CN101845423B - Method for preparing beta-glucanase and special bacterial strain thereof - Google Patents
Method for preparing beta-glucanase and special bacterial strain thereof Download PDFInfo
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Abstract
The invention discloses a method for preparing beta-glucanase and a special bacterial strain thereof. The bacterial strain is Bacillus subtilis TT-68, and the preservation number is CGMCC No.3340. Experiments prove that the bacterial strain has higher capability of producing the beta-glucanase. Through bottle shaking fermentation culture of the method, the enzyme production level is 430 to 3,000U/mL; and through 30h amplification culture of a 5L fermentation tank, the enzyme activity can reach 2,500 to 5,000U/ml. Therefore, the method for producing the beta-glucanase has the advantages of high level, short fermentation period, low production cost and good industrialized application prospect.
Description
Technical field
The present invention relates to a kind of method and special strain therefore thereof for preparing beta-glucanase.
Background technology
VISOSE be by glucose through the base polymer that the different sugar glycosidic bond couples together, be the abundantest glycan of occurring in nature content.VISOSE is with the tool physiologically active of beta-glucan.Wherein by β-1,3-and β-1, and the straight chain glycan that 4-mixing sugar glycosidic bond couples together is β-1,3-1,4-VISOSE.Most of cereal all contains β-1,3-1, and the 4-VISOSE, wherein the content in the barley is up to 5-8%, mainly is present in the barley endosperm cell walls.
Inscribe β-1,3-1,4-D-LSD (endo-β-1,3-1,4-D-glucanase; EC 3.2.1.73) be called for short beta-glucanase (β-glucanase) is a kind of lytic enzyme, the β-1 in can hydrolysis beta-glucan molecule, 3-and β-1, the 4-glycosidic link makes it to be degraded to small molecules.Beta-glucanase has two kinds of main sources: mikrobe and plant.Microbe-derived maximum be bacillus, comprise subtilis, bacillus amyloliquefaciens, soak numb genus bacillus, bacillus polymyxa, Bacillus licheniformis and bacillus pumilus etc.The majority in non-bacillus source is rumen microorganism and some fungies.The plant origin of beta-glucanase is mainly cereal cash crop such as barley, wheat, rye and Chinese sorghum.Beta-glucanase all has important effect aspect the nutrition of squeezing output, bakery product and animal-feed that improves fruit juice, beer, oil.Microbe-derived beta-glucanase has active high, distinguishing features such as cost is low, steady sources, extraction convenience, has broad application prospects.
The research of beta-glucanase starts from nineteen sixties, to 70, the eighties has been widely used in brewing industry, is used for shortening wheat juice and beer filtration time.After the nineties both at home and abroad especially country such as North America, Europe beta-glucanase is added in the feed as a kind of new feed enzyme.The subject matter that exists in the beta-glucanase research is that the thermostability of enzyme is relatively poor, and the enzymatic production level is on the low side.
Summary of the invention
An object of the present invention is to provide a kind of method for preparing beta-glucanase.
The method for preparing beta-glucanase provided by the present invention, the subtilis that comprises the steps: to ferment (Bacillus subtilis) TT-68 CGMCC No.3340 obtains beta-glucanase.
In the aforesaid method, the composition of the substratum that said fermentation is used is following: be made up of carbon source, nitrogenous source, inorganic salt and water;
Said carbon source is at least a in the following material: bagasse, wheat bran, straw, corn cob, corn stalk, oatmeal, barley meal, Chinese liquor distiller grains, beer vinasse;
Said nitrogenous source is at least a in the following material: the mixture of yeast extract and Tryptones, yeast extract, Tryptones, soy peptone, beef peptone, casein, bean cake powder and urea;
Said inorganic salt are at least a in the following material: KH
2PO
4, MgSO
4.7H
2O and CaCl
2
In the aforesaid method, said substratum is following 1) or 2) shown in:
1) said nitrogenous source is yeast extract and Tryptones; Said carbon source is at least a in the following material: bagasse, wheat bran, straw, corn cob, corn stalk, oatmeal, barley meal, Chinese liquor distiller grains, beer vinasse; Said inorganic salt are KH
2PO
4, MgSO
4.7H
2O and CaCl
2
2) said nitrogenous source is at least a in the following material: yeast extract and Tryptones etc. quality than mixture, yeast extract, Tryptones, soy peptone, beef peptone, casein, bean cake powder, urea; Said carbon source is an oatmeal; Said inorganic salt are KH
2PO
4, MgSO
4.7H
2O and CaCl
2
In the aforesaid method; The concentration of said carbon source in said substratum is 5g/L-50g/L or 15g/L-25g/L or 15g/L-40g/L or 15g/L-35g/L, is specially 5g/L, 10g/L, 15g/L, 20g/L, 25g/L, 30g/L, 35g/L, 40g/L, 45g/L or 50g/L; The concentration of said nitrogenous source in said substratum is 5g/L-30g/L, is specially 5g/L, 10g/L or 30g/L;
Said KH
2PO
4Concentration in substratum is 3g/L-7g/L, is specially 3g/L, 5g/L or 7g/L; Said MgSO
4.7H
2The concentration of O in substratum is 0.2g/L-0.4g/L, is specially 0.2g/L, 0.3g/L or 0.4g/L; Said CaCl
2Concentration in substratum is 0.2g/L-0.4g/L, is specially 0.2g/L, 0.3g/L or 0.4g/L.
In the aforesaid method, said 1) substratum is shown in following I or the II shown in:
I, said nitrogenous source are yeast extract and Tryptones; Said carbon source is at least a in the following material: bagasse, wheat bran, straw, corn cob, corn stalk, oatmeal, barley meal, Chinese liquor distiller grains, beer vinasse; Said inorganic salt are KH
2PO
4, MgSO
4.7H
2O and CaCl
2
The concentration of said yeast extract in substratum is 4-6g/L, is specially 4g/L, 5g/L or 6g/L; The concentration of said Tryptones in substratum is 4-6g/L, is specially 4g/L, 5g/L or 6g/L; The concentration of said carbon source in substratum is 9g/-11g/L, is specially 9g/L, 10g/L or 11g/L; Said KH
2PO
4Concentration in substratum is 3g/L-7g/L, is specially 3g/L, 5g/L or 7g/L; Said MgSO
4.7H
2The concentration of O in substratum is 0.2g/L-0.4g/L, is specially 0.2g/L, 0.3g/L or 0.4g/L; Said CaCl
2Concentration in substratum is 0.2g/L-0.4g/L, is specially 0.2g/L, 0.3g/L or 0.4g/L;
II, said nitrogenous source are yeast extract and Tryptones; Said carbon source is an oatmeal; Said inorganic salt are KH
2PO
4, MgSO
4.7H
2O and CaCl
2
The concentration of said yeast extract in substratum is 4-6g/L, is specially 4g/L, 5g/L or 6g/L; The concentration of said Tryptones in substratum is 4-6g/L, is specially 4g/L, 5g/L or 6g/L; The concentration of said oatmeal in substratum is 5g/L-50g/L, is specially 5g/L, 10g/L, 15g/L, 20g/L, 25g/L, 30g/L, 35g/L, 40g/L, 45g/L, 50g/L; Said KH
2PO
4Concentration in substratum is 3g/L-7g/L, is specially 3g/L, 5g/L or 7g/L; Said MgSO
4.7H
2The concentration of O in substratum is 0.2g/L-0.4g/L, is specially 0.2g/L, 0.3g/L or 0.4g/L; Said CaCl
2Concentration in substratum is 0.2g/L-0.4g/L, is specially 0.2g/L, 0.3g/L or 0.4g/L;
In the aforesaid method, said 2) substratum is shown in following III, IV, V, VI or the VII shown in:
III, said nitrogenous source are at least a in the following material: yeast extract and Tryptones etc. quality than mixture, yeast extract, Tryptones, soy peptone, beef peptone, casein, bean cake powder, urea; Said carbon source is an oatmeal; Said inorganic salt are KH
2PO
4, MgSO
4.7H
2O and CaCl
2
The concentration of said oatmeal in substratum is 15g/L-25g/L, is specially 15g/L, 20g/L or 25g/L; The concentration of said nitrogenous source in substratum is 9g/L-11g/L, is specially 9g/L, 10g/L or 11g/L; Said KH
2PO
4Concentration in substratum is 3g/L-7g/L, is specially 3g/L, 5g/L or 7g/L; Said MgSO
4.7H
2The concentration of O in substratum is 0.2g/L-0.4g/L, is specially 0.2g/L, 0.3g/L or 0.4g/L; Said CaCl
2Concentration in substratum is 0.2g/L-0.4g/L, is specially 0.2g/L, 0.3g/L or 0.4g/L;
IV, said nitrogenous source are soy peptone; Said carbon source is an oatmeal; Said inorganic salt are KH
2PO
4, MgSO
4.7H
2O and CaCl
2
The concentration of said oatmeal in substratum is 15g/L-25g/L, is specially 15g/L, 20g/L or 25g/L; The concentration of said soy peptone in substratum is 5g/L-15g/L, is specially 5g/L, 10g/L or 15g/L; Said KH
2PO
4Concentration in substratum is 3g/L-7g/L, is specially 3g/L, 5g/L or 7g/L; Said MgSO
4.7H
2The concentration of O in substratum is 0.2g/L-0.4g/L, is specially 0.2g/L, 0.3g/L or 0.4g/L; Said CaCl
2Concentration in substratum is 0.2g/L-0.4g/L, is specially 0.2g/L, 0.3g/L or 0.4g/L;
The pH of substratum is 4-8, is specially 4,4.5,5,5.5,6,6.5,7,7.5 or 8;
V, said nitrogenous source are soy peptone; Said carbon source is an oatmeal; Said inorganic salt are KH
2PO
4, MgSO
4.7H
2O and CaCl
2
The concentration of said oatmeal in substratum is 20g/L; The concentration of said soy peptone in substratum is 10g/L; Said KH
2PO
4Concentration in substratum is 5g/L; Said MgSO
4.7H
2The concentration of O in substratum is 0.3g/L; Said CaCl
2Concentration in substratum is 0.3g/L;
The pH of substratum is 5;
VI, said nitrogenous source are soy peptone; Said carbon source is an oatmeal; Said inorganic salt are KH
2PO
4, MgSO
4.7H
2O and CaCl
2
The concentration of said oatmeal in substratum is 20g/L; The concentration of said soy peptone in substratum is 20g/L; Said KH
2PO
4Concentration in substratum is 5g/L; Said MgSO
4.7H
2The concentration of O in substratum is 0.3g/L; Said CaCl
2Concentration in substratum is 0.3g/L; The pH of substratum is 5;
VII, said nitrogenous source are soy peptone; Said carbon source is an oatmeal; Said inorganic salt are KH
2PO
4, MgSO
4.7H
2O and CaCl
2
The concentration of said oatmeal in substratum is 15g/L-25g/L, is specially 15g/L, 20g/L or 25g/L; The concentration of said soy peptone in substratum is 5g/L-15g/L, is specially 5g/L, 10g/L or 15g/L; Said KH
2PO
4Concentration in substratum is 3g/L-7g/L, is specially 3g/L, 5g/L or 7g/L; Said MgSO
4.7H
2The concentration of O in substratum is 0.2g/L-0.4g/L, is specially 0.2g/L, 0.3g/L or 0.4g/L; Said CaCl
2Concentration in substratum is 0.2g/L-0.4g/L, is specially 0.2g/L, 0.3g/L or 0.4g/L;
The pH of substratum is 4.5-5.5, is specially 4.5,5 or 5.5.
In the aforesaid method, the condition of said fermentation comprises: temperature is 40 ℃-55 ℃, is specially 40 ℃, 50 ℃ or 55 ℃.
In the aforesaid method, the condition of said fermentation comprises: air flow is that the pressure in 1vvm-2vvm and the fermenting container is 0.4-0.8MPa; Said air flow is specially 1vvm, 1.2vvm, 1.5vvm or 2vvm.
Air flow be the volume of air through the unit volume substratum in the PM (V/Vmin, vvm).
In the aforesaid method, the condition of said fermentation comprises: inoculum size is 1%-3% (volume percent), is specially 1%, 2% or 3%.
During inoculation, use is in the bacterium of logarithmic growth middle and later periods to be inoculated, and this moment, the OD600 value of seed culture fluid was 10.5.
In the aforesaid method, said fermentation comprises following 1) or 2) said condition: 1) with the speed oscillation of 200rpm, rotation radius is 12mm; 2) speed with 300rpm-600rpm stirs, and rotation radius is 100mm, and the speed of stirring is specially 300rpm, 450rpm, 500rpm or 600rpm.
Subtilis (Bacillus subtilis) TT-68, deposit number is that CGMCC No.3340 also belongs to protection scope of the present invention.
Experiment showed, that bacterial strain of the present invention has the ability of very high product beta-glucanase.Cultivate through shake flask fermentation with the inventive method, the product enzyme level is 430-3000U/mL; Carry out 5L fermentor tank enlarged culturing 30h, enzyme work can reach 2500-5000U/ml.(oatmeal, the soy peptone) in the present invention source is extensive, and low price has reduced production cost.Therefore, the level that the inventive method is produced beta-glucanase is high, and fermentation period is short, and production cost is low, has good industrialized application prospect.
Description of drawings
Fig. 1 is different carbon sources are produced beta-glucanase to subtilis TT-68 shake flask fermentation influence.
Fig. 2 is oatmeal concentration is produced beta-glucanase to subtilis TT-68 shake flask fermentation influence.
Fig. 3 is different nitrogen sources is produced beta-glucanase to subtilis TT-68 shake flask fermentation influence.
Fig. 4 is initial pH produces beta-glucanase to subtilis TT-68 shake flask fermentation influence.
Fig. 5 is incubation time produces beta-glucanase to subtilis TT-68 shake flask fermentation influence.
Fig. 6 is incubation time produces beta-glucanase in the 5L fermentor tank to subtilis TT-68 influence.
Embodiment
Employed experimental technique is ordinary method like no specified otherwise among the following embodiment.
Used material, reagent etc. like no specified otherwise, all can obtain from commercial sources among the following embodiment.
Main raw material and reagent: the barley beta-glucan is available from SIGMA company; Yeast extract, Tryptones are available from Britain OXOID company; Agar, soy peptone, beef peptone, casein, bean cake powder are available from Beijing Kang Mingwei substratum technology Ltd; KH
2PO
4, MgSO
4.7H
2O, CaCl
2, NaOH, phenol, Na
2SO
3, CuSO
4.5H
2O, soluble tartrate, Na
2CO
3, Sodium desoxycholate, trichoroacetic acid(TCA) analytical pure, available from the Beijing Chemical Plant; 3, the 5-dinitrosalicylic acid is available from Shanghai sail preparation far away factory; Vegetable fibre materials such as Chinese liquor distiller grains, corn cob, bagasse, wheat stalk sieve after crushed, and it is subsequent use to get 0.18-0.3mm.
Separation of embodiment 1, bacterium and evaluation
One, the separation of bacterium
Subtilis TT-68 provided by the invention screens from the soil rotted leaf and obtains.
Isolation medium is configured according to following method: oatmeal (0.18-0.3mm) 10g, yeast extract 5g, Tryptones 5g, KH
2PO
45g, MgSO
4.7H
2O 0.3g, CaCl
20.3g agar 20g adds water to 1L, natural pH.
Primary dcreening operation substratum: oatmeal (0.18-0.3mm) 10g, yeast extract 5g, Tryptones 5g, KH
2PO
45g, MgSO
4.7H
2O 0.3g, CaCl
20.3g, Congo red 0.05g, agar 20g adds water to 1L, natural pH.
Fermention medium: oatmeal (0.18-0.3mm) 10g, yeast extract 5g, Tryptones 5g, KH
2PO
45g, MgSO
4.7H
2O 0.3g, CaCl
20.3g, add water to 1L, natural pH.
Coat in the isolation medium after the fresh soil sample dilution, in 50 ℃ of constant incubators, cultivate.Treat to have in the isolation medium bacterium colony to grow, in the primary dcreening operation substratum, allly can make Congo red fading form the bacterial strain of transparent circle inoculation, be inoculated in the fermention medium in periphery of bacterial colonies, 50 ℃ of culture condition, 200rpm cultivates 36h.Get the 1mL fermented liquid after the fermentation ends in the 1.5ml centrifuge tube, centrifugal (12000rpm 10min), gets supernatant, measures the beta-glucanase enzyme and lives.Choose enzyme higher bacterial strain TT-68 alive and carry out follow-up study as starting strain.
Two, the evaluation of bacterium
Bacterial characteristics is: bacterium colony is light yellow, circle or subcircular, and the edge is more neat, central uplift, dry fold; Opaque, chromogenesis does not have obvious gemma smell, colony diameter 5mm; Microscope inspection is a rod-short, long 3.5-5 μ m, wide 0.6-0.8 μ m, terminal spore.
Physiological and biochemical property is: Gram-positive, aerobic, chemoheterotrophic bacteria, and V-P reaction, catalase, glucose fermentation, nitrate reduction are positive, and clark and Lubsreaction is negative, its growth ability hydrolyzed starch, gelatin.
Comprehensive above characteristic proves that the bacterial strain TT-68 that the present invention obtains is a subtilis.Bacterial strain of the present invention is subtilis (Bacillus subtilis) TT-68; This bacterial strain is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on October 15th, 2009 and (is called for short CGMCC; Address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City; Institute of Microorganism, Academia Sinica, postcode 100101), preserving number is CGMCC No.3340.
One, the screening of carbon source in the fermention medium
1, actication of culture
The t bacteria T-68 of preservation is carried out activation on dull and stereotyped activation medium.
Activation medium is configured according to following method: oatmeal (0.18-0.3mm) 10g, yeast extract 5g, Tryptones 5g, KH
2PO
45g, MgSO
4.7H
2O 0.3g, CaCl
20.3g agar 20g adds water to 1L, natural pH.
2, seed liquor preparation
With the fresh inoculation after the activation in seed culture medium, on 50 ℃ of reciprocating type shaking tables of constant temperature air bath with 200r/min shaking culture (rotation radius is 12mm), to the OD600 value of culture system be 10.5 (they being that bacterium is in the logarithmic growth middle and later periods).
Seed culture medium is prepared according to following method: oatmeal (0.18-0.3mm) 10g, yeast extract 5g, Tryptones 5g, KH
2PO
45g, MgSO
4.7H
2O 0.3g, CaCl
20.3g, add water to 1L, natural pH.
3, shake bottle liquid fermenting and produce beta-glucanase
The 50mL fermention medium of in the 250mL triangular flask, packing into, inoculum size 2% (volume percent), on 50 ℃ of reciprocating type shaking tables of constant temperature air bath with 200r/min shaking culture 36h.Get the centrifugal 10min of fermented liquid 12000r/min, draw supernatant and promptly get crude enzyme liquid, carry out the beta-glucanase enzyme activity determination.
Fermention medium is prepared according to following method: carbon source (0.18-0.3mm) 10g, yeast extract 5g, Tryptones 5g, KH
2PO
45g, MgSO
4.7H
2O 0.3g, CaCl
20.3g, add water to 1L, natural pH.The experimental basis carbon source kind is different, establishes 9 kinds of substratum, and its carbon source is respectively bagasse, wheat bran, straw, corn cob, corn stalk, oatmeal, barley meal, Chinese liquor distiller grains, beer vinasse.
Straw, corn stalk, wheat bran, corn cob are all available from Xinxiang, Henan; Bagasse is available from Nanhua, Longzhou; Straw is available from Liaoning Panjin; The beer vinasse are available from factory of Yanjing Brewery; Chinese liquor distiller grains is available from Rui Jin, Shenyang spring brewery; Oatmeal, barley meal are to use oat and the barley pulverizing sent out available from supermarket, Beijing to make.
The enzyme activity determination method: (3,5-dinitrosalicylic acid and reducing sugar solution are reduced into henna aminocompound after the heat altogether, and within the specific limits, the shade of the amount of reducing sugar and red-brown thing is the certain proportion relation to adopt the DNS method.Under the 540nm wavelength, measure the absorbance of red-brown material, just can obtain the content of reducing sugar in the sample).Reference literature (Miller, G.L.1959.Use of dinitrosalicylic acid reagent for determination ofreducing sugars [J] .Analytical Chemistry.1959,31:426-428.) described in.Concrete grammar is following: get the enzyme liquid of 50 μ L dilution, join in 150 μ L 1% (50mM, the preparation of pH 6.0 citrate buffer solutions) the barley substrate; Adding 200 μ L DNS reagent behind 60 ℃ of water-bath 10min (1%3,5-dinitrosalicylic acid, 1%NaOH; 0.2% phenol; Use the zero(ppm) water preparation as storage liquid, add 0.05% sodium sulphite anhydrous 99.3 before using), add the saturated potassium sodium tartrate solution of 200 μ L after boiling 15min; The 540nm absorbance is measured in cooling back, with glucose as standard.Under these conditions, to generate the needed enzyme amount of 1 μ mol glucose be 1 unit of enzyme activity (U) to PM.
The experiment triplicate, carbon source is as shown in Figure 1 to the influence of TT-68 product 1,4 beta-glucanase activity, for carbon source substratum product enzyme is the highest, can reach the 430U/ml crude enzyme liquid with the oatmeal.
The detection method of protein contnt: reference literature (Lowry; O.H., Rosebrough, N.J.; Farr; A.L.andRandall, R.J.1951.Protein measurement with the folin phenol reagent [J] .Journal ofBiological Chemistry.193,265-275.) described in.(phospho-molybdic acid and phospho-wolframic acid are under alkaline condition, and the tyrosine reduction that is prone to contained in the protein for phenolic group produces navy blue mixture to adopt the Lowry method.Within the specific limits, the shade of protein content and mazarine mixture is the certain proportion relation.Under the 750nm wavelength, measure the absorbance of mazarine material, just can obtain protein content in the sample).
The preparation of CTC stoste: containing 0.2%CuSO
4.5H
2The Na of slow adding isopyknic 20% in O and the 0.2% soluble tartrate mixing solutions
2CO
3Solution stirs rapidly.
In the enzyme liquid of 200 μ L dilution, add 20 μ L, 0.15% Sodium desoxycholate, leave standstill the trichoroacetic acid(TCA) that adds 20 μ L72% behind the 10min, the centrifugal 10min of 10000rpm removes supernatant.In deposition, add 200 μ L zero(ppm) water, add 200 μ L CTC solution (following solution and the mixing of CTC stoste equal-volume being obtained: zero(ppm) water, 0.8M NaOH, 10%SDS) behind the vibration dissolution precipitation and leave standstill 10min.Add 100 μ L, 20% forint phenol reagent at last, leave standstill 30min, under 750nm, measure light absorption value.
Two, the screening of carbon source concentration in the fermention medium
1, actication of culture: with consistent described in the screening of carbon source among the embodiment 2 one, in the fermention medium.
2, the preparation of seed liquor: with consistent described in the screening of carbon source among the embodiment 2 one, in the fermention medium.
3, shake bottle liquid fermenting and produce a beta-glucanase: consistent described in the screening of carbon source among method and the embodiment 2 one, in the fermention medium, difference is the fermention medium difference of said usefulness, and all the other steps are identical.
The fermention medium of this step is prepared according to following method: oatmeal 5-50g, yeast extract 5g, Tryptones 5g, KH
2PO
45g, MgSO
4.7H
2O 0.3g, CaCl
20.3g, add water to 1L, natural pH.This experimental basis carbon source concentration is different, establishes 10 kinds of substratum, and oatmeal concentration (g/L) is respectively 5,10,15,20,25,30,35,40,45,50.
Three repetitions are established in experiment.Enzyme activity determination method according in the screening of carbon source among the embodiment 2 one, in the fermention medium is carried out beta-glucanase enzyme activity determination and determining the protein quantity.Oatmeal concentration is as shown in Figure 2 to the influence that TT-68 produces 1,4 beta-glucanase activity, and it is the highest to cultivate the product enzyme when being 20g/L with oatmeal concentration, can reach the 1010U/ml crude enzyme liquid.
Three, the screening of nitrogenous source in the fermention medium
1, actication of culture: with consistent described in the screening of carbon source among the embodiment 2 one, in the fermention medium.
2, the preparation of seed liquor: with consistent described in the screening of carbon source among the embodiment 2 one, in the fermention medium.
3, shake bottle liquid fermenting and produce a beta-glucanase: consistent described in the screening of carbon source among method and the embodiment 2 one, in the fermention medium, difference is the fermention medium difference of said usefulness, and all the other steps are identical.
The fermention medium of this step is prepared according to following method: oatmeal 20g, nitrogenous source 10g, KH
2PO
45g, MgSO
4.7H
2O 0.3g, CaCl
20.3g, add water to 1L, natural pH.This experimental basis nitrogenous source kind is different; If 7 kinds of substratum; Its nitrogenous source is respectively Tryptones, yeast extract, yeast extract: Tryptones=1: 1 (being yeast extract 5g, Tryptones 5g), bean cake powder, soy peptone, beef peptone, urea.
Three repetitions are established in experiment.Enzyme activity determination method according in the screening of carbon source among the embodiment 2 one, in the fermention medium is carried out beta-glucanase enzyme activity determination and determining the protein quantity.Nitrogenous source is as shown in Figure 3 to the influence that TT-68 produces 1,4 beta-glucanase activity, cultivates with the soy peptone that to produce enzyme the highest during for nitrogenous source, can reach the 1470U/ml crude enzyme liquid.
Four, the screening of the initial pH of fermention medium
1, actication of culture: with consistent described in the screening of carbon source among the embodiment 2 one, in the fermention medium.
2, the preparation of seed liquor: with consistent described in the screening of carbon source among the embodiment 2 one, in the fermention medium.
3, shake bottle liquid fermenting and produce a beta-glucanase: consistent described in the screening of carbon source among method and the embodiment 2 one, in the fermention medium, difference is the fermention medium of said usefulness and initial pH difference thereof, and all the other steps are identical.
The fermention medium of this step is prepared according to following method: oatmeal 20g, soy peptone 10g, KH
2PO
45g, MgSO
4.7H
2O 0.3g, CaCl
20.3g, add water to 1L.The initial pH of this experimental basis is different, establishes 9 kinds of substratum, and initial pH is respectively 4,4.5,5,5.5,6,6.5,7,7.5,8.
Three repetitions are established in experiment.Enzyme activity determination method according in the screening of carbon source among the embodiment 2 one, in the fermention medium is carried out beta-glucanase enzyme activity determination and determining the protein quantity.Initial pH is as shown in Figure 4 to the influence that TT-68 produces 1,4 beta-glucanase activity, is that 5 o'clock cultivation product enzymes are the highest with initial pH, can reach the 1550U/ml crude enzyme liquid.All material notes in the container are made fermented liquid.
Five, the screening of fermented incubation time
1, actication of culture: with consistent described in the screening of carbon source among the embodiment 2 one, in the fermention medium.
2, the preparation of seed liquor: with consistent described in the screening of carbon source among the embodiment 2 one, in the fermention medium.
3, shake bottle liquid fermenting and produce a beta-glucanase: among method and the embodiment 2 four, consistent described in the screening of the initial pH of fermention medium, difference is the fermentation time difference, and all the other steps are identical.
The fermention medium of this step is prepared according to following method: oatmeal 20g, soy peptone 10g, KH
2PO
45g, MgSO
4.7H
2O 0.3g, CaCl
20.3g, adding water to 1L, initial pH is 5.This experimental basis incubation time (h) is respectively: 12,24,36,48,60,72,84,96.
Three repetitions are established in experiment.Enzyme activity determination method according in the experiment one is carried out beta-glucanase enzyme activity determination and determining the protein quantity.Incubation time is as shown in Figure 5 to the influence that TT-68 produces 1,4 beta-glucanase activity, and enzymatic production is the highest during with cultivation 72h, can reach the 3000U/ml crude enzyme liquid.During 72h, the zymoprotein of generation is maximum, is the 0.83mg/ml crude enzyme liquid.
Embodiment 3, ferment tank prepare the condition of beta-glucanase
1, actication of culture: consistent with actication of culture method in the screening of carbon source among the embodiment 2 one, in the fermention medium.
2, the preparation of seed liquor: consistent with the preparation of seed liquor in the screening of carbon source among the embodiment 2 one, in the fermention medium.
3, the fermentor tank liquid fermenting is produced beta-glucanase
In 5L fermentor tank (substratum is 2.5L), insert the seed liquor of 1% (volume percent), 50 ℃ of cultivations, air flow is 1.2vvm, and stir speed (S.S.) is 500rpm (the stirring radius is 100mm), and fermentation period is 30h, and three repetitions are established in experiment.This experimental basis incubation time (h) is respectively: 18,19,20,21,22,23,24,25,26,27,28,29,30.
Fermention medium is as follows: oatmeal 20g, soy peptone 20g, KH
2PO
45g, MgSO4.7H
2O0.3g, CaCl
20.3g, add water to 1L, pH5.
Three repetitions are established in experiment.Enzyme activity determination method according in the screening of carbon source among the embodiment 2 one, in the fermention medium is carried out beta-glucanase enzyme activity determination and determining the protein quantity.Incubation time is as shown in Figure 6 to the influence that TT-68 produces 1,4 beta-glucanase activity, and enzymatic production is the highest during with cultivation 29h, can reach the 2470U/ml crude enzyme liquid.During 28h, the zymoprotein of generation is maximum, is the 0.78mg/ml crude enzyme liquid.
1, actication of culture: identical with the actication of culture method of embodiment 2.
2, the seed liquor preparation method of the preparation of seed liquor: embodiment 2 is identical.
3, the fermentor tank liquid fermenting is produced beta-glucanase
In 5L fermentor tank (substratum is 2.5L), insert the seed liquor of 1%, 2%, 3% (volume percent); (40,50,55 ℃) are cultivated down under differing temps; Air flow is 1,1.5 and 2vvm, and stir speed (S.S.) is 300,450 and 600rpm, and the stirring radius is 100mm; Fermentation period is 30h, and three repetitions are established in experiment.The processing setting of different vaccination amount, temperature, air flow and stir speed (S.S.) is seen table 1.
Fermention medium is shown in the following I:
I, according to the preparation of following method: oatmeal 20g, soy peptone 10g, KH
2PO
45g, MgSO
4.7H
2O0.3g, CaCl
20.3g, add water to 1L, pH5.
Enzyme activity determination method according in the screening of carbon source among the embodiment 2 one, in the fermention medium is carried out beta-glucanase enzyme activity determination and determining the protein quantity.
Different condition sees Table 1 to the influence that the TT-685L ferment tank produces 1,4 beta-glucanase activity.Thermophilic subtilis TT-68 30h in the 5L fermentor tank produces 1,4 beta-glucanase activity and can reach the 2500-5000U/ml crude enzyme liquid.
Table 1 different fermentations condition is produced the influence of beta-glucanase in the 5L fermentor tank to thermophilic subtilis TT-68
Claims (10)
1. method for preparing beta-glucanase, subtilis (Bacillussubtilis) the TT-68 CGMCC No.3340 that comprises the steps: to ferment obtains beta-glucanase.
2. method according to claim 1 is characterized in that: the composition of the substratum that said fermentation is used is following: be made up of carbon source, nitrogenous source, inorganic salt and water;
Said carbon source is at least a in the following material: bagasse, wheat bran, straw, corn cob, corn stalk, oatmeal, barley meal, Chinese liquor distiller grains, beer vinasse;
Said nitrogenous source is at least a in the following material: the mixture of yeast extract and Tryptones, yeast extract, Tryptones, soy peptone, beef peptone, casein, bean cake powder and urea;
Said inorganic salt are at least a in the following material: KH
2PO
4, MgSO
4.7H
2O and CaCl
2
3. method according to claim 2 is characterized in that: said substratum is following 1) or 2) shown in:
1) said nitrogenous source is yeast extract and Tryptones; Said carbon source is at least a in the following material: bagasse, wheat bran, straw, corn cob, corn stalk, oatmeal, barley meal, Chinese liquor distiller grains, beer vinasse; Said inorganic salt are KH
2PO
4, MgSO
4.7H
2O and CaCl
2
2) said nitrogenous source is at least a in the following material: the mixture of yeast extract and Tryptones, yeast extract, Tryptones, soy peptone, beef peptone, casein, bean cake powder, urea; Said carbon source is an oatmeal; Said inorganic salt are KH
2PO
4, MgSO
4.7H
2O and CaCl
2
4. method according to claim 3 is characterized in that: the concentration of said carbon source in said substratum is 5g/L-50g/L; The concentration of said nitrogenous source in said substratum is 5g/L-30g/L;
Said KH
2PO
4Concentration in substratum is 3g/L-7g/L; Said MgSO
4.7H
2The concentration of O in substratum is 0.2g/L-0.4g/L; Said CaCl
2Concentration in substratum is 0.2g/L-0.4g/L.
5. method according to claim 4 is characterized in that:
Said 1) substratum shown in is shown in the following I:
I, said nitrogenous source are yeast extract and Tryptones; Said carbon source is at least a in the following material: bagasse, wheat bran, straw, corn cob, corn stalk, oatmeal, barley meal, Chinese liquor distiller grains, beer vinasse; Said inorganic salt are KH
2PO
4, MgSO
4.7H
2O and CaCl
2
The concentration of said yeast extract in substratum is 4-6g/L; The concentration of said Tryptones in substratum is 4-6g/L; The concentration of said carbon source in substratum is 9g/-11g/L; Said KH
2PO
4Concentration in substratum is 3g/L-7g/L; Said MgSO
4.7H
2The concentration of O in substratum is 0.2g/L-0.4g/L; Said CaCl
2Concentration in substratum is 0.2g/L-0.4g/L;
Said 2) substratum shown in is shown in the following III:
III, said nitrogenous source are at least a in the following material: the mixture of yeast extract and Tryptones, yeast extract, Tryptones, soy peptone, beef peptone, casein, bean cake powder, urea; Said carbon source is an oatmeal; Said inorganic salt are KH
2PO
4, MgSO
4.7H
2O and CaCl
2
The concentration of said oatmeal in substratum is 15g/L-25g/L; The concentration of said nitrogenous source in substratum is 9g/L-11g/L; Said KH
2PO
4Concentration in substratum is 3g/L-7g/L; Said MgSO
4.7H
2The concentration of O in substratum is 0.2g/L-0.4g/L; Said CaCl
2Concentration in substratum is 0.2g/L-0.4g/L.
6. method according to claim 5 is characterized in that:
Said 1) substratum shown in is shown in the following II:
II, said nitrogenous source are yeast extract and Tryptones; Said carbon source is an oatmeal; Said inorganic salt are KH
2PO
4, MgSO
4.7H
2O and CaCl
2
The concentration of said yeast extract in substratum is 4-6g/L; The concentration of said Tryptones in substratum is 4-6g/L; The concentration of said oatmeal in substratum is 5g/L-50g/L; Said KH
2PO
4Concentration in substratum is 3g/L-7g/L; Said MgSO
4.7H
2The concentration of O in substratum is 0.2g/L-0.4g/L; Said CaCl
2Concentration in substratum is 0.2g/L-0.4g/L;
Said 2) substratum shown in is shown in following IV, V, VI or the VII:
IV, said nitrogenous source are soy peptone; Said carbon source is an oatmeal; Said inorganic salt are KH
2PO
4, MgSO
4.7H
2O and CaCl
2
The concentration of said oatmeal in substratum is 15g/L-25g/L; The concentration of said soy peptone in substratum is 5g/L-15g/L; Said KH
2PO
4Concentration in substratum is 3g/L-7g/L; Said MgSO
4.7H
2The concentration of O in substratum is 0.2g/L-0.4g/L; Said CaCl
2Concentration in substratum is 0.2g/L-0.4g/L;
The pH of substratum is 4-8;
V, said nitrogenous source are soy peptone; Said carbon source is an oatmeal; Said inorganic salt are KH
2PO
4, MgSO
4.7H
2O and CaCl
2
The concentration of said oatmeal in substratum is 20g/L; The concentration of said soy peptone in substratum is 10g/L; Said KH
2PO
4Concentration in substratum is 5g/L; Said MgSO
4.7H
2The concentration of O in substratum is 0.3g/L; Said CaCl
2Concentration in substratum is 0.3g/L;
The pH of substratum is 5;
VI, said nitrogenous source are soy peptone; Said carbon source is an oatmeal; Said inorganic salt are KH
2PO
4, MgSO
4.7H
2O and CaCl
2
The concentration of said oatmeal in substratum is 20g/L; The concentration of said soy peptone in substratum is 20g/L; Said KH
2PO
4Concentration in substratum is 5g/L; Said MgSO
4.7H
2The concentration of O in substratum is 0.3g/L; Said CaCl
2Concentration in substratum is 0.3g/L; The pH of substratum is 5;
VII, said nitrogenous source are soy peptone; Said carbon source is an oatmeal; Said inorganic salt are KH
2PO
4, MgSO
4.7H
2O and CaCl
2
The concentration of said oatmeal in substratum is 15g/L-25g/L; The concentration of said soy peptone in substratum is 5g/L-15g/L; Said KH
2PO
4Concentration in substratum is 3g/L-7g/L; Said MgSO
4.7H
2The concentration of O in substratum is 0.2g/L-0.4g/L; Said CaCl
2Concentration in substratum is 0.2g/L-0.4g/L;
The pH of substratum is 4.5-5.5.
7. according to arbitrary described method among the claim 1-6, it is characterized in that: the condition of said fermentation comprises: temperature is 40 ℃-55 ℃.
8. method according to claim 7 is characterized in that: the condition of said fermentation comprises: air flow is that the pressure in 1vvm-2vvm and the fermenting container is 0.4-0.8MPa.
9. method according to claim 8 is characterized in that: the condition of said fermentation comprises: inoculum size is the 1%-3% volume percent.
10. method according to claim 9 is characterized in that: said fermentation comprises following 1) or 2) said condition: 1) with the speed oscillation of 200rpm, rotation radius is 12mm, and incubation time is 12 hours-96 hours; 2) speed with 300rpm-600rpm stirs, and rotation radius is 100mm, and incubation time is 18 hours-30 hours.
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| 何国庆等.枯草芽孢杆菌ZJF-1A5 β-葡聚糖酶基因的克隆、序列分析和表达.《农业生物技术学报》.2006,第14卷(第1期),全文. * |
| 胥兵.枯草芽孢杆菌C-36内切葡聚糖酶基因在巨大芽孢杆菌中的表达.《中国优秀硕士学位论文全文数据库》.2008,(第2期),全文. * |
| 黄为一等.枯草芽孢杆菌产生β-葡聚糖酶的研究：高产突变株CW-06的选育及其发酵条件的优化.《中国医药工业杂志》.1994,第25卷(第8期),全文. * |
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