CN111304182A - 一种β-葡萄糖苷酶及其制备方法与应用 - Google Patents
一种β-葡萄糖苷酶及其制备方法与应用 Download PDFInfo
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Abstract
本发明公开了一种β‑葡萄糖苷酶及其制备方法与应用。本发明所公开的β‑葡萄糖苷酶的氨基酸序列如SEQ ID NO:1所示。所公开的制备方法包括基因组DNA的提取、目的基因扩增、载体构建、转化、表达和纯化。本发明的β‑葡萄糖苷酶比活力达到了59.19μkat/g,约为3550U/g,高于经传统方法表达的β‑葡萄糖苷酶的比活力,且热稳定性和pH稳定性良好,可用于葡萄酒酿制。
Description
技术领域
本发明涉及生物制酶技术领域,特别是涉及一种酒类酒球菌来源的β-葡萄糖苷酶及其制备方法和应用。
背景技术
β-葡萄糖苷酶作为一种关键的糖苷水解酶,在葡萄酒增香的过程中扮演着重要的角色,但是β-葡萄糖苷酶的制备方法繁琐,费时费力,产量低下,难以满足生产要求。
发明内容
针对现有技术的缺陷或不足,本发明的目的之一是提供一种β-葡萄糖苷酶。
本发明所提供的β-葡萄糖苷酶的氨基酸序列如SEQ ID NO:1所示。
同时本发明提供了一种β-葡萄糖苷酶的制备方法。本发明所提供的制备方法包括以下步骤:
(1)基因组DNA的提取:提取酒类酒球菌SD-2a的基因组DNA;
(2)目的基因扩增:以步骤(1)得到的基因组DNA为模板,进行聚合酶链式反应,扩增目的基因;
(3)载体构建:将扩增得到的目的基因插入表达载体PcoldI构建表达质粒PcoldI-0224;
(4)转化:将表达质粒PcoldI-0224导入大肠杆菌感受态细胞DH5α,得到转化子;
(5)表达:将转化子进行扩大培养,提取培养物中的重组表达质粒PcoldI-0224导入感受态细胞BL21,之后进行扩大培养;
(6)纯化:对步骤(5)的培养物依次进行裂解、过滤,之后依次采用His重力镍柱和超滤管收集β-葡萄糖苷酶。
优选的,本发明制备方法中步骤(1)中提取对数中期酒类酒球菌SD-2a的基因组DNA。
优选的,本发明制备方法中步骤(2)扩增时所用引物序列分别为:
上游引物:5’-CTCCATATGATGAATAAACTTTTTTTGCCGAAA-3’,
下游引物:5’-TGAGGATCCTTAATCTAATTGACTGCCGTTTGA-3’。
优选的,本发明制备方法步骤(2)中所用的DNA聚合酶为PrimeSTAR Max DNAPolymerase,PCR反应程序为:95℃预变性10min;98℃变性10s,55℃退火15s,72℃延伸60s,30次循环;72℃保温8min。
本发明的β-葡萄糖苷酶可用于酿制葡萄酒,相比于现有的同类酶,该酶的催化性能增加,在用于酿制葡萄酒时,可增加酒中香气物质的种类和含量、改善葡萄酒的风味、提高葡萄酒产品质量。
与传统方法相比,本发明直接将目的基因与表达载体双酶切后在DNA连接酶的作用下连接,不必经过传统的T-A克隆程序,极大地节省了时间。
本发明重组酶的比活力达到了59.19μkat/g,约为3550U/g,高于经传统方法表达的β-葡萄糖苷酶的比活力,且热稳定性和pH稳定性良好。
附图说明
图1为本发明实施例中制备的β-葡萄糖苷酶和酒酒球菌PSU-1中的β-葡萄糖苷酶蛋白序列比对;
图2为本发明实施例中表达质粒PcoldI-0224的构建过程图;
图3为本发明对比例中表达质粒PcoldI-0224的构建过程图;
图4为本发明中重组蛋白BGL0224表达的SDS-PAGE图;图中:M:Protein marker、1:空载大肠杆菌BL21(DE3)、2:PcoldI-0224表达结果(实施例)、3:PcoldI-0224表达结果(对比例);
图5为本发明实施例中重组酶BGL0224的最适温度;
图6为本发明对比例中重组酶BGL0224的最适温度;
图7为本发明实施例中重组酶BGL0224的热稳定性;
图8为本发明对比例中重组酶BGL0224的热稳定性;
图9为本发明实施例中重组酶BGL0224最适pH及pH稳定性。
图10为本发明对比例中重组酶BGL0224最适pH及pH稳定性。
具体实施方式
本发明的β-葡萄糖苷酶的目的基因OEOE-0224(Gene ID:4416011)来源于酒类酒球菌标准菌株PSU-1,根据该基因序列设计引物,并以酒类酒球菌SD-2a中的DNA为模板,实现该基因的克隆表达。
为使本发明的上述目的、特征和优点能够更加明显易懂,下面结合附图对本发明的具体实施方式做详细的说明。本发明所要保护的范围不限于以下以下实施例中所用实验材料均为市售产品。酶活测定方法采用文献1(李华,高丽,β-葡萄糖苷酶活性测定方法的研究进展,食品与生物技术学报,2007年3月,第26卷第2期)中公开的方法——比色法。
实施例1:
1、酒类酒球菌SD-2a的培养:
培养基选择ATB番茄培养基,具体成分如下:葡萄糖10g/L,酵母浸粉5g/L,蛋白胨10g/L,MgSO4·7H2O 0.2g/L,MnSO4·4H2O 0.05g/L,盐酸半胱氨酸0.5g/L,番茄汁250mL/L,用NaOH调pH至4.8,121℃灭菌15min。约40h左右,酒类酒球菌SD-2a在此培养基中生长至对数中期,收集细菌至2mL离心管,离心(12000xg,10min),备用。
2、基因组DNA的提取:采用CTAB/NaCl法,具体步骤如下:
(1)用570μL的TE buffer充分悬浮上述步骤1中的菌体沉淀;
(2)向步骤(1)的菌悬液中加入10μL的溶菌酶溶液(20mg/mL),颠倒离心管数次以充分混匀。37℃水浴60min。加入6μL的蛋白酶K溶液(10mg/mL)及30μL的浓度为10%的SDS溶液。充分混匀,37℃水浴60min;
(3)向步骤(2)的菌悬液中加入100μL的NaCl溶液(5mol/L),混匀后65℃水浴2min。加入80μL的CTAB/NaCl溶液(使用前65℃预热),混匀后65℃水浴10min;
(4)向步骤(3)的菌悬液中加入等体积(约800μL)的氯仿/异戊醇溶液(24:1),12000xg离心5min。将上层水相(上清液1,含DNA)转移至另一洁净的2mL离心管中;
(5)向上清液1中加入10μLRNA酶,静置15min后,加入等体积(约800μL)的苯酚/氯仿/异戊醇混合液(25:24:1),12000xg离心5min。将上层水相(上清液2,含DNA)转移至另一洁净的2mL离心管中;
(6)向上清液2中加入等体积(约800μL)的氯仿/异戊醇混合液(24:1),12000xg离心5min。将上层水相(上清液3,含DNA)转移至另一洁净的1.5mL离心管中;
(7)向上清液3中加入0.7倍体积(约560μL)的异丙醇,轻轻颠倒离心管数次。室温放置30min以沉淀DNA。12000xg离心15min,去上清,此时可在离心管底部看见白色沉淀;
(8)加入500μL70%的无水乙醇洗涤沉淀,12000xg离心15min,去上清,滤纸除去管壁附着的水分。加入100μL的TE buffer溶解DNA沉淀,用手指轻弹试管,使DNA充分溶解;
(9)0.7%琼脂糖凝胶电泳检测所提取DNA的完整性。DNA溶液放于-20℃冰箱中保存备用。
3、目的基因的扩增:
以步骤2中得到的基因组DNA为模板,采用上、下游引物进行聚合酶链式反应(PCR),以扩增目的基因;
所用的DNA聚合酶为PrimeSTAR Max DNA Polymerase,PCR反应程序为:95℃预变性10min;98℃变性10s,55℃退火15s,72℃延伸60s,30次循环;72℃保温8min。PCR产物经琼脂糖凝胶电泳检测后回收;
扩增时所用引物采取以下方法设计:以酒类酒球菌标准菌株PSU-1中的β-葡萄糖苷酶基因OEOE-0224为目的基因设计引物。目的基因序列通过登录NCBI数据库(https://www.ncbi.nlm.nih.gov/)下载得到,全长1443bp,设计引物时在上下游两端分别加入NdeI和BamHI酶切位点(以下划线标出),方便后续与PcoldI表达载体的连接;具体引物序列为:
上游引物:5’-CTCCATATGATGAATAAACTTTTTTTGCCGAAA-3’
下游引物:5’-TGAGGATCCTTAATCTAATTGACTGCCGTTTGA-3’。
4、载体构建
扩增得到的目的基因与表达载体PcoldI双酶切:采用快切酶NdeI和BamhI对目的基因和表达载体进行双酶切,双酶切反应体系(20μL)均为:10xQuickout buffer 2μL;OEOE-0224/PcoldI 2μL;NdeI 1μL;BamhI 1μL;ddH2O 14μL。将该体系置于37℃水浴反应30min后,上样1%琼脂糖凝胶电泳检测条带并进行胶回收;
扩增得到的目的基因与表达载体PcoldI连接:将胶回收得到的目的基因与表达载体进行连接,得到表达质粒PcoldI-0224,具体连接体系(10μL)为:PcoldI 1μL;OEOE-02241μL;DNA Ligation Mix 5μL;ddH2O 3μL。将该连接体系混匀后置于16℃反应30min,整体连接流程图如图2所示,构建的质粒序列如SEQ ID NO:2所示;
5、转化:
将连接体系全量(10μL)加入至100μL的DH5α感受态细胞中,轻柔混合然后冰上放置30min,完成后,42℃水浴45s,迅速转入冰中放置1min;然后加入890μL的SOC培养基(预先在37℃保温),37℃震荡培养60min;取100μL转化液涂在含有Amp的LB琼脂平板培养基上,37℃过夜培养;
之后,挑取单菌落接种摇瓶扩大培养,然后收集菌液提取重组表达质粒PcoldI-0224;质粒提取采用北京聚合美生物科技有限公司生产的质粒小提试剂盒进行,具体操作按照其说明书;
将提取的重组表达质粒进行双酶切体系验证并测序,测序结果翻译成蛋白序列后与酒酒球菌标准菌株PSU-1中的β-葡萄糖苷酶序列进行比对,结果如图1所示,比对结果显示重组蛋白序列与标准序列相比有6个氨基酸不同,分别位于第1、142、157、234、387、472位。
6、表达:
将步骤5中提取的表达质粒PcoldI-0224适当稀释至终浓度约为1ng/μL,取10μL加入至100μL的BL21(DE3)感受态细胞中,混合然后冰上放置30min,完成后,42℃水浴45s,迅速转入冰中放置1~2min;
然后加入890μL的SOC培养基(预先在37℃保温),37℃震荡培养60min;
取100μL转化液涂在含有Amp的LB琼脂平板培养基上,37℃过夜培养;
挑取转化子到含有100μg/ml Amp的LB培养液中,37℃振荡培养;
当培养液的OD600达到0.4~0.5时,在冰水中迅速冷却培养液到15℃,放置30分钟;然后添加IPTG到终浓度0.4mM,15℃振荡培养24小时;
培养完成后,利用SDS-PAGE测定确认目的产物的有无、表达量和可溶性,SDS-PAGE结果如图4所示。
7、纯化:
收集步骤6中的表达菌株BL21(DE3)培养物(12000xg,10min),加入裂解缓冲液重悬菌体后进行超声破碎,超声时间20min,功率100W,间隔3s;
超声破碎完成后离心(12000xg,10min),可溶性重组蛋白存在于上清液中,收集上清,过0.22μm滤膜;
PcoldI表达载体自身序列含有His标签,将过滤后的重组蛋白BGL0224加入到His重力镍柱中,4℃作用1h后流穿重力柱去除不能挂柱的杂蛋白,然后用300mM咪唑将挂在柱子上的重组蛋白洗脱下来;
将洗脱液通过30kDa超滤管(10000xg,20min)以除去大量的咪唑,并将重组蛋白溶于磷酸盐缓冲液中,冷冻干燥后保存蛋白备用。纯化方案如表1所示,结果显示纯化得到的β-葡萄糖苷酶(重组酶BGL0224)比活力为59.19μkat/g。
酶学性质测定:
重组酶酶活测定采用比色法,以对硝基苯基β-D-吡喃葡萄糖苷(pNPG)为底物,经重组酶BGL0224催化后产物为对硝基苯酚pNP,pNP在碱性条件下显黄色,可在420nm紫外波长下检测。酶活力U的定义为在1分钟内转化1微摩尔底物所需要的酶量称为1个酶活单位。以该方法为基础,测定了重组酶BGL0224的最适温度、热稳定性、最适pH及pH稳定性。
结果如图5、7、9所示:图5表明重组酶BGL0224的最适温度为50℃;图7表明重组酶BGL0224在30℃~50℃时热稳定性较好,在60℃时热稳定性有所降低,70℃时热稳定性较差;图9表明重组酶BGL0224的最适pH为5.0,pH在4.5~6.0之间时,重组酶BGL0224的pH稳定性较好,当pH大于6.0或者小于4.0时,该酶的pH稳定性降低。
对比例:
该对比例采用传统方法对目的基因OEOE-0224进行克隆表达进而制备β-葡萄糖苷酶BGL0224,采用的克隆载体为T-Vector PMD19,表达载体同实施例1一致,均为PcoldI冷表达载体,该对比例的目的在于说明实施例中的操作更简便,且比传统的克隆表达方法得到的结果更好。
具体操作如下:
1、酒类酒球菌SD-2a的培养、基因组DNA的提取、目的基因的扩增及所用引物同实施例1;
2、之后进行T-A克隆:将PCR得到的末端平滑的目的基因片段的3’末端添加“A”尾后进行T-A克隆。
在微量离心管中配制如下的加“A”反应液,全量为50μL:10xA-Tailing Buffer 5μL;dNTP Mixture 4μL;A-Tailing Enzyme 0.5μL;末端平滑的目的基因片段5μL;ddH2O36.5μL。将以上体系置于72℃反应20分钟后冰上静置2分钟;
然后,将A-Tailing目的基因片段与克隆载体T-Vector PMD19连接转化。在微量离心管中配制如下反应液:T-Vector PMD19 1μL;A-Tailing目的基因片段1μL;DNA LigationMix 5μL;ddH2O 3μL。将该连接体系混匀后置于16℃反应30min。将连接体系全量(10μL)加入至100μL的DH5α感受态细胞中,轻柔混合然后冰上放置30min;
完成后,42℃水浴45s,迅速转入冰中放置1min。然后加入890μL的SOC培养基(预先在37℃保温),37℃震荡培养60min。取100μL转化液涂在含有X-Gal、Amp的LB琼脂平板培养基上,37℃过夜培养;
挑取长出的白色菌落(阳性克隆子)进行扩大培养,然后收集菌液提取克隆质粒PMD19T-0224。
3、载体构建:
克隆质粒PMD19T-0224双酶切:采用快切酶NdeI和BamhI对克隆质粒进行双酶切。双酶切反应体系(20μL)为:10xQuickout buffer 2μL;PMD19T-0224 2μL;NdeI 1μL;BamhI1μL;ddH2O 14μL。将该体系置于37℃水浴反应30min后,上样1%琼脂糖凝胶电泳检测并对目的基因条带进行胶回收;
表达载体PcoldI双酶切:采用快切酶NdeI和BamhI对表达载体进行双酶切。双酶切反应体系(20μL)为:10xQuickout buffer 2μL;PcoldI 2μL;NdeI 1μL;BamhI 1μL;ddH2O14μL。将该体系置于37℃水浴反应30min后,上样1%琼脂糖凝胶电泳检测条带并进行胶回收;
扩增的目的基与表达载体PcoldI连接:将回收得到的目的基因和PcoldI质粒进行连接,具体操作同实施例1。本对比例中表达质粒PcoldI-0224的构建过程图如图3所示。
之后采用实施例1相同的方法进行转化和表达,表达完成后,利用SDS-PAGE测定确认目的产物的有无、表达量和可溶性。
该对比例中SDS-PAGE结果如图4所示。1号泳道为大肠杆菌BL21空载,2号泳道为实施例中目的蛋白BGL0224表达结果,3号泳道为对比例中目的蛋白BGL0224的表达结果。
纯化操作同实施例1,具体纯化方案如表1所示。结果显示,对比例中纯化得到的重组酶BGL0224比活力为47.38μkat/g,低于实施例1中纯化得到的重组酶BGL0224的比活力。
表1
对比例酶学性质测定结果如图6、8、10所示:图6表明对比例中重组酶BGL0224的最适温度为45℃;图8表明该重组酶BGL0224在30℃~40℃时热稳定性较好,在50℃时热稳定性有所降低,60℃~70℃时热稳定性较差;图10表明对比例中重组酶BGL0224的最适pH为5.0,pH在4.5~6.0之间时,重组酶BGL0224的pH稳定性较好,当pH大于6.0或者小于4.0时,该酶的pH稳定性降低;该对比例中重组酶BGL0224较实施例中的重组酶BGL0224的热稳定性差,pH稳定性基本一致。
核苷酸序列表电子文件
<110>西北农林科技大学
<120>一种β-葡萄糖苷酶及其制备方法与应用
<210>1
<211>480
<212>氨基酸
<213>
<220>β-葡萄糖苷酶
<400>1
MNKLFLPKNFLWGGAVAANQLEGGWDQDNKGLSVADIMTAGANGKAREITDGIVKGKYYPNHEAIDFYHRYKEDIKLFAEMGFKCFRTSIAWTRIFPNGDEEQPNEAGLKFYDQLFDECHKYGIEPVITLSHFEMPYHLVKVYGGWRNRKLIDFFVHFAKTVFKRYKDKVSYWMTFNEIDNQTDYTNRFLMATNSGLILKNDQSDAESLMYQAAHYELVASALAVKLGHSINPDFQIGCMINMTPVYPASSKPADIFQAEKAMQRRYWFSDIHALGKYPENMEVFLKQNNFRPDITSEDRIVLKEGTVDYIGLSYYNSMTVQSKESNPGFHFIGPELTVDNPNVEKSDWGWPIDPLGLRYSLNWLADHYHKPLFIVENGLGAYDKVENNQQIHDPYRIAYLKAHIQAMIDAVQEDGVKVIGYTPWGCIDLVSAGTGQMSKRYGFIYVDKDDQGKGSLKRLKKDSFFWYQQVIKSNGSQLD
<210>2
<211>5832
<212>DNA
<213>
<220>表达质粒PcoldI-0224基因序列
<400>2
AAGGAATGGTGTGGCCGATTAATCATAAATATGAAAAATAATTGTTGCATCACCCGCCAATGCGTGGCTTAATGCACATCAAATTGTGAGCGGATAACAATTTGATGTGCTAGCGCATATCCAGTGTAGTAAGGCAAGTCCCTTCAAGAGTTATCGTTGATACCCCTCGTAGTGCACATTCCTTTAACGCTTCAAAATCTGTAAAGCACGCCATATCGCCGAAAGGCACACTTAATTATTAAGAGGTAATACACCATGAATCACAAAGTGCATCATCATCATCATCATATCGAAGGTAGGCATATGATGAATAAACTTTTTTTGCCGAAAAATTTTTTGTGGGGAGGTGCCGTAGCGGCCAATCAATTAGAGGGCGGCTGGGACCAAGACAACAAAGGCCTCAGTGTAGCCGACATTATGACTGCCGGAGCCAATGGAAAAGCACGGGAGATTACCGATGGAATTGTTAAAGGCAAGTATTACCCCAATCACGAGGCCATCGACTTTTATCATCGCTATAAAGAAGATATCAAGTTATTCGCCGAGATGGGTTTTAAATGTTTTCGAACCTCGATTGCCTGGACGAGGATCTTTCCCAATGGAGACGAAGAGCAACCCAACGAAGCCGGCTTGAAGTTTTACGACCAGCTATTTGATGAATGCCACAAGTACGGTATCGAACCGGTCATTACCCTCTCACATTTTGAAATGCCCTATCATTTGGTTAAAGCCTACGGCGGCTGGCGTAACCGAAAACTAATCGATTTCTTTGTTCGCTTTGCCAAGACGGTCTTTAAACGTTATAAAGACAAAGTTAGCTACTGGATGACTTTTAATGAGATCGACAACCAAACCGATTATACAAATCGCTTCTTAATGGCTACTAATTCCGGTTTGATATTAAAAAATGATCAAAGTGATGCGGAAAGCTTAATGTATCAGGCGGCTCATTACGAATTGGTTGCCAGTGCTCTAGCCGTCAAGCTTGGCCACAGTATTAATCCTAATTTTCAGATCGGCTGCATGATCAACATGACGCCTGTTTACCCGGCTTCCTCAAAACCAGCTGATATCTTTCAAGCAGAAAAAGCAATGCAAAGGCGCTACTGGTTTTCCGACATTCACGCTCTGGGAAAATATCCGGAAAACATGGAAGTATTTTTGAAACAAAACAATTTTCGCCCGGATATCACGAGCGAGGACCGAATAGTATTAAAAGAAGGAACTGTCGACTATATTGGATTGAGTTATTACAATTCAATGACCGTTCAATCAAAAGAAAGCAACCCGGGCTTTCATTTCATCGGTCCGGAACTGACCGTTGATAATCCAAATGTTGAAAAAAGCGATTGGGGATGGCCGATCGATCCGTTGGGACTTAGGTATTCTTTAAACTGGCTGGCCGACCACTATCACAAGCCCTTGTTCATTGTTGAAAACGGTCTGGGAGCCTATGACAAAGTCGAAAATAGCCAACAGATCCATGACCCTTATCGAATCGCTTATCTAAAAGCTCATATCCAGGCAATGATCGATGCGGTTCAAGAAGACGGGGTTAAGGTCATTGGTTATACGCCCTGGGGTTGTATCGATCTGGTTTCCGCCGGAACCGGACAGATGTCCAAAAGGTACGGTTTTATCTATGTCGATAAAGACGACCAGGGCAAAGGCAGCTTAAAAAGACTGAAAAAGGATTCCTTTTTCTGGTATCAACAGGTTATTCAGTCAAACGGCAGTCAATTAGATTAAGGATCCGAATTCAAGCTTGTCGACCTGCAGTCTAGATAGGTAATCTCTGCTTAAAAGCACAGAATCTAAGATCCCTGCCATTTGGCGGGGATTTTTTTATTTGTTTTCAGGAAATAAATAATCGATCGCGTAATAAAATCTATTATTATTTTTGTGAAGAATAAATTTGGGTGCAATGAGAATGCGCAGGCCCTTTCGTCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATAAAATTGTAAACGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGCCCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCCACTACGTGAACCATCACCCAAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTACTATGGTTGCTTTGACGTATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATAGTCATGCCCCGCGCCCACCGGAAGGAGCTGACTGGGTTGAAGGCTCTCAAGGGCATCGGTCGAGATCCCGGTGCCTAATGAGTGAGCTAACTTACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGGTTTTTCTTTTCACCAGTGAGACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTCCACGCTGGTTTGCCCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTAACGGCGGGATATAACATGAGCTGTCTTCGGTATCGTCGTATCCCACTACCGAGATATCCGCACCAACGCGCAGCCCGGACTCGGTAATGGCGCGCATTGCGCCCAGCGCCATCTGATCGTTGGCAACCAGCATCGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTTTGTTGAAAACCGGACATGGCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCTGAATTTGATTGCGAGTGAGATATTTATGCCAGCCAGCCAGACGCAGACGCGCCGAGACAGAACTTAATGGGCCCGCTAACAGCGCGATTTGCTGGTGACCCAATGCGACCAGATGCTCCACGCCCAGTCGCGTACCGTCTTCATGGGAGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGACATCAAGAAATAACGCCGGAACATTAGTGCAGGCAGCTTCCACAGCAATGGCATCCTGGTCATCCAGCGGATAGTTAATGATCAGCCCACTGACGCGTTGCGCGAGAAGATTGTGCACCGCCGCTTTACAGGCTTCGACGCCGCTTCGTTCTACCATCGACACCACCACGCTGGCACCCAGTTGATCGGCGCGAGATTTAATCGCCGCGACAATTTGCGACGGCGCGTGCAGGGCCAGACTGGAGGTGGCAACGCCAATCAGCAACGACTGTTTGCCCGCCAGTTGTTGTGCCACGCGGTTGGGAATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTTTCGCAGAAACGTGGCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAGACACCGGCATACTCTGCGACATCGTATAACGTTACTGGTTTCACATTCACCACCCTGAATTGACTCTCTTCCGGGCGCTATCATGCCATACCGCGAAAGGTTTTGCGCCATTCGATGGTGTCCGGGATCTCGACGCTCTCCCTTATGCGACTCCTGCATTAGGAAGCAGCCCAGTAGTAGGTTGAGGCCGTTGAGCACCGCCGCCGC
<210>3
<211>33
<212>DAN
<213>
<220>上游引物
<400>3
5’- CTCCATATGATGAATAAACTTTTTTTGCCGAAA -3’
<210>4
<211>33
<212>DNA
<213>
<220>下游引物
<400>4
5’- TGAGGATCCTTAATCTAATTGACTGCCGTTTGA -3’
Claims (6)
1.一种β-葡萄糖苷酶,其特征在于,所述β-葡萄糖苷酶的氨基酸序列如SEQ ID NO:1所示。
2.权利要求1所述β-葡萄糖苷酶的制备方法,其特征在于,包括以下步骤:
(1)基因组DNA的提取:提取酒类酒球菌SD-2a的基因组DNA;
(2)目的基因扩增:以步骤(1)得到的基因组DNA为模板,进行聚合酶链式反应,扩增目的基因;
(3)载体构建:将扩增得到的目的基因插入表达载体PcoldI构建表达质粒PcoldI-0224;
(4)转化:将表达质粒PcoldI-0224导入大肠杆菌感受态细胞DH5α,得到转化子;
(5)表达:将转化子进行扩大培养,提取培养物中的重组表达质粒PcoldI-0224导入感受态细胞BL21,之后进行扩大培养;
(6)纯化:对步骤(5)的培养物依次进行裂解、过滤,之后依次采用His重力镍柱和超滤管收集β-葡萄糖苷酶。
3.如权利要求2所述的β-葡萄糖苷酶的制备方法,其特征在于,步骤(1)中提取对数中期酒类酒球菌SD-2a的基因组DNA。
4.如权利要求2中所述的β-葡萄糖苷酶的制备方法,其特征在于,步骤(2)扩增时所用引物序列分别为:
上游引物:5’-CTCCATATGATGAATAAACTTTTTTTGCCGAAA-3’,
下游引物:5’-TGAGGATCCTTAATCTAATTGACTGCCGTTTGA-3’。
5.如权利要求2所述的β-葡萄糖苷酶的制备方法,其特征在于,步骤(2)中所用的DNA聚合酶为PrimeSTAR Max DNA Polymerase,PCR反应程序为:95℃预变性10min;98℃变性10s,55℃退火15s,72℃延伸60s,30次循环;72℃保温8min。
6.权利要求1所述β-葡萄糖苷酶用于葡萄酒酿制的应用。
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Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN112143607A (zh) * | 2020-11-16 | 2020-12-29 | 辽宁省农业科学院 | 南果梨白兰地低温回香制备方法 |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103881958A (zh) * | 2014-03-31 | 2014-06-25 | 上海交通大学 | 基于β-葡萄糖苷酶的工程菌及其实现方法 |
WO2015119219A1 (ja) * | 2014-02-06 | 2015-08-13 | 国立大学法人東北大学 | 新規β-グルコシダーゼおよび同酵素を用いる易分解性セサミノール配糖体の製造方法 |
CN105754923A (zh) * | 2016-05-04 | 2016-07-13 | 云南农业大学 | 一种基于β-葡萄糖苷酶的工程菌及其实现方法 |
CN107828806A (zh) * | 2017-08-15 | 2018-03-23 | 广东药科大学 | 一种新型耐葡萄糖的β‑葡萄糖苷酶基因及其应用 |
-
2019
- 2019-11-21 CN CN201911146480.1A patent/CN111304182A/zh active Pending
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2015119219A1 (ja) * | 2014-02-06 | 2015-08-13 | 国立大学法人東北大学 | 新規β-グルコシダーゼおよび同酵素を用いる易分解性セサミノール配糖体の製造方法 |
CN103881958A (zh) * | 2014-03-31 | 2014-06-25 | 上海交通大学 | 基于β-葡萄糖苷酶的工程菌及其实现方法 |
CN105754923A (zh) * | 2016-05-04 | 2016-07-13 | 云南农业大学 | 一种基于β-葡萄糖苷酶的工程菌及其实现方法 |
CN107828806A (zh) * | 2017-08-15 | 2018-03-23 | 广东药科大学 | 一种新型耐葡萄糖的β‑葡萄糖苷酶基因及其应用 |
Non-Patent Citations (4)
Title |
---|
GENBANK: ""6-phospho-beta-glucosidase [Oenococcus oeni]",Accession Number:WP_002822493.1", 《GENBANK》 * |
HYO-MIN PYEON ET AL.: ""Cloning, purification, and characterization of GH3 β-glucosidase, MtBgl85, from Microbulbifer thermotolerans DAU221"", 《PEERJ》 * |
YANG,S.: ""Oenococcus oeni strain 31-DH 6-phospho-beta-glucosidase gene, complete cds",Accession Number:KM435260.1", 《GENBANK》 * |
李亚辉: ""酒类酒球菌SD-2a和31MBR的β-D-葡萄糖苷酶研究"", 《中国博士学位论文全文数据库 工程科技Ⅰ辑》 * |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN112143607A (zh) * | 2020-11-16 | 2020-12-29 | 辽宁省农业科学院 | 南果梨白兰地低温回香制备方法 |
CN112143607B (zh) * | 2020-11-16 | 2023-12-01 | 辽宁省农业科学院 | 南果梨白兰地低温回香制备方法 |
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