CN114854724B - 一组gh10家族木聚糖酶的n-糖基化突变体及其应用 - Google Patents
一组gh10家族木聚糖酶的n-糖基化突变体及其应用 Download PDFInfo
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- CN114854724B CN114854724B CN202210588930.8A CN202210588930A CN114854724B CN 114854724 B CN114854724 B CN 114854724B CN 202210588930 A CN202210588930 A CN 202210588930A CN 114854724 B CN114854724 B CN 114854724B
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- xylanase
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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- 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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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
- C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
- C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
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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
- C12P19/02—Monosaccharides
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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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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
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- Health & Medical Sciences (AREA)
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- Molecular Biology (AREA)
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- Chemical Kinetics & Catalysis (AREA)
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- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
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- Enzymes And Modification Thereof (AREA)
Abstract
一组GH10家族木聚糖酶的N‑糖基化突变体及其应用,对位于Loop区N141‑N153且处于GH10家族木聚糖酶基因序列保守区的相同氨基酸位点N147和A142进行定点突变,所述突变位点以4XX6为模板的N147位点,获得木聚糖酶突变体4XX6‑N147Q;以XYL10C∆N为模板的A142位点,获得木聚糖酶突变体XYL10C∆N‑A142N;其中木聚糖酶突变体4XX6‑N147Q的氨基酸序列如SEQ ID NO.3所示;木聚糖酶突变体XYL10C∆N‑A142N的氨基酸序列如SEQ ID NO.4所示。综合分析发现Ala142位点构建的N‑糖基化具有促进GH10家族木聚糖酶热稳定性的作用。本发明对利用N‑糖基化改良GH10家族木聚糖酶的热稳定性具有重要的指导意义。
Description
技术领域
本发明涉及基因工程、蛋白质工程领域,具体涉及一组GH10家族木聚糖酶的N-糖基化突变体及其应用。
背景技术
纤维素类物质包括纤维素、半纤维素和木质素,是自然界中常见的、最丰富的可再生资源。木聚糖是半纤维素中含量最高的多糖,广泛存在于在农业副产物如玉米芯、麦麸和甘蔗渣等中。木聚糖酶被广泛应用于降解木聚糖以促进半纤维素的应用。
木聚糖酶存在于很多家族中,其中,GH10家族的木聚糖酶是一种具有真正严格意义上的内切-1,4-β-木聚糖酶的催化活性和催化结构域。木聚糖酶被应用于造纸行业以减少污染和降低漂白过程中漂白剂的使用量。其还是木质纤维素类生物质糖化为生物燃料生物乙醇的关键酶。木聚糖酶被广泛应用于降解木聚糖,但是其热稳定性的劣势限制了应用价值。
N-糖基化是真核细胞中最常见的翻译后修饰方法之一,其是新生肽链的共翻译或翻译后修饰方法,特征序列为N-X-S/T,其中X不为P。其会影响蛋白质的生物学功能,如蛋白质折叠、细胞识别等,从而影响酶的活性、热稳定性和分泌效率。因此,基于N-糖基化的理论和构建方法,通过理性或半理性的分析方式,在重组酶的特定位点引入或去除N-糖基化可以改善重组酶的热稳定性等酶学性质。
针对生物质降解和造纸业等工业的需求,改良木聚糖酶的热稳定性仍具有重大意义。
发明内容
解决的技术问题:本发明提供了一组GH10家族木聚糖酶的N-糖基化突变体的构建方法与应用,该突变体为通过N-糖基化改造后筛选获得。去除N-糖基化的突变体4XX6-N147Q在80℃下的半衰期为19min,是野生型4XX6的0.56倍;引入N-糖基化的突变体XYL10CΔN-A142N在85℃下的半衰期为18min,是野生型XYL10CΔN的2.76倍,且能在偏酸性条件下具有80%以上的酶活力。
技术方案:一组GH10家族木聚糖酶的N-糖基化突变体,对位于Loop区N141-N153且处于GH10家族木聚糖酶基因序列保守区的相同氨基酸位点N147和A142进行定点突变,所述突变位点以4XX6为模板的N147位点,获得木聚糖酶突变体4XX6-N147Q;以XYL10CΔN为模板的A142位点,获得木聚糖酶突变体XYL10CΔN-A142N;其中木聚糖酶突变体4XX6-N147Q的氨基酸序列如SEQ ID NO.3所示;木聚糖酶突变体XYL10CΔN-A142N的氨基酸序列如SEQ IDNO.4所示。
编码木聚糖酶突变体4XX6-N147Q的核苷酸序列如SEQ ID NO.1所示。
编码木聚糖酶突变体XYL10CΔN-A142N的核苷酸序列如SEQ ID NO.2所示。
含有SEQ ID NO.1或SEQ ID NO.2所示核苷酸的质粒。
一种重组载体,含有上述质粒。
一种重组菌株,表达上述重组载体。
上述生物材料在降解木聚糖中的应用。
上述重组菌株的构建方法,包括以下步骤:
步骤1,分别将木聚糖酶4XX6和XYL10CΔN作为模板,通过定点突变的方法构建重组质粒;
步骤2,将木聚糖酶突变体4XX6-N147Q或XYL10CΔN-A142N的重组质粒转入DMT感受态,得重组载体;
步骤3,将突变重组载体进行线性化处理,通过电击法转入GS115毕赤酵母感受态,诱导表达后筛选有催化活力的突变菌株;
步骤4,培养重组菌株,大量表达木聚糖酶突变体;
步骤5,回收并且纯化所表达的有催化活力的重组菌株。
有益效果:在热稳定性方面,去除N-糖基化的突变体4XX6-N147Q在80℃下的半衰期(t1/2)为19min,是野生酶4XX6(34min)的0.55倍;4XX6-N147Q的T50值为78℃,相较野生酶4XX6(83℃)降低5℃;引入N-糖基化的突变体XYL10CΔN-A142N在85℃下的半衰期(t1/2)为18min,是野生酶XYL10CΔN(6.5min)的2.76倍;XYL10CΔN-A142N的T50值为88℃,相较野生酶XYL10CΔN(83℃)提高5℃。综合分析发现,Ala142位点引入的N-糖基化具有促进GH10家族木聚糖酶热稳定性的作用。突变体4XX6-N147Q和XYL10CΔN-A142N的最适pH和最适温度与野生型基本保持一致,符合工业应用要求。
附图说明
图1为木聚糖酶4XX6、XYL10CΔN及其突变体的聚丙烯酰胺凝胶电泳;
图2为木聚糖酶4XX6及其突变体的最适pH和最适温度;其中A为最适pH,B为最适温度;
图3为木聚糖酶XYL10CΔN及其突变体的最适pH和最适温度;其中A为最适pH,B为最适温度;
图4为木聚糖酶4XX6及其突变体的半衰期(t1/2)和T50值;其中A为半衰期(t1/2),B为T50值;
图5为木聚糖酶XYL10CΔN及其突变体的半衰期(t1/2)和T50值;其中A为半衰期(t1/2),B为T50值。
具体实施方式
将结合附图和实施例详细描述本发明的内容。
1、菌株及载体:Pichia pastoris GS115作为宿主,pPIC9γ作为质粒载体,均为市售材料;
2、酶类及其它生化试剂:Taq酶和Pfu酶均购自全式金生物科技公司,榉木木聚糖购自源叶公司;分析纯试剂均从国药集团购买;
3、培养基:
(1)LB培养基:0.5%酵母提取物,1%蛋白胨,1%NaCl,pH 7.0;
(2)YPD培养基:2%蛋白胨,1%酵母提取物,2%葡萄糖;
(3)MD固体培养基:2%葡萄糖,1.5%琼脂糖,1.34%YNB,0.00004%Biotin;
(4)BMGY培养基:1%酵母提取物,2%蛋白胨,1%甘油(V/V),1.34%YNB,0.00004%Biotin;
(5)BMMY培养基:1%酵母提取物,2%蛋白胨,0.5%甲醇(V/V),1.34%YNB,0.00004%Biotin。
实施例1木聚糖酶4XX6和XYL10CΔN及突变体的构建
合成基因序列4xx6(NCBI参考序列:EPQ55122.1)和XYL10CΔN(NCBI参考序列:ACS96449.1)并表达,分别获得GH10家族木聚糖酶4XX6和XYL10CΔN。4XX6的N147位点与XYL10CΔN的A142位点为相同的氨基酸位点且处于GH10家族木聚糖酶基因序列的保守区。以木聚糖酶4XX6为出发材料,采用定点突变的方法将N147位点突变为Q147以去除N-糖基化。以木聚糖酶XYL10CΔN为出发材料,采用定点突变的方法将A142位点突变为N142以引入N-糖基化。定点突变所用引物序列如表1所示,定点突变方法和克隆方法参考文献(You,etal.,2019)。
表1引物合成清单
实施例2木聚糖酶4XX6和XYL10CΔN及突变体的制备
将PCR获得的线性重组表达载体直接转化DMT感受态,菌落PCR验证,获得突变体4XX6-N147Q和XYL10CΔN-A142N的核苷酸序列,将重组质粒线性化后转化毕赤酵母GS115,获得重组酵母菌株4XX6-N147Q和XYL10CΔN-A142N。
用含有2mL BMGY培养基的10mL试管培养含有重组质粒的酵母菌株,将试管置于30℃摇床以220rpm转速培养48h。以3000G的转速离心培养液,离心5min后弃去上清液。配置含有0.5%甲醇的2mL BMMY培养基,使用BMMY培养基重悬沉淀。将菌体与培养基混合均匀,之后将试管放置于30℃的摇床以220rpm的转速培养48h。取上清离心后用于酶活性检测,筛选到具有催化活性的突变体4XX6-N147Q和XYL10CΔN-A142N。
将重组酵母菌株4XX6-N147Q和XYL10CΔN-A142N放大发酵体系,首先接种于YPD培养基中获得种子培养液。从种子培养液中取1%接种于l L三角瓶中,其中含有200mL BMGY培养基。将其放置于30℃的摇床中,以220rpm的转速培养48h;以3000G的转速离心培养液,离心5min后弃去上清液。配置含有0.5%甲醇的200mL BMMY培养基,使用BMMY培养基重悬沉淀。将菌体与培养基混合均匀,之后将试管放置于30℃的摇床以220rpm的转速培养48h。以开始培养菌体为准,每隔12h向培养基中加一次1mL的甲醇。每次加甲醇前,取一部分上清液离心后进行酶活性检测。纯化蛋白使用的是阴离子交换法。所表达的葡聚糖酶经过纯化之后,聚丙烯酰胺凝胶电泳(图1)显示其蛋白质的含量达到总蛋白的90%以上,其中M为Marker,1、2、3、4分别为4XX6、4XX6-N147Q、XYL10CΔN、XYL10CΔN-A142N。
实施例3木聚糖酶4XX6和XYL10CΔN及突变体的酶学性质分析
一、木聚糖酶的酶活力测定
以1%(w/v)的大麦葡聚糖作为底物测定木聚糖酶4XX6和XYL10CΔN及突变体的活力。方法根据Yang等人所报道的(Yang 10.1021/jf800303b)。
以下为具体的方法:1mL的反应体系中含有100μL酶液和900μL木聚糖底物。在给定的pH和温度条件下,将木聚糖酶与底物混合后水浴反应10min。反应结束后加入1.5mL DNS终止反应。在沸水中水浴5min以促进其显色。试管冷却后在酶标仪中测定其540nm吸光度时的OD值。上述条件下,每分钟生成1μmol的还原糖所需的酶量定义为木聚糖酶活力单位(U)。
二、木聚糖酶4XX6和XYL10CΔN及突变体的性质测定
1、木聚糖酶4XX6及其突变体的最适温度和最适pH测定如下:
将实施例2中纯化的4XX6和4XX6-N147Q在不同的pH下进行酶促反应,测定完成后分析其最适pH。用0.1mol/L柠檬酸-磷酸氢二钠配置成不同pH的缓冲液。用缓冲液稀释底物(榉木木聚糖)后配置成可以使用的底物溶液。在最适温度下对木聚糖酶4XX6及其突变体进行酶活力测定。结果(图2.A)表明,木聚糖酶4XX6及其突变体的最适pH值均为4.5。突变体4XX6-N147Q在pH 3.0-6.0范围内的酶催化活性降低。
将实施例2纯化的4XX6和4XX6-N147Q在不同的温度下进行酶促反应,测定完成后分析其最适温度。突变体4XX6-N147Q酶促反应的最适温度测定结果(图2.B)表明,野生型木聚糖酶4XX6和突变体4XX6-N147Q的最适温度都是80℃。
2、木聚糖酶XYL10CΔN及其突变体的最适温度和最适pH测定如下:
将实施例2中纯化的XYL10CΔN和XYL10CΔN-A142N在不同的pH下进行酶促反应,测定完成后分析其最适pH。用0.1mol/L柠檬酸-磷酸氢二钠配置成不同pH的缓冲液。用缓冲液稀释底物(榉木木聚糖)后配置成可以使用的底物溶液。在最适温度下对木聚糖酶XYL10CΔN及其突变体进行酶活力测定。结果(图3.A)表明,木聚糖酶XYL10CΔN及其突变体的最适pH值均为4.5。突变体XYL10CΔN-A142N在pH 2.0-3.5范围内的酶催化活性提高。
将实施例2纯化的XYL10CΔN和XYL10CΔN-A142N在不同的温度下进行酶促反应,测定完成后分析其最适温度。突变体XYL10CΔN-A142N酶促反应的最适温度测定结果(图3.B)表明,野生型木聚糖酶XYL10CΔN和突变体XYL10CΔN-A142N的最适温度都是90℃。
3、木聚糖酶4XX6及其突变体的热稳定性测定如下:
将木聚糖酶突变体4XX6-N147Q和野生型4XX6在80℃处理一定时间后热稳定性逐渐降低,突变体4XX6-N147Q的热稳定性不如野生型4XX6。通过拟合曲线(图4)可以发现,在80℃下,突变体4XX6-N147Q的半衰期(t1/2)为19min,是野生型酶t1/2值(34min)的0.55倍。通过拟合曲线可以发现,突变体4XX6-N147Q的T50值为78℃,较野生型酶T50值(83℃)降低5℃。比较突变体与野生型的半衰期和T50值发现,去除N-糖基化使酶的热稳定性降低。
4、木聚糖酶XYL10CΔN及其突变体的热稳定性测定如下:
将木聚糖酶突变体XYL10CΔN-A142N和野生型XYL10CΔN在85℃处理一定时间后热稳定性逐渐降低,突变体XYL10CΔN-A142N的热稳定性优于野生型XYL10CΔN。通过拟合曲线(图5)可以发现,在85℃下,突变体XYL10CΔN-A142N的半衰期(t1/2)为18min,是野生型酶t1/2值(6.5min)的2.76倍。通过拟合曲线可以发现,突变体XYL10CΔN-A142N的T50值为88℃,较野生型酶T50值(83℃)提高5℃。比较突变体与野生型的半衰期和T50值发现,引入N-糖基化使酶的热稳定性增强。
综合分析4XX6-N147Q和XYL10CΔN-A142N的热稳定性变化发现,位于GH10家族木聚糖酶保守区Ala142位点的N-糖基化具有增强热稳定性的作用。
序列表
<110> 江苏科技大学
<120> 一组GH10家族木聚糖酶的N-糖基化突变体及其应用
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Lys Glu Ser Phe Asn Ile Tyr Ala Trp Asp Val Val Asn Glu Ala Phe
130 135 140
Asn Asp Gln Gly Thr Tyr Arg Glu Asn Val Trp Tyr Thr Gln Leu Gly
145 150 155 160
Pro Asp Tyr Ile Pro Asn Ala Tyr Ala Val Ala Arg Ser Val Asn Thr
165 170 175
Pro Ser Lys Leu Tyr Ile Asn Asp Tyr Asn Thr Glu Gly Ile Asn Asn
180 185 190
Lys Ser Asp Ala Leu Leu Ala Val Val Gln Ser Met Lys Ala His Asn
195 200 205
Leu Val Asp Gly Val Gly Phe Gln Cys His Phe Phe Val Gly Glu Leu
210 215 220
Pro Pro Asp Leu Glu Gln Asn Phe Ala Arg Phe Val Ala Ala Gly Val
225 230 235 240
Glu Ile Ala Val Thr Glu Leu Asp Ile Arg Met Asn Leu Pro Pro Ser
245 250 255
Gln Ala Asp Ile Glu Gln Gln Ala Arg Asp Tyr Ala Thr Val Val Asn
260 265 270
Ala Cys Lys Ser Gln Gly Ala Ala Cys Val Gly Ile Thr Thr Trp Gly
275 280 285
Ile Thr Asp Leu Tyr Ser Trp Ile Pro Ser Thr Tyr Pro Gly Glu Gly
290 295 300
Tyr Ala Leu Leu Phe Asp Asp Asn Tyr Val Pro His Pro Ala Phe Asn
305 310 315 320
Ala Thr Ile Gln Ala Leu Leu Ala
325
<210> 4
<211> 340
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 4
Trp Gly Leu Asn Asn Ala Ala Arg Ala Asp Gly Lys Leu Trp Phe Gly
1 5 10 15
Thr Ala Ala Asp Ile Pro Gly Leu Glu Gln Asp Asp Arg Tyr Tyr Met
20 25 30
Lys Glu Tyr Asn Asn Thr His Asp Phe Gly Gly Thr Thr Pro Ala Asn
35 40 45
Ile Met Lys Phe Met Phe Thr Glu Pro Glu Gln Asn Val Phe Asn Phe
50 55 60
Thr Gly Ala Gln Glu Phe Leu Asp Ile Ala Phe Ala Ser His Lys Leu
65 70 75 80
Val Arg Cys His Asn Leu Ile Trp Gln Ser Glu Leu Pro Thr Trp Val
85 90 95
Thr Asn Pro Thr Thr Asn Trp Thr Asn Glu Thr Leu Ser Lys Val Leu
100 105 110
Gln Asn His Val Tyr Thr Leu Val Ser His Phe Gly Asp Gln Cys Tyr
115 120 125
Ser Trp Asp Val Val Asn Glu Ala Leu Ser Asp Asp Pro Asn Gly Ser
130 135 140
Tyr Gln Asn Asn Ile Trp Phe Asp Thr Ile Gly Pro Glu Tyr Val Ala
145 150 155 160
Met Ala Phe Glu Tyr Ala Glu Lys Ala Val Lys Asp His Lys Leu Asn
165 170 175
Val Lys Leu Tyr Tyr Asn Asp Tyr Asn Ile Glu Tyr Pro Gly Pro Lys
180 185 190
Ser Thr Ala Ala Gln Asn Ile Val Lys Glu Leu Lys Ala Arg Asn Ile
195 200 205
Gln Ile Asp Gly Val Gly Leu Glu Ser His Phe Ile Ala Gly Glu Thr
210 215 220
Pro Ser Gln Ala Thr Gln Ile Thr Asn Met Ala Asp Phe Thr Ser Leu
225 230 235 240
Asp Ile Asp Val Ala Val Thr Glu Leu Asp Val Arg Leu Tyr Leu Pro
245 250 255
Pro Asn Ala Thr Ser Glu Ala Gln Gln Val Ala Asp Tyr Tyr Ala Thr
260 265 270
Val Ala Ala Cys Ala Ala Thr Glu Arg Cys Ile Gly Ile Thr Val Trp
275 280 285
Asp Phe Asp Asp Thr Tyr Ser Trp Val Pro Ser Thr Phe Ala Gly Gln
290 295 300
Gly Tyr Ala Asp Leu Phe Phe Gln Pro Asp Gly Pro Asn Thr Pro Leu
305 310 315 320
Val Lys Lys Ala Ala Tyr Asp Gly Cys Leu Gln Ala Leu Gln His Lys
325 330 335
Ala Glu Ser Pro
340
<210> 5
<211> 987
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
tcacccctcg cacggcaact gcccacgtcc ccgttcgaga cgctgagggc agcagcggca 60
ccgcgctact ttggtgcagc tctgggtgtc ccccacctgt tgaatttcac gcatgatccg 120
ctgtttgatg tgactgctgt cttgcagttc aacggtgcca cgccggagaa cgagatgaaa 180
tgggcgtaca tcgagccgga gcggaaccag ttcaacttta ctggtggcga catcgttgct 240
gcgttctccg ccgccaacga ctatgtcctg cgcggtcaca atctcgtctg gtaccaggag 300
ctcgcaccgt gggtggagac cctgacgggt gaggacctat ggaacgctac tgtgaatcac 360
atcacgactg tgatgacaca ctacaaggag agcttcaata tctacgcttg ggacgttgtc 420
aacgaggctt tcaacgacaa cggtacctac cgggagaacg tttggtacac ccagctcgga 480
ccggattaca tcccgaacgc gtacgccgta gccagatccg tgaacacgcc gtctaagctg 540
tacatcaacg actacaatac tgagggcatc aacaacaagt ccgatgcact gctcgccgtt 600
gtgcagagca tgaaagcaca taacttggtt gacggtgttg gcttccaatg ccacttcttc 660
gtcggcgagc tccccccgga cctcgagcag aacttcgcgc ggtttgtggc cgcgggcgtc 720
gagatcgccg tcaccgaact cgatatcagg atgaacctcc cgccttcaca ggctgacatt 780
gagcagcagg cccgcgacta cgccacagtc gtgaatgcat gcaaatcaca gggtgctgcc 840
tgcgttggga tcaccacctg gggtatcacc gacctttact catggattcc ctccacgtat 900
cccggcgagg gatatgccct gctcttcgat gacaattatg ttccccaccc ggcattcaac 960
gcgactattc aggccttgct cgcttga 987
<210> 6
<211> 1023
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
tggggtctta ataatgcagc tcgagccgat ggcaagcttt ggtttggaac tgctgcagat 60
atccccggtt tagagcagga tgatcgctat tacatgaagg aatacaacaa tacgcatgat 120
tttggtggta ccacacccgc gaatattatg aaattcatgt tcacggagcc agagcaaaac 180
gtttttaatt tcaccggcgc gcaggagttc ctggacattg cctttgcgtc gcacaagctt 240
gttcgttgcc acaatcttat ctggcaatcc gagcttccca catgggttac taaccctacc 300
acaaattgga caaacgaaac cttgagcaag gtgctacaaa atcatgtata tactctagtc 360
tcacattttg gagatcagtg ctatagctgg gatgtggtta acgaagccct ctctgatgac 420
ccagccggat cgtatcaaaa caatatctgg ttcgacacta ttggtcccga gtacgttgcg 480
atggcattcg agtatgccga gaaagccgtc aaagaccata agttgaatgt taagctctac 540
tacaatgact acaacattga atatcctggg cccaaatcta cagcagcaca gaatattgtc 600
aaggagctta aagcaaggaa catccaaata gatggcgtcg gccttgagtc ccacttcatc 660
gctggtgaaa ctccgtctca ggctacgcaa atcacaaaca tggctgattt cacttctctt 720
gacattgacg ttgctgttac cgagctcgat gtacgtcttt atctgcctcc aaatgctacc 780
agcgaggccc agcaagttgc cgactattac gccaccgtcg cagcctgtgc tgcaacagaa 840
cgctgtatcg gtataactgt ctgggatttt gacgatacat attcatgggt gcccagcacg 900
ttcgccggcc aagggtatgc ggatctgttc ttccagccag acggccccaa cactccccta 960
gtgaaaaaag cggcgtacga cggttgccta caggctttgc aacataaggc ggaaagtcca 1020
tga 1023
<210> 7
<211> 31
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
caaggtacct accgggagaa cgtttggtac a 31
<210> 8
<211> 29
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
ccggtaggta ccttggtcgt tgaaagcct 29
<210> 9
<211> 34
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
aacggatcgt atcaaaacaa tatctggttc gaca 34
<210> 10
<211> 31
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
tttgatacga tccgtttggg tcatcagaga g 31
Claims (5)
1.一种GH10家族木聚糖酶的N-糖基化突变体,其特征在于,所述突变体为XYL10C∆N-A142N,其氨基酸序列如SEQ ID NO.4所示。
2.根据权利要求1所述的一种GH10家族木聚糖酶的N-糖基化突变体,其特征在于,编码突变体XYL10C∆N-A142N的核苷酸序列如SEQ ID NO.2所示。
3.含有SEQ ID NO.2所示核苷酸的质粒。
4.一种重组菌株,其特征在于,含有权利要求3所述的质粒。
5.权利要求4所述重组菌株在降解木聚糖中的应用。
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