CN113337528B - 一种甘露糖苷酶的工程菌株及其应用 - Google Patents
一种甘露糖苷酶的工程菌株及其应用 Download PDFInfo
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Abstract
本发明公开了一种甘露糖苷酶的工程菌株及其应用,属于生物工程技术领域。本发明采用来源于嗜热菌Thermobifida fusca来源的甘露糖苷酶编码TFMS,重组至大肠杆菌表达系统实现了高效活性表达,重组表达的TFMS酶可以水解魔芋甘露聚糖、瓜尔豆胶、刺槐豆胶以及棕榈甘露聚糖等底物,产生的甘露寡聚糖产物主要包括甘露糖、二糖、三糖以及寡糖。本发明为高效制备甘露寡糖奠定了一定的基础,适合于工业化生产应用。
Description
技术领域
本发明涉及一种甘露糖苷酶的工程菌株及其应用,属于生物工程技术领域。
背景技术
甘露聚糖(Mannan)是一种广泛存在于自然界中的半纤维素聚多糖。广泛存在于魔芋粉、瓜儿豆胶、田著胶和石斛等植物体及多种微生物细胞壁内。依据来源不同,可将其分为4个子家族,分别为纯甘露聚糖、半乳甘露聚糖、葡甘露聚糖、半乳葡甘露聚糖。甘露聚糖是一种优良的低热量、高纤维素的水溶性膳食纤维,已在造纸纺织、石油工业、化妆品、食品和医药等行业广泛应用。甘露聚糖作为一种膳食补充剂和食品添加剂,被广泛应用于调节溶解度,甜味剂和改善食品风味等。甘露寡糖作为生物体内重要的代谢中间产物,广泛参与合成糖蛋白和免疫调节。甘露糖具有一定的免疫原性,能够刺激机体免疫应答,从而增强动物体的细胞和体液免疫反应。甘露寡糖也能可通过刺激肝脏分泌甘露结合蛋白,激活补体系统发挥调理和天然抗感染免疫的功能,对肠道病原微生物有很好的识别、黏附和排除作用,在畜牧生产中有效代替抗生素添加剂的滥用弊端。而最新研究表明,甘露寡糖可以显著抑制肿瘤的生长,在体内甘露糖联合化疗药物如药阿霉素等使用,可以显著提高抗癌效果,并可明显延长机体生存期。
由于天然甘露聚糖分子量高、粘稠度大,使其在医药和食品加工领域的使用受到严重限制。提纯后的产品还需要进一步通过酸水解、酶法水解或者物理降解等方法制备为低聚甘露糖使用,而水解程度的不一也会对产品的质量稳定性产生较大的影响。因而这些因素导致了医药级和食品级的甘露聚糖生产成本居高不下。两种主要的甘露聚糖降解酶是β-甘露聚糖酶(1,4-β-D-甘露聚糖水解酶,EC 3.2.1.78)和β-甘露糖苷酶(1,4-β-D-甘露糖苷水解酶,EC3.2.1.25)。β-甘露聚糖酶是一种内切酶,催化甘露聚糖主链上β-1,4键的随机水解,产生不同分子量的寡聚糖产物。而β-甘露糖苷酶是一种外切酶,可从链的非还原端裂解β-1,4-连接的甘露糖苷键。而两种酶在底物偏好上存在区别,前者主要作用于长链底物,后者主要作用于甘露寡聚糖底物。因此两种酶协同作用于甘露聚糖和甘露寡糖,释放甘露寡糖和单糖甘露糖。然而,迄今为止,大多数被报道和表征的β-甘露聚糖水解酶都是随机性内切甘露聚糖酶。因此挖掘性能优异的β-甘露糖苷酶外切酶用于甘露寡糖及其甘露糖的酶法催化生产具有重要的应用价值和经济效益。
发明内容
[技术问题]
提供一种催化甘露聚糖获得单糖的β-1,4外切甘露糖苷酶的编码基因。
[技术方案]
本发明提供了一种编码β-1,4外切甘露糖苷酶的基因,所述核苷酸序列如SEQ IDNO.2所示。
在一种实施方式中,所述β-1,4外切甘露糖苷酶的氨基酸序列如SEQ ID NO.1所示。
本发明提供了携带权利要求1所述基因的载体。
本发明提供了表达权利要求2所述载体的基因工程菌。
在一种实施方式中,所述基因工程菌以pET序列为载体。
在一种实施方式中,所述基因工程菌以大肠杆菌为宿主,包括但不限于:E.coliBL21、E.coli BL21(DE3)、E.coli JM109、E.coli DH5α或E.coli TOP10。
本发明提供了一种制备甘露寡糖的方法,所述方法以甘露聚糖为底物,以上述基因工程菌或上述基因工程菌的代谢产物作为催化剂,进行转化反应。
在一种实施方式中,所述代谢产物是上述基因工程菌发酵生产的β-1,4外切甘露糖苷酶。
在一种实施方式中,所述底物包括含甘露聚糖的高分子多糖及其混合物。
在一种实施方式中,所述反应体系中,甘露聚糖浓度为0.2-20g/L。
在一种实施方式中,所述反应体系中,反应温度为60-80℃、pH为7.0-11.0。
本发明还提供了上述基因编码的甘露糖苷酶或含有编码甘露糖苷酶的基因的载体或携带编码甘露糖苷酶的基因的基因工程菌在制备甘露寡糖或含甘露寡糖的产品中的应用。
有益效果:
(1)本发明在将嗜热菌Thermobifida fusca来源的甘露糖苷酶编码基因TFMS重组大肠杆菌异源表达,实现了甘露糖苷酶的高效活性表达,由于大肠杆菌拥有较强的蛋白表达能力,易于TFMS酶的大规模发酵制备同时,以本发明重组表达的嗜热甘露糖苷酶TFMS可以直接用于甘露聚糖(如魔芋甘露聚糖、瓜尔豆胶、棕榈粕等)的高效水解,实现甘露寡糖甚至甘露单糖规模化酶法催化制备。该发明操作过程简单,且产品纯度高,易于实现工业化生产制备甘露寡糖,产品易于分离纯化。
(2)本发明利用大肠杆菌重组产生的嗜热甘露糖苷酶,与其他甘露糖苷酶相比,该发明具有非常大的应用优势,该酶具耐高温特性,高活性;其次,以高浓度甘露聚糖为反应底物,可高效水解产生甘露寡糖产物。
附图说明
图1:SDS-PAGE蛋白电泳分析TFMS表达与纯化。
图2:TFMS酶学特性表征。
图3:荧光电泳检测TFMS水解魔芋甘露聚糖产物图。
具体实施方式
下述实施例当中涉及的pET21a质粒和大肠杆菌E.coli BL21(DE3)均购自Stratagene,La Jolla,CA,USA。
下述实施例中涉及的培养基如下:
LB液体培养基:酵母粉5g/L,蛋白胨10g/L,氯化钠10g/L,氨苄浓度为100μg/mL。
LB固体培养基:酵母粉5g/L,蛋白胨10g/L,氯化钠10g/L,氨苄浓度为100μg/mL,琼脂15g/L。
甘露糖苷酶酶活测定:
酶活定义:在37℃下,每1分钟内能转化1微摩尔底物的酶量定义为一个单位酶活(U)。
酶活测定:甘露糖苷酶可以将甘露聚糖分解为甘露寡糖,甘露寡糖具有还原性,可以与DNS试剂在碱性条件下发生一定的化学反应,生成在煮沸条件下呈棕红色的3-amin-5-nitrosalicylic acid,可以用分光光度计测量生成的还原糖量,它的颜色的深浅程度与还原糖含量有关。
酶活测定的具体步骤为:总反应体系2mL,底物为4g/L的魔芋粉或者4g/L瓜尔豆胶溶液1mL,500mM的Tris-HCl缓冲液,200μL,加入10μL酶液,对照组加入10μL蒸馏水,所有反应液总体积补水至2mL。混合反应液在37℃孵育30min。取出后沸水浴1min使酶失活。每管中加入2mL DNS溶液,沸水浴3min,迅速冷却,加1mL蒸馏水定容至5mL。在540nm波长下测量吸光值,确定酶活。
甘露糖寡糖凝胶荧光电泳
试剂准备:配制DMSO(乙酸:水:DMSO=3:17:20)、0.2M ANTS(溶于水:乙酸=17:3)、1M NaCNBH3(溶于DMSO溶液);
样品处理:80uL甘露寡糖水解物和80uL蔗糖的水溶液于EP管中,用移液枪向各EP管中准确吸入10uL DMSO、5uL ANTS、5uLNaCNBH3,将EP管至于40℃水浴锅中水浴16h;烘箱调到45℃,将步骤EP管盖子打开放入烘箱6h,待样品体积减少到原来的二分之一时,样品加入3M 100ul尿素,标准样品加入6M 10uL尿素,振荡混匀于-20℃冰箱中放置过夜。
分离胶制作:30%丙烯酰胺5.33mL、1.5M Tirs-Hcl(pH8.8)1mL、蒸馏水1.67mL、10%过硫酸铵30uL、TEMEO 10uL,混匀制胶。
电泳完成后把取出的胶放到紫外灯下观察条带。
实施例1:TFMS的获取及重组大肠杆菌的表达
1、异源表达TFMS基因
以嗜热菌Thermobifida fusca中TFMS基因序列为模板,根据密码子的兼并性对密码子进行优化合成大小为1362bp的TFMS基因(核苷酸序列如SEQ ID NO.2所示),并引入NdeI和XhoI酶切位点,将合成产物用NdeI和XhoI双酶切,纯化回收酶切片段。然后将pET21a空质粒用同样的酶双酶切,纯化回收后与前面得到的片段连接,连接产物转化到E.coliBL21(DE3)。取适量转化液涂布在LB培养基平板上,37℃过夜培养,菌落PCR正确后挑取单菌落摇瓶培养、提取质粒。经进一步测序验证正确后,得到重组质粒TFMS-pET21a,含有重组质粒TFMS-pET21a的菌株即为重组菌TFMS-pET21a/BL21(DE3)。
2、重组菌蛋白的诱导表达与检测
将步骤1构建的重组菌TFMS-pET21a/BL21(DE3)接种至5mL的LB培养基(含100ug/mL氨苄青霉素)中,在37℃、200r/min摇瓶培养16h,再以体积比1%的接种量转接到的100mLLB液体培养基中(含100ug/mL氨苄青霉素),37℃、200r/min摇瓶培养至OD600为0.6后,添加1/1000 0.1mM的IPTG,在37℃、200rpm条件下振荡培养48h。获得的发酵液以10000×g、4℃离心10min,去除上清,收集菌体。用50mM的PBS缓冲液重悬菌体,超声破碎后,4℃、16000×g离心20min,去除沉淀,收集上清液用于TFMS酶的纯化备用。重组蛋白TFMS含有一个6xHis标签,通过固定化金属亲和层析进行高度均一的纯化。6×His标记的TFMS蛋白是通过镍-NTA柱纯化的。用低浓度咪唑缓冲液洗去背景蛋白后,用500mM咪唑洗脱目标重组蛋白,如图1所示,目标蛋白的纯度较高,在50KD处有明显的表达条带。纯化后的蛋白经过透析后冷冻干燥制成酶粉,制备的TFMS用于下一步。
实施例2甘露糖苷酶TFMS的活性表征
以魔芋葡甘聚糖为底物,对TFMS的酶学性质进行了表征研究。
1、分别称取0.2、0.4、0.8、1.6、1.8、2.0、2.2、2.4和2.8g魔芋粉溶于1L蒸馏水中,配制出魔芋甘露聚糖溶液,随后加入10μL酶液,37℃反应1h。其中酶液为实施例1制得的TFMS的酶粉用去离子水稀释,稀释液浓度至50μg/mL。如图2a所示,随着魔芋葡甘聚糖初始浓度从0.2克/升增加到2.2克/升,TFMS的催化活性迅速增加。这些结果表明果糖生物转化中没有明显的底物抑制。
2、最适温度测定:配制2g/L的魔芋葡甘聚糖作为反应底物,反应体系中加入1mL2g/L的魔芋葡甘聚糖,加入500mM的Tris-HCl缓冲液200μL,加入10μL酶液,对照组加入10μL蒸馏水,所有反应液总体积补水至2mL,,混合均匀后分别置于20℃、30℃、40℃、50℃、60℃、70℃、80℃、90℃水浴环境中温育30min。其中酶液为实施例1制得的TFMS的酶粉用去离子水稀释,稀释液浓度至50μg/mL。反应采用DNS-还原糖法测定各反应体系中新生成还原糖的浓度(OD540),并计算平均值,进行偏差分析。最大吸收值对应的反应温度为重组酶的最适温度,相对酶活(RA)定义为:各吸收值与最大吸收值的百分比。
结果如图2b所示,TFMS显示出80℃的最佳温度,并且在60℃和80℃之间的温度下显示出超过85%的活性。活性在90℃以上显著降低。温度稳定性显示TFMS在70℃以下保持超过86%的活性,特别是在低于60℃的180分钟内没有检测到活性损失,表明该酶是高度热稳定的。这种甘露聚糖酶的热稳定性明显高于其他来源的甘露聚糖酶,如来源于真菌Bispora sp.MEY-1的β-mannanase(Luo et al.,2009,Applied Microbiology&Biotechnology,82,453-461)。这些结果表明,TFMS可作为一种优良的高温生物催化酶。
3、最适pH测定:
配制不同pH的500mM缓冲液:柠檬酸缓冲液(pH 2、3、4、5、6)、磷酸缓冲液(pH6、7、8)、Tris-HCl缓冲液(pH 8、9、10、11)。
配制2g/L的魔芋葡甘聚糖作为反应底物,反应体系中加入1mL 2g/L的魔芋葡甘聚糖,用不同的pH缓冲液补充至2mL,每组设置3管,其中2管中加入10μL(50μg/mL)酶液,剩余1管加入10μL蒸馏水,测定不同pH下底物自分解情况,37℃孵育30min。取出后沸水浴1min使酶失活。每管中加入2mL DNS溶液,沸水浴3min,迅速冷却。其中酶液为实施例1制得的TFMS的酶粉用去离子水稀释,稀释液浓度至50μg/mL。
用去离子水调零,在540nm波长下测量吸光值,参考标准曲线,计算酶活力,绘制不同pH值反应条件与酶活性曲线关系图,确定最适pH。最大吸收值对应的反应温度为重组酶的最适温度,相对酶活(RA)定义为:各吸收值与最大吸收值的百分比。
结果如图2c所示,TFMS在pH 9.0时显示最大活性,在pH7.0至10.0范围内显示超过90%的活性。TFMS被发现是一种碱性外切甘露聚糖酶,形成一个独特的分支,不同于GH5甘露聚糖酶家族,适用于酸性环境。60分钟后,在不同的酸碱度下孵育后,酶的稳定性具有最宽的工作酸碱度范围7.0-11.0,剩余活性超过最大活性的85%。酸碱度稳定性试验的结果表明,TFMS也是高度稳定的广泛的酸碱度范围,这有利于潜在的应用。
实施例3利用TFMS酶液及高效制备甘露寡糖产物
具体步骤如下:
称取0.5g魔芋粉溶于50ml蒸馏水中,配制出魔芋甘露聚糖溶液,随后加入5mlTFMS的酶液,37℃反应1h。其中酶液为实施例1制得的TFMS的酶粉用去离子水稀释,稀释液浓度至50μg/mL。
样品经处理后进行凝胶荧光电泳,通过在紫外灯下的观察,结果如图3所示:以甘露糖和蔗糖作为标准对照,加入TFMS的细胞破碎液可以将魔芋甘露聚糖水解成单糖、二糖、三糖,即重组表达的嗜热甘露糖苷酶TFMS对甘露聚糖底物实现了有效水解,可以一步直接制备获得低分子量甘露寡糖甚至单糖产物。
对比例
采用实施例1相同的构建方法,克隆了其他两个来源的甘露糖苷酶基因并进行了重组表达,分别为Thermobifida halotolerans来源的β-mannosidase ThMS(NCBI登录号:WP_068692523.1)和Thermobifida alba来源的endo-1,4-β-mannosidase TAMS(NCBI登录号:BBA57841.1)。
结果显示,ThMS和TAMS重组酶产物对魔芋甘露聚糖进行水解反应,经检测后发现两者的水解终产物均为寡聚糖,未检测到单糖的出现。
虽然本发明已以较佳实施例公开如上,但其并非用以限定本发明,任何熟悉此技术的人,在不脱离本发明的精神和范围内,都可做各种的改动与修饰,因此本发明的保护范围应该以权利要求书所界定的为准。
SEQUENCE LISTING
<110> 浙江农林大学
<120> 一种甘露糖苷酶的工程菌株及其应用
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Claims (4)
1.一种制备甘露寡糖的方法,其特征在于,所述方法是以甘露聚糖为底物,以基因工程菌或基因工程菌的代谢产物作为催化剂,进行转化反应,温度为60~80℃、pH为7~11;
所述基因工程菌表达核苷酸序列如SEQ ID NO.2所示的β-1,4外切甘露糖苷酶;
所述代谢产物是所述基因工程菌发酵生产的β-1,4外切甘露糖苷酶。
2.如权利要求1所述的方法,其特征在于,所述底物包括含甘露聚糖的高分子多糖及其混合物。
3.如权利要求1所述的方法,其特征在于,甘露聚糖底物浓度为0.2~20 g/L,反应0.75~1.5 h。
4.核苷酸序列如SEQ ID NO.2所示的β-1,4外切甘露糖苷酶或携带核苷酸序列如SEQID NO.2所示的β-1,4外切甘露糖苷酶的载体或表达核苷酸序列如SEQ ID NO.2所示的β-1,4外切甘露糖苷酶的基因工程菌在制备甘露寡糖或含甘露寡糖的产品中的应用,温度为60~80℃、pH为7~11。
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CN103642779B (zh) * | 2013-12-26 | 2015-09-30 | 中国农业科学院饲料研究所 | 一种高比活酸性β-甘露聚糖酶Man5D及其基因和应用 |
CN103789289B (zh) * | 2014-01-22 | 2016-01-20 | 湖北汇龙食品有限责任公司 | 一种耐碱高温甘露聚糖酶ManB的制备方法及其基因和应用 |
CN109385411B (zh) * | 2017-08-04 | 2021-10-26 | 东莞泛亚太生物科技有限公司 | 一种β-甘露糖苷酶及其应用 |
CN109929861B (zh) * | 2017-12-15 | 2022-11-22 | 中国科学院大连化学物理研究所 | 一种葡甘聚糖酶编码基因及酶和制备与应用 |
CN109055333A (zh) * | 2018-07-26 | 2018-12-21 | 天津科技大学 | 一种糖苷水解酶及其复合酶在半乳甘露聚糖降解中的应用 |
CN110819610A (zh) * | 2019-10-16 | 2020-02-21 | 江苏大学 | 一种极耐热甘露聚糖酶及其制备方法与应用 |
CN113046376A (zh) * | 2021-03-22 | 2021-06-29 | 武汉轻工大学 | 一种甘露糖酶基因VbMan26A、重组质粒、重组菌株、甘露糖酶及其应用 |
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