CN105950592A - 耐盐耐乙醇耐胰蛋白酶的木糖苷酶jb13gh39及其制备方法 - Google Patents
耐盐耐乙醇耐胰蛋白酶的木糖苷酶jb13gh39及其制备方法 Download PDFInfo
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- CN105950592A CN105950592A CN201610559679.7A CN201610559679A CN105950592A CN 105950592 A CN105950592 A CN 105950592A CN 201610559679 A CN201610559679 A CN 201610559679A CN 105950592 A CN105950592 A CN 105950592A
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- Prior art keywords
- jb13gh39
- xylosidase
- resistance
- resistant
- ethanol
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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Abstract
本发明涉及基因工程技术领域,具体地说是一种耐盐耐乙醇耐胰蛋白酶的木糖苷酶JB13GH39及其制备方法,其氨基酸序列如SEQ No.1所示。木糖苷酶JB13GH39具有以下性质:最适pH4.5;经pH4.0–9.0的缓冲液处理1h,该酶酶活剩余达70%以上;最适温度50℃,在0–70℃内都具有酶活,在20℃时具有52.8%的酶活;该酶在37℃和60℃下该酶稳定,在70℃下该酶快速失活;胰蛋白酶及大部分金属离子对其活性无影响或影响微弱;可水解木二糖、木三糖、木四糖、木五糖及木六糖,水解产物主要为木糖。本发明的木糖苷酶可应用于饲料、食品和生物能源等行业。
Description
技术领域
本发明涉及基因工程技术领域,具体地说是一种耐盐耐乙醇耐胰蛋白酶的木糖苷酶JB13GH39及其制备方法。
背景技术
木聚糖是植物半纤维素的主要成份,是自然界中最为丰富的可再生资源之一,其主链由吡喃木糖以β-1,4糖苷键连接而成,侧链上具有***糖、葡糖醛酸、***、香豆酸、肉桂酸等(Collins et al.FEMS Microbiol Rev,2005,29:3–23.)。内切木聚糖酶(endo-1,4-β-d-xylanase,EC 3.2.1.8)随机地切割木聚糖的主链骨架,生成低聚木糖;木糖苷酶(β-d-xylosidase,EC 3.2.1.37)可降低低聚木糖对内切木聚糖酶的抑制作用,降解低聚木糖生成木糖;内切木聚糖酶和木糖苷酶之间存在协同作用,以内切木聚糖酶为主、木糖苷酶为辅对木聚糖主链进行完全降解(Collins et al.FEMSMicrobiol Rev,2005,29:3–23.)。木糖苷酶在医药、食品、酿酒、能源及造纸等领域都具有应用价值(Zhang et al.Process Biochem,2014,49:1422–1428.)。根据氨基酸序列同源性,木糖苷酶可归类于糖苷水解酶第1、3、5、30、39、43、51、52、54、116和120家族,其可来源于细菌、真菌及植物等(Lombard et al.,Nucleic AcidsRes,2014,42:D490–D495.)。
耐盐酶在高浓度NaCl下仍然具有催化活性,可应用于高盐食品和海产品加工及其它高盐环境生物技术领域(如酱油发酵),在高盐环境下加工食品还可以防止微生物的污染、节省灭菌等所消耗的能源(Madern et al.Extremophiles,2000,4:91–98);耐乙醇的酶在同步糖化发酵中可提高乙醇产量和生物质利用率、缩短发酵时间(Sato et al.J Biosci Bioeng,2010,110:679–683);耐蛋白酶的酶可应用于食品及饲料等多种行业(Zhou et al.J Ind MicrobiolBiot,2012,39:965–975)。
发明内容
本发明的目的是提供一种耐盐耐乙醇耐胰蛋白酶的木糖苷酶JB13GH39及其制备方法。
为实现上述目的,本发明采用的技术方案是:一种耐盐耐乙醇耐胰蛋白酶的木糖苷酶JB13GH39,其氨基酸序列如SEQ No.1所示。
本发明还提供一种编码木糖苷酶JB13GH39的基因jB13GH39,所述基因jB13GH39的核苷酸序列如SEQ No.2所示。
本发明还提供一种包含木糖苷酶基因jB13GH39的重组载体。
本发明还提供一种包含木糖苷酶基因jB13GH39的重组菌株。
本发明所述耐盐耐乙醇耐胰蛋白酶的木糖苷酶JB13GH39可得自鞘氨醇单胞菌(Sphingomonas sp.)。木糖苷酶JB13GH39总共含538个氨基酸,理论分子量为60.3kDa,其中N端19个氨基酸为预测信号肽序列“MAMGRSIMIRRMAMCVALA”,成熟的木糖苷酶JB13GH39含519个氨基酸。该木糖苷酶JB13GH39全序列与GenBank中Sphingomonas sp.PR090111-T3T-6A来源的潜在木糖苷酶(WP_051103436)全序列具有最高的氨基酸序列一致性,为70.8%。
木糖苷酶JB13GH39的最适pH值为4.5;经pH4.0–9.0的缓冲液处理1h,该酶酶活剩余达70%以上;该酶最适温度为50℃,在0–70℃内都具有酶活;该酶在37℃和60℃下稳定,在70℃下快速失活;在反应体系中加入3.0–20.0%(w/v)的NaCl,该酶的活性不受影响;经3.0–30.0%(w/v)的NaCl在37℃下处理60min,该酶仍能保持80%以上的活性;在15.0%(v/v)的乙醇中,该酶具有55.2%的活性;经3.0–20.0%(v/v)的乙醇在37℃下处理60min,该酶仍能保持74%以上的活性;经2.2–87.0mg/mL的胰蛋白酶在37℃下处理1h,该酶的酶活基本保持不变;该酶能水解木二糖、木三糖、木四糖、木五糖及木六糖,水解产物主要为木糖。
本发明提供了编码上述木糖苷酶JB13GH39的基因jB13GH39,该基因序列如SEQ ID NO.2所示。
本发明通过基因组测序的方法获得木糖苷酶JB13GH39的编码基因jB13GH39,其全长1617bp,起始密码为ATG,终止密码为TAA。
本发明还提供了包含上述木糖苷酶基因jB13GH39的重组载体,优选为pEasy-E2-jB13GH39。将本发明的木糖苷酶基因***到表达载体中,使其核苷酸序列与表达调控序列相连接。作为本发明的一个最优选的实施方案,将本发明的木糖苷酶基因和表达载体pEasy-E2通过T-A方式相连接,得到重组大肠杆菌表达质粒pEasy-E2-jB13GH39。
本发明还提供了包含上述木糖苷酶基因jB13GH39的重组菌株,优选所述菌株为大肠杆菌、酵母菌、芽孢杆菌或乳酸杆菌,优选为重组菌株BL21(DE3)/jB13GH39。
本发明制备木糖苷酶JB13GH39的方法按以下步骤进行:
1)用上述的重组载体转化宿主细胞,得重组菌株;
2)培养重组菌株,诱导重组木糖苷酶JB13GH39表达;
3)回收并纯化所表达的木糖苷酶JB13GH39。
其中,优选所述宿主细胞为大肠杆菌细胞,优选将重组大肠杆菌表达质粒转化大肠杆菌细胞BL21(DE3),得到重组菌株BL21(DE3)/jB13GH39。
本发明提供了一个新的木糖苷酶基因,其编码的木糖苷酶最适pH4.5;最适温度50℃;良好的耐盐耐乙醇耐胰蛋白酶特性。本发明的木糖苷酶可应用于饲料、食品和生物能源等行业。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动性的前提下,还可以根据这些附图获得其他的附图。
图1:在大肠杆菌中表达的木糖苷酶JB13GH39的SDS-PAGE分析,其中,M:蛋白质Marker;P:纯化的重组木糖苷酶JB13GH39。
图2:纯化的重组木糖苷酶JB13GH39的pH活性。
图3:纯化的重组木糖苷酶JB13GH39的pH稳定性。
图4:纯化的重组木糖苷酶JB13GH39的热活性。
图5:纯化的重组木糖苷酶JB13GH39的热稳定性。
图6:纯化的重组木糖苷酶JB13GH39的NaCl抗性。
图7:纯化的重组木糖苷酶JB13GH39的NaCl稳定性。
图8:纯化的重组木糖苷酶JB13GH39的乙醇抗性。
图9:纯化的重组木糖苷酶JB13GH39的乙醇稳定性。
图10:纯化的重组木糖苷酶JB13GH39的胰蛋白酶抗性。
图11:纯化的重组木糖苷酶JB13GH39水解木二糖(X2)、木三糖(X3)、木四糖(X4)、木五糖(X5)及木六糖(X6)的产物分析,其中,X1:木糖;CK:底物和失活的酶(煮沸10min);S:反应组。
具体实施方式
下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
试验材料和试剂
1、菌株及载体:鞘氨醇单胞菌(Sphingomonas sp.)分离于云南省红河哈尼族彝族自治州个旧市磷矿土土样,保藏于中国普通微生物菌种保藏管理中心,保藏号为CGMCC 1.10968;大肠杆菌Escherichia coli BL21(DE3)和表达载体pEasy-E2购于北京全式金生物技术有限公司。
2、试剂:DNA聚合酶和dNTP购自TaKaRa公司;pNP(p-nitrophenol)、pNPX(p-nitrophenyl-β-d-xylopyranoside)、p-nitrophenyl-α-L-arabinofuranoside、桦木木聚糖、山毛榉木聚糖、羧甲基纤维素纳和β-葡聚糖购自Sigma公司,***木聚糖、木二糖、木三糖、木四糖、木五糖及木六糖购自Megazyme公司,Genomic DNA Clean&Concentration试剂盒购自Zymo Research公司,Tureseq DNA Sample Preparation Kit购自Illumima公司,其它都为国产试剂(均可从普通生化试剂公司购买得到)。
3、培养基:
LB培养基:Peptone 10g,Yeast extract 5g,NaCl 10g,加蒸馏水至1000ml,pH自然(约为7)。固体培养基在此基础上加2.0%(w/v)琼脂。
说明:以下实施例中未作具体说明的分子生物学实验方法,均参照《分子克隆实验指南》(第三版)J.萨姆布鲁克一书中所列的具体方法进行,或者按照试剂盒和产品说明书进行。
实施例1:木糖苷酶基因jB13GH39的克隆
提取鞘氨醇单胞菌基因组DNA:将培养2d的液体菌液离心取菌体,加入1mL溶菌酶,37℃处理60min,再加入裂解液,裂解液组成为:50mM Tris,20mM EDTA,NaCl 500mM,2%SDS(w/v),pH8.0,70℃水浴裂解60min,每隔10min混匀一次,在4℃下10000rpm离心5min。取上清于酚/氯仿中抽提除去杂蛋白,再取上清加入等体积异丙醇,于室温静置5min后,4℃下10000rpm离心10min。弃上清,沉淀用70%的乙醇洗涤两次,真空干燥,加入适量TE溶解,置于-20℃备用。
用超声打断仪Biorupter将5μg的鞘氨醇单胞菌基因组打断为400–600bp的片段,用Genomic DNA Clean&Concentration试剂盒对打断的DNA片段进行纯化,纯化后用TureseqTM DNA SamplePreparation Kit进行DNA片段的末端补平、3'端加A碱基和加接头、及DNA片段的PCR扩增(操作按试剂盒说明书进行)。用MiSeq基因组测序仪(Illumima公司)对上述制备好的文库进行基因组测序。
基因组测序得到的数据经读码框预测和本地BLAST比对,得到木糖苷酶基因jB13GH39,该基因序列如SEQ ID NO.2所示。
实施例2:重组木糖苷酶JB13GH39的制备
以5'GCAACTCTCTGCACGGCTCCGG 3'和5'CTTTCGCTCCTTGGGTGCAATTGAC 3'为引物对,鞘氨醇单胞菌基因组DNA为模板,进行PCR扩增。PCR反应参数为:94℃变性5min;然后94℃变性30sec,55℃退火30sec,72℃延伸1min 30sec,30个循环后72℃保温10min。PCR结果得到木糖苷酶基因jB13GH39,并在该基因3’端引入突出的A碱基。将木糖苷酶基因jB13GH39和表达载体pEasy-E2通过T-A方式相连接,获得含有jB13GH39的重组表达质粒pEasy-E2-jB13GH39。将pEasy-E2-jB13GH39转化大肠杆菌BL21(DE3),获得重组大肠杆菌菌株BL21(DE3)/jB13GH39。
取含有重组质粒pEasy-E2-jB13GH39的重组大肠杆菌菌株BL21(DE3)/jB13GH39,以0.1%的接种量接种于LB(含100μg mL-1Amp)培养液中,37℃快速振荡16h。然后将此活化的菌液以1%接种量接种到新鲜的LB(含100μg mL-1Amp)培养液中,快速振荡培养约2–3h(OD600达到0.6–1.0)后,加入终浓度0.7mM的IPTG进行诱导,于20℃继续振荡培养约20h或26℃振荡培养约8h。12000rpm离心5min,收集菌体。用适量的pH7.0McIlvaine缓冲液悬浮菌体后,于低温水浴下超声波破碎菌体。以上胞内浓缩的粗酶液经12,000rpm离心10min后,吸取上清并用Nickel-NTA Agarose和0–500mM的咪唑分别亲和和洗脱目的蛋白。SDS-PAGE结果(图1)表明,重组木糖苷酶JB13GH39得到了纯化,产物为单一条带。
实施例3:纯化的木糖苷酶JB13GH39的性质测定
1、纯化的重组木糖苷酶JB13GH39的活性分析:
实施例2纯化的重组木糖苷酶JB13GH39的活性测定方法采用pNP法:将pNPX溶于缓冲液中,使其终浓度为2mM;反应体系含50μL适量酶液,450μL的2mM底物;底物在反应温度下预热5min后,加入酶液再反应10min,然后加2mL 1M Na2CO3终止反应,冷却至室温后在405nm波长下测定释放出的pNP;1个酶活单位(U)定义为每分钟分解底物产生1μmol pNP所需的酶量。对底物p-nitrophenyl-α-L-arabinofuranoside的测定也采用pNP法。对底物山毛榉木聚糖、羧甲基纤维素纳、普鲁兰多糖和β-葡聚糖的活性测定方法采用3,5-二硝基水杨酸(DNS)法:将底物溶于缓冲液中,使其终浓度为0.5%;反应体系含100μL适量酶液,900μL底物;底物在反应温度下预热5min后,加入酶液后再反应10min,然后加2.0mL DNS终止反应,沸水煮5min,冷却至室温后在540nm波长下测定OD值;1个酶活单位(U)定义为在给定的条件下每分钟分解底物产生1μmol还原糖(以木糖计)所需的酶量。
2、纯化的重组木糖苷酶JB13GH39的pH活性和pH稳定性测定:
酶的最适pH测定:将木糖苷酶JB13GH39在37℃下和pH3.0–8.0的缓冲液中进行酶促反应。酶的pH稳定性测定:将纯化的酶液置于pH3.0–10.0的缓冲液中,在37℃下处理60min,然后在pH4.5及37℃下进行酶促反应,以未处理的酶液作为对照。缓冲液为:McIlvainebuffer(pH3.0–8.0)和0.1M glycine–NaOH(pH9.0–10.0)。以pNPX为底物,反应10min,测定纯化的木糖苷酶JB13GH39的酶学性质。结果表明:JB13GH39的最适pH为4.5(图2);经pH4.0–9.0的缓冲液处理1h,该酶酶活剩余达70%以上(图3)。
3、纯化的重组木糖苷酶JB13GH39的热活性及热稳定性测定:
酶的热活性测定:在pH4.5的缓冲液中,于0–70℃下进行酶促反应。酶的热稳定性测定:将同样酶量的酶液分别置于37℃、60℃和70℃中,处理0–60min后,在pH4.5及37℃下进行酶促反应,以未处理的酶液作为对照。以pNPX为底物,反应10min,测定纯化的JB13GH39的酶学性质。结果表明:JB13GH39的最适温度为50℃,在0–70℃内都具有酶活,在20℃时具有52.8%的酶活(图4);该酶在37℃和60℃下稳定,在70℃下快速失活(图5)。
4、纯化的重组木糖苷酶JB13GH39的动力学参数测定:
酶的动力学参数一级反应时间测定:在pH4.5及50℃下,以1mMpNPX为底物,依次在酶促反应的1–10min内终止反应并测定酶活性,计算出酶活性与反应时间的比值,若在一定时间内该比值保持稳定,则此时间为一级反应时间。用0.05–2.0mM pNPX为底物,在pH4.5、50℃和一级反应时间下,根据Lineweaver–Burk方法测定Km、Vmax和kcat。经测定,在50℃及pH4.5条件下,JB13GH39对pNPX的Km、Vmax和kcat分别为3.44mM、90.32μmol min-1mg-1和90.77s-1。
5、不同金属离子及化学试剂对纯化的重组木糖苷酶JB13GH39活力的影响:
在酶促反应体系中加入一定终浓度的金属离子及化学试剂,研究其对酶活性的影响。在37℃及pH4.5条件下,以pNPX为底物测定酶活性。结果(表2)表明:SDS完全抑制JB13GH39;AgNO3、10.0mM的NiSO4、CuSO4及HgCl2对JB13GH39的抑制较强;添加10.0mM的CoCl2,JB13GH39受到部分抑制;而10.0mM的FeSO4及1.0%(v/v)的Triton X-100和Tween 80对JB13GH39有明显的促进作用,提高JB13GH39的酶活约为0.5倍;其余金属离子及化学试剂对该酶活性无影响或影响微弱。
表2.金属离子及化学试剂对纯化的重组JB13GH39活力的影响
7、纯化的重组木糖苷酶JB13GH39的NaCl抗性及NaCl稳定性测定:
酶的NaCl抗性测定:在酶促反应体系中加入3.0–30.0%(w/v)NaCl,于pH4.5及50℃下进行酶促反应。酶的NaCl稳定性测定:将纯化的酶液置于3.0–30.0%(w/v)的NaCl水溶液中,在37℃下处理60min,然后在pH4.5及50℃下进行酶促反应,以未加NaCl但在37℃下保温60min的酶液作为对照。以pNPX为底物,反应10min,测定纯化的JB13GH39的酶学性质。结果表明:在反应体系中加入3.0–20.0%(w/v)的NaCl,JB13GH39的活性不受影响,加入25.0%(w/v)和30.0%(w/v)NaCl,JB13GH39仍然分别具有52.6%和30.0%的活性(图6);经3.0–30.0%(w/v)的NaCl在37℃下处理60min,该酶仍能保持80%以上的活性(图7)。
8、纯化的重组木糖苷酶JB13GH39的乙醇抗性及乙醇稳定性测定:
酶的乙醇抗性测定:在酶促反应体系中加入3.0–30.0%(v/v)乙醇,于pH4.5及50℃下进行酶促反应。酶的乙醇稳定性测定:将纯化的酶液置于3.0–30.0%(v/v)的乙醇中,在37℃下处理60min,然后在pH4.5及50℃下进行酶促反应,以未加乙醇但在37℃下保温60min的酶液作为对照。以pNPX为底物,反应10min,测定纯化的JB13GH39的酶学性质。结果表明:随着乙醇含量的增加,JB13GH39活性逐渐降低,在15.0%(v/v)的乙醇中,该酶具有55.2%的活性(图8);经3.0–20.0%(v/v)的乙醇在37℃下处理60min,该酶仍能保持74%以上的活性(图9)。
9、纯化的重组木糖苷酶JB13GH39的胰蛋白酶抗性测定:
酶的胰蛋白酶抗性:用不同浓度的胰蛋白酶(pH7.5)在37℃下对重组酶处理1h,然后在pH4.5及50℃下进行酶促反应,以置于蛋白酶对应pH缓冲液中但未加蛋白酶的酶液作为对照。结果表明:经2.2–87.0mg/mL的胰蛋白酶在37℃下处理1h,JB13GH39的酶活基本保持不变(图10)。
10、纯化的重组木糖苷酶JB13GH39对底物的降解:
在pH4.5及50℃下,该酶对pNPX的比活为37.78±0.86U mg-1,对底物p-nitrophenyl-α-L-arabinofuranoside、桦木木聚糖、山毛榉木聚糖、***木聚糖、羧甲基纤维素纳、普鲁兰多糖和β-葡聚糖皆无活性。
11、纯化的重组木糖苷酶JB13GH39水解木寡糖的产物分析:
对木二糖、木三糖、木四糖、木五糖及木六糖的产物分析方法采用薄层层析法(TLC),反应体系含45μL 0.5%(w/v)的底物,5μL适当稀释酶液(约0.04U酶液),在pH4.5及50℃下,反应150min后终止反应并分析水解产物(使用青岛海洋化工有限公司的高效薄层层析硅胶板G型)。
薄层层析步骤如下所示:
(1)配制展开剂(冰醋酸20mL,双蒸水20mL,正丁醇40mL,混匀),取适量倒入展开槽,静置30min左右;
(2)将硅胶板放在110℃烘箱中活化30min,冷却后划线,点样(每次0.5μL,吹干,共点3次);
(3)将点样的一端硅胶板朝下放入展开槽中,点样点不要没入展开剂;
(4)待展开剂到距硅胶板上沿1.5cm时,取出硅胶板,吹干,再展开一次;
(5)第二次展开结束后,硅胶板直接浸入适量显色剂(1g二苯胺溶于50mL丙酮中,溶解后加入1mL苯胺及5mL 85%的磷酸,混匀,现用现配);
(6)几秒钟后,立即取出硅胶板并放置于90℃烘箱中10–15min,使斑点显色。
结果表明:JB13GH39能水解木二糖、木三糖、木四糖、木五糖及木六糖,水解产物主要为木糖(图11)。
本技术领域技术人员可以理解,除非另外定义,这里使用的所有术语(包括技术术语和科学术语)具有与本发明所属领域中的普通技术人员的一般理解相同的意义。还应该理解的是,诸如通用字典中定义的那些术语应该被理解为具有与现有技术的上下文中的意义一致的意义,并且除非像这里一样定义,不会用理想化或过于正式的含义来解释。
最后所应说明的是:以上实施例仅用以说明而非限制本发明的技术方案,尽管参照上述实施例对本发明进行了详细说明,本领域的普通技术人员应该理解:依然可以对本发明进行修改或者等同替换,而不脱离本发明的精神和范围的任何修改或局部替换,其均应涵盖在本发明的权利要求范围当中。
Claims (8)
1.一种耐盐耐乙醇耐胰蛋白酶的木糖苷酶JB13GH39,其特征在于,其氨基酸序列如SEQ No.1所示。
2.一种编码权利要求1所述的木糖苷酶JB13GH39的基因jB13GH39,其特征在于,所述基因jB13GH39的核苷酸序列如SEQ No.2所示。
3.一种包含权利要求2所述的木糖苷酶基因jB13GH39的重组载体。
4.一种包含权利要求2所述的木糖苷酶基因jB13GH39的重组菌株。
5.根据权利要求1所述耐盐耐乙醇耐胰蛋白酶的木糖苷酶JB13GH39在饲料工业中的应用。
6.根据权利要求1-5任一项所述木糖苷酶JB13GH39的制备方法,按以下步骤进行:
1)用上述的重组载体转化宿主细胞,得重组菌株;
2)培养重组菌株,诱导重组木糖苷酶JB13GH39表达;
3)回收并纯化所表达的木糖苷酶JB13GH39。
7.根据权利要求1所述耐盐耐乙醇耐胰蛋白酶的木糖苷酶JB13GH39在食品加工中的应用。
8.根据权利要求1所述耐盐耐乙醇耐胰蛋白酶的木糖苷酶JB13GH39在生物能源工业中的应用。
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