CN110184259A - 一种厌氧芽孢杆菌来源普鲁兰酶突变体及其应用 - Google Patents

一种厌氧芽孢杆菌来源普鲁兰酶突变体及其应用 Download PDF

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CN110184259A
CN110184259A CN201910612907.6A CN201910612907A CN110184259A CN 110184259 A CN110184259 A CN 110184259A CN 201910612907 A CN201910612907 A CN 201910612907A CN 110184259 A CN110184259 A CN 110184259A
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周哲敏
周丽
崔文璟
刘中美
庞博
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Abstract

本发明公开了一种厌氧芽孢杆菌来源普鲁兰酶突变体及其应用，属于基因工程和酶工程技术领域。本发明构建的普鲁兰酶突变体K419R，在热稳定性与野生型普鲁兰酶相差不大的情况下，其比酶活是野生型普鲁兰酶的1.30倍；普鲁兰酶突变体K419R/T245C/A326C，在比酶活与野生型普鲁兰酶相同的情况下，其热稳定性较野生型大幅提升；而突变体K419R/T245C/A326C/W651C/V707C的比酶活是野生型的1.47倍，且具有良好的热稳定性。因此本发明的普鲁兰酶突变体能够满足淀粉脱支过程高温条件的要求，同时提高淀粉脱支和水解效率，对于普鲁兰酶的工业化应用至关重要。

Description

一种厌氧芽孢杆菌来源普鲁兰酶突变体及其应用
技术领域
本发明涉及一种厌氧芽孢杆菌来源普鲁兰酶突变体及其应用，属于基因工程和酶工程技术领域。
背景技术
普鲁兰酶是一种重要的淀粉脱支酶，能够专一、高效地切割普鲁兰多糖、支链淀粉、α，β-极限糊精中的α-1,6-糖苷键，广泛地应用于现代淀粉糖工业中，是高品质糖浆生产过程中的关键酶制剂。普鲁兰酶经常在淀粉糖化过程中添加来发挥脱支作用，而糖化需要在高温(60-65℃)下进行，时间一般为48-60h。因此，普鲁兰酶必须在高温下保持高催化活力和稳定性。
近年来，国内外对普鲁兰酶的研究逐渐升温，取得了许多良好的研究成果，但是对高活性、耐热普鲁兰酶的开发仍然存在一些问题，比如Exiguobacterium sp.SH3来源普鲁兰酶比酶活仅为20.3U/mg，Bacillus acidopullulyticus来源普鲁兰酶在60℃的半衰期仅为34.9min，Bacillus pseudofirmus 703和Shewanella arctica来源普鲁兰酶在60℃几乎没有活力。因此提供一种能够在高温下保持高催化活力和稳定性的普鲁兰酶对于普鲁兰酶的工业化应用至关重要。
发明内容
为了解决上述存在的技术问题，本发明通过对来源于厌氧芽孢杆菌Anoxybacillus sp.WB42的野生型普鲁兰酶PulA进行突变改造，获得了三株突变体K419R、K419R/T245C/A326C和K419R/T245C/A326C/W651C/V707C，该突变体具有高酶活和热稳定性，能够满足淀粉脱支过程高温条件的要求，同时提高淀粉脱支和水解效率。
本发明的第一个目的是提供一种普鲁兰酶突变体，所述所述突变体的氨基酸序列包括：在SEQ ID NO.1所示的氨基酸序列的基础上，将第419位赖氨酸突变为精氨酸后得到的氨基酸序列。
在一种实施方式中，所述突变体是K419R，其氨基酸序列如SEQ ID NO.3所示。
在一种实施方式中，所述突变体是K419R/T245C/A326C，其是在突变体K419R的基础上将第245位苏氨酸、第326位丙氨酸分别突变为半胱氨酸，其氨基酸序列如SEQ ID NO.4所示。
在一种实施方式中，所述突变体是K419R/T245C/A326C/W651C/V707C，其是在突变体K419R/T245C/A326C的基础上将第651位色氨酸、第707位缬氨酸分别突变为半胱氨酸，其氨基酸序列如SEQ ID NO.5所示。
本发明的第二个目的是提供编码所述突变体的基因。
本发明的第三个目的是提供携带所述基因的载体或细胞。
本发明的第四个目的是提供表达所述突变体的基因工程菌。
在一种实施方式中，所述的基因工程菌，是以大肠杆菌为宿主。
在一种实施方式中，所述的基因工程菌，是以pET-22b(+)为表达载体。
本发明的第五个目的是提供一种切割底物中α-1,6-糖苷键的方法，是以所述突变体或含有所述突变体的全细胞为催化剂，切割底物中的α-1,6-糖苷键。
本发明还提供所述的突变体或所述的基因工程菌在医药生产、化工或食品领域的应用。
本发明的有益效果：
(1)本发明构建的普鲁兰酶突变体K419R，在热稳定性与野生型普鲁兰酶相差不大的情况下，其比酶活为86.4U/mg，是野生型普鲁兰酶(66.69U/mg)的1.30倍；
(2)本发明构建的普鲁兰酶突变体K419R/T245C/A326C，在比酶活与野生型普鲁兰酶相同的情况下(均为66.69U/mg)，其热稳定性较野生型大幅提升，在65℃保温30min残余酶活为94％，在67℃保温30min残余酶活为82％；而野生型普鲁兰酶在65℃保温30min残余酶活仅为60％，在67℃保温30min残余酶活仅为33％；
(3)本发明构建的普鲁兰酶突变体K419R/T245C/A326C/W651C/V707C，不仅比酶活是野生型的1.47倍，为98.2U/mg，且热稳定性较野生型大幅提升，65℃保温30min残余酶活约85％，在67℃保温30min残余酶活为70％；而野生型普鲁兰酶在65℃保温30min残余酶活仅为60％，在67℃保温30min残余酶活仅为33％；
(4)突变体K419R、K419R/T245C/A326C、K419R/T245C/A326C/W651C/V707C在pH6.6柠檬酸缓冲液中处理4h，剩余酶活为100％，而野生型剩余酶活仅为83％；三株突变体在pH 7.0柠檬酸缓冲液中处理4h，剩余酶活为90％以上，而野生型剩余酶活仅为66％。
附图说明
图1：pET-22b(+)-pulA定点突变全质粒PCR结果；其中1表示DNA marker；2表示pET-22b(+)-pulA定点突变全质粒PCR结果。
图2：普鲁兰酶的纯化结果；其中M表示蛋白质marker；1表示纯化后的野生型普鲁兰酶；2表示纯化后的普鲁兰酶突变体K419R；3表示纯化后的普鲁兰酶突变体K419R/T245C/A326C；4表示纯化后的普鲁兰酶突变体K419R/T245C/A326C/W651C/V707C。
图3：普鲁兰酶最适温度的测定。
图4：普鲁兰酶热稳定性的测定。
图5：普鲁兰酶最适pH的测定。
图6：普鲁兰酶pH耐受性的测定。
具体实施方式
LB培养基：蛋白胨10g/L，酵母浸膏5g/L，氯化钠10g/L。
LB固体培养基：蛋白胨10g/L，酵母浸膏5g/L，氯化钠10g/L，琼脂粉20g/L。
LB液体培养基：蛋白胨10g/L，酵母浸膏5g/L，氯化钠10g/L。
结合缓冲溶液：50mmol/L Na2HPO4、50mmol/L NaH2PO4、500mmol/L NaCl、20mmol/L咪唑。
磷酸盐缓冲液：50mmol/L Na2HPO4、50mmol/L NaH2PO4、500mmol/L NaCl。
普鲁兰酶酶活测定方法：100μL的反应体系(10μL终浓度50mM pH 6.6柠檬酸缓冲液、50μL终浓度3％普鲁兰多糖和40μL纯酶液)，在65℃反应15min，加入100μL DNS反应液，沸水浴6min，流水速冷至室温，加蒸馏水稀释，混匀，测定OD476。
1个酶活力单位(U)定义为：在测定条件下，每分钟释放相当于1μmol葡萄糖还原力的还原糖所需要的酶量。
实施例1：重组菌构建
根据来源于Anoxybacillus sp.WB42(Wang J，Liu Z，Zhou Z.Cloning andcharacterization of a novel thermophilic amylopullulanase with a type Ipullulanase structure from Anoxybacillus sp.WB42[J].Starch-2018，70(5-6)：1700265.公开日：2018-12-31)的普鲁兰酶如SEQ ID NO.2所示的碱基序列，设计普鲁兰酶的克隆引物如表1所示，用于构建大肠杆菌的质粒是pET-22b(+)，带有Nde I、Xho I酶切位点。将普鲁兰酶克隆至载体pET-22b(+)的Nde I、Xho I酶切位点之间，得到重组质粒pET-22b(+)-pulA。步骤如下：
(1)以Anoxybacillus sp.WB42基因组为模板，利用引物pulA-F和pulA-R对基因pulA进行PCR克隆；
(2)将上述PCR产物进行回收纯化，用限制性内切酶Nde I、Xho I分别对纯化后的PCR产物和载体pET-22b(+)进行双酶切，之后对酶切产物回收纯化；
(3)将酶切纯化后的PCR产物和载体在DNA连接酶的作用下连接过夜，转化大肠杆菌JM109，挑取单菌落进行测序，得到重组菌E.coli JM109/pET-22b(+)-pulA。
表1引物序列表
实施例2：普鲁兰酶突变体的制备
(1)单突变体
根据如SEQ ID NO.2所示的普鲁兰酶的基因序列，设计并合成引入突变体K419R的引物，利用快速PCR技术，以实施例1中得到的携带编码野生型普鲁兰酶的基因的重组质粒pET-22b(+)-pulA为模板，对普鲁兰酶基因进行定点突变，测定DNA编码序列，鉴别出第419位赖氨酸密码子变成精氨酸密码子，得到单突变体普鲁兰酶K419R，该突变体的氨基酸序列如SEQ ID NO.3所示。
表2引入K419R突变的定点突变引物
注：下划线为突变碱基
PCR产物用琼脂糖凝胶电泳进行检测，结果如图1。将验证正确的PCR产物经DpnⅠ消化，转化大肠杆菌JM109感受态，感受态细胞在LB固体培养基(含100μg/mL氨苄青霉素)上，挑取阳性克隆于LB液体培养基(含100μg/mL氨苄青霉素)中培养后提取质粒，突变质粒测序正确，得到的重组菌命名为E.coli JM109/pET-22b(+)-pulA(K419R)。
(2)普鲁兰酶迭代突变体
以步骤(1)中得到的单突变体K419R编码基因为模板，设计并合成引入T245C和A326C突变的引物，利用快速PCR技术，以携带编码单突变体K419R的基因的重组载体pET-22b(+)-pulA(K419R)为模板，对普鲁兰酶基因进行定点突变，测定DNA编码序列，鉴别出第245位苏氨酸密码子变成半胱氨酸密码子、第326位丙氨酸密码子变成半胱氨酸密码子的迭代突变体K419R/T245C/A326C，该突变体的氨基酸序列如SEQ ID NO.4所示。
表3引入T245C/A326C突变的定点突变引物
注：下划线为突变碱基
将验证正确的PCR产物经DpnⅠ消化，转化大肠杆菌JM109感受态，感受态细胞在LB固体培养基(含100μg/mL氨苄青霉素)培养过夜后，挑克隆于LB液体培养基(含100μg/mL氨苄青霉素)中培养后提取质粒，突变质粒测序正确，得到的重组菌命名为E.coli JM109/pET-22b(+)-pulA(K419R/T245C/A326C)。
(3)普鲁兰酶二次迭代突变体
以步骤(2)中得到的迭代突变体K419R/T245C/A326C编码基因为模板，设计并合成引入W651C和V707C突变的引物，利用快速PCR技术，以携带编码迭代突变体K419R/T245C/A326C的基因的重组载体pET-22b(+)-pulA(K419R/T245C/A326C)为模板，对普鲁兰酶基因进行定点突变，测定DNA编码序列，鉴别出第651位色氨酸密码子变成半胱氨酸密码子、第707位缬氨酸密码子变成半胱氨酸密码子的二次迭代突变体K419R/T245C/A326C/W651C/V707C，该突变体的氨基酸序列如SEQ ID NO.5所示。
表4引入W651C/V707C突变的定点突变引物
注：下划线为突变碱基
将验证正确的PCR产物经DpnⅠ消化，转化大肠杆菌JM109感受态，感受态细胞在LB固体培养基(含100μg/mL氨苄青霉素)培养过夜后，挑克隆于LB液体培养基(含100μg/mL氨苄青霉素)中培养后提取质粒，突变质粒测序正确，得到的重组菌命名为E.coli JM109/pET-22b(+)-pulA(K419R/T245C/A326C/W651C/V707C)。
分别将实施例1中得到的重组菌E.coli JM109/pET-22b(+)-pulA以及步骤(1)、(2)、(3)中得到的重组菌接种至LB培养基，过夜培养，提取得到质粒pET-22b(+)-pulA、pET-22b(+)-pulA(K419R/T245C/A326C)、pET-22b(+)-pulA(K419R/T245C/A326C/W651C/V707C)，将质粒分别转化表达宿主大肠杆菌BL21(DE3)感受态细胞，得到的重组菌分别命名为E.coliBL21(DE3)/pET-22b(+)-pulA(K419R)、E.coliBL21(DE3)/pET-22b(+)-pulA(K419R/T245C/A326C)、E.coliBL21(DE3)/pET-22b(+)-pulA(K419R/T245C/A326C/W651C/V707C)。
实施例3：普鲁兰酶的纯化
分别挑取实施例2中得到的重组菌E.coliBL21(DE3)/pET-22b(+)-pulA、E.coliBL21(DE3)/pET-22b(+)-pulA(K419R)、E.coliBL21(DE3)/pET-22b(+)-pulA(K419R/T245C/A326C)、E.coliBL21(DE3)/pET-22b(+)-pulA(K419R/T245C/A326C/W651C/V707C)接种于5mL的LB培养基(含100μg/mL氨苄抗生素)中，37℃、200r/min振荡过夜培养。将过夜培养物按1％(v/v)的接种量接种于100mL LB培养基(含100μg/mL氨苄抗生素)中，37℃、200r/min振荡培养至OD600至0.6-0.8时，加入10μM诱导剂IPTG，16℃诱导16-18h得到菌体，4℃、8000r/min的转速离心收集重组菌体。
将上述离心收集的重组菌体溶于20mL结合缓冲溶液，超声破碎，13000g离心25min后，将上清液用0.22μm滤膜过滤。然后用10倍柱体积的结合缓冲溶液平衡1mL的His TrapHP柱，用15倍柱体积的结合缓冲溶液洗去非特异性吸附的蛋白，分别用8倍柱体积的150、300和500mmol/L咪唑的缓冲液洗脱蛋白，收集样品，4℃、2L磷酸盐缓冲透析20h后，即得到野生型的纯化普鲁兰酶以及普鲁兰酶突变体K419R、K419R/T245C/A326C、K419R/T245C/A326C/W651C/V707C，SDS-PAGE分析鉴定结果如图2所示。
实施例4：普鲁兰酶酶活测定
测定实施例3中得到的普鲁兰酶野生型、突变体K419R、K419R/T245C/A326C、K419R/T245C/A326C/W651C/V707C的普鲁兰酶酶活。
表5普鲁兰酶酶活测定
实施例5：普鲁兰酶酶学性质的测定
(1)酶的最适温度
酶最适温度的测定方法：50μL终浓度3％普鲁兰多糖与10μL终浓度50mM pH 6.6柠檬酸缓冲液混匀，在35-80℃条件下保温5min，加入40μL纯酶液反应15min，迅速冷却至4℃，加入100μL DNS反应液，沸水浴6min，流水速冷至室温，加蒸馏水稀释，混匀，测定OD476，计算普鲁兰酶酶活。
结果表明，野生型普鲁兰酶的最适温度是65℃，突变体K419R的最适温度是65℃，突变体K419R/T245C/A326C的最适温度是65℃，突变体K419R/T245C/A326C/W651C/V707C的最适温度是65℃。
(2)酶热稳定性
酶热稳定性的测定方法：40μL纯酶液与10μL终浓度50mM pH 6.6柠檬酸缓冲液混匀，在58-70℃条件下分别保温30min，迅速冷却至4℃，加入50μL终浓度3％普鲁兰多糖于65℃反应15min，加入100μL DNS反应液，沸水浴6min，流水速冷至室温，加蒸馏水稀释，混匀，测定OD476，计算普鲁兰酶剩余酶活。
结果表明：野生型的普鲁兰酶65℃保温30min残余酶活约60％，突变体K419R/T245C/A326C/W651C/V707C在65℃保温30min剩余酶活达83.47U/mg，为保温前的85％，因此，突变体K419R/T245C/A326C/W651C/V707C的剩余酶活明显高于野生型，具有更好的热稳定性。
表6普鲁兰酶热稳定性测定
(3)酶的最适pH
酶最适pH的测定方法：50μL终浓度3％普鲁兰多糖与10μL终浓度50mM pH 3.8-7.8柠檬酸缓冲液混匀，在65℃条件下保温5min，加入40μL纯酶液反应15min，迅速冷却至4℃，加入100μL DNS反应液，沸水浴6min，流水速冷至室温，加蒸馏水稀释，混匀，测定OD476，计算普鲁兰酶酶活。
结果表明：野生型普鲁兰酶的最适pH为6.6、突变体K419R的最适pH为6.2，突变体K419R/T245C/A326C的最适pH为6.2，突变体K419R/T245C/A326C/W651C/V707C的最适p H为6.2，有向酸性范围偏移的趋势。
(4)酶pH耐受性
酶pH耐受性的测定方法：40μL纯酶液与10μL终浓度50mM pH 3.8-7.8柠檬酸缓冲液混匀，4℃条件下处理4h，加入50μL终浓度3％普鲁兰多糖65℃反应15min，迅速冷却至4℃，加入100μL DNS反应液，沸水浴6min，流水速冷至室温，加蒸馏水稀释，混匀，测定OD476，计算普鲁兰酶剩余酶活。
结果表明：突变体K419R/T245C/A326C/W651C/V707C对碱性环境的耐受性优于野生型普鲁兰酶。
表7普鲁兰酶pH耐受性测定
(5)酶动力学参数
酶动力学参数的测定方法：50μL终浓度1.25，2.5，5，10，20，40mg/mL普鲁兰多糖分别与10μL终浓度50mM柠檬酸缓冲液混匀，在65℃条件下保温5min，加入40μL已知浓度的纯酶液反应15min，迅速冷却至4℃，加入100μL DNS反应液，沸水浴6min，流水速冷至室温，加蒸馏水稀释，混匀，测定OD476，计算酶动力学参数。
表8野生型和突变体普鲁兰酶动力学参数
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。
SEQUENCE LISTING
<110> 江南大学
<120> 一种厌氧芽孢杆菌来源普鲁兰酶突变体及其应用
<160> 13
<170> PatentIn version 3.3
<210> 1
<211> 711
<212> PRT
<213> Anoxybacillus sp.WB42
<400> 1
Met Leu Thr Val His Arg Thr Phe Glu Ala Tyr Leu Asp Thr Met Glu
1 5 10 15
Thr Ile Thr Ile Leu Val Pro Lys Ser Tyr Cys Gln Gly Ile Val Arg
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Ser Phe Thr Ile Gln Thr Pro Asn Gly Glu Arg Arg Ala Leu Gln Ile
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Thr Lys Arg Glu Asp Leu Trp Thr Ser Ile Lys Tyr Glu Cys Ile Pro
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Asp Val Pro Val Glu Ile Gly Lys Ser Tyr Phe Ile Tyr Glu Glu His
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Gly Ala Phe Thr Asp Leu Gln Met Gly Ala Val Ile Arg Thr Glu Ala
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Phe Asp Glu Gln Phe Tyr Tyr Asp Gly Asp Leu Gly Ile Thr Tyr Ser
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Asn Asp Ala Thr Thr Phe Lys Leu Trp Ala Pro Thr Ala Thr Glu Val
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Lys Leu Lys Leu Ile Asn Lys Ala Gly Lys Glu Glu Gln Ile Ser Met
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Gln Arg Gly Glu Lys Gly Val Trp Cys Ala Thr Val Leu Gly Asn Leu
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Asp Gly Ile Tyr Tyr Thr Tyr Leu Ala Cys Ile Asn Leu Val Trp Arg
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Glu Ala Val Asp Pro Tyr Ala Val Ala Val Ser Val Asn Gly Glu Tyr
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Gly Val Val Val Asp Leu Ala Lys Thr Arg Val Pro Lys Pro Ala Leu
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Pro Pro Leu Thr Ala Pro Val Asp Ala Ile Ile Tyr Glu Met His Val
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Arg Asp Phe Thr Ile Asp Pro His Ser Gly Val Val His Lys Gly Lys
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Tyr Leu Gly Leu Thr Glu Phe Pro Thr Thr Glu Pro Ser Gly Thr Val
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Thr Gly Leu Ala Tyr Leu Lys Gln Leu Gly Val Thr His Val Glu Leu
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Leu Pro Val Asn Asp Phe Ala Gly Val Asp Glu Arg Glu Pro Glu Lys
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Glu Tyr Asn Trp Gly Tyr Asn Pro Leu His Tyr Asn Ala Pro Glu Gly
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Ser Tyr Ala Thr Asp Pro Tyr Asp Pro Tyr Ala Arg Ile His Glu Leu
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Lys Arg Ala Ile Arg Ala Leu Gln Gln Glu Gly Ile Arg Val Ile Leu
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Asp Val Val Tyr Asn His Val Tyr Ile Arg Glu Gln Ser Ser Leu Glu
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Lys Leu Val Pro Gly Tyr Tyr Phe Arg His Asp Ile Tyr Gly Met Pro
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Ser Asn Gly Thr Gly Val Gly Asn Asp Leu Ala Pro Glu Arg Lys Met
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Val Arg Lys Leu Ile Val Asp Ser Val Arg Phe Trp Leu Thr Glu Tyr
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Gly Ile Asp Gly Phe Arg Phe Asp Leu Met Gly Ile Leu Asp Ile Asp
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Thr Met Lys Glu Val Glu Ala Val Val Arg Ala Leu His Pro Ser Ala
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Leu Leu Leu Gly Glu Gly Trp Asp Leu Pro Thr Pro Leu Pro Ser Glu
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Phe Asn Asp Arg Phe Arg Asp Tyr Val Lys Gly Ser Thr Phe His Leu
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Lys Arg Val Ile Glu Gly Ser His His Leu Phe Ser Gln Pro Thr Gln
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Thr Val Asn Tyr Val Glu Ser His Asp Asn His Thr Leu Trp Asp Lys
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Met Ser Ile Ala Asn Tyr Tyr Glu Arg Glu Thr Ile Arg Lys Lys Arg
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Gln Lys Leu Ala Thr Ala Met Thr Leu Leu Ala Gln Gly Ile Pro Phe
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Leu His Ser Gly Gln Glu Phe Tyr Arg Thr Lys Gln Gly Val Glu Asn
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Ser Tyr Asn Ala Pro Asp Asp Ile Asn Arg Ile Asp Trp Thr Arg Lys
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Ser Met His Glu Gln Asp Val Arg Tyr Val Gln Gly Leu Ile Arg Leu
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Arg Lys Trp His Gly Ala Phe Arg Phe Gln Thr Val Glu Glu Ile Arg
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Asn His Leu Val Trp Leu Glu Pro Met Pro Ser Thr Val Leu Ala Phe
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His Leu Tyr Asp Val Ser Ala Tyr Gly Pro Trp Arg Asp Ile Ile Val
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Ile His His Asn Glu Glu Thr Arg Leu Ala Val Ala Leu Pro Asp Glu
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Glu Arg Trp Tyr Val Val Cys Asp Glu Thr Arg Ser Gly Ile Asp Pro
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Leu Tyr Ala Ala Thr Lys Lys Ile Glu Leu Gln Gly Ile Gly Thr Val
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Val Leu Val Lys Gly Leu Thr
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<210> 2
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<212> DNA
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atgctaacgg ttcatcggac gtttgaagca tatttagata caatggaaac gattacgatt 60
ttagtaccga aatcgtattg ccaagggata gtgcgctcat ttaccatcca aacgccaaac 120
ggagaacgac gcgcactaca aattacgaaa cgcgaagatt tatggacgag cattaaatat 180
gagtgcatcc ccgatgttcc ggtggaaatc ggaaaaagtt atttcattta tgaagaacat 240
ggggcgttta ccgatttgca aatgggagcg gttattcgca ccgaagcatt tgatgagcag 300
ttttattatg acggtgacct tggcattacg tacagcaacg atgccacaac gtttaaactt 360
tgggcaccga cagcaacaga ggtaaaacta aagctcatta acaaagcagg aaaagaagaa 420
caaatctcga tgcagcgcgg agaaaaaggg gtatggtgcg cgacagtact cggtaattta 480
gatgggatat actatacgta tttagcgtgt attaaccttg tttggcgaga agcggtcgac 540
ccgtatgctg tagcggtatc ggtgaatgga gaatacgggg tcgtcgtcga tctcgccaaa 600
acgcgagtgc ccaaaccagc gctacctccg cttactgccc cagtcgatgc cattatttat 660
gaaatgcacg ttcgtgattt tacaattgat cctcatagcg gcgttgtcca taaaggaaag 720
tatctaggac tgactgaatt tccaacaaca gaaccgtctg gaacagtgac ggggcttgct 780
tatttgaaac aacttggggt tacacacgta gagctgcttc cggtgaacga ttttgccggg 840
gtcgatgaac gtgaaccgga aaaagagtac aactgggggt acaatccgct tcactacaac 900
gcgccggaag gaagttatgc gaccgatcct tacgatccgt atgcgcgtat tcatgaattg 960
aaacgagcaa ttcgcgcttt gcagcaagaa ggtattcgtg tcattctcga tgtcgtttat 1020
aaccacgttt acattcgcga acagtcgtca ctagaaaaac tcgtgcctgg gtattatttt 1080
cgccatgata tttacggtat gccatcgaac gggacagggg tcggaaatga tcttgccccc 1140
gagcgcaaaa tggttcgcaa gctcattgtc gattccgttc gtttctggct tacagagtat 1200
ggcattgatg ggtttcgttt tgatttaatg ggcattctcg atattgacac gatgaaagaa 1260
gtcgaggcag ttgttcgtgc gctccatcca tccgctctat tgctcggcga aggatgggat 1320
ttgccgacac cattgccgtc ggaaaaaaaa gcaacgatgc aaaatgccca ccttctgccg 1380
acgattgcct tttttaacga tcggtttcgc gattatgtca aagggagtac gtttcattta 1440
ggggaacaag gatttgttct aggaaacagc gcacatcgcg aacaagtgaa acgagtaatc 1500
gaaggaagcc atcatttgtt ttctcaacca acgcaaacgg ttaactacgt cgaatcacac 1560
gacaaccata cgctttggga caaaatgagc atcgccaact attacgagcg agaaaccatt 1620
cgtaaaaaac gacaaaaatt agcgacagcg atgaccttat tagcacaagg cattccattt 1680
ttgcatagcg gccaagaatt ttaccgtaca aaacaaggag tagaaaatag ttataacgct 1740
ccagatgaca ttaaccgcat cgattggacg agaaaaagca tgcacgaaca agacgtccgt 1800
tacgtgcaag gattgattcg gctgcggaaa tggcacggtg cttttcgttt tcaaacagtg 1860
gaagaaataa gaaaccatct tgtatggctt gaaccgatgc cgtcgacagt gctcgctttt 1920
catctttacg acgtatcagc gtatgggccg tggcgtgata ttattgtcat tcaccataat 1980
gaagaaacac ggctagcagt tgcgctccct gatgaagaaa gatggtatgt cgtatgtgat 2040
gaaacaagga gtggaatcga tcctctttac gcggcgacaa aaaaaatcga gctgcaagga 2100
attggaacag tcgtgcttgt gaaaggactg acttaa 2136
<210> 3
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<212> PRT
<213> 人工合成
<400> 3
Met Leu Thr Val His Arg Thr Phe Glu Ala Tyr Leu Asp Thr Met Glu
1 5 10 15
Thr Ile Thr Ile Leu Val Pro Lys Ser Tyr Cys Gln Gly Ile Val Arg
20 25 30
Ser Phe Thr Ile Gln Thr Pro Asn Gly Glu Arg Arg Ala Leu Gln Ile
35 40 45
Thr Lys Arg Glu Asp Leu Trp Thr Ser Ile Lys Tyr Glu Cys Ile Pro
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Asp Val Pro Val Glu Ile Gly Lys Ser Tyr Phe Ile Tyr Glu Glu His
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Gly Ala Phe Thr Asp Leu Gln Met Gly Ala Val Ile Arg Thr Glu Ala
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Phe Asp Glu Gln Phe Tyr Tyr Asp Gly Asp Leu Gly Ile Thr Tyr Ser
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Asn Asp Ala Thr Thr Phe Lys Leu Trp Ala Pro Thr Ala Thr Glu Val
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Lys Leu Lys Leu Ile Asn Lys Ala Gly Lys Glu Glu Gln Ile Ser Met
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Gln Arg Gly Glu Lys Gly Val Trp Cys Ala Thr Val Leu Gly Asn Leu
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Asp Gly Ile Tyr Tyr Thr Tyr Leu Ala Cys Ile Asn Leu Val Trp Arg
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Glu Ala Val Asp Pro Tyr Ala Val Ala Val Ser Val Asn Gly Glu Tyr
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Gly Val Val Val Asp Leu Ala Lys Thr Arg Val Pro Lys Pro Ala Leu
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Pro Pro Leu Thr Ala Pro Val Asp Ala Ile Ile Tyr Glu Met His Val
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Arg Asp Phe Thr Ile Asp Pro His Ser Gly Val Val His Lys Gly Lys
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Tyr Leu Gly Leu Thr Glu Phe Pro Thr Thr Glu Pro Ser Gly Thr Val
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Thr Gly Leu Ala Tyr Leu Lys Gln Leu Gly Val Thr His Val Glu Leu
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Leu Pro Val Asn Asp Phe Ala Gly Val Asp Glu Arg Glu Pro Glu Lys
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Glu Tyr Asn Trp Gly Tyr Asn Pro Leu His Tyr Asn Ala Pro Glu Gly
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Ser Tyr Ala Thr Asp Pro Tyr Asp Pro Tyr Ala Arg Ile His Glu Leu
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Lys Arg Ala Ile Arg Ala Leu Gln Gln Glu Gly Ile Arg Val Ile Leu
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Asp Val Val Tyr Asn His Val Tyr Ile Arg Glu Gln Ser Ser Leu Glu
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Lys Leu Val Pro Gly Tyr Tyr Phe Arg His Asp Ile Tyr Gly Met Pro
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Ser Asn Gly Thr Gly Val Gly Asn Asp Leu Ala Pro Glu Arg Lys Met
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Val Arg Lys Leu Ile Val Asp Ser Val Arg Phe Trp Leu Thr Glu Tyr
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Gly Ile Asp Gly Phe Arg Phe Asp Leu Met Gly Ile Leu Asp Ile Asp
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Thr Met Arg Glu Val Glu Ala Val Val Arg Ala Leu His Pro Ser Ala
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Leu Leu Leu Gly Glu Gly Trp Asp Leu Pro Thr Pro Leu Pro Ser Glu
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Lys Lys Ala Thr Met Gln Asn Ala His Leu Leu Pro Thr Ile Ala Phe
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Phe Asn Asp Arg Phe Arg Asp Tyr Val Lys Gly Ser Thr Phe His Leu
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Gly Glu Gln Gly Phe Val Leu Gly Asn Ser Ala His Arg Glu Gln Val
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Lys Arg Val Ile Glu Gly Ser His His Leu Phe Ser Gln Pro Thr Gln
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Thr Val Asn Tyr Val Glu Ser His Asp Asn His Thr Leu Trp Asp Lys
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Met Ser Ile Ala Asn Tyr Tyr Glu Arg Glu Thr Ile Arg Lys Lys Arg
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Gln Lys Leu Ala Thr Ala Met Thr Leu Leu Ala Gln Gly Ile Pro Phe
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Leu His Ser Gly Gln Glu Phe Tyr Arg Thr Lys Gln Gly Val Glu Asn
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Ser Tyr Asn Ala Pro Asp Asp Ile Asn Arg Ile Asp Trp Thr Arg Lys
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Ser Met His Glu Gln Asp Val Arg Tyr Val Gln Gly Leu Ile Arg Leu
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Arg Lys Trp His Gly Ala Phe Arg Phe Gln Thr Val Glu Glu Ile Arg
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Asn His Leu Val Trp Leu Glu Pro Met Pro Ser Thr Val Leu Ala Phe
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His Leu Tyr Asp Val Ser Ala Tyr Gly Pro Trp Arg Asp Ile Ile Val
645 650 655
Ile His His Asn Glu Glu Thr Arg Leu Ala Val Ala Leu Pro Asp Glu
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Glu Arg Trp Tyr Val Val Cys Asp Glu Thr Arg Ser Gly Ile Asp Pro
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Leu Tyr Ala Ala Thr Lys Lys Ile Glu Leu Gln Gly Ile Gly Thr Val
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Val Leu Val Lys Gly Leu Thr
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<210> 4
<211> 711
<212> PRT
<213> 人工合成
<400> 4
Met Leu Thr Val His Arg Thr Phe Glu Ala Tyr Leu Asp Thr Met Glu
1 5 10 15
Thr Ile Thr Ile Leu Val Pro Lys Ser Tyr Cys Gln Gly Ile Val Arg
20 25 30
Ser Phe Thr Ile Gln Thr Pro Asn Gly Glu Arg Arg Ala Leu Gln Ile
35 40 45
Thr Lys Arg Glu Asp Leu Trp Thr Ser Ile Lys Tyr Glu Cys Ile Pro
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Asp Val Pro Val Glu Ile Gly Lys Ser Tyr Phe Ile Tyr Glu Glu His
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Gly Ala Phe Thr Asp Leu Gln Met Gly Ala Val Ile Arg Thr Glu Ala
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Phe Asp Glu Gln Phe Tyr Tyr Asp Gly Asp Leu Gly Ile Thr Tyr Ser
100 105 110
Asn Asp Ala Thr Thr Phe Lys Leu Trp Ala Pro Thr Ala Thr Glu Val
115 120 125
Lys Leu Lys Leu Ile Asn Lys Ala Gly Lys Glu Glu Gln Ile Ser Met
130 135 140
Gln Arg Gly Glu Lys Gly Val Trp Cys Ala Thr Val Leu Gly Asn Leu
145 150 155 160
Asp Gly Ile Tyr Tyr Thr Tyr Leu Ala Cys Ile Asn Leu Val Trp Arg
165 170 175
Glu Ala Val Asp Pro Tyr Ala Val Ala Val Ser Val Asn Gly Glu Tyr
180 185 190
Gly Val Val Val Asp Leu Ala Lys Thr Arg Val Pro Lys Pro Ala Leu
195 200 205
Pro Pro Leu Thr Ala Pro Val Asp Ala Ile Ile Tyr Glu Met His Val
210 215 220
Arg Asp Phe Thr Ile Asp Pro His Ser Gly Val Val His Lys Gly Lys
225 230 235 240
Tyr Leu Gly Leu Cys Glu Phe Pro Thr Thr Glu Pro Ser Gly Thr Val
245 250 255
Thr Gly Leu Ala Tyr Leu Lys Gln Leu Gly Val Thr His Val Glu Leu
260 265 270
Leu Pro Val Asn Asp Phe Ala Gly Val Asp Glu Arg Glu Pro Glu Lys
275 280 285
Glu Tyr Asn Trp Gly Tyr Asn Pro Leu His Tyr Asn Ala Pro Glu Gly
290 295 300
Ser Tyr Ala Thr Asp Pro Tyr Asp Pro Tyr Ala Arg Ile His Glu Leu
305 310 315 320
Lys Arg Ala Ile Arg Cys Leu Gln Gln Glu Gly Ile Arg Val Ile Leu
325 330 335
Asp Val Val Tyr Asn His Val Tyr Ile Arg Glu Gln Ser Ser Leu Glu
340 345 350
Lys Leu Val Pro Gly Tyr Tyr Phe Arg His Asp Ile Tyr Gly Met Pro
355 360 365
Ser Asn Gly Thr Gly Val Gly Asn Asp Leu Ala Pro Glu Arg Lys Met
370 375 380
Val Arg Lys Leu Ile Val Asp Ser Val Arg Phe Trp Leu Thr Glu Tyr
385 390 395 400
Gly Ile Asp Gly Phe Arg Phe Asp Leu Met Gly Ile Leu Asp Ile Asp
405 410 415
Thr Met Arg Glu Val Glu Ala Val Val Arg Ala Leu His Pro Ser Ala
420 425 430
Leu Leu Leu Gly Glu Gly Trp Asp Leu Pro Thr Pro Leu Pro Ser Glu
435 440 445
Lys Lys Ala Thr Met Gln Asn Ala His Leu Leu Pro Thr Ile Ala Phe
450 455 460
Phe Asn Asp Arg Phe Arg Asp Tyr Val Lys Gly Ser Thr Phe His Leu
465 470 475 480
Gly Glu Gln Gly Phe Val Leu Gly Asn Ser Ala His Arg Glu Gln Val
485 490 495
Lys Arg Val Ile Glu Gly Ser His His Leu Phe Ser Gln Pro Thr Gln
500 505 510
Thr Val Asn Tyr Val Glu Ser His Asp Asn His Thr Leu Trp Asp Lys
515 520 525
Met Ser Ile Ala Asn Tyr Tyr Glu Arg Glu Thr Ile Arg Lys Lys Arg
530 535 540
Gln Lys Leu Ala Thr Ala Met Thr Leu Leu Ala Gln Gly Ile Pro Phe
545 550 555 560
Leu His Ser Gly Gln Glu Phe Tyr Arg Thr Lys Gln Gly Val Glu Asn
565 570 575
Ser Tyr Asn Ala Pro Asp Asp Ile Asn Arg Ile Asp Trp Thr Arg Lys
580 585 590
Ser Met His Glu Gln Asp Val Arg Tyr Val Gln Gly Leu Ile Arg Leu
595 600 605
Arg Lys Trp His Gly Ala Phe Arg Phe Gln Thr Val Glu Glu Ile Arg
610 615 620
Asn His Leu Val Trp Leu Glu Pro Met Pro Ser Thr Val Leu Ala Phe
625 630 635 640
His Leu Tyr Asp Val Ser Ala Tyr Gly Pro Trp Arg Asp Ile Ile Val
645 650 655
Ile His His Asn Glu Glu Thr Arg Leu Ala Val Ala Leu Pro Asp Glu
660 665 670
Glu Arg Trp Tyr Val Val Cys Asp Glu Thr Arg Ser Gly Ile Asp Pro
675 680 685
Leu Tyr Ala Ala Thr Lys Lys Ile Glu Leu Gln Gly Ile Gly Thr Val
690 695 700
Val Leu Val Lys Gly Leu Thr
705 710
<210> 5
<211> 711
<212> PRT
<213> 人工合成
<400> 5
Met Leu Thr Val His Arg Thr Phe Glu Ala Tyr Leu Asp Thr Met Glu
1 5 10 15
Thr Ile Thr Ile Leu Val Pro Lys Ser Tyr Cys Gln Gly Ile Val Arg
20 25 30
Ser Phe Thr Ile Gln Thr Pro Asn Gly Glu Arg Arg Ala Leu Gln Ile
35 40 45
Thr Lys Arg Glu Asp Leu Trp Thr Ser Ile Lys Tyr Glu Cys Ile Pro
50 55 60
Asp Val Pro Val Glu Ile Gly Lys Ser Tyr Phe Ile Tyr Glu Glu His
65 70 75 80
Gly Ala Phe Thr Asp Leu Gln Met Gly Ala Val Ile Arg Thr Glu Ala
85 90 95
Phe Asp Glu Gln Phe Tyr Tyr Asp Gly Asp Leu Gly Ile Thr Tyr Ser
100 105 110
Asn Asp Ala Thr Thr Phe Lys Leu Trp Ala Pro Thr Ala Thr Glu Val
115 120 125
Lys Leu Lys Leu Ile Asn Lys Ala Gly Lys Glu Glu Gln Ile Ser Met
130 135 140
Gln Arg Gly Glu Lys Gly Val Trp Cys Ala Thr Val Leu Gly Asn Leu
145 150 155 160
Asp Gly Ile Tyr Tyr Thr Tyr Leu Ala Cys Ile Asn Leu Val Trp Arg
165 170 175
Glu Ala Val Asp Pro Tyr Ala Val Ala Val Ser Val Asn Gly Glu Tyr
180 185 190
Gly Val Val Val Asp Leu Ala Lys Thr Arg Val Pro Lys Pro Ala Leu
195 200 205
Pro Pro Leu Thr Ala Pro Val Asp Ala Ile Ile Tyr Glu Met His Val
210 215 220
Arg Asp Phe Thr Ile Asp Pro His Ser Gly Val Val His Lys Gly Lys
225 230 235 240
Tyr Leu Gly Leu Cys Glu Phe Pro Thr Thr Glu Pro Ser Gly Thr Val
245 250 255
Thr Gly Leu Ala Tyr Leu Lys Gln Leu Gly Val Thr His Val Glu Leu
260 265 270
Leu Pro Val Asn Asp Phe Ala Gly Val Asp Glu Arg Glu Pro Glu Lys
275 280 285
Glu Tyr Asn Trp Gly Tyr Asn Pro Leu His Tyr Asn Ala Pro Glu Gly
290 295 300
Ser Tyr Ala Thr Asp Pro Tyr Asp Pro Tyr Ala Arg Ile His Glu Leu
305 310 315 320
Lys Arg Ala Ile Arg Cys Leu Gln Gln Glu Gly Ile Arg Val Ile Leu
325 330 335
Asp Val Val Tyr Asn His Val Tyr Ile Arg Glu Gln Ser Ser Leu Glu
340 345 350
Lys Leu Val Pro Gly Tyr Tyr Phe Arg His Asp Ile Tyr Gly Met Pro
355 360 365
Ser Asn Gly Thr Gly Val Gly Asn Asp Leu Ala Pro Glu Arg Lys Met
370 375 380
Val Arg Lys Leu Ile Val Asp Ser Val Arg Phe Trp Leu Thr Glu Tyr
385 390 395 400
Gly Ile Asp Gly Phe Arg Phe Asp Leu Met Gly Ile Leu Asp Ile Asp
405 410 415
Thr Met Arg Glu Val Glu Ala Val Val Arg Ala Leu His Pro Ser Ala
420 425 430
Leu Leu Leu Gly Glu Gly Trp Asp Leu Pro Thr Pro Leu Pro Ser Glu
435 440 445
Lys Lys Ala Thr Met Gln Asn Ala His Leu Leu Pro Thr Ile Ala Phe
450 455 460
Phe Asn Asp Arg Phe Arg Asp Tyr Val Lys Gly Ser Thr Phe His Leu
465 470 475 480
Gly Glu Gln Gly Phe Val Leu Gly Asn Ser Ala His Arg Glu Gln Val
485 490 495
Lys Arg Val Ile Glu Gly Ser His His Leu Phe Ser Gln Pro Thr Gln
500 505 510
Thr Val Asn Tyr Val Glu Ser His Asp Asn His Thr Leu Trp Asp Lys
515 520 525
Met Ser Ile Ala Asn Tyr Tyr Glu Arg Glu Thr Ile Arg Lys Lys Arg
530 535 540
Gln Lys Leu Ala Thr Ala Met Thr Leu Leu Ala Gln Gly Ile Pro Phe
545 550 555 560
Leu His Ser Gly Gln Glu Phe Tyr Arg Thr Lys Gln Gly Val Glu Asn
565 570 575
Ser Tyr Asn Ala Pro Asp Asp Ile Asn Arg Ile Asp Trp Thr Arg Lys
580 585 590
Ser Met His Glu Gln Asp Val Arg Tyr Val Gln Gly Leu Ile Arg Leu
595 600 605
Arg Lys Trp His Gly Ala Phe Arg Phe Gln Thr Val Glu Glu Ile Arg
610 615 620
Asn His Leu Val Trp Leu Glu Pro Met Pro Ser Thr Val Leu Ala Phe
625 630 635 640
His Leu Tyr Asp Val Ser Ala Tyr Gly Pro Cys Arg Asp Ile Ile Val
645 650 655
Ile His His Asn Glu Glu Thr Arg Leu Ala Val Ala Leu Pro Asp Glu
660 665 670
Glu Arg Trp Tyr Val Val Cys Asp Glu Thr Arg Ser Gly Ile Asp Pro
675 680 685
Leu Tyr Ala Ala Thr Lys Lys Ile Glu Leu Gln Gly Ile Gly Thr Val
690 695 700
Val Leu Cys Lys Gly Leu Thr
705 710
<210> 6
<211> 35
<212> DNA
<213> 人工合成
<400> 6
ggaattccat atgctaacgg ttcatcggac gtttg 35
<210> 7
<211> 33
<212> DNA
<213> 人工合成
<400> 7
ccgctcgaga gtcagtcctt tcacaagcac gac 33
<210> 8
<211> 32
<212> DNA
<213> 人工合成
<400> 8
gcattctcga tattgacacg atgcgtgaag tc 32
<210> 9
<211> 29
<212> DNA
<213> 人工合成
<400> 9
acaactgcct cgacttcacg catcgtgtc 29
<210> 10
<211> 30
<212> DNA
<213> 人工合成
<400> 10
ggaaagtatc taggactgtg tgaatttcca 30
<210> 11
<211> 30
<212> DNA
<213> 人工合成
<400> 11
aataccttct tgctgcaaac agcgaattgc 30
<210> 12
<211> 30
<212> DNA
<213> 人工合成
<400> 12
cagcgtatgg gccgtgtcgt gatattattg 30
<210> 13
<211> 30
<212> DNA
<213> 人工合成
<400> 13
ttaagtcagt cctttacaaa gcacgactgt 30

Claims (10)

1.一种普鲁兰酶突变体，其特征在于，所述突变体的氨基酸序列包括：在SEQ ID NO.1所示的氨基酸序列的基础上，将第419位赖氨酸突变为精氨酸后得到的氨基酸序列。
2.根据权利要求1所述的突变体，其特征在于，所述突变体在权利要求1的基础上做了如下改造：将第245位苏氨酸、第326位丙氨酸分别突变为半胱氨酸。
3.根据权利要求2所述的突变体，其特征在于，所述突变体在权利要求2的基础上做了如下改造：将第651位色氨酸、第707位缬氨酸分别突变为半胱氨酸。
4.编码权利要求1～3任一所述突变体的基因。
5.携带权利要求4所述基因的载体或细胞。
6.表达权利要求1～3任一所述突变体的基因工程菌。
7.根据权利要求6所述的基因工程菌，其特征在于，以大肠杆菌为宿主。
8.根据权利要求6所述的基因工程菌，其特征在于，以pET-22b(+)为表达载体。
9.一种切割底物中α-1,6-糖苷键的方法，其特征在于，以权利要求1～3任一所述突变体或含有所述突变体的全细胞为催化剂，切割底物中的α-1,6-糖苷键。
10.权利要求1～3任一所述的突变体或权利要求6所述的基因工程菌在医药生产、化工或食品领域的应用。
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