Hexosamine Dehydrogenase Membrane Bound

D-Glucosamine ^ D-glucosaminate

D-Galactosamine ^ D-galactosaminate

D-Mannosamine ^ D-mannosaminate

In most strains of acetic acid bacteria, membrane-bound D-hexosamine dehydrogenase (tentatively named) oxidizes D-hexosamines to the corresponding D-

hexosaminates stoichiometrically. Conversion of D-hexosamines into D-hexosaminates is observed with growing cells of acetic acid bacteria and D-hexos-aminate is accumulated in the culture medium after D-hexosamine is exhausted. Since the responsible enzyme is accommodated on the outer surface of the cytoplasmic membrane and the enzyme activity is linked to the respiratory chain of the organisms, resting cells, dried cells, and immobilized cells of acetic acid bacteria are effective catalyst for D-hexosaminate production. D-Mannosaminate and D-galactosaminate, commercially unavailable compounds, can be prepared with ease with acetic acid bacteria. The respective three different D-hexosaminates are shown to be separated from each other by chromatography [95].

The first report of D-glucosaminate formation by acetic acid bacteria and other aerobic bacteria like pseudomonads was carried out in experiments with resting cells and D-glucosaminate was indirectly suggested to be formed as the oxidation product of D-glucosamine [96]. Recently, bioconversion of D-glucosamine to D-glucosaminate by a strain of Acinetobacter sp. isolated by enrichment techniques on D-glucosamine was briefly reported [97]. Although acetic acid bacteria are different from Acinetobacter in many respects, the enzyme functioning in D-glucosamine oxidation in Acinetobacter sp. is supposed to be the same as in acetic acid bacteria. There is no information about the substrate specificity of quinoprotein D-glucose dehydrogenase (m-GDH) or whether the enzyme is capable of D-glucosamine oxidation.

The enzyme activity of D-glucosamine dehydrogenase can be measured using either a combination of PMS and DCIP assayed in 50 mmol L-1 potassium phosphate, pH 7.0, or potassium ferricyanide assayed in 50 mmol L-1 glycine-NaOH buffer, pH 9.0, under essentially the same conditions as described by Ameyama [3]. Identification and measurement of D-hexosaminate can be done enzymati-cally with D-glucosaminate dehydratase (EC purified from the cell-free extract of P. fluorescens IFO 14808 according to Iwamoto and Imanaga [98].

When the growth profile of G. frateurii IFO 3264 with high D-hexosamine oxidase activity was examined in 0.5% D-glucosamine medium and monitored both by measuring turbidity and by direct viable cell counting, the organism survived even though cultivation was prolonged to the late stationary phase, where the D-glucosamine was exhausted and converted to D-glucosaminate [95]. Unlike the case of Acinetobacter sp. [97], it is characteristic to see that the majority of the cells of G. frateurii IFO 3264 survived over the prolonged cultivation after complete oxidation of D-glucosamine to D-glucosaminate, as demonstrated by viable cell counting throughout the cultivation. D-Glucosamine initially added to the culture medium was converted to D-glucosaminate almost stoichiometrically, indicating that the oxidation products of D-glucosamine stayed stable without any significant breakdown by the organism. This is a noticeable difference from the case of pseudomonads or other Gram-negative bacteria, in which an appreciable amount of D-glucosaminate was further assimilated, as also suggested by Takahashi and Kayamori [96].

Encouraged by the clear data on D-hexosamine oxidation as shown above, workers have examined the microbial conversion of D-mannosamine and D-

galactosamine to yield corresponding D-hexosaminates [95]. Freshly harvested cells (10 mg mL-1) of G. frateurii IFO 3264 grown to the stationary phase were mixed with either D-mannosamine-HCl or D-galactosamine-HCl in 5 mmol L-1 potassium phosphate, pH 7.0, and the total volume was adjusted to 10 mL. The mixtures were kept at 30 °C with shaking and the remaining D-hexosamine in the reaction mixture was checked periodically with a purified D-glucosamine dehydrogenase. D-Mannosamine oxidation took 2 h and D-galactosamine oxidation took 8 h under the above conditions. As additional new information, D-glucosami-nate dehydratase was just as effective as the three different D-hexosaminates. For example, 136% and 118% of the relative reaction rate to D-glucosaminate were observed for D-mannosaminate and D-galactosamainate, respectively, when individual oxidation products were assayed with D-glucosaminate dehydratase under steady state conditions. Thus, production of D-mannosaminate and D-galactosaminate was indicated successfully for the first time, accompanied by the enzymatic identification of the individual oxidation products.

We have to mention the reason why D-hexosamine oxidizing enzyme is classified as an FAD-dependent dehydrogenase. The main reason, after solubilization and partial purification of the respective enzyme, is that potassium ferricyanide was still valid as the electron acceptor. Second, the solubilized enzyme was highly resistant to EDTA treatment indicating either that it contains a covalently bound FAD or that the PQQ binding to the enzyme is unusually strong. The final answers will be found soon.

Heal Yourself With Qi Gong

Heal Yourself With Qi Gong

Qigong also spelled Ch'i Kung is a potent system of healing and energy medicine from China. It's the art and science of utilizing breathing methods, gentle movement, and meditation to clean, fortify, and circulate the life energy qi.

Get My Free Ebook

Post a comment