CN111560360A - 环糊精葡萄糖基转移酶突变体及其应用 - Google Patents
环糊精葡萄糖基转移酶突变体及其应用 Download PDFInfo
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- CN111560360A CN111560360A CN202010377685.7A CN202010377685A CN111560360A CN 111560360 A CN111560360 A CN 111560360A CN 202010377685 A CN202010377685 A CN 202010377685A CN 111560360 A CN111560360 A CN 111560360A
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- ala
- cyclodextrin glucosyltransferase
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Abstract
本发明公开了一种环糊精葡萄糖基转移酶突变体及其应用,属于基因工程和酶工程领域。本发明通过对环糊精葡萄糖基转移酶进行突变,得到了环糊精葡萄糖基转移酶歧化活力更高的突变体。突变体V6D/S90G/T168A/T171A/T383A/G608A的摇瓶发酵酶歧化活力为野生酶的3.16倍。本发明对环糊精葡萄糖基转移酶工业化生产具有一定意义,并提高了该酶的在食品、医药、化工行业中的应用潜力。
Description
技术领域
本发明涉及环糊精葡萄糖基转移酶突变体及其应用,属于基因工程和酶工程领域。
背景技术
环糊精葡萄糖基转移酶(Cyclodextrin Glycosyltransferase,简称CGTase,EC2.4.1.19)是α-淀粉酶家族(GH13)中的主要成员。它作为一种多功能酶,可催化四种不同的反应,包括三种转糖基(transfructosylation,又称转糖苷)反应-歧化反应、环化反应和偶合反应以及一种水解反应。其中,歧化反应占据主导地位,该反应是将直链低聚糖被切断的部分转移到另一受体上,是一种分子外转糖基反应;环化反应作为一种分子内转糖基反应,这是CGTase的特征反应,其原理是将直链麦芽低聚糖上非还原端的O4或C4上的糖苷转移到同一直链还原端的C1或O1上。环化反应的逆反应就是偶合反应,它可以打开环糊精的环,将糖苷转移到直链麦芽低聚物上。CGTase同时具备催化环化反应和耦合反应的活性,是导致其在催化淀粉发生反应的过程中,随着时间延长,产物逐渐由α-环糊精向β-环糊精转变的原因。CGTase催化淀粉发生水解反应时,是将直链淀粉分子切断以后,将断开的两端分子都转移到水分子上,完成水解反应;CGTase催化水解反应的活性较弱。
CGTase的应用比较广泛,最常见的就是通过环化反应把淀粉转化为环糊精。根据生成的环糊精种类的不同,可以把CGTase分为α-CGTase、β-CGTase、γ-CGTase,此外,还有报道显示CGTase能催化淀粉生产8个葡萄糖单元以上的环糊精。通过歧化和耦合反应,CGTase可以将转化淀粉或环糊精生成的单糖或低聚糖作为供体提供给各种受体分子,以改善其特性。例如,通过CGTase的歧化和偶合反应将小分子糖转移到蔗糖或果糖上而生产抗龋齿功能偶合糖;对甜菊苷、芸香苷、鼠李糖等物质进行糖基化修饰,显著改善其性能,使之可更加广泛地应用于食品、医药、化工等领域。
Bacillus circulans 251来源的CGTase是一种典型的β-CGTase,其优秀的转糖基性能可被应用多个领域。但是,该CGTase歧化活力(约40U/mL)较其他来源的CGTase低,提高该酶歧化活力势在必行。
发明内容
发明所要解决的一个技术问题是提供一种环糊精葡萄糖基转移酶的突变体,所述突变体是将氨基酸序列如SEQ ID NO.2所示的环糊精葡萄糖基转移酶的一个或多个氨基酸位点进行突变;这些位点包括:第6位的缬氨酸(Val)、90位的丝氨酸(Ser)、168位的苏氨酸(Thr)、171位的苏氨酸(Thr)、383位的苏氨酸(Thr)、608位的甘氨酸(Gly)。所述突变体催化歧化反应的活力有所提高。
在本发明的一种实施方式中,所述来源于环状芽孢杆菌(B.circulans)的环糊精葡萄糖基转移酶的氨基酸序列如SEQ ID NO.2所示。编码所述来源于环状芽孢杆菌(B.circulans)的环糊精葡萄糖基转移酶基因的核苷酸序列如SEQ ID NO.1所示。
在本发明的一种实施方式中,所述突变体是将氨基酸序列如SEQ ID NO.2所示的环糊精葡萄糖基转移酶中第6位的缬氨酸(V)突变为天冬氨酸,命名为V6D。
在本发明的一种实施方式中,所述突变体是将氨基酸序列如SEQ ID NO.2所示的环糊精葡萄糖基转移酶中第90位的丝氨酸(T)突变为甘氨酸,命名为S90G。
在本发明的一种实施方式中,所述突变体是将氨基酸序列如SEQ ID NO.2所示的环糊精葡萄糖基转移酶中第168位的苏氨酸(T)突变为丙氨酸,命名为T168A。
在本发明的一种实施方式中,所述突变体是将氨基酸序列如SEQ ID NO.2所示的环糊精葡萄糖基转移酶中第171位的苏氨酸(T)突变为丙氨酸,命名为T171A。
在本发明的一种实施方式中,所述突变体是将氨基酸序列如SEQ ID NO.2所示的环糊精葡萄糖基转移酶中第383位的苏氨酸(T)突变为丙氨酸,命名为T383A。
在本发明的一种实施方式中,所述突变体是将氨基酸序列如SEQ ID NO.2所示的环糊精葡萄糖基转移酶中第608位的甘氨酸(G)突变为丙氨酸,命名为G608A。
在本发明的一种实施方式中,所述突变体是将氨基酸序列如SEQ ID NO.2所示的环糊精葡萄糖基转移酶中第228位的第6位的缬氨酸(V)突变为天冬氨酸,同时将第90位的丝氨酸(T)突变为甘氨酸,第168位的苏氨酸(T)突变为丙氨酸,第171位的苏氨酸(T)突变为丙氨酸,第383位的苏氨酸(T)突变为丙氨酸,第608位的甘氨酸(G)突变为丙氨酸,命名为V6D/S90G/T168A/T171A/T383A/G608A。
本发明所要解决的另一个技术问题是提供一种环糊精葡萄糖基转移酶的突变体的制备方法,包括如下步骤:
(1)在Bacillus circulans 251来源的环糊精葡萄糖基转移酶氨基酸序列的基础上确定突变位点;设计定点突变的突变引物,以携带环糊精葡萄糖基转移酶基因的载体为模板进行定点突变;构建含突变体的质粒载体;
(2)将突变体质粒转化进宿主细胞;
(3)挑选阳性克隆进行发酵培养,并纯化环糊精葡萄糖基转移酶突变体。
在本发明的一种实施方式中,所述质粒载体为pUC系列,pET系列,或pGEX中的任意一种。
在本发明的一种实施方式中,所述宿主细胞为细菌和真菌细胞,其也为本发明的保护范围。
在本发明的一种实施方式中,所述宿主细胞为枯草芽孢杆菌、大肠杆菌或短小芽孢杆菌。
在本发明的一种实施方式中,所述的细菌为革兰氏阴性菌或革兰氏阳性菌。
本发明的有益效果:
本发明得到了一种环糊精葡萄糖基转移酶突变体,突变体V6D、S90G、T168A、T171A、T383A、G608A和V6D/S90G/T168A/T171A/T383A/G608A的摇瓶发酵酶歧化活力分别为野生酶的1.89倍、1.21倍、1.21倍、1.22倍、1.32倍、2.03倍、3.16倍。本发明对环糊精葡萄糖基转移酶工业化生产具有一定意义,并提高了该酶的在食品、医药、化工行业中的应用潜力。
具体实施方式
本发明的实施例仅作为本发明内容的进一步说明,不能作为本发明的限定内容或范围。
下述实施例中涉及的培养基和检测方法如下:
LB培养基(g·L-1):胰蛋白胨10,酵母粉5,氯化钠10。
TB培养基(g·L-1):胰蛋白胨12,酵母粉24,甘油5,KH2PO4 2.31,K2HPO4·3H2O16.43,甘氨酸7.5。
环糊精葡萄糖基转移酶催化歧化反应的活力的测定方法:以50mmol/L pH 5.5的磷酸盐缓冲液为溶剂,分别配置12mM的EPS(4,6-亚乙基-对硝基苯-α-D-麦芽七糖苷)和20mM麦芽糖溶液,各取300μL的12mM EPS和20mM麦芽糖溶液置于50℃水浴锅中预热,加入100μL稀释后的酶液,精确反应10min后,立刻煮沸10min终止反应,加入30μL稀释后的粗酶液,精确反应75min,于沸水中煮沸10min,冷却,加入100μLα-葡萄糖苷酶和100μL去离子水,混匀,于60℃水浴锅反应60min以上,加入100μL 1M Na2CO3溶液,混匀,最后于400nm测定吸光值。环糊精葡萄糖基转移酶催化歧化反应的活力定义为每分钟转化一微摩尔EPS的酶量。(歧化活力测定方法参见van der Veen B A,Leemhuis H,Kralj S,et al.Hydrophobicamino acid residues in the acceptor binding site are main determinants forreaction mechanism and specificity of cyclodextrin-glycosyltransferase[J].Journal of Biological Chemistry,2001,276(48):44557-44562)
实施例1:野生型环糊精葡萄糖基转移酶的表达
从实验室前期保藏的甘油管中,接种Cgt/pET20b(+)/BL21(DE3)(杨玉路,王蕾,陈晟,等.重组β-环糊精葡萄糖基转移酶生产β-环糊精的工艺条件优化[J].生物技术通报,2014,8:175-181.)于LB液体培养基(含100mg/L氨苄青霉素)生长8h,按5%接种量将种子液接入TB液体发酵培养基(含100mg/L氨苄青霉素)。大肠杆菌在25℃摇床培养发酵48h后,将一定体积发酵液于4℃、12000rpm离心15min,取发酵上清,即为野生酶的粗酶液。
实施例2:环糊精葡萄糖基转移酶单突变体的制备及表达
(1)环糊精葡萄糖基转移酶的单突变制备
根据Bacillus circulans环糊精葡萄糖基转移酶的基因序列,分别设计并合成引入单突变的引物,对环糊精葡萄糖基转移酶基因Cgt进行定点突变,分别测序确认环糊精葡萄糖基转移酶突变体的编码基因是否正确;将携带突变体基因的载体导入大肠杆菌中进行表达,得到单突变环糊精葡萄糖基转移酶。
定点突变体编码基因的PCR扩增:利用快速PCR技术,以携带编码野生型环糊精葡萄糖基转移酶的基因的表达载体Cgt/pET-20b(+)为模板。
引入V6D突变的定点突变引物为:
核苷酸序列如SEQ ID NO.3所示的正向引物:
5’-CCGGATACCAGCGATAGCAACAAGCAG-3’(下划线为突变碱基)
核苷酸序列如SEQ ID NO.4所示的反向引物:
5’-CTGCTTGTTGCTATCGCTGGTATCCGG-3’(下划线为突变碱基)
引入S90G突变的定点突变引物为:
核苷酸序列如SEQ ID NO.5所示的正向引物:
5’-CTATAGCATTATCAACTACGGCGGTGTGAATAATACGG-3’(下划线为突变碱基)
核苷酸序列如SEQ ID NO.6所示的反向引物:
5’-CCGTATTATTCACACCGCCGTAGTTGATAATGCTATAG-3’(下划线为突变碱基)
引入T168A突变的定点突变引物为:
核苷酸序列如SEQ ID NO.7所示的正向引物:
5’-CTGGGCGGTTATGCCAATGACACCC-3’(下划线为突变碱基)
核苷酸序列如SEQ ID NO.8所示的反向引物:
5’-CTGGGCGGTTATGCCAATGACACCC-3’(下划线为突变碱基)
引入T171A突变的定点突变引物为:
核苷酸序列如SEQ ID NO.9所示的正向引物:
5’-CGGTTATACCAATGACGCCCAAAATCTGTTTC-3’(下划线为突变碱基)
核苷酸序列如SEQ ID NO.10所示的反向引物:
5’-GAAACAGATTTTGGGCGTCATTGGTATAACCG-3’(下划线为突变碱基)
引入T383A突变的定点突变引物为:
核苷酸序列如SEQ ID NO.11所示的正向引物:
5’-CCAAGTTTTAGCGCGAGCACGACGG-3’(下划线为突变碱基)
核苷酸序列如SEQ ID NO.12所示的反向引物:
5’-CCGTCGTGCTCGCGCTAAAACTTGG-3’(下划线为突变碱基)
引入G608A突变的定点突变引物为:
核苷酸序列如SEQ ID NO.13所示的正向引物:
5’-CAAAATGTGTATCTGACGGCCAGCGTGAGCGAACTGGG-3’(下划线为突变碱基)
核苷酸序列如SEQ ID NO.14所示的反向引物:
5’-CCCAGTTCGCTCACGCTGGCCGTCAGATACACATTTTG-3’(下划线为突变碱基)
PCR反应体系均为:20μM正向引物和反向引物各0.5μL,dNTPs Mix 4μL,5×PSBuffer10μL,2.5U/μL的PrimeStar聚合酶0.5μL,模板0.5μL,加双蒸水补齐50μL。
PCR条件为:94℃预变性4min;随后进行25个循环(94℃10s,55℃5s,72℃7min50s)72℃延伸10min;最后4℃保温。PCR产物用1%琼脂糖凝胶电泳进行检测。
将上述验证正确的PCR产物进行Dpn I消化,转入大肠杆菌JM 109感受态细胞,转化产物涂布于含100mg/L氨苄青霉素的LB平板上,经37℃过夜培养,平板上挑取2个单菌落,接入LB液体培养基,8h后提取质粒并测序,结果正确。将测序正确的质粒转入大肠杆菌BL21(DE3)得到表达单突变体的重组大肠杆菌。
(2)突变体的表达
将本实施例步骤(1)制备得到的表达单突变体的重组大肠杆菌分别接种于LB液体培养基(含100mg/L氨苄青霉素),培养生长8h,按5%接种量将种子接入TB液体发酵培养基(含100mg/L氨苄青霉素)。大肠杆菌在25℃摇床培养发酵48h后,将一定体积发酵液于4℃、12000rpm离心15min,取发酵上清,即为单突变体的粗酶液。
实施例3:环糊精葡萄糖基转移酶六突变体的制备及表达
(1)环糊精葡萄糖基转移酶的六突变制备
以实施例2构建的携带编码突变体V6D的基因的质粒作为六突变的模板,并根据实施例2设计的S90G、T168A、T383A、G608A定点突变的引物,使用快速PCR技术,对携带编码突变体V6D的基因的质粒进行定点突变,得到环糊精葡萄糖基转移酶V6D/S90G/T168A/T383A/G608A五突变体。之后,以V6D/S90G/T168A/T383A/G608A五突变体的质粒为模板,设计新的引物,引入T171A突变,构建环糊精葡萄糖基转移酶V6D/S90G/T168A/T171A/T383A/G608A六突变体。
引入T171A突变的定点突变引物变更为:
核苷酸序列如SEQ ID NO.15所示的正向引物:
5’-CGGTTATGCCAATGACGCCCAAAATCTGTTTC-3’(下划线为突变碱基)
核苷酸序列如SEQ ID NO.16所示的反向引物:
5’-GAAACAGATTTTGGGCGTCATTGGCATAACCG-3’(下划线为突变碱基)
(2)突变体的表达
将本实施例步骤(1)制备得到的表达突变体的重组大肠杆菌分别接种于LB液体培养基(含100mg/L氨苄青霉素),培养生长8h,按5%接种量将种子接入TB液体发酵培养基(含100mg/L氨苄青霉素)。大肠杆菌在25℃摇床培养发酵48h后,将一定体积发酵液于4℃、12000rpm离心15min,取发酵上清,即为六突变体的粗酶液。
实施例4:环糊精葡萄糖基转移酶歧化活力分析
将实施例1、实施例2和实施例3所得的发酵上清粗酶液分别进行歧化活力测定。野生型环糊精葡萄糖基转移酶(WT)和突变体瓶培养48h的OD600nm及酶歧化活力列于表1,结果表明,所有突变体的酶歧化活力均高于野生型。突变体V6D、S90G、T168A、T171A、T383A、G608A和V6D/S90G/T168A/T171A/T383A/G608A的摇瓶发酵酶歧化活力分别为野生酶的1.89倍、1.21倍、1.21倍、1.22倍、1.32倍、2.03倍、3.16倍。
表1环糊精葡萄糖基转移酶的野生及突变体酶的摇瓶OD600nm和酶歧化活力
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> 环糊精葡萄糖基转移酶突变体及其应用
<160> 16
<170> PatentIn version 3.3
<210> 1
<211> 2061
<212> DNA
<213> Bacillus circulans 251
<400> 1
gcaccggata ccagcgttag caacaagcag aatttcagca cggatgtgat ctatcagatc 60
ttcacggacc gcttcagcga tggtaacccg gcgaacaacc caacgggcgc agcattcgat 120
ggcacctgca ccaatctgcg tctgtactgt ggtggtgact ggcagggcat catcaacaag 180
atcaacgatg gttacctgac cggtatgggt gttacggcaa tctggatcag ccaaccagtg 240
gaaaatatct atagcattat caactacagc ggtgtgaata atacggcata ccacggctat 300
tgggcccgtg atttcaaaaa aaccaatccg gcgtatggca cgatcgcgga ttttcagaat 360
ctgattgcag cggcacatgc aaaaaacatt aaagtgatta tcgattttgc gccgaatcac 420
accagcccag cgagcagcga tcaaccgagc ttcgcggaaa acggtcgcct gtatgacaat 480
ggtaccctgc tgggcggtta taccaatgac acccaaaatc tgtttcatca caacggtggt 540
accgatttta gcaccaccga gaatggtatt tacaagaacc tgtacgatct ggcggatctg 600
aaccataata atagcacggt tgacgtttat ctgaaagatg cgattaagat gtggctggat 660
ctgggcattg acggcattcg tatggatgcg gttaaacaca tgccattcgg ttggcaaaag 720
agctttatgg ccgcagttaa caattacaag ccggttttca cctttggcga atggttcctg 780
ggcgtgaatg aagtgagccc ggagaaccac aagtttgcga atgagagcgg tatgagcctg 840
ctggacttcc gtttcgcgca gaaagtgcgt caagtttttc gtgataacac ggataatatg 900
tatggcctga aggcgatgct ggaaggtagc gccgcagact atgcgcaagt tgacgatcaa 960
gtgaccttca ttgacaatca cgatatggaa cgcttccatg cgagcaacgc gaatcgtcgc 1020
aagctggaac aagcgctggc gtttaccctg acgagccgcg gtgttccggc gatctactat 1080
ggtacggaac agtatatgag cggtggcacc gacccggaca atcgtgcgcg tatcccaagt 1140
tttagcacga gcacgacggc ctaccaggtg attcagaaac tggcaccact gcgcaaatgt 1200
aacccagcca ttgcgtacgg tagcacgcaa gaacgttgga ttaacaacga cgttctgatc 1260
tacgaacgta aatttggcag caacgttgcc gttgttgcgg tgaaccgtaa cctgaacgca 1320
ccggcaagca tcagcggcct ggtgaccagc ctgccacaag gcagctataa cgatgttctg 1380
ggtggtctgc tgaacggtaa cacgctgagc gttggtagcg gcggtgcagc aagcaatttt 1440
acgctggcag ccggcggcac ggcagtttgg caatatacgg ccgcaaccgc gacgccgacc 1500
attggccatg tgggtccaat gatggcgaag ccaggtgtga ccattacgat tgatggtcgc 1560
ggcttcggca gcagcaaagg caccgtttac tttggtacga ccgccgttag cggtgcggat 1620
attacgagct gggaggatac ccaaatcaaa gttaagatcc cagccgttgc gggtggcaac 1680
tataacatca aggttgcgaa cgcggcaggt accgccagca atgtttacga caatttcgag 1740
gttctgagcg gcgaccaagt tagcgtgcgc tttgtggtga acaatgcaac cacggcgctg 1800
ggtcaaaatg tgtatctgac gggcagcgtg agcgaactgg gtaattggga cccggccaaa 1860
gcgatcggcc cgatgtacaa ccaagtggtg tatcagtatc cgaattggta ctatgatgtg 1920
agcgtgccag ccggtaaaac gatcgagttc aagttcctga agaaacaggg cagcaccgtg 1980
acgtgggaag gtggtagcaa tcatacgttt acggccccaa gcagcggtac ggccacgatt 2040
aacgtgaatt ggcaaccgta a 2061
<210> 2
<211> 686
<212> PRT
<213> Bacillus circulans 251
<400> 2
Ala Pro Asp Thr Ser Val Ser Asn Lys Gln Asn Phe Ser Thr Asp Val
1 5 10 15
Ile Tyr Gln Ile Phe Thr Asp Arg Phe Ser Asp Gly Asn Pro Ala Asn
20 25 30
Asn Pro Thr Gly Ala Ala Phe Asp Gly Thr Cys Thr Asn Leu Arg Leu
35 40 45
Tyr Cys Gly Gly Asp Trp Gln Gly Ile Ile Asn Lys Ile Asn Asp Gly
50 55 60
Tyr Leu Thr Gly Met Gly Val Thr Ala Ile Trp Ile Ser Gln Pro Val
65 70 75 80
Glu Asn Ile Tyr Ser Ile Ile Asn Tyr Ser Gly Val Asn Asn Thr Ala
85 90 95
Tyr His Gly Tyr Trp Ala Arg Asp Phe Lys Lys Thr Asn Pro Ala Tyr
100 105 110
Gly Thr Ile Ala Asp Phe Gln Asn Leu Ile Ala Ala Ala His Ala Lys
115 120 125
Asn Ile Lys Val Ile Ile Asp Phe Ala Pro Asn His Thr Ser Pro Ala
130 135 140
Ser Ser Asp Gln Pro Ser Phe Ala Glu Asn Gly Arg Leu Tyr Asp Asn
145 150 155 160
Gly Thr Leu Leu Gly Gly Tyr Thr Asn Asp Thr Gln Asn Leu Phe His
165 170 175
His Asn Gly Gly Thr Asp Phe Ser Thr Thr Glu Asn Gly Ile Tyr Lys
180 185 190
Asn Leu Tyr Asp Leu Ala Asp Leu Asn His Asn Asn Ser Thr Val Asp
195 200 205
Val Tyr Leu Lys Asp Ala Ile Lys Met Trp Leu Asp Leu Gly Ile Asp
210 215 220
Gly Ile Arg Met Asp Ala Val Lys His Met Pro Phe Gly Trp Gln Lys
225 230 235 240
Ser Phe Met Ala Ala Val Asn Asn Tyr Lys Pro Val Phe Thr Phe Gly
245 250 255
Glu Trp Phe Leu Gly Val Asn Glu Val Ser Pro Glu Asn His Lys Phe
260 265 270
Ala Asn Glu Ser Gly Met Ser Leu Leu Asp Phe Arg Phe Ala Gln Lys
275 280 285
Val Arg Gln Val Phe Arg Asp Asn Thr Asp Asn Met Tyr Gly Leu Lys
290 295 300
Ala Met Leu Glu Gly Ser Ala Ala Asp Tyr Ala Gln Val Asp Asp Gln
305 310 315 320
Val Thr Phe Ile Asp Asn His Asp Met Glu Arg Phe His Ala Ser Asn
325 330 335
Ala Asn Arg Arg Lys Leu Glu Gln Ala Leu Ala Phe Thr Leu Thr Ser
340 345 350
Arg Gly Val Pro Ala Ile Tyr Tyr Gly Thr Glu Gln Tyr Met Ser Gly
355 360 365
Gly Thr Asp Pro Asp Asn Arg Ala Arg Ile Pro Ser Phe Ser Thr Ser
370 375 380
Thr Thr Ala Tyr Gln Val Ile Gln Lys Leu Ala Pro Leu Arg Lys Cys
385 390 395 400
Asn Pro Ala Ile Ala Tyr Gly Ser Thr Gln Glu Arg Trp Ile Asn Asn
405 410 415
Asp Val Leu Ile Tyr Glu Arg Lys Phe Gly Ser Asn Val Ala Val Val
420 425 430
Ala Val Asn Arg Asn Leu Asn Ala Pro Ala Ser Ile Ser Gly Leu Val
435 440 445
Thr Ser Leu Pro Gln Gly Ser Tyr Asn Asp Val Leu Gly Gly Leu Leu
450 455 460
Asn Gly Asn Thr Leu Ser Val Gly Ser Gly Gly Ala Ala Ser Asn Phe
465 470 475 480
Thr Leu Ala Ala Gly Gly Thr Ala Val Trp Gln Tyr Thr Ala Ala Thr
485 490 495
Ala Thr Pro Thr Ile Gly His Val Gly Pro Met Met Ala Lys Pro Gly
500 505 510
Val Thr Ile Thr Ile Asp Gly Arg Gly Phe Gly Ser Ser Lys Gly Thr
515 520 525
Val Tyr Phe Gly Thr Thr Ala Val Ser Gly Ala Asp Ile Thr Ser Trp
530 535 540
Glu Asp Thr Gln Ile Lys Val Lys Ile Pro Ala Val Ala Gly Gly Asn
545 550 555 560
Tyr Asn Ile Lys Val Ala Asn Ala Ala Gly Thr Ala Ser Asn Val Tyr
565 570 575
Asp Asn Phe Glu Val Leu Ser Gly Asp Gln Val Ser Val Arg Phe Val
580 585 590
Val Asn Asn Ala Thr Thr Ala Leu Gly Gln Asn Val Tyr Leu Thr Gly
595 600 605
Ser Val Ser Glu Leu Gly Asn Trp Asp Pro Ala Lys Ala Ile Gly Pro
610 615 620
Met Tyr Asn Gln Val Val Tyr Gln Tyr Pro Asn Trp Tyr Tyr Asp Val
625 630 635 640
Ser Val Pro Ala Gly Lys Thr Ile Glu Phe Lys Phe Leu Lys Lys Gln
645 650 655
Gly Ser Thr Val Thr Trp Glu Gly Gly Ser Asn His Thr Phe Thr Ala
660 665 670
Pro Ser Ser Gly Thr Ala Thr Ile Asn Val Asn Trp Gln Pro
675 680 685
<210> 3
<211> 27
<212> DNA
<213> 人工合成
<400> 3
ccggatacca gcgatagcaa caagcag 27
<210> 4
<211> 27
<212> DNA
<213> 人工合成
<400> 4
ctgcttgttg ctatcgctgg tatccgg 27
<210> 5
<211> 38
<212> DNA
<213> 人工合成
<400> 5
ctatagcatt atcaactacg gcggtgtgaa taatacgg 38
<210> 6
<211> 38
<212> DNA
<213> 人工合成
<400> 6
ccgtattatt cacaccgccg tagttgataa tgctatag 38
<210> 7
<211> 25
<212> DNA
<213> 人工合成
<400> 7
ctgggcggtt atgccaatga caccc 25
<210> 8
<211> 25
<212> DNA
<213> 人工合成
<400> 8
ctgggcggtt atgccaatga caccc 25
<210> 9
<211> 32
<212> DNA
<213> 人工合成
<400> 9
cggttatacc aatgacgccc aaaatctgtt tc 32
<210> 10
<211> 32
<212> DNA
<213> 人工合成
<400> 10
gaaacagatt ttgggcgtca ttggtataac cg 32
<210> 11
<211> 25
<212> DNA
<213> 人工合成
<400> 11
ccaagtttta gcgcgagcac gacgg 25
<210> 12
<211> 25
<212> DNA
<213> 人工合成
<400> 12
ccgtcgtgct cgcgctaaaa cttgg 25
<210> 13
<211> 28
<212> DNA
<213> 人工合成
<400> 13
gttcaagttc ctggagaaac agggcagc 28
<210> 14
<211> 28
<212> DNA
<213> 人工合成
<400> 14
gctgccctgt ttctccagga acttgaac 28
<210> 15
<211> 32
<212> DNA
<213> 人工合成
<400> 15
cggttatgcc aatgacgccc aaaatctgtt tc 32
<210> 16
<211> 32
<212> DNA
<213> 人工合成
<400> 16
gaaacagatt ttgggcgtca ttggcataac cg 32
Claims (8)
1.一种环糊精葡萄糖基转移酶的突变体,其特征在于,将氨基酸序列如SEQ ID NO.2所示的环糊精葡萄糖基转移酶的第6位的缬氨酸突变为天冬氨酸,同时将第90位的丝氨酸突变为甘氨酸,第168位的苏氨酸突变为丙氨酸,第171位的苏氨酸突变为丙氨酸,第383位的苏氨酸突变为丙氨酸,第608位的甘氨酸突变为丙氨酸,命名为V6D/S90G/T168A/T171A/T383A/G608A。
2.表达权利要求1所述突变体的构建方法,其特征在于,包括以下步骤:
(1)根据确定的突变位点,设计定点突变的突变引物,以携带环糊精葡萄糖基转移酶基因的载体为模板进行定点突变;构建含编码突变体的基因的质粒载体;
(2)将突变体质粒转化进宿主细胞;
(3)挑选阳性克隆进行发酵培养,离心上清即为环糊精葡萄糖基转移酶突变体的粗酶液。
3.根据权利要求2所述方法,其特征在于,所述质粒载体为pUC系列,pET系列,或pGEX中的任意一种。
4.根据权利要求2所述方法,其特征在于,所述质粒为cgt/pET20b(+)。
5.根据权利要求2所述方法,其特征在于,所述宿主细胞为细菌或真菌细胞。
6.编码权利要求1所述突变体的基因。
7.携带权利要求6所述基因的质粒或细胞。
8.权利要求1所述突变体在食品、医药、化工领域的应用。
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