CN105986004A - Method of quickly measuring enzyme activity of feed-use cellulase - Google Patents
Method of quickly measuring enzyme activity of feed-use cellulase Download PDFInfo
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- CN105986004A CN105986004A CN201510043706.0A CN201510043706A CN105986004A CN 105986004 A CN105986004 A CN 105986004A CN 201510043706 A CN201510043706 A CN 201510043706A CN 105986004 A CN105986004 A CN 105986004A
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Abstract
The invention discloses a method of quickly measuring enzyme activity of feed-use cellulase, which includes the following steps: adding straw powder used for measuring the enzyme activity of the feed-use cellulase to a liquid containing to-be-tested feed-use cellulase to form an enzymolysis reaction liquid; and performing an enzymolysis reaction to the enzymolysis reaction liquid, and measuring the mass of soluble substances generated in the enzymolysis reaction to obtain the enzyme activity of the feed-use cellulase. The method not only can quickly measure the enzyme activity of the feed-use cellulase in a buffer solution, but also can quickly detect the enzyme activity of the feed-use cellulase in an in-vitro animal digestive fluid. On the basis of the method, a feed production enterprise and a breeding farmer can analyze and compare the enzyme activity of cellulase. The method is suitable for researching, production and application of the feed-use cellulase.
Description
Technical field
The present invention relates to a kind of method of quick mensuration feedstuff cellulose enzyme vigor in field of fodder.
Background technology
Cellulase is to apply the widest monomeric enzyme in current feed industry, is also the most ripe a kind of feed enzyme, accounts for
About the 15% of the whole feed enzyme output value.In animal feed, add appropriate cellulase can improve efficiency of feed utilization, promoting
Enter growth of animal, reduce feces discharge, improving the ecological environment and preventing and treating the aspects such as Animal diseases all has a positive effect, and
Largely avoid because adding negative effect produced by the materials such as antibiotic, hormone and high-copper, there is obvious warp
Ji benefit and environment protection significance.
Along with the cellulase extensive application in feedstuff, to the demand of cellulose enzyme vigor evaluation methodology the most increasingly
Urgently.For a long time, the analysis of cellulose enzyme vigor measures and is always controversial topic in feed industry field,
Practicality and the repeatability of the analytical data that existing analysis method is provided are the most undesirable.At present, the enzyme of cellulase is lived
Mainly with sodium carboxymethyl cellulose (abbreviation CMC sodium salt), as substrate, sodium carboxymethyl cellulose purity used is high in power evaluation
And soluble in water, but feed ingredient is relatively complicated, and the natural cellulose wherein contained is essentially all not
It is dissolved in the high polymeric compound of water, and these compounds are generally with other compositions in feedstuff, and (lignin, Portugal are poly-
Sugar and xylan etc.) be cross-linked with each other together with, form baroque polymer, the therefore carboxymethyl cellulose of purification
Sodium can not mimic cellulose enzyme when acting in vivo faced by complex substrate composition.Although Animal experiment is directly perceived,
Reliability is relatively good, but the time oversize (at least needing 2 months) of test, and cost is the highest, in actual production
Journey is difficult to promote, differs greatly with the requirement of quickly analyzing in process of scientific research.
It addition, cellulase also has a lot of isozyme, they adaptabilities to acid or alkali environment, the toleration to temperature with
And the most variant to the resistance of animal alimentary canal environment, it is the most indistinguishable with the laboratory analysis methodologies of current standard,
And production application is sought after these data.
Therefore, a kind of more science, the vitro detection side of objective and easy evaluation cellulose enzyme vigor are explored and set up
Method in the urgent need to.
Summary of the invention
The technical problem to be solved is the enzyme the most quickly, more meeting and measuring feedstuff cellulase practically
Vigor.
For solving above-mentioned technical problem, present invention firstly provides a kind of method measuring feedstuff cellulose enzyme vigor.
A kind of method measuring feedstuff cellulose enzyme vigor provided by the present invention, comprises the steps: to be used for
The powder of straw measuring feedstuff cellulose enzyme vigor joins in the liquid containing feedstuff cellulase to be measured, it is thus achieved that
Enzyme digestion reaction liquid；Described enzyme digestion reaction liquid is carried out enzyme digestion reaction, measures the soluble matters of described enzyme digestion reaction generation
Quality, obtains the enzyme activity of described feedstuff cellulase to be measured.
In said method, the described powder of straw for measuring feedstuff cellulose enzyme vigor is to be pulverized by the stem of plant
The powder arrived.In the described powder of straw for measuring feedstuff cellulose enzyme vigor, the particle diameter of stalk particle can be
370-850μm。
In said method, the described liquid containing feedstuff cellulase to be measured can be containing feedstuff cellulase to be measured
Buffer solution or containing the in vitro animal digestive juice of feedstuff cellulase to be measured.Described containing feedstuff fiber to be measured
The buffer solution of element enzyme described can contain with described feedstuff cellulase to be measured and NaAc_HAc buffer solution preparation
The in vitro animal digestive juice of feedstuff cellulase to be measured can be with described feedstuff cellulase to be measured and described in vitro animal
Digestive system is prepared.Described in vitro animal digestive juice concretely in vitro animal gastric juice, described in vitro animal gastric juice specifically may be used
For in vitro bovine rumen liquid.
In said method, in described enzyme digestion reaction liquid, the concentration of described cellulase can be 0.164-0.167mg/mL,
Concretely 0.164mg/mL or 0.167mg/mL；The concentration of described powder of straw can be 16.4-16.7mg/mL, specifically
Can be 16.4mg/mL or 16.7mg/mL；Described cellulase can be 1:100 with the mass ratio of described powder of straw.
In said method, the reaction temperature of described enzyme digestion reaction can be 37-39.5 DEG C, concretely 37 DEG C.
In said method, the pH value of described enzyme digestion reaction liquid can be 5.5-6.8, concretely 5.5 or 6.8；Described
The enzyme digestion reaction time can be 30-240min, concretely: 30-100min, 100-240min, 30min, 60min,
90min, 100min, 120min, 150min, 180min, 210min or 240min.
In said method, the quality of described soluble matters can obtain according to formula 3, and described formula 3 is: soluble matters
Quality=powder of straw gross mass-residue quality；Described powder of straw gross mass is for being used for measuring feedstuff cellulose enzyme
The dry weight of the powder of straw of vigor；Described residue quality is relatively by reacted for described cellulase degradation solution
Centrifugal force is the residue dry weight after the enzyme digestion reaction that centrifugal 3min obtains under the conditions of 500g；Described soluble matters
Quality is in terms of dry weight.
In said method, the described powder of straw for measuring feedstuff cellulose enzyme vigor can be according to following for measuring
Prepared by the preparation method of the powder of straw of feedstuff cellulose enzyme vigor.
For solving above-mentioned technical problem, present invention also offers the above-mentioned straw for measuring feedstuff cellulose enzyme vigor
The preparation method of stalk powder.
The preparation method of the powder of straw for measuring feedstuff cellulose enzyme vigor provided by the present invention, including as follows
Step: pulverized by the stem of plant, obtain the powder that particle diameter is 370-850 μm, this powder is described for measuring feedstuff use
The powder of straw of cellulose enzyme vigor.
Above, described straw can be the stem of ripe crops, as become the stem of cooked maize, the ripe stem of Sorghum vulgare Pers., maturation
The stem of Oryza sativa L. and the stem of ripe Semen Tritici aestivi.
The said method provided by the present invention application in differentiating feedstuff cellulose enzyme vigor falls within the present invention's
Protection domain.
It is demonstrated experimentally that the present invention measure feedstuff cellulose enzyme vigor method can be directly perceived, easy, objective entirely
Ground, face measures the enzyme activity of feedstuff cellulase.The present invention measures the feedstuff method of cellulose enzyme vigor, with
Stalk particle particle size range be the powder of straw of 370-850 μm be substrate, both can quickly detect feedstuff cellulase and exist
Enzyme activity in buffer solution, can quickly detect again feedstuff cellulase enzyme activity in vitro animal digestive juice.
Feedstuff Enterprises and raiser are referred to the method for the present invention and are analyzed the enzyme activity of cellulase and compare,
It is applicable to the research of feedstuff cellulase, produces and apply.
Detailed description of the invention
Below in conjunction with detailed description of the invention, the present invention is further described in detail, the embodiment be given only for
Illustrate the present invention rather than in order to limit the scope of the present invention.
Experimental technique in following embodiment, if no special instructions, is conventional method.
Material used in following embodiment, reagent etc., if no special instructions, the most commercially obtain.
Cellulase A in following embodiment is the feeding cellulase that biological company limited is raised by Wuhan Xinhua, fiber
Element enzyme B is the product (CS10000) of Nuo Neng bio tech ltd, Jinan, and cellulase C is to praise in Beijing flourishing age
The product (NXE10000) of bright Science and Technology Development Co., Ltd., cellulase D is that Nanjing adds the limited public affairs of good biotechnology
The cellulase of department.
Embodiment 1, for measuring the preparation of powder of straw of feedstuff cellulose enzyme vigor
Take the corn straw 200kg of maturation, remove Ye Hegen, leave cane, pulverize with pulverizer, obtain straw
Powder.Powder of straw is sieved through 20 mesh sieves for the first time, collected the powder of straw sieving of 20 mesh sieves, it is thus achieved that powder of straw
Sieving 56kg for the first time.By powder of straw sieving for the first time again with 40 mesh sieve second time screenings, retain particle diameter
Powder of straw granule between 20 mesh to 40 mesh, this powder of straw granule is for measuring feedstuff cellulose enzyme
The powder of straw of vigor, its weight is 16kg.This particle being used for measuring the powder of straw of feedstuff cellulose enzyme vigor
Footpath is 370-850 μm, and this powder of straw being used for measuring feedstuff cellulose enzyme vigor is divided in airtight container
In, keep being dried and low temperature environment.
Embodiment 2, feedstuff cellulase enzyme activity determination in NaAc_HAc buffer solution
The preparation of NaAc_HAc buffer solution: add 9.0mL glacial acetic acid and 69.70g in 10000mL distilled water
Anhydrous sodium acetate, stirs 10min, makes sodium acetate thoroughly dissolve and obtain NaAc_HAc buffer solution, and this acetic acid-
In sodium acetate buffer, the concentration of acetate ion is 0.1mol/L, and pH value is 5.5.
Containing the feedstuff preparation of the buffer solution of cellulase A: add 1.000g mash feed in beaker fine
Dimension element enzyme A, is subsequently adding the above-mentioned NaAc_HAc buffer solution of 50mL, transfers to 100mL after magnetic agitation 10min
In volumetric flask, and being settled to 100mL with above-mentioned NaAc_HAc buffer solution, being configured to mass percentage concentration is
The feedstuff of 1% buffer solution of cellulase A.
Taking the triangular flask of 8 1000mL, number consecutively is 1,2,3,4,5,6,7 and 8, respectively to each
Triangular flask adds the above-mentioned NaAc_HAc buffer solution of 600mL, then is separately added into 10.0g in each triangular flask
The powder of straw for measuring feedstuff cellulose enzyme vigor of embodiment 1, places in 37 DEG C of waters bath with thermostatic control
30min, in 8 triangular flasks, order from 1 to 8 is separately added into 10mL percent mass successively by number the most again
Concentration is the buffer solution of the feedstuff cellulase A of 1%, respectively obtains the enzyme of 1-8 feedstuff cellulase A
Solve reactant liquor.
8 triangular flasks equipped with the enzyme digestion reaction liquid of feedstuff cellulase A are placed in 37 DEG C of waters bath with thermostatic control,
According to number order (with adding the feedstuff sequence consensus of cellulase solution A) from 1-8 triangular flask, every
30min one triangular flask of extraction (30min extracts No. 1 triangular flask, and 60min extracts No. 2 triangular flasks, the
90min extracts No. 3 triangular flasks, and 120min extracts No. 4 triangular flasks, and 150min extracts No. 5 triangular flasks,
180min extracts No. 6 triangular flasks, and 210min extracts No. 7 triangular flasks, and 240min extracts No. 8 triangles
Bottle), the centrifugal 3min under the conditions of relative centrifugal force(RCF) is 500g of the solution after the enzyme digestion reaction in triangular flask is obtained
Residue after enzyme digestion reaction, obtains the residue after the enzyme digestion reaction in No. 1 triangular flask, No. 2 triangular flasks successively
In enzyme digestion reaction after residue, the residue after enzyme digestion reaction in No. 3 triangular flasks, in No. 4 triangular flasks
Residue after enzyme digestion reaction, the residue after enzyme digestion reaction in No. 5 triangular flasks, the enzymolysis in No. 6 triangular flasks
Reacted residue, the residue after enzyme digestion reaction in No. 7 triangular flasks and the enzyme digestion reaction in No. 8 triangular flasks
After residue.Residue after the enzyme digestion reaction each numbered respectively is placed in pallet, carries out labelling, at 80 DEG C
In calorstat, equal pneumatic conveying drying 4h, obtains dried residue, measures the quality of dried residue, and calculating can
The quality of solute, degradation rate and degradation speed, wherein:
Degradation rate=(powder of straw gross mass-residue quality)/powder of straw gross mass × 100% formula 1
Degradation speed=(powder of straw gross mass-residue quality)/degradation time formula 2
Soluble matters quality=powder of straw gross mass-residue mass formula 3
Powder of straw gross mass in formula 1, formula 2 and formula 3 is the fine for measuring feedstuff of embodiment 1
Tieing up the dry weight of the powder of straw of element enzyme enzyme activity, residue quality and soluble matters quality are dry weight, result such as table 1
Shown in.
Table 1, the enzyme activity determination result of NaAc_HAc buffer solution (pH value is 5.5) cellulase A
| Time (min) | 30 | 60 | 90 | 120 | 150 | 180 | 210 | 240 |
| Residue quality (g) | 9.76 | 9.59 | 9.44 | 9.29 | 9.17 | 9.08 | 9.00 | 8.94 |
| Soluble matters quality (g) | 0.24 | 0.41 | 0.56 | 0.71 | 0.83 | 0.92 | 1.00 | 1.06 |
| Degradation rate (%) | 2.4 | 4.1 | 5.6 | 7.1 | 8.3 | 9.2 | 10.0 | 10.6 |
| Degradation speed (g/h) | 0.48 | 0.41 | 0.37 | 0.36 | 0.33 | 0.31 | 0.29 | 0.27 |
In the present embodiment, an enzyme activity unit of feedstuff cellulase A refers to that at 37 DEG C and pH value be 5.5
Under the conditions of, per minute discharge 1 from embodiment 1 for measuring the powder of straw of feedstuff cellulose enzyme vigor
Feedstuff required for the μm ol soluble matters amount of cellulase A.
Along with the prolongation of enzyme digestion reaction time, the quality of the soluble matters of generation is continuously increased, and is used for measuring feedstuff and uses
The degradation rate of the powder of straw of cellulose enzyme vigor is also continuously increased；Along with the prolongation of enzyme digestion reaction time, it is used for surveying
The degradable substance ratio determined in the powder of straw of feedstuff cellulose enzyme vigor constantly reduces so that degradation speed is not
Disconnected decline, this situation meets reality.
Embodiment 3, the feedstuff cellulase enzyme activity determination in vitro bovine rumen liquid
Collect 5 beef cattle cuds (body weight of beef cattle is at about 500kg) in beef cattle slaughterhouse, collect beef cattle cud
Interior content, mix homogeneously, filters the bovine rumen content after mixing with 8 layers of hospital gauzes extruding, collected
Filtrate, it is thus achieved that 6000mL beef cattle cud chyme filtrate, then by beef cattle cud chyme filtrate the most centrifugal
Power is centrifugal 2min under the conditions of 500g, it is thus achieved that 3600mL beef cattle cud chyme filtrate supernatant also carries out pH value
Measuring, pH value is 6.8, as the in vitro bovine rumen liquid analyzing feedstuff cellulose enzyme vigor.
Take 4 1000mL triangular flasks, be separately added into 0.1g feedstuff cellulase A, feedstuff cellulase B,
Feedstuff cellulase C and feedstuff cellulase D (being powdery enzyme), only adds a kind of enzyme in each triangular flask,
The numbered A of triangular flask of feedstuff cellulase A will be only added, the triangle of feedstuff cellulase B will be only added
The numbered B of bottle, will only add the numbered C of triangular flask of feedstuff cellulase C, will only add feedstuff fiber
The numbered D of triangular flask of element enzyme D；600mL is respectively added the most respectively in the triangular flask of numbered A, B, C and D
The above-mentioned in vitro bovine rumen liquid as analysis feedstuff cellulose enzyme vigor, 37 DEG C of perseverances after magnetic agitation 2min
Tepidarium insulation 2min so that feedstuff cellulase fully dissolves, and obtains the in vitro cattle tumor containing cellulase A
Gastric juice, containing the in vitro bovine rumen liquid of cellulase B, in vitro bovine rumen liquid containing cellulase C and containing fibre
The in vitro bovine rumen liquid of dimension element enzyme D.To the in vitro bovine rumen liquid containing cellulase A, containing cellulase B's
In vitro bovine rumen liquid, the in vitro bovine rumen liquid containing cellulase C and the in vitro bovine rumen liquid containing cellulase D
In be sequentially added into the powder of straw for measuring feedstuff cellulose enzyme vigor of 10.0g embodiment 1, it is thus achieved that feedstuff
With the enzyme digestion reaction liquid of cellulase A, the enzyme digestion reaction liquid of feedstuff cellulase B, feedstuff cellulase C
Enzyme digestion reaction liquid and the feedstuff enzyme digestion reaction liquid of cellulase D.100min is reacted in 37 DEG C of waters bath with thermostatic control,
Obtain feedstuff cellulase A enzyme digestion reaction after solution, feedstuff cellulase B enzyme digestion reaction after molten
Liquid, feedstuff cellulase C enzyme digestion reaction after solution and feedstuff cellulase D enzyme digestion reaction after molten
Liquid.By feedstuff solution after the enzyme digestion reaction of cellulase A, feedstuff cellulase B enzyme digestion reaction after
After solution, the feedstuff enzyme digestion reaction with the solution after the enzyme digestion reaction of cellulase C and feedstuff cellulase D
Solution centrifugal 3min under the conditions of relative centrifugal force(RCF) is 500g successively, it is thus achieved that the enzymolysis of feedstuff cellulase A is anti-
Residue after should, the residue after the enzyme digestion reaction of feedstuff cellulase B, the enzyme of feedstuff cellulase C
Solve the residue after the enzyme digestion reaction of reacted residue and feedstuff cellulase D, collect above-mentioned all feeds
With the residue after the enzyme digestion reaction of cellulase.Residual by after the enzyme digestion reaction of all feeds cellulase respectively
Staying thing to be placed in pallet, carry out labelling, in 80 DEG C of calorstats, equal pneumatic conveying drying 4h, obtains dried residual
Thing, measures the quality of dried residue, calculates quality and the degradation rate of soluble matters, wherein:
Degradation rate=(powder of straw gross mass-residue quality)/powder of straw gross mass × 100% formula 1
Soluble matters quality=powder of straw gross mass-residue mass formula 3
Powder of straw gross mass in formula 1 and formula 3 be embodiment 1 for measuring feedstuff cellulose enzyme
The dry weight of the powder of straw of vigor, residue quality and soluble matters quality are dry weight, and result is as shown in table 2.
The enzyme activity determination result of four kinds of feedstuff cellulase in table 2, in vitro bovine rumen liquid
| Numbering | Action time (min) | Residue quality (g) | Soluble matters quality (g) | Degradation rate (%) |
| A | 100 | 9.13 | 0.87 | 8.70 |
| B | 100 | 7.45 | 2.55 | 25.5 |
| C | 100 | 8.73 | 1.27 | 12.7 |
| D | 100 | 7.92 | 2.08 | 20.8 |
In the present embodiment, an enzyme activity unit of feedstuff cellulase refers to that at 37 DEG C and pH value be the bar of 6.8
Under part, per minute discharge 1 μ from embodiment 1 for measuring the powder of straw of feedstuff cellulose enzyme vigor
The amount of the feedstuff cellulase required for mol soluble matters.
Table 2 discharges under same experimental conditions the degradation rate table of soluble matters according to four kinds of feedstuff cellulase
Bright, effect of feedstuff cellulase B is the most notable.
Claims (6)
1. the method measuring feedstuff cellulose enzyme vigor, comprises the steps: to measure feedstuff fibre by being used for
The powder of straw of dimension element enzyme enzyme activity joins in the liquid containing feedstuff cellulase to be measured, it is thus achieved that enzyme digestion reaction liquid；
Described enzyme digestion reaction liquid is carried out enzyme digestion reaction, measures the quality of the soluble matters that described enzyme digestion reaction produces, obtain institute
State the enzyme activity of feedstuff cellulase to be measured.
Method the most according to claim 1, it is characterised in that: the reaction temperature of described enzyme digestion reaction is
37-39.5℃。
Method the most according to claim 1, it is characterised in that: the pH value of described enzyme digestion reaction liquid is 5.5-6.8.
4. according to described method arbitrary in claims 1 to 3, it is characterised in that: described fine for measuring feedstuff
The preparation method of the powder of straw of dimension element enzyme enzyme activity includes pulverizing the stem of plant, and obtaining particle diameter is 370-850 μm
Powder, this powder is described for measuring the powder of straw of feedstuff cellulose enzyme vigor.
5. the preparation method of the powder of straw for measuring feedstuff cellulose enzyme vigor described in claim 4.
6. arbitrary described method application in differentiating feedstuff cellulose enzyme vigor in claim 1-5.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108559769A (en) * | 2018-04-12 | 2018-09-21 | 上海欧耐施生物技术有限公司 | A kind of appraisal procedure of fodder enzyme |
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|---|---|---|---|---|
| CN102212608A (en) * | 2011-04-14 | 2011-10-12 | 吉林大学 | High-throughput screening method of high-activity cellulase used for degrading straw |
| CN102517357A (en) * | 2011-12-14 | 2012-06-27 | 重庆理工大学 | Method for hydrolyzing celluloses through cellulase |
| CN102590013A (en) * | 2012-01-12 | 2012-07-18 | 中国农业大学 | Method for quickly detecting titer of non-starch polysaccharides for feeds |
-
2015
- 2015-01-28 CN CN201510043706.0A patent/CN105986004A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102212608A (en) * | 2011-04-14 | 2011-10-12 | 吉林大学 | High-throughput screening method of high-activity cellulase used for degrading straw |
| CN102517357A (en) * | 2011-12-14 | 2012-06-27 | 重庆理工大学 | Method for hydrolyzing celluloses through cellulase |
| CN102590013A (en) * | 2012-01-12 | 2012-07-18 | 中国农业大学 | Method for quickly detecting titer of non-starch polysaccharides for feeds |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108559769A (en) * | 2018-04-12 | 2018-09-21 | 上海欧耐施生物技术有限公司 | A kind of appraisal procedure of fodder enzyme |
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