CN117384886A - 一种高比活碱性木聚糖酶突变体 - Google Patents
一种高比活碱性木聚糖酶突变体 Download PDFInfo
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- CN117384886A CN117384886A CN202311342665.6A CN202311342665A CN117384886A CN 117384886 A CN117384886 A CN 117384886A CN 202311342665 A CN202311342665 A CN 202311342665A CN 117384886 A CN117384886 A CN 117384886A
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- xylanase
- specific activity
- mutant
- enzyme
- xylanase mutant
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- C12N9/2477—Hemicellulases not provided in a preceding group
- C12N9/248—Xylanases
- C12N9/2482—Endo-1,4-beta-xylanase (3.2.1.8)
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- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
- C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
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- Medicinal Chemistry (AREA)
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及其应用。本发明以野生型木聚糖酶H1为基础，提供了包含I37V、A59S、D63E、D104Y、T107M/K、N167G、D192E中至少一个突变位点的突变体。与野生型木聚糖酶H1相比，本发明提供的木聚糖酶突变体的比活力普遍提高了8.5%‑32.8%；其中，含D192E单点突变的木聚糖酶突变体的比活力最高，达1625.11 U/mg，有利于降低该酶的生产成本，促进其在工业领域的广泛应用。
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
木聚糖是自然界中广泛存在的一种五碳糖，木聚糖酶是一类能将木聚糖降解为木二糖、木二糖以上的寡聚木糖及少量木糖等的酶，是在木聚糖降解过程中起关键作用的酶。由于木聚糖的组成成分复杂，其水解需要多种酶协同作用，所以广义上的木聚糖酶是指能够将木聚糖水解成寡糖或单糖的一系列酶的总称，包括内切β-1,4-D-木聚糖酶、β-D-木糖苷酶、α-L-阿拉伯糖苷酶、α-D-葡糖苷酸酶、乙酰基木聚糖酶、酚酸酯酶等，狭义上的木聚糖酶指内切β-1,4-D-木聚糖酶。木聚糖酶来源非常广泛，可以由不同种类的微生物产生。根据木聚糖酶对酸碱环境的耐受度，可以将其分为碱性、中性和酸性。
碱性木聚糖酶在造纸工业、饲料行业及食品工业中都起着十分重要的作用，尤其在造纸的制浆、促进漂白及废纸脱墨等的工业生产中，碱性木聚糖酶能显著降低造纸过程中的污染排放，提高产品的质量。使用木聚糖酶AU-PE89对碱法麦草浆进行漂前预处理，使得成品浆A等品率提升1.43%，细浆得率提高1.48%，抄片强度指标均有所提升，降低了洗漂段纤维的损伤。有研究在纸浆漂白过程中加入了产自节杆菌(Arthrobacter sp．)MTCC5214的木聚糖酶，结果发现硫酸盐浆的卡伯值降低了20%，相当于漂白过程中用氯减少了29%，且与未处理的纸浆相比，酶处理使得纸浆的亮度提高了9.6%。
为了扩大碱性木聚糖酶在生产应用中的适用范围，近年来许多学者利用克隆技术和基因工程技术，对自然界中分离出来的碱性木聚糖酶的基因进行表达纯化及扩大培养，目前已取得较大进展。例如，Bai等对来自嗜碱枯草芽孢杆菌( Bacillus subtilis) SN5的木聚糖酶 Xyn11A-LC进行结构比较和突变分析，发现在pH较高的条件下，碱性木聚糖酶带电残基含量增加，且相对于中性和酸性木聚糖酶，碱性木聚糖酶的丝氨酸、苏氨酸和酪氨酸数量较少，突变分析显示至少有六个氨基酸（Glu16，Trp18，Asn44，Leu46，Arg48，Ser187）的参与使酶在碱性条件下具有更高的活性。Long等采用双质粒共同表达的方法将来自黑曲霉(Aspergillus niger)的木聚糖酶基因转入毕赤酵母( Pichia pastoris) 中表达，使其表达能力提高了33%，通过优化培养条件，表达能力较摇瓶培养提高了2.4倍，为生产中提高木聚糖酶产量提供了新的方法。刘俊等通过易错PCR法和双酶切载体重构建法建立了木聚糖酶基因随机突变文库，从中筛选出四个在pH 8.0 ~ 9.5 范围内相对酶活性均比出发菌株酶高15%左右的突变体，再将四个突变位点进行组合突变，各个组合突变体酶与底物的亲和力和催化效率都比野生型出发菌株酶高，pH稳定性也显著高于野生型酶。
目前使用的木聚糖酶存在比酶活低、不稳定、成本高，不能满足生产需要等问题，需要通过分子改造进行性质优化。本发明即提供了一种具有高比酶活的碱性木聚糖酶，可使其更适合于在工业领域中的实际应用。
发明内容
本发明的目的是提供一种碱性木聚糖酶突变体。所述突变体的比活力比野生型得到显著提高，有利于其在工业领域的广泛应用。
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：37，59，63，104，107，167，192。
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：I37V、A59S、D63E、D104Y、T107M/K、N167G、D192E。
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分子的重组表达载体。
本发明还涉及一种宿主细胞，包含上述重组表达载体。
将上述的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。
在本发明的一些实施例中，宿主细胞为毕赤酵母（Pichia pastoris）。
在本发明的一些实施例中，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。
本发明以野生型木聚糖酶H1为基础，提供了包含I37V、A59S、D63E、D104Y、T107M/K、N167G、D192E中至少一个突变位点的突变体。与野生型木聚糖酶H1相比，本发明提供的木聚糖酶突变体的比活力普遍提高了8.5%-32.8%；其中，含D192E单点突变的木聚糖酶突变体的比活力最高，达1625.11 U/mg，取得了意料不到的技术效果。
综上，本发明提供的木聚糖酶突变体的比活力得到显著提高，从而有利于降低木聚糖酶的生产成本，促进其在工业领域中的广泛应用。
具体实施方式
本发明公开了一种碱性木聚糖酶突变体、其制备方法及应用、编码该碱性木聚糖酶突变体的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 重组质粒的构建
将来源于拟青霉（Paecilomyces. sp）的木聚糖酶基因（GeneBank ACS26244. 1）根据毕赤酵母密码子偏好性进行优化，并在其起始密码子ATG前增加6个碱基GAATTC（EcoRI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H1，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），用氨苄青霉素进行选择。为确保准确，对若干克隆进行测序（Invitrogen）。
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，获得1个重组质粒，将其命名为pPIC9K-H1。
实施例2 高比活木聚糖酶突变体的筛选
为了进一步提高木聚糖酶H1的酶活性，申请人对其进行蛋白结构分析。该蛋白是GH11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H1-F1、H1-R1：
H1-F1：GGCGAATTCATGATGATTGGTATCACTTCTTTTGC（下划线为限制性内切酶EcoRI识别位点）；
H1-R1：ATAGCGGCCGC TTAACCGACGTCTGCAACGGTAATTC（下划线为限制性内切酶NotI识别位点）。
以H1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I37V、A59S、D63E、D104Y、T107M、T107K、N167G、D192E。
在上述野生型木聚糖酶H1的基础上，本发明提供了分别含I37V、A59S、D63E、D104Y、T107M/K、N167G、D192E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偏爱性分别对木聚糖酶H1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H1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H1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。
XD =[(AE- AB)×K+ C0] ×N×1000/(M×t) 。
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；
C0—标准曲线的截距；
M—木糖的摩尔质量M（C5XYN110O5）= 150.2 g/mol；
t—酶解反应时间，min；
N—酶液稀释倍数;
1000—转化因子，1 mmol = 1000 μmol。
（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H1及其突变体的毕赤酵母重组菌株发酵上清液的酶活为420-750 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H1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0.34-0.5 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）。
具体结果见表1。
表1 碱性木聚糖酶突变体比活力比较
| 木聚糖酶及其单点突变体 | 比活力（U/mg） |
| 野生型H1 | 1223.00 |
| I37V | 1328.92 |
| A59S | 1615.01 |
| D63E | 1395.12 |
| D104Y | 1397.25 |
| T107M | 1319.89 |
| T107K | 1379.92 |
| N167G | 1462.18 |
| D192E | 1625.11 |
从表1的结果可以看出，与野生型木聚糖酶H1相比，本发明提供的碱性木聚糖酶突变体的比活力普遍提高了8.5%-32.8%；其中，含D192E单点突变的碱性木聚糖酶突变体的比活力最高，达1625.11 U/mg，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。
Claims (6)
1.一种木聚糖酶突变体，其特征在于，所述突变体是氨基酸序列为SEQ ID NO:1的木聚糖酶的第167位氨基酸由Asn变为Gly。
2.编码权利要求1所述木聚糖酶突变体的DNA分子。
3.包含权利要求2所述DNA分子的重组表达质粒。
4.一种宿主细胞，其特征在于，所述的宿主细胞包含权利要求3所述的重组表达质粒。
5.如权利要求4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）或里氏木霉（Trichoderma reesei）。
6.权利要求1所述木聚糖酶突变体在造纸领域中的应用。
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