CN114410610A - 一种环麦芽糊精酶及其制备方法与应用 - Google Patents
一种环麦芽糊精酶及其制备方法与应用 Download PDFInfo
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- CN114410610A CN114410610A CN202111492517.3A CN202111492517A CN114410610A CN 114410610 A CN114410610 A CN 114410610A CN 202111492517 A CN202111492517 A CN 202111492517A CN 114410610 A CN114410610 A CN 114410610A
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- enzyme
- cyclodextrin
- cyclomaltodextrin
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Abstract
本发明公开了一种环麦芽糊精酶及其制备方法与应用。所述的酶序列见SEQ ID NO：2。本发明基于喜热厌氧菌基因组测序数据分析获得环麦芽糊精酶CtCD基因序列，并对CtCD基因序列进行优化，通过人工合成的方法获得优化后的环麦芽糊精酶基因序列，并添加酶切位点SacI和XhoI，将该优化后的基因克隆入pET28a‑SUMO表达载体上，在大肠杆菌E.coli Rosette中进行诱导表达，纯化得到较高纯度的重组环麦芽糊精酶蛋白。该酶对三种环糊精(α‑环糊精、β‑环糊精和γ‑环糊精)均有很强的水解作用，该酶可用于制备麦芽六糖、麦芽七糖或麦芽八糖；该酶对致病菌(金黄色葡萄球菌和铜绿假单胞菌)的生物膜形成有明显抑制作用，在和抗生素联用治疗生物膜引起的耐药性致病菌感染方面具有良好的应用前景。
Description
技术领域
本发明涉及采用基因工程手段获得一种环麦芽糊精酶及其制备方法与应用，属于生物工程技术领域。
背景技术
环糊精是由淀粉或淀粉衍生物产生的一组具有六个或更多吡喃葡萄糖单元通过α-1,4糖苷键连接的环状低聚糖。环糊精分子呈截锥状结构，其亲水基团位于结构外表面，而疏水基团位于结构内部空腔。因环糊精结构特殊，其可溶于水，并能同时容纳亲脂性客体分子，被广泛应用于食品、农业、化妆品、制药等领域。由于环糊精在食品、医药等多个行业的大量使用，人们对其使用安全性进行了不少研究。根据环糊精的药代动力学研究，发现口服环糊精的生物利用度较低，高剂量的α-环糊精和β-环糊精可能具有肾毒性和肝毒性，因此环糊精的代谢越来越引起了人们的关注。
环麦芽糊精酶属于糖苷水解酶GH13家族的成员，它对环糊精水解能力最强，也能够水解普鲁兰糖和淀粉，而传统的α-淀粉酶几乎不能水解环糊精和普鲁兰糖。环麦芽糊精酶在初始阶段先断裂环糊精的α-1,4糖苷键，将环糊精开环生成单一麦芽低聚糖，再将麦芽低聚糖彻底水解为麦芽糖和葡萄糖。利用环麦芽糊精酶的水解特性，可用于制备单一或混合聚合度的低聚麦芽糖。低聚麦芽糖由于其水溶性好、易消化、保湿等优良特性被广泛应用于食品、医药和化妆品等行业。近来研究发现，基于细菌特异性的麦芽糊精转运途径，利用麦芽六糖修饰的胆固醇和细菌响应脂质成分开发的智能纳米脂质体平台可以特异性地靶向检测细菌感染部位。此外，研究还发现麦芽糖淀粉酶(可降解β-环糊精)/β-环糊精介导的药物控释系统在低水溶性药物或疏水性化合物的控释方面具有潜在的应用前景。
与传统淀粉酶相比，环麦芽糊精酶研究较少。目前报道的环麦芽糊精酶主要来源于芽孢杆菌属、梭菌属、类芽孢杆菌属、厌氧芽孢杆菌属、脂环酸芽孢杆菌属和热球菌属的成员，这些环麦芽糊精酶大部分仅能水解一种或两种环糊精，极少能够对三种环糊精(α-环糊精、β-环糊精和γ-环糊精)均有很强的水解作用。此外，目前已鉴定的环麦芽糊精酶多用于麦芽寡糖制备，且其底物特异性和热稳定性较差。开发高活力、底物特异性强和热稳定性好的新型环麦芽糊精酶应用前景广阔。
发明内容
本发明的主要目的是针对现有技术的不足，提供一种环麦芽糊精酶(命名为CtCD)。
该酶的最适温度为60℃，最适pH为6.5；该酶在55℃和60℃条件下非常稳定，在55℃保温32h和60℃保温28h，其酶活力均无下降，在65℃保温3h其酶活力也没有明显变化；该酶对三种环糊精(α-环糊精、β-环糊精和γ-环糊精)均有很强的水解作用，在初始阶段先将三种环糊精开环，最终将三种环糊精完全水解为麦芽糖和葡萄糖。
本发明的另一目的，在于提供一种环麦芽糊精酶的制备方法与应用，它包括如下步骤：
(1)环麦芽糊精酶基因的设计及获得
基于喜热厌氧菌(Caloranaerobacter sp.MCCC 1A00790)基因组测序数据分析获得环麦芽糊精酶CtCD基因序列，并对所述基因序列进行优化，使其适合在大肠杆菌中进行表达，通过人工合成的方法获得优化后的环麦芽糊精酶CtCD基因序列，并添加酶切位点SacI和XhoI。
(2)重组质粒的构建
人工合成的带有SacI和XhoI酶切位点的环麦芽糊精酶基因的DNA片段经限制性内切酶SacI和XhoI酶切后，被连接到经同样限制性内切酶酶切的表达载体pET28a-SUMO上，将连接产物转化大肠杆菌Escherichia coli TOP10受体菌株，筛选含有环麦芽糊精酶基因的重组质粒，并进行双酶切和测序验证。
(3)环麦芽糊精酶的表达与纯化
将含有环麦芽糊精酶基因的重组质粒转化入大肠杆菌E.coli Rosette受体菌株，用IPTG诱导环麦芽糊精酶蛋白表达，采用Ni-Sepharose亲和层析(Ni SepharoseTM6FastFlow试剂盒)纯化重组蛋白。纯化后的重组环麦芽糊精酶(rCtCD)的SDS-PAGE电泳检测结果如图1所示。
本发明的环麦芽糊精酶具有良好的热稳定性，对三种环糊精均有很强的水解作用，利用其初始阶段对环糊精的开环反应，在制备单一麦芽寡糖(麦芽六糖、麦芽七糖或麦芽八糖)方面具有应用潜力；此外，该酶明显抑制致病菌金黄色葡萄球菌和铜绿假单胞菌的生物膜形成，可用于和抗生素联用治疗生物膜引起的耐药性致病菌感染。
附图说明
图1为纯化后的酶蛋白的SDS-PAGE电泳；
图2为不同pH对酶活力的影响；
图3为不同温度对酶活力的影响；
图4为酶在不同温度条件下的稳定性检测；
图5为酶对不同底物水解作用的效果；
图6为酶的完全水解产物分析；
图7为酶的开环反应水解产物分析；
图8为酶对金黄色葡萄球菌和铜绿假单胞菌的生物膜形成的影响
具体实施方式
实施例1环麦芽糊精酶的制备
步骤如下：
实验材料
喜热厌氧菌(Caloranaerobacter sp.MCCC 1A00790)(中国海洋微生物菌种保藏管理中心保藏，保藏编号为1A00790，可通过购买方式得到)，金黄色葡萄球菌(Staphylococcus aureus ATCC 6538)和铜绿假单胞菌(Pseudomonas aeruginosa ATCC10145)(购自美国菌种保藏中心)，大肠杆菌E.coil TOP10、大肠杆菌E.coil Rosetta(购自ThermoFisher公司)、表达载体pET28a-SUMO(购自Novagen公司)；限制性内切酶SacI和XhoI(购自全式金公司)；T4连接酶(购自Takara公司)；LB培养基(每升含1％蛋白胨，0.5％酵母抽提物，1％NaCl)；TSB培养基(每升含1.7％胰酪蛋白胨，0.3％大豆蛋白胨，0.25％葡萄糖，0.5％NaCl，0.25％K2HPO4)；THB培养基(每升含2％细菌蛋白胨，0.31％牛心浸粉，0.2％葡萄糖，0.25％Na2CO3，0.2％NaCl，0.04％Na2HPO4)；结合缓冲液(磷酸盐缓冲液：20mM磷酸盐，pH7.2～7.4)；漂洗缓冲液(500mM NaCl,10～50mM咪唑，20mM磷酸盐，pH7.4)；洗脱缓冲液(500mM NaCl，500mM咪唑，20mM磷酸盐，pH7.4)；3，5－二硝基水杨酸(DNS)(购自上海生工)；卡那霉素和氯霉素(购自上海生工)；Ni SepharoseTM6Fast Flow试剂盒(购自泰京公司)；8～14kD透析袋(购自泰京公司)；α-环糊精(α-CD)、β-环糊精(β-CD)、γ-环糊精(γ-CD)、可溶性淀粉(Soluble starch)和糊精(Dextrin)(购自上海生工)；普鲁兰糖(Pullulan)(购自Sigma公司)；葡萄糖(G1)和麦芽糖(G2)(购自上海生工)；麦芽三糖(G3)、麦芽四糖(G4)、麦芽五糖(G5)、麦芽六糖(G6)和麦芽七糖(G7)标准品(购自阿拉丁公司)。本发明中使用的含卡那霉素和氯霉素的LB培养基，其卡那霉素和氯霉素浓度分别为30μg/mL和34μg/mL。
实验步骤(1)环麦芽糊精酶基因的设计及获得
基于喜热厌氧菌(Caloranaerobacter sp.MCCC 1A00790)基因组测序数据分析获得环麦芽糊精酶CtCD基因序列，并对所述基因序列进行优化，使其适合在大肠杆菌中进行表达，通过人工合成的方法获得优化后的环麦芽糊精酶CtCD基因序列，并添加酶切位点SacI和XhoI。
优化后的编码环麦芽糊精酶CtCD的核苷酸序列如下：(SEQ ID NO:1)
该酶的氨基酸序列如下：(SEQ ID NO:2)
(2)重组质粒的构建
将人工合成的带有SacI和XhoI酶切位点的环麦芽糊精酶基因的DNA片段经限制性内切酶SacI和XhoI酶切后进行胶回收，并对pET28a-SUMO载体也用限制性内切酶SacI和XhoI进行酶切和胶回收，将酶切后的环麦芽糊精酶基因的DNA片段和pET28a-SUMO载体用T4连接酶进行连接；将连接产物与受体菌E.coli Top10感受态混合，冰上放置30min，42℃热激90s后，加入500μL LB液体培养基，37℃、150rpm复苏45min，离心后涂布于含30μg/mL卡那霉素的LB固体培养基上，37℃过夜培养，筛选重组质粒，并对重组质粒进行双酶切和测序验证，获得含有环麦芽糊精酶基因的重组质粒。
(3)基因的诱导表达及粗酶液的制备
将含有环麦芽糊精酶基因的重组质粒转化入E.coli Rosetta中，获得含有环麦芽糊精酶基因的重组菌株。将重组菌株接种于5mL含30μg/ml卡那霉素和34μg/ml氯霉素的LB液体培养基中过夜培养，按1％接种量接种于500mL含卡那霉素和氯霉素的LB液体培养基中，37℃、200rpm培养至OD600为0.8左右，加入IPTG使其终浓度为1mmol/L进行诱导，18℃、180rpm条件下诱导表达18h，5000rpm离心10min收集菌体。将菌体用结合缓冲液(pH7.2)清洗3次，然后将菌体重悬于60mL结合缓冲液中，加入适量溶菌酶和蛋白酶抑制剂，4℃放置2h后，以功率300W，工作/间隙时间为3s/4s超声破碎20min，4℃、8000rpm离心10min，收集上清即得到粗酶液。
(4)酶的纯化
将制备的粗酶液加入至预先平衡好的His纯化柱Ni SepharoseTM6Fast Flow，4℃混匀2～3h，然后弃去废液。之后先用20mL含有10mM咪唑的漂洗缓冲液漂洗两次，再用20mL含有20mM咪唑的漂洗缓冲液漂洗两次，最后用20mL含有50mM咪唑的漂洗缓冲液漂洗两次。漂洗结束后用含有500mM咪唑的洗脱缓冲液洗脱三次(第一次洗脱体积为3mL，第二次洗脱体积为4mL，第三次洗脱体积为2mL)，分批收集洗脱液，对洗脱液进行SDS-PAGE电泳检测其纯度。用8～14kD透析袋将洗脱液透析过夜以去除咪唑和高浓度的NaCl，获得纯化好的酶液。
实施例2
环麦芽糊精酶的酶学性质
(1)环麦芽糊精酶活性测定方法
采用DNS法测定还原糖含量。将50μL稀释的酶液和200μL 1％的α-环糊精底物混合，在60℃条件下反应10min。反应完成后加入500μL DNS试剂，煮沸5min，立即置于冰水中冷却，短暂离心后取上清，测定OD540值(以灭活酶液作为对照)，根据葡萄糖和麦芽糖标准曲线计算还原糖量和酶活力。酶活力单位定义：在上述测定条件下，每分钟水解α-环糊精底物产生1μmol还原糖所需酶量定义为一个酶活力单位(U)。
(2)酶的作用pH
将稀释的酶液在60℃下，以不同缓冲液(100mM Na-acetate缓冲液，pH4.0～6.0；100mM Na-phosphate缓冲液，pH6.0～8.0；100mM Tris-HCl缓冲液，pH7.0～9.0；100mMGlycine-NaOH，pH9.0～10.0)配制的1％的α-环糊精作为底物进行酶活测定，以最大酶活力为100％，计算不同pH下该酶的相对活力。结果表明，该酶的最适pH为6.5，在pH5.5～7.0范围内酶活力较高；在pH6.0～8.0范围内，用Na-phosphate缓冲液检测酶活性，该酶活力均保持58％以上；在相同pH条件下，Na-phosphate缓冲液优于Tris-HCl缓冲液。结果见图2。
(3)酶的作用温度
将稀释的酶液在25～80℃范围内，以100mM Na-phosphate(pH 6.5)缓冲液配制的1％的α-环糊精作为底物进行酶活测定，以最大酶活力为100％，计算不同温度下的相对酶活力。结果表明，该酶最适反应温度为60℃，在50～65℃范围内酶活力较高，其相对酶活力均保持80％以上；在40～70℃范围内，其相对酶活力均保持50％以上。结果见图3。
(4)酶在不同温度条件下的稳定性
将酶液分别在55℃和60℃条件下共保温44h，每4h测定一次酶活力；将酶液在65℃条件下共保温14h，每1h测定一次酶活力。分别将在不同温度下保温不同时间的酶液与100mM Na-phosphate(pH 6.5)缓冲液配制的1％的α-环糊精反应进行酶活测定，以未经处理的酶的活力为100％，计算不同处理的酶的相对活力。结果表明，该酶在55℃和60℃条件下非常稳定，在55℃保温32h和60℃保温28h，其酶活力均无下降；在55℃保温16～32h，其酶活力有明显升高；在55℃保温36h和60℃保温32h，其酶活力保持90％以上；在55℃和60℃分别保温40h，其酶活力还保持50％以上；该酶在65℃也比较稳定，在65℃保温3h，其酶活力没有明显变化；在65℃保温4h，其酶活力保持80％以上；在65℃保温8h，其酶活力仍保持50％以上。结果见图4。
(5)酶的底物特异性
在60℃、pH 6.5条件下，分别以1％的α-环糊精、β-环糊精、γ-环糊精、可溶性淀粉、糊精和普鲁兰糖为底物进行酶活测定，以最大酶活力为100％，计算环麦芽糊精酶水解不同底物的相对活力。结果表明，该酶对α-环糊精、β-环糊精和γ-环糊精均有很强的水解作用，其中对α-环糊精水解能力最强(其酶活力可达540U/mg)，对β-环糊精水解的相对酶活力达到97％以上，对γ-环糊精水解的相对酶活力也达86％以上；此外，该酶对可溶性淀粉、糊精和普鲁兰糖也有一定的水解作用。结果见图5。
(6)水解产物分析
采用薄层层析(TLC)法分析环麦芽糊精酶完全水解产物。
将50μL适量稀释的纯酶加入200μL的含有1％底物(α-环糊精/β-环糊精/γ-环糊精)的100mM Na-phosphate(pH 6.5)缓冲液中，混匀后于60℃条件下过夜反应，采用TLC法检测环麦芽糊精酶完全水解产物，上样量为1μL。展开剂为异丙醇：乙酸乙酯：水(3:1:1，v/v/v)，显色剂为N-(1-萘基)乙二胺：硫酸：甲醇(0.3:5:100，w/v/v)。结果显示，该酶可完全水解α-环糊精、β-环糊精和γ-环糊精，主要水解产物为麦芽糖和葡萄糖，说明该酶可特异性断裂α-1,4糖苷键。结果见图6。
实施例3
环麦芽糊精酶的用途
(1)麦芽寡糖的制备
将纯酶(0.5U)加入1mL的含有4％环糊精(α-环糊精/β-环糊精/γ-环糊精)的100mM Na-phosphate(pH 6.5)缓冲液中，混匀后于60℃、120rpm条件下反应4h，将反应产物稀释4倍后采用TLC法检测环麦芽糊精酶开环水解产物，上样量为1μL。所用展开剂和显色剂均与上述检测完全水解产物所述的展开剂和显色剂一样。结果表明，初始阶段该酶先将α-环糊精、β-环糊精和γ-环糊精开环，分别生成麦芽六糖、麦芽七糖和麦芽八糖，显示该酶在制备单一麦芽寡糖方面的应用潜力。结果见图7。
(2)抑制致病菌的生物膜形成
采用微孔板法检测环麦芽糊精酶对致病菌金黄色葡萄球菌和铜绿假单胞菌生物膜形成的影响。将金黄色葡萄球菌和铜绿假单胞菌的过夜培养物按1％的接种量分别接种至TSB和THB培养基中，并转移到无菌的96孔聚苯乙烯微孔板(200μL/孔)，然后分别加入不同酶量的环麦芽糊精酶(3U、6U、12U、24U和48U)，并以未添加酶液的培养物作为阳性对照和以未添加酶液的培养基作为阴性对照。在37℃静置培养24h后，去除漂浮的菌液，用超纯水清洗生物膜2～3次，然后用200μL 0.1％的结晶紫溶液染色20min，再用超纯水清洗2～3次，之后用200μL 95％的乙醇溶解生物膜，并于590nm波长下检测金黄色葡萄球菌和铜绿假单胞菌的生物膜形成情况。结果显示，该酶对金黄色葡萄球菌和铜绿假单胞菌的生物膜形成均有明显抑制作用，在添加酶量为24U时，该酶对两种致病菌的生物膜形成抑制率为78％；在添加酶量为48U时，该酶对两种致病菌金黄色葡萄球菌和铜绿假单胞菌的生物膜形成抑制率分别可达87％和90％，在和抗生素联用治疗生物膜引起的耐药性致病菌感染方面具有良好的应用前景。
序列表
<110> 自然资源部第三海洋研究所
<120> 一种环麦芽糊精酶及其制备方法与应用
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1734
<212> DNA
<213> 人工序列（Artificial Sequence）
<400> 1
atgaacctgc aggcgatcta ccacaaaccg aaatctaact tctgctacgc gtacgatgaa 60
aacgttatcc acattcgtct gcgtgcggct aaaggtgaca ttaaaaaagc agttctgatc 120
tacggtgaca aataccagtg ggacaaacgt caggaactgg tgatggaact gaccggcagc 180
gacgatctgt acgattactt caccgtggaa gtgtccccga aaaacaaacg tctggcttac 240
tacttcaaag tgcagggcga cgcgggcgaa ttctacttta ccgagtgggg tctgatcaaa 300
gatatcgacg aaaaagaagt ctacctgcac ttcttccagt acccgtacat gaacaaagaa 360
gatattcaca aagtgccgga atgggttaaa gataccgtgt tctaccagat tttcccggag 420
cgtttcttca acggtgatac ctctaacgac ccggacaacc tgaccaaatg gggtgaactg 480
ccgacttcca acagcttcta tggcggcgac ctgaaaggca tcatcgaaaa actggactac 540
ctggaagaac tgggcatcaa cggcatctac ctgaccccga ttttcgaaag cccgaccaat 600
cacaaatacg acaccaaaga ctacttcaaa atcgatccgc acttcggtga tctgcagacc 660
ctgaaagagc tggtttctaa atgccacgaa cgcggcattc gcgtgatcct ggatgcagtg 720
ttcaaccact gcggctactt cttcgaaccg ttccaggacg tgatcgaaaa gggcaaagaa 780
tccccgtact acaattggtt ccacatccac aaatggccga tcgaaacgaa cccgccgagc 840
tacgacacct tcgccttcgt gtatcatatg ccgaaactga acaccgacaa cccggaagtt 900
cgcgaatatc tgctgaaggt tgcgcgttac tggatcgaag aagcggatat cgatggctgg 960
cgcctggatg tgtccgatga agtgagccac gacttttggc gcgaatttcg taaaacggtt 1020
aaaggcgcca aagaagatgc ttatatcgtg ggtgaactgt ggctggacgc gtatccgtgg 1080
ctgcgtggcg atcagtttga tgcggttatg aactacccgg ttatgcgtgc cctgctgcag 1140
tatttcgcat acgaaaacat ttctgatcgc cagttcaaag aactgatcaa caaaacccgc 1200
atgcgcaaca ccgaacaggt taacgaggtc atgctgaacc tgatcgaatc tcatgatacc 1260
tcccgttacc tgaccgaatg caacaaagac attaacaaac tgatgatgac cgcgaccttt 1320
ctgctgacct atgttggtac cccgtgcatc tactacggta ccgaaattgg catggaaggt 1380
ggccacgatc cggactgccg tcgtaccatg gattggaaca aagaaaactg ggacctggaa 1440
ctgttcaact actacaagaa actgatccgt ctgcgtaaac agtacaaagc gctgcgtcgt 1500
ggcaaattca aatggatcga taacatcgaa ggtattatcg gcttcatccg tgaatacgaa 1560
aacgaaaaaa tgttcatcct gatcaacaac cacaacttcg atcgtgaagt tatcctgaac 1620
aacaaaggta aatacatcga tggcctgacc ggcgacgtgt tcaaatccga taaagcactg 1680
tacgtgaacg tgccgaaata cagcgcgcgt atcctggttt ctagcgaaga ttaa 1734
<210> 2
<211> 577
<212> PRT
<213> 喜热厌氧菌（Caloranaerobacter sp. MCCC 1A00790 ）
<400> 2
Met Asn Leu Gln Ala Ile Tyr His Lys Pro Lys Ser Asn Phe Cys Tyr
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Ala Tyr Asp Glu Asn Val Ile His Ile Arg Leu Arg Ala Ala Lys Gly
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Asp Ile Lys Lys Ala Val Leu Ile Tyr Gly Asp Lys Tyr Gln Trp Asp
35 40 45
Lys Arg Gln Glu Leu Val Met Glu Leu Thr Gly Ser Asp Asp Leu Tyr
50 55 60
Asp Tyr Phe Thr Val Glu Val Ser Pro Lys Asn Lys Arg Leu Ala Tyr
65 70 75 80
Tyr Phe Lys Val Gln Gly Asp Ala Gly Glu Phe Tyr Phe Thr Glu Trp
85 90 95
Gly Leu Ile Lys Asp Ile Asp Glu Lys Glu Val Tyr Leu His Phe Phe
100 105 110
Gln Tyr Pro Tyr Met Asn Lys Glu Asp Ile His Lys Val Pro Glu Trp
115 120 125
Val Lys Asp Thr Val Phe Tyr Gln Ile Phe Pro Glu Arg Phe Phe Asn
130 135 140
Gly Asp Thr Ser Asn Asp Pro Asp Asn Leu Thr Lys Trp Gly Glu Leu
145 150 155 160
Pro Thr Ser Asn Ser Phe Tyr Gly Gly Asp Leu Lys Gly Ile Ile Glu
165 170 175
Lys Leu Asp Tyr Leu Glu Glu Leu Gly Ile Asn Gly Ile Tyr Leu Thr
180 185 190
Pro Ile Phe Glu Ser Pro Thr Asn His Lys Tyr Asp Thr Lys Asp Tyr
195 200 205
Phe Lys Ile Asp Pro His Phe Gly Asp Leu Gln Thr Leu Lys Glu Leu
210 215 220
Val Ser Lys Cys His Glu Arg Gly Ile Arg Val Ile Leu Asp Ala Val
225 230 235 240
Phe Asn His Cys Gly Tyr Phe Phe Glu Pro Phe Gln Asp Val Ile Glu
245 250 255
Lys Gly Lys Glu Ser Pro Tyr Tyr Asn Trp Phe His Ile His Lys Trp
260 265 270
Pro Ile Glu Thr Asn Pro Pro Ser Tyr Asp Thr Phe Ala Phe Val Tyr
275 280 285
His Met Pro Lys Leu Asn Thr Asp Asn Pro Glu Val Arg Glu Tyr Leu
290 295 300
Leu Lys Val Ala Arg Tyr Trp Ile Glu Glu Ala Asp Ile Asp Gly Trp
305 310 315 320
Arg Leu Asp Val Ser Asp Glu Val Ser His Asp Phe Trp Arg Glu Phe
325 330 335
Arg Lys Thr Val Lys Gly Ala Lys Glu Asp Ala Tyr Ile Val Gly Glu
340 345 350
Leu Trp Leu Asp Ala Tyr Pro Trp Leu Arg Gly Asp Gln Phe Asp Ala
355 360 365
Val Met Asn Tyr Pro Val Met Arg Ala Leu Leu Gln Tyr Phe Ala Tyr
370 375 380
Glu Asn Ile Ser Asp Arg Gln Phe Lys Glu Leu Ile Asn Lys Thr Arg
385 390 395 400
Met Arg Asn Thr Glu Gln Val Asn Glu Val Met Leu Asn Leu Ile Glu
405 410 415
Ser His Asp Thr Ser Arg Tyr Leu Thr Glu Cys Asn Lys Asp Ile Asn
420 425 430
Lys Leu Met Met Thr Ala Thr Phe Leu Leu Thr Tyr Val Gly Thr Pro
435 440 445
Cys Ile Tyr Tyr Gly Thr Glu Ile Gly Met Glu Gly Gly His Asp Pro
450 455 460
Asp Cys Arg Arg Thr Met Asp Trp Asn Lys Glu Asn Trp Asp Leu Glu
465 470 475 480
Leu Phe Asn Tyr Tyr Lys Lys Leu Ile Arg Leu Arg Lys Gln Tyr Lys
485 490 495
Ala Leu Arg Arg Gly Lys Phe Lys Trp Ile Asp Asn Ile Glu Gly Ile
500 505 510
Ile Gly Phe Ile Arg Glu Tyr Glu Asn Glu Lys Met Phe Ile Leu Ile
515 520 525
Asn Asn His Asn Phe Asp Arg Glu Val Ile Leu Asn Asn Lys Gly Lys
530 535 540
Tyr Ile Asp Gly Leu Thr Gly Asp Val Phe Lys Ser Asp Lys Ala Leu
545 550 555 560
Tyr Val Asn Val Pro Lys Tyr Ser Ala Arg Ile Leu Val Ser Ser Glu
565 570 575
Asp
Claims (10)
1.一种表达环麦芽糊精酶的基因序列，其特征在于，其核苷酸序列如SEQ ID NO:1所示。
2.一种重组载体，其含有权利要求1所述的一种表达环麦芽糊精酶的基因序列。
3.一种重组菌，其含有权利要求2所述的重组载体。
4.一种环麦芽糊精酶，其特征在于：其蛋白序列如SEQ ID NO:2所示。
5.如权利要求4所述的环麦芽糊精酶在麦芽寡糖制备中的应用。
6.如权利要求5所述的应用，其特征在于，所述的环麦芽糊精酶将α-环糊精、β-环糊精和γ-环糊精中的至少一种水解为麦芽寡糖。
7.如权利要求4所述的环麦芽糊精酶在制备抗耐药性致病菌药物中的应用。
8.如权利要求7所述的应用，其特征在于，所述的耐药性致病菌包括金黄色葡萄球菌和铜绿假单胞菌中的至少一种。
9.重组菌突变体，其特征在于：其外源序列编码的蛋白序列与权利要求4所述的蛋白序列的相似性大于95％。
10.一种环麦芽糊精酶的制备方法，包括如下步骤：
(1)环麦芽糊精酶基因的设计及获得：利用喜热厌氧菌Caloranaerobacter sp.MCCC1A00790基因组测序数据，设计并人工合成序列优化后的环麦芽糊精酶基因序列，并添加酶切位点SacI和XhoI；
(2)重组质粒的构建：将步骤(1)的带有SacI和XhoI酶切位点的人工合成的环麦芽糊精酶基因的DNA片段经限制性内切酶SacI和XhoI酶切后，被连接到经同样限制性内切酶酶切的表达载体pET28a-SUMO上，将连接产物转化大肠杆菌Escherichia coli TOP10受体菌株，筛选含有环麦芽糊精酶基因的重组质粒，并进行双酶切和测序验证；
(3)环麦芽糊精酶的表达与纯化：将含有环麦芽糊精酶基因的重组质粒转化入大肠杆菌E.coli Rosette受体菌株，诱导环麦芽糊精酶蛋白表达并纯化，获得纯化后的环麦芽糊精酶。
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