CN107828809A - 寡褐藻胶裂解酶OalC6的制备方法与应用 - Google Patents

寡褐藻胶裂解酶OalC6的制备方法与应用 Download PDF

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CN107828809A
CN107828809A CN201711282841.6A CN201711282841A CN107828809A CN 107828809 A CN107828809 A CN 107828809A CN 201711282841 A CN201711282841 A CN 201711282841A CN 107828809 A CN107828809 A CN 107828809A
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陈雪红
李尚勇
韩彦弢
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Abstract

本发明公开了一种外切型寡褐藻胶裂解酶及其编码基因与应用。本发明从海洋细菌Cellulophaga sp.SY116中克隆得到新的寡褐藻胶裂解酶基因OalC6，大小2,328 bp，编码775个氨基酸，属于多糖裂解酶PL‑6家族。将该基因在大肠杆菌中进行表达和纯化，得到重组OalC6，分子量大小约为85.9kDa。该酶对褐藻酸钠和褐藻寡糖具有活性，对古洛糖醛酸片段（polyG）具有偏好性。本发明的寡褐藻胶裂解酶在生产生物乙醇开发新能源方面具有潜在的应用价值。

Description

寡褐藻胶裂解酶OalC6的制备方法与应用
技术领域
本发明属于生物工程领域，涉及一种寡褐藻胶裂解酶OalC6的制备方法及应用。具体地，本发明涉及一种利用重组大肠杆菌制备寡褐藻胶裂解酶OalC6的方法及其应用。
背景技术
我国褐藻胶产量位居全球第一。褐藻胶是多种海藻的细胞壁成分，其基本组成单元为单糖，是由互为差向异构体的甘露糖醛酸（M）和古洛糖醛酸（G）通过1,4糖苷键连接而成的线性多糖。根据其结构特点，褐藻胶多糖可分为三种片段，由多聚甘露糖醛酸构成的PolyM片段、多聚古洛糖醛酸构成的PolyG片段和甘露糖醛酸和古洛糖醛酸交替排列而成的PolyMG片段。
褐藻胶是一种重要的海洋多糖类物质，为多种微生物提供能源物质。绝大多数的海洋细菌通过以下途径进行褐藻胶的利用：首先，细菌分泌至胞外的内切的褐藻胶裂解酶将大分子褐藻胶降解为小分子褐藻胶寡糖；褐藻胶寡糖通过细菌的细胞膜进入细菌体内，在外切的寡褐藻胶裂解酶作用下降解为单糖；单糖经非酶催化作用形成α-酮酸，然后进入ED（Entner-Doudoroff）途径或类似ED途径进一步利用，生成乙醇等。
褐藻胶裂解酶根据其一级序列及三维结构的不同，在碳水化合物多糖裂解酶家族中被划分为7个不同的家族：PL-5、6、7、14、15、17 和18（Tan G et al. Nucleic AcidsRes, 2005, 33: e122）。目前发现的褐藻胶裂解酶大多数为PL-5和PL-7家族。本发明的寡褐藻胶裂解酶OalC6属于PL-6家族，为本家族中第三个寡褐藻胶裂解酶，且其生物活性最高。目前报道的寡褐藻胶裂解酶的降解方式均为外切。
褐藻胶单糖指饱和的和不饱和的古洛糖醛酸和甘露糖醛酸，可用于发酵生产生化试剂或生物燃料。随着人口、经济发展的双重压力，地球生态环境遭到极大的破坏，地球能源日渐枯竭。生物能源作为一种重要的替代能源，世界各国空前关注。目前生物能源原料多为陆生农作物，我国人口众多，粮食需求压力长期存在，不可能大量利用粮食生产生物燃料。而利用褐藻胶作为新能源生产生物乙醇已经成为一种全新的尝试。利用褐藻胶为原料生产生物乙醇过程中将褐藻胶转化为单糖是其中必须的一步。Wargacki等将褐藻胶降解相关基因转化入大肠杆菌和酵母菌中，利用大肠杆菌和酿酒酵母以褐藻胶为能源产生生物乙醇（Wargacki AJ et al. Science, 2012, 335: 308-313）。我国在此领域还属起步阶段，本发明的寡褐藻胶裂解酶生物活性高，具有良好的应用前景。
发明内容
本发明的第一个目的是提供一种新型寡褐藻胶裂解酶OalC6。
本发明的第二个目的是提供包含上述寡褐藻胶裂解酶的基因工程菌及其构建方法。
本发明的另一个目的是提供上述寡褐藻胶裂解酶的应用。
本发明的目的是通过以下技术方案予以实现的：
一种寡褐藻胶裂解酶基因oalC6，其核苷酸序列如SEQ ID NO.1所示。
上述寡褐藻胶裂解酶基因oalC6编码的寡褐藻胶裂解酶OalC6，其氨基酸序列如SEQ ID NO.2所示。
包含上述寡褐藻胶裂解酶的基因工程菌，该菌株中导入了寡褐藻胶裂解酶OalC6的编码基因，所述的寡褐藻胶裂解酶基因oalC6的核苷酸序列如SEQ ID NO.1所示。
上述产寡褐藻胶裂解酶OalC6的基因工程菌的构建方法，其特征在于，包括如下步骤：
(1)将寡褐藻胶裂解酶OalC6的编码基因克隆到质粒中，得到重组载体；
(2)将重组载体转化宿主菌，得到产寡褐藻胶裂解酶OalC6的基因工程菌。
步骤(1)中，所述的质粒为pET28a(+)。
步骤(2)中，所述的宿主菌为大肠杆菌BL21 (DE3)。
上述寡褐藻胶裂解酶OalC6在裂解褐藻酸钠和褐藻胶寡糖中的应用在本发明的保护范围之内。
有益效果：
本发明所述的寡褐藻胶裂解酶OalC6来源于海洋细菌Cellulophagasp. SY116，通过设计同源引物，获得编码寡褐藻胶裂解酶OalC6的DNA序列，该基因编码区长2,328 bp，编码775个氨基酸，理论分子量为85.9 kDa，属于多糖裂解酶PL-6家族。大肠杆菌重组表达获得的OalC6，对古洛糖醛酸片段（polyG）具有偏好性，40 ℃、pH7.0条件下，在polyG底物中测得的酶活为76.8 U/mg。本发明的褐藻胶裂解酶在生产生物乙醇开发新能源方面具有潜在的应用价值。
附图说明
图1：寡褐藻胶裂解酶OalC6的多重氨基酸序列比对图。
图2：重组寡褐藻胶裂解酶OalC6表达纯化的聚丙烯酰氨凝胶电泳图(SDS-PAGE)。
图3：温度、pH对于寡褐藻胶裂解酶OalC6的活性及稳定性影响曲线（A，不同反应温度对酶活力影响；B，酶的温度稳定性；C，不同反应pH对酶活力影响；D，酶的pH稳定性）。
图4：寡褐藻胶裂解酶降解褐藻胶所得产物的电喷雾质谱(ESI-MS)分析图（A，酶解褐藻胶产物；B，酶解褐藻胶二糖；C，ESI-MS分析酶解产物）。
具体实施方式
下面结合说明书附图和具体实施例，进一步阐述本发明，但本发明所保护范围不限于此。
实施例1：寡褐藻胶裂解酶OalC6编码基因的克隆与鉴定
根据与Cellulophagasp. SY116基因组中可能的寡褐藻胶裂解酶基因设计如下扩增引物：上游引物(5’-CAAAAACATACATTATAC -3’)和下游引物(5’- CTAATTAATCCTAAATTTTT-3’)。以提取的菌株基因组为模板，进行PCR扩增得到寡褐藻胶裂解酶OalC6基因全长序列。PCR条件为：94 ℃预变性5 min，随后以94 ℃ 30s、54.8 ℃ 30s、72 ℃ 1 min 30 s进行30个循环，最后在72 ℃延伸10 min。
经基因测序后，得到寡褐藻胶裂解酶OalC6基因的全核苷酸序列全长2,328 bp，核苷酸序列如SEQ ID NO.1所示；编码775个氨基酸，氨基酸序列如SEQ ID NO.2所示，蛋白理论分子量为85.9 kDa。获得的基因oalC6的核苷酸序列如SEQ ID NO.1所示。其编码的蛋白的氨基酸序列如SEQ ID NO.2所示。
将上述所得PCR产生进行胶回收后，连接至pMD18-T载体上，转化至E. Coli DH5α感受态细胞中，蓝白斑筛选挑取阳性克隆，提取质粒后PCR鉴定阳性克隆，将阳性克隆进行序列测定，将序列提交至NCBI。利用软件对OalC6与已经报道的寡藻胶裂解酶序列进行比对，并利用MEGA 7.1软件构建进化树。如图1所示，寡和藻胶裂解酶归属于多糖降解酶第6家族。
实施例2：寡褐藻胶裂解酶OalC6重组表达载体的构建
根据寡褐藻胶裂解酶基因全序列设计上游引物(5’- CAAAAACATACATTATAC -3’)和下游引物(5’- CTAATTAATCCTAAATTTTT-3’)。进行PCR扩增得到褐藻胶裂解酶OalC6基因全长序列。PCR条件为：94 ℃预变性5 min，随后以98 ℃ 30 s、58 ℃ 30 s、68 ℃ 2 min 进行30个循环，最后在68 ℃延伸10 min。PCR产物和大肠杆菌表达载体pET28a(+)进行酶切，连接后转化至E.coliDH5α感受态细胞，挑取单克隆在含有抗性的LB液体中培养，提取质粒并酶切鉴定阳性克隆。将此重组质粒命名为pET28a- oalC6。将阳性质粒转化进入表达宿主E.coliBL21感受态细胞中。
实施例3：寡褐藻胶裂解酶OalC6基因在大肠杆菌中的重组表达与纯化制备
将重组表达质粒pET28a-oalC6转化进入表达宿主E.coliBL21感受态细胞中。将重组E.coliBL21细菌挑取单克隆至相同抗性的LB液体中，37 ℃振荡培养至OD600约0.6时加入IPTG进行诱导（终浓度为0.5 mM），25℃下进行诱导表达20 h。利用Ni亲和层析对目的蛋白进行分离纯化。用聚丙烯酰胺凝胶电泳检测寡褐藻胶裂解酶OalC6的纯化情况，结果如图2所示，纯化后的OalC6在电泳胶上呈现单一条带，且位置与预测的分子量相吻合。
实施例4：寡褐藻胶裂解酶OalC6的酶学性质研究
1、SDS、EDTA、金属离子对重组酶活性的影响
在酶活力检测体系中分别加入终浓度为1 mM的金属离子以及去污剂SDS、金属离子螯合剂EDTA，在该体系下检测各组的酶活力，以未添加任何离子的酶活力检测组为空白对照组（100%）。结果见表1。实验结果表明，除了Ca2+，其他二价和三价金属离子会明显抑制该酶活性；EDTA、SDS严重抑制该酶的活性；而Na+则会显著促进酶活。
表1不同金属离子及去污剂对酶活力的影响
离子 浓度(mM) 相对活性 (%)
Noneb - 100± 1.9
1 129.2 ± 1.2
10 142.5 ± 3.3
NaCl 100 186.4 ± 5.1
300 305.4 ± 3.5
500 256.4 ± 2.5
KCl 1 114.9 ± 2.3
MnCl2 1 62.6 ± 0.7
CaCl2 1 117.5 ± 4.2
ZnCl2 1 61.2 ± 3.3
MgCl2 1 82.8 ± 1.8
NiCl2 1 57.3 ± 2.2
CuCl2 1 39.8 ± 2.1
AlCl3 1 35.3 ± 1.8
FeCl3 1 35.8 ± 0.3
SDS 1 68.1 ± 1.4
EDTA 1 22.0 ± 1.2
2、重组酶的最适反应温度及温度对重组酶稳定性的影响
将1％褐藻酸钠底物分别置于不同温度下（10 ℃-70 ℃）孵育10 min，然后将适当稀释的寡褐藻胶裂解酶OalC6在该温度下反应10 min，利用DNS法检测酶活力，根据酶活力的大小计算酶的反应最适温度。结果显示OalC6在40℃时达到最大活力，表明OalC6的最适反应温度为40℃(如图3-A)。
适当浓度的寡褐藻胶裂解酶OalC6放置到不同温度下（10 ℃-70 ℃）孵育1 h，迅速将其冷却至4 ℃。在酶的最适反应温度40℃下检测酶的活力。以放置之前的酶活力为100%，检测酶在不同温度下的稳定性。结果表明OalC6在低于40℃的温度下具有较好的热稳定性（如图3-B）。
3、重组酶的最适反应pH及pH对重组酶稳定性的影响
将寡褐藻胶裂解酶OalC6（溶解于20 mM pH 7.0的磷酸盐缓冲液）稀释后加入到缓冲液体系为50 mM Na2HPO4-柠檬酸(pH 4.6~7.0)，50 mM 的Na2HPO4-NaH2PO4 ( pH 6.6~7.6)，50mM的Tris-HCl ( pH 7.6~8.6)，50 mM的甘氨酸-NaOH (pH 8.6~10.6)，测定OalC6在不同反应体系，不同pH条件下的酶活力。结果显示OalC6在pH 7.8时达到最大活力，表明OalC6的最适反应pH为7.8(如图3-C)。
将寡褐藻胶裂解酶OalC6放置在以上pH值下4℃保温24 h，然后按照酶活力测定方法对上述酶进行酶活力测定，以此来检测OalC6在以上pH值下的稳定性。以放置之前的活力为100%。结果显示在pH5.0～9.0的范围内，OalC6酶活仍保持60％以上，表明OalC6对pH值耐受范围较广（如图3-D）。
实施例5：寡褐藻胶裂解酶OalC6的降解产物分析
将18 U/mg 的寡褐藻胶裂解酶加入到1 ml的0.3%藻酸钠（溶解在20 mM的pH 7.0的磷酸盐缓冲液中）。将上述体系在40℃下反应过夜，充分降解。将样品至TLC薄层层析TLC板中，展开剂为（正丁醇：冰乙酸：水=2:1:1），130℃ 烘烤5 min显色，显色剂为苯胺二苯胺试剂。分析其降解终产物。结果显示，OalC6对polyG有很强的偏好性，降解终产物为单糖（如图4-A）。
将30 U/ml寡褐藻胶裂解酶OalC6加入到1 ml的0.1%褐藻胶二糖（溶解在20 mM pH7.0的磷酸盐缓冲液中）。将上述体系在40℃下反应过夜，充分降解。将样品至TLC薄层层析TLC板中，分析其对和藻胶寡糖降解产物。结果显示，OalC6对能够降解褐藻胶二糖为单糖（如图4-B）。
用18 U/ml的酶OalC6降解polyG底物（0.5 g polyG，0.2 M NaCl溶于20 mM pH7.0的磷酸盐缓冲液），通过HPLC方法进行纯化制备，将制备的主产物组分用旋蒸仪蒸干，取少量溶于溶剂（水：乙腈=1：1）中离心取上清进行MS阴离子高分辨分析，将剩下的样品（约20mg）重新溶解于重水中，离心后取上清打NMR谱（1H, deptQ, H-H cosy）进行结构鉴定（如图4-C）。
SEQUENCE LISTING
<110> 青岛大学
<120> 寡褐藻胶裂解酶OalC6的制备方法与应用
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 2328
<212> DNA
<213> 海洋细菌Cellulophaga sp. SY116
<400> 1
atgcaaaaac atacattata cttagctttt atactacttg ttagtattac ttcatgcaca 60
caaaaaaaga ataccgtagc tactttacaa caatttaaca ctgcagtaaa aaatgcacaa 120
cctggagatg taattacact ggctaatggc gtatggaaca ataccgaatt actttttgct 180
gcaaaaggca cagcagaaaa gccaataact ttaactgtag aagaaaaagg taaggtaact 240
ttagaagggc aaagtaactt acgtattgca ggtgaacact taattattaa tggtttggta 300
tttaaaaatg gttatactcc cactaatgaa gttatctctt ttaaacaaaa tgctaaaact 360
cttgcaaata acactagact aacagaatgt gttatagata attttagtaa cccagaacgt 420
cacgaaccag atacttgggt tgctatatac ggtaaaaata accgaattga ccacaaccat 480
ttaactggta aaaaaaatag aggtgttact atgacggtta ggttaaattc taaagaaagt 540
caagacaaca atcataaaat agatcacaac tattttggac ctagacaaaa cttaggctct 600
aacggtggtg agactttacg tattggaact agccattttt ctagaacaaa ctctaatact 660
attgtagaaa ataattattt tgataggtgt aacggagaac atgaaataat ctctaacaaa 720
tcatgtcaaa acacatacaa aaacaatgtt ttttatgagt gtacaggtac attaactatg 780
cgccacggta acagaactat ggtagatggc aacatattta ttggcaacaa taaacctagc 840
acaggtggta ttaggattat taatggtgag caaactgtta taaataatta tggtattgga 900
ttaactggtt accgttttag aggtgctttt gttattatga acggaattta caattctcct 960
attaataggt atgaccaagt taaagatgct gttgtaaaaa ataacacttt tgttaatagt 1020
aaccacattc agttatgtgc aggtagtgat gaagaacgta gtgctcctcc tgtaaactct 1080
gttatggaaa ataatatttt ttataatgag agtaaagatt caatttttac ggtttatgat 1140
gatattaatg gtattacatt tacaaataac attattagta aaaacataaa accaattact 1200
gatactggat ttgtaagtaa agatattgag tgggtaaaaa atacagacgg attgctagtt 1260
cctgctaact ctaacttaag agctggtgct aaattaccag gtgaaattct taaaaaagaa 1320
gatacaggtg ttaactggta ctctaaaaaa aatactgctg ttgcctttgg tactggtaaa 1380
aaaattacag tagaaccagg tttaaatact ttgtttgaag ctgtaaataa ttcttcagcc 1440
ggagacgtag tagagcttac tgctggcgaa acttatactt tatcaaaaac tatagctata 1500
aatcatccta ttactatcct ttctaattca gataaaaaac ctactatttt atttgaaaag 1560
aaaaccttat ttgaaattaa caatggaggc agcttaaaat taactggaat taattttgat 1620
ggtgaaagcg ccccagacag aacaggaaat tctgtaatta ctactagtaa atactcaatg 1680
aacaaaaatt acaaattgtt tatagataat tgtgactttg taaacttaga tgttaaccat 1740
tcttttgatg ctgtaatggt gtacaaaaac acatttgcag acacaattag tattaaaaat 1800
tcaaaattta atactattag tggtaacgtt ttggctttag ataaagaaac tgaagatatt 1860
ggtatctata atgcagaata tgtaatctta aaaaataata gttttaacaa tgtaaatggt 1920
gctgttttaa gtttacacag aggtggtaag gatgaaagta cttttggtcc gtttttagaa 1980
ttagaccata acacttttga cgctattggt tacggtaaaa gaaataaata caatgctgca 2040
attaacttat atggtgtgca agtaaatact attaccaaca atatatttaa caacacaaaa 2100
tcgttaaaaa tgcatttagt tgttggtgag ccaatagtag atattttaaa caataatttt 2160
tacaaatctg atggtttaga agttactggt gatcaaaaat acaatgttaa aaacctatgg 2220
acattagatc ctaaatttaa agaaggtact tatacattat ctaaaaattc tgacctttta 2280
aataaaggta cagatggcaa aaatttagga ttaattagta ataactag 2328
<210> 2
<211> 2328
<212> PRT
<213> 海洋细菌Cellulophaga sp. SY116
<400> 2
Ala Thr Gly Cys Ala Ala Ala Ala Ala Cys Ala Thr Ala Cys Ala Thr
1 5 10 15
Thr Ala Thr Ala Cys Thr Thr Ala Gly Cys Thr Thr Thr Thr Ala Thr
20 25 30
Ala Cys Thr Ala Cys Thr Thr Gly Thr Thr Ala Gly Thr Ala Thr Thr
35 40 45
Ala Cys Thr Thr Cys Ala Thr Gly Cys Ala Cys Ala Cys Ala Ala Ala
50 55 60
Ala Ala Ala Ala Gly Ala Ala Thr Ala Cys Cys Gly Thr Ala Gly Cys
65 70 75 80
Thr Ala Cys Thr Thr Thr Ala Cys Ala Ala Cys Ala Ala Thr Thr Thr
85 90 95
Ala Ala Cys Ala Cys Thr Gly Cys Ala Gly Thr Ala Ala Ala Ala Ala
100 105 110
Ala Thr Gly Cys Ala Cys Ala Ala Cys Cys Thr Gly Gly Ala Gly Ala
115 120 125
Thr Gly Thr Ala Ala Thr Thr Ala Cys Ala Cys Thr Gly Gly Cys Thr
130 135 140
Ala Ala Thr Gly Gly Cys Gly Thr Ala Thr Gly Gly Ala Ala Cys Ala
145 150 155 160
Ala Thr Ala Cys Cys Gly Ala Ala Thr Thr Ala Cys Thr Thr Thr Thr
165 170 175
Thr Gly Cys Thr Gly Cys Ala Ala Ala Ala Gly Gly Cys Ala Cys Ala
180 185 190
Gly Cys Ala Gly Ala Ala Ala Ala Gly Cys Cys Ala Ala Thr Ala Ala
195 200 205
Cys Thr Thr Thr Ala Ala Cys Thr Gly Thr Ala Gly Ala Ala Gly Ala
210 215 220
Ala Ala Ala Ala Gly Gly Thr Ala Ala Gly Gly Thr Ala Ala Cys Thr
225 230 235 240
Thr Thr Ala Gly Ala Ala Gly Gly Gly Cys Ala Ala Ala Gly Thr Ala
245 250 255
Ala Cys Thr Thr Ala Cys Gly Thr Ala Thr Thr Gly Cys Ala Gly Gly
260 265 270
Thr Gly Ala Ala Cys Ala Cys Thr Thr Ala Ala Thr Thr Ala Thr Thr
275 280 285
Ala Ala Thr Gly Gly Thr Thr Thr Gly Gly Thr Ala Thr Thr Thr Ala
290 295 300
Ala Ala Ala Ala Thr Gly Gly Thr Thr Ala Thr Ala Cys Thr Cys Cys
305 310 315 320
Cys Ala Cys Thr Ala Ala Thr Gly Ala Ala Gly Thr Thr Ala Thr Cys
325 330 335
Thr Cys Thr Thr Thr Thr Ala Ala Ala Cys Ala Ala Ala Ala Thr Gly
340 345 350
Cys Thr Ala Ala Ala Ala Cys Thr Cys Thr Thr Gly Cys Ala Ala Ala
355 360 365
Thr Ala Ala Cys Ala Cys Thr Ala Gly Ala Cys Thr Ala Ala Cys Ala
370 375 380
Gly Ala Ala Thr Gly Thr Gly Thr Thr Ala Thr Ala Gly Ala Thr Ala
385 390 395 400
Ala Thr Thr Thr Thr Ala Gly Thr Ala Ala Cys Cys Cys Ala Gly Ala
405 410 415
Ala Cys Gly Thr Cys Ala Cys Gly Ala Ala Cys Cys Ala Gly Ala Thr
420 425 430
Ala Cys Thr Thr Gly Gly Gly Thr Thr Gly Cys Thr Ala Thr Ala Thr
435 440 445
Ala Cys Gly Gly Thr Ala Ala Ala Ala Ala Thr Ala Ala Cys Cys Gly
450 455 460
Ala Ala Thr Thr Gly Ala Cys Cys Ala Cys Ala Ala Cys Cys Ala Thr
465 470 475 480
Thr Thr Ala Ala Cys Thr Gly Gly Thr Ala Ala Ala Ala Ala Ala Ala
485 490 495
Ala Thr Ala Gly Ala Gly Gly Thr Gly Thr Thr Ala Cys Thr Ala Thr
500 505 510
Gly Ala Cys Gly Gly Thr Thr Ala Gly Gly Thr Thr Ala Ala Ala Thr
515 520 525
Thr Cys Thr Ala Ala Ala Gly Ala Ala Ala Gly Thr Cys Ala Ala Gly
530 535 540
Ala Cys Ala Ala Cys Ala Ala Thr Cys Ala Thr Ala Ala Ala Ala Thr
545 550 555 560
Ala Gly Ala Thr Cys Ala Cys Ala Ala Cys Thr Ala Thr Thr Thr Thr
565 570 575
Gly Gly Ala Cys Cys Thr Ala Gly Ala Cys Ala Ala Ala Ala Cys Thr
580 585 590
Thr Ala Gly Gly Cys Thr Cys Thr Ala Ala Cys Gly Gly Thr Gly Gly
595 600 605
Thr Gly Ala Gly Ala Cys Thr Thr Thr Ala Cys Gly Thr Ala Thr Thr
610 615 620
Gly Gly Ala Ala Cys Thr Ala Gly Cys Cys Ala Thr Thr Thr Thr Thr
625 630 635 640
Cys Thr Ala Gly Ala Ala Cys Ala Ala Ala Cys Thr Cys Thr Ala Ala
645 650 655
Thr Ala Cys Thr Ala Thr Thr Gly Thr Ala Gly Ala Ala Ala Ala Thr
660 665 670
Ala Ala Thr Thr Ala Thr Thr Thr Thr Gly Ala Thr Ala Gly Gly Thr
675 680 685
Gly Thr Ala Ala Cys Gly Gly Ala Gly Ala Ala Cys Ala Thr Gly Ala
690 695 700
Ala Ala Thr Ala Ala Thr Cys Thr Cys Thr Ala Ala Cys Ala Ala Ala
705 710 715 720
Thr Cys Ala Thr Gly Thr Cys Ala Ala Ala Ala Cys Ala Cys Ala Thr
725 730 735
Ala Cys Ala Ala Ala Ala Ala Cys Ala Ala Thr Gly Thr Thr Thr Thr
740 745 750
Thr Thr Ala Thr Gly Ala Gly Thr Gly Thr Ala Cys Ala Gly Gly Thr
755 760 765
Ala Cys Ala Thr Thr Ala Ala Cys Thr Ala Thr Gly Cys Gly Cys Cys
770 775 780
Ala Cys Gly Gly Thr Ala Ala Cys Ala Gly Ala Ala Cys Thr Ala Thr
785 790 795 800
Gly Gly Thr Ala Gly Ala Thr Gly Gly Cys Ala Ala Cys Ala Thr Ala
805 810 815
Thr Thr Thr Ala Thr Thr Gly Gly Cys Ala Ala Cys Ala Ala Thr Ala
820 825 830
Ala Ala Cys Cys Thr Ala Gly Cys Ala Cys Ala Gly Gly Thr Gly Gly
835 840 845
Thr Ala Thr Thr Ala Gly Gly Ala Thr Thr Ala Thr Thr Ala Ala Thr
850 855 860
Gly Gly Thr Gly Ala Gly Cys Ala Ala Ala Cys Thr Gly Thr Thr Ala
865 870 875 880
Thr Ala Ala Ala Thr Ala Ala Thr Thr Ala Thr Gly Gly Thr Ala Thr
885 890 895
Thr Gly Gly Ala Thr Thr Ala Ala Cys Thr Gly Gly Thr Thr Ala Cys
900 905 910
Cys Gly Thr Thr Thr Thr Ala Gly Ala Gly Gly Thr Gly Cys Thr Thr
915 920 925
Thr Thr Gly Thr Thr Ala Thr Thr Ala Thr Gly Ala Ala Cys Gly Gly
930 935 940
Ala Ala Thr Thr Thr Ala Cys Ala Ala Thr Thr Cys Thr Cys Cys Thr
945 950 955 960
Ala Thr Thr Ala Ala Thr Ala Gly Gly Thr Ala Thr Gly Ala Cys Cys
965 970 975
Ala Ala Gly Thr Thr Ala Ala Ala Gly Ala Thr Gly Cys Thr Gly Thr
980 985 990
Thr Gly Thr Ala Ala Ala Ala Ala Ala Thr Ala Ala Cys Ala Cys Thr
995 1000 1005
Thr Thr Thr Gly Thr Thr Ala Ala Thr Ala Gly Thr Ala Ala Cys
1010 1015 1020
Cys Ala Cys Ala Thr Thr Cys Ala Gly Thr Thr Ala Thr Gly Thr
1025 1030 1035
Gly Cys Ala Gly Gly Thr Ala Gly Thr Gly Ala Thr Gly Ala Ala
1040 1045 1050
Gly Ala Ala Cys Gly Thr Ala Gly Thr Gly Cys Thr Cys Cys Thr
1055 1060 1065
Cys Cys Thr Gly Thr Ala Ala Ala Cys Thr Cys Thr Gly Thr Thr
1070 1075 1080
Ala Thr Gly Gly Ala Ala Ala Ala Thr Ala Ala Thr Ala Thr Thr
1085 1090 1095
Thr Thr Thr Thr Ala Thr Ala Ala Thr Gly Ala Gly Ala Gly Thr
1100 1105 1110
Ala Ala Ala Gly Ala Thr Thr Cys Ala Ala Thr Thr Thr Thr Thr
1115 1120 1125
Ala Cys Gly Gly Thr Thr Thr Ala Thr Gly Ala Thr Gly Ala Thr
1130 1135 1140
Ala Thr Thr Ala Ala Thr Gly Gly Thr Ala Thr Thr Ala Cys Ala
1145 1150 1155
Thr Thr Thr Ala Cys Ala Ala Ala Thr Ala Ala Cys Ala Thr Thr
1160 1165 1170
Ala Thr Thr Ala Gly Thr Ala Ala Ala Ala Ala Cys Ala Thr Ala
1175 1180 1185
Ala Ala Ala Cys Cys Ala Ala Thr Thr Ala Cys Thr Gly Ala Thr
1190 1195 1200
Ala Cys Thr Gly Gly Ala Thr Thr Thr Gly Thr Ala Ala Gly Thr
1205 1210 1215
Ala Ala Ala Gly Ala Thr Ala Thr Thr Gly Ala Gly Thr Gly Gly
1220 1225 1230
Gly Thr Ala Ala Ala Ala Ala Ala Thr Ala Cys Ala Gly Ala Cys
1235 1240 1245
Gly Gly Ala Thr Thr Gly Cys Thr Ala Gly Thr Thr Cys Cys Thr
1250 1255 1260
Gly Cys Thr Ala Ala Cys Thr Cys Thr Ala Ala Cys Thr Thr Ala
1265 1270 1275
Ala Gly Ala Gly Cys Thr Gly Gly Thr Gly Cys Thr Ala Ala Ala
1280 1285 1290
Thr Thr Ala Cys Cys Ala Gly Gly Thr Gly Ala Ala Ala Thr Thr
1295 1300 1305
Cys Thr Thr Ala Ala Ala Ala Ala Ala Gly Ala Ala Gly Ala Thr
1310 1315 1320
Ala Cys Ala Gly Gly Thr Gly Thr Thr Ala Ala Cys Thr Gly Gly
1325 1330 1335
Thr Ala Cys Thr Cys Thr Ala Ala Ala Ala Ala Ala Ala Ala Thr
1340 1345 1350
Ala Cys Thr Gly Cys Thr Gly Thr Thr Gly Cys Cys Thr Thr Thr
1355 1360 1365
Gly Gly Thr Ala Cys Thr Gly Gly Thr Ala Ala Ala Ala Ala Ala
1370 1375 1380
Ala Thr Thr Ala Cys Ala Gly Thr Ala Gly Ala Ala Cys Cys Ala
1385 1390 1395
Gly Gly Thr Thr Thr Ala Ala Ala Thr Ala Cys Thr Thr Thr Gly
1400 1405 1410
Thr Thr Thr Gly Ala Ala Gly Cys Thr Gly Thr Ala Ala Ala Thr
1415 1420 1425
Ala Ala Thr Thr Cys Thr Thr Cys Ala Gly Cys Cys Gly Gly Ala
1430 1435 1440
Gly Ala Cys Gly Thr Ala Gly Thr Ala Gly Ala Gly Cys Thr Thr
1445 1450 1455
Ala Cys Thr Gly Cys Thr Gly Gly Cys Gly Ala Ala Ala Cys Thr
1460 1465 1470
Thr Ala Thr Ala Cys Thr Thr Thr Ala Thr Cys Ala Ala Ala Ala
1475 1480 1485
Ala Cys Thr Ala Thr Ala Gly Cys Thr Ala Thr Ala Ala Ala Thr
1490 1495 1500
Cys Ala Thr Cys Cys Thr Ala Thr Thr Ala Cys Thr Ala Thr Cys
1505 1510 1515
Cys Thr Thr Thr Cys Thr Ala Ala Thr Thr Cys Ala Gly Ala Thr
1520 1525 1530
Ala Ala Ala Ala Ala Ala Cys Cys Thr Ala Cys Thr Ala Thr Thr
1535 1540 1545
Thr Thr Ala Thr Thr Thr Gly Ala Ala Ala Ala Gly Ala Ala Ala
1550 1555 1560
Ala Cys Cys Thr Thr Ala Thr Thr Thr Gly Ala Ala Ala Thr Thr
1565 1570 1575
Ala Ala Cys Ala Ala Thr Gly Gly Ala Gly Gly Cys Ala Gly Cys
1580 1585 1590
Thr Thr Ala Ala Ala Ala Thr Thr Ala Ala Cys Thr Gly Gly Ala
1595 1600 1605
Ala Thr Thr Ala Ala Thr Thr Thr Thr Gly Ala Thr Gly Gly Thr
1610 1615 1620
Gly Ala Ala Ala Gly Cys Gly Cys Cys Cys Cys Ala Gly Ala Cys
1625 1630 1635
Ala Gly Ala Ala Cys Ala Gly Gly Ala Ala Ala Thr Thr Cys Thr
1640 1645 1650
Gly Thr Ala Ala Thr Thr Ala Cys Thr Ala Cys Thr Ala Gly Thr
1655 1660 1665
Ala Ala Ala Thr Ala Cys Thr Cys Ala Ala Thr Gly Ala Ala Cys
1670 1675 1680
Ala Ala Ala Ala Ala Thr Thr Ala Cys Ala Ala Ala Thr Thr Gly
1685 1690 1695
Thr Thr Thr Ala Thr Ala Gly Ala Thr Ala Ala Thr Thr Gly Thr
1700 1705 1710
Gly Ala Cys Thr Thr Thr Gly Thr Ala Ala Ala Cys Thr Thr Ala
1715 1720 1725
Gly Ala Thr Gly Thr Thr Ala Ala Cys Cys Ala Thr Thr Cys Thr
1730 1735 1740
Thr Thr Thr Gly Ala Thr Gly Cys Thr Gly Thr Ala Ala Thr Gly
1745 1750 1755
Gly Thr Gly Thr Ala Cys Ala Ala Ala Ala Ala Cys Ala Cys Ala
1760 1765 1770
Thr Thr Thr Gly Cys Ala Gly Ala Cys Ala Cys Ala Ala Thr Thr
1775 1780 1785
Ala Gly Thr Ala Thr Thr Ala Ala Ala Ala Ala Thr Thr Cys Ala
1790 1795 1800
Ala Ala Ala Thr Thr Thr Ala Ala Thr Ala Cys Thr Ala Thr Thr
1805 1810 1815
Ala Gly Thr Gly Gly Thr Ala Ala Cys Gly Thr Thr Thr Thr Gly
1820 1825 1830
Gly Cys Thr Thr Thr Ala Gly Ala Thr Ala Ala Ala Gly Ala Ala
1835 1840 1845
Ala Cys Thr Gly Ala Ala Gly Ala Thr Ala Thr Thr Gly Gly Thr
1850 1855 1860
Ala Thr Cys Thr Ala Thr Ala Ala Thr Gly Cys Ala Gly Ala Ala
1865 1870 1875
Thr Ala Thr Gly Thr Ala Ala Thr Cys Thr Thr Ala Ala Ala Ala
1880 1885 1890
Ala Ala Thr Ala Ala Thr Ala Gly Thr Thr Thr Thr Ala Ala Cys
1895 1900 1905
Ala Ala Thr Gly Thr Ala Ala Ala Thr Gly Gly Thr Gly Cys Thr
1910 1915 1920
Gly Thr Thr Thr Thr Ala Ala Gly Thr Thr Thr Ala Cys Ala Cys
1925 1930 1935
Ala Gly Ala Gly Gly Thr Gly Gly Thr Ala Ala Gly Gly Ala Thr
1940 1945 1950
Gly Ala Ala Ala Gly Thr Ala Cys Thr Thr Thr Thr Gly Gly Thr
1955 1960 1965
Cys Cys Gly Thr Thr Thr Thr Thr Ala Gly Ala Ala Thr Thr Ala
1970 1975 1980
Gly Ala Cys Cys Ala Thr Ala Ala Cys Ala Cys Thr Thr Thr Thr
1985 1990 1995
Gly Ala Cys Gly Cys Thr Ala Thr Thr Gly Gly Thr Thr Ala Cys
2000 2005 2010
Gly Gly Thr Ala Ala Ala Ala Gly Ala Ala Ala Thr Ala Ala Ala
2015 2020 2025
Thr Ala Cys Ala Ala Thr Gly Cys Thr Gly Cys Ala Ala Thr Thr
2030 2035 2040
Ala Ala Cys Thr Thr Ala Thr Ala Thr Gly Gly Thr Gly Thr Gly
2045 2050 2055
Cys Ala Ala Gly Thr Ala Ala Ala Thr Ala Cys Thr Ala Thr Thr
2060 2065 2070
Ala Cys Cys Ala Ala Cys Ala Ala Thr Ala Thr Ala Thr Thr Thr
2075 2080 2085
Ala Ala Cys Ala Ala Cys Ala Cys Ala Ala Ala Ala Thr Cys Gly
2090 2095 2100
Thr Thr Ala Ala Ala Ala Ala Thr Gly Cys Ala Thr Thr Thr Ala
2105 2110 2115
Gly Thr Thr Gly Thr Thr Gly Gly Thr Gly Ala Gly Cys Cys Ala
2120 2125 2130
Ala Thr Ala Gly Thr Ala Gly Ala Thr Ala Thr Thr Thr Thr Ala
2135 2140 2145
Ala Ala Cys Ala Ala Thr Ala Ala Thr Thr Thr Thr Thr Ala Cys
2150 2155 2160
Ala Ala Ala Thr Cys Thr Gly Ala Thr Gly Gly Thr Thr Thr Ala
2165 2170 2175
Gly Ala Ala Gly Thr Thr Ala Cys Thr Gly Gly Thr Gly Ala Thr
2180 2185 2190
Cys Ala Ala Ala Ala Ala Thr Ala Cys Ala Ala Thr Gly Thr Thr
2195 2200 2205
Ala Ala Ala Ala Ala Cys Cys Thr Ala Thr Gly Gly Ala Cys Ala
2210 2215 2220
Thr Thr Ala Gly Ala Thr Cys Cys Thr Ala Ala Ala Thr Thr Thr
2225 2230 2235
Ala Ala Ala Gly Ala Ala Gly Gly Thr Ala Cys Thr Thr Ala Thr
2240 2245 2250
Ala Cys Ala Thr Thr Ala Thr Cys Thr Ala Ala Ala Ala Ala Thr
2255 2260 2265
Thr Cys Thr Gly Ala Cys Cys Thr Thr Thr Thr Ala Ala Ala Thr
2270 2275 2280
Ala Ala Ala Gly Gly Thr Ala Cys Ala Gly Ala Thr Gly Gly Cys
2285 2290 2295
Ala Ala Ala Ala Ala Thr Thr Thr Ala Gly Gly Ala Thr Thr Ala
2300 2305 2310
Ala Thr Thr Ala Gly Thr Ala Ala Thr Ala Ala Cys Thr Ala Gly
2315 2320 2325

Claims (7)

1.一种寡褐藻胶裂解酶基因oalC6，其核苷酸序列如SEQ ID NO.1所示。
2.权利要求1所述寡褐藻胶裂解酶基因oalC6编码的寡褐藻胶裂解酶OalC6，其氨基酸序列如SEQ ID NO.2所示。
3.包含权利要求2所述寡褐藻胶裂解酶的基因工程菌，其特征在于，该菌株中导入了寡褐藻胶裂解酶基因oalC6，所述的褐藻胶裂解酶基因的核苷酸序列如SEQ ID NO.1所示。
4.权利要求3所述的产寡褐藻胶裂解酶的基因工程菌的构建方法，其特征在于，包括如下步骤：
(1)将寡褐藻胶裂解酶基因oalC6克隆到质粒中，得到重组载体；
(2)将重组载体转化宿主菌株，得到产寡褐藻胶裂解酶OalC6的基因工程菌。
5.根据权利要求4所述产褐藻胶裂解酶的基因工程菌的构建方法，其特征在于，步骤(1)中，所述的质粒为pET28a(+)。
6.根据权利要求4所述产褐藻胶裂解酶OalC6的基因工程菌的构建方法，其特征在于，步骤(2)中，所述的宿主菌为大肠杆菌BL21 (DE3)。
7.权利要求2所述寡褐藻胶裂解酶OalC6和/或权利要求1所述寡褐藻胶裂解酶基因oalC6在裂解褐藻酸钠和褐藻寡糖中的应用。
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CN108753642B (zh) * 2018-05-10 2020-02-11 江南大学 一株产褐藻胶裂解酶菌株约氏黄杆菌
CN109022408A (zh) * 2018-09-25 2018-12-18 青岛大学 一种新型褐藻胶裂解酶Aly08及其应用
CN109022408B (zh) * 2018-09-25 2019-05-21 青岛大学 一种新型褐藻胶裂解酶Aly08及其应用

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