CN114774394A - 一种热稳定β半乳糖苷酶及其合成甘油半乳糖苷的应用 - Google Patents
一种热稳定β半乳糖苷酶及其合成甘油半乳糖苷的应用 Download PDFInfo
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- CN114774394A CN114774394A CN202210420830.4A CN202210420830A CN114774394A CN 114774394 A CN114774394 A CN 114774394A CN 202210420830 A CN202210420830 A CN 202210420830A CN 114774394 A CN114774394 A CN 114774394A
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- galactosidase
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Abstract
本发明提供了一种热稳定的β半乳糖苷酶及其相关重组工程菌株,涉及重组酶催化工程技术领域。本发明所述β半乳糖苷酶BtGal42可在50℃以半乳糖作为糖基供体,甘油为糖基受体,进行酶法合成甘油半乳糖苷,该温度下酶稳定性良好,相对于现有技术酶的使用量显著降低,降低了生产成本。使用本发明所述β半乳糖苷酶用于合成反应时可以通过提高反应温度,从而降低染菌风险,提高底物的反应效率,进而提高甘油半乳糖苷的生产效率,能够完全满足工业应用需求。
Description
技术领域
本发明属于重组酶催化工程技术领域,具体涉及一种热稳定β半乳糖苷酶及其合成甘油半乳糖苷的应用。
背景技术
甘油半乳糖苷是一种用途广泛的天然小分子醇溶性半乳糖苷,其由半乳糖基和甘油组成,在红藻和赤潮藻细胞内多有发现。甘油半乳糖苷及其酯类衍生物在食品、化妆品、保健品甚至抗肿瘤药物中都有令人关注的用途。相比于具有半缩醛羟基的单糖,甘油半乳糖苷对强酸强碱等环境具有更高的稳定性,在藻类细胞内可有助于细胞的渗透适应,提高细胞抗冻或耐热作用,帮助细胞抵御极端环境。由于甘油半乳糖苷可生物降解,对环境没有毒害作用,且具有良好的保湿效果,因而在化妆品中多有应用。Colombo等人的研究揭示了甘油半乳糖苷的抗肿瘤活性,甘油半乳糖苷对Epstein–Barr病毒的激活和皮肤癌的促进具有抑制作用,故在抗肿瘤药物中也有一定应用。
甘油半乳糖苷可与脂肪酸一同作为底物,通过脂肪酶催化的选择性转酯化反应合成糖脂类物质。糖脂广泛存在于各种生物体中,如蔷薇中的但半乳糖甘油二酯,菠菜中的磺基-6-脱氧葡糖基二脂酰甘油、单半乳糖基二脂酰甘油、二半乳糖基二脂酰甘油等。糖脂具有多种重要的生物学功能,如对肿瘤细胞的抑制作用、抗炎活性、抗氧化、抗病毒、增强机体免疫功能等。甘油糖脂虽然分布广泛,但分离困难,天然提取法存在回收率低、产物不纯的缺陷,不能满足市场需求,故需要人工合成以提高产量。
作为糖脂合成的前体物质,甘油半乳糖苷的合成主要有化学合成和酶法合成。化学法得到的产品一般为有多种副产物,且合成工艺步骤繁琐,需经过保护、偶联、去保护等过程才能合成纯度较高的产品。酶法合成条件温和,步骤简单,主要有转糖苷法和逆水解法两种。转糖苷法合成通常使用农业废弃物乳糖作为糖基供体,反应成本较低,但乳糖可同时作为糖基受体合成低聚半乳糖,导致产物成分复杂,加重纯化负担。Wei Wei等人(Synthesis and characterisation of galactosyl glycerol by b-galactosidasecatalysed reverse hydrolysis of galactose and glycerol),公开了利用源于Kluyveromyces lactis的半乳糖苷酶以半乳糖和甘油作为底物进行逆水解法合成甘油半乳糖苷,在温度40℃最佳反应条件下,甘油半乳糖苷的产量达116.47mg/ml(半乳糖转化率55.88%),但其合成体系中用酶量很大,达到240U/ml,且40℃的反应温度存在染菌风险。
发明内容
本发明通过对多种来源半乳糖苷酶的筛选与研究,获得了一种来源于Bifidobacterium thermophilum NJ-5的β半乳糖苷酶(命名为BtGal42),具有良好的耐热稳定性。
本发明具体技术方案如下:
一种β半乳糖苷酶,所述酶分子包含SEQ ID NO:1所示含氨基酸序列。
本发明另一目的在于提供一种DNA分子,所述DNA分子编码权利要求1所述的β半乳糖苷酶。优选核苷酸序列如SEQ ID NO:2所示。
本发明另一目的在于提供一种β半乳糖苷酶的表达载体,表达本发明所述的β半乳糖苷酶。所述表达载体可以为质粒、噬菌体、病毒或宿主细胞。
所述宿主细胞为原核细胞或真核细胞,可以为大肠杆菌、酵母、芽孢杆菌、乳酸杆菌、曲霉或木霉,优选大肠杆菌。
本发明另一目的在于提供本发明所述的β半乳糖苷酶,DNA分子或表达载体在甘油半乳糖苷中的应用。
本发明优点:
现有技术下,β半乳糖苷酶的最佳反应温度为40℃,当温度高于50℃时,甘油半乳糖苷产率迅速下降。然而40℃的温度下存在杂菌生长的情况,导致反应底物或产物被消耗。此外,研究表明,当反应时间延长至48小时,甘油半乳糖苷的产率出现降低以及被降解。因此现有技术反应中需要极高的β半乳糖苷酶的用量(240U/ml)才能保证理想的产率。
本发明为解决甘油半乳糖苷合成过程中用酶量大、生产温度低的问题,对新酶进行挖掘,得到了一株新β半乳糖苷酶BtGal42。所述β半乳糖苷酶的最适温度为50℃,其对于高温度耐受良好,在50℃条件下保温2h后,活力仍有81%。本发明所述β半乳糖苷酶合成甘油半乳糖苷的催化用量仅为10U/ml,与现有技术相比有了显著的降低,极大地减少了生产成本。将本发明所述β半乳糖苷酶应用于甘油半乳糖苷制备,结果显示在酶解48h后产量趋于稳定,甘油半乳糖苷的最高产量为62.2g/L,底物转化率为41.2%。且随着反应时间的延长,产量维持稳定,未见下降。这一结果表明,应用本发明所述半乳糖苷酶的生产工艺与现有技术相比,适应更高的反应温度,以及更长的反应时间,该温度下可有效抑制杂菌生长,减少反应底物的消耗损失,能够完全满足工业应用需求,将其应用于甘油半乳糖苷的合成反应中能够提高产物的生产效率。
附图说明
构成本申请的一部分的附图用来提供对本发明的进一步理解,本发明的示意性实施例及其说明用于解释本发明,并不构成对本发明的不当限定。
图1为PCR扩增β半乳糖苷酶BtGal42基因的核酸电泳分析。
图2为β半乳糖苷酶BtGal42的SDS-PAGE电泳分析。泳道1为空白对照,泳道2为胞内表达的可溶性粗酶液,泳道3为纯化后的纯酶。
图3为β半乳糖苷酶BtGal42的最适温度分析。
图4为β半乳糖苷酶BtGal42的温度稳定性分析。
图5为β半乳糖苷酶BtGal42合成甘油半乳糖苷的时间曲线
具体实施方式
为了使本领域的技术人员更好地理解本发明方案,下面结合附图和实施实例对本发明做进一步说明。需要指出的是,本实施例仅用于解释本发明,而非对本发明范围的限制。显然,所描述的实施例仅仅是本发明的一部分实施例,而不是全部的实施例,基于本发明的实施例,本领域技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都应当属于本发明保护的范围。
实施例1用于表达半乳糖苷酶的工程菌的构建
本发明所述β-半乳糖苷酶来源菌株为Bifidobacterium thermophilum NJ-5,其氨基酸序列如SEQ ID NO:1所示,为提高蛋白表达量,构建重组大肠杆菌表达载体。
以菌株Bifidobacterium thermophilum NJ-5基因组DNA为模板,高保真酶2×Phanta Max Master Mix(南京诺维赞生物科技有限公司),和引物对B-F(5′CGGGATCCATGACAGCACGCAGAACACATCG 3′,BamH I,SEQ ID NO:3)和B-R(5′CCGCTCGAGTCAACCCATGCTGACGATGACG 3′Xho I,SEQ ID NO:4)进行PCR扩增,实验操作参照Vazyme生物产品及操作手册。PCR产物核酸电泳见图1,扩增得到的编码基因DNA片段应为2000bp,核酸电泳验证条带长度正确,其碱基序列如SEQ ID NO:2所示。扩增得到的DNA片段经Dpn I进行酶切处理,消化模板DNA。
消化结束后对PCR产物进行纯化,去除体系中的引物、酶蛋白、单核苷酸等,本步骤使用AxyPrep PCR Clean up试剂盒完成。
纯化得到的DNA片段与载体pET28a(+)共同进行双酶切,使用的限制性内切酶为BamH I和Xho I(宝日医生物技术有限公司),酶切操作菌采用50μl体系,按照DNA限制性内切酶生产商的说明书进行配制和酶切反应。酶切完成后使用凝胶回收,载体DNA与片段DNA的连接按物质的量比1:3进行连接,连接体系按照10μl配制,采用T4连接酶催化连接反应,16℃连接过夜,得到质粒pET28a-BtGal42。连接结束后,将连接液采用热激法转化至大肠杆菌感受态细胞E.coli BL21(DE3),并涂布于含有100μg/ml硫酸卡那霉素LB琼脂平板上,37℃培养14-16h。经序列测定(由安徽通用生物公司完成)验证测序结果,获得相应重组菌株E.coli BL21(DE3)-BtGal42。
实施例2β半乳糖苷酶BtGal42在大肠杆菌中的表达
将实施例1构建得到的重组菌株接种至含有100μg/mL硫酸卡那霉素的50mL的LB液体培养基中,37℃,180rpm过夜培养;以2%的接种量将种子液,接种至新鲜的50mL的LB液体培养基中,37℃,180rpm培养至OD600为0.6~1.0时,取出经冰水浴冷却5min,加入诱导剂IPTG(异丙基-β-D硫代半乳糖苷)(终浓度0.5mmol/L),20℃,150rpm诱导表达20h。
取诱导表达的发酵液,12000rpm离心20min,弃去上清,然后用50mM Na2HPO4-KH2PO4(pH 7.0)缓冲重悬清洗菌体,12000rpm离心20min,弃去上清,用缓冲再次重悬,然后超声破碎。破碎液离心12000rpm,20min,取上清进行SDS-PAGE电泳检测,浓缩胶浓度为4%,分离胶浓度为12.5%,样品与上样缓冲按照3:1的比例混合,沸水浴反应5min进行上样电泳。电泳仪设定初始电压为120V,当样品移动至分离胶时提高电压至230V,直至样品移动到电泳槽底部时结束电泳。
上述粗酶液SDS-PAGE电泳结果如图2所示,BtGal42分子量为77kDa,与计算得到的分子量一致,表明BtGal42成功诱导表达,约占可溶总蛋白的60%左右。
实施例3半乳糖苷酶BtGal42酶活的测定程序
将实施例1中构建的重组菌株按实施例2方法发酵培养,获得的蛋白粗酶液以β-oNPG为底物测定酶活变化,测定方法如下:
酶活力单位定义:一个酶活力单位即为在50℃,pH 7.0条件下,每分钟催化β-oNPG水解生成1μmol oNP所需的酶量。
准确称取30mgβ-oNPG,溶于10mL的Na2HPO4-KH2PO4缓冲中(50mM,pH 7.0),搅拌混匀后得到10mmol/L浓度的底物溶液。准确吸取240μl底物溶液至96孔板,加入10μl适当稀释的酶液,以灭活的酶反应液为对照,50℃反应10min后,用酶标仪测定410nm波长处吸光值。绘制吸光值与反应液中oNP浓度的标准曲线以进行酶活计算。
实施例4β半乳糖苷酶BtGal42的纯化
由于β半乳糖苷酶BtGal42的N端融合了六个组氨酸(His)标签,Ni柱中的氯化镍可以与含有His标签的蛋白结合,也可以与咪唑结合,因此使用镍柱分别对目的蛋白进行纯化。实施例2发酵表达所得的的菌液经离心破碎后,取上清液经0.22μm滤膜过滤。用BufferA(20mM Tris-HCl,pH 7.5)以2mL/min的流速冲洗镍柱至平衡,用注射器将蛋白样品打入进样环中,收集样品的穿透峰蛋白,再次用Buffer A(20mM Tris-HCl,pH 7.5)冲洗镍柱,直至无蛋白被洗脱出来。使用梯度洗脱法,每个梯度用至少5倍体积的Buffer B(20mMTris-HCl,500mM咪唑,pH 7.5)冲洗镍柱,收集每个梯度的吸收峰蛋白,直至无蛋白被洗脱出来。镍柱纯化得到的蛋白溶液使用GE公司预装的脱盐柱,以50mM Na2HPO4-KH2PO4(pH 7.0)缓冲置换含有咪唑的Buffer,去除蛋白液中的咪唑。将收集得到的不同梯度的蛋白溶液进行SDS-PAGE验证,20%Buffer B洗脱时得到的目的蛋白纯度较高,见图2。
实施例5大肠杆菌表达半乳糖苷酶的稳定性分析
为了确定逆水解反应的最佳反应温度,测定β半乳糖苷酶BtGal42的最适反应温度和温度稳定性。
最适温度:将实施例4得到的纯化酶进行适当稀释后,分别加入实施例3中配制的底物溶液,放入30℃、35℃、40℃、45℃、50℃、55℃、60℃、70℃水浴锅中反应10min后,测定410nm波长下吸收,并按标准曲线计算各温度下酶活,如图3所示,BtGal42的最适温度为50℃。
温度稳定性:将实施例4得到的纯化酶进行适当稀释后,分别于30℃、40℃、45℃、50℃、55℃、60℃、65℃、70℃水浴锅中孵育2h,孵育结束后,取样按照实施例3中酶活测定方法检测其剩余的酶活力。以0h最高的酶活为100%,计算各温度孵育后的相对酶活,绘制不同孵育条件下残余酶活的变化曲线。结果如图4所示,BtGal42在30~7℃范围内保温2h后,酶活损失随温度升高而加剧。在50℃及以下保温2h时,原始酶酶活损失较少,保留80%的酶活力,55℃孵育后残余酶活低于50%,60℃及以上孵育后BtGal42完全失活。综合考虑最适温度和温度稳定性数据,选定50℃作为合成反应的反应温度。
实施例6半乳糖苷酶应用于甘油半乳糖苷合成
将实施例3得到的纯酶,按实施例2所述方法检测酶活,取10U/ml酶液配制反应体系。反应体系中加入0.6mmol底物半乳糖,6mmol底物甘油,以50mM Na2HPO4-KH2PO4(pH 7.0)补足10ml,以失活酶作为对照。据实施例4的最适温度和热稳定性数据选定50℃作为反应温度,在50℃,200rpm下反应72小时,定期取出少量样品,通过HPLC法测定转化率。HPLC检测方法:色谱柱为Bio-Rad Aminex HPX-87H色谱柱(300mm×7.8mm),流动相5mMH2SO4,流速0.3ml·min-1;示差折光检测器;柱温:50℃。甘油半乳糖苷出峰时间:18.5min,半乳糖出峰时间:19.6min,甘油出峰时间:27.1min。反应进程曲线见图5,0-24h内产物甘油半乳糖苷迅速积累,24h后积累速度降低,至48h达到反应平衡,48-96h产率维持在温度状态,甘油半乳糖苷的最高产量为62.2g/L(半乳糖转化率41.2%)。
以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
序列表
<110> 南京工业大学
<120> 一种热稳定β半乳糖苷酶及其合成甘油半乳糖苷的应用
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 695
<212> PRT
<213> Artificial Sequence
<400> 1
Met Thr Ala Arg Arg Thr His Arg Trp Pro Arg Pro Leu Asp Asn Ala
1 5 10 15
Ala Arg Arg Ile Trp Tyr Gly Gly Asp Tyr Asn Pro Glu Gln Trp Pro
20 25 30
Glu Ser Val Trp Asp Glu Asp Ile Glu Leu Met Asn Gln Ala Gly Val
35 40 45
Asn Ile Val Ser Leu Gly Ile Phe Ala Trp Ser Ala Ile Glu Pro Glu
50 55 60
Glu Gly Val Tyr Asp Phe Gly Trp Leu Asp Arg Ile Ile Asp Lys Leu
65 70 75 80
Tyr His Ala Gly Ile Ala Val Asp Leu Ala Ser Ala Thr Ala Ser Pro
85 90 95
Pro Leu Trp Leu Thr Gln Lys His Pro Glu Val Leu Trp Lys Asp Glu
100 105 110
Arg Gly Asp Ala Cys Trp Pro Gly Ala Arg Gln His Trp Arg Pro Thr
115 120 125
Ser Pro Ile Phe Arg Glu Tyr Ala Leu Arg Leu Cys Arg Ala Met Ala
130 135 140
Gln His Tyr Arg Asp Asn Pro Ala Val Val Ala Trp His Val Ser Asn
145 150 155 160
Glu Tyr Gly Cys His Asn Arg Phe Asp Tyr Ser Asp Asp Ala Leu Arg
165 170 175
Ala Phe Gln Arg Trp Cys Lys Glu Arg Tyr Gly Asp Ile Glu Ala Val
180 185 190
Asn Asp Ala Trp Gly Thr Tyr Phe Trp Ser Gln Arg Met Thr Asp Phe
195 200 205
Ser Gln Ile Ile Pro Pro Arg Tyr Ile Gly Glu Gly Asn Phe Met Asn
210 215 220
Pro Gly Lys Leu Leu Asp Phe Lys Arg Phe Cys Ser Asp Ala Leu Lys
225 230 235 240
Glu Phe Phe Lys Ala Glu Arg Asp Ala Leu Ala Glu Ile Thr Pro Asn
245 250 255
Ile Pro Leu Thr Thr Asn Phe Met Val Ser Ala Pro Gly Asn Ala Leu
260 265 270
Asp Tyr Asp Asp Trp Gly Asp Glu Val Asp Phe Val Ser Asn Asp His
275 280 285
Tyr Phe Thr Pro Gly Arg Arg His Phe Asp Glu Leu Ala Tyr Ser Ser
290 295 300
Ser Leu Val Asp Gly Ile Ser Arg Lys Gln Pro Trp Phe Leu Met Glu
305 310 315 320
His Ser Thr Ser Ala Val Asn Trp Arg Ser Ile Asn Tyr Arg Lys Glu
325 330 335
Pro Gly Gln Leu Glu Arg Asp Ala Met Ala His Leu Ala Met Gly Ala
340 345 350
Asp Ala Ile Cys Phe Phe Gln Trp Arg Gln Ser Gln Ala Gly Ala Glu
355 360 365
Lys Phe His Ser Gly Met Val Pro His Ala Gly Arg Asn Ser Gln Val
370 375 380
Tyr Arg Asp Val Cys Ala Leu Gly Asn Asp Leu Asp Thr Leu Ser Gln
385 390 395 400
Ala Gly Leu Pro Gly Thr Thr Leu Ser Gln Ala Arg Ile Ala Val Val
405 410 415
Tyr Asp Tyr Ala Ser Glu Trp Ala Thr Glu His Thr Ala Thr Pro Thr
420 425 430
Gln Gln Val Arg His Trp Thr Glu Pro Leu Asp Trp Phe Thr Ala Leu
435 440 445
Ala Asp Gln Gly Leu Thr Ala Asp Val Val Pro Leu Ala Gly Asp Trp
450 455 460
Asp Arg Tyr Glu Met Val Val Leu Pro Ser Val Tyr Leu Leu Gly Glu
465 470 475 480
Asn Asp Ala Arg Arg Val Arg Asp Tyr Val Ala Ala Gly Gly Lys Leu
485 490 495
Phe Ala Thr Tyr Tyr Thr Gly Ile Ser Asp Glu Arg Asp His Val Trp
500 505 510
Leu Gly Gly Tyr Pro Gly Ala Ile Arg Asp Val Val Gly Val Arg Ser
515 520 525
Glu Glu Phe Ala Pro Met Gly Asp Asp Asp Gly Val Leu Asp His Leu
530 535 540
Asp Leu Ser Asn Gly Thr Val Ala His Asp Ile Ala Asp Val Ile Thr
545 550 555 560
Ser Thr Ala Asp Ser Ala Arg Ile Leu Ala Thr Tyr Gln Ala Asp Ser
565 570 575
Trp Thr Gly Met Asn Gly Val Pro Ala Ile Thr Val Asn Thr Tyr Gly
580 585 590
Glu Gly Arg Ala Ala Tyr Val Gly Cys Arg Leu Gly Ala Asp Gly Leu
595 600 605
Ala Ala Ser Leu Pro Ala Met Phe Glu Ala Met Asp Val Ser Val Pro
610 615 620
Ala Trp Gln Gly Ser Gly Asp Ile Leu Arg Val Val Arg Glu Gly Arg
625 630 635 640
Gly Asp Gly Glu His Glu Ala Pro Arg Phe Thr Phe Gln Phe Asn Arg
645 650 655
Thr His His Pro Val Ala Ala Asp Ile Glu Gly Arg Thr Ile Val Ala
660 665 670
Ser Leu Ala Glu Asp His Gly Asp Gly Thr Ala Thr Ile Ala Pro Asn
675 680 685
Gly Val Ile Val Ser Met Gly
690 695
<210> 2
<211> 2088
<212> DNA
<213> Artificial Sequence
<400> 2
atgacagcac gcagaacaca tcgctggcca agaccgctgg acaatgccgc ccggcgtatc 60
tggtacggcg gagactacaa tcccgaacaa tggccggaat ccgtctggga tgaagatatc 120
gagctcatga accaggccgg ggtcaatatc gtgtcccttg gtattttcgc atggagtgcg 180
atagaacccg aggaaggcgt ctacgatttc gggtggctcg accggatcat cgacaagctg 240
taccatgccg gcatcgccgt cgaccttgca tccgcgaccg ccagcccgcc gctgtggctg 300
acgcagaagc acccggaagt gctgtggaag gacgagcgcg gcgacgcctg ctggcccgga 360
gcgcgacagc attggcgtcc caccagcccg attttccgcg agtacgcctt gcggctgtgc 420
cgtgccatgg cacagcatta ccgggacaac ccggccgtcg tggcctggca tgtgagcaac 480
gagtacggct gccacaaccg gtttgactat tccgatgacg ccctgcgcgc cttccagcgc 540
tggtgcaagg aacgctacgg ggacatcgag gcggtcaacg acgcatgggg cacctacttc 600
tggtcccagc gcatgaccga tttctcccag atcatcccgc cgcggtacat cggggaaggc 660
aatttcatga acccgggcaa actgcttgat ttcaagcggt tctgctccga cgcgctcaag 720
gagttcttca aggcggagcg tgacgcgctc gccgaaatca ccccgaatat ccccctgacc 780
acaaacttca tggtcagcgc gcccggcaac gcactcgatt atgacgattg gggcgatgaa 840
gtcgatttcg tgtcgaatga ccactacttc accccgggac gccggcattt tgatgagttg 900
gcctactcgt cctcgctggt cgacggaatc agccgtaaac agccgtggtt cctcatggaa 960
cattcgacca gcgccgtcaa ctggcgtagt atcaactatc gcaaggaacc cgggcagctg 1020
gaacgcgacg cgatggcgca tctggccatg ggcgcggacg ccatctgctt cttccagtgg 1080
cggcagtccc aagcgggtgc cgagaaattc cacagcggca tggtgcctca cgcgggacgg 1140
aacagccagg tctaccgtga cgtctgcgcg ctcggcaatg acctggacac gctctcgcag 1200
gcgggattgc ccggcactac actgagccag gcacggattg ccgtcgtcta cgattacgcg 1260
agcgaatggg ccaccgagca tacggcgaca cccacccagc aggtgcggca ttggacggaa 1320
ccgctcgact ggttcaccgc gctggctgat caggggctga ccgccgatgt ggtgccgctg 1380
gcgggcgatt gggatcgcta cgagatggtc gttctgccga gcgtgtacct gctgggcgaa 1440
aacgatgcgc ggcgcgtgcg tgattatgtc gcggcaggcg gcaagctgtt cgccacctac 1500
tacactggta tcagcgacga acgcgaccac gtgtggctcg gcggctatcc gggggccatc 1560
cgcgatgttg tcggcgtgcg ttccgaggaa ttcgccccga tgggtgacga cgacggcgtg 1620
cttgatcacc tcgacctgag caacggcacc gtggcacacg acatcgcgga cgtcatcacc 1680
tcgaccgcgg attccgcacg tatacttgcc acctaccaag ccgattcgtg gaccggtatg 1740
aacggcgtcc ccgccatcac ggtcaacaca tatggcgagg ggcgtgccgc ctacgtcggt 1800
tgccgattgg gcgctgacgg gcttgccgca agcctgccgg ccatgttcga ggccatggac 1860
gtgagcgtac cggcatggca aggttccggc gacattctgc gcgtggtgcg tgaggggcgc 1920
ggcgacggcg aacatgaggc gccgcggttc acgttccagt tcaaccgtac gcaccatccg 1980
gtcgcagcgg atatcgaagg gcgtaccatc gtcgcatctc tcgccgaaga ccatggcgac 2040
ggaacggcga ccatcgcccc gaacggcgtc atcgtcagca tgggttga 2088
<210> 3
<211> 31
<212> DNA
<213> Artificial Sequence
<400> 3
cgggatccat gacagcacgc agaacacatc g 31
<210> 4
<211> 31
<212> DNA
<213> Artificial Sequence
<400> 4
ccgctcgagt caacccatgc tgacgatgac g 31
Claims (8)
1.一种β半乳糖苷酶,其特征在于所述酶包含SEQ ID NO:1所示氨基酸序列。
2.一种DNA分子,其特征在于,所述DNA分子编码权利要求1所述的β半乳糖苷酶。
3.一种β半乳糖苷酶的表达载体,其特征在于表达权利要求2所述的β半乳糖苷酶基因。
4.如权利要求3所述的表达载体,其特征在于含有权利要求2所述的DNA分子。
5.如权利要求4所述的表达载体,其特征在于所述表达载体为质粒、噬菌体、病毒或宿主细胞。
6.根据权利要求5所述的表达载体,其特征在于所述宿主细胞为原核细胞或真核细胞。
7.根据权利要求5所述的表达载体,其特征在于所述宿主细胞选自大肠杆菌、酵母、芽孢杆菌、乳酸杆菌、曲霉或木霉。
8.根据权利要求1所述的β半乳糖苷酶,权利要求2所述的DNA分子或权利要求3-7任一项所述的β半乳糖苷酶的表达载体在甘油半乳糖苷合成中的应用。
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CN102559571A (zh) * | 2012-01-18 | 2012-07-11 | 福建农林大学 | 可表达耐热β-半乳糖苷酶的重组菌及构建方法和应用 |
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CN101993864A (zh) * | 2009-08-13 | 2011-03-30 | 中国农业大学 | 一种耐高温β-半乳糖苷酶及其编码基因与应用 |
CN102559571A (zh) * | 2012-01-18 | 2012-07-11 | 福建农林大学 | 可表达耐热β-半乳糖苷酶的重组菌及构建方法和应用 |
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