Development of a multienzyme reactor for dopamine synthesis: II. Reactor engineering and simulation

Aspects of reaction engineering associated with multienzyme reactions have been studied in a system where dopamine is produced from catechol, pyruvate and ammonium by sequential enzymatic reactions catalyzed by tyrosine phenol lyase (TPL) and tyrosine decarboxylase (TDC). Microbial cells containing TPL activity (Erwinia herbicola) and TDC activity (Streptococcus faecalis) were coimmobilized in glutaraldehyde cross-linked porcine gelatin beads with a mean diameter of 2.8 mm for use in the reactions. Measurement of the transport properties in the beads indicate that the gelatin matrix does not significantly increase the diffusion resistance and that dopamine partitions into the matrix (K = 2). A packed-bed reactor containing the coimmobilized cell beads successfully produced dopamine, although with a low conversion. Using computer simultaneous it is shown that separate, sequential TPL and TDC, rather than simultaneous, reactions, would require smaller reactors overall for the same conversion. (c) 1992 John Wiley & Sons, Inc.     

Characterization of free and alginate-polylysine-alginate microencapsulated Erwinia herbicola for the conversion of ammonia, pyruvate, and phenol into L-tyrosine

The whole cell tyrosine phenol-lyase (TPL, E.C. 4.1.99.2) activity of Erwinia herbicola (ATCC 21434) was microen-capsulated. We studied the use of this for the conversion of ammonia and pyruvate along with phenol or catechol, respectively, into L-tyrosine or dihydroxyphenyl-L-alanine (L-dopa). The reactions are relevant to the development of new methods for the production of L-tyrosine and L-dopa. The growth of E. herbicola at temperatures from 22 degrees C to 32 degrees C is stable, since at these temperatures the cells grow up to the stationary phase and remain there for at least 10 h. At 37 degrees C the cells grow rapidly, but they also enter the death phase rapidly. There is only limited growth of E. herbicola at 42 degrees C. Whole cells of E. herbicola were encapsulated within alginate-polylysine-alginate microcapsules (916 +/- 100 mum, mean +/- std. dev.). The TPL activity of the cells catalyzed the production of L-tyrosine or dihydroxyphenyl-L-alanine (L-dopa) from ammonia, pyruvate, and phenol or catechol, respectively. In the production of tyrosine, an integrated equation based on an ordered ter-uni rapid equilibrium mechanism can be used to find the kinetic parameters of TPL. In an adequately stirred system, the apparent values of-the kinetic parameters of whole cell TPL are equal whether the cells are free or encapsulated. The apparent K(M) of tyrosine varies with the amount of whole cells in the system, ranging from 0.2 to 0.3 mM. The apparent K(M) for phenol is 0.5 mM. The apparent K(M) values for pyruvate and ammonia are an order of magnitude greater for whole cells than they are for the cell free enzyme. (c) 1995 John Wiley & Sons, Inc.     

Production of racemic lactic acid in Pediococcus cerevisiae cultures by two lactate dehydrogenases.

Nicotinamide adenine dinucleotide (NAD)-dependent d(minus)-and l(plus)-lactate dehydrogenases have been partially purified 89- and 70-fold simultaneously from cell-free extracts of Pediococcus cerevisiae. Native molecular weights, as estimated from molecular sieve chromatography and electrophoresis in nondenaturing polyacrylamide gels, are 71,000 to 73,000 for d(minus)-lactate dehydrogenase and 136,000 to 139,000 for l(plus)-lactate dehydrogenase. Electrophoresis in sodium dodecyl sulfate-containing gels reveals subunits with approximate molecular weights of 37,000 to 39,000 for both enzymes. By lowering the pyruvate concentration from 5.0 to 0.5 mM, the pH optimum for pyruvate reduction by d(minus)-lactate dehydrogenase decreases from pH 8.0 to 3.6. However, l(plus)-lactate dehydrogenase displays an optimum for pyruvate reduction between pH 4.5 and 6.0 regardless of the pyruvate concentration. The enzymes obey Michaelis-Menten kinetics for both pyruvate and reduced NAD at pH 5.4 and 7.4, with increased affinity for both substrates at the acid pH. alpha-Ketobutyrate can be used as a reducible substrate, whereas oxamate has no inhibitory effect on lactate oxidation by either enzyme. Adenosine triphosphate causes inhibition of both enzymes by competition with reduced NAD. Adenosine diphosphate is also inhibitory under the same conditions, whereas NAD acts as a product inhibitor. These results are discussed with relation to the lactate isomer production during the growth cycle of P. cerevisiae.     

L-dopa and dopamine enhance the formation of aggregates under proteasome inhibition in PC12 cells

The formation of inclusion bodies in dopaminergic neurons is associated with the pathogenesis of Parkinson's disease. In order to clarify the role of dopamine/L-dopa in the formation of protein aggregates, we investigated dopamine/L-dopa-related aggregation using an experimental inclusion model. The inhibition of tyrosine hydroxylase (TH) by alpha-methyltyrosine dramatically decreased MG132-induced aggregate formation. In addition, the inhibition of TH caused the upregulation of proteasomes in cultured cells and the dopamine/L-dopa induced non-enzymatic polymerization of ubiquitin. This inhibition did not affect cell viability. These results suggest that dopamine/L-dopa might enhance aggregate formation, and that intracellular aggregates may not be toxic to cells.     

酪氨酸羟化酶和左旋芳香族氨基酸脱羧酶基因的细胞表达及活性检测

由于在帕金森病中合成多巴胺所需的酪氨酸羟化酶(tyrosine hydroxylase,TH)和左旋芳香族氨基酸脱羧酶(aromatic L-amino acid decarboxylase,AADC)活性明显降低,所以补充多巴胺合成酶成为基因治疗帕金森病研究的主要手段。我们分别构建了重组逆转录病毒载体pLHCX／TH及pLNCX2／AADC,通过脂质体介导将带有目的基因的载体分别转到包装细胞PA317中,经筛选得到产病毒的细胞PA317／TH和PA317／AADC,采用免疫组化、原位杂交方法检测目的基因表达;细胞经裂解后进行的酶促反应产物多巴胺以高压液相电化学方法检测证明所克隆的T‘H及AADC基因具有功能活性;这两种基因工程改造细胞可以完成酶促动力学的功能,使L-dopa及多巴胺产生明显增加。本研究为用TH和AADC双基因对帕金森病进行基因治疗提供了一定的依据。     

Polyphenol oxidases from latex of Hevea brasiliensis: purification and characterization

Polyphenol oxidase (PPO) was isolated from the B-serum obtained after repetitive freeze-thawing of the bottom fraction isolated from ultracentrifuged fresh latex. The B-serum was subjected to acetone precipitation and CM-Sepharose chromatography, affording two PPOs, PPO-I and PPO-II, which, upon SDS-PAGE, were 32 and 34 kDa, respectively. Both PPOs possessed the same pI (9.2), optimum pH (7) and optimum temperature (35-45 degrees C). They are stable up to 60 degrees C and active at broad pH ranges from 4-9. The K(m) values of PPO-I for dopamine, L-dopa and catechol as substrates are 2.08, 8.33 and 9.09 mM, while those for PPO-II are 2.12, 4.76 and 7.14 mM, respectively. Among various PPO inhibitors tested, 4-hexylresorcinol was the most potent. Anionic detergents were among the most effective activators of the enzymes, while cationic and nonionic detergents showed little and no effect on the PPO activities, respectively.     

Inhibition of the soluble form of testis adenylate cyclase by catechol estrogens and other catechols

The soluble form of rat germ cell adenylate cyclase was inhibited by compounds with a catechol moiety. Among the naturally occurring catechols tested, catechol estrogens were the most potent inhibitors. Catechol estrogens at 2-6 microM inhibited enzyme activity by 50% and almost completely at 30-100 microM concentration. The inhibitory activity of catechol estrogens depends on the catechol moiety of the molecule. Catechol per se also inhibited the activity of this enzyme, 50% inhibition being achieved at about 11 microM. The two hydroxyls of the catechol moiety are essential for the inhibitory interaction with the enzyme. Thus, aromatic compounds containing only one hydroxyl group in the benzene ring, such as tyrosine, phenylephrine, estradiol, and 6 alpha-hydroxyestradiol were either completely inactive or had marginal inhibitory activity at concentrations up to 0.3-1 mM. Moreover, methylation of the hydroxyl groups of the catechol moiety of the catechol estrogens as in 2-methoxyestradiol 3-methyl ether rendered the catechol estrogens inactive. The inhibitory potency of these compounds varied greatly depending on the structure associated with the catechol ring. Thus, compounds in which catechol is associated with an aliphatic side chain, such as dopamine, L-dopa, norepinephrine, and isoproterenol, were about 11- to 34-fold less potent than catechol. On the other hand, compounds in which catechol is associated either with a hydroaromatic ring system, as in apomorphine, or with an alicyclic ring system, as in catechol estrogens, were about 2- to 5-fold more potent than catechol. The inhibitory effect of dopamine, apomorphine, and catechol estrogens was not affected by specific D-1 or D-2 antagonist, indicating that they do not act via receptors for dopamine.     

Studies of regulatory metabolism in Moniezia expansa: the role of phosphofructokinase (with a note on pyruvate kinase).

Some of the properties of a partially purified preparation of phosphofructokinase (PFK) from Moniezia expansa are described. PFK has a pH optimum between 7·4 and 8·0, and is activated by magnesium and divalent manganese ions. It exhibits sigmoid kinetics with fructose-6-phosphate, and ATP decreases the affinity of the enzyme for F6P. This inhibition is partially relieved by F6P, AMP and ammonium ions. GTP and ITP act as substrates for the PFK reaction but do not exert the same inhibitory effects. The effect of ATP on pyruvate kinase was also examined, and was found to inhibit both the activated and inactivated enzyme. Apparent K_m's for both enzymes are presented.Generally, PFK and pyruvate kinase from M. expansa show properties similar to the enzymes from mammalian sources. The presence of sigmoid kinetics for F6P and ATP at pH8 is, however, a significant departure from what is observed in PFK from mammalian sources. Possibilities exist in M. expansa for controls of metabolism similar to those found in mammalian tissues.     

Concomitant elevation of tyrosine hydroxylase and dopamine beta-hydroxylase by cyclic AMP in cultured mouse neuroblastoma cells.

The effects of cyclic AMP analogues and of phosphodiesterase inhibitors were investigated in neuroblastoma cells (NBD-2) cloned from the C-1300 tumor. 8Br-cAMP and phosphodiesterase inhibitors that elevated cAMP induced large (greater than 15 fold) and specific increases in tyrosine hydroxylase and dopamine beta-hydroxylase activity. In contrast, catechol O-methyltransferase, monoamine oxidase and aromatic-l -amino-acid decarboxylase were unaffected by the cAMP altering drugs. Similarly, AChE was unaffected and only a small increase in choline acetyltransferase (3 fold) was observed. The increases in tyrosine hydroxylase and dopamine beta-hydroxylase were similar with respect to dose response relationships and with respect to time course of onset. Only those phosphodiesterase inhibitors that elevated cAMP (papaverine and Ro20-1724 as opposed to theophylline) were effective in elevating tyrosine hydroxylase and dopamine beta-hydroxylase. Further, the doses optimal for elevating cAMP coincided with the optimal doses for elevating the two enzymes. Theophylline had no influence either upon NBD-2 cell cAMP levels or upon tyrosine hydroxylase and dopamine beta-hydroxylase activity. The changes in protein synthesis rates produced by the cAMP altering drugs were temporally distinct from the changes in either tyrosine hydroxylase or dopamine beta-hydroxylase. These results suggest that the intracellular messenger compound cAMP is involved in the specific regulation of both tyrosine hydroxylase and dopamine beta-hydroxylase in adrenergic cells.     

Signaling cascade of insulin-induced stimulation of L-dopa uptake in renal proximal tubule cells

The inward l-dihydroxyphenylalanine (L-dopa) transport supplies renal proximal tubule cells (PTCs) with the precursor for dopamine synthesis. We have previously described insulin-induced stimulation of L-dopa uptake into PTCs. In the present paper we examined insulin-related signaling pathways involved in the increase of l-dopa transport into isolated rat PTCs. Insulin (50-500 microU/ml) increased L-dopa uptake by PTCs, reaching the maximal increment (60% over the control) at 200 microU/ml. At this concentration, insulin also increased insulin receptor tyrosine phosphorylation. Both effects were abrogated by the tyrosine kinase inhibitor genistein (5 microM). In line, inhibition of the protein tyrosine phosphatase by pervanadate (0.2-100 microM) caused a concentration-dependent increase in both the uptake of L-dopa (up to 400%) and protein tyrosine phosphorylation. A synergistic effect between pervanadate and insulin on L-dopa uptake was observed only when threshold (0.2 microM), but not maximal (5 microM), concentrations of pervanadate were assayed. Insulin-induced stimulation of L-dopa uptake was also abolished by inhibition of phosphatidylinositol 3-kinase (PI3K; 100 nM wortmannin, and 25 microM LY-294002) and protein kinase C (PKC; 1 microM RO-318220). Insulin-induced activation of PKC-zeta was confirmed in vitro by its translocation from the cytosol to the membrane fraction, and in vivo by immunohistochemistry studies. Insulin caused a wortmannin-sensitive increase in Akt/protein kinase B (Akt/PKB) phosphorylation and a dose-dependent translocation of Akt/PKB to the membrane fraction. Our findings suggest that insulin activates PKC-zeta, and Akt/PKB downstream of PI3K, and that these pathways contribute to the insulin-induced increase of L-dopa uptake into PTCs.     

The effects of alpha-adrenergic stimulation on the regulation of the pyruvate dehydrogenase complex in the perfused rat liver

The regulation of the pyruvate dehydrogenase multienzyme complex was investigated during alpha-adrenergic stimulation with phenylephrine in the isolated perfused rat liver. The metabolic flux through the pyruvate dehydrogenase reaction was monitored by measuring the production of 14CO2 from infused [1-14C] pyruvate. In livers from fed animals perfused with a low concentration of pyruvate (0.05 mM), phenylephrine infusion significantly inhibited the rate of pyruvate decarboxylation without affecting the amount of pyruvate dehydrogenase in its active form. Also, phenylephrine caused no significant effect on tissue NADH/NAD+ and acetyl-CoA/CoASH ratios or on the kinetics of pyruvate decarboxylation in 14CO2 washout experiments. Phenylephrine inhibition of [1-14C]pyruvate decarboxylation was, however, closely associated with a decrease in the specific radioactivity of perfusate lactate, suggesting that the pyruvate decarboxylation response simply reflected dilution of the labeled pyruvate pool due to phenylephrine-stimulated glycogenolysis. This suggestion was confirmed in additional experiments which showed that the alpha-adrenergic-mediated inhibitory effect on pyruvate decarboxylation was reduced in livers perfused with a high concentration of pyruvate (1 mM) and was absent in livers from starved rats. Thus, alpha-adrenergic agonists do not exert short term regulatory effects on pyruvate dehydrogenase in the liver. Furthermore, the results suggest either that the rat liver pyruvate dehydrogenase complex is insensitive to changes in mitochondrial calcium or that changes in intramitochondrial calcium levels as a result of alpha-adrenergic stimulation are considerably less than suggested by others.     

Metabolic regulation of carbon and electron flow as a function of pH during growth of Sarcina ventriculi

The physiology and biochemistry of Sarcina ventriculi was studied in order to determine adaptations made by the organism to changes in environmental pH. The organism altered carbon and electron flow from acetate, formate and ethanol production at neutral pH, to predominantly ethanol production at pH 3.0. Increased levels of pyruvate dehydrogenase (relative to pyruvate decarboxylase) and acetaldehyde dehydrogenase occurred when the organism was grown at neutral pH, indicating the predominance of carbon flux through the oxidative branch of the pathway for pyruvate metabolism. When the organism was grown at acid pH, there was a significant increase in pyruvate decarboxylase levels and a decrease in acetaldehyde dehydrogenase, causing flux through the non-oxidative branch of the pathway. CO₂ reductase and formate dehydrogenase were not regulated as a function of growth pH. Pyruvate dehydrogenase possessed Michaelis-Menten kinetics for pyruvate with an apparent K _m of 2.5 mM, whereas pyruvate decarboxylase exhibited sigmoidal kinetics, with a S_0.5 of 12.0 mM. Differences in total protein banding patterns from cells grown at pH extremes suggested that synthesis of pyruvate decarboxylase and other enzymes was in part responsible for metabolic regulation of the fermentation products formed.     

Structure and mechanism of tryptophan indole-lyase and tyrosine phenol-lyase

Tyrosine phenol-lyase (TPL) and tryptophan indole-lyase (Trpase) catalyse the reversible hydrolytic cleavage of L-tyrosine or L-tryptophan to phenol or indole, respectively, and ammonium pyruvate. These enzymes are very similar in sequence and structure, but show strict specificity for their respective physiological substrates. We have mutated the active site residues of TPL (Thr(124), Arg(381), and Phe(448)) to those of Trpase and evaluated the effects of the mutations. Tyr(71) in Citrobacter freundii TPL, and Tyr(74) in E. coli Trpase, are essential for activity with both substrates. Mutation of Arg(381) of TPL to Ala, Ile, or Val (the corresponding residues in the active site of Trpase) results in a dramatic decrease in L-Tyr beta-elimination activity, with little effect on the activity of other substrates. Arg(381) may be the catalytic base with pK(a) of 8 seen in pH-dependent kinetic studies. T124D TPL has no measureable activity with L-Tyr or 3-F-L-Tyr as substrate, despite having high activity with SOPC. T124A TPL has very low but detectable activity, which is about 500-fold less than wild-type TPL, with L-Tyr and 3-F-L-Tyr. F448H TPL also has very low activity with L-Tyr. None of the mutant TPLs has any detectable activity with L-Trp as substrate. H463F Trpase also exhibits low activity with L-Trp, but retains high activity with other substrates. Thus, additional residues remote from the active site may be needed for substrate specificity. Both Trpase and TPL may react by a rare S(E)2-type mechanism.     

First evidence of a membrane‐bound,tyramine and β‐phenylethylamine producing,tyrosine decarboxylase in Enterococcus faecalis: A two‐dimensional electrophoresis proteomic study

The soluble and membrane proteome of a tyramine producing Enterococcus faecalis, isolated from an Italian goat cheese, was investigated. A detailed analysis revealed that this strain also produces small amounts of β‐phenylethylamine. Kinetics of tyramine and β‐phenylethylamine accumulation, evaluated in tyrosine plus phenylalanine‐enriched cultures (stimulated condition), suggest that the same enzyme, the tyrosine decarboxylase (TDC), catalyzes both tyrosine and phenylalanine decarboxylation: tyrosine was recognized as the first substrate and completely converted into tyramine (100% yield) while phenylalanine was decarboxylated to β‐phenylethylamine (10% yield) only when tyrosine was completely depleted. The presence of an aspecific aromatic amino acid decarboxylase is a common feature in eukaryotes, but in bacteria only indirect evidences of a phenylalanine decarboxylating TDC have been presented so far. Comparative proteomic investigations, performed by 2‐DE and MALDI‐TOF/TOF MS, on bacteria grown in conditions stimulating tyramine and β‐phenylethylamine biosynthesis and in control conditions revealed 49 differentially expressed proteins. Except for aromatic amino acid biosynthetic enzymes, no significant down‐regulation of the central metabolic pathways was observed in stimulated conditions, suggesting that tyrosine decarboxylation does not compete with the other energy‐supplying routes. The most interesting finding is a membrane‐bound TDC highly over‐expressed during amine production. This is the first evidence of a true membrane‐bound TDC, longly suspected in bacteria on the basis of the gene sequence.     

Citrobacter freundii休止细胞催化合成L-多巴

以在L-酪氨酸诱导下高效表达酪氨酸酚解酶的菌株Citrobacter freundii 48003-3的休止细胞为生物催化剂,以邻苯二酚、丙酮酸钠、醋酸铵为前体,选择性合成L-DOPA。研究了反应温度、pH和前体浓度等对合成L-DOPA的影响。最优反应条件下,反应12h,L-DOPA的量可达到9．5g／L。     

Pancreatic acinar cells utilize tyrosine to synthesize L-dihydroxyphenylalanine

The pancreatic β cells can synthesize dopamine by taking L-dihydroxyphenylalanine, but whether pancreatic acinar cells synthesize dopamine has not been confirmed. By means of immunofluorescence, the tyrosine hydroxylase -immunoreactivity and aromatic amino acid decarboxylase (AADC)- immunoreactivity were respectively observed in pancreatic acinar cells and islet β cells. Treatment with L-dihydroxyphenylalanine, not tyrosine, caused the production of dopamine in the incubation of INS-1 cells (rat islet β cell line) and primary isolated islets, which was blocked by AADC inhibitor NSD-1015. However, only L-dihydroxyphenylalanine, but not dopamine, was detected when AR42J cells (rat pancreatic acinar cell line) were treated with tyrosine, which was blocked by tyrosine hydroxylase inhibitor AMPT. Dopamine was detected in the coculture of INS-1 cells with AR42J cells after treatment with tyrosine. In an in vivo study, pancreatic juice contained high levels of L-dihydroxyphenylalanine and dopamine. Both L-dihydroxyphenylalanine and dopamine accompanied with pancreatic enzymes and insulin in the pancreatic juice were all significantly increased after intraperitoneal injection of bethanechol chloride and their increases were all blocked by atropine. Inhibiting TH with AMPT blocked bethanechol chloride-induced increases in L-dihydroxyphenylalanine and dopamine, while inhibiting AADC with NSD-1015 only blocked the dopamine increase. Bilateral subdiaphragmatic vagotomy of rats leads to significant decreases of L-dihydroxyphenylalanine and dopamine in pancreatic juice. These results suggested that pancreatic acinar cells could utilize tyrosine to synthesize L-dihydroxyphenylalanine, not dopamine. Islet β cells only used L-dihydroxyphenylalanine, not tyrosine, to synthesize dopamine. Both L-dihydroxyphenylalanine and dopamine were respectively released into the pancreatic duct, which was regulated by the vagal cholinergic pathway. The present study provides important evidences for the source of L-dihydroxyphenylalanine and dopamine in the pancreas.     

Tyrosine Decarboxylase and Dopa Decarboxylase in Drosophila virilis Under Normal Conditions and Heat Stress: Genetic and Physiological Aspects

The activity of tyrosine decarboxylase (TDC) and dopa decarboxylase (DDC) was studied in adults of two lines of Drosophila virilis,contrasting in their reaction to stress conditions. Differences were found in the activity of both enzymes between individuals of the examined lines. Genetic analysis of these differences was made. Each of the two enzymes was found to be controlled by a single gene or, possibly, by a block of closely linked genes. The gene responsible for TDC activity is located on one of the autosomes (excluding chromosome II). DDC activity in D. virilisis regulated by a gene located, apparently, on chromosome II. Adults of the line responding to stress by a stress reaction (r-line) were shown to react to a short-term heat stress (38°C, 60 min) by a decrease in TDC activity. TDC activity in flies of the line incapable of the stress reaction (nr-line) did not alter in such conditions. DDC activity of adults of both lines was found to be unchangeable under stress conditions.     

Characterization of SafC, a catechol 4-O-methyltransferase involved in saframycin biosynthesis

Members of the saframycin/safracin/ecteinascidin family of peptide natural products are potent antitumor agents currently under clinical development. Saframycin MX1, from Myxococcus xanthus, is synthesized by a nonribosomal peptide synthetase, SafAB, and an O-methyltransferase, SafC, although other proteins are likely involved in the pathway. SafC was overexpressed in Escherichia coli, purified to homogeneity, and assayed for its ability to methylate a variety of substrates. SafC was able to catalyze the O-methylation of catechol derivatives but not phenols. Among the substrates tested, the best substrate for SafC was L-dihydroxyphenylalanine (L-dopa), which was methylated specifically in the 4'-O position (k(cat)/K(m) = 5.5 x 10(3) M(-1) s(-1)). SafC displayed less activity on other catechol derivatives, including catechol, dopamine, and caffeic acid. The more labile l-5'-methyldopa was an extremely poor substrate for SafC (k(cat)/K(m) = approximately 2.8 x 10(-5) M(-1) s(-1)). L-dopa thioester derivatives were also much less reactive than L-dopa. These results indicate that SafC-catalyzed 4'-O-methylation of L-dopa occurs prior to 5'-C-methylation, suggesting that 4'-O-methylation is likely the first committed step in the biosynthesis of saframycin MX1. SafC has biotechnological potential as a methyltransferase with unique regioselectivity.     

Structure and function of L-lactate dehydrogenases from thermophilic and mesophilic bacteria. I) Isolation and characterization of lactate dehydrogenases from thermophilic and mesophilic bacilli.

Lactate dehydrogenases from thermophilic bacilli (Bacillus stearothermophilus, Bacillus caldotenax) and from mesophilic bacilli (Bacillus X1, Bacillus subtilis) have been isolated by a two-step purification procedure. Only one type (LDH-P4) composed of four identical subunits (Mr 34 000 or 36 000) was found in each bacillus. The tetrameric enzymes were characterized with respect to thermostability, pH and temperature dependence of the pyruvate reduction and the L-lactate oxidation, substrate specificity, saturation kinetics (Km values of pyruvate, lactate, NAD, NADH), pyruvate and oxamate inhibition, and activation by fructose bisphosphate. The thermophilic and mesophilic enzymes differ characteristically in these parameters. Preliminary structural data (amino acid composition, comparative N-terminal sequence analysis) show the expected close phylogenetic relationship (high degree of sequence homology), but also typical differences between thermophilic and mesophilic dehydrogenases, a suitable basis for further comparative studies.     

Enzymatic synthesis of 3,4-dihydroxyphenyl-L-alanine by free and immobilized Citrobacter freundii cells

The Citrobacter freundii 62 cells immobilized in PAAG and possessing the tyrosine-phenol-lyase (TPL) activity catalyse the synthesis of 3,4-dihydroxyphenyl-L-alanine (DOPA) from pyrocatechol and ammonium pyruvate. The synthesis of DOPA was studied using both free and immobilized bacterial cells. When the concentration of pyrocatechol is over 0.1 M the TPL activity of the cells is inhibited. The concentration of pyrocatechol can be increased up to 0.3 M by using an equimolar mixture of pyrocatechol and boric acid. The addition of ascorbic acid as an antioxidant results in a lower TPL activity of both free and immobilized bacterial cells.