CN101974492A - Complex enzyme capable of degrading cottonseed hulls, preparation method thereof using xylan for induction and application - Google Patents
Complex enzyme capable of degrading cottonseed hulls, preparation method thereof using xylan for induction and application Download PDFInfo
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Abstract
The invention relates to a complex enzyme capable of degrading cottonseed hulls, a preparation method thereof using xylan for induction and an application. The complex enzyme comprises the components of cellulolytic enzyme, xylanase and pectinase vigor. The preparation method comprises: inoculating a Trichoderma reesei seed into a seed culture medium; culturing for 24-48 hours under the condition that the temperature is 25-30 DEG C and the revolving speed is 120-250r/min; switching the cultured seed with the inoculation amount of 4-10% to a fermentation culture medium containing1-10% of xylan; in the culture process, adding xylan for further inducing for producing enzyme; culturing for 4-8 days under the conditions that the temperature is 25-30 DEG C and the revolving speed is 120-250r/min; and filtering, and centrifuging to obtain crude enzyme liquid. The complex enzyme prepared by the invention has high enzyme activity, is more suitable for the complex enzyme system degrading the cotton seed hulls, is safe, protects environment and has simple operation and strong controllability; and compared with normal alkali processing, the invention has moderate reaction condition, is friendly to environment and has good application prospect.
Description
Technical field
The invention belongs to the fermentative preparation and the Application Areas thereof of prozyme in the microbial technique, particularly a kind of prozyme of degradable cottonseed shell and with xylan inductive preparation method and application.
Background technology
The chemical ingredients of cotton seed hulls, color and form are different from cotton fibre and fabric, influence the feel and the outward appearance of painted uniformity coefficient and fabric, must remove before carrying out coloration of textile materials and arrangement.But the compact structure of cotton seed hulls, complicated component are the non-cellulosic impurities of difficult removal in the cotton fabric refining process.Compare with other impurity in the cotton fibre, its degraded needs more violent treatment condition and longer time usually, and cotton seed hulls therefore how to remove surface of cotton fabric is the difficult problem in the dyeing and finishing work always.
Current in order to tackle the low-carbon economy requirement of China's traditional industry transition and energy-saving and emission-reduction, textile industry is developing the concise novel pre-treating technology of cotton fabric biological enzyme.The removal of cotton seed hulls is the key that realizes the Cotton Fabric Enzymatic Scouring Process heavy industrialization.The complex chemical composition of cotton fibre Symbiont and cotton seed hulls, and enzyme has height substrate catalysis specificity, the different types of enzyme different impurity composition of can only degrading.Using enzyme carries out concise to cotton fabric, the single enzyme preparation is difficult to finish the degraded of cotton seed hulls, be difficult to reach ideal clearance and scouring result, therefore the enzyme of a certain component needs screening to find to be suitable for most to degrade, carry out these enzymes composite again, guarantee simultaneously that under identical conditions different enzyme in the prozyme system can bring into play effect separately, could utilize the collaborative compound action of plurality of enzymes preparation to promote the degraded and the removal of cotton seed hulls.Studies show that cellulase and zytase have certain Degradation to the cotton seed hulls in the cotton fabric.Form by appearance cortex, outer pigmentary layer, colorless layer, palisade layer and interior pigmentary layer by studying as can be known cotton seed hulls; The appearance cortex cutin (lipoid) that distributing equably, Mierocrystalline cellulose, hemicellulose, xylogen (aromatic ring class, Polyphenols) and pectin also are the main components of appearance cortex in addition; Palisade layer is mainly based on pectin, hemicellulose and xylogen; Interior pigmentary layer and outer pigmentary layer are identical, main to have the xylogen of phenolic hydroxyl group compounds on structure and chemical ingredients.Mainly comprise hemicellulose (about 25%), xylogen (about 29%) such as Mierocrystalline cellulose (about 37%), pectin and xylan on the chemical composition, contain chemical substances such as ash content (about 3%), a small amount of protein and lipid in addition.In three big chemical compositions of cotton seed hulls, there is chemical bond in hemicelluloses such as xylan usually and between the xylogen, constituted xylogen---carbohydrate complex body (LCC), zytase can be removed the xylan in the cotton seed hulls, thereby destroy the LCC structure, help the stripping xylogen, and a spot of cellulase also help to degrade Mierocrystalline cellulose in the cotton seed hulls, finally play softening cotton seed hulls, be beneficial to and in washing, remove cotton seed hulls.
Present key issue is, though it is composite to improve the degraded of cotton seed hulls that many scholars and mechanism have adopted single pure enzyme, for example that cellulase and zytase is composite, or zytase and polygalacturonase is composite, but still but less than removal of ideal cotton seed hulls and scouring result.This is because cotton seed hulls is a kind of containing to comprise that the grade mixture of complicated ingredient of Mierocrystalline cellulose, hemicellulose, xylogen, pectin, cutin, protein and ash, the single-minded characteristic of height substrate catalysis of enzyme make different types of enzyme different separately impurity composition of can only degrading.Even adopted the compounded technology of enzyme, because the characteristic difference of the enzyme of different biogenetic derivations, catalytic condition is widely different, and the proportioning of adding various enzymes is difficult to grasp, and does not therefore reach the optimum synergistic treatment effect.The vigor of different enzymes and result of use and working conditions have sizable relation.The optimal reaction pH value of different enzymes and temperature and stable pH value and temperature etc. are all had nothing in common with each other, under identical conditions, the vigor of various enzymes in the performance prozyme guarantees that under identical conditions different enzyme in the prozyme system can bring into play effect separately, is technological difficulties of this technology.
Occurring in nature certain micro-organisms self just can independently be finished the degraded of the complicated lignocellulose material that comprises cotton seed hulls, therefore screening is found and is suitable for the microorganism of cotton seed hulls of degrading most, and to obtain best prozyme system by fermentation control be a good approach with the degraded that is used for cotton seed hulls.This technology can guarantee that under same catalytic reaction condition enzymes different in the prozyme system can be fully and play consistently separately effect, utilizes the collaborative compound action of each enzyme to come efficient catalytic hydrolysis cotton seed hulls.Trichodermareesei can synthesize multiple lignocellulose degradation enzyme simultaneously, so the present invention's degraded of proposing to induce this bacterium enzymatic production and various enzyme work in this enzyme system being regulated and control to be beneficial to cotton seed hulls.
Adopting high-performance bio catalyzer group is the alternative traditional chemical treatment technology of enzyme treatment process of main body, can significantly reduce water consumption, energy consumption, and wastewater flow rate significantly reduces and handles easily; Save dyestuff and obtain more stable Color; The pliability of fabric is better, intensity is higher; Operation safe and easy; Because consumption is few, also has competitive power on use cost, thereby becomes the future thrust of textile industry clearer production technology.
Summary of the invention
Technical problem to be solved by this invention provides a kind of prozyme of degradable cottonseed shell and with xylan inductive preparation method and application, the prozyme that present method makes has the prozyme system that higher enzyme is lived, is more suitable for the cotton seed hulls degraded, and safety and environmental protection, simple to operate, controllability is strong, compare conventional alkaline purification reaction conditions gentleness, environmentally friendly, have a good application prospect.
The prozyme of a kind of degradable cottonseed shell of the present invention, its component mainly comprises: cellulase, zytase and polygalacturonase, and small part lipase, proteolytic enzyme etc.; Described prozyme is made by following method, comprising:
The Trichodermareesei seed is inoculated into seed culture based on 25～30 ℃, cultivated under 120～250r/min condition 24～48 hours, be forwarded to the fermention medium that contains 1～10% xylan with 4～10% inoculum sizes and produce enzyme, further induce the product enzyme by the xylan that adds relative fermention medium 1～2% in the culturing process, at 25～30 ℃, cultivated under 120～250r/min condition 4～8 days, and filtered, the centrifugal crude enzyme liquid that gets;
Wherein, seed culture medium comprises: 1% glucose, and 0.1～1% peptone, 0.05% citric acid, 0.015%Tween 80, the Vogel substratum of 2%Vogel ' s medium N, pH5.0; Or comprising 1% glucose, 0.1～1% peptone, final concentration are the citrate buffer solution of 0.05M, 0.015%Tween 80,0.14% (NH
4)
2SO
4, 0.2%KH
2PO
4, 0.03% urea, 0.04%CaCl
22H
2O, 0.03%MgSO
47H
2O, 0.0005%FeSO
47H
2O, 0.00016%MnSO
4H
2O, 0.00014%ZnSO
47H
2O, 0.00037%CoCl
26H
2The Mandels substratum of O, pH4.8-5.0;
Fermention medium comprises: 0.1～1% glucose, and 0.1～1% peptone, 0.05% citric acid, 0.015%Tween 80, and 2%Vogel ' s medium N adds 1～10% xylan, pH5.0; Or containing 0.1～1% glucose, 0.1～1% peptone, final concentration are the citrate buffer solution of 0.05M, 0.015%Tween 80,0.14% (NH
4)
2SO
4, 0.2%KH
2PO
4, 0.03% urea, 0.04%CaCl
22H
2O, 0.03%MgSO
47H
2O, 0.0005%FeSO
47H
2O, 0.00016%MnSO
4H
2O, 0.00014%ZnSO
47H
2O, 0.00037%CoCl
26H
2The Mandels substratum of O adds 1～10% xylan, pH4.8-5.0.
Of the present inventionly a kind ofly induce the method for preparing degradable cottonseed shell prozyme, comprising with xylan:
The fermentative preparation of Trichodermareesei degraded cotton seed hulls enzyme system
The Trichodermareesei seed is inoculated into seed culture based on 25～30 ℃, cultivated under 120～250r/min condition 24～48 hours, be forwarded to the fermention medium that contains 1～10% xylan with 4～10% inoculum sizes and produce enzyme, further induce the product enzyme by the xylan that adds relative fermention medium 1～2% in the culturing process, at 25～30 ℃, cultivated under 120～250r/min condition 4～8 days, and filtered, the centrifugal crude enzyme liquid that gets;
Wherein, seed culture medium comprises: 1% glucose, and 0.1～1% peptone, 0.05% citric acid, 0.015%Tween 80, the Vogel substratum of 2%Vogel ' s medium N, pH5.0; Or comprising 1% glucose, 0.1～1% peptone, final concentration are the citrate buffer solution of 0.05M, 0.015%Tween 80,0.14% (NH
4)
2SO
4, 0.2%KH
2PO
4, 0.03% urea, 0.04%CaCl
22H
2O, 0.03%MgSO
47H
2O, 0.0005%FeSO
47H
2O, 0.00016%MnSO
4H
2O, 0.00014%ZnSO
47H
2O, 0.00037%CoCl
26H
2The Mandels substratum of O, pH4.8-5.0;
Fermention medium comprises: 0.1～1% glucose, and 0.1～1% peptone, 0.05% citric acid, 0.015%Tween 80, and 2%Vogel ' s medium N adds 1～10% xylan, pH5.0; Or containing 0.1～1% glucose, 0.1～1% peptone, final concentration are the citrate buffer solution of 0.05M, 0.015%Tween 80,0.14% (NH
4)
2SO
4, 0.2%KH
2PO
4, 0.03% urea, 0.04%CaCl
22H
2O, 0.03%MgSO
47H
2O, 0.0005%FeSO
47H
2O, 0.00016%MnSO
4H
2O, 0.00014%ZnSO
47H
2O, 0.00037%CoCl
26H
2The Mandels substratum of O adds 1～10% xylan, pH4.8-5.0.
Preferably add that with 0.1% glucose 1～10% xylan is the quick-acting carbon of the best and the product enzyme carbon source proportioning of inductor in the fermention medium.
Described in fermention medium produces enzyme process, employing be the cultural method of batch-type, cultured continuously formula or batch feeding formula intermittently.
Measure cellulase, zytase and pectinase activity
1. cellulase activity is measured
(a) mensuration of reducing sugar in the crude enzyme liquid: 0.2mL crude enzyme liquid+0.8mL distilled water+3mL 3,5-dinitrosalicylic acid (DNS), boiling water bath 5min, cooling back adding distil water is to 25mL, and the 550nm place records OD
1
(b) mensuration of crude enzyme liquid and Xylo-Mucine (CMC-Na) reaction back total reducing sugars: 0.2mL crude enzyme liquid+0.8mL 1%CMC-Na solution (using 50mM, the acetate buffer solution preparation of pH4.8) adds 3mLDNS behind 50 ℃ of water-bath 10min, boiling water bath 5min; Cooling back adding distil water is to 25mL, and the 550nm place records OD
2
(c) Δ OD=OD
2-OD
1, 1 enzyme activity unit (1U) is defined as the enzyme amount that per minute hydrolyzed carboxymethylcellulo, e sodium produces 1 μ mol reducing sugar (with glucose meter),
In the formula, K is the glucose slope of standard curve, and N is an extension rate, and 1000 for mg is converted into the coefficient of μ g, and 180 is the molecular weight of glucose, and 10 is reaction times (min).
2. the polygalacturonase enzyme mensuration of living
(a) substrate blank: 0.2mL0.1M citrate buffer solution (PH5.0)+0.8mL 5g/L pectin+3mLDNS, boiling water boils 5min, and cooling adds water to 25mL, and 550nm surveys light absorption value, is designated as OD
1
(b) the enzyme liquid air is white: 0.2mL enzyme liquid+0.8mL damping fluid+3mLDNS, and boiling water boils 5min, and cooling adds water to 25mL, and 550nm surveys light absorption value, is designated as OD
2
(c) reaction solution: 0.2mL enzyme liquid+0.8mL pectin;
(d) 50 ℃ of water-bath 60min add 3mLDNS then rapidly, and boiling water boils 5min, adds water to 25mL after the cooling, and the 550nm place surveys light absorption value, is designated as OD
3
(e) Δ OD=OD
3-OD
2-OD
1, a pectinase activity unit (IU) is defined as per minute and generates the required enzyme amount of 1 μ mol D-galacturonic acid.
In the formula: K is a D-galacturonic acid slope of standard curve, and N is an extension rate, and 1000 for mg is converted into the coefficient of μ g, and 212 is the molecular weight of D-galacturonic acid, 60 be the reaction times (minute).
3. xylanase activity is measured
(a) in 25mL scale test tube, add the enzyme liquid of the suitable dilution of 0.5mL and 1% (w/v) oat xylan (manufacturing of Sigma company) solution that 1mL prepares with 0.1M (pH4.8) citric acid; Each enzyme sample is done 3 different extent of dilution at least, makes the wood sugar amount that records under the reaction conditions about 2mg;
(b) 50 ℃ of insulation 30min;
(c) add DNS reagent 3mL, boil 5min in the boiling water, add water to 25mL after the cooling, survey light absorption value at 550nm behind the mixing, should do enzyme at every turn and not have substrate and have substrate not have the blank test of enzyme;
(d), find out the wood sugar amount (deduction blank value) that reaction produces according to the wood sugar typical curve;
(e) on semi-logarithmic coordinate paper, search the needed enzyme amount of release 2mg wood sugar, be calculated as follows enzyme activity:
An xylanase activity unit of force (U) is defined as per minute and generates the required enzyme amount of 1 μ mol wood sugar.
4. the fermented liquid residual sugar is measured
(a) mensuration of reducing sugar in the crude enzyme liquid: 0.4mL crude enzyme liquid+0.6mL distilled water+3mL 3,5-dinitrosalicylic acid (DNS), boiling water bath 5min, cooling back adding distil water is to 25mL, and the 550nm place records OD
1
(b) 1mL distilled water+3mLDNS, boiling water bath 5min, cooling back adding distil water is to 25mL, and the 550nm place records OD
2
(c) Δ OD=OD
1-OD
2,, calculate the amount of reducing sugar in the fermented liquid according to the grape typical curve.
The application of prozyme provided by the invention in the hydrolysis cotton seed hulls comprises,
Crude enzyme liquid hydrolysis cotton seed hulls with the fermentation acquisition
(a) with the ceramic crucible of having dried to constant weight, quality is G0, weighs behind a certain amount of cotton seed hulls particle (less than 40 orders) of packing into, and quality is G1 very, and 105 ℃ dry to constant weight, and weighs balance half an hour in moisture eliminator, and quality is designated as G2.
(b) the crude enzyme liquid stoste that obtains at 10mL or in the enzyme liquid of 1～8 times of dilution adds the 1g cotton seed hulls, and water-bath oscillatory reaction 10h under pH3.0-5.0,40-60 ℃, 100-200rpm condition surveys the amount of reducing sugar in the enzymolysis solution every sampling in 1 hour.
Cellulose is about 37% in the cotton seed hulls, and hemicellulose is about 25%, so the theoretical total amount of reducing sugar is cotton seed hulls quality * 62% before the hydrolysis, the theoretical total amount (g) * 100% of the total amount (g) of reducing sugar in reducing sugar yield=hydrolysis posthydrolysis liquid/reducing sugar.
(c) behind the hydrolysis 10h, with the G that has dried to constant weight
3Glass Hessian crucible quality is designated as M1, collects the enzymolysis residue, and 105 ℃ dry to constant weight, and takes out in moisture eliminator balance half an hour, weighs, and quality is designated as M2, calculates the cotton seed hulls percent hydrolysis.Only to do reference with what damping fluid was handled.
Described cotton seed hulls is available from Distributions in Liaocheng of Shandong Province, pulverizes through pulverizer after air-dry, with sieve cotton seed hulls is separated with linters, collects less than 40 purpose cotton seed hulls particles.
The present invention is directed to various compositions in the cotton seed hulls, is that raw material is induced the product enzyme to bacterial classification with the xylan, obtains in the degradable cottonseed shell multi-component prozyme and does in order to reach optimum degradation effect with multienzyme synergism.The output of hemicellulase such as Trichodermareesei zytase is higher among the present invention, and can produce cellulase and polygalacturonase, can reach the effect of best degraded cotton seed hulls by regulating carbon-nitrogen ratio and the ratio of glucose and xylan and then the vigor ratio of regulation and control cellulase and zytase of producing in the enzyme substratum.
Beneficial effect
Utilization of the present invention conveniently is easy to get and cheap xylan is induced Trichodermareesei production hydrolysis cotton seed hulls prozyme system for raw material.By control, produce the mixed enzyme that is suitable for the hydrolysis cotton seed hulls, and, provide new thinking and approach for removing cotton seed hulls in the textile industry with the enzymic hydrolysis cotton seed hulls of producing to fermentation raw material and each composition proportion thereof.Utilize the inductive crude enzyme liquid to handle cotton seed hulls, than the alkaline purification reaction conditions gentleness of routine; Environmentally friendly.Present method can obtain the prozyme system that higher enzyme is lived, is more suitable for the cotton seed hulls degraded, compares with composite commercial goods enzyme, and this complex enzyme degradation cotton seed hulls effect is remarkable, and safety and environmental protection, and simple to operate, controllability is strong.
Description of drawings
Fig. 1 is a cellulase activity measurement result among the embodiment 1-3;
Fig. 2 is a pectinase activity measurement result among the embodiment 1-3;
Fig. 3 is an Xylanase activity measurement result among the embodiment 1-3;
Fig. 4 is that the fermented liquid residual sugar changes among the embodiment 1-3;
Fig. 5 is the variation that the crude enzyme liquid hydrolysis cotton seed hulls of embodiment 1-3 acquisition generates concentration of reduced sugar;
Wherein, among Fig. 1-5, (◆) represents embodiment 1, (■) represents embodiment 2, and (▲) represents embodiment 3.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after the content of having read the present invention's instruction, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
1. the seed culture of Trichodermareesei
(1) Trichodermareesei seed culture medium: 1% glucose, 0.1% peptone, 0.05% citric acid, 0.015%Tween 80,2%Vogel ' s medium N (125g/L Sodium Citrate, usp, Dihydrate Powder, 250g/L KH
2PO
4, 100g/L NH
4NO
3, 10g/LMgSO
47H
2O, 250 μ g/L vitamin Hs, 5g/L CaCl
22H
2O, the 5mL/L trace element solution); Wherein, trace element solution contains the 50g/L Citric acid monohydrate Food grade, 50g/L ZnSO
47H
2O, 10g/L Fe (NH
4)
2(SO
4)
26H
2O, 2.5g/L CuSO
45H
2O, 0.5g/L MnSO
4H
2O, 0.5g/L H
3B0
3, 0.5g/L Na
2MoO
42H
2O;
(2) serve as to produce bacterial strain with Trichodermareesei (T.reesei) Rut C30,, 30 ℃, cultivate 36h under the 200r/min condition at pH5.0.
2. Trichodermareesei enzymatic production
(1) Trichodermareesei produces the enzyme substratum: 0.1% glucose, and 0.1% peptone, 0.05% citric acid, 0.015%Tween 80, and 2%Vogel ' s medium N adds 2% xylan, pH5.0;
(2) with 10% inoculum size, the cultivation that does not add xylan, is cultivated 8d and is induced the product enzyme at 28 ℃ for contrast under the 160r/min condition;
(3) filter or the fermented liquid clear liquid of the centrifugal collection cotton seed hulls prozyme liquid of degrading exactly.
3. cellulase activity is measured
(1) mensuration of reducing sugar in the crude enzyme liquid: 0.2mL crude enzyme liquid+0.8mL distilled water+3mL 3,5-dinitrosalicylic acid (DNS), boiling water bath 5min, cooling back adding distil water is to 25mL, and the 550nm place records OD
1
(2) mensuration of crude enzyme liquid and Xylo-Mucine (CMC-Na) reaction back total reducing sugars: 0.2mL crude enzyme liquid+0.8mL1%CMC-Na solution (using 50mM, the acetate buffer solution preparation of pH4.8) adds 3mL DNS, boiling water bath 5min behind 50 ℃ of water-bath 10min; Cooling back adding distil water is to 25mL, and the 550nm place records OD
2
(3) Δ OD=OD
2-OD
1, 1 enzyme activity unit (1U) is defined as the enzyme amount that per minute hydrolyzed carboxymethylcellulo, e sodium produces 1 μ mol reducing sugar (with glucose meter),
In the formula, K is the glucose slope of standard curve, and N is extension rate (5 times), and 1000 for mg is converted into the coefficient of μ g, and 180 is the molecular weight of glucose, and 10 is reaction times (min).
4. the polygalacturonase enzyme mensuration of living
(1) substrate blank: 0.2mL0.1M citrate buffer solution (PH5.0)+0.8mL 5g/L pectin+3mLDNS, boiling water boils 5min, and cooling adds water to 25mL, and 550nm surveys light absorption value, is designated as OD
1:
(2) the enzyme liquid air is white: 0.2mL enzyme liquid+0.8mL damping fluid+3mLDNS, and boiling water boils 5min, and cooling adds water to 25mL, and 550nm surveys light absorption value, is designated as OD
2:
(3) reaction solution: 0.2mL enzyme liquid+0.8mL pectin
(4) 50 ℃ of water-bath 60min add 3mLDNS then rapidly, and boiling water boils 5min, adds water to 25mL after the cooling, and the 550nm place surveys light absorption value, is designated as OD
3
(5) Δ OD=OD
3-OD
2-OD
1, a pectinase activity unit (IU) is defined as per minute and generates the required enzyme amount of 1 μ mol D-galacturonic acid.
In the formula: K is a D-galacturonic acid slope of standard curve, and N is an extension rate, and 1000 for mg is converted into the coefficient of μ g, and 212 is the molecular weight of D-galacturonic acid, 60 be the reaction times (minute).
5. xylanase activity is measured
(1) in 25mL scale test tube, adds the enzyme liquid of the suitable dilution of 0.5mL and 1% (w/v) oat xylan (manufacturing of sigma company) solution that 1mL prepares with 0.1M (pH4.8) citric acid.Each enzyme sample is done 3 different extent of dilution at least, makes the wood sugar amount that records under the reaction conditions about 2mg.
(2) 50 ℃ of insulation 30min.
(3) add DNS reagent 3mL, boil 5min in the boiling water, add water to 25mL after the cooling, survey light absorption value at 550nm behind the mixing, should do enzyme thing substrate at every turn and have substrate not have the blank test of enzyme.
(4), find out the wood sugar amount (deduction blank value) that reaction produces according to the wood sugar typical curve.
(5) on semi-logarithmic coordinate paper, search the needed enzyme amount of release 2mg wood sugar, be calculated as follows enzyme activity:
An xylanase activity unit of force (U) is defined as per minute and generates the required enzyme amount of 1 μ mol wood sugar.
6. the fermented liquid residual sugar is measured
(1) mensuration of reducing sugar in the crude enzyme liquid: 0.4mL crude enzyme liquid+0.6mL distilled water+3mL 3,5-dinitrosalicylic acid (DNS), boiling water bath 5min, cooling back adding distil water is to 25mL, and the 550nm place records OD
1
(2) 1mL distilled water+3mLDNS, boiling water bath 5min, cooling back adding distil water is to 25mL, and the 550nm place records OD
2
(3) Δ OD=OD
1-OD
2,, calculate the amount of reducing sugar in the fermented liquid according to the grape typical curve.
7. crude enzyme liquid hydrolysis cotton seed hulls
(1) with the ceramic crucible of having dried to constant weight, quality is G0, weighs behind a certain amount of cotton seed hulls of packing into, and quality is G1 very, and 105 ℃ dry to constant weight, and weighs balance half an hour in moisture eliminator, and quality is designated as G2.
(2) will ferment the crude enzyme liquid that obtains with 0.1M citrate buffer solution (pH5.0) suitably after the dilution, get 10mL enzyme liquid, add 1g cotton seed hulls particle, water-bath oscillatory reaction 10h under pH5.0,50 ℃, 100rpm condition surveys the amount of reducing sugar in the enzymolysis solution every sampling in 1 hour.
Cellulose is about 37% in the cotton seed hulls, and hemicellulose is about 25%, so the theoretical total amount of reducing sugar is cotton seed hulls quality * 62% before the hydrolysis, the theoretical total amount (g) * 100% of the total amount (g) of reducing sugar in reducing sugar yield=hydrolysis posthydrolysis liquid/reducing sugar.
(3) behind the hydrolysis 10h, with the G that has dried to constant weight
3Glass Hessian crucible quality is designated as M1, collects the enzymolysis residue, and 105 ℃ dry to constant weight, and takes out in moisture eliminator balance half an hour, weighs, and quality is designated as M2, calculates the cotton seed hulls percent hydrolysis.Only to do reference with what damping fluid was handled.
Experimental result is seen Fig. 1-Fig. 4, and zymogenic bacteria can utilize the plain enzyme of these oat xylan eccrine fibers than being easier to, and cellulose enzyme activity is higher, and the highest enzyme work reaches 3.16U/mL (Fig. 1); Cultivate after 2 days, it is the highest that pectinase activity reaches, and is 0.04U/mL, slightly descends later on but tend towards stability (Fig. 2); Xylan is induced and can be obtained higher zytase, and the highest enzyme work can reach 5U/mL (Fig. 3).Remaining sugar concentration approaches zero after 4 days, and variation very little (Fig. 4).
1. the seed culture of Trichodermareesei
(1) Trichodermareesei seed culture medium: 1% glucose, 0.1% peptone, 0.05% citric acid, 0.015%Tween 80,2%Vogel ' s medium N;
(2) serve as to produce bacterial classification with Trichodermareesei Rut C30,, 30 ℃, cultivate 36h under the 200r/min condition at pH5.0.
2. Trichodermareesei enzymatic production
(1) Trichodermareesei produces the enzyme substratum: 0.5% glucose, and 0.1% peptone, 0.05% citric acid, 0.015%Tween 80, and 2%Vogel ' s medium N adds 2% xylan, pH5.0;
(2) with 5% inoculum size, the cultivation that does not add xylan, is cultivated 8d and is induced the product enzyme at 30 ℃ for contrast under the 160r/min condition;
(3) the fermented liquid clear liquid of filtration or centrifugal collection is exactly the prozyme liquid of hydrolysis cotton seed hulls.
3. cellulase activity is measured, and sees embodiment 1 for details.
4. Xylanase activity is measured, and sees embodiment 1 for details.
5. pectinase activity is measured, and sees embodiment 1 for details.
6. the fermented liquid residual sugar is measured, and sees embodiment 1 for details.
7. crude enzyme liquid hydrolysis cotton seed hulls sees embodiment 1 for details.Hydrolysising condition is: water-bath oscillatory reaction 10h under pH5.0,50 ℃, 200rpm condition, survey the amount of reducing sugar in the enzymolysis solution every sampling in 1 hour.
Experimental result is seen Fig. 1-Fig. 4, and embodiment 2 has improved 5 times than embodiment 1 quick-acting carbon (glucose) concentration, but both cellulose enzyme activities are more or less the same, and variation tendency too; But in whole culturing process, the polygalacturonase that embodiment 1 produces and the vigor of zytase are all than the height of embodiment 2.
1. the seed culture of Trichodermareesei
(1) Trichodermareesei seed culture medium: 10g/L glucose, 1g/L peptone, final concentration are the citrate buffer solution of 0.05M, 0.15g/L Tween 80,1.4g/L (NH
4)
2SO
4, 2.0g/L KH
2PO
4, 0.3g/L urea, 0.4g/L CaCl
22H
2O, 0.3g/L MgSO
47H
2O, 0.005g/L FeSO
47H
2O, 0.0016g/L MnSO
4H
2O, 0.0014g/LZnSO
47H
2O, 0.0037g/L CoCl
26H
2The Mandels substratum of O, pH4.8-5.0;
(2) serve as to produce bacterial classification with Trichodermareesei Rut C30,, 30 ℃, cultivate 36h under the 200r/min condition at pH5.0.
2. Trichodermareesei enzymatic production
(1) Trichodermareesei produces the enzyme substratum: 10g/L glucose, and 1g/L peptone, final concentration are the citrate buffer solution of 0.05M, 0.015%Tween 80,1.4g/L (NH
4)
2SO
4, 2.0g/L KH
2PO
4, 0.3g/L urea, 0.4g/L CaCl
22H
2O, 0.3g/L MgSO
47H
2O, 0.005g/L FeSO
47H
2O, 0.0016g/L MnSO
4H
2O, 0.0014g/LZnSO
47H
2O, 0.0037g/L CoCl
26H
2The Mandels substratum of O adds 2% pectin, pH4.8-5.0;
(2) with 10% inoculum size, the cultivation that does not add xylan, is cultivated 8d and is induced the product enzyme at 28 ℃ for contrast under the 160r/min condition;
(3) the fermented liquid clear liquid of filtration or centrifugal collection is exactly the prozyme liquid of hydrolysis cotton seed hulls.
3. cellulase activity is measured, and sees embodiment 1 for details.
4. Xylanase activity is measured, and sees embodiment 1 for details.
5. pectinase activity is measured, and sees embodiment 1 for details.
6. the fermented liquid residual sugar is measured, and sees embodiment 1 for details.
7. crude enzyme liquid hydrolysis cotton seed hulls sees embodiment 1 for details.
Experimental result is seen Fig. 1-Fig. 4, and the monosaccharide concentration of embodiment 3 is higher 10 times than embodiment 1, but the difference of the cellulase that produces and preceding two embodiment little (Fig. 1); Polygalacturonase (Fig. 2) and zytase are starkly lower than the above two, and the basic survey in preceding 3 days of not fermenting xylanase activity, and maximum enzyme work is 1.75U/mL (Fig. 3) after appearing at and cultivating 8d.This may be since among the embodiment 3 monosaccharide concentration, do not need self to produce high vigor degrading enzyme and anhydrate and separate glycan and obtain monose for zymogenic bacteria growth provides competent nutrient than higher.Remaining sugar concentration approached zero after 5 days, and the later stage changes little (Fig. 4).
Enzyme activity determination result by comparing embodiment 1 to embodiment 3 (sees Fig. 1, Fig. 2, Fig. 3) as can be known, Trichodermareesei can be induced the prozyme system that produces the degraded cotton seed hulls by add xylan in substratum, with the enzyme liquid hydrolysis cotton seed hulls of inducing generation, hydrolysis effect is remarkable, percent hydrolysis can reach 7.5-18.6% (table 1), the concentration of reduced sugar the highest (Fig. 5) that the complex enzyme degradation cotton seed hulls that embodiment 1 produces produces; Not can not show a candle to the enzyme liquid of inducing generation and add xylan inductive crude enzyme liquid treatment effect.In addition, the ratio of carbon-nitrogen ratio in the fermention medium and glucose and xylan also can have influence on the output of cellulase and zytase, and the ratio that can adjust carbon-nitrogen ratio and glucose and xylan in the production as required is to obtain best enzyme system.As table 1, initial sugar is dense can to obtain higher Xylanase activity when relatively lower (embodiment 1), and the effect of degraded cotton seed hulls is also optimum.
Table 1 cotton seed hulls hydrolysis effect relatively
| Percent hydrolysis (%) | Reducing sugar yield (%) | |
| |
18.6 | 22.8 |
| |
12.4 | 14.0 |
| |
7.5 | 7.6 |
1. the seed culture of Trichodermareesei
(1) Trichodermareesei seed culture medium: 10g/L glucose, 10g/L peptone, final concentration are the citrate buffer solution of 0.05M, 0.15g/L Tween 80,1.4g/L (NH
4)
2SO
4, 2.0g/L KH
2PO
4, 0.3g/L urea, 0.4g/L CaCl
22H
2O, 0.3g/L MgSO
47H
2O, 0.005g/L FeSO
47H
2O, 0.0016g/L MnSO
4H
2O, 0.0014g/LZnSO
47H
2O, 0.0037g/L CoCl
26H
2The Mandels substratum of O, pH4.8-5.0;
(2) serve as to produce bacterial classification with Trichodermareesei Rut C30,, 25 ℃, cultivate 24h under the 120r/min condition at pH5.0.
2. Trichodermareesei enzymatic production
(1) Trichodermareesei produces the enzyme substratum: 10g/L glucose, 10g/L peptone, final concentration are the citrate buffer solution of 0.05M, 0.15g/L Tween 80,1.4g/L (NH
4)
2SO
4, 2.0g/L KH
2PO
4, 0.3g/L urea, 0.4g/L CaCl
22H
2O, 0.3g/L MgSO
47H
2O, 0.005g/L FeSO
47H
2O, 0.0016g/L MnSO
4H
2O, 0.0014g/LZnSO
47H
2O, 0.0037g/L CoCl
26H
2The Mandels substratum of O adds 2% pectin, pH4.8-5.0;
(2) with 5% inoculum size, the cultivation that does not add xylan, is cultivated 4d and is induced the product enzyme at 30 ℃ for contrast under the 250r/min condition, further induce the product enzyme by the xylan that adds relative fermention medium 1～2% in the culturing process;
(4) the fermented liquid clear liquid of filtration or centrifugal collection is exactly the prozyme liquid of hydrolysis cotton seed hulls.
3. cellulase activity is measured, and sees embodiment 1 for details.
4. Xylanase activity is measured, and sees embodiment 1 for details.
5. pectinase activity is measured, and sees embodiment 1 for details.
6. the fermented liquid residual sugar is measured, and sees embodiment 1 for details.
7. crude enzyme liquid hydrolysis cotton seed hulls sees embodiment 1 for details.
Claims (6)
1. the prozyme of a degradable cottonseed shell, its component mainly comprises: cellulase, zytase, polygalacturonase, lipase and proteolytic enzyme;
Described prozyme is made by following method, comprising:
The Trichodermareesei seed is inoculated into seed culture based on 25～30 ℃, cultivated under 120～250r/min condition 24～48 hours, be forwarded to the fermention medium that contains 1～10% xylan with 4～10% inoculum sizes and produce enzyme, further induce the product enzyme by the xylan that adds relative fermention medium 1～2% in the culturing process, at 25～30 ℃, cultivated under 120～250r/min condition 4～8 days, and filtered, the centrifugal crude enzyme liquid that gets;
Wherein, seed culture medium comprises: 1% glucose, and 0.1～1% peptone, 0.05% citric acid, 0.015%Tween 80, the Vogel substratum of 2%Vogel ' s medium N, pH5.0; Or comprising 1% glucose, 0.1～1% peptone, final concentration are the citrate buffer solution of 0.05M, 0.015%Tween 80,0.14% (NH
4)
2SO
4, 0.2%KH
2PO
4, 0.03% urea, 0.04%CaCl
22H
2O, 0.03%MgSO
47H
2O, 0.0005%FeSO
47H
2O, 0.00016%MnSO
4H
2O, 0.00014%ZnSO
47H
2O, 0.00037%CoCl
26H
2The Mandels substratum of O, pH4.8-5.0;
Fermention medium comprises: 0.1～1% glucose, and 0.1～1% peptone, 0.05% citric acid, 0.015%Tween 80, and 2%Vogel ' s medium N adds 1～10% xylan, pH5.0; Or containing 0.1～1% glucose, 0.1～1% peptone, final concentration are the citrate buffer solution of 0.05M, 0.015%Tween 80,0.14% (NH
4)
2SO
4, 0.2%KH
2PO
4, 0.03% urea, 0.04%CaCl
22H
2O, 0.03%MgSO
47H
2O, 0.0005%FeSO
47H
2O, 0.00016%MnSO
4H
2O, 0.00014%ZnSO
47H
2O, 0.00037%CoCl
26H
2The Mandels substratum of O adds 1～10% xylan, pH4.8-5.0.
2. the preparation method of the prozyme of a kind of degradable cottonseed shell as claimed in claim 1 comprises:
The fermentative preparation of Trichodermareesei degraded cotton seed hulls enzyme system
The Trichodermareesei seed is inoculated into seed culture based on 25～30 ℃, cultivated under 120～250r/min condition 24～48 hours, be forwarded to the fermention medium that contains 1～10% xylan with 4～10% inoculum sizes and produce enzyme, further induce the product enzyme by the xylan that adds relative fermention medium 1～2% in the culturing process, at 25～30 ℃, cultivated under 120～250r/min condition 4～8 days, and filtered, the centrifugal crude enzyme liquid that gets;
Wherein, seed culture medium comprises: 1% glucose, and 0.1～1% peptone, 0.05% citric acid, 0.015%Tween 80, the Vogel substratum of 2%Vogel ' s medium N, pH5.0; Or comprising 1% glucose, 0.1～1% peptone, final concentration are the citrate buffer solution of 0.05M, 0.015%Tween 80,0.14% (NH
4)
2SO
4, 0.2%KH
2PO
4, 0.03% urea, 0.04%CaCl
22H
2O, 0.03%MgSO
47H
2O, 0.0005%FeSO
47H
2O, 0.00016%MnSO
4H
2O, 0.00014%ZnSO
47H
2O, 0.00037%CoCl
26H
2The Mandels substratum of O, pH4.8-5.0;
Fermention medium comprises: 0.1～1% glucose, and 0.1～1% peptone, 0.05% citric acid, 0.015%Tween 80, and 2%Vogel ' s medium N adds 1～10% xylan, pH5.0; Or containing 0.1～1% glucose, 0.1～1% peptone, final concentration are the citrate buffer solution of 0.05M, 0.015%Tween 80,0.14% (NH
4)
2SO
4, 0.2%KH
2PO
4, 0.03% urea, 0.04%CaCl
22H
2O, 0.03%MgSO
47H
2O, 0.0005%FeSO
47H
2O, 0.00016%MnSO
4H
2O, 0.00014%ZnSO
47H
2O, 0.00037%CoCl
26H
2The Mandels substratum of O adds 1～10% xylan, pH4.8-5.0.
3. the preparation method of the prozyme of a kind of degradable cottonseed shell as claimed in claim 2 is characterized in that: described in fermention medium produces enzyme process, employing be the cultural method of batch-type, cultured continuously formula or batch feeding formula intermittently.
4. the preparation method of the prozyme of a kind of degradable cottonseed shell as claimed in claim 2, it is characterized in that: described fermention medium comprises: 0.1% glucose, 0.1～1% peptone, 0.05% citric acid, 0.015%Tween 80,2%Vogel ' smedium N adds 1～10% xylan, pH5.0; Or containing 0.1～1% glucose, 0.1～1% peptone, final concentration are the citrate buffer solution of 0.05M, 0.015%Tween 80,0.14% (NH
4)
2SO
4, 0.2%KH
2PO
4, 0.03% urea, 0.04%CaCl
22H
2O, 0.03%MgSO
47H
2O, 0.0005%FeSO
47H
2O, 0.00016%MnSO
4H
2O, 0.00014%ZnSO
47H
2O, 0.00037%CoCl
26H
2The Mandels substratum of O adds 1～10% xylan, pH4.8-5.0.
5. the application of prozyme as claimed in claim 1 in the hydrolysis cotton seed hulls comprises,
Crude enzyme liquid hydrolysis cotton seed hulls with the fermentation acquisition
In 10mL crude enzyme liquid stoste or in the enzyme liquid of 1～8 times of dilution, add the 1g cotton seed hulls, water-bath oscillatory reaction 10h under pH3.0-5.0,40-60 ℃, 100-200rpm condition.
6. the application of prozyme as claimed in claim 5 in the hydrolysis cotton seed hulls is characterized in that: described cotton seed hulls is that the order number was less than 40 orders under 40 mesh sieves were held back.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102154840A (en) * | 2011-05-23 | 2011-08-17 | 湖州美欣达生物科技有限公司 | Enzyme preparation and application thereof |
| CN110004091A (en) * | 2019-04-15 | 2019-07-12 | 微生物肥料技术研究推广中心 | A kind of complex microbial inoculum, soil-repairing agent and preparation method and application |
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| CN1210147A (en) * | 1997-08-21 | 1999-03-10 | 弗·哈夫曼-拉罗切有限公司 | Enzyme complex |
| CN101186907A (en) * | 2007-12-07 | 2008-05-28 | 东华大学 | Fermentation inducement preparation method for ramee degumming composite enzyme |
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2010
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| EP0455928A1 (en) * | 1990-05-08 | 1991-11-13 | Lenzing Aktiengesellschaft | Method of production of a xylanase-rich and a cellulase-poor enzyme solution |
| CN1210147A (en) * | 1997-08-21 | 1999-03-10 | 弗·哈夫曼-拉罗切有限公司 | Enzyme complex |
| CN101186907A (en) * | 2007-12-07 | 2008-05-28 | 东华大学 | Fermentation inducement preparation method for ramee degumming composite enzyme |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102154840A (en) * | 2011-05-23 | 2011-08-17 | 湖州美欣达生物科技有限公司 | Enzyme preparation and application thereof |
| CN110004091A (en) * | 2019-04-15 | 2019-07-12 | 微生物肥料技术研究推广中心 | A kind of complex microbial inoculum, soil-repairing agent and preparation method and application |
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