CN109402092A - 一种海洋环境来源的几丁质酶及其基因 - Google Patents
一种海洋环境来源的几丁质酶及其基因 Download PDFInfo
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- CN109402092A CN109402092A CN201811332636.0A CN201811332636A CN109402092A CN 109402092 A CN109402092 A CN 109402092A CN 201811332636 A CN201811332636 A CN 201811332636A CN 109402092 A CN109402092 A CN 109402092A
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- chitinase
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- encoding gene
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Abstract
本发明提供一种海洋环境来源的几丁质酶及其基因，以及将其在大肠杆菌和酵母细胞中克隆表达的方法，所述几丁质酶基因核苷酸序列的如SEQ ID NO.1所示；几丁质酶的氨基酸序列如SEQ ID NO.2所示；含所述基因的载体是大肠杆菌质粒或酵母质粒；含所述基因的细胞由所述载体转化而得；所述几丁质酶的基因的细胞是包含所述核苷酸分子或用所述载体转化的大肠杆菌或包含所述核酸分子或用所述载体转化的毕氏酵母。
Description
技术领域
本发明属于生物技术领域，具体涉及一种海洋环境来源的几丁质酶及其基因。
技术背景
基于宏基因组学研究样品微生物的发展使我们能够解锁很多功能多样性，从而获得新的基因和有用的生物分子。几丁质编码基因资源非常丰富，但是99%的微生物都是不可培养的，给获取几丁质基因带来了巨大的困难。采用宏基因组法可以绕过微生物的培养环节直接研究所有微生物的基因信息。2014年，Karin Hjort等人使用土壤宏基因组构建的文库，针对未培养的细菌群落的几丁质酶，发现了一种新型的细菌几丁质酶Chi18H8，Chi18H8是从宏基因组文库中得到的第一个纯化酶，并具有作为控制植物真菌病害制剂的潜力。2015年，Mariana Silvia Cretoiu等人从土壤的宏基因组文库筛选编码新型几丁质分解酶的基因，成功扩增了5条完整的几丁质酶基因，克隆并表达了一种耐盐性几丁质酶。2017年，Thimoteo 等人通过使用含有胶体几丁质的平板对使用大肠杆菌构建的宏基因组库进行基于功能的筛选，从废水污染的土壤中鉴定出一种几丁质酶。2015年Yuchun Liu等人利用genomic walking技术从猪粪的宏基因组获得了一种新的外切几丁质酶基因。
根据几丁质酶的作用方式分类，分为几丁质内切酶、几丁质外切酶和N-乙酰基葡糖苷酶。几丁质内切酶随机切割几丁质的β-1,4-糖苷键，产生几丁二糖和可溶的的低分子量的不同聚合度的几丁寡糖，外切几丁质酶从还原和非还原端切割链以形成几丁二糖（GlcNAc2），N-乙酰基葡糖苷酶将GlcNAc2水解成GlcNAc或从N-乙酰-寡糖的非还原端产生N-乙酰-D-氨基葡萄糖（GLcNAc）单体。
根据氨基酸序列同源性，几丁质酶分属于18、19和20家族葡萄糖苷水解酶。18家族包括来自病毒、细菌、真菌、动物和某些植物几丁质酶的几丁质酶。19家族包含一些植物几丁质酶和链霉菌几丁质酶。家族18和19不具有氨基酸序列的相似性，它们具有完全不同的3D结构，因此据说是从不同的祖先进化而来。家族20涉及来自细菌、某些真菌和人类的N-乙酰氨基葡糖苷酶。序列同源性的分析已经将18家族几丁质酶分成亚科A，B和C，迄今为止，亚家族B和C几丁质酶仅在少数细菌中被鉴定出来，而亚家族A几乎遍及陆地和水生栖息地，后者包括分子量为20-115KDa，最适温度18-90℃，pH值2.0-10.5，等电点为3.5-8.0的蛋白质。
现有在医学、农业和工业上有用的酶和天然产物，主要来源于容易培养的微生物。然而，在标准的实验室条件下，绝大多数环境中的微生物是不可培养的。宏基因组文库的表达筛选可获得不可培养的微生物群的功能多样性以及发现对生物技术应用有用的新型酶。
发明内容
本发明的主要目的在于提供一种海洋环境样品宏基因组来源的几丁质酶及其基因，以及将其在大肠杆菌和酵母细胞中中克隆表达的方法，提供具有潜在应用价值的新酶源。
为实现上述目的，采用以下技术方案：
本发明的几丁质酶，所述几丁质酶的基因核苷酸序列如SEQ ID NO.1所示；几丁质酶的氨基酸序列如SEQ ID NO.2所示。
含有几丁质酶基因的载体是大肠杆菌质粒或酵母质粒；所述的载体为pET28a(+)-Chitinase或pPIC9k-Chitinase。
含有几丁质酶的基因的细胞选大肠杆菌细胞或酵母细胞；所述细胞由所述载体转化而得；所述细胞是包含所述核苷酸分子或用所述载体转化的大肠杆菌或包含所述核酸分子或用所述载体转化的毕氏酵母。
具体为：
本发明涉及利用宏基因组学方法从漳州红树林滩涂环境中筛选几丁质酶基因及克隆几丁质酶基因。实施方式之一，本发明提取环境样品的宏基因组DNA，利用18家族亚家族A几丁质酶氨基酸保守片段的简并引物，获取一段大小250bp左右，存在核酸序列差异的几丁质酶的编码基因；构建几丁质酶基因文库，经序列测定及在NCBI数据库中的比对分析，选取数个几丁质酶基因片段；再通过 genome walking技术获得全长，并在NCBI数据库中进行比对分析，得到Chitinase基因。实施方式之一中，所述编码序列包含如SEQ ID NO.1所示的核酸序列，称之为Chitinase。
本发明还涉及包含所述Chitinase编码序列的重组载体，例如由各种本领域常用的表达载体制备的重组载体，其中，所述编码序列不包含其来源微生物的内源性信号肽序列。实施方式之一中，将不带内源性信号肽的编码序列即本发明Chitinase编码序列经SacI和XhoI双酶切后与SacI和XhoI双酶切的pET28a(+)载体连接，得到大肠重组表达载体pET28a(+)-Chitinase。另一实施方式中，将不带内源性信号肽编码序列的本发明Chitinase编码序列经SacI和XhoI双酶切后与SacI和XhoI双酶切的pPIC9k载体连接，得到酵母重组表达载体pPIC9k- Chitinase。
本发明还制备包含本发明Chitinase编码序列的细胞。实施方式之一中，所述细胞是用上述本发明重组载体转化来构建的。所述细胞优选各种利于基因产物表达的细胞，此类细胞已为本领域熟知并常用，例如各种大肠杆菌细胞和酵母细胞。在本发明的实施方式之一中，选用大肠杆菌BL21(DE3) 和毕氏酵母GS115构建表达Chitinase的重组细胞。
本发明还提供了表达Chitinase的方法，包括：培养前文所述包含本发明Chitinase编码序列的细胞或所述经转化的细胞，诱导其表达，收获表达产物，还包括纯化表达产物的步骤。实施方式之一中，本发明通过包含本发明Chitinase编码序列的大肠杆菌诱导表达来生产Chitinase几丁质酶，并通过镍柱纯化得到了纯酶形式的目的蛋白。
本发明利用基因工程手段制备了能够高效表达并分泌Chitinase的重组生产株，获得了优质的Chitinase产品。本发明通过酶活测定，酶学性质分析，底物特异性的分析。证明本发明的Chitinase是一种低温的几丁质酶，且对胶体几丁质具有严格的底物特异性。
本发明的优点在于：本发明的几丁质酶温度适应性强，在低于最适温度的条件下仍具有较高活性和持久的稳定性。可以不受温度限制，在较低温条件下保持较高活性持久进行反应。
附图说明
图1 环境样品宏基因组来源的Chitinase在质粒pET28a(+)和pPIC9k上的重组子结构图。
图2环境样品宏基因组来源的Chitinase的SDS-PAGE。M蛋白质Marker；1、3:IPTG诱导空质粒菌体的不可溶性的和可溶性部分；2、4:IPTG诱导含有Chitinase基因的菌体的不可溶性和可溶性部分；5：重组Chitinase镍柱纯化产物。
图3 环境样品宏基因组来源的Chitinase的最适pH和pH稳定性。a：pH对重组Chitinase的活性影响；b:重组Chitinase的pH稳定性
图4 环境样品宏基因组来源的Chitinase的最适温度和温度稳定性。a：温度对重组Chitinase的活性影响；b:重组Chitinase的温度稳定性稳定性
具体实施方式
以下根据具体的实施例充分说明本发明。
实验材料和试剂
1.菌株和载体：
大肠杆菌BL21（DE3）、Top10及表达载体pET28a(+)购自Novagen公司，毕氏酵母GS115及表达载体pPIC9k均购自Invitrogen公司（Carlsbad，CA，USA）。
2.酶类及其他生化试剂：
限制性内切酶、DNA Maker、Protein Maker均购自Fermentas（MBI），genome walkingkit购自TaKaRa公司。
3.培养基：
使用的培养基：LB培养基，YPD，YPAD，BMDY，BNNY，MM，MD培养基均参照Invitrogen毕氏酵母操作手册。
4.本发明中所用到的生物化学技术均为本领域中的常规技术。在以下实施例中，除非特殊说明，所有实验操作均按照以下实验手册或文献中的相关章节或部分进行，包括：[美]J.莎姆布鲁克等，分子克隆实验指南；赵永芳等，生物化学技术原理及其应用（第二版）；朱检等，生物化学实验[M]。
5.本发明中所有相关的酶活、酶活力、酶活性均是指Chitinase酶活性，均采用DNS法并按照所述的方法进行测定及计算。
实施例1 Chitinase基因的获得
根据genome walking kit进行操作。
（1）红树林滩涂土样宏基因组的提取：使用OMEGA Soil DNA kit 提取土样宏基因组DNA。
（2）Chitinase保守序列的获取
对NCBI已有的18家族亚家族A的Chitinase基因进行分析，设计简并引物以扩增Chitinase基因保守序列。简并引物序列如下：
ChiA F：5' GGTGGACATCGACTGGGARTWYCC 3'；
ChiA R：5' CCCAGGCGCCGTAGARRTCRTARSWCA 3'。
PCR程序为： 95℃预变性5min；94℃变性30s，50℃退火30s，72℃延伸1min，循环扩增35次；最后72℃延伸10min。扩增结束后取PCR产物进行电泳检测，并回收凝胶中的目标基因。
（3）几丁质酶保守序列文库的构建
PCR产物连接：将纯化后的PCR扩增产物与载体pMD18-T连接，于4℃过夜连接反应。
用化学转化法转化到受体菌Top 10中，在含有100mg/mL Amp的LB平板上37℃培养过夜。挑取多个单克隆菌落分别接种到2ml含有100mg/mL Amp的LB液体培养基中，
几丁质酶基因片段文库阳性重组子的鉴定：利用PCR的方法鉴定阳性重组子，25 uL反应体系中取1 uL培养的菌液直接作为PCR反应的模板，用上述Chitinase简并引物（ChiA F和ChiA R）鉴定重组子。将PCR产物用1.5 %的琼脂糖凝胶电泳分析，检测大小。如果是重组子的话，大小是250 bp。
将鉴定为阳性重组子的克隆进行DNA测序(上海生工科技有限公司)。使用NCBI中的序列比对工具BLASTx与GenBank中的几丁质酶序列有较高相似性的片段序列鉴定为几丁质酶的基因片段。
（4）genome walking 获取目的基因
以获得的Chitinase保守序列为模板，根据genome walking kit进行操作。扩增结束后取PCR产物进行电泳检测，并回收凝胶中的目标基因。
（5） PCR获取目的基因
以由genome walking扩增得到的最终序列为模板，设计上下游引物P1和P2。上、下游引物分别含有SacI和XhoI酶切位点，由上海生工合成，引物序列如下：
P1：5' ttcgagctcCAAGAACCCGTCTATCGAGTC 3'；
P2：5' gtgctcgagTATCGCTGTTGAGCTGATCG 3'。
PCR程序为： 94℃预变性4min；94℃变性30s，52℃退火30s，72℃延伸2min，循环扩增30次；最后72℃延伸10min。扩增结束后取PCR产物进行电泳检测，并回收凝胶中的目标基因。
（5）cDNA亚克隆：
制备好的双链cDNA插入到载体系统pMD18-T上，得到重组质粒pMD18-Chitinase（如图2所示），用化学转化法转化受体菌Top 10，在含有100mg/mL Amp的LB平板上37℃培养过夜。挑取的单克隆菌落接种到2ml含有100mg/mL Amp的LB液体培养基中，37℃ 200rpm培养6-10h，10000rpm离心10min收集菌体，提取质粒，酶切回收目的基因备用（质粒提取和胶回收分别用OMEGA公司的E.Z.N.A. Plasmid Mini Kit I和E.Z.N.A. Gel Extraction Kit试剂盒）。将所得目的基因进行DNA序列测定（Invitrogen公司），并在NCBI数据库中进行比对分析，结果如SEQ ID NO.1-2所示。
由此所得到的Chitinase的编码序列共有1146bp（SEQ ID NO:1），其中第1144-1146位为终止密码子TGA、第1-1146位编码不含信号肽的成熟蛋白，该成熟蛋白含有278个氨基酸（SEQ ID NO:2）。根据在NCBI数据库里同源性比对的结果初步确定所得到的基因片段为Chitinase。
实施例2 Chitinase编码基因在大肠杆菌中的表达和扩增
将实施例1-(5)中所得到的目的基因，与经过到SacI和XhoI双酶切的pET28a(+)质粒连接，得到重组质粒pET28a(+)-Chitinase（如图1所示）。
取10uL构建好的质粒DNA，加入到100uL制备好的感受态细大肠杆菌BL21(DE3)中，摇匀置于冰上，冰浴30min；置于42℃水浴中热击45s；将离心管快速移至冰水混合物中冰浴2min；每管加入800uL LB培养基，用移液器轻吸打散后于37℃摇床上复苏1h（80rpm～200rpm）；离心，4000rpm×5min，除去700uL上清，剩余部分混匀；涂平板（LB-agar平板，含100ug/mLAmp），37℃正置1h后，倒置培养过夜，在抗性平板上生长的为含有重组质粒的阳性克隆子。
取重组大肠杆菌菌株BL21(DE3)，接种于200ml LB培养液中（1000ml三角瓶，100ug/mLAmp），37℃ 200rpm振荡培养1-1.5h，加入IPTG诱导（终浓度为1mmol/L），20℃200rpm再振荡培养10h。取培养液10000rpm离心10min，收集菌体，再加入等体积的无菌水重新悬浮菌体，12000rpm离心10min，取沉淀用1/20体积pH7.4、20mM的Tris-HCl缓冲液悬浮菌体，进行超声波破碎，破碎条件为：60%功率，间隔5s破碎10min，停止10min，再破碎10min。12000rpm离心，收集上清液分析Chitinase活力，并通过SDS-PAGE电泳分析目的蛋白的表达量，结果表明Chitinase的编码基因所编码的Chitinase可在大肠杆菌中表达，且有一定的Chitinase活性，测得酶活力为1 U/ml。
实施例3 重组 Chitinase的纯化
将实施例2所制备的发酵培养液12000rpm离心10min，再用20 mM Tris-HCl缓冲液（pH7.4）重悬细胞，超声破碎。将细胞破碎12000rpm离心10min，收集上清液。将粗酶液用缓冲液A（20mM Tris-HCl，20mM咪唑，500mM NaCl,pH 7.4）稀释5倍，以1mL/min的流速加载到镍柱中，用缓冲液A洗脱弱结合的蛋白至柱平衡，再用缓冲液B（20mM Tris-HCl，200mM咪唑，500mM NaCl,pH 7.4）以1mL/min的流速洗脱目的蛋白，收集洗脱液，SDS-PAGE测定目的蛋白纯度。
纯化完成后，获得的几丁质酶Chitinase比活性从粗酶液的0.14U/mg提高到纯酶的0.63U/mg，纯化倍数为4.23倍，得率为62.3%。SDS-PAGE结果如图2。
实施例4 重组Chitinase生化性质表征
1、重组酶的最适反应Ph和Ph稳定性的测定
重组酶最适反应pH测定：将纯化后的重组酶在50℃条件下，于不同pH（pH 3.0、4.0、5.0、6.0、7.0、8.0、9.0、10.0、11.0）的底物中测酶活，以确定其最适反应pH。
重组酶pH稳定性测定：将纯化好的重组酶在不同pH值（pH 3.0、4.0、5.0、6.0、7.0、8.0、9.0、10.0、11.0）的缓冲液中于37℃下水浴保温60 min后，取酶液于最适pH和温度下分别测定其酶活力。结果显示重组Chitinase最适反应pH为pH6，在pH5-10范围内稳定，实验结果如图3所示。
2、重组酶最适反应温度和热稳定性的测定
重组酶最适反应温度的测定：将纯化后的重组酶在最适pH底物中，于不同温度（30℃、35℃、40℃、45℃、50℃、55℃、60℃）条件下反应测定酶活力，以确定其最适反应温度。
重组酶热稳定性测定：将纯化好的重组酶在不同温度（35℃、40℃、45℃）下水浴保温处理，并在不同时间点2 min，5 min，10 min，20 min，30 min，60 min取样后迅速置于冰上，在最适pH和温度条件下分别测定其酶活力。结果显示Chitinase最适反应温度为45℃，在30℃仍可以保持80%以上的酶活。实验结果如图4所示。
3、重组酶的底物特异性研究
配制浓度为0.5%（w/v）胶体几丁质、细粉几丁质、壳聚糖、羧甲基纤维素钠（CMC-Na），在最适反应条件下测定重组酶活力。以底物为胶体几丁质的酶活设为100%，计算其他底物的相对酶活力。结果显示，Chitinase对胶体几丁质具有严格的底物特异性。实验结果如表1所示。
表1 环境样品宏基因组来源的Chitinase的底物特异性。
SEQUENCE LISTING
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accaacttca acgcgccgct ctacccctcc tcagatgatg cgaacacgga tgacgcgcgc 720
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50 55 60
Ala Asp Ala Pro Tyr Leu Gly Asn Phe His Gln Leu Asn Val Leu Lys
65 70 75 80
Glu Gln Tyr Pro Gln Ile Lys Thr Leu Ile Ser Val Gly Gly Trp Thr
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100 105 110
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Glu Leu Arg Ala Gln Leu Asp Ala Gln Gly Glu Gln Asp Asn Gly Thr
165 170 175
His Tyr Leu Leu Thr Ile Ala Ala Pro Ala Gly Pro Asn Gln Tyr Arg
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Asn Ile Gln Leu Asp Arg Ile His Pro Tyr Leu Asp Trp Ile Asn Val
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Met Thr Tyr Asp Ser His Gly Gly Trp Asp Lys Thr Thr Asn Phe Asn
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Ala Pro Leu Tyr Pro Ser Ser Asp Asp Ala Asn Thr Asp Asp Ala Arg
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Leu Thr Ala Asp Ala Ala Leu Gln Ala Tyr Leu Ala Ala Gly Ile Pro
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Ala Glu Lys Leu Val Met Gly Val Pro Phe Tyr Gly Arg Gly Trp Ala
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Gly Val Pro Asp Thr His Asn Gly Leu Phe Gln Pro Ala Thr Arg Ile
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Pro Ala Gly Thr Trp Glu Pro Gly Ser Phe Gly Tyr Ser Asp Leu Val
290 295 300
Ala Asn Tyr Leu Pro Thr Met Thr Arg Tyr Trp His Ala Glu Ala Ser
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Val Pro Trp Leu Tyr Asp Ala Asp Gln Gln Leu Met Ile Ser Tyr Asp
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Asp Pro Glu Ser Leu Arg Ile Lys Ala Asp Tyr Val Lys Asn Ala Gly
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Claims (9)
1.一种几丁质酶，其特征在于：所述几丁质酶的氨基酸序列如SEQ ID NO.2所示。
2.根据权利要求1所述几丁质酶基因，其特征在于：其基因序列如 SEQ ID NO.1所示。
3.一种如权利要求2所述的几丁质酶基因的重组载体，其特征在于：所述载体是大肠杆菌质粒或酵母质粒。
4.根据权利要求3所述的重组载体，其特征在于：所述载体为pET28a(+)-Chitinase或pPIC9k-Chitinase。
5.一种如权利要求2所述编码几丁质酶基因的细胞，其特征在于：所述细胞选大肠杆菌细胞或酵母细胞；所述细胞由所述载体转化而得。
6.根据权利要求5所述的细胞，其特征在于：所述细胞是包含所述编码几丁质酶基因或用载体转化的大肠杆菌或包含所述编码几丁质酶基因或用所述载体转化的毕赤酵母。
7.一种如权利要求2所述编码几丁质酶基因的制备方法，其特征在于：提取红树林滩涂土样的宏基因组；利用18家族亚家族A的几丁质酶氨基酸保守片段的简并引物，获得250bp序列不同的几丁质酶的编码基因；构建几丁质酶的编码基因文库，选择其中单一克隆， 再通过genome walking获得其全长，并在NCBI数据库中进行比对分析，得到几丁质酶基因。
8.一种如权利要求3所述重组载体的制备方法，其特征在于：采用权利要求2所述基因序列经SacI和XhoI双酶切后与SacI和XhoI双酶切的pET28a(+)载体连接，得到大肠重组表达载体pET28a(+)-Chitinase或采用所述的编码几丁质酶基因序列经SacI和 XhoI双酶切后与SacI和 XhoI双酶切的pPIC9k载体连接，得到酵母重组表达载体pPIC9k-Chitinase。
9.一种如权利要求1所述的几丁质酶的制备方法，其特征在于：培养所述包含编码几丁质酶基因的细胞或所述经转化的细胞，诱导其表达，收获表达产物，并通过镍柱纯化得到了纯酶形式的目的蛋白。
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