CN105505901A - Composition for inducing fungi to realize high-yield production of cellulase and application method thereof - Google Patents
Composition for inducing fungi to realize high-yield production of cellulase and application method thereof Download PDFInfo
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- CN105505901A CN105505901A CN201410500338.3A CN201410500338A CN105505901A CN 105505901 A CN105505901 A CN 105505901A CN 201410500338 A CN201410500338 A CN 201410500338A CN 105505901 A CN105505901 A CN 105505901A
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Abstract
The invention relates to the field of cellulase fermentation, especially to a composition for inducing fungi to realize high-yield production of cellulase and an application method thereof. The composition for inducing fungi to realize high-yield production of cellulose comprises, by weight, 0.5 to 1.5 parts of lignocellulose and 6 to 9.5 parts of microcrystalline cellulose. The application method for the composition comprises a step of adding the composition into a fungus medium, wherein the addition amount of the composition is 30 to 35 g/L. The composition for inducing fungi to realize high-yield production of cellulose is simple in composition, utilizes easily available raw materials, has low cost and is a novel cheap inducer for cellulase synthesis; and the application method for the composition has the advantages of simple process, mild conditions and environment friendliness and provides a novel approach for high-efficiency low-cost production of cellulase.
Description
Technical field
The present invention relates to cellulase fermentations field, particularly a kind of compoistion and method of use of inducing fungal High Cellulase Production.
Background technology
Carbon source is the primary factor affecting cellulose enzyme gene expression, and the cellulase of fungi needs to deposit in cellulosic substrate just can be induced out in case.Natural cellulosic feedstocks is the best substrate of Cellulase-producing Fungi, and it can promote the generation of cellulase effectively.Natural cellulosic feedstocks itself directly can not trigger the inducing action to cellulase as insoluble carbon source, but the oligosaccharides of solubility is discharged by the cellulase hydrolysis Mierocrystalline cellulose that basal level is expressed, oligosaccharides enters in cell and then cellulase induction great expression.Study a lot of few carbohydrates and their derivative at present, find cellobiose, sophorose, gentiobiose, the production to cellulase such as lactose has inducing action.But directly add inducing fungal in substratum with this oligosaccharides and produce cellulase, high cost, and two sugar mutual-cellulose enzymes there is feedback inhibition, affect the Degradation of cellulase.So adopt two sugar substances to be inappropriate as the inductor of cellulase production in the medium.Urgently find the inductor of the cellulase synthesis of novel cheapness at present, produce for cellulase high efficiency, low cost and new approach is provided.
Summary of the invention
One of the object of the invention is the composition providing a kind of inducing fungal High Cellulase Production, for solving existing cellulase induction high cost, the deficiency that inductor effect is general.Two of the object of the invention is the using method of the composition providing inducing fungal High Cellulase Production, for providing the reasonable method of the composition using inducing fungal High Cellulase Production of the present invention, give play to the best effect of the composition of inducing fungal High Cellulase Production.
For achieving the above object, the technical solution used in the present invention is:
A composition for inducing fungal High Cellulase Production, is characterized in that, by weight, comprises following component: lignocellulose 0.5 ~ 1.5 and Microcrystalline Cellulose 6 ~ 9.5.
Preferably, by weight, following component is comprised: lignocellulose 1 ~ 1.5 and Microcrystalline Cellulose 7 ~ 8.
Preferably, by weight, following component is comprised: lignocellulose 1 and Microcrystalline Cellulose 7.5.
Preferably, described lignocellulose is any one in laver, paddy rice or wheat.
Preferably, described lignocellulose is laver.
A using method for the composition of inducing fungal High Cellulase Production, is characterized in that, adds described composition in fungi culture medium, and the addition of described composition is 30 ~ 35g/L.
Preferably, the addition of described composition is 33g/L.
Preferably, described fungi culture medium is liquid nutrient medium, and described liquid nutrient medium also comprises nitrogenous source needed for fungal growth and inorganic ion is: massfraction is the Dried Corn Steep Liquor Powder of 1.7%, and massfraction is the (NH of 0.5%
4)
2sO
4, massfraction is the MgSO of 0.1%
4, massfraction is the glycerine of 0.25%, and massfraction is the CaCO of 0.25%
3, and regulate pH to be 4.5-5.5.
Preferably, further comprising the steps of:
In described liquid nutrient medium, access the spore suspension of fungi, under the condition of 28-30 DEG C, cultivate 96-120h with concussion on the shaking table of rotating speed 170-200 rev/min speed.
Preferably, described fungi is the one in Chinese juniper shape mould or Trichodermareesei.
The invention has the beneficial effects as follows:
Lignocellulose and Microcrystalline Cellulose proportion and realize making full use of lignocellulose by the inventive method employing, economize on resources;
Lignocellulose is any one in laver, paddy rice or wheat, is common common, and the resource very easily obtained, cost is low, and solves in daily life the difficult problem processing lignocellulose;
The polysaccharide that lignocellulose is hydrolyzed into is that fungi amount reproduction and its cellulase synthesis of induction are supplied raw materials, instead of to gather directly to substratum and add the way of two sugar substances as the inductor of cellulase production, reduce production cost, and the feedback inhibition that also can not occur cellulase, also can not affect the Degradation of cellulase.
In a word, the composition proportioning of inducing fungal cellulase-producing of the present invention is simple, and raw material is easy to obtain, and cost is low, is the inductor of the cellulase synthesis of novel cheapness; The using method of said composition has technique simply, mild condition and eco-friendly advantage, and producing for cellulase high efficiency, low cost provides new approach.
Embodiment
Below in conjunction with embodiment, the present invention is described in further detail, can implement according to this with reference to specification sheets word to make those skilled in the art.
Embodiment 1
Step one, laver is dried to constant weight, pulverizes with pulverizer afterwards, used by the laver of pulverizing 80 mesh standard sieve to carry out process of sieving, for subsequent use;
Step 2, by Microcrystalline Cellulose with sieve after laver mix according to 9.5: 0.5, the composition as the composition inducing fungal cellulase-producing of inducing fungal cellulase-producing adds in fungi liquid substratum, and addition is 30g/L;
Step 3, the N source adding fungal growth needs in liquid nutrient medium and inorganic ion, Dried Corn Steep Liquor Powder 1.7%, (NH
4)
2sO
40.5%, MgSO
40.1%, glycerine 0.25%, CaCO30.25%, regulates pH4.5-5.5;
The spore suspension of step 4, access Chinese juniper shape mould (Penicilliumpiceum) in the medium, under the condition of 30 DEG C, cultivates 96-120h with concussion on the shaking table of rotating speed 170-200 rev/min speed.
After fermentation 120h, the International Standards Method adopting IUPAC (IUPAC) to recommend measures fermentation broth enzyme vigor, with only induce Chinese juniper shape mould enzyme liquid phase ratio with Microcrystalline Cellulose, utilize beta-glucuroide enzyme activity in the composition induction Chinese juniper shape mould enzyme liquid of inducing fungal cellulase-producing to improve 46.2%.Protein concentration improves 35.7%, and excision enzyme enzyme activity improves 47.6%, and filter paper enzyme activity improves 14.2%.
Embodiment 2
Step one, laver is dried to constant weight, pulverizes with pulverizer afterwards, used by the laver of pulverizing 80 mesh standard sieve to carry out process of sieving, for subsequent use;
Step 2, by Microcrystalline Cellulose with sieve after laver mix according to 9: 1, the composition as inducing fungal cellulase-producing adds in fungi liquid substratum, and addition is 33g/L;
Step 3, the N source adding fungal growth needs in liquid nutrient medium and inorganic ion, Dried Corn Steep Liquor Powder 1.7%, (NH
4)
2sO
40.5%, MgSO
40.1%, glycerine 0.25%, CaCO30.25%, regulates pH4.5-5.5;
The spore suspension of step 4, access Chinese juniper shape mould (Penicilliumpiceum) in the medium, under the condition of 30 DEG C, cultivates 96-120h with concussion on the shaking table of rotating speed 170-200 rev/min speed.
After fermentation 120h, the International Standards Method adopting IUPAC (IUPAC) to recommend measures fermentation broth enzyme vigor, with only induce Chinese juniper shape mould enzyme liquid phase ratio with Microcrystalline Cellulose, utilize beta-glucuroide enzyme activity in the composition induction Chinese juniper shape mould enzyme liquid of inducing fungal cellulase-producing to improve 61.2%.Protein concentration improves 56.7%, and excision enzyme enzyme activity improves 61.4%, and filter paper enzyme activity improves 19.5%.
Embodiment 3
Step one, laver is dried to constant weight, pulverizes with pulverizer afterwards, used by the laver of pulverizing 80 mesh standard sieve to carry out process of sieving, for subsequent use;
Step 2, by Microcrystalline Cellulose with sieve after laver mix according to 8.5: 1, the composition as inducing fungal cellulase-producing adds in fungi liquid substratum, and addition is 35g/L;
Step 3, the N source adding fungal growth needs in liquid nutrient medium and inorganic ion, Dried Corn Steep Liquor Powder 1.7%, (NH
4)
2sO
40.5%, MgSO
40.1%, glycerine 0.25%, CaCO30.25%, regulates pH4.5-5.5;
The spore suspension of step 4, access Chinese juniper shape mould (Penicilliumpiceum) in the medium, under the condition of 30 DEG C, cultivates 96-120h with concussion on the shaking table of rotating speed 170-200 rev/min speed.
After fermentation 120h, the International Standards Method adopting IUPAC (IUPAC) to recommend measures fermentation broth enzyme vigor, with only induce Chinese juniper shape mould enzyme liquid phase ratio with Microcrystalline Cellulose, utilize beta-glucuroide enzyme activity in the composition induction Chinese juniper shape mould enzyme liquid of inducing fungal cellulase-producing to improve 67.9%.Protein concentration improves 59.1%, and excision enzyme enzyme activity improves 100%, and filter paper enzyme activity improves 39.2%.
Embodiment 4
Step one, laver is dried to constant weight, pulverizes with pulverizer afterwards, used by the laver of pulverizing 80 mesh standard sieve to carry out process of sieving, for subsequent use;
Step 2, by Microcrystalline Cellulose with sieve after laver mix according to 7.5: 1.5, the composition as inducing fungal cellulase-producing adds in fungi liquid substratum, and addition is 30g/L;
Step 3, the N source adding fungal growth needs in liquid nutrient medium and inorganic ion, Dried Corn Steep Liquor Powder 1.7%, (NH
4)
2sO
40.5%, MgSO
40.1%, glycerine 0.25%, CaCO30.25%, regulates pH4.5-5.5;
The spore suspension of step 4, access Chinese juniper shape mould (Penicilliumpiceum) in the medium, under the condition of 30 DEG C, cultivates 96-120h with concussion on the shaking table of rotating speed 170-200 rev/min speed.
After fermentation 120h, the International Standards Method adopting IUPAC (IUPAC) to recommend measures fermentation broth enzyme vigor, with only induce Chinese juniper shape mould enzyme liquid phase ratio with Microcrystalline Cellulose, utilize beta-glucuroide enzyme activity in the composition induction Chinese juniper shape mould enzyme liquid of inducing fungal cellulase-producing to improve 57.2%.Protein concentration improves 54.7%, and excision enzyme enzyme activity improves 70.0%, and filter paper enzyme activity improves 19.3%.
Embodiment 5
Step one, laver is dried to constant weight, pulverizes with pulverizer afterwards, used by the laver of pulverizing 80 mesh standard sieve to carry out process of sieving, for subsequent use;
Step 2, by Microcrystalline Cellulose with sieve after laver mix according to 6: 0.5, the composition as inducing fungal cellulase-producing adds in fungi liquid substratum, and addition is 33g/L;
Step 3, the N source adding fungal growth needs in liquid nutrient medium and inorganic ion, Dried Corn Steep Liquor Powder 1.7%, (NH
4)
2sO
40.5%, MgSO
40.1%, glycerine 0.25%, CaCO30.25%, regulates pH4.5-5.5;
The spore suspension of step 4, access Chinese juniper shape mould (Penicilliumpiceum) in the medium, under the condition of 30 DEG C, cultivates 96-120h with concussion on the shaking table of rotating speed 170-200 rev/min speed.
After fermentation 120h, the International Standards Method adopting IUPAC (IUPAC) to recommend measures fermentation broth enzyme vigor, with only induce Chinese juniper shape mould enzyme liquid phase ratio with Microcrystalline Cellulose, utilize beta-glucuroide enzyme activity in the composition induction Chinese juniper shape mould enzyme liquid of inducing fungal cellulase-producing to improve 21.2%.Protein concentration improves 14.2%, and excision enzyme enzyme activity improves 24.5%, and filter paper enzyme activity improves 11.6%.
Embodiment 6
Step one, laver is dried to constant weight, pulverizes with pulverizer afterwards, used by the laver of pulverizing 80 mesh standard sieve to carry out process of sieving, for subsequent use;
Step 2, by Microcrystalline Cellulose with sieve after laver mix according to 9: 1, the composition as inducing fungal cellulase-producing adds in fungi liquid substratum, and addition is 35g/L;
Step 3, the N source adding fungal growth needs in liquid nutrient medium and inorganic ion, Dried Corn Steep Liquor Powder 1.7%, (NH
4)
2sO
40.5%, MgSO
40.1%, glycerine 0.25%, CaCO30.25%, regulates pH4.5-5.5;
Step 4, access the spore suspension of Trichodermareesei (T.reesei) in the medium, under the condition of 30 DEG C, cultivate 96-120h with concussion on the shaking table of rotating speed 170-200 rev/min speed.
After fermentation 120h, the International Standards Method adopting IUPAC (IUPAC) to recommend measures fermentation broth enzyme vigor, with only induce Trichodermareesei enzyme liquid phase ratio with Microcrystalline Cellulose, utilize beta-glucuroide enzyme activity in the composition induction Trichodermareesei enzyme liquid of inducing fungal cellulase-producing to improve 104%.Protein concentration improves 24%, and excision enzyme enzyme activity improves 17.2%, and Xylanase activity improves 479%.
Embodiment 7
Step one, laver is dried to constant weight, pulverizes with pulverizer afterwards, used by the laver of pulverizing 80 mesh standard sieve to carry out process of sieving, for subsequent use;
Step 2, by Microcrystalline Cellulose with sieve after laver mix according to 7.5: 1, the composition as inducing fungal cellulase-producing adds in fungi liquid substratum, and addition is 30g/L;
Step 3, the N source adding fungal growth needs in liquid nutrient medium and inorganic ion, Dried Corn Steep Liquor Powder 1.7%, (NH
4)
2sO
40.5%, MgSO
40.1%, glycerine 0.25%, CaCO30.25%, regulates pH4.5-5.5;
Step 4, access the spore suspension of Trichodermareesei (T.reesei) in the medium, under the condition of 30 DEG C, cultivate 96-120h with concussion on the shaking table of rotating speed 170-200 rev/min speed.
After fermentation 120h, the International Standards Method adopting IUPAC (IUPAC) to recommend measures fermentation broth enzyme vigor, with only induce Trichodermareesei enzyme liquid phase ratio with Microcrystalline Cellulose, utilize beta-glucuroide enzyme activity in the composition induction Trichodermareesei enzyme liquid of inducing fungal cellulase-producing to improve 112%.Protein concentration improves 21.4%, and excision enzyme enzyme activity improves 54.1%, and Xylanase activity improves 521%.
Embodiment 8
Step one, laver is dried to constant weight, pulverizes with pulverizer afterwards, used by the laver of pulverizing 80 mesh standard sieve to carry out process of sieving, for subsequent use;
Step 2, by Microcrystalline Cellulose with sieve after laver mix according to 8.5: 1.5, the composition as inducing fungal cellulase-producing adds in fungi liquid substratum, and addition is 33g/L;
Step 3, the N source adding fungal growth needs in liquid nutrient medium and inorganic ion, Dried Corn Steep Liquor Powder 1.7%, (NH
4)
2sO
40.5%, MgSO
40.1%, glycerine 0.25%, CaCO30.25%, regulates pH4.5-5.5;
Step 4, access the spore suspension of Trichodermareesei (T.reesei) in the medium, under the condition of 30 DEG C, cultivate 96-120h with concussion on the shaking table of rotating speed 170-200 rev/min speed.
After fermentation 120h, the International Standards Method adopting IUPAC (IUPAC) to recommend measures fermentation broth enzyme vigor, with only induce Trichodermareesei enzyme liquid phase ratio with Microcrystalline Cellulose, utilize beta-glucuroide enzyme activity in the composition induction Trichodermareesei enzyme liquid of inducing fungal cellulase-producing to improve 108%.Protein concentration improves 15.7%, and excision enzyme enzyme activity improves 50%, and Xylanase activity improves 495%.
Embodiment 9
Step one, paddy rice bar is dried to constant weight, pulverizes with pulverizer afterwards, used by the paddy rice of pulverizing 80 mesh standard sieve to carry out process of sieving, for subsequent use;
Step 2, by Microcrystalline Cellulose with sieve after paddy rice bar mix according to 9: 1, the composition as inducing fungal cellulase-producing adds in fungi liquid substratum, and addition is 35g/L;
Step 3, the N source adding fungal growth needs in liquid nutrient medium and inorganic ion, Dried Corn Steep Liquor Powder 1.7%, (NH
4)
2sO
40.5%, MgSO
40.1%, glycerine 0.25%, CaCO30.25%, regulates pH4.5-5.5;
Step 4, access the spore suspension of Trichodermareesei (T.reesei) in the medium, under the condition of 30 DEG C, cultivate 96-120h with concussion on the shaking table of rotating speed 170-200 rev/min speed.
After fermentation 120h, the International Standards Method adopting IUPAC (IUPAC) to recommend measures fermentation broth enzyme vigor, with only induce Trichodermareesei enzyme liquid phase ratio with Microcrystalline Cellulose, utilize filter paper enzyme activity in the composition induction Trichodermareesei enzyme liquid of inducing fungal cellulase-producing to improve 59.5%.Restriction endonuclease enzyme activity improves 72.5%, and excision enzyme enzyme activity improves 103.5%.
Embodiment 10
Step one, wheat straw is dried to constant weight, pulverizes with pulverizer afterwards, used by the paddy rice of pulverizing 80 mesh standard sieve to carry out process of sieving, for subsequent use;
Step 2, by Microcrystalline Cellulose with sieve after wheat straw mix according to 9: 1, the composition as inducing fungal cellulase-producing adds in fungi liquid substratum, and addition is 30g/L;
Step 3, the N source adding fungal growth needs in liquid nutrient medium and inorganic ion, Dried Corn Steep Liquor Powder 1.7%, (NH
4)
2sO
40.5%, MgSO
40.1%, glycerine 0.25%, CaCO30.25%, regulates pH4.5-5.5;
Step 4, access the spore suspension of Trichodermareesei (T.reesei) in the medium, under the condition of 30 DEG C, cultivate 96-120h with concussion on the shaking table of rotating speed 170-200 rev/min speed.
After fermentation 120h, the International Standards Method adopting IUPAC (IUPAC) to recommend measures fermentation broth enzyme vigor, with only induce Trichodermareesei enzyme liquid phase ratio with Microcrystalline Cellulose, utilize filter paper enzyme activity in the composition induction Trichodermareesei enzyme liquid of inducing fungal cellulase-producing to improve 41.3%.Restriction endonuclease enzyme activity improves 68.7%.
Embodiment 11
Step one, Chinese silvergrass bar is dried to constant weight, pulverizes with pulverizer afterwards, used by the paddy rice of pulverizing 80 mesh standard sieve to carry out process of sieving, for subsequent use;
Step 2, by Microcrystalline Cellulose with sieve after Chinese silvergrass bar mix according to 9: 1, the composition as inducing fungal cellulase-producing adds in fungi liquid substratum, and addition is 33g/L;
Step 3, the N source adding fungal growth needs in liquid nutrient medium and inorganic ion, Dried Corn Steep Liquor Powder 1.7%, (NH
4)
2sO
40.5%, MgSO
40.1%, glycerine 0.25%, CaCO30.25%, regulates pH4.5-5.5;
Step 4, access the spore suspension of Trichodermareesei (T.reesei) in the medium, under the condition of 30 DEG C, cultivate 96-120h with concussion on the shaking table of rotating speed 170-200 rev/min speed.
After fermentation 120h, the International Standards Method adopting IUPAC (IUPAC) to recommend measures fermentation broth enzyme vigor, with only induce Trichodermareesei enzyme liquid phase ratio with Microcrystalline Cellulose, utilize filter paper enzyme activity in the composition induction Trichodermareesei enzyme liquid of inducing fungal cellulase-producing to improve 38.4%, excision enzyme enzyme activity improves 72.1%.
Although embodiment of the present invention are open as above, but it is not restricted to listed in specification sheets and embodiment utilization, it can be applied to various applicable the field of the invention completely, for those skilled in the art, can easily realize other amendment, therefore do not deviating under the universal that claim and equivalency range limit, the present invention is not limited to specific details and shown here embodiment.
Claims (10)
1. a composition for inducing fungal High Cellulase Production, is characterized in that, by weight, comprises following component: lignocellulose 0.5 ~ 1.5 and Microcrystalline Cellulose 6 ~ 9.5.
2. the composition of inducing fungal High Cellulase Production as claimed in claim 1, is characterized in that, by weight, comprise following component: lignocellulose 1 ~ 1.5 and Microcrystalline Cellulose 7 ~ 8.
3. the composition of inducing fungal High Cellulase Production as claimed in claim 2, is characterized in that, by weight, comprise following component: lignocellulose 1 and Microcrystalline Cellulose 7.5.
4. the composition of inducing fungal High Cellulase Production as claimed in claim 3, it is characterized in that, described lignocellulose is any one in laver, paddy rice, wheat or Chinese silvergrass.
5. the composition of inducing fungal High Cellulase Production as claimed in claim 4, it is characterized in that, described lignocellulose is laver.
6. a using method for the composition of inducing fungal High Cellulase Production as claimed in claim 1, is characterized in that, adds described composition in fungi culture medium, and the addition of described composition is 30 ~ 35g/L.
7. the using method of the composition of inducing fungal High Cellulase Production as claimed in claim 6, it is characterized in that, the addition of described composition is 33g/L.
8. the using method of the composition of inducing fungal High Cellulase Production as claimed in claim 7, it is characterized in that, described fungi culture medium is liquid nutrient medium, described liquid nutrient medium also comprises nitrogenous source needed for fungal growth and inorganic ion is: massfraction is the Dried Corn Steep Liquor Powder of 1.7%, and massfraction is the (NH of 0.5%
4)
2sO
4, massfraction is the MgSO of 0.1%
4, massfraction is the glycerine of 0.25%, and massfraction is the CaCO of 0.25%
3, and regulate pH to be 4.5-5.5.
9. the using method of the composition of inducing fungal High Cellulase Production as claimed in claim 8, is characterized in that, further comprising the steps of:
In described liquid nutrient medium, access the spore suspension of fungi, under the condition of 28-30 DEG C, cultivate 96-120h with concussion on the shaking table of rotating speed 170-200 rev/min speed.
10. the using method of the composition of inducing fungal High Cellulase Production as claimed in claim 9, is characterized in that, described fungi is the one in Chinese juniper shape mould or Trichodermareesei.
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