CN112391368A - 一种甘露聚糖酶突变体及其制备方法和应用 - Google Patents
一种甘露聚糖酶突变体及其制备方法和应用 Download PDFInfo
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- CN112391368A CN112391368A CN202011041012.0A CN202011041012A CN112391368A CN 112391368 A CN112391368 A CN 112391368A CN 202011041012 A CN202011041012 A CN 202011041012A CN 112391368 A CN112391368 A CN 112391368A
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- mannanase
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- gly
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Abstract
本发明公开一种甘露聚糖酶突变体及其制备方法和应用。所述甘露聚糖酶突变体为将甘露聚糖酶的氨基酸序列进行突变后得到的蛋白质,所述突变为在甘露聚糖酶的C端添加6个精氨酸、6个赖氨酸或6个天冬氨酸。本发明构建获得高效分泌表达所述突变体的毕赤酵母工程菌,与表达野生型甘露聚糖酶的重组菌相比,表达甘露聚糖酶突变体重组菌的胞内酶活均有下降,而上清液的酶活均有提高,其中X33/ManA‑R6上清液的酶活力提高幅度最大,达549U/mL,提高了1.52倍。本发明的突变体可减少该甘露聚糖酶分泌表达时在胞内的滞留,提高分泌效率和分泌量。
Description
技术领域
本发明涉及生物技术领域,具体涉及一种甘露聚糖酶突变体及其制备方法和应用。
背景技术
甘露聚糖在很多植物如大豆、玉米、角豆、魔芋等中含量较为丰富,然而植物细胞壁中的甘露聚糖在水环境中极易吸水膨胀,且黏度大大增加,导致其水解困难,因此会对上述植物资源的开发和利用造成不利的影响。β-甘露聚糖酶(β-mannanase)可水解甘露寡糖和甘露多糖(包括直链甘露聚糖、半乳甘露聚糖、葡甘露聚糖、半乳葡甘露聚糖等)主链中的β-1,4-D-甘露糖苷键,是甘露聚糖降解过程中的关键酶,将其添加至富含甘露聚糖的植物资源时可有效降解甘露聚糖,从而促进富含甘露聚糖植物资源的开发和利用,例如将β-甘露聚糖酶添加至动物的饲料中,可破坏植物细胞壁中的甘露聚糖,有助于释放细胞内的营养物质,降低饲料黏度,从而增加饲料的利用率和提高动物生长速度。目前β-甘露聚糖酶已广泛应用于饲料、食品、纺织、洗涤、造纸、制药等多个行业。
β-甘露聚糖酶广泛存在于植物、动物和微生物中,产甘露聚糖酶的原始菌株通常产量低且酶学性质不突出,基因工程技术的快速发展可以有效弥补上述缺陷,实现高效表达。目前已有多种不同来源的β-甘露聚糖酶在多个表达宿主中成功进行异源表达,如大肠杆菌、枯草芽孢杆菌、乳酸杆菌、毕赤酵母等。然而随着β-甘露聚糖酶的应用越来越广泛,β-甘露聚糖酶的产量有待进一步提高,因此提高β-甘露聚糖酶的表达量满足工业化生产需求,降低工业化生产成本具有重要意义。
发明内容
有鉴于此,本发明目的在于提供一种甘露聚糖酶突变体,以提高甘露聚糖酶的分泌效率,并构建得到高效分泌表达该突变体的毕赤酵母工程菌,为该突变体的广泛应用奠定基础。
为了实现上述目的,本发明提供一种甘露聚糖酶突变体,其为将甘露聚糖酶的氨基酸序列进行突变后得到的蛋白质,所述突变为在甘露聚糖酶的C端添加6个精氨酸(R6)、6个赖氨酸(K6)或6个天冬氨酸(D6)。
进一步地,其中所述甘露聚糖酶来自于嗜热好氧细菌,其氨基酸序列如SEQ IDNO:1所示;编码所述甘露聚糖酶的基因的核苷酸序列如GeneBank Accession No.KJ806637所示。
进一步地,其中所述嗜热好氧细菌为嗜热纤维杆菌Caldibacilluscellulovorans。
进一步地,其中当所述突变为在甘露聚糖酶的C端添加6个精氨酸(R6)时,所述甘露聚糖酶突变体为SEQ ID NO:2所示的氨基酸序列。
进一步地,其中当所述突变为在甘露聚糖酶的C端添加6个赖氨酸(K6)时,所述甘露聚糖酶突变体为SEQ ID NO:3所示的氨基酸序列。
进一步地,其中当所述突变为在甘露聚糖酶的C端添加6个天冬氨酸(D6)时,所述甘露聚糖酶突变体为SEQ ID NO:4所示的氨基酸序列。
为了实现上述目的,本发明还提供一种核苷酸序列,所述核苷酸序列编码上述任意一种甘露聚糖酶突变体。
进一步地,其中所述核苷酸序列为SEQ ID NO:5、SEQ ID NO:6或SEQ ID NO:7所示的序列。
为了实现上述目的,本发明还提供一种重组质粒,所述重组质粒含有上述的核苷酸序列。
为了实现上述目的,本发明还提供了一种重组质粒的制备方法,包括以下步骤:将上述的核苷酸序列***到表达载体的多克隆位点处,即得到所述重组质粒。
进一步地,其中所述表达载体为pPIC9K、pPICZαA、pPICZαB、pPICZαC、pGAPZαA、pGAPZαB或pGAPZαC。
为了实现上述目的,本发明还提供一种基因工程菌株的制备方法,包括以下步骤:将上述的重组质粒转化至酵母宿主细胞,即得到所述基因工程菌株。
进一步地,其中所述酵母宿主细胞为Pichia pastoris SMD1168、Pichiapastoris GS115、Pichia pastoris X33或Pichia pastoris KM71。
为了实现上述目的,本发明还提供一种基因工程菌株,所述工程菌株是通过上述的制备方法制得的。
为了实现上述目的,本发明还提供一种上述甘露聚糖酶突变体的制备方法,对上述基因工程菌株进行培养,收集上清后分离纯化即得到所述甘露聚糖酶的突变体。
为了实现上述目的,本发明还提供一种上述甘露聚糖酶突变体在饲料、纺织、造纸、洗涤或食品中的应用。
本发明的有益效果为:本发明的甘露聚糖酶来源于嗜热好氧细菌,所述的甘露聚糖酶突变体是在甘露聚糖酶的C端添加R6、K6或D6获得的,并构建获得高效分泌表达该突变体的毕赤酵母工程菌,与表达野生型甘露聚糖酶的重组菌相比,表达甘露聚糖酶突变体重组菌的上清液的酶活均有提高,其中X33/ManA-R6上清液的酶活力提高幅度最大,达到549U/mL,提高了1.52倍,且表达突变体重组菌的胞内酶活均有不同程度的下降。本发明的突变体可减少该甘露聚糖酶分泌表达时在胞内的滞留,提高分泌效率和分泌量,降低了工业上生产耐热甘露聚糖酶的成本,同时也为毕赤酵母高效分泌表达外源蛋白奠定了一定的基础。
上述说明仅是本发明技术方案的概述,为了能够更清楚了解本发明的技术手段,并可依照说明书的内容予以实施,以下以本发明的较佳实施例详细说明如后。
附图说明
图1:甘露聚糖酶ManA突变体分泌表达重组载体的示意图。
图2:ManA、ManA-R6、ManA-K6和ManA-D6在毕赤酵母中分泌表达上清酶活的比较。
图3:ManA、ManA-R6、ManA-K6和ManA-D6在毕赤酵母中分泌表达胞内滞留酶活的比较。
图4:ManA、ManA-R6、ManA-K6和ManA-D6在毕赤酵母中分泌表达后上清液的SDS-PAGE电泳检测结果。
具体实施方式
为更进一步阐述本发明为达成预定发明目的所采取的技术手段及功效,以下结合较佳实施例,对依据本发明提出的一种甘露聚糖酶突变体及其制备方法和应用其具体实施方式、特征及其功效,详细说明如后。在下述说明中,不同的“一实施例”或“实施例”指的不一定是同一实施例。此外,一或多个实施例中的特定特征或特点可由任何合适形式组合。下列实施例中未注明具体条件的实验方法,按照常规方法和条件,或按照商品说明书选择。本发明中的实验方法如无特别说明均为常规方法,基因克隆操作具体可参考J.萨姆布鲁克等编的《分子克隆实验指南》和Invitrogen公司的《毕赤酵母表达操作手册》。
以下材料或试剂,如非特别说明,均为市购。
实施例1:含野生型甘露聚糖酶基因的重组菌株的构建
(1)甘露聚糖酶ManA基因的克隆
甘露聚糖酶ManA的编码基因如GeneBank Accession No.KJ806637所示,根据上述序列及表达载体pPICZαA上多克隆位点的特征,设计合成引物:
P1:5’-GACGAATTCTCTGGTGGACCAAACTTGT-3’(SEQ ID NO:8)
P2:5’-TAGATAGCGGCCGCAGAGGTACCGATAGCGT-3’(SEQ ID NO:9)
其中引物P1下划线部分为EcoRⅠ酶切位点,引物P2下划线部分为NotⅠ酶切位点。以委托金斯瑞生物科技公司全基因合成的重组质粒pUC18/ManA为模板,以P1/P2为引物,通过PCR扩增获得甘露聚糖酶ManA的基因片段。
(2)重组质粒pPICZαA/ManA的构建
将PCR扩增获得的甘露聚糖酶ManA的基因片段用EcoRⅠ和NotⅠ双酶切,同时将表达载体pPICZαA也用EcoRⅠ和NotⅠ双酶切,然后将ManA的双酶切产物和pPICZαA的双酶切产物进行连接,并用化学法(CaCl2法)转化至自制E.coli Top10感受态细胞中,涂布于LBLZ抗性平板(Zeocin终浓度为25μg/mL)上进行筛选,挑取转化子于LBLZ液体培养基中,在37℃、200rpm摇床中过夜培养,提取质粒后使用EcoRⅠ和NotⅠ进行双酶切鉴定并测序验证,成功获得重组质粒pPICZαA/ManA。
(3)重组菌X33/ManA的构建
对重组质粒pPICZαA/ManA进行线性化(使用的限制性内切酶为SacⅠ)后,使用电转化法(电击电压为1.5KV、电击时间为5ms)转化至Pichia pastorisX33感受态细胞(市购)中,涂布于YPDSZ平板(Zeocin终浓度为100μg/mL)上进行筛选,挑取转化子于YPD液体培养基中,在30℃、250rpm摇床中过夜培养,以P1/P2为引物进行菌液PCR鉴定,结果表明甘露聚糖酶ManA的基因序列整合至Pichia pastorisX33基因组中,获得的产甘露聚糖酶ManA的重组菌株命名为X33/ManA。
实施例2:含突变型甘露聚糖酶基因的重组菌株的构建
(1)甘露聚糖酶ManA突变体基因的克隆
在甘露聚糖酶ManA的C端添加6个精氨酸(R6)、6个赖氨酸(K6)或6个天冬氨酸(D6),设计引物如下:
P1:5’-GACGAATTCTCTGGTGGACCAAACTTGT-3’(SEQ ID NO:8)
P3:5’-TAGATAGCGGCCGCCGTCGTCGTCGTCGTCGTAGAGGTACCGATAG CGT-3’(SEQ IDNO:10)
P4:5’-TAGATAGCGGCCGCAAGAAGAAGAAGAAGAAGAGAGGTACCGATAGC GT-3’(SEQ IDNO:11)
P5:5’-TAGATAGCGGCCGCGATGATGATGATGATGATAGAGGTACCGATAGCGT-3’(SEQ ID NO:12)
其中引物P1下划线部分为EcoRⅠ酶切位点,引物P3、P4和P5下划线部分为NotⅠ酶切位点,引物P3、P4和P5斜体部分为添加的R6、K6和D6对应的基因片段。以委托金斯瑞生物科技公司全基因合成的重组质粒pUC18/ManA为模板,分别以P1/P3、P1/P4和P1/P5为引物,通过PCR扩增获得甘露聚糖酶突变体ManA-R6、ManA-K6和ManA-D6的基因片段。
(2)含ManA突变体基因的重组质粒的构建
将获得的ManA-R6、ManA-K6和ManA-D6的基因片段分别用EcoRⅠ和NotⅠ双酶切,同时将表达载体pPICZαA也用EcoRⅠ和NotⅠ双酶切,然后将ManA-R6、ManA-K6和ManA-D6的双酶切产物分别与pPICZαA的双酶切产物进行连接,并用化学法(CaCl2法)转化至自制E.coliTop10感受态细胞中,涂布于LBLZ抗性平板(Zeocin终浓度为25μg/mL)上进行筛选,挑取转化子于LBLZ液体培养基中,在37℃、200rpm的摇床中过夜培养,提取质粒后使用EcoRⅠ和NotⅠ进行双酶切鉴定并测序验证,成功获得重组质粒pPICZαA/ManA-R6、pPICZαA/ManA-K6和pPICZαA/ManA-D6,重组质粒的示意图如图1所示。
(3)含ManA突变体基因的重组菌的构建
对重组质粒pPICZαA/ManA-R6、pPICZαA/ManA-K6和pPICZαA/ManA-D6进行线性化(使用的限制性内切酶为SacⅠ)后,使用电转化法(电击电压为1.5KV、电击时间为5ms)转化至自制Pichia pastoris X33感受态细胞中,涂布于YPDSZ平板(Zeocin终浓度为100μg/mL)上进行筛选,挑取转化子于YPD液体培养基中,在30℃、250rpm摇床中过夜培养,分别以P1/P3、P1/P4和P1/P5为引物进行菌液PCR鉴定,结果表明ManA-R6、ManA-K6和ManA-D6的基因序列均整合至Pichia pastorisX33基因组中,获得的产ManA突变体的重组菌株分别命名为X33/ManA-R6、X33/ManA-K6和X33/ManA-D6。
实施例3:甘露聚糖酶野生型ManA及突变体ManA-R6、ManA-K6和ManA-D6的获得
(1)各重组菌的诱导表达
对构建获得的4株重组菌X33/ManA、X33/ManA-R6、X33/ManA-K6和X33/ManA-D6分别进行摇瓶水平的发酵培养。将各重组菌接种于25mL BMGY培养基中,30℃、250rpm条件下过夜培养至OD600值到2-6,室温、6000rpm离心收集菌体重悬至25mL BMMY液体培养基中,控制起始OD600值为1,30℃、250rpm培养至120h,每隔24h补加1wt%的甲醇进行诱导表达。
(2)各重组菌发酵上清液酶活力的测定
发酵结束后取各重组菌上清液,利用DNS法测定酶活力,具体做法如下:将稀释后的酶液100μL与0.5wt%的甘露聚糖底物(购于Sigma公司)900μL混匀,75℃反应10min后立即加入1.5mL DNS终止反应并煮沸5min显色,将试管置于冷水浴中完全冷却后,测得OD540的值,计算酶活力。1个酶活力单位定义为在最适温度和pH条件下,1min内水解0.5wt%甘露聚糖底物产生1μmol甘露糖所需要的甘露聚糖酶的量。
(3)各重组菌发酵结束时胞内酶活力的测定
各重组菌发酵结束后,室温、10000rpm离心1min后收集0.01g菌体,用1mLNa2HPO4-柠檬酸缓冲液(pH 6.0)洗涤菌体3次,重悬至Minibeadbeater-16细胞破碎仪的配套试管中,加入0.73g酸性玻璃珠(购于Sigma公司),置于细胞破碎仪中完成细胞破碎后,室温、10000rpm离心1min后收集上清即为各重组菌胞内样品,再按照上清酶活测定方法进行胞内酶活的检测。
各重组菌发酵上清液酶活测定结果如图2所示,与表达野生型ManA的重组菌(361U/mL)相比,表达突变体的重组菌X33/ManA-R6、X33/ManA-K6和X33/ManA-D6上清液的酶活分别为549U/mL、451U/mL和465U/mL,分别提高了1.52倍、1.25倍和1.29倍。各重组菌胞内酶活测定结果如图3所示,与表达野生型ManA的重组菌相比,表达突变体的重组菌的胞内酶活均有不同程度的下降。上述结果表明在ManA的C端添加R6、K6或D6可减少该酶分泌表达时在胞内的滞留,提高胞外分泌效率和酶活。
(4)各重组菌发酵上清液SDS-PAGE分析
各重组菌发酵结束后,取等体积上清液进行SDS-PAGE电泳分析,结果如图4所示,各重组菌的上清液均在30KDa处有明显的条带,且条带深浅变化趋势与酶活变化趋势一致,进一步说明在ManA的C端添加R6、K6或D6可提高ManA的分泌表达水平。
实施例4:甘露聚糖酶野生型ManA及突变体ManA-R6、ManA-K6和ManA-D6的基本酶学性质
各重组菌发酵结束后,室温、10000rpm离心后取上清液样品测定基本酶学性质。
在40-90℃的温度范围,pH 6.0的条件下,分别测定ManA及其突变体的酶活,检测温度对ManA及其突变体活力的影响。
在pH 3.0-7.0的范围,75℃的条件下,分别测定ManA及其突变体的酶活,检测pH对ManA及其突变体活力的影响。
结果显示ManA、ManA-R6、ManA-K6和ManA-D6的最适反应温度均为75℃,属于高温酶,最适反应pH值均为6.0,说明对ManA的突变未改变其基本酶学性质。
本发明的实施例中所用到的常见培养基的配方如下所示:
LBLZ培养基:0.5%NaCl,1%蛋白胨,0.5%酵母浸粉,抗生素Zeocin终浓度为25μg/mL,固体培养基再加入2%琼脂粉。
YPDSZ平板:2%葡萄糖,2%蛋白胨,1%酵母浸粉,18.2%山梨醇,2%琼脂粉,抗生素Zeocin终浓度为100μg/mL。
YPD液体培养基:2%葡萄糖,2%蛋白胨,1%酵母浸粉。
BMGY培养基:1%酵母浸粉,2%胰化蛋白胨,1.34%YNB,4×10-5%生物素,1%甘油,100mM pH6.0的磷酸盐缓冲液。
BMMY培养基:1%酵母浸粉,2%胰化蛋白胨,1.34%YNB,4×10-5%生物素,1%甲醇,100mM pH6.0的磷酸盐缓冲液。
本发明的说明书中,说明了大量具体细节。然而,能够理解,本发明的实施例可以在没有这些具体细节的情况下实践。在一些实施例中,并未详细示出公知的方法、结构和技术,以便不模糊对本说明书的理解。
另外需要说明的是,在上述具体实施方式中所描述的各个具体技术特征,在不矛盾的情况下,可以通过任何合适的方式进行组合,为了避免不必要的重复,本发明对各种可能的组合方式不再另行说明。
此外,本发明的各种不同的实施方式之间也可以进行任意组合,只要其不违背本发明的思想,其同样应当视为本发明所公开的内容。
以上所述,仅是本发明的较佳实施例而已,并非对本发明作任何形式上的限制,依据本发明的技术实质对以上实施例所作的任何简单修改、等同变化与修饰,均仍属于本发明技术方案的范围内。
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Claims (12)
1.一种甘露聚糖酶突变体,其特征在于,其为将甘露聚糖酶的氨基酸序列进行突变后得到的蛋白质,所述突变为在甘露聚糖酶的C端添加6个精氨酸、6个赖氨酸或6个天冬氨酸。
2.如权利要求1所述的甘露聚糖酶突变体,其特征在于,所述甘露聚糖酶来自于嗜热好氧细菌,其氨基酸序列如SEQ ID NO:1所示;编码所述甘露聚糖酶的基因的核苷酸序列如GeneBank Accession No.KJ806637所示。
3.如权利要求1所述的甘露聚糖酶突变体,其特征在于,当所述突变为在甘露聚糖酶的C端添加6个精氨酸时,所述甘露聚糖酶突变体为SEQ ID NO:2所示的氨基酸序列。
4.如权利要求1所述的甘露聚糖酶突变体,其特征在于,当所述突变为在甘露聚糖酶的C端添加6个赖氨酸时,所述甘露聚糖酶突变体为SEQ ID NO:3所示的氨基酸序列。
5.如权利要求1所述的甘露聚糖酶突变体,其特征在于,当所述突变为在甘露聚糖酶的C端添加6个天冬氨酸时,所述甘露聚糖酶突变体为SEQ ID NO:4所示的氨基酸序列。
6.一种核苷酸序列,其特征在于,所述核苷酸序列编码如权利要求1所述的甘露聚糖酶的突变体,所述核苷酸序列为SEQ ID NO:5、SEQ ID NO:6或SEQ ID NO:7所示的序列。
7.一种重组质粒,其特征在于,所述重组质粒含有权利要求6所述的核苷酸序列。
8.一种权利要求7所述的重组质粒的制备方法,其特征在于,包括以下步骤:将权利要求6所述的核苷酸序列***到表达载体的多克隆位点处,即得到所述重组质粒;所述表达载体为pPIC9K、pPICZαA、pPICZαB、pPICZαC、pGAPZαA、pGAPZαB或pGAPZαC。
9.一种基因工程菌株的制备方法,其特征在于,包括以下步骤:将权利要求7所述的重组质粒转化至酵母宿主细胞,即得到所述基因工程菌株;所述酵母宿主细胞为Pichiapastoris SMD1168、Pichia pastoris GS115、Pichia pastoris X33或Pichia pastorisKM71。
10.一种基因工程菌株,其特征在于,所述工程菌株是通过权利要求9所述的制备方法制得的。
11.一种权利要求1-5任一项所述的甘露聚糖酶突变体的制备方法,其特征在于,培养权利要求10所述的基因工程菌株,收集上清后分离纯化即得到所述甘露聚糖酶的突变体。
12.一种权利要求1-5任一项所述的甘露聚糖酶突变体在饲料、纺织、造纸、洗涤或食品中的应用。
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