Collagenase production by Achromobacter iophagus.

Achromobacter iophagus produced collagenase (EC 3.4.24.3) when cultured aerobically in buffer containing 5% peptone. The bacterium is non-pathogenic and tests on rabbits indicated that the culture medium was atoxic. The collagenase, which hydrolyzed insoluble and soluble native collagen, was purified by (NH4)2 SO4 precipitation, starch block electrophoresis, and gel filtration. It was shown to be serologically distinct from Clostridium histolyticum collagenase and to have molecular weight and sedimentation coefficient values of approx. 112 000 and 5.3 S, respectively.     

Cellulase-free xylanase production from an alkalophilicBacillus species

An alkalophilicBacillus (NCL-87-6-10, NCIM 2128), with a high productivity for extracellular xylanase (EC 3.2.1.8) and free of cellulase, was isolated from soil containing coconut fibre detritus. When grown on a wheat bran/yeast extract medium in submerged culture for 48 h, it produced 100 to 120 IU of enzyme activity per ml. The crude enzyme consists of two fractions of apparent mol sizes of approx 10.4 and 29 kDa in the proportion of 90:10, as determined by native gel exclusion chromatography. Optimum activity of the xylanase was at 60°C and pH 8.0. A two-fold increase in enzyme activity was obtained when reducing agents, thioethanol and dithiothreitol, were included in the assay.NCL Communication No. 5381.     

Pigment production by Pseudomonas reptilivora

Summary A study about the effect of iron concentration and the presence of different organic nitrogen sources on fluorescent pigment production by Pseudomonas reptilivora and Pseudomonas fluorescens has been investigated. An inverse proportionality between pigment formation and iron concentration for both species has been deduced. However, P. reptilivora responds to increasing iron concentrations more sensitive than P. fluorescens, i.e. it forms at equal iron concentration less pigment. P. reptilivora needs iron even for growth contrary to P. fluorescens. The differences in pigment production on different peptones are due to the iron contained in these media.     

Nature of factors stimulating cobalamin production by Achromobacter cobalamini

The object of this work was to study the nature of factors contained in molasses and maize extract and stimulating cobalaminogenesis in Achromobacter cobalamini. The activity of substrate fractions was analyzed to show that the stimulating substance was precipitated on the cation exchanger and eluted from it with HCl. The factor was found to be an organic nitrogen base readily soluble in water and ethanol but insoluble in ether, chloroform and methanol. It was stable upon heating in concentrated HCl. Betaine in the composition of molasses and choline in the composition of maize extract had similar properties. Their addition to the growth medium produced the same effect as that of molasses and maize extract. It is concluded therefore that cobalaminogenesis is stimulated in A. cobalamini by betaine in molasses and by choline in maize extract.     

Continuous production of urocanic acid by immobilized Achromobacter liquidum cells

Several microorganisms having higher L -histidine ammonia-lyase activity were immobilized into polyacrylamide gel lattice. The yield of enzyme activity by immobilization was highest in Achromobacter liquidum IAM 1667. As A. liquidum has urocanase activity, the cells were heat-treated at 70°C for 30 min to inactivate the urocanase. Enzymatic properties of the immobilized A. liquidum cells were investigated and compared with those of the intact cells. No difference was observed between the pH activity curve and optimal temperature for the intact and immobilized cells. The permeability of substrate or product through the cell wall was increased by immobilization of the cells. When an aqueous solution of 0.25M L -histidine (pH 9.0) containing 1mM Mg²⁺ was passed through a column packed with the immobilized A. liquidum cells at a flow rate of SV = 0.06 at 37°C, L -histidine was completely converted to urocanic acid. The L -histidine ammonia-lyase activity of the immobilized cell column was stable over 40 days at 37°C. From the effluent of the immobilized cell column, Urocanic acid was easily obtained in a good yield.     

Continuous production of NADP by immobilized Achromobacter aceris cells.

Several microorganisms having higher nicotinamide adenine dinucleotide kinase (NAD kinase, EC 2.7.1.23) activity were immobilized into polyacrylamide gel lattices. The enzyme activity field by immobilization was highest in Achromobacter aceris AKU 0120. By the incubation of the immobilized A. aceris cells at pH 4.0, the NAD kinase activity increased and the adenosine triphosphate (ATP)-degradation activity disappeared completely. Enzymatic properties of the immobilized A. aceris cells were investigated and compared with those of intact cells. The optimal pH and the optimal temperature of immobilized cells were the same as those of intact cells. Immobilized cell NAD kinase was more stable than that of intact cells. The operational half-life of immobilized cells was 20 days when the substrate solution was passed through a column packed with immobilized cells at a flow rate which gives a space velocity (SV) of 0.1 hr-1 at 37 degrees C. On the other hand, the half-life of the intact cells was only 6 hr.     

Lysine production from hydrocarbon by micrococcus species

A bacterium isolated from Assam (India) soil was found to accumulate L-lysine in the mineral salt-hydrocarbon medium and identified to be a strain of Micrococcus luteus. The strain is able to grow and accumulate l-lysine in a purely synthetic medium, but supplementation of the synthetic medium with casamino acid or yeast extract or both, improves the yield. The entire fermentation period can be divided into a growth phase and a production phase, which can be prolonged by adjustment of the pH to the neutral range. Among the different hydrocarbon and nitrogen sources tested straight run gas oil (47percnt;) and ammonium sulphate (0.4%), respectively, were found most suitable. Erythromycin at 1 μg/ml level inhibited growth bu¸t stimulated lysine excretion. An inoculum level of 10% (v/v) of the medium was optimal for lysine production. The yield of lysine under optimal conditions was found to be 3.25 g per litre of the medium. Lysine has been isolated in crystalline form from the fermented broth by ion exchange resin chromatography and found to be pure sample of L-isomer.     

Continuous production of glucose-6-phosphate by immobilized Achromobacter butyri cells

Summary Whole cells of Achromobacter butyri OUT 8004 having polyphosphate glucokinase activity were immobilized in polyacrylamide gel. The immobilized cells were activated by organic solvents, especially acetone. The immobilization resulted in increased stability of polyphosphate glucokinase. Continuous high yield production of G-6-P from glucose and metaphosphate was performed with an immobilized cell column, which had a half-life of approximately 20 days.Abbreviations G-6-P glucose-6-phosphate - G-1-P glucose-1-phosphate - Cation-S stearyl trimethyl ammonium chloride - SDS sodium dodecyl sulfate - Tris tris(hydroxymethyl)-aminomethane; p-NPP, p-nitrophenyl phosphate - S.V. space velocity     

Enhancement of tryptophan production by Escherichia coli as an application of genetic engineering

Summary A feedback resistant trp operon plasmid that transformed a multiple mutant (trpR tnaA) of Escherichia coli was found to enhance remarkably the production of tryptophan in a bench-scale fermentor. 5.5 g of tryptophan was accumulated per litre of culture medium at 24th hr in batch. The productivity was 0.229 g/l/hr. This productivity is the highest among those ever reported by other workers. The recombinant plasmid (Tc^r Trp⁺ I^-) used was completely stable in each run when tetracycline was added by 10 g/l into the medium.     

The production of tryptamine from tryptophan by Bacillus cereus (KVT)

1. A strain of Bacillus cereus has been isolated that can produce tryptamine when grown in a broth containing tryptophan. 2. The conditions of culture under which this conversion is optimum, as well as the general pathways of tryptophan metabolism by this micro-organism, have been examined, and the information obtained has been used to obtain the first demonstration of cell-free tryptophan-carboxy-lyase activity. 3. The significance of these findings both to the current attempts to elucidate the pathways of metabolism of tryptophan in higher plants and to the published generalizations about the previously studied amino acid carboxy-lyases is discussed.     

Modification of tryptophan and tryptophan residues in proteins by reactive nitrogen species.

Formation of 3-nitrotyrosine by the reaction between reactive nitrogen species (RNS) and tyrosine residues in proteins has been analyzed extensively and it is used widely as a biomarker of pathophysiological and physiological conditions mediated by RNS. In contrast, few studies on the nitration of tryptophan have been reported. This review provides an overview of the studies on tryptophan modifications by RNS and points out the possible importance of its modification in pathophysiological and physiological conditions. Free tryptophan can be modified to several nitrated products (1-, 4-, 5-, 6-, and 7-), 1-N-nitroso product, and several oxidized products by reaction with various RNS, depending on the conditions used. Among them, 1-N-nitrosotryptophan and 6-nitrotryptophan (6-NO(2)Trp) have been found as the abundant products in the reaction with peroxynitrite, and 6-NO(2)Trp has been the most abundant product in the reaction with the peroxidase/hydrogen peroxide/nitrite systems. 6-NO(2)Trp has also been observed as the most abundant nitrated product of the reactions between peroxynitrite or myeloperoxidase/hydrogen peroxide/nitrite and tryptophan residues both in human Cu,Zn-superoxide dismutase and in bovine serum albumin, as well as the reaction of peroxynitrite with myoglobin and hemoglobin. Several oxidized products have also been identified in the modified Cu,Zn-SOD. However, no 1-N-nitrosotryptophan and 1-N-nitrotryptophan has been observed in the proteins reacted with peroxynitrite or the myeloperoxidase/H(2)O(2)/nitrite system. The modification of tryptophan residues in proteins may occur at a more limited number of sites in vivo than that of tyrosine residues, since tryptophan residues are more buried inside proteins and exist less frequently in proteins, generally. However, surface-exposed tryptophan residues tend to participate in the interaction with the other molecules, therefore the modification of those tryptophans may result in modulation of the specific interaction of proteins and enzymes with other molecules.     

Pigment production by Streptococcus agalactiae in quasi-defined media

A quasi-defined medium that supports the growth of Streptococcus agalactiae as pigmented colonies has been developed. The medium contains starch, a peptic digest of albumin, amino acids, nucleosides, vitamins, and salts. The presence of free cysteine, which could be replaced with other sulphur-containing compounds and to a lesser degree by reducing agents, was required for pigment formation.     

NADPH production from NADP+ using malic enzyme of Achromobacter parvulus IFO-13182

A sonicate of Achromobacter parvulus IFO-13182 produced NADPH from NADP⁺by an NADP⁺-linked malic enzyme [l-malate: NAD(P)⁺oxidoreductase, EC 1.1.1.39–40] reaction in the presence of l-malic acid and divalent metal ions. Malic enzyme of A. parvulus was stabilized by 5% l-malic acid, and activity was maintained at 60°C for 1 h. Contaminating phosphatase (orthophosphoricmonoester phosphohydrolase, EC 3.1.3.1–2) was completely inactivated by this treatment. Among the conditions tested, the optimum NADPH production was done using 36 μmol NADP⁺, 67 μmol l-malic acid, 63 μmol MgCl₂ and 1 unit of the malic enzyme in 3 ml of 55 mm phosphate buffer (pH 7.8). Conversion ratio of NADPH from NADP⁺ reached 100% after 4 h incubation at 30°C and the amount of NADPH accumulated was ～12 μmol ml^?1of the reaction mixture. No dephosphorylation of NADP⁺to NAD⁺or of NADPH to NADH was found by high performance liquid chromatography. The NADPH produced by such enzymatic reduction was purified by ethanol precipitation and dried in vacuo in powdered form with 97% purity, judged from the ratio of the absorbances at 340 and 260 nm. The purity of the NADPH produced was determined to be 95% from its coenzyme activity with NAD(P)⁺-linked glutathione reductase [NAD(P)H: oxidized-glutathione oxidoreductase, EC 1.6.4.2].