Figure 11. Regulation of metabolic pathway.

Repression of enzyme generation

Inhibition of enzyme activity

Guanosine. From B. subtilis, which is capable of producing inosine, in which PRPP amidotransferase IMP dehydrogenase (IMP -> XMP), and GMP synthetase (XMP -* GMP) are inhibited or repressed by GMP and guanine, methionine sulfoxide—, psicofuranine-, and decoyinine-resistant mutants were derived (36). The key to increasing guanosine is the loss of repression and feedback inhibition of IMP dehydrogenase, GMP synthetase, SAMP lyase, and PRPP amidotransferase. This strain showed the accumu lation of 16 g/L of guanine and both hypoxanthine and inosine in the culture, glucose 80 g/L and L-histidine 0.3 g/L (72 h at 34°C).

Phosphorylation by the Chemical Method. For nucleosides, direct reaction with POCl3 resulted in phosphorylation of not only a 5'-OH bond but also 2'- and 3' OH bonds, simultaneously. So it is difficult to selectively obtain 5'-nucleotide. Obtaining 5'-nucleotide, 2'- and 3'-OH in the ribose of nucleosides was protected previously by acetone or benzaldehyde to form an isopropiridene bond or benzir-idene bond; the product was then reacted with POCl3 to substitute the phosphate bond for 5'-OH on the riboside, at lower temperature (0 to 5 °C), and then a removal reaction of protected groups for the 2'- and 3'-bonds on the riboside was performed. The improved process is reported as follows: in the appropriate solvent with a small amount of water, POCl3 was added directly to nucleoside; the protection of 2'- and 3'-OH bonds and 5'-phosphorylation occurred, simultaneously, and a highly pure 5'-nucleotide solution was obtained. To this solution, a large amount of water was added to stop the excess phosphorylation reaction, and then sodium hydroxide was added to crystallized the disodium 5'-nucleotide, producing, for example, Na2GMP and Na2IMP crystals.

Direct Fermentation

5' -Inosinic Acid. The adenine-leaky auxotrophic strain of B. ammoniagenes showed the ability to accumulate IMP; 12.8 g/L in broth was obtained (37), which is the first report exhibiting the possibility of direct fermentation of IMP. This strain showed a high sensitivity for Mn2+ ions and optimum concentration; an extremely low and narrow range of value (10 to 20 /¿g/L; and a change in cell shape and a decrease in vital cell number when it contained Mn2+ at a concentration suitable for IMP accumulation during cultivation. By adding the nitrosoguanizine-resis-tance ability, this strain mutated to Mn2+-nonsensitive, and no change in cell shape was observed during cultivation, which accompanied the change in the mechanism of leakage of IMP, due to the improvement of the permeability of IMP through cell membrane associated with inhibition of the conjugated decomposition system (IMP -» Hx) by glucose in the cell membrane.

These results lead to the conclusion that the leakage of IMP through the cell membrane attributes to the energy-dependent reaction.

5' -Cuanylic Acid. So far GMP is not successfully produced by the direct fermentation method, mainly because of the intrinsic problem concerning the metabolic pathway; that is, no base corresponding to GMP for salvage synthesis exists, such as hypoxanthine, xanthine for IMP, or guanine for XMP (xanthosine monophosphate). Therefore, it is impossible to obtain the mutant-blocked pathway to GMP, and GMP itself is a cell-constituted component, different from IMP and XMP; then there exists a strict barrier in the cell membrane that inhibits GMP from passing through (38).

From the industrial viewpoint, the following three-step fermentation method is employed. First, XMP was produced by using the mutant of B. ammoniagenes, and then it was transformed into GMP by the enzymatic method using the other mutant of B. ammoniagenes (39).

1. XMP fermentation. In the case of XMP, B. ammoniagenes mutated to have both an adenine requirement (leakage type) and a guanine requirement. Nucleotide activity weakened, showing high productivity of XMP; also, no cell membrane barrier existed for XMP, which is different from IMP.

2. Enzyme for conversion of XMP to GMP. The nucleotide weakened B. ammoniagenes with docoynine re sistance, which strengthens the activity of the GMP synthetase that was obtained. This strain showed an increased ability to convert XMP to GMP, with no cleavage of GMP and XMP. The cell membrane permeability of XMP and GMP was improved by adding a cationic surface-active agent, polyethylene stearyl-amine (40).

3. Conjugated reaction with ATP energy system. The XMP aminase reaction is a one-step reaction for conversion of XMP to GMP, conjugated with energy regenerated by the conversion of ATP to AMP and pyrophosphoric acid (PPi), which required an indispensable cofactor, the Mg2 + ion:

Brevibacterium sp. treated with an active surfactant have an increased ability to synthesize ATP through the Krebs cycle from glucose. By using this process, in place of ATP, glucose was used as an energy supply source; conjugated with this process, the conversion of XMP to GMP was achieved economically. The enzymatic process was as follows:

PPi -> 2Pi (pyrophosphatase)

AMP + ATP 2ADP (AMP kinase)

(ATP-generating enzyme system)

These enzymes, contained in a cell, are must be regulated appropriately to prevent the subreaction from occurring. Controlling the temperature to repress the activity of nucleotide monophosphate kinase and the concentration of phosphate ion (PO3-4) increases the ability of the ATP-generating system. If Mg2"1" and PPi form insoluble magnesium pyrophosphate (Mg2PPi), the preceding reactions cannot proceed smoothly. Phytic acid, a chelating agent, was able to prevent the formation of this Mg2PPi.

When using purified XMP as raw material, overall conversion to GMP was more than 80% after 22 h (41).

Increasing the activity of XMP aminase is expected to reduce the number of cells required for conversion when whole cells are used as the enzyme sources, while reducing to a minimum the degradation of products and by-products GDP and GTP. The proliferation of the XMP aminase strain of Escherichia coli using genetic engineering techniques was attempted, which involved the subcloning of the guaA gene for XMP aminase into pBR322, followed by the construction of a plasmid utilizing the trp promoter. The amount of XMP aminase produced by the strain carrying the gauA gene accounted for 10% of total cellular protein, and the E. coli K294/XAR33 strain showed an increase in activity of about 80 times compared with E. coli K294. The conversion ratio from XMP to GMP was more than 90% after 7 h (42).

How To Reduce Acne Scarring

How To Reduce Acne Scarring

Acne is a name that is famous in its own right, but for all of the wrong reasons. Most teenagers know, and dread, the very word, as it so prevalently wrecks havoc on their faces throughout their adolescent years.

Get My Free Ebook

Post a comment