CN116064477B - 一种糖苷酶家族43的β-糖苷酶及其编码基因与应用 - Google Patents
一种糖苷酶家族43的β-糖苷酶及其编码基因与应用 Download PDFInfo
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Abstract
本发明涉及一种糖苷酶家族43的β‑糖苷酶及其编码基因与应用。所述糖苷酶家族43的β‑糖苷酶来源于山东泰安土壤宏基因组,核苷酸序列如SEQ ID No.1所示,氨基酸序列如SEQ ID No.2所示。本发明提供了一种新型GH43的β‑糖苷酶基因,为低聚半乳糖、烷基半乳糖苷、酪醇半乳糖苷等产品的合成提供了新的工具酶。本发明提供的β‑糖苷酶能以半乳糖为底物直接合成低聚半乳糖,与传统的以乳糖底物合成寡糖的工艺显著不同,使用本发明β‑糖苷酶反应获得的低聚半乳糖粗品不含葡萄糖,容易纯化获得纯品供糖尿病患者使用,拓展应用范围;该酶还能够以半乳糖为糖基供体、多种羟基化合物为受体合成糖苷产物,在食品、医药、化工等领域有重要应用价值。
Description
技术领域
本发明涉及一种糖苷酶家族43的β-糖苷酶及其编码基因与应用,属于基因工程技术领域。
背景技术
酶法催化反应具有高效、特异、绿色环保等优点,广泛应用于制药、食品、化妆品、农业、环境保护和能源生产等领域。糖苷酶(EC3.2.1)是生物体内重要的糖苷水解酶,在体外还能用于催化合成反应。糖类分子由于具有多个可以反应的羟基,使得化学法合成过程比较复杂,需要多步的基团保护和去保护作用,才能实现特定糖苷键的合成,而酶法合成具有立体选择性和区域选择性,能够一步合成糖苷产物,步骤简单,适合于规模化合成反应。
含β-半乳糖苷键的具有重要功能的低聚半乳糖和半乳糖苷可由β-半乳糖苷酶(EC3.2.1.23)催化合成。β-半乳糖苷酶是一类重要的糖苷水解酶,按照氨基酸和结构的同源性归属于GH1、GH2、GH35、GH42、GH59、GH147。该类酶具有β-半乳糖苷键的水解活性,当以乳糖为底物时,水解底物释放出葡萄糖和半乳糖,该特性已被用于食品工业降解乳糖来改善乳制品的甜度、溶解性以及消化率,缓解人群中乳糖不耐受的症状。此外,装配有β-半乳糖苷酶的生物传感器,可用于检测乳制品中的乳糖含量。除了水解活性,有的来源的β-半乳糖苷酶还具有转糖基活性,能够以乳糖或硝基苯-β-半乳糖苷为糖基供体,催化半乳糖基从糖基供体底物上转移到受体分子上,形成新的β-半乳糖苷键,合成食品、药品及化妆品行业重要的寡糖及糖苷化合物。
β-半乳糖苷酶通过双置换机制催化反应,酶分子两个关键氨基酸如谷氨酸在催化过程中发挥着至关重要的作用,一个作为酸/碱催化剂,另一个作为亲核体。反应第一步是酶的半乳糖基化,亲核体羧基直接作用于底物,形成共价键的糖基-酶中间物,酸碱羧基先进行酸催化,提供质子,促进底物离去基团离去。反应第二步是酶的去糖基化,酸碱羧基再进行碱催化,激活受体分子,发生以水为受体的水解反应和以糖为受体的糖苷合成反应。该反应如果以乳糖为底物分子,则会发生水解反应;具有转糖基活性的酶还能以水解产物即葡萄糖、半乳糖及乳糖底物自身为受体合成重要的益生元——β-低聚半乳糖;若以乳糖为糖基供体,其他羟基化合物为受体,则合成其他寡糖或糖苷。
值得注意的是,上述β-低聚半乳糖的合成反应,存在乳糖水解副反应生成葡萄糖和半乳糖,其中葡萄糖的存在不利于糖尿病患者使用;对于采用乳糖或硝基苯半乳糖为糖基供体、羟基化合物为受体的转糖基反应,同样存在糖基供体的水解副产物,导致产物纯化复杂,高纯度半乳寡糖和糖苷的获得比较困难,在一定程度上限制了半乳糖苷产物在生物、医药领域的应用。因而,挖掘新的能以半乳糖为糖基供体合成β-糖苷键的糖苷酶具有重要意义,由于单糖与糖基受体直接发生糖苷合成反应,因而不存在以双糖及糖苷为糖基供体的水解副反应。
目前,现有技术中没有糖苷酶家族43(GH43)具有合成β-半乳糖苷键活性的报道。
发明内容
针对现有技术的不足,本发明提供一种糖苷酶家族43的β-糖苷酶及其编码基因与应用。
本发明的技术方案如下:
一种糖苷酶家族43的β-糖苷酶,其氨基酸序列如SEQ ID No.2所示。
上述糖苷酶家族43的β-糖苷酶的编码基因,其核苷酸序列如SEQ ID No.1所示。
根据本发明优选的,所述编码基因来源于山东泰安土壤宏基因组。
一种重组表达载体,在表达载体中插入了上述糖苷酶家族43的β-糖苷酶的编码基因。
根据本发明优选的,所述表达载体为pET-21b表达载体。
一种重组细胞,在宿主细胞中转入了含有上述糖苷酶家族43的β-糖苷酶的编码基因的载体。
根据本发明优选的,所述宿主细胞为大肠杆菌BL21(DE3)。
利用上述β-糖苷酶的核苷酸序列制备重组β-糖苷酶的方法,步骤如下:
(1)将β-糖苷酶的基因序列SEQ ID No.1克隆到大肠杆菌表达载体pET-21b中,构建重组表达载体pET-ebg-39308,将表达载体转化到大肠杆菌BL21(DE3)中,用氨苄青霉素抗性筛选出含β-糖苷酶的重组菌株;
(2)将步骤(1)筛选出的重组菌株接种于LB液体培养基中,在37℃、180rpm下培养过夜,获得种子液;将种子液以体积比0.5~1.5%的量接种于LB液体培养基中,待生长到OD600为0.6时,加入终浓度1mM的IPTG诱导,诱导温度为37℃,诱导时间为5h;
(3)收集步骤(2)诱导后获得的菌细胞,超声破碎后,离心所得包涵体沉淀即为重组β-糖苷酶。
上述糖苷酶家族43的β-糖苷酶在合成低聚半乳糖、烷基半乳糖苷、酪醇半乳糖苷中的应用。
本发明提供的上述β-糖苷酶具有如下酶学性质:
β-糖苷酶单亚基的分子量为37±5kDa;β-糖苷酶对邻硝基苯-β-D-吡喃半乳糖苷(o-Nitrophenyl β-D-galactopyranoside,oNPG)适宜反应的温度为40~55℃,适宜反应的pH为7.0~10.0;在25~50℃范围内保温2h,能够保留80%以上的活性;在pH 7.0~8.0的范围内4℃保存2h可以保留80%以上的活性;酶对邻硝基苯-β-D-吡喃半乳糖苷的Km值为0.64mM。
有益效果
1、本发明提供了一种新型GH43的β-糖苷酶基因,并通过高效重组表达提供了大量的β-糖苷酶,为低聚半乳糖、烷基半乳糖苷、酪醇半乳糖苷等产品的合成提供了新的工具酶。
2、本发明将β-糖苷酶基因克隆到pET-21b表达载体,转化大肠杆菌BL21(DE3),IPTG诱导表达,得到β-糖苷酶。本发明提供的β-糖苷酶能以半乳糖为底物直接合成低聚半乳糖,与传统的以乳糖底物合成寡糖的工艺显著不同,采用本发明β-糖苷酶反应获得的低聚半乳糖粗品不含葡萄糖,容易纯化获得高纯度产品供糖尿病患者使用,拓展应用范围。此外,该酶还能够以半乳糖为糖基供体、多种羟基化合物为受体合成糖苷产物,在食品、制药和化工行业具有重要应用价值。
附图说明
图1为实施例1所述的样本宏基因组测序结果的生物信息学门分类水平上的共线性分析。
图2为实施例1所述的样本宏基因组测序结果的生物信息学门分类学水平上的相对丰度直方图。
图3为实施例1所述的样本宏基因组测序结果的生物信息学为CAZy功能分类条形图。
图4为β-糖苷酶基因ebg-39308在大肠杆菌表达的重组酶聚丙烯酰胺凝胶电泳图。
图中,M为蛋白分子量标准;1和2分别为重组大肠杆菌IPTG诱导前和诱导后全细胞粗酶液;3为重组Ebg-39308酶。
图5为温度对重组Ebg-39308酶活性的影响。
图6为pH对重组Ebg-39308酶活性的影响。
图7为温度对重组Ebg-39308酶稳定性的影响。
图8为pH对重组Ebg-39308酶稳定性的影响。
图9为利用重组Ebg-39308酶合成的低聚半乳糖产物的质谱图。
图10为利用重组Ebg-39308酶合成的烷基半乳糖苷产物的质谱图。
图11为利用重组Ebg-39308酶合成的酪醇半乳糖苷产物的质谱图。
具体实施方式
下面结合说明书附图和实施例对本发明做进一步说明,但本发明所保护范围不限于此。实施例中未明确限定的均可按本领域现有技术。
实施例1:样本宏基因组的获得、测序及生物信息学分析
采集山东省泰安市土壤样本,采用Soil Genomic DNA Kit试剂盒(康为世纪)提取基因组,命名为样品1,然后通过琼脂糖凝胶电泳检测验证提取效果后进行宏全基因组测序。根据测序结果,通过对宏全基因组测序中的CAZy数据库注释,预测糖苷水解酶基因,结果如图1~3所示。
通过NCBI Protein BLAST以及MEGAX分析,将上述样本宏全基因组测序所得目的基因同数据库中的已知酶基因进行比对,重点分析预测与现有酶同源性较低的β-半乳糖苷加工相关酶类,发现其中一种编号为ebg-39308所编码的酶来自于GH43,预测为exo-β-1,3-galactanase(EC 3.2.1.145),该家族的酶尚未报道有β-半乳糖苷键合成活性,其核苷酸序列如SEQ ID No.1所示,共有957个碱基,编码氨基酸序列如SEQ ID No.2所示,该酶蛋白大小为318个氨基酸,命名为Ebg-39308。
所述Ebg-39308蛋白与GH43家族的糖苷酶如MBE3134935.1(大小为472个氨基酸)、MBY0495524.1(大小为502个氨基酸)、MCL4847321.1(大小为531个氨基酸)表现出相对较高的同源性,但氨基酸一致性均低于75%,而且这些同源酶均未注释酶的功能。
实施例2:ebg-39308基因表达载体的构建
1、以实施例1中的土壤DNA基因组(样品1)为模板,进行PCR扩增,所述PCR扩增引物序列如下:
39308-F:5'-ATGGAGAGCTTCTCGCAGGAAG-3'
39308-R:5'-TCAGTCGAGTGCACGATAGACG-3'
PCR扩增体系为:2×Rapid Taq Master Mix(Vazyme)25μL、上下游引物(10mM)各1μL、模板2μL,超纯水补足至50μL。
PCR扩增条件:95℃预变性3min,反应30个循环(95℃变性15s,60℃退火15s,72℃延伸30s),30个循环结束后72℃延伸5min,4℃暂存。
将PCR产物经琼脂糖凝胶电泳检测,大小约为1000bp,与理论值957bp相符。采用DNA胶回收试剂盒对目的基因条带进行回收,所得产物即为ebg-39308基因序列。
2、根据同源重组原理,设计一对PCR引物rD-39308-F/R,采用该引物对ebg-39308基因序列进行扩增,具体引物序列如下:
rD-39308-F:5'-GACAGCAAATGGGTCGGGATCCGATGGAGAGCTTCTCGCAGGAAG-3'
rD-39308-R:5'-TTGTCGACGGAGCTCGAATTCGGTCAGTCGAGTGCACGATAGACG-3'
PCR扩增条件和PCR产物回收方法同本实施例步骤1。PCR产物大小为1006bp。所得产物即为用于与质粒载体构建的ebg-39308基因序列。
3、取大肠杆菌DH5α/pET-21b,接种于5mL的LB液体培养基中,37℃摇床培养过夜,摇床转速为180rpm,提取pET-21b质粒载体,添加限制性内切酶EcoR I、BamH I(Takara)对pET-21b质粒载体进行双酶切处理,再将用于与质粒载体构建的ebg-39308基因序列与线性化pET-21b载体混合,采用无缝克隆试剂盒(Vazyme)于37℃同源重组30min。然后采用CaCl2转化法转化宿主菌大肠杆菌DH5α,转化菌液涂布含氨苄青霉素的LB固体培养基,37℃过夜培养。挑取单菌落接种于5mL含50μg/mL氨苄青霉素的LB培养液中,37℃摇床培养过夜,摇床转速为180rpm,提取重组质粒,测序验证重组质粒构建成功,即为重组质粒pET-ebg-39308。
实施例3:ebg-39308基因在大肠杆菌中的重组表达
将实施例2所述的重组质粒pET-ebg-39308采用CaCl2转化法转化到宿主菌大肠杆菌BL21(DE3),再将转化菌液涂布氨苄青霉素平板,37℃过夜培养。然后提取阳性菌落质粒,通过限制性内切酶EcoRI将重组质粒pET-ebg-39308线性化,琼脂糖凝胶电泳检测,验证转化成功。将转化成功的BL21(DE3)/pET-ebg-39308接种于5mL含50μg/mL氨苄青霉素的LB液体培养基中,37℃、180rpm摇床培养过夜。将菌液按1%接种量接种于350mL的LB液体培养基中,37℃、180rpm摇床培养至OD600约为0.6,加入终浓度为1mM的IPTG诱导表达,继续在37℃、180rpm摇床培养5h。随后将菌液在11000rpm下离心3min,弃去上清,用50mM的磷酸钾缓冲液(KPB,pH 7.5)清洗菌体沉淀2~3次,收集菌体,用50mM的KPB缓冲液(pH 7.5)重悬菌体沉淀,并定容至培养液总体积的1%,将装有菌体的离心管置于冰水浴中,使用超声波细胞破碎机进行破壁,获得重组大肠杆菌细胞破壁液;再将破壁液在11000rpm下离心30min,分离上清与包涵体沉淀,用5mL浓度为50mM的KPB缓冲液(pH 7.5)重悬白色沉淀,即得目的蛋白,通过聚丙烯酰胺凝胶电泳对目的蛋白进行检测,检测结果如图4所示,β-糖苷酶Ebg-39308包涵体蛋白在电泳上呈单一条带,且位置与预测的分子量相吻合。酶活测定结果显示,该酶蛋白具有β-半乳糖苷键的水解活性。
所述LB液体培养基成分:蛋白胨10g/L,酵母粉5g/L,NaCl 7g/L,pH 7.0~7.5,121℃灭菌30min。
上述测定酶活的具体方法为:
取50μL的β-糖苷酶酶液,加2mM的β-半乳糖苷(oNPG)溶液450μL,在37℃反应10min,加1mL浓度为0.5M的Na2CO3溶液终止反应,在12000rpm下离心1min,取200μL上清液于96孔板中,酶标仪检测OD420。
酶的活力单位规定:以1min水解oNPG释放1μmol对硝基苯酚的酶量为一个酶活力单位(U)。
实施例4:β-糖苷酶Ebg-39308的酶学性质分析
1、温度对酶活性的影响
分别在25、30、35、40、45、50、55、60和65℃条件下测定β-糖苷酶Ebg-39308的酶活,以确定酶的适宜反应温度,结果如图5所示。
上述酶活测定方法按照实施例3进行,所不同的是温度分别选取上述温度。
由图5可知,β-糖苷酶Ebg-39308适宜反应的温度为40~55℃,最适反应温度为50℃。
2、pH对酶活性的影响
配制pH分别为3.0、4.0、5.0、6.0、6.4、7.0、7.4、8.0的Na2HPO4-柠檬酸缓冲液,以及pH分别为8.6、9.0、9.4、10.0、10.4的甘氨酸-NaOH缓冲液,然后分别在梯度pH的缓冲液中按照实施例3中的方法测定β-糖苷酶Ebg-39308的酶活,以确定酶的适宜反应pH值,结果如图6所示。
由图6可知,β-糖苷酶Ebg-39308适宜反应pH为7.0~10.0,最适反应pH为7.0。
3、酶活稳定性分析
将酶液在25、30、35、40、45、50、55和60℃条件下孵育2h,按照实施例3中的方法测定β-糖苷酶Ebg-39308的酶活,结果如图7所示。
由图7可知,β-糖苷酶Ebg-39308在25~50℃范围内能够保留80%以上的活性,55℃以上失活较快,55~60℃失去所有的酶活。
将酶液在pH分别为5.0、6.0、7.0、8.0、9.0的缓冲液中于4℃下保存2h,按照实施例3中的方法测定β-糖苷酶Ebg-39308的酶活,结果如图8所示。
由图8可知,β-糖苷酶Ebg-39308在pH 7.0~8.0范围内稳定。
4、酶的动力学参数
将50μL酶液与450μL梯度浓度为0.4、0.8、1.2、1.6、2.0、4.0、6.0、8.0、10、12、14、16、18、20mM的oNPG混合,于37℃反应10min,加1mL浓度为0.5M的Na2CO3溶液终止反应,12000rpm离心1min,取200μL上清液于96孔板,酶标仪测定OD420,计算水解速率,用GraphPad Prism绘图测得酶对oNPG的Km值为0.64mM。
实施例5:利用β-糖苷酶Ebg-39308以单糖为糖基供体的糖苷合成反应
1、以半乳糖为底物合成低聚半乳糖
采用浓度为50mM、pH 7.5的磷酸缓冲液分别配制浓度为60%的半乳糖、浓度为20U/mL的β-糖苷酶的反应体系,反应体系于50℃条件下反应12h,然后100℃煮沸10min灭活酶液终止反应,在12000rpm离心10min,取上清液稀释后进行薄层层析(TLC)检测,然后进行质谱分析验证,结果如图9所示。
所述薄层层析检测方法为:
薄层层析板点样后在展层剂中展开,雾喷显色剂后,于120℃烘烤5min使糖斑点显色;
上述展层剂由正丁醇、无水乙醇和水按体积比5:3:2混合配制;显色剂是由体积百分比为20%的硫酸溶液配制的质量浓度为0.5%的3,5-二羟基甲苯溶液。
所述质谱分析所用仪器为Bruker质谱仪(德国)。
由图9可知,以半乳糖为底物,采用β-糖苷酶Ebg-39308成功合成低聚半乳糖,所合成的低聚半乳糖组成为半乳二糖和半乳三糖。
2、以半乳糖为糖基供体、以烷基醇为糖基受体合成烷基半乳糖苷
采用浓度为50mM、pH 7.5的磷酸缓冲液分别配制浓度为1M的半乳糖,浓度均为35%的甲醇、乙醇、异丙醇、正丁醇、正戊醇或正己醇,浓度为20U/mL的β-糖苷酶的反应体系,反应体系分别于40℃条件下反应12h,然后100℃煮沸10min终止反应,在12000rpm离心10min,取上清液稀释后进行薄层层析检测,然后进行质谱分析验证,结果如图10所示。
由图10可知,以半乳糖为糖基供体,以烷基醇为糖基受体,采用β-糖苷酶Ebg-39308成功合成甲基半乳糖苷、乙基半乳糖苷、异丙基半乳糖苷、正丁基半乳糖苷、正戊基半乳糖苷、正己基半乳糖苷。
TLC分析方法及质谱仪器条件同上。
3、以半乳糖为糖基供体、以酪醇为糖基受体合成酪醇半乳糖苷反应
采用浓度为为50mM、pH 7.5的磷酸缓冲液分别配制浓度为1M的半乳糖,浓度为200mM的酪醇、浓度为20U/mL的β-糖苷酶的反应体系,反应体系于50℃条件下反应12h,然后100℃煮沸5min终止反应,在12000rpm离心10min,取上清液稀释后进行薄层层析检测,然后进行质谱分析验证,结果如图11所示。
由图11可知,以半乳糖为供体、以酪醇为糖基受体,采用β-糖苷酶Ebg-39308成功合成酪醇半乳糖苷。
TLC分析方法及质谱仪器条件同上。
Claims (8)
1.一种糖苷酶家族43的β-糖苷酶,其特征在于,氨基酸序列如 SEQ ID No.2所示。
2.权利要求1所述的糖苷酶家族43的β-糖苷酶的编码基因,其特征在于,核苷酸序列如SEQ ID No.1所示。
3.一种重组表达载体,其特征在于,在表达载体中插入了权利要求2所述的糖苷酶家族43的β-糖苷酶的编码基因。
4.如权利要求3所述的重组表达载体,其特征在于,所述表达载体为pET-21b表达载体。
5.一种重组细胞,其特征在于,在宿主细胞中转入了权利要求3所述的重组表达载体。
6.如权利要求5重组细胞,其特征在于,所述宿主细胞为大肠杆菌BL21(DE3)。
7.利用权利要求2所述的β-糖苷酶的核苷酸序列制备重组β-糖苷酶的方法,其特征在于,步骤如下:
(1)将β-糖苷酶的基因序列SEQ ID No.1克隆到大肠杆菌表达载体pET-21b中,构建重组表达载体pET-ebg-39308,将表达载体转化到大肠杆菌BL21(DE3)中,用氨苄青霉素抗性筛选出含β-糖苷酶的重组菌株;
(2)将步骤(1)筛选出的重组菌株接种于LB液体培养基中,在37℃、180rpm下培养过夜,获得种子液;将种子液以体积比0.5~1.5%的量接种于LB液体培养基中,待生长到OD600为0.6时,加入终浓度1 mM的IPTG诱导,诱导温度为37℃,诱导时间为5h;
(3)收集步骤(2)诱导后获得的菌细胞,超声破碎后,离心所得包涵体沉淀即为重组β-糖苷酶。
8.权利要求1所述的糖苷酶家族43的β-糖苷酶在合成低聚半乳糖、烷基半乳糖苷、酪醇半乳糖苷中的应用。
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