CN101451116A - 一种表达高温α-淀粉酶的基因工程菌株 - Google Patents
一种表达高温α-淀粉酶的基因工程菌株 Download PDFInfo
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Abstract
本发明公开了一种表达高温α－淀粉酶的基因工程菌株。该工程菌是是将表达透明颤菌血红蛋白基因的重组载体导入地衣芽孢杆菌(Bacillus Licheniformis)中，筛选得到的高温α－淀粉酶表达量提高的工程菌株；所述透明颤菌血红蛋白基因具有GENBANK Accession Number为L21670的5′端第101－541位核苷酸序列。该工程具有比目前国内生产菌株更高的高温α－淀粉酶产酶能力，具有独特的鉴定标签，是一株极具生产应用价值的高温α－淀粉酶的地衣芽孢杆菌生产菌株。
Description
技术领域
本发明涉及一种表达高温α-淀粉酶的基因工程菌株。
背景技术
高温α-淀粉酶，即能水解淀粉、可溶性糊精及低聚糖中的a—1，4葡萄糖苷键的α-淀粉酶，因为其最适反应温度较高，一般为60摄氏度以上，因此称为高温α-淀粉酶。
高温α-淀粉酶广泛应用于酒类、味精及淀粉糖等工业生产，代替传统工艺既省工省厂房设备，又能提高出品率，降低成本，增加效益。地衣杆菌α-淀粉酶是我国产量最大用途最广的一种高效液化型淀粉酶，它是由耐热性能高的地衣杆菌诱变后逐级扩大培养再经提纯而获得的一种酶制剂，主要作用是能将原料中的淀粉质液化为糊精。
透明颤菌血红蛋白(vitreoscilla hemoglobin，VHb)是20世纪70年代后期发现的一种血红蛋白，该蛋白质能使透明颤菌在低氧的环境下生存，并保持较高的生长速率。透明颤菌为专性好氧的革兰氏阴性菌，为贝日阿托氏菌属(Beggiatoa)，能在贫氧条件下合成一种可溶性的血红蛋白以满足其对氧的需求。1986年Wakabayashi综合研究其光谱学特征、氧结合特性以及蛋白质氨基酸顺序，表明该蛋白与真核生物的血红蛋白具有相当高的同源性和相似性，故将其命名为透明颤菌血红蛋白(WakabayashiS，MatsubaraH，WebsterDA.Nature，1986，322：481～483)。1988年，美国IIT和CIT两个实验室分别成功地从透明颤菌染色体上克隆了透明颤菌血红蛋白的基因(vitreoscillahemoglobingene，vgb)，测定了核苷酸序列并在大肠杆菌中进行了表达，由于VHb的存在，使细菌能够在限氧条件下生存，促进了细胞总蛋白的合成和外源蛋白的积聚，改善了生长条件(DikshitK L，WebsterD A.Cloning，Gene，1988，70：377～386)。自从VHb在大肠杆菌中成功地进行表达以来，VHb已在假单胞杆菌、链霉菌、霉菌和酵母等多种生物体内实现克隆。
发明内容
本发明的目的是提供一种表达高温α-淀粉酶的基因工程菌株。
本发明所提供的可表达高温α-淀粉酶的工程菌，是将表达透明颤菌血红蛋白基因的重组载体导入地衣芽孢杆菌(Bacillus Licheniformis)中，筛选得到的高温α-淀粉酶表达量提高的工程菌株；所述透明颤菌血红蛋白基因具有GENBANK AccessionNumber为L21670的5′端第101—541位核苷酸序列。
所述表达透明颤菌血红蛋白基因的重组载体中所述透明颤菌血红蛋白基因的5′端连接有sacB基因启动子和信号肽编码基因的DNA片段；所述sacB基因启动子和信号肽编码基因的DNA片段具有Genebank Accession Number为X02730的5′端第208-463位核苷酸序列。
所述地衣芽孢杆菌(Bacillus Licheniformis)为地衣芽孢杆菌(BacillusLicheniformis)CICC10181。所述可表达高温α-淀粉酶的工程菌优选为地衣芽孢杆菌(Bacillus Licheniformis)WY CGMCC No.2830。
所述地衣芽孢杆菌(Bacillus Licheniformis)WY CGMCC No.2830，已于2008年12月25日保藏于中国微生物菌种保藏管委理员会普通微生物中心(简称CGMCC，地址为：中国北京市朝阳区大屯路)，保藏号为CGMCC No.2830。
本发明的可表达高温α-淀粉酶的工程菌，特别是地衣芽孢杆菌(BacillusLicheniformis)WY CGMCC No.2830具有比目前国内生产菌株更高的高温α-淀粉酶产酶能力，具有独特的鉴定标签，是一株极具生产应用价值的高温α-淀粉酶的地衣芽孢杆菌生产菌株。实验证明，本发明的高温淀粉酶基因工程菌株与野生型菌株在相同条件下发酵诱导后，发酵菌体生物量比野生菌高1.3倍；高温淀粉酶酶活比野生菌提高2.1倍。
附图说明
图1为pAXOI-SacB-vgb的结构图。
具体实施方式
下述实施例中所用方法如无特别说明均为常规方法。
实施例1、表达血红蛋白基因的地衣芽孢杆菌整合载体的构建
采用常规分子生物学融合PCR技术，将枯草芽胞杆菌SacB基因(果聚糖蔗糖酶levansucrase)的启动子及信号肽部分(Genebank Accession Number为X02730的5′端第208-463位核苷酸序列)和带有透明颤菌(Vitreoscilla)血红蛋白基因(VHb)(Genebank Accession Number为L21670的5′端第101—541位核苷酸序列)连接在一起。
以地衣芽孢杆菌(B.Licheniformis-181)CICC10181基因组DNA为模板进行PCR扩增，上游引物为引物1(5’cgggatcccccgtagtctgcaaatcctt 3’(下划线标注部分为BamHI酶切位点))和下游引物为引物2
(5’atggtttgctggtctaacatcgttcatgtctcctttttta 3’(下划线标注的20个碱基对与血红蛋白编码基因的5’端互补，其余20个碱基对和SacB启动子3’互补))。
扩增条件：预变性94℃ 5分钟，然后变性94℃ 1分钟，退火56℃一分钟，延伸72℃ 1分30秒。完成30个循环后，72℃保温10分钟。
扩增产物为含sacB基因启动子和信号肽的DNA序列。将PCR产物用PCR产物胶回收试剂盒纯化，进行测序表明该片段具有Genebank Accession Number为X02730的5′端第208-463位核苷酸序列为含sacB基因启动子和信号肽的DNA序列的片段。将该含sacB基因启动子和信号肽的DNA序列的基因片段命名为sacB。
以透明颤菌vitreoscilla(由美国ATCC购得，保藏编号为ATCC 15551，KhoslaC Mol Gen Genet.1988 Sep；214(1)：158-61，北京中天诺亚体育新技术发展有限公司)基因组DNA为模板进行PCR扩增，上游引物为引物3(TAAAAAAGGAGACATGAACGATGTTAGACCAGCAAACCAT(下划线标注的20个碱基对与血红蛋白编码基因的5’端互补，其余20个碱基对和SacB启动子3’互补)和下游引物为引物4(5’tccatccgcggttattcaacctcttg(下划线部分为SacII酶切位点)。
扩增条件：预变性94℃ 5分钟，然后变性94℃ 1分钟，退火58℃一分钟，延伸72℃ 1分钟。完成30个循环后，72℃保温10分钟。
将PCR产物凝胶回收试剂盒纯化，将回收得到的基因片段进行测序，结果表明该基因片段含有透明颤菌血红蛋白基因vgb，具有Genebank Accession Number为L21670的5′端第101—541位核苷酸序列，将该片段命名为vgb。
采用融合PCR技术，将上述得到的sacB和vgb二个基因片段连接，按照分子比1：1混匀为模板，以引物1和引物4为引物进行PCR扩增。
扩增条件：不加引物仅加模板，dNTP和DNA聚合酶扩增循环：预变性94℃ 5分钟，再进行5个循环：变性94℃ 1分钟，退火56℃一分钟，延伸72℃ 1分30秒。完成5个循环后再加引物1和引物4，然后再进行25个循环。
扩增产物为约800bp的DNA片段。回收产物后进行SacII和BanHI双酶切回收大片段，用SacII和BanHI酶切载体pAXOI(从美国俄亥俄州立大学菌种保存中心(Bacillus Genetic Stock Center(BGSC)The Ohio State University购得)，回收载体片段，然后将两个片段采用T4 DNA连接酶在4℃进行连接反应16小时，将连接反应产物转化E.coli TOP 10(购自北京天根生化科技有限公司)的感受态细胞，挑选阳性克隆，在LB培养基中37℃过夜培养后提取质粒进行酶切和电泳验证，得到含有sacB的启动子和信号肽和vgb的地衣芽胞杆菌整合质粒，将其进行测序验证，将测序表明含有sacB的启动子和信号肽和vgb正确的重组载体命名为pAXOI-sacB-vgb(其结构如图1所示)。
实施例2、表达高温α-淀粉酶的工程菌WY的构建
将实施例1构建的带有sacB启动子、信号肽、vgb的地衣芽胞杆菌整合质粒pAXOI-SacB-vgb以原生质体加电穿孔法(Romero D，Journal of MicrobiologyMethods，2006 Vol 66 p556-559)转化产高温淀粉的地衣芽孢杆菌CICC10181(购于中国工业微生物菌种保藏管理中心，保藏号为CICC10181)，具体方法如下所述：
采用Romero D地衣芽孢杆菌原生质体制备方法(Romero D，Journal ofMicrobiology Methods，2006 Vol 66 p556-559)获得地衣芽孢杆菌CICC10181原生质体后，取5ul(2ug)纯化后的质粒pAXOI-SacB-vgb于一个1.5ml的离心管中，将其和0.2CM的电击杯放在冰上预冷，将120ul制备好的原生质体转入至上述装有pAXOI-SacB-vgb的1.5ml的离心管中，小心混匀，冰上放置10min，然后开启电转仪，调电压到600V；将质粒和原生质体细胞混合液移到已经预冷的电击杯中，用干滤纸擦干电击杯，注意混合液中不得有气泡。将电击杯放入电转仪的杯槽中，按下电击键，有放电声出现后，立即向电转杯中加入1ml细胞复苏缓冲液(Romero D，Journal of Microbiology Methods，2006 Vol 66p556-559)，重悬细胞后，转移到1.5ml的离心管中。
将上述装有转化后的细胞的离心管放置37℃，100rpm摇床培养12-16小时，取150ul菌液涂布于含有10ug/ml红霉素的DM3固体培养基(含8g/L琼脂，5g/L水解酪蛋白，5g/L酵母粉，1.5g/L KH2PO4，3.5g/L K2HPO4，45.5g/L山梨醇和10g/L淀粉的培养基)平板，放入37℃培养箱培養72—96小时后出现地衣芽孢杆菌的单菌落，挑取单菌落即为可在含10ug/ml红霉素平板上生长的阳性克隆，将得到的阳性菌株提取基因组基因，用引物3和引物4进行PCR验证，扩增产物为约800bp的DNA片段的菌株为阳性菌株，结果表明得到一株表明正确的导入pAXOI-SacB-vgb的重组菌株，将该菌株命名为地衣芽孢杆菌(BacillusLicheniformis)WY。该地衣芽孢杆菌(Bacillus Licheniformis)WY CGMCC No.2830，已于2008年12月25日保藏于中国微生物菌种保藏管委理员会普通微生物中心(简称CGMCC，地址为：中国北京市朝阳区大屯路)，保藏号为CGMCC No.2830。
实施例3、基因重组地衣芽孢杆菌WY高温淀粉酶表达验证
采用500毫升带档板的三角瓶，装培养基的量为50ml，将实施例2所得地衣芽孢杆菌WY或地衣芽孢杆菌(B.Licheniformis-181)CICC10181(野生菌)分别按照0.01g菌体/ml培养基的量接种于一个三角瓶中，在37℃摇床培养，摇床转速为200rpm。发酵48h时，向摇瓶内加终浓度为20g/L的蔗糖诱导，发酵96h，终止发酵。两组处理分别设三个重复。
所述培养基的组成为：豆饼粉26.1g/L，棉籽饼粉21.1g/L，氯化钙0.46g/L，硫酸铵5g/L，柠檬酸钠2g/L，磷酸氢二钾14g/L，磷酸二氢钾6g/L，玉米浆21.1g/L，乳糖105g/L(独立灭菌)，pH值为6.7，其余为水。
发酵过程中，取发酵上清液进行酶活检测。高温淀粉酶活检测采用中华人民共和国家标准法测定(QB/T2306-1997)；酶活的定义为在70℃，pH6.0条件下，1分钟液化1毫克可溶性淀粉成为糊精所需的酶量，即为一个酶活力单位，以U/ml或U/g表示。
结果表明，在发酵培养96小时，重组地衣芽孢杆菌WY的酶活达到9100U/ml发酵液，生物量达到20g菌/L发酵液，而野生菌地衣芽孢杆菌(B.Licheniformis-181)CICC10181酶活为4200U/ml，生物量为12g/L发酵液。地衣芽孢杆菌WY酶活(9100U/ml发酵液)比野生菌提高2.1倍。
Claims (8)
1、表达高温α-淀粉酶的工程菌，是将表达透明颤菌血红蛋白基因的重组载体导入地衣芽孢杆菌(Bacillus Licheniformis)中，筛选得到的高温α-淀粉酶表达量提高的工程菌株；所述透明颤菌血红蛋白基因具有GENBANK Accession Number为L21670的5′端第101—541位核苷酸序列。
2、根据权利要求1所述的工程菌，其特征在于：所述表达透明颤菌血红蛋白基因的重组载体中所述透明颤菌血红蛋白基因的5′端连接有sacB基因启动子和信号肽编码基因的DNA片段；所述sacB基因启动子和信号肽编码基因的DNA片段具有Genebank Accession Number为X02730的5′端第208-463位核苷酸序列。
3、根据权利要求2所述的工程菌，其特征在于：所述地衣芽孢杆菌(BacillusLicheniformis)为地衣芽孢杆菌(Bacillus Licheniformis)CICC10181。
4、根据权利要求3所述的工程菌，其特征在于：所述表达高温α-淀粉酶的工程菌为地衣芽孢杆菌(Bacillus Licheniformis)WY CGMCC No.2830。
5、一种表达高温α-淀粉酶的方法，是发酵权利要求1-4中任意一项所述的表达高温α-淀粉酶的工程菌，在发酵过程中加入蔗糖诱导培养得到高温α-淀粉酶。
6、根据权利要求5所述的方法，其特征在于：所述发酵用培养基为含有26.1g/L豆饼粉，21.1g/L棉籽饼粉，0.46g/L氯化钙，5g/L硫酸铵，2g/L柠檬酸钠，14g/L磷酸氢二钾，6g/L磷酸二氢钾，21.1g/L玉米浆，105g/L乳糖的液体培养基。
7、根据权利要求6所述的方法，其特征在于：所述蔗糖加入的质量百分比终浓度为2％-4％。
8、根据权利要求7所述的方法，其特征在于：所述蔗糖加入的时间是所述发酵培养后40—48小时；所述发酵的时间为72～96小时，所述发酵的温度为37℃。
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102363761A (zh) * | 2011-11-18 | 2012-02-29 | 山东安克生物工程有限公司 | 一种产耐高温α-淀粉酶菌种的优化方法 |
| CN101633898B (zh) * | 2009-06-25 | 2013-04-10 | 昆明理工大学 | 高温地衣芽孢杆菌及其产高温淀粉酶 |
| CN103739080A (zh) * | 2014-01-13 | 2014-04-23 | 黑龙江省科学院大庆分院 | 一种高纬度城市生活污水植物水处理方法 |
| CN103834606A (zh) * | 2014-01-16 | 2014-06-04 | 北京中科星冠生物技术有限责任公司 | 一种表达耐酸高温α-淀粉酶基因突变体的工程菌株 |
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| CN103146629B (zh) * | 2013-03-05 | 2014-03-19 | 绿康生化股份有限公司 | 携带透明颤菌血红蛋白基因的地衣芽孢杆菌的菌株、构建方法及应用 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101633898B (zh) * | 2009-06-25 | 2013-04-10 | 昆明理工大学 | 高温地衣芽孢杆菌及其产高温淀粉酶 |
| CN102363761A (zh) * | 2011-11-18 | 2012-02-29 | 山东安克生物工程有限公司 | 一种产耐高温α-淀粉酶菌种的优化方法 |
| CN103739080A (zh) * | 2014-01-13 | 2014-04-23 | 黑龙江省科学院大庆分院 | 一种高纬度城市生活污水植物水处理方法 |
| CN103739080B (zh) * | 2014-01-13 | 2016-01-13 | 黑龙江省科学院大庆分院 | 一种高纬度城市生活污水植物水处理方法 |
| CN103834606A (zh) * | 2014-01-16 | 2014-06-04 | 北京中科星冠生物技术有限责任公司 | 一种表达耐酸高温α-淀粉酶基因突变体的工程菌株 |
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