CN101979525B - Complex enzyme capable of degrading cottonseed hulls, method for preparing same under induction of pectin and application thereof - Google Patents
Complex enzyme capable of degrading cottonseed hulls, method for preparing same under induction of pectin and application thereof Download PDFInfo
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Abstract
The invention relates to the complex enzyme capable of degrading cottonseed hulls, a method for preparing the same under the induction of pectin, and application thereof. The complex enzyme comprises the components of cellulase, xylanase, and pectinase. The preparation method comprises the following steps of: inoculating trichoderma reesei seeds into a seed culture medium to perform culture under the conditions of 25 to 30 DEG C and 120 to 250 r/min for 24 to 48 hours, transferring 4 to 10 percent of inoculated seeds to a fermentation culture medium containing 1 to 5 percent pectin for enzyme production, performing culture under the conditions of 25 to 30 DEG C and 120 to 250 r/min for 4 to 8 days, and performing filtration and centrifugation to obtain crude enzyme, wherein the pectin can be added to further induce the enzyme production in the culture process. The complex enzyme prepared by the method has relatively higher enzyme activity, is more suitable for a complex enzyme system for degrading the cottonseed hulls, safe, environmentally-friendly, convenient to operate and highly controllable, and compared with conventional alkaline treatment, is mild in reaction conditions, environmentally-friendly and vast in application prospect.
Description
Technical field
The invention belongs to fermentation preparation and the Application Areas thereof of prozyme in the microbial technique, particularly a kind of prozyme of degradable cottonseed shell and the preparation method and application that induce with pectin thereof.
Background technology
The chemical ingredients of cotton seed hulls, color and form are different from cotton fibre and fabric, and the uniformity coefficient of impact dyeing and feel and the outward appearance of fabric must be removed before the dyeing of carrying out fabric 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.With other impurity phase ratio 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 pretreatment process of cotton fabric with enzymes.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 the clearance and the scouring result that reach desirable, 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 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.Cotton seed hulls is comprised of appearance cortex, outer pigmentary layer, colorless layer, palisade layer and interior pigmentary layer by studying as can be known; 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 take pectin, hemicellulose and xylogen as main; Interior pigmentary layer and outer pigmentary layer are identical on structure and chemical ingredients, mainly take with the xylogen of phenolic hydroxyl group compounds as main.Mainly comprise hemicellulose (about 25%), the xylogen (about 29%) such as Mierocrystalline cellulose (about 37%), pectin and xylan on the chemical composition, contain in addition the chemical substances such as ash content (about 3%), a small amount of protein and lipid.In three large chemical compositions of cotton seed hulls, there is chemical bond in the hemicelluloses such as xylan usually and between the xylogen, consisted of xylogen---carbohydrate complex body (LCC), zytase can be removed the xylan in the cotton seed hulls, thereby destroy the LCC structure, help dissolved lignin, and a small amount 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, although 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 desirable cotton seed hulls removal and scouring result.This is because cotton seed hulls is a kind of containing to comprise the grade mixture of complicated ingredient of Mierocrystalline cellulose, hemicellulose, xylogen, pectin, cutin, protein and ash, and the single-minded characteristic of height substrate catalysis of enzyme is different separately impurity compositions so that different types of enzyme can only be degraded.Even adopted the compounded technology of enzyme, because the characteristic of the enzyme of different biogenetic derivations is different, 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.Different enzyme activities 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, various enzyme activities in the performance prozyme guarantee that under identical conditions different enzyme in the prozyme system can bring into play effect separately, are technological difficulties of this technique.
Occurring in nature certain micro-organisms self comprises the degraded of the complicated lignocellulose material of cotton seed hulls with regard to the energy complete independently, therefore the microorganism of the cotton seed hulls that is suitable for most degrading is found in screening, and to obtain best prozyme by fermentation control be to 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 processes 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 the preparation method and application that induce with pectin thereof, 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, 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, polygalacturonase, and small part lipase, proteolytic enzyme etc.; Described prozyme is made by following methods, 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 1～5% pectin with 4～10% inoculum sizes and produce enzyme, further induce the product enzyme by the pectin 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 1～5% pectin, 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 1～5% pectin, pH4.8-5.0.
Of the present inventionly a kind ofly induce the method for preparing degradable cottonseed shell prozyme with pectin, comprising:
The fermentation 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 1～5% pectin with 4～10% inoculum sizes and produce enzyme, further induce the product enzyme by the pectin 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 1～5% pectin, 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 1～5% pectin, pH4.8-5.0.
Add that with preferred 0.1% glucose 1～5% pectin is the quick-acting carbon of the best and the product enzyme carbon source proportioning of inductor in the fermention medium.
Described pectin is available from Beijing extensive and profound in meaning star biotechnology responsibility company limited.
Described in fermention medium produces enzyme process, employing be the cultural method of batch-type, cultured continuously formula or fed-batch type 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, adding distil water is to 25mL after the cooling, and the 550nm place records OD
1
(b) mensuration of total reducing sugars after crude enzyme liquid and Xylo-Mucine (CMC-Na) reaction: 0.2mL crude enzyme liquid+0.8mL 1%CMC-Na solution (is used 50mM, the acetate buffer solution preparation of pH4.8), add 3mLDNS behind 50 ℃ of water-bath 10min, boiling water bath 5min; Adding distil water is to 25mL after the cooling, 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 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: 0.2mL 0.1M citrate buffer solution (
PH5.0)+and 0.8mL 5g/L pectin+3mLDNS, boiling water boiling 5min, 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 boiling 5min, 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) then 50 ℃ of water-bath 60min add rapidly 3mLDNS, 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 (U) is defined as per minute and generates the required enzyme amount of 1 μ mol D-galacturonic acid.
In the formula: K is D-galacturonic acid slope of standard curve, and N is 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 without substrate at every turn and substrate be arranged without the blank test of enzyme;
(d) according to the wood sugar typical curve, find out the wood sugar amount (deduction blank value) that reaction produces;
(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. fermented liquid residual glucose
(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, adding distil water is to 25mL after the cooling, and the 550nm place records OD
1
(b) 1mL distilled water+3mLDNS, boiling water bath 5min, adding distil water is to 25mL after the cooling, and the 550nm place records OD
2
(c) Δ OD=OD
1-OD
2, according to the grape typical curve, calculate the amount of reducing sugar in the fermented liquid.
The application of prozyme provided by the invention in the hydrolysis cotton seed hulls comprises,
The crude enzyme liquid that obtains with fermentation is hydrolyzed cotton seed hulls
(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 designated as G1, and 105 ℃ dry to constant weight, and weighs balance half an hour in moisture eliminator, and quality is designated as G2.
(b) in the crude enzyme liquid stoste of 10mL acquisition or in the enzyme liquid of 1～8 times of dilution, add the 1g cotton seed hulls, in pH3.0-5.0,40-60 ℃, 100-200rpm Water Under bath oscillatory reaction 10h, the amount of reducing sugar in 1 hour takes a sample the survey enzymolysis solution.
Cellulose about 37% in the cotton seed hulls, hemicellulose about 25%, therefore the theoretical total amount of reducing sugar is the front cotton seed hulls quality of hydrolysis * 62%, the theoretical total amount (g) * 100% of the total amount (g) of reducing sugar/reducing sugar in the hydrolyzed solution after Reducing sugar=hydrolysis.
(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 processed.
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, take pectin as raw material bacterial classification is induced the product enzyme, obtain in the degradable cottonseed shell multi-component prozyme and do in order to reach optimum degradation effect with multienzyme synergism.The output of the hemicellulase such as Xylanase from Trichoderma reesei is higher among the present invention, and can produce cellulase and polygalacturonase, can by ratio and then regulation and control cellulase and the xylan enzyme activity ratio of the carbon-nitrogen ratio in the adjusting culture medium and glucose and pectin, reach the effect of best degraded cotton seed hulls.
Beneficial effect
Utilization of the present invention conveniently is easy to get and cheap pectin is induced Trichodermareesei production hydrolysis cotton seed hulls prozyme system for raw material.By the control to fermentation raw material and each composition proportion thereof, produce the mixed enzyme that is suitable for being hydrolyzed cotton seed hulls, and with the enzymic hydrolysis cotton seed hulls of producing, provide new thinking and approach for removing cotton seed hulls in the textile industry.The crude enzyme liquid that utilizes fermentation to make is processed cotton seed hulls, and is 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, and safety and environmental protection, and simple to operate, controllability is strong.
Description of drawings
Fig. 1 is embodiment 1-3 cellulase vitality test result;
Fig. 2 is pectinase activity measurement result among the embodiment 1-3;
Fig. 3 is 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 that the crude enzyme liquid that embodiment 1-3 obtains is hydrolyzed the variation that cotton seed hulls 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 for explanation the present invention 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 limited range 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) take Trichodermareesei (T.reesei) Rut C30 as producing bacterial strain, at pH5.0,30 ℃, cultivate 36h under the 200r/min condition.
2. Trichodermareesei enzymatic production
(1) Trichodermareesei culture medium: 0.1% glucose, 0.1% peptone, 0.05% citric acid, 0.015%Tween 80, and 2%Vogel ' s medium N adds 1% pectin, pH5.0;
(2) with 10% inoculum size, do not add the cultivation of pectin for contrast, at 28 ℃, cultivate 8d under the 160r/min condition and induce the product enzyme;
(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, adding distil water is to 25mL after the cooling, and the 550nm place records OD
1
(2) mensuration of total reducing sugars after crude enzyme liquid and Xylo-Mucine (CMC-Na) reaction: 0.2mL crude enzyme liquid+0.8mL1%CMC-Na solution (is used 50mM, the acetate buffer solution preparation of pH4.8), add 3mL DNS, boiling water bath 5min behind 50 ℃ of water-bath 10min; Adding distil water is to 25mL after the cooling, 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 is blank: 0.2mL0.1M citrate buffer solution (PH5.0)+0.8mL 5g/L pectin (manufacturing of sigma company)+3mLDNS, and boiling water boiling 5min, 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 boiling 5min, 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;
Then (4) 50 ℃ of water-bath 60min add rapidly 3mLDNS, 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 (U) is defined as per minute and generates the required enzyme amount of 1 μ mol D-galacturonic acid,
In the formula: K is D-galacturonic acid slope of standard curve, and N is 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 substrate be arranged without the blank test of enzyme;
(4) according to the wood sugar typical curve, find out the wood sugar amount (deduction blank value) that reaction produces;
(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. fermented liquid residual glucose
(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, adding distil water is to 25mL after the cooling, and the 550nm place records OD
1
(2) 1mL distilled water+3mLDNS, boiling water bath 5min, adding distil water is to 25mL after the cooling, and the 550nm place records OD
2
(3) Δ OD=OD
1-OD
2, according to the grape typical curve, calculate the amount of reducing sugar in the fermented liquid.
7. crude enzyme liquid is hydrolyzed 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) after suitably diluting, get 10mL enzyme liquid, hydrolysis 1g cotton seed hulls (less than 40 orders), in pH5.0,50 ℃, 100rpm Water Under bath oscillatory reaction 10h, the amount of reducing sugar in 1 hour takes a sample the survey enzymolysis solution.
Cellulose about 37% in the cotton seed hulls, hemicellulose about 25%, therefore the theoretical total amount of reducing sugar is the front cotton seed hulls quality of hydrolysis * 62%, the theoretical total amount (g) * 100% of the total amount (g) of reducing sugar/reducing sugar in the hydrolyzed solution after Reducing sugar=hydrolysis.
(3) behind the hydrolysis 10h, with the G that has dried to constant weight
3The glass Hessian crucible is collected the enzymolysis residue, and 105 ℃ dry to constant weight, and calculates the cotton seed hulls percent hydrolysis.Only to do reference with what damping fluid was processed.
Experimental result is seen Fig. 1-Fig. 4, the pectin of preparation take the fruit skin as raw material, molecular weight is not very large, zymogenic bacteria can more readily digested these pectin, thus eccrine fiber element enzyme, after cultivating 2 days, cellulase activity just reaches 2.62U/mL (Fig. 1), and rising, the highest enzyme work is after fermenting 8 days, to be 3.47U/mL always; Pectin content is high in the pectin, and zymogenic bacteria can take full advantage of these carbon sources and secrete polygalacturonase albumen, and the pectinase activity maximum appears at cultivated after 4 days, reached 0.17U/mL (Fig. 2); The Xylanase activity that produces reaches maximum value after cultivating 5 days, be 0.13U/mL (Fig. 3).Remaining sugar concentration changes very little (Fig. 4) after 2 days.
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) take Trichodermareesei Rut C30 as producing bacterial classification, at pH5.0,30 ℃, cultivate 36h under the 200r/min condition.
2. Trichodermareesei enzymatic production
(1) Trichodermareesei culture medium: 0.5% glucose, 0.1% peptone, 0.05% citric acid, 0.015%Tween 80, and 2%Vogel ' s medium N adds 2% pectin, pH5.0;
(2) with 5% inoculum size, do not add the cultivation of pectin for contrast, at 30 ℃, cultivate 8d under the 160r/min condition and induce the product enzyme;
(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 glucose sees embodiment 1 for details.
7. crude enzyme liquid hydrolysis cotton seed hulls sees embodiment 1 for details.Hydrolysising condition is: in pH5.0,50 ℃, 200rpm Water Under bath oscillatory reaction 10h, the amount of reducing sugar in 1 hour takes a sample the survey enzymolysis solution.
Experimental result is seen Fig. 1-Fig. 4, embodiment 2 has improved 5 times than embodiment 1 quick-acting carbon (glucose) concentration, at front 3 days that cultivate, cellulase activity rises very fast, the back just increasess slowly, and vigor is obviously than embodiment 1 height after cultivating three days, and its highest enzyme work can reach 3.80U/mL (Fig. 1); Polygalacturonase is suitable with the former, and variation tendency is also basic identical, and the highest enzyme work is 0.16U/mL cultivating afterwards appearance in 4 days; Zytase reaches maximum after cultivating the 7th day, be 0.065U/mL.
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) take Trichodermareesei Rut C30 as producing bacterial classification, at pH5.0,30 ℃, cultivate 36h under the 200r/min condition.
2. Trichodermareesei enzymatic production
(1) Trichodermareesei 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 adds 2% pectin, pH4.8-5.0;
(2) with 10% inoculum size, do not add the cultivation of pectin for contrast, at 28 ℃, cultivate 8d under the 160r/min condition and induce the product enzyme;
(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 glucose 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 embodiment 3 monosaccharide concentration are higher 10 times than embodiment 1, but at front 4 days that cultivate, the cellulase difference that both produce was little, and afterwards just obviously than embodiment 1 height, its maximum enzyme work reached 4.08U/mL (Fig. 1) at 5 days; After the work of polygalacturonase maximum enzyme appears at and cultivated four days, be 0.18U/mL (Fig. 2), and the work of zytase maximum enzyme is to cultivate after 5 days, is 0.059U/mL (Fig. 3) that xylanase activity is lower than the above two.Remaining sugar concentration changed little (Fig. 4) afterwards at 4 days.
Enzyme activity determination result (seeing Fig. 1, Fig. 2, Fig. 3) by comparing embodiment 1 to embodiment 3 as can be known, Trichodermareesei can be induced the prozyme system that produces the degraded cotton seed hulls by add pectin in substratum, with the enzyme liquid hydrolysis cotton seed hulls of inducing generation, hydrolysis effect is remarkable, percent hydrolysis can reach 4.2-7.1% (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 the crude enzyme liquid treatment effect that cotton seed hulls induces.In addition, the ratio of the carbon-nitrogen ratio in the fermention medium and glucose and pectin also can have influence on the output of cellulase and zytase, and the ratio of carbon-nitrogen ratio and glucose and pectin of can adjusting as required in the production is to obtain best enzyme system.Such 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 (%) | |
| |
7.1 | 9.5 |
| |
5.5 | 6.9 |
| |
4.2 | 3.8 |
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/LCaCl
22H
2O, 0.3g/L MgSO
47H
2O, 0.005g/L FeSO
47H
2O, 0.0016g/L MnSO
4H
2O, 0.0014g/L ZnSO
47H
2O, 0.0037g/L CoCl
26H
2The Mandels substratum of O, pH4.8-5.0;
(2) take Trichodermareesei Rut C30 as producing bacterial classification, at pH5.0,25 ℃, cultivate 24h under the 120r/min condition;
2. Trichodermareesei enzymatic production
(1) Trichodermareesei 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 adds 5% pectin, pH4.8-5.0;
(2) with 5% inoculum size, do not add the cultivation of pectin for contrast, at 30 ℃, cultivate 4d under the 250r/min condition and induce the product enzyme, further induce the product enzyme by the pectin that adds relative fermention medium 1～2% in the culturing process;
(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 glucose 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, described prozyme is made by following methods, 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 1～5% pectin with 4～10% inoculum sizes and produce enzyme, further induce the product enzyme by the pectin 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 mediumN, 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 mediumN adds 1～5% pectin, 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 1～5% pectin, 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 fermentation 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 1～5% pectin with 4～10% inoculum sizes and produce enzyme, further induce the product enzyme by the pectin 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 mediumN, 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 1～5% pectin, 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 1～5% pectin, 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: describedly produce in the enzyme process at fermention medium, employing be the cultural method of batch-type, cultured continuously formula or fed-batch type 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 ' smediumN, add 1～5% pectin, pH5.0; Or containing 0.1% 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 1～5% pectin, pH4.8-5.0.
5. the application of prozyme as claimed in claim 1 in the hydrolysis cotton seed hulls comprises,
The crude enzyme liquid that obtains with fermentation is hydrolyzed cotton seed hulls
In 10mL crude enzyme liquid stoste or in the enzyme liquid of 1～8 times of dilution, add the 1g cotton seed hulls, at pH3.0-5.0,40-60 ℃, 100-200rpm Water Under bath oscillatory reaction 10h.
6. the application of prozyme as claimed in claim 5 in the hydrolysis cotton seed hulls, it 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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| 程池.对里氏木霉Rut C-30所产非淀粉多糖酶系的分析.《食品与发酵工业》.2004,第30卷(第6期),64-67. * |
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