CN114574460B - 一种利用糖基转移酶ugt76g1突变体高效生物合成莱鲍迪苷m的方法 - Google Patents
一种利用糖基转移酶ugt76g1突变体高效生物合成莱鲍迪苷m的方法 Download PDFInfo
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- CN114574460B CN114574460B CN202210253543.9A CN202210253543A CN114574460B CN 114574460 B CN114574460 B CN 114574460B CN 202210253543 A CN202210253543 A CN 202210253543A CN 114574460 B CN114574460 B CN 114574460B
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- glycosyltransferase
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Abstract
本发明公开了一种利用糖基转移酶UGT76G1突变体高效生物合成莱鲍迪苷M的方法,属于酶工程成领域。本发明通过定向进化得到高催化活性突变体UGT76G1‑T284S/M88L/L200A。并将糖基转移酶突变体UGT76G1‑T284S/M88L/L200A与拟南芥来源的蔗糖合酶AtSuSy构建偶联反应,实现以莱鲍迪苷D为底物高效催化合成莱鲍迪苷M,通过补料添加底物Reb D以22.58g/L(20mM)Reb D为底物反应7h,高效合成23.37g/L的Reb M,Reb M产率达到90.5%,为Reb M的生产提供了一条高效且绿色的新途径。
Description
技术领域
本发明涉及一种利用糖基转移酶UGT76G1突变体高效生物合成莱鲍迪苷M的方法,属于酶工程领域。
背景技术
由于高热量糖类的过量摄入导致世界范围内严重的过度肥胖、糖尿病、高血压和心脑血管等疾病。因此,来自甜叶菊的甜菊糖苷类化合物因其甜度高、热量低且安全性高而受到广泛关注。其中,含量较为丰富的甜菊糖、莱鲍迪苷A(Reb A)等作为甜味剂已广泛应用于饮料、食品等领域。它们具有相较于蔗糖250-300倍的甜度,但是口感上存在甜味之外的苦后味严重影响它们作为甜味剂的口感。而在甜菊糖苷中含量较少的莱鲍迪苷M(Reb M)相较于Reb A和甜菊糖具有更高的甜度,并且减少了甜味之外的后苦味,且甜味更快而因此作为甜味剂具有更好的口感,被认为是非常具有潜力的下一代甜味剂。其中,Reb M相较于RebD具有更高的商业价值。然而,Reb M在甜叶菊干叶中的含量仅为0.4%-0.5%,仅为Reb A含量的十分之一左右,这也使得通过传统的叶片提取Reb A的方法并不适用于Reb M的提取。繁琐且复杂的提取方法使得仅从甜叶菊叶子中提取难以实现规模化生产,也难以满足市场需求。
目前,通过相关科学家不断探索和挖掘,通过酶催化方法利用Reb D合成Reb M被认为是一种可行的提高Reb M生产量的技术路线。然而,负责催化Reb D合成Reb M糖基转移酶UGT76G1存在酶活低、异源表达水平低和催化过程中催化Reb A生成副产物Reb I等缺点,因此UGT76G1成为酶催化方法合成Reb M过程中关键的限速步骤,使其难以实现规模化生产以满足市场需求。目前,已有文献报道通过融合表达提高UGT76G1异源表达水平,酶固定化方法提高酶UGT76G1的稳定性和催化效率,以及通过定点突变获得活性提高的突变体UGT76G1-T284S并且减少了副产物Reb I的生成。然而这些方法不足以使UGT76G1具有足够的酶活满足规模化生产Reb M的要求。因此,通过基于蛋白质晶体结构的定向进化改造UGT76G1提高其活性以满足Reb M的规模化生产具有重要意义。
发明内容
为解决上述问题,本发明通过对糖基转移酶UGT76G1进行基于蛋白质晶体结构的定向进化,成功获得高效突变体,并通过构建尿苷二磷酸葡萄糖UDPG循环体系,利用体外纯酶反应体系实现高效生物合成Reb M,为Reb M生物合成提供一种新的方法。
为了解决上述技术问题,本发明的技术方案如下:
本发明的第一个目的是提供一种糖基转移酶UGT76G1突变体,为如下(a)或(b)的蛋白质:
(a)氨基酸序列如SEQ ID NO.1所示的蛋白质;
(b)在如SEQ ID NO.1所示的氨基酸序列中经过取代、缺失或添加一个或几个氨基酸序列并且具有催化Reb D为Reb M的酶活性的由(a)衍生的蛋白质。
本发明的第二个目的是提供表达上述糖基转移酶UGT76G1突变体的基因。
本发明的第三个目的是提供携带上述糖基转移酶UGT76G1突变体的基因的载体。
本发明的第四个目的是提供表达上述糖基转移酶UGT76G1突变体的重组菌。
在一种实施方式中,所述重组菌还可以表达蔗糖合酶。
在一种实施方式中,所述蔗糖合酶的氨基酸序列可以是任意来源的具有蔗糖合酶活性的氨基酸序列。
在一种实施方式中,所述蔗糖合酶的氨基酸序列的NCBI登录号为NP_001031915。
在一种实施方式中,所述重组菌以大肠杆菌为宿主细胞,以pETDuet-1为表达载体。
本发明的第五个目的是提供一种催化合成Reb M的方法,所述方法为以Reb D为底物,利用所述糖基转移酶UGT76G1突变体和蔗糖合酶偶联进行催化反应。
在一种实施方式中,所述糖基转移酶UGT76G1突变体和蔗糖合酶的摩尔浓度比为1:1。
在一种实施方式中,所述糖基转移酶UGT76G1突变体和蔗糖合酶的摩尔浓度为7~9μM。
在一种实施方式中,所述催化反应的第0.5h,1h和2h分别添加5mM Reb D。
在一种实施方式中,所述蔗糖合酶的氨基酸序列可以是任意来源的具有蔗糖合酶活性的氨基酸序列。
在一种实施方式中,所述蔗糖合酶的氨基酸序列的NCBI登录号为NP_001031915。
在一种实施方式中,所述催化反应的条件为:以1-20mM Reb D、0-1000mM蔗糖、50mM K2HPO4-KH2PO4缓冲液为反应体系,20-45℃糖基化反应0-24h。
在一种实施方式中,所述缓冲液pH为5.5-10.0。
在一种实施方式中,所述缓冲液为50mM Tris-HCl。
本发明还保护上述糖基转移酶或上述基因或上述表达载体或上述重组菌或上述方法在制备含有Reb M的产品中的应用。
有益效果:
(1)本发明将糖基转移酶UGT76G1的氨基酸序列进行定点突变,在以UDPG为糖基供体,得到的突变体UGT76G1-T284S/M88L/L200A显著提高催化Reb D合成Reb M的效率,催化活性大幅提升,相比野生型酶UGT76G1-T284S提高了2.38倍。
(2)本发明将编码糖基转移酶突变体UGT76G1-T284S/M88L/L200A的核苷酸序列与编码蔗糖合酶AtSuSy核苷酸序列分别于大肠杆菌中进行异源表达纯化(图1)。通过偶联反应体系条件优化,实现利用22.58g/L(20mM)Reb D以90.50%的高产率合成23.37g/L的RebM。
附图说明
图1为糖基转移酶和蔗糖合酶偶联反应示意图。
图2为实施例3中糖基转移酶UGT76G1-T284S催化Reb D合成Reb M的UPLC分析图。
图3为实施例4中糖基转移酶浓度对糖基化偶联反应的影响。
图4为实施例4中蔗糖合酶浓度对糖基化偶联反应的影响。
图5为实施例5中温度对糖基化偶联反应的影响。
图6为实施例6中pH对糖基化偶联反应缓冲溶液的影响。
图7为实施例7中蔗糖浓度对糖基化偶联反应的影响。
图8为实施例8中UDP浓度对糖基化偶联反应的影响。
图9为实施例9中补料添加底物制备Reb M。
具体实施方式
以下结合说明书附图和具体实施例来进一步说明本发明,但实施例并不对本发明做任何形式的限定。除非特别说明,本发明采用的方法和设备为本技术领域常规方法和设备。
除非特别说明,以下实施例所用试剂和材料均为市售商品或者可以通过已知方法制备。
实施例1糖基转移酶UGT76G1基因的获取及突变体的构建
从Genbank中下载甜叶菊来源的糖基转移酶UGT76G1氨基酸序列(登录号:Q6VAB4)及核酸序列(登录号:PRO_0000434465),并添加突变点T284S,由亦欣生物科技有限公司进行基因合成和密码子优化并连接至载体pETDuet-1的多克隆酶切位点,得到重组质粒pETDuet-1-UGT76G1-T284S。从Genbank中下载来源于拟南芥的蔗糖合酶AtSuSy氨基酸序列(登录号:NP_001031915)以及其核酸序列(登录号:NM_001036838.2),由亦欣生物科技有限公司进行大肠杆菌偏好的密码子优化及基因合成。
以重组质粒pETDuet-1-UGT76G1-T284S为模板,利用引物M88L-F/M88L-R和L200A-F/L200A-R,进行全质粒PCR(引物如表1所示),构建携带突变体的重组质粒pETDuet-1-UGT76G1-T284S/M88L/L200A。
将所得质粒pETDuet-1-UGT76G1-T284S及pETDuet-1-UGT76G1-T284S/M88L/L200A进行测序鉴定并分别转化至大肠杆菌E.coli BL21(DE3)感受态细胞中,采用含有100μg/mL氨苄青霉素的LB固体平板(10g/L蛋白胨,5g/L酵母粉,10g/L NaCl,20g/L琼脂粉)进行筛选,得到重组菌株E.coli BL2l(DE3)pETDuet-1-UGT76G1-T284S和E.coli BL2l(DE3)pETDuet-1-UGT76G1-T284S/M88L/L200A。
表1引物名称及引物序列
引物名称 | 引物序列 |
M88L-F | TGGCCGGACTTCGTATCCCGATCATCAATGAACACG |
M88L-R | GGATACGAAGTCCGGCCAGAGGACCATG |
L200A-F | GGCAAATTGCTAAAGAGATTTTGGGAAAGATGATCAAACAAACT |
L200A-R | ATCTCTTTAGCAATTTGCCAATTTGAATAAGCACTTTTAATGTCT |
实施例2重组菌株的诱导表达及目的蛋白纯化
将实施例1中构建的重组菌株E.coli BL2l(DE3)pETDuet-1-UGT76G1-T284S和E.coli BL2l(DE3)pETDuet-1-UGT76G1-T284S/M88L/L200A分别接种至含有100μg/mL氨苄青霉素的1L TB液体培养基(12g/L蛋白胨,24g/L酵母粉,5g/L甘油,2.32g/L KH2PO4,12.53g/L K2HPO4)中,并在115rpm,37℃条件下培养至OD600为0.6-0.8后,培养温度降至18℃,加入终浓度为0.1mM的异丙基-β-硫代半乳糖苷(IPTG),诱导培养8h。
将诱导表达的菌液离心(7000rpm,7min,4℃),弃上清,收集菌体。将菌体用裂解缓冲液(50mM Tris-HCl pH 8.0,300mM NaCl,10mM咪唑,10%甘油)按照1g菌体每10mL裂解液进行重悬。利用高压匀浆机进行破碎,然后将破碎后的菌液进行离心(40000×g,30min),取上清即获得粗酶液。
将粗酶液利用Ni+柱进行亲和层析纯化,上样结束后,10倍体积裂解液冲洗杂蛋白,用洗脱缓冲液进行目的蛋白洗脱。收集洗脱的目的蛋白,通过脱盐柱(HistrpTM 5mLDesalting)进行脱盐,脱盐缓冲液(25mM Tris-HCl,150mM NaCl,10%甘油)。脱盐后浓缩至10mg/mL,进行后续反应。纯化的蛋白通过10%SDS-PAGE凝胶电泳进行检测,成功获得目的条带清晰蛋白大小准确的纯酶,测定野生型酶UGT76G1-T284S及突变体酶UGT76G1-T284S/M88L/L200A酶活分别为103mU/mg、274mU/mg。
蔗糖合酶AtSuSy采用相同的方法进行诱导表达和蛋白纯化。
实施例3 UGT76G1-T284S和UGT76G1-T284S/M88L/L200A催化Reb D反应合成Reb M的糖基化反应
将实施例2获得的纯化后的野生型酶及突变体UGT76G1-T284S/M88L/L200A进行糖基化反应。
糖基化反应在200μL反应体系中进行,反应体系如下:50mM Tris-HCl pH 8.0,5mMUDPG,2mM Reb D,实施例2中所获得纯酶UGT76G1-T284S或UGT76G1-T284S/M88L/L200A浓度为2.5μM。在35℃下反应10min。反应结束后,95℃加热10min,用3倍体积甲醇稀释,20000×g离心5min,0.22μM滤膜进行抽滤后进行超高效液相色谱(UPLC)进行检测分析(图2)。UPLC采用waters公司BEH C18 1.7μM反向柱,进样量4μL,柱温40℃,流动相采用A管路:乙腈,B管路:1.38g/L NaH2PO4缓冲液(pH2.6),流速0.3mL/min,具体程序如表2所示:
表2 UPLC反应程序
结果如图2所示,与Reb D和Reb M标准品对比可知,在反应体系中有明显的与RebM具有相同保留时间的新产物生成。
实施例4糖基转移酶和蔗糖合酶浓度比例对偶联反应的影响
将实施例2中纯化的糖基转移酶突变体UGT76G1-T284S/M88L/L200A和蔗糖合酶AtSuSy采用不同的比例进行反应,测定不同酶比例对偶联反应的影响。首先选择糖基转移酶的浓度为1、3、5、7、9μM,AtSuSy浓度为9μM进行测试。选择糖基转移酶的浓度为9μM,AtSuSy的浓度为1、3、5、7、9μM进行测试。
糖基化偶联反应体系为200μL,其中含有不同比例的糖基转移酶和蔗糖合酶纯酶、5mM Reb D、200mM蔗糖、1mM UDP和5%DMSO(v/v),50mM Tris-HCl pH 8.0缓冲液;35℃条件下反应20min。反应结束后,95℃加热10min终止反应,加入5倍体积甲醇进行稀释,20000×g离心5min,0.22μM滤膜进行抽滤后,UPLC进行检测分析。液相检测方法按照实施例3中所述进行,并计算Reb M的产率,结果显示当糖基转移酶UGT76G1-T284S/M88L/L200A为7μM,AtSuSy为9μM时Reb M产率达到最高为58.80%(图3)。结果显示当糖基转移酶UGT76G1-T284S/M88L/L200A为9μM,AtSuSy为7μM时Reb M产率达到最高为58.50%(图4)。因此确定糖基转移酶和蔗糖合酶偶联反应中的酶浓度UGT76G1-T284S/M88L/L200A 为7μM,AtSuSy为7μM。
实施例5温度对糖基转移酶和蔗糖合酶糖基化偶联反应的影响
将糖基化偶联反应体系置于不同温度(20~45℃)中进行反应,测定温度对糖基转移酶和蔗糖合酶糖基化偶联反应的影响。
按照实施例2所述纯化的糖基转移酶和蔗糖合酶以及实施例4中确定的糖基转移酶和蔗糖合酶的酶浓度比例,糖基化偶联反应体系为200μL,其中含有7μM UGT76G1-T284S/M88L/L200A、7μM AtSuSy、5mM Reb D、200mM蔗糖、1mM UDP和5%DMSO(v/v),50mM Tris-HClpH 8.0缓冲液;不同温度条件下反应20min。反应结束后,95℃加热10min终止反应,加入5倍体积甲醇进行稀释,20000×g离心5min,0.22μM滤膜进行抽滤后,UPLC进行检测分析。液相检测方法按照实施例3中所述进行,并计算Reb M的产率,结果显示当温度在30-35℃时,RebM的产率可以达到50%以上(图5)。
实施例6 pH对糖基转移酶和蔗糖合酶糖基化偶联反应的影响
将糖基化偶联反应体系置于不同pH值的缓冲液中进行反应,测定pH对糖基转移酶和蔗糖合酶糖基化偶联反应的影响,选择的缓冲液为50mM Bis-Tris pH 5.5-7.0;50mMKPi pH6.0-8.0;50mM Tris-HCl pH 7.0-9.0和50mM Glycine-NaOH pH 5.5-7.0。
按照实施例2所述纯化的糖基转移酶和蔗糖合酶以及实施例4中确定的糖基转移酶和蔗糖合酶的酶浓度比例,糖基化偶联反应体系为200μL,其中含有7μM UGT76G1-T284S/M88L/L200A、7μM AtSuSy、5mM Reb D、200mM蔗糖、1mM UDP和5%DMSO(v/v);30℃条件下反应20min。反应结束后,95℃加热10min终止反应,加入5倍体积甲醇进行稀释,20000×g离心5min,0.22μM滤膜进行抽滤后,UPLC进行检测分析。液相检测方法按照实施例3中所述进行,并计算Reb M的产率,结果显示当缓冲液为50mM KPi pH 8.0时,Reb M的产率可以达到76.10%(图6)。
实施例7蔗糖浓度对糖基转移酶和蔗糖合酶偶联反应的影响
将糖基化偶联反应体系中添加不同浓度的蔗糖(0-1000mM)中进行反应,测定蔗糖浓度对糖基转移酶和蔗糖合酶糖基化偶联反应的影响。
按照实施例2所述纯化的糖基转移酶和蔗糖合酶以及实施例4中确定的糖基转移酶和蔗糖合酶的酶浓度比例,糖基化偶联反应体系为200μL,其中含有7μM UGT76G1-T284S/M88L/L200A、7μM AtSuSy、5mM Reb D、5%DMSO(v/v)、1mM UDP和50mM Tris-HCl pH 8.0缓冲液;30℃条件下反应20min。反应结束后,95℃加热10min终止反应,加入5倍体积甲醇进行稀释,20000×g离心5min,0.22μM滤膜进行抽滤后,UPLC进行检测分析。液相检测方法按照实施例3中所述进行,并计算Reb M的产率,结果显示当蔗糖浓度为500~700mM时,Reb M的产率可以达到60%以上(图7)。
实施例8 UDP浓度对糖基转移酶和蔗糖合酶偶联反应底物的影响
将糖基化偶联反应体系中添加不同浓度的UDP(0.1-5mM)中进行反应,测定UDP浓度对糖基转移酶和蔗糖合酶糖基化偶联反应的影响。
按照实施例2所述纯化的糖基转移酶和蔗糖合酶以及实施例4中确定的糖基转移酶和蔗糖合酶的酶浓度比例,糖基化偶联反应体系为200μL,其中含有7μM UGT76G1-T284S/M88L/L200A、7μM AtSuSy、5mM Reb D、200mM蔗糖和5%DMSO(v/v),50mM Tris-HCl pH 8.0缓冲液;30℃条件下反应20min。反应结束后,95℃加热10min终止反应,加入5倍体积甲醇进行稀释,20000×g离心5min,0.22μM滤膜进行抽滤后,UPLC进行检测分析。液相检测方法按照实施例3中所述进行,并计算Reb M的产率,结果显示在UDP浓度为0.8mM时,Reb M的产率可以达到60.87%(图8)。
实施例9底物补料制备Reb M
由于底物Reb D的溶解度较差,因此将糖基化偶联反应体系反应不同的时间进行补加底物(0-7h),从而制备Reb M。
按照实施例2所述纯化的糖基转移酶和蔗糖合酶以及实施例4中确定的糖基转移酶和蔗糖合酶的酶浓度比例,糖基化偶联反应体系为2mL,其中含有7μM UGT76G1-T284S/M88L/L200A、7μM AtSuSy、5mM Reb D、600mM蔗糖和5%DMSO(v/v),0.8mM UDP和50mM Tris-HCl pH 8.0缓冲液;30℃条件下反应。在0.5h,1h和2h分别添加终浓度为5mM Reb D,并按照时间进行取样检测,95℃加热10min终止反应,加入20倍体积甲醇进行稀释,20000×g离心5min,0.22μM滤膜进行抽滤后,UPLC进行检测分析。液相检测方法按照实施例3中所述进行,并计算产物Reb M的浓度最终以90.50%的产率催化22.58g/L Reb D合成23.37g/L Reb M(图9),高于文献报道的关于Reb M的产量。
对比例1:
在突变体筛选过程中对比以文献报道的UGT76G1-T284S为出发酶进行突变,筛选到突变体UGT76G1-T284S/M88L和UGT76G1-T284S/L200A其催化活性分别为UGT76G1-T284S的1.67倍和2.09倍。随后将两个突变位点进行组合突变得到突变体UGT76G1-T284S/M88L/L200A,其催化活性是UGT76G1-T284S的2.38倍(表3)。因此选取UGT76G1-T284S/M88L/L200A用于后续级联反应制备Reb M。
表3 UGT76G1突变体催化活性
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> 江南大学
<120> 一种利用糖基转移酶UGT76G1突变体高效生物合成莱鲍迪苷M的方法
<130> BAA220196A
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 458
<212> PRT
<213> 人工序列
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Leu Phe Pro Val Pro Phe Gln Gly His Ile Asn Pro Ile Leu Gln Leu
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Ala Asn Val Leu Tyr Ser Lys Gly Phe Ser Ile Thr Ile Phe His Thr
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Asn Phe Asn Lys Pro Lys Thr Ser Asn Tyr Pro His Phe Thr Phe Arg
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Phe Ile Leu Asp Asn Asp Pro Gln Asp Glu Arg Ile Ser Asn Leu Pro
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Thr His Gly Pro Leu Ala Gly Leu Arg Ile Pro Ile Ile Asn Glu His
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Gly Ala Asp Glu Leu Arg Arg Glu Leu Glu Leu Leu Met Leu Ala Ser
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Glu Glu Asp Glu Glu Val Ser Cys Leu Ile Thr Asp Ala Leu Trp Tyr
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Phe Ala Gln Ser Val Ala Asp Ser Leu Asn Leu Arg Arg Leu Val Leu
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Met Thr Ser Ser Leu Phe Asn Phe His Ala His Val Ser Leu Pro Gln
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Phe Asp Glu Leu Gly Tyr Leu Asp Pro Asp Asp Lys Thr Arg Leu Glu
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Glu Gln Ala Ser Gly Phe Pro Met Leu Lys Val Lys Asp Ile Lys Ser
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Ala Tyr Ser Asn Trp Gln Ile Ala Lys Glu Ile Leu Gly Lys Met Ile
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Lys Gln Thr Lys Ala Ser Ser Gly Val Ile Trp Asn Ser Phe Lys Glu
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Leu Glu Glu Ser Glu Leu Glu Thr Val Ile Arg Glu Ile Pro Ala Pro
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Ser Phe Leu Ile Pro Leu Pro Lys His Leu Thr Ala Ser Ser Ser Ser
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Leu Leu Asp His Asp Arg Thr Val Phe Gln Trp Leu Asp Gln Gln Pro
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Pro Ser Ser Val Leu Tyr Val Ser Phe Gly Ser Ser Ser Glu Val Asp
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Glu Lys Asp Phe Leu Glu Ile Ala Arg Gly Leu Val Asp Ser Lys Gln
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Claims (10)
1.一种糖基转移酶突变体,其特征在于,为氨基酸序列如SEQ ID NO.1所示的蛋白质。
2.编码权利要求1所述糖基转移酶突变体的基因。
3.携带权利要求2所述基因的表达载体。
4.表达权利要求1所述糖基转移酶突变体的重组菌。
5.一种催化合成莱鲍迪苷M的方法,其特征在于,以莱鲍迪苷D为底物,利用权利要求1所述糖基转移酶突变体与蔗糖合酶偶联进行催化反应。
6.根据权利要求5所述的方法,其特征在于,所述蔗糖合酶的氨基酸序列可以是任意来源的具有蔗糖合酶活性的氨基酸序列。
7.根据权利要求5所述的方法,其特征在于,所述糖基转移酶突变体和蔗糖合酶的摩尔浓度比为1:1。
8.根据权利要求5~7任一项所述的方法,其特征在于,所述催化反应的条件为:1-20 mM莱鲍迪苷D、500-700 mmol/L 蔗糖、0.1-1 mM UDP、50 mM K2HPO4-KH2PO4缓冲液为反应体系,20-45℃反应20 min-24 h。
9.根据权利要求5所述的方法,其特征在于,所述催化反应的第0.5 h,1 h和2 h分别添加终浓度5 mM的莱鲍迪苷D。
10.权利要求1所述糖基转移酶突变体或权利要求2所述基因或权利要求3所述表达载体或权利要求4所述重组菌或权利要求5~9任一所述方法在制备含有莱鲍迪苷M的产品中的应用。
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