1 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  Na 2 -IMP  Na 2 -GMP -Sup -Sauces Used as food additives Combined with Na-glutamat %

-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 & sitostatic  purin analogue : - 8-Azaguanin - 6-Merkaptopurin  β-D-arabifuranosiladenine  efective for Herpes 2 Cancer teurapy

3 Structure: Biosynthesis: -From PRPP (formed from ribosa 5-fosfat & ATP) -derivated purin :  as. Inosinat (5’-IMP) Precusor 5’-AMP, 5’-XMP, 5’-GMP

4 Regulation: -overproduction of purin nucleotides was inhibited by feedback inhibition.  comercial production, partial elimination of the regulation mutant auxothrophs resistant to purin analogues Production: 1. Enzymatic or chemical hydrolysis a. Enzymatic hydrolysis from yeast RNA b. RNA hydrolysis using endogenous enzymes  excretion c. Chemical hydrolysis from yeast RNA with phosphorilation 2. Fermentation: use mutant  blocked biosynthesis of nucleotides  no end-products regulation a. Fermentation & chemical phosphorilation b. Nucleoside conversion to be 5’-nucleotide 1a, 1c, 2a, 2b  used for production of 5’- IMP & 5’- GMP

5 *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 RNA 2. Extraction of RNA 3. Production of hydrolitic enzymes 4. RNA Hydrolysis 5. 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 utilis Saccharomyces cerevisiae -Content of RNA depend on culture condition:  begining of Log phase : high RNA  low C/N ratio : high RNA  ppm Zn ion & 0.15 % phosphate (mollase or glucose)

6  Yeast cells were separated & dried  RNA was extracted with hot alkaline saline solution (8-20 % NaCl; 8 jam; 100 o C)  RNA was precipitated with HCl or ethanol  Dried ( content of RNA: 70 – 90 %; BM: ) Enzymatic Hydrolysis Sources of the enzymes : - Penicillium citrinum - Streptomyces aureus

7 *Production of 5’-IMP & 5’-GMP with chemical Hydrolysis -hydrolysis at alkaline condition -Mixture of 2’- & 3’- nucleotides  not 5’-nucleotides -Heating at 130 o C 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’-IMP 2. Direct Fermentation to produce 5’-IMP 3. Production of adenosine or 5’-AMP  enzymatic conversion  5’-IMP 4. 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 (PCl 3)

8 Fermentation of inosine Brevibacterium ammoniagenes KY 13761

9 Direct Fermentation of 5’-IMP:  Mutant should be: - do not have SAMP sinthetase  to eliminate AMP regulation at level of PRPP amidotransferase - activity of 5’-IMP degrading enzymes is low - membrane is permeable for excretion of 5’-IMP

10 *Production of 5’-GMP using Fermentation  Excretion of 5’-GMP is rare in wild types  effect of regulation at PRPP amidotransferase, IMP dehidrogenase, and GMP sinthetase Methode: 1. Fermentation from AICAR  chemical conversion  5’-GMP 2. Production of guanosine  chemical phosphorilation  5’-GMP 3. Production of xanthine or 5’-XMP  enzymatic conversion  5’-GMP 4. Direct Fermentation  5’-GMP *no 1 & 2 commercially used

Characteristics: Fermentation from AICAR: Microbes produce AICAR (5-amino-4-imidazole karboksamida ribosida) E. coli B. subtilis B. megaterium Brevibacterium flavum - purin auxotroph strains  block reaction AICARP formiltransferase (AICARP  FAICARP) - No activity of AICA-riboside hydrolytic enzimes - enzymes catalysing biosynthesis of AICAR is not sensitive to intracelular regulation of purin nucleotides -Production was affected by sporulation - sporulation  decrease production of AICAR Suppressed by : - inhibitor (Butiric acid) - supply of O 2 -AICARP was excreted in the form of dephosphorilated (AICAR) - AICAR  converted to guanosin  phosphorilation to 5’-GMP 11

12 Fermentation of Guanosin: -Bacterial strains excreting guanosin shlould be:  do not have SAMP sinthetase  do not have GMP reduktase  activity nucleosidase was reduced  enzymes for biosynthesis of GMP are unregulated -Bacillus subtilis -Bacillus pumilus -Bacillus licheniformis -Corynebacterium petrophilum -Corinebacterium guanofaciens -Streptomyces griseus

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