Production of d-lactate using a pyruvate-producing Escherichia coli strain

To generate an organism capable of producing d-lactate, NAD⁺-dependent d-lactate dehydrogenase was expressed in our pyruvate-producing strain, Escherichia coli strain LAFCPCPt-accBC-aceE. After determining the optimal culture conditions for d-lactate production, 18.4 mM d-lactate was produced from biomass-based medium without supplemental mineral or nitrogen sources. Our results show that d-lactate can be produced in simple batch fermentation processes.     

Structure-Based Modification of D-Alanine-D-Alanine Ligase from Thermotoga maritima ATCC 43589 for Depsipeptide Synthesis

Depsipeptides are peptide-like polymers consisting of amino acids and hydroxy acids, and are expected to be new functional materials for drug-delivery systems and polymer science. In our previous study, D-alanyl-D-lactate, a type of depsipeptide, was enzymatically synthesized using D-alanine-D-alanine ligase from Thermotoga maritima ATCC 43589 (TmDdl) by Y207F substitution. Thereafter, in this study, further mutagenesis was introduced, based on structural comparison between TmDdl and a well-characterized D-alanine-D-alanine ligase from Escherichia coli. The S137A/Y207F mutant showed higher D-alanyl-D-lactate and lower D-alanyl-D-alanine synthesizing activity than the Y207F mutant. This suggests that substitution at the S137 residue contributes to product selectivity. Saturated mutagenesis on S137 revealed that the S137G/Y207F mutant showed the highest D-alanyl-D-lactate synthesizing activity. Moreover, the mutant showed broad substrate specificity toward D-amino acid and recognized D-lactate and D,L-isoserine as substrates. On the basis of these characteristics, various depsipeptides can be produced using S137G/Y207F-replaced TmDdl.     

Growth of Yeasts on n-Alkanes: Inhibition by a Lactonic Sophorolipid Produced by

The effects of amino acids on IMP production were examined with a mutant strain, KY10895, derived from Corynebacterium ammoniagenes KY13374. l-Proline improved the productivity of IMP more than any other amino acid. The optimum concentration of l-proline for IMP production was 1–2% and the IMP productivity was about 70% more than that in the control medium. The effects of l-proline analogs on IMP production were also examined with the mutant KY10895. DL-3,4-Dehydroproline inhibited IMP production. Mutants resistant to growth inhibition by dl-3,4-dehydroproline were derived from strain KY10895. Among mutants thus obtained, strain H-7335 had the highest productivity. The intracellular concentrations of l-proline in strain H-7335 were higher than those of the parental strain, KY10895. These findings indicated that an increase in intracellular l-proline was linked with an increase of IMP productivity and strengthening the l-proline synthesis of a strain was an effective method for obtaining a hyper-producer of IMP.     

Cell Surface Proteins of Agrobactevium tumefaciens Involved in the Transfer of T-DNA to the Host

We investigated the taste synergy between L-theanine and the flavour enhancer, inosine 5′-monophosphate (IMP), by using a human sensory evaluation. When L-theanine was added to IMP, only the umami taste was enhanced. We then investigated this synergistic effect of L-theanine in mice by gustatory nerve recording. We confirmed the synergism between L-theanine and IMP for the umami taste.     

Functional Characterization of D-Galacturonic Acid Reductase,a Key Enzyme of the Ascorbate Biosynthesis Pathway,from Euglena gracilis

D-Galacturonic acid reductase, a key enzyme in ascorbate biosynthesis, was purified to homogeneity from Euglena gracilis. The enzyme was a monomer with a molecular mass of 38–39 kDa, as judged by SDS–PAGE and gel filtration. Apparently it utilized NADPH with a Km value of 62.5±4.5 μM and uronic acids, such as D-galacturonic acid (Km=3.79±0.5 mM) and D-glucuronic acid (Km=4.67±0.6 mM). It failed to catalyze the reverse reaction with L-galactonic acid and NADP⁺. The optimal pH for the reduction of D-galacturonic acid was 7.2. The enzyme was activated 45.6% by 0.1 mM H₂O₂, suggesting that enzyme activity is regulated by cellular redox status. No feedback regulation of the enzyme activity by L-galactono-1,4-lactone or ascorbate was observed. N-terminal amino acid sequence analysis revealed that the enzyme is closely related to the malate dehydrogenase families.     

Purification and Properties of l-Arginase from Bacillus subtilis

l-Arginase (l-arginine amidinohydrolase, EC 3.5.3.1) was purified in a crystalline form from cells of Bacillus subtilis KY 3281 with an overall yield of 23.2%. The crystalline enzyme had a specific activity of 858 i.u./mg-protein and was ultracentrifugally homogeneous. It was estimated to have a molecular weight of 115,000±5000 by the method of Yphantis.

The enzyme highly specific for l-arginine showed the maximum activity at pH 10 with Mn²⁺ ion. The Km for l-arginine was 1.35 × 10^?2 m The activity was competitively inhibited by l-lysine, but not by l-ornithine and increased by the addition of Mn²⁺ or Co²⁺ ions. The stable pH and temperature ranges became wider in the presence of Mn²⁺ ion and l-threonine.     

Improvement of L-Lactate Production by CYB2 Gene Disruption in a Recombinant Saccharomyces cerevisiae Strain under Low pH Condition

Using a DNA microarray, we found that expression of the genes related to lactate metabolism was upregulated in a lactate-producing recombinant Saccharomyces cerevisiae strain. Disruption of the CYB2 gene encoding L-lactate dehydrogenase improved the L-lactate production by S. cerevisiae under low pH condition.     

Production of l-rhamnulose,a rare sugar,from l-rhamnose using commercial immobilized glucose isomerase

Abstract

A commercial immobilized d-glucose isomerase from Streptomyces murines (Sweetzyme) was used to produce l-rhamnulose from l-rhamnose in a packed-bed reactor. The optimal conditions for l-rhamnulose production from l-rhamnose were determined as pH 8.0, 60?°C, 300?g L^?1 l-rhamnose as a substrate, and 0.6?h^?1 dilution rate. The half-life of the immobilized enzyme at 60?°C was 809?h. Under the optimal conditions, the immobilized enzyme produced an average of 135?g L^?1 l-rhamnulose from 300?g L^?1 l-rhamnose after 16 days at pH 8.0, 60?°C, and 0.6?h^?1 dilution rate, with a productivity of 81?g/L/h and a conversion yield of 45% in a packed-bed reactor.     

Amino Acid Sequence of an Active Center Peptide Containing Serine Isolated from Alkaline Protease of Bacillus No. 221

The substrate specificity of pig liver acid α-glucosidase was investigated. The enzyme showed a wide specificity on various substrates. The Km values for maltose, malto-triose, -tetraose, -pentaose, -hexaose and -heptaose, and maltodextrin (mean degree of polymerization, 13) were 6.7 mm, 4.4 mm, 5.9 mm, ll mm, 4.0 mm, 5.6 mm and 7.1 mm, respectively. The relative maximum velocities for maltooligosaccharides consisting of three or more glucose units were 82.6 to 92.3% of the maximum velocity for maltose. For disaccharides, the rates of hydrolysis decreased in the following order: maltose > nigerose > kojibiose > isomaltose. The acid α-glucosidase also hydrolyzed several α-glucans, such as glycogen, soluble starch, β-limit dextrin and amylopectin. The Km value for β-limit dextrin was the lowest of those for α-glucans.

The nature of the active site catalyzing the hydrolyses of maltose and glycogen was investigated by kinetic methods. In experiments with mixed substrates, maltose and glycogen, the kinetic features agreed very closely with those theoretically predicted for a single active site catalyzing the hydrolyses of both substrates. Cations, Na⁺, K⁺ and Mg⁺⁺, were about equally effective in the activation of the enzyme action on maltose and glycogen. The inhibitor constants of tris(hydroxymethyl)aminomethane (Tris) and turanose were nearly the same for maltase activity as those for glucoamylase activity. From these results, the enzyme was concluded to attack maltose and glycogen by a single active site mechanism.     

Comparison of Structure and Antitumor Activity of Polysaccharides Isolated from Fukurotake,the Fruiting Body of Volvariella volvacea

An α-linked d-manno-d-galactan, glycogen, and three kinds of branched (1→3)-γ-d-glucans were isolated from Fukurotake, the fruiting body of Volvariella volvacea by successive extractions with cold and hot water, cold and hot alkali, and dimethyl sulfoxide. The mannogalactan, purified from the cold water extract, had a MW of 4x 10⁵, and consisted of an α-(1→6) linked d-galactose backbone, one out of every three d-galactose residues being substituted with a single α-d-mannosyl group. The glycogen, isolated from the hot water extract, had a MW of 12 × 10⁵, and 14 ~ 15 d-glucose residues as an average chain length, as revealed by methylation analysis. The α-(1→4)-linked unit chains of this glycogen were distributed from DP 6 to 13, approximately in equal numbers.

There were three kinds of branched (1→3)-γ-d-glucans, isolated from alkali and dimethyl sulfoxide extractions. They contain a backbone of (1→3)-linked d-glucose residues with side chains of single d-glucosyl groups, but having different degrees of branching. In addition, alkali-extracted glucans contain small but significant proportions of (1→6)-linked sugar units.

Among these polysaccharides, the cold alkali-extracted glucan (degree of branching, 1:5) showed a potent antitumor activity against Sarcoma 180 solid tumors implanted in mice, and chemical modifications changed its original activity, confirming our previous results. Other polysaccharides, such as the mannogalactan and other (1→3)-γ-d-glucans, showed no or lower antitumor activity.     

Enzymatic Synthesis of α-d-Glucopyranosyl-2-deoxy-d-glucoses by Transglucosylation Reaction of Buckwheat α-Glucosidase

The transglucosylation reaction of buckwheat α-glucosidase was examined under the coexistence of 2-deoxy-d-glucose and maltose. As the transglucosylation products, two kinds of new disaccharide were chromatographically isolated in a crystalline form (hemihydrate). It was confirmed that these disaccharides were 3-O-α-d-glucopyranosyl-2-deoxy-d-glucose ([α]_d + 132°, mp 130 ~ 132°C, mp of ±-heptaacetate 151 ~ 152°C) and 4-O-±-d-glucopyranosyl-2-deoxy-d-glucose ([±]_d + 136°, mp 168 ~ 170°C), respectively. The principal product formed in the enzyme reaction was 3-O-±-d-glucopyranosyl-2-deoxy-d-glucose.     

Effect of Ca2+ Ions on the Viscometric Behavior of F-actin

Changes in the viscosity of the F-actin solutions which occur on addition of Ca²⁺ ions were investigated. The viscosity of F-actin decreased on addition of Ca²⁺ ions. The amount of Ca²⁺ ions needed to decrease the viscosity changed with pH of the solution, namely, 20~30 mm at pH 7, 15~20 mm at pH 6 and 5~10mm at pH 5.5. Other divalent cations had the same action on F-actin, but monovalent cations did not affect the F-actin viscosity even at the concentration as high as 1 m. Intrinsic viscosity of F-actin with and without Ca²⁺ions was 250 ±40 (ml/g) and 670 ±80 (ml/g), respectively. The cause of this viscosity change was discussed from the results of electron microscopic observation and light scattering measurements.     

Genetic Engineering of Candida utilis Yeast for Efficient Production of L-Lactic Acid

Polylactic acid is receiving increasing attention as a renewable alternative for conventional petroleum-based plastics. In the present study, we constructed a metabolically-engineered Candida utilis strain that produces L-lactic acid with the highest efficiency yet reported in yeasts. Initially, the gene encoding pyruvate decarboxylase (CuPDC1) was identified, followed by four CuPDC1 disruption events in order to obtain a null mutant that produced little ethanol (a by-product of L-lactic acid). Two copies of the L-lactate dehydrogenase (L-LDH) gene derived from Bos taurus under the control of the CuPDC1 promoter were then integrated into the genome of the CuPdc1-null deletant. The resulting strain produced 103.3 g/l of L-lactic acid from 108.7 g/l of glucose in 33 h, representing a 95.1% conversion. The maximum production rate of L-lactic acid was 4.9 g/l/h. The optical purity of the L-lactic acid was found to be more than 99.9% e.e.     

Mutual Binding Inhibition of Tetrodotoxin and Saxitoxin to Their Binding Protein from the Plasma of the Puffer Fish,Fugu pardalis

The mutual binding inhibition of tetrodotoxin and saxitoxin to their binding protein from the plasma of Fugu pardalis was investigated by HPLC. The values for the half inhibitory concentration of tetrodotoxin (1.6 μM) binding to this protein (1.2 μM) for saxitoxin, and of saxitoxin (0.47 μM) binding to that (0.30 μM) for tetrodotoxin were 0.35±0.057 μM and 81±16 μM (n=2), respectively.     

Preparation of Zeaxanthin from Berries of Boxthorn,Lycium chinensis

Purification and properties of a new alkaline protease of rat skeletal muscle have been reported. The purification procedure of the enzyme is as follows: skeletal muscle tissue was extracted successively with Hasselbach-Schneider solution, 5 m urea solution and 2% sodium deoxycholate solution. After then, the enzyme was extracted from the residue with 1.1 m potassium iodide solution. This enzyme solution was treated with n-butanol, and dialyzed against water. The enzyme precipitated during dialysis was collected and dissolved in 1.1 m potassium iodide solution. The enzyme solution was fractionated with acetone, and chromatographed on Sephadex G-200. The final preparation showed over 20,000 times of purity.

The optimum pH range of the enzyme activity is 9.5~10.5, and the maximum reaction rate occurs at 47~57°C. The enzyme is stable below 47°C at pH 7.3. At 37°C, the enzyme is stable during 30 min at least, in the pH range of 5.5~10.0. Below pH 5.0, it is relatively labile. Hg²⁺, Ca²⁺, Mg²⁺, Mn²⁺, Co²⁺, and Zn²⁺ scarcely affect the enzyme activity at the concentration of 1 mm. Ethylenediaminetetraacetate shows little effect on the activity at the concentration of 10 mm, and iodoacetamide, 2,4-dinitrophenol, p-chloromercuribenzoate show the similar effect at the concentration of 1 mm. Diisopropyl-flurophosphate inhibits the enzyme activity. From the results obtained, this enzyme is presumed to be responsible for the activity of autolytic breakdown of rat skeletal muscle proteins in the alkaline pH range.     

Continuous Production of l-Alanine with NADH Regeneration by a Nanofiltration Membrane Reactor

A conjugated enzyme system, alanine dehydrogenase (AIDH) for stereospecific reduction of pyruvate to l-alanine and glucose dehydrogenase (GDH) for regeneration of NADH, were coimmobilized in a nanofiltration membrane bioreactor (NFMBR) for the continuous production of l-alanine from pyruvate with NADH regeneration. Since pyruvate was proved to be unstable at neutral pH, it was kept under acidic conditions and supplied to NFMBR separately from the other substrates. As 0.2 m pyruvate in HCl solution (pH 4), 10 mm NAD, 0.2 m glucose, and 0.2 m NH₄Cl in 0.5 m Tris buffer (pH 8) were continuously supplied to NFMBR with immobilized AIDH (100 U/ml) and GDH (140 U/ml) at the retention time of 80 min, the maximum conversion, reactor productivity, and NAD regeneration number were 100%, 320 g/liter/d, and 20,000, respectively. To avoid the effect of pyruvate instability, a consecutive reaction system, lactate dehydrogenase (l-LDH) and AIDH, was also used. In this system, the l-LDH provides pyruvate, the substrate for the AIDH reaction, from l-lactate regenerating NADH simultaneously, so the pyruvate could be consumed as soon as it was produced. As 0.2 m l-lactate, 10 mm NAD, 0.2 m NH₄Cl in 0.5 m Tris buffer (pH 8) were continuously supplied to NFMBR with immobilized l-LDH (100 U/ml) and AIDH (100 U/ml) at the retention time of 160 min, the maximum conversion, reactor productivity, and the NAD regeneration number were 100%, 160 g/Iiter/d, and 20,000, respectively.     

Production of l-Lysine by Leucine Auxotrophs Derived from AEC Resistant Mutant of Brevibacterium lactofermentum

The effect of amino acids was examined on the production of l-lysine by AEC resistant mutant of B. lactofermentum. Among amino acids tested, only leucine showed strong specific inhibition. In order to release the production of l-lysine from this negative effect of leucine, leucine auxotrophs were derived from AEC resistant strain of B. lactofermentum. Most of these leucine auxotrophs produced larger amount of l-lysine (maximally 41 mg/ml) than the parental strain which produced about 18 mg/ml of l-lysine. It was confirmed that leucine auxotrophs derived from AEC resistant mutant of other glutamate producing bacteria, B. saccharolyticum and Corynebacterium glutamicum. These results suggested that leucine might directly or indirectly affect the biosynthesis of lysine.

However, this increase in lysine productivity of leucine auxotrophs could not be explained by the alteration of aspartokinase (EC 2.7.2.4) and homoserine dehydrogenase (EC 1.1.1.3). These enzymes are key enzymes in lysine and threonine biosynthesis, respectively.     

Properties of Tyrosinase from Pseudomonas melanogenum

The properties of the tyrosinase from Pseudomonas melanogenum was investigated with the crude enzyme preparation. Optimum temperature and pH of the enzyme were 23°C and 6.8, respectively. l-Tyrosine, d-tyrosine, m-tyrosine, N-acetyl-l-tyrosine and l-DOPA were utilized as a substrate by the enzyme. The value for Km obtained were as follows: l-tyrosine 6.90 × 10^?4 m, d-tyrosine 1.43 ×10^?3 m and l-DOPA 9.90 × 10^?4 m. The enzyme was inhibited by chelating agents of Cu²⁺ l-cysteine, l-homocysteine, thiourea and diethyl-dithiocarbamate and the inhibition was completely reversed by the addition of excess Cu²⁺ From these results it is concluded that the enzyme is a copper-containing oxidase.     

Enzymatic activity of L-amino acid oxidase from snake venom Crotalus adamanteus in supercritical CO2

L-amino acid oxidase (L-AAO) from snake venom Crotalus adamanteus was successfully tested as a catalyst in supercritical CO₂ (SC-CO₂). The enzyme activity was measured before and after exposure to supercritical conditions (40°C, 110 bar). It was found that L-AAO activity slightly increased after SC-CO₂ exposure by up to 15%. L-AAO was more stable in supercritical CO₂ than in phosphate buffer under atmospheric pressure, as well as in the enzyme membrane reactor (EMR) experiment. 3,4-Dihydroxyphenyl-L-alanine (L-DOPA) oxidation was performed in a batch reactor made of stainless steel that could withstand the pressures of SC-CO₂, in which L-amino acid oxidase from C. adamanteus was able to catalyze the reaction of oxidative deamination of L-DOPA in SC-CO₂. For the comparison L-DOPA oxidation was performed in the EMR at 40°C and pressure of 2.5 bar. Productivity expressed as mmol-s of converted L-DOPA after 3?h per change of enzyme activity after 3?h was the highest in SC-CO₂ (1.474?mmol?U^?1), where catalase was present, and the lowest in the EMR (0.457?mmol?U^?1).     

Studies on d-Glucose-isomerizing Activity of d-Xylose-grown Cells from Bacillus coagulans,Strain HN-68

d-Glucose-isomerizing enzyme has been extracted in high yield from d-xylose-grown cells of Bacillus coagulans, strain HN-68, by treating with lysozyme, and purified approximately 60-fold by manganese sulfate treatment, fractionation with ammonium sulfate and chromatography on DEAE-Sephadex column. The purified d-glucose-isomerizing enzyme was homogeneous in polyacrylamide gel electrophoresis and ultracentrifugation and was free from d-glucose-6-phosphate isomerase. Optimum pH and temperature for activity were found to be pH 7.0 and 75°C, respectively. The enzyme required specifically Co⁺⁺ with suitable concentration for maximal activity being 10^?3 m. In the presence of Co⁺⁺, enzyme activity was inhibited strongly by Cu⁺⁺, Zn⁺⁺, Ni⁺⁺, Mn⁺⁺ or Ca⁺⁺. At reaction equilibrium, the ratio of d-fructose to d-glucose was approximately 1.0. The enzyme catalyzed the isomerization of d-glucose, d-xylose and d-ribose. Apparent Michaelis constants for d-glucose and d-xylose were 9×10^?2 m and 7.7×10^?2 m, respectively.