CN102796673A - Feruloyl esterase production strain and method for producing feruloyl esterase by using same - Google Patents

Feruloyl esterase production strain and method for producing feruloyl esterase by using same Download PDF

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CN102796673A
CN102796673A CN2012103075462A CN201210307546A CN102796673A CN 102796673 A CN102796673 A CN 102796673A CN 2012103075462 A CN2012103075462 A CN 2012103075462A CN 201210307546 A CN201210307546 A CN 201210307546A CN 102796673 A CN102796673 A CN 102796673A
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liquid
feruloyl esterase
ferulic acid
ion
fermentation
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CN102796673B (en
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徐泽平
马韵升
史庆苓
杨传伦
张心青
王秀芝
付慧君
虞凤慧
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Chambroad Chemical Industry Research Institute Co Ltd
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Chambroad Chemical Industry Research Institute Co Ltd
Shandong Chambroad Holding Group Co Ltd
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Abstract

The invention belongs to the technical field of bioengineering, and provides a feruloyl esterase high-producing strain and a method for preparing feruloyl esterase through liquid fermentation by using the same. The feruloyl esterase production strain provided by the invention is a filamentous fungus; 16S rDNA identification shows that the feruloyl esterase production strain is a Trichodermaviride; the strain code is JBSH-001; and the preservation number of China General Microbiological Culture Collection Center is CGMCC NO.5612. Feruloyl esterase can be produced through liquid fermentation by using the strain. The enzyme activity of the feruloyl esterase obtained through fermentation according to the method can reach 200 mU/ml or above; and the enzyme activity of the purified feruloyl esterase can reach 50000 mU/g or above.

Description

One strain feruloyl esterase is produced bacterial strain and is used the method that this bacterial strain is produced feruloyl esterase
Technical field
The invention belongs to technical field of bioengineering, the microorganism strains that provides a plant height to produce feruloyl esterase, particularly a strain green trichoderma and utilize this bacterium to produce the method for feruloyl esterase.
Background technology
Feruloyl esterase also is called as the styracin esterase, is a subclass of carboxylic ester hydrolase, also is a kind of extracellular enzyme, and it can the hydrolysis Ferulic acid methylester, the ester bond in oligosaccharide ferulic acid ester and the polysaccharide ferulic acid ester, and FLA is dissociated out.FLA plays an important role in the vegetable cell wall construction; It can between xylogen and the semicellulose, form handing-over between semicellulose and the semicellulose between the xylogen and xylogen of plant cell wall; Thereby constitute a skeleton structure, make whole cell walls become hard.Feruloyl esterase can be opened the fine and close cross-linked network that FLA in the cell wall lignin, P-coumaric acid and dimerization FLA equimolecular and semicellulose prop up chain formation; Effective degraded of Mierocrystalline cellulose and semicellulose the pair cell wall from the space, so feruloyl esterase has been thought one of key enzyme of eliminating the cell wall degradation limiting factor by supposition.
In paper industry, through the raw material that feruloyl esterase is handled, xylogen removes more easily, can reduce the pharmaceutical chemicals consumption of pulping process.And the fiber finer cell wall is after feruloyl esterase is handled, and it is loose that the surface becomes, and can improve the physical strength of paper product when becoming paper.Utilize the FLA that produces after the feruloyl esterase pulp treatment to can be used as the fine inhibitor, health-care effect such as in addition, that FLA also has is antitumor, anti-inflammatory, promotion wound are healed is so FLA can be used as the food of functional factor development functionality.Simultaneously, FLA can be used as the precursor of synthesis of natural vanillin food grade,1000.000000ine mesh, is raw material with the FLA, utilizes the vanillin food grade,1000.000000ine mesh of method of microorganism production to compare with the synthetic vanillin food grade,1000.000000ine mesh to have toxicity low, safe characteristics, can be as the spices of foods and cosmetics industry.
Patent CN200810056477.6 discloses a kind of process for extracting of feruloyl esterase; But operation is comparatively complicated; Research about feruloyl esterase; Having a lot of problems to need to solve, mainly is the requirement that the secretory volume of the mikrobe of the secretion feruloyl esterase that filters out does not at present still reach the suitability for industrialized production feruloyl esterase; Secondly, the feruloyl esterase process for extracting haves much room for improvement, and its suitability for industrialized production is restricted.
Summary of the invention
The present invention is directed to the deficiency that above-mentioned technology exists; A kind of method of utilizing viride (Trichoderma viride) to produce feruloyl esterase through liquid fermenting is provided; The existing bibliographical information of yield has improved several times; The bacterial strain code that is adopted is JBSH-OO1, and the preserving number at China Committee for Culture Collection of Microorganisms common micro-organisms center is CGMCC No.5612; This bacterial strain can be applicable to produce feruloyl esterase, and this method fermentation gained feruloyl esterase enzyme work can reach more than the 200mU/ml, and the feruloyl esterase enzyme work behind the purifying can reach more than the 50000mU/g.
Concrete technical scheme provided by the invention is:
At first obtained the new bacterial strain of a strain, the preserving number of this bacterial strain at China Committee for Culture Collection of Microorganisms common micro-organisms center is CGMCC No.5612, and its concrete preparation method is following:
(1) starting strain: as initial strain, comprising multiple wood-decay fungi, soft-rot bacterium, Fusarium oxysporum and other moulds, classification is cultivated and is set up strain library with the various bacterial strains of this laboratory collection.
(2) screening method: prepare plate culture medium with Ferulic acid methylester as sole carbon source, insert above-mentioned bacterial strains and cultivate under optimum conditions, carry out the one-level screening according to whether producing transparent circle.Preliminary screening has the bacterial strain that produces the feruloyl esterase ability, then the primary dcreening operation bacterial strain is carried out shake-flask culture, filters out the highest bacterial strain of wherein 2 strain inulinase-producing activities as naturalized strain according to the active size of feruloyl esterase in the liquid culture.
(3) to 2 strains treat naturalized strain physio-biochemical characteristics, produce enzyme performance etc. and compare further investigation, finishing screen is selected the bacterial strain that a strain enzymatic productivity is strong, be easy to cultivate and have the stable characteristic that goes down to posterity, and is JBSH01 with its preliminary designation.
(4) be starting strain with this bacterial strain, adopt conventional mutafacient system, like UV, DMS, MMS, NTG etc., in conjunction with the low energy ion injection method, repeated multiple times mutagenesis, multiple sieve has finally obtained superior strain JBSH-001.
The contriver has carried out biological preservation to this bacterial strain at Chinese microorganism strain preservation conservator common micro-organisms center, and deposit number is CGMCC No.5612, is existing state through detecting it.
Above-mentioned JBSH-001 bacterial strain, its morphological specificity is following:
Mycelia is very thin colourless, and tool is separated, multi-branched.Conidiophore bears from the side shoot of mycelia, to giving birth to or alternate, branch is arranged generally 2-3 time, sporogenic stigma doleiform estranged or taper.It is spherical mostly conidium is, sporoderm tool pustule, green.Bacterium colony just is the white cotton fiber shape on the PDA substratum, and the back is a sap green.Above-mentioned morphological specificity is consistent with the cultural characters of viride Trichoderma viride.
The contriver has carried out 16S rDNA order-checking to it simultaneously, and its gene order is shown in Seq ID No:1, and this sequence is the complete sequence of the 16S rDNA of bacterial strain JBSH-OO1.
Carry out the BLSTN comparison with measured 16S rDNA sequence; The result shows; The nucleotides sequence of the nucleotide sequence of the 16S rDNA of bacterial strain JBSH-OO1 and Trichoderma Trichoderma spp. different strains is shown the homology greater than 99%; With the 5 strain bacterial strains that wherein clearly are labeled as viride Trichoderma viride 100% homology is arranged, thereby confirm that further bacterial strain provided by the present invention is a strain viride bacterial strain.
The bacterial strain viride Trichoderma viride JBSH-OO1 that the present invention obtained; Can be applied in the middle of the daily production; Particularly can be applied to the production of feruloyl esterase; Concrete steps mainly comprise the extraction separation and the purification step of trichoderma viride liquid fermenting and feruloyl esterase, it is characterized in that: in the trichoderma viride liquid fermenting, use feruloyl esterase generation and accumulation and induce operation
Described feruloyl esterase produces and accumulation induces the operation concrete steps following:
A. inducible factor screening:
From the regulation and control aspect screening inducible factor of ferulic acid derivative, growth factor, enzyme system, inducible factor is selected from vitamins or ferulic acid derivative or induces ion or other inducible factors or its mixture;
B. inducible factor method of use:
Vitamins or ferulic acid derivative or induce ion to be added in the fermention medium in conjunction with adding carbon source and nitrogenous source mid-term, are used to induce and promote to produce feruloyl esterase.
At first to cultivate and make the Trichoderma bulk-growth with routine; Get into to produce that the enzyme after date is added carbon source such as glucose, sucrose more successively and peptone, yeast soak nitrogenous sources such as powder, yeast extract; Make thalli growth through continuous feed supplement, thereby increase the expression and the secretion of feruloyl esterase.
The ion of inducing in the wherein said inducible factor is selected from one or more the mixture in boron ion or mg ion or cobalt ion or the molybdenum ion; Vitamins is selected from vitamins B or folic acid; Ferulic acid derivative is Ferulic acid methylester or Ferulic acid ethylester; Other inducible factor is one or more the mixture in chitin or chitosan or rice bran or the wheat bran.
Why select above-mentioned material as inducible factor; The contriver finds to bacterial strain of the present invention through the research back; Make it produce gliocladin; The method of induction regulating controlling is mainly through three kinds of modes: 1, replenish the substrate of expression of enzymes, like Ferulic acid methylester, Ferulic acid ethylester, chitin, chitosan, wheat bran, thereby induce bacterium to produce feruloyl esterase; 2, replenish the necessary nutritional factor of biological growth, like vitamins B, folic acid etc.; 3, replenish the coenzyme ion that various enzymes are in the thalline; Mainly select from metals ion or other examples; Generally can adopt the form of its soluble metallic salt; The contriver finds through three kinds of above-mentioned inducible factors, in conjunction with the control for fermenting process, can realize that the output of feruloyl esterase improves.
More concrete production stage is following:
(1) trichoderma viride liquid fermenting:
A. actication of culture;
B. liquid seeds preparation;
C. fermentation: behind the actication of culture, expand numerous seed liquor, utilize liquid submerged fermentation method to obtain the feruloyl esterase fermented liquid again through liquid fermenting;
Fermenting process wherein: liquid seeds is received in the fermention medium with the inoculum size of 2-10% (v/w), cultivated 48h, add the carbon source of 0.5-1% (v/w) and the nitrogenous source of 0.05-0.2% (v/w) for 26 ℃;
(2) Preparation of Ferulic Acid Esterase:
A. solid-liquid separation: after solid-liquid separation, clear liquid is used to prepare feruloyl esterase with the feruloyl esterase fermented liquid of above-mentioned fermentation;
B. fermentation clear liquid adopts micro-filtration to remove colloid and insoluble particle, after ultra-filtration membrane concentrates, adds protective material, the spray-dried feruloyl esterase bullion that obtains;
And in order to obtain the pure article of feruloyl esterase, the stillness of night that the contriver obtains above-mentioned steps (2) is directly after solid-liquid separation; Adopt micro-filtration to remove colloid and insoluble particle, after ultra-filtration membrane concentrates, with the method for saltouing; Obtain the thick enzyme of feruloyl esterase, after redissolving, the enzyme liquid of preparation 0.5-2%; Through chromatography purification, after the ultrafiltration desalination, obtain the pure article of feruloyl esterase through lyophilize.
Solid-liquid separation in the wherein said step (2) is a Plate Filtration or tripodia is centrifugal or disk centrifugal; The aperture of microfiltration membrane is 0.1-0.5 μ m in the described step (3), and the aperture of ultra-filtration membrane is 2000-10000Dalton; Described salting medium is sodium-chlor or sodium sulfate or sal epsom or ammonium sulfate; What chromatography adopted in the described step (3) is ion exchange chromatography, and the chromatography media that uses is DOWEX MXA-1 or Q-Sepharose FF or DEAE-Sephacel or DEAE-Sepharose FF or 717 strong basic ion exchange resins or D201 macroporous strong basic anionite-exchange resin.
More concrete process is following:
(1) actication of culture: the test tube slant bacterial classification that is kept under 4 ℃ of conditions on the PDA substratum is moved to (20 ℃-25 ℃) activation 4h-8h under the room temperature condition;
(2) liquid seeds preparation: on the aseptic technique platform; To pass through activatory test tube slant bacterial classification with the zero(ppm) water after the 10mL sterilization and process thallus suspension liquid; Flushing goes into to be equipped with in the triangular flask of sterilising liq substratum, 1 triangular flask of 1 test tube strains inoculation, 24 ℃ of culture temperature; Shaking speed 200r/min, shaking culture 24-48h prepares seed liquor;
(3) ferment, induce process: in fermention medium, add accounting for substratum gross weight 0.01-0.05% metals ion (like one or more mixtures in mg ion or cobalt ion or molybdenum ion or the boron ion); And account for the growth factor (like vitamins B or folic acid or its mixture) of substratum gross weight 0.005-0.02%; Liquid seeds is received in the fermention medium under the condition of aseptic technique with the inoculum size of 2-10% (v/w); Cultivate 48h for 26 ℃; Add the carbon source (like glucose or one or more mixtures of sucrose) of 0.5-1% (v/w) and the nitrogenous source (soaking one or more mixtures in powder or the yeast powder) of 0.05-0.2% like peptone or yeast; Cultivate 96-144h altogether, obtain the feruloyl esterase fermented liquid, after solid-liquid separation, obtain fermentation clear liquid.
(4) extraction of feruloyl esterase: fermentation clear liquid is after the 4000-6000r/min spinning; Clear liquid adopts the micro-filtration membrane module of 0.1-0.22 μ m to filter settled solution; And then with the hyperfiltration membrane assembly ultrafiltration and concentration of 2000-60000Dalton; Being concentrated to concentration is 5-10% (v/v), adds the protective material (w/v) of 5-10%, the spray-dried feruloyl esterase bullion that obtains.
(5) purifying of feruloyl esterase
Fermentation clear liquid is after the 4000-6000r/min spinning, and clear liquid adopts the micro-filtration membrane module settled solution of 0.1-0.22 μ m, and then concentrates with the hyperfiltration membrane assembly of 2000-60000Dalton; Being concentrated to concentration is 5-10% (v/v); With weight fraction 50-70% ammonium sulfate precipitation, after 2000-6000r/min was centrifugal, deposition use pH was 6.86 phosphate buffer salt preparation 0.5-1% (w/v; Weight in wet base) enzyme liquid; Adopt QFF sepharose chromatography purification, after the ultrafiltration desalination, obtain the pure article of feruloyl esterase through lyophilize.
Wherein, the feruloyl esterase bullion cost that step (4) obtains is lower, can directly be used for feed, processing of farm products, and the pure article purity of the feruloyl esterase that step (4) obtains is high, can be used for aspects such as food, papermaking.
Wherein the working condition of bacterial strain product feruloyl esterase is definite, and its method is following:
(1) adopts one-factor experiment and orthogonal test,, confirm liquid seeds optimal culture condition and liquid fermenting optimal culture condition through changing temperature, original ph, inoculum size, charge and incubation time;
(2) adopt one-factor experiment and orthogonal test, confirm that through changing carbon nitrogen source and the inorganic salt composition cultivated this strain liquid seed optimal medium is formed and the optimal medium of liquid fermenting is formed.
Following through above-mentioned one-factor experiment and bacterial strain that orthogonal test obtains, liquid seed culture medium composition and fermentation condition:
Liquid seed culture medium is formed, and (w/w) is by weight: sucrose 2%~4%, yeast soak powder 0.2%~1%, sal epsom 0.02-0.1%, and potassium primary phosphate 0.01-0.05% adds water to 1000mL;
The liquid seeds culture condition: inoculum size is 1 bacterial strain slant culture of each triangular flask inoculation, pH value nature, and 24~28 ℃ of culture temperature, shaking speed 75-200r/mi n, incubation time are 24~48h;
Through above-mentioned one-factor experiment and orthogonal test obtain strain liquid fermention medium composition and fermentation condition following:
Liquid nutrient medium is formed, and (w/w) is by weight: sucrose 2%~4%, peptone 0.2~1%, wheat bran: 1-4%; Murphy juice 1-4%; Sal epsom 0.02-0.1%, potassium primary phosphate 0.01-0.05%, zinc sulfate 0.001-0.005%, cobalamin 0.005% adds water to 1000mL;
Liquid fermentation condition: inoculum size is 5%-10% (v/w), pH value nature, and 24~28 ℃ of culture temperature, shaking speed 75-200r/min, incubation time are 96-144h;
Utilize above-mentioned green trichoderma fermentation, feruloyl esterase enzyme work behind the liquid fermenting can be reached more than the 200mU/ml, the feruloyl esterase enzyme work behind the purifying can reach more than the 50000mU/g.
Utilize this bacterial strain to produce feruloyl esterase following advantage arranged:
1, utilize this strain fermentation to prepare the work of feruloyl esterase enzyme and can reach more than the 200mU/ml, the feruloyl esterase enzyme work behind the purifying can reach more than the 50000mU/g;
2, the meta-bolites feruloyl esterase of this bacterial strain is an extracellular enzyme, reduces the cell walls shattering process, cuts down the consumption of energy greatly, reduces production costs.
In sum; The invention provides a strain feruloyl esterase superior strain and utilize this bacterial strain to carry out the method that liquid fermenting prepares feruloyl esterase; This bacterial strain can be applicable to produce feruloyl esterase, and this method fermentation gained feruloyl esterase enzyme live birth rate can reach more than the 200mU/ml.
Preservation information
The preservation time: on December 16th, 2011
Depositary institution's title: China Committee for Culture Collection of Microorganisms common micro-organisms center
Deposit number: CGMCC No.5612
Depositary institution address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City institute of microbiology of the Chinese Academy of Sciences
Classification name: viride (Trichoderma viride)
Embodiment
Below, foregoing of the present invention is done further detailed description, but should this be interpreted as that the scope of the above-mentioned theme of the present invention only limits to following instance through the embodiment of embodiment form.All technology that realizes based on foregoing of the present invention all belong to scope of the present invention, and the bacterial classification that is adopted among the following embodiment is the bacterial classification that preserving number is CGMCC No.5612.
Embodiment 1
(1) actication of culture: the test tube slant bacterial classification that is kept under 4 ℃ of conditions on the PDA substratum is moved to (20-25 ℃) activation 4h under the room temperature condition;
(2) liquid seeds preparation: on the aseptic technique platform; To pass through activatory test tube slant bacterial classification with the zero(ppm) water after the 10mL sterilization and process thallus suspension liquid, flushing goes into to be equipped with in the triangular flask of sterilization seed culture medium under aseptic condition, 1 triangular flask of 1 test tube strains inoculation; PH value nature; 24 ℃ of culture temperature, shaking speed 200r/min, incubation time are 48h;
Seed culture medium is formed, and (w/w) is by weight: sucrose 2%, yeast soak powder 0.2%, sal epsom 0.05%, and potassium primary phosphate 0.025% adds water to 1000mL; Sterilising conditions is 121 ℃, the 0.15Mpa 20min that sterilizes;
(3) fermentation, regulation process: liquid seeds is received in the fermention medium with the inoculum size of 10% (v/v); Cultivate 48h for 26 ℃; Add the peptone of peptone sterile solution 5mL0.1% (w/v) of glucose and 20% (w/v) of the glucose sterile solution 25mL1% (w/v) of 40% (w/v), cultivate 96h altogether.
Fermention medium is formed, and (w/w) is by weight: sucrose 4%, peptone 1%, wheat bran 4%; Sal epsom 0.1%, potassium primary phosphate 0.05%, NSC 51149 0.001%, cobalamin 0.005% adds water to 1000mL; Sterilising conditions is 121 ℃, 0.15Mpa sterilization 20mi n;
After the fermentation ends; Fermentation broth enzyme is lived and is 248mU/mL, and fermented liquid is behind the centrifugal 10min of 4000r/min, and clear liquid adopts the ceramic micro filter membrane module of 0.22 μ m to filter; Filtrating concentrates with the hyperfiltration membrane assembly of 10000Dalton; Being concentrated to weight concentration is 5% (v/v), adds the W-Gum of 10% (w/v), the spray-dried feruloyl esterase bullion 25.3g that obtains.Enzyme 7200mU/g alive.
Embodiment 2
(1) actication of culture: the test tube slant bacterial classification that is kept under 4 ℃ of conditions on the PDA substratum is moved to (20-25 ℃) activation 4h under the room temperature condition;
(2) liquid seeds preparation: on the aseptic technique platform; To pass through activatory test tube slant bacterial classification with the zero(ppm) water after the 10mL sterilization and process thallus suspension liquid, flushing goes into to be equipped with in the triangular flask of sterilization seed culture medium under aseptic condition, 1 triangular flask of 1 test tube strains inoculation; PH value nature; 24 ℃ of culture temperature, shaking speed 200r/min, incubation time are 48h;
Seed culture medium is formed (w/w): (w/w) is by weight: sucrose 2%, yeast soak powder 0.6%, sal epsom 0.05%, and potassium primary phosphate 0.025% adds water to 1000mL; Sterilising conditions is 121 ℃, the 0.15Mpa 20min that sterilizes;
(3) fermentation, regulation process: liquid seeds is received in the fermention medium with the inoculum size of 5% (v/v); Cultivate 48h for 26 ℃; The yeast of adding the sucrose sterile solution 25mL and 20% (w/v) of 40% (w/v) soaks the sucrose sugar of powder sterile solution 5mL1% (w/v) and the yeast of 0.1% (w/v) soaks powder, cultivates 120h altogether.
Fermention medium is formed, and (w/w) is by weight: sucrose 4%, peptone 1%, Ferulic acid ethylester 1%; Sal epsom 0.1%, potassium primary phosphate 0.05%, NSC 51149 0.001%, Y factor 0.005% adds water to 1000mL; Sterilising conditions is 121 ℃, the 0.15Mpa 20min that sterilizes;
After the fermentation ends, fermented liquid is through the centrifugal 10min of 4000r/min, and clear liquid adopts the ceramic micro filter membrane module of 0.22 μ m to filter; Filtrating concentrates with the hyperfiltration membrane assembly of 6000Dalton, is concentrated to 10% (v/v) of original volume, with 60% (w/v) ammonium sulfate precipitation; After 4000r/min was centrifugal, it was that 6.86 phosphate buffer salt is mixed with the enzyme liquid of 0.5% (w/v, weight in wet base) that deposition is used pH; Adopt DEAE-Sepharose FF medium to carry out column chromatography purification; After the elutriant ultrafiltration desalination, obtain the pure article 470mg of feruloyl esterase, enzyme 86450mU/g alive through lyophilize.
Embodiment 3
(1) actication of culture: the test tube slant bacterial classification that is kept under 4 ℃ of conditions on the PDA substratum is moved to (20-25 ℃) activation 4h under the room temperature condition;
(2) liquid seeds preparation: on the aseptic technique platform; To pass through activatory test tube slant bacterial classification with the zero(ppm) water after the 10mL sterilization and process thallus suspension liquid, flushing goes into to be equipped with in the triangular flask of sterilization seed culture medium under aseptic condition, 1 triangular flask of 1 test tube strains inoculation; PH value nature; 24 ℃ of culture temperature, shaking speed 200r/min, incubation time are 48h;
Seed culture medium is formed, and (w/w) is by weight: sucrose 2%, yeast soak powder 0.2%, sal epsom 0.05%, and potassium primary phosphate 0.025% adds water to 1000mL; Sterilising conditions is 121 ℃, the 0.15Mpa 20min that sterilizes;
(3) fermentation, regulation process: liquid seeds is received in the fermention medium with the inoculum size of 5% (v/v); Cultivate 48h for 26 ℃; The yeast of adding the sucrose sterile solution 25mL and 20% (w/v) of 40% (w/v) soaks the yeast that powder sterile solution 5mL adds the sucrose and 0.1% (w/v) of 1% (w/v) and soaks powder, cultivates 120h altogether.
Fermention medium is formed, and (w/w) is by weight: sucrose 4%, yeast soak powder 1%, Ferulic acid methylester 2%; Sal epsom 0.1%, potassium primary phosphate 0.05%, zinc sulfate 0.005%, ammonium molybdate 0.001%, vitaminB10 .01% adds water to 1000mL;
After the fermentation ends; It is that the 248mU/mL fermented liquid is through the centrifugal 10min of 4000r/min that enzyme is lived; Clear liquid adopts the ceramic micro filter membrane module of 0.1 μ m to filter, and filtrating concentrates with the hyperfiltration membrane assembly of 10000Dalton, and being concentrated to weight concentration is 8% (v/v); Add the starch of 10% (w/v), the spray-dried feruloyl esterase bullion 55.3g that obtains.Measure its enzyme 3800mU/g of being alive.
Embodiment 4
(1) actication of culture: the test tube slant bacterial classification that is kept under 4 ℃ of conditions on the PDA substratum is moved to (20-25 ℃) activation 4h under the room temperature condition;
(2) liquid seeds preparation: on the aseptic technique platform; To pass through activatory test tube slant bacterial classification with the zero(ppm) water after the 10mL sterilization and process thallus suspension liquid, flushing goes into to be equipped with in the triangular flask of sterilization seed culture medium under aseptic condition, 1 triangular flask of 1 test tube strains inoculation; PH value nature; 24 ℃ of culture temperature, shaking speed 200r/min, incubation time are 48h;
Seed culture medium is formed, and (w/w) is by weight: sucrose 2%, yeast soak powder 0.2%, sal epsom 0.05%, and potassium primary phosphate 0.025% adds water to 1000mL; Sterilising conditions is 121 ℃, the 0.15Mpa 20min that sterilizes;
(3) fermentation, regulation process: liquid seeds is received in the fermention medium with the inoculum size of 5% (v/v); Cultivate 48h for 26 ℃; The yeast of adding the sucrose sterile solution 25mL and 20% (w/v) of 40% (w/v) soaks the yeast that powder sterile solution 5mL adds the sucrose and 0.1% (w/v) of 1% (w/v) and soaks powder, cultivates 120h altogether.
Fermention medium is formed, and (w/w) is by weight: sucrose 4%, yeast soak powder 1%, Ferulic acid methylester 2%; Sal epsom 0.1%, potassium primary phosphate 0.05%, zinc sulfate 0.005%, ammonium molybdate 0.001%, vitaminB10 .01% adds water to 1000mL;
After the fermentation ends, fermented liquid is behind the centrifugal 10min of 4000r/min, and clear liquid adopts the ceramic micro filter membrane module of 0.10 μ m to filter; Filtrating concentrates with the hyperfiltration membrane assembly of 6000Dalton, is concentrated to 10% of former filtrate volume, the ammonium sulfate precipitation with 70%; After 4000r/min was centrifugal, it was the enzyme liquid of 6.86 phosphate buffer salt preparation 0.5% (w/v, weight in wet base) that deposition is used pH; Adopt QFF sepharose column chromatography purification; After the ultrafiltration desalination, lyophilize obtains the pure article 395mg of feruloyl esterase again, measures its enzyme 92600mU/g alive.
Figure IDA00002053025800011

Claims (10)

1. a strain feruloyl esterase superior strain, its preserving number is CGMCC No.5612.
2. bacterial strain according to claim 1 is characterized in that: this bacterial strain is the viride bacterial strain, and the gene order that this its 16S rDNA is 16S rDNA is shown in Seq ID No:1.
3. method of utilizing the said bacterial strain of claim 1 to produce feruloyl esterase; The extraction separation and the purification step that mainly comprise trichoderma viride liquid fermenting and feruloyl esterase; It is characterized in that: in the trichoderma viride liquid fermenting, use feruloyl esterase generation and accumulation and induce operation
Described feruloyl esterase produces and accumulation induces the operation concrete steps following:
A. inducible factor screening:
From the regulation and control aspect screening inducible factor of ferulic acid derivative, vitamins growth factor, enzyme system, inducible factor is selected from vitamins or ferulic acid derivative or induces ion or other its inducible factors or its mixture;
B. inducible factor method of use:
Vitamins or ferulic acid derivative or induce ion to be added in the fermention medium in conjunction with adding carbon source and nitrogenous source mid-term, are used to induce and promote to produce feruloyl esterase.
4. according to the said preparation method of claim 3, it is characterized in that: the ion of inducing in the said inducible factor is selected from one or more the mixture in boron ion or mg ion or cobalt ion or the molybdenum ion; Vitamins is selected from vitamins B or folic acid; Ferulic acid derivative is Ferulic acid methylester or Ferulic acid ethylester; Other inducible factor is one or more the mixture in chitin or chitosan or rice bran or the wheat bran.
5. according to the said preparation method of claim 3, it is characterized in that: concrete steps are following:
(1) trichoderma viride liquid fermenting:
A. actication of culture;
B. liquid seeds preparation;
C. fermentation: behind the actication of culture, expand numerous seed liquor, utilize liquid submerged fermentation method to obtain to contain the fermented liquid of feruloyl esterase again through liquid fermenting;
Fermenting process wherein: liquid seeds is received in the fermention medium with the inoculum size of 2-10v/v%, cultivated 48h, add the carbon source material of 0.5-1w/v% and the nitrogen source of 0.05-0.2w/v% for 26 ℃;
(2) Preparation of Ferulic Acid Esterase:
A. solid-liquid separation: after solid-liquid separation, clear liquid is used to prepare feruloyl esterase with the fermented liquid that contains feruloyl esterase of above-mentioned fermentation;
B. fermentation clear liquid adopts micro-filtration to remove colloid and insoluble particle, after ultra-filtration membrane concentrates, adds protective material, the spray-dried feruloyl esterase bullion that obtains.
6. according to the said preparation method of claim 3, it is characterized in that: concrete steps are following:
(1) trichoderma viride liquid fermenting:
A. actication of culture;
B. liquid seeds preparation;
C. fermentation: behind the actication of culture, expand numerous seed liquor, utilize liquid submerged fermentation method to obtain to contain the fermented liquid of feruloyl esterase again through liquid fermenting;
Fermenting process wherein: liquid seeds is received in the fermention medium with the inoculum size of 2-10v/v%, cultivated 48h, add the carbon source material of 0.5-1w/v% and the nitrogen source of 0.05-0.2w/v% for 26 ℃;
(2) Preparation of Ferulic Acid Esterase:
A. solid-liquid separation: after solid-liquid separation, clear liquid is used to prepare feruloyl esterase with the fermented liquid that contains feruloyl esterase of above-mentioned fermentation;
B. the clear liquid that above-mentioned steps A is obtained adopts micro-filtration to remove colloid and insoluble particle, after ultra-filtration membrane concentrates; Method with saltouing obtains the thick enzyme of feruloyl esterase, after redissolving again; Be mixed with the enzyme liquid of 0.5-2w/v%; Through column chromatography purification, after the ultrafiltration desalination, lyophilize obtains the pure article of feruloyl esterase.
7. according to claim 5 or 6 said preparing methods, it is characterized in that: the solid-liquid separation in the said step (2) is a Plate Filtration or link-suspended basket centrifuge is centrifugal or disc centrifuge is centrifugal; The aperture of microfiltration membrane is 0.1-0.5 μ m in the described step (3), and the aperture of ultra-filtration membrane is the molecular weight 2000-10000Dalton that holds back material; Described salting medium is sodium-chlor or sodium sulfate or sal epsom or ammoniumsulphate soln.
8. according to claim 5 or 6 said preparing methods; It is characterized in that: what column chromatography adopted in the described step (3) is ion exchange chromatography, and the chromatography media that uses is DOWEX MXA-1 or Q-Sepharose FF or DEAE-Sephacel or DEAE-Sepharose FF or 717 strong basic ion exchange resins or D201 macroporous strong basic anionite-exchange resin.
9. according to claim 5 or 6 said preparing methods, it is characterized in that: the mycelial fermentative prepn concrete steps of described trichoderma viride are: the test tube slant bacterial classification that will be kept on the PDA substratum moves to activation 4h-8h under 20 ℃ of-25 ℃ of conditions; On the aseptic technique platform; To pass through activatory test tube slant bacterial classification with the zero(ppm) water after the 10mL sterilization and process thallus suspension liquid; Flushing goes into to be equipped with in the triangular flask of sterilization seed culture medium 24 ℃ of culture temperature, shaking speed 200r/min under aseptic condition; Incubation time is 48h, the preparation liquid seeds;
In fermention medium, add to account for and induce ionic salt containing of substratum gross weight 0.01-0.05w/w%; Account for the vitamins of substratum gross weight 0.005-0.02w/w%, and account for ferulic acid derivative or other its inducible factors or its mixture of substratum gross weight 0.1-4w/w%; Liquid seeds is received in the fermention medium under the condition of aseptic technique with the inoculum size of 2-10v/v%, cultivated 48h for 26 ℃, add the carbon source material of 0.5-1w/v% and the nitrogen source of 0.05-0.2w/v%, cultivate 96-144h altogether.
10. according to the said preparation method of claim 9, it is characterized in that: described carbon source material is one or more the mixture in glucose or sucrose or the starch; Described nitrogen source peptone or yeast soak one or more the mixture in powder or yeast powder or the steeping water.
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