CN101230384A - Method for measuring activity of ruminant tumor gastric juice cellulase - Google Patents

Abstract

The invention belongs to the technical field of animal husbandry, and is a method of detecting the activity of cellulose in tumor gastric juice of ruminant. Testing subjects can be three fistula cows, and tumor gastric juice can be sampled 2h after the early feeding to be used for conducting the external detection of the activity of cellulose. Cellulose mainly contains: (1) microcrystalline cellulose, (2) carboxymethyl cellulose and (3) salicin.

Description

A kind of measuring method of activity of ruminant tumor gastric juice cellulase

Technical field

The invention relates to the measuring method of cellulase activity, more particularly, the present invention is a kind of method of measuring ruminant tumor gastric juice inner cellulose enzymic activity.

Background technology

Prior art shows, cellulose-decomposing bacteria is before decomposing cell wall substance, at first require fibrous matter generation oxidation and physics, changes of chemical structures, some drawing up property material should be excluded earlier, before enzyme formally entered, fibrous matter should expand, and had only simultaneously when cellulose-decomposing bacteria reaches enough big to the fiber adhesive capacity, and with the generation of capacity enzyme, real cellulose decomposition could take place.The digestion of carbohydrate is actually the microbial consumption soluble-carbohydrate in the cud, constantly produces a continuous working cycle of cellulolytic enzyme class degrade coarse fibers.The plain enzyme of known fiber is for meeting enzyme system at present, and mainly contain 3 types: the first kind comprises: circumscribed cellulase, or claim circumscribed B β-1,4-dextranase, avicelase, cellobiohydrolase, C ₁Cellulase is the glucogenic multienzyme complex of energy synergetic hydrolysis crystalline cellulose powder; Second class comprises: endo cellulase, or claim inscribe β-1,4-dextranase, carboxymethylcelluloenzyme enzyme, Cx cellulase, it is the dextran chain that can act on the Mierocrystalline cellulose noncrystalline domain, cut off β-1,4 glycosidic link in the chain at random, generate the enzyme of glucose, cellobiose or cellodextrin; The 3rd class comprises: β-1,4-Glycosylase, or claim cellobiase, aryl beta-glucosidase enzyme is β-1,4 glycosidic link that can cut cellobiose and generate the enzyme of free glucose.

The mensuration of cellulase activity is detected in papermaking and fodder industry enzyme mensuration alive at present, domestic still do not have an active measuring method of relevant cud Mierocrystalline cellulose enzyme of generally acknowledging, measure the activity of rumen fluid cellulase, can directly weigh fibrous feedstuff mass degradation degree in the cud, indirect reaction goes out microbe population that produces cellulase in the cud and the variation of being correlated with fauna, provide reliable basis to illustrate the zymetology mechanism that fiber substance degrades in cud, therefore have important practical value.

Summary of the invention

The present invention is achieved by following technical proposals: with 3 fistula milk cows is subjects, gets during sampling early to raise back 2h rumen fluid sample and carry out external cellulase activity and measure.Its method is as follows:

The ball milling filter paper substrate solution of getting 1mL0.5% (pH=6.0) is in 15mL scale test tube, 30min vibrates in 39 ℃ of water-baths, add the enzyme liquid that 0.2mL handles well, and pick up counting, continue vibration 60min, add DNS solution 2mL termination reaction immediately, heating 10min makes and reacts completely on boiling water bath, cool off with cold water rapidly, water is settled to 15mL, uses the 1cm cuvette, and 550nm wavelength place measures absorbancy, with reference to finding sample solution concentration on the glucose standard working curve, calculate cellulase activity.

Embodiment

The present invention respectively gets kind of a typical enzyme with above-mentioned three types of cellulases, is further described.

1. active measuring method of rumen fluid avicelase and step:

A. the preparation of solution:

(1) 0.2mol/L phosphate buffer solution: 27.36g SODIUM PHOSPHATE, MONOBASIC (NaH2PO42H2O) and 6.61g Sodium phosphate dibasic (Na2HPO4) are dissolved in the 100mL water, transfer pH=6.0.

(2) 0.5% ball milling filter paper substrate solutions: (WhatwanNo.1) is cut into fine strip shape with filter paper, take by weighing 0.5g in triangular flask, add 100mL 0.2mol./L phosphate buffer solution and an amount of granulated glass sphere, build bottleneck, the vibration 12h or the 72h that at room temperature vibrates on 65 ℃ of shaking baths are until being even oar.

(3) 0.5% saligenin (silicin) substrate solutions: it is in the 0.2mol/L phosphate buffer solution that the 0.5g saligenin is dissolved in 100mL concentration.

(4) 0.5% Xylo-Mucine substrate solutions: (CMC Na) is dissolved in the 100mL0.2mo/L phosphate buffer solution with the 0.5g Xylo-Mucine.

(5) 3,5-dinitrosalicylic acid DNS solution: (6.9g distillation phenol is dissolved in 15mL 10% sodium hydroxide solution with first solution, add water to 70mL, add 6.9 sodium bisulfites) (the 255g Seignette salt is dissolved in the 300mL 10% potassium hydroxide sodium solution with second solution, add 850mL 1%3,5-dinitrosalicylic acid sodium solution) mix, solution is yellowish brown.Use after placing 7～10d, in brown bottle, preserve 1 year effectively.

(6) glucose standardized solution: it is water-soluble and be settled to 1000mL, i.e. 5 μ mol/mL glucose standardized solution to take by weighing 0.9000g glucose.

B. the enzyme mensuration of living

(1) standard working curve: get 7 10mL volumetric flasks, add 0,1.0 respectively, 2.0,4.0,8.0,10.0mL5 μ mol/mL glucose, be diluted with water to scale, after fully shaking up, in each test tube, add 1.5mL DNS solution, shake up back boiling water bath 5min, take out the cooling back and be settled to 20mL, fully mixing with distilled water.Under the 540nm wavelength, as blank, zeroising is measured other and is respectively managed the optical density value of solution and write down the result with No. 1 test tube solution.With glucose content (mg) is X-coordinate, is ordinate zou with the optical density value of correspondence, draws out the glucose typical curve on graph paper.

(2) preparation of enzyme liquid: the 100mL triangular flask solid-liquid intersection in cud with sterilization is gathered rumen content 100mL, with sterilization plug jam-pack, filters with four layers of sterile gauze rapidly.

(3) enzyme mensuration alive

Get 1mL0.5% ball milling filter paper substrate solution in 15mL scale test tube, the 30min that vibrates in 39 ℃ of water-baths adds the enzyme liquid that 0.2mL handles well, and pick up counting, continue vibration 60min, add DNS solution 2mL termination reaction immediately, heating 10min makes and reacts completely on boiling water bath, cool off with cold water rapidly, water is settled to 15mL, uses the 1cm cuvette, and 550nm wavelength place measures absorbancy, find sample solution concentration from the glucose standard working curve, and calculate enzymic activity.

C. enzyme work is calculated

With Microcrystalline Cellulose suspension is substrate, and enzyme unit alive (IU) is defined as the amount (μ mol) that the every mL enzyme of every min liquid acts on the glucose of substrate generation.

In the formula, c-is glucose concn from the sample that standard working curve checks in, μ mol/mL;

V1-institute sample thief enzymic activity liquor capacity, mL;

Constant volume liquor capacity after the V2-termination reaction, mL;

The T-reaction times, min;

2. active measuring method of rumen fluid carboxymethylcelluloenzyme enzyme and step:

Unit of enzyme activity, measuring method and standard working curve are identical with the active mensuration of avicelase, but substrate changes 0.5% Xylo-Mucine (CMC-Na) substrate solution into.

3. the measuring method of rumen fluid saligenin enzymic activity and step:

Enzyme unit, measuring method and standard working curve and the active same measured of avicelase alive, but substrate changes 0.5% saligenin substrate solution into.

Claims (1)

1. method of measuring ruminant tumor gastric juice inner cellulose enzymic activity is characterized in that getting the rumen fluid that the fistula milk cow is early raised back 2h, and at external 39 ℃, the pH value is to measure under 6.0 the condition, and its method is as follows:

The ball milling filter paper substrate solution of getting 1mL0.5% (pH=6.0) is in 15mL scale test tube, 30min vibrates in 39 ℃ of water-baths, add the enzyme liquid that 0.2mL handles well, and pick up counting, continue vibration 60min, add DNS solution 2mL termination reaction immediately, heating 10min makes and reacts completely on boiling water bath, cool off with cold water rapidly, water is settled to 15mL, uses the 1cm cuvette, and 550nm wavelength place measures absorbancy, with reference to finding sample solution concentration on the glucose standard working curve, calculate cellulase activity.