CN104630186A - Preparation method and application of saccharide mixed solution for induced production of cellulase - Google Patents

Preparation method and application of saccharide mixed solution for induced production of cellulase Download PDF

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CN104630186A
CN104630186A CN201510065650.9A CN201510065650A CN104630186A CN 104630186 A CN104630186 A CN 104630186A CN 201510065650 A CN201510065650 A CN 201510065650A CN 104630186 A CN104630186 A CN 104630186A
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acid
mixed solution
cellulase
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李勇昊
赵心清
白凤武
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Dalian University of Technology
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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
    • C12N9/2434—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 relates to a preparation method and application of a saccharide mixed solution for induced production of cellulase, belonging to the technical field of microbial fermentation. The preparation method comprises the following steps: reacting glucoside-containing substances with an acid solution to obtain a saccharide mixture, and fermenting by using the saccharide mixture as a Trichoderma reesei inducer to produce the cellulase. The soluble inducer is utilized to solve the problems of uncontrollable fermentation, overhigh energy consumption and overhigh production cost when the high-concentration insoluble cellulose is utilized in the cellulase production process. The cheap mixed saccharide soluble inducer is utilized to induce the production of cellulase at high level.

Description

A kind of for inducing preparation method and the application of the sugared mixed solution of production of cellulose enzyme
Technical field
The present invention relates to a kind of for inducing preparation method and the application of the sugared mixed solution of production of cellulose enzyme, belonging to technical field of microbial fermentation.
Background technology
Mierocrystalline cellulose and hemicellulose are renewable energy sources the abundantest in the world; cellulase and hemicellulase is utilized to be hydrolyzed to glucose and xylose; bioenergy and the bio-based chemical such as ethanol, butanols, Xylitol and furfural is further converted to by biological process or chemical method; for the crisis of solution future source of energy, the Sustainable development maintaining society is significant.
The cellulosic bacterium of filamentous fungus Sum decomposition can generate born of the same parents' outer fiber element enzyme, and this gives this biological hydrolysis Mierocrystalline cellulose β-Isosorbide-5-Nitrae glycosidic link thus generates the ability of cell-oligosaccharide or glucose, and these enzymes provide the ability utilizing Mierocrystalline cellulose to grow to biology.
Trichodermareesei ( trichoderma reesei) be one of the most effective cellulase production bacterium found at present, and developed the product of trichoderma reesei cellulase, these products are widely used in the fields such as bioenergy, washing composition and cloth.In addition, have also been developed the ability that Trichodermareesei produces heterologous protein.It is exercisable that to be connected to Trichodermareesei derivable cbh1after promotor, the gene of encoding target albumen can be conditioned.Wherein, cbh1promotor is the promotor that ability to express is the strongest in Trichodermareesei found up to now, but cbh1can promotor be inducible promoter, use which kind of inductor to be the key that realize gene great expression.And main secretor type cellulose enzyme gene is induction type in Trichodermareesei, such as cbh1, cbh2, eg1, eg2, eg3, xyn1, xyn2with bgl1deng, it is expressed and generates and determined by inductor, has been found that sophorose is its most strong liquid inductor, its inducibility is 2500 times of cellobiose, be 233 times of lactose, and inducibility is higher than Mierocrystalline cellulose (J. Bacteriol. 1962,83,400-408).And glucose is the inhibition that these cellulose enzyme genes are expressed, but some Trichodermareesei mutant, such as Rut-C30 past release glucose suppression, find that in Trichodermareesei, CRE1 albumen is the major protein that glucose is suppressed simultaneously, knocked out or suddenlyd change, contributing in dextrose culture-medium, make cellulase and hemicellulase genes express.
The maximum problem of commercially producing of cellulase provides suitable inductor to wood is mould.Generally adopt cellulose substances or lactose fed-batch fermentation cellulase-producing in next life at present.But utilize these two kinds of inductor cellulase-producings in next life all to have certain defect, utilize cellulose substances production of cellulose enzyme, solid matter content in liquid nutrient medium can be made too high, cause fermentor tank restive, excessive fermentor tank rotating speed consumes the energy, solid matter is not easy to feeding culture simultaneously, and these materials could use after usually needing certain pre-treatment; And lactose is expensive in some countries and regions, be not suitable for scale operation, these factors cause cellulase production high cost.
Investigator is had to confirm (Agric. Biol. Chem., 1987,51 (8), 2255-2256), steviol glycoside utilizes certain density hydrochloric acid at a certain temperature, after hatching certain hour, and separation and purification, utilize activated carbon chromatography can prepare the higher sophorose of purity, and sophorose is the most potent inducer of cellulase.But in this step, namely the abstraction and purification of sophorose takes time and effort, and turn increases production cost.In addition, glucose in hydrolyzed solution is not utilized, so this work directly utilizes glucose and sophorose mixed solution to carry out inducing cellulase gene expression, glucose can as the carbon source of microorganism growth, and sophorose can as inductor, The method avoids the cost of purified sophorose, simplify the production process of inductor, possess novelty.
By using soluble substrate and inductor (as lactose and sophorose), can overcome and the series of problems using Mierocrystalline cellulose to accompany as inductor.Sophorose is inductor more more effective than Mierocrystalline cellulose, but sophorose expensive being difficult to produces.Therefore, than solid cellulose easier control and process while, sophorose still makes the cost intensive of production of cellulose enzyme and is difficult to accept, and is therefore unpractical in order to suitability for industrialized production cellulase.Obviously, soluble substrate easily is still needed and the method for cheap cellulase induction in filamentous fungus (as Trichodermareesei).In addition, by the ability that the inductor of cheapness regulates inducible promoter to express native gene or foreign gene, there is huge commercial value.
Summary of the invention
The invention provides a kind of for inducing the preparations and applicatio of the sugared mixed solution of production of cellulose enzyme, to solve the high cost problem utilizing the plain fermentation caused of concentration cellulose difficult control, energy consumption problems of too etc. to cause in cellulase production or foreign protein production process.By the mixing sugar solubility inductor of cheapness, high-caliber production of cellulose enzyme or the albumen from different heterologous constructs.
Technical scheme of the present invention is: a kind of preparation method of the sugared mixed solution for inducing production of cellulose enzyme comprises the steps:
(1) sugary glycoside material being made into concentration is 5%-20%(w/w) aqueous solution, then add mineral acid or organic acid, make the acid concentration of mixture be 0.01 mol/L-1 mol/L; Described sugary glycoside material is steviol glycoside (Stevioside), rebaudioside A (Rebaudioside A), RB (Rebaudioside B) or dulcoside B (Rebaudioside C) or its mixture; Described mineral acid is hydrochloric acid, sulfuric acid, hypochlorous acid, nitric acid or phosphoric acid, and described organic acid is formic acid, acetic acid, propionic acid, toxilic acid, oxysuccinic acid or citric acid;
(2) at 25-250 DEG C, hatch 1 min-100 h, cooling, filtered fluid is the sugared mixed solution containing sophorose and glucose, wherein acid concentration is higher, spendable temperature of reaction is lower, when temperature of reaction reaches more than 220 DEG C, does not add any acid to be hydrolyzed stevioside.
A kind of application of the sugared mixed solution for inducing production of cellulose enzyme comprises the following steps:
(1) slant culture: the wild-type cell comprising inducible promoters or the construct comprising cellulase promoter cultivate 7 d in 3% Fructus Hordei Germinatus leaching powder solid medium at 28 DEG C, with spore under aseptic washing, for subsequent use at being put in 4 DEG C;
(2) seed culture: by the spore under washing, be inoculated in 10 g l -1corn steep liquor, 5 g l -1in dextrose culture-medium, cultivate 1 d at 28 DEG C, as seed liquor;
(3) fermentation culture: by seed liquor with 10% volume ratio be inoculated in fermention medium, in 250 ml triangular flasks, load 50 ml fermention mediums, 28 DEG C, 150 r/min condition bottom fermentations cultivate 7 d;
Described fermentative medium formula is: sugared mixed solution 10-15 g l -1, peptone 1 g l -1, urea 0.3 g l -1, (NH 4) 2sO 41.4 g l -1, KH 2pO 42 g l -1, MgSO 47H 2o 0.3 g l -1, CaCl 20.4 g l -1, FeSO 47H 2o 5 mg l -1, MnSO 4h 2o 1.7 mg l -1, ZnSO 47H 2o1.4 mg l -1, CoCl 22 mg l -1, tween 80 0.2 ml l -1, 500 ml l -10.2 M pH 5.0 Na 2hPO 4-citrate buffer solution.
The another kind of application for the sugared mixed solution of inducing production of cellulose enzyme comprises the following steps:
(1) slant culture: the wild-type cell comprising inducible promoters or the construct comprising cellulase promoter cultivate 7 d in 3% Fructus Hordei Germinatus leaching powder solid medium at 28 DEG C, with spore under aseptic washing, for subsequent use at being put in 4 DEG C;
(2) seed culture: by the spore under washing, be inoculated in 10 g l -1corn steep liquor, 5 g l -1in dextrose culture-medium, cultivate 1 d at 28 DEG C, as seed liquor;
(3) fermentation culture: being seeded to liquid amount by 10% inoculum size is in 7.5 L fermentor tanks of 3 L fermention mediums, before fermentation, 48 h are thalli growth main phase, controlling leavening temperature is 28 DEG C, pH utilizes ammoniacal liquor to control as being not less than 4, feed supplement is started after sugar consumption in fermentor tank is complete, described feed supplement material is sugar mixture, ensures that in fermentor tank, sugared concentration is 1 g l -1-2 g l -1, wherein fermentor tank rotating speed mixes as index to reach fermented liquid, and dissolved oxygen is 20% by control oxygen and air inlet ratio;
Described fermentative medium formula is: sugared mixed solution 10-15 g l -1, peptone 1 g l -1, urea 0.3 g l -1, (NH 4) 2sO 41.4 g l -1, KH 2pO 42 g l -1, MgSO 47H 2o 0.3 g l -1, CaCl 20.4 g l -1, FeSO 47H 2o 5 mg l -1, MnSO 4h 2o 1.7 mg l -1, ZnSO 47H 2o1.4 mg l -1, CoCl 22 mg l -1, tween 80 0.2 ml l -1, 500 ml l -10.2 M pH 5.0 Na 2hPO 4-citrate buffer solution.
The described wild-type cell comprising inducible promoters is the Trichodermareesei comprising sophorose inducible promoters and glucose inducible promoters; The described construct comprising cellulase promoter is comprise Trichodermareesei cbh1the bacterium of promotor, described bacterium is streptomyces, hot zygosaccharomyces, bacillus or Cellulomonas.
Have been found that now and utilize aqueous acid hydrolysis steviol glycoside, after hatching certain hour at a certain temperature, the sugar mixture of the inductor that the considerable cellulose enzyme gene of content is expressed can be produced.This sugar mixture is containing the 1-30 g l that has an appointment -1sophorose, and 20-70 g l -1glucose.An attracting advantage is, this mixture need not the complicated step such as purifying, and after only need adjusting pH, whether (concentrated optional) can the generation of cellulase induction.The discovery provides the cheap substitute of lactose or the purified sophorose industrially needed, and than the substitute more easily of the solid cellulose for producing the albumen regulated and controled by inducible promoter in filamentous fungus.Viewpoint is more specifically, and sugar mixture of the present invention can be used for the cellulase production of Trichoderma.
The technical scheme preparing sugar mixture is that steviol glycoside is hydrolyzed 1 min-100 h, for genetic expression after filtered of solid materials isosteviol under acid effect.Wherein steviol glycoside concentration is 5%-20%, and acid concentration is 0.1 mol l -1-1 mol l -1, temperature be normal temperature to 250 DEG C, incubation time is 1min to 100h, and sour kind is common mineral acid or organic acid, includes but not limited to hydrochloric acid, sulfuric acid or citric acid.
The method of fermentation production of protein matter of the present invention utilizes sugar mixture as carbon source, adopts batch fermentation, fed-batch fermentation or the inducible gene expression that continuously ferments, and produces protein.Described sugar mixture is 0.1 g l as the starting point concentration of carbon source -1-50 g l -1, be wherein preferably 5 g l -1-20 g l -1.According to fed-batch fermentation mode, can control sugared concentration in fermented liquid is 0.01 g l -1-5 g l -1, wherein preferred version is that sugared concentration is more low better.Or do not adopt the batch feeding mode controlling sugared concentration, but still carry out the fermentation mode of feed supplement using sugar mixture as carbon source.
Described bacterial strain is the bacterium of filamentous fungus or biodegradable fiber element, and wherein preferred bacterial strain is Trichodermareesei.
Wherein, initial medium composition and supplemented medium composition can be identical or different, as can according to the common practise of this area, and the component of Selective agar medium.As added the common inducible gene expression of suitable wheat bran, tween 80 or cellulose substances.
The invention has the beneficial effects as follows: the method utilizes sugary glycoside material and acid solution reaction to obtain sugar mixture, using this sugar mixture as the inductor of Trichodermareesei, fermentative production cellulase.Solve the high cost problem that fermentation difficult control, the energy consumption problems of too etc. that to utilize concentration cellulose element or the insoluble inductor of other solids to cause in cellulase production or foreign protein production process cause.By the mixing sugar solubility inductor of cheapness, high-caliber production of cellulose enzyme or the albumen from different heterologous constructs.
Accompanying drawing explanation
The chromatography of ions detected result of sophorose in Fig. 1 sugar mixing inductor.
Fig. 2 sugar inductor and other conventional inductor induce Trichodermareesei cellulase-producing ability to contrast.
Fig. 3 sugar inductor is to the induction of the trichoderma reesei cellulase of shake-flask culture.
Fig. 4 sugar inductor is to the induction of the trichoderma reesei cellulase of fermentor cultivation.
Embodiment
The following examples are used for explaining instead of restriction claimed invention.
embodiment 1
The present embodiment explains how for the preparation of the inducibility sugar mixture of induction trichoderma reesei expression cellulose enzyme gene.Table 1 is treatment temp and the different condition in treatment time under different substrate and different acid concentration condition.
Table 1 is solvable inductor preparation condition.
Different condition prepared by inductor and result as follows:
(1) by the upper table certain density steviol glycoside of configuration or the total glycosides of stevioside;
(2) pouring liquids is cooled after reacting the regular hour under certain condition by upper table mixed solution for subsequent use.Wherein solid is isosteviol.Utilize ion chromatography liquid wherein, result is (No. 5 reactions) as shown in Figure 1, have sophorose to generate; Remaining reaction all can detect sophorose according to same method;
(3) wherein No. 1, No. 6 and No. 10 reaction sophorose concentration be respectively 3.02 g l -1, 4.67 g l -1with 5.48 g l -1, illustrate that different concentration of substrate all can generate sophorose under same acids condition and reaction conditions;
(4) No. 2 to No. 7 reactions all can generate sophorose, and wherein sophorose concentration first increases rear reduction, when concentration of hydrochloric acid reaches 0.06 mol l -1time, sophorose concentration is maximum, but in No. 3 final liquid of reaction, sophorose concentration accounting example is maximum, reaches 13%, higher than 10% of No. 5 reactions; The increase along with acid concentration under lower concentration acid condition is described, sophorose and glucose concn improve along with acid concentration and improve, but when acid concentration acquires a certain degree, acid concentration improves glucose concn again and improves, but sophorose concentration reduces;
Hydrochloric acid is changed to organic acid acetic acid by (5) No. 10 reactions, and can reach the result of basic simlarity, sophorose concentration reaches 4.28 g l -1, no matter mineral acid or organic acid are described, all can be hydrolyzed stevioside is sophorose;
(6) No. 7 reactions are when acid concentration is lower or only have the aqueous solution, and can improve temperature of reaction to reach hydrolysis stevioside is the object of sophorose, temperature of reaction can be brought up to more than 200 degrees Celsius and generate sophorose;
(7) No. 8 reaction when improve acid concentration to a certain extent time, can by reduce temperature of reaction be hydrolyzed stevioside reach generation sophorose object, final sophorose concentration is 1.06 g l -1;
Steviol glycoside sterling can be changed to the total glycosides of stevioside by (8) No. 11 reactions, comprising steviol glycoside (Stevioside), rebaudioside A (Rebaudioside A), RB (Rebaudioside B) or dulcoside B (Rebaudioside C) etc., can reach similar result equally, wherein sophorose concentration is 4.52 g l -1.
embodiment 2
The sugared mixed solution batch fermentation production of cellulose enzyme how the induction performance that the following examples describe sugared inductor utilizes embodiment 1 to prepare higher than conventional cellulase induction thing lactose and pretreated maize straw.
(1) slant culture: Trichodermareesei cultivates 7 d in 3% Fructus Hordei Germinatus leaching powder solid medium at 28 DEG C, with spore under aseptic washing, for subsequent use at being put in 4 DEG C,
(2) seed culture: the spore under step 1 being washed, is inoculated in 10 g l -1corn steep liquor, 5 g l -1in dextrose culture-medium, cultivate 1 d at 28 DEG C, as seed liquor;
(3) fermentation culture: by seed liquor with 10%(volume ratio) be inoculated in fermention medium, in 250 ml triangular flasks, load 50 ml fermention mediums, 28 DEG C, 150 r/min condition bottom fermentations cultivate 7 d, fermentation level is 5.0 IU/ml.Carbon source concentration wherein for different inductor contrast experiment is 10 g l -1.The preferred carbon source concentration of batch fermentation is 15 g l -1, different carbon source comparing result as Fig. 2,15 g l -1the fermentation diagram of sugared mixed solution induction cellulase-producing as Fig. 3.
Fermentative medium formula wherein described in step (3) is:
Sugar mixed solution 15 g l -1(contrast experiment is 10 g l -1), peptone 1 g l -1, urea 0.3 g l -1, (NH 4) 2sO 41.4 g l -1, KH 2pO 42 g l -1, MgSO 47H 2o 0.3 g l -1, CaCl 20.4 g l -1, FeSO 47H 2o 5 mg l -1, MnSO 4h 2o 1.7 mg l -1, ZnSO 47H 2o1.4 mg l -1, CoCl 22 mg l -1, tween 80 0.2 ml l -1, 500 ml l -10.2 M pH 5.0 Na 2hPO 4-citrate buffer solution.
Wherein described in (3) is lactose, pretreated maize straw, cellobiose and glucose for the inductor contrasted, pretreated maize straw be 2% NaOH under 121 DEG C of conditions, process 10% maize straw after 60 minutes through washing material.
Fermentation different time sampling and measuring cellulose enzyme activity, cellulose enzyme activity uses International Standards Method, adopt 50 mg Whatman No.1 filter paper as substrate, under the optimal reactive temperature and pH condition of enzyme, 0.5 milliliter of enzyme liquid and filter paper test suitably diluted is after 1 hour, and the amount according to the reducing sugar generated calculates filter paper enzyme activity.
A filter paper enzyme activity unit of activity (IU/ milliliter) is defined as: the glucose amount (calculating with reducing sugar gauge) that 1 milliliter of enzyme liquid per minute generates 1 μm of ol is defined as 1 IU/ milliliter.
The mensuration of reducing sugar adopts DNS method, and detailed process is as follows.
DNS reagent collocation method:
1. NaOH 104g is dissolved in 1300ml distilled water;
2. 3,5-dinitrosalicylic acid 30g add mixing 1., heating for dissolving;
3. Seignette salt 910g is dissolved in 2500ml distilled water;
4. 25g re-distilled phenol, adds 25g sodium sulphite anhydrous 99.3, adds 3.;
By above each step mixing, add 1200ml distilled water, in brown reagent bottle, can use after depositing one week.
DNS using method: the DNS reagent adding 2 mL in 1.5 mL enzymes live reaction system, adds the distilled water of 9 mL, measure at OD in boiling water bath after boiling 10 min after being cooled to room temperature 540light absorption value under condition.With 1 g l -1glucose standard, according to above-mentioned same method drawing standard curve, then calculates concentration of reduced sugar according to light absorption value.
As seen from Figure 2, when all fermenting using inductor selected by unique as carbon source, the sugar mixture prepared by the present invention has the highest filter paper enzyme activity, even if when containing a large amount of glucose, enzyme work reached 3.46 IU/ml at the 5th day, more than a times of lactose 1.57 IU/ml and pretreated maize straw 1.53 IU/ml, show extremely strong inducibility; And another two kinds of liquid induction fibres disaccharides and glucose, all show lower inducibility, enzyme is lived and is respectively 0.47 IU/ml and 0.14 IU/ml.Absolutely prove that sugar mixture prepared by the present invention has very large application potential as neat liquid inductor.And the concentration of sugar mixture is brought up to 15 g l -1time, final filter paper enzyme activity reached 5 IU/ml at the 7th day, and result is as shown in Figure 3.
embodiment 3
Example below describes how to utilize sugar mixture batch feeding production of cellulose enzyme in fermentor tank, wherein slant culture and seed culture and example 2 process completely the same, and fermention medium is also completely the same with example 2 fermention medium.
Fermentation culture: being seeded to liquid amount by 10% inoculum size is in 7.5 L fermentor tanks of 3 L substratum, before fermentation, 48 h are thalli growth main phase, controlling leavening temperature is 28 DEG C, pH utilizes ammoniacal liquor to control as being not less than 4, feed supplement is started after sugar consumption in fermentor tank is complete, wherein feed supplement material is sugar mixture prepared by example 1, ensures that in fermentor tank, sugared concentration is 1 g l -1-2 g l -1, wherein fermentor tank rotating speed mixes as index to reach fermented liquid, and dissolved oxygen is 20% by control oxygen and air inlet ratio control, and fermentation results as shown in Figure 4.Ferment the 8th day, mixing sugar inductor makes cellulose enzyme activity unit reach 11.4 IU/ml, illustrates compared with shake-flask culture, controls the induction production level that feed profile and dissolved oxygen can improve cellulase protein in fermentor tank.

Claims (4)

1., for inducing a preparation method for the sugared mixed solution of production of cellulose enzyme, it is characterized in that: comprise the steps:
(1) sugary glycoside material being made into concentration is 5%-20%(w/w) aqueous solution, then add mineral acid or organic acid, make the acid concentration of mixture be 0.01 mol l -1-1 mol l -1; Described sugary glycoside material is steviol glycoside (Stevioside), rebaudioside A (Rebaudioside A), RB (Rebaudioside B) or dulcoside B (Rebaudioside C) or its mixture; Described mineral acid is hydrochloric acid, sulfuric acid, hypochlorous acid, nitric acid or phosphoric acid, and described organic acid is formic acid, acetic acid, propionic acid, toxilic acid, oxysuccinic acid or citric acid;
(2) at 25-250 DEG C, hatch 1 min-100 h, cooling, filtered fluid is the sugared mixed solution containing sophorose and glucose, wherein acid concentration is higher, spendable temperature of reaction is lower, when temperature of reaction reaches more than 220 DEG C, can not add any acid to be hydrolyzed stevioside.
2. according to claim 1 a kind of for inducing the application of the sugared mixed solution of production of cellulose enzyme, it is characterized in that, comprise the following steps:
(1) slant culture: the wild-type cell comprising inducible promoters or the construct comprising cellulase promoter cultivate 7 d in 3% Fructus Hordei Germinatus leaching powder solid medium at 28 DEG C, with spore under aseptic washing, for subsequent use at being put in 4 DEG C;
(2) seed culture: by the spore under washing, be inoculated in 10 g l -1corn steep liquor, 5 g l -1in dextrose culture-medium, cultivate 1 d at 28 DEG C, as seed liquor;
(3) fermentation culture: by seed liquor with 10% volume ratio be inoculated in fermention medium, in 250 ml triangular flasks, load 50 mL fermention mediums, at 28 DEG C, 150 r/min condition bottom fermentation 7 d;
Described fermentative medium formula is: sugared mixed solution 10-15 g l -1, peptone 1 g l -1, urea 0.3 g l -1, (NH 4) 2sO 41.4 g l -1, KH 2pO 42 g l -1, MgSO 47H 2o 0.3 g l -1, CaCl 20.4 g l -1, FeSO 47H 2o 5 mg l -1, MnSO 4h 2o 1.7 mg l -1, ZnSO 47H 2o1.4 mg l -1, CoCl 22 mg l -1, tween 80 0.2 ml l -1, 500 ml l -10.2 M pH 5.0 Na 2hPO 4-citrate buffer solution.
3. according to claim 1 a kind of for inducing the application of the sugared mixed solution of production of cellulose enzyme, it is characterized in that, comprise the following steps:
(1) slant culture: the wild-type cell comprising inducible promoters or the construct comprising cellulase promoter cultivate 7 d in 3% Fructus Hordei Germinatus leaching powder solid medium at 28 DEG C, with spore under aseptic washing, for subsequent use at being put in 4 DEG C;
(2) seed culture: by the spore under washing, be inoculated in 10 g l -1corn steep liquor, 5 g l -1in dextrose culture-medium, cultivate 1 d at 28 DEG C, as seed liquor;
(3) fermentation culture: being seeded to liquid amount by 10% inoculum size is in 7.5 L fermentor tanks of 3 L fermention mediums, before fermentation, 48 h are thalli growth main phase, controlling leavening temperature is 28 DEG C, pH utilizes ammoniacal liquor to control to be not less than 4, feed supplement is started after sugar consumption in fermentor tank is complete, described feed supplement material is sugar mixture, ensures that in fermentor tank, sugared concentration is 1 g l -1-2 g l -1, wherein fermentor tank rotating speed is to reach for the purpose of fermented liquid mixes, and dissolved oxygen is 20% by oxygen and air inlet ratio control;
Described fermentative medium formula is: sugared mixed solution 10-15 g l -1, peptone 1 g l -1, urea 0.3 g l -1, (NH 4) 2sO 41.4 g l -1, KH 2pO 42 g l -1, MgSO 47H 2o 0.3 g l -1, CaCl 20.4 g l -1, FeSO 47H 2o 5 mg l -1, MnSO 4h 2o 1.7 mg l -1, ZnSO 47H 2o1.4 mg l -1, CoCl 22 mg l -1, tween 80 0.2 ml l -1, 500 ml l -10.2 M pH 5.0 Na 2hPO 4-citrate buffer solution.
4. a kind of for inducing the application of the sugared mixed solution of production of cellulose enzyme according to Claims 2 or 3, is characterized in that: described in comprise inducible promoters wild-type cell be the Trichodermareesei comprising sophorose inducible promoters and glucose inducible promoters; The described construct comprising cellulase promoter is comprise Trichodermareesei cbh1the bacterium of promotor, described bacterium is streptomyces, hot zygosaccharomyces, bacillus or Cellulomonas.
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US10611997B2 (en) 2015-03-31 2020-04-07 Xyleco, Inc. Compositions for enhanced enzyme production
CN105420217A (en) * 2015-12-17 2016-03-23 山东龙力生物科技股份有限公司 Production method and application of high-efficient cellulase mixture
CN105925551A (en) * 2016-05-11 2016-09-07 上海交通大学 Method for efficiently producing cellulose based on preparation of mixture through glucose glucoside conversion reaction
CN110325647A (en) * 2017-03-15 2019-10-11 科莱恩国际有限公司 Method for producing protein under inductive condition
CN112313341A (en) * 2018-07-04 2021-02-02 花王株式会社 Method for producing protein
CN112175842A (en) * 2020-11-13 2021-01-05 上海汉禾生物新材料科技有限公司 Method for producing cellulase with high yield by fermenting trichoderma reesei
CN112939239A (en) * 2021-02-19 2021-06-11 杭州楠大环保科技有限公司 Compound microbial preparation and application thereof in sewage treatment
CN112939239B (en) * 2021-02-19 2022-05-24 杭州楠大环保科技有限公司 Compound microbial preparation and application thereof in sewage treatment
CN113388532A (en) * 2021-06-16 2021-09-14 华东理工大学 Recombinant trichoderma reesei for producing asparaginase and construction method and application thereof
CN114703164A (en) * 2022-03-03 2022-07-05 上海交通大学 Efficient cellulase inducer and preparation and application methods thereof

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