CN114836397B - 一种环糊精葡萄糖基转移酶突变体及应用 - Google Patents
一种环糊精葡萄糖基转移酶突变体及应用 Download PDFInfo
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Abstract
本发明提供了一种环糊精葡萄糖基转移酶突变体及应用，属于基因工程和酶工程技术领域，具体为氨基酸突变位点为环糊精葡萄糖基转移酶氨基酸序列SEQ ID NO.2的第33、119、122、216、255、258、394、566位氨基酸之一或两者以上，主要突变体为N33K、N33R、Y119R、Y122E、L216Q、H255Y、E258Y、E258Q、E566H、N33K/Y122E/E258Y/P394R，本发明中突变体的歧化反应活性相较于野生型明显提高，本发明对于环糊精葡萄糖基转移酶工业化生产具有一定意义，并提高该酶在医药、食品、生物行业中的应用前景。
Description
技术领域
本发明属于基因工程和酶工程技术领域，具体涉及一种环糊精葡萄糖基转移酶突变体及应用。
背景技术
环糊精葡萄糖基转移酶(Cyclodextrin glycosyl transfer,CGTase,EC2.4.1.19)属于α-淀粉酶家族(糖苷水解酶13_2,GH13_2)，是一种胞外酶，同时也是一种多功能酶，能够以淀粉、麦芽糊精等为底物催化转糖苷反应(歧化、环化和耦合反应)和水解反应。其中，歧化反应是两个不同分子间发生的转糖苷反应，即将直链低聚糖被切断的部分转移到另一受体上。环化反应是分子内发生的转糖苷反应，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，这是CGTase的特征反应。偶合反应是环化反应的逆反应，可以打开环糊精的环，将糖苷转移到直链麦芽低聚物上。
CGTase的应用十分广泛，常见的是利用其环化反应把淀粉转化成环糊精。另外，也常利用CGTase歧化反应生成稳定性提高的糖苷化合物，如将小分子的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、鼠李糖、芸香苷、L-抗坏血酸等物质进行糖基化修饰，显著改善其性能。
现有技术中，CGTase具有较优秀的转糖基性能，可被应用到多个领域之中，但其歧化反应酶活通过突变体很难明显提高。
发明内容
针对现有技术的不足，本发明提供了一种环糊精葡萄糖基转移酶突变体及应用。
本发明所要解决的技术问题是通过对来源于Bacillus sp.G1的β-CGTase进行改造，以此来获得歧化活性效率提高的突变体。
本发明中所述来源于芽孢杆菌属(Bacillus sp.G1)的环糊精葡萄糖基转移酶的编码核苷酸序列如SEQ ID NO.1所示，所述环糊精葡萄糖基转移酶的氨基酸序列如SEQ IDNO.2所示。
本发明的技术方案如下：
一种环糊精葡萄糖基转移酶的突变体，氨基酸突变位点为环糊精葡萄糖基转移酶氨基酸序列SEQ ID NO.2的第33、119、122、216、255、258、394、566位氨基酸之一或两者以上。
根据本发明优选的，所述突变体为：
第33位的天冬酰胺(N)突变为赖氨酸(K)或精氨酸(R)，分别命名为N33K、N33R；
第119位的酪氨酸(Y)突变为精氨酸(R)，命名为Y119R；
第122位的酪氨酸(Y)突变为谷氨酸(E)，命名为Y122E；
第216位的亮氨酸(L)突变为谷氨酰胺(Q)，命名为L216Q；
第255位的组氨酸(H)突变为酪氨酸(Y)，命名为H255Y；
第258位的谷氨酸(E)突变为酪氨酸(Y)或谷氨酰胺(Q)，分别命名为E258Y或E258Q；
第394位的脯氨酸(P)突变为精氨酸(R)或谷氨酰胺(Q)，分别命名为P394R或P394Q；
或第566位的谷氨酸(E)突变为组氨酸(H)，命名为E566H。
根据本发明优选的，所述突变体为第33位的天冬酰胺(N)突变为赖氨酸(K)、第122位的酪氨酸(Y)突变为谷氨酸(E)、第258位的谷氨酸(E)突变为酪氨酸(Y)和第394位的脯氨酸(P)突变为精氨酸(R)，命名为N33K/Y122E/E258Y/P394R。
上述突变体的编码基因，根据氨基酸的突变位点，在环糊精葡萄糖基转移酶的编码核苷酸序列SEQ ID NO.1上，进行定点突变获得。
一种重组表达载体，包含上述突变体的编码基因。
一种重组菌株，包含上述突变体的编码基因。
上述突变体的编码基因、重组表达载体或重组菌株在制备环糊精葡萄糖基转移酶中的应用。
上述突变体在制备偶合糖中的应用。
上述突变体在制备海藻糖中的应用。
有益效果
本发明提供的环糊精葡萄糖基转移酶突变体的歧化反应活性相较于野生型明显提高，本发明对于环糊精葡萄糖基转移酶工业化生产具有一定意义，并提高该酶在医药、食品、生物行业中的应用前景。
附图说明
图1为单点突变基因电泳检测结果图；
图中：1、2、3、4、5、6、7、8、9、10、11泳道分别代表N33K、N33R、Y119R、Y122E、L216Q、H255Y、E258Y、E258Q、P394R、P394Q和E566H位点突变PCR验证条带。
图2为多点突变基因N33K/Y122E/E258Y/P394R PCR电泳检测结果图。
具体实施方式
下面结合实施例对本发明的技术方案做进一步阐述，但本发明所保护范围不限于此。
下述实施例中涉及的培养基和检测方法如下：
LB培养基：胰蛋白胨10g/L，酵母浸粉5g/L，氯化钠10g/L，余量水。
TB培养基：胰蛋白胨12g/L，酵母浸粉24g/L，磷酸氢二钾12.54g/L，磷酸二氢钾2.31g/L，甘油4mL/L，余量水。
环糊精葡萄糖基转移酶催化歧化反应活力的测定方法：
以50mmol/L pH 6.0的磷酸盐缓冲液为溶剂，分别配制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，加入50μL3M HCl，5min后加入3M NaOH中和，然后加入100μLα-葡萄糖苷酶于37℃水浴锅静置反应60min以上，加入100μL 1M Na2CO3溶液将pH调节到8.0以上，最后于401nm测定吸光值。环糊精葡萄糖基转移酶的歧化活力酶活定义为，每分钟转化1μmol EPS所需要的的酶量。
实施例1
野生型环糊精葡萄糖基转移酶的制备及表达
来源于Bacillus sp.G1的环糊精葡萄糖基转移酶的基因来源于人工合成，核苷酸序列如SEQ ID NO.1所示，所述环糊精葡萄糖基转移酶的氨基酸序列如SEQ ID NO.2所示，并构建载体导入大肠杆菌中进行表达，得到野生型环糊精葡萄糖基转移酶。于LB液体培养基中(含100mg/L卡那霉素)培养10h，按体积比5％接种量将种子液接入TB液体发酵培养基(含100mg/L卡那霉素)，37℃培养2h后加入终浓度0.2mM IPTG，转移到25℃摇床培养8h，将一定体积发酵液于4℃、12000rpm离心10min，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的单突变制备及表达
(1)根据Bacillus sp.G1环糊精葡萄糖基转移酶的核苷酸序列，如SEQ ID NO.1所示，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pET-28a(+)/Cgt为模板。
引入N33K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'-GTTACAAAGAAAGTCAATTATTCTAAGGATGTGATTTACCA-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'-TGACTTTCTTTGTAACATCTGCTTCAGCTACTG-3'(下划线为突变碱基)；
引入N33R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'-GTTACAAGGAAAGTCAATTATTCTAAGGATGTGATTTACCA-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'-GACTTTCCTTGTAACATCTGCTTCAGCTACTG-3'(下划线为突变碱基)；
引入Y119R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'-TCACGTCATGGCTATTGGGCAAGAGA-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'-GCCATGACGTGATGTATAGCCGCTCGG-3'(下划线为突变碱基)；
引入Y122E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'-GCGAATGGGCAAGAGATTATAAAAAAACAAATCCGT-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'-TTGCCCATTCGCCATGATATGATGTATAGCCGC-3'(下划线为突变碱基)；
引入L216Q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'-AGAAATCAGTATGATTTGGCAGACTACGAT-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'-GCCAAATCATACTGATTTCTATAAATTGAATCTTCATATGATG-3'(下划线为突变碱基)；
引入H255Y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'-GTTAAATACATGTCAGAAGGCTGGCAAA-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'-CTGACATGTATTTAACTGCATCAACTCTAATGCCAT-3'(下划线为突变碱基)；
引入E258Y突变的定点突变引物为：
核苷酸序列如SEQ ID NO.15所示的正向引物：
5'-GTCATACGGCTGGCAAACATCACTG-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'-CAGCCGTATGACATATGTTTAACTGCATCAACTC-3'(下划线为突变碱基)；
引入E258Q突变的定点突变引物为：
核苷酸序列如SEQ ID NO.17所示的正向引物：
5'-GTCACAAGGCTGGCAAACATCACTG-3'(下划线为突变碱基)；
核苷酸序列如SEQ ID NO.18所示的反向引物：
5'-CAGCCTTGTGACATATGTTTAACTGCATCAACTCT-3'(下划线为突变碱基)；
引入P394R突变的定点突变引物为：
核苷酸序列如SEQ ID NO.19所示的正向引物：
5'-GATCGGGAAAATAGAAAACCGCTGAAAACA-3'(下划线为突变碱基)；
核苷酸序列如SEQ ID NO.20所示的反向引物：
5'-CTATTTTCCCGATCATTGCCGCCCGTAACA-3'(下划线为突变碱基)；
引入P394Q突变的定点突变引物为：
核苷酸序列如SEQ ID NO.21所示的正向引物：
5'-CAATGATCAGGAAAATAGAAAACCGCTGAAAACA-3'(下划线为突变碱基)；
核苷酸序列如SEQ ID NO.22所示的反向引物：
5'-ATTTTCCTGATCATTGCCGCCCGTAAC-3'(下划线为突变碱基)；
引入E566H突变的定点突变引物为：
核苷酸序列如SEQ ID NO.23所示的正向引物：
5'-CAAATACACACATTTCAGTTAAAGTTCCGAATGTTGCGG-3'(下划线为突变碱基)；
核苷酸序列如SEQ ID NO.24所示的反向引物：
5'-GAAATGTGTGTATTTGACCATGAAATAATTTCTGATGATG-3'(下划线为突变碱基)。
PCR反应体系均为：10μM正向引物和反向引物各2.5μL，2×Phanta Max MasterMix 25μL，模板2.5μL，加双蒸水补齐50μL。
PCR条件为：95℃预变性5min；随后进行25个循环(95℃15s，55℃15s，72℃3min50s)72℃延伸5min；最后4℃保存。PCR产物用1％琼脂糖凝胶电泳进行检测，突变基因PCR验证结果见图1。
将上述验证正确的PCR产物进行DpnⅠ消化，转入大肠杆菌DH5α感受态细胞，转化产物涂布于含100mg/L卡那霉素的LB平板上，经37℃过夜培养，平板上挑取10个单菌落，转入含100mg/L卡那霉素的LB液体培养基，8h后提取质粒并验证，选取3个验证正确的质粒测序，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含100mg/L卡那霉素的LB液体培养基，培养10h，按体积比5％接种量将种子液接入TB液体发酵培养基(含100mg/L卡那霉素)，37℃培养2h后加入终浓度0.2mM IPTG，转移到25℃摇床培养8h，将一定体积发酵液于4℃、12000rpm离心10min，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四突变体的制备及表达
(1)环糊精葡萄糖基转移酶的四突变体制备
以实施例2构建的携带编码突变体N33K的基因的质粒作为四突变体的模板，并根据实施例2设计的Y122E、E258Y、P394R定点突变的引物，使用快速PCR技术，对携带编码突变体N33K的基因的质粒进行依次定点突变，突变基因PCR验证结果见图2。得到环糊精葡萄糖基转移酶N33K/Y122E/E258Y/P394R四突变体，并依照实施例2中的方法制备表达四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)制备得到的表达突变体的重组大肠杆菌接种于含100mg/L卡那霉素的LB液体培养基，培养10h，按体积比5％接种量将种子液接入TB液体发酵培养基(含100mg/L卡那霉素)，37℃培养2h后加入终浓度0.2mM IPTG，转移到25℃摇床培养8h，将一定体积发酵液于4℃、12000rpm离心10min，取发酵上清，即为四突变体的粗酶液。
实施例4
环糊精葡萄糖基转移酶的歧化反应活性分析
将实施例1、实施例2和实施例3所得到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(BsCGT)和突变体摇瓶诱导发酵8h的歧化活性列于表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N33K、N33R、Y119R、Y122E、L216Q、H255Y、E258Y、E258Q、P394R、P394Q、E566H和N33K/Y122E/E258Y/P394R的摇瓶发酵歧化活力分别为野生型的2.10倍、1.43倍、1.35倍、1.32倍、1.21倍、1.21倍、1.53倍、2.03倍、1.80倍、1.71倍、1.45倍、2.32倍。
表1
本发明构建的突变体其歧化反应催化效率与野生酶相比有所提高，尤其是N33K突变体、E258Q突变体、N33K/Y122E/E258Y/P394R突变体酶活提高明显，应用于工业化生产可降低生产成本，并由于歧化反应活性的提高，对于其在转糖苷化合物，如在生产海藻糖中的应用，在L-抗坏血酸、甜菊苷等物质改性上的应用，有效提高了其应用前景。
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> 齐鲁工业大学
<120> 一种环糊精葡萄糖基转移酶突变体及应用
<160> 24
<170> PatentIn version 3.5
<210> 1
<211> 2109
<212> DNA
<213> 人工序列
<400> 1
atgaatgatc tgaatgattt tctgaaaaca atttcactgt catttatttt ttttctgctg 60
ctgtcactgc cgacagtagc tgaagcagat gttacaaata aagtcaatta ttctaaggat 120
gtgatttacc aagttgttac agatagattt tcagatggca atccgggcaa taatccgagc 180
ggcgcaattt tttcacaaaa ttgcattgat ctgcataaat attgcggcgg cgattggcaa 240
ggcattattg ataaaattaa tgatggctat ctgacagatc tgggcattac agcactgtgg 300
atttcacaac cggttgaaaa tgtttatgca ctgcatccga gcggctatac atcatatcat 360
ggctattggg caagagatta taaaaaaaca aatccgtatt atggcaattt tgatgatttt 420
gatagactga tgtcaacagc acattcaaat ggcattaaag ttattatgga ttttacaccg 480
aatcattcat caccggcact ggaaacaaat ccgaattatg ttgaaaatgg cgcaatttat 540
gataatggca cactgctggg caattattca aatgatcaac aaaatctgtt tcatcataat 600
ggcggcacag atttttcatc atatgaagat tcaatttata gaaatctgta tgatttggca 660
gactacgatc tgaataacac ggttatggat caatatttga aagaatcaat taaattttgg 720
ctggataaag gcattgatgg cattagagtt gatgcagtta aacatatgtc agaaggctgg 780
caaacatcac tgatgtcaga aatttattca cataaaccgg tttttacatt tggcgaatgg 840
tttctgggca gcggcgaagt tgatccgcaa aatcatcatt ttgcaaatga aagcggcatg 900
tcactgctgg attttcaatt tggccaaacc attagaaatg ttctgaaaga ccgcacttca 960
aattggtacg actttaacga aatgattaca tcaaccgaaa aggaatacaa tgaagttatt 1020
gaccaagtaa catttatcga taatcatgat atgtcaagat tttcagttgg ctcatcatca 1080
aatagacaaa cagatatggc actggcagtt ctgctgacat caagaggcgt tccgacaatt 1140
tattatggca cagaacaata tgttacgggc ggcaatgatc cggaaaatag aaaaccgctg 1200
aaaacatttg atagatcgac aaattcatac caaattattt caaaactggc atcactgaga 1260
caaacaaatt cagcactggg ctatggcaca acaacagaaa gatggctgaa tgaagatatt 1320
tatatttatg aaagaacatt tggcaattca attgttctga cagcagttaa ttcatcaaat 1380
tcaaatcaaa caattacaaa tctgaataca tcactgccgc aaggcaatta tacagatgaa 1440
ctgcaacaaa gactggatgg gaatacaatc acagttaatg caaatggcgc agttaactca 1500
ttccaactga gagcaaattc agttgcagtt tggcaagttt caaatccgtc aacatcaccg 1560
ctgattggcc aagttggccc gatgatgggc aagtctggca atacaattac agtatctggc 1620
gaaggctttg gcgatgaaag aggctcagtt ctgtttgatt caacatcatc agaaattatt 1680
tcatggtcaa atacagaaat ttcagttaaa gttccgaatg ttgcgggcgg ctattatgat 1740
ctgtcagttg ttacagcagc aaatctgaaa tcaccgacat ataaagaatt tgaagttctg 1800
agcggcaatc aagtttcagt tagatttggc gttaataatg caacaacatc accgggcaca 1860
aatctgtata ttgttggcaa tgtttcagaa ctgggcaatt gggatgcaga taaagcaatt 1920
ggcccgatgt ttaatcaagt tatgtatcaa tatccgacat ggtattatga tatttcagtt 1980
ccggcgggca aaaatctgga atataaatat attaaaaaag atcaaaatgg caatgttgtt 2040
tggcagtccg gcaataatcg gacatataca tcaccgacaa cgggcacaga tacagttatg 2100
attaattgg 2109
<210> 2
<211> 703
<212> PRT
<213> 人工序列
<400> 2
Met Asn Asp Leu Asn Asp Phe Leu Lys Thr Ile Ser Leu Ser Phe Ile
1 5 10 15
Phe Phe Leu Leu Leu Ser Leu Pro Thr Val Ala Glu Ala Asp Val Thr
20 25 30
Asn Lys Val Asn Tyr Ser Lys Asp Val Ile Tyr Gln Val Val Thr Asp
35 40 45
Arg Phe Ser Asp Gly Asn Pro Gly Asn Asn Pro Ser Gly Ala Ile Phe
50 55 60
Ser Gln Asn Cys Ile Asp Leu His Lys Tyr Cys Gly Gly Asp Trp Gln
65 70 75 80
Gly Ile Ile Asp Lys Ile Asn Asp Gly Tyr Leu Thr Asp Leu Gly Ile
85 90 95
Thr Ala Leu Trp Ile Ser Gln Pro Val Glu Asn Val Tyr Ala Leu His
100 105 110
Pro Ser Gly Tyr Thr Ser Tyr His Gly Tyr Trp Ala Arg Asp Tyr Lys
115 120 125
Lys Thr Asn Pro Tyr Tyr Gly Asn Phe Asp Asp Phe Asp Arg Leu Met
130 135 140
Ser Thr Ala His Ser Asn Gly Ile Lys Val Ile Met Asp Phe Thr Pro
145 150 155 160
Asn His Ser Ser Pro Ala Leu Glu Thr Asn Pro Asn Tyr Val Glu Asn
165 170 175
Gly Ala Ile Tyr Asp Asn Gly Thr Leu Leu Gly Asn Tyr Ser Asn Asp
180 185 190
Gln Gln Asn Leu Phe His His Asn Gly Gly Thr Asp Phe Ser Ser Tyr
195 200 205
Glu Asp Ser Ile Tyr Arg Asn Leu Tyr Asp Leu Ala Asp Tyr Asp Leu
210 215 220
Asn Asn Thr Val Met Asp Gln Tyr Leu Lys Glu Ser Ile Lys Phe Trp
225 230 235 240
Leu Asp Lys Gly Ile Asp Gly Ile Arg Val Asp Ala Val Lys His Met
245 250 255
Ser Glu Gly Trp Gln Thr Ser Leu Met Ser Glu Ile Tyr Ser His Lys
260 265 270
Pro Val Phe Thr Phe Gly Glu Trp Phe Leu Gly Ser Gly Glu Val Asp
275 280 285
Pro Gln Asn His His Phe Ala Asn Glu Ser Gly Met Ser Leu Leu Asp
290 295 300
Phe Gln Phe Gly Gln Thr Ile Arg Asn Val Leu Lys Asp Arg Thr Ser
305 310 315 320
Asn Trp Tyr Asp Phe Asn Glu Met Ile Thr Ser Thr Glu Lys Glu Tyr
325 330 335
Asn Glu Val Ile Asp Gln Val Thr Phe Ile Asp Asn His Asp Met Ser
340 345 350
Arg Phe Ser Val Gly Ser Ser Ser Asn Arg Gln Thr Asp Met Ala Leu
355 360 365
Ala Val Leu Leu Thr Ser Arg Gly Val Pro Thr Ile Tyr Tyr Gly Thr
370 375 380
Glu Gln Tyr Val Thr Gly Gly Asn Asp Pro Glu Asn Arg Lys Pro Leu
385 390 395 400
Lys Thr Phe Asp Arg Ser Thr Asn Ser Tyr Gln Ile Ile Ser Lys Leu
405 410 415
Ala Ser Leu Arg Gln Thr Asn Ser Ala Leu Gly Tyr Gly Thr Thr Thr
420 425 430
Glu Arg Trp Leu Asn Glu Asp Ile Tyr Ile Tyr Glu Arg Thr Phe Gly
435 440 445
Asn Ser Ile Val Leu Thr Ala Val Asn Ser Ser Asn Ser Asn Gln Thr
450 455 460
Ile Thr Asn Leu Asn Thr Ser Leu Pro Gln Gly Asn Tyr Thr Asp Glu
465 470 475 480
Leu Gln Gln Arg Leu Asp Gly Asn Thr Ile Thr Val Asn Ala Asn Gly
485 490 495
Ala Val Asn Ser Phe Gln Leu Arg Ala Asn Ser Val Ala Val Trp Gln
500 505 510
Val Ser Asn Pro Ser Thr Ser Pro Leu Ile Gly Gln Val Gly Pro Met
515 520 525
Met Gly Lys Ser Gly Asn Thr Ile Thr Val Ser Gly Glu Gly Phe Gly
530 535 540
Asp Glu Arg Gly Ser Val Leu Phe Asp Ser Thr Ser Ser Glu Ile Ile
545 550 555 560
Ser Trp Ser Asn Thr Glu Ile Ser Val Lys Val Pro Asn Val Ala Gly
565 570 575
Gly Tyr Tyr Asp Leu Ser Val Val Thr Ala Ala Asn Leu Lys Ser Pro
580 585 590
Thr Tyr Lys Glu Phe Glu Val Leu Ser Gly Asn Gln Val Ser Val Arg
595 600 605
Phe Gly Val Asn Asn Ala Thr Thr Ser Pro Gly Thr Asn Leu Tyr Ile
610 615 620
Val Gly Asn Val Ser Glu Leu Gly Asn Trp Asp Ala Asp Lys Ala Ile
625 630 635 640
Gly Pro Met Phe Asn Gln Val Met Tyr Gln Tyr Pro Thr Trp Tyr Tyr
645 650 655
Asp Ile Ser Val Pro Ala Gly Lys Asn Leu Glu Tyr Lys Tyr Ile Lys
660 665 670
Lys Asp Gln Asn Gly Asn Val Val Trp Gln Ser Gly Asn Asn Arg Thr
675 680 685
Tyr Thr Ser Pro Thr Thr Gly Thr Asp Thr Val Met Ile Asn Trp
690 695 700
<210> 3
<211> 41
<212> DNA
<213> 人工序列
<400> 3
gttacaaaga aagtcaatta ttctaaggat gtgatttacc a 41
<210> 4
<211> 33
<212> DNA
<213> 人工序列
<400> 4
tgactttctt tgtaacatct gcttcagcta ctg 33
<210> 5
<211> 41
<212> DNA
<213> 人工序列
<400> 5
gttacaagga aagtcaatta ttctaaggat gtgatttacc a 41
<210> 6
<211> 32
<212> DNA
<213> 人工序列
<400> 6
gactttcctt gtaacatctg cttcagctac tg 32
<210> 7
<211> 26
<212> DNA
<213> 人工序列
<400> 7
tcacgtcatg gctattgggc aagaga 26
<210> 8
<211> 27
<212> DNA
<213> 人工序列
<400> 8
gccatgacgt gatgtatagc cgctcgg 27
<210> 9
<211> 36
<212> DNA
<213> 人工序列
<400> 9
gcgaatgggc aagagattat aaaaaaacaa atccgt 36
<210> 10
<211> 33
<212> DNA
<213> 人工序列
<400> 10
ttgcccattc gccatgatat gatgtatagc cgc 33
<210> 11
<211> 30
<212> DNA
<213> 人工序列
<400> 11
agaaatcagt atgatttggc agactacgat 30
<210> 12
<211> 43
<212> DNA
<213> 人工序列
<400> 12
gccaaatcat actgatttct ataaattgaa tcttcatatg atg 43
<210> 13
<211> 28
<212> DNA
<213> 人工序列
<400> 13
gttaaataca tgtcagaagg ctggcaaa 28
<210> 14
<211> 36
<212> DNA
<213> 人工序列
<400> 14
ctgacatgta tttaactgca tcaactctaa tgccat 36
<210> 15
<211> 25
<212> DNA
<213> 人工序列
<400> 15
gtcatacggc tggcaaacat cactg 25
<210> 16
<211> 34
<212> DNA
<213> 人工序列
<400> 16
cagccgtatg acatatgttt aactgcatca actc 34
<210> 17
<211> 25
<212> DNA
<213> 人工序列
<400> 17
gtcacaaggc tggcaaacat cactg 25
<210> 18
<211> 35
<212> DNA
<213> 人工序列
<400> 18
cagccttgtg acatatgttt aactgcatca actct 35
<210> 19
<211> 30
<212> DNA
<213> 人工序列
<400> 19
gatcgggaaa atagaaaacc gctgaaaaca 30
<210> 20
<211> 30
<212> DNA
<213> 人工序列
<400> 20
ctattttccc gatcattgcc gcccgtaaca 30
<210> 21
<211> 34
<212> DNA
<213> 人工序列
<400> 21
caatgatcag gaaaatagaa aaccgctgaa aaca 34
<210> 22
<211> 27
<212> DNA
<213> 人工序列
<400> 22
attttcctga tcattgccgc ccgtaac 27
<210> 23
<211> 39
<212> DNA
<213> 人工序列
<400> 23
caaatacaca catttcagtt aaagttccga atgttgcgg 39
<210> 24
<211> 40
<212> DNA
<213> 人工序列
<400> 24
gaaatgtgtg tatttgacca tgaaataatt tctgatgatg 40
Claims (8)
1. 一种环糊精葡萄糖基转移酶的突变体，其特征在于，所述突变体为环糊精葡萄糖基转移酶氨基酸序列SEQ ID NO.2的第33位的天冬酰胺（N）突变为赖氨酸（K）或精氨酸（R），分别命名为N33K、N33R；
所述突变体为环糊精葡萄糖基转移酶氨基酸序列SEQ ID NO.2的第119位的酪氨酸（Y）突变为精氨酸（R），命名为Y119R；
所述突变体为环糊精葡萄糖基转移酶氨基酸序列SEQ ID NO.2的第122位的酪氨酸（Y）突变为谷氨酸（E），命名为Y122E；
所述突变体为环糊精葡萄糖基转移酶氨基酸序列SEQ ID NO.2的第216位的亮氨酸（L）突变为谷氨酰胺（Q），命名为L216Q；
所述突变体为环糊精葡萄糖基转移酶氨基酸序列SEQ ID NO.2的第255位的组氨酸（H）突变为酪氨酸（Y），命名为H255Y；
所述突变体为环糊精葡萄糖基转移酶氨基酸序列SEQ ID NO.2的第258位的谷氨酸（E）突变为酪氨酸（Y）或谷氨酰胺（Q），分别命名为E258Y或E258Q；
所述突变体为环糊精葡萄糖基转移酶氨基酸序列SEQ ID NO.2的第394位的脯氨酸（P）突变为精氨酸（R）或谷氨酰胺（Q），分别命名为P394R或P394Q；
或第566位的谷氨酸（E）突变为组氨酸（H），命名为E566H。
2. 一种环糊精葡萄糖基转移酶的突变体，其特征在于，所述突变体为环糊精葡萄糖基转移酶氨基酸序列SEQ ID NO.2的第33位的天冬酰胺（N）突变为赖氨酸（K）、第122位的酪氨酸（Y）突变为谷氨酸（E）、第258位的谷氨酸（E）突变为酪氨酸（Y）和第394位的脯氨酸（P）突变为精氨酸（R），命名为N33K/Y122E/E258Y/P394R。
3. 权利要求1或权利要求2所述突变体的编码基因，其特征在于，根据氨基酸突变位点，在环糊精葡萄糖基转移酶的编码核苷酸序列SEQ ID NO.1上进行定点突变获得。
4.一种重组表达载体，其特征在于，包含权利要求3所述突变体的编码基因。
5.一种重组菌株，其特征在于，包含权利要求3所述突变体的编码基因。
6.权利要求3所述编码基因、权利要求4所述重组表达载体或权利要求5所述重组菌株在制备环糊精葡萄糖基转移酶中的应用。
7.权利要求1或权利要求2所述突变体在制备偶合糖中的应用。
8.权利要求1或权利要求2所述突变体在制备海藻糖中的应用。
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| CN109486786A (zh) * | 2018-12-07 | 2019-03-19 | 江南大学 | 一种环糊精葡萄糖基转移酶突变体 |
| CN110982865A (zh) * | 2019-12-31 | 2020-04-10 | 浙江工业大学 | 一种碱性环糊精葡萄糖基转移酶在生产α-葡萄糖基橙皮苷中的应用 |
| CN112301012A (zh) * | 2020-10-15 | 2021-02-02 | 江南大学 | 一种环糊精葡萄糖基转移酶突变体及其构建方法 |
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