CN101250511B - Method for promoting cellulolytic enzymes synthesis of trichoderma reesei by using dilute sulfuric acid treated pulp as carbon source - Google Patents
Method for promoting cellulolytic enzymes synthesis of trichoderma reesei by using dilute sulfuric acid treated pulp as carbon source Download PDFInfo
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- CN101250511B CN101250511B CN2008100234780A CN200810023478A CN101250511B CN 101250511 B CN101250511 B CN 101250511B CN 2008100234780 A CN2008100234780 A CN 2008100234780A CN 200810023478 A CN200810023478 A CN 200810023478A CN 101250511 B CN101250511 B CN 101250511B
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Abstract
The invention relates to a method for taking paper pulp as raw materials and as a carbon source which is needed for synthesizing cellulose enzyme by trichoderma reesei after the paper pulp is processed with dilute sulfuric acid. Compared with a method for taking pure cellulose as the carbon source, the filter paper enzyme activity can be increased by 40% through using the method for preparing thecellulose enzyme, and the problems that somatic cell is lack of the ability to utilize cellulose and the activity of the cellulose enzyme is not high when the paper pulp and dextrose are taken as thecarbon source to prepare culture medium are solved.
Description
One, technical field
The invention belongs to microorganism culturing technical field in the biological chemistry, particularly a kind of using dilute sulfuric acid treated pulp promotes the method for Trichodermareesei synthetic cellulose enzyme as carbon source.
Two, background technology
Prior art: cellulase (cellulase) is the general name that is used for one group of enzyme of degraded cellulose, and main ingredient is endoglucanase (endoglucanase), exoglucanase (exoglucanase) and beta-glucosidase (β-glucosidase).The cellulosic mechanism of action of cellulose degraded is: the endoglucanase plain macromole of staple fiber at random becomes the short chain Mierocrystalline cellulose, exoglucanase cuts off terminal cellobiose or the oligosaccharides of forming of short chain Mierocrystalline cellulose, and beta-glucosidase acts on cellobiose and oligosaccharides forms glucose.The bacterial classification that can be used for the production of cellulose enzyme mainly contains Trichoderma (Trichoderma), Aspergillus (Aspergillus) and Penicillium (Penicillium).Wherein studying one of more bacterial strain is Trichodermareesei (Trichoderma reesei).
In the process of Trichodermareesei production of cellulose enzyme, Mierocrystalline cellulose is a main source of carbon.Use pure cellulose as carbon source, can induce the cellulase that produces higher vigor, but cost an arm and a leg.And use cheap lignocellulose (for example paper pulp) as carbon source, the output of cellulase is lower.
Trichodermareesei utilizes carbon source production of cellulose enzyme mainly to divide three phases: one, Trichodermareesei utilizes the carbon source of easily utilizing in the substratum, and somatic cells is grown fast, does not secrete or the plain enzyme of a small amount of eccrine fiber; Two, Trichodermareesei utilizes in the substratum difficulty to utilize carbon source, somatic cells concentration kept stable, and begin the plain enzymes of a large amount of eccrine fibers; Three, carbon source in the substratum and nitrogenous source are consumed substantially, and somatic cells begins death, and intracellular enzyme discharges gradually, and cellulase is inactivation gradually.
The carbon source of producing in the enzyme substratum is formed very big to the synthetic influence of cellulase.The substratum medium staple fibre is too much plain, and microorganism cells is difficult to be utilized, and causes the enzymatic production time lengthening, produces the enzyme yield and descends; The substratum middle short fiber is too much plain, and the microorganism cells hypertrophy causes a large amount of carbon sources to be used for the fungus growth, and the enzymatic productivity deficiency.
In the process of Trichodermareesei production of cellulose enzyme, the reasonable composition of carbon source is most important.Lower, the component that more easily is utilized of the polymerization degree can promote somatic cells to grow fast in the carbon source, for utilizing the somatic cells that the polymerization degree is higher, the difficult component of utilizing provides enough concentration thereafter in a large number.Higher, the difficult component of utilizing of the polymerization degree then is to induce cellulase synthetic main body in the carbon source, has determined the height of final cellulase activity.
General lignocellulosic material (for example paper pulp) is difficult to satisfy the requirement that these two kinds of components are provided simultaneously.Often add monose such as glucose in the production promoting the somatic cells growth, but be that somatic cells that carbon source for growth gets up lacks and utilizes cellulosic ability with glucose, cause cellulase activity not high.
Up to now, do not see the method that cost is lower, effect quite or better substitutes carbon source promotion Trichodermareesei synthetic cellulose enzyme as yet.
Three, summary of the invention
Technical problem: the invention provides a kind of using dilute sulfuric acid treated pulp promotes Trichodermareesei synthetic cellulose enzyme as carbon source method.This method is carried out suitable processing to paper pulp, makes the paper cellulose mild hydrolysis to produce the cellulosic component that a part of polymerization degree is lower, more easily be utilized.
Technical solution of the present invention is: a kind of method of using paper pulp as carbon source promotion Trichodermareesei synthetic cellulose enzyme, it is characterized in that paper pulp in advance through the dilute sulphuric acid processing, and concrete treatment process is:
A. paper pulp is pulverized, added mass concentration and be 0.75～4.0% sulphuric acid soln, the pulp powder granular mass is 1g: 5～20mL with the ratio of sulphuric acid soln volume, soak at room temperature 1～5 hour, then with mixed solution at 110～130 ℃ of following hydrolysis 40～80min;
B. the pulp powder particle after water is handled dilute sulphuric acid is washed till neutrality, and suction filtration is to pulp powder pellet moisture content to 50%～about 80%.
With common paper pulp and using dilute sulfuric acid treated pulp is carbon source preparation Trichodermareesei cellulase-producing substratum, and the amount ratio of common paper pulp and using dilute sulfuric acid treated pulp is 3: 1～2.
Beneficial effect: lower, the cellulosic component that more easily is utilized of the polymerization degree in mild hydrolysis's paper pulp can promote the growth of Trichodermareesei somatic cells, and induce the ability of its synthetic cellulose enzyme simultaneously; Higher, the difficult cellulosic component that utilizes of the polymerization degree by the somatic cells utilization of enough concentration, can be induced the synthetic of cellulase in a large number.Like this, just can prepare cellulase as carbon source with the paper pulp of cheapness.Use the paper pulp after 0.75～4.0% dilute sulphuric acid is handled to produce a large amount of fiber fines, fiber shortens simultaneously, and fibrous bundle surface roughen helps microorganism and utilizes.
Compare as carbon source with pure cellulose, prepare cellulase in this way and can make filter paper enzyme activity improve 40%; Solved also that somatic cells lacks the cellulosic ability, the difficult problem that cellulase activity is not high utilized when making carbon source preparation substratum with paper pulp+glucose.
Four, description of drawings
Fig. 1 is for being the course of carbon source Trichodermareesei cellulase-producing with the commodity pure cellulose;
Fig. 2 is the paper pulp electron microscope scanning photo that is untreated;
Fig. 3 is the paper pulp electron microscope scanning photo of handling through dilute sulphuric acid;
Fig. 4 is for being carbon source with paper pulp and using dilute sulfuric acid treated pulp, the course of Trichodermareesei cellulase-producing.
Five, embodiment
Embodiment 1:
With the commodity pure cellulose is carbon source Trichodermareesei cellulase-producing
Producing the enzyme substratum is: commodity pure cellulose (delignification's Mierocrystalline cellulose, SolkcFlock200) 12g/L, glucose 1g/L, peptone 1g/L, ammonium sulfate 2.2g/L, urea 0.5g/L, calcium chloride 0.3g/L, sal epsom 0.3g/L, ferrous sulfate 0.005g/L, manganous sulfate 0.0016g/L, sulfuric acid 0.0014g/L, cobalt chloride 0.0037g/L, 2/50mL of tween 80.It is 4.5～5.0 that substratum is regulated the pH value with citrate buffer solution.
Condition of enzyme production: to producing enzyme culture medium inoculated Trichodermareesei mycelium, Trichodermareesei mycelium inoculum size accounts for 10% of culture volume.26～32 ℃ of culture temperature, rotating speed are 150～250r/min.Sampling every other day, 3000r/min is centrifugal, gets supernatant liquor and measures pH value, filter paper enzyme activity and protein concn respectively.
The mensuration of filter paper enzyme activity: the standard method of adopting international theory and applied chemistry association (IUPAC) to recommend is measured.Experiment condition is: substrate uses Whatman filter paper 50mg, adds the enzyme liquid of suitable extension rate, and at 50 ℃, 80r/min is reaction 1h down, measures the glucose amount that enzymolysis produces.The international unit of a filter paper enzyme activity (IU) is defined as the enzyme amount that per minute under the standard reaction condition generates 1 μ mol glucose amount.
The result shows that the commodity in use pure cellulose is a carbon source, and the filter paper enzyme activity maximum is 2.23IU/mL, and zymoprotein concentration is 1.1g/L, as shown in Figure 1.
Embodiment 2:
The dilute sulphuric acid of paper pulp is handled
The general goods pulp powder is broken into particle about diameter 1mm, in solid-to-liquid ratio be 1: 10 ratio to add concentration be 0.75～4.0% sulphuric acid soln, soak 1～5h, then at 110～130 ℃ of following hydrolysis 40～80min.
Paper pulp after dilute sulphuric acid is handled washes with water to neutrality, and suction filtration to water ratio is about 50～80%.
Paper pulp and using dilute sulfuric acid treated pulp are carried out electron microscope (SEM) analysis, and electron microscopic mirror device and operation steps are as follows:
Equipment: Philip quanta200 electron microscope, the HCP-2 of Hitachi critical evaporator, the E-1010 of Hitachi ion sputtering instrument.
Using dilute sulfuric acid treated pulp sample preparation steps: fixing, gradient dehydration, critical point drying, sticking platform, conductive processing.
Electron microscopic analysis shows, compares with undressed paper pulp (accompanying drawing 2), and the paper pulp (accompanying drawing 3) after dilute sulphuric acid is handled produces a large amount of fiber fines, and fiber shortens simultaneously, and fibrous bundle surface roughen helps microorganism and utilizes.
In paper pulp dilute sulphuric acid treating processes, temperature cross low or soaking time too short, fully stripping of the hemicellulose in the paper pulp, the fibrous bundle degree of fragmentation is not high; Temperature is too high or soaking time is long, and a large amount of cellulose hydrolysises become monose and oligose, is unfavorable for inducing the synthetic of cellulase.Only 110～130 ℃ of temperature, the reaction times is under the condition of 40～80min, and the hemicellulose in the paper pulp could fully be removed, and fibrous bundle fragments into more fiber fines simultaneously.
Embodiment 3:
With common paper pulp and using dilute sulfuric acid treated pulp is carbon source preparation Trichodermareesei cellulase-producing substratum.
Common paper pulp and using dilute sulfuric acid treated pulp are pressed used pure cellulose in 3: 1～2 the usage ratio alternate embodiment 1, and all the other are with embodiment 1, and condition of enzyme production is also with embodiment 1.
By accompanying drawing 4 as can be known, use paper pulp and using dilute sulfuric acid treated pulp to be carbon source, the filter paper enzyme activity maximum is 3.12IU/mL, and zymoprotein concentration is 1.34g/L.(delignification's Mierocrystalline cellulose SolkcFlock200) is a kind of cellulase synthetic carbon source of inducing preferably to the commodity pure cellulose, but costs an arm and a leg, and can only be used for laboratory study.Embodiment 1 usefulness commodity pure cellulose 12g/L is a carbon source, and the filter paper enzyme activity maximum is 2.23IU/mL.Present embodiment is a carbon source preparation Trichodermareesei cellulase-producing substratum with the mixture of cheap relatively common paper pulp and using dilute sulfuric acid treated pulp, the carbon source total amount does not increase, but the filter paper enzyme activity maximum has improved 40% than embodiment 1, significantly improved cellulase yield, simultaneously, zymoprotein concentration also increases.
Claims (2)
1. one kind promotes the method for Trichodermareesei (Trichoderma reesei) synthetic cellulose enzyme with paper pulp as carbon source, it is characterized in that paper pulp handles through dilute sulphuric acid in advance, and concrete treatment process is:
A. paper pulp is pulverized, added mass concentration and be 0.75～4.0% sulphuric acid soln, the pulp powder granular mass is 1g: 5～20mL with the ratio of sulphuric acid soln volume, soak at room temperature 1～5 hour, then with mixed solution at 110～130 ℃ of following hydrolysis 40～80min;
B. the pulp powder particle after water is handled dilute sulphuric acid is washed till neutrality, and suction filtration is to pulp powder pellet moisture content to 50%～about 80%.
2. the method that promotes Trichodermareesei synthetic cellulose enzyme with paper pulp as carbon source as claimed in claim 1, it is characterized in that the paper pulp with common paper pulp and a, the processing of b step dilute sulphuric acid in claim 1 is carbon source preparation Trichodermareesei cellulase-producing substratum, common paper pulp is 3: 1～2 with the amount ratio of the paper pulp that a in claim 1, b step dilute sulphuric acid are handled.
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| KR20170132190A (en) * | 2015-03-31 | 2017-12-01 | 질레코 인코포레이티드 | Compositions for Improved Enzyme Production |
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| CN113122522B (en) * | 2019-12-31 | 2022-05-03 | 中国石油化工股份有限公司 | Method for promoting biological fermentation to produce cellulase |
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