1 / 32

BIOCHEMICAL ENGINEERING

PTT 203. SEMESTER 1 (2012/2013). BIOCHEMICAL ENGINEERING . ENZYMES (LAB). PN. NURUL AIN HARMIZA ABDULLAH. COURSE OUTCOME 1 : Ability to differentiate types of enzymes and analyze its kinetics study and catalysis. . FORMAT OF LABORATORY REPORT. Objectives 5% Method 10% Result 15%

kermit
Download Presentation

BIOCHEMICAL ENGINEERING

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. PTT 203 SEMESTER 1 (2012/2013) BIOCHEMICAL ENGINEERING ENZYMES (LAB) PN. NURUL AIN HARMIZA ABDULLAH • COURSE OUTCOME 1 : • Ability to differentiate types of enzymes and analyze its kinetics study and catalysis.

  2. FORMAT OF LABORATORY REPORT • Objectives 5% • Method 10% • Result 15% • Discussion 40% • Conclusion 10% • Lab sheet 20% (If the experiment does not have questions, the mark will be evaluated in discussion – then the mark will be 60%)

  3. LABSHEET

  4. The report writing is group based. The lab report for each session should be submitted within ONE week after the experiment was performed. The Lab Sheet (exercise) should be filled and signed by the teaching engineer at the end of each lab session.

  5. Lab test will be given after complete all the experiments in the lab manual. • It is individual and will be based on all the experiments that have been carried out. Therefore, students are advised to give full attention during lab session.

  6. BRING ALONG: • Lab manual • Lab coat • Shoes (no sandals allowed) • Neat and suitable clothes • Calculator • Marker pen

  7. TASK DISTRIBUTION Total students per group = 3 ; • Engineer Tasks: • Fill up the OBJECTIVES and METHOD (flowchart) parts of the report before coming to lab. • Write lab report (submit on next lab session) • Assistant Engineer Task: • Do the experiment (hands-on) • Technician Task: • Fill up lab sheet and answer all questions then submit to TE for signature.

  8. LAB REPORT TEMPLATE

  9. Shuler, M. L. and Kargi. (2002). Bioprocess Engineering: Basic Concept. 2nd Ed. Upper Saddle River, NJ: Prentice Hall PTR . CHAPTER 3. LIST OF EXPERIMENTS • EXPERIMENT 1: ENZYME KINETICS: STANDARD CURVE OF ENZYME • EXPERIMENT 2: TIME COURSE OF ENZYME ACTIVITY • EXPERIMENT 3: EVALUATION OF KINETIC CONSTANTS OF PURE ENZYME • EXPERIMENT 4: ENZYME ACTIVITY: EFFECT OF TEMPERATURE • EXPERIMENT 5: ENZYME ACTIVITY: EFFECT OF ENZYME INHIBITOR • EXPERIMENT 6: ENZYME ACTIVITY: EFFECT OF pH • EXPERIMENT 7: YEAST CULTIVATION METHOD • EXPERIMENT 8: GROWTH KINETICS OF YEAST • EXPERIMENT 9: FUNGUS CULTIVATION METHOD • EXPERIMENT 10: GROWTH KINETICS OF FUNGUS • EXPERIMENT 11: ASEPTIC TECHNIQUE IN PLANT CELL CULTIVATION • EXPERIMENT 12: EXTRACELLULAR/INTRACELLULAR ENZYME RECOVERY • EXPERIMENT 13: OPEN-ENDED: BIOCONVERSION TECHNOLOGY • EXPERIMENT 14: OPEN-ENDED: BIOSEPARATION METHOD

  10. EXPERIMENT 1-6 (ENZYME ASSAY)

  11. STARCH • Plants store glucose as the polysaccharide starch. The cereal grains (wheat, rice, corn, oats, barley) as well as tubers such as potatoes are rich in starch. • Starchis a carbohydrate consisting of a large number of glucose units joined by glycosidic bonds. • Starch can be separated into two fractions--amylose and amylopectin. Natural starches are mixtures of amylose (10-20%) and amylopectin (80-90%).

  12. TYPES OF AMYLASE • α-Amylase • β-Amylase • γ-Amylase

  13. α-amylase • α-amylase breaks down long-chain carbohydrates, ultimately yielding maltotriose and maltose from amylose, or maltose, glucose and "limitdextrin" from amylopectin. • Because it can act anywhere on the substrate, α-amylase tends to be faster-acting than β-amylase.

  14. β-Amylase • β-amylase catalyzes the hydrolysis of the second α-1,4 glycosidic bond, cleaving off two glucose units (maltose) at a time.

  15. γ-Amylase • γ-Amylase will cleave α (1-6) glycosidic linkages, as well as the last α(1-4) glycosidiclinkages at the nonreducing end of amylose and amylopectin, yielding glucose.

  16. Amylase which catalyzes the digestion (hydrolysis) of the polysaccharide starch to the disaccharide maltose. • Amylase is produced by a wide range of organisms, including humans. The amylase used in this exercise has been derived from a microorganism.

  17. Most substrate and products of enzymatic reactions are colorless. This is true for the reaction conducted in lab. In the laboratory, colorimetric procedures are performed to determine the disappearance of substrate (s) or the appearance of the product(s) in the reaction mixture. These kinds of chemical identifications of enzyme substrate and products are called enzyme assays.

  18. TYPES OF AMYLASE ASSAY • Reaction with iodine • Presence of maltose

  19. In this assay, the presence of maltose, a reducing sugar, is detected by performing an oxidation-reduction reaction. • Here, the reaction mixture (water, amylase, undigested starch, and maltose) is mixed with an alkaline solution of 3, 5-dinitrosalicylic acid (DNS) and boiled for 5-10 minutes.

  20. Due to the presence of a carbonyl group (C=O), maltose participates in an oxidation-reduction reaction with DNS. The carbonyl group of maltose gets oxidized and changed into a carboxyl group and the 3, 5-dinitrosalicylic acid is reduced and changed into 3-amino, 5-nitrosalicylic acid (simply can be termed as reduced DNS). • This reaction cause a change in color form yellow to orange or red, depending the concentration of maltose produced.

  21. As you might expect, there will be no color change on a control without the enzyme, Using the controls as the “blank” for the absorbance in a colorimeter, the value of absorbance is measured on the “samples” where the enzyme was present and is interpreted as the amount of maltose produced, thus showing the extent of amylase activity.

  22. Amylase is the enzyme that catalyzes the hydrolysisof the polysaccharide starch in to the disaccharide maltose: • This changes color to from yellow to orange to red.

  23. You will use the redox reactions between maltose and DNS to study the extent of the amylase activity. • DNS is the oxidizing again (it is the electron acceptor). • Maltose is the reducing agent (it is the electron donor) because it contains the carbonyl (C=O) functional group. • It produces reduced DNS, which cause a color change from yellow to orange to red (depending on the concentration of maltose). • The color or of your reaction indicates the extent of amylase activity.

  24. If you have a high absorbance of reduce then more maltose produced because higher amylase activity. • Increased amylase activity  increase maltose  increased redox reaction  increase reduced DNS  increased color change  more abs • In the optimal pH or temperature, the most rapid hydrolysis of starch to maltose occurs.

  25. CALCULATIONS • Refer handouts from Pn Naha…

  26. THANK YOU

More Related