How to prepare salivary amylase and precautions for the observation of salivary amylase activity

Preparation of salivary amylase application solution:1. Each person takes a clean drinking cup and fills it with distilled water.

2. Rinse your mouth with distilled water to remove the food residue in your mouth.

3. Fill about 20ml of distilled water in your mouth and chew for 1 2min to secrete more saliva.

1. Weigh the soluble starch, add a small amount of pre-cooled distilled water, and mix it into a paste in a mortar.

2. Weigh the i g ki, add a small amount of distilled water to dissolve, and then add distilled water to 200ml.

3. Weigh Na2HPO4·12H2O, dissolve it in distilled water and set the volume to 1000ml.

4. Weigh anhydrous citric acid.

Dissolve it in distilled water and set the volume to 1000ml.

5. Buffer.

It is made by taking a liquid and mixing it.

6. Buffer - take a liquid and mix it.

7. Buffer - take a liquid and mix it.

1.Rinse the food scraps in your mouth first.

2.Drink a large sip of cold boiled water (or distilled water), do not swallow it, do chewing exercises for 2 minutes, and then spit it into a beaker.

Drink distilled water to rinse the food residue in your mouth, then take a sip of water in your mouth and spit it into a beaker after a few minutes.

Open your mouth and let the saliva flow out, and you're done.

Precautions:Ptyalin.

The optimal temperature is 37, the temperature is higher or lower than the optimal temperature, the activity of the enzyme is reduced, and three sets of experiments can be designed, and the temperature is saliva-catalyzed hydrolysis of starch.

1) The optimal pH of salivary amylase is to make the experimental results more obvious.

2) At this time, the starch is completely decomposed, and after iodization, it takes on the color of iodine solution.

3) Increase the dilution factor of saliva.

4) The optimal temperature of salivary amylase was between 27 and 47; Temperatures that are too high or too low can affect the activity of salivary amylase.

Brief introduction. - Amylase is widely distributed in animal coarse eggplant (Honayu saliva, pancreas.

etc.), plants (malt, mountain weed) and microorganisms. Enzymes of microorganisms are almost always secretory. This enzyme uses Ca2+ as an essential factor and acts as a stabilizing factor to act on both amylose and amylopectin, cleaving -1,4-chain indiscriminately.

Thus, it is characterized by causing a sharp decrease in the viscosity of the substrate solution and the disappearance of the iodine reaction, and the final product is maltose when breaking down amylose.

Mainly. In addition, there are maltotrioses and a small amount of glucose.

On the other hand, when decomposing amylopectin, in addition to maltose and glucose, -limiting dextrin with -1,6-bond in the branch part is also generated. Generally, the decomposition limit is 35-50% based on glucose, but in bacterial amylase, there are also cases with a decomposition limit of up to 70% (final free glucose).

Precautions for the observation of salivary amylase activity

1. The activity of enzymes is affected by temperature. Within a certain temperature range, as the temperature increases, the activity of the enzyme also increases. When the maximum value is reached, the chain or time temperature is the optimal temperature of the enzyme, and because the temperature is too high, the enzyme begins to inactivate, resulting in a decrease in the efficiency of the enzyme, and finally complete inactivation.

2. Enzymes are biological catalysts with extremely high catalytic efficiency, and their catalytic efficiency is higher than that of general catalysts.106 1013Catalase can catalyze the decomposition of H2O2 into H2O and O2 in living organisms, and the decomposition of H2O2 in iron powder also has a catalytic effect, but its efficiency is much lower than that of enzymes.

3. The activity of the enzyme is affected by the pH value. The enzyme is only active at a certain pH value, and higher or lower than the optimal pH will reduce the activity of the enzyme.

4. Enzyme activity is often affected by certain substances. Some substances can increase the activity of enzymes, called activators, and some substances can reduce the activity of enzymes, called inhibitors.

5. Different color changes of iodine solution indicating the degree of starch hydrolysis: amylase, purple dextrin amylase, dark brown dextrin amylase, red dextrin amylase, maltose + a small amount of glucose After iodization: blue, purple red, dark brown, reddish-brown, yellow.

1. Experimental principle.

Starch can be hydrolyzed into maltose under the catalysis of salivary amylase. Under boiling conditions, the Feilin reagent can oxidize the maltose and reduce itself to a brick-red cuprous oxide precipitate.

Therefore, the Filin reagent can be used to identify the presence of maltose in the solution, and then it can be seen whether salivary amylase can only catalyze the hydrolysis of starch and cannot catalyze the hydrolysis of other sugars (such as sucrose).

2. Purpose requirements.

1. Initially learn how to do enzyme specificity experiments.

2 Understand that enzymes are specific.

3. Materials and utensils.

Fresh saliva, sterilized absorbent cotton, tweezers, test tube, small beaker, measuring Jane, glass rod, alcohol lamp, matches, soluble starch solution with 3% mass concentration, sucrose solution with 3% mass concentration, newly configured Feilin reagent, water.

Fourth, the method and steps.

1. Rinse your mouth with water, and contain a piece of detoxified absorbent cotton in your mouth. Remove the absorbent cotton with tweezers so that the saliva in it collects into a small beaker.

2. Take 3ml of saliva, pour it into another small beaker, add 30ml of distilled water, stir well with a glass rod, and make diluted saliva for later use.

3. Take two clean test tubes, numbered, and add reagents according to the table below

Tube No. 1: 2ml of 3% starch solution plus 2ml of diluted saliva

Tube No. 2: 2ml of 3% sucrose solution plus 2ml of diluted saliva

Shake well and keep warm for 5 min at 37 points

4. Add 2ml of newly configured Feilin reagent to the two test tubes, shake well, and keep warm at 100 for 3min

Phenomenon: Test tube No. 1: brick-red precipitate; Tube 2: Blue precipitate appears.

5. Experimental conclusions.

Salivary amylase can only catalyze the hydrolysis of starch, but cannot catalyze the hydrolysis of other sugars (such as sucrose), which proves that salivary amylase has specificity.

Salivary amylase is a catalytically active protein contained in saliva, and the amylase in it is mainly -amylase.

Amylase acts on both amylose and amylopectin, indiscriminately cutting -1,4-chain in starch.

Salivary amylase catalyzes the breakdown of starch into maltose (a disaccharide) and glucose (a monosaccharide) and also produces a subset of -1,6-bond -limiting dextrin (a polysaccharide).

That is, salivary amylase can catalyze the breakdown of starch into monosaccharides, disaccharides, and a subset of polysaccharides.

1 Pinyin 2 English references.

3 Overview 4 Medical examination of salivary amylase.

Check the name. The principle of determination of salivary amylase was taken by classification.

How to operate the reagent.

Normal test results are clinically significant.

Notes: tuò yè diàn fěn méi

salivary amylase

Amylase AMS has 2 and 2 kinds, which belong to glycoside chain hydrolase with a molecular weight of 4000 50000d. Human AMS is the type, mainly in the pancreas and salivary glands, and a small amount of proximal duodenum, lung, uterus, lactation and other organs also secrete, AMS plays an important role in the digestion of polysaccharide compounds in food. Due to its small molecular weight, it is easily excreted in the urine.

Salivary amylase sedan state bar.

Examination of body fluids and excretion > Saliva and tears.

Saliva. The starch in the matrix is hydrolyzed by amylase in the specimen to produce glucose, maltose, and dextrin. The remaining starch is combined with iodine to form a blue complex, the shade of which is inversely proportional to the enzyme activity.

1) Matrix buffer (starch): weigh 9g of NaCl, Na2HPO4 and KH2PO4, dissolve in about 500ml of distilled water, and heat to boil. Take another small beaker, accurately weigh the soluble starch, add about 10ml of water to the trace beam, make it suspend and then add it to the above boiling solution, and pour it into the washing beaker together.

After cooling to room temperature, add 5ml of 37% formaldehyde solution, dilute it with distilled water, and store in the refrigerator.

2) Iodine storage solution (: weigh potassium iodate, potassium iodide dissolved in 400ml of water. Slowly add concentrated hydrochloric acid, stir while adding, dilute to 500ml with distilled water, mix well and put it in a brown bottle, and store it in the refrigerator.

3) Iodine application solution (take iodine storage solution, dilute it 10 times with distilled water, store it in a brown bottle, and store it in a refrigerator for 1 month.

Use iodine starch colorimetric manipulation.

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Decreased: pancreatitis.

1) The results of saliva and heparin anticoagulant plasma were consistent, while EDTANA2, oxalate, citrate, and sodium fluoride could inhibit the activity of AMS, making the results low.

2) The enzyme activity is significantly increased, that is, when the absorbance of the measuring tube is less than half of the absorbance of the blank tube, due to the relative shortage of substrate, the dilution factor of the specimen should be increased, or the amount of specimen added should be reduced, and the measurement result should be multiplied by the dilution factor.

3) The activity of AMS in saliva is high and should be prevented from entering.

4) If the starch matrix solution is turbid or flocculent, it indicates deterioration or contamination and should be reformulated.