CN101906430B - 一种蔗糖异构酶基因及其高效表达方法 - Google Patents
一种蔗糖异构酶基因及其高效表达方法 Download PDFInfo
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Abstract
本发明公开了一种蔗糖异构酶基因,pal I基因,其核苷酸序列如SEQ ID NO:1所示。本发明还公开了上述基因的编码蛋白质,SIase酶。本发明还公开了包含上述基因的表达载体及宿主细胞,以及该基因的高效表达方法。包含上述蔗糖异构酶基因的重组菌具有异构的活性,能转化蔗糖生成异麦芽酮糖。该重组菌稳定性好,催化效率高,产物特异性明显改善,可应用于异麦芽酮糖的工业生产中。
Description
技术领域
本发明属于基因工程和酶工程领域,具体涉及一种蔗糖异构酶基因及其高效表达方法。
背景技术
蔗糖异构酶(Sucrose isomerase,简称SIase)是生产功能性甜味剂异麦芽酮糖(醇)和海藻酮糖的关键酶。异麦芽酮糖醇是近年来新兴的一种功能性糖醇,它是由异麦芽酮糖经镍催化加氢制得。它除了具有一般糖醇的共同特点外,还具有木糖醇和麦芽糖醇等功能糖醇无法比拟的极低的吸湿性和纯正的口感。由于其独特的性能,受到了食品界制取无糖甜食品的欢迎,特别是近年来,随着人们对自身健康的重视,异麦芽酮糖醇的生产和开发利用受到了广泛关注。在欧洲上市不到5年,销售量已经达到10万吨以上,国内市场刚刚起步,预计其需求量年增长率在10%以上。
目前国内外生产异麦芽酮糖普遍采用SIase催化蔗糖而进行的。该反应可以同时生成异麦芽酮糖和海藻酮糖两种同分异构体,根据主产物的不同分为异麦芽酮糖主产型和海藻酮糖主产型。所报道的SIase生产菌种中,大黄欧文菌(Erwinia rhapontici),沙雷氏菌(serratia plymuthica),克雷伯氏菌(Klebsiella sp.LX-3)和多源发散菌(Pantoeadispera)可以生成70~85%的异麦芽酮糖,而嗜中酸性假单胞菌(Pseudomonasmesoacidophila MX-45),放射性土壤杆菌(Agrobacterium radiobacterMX-232)生成90%以上的海藻酮糖。
目前,许多SIase酶已经得到了纯化,相应SIase的编码基因得到了克隆。由于分泌SIase的野生菌株的生产能力普遍较低,为了克服野生菌的低生产能力,采用基因工程菌高效表达SIase引起了人们极大的兴趣。在国内,SIase的克隆与表达迄今为止鲜有报道,国内有限的研究仍主要致力于提高原始菌株的酶产量。因此构建高效的基因工程菌是降低SIase生产成本的关键。
发明内容
本发明所要解决的技术问题是提供一种优质的蔗糖异构酶基因。
本发明还要解决的技术问题是提供上述蔗糖异构酶基因所编码的蛋白质。
本发明还要解决的技术问题是提供包含上述蔗糖异构酶基因的表达载体。
本发明还要解决的技术问题是提供包含上述表达载体的重组大肠杆菌。
本发明还要解决的技术问题是提供利用上述重组大肠杆菌高效表达蔗糖异构酶基因的方法。
本发明还要解决的技术问题是提供上述重组大肠杆菌的应用。
为解决上述技术问题,本发明采用的技术方案如下:
一种蔗糖异构酶基因(即pal I基因),其核苷酸序列如SEQ ID NO:1所示。该基因是从大黄欧文菌(Erwinia rhapontici)NX-5(CGMCC NO:2222)(200710190755.2)中提取获得,共1803bp。
一种权利要求1所述基因的编码蛋白质(即蔗糖异构酶,缩写SIase),其氨基酸序列如SEQ ID NO:2所示,共601个氨基酸。
一种表达载体,包含如SEQ ID NO:1所示的蔗糖异构酶基因。
上述表达载体,优选为重组表达载体pET22b(+)。
一种重组大肠杆菌,包含如SEQ ID NO:1所示的蔗糖异构酶基因。
上述重组大肠杆菌,优选为包含有重组质粒pal I/pET22b(+)的E.coli BL21(DE3)。
利用上述重组大肠杆菌细胞高效表达pal I基因的方法,即上述重组大肠杆菌细胞自诱导产酶的方法,是以糖蜜水解液为诱导剂诱导培养重组大肠杆菌产酶。糖蜜水解液即作为碳源又作为诱导剂。
利用上述重组大肠杆菌细胞高效表达pal I基因的方法,具体为:将重组大肠杆菌在含有50μg/mL氨苄青霉素的LB培养基中30~40℃液体培养8~20h,转接至发酵培养基培养5~7h后降温至25~30℃再诱导培养6~12h;所述的发酵培养基为:糖蜜水解液10~50mL/L,(NH4)2SO4 5~10g/L,NaCl 5~10g/L,KH2PO4 1~3g/L,MgSO4 0.1~1g/L。
上述宿主细胞自诱导产酶的方法优选为:将重组大肠杆菌细胞在含有50μg/mL氨苄青霉素的LB培养基中37℃液体培养12h,转接至发酵培养基培养5~7h后降温至25~30℃再诱导培养6~10h;所述的发酵培养基为:糖蜜水解液40mL/L(50g糖蜜溶解于100mL水中,调pH至1.8,90℃酸水解3h,KOH回调pH至7.0制得糖蜜水解液),(NH4)2SO4 8g/L,NaCl 10g/L,KH2PO4 2g/L,MgSO4 0.5g/L。
本发明所述的糖蜜按如下方法制备:对于每100mL水,加入20~80g糖蜜溶解,在酸性条件下80~100℃水解1~5h,水解结束后加碱调至中性,即为糖蜜水解液。具体来说,对于每100mL水,加入20~80g糖蜜溶解,混匀后离心去除不溶物,室温下用盐酸调pH值至1~3左右,于80~100℃水解1~5h,水解结束后用浓碱回调pH至7.0,即为糖蜜水解液。糖蜜主要成分蔗糖水解为等摩尔的葡萄糖和果糖,少量(1-2%)棉子糖水解为等摩尔的半乳糖、葡萄糖和果糖。
常规的诱导方法是将含有质粒pal I/pET22b(+)的E.coli BL21(DE3),在含有50μg/mL氨苄青霉素的LB培养基中37℃液体培养过夜,转接一次,37℃液体培养至OD达0.5~0.8后用终浓度为0.2~1.0mmol/L的异丙基硫代-β-D-半乳糖苷(或0.2-1.0mmol/L乳糖)在25~30℃条件下诱导10~20h,产SIase酶活为10~15U/mL。
通过本发明自诱导产酶的方法(即pal I基因高效表达方法),重组大肠杆菌产SIase酶达20~30U/mL。而以常规诱导方法即以外加异丙基硫代-β-D-半乳糖苷或乳糖为诱导剂,则宿主细胞产SIase酶活为10~15U/mL。
上述宿主细胞在发酵生产异麦芽酮糖中的应用。具体为:将游离的或固定化后的宿主细胞填充至反应罐中,加入450~600g/L的蔗糖溶液,进行酶转化反应,反应温度25~35℃,转化时间2~10h;或者将固定化后的宿主细胞装入填充床式柱反应器中,该反应器以0.5~5mL/min的流速单向流加或循环连续流加400~600g/L的蔗糖溶液,进行酶转化反应,反应温度25~35℃,每批转化时间2~10h。
有益效果:本发明方法与现有技术相比具有如下优势:
1、本发明提供了pal I基因的高效表达方法。以质粒pET22b(+)为表达载体,以E.coli BL21(DE3)为表达宿主,可实现pal I的高效表达,发酵液酶活达20U/mL以上,而野生菌(Erwinia rhaponticiNX-5)的发酵液酶活仅为1.3U/mL,重组酶具有转化蔗糖生成异麦芽酮糖的能力。
2、本发明提供了一种适合于重组大肠杆菌(含pET系统)的以糖蜜水解液为碳源的自诱导产酶体系,糖蜜经酸水解后,主要成分为葡萄糖和果糖,可以作为菌体生长的碳源,糖蜜中还含有少量的棉子糖(1-2%),经酸水解后分解为等量的半乳糖、葡萄糖和果糖,其中的半乳糖可充当pET系统中T7启动子的诱导剂,该体系免去了昂贵的诱导剂(异丙基硫代-β-D-半乳糖苷,IPTG)的加入环节,诱导时间短,使用方便廉价。
附图说明
图1用于生产重组SIase的pal I/pET22b(+)示意图。
图2SIase纯化过程电泳图。其中1:标准蛋白,2:重组菌超声破碎后的上清液,3:经过Ni亲合层析后的SIase。
具体实施方式
根据下述实施例,可以更好地理解本发明。然而,本领域的技术人员容易理解,实施例所描述的具体的物料配比、实验操作条件及其结果仅用于说明本发明,而不应当也不会限制权利要求书中所详细描述的本发明。
实施例1:大黄欧文菌(Erwinia rhapontici)NX-5总DNA的提取。
E.rhapontici NX-5菌株(CCTCC NO:2222)在SB液体培养基(蛋白胨10g/L,酵母膏5g/L,NaCl 10g/L)中培养12h,8000r/min离心收集菌体,无菌水洗涤,收集沉淀悬浮于500μL Tris-EDTA(三羟甲基氨基甲烷-乙二胺四乙酸)缓冲液,加入5μLRNA酶,37℃下保温30min,加入30μL 10%SDS(十二烷基硫酸钠)和15μL蛋白酶K,37℃下保温60min,加入100μL 5M的NaCl溶液和80μL CTAB(十六烷基三甲基溴化铵),65℃下保温20min,用700μL的酚∶氯仿∶异戊醇体积比25∶24∶1混合溶液抽提,10000r/min离心,上清液用700μL的氯仿∶异戊醇体积比24∶1混合溶剂抽提,10000r/min离心,上清液与1400μL的冰异戊醇混合,-20℃沉淀30min,10000r/min离心,沉淀加200μL 70%乙醇清洗,10000r/min离心,沉淀用Tris-EDTA缓冲液溶解,即得到Erwinia rhapontici NX-5总DNA。
实施例2:SIase编码基因的克隆。
以实施例1得到的E.rhapontici NX-5总DNA为模板,以下列核苷酸序列作为引物:
引物1:TTAAGCTT CCATGGATTCTCAAGGATT,分别引入HindIII、Nco I酶切位点(下划线部分)。
引物2:GTAAATATTTGAATTAGGCGAGCTC CCTAGGTT,分别引入BamH I和Xho I双酶切位点(下划线部分)。
PCR反应在20μL体系中进行:10×PCR缓冲液2μL、2.5mmol/LdNTP 2μL、10μmol/L引物1和引物2各2μL、模板DNA 2μL、25mmol/LMgCl2 2μL、TaqDNA聚合酶1μL,补加双蒸水至20μL。
PCR反应条件:95℃变性3min,后开始循环,(94℃ 30s;45℃ 30s;72℃ 2min;72℃ 5min),共25次循环,扩增得到1800bp的PCR片段,切胶回收,回收片段经HindIII和BamH I双酶切,采用DNA片段纯化试剂盒纯化,与经同样双酶切的pUC18载体连接。转化至E.coli DH5α,转化产物涂布含100μg/ml氨苄青霉素的LB平板,经37℃培养过夜,平板上长了约30个菌落,挑选阳性转化子,接入LB液体培养基,10h后提取质粒并进行酶切验证后测序。结果表明插入片段含有一个长1803bp(如SEQ ID NO:1所示)的开放阅读框(ORF),编码一个由601个氨基酸编码的蛋白质(如SEQ ID NO:2所示)。
实施例3:pal I基因在表达载体上的构建。
用于构建表达载体的质粒是pET22b(+),带有pelB信号肽和His-tag标记。将pET22b(+)质粒和pal I基因进行Xho I和Nco I双酶切,切胶回收pal I后经T4连接酶16℃连接过夜,连接产物转化E.coli BL21(DE3)感受态细胞,经37℃培养过夜,挑选转化子于100μg/mL氨苄青霉素的LB进行液体培养,然后抽提质粒,得到富集的pal I/pET22b(+)质粒(图1)。
实施例4:大肠杆菌宿主转化。
E.coli BL21(DE3)宿主菌在LB液体培养基中培养12h,按5%的接种量转移至新鲜的LB液体培养基中,37度培养2h,取1mL培养液加入1.5ml离心管中,5000r/min离心5min(4℃),倾去上清液加入用冰遇冷的0.1mol/L CaCl2500μL振荡混匀,低温离心5min(5000r/min),倾去上清,再加CaCl2500μL振荡混匀,低温离心5min(5000r/min),收集菌体加200μl CaCl2混匀,分装成两管(100μL),制成感受态细胞。吸取5μLpal I/pET22b(+),加入100μL感受态细胞溶液中,冰浴30min,42℃水浴90s,迅速将离心管转移至冰浴中,使细胞冷却1-2min,然后加入1ml LB培养基,37℃培养45min后,吸取200μL已转化的感受态细胞转移至含氨苄抗性的LB平板上,37℃培养24-36h。挑选转化子进行酶切鉴定。
实施例5:含pal I/pET22b(+)的E.coli BL21(DE3)经常规方法诱导产酶。
将含有质粒pal I/pET22b(+)的E.coli BL21(DE3),在氨苄青霉素(100μg/mL)LB平板上经37℃培养过夜,挑选转化子(含pal I/pET22b(+)的E.coli BL21(DE3))在LB液体培养基中37℃培养过夜,后转接至LB发酵培养基中37℃培养至OD 0.6后用终浓度为0.8mmol/L的IPTG(异丙基硫代-β-D-半乳糖苷)在20℃条件下诱导14h,SIase酶活力为10U/mL;或采用0.5mmol/L乳糖,在24℃条件下诱导12h,SIase酶活力为12U/mL。酶活的检测方法为:取经诱导表达后的细胞培养液1mL,8000r/min离心5min收集菌体,生理盐水洗涤一遍,用200μl磷酸钾缓冲液(pH 5.8,0.025mmol/L)重悬,加入800μL蔗糖溶液(500g/L)作为底物,30℃下转化1h,100℃沸水浴10min终止反应,12000r/min离心10min,取上清,使用高效液相色谱测定异麦芽酮糖在产物中的含量,(HPLC条件:Agilent1200型HPLC系统,ShodexR101示差折光检测器,色谱柱:Rezex RCM-Monosaccharide Ca2+柱,流动相为纯水,流速0.5ml/min,柱温80℃)以30℃条件下1mL培养液中的细胞每分钟催化蔗糖生成1μmol异麦芽酮糖为1个酶活单位(U),以下相同。
实施例6:糖蜜水解液的制备方法。
称取50g糖蜜溶解于100ml蒸馏水中,混匀后于8000r/min离心20min,去除不溶物,室温下用37%(v/v)的盐酸调pH值至1.8左右,于90℃水解3h,水解结束后用5mol/LKOH回调pH至7.0,即为糖蜜水解液。糖蜜主要成分蔗糖水解为等摩尔的葡萄糖和果糖,少量(1-2%)棉子糖水解为等摩尔的半乳糖、葡萄糖和果糖。糖蜜水解液具体成分:葡萄糖18~20%(即100g糖蜜中含18~20g葡萄糖,以下定义相同),果糖23~25%,半乳糖0.4~0.6%,检测方法:HPLC法,具体条件同实施例5。
实施例7:含pal I/pET22b(+)的E.coli BL21(DE3)以糖蜜水解液为碳源,形成SIase的自诱导表达体系。
将含有质粒pal I/pET22b(+)的E.coli BL21(DE3),在含有50μg/mL氨苄青霉素的LB培养基中37℃液体培养过夜,转接至发酵培养基(糖蜜水解液40mL/L,(NH4)2SO48g/L,NaCl 10g/L,KH2PO4 2g/L,MgSO4 0.5g/L,自然pH),培养5-7h后降温至25℃诱导培养,8h产SIase酶达23U/mL。
实施例8:SIase的纯化和特性。
将上述SIase发酵液于4℃、10000r/min离心20min收集菌体,生理盐水洗涤一遍,重新悬浮于缓冲液中,冰浴条件下超声破碎菌体(400W,20min)。缓冲液组成为25mmol/L磷酸钠(pH=5.8),超声后离心(10000r/min,40min),取上清液,得SIase粗酶液。Ni亲和柱用缓冲液A(25mmol/L的磷酸钠缓冲液,pH 5.8)平衡后,将上样样品吸入Ni柱,使之完全吸附后,分别用缓冲液A、含20mmol/L咪唑的缓冲液A、含250mmol/L咪唑的缓冲液A洗脱,流速1mL/min,检测波长为280nm,收集SIase酶活洗脱液液,活力组分在pH5.8、25mmol/L磷酸钠缓冲液中透析过夜后,得纯化SIase酶制品。纯化过程电泳图见图2。
实施例9:
按实施例6所述方法,将成功表达SIase基因的E.coli BL21(DE3)细胞收集,25mM的磷酸钠缓冲液(pH5.8)洗涤两次,离心收集进行游离细胞转化,称取10g的细胞加入500mL 500g/L的蔗糖溶液中,在30℃的条件下,于摇瓶中振荡转化生产异麦芽酮糖,反应时间2h,反应中止后测底物转化率和产物浓度。蔗糖转化率为99.5%,异麦芽酮糖转化率达90%。
对比例1:
斜面培养基:蛋白胨10g/L,牛肉膏3g/L,NaCl5g/L,琼脂20g/L,pH7.0。
摇瓶培养基:蔗糖50g/L,酵母膏10g/L,Na2HPO4.12H2O 5g/L,pH7.0。
把大黄欧文氏菌NX-5CGMCC No.2222在斜面培养基上30℃培养24h,然后接一环此菌于摇瓶培养基中,30℃培养10h,摇瓶转速200r/min,得到的发酵液中菌体的含量为20g/L,产酶达2.5~5.0U/ml,以游离细胞转化,称取10g的细胞加入500mL 500g/L的蔗糖溶液中,在30℃的条件下,于摇瓶中振荡转化生产异麦芽酮糖,反应时间20h,反应中止后测底物转化率和产物浓度。蔗糖转化率为90%,异麦芽酮糖转化率达80%。
SEQUENCE LISTING
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Claims (1)
1.一种重组大肠杆菌生物催化生产异麦芽酮糖的方法,其特征在于,将含有质粒pal I/pET22b(+)的E.coli BL21(DE3),在含有50μg/mL氨苄青霉素的LB培养基中37℃液体培养过夜,转接至发酵培养基培养5-7h后降温至25℃诱导培养8h;将诱导后的细胞收集,25mM pH5.8的磷酸钠缓冲液洗涤两次,离心收集进行游离细胞转化,称取10g的细胞加入500mL 500g/L的蔗糖溶液中,在30℃的条件下,于摇瓶中振荡转化生产异麦芽酮糖,反应时间2h;
所述的pal I基因的核苷酸序列如SEQ ID NO:1所示;
所述的发酵培养基为:糖蜜水解液40mL/L,(NH4)2SO48g/L,NaCl 10g/L,KH2PO42g/L,MgSO40.5g/L;
所述的糖蜜水解液按如下方法制备:称取50g糖蜜溶解于100ml蒸馏水中,混匀后于8000r/min离心20min,去除不溶物,室温下用体积百分浓度37%的盐酸调pH值至1.8左右,于90℃水解3h,水解结束后用5mol/L KOH回调pH至7.0。
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