CN110093331A - 一种高产的耐高温宽pH稳定性的甘露聚糖酶ManGold及基因与应用 - Google Patents
一种高产的耐高温宽pH稳定性的甘露聚糖酶ManGold及基因与应用 Download PDFInfo
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- CN110093331A CN110093331A CN201910371952.7A CN201910371952A CN110093331A CN 110093331 A CN110093331 A CN 110093331A CN 201910371952 A CN201910371952 A CN 201910371952A CN 110093331 A CN110093331 A CN 110093331A
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Abstract
本发明属于基因工程技术领域,涉及一种高产的耐高温宽pH稳定性的甘露聚糖酶ManGold及基因与应用。所述甘露聚糖酶ManGold的氨基酸序列含有SEQ ID NO:1所示的氨基酸序列。所述甘露聚糖酶基因mangold的碱基序列与SEQ ID NO:3所示的碱基序列具有95%以上的同一性。本发明显著提高了人工合成的甘露聚糖酶基因mangold在毕赤酵母细胞中的表达量,获得了高水平表达的、耐高温、宽pH适应性的甘露聚糖酶及基因。
Description
技术领域
本发明属于基因工程技术领域,更具体地,涉及一种高产的、耐高温和宽pH稳定性的甘露聚糖酶ManGold及基因序列、以及重组表达载体、重组表达菌株、该甘露聚糖酶ManGold的应用和一种水解甘露聚糖的方法。
背景技术
甘露聚糖酶(β-1,4-D-甘露聚糖酶,EC 3.2.1.78)能够水解甘露聚糖和半乳甘露聚糖等多糖中的β-1,4-甘露聚糖苷键,促进该类物质的降解和利用。
甘露聚糖酶具有广阔的应用前景。在饲料中,甘露聚糖及其衍生物(β-半乳甘露聚糖和β-葡萄糖甘露聚糖)是豆科植物细胞壁的组成成分,由于其结构复杂,是单胃动物的抗营养因子,降低饲料利用率。甘露聚糖酶能有效分解豆类原料中的甘露聚糖,提高饲料的营养价值;在食品和医药中,甘露聚糖酶能将魔芋粉等甘露聚糖水解为低聚甘露糖,从而改善肠道环境,有利于双歧杆菌等有益菌群的增殖,有效降低人体胆固醇和血糖水平。在造纸工业中,甘露聚糖酶与其它酶的协同作用下,能更有效的降解纸浆中的半纤维素,降低污染、提高效率。
尽管甘露聚糖酶在食品、饲料、造纸、医药等领域具有广泛的应用前景,但仍然存在需要解决的关键问题:1)甘露聚糖酶的比活力低,对甘露聚糖的水解效率低。2)甘露聚糖酶的工业生产水平较低,生产成本和使用成本相对较高。3)甘露聚糖酶的高温稳定性和pH稳定性较差,低别是在饲料工业中,高温制粒工艺易使不耐热的甘露聚糖酶失去活性,胃中的酸性环境和肠道的碱性环境易使甘露聚糖酶失去活性。
因此,需要对甘露聚糖酶进行改造来提高酶对温度和pH的稳定性,提高甘露聚糖酶的表达水平,降低生产和使用成本。
发明内容
本发明的目的是提供一种甘露聚糖酶ManGold及基因序列、以及重组表达载体、重组表达菌株、该甘露聚糖酶ManGold的应用和一种水解甘露聚糖的方法,旨在获得一种高产的、耐高温的、宽pH耐受性的甘露聚糖酶,为其产业化应用奠定基础。
为了实现上述目的,本发明提供一种高产的耐高温宽pH稳定性的甘露聚糖酶ManGold,所述甘露聚糖酶ManGold的氨基酸序列含有SEQ ID NO:1所示的氨基酸序列。根据本发明,SEQ ID NO:1所示的氨基酸序列为核心序列,所述甘露聚糖酶ManGold的氨基酸序列含有SEQ ID NO:1所示的氨基酸序列即可实现本发明的效果。进一步优选地,所述甘露聚糖酶ManGold的氨基酸序列为SEQ ID NO:1所示的氨基酸序列。
根据本发明,所述甘露聚糖酶ManGold由甘露聚糖酶Man通过点突变得到,所述甘露聚糖酶Man的氨基酸序列如SEQ ID NO:2所示。具体的突变包括:将原始甘露聚糖酶Man中50位的天冬酰氨突变为酪氨酸(N50Y)、167位的甘氨酸突变为色氨酸(G167W)、308位的丙氨酸突变为酪氨酸(A308Y)。所述点突变可采用本领域常规的各种突变方法,其具体操作步骤为本领域技术人员公知,在此不再赘述。
本发明的第二方面提供一种甘露聚糖酶基因mangold,用于编码甘露聚糖酶ManGold。所述甘露聚糖酶基因mangold的碱基序列与SEQ ID NO:3所示的碱基序列具有95%以上的同一性。优选地,所述的甘露聚糖酶基因mangold的碱基序列含有SEQ ID NO:3所示的碱基序列。在最优选情况下,本发明所述甘露聚糖酶基因mangold的碱基序列为SEQID NO:3所示的碱基序列。
本发明提供的甘露聚糖酶基因mangold,系基于甘露聚糖酶氨基酸序列人工设计。与原始的甘露聚糖酶基因man(SEQ ID NO:4)相比,本发明的甘露聚糖酶基因mangold采用了在毕赤酵母中高频使用的密码子,从而显著提高了人工设计的甘露聚糖酶基因mangold在毕赤酵母细胞中的表达量。本发明根据甘露聚糖酶的氨基酸序列,人工设计出一条全新的甘露聚糖酶基因序列,并通过人工合成的方法获得所述甘露聚糖酶基因片段,其碱基序列如SEQ ID NO:3所示,命名为mangold,所述甘露聚糖酶基因mangold编码出的甘露聚糖酶的氨基酸序列如SEQ ID NO:1所示。本发明的甘露聚糖酶基因mangold表达出的甘露聚糖酶ManGold的适宜温度为70~80℃,最优选为80℃,适宜pH值为3-5,最优选为4。
本发明的第三方面提供一种重组表达载体,其包括上述甘露聚糖酶基因mangold,还可包括其他功能单元。在甘露聚糖酶ManGold的氨基酸序列和甘露聚糖酶基因mangold的碱基序列确定的情况下,本领域技术人员能够选择合适的重组表达载体以及其他功能单元,例如,毕赤酵母表达载体。
本发明对所述重组表达载体的制备方法没有特别限定,可根据本领域常规技术手段确定。
根据本发明一种具体实施方式,所述重组表达载体的制备方法包括以下步骤:将合成的甘露聚糖酶基因mangold连接至表达载体中。在本发明的一些实施例中,所述表达载体为毕赤酵母表达载体pPICZαA。当然,在本发明其他实施例中,也可选用其他毕赤酵母表达载体。
根据本发明一种更加具体的实施方式,所述重组表达载体的制备方法包括如下步骤:将合成的甘露聚糖酶基因mangold通过限制性内切酶EcoRI和Xba I酶切,随后插入同样经限制性内切酶EcoR I和Xba I酶切后的毕赤酵母表达载体pPICZαA中,获得pPIC-mangold重组表达质粒。
本发明的第四方面提供一种重组表达菌株,其表达产物为上述甘露聚糖酶ManGold。在甘露聚糖酶ManGold的氨基酸序列确定的情况下,本领域技术人员能够获得合适的重组表达菌株。
通常来说,所述宿主细胞可以是大肠杆菌、芽孢杆菌、曲霉,也可以是酵母等细胞,或者是其他类型的细胞例如动物细胞等。优选地,所述重组表达菌株的宿主细胞为毕赤酵母。同时,本发明提供的甘露聚糖酶基因mangold经过密码子优化,采用了适合于毕赤酵母进行转录翻译表达出甘露聚糖酶的高频密码子,提高了甘露聚糖酶基因mangold在毕赤酵母细胞中的表达量。
本发明对所述重组表达菌株的制备方法没有特别限定,可根据本领域常规技术手段确定。
根据本发明一种具体实施方式,所述重组表达菌株的制备方法包括如下步骤:将重组表达载体通过限制性内切酶线性化;将上述线性化片段导入毕赤酵母中,获得重组表达菌株。可选地,通过电转将重组表达载体导入毕赤酵母宿主细胞中,获得重组表达菌株。在本发明的一些实施例中,所述毕赤酵母为巴斯德毕赤酵母X-33,当然,在本发明其他实施例中,也可选用其他毕赤酵母菌株。
根据本发明一种更加具体的实施方式,所述重组表达菌株的制备方法包括如下步骤:将pPIC-mangold重组表达质粒通过Bgl II限制性内切酶线性化;通过电转化法将线性化的pPIC-mangold导入毕赤酵母宿主细胞中,获得重组表达菌株。
本发明中,重组表达菌株制备甘露聚糖酶的方法可以采用本领域常规的方法。根据本发明一种具体实施方式,生产甘露聚糖酶的方法包括:通过发酵培养上述重组表达菌株,从发酵上清液中提纯获得甘露聚糖酶。在一些实施方式中,也可以不经过提纯处理,直接将发酵上清液视为甘露聚糖酶产物。
根据本发明一种更加具体的实施方式,由上述重组表达菌株制备甘露聚糖酶的方法包括如下步骤:挑取重组表达菌株至培养基中培养约24小时,并收获菌体;将菌体转入新鲜的培养基中,每24小时加入约1%的甲醇,诱导甘露聚糖酶ManGold的表达。诱导表达约96小时后,离心收集上清液,获得含甘露聚糖酶ManGold的溶液。
本发明的第五方面提供上述甘露聚糖酶ManGold的应用。
具体地,本发明的第六方面提供一种水解甘露聚糖的方法,该方法包括:将上述甘露聚糖酶ManGold与含甘露聚糖物料接触。所述含甘露聚糖物料可以为甘露聚糖,也可以为其他含甘露聚糖的物料,例如魔芋精粉。
本发明提供的甘露聚糖酶ManGold系由原始甘露聚糖酶突变而来,基因mangold依照ManGold的氨基酸序列进行人工设计,根据毕赤酵母密码子的偏好性,选用毕赤酵母中高频使用的密码子,降低了mangold基因mRNA二级结构的自由能,从而显著提高了人工合成的甘露聚糖酶基因mangold在毕赤酵母细胞中的表达量,显著提高了甘露聚糖酶的活性、温度稳定性并加宽了其pH适应性,获得了在毕赤酵母中高水平表达的、耐高温、宽pH适应性的甘露聚糖酶及基因。
本发明的其它特征和优点将在随后具体实施方式部分予以详细说明。
附图说明
通过结合附图对本发明示例性实施方式进行更详细的描述,本发明的上述以及其它目的、特征和优势将变得更加明显。
图1A-图1B为本发明实施例1中以PCR介导的原始甘露聚糖酶基因的点突变的电泳结果图。
图2为实施例2中原始甘露聚糖酶基因和优化后甘露聚糖酶基因mRNA二级结构图。其中,左图为原始甘露聚糖酶基因man基因mRNA的二级结构。最小自由能为-29.84kcal/mol;右图为mangold基因mRNA的二级结构,最小自由能为-20.70kcal/mol。
图3A-图3C为实施例3重组表达载体构建过程中进行酶切验证的电泳结果图。
图4A和图4B示出了本发明实施例4中高水平表达菌株发酵上清液的SDS-PAGE测试结果。
图5示出了本发明实施例4中发酵上清液的酶活随时间的变化曲线。
图6A为本发明实施例5中甘露聚糖酶ManGold活性随温度变化曲线,图6B为本发明实施例5中甘露聚糖酶ManGold活性随pH变化曲线。
图7示出了本发明实施例6中甘露聚糖酶水解甘露聚糖的薄层层析测试结果。
具体实施方式
下面将更详细地描述本发明的优选实施方式。虽然以下描述了本发明的优选实施方式,然而应该理解,可以以各种形式实现本发明而不应被这里阐述的实施方式所限制。实施例中未注明具体条件者,按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者,均为可以通过市售购买获得的常规产品。
实施例1
本实施例用于说明甘露聚糖酶AoMan的定点突变。
以甘露聚糖酶基因man为出发基因,采用定点突变技术,将原始甘露聚糖酶Man中50位的天冬酰氨突变为酪氨酸(N50Y)、167位的甘氨酸突变为色氨酸(G167W)、308位的丙氨酸突变为酪氨酸(A308Y)。具体实施步骤包括:
(1)采用重叠延伸PCR技术,向原始的甘露聚糖酶基因man基因(SEQID NO:4)中引入N50Y突变。以man基因为模板分别采用引物PF(SEQID NO:5,5’-GAATTCATGAAGCTTAACCCTTCACTC-3’)和NQR1(SEQID NO:6,5’-CTTGCCATCGATCACAAAGTAGAGACCTGAAGTAG-3’),扩增出man基因的前半段F1,再采用引物NQF2(SEQ ID NO:7,5’-CTACTTCAGGTCTCTACTTTGTGATCGATGGCAAG-3’)和PR(SEQ ID NO:8,5’-GCGGCCGCTCACTTACGACTGTTGATGG-3’),扩增出man基因的后半段F2;分别以F1和F2为模板,再采用引物PF和PR扩增出全长的突变基因,完成N50Y的突变;
(2)在上述突变基因的基础上,采用重叠延伸PCR技术向甘露聚糖酶基因man中引入G167W突变。采用引物PF(SEQ ID NO:5,5’-GAATTCATGAAGCTTAACCCTTCACTC-3’)和GWR1(SEQ ID NO:9,5’-CTCCTTCGACCCCCAGAAGGCCTTGACATAG-3’),扩增得到上游片段EWF1,再采用引物GWF2(SEQ ID NO:10,5’-CTA TGT CA AGG CCTT CTGG GGG TCG AAG GAG-3’)和引物PR(SEQ ID NO:8,5’-GCGGCCGCTCACTTACGACTGTTGATGG-3’),扩增得到下游片段EWF2;再以前半段基因和后半段基因为模板,以PF和PR为引物,PCR扩增并获得全长的突变基因片段。
(3)在上述突变基因的基础上,采用重叠延伸PCR技术向甘露聚糖酶基因man中引入A308Y突变。采用引物PF(SEQ ID NO:5,5’-GAATTCATGAAGCTTAACCCTTCACTC-3’)和AYR1(SEQ ID NO:11,5’-GAACAGGCAAGGCTTTCCGTACGCCTTGCAGGCTG-3’),扩增得到上游片段,再采用引物AYF2(SEQ ID NO:12,5’-CAGCCTGCAAGGCGTACGGAAAGCCTTGCCTGTTC-3’)和引物PR(SEQ ID NO:8,5’-GCGGCCGCTCACTTACGACTGTTGATGG-3’),扩增得到下游片段EWF2;再以前半段基因和后半段基因为模板,以PF和PR为引物,PCR扩增并获得全长的突变基因片段。
图1A-图1B为本发明实施例1中以PCR介导的原始甘露聚糖酶基因的点突变的电泳结果图。其中,M为DL5000DNA Marker,图1A中泳道1为突变N50Y的上半段序列PCR产物,泳道2为突变N50Y的下半段序列PCR产物;泳道3为突变G167W的上半段序列PCR产物,泳道4为突变G167W的下半段序列PCR产物;泳道5为突变A308Y的上半段序列PCR产物,泳道2为突变A308Y的下半段序列PCR产物。图1B中,M为DL5000DNA Marker,泳道1为全长的突变基因片段。
经测序,确定突变成功。
实施例2
本实施例用于说明甘露聚糖酶经密码子的优化后获得基因mangold。
基于实施例1所获得的突变的甘露聚糖酶的氨基酸序列,为提高突变后甘露聚糖酶基因的产量,在计算机的辅助下,人工重新设计了甘露聚糖酶基因的核苷酸序列。具体包括:根据毕赤酵母密码子使用的偏好性,采用在新的宿主中的高频率使用的密码子来替代低频率的密码子;降低新设计基因编码的mRNA的二级结构的复杂度和最小自由能;消失不必要的限制性内切酶位点。从而获得了人工设计的甘露聚糖酶基因mangold序列。
根据甘露聚糖酶的氨基酸序列人工设计出的全新的甘露聚糖酶基因,命名为mangold,其碱基序列如SEQ ID NO:3所示。所述甘露聚糖酶基因mangold编码的甘露聚糖酶的氨基酸序列如SEQ ID NO:1所示。
图2为本发明实施例2中原始甘露聚糖酶基因和优化后甘露聚糖酶基因mRNA二级结构图。其中,左图为原始甘露聚糖酶基因man基因mRNA的二级结构。最小自由能为-29.84kcal/mol;右图为mangold基因mRNA的二级结构,最小自由能为-20.70kcal/mol。
实施例3
本实施例用于说明甘露聚糖酶基因mangold重组表达菌株的构建。
采用毕赤酵母表达载体克隆甘露聚糖酶基因mangold。重组载体的构建过程包括以下步骤:
(1)在甘露聚糖酶基因mangold两端加上酶切位点EcoR I和Not I酶切位点;甘露聚糖酶基因mangold和载体pPICZαA均经EcoR I和Not I酶切。酶切体系为:3mg DNA、10UEcoR I、10U Not I、20μL的Buffer H加水补齐至200μL体积。酶切结果如图3A-图3B所示(图3A和图3B中:M为DL5000DNA Marker,1泳道为酶切后的mangold片段,2泳道为酶切后的pPICZαA重组表达载体;由图3A-图3B可知,pPICZαA和mangold片段的大小分别为3.7kb和1.2kb左右,与实际相符。
(2)通过T4DNA连接酶连接将mangold和pPICZαA酶切片段连接并获得pPIC-mangold重组表达载体。连接体系为:150ng mangold、50ngpPICZαA、1μL T4buffer、5UT4DNA连接酶。提取重组表达载体pPIC-mangold,进行双酶切验证(EcoR I、Not I),结果如图3C所示(图中:M为DL5000DNA Marker,3泳道为单酶切后的pPIC-mangold重组表达载体,4泳道为双酶切后的pPIC-mangold重组表达载体)。图3C中,在1200bp位置有条带,说明甘露聚糖酶基因mangold成功连接至pPICZαA重组表达载体上。
(3)将重组表达载体pPIC-mangold电转化入巴斯德毕赤酵母(Pichiapastoris)X-33中获得含mangold的重组表达菌株。电转化方法为:分别取3μg pPIC-mangold与90μL巴斯德毕赤酵母X-33混匀,置于冰上5min,然后用电转化仪(BioRad公司生产)电击。
实施例4
本实施例用于说明甘露聚糖酶ManGold在台式发酵罐条件下的发酵生产。具体包括以下步骤:
(1)将实施例3中获得的甘露聚糖酶ManGold重组表达菌株接种到700mL含酵母粉和蛋白胨的培养基中,培养20h,获得种子液。
(2)将上述种子液接种至含7升发酵培养基的发酵罐中进行发酵培养。每升发酵培养基包括:40g KH2PO4、1g CaSO4、3.5g(NH4)2SO4、8.2gMgSO4、23g K2SO4和80g甘油。
(3)在发酵罐中的培养可分为营养生长和甲醇诱导表达两个阶段。在营养生长阶段,发酵参数控制为:温度28℃,pH=6.0,转速300-600rpm,通气量2.0-6.0L/min;在甲醇诱导表达阶段,溶氧保持在15-350%之间,温度25℃,pH=5.0,甲醇流加速度为2.5-4.0mL/L/h。每隔一段时间取样测定发酵液的甘露聚糖酶活性。
(4)发酵液中甘露聚糖酶的产量和活力的测定:发酵液中蛋白质含量的测定采用Bradford蛋白质定量法;发酵液中蛋白质组份的检测采用SDS-PAGE法检测,结果如图4A和图4B所示。可以看出,随着诱导表达时间的增长,发酵液中蛋白质含量随之增加。当诱导表达时间为96小时,发酵液中蛋白质的含量达3.4mg/mL。
甘露聚糖酶活性采用还原糖法。其步骤如下:
a、取0.5mL稀释500倍的发酵上清液,加入到0.5mL的刺槐豆胶溶液(1%)和1mLpH5.0的醋酸缓冲液中,于70℃水浴反应5min;随后,加入2mL的DNS(3,5-二硝基水杨酸)终止反应,并置于沸水中水浴5min让还原糖充分显色;冷却至室温后用蒸馏水定容至25mL,混合均匀后测试520nm处的吸光度;
b、根据所测得的吸光度,参照酶活公式计算出甘露聚糖酶活力。酶活(U/mL)计算公式为:U=r×Df×1000/150.14/t,其中,Df为上清液稀释倍数;r为根据OD520吸光度值,得到的甘露糖的摩尔浓度(μmol/mL);t为酶解反应时间(min)。酶活单位定义为:每分钟水解甘露聚糖形成1μmol甘露糖所需的酶量为1个酶活力单位(U)。
(5)结果如图5所示。由图5可知,甘露聚糖酶生产水平高,随着甲醇诱导时间的延长,发酵液中甘露聚糖酶的产量逐渐增高,在培养时间为96h时,甘露聚糖酶的产量最高为36,000U/mL发酵液。
实施例5
本实施例用于说明甘露聚糖酶ManGold最适温度和最适pH值的测定。
(1)甘露聚糖酶ManGold水解反应体系为:甘露聚糖0.001g,用50mmoL pH 5.0的NaAC-HAC溶液配制成1%浓度的甘露聚糖溶液,加入0.1mL适量稀释后的甘露聚糖酶溶液。
(2)分别设置30℃、40℃、50℃、60℃、70℃、80℃和90℃等温度梯度,将上述反应体系置入所设置的不同温度中,反应10分钟,反应结束后,采用如实施例4中的DNS法测定反应产生的还原糖含量并计算剩余的酶活性。
(3)根据不同温度下测定的酶活绘制出甘露聚糖酶活与反应温度的曲线,获得最适反应温度。结果如图6A所示,随着温度的升高,甘露聚糖酶ManGold的表观活性逐渐升高,当温度达到80℃时活性最高。可见,本发明的耐高温甘露聚糖酶的最适温度为80℃。
(2)最适pH:以乙酸-乙酸钠缓冲液pH3.0、pH4.0、pH5.0、pH6.0、pH7.0、pH8.0、pH9.0、pH10.0分别配制0.5%的甘露聚糖底物溶液,以甘氨酸-氢氧化钠为缓冲剂配制pH9.0和pH10.0的甘露聚糖底物溶液。在最适温度80℃下反应5min,测定甘露聚糖酶活性。根确不同pH值下测定的酶活绘制出甘露聚糖酶活与反应温度的曲线,获得最适pH值。结果如图6B所示,随着pH值从2至12逐渐增加,pH值为4时达到最高酶活性;此后,随着pH值的升高,甘露聚糖酶ManGold的表观活性逐渐降低。可见,本发明的耐高温甘露聚糖酶的最适pH值为4。
实施例6
本实施例用于说明采用甘露聚糖酶ManGold水解甘露聚糖。
(1)取1mL实施例4制备的发酵上清液,用蒸馏水稀释100倍,取0.5mL加入到0.5mL浓度为2%的魔芋精粉溶液和1mL醋酸缓冲液(pH为5.0)中,并于70℃水浴反应10min。
(2)采用薄层层析法检测甘露聚糖水解产物:取上述步骤(1)中的水解产物9μL点于硅胶板上,放入展开剂中展层60min;取出硅胶板,干燥,并均匀喷涂显色剂,放入108℃的烘箱中烘干显色。结果如图7所示。图7中,1列为标样,2列为未添加甘露聚糖酶的魔芋精粉(主要成分为甘露聚糖)底物,3-6列分别为不同浓度的魔芋精粉(浓度分别为0.5%、1%、1.5%、2%)被甘露聚糖酶酶解后的产物。由图7可知,本发明提供的甘露聚糖酶可以将魔芋精粉完全水解。
以上实验结果表明,甘露聚糖酶ManGold能高效地将魔芋精粉水解为单糖和寡聚糖。
综上所述,本发明采用定点突变技术成功地获得了高产、耐高温、宽pH稳定性的甘露聚糖酶ManGold;基于甘露聚糖酶ManGold的氨基酸序列,针对毕赤酵母对密码子的偏好性设计并合成了甘露聚糖酶基因mangold。该基因在毕赤酵母中实现了高水平表达,从而获得了高产、耐高温、宽pH稳定性的甘露聚糖酶生产菌株。其在发酵罐中诱导表达96小时后,上清液中甘露聚糖酶的酶活可达36,000U/mL。本发明获得的耐高温甘露聚糖酶的最适温度为80℃,最适pH值为4,且对甘露聚糖具有高效的水解能力。
以上已经描述了本发明的各实施例,上述说明是示例性的,并非穷尽性的,并且也不限于所披露的各实施例。在不偏离所说明的各实施例的范围和精神的情况下,对于本技术领域的普通技术人员来说许多修改和变更都是显而易见的。
序列表
<110> 武汉轻工大学
中国大洋矿产资源研究开发协会(中国大洋事务管理局)
<120> 一种高产的耐高温宽pH稳定性的甘露聚糖酶ManGold及基因与应用
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gcgtacgtta aagcgttctg gggttctaaa gaaggtttct acaccaacga cgcgatgcag 540
gcggcgtacc gtgcgtacat caaagcggtt atctctcgtt actctgactc taccgcgatc 600
ttcgcgtggg aactggcgaa cgaaccgcgt tgccagggtt gcgaaaccac cgttctgtac 660
aactggatcg aatctacctc tcagtacatc aaatctctgg actctaaaca cctggtttgc 720
atcggtgacg aaggtttcgg tctggacacc ggttctgacg gttcttaccc gtaccagtac 780
tctgaaggtt ctgacttcgc gaaaaacctg gcgatcccga ccatcgactt cggtaccttc 840
cacctgtacc cgtcttcttg gggtaccacc aacgactggg gtaacggttg ggttacctct 900
cacggtgcgg cgtgcaaagc gtacggtaaa ccgtgcctgt tcgaagaata cggtgttacc 960
tctgaccact gcgcggttga aaaaccgtgg cagaacaccg cgctgaacac caccgcgatc 1020
tctggtgacc tgtactggca gtacggtgac cagctgtctg gtggtccgtc tccggacgac 1080
ggtaacacct tctactacgg taccgacgac ttcaaatgcc tggttaccga ccacatcgcg 1140
gcgatcaact ctcgtaaata a 1161
<210> 4
<211> 1161
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
atgaagctta acccttcact cctcaccgca gccggcctgg tgtcggccca attggcctct 60
gctcttcccc aagcgtcatc ctcaactgtc tcgcccagtc cgagtccatc cgccacaccc 120
ggttctttcg ttactacttc aggtctcaac tttgtgatcg atggcaagac aggatatttt 180
gccggttcca actcatactg gattggattc cagaagaaca acgacgatgt ggatctcgtg 240
ttcagtcact tgcaggaatc cggtctcaag atccttcgtg tctgggggtt caatgatgtc 300
aaccagaagc ccaccgatgg ctcggtctac taccatctgc tggcagatgg cacggcaacc 360
gtcaatgagg gtgaagatgg cctccagcga ctggactacg tggtctcctc ggcagagaag 420
cacggtatta agttgatcat taacttcgtc aacttctggg atgactatgg tggtatcaat 480
gcctatgtca aggccttcgg tgggtcgaag gagggctttt acaccaacga cgccatgcag 540
gcagcctacc gcgcttatat caaggcagtc atttcgcggt acagcgactc taccgcgatc 600
tttgcatggg aactggccaa tgagccccgt tgccagggct gtgagaccac cgtgttgtat 660
aactggatcg agagtaccag ccagtatatc aagtcgttgg actcgaagca tctggtttgc 720
atcggtgacg agggcttcgg cctcgacact ggttcggatg gaagctatcc gtaccaatac 780
tctgaaggaa gtgattttgc caagaacctg gctattccca cgattgactt cggaaccttc 840
catctctatc caagcagctg gggaaccacg aatgactggg gcaacggctg ggtcacctcg 900
cacggggcag cctgcaaggc ggctggaaag ccttgcctgt tcgaggagta tggagtgacg 960
tcggaccatt gtgccgttga gaaaccttgg caaaacacgg cacttaacac gactgccatc 1020
tcaggggacc tctactggca gtacggagat cagctgagcg gtggaccatc accagacgat 1080
ggcaacacct tctactatgg gacggatgat ttcaagtgtc tcgtgactga ccatattgcg 1140
gccatcaaca gtcgtaagtg a 1161
<210> 5
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
gaattcatga agcttaaccc ttcactc 27
<210> 6
<211> 35
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
cttgccatcg atcacaaagt agagacctga agtag 35
<210> 7
<211> 35
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
ctacttcagg tctctacttt gtgatcgatg gcaag 35
<210> 8
<211> 28
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
gcggccgctc acttacgact gttgatgg 28
<210> 9
<211> 31
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
ctccttcgac ccccagaagg ccttgacata g 31
<210> 10
<211> 31
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
ctatgtcaag gccttctggg ggtcgaagga g 31
<210> 11
<211> 35
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
gaacaggcaa ggctttccgt acgccttgca ggctg 35
<210> 12
<211> 35
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
cagcctgcaa ggcgtacgga aagccttgcc tgttc 35
Claims (10)
1.一种高产的耐高温宽pH稳定性的甘露聚糖酶ManGold,其特征在于,所述甘露聚糖酶ManGold的氨基酸序列含有SEQ ID NO:1所示的氨基酸序列。
2.根据权利要求1所述的甘露聚糖酶ManGold,其特征在于,所述甘露聚糖酶ManGold的氨基酸序列为SEQ ID NO:1所示的氨基酸序列。
3.根据权利要求2所述的甘露聚糖酶ManGold,其特征在于,所述甘露聚糖酶ManGold由甘露聚糖酶Man通过点突变得到,所述甘露聚糖酶Man的氨基酸序列如SEQ ID NO:2所示。
4.一种甘露聚糖酶基因mangold,用于编码权利要求1-3中任意一项所述的甘露聚糖酶ManGold,其特征在于,所述甘露聚糖酶基因mangold的碱基序列与SEQ ID NO:3所示的碱基序列具有95%以上的同一性。
5.根据权利要求4所述的甘露聚糖酶基因mangold,其特征在于,所述甘露聚糖酶基因mangold的碱基序列含有SEQ ID NO:3所示的碱基序列。
6.根据权利要求5所述的甘露聚糖酶基因mangold,其特征在于,所述甘露聚糖酶基因mangold的碱基序列为SEQ ID NO:3所示的碱基序列。
7.一种重组表达载体,其特征在于,其包括权利要求4-6中任意一项所述的甘露聚糖酶基因mangold。
8.一种重组表达菌株,其特征在于,其表达产物为权利要求1-3中任意一项所述的甘露聚糖酶ManGold;所述重组表达菌株的宿主细胞为毕赤酵母。
9.权利要求1-3中任意一项所述的甘露聚糖酶ManGold的应用。
10.一种水解甘露聚糖的方法,其特征在于,该方法包括:将权利要求1-3中任意一项所述的甘露聚糖酶ManGold与含甘露聚糖物料接触。
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CN112270956A (zh) * | 2020-10-26 | 2021-01-26 | 福建师范大学 | 一种适合毕赤酵母高表达的基因序列优化方法 |
RU2747782C1 (ru) * | 2020-06-18 | 2021-05-14 | Федеральное государственное бюджетное учреждение "Государственный научно-исследовательский институт генетики и селекции промышленных микроорганизмов Национального исследовательского центра "Курчатовский институт" (НИЦ "Курчатовский институт" - ГосНИИгенетика) | Рекомбинантный штамм дрожжей Ogataea haglerorum, продуцирующий бета-маннаназу Bacillus subtilis |
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RU2764793C1 (ru) * | 2020-10-20 | 2022-01-21 | Федеральное государственное бюджетное учреждение "Государственный научно-исследовательский институт генетики и селекции промышленных микроорганизмов Национального исследовательского центра "Курчатовский институт" (НИЦ "Курчатовский институт" - ГосНИИгенетика) | Трансформант дрожжей Ogataea haglerorum, продуцирующий бета-маннаназу, содержащий в составе хромосомы синтетический ген MANS |
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RU2764793C1 (ru) * | 2020-10-20 | 2022-01-21 | Федеральное государственное бюджетное учреждение "Государственный научно-исследовательский институт генетики и селекции промышленных микроорганизмов Национального исследовательского центра "Курчатовский институт" (НИЦ "Курчатовский институт" - ГосНИИгенетика) | Трансформант дрожжей Ogataea haglerorum, продуцирующий бета-маннаназу, содержащий в составе хромосомы синтетический ген MANS |
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CN114250211B (zh) * | 2021-12-24 | 2024-01-26 | 内蒙古科为博生物科技有限公司 | 一种甘露聚糖酶及其基因与应用 |
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