CN111621483B - 一种蔗糖磷酸化酶突变体及其应用 - Google Patents
一种蔗糖磷酸化酶突变体及其应用 Download PDFInfo
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Abstract
本发明公开了一种蔗糖磷酸化酶突变体及其应用，属于酶工程和微生物工程技术领域。本发明的蔗糖磷酸化酶突变体的热稳定性较野生型有了明显的改善，蔗糖磷酸化酶突变体T219L和I31F/T219L/S360A/T263L在50℃下具有较好的热稳定性，半衰期大约为50min，相比野生型提高了35min左右。
Description
技术领域
本发明涉及一种蔗糖磷酸化酶突变体及其应用，属于酶工程和微生物工程技术领域。
背景技术
蔗糖磷酸化酶(EC 2.4.1.7，Sucrose phosphorylase，SPase)催化蔗糖磷酸解反应生成α-D-葡萄糖-1-磷酸和D-果糖及其可逆反应。该酶具有葡萄糖基转移能力，可以将蔗糖分子的一个葡萄糖基转移至不同的受体上。近年来，利用该酶生产α-熊果苷和α-葡萄糖基甘油(α-glucosyl glycerol,αGG)等精细化学品受到了越来越多的关注。
对于工业应用，为了避免微生物污染，碳水化合物的转化最好在60℃或更高温度下进行。但是大多数SPase并不是嗜热型的酶。现有的SPase热稳定性差，例如，来源于streptococcus mutants的野生型SPase，其在55℃条件下处理20min酶活仅为初始活性的11.3％(具体可见参考文献：Fujii K,Iiboshi M,Yanase M,et al.Enhancing theThermal Stability of Sucrose Phosphorylase from Streptococcus mutans byRandom Mutagenesis[J].Journal of Applied Glycoence,2006,53(2):91-97.)。
因此，急需找到一种热稳定性好的蔗糖磷酸化酶突变体。
发明内容
为解决上述技术问题，本发明提供了一种蔗糖磷酸化酶突变体，所述蔗糖磷酸化酶突变体与氨基酸序列如SEQ ID NO.22所示的蔗糖磷酸化酶相比，第219位氨基酸由苏氨酸突变为了亮氨酸；
或者，所述蔗糖磷酸化酶突变体与氨基酸序列如SEQ ID NO.22所示的蔗糖磷酸化酶相比，第31位氨基酸由异亮氨酸突变为了苯丙氨酸，第219位氨基酸由苏氨酸突变为了亮氨酸，第360位氨基酸由丝氨酸突变为了丙氨酸，第263位氨基酸由苏氨酸突变为了亮氨酸。
在本发明的一种实施方式中，编码所述蔗糖磷酸化酶突变体的原始酶的核苷酸序列如SEQ ID NO.1所示。
在本发明的一种实施方式中，所述蔗糖磷酸化酶来源于肠膜明串珠菌ATCC12291。
本发明还提供了携带上述基因的重组质粒。
在本发明的一种实施方式中，在本发明的一种实施方式中，所述重组质粒的载体为pET-28a质粒。
本发明还提供了携带上述基因或上述重组质粒的宿主细胞。
在本发明的一种实施方式中，所述宿主细胞为细菌或真菌。
在本发明的一种实施方式中，所述宿主细胞为大肠杆菌。
本发明还提供了产上述蔗糖磷酸化酶突变体的制备方法，所述方法为将1-5％接种量接入到TB液体培养基中，于35-39℃，200-220r/min培养至菌密度OD达到0.5-0.7后，加入IPTG诱导剂于23-27℃，200-220r/min诱导20-30h后收集菌液，离心收集菌体，重悬后破碎菌体，离心收集上清，分离上清液中的蔗糖磷酸化酶突变体。
本发明还提供了应用上述方法制备得到的蔗糖磷酸化酶突变体。
本发明还提供了上述蔗糖磷酸化酶突变体或上述基因或上述重组质粒或上述宿主细胞或上述制备方法在制备食品、药品、保健品、化妆品中的应用。
[有益效果]
本发明的蔗糖磷酸化酶突变体的热稳定性较野生型有了明显的改善，蔗糖磷酸化酶突变体T219L和I31F/T219L/S360A/T263L在50℃下具有较好的热稳定性，半衰期大约为50min，相比野生型提高了35min左右。
附图说明
图1：野生型蔗糖磷酸化酶和蔗糖磷酸化酶突变体表达的SDS-PAGE分析；其中，M：marker；WT：野生型，1：I31F；2：Q453G；3：G252L；4：A232M；5：T152G；6：N158C；7：T219L；8：S360A；9：N249A；10：T263L。
图2：野生型和突变体粗酶液的比酶活和50℃保温10min后的残余酶活；其中，WT：野生型，1：I31F；2：Q453G；3：G252L；4：A232M；5：T152G；6：N158C；7：T219L；8：S360A；9：N249A；10：T263L；组合：I31F/T219L/S360A/T263L。
图3：野生型和正向突变体粗酶液和纯酶的SDS-PAGE；其中，1为WT粗酶液；2为WT纯酶；3为I31F粗酶液；4为I31F纯酶；5为T219L粗酶液；6为T219L纯酶；7为S360A粗酶液；8为S360A纯酶；9为T263L粗酶液；10为T263L纯酶；11为I31F/T219L/S360A/T263L粗酶液；12为I31F/T219L/S360A/T263L纯酶。
图4：蔗糖磷酸化酶突变体I31F、T219L、S360A、T263L和I31F/T219L/S360A/T263L的CD光谱。
图5：野生型和突变体50℃下的半衰期。
图6：差示扫描量热法确定Tm。
具体实施方式
下面结合具体实施例，对本发明进行进一步的阐述。
下述实施例中涉及的培养基如下：
LB培养基：5g·L-1酵母粉，10g·L-1蛋白胨，10g·L-1NaCl，固体培养基另加1.5～2.0％琼脂粉。培养基灭菌温度为121℃，时间为15min。
TB培养基：24g·L-1酵母粉，12g·L-1蛋白胨，4mL·L-1甘油，2.31g·L-1KH2PO4，16.43g·L-1K2HPO4·3H2O。培养基灭菌温度为121℃，时间为15min。
下述实施例中涉及的检测方法如下：
蔗糖磷酸化酶酶活的测定方法：
(1)果糖标准曲线的测定
取7个5mL离心管将果糖标准液按表2-2配制不同果糖浓度的溶液，体系为1mL，然后加入DNS 1.5mL，混匀后放入沸水浴中煮沸15min，放入冰水中冷却后用紫外分光光度计测定540nm下的吸光值，以果糖浓度为横坐标，以OD540处的吸光值为纵坐标，绘制果糖标准曲线。
(2)蔗糖磷酸化酶酶活的测定
反应体系如下：5％蔗糖溶液500μL，50mM磷酸盐缓冲液(pH 6.5)450μL，重组SPase50μL，30℃准确反应10min后，立即加入1.5mL DNS沸水浴15min，540nm下测定吸光值，计算反应液中生成果糖的含量(以空载为对照组，其他条件不变)。
蔗糖和磷酸盐在SPase的催化下生成葡萄糖-1-磷酸和果糖。测定生成产物果糖的量从而计算出SPase的酶活，酶活计算公式如下：
式中，A：吸光值，B：截距，n：稀释倍数，M：果糖分子质量，k：斜率，t：反应时间(min)；
酶活定义：将每分钟水解蔗糖生成1μmol的果糖所需酶量定义为SPase的一个酶活力单位。
(3)蔗糖磷酸化酶比酶活的测定
利用Brandford法(Bradford M M.A rapid and sensitive method for thequantitation of microgram quantities of protein utilizing the principle ofprotein-dye binding[J].Analytical biochemistry,1976,72:248-254.)测定所制备酶液中蛋白质的含量，比酶活计算公式如下：
比酶活＝酶活/酶液中蛋白浓度。
比酶活定义：每毫克蛋白所具有的酶活力。
实施例1：蔗糖磷酸化酶野生酶的表达
(1)从NCBI中获取肠膜明串珠菌ATCC 12291的SPase基因(GenBank登录号：D90314)的基因序列，根据大肠杆菌密码子的偏好性，对其进行优化，并将两个酶切位点NcoI、XhoI加到经过优化后的SPase基因两端，送到金唯智生物科技有限公司合成得到编码核苷酸序列如SEQ ID NO.1所示的蔗糖磷酸化酶。
(2)基因表达载体的构建及转化
将编码核苷酸序列如SEQ ID NO.1所示的蔗糖磷酸化酶SPase和pET-28a载体用限制性内切酶NcoI和XhoI双酶切，酶切后产物用Solution I连接，得到重组载体pET-28a-SPase，然后将重组载体pET-28a-SPase转入大肠杆菌BL21(DE3)中表达，挑取4个转化子提质粒用NcoI、XhoI进行酶切验证，得到重组大肠杆菌BL21/pET-28a-SPase。
实施例2：蔗糖磷酸化酶突变体的制备及表达
具体步骤如下：
利用全质粒PCR技术，以实施例1获得的重组质粒pET-28a-spase为模板进行定点突变，获得携带编码蔗糖磷酸化酶突变体I31F、Q453G、G252L、A232M、T152G、N158C、T219L、S360A、N249A、T263L以及T31F\T219L\S360A\T263L的基因的重组质粒pET-28a-SPase1～pET-28a-SPase11；
其中，突变I31F是通过将氨基酸序列如SEQ ID NO.22所示的蔗糖磷酸化酶的第31位氨基酸由异亮氨酸突变为苯丙氨酸得到的，所用引物如下：
I31F-1：5’-GTTCTGAAAGAAGACTTCGGTGACGCTA-3’(SEQ ID NO.2)；
I31F-2：5’-ACCGATAGCG TCACCGAAGTCTTCTTTC-3’(SEQ ID NO.3)；
突变Q453G是通过将氨基酸序列如SEQ ID NO.22所示的蔗糖磷酸化酶的第453位氨基酸由谷氨酰胺突变为甘氨酸得到的，所用引物如下：
Q453G-1：5’-ATCGTTGTTACCCGTGGCGACGAAAACG-3’(SEQ ID NO.4)；
Q453G-2：5’-CTGACCGTTT TCGTCGCCACGGGTAACA-3’(SEQ ID NO.5)；
突变G252L是通过将氨基酸序列如SEQ ID NO.22所示的蔗糖磷酸化酶的第252位氨基酸由甘氨酸突变为亮氨酸得到的，所用引物如下：
G252L-1：5’-AAAATCAACGACCACCTTTACTTCACCT-3’(SEQ ID NO.6)；
G252L-2：5’-GTCGTAGGTGAAGTAAAGGTGGTCGTTG-3’(SEQ ID NO.7)；
突变A232M是通过将氨基酸序列如SEQ ID NO.22所示的蔗糖磷酸化酶的第232位氨基酸由丙氨酸突变为蛋氨酸得到的，所用引物如下：
A232M-1：5’-CTGACCCCGCTGAAAATGGAAATCCTGC-3’(SEQ ID NO.8)；
A232M-2：5’-TTCCGGCAGG ATTTCCATTTTCAGCGGG-3’(SEQ ID NO.9)；
突变T152G是通过将氨基酸序列如SEQ ID NO.22所示的蔗糖磷酸化酶的第152位氨基酸由苏氨酸突变为甘氨酸得到的，所用引物如下：
T152G-1：5’-ACCTTCGACGACGGTGGTACCGAAAACC-3’(SEQ ID NO.10)；
T152G-2：5’-CCACAGGTTT TCGGTACCACCGTCGTCG-3’(SEQ ID NO.11)；
突变N158C是通过将氨基酸序列如SEQ ID NO.22所示的蔗糖磷酸化酶的第158位氨基酸由天冬酰胺突变为半胱氨酸得到的，所用引物如下：
N158C-1：5’-ACCGAAAACCTGTGGTGCACCTTCGGTG-3’(SEQ ID NO.12)；
N158C-2：5’-TTCTTCACCG AAGGTGCACC ACAGGTTT-3’(SEQ ID NO.13)；
突变T219L是通过将氨基酸序列如SEQ ID NO.22所示的蔗糖磷酸化酶的第219位氨基酸由苏氨酸突变为亮氨酸得到的，所用引物如下：
T219L-1：5’-CCGGAAATCTGGGACCTCCTGAACGAAG-3’(SEQ ID NO.14)；
T219L-2：5’-ACGAACTTCG TTCAGGAGGTCCCAGATT-3’(SEQ ID NO.15)；
突变S360A是通过将氨基酸序列如SEQ ID NO.22所示的蔗糖磷酸化酶的第360位氨基酸由丝氨酸突变为丙氨酸得到的，所用引物如下：
S360A-1：5’-GCTGCTTACCTGCTGGCGCGTGTTTTCC-3’(SEQ ID NO.16)；
S360A-2：5’-AACCTGGAAAACACGCGCCAGCAGGTAA-3’(SEQ ID NO.17)；
突变N249A是通过将氨基酸序列如SEQ ID NO.22所示的蔗糖磷酸化酶的第249位氨基酸由天冬酰胺突变为了丙氨酸得到的，所用引物如下：
N249A-1：5’-ATCCCGAAAAAAATCGCCGACCACGGTT-3’(SEQ ID NO.18)；
N249A-2：5’-GAAGTAACCGTGGTCGGCGATTTTTTTC-3’(SEQ ID NO.19)；
突变T263L是通过将氨基酸序列如SEQ ID NO.22所示的蔗糖磷酸化酶的第263位氨基酸由苏氨酸突变为亮氨酸得到的，所用引物如下：
T263L-1：5’-TTCGCTCTGCCGATGCTCACCCTGTACA-3’(SEQ ID NO.20)；
T263L-2：5’-CAGGGTGTACAGGGTGAGCATCGGCAGA-3’(SEQ ID NO.21)；
突变T31F\T219L\S360A\T263L是通过将氨基酸序列如SEQ ID NO.22所示的蔗糖磷酸化酶的第31位氨基酸由异亮氨酸突变为苯丙氨酸，第219位氨基酸由苏氨酸突变为亮氨酸，第360位氨基酸由丝氨酸突变为丙氨酸，第263位氨基酸由苏氨酸突变为亮氨酸得到的，所用引物如下：
T219L-1：CCGGAAATCTGGGACCTCCTGAACGAAG(SEQ ID NO.14)；
S360A-2：AACCTGGAAAACACGCGCCAGCAGGTAA(SEQ ID NO.17)
T263L-1：TTCGCTCTGCCGATGCTCACCCTGTACA(SEQ ID NO.20)
T263L-2：CAGGGTGTACAGGGTGAGCATCGGCAGA(SEQ ID NO.21)
定点突变PCR程序(两步法)：98℃预变性3min；98℃，变性10s，68℃退火延伸7min，循环25次；72℃延伸10min，16℃保温。
DpnI消化，体系如下：PCR产物17.5μL，Buffer 2μL，DpnI 0.5μL。放置于37℃培养箱2h。
将重组载体pET-28a-SPase1～pET-28a-SPase11转入大肠杆菌BL21(DE3)中表达，挑取4个转化子提质粒用NcoI、XhoI进行酶切验证，得到重组大肠杆菌BL21/pET-28a-SPase1～BL21/pET-28a-SPase11。
将实施例1和实施例2获得的重组大肠杆菌BL21/pET-28a-SPase以及重组大肠杆菌BL21/pET-28a-SPase1～BL21/pET-28a-SPase11分别在卡那霉素浓度为100μg·mL-1的LB固体培养基上划线，挑取单菌落接种于含卡那霉素浓度为50μg·mL-1的20mL LB液体培养基中，于37℃，200r·min-1过夜培养后，按1％接种量接入到含卡那霉素浓度为50μg·mL-1的50mL TB液体培养基中，于37℃，200r·min-1培养至菌密度OD600达到0.6后，加入终浓度为0.5mmol·L-1的IPTG诱导剂于25℃，200r·min-1诱导24h后收集菌液并离心，收集离心后的菌体，加入一定量50mmol·L-1K2HPO4-KH2PO4缓冲液(pH 6.5)，轻轻混匀后，离心弃掉上清，重复一次，用缓冲液洗两次后再收集菌体。将收集好的湿菌体加入20mL磷酸盐缓冲液，涡旋震荡混合均匀制成菌悬液，然后用超声波破碎菌体。超声波破碎仪工作时间：2s，间歇时间：4s，总时间：60min。将破碎后的液体于4℃，10000r·min-1的低温冷冻离心机下离心30min，收集上清即获得野生型蔗糖磷酸化酶以及蔗糖磷酸化酶突变体I31F、Q453G、G252L、A232M、T152G、N158C、T219L、S360A、N249A、T263L以及T31F\T219L\S360A\T263L的粗酶液。
将得到的含有蔗糖磷酸化酶突变体I31F、Q453G、G252L、A232M、T152G、N158C、T219L、S360A、N249A、T263L以及T31F\T219L\S360A\T263L粗酶液进行SDS-PAGE凝胶电泳分析，分析结果见图1。
由图1可知，上述突变体在55kDa附近均有很明显的条带，表明这些突变体蛋白均正常表达。但其中突变体A232M的目的蛋白表达量明显较少，表明即使是单点突变也会影响蛋白的表达。
在最适条件下测定各个SPase突变体粗酶液的比酶活，然后将其在50℃保温10min后，测定其相对残余酶活(样品蛋白浓度调整到一致后再进行热处理，蛋白浓度为0.010mg·mL-1)
如图2所示，根据各个突变体与野生型比较可知，1、7、8、10对应的突变体I31F、T219L、S360A及T263L比野生型具有较好的稳定性，所以后续选择这几个突变位点构建组合突变体，由结果可知，组合突变也具有较好的稳定性。所以选择蔗糖磷酸化酶突变体I31F、T219L、S360A、T263L及T31F\T219L\S360A\T263L进行下一步分析。
实施例3：不同蔗糖磷酸化酶突变体的分离纯化
具体步骤如下：
分别将实施例2获得的含有蔗糖磷酸化酶突变体I31F、T219L、S360A、T263L及T31F\T219L\S360A\T263L的粗酶液进行纯化，在SPase C末端连接了His标签，利用镍柱亲和层析纯化蛋白。
(1)提前准备好新鲜的含有蔗糖磷酸化酶突变体I31F、T219L、S360A、T263L及T31F\T219L\S360A\T263L的粗酶液放置于冰上(纯化过程都尽量在低温下进行)；
(2)用结合缓冲液以l mL·min-1的流速平衡柱子，随后将含有蔗糖磷酸化酶突变体I31F、T219L、S360A、T263L及T31F\T219L\S360A\T263L的粗酶液以0.5mL·min-1流速上样，并用结合缓冲液洗去未结合蛋白，然后用洗脱缓冲液洗脱并收集含有重组SPase的组分；
(3)用Hitrap Desalting柱去咪唑，平衡缓冲液(50mM K2HPO4-KH2PO4，pH 6.5)以5mL·min-1的流速平衡纯化柱，随后将镍柱收集到的SPase样品以相同流速上样，并用平衡缓冲液洗脱，收集得到纯化后的蔗糖磷酸化酶突变体I31F、T219L、S360A、T263L及T31F\T219L\S360A\T263L酶液，并用SDS-PAGE电泳验证(如图3)。
实施例4：不同蔗糖磷酸化酶突变体的二级结构
利用CD测定野生型和各突变体的二级结构，将实施例3获得的纯化后的蔗糖磷酸化酶突变体I31F、T219L、S360A、T263L及T31F\T219L\S360A\T263L酶液用磷酸盐缓冲液调节蛋白浓度至0.2mg·mL-1，送至实验平台测定。由于蛋白质的肽键在特定波长下具有圆二色性，利用CD可以反映出蛋白质的二级结构。将样品蛋白浓度调为0.02mg·mL-1后进行测定。结果如图4所示，从图4可以看出，蔗糖磷酸化酶突变体I31F、T219L、S360A、T263L及T31F\T219L\S360A\T263L与野生型的曲线重合度比较高，由此可知，蔗糖磷酸化酶突变体I31F、T219L、S360A、T263L及T31F\T219L\S360A\T263L与野生型的二级结构并没有很大的差异，可见，蔗糖磷酸化酶突变后对蔗糖磷酸化酶的二级结构影响不大。
实施例5：不同蔗糖磷酸化酶突变体的热稳定性
具体步骤如下：
首先将蔗糖磷酸化酶突变体I31F、T219L、S360A、T263L及T31F\T219L\S360A\T263L酶液的蛋白浓度调为0.014mg·mL-1，在最适pH的条件下，分别将蔗糖磷酸化酶突变体I31F、T219L、S360A、T263L及T31F\T219L\S360A\T263L纯酶放置在温度50℃金属浴保温，每隔10min测定相对残余酶活，以未处理酶液的酶活为100％对照。用指数函数拟合计算半衰期。由图5和表2可知，野生型蔗糖磷酸化酶、蔗糖磷酸化酶突变体I31F、蔗糖磷酸化酶突变体S360A和蔗糖磷酸化酶突变体T263L在50℃条件下的热稳定性没有太大的差异，保温不到20min，相对残余酶活已经降到50％。
蔗糖磷酸化酶突变体T219L和蔗糖磷酸化酶突变体突变体T31F\T219L\S360A\T263L在50℃下具有较好的热稳定性，半衰期大约为50min，相比野生型提高了35min左右。
表2蔗糖磷酸化酶野生型和突变体50℃下的半衰期
利用DSC测定蛋白质的结构稳定性，温度范围设置为20～100℃，确定蛋白质的Tm，将纯化后的蔗糖磷酸化酶突变体I31F、T219L、S360A、T263L及T31F\T219L\S360A\T263L酶液调节蛋白浓度为0.5mg·mL-1后进行测定。由图6和表3可知，蔗糖磷酸化酶突变体T219L和T31F\T219L\S360A\T263L的Tm值有所提高，提高了4℃左右，通过这个结果进一步证明了蔗糖磷酸化酶突变体T219L和T31F\T219L\S360A\T263L的热稳定性确实有所提高。
表3蔗糖磷酸化酶野生型和突变体的Tm
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> 一种蔗糖磷酸化酶突变体及其应用
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<170> PatentIn version 3.3
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gacgctgctt tcggtgactg ggctgacgtt gaagctctgg gtgaagaata ctacctgatg 240
ttcgacttca tgatcaacca catctctcgt gaatctgtta tgtaccagga cttcaaaaaa 300
aaccacgacg actctaaata caaagacttc ttcatccgtt gggaaaaatt ctgggctaaa 360
gctggtgaaa accgtccgac ccaggctgac gttgacctga tctacaaacg taaagacaaa 420
gctccgaccc aggaaatcac cttcgacgac ggtactaccg aaaacctgtg gaacaccttc 480
ggtgaagaac agatcgacat cgacgttaac tctgctatcg ctaaagaatt tatcaaaacc 540
accctggaag acatggttaa acacggtgct aacctgatcc gtctggacgc tttcgcttac 600
gctgttaaaa aagttgacac caacgacttc ttcgttgaac cggaaatctg ggacaccctg 660
aacgaagttc gtgaaatcct gaccccgctg aaagctgaaa tcctgccgga aatccacgaa 720
cactactcta tcccgaaaaa aatcaacgac cacggttact tcacctacga cttcgctctg 780
ccgatgacca ccctgtacac cctgtactct ggtaaaacca accagctcgc taaatggctg 840
aaaatgtctc cgatgaaaca gttcaccacc ctggacaccc acgacggtat cggtgttgtt 900
gacgctcgtg acatcctgac cgacgacgaa atcgactacg cttctgaaca gctctacaaa 960
gttggtgcta acgttaaaaa aacctactct tctgcttctt acaacaacct ggacatctac 1020
cagatcaact ctacctacta ctctgctctg ggtaacgacg acgctgctta cctgctgtct 1080
cgtgttttcc aggttttcgc tccgggtatc ccgcagatat actacgttgg tctgctggct 1140
ggtgaaaacg acatcgctct gctggaatct accaaagaag gtcgtaacat caaccgtcac 1200
tactacaccc gtgaagaagt taaatctgaa gttaaacgtc cggttgttgc taacctgctg 1260
aaactgctgt cttggcgtaa cgaatctccg gctttcgacc tggctggttc tatcaccgtt 1320
gacaccccga ccgacaccac catcgttgtt acccgtcagg acgaaaacgg tcagaacaaa 1380
gctgttctga ccgctgacgc tgctaacaaa accttcgaaa tcgttgaaaa cggtcagacc 1440
gttatgtctt ctgacaacct gacccagaac taa 1473
<210> 2
<211> 28
<212> DNA
<213> 人工序列
<400> 2
gttctgaaag aagacttcgg tgacgcta 28
<210> 3
<211> 28
<212> DNA
<213> 人工序列
<400> 3
accgatagcg tcaccgaagt cttctttc 28
<210> 4
<211> 28
<212> DNA
<213> 人工序列
<400> 4
atcgttgtta cccgtggcga cgaaaacg 28
<210> 5
<211> 28
<212> DNA
<213> 人工序列
<400> 5
ctgaccgttt tcgtcgccac gggtaaca 28
<210> 6
<211> 28
<212> DNA
<213> 人工序列
<400> 6
aaaatcaacg accaccttta cttcacct 28
<210> 7
<211> 28
<212> DNA
<213> 人工序列
<400> 7
gtcgtaggtg aagtaaaggt ggtcgttg 28
<210> 8
<211> 28
<212> DNA
<213> 人工序列
<400> 8
ctgaccccgc tgaaaatgga aatcctgc 28
<210> 9
<211> 28
<212> DNA
<213> 人工序列
<400> 9
ttccggcagg atttccattt tcagcggg 28
<210> 10
<211> 28
<212> DNA
<213> 人工序列
<400> 10
accttcgacg acggtggtac cgaaaacc 28
<210> 11
<211> 28
<212> DNA
<213> 人工序列
<400> 11
ccacaggttt tcggtaccac cgtcgtcg 28
<210> 12
<211> 28
<212> DNA
<213> 人工序列
<400> 12
accgaaaacc tgtggtgcac cttcggtg 28
<210> 13
<211> 28
<212> DNA
<213> 人工序列
<400> 13
ttcttcaccg aaggtgcacc acaggttt 28
<210> 14
<211> 28
<212> DNA
<213> 人工序列
<400> 14
ccggaaatct gggacctcct gaacgaag 28
<210> 15
<211> 28
<212> DNA
<213> 人工序列
<400> 15
acgaacttcg ttcaggaggt cccagatt 28
<210> 16
<211> 28
<212> DNA
<213> 人工序列
<400> 16
gctgcttacc tgctggcgcg tgttttcc 28
<210> 17
<211> 28
<212> DNA
<213> 人工序列
<400> 17
aacctggaaa acacgcgcca gcaggtaa 28
<210> 18
<211> 28
<212> DNA
<213> 人工序列
<400> 18
atcccgaaaa aaatcgccga ccacggtt 28
<210> 19
<211> 28
<212> DNA
<213> 人工序列
<400> 19
gaagtaaccg tggtcggcga tttttttc 28
<210> 20
<211> 28
<212> DNA
<213> 人工序列
<400> 20
ttcgctctgc cgatgctcac cctgtaca 28
<210> 21
<211> 28
<212> DNA
<213> 人工序列
<400> 21
cagggtgtac agggtgagca tcggcaga 28
<210> 22
<211> 490
<212> PRT
<213> 人工序列
<400> 22
Met Glu Ile Gln Asn Lys Ala Met Leu Ile Thr Tyr Ala Asp Ser Leu
1 5 10 15
Gly Lys Asn Leu Lys Asp Val His Gln Val Leu Lys Glu Asp Ile Gly
20 25 30
Asp Ala Ile Gly Gly Val His Leu Leu Pro Phe Phe Pro Ser Thr Gly
35 40 45
Asp Arg Gly Phe Ala Pro Ala Asp Tyr Thr Arg Val Asp Ala Ala Phe
50 55 60
Gly Asp Trp Ala Asp Val Glu Ala Leu Gly Glu Glu Tyr Tyr Leu Met
65 70 75 80
Phe Asp Phe Met Ile Asn His Ile Ser Arg Glu Ser Val Met Tyr Gln
85 90 95
Asp Phe Lys Lys Asn His Asp Asp Ser Lys Tyr Lys Asp Phe Phe Ile
100 105 110
Arg Trp Glu Lys Phe Trp Ala Lys Ala Gly Glu Asn Arg Pro Thr Gln
115 120 125
Ala Asp Val Asp Leu Ile Tyr Lys Arg Lys Asp Lys Ala Pro Thr Gln
130 135 140
Glu Ile Thr Phe Asp Asp Gly Thr Thr Glu Asn Leu Trp Asn Thr Phe
145 150 155 160
Gly Glu Glu Gln Ile Asp Ile Asp Val Asn Ser Ala Ile Ala Lys Glu
165 170 175
Phe Ile Lys Thr Thr Leu Glu Asp Met Val Lys His Gly Ala Asn Leu
180 185 190
Ile Arg Leu Asp Ala Phe Ala Tyr Ala Val Lys Lys Val Asp Thr Asn
195 200 205
Asp Phe Phe Val Glu Pro Glu Ile Trp Asp Thr Leu Asn Glu Val Arg
210 215 220
Glu Ile Leu Thr Pro Leu Lys Ala Glu Ile Leu Pro Glu Ile His Glu
225 230 235 240
His Tyr Ser Ile Pro Lys Lys Ile Asn Asp His Gly Tyr Phe Thr Tyr
245 250 255
Asp Phe Ala Leu Pro Met Thr Thr Leu Tyr Thr Leu Tyr Ser Gly Lys
260 265 270
Thr Asn Gln Leu Ala Lys Trp Leu Lys Met Ser Pro Met Lys Gln Phe
275 280 285
Thr Thr Leu Asp Thr His Asp Gly Ile Gly Val Val Asp Ala Arg Asp
290 295 300
Ile Leu Thr Asp Asp Glu Ile Asp Tyr Ala Ser Glu Gln Leu Tyr Lys
305 310 315 320
Val Gly Ala Asn Val Lys Lys Thr Tyr Ser Ser Ala Ser Tyr Asn Asn
325 330 335
Leu Asp Ile Tyr Gln Ile Asn Ser Thr Tyr Tyr Ser Ala Leu Gly Asn
340 345 350
Asp Asp Ala Ala Tyr Leu Leu Ser Arg Val Phe Gln Val Phe Ala Pro
355 360 365
Gly Ile Pro Gln Ile Tyr Tyr Val Gly Leu Leu Ala Gly Glu Asn Asp
370 375 380
Ile Ala Leu Leu Glu Ser Thr Lys Glu Gly Arg Asn Ile Asn Arg His
385 390 395 400
Tyr Tyr Thr Arg Glu Glu Val Lys Ser Glu Val Lys Arg Pro Val Val
405 410 415
Ala Asn Leu Leu Lys Leu Leu Ser Trp Arg Asn Glu Ser Pro Ala Phe
420 425 430
Asp Leu Ala Gly Ser Ile Thr Val Asp Thr Pro Thr Asp Thr Thr Ile
435 440 445
Val Val Thr Arg Gln Asp Glu Asn Gly Gln Asn Lys Ala Val Leu Thr
450 455 460
Ala Asp Ala Ala Asn Lys Thr Phe Glu Ile Val Glu Asn Gly Gln Thr
465 470 475 480
Val Met Ser Ser Asp Asn Leu Thr Gln Asn
485 490
Claims (10)
1.一种蔗糖磷酸化酶突变体，其特征在于，所述蔗糖磷酸化酶突变体与氨基酸序列如SEQ ID NO.22所示的蔗糖磷酸化酶相比，第219位氨基酸由苏氨酸突变为了亮氨酸；
或者，所述蔗糖磷酸化酶突变体与氨基酸序列如SEQ ID NO.22所示的蔗糖磷酸化酶相比，第31位氨基酸由异亮氨酸突变为了苯丙氨酸，第219位氨基酸由苏氨酸突变为了亮氨酸，第360位氨基酸由丝氨酸突变为了丙氨酸，第263位氨基酸由苏氨酸突变为了亮氨酸。
2.如权利要求1所述的一种蔗糖磷酸化酶突变体，其特征在于，编码所述蔗糖磷酸化酶的核苷酸序列如SEQ ID NO.1所示。
3.编码权利要求1或2所述的蔗糖磷酸化酶突变体的基因。
4.携带权利要求3所述基因的重组质粒。
5.如权利要求4所述的重组质粒，其特征在于，所述重组质粒的载体为pET-28a质粒。
6.携带权利要求3所述基因，或权利要求4或5所述重组质粒的宿主细胞。
7.如权利要求6所述的宿主细胞，其特征在于，所述宿主细胞为细菌或真菌。
8.如权利要求7所述的宿主细胞，其特征在于，所述宿主细胞为大肠杆菌。
9.权利要求1所述蔗糖磷酸化酶突变体的制备方法，其特征在于，所述方法为将权利要求6或7所述的宿主细胞按1-5％接种量接入到TB液体培养基中，于35-39℃，200-220r/min培养至菌密度OD达到0.5-0.7后，加入IPTG诱导剂于23-27℃，200-220r/min诱导20-30h后收集菌液，离心收集菌体，重悬后破碎菌体，离心收集上清，分离上清液中权利要求1所述的蔗糖磷酸化酶突变体。
10.权利要求1所述蔗糖磷酸化酶突变体，或权利要求3所述基因，或权利要求4所述重组质粒，或权利要求6所述宿主细胞，或权利要求9所述制备方法在制备果糖中的应用。
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| CN109423485A (zh) * | 2017-08-25 | 2019-03-05 | 中国科学院微生物研究所 | 蔗糖磷酸化酶突变体及其应用 |
| CN110734899A (zh) * | 2019-10-31 | 2020-01-31 | 江南大学 | 一种酶活提高的蔗糖磷酸化酶突变体及其构建方法和应用 |
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