CN105018445A - Cellulase preparation for composite biological fungus fertilizer and preparation method thereof - Google Patents
Cellulase preparation for composite biological fungus fertilizer and preparation method thereof Download PDFInfo
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- CN105018445A CN105018445A CN201510471911.7A CN201510471911A CN105018445A CN 105018445 A CN105018445 A CN 105018445A CN 201510471911 A CN201510471911 A CN 201510471911A CN 105018445 A CN105018445 A CN 105018445A
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- 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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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
- C05F11/00—Other organic fertilisers
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- 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
- C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
- C12N9/2411—Amylases
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- 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)
- C12N9/2477—Hemicellulases not provided in a preceding group
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/58—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from fungi
- C12N9/62—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from fungi from Aspergillus
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Abstract
The invention discloses a cellulase preparation and a preparation method thereof. The cellulase preparation comprises cellulase, hemicellulase, amylase, protease and pectinase. Trichoderma koningii strains and Aspergillus niger strains are cultured respectively, drying and smashing are directly conducted on the cultured Trichoderma koningii strains and Aspergillus niger strains without extracting and adding carriers, and the cellulase preparation is prepared after mixing is conducted. The preparation method is simple, the cellulase is easy to separate, improvement of the stability of the cellulase is facilitated, the activity of the obtained cellulase is high, the usage rate of enzymes is high, the cost is low, and long-time repeated continuous reaction can be achieved.
Description
Technical field
The present invention relates to field of microbial fermentation.More specifically, a kind of cellulase preparation for composite biological fertilizer and preparation method thereof is related to.
Background technology
Agricultural crop straw is very important organic fertilizer resource.Its nutrient enriches, and source is wide, and quantity is many, can direct returning to farmland.It is the important source material of bio-bacterial manure.The annual total growing amount average out to more than 400,000,000 ton of China's Primary Crop Straw, by straw incorporation rate 30%, wheat straw returning rate 45%, corn stalk also field rate 20% calculates, the stalk that can be used as organic fertilizer every year just has more than 1.3 hundred million ton, about can provide nitrogen 660,000 tons, phosphorus element 400,000 tons, 10.6 ten thousand tons, potassium, agricultural crop straw main component is Mierocrystalline cellulose.Cellulase preparation promotes the cellulosic rapid decomposition of soil, contributes to the formation of agron and the release of carbon element nutrient, and therefore, cellulosic decomposition and utilization are of great significance increasing soil fertility and increasing crop yield.
Cellulase preparation is the general name of the class of enzymes of energy degraded cellulose, one that is made up of multiple enzyme complicated enzyme system, mainly come from fungus and bacterium. existing oneself determines that cellulase preparation contains three kinds of main ingredients: (1) Portugal is poly-wards off restriction endonuclease: also claim Cx enzyme, CMC enzyme, from the abbreviation EG of fungi, from the abbreviation Len of bacterium.It can be hydrolyzed the derivatived cellulose of dissolving, and expands and Partial digestion Mierocrystalline cellulose, but can not act on the Mierocrystalline cellulose of crystallization.Can by long chain cellulose molecular cut.Produce in a large number with the small molecules Mierocrystalline cellulose of non-reducing end.Its primary product is cellodextrin, cellobiose and procellose.(2) 1,4-BETA-D-glucancellobio-hydrolase: also claim Cl enzyme, from the abbreviation CBH of fungi, from the abbreviation Cex of bacterium.Be the important component in cellulase system, play a leading role in the degradation process of natural cellulose, it can cut from the non reducing end of cellulose chain, hydrolysis sugar former times key, cut next cellobiose molecule at every turn, generate solvable cellodextrin and cellobiose, therefore claim cellobiohydrolase.(3) the general enzyme of β-glucose: be called for short BG, the cell-oligosaccharide of its energy hydrolysis fiber disaccharides and short chain generates glucose, very fast to the hydrolysis of cellobiose and procellose, along with the increase of glucose polymerization degree, hydrolysis rate declines, and the specificity of this enzyme is poor.
Occurring in nature has a lot of microorganism can decomposition of cellulose, and the microorganism of cellulase-producing belongs to fungi mostly, and wherein studying more monoid is filamentous fungus, especially the most remarkable with aspergillus, wood ability that is mould, mould.Viride (Trichoderma viride), trichoderma pseudokiningii (Trichodermapsoudokoningi) koning trichoderma (Trichoderma koniggii), Trichodermareesei (Trichodermaressiei), aspergillus niger (Aspergillus niger) etc. in the mould monoid of wood highly actively represent bacterial classification.
At present, the domestic research about cellulase focuses mostly in solid state fermentation, and it is coarse that this fermentation process has product appearance, quality is unstable, and foreign matter content is high, and some component is usually adsorbed on solid residue, add extraction difficulty, be unfavorable for the shortcomings such as modernization streamline operration.The shortcomings such as liquid state fermentation has that enzyme component is undesirable, separation and Extraction workload is large, it is high to consume energy and investment is many.And China's liquid state fermentation cellulase is also at the experimental stage.The present invention compensate for the deficiencies in the prior art, and the preparation of cellulase preparation is after being cultivated respectively by two kinds of bacterial strains, and without extraction, no carrier added(NCA), pulverizes its culture convection drying, mix by a certain percentage.
Summary of the invention
First object of the present invention is to provide a kind of cellulase preparation.
Second object of the present invention is the preparation method providing a kind of cellulase preparation.The cellulase preparation of preparation method's gained of the present invention has given full play to the synergy between enzyme component, strengthens enzyme activity, improves the output of enzyme simultaneously; And gained cellulase stability is better, easily separates with substrate.Meanwhile, this preparation method can successive reaction repeatedly for a long time.
3rd object of the present invention is to provide the application of a kind of cellulase preparation in composite biological fertilizer.
For reaching above-mentioned first object, the present invention adopts following technical proposals:
A kind of cellulase preparation, comprises cellulase, hemicellulase, amylase, proteolytic enzyme and polygalacturonase.
For reaching above-mentioned second object, the present invention adopts following technical proposals:
A preparation method for cellulase preparation, is characterized in that, comprises the steps:
Koning trichoderma bacterial strain and black mold bacterial strain are cultivated respectively, without extraction after cultivation, no carrier added(NCA), convection drying is pulverized, and namely obtains cellulase preparation after mixing.
Preferably, in described fermenting process, PH during strain fermentation is 3.0-6.0, and more preferably, described PH is 4-5.5.
Preferably, in described fermenting process, temperature during strain fermentation is 20-60 DEG C, and more preferably, described temperature is 20-28 DEG C.
Preferably, the blending ratio of described koning trichoderma bacterial strain and Aspergillus niger strain is 40%-85%.
The selection of above-mentioned bacterial strains and the cooperation of cultivation PH and temperature, compared with replacing wherein any one proportioning, in gained cellulase preparation, the activity of enzyme is better, and yield of enzyme is higher, has the effect that good synergistic produces enzyme and improves enzymic activity.
For reaching above-mentioned 3rd object, the application of a kind of cellulase preparation in composite biological fertilizer, preferably, described cellulase preparation is used for the cellulosic fermentative degradation of soil.
Beneficial effect of the present invention is as follows:
1. preparation method of the present invention very easily by Mierocrystalline cellulose enzyme-to-substrate, product separately.
2. preparation method of the present invention can carry out batch reactions and successive reaction in a long time repeatedly.
3. preparation method of the present invention improves the stability of cellulase.
4. enzyme residual less in preparation method's reaction mixture of the present invention, simplifies purifying technique.
5. the cellulase prepared of the present invention, its enzyme activity is high, and the rate of utilization of enzyme is high, reduces costs.
6. the present invention all adopts waste as culture medium raw material, environmental protection.
Embodiment
In order to be illustrated more clearly in the present invention, below in conjunction with preferred embodiment, the present invention is described further.It will be appreciated by those skilled in the art that specifically described content is illustrative and nonrestrictive, should not limit the scope of the invention with this below.
Embodiment 1
A preparation method for cellulase preparation, comprises the steps:
1) bacterial strain obtains
Bacterial strain uses therefor of the present invention is Kang Shi wood enzyme bacterial strain and Aspergillus niger strain.These two kinds of bacterial strains are all that the purchase of safe true tumor Technology Co., Ltd. obtains from Jilin Province.
2) starting material
With rice straw, Semen Maydis powder, wheat bran, ammonium sulfate, urea etc. are main raw material(s).Require to become, without silt, without other dirts without enzyme.
3) Kang Shi wood enzyme fermentation process
Main agents: DNS reagent; L% acetic acid one sodium acetate buffer solution; L% carboxymethylcellulose sodium solution.Substratum: peeled potatoes 200g, sucrose 20g, agar 15g, water 1000ml, 121 DEG C of high pressure steam sterilization 30min.Solid fermentation substratum: straw, wheat bran, urea; Changing solid medium initial pH is 5.5, in room temperature (under the inconstant condition of temperature and humidity), cultivates 72 hours.This after measured, time produce enzyme activity maximum, reach 330U/g.
4) black mold fermenting process
Substratum: sodium cellulose glycolate (CMC); Yeast extract paste; (NH
4)
2sO
4; Agar; PH=5.5.Seed culture medium: rice straw powder; Testa Tritici; (NH
4)
2sO
4; Semen Maydis powder; CaCl
2; PH=5.5.Fermention medium: rice straw powder; Testa Tritici; Soya bean; Cake powder; Corn steep liquor; KH
2pO
4; CaCl
2; PH=5.5.Fermentation raw material: rice straw: pulverized 60 mesh sieves; Wheat bran: cross 60 mesh sieves; Semen Maydis powder: cross 60 mesh sieves; (NH
4)
2sO
4; CaCl
2; KH
2pO
4deng being analytical pure.30 DEG C of cultivations, enzymic activity reaches 198U/mL.
5) composite biological fertilizer zymin obtains
After koning trichoderma bacterial strain and Aspergillus niger strain are cultivated respectively, without extraction, no carrier added(NCA), pulverizes its culture convection drying, is mixed and made into cellulase preparation according to the ratio of 40:100.Cellulase preparation institute cellulase, hemicellulase, amylase, proteolytic enzyme and polygalacturonase.For the fermentative degradation that soil is cellulosic, improve organic composition in soil, strengthen soil fertility.
6) detection of filter paper enzyme activity
The standard method that filter paper enzyme activity (FPA) is recommended according to international theory and applied chemistry association (IUPAC) measures, and represents with international unit IU.The WhatlnanNo.1 filter paper one of 1 × 6cm (about 50mg) is put in test tube, add cellulase solution and 1mL citrate buffer solution (0.05M, pH4.8) that 0.5mL suitably dilutes, by abundant for filter paper bar submergence, at 50 DEG C, be incubated 30min.Dezymotize in blank test outside the prior deactivation of liquid, all the other conditions are constant.It is 20% that reaction terminates rear DNS method mensuration reducing sugar percentage composition.
Embodiment 2
Repeat embodiment 1, difference is, by step 3) in the PH of solid fermentation substratum be changed to 3.0, culture temperature is changed to 20 DEG C.
Embodiment 3
Repeat embodiment 1, difference is, by step 3) in the PH of solid fermentation substratum be changed to 6.0, culture temperature is changed to 60 DEG C.
Embodiment 4
Repeat embodiment 2, difference is, by step 3) in the PH of solid fermentation substratum be changed to 4.0, culture temperature is changed to 28 DEG C.
Embodiment 5
Repeat embodiment 4, difference is, by step 3) in the PH of solid fermentation substratum be changed to 5.5.
Embodiment 6
Repeat embodiment 1, difference is, by step 4) in the PH of substratum, seed culture medium and fermention medium be changed to 3.0, culture temperature is changed to 20 DEG C.
Embodiment 7
Repeat embodiment 1, difference is, by step 4) in the PH of substratum, seed culture medium and fermention medium be changed to 6.0, culture temperature is changed to 60 DEG C.
Embodiment 8
Repeat embodiment 2, difference is, by step 4) in the PH of substratum, seed culture medium and fermention medium be changed to 4.0, culture temperature is changed to 28 DEG C.
Embodiment 9
Repeat embodiment 4, difference is, by step 4) in the PH of substratum, seed culture medium and fermention medium be changed to 5.5.
Obviously; the above embodiment of the present invention is only for example of the present invention is clearly described; and be not the restriction to embodiments of the present invention; for those of ordinary skill in the field; can also make other changes in different forms on the basis of the above description; here cannot give exhaustive to all embodiments, every belong to technical scheme of the present invention the apparent change of extending out or variation be still in the row of protection scope of the present invention.
Claims (6)
1. a cellulase preparation, is characterized in that, comprises cellulase, hemicellulase, amylase, proteolytic enzyme and polygalacturonase.
2. the preparation method of a kind of cellulase preparation according to claim 1, is characterized in that, comprise the steps:
By koning trichoderma bacterial strain and black mold bacterial strain fermentation culture respectively, without extraction after cultivation, no carrier added(NCA), convection drying is pulverized, and namely obtains cellulase preparation after mixing.
3. the preparation method of a kind of cellulase preparation according to claim 2, is characterized in that, in described fermenting process, PH during strain fermentation is 3.0-6.0, and preferably, described PH is 4-5.5.
4. the preparation method of a kind of cellulase preparation according to claim 2, is characterized in that, in described fermenting process, temperature during strain fermentation is 20-60 DEG C, and preferably, described temperature is 20-28 DEG C.
5. the preparation method of a kind of cellulase preparation according to claim 2, is characterized in that, the blending ratio of described koning trichoderma bacterial strain and Aspergillus niger strain is 40%-85%.
6. the application of a kind of cellulase preparation in composite biological fertilizer according to claim 1-5, is characterized in that, described cellulase preparation is used for the cellulosic fermentative degradation of soil.
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Citations (8)
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| CN101786911A (en) * | 2010-02-04 | 2010-07-28 | 北京微邦生物工程有限公司 | Microorganism and enzyme powder composite biological agent for microbial organic fertilizer |
| CN102018146A (en) * | 2010-11-22 | 2011-04-20 | 李�权 | Straw fermenting agent for raising pigs |
| CN103060206A (en) * | 2013-01-10 | 2013-04-24 | 浙江大学 | Fermentation bacteria agent and preparation method and application thereof |
| CN103374551A (en) * | 2012-04-17 | 2013-10-30 | 安琪酵母股份有限公司 | Compound enzyme for molasses fermentation and preparation method and application of compound enzyme |
| CN104232536A (en) * | 2014-09-03 | 2014-12-24 | 稼禾生物股份有限公司 | Preparation method of fermentation bacterium agent |
| CN104403971A (en) * | 2014-11-28 | 2015-03-11 | 蒋小春 | Straw decomposing agent and preparation method thereof |
| WO2015035029A1 (en) * | 2013-09-04 | 2015-03-12 | Novozymes A/S | Processes for increasing enzymatic hydrolysis of cellulosic material |
-
2015
- 2015-08-05 CN CN201510471911.7A patent/CN105018445A/en active Pending
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1522591A (en) * | 2003-09-11 | 2004-08-25 | 北京中科群星科技有限责任公司 | Microorganism compolex enzyme environmental protection feed promoting agent and its preparation method |
| CN101786911A (en) * | 2010-02-04 | 2010-07-28 | 北京微邦生物工程有限公司 | Microorganism and enzyme powder composite biological agent for microbial organic fertilizer |
| CN102018146A (en) * | 2010-11-22 | 2011-04-20 | 李�权 | Straw fermenting agent for raising pigs |
| CN103374551A (en) * | 2012-04-17 | 2013-10-30 | 安琪酵母股份有限公司 | Compound enzyme for molasses fermentation and preparation method and application of compound enzyme |
| CN103060206A (en) * | 2013-01-10 | 2013-04-24 | 浙江大学 | Fermentation bacteria agent and preparation method and application thereof |
| WO2015035029A1 (en) * | 2013-09-04 | 2015-03-12 | Novozymes A/S | Processes for increasing enzymatic hydrolysis of cellulosic material |
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| CN104403971A (en) * | 2014-11-28 | 2015-03-11 | 蒋小春 | Straw decomposing agent and preparation method thereof |
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Application publication date: 20151104 |