Rubrivivax Benzoatilyticus JA2 Research Articles

Stable isotope-assisted metabolite profiling reveals new insights into L-tryptophan chemotrophic metabolism of Rubrivivax benzoatilyticus

Anoxygenic photosynthetic bacteria (APB) are metabolically versatile, capable of surviving with an extended range of carbon and nitrogen sources. This group of phototrophic bacteria have remarkable metabolic plasticity in utilizing an array of organic compounds as carbon source/electron donors and nitrogen sources with sophisticated growth modes. Rubrivivax benzoatilyticus JA2 is one such photosynthetic bacterium utilizes L-tryptophan as nitrogen source under phototrophic growth mode and produces an array of indolic compounds of biotechnological significance. However, chemotrophic L-tryptophan metabolism is largely unexplored and studying L-tryptophan metabolism under chemotrophic mode would provide new insights into metabolic potential of strain JA2. In the present study, we employed stable-isotopes assisted metabolite profiling to unravel the L-tryptophan catabolism in Rubrivivax benzoatilyticus strain JA2 under chemotrophic (dark aerobic) conditions. Utilization of L-tryptophan as a nitrogen source for growth and simultaneous production of indole derivatives was observed in strain JA2. Liquid chromatography mass spectrometry (LC-MS) analysis of exo-metabolite profiling of carbon labeled L-tryptophan (13C11) fed cultures of strain JA2 revealed at least seventy labeled metabolites. Of these, only fourteen metabolites were confirmed using standards, while sixteen were putative and forty metabolites remained unidentified. L-tryptophan chemotrophic catabolism revealed multiple catabolic pathways and distinct differential catabolism of L-tryptophan under chemotropic state as compared to photo-catabolism of L-tryptophan in strain JA2.

Dynamics of proteo-metabolome from Rubrivivax benzoatilyticus JA2 reveals a programmed switch-off of phototrophic growth, leading to a non-cultivable state as a hyperglycemic effect

Anoxygenic phototrophic bacteria display phenomenal metabolic plasticity leading to distinct phenotypes. Extracellular elevated glucose levels limit photosynthesis in photosynthetic organisms; diversely, cause oxidative stress with ROS generation and “diabetic” like situation in non-photosynthetic organisms. In this study, longer incubations of externally provided glucose (22 mM) inhibited photosynthetic machinery in a phototrophic bacterium, Rubrivivax benzoatilyticus. Data analysis at three time points- exponential, early and late stationary phase, uncovered dynamic protein and metabolite abundance implying metabolic rewiring led non-cultivable state in response to glucose. Protein dynamics datum suggested that proteins related to primary metabolism down-regulated prior to those of secondary metabolism. Numerous proteins for metabolism and energy generation were highly expressed during exponential phase whereas those for membrane transport/translocation and DNA repair accumulated at early and late stationary phase respectively, suggesting a programmed knock-off of phototrophic growth mode and a switch to non-cultivable state. Overall, the omics analyses explicated the metabolic adjustment associated with glucose grown cells of R. benzoatilyticus. Further, our investigation unravelled creation of oxidative stress suggesting physiological stress (oxygen limitation) might be a key player leading to a non-cultivable state in this phototrophic organism. The study, emphasizing microbial glucose intolerance, unlocks the doorway to perceive microorganisms with new perspective. SignificanceAnoxygenic photosynthetic bacteria (APB), thriving under diverse habitat, exhibits magnificent metabolic flexibility. Generally, phototrophy is the preferred growth mode and energy generating route for APB. But, our analyses implicated that the glucose, under phototrophic growth conditions, triggered photobleaching in an APB member, Rubrivivax benzoatilyticus. However, retention of growth along with pigmentation under chemotrophic growth mode supports that glucose gradually knocked off the phototrophic growth mode of R. benzoatilyticus and switched to an alternate energy driving route or less energy demanding non-cultivabile state. Thus, the change in lifestyle i.e. photoheterotrophic growth instead of chemotrophic perhaps, might be the prime culprit and key player in inducing the said state of non-cultivability, akin to diabetes. The study, shedding light on the plausible regulation of cultivability, unveils the programmed regulated switching between different growth modes of the organism and illuminates the importance of glucose intolerance by microorganisms. Through this investigation, we appeal that the studies on ‘glucose intolerance in microorganisms’ also need due attention that will perhaps change our outlook to perceive micro-organisms in relation to their physiological life style.

Insights into discrepancy in power generation among glucose and malate grown Rubrivivax benzoatilyticus JA2 microbial fuel cells

In this study, Rubrivivax benzoatilyticus JA2T (=ATCC BAA-35T = JCM 13220T = MTCC 7087T), an anoxygenic photosynthetic bacterium, was subjected to altered conditions and observed for changes in power outcome in the two chambered microbial fuel cells (MFCs), the basis of which was established using metabolomic studies. This is an extension to our previous studies, which showed that, under photo heterotrophic conditions, glucose in the form of a solitary carbon resource in minimal media, caused the strain JA2 to exhibit altered growth rates, progressive loss of pigmentation and reduced cell size (3–4 μm), compared to malate grown cells (6–7 μm). When R. benzoatilyticus JA2 cells were grown in malate bio-anodes, they presented higher potentials (289.22 ± 4.6 mV or 436.22 OCV per mg dry weight) compared to glucose bioanodes (163 ± 5.5 mv or 188.98 OCV per mg dry weight). Insights from the metabolomic footprints and fingerprints have revealed differential regulation of key components in the central metabolic pathway such as fumarate, citrate and succinate, which are significantly increased in malate grown bio anodes. Strain JA2 cells when grown with malate as substrate are densely grown on the electrodes and exhibited reduced size, when observed under SEM, which contrasts with control cells grown on malate broth. The artificial selection pressure of the MFC and the different metabolic pathways followed by these bacteria are the reasons for such discrepancy in the power production by the strain JA2. These adaptations may indicate survival advantage during the electron transfer and growth in bio anodes. The study throws light on what types of effluents would be more suitable as substrates for R. benzoatilyticus JA2 microbial fuel cells.

Tryptophan, a non-canonical melanin precursor: New L-tryptophan based melanin production by Rubrivivax benzoatilyticus JA2

Melanins are chemically diverse ubiquitous pigments found across the life forms synthesized via different biochemical pathways mainly from L-tyrosine or acetyl CoA. Though few reports suggest the possibility of tryptophan-based melanin synthesis, however, such tryptophan-based melanin and its biosynthesis remained a biochemical riddle. Here we report tryptophan-based melanin production by bacterium, Rubrivivax benzoatilyticus JA2. Aerobic cultures of strain JA2 produced brown pigment when grown on L-tryptophan-containing media. Purified pigment showed typical physico-chemical properties of melanin. Further, extensive spectroscopic studies revealed that pigment is an amorphous, indole-type polymer with stable free radical centers. Further, hydrolysis of the brown pigment revealed the presence of indole moiety, confirming the indolic nature of the pigment. Demonstration of in vitro and in vivo pigment synthesis directly from L-tryptophan or hydroxytryptophan confirms tryptophan-based melanin synthesis in strain JA2. Interestingly, canonical melanin biosynthetic inhibitors did not affect the pigment synthesis indicating possible non-canonical tryptophan-based melanin biosynthesis in strain JA2. Further, the exometabolite profiling and precursor feeding studies suggests that L-tryptophan converted to hydroxytryptophan/hydroxyindoles and their subsequent polymerization lead to the formation of melanin. The current study sheds light on biosynthetic diversity of melanins and L-tryptophan can be a potential precursor for melanin synthesis in life forms.

New insights into aniline toxicity: Aniline exposure triggers envelope stress and extracellular polymeric substance formation in Rubrivivax benzoatilyticus JA2

Aniline is a major environmental pollutant of serious concern due to its toxicity. Although microbial metabolism of aniline is well-studied, its toxic effects and physiological responses of microorganisms to aniline are largely unexplored. Rubrivivax benzoatilyticus JA2, an aniline non-degrading bacterium, tolerates high concentrations of aniline and produces extracellular polymeric substance(EPS). Surprisingly, strain JA2 forms EPS only when exposed to aniline and other toxic compounds like organic solvents and heavy metals indicating that EPS formation is coupled to cell toxicity. Further, extensive reanalysis of the previous proteomic data of aniline exposed cells revealed up-regulation of envelope stress response(ESR) proteins such as periplasmic protein folding, envelope integrity, transmembrane complex, and cell-wall remodelling proteins. In silico analysis and molecular modeling of three highly up-regulated proteins revealed that these proteins were homologous to CpxARP proteins of ESR signalling pathway. Furthermore, EPS formation to known ESR activators(Triton-X-100, EDTA) suggests that envelope stress possibly regulating the EPS production. The present study suggests that aniline triggers envelope stress; to counter this strain JA2 activates ESR pathway and EPS production. Our study revealed the hitherto unknown toxic effects of aniline as an acute envelope stressor thus toxicity of aniline may be more profound to life-forms than previously thought.

Precursor-feeding and altered-growth conditions reveal novel blue pigment production by Rubrivivax benzoatilyticus JA2.

To explore the secondary metabolite biosynthetic potential of Rubrivivax benzoatilyticus JA2 using a new metabolite mining strategy. Combination of precursor-feeding and altered growth conditions were used to mine new biomolecules. Strain JA2 utilised L-phenylalanine as sole source of nitrogen and showed pigments production only under phenylalanine-amended aerobic cultures. Stable isotope based precursor feeding studies indicated the blue pigment consists of 4-phenyl rings derived from L-phenylalanine. The purified blue pigment displayed characteristic visible-absorption and pH-dependent color variations. Precursor-feeding under altered growth conditions activated the plausible novel aromatic pigment production in strain JA2. Our approach unraveled the previously unknown pigment synthesis in strain JA2 and demonstrated the potential of mining strategy in discovering the hidden secondary metabolite repertoire in microorganisms.

ITRAQ-based quantitative proteomics reveals insights into metabolic and molecular responses of glucose-grown cells of Rubrivivax benzoatilyticus JA2

Anoxygenic photosynthetic bacteria thrive under diverse habitats utilising an extended range of inorganic/organic compounds under different growth modes. Although they display incredible metabolic flexibility, their responses and adaptations to changing carbon regimes is largely unexplored. In the present study, we employed iTRAQ-based global proteomic profiling and physiological studies to uncover the adaptive strategies of a phototrophic bacterium, Rubrivivax benzoatilyticus JA2 to glucose. Strain JA2 displayed altered growth rates, reduced cell size and progressive loss of pigmentation when grown on glucose compared to malate under photoheterotrophic condition. A ten-fold increase in the saturated to unsaturated fatty acid ratio of glucose-grown cells indicates a possible membrane adaptation. Proteomic profiling revealed extensive metabolic remodelling in the glucose-grown cells wherein signal-transduction, selective-transcription, DNA-repair, transport and protein quality control processes were up-regulated to cope with the changing milieu. Proteins involved in DNA replication, translation, electron-transport, photosynthetic machinery were down-regulated possibly to conserve the energy. Glycolysis/gluconeogenesis, TCA cycle and pigment biosynthesis were also down-regulated. The cell has activated alternative energy metabolic pathways viz., fatty acid β-oxidation, glyoxylate, acetate-switch and Entner-Doudoroff pathways. Overall, the present study deciphered the molecular/metabolic events associated with glucose-grown cells of strain JA2 and also unraveled how a carbon source modulates the metabolic phenotypes. SignificanceAnoxygenic photosynthetic bacteria (APB) exhibit incredible metabolic flexibility leading to diverse phenotypes. They thrive under diverse habitat using an array of inorganic/organic compounds as carbon sources, yet their metabolic adaptation to varying carbon regime is mostly unexplored. Present study uncovered the proteomic insights of the cellular responses of strain JA2 to changing carbon sources viz. malate and glucose under photoheterotrophic conditions. Our study suggests that carbon source can also determine the metabolic fate of the cells and reshape the energy dynamics of APB. Here, for the first time study highlighted the plausible carbon source (glucose) mediated regulation of photosynthesis in APB. The study sheds light on the plausible cellular events and adaptive metabolic strategies employed by strain JA2 in presence of non-preferred carbon source. It also revealed new insights into the metabolic plasticity of APB to the changing milieu.

Pyomelanin production: Insights into the incomplete aerobic l-phenylalanine catabolism of a photosynthetic bacterium, Rubrivivax benzoatilyticus JA2

Rubrivivax benzoatilyticus JA2 is a metabolically versatile bacterium, thrives on a wide array of organic compounds under different growth modes. Though genomic insights revealed the aromatic compound catabolic potential of strain JA2 under anaerobic/aerobic conditions, the studies are largely restricted to anaerobic metabolism. The previous study on phenylalanine metabolism in strain JA2 indicated melanin-like pigment production under aerobic conditions; however, characterization of pigment and its biosynthetic pathway is not explored. The current study aims at the characterization of pigment and elucidation of its biosynthetic pathway. Strain JA2 utilized l-phenylalanine as source of nitrogen under anaerobic/aerobic conditions but not as a carbon source. Strain JA2 produced a brown-pigment under phenylalanine-amended aerobic conditions. Spectroscopic and physicochemical analysis identified the purified brown-pigment as a melanin. Further, the genomic insights revealed the presence of a complete set of genes related to pyomelanin synthesis. Identification of key metabolites l-tyrosine, 4-hydroxyphenylpyruvic acid and homogentisic acid and their respective enzyme activities further supports the pyomelanin synthesis. Moreover, the precursors feeding, pathway specific inhibitor studies confirmed the pyomelanin synthesis in strain JA2. Our study revealed an incomplete catabolism of phenylalanine; absence of ring cleavage gene, homogentisate dioxygenase leading to homogentisate accumulation thereby pyomelanin synthesis in strain JA2.

Stable Isotope-Assisted Metabolic Profiling Reveals Growth Mode Dependent Differential Metabolism and Multiple Catabolic Pathways of l-Phenylalanine in Rubrivivax benzoatilyticus JA2.

Anoxygenic phototrophic bacteria are metabolically versatile and survive under different growth modes using diverse organic compounds, yet their metabolic diversity is largely unexplored. In the present study, we employed stable-isotope-assisted metabolic profiling to unravel the l-phenylalanine catabolism in Rubrivivax benzoatilyticus JA2 under varying growth modes. Strain JA2 grows under anaerobic and aerobic conditions by utilizing l-phenylalanine as a nitrogen source. Furthermore, ring-labeled 13C6-phenylalanine feeding followed by liquid chromatography-mass spectrometry exometabolite profiling revealed 60 labeled metabolic features (M + 6, M + 12, and M + 18) derived solely from l-phenylalanine, of which 11 were identified, 7 putatively identified, and 42 unidentified under anaerobic and aerobic conditions. However, labeled metabolites were significantly higher in aerobic compared to anaerobic conditions. Furthermore, detected metabolites and enzyme activities indicated multiple l-phenylalanine catabolic routes mainly Ehrlich, homogentisate-dependent melanin, benzenoid, and unidentified pathways operating under anaerobic and aerobic conditions in strain JA2. Interestingly, the study indicated l-phenylalanine-dependent and independent benzenoid biosynthesis in strain JA2 and a differential flux of l-phenylalanine to Ehrlich and benzenoid pathways under anaerobic and aerobic conditions. Additionally, unidentified labeled metabolites strongly suggest the presence of unknown phenylalanine catabolic routes in strain JA2. Overall, the study uncovered the l-phenylalanine catabolic diversity in strain JA2 and demonstrated the potential of stable isotope-assisted metabolomics in unraveling the hidden metabolic repertoire.

Isolation and Characterization of L-Tryptophan Ammonia Lyase from Rubrivivax benzoatilyticus Strain JA2.

Ammonia lyase belongs to the family of enzymes that catalyzes the deamination of amino acids. Depending on the relative activity towards the substrates, L-tryptophan ammonia lyase converts L-tryptophan to indole 3-acrylic acid and ammonia. Here, we isolated, purified, and characterized an L-tryptophan ammonia lyase from phototrophic purple non-sulfur bacterium Rubrivivax benzoatilyticus JA2. The isolated L-tryptophan ammonia lyase found to catalyze the reaction of L-tryptophan to produce indole 3-acrylic acid and NH3. The enzyme is a heterotetramer and has the highest affinity to L-tryptophan. The optimum pH and temperature for the enzymatic action were 7.5 and 35°C, respectively and the Km and Vmax were 40.4 ± 23.1 nM and 0.964±0.2046 s(-1), respectively. These results suggest that the isolated enzyme is highly bioactive and could be a new class. Further molecular analyses are required to confirm the novelty of the enzyme.

Integrated metabolomic and proteomic analysis reveals systemic responses of Rubrivivax benzoatilyticus JA2 to aniline stress.

Aromatic amines are widely distributed in the environment and are major environmental pollutants. Although degradation of aromatic amines is well studied in bacteria, physiological adaptations and stress response to these toxic compounds is not yet fully understood. In the present study, systemic responses of Rubrivivax benzoatilyticus JA2 to aniline stress were deciphered using metabolite and iTRAQ-labeled protein profiling. Strain JA2 tolerated high concentrations of aniline (30 mM) with trace amounts of aniline being transformed to acetanilide. GC-MS metabolite profiling revealed aniline stress phenotype wherein amino acid, carbohydrate, fatty acid, nitrogen metabolisms, and TCA (tricarboxylic acid cycle) were modulated. Strain JA2 responded to aniline by remodeling the proteome, and cellular functions, such as signaling, transcription, translation, stress tolerance, transport and carbohydrate metabolism, were highly modulated. Key adaptive responses, such as transcription/translational changes, molecular chaperones to control protein folding, and efflux pumps implicated in solvent extrusion, were induced in response to aniline stress. Proteo-metabolomics indicated extensive rewiring of metabolism to aniline. TCA cycle and amino acid catabolism were down-regulated while gluconeogenesis and pentose phosphate pathways were up-regulated, leading to the synthesis of extracellular polymeric substances. Furthermore, increased saturated fatty acid ratios in membranes due to aniline stress suggest membrane adaptation. The present study thus indicates that strain JA2 employs multilayered responses: stress response, toxic compound tolerance, energy conservation, and metabolic rearrangements to aniline.

Aniline Is an Inducer, and Not a Precursor, for Indole Derivatives in Rubrivivax benzoatilyticus JA2

Rubrivivax benzoatilyticus JA2 and other anoxygenic photosynthetic bacteria produce indole derivatives when exposed to aniline, a xenobiotic compound. Though this phenomenon has been reported previously, the role of aniline in the production of indoles is still a biochemical riddle. The present study aims at understanding the specific role of aniline (as precursor or stimulator) in the production of indoles and elucidating the biochemical pathway of indoles in aniline-exposed cells by using stable isotope approaches. Metabolic profiling revealed tryptophan accumulation only in aniline exposed cells along with indole 3-acetic acid (IAA) and indole 3-aldehyde (IAld), the two major catabolites of tryptophan. Deuterium labelled aniline feeding studies revealed that aniline is not a precursor of indoles in strain JA2. Further, production of indoles only in aniline-exposed cells suggests that aniline is an indoles stimulator. In addition, production of indoles depended on the presence of a carbon source, and production enhanced when carbon sources were added to the culture. Isotope labelled fumarate feeding identified, fumarate as the precursor of indole, indicating de novo synthesis of indoles. Glyphosate (shikimate pathway inhibitor) inhibited the indoles production, accumulation of tryptophan, IAA and IAld indicating that indoles synthesis in strain JA2 occurs via the de novo shikimate pathway. The up-regulation of anthranilate synthase gene and induction of anthranilate synthase activity correlated well with tryptophan production in strain JA2. Induction of tryptophan aminotransferase and tryptophan 2-monooxygenase activities corroborated well with IAA levels, suggesting that tryptophan catabolism occurs simultaneously in aniline exposed cells. Our study demonstrates that aniline (stress) stimulates tryptophan/indoles synthesis via the shikimate pathway by possibly modulating the metabolic pathway.

Carbon Catabolite Repression-Independent and pH-Dependent Production of Indoles by Rubrivivax benzoatilyticus JA2

Rubrivivax benzoatilyticus JA2 produces indole derivatives (indoles) from aniline, anthranilate or L-tryptophan. Glucose repressed indole production in R. benzoatilyticus JA2, while malate had no effect. Growth of R. benzoatilyticus JA2 on glucose resulted in decrease in culture pH (6.4) compared with malate (8.4). Growth of R. benzoatilyticus JA2 on sugar carbon sources decreased culture pH (6.4-6.6) and indole production. Further, culture pH of 6.4 repressed the indole production, and pH 8.4 promoted the production irrespective of carbon sources used for growth. Moreover, correlation between indole production and culture pH was observed, where acidic pH inhibited indole production, while alkaline pH promoted the production, suggesting the role of pH in indole production. Tryptophan-catabolizing enzyme activities are significantly high in malate-grown cultures (pH 8.4) compared with that of the glucose (pH 6.4)-grown cultures and corroborated well with indole production, indicating their role in indole production. These results confirm that indole production in R. benzoatilyticus JA2 is pH dependent rather than carbon catabolite repression.

L-Phenylalanine catabolism and l-phenyllactic acid production by a phototrophic bacterium, Rubrivivax benzoatilyticus JA2

A phototrophic bacterium (Rubrivivax benzoatilyticus JA2) grows at the expense of l-phenylalanine as sole source of nitrogen but not as carbon source. Near stoichiometric yields of l-phenylpyruvic acid (0.4mM) and l-phenyllactate (0.4mM) were observed from l-phenylalanine (0.9mM consumed). Aminotransfarase and dehydrogenase activities involved in the formation of l-phenylpyruvic acid and l-phenyllactate were demonstrated unequivocally in Rubrivivax benzoatilyticus JA2. Growth conditions and carbon sources had an influence on l-phenyllactate production. The process yielded a maximum of 0.92mM l-phenyllactate from l-phenylalanine (1mM) when fructose served as carbon source for R. benzoatilyticus JA2.

Production of indole-3-acetic acid and related indole derivatives from L-tryptophan by Rubrivivax benzoatilyticus JA2

Rubrivivax benzoatilyticus JA2 produces indoles with simultaneous utilization of L-tryptophan. Fifteen chromatographically distinct indole derivatives were detected from the L-tryptophan-supplemented cultures of R. benzoatilyticus JA2. Nine of these were identified as, indole 3-acetamide, Methoxyindole-3-aldehyde, indole 3-aldehyde, methoxyindole-3-acetic acid, indole 3-acetic acid, indole-3-carboxylic acid, indole-3-acetonitrile, indole, and trisindoline. Tryptophan stable isotope feeding confirmed the indoles produced are from the supplemented L-tryptophan. Indole 3-acetic acid is one of the major products of L-tryptophan catabolism by R. benzoatilyticus JA2 and its production was influenced by growth conditions. Identification of indole 3-acetamide and tryptophan monooxygenase activity suggests indole 3-acetamide routed IAA biosynthesis in R. benzoatilyticus JA2. The study also indicated the possible multiple pathways of IAA biosynthesis in R. benzoatilyticus JA2.

Rubrivivaxin, a new cytotoxic and cyclooxygenase-I inhibitory metabolite from Rubrivivax benzoatilyticus JA2

A new phenol terpenoid ester metabolite was isolated from the culture supernatant of Rubrivivax benzoatilyticus JA2 when the organism grown on L-phenylalanine as sole source of nitrogen under photoheterotrophic condition. The compound was characterized by HPLC, LCMS, IR, NMR (1H, 13C) and Mass spectrometry, as a 3, 4-dihydroxybenzoyl-terpenoid ester [IUPAC: 3, 4-dihydroxy, 5-carboxy, 3-methylpentyl ester], which has a molecular mass (m/z) of 298 and we designated as rubrivivaxin. The significance of cyclooxygenase-I (COX-I) inhibitory, antimicrobial, cytotoxic activities against U937 (Human leukemic monocyte lymphoma) and Jurkat (T lymphocyte) cell lines conferred by rubrivivaxin.

L-Tryptophan catabolism by Rubrivivax benzoatilyticus JA2 occurs through indole 3-pyruvic acid pathway

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