The vitality determination and speciality experiment of amylase.

I. ObjectiveUnderstanding the specificity of amylase to different substrates
Mastering the principles and basic methods for determining amylase vitality 2. Principle
One of the main differences between enzymes and general catalysts is that it has a high degree of specificity. The so-called specificity refers to an enzyme can only one
compound
or a class of compounds (usually in the structure of these compounds have the same chemical bonds) to play a certain catalytic effect, but not to other substances catalytic reaction.
For example, amylase only actes on the a-1,4 glucoside bond in starch, not on the glycoside bond between C1 and b-D-furan fructose C2 in sucrose molecules.
Sucrose is non-reductive, starch reduction is also very small, they have a negative reaction to 3,5-dinitrosic acid
reagents
(DNS reagents), while salivary amylase (mainly containing a-starchase) hydrolyzed starch products are reduced sugar, with DNS reagents cothermal positive reaction, producing a red-brown. Based on this, it can be tested whether sucrose and starch are hydrolyzed by salivary amylase, so as to understand the specificity of enzyme action.
enzyme vitality, also known as enzyme activity, is expressed by the initial speed of the enzyme catalyzing certain chemical reactions under the conditions of the most suitable temperature and pH.
This experiment is based on a certain amount of salivary amylase, at 37 degrees C, pH6.8 conditions, in a certain initial action time to reduce starchas a reduced sugar, and then through the DNS reagent action, color measurement, to find the amount of reduced sugar production, so as to calculate the initial speed of the enzyme reaction, that is, the vitality of the enzyme. It is specified here that a amylase vitality unit is defined as the amount of enzyme required to hydrolyzed starch to produce 1 mg of reduced sugar per minute at a condition of 37 degrees C and pH6.8.III. Experimental materials and equipment
1. Materials
saliva
2. Instrument
hydrolight meter string temperature
water bath boiling water bath
3. Equipment
scale
test tube
: 25mL×10
capacity Bottle: 100mL×1
pipe: 1mL×4 2mL×2
Berries: 250mL×1
Drops: 2
Washing Balls: 2
Washing Bottles
, Test Tube Racks, Pipe Tube Racks, Glass Bars: 1 4 each. Reagent preparation
1. Starch solution (0.5%)
called 5g soluble starch, dissolved in 1000mL hot water. (Pre-use preparation)
2. Sucrose solution (1%)
3.3,5-nitrosic acid reagents (DNS reagents)
dissolved 5.0g 3,5-nitrosyanic acid in 200mL 2N NaOH solution (not suitable for high temperature solubility), followed by 500mL containing 130g potassium sodium staic acid, mixed. Then add 5g crystalline phenol and 5g sodium sulphate, stir dissolved, and settled to 1000mL. Save the spare in the dark.
Phosphate buffer (0.2mol/L, pH6.8)
. 5. NaOH solution (6mol/L)
6. NaCl solution (0.3%) 5. Operation step 1. Preparation of salivary amylase
(1) Extraction: The experimenter first cleans the mouth with a mouthwash, then a small mouthful (about 5mL) distilled water in the mouth for one or two minutes.
(2)
filtration
: the enzyme extract in the mouth is filtered with a layer of skimmed cotton.
(3) dilution: take the filter 1mL, water capacity to 100mL. As a sample fluid of amylase.
(because the activity of salivary amylase collected at different times by different people or the same person is not the same, the dilution multiple can be 50 to 300 times, or even more than this range.) The
experiment with salivary amylase
took four test tubes, numbered and operated according to table one, to record the observed color.
3. The determination of salivary amylase vitality
took 5 test tubes of 25mL scale, numbered and operated according to table 2, and recorded the experimental results.
. Results are processed . 1. Explain the results of the experiment in Table 1.
2. Based on the experimental results of Table II, and using the standard curve produced in experiment IV "3,5-nitrosic acid color to determine the sugar content", the dynamic unit containing amylase per milliliter of fresh saliva was calculated. .