CN113604460A - 一种新型褐藻胶裂解酶by17pv7及其应用 - Google Patents
一种新型褐藻胶裂解酶by17pv7及其应用 Download PDFInfo
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- CN113604460A CN113604460A CN202111029734.9A CN202111029734A CN113604460A CN 113604460 A CN113604460 A CN 113604460A CN 202111029734 A CN202111029734 A CN 202111029734A CN 113604460 A CN113604460 A CN 113604460A
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- by17pv7
- alginate
- alginate lyase
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- lyase
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Abstract
本发明涉及一种新型褐藻胶裂解酶BY17PV7及其应用。褐藻胶裂解酶BY17PV7来源于Microbulbifer sp.BY17,其氨基酸序列如SEQ ID NO.1所示,其编码基因by17pv7的核苷酸序列如SEQ ID NO.2所示。重组褐藻胶裂解酶BY17PV7酶活力可达124.7 U/mg,其最适反应温度为43℃,最适反应pH为8.9,在20‑40℃、pH 7.0‑8.9条件下保持相对稳定,对海藻酸钠、聚甘露糖醛酸、聚古洛糖醛酸均具有降解活性,酶解产生的褐藻寡糖为三糖、四糖,具有抗氧化活性。褐藻胶裂解酶BY17PV7可应用于褐藻寡糖生产及工业、食品及医药等领域。
Description
技术领域
本发明具体涉及一种新型褐藻胶裂解酶BY17PV7及其应用,属于生物工程技术领域。
背景技术
褐藻胶是一种酸性的水溶性多糖,广泛存在于马尾藻、褐藻、龙须菜、海带等海洋藻类植物中,我国沿海地区如山东、福建等省份是其主要产区。褐藻胶是由α-L-古洛糖醛酸(G)和β-D-甘露糖醛酸(M)通过C1和C4上的糖苷键连接起来形成的聚合多糖,根据其组合方式的不同,可分为仅由α-L-古洛糖醛酸(G)聚合而成的共聚多糖(polyG),仅由β-D-甘露糖醛酸(M)聚合而成的共聚多糖(polyM)以及由α-L-古洛糖醛酸(G)和β-D-甘露糖醛酸(M)聚合而成的杂聚多糖(polyMG)。褐藻胶广泛应用于医药、食品、工业等领域。近年来,研究表明,低聚褐藻寡糖具有多种生物活性,如清除自由基、通过促进促进生物体免疫功能实现其抗肿瘤作用、降低血糖及减少脂质作用、促进植物根系生长、提高植物的抗逆性等。并且,低聚G寡糖具有较好的抗病原菌活性。相比于褐藻胶,褐藻寡糖(Alginateoligosaccharides,AOS)具有广阔的应用前景。
目前,褐藻寡糖的制备方法分为物理降解法、化学降解法和生物酶解法。物理法耗时短,过程易控,但降解产生的副产物多,产物的分子量高,能耗高。化学法成本低廉,工艺简单,产物性质稳定但反应过程不可控,环境污染大。相比于上述两种方法,生物酶解法具有专一高效,反应过程温和,操作简单,产物聚合度低,对环境友好等优点,在制备褐藻寡糖方面更具优势。由于从野生菌中分离提取褐藻胶裂解酶过程较为繁琐,且产量低,通过基因工程的手段实现褐藻胶裂解酶的异源表达成为主流研究方向。
因此,提供一种新型的褐藻胶裂解酶基因并实现重组表达,研究其酶学性质,测定制备的褐藻寡糖的抗氧化性,对于海洋资源的开发利用和褐藻寡糖的深入研究具有重要意义。
发明内容
本发明的第一目的是提供一种新型褐藻胶裂解酶BY17PV7及其编码基因。
本发明的第二目的是提供一种新型褐藻胶裂解酶BY17PV7的制备方法。
本发明的第三目的是提供新型褐藻胶裂解酶BY17PV7重组表达质粒和重组基因工程菌株。
本发明的第四目的是提供新型褐藻胶裂解酶BY17PV7在裂解海藻酸钠、多聚古洛糖醛酸、多聚甘露糖醛酸中的应用。
本发明的第五目的是提供新型褐藻胶裂解酶BY17PV7在制备褐藻寡糖中的应用。
为实现上述目的,本发明采用如下技术方案:
一种新型褐藻胶裂解酶BY17PV7,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by17pv7的核苷酸序列如SEQ ID NO.2所示。
一种携带上述褐藻胶裂解酶基因by17pv7的重组表达质粒。
上述重组表达质粒为pGEX-4T-by17pv7。
一种包含上述重组表达质粒的重组基因工程菌株。
上述重组基因工程菌株为BL21-pGEX-BY17PV7。
一种制备上述褐藻胶裂解酶BY17PV7的方法,包括以下步骤:培养上述基因工程菌株BL21-pGEX-BY17PV7,诱导重组褐藻胶裂解酶BY17PV7的表达;通过GST磁珠回收纯化所表达的褐藻胶裂解酶BY17PV7。
上述一种褐藻胶裂解酶BY17PV7在裂解海藻酸钠、聚古洛糖醛酸、聚甘露糖醛酸中的应用。
上述一种褐藻胶裂解酶BY17PV7在制备褐藻寡糖中的应用。
本发明的优点在于:
(1)本发明提供的褐藻胶裂解酶BY17PV7为多糖裂解酶PL7家族新成员,其氨基酸序列与已公开的褐藻胶裂解酶(CP014864.1)序列相似度仅为78%。
(2)本发明提供的制备褐藻胶裂解酶BY17PV7的方法,利用基因工程手段实现该酶的重组表达,可通过GST磁珠进行纯化回收。
(3)本发明提供的褐藻胶裂解酶BY17PV7最适反应pH为8.9,为碱性酶,偏好于降解polyG,降解产物为三糖和四糖,可作为制备褐藻寡糖的工具,在医药、食品及农业等方面进行应用。
附图说明
图1为褐藻胶裂解酶BY17PV7与PL7家族的褐藻胶裂解酶之间的多重序列比对分析。
图2为重组褐藻胶裂解酶BY17PV7分离纯化的SDS-PAGE电泳图;其中,泳道M为marker,泳道1和2为纯化所得褐藻胶裂解酶BY17PV7。
图3为温度对重组褐藻胶裂解酶BY17PV7的影响;其中,3A为温度对重组褐藻胶裂解酶BY17PV7活力的影响,3B为温度对重组褐藻胶裂解酶BY17PV7稳定性的影响。
图4为pH对重组褐藻胶裂解酶BY17PV7的影响;其中,4A为pH对重组褐藻胶裂解酶BY17PV7活力的影响,4B为pH对重组褐藻胶裂解酶BY17PV7的稳定性的影响。
图5为金属离子、SDS、EDTA和NaCl对重组褐藻胶裂解酶BY17PV7的影响;其中,5A为金属离子、SDS和EDTA对重组褐藻胶裂解酶BY17PV7活力的影响,5B为NaCl对重组褐藻胶裂解酶BY17PV7活力的影响。
图6为重组褐藻胶裂解酶BY17PV7的底物偏好性。
图7为薄层层析法检测重组褐藻胶裂解酶BY17PV7酶解海藻酸钠产物。
图8为四级三杆飞行质谱检测重组褐藻胶裂解酶BY17PV7酶解海藻酸钠产物;其中,8A为酶解产物洗脱曲线,8B为Q-TOF-MS检测结果。
图9为重组褐藻胶裂解酶BY17PV7酶解海藻酸钠产生的褐藻寡糖的抗氧化性测定;其中,9A为BY17PV7酶解产生的褐藻寡糖对羟基自由基的清除率,9B为BY17PV7酶解产生的褐藻寡糖对DPPH自由基的清除率,9C为BY17PV7酶解产生的褐藻寡糖对ABTS自由基的清除率,9D为BY17PV7酶解产生的褐藻寡糖的浓度与总还原力的关系。
具体实施方式
为了使本发明所述的内容更加便于理解,下面结合具体实施方式对本发明所述的技术方案做进一步的说明。应当理解,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BY17PV7的来源及序列分析
本实施例中所用到的微泡菌Microbulbifer sp.BY17由福州大学提供。
本发明所述的褐藻胶裂解酶BY17PV7由by17pv7基因表达获得,其氨基酸序列如SEQ ID No.1所示,其编码基因的核苷酸序列如SEQ ID No.2所示。
利用V7保守引物扩增Microbulbifer sp.BY17的保守区片段,并通过SiteFingding PCR扩增得到by17pv7基因的核苷酸序列SEQ ID No.1,该序列全长681bp,编码227个氨基酸,将SEQ ID No.1所示序列上传至NCBI获得序列号KY783478.1。V7保守引物的序列如下:
V7F(SEQ ID No.3):5’-GTTGTTGTCGGNCARATHCAYGCN-3’;
V7R(SEQ ID No.4):5’-TTGACCRTAAGCNCCNGCYTTRAARTA-3’。
通过NCBI中保守结构(http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi)分析表明,褐藻胶裂解酶BY17PV7含有Alginate lyase 2超家族结构域。通过NCBI的Blast比对表明,褐藻胶裂解酶BY17PV7与来源于Microbulbifer thermotolerans strain DAU221的褐藻胶裂解酶(CP014864.1)具有最高相似度(identity),仅为78%。
从Cazy数据库中下载4种PL7家族褐藻胶裂解酶,将上述酶(CP014864.1)及褐藻胶裂解酶BY1PV7的氨基酸序列进行ClustalW比对,比对结果上传至在线网站ESPript3.0(http://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi)进行多重序列比对分析,发现褐藻胶裂解酶BY17PV7含有保守序列“QIH”(图1)。以上结果综合表明重组酶BY17PV7为PL7家族新成员。褐藻胶裂解酶BY17PV7的氨基酸序列如SEQ ID No.4所示。
实施例2 褐藻胶裂解酶BY17PV7的重组表达
以BamH I和Xho I为酶切位点,设计引物使实施例1获得的by17pv7基因序列两端带上酶切位点。
正向引物BY17PV7FB(SEQ ID No.5): 5’-GCGGATCCATGGTATTCCACTGCCCGAT-3’(下划线处为BamH I酶切位点);
反向引物BY17PV7RX(SEQ ID No.6): 5’-CCCTCGAGGTCGGCGTAGCCGGTGTGGCT-3’(下划线处为Xho I酶切位点)。
PCR反应按照以下条件进行:95℃预变性5 min;95℃变性30 s;50℃退火20s;72℃延伸60 s,共进行30个循环,之后72℃延伸10 min。PCR反应所用的PCR SuperMix购自北京全式金生物技术有限公司。
使用购自宝生物工程(大连)有限公司的限制性内切酶BamHⅠ和XhoI对PCR产物及质粒pGEX-4T-1进行双酶切,琼脂糖凝胶电泳后回收目的片段。酶切所用酶和底物反应体系参照宝生物工程(大连)有限公司提供的产品说明执行。
双酶切处理后的PCR产物和pGEX-4T-1质粒载体参照DNA连接酶说明书(宝生物工程(大连)有限公司)进行连接;连接产物转化至大肠杆菌DH5α,涂布在含有终浓度50 ug/ml氨苄青霉素(Amp+)的LB培养基固体平板上,37℃温箱中培养12-16 h后,挑取阳性克隆子,获得重组质粒pGEX-4T-by17pv7;将重组质粒转化至大肠杆菌BL21,重组大肠杆菌菌株命名为BL21-pGEX-BY17PV7,保存在-80℃备用。
实施例3褐藻胶裂解酶BY17PV7的诱导发酵
复苏重组褐藻胶裂解酶表达菌BL21-pGEX-BY17PV7,按1%体积比接种量转接至1L含终浓度50 ug/ml Amp+的LB液体培养基中,于37℃培养4 h后,加入培养基终浓度0.4 mM的异丙基硫代半乳糖苷(isopropyl-β-D-galactopyraNoside,IPTG),20℃下诱导18 h。10000 rpm、4℃离心8 min,预冷的蒸馏水重悬菌体,10000 rpm、4℃离心15 min,去离子水重悬(重复三次),将培养基溶液完全去除。沉淀用buffer1溶解,超声破碎35 min。然后在12000 rpm、4℃条件下离心15 min,收集上清,得到粗酶液。
通过3,5-二硝基水杨酸(DNS)法测定释放的还原糖的量来测定褐藻胶裂解酶的酶活力。取120 μL粗酶液加入到80 μL体积分数为0.75%的海藻酸钠溶液(pH=7.5)中,30℃金属浴30 min,然后立即加入100 μL的DNS试剂并煮沸10min来终止反应,冷却至室温后,在540 nm处监测还原糖的释放量。酶活力单位定义为:每分钟降解海藻酸钠底物产生1 μg还原糖所需的酶量,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 褐藻胶裂解酶BY17PV7的分离纯化
本实施例中的磁性分离利用磁力架进行。将能够结合待分离样品的GST磁珠加入到离心管中含有待分离样品的溶液中,充分混匀,并放置在磁力架上,此时,磁性材料在磁力架的磁铁作用下,被吸附到靠磁力架的磁铁一侧的管壁上,然后用移液器吸走未吸附的溶液,进而实现待分离样品与其他样品的分离。
取40 ml GST磁珠(购自海狸生物)于离心管中,磁性分离后,去除上清液。加入40ml的buffer1于离心管,震荡重悬,磁性分离并去除(重复两次)。向离心管中加入40 ml粗酶液,震荡15 s后,将离心管置于旋转混合仪上,旋转1 h,磁性分离,去除上清液。加入40 ml的buffer 1于离心管,震荡重悬,磁性分离并去除(重复两次)。加入10 mL buffer 2进行洗脱,置于旋转混合仪上,室温旋转10 min,磁性分离,收集上清液,上清液为纯化蛋白液。
取50 μL纯化蛋白液,加入等体积的蛋白处理液(1.25 mL 5 mol/L pH6.8的Tris-HCl 、2 mL甘油、2 mL体积分数为10%的SDS 、1 mL β-巯基乙醇、0.5 mL体积分数为0.1%的溴酚蓝混合,加蒸馏水定容至10 mL),混匀后煮沸10 min,进行SDS-PAGE电泳。如图2所示,重组蛋白大小为48 kDa,与预测结果一致(预测蛋白大小为22 kDa,载体pGEX-4T-1含有大小为26 kDa的谷胱甘肽巯基转移酶标签)。
实施例5 重组褐藻胶裂解酶BY17PV7的酶学性质
(1)温度对褐藻胶裂解酶BY17PV7的影响
①取120 μL粗酶液加入到80 μL体积分数为0.75%的海藻酸钠溶液(pH=7.5)中,分别放置在35,40,43,45,50,55℃水浴锅中孵育30 min,测定酶活力,以最高酶活力为100%,计算其余温度条件下重组酶的相对酶活力。如图3A所示,重组褐藻胶裂解酶BY17PV7最适反应温度为43℃。
②分装粗酶液至不同的EP管中,于20,30,40,50,60℃条件下孵育1 h,立即冰浴5min,然后取120 μL与80 μL体积分数为0.75%的海藻酸钠溶液(pH=7.5)混合,震荡混匀后在43℃下反应30 min,测定酶活力,设未热孵育的酶相同条件下测定的酶活力为100%,测定重组酶的相对残余酶活力。如图3B所示,重组褐藻胶裂解酶BY17PV7在20-40℃条件下保持相对稳定,表明重组酶BY17PV7为冷适应性酶,热稳定性较差。
(2)pH对褐藻胶裂解酶BY17PV7的影响
①取120 μL粗酶液加入到80 μL体积分数为0.75%的海藻酸钠溶液中,测定使用的底物缓冲液为:Citrate(pH=4-6)、Na2HPO4-NaH2PO4(pH=6-8)、Tris-HCl(pH=7-8.9)、Gly-NaOH(pH=9-10),43℃下反应30 min,测定酶活力,以最高酶活力为100%,计算其余pH条件下重组酶的相对酶活力。底物缓冲液的配置见表1和表2。如图4A所示,褐藻胶裂解酶BY17PV7的最适反应pH为8.9。
表1 缓冲溶液配置表(pH 4-8)
表2 缓冲溶液配置表(pH 7-10)
②分装粗酶液,按照体积比为1:1加入上述底物缓冲液中,4℃条件下孵育1h,孵育结束后,取120 μL加入到80 μL体积分数为0.75%的海藻酸钠溶液(pH=8.9)中,43℃下反应30 min,测定酶活力,以未经处理的酶活性定义为100%,计算重组酶的pH稳定性。如图4B所示,重组褐藻胶裂解酶BY17PV7在pH 7.0-8.9条件下保持相对稳定,表明重组酶BY17PV7为碱性酶。
(3)金属离子、SDS和EDTA对褐藻胶裂解酶BY17PV7的影响
①取120 μL粗酶液加入到80 μL体积分数为0.75%的海藻酸钠溶液(pH=7.5)中,分别加入终浓度为10 mM或100 mM的不同金属离子及金属螯合剂,混匀后于43℃孵育30 min,测定酶活力。如图5A所示,终浓度为10 mM的Na+、K+、Ca2+、Fe3+、Co2+、Mn2+提高BY17PV7的酶活力,终浓度为10 mM及100 mM的Mg2+、Zn2+、Ba2+、Cu2+、EDTA和SDS均抑制BY17PV7的酶活力。
②取120 μL粗酶液加入到80 μL体积分数为0.75%的海藻酸钠溶液(pH=7.5)中,加入终浓度为20、40、60、80、100、120 mM的NaCl,混匀后于43℃孵育30 min,测定酶活力,设未加NaCl体系测得的酶活力为100%,计算不同终浓度NaCl对重组褐藻胶裂解酶BY17PV7酶活力的影响。如图5B所示,终浓度为80 mM的NaCl使BY17PV7的活性增加到210%,显著促进BY17PV7的酶活力。
(4)褐藻胶裂解酶BY17PV7的底物偏好性
取实施例4 获得的纯化目的蛋白溶液100 μL与等量的底物溶液混合,底物溶液及其体积分数分别为:海藻酸钠溶液2%、聚古洛糖醛酸2%、聚甘露糖醛酸2%。于43℃下孵育30min后,测定酶活力,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BY17PV7属于双功能酶,相比于polyM,褐藻胶裂解酶BY17PV7对海藻酸钠和polyG具有更高的偏好性,降解效率更高。
使用考马斯亮蓝法测定实施例4获得的蛋白溶液浓度,采用Lineweaver-Burk法,以反应速率倒数为纵坐标,底物浓度倒数为横坐标作图,计算K m 、V max 、K cat 以及K cat /K m ,结果见表3,BY17PV7对海藻酸钠、polyM、polyG的K m 值分别为2.87,3.5,1.5,而K m 值越低,酶对底物的亲和力越高,故BY17PV7对polyG具有相对较好的亲和力。
表3重组酶BY17PV7的酶动力学参数
实施例6 褐藻胶裂解酶BY17PV7酶解产物的制备及TLC
(1)在20 mL体积分数为2%的海藻酸钠溶液(pH=7.5)中加入等体积纯化的目的蛋白溶液于40℃下孵育,酶解24 h后,煮沸20 min,12000 rpm离心20 min,取上清液进行旋蒸浓缩,浓缩液中加入等体积体积分数为20%的乙醇溶液醇沉过夜,12000 rpm离心15 min,取上清液旋蒸浓缩,加入2倍体积的无水乙醇,醇沉过夜,12000 rpm离心15 min后收集沉淀,沉淀用去离子水溶解,12000 rpm离心15 min,去除不溶物,将上清液冻干,即得到褐藻寡糖。
(2)制备浓度为50 mg/ml褐藻寡糖溶液,将预制的薄层层析板裁剪为合适的大小,85℃烘干1 h,在距离层析板的下方1.5 cm处同一水平线上间隔1 cm点样,每个样点样3次,风干后于层析缸中展开45 min,90℃烘箱中放置8 min显色。展开剂为正丁醇:甲酸:水=4:6:1(v/v/v),室温下风干后喷洒显色剂。显色剂为苯胺2 mL、二苯胺2 g、体积分数为85%的磷酸10 mL、体积分数为38%的盐酸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7所示,BY17PV7酶解海藻酸钠产生的褐藻寡糖为三糖和四糖。
实施例7 褐藻胶裂解酶BY17PV7酶解产物的Q-TOF-MS
将实施例6获得的褐藻寡糖制备成100 mg/mL溶液,流动相为超纯水,待柱平衡后,按照0.5 mL/min的速度上样,上样量1 mL,每5 min收集一次,检测235 nm下波长,合并收集到的相同组分并冻干。如图8A所示,随着洗脱时间的增加,洗脱液在A235处见明显峰值,合并收集到的洗脱液,浓缩冻干。将脱盐冻干后的AOS制备成浓度为50 mg/ml的样品溶液,进行Q-TOF-MS检测,结果如图8B所示,酶解产物在负离子模式下,鉴定出4个明显特征峰,其中三糖m/z为527.8170([DP3-H]-)、549.0744([DP3-2H+1Na]-)和571.0554([DP3-3H+2Na]-),四糖m/z为703.8521([DP4-H]-),表明BY17PV7通过内切的方式酶解海藻酸钠,产生不饱和三糖和四糖。
实施例8 褐藻寡糖的抗氧化性分析
(1)取实施例6获得的褐藻寡糖,制备浓度为0.5、1、2、3、4 mg/mL的溶液,取0.1 mL上述不同浓度的褐藻寡糖溶液于试管,分别加入0.1 mL硫酸亚铁(9 mmol/L)、0.1 mL水杨酸(9 mmol/L)、0.1 mL过氧化氢(8.8 mmol/L),去离子水补足至1 mL,震荡混匀后37℃反应15 min,然后测定510 nm处的吸光值As,对照组为去离子水代替样品同样处理并测定510nm处的吸光值Ad,测定不加过氧化氢的样品(以去离子水代替)处理后的吸光值Ah,按照下列公式计算AOS的羟自由基清除率。如图9A所示,BY17PV7酶解产生的AOS对羟自由基具有清除作用。
(2)避光环境下分别取1 mL 0.5、1、2、3、4 mg/mL褐藻寡糖溶液于棕色离心管中,向其中加入1 mL 0.15 mmol/L的DPPH溶液,暗室反应30 min后,于517 nm下测定吸光值As。对照组为去离子水于同样条件下反应30 min后测定吸光值Ad,按照下列公式计算AOS的DPPH自由基清除率。如图9B所示,BY17PV7酶解产生的AOS对DPPH具有清除作用。
(3)分别取0.1 mL 0.5、1、2、3、4 mg/mL褐藻寡糖,向其中加入0.9 mL稀释后ABTS溶液(0.384 g ABTS溶于0.1 L超纯水中,0.134 g过硫酸钾溶于0.1 L超纯水,ABTS溶液和过硫酸钾溶液1:1体积比混匀后,避光储存12 h,使用时用超纯水稀释20倍),30℃反应15min后,测定734 nm处的吸光值As。对照组为去离子水于同样条件下反应15 min后测定吸光值Ad,按照下列公式计算AOS的ABTS自由基清除率。如图9C所示,BY17PV7酶解产生的AOS对ABTS自由基具有清除作用。
(4)分别取0.1 mL 0.5、1、2、3、4 mg/mL褐藻寡糖,然后依次加入0.1 mL pH6.60.1 mol/L磷酸盐溶液、0.1 mL质量分数为1%的铁氰化钾,震荡混匀后于50℃水浴锅中反应20 min,取出后依次加入0.1 mL体积分数为10%的三氯乙酸、0.5 mL质量分数为0.1%的氯化铁,混匀后测定700 nm处的吸光值As。对照组为去离子水于同样条件下反应后测定吸光值Ad,构建不同AOS浓度与总还原力的关系。如图9D所示,BY17PV7酶解产生的AOS对Fe3+具有还原力。
以上所述仅为本发明的较佳实施例,凡依本发明申请专利范围所做的均等变化与修饰,皆应属本发明的涵盖范围。
SEQUENCE LISTING
<110> 福州大学
<120> 一种新型褐藻胶裂解酶BY17PV7及其应用
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Claims (8)
1.一种新型褐藻胶裂解酶BY17PV7,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by17pv7的核苷酸序列如SEQ ID NO.2所示。
2.一种携带如权利要求1所述褐藻胶裂解酶基因by17pv7的重组表达质粒。
3.根据权利要求2所述的重组表达质粒,其特征在于:所述重组表达质粒为pGEX-4T-by17pv7。
4.一种包含如权利要求3所述重组表达质粒的重组基因工程菌株。
5.根据权利要求4所述的重组基因工程菌株,其特征在:所述重组基因工程菌株为BL21-pGEX-BY17PV7。
6.一种制备如权利要求1所述的褐藻胶裂解酶BY17PV7的方法,其特征在于:包括以下步骤:培养重组基因工程菌株BL21-pGEX-BY17PV7,诱导重组褐藻胶裂解酶BY17PV7的表达;通过GST磁珠回收纯化所表达的褐藻胶裂解酶BY17PV7。
7.如权利要求1所述的褐藻胶裂解酶BY17PV7在裂解海藻酸钠、聚古洛糖醛酸、聚甘露糖醛酸中的应用。
8.如权利要求1所述的褐藻胶裂解酶BY17PV7在制备褐藻寡糖中的应用。
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