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1 Glycolysis and Ethanol Fermentation Volume EC No. Thermostability Example of use product Glucokinase YK1 glucose 7.5~ ml phosphorylation Stable after incubation at 80 o C for 60 min GLK *Ethanol Production Kit glucose-6-phosphate ~80 Glucosephosphate isomerase YK1 glucose-6-phosphate 6.0~ ml isomerization - GPI *Ethanol Production Kit fructose-6-phosphate - Phosphofructokinase YK1 fructose-6-phosphate 9.0~ ml phosphorylation Stable after incubation at 60 o C for 60 min PFK *Ethanol Production Kit fructose-1,6-bisphosphate ~60 Fructose-1,6-bisphosphatase fructose-1,6-bisphosphate hydrolysis - FBP fructose-6-phosphate ~65 Fructose-1,6-bisphosphate aldolase HI1 fructose-1,6-bisphosphate 5.0~8.0 2 ml aldol cleavage reaction Stable after incubation at 100 o C for 60 min FBA *Ethanol Production Kit dihydroxyacetone phosphate + glyceraldehyde-3-phosphate ~100 Triosephosphate isomerase YK1 dihydroxyacetone phosphate 5.0~ ml isomerization Stable after incubation at 95 o C for 60 min TPI *Ethanol Production Kit glyceraldehyde-3-phosphate ~95 Glyceraldehyde-3-phosphate dehydrogenase YK1 glyceraldehyde-3-phosphate 8.0~ ml oxidation and reduction (NAD + ) Stable after incubation at 70 o C for 60 min GAP *Ethanol Production Kit 1,3-bisphosphoglycerate ~70 Phosphoglycerate kinase YK1 1,3-bisphosphoglycerate 8.0~ ml phosphorylation Stable after incubation at 85 o C for 60 min PGK *Ethanol Production Kit 3-phosphoglycerate ~85 Phosphoglycerate mutase HI1 3-phosphoglycerate 6.0~ ml isomerization Stable after incubation at 75 o C for 60 min PGM *Ethanol Production Kit 2-phosphoglycerate ~75 Enolase YK1 2-phosphoglycerate 5.5~ ml dehydration reaction Stable after incubation at 80 o C for 60 min ENL *Ethanol Production Kit phosphoenolpyruvic acid ~80 Pyruvate kinase HI1 phosphoenolpyruvic acid 5.0~8.0 1 ml phosphorylation Stable after incubation at 75 o C for 60 min PYK pyruvic acid ~75 Pyruvate kinase KS1 phosphoenolpyruvic acid 5.0~8.0 1 ml phosphorylation Stable after incubation at 85 o C for 60 min PYK *Ethanol Production Kit pyruvic acid ~85 Pyruvate decarboxylase YK1 pyruvic acid 5.0~ ml decarboxylation (TPP) Stable after incubation at 50 o C for 60 min PDC *Ethanol Production Kit acetaldehyde ~50

2 Glycolysis and Ethanol Fermentation Volume EC No. Thermostability Example of use Alcohol dehydrogenase YK1 enanthaldehyde (aldehyde) 4.5~7.0 1 ml oxidation and reduction (NADPH) Stable after incubation at 85 o C for 60 min ADH heptanol (alcohol) ~85 Alcohol dehydrogenase HI1 propionaldehyde (aldehyde) 4.5~7.0 1 ml oxidation and reduction (NADH) Stable after incubation at 65 o C for 60 min ADH *Ethanol Production Kit 1-propanol (alcohol) ~65 Alcohol dehydrogenase HI2 butyraldehyde (aldehyde) 4.5~6.5 1 ml oxidation and reduction (NADH) - ADH butanol (alcohol) ~65 Lactate dehydrogenase 2 pyruvic acid 6.0~ ml oxidation and reduction Stable after incubation at 70 o C for 60 min LDH Lactic acid ~70 Secondary alcohol dehydrogenase A 1,2-propanediol (secondary alcohol) 8.5~ ml oxidation and reduction Stable after incubation at 70 o C for 60 min SAD hydroxyacetone (ketone) ~70 Secondary alcohol dehydrogenase B 1,2-propanediol (secondary alcohol) 8.0~ ml oxidation and reduction Stable after incubation at 85 o C for 60 min SAD hydroxyacetone (ketone) ~85 reduction of long chain aldehyde asymmetric reduction of ketone coenzyme regenerating system asymmetric reduction of ketone coenzyme regenerating system oxidation of secondary alcohols oxidation of secondary alcohols

3 Pentose Phosphate Pathway Volume EC No. Thermostability Example of use Deoxyribose-phosphate aldolase 2-deoxy-D-ribose 5-phosphate 5.0~ Condensation reaction - TRA D-glyceraldehyde-3-phosphate + acetaldehyde ~50 synthesis of NADPH, or organic compounds Glucose 1-dehydrogenase glucose 5.0~8.0 1 ml oxidation and reduction (NADP + ) Stable after incubation at 70 o C for 60 min GCD gluconolactone ~70 Glucose-6-phosphate dehydrogenase glucose-6-phosphate 6.5~8.0 1 ml oxidation and reduction (NADP + & NAD + ) Stable after incubation at 70 o C for 60 min GLD phosphogluconate ~70 glucose measurement or enzyme electrodes 6-Phosphogluconate dehydrogenase 6-phosphogluconate oxidation and reduction (NADP 6.0~9.0 1 ml ) & PGD ribulose 5-phosphate + CO decarboxylation 2 ~70 Stable after incubation at 70 o C for 60 min Ribose-5-phosphate isomerase ribose 5-phosphate 4.5~ ml isomerization - PRI ribulose 5-phosphate ~85 Ribulose-5-phosphate epimerase ribulose 5-phosphate 7.0~ ml optical isomerization - RPE xylulose 5-phosphate ~50 synthesis of NADPH synthesis of NADPH (Substrate 1) erythrose 4-phosphate + xylulose 5-phosphate Transketolase 7.0~9.0 (Product 1) fructose-6-phosphate + glyceraldehyde-3-phosphate transferring ketole group (Substrate 2) ribose 5-phosphate + xylulose 5-phosphate TRK ~50 (Product 2) sedoheptulose 7-phosphate + glyceraldehyde-3-phosphate - synthesis of NADPH Xylose isomerase A (Glucose isomerase A ) xylose 6.0~ ml isomerization - XYI xylulose ~90 Xylulokinase A xylulose 7.0~9.0 1 ml phosphorylation - XYK xylulose 5-phosphate ~60 Xylulokinase B xylulose 7.0~9.0 1 ml phosphorylation - XYK xylulose 5-phosphate ~60 synthesis of xylulose

4 Saccharification Volume EC No. Thermostability Example of use β-1,3-glucanase A β-1,3-glucan 4.0~6.5 1 ml hydrolysis (β-1,3) Stable after incubation at 85 o C for 60 min TGL β-1,3-glucan oligomer ~85 laminarin β-1,3-glucanase B β-1,3-glucan 4.0~8.0 1 ml hydrolysis (β-1,3) Stable after incubation at 90 o C for 60 min TGL β-1,3-glucan oligomer ~90 β-1,3-glucanase C β-1,3-glucan 4.5~7.5 1 ml hydrolysis (β-1,3) Stable after incubation at 50 o C for 60 min TGL β-1,3-glucan oligomer ~50 Cellulase Cel.1 β-1,4-glucan(cellulose) 4.5~ ml hydrolysis (β-1,4) - EGL cellobiose ~90 Cellulase Cel.5 β-1,4-glucan(cellulose) 4.5~7.0 2 ml hydrolysis (β-1,4) - EGL cellobiose ~90 Cellulase HT1 β-1,4-glucan(cellulose) 4.5~8.5 EGL cellobiose ~75 Cellulase HT2 β-1,4-glucan(cellulose) 5.0~7.0 EGL cellobiose ~80 Cellulase HT3 β-1,4-glucan(cellulose) 5.5~7.0 EGL cellobiose ~75 Cellulase HT4 β-1,4-glucan(cellulose) 4.0~6.0 EGL cellobiose ~80 Cellobiohydrolase HT1 β-1,4-glucan 5.0~ ml hydrolysis (β-1,4) - CBH cellobiose ~80 β-glucosidase HT1 β-1,4-glucan 4.0~6.5 1 ml hydrolysis (β-1,4) - BGL glucose ~65 Xylanase YK1 xylan 3.0~ ml hydrolysis (β-1,4) Stable after incubation at 50 o C for 60 min XYL xylo-oligosaccharides ~80 β-xylosidase YK1 xylo-oligosaccharides 4.0~8.0 1 ml hydrolysis (β-1,4) Stable after incubation at 80 o C for 60 min XYS xylose ~100 Chitinase chitin 3.0~8.0 1 ml hydrolysis - CTN chitobiose ~85 Pullulanase KS1 (solution and spray-dried products are available) pullulan 4.5~ ml hydrolysis (α-1,6) - PLN maltotriose ~90 Amylopullulanase pullulan or amylose 4.0~ ml hydrolysis (α-1,6 & α-1,4) - APN malto-oligosaccharides ~90 yeast β-glucan laminarin α, β- chitin Saccharification of pullulan and starch Saccharification of pullulan and starch

5 Saccharification Volume EC No. Thermostability Example of use β-1,4-mannanase TT1 mannan 4.5~ ml hydrolysis Stable after incubation at 90 o C for 60 min MNN manno-origosaccaharides mannose ~100 Amyloglucosidase α-1,4-glucan 4.0~ ml hydrolysis Stable after incubation at 80 o C for 60 min AGL glucose ~80 Dissolving glucomannan and galactomannan Saccharification of pullulan and starch

6 Volume EC No. Thermostability Example of use Ape DNA Ligase (with Buffer) ATP + (deoxyribonucleotide) n + (deoxyribonucleotide) m ml Forming phosphoric ester bonds DLG AMP + diphosphate + (deoxyribonucleotide) n+m ~100 Stable after incubation at 100 for 60 min Glycerol dehydrogenase HI1 1,2-propanediol (or glycerol) 7.0~ ml oxidation and reduction (NAD + ) Stable after incubation at 85 for 60 min GDH hydroxyacetone (or dihydroxyacetone) ~100 myo -Inositol-1-phosphate synthase glucose-6-phosphate 7.5~9.5 1 ml isomerization (NAD + ) - IPS myo-inositol 1-phosphate ~95 Malate dehydrogenase malic acid 9.0~ ml oxidation and reduction (NAD + or NADP + ) Stable after incubation at 85 for 60 min MDH oxaloacetic acid ~85 Malate dehydrogenase (decaroboxylating) malic acid or oxaloacetic acid 7.0~ ml decarboxylation (NAD + ) Stable after incubation at 70 for 60 min MDH pyruvic acid + CO 2 ~70 Laccase YK1 benzenediol 3.0~5.0 2 ml oxidation and reduction (CuSO 4 ) Stable after incubation at 85 for 60 min LAC benzosemiquinone ~95 Others Production of inositol neutral fat Oxidation of phenol Expansin ml ml EXP Acetate kinase acetyl phosphate + ADP 6.0~ phosphorylation Stable after incubation at 80 o C for 60 min ACK acetate + ATP ~80 Adenylate kinase 2ADP 4.5~ phosphorylation Stable after incubation at 90 o C for 60 min ADK ATP + AMP ~90 Aldehyde dehydrogenase aldehyde 6.0~ oxidation and reduction (NAD + ) Stable after incubation at 55 o C for 60 min ALD carboxylate ~55 Alditol oxidase sorbitol (or xylitol) 5.5~ oxidation and reduction Stable after incubation at 75 o C for 60 min ALO glucose (or xylose) ~75 Glycerol-3-phosphate dehydrogenase A dihydroxyacetone phosphate 6.5~ oxidation and reduction (NADPH) Stable after incubation at 75 o C for 60 min GLP glycerol-3-phosphate ~75 Glycerol-3-phosphate dehydrogenase B glycerol-3-phosphate 6.0~ oxidation and reduction Stable after incubation at 75 o C for 60 min GLP dihydroxyacetone phosphate ~70 KDG aldolase pyruvate + aldehyde 4.0~ aldol reaction Stable after incubation at 85 o C for 60 min KAL aldol product ~85 The additive which promotes cellulose degradation ATP regenaration system ATP regenaration system aldehyde sorbitol (xylitol) reduction of dihydroxyacetone phosphate oxidation of glycerol-3- phosphate synthesis of chemical products

7 Volume EC No. Thermostability Nitrite reductase nitrite 6.0~ oxidation and reduction Stable after incubation at 80 o C for 60 min NRN nitric oxide ~80 Polyphosphate kinase polyphosphate (n) + ADP 4.5~ phosphorylation Stable after incubation at 80 o C for 60 min PPK polyphosphate (n-1) + ATP ~80 Urate oxidase urate 6.5~ oxidation and reduction Stable after incubation at 55 o C for 60 min URI Hydroxyisourate ~55 Bilirubin oxidase bilirubin + O 2 3.0~ oxidation and reduction Stable after incubation at 60 o C for 30 min BOX biliverdin + H 2 O ~70 Others Example of use nitrite removal ATP regenaration system uric acid uric acid Name Enzymatic Ethanol Production Kit KETP01 Kit Contets Enzyme Mix (11 kinds of enzymes) Alcohol dehydrogenase 10 buffer Glucose solution Kit Summary 12 kinds of enzymes take part in conversion from glucose to ethanol. This kit is an experimental kit to enable conversion from glucose to ethanol by the continuous enzyme reaction with those 12 kinds of thermostable enzymes in vitro.

Review of Carbohydrate Digestion

Review of Carbohydrate Digestion Glycolysis Glycolysis is a nine step biochemical pathway that oxidizes glucose into two molecules of pyruvic acid. During this process, energy is released and some of it