CN103992954B - A kind of aspergillus niger of high yield zytase and application thereof - Google Patents
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Abstract
The invention discloses a kind of aspergillus niger and application thereof of high yield zytase. Aspergillus niger (Aspergillus? niger) SM24/a, does is deposit number: CGMCC? No.8671. Aspergillus niger SM24/a provided by the invention, its can produce a kind of for ethanol, acetic acid, furfural, vanillic aldehyde, forulic acid with and composition thereof there is the zytase of better tolerance, and it is high that this bacterium producing multi enzyme preparation produces Xylanase activity, the solid fermentation enzyme work of this zytase reaches as high as the amount that 10801IU/g(catalysis in 1min produces 1 μ mol wood sugar and is defined as an enzyme unit), optimal reaction pH value is 5.6, optimal reactive temperature is 37 DEG C, and 50 DEG C time, enzyme residual rate alive is 79.01%. In addition, 5 hydroxymethyl furfural is inhibited for this enzyme. 5 hydroxymethyl furfural is in the time that individualism or mixing exist, and its concentration and inhibiting rate present certain linear relationship. Therefore, zytase of the present invention also can be used as the qualitative and even quantitative indicator of the concentration of 5 hydroxymethyl furfural.
Description
Technical field:
The invention belongs to microorganism fermentation and enzyme engineering application thereof, be specifically related to a kind of aspergillus niger of high yield zytase(Aspergillusniger) SM24/a and this zytase suppress method fast detecting 5 hydroxymethyl furfural concentration at Application and preparation enzymePreparation in application.
Background technology:
Zytase, English name xylanase, is called for short XYL, is a kind of pyrrole of energy catalyzing hydrolysis β-Isosorbide-5-Nitrae xylan glycosidic bondThe wood sugar of muttering, thus discharge the enzyme of wood sugar and wood oligose. In the process of hemicellulose raw material and even cellulosic material, addAdding zytase can significantly accelerated reaction process, improves the operating characteristic of product, reduces cost, energy consumption and the environment of production technologyPollute. Therefore, zytase is widely applied to traditional field and the wood fibres such as paper pulp papermaking, food, feed and weavingElement ethanol field.
Zytase has important Research Significance and great application prospect in lignocellulosic biorefining field. PryorSW(2012) think and add after zytase, can promote the cellulase degradation of bagasse after immersing in liquid nitrogen. SillsDL(2012)By adding zytase in the switchgrass after alkali treatment, can further improve the yield of reduced sugar. Zytase is shortEntering the pretreated effect of cellulase degradation is approved widely. Even, BillardH(2012) think,Be only and reduce lignocellulosic field enzyme cost and improve enzymolysis efficiency by adding the cellulose mixture enzyme of the enzyme such as zytaseDeciding factor.
But, being applied in lignocellulosic biorefining field at zytase, zytase also will face complicated enzymeSeparate conditions and environment. It should be noted that the impact of the mortifier producing after pretreatment on zytase enzymolysis performance. But,Up to now, rarely seen mortifier is for the effect research of zytase, and studied mortifier kind is on the low side.
Not every zytase all have preferably for the mixture of multiple single mortifier or multiple mortifier orGood tolerance. Good zytase of tolerance that neither be all, its bacterial strain can high yield zytase. As deSouzaStudies show that Moreira(2013), vanillic aldehyde and forulic acid have certain inhibition for the zytase that derives from Aspergillus terreusEffect, but its Aspergillus terreus bacterial strain used product xylanase activity is extremely low.
Summary of the invention:
First object of the present invention is to provide a kind of aspergillus niger (Aspergillusniger) SM24/a of high yield zytase, shouldBacterium 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, and deposit number is: CGMCCNo.8671.
Aspergillus niger of the present invention (Aspergillusniger) SM24/a is that screening and culturing is out from the material such as deadwood and rotten leaf, soil.Its classification of fungi is learned and is shown: this bacterium mycelium is white in color, and bacterium colony is little and fine and close, can produce black spore. PDA medium culture 1It starts long white hypha, cultivates 36h left and right and starts long black spore.
The taxonomy of aspergillus niger of the present invention (Aspergillusniger) SM24/a is determined according to following method:
Conventional method is extracted the 18srDNA of fungi, and its nucleotide sequence is as shown in SEQIDNO.1. Through comparative analysis and utilizationBIOLOG identification systems, aspergillus niger of the present invention (Aspergillusniger) SM24/a belongs to aspergillus, by black its called afterAspergillus (Aspergillusniger) SM24/a, this bacterium was preserved in Chinese microorganism strain preservation management on December 31st, 2013Committee's common micro-organisms center (CGMCC), deposit number is: CGMCCNo.8671.
Utilize xylan (Beechwood, SIGMA) to measure xylanase activity, find aspergillus niger (Aspergillus of the present inventionNiger) SM24/a energy high yield zytase, the solid fermentation enzyme work of this zytase reaches as high as 10801IU/g(at 1minThe amount that interior catalysis produces 1 μ mol wood sugar is defined as an enzyme unit), optimal reaction pH value is 5.6, optimal reactive temperature is 37 DEG C,50 DEG C time, enzyme residual rate alive is 79.01%.
Therefore, second object of the present invention is to provide a kind of zytase, it is characterized in that, with aspergillus niger (AspergillusNiger) SM24/aW2 is fermentation strain, makes through fermentation.
The 3rd object of the present invention is to provide aspergillus niger (Aspergillusniger) SM24/a in the application of producing on zytase.
The 4th object of the present invention is to provide zytase and suppresses method fast detecting 5 hydroxymethyl furfural concentration at Application and preparation enzymeApplication in preparation.
Aspergillus niger provided by the invention (Aspergillusniger) SM24/a, its can produce a kind of for ethanol, acetic acid, furfural,Vanillic aldehyde, forulic acid with and composition thereof there is the zytase of better tolerance, and this bacterium producing multi enzyme preparation produces Xylanase activity utmost pointHeight, the solid fermentation enzyme work of this zytase reaches as high as 10801IU/g(catalysis in 1min, and to produce the amount of 1 μ mol wood sugar fixedJustice is an enzyme unit), optimal reaction pH value is 5.6, and optimal reactive temperature is 37 DEG C, and 50 DEG C time, enzyme residual rate alive is 79.01%.In addition, 5 hydroxymethyl furfural is inhibited for this enzyme. 5 hydroxymethyl furfural in the time that individualism or mixing exist, itsConcentration and inhibiting rate present certain linear relationship. Therefore, zytase of the present invention also can be used as the concentration of 5 hydroxymethyl furfuralQualitative and even quantitative indicator.
Aspergillus niger of the present invention (Aspergillusniger) SM24/a is preserved in Chinese microbial bacteria on December 31st, 2013Plant preservation administration committee common micro-organisms center (CGMCC), preservation address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3Number, deposit number is: CGMCCNo.8671.
Brief description of the drawings:
Fig. 1 is that pH value is produced xylanase activity as fermentation parameter to aspergillus niger of the present invention (Aspergillusniger) SM24/aImpact;
Fig. 2 is the pH enzymatic property that aspergillus niger of the present invention (Aspergillusniger) SM24/a produces zytase;
Fig. 3 is the temperature enzymatic property that aspergillus niger of the present invention (Aspergillusniger) SM24/a produces zytase;
Fig. 4 is containing the relation between variable concentrations and the xylanase activity retention rate of the mixing mortifier of 5 hydroxymethyl furfural;
Fig. 5 is the phylogenetic tree that the 18SrDNA of application aspergillus niger of the present invention (Aspergillusniger) SM24/a builds.
Detailed description of the invention:
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 xylanPrimary dcreening operation culture medium, the formula of this culture medium is: in every liter of culture medium, contain KH2PO40.5g，(NH4)2SO42.0g，MgSO4·7H2O0.25g, xylan (purchased from source, Shanghai consor thing) 5.0g, agar 18～20g, surplus is water, pH5.6. 30 DEG CCultivate 4～7d, the larger bacterium colony purifying on PDA culture medium of picking hydrolysis transparent circle, the bacterial strain after purifying carries out PDA inclined-planePreserve.
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 againIn liquid medium, 30 DEG C, 120rpm, cultivates 6d. According to Bailey(1992) etc. people assay 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 2.5hRear constant volume is 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 preheatings5min, accurately adds enzyme liquid (zymotic fluid of the purifying bacterial strain) 0.2mL of suitable dilution, with the accurate clock reaction 30min of stopwatch,Add immediately 2mLDNS cessation reaction, boiling water bath 5min, in 540nm place photometry absorption value (OD). Then select xylanThe bacterial strain that enzyme activity vigor is higher carries out properly preservation. It is Mandel ' the s nutrition of revising that liquid medium is sieved in described fermentation againLiquid, it is on the basis of former Mandel ' s nutrient solution, to remove dusty yeast and peptone adds maize cob meal again, makes its final concentration be30g/L。
Obtain the high bacterial strain of 1 strain Xylanase activity through screening, by this bacterial strain called after bacterial strain SM24/a.
Bacterial strain SM24/a mycelium is white in color, and bacterium colony is little and fine and close, can produce black spore. PDA medium culture starts for 1 dayLong white hypha, cultivates 36h left and right and starts long black spore.
Adopt modified CTAB method to extract the total DNA of bacterial strain, select universal primer the ITS1:5 '-TCC of amplification fungi ITS sequenceGTAGGTGAACCTGCGG-3 ' and ITS4:5 '-TCCTCCGCTTATTGATATGC-3 ' are to aspergillus niger(Aspergillusniger) the total DNA of SM24/a carries out the amplification of sequence. In the PCR reaction system of 20 μ L, contain10 × Buffer2 μ L(is containing MgCl2,2.5mmol/L), dNTP(10mmol/L) 0.4 μ L, upstream and downstream 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 ultra-pure water. Pcr amplification conditionFor: 95 DEG C of denaturation 3min; 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 prolongedStretch 10min. Pcr amplification product adopts DNA gel to reclaim the kit recovery of tap rubber, and checks order, and its sequence is as SEQIDShown in NO.1. Known array in this sequence and GenBank database is carried out to BLAST comparative analysis, and obtain from databaseThe 18SrDNA sequence of the kind of must being correlated with, phylogenetic tree construction, as shown in Figure 5, through comparative analysis and in conjunction with BIOLOGQualification result, this Pseudomonas is in aspergillus niger, and by its called after aspergillus niger (Aspergillusniger) SM24/a, this bacterium is in 2013Be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), address: north on December 31, inNo. 3, No. 1, North Star West Road, Jing Shi Chaoyang District institute, Institute of Microorganism, Academia Sinica, its deposit number is: CGMCCNo.8671.
Embodiment 2: the preparation of zytase and enzyme activity thereof, zymologic property are measured
1. the preparation of xylanase preparation: by aspergillus niger (Aspergillusniger) SM24/a activation, through seed culture, plantSub-culture medium is Mandel ' the s nutrient solution (with embodiment 1) of revising, then adds xylan, and making its final concentration is 5g/L, 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. WillSeed culture fluid is with 7.5%(v/w) inoculum concentration be inoculated in solid fermentation culture medium, the formula of this solid fermentation culture medium is:The mass ratio 1:5 of corncob and wheat bran, taking the mixture of corncob and wheat bran as substrate and backing material, with 1.4g/L (NH4)2SO4，2.0g/LKH2PO4，0.3g/LCaCl2，0.3g/LMgSO4, add appropriate trace element (FeSO4·7H2O，5mg/L；CoCl2,20mg/L；MnSO4,1.6mg/L；ZnSO4, 1.4mg/L) and be configured to nutrient solution, solvent isWater, the initial pH of this nutrient solution be 5.0(as Fig. 1, this pH value is determined after a series of single factors optimizations experiments), withAfter configuring solid-state fermentation culture medium, material-water ratio (mixture of corncob and wheat bran: nutrient solution) 1:3.5 carries out sterilizing. Inoculum concentrationFor 7.5%v/v ferments. After fermentation 92-108h, its zymotic fluid is xylanase preparation, carries out xylanase activity surveyFixed.
In triangular flask, add the acetate buffer solution of 40mLpH4.8, the solid fermentation culture medium of smashing caking with glass bar to pieces is (sameOn), sway 60min in 110rpm30 DEG C, directly draw leaching liquor 0.4mL and carry out gradient dilution, until reach suitable doublyCount, then carry out the mensuration of xylanase activity.
2. the enzyme activity determination of zytase: according to Bailey(1992) etc. people assay method and suitably revise. Take1g beech wood glycan is placed in after 100mL beaker, pours the acetate buffer solution stirring at low speed 2.5h of 60mLpH4.8 into, then fixedHold 100mL volumetric flask and be configured to 1% beech xylan solution. Draw 0.9mL1% beech xylan solution (BeechWood, SIGMA) as substrate, preheating 5min adds the suitably xylanase preparation of dilution of 0.2mL again, 50 DEG C of reactions30min, adds 2mLDNS solution cessation reaction immediately, and boiling water bath 5min, in 540nm place photometry absorption value (OD). WarpMeasure the work of the zytase in the xylanase preparation that aspergillus niger (Aspergillusniger) SM24/a that step 1 obtains producesPower reaches 10801IU/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 an enzyme unit.
3. the mensuration of the zymologic property of zytase: press said determination method, in the constant situation of other conditions, regulate buffer solutionCarry out enzyme activity determination reaction to different pH, different temperature, to record the highest enzyme work as 100%, this condition is poly-for recording woodCarbohydrase optimum reaction conditions. Utilize corresponding buffer solution to carry out gradient dilution, at identical temperature, measure best pH. At 50 DEG CUnder best pH condition, set different temperatures to measure the optimum temperature of enzyme.
Result shows, the optimum response pH of the zytase that aspergillus niger of the present invention (Aspergillusniger) SM24/a produces is5.6(Fig. 2), and it is stable under the condition of pH5.6, to keep enzyme to live, and optimal reactive temperature is 37 DEG C (as shown in Figure 3).
Embodiment 3: zytase is for the tolerance performance of fermentation inhibitor
1. the tolerance performance of zytase to single mortifier
The variable concentrations of zytase to same fermentation inhibitor (as ethanol, acetic acid, furfural, vanillic aldehyde and forulic acid),Its relative enzyme work presents certain difference, still, overall on, all retained the higher enzyme residual rate of living. In ethanol, secondThe concentration of acid is while being 5g/L, 15g/L and 30g/L, and furfural (2.5g/L, 1.25g/L and 0.4g/L), forulic acid (1.2g/L,0.6g/L and 0.2g/L) and when the concentration range of vanillic aldehyde (1.3g/L, 0.65g/L and 0.2g/L), zytase of the present inventionEnzyme live retention rate 93～108%.
2. zytase is to being added with the tolerance performance of mortifier of mixing of 5 hydroxymethyl furfural:
As shown in Figure 4, in the mixing mortifier system of higher concentration (ethanol, acetic acid, furfural, vanillic aldehyde, forulic acid andThe concentration of 5 hydroxymethyl furfural is respectively (30g/L, 30g/L, 2.1g/L, 1.2g/L, 1.27g/L and 1.24g/L), the present inventionZytase enzyme live retention rate be 83.30%; In the mixing mortifier system of intermediate concentration (ethanol, acetic acid, furfural,The concentration of vanillic aldehyde, forulic acid and 5 hydroxymethyl furfural be respectively (15g/L, 15g/L, 1.1g/L, 0.6g/L, 0.6g/L and0.6g/L), the enzyme of zytase of the present invention retention rate alive is 90.10%; In the mixing mortifier system of low concentration (ethanol,The concentration of acetic acid, furfural, vanillic aldehyde, forulic acid and 5 hydroxymethyl furfural be respectively 5g/L, 5g/L, 0.35g/L, 0.2g/L,0.2g/L and 0.2g/L), the enzyme retention rate alive of zytase of the present invention is 95.59%. Therefore, can utilize zytase enzymeInhibition method is indicated the mixing mortifier that contains 5 hydroxymethyl furfural.
Zytase of the present invention completely different from zytase of the prior art to the tolerance of mortifier, this toleranceZytase there are no cross report.
Zytase of the present invention has linear indicative function for the 5 hydroxymethyl furfural mixing in mortifier, this linear instructionEffect is there are no crossing report. Therefore, the application of zytase of the present invention belongs to a brand-new field.
Above-mentioned detailed description is for the illustrating of possible embodiments of the present invention, and this embodiment is not of the present invention in order to limitThe scope of the claims, 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 (2)
1. aspergillus niger (Aspergillusniger) SM24/a, its deposit number is: CGMCCNo.8671.
2. aspergillus niger claimed in claim 1 (Aspergillusniger) SM24/a is in the application of producing in zytase.
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| CN201410125861.2A CN103992954B (en) | 2014-03-28 | 2014-03-28 | A kind of aspergillus niger of high yield zytase and application thereof |
| PCT/CN2015/072766 WO2015143960A1 (en) | 2014-03-28 | 2015-02-11 | High xylanase yield aspergillus niger and application thereof |
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| CN105063170B (en) * | 2015-08-03 | 2018-11-13 | 中国热带农业科学院海口实验站 | A kind of production zytase microorganism screens culture medium and its cultural method |
| CN107760607B (en) * | 2016-08-18 | 2020-12-01 | 青岛蔚蓝生物集团有限公司 | Aspergillus niger mutant strain and application thereof |
| 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 |
| 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 |
| CN114437938B (en) * | 2022-01-14 | 2023-06-06 | 山东隆科特酶制剂有限公司 | Strain for high-yield high-temperature-resistant acid beta-mannase and application thereof |
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Effective date of registration: 20180130 Address after: 712100 room 227 of the two floor of the comprehensive service hall of the Yangling demonstration zone, Shaanxi Province Patentee after: Yangling future Zhongke Environmental Protection Technology Co., Ltd. Address before: Guangzhou City, Guangdong province 510640 energy road No. 2 Tianhe District Wushan Patentee before: Guangzhou Institute of Energy Conversion, Chinese Academy of Sciences |