CN108018275A - 一种极端耐热木聚糖酶1vbr的突变体xynr及其用途 - Google Patents
一种极端耐热木聚糖酶1vbr的突变体xynr及其用途 Download PDFInfo
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- CN108018275A CN108018275A CN201810101058.3A CN201810101058A CN108018275A CN 108018275 A CN108018275 A CN 108018275A CN 201810101058 A CN201810101058 A CN 201810101058A CN 108018275 A CN108018275 A CN 108018275A
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- xynr
- mutant
- 1vbr
- zytase
- amino acid
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Abstract
本发明提供了一种极端耐热木聚糖酶1VBR的突变体XYNR,由木聚糖酶1VBR的氨基酸序列第290位苯丙氨酸突变为缬氨酸而得,其氨基酸序列如SEQ ID NO.1所示。本发明还提供了一种编码上述突变体XYNR的基因,其核苷酸序列如SEQ ID NO.2所示。本发明还提供了一种包含上述编码突变体XYNR基因的重组表达载体pPIC9K‑XYNR;更提供了一种包含上述重组表达载体的重组菌株。本发明中将木聚糖酶1VBR的氨基酸序列定点突变后,获得更高耐热性的突变体XYNR,并在毕赤酵母表达***中高效表达,可在饲料添加剂、保健食品、造纸、洗涤、酿造、纺织和医药等领域具有广阔的应用前景。
Description
技术领域
本发明属于蛋白质工程和基因工程领域,具体涉及一种极端耐热木聚糖酶的突变体XYNR氨基酸序列及其应用。
背景技术
木聚糖是植物细胞壁中最主要的半纤维素,约占植物细胞干重的35%左右,是自然界中除纤维素之外含量最丰富的多糖。木聚糖是由木糖通过β-1,4-糖苷键聚合而成的主链和一些侧链基团共同构成的一类杂合多聚糖,作为一种丰富的生物质资源,在木聚糖酶的作用下可被降解为国际市场上急需的低聚木糖和木糖。然而自然界中很大一部分木聚糖酶尚未被有效利用,造成了该资源的很大浪费。
微生物木聚糖酶(EC 3.2.1.8)是重要的工业用酶,随机催化水解木聚糖内部的β-1,4-D-木糖苷键生成木寡糖。木寡糖作为底物可被其他木聚糖酶类进一步降解,如β-D-木糖苷酶、α-L-***呋喃糖苷酶和D-葡萄糖醛酸酶等(Khandeparker R,NumanMT.Bifunctionalxylanase and their potential use in biotechnology.J IndMicrobiol biotechnol2008,35:635-644.)。基于功能上初级和三级结构和模型的显著差异,木聚糖酶主要分类为糖苷水解酶类第10和11家族,然而在糖苷水解酶类第5、7、8、16、26、30、43、52和62家族的酶中也发现了具有水解木聚糖的活性(http://www.cazy.org/fam/acc_GH.html)(Collins T,Gerday C,Feller G.Xylanase,xylanase families andextremophilic xylanases.FEMS Microbiol Rev 2005,29:3-23.)。其中GH 10家族木聚糖酶相对分子量较大(>30000),结构复杂,通常有多个结构域组成,包含一个催化结构域(Catalysis Domain,CD)是木聚糖酶主要组成部分,承担着木聚糖酶的水解特性。虽然他们在氨基酸数量和组成方面差异很大,但其催化结构在大小上都很接近,主要以α-螺旋和α-折叠片重复出现的结构,因为与TIM结构相近,属于一个家族,称为(α/β)8折叠结构,其中特定位置的谷氨酸和天冬氨酸对催化特性影响很大。该酶还含有非催化活性的结构域,如多糖底物结合域,热稳定结构域,以及多个催化结构域等,赋予该酶分解可溶性木聚糖,不可溶性木聚糖酶和其他底物等功能。
木聚糖酶的应用起源于1980年在动物饲料加工中的使用,随之被逐渐应用于造纸、食品、制药、酿造、纺织和生物燃料等领域。目前该酶主要应用于制浆造纸,饲料和食品等行业,并在现代工业中的应用地位越来越明显。木聚糖酶是利用非淀粉质原料生产酒精过程中的关键酶之一。随着生物能源事业发展的需要,木聚糖酶势必应用于更广泛的领域(Fawzi EM.Highly thermostable purified xylanase from Rhizomucor mieheiNRRL3169.Ann Microbiol 2010,60:363-368.)。在工业生产过程中,往往存在高温等极端环境,高温环境能加快酶促反应速度,提高液体物料的流动性能,防止有害微生物在过程中的生长繁殖等等。普通中温木聚糖酶在高温下会发生结构变化,从而大幅度地丧失活性。为防止这个热灭活过程,就需要添加一些化学物质等方法保护酶,这样不仅增加生产成本且对产品质量有不利影响。应用海栖热袍菌Thermotoga maritima MSB8所生产的耐热木聚糖酶进行水解,能从根本上解决高温环境使加入的酶很快失活的问题。
来源于细菌的木聚糖酶普遍比来源于真菌的木聚糖酶具有更高的热稳定性,且毕赤酵母具有高效分泌、正确折叠蛋白和极高细胞浓度培养的潜能常作为大规模生产外源蛋白质的表达***,将来源于细菌的木聚糖酶基因经序列优化后应用于可高密度培养的毕赤酵母,更适宜于工业化生产。来源于海栖热袍菌Thermotoga maritima MSB8的木聚糖酶1VBR由具有较优良的酶学性质(Winterhalter C,Liebl W.Two extremely thermostablexylanases of the hyperthermophilic bacterium Thermoyoga maritima MSB8.ApplEnviron Microbiol.1995,61(5):1810-1815.),属于糖苷水解酶类第10家族,其基因大小为984bp,氨基酸序列大小为328aa,预测其分子量为40kDa,其将木聚糖主要分解为木二糖与木糖,结晶体三维结构和功能等也进行了深入分析研究,并具有极端热稳定性,在广泛pH条件下均能维持较高酶活性。
发明内容
本发明中将木聚糖酶1VBR的氨基酸序列定点突变后,获得更高耐热性的木聚糖酶突变体XYNR,并在毕赤酵母表达***中高效表达,可在饲料添加剂、保健食品、造纸、洗涤、酿造、纺织和医药等领域具有广阔的应用前景。
本发明首先提供了一种极端耐热木聚糖酶1VBR的突变体XYNR,由木聚糖酶1VBR的氨基酸序列第290位苯丙氨酸突变为缬氨酸而得,其氨基酸序列如SEQ ID NO.1所示。
本发明还提供了一种编码上述突变体XYNR的基因,其核苷酸序列如SEQ ID NO.2所示。
本发明还提供了一种包含上述编码突变体XYNR基因的重组表达载体pPIC9K-XYNR。
本发明更提供了一种包含上述重组表达载体pPIC9K-XYNR的重组菌株。所述的菌株为重组大肠杆菌或重组酵母菌。
本发明首先通过全基因合成木聚糖酶1VBR基因,包括其终止子序列,及其两端酶切位点序列。通过双酶切、连接将全基因合成木聚糖酶基因***表达载体pPIC9K中,构建重组表达质粒pPIC9K-1VBR。将该重组表达质粒转化大肠杆菌DH5α感受态细胞,以PCR验证法筛选出阳性克隆菌株。
通过Swiss-Pdb结构预测,通过基因定点突变的方法,将木聚糖酶1VBR的第806位氨基酸由苯丙氨酸改变为缬氨酸。参考Invitrogen毕赤酵母表达手册,提取阳性克隆菌株内的重组表达质粒pPIC9K-XYNR,以限制性内切酶Bgl II线性化后,电击转化毕赤酵母GS115感受态细胞,采用遗传霉素G418浓度梯度法增加选择压力和相对酶活力检测等方法,筛选出多拷贝高效表达木聚糖酶突变体XYNR的毕赤酵母基因工菌株XYNR-GS115。
与现有技术相比,本发明具有以下优点:1、本发明首先解决来源于细菌的极端耐热木聚糖酶基因1VBR在真核表达***中的表达,并通过基因定点突变获得了更耐热的木聚糖酶突变体XYNR。该酶在宽广的pH范围内具有较强的稳定性,最适pH为5.0,在pH 4.0~9.0条件下处理1h后仍能维持80%以上相对酶活力,最适温度为100℃,在最适pH条件下100℃处理的半数死亡时间长达3h,对在极端高温环境的工业应用具有极佳的适应能力。
2、本发明所使用宿主菌为巴斯德毕赤酵母GS115,适度糖基化修饰表达的外源蛋白质,并促使蛋白质的正确折叠,能够有效的提高外源蛋白的稳定性。经过N-糖基化分析预测该酶仅有一个糖基化位点,同时,SDS-PAGE结果显示木聚糖酶突变体XYNR的分子量大小约为40kDa,与预测的该酶分子量一致,表明该酶不存在过度修饰,不影响该酶与底物亲和力。
3、本发明提供的产木聚糖酶突变体XYNR的基因工程菌株XYNR-GS115,所分泌木聚糖酶突变体XYNR在发酵液中的蛋白质含量可达电泳纯级别,几乎不需要纯化,降低生产成本。
附图说明
图1为木聚糖酶突变体XYNR的SDS-PAGE分析图;
图2为木聚糖酶突变体XYNR的热致死曲线图(pH 5.5,100℃);
图3为木聚糖酶突变体XYNR的pH稳定性图(90℃,1小时);
图4为木聚糖酶突变体XYNR的最适温度图(pH 5,10分钟);
图5为木聚糖酶突变体XYNR的最适pH图(90℃,10分钟)。
具体实施方式
下面结合附图及具体实施例对本发明的技术方案进行详细具体的说明。
实验材料:
1)菌株和质粒:大肠杆菌(Escherichia coli)DH5α和毕赤酵母GS115获赠于中国农业科学院饲料研究所;pPIC9K分泌型表达载体购自Invitrogen公司。
2)酶和试剂盒:限制性内切酶、Taq酶、Pyrobest DNA聚合酶、T4DNA连接酶等工具酶购TaKaRa公司;DNA纯化试剂盒购自爱思进生物技术有限公司。
3)生化试剂:G418购自Invitrogen公司;蛋白质分子量标准购自上海生物化学研究所;IPTG、X-Gal、SDS及角豆胶购自Sigma公司;TEMED、过硫酸铵、丙烯酰胺、甲叉双丙烯酰胺为国药试剂。
50mM磷酸氢二钠-柠檬酸缓冲液:取7.10g磷酸氢二钠溶解于800mL双蒸水,用柠檬酸调节pH为4.0~7.5范围内任一值后,定容至1L。
50mM Tris-HCl缓冲液:取6.06g Tris溶解于800mL双蒸水,用1M HCl调节pH至7.5~9.0范围内任一值后,定容至1L。
50mM甘氨酸-氢氧化钠缓冲液:取7.50g甘氨酸溶解于800mL双蒸水,用1M氢氧化钠溶液调pH至9.0~12范围内任一值后,定容至1L。
下述实施例中的实验方法,如无特别说明,均为常规方法。
下述实施例中的百分含量,如无特别说明,均为质量百分含量。
实施例1:木聚糖酶突变体XYNR基因序列的获得
依据序列比对,PDB中发表的来源于海栖热袍菌Thermotoga maritima MSB8的木聚糖酶1VBR氨基酸序列与Thermotoga maritima MSB8基因组序列(CP007013.1:1,869,644bp)中预测木聚糖酶(EHA58720.1)的氨基酸序列相似性为100%。该预测木聚糖酶基因序列是Thermotoga maritima MSB8基因组序列中一段基因序列(873,589→874,572bp)。
基于巴斯德毕赤酵母密码子使用偏好性,将其中稀有密码子转换为高频表达密码子,同时对照木聚糖酶1VBR的氨基酸序列,对预测木聚糖酶(EHA58720.1)基因序列进行密码子优化,获得优化后木聚糖酶1VBR基因序列。以此模板为基础,通过基因定点突变的方法,将木聚糖酶1VBR的氨基酸序列中第290位氨基酸由苯丙氨酸改变为缬氨酸,获得木聚糖酶突变体XYNR的基因序列,如SEQ ID NO.2所示。
实施例2:含有木聚糖酶突变体XYNR基因的重组表达质粒pPIC9K-XYNR的构建
在木聚糖酶突变体XYNR基因3′端添加毕赤酵母偏好的终止子序列,并在5′端和3′端分别引入限制性内切酶EcoR I和Not I位点,将该基因序列交与武汉擎科创新生物科技有限公司完成全基因合成。采用限制性内切酶EcoR I和Not I完成优化的木聚糖酶突变体XYNR基因和分泌型表达载体pPIC9K双酶切,再使用连接酶将两者连接,构建重组表达质粒pPIC9K-XYNR。将该重组表达质粒转化大肠杆菌DH5α感受态细胞,以PCR验证法筛选出阳性克隆菌株pPIC9K-XYNR-DH5α。
实施例3:高效分泌表达木聚糖酶突变体XYNR的毕赤酵母基因工菌株的构建
采用LB液体培养基活化培养菌株pPIC9K-XYNR-DH5α,提取重组质粒pPIC9K-XYNR。采用限制性内切酶Bgl II线性化该重组质粒,并回收酶切产物。参考EasySelectTM PichiaExpression Kit制备毕赤酵母GS115感受态细胞。取约10μg线性化质粒与80μL的感受态细胞轻柔混匀后置于冰上15min,转移至预冷的0.2cm电转杯中,1500V电击完成后立即加入1mL预冷的1mol/L山梨醇,在30℃培养箱中静置1h,涂布于MD平板上,于30℃倒置培养约48h至转化子出现。
挑取单菌落按相应编号依次分别接种于含0.25,0.5,1.0,2.0,3.0和4.0mg/mLG418的MD平板上,于30℃倒置培养约48h至单菌落出现。筛选抗性最强的重组菌株,接种于相应编号的含有3mL BMGY培养基中,30℃,200rpm摇床培养约48h,至OD600达1.8~6.0。收集菌体,重悬于1mL BMMY培养基,0.5%甲醇诱导培养约48h。收集上清液,采用DNS法检测酶活力以筛选产酶量较高的阳性重组菌株GS115-XYNR。
实施例4:毕赤酵母基因工程菌XYNR-GS115的发酵罐高密度发酵
采用毕赤酵母基因工程菌XYNR-GS115在10L发酵罐中高密度发酵生产木聚糖酶。发酵过程具体如下:
1)种子液制备阶段。将保存的毕赤酵母基因工程菌XYNR-GS115接种于接种于YPD培养基,28℃,200rpm摇床培养约12h,获得种子液。
2)发酵前期培养阶段。以2%接种量将种子液接入BSM无机盐甘油培养基,接种前使用氨水调节pH至5.5,通气搅拌培养,在培养过程中随着菌株的生长,培养基中的溶氧量由100%逐渐降低。当碳源消耗完后溶氧量会再度升高,当溶氧升高至80%以上时,开始碳源流加补料发酵阶段。
其中,BSM培养基由以下成分组成:85%H3PO4 26.7mL/L,CaSO4·2H2O 0.93g/L,K2SO4 18.2g/L,MgSO4·2H2O 14.9g/L,KOH 4.13g/L,甘油40g/L,PMT1 4.0mL/L。所述PMT1配方:CuSO4·5H2O 6.0g/L,KI 0.088g/L,MnSO4·H2O 3.0g/L,Na2MoO4·2H2O 0.2g/L,H3BO30.02g/L,CoCl2·6H2O 0.5g/L,ZnCl2 20.0g/L,FeSO4·7H2O 65.0g/L,Biotin 0.2g/L,浓H2SO4 5.0mL/L(过滤除菌)。
3)流加补料发酵阶段。流加40%甘油溶液(含12mL/L PTM1),同时控制溶氧在60%左右,当转速和风量达到最大,溶氧出现较大浮动的下降时,停止甘油补料。
4)诱导表达阶段。流加甲醇(含12mL/L PTM1)诱导产酶,同时调节甲醇补料速度将溶氧维持在15%以上,pH维持在5.5左右,每24h取样检测OD值、湿重、酶活等,当木聚糖酶活力不再升高时即可停止诱导。
发酵液经SDS-PAGE检测,结果显示,发酵液中木聚糖酶突变体XYNR的蛋白质含量达到电泳纯级别,分子量大小约为40kDa,如图1所示。
实施例5:木聚糖酶突变体XYNR的性质分析
1)木聚糖酶突变体XYNR热致死曲线的测定:将木聚糖酶溶液经pH 5.5缓冲液稀释后,置于100℃水浴中,分别处理0,30,60,120和180min后,于最适反应条件下检测木聚糖酶活力,据此计算出木聚糖酶的半衰期。结果显示,在最适pH条件下,于100℃处理3h后,突变体XYNR仍能保持50%以上的相对酶活力,较原始木聚糖酶1VBR的耐热性更好,如图2所示。
2)木聚糖酶突变体XYNR的pH稳定性测定:于90℃下,将酶液在不同pH缓冲液中准确处理1h,迅速置于冰上冷却。于100℃,pH 5.0下,该酶与0.5%木聚糖溶液分别在不同温度下反应15min,加入2.5mL DNS试剂,沸水浴5min,冷却至室温,加水定容至12.5mL。于分光光度计中检测OD540。结果显示,木聚糖酶突变体XYNR在pH 4.0~9.0条件下处理1h后仍能维持80%以上相对酶活力,如图3所示。
3)木聚糖酶突变体XYNR最适温度的测定:于pH 5.5下,该酶与0.5%木聚糖溶液分别在不同温度下反应15min,加入2.5mL DNS试剂,沸水浴5min,冷却至室温,加水定容至12.5mL。于分光光度计中检测OD540。以最高酶活力为100%,计算其他条件下的相对残余酶活力。结果显示,木聚糖酶突变体XYNR最适反应温度为100℃,作为极端耐高温酶在80℃~100℃下木聚糖酶突变体XYNR能够维持50%以上的相对酶活力,如图4所示。
4)木聚糖酶突变体XYNR最适pH的测定:采用DNS法检测该木聚糖酶最适pH,100℃时,木聚糖酶突变体XYNR在不同pH缓冲液条件下与0.5%木聚糖溶液准确反应15min,加入2.5mL DNS试剂,沸水浴5min,冷却至室温,加水定容至12.5mL,于分光光度计中检测OD540。结果显示,木聚糖酶突变体XYNR最适pH为5.5,在pH5.0~7.5范围内均能保持70%以上的相对酶活力,如图5所示。
以上结果证实,本发明中所提供的突变体XYNR在宽广的pH范围内具有较强的稳定性,最适pH为5.0,在pH 4.0~9.0条件下处理1h后仍能维持80%以上相对酶活力,最适温度为100℃,在最适pH条件下100℃处理的半数死亡时间长达3h,对在极端高温环境的工业应用具有极佳的适应能力。
序列表
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Claims (6)
1.一种极端耐热木聚糖酶1VBR的突变体XYNR,其特征在于:由木聚糖酶1VBR的氨基酸序列第290位苯丙氨酸突变为缬氨酸而得,其氨基酸序列如SEQ ID NO.1所示。
2.编码权利要求1所述突变体XYNR的基因,其核苷酸序列如SEQ ID NO.2所示。
3.包含权利要求2所述编码突变体XYNR基因的重组表达载体pPIC9K-XYNR。
4.包含权利要求3所述重组表达载体pPIC9K-XYNR的重组菌株。
5.根据权利要求4所述的重组菌株,其特征在于:所述的菌株为重组大肠杆菌或重组酵母菌。
6.权利要求1所述的突变体XYNR在高温环境下水解木聚糖中的用途。
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Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN109295039A (zh) * | 2018-10-11 | 2019-02-01 | 山东隆科特酶制剂有限公司 | 一种耐热木聚糖酶突变体及其毕赤酵母工程菌 |
CN110117586A (zh) * | 2019-05-06 | 2019-08-13 | 武汉轻工大学 | 一种超耐热的木聚糖酶XynGold及基因与应用 |
CN111549016A (zh) * | 2020-05-25 | 2020-08-18 | 中南民族大学 | 一种极端耐热木聚糖酶xyna及其突变体基因、应用和制备方法 |
CN111621486A (zh) * | 2020-05-25 | 2020-09-04 | 中南民族大学 | 一种低温下高酶活的耐热木聚糖酶xynb及其突变体基因、应用和基因序列制备方法 |
WO2021126966A1 (en) * | 2019-12-16 | 2021-06-24 | Novozymes A/S | Processes for producing fermentation products |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102260659A (zh) * | 2010-05-31 | 2011-11-30 | 中国科学院成都生物研究所 | 一种1, 4-β-D-木聚糖酶突变体 |
CN102690832A (zh) * | 2012-06-05 | 2012-09-26 | 江南大学 | 一种提高第10家族木聚糖酶最适温度的方法 |
-
2018
- 2018-02-01 CN CN201810101058.3A patent/CN108018275B/zh active Active
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102260659A (zh) * | 2010-05-31 | 2011-11-30 | 中国科学院成都生物研究所 | 一种1, 4-β-D-木聚糖酶突变体 |
CN102690832A (zh) * | 2012-06-05 | 2012-09-26 | 江南大学 | 一种提高第10家族木聚糖酶最适温度的方法 |
Non-Patent Citations (1)
Title |
---|
IHSANAWATI等: "Structural Basis of the Substrate Subsite and the Highly Thermal Stability of Xylanase 10B From Thermotoga maritima MSB8", 《PROTEINS: STRUCTURE, FUNCTION, AND BIOINFORMATICS》 * |
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CN109295039A (zh) * | 2018-10-11 | 2019-02-01 | 山东隆科特酶制剂有限公司 | 一种耐热木聚糖酶突变体及其毕赤酵母工程菌 |
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WO2021126966A1 (en) * | 2019-12-16 | 2021-06-24 | Novozymes A/S | Processes for producing fermentation products |
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