Enzymatic production of pyruvate from fumarate — an application of microbial cyclic-imide-transforming pathway

For the purpose of producing pyruvate from fumarate through microbial cyclic-imide-transforming pathway, various cyclic-imide-utilizing microorganisms were isolated from soil. Among them, strain g31 was the best producer and was identified as Pseudomonas sp. With the resting cells of the strain, the conditions were optimized for pyruvate production from fumarate. The cells cultivated in the medium containing 2% (w/v) of fumarate showed the highest production with sufficient yield. The optimized wet-cell concentration, pH and temperature of the reaction were 1% (w/v), pH 6 to 7, and 30°C, respectively. Aeration was found to be an effective factor, and vigorous shaking during the reaction mixture resulted in higher production. Under the optimized reaction conditions, 100 mM of fumarate was almost stoichiometrically converted into pyruvate (94 mM) in 24 h.     

A biocatalyst for pyruvate preparation from -lactate: lactate oxidase in a Pseudomonas sp.

For the purpose of producing pyruvate from -lactate by enzymatic methods, four microorganism strains that produce lactate oxidase (LOD) were screened and isolated from many soil samples. Among them, strain SM-6, which showed high potential for pyruvate production, was chosen for further research. Physiological studies and 16S rDNA relationship reveal that SM-6 belongs to Pseudomonas putida. The optimized pH and temperature of the enzyme-catalyzed reaction were pH 7.2, and 39 °C, respectively. Low-concentration EDTA (1 mM) could improve the stability of pyruvate and conversion ratio of lactate oxidase. V_max and K_m value for -lactate were 2.46 μmol/(min mg) protein and 9.53 mM, respectively. On preparation scale, cell-free extract from SM-6, containing 300 mg/l of crude enzyme (4037 U/ml lactate oxidase), could convert 66% of 116 mM of -lactate into 76.6 mM pyruvate in 18 h, and 82% of substrate was transformed after 48 h, giving 95.0 mM (10.5 mg/ml) of pyruvate. The ratio of product to biocatalyst was 34.8:1 (g/g).     

Biotransformation of aromatic and heterocyclic amides by amidase of whole cells of Rhodococcus sp. MTB5: Biocatalytic characterization and substrate specificity

In this study, an amidohydrolase activity of amidase in whole cells of Rhodococcus sp. MTB5 has been used for the biotransformation of aromatic, monoheterocyclic and diheterocyclic amides to corresponding carboxylic acids. Benzoic acid, nicotinic acid and pyrazinoic acid are carboxylic acids which have wide industrial applications. The amidase of this strain is found to be inducible in nature. The biocatalytic conditions for amidase present in the whole cells of MTB5 were optimized against benzamide. The enzyme exhibited optimum activity in 50?mM potassium phosphate buffer pH 7.0. The optimum temperature and substrate concentrations for this enzyme were 50?°C and 50?mM, respectively. The enzyme was quite stable for more than 6?h at 30?°C. It showed substrate specificity against different amides, including aliphatic, aromatic and heterocyclic amides. Under optimized reaction conditions, the amidase is capable of converting 50?mM each of benzamide, nicotinamide and pyrazinamide to corresponding acids within 100, 160 and 120?min, respectively, using 5?mg dry cell mass (DCM) per mL of reaction mixture. The respective percent conversion of these amides was 95.02%, 98.00% and 98.44% achieved by whole cells. The amidase in whole cells can withstand as high as 383?mM concentration of product in a reaction mixture and above which it undergoes product feedback inhibition. The results of this study suggest that Rhodococcus sp. MTB5 amidase has the potential for large-scale production of carboxylic acids of industrial value.     

An enzymatic route to produce pyruvate from lactate

A bacterial strain of Acinetobacter sp., which was capable of enzymatic production of pyruvate from lactate, was cultured in a 5-l reactor with a basal salt medium. After 14 h of fed-batch fermentation, 9.56 g l^–1 cell concentration in the broth was obtained with 20 g l^–1 (178 mM) sodium lactate and 4 g l^–1 NH₄Cl in the medium; and the biotransformation ability was 2.51 units ml^–1. The cells were harvested from one reactor and then used for pyruvate production from lactate in the same reactor. l-lactate at a concentration about 527 mM was almost stoichiometrically converted to pyruvate in 28 h. After a total 42 h of cell culture and biotransformation, the transformative yield was about 0.72 g g^–1 pyruvate from lactate and the rate of pyruvate production was calculated as 1.33 g l^–1 h^–1 during the process. The results suggested this simple enzymatic production of pyruvate from lactate should be a promising process and may bring a yield higher than that by microbial fermentation. By this process, the recovery of pyruvate from such a simple reaction liquid is relatively easy and inexpensive to perform.     

Biotransformation of dl-lactate to pyruvate by a newly isolated Serratia marcescens ZJB-07166

The production of pyruvate using biotransformation from dl-lactate has been recently drawn more and more attentions due to the wide applications of pyruvate in chemicals, drugs, and agrochemicals industries. In the current study, a strain ZJB-07166, which was capable of converting dl-lactate to pyruvate, was newly isolated and characterized and later identified as Serratia marcescens based on the morphology, physiological tests, ATB system and its 16S rDNA sequence. The strain S. marcescens ZJB-07166 was applied in biotransformation of dl-lactate to pyruvate and the detailed time courses for cultivation and biotransformation were investigated. The optimum nitrogen source and carbon source in the microorganism culture for production of lactate dehydrogenase were NH₄Cl and dl-lactate, respectively. The optimum substrate concentration for biotransformation was around 40 mM and EDTA had an obvious stabilizing effect on pyruvate in biotransformation process. The pyruvate production concentration of 210 mM was achieved under the optimum conditions. These results demonstrated that the newly isolated S. marcescens ZJB-07166 was a promising strain for pyruvate production in industrial scale.     

羰基还原酶产生菌Candida ontarioensis制备(R)-2-氯-1-(3-氯苯基)乙醇

从实验室保藏的菌株中筛选获得Candida sp.PT2A,并通过18S rRNA鉴定为安大略假单胞菌Candida on-tarioensis。对C.ontarioensis不对称还原合成(R)-2-氯-1-(3-氯苯基)乙醇的发酵产酶条件和转化条件进行优化,确定了最适的发酵产酶条件和转化条件:温度30℃,初始pH 6.5,摇床转速180 r/min,菌体质量浓度200 g/L。采用2-氯-1-(3-氯苯基)乙酮质量浓度为10 g/L时,还原反应72 h,(R)-2-氯-1-(3-氯苯基)乙醇的e.e.值为99.9%,产率为99%;底物质量浓度提高至30 g/L时,产率下降为84.3%。采用十六烷基三甲基溴化铵(CTAB)对C.ontarioensis细胞进行通透性处理(CTAB g/L,4℃下处理20 min),在30 g/L底物下反应24 h,产物的e.e.和产率分别达到99.9%和97.5%。     

Optimisation of reaction conditions for production ofS-(−)-2-hydroxypropiophenone byAcinetobacter calcoaceticus

Summary Whole cells and cell-free extracts ofAcinetobacter calcoaceticus containing benzoylformate decarboxylase efficiently condensed benzoylformate and acetaldehyde to produce the acyloin compoundS-(–)-2-hydroxypropiophenone. Optimal concentrations of acetaldehyde cosubstrate for this reaction were found to be 1600 and 800 mM when whole cells and cell-free extracts were used respectively as biocatalysts. In both cases, optimal benzoylformate concentration was 100 mM. Temperature and pH optima for the biotransformation reaction were 30°C and 6.0 respectively. Under optimised conditions, maximum production of 2-hydroxypropiophenone, amounting to 8.4 g L^–1, occurred after a 2-h incubation. Product formation equivalent to 6.95 g in 1 h corresponded to a productivity of 267 mg acyloin per g dry cells per h.     

The role of glucose,pyruvate and lactate in ATP production by rat spermatocytes and spermatids

The ATP content of pachytene spermatocytes and round spermatids, isolated from rat testes, was not maintained during incubation of the germ cells in the presence of glucose. Glucose was metabolized via glycolysis at a considerable rate, but the rate of oxidation of the resulting endogenous pyruvate in the mitochondria was too low to support fully ATP production. Exogenous pyruvate (0.25 mM) or exogenous l-lactate (3–6 mM), however, were effective energy substrates. The lactate dehydrogenase reaction in isolated germ cells favoured the rapid conversion of pyruvate to lactate, at the expense of reducing equivalents from mitochondrial NADH. Hence, to support ATP production by the germ cells via mitochondrial metabolism of endogenous pyruvate, a relatively high concentration of exogenous lactate may be essential. In the spermatogenic microenvironment in vivo, such high concentrations of lactate could result from the net production of lactate by Sertoli cells. The mitochondria of the isolated germ cells produced ATP probably at a close to maximal rate, and spermatogenesis therefore may be extremely sensitive to compounds which interfere with mitochondrial energy metabolism and respiratory control.     

The two-step biotransformation of monosodium glutamate to GABA by Lactobacillus brevis growing and resting cells

Gama-aminobutyric acid (GABA) is a natural functional amino acid. In the current study, Lactobacillus brevis TCCC13007, a high GABA-producing strain, was isolated from naturally pickled Chinese vegetables. A two-step cellular bioconversion process was established using L. brevis TCCC13007 for the production of GABA. First, L. brevis cells were grown anaerobically in 7% monosodium glutamate (MSG)-containing medium at an initial pH of 6.0 and a controlled pH of 4.6 for 16 to 66 h; approximately 38 g L(-1) of GABA was obtained after 66 h of fermentation at a conversion rate of 98.6%. In the second stage of the process, about 7.6 g L(-1) of GABA was produced three more times at a conversion rate of 92.2% using the same batch of resting cells in the substrate-containing buffer under optimized conditions. Thus, the total GABA yield reached 61 g L(-1). A model system for the biotransformation of MSG to GABA was established using L. brevis TCCC13007 resting cells. The reaction rates were found to follow the classic Michaelis-Menten equation at low substrate concentrations (<80 mM). Kinetic analysis of the biotransformation revealed that L. brevis TCCC13007 resting cells produced GABA similar to that produced by purified glutamate decarboxylase from L. brevis.     

Biotransformation of aromatic aldehydes bySaccharomyces cerevisiae: Investigation of reaction rates

Summary The rate of production ofl-phenylacetyl carbinol bySaccharomyces cerevisiae in reaction mixtures containing benzaldehyde with sucrose or pyruvate as cosubstrate was investigated in short 1 h incubations. The effect of yeast dose rate, sucrose and benzaldehyde concentration and pH on the rate of reaction was determined. Maximum biotransformation rates were obtained with concentrations of benzaldehyde, sucrose and yeast of 6 g, 40 g and 60 g/l, respectively. Negligible biotransformation rates were observed at a concentration of 8 g/l benzaldehyde. The reaction had a pH optimum of 4.0–4.5. Rates of bioconversion of benzaldehyde and selected substituted aromatic aldehydes using both sucrose and sodium pyruvate as cosubstrate were compared. The rate of aromatic alcohol production was much higher when sucrose was used rather than pyruvate.o-Tolualdehyde and 1-chlorobenzaldehyde were poor substrates for aromatic carbinol formation although the latter produced significant aromatic alcohol in sucrose-containing media. Yields of 2.74 and 3.80 g/l phenylacetyl carbinol were produced from sucrose and pyruvate, respectively, in a 1 h reaction period.     

Enzymatic (R)-phenylacetylcarbinol production in benzaldehyde emulsions

(R)-Phenylacetylcarbinol [(R)-PAC)] is the chiral precursor for the production of the pharmaceuticals ephedrine and pseudoephedrine. Reaction conditions were improved to achieve increased (R)-PAC levels in a simple batch biotransformation of benzaldehyde emulsions and pyruvate, using partially purified pyruvate decarboxylase (PDC) from the filamentous fungus Rhizopus javanicus NRRL 13161 as the catalyst. Lowering the temperature from 23 degrees C to 6 degrees C decreased initial rates but increased final (R)-PAC concentrations. Addition of ethanol, which increases benzaldehyde solubility, was not beneficial for (R)-PAC production. It was established that proton uptake during biotransformation increases the pH above 7 thereby limiting (R)-PAC production. For small-scale studies, biotransformations were buffered with 2-2.5 M MOPS (initial pH 6.5). High concentrations of MOPS as well as some alcohols and KCl stabilised PDC. A balance between PDC and substrate concentrations was determined with regards to ( R)-PAC production and yields on enzyme and substrates. R. javanicus PDC (7.4 U/ml) produced 50.6 g/l (337 mM) ( R)-PAC in 29 h at 6 degrees C with initial 400 mM benzaldehyde and 600 mM pyruvate. Molar yields on consumed benzaldehyde and pyruvate were 97% and 59%, respectively, with 17% pyruvate degraded and 24% converted into acetaldehyde and acetoin; 43% PDC activity remained, indicating reasonable enzyme stability at high substrate and product concentrations.     

Biotransformation of benzaldehyde into (R)-phenylacetylcarbinol by filamentous fungi or their extracts

Extracts of 14 filamentous fungi were examined regarding their potential for production of (R)-phenylacetylcarbinol [(R)-PAC], which is the chiral precursor in the manufacture of the pharmaceuticals ephedrine and pseudoephedrine. Benzaldehyde and pyruvate were transformed at a scale of 1.2 ml into PAC by cell-free extracts of all selected strains, covering the broad taxonomic spectrum of Ascomycota, Zygomycota and Basidiomycota. Highest final PAC concentrations were obtained with the extracts of Rhizopus javanicus and Fusarium sp. [78-84 mM (11.7-12.6 g/l) PAC within 20 h from initial substrate concentrations of 100 mM benzaldehyde and 150 mM pyruvate]. (R)-PAC was in about 90-93% enantiomeric excess. Rhizopus javanicus had the advantage of faster growth than Fusarium sp. Rhizopus javanicus mycelia were used as an example in a biotransformation process based on whole cells and benzaldehyde and glucose as substrates. The substrate pyruvate was generated through the fungal fermentation of glucose. Only 19 mM PAC (2.9 g/l) were produced within 8 h from 80 mM benzaldehyde. with evidence of significant benzyl alcohol production.     

Production of pyruvate by isolated mouse cumulus cells

Cumulus cells were isolated by hyaluronidase treatment of whole cumulus masses from superovulated, non-mated mice. The cells, in groups of approximately 200, were incubated for up to 4 h in 50 nl medium M2 at 37 degrees C, and serial 3-nl samples assayed for pyruvate using an ultramicrofluorescence technique. With 5.55 mM glucose, 23.3 mM lactate, or a mixture of the two substrates, the cumulus cells formed pyruvate at rates of 10.2, 9.6, and 8.9 fmol/cell/h, respectively. The concentrations of glucose, pyruvate, and lactate, as measured in 3-nl aliquots of rabbit oviduct fluid were 1.5 mM, 0.3 mM, and 3.7 mM, respectively. When incubated with 1 mM glucose and 3 mM lactate, mouse cumulus cells formed 7.5 fmol pyruvate/cell/h. The mean number of cumulus cells per ovum within a cumulus mass was 2,060. Intact cumulus masses from mated and non-mated superovulated mice, incubated with 1 mM glucose and 3 mM lactate, formed 22.6 and 23.3 pmol pyruvate/ovum/h, respectively. The results suggest that pyruvate production by cumulus cells may be important in supporting the nutrition of unfertilized and fertilized ova, and of spermatozoa, within the oviduct lumen.     

Asymmetric synthesis of chiral amines with omega-transaminase.

The asymmetric synthesis of chiral amines using prochiral ketones was carried out with (S)-specific omega-transaminase (omega-TA) from Vibrio fluvialis JS17. This reaction is inhibited severely by both products, (S)-amine and deaminated ketone. In addition, thermodynamic equilibrium strongly favored the reverse reaction. L-Alanine proved to be the best amino donor based on easy removal of the products. Optimal pH of the reactions with both whole cells and cell-free extract was 7. Amino acceptor reactivities of ketone substrates and reaction profiles of the asymmetric synthesis showed that the initial rate as well as the reaction yield were lower when the resulting (S)-amine from a prochiral ketone substrate was a more reactive amino donor. The yield could be increased dramatically by removing pyruvate, which is a more inhibitory product than (S)-alpha-methylbenzylamine [(S)-alpha-MBA] when acetophenone and L-alanine are used as an amino acceptor and donor, respectively. The removal of pyruvate was carried out by incorporating lactate dehydrogenase (LDH) in cell-free extract or by using whole cells. The whole cell reaction yielded a much better result. When 25 mM benzylacetone and 30 mM acetophenone were used as an amino acceptor with 300 mM L-alanine, 90.2% and 92.1% of the reaction yields after 1 day were obtained with whole cells, respectively. Enantiomeric excesses of both (S)-alpha-MBA and (S)-1-methyl-3-phenylpropylamine [(S)-MPPA] were all above 99%.     

Efficient production of pyruvate from DL-lactate by the lactate-utilizing strain Pseudomonas stutzeri SDM

Background

The platform chemical lactate is currently produced mainly through the fermentation of sugars presented in biomass. Besides the synthesis of biodegradable polylactate, lactate is also viewed as a feedstock for the green chemistry of the future. Pyruvate, another important platform chemical, can be produced from lactate through biocatalysis.

Methodology/Principal Findings

It was established that whole cells of Pseudomonas stutzeri SDM catalyze lactate oxidation with lactate-induced NAD-independent lactate dehydrogenases (iLDHs) through the inherent electron transfer chain. Unlike the lactate oxidation processes observed in previous reports, the mechanism underlying lactate oxidation described in the present study excluded the costliness of the cofactor regeneration step and production of the byproduct hydrogen peroxide.

Conclusions/Significance

Biocatalysis conditions were optimized by using the cheap dl-lactate as the substrate and whole cells of the lactate-utilizing P. stutzeri SDM as catalyst. Under optimal conditions, the biocatalytic process produced pyruvate at a high concentration (48.4 g l⁻¹) and a high yield (98%). The bioconversion system provides a promising alternative for the green production of pyruvate.     

Relationship between hydroxypyruvate and the production of oxalate in vitro

Chicken liver lactate dehydrogenase (l-lactate: NAD⁺ oxidoreductase, EC 1.1.1.27) catalyses the reversible reduction reaction of hydroxypyruvate to l-glycerate. It also catalyses the oxidation reaction of the hydrated form of glyoxylate to oxalate and the reduction of the non-hydrated form to glycolate. At pH 8, these latter two reactions are coupled. The coupled system equilibrium is attained when the NAD⁺/NADH ratio is greater than unity.Hydroxypyruvate binds to the enzyme at the same site as the pyruvate. When there are substances with greater affinity to this site in the reaction medium and their concentration is very high, hydroxypyruvate binds to the enzyme at the l-lactate site. In vitro and with purified preparation of lactate dehydrogenase, hydroxypyruvate stimulates the production of oxalate from glyoxylate-hydrated form and from NAD; the effect is due to the fact that hydroxypyruvate prevents the binding of non-hydrated form of glyoxylate to the lactate dehydrogenase in the pyruvate binding site. At pH 8, the l-glycerate stimulates the production of glycolate from glyoxylate-non-hydrated form and NADH since hydroxypyruvate prevents the binding of glyoxylate-hydrated form to the enzyme.     

Conversion of linoleic acid into 10-Hydroxy-12(Z)-octadecenoic acid by whole cells of Stenotrophomonas nitritireducens

The conversion of linoleic acid into 10-hydroxy-12(Z)-octadecenoic acid by whole cells of Stenotrophomonas nitritireducens as an isolated bacterium was optimized, and the optimal temperature, pH, and cell and substrate concentrations were 30 degrees C, 7.5, and 20 and 20 g/L, respectively. Under these conditions, whole cells in a bioreactor produced 15 g/L 10-hydroxy-12(Z)-octadecenoic acid in 2 h of reaction time without detectable byproducts. Using 2 g/L linoleic acid, the cells produced 1.92 g/L 10-hydroxy-12(Z)-octadecenoic acid. These are the highest concentration and yield of 10-hydroxy-12(Z)-octadecenoic acid ever reported.     

静息细胞转化生产3-羟基丙酸的条件优化

本文研究了静息细胞生物转化生产3-羟基丙酸的反应体系。考察了以甘油为底物,利用静息细胞转化生产3一羟基丙酸的相关因素,确定了最佳的转化条件：细胞浓度20g／L,甘油浓度20g／L,辅酶VB12浓度10mg／L,NAD＋浓度0．15mmol／L,温度35℃,反应体系为0．05mol／LpH7．0Tris—HCl缓冲液。在上述条件下反应6h后,3-羟基丙酸的产量达到为3．17g／L,底物转化率为28．33％。由上述结果可知,采用静息细胞转化法为3-HP的生物合成提供了一种可能的方法。     

Biotransformation of 4-halophenols to 4-halocatechols using Escherichia coli expressing 4-hydroxyphenylacetate 3-hydroxylase

Escherichia coli cells, expressing 4-hydroxyphenylacetate 3-hydroxylase, fully transformed 4-halogenated phenols to their equivalent catechols as single products in shaken flasks. 4-Fluorophenol was transformed at a rate 1.6, 1.8, and 3.4-fold higher than the biotransformation of 4-chloro-, 4-bromo-, and 4-iodo-phenol, respectively. A scale-up from shaken flask to a 5 L stirred tank bioreactor was undertaken to develop a bioprocess for the production of 4-substituted halocatechols at higher concentrations and scale. In a stirred tank reactor, the optimized conditions for induction of 4-HPA hydroxylase expression were at 37 °C for 3 h. The rate of biotransformation of 4-fluorophenol to 4-fluorocatechol by stirred tank bioreactor grown cells was the same at 1 and 4.8 mM (5.13 μmol/min/g CDW) once the ratio of biocatalyst (E. coli CDW) to substrate concentration (mM) was maintained at 2:1. At 10.8 mM 4-fluorophenol, the rate of 4-fluorocatechol formation decreased by 4.7-fold. However, the complete transformation of 1.3 g of 4-fluorophenol (10.8 mM) to 4-fluorocatechol was achieved within 7 h in a 1 L reaction volume. Similar to 4-fluorophenol, other 4-substituted halophenols were completely transformed to 4-halocatechols at 2 mM within a 1–2 h period. An increase in 4-halophenol concentration to 4.8 mM resulted in a 2.5–20-fold decrease in biotransformation efficiency depending on the substrate tested. Organic solvent extraction of the 4-halocatechol products followed by column chromatography resulted in the production of purified products with a final yield of between 33% and 38%.