CN103923840B - A kind of aspergillus niger of high yield zytase and application thereof - Google Patents

A kind of aspergillus niger of high yield zytase and application thereof Download PDF

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CN103923840B
CN103923840B CN201410124121.7A CN201410124121A CN103923840B CN 103923840 B CN103923840 B CN 103923840B CN 201410124121 A CN201410124121 A CN 201410124121A CN 103923840 B CN103923840 B CN 103923840B
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zytase
enzyme
aspergillus niger
aspergillusniger
fermentation
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CN103923840A (en
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袁振宏
许敬亮
何敏超
张宇
孔晓英
陈小燕
粱翠谊
郭颖
刘云云
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Guangzhou Institute of Energy Conversion of CAS
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
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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)
    • C12N9/2477—Hemicellulases not provided in a preceding group
    • C12N9/248—Xylanases
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    • 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)
    • C12Y302/01008—Endo-1,4-beta-xylanase (3.2.1.8)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/66—Aspergillus
    • C12R2001/685—Aspergillus niger

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Abstract

The invention discloses a kind of aspergillus niger and application thereof of high yield zytase.Aspergillus niger (Aspergillus? niger) SM751, does is its preserving number: CGMCC? No.8670.The zytase that this bacterium produces is under the mixing effect of multiple inhibition, and enzyme is lived and activated, and compares blank, and enzyme the highest activity ratio alive reaches 33.43%.Aspergillus niger (Aspergillus provided by the invention? niger) SM751, zytase that its energy high yield height enzyme is lived, that can tolerate pretreatment secondary fermentation inhibition, therefore has a wide range of applications in lignocellulose enzymolysis field.

Description

A kind of aspergillus niger of high yield zytase and application thereof
Technical field:
The invention belongs to fermentable and enzyme engineering Application Areas thereof, aspergillus niger (Aspergillusniger) SM751 being specifically related to a kind of high yield zytase and the zytase produced, and the application of this zytase hemicellulose enzymolysis in ligno-cellulose hydrolysate.
Background technology:
The annual hemicellulose output in the whole world is 6 × 10 10ton is only second to cellulosic important renewable carbon resources bank, can be used for widely manufacturing biofuel and many biochemical refining product.In the plant material course of processing being rich in hemicellulosic material, adding zytase can significantly accelerated reaction process, promotes that other enzyme is as the hydrolysis result of the enzymes such as cellulase, polygalacturonase and mannase, significantly improves the service performance of product.Therefore, zytase is widely applied to traditional field and the lignocellulose ethanol fields such as paper pulp papermaking, food, feed and weaving.
Zytase has important using value in lignocellulose enzymolysis field.There is scholar to think, be only the deciding factor reducing lignocellulose field enzyme cost and improve enzymolysis efficiency by the cellulose mixture enzyme adding the enzymes such as zytase.VarnaiA(2011) result of study shows, for the lignocellulose deriving from cork, hardwood and farm crop, zytase as the secondary enzyme of cellulase can reduce significantly cellulase with enzyme cost, the enzymolysis efficiency improving cellulase.Its possible mechanism is that blocking action by eliminating xylan or the degraded wood oligose obstruction of adsorbing for cellulase is to promote the enzymolysis of cellulase.Alvira(2011) result of study shows, zytase can reach 29.9% with the degree of cooperating of cellulase, and can increase by the hydrolysis sugar of 10%.Even have scholar HuJ(2011) think, the replacement cellulase of zytase part can be utilized.
Zytase has important use value in lignocellulosic material producd fibers ethanol.But, not all zytase can be applicable to pre-treatment after lignocellulose.Lignocellulose after pre-treatment, often containing fermentation inhibitor more or less in its pretreatment fluid.Fermentation inhibitor has acetic acid, furfural, 5 hydroxymethyl furfural, Vanillin, forulic acid etc.In the zytase research report of domestic and international thousands of sections, several sections of researchs are only had to involve the impact of fermentation inhibitor for zytase.DeSouzaMoreira(2013) research shows, Vanillin and forulic acid have certain restraining effect or activation for the zytase deriving from terreus, but the xylanase activity that terreus used in this research produces is extremely low.And about furfural, acetic acid, with 5 hydroxymethyl furfural, for the impact of the pretreated zytase enzymolysis of lignocellulose, there is not been reported.In addition, Yeast fermentation process aborning ethanol also can produce restraining effect to the enzymolysis of zytase.
Current extremely rare about the influence research report of pretreatment secondary fermentation inhibition to zytase enzymolysis performance.
Summary of the invention:
First object of the present invention is to provide a kind of aspergillus niger (Aspergillusniger) SM751 of high yield zytase, this bacterium is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) on December 31st, 2013, preservation address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, deposit number is: CGMCCNo.8670.
Aspergillus niger of the present invention (Aspergillusniger) SM751 is from Guangxi province wood by screening and culturing in the material such as deadwood and rotten leaf, soil protective belt out.Its fungal taxonomy shows: this bacterium mycelium is originally in white, and after about 36-48h, colony colour becomes grey black gradually, finally can produce vigorous, dense black spore at 48h.Ordinary method extracts the 18srDNA of fungi, its nucleotide sequence is as shown in SEQIDNO.1, show through 18srDNA sequential analysis, it belongs to aspergillus niger (Aspergillusniger), by its called after aspergillus niger (Aspergillusniger) SM751, this bacterium is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) on December 31st, 2013, preservation address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, deposit number is: CGMCCNo.8670.
Utilize xylan (Beechwood, SIGMA) xylanase activity is measured, find aspergillus niger (Aspergillusniger) SM751 energy high yield zytase, the solid fermentation enzyme of this enzyme is lived and is reached as high as 10446IU/g, optimal reaction pH value is 5.0, between pH3.0 ~ 6.0, enzyme lives residual rate up to 93-99.8%.When pH6.5, enzyme work is 78% of the work of Optimal pH enzyme.Therefore, this enzyme has very strong pH stability between pH3.0 ~ 6.0.Optimal reactive temperature is 45 DEG C; 37 DEG C, 50 DEG C are respectively 80.41%, 98.96% and 79.01% with the residual rate alive of enzyme when 55 DEG C.
Therefore, second object of the present invention is to provide a kind of zytase, it is characterized in that, obtains through fermentation for fermentation strain with aspergillus niger (Aspergillusniger) SM751.
3rd object of the present invention is to provide aspergillus niger (Aspergillusniger) SM751 and is producing the application on zytase.
Zytase of the present invention can tolerate pretreatment secondary fermentation inhibition.The zytase that this bacterium produces is under the mixing effect of multiple inhibition, and enzyme is lived and activated, and compares blank, and enzyme the highest activity ratio alive reaches 33.43%.
Therefore, the 4th object of the present invention is to provide the application of zytase hemicellulose enzymolysis in ligno-cellulose hydrolysate.
Aspergillus niger provided by the invention (Aspergillusniger) SM751, zytase that its energy high yield height enzyme is lived, that can tolerate pretreatment secondary fermentation inhibition, therefore has a wide range of applications in lignocellulose enzymolysis field.
Aspergillus niger of the present invention (Aspergillusniger) SM751, this bacterium is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) on December 31st, 2013, preservation address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, deposit number is: CGMCCNo.8670.
Accompanying drawing illustrates:
Fig. 1 is pH value produces xylanase activity on aspergillus niger of the present invention (Aspergillusniger) SM751 impact as fermentation parameter;
Fig. 2 is the pH enzymatic property that aspergillus niger of the present invention (Aspergillusniger) SM751 produces zytase;
Fig. 3 is the temperature enzymatic property that aspergillus niger of the present invention (Aspergillusniger) SM751 produces zytase;
Fig. 4 is that various different fermentation inhibitor produces the effect of zytase to aspergillus niger of the present invention (Aspergillusniger) SM751;
Fig. 5 is the phylogenetic tree that the 18SrDNA of application aspergillus niger of the present invention (Aspergillusniger) SM751 builds.
Embodiment:
Following examples further illustrate of the present invention, instead of limitation of the present invention.
Embodiment 1: the screening of bacterial strain
Screening sample is the material such as deadwood and rotten leaf, soil.Screening sample is after suitably processing, and gradient dilution coats xylan primary dcreening operation substratum, and the formula of this substratum is: containing KH in often liter of substratum 2pO 40.5g, (NH 4) 2sO 42.0g, MgSO 47H 2o0.25g, xylan (purchased from source, Shanghai consor thing) 5.0g, agar 18 ~ 20g, surplus is water, pH5.6.Cultivate 4 ~ 7d for 30 DEG C, bacterium colony purifying on PDA substratum that picking hydrolysis transparent circle is larger, the bacterial strain after purifying carries out the preservation of PDA inclined-plane.
Produce zytase strain fermentation vigor to sieve again: the fermentation that the purifying bacterial strain that PDA inclined-plane is preserved is inoculated into equivalent is respectively sieved liquid medium, 30 DEG C, 120rpm again, cultivates 6d, obtain fermented liquid (i.e. enzyme liquid).According to Bailey(1992) etc. people measuring method and suitably revise.Concrete grammar is: accurately take xylan 1g, at pH4.8(0.2mol/L) acetic acid ~ sodium-acetate buffer stirring at low speed 2.5h after constant volume be configured to 1% substrate.The xylan substrate getting 0.9mL1% is placed in 15mL scale test tube, in 50 DEG C of constant temperature preheating 5min.Accurately add the enzyme liquid 0.2mL of suitably dilution, with the accurate clock reaction 30min of stopwatch, add 2mLDNS termination reaction immediately, boiling water bath 5min, in 540nm place light-metering absorption value (OD).Then the higher bacterial strain of Xylanase activity is selected to carry out appropriate preservation.It is Mandel ' the s nutritive medium revised that liquid medium is sieved in described fermentation again, and it on the basis of former Mandel ' s nutritive medium, removes yeast powder and peptone adds corn cob meal again, makes its final concentration be 30g/L.
Through the bacterial strain that screening acquisition 1 strain Xylanase activity is high, be bacterial strain SM751 by this Strain Designation.
Adopt modified CTAB method to extract bacterial strain SM751 STb gene, select universal primer the ITS1:5 '-TCCGTAGGTGAACCTGCGG-3 ' of amplification fungi ITS sequence and ITS4:5 '-TCCTCCGCTTATTGATATGC-3 ' to carry out the amplification of sequence to aspergillus niger (Aspergillusniger) SM751 STb gene.MgCl is contained containing 10 × Buffer2 μ L(in the PCR reaction system of 20 μ L 2, 2.5mmol/L), dNTP(10mmol/L) 0.4 μ L, primer amount is 10pmol, rTag(5U/ μ L) 0.2 μ L, the template DNA of about 50ng, all the other volumes are supplied with aseptic ultrapure water.Pcr amplification condition is: 95 DEG C of denaturation 3min, 94 DEG C of sex change 1min, 52 DEG C of annealing 50s, and 72 DEG C extend 50s, 35 circulations, and 72 DEG C extend 10min.Pcr amplification product adopts DNA gel recovery test kit to carry out rubber tapping and reclaims.Check order.Through order-checking, its sequence is as shown in SEQIDNO.1.Known array in this sequence and GenBank database is carried out BLAST comparative analysis, and the 18SrDNA sequence of related species is obtained from database, phylogenetic tree construction, as shown in Figure 5, through comparative analysis and in conjunction with BIOLOG qualification result, this Pseudomonas is in aspergillus niger, by its called after aspergillus niger (Aspergillusniger) SM751, this bacterium is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) on December 31st, 2013, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, its deposit number is: CGMCCNo.8670.
Embodiment 2: the preparation of zytase and enzyme activity, zymologic property mensuration
1. the preparation of xylanase preparation: by postactivated through seed culture for aspergillus niger (Aspergillusniger) SM751.Seed culture medium is Mandel ' the s nutritive medium (with embodiment 1) revised, and adds the xylan of 5g/L, initial pH5.6.Sterilizing 30min at 115 DEG C.The parameter of setting shaking table is: 30 DEG C, 120rpm, cultivates and obtain seed culture fluid after 3 days.By seed culture fluid with 10%(v/w) inoculum size be inoculated in solid fermentation substratum.The formula of solid-state fermentation culture medium is: the mass ratio 1:5 of corn cob and wheat bran, with the mixture of corn cob and wheat bran for substrate and propping material, with 1.4g/L (NH4) 2sO 4, 2.0g/LKH 2pO 4, 0.3g/LCaCl 2, 0.3g/LMgSO 4, add appropriate trace element (FeSO 47H 2o, 5mg/L; CoCl 2, 20mg/L; MnSO 4, 1.6mg/L; ZnSO 4, 1.4mg/L) and be configured to nutritive medium, solvent is water.The initial pH of this nutritive medium be 3.5(as Fig. 1, this pH value is determined after a series of single factor test optimization experiment), carry out sterilizing after configuring solid-state fermentation culture medium with material-water ratio (mixture of corn cob and wheat bran: nutritive medium) 1:3.5.Inoculum size is that 7.5%v/v ferments.After fermentation 92-108h, its fermented liquid is xylanase preparation, carries out xylanase activity mensuration.
The acetate buffer solution of 40mLpH4.8 is added in triangular flask, the solid fermentation substratum (the same) of caking is smashed to pieces with glass stick, 60min is swayed in 110rpm30 DEG C, direct absorption carries out gradient dilution without vat liquor 0.4mL that is centrifugal and that filter, until reach suitable multiple, then carry out the mensuration of xylanase activity.
2. the enzyme activity determination of zytase: according to Bailey(1992) etc. people measuring method and suitably revise.Take after 1g beech wood glycan is placed in 100mL beaker, pour the acetate buffer solution stirring at low speed 2.5h of 60mLpH4.8 into, then constant volume 100mL volumetric flask is configured to 1% beech xylan solution.Draw 0.9mL1% beech xylan solution (Beechwood, SIGMA) as substrate, preheating 5min adds the zytase that 0.2mL suitably dilutes again, at 50 DEG C of reaction 30min, add 2mLDNS solution termination reaction immediately, boiling water bath 5min, in 540nm place light-metering absorption value (OD).After measured, the vigor of the zytase that aspergillus niger (Aspergillusniger) SM751 that step 1 obtains produces reaches 10446IU/g, has high enzyme activity.
Enzyme is lived definition: in 1min, catalysis produces 1 μm of ol and reduces the amount of wood sugar and be defined as a unit of enzyme.
3. the mensuration of the zymologic property of zytase: by said determination method, when other condition is constant, damping fluid is regulated to carry out enzyme activity determination reaction to different pH, different temperature, to record the work of the highest enzyme for 100%, be multiplied by 100% after the enzyme under other pH value or at temperature is lived and the work of the highest enzyme is divided by and be the work of relative enzyme.Utilize corresponding damping fluid to carry out gradient dilution, at 50 DEG C, measure Optimal pH.Under the pH condition of the best, setting differing temps is to measure the optimum temps of enzyme.
Result shows, the optimum response pH of zytase that aspergillus niger of the present invention (Aspergillusniger) SM751 produces be 5.0(as shown in Figure 2), and under the condition of pH5.0, keep enzyme to live stable, and optimal reactive temperature is 45 DEG C (as shown in Figure 3).This enzyme has excellent pH stability between pH3.5 ~ 6.0, and its relative enzyme work is 93.45% ~ 99.84%.
Embodiment 3: fermentation inhibitor is for the effect of zytase
1. single fermentation inhibitor is for the restraining effect of zytase (zytase that aspergillus niger (Aspergillusniger) SM751 produces):
Different fermentation inhibitors is different to the restraining effect of zytase.As shown in Figure 4, when alcohol concn is 10.00g/L, the enzyme residual rate alive of zytase is 102.15%, and enzyme is lived and improved about 2.15%.When acetic acid concentration is 10.10g/L, the enzyme residual rate alive of zytase is 99.05%.When furfural content is 1.40g/L, the residual rate of zytase is 100.08%.When 5 hydroxymethyl furfural concentration is 1.20g/L, the residual rate of zytase is 95.64%.When the concentration of Vanillin is 1.10g/L, the residual rate of zytase is respectively 98.16%.When the concentration of forulic acid is 1.10g/L, the residual rate of zytase is 72.99%.Therefore, zytase of the present invention has good tolerance for fermentation inhibitor.
2. mix the restraining effect of inhibition for zytase (zytase that aspergillus niger (Aspergillusniger) SM751 produces):
The mixture of ethanol (11.90g/L), acetic acid (13.60g/L), furfural (1.40g/L), 5 hydroxymethyl furfural (1.10g/L), forulic acid (1.20g/L) and Vanillin (1.30g/L) has activation for zytase.Enzyme residual rate alive is 133.39%, and that is, under the effect of this fermentation inhibitor, xylanase activity is 11008IU/g, and activity ratio can reach 33.39%.
As can be seen here, the xylanase activity power that aspergillus niger of the present invention (Aspergillusniger) SM751 produces is higher, and enzyme work is up to 10446IU/g.When being applied to ligno-cellulose hydrolysate enzymolysis, the mixture for various single fermentation inhibitor or multiple fermentation inhibitor has excellent tolerance.
Zytase of the present invention completely different from zytase of the prior art to the tolerance of inhibition, the zytase of this tolerance is there are no crossing report.
The zytase utilizing aspergillus niger (Aspergillusniger) SM751 to produce takes the pretreated bagasse of enzymolysis high temperature liquid water.The condition of high-temperature liquid state water pretreatment bagasse is: 180 degree, nitrogen pressurization 4MPA, 20min, solid-to-liquid ratio 1:20.After adding the zytase enzymolysis 24h of 600IU, wood sugar yield is 75.63%.
Above-mentioned detailed description is illustrating for possible embodiments of the present invention, and this embodiment is also not used to limit the scope of the claims of the present invention, all do not depart from of the present invention equivalence implement or change, all should be contained in the scope of the claims of the present invention.

Claims (4)

1. aspergillus niger (Aspergillusniger) SM751, its preserving number is: CGMCCNo.8670.
2. a zytase, is characterized in that, obtains through fermentation for fermentation strain with aspergillus niger according to claim 1 (Aspergillusniger) SM751.
3. the application of aspergillus niger according to claim 1 (Aspergillusniger) SM751 on the zytase described in production claim 2.
4. the application of zytase according to claim 2 hemicellulose enzymolysis in ligno-cellulose hydrolysate.
CN201410124121.7A 2014-03-28 2014-03-28 A kind of aspergillus niger of high yield zytase and application thereof Expired - Fee Related CN103923840B (en)

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