CN101451115A - 一种表达中温α-淀粉酶的基因工程菌株 - Google Patents
一种表达中温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中温α－淀粉酶的基因工程菌株。该工程菌是表达中温α－淀粉酶的工程菌，是将表达中温α－淀粉酶基因的重组载体导入枯草芽孢杆菌(BacillusSubtilis)中，筛选得到的中温α－淀粉酶表达量提高的工程菌株；所述中温α－淀粉酶基因具有具有GENBANK ACCESSION Number为AM409180的5′端第7－1551位核苷酸序列。本发明的方法，是发酵所述的表达中温α－淀粉酶的工程菌，得到中温α－淀粉酶。该工程菌具有比目前国内生产菌株更高的中温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中温α－淀粉酶的枯草芽孢杆菌生产菌株。
Description
技术领域
本发明涉及一种表达中温α-淀粉酶的基因工程菌株。
背景技术
中温α-淀粉酶是一种内切酶，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，其一般反应温度为50-70摄氏度，因此称为中温α-淀粉酶。
随着酿造和发酵工业的发展.α-淀粉酶广泛应用于酱油、酱类、食醋、酒类、味精及糖色等工业生产，代替传统的制曲。应用酶制剂与制曲相比，既省工省厂房设备，又能提高出品率，降低成本，增加效益。枯草杆菌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枯草杆菌诱变后逐级扩大培养再经提纯而获得的一种浅灰褐色粉来状有臭味的酶制剂，主要作用是能将原料中的淀粉质液化为糊精。
我国目前生产的α-淀粉酶所用菌种均是BF7658的变异种，如BF7658-209、8a5、86315、K211。目前国内工业用α-淀粉酶，是将发酵液加入硫酸铵，使淀粉酶连同菌丝体等一起析出，经过滤、干燥和粉碎即得工业用α-淀粉酶酶粉。也有将发酵液加稳定剂，直接喷雾干燥制成工业酶粉[谷军：生物技术4(3)：1-5.1994]。
我国对α-淀粉酶菌种的选育、研究和生产做了大量的工作，1957年陈琦等报导了产生淀粉酶能力强的枯草杆菌S17和S56[陈琦等：微生物学报5(3)：256-261.1957]，1965年我国开始应用解淀粉芽孢杆菌BF7658生产α-淀粉酶，以前曾误称为枯草芽孢杆菌BF7658α-淀粉酶，王桂芬证明了原称为枯草芽孢杆菌(BacillusSubtilis)BF7658产生的α-淀粉酶与解淀粉芽孢杆菌的液化型α-淀粉酶完全一样[王桂芬：微生物通报29(2)：124-126.1989]。70年代，无锡酶制剂厂与中科院遗传所等单位合作，以枯草杆菌06-11为出发株经热处理、UV照射，亚硝基胍处理和Co60、γ射线、诱变育种得苗株209。在10吨及20吨罐扩试中，发酵单位提高一倍，产品稳定在300U/ml，最高可达400U/ml。高定华等利用x射线、γ射线及激光等物理因素诱变，获得BF7658的变异株K211，酶活力由原来的260U/ml提高到500-600U/ml，20升发酵罐酶活力达420U/ml[高定华：食品与发酵工业(6).1986]。邬显章等从BF7658变异株209出发，经硫酸二乙酯和5-氟尿嘧啶、亚硝基胍等诱变选育出796变异株，在麦芽糖培养基中可获得较高的α-淀粉酶活性，达477U/ml[邬显章：生物工程学报4(2).115-126.1985]。
通过对α-淀粉酶菌种进行传统的物理化学诱变已经取得了很大的成就，但是若想再进一步提高其产淀粉酶水平就变得非常困难。伴随着分子生物学理论和基因改造技术的飞速发展，使对菌株进行基因方面的改造成为可能。但是国内对枯草芽孢杆菌(Bacillus Subtilis)BF7658的改造主要还是停留在技术水平上，有人将带有淀粉酶基因的克隆片段，在枯草杆菌中表达[蔡恒等：食品与发酵工业31(10)：33-35.2005]，或者将枯草杆菌α-淀粉酶基因在大肠杆菌中的表达。而对BF7658直接进行基因改造，使其淀粉酶产酶效率提升则没有报道。
发明内容
本发明的目的是提供一种表达中温α-淀粉酶的基因工程菌株。
本发明所提供的表达中温α-淀粉酶的工程菌，是将表达中温α-淀粉酶基因的重组载体导入枯草芽孢杆菌(Bacillus Subtilis)中，筛选得到的中温α-淀粉酶表达量提高的工程菌株；所述中温α-淀粉酶基因具有具有GENBANK ACCESSION Number为AM409180的5′端第7-1551位核苷酸序列。
所述枯草芽孢杆菌(Bacillus Subtilis)为枯草芽孢杆菌(Bacillus subtilis)264CICC10264。
所述表达中温α-淀粉酶的工程菌为枯草芽孢杆菌(Bacillus Subtilis)ZHWYCGMCC No.2829。
所述枯草芽孢杆菌(Bacillus subtilis)ZHWY CGMCC No.2829，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.2829。
本发明所提供的一种表达中温α-淀粉酶的方法，是发酵权利要求13中任意一项所述的表达中温α-淀粉酶的工程菌，得到中温α-淀粉酶。
所述发酵用培养基为含75-86g/L的玉米粉，35-45g/L的豆饼粉，1-3g/L的氯化钙，6.5-8.5g/L的磷酸氢二钠，3.5-5.5g/L的硫酸铵的液体培养基。
所述发酵的温度为36.0-38.5℃，优选为37℃。
本发明利用基因工程技术构建带有中温淀粉基因的整合载体，经转化后得到增加了一个中温淀粉基因拷贝工程菌，即本发明的表达中温α-淀粉酶的工程菌。本发明的表达中温α-淀粉酶的工程菌特别是枯草芽孢杆菌(Bacillus subtilis)ZHWY CGMCCNo.2829具有比目前国内生产菌株更高的中温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中温α-淀粉酶的枯草芽孢杆菌生产菌株。本发明的工程菌，发酵表达量高，相同条件下发酵，较野生菌酶活提高了83％。
附图说明
图1为pAXOI-amyl的结构示意图。
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、表达中温α-淀粉酶的携带LacA整合臂穿梭载体pAXOI-amyl的构建
以枯草芽孢杆菌(Bacillus subtilis)264(购自中国工业微生物菌种保藏中心，保藏号为CICC10264，是一株目前广泛应用于工业生产中温α-淀粉酶的枯草芽孢杆菌)的总DNA为模板进行PCR扩增中温α-淀粉酶的基因(amyl)(具有GENBANK ACCESSIONNumber为AM409180的5′端第7—1551位核苷酸序列)。扩增引物为：上游引物5′cgggatccATGATTCAAAAACGAAAGC 3′(下划线标注部分为BamHI酶切位点)和下游引物5′tccatccgcggTTATTTCTGAACATAAATG 3′(下划线标注部分为SacII，酶切位点)。
上述扩增得到1545bp的片段，经过测序表明，该片段具有GENBANKACCESSIONNumber为AM409180的5′端第7—1551位核苷酸序列，该片段包括中温α-淀粉酶基因(amyl)的启动子、信号肽编码序列和结构基因，将该片段命名为amyl。
分别用BamHI和SacII双酶切PCR产物amyl和穿梭整合质粒pAXOI(购自从美国俄亥俄州立大学菌种保存中心，Bacillus Genetic Stock Center(BGSC)The Ohio StateUniversity)，将酶切产物分别用PCR产物回收试剂盒回收纯化后，在4℃采用T4 DNA连接酶进行连接反应16小时。将连接反应物转化宿主菌E.coli Top10(购自北京天根生化科技有限公司)感受态细胞，涂布LB平板(Amp+)，挑选阳性克隆，进行摇瓶培养16小时。收获细胞，提取小量质粒进行酶切和测序验证。将验证正确的穿梭质粒即携带中温淀粉酶的启动子、信号肽和结构基因的表达穿梭整合质粒命名为pAXOI-amyl(结构图如图1所示)。
实施例2、表达中温α-淀粉酶重组枯草芽胞杆菌(ZHWY)的构建
将实施例1构建的携带中温淀粉酶的启动子、信号肽和结构基因的表达穿梭整合质粒pAXOI-amyl以原生质体加电穿孔法(Romero D，Journal of MicrobiologyMethods，2006 Vol 66 p556-559)转化野生菌枯草芽孢杆菌(Bacillus subtilis)264(购自中国工业微生物菌种保藏中，保藏号为CICC10264，是一株目前广泛应用于工业生产中温α-淀粉酶的枯草芽孢杆菌)，具体方法如下所述：
采用Romero D枯草芽孢杆菌原生质体制备方法(Romero D，Journal ofMicrobiology Methods，2006 Vol 66 p556-559)获得枯草芽孢杆菌(Bacillus subtilis)264CICC10264原生质体后，取5ul纯化后的质粒于一个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转杯中，用干滤纸擦干电转杯，注意混合液中不得有气泡。将电转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 66p556559)，重悬细胞后，转移到1.5ml的离心管中。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固体培养基平板(含8g/L琼脂，5g/L水解酪蛋白，5g/L酵母粉，1.5g/L KH2PO4，3.5g/LK2HPO4，45.5g/L山梨醇和10g/L淀粉的培养基)，放入37℃培养箱培养72—96小时后，挑取出现枯草芽孢杆菌的单菌落，提取基因组基因，用实施例1所述的扩增中温α-淀粉酶的基因(amyl)的上下游引物进行PCR鉴定，扩增得到1545bp的片段的菌株即为阳性菌株，结果得到一株验证表明正确的导入pAXOI-amyl得到基因重组菌，将该菌株命名为枯草芽孢杆菌(Bacillus subtilis)ZHWY。该枯草芽孢杆菌(Bacillus subtilis)ZHWY，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.2829。
实施例3、基因重组枯草芽孢杆菌(Bacillus subtilis)ZHWY中温α-淀粉酶表达效果验证
将实施例2所得重组枯草芽孢杆菌(Bacillus subtilis)ZHWY CGMCC No.2829和枯草芽孢杆菌(Bacillus subtilis)264 CICC10264(野生菌)活化做种子。活化条件为将重组枯草芽孢杆菌ZHWY或枯草芽孢杆菌(Bacillus subtilis)264 CICC10264(野生菌)分别接种于装有25ml活化培养基的250mL三角瓶中，37℃，200rpm摇床培养16小时得到重组枯草芽孢杆菌(Bacillus subtilis)ZHWY CGMCC No.2829或枯草芽孢杆菌(Bacillus subtilis)264 CICC10264(野生菌)种子液。
活化培养基组分为：蛋白胨10g/L，酵母粉5/L，氯化钠10g/L，pH7.0-7.2，其余为水。
将上述获得的重组枯草芽孢杆菌(Bacillus subtilis)ZHWY CGMCC No.2829或枯草芽孢杆菌(Bacillus subtilis)264 CICC10264(野生菌)种子液分别按照体积百分含量为24％(请将该数值范围替换成具体实施的数据)的接种量接种于装有50ml发酵培养基的500毫升带带档板的三角瓶中，37℃，200rpm摇床培养，发酵培养76小时，终止发酵。
上述发酵培养基的组成为：玉米粉85g/L，豆饼粉40g/L，氯化钙2/L，磷酸氢二钠8g/L，硫酸铵4g/L，pH7.0，其余为水。
发酵过程中，取样发酵液离心(12000r/min，5min)，取上清液进行检测酶活，中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1g酶粉或1ml酶液于60℃，pH6.0条件下，以1h液化可溶性淀粉的克数来表示(克可溶性淀粉/克.小时或克可溶性淀粉/毫升.小时)(中华人民共和国国家标准，GB8275-87)。
结果表明，在发酵培养76小时，重组菌枯草芽孢杆菌(Bacillus subtilis)ZHWY的酶活达到731U/毫升发酵液，而野生菌酶活为400U/毫升发酵液，重组菌枯草芽孢杆菌(Bacillus subtilis)ZHWY CGMCC No.2829酶活(731U/毫升)比野生菌提高83％。
Claims (7)
1、表达中温α-淀粉酶的工程菌，是将表达中温α-淀粉酶基因的重组载体导入枯草芽孢杆菌(Bacillus Subtilis)中，筛选得到的中温α-淀粉酶表达量提高的工程菌株；所述中温α-淀粉酶基因具有具有GENBANK ACCESSION Number为AM409180的5′端第7—1551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：所述枯草芽孢杆菌(BacillusSubtilis)为枯草芽孢杆菌(Bacillus subtilis)264CICC10264。
3、根据权利要求2所述的工程菌，其特征在于：所述表达中温α-淀粉酶的工程菌为枯草芽孢杆菌(Bacillus subtilis)ZHWY CGMCC No.2829。
4、一种表达中温α-淀粉酶的方法，是发酵权利要求1-3中任意一项所述的表达中温α-淀粉酶的工程菌，得到中温α-淀粉酶。
5、根据权利要求4所述的方法，其特征在于：所述发酵用培养基为含75-86g/L的玉米粉，35-45g/L的豆饼粉，1-3g/L的氯化钙，6.5-8.5g/L的磷酸氢二钠，3.5-5.5g/L的硫酸铵的液体培养基。
6、根据权利要求5所述的方法，其特征在于：所述发酵的温度为36.0-38.5℃。
7、根据权利要求6所述的方法，其特征在于：所述发酵的温度为37℃。
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| CN102127514A (zh) * | 2010-12-01 | 2011-07-20 | 江南大学 | 一株强稳定性中温中性α-淀粉酶高产菌及其酶学性质 |
| CN102533584A (zh) * | 2011-11-05 | 2012-07-04 | 山西卫氏鱼康实业有限公司 | 一种抗水霉解淀粉芽孢杆菌的发酵培养基 |
| CN101962633B (zh) * | 2009-07-23 | 2012-09-05 | 福建福大百特科技发展有限公司 | α-淀粉酶,其编码基因及其表达 |
| CN105348384A (zh) * | 2015-12-01 | 2016-02-24 | 山东隆科特酶制剂有限公司 | 透明颤菌血红蛋白突变体及其在基因工程菌中的可控表达 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101962633B (zh) * | 2009-07-23 | 2012-09-05 | 福建福大百特科技发展有限公司 | α-淀粉酶,其编码基因及其表达 |
| CN102127514A (zh) * | 2010-12-01 | 2011-07-20 | 江南大学 | 一株强稳定性中温中性α-淀粉酶高产菌及其酶学性质 |
| CN102127514B (zh) * | 2010-12-01 | 2012-10-03 | 江南大学 | 一株强稳定性中温中性α-淀粉酶高产菌及其酶学性质 |
| CN102533584A (zh) * | 2011-11-05 | 2012-07-04 | 山西卫氏鱼康实业有限公司 | 一种抗水霉解淀粉芽孢杆菌的发酵培养基 |
| CN105348384A (zh) * | 2015-12-01 | 2016-02-24 | 山东隆科特酶制剂有限公司 | 透明颤菌血红蛋白突变体及其在基因工程菌中的可控表达 |
| CN105348384B (zh) * | 2015-12-01 | 2018-12-18 | 山东隆科特酶制剂有限公司 | 透明颤菌血红蛋白突变体及其在基因工程菌中的可控表达 |
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