Evaluation of D-amino acid oxidase from Rhodotorula gracilis for the production of alpha-keto acids: a reactor system

The study reports on the development of a bioreactor for the production of alpha-keto acids from D,L- or D-amino acids using Rhodotorula gracilis D-amino acid oxidase. D-Amino acid oxidase was co-immobilized with catalase on Affi-Gel 10 matrix, and the reactor was operated as a continuous-stirred tank reactor (CSTR) or stirred tank with medium recycling conditions. The optimum substrate concentration and quantity of biocatalyst were determined (5 mM and 1.2 mg/L, respectively). Under optimum operating conditions, product formation was linearly related to both substrate and enzyme concentration, showing the system to be highly flexible. Under these conditions, in a stirred tank, over 90% conversion was achieved in 30 min with a maximum production of 0.23 g of pyruvic acid/day/enzyme units. Product was recovered by ion exchange chromatography. The operational stability of the reactor was high (up to 9.5 h of operation without loss of activity) and the inactivation half-life was not reached even after 18 h or 36 bioconversion cycles. This represents the first case of a reactor developed successfully with a D-amino acid oxidase. (c) 1994 John Wiley & Sons, Inc.     

Characterization of an industrial biocatalyst: immobilized glutaryl-7-ACA acylase

A batch of the immobilized industrial biocatalyst glutaryl-7-ACA acylase (GA), one of the two enzymes involved in the biotransformation of cephalosporin C (CefC) into 7-aminocephalosporanic acid (7-ACA), was characterized. K(m) value for glutaryl-7-ACA was 5 mM. Enzyme activity was found to be optimal at pH between 7 and 9.5 and to increase with temperature and in buffered solutions. To avoid product degradation, optimal reaction conditions were obtained working at 25 degrees C using a 50-mM phosphate buffer, pH 8.0. Immobilized GA showed good stability at pH value below 9 and at temperature up to 30 degrees C. The inactivation of immobilized GA in the presence of different amounts of H(2)O(2), a side product that might be present in the plant-scale industrial solutions of glutaryl-7-ACA, was also investigated, but the deactivation rates were negligible at H(2)O(2) concentration that might be reached under operative conditions. Finally, biocatalyst performance in the complete two-step enzymatic conversion process from CefC to 7-ACA was determined on a laboratory scale. Following the complete conversion of a 75 mM solution of CefC into glutaryl-7-ACA catalyzed by an immobilized D-amino acid oxidase (DAAO), immobilized GA was used for the transformation of this intermediate into the final product 7-ACA. This reaction was repeated for 42 cycles. An estimation of the residual activity of the biocatalyst showed that 50% inactivation of immobilized GA was reached after approximately 300 cycles, corresponding to an enzyme consumption of 0.4 kU per kg of isolated 7-ACA.     

Improvement of the enzymatic synthesis of ethyl valerate by esterification reaction in a solvent system

The present study reports the improved enzymatic synthesis of ethyl valerate (green apple flavor) by esterification reaction of ethanol and valeric acid in heptane medium. Lipase from Thermomyces lanuginosus (TLL) was immobilized by physical adsorption on polyhydroxybutyrate (PHB) particles and used as a potential biocatalyst. The effect of certain parameters that influence the ester synthesis was evaluated by factorial design. The experimental conditions that maximized the synthesis of ethyl valerate were 30.5°C, 18% m/v of biocatalyst (TLL–PHB), absence of molecular sieves, agitation of 234?rpm, and 1,000?mM of each reactant (ethanol and valeric acid). Under these conditions, conversion percentage ≈92% after 105?min of reaction was observed. Soluble TLL was also used as biocatalyst and the highest conversion was of 82% after 120?min of reaction. Esterification reaction performed in a solvent-free system exhibited conversion of 13% after 45?min of reaction catalyzed by immobilized lipase, while the soluble lipase did not exhibit catalytic activity. The synthesis of the ester was confirmed by Fourier transform infrared spectroscopy and gas chromatography–mass spectrometry analyses. After six consecutive cycles of ethyl valerate synthesis, the prepared biocatalyst retained ≈86% of its original activity.     

Adsorptive control of water in esterification with immobilized enzymes: I. Batch reactor behavior

Reducing the influence of an undesired product in an enzymatic reaction could have a significant impact on the productivity of such systems. Here, we focus on the removal of water formed during an enzymatic esterification in a batch reactor. A commercial immobilized lipase preparation, known as Lipozyme, is used as the biocatalyst and propionic acid and isoamyl alcohol dissolved in hexane are the substrates. In this system, the water formed will partition between the catalyst and the medium. As the more polar reactants are converted into the less polar ester product, the water is partitioned more towards the biocatalyst and the accumulation of water eventually causes lower reaction rates. Addition of a strong-acid cation exchange resin in sodium form is found to control the water accumulation on the biocatalyst without stripping the essential water needed for the enzyme to function and substantial improvements in conversion are achieved. A mathematical model is developed to describe the batch reaction behavior with and without added absorbent, which successfully predicts the behavior of water and its effects.     

Enzymatic reaction of beta-N-methylaminoalanine with L-amino acid oxidase

The reaction of beta-N-methylaminoalanine (BMAA) with L-amino acid oxidase (L-AAO) in the presence of catalase yields ammonia and beta-N-methylaminopyruvate, which was trapped as its 2,4-dinitrophenylhydrazone, as products. Incubation of BMAA with L-AAO in the presence of semicarbazide led to the formation of a semicarbazone, indicating intermediate iminium ion formation; when potassium cyanide (5 mM) was added, semicarbazone formation was blocked. The formation of beta-N-methylaminopyruvate was decreased by omission of catalase and was reduced in the presence of hydrogen peroxide (100 mM). These results indicate that BMAA is converted by L-AAO to the corresponding alpha-imino acid, which undergoes hydrolysis to beta-N-methylaminopyruvate. The alpha-keto acid is readily oxidized to N-methylglycine by hydrogen peroxide.     

Enzymatic synthesis of unsaturated fatty acid glucoside esters for dermo-cosmetic applications.

Unsaturated fatty acid alpha-butylglucoside esters were prepared by enzymatic esterification of alpha-butylglucoside in nonaqueous media. Conditions were firstly optimized using oleic acid as acyl group. Synthesis was possible in several solvents but the presence of water co-product in the medium limited the reaction to a thermodynamic equilibrium corresponding to a maximal conversion yield of 62%. In pure molten substrates, the removal of water under reduced pressure enabled yields superior to 95% to be obtained. Product profiles depended on enzyme origin : whatever the support, immobilized lipase B from Candida antarctica proved to be far more regioselective for the primary hydroxyl group of glucose than immobilized lipase from Rhizomucor miehei. Results obtained could be easily transposed to the acylation of alpha-butylglucoside with a commercial mixture of unsaturated fatty acids containing more than 60% of linoleic acid. The biocatalyst could be recycled more than ten times without any significant activity loss.     

Enzymatic oxidation of L-homocysteine

Homocyst(e)ine, a normal metabolite, accumulates in certain inborn errors of sulfur amino acid metabolism. Since many amino acids are converted by enzymatic oxidation and by transamination to the corresponding alpha-keto acid analogs and related products, which may exert inhibitory effects on metabolism, and because the alpha-keto acid analog of homocysteine has not yet been prepared, the enzymatic oxidation of homocysteine was investigated with the aim of obtaining alpha-keto-gamma-mercaptobutyric acid. Oxidation of DL-homocysteine by L-amino acid oxidase led to formation of at least seven products that react with 2,4-dinitrophenylhydrazine; of these, five were identified: alpha-keto-gamma-mercaptobutyrate, the mono and diketo analogs of homolanthionine, and the mono and diketo analogs of homocystine. In addition, one product was tentatively identified as alpha-ketomercaptobutyric acid gamma-thiolactone. In the course of this work alpha-keto-gamma-mercaptobutyrate was found to be a substrate of lactate dehydrogenase. L-Homocysteine and its alpha-keto acid analog were shown to be substrates of glutamate dehydrogenase and kidney glutamine transaminase. DL-Homocysteine reacts readily with alpha-keto acids to form stable hemithioketals, which were found to be substrates of L- and D-amino acid oxidases. A scheme is presented which integrates some of the complexities involved in the oxidation metabolism of homocyst(e)ine. The significance of these findings is considered in relation to the toxicity of homocysteine, which accumulates in certain pathological states.     

Ability of thermophilic lactic acid bacteria to produce aroma compounds from amino acids

Although a large number of key odorants of Swiss-type cheese result from amino acid catabolism, the amino acid catabolic pathways in the bacteria present in these cheeses are not well known. In this study, we compared the in vitro abilities of Lactobacillus delbrueckii subsp. lactis, Lactobacillus helveticus, and Streptococcus thermophilus to produce aroma compounds from three amino acids, leucine, phenylalanine, and methionine, under mid-pH conditions of cheese ripening (pH 5.5), and we investigated the catabolic pathways used by these bacteria. In the three lactic acid bacterial species, amino acid catabolism was initiated by a transamination step, which requires the presence of an alpha-keto acid such as alpha-ketoglutarate (alpha-KG) as the amino group acceptor, and produced alpha-keto acids. Only S. thermophilus exhibited glutamate dehydrogenase activity, which produces alpha-KG from glutamate, and consequently only S. thermophilus was capable of catabolizing amino acids in the reaction medium without alpha-KG addition. In the presence of alpha-KG, lactobacilli produced much more varied aroma compounds such as acids, aldehydes, and alcohols than S. thermophilus, which mainly produced alpha-keto acids and a small amount of hydroxy acids and acids. L. helveticus mainly produced acids from phenylalanine and leucine, while L. delbrueckii subsp. lactis produced larger amounts of alcohols and/or aldehydes. Formation of aldehydes, alcohols, and acids from alpha-keto acids by L. delbrueckii subsp. lactis mainly results from the action of an alpha-keto acid decarboxylase, which produces aldehydes that are then oxidized or reduced to acids or alcohols. In contrast, the enzyme involved in the alpha-keto acid conversion to acids in L. helveticus and S. thermophilus is an alpha-keto acid dehydrogenase that produces acyl coenzymes A.     

A possible involvement of sulfhydryl groups in the conversion of lysine monooxygenase to an oxidase.

Lysine monooxygenase catalyzes the oxygenation of lysine and arginine, and produces delta-amino-n-valeramide and gamma-guanidinobutyramide, respectively, concomitant with decarboxylation. In a preliminary communication, treatment of the native enzyme with p-chloromercuribenzoate was shown to inactivate the oxygenase and to induce an oxidase activity. The modified enzyme catalyzed predominantly the oxidative deamination of lysine and arginine resulting in the formation of the corresponding alpha-keto acid, ammonia, and hydrogen peroxide (YAMAUCHI, T., YAMAMOTO, S., and HAYAISHI, O.(1973) J. Biol. Chem. 2j8, 3750-3752). Paper electrophoresis, cellulose thin layer chromatography, and chemical degradation of the reaction products from lysine and arginine, provided further evidence for their identity with alpha-keto-epsilon-aminocaproate and alpha-keto-delta-guanidinovalerate, respectively. Further studies were carried out to establish the involvement of sulfhydryl groups in this conversion of the enzyme activities. Various sulfhydryl reagents including certain mercurials, alkylating, and oxidizing reagents, showed essentially identical effects on the enzyme. Dithiothreitol treatment reversed the conversion produced by various mercurials; the oxidase activity disappeared and the oxygenase activity was recovered. When p-chloromercuribenzoate was added to the enzyme and the increase in the absorbance at 250 nm was followed, 3.6 of the 6.5 half-cystine residues present per enzyme-bound FAD were readily titrated within 3 to 4 min. The inactivation of the oxygenase and the induction of the oxidase activity were almost maximal with 4 to 5 mol of p-chloromercuribenzoate/mol of enzyme, and these effects occurred within 3 to 4 min. These results together with other properties of the modified enzyme provided evidence for a possible involvement of these reactive sulfhydryl groups during the conversion of the oxygenase to an oxidase.     

Role of renal D-amino-acid oxidase in pharmacokinetics of D-leucine

d-Amino acids are now recognized to be widely present in mammals. Renal d-amino-acid oxidase (DAO) is associated with conversion of d-amino acids to the corresponding alpha-keto acids, but its contribution in vivo is poorly understood because the alpha-keto acids and/or l-amino acids formed are indistinguishable from endogenous compounds. First, we examined whether DAO is indispensable for conversion of d-amino acids to their alpha-keto acids by using the stable isotope tracer technique. After a bolus intravenous administration of d-[(2)H(7)]leucine to mutant mice lacking DAO activity (ddY/DAO(-)) and normal mice (ddY/DAO(+)), elimination of d-[(2)H(7)]leucine and formation of alpha-[(2)H(7)]ketoisocaproic acid ([(2)H(7)]KIC) and l-[(2)H(7)]leucine in plasma were determined. The ddY/DAO(-) mice, in contrast to ddY/DAO(+) mice, failed to convert d-[(2)H(7)]leucine to [(2)H(7)]KIC and l-[(2)H(7)]leucine. This result clearly revealed that DAO was indispensable for the process of chiral inversion of d-leucine. We further investigated the effect of renal mass reduction by partial nephrectomy on elimination of d-[(2)H(7)]leucine and formation of [(2)H(7)]KIC and l-[(2)H(7)]leucine. Renal mass reduction slowed down the elimination of d-[(2)H(7)]leucine. The fraction of conversion of d-[(2)H(7)]leucine to [(2)H(7)]KIC in sham-operated rats was 0.77, whereas that in five-sixths-nephrectomized rats was 0.25. The elimination behavior of d-[(2)H(7)]leucine observed in rats suggested that kidney was the principal organ responsible for converting d-leucine to KIC.     

Enhanced enzyme-catalyzed synthesis of l-methionine with ionic liquid additives

The low solubility of l-methionine and low activity of enzyme are the major hurdles during l-methionine production by the enzymatic conversion approach. In this study, we investigated various ionic liquids (ILs) as additives for the enzyme-catalyzed production of l-methionine from O-acetyl L-homoserine and methyl mercaptan. Among the ILs evaluated, we found that tetraalkylammonium hydroxide ILs enhanced the solubility of l-methionine as well as the activity of the enzyme. Methionine solubility decreased with increasing alkyl chain length but increased with increasing IL concentration. l-methionine could be dissolved up to 232 g/L in 10% tetramethylammonium hydroxide solution. The enzyme O-acetylhomoserine aminocarboxypropyltransferase reached its maximum activity when the IL concentration was 2.5% (3 times higher than that without ILs) and significantly decreased with increasing IL concentration. The stability of the enzyme also decreased rapidly after 2 h of incubation regardless of the presence or absence of ILs. Nevertheless, 74 g/L of l-methionine could be produced in a reaction media containing 2.5% tetraethylammonium hydroxide compared to 35 g/L of l-methionine obtained in a reaction system without ILs.     

Spores of Aspergillus niger as reservoir of glucose oxidase synthesized during solid-state fermentation and their use as catalyst in gluconic acid production

AIMS: To exploit conidiospores of Aspergillus niger as a vector for glucose oxidase extraction from solid media, and their direct use as biocatalyst in the bioconversion of glucose to gluconic acid. METHODS AND RESULTS: Spores of A. niger (200 h old) were shown to fully retain all the glucose oxidase synthesized by the mycelium during solid-state fermentation (SSF). They acted as catalyst and carried out the bioconversion reaction effectively, provided they were permeabilized by freezing and thawing. Glucose oxidase activity was found retained in the spores even after repeated washings. Average rate of reaction was 1.5 g l(-1) h(-1) with 102 g l(-1) of gluconic acid produced out of 100 g l(-1) glucose consumed after approx. 100 h reaction, which corresponded to a molar yield close to 93%. These results were obtained with permeabilized spores in the presence of a germination inhibitor, sodium azide. CONCLUSIONS: Spores of A. niger served as efficient catalyst in the model bioconversion reaction after permeabilization. SIGNIFICANCE AND IMPACT OF THE STUDY: To our knowledge, this is the first detailed study on the ability of A. niger spores to act as reservoir of enzyme synthesized during SSF without its release into solid media. Use of this material served as an innovative concept for enzyme extraction and purification from a solid medium. Moreover, this approach could compete efficiently with the conventional use of mycelial form of the fungus in gluconic acid production.     

Mathematical modelling of the dehydrogenase catalyzed hexanol oxidation with coenzyme regeneration by NADH oxidase

The hexanol oxidation catalyzed by alcohol dehydrogenase from baker's yeast (YADH) has been investigated with two different forms of the biocatalyst: the isolated YADH as well as the YADH in the permeabilized whole cells. It was found that in this reaction, equilibrium is shifted to the reduction side. Hence, to increase the conversion it was necessary to regenerate NAD⁺. For that purpose, enzyme NADH oxidase isolated from Lactobacillus brevis was used. All biocatalysts were kinetically characterized. The overall reaction rate was described by the mathematical model which consisted of kinetics and balance equations. Due to the deactivation of NADH oxidase, only 50–58% hexanol was converted to hexanal in the batch reactor where the hexanol oxidation was catalyzed by isolated YADH. In the case of permeabilized baker's yeast cells, no enzyme deactivation occurred and 100% hexanol conversion in the hexanoic acid was detected.     

Branched-chain alpha-keto acid catabolism via the gene products of the bkd operon in Enterococcus faecalis: a new, secreted metabolite serving as a temporary redox sink

Recently the bkd gene cluster from Enterococcus faecalis was sequenced, and it was shown that the gene products constitute a pathway for the catabolism of branched-chain alpha-keto acids. We have now investigated the regulation and physiological role of this pathway. Primer extension analysis identified the presence of a single promoter upstream of the bkd gene cluster. Furthermore, a putative catabolite-responsive element was identified in the promoter region, indicative of catabolite repression. Consistent with this was the observation that expression of the bkd gene cluster is repressed in the presence of glucose, fructose, and lactose. It is proposed that the conversion of the branched-chain alpha-keto acids to the corresponding free acids results in the formation of ATP via substrate level phosphorylation. The utilization of the alpha-keto acids resulted in a marked increase of biomass, equivalent to a net production of 0.5 mol of ATP per mol of alpha-keto acid metabolized. The pathway was active under aerobic as well as anaerobic conditions. However, under anaerobic conditions the presence of a suitable electron acceptor to regenerate NAD(+) from the NADH produced by the branched-chain alpha-keto acid dehydrogenase complex was required for complete conversion of alpha-ketoisocaproate. Interestingly, during the conversion of the branched-chain alpha-keto acids an intermediate was always detected extracellularly. With alpha-ketoisocaproic acid as the substrate this intermediate was tentatively identified as 1, 1-dihydroxy-4-methyl-2-pentanone. This reduced form of alpha-ketoisocaproic acid was found to serve as a temporary redox sink.     

Production of α-keto acids: 2. Immobilized whole cells of Providencia sp. PCM 1298 containing l-amino acid oxidase

A number of bacteria belonging to the genera Proteus, Providencia, Pseudomonas and Erwinia have been tested for their capacity to oxidize l-amino acids to their corresponding α-keto acids. Members of the Proteus and the Providencia genera were active towards various l-amino acids. Immobilized cell preparations of Providencia sp. PCM 1298 were shown to form up to 80 mg α-keto-γ-methiol butyric acid from l-methionine per g of gel preparation (containing 4% w/w cells) per day. The productivity was highly dependent on the size of the beads. Oxygen appeared to be the rate-limiting substrate and oxygen transfer rates of 3–4 μmol cm^?2h^?1were calculated. The entrapment of activated charcoal to remove H₂O₂ formed during the oxidation extended the half-life of the immobilized biocatalyst considerably. A decrease in l-amino acid oxidase [l-amino acid: oxygen oxidoreductase (deaminating); EC 1.4.3.2] activity during operation could be compensated for by reinoculation of the alginate-entrapped cells in fresh growth medium, allowing use of these preparations of immobilized bacterial cells for more than one month.     

Stepwise engineering of a <Emphasis Type="Italic">Pichia pastoris</Emphasis> D-amino acid oxidase whole cell catalyst

Background  

Trigonopsis variabilis D -amino acid oxidase (Tv DAO) is a well characterized enzyme used for cephalosporin C conversion on industrial scale. However, the demands on the enzyme with respect to activity, operational stability and costs also vary with the field of application. Processes that use the soluble enzyme suffer from fast inactivation of Tv DAO while immobilized oxidase preparations raise issues related to expensive carriers and catalyst efficiency. Therefore, oxidase preparations that are more robust and active than those currently available would enable a much broader range of economically viable applications of this enzyme in fine chemical syntheses. A multi-step engineering approach was chosen here to develop a robust and highly active Pichia pastoris Tv DAO whole-cell biocatalyst.     

Preparation of L-aspartic acid by means of immobilized Alcaligenes metalcaligenes cells

Two types of biocatalysts based on immobilized cells of Alcaligenes metalcaligenes exhibiting aspartate ammonia-lyase activity (EC 4.3.1.1) were developed for the enzymic preparation of L-aspartic acid from ammonium fumarate. The first type of the biocatalyst consists in individual covalently crosslinked and permeabilized cells(I), while the second type is represented by cell aggregates (II). For the above preparation, biocatalyst I can be used only discontinuously in a mixed reactor. After termination of the reaction between individual cycles of its use, the biocatalyst is returned to the reactor in the form of a highly concentrated cell suspension or paste. Biocatalyst II can be used discontinuously or continuously in a fixed-bed column of the catalyst. The effects of pH, substrate concentration and temperature on the reaction velocity and effectivity of enzymic conversion was investigated. Optimal parameters of the reaction are as follows: pH 8.5, initial substrate concentration, 1.35 mol/L, temperature for discontinuous process, 37 degrees C, and temperature for continuous process, 25 degrees C. Under these conditions the enzymic conversion of substrate to product is quantitative. Under optimal toring conditions, the specific activity of both catalysts does not change within a period of one year. The operational half-life of the biocatalyst II during continuous use in a fixed-bed column of the catalyst under standard reaction conditions depends on the quality of the substrate. The discontinuous preparation of L-asparatic acid with the aid of biocatalyst I and continuous preparation of this product with the aid of biocatalyst II have been verified under pilot-plant conditions.     

Overexpression of a recombinant wild-type and His-tagged Bacillus subtilis glycine oxidase in Escherichia coli.

We have cloned the gene coding for the Bacillus subtilis glycine oxidase (GO), a new flavoprotein that oxidizes glycine and sarcosine to the corresponding alpha-keto acid, ammonia and hydrogen peroxide. By inserting the DNA encoding for GO into the multiple cloning site of the expression vector pT7.7 we produced a recombinant plasmid (pT7-GO). The pT7-GO encodes a fully active fusion protein with six additional residues at the N-terminus of GO (MARIRA). In BL21(DE3)pLysS Escherichia coli cells, and under optimal isopropyl thio-beta-D-galactoside induction conditions, soluble and active chimeric GO was expressed up to 1.14 U g(-1) of cell (and a fermentation yield of 3.82 U x L(-1) of fermentation broth). An N-terminal His-tagged protein (HisGO) was also successfully expressed in E. coli as a soluble protein and a fully active holoenzyme. HisGO represents approximately 3.9% of the total soluble protein content of the cell. The His-tagged GO was purified in a single step by nickel-chelate chromatography to a specific activity of 1.06 U x mg(-1) protein at 25 degrees C and with a yield of 98%. The characterization of the purified enzyme showed that GO is a homotetramer of approximately 180 kDa with the spectral properties typical of flavoproteins. GO exhibits good thermal stability, with a Tm of 46 degrees C after 30 min incubation; its stability is maximal in the 7.0-8.5 pH range. A comparison of amino-acid sequence and substrate specificity indicates that GO has similarities to other flavoenzymes acting on primary amines and on D-amino acids.     

Reactivation of biocatalysts after long-term storage and startup of the DEAMOX process

Studies of our laboratory have led to elaboration of the DEAMOX (DEnitrifying AMmonia OXidation) technology intended for removal of nitrogen contaminants from wastewater. The DEAMOX process comprises two anaerobic stages implemented by the same sludge biocatalyst, namely, denitratation (conversion of nitrate to nitrite) and anammox reaction (ANaerobic AMmonium nitrogen OXidation by nitrite). The results of reactivation of biocatalysts after their long-term storage (5 and 16 months) and successful startup of the DEAMOX process in two modifications (S- and O-) are described. An S-DEAMOX process was launched using a sludge biocatalyst with restored anammox activity of 20.1 mg N/g VSS/day; this process provided removal of 78% of nitrogen in reactor over 20 days. The launched O-DEAMOX process with the sludge biocatalyst with anammox activity of 6.1 mg N/g VSS/day provided for 87% removal of the total nitrogen compounds over 30 days. Two different electron donors were used at the stage of nitrate conversion to nitrite, namely, an inorganic donor, sulfide (S-DEAMOX), and an organic one, acetate (O-DEAMOX).