WO2015143961A1 - 一种高产木聚糖酶的黑曲霉及其应用 - Google Patents

一种高产木聚糖酶的黑曲霉及其应用 Download PDF

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WO2015143961A1
WO2015143961A1 PCT/CN2015/072777 CN2015072777W WO2015143961A1 WO 2015143961 A1 WO2015143961 A1 WO 2015143961A1 CN 2015072777 W CN2015072777 W CN 2015072777W WO 2015143961 A1 WO2015143961 A1 WO 2015143961A1
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xylanase
aspergillus niger
enzyme activity
fermentation
application
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French (fr)
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袁振宏
许敬亮
何敏超
张宇
孔晓英
陈小燕
梁翠谊
郭颖
刘云云
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中国科学院广州能源研究所
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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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/145—Fungal isolates
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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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    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01008—Endo-1,4-beta-xylanase (3.2.1.8)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/66—Aspergillus
    • C12R2001/685—Aspergillus niger

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  • the invention belongs to the field of microbial fermentation and enzyme engineering application, and particularly relates to a high-yield xylanase Aspergillus niger SM751 and a xylanase produced thereby, and the xylanase hydrolyzed in lignocellulose The application of enzymatic hydrolysis of hemicellulose in liquid.
  • xylanase is widely used in traditional fields such as pulp and paper, food, feed and textile, and lignocellulosic ethanol.
  • Xylanase has important application value in the field of lignocellulose hydrolysis.
  • Some researchers believe that the addition of cellulase enzymes such as xylanase is the decisive factor in reducing the cost of enzymes in the field of lignocellulose and improving the efficiency of enzymatic hydrolysis.
  • the results of Varnai A (2011) show that for lignocellulose derived from softwood, hardwood and crops, xylanase as a secondary enzyme of cellulase can significantly reduce the enzyme cost of cellulase and increase cellulase. Enzymatic efficiency.
  • the possible mechanism is to promote the enzymatic hydrolysis of cellulase by eliminating the blocking effect of xylan or degrading the inhibition of cellulase adsorption by xylooligosaccharides.
  • Alvira (2011) showed that xylanase can cooperate with cellulase to 29.9% and increase hydrolyzed sugar by 10%. Even some scholars Hu J (2011) believe that xylanase can be used to partially replace cellulase.
  • Xylanase has important value in the production of fiber ethanol from lignocellulosic feedstock.
  • xylanases can be applied to lignocellulose after pretreatment.
  • the lignocellulose after pretreatment often contains more or less fermentation inhibitors in the pretreatment liquid.
  • the fermentation inhibitors are acetic acid, furfural, 5-hydroxymethylfurfural, vanillin, ferulic acid, and the like.
  • xylanase studies reported at home and abroad, only a few studies have implications for the effects of fermentation inhibitors on xylanase.
  • De Souza Moreira (2013) showed that vanillin and ferulic acid are derived from xylanase derived from Aspergillus terreus.
  • a first object of the present invention is to provide a high-yield xylanase-producing Aspergillus niger SM751 which was deposited on December 31, 2013 at the General Microbiology Center (CGMCC) of the China Microbial Culture Collection Management Committee.
  • CGMCC General Microbiology Center
  • the deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, China, Institute of Microbiology, Chinese Academy of Sciences, and the deposit number is CGMCC No.8670.
  • the Aspergillus niger SM751 of the present invention is screened and cultured from materials such as dead leaves and soil in the wood conservation area of Guangxi province. Its fungal taxonomy showed that the mycelium of the fungus was initially white, and the color of the colony gradually turned grayish black after about 36-48 hours, and finally, strong and dense black spores could be produced at 48 hours.
  • the 18s rDNA of fungi was extracted by a conventional method, and the nucleotide sequence thereof is shown in SEQ ID NO. 1, and it was analyzed by 18s rDNA sequence, which belongs to Aspergillus niger, and was named as Aspergillus niger SM751.
  • the fungus was deposited with the General Microbiology Center (CGMCC) of the China Microbial Culture Collection Management Committee on December 31, 2013.
  • CGMCC General Microbiology Center
  • the deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, China.
  • the Institute of Microbiology, Chinese Academy of Sciences, with the preservation number is : CGMCC No. 8670.
  • the xylanase activity was determined by using xylan (Beech wood, SIGMA). It was found that Aspergillus niger SM751 can produce high xylanase, and the solid fermentation activity of the enzyme can reach up to 10446 IU/g, which is optimal.
  • the pH value is 5.0, and the residual rate of the enzyme activity is as high as 93-99.8% between pH 3.0 and 6.0. At pH 6.5, the enzyme activity was 78% of the optimum pH enzyme activity. Therefore, the enzyme has a strong pH stability between pH 3.0 and 6.0.
  • the optimum reaction temperature was 45 °C; the residual rates of enzyme activity at 37 °C, 50 °C and 55 °C were 80.41%, 98.96% and 79.01%, respectively.
  • a second object of the present invention is to provide a xylanase which is obtained by fermentation using Aspergillus niger SM751 as a fermentation strain.
  • a third object of the present invention is to provide Aspergillus niger SM751 in the production of xylan Enzyme application.
  • the xylanase of the invention is capable of tolerating pre-treatment fermentation inhibitors.
  • the xylanase produced by the strain was activated by a mixture of various inhibitors, and the highest activation rate of the enzyme activity was 33.43% compared with the blank control.
  • a fourth object of the present invention is to provide an application of a xylanase for enzymatic hydrolysis of hemicellulose in a lignocellulosic hydrolysate.
  • the Aspergillus niger SM751 provided by the invention can produce high and high enzyme activity xylanase capable of withstanding pre-treatment fermentation inhibitor, and thus has wide application in the field of lignocellulose digestion.
  • the Aspergillus niger SM751 of the present invention is deposited on the General Microbiology Center (CGMCC) of the China Microbial Culture Collection Management Committee on December 31, 2013, and the deposit address is No. 1 Courtyard of Beichen West Road, Chaoyang District, Beijing. No., Institute of Microbiology, Chinese Academy of Sciences, deposit number: CGMCC No. 8670.
  • CGMCC General Microbiology Center
  • Figure 1 is a graph showing the effect of pH as a fermentation parameter on the xylanase enzymatic activity of Aspergillus niger SM751 of the present invention
  • Figure 3 is a temperature enzymatic property of a xylanase produced by Aspergillus niger SM751 of the present invention
  • Figure 4 is a graph showing the effects of various fermentation inhibitors on the xylanase produced by Aspergillus niger SM751 of the present invention
  • Figure 5 is a phylogenetic tree constructed using the 18S rDNA of Aspergillus niger SM751 of the present invention.
  • the screening samples are materials such as dead leaves and soil.
  • the gradient dilution is applied to the xylan screening medium, which is formulated to contain KH 2 PO 4 0.5 g, (NH 4 ) 2 SO 4 2.0 g per liter of the medium.
  • Fermentation activity of xylanase-producing strains The purified strains stored on the PDA slant surface were inoculated separately into an equal amount of fermented rescreening medium, and cultured at 30 ° C, 120 rpm for 6 days to obtain a fermentation broth (ie, an enzyme solution).
  • a fermentation broth ie, an enzyme solution.
  • the specific method is as follows: 1 g of xylan is accurately weighed, and the mixture is adjusted to a 1% substrate after stirring at a low speed of pH 4.8 (0.2 mol/L) of acetic acid-sodium acetate buffer for 2.5 h.
  • the fermented rescreening medium is a modified Mandel's nutrient solution, which is based on the original Mandel's nutrient solution and the yeast powder and peptone are removed, and the corn cob powder is added to a final concentration of 30 g/L.
  • strain SM751 A strain with extremely high xylanase activity was obtained by screening, and the strain was named as strain SM751.
  • the total DNA of strain SM751 was extracted by modified CTAB method, and the universal primers for amplifying fungal ITS sequences were selected: ITS1: 5'-TCC GTA GGT GAA CCT GCG G-3' and ITS4: 5'-TCC TCC GCT TAT TGA TAT GC-3' Amplification of the total DNA of Aspergillus niger SM751 was carried out.
  • PCR reaction system 10 ⁇ Buffer 2 ⁇ L (containing MgCl 2 , 2.5 mmol/L), dNTP (10 mmol/L) 0.4 ⁇ L, primer amount 10 pmol, rTag (5 U/ ⁇ L) 0.2 ⁇ L, and about 50 ng of template DNA, the rest of the volume is complemented with sterile ultrapure water.
  • the PCR amplification conditions were: pre-denaturation at 95 ° C for 3 min, denaturation at 94 ° C for 1 min, annealing at 52 ° C for 50 s, extension at 72 ° C for 50 s, 35 cycles, and extension at 72 ° C for 10 min.
  • the PCR amplification product was subjected to tapping recovery using a DNA gel recovery kit. Sequencing was performed. After sequencing, the sequence is shown in SEQ ID NO. The sequence was compared with the known sequence in the GenBank database, and the 18S rDNA sequence of the relevant species was obtained from the database to construct a phylogenetic tree, as shown in Figure 5. After comparison and analysis, combined with BIOLOG identification results, the strain It belongs to Aspergillus niger and is named as Aspergillus niger SM751. It was deposited on December 31, 2013 at the General Microbiology Center (CGMCC) of China Microbial Culture Collection Management Committee. Address: Beichen West Road, Chaoyang District, Beijing No. 3, No. 3, Institute of Microbiology, Chinese Academy of Sciences, with the preservation number: CGMCC No. 8670.
  • CGMCC General Microbiology Center
  • Example 2 Preparation of xylanase and determination of its enzyme activity and enzymatic properties
  • xylanase preparation Aspergillus niger SM751 was activated by seed culture.
  • the seed medium was a modified Mandel's nutrient solution (same as in Example 1), and 5 g/L of xylan was added at a starting pH of 5.6. Sterilize at 115 ° C for 30 min.
  • the parameters of the shaker were set to 30 ° C, 120 rpm, and the seed culture solution was obtained after 3 days of culture.
  • the seed culture was inoculated into the solid fermentation medium at an inoculation amount of 10% (v/w).
  • the solid fermentation medium is formulated as follows: the mass ratio of corn cob to bran is 1:5, and the mixture of corn cob and bran is used as the substrate and support material, with 1.4 g/L (NH4) 2 SO 4 , 2.0 g/ L KH 2 PO 4 , 0.3g/L CaCl 2 , 0.3g/L MgSO 4 , add appropriate amount of trace elements (FeSO 4 ⁇ 7H 2 O, 5mg/L; CoCl 2 , 20mg/L; MnSO 4 , 1.6mg/ L; ZnSO 4 , 1.4 mg / L) is configured as a nutrient solution, and the solvent is water.
  • the mass ratio of corn cob to bran is 1:5
  • the mixture of corn cob and bran is used as the substrate and support material, with 1.4 g/L (NH4) 2 SO 4 , 2.0 g/ L KH 2 PO 4 , 0.3g/L CaCl 2 , 0.3g/L MgSO 4
  • the initial pH of the nutrient solution is 3.5 (as shown in Figure 1, which is determined after a series of single factor optimization experiments), with a ratio of feed to water (mixture of corn cobs and bran: nutrient solution) 1:3.5 Sterilize after solid fermentation medium.
  • the inoculum amount was 7.5% v/v for fermentation.
  • the fermentation broth is a xylanase preparation, and the xylanase activity assay is performed.
  • Enzyme activity definition The amount of 1 ⁇ mol of reduced xylose catalyzed by 1 min was defined as one enzyme unit.
  • the temperature is 45 ° C (as shown in Figure 3).
  • the enzyme has excellent pH stability between pH 3.5 and 6.0, and its relative enzyme activity is 93.45% to 99.84%.
  • xylanase Different fermentation inhibitors have different inhibitory effects on xylanase. As shown in Fig. 4, when the ethanol concentration was 10.00 g/L, the enzyme activity residual ratio of xylanase was 102.15%, and the enzyme activity was increased by about 2.15%. When the acetic acid concentration was 10.10 g/L, the enzyme activity residual ratio of xylanase was 99.05%. When the furfural concentration was 1.40 g/L, the residual ratio of xylanase was 100.08%. When the concentration of 5-hydroxymethylfurfural was 1.20 g/L, the residual ratio of xylanase was 95.64%.
  • the xylanase of the present invention has good tolerance to fermentation inhibitors.
  • Ethanol 11.90g / L
  • acetic acid 13.60g / L
  • furfural (1.40g / L)
  • 5-hydroxymethylfurfural (1.10g / L)
  • ferulic acid (1.20g / L)
  • vanillin The mixture of 1.30 g/L
  • the enzyme activity residual rate was 133.39%, that is to say, under the action of the fermentation inhibitor, the xylanase activity was 11008 IU/g, and the activation rate was 33.39%.
  • the Aspergillus niger SM751 of the present invention has a high xylanase activity and an enzyme activity of up to 10446 IU/g. When applied to the hydrolysis of lignocellulosic hydrolysate, it has excellent tolerance to various single fermentation inhibitors or mixtures of various fermentation inhibitors.
  • the tolerance of the xylanase of the present invention to an inhibitor is completely different from that of the xylanase of the prior art, and this tolerated xylanase has not been reported.
  • the xylanase produced by Aspergillus niger SM751 was used to digest the bagasse pretreated with high temperature liquid water.
  • the conditions for pretreatment of bagasse with high temperature liquid water are: 180 degrees, nitrogen pressure 4MPA, 20 min, solid-liquid ratio 1:20. After adding 600 IU of xylanase for 24 h, the yield of xylose was 75.63%.

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Abstract

提供了一种高产木聚糖酶的黑曲霉及其应用，所述黑曲霉为黑曲霉(Aspergillus niger)SM751，其保藏号为：CGMCC No.8670。该菌所产生的木聚糖酶在多种抑制物的混合作用下，酶活受到激活，相较空白对照，酶活最高激活率达到了33.43％。

Description

一种高产木聚糖酶的黑曲霉及其应用 技术领域：
本发明属于微生物发酵及其酶工程化应用领域，具体涉及一种高产木聚糖酶的黑曲霉(Aspergillus niger)SM751及所产的木聚糖酶，以及该木聚糖酶在木质纤维素水解液中酶解半纤维素的应用。
背景技术：
全球每年的半纤维素产量为6×1010吨，是仅次于纤维素的重要可再生碳资源库，可广泛的用来制造生物燃料和诸多的生化精炼品。在富含半纤维素原料的植物原料加工过程中，添加木聚糖酶可显著加速反应进程，促进其它酶如纤维素酶、果胶酶和甘露聚糖酶等酶的酶解效果，显著改善产品的使用特性。因此，木聚糖酶被广泛地应用到纸浆造纸、食品、饲料和纺织等传统领域和木质纤维素乙醇领域。
木聚糖酶在木质纤维素酶解领域具有重要的应用价值。有学者认为，通过添加木聚糖酶等酶的混合纤维素酶才是降低木质纤维素领域用酶成本和提高酶解效率的决定性因素。Varnai A(2011)的研究结果表明，针对来源于软木、硬木和农作物的木质纤维素，木聚糖酶作为纤维素酶的副酶可以显著地降低纤维素酶的用酶成本、提高纤维素酶的酶解效率。其可能的机理是通过消除木聚糖的阻塞作用或者降解木寡糖对于纤维素酶吸附的阻碍以促进纤维素酶的酶解。Alvira(2011)的研究结果表明，木聚糖酶与纤维素酶的协作程度可以达到29.9％，并且能够增加10％的水解糖。甚至有学者Hu J(2011)认为，可以利用木聚糖酶来部分的代替纤维素酶。
木聚糖酶在木质纤维素原料生产纤维乙醇工艺中具有重要的使用价值。不过，并不是所有的木聚糖酶都可以适用于预处理之后的木质纤维素。预处理之后的木质纤维素，其预处理液中往往含有或多或少的发酵抑制物。发酵抑制物有乙酸、糠醛、5-羟甲基糠醛、香草醛、阿魏酸等。国内外数千篇的木聚糖酶研究报告中，仅有少数几篇研究牵涉到发酵抑制物对于木聚糖酶的影响。de Souza Moreira(2013)的研究表明，香草醛和阿魏酸对于来源于土曲霉的木聚糖酶具 有一定的抑制作用或者激活作用，但该研究中所用的土曲霉所产的木聚糖酶酶活极低。而关于糠醛、乙酸以5-羟甲基糠醛对于木质纤维素预处理后的木聚糖酶酶解作用的影响尚未见报道。此外，酵母发酵过程所产生中的乙醇也会对木聚糖酶的酶解作用产生抑制作用。
当前关于预处理后发酵抑制物对木聚糖酶酶解性能的影响研究报导极其少见。
发明内容：
本发明的第一个目的是提供一种高产木聚糖酶的黑曲霉(Aspergillus niger)SM751，该菌于2013年12月31日保藏于中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)，保藏地址为北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所，保藏编号为：CGMCC No.8670。
本发明的黑曲霉(Aspergillus niger)SM751是从广西省木论保护区中的枯枝烂叶、土壤等材料中筛选培养出来。其真菌分类学表明：该菌菌丝体起初呈白色，约36-48h后菌落颜色逐渐变成灰黑色，最终在48h可产旺盛、浓密的黑色孢子。常规方法提取真菌的18s rDNA，其核苷酸序列如SEQ ID NO.1所示，经18s rDNA序列分析表明，其属于黑曲霉(Aspergillus niger)，将其命名为黑曲霉(Aspergillus niger)SM751，该菌于2013年12月31日保藏于中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)，保藏地址为北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所，保藏编号为：CGMCC No.8670。
利用木聚糖(Beech wood，SIGMA)测定木聚糖酶酶活，发现黑曲霉(Aspergillus niger)SM751能高产木聚糖酶，该酶的固体发酵酶活最高可达10446IU/g，最适反应pH值为5.0，在pH3.0～6.0之间，酶活残留率高达93-99.8％。在pH6.5时酶活为最佳pH酶活的78％。因此，该酶在pH3.0～6.0之间具有很强的pH稳定性。最适反应温度为45℃；37℃、50℃与55℃时的酶活残留率分别为80.41％、98.96％与79.01％。
因此，本发明的第二个目的是提供一种木聚糖酶，其特征在于，以黑曲霉(Aspergillus niger)SM751为发酵菌株，经发酵而制得。
本发明的第三个目的是提供黑曲霉(Aspergillus niger)SM751在生产木聚糖 酶上的应用。
本发明的木聚糖酶能够耐受预处理后发酵抑制物。该菌所产生的木聚糖酶在多种抑制物的混合作用下，酶活受到激活，相较空白对照，酶活最高激活率达到了33.43％。
因此，本发明的第四个目的是提供木聚糖酶在木质纤维素水解液中酶解半纤维素的应用。
本发明提供的黑曲霉(Aspergillus niger)SM751，其能高产高酶活的、能耐受预处理后发酵抑制物的木聚糖酶，因此在木质纤维素酶解领域具有广泛的应用。
本发明的黑曲霉(Aspergillus niger)SM751，该菌于2013年12月31日保藏于中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)，保藏地址为北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所，保藏编号为：CGMCC No.8670。
附图说明：
图1是pH值作为发酵参数对本发明的黑曲霉(Aspergillus niger)SM751产木聚糖酶酶活性的影响；
图2是本发明黑曲霉(Aspergillus niger)SM751产木聚糖酶的pH酶学特性；
图3是本发明黑曲霉(Aspergillus niger)SM751产木聚糖酶的温度酶学特性；
图4是各种不同的发酵抑制物对本发明的黑曲霉(Aspergillus niger)SM751产木聚糖酶的作用；
图5是应用本发明的黑曲霉(Aspergillus niger)SM751的18S rDNA构建的系统发育树。
具体实施方式：
以下实施例是对本发明的进一步说明，而不是对本发明的限制。
实施例1：菌株的筛选
筛选样品为枯枝烂叶、土壤等材料。筛选样品经过适当处理后，梯度稀释涂布于木聚糖初筛培养基，该培养基的配方是：每升培养基中含有KH2PO40.5g， (NH4)2SO42.0g，MgSO4·7H2O 0.25g，木聚糖(购自上海源聚生物)5.0g，琼脂18～20g，余量为水，pH5.6。30℃培养4～7d，挑取水解透明圈较大的菌落在PDA培养基上纯化，纯化后的菌株进行PDA斜面保存。
产木聚糖酶菌株发酵活力复筛：将PDA斜面保存的纯化菌株分别接种到等量的发酵复筛培养基液，30℃，120rpm，培养6d，得到发酵液(即酶液)。依据Bailey(1992)等人的测定方法并进行适当修改。具体方法为：准确称取木聚糖1g，在pH4.8(0.2mol/L)的醋酸～醋酸钠缓冲液低速搅拌2.5h后定容配置成1％的底物。取0.9mL 1％的木聚糖底物置于15mL刻度试管，于50℃恒温预热5min。准确加入适当稀释的酶液0.2mL，用秒表精确计时反应30min，立即加入2mLDNS终止反应，沸水浴5min，于540nm处测光吸收值(OD)。然后选择木聚糖酶活力较高的菌株进行妥善保藏。所述的发酵复筛培养基液为修改过的Mandel’s营养液，其是在原Mandel’s营养液的基础上去掉酵母粉和蛋白胨再加入玉米芯粉，使其终浓度为30g/L。
经筛选获得1株木聚糖酶活力极高的菌株，将该菌株命名为菌株SM751。
采用改良CTAB法提取菌株SM751总DNA，选择扩增真菌ITS序列的通用引物ITS1:5’-TCC GTA GGT GAA CCT GCG G-3’和ITS4:5’-TCC TCC GCT TAT TGA TAT GC-3’对黑曲霉(Aspergillus niger)SM751总DNA进行序列的扩增。在20μL的PCR反应体系中含有10×Buffer 2μL(含MgCl2，2.5mmol/L)，dNTP(10mmol/L)0.4μL，引物量为10pmol，rTag(5U/μL)0.2μL，约50ng的模板DNA，其余体积以无菌超纯水补足。PCR扩增条件为：95℃预变性3min，94℃变性1min，52℃退火50s，72℃延伸50s，35个循环，72℃延伸10min。PCR扩增产物采用DNA凝胶回收试剂盒进行割胶回收。进行测序。经测序，其序列如SEQ ID NO.1所示。将该序列与GenBank数据库中的已知序列进行BLAST比较分析，并从数据库获得相关种属的18S rDNA序列，构建系统发育树，如图5所示，经比较分析并结合BIOLOG鉴定结果，该菌属于黑曲霉，将其命名为黑曲霉(Aspergillus niger)SM751，该菌于2013年12月31日保藏于中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)，地址：北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所，其保藏编号为：CGMCC No.8670。
实施例2：木聚糖酶的制备及其酶活力、酶学性质测定
1.木聚糖酶制剂的制备：将黑曲霉(Aspergillus niger)SM751经种子培养后活化。种子培养基为修改过的Mandel’s营养液(同实施例1)，加入5g/L的木聚糖，起始pH5.6。在115℃下灭菌30min。设定摇床的参数为：30℃，120rpm，培养3天后获得种子培养液。将种子培养液以10％(v/w)的接种量接种到固体发酵培养基中。固态发酵培养基的配方为：玉米芯与麸皮的质量比1:5，以玉米芯和麸皮的混合物为底物和支撑材料，以1.4g/L(NH4)2SO4，2.0g/L KH2PO4，0.3g/L CaCl2，0.3g/L MgSO4，添加适量的微量元素(FeSO4·7H2O，5mg/L；CoCl2,20mg/L；MnSO4,1.6mg/L；ZnSO4，1.4mg/L)配置成营养液，溶剂为水。该营养液的起始pH为3.5(如图1，该pH值是在一系列单因素优化实验后确定的)，以料水比(玉米芯和麸皮的混合物：营养液)1:3.5配置好固态发酵培养基后进行灭菌。接种量为7.5％v/v进行发酵。发酵92-108h后，其发酵液即为木聚糖酶制剂，进行木聚糖酶酶活测定。
在三角瓶中加入40mL pH4.8的醋酸缓冲液，用玻璃棒捣碎结块的固体发酵培养基(同上)，于110rpm 30℃摇荡60min，直接吸取未经离心及过滤的浸提液0.4mL进行梯度稀释，直至达到适当的倍数，然后进行木聚糖酶酶活的测定。
2.木聚糖酶的酶活力测定：依据Bailey(1992)等人的测定方法并进行适当修改。称取1g山毛榉木聚糖置于100mL烧杯后，倒入60mL pH4.8的醋酸缓冲液低速搅拌2.5h，然后定容100mL容量瓶配置成1％山毛榉木聚糖溶液。吸取0.9mL 1％山毛榉木聚糖溶液(Beech wood，SIGMA)作为底物，预热5min再加入0.2mL适当稀释的木聚糖酶，在50℃反应30min，立即加入2mL DNS溶液终止反应，沸水浴5min，于540nm处测光吸收值(OD)。经测定，步骤1得到的黑曲霉(Aspergillus niger)SM751产的木聚糖酶的活力达到10446IU/g，具有极高的酶活力。
酶活定义：在1min内催化产生1μmol还原木糖的量定义为一个酶单位。
3.木聚糖酶的酶学性质的测定：按上述测定方法，其它条件不变的情况下，调节缓冲液到不同的pH、不同的温度进行酶活测定反应，以测得最高酶活为100％，其它pH值下或者温度下的酶活与最高酶活相除之后乘以100％即为相对酶活。利用相应的缓冲液进行梯度稀释，在50℃下测定最佳pH。在最佳的pH 条件下，设定不同温度以测定酶的最佳温度。
结果表明，本发明的黑曲霉(Aspergillus niger)SM751产的木聚糖酶的最佳反应pH为5.0(如图2所示)，且在pH5.0的条件下保持酶活稳定，最适反应温度为45℃(如图3所示)。该酶在pH3.5～6.0之间具有优良的pH稳定性，其相对酶活为93.45％～99.84％。
实施例3：发酵抑制物对于木聚糖酶的作用
1.单一的发酵抑制物对于木聚糖酶(黑曲霉(Aspergillus niger)SM751产的木聚糖酶)的抑制作用：
不同的发酵抑制物对木聚糖酶的抑制作用不同。如图4所示，在乙醇浓度为10.00g/L时，木聚糖酶的酶活残留率为102.15％，酶活提高约2.15％。在乙酸浓度为10.10g/L时，木聚糖酶的酶活残留率为99.05％。糠醛浓度为1.40g/L时，木聚糖酶的残留率为100.08％。5-羟甲基糠醛浓度为1.20g/L时，木聚糖酶的残留率为95.64％。香草醛的浓度为1.10g/L时，木聚糖酶的残留率分别为98.16％。阿魏酸的浓度为1.10g/L时，木聚糖酶的残留率为72.99％。因此，本发明的木聚糖酶对于发酵抑制物具有良好的耐受性。
2.混合抑制物对于木聚糖酶(黑曲霉(Aspergillus niger)SM751产的木聚糖酶)的抑制作用：
乙醇(11.90g/L)、乙酸(13.60g/L)、糠醛(1.40g/L)、5-羟甲基糠醛(1.10g/L)、阿魏酸(1.20g/L)和香草醛(1.30g/L)的混合物对于木聚糖酶具有激活作用。酶活残留率为133.39％，也就是说，在此发酵抑制物的作用下，木聚糖酶酶活为11008IU/g，激活率可达33.39％。
由此可见，本发明的黑曲霉(Aspergillus niger)SM751产的木聚糖酶酶活力较高，酶活高达10446IU/g。在应用于木质纤维素水解液酶解时，对于各种单一的发酵抑制物或者多种发酵抑制物的混合物具有优良的耐受性。
本发明的木聚糖酶的对抑制物的耐受性与现有技术中的木聚糖酶完全不同，这种耐受性的木聚糖酶未见有过报道。
利用黑曲霉(Aspergillus niger)SM751产的木聚糖酶来酶解高温液态水预处理后的甘蔗渣。高温液态水预处理甘蔗渣的条件为：180度，氮气加压4MPA，20min，固液比1:20。加入600IU的木聚糖酶酶解24h之后，木糖得率为75.63％。
上述详细说明是针对本发明的可行实施例的具体说明，该实施例并非用以限制本发明的专利范围，凡未脱离本发明的等效实施或变更，均应包含于本发明的专利范围中。
Figure PCTCN2015072777-appb-000001

Claims (4)

  1. 一种黑曲霉(Aspergillus niger)SM751，其保藏号为：CGMCC No.8670。
  2. 一种木聚糖酶，其特征在于，以权利要求1所述的黑曲霉(Aspergillus niger)SM751为发酵菌株，经发酵而制得。
  3. 权利要求1所述的黑曲霉(Aspergillus niger)SM751在生产权利要求2所述的木聚糖酶上的应用。
  4. 权利要求2所述的木聚糖酶在木质纤维素水解液中酶解半纤维素的应用。
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