CN116265580A - 高比活碱性木聚糖酶突变体 - Google Patents
高比活碱性木聚糖酶突变体 Download PDFInfo
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- CN116265580A CN116265580A CN202111546831.5A CN202111546831A CN116265580A CN 116265580 A CN116265580 A CN 116265580A CN 202111546831 A CN202111546831 A CN 202111546831A CN 116265580 A CN116265580 A CN 116265580A
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- xylanase
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- enzyme
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Abstract
本发明涉及基因工程与蛋白质改造技术领域,具体涉及一种高比活碱性木聚糖酶突变体。所述木聚糖酶突变体包含与SEQ ID NO:1具有至少90%同一性的氨基酸序列,且与SEQ ID NO:1相比在选自下组中的至少一个位置上包含氨基酸的取代:7,12,21,23,24,25,26,29,30,31,34,37,38,39,40,41,42,43,44,51,54,91,92,100,102,103,109,118,119,122,123,132,134,141,142,144,145,150,151,152,156,167,168,169,172,173,175,183,184,186。与野生型相比,所述突变体的比活力得到显著提高,有利于降低该酶的生产成本,促进其在工业领域,尤其是造纸领域的广泛应用。
Description
技术领域
本发明涉及基因工程和蛋白质工程技术领域,具体涉及一种高比活碱性木聚糖酶突变体及其应用。
背景技术
木聚糖(Xylan)是一种多聚五碳糖,是植物半纤维素的重要组分,它占植物碳水化合物总量的三分之一,在自然界中是继纤维素之后含量第二丰富的可再生物质资源。它存在于植物的细胞壁中以及几乎所有部位。
木聚糖酶是可将木聚糖降解成低聚糖或木糖的一类酶的总称。一个木聚糖分子的完全酶解需要几步酶促反应,作用于主链的有两种酶:β-1,4-木聚糖酶(l,4-β-D-xylanohydrolase: EC3.1.2.8)和β-木糖苷酶(1,4-β-D-xylanxylohydrolase:EC3.2.1.37)。一般而言,前者从主链内部作用于木糖苷键,将木聚糖分解成低聚糖,而后者则作用于低聚木糖的末端,释放出木糖。
许多微生物可产生木聚糖酶。木聚糖酶的生物化学性质的获得主要来自于细菌和真菌木聚糖酶的研究。细菌所产木聚糖酶可大体分为两类:高分子量的耐酸木聚糖酶,低分子量的耐碱木聚糖酶。但在真菌中却没有这种差别,不过,低分子量木聚糖酶的耐碱性却是共同的。
饲料中的木聚糖本身难以被单胃动物消化,同时结合大量的水,使采食动物消化道中食糜的体积增大、粘度增加、养分与消化道内源酶的作用降低,从而阻碍营养物质,尤其是脂肪和蛋白质的消化吸收,降低饲料的利用率。研究结果表明,饲料中如果添加木聚糖酶,就可显著降低阿拉伯木聚糖分子大小,将其分解成较小聚合度的低聚木糖,从而改善饲料性能,消除或降低因粘度增加而引起的抗营养作用。
木聚糖酶可作为生物漂白剂用于造纸工业。木聚糖酶在造纸和纸浆工业中的重要性在于其取代了有毒的化学物质,同时通过酶法预处理可以回收该行业中有用的副产品。其漂白作用在于切开木质素和糖之间的键,松弛纸浆结构。扫描电镜研究显示,用木聚糖酶预处理后的纸浆在纸浆纤维的孔隙上有所提高,有助于其与起漂白作用的化合物的亲和。
在葡萄酒和日本大麦烧酒的生产中,已经有了木聚糖酶的应用研究。日本研究人员将耐酸性木聚糖酶Xy1C应用于日本大麦烧酒酿造中,结果发现该酶有助于提高发酵效率,增加酒精的产率。
木聚糖酶在造纸、食品、能源、饲料以及环境等领域的应用价值,已经得到了肯定,然而由于在天然材料中表达水平低、难以大规模生产、且产物难以纯化以及木聚糖的一些性质不能完全满足应用的要求。随着基因工程技术的发展,通过基因工程这一手段,利用生物反应器提高它的表达量并在分子水平上对木聚糖酶基因进行改造以解决一些木聚糖酶活性(如比酶活、抗逆性、pH值、热稳定性等)的缺陷已成为目前的研究热点。
发明内容
本发明的目的是提供一种高比活碱性木聚糖酶突变体。所述突变体的比活力得到显著提高,有利于降低该酶的生产成本,促进其广泛应用。
为了实现上述发明目的,本发明提供以下技术方案:
本发明涉及一种木聚糖酶突变体,其包含与SEQ ID NO:1具有至少90%同一性的氨基酸序列,且与SEQ ID NO:1相比在选自下组中的至少一个位置上包含氨基酸的取代:7,12,21,23,24,25,26,29,30,31,34,37,38,39,40,41,42,43,44,51,54,91,92,100,102,103,109,118,119,122,123,132,134,141,142,144,145,150,151,152,156,167,168,169,172,173,175,183,184,186。
在本发明的一些实施例中,所述突变体的氨基酸序列与SEQ ID NO:1相比具有至少91%,92%,93%,94%,95%,96%,97%,98%,或至少99%的同一性。
在一些更具体的实施例中,所述突变体的氨基酸序列与SEQ ID NO:1相比具有至少99.1%,99.2%,99.3%,99.4%,99.5%,99.6%,99.7%,99.8%,或至少99.9%的同一性。
在本发明的一些实施例中,所述突变体包含下组中至少一个氨基酸的取代:N7S,D12F,T21S,A23G,P24C/G,G25A,T26D,M29/S/V,C30V,L31C/F/N/V,G34A,Y37F,S38H/T,T39V/F,Q40T,W41F,R42D/H/S,N43S,T44V/S,K51Q,A54H/N,V91I,E92D,T100S,T102Q,Y103T,T109V,E118Q/R,T119S/D,Y122V/F/T/S,N123D/K/Y,T132S,Q134N/D,Q141S/A/E/F/I/L/M/P/W,Q142H/S,R144H/K/Q,T145S/P/V,T150N,V151F/S/T,G152D,D156S,S167T,H168N/S/V,D169N,I172V,L173I,T175V,S183D,S184A,V186I。
本发明还涉及编码上述木聚糖酶突变体的DNA分子。
本发明还涉及包含上述DNA分子的重组表达质粒。
本发明还涉及一种宿主细胞,包含上述重组表达质粒。
将上述质粒转入宿主细胞中,重组表达的木聚糖酶突变体的比活力得到显著提升。
所述的宿主细胞为毕赤酵母(Pichia pastoris)。
所述的宿主细胞为里氏木霉(Trichoderma reesei)。
本发明还提供了上述木聚糖酶突变体在造纸领域中的应用。
与野生型木聚糖酶MXYL11相比,本发明提供的木聚糖酶单点突变体的比活力均得到显著提高。其中,含T21S单点突变的木聚糖酶突变体的比活力最高,达2322.8 U/mg,是野生型木聚糖酶的两倍,取得了意料不到的技术效果。
本发明提供的碱性木聚糖酶突变体的比活力得到显著提高,从而有利于降低该酶的生产成本,促进其在工业领域,尤其是造纸领域中的广泛应用。
具体实施方式
本发明公开了一种木聚糖酶突变体、其制备方法及应用、编码该木聚糖酶突变体的DNA分子、载体、宿主细胞,本领域技术人员可以借鉴本文内容,适当改进工艺参数实现。本发明的方法及应用已经通过较佳实施例进行了描述,相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法和应用进行改动或适当变更与组合,来实现和应用本发明技术。
本发明用到了遗传工程和分子生物学领域使用的常规技术和方法,例如MOLECΜLAR CLONING:A LABORATORY MANUAL,3nd Ed. (Sambrook, 2001)和CURRENT PROTOCOLSIN MOLECΜLAR BIOLOGY (Ausubel, 2003)中所记载的方法。这些一般性参考文献提供了本领域技术人员已知的定义和方法。但是,本领域的技术人员可以在本发明所记载的技术方案的基础上,采用本领域其它常规的方法、实验方案和试剂,而不限于本发明具体实施例的限定。例如,本发明可选用如下实验材料和试剂:
菌株与载体:大肠杆菌DH5α、毕赤酵母GS115、载体pPIC9k、Amp、G418购自Invitrogen公司。
酶与试剂盒:PCR酶及连接酶购买自Takara公司,限制性内切酶购自Fermentas公司,质粒提取试剂盒及胶纯化回收试剂盒购自Omega公司,GeneMorph II随机诱变试剂盒购自北京博迈斯生物科技有限公司。
培养基配方:
大肠杆菌培养基(LB培养基):0.5%酵母提取物,1%蛋白胨,1%NaCl,pH7.0;
酵母培养基(YPD培养基):1%酵母提取物,2%蛋白胨,2%葡萄糖;
酵母筛选培养基(MD培养基):2%蛋白胨,2%琼脂糖;
BMGY培养基:2%蛋白胨,1%酵母提取物,100 mM磷酸钾缓冲液(pH6.0),1.34% YNB,4×10-5 %生物素,1%甘油;
BMMY培养基:2%蛋白胨,1%酵母提取物,100 mM磷酸钾缓冲液(pH6.0),1.34% YNB,4×10-5 %生物素,0.5%甲醇;
LB-AMP培养基:0.5%酵母提取物,1%蛋白胨,1%NaCl,100μg/mL氨苄青霉素,pH7.0;
LB-AMP平板:0.5%酵母提取物,1%蛋白胨,1%NaCl,1.5%琼脂,100μg/mL氨苄青霉素,pH7.0;
上层培养基:0.1%MgSO4,1%KH2PO4,0.6%(NH4)2SO4,1%葡萄糖,18.3%山梨醇,0.35%琼脂糖;
下层培养基平板:2%葡萄糖,0.5%(NH4)2SO4,1.5%KH2PO4,0.06%MgSO4,0.06%CaCl2,1.5%琼脂。
下面结合实施例,进一步阐述本发明:
实施例1 表达质粒的构建
将来源于放线杆菌(Actinobacteria bacterium)的木聚糖酶基因(GenBankPZN45477.1)根据毕赤酵母密码子偏好性进行密码子优化,并在其起始密码子ATG前增加6个碱基GAATTC(EcoR I酶切位点),在其终止密码子TAA后增加GCGGCCGC(Not I酶切位点)。优化后的核苷酸序列由上海捷瑞生物工程有限公司合成。将该木聚糖酶命名MXYL11,其氨基酸序列为SEQ ID NO:1,编码核苷酸序列为SEQ ID NO:2。
用限制性内切酶EcoR I和Not I(Fermentas)对木聚糖酶基因进行酶切;同时,用限制性内切酶EcoR I和Not I对质粒pPIC9K进行酶切。使用凝胶纯化试剂盒将酶切产物纯化,并用T4 DNA连接酶(Fermentas)将上述两个酶切产物连接。将连接产物转化进DH5α大肠杆菌(Invitrogen),涂布于LB+Amp平板,37℃倒置培养,待转化子出现后,菌落PCR(反应体系:模板挑取的单克隆,rTaqDNA聚合酶 0.5μl,10×Buffer 2.0μL,dNTPs(2.5mM)2.0μL,5’AOX引物(10mM):0.5μL,3’AOX引物:0.5μL,ddH2O 14.5μL,反应程序:95℃预变性5min,30个循环:94℃ 30sec,55℃ 30sec,72℃ 2min,72℃ 10min),验证阳性克隆子,并进行测序分析。使用质粒小量制备试剂盒(Omega)从测序结果正确的大肠杆菌克隆中纯化质粒,测序结果显示获得的DNA序列为SEQ ID NO:2,其编码的氨基酸序列为SEQ ID NO:1,从而说明表达质粒构建成功,命名为pPIC9K-MXYL11。
实施例2 高比活突变体的筛选
为了进一步提高木聚糖酶MXYL11的比活力,申请人对其进行蛋白结构分析。该蛋白是11家族木聚糖酶,其结构为β-果冻卷的结构。申请人通过定向进化技术对该酶进行了大量突变的筛选。
1.1设计PCR引物MXYL11-F1、MXYL11-R1:
MXYL11-F1:GGCGAATTCGATACTTGTATCACTCAAAACCAAAC(下划线为限制性内切酶EcoRI识别位点);
MXYL11-R1:ATAGCGGCCGCTTATCCAATGGTAACAGTTGATGATCC(下划线为限制性内切酶NotI识别位点)。
以MXYL11基因(SEQ ID NO:1)为模板,利用上述引物用GeneMorph II随机突变PCR试剂盒((博迈斯))进行PCR扩增,胶回收PCR产物,EcoRI、NotI进行酶切处理后与经同样酶切后的pET21a载体连接,转化至大肠杆菌BL21(DE3)中,涂布于LB+Amp平板,37℃倒置培养,待转化子出现后,用牙签逐个挑至96孔板,每个孔中加入150μl含有0.1mM IPTG的LB+Amp培养基,37℃、220rpm培养6 h左右,离心弃上清,菌体用缓冲液重悬,反复冻融破壁,获得含有木聚糖酶的大肠杆菌细胞裂解液。
分别取出30 μl裂解液至两块新的96孔板;将其中一块96孔板加入30 μl底物,于37℃反应30 min后,DNS法测定生成的还原糖,另一块板加入150μl考马斯亮蓝溶液,静置10min,考马斯亮蓝(Bradford)结合法测定蛋白质含量,分别计算不同突变子酶活水平及蛋白含量。最终,申请人从两万多个转化子中筛选出显著提高木聚糖酶比活力的单点突变体N7S,D12F,T21S,A23G,P24C,P24G,G25A,T26D,M29L,M29S,M29V,C30V,L31C,L31F,L31N,L31V,G34A,Y37F,S38H,S38T,T39V,T39F,Q40T,W41F,R42D,R42H,R42S,N43S,T44V,T44S,,K51Q,A54H,A54N,V91I,E92D,T100S,T102Q,Y103T,T109V,E118Q,E118R,T119S,T119D,Y122V,Y122F,Y122T,Y122S,N123D,N123K,N123Y,T132S,Q134N,Q134D,Q141S,Q141A,Q141E,Q141F,Q141I,Q141L,Q141M,Q141P,Q141W,Q142H,Q142S,R144H,R144K,R144Q,T145S,T145P,T145V,T150N,V151F,V151S,V151T,G152D,D156S,S167T,H168N,H168S,H168V,D169N,I172V,L173I,T175V,S183D,S184A,V186I。
在野生型木聚糖酶MXYL11的基础上,本发明提供了分别含上述单个突变位点的突变体。
实施例3 木聚糖酶在毕赤酵母中的表达,
3.1表达载体的构建
依照毕赤酵母的密码子偏爱性分别对木聚糖酶MXYL11及其突变体的基因序列进行优化,由上海捷瑞生物工程有限公司合成,并且在合成序列5’和3’两端分别加上EcoRI和NotI两个酶切位点。
按照实施例1中所述方法,将合成的木聚糖酶MXYL11及其突变体的基因序列分别进行EcoRI和NotI双酶切,然后与经同样酶切后的pPIC-9K载体16℃过夜连接,并转化大肠杆菌DH5a,涂布于LB+Amp平板,37℃倒置培养,待转化子出现后,菌落PCR(反应体系:模板挑取的单克隆,rTaqDNA聚合酶 0.5μl,10×Buffer 2.0μL,dNTPs(2.5mM) 2.0μL,5’AOX引物(10mM):0.5μL,3’AOX引物:0.5μL,ddH2O 14.5μL,反应程序:95℃预变性5min,30个循环:94℃ 30sec,55℃ 30sec,72℃ 2min,72℃ 10min)。验证阳性克隆子,经测序验证后获得了正确的重组表达质粒。
3.2毕赤酵母工程菌株的构建
3.2.1酵母感受态制备
将毕赤酵母GS115菌株进行YPD平板活化,30℃培养48 h后接种活化的GS115单克隆于6 mL YPD液体培养基中,30℃、220 rpm,培养约12 h后转接菌液于装有30mL YPD液体培养基的三角瓶中,30℃、220 rpm培养约5h,经紫外分光光度计检测其菌体密度,待其OD600值在1.1–1.3范围后,4℃ 9000 rpm离心2 min分别收集4mL菌体至灭菌EP管中,轻轻弃上清,用灭菌的滤纸吸干残留的上清后用预冷的1 mL灭菌水重悬菌体,4℃、9000 rpm离心2 min,轻轻弃上清,重复用1mL灭菌水洗一遍后,4℃、9000 rpm离心2 min,轻轻弃上清,预冷的1mL山梨醇(1 mol/L)重悬菌体;4℃、9000 rpm离心2 min,轻轻弃上清,预冷的100-150μl山梨醇(1 mol/L)轻柔重悬菌体。
3.2.2转化和筛选
分别将3.1构建得到的重组表达质粒用Sac I进行线性化,线性化片段纯化回收后通过电穿孔法分别转化毕赤酵母GS115,在MD平板上筛选得到毕赤酵母重组菌株,然后在含不同浓度遗传霉素的YPD平板(0.5mg/mL-8mg/mL)上筛选多拷贝的转化子。
将获得的转化子分别转接于BMGY培养基中,30℃、250rpm振荡培养1d;再转入BMMY培养基中,30℃、250rpm振荡培养;每天添加0.5%的甲醇,诱导表达4 d;9000rpm离心10min去除菌体,即得到分别含木聚糖酶MXYL11和木聚糖酶突变体的发酵上清液。
1、木聚糖酶酶活测定方法
(1)木聚糖酶酶活单位的定义
在温度为50℃、pH为8.0的条件下,每分钟从浓度为5mg/ml的木聚糖溶液中降解释放1μmol还原糖所需要的酶量即为一个酶活性单位,以U表示。
(2)木聚糖酶酶活测定方法
吸取10.0 ml木聚糖溶液,50℃平衡20 min。
吸取10.0 ml经过适当稀释的酶液,50℃平衡5 min。
空白样品测定:吸取2.00 ml经过适当稀释的酶液(已经过50℃平衡),加入到刻度试管中,再加入5ml DNS试剂,电磁振荡3 s。然后加入2.0 ml木聚糖溶液,50℃平衡30 min,沸水浴加热5 min。用自来水冷却至室温,加水定容至25 ml,电磁振荡3 s~5s。以标准空白样为空白对照,在540 nm处测定吸光度AB。
样品测定:吸取2.00 ml经过适当稀释的酶液(已经过50℃平衡),加入到刻度试管中,再加入2.0 ml木聚糖溶液(已经过50℃平衡),电磁振荡3 s,50℃精确保温30 min。加入5.0 ml DNS试剂,电磁振荡3 s,以终止酶解反应。沸水浴加热5 min,用自来水冷却至室温,加水定容至25 ml,电磁振荡3 s。以标准空白样为空白对照,在540 nm处测定吸光度AE。
式中:
XD —试样稀释液中木聚糖酶的活力,U/ml;
AE —酶反应液的吸光度;
AB —酶空白样的吸光度;
K —标准曲线的斜率;
CO —标准曲线的截距;
M —木糖的摩尔质量M(C5MXYL110O5)= 150.2 g/mol;
t —酶解反应时间,min;
1000 —转化因子,1 mmol = 1000 μmol;
XD值应在0.04—0.10 U/ml之间。如果不在这个范围内,应重新选择酶液的稀释度,再进行分析测定。
X = XD·Df (2)
X —试样中木聚糖酶的活力,U/ml;
Df —试样的稀释倍数。
(3)测定结果
按照上述方法进行酶活检测,结果显示:上述构建得到的重组表达木聚糖酶MXYL11及其突变体的毕赤酵母重组菌株发酵上清液的酶活为300-600 U/mL。
2、蛋白含量测定方法
考马斯亮蓝(Bradford)结合法测定蛋白质含量是比色法与色素法结合的复合方法。考马斯亮兰G-250在酸性溶液时呈棕红色,当与蛋白质结合后变为蓝色,且在蛋白质一定浓度范围内符合比尔定律,可在595nm处比色测定。在3~5分钟即成大量吸收,至少稳定1小时。在10~1000μg/mL范围内,吸光值与蛋白质浓度成正比。
按照酶液和考马斯亮蓝溶液体积比1:5的比例进行混合,静置10mim,考马斯亮蓝(Bradford)结合法测定蛋白质含量
按照上述方法进行蛋白含量的检测。结果显示:上述构建得到的重组表达木聚糖酶MXYL11及其突变体的毕赤酵母重组菌株发酵上清液的蛋白含量为0.321-0.408 mg/mL。
3、比活力计算
“比活力 (Specific Activity)”是指:单位重量的蛋白质中所具有酶的活力单位数,一般用U/mg蛋白质来表示。
比活力计算公式:比活力(U/mg)=酶活(U/mL)/ 蛋白含量(mg/mL)。
具体的计算结果见表1。
表1 野生型木聚糖酶及其突变体比活力比较
野生型及其单点突变体 | 比活力(U/mg) |
MXYL11 | 1161.4 |
N7S | 1300.8 |
D12F | 1289.1 |
T21S | 2322.8 |
A23G | 1324.0 |
P24C | 1428.5 |
P24G | 1405.3 |
G25A | 1393.7 |
T26D | 1358.8 |
M29L | 1318.2 |
M29S | 1365.8 |
M29V | 1420.4 |
C30V | 1393.7 |
L31C | 1382.1 |
L31F | 1370.4 |
L31N | 1335.6 |
L31V | 1353.0 |
G34A | 1361.2 |
Y37F | 1409.9 |
S38H | 1544.7 |
S38T | 1475.0 |
T39V | 1513.3 |
T39F | 1243.9 |
Q40T | 1322.8 |
W41F | 1275.2 |
R42D | 1400.6 |
R42H | 1391.3 |
R42S | 1276.4 |
N43S | 1441.3 |
T44V | 1420.4 |
T44S | 1401.8 |
K51Q | 1319.3 |
A54H | 1329.8 |
A54N | 1671.2 |
V91I | 1367.9 |
E92D | 1393.8 |
T100S | 1254.3 |
T102Q | 1277.3 |
Y103T | 1383.3 |
T109V | 1374.3 |
E118Q | 1330.5 |
E118R | 1343.4 |
T119S | 1320.0 |
T119D | 1357.4 |
Y122V | 1277.4 |
Y122F | 1392.0 |
Y122T | 1369.2 |
Y122S | 1392.0 |
N123D | 1394.7 |
N123K | 1780.4 |
N123Y | 1716.2 |
T132S | 1315.3 |
Q134N | 1349.9 |
Q134D | 1338.2 |
Q141S | 1411.8 |
Q141A | 1393.7 |
Q141E | 1393.3 |
Q141F | 1382.0 |
Q141I | 1332.1 |
Q141L | 1344.6 |
Q141M | 1349.7 |
Q141P | 1316.2 |
Q141W | 1394.9 |
Q142H | 1573.7 |
Q142S | 1509.7 |
R144H | 1342.0 |
R144K | 1445.4 |
R144Q | 1367.4 |
T145S | 1353.3 |
T145P | 1803.4 |
T145V | 1363.7 |
T150N | 1443.7 |
V151F | 1418.2 |
V151S | 1388.6 |
V151T | 1353.4 |
G152D | 1363.6 |
D156S | 1349.8 |
S167T | 1277.3 |
H168N | 1290.3 |
H168S | 1292.6 |
H168V | 1296.5 |
D169N | 1304.4 |
I172V | 1327.7 |
L173I | 1338.3 |
T175V | 1355.7 |
S183D | 1370.2 |
S184A | 1313.7 |
V186I | 1330.4 |
从表1的结果可以看出,与野生型木聚糖酶MXYL11相比,本发明提供的木聚糖酶单点突变体的比活力均得到显著提高。其中,含T21S单点突变的木聚糖酶突变体的比活力最高,达2322.8 U/mg,是野生型木聚糖酶的两倍,取得了意料不到的技术效果。
综上,本发明提供的碱性木聚糖酶突变体的比活力得到显著提高,从而有利于降低该酶的生产成本,促进其在工业领域,尤其是造纸领域中的广泛应用。
序列表
<110> 青岛蔚蓝生物集团有限公司
<120> 高比活碱性木聚糖酶突变体
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 189
<212> PRT
<213> 放线杆菌(Actinobacteria bacterium)
<400> 1
Asp Thr Thr Ile Thr Gln Asn Gln Thr Gly Tyr Asp Asn Gly Tyr Phe
1 5 10 15
Tyr Ser Phe Trp Thr Asp Ala Pro Gly Thr Val Ser Met Thr Leu His
20 25 30
Ser Gly Gly Ser Tyr Ser Thr Gln Trp Arg Asn Thr Gly Asn Phe Val
35 40 45
Ala Gly Lys Gly Trp Ala Thr Gly Gly Arg Arg Thr Val Thr Tyr Asn
50 55 60
Ala Thr Phe Asn Pro Ser Gly Asn Ala Tyr Leu Ala Leu Tyr Gly Trp
65 70 75 80
Thr Arg Asn Pro Leu Val Glu Tyr Tyr Ile Val Glu Ser Trp Gly Thr
85 90 95
Tyr Arg Pro Thr Gly Thr Tyr Lys Gly Thr Val Thr Thr Asp Gly Gly
100 105 110
Thr Tyr Asp Ile Tyr Glu Thr Trp Arg Tyr Asn Ala Pro Ser Ile Glu
115 120 125
Gly Thr Arg Thr Phe Gln Gln Phe Trp Ser Val Arg Gln Gln Lys Arg
130 135 140
Thr Ser Gly Thr Ile Thr Val Gly Asn His Phe Asp Ala Trp Ala Arg
145 150 155 160
Ala Gly Leu Asn Leu Gly Ser His Asp Tyr Gln Ile Leu Ala Thr Glu
165 170 175
Gly Tyr Gln Ser Ser Gly Ser Ser Thr Val Thr Ile Gly
180 185
<210> 2
<211> 567
<212> DNA
<213> 放线杆菌(Actinobacteria bacterium)
<400> 2
gatactacta tcactcaaaa ccaaactgga tacgataacg gatactttta ctctttttgg 60
actgatgctc caggaactgt ttctatgact ttgcactctg gaggatctta ctctactcaa 120
tggagaaaca ctggtaactt cgttgctggt aagggttggg ctactggtgg tagaagaacc 180
gttacttaca atgctacttt taacccatct ggtaacgctt acttggcttt gtacggttgg 240
acaagaaacc cattggttga gtactatatt gttgaatctt ggggtactta cagacctact 300
ggaacttaca agggaactgt tactactgat ggaggtactt acgatattta cgagacttgg 360
agatacaacg ctccatctat tgaaggtact agaacctttc agcaattttg gtctgttaga 420
caacaaaaga gaacctctgg aactattaca gttggtaacc attttgatgc ttgggctaga 480
gctggattga acttgggatc tcatgattac caaattttgg ctactgaggg ttaccaatct 540
tccggatcat caactgttac cattgga 567
Claims (10)
1.一种木聚糖酶突变体,其特征在于,所述突变体包含与SEQ ID NO:1具有至少90%同一性的氨基酸序列,且与SEQ ID NO:1相比在选自下组中的至少一个位置上包含氨基酸的取代:7,12,21,23,24,25,26,29,30,31,34,37,38,39,40,41,42,43,44,51,54,91,92,100,102,103,109,118,119,122,123,132,134,141,142,144,145,150,151,152,156,167,168,169,172,173,175,183,184,186。
2.如权利要求1所述的突变体,其特征在于,所述突变体的氨基酸序列与SEQ ID NO:1相比具有至少91%,92%,93%,94%,95%,96%,97%,98%,或至少99%的同一性。
3.如权利要求1所述的突变体,其特征在于,所述突变体的氨基酸序列与SEQ ID NO:1相比具有至少99.1%,99.2%,99.3%,99.4%,99.5%,99.6%,99.7%,99.8%,或至少99.9%的同一性。
4.如权利要求1-3任一所述的突变体,其特征在于,所述的突变体包含下组中至少一个氨基酸的取代:N7S,D12F,T21S,A23G,P24C/G,G25A,T26D,M29/S/V,C30V,L31C/F/N/V,G34A,Y37F,S38H/T,T39V/F,Q40T,W41F,R42D/H/S,N43S,T44V/S,K51Q,A54H/N,V91I,E92D,T100S,T102Q,Y103T,T109V,E118Q/R,T119S/D,Y122V/F/T/S,N123D/K/Y,T132S,Q134N/D,Q141S/A/E/F/I/L/M/P/W,Q142H/S,R144H/K/Q,T145S/P/V,T150N,V151F/S/T,G152D,D156S,S167T,H168N/S/V,D169N,I172V,L173I,T175V,S183D,S184A,V186I。
5.编码权利要求4所述木聚糖酶突变体的DNA分子。
6.包含权利要求5所述DNA分子的重组表达质粒。
7.一种宿主细胞,其特征在于,所述的宿主细胞包含权利要求6所述的重组表达质粒。
8.如权利要求7所述的宿主细胞,其特征在于,所述的宿主细胞为毕赤酵母(Pichia pastoris)。
9.如权利要求7所述的宿主细胞,其特征在于,所述的宿主细胞为里氏木霉(Trichoderma reesei)。
10.权利要求1-4任一所述木聚糖酶突变体在造纸领域中的应用。
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CN116410960B (zh) * | 2023-03-10 | 2024-02-23 | 云南师范大学 | 嗜盐适冷及pH适应性改良的β-木糖苷酶突变体D41G及其应用 |
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