CN108424896A - A kind of method of mixed fungus fermentation maize straw furfural dregs production cellulase - Google Patents

A kind of method of mixed fungus fermentation maize straw furfural dregs production cellulase Download PDF

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CN108424896A
CN108424896A CN201710077685.3A CN201710077685A CN108424896A CN 108424896 A CN108424896 A CN 108424896A CN 201710077685 A CN201710077685 A CN 201710077685A CN 108424896 A CN108424896 A CN 108424896A
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mixed
furfural dregs
cellulase
maize straw
fermentation
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CN108424896B (en
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张宗超
刘会芳
刘秀梅
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Dalian Institute of Chemical Physics of CAS
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    • 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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    • 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
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    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01004—Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase
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    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01021—Beta-glucosidase (3.2.1.21)
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    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01091—Cellulose 1,4-beta-cellobiosidase (3.2.1.91)

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Abstract

The present invention provides a kind of mixed fungus fermentation maize straw furfural dregs produce cellulase method, this method using trichoderma reesei CICC13052 and CICC40360 be with maize straw furfural dregs induction carbon source culture medium in progress mixed fermentation cellulase-producing.The method of the present invention raw material is cheap and easy to get, overcomes the low disadvantage of single bacterial strain producing enzyme, improves production efficiency, and both of mixed bacterium comes under trichoderma reesei, growth condition is identical, no antagonism, is conducive to mixed culture.Operation is simple for the method for the present invention, at low cost, is expected to provide the zymotechnique of simple economy for the industrialization of Production by Microorganism Fermentation cellulase.

Description

A kind of method of mixed fungus fermentation maize straw furfural dregs production cellulase
Technical field
The invention belongs to bioenergies, and field, more particularly to a kind of mixed fungus fermentation maize straw furfural dregs to be utilized to produce fiber The method of plain enzyme.
Background technology
Cellulase is in textile industry, feed industry, brewing, food processing and taps a new source of energy etc. and to have good application Foreground.In recent years, increasingly serious with energy problem, utilize cellulose degraded stalk production fibre fuel ethyl alcohol etc. The proposition of novel energy is even more to refer in schedule, but on the high side hinder its extensive use.Therefore it produces efficiently low The cellulase of valence has epoch-making meaning for finally solving the energy deficiency that the mankind are faced.
Cellulase source is very extensive, and bacterium, fungi, can generate cellulase at actinomyces in animal body etc..It is different The cellulase of Microbe synthesis has significant difference in composition, also differs widely to the degradation capability of cellulose.Actinomyces Yield of cellulase it is extremely low, research is few.The yield of bacterium is not also high, and mainly endoglucanase, in addition, produced Enzyme be endocellular enzyme or be adsorbed on cell wall, increase purification difficulty, seldom industrially use.And filamentous fungi has producing enzyme Plurality of advantages：The cellulase of generation is ectoenzyme, convenient for extraction；Producing enzyme is efficient, and enzyme system is more reasonable etc..It is presently used for giving birth to The microorganism fungus kind of cellulase-producing is all filamentous fungi mostly, has trichoderma (Trichodema), aspergillus than more typical (Aspergillus) and Penicillium (Penicillium), wherein trichoderma reesei because its producing enzyme vigor is high, enzyme system completely, growth ring The features such as border is extensive, enzyme easily extracts and bacterial strain is safe and non-toxic as most industrial application value cellulase production bacterium.
Cellulase is a kind of multi-component complex enzyme, generally includes endo-type -1,4 beta-glucanase (Cx enzymes), circumscribed-type - 1,4 beta-glucanase (C1 enzymes) and beta-glucosidase (CB enzymes).When by native cellulolytic at glucose, it is necessary in dependence Stating the synergistic effect of 3 kinds of components could complete：Excision enzyme (cellobiohydrolase) can be with hydrocellulose crystal region, (CBH I) the continued hydrolysis since the reducing end of cellulose chain or (CBH II) non-reducing end discharges cellobiose；Restriction endonuclease is mainly made For the noncrystalline domain of cellulose, the glycosidic bond in random hydrolysis cellulose chain cuts off cellulose long-chain, transforms into a large amount of The cellulose short chain of different polymerization degree so that the degree of polymerization of cellulosic molecule reduces, for the cellulose last-in-chain(LIC) of circumscribed enzyme effect Number is held to increase；Beta-glucosidase then main hydrolysis fiber disaccharides and soluble cellooligsaccharides, finally converting cellulose into can The glucose utilized.
Single bacterial strain fermentation there are enzyme activity low, the incomplete disadvantage of component of enzyme system, these can all seriously affect enzymolysis effect Rate, the research for improving cellulase activity and improving enzyme system composition is now very active, and there are many method of proposition：Pass through gene Engineering structure polygenes bacterial strain expresses a variety of enzyme components simultaneously, but it is of high cost, time-consuming；Comparatively, mixed fungus fermentation does not lose then For a kind of most simple effective method.Mutualism between mixed bacterium, makes enzyme system eurythmy, avoids fermenting during mixed fungus fermentation A certain intermediate product, which largely accumulates, in journey generates feedback inhibition so that mixed fungus fermentation enzymatic productivity is much higher than single bacterial strain, carries High yield.101100660 A of CN propose a kind of side using trichodermaharzianum and Rhizopus oryzae mixed fermentation cellulase-producing Method, but two plants of bacterium growth conditions are not quite similar, and Rhizopus oryzae is faster than the trichodermaharzianum speed of growth, it is easy to cause to be mixed Rhizopus oryzae dominant growth in journey, such case make filter paper enzyme activity decline instead, it is therefore desirable to stringent control training method and item Part, two plants of bacterium need to be inoculated with stage by stage, and operation is more complex, and also unavoidable nutrition when two plants of bacterium of phase exist simultaneously after fermentation The problems such as competition；Although beta-glucosidase enzyme activity can be improved using Trichoderma viride and aspergillus niger mixed fermentation in 102154243 A of CN Power, but equally exist identical problem；High star etc. is using trichoderma reesei and aspergillus niger mixed fermentation, in addition to due to growth conditions Be not quite similar and there are certain antagonism outside, the addition of aspergillus niger changes the pH value of fermentation system, be unfavorable for Richter scale wood Mould cellulase-producing leads to the filter paper enzyme activity of mixed fermentation also lower than single trichoderma reesei (Food Science, 2012,33 (19): 193-198).For the above deficiency, the present invention provides a kind of new mixed fermentation methods, using two plants of trichoderma reesei mixing hairs Ferment carries out producing enzyme, and two plants of bacterium growth conditions are identical, easy to operate.
During furfural production, being generated with a large amount of furfuraldehyde waste slags, 10 tons or more residues are discharged in furfural product per ton, I About several ten million tons of furfuraldehyde waste slag discharges every year in state, and very big pressure is brought to environment and enterprise.Containing a large amount of high additional in furfural dregs The cellulose of value, enzymatic saccharification processing is such as carried out to it makes its saccharification that can turn waste into wealth, and generates great economic benefit.Furfural Production mostly use weak acid water solution greatly, while having detached most of hemicellulose, the original cellulose of raw material and half fiber Complicated reticular structure between dimension element, lignin can save complicated plant fiber material pretreatment by a degree of destruction Process, to provide advantage using furfural dregs cellulose bioconversion.The present invention selects chaff resourceful, that structure is special Aldehyde slag has double benefit economical and environmentally friendly as cellulosic material fermentation cellulase-producing.
Invention content
In view of the deficiencies of the prior art, it is given birth to using mixed fungus fermentation maize straw furfural dregs the object of the present invention is to provide a kind of The method of cellulase-producing.
Production strain of the present invention is 13052 Hes of trichoderma reesei (Trichoderma reesei) CICC CICC40360 is purchased from Chinese industrial Microbiological Culture Collection administrative center (China Center of Industrial Culture Collection, CICC).
The purpose of the present invention is by following technical solution to complete：
A method of cellulase being produced using mixed fungus fermentation maize straw furfural dregs, this method is by trichoderma reesei (Trichoderma reesei) CICC 13052 and CICC 40360 is in being to induce the culture medium of carbon source with furfural dregs Mixed fermentation produces cellulase.
The method for producing cellulase using mixed fungus fermentation furfural dregs of the present invention, preferred technical solution include following step Suddenly：
(1) bacterium powder is lyophilized in the trichoderma reesei of purchase (Trichoderma reesei) CICC 13052 and CICC 40360 It is activated with slant activation culture medium：The slant tubes of above-mentioned bacterial strains stationary culture 5-7 days at 28 DEG C are coated with, until Grow spore, later with same method carry out secondary culture activation (due to strain after preservation is lyophilized in a dormant state, one Incubation time need to be appropriately extended for strain, be forwarded to 2-3 generation can rejuvenate).
(2) trichoderma reesei of activation (Trichoderma reesei) CICC 13052 and 40360 inclined-planes CICC are distinguished Appropriate sterile saline is added, mixing 2min is prepared into a concentration of 1 × 106-7The spore suspension of a/ml.By spore suspension with 5% inoculum concentration is inoculated in fresh seed culture medium, 24~48h of shaking table culture at 28 DEG C, 180rpm, is obtained respectively described The seed culture fluid of two kinds of bacterium；
(3) the seed mixture culture solution of two kinds of bacterium is seeded to by 1~10% volume ratio in culture medium, 28 DEG C, carry out enzymatic production under conditions of 180rpm.
The inoculative proportion of CICC 40360 and CICC 13052 are 1~10 in the wherein described seed mixture culture solution:10~ 1。
Further, it is preferable to which ratio is 1:3.
Further, the furfural dregs initial concentration of mixed fermentation is 20~200g/L, preferably 100g/L.
Culture medium employed in the above-mentioned method for producing cellulase using mixed fungus fermentation furfural dregs is as follows：
Activation medium：Potato extracting solution 1.0L, glucose 20.0g, agar 15.0g, pH is natural.
Seed culture medium：Microcrystalline cellulose 10g, glucose 10g, urea 0.3g, KH2PO42.0g, (NH4)2SO4 1.4g, MgSO4-7H2O 0.3g, peptone 0.75g, yeast powder 0.25g, CaCl2-2H2O0.4g, water 1L, trace element:CoCl2 0.002g, ZnSO4-7H2O 0.0014g, MnS04-7H2O 0.0016g, FeS04-7H2O 0.005g。
Culture medium：Maize straw furfural dregs 20-200g, urea 0.3g, KH2PO42.0g, (NH4)2SO41.4g MgSO4-7H2O 0.3g, peptone 0.75g, yeast powder 0.25g, CaCl2-2H2O 0.4g, water 1L, trace element:CoCl2 0.002g, ZnSO4-7H2O 0.0014g, MnS04-7H2O 0.0016g, FeS04-7H2O 0.005g。
The present invention has the advantages that compared with the existing technology：
(1) Technical furfural residue resource amount is big, cheap, and containing abundant cellulose, cellulosic degree of polymerization is relatively low And it is loosely organized, it is the preferred raw material for preparing cellulose bioconversion.The present invention was both saved using maize straw furfural dregs as raw material Cost, also solves environmental problem, and while developing and using the native celluloses resources such as stalk, non-environmental-pollution is to promote Into the effective means of straw ethanol industrialization.
(2) mixed fungus fermentation has cellulose utilization rate more higher than single culture fermentation and producing enzyme efficiency.
(3) mixed fungus fermentation tolerance concentration of substrate is high, can tolerate 200g/L maize straw furfural dregs.
(4) it is aerobic fungi-trichoderma reesei the single bacterium that bacterium is included to be mixed in the present invention, and growth condition is identical, is convenient for It co-cultures.
The method of the present invention solves the problems, such as that furfural dregs utilize, and improves cellulase production efficiency, at low cost, technique letter It is single, it is environmental-friendly, suitable for industrial applications, there is good economic and social benefit, be a kind of there is prospects for commercial application The method for producing cellulase.
Description of the drawings
Fig. 1 mixes bacterium and utilizes fermentation of furfural residues producing enzyme situation；
Fig. 2 trichoderma reeseis CICC 40360 utilizes fermentation of furfural residues producing enzyme situation；
Fig. 3 trichoderma reeseis CICC 13052 utilizes fermentation of furfural residues producing enzyme situation；
Influence of Fig. 4 concentration of substrate to mixed fungus fermentation producing enzyme；
Different initial influences of the pH to mixed fungus fermentation producing enzyme of Fig. 5；
Influence of Fig. 6 different vaccinations amount to mixed fungus fermentation producing enzyme；
Influence of Fig. 7 different vaccinations ratio to mixed fungus fermentation producing enzyme；
Fig. 8 differences assist influence of the inducer to mixed fungus fermentation producing enzyme.
Specific implementation mode
Following non-limiting embodiments can make those skilled in the art be more fully understood the present invention, but not with Any mode limits the present invention.In following embodiments, unless otherwise specified, used experimental method is conventional method, institute With reagent etc. can chemically or biological reagent company purchase.
1. microculture and fermentation：
Actication of culture：The bacterial strain of purchase is activated with slant activation culture medium, is coated with the slant tube of bacterium solution Stationary culture 5-7 days at 28 DEG C carries out secondary culture 2-3 generation acquisition activation bacterium with same method later until growing spore Strain.
Seed culture：By equipped with 20mL seed culture mediums 100mL triangular flasks tampon and bottleneck film sealing, in 115 DEG C Lower sterilizing 20 minutes, accesses the spore suspension of the inclined-plane microorganism, 24- is cultivated in 28 DEG C, 180rpm shaking tables after cooling 48h obtains seed culture fluid.
Enzymatic production：The seed culture fluid of culture is transferred to 1~10% inoculum concentration containing 20mL culture mediums In Oscillating bottles of 100mL, 28 DEG C, 3-11d is cultivated in 180rpm shaking tables, timing sampling measures enzyme activity.
2. analysis method：It is measured using the International Standards Method that International Federation of Theoretical and Applied Chemistry (IUPAC) is recommended Zymotic fluid enzyme activity.
Embodiment 1
Mixed fungus fermentation cellulase-producing
The trichoderma reesei CICC13052 and CICC40360 of activated inclined plane preservation respectively, with 5% inoculum concentration respectively from spore Suspension is forwarded to seed culture fluid in shake culture 24-48h on 28 DEG C, the shaking table of 180rpm.Again with inoculative proportion 1:1, it always connects Kind amount is transferred to for 5% in the culture medium containing 50g/L furfural dregs, and timing sampling measures enzyme activity.The results are shown in Figure 1, For mixed bacterium when being induction carbon source with 50g/L maize straw furfural dregs, cellulose enzyme activity is up to 0.82FPU/mL.
Comparative example 1
The fermentation cellulase-producings of single bacterium CICC 40360
The trichoderma reesei CICC40360 of activated inclined plane preservation is forwarded to seed culture fluid with 5% inoculum concentration from spore suspension In in shake culture 24-48h on 28 DEG C, the shaking table of 180rpm.It is transferred to again containing 50g/L furfural dregs with 5% inoculum concentration In culture medium, timing sampling measures enzyme activity, and the results are shown in Figure 2, and trichoderma reesei CICC 40360 is with 50g/L corn stalks When stalk furfural dregs are induction carbon source, cellulose enzyme activity is only 0.47FPU/mL, and lower than mixed fungus fermentation 42.68%.
Comparative example 2
The fermentation cellulase-producings of single bacterium CICC 13052
According to the method described in comparative example 1, trichoderma reesei CICC 13052 is connected to the training of the producing enzyme containing 50g/L furfural dregs It supports and ferments in base, timing sampling measures enzyme activity.The results are shown in Figure 3, it can be seen from the figure that CICC 13052 with When 50g/L maize straw furfural dregs are induction carbon source, cellulase activity is higher than CICC 40360 up to 0.67FPU/mL, than Mixed fungus fermentation low 18.29%.
Embodiment 2
Influence of the different concentration of substrate to mixed fungus fermentation producing enzyme
According to 1 the method for embodiment, by mixed bacterium with inoculative proportion 1:1, total inoculum concentration is transferred to different furfural dregs for 5% In the culture medium of concentration, timing sampling measures enzyme activity, explores its optimal carbon source content and producing enzyme situation.As a result such as Fig. 4 institutes Show, as seen from the figure, mix bacterium have preferable substrate tolerance, even if under 200g/L furfural dregs concentration also can with enzymatic production, When being induction carbon source with 100g/L furfural dregs, cellulase activity maximum is up to 0.87FPU/mL, therefore follow-up test substrate The preferred 100g/L of concentration.
Embodiment 3
Different initial influences of the pH to mixed fungus fermentation producing enzyme
The pH of culture medium and the growth and breeding of cell and enzymatic production are in close relations, during the fermentation, with cell Growth and breeding and metabolism product accumulation, the pH of culture medium often changes, therefore this experiment only controls just Beginning pH.
According to 2 the method for embodiment, by mixed bacterium with inoculative proportion 1:1, total inoculum concentration is 5% to be transferred to initial pH and be 2.80, it ferments in 4.32,4.80,6.00,7.00,8.00 culture medium containing 100g/L furfural dregs, periodically takes Sample measures enzyme activity, observes influences of the initial pH to mixed fungus fermentation producing enzyme, explores its optimal initial pH and producing enzyme situation.As a result as schemed Shown in 5, the mixed bacterium of trichoderma reesei fermentation cellulase-producing situation difference under the conditions of different initial pH is larger, and cellulase is formed in Under meta-acid environment, but pH too low (pH=2.8) is unfavorable for the formation of cellulase, and in neutral meta-alkali condition (pH=7,8) The formation of lower cellulase is also at disadvantage, it is seen that pH is too low or the generation of excessively high cellulase can be impacted.By experiment number According to it is found that the preferred pH4.8 of initial pH that cellulase is formed, than shortening the producing enzyme time before optimization, producing enzyme peak is reachable in 7d 0.91FPU/mL, than 13052 single bacteriums of CICC fermentation 3 days in advance, than 40360 single bacteriums of CICC fermentation 2 days in advance, and enzyme activity carried It is high.
Embodiment 4
Influence of the different vaccination amount to mixed fungus fermentation producing enzyme
According to 3 the method for embodiment, with inoculative proportion for 1:1, total inoculum concentration is 1%, 2.5%, 5%, 8%, 10%, Respectively from initial pH is forwarded in seed liquor to carry out enzymatic production in 4.8 culture medium, timing sampling measures enzyme activity, visits Its optimal inoculum concentration of rope.
Influence as shown in fig. 6, mixed bacterium different vaccination amount under the conditions of ferment production of the different vaccination amount to mixed fungus fermentation producing enzyme The case where cellulase, difference was larger.Inoculum concentration is too small, and cell concentration is insufficient, and the generation of cellulase is interrupted；Inoculum concentration mistake Greatly, cell concentration is excessively high, and dissolved oxygen is insufficient, and substrate nutritional deficiency, and the formation of cellulase is also suppressed, by experimental data It is found that it is preferred that 5% inoculum concentration.
Embodiment 5
Influence of the different vaccination ratio to mixed fungus fermentation producing enzyme
Inoculative proportion is a critically important parameter in mixed fungus fermentation.According to 4 the method for embodiment, with total inoculum concentration It is respectively CICC 40360 for 5%, inoculative proportion：CICC 13052=1:1、1:2、2:1、1:3、3:1、1:4、4:1、1:5、5: 1 method, which is seeded to from seed liquor in the culture medium that initial pH is 4.8, ferments, and timing sampling measures enzyme activity, explores Its optimal inoculative proportion.Influence of the different vaccination ratio to mixed fungus fermentation producing enzyme is as shown in fig. 7, with CICC40360 institutes accounting Example increases, and the enzymatic activity for mixing bacterium generation is relatively low, and as CICC13052 proportions increase, enzymatic activity increased.Wherein, CICC 40360:CICC 13052=1:3 producing enzymes higher (0.98FPU/mL), very fast and enzymatic activity are relatively stablized, and industry is conducive to Production.Therefore preferably inoculative proportion is CICC 40360:CICC 13052=1:3.
Embodiment 6
Influence of the difference auxiliary inducer to mixed fungus fermentation cellulase-producing
Cellulase is co-induction enzyme, therefore devises and add different auxiliary on the basis of being induction carbon source with furfural dregs Help the experiment of inducer.
According to 5 the method for embodiment, it is according to inoculative proportion by the CICC 40360 of culture, 13052 seed liquors of CICC CICC40360:CICC13052=1:3, inoculum concentration 5%, it is 4.8 to be seeded to the initial pH containing different inducers respectively Enzymatic production culture is carried out in culture medium, is sampled detection filter paper enzyme activity at regular intervals, is observed different inducers Influence to strain fermentation producing enzyme.
Wherein, CK groups be do not add helper-inducer object carbon source be 100g/L furfural dregs control, test group be in CK groups On the basis of add respectively 0.5% glucose, lactose, xylose, cellobiose, microcrystalline cellulose (MCC), carboxymethyl cellulose Sodium (CMC).
For test result as shown in figure 8, compared with CK, each helper-inducer object does not play the role of positive induction, illustrates corn Stalk furfural dregs can induce alone mixed bacterium cellulase-producing, without adding other helper-inducer objects, and then save producing enzyme cost, Conducive to industrial production.

Claims (5)

1. a kind of method of mixed fungus fermentation maize straw furfural dregs production cellulase, which is characterized in that utilize two plants of Richter scale wood It is mould be with maize straw furfural dregs induce carbon source culture medium in mixed fermentation produce cellulase.
2. the method for mixed fungus fermentation maize straw furfural dregs production cellulase according to claim 1, which is characterized in that The trichoderma reesei is CICC 13052 and CICC 40360.
3. the method for mixed fungus fermentation maize straw furfural dregs production cellulase according to claim 1, which is characterized in that The inoculative proportion of the trichoderma reesei CICC 13052 and CICC 40360 is 1~10:10~1, preferably CICC 40360: CICC 13052=1:3.
4. the method for mixed fungus fermentation maize straw furfural dregs production cellulase according to claim 1, which is characterized in that The induction carbon source of culture medium is the maize straw furfural dregs after this Laboratory Production furfural；Mixed bacterium tolerance concentration of substrate is high, Tolerable 200g/L maize straw furfural dregs, preferred concentration 100g/L.
5. the method for mixed fungus fermentation maize straw furfural dregs production cellulase according to claim 1, which is characterized in that The initial pH of mixed fermentation is 2~8, preferably pH4.8；Inoculum concentration is 1~10%, preferably 5%.
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112442495A (en) * 2018-08-06 2021-03-05 杭州园泰生物科技有限公司 Enzyme production process by mixed fermentation
CN112522342A (en) * 2020-12-17 2021-03-19 新疆希普生物科技股份有限公司 Method for efficiently performing enzymolysis on straws
CN115975819A (en) * 2022-11-21 2023-04-18 福建师范大学 Furfural-tolerant trichoderma reesei mutant strain and application thereof

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