Regulation of the biosynthesis of insulin-secretory-granule proteins. Co-ordinate translational control is exerted on some, but not all, granule matrix constituents.

The enzyme 4-ethylphenol methylenehydroxylase was purified from Pseudomonas putida JD1 grown on 4-ethylphenol. It is a flavocytochrome c for which the Mr was found to be 120,000 by ultracentrifuging and 126,000 by gel filtration. The enzyme consists of two flavoprotein subunits each of Mr 50,000 and two cytochrome c subunits each of Mr 10,000. The redox potential of the cytochrome is 240 mV. Hydroxylation proceeds by dehydrogenation and hydration to give 1-(4'-hydroxyphenyl)ethanol, which is also dehydrogenated by the same enzyme to 4-hydroxyacetophenone. The enzyme will hydroxylate p-cresol but is more active with alkylphenols with longer-chain alkyl groups. It is located in the periplasm of the bacterium.     

4-Ethylphenol metabolism by Aspergillus fumigatus.

Aspergillus fumigatus ATCC 28282 was found to be capable of growth on 4-ethylphenol as its sole carbon and energy source. A pathway for the metabolism of this compound has been proposed. The initial step involves hydroxylation of the methylene group of 4-ethylphenol to form 1-(4'-hydroxyphenyl)ethanol, followed by oxidation to 4-hydroxyacetophenone. The hydroxylase was NADPH and oxygen dependent, which is a characteristic of a monooxygenase type of enzyme. The 1-(4'-hydroxyphenyl)ethanol isolated from growth medium was a racemic mixture of R-(+) and S-(-) enantiomers. 4-Hydroxyacetophenone undergoes an NADPH-dependent Baeyer-Villiger type of oxygenation to give 4-hydroxyphenyl acetate, which is hydrolyzed to form hydroquinone (1,4-dihydroxybenzene). Hydroxylation of hydroquinone by an NADPH-dependent enzyme produces 1,2,4-trihydroxybenzene, the ring fission substrate, which is cleaved by ortho fission to form maleylacetate. The pathway was elucidated by various kinds of investigations. Analysis of culture medium sampled during growth on 4-ethylphenol revealed the transient appearance of 1-(4'-hydroxyphenyl)ethanol, 4-hydroxyacetophenone, and hydroquinone. Cells grown on 4-ethylphenol were able to oxidize all of these compounds immediately, whereas oxidation by succinate-grown cells showed a lag period. Extracts prepared from cells grown on 4-ethylphenol contained enzyme activities for all of the proposed steps. Apart from a low level of esterase activity towards 4-hydroxyphenyl acetate, extracts prepared from cells grown on succinate did not contain any of these enzyme activities.     

Regio- and Stereospecific Conversion of 4-Alkylphenols by the Covalent Flavoprotein Vanillyl-Alcohol Oxidase

The regio- and stereospecific conversion of prochiral 4-alkylphenols by the covalent flavoprotein vanillyl-alcohol oxidase was investigated. The enzyme was active, with 4-alkylphenols bearing aliphatic side chains of up to seven carbon atoms. Optimal catalytic efficiency occurred with 4-ethylphenol and 4-n-propylphenols. These short-chain 4-alkylphenols are stereoselectively hydroxylated to the corresponding (R)-1-(4′-hydroxyphenyl)alcohols (F. P. Drijfhout, M. W. Fraaije, H. Jongejan, W. J. H. van Berkel, and M. C. R. Franssen, Biotechnol. Bioeng. 59:171–177, 1998). (S)-1-(4′-Hydroxyphenyl)ethanol was found to be a far better substrate than (R)-1-(4′-hydroxyphenyl)ethanol, explaining why during the enzymatic conversion of 4-ethylphenol nearly no 4-hydroxyacetophenone is formed. Medium-chain 4-alkylphenols were exclusively converted by vanillyl-alcohol oxidase to the corresponding 1-(4′-hydroxyphenyl)alkenes. The relative cis-trans stereochemistry of these reactions was strongly dependent on the nature of the alkyl side chain. The enzymatic conversion of 4-sec-butylphenol resulted in two (4′-hydroxyphenyl)-sec-butene isomers with identical masses but different fragmentation patterns. We conclude that the water accessibility of the enzyme active site and the orientation of the hydrophobic alkyl side chain of the substrate are of major importance in determining the regiospecific and stereochemical outcome of vanillyl-alcohol oxidase-mediated conversions of 4-alkylphenols.     

Conversion of 4-hydroxyacetophenone into 4-phenyl acetate by a flavin adenine dinucleotide-containing Baeyer-Villiger-type monooxygenase

An arylketone monooxygenase was purified from Pseudomonas putida JD1 by ion exchange and affinity chromatography. It had the characteristics of a Baeyer-Villiger-type monooxygenase and converted its substrate, 4-hydroxyacetophenone, into 4-hydroxyphenyl acetate with the consumption of one molecule of oxygen and oxidation of one molecule of NADPH per molecule of substrate. The enzyme was a monomer with an M(r) of about 70,000 and contained one molecule of flavin adenine dinucleotide (FAD). The enzyme was specific for NADPH as the electron donor, and spectral studies showed rapid reduction of the FAD by NADPH but not by NADH. Other arylketones were substrates, including acetophenone and 4-hydroxypropiophenone, which were converted into phenyl acetate and 4-hydroxyphenyl propionate, respectively. The enzyme displayed Michaelis-Menten kinetics with apparent K(m) values of 47 microM for 4-hydroxyacetophenone, 384 microM for acetophenone, and 23 microM for 4-hydroxypropiophenone. The apparent K(m) value for NADPH with 4-hydroxyacetophenone as substrate was 17.5 microM. The N-terminal sequence did not show any similarity to other proteins, but an internal sequence was very similar to part of the proposed NADPH binding site in the Baeyer-Villiger monooxygenase cyclohexanone monooxygenase from an Acinetobacter sp.     

Alkylphenol hydroxylases in the oxidation of p-cresol, 4-ethylphenol and 4-n-propylphenol by Pseudomonas putida JD1

Abstract Growth of Pseudomonas putida JD1 on 4-ethylphenol results in the production of the flavocytochrome c, 4-ethylphenol methylenehydroxylase. Both p -cresol and 4- n -propylphenol are substrates for this enzyme. 4-Ethylphenol methylenehydroxylase is also produced by the organism when grown with 4- n -propylphenol. However, when grown with p -cresol, a different hydroxylase is produced which shows greater activity towards p -cresol than towards 4-ethylphenol, and is not active towards 4- n -propylphenol.     

Anaerobic co-metabolic oxidation of 4-alkylphenols with medium-length or long alkyl chains by <Emphasis Type="Italic">Thauera</Emphasis> sp., strain R5

A 4-alkylphenol-degrading facultative anaerobic bacterium, strain R5, was isolated from paddy soil after enrichment with 4-n-propylphenol, 4-n-butylphenol and 4-hydroxybenzoate (4-HBA) under nitrate-reducing conditions. Strain R5 is a Gram-negative rod bacillus grown on phenolic compounds with short alkyl chains (≤C2), organic acids and ethanol. The sequence of the 16S ribosomal RNA gene revealed that the strain is affiliated with Thauera sp. In the presence of 4-HBA as a carbon source, the strain transformed 4-n-alkylphenols with a medium or long-length alkyl chain (C3–C8) to the corresponding oxidised products as follows: 1-(4-hydroxyphenyl)-1-alkenes, -(4-hydroxyphenyl)-1-alkanones and/or 1-(4-hydroxyphenyl)-1-alcohols. The strain also transformed 4-i-propylphenol and 4-sec-butylphenol to (4-hydroxyphenyl)-i-propene and (4-hydroxyphenyl)-sec-butene but not 4-alkylphenols with tertiary alkyl chains (4-t-butylphenol or 4-t-octylphenol). The biotransformation did not proceed without another carbon source and was coupled with nitrate reduction. Biotransformation activity was high in the presence of p-cresol, 4-ethylphenol, 4′-hydroxyacetophenone and 4-HBA as carbon sources and low in the presence of organic acids and ethanol. We suggest that strain R5 co-metabolically transforms alkylphenols to the corresponding metabolites with oxidised alpha carbon in the alkyl chain during coupling with nitrate reduction.     

Alkylphenol biotransformations catalyzed by 4-ethylphenol methylenehydroxylase

4-ethylphenol methylenehydroxylase from Pseudomonas putida JD1 acts by dehydrogenation of its substrate to give a quinone methide, which is then hydrated to an alcohol. It was shown to be active with a range of 4-alkylphenols as substrates. 4-n-propylphenol, 4-n-butylphenol, chavicol, and 4-hydroxydiphenylmethane were hydroxylated on the methylene group next to the benzene ring and produced the corresponding chiral alcohol as the major product. The alcohols 1-(4'-hydroxyphenyl)propanol and 1-(4'-hydroxyphenyl)-2-propen-1-ol, produced by the biotransformation of 4-n-propylphenol and chavicol, respectively, were shown to be R(+) enantiomers. 5-Indanol, 6-hydroxytetralin, 4-isopropylphenol, and cyclohexylphenol, with cyclic or branched alkyl groups, gave the corresponding vinyl compounds as their major products.     

Substrate specificity and enantioselectivity of 4-hydroxyacetophenone monooxygenase

The 4-hydroxyacetophenone monooxygenase (HAPMO) from Pseudomonas fluorescens ACB catalyzes NADPH- and oxygen-dependent Baeyer-Villiger oxidation of 4-hydroxyacetophenone to the corresponding acetate ester. Using the purified enzyme from recombinant Escherichia coli, we found that a broad range of carbonylic compounds that are structurally more or less similar to 4-hydroxyacetophenone are also substrates for this flavin-containing monooxygenase. On the other hand, several carbonyl compounds that are substrates for other Baeyer-Villiger monooxygenases (BVMOs) are not converted by HAPMO. In addition to performing Baeyer-Villiger reactions with aromatic ketones and aldehydes, the enzyme was also able to catalyze sulfoxidation reactions by using aromatic sulfides. Furthermore, several heterocyclic and aliphatic carbonyl compounds were also readily converted by this BVMO. To probe the enantioselectivity of HAPMO, the conversion of bicyclohept-2-en-6-one and two aryl alkyl sulfides was studied. The monooxygenase preferably converted (1R,5S)-bicyclohept-2-en-6-one, with an enantiomeric ratio (E) of 20, thus enabling kinetic resolution to obtain the (1S,5R) enantiomer. Complete conversion of both enantiomers resulted in the accumulation of two regioisomeric lactones with moderate enantiomeric excess (ee) for the two lactones obtained [77% ee for (1S,5R)-2 and 34% ee for (1R,5S)-3]. Using methyl 4-tolyl sulfide and methylphenyl sulfide, we found that HAPMO is efficient and highly selective in the asymmetric formation of the corresponding (S)-sulfoxides (ee > 99%). The biocatalytic properties of HAPMO described here show the potential of this enzyme for biotechnological applications.     

Enzyme-catalyzed synthesis of optically pure R(+)-phenylethanol

Summary A dehydrogenase catalyzing the NADPH-dependent reduction of acetophenone to R(+)-phenylethanol was found inLactobacillus kefir. A continuous conversion of 10 mM acetophenone with a yield of up to 90% was achieved in an enzyme-membrane-reactor with simultaneous NADPH-regeneration using glucose-6-phosphate and glucose-6-phosphate dehydrogenase. Enzyme-catalyzed oxidation of phenylethanol occurred with the R(+)-enantiomer only, whereas S(–) was not converted by the enzyme. The formation of R(+)-phenylethanol with an enantiomeric excess of 100% was confirmed by chiral HPLC.     

Overlapping substrate specificities of benzaldehyde dehydrogenase (the xylC gene product) and 2-hydroxymuconic semialdehyde dehydrogenase (the xylG gene product) encoded by TOL plasmid pWW0 of Pseudomonas putida.

Two aldehyde dehydrogenases involved in the degradation of toluene and xylenes, namely, benzaldehyde dehydrogenase and 2-hydroxymuconic semialdehyde dehydrogenase, are encoded by the xylC and xylG genes, respectively, on TOL plasmid pWW0 of Pseudomonas putida. The nucleotide sequence of xylC was determined in this study. A protein exhibiting benzaldehyde dehydrogenase activity had been purified from cells of P. putida (pWW0) (J. P. Shaw and S. Harayama, Eur. J. Biochem. 191:705-714, 1990); however, the amino-terminal sequence of this protein does not correspond to that predicted from the xylC sequence but does correspond to that predicted from the xylG sequence. The protein purified in the earlier work was therefore 2-hydroxymuconic semialdehyde dehydrogenase (the xylG gene product). This conclusion was confirmed by the fact that this protein oxidized 2-hydroxymuconic semialdehyde (kcat/Km = 1.6 x 10(6) s-1 M-1) more efficiently than benzaldehyde (kcat/Km = 3.2 x 10(4) s-1 M-1). The xylC product, the genuine benzaldehyde dehydrogenase, was purified from extracts of P. putida (pWW0-161 delta rylG) which does not synthesize 2-hydroxymuconic semialdehyde dehydrogenase. The amino-terminal sequence of the purified protein corresponds to the amino-terminal sequence deduced from the xylC sequence. This enzyme efficiently oxidized benzaldehyde (kcat/Km = 1.7 x 10(7) s-1 M-1) and its analogs but did not oxidize 2-hydroxymuconic semialdehyde or its analogs.     

Stereospecific hydroxylation of indan by Escherichia coli containing the cloned toluene dioxygenase genes from Pseudomonas putida F1.

Escherichia coli JM109(pDTG601), containing the todC1C2BA genes encoding toluene dioxygenase from Pseudomonas putida F1, oxidizes indan to (-)-(1R)-indanol (83% R) and trans-1,3-indandiol. Under similar conditions, P. putida F39/D oxidizes indan to (-)-(1R)-indanol (96% R), 1-indanone, and trans-1,3-indandiol. The differences in the enantiomeric composition of the 1-indanols formed by the two organisms are due to the presence of a 1-indanol dehydrogenase in P. putida F39/D that preferentially oxidizes (+)-(1S)-indanol.     

D-Malic enzyme of Pseudomonas fluorescens

By the enrichment culture technique 14 gram-negative bacteria and two yeast strains were isolated that used D(+)-malic acid as sole carbon source. The bacteria were identified as Pseudomonas putida, Pseudomonas fluorescens, Pseudomonas aeruginosa and Klebsiella aerogenes. In cell-free extracts of P. fluorescens and P. putida the presence of malate dehydrogenase, D-malic enzyme (NAD-dependent) and L-malic enzyme (NADP-dependent) was demonstrated. D-Malic enzyme from P. fluorescens was purified. Stabilization of the enzyme by 50 mM ammonium sulphate an 1 mM EDTA was essential. Preparation of D-malic enzyme that gave one band with disc gel electrophoresis showed a specific activity of 4-5 U/mg. D-Malic enzyme requires divalent cations. The Km values were for malate Km = 0.3 mM and for NAD Km = 0.08 mM. The pH optimum for the reaction was found to be in the range of pH 8.1 to pH 8.8. D-Malic enzyme is partially inhibited by oxaloacetic acid, meso-tartaric acid, D-lactic acid and ATP. Determined by gel filtration and gradient gel electrophoresis, the molecular weight was approximately 175 000.     

Stereospecific hydroxylation of indan by Escherichia coli containing the cloned toluene dioxygenase genes from Pseudomonas putida F1.

Escherichia coli JM109(pDTG601), containing the todC1C2BA genes encoding toluene dioxygenase from Pseudomonas putida F1, oxidizes indan to (-)-(1R)-indanol (83% R) and trans-1,3-indandiol. Under similar conditions, P. putida F39/D oxidizes indan to (-)-(1R)-indanol (96% R), 1-indanone, and trans-1,3-indandiol. The differences in the enantiomeric composition of the 1-indanols formed by the two organisms are due to the presence of a 1-indanol dehydrogenase in P. putida F39/D that preferentially oxidizes (+)-(1S)-indanol.     

Toluene degradation by Pseudomonas putida F1. Nucleotide sequence of the todC1C2BADE genes and their expression in Escherichia coli

The nucleotide sequence of the todC1C2BADE genes which encode the first three enzymes in the catabolism of toluene by Pseudomonas putida F1 was determined. The genes encode the three components of the toluene dioxygenase enzyme system: reductaseTOL (todA), ferredoxinTOL (todB), and the two subunits of the terminal dioxygenase (todC1C2); (+)-cis-(1S, 2R)-dihydroxy-3-methylcyclohexa-3,5-diene dehydrogenase (todD); and 3-methylcatechol 2,3-dioxygenase (todE). Knowledge of the nucleotide sequence of the tod genes was used to construct clones of Escherichia coli JM109 that overproduce toluene dioxygenase (JM109(pDT-601]; toluene dioxygenase and (+)-cis-(1S, 2R)-dihydroxy-3-methylcyclohexa-3,5-diene dehydrogenase (JM109(pDTG602]; and toluene dioxygenase, (+)-cis-(1S, 2R)-dihydroxy-3-methylcyclohexa-3,5-diene dehydrogenase, and 3-methylcatechol 2,3-dioxygenase (JM109(pDTG603]. The overexpression of the tod-C1C2BADE gene products was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The three E. coli JM109 strains harboring the plasmids pDTG601, pDTG602, and pDTG603, after induction with isopropyl-beta-D-thiogalactopyranoside, oxidized toluene to (+)-cis-(1S, 2R)-dihydroxy-3-methylcyclohexa-3,5-diene, 3-methylcatechol, and 2-hydroxy-6-oxo-2,4-heptadienoate, respectively. The tod-C1C2BAD genes show significant homology to the reported nucleotide sequence for benzene dioxygenase and cis-1,2-dihydroxycyclohexa-3,5-diene dehydrogenase from P. putida 136R-3 (Irie, S., Doi, S., Yorifuji, T., Takagi, M., and Yano, K. (1987) J. Bacteriol. 169, 5174-5179). In addition, significant homology was observed between the nucleotide sequences for the todDE genes and the sequences reported for cis-1,2-dihydroxy-6-phenylcyclohexa-3,5-diene dehydrogenase and 2,3-dihydroxybiphenyl-1,2-dioxygenase from Pseudomonas pseudoalcaligenes KF707 (Furukawa, K., Arimura, N., and Miyazaki, T. (1987) J. Bacteriol. 169, 427-429).     

Occurrence of a novel NADP(+)-linked alcohol dehydrogenase in Euglena gracilis

An NADP(+)-dependent alcohol dehydrogenase was found in Euglena gracilis Z grown on 1-hexanol, while it was detected at low activity in cells grown on ethanol or glucose as a carbon source, indicating that the enzyme is induced by the addition of 1-hexanol into the medium as a carbon source. This enzyme was extremely unstable, even at 4 degrees C, unless 20% ethylene glycol was added. The optimal pH was 8.8-9.0 for oxidation reaction. The apparent K(m) values for 1-hexanol and NADP(+) were found to be 6.79 mM and 46.7 microM for this enzyme, respectively. The substrate specificity of this enzyme was very different from that of already purified NAD(+)-specific ethanol dehydrogenase by showing the highest activity with 1-hexanol as a substrate, followed by 1-pentanol and 1-butanol, and there was very little activity with ethanol and 1-propanol. This enzyme was active towards the primary alcohols but not secondary alcohols. Accordingly, since the NADP(+)-specific enzyme was separated on DEAE cellulose column, Euglena was confirmed to contain a novel enzyme to be active towards middle and long-chain length of fatty alcohols.     

Allantoin racemase: a new enzyme from Pseudomonas species.

1. Allantoin racemase is a novel enzyme which catalyzes the conversion of S(+)-and R(minus)-allantoin into the racemate. 2. The enzyme is present in Pseudomonas testosteroni, Pseudomonas putida and five biotypes of Pseudomonas fluorescens, but absent in a number of other Pseudomonas species. 3. The enzyme of Ps. testosteroni was purified 133-fold and exposes optimal activity at pH 8.0-8.2 and 50 degrees C. The enzyme is stable on heating for 15 min at 70 degrees C. 4. The enzyme appeared to be specific for the optical isomers of allantoin and no cofactors are involved in the reaction. 5. The optical aspecificity of allantoinase of Proteus rettgeri was reaffirmed.     

Stereochemistry of 1-(4''-hydroxyphenyl)ethanol produced by hydroxylation of 4-ethylphenol by p-cresol methylhydroxylase.

Enzymic hydroxylation of 4-ethylphenol by (a) Pseudomonas putida and (b) highly purified p-cresol methylhydroxylase gave optically active 1-(4'-hydroxyphenyl)-ethanol. The products were transformed into the phenolic methyl ethers and shown to contain 69.5% and 65.6%, respectively, of the (S)-(-)-isomer. The stereochemistry of the reaction is discussed in terms of three distinct steps occurring at the active site of the enzyme.     

Transient kinetic studies of substrate inhibition in the horse liver alcohol dehydrogenase reaction

The rate-limiting step of ethanol oxidation by alcohol dehydrogenase (E) at substrate inhibitory conditions (greater than 500 mM ethanol) is shown to be the dissociation rate of NADH from the abortive E-ethanol-NADH complex. The dissociation rate constant of NADH decreased hyperbolically from 5.2 to 1.4 s-1 in the presence of ethanol causing a decrease in the Kd of NADH binding from 0.3 microM for the binary complex to 0.1 microM for the abortive complex. Correspondingly, ethanol binding to E-NADH (Kd = 37 mM) was tighter than to enzyme (Kd = 109 mM). The binding rate of NAD+ (7 X 10(5) M-1s-1) to enzyme was not affected by the presence of ethanol, further substantiating that substrate inhibition is totally due to a decrease in the dissociation rate constant of NADH from the abortive complex. Substrate inhibition was also observed with the coenzyme analog, APAD+, but a single transient was not found to be rate limiting. Nevertheless, the presence of substrate inhibition with APAD+ is ascribed to a decrease in the dissociation rate of APADH from 120 to 22 s-1 for the abortive complex. Studies to discern the additional limiting transient(s) in turnover with APAD+ and NAD+ were unsuccessful but showed that any isomerization of the enzyme-reduced coenzyme-aldehyde complex is not rate limiting. Chloride increases the rate of ethanol oxidation by hyperbolically increasing the dissociation rate constant of NADH from enzyme and the abortive complex to 12 and 2.8 s-1, respectively. The chloride effect is attributed to the binding of chloride to these complexes, destabilizing the binding of NADH while not affecting the binding of ethanol.     

NAD(+)-dependent (S)-specific secondary alcohol dehydrogenase involved in stereoinversion of 3-pentyn-2-ol catalyzed by Nocardia fusca AKU 2123

An NAD(+)-dependent alcohol dehydrogenase was purified to homogeneity from Nocardia fusca AKU 2123. The enzyme catalyzed (S)-specific oxidation of 3-pentyn-2-ol (PYOH), i.e., part of the stereoinversion reaction for the production of (R)-PYOH, which is a valuable chiral building block for pharmaceuticals, from the racemate. The enzyme used a broad variety of secondary alcohols including alkyl alcohols, alkenyl alcohols, acetylenic alcohols, and aromatic alcohols as substrates. The oxidation was (S)-isomer specific in every case. The K(m) and Vmax for (S)-PYOH and (S)-2-hexanol oxidation were 1.6 mM and 53 mumol/min/mg, and 0.33 mM and 130 mumol/min/mg, respectively. The enzyme also catalyzed stereoselective reduction of carbonyl compounds. (S)-2-Hexanol and ethyl (R)-4-chloro-3-hydroxybutanoate in high optical purity were produced from 2-hexanone and ethyl 4-chloro-3-oxobutanoate by the purified enzyme, respectively. The K(m) and Vmax for 2-hexanone reduction were 2.5 mM and 260 mumol/min/mg. The enzyme has a relative molecular mass of 150,000 and consists of four identical subunits. The NH2-terminal amino acid sequence of the enzyme shows similarity with those of the carbonyl reductase from Rhodococcus erythropolis and phenylacetaldehyde reductase from Corynebacterium sp.     

Conversion of alpha-ketoglutarate into L-glutamic acid with urea as ammonium source using multienzyme systems and dextran-NAD+ immobilized by microencapsulation within artificial cells in a bioreactor

Urea could be effectively converted into L-glutamic acid with semipermeable nylon-polyethylenimine artificial cells containing L-glutamic dehydrogenase (EC 1.4.1. 3), yeast alcohol dehydrogenase (EC 1.1.1.1), urease (EC 3.5.1. 5) and soluble dextran-NAD(+). For batch conversion, the artificial cell suspension to total reaction volume ratios ranged from 1 in 5 to 1 in 60. From 22.6 to 53.4 mumol of L-glutamic acid could be produced by 0.4 mL artificial cell suspension within 2 h. The corresponding conversion ratios were 56.5-11. 1%. The L-glutamic dehydrogenase multienzyme system showed a good storage stability: 66.0% of the original activity was retained after 1 month of storage at 4 degrees C. A small bioreactor was prepared to contain 4.0 mL artificial cells. At a flow rate of SV = 1.5 h(-1), the maximum conversion rate was 49.6 mumol L-glutamic acid/p h. Thirty-eight percent of the maximum activity was retained when continuously used for four days at 22 degrees C. A kinetic analysis for the L-glutamic dehydrogenase multienzyme system was studied. The Michaelis constants are as follows: alpha-ketoglutarate is 0.838 mM; urea is 1.90 mM; dextran- NAD(+) is 0.345 mM; and ethanol is 5.31 mM.