CN111334493B - 中低温内切β-甘露聚糖酶及其编码基因和应用 - Google Patents

中低温内切β-甘露聚糖酶及其编码基因和应用 Download PDF

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CN111334493B
CN111334493B CN202010254870.7A CN202010254870A CN111334493B CN 111334493 B CN111334493 B CN 111334493B CN 202010254870 A CN202010254870 A CN 202010254870A CN 111334493 B CN111334493 B CN 111334493B
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李霜
吕亮
陶惟一
王丹
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Abstract

本发明涉及中低温内切β‑甘露聚糖酶及其编码基因和应用,其核苷酸序列如SEQ ID NO:1或SEQ ID NO:2所示。本发明的β‑甘露聚糖酶来源于枯草芽孢杆菌Bacillus subtilis BL‑27,通过重组菌表达的β‑甘露聚糖酶酶活很低,可以快速降低瓜尔胶粘度却仅产生极少量的甘露糖寡糖,与现有β‑甘露聚糖酶酶学性质存在巨大差异;β‑甘露聚糖酶ManBL27‑1在未破胞的情况下,能够在1h以内将40‑50℃下的水基压裂液粘度从5000mpa.s以上降到500mpa.s,随着时间的增加能够完全降低粘度,β‑甘露聚糖酶ManBL27‑2基因工程菌,形成了菌体的“自包埋”,在相同条件下对水基压裂液的降粘速率有明显的减缓趋势,实现了甘露聚糖酶在水基压裂液体系中的缓释降粘效果。

Description

中低温内切β-甘露聚糖酶及其编码基因和应用
技术领域
本发明属于基因工程技术领域,具体地说,本发明涉及的是具有中低温活性、耐碱的内切β-甘露聚糖酶及其基因以及该β-甘露聚糖酶作为水基压裂液破胶剂的应用。
背景技术
随着我国现在石油的探明和开发,低渗透油藏的储量占总石油储量的三分之二以上,且呈逐年上升的趋势;且现在大部分石油的消耗也都来自于低渗透油藏,自此,开发利用低渗透油藏是如今石油开采中的重大目标。诞生于1949年的水力压裂技术已成为低渗透油藏开采的通用技术,能够有效地刺激低渗透油井,使其恢复产油能力[侯晓晖,王旭,王玉斌.水基压裂液聚合物增稠剂的应用状况及展望[J].西南石油学院学报),2003,15:45-47]。在水力压裂技术体系中,增稠剂和破胶剂都是必要元素;压裂完成后,破胶剂将增稠剂的大分子结构破坏从而降低粘度,破胶液返排出油井,这样压裂储层才能形成导流能力良好的支撑裂缝。
瓜尔胶及其衍生产品是压裂液的常用增稠剂。传统用的破胶方法一般为化学方法,利用过硫酸盐等氧化剂,依靠氧化还原反应产生高活性的自由基,迅速破坏大分子增稠剂主链,达到破胶目的。由于这种反应对温度和时间有要求,反应的最低温度为50℃时才能实现在1h内破胶,仅仅只能适用于中高温油藏,对于温度低于50℃的中低温油藏来说,化学破胶剂在工程应用上并不适合。此外,化学破胶所需的氧化反应存在随机性,会与管道、地基材料、烃类等反应,产生污染物,对环境不友好,存在很多局限性[庄照锋,张士诚,张劲,et al.中高温低浓度压裂液研究与应用[J].石油化学,2007,24(2):120-123]。
瓜尔胶分子结构由甘露糖通过β-1,4糖苷键连接而成的主链和α-1,6糖苷键连接的半乳糖侧链构成。β-甘露聚糖酶可水解瓜尔胶的主链糖苷键,破坏瓜尔胶的大分子结构。为解决低温油藏环境条件下的破胶剂瓶颈,耐低温、耐碱的高活性β-甘露聚糖酶具有不可替代性。与传统破胶剂相比,酶破胶剂只会与特定的底物瓜尔胶起反应,不会产生其他附加伤害,环境友好;且具有在低浓度添加量下快速降粘的效果,所以生物酶制剂在油田破胶中更受欢迎。尤其是对于温度低于50℃的中低温油田,采用酶法破胶是替代化学破胶的重要技术手段。
目前,碱性β-甘露聚糖酶在国外已广泛应用于油气田压裂工艺,尤其适于中低温油气井的压裂破胶(20-50℃);国内,也有低温甘露聚糖酶及破胶特性的相关专利及研究论文发表[一种生产中低温β-甘露聚糖酶的菌株及其应用,申请号201310539350.0;一种利用肠杆菌生产耐低温β-甘露聚糖酶的方法,申请号201410723219.4;王丽仙,李良川,刘彝,etal.嗜碱杆菌甘露聚糖酶环境适应性及其对瓜尔胶的降解作用[J].华东理工大学学报(自然科学版),2018,44(05):47-55.Li C,Liu F F,Ye J,et al.A low-temperature activeendo-β-1,4-mannanase from Bacillus subtilis TD7 and its gene expression inEscherichia coli[J].Applied Environmental Biotechnology,2018,3(2):17-25.]。适用于破胶剂的β-甘露聚糖酶需具备中低温活性和耐碱性等特征,开发高活性的低温碱性甘露聚糖酶是实现低温破胶的必要条件。
发明内容
本发明目的在于提供具有低温活性的碱性内切β-甘露聚糖酶及其编码基因和应用,通过对该编码基因进行两种方式(无信号肽序列)的重组表达,进一步开发了其用于水基压裂液破胶的应用方式,有信号肽序列的大肠杆菌工程菌无需破胞即可用于压裂液降黏,而去除信号肽序列的大肠杆菌工程菌对酶具有包埋缓释作用。
为实现上述技术目的,本发明采用如下技术方案:
本发明所提供的内切β-甘露聚糖酶及其编码基因,来源于枯草芽孢杆菌Bacillussubtilis BL-27,从中扩增出完整的β-甘露聚糖酶ManBL27编码基因(命名为manBL27-1),其核苷酸序列如SEQ ID NO:1所示,对应的氨基酸序列如SEQ ID NO:3所示。
本发明通过PCR方法克隆了来自枯草芽孢杆菌Bacillus subtilis BL-27的β-甘露聚糖酶的完整编码基因manBL27-1,全长为1089bp,其所表达的β-甘露聚糖酶ManBL27-1总共含363个氨基酸,理论分子量为40.3kDa;β-甘露聚糖酶ManBL27-1的N端27个氨基酸为信号肽序列“MFKKHTISLLILFLLASAVLAKPIEAH”,去除信号肽的β-甘露聚糖酶ManBL27-2氨基酸序列如SEQ ID NO:4所示,含有336个氨基酸,对应的去除信号肽序列的编码基因manBL27-2核苷酸序列如SEQ ID NO:2所示,全长为1008bp,起始密码子都为ATG,终止密码子也都为TGA。
本发明的另一目的在于提供上述内切β-甘露聚糖酶作为水基压裂液破胶剂的应用。
具体的,本发明通过在水基压裂液中加入β-甘露聚糖酶粗酶液或重组菌发酵液,实现水基压裂液破胶。
所述粗酶液制备方式为,将β-甘露聚糖酶基因导入宿主菌构建重组菌,将重组菌诱导表达,获取所述β-甘露聚糖酶粗酶液;所述重组菌发酵液获取方式为,将所述的重组菌发酵,获取所述重组菌发酵液。优选的,发酵培养基组分为:蛋白胨15g/L,酵母粉25g/L,NaCl 10g/L,甘油10g/L,葡萄糖2g/L,乳糖2g/L。
本发明所述的宿主菌选用大肠杆菌,重组菌的构建方式为:以pET28a为表达载体,在设计的上下游引物中分别加入NcoI和XhoI酶切位点作为构建重组表达载体的作用位点;将PCR胶回收后的基因和表达载体pET28a分别用NcoI和XhoI进行双酶切,酶切产物胶回收后,用T4 DNA连接酶连接过夜;取连接产物转化至大肠杆菌E.coli BL21(DE3)感受态细胞,获取重组菌;
所述的上下游引物序列为:
对于SEQ ID NO:1所示基因序列:
BL27man1-F:5’-CATGCCATGGCCTTTAAGAAACATACGATCTCTT-3’;
BL27man1-R:5’-CCGCTCGAGTTCAACGATTGGCGTTAAAGAA-3;
对于SEQ ID NO:2所示基因序列:
BL27man2-F:5’-CATGCCATGGCCACTGTGTCGCCTGTGAATCCTA-3’;
BL27man2-R:5’-CCGCTCGAGTTCAACGATTGGCGTTAAAGAA-3’。
进一步的,所述粗酶液或重组菌发酵液添加量为0.125%(v/v)。
进一步的,所述水基压裂液中底物浓度为8g/L。
进一步的,所述底物为瓜尔胶、羟丙基瓜尔胶或阳离子瓜尔胶。
进一步的,所述水基压裂液成分为:每200mL含Gly-NaOH缓冲液100mM,底物1.6g,KCl 4g,Na2S2O3 2g,25%戊二醛100μL,硼砂0.6mg,pH7.0-9.0。
进一步的,对于SEQ ID NO:1所示基因表达的β-甘露聚糖酶(ManBL27-1),作用温度为35-50℃,pH7-8;对于SEQ ID NO:2所示基因表达的β-甘露聚糖酶(ManBL27-2),作用温度为20-50℃,pH7-9。
本发明提供了具有中低温活性、耐碱的β-甘露聚糖酶ManBL27-1和ManBL27-2及其编码基因manBL27-1和manBL27-2。β-甘露聚糖酶ManBL27-1的适用条件为温度35-50℃、pH7-8;β-甘露聚糖酶ManBL27-2的适用条件为温度20-50℃、pH7-9。在上述条件下,该酶作为破胶剂作用于胍胶基水基压裂液时,使用量仅为0.125%(v/v),经济效益显著。此外,本发明的β-甘露聚糖酶耐受水基压裂液各种组分,对瓜尔胶、羟丙基瓜尔胶、阳离子瓜尔胶作为稠化剂的水基压裂液都具有良好的破胶效果,并可适用于高粘度水基压裂液破胶,最适底物浓度为8g/L。
本发明重组菌的β-甘露聚糖酶酶活(DNS法)很低(<10U/mL),比现有同类β-甘露聚糖酶酶活(一般为500-5000U/mL)低很多,可以快速降低瓜尔胶粘度却仅产生极少量的甘露糖寡糖,酶学性质存在巨大差异;β-甘露聚糖酶ManBL27-1在未破胞的情况下,能够在1h以内将40-50℃下的水基压裂液粘度从5000mpa.s以上降到500mpa.s,随着时间的增加能够完全降低粘度,实现了中低温条件下的全细胞催化瓜尔胶降粘。另外,本发明中的β-甘露聚糖酶ManBL27-2基因工程菌,形成了菌体的“自包埋”,在相同条件下对水基压裂液的降粘速率有明显的减缓趋势,实现了甘露聚糖酶在水基压裂液体系中的缓释降粘效果。
附图说明
图1为重组菌pET28a-BL27man1-BL21(DE3)菌落PCR验证图。
图2为重组菌pET28a-BL27man2-BL21(DE3)菌落PCR验证图。
图3为β-甘露聚糖酶ManBL27-1表达的SDS-PAGE电泳图(M:marker,1:表达的ManBL27-1)。
图4为β-甘露聚糖酶ManBL27-2表达的SDS-PAGE电泳图(M:marker,1:表达的ManBL27-2)。
图5为甘露糖标准曲线。
图6-图9为25-70℃温度下β-甘露聚糖酶ManBL27-1和ManBL27-2对水基压裂液的降粘效果。
图10-图11为碱性条件下β-甘露聚糖酶ManBL27-1和ManBL27-2对水基压裂液的降粘效果。
图12-图13为重组菌全细胞催化水基压裂液的降粘效果。
具体实施方式
实施例涉及的枯草芽孢杆菌Bacillus subtilis BL-27已于申请人在先申请的专利CN109055261A中公开,保藏编号为CCTCC NO:M2018402。
LB培养基配方为:5g/L酵母粉、10g/L NaCl、10g/L胰蛋白胨。
水基压裂液配方为:Gly-NaOH缓冲液(100mM,pH7.0-9.0)200mL、瓜尔胶1.6g、KCl4g、Na2S2O3 2g、25%戊二醛100μL、硼砂0.6mg。破胶剂添加量0.125%(v/v)。
实施例1中低温内切β-甘露聚糖酶ManBL27-1、ManBL27-2的基因克隆
参照细菌基因组DNA提取试剂盒(离心柱型Cat#:DP2001)操作步骤提取枯草芽孢杆菌Bacillus subtilis BL-27的基因组DNA。将枯草芽孢杆菌Bacillus subtilis BL-27的全基因序列与NCBI数据库中的β-甘露聚糖酶基因序列进行比较分析后,设计引物BL27man1-F:5’-CATGCCATGGCCTTTAAGAAACATACGATCTCTT-3’和BL27man1-R:5’-CCGCTCGAGTTCAACGATTGGCGTTAAAGAA-3’用于扩增带有信号肽的完整编码基因;设计引物BL27man2-F:5’-CATGCCATGGCCACTGTGTCGCCTGTGAATCCTA-3’和BL27man2-R:5’-CCGCTCGAGTTCAACGATTGGCGTTAAAGAA-3’用于扩增去除信号肽的编码基因。以提取的枯草芽孢杆菌Bacillus subtilis BL-27的基因组DNA为模板,扩增编码β-甘露聚糖酶BL27Man的基因序列,含有信号肽的基因manBL27-1和不含信号肽的基因manBL27-2,其PCR反应条件分别为PCR1和PCR2。PCR1反应条件为:95℃3min,1个循环;95℃15S,56℃15S,72℃33S,30个循环;72℃5min,1个循环。PCR2反应条件为95℃3min,1个循环;95℃15S,59℃15S,72℃30S,30个循环;72℃5min,1个循环。PCR产物进行琼脂糖凝胶电泳后,对目的基因进行切胶回收。
实施例2基因manBL27-1和manBL27-2在大肠杆菌中BL21(DE3)中的重组表达
以pET28a为表达载体,在设计的上下游引物中分别加入NcoI和XhoI酶切位点作为构建重组表达载体的作用位点。将PCR胶回收后的基因manBL27-1、manBL27-2和表达载体pET28a分别用NcoI和XhoI进行双酶切(50μL双酶切体系:30μL片段或质粒、2μL NcoI快切酶、2μLXhoI快切酶、10μL快切酶Buffer、6μL ddH2O),酶切产物胶回收后,用T4 DNA连接酶连接(25μL连接体系:T4 DNA Ligase 1μL、10×T4 DNA Ligase Buffer 2.5μL、DNA片段约0.3pmol、载体DNA约0.03pmol、ddH2O up to 25μL),16℃连接过夜。取10μL连接产物转化50μL的E.coli BL21(DE3)感受态细胞,涂布在含50μg/mL卡那霉素的固体LB平板上,37℃培养过夜。挑取单菌落,进行菌落PCR验证(PCR1反应条件为95℃6min,1个循环;94℃30S,56℃30S,72℃65S,25个循环;72℃7min,1个循环;PCR2反应条件为95℃6min,1个循环;94℃30S,59℃30S,72℃59S,25个循环;72℃7min,1个循环),验证图如图1、图2所示。将验证正确的单菌落提取质粒送去测序。测序结果正确,表明重组菌构建成功,将重组菌命名为pET28a-BL27man1-BL21(DE3)和pET28a-BL27man2-BL21(DE3)。
将pET28a-BL27man1-BL21(DE3)和pET28a-BL27man2-BL21(DE3)这两个重组菌在20℃,0.1mM IPTG,160rpm条件下进行诱导表达12h,之后将诱导结束的菌体进行超声破胞后离心收取上清粗酶液,用聚丙烯酰胺凝胶电泳检测β-甘露聚糖酶ManBL27-1和ManBL27-2的表达情况,结果如图3、图4所示,存在目标蛋白且与预测蛋白大小吻合。
实施例3重组表达β-甘露聚糖酶的酶活力测定(DNS法)
甘露糖标准曲线制作:取洁净的刻度试管并标号,分别吸取100,200,300,400,500,600,700,800,900,1000μL的1g/L的标准甘露糖溶液于10mL试管中,以不加甘露糖的作为空白对照,每个样做三个平行。向各试管分别加入蒸馏水补足至1mL,然后各加入3mL的DNS试剂,煮沸4min后用流水冷却,再用蒸馏水定容到15mL,在540nm波长处测定吸光度。根据数据制成标准曲线,如图5所示。
酶活性测定:选用槐豆胶、魔芋胶、田菁胶、瓜尔胶、羟丙基瓜尔胶、阳离子瓜尔胶这6种底物,先用50mM的pH7磷酸盐缓冲液配成8g/L的胶溶液。在900μL的各种胶溶液中加入100μL的粗酶液,在50℃的水浴中反应10min,加入3mL的DNS试剂,煮沸4min后用流水冷却,再用蒸馏水定容到15mL,在540nm波长处测定吸光度。酶活单位定义:在所选取的反应条件下,以每分钟水解底物产生相当于1μmol甘露糖所需要的酶液量定义为1个酶活力单位(U/mL)。
结果如表1所示,重组表达的β-甘露聚糖酶ManBL27-1和ManBL27-2对田菁胶、羟丙基瓜尔胶没有活性,对魔芋胶、槐豆胶、瓜尔胶、阳离子瓜尔胶有很低的酶活。
表1利用不同底物测定重组β-甘露聚糖酶ManBL27-1和ManBL27-2的酶活结果
Figure GDA0003321288970000061
来自于枯草芽孢杆菌Bacillus subtilis BL-27的β-甘露聚糖酶ManBL27-1和ManBL27-2,可快速降低瓜尔胶粘度却仅产生极少量的甘露糖寡糖;因此,可认为β-甘露聚糖酶ManBL27-1和ManBL27-2为内切β-甘露聚糖酶。
实施例4重组大肠杆菌基因工程菌产β-甘露聚糖酶的发酵条件
将构建的2株重组大肠杆菌基因工程菌分别接入LB液体培养基(含50μg/mL卡那霉素)中,37℃,200rpm培养12h,成为种子液;然后按照2%(v/v)接种量转接入产酶发酵培养基(含50μg/mL卡那霉素)中,产酶发酵培养基组分(g/L)为:蛋白胨15,酵母粉25,NaCl10,甘油10,葡萄糖2,乳糖2。使用小型发酵罐(5L)进行产酶发酵,装液量为3.5L,通气量为1.5vvm,搅拌转速200rpm,发酵温度为25℃,发酵36h。
实施例5重组表达β-甘露聚糖酶在不同温度下对水基压裂液的降黏效果
配置pH9的水基压裂液,先将20mL的水基压裂液分别在20℃、25℃、30℃、35℃、40℃、45℃、50℃、55℃、60℃、65℃、70℃下预热30min,预热完成后加入0.125%的破胞重组菌发酵液,在此温度条件下测定水基压裂液的粘度变化。结果如图6、图7、图8、图9所示:含信号肽的重组酶ManBL27-1的最适破胶作用温度为45℃,在40-55℃之间活性较高,温度高于60℃时活性大幅下降;不含信号肽的重组酶ManBL27的最适破胶作用温度为40℃,在20-50℃之间活性稳定,温度高于55℃时破胶活性大幅下降。尤其值得注意的是,不含信号肽的重组酶ManBL27-2在温度为20-25℃时体现了较好的破胶活性,1h后压裂液体系粘度由5000mpa.s降低至500mpa.s以下。
实施例6重组表达β-甘露聚糖酶在碱性条件下对水基压裂液的降粘效果
由于本发明的重组β-甘露聚糖酶ManBL27-1和ManBL27-2用作水基压裂液的破胶剂,考虑到水基压裂液的碱性环境条件,重点考察该酶在碱性环境条件下对瓜尔胶的降黏效果。配制pH7、8、9、10的水基压裂液,保持水基压裂液的温度40℃一定,分别测定不同pH水基压裂液体系的粘度变化。先将20mL各种pH水基压裂液在40℃下预热,之后加入0.125%破胞酶液,测定水基压裂液体系的粘度变化。结果如图10、图11所示:带有信号肽的重组酶ManBL27-1的最适反应pH8,在pH7-8之间活性稳定;不含信号肽的重组酶ManBL27-2最适反应pH8,在pH7-9之间活性稳定。
实施例7重组表达β-甘露聚糖酶在对各种瓜尔胶基水基压裂液的降粘效果
以瓜尔胶、羟丙基瓜尔胶以及阳离子瓜尔胶等常见稠化剂(8g/L),配制pH7、8、9的水基压裂液,先将20mL各种pH水基压裂液在40℃下预热,之后加入0.125%(v/v)破胞酶液,测定水基压裂液体系的粘度变化,考察重组β-甘露聚糖酶ManBL27-1和ManBL27-2对各种瓜尔胶基水基压裂液的降黏效果。结果如表2所示。重组β-甘露聚糖酶ManBL27-1和ManBL27-2对瓜尔胶、羟丙基瓜尔胶和阳离子瓜尔胶作为稠化剂的压裂液都具有显著的降黏效果,重组β-甘露聚糖酶ManBL27-1对pH7-8的各种压裂液降黏效果最好,重组β-甘露聚糖酶ManBL27-2对pH7-9的各种压裂液都具有良好的降黏效果。
表2重组β-甘露聚糖酶ManBL27-1和ManBL27-2对不同瓜尔胶基水基压裂液降黏效果
Figure GDA0003321288970000071
实施例8重组菌全细胞催化水基压裂液的降黏效果
重组β-甘露聚糖酶ManBL27-1在N端含有信号肽,可以将酶分泌到周质空间,有助于接触底物;而不含信号肽重组β-甘露聚糖酶ManBL27-2为胞内表达,相当于酶被菌体细胞“自包埋”。考察两种重组菌全细胞体系对水基压裂液的破胶效果有助于降低成本。
配制pH9的油田用水基压裂液,考察在中低温20-40℃条件下的全细胞破胶效果。将20mL水基压裂液在不同温度下预热30min,分别加入0.125%(v/v)的重组菌发酵液,测定水基压裂液体系的粘度变化。
结果如图12、图13所示:在未破胞的情况下,添加量为0.125%(v/v)的重组菌pET28a-BL27man1-BL21(DE3)发酵液,能够在1h以内将中低温水基压裂液粘度从5000mpa.s以上降到1000mpa.s,随着时间的增加能够完全降低粘度,可实现中低温条件下的全细胞催化水基压裂液降粘。在未破胞的情况下,添加量为0.125%(v/v)的重组菌pET28a-BL27man2-BL21(DE3)发酵液对水基压裂液的降粘速率有明显的减缓趋势,表明菌体细胞“自包埋”对酶起到了缓释效果,作用时间延长后对水基压裂液的依然具有良好降黏效果。
序列表
<110> 南京工业大学
<120> 中低温内切β-甘露聚糖酶及其编码基因和应用
<130> xb20040201
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<170> SIPOSequenceListing 1.0
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ggagcgttcg gaggttacag tcatgacaca ttttctatgg ctgaggctga tagaatccga 240
agcgccaccg ggcaatcgcc tgctatttac ggctgcgatt atgccagagg atggcttgaa 300
acagcaaata ttgaagattc aatagatgta agctgcaaca gcgatttaat atcgtattgg 360
aaaaatggtg gaatcccgca aatcagcatg cacctggcga atcctgcttt tcagtcaggg 420
cattttaaaa caccgattac aaacgatcag tataaaaaaa tactagattc ttcaacagca 480
gaagggaagc ggctgaatgc catgctcagc aaaattgctg acggacttca agagctggag 540
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tggggactta catcatataa ccaaaaggat aatgaaagaa tctctctata taaacagctc 660
tacaagaaaa tctatcatta tatgaccgac acaagaggac ttgatcattt gctttgggtt 720
tactctcccg acgccaaccg agattttaaa actgattttt acccgggcgc gtcttacgtg 780
gatattgtcg gattagatgc gtattttcaa gatgcctact cgatcaatgg atacgatcag 840
ctaacagcgc ttaataaacc atttgctttt acagaagtcg gcccgcaaac agcaaacggt 900
agcttagatt acagcctatt tatcaatgca ataaaacaaa gatatcctaa aaccatttac 960
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catgatagct ggacactcaa caagggagaa atttggaatg gtgattcttt aacgccaatc 1080
gttgaatga 1089
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actgtgtcgc ctgtgaatcc taatgcacag cagacaacaa aagcagtgat gaactggctt 60
gcgcacctgc cgaaccgaac ggaaaacaga gtcctttccg gagcgttcgg aggttacagt 120
catgacacat tttctatggc tgaggctgat agaatccgaa gcgccaccgg gcaatcgcct 180
gctatttacg gctgcgatta tgccagagga tggcttgaaa cagcaaatat tgaagattca 240
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atcagcatgc acctggcgaa tcctgctttt cagtcagggc attttaaaac accgattaca 360
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caaaaggata atgaaagaat ctctctatat aaacagctct acaagaaaat ctatcattat 600
atgaccgaca caagaggact tgatcatttg ctttgggttt actctcccga cgccaaccga 660
gattttaaaa ctgattttta cccgggcgcg tcttacgtgg atattgtcgg attagatgcg 720
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Claims (8)

1.一种中低温内切β-甘露聚糖酶作为水基压裂液破胶剂的应用,其特征在于,在水基压裂液中加入β-甘露聚糖酶粗酶液或重组菌发酵液;
所述β-甘露聚糖酶粗酶液或重组菌发酵液的获取方式为:将β-甘露聚糖酶基因导入宿主菌构建重组菌,将重组菌利用乳糖诱导表达,获取所述β-甘露聚糖酶粗酶液;或将重组菌发酵获取所述重组菌发酵液;
编码所述中低温内切β-甘露聚糖酶的基因的核苷酸序列如SEQ ID NO:1或SEQ ID NO:2所示。
2.根据权利要求1所述的应用,其特征在于,所述重组菌的构建方法为:以pET28a为表达载体,在设计的上下游引物中分别加入NcoI和XhoI酶切位点作为构建重组表达载体的作用位点;将PCR胶回收后的基因和表达载体pET28a分别用NcoI和XhoI进行双酶切,酶切产物胶回收后,用T4 DNA连接酶连接过夜;取连接产物转化至大肠杆菌E.coli BL21(DE3)感受态细胞,获取重组菌;
所述设计的上下游引物序列为:
对于SEQ ID NO:1所示基因序列:
BL27man1-F:5’- CATGCCATGGCCTTTAAGAAACATACGATCTCTT -3’;
BL27man1-R:5’- CCGCTCGAGTTCAACGATTGGCGTTAAAGAA -3;
对于SEQ ID NO:2所示基因序列:
BL27man2-F: 5’- CATGCCATGGCCACTGTGTCGCCTGTGAATCCTA -3’;
BL27man2-R:5’- CCGCTCGAGTTCAACGATTGGCGTTAAAGAA-3’。
3.根据权利要求1所述的应用,其特征在于,发酵培养基组分为:蛋白胨15g/L,酵母粉25g/L,NaCl 10g/L,甘油10g/L,葡萄糖2g/L,乳糖2g/L,其余为水。
4.根据权利要求1所述的应用,其特征在于,所述粗酶液或重组菌发酵液添加量为0.125%(v/v)。
5.根据权利要求1所述的应用,其特征在于,所述水基压裂液中底物浓度为8g/L。
6.根据权利要求1所述的应用,其特征在于,所述水基压裂液中底物为瓜尔胶、羟丙基瓜尔胶或阳离子瓜尔胶。
7.根据权利要求5或6所述的应用,其特征在于,所述水基压裂液成分为:每200mL含Gly-NaOH缓冲液100mM,底物1.6g,KCl 4g,Na2S2O3 2g,25%戊二醛100μL,硼砂0.6mg,pH7.0-9.0,其余为水。
8.根据权利要求2所述的应用,其特征在于,对于SEQ ID NO:1所示基因表达的β-甘露聚糖酶,作用温度为35-50℃,pH7-8;对于SEQ ID NO:2所示基因表达的β-甘露聚糖酶,作用温度为20-50℃,pH7-9。
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