CN115261350A - 一种淀粉蔗糖酶的突变体及其在生产α-熊果苷中的应用 - Google Patents
一种淀粉蔗糖酶的突变体及其在生产α-熊果苷中的应用 Download PDFInfo
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Abstract
本发明涉及一种淀粉蔗糖酶的突变体及其在生产α‑熊果苷中的应用。本发明将氨基酸序列如SEQ ID NO.1所示的野生淀粉蔗糖酶XcAS的第25位的丙氨酸突变为谷氨酸、第326位的缬氨酸突变为丙氨酸、第245位的苯丙氨酸突变为、第285位的酪氨酸突变为丙氨酸得到淀粉蔗糖酶突变体，并在大肠杆菌中表达所述淀粉蔗糖酶突变体，得到重组基因工程菌。以基因工程菌经过诱导表达后获得的湿菌体作为生物催化剂，以对苯二酚和蔗糖作为底物，通过底物梯度投料方式催化反应生产α‑熊果苷，150g/L湿菌体可以生产161.8g/Lα‑熊果苷，对苯二酚的摩尔转化率达到99.05％，为α‑熊果苷的工业化大规模生产提供了良好的技术支持。
Description
技术领域
本发明公开了一种淀粉蔗糖酶的突变体及其在生产α-熊果苷中的应用，属于酶工程技术领域。
背景技术
α-熊果苷(α-arbutin)，化学名称4-羟基苯-α-D-吡喃葡萄糖苷(4-hydroxyphenyl-α-D-glucopyranoside)，是一种高效安全的美白添加剂，广泛应用于化妆品中。它能够通过抑制酪氨酸酶活性减少体内黑色素的合成从而减少皮肤黑色素沉积达到美白护肤的作用。其生物活性10倍高于其同分异构体β-熊果苷(β-arbutin)。另外α-熊果苷还具有良好的抗氧化，抗菌和消炎作用，因此其作为一种安全高效的美白添加剂，具有广阔的应用前景。α-熊果苷的制备方法有化学合成法、发酵法和生物催化法。其中，化学合成法因为其反应条件苛刻，立体选择性低，催化剂对环境的污染及大量产生的副产物等问题制约了其工业话应用；发酵法生产α-熊果苷由于底物转化率低，发酵周期较长，产量较低，生产成本高，同样不适用于大规模生产；而生物催化法由于α-熊果苷产量高，副产物少，产品提取收率高，生产过程绿色环保，是目前生产α-熊果苷最常用的方法。
生物催化法合成α-熊果苷所用的酶主要有蔗糖磷酸化酶、葡聚糖蔗糖酶、蔗糖异构酶、脂肪酶和淀粉蔗糖酶，均以对苯二酚作为糖苷受体，以不同的糖类作为葡萄糖苷供体催化转糖苷反应合成α-熊果苷。专利CN 112375724 A公布了将经过定点饱和突变并筛选得到的蔗糖磷酸化酶(SmSP)重组到枯草芽孢杆菌中，利用重组全细胞催化，α-熊果苷的产量达到115.8g/L，底物HQ的摩尔转化率为93.6％；专利CN 112300977 A利用表达蔗糖磷酸化酶(SmSP)的重组枯草芽孢杆菌全细胞催化生产α-熊果苷，α-熊果苷产量为61.1g/L，底物对苯二酚的摩尔转化率为49.4％。专利CN 106148256 A公布了将淀粉蔗糖酶基因重组到导入大肠杆菌中，构建基因工程菌，用其来生产α-熊果苷，底物的转化效率达到99％,α-熊果苷的产量为45.36g/L。
尽管目前已经报道了多种来源的酶用于合成α-熊果苷，在催化过程中存由于酶活性低等问题，导致底物对苯二酚转化率低、残留量大、产物含量低等问题，限制了应用。而利用纯酶工艺催化制备α-熊果苷需要对酶进行纯化，生产成本高，不利于工业化生产。因此，如何构建高效的微生物细胞并且匹配有效的工艺来高效的催化合成α-熊果苷，是本领域亟待解决的问题。
发明内容
针对目前存在的淀粉蔗糖酶活较低、对苯二酚转化率低、α-熊果苷产量低等问题，本发明通过对来源于野油菜黄单胞菌(Xanthomonas campestris pv.campestrisstr.ATCC 33913)的淀粉蔗糖酶XcAS进行改造，以获得酶活及苯酚转化率提高的突变体，将突变体应用于的α-熊果苷生产，以提高α-熊果苷的产量和对苯二酚转化率。
本发明第一个目的是提供了淀粉蔗糖酶突变体A25E-V326A-F245A-Y285A，是将氨基酸序列如SEQ ID NO.1所示的野生淀粉蔗糖酶XcAS的第25位的丙氨酸突变为谷氨酸，第326位的缬氨酸突变为丙氨酸，第245位的苯丙氨酸突变为丙氨酸，第285位的酪氨酸突变为丙氨酸。
SEQ ID No.1序列如下：
Met Ile Ala Ser Ser Pro Ile Asp Ala Ala Ala Leu Arg Ala Ser Val AlaAla Ala Leu Asp Pro Ser Thr Ala Val Ala Thr Leu Ala Arg Phe Asp Thr His AlaPro Arg Leu Leu Asp Ala Leu Ser Thr Leu Tyr Gly Asp His Ala Asp Tyr Val ThrTrp Leu Pro Gln Trp Leu Ser Ala Leu Gly Val Val Ala Gln Ala Arg Pro Ala AlaLeu Arg His Leu Asp Asp Ser Arg Ala Pro Gly Trp Phe Gly Gln Gln Asp Met LeuGly Tyr Ser Ala Tyr Val Asp Arg Phe Ala Gly Thr Leu Arg Gly Val Ala Glu ArgVal Pro Tyr Leu Gln Glu Leu Gly Val Arg Tyr Leu His Leu Leu Pro Phe Leu ArgAla Arg Ala Gly Asp Asn Asp Gly Gly Phe Ala Val Ser Asp Tyr Gly Gln Val GluPro Ala Leu Gly Ser Asn Asp Asp Leu Val Ala Leu Thr Ala Arg Leu Arg Ala AlaAsn Ile Ser Leu Cys Ala Asp Phe Val Leu Asn His Thr Ala Asp Asp His Ala TrpAla Gln Ala Ala Arg Ala Gly Asp Thr Arg Tyr Leu Asp Tyr Tyr His His Phe AlaAsp Arg Asn Ala Pro Asp Gln Tyr Asp Thr Thr Leu Val Gln Val Phe Pro Gln ThrAla Pro Gly Asn Phe Thr Trp Val Asp Glu Thr Arg Gln Trp Met Trp Thr Thr PheTyr Pro Tyr Gln Trp Asp Leu Asn Trp Ser Asn Pro Ala Val Phe Gly Glu Met AlaLeu Ala Met Leu Glu Leu Ala Asn Leu Gly Val Glu Ala Phe Arg Leu Asp Ser ThrAla Tyr Leu Trp Lys Arg Pro Gly Thr Asn Cys Met Asn Gln Pro Glu Ala His ThrIle Leu Val Ala Leu Arg Ala Val Ala Asp Ile Val Ala Pro Ser Val Val Met LysAla Glu Ala Ile Val Pro Met Ala Glu Leu Pro Pro Tyr Phe Gly Ser Gly Val GlnArg Gly His Glu Cys His Leu Ala Tyr His Ser Thr Leu Met Ala Ala Gly Trp SerAla Leu Ala Leu Gln Arg Gly Asp Ile Leu Gln Asp Val Ile Ala His Ser Pro ProLeu Pro Pro Asn Cys Ala Trp Leu Ser Tyr Val Arg Cys His Asp Asp Ile Gly TrpAsn Val Leu Gln His Glu Ala Ala Gly Thr Ala Ala Gln Pro Pro Phe Ser Leu ArgGlu Val Ala Gln Phe Tyr Ala Asn Ala Val Pro Gly Ser Tyr Ala Arg Gly Glu SerPhe Gln Ser Ser Gly Asp Gly Val His Gly Thr Asn Gly Met Ser Ala Ala Leu ValGly Val Gln Ala Ala His Glu His Ala Asp Ala Ala Ala Ala Ala Arg Ala Val AspArg Leu Val Leu Leu Tyr Ala Val Ser Leu Ala Met Pro Gly Val Pro Leu Ile TyrMet Gly Asp Glu Leu Ala Leu Pro Asn Asp Thr Ala Tyr Leu Asp Asp Ala Gln ArgArg His Glu Gly Arg Trp Leu His Arg Pro Ala Met Ala Trp Glu Leu Ala Ala GlnArg His Asp Ala Ser Thr Leu Ala Gly ThrVal Tyr Thr Arg Leu Arg Ala Leu IleArg Leu Arg Ala Gly Leu Pro Ala Leu Ala Ala Thr Gln Ser Leu Gly Ser Val AlaLeu Gly Asp Ala Arg Leu Phe Ala Leu Thr Arg Gly Asp Ser Phe Leu Ala Val HisAsn Phe Ser Asp Val Pro Leu Pro Val Asp Leu Thr Gln Thr Gly His Ala Leu TrpAla Val Leu Asp Thr Asp Gly Thr Gly Asp Ala Pro Glu Pro His Thr Glu Leu LeuLeu Pro Ala Tyr Gly Val Arg Trp Leu Gln Arg Arg
本发明的第二个目的是提供编码所述淀粉蔗糖酶突变体的氨基酸序列和基因核苷酸序列。
进一步地，所述的淀粉蔗糖酶突变体的氨基酸序列如SEQ ID NO.2所示。
SEQ ID No.2序列如下：
Met Ile Ala Ser Ser Pro Ile Asp Ala Ala Ala Leu Arg Ala Ser Val AlaAla Ala Leu Asp Pro Ser Thr Glu Val Ala Thr Leu Ala Arg Phe Asp Thr His AlaPro Arg Leu Leu Asp Ala Leu Ser Thr Leu Tyr Gly Asp His Ala Asp Tyr Val ThrTrp Leu Pro Gln Trp Leu Ser Ala Leu Gly Val Val Ala Gln Ala Arg Pro Ala AlaLeu Arg His Leu Asp Asp Ser Arg Ala Pro Gly Trp Phe Gly Gln Gln Asp Met LeuGly Tyr Ser Ala Tyr Val Asp Arg Phe Ala Gly Thr Leu Arg Gly Val Ala Glu ArgVal Pro Tyr Leu Gln Glu Leu Gly Val Arg Tyr Leu His Leu Leu Pro Phe Leu ArgAla Arg Ala Gly Asp Asn Asp Gly Gly Phe Ala Val Ser Asp Tyr Gly Gln Val GluPro Ala Leu Gly Ser Asn Asp Asp Leu Val Ala Leu Thr Ala Arg Leu Arg Ala AlaAsn Ile Ser Leu Cys Ala Asp Phe Val Leu Asn His Thr Ala Asp Asp His Ala TrpAla Gln Ala Ala Arg Ala Gly Asp Thr Arg Tyr Leu Asp Tyr Tyr His His Phe AlaAsp Arg Asn Ala Pro Asp Gln Tyr Asp Thr Thr Leu Val Gln Val Phe Pro Gln ThrAla Pro Gly Asn Phe Thr Trp Val Asp Glu Thr Arg Gln Trp Met Trp Thr Thr AlaTyr Pro Tyr Gln Trp Asp Leu Asn Trp Ser Asn Pro Ala Val Phe Gly Glu Met AlaLeu Ala Met Leu Glu Leu Ala Asn Leu Gly Val Glu Ala Phe Arg Leu Asp Ser ThrAla Ala Leu Trp Lys Arg Pro Gly Thr Asn Cys Met Asn Gln Pro Glu Ala His ThrIle Leu Val Ala Leu Arg Ala Val Ala Asp Ile Val Ala Pro Ser Val Val Met LysAla Glu Ala Ile Ala Pro Met Ala Glu Leu Pro Pro Tyr Phe Gly Ser Gly Val GlnArg Gly His Glu Cys His Leu Ala Tyr His Ser Thr Leu Met Ala Ala Gly Trp SerAla Leu Ala Leu Gln Arg Gly Asp Ile Leu Gln Asp Val Ile Ala His Ser Pro ProLeu Pro Pro Asn Cys Ala Trp Leu Ser Tyr Val Arg Cys His Asp Asp Ile Gly TrpAsn Val Leu Gln His Glu Ala Ala Gly Thr Ala Ala Gln Pro Pro Phe Ser Leu ArgGlu Val Ala Gln Phe Tyr Ala Asn Ala Val Pro Gly Ser Tyr Ala Arg Gly Glu SerPhe Gln Ser Ser Gly Asp Gly Val His Gly Thr Asn Gly Met Ser Ala Ala Leu ValGly Val Gln Ala Ala His Glu His Ala Asp Ala Ala Ala Ala Ala Arg Ala Val AspArg Leu Val Leu Leu Tyr Ala Val Ser Leu Ala Met Pro Gly Val Pro Leu Ile TyrMet Gly Asp Glu Leu Ala Leu Pro Asn Asp Thr Ala Tyr Leu Asp Asp Ala Gln ArgArg His Glu Gly Arg Trp Leu His Arg Pro Ala Met Ala Trp Glu Leu Ala Ala GlnArg His Asp Ala Ser Thr Leu Ala Gly Thr Val Tyr Thr Arg Leu Arg Ala Leu IleArg Leu Arg Ala Gly Leu Pro Ala LeuAlaAla Thr Gln Ser Leu Gly Ser Val Ala LeuGly Asp Ala Arg Leu Phe Ala Leu Thr Arg Gly Asp Ser Phe Leu Ala Val His AsnPhe SerAsp Val Pro Leu Pro ValAsp Leu Thr Gln Thr Gly His Ala Leu Trp Ala ValLeu Asp Thr Asp Gly Thr Gly Asp Ala Pro Glu Pro His Thr Glu Leu Leu Leu ProAla Tyr Gly Val Arg Trp Leu Gln Arg Arg
进一步地，编码所述淀粉蔗糖酶突变体的核苷酸序列如SEQ IDNO.3所示。
SEQ ID No.3序列如下：
ATGATCGCTTCCTCCCCCATCGATGCCGCTGCACTGCGCGCCAGCGTTGCCGCCGCACTGGATCCGTCCACTGAAGTAGCAACACTGGCGCGTTTCGATACGCATGCCCCGCGCCTGCTCGACGCGCTGTCCACGCTCTACGGCGACCATGCCGACTACGTCACCTGGTTGCCACAGTGGCTGAGCGCACTGGGCGTGGTCGCACAGGCACGGCCAGCGGCCTTGCGGCACCTCGACGACAGCCGCGCACCGGGCTGGTTCGGCCAGCAGGACATGCTGGGCTACAGCGCCTACGTGGACCGTTTTGCCGGCACGCTGCGCGGTGTTGCCGAGCGCGTTCCGTATCTGCAGGAACTGGGCGTGCGGTATCTGCACCTGCTGCCATTCCTGCGCGCACGCGCCGGCGACAACGACGGCGGTTTTGCGGTCAGCGATTACGGCCAGGTGGAACCGGCCCTGGGCAGCAACGACGATCTGGTCGCACTCACCGCACGCCTACGCGCGGCCAACATCAGCCTGTGCGCAGATTTCGTGCTCAACCACACCGCCGACGACCACGCCTGGGCACAGGCCGCACGCGCTGGCGATACGCGTTATCTCGACTACTACCATCACTTCGCCGACCGCAACGCACCTGACCAGTACGACACCACCCTGGTGCAGGTGTTCCCGCAGACTGCGCCCGGCAACTTCACCTGGGTGGACGAGACCCGGCAGTGGATGTGGACCACGGCCTATCCCTATCAGTGGGATTTGAACTGGAGCAACCCGGCGGTGTTCGGCGAGATGGCATTGGCGATGCTGGAGCTGGCCAATCTGGGCGTGGAAGCGTTCCGACTCGATTCCACGGCGGCCCTGTGGAAACGCCCGGGCACCAACTGCATGAACCAGCCCGAGGCGCACACCATCTTGGTGGCATTGCGTGCCGTGGCCGACATCGTGGCGCCCTCGGTGGTGATGAAGGCCGAAGCCATCGCGCCGATGGCGGAGCTGCCGCCCTACTTCGGCAGCGGCGTGCAGCGCGGGCACGAGTGTCACCTGGCCTATCACAGCACGCTGATGGCGGCTGGCTGGTCGGCACTGGCCCTGCAACGCGGCGACATCCTGCAGGACGTGATCGCGCACAGCCCACCGCTGCCGCCCAACTGCGCCTGGCTCAGCTACGTGCGCTGCCATGACGACATCGGCTGGAACGTGTTGCAGCACGAGGCCGCCGGCACTGCCGCGCAACCGCCGTTCTCGCTGCGCGAAGTGGCGCAGTTCTACGCCAACGCCGTGCCCGGCAGTTACGCGCGCGGCGAAAGTTTCCAGAGCAGCGGCGATGGCGTGCATGGCACCAATGGCATGAGCGCGGCATTGGTGGGCGTGCAGGCTGCGCACGAACACGCCGATGCGGCGGCGGCCGCCCGTGCGGTGGATCGCCTGGTGTTGCTCTACGCCGTGTCGCTGGCGATGCCGGGTGTTCCGCTGATCTACATGGGCGATGAACTCGCGCTACCCAACGACACCGCCTACCTGGACGATGCGCAGCGACGCCACGAAGGACGCTGGCTGCATCGCCCGGCGATGGCGTGGGAGCTGGCCGCGCAGCGGCATGACGCCTCCACGTTGGCAGGTACGGTGTACACGCGCTTGCGTGCATTGATCCGGCTGCGTGCGGGCCTGCCTGCGCTGGCTGCCACGCAATCGCTGGGCAGCGTCGCCCTCGGTGATGCCCGGCTGTTTGCACTGACGCGTGGCGACAGTTTCCTGGCGGTGCACAATTTCAGCGACGTGCCACTGCCGGTGGATCTCACCCAGACCGGACATGCCCTATGGGCTGTGCTGGACACCGACGGCACTGGCGATGCGCCCGAACCGCATACGGAGCTGTTGCTTCCGGCCTATGGCGTGCGCTGGTTGCAGCGTCGTTGA
本发明还涉及含有所述淀粉蔗糖酶突变体的编码基因的重组表达载体以及基因工程菌。
具体的，所述重组载体优选为重组质粒，所述重组质粒通过如下方法构建获得：将SEQ ID No.3所示基因克隆到pET-28a(+)表达载体上，得到重组质粒pET-28a-XcAS。基因工程菌构建方法具体为：将表达重组质粒转化至大肠杆菌BL21感受态细胞，获得重组大肠杆菌E.coli BL21-pET-28a-DXcAS，即所述基因工程菌。
本发明还涉及所述基因工程菌在制备α-熊果苷中的应用。
具体的，所述应用为：以蔗糖和对苯二酚作为底物，以所述基因工程菌发酵获得的湿菌体为催化剂，在反应介质中进行全细胞转化制备α-熊果苷。所述湿菌体的添加量不低于120g/L。
所述反应介质为pH6.0～8.0的缓冲液，反应温度为25℃～45℃。
底物采用递减梯度补料的方法流加入反应介质中。具体的，初始的对苯二酚浓度为180.0mmol/L、蔗糖浓度为720mmol/L、Vc 1mM，4时后再补加150.0mmol/L的对苯二酚和600.0mmol/L的蔗糖，8小时后再补加120.0mmol/L的对苯二酚和480.0mmol/L的蔗糖，12小时再补加90.0mmol/L的对苯二酚和360.0mmol/L的蔗糖，16小时再补加60.0mmol/L的对苯二酚和240.0mmol/L的蔗糖。
所述基因工程菌湿菌体通常需要经过预培养，具体培养方法如下：
将重组重组大肠杆菌E.coli BL21-PET-28a-DXcAS经种子培养基活化后转入发酵培养基，于30～37℃、pH 6.0～8.0条件下培养，OD600达到15～25时，培养温度降到20～25℃，加入IPTG诱导淀粉蔗糖酶酶突变体的表达，持续诱导至OD不在增高停止诱导
进一步地，种子培养基按g/L计含有：蛋白胨5～15，酵母粉3～7，NaCl 8～12。
进一步地，发酵培养基按g/L计含有：甘油10～15，蛋白胨10～20，酵母粉15～30，磷酸氢二钾10～20，磷酸二氢钾2～36，甘油流加，氨水用于调节pH。
进一步地，所述的IPTG的浓度为0.05～0.5mmol/L。
与现有技术相比，本发明有益效果主要体现在：本发明通过对来源于野油菜黄单胞菌(Xanthomonas campestris pv.campestris str.ATCC33913)的淀粉蔗糖酶XcAS进行改造，获得酶活及苯酚转化率提高的突变体。通过对底物对苯二酚进行梯度递减补料策略，α-熊果苷产量达到161.8g/L，底物对苯二酚的转化率达到99.05％，为α-熊果苷的工业化大规模生产提供了良好的技术支持。
具体实施方式
下面结合实施例对本发明的技术方案进行详细说明。以下采用的试剂和生物材料如未特别说明，均为商业化产品。
实施例1：野生型淀粉蔗糖酶基因的合成和表达载体的构建：
根据NCBI报道的野油菜黄单胞菌(Xanthomonas campestris pv.CampestrisATCC 33913)的淀粉蔗糖酶基因XcAS(GeneBank NC_003902.1REGION:4002308…4004221)，按照大肠杆菌密码子偏好性交由生工生物工程(上海)股份有限公司优化合成编码基因，并将其构建到pET-28a(+)表达载体上，得到重组质粒pET-28a-XcAS。淀粉蔗糖酶基因XcAS编码氨基酸序列如SEQ ID NO1所示。
实施例2：淀粉蔗糖酶突变体A25E-V326A-F245A-Y285A的制备(1)突变体酶A25E的制备
(a)设计定点突变的正向引物(pA25E-F：5’-CTGGATCCGTCCACTGAAGTAGCAACACTGGCG-3’，下划线为突变碱基)和反向引物(pA25E-R：5’-CGCCAGTGTTGCTACTTCAGTGGACGGATCCAG-3’，下划线为突变碱基)。
(b)PCR反应体系(50μL)为：5μL10*DNAPolymerase Buffer，1.5μL正向引物pA25E-F(10umol/L)，1.5μL下游引物pA25E-R(10umol/L)，1μL模板pET-28a-DXcAS，其余为无菌水。
PCR反应程序为95℃预变性2min；30个循环(95℃变性20s，580℃退火20s，72℃延伸5min)；72℃延伸10min，最后16℃保温。
(c)PCR产物经Dpn I(购于Takara公司)消化，热激转化大肠杆菌BL21感受态细胞，涂布于LB固体培养基(0.5％氯化钠，0.5％酵母提取物，1％胰蛋白胨，2％琼脂粉，含有30μg/mL的Kan抗性)，37℃过夜培养。挑取单克隆至LB试管培养，提取质粒进行测序，突变质粒测序正确。突变质粒命名为pET-28a-XcAS-A25E，突变体A25E如SEQ ID NO.4所示。
SEQ ID NO.4：
Met Ile Ala Ser Ser Pro Ile Asp Ala Ala Ala Leu Arg Ala Ser Val AlaAla Ala Leu Asp Pro Ser Thr Glu Val Ala Thr Leu Ala Arg Phe Asp Thr His AlaPro Arg Leu Leu Asp Ala Leu Ser Thr Leu Tyr Gly Asp His Ala Asp Tyr Val ThrTrp Leu Pro Gln Trp Leu Ser Ala Leu Gly Val Val Ala Gln Ala Arg Pro Ala AlaLeu Arg His Leu Asp Asp Ser Arg Ala Pro Gly Trp Phe Gly Gln Gln Asp Met LeuGly Tyr Ser Ala Tyr Val Asp Arg Phe Ala Gly Thr Leu Arg Gly Val Ala Glu ArgVal Pro Tyr Leu Gln Glu Leu Gly Val Arg Tyr Leu His Leu Leu Pro Phe Leu ArgAla Arg Ala Gly Asp Asn Asp Gly Gly Phe Ala Val Ser Asp Tyr Gly Gln Val GluPro Ala Leu Gly Ser Asn Asp Asp Leu Val Ala Leu Thr Ala Arg Leu Arg Ala AlaAsn Ile Ser Leu Cys Ala Asp Phe Val Leu Asn His Thr Ala Asp Asp His Ala TrpAla Gln Ala Ala Arg Ala Gly Asp Thr Arg Tyr Leu Asp Tyr Tyr His His Phe AlaAsp Arg Asn Ala Pro Asp Gln Tyr Asp Thr Thr Leu Val Gln Val Phe Pro Gln ThrAla Pro Gly Asn Phe Thr Trp Val Asp Glu Thr Arg Gln Trp Met Trp Thr Thr PheTyr Pro Tyr Gln Trp Asp Leu Asn Trp Ser Asn Pro Ala Val Phe Gly Glu Met AlaLeu Ala Met Leu Glu Leu Ala Asn Leu Gly Val Glu Ala Phe Arg Leu Asp Ser ThrAla Tyr Leu Trp Lys Arg Pro Gly Thr Asn Cys Met Asn Gln Pro Glu Ala His ThrIle Leu Val Ala Leu Arg Ala Val Ala Asp Ile Val Ala Pro Ser Val Val Met LysAla Glu Ala Ile Val Pro Met Ala Glu Leu Pro Pro Tyr Phe Gly Ser Gly Val GlnArg Gly His Glu Cys His Leu Ala Tyr His Ser Thr Leu Met Ala Ala Gly Trp SerAla Leu Ala Leu Gln Arg Gly Asp Ile Leu Gln Asp Val Ile Ala His Ser Pro ProLeu Pro Pro Asn Cys Ala Trp Leu Ser Tyr Val Arg Cys His Asp Asp Ile Gly TrpAsn Val Leu Gln His Glu Ala Ala Gly Thr Ala Ala Gln Pro Pro Phe Ser Leu ArgGlu Val Ala Gln Phe Tyr Ala Asn Ala Val Pro Gly Ser Tyr Ala Arg Gly Glu SerPhe Gln Ser Ser Gly Asp Gly Val His Gly Thr Asn Gly Met Ser Ala Ala Leu ValGly Val Gln Ala Ala His Glu His Ala Asp Ala Ala Ala Ala Ala Arg Ala Val AspArg Leu Val Leu Leu Tyr Ala Val Ser Leu Ala Met Pro Gly Val Pro Leu Ile TyrMet Gly Asp Glu Leu Ala Leu Pro Asn Asp Thr Ala Tyr Leu Asp Asp Ala Gln ArgArg His Glu Gly Arg Trp Leu His Arg Pro Ala Met Ala Trp Glu Leu Ala Ala GlnArg His Asp Ala Ser Thr Leu Ala Gly ThrVal Tyr Thr Arg Leu Arg Ala Leu IleArg Leu Arg Ala Gly Leu Pro Ala Leu Ala Ala Thr Gln Ser Leu Gly Ser Val AlaLeu Gly Asp Ala Arg Leu Phe Ala Leu Thr Arg Gly Asp Ser Phe Leu Ala Val HisAsn Phe Ser Asp Val Pro Leu Pro Val Asp Leu Thr Gln Thr Gly His Ala Leu TrpAla Val Leu Asp Thr Asp Gly Thr Gly Asp Ala Pro Glu Pro His Thr Glu Leu LeuLeu Pro Ala Tyr Gly Val Arg Trp Leu Gln Arg Arg
(2)双突变体酶A25E-V326A的制备
(a)设计定点突变的正向引物(pV326A-F：5’-AAGGCCGAAGCCATCGCGCCGATGGCGGAGCT-3’，下划线为突变碱基)和反向引物(pV326A-R：5’-AGCTCCGCCATCGGCGCGATGGCTTCGGCCTT-3’，下划线为突变碱基)。
(b)PCR反应体系(50μL)为：5μL10*DNAPolymerase Buffer，1.5μL正向引物pV326A-F(10umol/L)，1.5μL下游引物pV326A-R(10umol/L)，1μL模板pET-28a-DXcAS-A25E，其余为无菌水。
PCR反应程序为95℃预变性2min；30个循环(95℃变性20s，580℃退火20s，72℃延伸5min)；72℃延伸10min，最后16℃保温。
(c)PCR产物经Dpn I(购于Takara公司)消化，热激转化大肠杆菌BL21感受态细胞，涂布于LB固体培养基(0.5％氯化钠，0.5％酵母提取物，1％胰蛋白胨，2％琼脂粉，含有30μg/mL的Kan抗性)，37℃过夜培养。挑取单克隆至LB试管培养，提取质粒进行测序，突变质粒测序正确。突变质粒命名为pET-28a-XcAS-A25E-V326A，突变体A25E-V326A如SEQ ID NO.5所示。
SEQ ID NO.5：
Met Ile Ala Ser Ser Pro Ile Asp Ala Ala Ala Leu Arg Ala Ser Val AlaAla Ala Leu Asp Pro Ser Thr Glu Val Ala Thr Leu Ala Arg Phe Asp Thr His AlaPro Arg Leu Leu Asp Ala Leu Ser Thr Leu Tyr Gly Asp His Ala Asp Tyr Val ThrTrp Leu Pro Gln Trp Leu Ser Ala Leu Gly Val Val Ala Gln Ala Arg Pro Ala AlaLeu Arg His Leu Asp Asp Ser Arg Ala Pro Gly Trp Phe Gly Gln Gln Asp Met LeuGly Tyr Ser Ala Tyr Val Asp Arg Phe Ala Gly Thr Leu Arg Gly Val Ala Glu ArgVal Pro Tyr Leu Gln Glu Leu Gly Val Arg Tyr Leu His Leu Leu Pro Phe Leu ArgAla Arg Ala Gly Asp Asn Asp Gly Gly Phe Ala Val Ser Asp Tyr Gly GlnVal GluPro Ala Leu Gly Ser Asn Asp Asp Leu Val Ala Leu Thr Ala Arg Leu Arg Ala AlaAsn Ile Ser Leu Cys Ala Asp Phe Val Leu Asn His Thr Ala Asp Asp His Ala TrpAla Gln Ala Ala Arg Ala Gly Asp Thr Arg Tyr Leu Asp Tyr Tyr His His Phe AlaAsp Arg Asn Ala Pro Asp Gln Tyr Asp Thr Thr Leu Val Gln Val Phe Pro Gln ThrAla Pro Gly Asn Phe Thr Trp Val Asp Glu Thr Arg Gln Trp Met Trp Thr Thr PheTyr Pro Tyr Gln Trp Asp Leu Asn Trp Ser Asn Pro Ala Val Phe Gly Glu Met AlaLeu Ala Met Leu Glu Leu Ala Asn Leu Gly Val Glu Ala Phe Arg Leu Asp Ser ThrAla Tyr Leu Trp Lys Arg Pro Gly Thr Asn Cys Met Asn Gln Pro Glu Ala His ThrIle Leu Val Ala Leu Arg Ala Val Ala Asp Ile Val Ala Pro Ser Val Val Met LysAla Glu Ala Ile Ala Pro Met Ala Glu Leu Pro Pro Tyr Phe Gly Ser Gly Val GlnArg Gly His Glu Cys His Leu Ala Tyr His Ser Thr Leu Met Ala Ala Gly Trp SerAla Leu Ala Leu Gln Arg Gly Asp Ile Leu Gln Asp Val Ile Ala His Ser Pro ProLeu Pro Pro Asn Cys Ala Trp Leu Ser TyrVal Arg Cys His Asp Asp Ile Gly TrpAsn Val Leu Gln His Glu Ala Ala Gly Thr Ala Ala Gln Pro Pro Phe Ser Leu ArgGlu Val Ala Gln Phe Tyr Ala Asn Ala Val Pro Gly Ser Tyr Ala Arg Gly Glu SerPhe Gln Ser Ser Gly Asp Gly Val His Gly Thr Asn Gly Met Ser Ala Ala Leu ValGly Val Gln Ala Ala His Glu His Ala Asp Ala Ala Ala Ala Ala Arg Ala Val AspArg Leu Val Leu Leu Tyr Ala Val Ser Leu Ala Met Pro Gly Val Pro Leu Ile TyrMet Gly Asp Glu Leu Ala Leu Pro Asn Asp Thr Ala Tyr Leu Asp Asp Ala Gln ArgArg His Glu Gly Arg Trp Leu His Arg Pro Ala Met Ala Trp Glu Leu Ala Ala GlnArg His Asp Ala Ser Thr Leu Ala Gly ThrVal Tyr Thr Arg Leu Arg Ala Leu IleArg Leu Arg Ala Gly Leu Pro Ala Leu Ala Ala Thr Gln Ser Leu Gly Ser Val AlaLeu Gly Asp Ala Arg Leu Phe Ala Leu Thr Arg Gly Asp Ser Phe Leu Ala Val HisAsn Phe Ser Asp Val Pro Leu Pro Val Asp Leu Thr Gln Thr Gly His Ala Leu TrpAla Val Leu Asp Thr Asp Gly Thr Gly Asp Ala Pro Glu Pro His Thr Glu Leu LeuLeu Pro Ala Tyr Gly Val Arg Trp Leu Gln Arg Arg
(3)突变体酶A25E-V326A-F245A的制备
(a)设计定点突变的正向引物(pF245A-F：5’-TGGATGTGGACCACGGCCTATCCCTATCAGTGGGAT-3’，下划线为突变碱基)和反向引物(pF245A-R：5’-ATCCCACTGATAGGGATAGGCCGTGGTCCACATCCA-3’，下划线为突变碱基)。
(b)PCR反应体系(50μL)为：5μL10*DNAPolymerase Buffer，1.5μL正向引物pF245A-F(10umol/L)，1.5μL下游引物pF245A-R(10umol/L)，1μL模板pET-28a-XcAS-A25E-V326A，其余为无菌水。PCR反应程序为95℃预变性2min；30个循环(95℃变性20s，580℃退火20s，72℃延伸5min)；72℃延伸10min，最后16℃保温。
(c)PCR产物经Dpn I(购于Takara公司)消化，热激转化大肠杆菌BL21感受态细胞，涂布于LB固体培养基(0.5％氯化钠，0.5％酵母提取物，1％胰蛋白胨，2％琼脂粉，含有30μg/mL的Kan抗性)，37℃过夜培养。挑取单克隆至LB试管培养，提取质粒进行测序，突变质粒测序正确。突变质粒命名为pET-28a-DXcAS-A25E-V326A-F245A，突变体A25E-V326A-F245A如SEQ ID NO.6所示。
SEQ ID NO.6：
Met Ile Ala Ser Ser Pro Ile Asp Ala Ala Ala Leu Arg Ala Ser Val AlaAla Ala Leu Asp Pro Ser Thr Glu Val Ala Thr Leu Ala Arg Phe Asp Thr His AlaPro Arg Leu Leu Asp Ala Leu Ser Thr Leu Tyr Gly Asp His Ala Asp Tyr Val ThrTrp Leu Pro Gln Trp Leu Ser Ala Leu Gly Val Val Ala Gln Ala Arg Pro Ala AlaLeu Arg His Leu Asp Asp Ser Arg Ala Pro Gly Trp Phe Gly Gln Gln Asp Met LeuGly Tyr Ser Ala Tyr Val Asp Arg Phe Ala Gly Thr Leu Arg Gly Val Ala Glu ArgVal Pro Tyr Leu Gln Glu Leu Gly Val Arg Tyr Leu His Leu Leu Pro Phe Leu ArgAla Arg Ala Gly Asp Asn Asp Gly Gly Phe Ala Val Ser Asp Tyr Gly GlnVal GluPro Ala Leu Gly Ser Asn Asp Asp Leu Val Ala Leu Thr Ala Arg Leu Arg Ala AlaAsn Ile Ser Leu Cys Ala Asp Phe Val Leu Asn His Thr Ala Asp Asp His Ala TrpAla Gln Ala Ala Arg Ala Gly Asp Thr Arg Tyr Leu Asp Tyr Tyr His His Phe AlaAsp Arg Asn Ala Pro Asp Gln Tyr Asp Thr Thr Leu Val Gln Val Phe Pro Gln ThrAla Pro Gly Asn Phe Thr Trp Val Asp Glu Thr Arg Gln Trp Met Trp Thr Thr AlaTyr Pro Tyr Gln Trp Asp Leu Asn Trp Ser Asn Pro Ala Val Phe Gly Glu Met AlaLeu Ala Met Leu Glu Leu Ala Asn Leu Gly Val Glu Ala Phe Arg Leu Asp Ser ThrAla Tyr Leu Trp Lys Arg Pro Gly Thr Asn Cys Met Asn Gln Pro Glu Ala His ThrIle Leu Val Ala Leu Arg Ala Val Ala Asp Ile Val Ala Pro Ser Val Val Met LysAla Glu Ala Ile Ala Pro Met Ala Glu Leu Pro Pro Tyr Phe Gly Ser Gly Val GlnArg Gly His Glu Cys His Leu Ala Tyr His Ser Thr Leu Met Ala Ala Gly Trp SerAla Leu Ala Leu Gln Arg Gly Asp Ile Leu Gln Asp Val Ile Ala His Ser Pro ProLeu Pro Pro Asn Cys Ala Trp Leu Ser Tyr Val Arg Cys His Asp Asp Ile Gly TrpAsn Val Leu Gln His Glu Ala Ala Gly Thr Ala Ala Gln Pro Pro Phe Ser Leu ArgGlu Val Ala Gln Phe Tyr Ala Asn Ala Val Pro Gly Ser Tyr Ala Arg Gly Glu SerPhe Gln Ser Ser Gly Asp Gly Val His Gly Thr Asn Gly Met Ser Ala Ala Leu ValGly Val Gln Ala Ala His Glu His Ala Asp Ala Ala Ala Ala Ala Arg Ala Val AspArg Leu Val Leu Leu Tyr Ala Val Ser Leu Ala Met Pro Gly Val Pro Leu Ile TyrMet Gly Asp Glu Leu Ala Leu Pro Asn Asp Thr Ala Tyr Leu Asp Asp Ala Gln ArgArg His Glu Gly Arg Trp Leu His Arg Pro Ala Met Ala Trp Glu Leu Ala Ala GlnArg His Asp Ala Ser Thr Leu Ala Gly ThrVal Tyr Thr Arg Leu Arg Ala Leu IleArg Leu Arg Ala Gly Leu Pro Ala Leu Ala Ala Thr Gln Ser Leu Gly Ser Val AlaLeu Gly Asp Ala Arg Leu Phe Ala Leu Thr Arg Gly Asp Ser Phe Leu Ala Val HisAsn Phe Ser Asp Val Pro Leu Pro Val Asp Leu Thr Gln Thr Gly His Ala Leu TrpAla Val Leu Asp Thr Asp Gly Thr Gly Asp Ala Pro Glu Pro His Thr Glu Leu LeuLeu Pro Ala Tyr Gly Val Arg Trp Leu Gln Arg Arg
(4)突变体酶A25E-V326A-F245A-Y285A的制备
(a)设计定点突变的正向引物(pY285A-F：5’-TCGATTCCACGGCGGCCCTGTGGAAAC-3’，下划线为突变碱基)和反向引物(pY285A-R：5’-GTTTCCACAGGGCCGCCGTGGAATCGA-3’，下划线为突变碱基)。
(b)PCR反应体系(50μL)为：5μL10*DNAPolymerase Buffer，1.5μL正向引物pF245A-F(10umol/L)，1.5μL下游引物pF245A-R(10umol/L)，1μL模板pET-28a-DXcAS-A25E-V326A-F245A，其余为无菌水。
PCR反应程序为95℃预变性2min；30个循环(95℃变性20s，580℃退火20s，72℃延伸5min)；72℃延伸10min，最后16℃保温。
(c)PCR产物经Dpn I(购于Takara公司)消化，热激转化大肠杆菌BL21感受态细胞，涂布于LB固体培养基(0.5％氯化钠，0.5％酵母提取物，1％胰蛋白胨，2％琼脂粉，含有30μg/mL的Kan抗性)，37℃过夜培养。挑取单克隆至LB试管培养，提取质粒进行测序，突变质粒测序正确。突变质粒命名为pET-28a-XcAS-A25E-V326A-F245A-Y285A，突变体A25E-V326A-F245A-Y285A如SEQ ID NO.2所示。
SEQ ID No.2：
Met Ile Ala Ser Ser Pro Ile Asp Ala Ala Ala Leu Arg Ala Ser Val AlaAla Ala Leu Asp Pro Ser Thr Glu Val Ala Thr Leu Ala Arg Phe Asp Thr His AlaPro Arg Leu Leu Asp Ala Leu Ser Thr Leu Tyr Gly Asp His Ala Asp Tyr Val ThrTrp Leu Pro Gln Trp Leu Ser Ala Leu Gly Val Val Ala Gln Ala Arg Pro Ala AlaLeu Arg His Leu Asp Asp Ser Arg Ala Pro Gly Trp Phe Gly Gln Gln Asp Met LeuGly Tyr Ser Ala Tyr Val Asp Arg Phe Ala Gly Thr Leu Arg Gly Val Ala Glu ArgVal Pro Tyr Leu Gln Glu Leu Gly Val Arg Tyr Leu His Leu Leu Pro Phe Leu ArgAla Arg Ala Gly Asp Asn Asp Gly Gly Phe Ala Val Ser Asp Tyr Gly Gln Val GluPro Ala Leu Gly Ser Asn Asp Asp Leu Val Ala Leu Thr Ala Arg Leu Arg Ala AlaAsn Ile Ser Leu Cys Ala Asp Phe Val Leu Asn His Thr Ala Asp Asp His Ala TrpAla Gln Ala Ala Arg Ala Gly Asp Thr Arg Tyr Leu Asp Tyr Tyr His His Phe AlaAsp Arg Asn Ala Pro Asp Gln Tyr Asp Thr Thr Leu Val Gln Val Phe Pro Gln ThrAla Pro Gly Asn Phe Thr Trp Val Asp Glu Thr Arg Gln Trp Met Trp Thr Thr AlaTyr Pro Tyr Gln Trp Asp Leu Asn Trp Ser Asn Pro Ala Val Phe Gly Glu Met AlaLeu Ala Met Leu Glu Leu Ala Asn Leu Gly Val Glu Ala Phe Arg Leu Asp Ser ThrAla Ala Leu Trp Lys Arg Pro Gly Thr Asn Cys Met Asn Gln Pro Glu Ala His ThrIle Leu Val Ala Leu Arg Ala Val Ala Asp Ile Val Ala Pro Ser Val Val Met LysAla Glu Ala Ile Ala Pro Met Ala Glu Leu Pro Pro Tyr Phe Gly Ser Gly Val GlnArg Gly His Glu Cys His Leu Ala Tyr His Ser Thr Leu Met Ala Ala Gly Trp SerAla Leu Ala Leu Gln Arg Gly Asp Ile Leu Gln Asp Val Ile Ala His Ser Pro ProLeu Pro Pro Asn Cys Ala Trp Leu Ser Tyr Val Arg Cys His Asp Asp Ile Gly TrpAsn Val Leu Gln His Glu Ala Ala Gly Thr Ala Ala Gln Pro Pro Phe Ser Leu ArgGlu Val Ala Gln Phe Tyr Ala Asn Ala Val Pro Gly Ser Tyr Ala Arg Gly Glu SerPhe Gln Ser Ser Gly Asp Gly Val His Gly Thr Asn Gly Met Ser Ala Ala Leu ValGly Val Gln Ala Ala His Glu His Ala Asp Ala Ala Ala Ala Ala Arg Ala Val AspArg Leu Val Leu Leu Tyr Ala Val Ser Leu Ala Met Pro Gly Val Pro Leu Ile TyrMet Gly Asp Glu Leu Ala Leu Pro Asn Asp Thr Ala Tyr Leu Asp Asp Ala Gln ArgArg His Glu Gly Arg Trp Leu His Arg Pro Ala Met Ala Trp Glu Leu Ala Ala GlnArg His Asp Ala Ser Thr Leu Ala Gly ThrVal Tyr Thr Arg Leu Arg Ala Leu IleArg Leu Arg Ala Gly Leu Pro Ala Leu Ala Ala Thr Gln Ser Leu Gly Ser Val AlaLeu Gly Asp Ala Arg Leu Phe Ala Leu Thr Arg Gly Asp Ser Phe Leu Ala Val HisAsn Phe Ser Asp Val Pro Leu Pro Val Asp Leu Thr Gln Thr Gly His Ala Leu TrpAla Val Leu Asp Thr Asp Gly Thr Gly Asp Ala Pro Glu Pro His Thr Glu Leu LeuLeu Pro Ala Tyr Gly Val Arg Trp Leu Gln Arg Arg
实施例3：野生型级突变的淀粉蔗糖酶的大肠杆菌重组细胞的构建
(1)大肠杆菌感受态的制备及转化：
a)挑取大肠杆菌E.coli BL21单菌落接种到5mL液体LB培养基中，于37℃，200rpm/min的摇床上培养12h；
b)按1％的量取试管中培养的种子，接种在50mL的三角烧瓶中，之后于37℃，200rpm的摇床上培养,至OD600在0.4-0.5之间时停止培养，置于冰上孵育30min；
c)使用冷冻离心机4℃，5000g离心10min，去除上清，以预冷的感受态Buffer(含有0.05mol/L Cacl2及15％甘油)重悬细胞，重复两次。
d)以预冷的感受态Buffer重悬菌体，分装于1.5mL离心管中，每管100ul感受态细胞，放置于-80℃保存也可直接用于转化。
(2)线性化表达载体电转化大肠杆菌
按《分子克隆实验指南》将质粒pET28a-XcAS-A25E-V326A-F245A-Y285A分别转化至50μL大肠杆菌BL21感受态细胞。涂布至含50mg/L卡那霉素的LB固体培养基平板，37℃培养过夜。长出的转化子挑选3～4个用含50mg/L卡那霉素的LB试管培养，获得的菌株送往生工生物工程(上海)股份有限公司测序，通过原始序列比对，可得到淀粉蔗糖酶突变株E.coli BL21-pET28a-XcAS-A25E-V326A-F245A-Y285A(淀粉蔗糖酶突变体的氨基酸序列如SEQ ID NO：2所示，核苷酸序列如SEQ ID NO：3所示)。
实施例4：淀粉蔗糖酶XcAS在大肠杆菌重组菌株中的诱导表达
将突变株E.coli BL21-pET28a-XcAS-A25E-V326A-F245A-Y285A的单菌落分别接入装有100mL种子培养基(蛋白胨10g/L、酵母粉5g/L，NaCl 8g/L)的500mL锥形瓶中培养，37℃、220rpm下培养12小时；按照2％接种量分别将培养好的亲本株和突变株种子液分别接入装有3L发酵培养基(甘油10g/L，蛋白胨15g/L，酵母粉15g/L，磷酸氢二钾10g/L，磷酸二氢钾5g/L)的两个不同的5L发酵罐中，37℃、pH＝7下培养；OD600达到15～25时，培养温度降到25℃，然后加入0.1mmol/LIPTG诱导淀粉蔗糖酶酶的表达，诱导期间会出现溶氧上升的情况，将补料与溶氧相偶联，设置DO>20％时进行自动补料(补料成分：700g/L甘油；60g/L磷酸氢二铵；氨水用于调节pH)，可观察到溶氧反馈。持续诱导35h后，亲本株的OD600达到97.8，突变株的OD600达到96.6。
实施例5：淀粉蔗糖酶XcAS突变株在制备α-熊果苷中的应用
将实施例4中培养好的突变株发酵液低温离心(6,000rpm，10min，4℃)以收集细胞。使用预冷的100mmol/LPBS缓冲液(pH 7.0)清洗3次，获得湿菌体。
(1)采用递减梯度补料的方法向反应溶液中流加底物。初始全细胞催化体系包括：湿菌体120g/L、对苯二酚180.0mmol/L、蔗糖720mmol/L、Vc 1mM、Tritonx-1004g/L，pH7.0，催化反应在3L发酵罐中进行，30℃，转速700rpm，通气量2L/min。反应4小时后再补加150.0mmol/L的对苯二酚和600.0mmol/L的蔗糖；反应8小时后再补加120.0mmol/L的对苯二酚和480.0mmol/L的蔗糖；反应12小时再补加90.0mmol/L的对苯二酚和360.0mmol/L的蔗糖；反应16小时再补加60.0mmol/L的对苯二酚和240.0mmol/L的蔗糖。经过16h的反应最终α-熊果苷产量达到159.6g/L，底物对苯二酚的转化率达到97.7％。
采用上述同样方法测定，野生型淀粉蔗糖酶的α-熊果苷产量达为128.7g/L，底物对苯二酚的转化率到78.78％。
(2)采用递减梯度补料的方法向反应溶液中流加底物。初始全细胞催化体系包括：湿菌体150g/L、对苯二酚180.0mmol/L、蔗糖720mmol/L、Vc 1mM、Tritonx-1004g/L，pH7.0，催化反应在3L发酵罐中进行，30℃，转速700rpm，通气量2L/min。反应4小时后再补加150.0mmol/L的对苯二酚和600.0mmol/L的蔗糖；反应8小时后再补加120.0mmol/L的对苯二酚和480.0mmol/L的蔗糖；反应12小时再补加90.0mmol/L的对苯二酚和360.0mmol/L的蔗糖；反应16小时再补加60.0mmol/L的对苯二酚和240.0mmol/L的蔗糖。经过16h的反应最终α-熊果苷产量达到161.8g/L，底物对苯二酚的转化率达到99.05％。
采用上述同样方法测定，野生型淀粉蔗糖酶的α-熊果苷产量达为134.5g/L，底物对苯二酚的转化率到82.23％。
上述仅为本发明的部分优选实施例，本发明并不仅限于实施例的内容。对于本领域中的技术人员来说，在本发明技术方案的构思范围内可以有各种变化和更改，所作的任何变化和更改，均在本发明保护范围之内。
Claims (9)
1.一种淀粉蔗糖酶突变体，其特征在于，所述淀粉蔗糖酶突变体由是将野生淀粉蔗糖酶XcAS的第25位的丙氨酸突变为谷氨酸、第326位的缬氨酸突变为丙氨酸、第245位的苯丙氨酸突变为、第285位的酪氨酸突变为丙氨酸；所述野生淀粉蔗糖酶XcAS的氨基酸序列如SEQ ID NO.1所示。
2.一种编码权利要求1所述的淀粉蔗糖酶突变体的编码基因，其特征在于，所述基因的核苷酸序列如SEQ ID NO.3所示。
3.一种携带权利要求2所述编码基因的重组表达载体。
4.一种表达权利要求1所述的淀粉蔗糖酶突变体的基因工程菌，其特征在于所述基因工程菌含有如权利要求2所述的编码基因，或者所述基因工程菌含有如权利要求3所述的重组表达载体。
5.根据权利要求4所述的重组菌，其特征在于，所述基因工程菌构建方法为：以大肠杆菌为宿主，重组表达编码所述淀粉蔗糖酶突变体的编码基因，得到基因工程菌。
6.一种生产α-熊果苷的方法，其特征在于，包括以下步骤：将权利要求4或5所述的基因工程菌经过诱导表达后获得的湿菌体作为生物催化剂，以对苯二酚和蔗糖作为底物，通过催化反应生产α-熊果苷。
7.根据权利要求6所述的生产α-熊果苷的方法，其特征在于：催化反应过程中以pH为6.0～8.0的缓冲液为反应介质，于25℃～45℃的反应温度条件下进行反应。
8.根据权利要求7所述的生产α-熊果苷的方法，其特征在于，所述底物通过梯度流加方法加入所述反应介质中。
9.根据权利要求7所述的生产α-熊果苷的方法，其特征在于，所述湿菌体的用量不少于120g/L。
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Non-Patent Citations (2)
| Title |
|---|
| "WP_011038457.1", GENBANK, 18 May 2022 (2022-05-18), pages 1 - 2 * |
| 刘明等: "淀粉蔗糖酶AcAS的工作机理研究与稳定性改造", 《生物技术通讯》, vol. 24, no. 03, 30 May 2013 (2013-05-30), pages 308 - 313 * |
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