CN103497941B - Method for preparing cellulase through trichoderma viride high-efficiency fermentation - Google Patents
Method for preparing cellulase through trichoderma viride high-efficiency fermentation Download PDFInfo
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- 108010059892 Cellulase Proteins 0.000 title claims abstract description 55
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- 238000000034 method Methods 0.000 title claims abstract description 23
- 241000223261 Trichoderma viride Species 0.000 title abstract description 6
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- 230000004913 activation Effects 0.000 claims abstract description 19
- 239000000843 powder Substances 0.000 claims abstract description 8
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- 229910019142 PO4 Inorganic materials 0.000 claims description 12
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- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims description 12
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- 239000010452 phosphate Substances 0.000 claims description 12
- 239000011591 potassium Substances 0.000 claims description 12
- 229910052700 potassium Inorganic materials 0.000 claims description 12
- 239000002904 solvent Substances 0.000 claims description 12
- 238000004659 sterilization and disinfection Methods 0.000 claims description 12
- 239000001888 Peptone Substances 0.000 claims description 6
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- 238000011081 inoculation Methods 0.000 claims description 6
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- 229920006393 polyether sulfone Polymers 0.000 claims description 6
- 150000003839 salts Chemical class 0.000 claims description 6
- 235000013619 trace mineral Nutrition 0.000 claims description 6
- 239000011573 trace mineral Substances 0.000 claims description 6
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- 241000223259 Trichoderma Species 0.000 claims description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims 2
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 claims 2
- 229920000053 polysorbate 80 Polymers 0.000 claims 2
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims 1
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- 238000004519 manufacturing process Methods 0.000 abstract description 14
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- 235000017166 Bambusa arundinacea Nutrition 0.000 abstract description 7
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- ZTOKUMPYMPKCFX-CZNUEWPDSA-N (E)-17-[(2R,3R,4S,5S,6R)-6-(acetyloxymethyl)-3-[(2S,3R,4S,5S,6R)-6-(acetyloxymethyl)-3,4,5-trihydroxyoxan-2-yl]oxy-4,5-dihydroxyoxan-2-yl]oxyoctadec-9-enoic acid Chemical compound OC(=O)CCCCCCC/C=C/CCCCCCC(C)O[C@@H]1O[C@H](COC(C)=O)[C@@H](O)[C@H](O)[C@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](COC(C)=O)O1 ZTOKUMPYMPKCFX-CZNUEWPDSA-N 0.000 description 1
- 241000228245 Aspergillus niger Species 0.000 description 1
- 241000221198 Basidiomycota Species 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
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- 241001465754 Metazoa Species 0.000 description 1
- 241000228143 Penicillium Species 0.000 description 1
- 241000193448 Ruminiclostridium thermocellum Species 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- 241000209140 Triticum Species 0.000 description 1
- 235000021307 Triticum Nutrition 0.000 description 1
- 240000008042 Zea mays Species 0.000 description 1
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 1
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 108010047754 beta-Glucosidase Proteins 0.000 description 1
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- 235000013305 food Nutrition 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 229930182478 glucoside Natural products 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
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- 230000000630 rising effect Effects 0.000 description 1
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
- C12N9/2437—Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
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Abstract
The invention discloses a method for preparing cellulase through trichoderma viride high-efficiency fermentation. The key points of the method are that: trichoderma viride is inoculated on a Czapek medium and is activated; a single colony is selected and is inoculated in a liquid activation culture medium and is subjected to amplification culture, such that a seed liquid is obtained. The seed liquid is inoculated into a liquid enzyme-production culture medium according to a volume ratio of 1:10-20; 5-20g/L of bamboo powder with a specification of 40-60 meshes is adopted as a fermentation substrate; liquid fermentation is carried out for 72-120h, such that cellulase is prepared. The method is stable and highly efficient. The method is especially suitable for producing cellulase through trichoderma viride high-efficiency fermentation. According to the invention, the entire set of liquid fermentation process is obtained through optimization aiming at trichoderma viride physiological characteristics, such that cellulase with high enzyme activity can be obtained. Also, the bamboo raw material is used as a resource and as a cellulase induction substrate, and the biological enzyme preparation with high added value is prepared. The invention provides an innovative idea for utilizing bamboo resource with high value. With the method, Bamboo area farmers' economic income can be effectively improved. The invention has important significance.
Description
Technical field
The present invention relates to a kind of method preparing cellulase, particularly a kind of high-efficiency fermenting viride prepares the method for cellulase, belongs to field of microbial culture technology in biological chemistry.
Background technology
Cellulase is one group can the glucoside bond of hydrocellulose molecular chain make it change into the general name of the polycomponent enzyme of glucose, be several enzyme complexs with different enzyme activity, mainly comprise exocellulase, endo cellulase, beta-glucosidase etc.Mierocrystalline cellulose need the common appearance of these enzymes and act synergistically, co-catalysis just can be completely degraded.Utilize that cellulose conversion is food by cellulase, the energy, chemical materials, to the many Environmental and resource issues solving facing mankind, there is great realistic meaning.
The bottleneck of the extensive efficiency utilization cellulase of the current obstruction mankind, one is lack High-Cellulase-Yielding bacterium, and two is that enzymatic production technique still treats further optimization.What the current microbial strains for the production of cellulase was more is filamentous fungus, and wherein, the bacterial classification that enzyme activity is stronger is that Trichoderma, Aspergillus and Penicillium, particularly viride and nearly edge bacterial strain thereof are comparatively typical, is generally acknowledged good cellulase production bacterium.In addition, ruminating animal relies on rumen microorganism digestible cellulose, as rumen bacteria and Clostridium thermocellum etc., but output and activity lower.The preparation method of domestic and international cellulase mainly contains solid fermentation method and solution fermentation two kinds.Solid fermentation apparatus less investment, but floor space greatly, easily contaminates miscellaneous bacteria, production stability is poor, and also technology is not easily grasped, and is difficult to carry out extension and produces, and most enterprise of current China still adopts solid fermentation process to prepare cellulase; Liquid fermenting has that production stability is high, technology is easily grasped, be convenient to the advantages such as suitability for industrialized production, but because cellulase is inducible enzyme, adopt corn stalk, wheat straw, straw etc. as inductor, effect is not very good, therefore enterprise adopts expensive timber as raw material one after another, considerably increase enterprise's production cost, constrain the promotion and application of cellulase production technology.
Cellulase field is prepared at organism of fermentation, Chinese patent (ZL200810023480.8) " a kind of trichoderma reesei cultivation method improving yield of cellulase " produces in enzyme process at Trichodermareesei and regulates ventilation, make product enzyme main phase dissolved oxygen concentration 20 ~ 30%, be beneficial to thalli growth, the cellulase of output comparatively fixes the filter paper enzyme activity raising 30% of ventilation volume production enzyme, produces enzyme time shorten 14%; Chinese patent (ZL200510009000.9) " high active cellulase and manufacture method thereof " is using basidiomycetes CGMCCNo.1294 as production bacterial classification, sterilizing access triangular flask liquid spawn, carry out liquid submerged fermentation, after filtration, concentrated and spraying dry makes cellulase powder, measuring and producing enzyme activity is 40000u/g, and process stability is good, not easily microbiological contamination, be convenient to large-scale industrial production; Chinese patent (ZL200610002014.2) " method of producing low temperature cellulase using microbe fermentation " will produce the microorganism domestication by low temperature step by step of cellulase, make its well-grown in low temperature environment, through enlarged culturing, enzymatic production, need according to difference and use object, by crude enzyme liquid concentrated, separation and purification further, prepare the cellulase preparation of different activities, purity and formulation; United States Patent (USP) (US61110732) " Hosts and fermentation processesfor cellulase production " using through gene-modified filamentous fungus as cultivation bacterial classification, using the hemicellulose of 25 ~ 100wt% source alcohol sugar and the glucose of 0 ~ 75wt%, glycerol or the mixture of the two as carbon source, the standby prozyme simultaneously containing cellulase and hemicellulose enzyme activity of fermentation.Mould for long shoot wood, Trichoderma lignorum, aspergillus niger, wooden aspergillus are cultivated and zymotechnique by different by United States Patent (USP) (US60920942) " Process for producing cellulase ", prepare cellulase and sophorolipid two kinds of products simultaneously.So far, yet there are no and utilize mao bamboon raw material as inducing substrate, the related process technologies that high-efficiency fermenting viride prepares cellulase occurs.
Summary of the invention
The bacterial classification preparing cellulase technological process in order to solve current fermentable produces the practical problemss such as enzyme efficiency is low, solid fermentation poor stability, inducing substrate effect are undesirable, recycling China's abundant, cheap bamboo resource simultaneously, preparation high added value biological enzyme formulation, the object of the invention is to provide a kind of method that high-efficiency fermenting viride prepares cellulase.
For achieving the above object, technical scheme of the present invention adopts following steps:
1) not activated viride is dissolved with sterilized water in gnotobasis, adopt plate streak by strain inoculation on czapek's solution, in the constant incubator of 24 ~ 32 DEG C, cultivating 72 ~ 168h, to growing single bacterium colony, obtaining the viride through overactivation;
2) viride through overactivation step 1) obtained is picking list bacterium colony in gnotobasis, is inoculated in liquid activation medium, 24 ~ 32 DEG C, cultivate 48 ~ 72h in the constant temperature oscillator of 180 ~ 260r/min, obtain the seed liquor of enlarged culturing;
3) by step 2) seed liquor of enlarged culturing that obtains is inoculated in liquid culture medium according to the inoculum size of volume ratio 1:10 ~ 20, Intake Quantity according to 60% loads in fermentor tank, produce enzyme cultivation and prepare cellulase, control fermented liquid pH value 3 ~ 7, leavening temperature 24 ~ 32 DEG C, air flow 1:0.4 ~ 1.2, fermentation time 72 ~ 120h, the fermented liquid obtained is centrifugal 10min in the refrigerated centrifuge of 4000r/min, adopting the poly (ether sulfone) film of molecular weight cut-off 100 ~ 500k to concentrate makes its concentration promote 20 times, obtains cellulase.
Described liquid activation culture based formulas is: glucose 10 ~ 20g/L, peptone 1 ~ 2g/L, Tween800.2 ~ 0.4g/L, ammonium sulfate 1 ~ 2g/L, potassium primary phosphate 1 ~ 3g/L, magnesium sulfate 0.1 ~ 0.3g/L, calcium chloride 0.1 ~ 0.3g/L, urea 0.4 ~ 0.8g/L, the Mandels nutritive salt accounting for liquid activation medium cumulative volume 10 ~ 20%, solvent are the PBS of pH=3 ~ 7,120 DEG C of high pressure steam sterilization 20min.
Described liquid culture medium formula is: glucose 4 ~ 6g/L, 40 to 60 order mao bamboon powder 5 ~ 20g/L, Tween800.2 ~ 0.6g/L, ammonium sulfate 1 ~ 3g/L, potassium primary phosphate 3 ~ 5g/L, magnesium sulfate 0.3 ~ 0.5g/L, calcium chloride 0.3 ~ 0.5g/L, urea 0.8 ~ 1.2g/L, the Mandels trace element, the solvent that account for liquid culture medium cumulative volume 0.1 ~ 0.3% are the PBS of pH=3 ~ 7,120 DEG C of high pressure steam sterilization 20min.
Compared with background technology, the beneficial effect that the present invention has is:
The present invention introduces viride in the microbial strains of fermentation for cellulase, and the physiological property optimization for viride obtains a set of efficient liquid zymotechnique, can obtain the cellulase of high enzymatic activity under this technique; The inducing substrate of recycling mao bamboon raw material as cellulase is proposed simultaneously, preparation high added value biological enzyme formulation, be that of higher value application bamboo resource opens one's minds, meet national development circular economy policy, and conscientiously can improve the income of China bamboo district peasant.
Accompanying drawing explanation
Fig. 1 is that embodiment 3 goes out the viride photo of single bacterium colony through czapek's solution activated growth;
Fig. 2 is the processing condition according to embodiment 3, in the CMC enzyme work of cellulase prepared by the different fermentations time period.
Embodiment
Below in conjunction with specific embodiment, the invention will be further described.
Embodiment 1:
1) dissolved with sterilized water in gnotobasis by not activated viride, strain inoculation on czapek's solution, being cultivated 72h, to growing single bacterium colony, being obtained the viride through overactivation by employing plate streak in the constant incubator of 24 DEG C;
2) viride through overactivation step 1) obtained is picking list bacterium colony in gnotobasis, is inoculated in liquid activation medium, 24 DEG C, cultivate 48h in the constant temperature oscillator of 180r/min, obtain the seed liquor of enlarged culturing; (liquid activation culture based formulas: glucose 10g/L, peptone 1g/L, Tween800.2g/L, ammonium sulfate 1g/L, potassium primary phosphate 1g/L, magnesium sulfate 0.1g/L, calcium chloride 0.1g/L, urea 0.4g/L, the Mandels nutritive salt accounting for liquid activation medium cumulative volume 10%, solvent are the PBS of pH=3,120 DEG C of high pressure steam sterilization 20min)
3) by step 2) seed liquor of enlarged culturing that obtains is inoculated in liquid culture medium according to the inoculum size of volume ratio 1:10, Intake Quantity according to 60% loads in fermentor tank, produce enzyme cultivation and prepare cellulase, control fermented liquid pH value 3, leavening temperature 24 DEG C, air flow 1:0.4, fermentation time 72h, the fermented liquid obtained is centrifugal 10min in the refrigerated centrifuge of 4000r/min, adopting the poly (ether sulfone) film of molecular weight cut-off 100k to concentrate makes its concentration promote 20 times, obtains cellulase (a).(liquid culture medium is filled a prescription: glucose 4g/L, 40 to 60 order mao bamboon powder 5g/L, Tween800.2g/L, ammonium sulfate 1g/L, potassium primary phosphate 3g/L, magnesium sulfate 0.3g/L, calcium chloride 0.3g/L, urea 0.8g/L, the Mandels trace element, the solvent that account for liquid culture medium cumulative volume 0.1% are the PBS of pH=3,120 DEG C of high pressure steam sterilization 20min)
Embodiment 2:
1) dissolved with sterilized water in gnotobasis by not activated viride, strain inoculation on czapek's solution, being cultivated 108h, to growing single bacterium colony, being obtained the viride through overactivation by employing plate streak in the constant incubator of 28 DEG C;
2) viride through overactivation step 1) obtained is picking list bacterium colony in gnotobasis, is inoculated in liquid activation medium, 28 DEG C, cultivate 60h in the constant temperature oscillator of 220r/min, obtain the seed liquor of enlarged culturing; (liquid activation culture based formulas: glucose 15g/L, peptone 1.5g/L, Tween800.3g/L, ammonium sulfate 1.5g/L, potassium primary phosphate 2g/L, magnesium sulfate 0.2g/L, calcium chloride 0.2g/L, urea 0.6g/L, the Mandels nutritive salt accounting for liquid activation medium cumulative volume 15%, solvent are the PBS of pH=5,120 DEG C of high pressure steam sterilization 20min)
3) by step 2) seed liquor of enlarged culturing that obtains is inoculated in liquid culture medium according to the inoculum size of volume ratio 1:15, Intake Quantity according to 60% loads in fermentor tank, produce enzyme cultivation and prepare cellulase, control fermented liquid pH value 5, leavening temperature 28 DEG C, air flow 1:0.8, fermentation time 96h, the fermented liquid obtained is centrifugal 10min in the refrigerated centrifuge of 4000r/min, adopting the poly (ether sulfone) film of molecular weight cut-off 300k to concentrate makes its concentration promote 20 times, obtains cellulase (b).(liquid culture medium is filled a prescription: glucose 5g/L, 40 to 60 order mao bamboon powder 10g/L, Tween800.4g/L, ammonium sulfate 2g/L, potassium primary phosphate 4g/L, magnesium sulfate 0.4g/L, calcium chloride 0.4g/L, urea 1.0g/L, the Mandels trace element, the solvent that account for liquid culture medium cumulative volume 0.2% are the PBS of pH=5,120 DEG C of high pressure steam sterilization 20min)
Embodiment 3:
1) dissolved with sterilized water in gnotobasis by not activated viride, strain inoculation on czapek's solution, being cultivated 144h, to growing single bacterium colony, being obtained the viride through overactivation by employing plate streak in the constant incubator of 30 DEG C;
2) viride through overactivation step 1) obtained is picking list bacterium colony in gnotobasis, is inoculated in liquid activation medium, 30 DEG C, cultivate 72h in the constant temperature oscillator of 240r/min, obtain the seed liquor of enlarged culturing; (liquid activation culture based formulas: glucose 20g/L, peptone 2g/L, Tween800.3g/L, ammonium sulfate 1.5g/L, potassium primary phosphate 2g/L, magnesium sulfate 0.3g/L, calcium chloride 0.2g/L, urea 0.8g/L, the Mandels nutritive salt accounting for liquid activation medium cumulative volume 15%, solvent are the PBS of pH=5,120 DEG C of high pressure steam sterilization 20min)
3) by step 2) seed liquor of enlarged culturing that obtains is inoculated in liquid culture medium according to the inoculum size of volume ratio 1:15, Intake Quantity according to 60% loads in fermentor tank, produce enzyme cultivation and prepare cellulase, control fermented liquid pH value 5, leavening temperature 30 DEG C, air flow 1:1, fermentation time 108h, the fermented liquid obtained is centrifugal 10min in the refrigerated centrifuge of 4000r/min, adopting the poly (ether sulfone) film of molecular weight cut-off 300k to concentrate makes its concentration promote 20 times, obtains cellulase (c).(liquid culture medium is filled a prescription: glucose 6g/L, 40 to 60 order mao bamboon powder 15g/L, Tween800.4g/L, ammonium sulfate 1.5g/L, potassium primary phosphate 4g/L, magnesium sulfate 0.5g/L, calcium chloride 0.4g/L, urea 1.2g/L, the Mandels trace element, the solvent that account for liquid culture medium cumulative volume 0.15% are the PBS of pH=5,120 DEG C of high pressure steam sterilization 20min)
Embodiment 4:
1) dissolved with sterilized water in gnotobasis by not activated viride, strain inoculation on czapek's solution, being cultivated 168h, to growing single bacterium colony, being obtained the viride through overactivation by employing plate streak in the constant incubator of 32 DEG C;
2) viride through overactivation step 1) obtained is picking list bacterium colony in gnotobasis, is inoculated in liquid activation medium, 32 DEG C, cultivate 72h in the constant temperature oscillator of 260r/min, obtain the seed liquor of enlarged culturing; (liquid activation culture based formulas: glucose 20g/L, peptone 2g/L, Tween800.4g/L, ammonium sulfate 2g/L, potassium primary phosphate 3g/L, magnesium sulfate 0.3g/L, calcium chloride 0.3g/L, urea 0.8g/L, the Mandels nutritive salt accounting for liquid activation medium cumulative volume 20%, solvent are the PBS of pH=7,120 DEG C of high pressure steam sterilization 20min)
3) by step 2) seed liquor of enlarged culturing that obtains is inoculated in liquid culture medium according to the inoculum size of volume ratio 1:20, Intake Quantity according to 60% loads in fermentor tank, produce enzyme cultivation and prepare cellulase, control fermented liquid pH value 7, leavening temperature 32 DEG C, air flow 1:1.2, fermentation time 120h, the fermented liquid obtained is centrifugal 10min in the refrigerated centrifuge of 4000r/min, adopting the poly (ether sulfone) film of molecular weight cut-off 500k to concentrate makes its concentration promote 20 times, obtains cellulase (d).(liquid culture medium is filled a prescription: glucose 6g/L, 40 to 60 order mao bamboon powder 20g/L, Tween800.6g/L, ammonium sulfate 3g/L, potassium primary phosphate 5g/L, magnesium sulfate 0.5g/L, calcium chloride 0.5g/L, urea 1.2g/L, the Mandels trace element, the solvent that account for liquid culture medium cumulative volume 0.3% are the PBS of pH=7,120 DEG C of high pressure steam sterilization 20min)
The CMC enzyme measuring four kinds of cellulases that embodiment 1,2,3,4 prepares is lived.Table 1 is the measurement result that the four kinds of cellulase CMC enzymes prepared by embodiment 1,2,3,4 are lived.From data in table 1, the CMC enzyme of the cellulase (a) adopting preparation method of the present invention to obtain, (b), (c), (d) is lived and is distributed in 2110 ~ 2640IU/mL, all there is higher cellulase activity, the technology category that the method for the present invention relates to meets high-efficiency fermenting and prepares cellulase is described.
As Fig. 1, the viride photo going out single bacterium colony through czapek's solution activated growth from embodiment 3 can be found out, it is the raw green mycelia of gas, and mycelia is longer and straight, and side shoot is less, growth middle and later periods contraction and produce fibrillae of spores, spore is little, in oval.As Fig. 2, according to the processing condition of embodiment 3, in the fermenting process of 72 ~ 120h, the cellulase CMC enzyme obtained is lived and is fallen after rising, and reaches the highest enzyme 2640IU/mL alive at 108h, and therefore the best product enzymic fermentation time of embodiment 3 is decided to be 108h.
Table 1
What more than enumerate is only specific embodiments of the invention.The invention is not restricted to above embodiment, many distortion can also be had.All distortion that those of ordinary skill in the art can directly derive from content disclosed by the invention or associate, all should think protection scope of the present invention.
Claims (1)
1. high-efficiency fermenting viride prepares a method for cellulase, it is characterized in that, comprises the following steps:
1) not activated viride (Trichoderma viviride) is dissolved with sterilized water in gnotobasis, adopt plate streak by strain inoculation on czapek's solution (czapck), 72 ~ 108h is cultivated in the constant incubator of 24 ~ 28 DEG C, to growing single bacterium colony, obtain the viride through overactivation;
2) by step 1) the picking list bacterium colony in gnotobasis of the viride through overactivation that obtains, be inoculated in liquid activation medium, at 24 ~ 28 DEG C, 60 ~ 72h is cultivated in the constant temperature oscillator of 180 ~ 260r/min, obtain the seed liquor of enlarged culturing, described liquid activation culture based formulas is: glucose 10 ~ 20g/L, peptone 1 ~ 2g/L, Tween 80 0.2 ~ 0.4g/L, ammonium sulfate 1 ~ 2g/L, potassium primary phosphate 1 ~ 3g/L, magnesium sulfate 0.1 ~ 0.3g/L, calcium chloride 0.1 ~ 0.3g/L, urea 0.4 ~ 0.8g/L, account for the Mandels nutritive salt of liquid activation medium cumulative volume 10 ~ 20%, solvent is pH=3 or 7 phosphoric acid buffers (PBS), 120 DEG C of high pressure steam sterilization 20min,
3) by step 2) seed liquor of enlarged culturing that obtains is inoculated in liquid culture medium according to the inoculum size of volume ratio 1:15 ~ 20, Intake Quantity according to 60% loads in fermentor tank, produce enzyme cultivation and prepare cellulase, control fermented liquid pH value 3 or 7, leavening temperature 24 ~ 28 DEG C, air flow 1:0.4 ~ 1.2, fermentation time 96 ~ 120h, the fermented liquid obtained is centrifugal 10min in the refrigerated centrifuge of 4000r/min, adopting the poly (ether sulfone) film of molecular weight cut-off 100 ~ 500k to concentrate makes its concentration promote 20 times, obtain cellulase, described liquid culture medium formula is: glucose 4 ~ 6g/L, 40 to 60 order mao bamboon powder 5 ~ 20g/L, Tween 80 0.2 ~ 0.6g/L, ammonium sulfate 1 ~ 3g/L, potassium primary phosphate 3 ~ 5g/L, magnesium sulfate 0.3 ~ 0.5g/L, calcium chloride 0.3 ~ 0.5g/L, urea 0.8 ~ 1.2g/L, account for the Mandels trace element of liquid culture medium cumulative volume 0.1 ~ 0.3%, solvent is pH=3 or 7PBS, 120 DEG C of high pressure steam sterilization 20min.
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| 利用X2射线衍射法测定竹材纤维素结晶度;杨淑敏等;《东北林业大学学报》;20100831;第38卷(第8期);摘要 * |
| 碳源对绿色木霉ZC产中性纤维素酶的影响;郑亚平、余晓斌;《无锡轻工大学学报》;20030331;第22卷(第2期);摘要，第31页右栏 * |
| 纤维素酶产生菌的选育及发酵条件优化;钟文文;《环境工程学报》;20071130;第1卷(第11期);第140页右栏至第141页左栏，第142页左栏 * |
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