Asymmetric synthesis of (<Emphasis Type="Italic">R</Emphasis>)-3-fluoroalanine from 3-fluoropyruvate using omega-transaminase

In this study, (R)-3-fluoroalanine was asymmetrically synthesized from 3-fluoropyruvate (F-pyruvate) and (S)-α-methylbenzylamine (MBA) using recombinant ω-transaminase (TA) from Vibrio fluvialis JS17. The reaction was severely inhibited by acetophenone (deaminated product of α-MBA). In the presence of 5 mM acetophenone, the reactivity of the enzyme towards F-pyruvate decreased by 78%. To overcome the product inhibition by acetophenone, a biphasic reaction was successfully used. The conversion of F-pyruvate into (R)-3-fluoroalanine (enatiomeric exess (e.e.) > 99%) was about 95% in the biphasic system (75 mM F-pyruvate, 100 mM (S)-α-MBA, and 3.0 U/mL), whereas 31% was obtained without product extraction. The use of racemic α-MBA as an amino donor instead of (S)-α-MBA can reduce the reaction cost and also produce chiral amines through kinetic resolution. When the kinetic resolution of racemic α-MBA (40 mM) was carried out with F-pyruvate (30 mM) and ω-TA (3.0 U/mL) in 100 mM phosphate buffer (pH 7.0), the e.e. of (R)-α-MBA reached 98.4% with 52.2% conversion for 10 h and 21 mM (R)-3-fluoroalanine was produced with 70% conversion and an e.e. > 99%.     

Cloning,Purification and Characterization of an NAD-Dependent <Emphasis Type="SmallCaps">d</Emphasis>-Arabitol Dehydrogenase from Acetic Acid Bacterium, <Emphasis Type="Italic">Acetobacter suboxydans</Emphasis>

d-Xylulose-forming d-arabitol dehydrogenase (aArDH) is a key enzyme in the bio-conversion of d-arabitol to xylitol. In this study, we cloned the NAD-dependent d-xylulose-forming d-arabitol dehydrogenase gene from an acetic acid bacterium, Acetobacter suboxydans sp. The enzyme was purified from A. suboxydans sp. and was heterogeneously expressed in Escherichia coli. The native or recombinant enzyme was preferred NAD(H) to NADP(H) as coenzyme. The active recombinant aArDH expressed in E. coli is a homodimer, whereas the native aArDH in A. suboxydans is a homotetramer. On SDS–PAGE, the recombinant and native aArDH give one protein band at the position corresponding to 28 kDa. The optimum pH of polyol oxidation and ketone reduction is found to be pH 8.5 and 5.5 respectively. The highest reaction rate is observed when d-arabitol is used as the substrate (K _m = 4.5 mM) and the product is determined to be d-xylulose by HPLC analysis.     

Heterologous expression and characterization of <Emphasis Type="Italic">Bacillus coagulans</Emphasis><Emphasis Type="SmallCaps">l</Emphasis>-arabinose isomerase

Bacillus coagulans has been of great commercial interest over the past decade owing to its strong ability of producing optical pure l-lactic acid from both hexose and pentose sugars including l-arabinose with high yield, titer and productivity under thermophilic conditions. The l-arabinose isomerase (L-AI) from Bacillus coagulans was heterologously over-expressed in Escherichia coli. The open reading frame of the L-AI has 1,422 nucleotides encoding a protein with 474 amino acid residues. The recombinant L-AI was purified to homogeneity by one-step His-tag affinity chromatography. The molecular mass of the enzyme was estimated to be 56 kDa by SDS-PAGE. The enzyme was most active at 70°C and pH 7.0. The metal ion Mn²⁺ was shown to be the best activator for enzymatic activity and thermostability. The enzyme showed higher activity at acidic pH than at alkaline pH. The kinetic studies showed that the K _m, V _max and k _cat/K _m for the conversion of l-arabinose were 106 mM, 84 U/mg and 34.5 mM⁻¹min⁻¹, respectively. The equilibrium ratio of l-arabinose to l-ribulose was 78:22 under optimal conditions. l-ribulose (97 g/L) was obtained from 500 g/l of l-arabinose catalyzed by the enzyme (8.3 U/mL) under the optimal conditions within 1.5 h, giving at a substrate conversion of 19.4% and a production rate of 65 g L⁻¹ h⁻¹.     

Kinetic biosynthesis of l-ascorbyl acetate by immobilized Thermomyces lanuginosus lipase (Lipozyme TLIM)

Thermomyces lanuginosus lipase (Lipozyme TLIM)-catalyzed esterification of l-ascorbic acid was studied. It was suggested that Lipozyme TLIM was a suitable biocatalyst for enzymatic esterification of l-ascorbic acid. Three solvents were investigated for the reaction, and acetone was found to be a suitable reaction medium. Furthermore, it was found that water activity could notably affect the conversion. Moreover, pH memory of Lipozyme TLIM lipase for catalyzing l-ascorbic acid esterification in acetone was observed and the effect of pH on the reaction was estimated. In addition, the influences of other parameters such as substrate mole ratio, enzyme loading, and reaction temperature and reusability of lipase on esterification of l-ascorbic acid were also analyzed systematically and quantitatively. Kinetic characterization of Lipozyme TLIM showed that K _m,a and V _max were 80.085 mM and 0.747 mM min⁻¹, respectively. As a result, Lipozyme TLIM-catalyzed esterification of l-ascorbic acid gave a maximum conversion of 99%.     

A novel <Emphasis Type="SmallCaps">l</Emphasis>-aspartate dehydrogenase from the mesophilic bacterium <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> PAO1: molecular characterization and application for <Emphasis Type="SmallCaps">l</Emphasis>-aspartate production

l-aspartate dehydrogenase (EC 1.4.1.21; l-AspDH) is a rare member of amino acid dehydrogenase superfamily and so far, two thermophilic enzymes have been reported. In our study, an ORF PA3505 encoding for a putative l-AspDH in the mesophilic bacterium Pseudomonas aeruginosa PAO1 was identified, cloned, and overexpressed in Escherichia coli. The homogeneously purified enzyme (PaeAspDH) was a dimeric protein with a molecular mass of about 28 kDa exhibiting a very high specific activity for l-aspartate (l-Asp) and oxaloacetate (OAA) of 127 and 147 U mg⁻¹, respectively. The enzyme was capable of utilizing both nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP) as coenzyme. PaeAspDH showed a T _m value of 48°C for 20 min that was improved to approximately 60°C by the addition of 0.4 M NaCl or 30% glycerol. The apparent K _m values for OAA, NADH, and ammonia were 2.12, 0.045, and 10.1 mM, respectively; comparable results were observed with NADPH. The l-Asp production system B consisting of PaeAspDH, Bacillus subtilis malate dehydrogenase and E. coli fumarase, achieved a high level of l-Asp production (625 mM) from fumarate in fed-batch process with a molar conversion yield of 89.4%. Furthermore, the fermentative production system C released 33 mM of l-Asp after 50 h by using succinate as carbon source. This study represented an extensive characterization of the mesophilic AspDH and its potential applicability for efficient and attractive production of l-Asp. Our novel production systems are also hopeful for developing the new processes for other compounds production.     

d-lactic acid production from cellooligosaccharides and β-glucan using l-LDH gene-deficient and endoglucanase-secreting Lactobacillus plantarum

In order to achieve direct fermentation of an optically pure d-lactic acid from cellulosic materials, an endoglucanase from a Clostridium thermocellum (CelA)-secreting plasmid was introduced into an l-lactate dehydrogenase gene (ldhL1)-deficient Lactobacillus plantarum (∆ldhL1) bacterial strain. CelA expression and its degradation of β-glucan was confirmed by western blot analysis and enzyme assay, respectively. Although the CelA-secreting ∆ldhL1 assimilated cellooligosaccharides up to cellohexaose (although not cellotetraose), the main end product was acetic acid, not lactic acid, due to the conversion of lactic acid to acetic acid. Cultivation under anaerobic conditions partially suppressed this conversion resulting in the production of 1.27 g/l of D-lactic acid with a high optical purity of 99.5% from a medium containing 2 g/l of cellohexaose. Subsequently, D-lactic acid fermentation from barley β-glucan was carried out with the addition of Aspergillus aculeatus β-glucosidase produced by recombinant Aspergillus oryzae and 1.47 g/l of D-lactic was produced with a high optical purity of 99.7%. This is the first report of direct lactic acid fermentation from β-glucan and a cellooligosaccharide that is a more highly polymerized sugar than cellotriose.     

<Emphasis Type="SmallCaps">l</Emphasis>-Tryptophan catabolism by <Emphasis Type="Italic">Rubrivivax benzoatilyticus</Emphasis> JA2 occurs through indole 3-pyruvic acid pathway

Rubrivivax benzoatilyticus JA2 utilizes l-tryptophan as the sole source of nitrogen for growth, and it has a doubling time of ~11 h (compared to 8 h with ammonium chloride). With cell free extracts in the presence of 2-oxoglutarate, indole-3-pyruvic acid, indole-3-acetaldehyde, indole-3-acetic acid, isatin, benzaldehyde, gallic acid and pyrogallol were identified using high performance liquid chromatography (HPLC) and liquid chromatography–mass spectroscopy (LC–MS) analysis. The conversion of l-tryptophan into indole 3-pyruvic acid and glutamate by an enzyme aminotransferase was confirmed and the catabolism of indole-3-pyruvic acid via side chain oxidation followed by ring oxidation, gallic acid and pyrogallol were confirmed as metabolites. In addition, the proposed pathway sequential conversion of indole-3-pyruvic acid to the end product of pyrogallol was identified, including an enzymatic step that would convert isatin to benzaldehyde by an enzyme yet to be identified. At this stage of the study, the enzyme tryptophan aminotransferase in R. benzoatilyticus JA2 was demonstrated.     

Characterization of a mannose-6-phosphate isomerase from Geobacillus thermodenitrificans that converts monosaccharides

A recombinant mannose-6-phosphate isomerase from Geobacillus thermodenitrificans (GTMpi) isomerizes aldose substrates possessing hydroxyl groups oriented in the same direction at the C2 and C3 positions such as the d- and l-forms of ribose, lyxose, talose, mannose, and allose. The activity of GTMpi for d-lyxose isomerization was optimal at pH 7.0, 70°C and 1 mM Co²⁺. Under these conditions, the k _cat and K _m values were 74,300 s⁻¹ and 390 mM for d-lyxose and 28,800 s⁻¹ and 470 mM for l-ribose, respectively. The half-lives of the enzyme at 60, 65, and 70°C were 388, 73, and 27 h, respectively. GTMpi catalyzed the conversion of d-lyxose to d-xylulose with a 38% conversion yield after 3 h, and converted l-ribose to l-ribulose with a 29% conversion yield.     

Asymmetric synthesis of unnaturall-amino acids using thermophilic aromaticl-amino acid transaminase

Aromaticl-amino acid transaminase is an enzyme that is able to transfer the amino group froml-glutamate to unnatural aromatic α-keto acids to generate α-ketoglutarate and unnatural aromaticl-amino acids, respectively. Enrichment culture was used to isolate thermophilicBacillus sp. T30 expressing this enzyme for use in the synthesis of unnaturall-amino acids. The asymmetric syntheses ofl-homophenylalanine andl-phenylglycine resulted in conversion yields of >95% and >93% from 150 mM 2-oxo-4-phenylbutyrate and phenylglyoxylate, respectively, usingl-glutamate as an amino donor at 60°C. Synthesizedl-homophenylalanine andl-phenylglycine were optically pure (>99% enantiomeric excess) and continuously pre-cipitated in the reaction solution due to their low solubility at the given reaction pH. While the solubility of the α-keto acid substrates is dependent on temperature, the solubility of the unnaturall-amino acid products is dependent on the reaction pH. As the solubility difference between substrate and product at the given reaction pH is therefore larger at higher temperature, the thermophilic transaminase was successfully used to shift the reaction equilibrium toward rapid product formation.     

Repeated batch and continuous syntheses of N-(benzyloxycarbonyl)-l-phenylalanyl-l-phenylalanine methyl ester with immobilized thermolysin

Summary N-(Benzyloxycarbonyl)-l-phenylalanyl-l-phenylalanine methyl ester was synthesized from N-(benzyloxycarbonyl)-l-phenylalanine and l-phenylalanine methyl ester in an aqueous solution (aqueous phasic reaction), in an aqueous/organic biphasic system (biphasic reaction), and in an organic solvent (organic phasic reaction) with immobilized thermolysin. In the aqueous phasic reaction with thermolysin immobilized on Amberlite XAD-7, the whole product was trapped inside the support; extraction with ethyl acetate was needed to recover the product, and the equilibrium yield was low (about 65%). With the biphasic and organic phasic reactions with ethyl acetate as an organic solvent, the yield was around 95%. Because of the high yield and feasibility of operation, repeated batch and continuous reactions were done in the biphasic and organic phasic systems, respectively. The half-lives of the activity for the immobilized enzyme used in the biphasic system at 40°C by repeated batch operation and in a plug flow reactor fed with substrate dissolved in ethyl acetate at 40°C and 30°C were estimated to be about 200 h (67 batches), 420 h, and 1100 h, respectively.     

Substrate specificity of a glucose-6-phosphate isomerase from <Emphasis Type="Italic">Pyrococcus furiosus</Emphasis> for monosaccharides

We purified recombinant glucose-6-phosphate isomerase from Pyrococcus furiosus using heat treatment and Hi-Trap anion-exchange chromatography with a final specific activity of 0.39 U mg⁻¹. The activity of the glucose-6-phosphate isomerase for l-talose isomerization was optimal at pH 7.0, 95°C, and 1.5 mM Co²⁺. The half-lives of the enzyme at 65°C, 75°C, 85°C, and 95°C were 170, 41, 19, and 7.9 h, respectively. Glucose-6-phosphate isomerase catalyzed the interconversion between two different aldoses and ketose for all pentoses and hexoses via two isomerization reactions. This enzyme has a unique activity order as follows: aldose substrates with hydroxyl groups oriented in the same direction at C2, C3, and C4 > C2 and C4 > C2 and C3 > C3 and C4. l-Talose and d-ribulose exhibited the most preferred substrates among the aldoses and ketoses, respectively. l-Talose was converted to l-tagatose and l-galactose by glucose-6-phosphate isomerase with 80% and 5% conversion yields after about 420 min, respectively, whereas d-ribulose was converted to d-ribose and d-arabinose with 53% and 8% conversion yields after about 240 min, respectively.     

l-Ribulose production by an Escherichia coli harboring l-arabinose isomerase from Bacillus licheniformis

Recombinant Escherichia coli harboring the l-arabinose isomerase (BLAI) from Bacillus licheniformis was used as a biocatalyst to produce l-ribulose in the presence of borate. Effects of substrate concentration, the borate to l-arabinose ratio, pH, and temperature on the conversion of l-arabinose to l-ribulose were investigated. l-Ribulose production was efficient when pH was higher than 9 and temperature was higher than 50 °C. Borate addition to the reaction mixture was essential for high conversion of l-arabinose to l-ribulose as it resulted in an equilibrium shift in favor of the product. Under the optimal conditions determined by response surface methodology, the E. coli harboring BLAI produced 375 g l⁻¹ L-ribulose from 500 g l⁻¹ l-arabinose at a reaction time of 60 min, corresponding to a conversion yield of 75% and productivity of 375 g l⁻¹ h⁻¹. When the resting recombinant E. coli cells were recycled, 85% of the yield was obtained even after seven cycles of reuse. The productivity and final concentration of l-ribulose obtained in the present study were the highest yet reported.     

Synthesis and characterization of hydroquinone fructoside using <Emphasis Type="Italic">Leuconostoc mesenteroides</Emphasis> levansucrase

Hydroquinone (HQ) functions as a skin-whitening agent, but it has the potential to cause dermatitis. We synthesized a HQ fructoside (HQ-Fru) as a potential skin-whitening agent by reacting levansucrase from Leuconostoc mesenteroides with HQ as an acceptor and sucrose as a fructofuranose donor. The product was purified using 1-butanol partition and silica-gel column chromatography. The structure of the purified HQ-Fru was determined by ¹H and ¹³C nuclear magnetic resonance, and the molecular ion of the product was observed at m/z 295 (C12 H16 O7 Na)⁺. The HQ-Fru was identified as 4-hydroxyphenyl-β-d-fructofuranoside. The optimum condition for HQ-Fru synthesis was determined using a response surface method (RSM), and the final optimum condition was 350 mM HQ, 115 mM sucrose, and 0.70 U/ml levansucrase, and the final HQ-Fru produced was 1.09 g/l. HQ-Fru showed anti-oxidation activities and inhibition against tyrosinase. The median inhibition concentration (IC₅₀) of 1,1-diphenyl-2-picrylhydrazyl scavenging activity was 5.83 mM, showing higher antioxidant activity compared to β-arbutin (IC₅₀ = 6.04 mM). The K _i value of HQ-Fru (1.53 mM) against tyrosinase was smaller than that of β-arbutin (K _i  = 2.8 mM), indicating that it was 1.8-times better as an inhibitor. The inhibition of lipid peroxidation by HQ-Fru was 105.3% that of HQ (100%) and 118.9 times higher than that of β-arbutin (0.89% of HQ).     

Continuous 2-keto-<Emphasis Type="SmallCaps">l</Emphasis>-gulonic acid fermentation from <Emphasis Type="SmallCaps">l</Emphasis>-sorbose by <Emphasis Type="Italic">Ketogulonigenium vulgare</Emphasis> DSM 4025

A single-stage continuous fermentation process for the production of 2-keto-l-gulonic acid (2KGA) from l-sorbose using Ketogulonigenium vulgare DSM 4025 was developed. The chemostat culture with the dilution rate that was calculated based on the relationship between the 2KGA production rate and the 2KGA concentration was feasible for production with high concentration of 2KGA. In this system, 112.2 g/L of 2KGA on the average was continuously produced from 114 g/L of l-sorbose. A steady state of the fermentation was maintained for the duration of more than 110 h. The dilution rate was kept in the range of 0.035 and 0.043 h⁻¹, and the 2KGA productivity was 3.90 to 4.80 g/L/h. The average molar conversion yield of 2KGA from l-sorbose was 91.3%. Under the optimal conditions, l-sorbose concentration was kept at 0 g/L. Meanwhile, the dissolved oxygen level was changing in response to the dilution rate and 2KGA concentration. In the dissolved oxygen (DO) range of 16% to 58%, it was revealed that the relationship between DO and D possessed high degree of positive correlation under the l-sorbose limiting condition (complete consumption of l-sorbose). Increasing D closer to the critical value for washing out point of the continuous fermentation, DO value tended to be gradually increased up to 58%. In conclusion, an efficient and reproducible continuous fermentation process for 2KGA production by K. vulgare DSM 4025 could be developed using a medium containing baker’s yeast without using a second helper microorganism.     

Enhanced synthesis of l - threo -3,4-dihydroxyphenylserine by high-density whole-cell biocatalyst of recombinant l -threonine aldolase from Streptomyces avelmitilis

l-threo-3,4-Dihydroxyphenylserine (DOPS) is a chiral unnatural β-hydroxy amino acid used for the treatment of Parkinson disease. We developed a continuous bioconversion system for DOPS production that uses whole-cell biocatalyst of recombinant Escherichia coli expressing l-threonine aldolase (l-TA) genes cloned from Streptomyces avelmitilis MA-4680. Maximum conversion rates were observed at 2 M glycine, 145 mM 3,4-dihydroxybenzaldehyde, 0.75% Triton-X, 5 g E. coli cells/l, pH 6.5 and 10°C. In the optimized condition, overall productivity was 8 g/l, which represents 40 times the synthesis yield possible with no optimization of conditions.     

Enzymatic synthesis of γ-glutamylglutamine,a stable glutamine analogue,by γ-glutamyltranspeptidase from <Emphasis Type="Italic">Escherichia coli</Emphasis> K-12

The optimal reaction conditions for the synthesis of γ-glutamylglutamine using γ-glutamyltranspeptidase from Escherichia coli were determined. The maximum yield of γ-glutamylglutamine (110 mM) was obtained using 250 mM l-glutamine and 1.1 U γ-glutamyltranspeptidase/ml at pH 10.5 and at 37°C for 7 h; the conversion of glutamine to γ-glutamylglutamine was 88%.     

Engineering <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> for the production of pyruvate

A Corynebacterium glutamicum strain with inactivated pyruvate dehydrogenase complex and a deletion of the gene encoding the pyruvate:quinone oxidoreductase produces about 19 mM l-valine, 28 mM l-alanine and about 55 mM pyruvate from 150 mM glucose. Based on this double mutant C. glutamicum △aceE △pqo, we engineered C. glutamicum for efficient production of pyruvate from glucose by additional deletion of the ldhA gene encoding NAD⁺-dependent l-lactate dehydrogenase (LdhA) and introduction of a attenuated variant of the acetohydroxyacid synthase (△C–T IlvN). The latter modification abolished overflow metabolism towards l-valine and shifted the product spectrum to pyruvate production. In shake flasks, the resulting strain C. glutamicum △aceE △pqo △ldhA △C–T ilvN produced about 190 mM pyruvate with a Y _P/S of 1.36 mol per mol of glucose; however, it still secreted significant amounts of l-alanine. Additional deletion of genes encoding the transaminases AlaT and AvtA reduced l-alanine formation by about 50%. In fed-batch fermentations at high cell densities with adjusted oxygen supply during growth and production (0–5% dissolved oxygen), the newly constructed strain C. glutamicum △aceE △pqo △ldhA △C–T ilvN △alaT △avtA produced more than 500 mM pyruvate with a maximum yield of 0.97 mol per mole of glucose and a productivity of 0.92 mmol g_(CDW)⁻¹ h⁻¹ (i.e., 0.08 g g_(CDW) ⁻¹ h⁻¹) in the production phase.     

Regioselective carboxylation of catechol by 3,4-dihydroxybenzoate decarboxylase of <Emphasis Type="Italic">Enterobacter cloacae</Emphasis> P

3,4-Dihydroxybenzoate decarboxylase in Enterobacter cloacae P241 was induced by adding 3,4-dihydroxybenzoic acid, 3-hydroxybenzoic acid, 3,4,5-trihydroxybenzoic acid or 4-acetamidobenzoic acid to the culture medium. After stabilizing the enzyme activity by adding 5 mM dithiothreitol and 20 mM Na₂S₂O₃ to a cell-free extract, catechol at 50 mM was carboxylated in the presence of 3 M KHCO₃ to 3,4-dihydroxybenzoic acid with a molar conversion ratio of 28% after 14 h at 30°C.     

Novel l-specific cleavage of the urethane bond of t -butoxycarbonylamino acids by whole cells of Corynebacterium aquaticum

Microorganisms capable of cleaving the urethane bond of t-butoxycarbonyl (Boc) amino acids in a whole-cell reaction were screened among stock cultures, and Corynebacterium aquaticum IFO12154 was the most promising. The conversion of Boc-Ala to Ala was stimulated by CoSO₄ in the medium and reaction mixture. The optimum whole-cell concentration was 25 mg lyophilized cells/ml. Boc-l-Met was the best substrate for this reaction, and other Boc-L-amino acids, as well as benzyloxycarbonyl-l-amino acids with hydrophobic residues, were also good substrates. Boc-d- and Z-d-amino acids were inert. When the reactions had proceeded for 24 h with each substrate at 10 mM, the molar conversion rates from Boc-l-, dl- and d-Met were 100%, 50%, and 0% respectively. From 150 mM Boc-l-Met, 143 mM l-Met was formed at a molar yield of 95.3%. Received: 3 September 1996 / Received last revision: 7 April 1997 / Accepted: 19 April 1997     

Mannose production from fructose by free and immobilized <Emphasis Type="SmallCaps">d</Emphasis>-lyxose isomerases from <Emphasis Type="Italic">Providencia stuartii</Emphasis>

A recombinant d-lyxose isomerase from Providencia stuartii was immobilized on Duolite A568 beads which gave the highest conversion of d-fructose to d-mannose among the various immobilization beads evaluated. Maximum activities of both the free and immobilized enzymes for fructose isomerization were at pH 7.5 and 45°C in the presence of 1 mM Mn²⁺. Enzyme half-lives were 14 and 30 h at 35°C and 3.4 and 5.1 h at 45°C, respectively. The immobilized enzyme in 300 g fructose/l (replaced hourly), produced 75 g mannose/l at 35°C = 25% (w/w) yield with a productivity of 75 g mannose l⁻¹ h⁻¹ after 23 cycles.