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Test One

Test One. Preparation of α-amylase. 1. Principle. α- amylase can act anywhere on the long-chain carbohydrates to produce various oligosaccharides. Bacillus subtilis is often used to produce α- amylase in fermentation industry. Medium preparation and sterilization

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Test One

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  1. Test One Preparation of α-amylase

  2. 1. Principle • α-amylase can act anywhere on the long-chain carbohydrates to produce various oligosaccharides. • Bacillus subtilis is often used to produce α-amylase in fermentation industry.

  3. Medium preparation and sterilization Inoculation and fermentation (2d) Centrifugation 3. Result The supernatant contains α-amylase and can be used in starch hydrolysis process. 2. Procedure

  4. Test Two Preparation of Glucoamylase

  5. 1. Principle • Glucoamylase can attack both α- [1,4] and α-[1,6]-linksbonds at non-reducing end to produce glucose. • Aspergillus nigeris often used to produce glucoamylase in industry.

  6. 2. Procedure • Medium preparation (corn meal) and sterilization • Inoculation (12 piece/flask) and fermentation (4d) • Centrifugation

  7. 3. Result The supernatant contains glucoamylase and can be used in starch hydrolysis process.

  8. Test Three Determination of Glucoamylase Activity

  9. 1. Principle • The glucoamylase can attacks [1,4] bonds at non-reducing end to produce glucose. Its activity can be determined by measuring the amount of glucose with DNS method. • The enzyme activity is the amount of enzyme necessary to produce 1 mg of glucose per hour under 40℃, pH4.5.

  10. Enzyme activity assay Uronic Acid3-amino-5-nitrosalicylic acid（brown-red） glucoamylase Starch glucose DNS 40 ℃, 30min 100 ℃, 3min Reducing sugars are oxidized to DNS is reduced to cooling ＋H2O measure A540nm

  11. 2. Procedure • Preheat (sample and CK) • Enzyme reaction • Stopping reaction (add alkali) and cooling • Add DNS , 100℃ coloration and colorimetry • 3. Calculation

  12. Test Four Preparation of Sugar Syrup

  13. 1. Principle • The process that starch can be hydrolyzed to glucose by amylase and glucoamylase is called enzyme hydrolysis. • There are two basic steps in enzymatic starch conversion: liquefaction and saccharification.

  14. 2. Procedure (1) Liquefaction Starch solution + CaCl2+ α-amylase dextrin 100℃, 5 min (Inactivating enzyme) iodine test brown 85~90 ℃,30min

  15. 48h (2) Saccharification • Liquefied starch + glucoamylase glucose (60 ℃, pH 4.0~4.5) • Iodine test can determine completion of hydrolysis when a color change does not occur.

  16. Test Five Fermentation and Crystallization of Citric acid

  17. 1. Principle • Aspergillus niger can produce both liquefying enzyme and glucoamylase. • It is a well-known production organism of citric acid.

  18. 2. Procedure • Heating and Centrifugation (removal of cell) • Neutralization (precipitation is wanted) • Acidifying(supernatant is wanted ) • Concentration( 70 ℃ over night ) • Crystallization(50 ℃ 40min, cooling down) 3. Calculation and Observation

  19. Test Six Paper chromatographyof citric acid

  20. 1. Principle Paper chromatography is a technique of separation and identification of chemical substances. Differential rates of migration of components of a mixture along the paper arises from the different affinities of the components for the solvent (mobile phase) and the paper ( stationary phase).

  21. Illustration of Chromatography Stationary Phase Separation Mobile Phase Mixture Components

  22. 2. Procedure • Preparation (paper and expanding agent) • Sample application • Paper chromatography • Colouration

  23. A a B b 标准 样品 比移值 Rf l

  24. 3. Results Draw the outline of the patches with a pencil. According to the following table, calculate the Rf values. Contrast the Rf values of the patches of “determination” and “control” with the Rf values of citric acid.

  25. 4. Attentions 1. Do not touch the chromatography filter paper, or else the fingerprints would be kept. 2. Use pencil to draw lines on the filter paper. 3. Pay much attention to fire when roasting. 4. Prevent cross pollution when dot solution with capillary tubes.

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