CN103289977A - Preparation and compounding methods of low-temperature neutral cellulase - Google Patents
Preparation and compounding methods of low-temperature neutral cellulase Download PDFInfo
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- CN103289977A CN103289977A CN2013102687288A CN201310268728A CN103289977A CN 103289977 A CN103289977 A CN 103289977A CN 2013102687288 A CN2013102687288 A CN 2013102687288A CN 201310268728 A CN201310268728 A CN 201310268728A CN 103289977 A CN103289977 A CN 103289977A
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- 108010059892 Cellulase Proteins 0.000 title claims abstract description 52
- 229940106157 cellulase Drugs 0.000 title claims abstract description 52
- 230000007935 neutral effect Effects 0.000 title claims abstract description 37
- 238000002360 preparation method Methods 0.000 title claims abstract description 37
- 238000000034 method Methods 0.000 title abstract description 10
- 238000013329 compounding Methods 0.000 title abstract 3
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- 230000001954 sterilising effect Effects 0.000 claims description 18
- 238000004659 sterilization and disinfection Methods 0.000 claims description 18
- 230000004913 activation Effects 0.000 claims description 11
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- 239000000463 material Substances 0.000 claims description 10
- 239000004094 surface-active agent Substances 0.000 claims description 10
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- 244000061456 Solanum tuberosum Species 0.000 claims description 8
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- 241000223259 Trichoderma Species 0.000 claims description 6
- 239000002253 acid Substances 0.000 claims description 6
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- 108010080698 Peptones Proteins 0.000 claims description 5
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims description 5
- 239000004202 carbamide Substances 0.000 claims description 5
- 239000001768 carboxy methyl cellulose Substances 0.000 claims description 5
- 235000010948 carboxy methyl cellulose Nutrition 0.000 claims description 5
- 239000008112 carboxymethyl-cellulose Substances 0.000 claims description 5
- 238000012262 fermentative production Methods 0.000 claims description 5
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- 235000019322 gelatine Nutrition 0.000 claims description 5
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- 239000000843 powder Substances 0.000 claims description 5
- 235000009566 rice Nutrition 0.000 claims description 5
- 210000000582 semen Anatomy 0.000 claims description 5
- 235000015099 wheat brans Nutrition 0.000 claims description 5
- 239000002131 composite material Substances 0.000 claims description 4
- MEIRRNXMZYDVDW-MQQKCMAXSA-N (2E,4E)-2,4-hexadien-1-ol Chemical compound C\C=C\C=C\CO MEIRRNXMZYDVDW-MQQKCMAXSA-N 0.000 claims description 3
- DNIAPMSPPWPWGF-GSVOUGTGSA-N (R)-(-)-Propylene glycol Chemical compound C[C@@H](O)CO DNIAPMSPPWPWGF-GSVOUGTGSA-N 0.000 claims description 3
- QCDWFXQBSFUVSP-UHFFFAOYSA-N 2-phenoxyethanol Chemical compound OCCOC1=CC=CC=C1 QCDWFXQBSFUVSP-UHFFFAOYSA-N 0.000 claims description 3
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 claims description 3
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- 239000004372 Polyvinyl alcohol Substances 0.000 claims description 3
- 125000001931 aliphatic group Chemical group 0.000 claims description 3
- AMAICRYCMCVAHT-UHFFFAOYSA-K calcium;sodium;trichloride Chemical compound [Na+].[Cl-].[Cl-].[Cl-].[Ca+2] AMAICRYCMCVAHT-UHFFFAOYSA-K 0.000 claims description 3
- 239000008367 deionised water Substances 0.000 claims description 3
- 229910021641 deionized water Inorganic materials 0.000 claims description 3
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- 150000002632 lipids Chemical class 0.000 claims description 3
- 229910001629 magnesium chloride Inorganic materials 0.000 claims description 3
- 229960005323 phenoxyethanol Drugs 0.000 claims description 3
- 229920001495 poly(sodium acrylate) polymer Polymers 0.000 claims description 3
- -1 polyoxyethylene Polymers 0.000 claims description 3
- 229940051841 polyoxyethylene ether Drugs 0.000 claims description 3
- 229920000056 polyoxyethylene ether Polymers 0.000 claims description 3
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- 239000001267 polyvinylpyrrolidone Substances 0.000 claims description 3
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 claims description 3
- NNMHYFLPFNGQFZ-UHFFFAOYSA-M sodium polyacrylate Chemical compound [Na+].[O-]C(=O)C=C NNMHYFLPFNGQFZ-UHFFFAOYSA-M 0.000 claims description 3
- 238000001914 filtration Methods 0.000 claims description 2
- 240000007594 Oryza sativa Species 0.000 claims 1
- 239000000835 fiber Substances 0.000 abstract description 8
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- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 4
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Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a preparation method of low-temperature neutral cellulase. The low-temperature neutral cellulase is prepared by taking trichoderma reesei as strains, and the preparation method comprises the following steps of: activating strains, and carrying out ultraviolet mutagenesis on the strains; then, cultivating and purifying the strains at a low temperature, and fermenting the purified strains at a temperature of 10-15 DEG C so as to prepare a seed solution; vaccinating the seed solution into a produced enzyme culture medium; and culturing the seed solution for 96-144 hours at a temperature of 10-15 DEG C, so that the low-temperature neutral cellulase is obtained. The invention also provides a compounding method of the low-temperature neutral cellulase. The methods disclosed by the invention have the beneficial effects that the operation is simple, the fermentation cycle is short, and the application temperature of enzyme can be reduced, so that the energy consumption for industrial applications is reduced, therefore, the methods are more environment-friendly, and have broad application prospects; in addition, the invention also provides a compounding method of the cellulase, which can be widely applied to the modification of papermaking fiber, and reduces the cost of papermaking fiber modification.
Description
Technical field
The present invention relates to biochemical field, be specifically related to a kind of preparation and preparation method of low temperature neutral cellulase.
Background technology
Cellulase (cellulase) is a kind of important enzyme product, mainly formed by circumscribed beta-glucanase, inscribe beta-glucanase and beta-glucosidase etc., energy degraded cellulose β-1,4 glycosidic links generate the general name of one group of enzyme of small-molecule substances such as cellobiose and glucose, be a kind of compound inducible enzyme, comprise multiple lytic enzyme.The industrial applications of cellulase mainly concentrates on bioenergy, brewery industry, and washing industry is in the industries such as paper industry and textile industry.Aspect bioenergy, cellulase decomposes cellulosic material completely, and its hydrolysate becomes monose, with its further fermentation, just can produce biofuel again; In brewery industry, can be used for the production of beer, increase the output of sugar fermentation, also can be used for brewageing of soy sauce, increase the output of soy sauce and improve its quality; In washing industry, cellulase is added in the washing composition, the hydrocellulose molecule thoroughly can be destroyed, and thoroughly removes the spot that is enclosed in fibrous inside, promotes washing effect; In paper industry, cellulase can make fiber surface modification, reduces the energy consumption of machinery making beating; In textile industry, be used for the back arrangement of linen-cotton, cellulase can reduce temperature of reaction, makes the reaction conditions gentleness, and the level and smooth sense of touch of textile finishing is good.
Though the research of cellulase is had the history in more than 50 year both at home and abroad, mainly concentrate on middle temperature, the plain enzyme of high temperature fiber, the research of relevant 10～15 ℃ of following cellulases is then fewer.Especially at home, can be used as business-like low temperature neutral cellulase and rarely have existence especially.But industrial use medium and high temperature cellulase is to technology and equipment requirements is high and relatively power consumption, and the preparation and application of therefore studying the low temperature neutral cellulase have great importance.
Summary of the invention
The preparation and the preparation method that the purpose of this invention is to provide a kind of low temperature neutral cellulase are to overcome the deficiency that temperature, the plain enzyme of high temperature fiber are brought in the present application.This method mainly is to utilize Li's Trichoderma strains, and by ultraviolet mutagenesis, low temperature is cultivated, and produces the low temperature neutral cellulase then in 10～15 ℃ fermented liquid, and has studied its preparation method and the application in paper industry.
The objective of the invention is to be achieved through the following technical solutions:
A kind of preparation method of low temperature neutral cellulase selects for use Trichodermareesei to prepare described low temperature neutral cellulase, and described preparation method may further comprise the steps:
(1) Li's Trichoderma strains is inoculated on the slant activation substratum activated spawn; Cultivate the bacterial classification after activating, picking list bacterium colony prepares spore suspension, and uses the uv irradiating spore suspension, and mutagenesis obtains the spore bacterium colony;
(2) the spore bacterium colony in (1) is coated in the primary dcreening operation substratum, cultivated 3～5 days down at 10～15 ℃, select this bacterial strain of primary dcreening operation bacterial strain and purifying;
(3) under 10～15 ℃, the purifying bacterial strain in (2) is inoculated in the fermention medium by 5% inoculum size, enlarged culturing prepares seed liquor step by step, and incubation time is 48～72 hours;
(4) inoculum size of the seed liquor in (3) by fermentating liquid volume 3～5% is inoculated in the product enzyme substratum, cultivated 96～144 hours for 10～15 ℃, namely Trichodermareesei fermentative production low temperature neutral cellulase finishes; Fermented liquid is centrifugal at 4000～6000rpm, and collecting gained liquid is crude enzyme liquid;
(5) crude enzyme liquid that (4) are obtained carries out the hyperconcentration filtration, obtains the concentrated enzyme liquid of 1000～2000UI/G.
Preferably, described primary dcreening operation substratum is selected 60.0～80.0g potato for use, 0.5g K
2HPO
4, 2.0～2.4g gelatin, the preparation of 20.0～25.0g agar and 1.0L distilled water, and the described primary dcreening operation substratum that will prepare is sterilization 30 minutes under 115 ℃～121 ℃ the high pressure steam in temperature.
Preferably, described fermention medium is selected 3.0～5.0g Semen Maydis powder for use, 0.5～1.0g peptone, 0.8～1.2g (NH
4)
2SO
4, 1.2～1.8g KH
2PO
4, 0.1～0.3g urea, 0.1～0.2g CaCl
2With 1.0L distilled water preparation, and the described fermention medium that will prepare is sterilization 30 minutes under 115 ℃～121 ℃ the high pressure steam in temperature.
Preferably, described product enzyme substratum is selected 70～80g wheat bran for use, 15～20g rice bran, 0.15～0.2g carboxymethyl cellulose, 0.05～0.1g (NH
4)
2SO
4, 0.05～0.1g MgSO
4, 0.1～0.15g K
2HPO
4With 1.48～1.52L distilled water, and the described product enzyme substratum that will prepare is sterilization 30 minutes under 115 ℃～121 ℃ the high pressure steam in temperature.
A kind of preparation method that adopts the low temperature neutral cellulase of aforementioned techniques scheme acquisition, one or more components in described concentrated enzyme liquid and surfactant, sanitas, the stablizer etc. are carried out composite, its mass percent is respectively: concentrate enzyme liquid 10～50%, surfactant 5～10%, sanitas 0.1～1%, stablizer 1～30%, other: deionized water namely is re-dubbed the low temperature neutral cellulase preparation that can be used for paper industry.
Preferably, described surfactant mainly is made up of one or more materials in fatty alcohol-polyoxyethylene ether, aliphatic alcohol polyethenoxy polyethenoxy ether, aliphatic acid polyethenoxy ether, lipid acid polyethenoxy ether and the alkylphenol polyoxyethylene; Described sanitas mainly is made up of one or more materials in cloth sieve bohr, glutaraldehyde, Ka Song and the Phenoxyethanol; Described stablizer mainly is made up of one or more materials in sodium-chlor, calcium chloride, magnesium chloride, glycerine, sorbyl alcohol, propylene glycol, polyvinylpyrrolidone, sodium polyacrylate and the polyvinyl alcohol.
The Trichodermareesei that uses among the present invention (Trichoderma reesei) is known microorganism, and the bacterial strain after the mutagenesis can be preserved 2 months in 4 ℃ of environment, in 10～20% Sorbitol Solution USP, but in subzero 80 ℃ of following prolonged preservation.
Beneficial effect of the present invention is: obtained a kind of low temperature neutral cellulase of high vigor, not only the preparation method is simple for it, and fermentation period is short, and reduced the application of temperature of enzyme, can reduce the power consumption of industrial application greatly, make its more environmental protection, have wide prospect in industrial application; In addition, the present invention also provides a kind of preparation method and application aspect papermaking thereof of low temperature neutral cellulase, makes this technology can be widely used in the modification of paper-making fibre, reduces the cost of paper industry fibre modification.
Embodiment
Embodiment one:
(1) medium preparation
1. activation medium: 200.0g potato, 25.0g glucose, 15.0g agar, 1.0L distilled water, pH7.0,115 ℃ of high pressure steam sterilizations 30 minutes.
2. primary dcreening operation substratum: 60.0g potato, 0.5g K
2HPO
4, 2.0g gelatin, 20.0g agar, 1.0L distilled water, pH6.3,115 ℃ of high pressure steam sterilizations 30 minutes.
3. fermention medium: 5.0g Semen Maydis powder, 1.0g peptone, 1.2g (NH
4)
2SO
4, 1.8g KH
2PO
4, 0.3g urea, 0.2g CaCl
2, 1.0L distilled water, pH7.0,115 ℃ of high pressure steam sterilizations 30 minutes.
4. produce the enzyme substratum: 70.0g wheat bran, 15.0g rice bran, 0.15g carboxymethyl cellulose, 0.05g (NH
4)
2SO
4, 0.05g MgSO
4, 0.1g K
2HPO
4, 1.48L distilled water, pH6.0,115 ℃ of high pressure steam sterilizations 30 minutes.
(2) Li's Trichoderma strains is inoculated on the slant activation substratum continuous passage three times, activated spawn; Coat on the flat board after the bacterial classification dilution with activation, observe the upgrowth situation of bacterial strain, utilize the size of transparent circle, choose the big single bacterium colony of transparent circle as the bacterium colony of mutagenesis.
(3) single bacterium colony that (2) are selected joins in the 10mL sterilized water, and vibration washes ripe spore, and the dispersed with stirring spore gets spore suspension, and spore suspension is placed under the ultraviolet lamp, and vertical range 30CM stirs irradiation, and mutagenesis gets the spore bacterium colony.Above-mentioned uv irradiating all must carry out under the condition of lucifuge.
(4) coat in the primary dcreening operation substratum after the spore bacterium colony dilution that (3) ultraviolet mutagenesis is obtained, cultivated 3～5 days down at 10～13 ℃, add an amount of congo red staining, picking produces the primary dcreening operation bacterial strain of transparent circle, through continuous this bacterial strain of plate streaking purifying.
(5) under 10～13 ℃, the purifying bacterial strain of (4) is inoculated in the fermention medium by 5% inoculum size, enlarged culturing prepares seed liquor step by step, and incubation time is 48～72 hours.
(6) seed liquor that (5) are prepared is inoculated in the product enzyme substratum of 10L by the inoculum size of fermentating liquid volume 3～5%, cultivates 130～144 hours for 10～13 ℃, and namely Trichodermareesei fermentative production low temperature neutral cellulase finishes.
(7) fermented liquid of (6) is centrifugal at 4000rpm, collecting gained liquid is crude enzyme liquid.
(8) according to different needs, the crude enzyme liquid that (7) can also be obtained further concentrates, separation and purification, is prepared into different activities, purity low-temperature cellulase.
Embodiment two:
(1) medium preparation
1. activation medium: 200.0g potato, 25.0g glucose, 15.0g agar, 1.0L distilled water, pH6.8,118 ℃ of high pressure steam sterilizations 30 minutes.
2. primary dcreening operation substratum: 70.0g potato, 0.5g K
2HPO
4, 2.2g gelatin, 22.0g agar, 1.0L distilled water, pH6.5,118 ℃ of high pressure steam sterilizations 30 minutes.
3. fermention medium: 3.0g Semen Maydis powder, 0.5g peptone, 0.8g (NH
4)
2SO
4, 1.2g KH
2PO
4, 0.1g urea, 0.1g CaCl
2, 1.0L distilled water, pH6.0,118 ℃ of high pressure steam sterilizations 30 minutes.
4. produce the enzyme substratum: 73g wheat bran, 17g rice bran, 0.17g carboxymethyl cellulose, 0.08g (NH
4)
2SO
4, 0.08g MgSO
4, 0.1g K
2HPO
4, 1.5L distilled water, pH6.4,118 ℃ of high pressure steam sterilizations 30 minutes.
(2) Li's Trichoderma strains is inoculated on the slant activation substratum continuous passage three times, activated spawn, coat on the flat board after the bacterial classification dilution with activation, observe the upgrowth situation of bacterial strain, utilize the size of transparent circle, choose the big single bacterium colony of transparent circle as the bacterium colony of mutagenesis.
(3) single bacterium colony that (2) are selected joins in the 10mL sterilized water, and vibration washes ripe spore, and the dispersed with stirring spore gets spore suspension, and the spore suspension that stirs is placed under the ultraviolet lamp, and vertical range 30CM stirs irradiation, and mutagenesis gets the spore bacterium colony.Above-mentioned uv irradiating all must carry out under the condition of lucifuge.
(4) coat in the primary dcreening operation substratum after the spore bacterium colony dilution that (3) ultraviolet mutagenesis is obtained, cultivated 3～5 days down at 13～15 ℃, add an amount of congo red staining, picking produces the primary dcreening operation bacterial strain of transparent circle, through continuous this bacterial strain of plate streaking purifying.
(5) under 13～15 ℃, (4) purifying bacterial strain is inoculated in the fermention medium by 5% inoculum size, enlarged culturing prepares seed liquor step by step, and incubation time is 48～72 hours.
(6) seed liquor that (5) are prepared is inoculated in the product enzyme substratum of 10L by the inoculum size of fermentating liquid volume 3～5%, cultivates 110～130 hours for 13～15 ℃, and namely Trichodermareesei fermentative production low temperature neutral cellulase finishes.
(7) fermented liquid of (6) is centrifugal at 5000rpm, collecting gained liquid is crude enzyme liquid.
(8) according to different needs, the crude enzyme liquid that (7) can also be obtained further concentrates, separation and purification, is prepared into different activities, purity low-temperature cellulase.
Embodiment three:
(1) medium preparation
1. activation medium: 200.0g potato, 25.0g glucose, 15.0g agar, 1.0L distilled water, pH7.2,121 ℃ of high pressure steam sterilizations 30 minutes.
2. primary dcreening operation substratum: 80.0g potato, 0.5g K
2HPO
4, 2.4g gelatin, 25.0g agar, 1.0L distilled water, pH7.0,121 ℃ of high pressure steam sterilizations 30 minutes.
3. fermention medium: 4.0g Semen Maydis powder, 0.7g peptone, 1.0g (NH
4)
2SO
4, 1.5g KH
2PO
4, 0.2g urea, 0.15g CaCl
2, 1.2L distilled water, pH6.5,121 ℃ of high pressure steam sterilizations 30 minutes.
4. produce the enzyme substratum: 80.0g wheat bran, 20.0g rice bran, 0.2g carboxymethyl cellulose, 0.1g (NH
4)
2SO
4, 0.1g MgSO
4, 0.15g K
2HPO
4, 1.52L distilled water, pH7.0,121 ℃ of high pressure steam sterilizations 30 minutes.
(2) Li's Trichoderma strains is inoculated on the slant activation substratum continuous passage three times, activated spawn, coat on the flat board after the bacterial classification dilution with activation, observe the upgrowth situation of bacterial strain, utilize the size of transparent circle, choose the big single bacterium colony of transparent circle as the bacterium colony of mutagenesis.
(3) single bacterium colony that (2) are selected joins in the 10mL sterilized water, and vibration washes ripe spore, and the dispersed with stirring spore gets spore suspension, and the spore suspension that stirs is placed under the ultraviolet lamp, and vertical range 30CM stirs irradiation, and mutagenesis gets the spore bacterium colony.Above-mentioned uv irradiating all must carry out under the condition of lucifuge.
(4) coat in the primary dcreening operation substratum after the spore bacterium colony dilution that (3) ultraviolet mutagenesis is obtained, cultivated 3～5 days down at 12～14 ℃, add an amount of congo red staining, picking produces the primary dcreening operation bacterial strain of transparent circle, through continuous this bacterial strain of plate streaking purifying.
(5) under 12～14 ℃, the purifying bacterial strain is inoculated in the fermention medium by 5% inoculum size, enlarged culturing prepares seed liquor step by step, and incubation time is 48～72 hours.
(6) seed liquor that (5) are prepared is inoculated in the product enzyme substratum of 10L by the inoculum size of fermentating liquid volume 3～5%, cultivates 96～110 hours for 12～14 ℃, and namely Trichodermareesei fermentative production low temperature neutral cellulase finishes.
(7) fermented liquid of (6) is centrifugal at 6000rpm, collecting gained liquid is crude enzyme liquid.
(8) according to different needs, the crude enzyme liquid that (7) can also be obtained further concentrates, separation and purification, is prepared into different activities, purity low-temperature cellulase.
According to the low temperature neutral cellulase of embodiment one, two, three described method preparations, average enzymic activity is 1400UI/G.Test result shows: work as pH6.5, temperature is between 20～30 ℃ the time, and this cellulase can keep 80% vigor; If when temperature is 15 ℃, still can keep 60% enzyme work; And when pH5.0～8.0, when temperature was 25 ℃, enzyme work was up to 80%.So it can be widely used in the modification of fiber under the paper industry cold condition.Therefore, be re-dubbed zymin by further cellulase being concentrated enzyme liquid and surfactant, stablizer, sanitas etc., the longer quality guaranteed period is arranged, can satisfy requirement on industrial application fully, and cost performance is more excellent.
Embodiment four:
The preparation method of low temperature neutral cellulase: one or more components that concentrate in enzyme liquid and surfactant, sanitas, the stablizer etc. are carried out composite, its mass percent is respectively: concentrate enzyme liquid 10～50%, surfactant 5～10%, sanitas 0.1～1%, stablizer 1～30%, other: deionized water namely is re-dubbed the low temperature neutral cellulase preparation that can be used for paper industry.
Described surfactant mainly is made up of one or more materials in fatty alcohol-polyoxyethylene ether, aliphatic alcohol polyethenoxy polyethenoxy ether, aliphatic acid polyethenoxy ether, lipid acid polyethenoxy ether, the alkylphenol polyoxyethylene; Described sanitas is mainly by cloth sieve bohr, and glutaraldehyde is blocked pine, and one or more materials in the Phenoxyethanol are formed; Described stablizer mainly is made up of one or more materials in sodium-chlor, calcium chloride, magnesium chloride, glycerine, sorbyl alcohol, propylene glycol, polyvinylpyrrolidone, sodium polyacrylate, the polyvinyl alcohol.
When using, reality can carry out with reference to following per-cent:
Compounded combination 1
Compounded combination 2
Compounded combination 3
Embodiment five:
The application of low temperature neutral cellulase preparation: 10～25 ℃ of water temperatures, pH5.0～8.0, PFI hollander 2500rpm, consumption 0.3～the 0.8kg/ton of low temperature neutral cellulase preparation, 1 hour action time, under certain situation of hollander revolution and time, beating degree has tangible increase; If keep same beating degree, can reduce the making beating revolution.
More than invention provides a kind of preparation method of low temperature neutral cellulase, and the composite and application method of this low temperature neutral cellulase is described in detail.Having used specific case herein sets forth principle of the present invention and embodiment; the explanation of above embodiment just is used for helping to understand method of the present invention and core concept; for the person of ordinary skill of the art; under the prerequisite that does not break away from the principle of the invention; also can carry out some improvement and modification to the present invention, these improvement and modification also fall in the protection domain of claim of the present invention.
Claims (6)
1. the preparation method of a low temperature neutral cellulase, it is characterized in that: select for use Trichodermareesei to prepare described low temperature neutral cellulase, described preparation method may further comprise the steps:
(1) Li's Trichoderma strains is inoculated on the slant activation substratum activated spawn; Cultivate the bacterial classification after activating, picking list bacterium colony prepares spore suspension, and uses the uv irradiating spore suspension, and mutagenesis obtains the spore bacterium colony;
(2) the spore bacterium colony in (1) is coated in the primary dcreening operation substratum, cultivated 3～5 days down at 10～15 ℃, select this bacterial strain of primary dcreening operation bacterial strain and purifying;
(3) under 10～15 ℃, the purifying bacterial strain in (2) is inoculated in the fermention medium by 5% inoculum size, enlarged culturing prepares seed liquor step by step, and incubation time is 48～72 hours;
(4) inoculum size of the seed liquor in (3) by fermentating liquid volume 3～5% is inoculated in the product enzyme substratum, cultivated 96～144 hours for 10～15 ℃, namely Trichodermareesei fermentative production low temperature neutral cellulase finishes; Fermented liquid is centrifugal at 4000～6000rpm, and collecting gained liquid is crude enzyme liquid;
(5) crude enzyme liquid that (4) are obtained carries out the hyperconcentration filtration, obtains the concentrated enzyme liquid of 1000～2000UI/G.
2. the preparation method of low temperature neutral cellulase according to claim 1, it is characterized in that: described primary dcreening operation substratum is selected 60.0～80.0g potato for use, 0.5g K
2HPO
4, 2.0～2.4g gelatin, the preparation of 20.0～25.0g agar and 1.0L distilled water, and the described primary dcreening operation substratum that will prepare is sterilization 30 minutes under 115 ℃～121 ℃ the high pressure steam in temperature.
3. the preparation method of low temperature neutral cellulase according to claim 1, it is characterized in that: described fermention medium is selected 3.0～5.0g Semen Maydis powder for use, 0.5～1.0g peptone, 0.8～1.2g (NH
4)
2SO
4, 1.2～1.8g KH
2PO
4, 0.1～0.3g urea, 0.1～0.2g CaCl
2With 1.0L distilled water preparation, and the described fermention medium that will prepare is sterilization 30 minutes under 115 ℃～121 ℃ the high pressure steam in temperature.
4. the preparation method of low temperature neutral cellulase according to claim 1, it is characterized in that: described product enzyme substratum is selected 70.0～80.0g wheat bran for use, 15.0～20.0g rice bran, 0.15～0.2g carboxymethyl cellulose, 0.05～0.1g (NH
4)
2SO
4, 0.05～0.1g MgSO
4, 0.1～0.15g K
2HPO
4With 1.48～1.52L distilled water preparation, and the described product enzyme substratum that will prepare is sterilization 30 minutes under 115 ℃～121 ℃ the high pressure steam in temperature.
5. the preparation method of the low temperature neutral cellulase that each described preparation method obtains in an employing such as the claim 1～4, it is characterized in that: one or more components in described concentrated enzyme liquid and surfactant, sanitas, the stablizer are carried out composite, its mass percent is respectively: concentrate enzyme liquid 10～50%, surfactant 5～10%, sanitas 0.1～1%, stablizer 1～30%, other: deionized water namely is re-dubbed low temperature neutral cellulase preparation.
6. the preparation method of low temperature neutral cellulase according to claim 5, it is characterized in that: described surfactant is made up of one or more materials in fatty alcohol-polyoxyethylene ether, aliphatic alcohol polyethenoxy polyethenoxy ether, aliphatic acid polyethenoxy ether, lipid acid polyethenoxy ether and the alkylphenol polyoxyethylene; Described sanitas is made up of one or more materials in cloth sieve bohr, glutaraldehyde, Ka Song and the Phenoxyethanol; Described stablizer is made up of one or more materials in sodium-chlor, calcium chloride, magnesium chloride, glycerine, sorbyl alcohol, propylene glycol, polyvinylpyrrolidone, sodium polyacrylate and the polyvinyl alcohol.
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| CN106119227A (en) * | 2016-06-28 | 2016-11-16 | 郭舒洋 | A kind of preparation method of low-temperature cellulase |
| CN106434447A (en) * | 2016-09-26 | 2017-02-22 | 浙江大学舟山海洋研究中心 | Strain capable of producing salt-tolerant neutral cellulase, and screening method and application thereof |
| US9605247B2 (en) | 2013-12-23 | 2017-03-28 | Hunan Hong Ying Biotech Co., Ltd. | Strain and a method to produce cellulase and its use |
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| CN101928702A (en) * | 2010-07-24 | 2010-12-29 | 江西金伟生物制品有限公司 | Method for preparing cellulase |
| CN102242067A (en) * | 2010-05-13 | 2011-11-16 | 上海康地恩生物科技有限公司 | Bacterial strain for producing low temperature neutral cellulase and method for preparing said cellulose |
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| CN106434447A (en) * | 2016-09-26 | 2017-02-22 | 浙江大学舟山海洋研究中心 | Strain capable of producing salt-tolerant neutral cellulase, and screening method and application thereof |
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