CN115125226A - 高比活淀粉酶突变体 - Google Patents
高比活淀粉酶突变体 Download PDFInfo
- Publication number
- CN115125226A CN115125226A CN202210884241.1A CN202210884241A CN115125226A CN 115125226 A CN115125226 A CN 115125226A CN 202210884241 A CN202210884241 A CN 202210884241A CN 115125226 A CN115125226 A CN 115125226A
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- Prior art keywords
- amylase
- mutant
- gly
- asp
- tyr
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- 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
- C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
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- C12R2001/84—Pichia
Abstract
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淀粉酶突变体及其应用。本发明提供了分别包含M96I、Y263F、M309L、M309F单点突变的淀粉酶突变体，其比活力比野生型提高了20.0%‑30.4%；其中，含M309F单点突变的淀粉酶突变体比活力最高，达1189.27U/mg，更有利于其在工业领域中的广泛应用。
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淀粉酶突变体及其应用。
背景技术
α-淀粉酶(α-amylase)是一种内切酶，它以在淀粉分子的内部随机切开α-1,4糖苷键的方式作用于淀粉从而淀粉分子迅速降解，失去粘性，同时使水解物的还原力增加。
α-淀粉酶是淀粉酶中应用最广泛的酶种，其可以随机地从内部切断淀粉、糖原内的α-1, 4-葡萄糖苷键，将淀粉水解为糊精、低聚糖和单糖。该类酶水解产物的末端残基碳原子的构型为A构型，所以称为α-淀粉酶。α-淀粉酶的pH适用范围十分广泛，pH在2.0–12.0之间均适用。根据催化作用温度的不同，α-淀粉酶可分为低温α-淀粉酶、中温α-淀粉酶和耐高温α-淀粉酶。相较于常温α-淀粉酶，耐高温α-淀粉酶有下述优点：(1)热稳定性更好，在工业生产的高温条件下稳定，能够长时间地发挥作用；(2)能够使淀粉的液化更加彻底，缩短糊化和过滤的时间，从而达到节约能源、降低成本的效果；(3)保存条件范围广、易于储存和运输等。因具有热稳定性和较宽的pH适应范围等特质，耐高温α-淀粉酶可直接在温度要求严苛的高温环境中发挥作用，有着较高的开发价值及应用价值。目前，耐高温α-淀粉酶已经逐渐发展为主流淀粉酶品种，广泛应用于淀粉制糖业、味精、啤酒等食品发酵行业以及纺织印染行业等。
最初用于工业生产的α-淀粉酶来自于真菌，曾被用作治疗消化系统疾病的药物辅助剂。随后的研究发现一些细菌来源的α-淀粉酶具有耐高温、耐酸或耐碱等极端环境条件的特性，能在高温工业生产的极端条件下保持催化性能。细菌中Bacillus属的48个种中有32个种可以产生α-淀粉酶，但是只有少数能够产生耐高温α-淀粉酶，常见的有地衣芽孢杆菌、嗜热芽孢杆菌(Bacillus thermophilus)、嗜热脂肪芽孢杆菌(Bacillusstearothermophilus)等。细菌α-淀粉酶目前仍是淀粉液化水解处理的高温工业过程中使用最广泛的工业酶。
发明内容
本发明的目的是提供一种比活力显著提高的淀粉酶突变体。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：
本发明一方面提供了一种淀粉酶，其氨基酸序列为SEQ ID NO:1。
本发明一方面提供了一种淀粉酶突变体，是氨基酸序列为SEQ ID NO:1的淀粉酶的第96位氨基酸由Met变为Ile。
本发明还提供了一种淀粉酶突变体，是氨基酸序列为SEQ ID NO:1的淀粉酶的第263位氨基酸由Tyr变为Phe。
本发明还提供了一种淀粉酶突变体，是氨基酸序列为SEQ ID NO:1的淀粉酶的第309位氨基酸由Met变为Leu或Phe。
本发明还涉及编码上述淀粉酶突变体的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。
所述宿主细胞为毕赤酵母（Pichia pastoris）。
将上述的质粒转入宿主细胞中，重组表达的淀粉酶突变体的比活力得到显著提升。
本发明还涉及所述宿主细胞在淀粉酶生产中的应用。
与野生型淀粉酶A1相比，本发明提供的分别包含M96I、Y263F、M309L、M309F单点突变的淀粉酶突变体比活力提高了20.0%-30.4%；其中，含M309F单点突变的淀粉酶突变体比活力最高，达1189.27U/mg，取得了意料不到的技术效果。
本发明所述淀粉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，促进其在工业领域中的广泛应用。
具体实施方式
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
将来源于嗜热脂肪土芽孢杆菌（Geobacillus stearothermophilus）的淀粉酶基因（GeneBank:747155414）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（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。将该淀粉酶命名A1，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）对淀粉酶基因进行酶切；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-A1。
实施例2 高比活突变体的筛选
淀粉酶A1是糖苷水解酶G11家族淀粉酶，为了进一步提高淀粉酶A1的酶活性，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A1-F1、A1-R1：
A1-F1：GGCGAATTC GCCGCACCGTTTAACCCAACCATG（下划线为限制性内切酶EcoRI识别位点）；
A1-R1：ATAGCGGCCGC CTATCTTTGGACATAAATTGAAAC（下划线为限制性内切酶NotI识别位点）。
以A1基因（SEQ ID NO：2）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，获得含有淀粉酶的大肠杆菌细胞裂解液。
分别取出10μl裂解液至两块新的96孔板；将其中一块96孔板加入190 μl底物，于60℃反应5 min后，取20μl反应体系至一块新的96孔板，加入160μl碘液和20μl Hcl（0.1mol/L）来终止并显色，碘显色法测定剩余的淀粉含量，计算水解掉的淀粉含量；另一块板加入190μl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。终，申请人筛选出显著提高淀粉酶A1比活力的突变位点：M96I、Y263F、M309L、M309F。
在上述野生型淀粉酶A1的基础上，本发明提供了分别含M96I、Y263F、M309L、M309F单个突变位点的突变体。
实施例3 淀粉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淀粉酶A1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，将合成的淀粉酶A1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，即得到分别含淀粉酶A1和淀粉酶突变体的发酵上清液。
1、淀粉酶酶活测定方法
（1）淀粉酶酶活单位的定义
1g固体酶粉，于60℃、pH值6.0条件下，1h液化1g可溶性淀粉，即为1个酶活力单位，以U/g表示。
（2）淀粉酶酶活测定方法
称取1g酶粉(精确至0.0001g)，用少量磷酸缓冲液充分溶解，将上清液小心倾入容量瓶中，若有剩余残渣，再加少量磷酸缓冲液充分研磨，最终样品全部移入容量瓶中，用磷酸缓冲液定容至刻度，摇匀。用四层纱布过滤，滤液待用。
吸取20.0 mL可溶性淀粉溶液于试管中，加人磷酸缓冲液5.00 mL，摇匀后，置于60℃ ± 0. 2℃,恒温水浴中预热8 min.
加入1.00 mL稀释好的待测酶液，立即计时，摇匀，准确反应5 min。
立即用自动移液器吸取1.00mL反应液，加到预先盛有0.5mL盐酸溶液和5.00mL稀碘液的试管中，摇匀，并以0.5 mL盐酸溶液和5.00 mL稀碘液为空白，于660 nm波长下，用10mm比色皿迅速测定其吸光度(A)。根据吸光度，求得测试酶液的浓度。
酶活力计算公式：X1 =c×n。
式中：
X1—样品的酶活力，U/mL(或U/g)；
c—测试酶样的浓度，U/mL(或U/g)；
n—样品的稀释倍数。
所得结果表示至整数。
（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野生型淀粉酶A1及其突变体的毕赤酵母重组菌株发酵上清液的酶活为370-450 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10 mim，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野生型淀粉酶A1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0.352-0.406 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 淀粉酶突变体比活力比较
| 淀粉酶及其单点突变体 | 比活力（U/mg） |
| 野生型A1 | 912.02 |
| M96I | 1140.03 |
| Y263F | 1094.42 |
| M309L | 1112.66 |
| M309F | 1189.27 |
从表1的结果可以看出，与野生型淀粉酶A1相比，本发明提供的分别包含M96I、Y263F、M309L、M309F单点突变的淀粉酶突变体比活力提高了20.0%-30.4%；其中，含M309F单点突变的淀粉酶突变体比活力最高，达1189.27U/mg，取得了意料不到的技术效果。
综上，本发明提供的淀粉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> 高比活淀粉酶突变体
<160> 2
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<213> 嗜热脂肪土芽孢杆菌(Geobacillus stearothermophilus)
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Ala Ala Pro Phe Asn Pro Thr Met Met Gln Tyr Phe Glu Trp Tyr Leu
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Pro Asp Asp Gly Thr Leu Trp Thr Lys Val Ala Asn Glu Ala Asn Asn
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Leu Ser Ser Leu Gly Ile Asn Ala Leu Trp Leu Pro Pro Ala Tyr Lys
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Gly Thr Ser Arg Ser Asp Val Gly Tyr Gly Val Tyr Asp Leu Tyr Asp
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Lys Ala Gln Tyr Leu Gln Ala Ile Gln Ala Ala His Ala Ala Gly Met
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Gln Val Tyr Ala Asp Val Val Phe Asp His Lys Gly Gly Ala Asp Gly
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Thr Glu Trp Val Asp Ala Val Glu Val Asn Pro Ser Asp Arg Asn Gln
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Glu Ile Ser Gly Thr Tyr Gln Ile Gln Ala Trp Thr Lys Phe Asp Phe
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Pro Gly Arg Gly Asn Thr Tyr Ser Ser Phe Lys Trp Arg Trp Asp His
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Phe Asp Gly Val Asp Trp Asp Glu Ser Arg Lys Leu Ser Arg Ile Tyr
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Lys Phe Arg Gly Lys Ala Trp Asp Trp Glu Val Asp Thr Glu Phe Gly
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Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Leu Asp Met Asp His Pro Glu
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Val Val Thr Glu Leu Lys Asn Trp Gly Lys Trp Tyr Val Asn Thr Thr
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Asn Ile Asp Gly Phe Arg Leu Asp Ala Val Lys His Ile Lys Phe Gln
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Phe Phe Pro Asp Trp Leu Ser Tyr Val Arg Ser Gln Thr Gly Lys Pro
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gacttgtatg acctcggcga gtttaatcaa aaagggaccg tccgcacaaa atatggaaca 240
aaagctcaat atcttcaagc cattcaagcc gcccacgccg ctggaatgca agtgtacgcc 300
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gtcaatccgt ccgaccgcaa ccaagaaatc tcgggcacct atcaaatcca agcatggacg 420
aaatttgatt ttcccgggcg gggcaacacc tactccagct ttaagtggcg ctgggatcat 480
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aaagcgtggg attgggaagt agacacagaa ttcggaaact atgactactt aatgtatgcc 600
gaccttgata tggatcatcc cgaagtcgtg accgagctga aaaactgggg gaaatggtat 660
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ggggaatatt ggagctatga catcaacaag ttgcacaatt acattacgaa aacagatgga 840
acgatgtctt tgtttgatgc cccgttacac aacaaatttt ataccgcttc caaatcaggg 900
ggcgcatttg atatgcgcac gttaatgacc aatactctca tgaaagatca accgcatttg 960
gccgtcacct tcgttgataa tcatgacacc gaacccggcc aagcgctgca gtcatgggtt 1020
gatccatggt tcaaaccgtt ggcttacgcc tttattctaa ctcggcagga aggatacccg 1080
tgcgtctttt atggtgacta ttatggcatt ccacaatata acattccttc gctgaaaagc 1140
aaaatcgatc cgctcctcat cgcgcgcagg gattatgctt acggaacgca acatgattat 1200
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aacagtgatg gatgggggga gtttaaagtc aatggcgggt cggtttcaat ttatgtccaa 1440
agatag 1446
Claims (9)
1.一种淀粉酶，其特征在于，所述淀粉酶的氨基酸序列为SEQ ID NO:1。
2.一种淀粉酶突变体，其特征在于，所述突变体是氨基酸序列为SEQ ID NO:1的淀粉酶的第96位氨基酸由Met变为Ile。
3.一种淀粉酶突变体，其特征在于，所述突变体是氨基酸序列为SEQ ID NO:1的淀粉酶的第263位氨基酸由Tyr变为Phe。
4.一种淀粉酶突变体，其特征在于，所述突变体是氨基酸序列为SEQ ID NO:1的淀粉酶的第309位氨基酸由Met变为Leu或Phe。
5.编码权利要求2-4任一所述淀粉酶突变体的DNA分子。
6.包含权利要求5所述的DNA分子的重组表达载体。
7.一种宿主细胞，其特征在于，所述宿主细胞包含权利要求6所述的重组表达载体。
8.如权利要求7所述的宿主细胞，其特征在于，所述宿主细胞为毕赤酵母（Pichia pastoris）。
9.权利要求7或8所述的宿主细胞在淀粉酶生产中的应用。
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| CN103068975A (zh) * | 2010-01-04 | 2013-04-24 | 诺维信公司 | α-淀粉酶变体和编码该变体的多核苷酸 |
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