Amylases mainly include α-amylase, β-amylase, glucoamylase and R-enzyme, which are widely present in the animal, plant and microbial community. Different sources of amylase have different properties. The most important amylases in plants are α-amylase and β-amylase. Alpha-amylase acts randomly on the α -1,4 glycosidic bonds of the amylose and amylopectin, and when used alone, it eventually produces oligomeric glucose, α-limit dextrin and a small amount of glucose. Ca 2+ can activate and stabilize α-amylase. It is relatively heat-resistant but not acid-resistant. It can be passivated below pH 3.6. β-amylase acts on α-1,4 glycosidic bonds from the non-reducing end, and stops when it encounters α-1,6 bonds of amylopectin. When acting alone, the products are maltose and β-limit dextrin. β-amylase is a sulfhydryl enzyme, does not need co-factors such as Ca 2+ and Cl —, the most suitable pH is acidic, contrary to α-amylase, it is not heat-resistant but feels acid-resistant. It can be kept at 60 ℃ for 15 minutes Passivation. Usually, α-amylase and β-amylase coexist in the extract. You can first determine the total activity of (α + β) amylase, then heat at 60 ℃ for 15 min, inactivate β-amylase, measure the activity of α-amylase, subtract the activity of α-amylase from the total activity, you can find Β-amylase activity. The activity of amylase can be measured by the color reaction of reducing sugar produced by starch and 3,5-dinitrosalicylic acid. Reducing sugar acts on yellow 3,5-dinitrosalicylic acid to produce brown-red 3-amino-5-nitrosalicylic acid. The color of the product is proportional to the amount of reducing sugar. The amount of reducing sugar (maltose) produced per gram of sample within a certain period of time represents the size of the enzyme activity. 1 Enzyme activity measurement method (1) Preparation of standard curve (see table below) ①Take 7 20 ml test tubes with stopper scale, clean and sterilize and dry in advance, number them, and add reagents according to the table. ②Shake well, boil in boiling water bath for 5 min. After taking it out, it was cooled by running water, and the volume was adjusted to 20 ml with distilled water, the No. 1 tube was used as a blank zero point, and the absorbance value was measured colorimetrically at a wavelength of 520 nm. And establish a regression equation for the content of maltose by the absorbance value. Reagent 1 2 3 4 5 6 7 Maltose standard solution (mL) 0 0.2 0.6 1.0 1.4 1.8 2.0 H2O (mL) 2.0 1.8 1.4 1.0 0.6 0.2 03,5-dinitrosalicylic acid (mL) 2.0 2.0 2.0 2.0 2.0 2.0 2.0 Maltose content (mg) 0 0.2 0.6 1.0 1.4 1.8 2.0OD520 (2) Determination of amylase activity of crude enzyme liquid ①Preparation of crude enzyme liquid to be tested: after 24 h fermentation, the fermentation broth was centrifuged at 4000 r / min for 10 min to remove the bacteria Add 65% saturated ammonium sulfate to the supernatant. After the ammonium sulfate is fully dissolved, salt it out at 4 ° C for 2h, and then centrifuge at 5000r / min for 20min to obtain a preliminary purified amylase. ②Operate in the following order: take the pre-cleaned sterilized and dried test tubes and number them. Take 1 ml of crude enzyme solution in each test tube, preheat 5 minutes in a 60 ° C water bath with citrate starch buffer and preheat in 60 ° C water for 5 minutes, → add 1ml of citrate starch buffer to the test tube, and bath in 60 ° C Incubate for 30 minutes → add 1.5 ml of 3,5-dinitrosalicylic acid, 5 minutes in boiling water, add sodium hydroxide solution to stop the reaction, and add distilled water to 20 ml. → Shake well and measure the OD520 nm value with a spectrophotometer. Under the above conditions, the enzyme activity required for a unit volume of sample to release 1 mg of maltose in 30 min is one maltose unit. Find out the corresponding maltose content on the standard curve and calculate the enzyme activity according to the following formula. The enzyme activity determination formula: amylase activity = maltose content (mg)? Total volume of amylase stock solution (mL) / quality of added starch. Each sample is as follows The steps shown in the operation, during the reaction, starting from adding substrate, the time interval for adding reagents to each tube must be absolutely consistent: Table 2 Sample Enzyme Activity Determination Step Reaction Sequence Sample (repeated 3) Sample blank Standard blank sample dilution Solution (ml) 1 1 0 Distilled water 0 0 Preheat at 160 ° C for 5min and add starch solution (ml) in sequence 1.5 1.5 1.5 Mix at 60 ° C for 30min then add DNS reagent (ml) 1.5 1.5 1.5 Mix at 100 ° C for 5min and add 0.4mol / L Stop the reaction with NaOH solution and add distilled water to the total volume (ml). 20 20 20 After the reaction, the sample is allowed to stand at room temperature for 10 minutes. If turbidity occurs, centrifuge at 4,000 rpm for 10 minutes on the centrifuge. The absorbance values ​​of the sample blank (A0) and the sample solution (A) are measured at a wavelength of 520 nm of the spectrophotometer. A-A0 is the measured absorbance value. The linear regression equation was used to calculate the sample amylase activity. 2 Activity calculation Definition of enzyme activity unit: at 60 ° C and pH 5.6, the amount of enzyme that releases 1 mg of maltose from a 2% soluble starch solution per hour is defined as 1 enzyme activity unit (U) amylase activity U Calculate as follows: U = × F where: U-sample amylase activity, U / ml; K-slope of standard curve; F-total amount of sample solution before reaction, ml; S-sample test amount; In Table 1, S = 1 ml; 60-60 minutes for 1 hour; 30-reaction time, min. Reagent (1) 2% starch solution (2) 0.4mol / L sodium hydroxide (3) pH5.6 citric acid buffer solution Weigh 20.01g of citric acid, dissolve to 1000mL, and make it as solution A. Weigh 29.41g of sodium citrate, dissolve to 1000mL, and make it as B liquid. Take 13.7mL of solution A and 26.3mL of solution B to mix, which is pH 5.6 buffer solution. (4) 3,5-Dinitrosalicylic acid Weigh accurately 1g 3,5-dinitrosalicylic acid dissolved in 20mL 1mol / L sodium hydroxide, add 50mL distilled water, then add 30g potassium sodium tartrate, to be dissolved Then dilute to 100Ml with distilled water, and close the stopper tightly to prevent CO 2 from entering.
Golf Club Set,Golf Club Protection,Golf Knitted Club Cover,Crocodile Pattern Golf Caps
Grand Dragon Sports Company Limited , https://www.golfyy.com