CN105886519B - 一种异淀粉酶及其基因、含有该基因的工程菌及其应用 - Google Patents
一种异淀粉酶及其基因、含有该基因的工程菌及其应用 Download PDFInfo
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Abstract
本发明属于应用工业微生物领域,公开了一种异淀粉酶及其基因、含有该基因的工程菌及其应用。本发明提供了淀粉水解酶系中关键酶之一的异淀粉酶基因,该基因全长为2436bp,G+C含量为66%,编码811个氨基酸,其核苷酸序列为:SEQ ID NO.1所编码的内切酶蛋白质氨基酸序列为:SEQ ID NO.2。利用该基因构建的工程菌株能高效表达异淀粉酶,该酶以土豆淀粉为测活底物,通过碘液检测异淀粉酶的活性,其比活力高达70600U/mg。利用基因生产的酶制剂可用于粮食加工、食品工业、酿造、发酵、纺织工业和医药等工业,在解决实际问题的同时还可以取得可观的经济效益。
Description
技术领域
本发明属于应用工业微生物领域,公开了一种异淀粉酶基因、含有该基因的工程菌及其应用。
背景技术
淀粉作为地球上含量最为丰富的聚合物之一,是由葡萄糖单体单元以葡萄糖苷键连接而成的,根据糖苷键的区别分为两种重要组分:直链淀粉(线性α‐1,4键连接的葡聚糖)和支链淀粉(线性α‐1,4键连接的葡聚糖上连有α‐1,6键的支链),虽然α‐1,6键只占总糖苷键的6%左右,使得淀粉糖链形成了分支结构。淀粉由于其结构的复杂性,单一酶类难以将其彻底的水解,因此在淀粉处理过程中通常需要多种酶协同作用,包括α‐淀粉酶以及淀粉脱支酶等。
异淀粉酶(EC 3.2.1.68)是一种主要的淀粉脱支酶,为糖原6‐葡聚糖水解酶,对糖原,支链淀粉以及极限糊精中的α‐1,6‐糖苷键具有不同程度的水解作用,但对普鲁兰多糖则没有水解作用。即仅能水解分支点的α‐1,6糖苷键。异淀粉酶在淀粉深加工工业具有重要的价值,可以与α‐1,4‐淀粉酶、糖化酶等淀粉酶配合使用生产葡萄糖、麦芽糖、部分麦芽寡糖、环糊精以及抗性淀粉等产品,提高淀粉的转化效率。基于异淀粉酶特殊的性质使其在不同领域具有广泛的应用前景,如在饲料中添加异淀粉酶可以提高日粮的消化利用率,在配合其他淀粉水解酶处理淀粉过程中可以提高产物的生成率,作为酶制剂和加工助剂应用于食品行业以及将异淀粉酶应用于淀粉膜改进方面的研究等。
作为一种具有重要工业应用价值的淀粉脱支酶,异淀粉酶研究在国外已有60多年的历史。最早报道的是在1951年日本学者Maruo等在酿酒酵母中发现了一种胞内异淀粉酶。Amemura等人在1988年首次对多支淀粉假单胞菌Pseudomonas amyloderamosa来源的异淀粉酶的基因进行克隆和表达,目前,只有P.amyloderamosa来源的异淀粉酶实现了工业化生产。我国异淀粉酶研究虽然相对较晚,但是也开展了广泛的研究,王武等人1993年研究和讨论了一株产异淀粉酶短杆菌的筛选、诱变,Duan等人2013年将异淀粉酶与α-环糊精酶联合使用,显著提高了环糊精的产量。目前,国内在不同来源的异淀粉酶产生菌以及异淀粉酶的性质等异淀粉酶研究方面上虽然取得了较多的进展,但是至今还未实现工业化生产。同时国内自主知识产权的异淀粉酶较少,受限于酶活力以及性质的限制,使得目前市场上的商业化产品主要为进口酶制剂。
发明内容
本发明的目的在于提供一种新的异淀粉酶基因,及其编码的蛋白质。
本发明的另一目的是提供含有该异淀粉酶基因的基因工程菌。
本发明的又一目的是提供该基因的应用。
异淀粉酶基因,其核苷酸序列为:SEQ ID NO.1,该基因全长(从起始密码子到终止密码子)为2436bp,G+C含量为66%,编码811个氨基酸。
本发明所述的异淀粉酶基因编码的异淀粉酶蛋白质,其氨基酸序列为:SEQ IDNO.2。
所述的异淀粉酶最适反应pH为7.0,最适反应温度为45℃,并在20℃‐40℃(1h)和pH 6.0-10.0(24h)之间保持活性稳定。
含本发明所述异淀粉酶基因的重组质粒。
所述的重组质粒优选将所述异淀粉酶基因克隆到pEFaA质粒中所得。
含本发明所述的重组质粒的重组微生物。
所述的重组微生物,优选以毕赤酵母为宿主菌。
本发明所述异淀粉酶蛋白质在淀粉加工或工业生产方面的应用。
本发明所述异淀粉酶蛋白质联合α-1,4淀粉酶内切酶在产麦芽六糖过程中的应用。
所述的α-1,4淀粉酶内切酶优选专利申请号为“201310043628.5”(国际申请号为PCT/CN2013/078680),名称为“一种α-淀粉酶及其基因、含有该基因的工程菌及其应用”中保护的α-1,4淀粉酶内切酶。该α-1,4淀粉酶内切酶的编码基因如该专利序列表SEQ IDNO.3所示,其氨基酸序列如该专利序列表SEQ ID NO.4所示。表达方法、酶学性质、功能在该专利中已公开,详见该专利的公开文本。
有益效果
1.本发明以粘细菌菌株EGB为材料,参考EGB基因组序列信息并结合PCR扩增,成功获得异淀粉酶基因序列。该基因全长(从起始密码子到终止密码子)为2436bp,G+C含量为66%,编码811个氨基酸。
2.通过PCR技术扩增末端含XhoI和XbaI酶切位点的完整的异淀粉酶基因片段,将它连接到毕赤酵母P.pastoris GS115高表达载体pEFaA(购自Novegen公司)的XhoI和XbaI酶切位点上,转化表达宿主菌P.pastoris GS115(购自Invitrogen公司),进行甲醇诱导表达,诱导表达量为0.5mg/L。
3.本发明对异淀粉酶基因表达的产物,通过碘液方法对异淀粉酶进行酶活性测定,该异淀粉酶能高效的水解可溶性淀粉、玉米淀粉、土豆淀粉和支链淀粉,在以土豆淀粉为底物时的的比活力高达高达70 600U/mg。
4.利用该基因构建的工程菌株能高效表达异淀粉酶,在淀粉加工过程中,该异淀粉酶联合α-1,4淀粉酶内切酶能够有效提高淀粉的水解效率,水解产物中麦芽六糖的含量相比较α-1,4淀粉酶内切酶单独处理组提高了60%,与商业化脱支酶D2的40%提高量相比较,显示出较好的水解效率和水解稳定性。
附图说明
图1异淀粉酶基因的PCR扩增电泳图
1:DL5000核酸Marker;2:异淀粉酶基因PCR扩增
图2异淀粉酶基因克隆的策略图
图3异淀粉酶基因在P.pastoris GS115(pEFaA)中高效表达实验方案图
图4重组异淀粉酶的SDS‐PAGE电泳图
M:标准蛋白Marker;1:酵母诱导上清酶液;2:以淀粉平板的酶谱分析
图5异淀粉酶酶学性质
a:异淀粉酶最适温度;b:温度稳定性;c:异淀粉酶最适pH;d:pH稳定性
图6异淀粉酶联合α-1,4淀粉酶内切酶在淀粉处理过程的应用
a:对照b:麦芽六糖淀粉酶处理;c:麦芽六糖淀粉酶与异淀粉酶联合处理
生物材料保藏信息
Corαllococcus coralloldes EGB,保藏于中国典型培养物保藏中心,保藏地址为中国武汉,武汉大学,保藏日期为2012年12月17日,保藏号为CCTCC NO:M2012528。
具体实施方式
实施例1异淀粉酶基因的克隆及表达载体的构建
1.1异淀粉酶基因的PCR扩增
参考粘细菌EGB基因组序列并结合NCBI基因组信息进行ORF预测,以全长序列设计异淀粉酶基因引物,以EGB菌(CCTCC NO:M2012528)的基因组DNA为模板,进行异淀粉酶基因全长的PCR扩增,得到异淀粉酶基因的全长序列,所用引物为F和R,结果见图1。具体过程参照图2。
F:ctcgagAAAAGAGAGGCTATGACCCCTCCCCGTCGTCAC(XhoI)(SEQ ID NO.5)
R:tctagaCTTGGCCACCAACACCAGC(XbaI)(SEQ ID NO.6)
1.2E.coli DH10B电转感受态的制备
从‐80℃冰箱中取菌种E.coli DH10B划线于新鲜的LB平板上,培养过夜,挑取直径约2mm菌落接入没有添加Mg2+的SOB试管,37℃培养至OD600到达1.0后,以1/100的接种量接入装有100ml SOB培养基的0.5L摇瓶,18℃,220rpm培养至OD600到达0.7~0.8之间;将摇瓶置于冰浴中,冷却10min之后,4℃4000rpm离心5min收集菌体沉淀;等体积的灭菌超纯水重悬、洗涤菌体后,4℃4000rpm离心5min收集菌体沉淀;重复洗涤一次;100ml 10%甘油重悬菌体,4℃4000rpm离心5min收集菌体沉淀;重复洗涤一次;小心弃上清,倒置离心瓶于灭菌吸水纸上沥干约1min。每1000ml培养物用2ml 10%甘油小心重悬,每管100μl分装于离心管后迅速放入‐80℃冰箱保存备用。
1.3毕赤酵母感受态细胞制备
从接种在新鲜的YPD斜面上的毕赤酵母GS115挑取单菌落;接种单菌落至50mL YPD培养基的250mL三角瓶中,28℃,200rpm,培养过夜至OD600值在1.0‐1.5之间;吸取培养液按5%接种量接种至50mL YPD培养基的250mL三角瓶中,28℃,200rpm,培养至OD600值在0.3‐0.5之间;4500rpm,离心5min,收集菌体;将菌体与新鲜配制的8mL酵母细胞感受态母液轻轻混匀后,室温放置30min,每10min轻轻摇匀一次;4500rpm,离心5min,收集菌体;用2mL的预冷的1M山梨醇重悬,然后于4℃条件下,4000rpm,离心5min,收集菌体,重复三次;然后用100μL预冷的1M山梨醇重悬,分装至1.5mL离心管中,每管80μL,于‐80℃下保存。
1.4重组质粒的构建
将1.1中异淀粉酶基因PCR扩增产物经过过柱回收之后与pMD19‐T vector(TaKaRaCode:D102A)在连接液作用下,16℃水浴过夜。酶连体系如下:
将10μl酶连产物加入到200μl在冰上融化后的E.coli DH10B感受态细胞中,冰浴30min,在42℃水浴锅中热激90s后。快速转移到冰浴中冷却1~2min,向每管中加入800μl液体LB培养基,37℃摇床80-90rpm温育45min,复苏细胞。4000rpm离心3min,剩余200μl感受态细胞涂布于含100mg/l氨苄青霉素的LB琼脂平板上,平板倒置于37℃培养箱培养。挑取经过测序正确的转化子并提取质粒。挑取单菌落在含氨苄青霉素的LB培养基中培养过夜,12000rpm离心10min收集菌体,利用质粒提取试剂盒提取质粒,送上海英潍捷基生物有限公司测序。结果该基因全长(从起始密码子到终止密码子)为2436bp,G+C含量为66%,序列为SEQ ID NO.1;该基因编码811个氨基酸,其氨基酸序列为SEQ ID NO.2。
并将提取的重组质粒用XbaI和XhoI双酶切,同时将质粒pEFαA也用相同的XbaI和XhoI进行双双酶切。
酶切体系:
在37℃水浴中,反应3h以上。酶切产物进行0.75%的琼脂糖凝胶电泳切胶回收
1.5酶连转化
体系如下:
将体系放置于16℃水浴锅中过夜反应。将酶连产物转化到E.coli DH10B中后,涂布于含有终浓度为25μg/mL的Zeocin的LB培养基平板上,挑取取转化子质粒并酶切验证,测序正确后保存于终浓度为15%甘油的‐80℃低温冰箱中;构建成功的重组质粒命名为pEFαA‐IaM。
1.6重组质粒pEFαA‐IaM的线性化
采用限制性内切酶Sca I对正确的重组质粒进行线性化,将体系放置于37℃水浴条件下反应8h后回收酶切产物。
反应体系如下:
本实验采用乙醇沉淀法纯化和回收线性化产物,具体方法如下:
1.加入预冷的1/10体积的3M醋酸钠(pH 5.2)和2倍体积的无水乙醇,轻轻混匀后放置于‐20℃下20min;
2.然后于4℃条件下12000rpm,离心10min,弃上清液;
3.向离心管中加入2倍体积的预冷70%乙醇,12000rpm,离心3min,重复一次,弃上清;
4.待乙醇挥发干净后,加入30μL无菌水溶解。
1.7电转化
将0.2cm电转化杯放置于冰上5min,打开电转化仪,调整所需电转化参数:电压1.5kV,电阻250Ω,电容25μF;向制备好的毕赤酵母GS115感受态细胞中加入1-3μg DNA片段,轻轻混匀后放置于冰上5min后转移至电转杯中;按照仪器使用说明书进行电转操作;电转化后立刻向电转杯中加入1mL预冷的1M山梨醇,转入1.5mL离心管中,放置在30℃培养箱中静置培养1h;室温下4000rpm下离心3min,弃上清,收集菌体后用200μL YPDS重悬;将100μL细胞悬浮液涂布在含有100μg/mL Zeocin的YPD平板上;将平板放置在30℃下培养3-4天,待菌体长出。
将平板上长出的单菌落挑至含有100μg/mL Zeocin的YPD平板上,待长出后使用目的基因的特征引物进行菌落PCR,以检测目的基因是否整合到酵母染色体中;25μL反应体系,退火温度60℃,延伸时间1min 30s。PCR产物用0.75%琼脂糖凝胶电泳检测DNA片段大小。阳性克隆命名为P.pastoris GS115(pEFaA‐IaM)。
实施例2.异淀粉酶基因在P.pastoris GS115(pEFaA‐Iam)中的高效表达
2.1异淀粉酶IaM的表达
将表达菌株P.pastoris GS115(pEFαA‐IaM)划线平板培养,挑取单菌落至50mL液体YPD三角瓶中,28℃,200rpm培养24h;然后在室温下4000rpm,离心5min,弃去上清液,将菌体用25mL BMMY培养基重悬,开始诱导酵母细胞的表达;继续28℃条件下,200rpm培养,每隔24h补加甲醇至终浓度为0.5%(v/v),共培养96h;待培养结束后,检测目的蛋白的酶活,结果发现该异淀粉酶的表达量达到0.5μg/mL。
2.2蛋白质电泳SDS‐PAGE鉴定和复性酶谱
蛋白样品在12%的聚丙烯酰胺凝胶电泳(SDS‐PAGE),电泳完成后,对含有酶样品的泳道进行割胶处理,放置于含有预冷的2.5%Triton‐X 100的平板中,4℃放置30min后,更换Triton‐X100,放置30min;然后将条带转移到含有预冷的0.1M pH 7.0Tris‐HCl的缓冲液中,4℃,30min后,更换缓冲液,继续放置30min;同样的操作放置在50mM pH 7.0Tris‐HCl缓冲液中,时间间隔为15min;将复性好的条带铺展在溶解于50mM pH 7.0Tris‐HCl缓冲液的可溶性淀粉固体平板上,37℃反应10min后,滴加碘液进行显色。由于异淀粉酶IaM能水解淀粉成低聚糖物质使得其不能和碘液发生颜色反应,所以条带上有活性的异淀粉酶IaM处不会产生蓝紫色反应,通过这一现象可检测电泳后条件的酶是否具有活性。图4显示表达后的异淀粉酶条带较为单一,酶谱结果显示该单一条带为异淀粉酶表达目的条带。
实施例3.异淀粉酶酶学性质的研究
3.1温度对酶活力的影响
最适反应温度的测定:在不同温度(20℃、30℃、40℃、45℃、50℃、55℃、60℃、70℃),pH7.0的条件下测定重组酶纯化酶的活性,将最高酶活力设定为100%(图5a)。热稳定性的测定:将重组酶纯化酶液在20℃、30℃、40℃、45℃、50℃、60℃、70℃,pH7.0下保温1h,每隔10min取样,于冰上迅速冷却,各自测定残余酶活力,以未保温的酶活力为100%(图5b)。经测定该淀粉酶最适反应温度为45℃,并在20℃-45℃之间保持稳定。
3.2pH对酶活力的影响
最适反应pH的测定:在不同pH值(5.0、6.0、7.0、8.0、9.0、10.0),50℃下测定重组酶纯化酶液的活性,将最高活力设定为100%(图5c)。pH稳定性的测定:将重组酶纯化酶液在pH3.0、4.0、5.0、6.0、7.0、8.0、9.0、10.0、11.0下,4℃保持24h,后各自测定其残余活力,以pH7.0的酶活力为100%(图5d)。经测定该淀粉酶最适反应pH为6.0,并在pH 6.0-11.0保持稳定。
3.3异淀粉酶底物特异性
以可溶性淀粉,土豆淀粉,玉米淀粉,支链淀粉,直链淀粉,普鲁兰多糖为底物,对表达的异淀粉酶进行底物特异性分析(如表1),结果发现该异淀粉酶对可溶性淀粉,土豆淀粉,玉米淀粉,支链淀粉等底物均具有活性,其中土豆淀粉为底物时活性最高。以土豆淀粉为测活底物,通过碘液检测异淀粉酶的活性,酶活单位定义为每分钟OD610值上升0.01为一个活力单位,测得异淀粉酶的比活力为70 600U/mg。
表1
底物 | 相对酶活(%) |
普鲁兰多糖 | 0 |
支链淀粉 | 21 |
可溶性淀粉 | 20 |
玉米淀粉 | 88 |
土豆淀粉 | 100 |
糖原 | 0 |
实施例4.异淀粉酶联合相关淀粉水解酶在淀粉加工过程中的应用
由于淀粉中含有大量的支链淀粉,导致淀粉酶解不完全,影响水解产物的含量,而异淀粉酶为专一性切割淀粉支链的水解酶,使淀粉的支链化程度减少,提高酶解效率。因此异淀粉酶配合其他淀粉水解酶在淀粉加工过程中的应用能够显著提高淀粉的利用效率,提高产物的生产量。
根据专利201310043628.5引述,α-1,4淀粉酶内切酶为一个高产麦芽六糖的淀粉水解酶。本专利采用该α-1,4淀粉酶内切酶与异淀粉酶配合使用,以提高淀粉处理产物中的麦芽六糖含量。以土豆淀粉为底物,每g土豆淀粉中加入8U的异淀粉酶和1U的α-1,4淀粉酶内切酶,45℃处理1h。同时商业化的普鲁兰酶D2作为处理对照,添加酶量和处理条件参考异淀粉酶。高效液相色谱检测水解产物中麦芽六糖的含量。液相色谱条件:安捷伦液相色谱,Cosmosil Sugar‐D氨基柱,ELSD ES2000蒸发光散射检测器,流动相为乙腈/水=65/35(v/v),流速是1mL/min,柱温30℃。图6所示,异淀粉酶与α-1,4淀粉酶内切酶联合处理,与单一α-1,4淀粉酶内切酶处理相比较,麦芽六糖的含量提高0.6倍,相比商业化普鲁兰酶0.4倍的提高量,本专利所述的异淀粉酶具有较为高效的催化效率,同时该异淀粉酶在联合加工淀粉过程中的水解形式在整个水解过程中与商业化普鲁兰酶相比较也较为稳定。
Claims (10)
1.一种异淀粉酶基因,其核苷酸序列为:SEQ ID NO.1。
2.权利要求1所述的异淀粉酶基因编码的异淀粉酶蛋白质,其氨基酸序列为:SEQ IDNO.2。
3.含权利要求1所述异淀粉酶基因的重组质粒。
4.根据权利要求3所述的重组质粒,其特征在于所述的重组质粒是将权利要求1所述异淀粉酶基因克隆到质粒pEFaA中所得。
5.含权利要求1所述的异淀粉酶基因的重组微生物。
6.根据权利要求5所述的重组微生物,其特征在于以毕赤酵母为宿主菌。
7.权利要求2所述异淀粉酶蛋白质联合其他淀粉水解相关酶在淀粉加工或工业生产方面的应用。
8.根据权利要求7所述的应用,其特征在于所述的其他淀粉水解相关酶为α-1,4淀粉酶内切酶。
9.权利要求1所述异淀粉酶基因在淀粉加工、食品以及饲料领域的基因工程应用。
10.权利要求2所述异淀粉酶蛋白质作为酶制剂在淀粉加工、食品以及饲料领域的生产应用。
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