CN114231514A - 一种重组褐藻胶裂解酶AlyL7及其应用 - Google Patents
一种重组褐藻胶裂解酶AlyL7及其应用 Download PDFInfo
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- CN114231514A CN114231514A CN202111240122.4A CN202111240122A CN114231514A CN 114231514 A CN114231514 A CN 114231514A CN 202111240122 A CN202111240122 A CN 202111240122A CN 114231514 A CN114231514 A CN 114231514A
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- CN
- China
- Prior art keywords
- alyl7
- alginate
- alginate lyase
- lyase
- recombinant
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Links
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Abstract
本发明涉及一种重组褐藻胶裂解酶AlyL7及其应用，所述褐藻胶裂解酶的氨基酸序列如SEQ ID NO.1所示，其编码基因的核苷酸序列如SEQ ID NO.2所示。所述重组褐藻胶裂解酶属于双功能型褐藻胶裂解酶，其最适反应温度为40℃，最适反应pH为9.0，在0‑25℃、pH 5.0‑10.0的条件时较稳定，对海藻酸钠、聚古洛糖醛酸、聚甘露糖醛酸均具有降解活性。所述重组褐藻胶裂解酶也属于内切酶，其酶解海藻酸钠的终产物包括不饱和1‑5糖，以不饱和2‑3糖为主。所述褐藻胶裂解酶反应速度快，效率高，具有良好的工业化应用潜质。
Description
技术领域
本发明具体涉及一种重组褐藻胶裂解酶AlyL7及其应用，属于生物工程技术领域。
背景技术
褐藻胶是由β-D-甘露糖醛酸（M）与其C5差向异构体α-L-古洛糖醛酸（G）通过1,4-O糖苷键连接形成的线性阴离子多糖聚合物。其因具有高粘度性和凝胶性已被应用于食品、生物材料和制药行业，但其溶解度低限制了褐藻胶的进一步开发应用。褐藻胶经降解后可获得聚合度低、溶解度高的褐藻寡糖，低聚褐藻胶寡糖具免疫调节、抗菌、抗肿瘤、抗炎、抗凝血、促进植物生长、提高植物抗逆性、作为果蔬保鲜剂延长果蔬货架期等多种生理活性。
目前降解褐藻胶的方法主要有三类：物理法、化学法、生物酶法。物理降解法存在降解效率低、成本高、产物聚合度高的缺点，化学降解法过程不易控制、副产物多不利于后续产物的分离纯化，生物酶法是通过褐藻胶裂解酶来特异性的降解褐藻胶，具有反应条件温和、易控制、产物纯度高等优点。相较物理法与化学法，通过生物酶法降解褐藻胶所得的褐藻寡糖聚合度更低且寡糖的非还原性末端含有不饱和双键，具有更好的生物活性。产褐藻胶裂解酶的野生型菌株中含有多种褐藻胶裂解酶，其本质上属于同工酶，但不同的编码基因产生的褐藻胶裂解酶具有不同的底物特异性，因此通过野生菌可控的产生结构均一的褐藻寡糖较为困难且野生型菌株产酶量较低无法工业化应用。通过异源表达手段专一性的表达一种褐藻胶裂解酶，不仅可以获得高产量的基因工程菌而且可以获得结构均一的褐藻寡糖，满足工业生产所需。
发明内容
本发明的目的在于提供一种重组褐藻胶裂解酶AlyL7及其应用。
为实现以上发明目的，本发明采用以下技术方案：
一种来源于黄杆菌的重组褐藻胶裂解酶AlyL7在制备褐藻寡糖中的应用，所述褐藻胶裂解酶的氨基酸序列如SEQ ID NO.1所示，其编码基因alyl7的核苷酸序列如SEQ IDNO.2所示。
上述应用具体为：采用褐藻胶裂解酶AlyL7降解海藻酸钠，形成褐藻寡糖。
上述降解条件为：温度40 ℃，pH 9.0。
一种制备褐藻胶裂解酶AlyL7的方法，所述方法具体为：将褐藻胶裂解酶基因alyl7克隆到表达载体中，得到重组质粒pGEX-4T-1-alyl7；将重组质粒pGEX-4T-1-alyl7转化宿主菌，得到产褐藻胶裂解酶AlyL7的基因工程菌E.coil BL21(DE3)-pGEX-4T-1-alyl7；诱导培养基因工程菌E.coil BL21(DE3)-pGEX-4T-1-alyl7，获得重组表达的褐藻胶裂解酶AlyL7。
本发明的优点在于：
（1）本发明提供的褐藻胶裂解酶AlyL7为多糖裂解酶PL7家族新成员，其核酸序列与已表征的褐藻胶裂解酶CaAly1（ADV51457.1）序列相似度仅为63.4%。
（2）本发明通过基因工程手段成功实现了褐藻胶裂解酶AlyL7的异源表达，通过GST磁珠对AlyL7进行纯化后测得对海藻酸钠的比活力高达16175.17 U/mg，与现有褐藻胶裂解酶相比，具有高酶活力、高催化效率，克服了野生菌催化效率低的缺点，同时褐藻胶裂解酶AlyL7可降解polyG和polyM，因此属于双功能型褐藻胶裂解酶。
（3）本发明提供的褐藻胶裂解酶AlyL7最适反应pH为9.0，为碱性酶，偏好降解海藻酸钠，降解产物为聚合度1-5的褐藻寡糖，已有文献表明低聚褐藻寡糖具有抗肿瘤、抗炎、降血脂、提高植物抗逆性等功效，同时还可应用于食品等领域，因此，褐藻胶裂解酶AlyL7具有良好的应用前景。
附图说明
图1：褐藻胶裂解酶AlyL7与PL7家族已表征褐藻胶裂解酶之间的多序列比对分析。
图2：重组褐藻胶裂解酶AlyL7分离纯化后的SAD-PAGE图，其中泳道M为Marker，泳道1、2为纯化所得褐藻胶裂解酶AlyL7 。
图3：温度对重组褐藻胶裂解酶酶学性质的影响（3A，温度对重组褐藻胶裂解酶AlyL7活力的影响；3B，温度对重组褐藻裂解酶AlyL7稳定性的影响）。
图4：pH对重组褐藻胶裂解酶AlyL7酶学性质的影响（4A，pH对重组褐藻胶裂解酶AlyL7活力的影响；4B，pH对重组褐藻胶裂解酶AlyL7 稳定性的影响）。
图5：金属离子、螯合剂对重组褐藻胶裂解酶AlyL7酶学性质的影响。
图6：重组褐藻胶裂解酶AlyL7的底物偏好性。
图7：重组褐藻胶裂解酶AlyL7的最终降解产物结果（7A，薄层层析结果；7B，阴离子电喷雾离子化质谱结果）。
具体实施方式
下面结合附图和实施例对本发明作进一步的说明解释。此处所描述的具体实施例仅用以解释本发明，并不用于限定本发明。
实施例1：褐藻胶裂解酶AlyL7的来源及序列分析
本实施例中所用到的黄杆菌由福州大学提供。
本发明的褐藻胶裂解酶alyl7基因为以黄杆菌Zobellia sp.基因组通过染色体步移手段扩增获得，此序列含有924个碱基（如SEQ ID NO.2所示），其编码307个氨基酸（如SEQID NO.1所示），将该碱基序列上传至NCBI获得序列登录号MW561203.1，同源性比对结果表明，其与已有褐藻胶裂解酶CaAly1（ADV51457.1）核苷酸序列之间相似度仅为63.4%。根据多序列比对结果表明重组褐藻胶裂解酶AlyL7氨基酸序列包括保守序列R（S/N）E（L/V）R、QIH、YFKAG（V/I）Y（N/P）O（图1），因此褐藻胶裂解酶AlyL7属于多糖水解酶第7家族（PL7）。
实施例2：褐藻胶裂解酶AlyL7的重组表达工程菌构建
以BamH Ⅰ和Xho Ⅰ为酶切位点，设计引物使实例1获得的alyl7基因序列两端带上酶切位点。
正向引物AlyL7F（SEQ ID NO.3）：
5'-CGCGGATCCATGCAGTTTTTAAGCAACT-3'（下划线处为BamH Ⅰ酶切位点）
反向引物AlyL7R（SEQ ID NO.4）：
5'-CCGCTCGAGGTGTATAACTTCTAAATCGT-3'（下划线处为Xho Ⅰ酶切位点）
PCR反应按以下条件进行：94 ℃预变性5 min；94 ℃变性30 s；55 ℃退火20 s；72℃延伸2 min，共进行30个循环，之后72 ℃延伸10 min。PCR产物经1%的琼脂糖核酸电泳后进行切胶回收，获得带酶切位点的目的基因，使用购自TAKATA公司的T/A克隆试剂盒将带酶切位点的目的基因连接至pMD-18T克隆载体上，连接体系及条件如下：胶回收目的条带5 μL；Solution Ⅰ 4.3 μL；pMD-18T 0.7 μL，连接体系置于16 ℃下连接过夜，次日将连接产物转化至E.coil(DH5α)感受态细胞，挑取阳性克隆进行DNA测序。抽提测序成功的pMD-18T-alyl7和pGEX-4T-1表达载体质粒（质粒提取试剂盒购自全式金公司），使用限制性内切酶BamH Ⅰ和Xho Ⅰ（购自TAKARA公司）对两种质粒进行双酶切，酶切产物经1%琼脂糖核酸电泳后切胶回收。使用T4连接酶（购自TAKARA）对胶回收后片段进行连接，16 ℃过夜连接后将连接产物转化至E.coil(DH5α)感受态细胞，挑取阳性克隆进行DNA测序，抽提测序成功的重组表达质粒转化至表达宿主E.coilBL21(DE3)，得到重组褐藻胶裂解酶表达菌，将其命名为E.coil BL21(DE3)-pGEX-4T-1-alyl7。
实施例3：重组褐藻胶裂解酶AlyL7的诱导发酵
将重组菌E.coilBL21(DE3)-pGEX-4T-1-alyl7在含有100 μg/mL Amp+的LB固体培养基平板上进行三区划线，挑取单菌落至5 mL含有100 μg/mL Amp+的LB液体培养基中，37℃摇床培养过夜，得到种子液。按1vol%的接种量将种子液接种至1 L含100 μg/mL Amp+的LB液体培养基中，培养至OD600为0.6-0.8时，向培养基中加入终浓度为0.8 mM的异丙基硫代半乳糖苷（IPTG），20 ℃诱导18 h。4 ℃ 12000 rpm离心15 min收集菌体，使用预冷的40 mLbuffer A（140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4；pH 7.4）进行重悬，超声破碎30 min后，4 ℃ 12000 rpm离心15 min收集上清，上清即为粗酶液。使用3,5-二硝基水杨酸（DNS）法测定粗酶液酶活力，具体操作如下：取120 μL粗酶液，向其中加入80μL 0.75 wt %海藻酸钠（溶剂为：20 mM PB缓冲液，pH 7.0），在30 ℃下孵育3 min，然后加入100 μL DNS试剂沸水浴10 min，后加700 μL超纯水，离心后取上清于OD540下检测其吸光值，根据标准曲线公式(1-1)计算粗酶液酶活力。酶活力单位定义：在上述实验条件下，每分钟催化底物产生1 μg还原糖所需的酶量作为一个酶活力单位（U）。酶活力计算公式为：
式中：
U―酶活力单位U/mL
A1―灭活酶吸光值
A2―重组酶吸光值
实施例4：重组褐藻胶裂解酶AlyL7的分离纯化
使用20 mL buffer A清洗20 mLGSH磁珠（购自海狸生物），将20 mL粗酶液与20 mL清洗后的GSH磁珠于4 ℃下结合1 h，磁性分离，去除上清液；再使用20 mLbuffer A清洗磁珠两次，去除上清，向磁珠中加入5-10 mL buffer B（50 mM Tris-HCl, 10 mM 还原性谷胱甘肽；pH 8.0）以洗脱目标蛋白，磁性分离，收集上清，即得到重组褐藻胶裂解酶AlyL7的纯酶液。取50 μL目标蛋白AlyL7纯酶液，向其中加入等体积的2×蛋白处理液（1.25 mL 5mol/L pH 6.8的Tris-HCl 、2 mL甘油、2 mL 10wt%的SDS 、1 mL β-巯基乙醇、0.5 mL0.1wt%的溴酚蓝混合均匀，加蒸馏水定容至10 mL），100 ℃煮沸10 min后，常温12000 rpm离心5 min，取上清，进行SDS-PAGE电泳，胶浓度为12%，浓缩胶电压为100 V，分离胶电压为120 V。电泳在考马斯亮蓝R-250下染色过夜，使用脱色液脱色后，拍照分析。如图2所示，重组褐藻胶裂解酶AlyL7为61 KDa，与预测结果一致（预测蛋白大小为35 KDa，GST标签蛋白大小为26 KDa）。以20 mM pH7.0的PB缓冲液为溶剂分别配制1wt%海藻酸钠、1wt%聚古洛糖醛酸和1wt%聚甘露糖醛酸作为酶促反应底物，采用DNS法测定重组酶AlyL7对上述三种底物的酶活力（单位：U/mL），通过考马斯亮蓝G-25测定纯化后ALyL7浓度（单位：mg/mL），并计算重组酶AlyL7的比活力（单位U/mg）。经计算所得重组酶AlyL7对1wt%海藻酸钠、1wt%聚古洛糖醛酸、1wt%聚甘露糖醛酸的比活力分别为16175.17 U/mg、8548.94 U/mg和4708.05 U/mg。
实施例5：重组褐藻胶裂解酶AlyL7的酶学性质
（1）温度对重组褐藻胶裂解酶AlyL7活力的影响
取120 μL AlyL7纯酶液，向其中加入80 μL 0.75wt%海藻酸钠（溶剂为：20 mM PB缓冲液，pH 7.0），分别置于20 ℃、25 ℃、30 ℃、35 ℃、40 ℃、45 ℃、50 ℃、55 ℃、60 ℃、65 ℃、70 ℃、75 ℃、80 ℃条件下反应3 min后，采用DNS法测定重组酶的酶活力，以最高酶活力为100%。如图3A所示，重组褐藻胶裂解酶AlyL7最适反应温度为40 ℃。
（2）温度对重组褐藻裂解酶AlyL7稳定性的影响
将AlyL7纯酶液分别置于20 ℃、25 ℃、30 ℃、35 ℃、40 ℃下孵育不同的时间后，采用DNS法在最适反应温度条件下（40 ℃）测定剩余酶活，以未孵育酶活力为100%。如图3B所示，重组褐藻胶裂解酶AlyL7在25 ℃以下可以保持相对稳定，表明重组酶AlyL7属于冷适应性酶。
（3） pH对重组褐藻胶裂解酶AlyL7活力的影响
分别配制底物缓冲液柠檬酸-柠檬酸钠缓冲液（pH 3.0-6.0）、PB缓冲液（pH 6.0-8.0）、Tris-盐酸缓冲液（pH 8.0-9.0）、甘氨酸-氢氧化钠缓冲液（pH 9.0-11.0），分别使用不同pH的底物缓冲液配制0.75wt%海藻酸钠，底物缓冲液的具体配方见下表。采用DNS法，将反应体系置于40 ℃下反应3 min后测定酶活力，以最高酶活力为100%。如图4A所示，重组褐藻胶裂解酶AlyL7的最适反应pH为pH 9.0。
表1 柠檬酸-柠檬酸钠缓冲液配方（pH 3.0-6.0）
表2 PB缓冲液配方（pH 6.0-8.0）
表3 Tris-盐酸缓冲液配方（pH 8.0-9.0）
表4 甘氨酸-氢氧化钠缓冲液配方（pH 9.0-11.0）
（4）pH对重组褐藻胶裂解酶AlyL7 稳定性的影响
将AlyL7 AlyL7纯酶液与上述不同pH的缓冲液等体积混合后，4 ℃下孵育2 h，采用DNS法进行酶活力测定，在最适温度（40 ℃）和最适pH（pH 9.0）条件下检测褐藻胶裂解酶的pH稳定性，以放置前的酶活力设为100%。如图4B所示，重组褐藻胶裂解酶AlyL7在pH 5.0-10.0下有较好的稳定性。
（5）金属离子和螯合剂对褐藻胶裂解酶AlyL7的影响
将80 μL AlyL7 AlyL7纯酶液与120 μL 0.75wt%海藻酸钠（溶剂为：20 mM PB缓冲液，pH 7.0）进行混合后，向其中分别加入终浓度为1 mM、5 mM、10 mM的金属离子或螯合剂，混匀后，置于最适温度下（40 ℃）反应3 min测定酶活力，以未添加任何离子的酶活力作为100%。如图5所示，1 mM的Na+、Zn+、Cu+和10 mM的Fe2+可提高重组酶AlyL7的酶活力，Ca2+、Fe3 +、Mn+、Ba+较大程度地抑制其酶活力，EDTA和SDS均抑制其酶活力。
（6）重组褐藻胶裂解酶的底物偏好性
以0.1M pH 9.0的Tris-HCl为溶剂分别配制1wt%海藻酸钠、1wt% 聚古洛糖醛酸（polyG）、1wt% 聚甘露糖醛酸（polyM）、1wt%卡拉胶、1wt% 琼胶、1wt% 壳聚糖和1wt% 几丁质。采用DNS法，取80 μL AlyL7纯酶液与120 μL底物溶液混合，在最适反应温度（40℃）下对褐藻胶裂解酶AlyL7进行酶活力测定，以酶活力最高值为100%，计算其余底物溶液条件下的相对酶活力。如图6所示，褐藻胶裂解酶AlyL7属于双功能酶，偏好降解海藻酸钠，对卡拉胶有较弱的降解能力，对琼胶、壳聚糖、几丁质无降解能力。
实施例6：重组褐藻胶裂解酶AlyL7的降解终产物
以0.1 M pH 9.0的Tris-HCl为溶剂配制2wt%海藻酸钠。取40 mL 2wt%海藻酸钠，向其中加入1 mg纯化后的褐藻胶裂解酶AlyL7，置于30 ℃摇床进行酶解，每隔6 h加1 mg纯化后的褐藻胶裂解酶AlyL7，降解96 h后煮沸30 min，室温12000 rpm离心15 min后收集上清液，加入终浓度为20wt%的预冷的无水乙醇，4 ℃沉淀过夜。次日在室温下12000 rpm离心15 min后，收集上清液进行旋转蒸发浓缩，向浓缩液中加入5倍体积预冷的无水乙醇，4 ℃下沉淀过夜。第三日在室温下12000 rpm离心15 min后，收集沉淀溶于20 mL去离子水中，在室温下12000 rpm离心15 min，收集上清进行冷冻干燥，即得到褐藻寡糖。使用Superdex G-25对冻干后的褐藻寡糖进行脱盐处理，然后使用薄层层析色谱（TLC）与四级杆飞行质谱（Q-TOF-MS）法分析酶解法产生不饱和寡糖的聚合度。
先将预制的薄层层析板置于90 ℃烘箱烘干1 h。在距离层析板底部1.5 cm除同一水平线上间隔0.8 cm点样，每个样品点样3次，风干后于层析缸中进行展开1 h (展开剂为正丁醇：甲酸：水=4：6：1（v/v/v）)，风干后喷洒显色剂（苯胺2 mL、二苯胺2 g、85%磷酸（v/v）、10 mL、盐酸1 mL，混合均匀后溶于100 mL丙酮，再次混合均匀至完全溶解）。如图7A所示，AlyL7酶解海藻酸钠产生聚合度1-5的褐藻寡糖。
将上述脱盐后的褐藻寡糖配制成10 mg/mL的样品溶液进行Q-TOF-MS检测。结果如图7B所示，酶解产物在负离子模式下，鉴定出5个明显特征峰。其中单糖m/z为175.0267（[ΔDP1-H]-），二糖m/z为351.5625（[ΔDP2-H]-），三糖m/z为264.0478（[ΔDP3-H]2-）、527.0976（[ΔDP3-H]-）、549.0782（[ΔDP3-2H+Na]-）和571.3519（[ΔDP3-3H+2Na]-），四糖m/z为703.1314（[ΔDP4-H]-），五糖m/z为879.165（[ΔDP5-H]-）。
SEQUENCE LISTING
<110> 福州大学
<120> 一种重组褐藻胶裂解酶AlyL7及其应用
<130>
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Val Leu Lys Asn Lys Ser Ala Ser Asp Gln Glu Ile Leu His Glu Asp
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Claims (4)
1.一种来源于黄杆菌的重组褐藻胶裂解酶AlyL7在制备褐藻寡糖中的应用，其特征在于：所述褐藻胶裂解酶的氨基酸序列如SEQ ID NO.1所示，其编码基因alyl7的核苷酸序列如SEQ ID NO.2所示。
2.根据权利要求1所述的应用，其特征在于：所述应用具体为：采用褐藻胶裂解酶AlyL7降解海藻酸钠，形成褐藻寡糖。
3.根据权利要求2所述的应用，其特征在于：所述降解条件为：温度40 ℃，pH 9.0。
4.一种制备如权利要求1所述的褐藻胶裂解酶AlyL7的方法，其特征在于：所述方法具体为：将褐藻胶裂解酶基因alyl7克隆到表达载体中，得到重组质粒pGEX-4T-1-alyl7；将重组质粒pGEX-4T-1-alyl7转化宿主菌，得到产褐藻胶裂解酶AlyL7的基因工程菌E.coilBL21(DE3)-pGEX-4T-1-alyl7；诱导培养基因工程菌E.coil BL21(DE3)-pGEX-4T-1-alyl7，获得重组表达的褐藻胶裂解酶AlyL7。
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