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DEBORA NANGIN AND AJI SUTRISNODEPARTMENT OF AGRICULTURAL PRODUCT TECHNOLOGYBRAWIJAYA UNIVERSITYMALANG , INDONESIA
ISOLATION AND CHARACTERIZATION OF RAW
STARCH DEGRADING AMYLASE FOR IMPROVING ENERGY EFFICIENCY OF
STARCH-BASED INDUSTRY
INTRODUCTION
Amylase25%
Gelatinization
Liquefaction
Saccharification
Raw Starch Degrading Amylase (RSDA)
INTRODUCTION
Industrial enzyme ????
Farm soil
Cassava waste
ISOLATION OF MICROBES
Farm soil
Cassava waste
Sample : Starch broth medium (1:2)37 OC, 120 rpm, 48 h
Platting on starch agar medium
Incubation 37 OC, 24-48 h
POTENTIAL ISOLATES
TK6f
TK6bTK6c
O5c
Yellowish oval colony
No endosporeCoccus shaped Gram positive
Isolates name
Amylolytic index
RSDA activity(U/ mL)
TK6f 2 0,986 TK6b 3 1,130 TK6c 1,75 0,906 O5c 1,5 0,710
PRODUCTION-PURIFICATION
Starch broth medium 37 OC, 120 rpm, 10 h Dialysis
RSDA enzyme
Ammonium sulphat 70%
RSDA PURIFICATION
StepTotal
Activity(U)
Total Protein(mg)
Specific Activity (U/ mg)
Purifi-cation (fold)
Reco-very (%)
Supernatant 934,48 8670 0,108 100 1Ammonium
sulfate precipitation
67,71 251,98 0,269 7,25 2,49
Dialysis 71,62 127,26 0,563 7,66 5,22
120 kDa
91 kDa
62 kDa
46 kDa
38 kDa
26 kDa
19 kDa
9 kDa
58,77 kDa
1 2 31: Marker, 2: SDS-PAGE
3: Zymogram
EFFECT OF TEMPERATURE
Effect of temperature on enzyme activity
Effect of temperature on enzyme stability
EFFECT OF pH
Effect of pH on enzyme activity
Effect of pH on enzyme stability
EFFECT OF STARCH AND ION
Effect of starch type on enzyme digestibility Effect of metal ions on
enzyme digestibility
CONCLUSIONS
• 4 isolates obtained (TK6f, TK6b, TK6c, and O5c) and TK6b showed highest RSDA activity and was selected for RSDA production.
• A 58,77 kDa enzyme was obtained after partial purification.
• The enzyme had highest activity at 40 0C and pH 7, and relative stable at 30-50 0C and pH 7-8.
• Rice starch had the highest digestibility by enzyme followed by corn, potato, and cassava starch.
• Enzyme activity was enhanced with the presence of calcium ion, while ferrous, barium, copper, sodium, and magnesium ions inhibited enzyme.
THANK YOU
DOCUMENTATION
