The Japanese Traditional Applied Microbiology may Contribute to the - PowerPoint PPT Presentation

The Japanese Traditional Applied Microbiology may Contribute to the Argentine Osao ADACHI & Kazunobu MATSUSHITA Laboratory of Applied Microbiology Department of Biological Chemistry Faculty of Agriculture, Yamaguchi University Yamaguchi 753-8515, Japan

Growth Habitats of Acetic Acid Bacteria Flowers (Pistils/Pollens) Fruits and Flowers (high sugars or sugar alcohols) Yeast, Fungi Lactic acid bacteria Zymomonas Fruit fly Vinegar fly Wine fly In high conc. of sugars and Competitive microbes sugar alcohols

Sushi based on Rice Vinegar

Static Rice Vinegar Fermentation

2-Keto- 5-Keto- 2,5-Diketo- D -Glucose D -Gluconate D -gluconate D -gluconate D -gluconate Periplasm Periplasm FAD Outer surface Outer surface FAD PQQ PQQ Cytoplasmic membrane membrane Cytoplasmic e - e - e - e - Hexosamine D -Mannitol FAD PQQ D -Fructose Hexosaminate e - e - D -Sorbitol Glycerol PQQ FAD L -Sorbose e - Dihydroxyacetone e - Cytosolic enzymes Cytoplasm Cytoplasm meso -Erythritol D -Sorbitol PQQ PQQ L -Sorbose L -Erythrulose e - e - D -Arabitol Cyclic alcohols PQQ PQQ D -Xylulose e - e - e - e - e - e - e - Cyclic ketones PQQ MoCo? PQQ FAD? PQQ Ethanol Acetate Ribitol D -Fructose Quinate Acetaldehyde L -Ribulose 5-Keto- D -fructose Dehydroquinate

Strong Enzymes in Acetic Acid Bacteria Membrane-bound Cytoplasmic Glucose dehydrogenase (NADP) Alcohol dehydrogenase (PQQ) 2-Ketogluconate reductase (NADPH) Glucose dehydrogenase (PQQ) Gluconate dehydrogenase (FAD) 5-Ketogluconate reductase (NADPH) Sorbitol dehydrogenase (PQQ) Aldehyde dehydrogenase (NAD/NADP) Glycerol dehydrogenase (PQQ) Dioxyacetone reductase (NADH/NADPH) Aldehyde dehydrogenase (Mo) 5-Ketofructose reductsae (NADPH) Fructose dehydrogenase (FAD) Shikimate dehydrogenase (NADP) Quinate dehydrogenase (PQQ) Mannitol dehydrogenase (NADP) Polyol dehydrogenase (PQQ) Sorbitol dehydrogenase (NAD) Coenzymes (NAD/NADP, NADH/NADPH, PQQ, FAD, CoQ 10 )

Fermentation-based Biotechnology of AAB Fermentation-based Biotechnology of AAB Physiological features Basic Research Industrial development Acetobacter ・ Gluconacetobacter Acetic acid Acetic acid Vinegar fermentation fermentation Gluconobacter Acetic acid resistance Ketogulonate Vitamin C fermentation Oxidative Fermentation Dihydroxyacetone DHA ( Cosmetics ) fermentation Tartaric acid 5-Ketogluconate ( Chemicals) fermentation Dehydroquinate Shikimate fermentation (Medicine)

Membrane-bound Enzymes in Acetic Acid Bacteria and Energy Generation by Substrate Oxidation 2-Keto- 5-Keto- 2,5-Diketo- D -Glucose D -Gluconate D -gluconate D -gluconate D -gluconate Periplasm Periplasm 2KGDH GADH GLDH GDH e - e - e - e - UQH 2 UQ Cytoplasmic Cytoplasmic membrane membrane 2H + 2H + + O Cytoplasm 2H + Cyt. bo 3 H 2 O 2H + Energy UQH 2 UQ generation e - e - e - e - ADH ALDH QDH SLDH Acetaldehyde D -Sorbitol Quinate Ethanol L -Sorbose 3-Dehydroquinate Acetate

CH 2 OH CH 2 OH CHO H C OH H OH C C O PQQ-GLDH HO C H HO H C FAD-SDH HO C H H C OH H OH H C OH C PQQ-SDH/SNDH FAD-SLDH C O SNDH HO H C OH C H PQQ-SDH/SNDH CH 2 OH CH 2 OH CH 2 OH NAD(P)-SNDH +H2/Cat L -Sorbose L -Sorbosone D -Sorbitol In vitro Reichstein-Grussner's method CHO H OH C HO C H COOH COOH CO COOH H OH C C O C O O HOC C FAD-2KGADH 2,5-DKGR in vitro H OH C O HO C H HO C H HOC HO C H CH 2 OH PQQ-GDH H OH C enzymatic HC FAD-GADH H C OH H C OH D -Glucose H OH C HOCH HO H C C O COOH CH 2 OH CH 2 OH CH 2 OH H OH C CH 2 OH 2-Keto- D -gluconate 2,5-Diketo- D -gluconate 2-Keto- L -gulonate L -Ascorbate HO C H H C OH H C OH COOH COOH Gray's method H OH H OH CH 2 OH C C Gray's method enzymatic PQQ-GLDH HO H HO C H C D -Gluconate in vitro H OH H C OH C HO C O C H CH 2 OH CH 2 OH 5-Keto- D -gluconate L -Idonate Different Routes for Vitamin C

CHO COOH CHO COOH HCOH HCOH HCNH 2 HCNH 2 HOCH HOCH HOCH HOCH HCOH HCOH HCOH HOCH HCOH HCOH HCOH HCOH CH 2 OH CH 2 OH CH 2 OH CH 2 OH D -Glucose D -Gluconate D -Galactosamine D -Galactosaminate CHO COOH CHO COOH HCNH 2 HCNH 2 H 2 NCH H 2 NCH HOCH HOCH HOCH HOCH HCOH HCOH HCOH HCOH HCOH HCOH HCOH HCOH CH 2 OH CH 2 OH CH 2 OH CH 2 OH D -Glucosaminate D -Mannosamine D -Glucosamine D -Mannosaminate Alternative Membrane-bound GDH from Gluconobacter

Hexosamine Oxidation Rf Glu N Oxd Glu N Man N Oxd Man N Gal N Oxd Gal N Glucosaminate

D-glucose 1-deoxynojirimycin Miglitol nojirimycin

OH O OH O Cyclobutanol Cyclobutanone Cyclohexanol Cyclohexanone O O OH OH O OH Cyclopentanol Cyclopentanone 1,2-Cyclohexanediol 1,2-Cycloxanedione OH O O OH OH O 1,3-Cyclopentanediol 1,3-Cyclopentanedione Cyclooctanol Cyclooctanone Cyclic Alcohol Oxidation

COOH HO Glucose Quinate HO OH OH QDH ( Quinoprotein ) Phosphoenol- Erythrose-4-P pyruvate COOH HO Dehydroquinate O OH DQD OH COOH Dehydroshikimate 3-Deoxy-7-phospho-D- arabinoheptulosonate O OH NH 2 O SKDH OH O CH 3 H O N H 3 C H H COOH H O Ansamycin Shikimate H 3 C CH 3 Rifamycin HO OH Oseltamivir (Tamiflu) OH Candicidin Chorismate Anthranilate Pyocyanine Nystatin Tryptophan Chloramphenicol Prephenate Nocardicin Actinomycin Novobiocin Polymyxin Tyrosine Phenylalanine Metabolic Map of Shikimate Pathway Metabolic Map of Shikimate Pathway

Nata de coco

Biocellulose and heteropolysaccharides of AAB Biocellulose and heteropolysaccharides of AAB S strain Pellicle Static Aerobic growth Biofirm Drug resistance Acetobacter lovaniensis Gluconacetobacter xylinus NBRC 3284 1 µm Acetobacter aceti AJ 12368 R strain （ S. Yamanaka et al., J. Materials Science 24, 3141-3145, 1989 ）

0.1 µmol Ferricyanide reduced (µmol) 0.4 0.2 Absorbance at 660 nm 0.05 µmol 0.2 0.1 0 20 40 60 80 Incubation period (min) Time Course of D-Fructose Oxidation

Time Course D -Gluconate Oxidation

Enzyme-based Biotechnology by using AAB Enzyme-based Biotechnology by using AAB Physiological features Basic Research Industrial development Cytoplasmic AAB NAD(P) enzymes Gluconobacter Chemicals ・ Oxidative Medicines fermentation (Bioconversion) PQQ ・ FAD Enzymes Biosensor Bio-fuel cell PQQ GDH FAD FDH Cyt c FDH-dependent battery K. Kano et al. (2007)

n -hexanal n -hexanal Off-flavor Elimination from Soy Bean Meal with Acetic Acid Bacteria

6 250 A. rancens SKU 1108 Acetate 4 150 Ethanol, Acetic acid(g/100 ml) Klett 2 50 Ethanol 0 0 A. rancens IFO 3298 4 Klett 150 2 50 0 0 A. aceti IFO 3283 200 4 100 2 0 0 0 40 80 Fermentation period (h)

(A) 30 C (B) 37 C 120 growth 200 Conversion rate (%) 80 Klett units 100 L -erythrulose 40 0 0 0 24 48 0 24 48 72 Fermentation period (h) L-Erythrulose Production by Growing Cells of G. frateurii CHM 43

Fungal Strains for Koji Preparation and Rice Grains Covered with Asp. oryzae (Koji-gaku, H.Murakami(1985)) Asp. oryzae 12 h Asp. oryzae 24 h Asp. sojae 46 h

Rice Koji

Oxidation of Various Amines by Fungal Amine Oxidase + O 2 + H 2 O 2 + NH 3 Imidazole Histamine acetaldehyde Relative Relative Substrate Substrate rate rate Monoamine Monoamine 4.0 100* Ethylamine Benzylamine 108.0 150.6 n- Propylamine Phenethylamine 195.2 n- Butylamine Diamine 194.8 0 n- Amylamine Ethylenediamine 194.5 0 n- Hexylamine Propylenediamine 75.1 13.2 Tyramine Putrescine 28.8 13.6 Tryptamine Cadaverine 0 13.9 Epinephrine Hexylenediamine 67.3 Norepinephrine Polyamine 42.0 0 Serotonin Spermine 89.0 0 Agmatine Spermidine 110.5 Histamine

Fungal Tannase and Release of Polyphenols

Coffee Pulp Koji

Coffee Pulp Chlorogenate Hydrolase HO COOH ＋ Quinate Caffeic acid OH HO OH Phosphoenolpyruvate Quinate Dehydrogenase （ QDH ） DOPA HO COOH 3-Deoxyarabino - Glucose 3-Dehydroquinate heptulosonate O OH OH 7-phosphate Dehydroquinate Dehydratase （ DQD ） Erythrose 4-phosphate COOH 3-Dehydroshikimate Shikimic acid O OH OH Pathway Shikimate Dehydrogenase （ SKDH ） COOH O NH 2 Shikimate O H O CH 3 HO OH N H 3 C OH H H H O H 3 C CH 3 Oseltamivir (Tamiflu) Several Antibiotics Aromatic Biodegradable Amino acids Herbicide/Agrochemicals