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Microbes: Nucleotide producers

Widely used as flavour enhancer

-purine ribonucleoside-5’-monophosphate:5’-GMP (Guanilic accid)5’-IMP (Inocinic acid)5’- XMP (Xanthilic acid)

-5’-AMP & isomer 2’ , 3’-5’-deoksiribonucleotide no effect-pirimidineNucleotide

Production: 5’-IMP & 5’-GMP

-Enzymatic : hydrolysis of yeast RNA

-developed in 1959 in Japan- Commercialized in 1961

Na2-IMPNa2-GMP

-Sup-Sauces

Used as food additives

Combined with Na-glutamat

0.005- 0.01 %

-Number of flavour enhancer consumed each meal:0.1 – 0.2 g consisted of:

- 5’- IMP = 8 – 12 %- 5’ – GMP = 1.5 – 2.0 %

Combined with glutamic acid

Production : - enzymatic hydrolysis- Fermentation

in Japan : 3000 ton/year

Studied for chemoterapeutic : antibiotics & sitostaticpurin analogue : - 8-Azaguanin

- 6-Merkaptopurinβ-D-arabifuranosiladenine efective for Herpes

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Cancer teurapy

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Structure:

Biosynthesis:

-From PRPP (formed fromribosa 5-fosfat & ATP)

-derivated purin :as. Inosinat (5’-IMP)

Precusor5’-AMP, 5’-XMP, 5’-GMP

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Regulation:

-overproduction of purin nucleotides wasinhibited by feedback inhibition.comercial production, partial eliminationof the regulation

mutant auxothrophsresistant to purin analogues

Production:

1. Enzymatic or chemical hydrolysisa. Enzymatic hydrolysis from yeast RNAb. RNA hydrolysis using endogenous enzymes excretionc. Chemical hydrolysis from yeast RNA with phosphorilation

2. Fermentation: use mutant blocked biosynthesis of nucleotidesno end-products regulation

a. Fermentation & chemical phosphorilationb. Nucleoside conversion to be 5’-nucleotide

1a, 1c, 2a, 2b used for production of 5’- IMP & 5’- GMP

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*Production of 5’-IMP & 5’-GMP with enzymatic hydrolysis of RNA

- first methode for production of commercial nucleotides-50 % product of 5’-nucleotides in Japan

Processing steps:1. Yeast cultures high RNA2. Extraction of RNA3. Production of hydrolitic enzymes4. RNA Hydrolysis5. Isolation and purification of 5’-IMP & 5’-GMP

Cell RNA :- 5 % mRNA- 10 – 15 % tRNA- 75 – 80 % rRNA

- Yeast low DNA content

The best source of RNA

Candida utilisSaccharomyces cerevisiae

-Content of RNA depend on culture condition:begining of Log phase : high RNAlow C/N ratio : high RNA+0.25 ppm Zn ion & 0.15 % phosphate (mollase or glucose)

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Yeast cells were separated & driedRNA was extracted with hot alkaline saline solution

(8-20 % NaCl; 8 jam; 100oC)RNA was precipitated with HCl or ethanolDried ( content of RNA: 70 – 90 %; BM:10000-150000)

Enzymatic Hydrolysis

Sources of the enzymes : - Penicillium citrinum- Streptomyces aureus

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*Production of 5’-IMP & 5’-GMP with chemical Hydrolysis

-hydrolysis at alkaline condition-Mixture of 2’- & 3’- nucleotidesnot 5’-nucleotides-Heating at 130oC for 3-4 hours in Ca(OH)2 produce nucleosides-Phosphorilation produce 5’-IMP dan 5’- GMP

*Production of 5’-IMP with Fermentation

Methode:1. Production of inocine chemical phosphorilation 5’-IMP2. Direct Fermentation to produce 5’-IMP3. Production of adenosine or 5’-AMP enzymatic conversion 5’-IMP4. chemical conversion of Hypoxanthine 5’-IMP

* Economics reason : no 1 & 2 commercially used

Fermentation to produce inosine:

-Cell is not permeable for nucleotides-Permeable for nukleosides

5’-IMP (not excreted from cells)

Inosine (excreted from cells)

dephosphorilation

-Firstly found in auxotroph mutants of adenin (ade-)- Bacillus- Brevibacterium- Corinebacterium- Streptomyces- Saccharomyces

*chemical phosphorilation using triacilphosphate (PCl3)

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Fermentation of inosineBrevibacterium ammoniagenes KY 13761

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Direct Fermentation of 5’-IMP:

Mutant should be:- do not have SAMP sinthetase to eliminate AMP regulationat level of PRPP amidotransferase

- activity of 5’-IMP degrading enzymes is low- membrane is permeable for excretion of 5’-IMP

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*Production of 5’-GMP using Fermentation

Excretion of 5’-GMP is rare in wild typeseffect of regulation at PRPP amidotransferase, IMP dehidrogenase,

and GMP sinthetase

Methode:1. Fermentation from AICARchemical conversion5’-GMP2. Production of guanosine chemical phosphorilation5’-GMP3. Production of xanthine or 5’-XMP enzymatic conversion 5’-GMP4. Direct Fermentation5’-GMP

*no 1 & 2 commercially used

Characteristics:

Fermentation from AICAR:

Microbes produce AICAR(5-amino-4-imidazole karboksamida ribosida)

E. coliB. subtilisB. megateriumBrevibacterium flavum

- purin auxotroph strains block reaction AICARPformiltransferase (AICARP FAICARP)

- No activity of AICA-riboside hydrolytic enzimes- enzymes catalysing biosynthesis of AICARis not sensitive to intracelular regulation ofpurin nucleotides

-Production was affected by sporulation- sporulation decrease production of AICAR

Suppressed by : - inhibitor (Butiric acid)- supply of O2

-AICARP was excreted in the form of dephosphorilated (AICAR)

- AICAR converted to guanosin phosphorilation to 5’-GMP

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Fermentation of Guanosin:

-Bacterial strains excreting guanosin shlould be:do not have SAMP sinthetasedo not have GMP reduktaseactivity nucleosidase was reducedenzymes for biosynthesis of GMP are unregulated

-Bacillus subtilis-Bacillus pumilus-Bacillus licheniformis-Corynebacterium petrophilum-Corinebacterium guanofaciens-Streptomyces griseus

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