CN102766615B - Method for preparing cellulase by bacilli - Google Patents
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Abstract
The invention provides a method for preparing cellulase by bacilli. The method includes the steps of using bacillus as a producing strain, subjecting the producing strain to slant culture, shaking culture and fermenting culture to obtain the cellulase. Fermenting culture medium comprises, by weight percent, 0.5-2% of starch, 1-5% of soybean cake meal, 8-12% of soybean cake meal hydrolysate, 0.3-0.8% of bran, 1-2% of inorganic salt, and 0-0.3% of foaming agent. The cellulase prepared by the method has high activity in neutral pH and alkali conditions and is better than acidic cellulase in terms of water abrasion enzyme application. Starch material is used as a carbon source by the method, and accordingly the cellulase is better than that of the fiber material in terms of transfer, feeding, sterilization and the like. The soybean cake meal hydrolysate is used as a soluble nitrogen source, and accordingly the cellulase is low in cost and convenient to prepare and use. The cellulase prepared by the method is low in cost, high in activity, wide in pH application range, high in stability and short in production cycle.
Description
Technical field
The invention belongs to biochemical field, be specially a kind of preparation method of cellulase.
Background technology
For many years, the bio-transformation cellulosic materials becomes the important research field that useful products is biotechnology always, but can directly decompose under natural condition, utilize microorganism that Mierocrystalline cellulose makes carbon source seldom, especially under neutrallty condition, can decompose the mushroom of cellulose raw material, rare especially.
At present about the research of cellulase aspect and the report produced, be mostly about mushroom that can the decomposition of cellulose raw material under acidic conditions, the almost still blank out under neutrallty condition.Very good of neutral cellulase market distant view.China is a weaving big country, needs more than 50,000 tons of textile fiber element enzymes every year, and neutral cellulase is mainly by import at present, needs that cost of development is reasonable, the neutral cellulase preparation method of easy handling badly.
Just produce and application facet, current domestic acidic cellulase some producer is produced, and washes with water on enzyme and occupy very large market in weaving, but acidicenzym is not to be well suited for washing.The one, relevant with its prozyme system composition, wherein effectively the enzyme component ratio is too little, and the 2nd, while under acidic conditions, washing, the antipollution situation is quite serious, and neutral cellulase has just just in time solved above-mentioned problem.Neutral enzymatic can be used under the condition of pH value 6-8.This specific pH value scope can not damaged the brute force of fabric.Tensio-active agent is the most effective in this pH value scope, thereby has saved the use of the tensio-active agent in the textile processing.Neutral cellulase sells well very much on weaving washing enzyme market at present, and price is also very high, and acid washing enzyme is difficult to and its competition, and Developing Tendency in the future certainly will be neutral enzymatic, now main by from external import.
In recent years, the successful Application of alkali cellulose enzyme on detergent industry, changed traditional decontamination method, set up a set of new decontamination mechanism, improved washing effect, and softness is arranged, increase the effects such as gorgeous, is washed agent industry and is referred to as a technological revolution; It is widely used as the additive of washing composition in Japan and Denmark.Alkali cellulose enzyme can improve washing effect, and the detergency mechanism people of alkali cellulose enzyme know very early.But still there is blank in domestic production alkali cellulose enzyme aspect.
Summary of the invention
The present invention is directed to the deficiencies in the prior art, work out a kind of production technique of high active cellulase, filled up the blank of this respect.
Another object of the present invention is to provide the cellulase for preparing gained by the method.
The technical scheme that realizes above-mentioned purpose of the present invention is:
A kind of method of utilizing genus bacillus production of cellulose enzyme, comprise step: take genus bacillus as producing bacterial strain, through slant culture, shaking culture, fermentation culture, obtain, wherein the substratum of fermentation culture comprises the water of starch 0.5-2%, soybean cake powder 1 ~ 5%, soybean cake powder hydrolyzed solution 8-12%, wheat bran 0.3 ~ 0.8%, inorganic salt 1-2%, defoamer 0-0.3% and the 70-90% of mass content.
Described genus bacillus is (Bacillus Cohn) DM-32 preferably.
A kind of genus bacillus (Bacillus Cohn) DM-32, deposit number is CGMCC No.6211.Be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, phone: (010) 64807355.Preservation date: on June 8th, 2012.
Described genus bacillus is that the contriver obtains by mutagenesis from separation screening soil.This bacterial strain has following characteristic: the DM-32 bacterial strain both can be grown on yeast extract paste, glucose synthetic medium, also can on the thick substratum that wheat bran, soybean cake powder, soybean cake powder hydrolyzed solution form, grow, and its main metabolites is cellulase.Growth pH value scope is 5.5 ~ 10.0, and growth temperature range is 25 ~ 42 ℃.The DM-32 somatic cells is shaft-like, and end side becomes short or long-chain.Produce gemma, gemma ellipse or cylindricality, middle life or nearly middle giving birth to.Aerobic type, Gram-positive.DM-32 is at dull and stereotyped (the extractum carnis 5g of bacteria culture medium, peptone 10g, sodium-chlor 5g, agar 20g, distilled water 1000ml, tune pH7.8-8.2) colony characteristics on is: form is for circular, and smooth surface is glossy, the shallow oyster white of neat in edge, flat, rapidly, this identification of strains is Bacillus sp., and called after Bacillus Cohn in growth.
Wherein, the substratum of described slant culture comprises the water of starch 0.3-0.8%, peptone 0.8-1.2%, yeast powder 0.8-1.2%, inorganic salt 1.5-1.8%, Xylo-Mucine 0.4-0.6% and the 90-95% of mass content.
Wherein, the temperature of described slant culture is 35-37 ℃, and the time of cultivation is 3-4 days.
Wherein, the substratum of described shaking culture comprises the water of starch 0.4-0.6%, peptone 0.8-1.2%, yeast powder 0.8-1.2%, wheat bran 0.3-0.8%, inorganic salt 1-2% and the 90-95% of mass content.
Wherein, the temperature of described shaking culture is 35-37 ℃, and incubation time 24-48 hour, the device rotary speed of shaking culture are 120-200r/min.
Wherein, described starch is one or more in W-Gum, sweet potato starch, yam starch, tapioca (flour).
Wherein, described inorganic salt are one or more in sodium-chlor, Sodium phosphate dibasic, SODIUM PHOSPHATE, MONOBASIC, dipotassium hydrogen phosphate, sodium carbonate, sodium sulfate.
Wherein, described soybean cake powder hydrolyzed solution is according to mass ratio, the water of the calcium oxide of the soybean cake powder of 10-18% and 0.5-1.5% or calcium hydroxide, surplus is mixed, and under temperature 110-130 ℃, processes to obtain in 0.5-2 hour.
Wherein, the inoculum size of described fermentation culture is 0.1-15%, and the temperature of cultivation is 33-37 ℃, and ventilation is that 0-0.8vvm(passes into that volume of air/fermentating liquid volume/min), incubation time is 24-120 hour, and the pressure of fermentation is 0.02-0.07MPa.
The cellulase that the method that the present invention proposes makes.
The application of cellulase of the present invention in textile processing.
Beneficial effect of the present invention is:
What 1. the method for the present invention's proposition obtained is the cellulase all be suitable under pH neutrality and alkaline condition, and domestic now general what produce is all cellulase applicable under acidic conditions, neutrality and alkali cellulose enzyme obviously are better than acidic cellulase in washing enzyme application facet.
2. the bacterial classification used is genus bacillus, with domestic general employing Trichoderma is different as the cellulase production bacterial classification at present.The genus bacillus that the method is used is that fast, as to be applicable to scale operation bacterial classification is preserved, increases, bred to a kind of being easy to, and can shorten the production cycle.
3. the culture medium adopted is different with general conventional culture medium.Fiber-like raw material carbon source all to be arranged as producing the enzyme induction substrate in the substratum of general cellulase-producing.Fiber-like raw materials cost costliness, and feeding intake, the aspect such as material sterilization, scale operation used inconvenience.And the method adopts starch materials as carbon source, cost is low, and transporting, feed intake, the aspect such as sterilization all is better than the fiber-like raw material.
4. in substratum, add the soybean cake powder hydrolyzed solution as the solubility nitrogenous source, cost is low, making and easy to use, nitrogen use efficiency is high.General production of cellulose enzyme substratum is with peptone or corn steep liquor as the solubility nitrogenous source, and cost is very high.
5. the prepared cellulase cost of the method is low, vigor is high, to pH good stability applied widely, with short production cycle.
Embodiment
Following examples are used for the present invention is described, but should be used for limiting the scope of the invention.
The mensuration that the CMC enzyme is lived: substrate is used 1% Xylo-Mucine (CMC), adds the enzyme liquid of suitable extension rate, 50 ℃, 80r/min, pH4 ~ 10 time reaction 30 minutes, measures the glucose amount that enzymolysis produces.A CMC enzyme activity unit is defined as: under the standard reaction condition per hour every milliliter of enzyme liquid generate the enzyme amount of one milligram of glucose, with u/ml, mean.
In embodiment, peptone used is purchased from Tianjin Da Mao chemical reagent factory, analytical pure; Defoamer opposes for bubble, purchased from Jinan chemical industry company limited of state nation, and PPG type technical grade; Zulkovsky starch is purchased from sky, Tianjin power chemical reagent company limited, analytical pure.
Embodiment 1: the triangular flask fermentation prepares cellulase
1, slant culture: under aseptic technique, by genus bacillus (Bacillus Cohn, deposit number CGMCC No.6211) bacterial strain transfering loop (internal diameter 2.5mm, taken amount 10 microlitres, choose an articulating down together) and enter in the test tube slant substratum, cultivated 3 days for 35 ℃; In slant medium, the mass percent of each composition is: Zulkovsky starch 0.5% peptone 1.0% yeast powder 1.0% sodium-chlor 0.5% dipotassium hydrogen phosphate 0.1%, CMC(Xylo-Mucine) 0.5%, sodium carbonate 1%, agar 2.0%; All the other are water.
2, shaking culture: under aseptic condition, slant strains is chosen in the seed culture medium that two articulatings enter triangular flask with transfering loop, put on shaking table shaking culture 36 hours, 36 ℃ of culture temperature, shaking speed are 150r/min.In seed culture medium, the mass percent of each composition is: Zulkovsky starch 0.5% peptone 1.0% yeast powder 1.0% wheat bran 0.5% sodium-chlor 0.5% dipotassium hydrogen phosphate 0.1% sodium carbonate 1%; All the other are water.
3 and then be in the 500ml triangular flask of 100ml, to do strain fermentation with genus bacillus to produce neutral cellulase at liquid amount, inoculum size is 0.1% volume ratio, leavening temperature 0-72 hour is 35-37 ℃, and 72-120 hour is 33-35 ℃, and shaking speed is 180-200r/min.Wherein in fermention medium, the mass percent of each composition is: W-Gum 1% soybean cake powder 2% soybean cake powder hydrolyzed solution 10% wheat bran 0.5% sodium-chlor 0.5%, and dipotassium hydrogen phosphate 0.1% sodium carbonate 1%, all the other are water.
4, wherein, the soybean cake powder hydrolyzed solution is that soybean cake powder obtains under alkaline condition after high temperature steaming, and its component is soybean cake powder 14%, lime 1%, and water 85%, treatment condition are 120-122 ℃, 1 hour.
5, enzyme activity determination: measurement result shows, pH6.0 ~ 9.0 o'clock, and this cellulase activity is the highest, and 9.0 o'clock is 459.2u/ml, is 382.5u/ml during pH value 6.0.PH is 5.0 and 10.0 o'clock, and its vigor drops to 80% of maximum.PH is 4.0 o'clock, and vigor drops to 50% of maximum.Experimental result shows, the cellulase that the present invention prepares all has very high vigor under neutral and alkaline condition.
Embodiment 2:10L fermentor tank prepares cellulase
1, slant culture: under aseptic technique, genus bacillus (Bacillus Cohn, deposit number CGMCC No.6211) bacterial strain is chosen to an articulating with transfering loop and enter in the test tube slant substratum, cultivated 3 days for 37 ℃; In slant medium, the mass percent of each composition is: Zulkovsky starch 0.3%, peptone 0.8%, yeast powder 0.8%, sodium-chlor 0.4%, dipotassium hydrogen phosphate 0.1%, CMC0.5%, sodium carbonate 1%, agar 2.0%, all the other are water.
2, shaking culture: under aseptic condition, slant strains is chosen in the seed culture medium that two articulatings enter triangular flask with transfering loop, put on shaking table shaking culture 36 hours, 36 ℃ of culture temperature, shaking speed are 150r/min.In seed culture medium, the mass percent of each composition is: Zulkovsky starch 0.4%, peptone 1.0%, yeast powder 0.8%, wheat bran 0.5%, sodium-chlor 0.4%, dipotassium hydrogen phosphate 0.1%, sodium carbonate 0.6%, all the other are water.
3, then in being the 10L stainless steel standard fermentor tank of 6L, charge amount does strain fermentation production of cellulose enzyme with genus bacillus, inoculum size is 1.5% volume ratio, leavening temperature was 35-37 ℃ within 0-72 hour, then at 72-120 hour, be 33-35 ℃, mixing speed is 200r/min, ventilate for 0.7vvm, tank pressure is 0.05MPa.Wherein in fermention medium, the mass percent of each composition is: W-Gum 0.5%, soybean cake powder 5%, soybean cake powder hydrolyzed solution 8%, wheat bran 0.3%, sodium-chlor 0.5%, dipotassium hydrogen phosphate 0.1%, sodium carbonate 1.2%, bubble enemy 0.01%, all the other are water.The every 4 hours various indexs of sampling and measuring of fermenting.
4, wherein, the soybean cake powder hydrolyzed solution is that soybean cake powder obtains under alkaline condition after high temperature steaming, and its component is soybean cake powder 14%, lime 1%, and water 85%, treatment condition are 120 ℃, boiling 1 hour.
5, enzyme activity determination: result shows, during pH6.5, this cellulase activity is 402.7u/ml.During pH8.5, this cellulase activity is 446.2u/ml.PH6.0 ~ 9.0 o'clock, this cellulase activity is the highest.
Embodiment 3:30m
3Fermentor tank prepares cellulase
1, slant culture: under aseptic technique, genus bacillus (Bacillus Cohn, deposit number CGMCC No.6211) bacterial strain is chosen to an articulating with transfering loop and enter in the test tube slant substratum, cultivate 3-4 days for 35-37 ℃; In slant medium, the mass percent of each composition is: Zulkovsky starch 0.8%, peptone 1.2%, yeast powder 1.2%, sodium-chlor 0.6%, dipotassium hydrogen phosphate 0.2%, CMC0.6%, sodium carbonate 1.2%, agar 2.0%, all the other are water.
2, shaking culture: under aseptic condition, slant strains is chosen in the seed culture medium that two articulatings enter triangular flask with transfering loop, put on shaking table shaking culture 36 hours, 37 ℃ of culture temperature, shaking speed are 150r/min.In seed culture medium, the mass percent of each composition is: Zulkovsky starch 0.6%, peptone 1.2%, yeast powder 1.2%, wheat bran 0.8%, sodium-chlor 0.3%, dipotassium hydrogen phosphate 0.1%, sodium carbonate 0.6%, all the other are water.
3, seeding tank spreads cultivation: 3m
3Seeding tank, liquid amount are 1.8m
3, inoculum size is 0.2%, culture temperature 35-37 ℃, and ventilation is 0.5vvm, mixing speed 180r/min, culture cycle 24 hours.In substratum, the mass percent of each composition is: Zulkovsky starch 0.5% peptone 1.0% yeast powder 1.0% wheat bran 0.5% sodium-chlor 0.5% potassium primary phosphate 0.1% sodium carbonate 1%; All the other are water.
4, at liquid amount, be then 18m
330m
3In the stainless steel standard fermentor tank, with fermentation of bacillus production of cellulose enzyme, inoculum size is 10% volume ratio, leavening temperature be 0-72 hour be 36 ℃, 72-120 hour is 35 ℃, mixing speed is 150r/min, ventilates for 0.8vvm, tank pressure is 0.07MPa.Wherein in fermention medium, the mass percent of each composition is: sweet potato starch 1%, soybean cake powder 2%, soybean cake powder hydrolyzed solution 10%, wheat bran 0.5%, sodium-chlor 0.5%, dipotassium hydrogen phosphate 0.1%, sodium carbonate 1%, bubble enemy 0.01%, all the other are water.The every 4 hours various indexs of sampling and measuring of fermenting.After fermentation ends, through the concentrated cellulase finished product that obtains of press filtration.
Wherein, the soybean cake powder hydrolyzed solution is that soybean cake powder obtains under alkaline condition after high temperature steaming, and its component is soybean cake powder 14%, calcium hydroxide 1%, and water 85%, treatment condition are 122 ℃, boiling 1 hour.
5, enzyme activity determination: result shows, during pH6.5, this cellulase activity is 394.5u/ml, and during pH8.5, this cellulase activity is 436.1u/ml.PH6.0 ~ 9.0 o'clock, this cellulase activity is the highest.
Claims (5)
1. method of utilizing genus bacillus production of cellulose enzyme, by following steps, formed: take genus bacillus as producing bacterial strain, through slant culture, shaking culture, fermentation culture, obtain, wherein the substratum of fermentation culture comprises starch 0.5-1%, soybean cake powder 2-5%, soybean cake powder hydrolyzed solution 8-10%, wheat bran 0.3-0.5%, sodium-chlor 0.5%, dipotassium hydrogen phosphate 0.1%, sodium carbonate 1.0%, the bubble enemy 0.01% of mass content, and all the other are water; Described genus bacillus is (Bacillus Cohn) DM-32, and its deposit number is CGMCC No.6211;
Wherein, the temperature of described slant culture is 35-37 ℃, and the time of cultivation is 3-4 days;
Wherein, the temperature of described shaking culture is 35-37 ℃, and incubation time 24-48 hour, the device rotary speed of shaking culture are 120-200r/min;
Wherein, the inoculum size of described fermentation culture is volume ratio 0.1-15%, and the temperature of cultivation is 33-37 ℃, and ventilation is 0-0.8vvm, and incubation time is 24-120 hour, and the pressure of fermentation is 0.02-0.07MPa.
2. the method for claim 1, it is characterized in that, in the substratum of described slant culture, the mass percent of each composition is: Zulkovsky starch 0.5%, peptone 1.0%, yeast powder 1.0%, sodium-chlor 0.5%, dipotassium hydrogen phosphate 0.1%, Xylo-Mucine 0.5%, sodium carbonate 1%, agar 2.0%, and all the other are water;
Or: Zulkovsky starch 0.3%, peptone 0.8%, yeast powder 0.8%, sodium-chlor 0.4%, dipotassium hydrogen phosphate 0.1%, CMC0.5%, sodium carbonate 1%, agar 2.0%, all the other are water;
Or: Zulkovsky starch 0.8%, peptone 1.2%, yeast powder 1.2%, sodium-chlor 0.6%, dipotassium hydrogen phosphate 0.2%, CMC0.6%, sodium carbonate 1.2%, agar 2.0%, all the other are water.
3. the method for claim 1, it is characterized in that, the mass percent of each composition of substratum of described shaking culture is: Zulkovsky starch 0.5%, peptone 1.0%, yeast powder 1.0%, wheat bran 0.5%, sodium-chlor 0.5%, dipotassium hydrogen phosphate 0.1%, sodium carbonate 1%, and all the other are water;
Or: Zulkovsky starch 0.4%, peptone 1.0%, yeast powder 0.8%, wheat bran 0.5%, sodium-chlor 0.4%, dipotassium hydrogen phosphate 0.1%, sodium carbonate 0.6%, all the other are water;
Or: Zulkovsky starch 0.6%, peptone 1.2%, yeast powder 1.2%, wheat bran 0.8%, sodium-chlor 0.3%, dipotassium hydrogen phosphate 0.1%, sodium carbonate 0.6%, all the other are water.
4. described method as arbitrary as claim 1-3, is characterized in that, described starch is one or more in W-Gum, sweet potato starch, yam starch, tapioca (flour).
5. the method for claim 1, it is characterized in that, described soybean cake powder hydrolyzed solution is according to mass ratio, the water of the calcium oxide of the soybean cake powder of 10-18% and 0.5-1.5% or calcium hydroxide, surplus is mixed, and under temperature 110-130 ℃, processes to obtain in 0.5-2.0 hour.
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| CN103468602A (en) * | 2013-07-04 | 2013-12-25 | 福州大学 | Bacterial strain H1 capable of producing cellulase and enzyme production culture medium of bacterial strain H1 |
| CN105039195B (en) * | 2015-05-22 | 2018-04-10 | 中国科学院水生生物研究所 | A kind of cellulase production bacterium and its application |
| CN105802880B (en) * | 2016-04-06 | 2019-07-30 | 安徽工程大学 | One plant of Bacillus alcalophilus and its application |
| CN108547163A (en) * | 2018-04-20 | 2018-09-18 | 裴文韬 | A kind of preparation method of weaving bleeding agent |
| CN109750018A (en) * | 2019-03-11 | 2019-05-14 | 大连大学 | A method of preparing cellulase |
| CN112410266A (en) * | 2020-12-09 | 2021-02-26 | 山东隆科特酶制剂有限公司 | Strain for high-yield heat-resistant alkaline cellulase and production method thereof |
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