CN103923840A - Aspergillus niger for largely producing xylanase and application thereof - Google Patents
Aspergillus niger for largely producing xylanase and application thereof Download PDFInfo
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Abstract
The invention discloses aspergillus niger for largely producing xylanase and application thereof. The collection number of the Aspergillus niger SM751 is CGMCC No. 8670. The xylanase generated by the Aspergillus niger SM751 is activated in enzyme activity under the action of mixing of multiple inhibitors; compared with blank control, the maximal activation rate of the enzyme activity achieves 33.43%. The Aspergillus niger SM751 provided by the invention can be used for largely producing xylanase which has high enzyme activity and can tolerate the pretreated fermented inhibitors, thereby having wide application in the field of lignocellulose enzymolysis.
Description
Technical field:
The invention belongs to microorganism fermentation and enzyme engineering Application Areas thereof, be specifically related to a kind of aspergillus niger (Aspergillus niger) SM751 of high yield zytase and the zytase producing, 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 to be only second to cellulosic important renewable carbon resource storehouse, can be used for widely manufacturing biofuel and many biochemical refining product.In the plant material course of processing that is rich in hemicellulose raw material, adding zytase can significantly accelerated reaction process, promotes other enzyme 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 by adding the cellulose mixture enzyme of the enzymes such as zytase the deciding factor that reduces lignocellulose field enzyme cost and improve enzymolysis efficiency.Varnai A(2011) result of study show, for the lignocellulose that derives from cork, hardwood and farm crop, zytase as the secondary enzyme of cellulase can reduce significantly cellulase with enzyme cost, improve the enzymolysis efficiency of cellulase.Its possible mechanism is blocking action by eliminating xylan or degraded wood oligose for the obstruction of cellulase absorption 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 10% hydrolysis sugar.Even have scholar Hu J(2011) think, can utilize the replacement cellulase of zytase part.
Zytase has important use value in lignocellulosic material producd fibers ethanol.But, not all zytase can be applicable to pre-treatment lignocellulose afterwards.Lignocellulose after pre-treatment, often contains 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, only there are several sections of researchs to involve the impact of fermentation inhibitor for zytase.De Souza Moreira(2013) research show, Vanillin and forulic acid have certain restraining effect or activation for the zytase that derives from terreus, but the xylanase activity that in this research, terreus used produces is extremely low.And there is not yet report with 5 hydroxymethyl furfural for the impact of the pretreated zytase enzymolysis of lignocellulose about furfural, acetic acid.In addition, yeast fermentation process aborning ethanol also can produce restraining effect to the enzymolysis of zytase.
Current extremely rare on the impact research report of zytase enzymolysis performance about pretreatment secondary fermentation inhibition.
Summary of the invention:
First object of the present invention is to provide a kind of aspergillus niger (Aspergillus niger) 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: CGMCC No.8670.
Aspergillus niger of the present invention (Aspergillus niger) SM751 is that in the material such as deadwood and rotten leaf, soil from Mu Lun protective belt, Guangxi province screening and culturing is out.Its classification of fungi is learned and is shown: originally this bacterium mycelium is white in color, and after about 36-48h, colony colour becomes grey black gradually, finally can produce vigorous, dense black spore at 48h.Ordinary method is extracted the 18s rDNA of fungi, its nucleotide sequence is as shown in SEQ ID NO.1, show through 18s rDNA sequential analysis, it belongs to aspergillus niger (Aspergillus niger), by its called after aspergillus niger (Aspergillus niger) 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: CGMCC No.8670.
Utilize xylan (Beech wood, SIGMA) measure xylanase activity, find aspergillus niger (Aspergillus niger) SM751 energy high yield zytase, the solid fermentation enzyme work of this enzyme reaches as high as 10446IU/g, optimal reaction pH value is 5.0, between pH3.0～6.0, enzyme is lived residual rate up to 93-99.8%.In the time of pH6.5, enzyme work is 78% of best pH enzyme work.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 enzymes during with 55 DEG C residual rate of living is respectively 80.41%, 98.96% and 79.01%.
Therefore, second object of the present invention is to provide a kind of zytase, it is characterized in that, taking aspergillus niger (Aspergillus niger), SM751, as fermentation strain, makes through fermentation.
The 3rd object of the present invention is to provide aspergillus niger (Aspergillus niger) SM751 in the application of producing 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 work is activated, and compares blank, and enzyme the highest activity ratio alive has reached 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 (Aspergillus niger) SM751, the high enzyme of its energy high yield zytase that live, that can tolerate pretreatment secondary fermentation inhibition, therefore has a wide range of applications in lignocellulose enzymolysis field.
Aspergillus niger of the present invention (Aspergillus niger) 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: CGMCC No.8670.
Brief description of the drawings:
Fig. 1 is pH value is produced xylanase activity on aspergillus niger of the present invention (Aspergillus niger) SM751 impact as fermentation parameter;
Fig. 2 is the pH enzymatic property that aspergillus niger of the present invention (Aspergillus niger) SM751 produces zytase;
Fig. 3 is the temperature enzymatic property that aspergillus niger of the present invention (Aspergillus niger) SM751 produces zytase;
Fig. 4 is various fermentation inhibitor produces zytase effect to aspergillus niger of the present invention (Aspergillus niger) SM751;
Fig. 5 is the phylogenetic tree that the 18S rDNA of application aspergillus niger of the present invention (Aspergillus niger) SM751 builds.
Embodiment:
Following examples are to further illustrate of the present invention, instead of limitation of the present invention.
Embodiment 1: the screening of bacterial strain
Screening sample is the materials such as deadwood and rotten leaf, soil.Screening sample is after suitable processing, and gradient dilution is coated xylan primary dcreening operation substratum, and the formula of this substratum is: in every liter of substratum, contain KH
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, the larger bacterium colony purifying on PDA substratum of picking hydrolysis transparent circle, the bacterial strain after purifying carries out the preservation of PDA inclined-plane.
Producing zytase strain fermentation vigor sieves again: the purifying bacterial strain that PDA inclined-plane is preserved is inoculated into respectively the fermentation of equivalent and sieves again liquid medium, and 30 DEG C, 120rpm, cultivates 6d, obtains fermented liquid (being 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 of 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 suitable dilution, with the accurate clock reaction 30min of stopwatch, add immediately 2mL DNS termination reaction, boiling water bath 5min, in 540nm place photometry absorption value (OD).Then select the bacterial strain that Xylanase activity is higher to carry out appropriate preservation.It is Mandel ' the s nutritive medium of revising that liquid medium is sieved in described fermentation again, and it is on the basis of former Mandel ' s nutritive medium, to remove yeast powder and peptone adds corn cob meal again, and making its final concentration is 30g/L.
Obtain the high bacterial strain of 1 strain Xylanase activity through screening, by this bacterial strain called after bacterial strain SM751.
Adopt modified CTAB method to extract the total DNA of bacterial strain SM751, select universal primer ITS1:5 '-TCC GTA GGT GAA CCT GCG G-3 ' and ITS4:5 '-TCC TCC GCT TAT TGA TAT GC-3 ' of amplification fungi ITS sequence to carry out the amplification of sequence to the total DNA of aspergillus niger (Aspergillus niger) SM751.In the PCR reaction system of 20 μ L, contain 10 × Buffer2 μ L(containing MgCl
2, 2.5mmol/L), dNTP(10mmol/L) 0.4 μ L, primer amount is 10pmol, rTag(5U/ μ L) and 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, and 94 DEG C of sex change 1min, 52 DEG C of annealing 50s, 72 DEG C are extended 50s, 35 circulations, 72 DEG C are extended 10min.Pcr amplification product adopts DNA gel to reclaim the test kit recovery of tapping rubber.Check order.Through order-checking, its sequence is as shown in SEQ ID NO.1.Known array in this sequence and GenBank database is carried out to BLAST comparative analysis, and obtain the 18S rDNA sequence of relevant kind 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 (Aspergillus niger) 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: CGMCC No.8670.
Embodiment 2: the preparation of zytase and enzyme activity thereof, zymologic property are measured
1. the preparation of xylanase preparation: by postactivated through seed culture aspergillus niger (Aspergillus niger) SM751.Seed culture medium is Mandel ' the s nutritive medium (with embodiment 1) of revising, 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, and taking the mixture of corn cob and wheat bran as substrate and propping material, with 1.4g/L (NH4)
2sO
4, 2.0g/L KH
2pO
4, 0.3g/L CaCl
2, 0.3g/L MgSO
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 factors optimizations experiments), 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.
In triangular flask, add the acetate buffer solution of 40mL pH4.8, smash the solid fermentation substratum (the same) of caking to pieces with glass stick, sway 60min in 110rpm30 DEG C, directly draw without vat liquor 0.4mL centrifugal and that filter and carry out gradient dilution, 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 1g beech wood glycan and be placed in after 100mL beaker, pour the acetate buffer solution stirring at low speed 2.5h of 60mL pH4.8 into, then constant volume 100mL volumetric flask is configured to 1% beech xylan solution.Draw 0.9mL1% beech xylan solution (Beech wood, SIGMA) as substrate, preheating 5min adds the suitably zytase of dilution of 0.2mL again, at 50 DEG C of reaction 30min, add immediately 2mL DNS solution termination reaction, boiling water bath 5min, in 540nm place photometry absorption value (OD).After measured, the vigor of the zytase that aspergillus niger (Aspergillus niger) SM751 that step 1 obtains produces reaches 10446IU/g, has high enzyme activity.
The enzyme definition of living: the amount that catalysis produces 1 μ mol reduction wood sugar in 1min is defined as a unit of enzyme.
3. the mensuration of the zymologic property of zytase: press said determination method, in the constant situation of other condition, regulate damping fluid to carry out enzyme activity determination reaction to different pH, different temperature, to record the highest enzyme work as 100%, the enzyme at the lower or temperature of other pH value is lived and is multiplied by 100% after the highest enzyme work is divided by and is relative enzyme and lives.Utilize corresponding damping fluid to carry out gradient dilution, at 50 DEG C, measure best pH.Under best pH condition, set differing temps to measure the optimum temps of enzyme.
Result shows, the optimum response pH of the zytase that aspergillus niger of the present invention (Aspergillus niger) SM751 produces be 5.0(as shown in Figure 2), and it is stable under the condition of pH5.0, to keep enzyme to live, optimal reactive temperature is 45 DEG C (as shown in Figure 3).This enzyme has good 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 (aspergillus niger (Aspergillus niger) SM751 produce zytase):
The restraining effect difference of different fermentation inhibitors to zytase.As shown in Figure 4, in the time that alcohol concn is 10.00g/L, the enzyme of zytase residual rate alive is 102.15%, and enzyme is lived and improved approximately 2.15%.In the time that acetic acid concentration is 10.10g/L, the enzyme of zytase residual rate alive 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 (Aspergillus niger) 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%, that is to say, 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 (Aspergillus niger) SM751 produces is higher, and enzyme work is up to 10446IU/g.In the time being applied to ligno-cellulose hydrolysate enzymolysis, there is good tolerance for the mixture of various single fermentation inhibitors or multiple fermentation inhibitor.
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 that utilizes aspergillus niger (Aspergillus niger) 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 for the illustrating of possible embodiments of the present invention, and this embodiment is not in order to limit the scope of the claims of the present invention, does not allly depart from equivalence of the present invention and implements or change, and all should be contained in the scope of the claims of the present invention.
Claims (4)
1. aspergillus niger (Aspergillus niger) SM751, its preserving number is: CGMCC No.8670.
2. a zytase, is characterized in that, taking aspergillus niger claimed in claim 1 (Aspergillus niger), SM751, as fermentation strain, makes through fermentation.
3. the application of aspergillus niger claimed in claim 1 (Aspergillus niger) SM751 on the zytase described in production claim 2.
4. the application of zytase claimed in claim 2 hemicellulose enzymolysis in ligno-cellulose hydrolysate.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410124121.7A CN103923840B (en) | 2014-03-28 | 2014-03-28 | A kind of aspergillus niger of high yield zytase and application thereof |
| AU2015237100A AU2015237100B2 (en) | 2014-03-28 | 2015-02-11 | High xylanase yield Aspergillus niger and application thereof |
| PCT/CN2015/072777 WO2015143961A1 (en) | 2014-03-28 | 2015-02-11 | High xylanase yield aspergillus niger and application thereof |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2015143961A1 (en) * | 2014-03-28 | 2015-10-01 | 中国科学院广州能源研究所 | High xylanase yield aspergillus niger and application thereof |
| CN110004070A (en) * | 2019-04-10 | 2019-07-12 | 南京工业大学 | One plant of production zytase aspergillus niger and its construction method and application |
| CN113913305A (en) * | 2021-11-22 | 2022-01-11 | 山东隆科特酶制剂有限公司 | Mutant strain of high-yield acidic xylanase and application thereof |
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| CN102041250A (en) * | 2009-10-19 | 2011-05-04 | 湖州礼来生物技术有限公司 | Method for producing xylanase by using aspergillus niger |
| CN103305428A (en) * | 2013-06-26 | 2013-09-18 | 武汉合缘绿色生物工程有限公司 | Aspergillus niger strain and application thereof |
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| GB9928968D0 (en) * | 1999-12-07 | 2000-02-02 | Danisco | Enzyme |
| CN101285044B (en) * | 2008-05-21 | 2010-07-14 | 浙江省农业科学院 | Xylanase for feeding and method for preparing same |
| EA201201126A1 (en) * | 2010-02-11 | 2013-05-30 | ДСМ АйПи АССЕТС Б.В. | POLYPEPTIDE, HAVING ACTIVITY OF CELL-BIOHYDROLASE, AND ITS APPLICATION |
| CN103923840B (en) * | 2014-03-28 | 2016-01-20 | 中国科学院广州能源研究所 | A kind of aspergillus niger of high yield zytase and application thereof |
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2014
- 2014-03-28 CN CN201410124121.7A patent/CN103923840B/en not_active Expired - Fee Related
-
2015
- 2015-02-11 AU AU2015237100A patent/AU2015237100B2/en not_active Ceased
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101591619A (en) * | 2009-07-03 | 2009-12-02 | 中国农业科学院饲料研究所 | Aspergillus niger strain and application thereof |
| CN102041250A (en) * | 2009-10-19 | 2011-05-04 | 湖州礼来生物技术有限公司 | Method for producing xylanase by using aspergillus niger |
| CN103305428A (en) * | 2013-06-26 | 2013-09-18 | 武汉合缘绿色生物工程有限公司 | Aspergillus niger strain and application thereof |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2015143961A1 (en) * | 2014-03-28 | 2015-10-01 | 中国科学院广州能源研究所 | High xylanase yield aspergillus niger and application thereof |
| CN110004070A (en) * | 2019-04-10 | 2019-07-12 | 南京工业大学 | One plant of production zytase aspergillus niger and its construction method and application |
| CN110004070B (en) * | 2019-04-10 | 2020-11-03 | 南京工业大学 | Xylanase-producing Aspergillus niger genetically engineered bacterium and construction method and application thereof |
| CN113913305A (en) * | 2021-11-22 | 2022-01-11 | 山东隆科特酶制剂有限公司 | Mutant strain of high-yield acidic xylanase and application thereof |
| CN113913305B (en) * | 2021-11-22 | 2023-10-13 | 山东隆科特酶制剂有限公司 | Mutant strain for high yield of acid xylanase and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| AU2015237100B2 (en) | 2018-05-31 |
| WO2015143961A1 (en) | 2015-10-01 |
| AU2015237100A1 (en) | 2016-11-10 |
| CN103923840B (en) | 2016-01-20 |
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