CN104862294B - 一种β-琼胶酶及其应用 - Google Patents
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Abstract
本发明的目的是提供一种β‑琼胶酶，其氨基酸序列为SEQ ID NO：1；编码上述β‑琼胶酶的基因，其一种核苷酸序列为SEQ ID NO：2。本发明的β‑琼胶酶酶能够特异性地降解琼胶产生唯一的降解产物新琼二糖。本发明的大肠杆菌重组株YM01‑5/pET24a(+)、BL21(DE3)所表达的重组β‑琼胶酶，表达水平高，易于纯化，所以通过本发明可以大量生产重组β‑琼胶酶，成本低。
Description
技术领域
本发明属于功能基因筛选技术领域，具体涉及一种β-琼胶酶及其应用。
背景技术
琼胶广泛存在于江蓠、石花菜等红藻的细胞壁，是一种重要的海藻多糖。琼胶结构复杂，由1,3连接的β-D-半乳吡喃糖和1,4连接的3,6-内醚-α-L-半乳吡喃糖残基反复交替连接组成骨架，并包含硫酸基、甲基等取代基。琼胶酶是指一类能够降解琼胶，生成琼胶寡糖的糖苷水解酶，其主要来源是海洋细菌，少部分来源于陆地环境中的细菌和海洋软体动物。琼胶酶分类方式多样，根据琼胶酶降解琼脂糖时作用的糖苷键和产物的不同，琼胶酶可分为两大类：α-琼胶酶和β-琼胶酶。α-琼胶酶作用于琼脂糖的α-1,3糖苷键，生成琼寡糖；β-琼胶酶作用于β-1,4糖苷键，降解产物为新琼寡糖。根据序列相似性，β-琼胶酶通常被划分到四个糖苷水解酶家族(GH，GlucosideHydrolase)：分别是GH16、GH50、GH86和GH118家族；而α-琼胶酶则属于GH96家族。根据作用方式不同，可以将琼胶酶分类为内切酶和外切酶。通常情况下，内切酶(大多数的GH16、GH86和GH118家族的琼胶酶)作用于多糖长链的内部，以较大聚合度的寡糖分子为降解的终产物；而外切酶(大多数的GH50家族的琼胶酶)作用于多糖或高聚合度寡糖的两端，产生低聚合度的寡糖或单糖供细胞进一步的降解利用，在多糖的降解过程中起重要作用。
琼胶酶降解琼胶所产生的琼胶寡糖具有多种生理活性，具有抑菌性，并且具有明显的抗肿瘤和免疫增强效应。新琼二糖对肠道菌群的生长还具有益生元效应。利用琼胶酶降解琼胶生产琼胶寡糖具有专一性和高效性、条件温和、产物易于回收等特点，是未来获取琼胶寡糖的主要方向。除此以外，利用琼胶酶降解琼脂糖凝胶回收胶条中的DNA或RNA、降解海藻细胞壁以制备原生质体、水解海藻多糖以分析多糖结构等，也是琼胶酶在分子生物学研究中的重要应用。由于能源危机和全球变暖等气候问题，生物乙醇作为一种可持续的生物燃料成为现在的研究热点。利用琼胶酶和α-新琼寡糖降解酶将结构复杂的琼胶多糖转化成单糖，再通过糖化作用和发酵作用将单糖转化为酒精。因此，将红藻高效地转化为生物酒精也是琼胶酶的一个重要应用。
发明内容
本发明的目的是提供一种β-琼胶酶及其应用，从而弥补现有技术的不足。
本发明提供一种β-琼胶酶，包含有
1)氨基酸序列为SEQ ID NO：1的β-琼胶酶；
2)在1)进行取代、缺失、添加一个或几个氨基酸形成的，且具有1)中酶学性质的β-琼胶酶；
编码上述β-琼胶酶的基因，其一种核苷酸序列为SEQ ID NO：2；
本发明还提供一种重组大肠杆菌，转化有携带上述基因的表达质粒。
上述的重组大肠杆菌，为大肠杆菌YM01-5(Escherichia coli YM01-5)，于2015年3月25日保藏于中国武汉武汉大学的中国典型培养物保藏中心，保藏编号为CCTCC NO:M2015164。
本发明的β-琼胶酶5用于制备新琼寡糖，例如新琼二糖。
本发明的β-琼胶酶酶能够特异性地降解琼胶产生唯一的降解产物——新琼二糖。本发明的大肠杆菌重组株YM01-5/pET24a(+)、BL21(DE3)所表达的重组β-琼胶酶，表达水平高，易于纯化，所以通过本发明可以大量生产重组β-琼胶酶，成本低。
附图说明
图1：薄层层析检测琼胶酶YM01-5的降解产物图，
图2：重组琼胶酶YM01-5降解产物(新琼二糖)的离子肼质谱图。
具体实施方式
下面结合实例对本发明的方法做进一步说明。
实施例1：β-琼胶酶基因YM01-5的获取
嗜琼胶卵链菌(Catenivulum agarivorans gen.nov.sp.nov.)YM01T由本实验室筛选、鉴定、保藏，通过对其全基因组进行测序分析得到15个琼胶酶基因及其基因序列，包括13个β-琼胶酶基因和2个α-琼胶酶基因，YM01-3是其中的一个β-琼胶酶基因。利用生物学软件Primer5.0设计上游引物(5’-GGATCCGATGAAATATCTAGATTACGGTTATAGCC-3’)和下游引物(5’-CCCTCGAGTTTGCTGTCAGCCTGCACTTG-3’)以基因组DNA为模板，进行PCR反应，PCR反应组成如下(10μl反应体系)：ddH2O 10.5μl、上游和下游引物各0.5μl，DNA模板1μl、2×MasterMix 12.5μl。反应条件为：94℃预变性5min，94℃变性1min，64℃退火1min，72℃延伸1.5min，72℃终延伸10min，共30个循环。反应结束后，将PCR产物回收得到β-琼胶酶基因YM01-5，其具体核苷酸序列为SEQ ID NO：2，其翻译的酶的氨基酸序列为SEQ ID NO：1。YM01-5和来自于Cellvibrio sp.BR的agarase(WP_007640756.1)(相似度63％)、Glaciecola agarilytica的beta-agarase B(WP_008306471.1)(相似度59％)，Thalassomonas agarivorans的beta-agarase(AGT98631.1)(相似度57％),Saccharophagus degradans 2-40的Exo-beta-agarase(4BQ2)(相似度58％)，Alteromonassp.E-1的beta-agarase(BAE97587.1)(相似度62％)。上述结果表明本发明筛选得到了一个新型的β-琼胶酶基因。
实施例2：大肠杆菌表达载体YM01-5/pET24a(+)的构建
将PCR产物和表达载体pET24a(+)分别用BamH I和Xhol I双酶切，酶切体系(20μl)如下：
反应体系1：ddH2O 8μl，PCR产物8μl，BamH I1μl，Xhol I1μl，10×Buffer 2μl
37℃酶切1小时，电泳后分别回收两个目的片段2409bp和5.3Kb，二者分别与β-琼胶酶YM01-5基因全长片段和表达载体PET24a(+)片段大小相同，用DNALigation Kit连接，连接体系(10μl)如下：SolutionⅠ5μl，DNA片段1.5μl，pET24a(+)载体1.1μl，ddH2O 2.4μl。16℃连接16小时即可获得大肠杆菌表达载体YM01-5/pET24a(+)，用于转化大肠杆菌BL21(DE3)。
实施例3:大肠杆菌重组株YM01-5/pET24a(+)/BL21(DE3)的构建
向200μlE.coliBL21感受态细胞中加入10μlYM01-5/pET24a(+)连接液，冰浴30min，42℃90s，冰浴2min，加入LB培养基800μl，37℃振荡培养1小时。菌液涂布于含有100μg/ml卡那霉素的LB平板上，37℃培养12-14h。挑取白色菌落提取质粒并做双酶切检测，酶切体系(10μl)如下：ddH2O 4μl，YM01-5/pET24a(+)质粒DNA 4μl，BamH I 0.5μl，Xhol I 0.5μl，10×Buffer 1μl。琼脂糖凝胶电泳中出现2409bp特异带者为阳性转化克隆。
实施例3：重组β-琼胶酶的制备
将重组表达工程菌YM01-5/pET24a(+)/BL21(DE3)接种至含有终浓度为100μg/ml的硫酸卡那霉素的LB液体培养基中，于37℃培养箱中150rpm振荡培养过夜。将过夜培养的菌液转接至新鲜LB液体培养基中，并加入终浓度相同的硫酸卡那霉素或者氨苄青霉素，于37℃培养箱中150rpm振荡培养，至细菌达到对数生长期，即OD600值在0.4～0.5之间。向菌液中加入终浓度为0.1mM的IPTG进行诱导表达，于16℃培养箱中150rpm振荡培养12-24h。将250ml发酵液中离心(4℃ 12,000rpm离心10min)获得的菌体用12.5ml的1×Ni柱结合缓冲液重悬。破碎菌体，离心收集上清。用灭菌后的0.22mm的滤器过滤收集的破碎上清，待用。将步骤1)中获得的破碎上清加入层析柱中，冰浴环境中水平震荡1h，并不断上下颠倒以便重组蛋白与Ni琼脂糖充分结合。用25ml的1×Ni柱洗涤缓冲液1(20mM Tris-HCl,pH8.0；20mM咪唑；0.5M NaCl；0.1％Tween X-100)洗涤，再用1×Ni柱洗涤缓冲液2(20mM Tris-HCl,pH8.0；50mM咪唑；0.5M NaCl)、1×Ni柱洗涤缓冲液3(20mM Tris-HCl,pH8.0；100mM咪唑；0.5M NaCl)、1×Ni柱洗涤缓冲液4各5ml(20mM Tris-HCl,pH8.0；150mM咪唑；0.5M NaCl)洗涤，用1×Ni柱洗涤缓冲液5(20mM Tris-HCl,pH8.0；250mM咪唑；0.5M NaCl)收集洗涤液。透析收集所得的样品，利用SDS-PAGE检测纯化所得的重组蛋白的纯度。用Bradford法测定目的蛋白的浓度，分装后将蛋白冻于-20℃保存以备用。
实施例4：重组β-琼胶酶活性检测及其酶学性质
取20μl纯化蛋白到2.5％的琼脂糖板上，将对照组同样处理。于37℃培养箱中正置3个小时后，加入卢戈氏碘液，30s后倒掉，然后用卢戈氏碘液染色，结果可在棕色背景上看到明显的透明圈，说明纯化的重组酶具有琼胶降解活性。用DNS试剂法测得此琼胶酶的酶学性质如下：
1、最适温度为37℃
2、温度稳定性：0℃-100℃下放置30min，琼胶酶YM01-5在0℃-37℃能保持80％以上的活性。
3、最适pH为9.0
4、pH稳定性：在pH6-10之间保持较高的pH稳定性，在该范围的pH缓冲液中放置12h后，YM01-5仍能保持80％以上的酶活力不同离子对酶活的影响：SDS、Ni2+对重组琼胶酶YM01-5有很强的抑制作用(>80％)。Na+、K+、和Urea在低浓度(1mM)下对重组琼胶酶的活力具有轻微的抑制作用，在高浓度(10mM)下具有明显的促进作用。Mg2+和Fe3+对YM01-5的活性具有促进作用，且随着浓度升高促进作用更加明显。除此以外，1mM的Cu2+和EDTA对重组琼胶酶YM01-5的活性具有促进作用，但是高浓度的Cu2+和EDTA(10mM)对其活性有抑制作用酶促动力学参数：Km＝222.2mg/ml；Vmax＝20.8U/mg
实施例5：表达产物用于制备新琼寡糖
在170μl琼脂糖溶液中(pH＝8，浓度为0.25％)加入30μl重组琼胶酶酶液50℃温浴不同时间(5min，10min，15min，30min，1h，3h，6h，18h，24h)后离心，取上清、点样于薄层层析板，
，用风筒吹干，置于自制层析缸中展层。展开剂配方为正丁醇：冰乙酸：水＝2:1:1(体积比)。待前沿线到达离层析板顶端1cm处时停止展层，烘箱中干燥。最后往层析板喷洒显色剂(2％二苯胺丙酮溶液5ml，2％苯胺丙酮溶液5ml，三氯乙酸5g)，105℃烘箱中显色。此重组琼胶酶降解的琼脂糖的产物只有一种，且产物浓度随着时间的延长而增大，因此推测YM01-5是一种外切酶(见图1)。
使琼胶酶作用于20mM Tris-HCl缓冲液(pH 8.0)0.25％琼脂糖溶液24h。12000rpm离心10min获得上清，进行柱分离层析，获得反应产物，冻干后进行质谱分析，确定终产物为新琼二糖(见图2)。
Claims (2)
1.氨基酸序列为SEQ ID NO：1的β-琼胶酶在制备新琼寡糖中的应用。
2.如权利要求1所述的应用，其特征在于，所述的新琼寡糖为新琼二糖。
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0743363A2 (en) * | 1995-04-27 | 1996-11-20 | Kabushiki Kaisha Yakult Honsha | Novel beta-agrase and microorganism generating the same |
| CN103194420A (zh) * | 2013-04-09 | 2013-07-10 | 中国海洋大学 | 一种β-琼胶酶及重组表达菌株 |
| CN103194435A (zh) * | 2013-04-09 | 2013-07-10 | 中国海洋大学 | 一种β-琼胶酶及其应用 |
| CN104152427A (zh) * | 2014-08-12 | 2014-11-19 | 山东大学 | 一种外切型琼胶酶及其编码基因与应用 |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0743363A2 (en) * | 1995-04-27 | 1996-11-20 | Kabushiki Kaisha Yakult Honsha | Novel beta-agrase and microorganism generating the same |
| CN103194420A (zh) * | 2013-04-09 | 2013-07-10 | 中国海洋大学 | 一种β-琼胶酶及重组表达菌株 |
| CN103194435A (zh) * | 2013-04-09 | 2013-07-10 | 中国海洋大学 | 一种β-琼胶酶及其应用 |
| CN104152427A (zh) * | 2014-08-12 | 2014-11-19 | 山东大学 | 一种外切型琼胶酶及其编码基因与应用 |
Non-Patent Citations (3)
| Title |
|---|
| "hypothetical protein[Catenovulum agarivorans]"，Accession Number：WP_016957584;genbank;《GenBank》;20130627;第1页 * |
| "Overexpression and Characterization of a Novel Thermostable β-Agarase YM01-3, from Marine Bacterium Catenovulum agarivorans YM01T";Fangyuan Cui et al.;《Mar.Drugs 》;20140512;第12卷;第2731-2747页 * |
| "嗜琼胶卵链菌β-琼胶酶基因YM01-3的克隆表达及酶学性质研究";董素洁;《中国优秀硕士学位论文全文数据库 农业科技辑》;20140315(第03期);前言第2段、第2页第1段、第24页第1.7.1节、第31-32页第1.9.7节和第33页表2-1 * |
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