoleucine, (2S,3R,4S)-4-hydroxyisoleucine, was again reacted with IDO and dehydrogenated into (2S,3R)-2-amino-3-methyl-4-ketopentanoate, which is also a metabolite found in B. thuringiensis 2e2. Interestingly, IDO catalyzed the sulfoxidation of some sulfur-containing l-amino acids and generated l-methionine sulfoxide and l-ethionine sulfoxide. Consequently, the effective production of various modified amino acids would be possible using IDO as the biocatalyst.