CN104250643A - 一种淀粉酶与其编码基因和应用 - Google Patents
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Abstract
本发明涉及两种淀粉酶蛋白及其编码基因,以及该蛋白作为淀粉水解酶在工业领域的应用。本发明以深海热液口来源样品Fosmid文库进行454测序后得到的Fosmid文库基因信息片段的注释信息为研究材料,从宏基因组文库筛选到对应淀粉酶基因簇中对应克隆子F_135,并对该Fosmid克隆子进行鸟枪法结合引物步移法测序和ORF预测,得到编码淀粉酶的基因序列,其DNA序列如序列表SEQ ID NO.1所示。将目的基因插入载体pET-32a(+)表达载体中,表达纯化得到该基因编码的多肽其成熟肽,其氨基酸序列如序列表中SEQ ID NO.2序列所示。对其所具有的酶活性质进行了初步研究。本发明的淀粉酶,由于其特有的活性及酶学特性,可应用于与淀粉水解相关的工业化生产中。
Description
技术领域
本发明涉及基因工程领域,特别是涉及一种淀粉酶与其编码基因及其应用。
背景技术
淀粉酶是一类能将淀粉分子水解为由葡萄糖组成的多聚体分子的酶,它被发现广泛分布于动物、植物和微生物有机体中。淀粉酶构成了工业酶的一大类,大约占有酶市场25%的份额,随着技术的发展,淀粉酶的应用范围从传统的淀粉降解、纺织、食品、发酵、造纸、酿造和蒸馏等工业领域,扩展到了临床、医药和分析化学等领域。
酶是一种生物催化剂,周围环境(温度、pH、有机试剂等)对它的活性影响很大,很多酶在一些比较苛刻的反映条件下(高温、低温或者强酸碱环境)均会失去活性,这就限制了其应用范围。为了适应更苛刻的工业生产环境需求,需要我们在尊重生物多样性的前提条件下,不断在环境中进行新的淀粉酶来源的探索。
深海热液口烟囱是通过来自地壳的熔浆与海水相互作用而形成的特殊的地质结构,该环境被认为与地球早期环境相似,为特异微生物群落的形成和演化提供了良好的环境和物质基础,使得群落显示出独特的极端环境耐受力,蕴藏了大量的生物活性物质。热液口微生物中具有很多特殊的基因和酶,在现代工业、医药、环保、材料、生物工程和国防等领域具有广阔的开发应用前景。又因其与地球上生命形成初期的环境又十分相似,因此热液口又被喻为是研究生命起源与进化的天然实验室。近年来宏基因组学技术体系的建立使从不可培养微生物样品中直接获得酶资源成为可能。
宏基因组学又叫环境基因组学或群体基因组学,是指特定环境中全部生物遗传物质的总和,利用现代基因组技术直接研究自然生态环境中的有机群落,而不需要分离、培养单一种类的微生物。宏基因组文库的构建沿用了分子克隆的基本原理和技术方法,然后根据不同环境样品的特点和建库目的采取了一些特殊的步骤和策略。其中主要包括以下几个关键因素:DNA提取质量、克隆载体的选择、宿主菌的选择。
发明内容
本发明的目的在于提供一种来源于深海热液口的淀粉酶基因簇克隆子F_93基因ORF编码的淀粉酶、其编码基因及其在工业化生产中的应用。
其一,本发明涉及一种淀粉酶,其特征在于,其氨基酸序列如序列表SEQ IDNO.2所示。
进一步地,本发明还涉及编码上述的淀粉酶的基因,其核苷酸序列具体为序列表SEQ ID NO.1所示。
进一步地,用于所述的核苷酸序列特异性扩增的引物,包括以下两条序列:
上游引物见序列表中SEQ ID No.3;
下游引物见序列表中SEQ ID No.4;
进一步地,本发明还包括一种表达载体,所述的表达载体含有上述核苷酸片段。
进一步地,所述的表达载体,其具体为pET-32a(+)M-F_135ORF1。
进一步地,本发明还包括一种表达工程菌,其携带有上所述的表达载体。
进一步地,所述的表达工程菌,其具体为转化的大肠杆菌菌株BL21(DE3)。
本发明所述的淀粉酶,为淀粉酶基因簇克隆子F_135的开放阅读框架ORF1编码的基因,所述淀粉酶基因簇克隆子F_135的获得方法为:通过从热液口来源样品的宏基因组数据中获取了含淀粉酶序列片段,通过对这些宏基因组序列信息设计引物,对2880个克隆子进行两步法筛选,最后筛选得到克隆子F_135。
本发明对Fosmid克隆子F_135测序结果显示,该克隆子中含一个编码淀粉酶的放阅读框,命名为ORF1,全长分别为1236、,其DNA序列如序列表中SEQID NO.1所示。蛋白理论分子量为49.24,其氨基酸序列如序列表中SEQ ID NO.2、所示;序列分析显示它为葡萄糖水解酶家族蛋白(GH-57家族)。
本发明根据淀粉酶ORF1基因序列,设计带酶切位点的扩增引物SEQ IDNO.3、SEQ ID NO.4,将扩增后的到的片段酶切后与线性化的载体pET-32a(+)连接,构建表达质粒并将其转化到大肠杆菌BL21(DE3)中构建成工程菌株。通过对工程菌株培养、诱导表达、超声破碎获得重组蛋白。
本发明的淀粉酶ORF1重组蛋白理论分子量分别为49.24kD,可以在构建的工程菌中,以包涵体形式大量表达,对包涵体进行蛋白透析复兴后,获得目标淀粉酶ORF1。
本发明通过DNS法,测得F_135中的ORF1的淀粉酶比活力为4.46U/mg。由此推断,本发明的淀粉酶在应用工业化生产等领域具有潜在的应用价值。
本发明还对所述淀粉酶进行了初步的酶学性质研究:淀粉酶F_135的ORF1最适温度均在45℃左右,最适pH为7.0,属中性淀粉酶。另外,Cu2+、Ni2+、Zn2+、K+对淀粉酶orf1的活性都具有不同程度的抑制作用,值得注意的是,Mg2+、Na+、Ca2+对淀粉酶orf1的活力均有不同程度的促进作用,与已发现的α-淀粉酶所具有的性质一致。去污剂SDS对该淀粉酶的催化活性有强抑制作用,Triton对它们却有一定的促进作用,而Tween20和Tween80能部分促进激活orf1的酶活力。
与现有技术相比,本发明的淀粉酶较强的酶活力,对金属离子和去污剂的敏感程度不同,在工业应用领域有很大的优势。
为了更好地理解和实施,下面结合附图详细说明本发明。
附图说明
图1:添加酶切位点后F_135的ORF1基因片段的PCR产物电泳结果;
1、2:目的片段ORF1的PCR产物;M:DL2000bp的DNA分子量标准。
图2:重组表达质粒pET-32a(+)M-F_135ORF1的构建图
图3:淀粉酶ORF1的表达和纯化电泳图
M:蛋白分子量标准;1:诱导前总细菌蛋白;2:IPTG诱导后总细菌蛋白;3:IPTG诱导后总细菌蛋白超声沉淀;4:IPTG诱导后超声上清总细菌蛋白;5:纯化后的融合蛋白BL21(DE3)-pET32a-135_ORF1
图4为融合蛋白BL21(DE3)-pET32a-135_orf1纯化验证结果。
M:蛋白分子量标准;1、2:纯化后的融合蛋白BL21(DE3)-pET32a-135_orf1
图5:葡萄糖标准曲线。用以测定淀粉酶纯化酶液酶活,其中横坐标为葡萄糖含量,纵坐标为吸光值;
图6:pH对淀粉酶活性的影响。
图7:温度对淀粉酶活性的影响。
具体实施方式
以下实施例用于说明本发明,但不用来限制本发明的范围。
一:含淀粉酶序列克隆子的筛选。
先将深海热液口宏基因组序列与NCBI non-redundant database进行信息比对,查找出与淀粉酶基因相似的信息;再根据注释信息查找对应的序列信息,设计特异性引物,从文库中筛选对应的克隆子。为了提高筛选效率,将参与454测序的2880克隆分为30组,即每个96孔板制备成一个混合质粒样品,另外再制备一个由2880个克隆混合而成的质粒样品作为筛选时的阳性对照,就这样先以那30个混合样作为PCR扩增模板进行第一轮筛选,找到该基因序列所对应的混合样编号,再以对应编号的96孔板为模板做第二轮筛选,最后筛选得Fosmid克隆子,命名为F_135。
PCR筛选筛选引物为:
CL6683Contig1_up:5’-CAGGTCGTCTGGGGCATAGCGGATT-3’
CL6683Contig1_down:5’-CCCGAGATATTCGCCGTAGTTGGTT-3’
二:目的克隆子的鸟枪法测序及ORF的预测。
对Fosmid克隆子F_135进行鸟枪法结合引物步移法测序,即先将鸟枪法测序所得序列利用TGICL软件进行拼接,对于拼接后序列两端设计引物进行步移法测序,直到不同拼接序列形成一条完整的序列。F_135测序结果显示,该克隆子插入片段为7903bp,G/C含量为52.99%,通过ORF预测软件Glimmer3.02进行预测发现,该克隆子共包含4个ORF。
三:淀粉酶表达载体的构建。
确定其中一个ORF序列,分别命名为F_135_ORF1,(后简写为ORF1),其中ORF1的序列如序列表SEQ ID NO.1所示,根据其序列,分别设计带酶切位点的扩增引物,如下:
F_135_ORF1_BamHⅠ_up:5’-CGCGGATCCATGAGCGAAGTTTATCA-3’(SEQ IDNO.3)
F_135_ORF1_XholⅠ_down:5’-CCGCTCGAGGAATTCCTTCCGGCTGG-3’(SEQ IDNO.4)
用PCR的方法,从实施例一中的深海热液口宏基因组序列文库中扩增得到淀粉酶克隆子F_135ORF1基因片断(含酶切位点),长度为1236bp,与预期大小一致(见图1)。如图2所示,将回收纯化的F_135ORF1基因PCR产物,用Bam HI和XhoI酶切后,与同样用Bam HI和XhoI切开的线性化载体pET-32a(+)连接,构成含F_135ORF1基因片段的表达载体pET-32a(+)M-F_135ORF1。
对纯化后的pET-32a(+)M-F_135ORF1重组质粒用T7和T7t引物进行双向测序,其序列与合成的模板序列结果完全一致,并且读码框正确。
四:重组深海热液口来源的淀粉酶F_135ORF1的表达。
将测序正确的重组表达质粒转化到表达宿主菌BL21(DE3)中构建成工程菌株。挑取单菌落接种于Amp+的LB液体培养基中,37℃振摇培养过夜,作为种子菌。先用50%甘油进行保种,再取剩余菌液按1∶100体积比接种于新鲜的Amp+LB培养基中,37℃剧烈振荡放大培养至OD600约为0.8时,加入IPTG至终浓度为0.1mmol/L,于18℃诱导表达过夜。4℃、5000rpm离心10min,收菌,并预留1ml菌液用于SDS-PAGE分析。用预冷的TE缓冲液(pH8.0)洗涤菌体后,再用预冷的超声缓冲液重悬菌体。以300W的功率,冰浴下超声30min~1hr使细菌细胞破碎,4℃、12000rpm离心30min后分别取上清和沉淀进行SDS-PAGE分析检测重组蛋白质的表达情况。
五:重组深海热液口来源的淀粉酶F_135ORF1的纯化及纯化效果验证。
ORF1的氨基酸序列如序列表SEQ ID NO.2所示。如图3所示,淀粉酶F_135ORF1诱导表达后,电泳结果显示ORF1在分子量58kD附近有一个明显的条带,且诱导后以包涵体形式大量表达。
ORF1包涵体进行蛋白透析复性:按照1g:10ml的比例用8M尿素-Tris缓冲液溶解包涵体,通过加入磁力搅拌器加速其溶解,室温12000rpm离心溶解液10min上清液取备用。将透析袋按合适长度剪成小段,用10mM NaHCO3溶液浸泡并煮沸5min,然后将透析袋转移至10mM EDTA溶液煮沸5min,更换新鲜10mM EDTA溶液再次煮沸5min,用去离子水漂洗数次,置于20%乙醇短期保存,使用前用去离子水漂洗数次除去乙醇即可。将溶解好包涵体的上清液装入已处理的透析袋;把透析袋转移至Buffer A,4℃透析24h,然后将透析袋转移至Buffer B,4℃透析24h,最后将透析袋转移至Buffer C,4℃透析12h,更换新鲜透析Buffer C,重复上述步骤,复性蛋白在4℃,12000rpm离心15min,取上清进行纯化和活性检测。通过Western blot验证此蛋白为我们所需的目的蛋白(图4)。
七:深海热液口来源的淀粉酶F_135ORF酶活与比活力测定。
(1)DNS法
取2个试管,分别为对照管和样品管。将两试管中各加入Tris缓冲液800μl和底物溶液100μl,37℃水浴保温5min,然后在对照管中加入灭活的纯化酶液(沸水浴煮沸10min)100μl,样品管中加入纯化酶液100μl,立即混匀计时,在37℃水浴中准确反应30min后加入等体积的DNS终止反应,沸水浴显色5min,冷却后在540nm分光光度计测定吸光值,每个样品重复三次,取平均值。
酶活力单位(1U)定义:在pH6.5,37℃条件下,以1min内催化产生1μmol还原糖所需的酶量。
(2)葡萄糖标准曲线的绘制
取7支试管编号,分别取0,0.2,0.4,0.6,0.8,1.0,1.2ml的葡萄糖标准液(1mg/ml),用蒸馏水补加至2ml,再加入1.5ml DNS,将各管摇匀,在沸水浴中准确加热5min,取出,冷却至室温,用蒸馏水定容至20ml,用第一支试管调零,测定在540nm处的吸光值。以葡萄糖含量为横坐标,吸光值为纵坐标,绘制标准曲线图5。
根据葡萄糖标准曲线,淀粉酶纯化酶液酶活(EA)计算公式如下:
EA=(OD540×1.934)/M葡萄糖×稀释倍数/反应时间
比活力=EA/纯化酶液蛋白浓度
根据测量,本发明的淀粉酶F_135_ORF1酶活为1.66U,比活力为4.46U/mg。
八:pH对深海热液口来源的淀粉酶F_135ORF活力的影响。
酶作用最适pH:分别用pH3.0,4.0,5.5,6.0(乙酸钠缓冲液),6.5,7.0,7.5,8.0,8.5(Tris-HCl缓冲液),9.0,9.5,10.0(Gly-NaOH缓冲液)的50mM缓冲液代替酶活测定体系中的Tris缓冲液,为减少纯化蛋白的使用量,反应体系由之前的1ml缩减为100μl(即Tris缓冲液80μl、底物溶液10μl、纯化酶液10μl),其它条件不变。酶-底物反应体系在37℃反应30min后加入等体积的DNS终止反应,沸水浴显色5min,测定吸光值,每个梯度重复三次,取平均值。以测得的最高酶活力为100%,其余所得酶活力折算成百分数,再对pH作图。
本实验为达到不同的pH值环境,经相关报道表明淀粉酶最适pH值在5-9.5之间,所以选用了三种不同的缓冲液体系,pH值范围为3.0-10.0。
结果见图6,淀粉酶ORF1在pH7.0时,反应酶活最高,说明它们分属中性淀粉酶,高于或低于最适pH时,酶活都有所下降,且在偏碱性条件下比在偏酸性条件下活性下降更加明显。
九:温度对深海热液口来源的淀粉酶F_135ORF活力的影响。
酶作用最适温度:将溶液A与底物溶液B的混合物,分别置于4℃,16℃,25℃,30℃,40℃,50℃,60℃温度下冰浴或预热5min后,加入10μl纯化酶液,分别在相应温度下反应10min,测定各温度下的酶活力。以测得的最高酶活力为100%,其余所得酶活力折算成百分数,再对温度作图。
根据实验室所具备的实验条件,结合文献报道,本实验选择的温度范围为0℃-65℃。结果见图7淀粉酶ORF1的最适温度为44.3℃,20℃时有较明显的酶活上升现象,当温度上升到52℃以上时,酶活力均有明显的下降趋势,综上分析,ORF属于中温酶。
十:金属离子及表面活性剂对深海热液口来源的淀粉酶F_135ORF活力的影响。
将选择的金属离子及表面活性剂用反应缓冲液配制成一定的浓度,加入底物溶液B及纯化酶液,使金属离子及其它物质的最终浓度为1mM,分别在37℃水浴中保温5min后,反应10min后测定酶的残留酶活,其中以不加任何物质的酶活力为100%。所选金属离子分别为Mg2+、Cu2+、Ni2+、Mn2+、Zn2+、K+、Na+、Ca2+(MgCl2、MgSO4、CuSO4、NiSO4、MnCl2、ZnCl2、KCl、NaCl、CaCl2),其它化学物质如EDTA、SDS、Tween20、Tween80。
结果见表1.可见,金属离子Cu2+、Ni2+、Zn2+、K+对淀粉酶orf1的活性有不同程度的抑制作用,其他离子对它们的活力影响皆不相同,值得注意的是,Mg2+、Na+、Ca2+对淀粉酶orf1的活力均有不同程度的促进作用,由之前的进化分析orf1很可能属于α-淀粉酶,通过多年研究,我们已经知道α-淀粉酶是一种Ca2+依赖性蛋白酶,钙离子能够保持酶分子适当的构象,从而维持其活性和稳定性。此实验的结果也说明了这一点。
表1金属离子对淀粉酶活性的影响
各种去污剂的加入,SDS对该淀粉酶的催化活性有强抑制作用,而Triton对它却有一定的促进作用,另外,Tween20和Tween80能部分促进激活orf1的酶活力,这些特点都可能成为此酶今后在应用领域中的优势,结果见表2。
表2各种去污剂对淀粉酶活性的影响
以上实验结果表明,本发明的淀粉酶蛋白F_135_ORF1理论分子量为49.24,最适温度为44.3℃,最适pH为7.0、对金属离子和去污剂的敏感程度不同,在工业应用领域有很大的优势。
以上仅为本发明的优选实施例而已,并不用于限制本发明,对于本领域的技术人员来说,凡在本发明的精神和原则之内所做的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (7)
1.一种淀粉酶,其特征在于,其氨基酸序列如序列表SEQ ID NO.2所示。
2.编码权利要求1所述的淀粉酶的基因,其核苷酸序列具体为序列表SEQ ID NO.1所示。
3.用于权利要求2所述的淀粉酶的基因特异性扩增的引物,包括以下两条序列:
上游引物见序列表中SEQ ID No.3;
下游引物见序列表中SEQ ID No.4。
4.一种表达载体,所述的表达载体含有权利要求3所述核苷酸片段。
5.权利要求4所述的表达载体,其具体为pET-32a(+)M-F_135ORF1。
6.一种表达工程菌,其携带有权利要求4或5所述的表达载体。
7.权利要求6所述的表达工程菌,其具体为转化的大肠杆菌菌株BL21(DE3)。
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