CN115074345A - 耐热β-1，4-内切木聚糖酶及其编码基因与应用 - Google Patents
耐热β-1，4-内切木聚糖酶及其编码基因与应用 Download PDFInfo
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- CN115074345A CN115074345A CN202110259035.7A CN202110259035A CN115074345A CN 115074345 A CN115074345 A CN 115074345A CN 202110259035 A CN202110259035 A CN 202110259035A CN 115074345 A CN115074345 A CN 115074345A
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- xylanase
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Abstract
本发明公开了耐热β‑1，4‑内切木聚糖酶及其编码基因与应用。实验证明，氨基酸序列如SEQ ID NO:1所示的木聚糖酶Xyn10A、氨基酸序列如SEQ ID NO:3所示的木聚糖酶XynAF0、氨基酸序列如SEQ ID NO:5所示的木聚糖酶XynAF1和氨基酸序列如SEQ ID NO:7所示的木聚糖酶XynAF1‑C均为β‑1，4‑内切木聚糖酶，木聚糖酶XynAF1‑C的耐热能力显著提高。本发明具有重要的应用价值。
Description
技术领域
本发明属于微生物领域，具体涉及耐热β-1，4-内切木聚糖酶及其编码基因与应用。
背景技术
木质纤维素是重要的可再生物质资源，主要来自于植物细胞壁，由纤维素、半纤维素和木质素构成。木质纤维素结构中，纤维素、半纤维素和木质素之间形成大量共价与非共价相互作用，其中半纤维素广泛存在于纤维素和木质素交界处，对维持木质纤维素整体稳定性具有重要帮助。半纤维素是一种由多聚糖组成的复合物，组成成分包括木聚糖、木葡聚糖、葡甘露聚糖、阿拉伯半乳聚糖等，其中木聚糖是构成半纤维素的主要成分。木聚糖不仅功能重要，而且生物量巨大，是自然界含量第二丰富的多聚糖成分，在一年生的草本植物中，木聚糖含量高达30％。
天然木聚糖是复杂的杂多糖，其侧链存在多种取代基修饰，因而木聚糖的高效水解往往需要多种酶的共同参与。目前研究较为广泛的水解酶包括β-1，4-内切木聚糖酶、木糖苷酶、乙酰木聚糖酯酶、阿拉伯呋喃糖苷酶、葡萄糖醛酸酶、阿魏酸酯酶等，这些酶通常以协同的方式共同催化降解木聚糖。其中β-1，4-内切木聚糖酶能够高效降解木聚糖主链，在木聚糖降解过程中发挥了主要作用。
β-1，4-内切木聚糖酶是重要的工业酶，广泛应用于食品、饲料、纺织、纸浆漂白及能源开发等领域，其应用环境通常要求耐高温，因此开发耐高温的β-1，4-内切木聚糖酶具有的应用前景。
发明内容
本发明的目的为提供耐热的β-1，4-内切木聚糖酶。
本发明首先保护蛋白质，可为C1)-C5)中的至少一种：
C1)氨基酸序列如SEQ ID NO:7所示的蛋白质；
C2)氨基酸序列如SEQ ID NO:5所示的蛋白质；
C3)氨基酸序列如SEQ ID NO:3所示的蛋白质；
C4)氨基酸序列如SEQ ID NO:1所示的蛋白质；
C5)在C1)-C4)任一所示的蛋白质的N端或/和C端连接标签得到的融合蛋白质。
SEQ ID NO:1可由397个氨基酸残基组成。SEQ ID NO:3可由378个氨基酸残基组成。SEQ ID NO:5可由325个氨基酸残基组成。SEQ ID NO:7可由325个氨基酸残基组成。
为了使C1)-C4)中的蛋白质便于纯化，可在SEQ ID NO:1、SEQ ID NO:3、SEQ IDNO:5或SEQ ID NO:7所示的蛋白质的氨基末端或羧基末端连接上如表1所示的标签。
表1.标签的序列
| 标签 | 残基 | 序列 |
| Poly-Arg | 5-6(通常为5个) | RRRRR |
| Poly-His | 2-10(通常为6个) | HHHHHH |
| FLAG | 8 | DYKDDDDK |
| Strep-tag II | 8 | WSHPQFEK |
| c-myc | 10 | EQKLISEEDL |
编码所述蛋白质的核酸分子也属于本发明的保护范围。
编码所述蛋白质的核酸分子可为b1)或b2)或b3)或b4)所示的DNA分子：
b1)编码区是SEQ ID NO：8、SEQ ID NO：6、SEQ ID NO：4或SEQ ID NO：2所示的DNA分子；
b2)核苷酸序列是SEQ ID NO：8、SEQ ID NO：6、SEQ ID NO：4或SEQ ID NO：2所示的DNA分子；
b3)与b1)或b2)限定的核苷酸序列具有75％或75％以上同一性，来源于Aspergillus fumigatus且所述蛋白质的DNA分子；
b4)在严格条件下与b1)或b2)限定的核苷酸序列杂交，来源于Aspergillusfumigatus且编码所述蛋白质的DNA分子。
其中，所述核酸分子可以是DNA，如cDNA、基因组DNA或重组DNA；所述核酸分子也可以是RNA，如mRNA或hnRNA等。
SEQ ID NO：8由978个核苷酸组成，编码SEQ ID NO：7所示的氨基酸序列。SEQ IDNO：6由978个核苷酸组成，编码SEQ ID NO：5所示的氨基酸序列。SEQ ID NO：4由1137个核苷酸组成，编码SEQ ID NO：3所示的氨基酸序列。SEQ ID NO：2由1194个核苷酸组成，编码SEQID NO：1所示的氨基酸序列。
本领域普通技术人员可以很容易地采用已知的方法，例如定向进化和点突变的方法，对本发明的编码所述蛋白质的核苷酸序列进行突变。那些经过人工修饰的，具有与本发明分离得到的所述蛋白质的核苷酸序列75％或者更高同一性的核苷酸，只要编码所述蛋白质，均是衍生于本发明的核苷酸序列并且等同于本发明的序列。
这里使用的术语“同一性”指与天然核酸序列的序列相似性。“同一性”包括与本发明的编码SEQ ID NO：1、SEQ ID NO：3、SEQ ID NO：5或SEQ ID NO：7所示的氨基酸序列组成的蛋白质的核苷酸序列具有75％或更高，或80％或更高，或85％或更高，或90％或更高，或95％或更高同一性的核苷酸序列。同一性可以用肉眼或计算机软件进行评价。使用计算机软件，两个或多个序列之间的同一性可以用百分比(％)表示，其可以用来评价相关序列之间的同一性。
含有所述核酸分子的表达盒、重组载体或重组微生物也属于本发明的保护范围。
所述重组载体可为向表达载体或克隆载体插入所述核酸分子得到的重组质粒。
所述表达载体可为表达载体pPICZαa。
所述重组载体具体可为实施例提及的重组质粒XynAF0、重组质粒XynAF1、重组质粒XynAF1-C。
所述重组微生物可为将上述任一所述重组载体导入出发微生物得到的重组菌。
所述出发微生物可为酵母、细菌、藻类或真菌。所述酵母可为Pichia pastoris X-33。
本发明还保护上述任一所述蛋白质或上述任一所述核酸分子在制备β-1，4-内切木聚糖酶产品中的应用。
本发明还保护上述任一所述蛋白质或上述任一所述核酸分子在作为β-1，4-内切木聚糖酶中的应用。
上述任一所述β-1，4-内切木聚糖酶具有耐热性。所述耐热的温度可为70℃以下(如70℃、65℃)。
本发明的发明人从丝状真菌Aspergillus fumigates Z5中克隆到编码木聚糖酶Xyn10A的Xyn10A基因，通过pPICZαa载体在Pichia pastorisX-33中实现表达，发现该酶的β-1，4-内切木聚糖酶酶活力相对较高，具有较好的工业应用价值。为进一步增加该酶的工业应用潜力，对该酶进行突变，成功构建耐热能力大幅提升的3个突变体木聚糖酶XynAF0、木聚糖酶XynAF1和木聚糖酶XynAF1-C。木聚糖酶XynAF0最大催化速率达到1110μmol*min-1*mg-1，木聚糖酶XynAF1最大催化速率达到1971μmol*min-1*mg-1，木聚糖酶XynAF1-C最大催化速率达到1670μmol*min-1*mg-1，均具有极高的催化速率。在70℃或65℃条件下，与木聚糖酶XynAF0或木聚糖酶XynAF1相比，木聚糖酶XynAF1-C的耐热能力均显著提高。本发明具有重要的应用价值。
附图说明
图1为木聚糖酶XynAF0的热稳定性检测结果。
图2为木聚糖酶XynAF1的热稳定性检测结果。
图3为木聚糖酶XynAF1-C的热稳定性检测结果。
具体实施方式
下面结合具体实施方式对本发明进行进一步的详细描述，给出的实施例仅为了阐明本发明，而不是为了限制本发明的范围。以下提供的实施例可作为本技术领域普通技术人员进行进一步改进的指南，并不以任何方式构成对本发明的限制。
下述实施例中的实验方法，如无特殊说明，均为常规方法，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
丝状真菌(Aspergillus fumigatus)Z5记载于如下文献中：LiuD.，ZhangR.，YangX.，Wu H.，XuD.，TangZ.，ShenQ..Thermostable cellulase production ofAspergillus fumigatus Z5 under solid-state fermentation and its applicationin degradation of agricultural wastes.International Biodeterioration&Biodegradation 2011，65(5)，717-725.在下文中，丝状真菌(Aspergillus fumigatus)Z5简称Z5。
表达载体pPICZαa为Invitrogen公司的产品。载体pPICZαa含有His-tag标签。
一步克隆试剂盒为诺唯赞公司的产品。
实施例1、重组β-1，4-内切木聚糖酶的制备
一、重组质粒的构建
1、Xyn10A基因的获得
以Z5的基因组DNA为模板，采用5'-ATGGTCCATCTATCTTCATTG-3'和5'-TTACAGGCACTGTGAGTACCA-3'组成的引物对进行PCR扩增，得到DNA片段Xyn10A。
DNA片段Xyn10A即为木聚糖酶Xyn10A的编码基因(即Xyn10A基因)。
木聚糖酶Xyn10A的氨基酸序列如SEQ ID NO:1所示。
Xyn10A基因的核苷酸序列如SEQ ID NO:2所示。
2、重组质粒XynAF0的构建
(1)以DNA片段Xyn10A为模板，采用5'-GCTGGCCTGAACACAGCAGCC-3'和5'-TTACAGGCACTGTGAGTACCA-3'组成的引物对进行PCR扩增，得到DNA片段XynAF0。
DNA片段XynAF0即为木聚糖酶XynAF0的编码基因(即XynAF0基因)。
木聚糖酶XynAF0的氨基酸序列如SEQ ID NO:3所示。木聚糖酶Xyn10A N端去除19个氨基酸后与木聚糖酶XynAF0完全相同。
XynAF0基因的核苷酸序列如SEQ ID NO:4所示。
(2)采用一步克隆试剂盒将表达载体pPICZαa和DNA片段XynAF0进行同源重组，得到重组质粒XynAF0。具体的，重组质粒XynAF0为将表达载体pPICZαa的分泌标签末端(EAEA)序列和His-tag标签之间的DNA短片段替换为DNA片段XynAF0，其它序列均不变，得到的重组质粒。
3、重组质粒XynAF1的构建
(1)以DNA片段Xyn10A为模板，采用5'-GCTGGCCTGAACACAGCAGCC-3'和5'-TGTGGTGCCGGAGCCGCTTGC-3'组成的引物对进行PCR扩增，得到DNA片段XynAF1。
DNA片段XynAF1即为木聚糖酶XynAF1的编码基因(即XynAF1基因)。
木聚糖酶XynAF1的氨基酸序列如SEQ ID NO:5所示。木聚糖酶Xyn10A N端去除19个氨基酸、C端去除53个氨基酸后与木聚糖酶XynAF1完全相同。
XynAF1基因的核苷酸序列如SEQ ID NO:6所示。
(2)采用一步克隆试剂盒将表达载体pPICZαa和DNA片段XynAF1进行同源重组，得到重组质粒XynAF1。具体的，重组质粒XynAF1为将表达载体pPICZαa的分泌标签末端(EAEA)序列和His-tag标签之间的DNA短片段替换为DNA片段XynAF1，其它序列均不变，得到的重组质粒。
4、重组质粒XynAF1-C的构建
(1)以DNA片段XynAF1为模板，采用5'-GCTGGCCTGAACACATGTGCCAAAGCCAAA-3'和5'-TGTGGTGCCGGAGCCGCTACATCCAAGACCCGCCATCAG-3'组成的引物对进行PCR扩增，得到DNA片段XynAF1-C。
DNA片段XynAF1-C即为木聚糖酶XynAF1-C的编码基因(即XynAF1-C基因)。
木聚糖酶XynAF1-C的氨基酸序列如SEQ ID NO:7所示。
木聚糖酶XynAF1和木聚糖酶XynAF1-C的唯一不同在于：前者N端第6位和第319位为丙氨酸，后者N端第6位和第319位为半胱氨酸。
XynAF1-C基因的核苷酸序列如SEQ ID NO:8所示。
(2)采用一步克隆试剂盒将表达载体pPICZαa和DNA片段XynAF1-C进行同源重组，得到重组质粒XynAF1-C。具体的，重组质粒XynAF1-C为将表达载体pPICZαa的分泌标签末端(EAEA)序列和His-tag标签之间的DNA短片段替换为DNA片段XynAF1-C，其它序列均不变，得到的重组质粒。
二、重组β-1，4-内切木聚糖酶的表达与纯化
重组β-1，4-内切木聚糖酶为木聚糖酶XynAF0、木聚糖酶XynAF1或木聚糖酶XynAF1-C。
1、木聚糖酶XynAF0的表达与纯化
(1)将重组质粒XynAF0转化Pichia pastorisX-33(Invitrogen)，得到重组菌，将该重组菌命名为X-33/XynAF0。
(2)取X-33/XynAF0单克隆，接种至5mLYPD培养基(溶质及其浓度为蛋白胨20g/L、酵母粉10g/L、葡萄糖20g/L、YNB 13.5g/L，溶剂为水)，28℃、220rpm振荡培养36h，得到培养菌液。
(3)取培养菌液，按体积比为1:100接种至0.5L BMGY培养基(溶质及其浓度为蛋白胨20g/L、酵母粉10g/L、YNB 13.5g/L、生物素4mg/L、甘油10g/L和100mM磷酸盐pH值为6.0，溶剂为水)，28℃、220rpm振荡培养5天；培养期间，每隔24h加入甲醇并使其在体系中的浓度为1％。
(4)完成步骤(3)后，离心，收集上清；之后用0.45μm滤膜抽滤上清，得到滤液。
(5)将步骤(4)得到的滤液依次进行硫酸铵沉淀、纯化和透析，得到纯化的木聚糖酶XynAF0。具体步骤如下：将步骤(4)得到的滤液置于冰上，之后加入硫酸铵粉末直至滤液中硫酸铵浓度为80％；4℃静置，离心收集沉淀；将沉淀用PBS缓冲液(溶质及其浓度为50mMNaH2PO4、0.9％NaCl，溶剂为水，pH值为6.0)重溶，之后用镍离子亲和层析进行纯化后，使用25℃的PBS缓冲液透析buffer透析去除亲和层析引入的咪唑等溶剂，获得木聚糖酶XynAF0。
2、木聚糖酶XynAF1的表达与纯化
按照步骤1的方法，将重组质粒XynAF0替换为重组质粒XynAF1，其它步骤均不变，得到木聚糖酶XynAF1。
3、木聚糖酶XynAF1-C的表达与纯化
按照步骤1的方法，将重组质粒XynAF0替换为重组质粒XynAF1-C，其它步骤均不变，得到木聚糖酶XynAF1-C。
实施例2、实施例1制备的重组β-1，4-内切木聚糖酶的动力学检测
重组β-1，4-内切木聚糖酶为木聚糖酶XynAF0、木聚糖酶XynAF1或木聚糖酶XynAF1-C。
1、木糖标准曲线的制备
(1)配制浓度分别为1、2、4、6、8、10mM木糖标准溶液。溶剂为水。
(2)分别取800μL木糖标准溶液，加入等体积DNS溶液(浓度为0.5％)，沸水浴5min。之后采用酶标仪检测A540nm处反应液的吸光度值。以木糖溶液浓度为横坐标，以A540nm吸光度值为纵坐标，绘制木糖标准曲线。
2、木聚糖酶动力学检测
分别配置浓度为0.02％、0.05％、0.1％、0.2％、0.4％、0.6％、0.8％、1％的木聚糖溶液(溶剂为水)，用于酶动力学测定。
(1)取780μL不同浓度的木聚糖溶液，90℃下预热10min，之后在溶液中加入20μL浓度为0.4μM的重组β-1，4-内切木聚糖酶，快速混合后，在90℃水浴锅中精确反应2min。反应结束后加入800μLDNS溶液终止反应，沸水浴5min后，将反应液稀释至酶标板测定A540nm吸光度值。
(2)根据木糖标准曲线，将A540nm吸光度值换算为还原糖的生成量，获得木聚糖酶在不同浓度底物中还原糖生成速率后，使用Origin软件进行非线性回归分析，并拟合Michaelis-Menten方程来计算蛋白的Kcat和Km值。
动力学检测时每个样品设置三个生物学重复。
检测结果见表1。
表1
| V<sub>max</sub>(μmol*min<sup>-1</sup>*mg<sup>-1</sup>) | K<sub>m</sub>(mg*mL<sup>-1</sup>) | K<sub>cat</sub>(s<sup>-1</sup>) | K<sub>cat</sub>/K<sub>m</sub>(mL*mg<sup>-1</sup>*s<sup>-1</sup>) | |
| XynAF0 | 1110.84±81.90 | 1.81±0.31 | 770.50 | 426.63 |
| XynAF1 | 1971.14±93.46 | 2.18±0.32 | 1188.41 | 546.36 |
| XynAF1-C | 1670.39±116.02 | 2.85±0.58 | 1006.90 | 353.67 |
结果表明，木聚糖酶XynAF0最大催化速率达到1110μmol*min-1*mg-1，木聚糖酶XynAF1最大催化速率达到1971μmol*min-1*mg-1，木聚糖酶XynAF1-C最大催化速率达到1670μmol*min-1*mg-1，均具有极高的催化速率。
实施例3、实施例1制备的重组β-1，4-内切木聚糖酶的热稳定性检测
重组β-1，4-内切木聚糖酶为木聚糖酶XynAF0、木聚糖酶XynAF1或木聚糖酶XynAF1-C。
木聚糖底物溶液：称取0.4g NaOH固体至800mL ddH2O中充分溶解，得到NaOH溶液；准确称取木聚糖10g加入NaOH溶液，磁力搅拌器搅拌1h后，加入磷酸调节至pH值至6.0，加入ddH2O定容至1000mL，得到木聚糖底物溶液。
1、重组蛋白溶液的制备
取实施例1制备的重组β-1，4-内切木聚糖酶，用PBS缓冲液稀释，得到浓度为0.4μM的酶液，该酶液即为重组蛋白溶液。
2、蛋白原始酶活检测
将50μL重组蛋白溶液和750μL木聚糖底物溶液混合，置于65℃、70℃、75℃、80℃或85℃反应5min；然后加入800μL DNS溶液，沸水浴5min。之后采用酶标仪检测A540nm处反应液的吸光度值。根据实施例2制备的木糖标准曲线，将A540nm吸光度值换算为还原糖的生成量，即获得蛋白原始酶活。
3、重组蛋白溶液温育
将1mL重组蛋白溶液置于65℃、70℃、75℃、80℃或85℃温育反应5min、10min、15min、20min、25min、30min、40min、50min、60min、2h、4h、8h、12h、18h或24h，得到热温育处理后的蛋白溶液。
4、蛋白热处理后酶活检测
取50μL热温育处理后的蛋白溶液和750μL木聚糖底物溶液混合，置于65℃、70℃、75℃、80℃或85℃反应5min；然后加入800μL DNS溶液，沸水浴5min。之后采用酶标仪检测A540nm处反应液的吸光度值。根据实施例2制备的木糖标准曲线，将A540nm吸光度值换算为还原糖的生成量，即获得蛋白热处理后酶活。
5、相对酶活的获得
相对酶活＝蛋白热处理后酶活/蛋白原始酶活
木聚糖酶XynAF0的热稳定性检测结果见图1。
木聚糖酶XynAF1的热稳定性检测结果见图2。
木聚糖酶XynAF1-C的热稳定性检测结果见图3。
6、半衰期的获得
根据相对酶活曲线图，计算重组β-1，4-内切木聚糖酶在特定温度下孵育后，剩余50％相对酶活时需孵育的时间，即重组β-1，4-内切木聚糖酶在特定温度下的半衰期。
重组β-1，4-内切木聚糖酶的半衰期检测结果见表2。
表2
| T<sub>50</sub>(min) | 65℃ | 70℃ | 75℃ | 80℃ | 85℃ |
| 木聚糖酶XynAF0 | 262 | 60 | 39 | 11 | 4 |
| 木聚糖酶XynAF1 | 244 | 63 | 35 | 9 | 4 |
| 木聚糖酶XynAF1-C | 540 | 145 | 45 | 15 | 7 |
结果表明，木聚糖酶XynAF0在65℃条件下温育262min后，剩余50％相对酶活，木聚糖酶XynAF1表现与之相似，木聚糖酶XynAF1-C在65℃条件下温育540min后保持50％相对酶活，即蛋白热稳定性提高1倍。同样，木聚糖酶XynAF1-C在70℃条件下，比木聚糖酶XynAF0与木聚糖酶XynAF1耐热能力提高超过1倍。
以上对本发明进行了详述。对于本领域技术人员来说，在不脱离本发明的宗旨和范围，以及无需进行不必要的实验情况下，可在等同参数、浓度和条件下，在较宽范围内实施本发明。虽然本发明给出了特殊的实施例，应该理解为，可以对本发明作进一步的改进。总之，按本发明的原理，本申请欲包括任何变更、用途或对本发明的改进，包括脱离了本申请中已公开范围，而用本领域已知的常规技术进行的改变。按以下附带的权利要求的范围，可以进行一些基本特征的应用。
<110> 中国农业科学院农业资源与农业区划研究所
<120> 耐热β-1，4-内切木聚糖酶及其编码基因与应用
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<212> DNA
<213> Artificial sequence
<400> 4
gctggcctga acacagcagc caaagccaaa ggactaaagt actttggttc cgccacggac 60
aatccagagc tcacggactc tgcgtatgtc gcgcaactga gcaacaccga tgattttggt 120
caaatcacac ccggaaactc catgaagtgg gatgccaccg agccttctca gaattctttt 180
tcgttcgcaa atggagacgc cgtggtcaat ctggcgaaca agaatggcca gctgatgcga 240
tgccatactc tggtctggca cagtcagcta ccgaactggg tctctagcgg gtcatggacc 300
aatgcgaccc ttttggcggc catgaagaat catatcacca atgtggttac tcactacaag 360
gggaagtgct acgcctggga tgttgtcaat gaagccctga acgaggacgg tactttccgt 420
aactctgtct tctaccagat catcggccca gcatacattc ctattgcgtt cgccacggct 480
gctgccgcag atcccgacgt gaaactctac tacaacgact acaacattga atactcaggc 540
gccaaagcga ctgctgcgca gaatatcgtc aagatgatca aggcctacgg cgcgaagatc 600
gacggcgtcg gcctccaggc acactttatc gtcggcagca ctccgagtca atcggatctg 660
acgaccgtct tgaagggcta cactgctctc ggcgttgagg tggcctatac cgaacttgac 720
atccgcatgc agctgccctc gaccgccgca aagctggccc agcagtccac tgacttccaa 780
ggcgtggccg cagcatgcgt tagcaccact ggctgcgtgg gtgtcactat ctgggactgg 840
accgacaagt actcctgggt ccccagcgtg ttccaaggct acggcgcccc attgccttgg 900
gatgagaact atgtgaagaa gccagcgtac gatggcctga tggcgggtct tggagcaagc 960
ggctccggca ccacaacgac cactactact acttctacta cgacaggagg tacggaccct 1020
actggagtcg ctcagaaatg gggacagtgt ggcggtattg gctggaccgg gccaacaact 1080
tgtgtcagtg gtaccacttg ccaaaagctg aatgactggt actcacagtg cctgtaa 1137
<210> 5
<211> 325
<212> DNA
<213> Artificial sequence
<400> 5
Ala Gly Leu Asn Thr Ala Ala Lys Ala Lys Gly Leu Lys Tyr Phe Gly
1 5 10 15
Ser Ala Thr Asp Asn Pro Glu Leu Thr Asp Ser Ala Tyr Val Ala Gln
20 25 30
Leu Ser Asn Thr Asp Asp Phe Gly Gln Ile Thr Pro Gly Asn Ser Met
35 40 45
Lys Trp Asp Ala Thr Glu Pro Ser Gln Asn Ser Phe Ser Phe Ala Asn
50 55 60
Gly Asp Ala Val Val Asn Leu Ala Asn Lys Asn Gly Gln Leu Met Arg
65 70 75 80
Cys His Thr Leu Val Trp His Ser Gln Leu Pro Asn Trp Val Ser Ser
85 90 95
Gly Ser Trp Thr Asn Ala Thr Leu Leu Ala Ala Met Lys Asn His Ile
100 105 110
Thr Asn Val Val Thr His Tyr Lys Gly Lys Cys Tyr Ala Trp Asp Val
115 120 125
Val Asn Glu Ala Leu Asn Glu Asp Gly Thr Phe Arg Asn Ser Val Phe
130 135 140
Tyr Gln Ile Ile Gly Pro Ala Tyr Ile Pro Ile Ala Phe Ala Thr Ala
145 150 155 160
Ala Ala Ala Asp Pro Asp Val Lys Leu Tyr Tyr Asn Asp Tyr Asn Ile
165 170 175
Glu Tyr Ser Gly Ala Lys Ala Thr Ala Ala Gln Asn Ile Val Lys Met
180 185 190
Ile Lys Ala Tyr Gly Ala Lys Ile Asp Gly Val Gly Leu Gln Ala His
195 200 205
Phe Ile Val Gly Ser Thr Pro Ser Gln Ser Asp Leu Thr Thr Val Leu
210 215 220
Lys Gly Tyr Thr Ala Leu Gly Val Glu Val Ala Tyr Thr Glu Leu Asp
225 230 235 240
Ile Arg Met Gln Leu Pro Ser Thr Ala Ala Lys Leu Ala Gln Gln Ser
245 250 255
Thr Asp Phe Gln Gly Val Ala Ala Ala Cys Val Ser Thr Thr Gly Cys
260 265 270
Val Gly Val Thr Ile Trp Asp Trp Thr Asp Lys Tyr Ser Trp Val Pro
275 280 285
Ser Val Phe Gln Gly Tyr Gly Ala Pro Leu Pro Trp Asp Glu Asn Tyr
290 295 300
Val Lys Lys Pro Ala Tyr Asp Gly Leu Met Ala Gly Leu Gly Ala Ser
305 310 315 320
Gly Ser Gly Thr Thr
325
<210> 6
<211> 978
<212> DNA
<213> Artificial sequence
<400> 6
gctggcctga acacagcagc caaagccaaa ggactaaagt actttggttc cgccacggac 60
aatccagagc tcacggactc tgcgtatgtc gcgcaactga gcaacaccga tgattttggt 120
caaatcacac ccggaaactc catgaagtgg gatgccaccg agccttctca gaattctttt 180
tcgttcgcaa atggagacgc cgtggtcaat ctggcgaaca agaatggcca gctgatgcga 240
tgccatactc tggtctggca cagtcagcta ccgaactggg tctctagcgg gtcatggacc 300
aatgcgaccc ttttggcggc catgaagaat catatcacca atgtggttac tcactacaag 360
gggaagtgct acgcctggga tgttgtcaat gaagccctga acgaggacgg tactttccgt 420
aactctgtct tctaccagat catcggccca gcatacattc ctattgcgtt cgccacggct 480
gctgccgcag atcccgacgt gaaactctac tacaacgact acaacattga atactcaggc 540
gccaaagcga ctgctgcgca gaatatcgtc aagatgatca aggcctacgg cgcgaagatc 600
gacggcgtcg gcctccaggc acactttatc gtcggcagca ctccgagtca atcggatctg 660
acgaccgtct tgaagggcta cactgctctc ggcgttgagg tggcctatac cgaacttgac 720
atccgcatgc agctgccctc gaccgccgca aagctggccc agcagtccac tgacttccaa 780
ggcgtggccg cagcatgcgt tagcaccact ggctgcgtgg gtgtcactat ctgggactgg 840
accgacaagt actcctgggt ccccagcgtg ttccaaggct acggcgcccc attgccttgg 900
gatgagaact atgtgaagaa gccagcgtac gatggcctga tggcgggtct tggagcaagc 960
ggctccggca ccacataa 978
<210> 7
<211> 325
<212> DNA
<213> Artificial sequence
<400> 7
Ala Gly Leu Asn Thr Cys Ala Lys Ala Lys Gly Leu Lys Tyr Phe Gly
1 5 10 15
Ser Ala Thr Asp Asn Pro Glu Leu Thr Asp Ser Ala Tyr Val Ala Gln
20 25 30
Leu Ser Asn Thr Asp Asp Phe Gly Gln Ile Thr Pro Gly Asn Ser Met
35 40 45
Lys Trp Asp Ala Thr Glu Pro Ser Gln Asn Ser Phe Ser Phe Ala Asn
50 55 60
Gly Asp Ala Val Val Asn Leu Ala Asn Lys Asn Gly Gln Leu Met Arg
65 70 75 80
Cys His Thr Leu Val Trp His Ser Gln Leu Pro Asn Trp Val Ser Ser
85 90 95
Gly Ser Trp Thr Asn Ala Thr Leu Leu Ala Ala Met Lys Asn His Ile
100 105 110
Thr Asn Val Val Thr His Tyr Lys Gly Lys Cys Tyr Ala Trp Asp Val
115 120 125
Val Asn Glu Ala Leu Asn Glu Asp Gly Thr Phe Arg Asn Ser Val Phe
130 135 140
Tyr Gln Ile Ile Gly Pro Ala Tyr Ile Pro Ile Ala Phe Ala Thr Ala
145 150 155 160
Ala Ala Ala Asp Pro Asp Val Lys Leu Tyr Tyr Asn Asp Tyr Asn Ile
165 170 175
Glu Tyr Ser Gly Ala Lys Ala Thr Ala Ala Gln Asn Ile Val Lys Met
180 185 190
Ile Lys Ala Tyr Gly Ala Lys Ile Asp Gly Val Gly Leu Gln Ala His
195 200 205
Phe Ile Val Gly Ser Thr Pro Ser Gln Ser Asp Leu Thr Thr Val Leu
210 215 220
Lys Gly Tyr Thr Ala Leu Gly Val Glu Val Ala Tyr Thr Glu Leu Asp
225 230 235 240
Ile Arg Met Gln Leu Pro Ser Thr Ala Ala Lys Leu Ala Gln Gln Ser
245 250 255
Thr Asp Phe Gln Gly Val Ala Ala Ala Cys Val Ser Thr Thr Gly Cys
260 265 270
Val Gly Val Thr Ile Trp Asp Trp Thr Asp Lys Tyr Ser Trp Val Pro
275 280 285
Ser Val Phe Gln Gly Tyr Gly Ala Pro Leu Pro Trp Asp Glu Asn Tyr
290 295 300
Val Lys Lys Pro Ala Tyr Asp Gly Leu Met Ala Gly Leu Gly Cys Ser
305 310 315 320
Gly Ser Gly Thr Thr
325
<210> 8
<211> 978
<212> DNA
<213> Artificial sequence
<400> 8
gctggcctga acacatgtgc caaagccaaa ggactaaagt actttggttc cgccacggac 60
aatccagagc tcacggactc tgcgtatgtc gcgcaactga gcaacaccga tgattttggt 120
caaatcacac ccggaaactc catgaagtgg gatgccaccg agccttctca gaattctttt 180
tcgttcgcaa atggagacgc cgtggtcaat ctggcgaaca agaatggcca gctgatgcga 240
tgccatactc tggtctggca cagtcagcta ccgaactggg tctctagcgg gtcatggacc 300
aatgcgaccc ttttggcggc catgaagaat catatcacca atgtggttac tcactacaag 360
gggaagtgct acgcctggga tgttgtcaat gaagccctga acgaggacgg tactttccgt 420
aactctgtct tctaccagat catcggccca gcatacattc ctattgcgtt cgccacggct 480
gctgccgcag atcccgacgt gaaactctac tacaacgact acaacattga atactcaggc 540
gccaaagcga ctgctgcgca gaatatcgtc aagatgatca aggcctacgg cgcgaagatc 600
gacggcgtcg gcctccaggc acactttatc gtcggcagca ctccgagtca atcggatctg 660
acgaccgtct tgaagggcta cactgctctc ggcgttgagg tggcctatac cgaacttgac 720
atccgcatgc agctgccctc gaccgccgca aagctggccc agcagtccac tgacttccaa 780
ggcgtggccg cagcatgcgt tagcaccact ggctgcgtgg gtgtcactat ctgggactgg 840
accgacaagt actcctgggt ccccagcgtg ttccaaggct acggcgcccc attgccttgg 900
gatgagaact atgtgaagaa gccagcgtac gatggcctga tggcgggtct tggatgtagc 960
ggctccggca ccacataa 978
Claims (10)
1.蛋白质，为C1)-C5)中的至少一种：
C1)氨基酸序列如SEQ ID NO:7所示的蛋白质；
C2)氨基酸序列如SEQ ID NO:5所示的蛋白质；
C3)氨基酸序列如SEQ ID NO:3所示的蛋白质；
C4)氨基酸序列如SEQ ID NO:1所示的蛋白质；
C5)在C1)-C4)任一所示的蛋白质的N端或/和C端连接标签得到的融合蛋白质。
2.编码权利要求1所述蛋白质的核酸分子。
3.如权利要求2所述的核酸分子，其特征在于：所述核酸分子为b1)或b2)或b3)或b4)所示的DNA分子：
b1)编码区是SEQ ID NO：8、SEQ ID NO：6、SEQ ID NO：4或SEQ ID NO：2所示的DNA分子；
b2)核苷酸序列是SEQ ID NO：8、SEQ ID NO：6、SEQ ID NO：4或SEQ ID NO：2所示的DNA分子；
b3)与b1)或b2)限定的核苷酸序列具有75％或75％以上同一性，来源于Aspergillusfumigatus且编码权利要求1所述蛋白质的DNA分子；
b4)在严格条件下与b1)或b2)限定的核苷酸序列杂交，来源于Aspergillus fumigatus且编码权利要求1所述蛋白质的DNA分子。
4.含有权利要求3所述核酸分子的表达盒、重组载体或重组微生物。
5.如权利要求4所述的重组载体，其特征在于：所述重组载体为向表达载体或克隆载体插入所述核酸分子得到的重组质粒。
6.如权利要求4所述的重组微生物，其特征在于：所述重组微生物为将权利要求5所述重组载体导入出发微生物得到的重组菌。
7.权利要求1所述蛋白质或权利要求2或3所述核酸分子在制备β-1，4-内切木聚糖酶产品中的应用。
8.权利要求1所述蛋白质或权利要求2或3所述核酸分子在作为β-1，4-内切木聚糖酶中的应用。
9.如权利要求1所述的蛋白质或权利要求7或8所述的应用，其特征在于：所述β-1，4-内切木聚糖酶具有耐热性。
10.如权利要求9所述的蛋白质或权利要求9所述的应用，其特征在于：所述耐热的温度为70℃以下。
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| WO2023225459A2 (en) | 2022-05-14 | 2023-11-23 | Novozymes A/S | Compositions and methods for preventing, treating, supressing and/or eliminating phytopathogenic infestations and infections |
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