Download presentation
Presentation is loading. Please wait.
Published byBrianna Stafford Modified over 9 years ago
1
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 0.005- 0.01 %
2
-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
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
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
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 +0.25 ppm Zn ion & 0.15 % phosphate (mollase or glucose)
6
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:10000-150000) Enzymatic Hydrolysis Sources of the enzymes : - Penicillium citrinum - Streptomyces aureus
7
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
8 Fermentation of inosine Brevibacterium ammoniagenes KY 13761
9
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
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
11
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
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
13
13
Similar presentations
© 2024 SlidePlayer.com Inc.
All rights reserved.