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, and is simple to operate; Controllability is strong; It is gentle to compare conventional alkaline purification reaction conditions, environmentally friendly, has 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 lack part lypase, 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, and be forwarded to the fermention medium that contains 1~10% xylan with 4~10% inoculum sizes and produce enzyme, further induce the product enzyme through 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% Hydrocerol A, 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% Hydrocerol A, 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 prepn 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, and be forwarded to the fermention medium that contains 1~10% xylan with 4~10% inoculum sizes and produce enzyme, further induce the product enzyme through 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% Hydrocerol A, 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% Hydrocerol A, 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 zero(ppm) 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 PM 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 is blank: 0.2mL0.1M citrate buffer solution (PH5.0)+0.8mL 5g/L pectin+3mLDNS, and 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 PM 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 (Sigma manufactured) solution that 1mL prepares with 0.1M (pH4.8) Hydrocerol A; Each enzyme appearance 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 PM 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 zero(ppm) 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 zero(ppm) 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 a hour in moisture eliminator, and quality is designated as G2.
(b) in crude enzyme liquid stoste that 10mL obtains 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, every in 1 hour sampling survey enzymolysis solution the amount of reducing sugar.
Cellulose is about 37% in the cotton seed hulls, and semicellulose 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 a 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.
a% is the water ratio of cotton seed hulls, and m is the quality before the cotton seed hulls hydrolysis.
Described cotton seed hulls is available from Distributions in Liaocheng of Shandong Province, pulverizes through kibbler 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 through regulating C/N 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.Through control, produce the mixed enzyme that is suitable for the hydrolysis cotton seed hulls, and, new thinking and approach is provided 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, gentleer than the alkaline purification reaction conditions 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.
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 the restriction scope of the present invention.Should be understood that in addition those skilled in the art can do various changes or modification to 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.
Embodiment 1
1. the seed culture of Trichodermareesei
(1) Trichodermareesei seed culture medium: 1% glucose, 0.1% peptone, 0.05% Hydrocerol A, 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% Hydrocerol A, 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 zero(ppm) 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 PM 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 is blank: 0.2mL0.1M citrate buffer solution (PH5.0)+0.8mL 5g/L pectin+3mLDNS, and 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 PM 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 (sigma manufactured) solution that 1mL prepares with 0.1M (pH4.8) Hydrocerol A.Each enzyme appearance 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 PM 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 zero(ppm) 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 zero(ppm) 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 a hour in moisture eliminator, and quality is designated as G2.
(2) will ferment after the crude enzyme liquid that obtains suitably dilutes with 0.1M citrate buffer solution (pH5.0); Get 10mL enzyme liquid; Add 1g cotton seed hulls particle, water-bath oscillatory reaction 10h under pH5.0,50 ℃, 100rpm condition, every in 1 hour sampling survey enzymolysis solution the amount of reducing sugar.
Cellulose is about 37% in the cotton seed hulls, and semicellulose 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 a 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.
a% is the water ratio of cotton seed hulls, and m is the quality before the cotton seed hulls hydrolysis.
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).
Embodiment 2
1. the seed culture of Trichodermareesei
(1) Trichodermareesei seed culture medium: 1% glucose, 0.1% peptone, 0.05% Hydrocerol A, 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% Hydrocerol A, 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, every in 1 hour sampling survey enzymolysis solution the amount of reducing sugar.
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.
Embodiment 3
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 possibly 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 through comparing embodiment 1 to embodiment 3 can know (seeing Fig. 1, Fig. 2, Fig. 3); Trichodermareesei can be induced the prozyme system that produces the degraded cotton seed hulls through in substratum, adding xylan; With the enzyme liquid hydrolysis cotton seed hulls of inducing generation; Hydrolysis effect is remarkable, and 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 C/N 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.Like 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 (%) |
Embodiment 1 |
18.6 |
22.8 |
Embodiment 2 |
12.4 |
14.0 |
Embodiment 3 |
7.5 |
7.6 |
Embodiment 4
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 through 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.