CN110592119B - 一种来源于类芽孢杆菌普鲁兰酶及其基因与应用 - Google Patents
一种来源于类芽孢杆菌普鲁兰酶及其基因与应用 Download PDFInfo
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- CN110592119B CN110592119B CN201910985018.4A CN201910985018A CN110592119B CN 110592119 B CN110592119 B CN 110592119B CN 201910985018 A CN201910985018 A CN 201910985018A CN 110592119 B CN110592119 B CN 110592119B
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- pullulanase
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Abstract
本发明公开了一种来源于类芽孢杆菌的普鲁兰酶及其编码基因与应用。所述来源于类芽孢杆菌的普鲁兰酶，其氨基酸序列如SEQ ID NO.2所示。所述普鲁兰酶的编码基因pulA，核苷酸序列如SEQ ID NO.1所示。该普鲁兰酶为Ⅰ型普鲁兰酶，能够在Escherichia coli BL21(DE3)中进行异源表达。该重组普鲁兰酶的分子量约为76.95ku，比酶活达508.8U/mg。本发明提供的来源于类芽孢杆菌的普鲁兰酶，可应用于淀粉加工、医药、化工、食品等相关行业。
Description
技术领域
本发明属于基因工程与酶工程技术领域，具体涉及一种来源于类芽孢杆菌普鲁兰酶及其基因与应用。
背景技术
普鲁兰酶(pullulanase，EC 3.2.1.41)是一种淀粉脱枝酶，可以专一性地作用于普鲁兰糖、支链淀粉、极限糊精中的α-l,6糖苷键，切下整个侧枝，形成直链淀粉。普鲁兰酶与糖化酶并用时，可提高葡萄糖的产量与纯度，使其DE值达到97％-98％；当其与β-淀粉酶共同作用时，能够将淀粉100％地转化成麦芽糖，从而获得超高麦芽糖浆；在啤酒酿造的糖化过程中加入普鲁兰酶，能大幅度地提高可发酵性糖的含量，保证啤酒的口感。普鲁兰酶的这些性质决定了它在淀粉加工业中的广泛应用。
目前报道出的自然界中产普鲁兰酶的菌株主要集中在芽孢杆菌、多形拟杆菌、克雷伯氏菌、热硫梭菌及链球菌等。普鲁兰酶的工业化生产菌很少，仅有丹麦Novo公司的Bacillus acidopullulyticus和美国Genencor公司的Bacillus deramificans等应用到实际生产中(Jensen B F,Norman B E.Bacillus acidopullulyticus Pullulanase:application and regulatory aspects for use in the food industry[J].ProcessBiochemistry,1984,19:129-134；Malviya S N,Malakar R,TiwariA.Pullulanase:a potential enzyme for industrial application[J].InternationalJournal of Materials Research,2010,1:10-20)。国内不断有产普鲁兰酶菌株的相关报道，但野生菌株的产酶活力普遍偏低，大多数基因工程菌的发酵水平也达不到商业生产需求。我国淀粉加工业中使用的普鲁兰酶主要依靠进口，价格较为高昂。因此，通过基因工程技术实现不同性质的普鲁兰酶的高效表达，为普鲁兰酶的工业化生产提供更多潜在资源，以适应更广泛的工业需求，具有较强的现实意义。
发明内容
为了克服现有技术存在的不足，本发明的目的是提供了一种来源于类芽孢杆菌普鲁兰酶及其基因与应用。
本发明提供了一种产酶水平较高，具有较大应用潜力的普鲁兰酶编码基因(记为pulA)。本发明提供了包含上述普鲁兰酶编码基因的重组质粒。本发明提供了包含上述基因、能够高效表达普鲁兰酶的重组菌株。
本发明的目的至少通过如下技术方案之一实现。
本发明提供的一种来源于类芽孢杆菌(Paenibacillus puldeungensis)的普鲁兰酶的编码基因pulA，核苷酸序列如SEQ ID NO.1所示。
本发明提供的一种来源于类芽孢杆菌的普鲁兰酶，氨基酸序列如SEQ IDNO.2所示。
进一步地，所述普鲁兰酶为I型普鲁兰酶。
本发明提供的一种重组质粒，插入了SEQ ID NO.1所述的核苷酸序列。
进一步地，所述重组质粒通过将编码基因pulA克隆至表达载体pET-28a(+)的多克隆位点所获得，记为pET-28a(+)-pulA。
进一步地，所述重组质粒包含了上述普鲁兰酶基因；该质粒为pET-28a(+)-pulA通过将上述普鲁兰酶基因pulA克隆至大肠杆菌表达载体pET-28a(+)的Bam HI和HindⅢ酶切位点之间，使目的基因序列与表达调控序列相连接，得到表达质粒pET-28a(+)-pulA。
本发明提供的一种基因工程菌株(重组菌株)，含有上述的重组质粒。所述重组菌株能够高效表达普鲁兰酶。
进一步地，所述的基因工程菌株，其宿主细胞为大肠杆菌BL21(DE3)。
本发明提供的来源于类芽孢杆菌的普鲁兰酶的编码基因pulA、所述的重组质粒及所述基因工程菌株，能够应用在制备所述来源于类芽孢杆菌的普鲁兰酶。
进一步地，所述的应用，包括如下的步骤：
(1)普鲁兰酶基因pulA的克隆；
(2)构建重组质粒；
(3)将步骤(2)中所述的重组质粒转化宿主细胞，得到重组菌株，即所述基因工程菌株；
(4)所述来源于类芽孢杆菌的普鲁兰酶的表达以及纯化。
本发明提供的来源于类芽孢杆菌的普鲁兰酶能应用于淀粉加工、医药、化工、食品等相关行业中。
本发明从淀粉厂污水中分离筛选出一株产I型普鲁兰酶的菌株LK18，将其鉴定为Paenibacillus puldeungensis，命名为Paenibacillus puldeungensis LK 18。目前国内外还没有来源于Paenibacillus puldeungensis的普鲁兰酶及其相关酶学性质的报道。本发明提供了Paenibacillus puldeungensis来源的普鲁兰酶的克隆表达及酶学性质，成功构建一株高效表达普鲁兰酶的重组菌株，为普鲁兰酶的酶库构建及工业化生产奠定了基础。
所述Paenibacillus puldeungensis LK 18于2019年4月29日在广东省微生物菌种保藏中心保藏，保藏编号为GDMCC No：60652；所述广东省微生物菌种保藏中心的地址为广东省广州市先烈中路100号大院59号楼5楼，广东省微生物菌种保藏中心的邮编号码为510075。
本发明利用PCR技术获得Paenibacillus puldeungensis LK18中的普鲁兰酶基因pulA，序列全长1968bp，编码655个氨基酸，起始密码子为TTG，终止密码子为TAG，其氨基酸序列如SEQ ID NO.2所示。在线分析软件(http://web.expasy.org/protparam/)表明：该普鲁兰酶的等电点pI为5.15，总原子数为10195，分子式为C 3286H 5013N 889O 987S 20，不稳定系数(Instability index)为38.88(数值小于40，蛋白质稳定)，脂溶指数(Aliphaticindex)为81.71；总平均亲水性(Grand average of hydropathicity:GRAVY)为-0.339。通过Clustal Omega在线软件(http://www.ebi.ac.uk/Tools/msa/clustalo/)比对该氨基酸序列，发现其与Paenibacillus barengoltzii CAU904(GenBank登录号：KP714732.1)的I型普鲁兰酶序列同源性最高，相似度为73％，说明该普鲁兰酶是一种新的普鲁兰酶。
本发明提供了该普鲁兰酶基因的克隆方法，首先通过ClustalX软件比对NCBI数据库所公布的类芽孢杆菌属的I型普鲁兰酶基因序列，确定其保守区域，设计兼并引物(PF1和PR1；PF2和PR2；PF3和PR3)，以Paenibacillus puldeungensis LK18全基因组为模板，扩增出其部分基因，然后将部分基因的测序结果用DNAMAN进行比对和序列拼接(如图1所示)，并将拼接的序列提交至NCBI上的ORF Finder进行分析，重新设计引物(Pul-F和Pul-R)，进一步获得pulA基因全长(见图2a、图2b、图2c及图2d)。
本发明提供了来源于Paenibacillus puldeungensis的有活性的普鲁兰酶，通过Ni柱亲和层析分离出纯度较高的重组蛋白，测定其比酶活为508.8U/mg，SDS-PAGE检测显示其分子量约为76.95ku(见图3)。该重组酶的最适反应温度为45℃(如图4所示)，在35℃-40℃范围下保温120min仍保持有60％以上酶活(如图5所示)；最适作用pH为6.0(如图6所示)，在pH 6.0-8.0条件下保温1h后，相对酶活仍剩余60％以上(如图7所示)；10mmol/L的K+和Mg2+对该重组酶具有激活作用，而Zn 2+、Mn 2+、Ni 2+、Fe 2+、Cu 2+、Co 2+、Ca 2+等对其有不同程度的抑制作用(图8)。本发明成功获得一株高效表达普鲁兰酶的重组菌株，所表达的普鲁兰酶可应用于淀粉加工、医药、化工、食品等相关行业。
与现有技术相比，本发明具有如下优点和有益效果：
(1)本发明提供了Paenibacillus puldeungensis来源的普鲁兰酶的克隆表达方法及酶学性质，本发明成功构建一株高效表达普鲁兰酶的重组菌株，能够为普鲁兰酶的酶库构建及工业化生产奠定基础；
(2)本发明提供的普鲁兰酶分子量较小，仅有655个氨基酸，更利于重组蛋白表达；
(3)本发明提供的普鲁兰酶可适应偏酸的反应环境，在工业应用中具有一定优势。
附图说明
图1为所述来源于类芽孢杆菌普鲁兰酶的基因序列拼接图。
图2a为引物PF1和PR1的扩增产物电泳图；
图2b为引物PF2和PR2的扩增产物电泳图；
图2c为引物PF3和PR3的扩增产物电泳图；
图2d为引物Pul-F和Pul-R扩增的普鲁兰酶基因全长序列电泳图；其中，M为DL5000DNA Marker。
图3为纯化后的所述来源于类芽孢杆菌普鲁兰酶SDS-PAGE分析图，其中M为蛋白Marker；泳道1为空载菌株破壁上清；泳道2为未纯化的重组普鲁兰酶(普鲁兰酶)；泳道3为纯化后的重组普鲁兰酶(普鲁兰酶)。
图4为温度对所述来源于类芽孢杆菌普鲁兰酶的酶活力影响图。
图5为所述来源于类芽孢杆菌普鲁兰酶的温度稳定性图。
图6为pH对所述来源于类芽孢杆菌普鲁兰酶的酶活力影响图。
图7为所述来源于类芽孢杆菌普鲁兰酶的pH稳定性图。
图8为金属离子对所述来源于类芽孢杆菌普鲁兰酶的酶活力影响图。
具体实施方式
以下结合附图和实例对本发明的具体实施作进一步说明，但本发明的实施和保护不限于此。需指出的是，以下若有未特别详细说明之过程，均是本领域技术人员可参照现有技术实现或理解的。所用试剂或仪器未注明生产厂商者，视为可以通过市售购买得到的常规产品。
实验材料与试剂
1、菌株与质粒：Paenibacillus puldeungensis LK18为实验室自筛野生菌；JM110感受态细胞，购自上海唯地生物技术有限公司；Escherichia coil BL21(DE3)感受态细胞、pEASY-Blunt Cloning vector，购自北京全式金生物技术有限公司；pET-28a(+)表达载体，购自Invitrogen生物公司。
2、酶与主要试剂：TransStart KD Plus DNA Polymerase、2×Easy TaqPCRSuperMix酶，购自北京全式金生物技术有限公司；限制性内切酶(Bam HI和Hind III)、T4 DNA连接酶，购自宝生物工程(大连)有限公司；普鲁兰糖购自美国Sigma公司；引物合成和测序工作由广州艾基生物技术有限公司完成。
3、培养基
LB培养基包括：蛋白胨1.0％(w/v)，酵母粉0.5％(w/v)，NaCl 0.5％(w/v)，pH7.0；固体培养基在此基础上加入1.5％(w/v)琼脂，w/v的单位为g/mL。
本发明的主要操作方法如下，未作详述处参考《分子克隆实验指南》(J.萨姆布鲁克,D.W.拉塞尔.分子克隆实验指南第三版[M].北京:科学出版社,2002.)或琼脂糖凝胶DNA纯化试剂盒说明书(天根生化科技有限公司)。
实施例1普鲁兰酶基因pulA的克隆
在NCBI数据库中选取多条类芽孢杆菌属来源的I型普鲁兰酶基因，使用ClustalX软件进行序列比对，确定其保守区，同时结合密码子偏好性，设计三对兼并引物：PF1和PR1；PF2和PR2；PF3和PR3，以Paenibacillus puldeungensis LK18的全基因组为模板，通过Touchdown PCR扩增其部分基因，反应条件为：94℃预变性5min；94℃变性30s；65℃退火30s，68℃延伸(延伸时间1min/kb)，每个循环退火温度降低0.5℃，共30个循环；然后94℃变性30s；50℃退火30s；68℃延伸(延伸时间1min/kb)；10个循环；最后68℃保温10min。将扩增产物进行1％(w/v，单位g/mL)琼脂糖凝胶电泳检测，胶回收目的条带后送公司测序。
上游引物PF1:5’-GCTTTCATYACKGCSTATTGCAAAGT-3’，即SEQ ID NO.3所示。
下游引物PR1:5’-TAATTCTTCGGATCDTASCCCCAGTT-3’，即SEQ ID NO.4所示。
上游引物PF2:5’-TGGAACGAGGCCGCCGAYCCNTAYGC-3’，即SEQ ID NO.5所示。
下游引物PR2:5’-AGGCCCATCAGGTCGAACCKRAANCCRTC-3’，即SEQ ID NO.6所示。
上游引物PF3:5’-GACGTGTAYAACCAYGTNTATGACGG-3’，即SEQ ID NO.7所示。
下游引物PR3:5’-TACCCYAGWATGAAKAGACCCGC-3’，即SEQ ID NO.8所示。
通过DNAMAN比对测序结果的重叠区域，进行序列拼接(如图1所示)，提交至NCBI上的ORF Finder进行分析，进一步获得pulA基因序列。根据pulA基因序列及表达载体pET-28a(+)(此载体购自Invitrogen生物公司)的多克隆位点重新设计引物(Pul-F、Pul-R)，扩增出pulA基因全长(如图2a、图2b、图2c及图2d所示)。PCR反应条件为：94℃预变性5min；94℃变性30s；59℃退火30s；68℃延伸2min，共30个循环；最后68℃保温10min。扩增产物通过1％(w/v，单位g/mL)琼脂糖凝胶电泳进行检测，将目的条带胶回收后连接pEASY-Blunt平末端载体(此载体购自北京全式金生物技术有限公司)，构建克隆质粒pEASY-Blunt-pulA，并转入JM110感受态细胞。经蓝白斑筛选挑取多个单菌落进行菌落PCR验证，将验证正确的菌株扩大培养后送至公司测序；
扩增上游引物Pul-F:5’-CGC GGATCC TTGTCAGTACAGAAGGAAAGCAATG-3’(划线部分为Bam HI酶切位点)，即SEQ ID NO.9。
扩增下游引物Pul-R:5’-CCC AAGCTT CTACTCCGCGATGCTCAGCAC-3’(划线部分为Hind III酶切位点)，即SEQ ID NO.10。
实施例2重组质粒的构建
将验证正确的阳性克隆子扩大培养后提取质粒pEASY-Blunt-pulA，用Bam HI和Hind Ⅲ进行双酶切，将其双酶切后的胶回收产物与pET-28a(+)双酶切胶回收产物用T4DNA连接酶于16℃条件下连接，连接时间为12h。将连接产物转化至E.coli BL21(DE3)感受态细胞中，涂布于含有50μg/mL卡那霉素的LB平板上，37℃培养12h。挑取单克隆进行菌落PCR验证，验证正确的菌株即为重组菌株BL21(DE3)/pET-28a(+)-pulA。
实施例3重组普鲁兰酶的制备
挑取重组菌株BL21(DE3)/pET-28a(+)-pulA接种到20mL的LB液体培养基(含50μg/mL卡那霉素)中，37℃、200r/min条件下培养12h。将菌液以1％(v/v)的接种量接种到50mLLB液体培养基(含50μg/mL卡那霉素)中，继续振荡培养至其OD 600达到0.6时，加入终浓度为0.5mmol/L的IPTG，于22℃、200r/min条件下培养12h后，离心收集菌体，并用0.1mol/L磷酸缓冲液(pH 8.0)洗涤菌体。然后加入缓冲液将其配成菌体浓度在50mg/mL-100mg/mL区间内的重悬液，冰上超声破壁20min后，4℃、12000r/min离心30min收集上清液，即为粗酶液。使用GE公司的镍柱HisTrap HP对粗酶液进行纯化，收集洗脱峰，采用Bradford法测定其蛋白含量，并以质量百分比浓度为5％浓缩胶，质量百分比浓度为10％分离胶进行SDS-PAGE检测。结果显示(如图3所示)：经Ni柱亲和层析纯化后，重组酶在76.95ku处有明显单一蛋白条带，测定其比酶活为508.8U/mg。
实施例4普鲁兰酶活力测定方法
本发明采用DNS法测定重组普鲁兰酶活力，具体操作如下：取100μL稀释适当倍数(稀释倍数为60-120倍，此处为100倍)的重组普鲁兰酶液加入到100uL用100mmol/L PBS缓冲液(pH 6.0)配置而成的1％(w/v，单位g/mL)普鲁兰糖溶液中，45℃下反应30min后，加入300μL DNS试剂终止反应，沸水浴10min。取出冷却后用蒸馏水定容至2.5mL，在540nm处测定反应液的吸光值。以煮沸10min灭活处理后的酶液作为对照。普鲁兰酶活力的定义为：在相应条件下，以每分钟生成1μmol葡萄糖所需的酶量定义为1个酶活力单位(U)。
实施例5重组普鲁兰酶的酶学性质分析
1、温度对重组普鲁兰酶活力的影响及温度稳定性
在pH 6.0条件下，将适当稀释(稀释倍数为60-120倍，此处为100倍)的重组普鲁兰酶液与1％(w/v，单位g/mL)普鲁兰糖分别置于不同温度(25、30、35、40、45、50、55、60℃)下反应30min，分别测定其普鲁兰酶活力，以酶活力最高者为100％对照，探索该酶的最适反应温度。将适当稀释(适当稀释倍数为60-120倍，此处为100倍)的重组普鲁兰酶液分别置于35、40、45、50℃条件下保温15、30、45、60、75、90、105、120min，然后加入到1％(w/v，单位g/mL)普鲁兰糖中于最适温度下反应30min，分别测定其剩余酶活力，以未保温处理的酶液的酶活作为对照，探究该普鲁兰酶的温度稳定性。结果显示：该重组酶的最适反应温度为45℃(如图4)，在35℃-40℃下较为稳定，保温120min后剩余酶活达60％以上，当保温温度高于45℃时，重组酶活力稳定性较差，在50℃下保温30min后相对酶活为43.65％(如图5)。
2、pH对重组普鲁兰酶活力的影响及pH稳定性
在45℃下，将适当稀释的酶液(稀释倍数为60-120倍，此处为100倍)分别与pH3.0-10.0的缓冲液配置而成的1％(w/v，单位g/mL)普鲁兰糖反应30min，测定其普鲁兰酶活力，以酶活力最高者为100％对照，研究该酶的最适反应pH。将适当稀释的酶液(适当稀释倍数为60-120倍，此处为100倍)分别置于pH 3.0-10.0缓冲液中室温保存1h后，于最适pH和最适温度下测定其剩余酶活力，以未处理的酶液的酶活力作为对照，探究该普鲁兰酶的pH稳定性。结果显示，该重组酶的最适反应pH是6.0(如图6)，在pH 6.0-8.0条件下稳定性较高，保温1h后酶活力仍剩余60％以上，在pH 9.0条件下保温1h后，剩余酶活为45.32％(如图7所示)。
3、金属离子对酶活力的影响
在1％(w/v，单位g/mL)普鲁兰糖中加入不同的金属离子，使其终浓度为10mmol/L，在45℃、pH 6.0条件下，与适当稀释的酶液(适当稀释倍数为60-120倍，此处为100倍)反应30min，测定相应的普鲁兰酶活力，以未加入金属离子的酶液的酶活力作为对照。结果显示(如图8所示)：10mmol/L的K+和Mg 2+对该重组酶有激活作用，使其酶活力分别激活至119.36％和123.68％；10mmol/L的Zn 2+、Mn 2+、Ni 2+、Fe 2+、Cu 2+、Co 2+、Ca 2+对重组酶有不同程度的抑制作用，其中Cu 2+对该重组酶的抑制作用最强，使其酶活力仅残留16.73％。
以上实施例仅为本发明较优的实施方式，仅用于解释本发明，而非限制本发明，本领域技术人员在未脱离本发明精神实质下所作的改变、替换、修饰等均应属于本发明的保护范围。
序列表
<110> 华南理工大学
<120> 一种来源于类芽孢杆菌新型普鲁兰酶及其基因与应用
<160> 10
<170> SIPOSequenceListing 1.0
<210> 2
<211> 1968
<212> DNA
<213> 产І型普鲁兰酶的菌株LK18(Paenibacillus puldeungensis LK18)
<400> 2
ttgtcagtac agaaggaaag caatgctgta gttgattacg gcgacctggc ggtaactgaa 60
gggatatccg ttttttccgg ggaattcgac gcaaaatata gttacaacag cgacgatctt 120
ggagcgacct acacgccggg gcagacgaag tttcgacttt gggcgccgac ggcttctgag 180
gcgaaggtga tattttacaa aacgtgggac ggggaaccgg agcgggaact gtccatgaag 240
cgggatgtgc agggaacttg gatactcacc gtcatggagg attgcgccaa tattttctac 300
acgtaccgtg ttaaggttgg cgatcagtgg aatgaagccg ctgacccgta tgccaaagcg 360
gtaggggtaa atggggataa agcggtagtc ctctatctgc gaagcacaga cccggaaggc 420
tggaacaagg agaagccact ttttgactct tccgtggacg cagtgattta tgagcttcat 480
gttcgcgatt tatcgattca tcctagcagc ggcattgatc ctcaaaacca aggaaagttt 540
ctgggccttg ctgaaagcgg tacaaaaggg cctggtggaa tcgccacagg tctcgatcat 600
attgccgggc ttggggtgac gcatgttcag cttctgccga tcttcgatta cgcaacggaa 660
agcgtggatg agcaaaagct cgaccagccg cactacaact gggggtacga tccgaaaaat 720
tacaacgtac ctgaaggctc ttacgcaacc gatccgtatt cccctgcgct gcgcattacg 780
gagctgaaac gaatgattca ggagctgcac gaccgggggc tccgggtcat tatggacgtg 840
gtgtataacc acgtatatga cgggtacctg acccacttca gcaaactagt tcccggttat 900
tacttgcgct acaaaccgga tggaactttt tcaaatggtg cgttttgcgg aaacgagtgt 960
gcctcggagc ggccgatgat gtcaaagtat atcgttgatt cagtacttca ctgggttcgc 1020
gagtaccata tcgatggctt ccggtttgac ctgatgggcc tgatcgatat aagtactatg 1080
aacgagattc gacggcagct tcaggagata gacccttcgt tgatgctgct cggcgaaggt 1140
tggatcatgg atacggttct tccggaagct gcgagggcca atcagactaa tgcggctcag 1200
ctgccgggta tcggtttctt taatgacgga cttcgggacg cggtcaaagg ggatattttt 1260
cagtttgaaa aaaccgggtt catcagtggg ggaggcggct ttgaggagag cgtcaagcgt 1320
ggcgtcgtcg gaggtatcga ttatggcggc acaatccggc aatttgccgt agaccctgga 1380
cagtcggtga actatgtcga gtgccacgac aaccacacat tgtgggacaa aatcgtgctg 1440
tctactcccg gagtgaatga cgaacatcgc cgtgcgatgc accgccttgc ctcagccatc 1500
gtgatgacta gccaggggat tccgtttatc catgccggac aggagtttat gcgaacgaaa 1560
gacggcgtgg aaaacagcta caaatcccca attgagatca actggctcga ttgggagcgc 1620
tgcgcagcac accagtatga cgtagcctat atgcggagcc tgatcgagct gcgcaaggcg 1680
catcgggcgt ttcgcctgcg aacggcggag gagattcggg agcatttaca gtttgaagat 1740
gctccgcctc ataccgtagc ctatacgctg cgggatcatg ccggaggcga tgcagctcgc 1800
cacttgtatg tgctctacaa cgcggcgtca ccggaggcgg tcaccttgcg cctaccagag 1860
cttggcgagt ggcaggtgcg ctatggtgga gagtttgtcc aaactctaag cggcaatcag 1920
ctagtcgtcc aaggcatcgg tatggtcgtg ctgagcatcg cggagtag 1968
<210> 1
<211> 655
<212> PRT
<213> 产І型普鲁兰酶的菌株LK18(Paenibacillus puldeungensis LK18)
<400> 1
Leu Ser Val Gln Lys Glu Ser Asn Ala Val Val Asp Tyr Gly Asp Leu
1 5 10 15
Ala Val Thr Glu Gly Ile Ser Val Phe Ser Gly Glu Phe Asp Ala Lys
20 25 30
Tyr Ser Tyr Asn Ser Asp Asp Leu Gly Ala Thr Tyr Thr Pro Gly Gln
35 40 45
Thr Lys Phe Arg Leu Trp Ala Pro Thr Ala Ser Glu Ala Lys Val Ile
50 55 60
Phe Tyr Lys Thr Trp Asp Gly Glu Pro Glu Arg Glu Leu Ser Met Lys
65 70 75 80
Arg Asp Val Gln Gly Thr Trp Ile Leu Thr Val Met Glu Asp Cys Ala
85 90 95
Asn Ile Phe Tyr Thr Tyr Arg Val Lys Val Gly Asp Gln Trp Asn Glu
100 105 110
Ala Ala Asp Pro Tyr Ala Lys Ala Val Gly Val Asn Gly Asp Lys Ala
115 120 125
Val Val Leu Tyr Leu Arg Ser Thr Asp Pro Glu Gly Trp Asn Lys Glu
130 135 140
Lys Pro Leu Phe Asp Ser Ser Val Asp Ala Val Ile Tyr Glu Leu His
145 150 155 160
Val Arg Asp Leu Ser Ile His Pro Ser Ser Gly Ile Asp Pro Gln Asn
165 170 175
Gln Gly Lys Phe Leu Gly Leu Ala Glu Ser Gly Thr Lys Gly Pro Gly
180 185 190
Gly Ile Ala Thr Gly Leu Asp His Ile Ala Gly Leu Gly Val Thr His
195 200 205
Val Gln Leu Leu Pro Ile Phe Asp Tyr Ala Thr Glu Ser Val Asp Glu
210 215 220
Gln Lys Leu Asp Gln Pro His Tyr Asn Trp Gly Tyr Asp Pro Lys Asn
225 230 235 240
Tyr Asn Val Pro Glu Gly Ser Tyr Ala Thr Asp Pro Tyr Ser Pro Ala
245 250 255
Leu Arg Ile Thr Glu Leu Lys Arg Met Ile Gln Glu Leu His Asp Arg
260 265 270
Gly Leu Arg Val Ile Met Asp Val Val Tyr Asn His Val Tyr Asp Gly
275 280 285
Tyr Leu Thr His Phe Ser Lys Leu Val Pro Gly Tyr Tyr Leu Arg Tyr
290 295 300
Lys Pro Asp Gly Thr Phe Ser Asn Gly Ala Phe Cys Gly Asn Glu Cys
305 310 315 320
Ala Ser Glu Arg Pro Met Met Ser Lys Tyr Ile Val Asp Ser Val Leu
325 330 335
His Trp Val Arg Glu Tyr His Ile Asp Gly Phe Arg Phe Asp Leu Met
340 345 350
Gly Leu Ile Asp Ile Ser Thr Met Asn Glu Ile Arg Arg Gln Leu Gln
355 360 365
Glu Ile Asp Pro Ser Leu Met Leu Leu Gly Glu Gly Trp Ile Met Asp
370 375 380
Thr Val Leu Pro Glu Ala Ala Arg Ala Asn Gln Thr Asn Ala Ala Gln
385 390 395 400
Leu Pro Gly Ile Gly Phe Phe Asn Asp Gly Leu Arg Asp Ala Val Lys
405 410 415
Gly Asp Ile Phe Gln Phe Glu Lys Thr Gly Phe Ile Ser Gly Gly Gly
420 425 430
Gly Phe Glu Glu Ser Val Lys Arg Gly Val Val Gly Gly Ile Asp Tyr
435 440 445
Gly Gly Thr Ile Arg Gln Phe Ala Val Asp Pro Gly Gln Ser Val Asn
450 455 460
Tyr Val Glu Cys His Asp Asn His Thr Leu Trp Asp Lys Ile Val Leu
465 470 475 480
Ser Thr Pro Gly Val Asn Asp Glu His Arg Arg Ala Met His Arg Leu
485 490 495
Ala Ser Ala Ile Val Met Thr Ser Gln Gly Ile Pro Phe Ile His Ala
500 505 510
Gly Gln Glu Phe Met Arg Thr Lys Asp Gly Val Glu Asn Ser Tyr Lys
515 520 525
Ser Pro Ile Glu Ile Asn Trp Leu Asp Trp Glu Arg Cys Ala Ala His
530 535 540
Gln Tyr Asp Val Ala Tyr Met Arg Ser Leu Ile Glu Leu Arg Lys Ala
545 550 555 560
His Arg Ala Phe Arg Leu Arg Thr Ala Glu Glu Ile Arg Glu His Leu
565 570 575
Gln Phe Glu Asp Ala Pro Pro His Thr Val Ala Tyr Thr Leu Arg Asp
580 585 590
His Ala Gly Gly Asp Ala Ala Arg His Leu Tyr Val Leu Tyr Asn Ala
595 600 605
Ala Ser Pro Glu Ala Val Thr Leu Arg Leu Pro Glu Leu Gly Glu Trp
610 615 620
Gln Val Arg Tyr Gly Gly Glu Phe Val Gln Thr Leu Ser Gly Asn Gln
625 630 635 640
Leu Val Val Gln Gly Ile Gly Met Val Val Leu Ser Ile Ala Glu
645 650 655
<210> 3
<211> 26
<212> DNA
<213> 人工合成(人工序列)
<400> 3
gctttcatya ckgcstattg caaagt 26
<210> 4
<211> 26
<212> DNA
<213> 人工合成(人工序列)
<400> 4
taattcttcg gatcdtascc ccagtt 26
<210> 5
<211> 26
<212> DNA
<213> 人工合成(人工序列)
<400> 5
tggaacgagg ccgccgaycc ntaygc 26
<210> 6
<211> 29
<212> DNA
<213> 人工合成(人工序列)
<400> 6
aggcccatca ggtcgaacck raanccrtc 29
<210> 7
<211> 26
<212> DNA
<213> 人工合成(人工序列)
<400> 7
gacgtgtaya accaygtnta tgacgg 26
<210> 8
<211> 23
<212> DNA
<213> 人工合成(人工序列)
<400> 8
tacccyagwa tgaakagacc cgc 23
<210> 9
<211> 34
<212> DNA
<213> 人工合成(人工序列)
<400> 9
cgcggatcct tgtcagtaca gaaggaaagc aatg 34
<210> 10
<211> 30
<212> DNA
<213> 人工合成(人工序列)
<400> 10
cccaagcttc tactccgcga tgctcagcac 30
Claims (8)
1.一种来源于类芽孢杆菌（Paenibacillus puldeungensis）的普鲁兰酶的编码基因pulA，其特征在于，核苷酸序列如 SEQ ID NO.1 所示。
2.一种来源于类芽孢杆菌的普鲁兰酶，其特征在于，氨基酸序列如 SEQ ID NO.2 所示。
3.一种重组质粒，其特征在于，插入了权利要求 1 中SEQ ID NO.1 所述的核苷酸序列。
4.根据权利要求3 所述的一种重组质粒，其特征在于，所述重组质粒通过将 pulA 的编码基因克隆至表达载体 pET-28a(+)的多克隆位点所获得，记为 pET-28a(+)- pulA 。
5.一种基因工程菌株，其特征在于，含有权利要求 4 所述的重组质粒。
6.根据权利要求 5 所述的基因工程菌株，其特征在于，宿主细胞为大肠杆菌 BL21(DE3)。
7.权利要求 1 所述来源于类芽孢杆菌的普鲁兰酶的编码基因 pulA 、权利要求 4 所述的重组质粒、权利要求 5 所述的基因工程菌株在制备权利要求 2 所述来源于类芽孢杆菌的普鲁兰酶的应用。
8.根据权利要求 7 所述的应用，其特征在于，包括如下的步骤：
（1）普鲁兰酶基因 pulA 的克隆；
（2）构建重组质粒；
（3）将步骤（2）中所述的重组质粒转化宿主细胞，得到重组菌株，即所述基因工程菌株；
（4）所述来源于类芽孢杆菌的普鲁兰酶的表达以及纯化。
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| CN110172468A (zh) | 2019-08-27 |
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