CN109825484B - 玉米赤霉烯酮水解酶zhd101突变体及利用该突变体水解玉米赤霉烯酮的方法 - Google Patents
玉米赤霉烯酮水解酶zhd101突变体及利用该突变体水解玉米赤霉烯酮的方法 Download PDFInfo
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Abstract
本发明涉及玉米赤霉稀酮水解酶ZHD101突变体及利用该突变体水解玉米赤霉烯酮的方法。所述突变体是通过在野生型玉米赤霉稀酮水解酶ZHD101的三个关键位点处引入突变而得到。本发明中的ZHD101突变体与野生型ZHD101相比,在酸性条件(pH 5.5)下对ZEN水解的比活提高了至少75%;并且,与野生型ZHD101相比,在酸性条件(pH 5.5)下对ZEN水解的催化效率(kcat/KM)也可提高多达40%。由此,为进一步提高酶催化活性和实现酸性条件下的工业应用奠定了基础。
Description
技术领域
本发明涉及玉米赤霉烯酮水解酶ZHD101突变体及利用该突变体水解玉米赤霉烯酮的方法。具体而言,本发明涉及一种经过基因改造和蛋白质工程化得到的在酸性条件下仍具有出色活性的玉米赤霉烯酮水解酶ZHD101突变体及利用该突变体水解玉米赤霉烯酮的方法。
背景技术
真菌毒素是真菌的次级代谢产物,很多真菌毒素具有致畸、致癌、产生神经毒性、引发免疫抑制的作用,对于全球食品安全的负面影响不容忽视(Kabak B,Dobson ADW,VarI.Strategies to Prevent Mycotoxin Contamination of Food and Animal Feed:AReview.Critical Reviews in Food Science and Nutrition 2006;46:593-619)。玉米赤霉烯酮(Zearalenone,ZEN)是由镰刀菌属(Fusarium)真菌产生的具有雌性激素毒性的非类固醇毒素,具有二羟基苯甲酸内酯结构(Zinedine A,Soriano JM,MoltóJC,J.Review on the toxicity,occurrence,metabolism,detoxification,regulations andintake of zearalenone:An oestrogenic mycotoxin.Food and Chemical Toxicology2007;45:1-18)。ZEN在全世界谷物中广泛分布且稳定存在,因而成为污染饲料和谷物深加工食品的主要真菌毒素。ZEN的热稳定性较好(Marin S,Ramos AJ,Cano-Sancho G,SanchisV.Mycotoxins:Occurrence,toxicology,and exposure assessment.Food and ChemicalToxicology 2013;60:218-237),在110℃下加热12天仍没有明显分解迹象(Lauren DR,Smith WA.Stability of the Fusarium mycotoxins nivalenol,deoxynivalenol andzearalenone in ground maize under typical cooking environments.FoodAdditives&Contaminants 2001;18:1011-1016)。ZEN对人和动物具有生殖毒性,人和动物长期摄入被ZEN污染的食物可引发雌性激素过多症,导致生殖器官的病变或功能障碍;ZEN还具有强致癌性,通过在生物体内聚积,会导致细胞内染色体异常,诱导肿瘤,促使癌细胞在组织器官中的形成与扩散,引起乳腺癌、食管癌等发病率增加(Yu Z,Hu D,Li Y.Effectsof zearalenone on mRNAexpression and activity of cytochrome P450 1A1and 1B1inMCF-7cells.Ecotoxicology and Environmental Safety 2004;58:187-193);ZEN本身还具有免疫毒性,能阻止淋巴细胞的正常增殖和发挥免疫功能(Berek L,Petri IB,MesterházyTéren J,Molnár J.Effects of mycotoxins on human immune functions invitro.Toxicology in Vitro 2001;15:25-30)。因此,解决ZEN污染问题已经成为保证食品安全和维护人体健康的当务之急。一方面,需要加强农作物生产收获各个时期的管理,尽量避免农作物感染真菌;另一方面,对已经产生的ZEN污染,需要积极开发使其降解与脱毒的技术。
目前,国内外的学者已经研究出了一系列的ZEN降解脱毒技术,包括物理法、化学法和生物转化法。由于物理和化学降解法具有破坏原料营养成分、高污染、高成本的缺点,其应用受到了很大限制;而生物转化法依靠微生物产生的具有ZEN水解活性的酶,具有高效、高特异性、反应条件温和、无毒副产物生成等优点。因此,ZEN的生物转化降解法得到了研究人员的广泛关注并已进行了深入研究。
ZHD101(Zearalenone hydrolase 101)是一种由粉红粘帚霉菌菌株Gliocladiumroseum IFO 7063中分离出来的ZEN内酯水解酶,该酶能够特异性地结合ZEN并使之高效水解(Takahashi-Ando N,Kimura M,Kakeya H,Osada H,Yamaguchi I.A novellactonohydrolase responsible for the detoxification of zearalenone:enzymepurification and gene cloning.Biochemical Journal 2002;365:1-6),其机理为ZHD101中的催化三联体结构(Ser102-His242-Glu126)能够催化水解ZEN中的二羟基苯甲酸内酯键,从而将ZEN的大环状结构打开成为直链结构中间产物,随后中间产物自发脱羧生成断裂产物(Peng W.等,Crystal structure and substrate-binding mode of themycoestrogen-detoxifying lactonase ZHD from Clonostachys rosea.RSC Adv 2014;4:62321-62325)。所生成的断裂产物不能与雌激素受体结合,因而毒性较ZEN而言大幅减弱。
当前研究表明,ZHD101在35-45℃、pH>6的环境中有较好的催化效率,但是在酸性体系下其催化效率不够理想,低于4.5的pH条件甚至会造成该酶的不可逆失活(Takahashi-Ando N等,Metabolism of Zearalenone by Genetically Modified OrganismsExpressing the Detoxification Gene from Clonostachys rosea.Applied andEnvironmental Microbiology 2004;70:3239-3245)。然而,由于工业物料(如玉米浆)的pH多为酸性,在工业生产中也存在降低其中的ZEN毒素含量的需求,因此迫切需要开发一种即便在酸性条件下也能够有效催化水解ZEN的内酯水解酶。
发明内容
为解决上述课题,本发明的目的是在已报道的玉米赤霉烯酮水解酶ZHD101的基础上,提高其在酸性条件下的催化活性。
就此而言,已发现的野生型ZHD101由264个氨基酸组成(SEQ ID NO.1)。经过本发明人的锐意研究,发现相比于野生型ZHD101,引入特定突变的ZHD101突变体在酸性条件下具有更出色的催化活性,在中性/碱性条件下也具有相当的催化活性。具体而言,本发明人对野生型ZHD101的氨基酸序列进行了深入研究和分析,从中挑选了多个潜在核心位点进行突变测试,合成编码基因并表达成功,由此确定了三个关键位点并获得了相应的在酸性条件下也可高效发挥作用的ZHD101突变体。
因此,本发明的第一方面在于提供一种玉米赤霉稀酮水解酶ZHD101突变体,其特征在于,所述ZHD101突变体在野生型ZHD101氨基酸序列(SEQ ID NO.1)中引入了如下突变中的一种或多种:第157位的D突变为K(下文可称为D157K);第133位的D突变为K(下文可称为D133K);以及第171位的E突变为K(下文可称为E171K)。
本发明的第二方面在于提供编码上述玉米赤霉稀酮水解酶ZHD101突变体的DNA分子。
本发明的第三方面在于提供包含上述DNA分子的表达载体。
本发明的第四方面在于提供导入上述表达载体的转化细胞。
本发明的第五方面在于提供含有上述玉米赤霉稀酮水解酶ZHD101突变体、DNA分子、表达载体和/或转化细胞的试剂盒。
本发明的第六方面在于提供制备上述玉米赤霉稀酮水解酶ZHD101突变体的方法,所述方法包括如下步骤:在培养基中培养上述转化细胞;以及收集所述玉米赤霉稀酮水解酶ZHD101突变体。
本发明的第七方面在于提供降解玉米赤霉烯酮ZEN的方法,所述方法包括使用上述玉米赤霉稀酮水解酶ZHD101突变体、DNA分子、表达载体、转化细胞和/或试剂盒。
本发明的第八方面在于提供上述玉米赤霉稀酮水解酶ZHD101突变体、DNA分子、表达载体、转化细胞和/或试剂盒在降解玉米赤霉烯酮中的用途。
有益效果
与现有技术相比,本发明具有如下优点:
(1)本发明的各种ZHD101突变体表达活性高,可在大肠杆菌(Escherichiacoli)细胞内高活性表达;(2)本发明的各种ZHD101突变体在酸性条件下的催化活性均较野生型ZHD101有所提高,为进一步提高酶活和实现酶法的工业化生产奠定了基础。
附图说明
图1为玉米赤霉稀酮水解酶ZHD101催化ZEN水解生成断裂产物的反应式。
图2为通过SDS凝胶电泳检验本发明的ZHD101及ZHD101突变体的纯化结果。其中,左侧两条泳道依次为ZHD101及ZHD101突变体;右侧泳道为蛋白Marker。
图3为通过HPLC检测ZHD101突变体降解ZEN生成产物的色谱图。
具体实施方式
下文将详细阐述本发明。
一.本发明的玉米赤霉稀酮水解酶ZHD101突变体及其活性测定
本发明的ZHD101突变体在野生型ZHD101氨基酸序列(SEQ ID NO.1)中引入了如下突变中的一种或多种:D157K;D133K;以及E171K。
在下文中,可将D157K、D133K以及E171K突变分别称为M1、M2以及M3。从而,可将携有D157K突变的突变体称为ZHD101M1(氨基酸序列如SEQ ID NO.3所示)、将携有D133K突变的突变体称为ZHD101M2(氨基酸序列如SEQ ID NO.5所示)、将携有E171K突变的突变体称为ZHD101M3(氨基酸序列如SEQ ID NO.7所示);相应地,可将携有D157K突变和D133K突变的突变体称为ZHD101M1M2(氨基酸序列如SEQ ID NO.9所示)、将携有D157K突变和E171K突变的突变体称为ZHD101M1M3(氨基酸序列如SEQ ID NO.11所示)、将携有D133K突变和E171K突变的突变体称为ZHD101M2M3(氨基酸序列如SEQ ID NO.13所示)、将携有全部三个D157K突变、D133K突变和E171K突变的突变体称为ZHD101M1M2M3(氨基酸序列如SEQ ID NO.15所示)。
在本发明中,“157位”、“133位”及“171位”并不是必然表示从所述玉米赤霉稀酮水解酶的N端起的绝对位置,而是表示与SEQ ID NO.1的氨基酸序列相比的相对位置。例如,在含有SEQ ID NO.1的氨基酸序列的玉米赤霉稀酮水解酶中,当该玉米赤霉稀酮水解酶157位的N端某一位置缺失了一个氨基酸,上述的157位即变为156位。即使在这一情况下,所述从N端残基起计数为156位的氨基酸在本发明中仍为“157位”的氨基酸。通过将感兴趣的玉米赤霉稀酮水解酶的氨基酸序列与SEQ ID NO.1的氨基酸序列进行比对而确定所述氨基酸的相对位置。
本发明的ZHD101M1、ZHD101M2、ZHD101M3、ZHD101M1M2、ZHD101M1M3、ZHD101M2M3以及ZHD101M1M2M3突变体可通过人工合成而得到;也可通过先合成其编码基因、再进行生物表达而得到。
与对应的野生型玉米赤霉稀酮水解酶(SEQ ID NO.1)催化水解ZEN的酶活性相比,本发明的各玉米赤霉稀酮水解酶突变体在中性/碱性条件下的酶活性相当,在酸性条件下的酶活性更高。
本领域技术人员知晓测定玉米赤霉稀酮水解酶活性的方法。例如,根据本发明的一个实施方式,可通过下述方法测定本发明的玉米赤霉稀酮水解酶突变体的活性:以一定浓度的ZEN作为底物,在特定pH条件下,使底物与酶在37℃恒温水浴下反应10min,反应结束后煮沸1min终止反应,通过HPLC检测反应产物。酶催化动力学参数(Vmax、KM、Vmax/KM)通过Lineweaver-Burk法将实验数据拟合为经典的米氏方程(Michaelis-Menten equation)得到。
二.编码本发明的玉米赤霉稀酮水解酶突变体的DNA分子
本发明的DNA分子是编码本发明的玉米赤霉稀酮水解酶突变体的DNA分子。根据本发明一个优选的实施方式,本发明的DNA分子可通过以下方式获得:例如采用PCR等方法获得野生型玉米赤霉稀酮水解酶基因(例如,如SEQ ID NO.2所示的核苷酸序列),通过定点突变法等将目的突变导入,制备编码各玉米赤霉稀酮水解酶突变体的DNA分子。
对于定点突变法并没有特别限定,例如,可以使用市售的QuikChange Site-Directed Mutagenesis Kit(Stratagene公司制造)等进行。作为引入定点突变的方法,例如,Gapped duplex法和Kunkel法都是已知的。
根据本发明另一个优选的实施方式,也可以通过化学合成得到本发明的DNA分子。根据本发明一个特别优选的实施方式,为提高突变体在宿主细胞内的表达效率,可将野生型玉米赤霉稀酮水解酶的编码序列(SEQ ID NO.2)替换为由宿主细胞偏爱密码子组成的DNA序列,引入目的突变后,再通过化学合成的方式制备本发明的DNA分子。根据本发明一个最优选的实施方式,所述宿主细胞为大肠杆菌,将野生型玉米赤霉稀酮水解酶的编码序列(SEQ ID NO.2)替换为由大肠杆菌偏爱密码子组成的DNA序列后得到的序列如SEQ IDNO.17所示。
优选地,作为编码本发明的玉米赤霉稀酮水解酶突变体的DNA分子,例如为具有如SEQ ID NO.4、SEQ ID NO.6、SEQ ID NO.8、SEQ ID NO.10、SEQ ID NO.12、SEQ ID NO.14和SEQ ID NO.16所示的核苷酸序列的DNA分子。但是,只要是编码本发明的玉米赤霉稀酮水解酶突变体的核苷酸序列,则并不限于这些DNA分子。
根据本发明另一个优选的实施方式,作为编码本发明的玉米赤霉稀酮水解酶突变体的DNA分子,也可以是下述DNA分子:与如SEQ ID NO.4、SEQ ID NO.6、SEQ ID NO.8、SEQID NO.10、SEQ ID NO.12、SEQ ID NO.14和SEQ ID NO.16所示的核苷酸序列的互补序列在严格条件下进行杂交、且编码具有上述特定突变并具有上述所希望的活性的玉米赤霉稀酮水解酶突变体。其中,严格条件是指形成所谓的特异性杂交而不形成非特异性杂交的条件。尽管该条件因核苷酸序列或其长度而不同,其实例包括具有高同源性(例如,具有不低于75%的同源性、优选不低于90%的同源性、进一步优选不低于95%的同源性、最优选不低于98%的同源性)的DNA分子相互杂交,而同源性低于上述标准的DNA分子不杂交的条件;或Southern杂交中用于漂洗的常用条件的杂交条件(60℃和1×SSC、0.1%SDS,优选0.1×SSC和相当于0.1%SDS的盐浓度)。
根据本发明另一个优选的实施方式,作为编码本发明的玉米赤霉稀酮水解酶突变体的DNA分子,也可以是与SEQ ID NO.4、SEQ ID NO.6、SEQ ID NO.8、SEQ ID NO.10、SEQ IDNO.12、SEQ ID NO.14和SEQ ID NO.16所表示的核苷酸序列具有90%以上的同源性(优选具有95%以上的同源性、更优选具有98%以上的同源性、进一步更优选具有99%以上的同源性)、且编码具有上述特定突变并具有上述所希望的活性的玉米赤霉稀酮水解酶突变体。
其中,SEQ ID NO.4、SEQ ID NO.6、SEQ ID NO.8、SEQ ID NO.10、SEQ ID NO.12、SEQ ID NO.14和SEQ ID NO.16由792个碱基组成,其开放阅读框(ORF)为第1-792位碱基,分别编码具有如SEQ ID NO.3、SEQ ID NO.5、SEQ ID NO.7、SEQ ID NO.9、SEQ ID NO.11、SEQID NO.13和SEQ ID NO.15所示的氨基酸序列的蛋白(分别为本发明的ZHD101M1、ZHD101M2、ZHD101M3、ZHD101M1M2、ZHD101M1M3、ZHD101M2M3以及ZHD101M1M2M3突变体)。
三.本发明的表达载体
本发明的表达载体是用于表达本发明的玉米赤霉稀酮水解酶突变体的表达载体。根据本发明一个优选的实施方式,所述表达载体可具有如下的结构:控制编码本发明的玉米赤霉稀酮水解酶突变体的DNA分子表达的启动子序列连接到所述DNA分子的上游。此外,还可以将终止子连接到所述DNA分子的下游。其它常规操作元件也可包含于表达载体中。
作为表达载体,可使用本领域已知的任何常规类型。从拷贝数和稳定性的角度来看,优选可在大肠杆菌细胞中发挥作用的pET30a(+)表达载体。例如,可借助常规基因工程手段,通过将本发明的DNA分子插入pET30a(+)载体的多克隆位点之间而获得本发明的表达载体。
根据本发明一个优选的实施方式,用于挑选所表达的突变体的选择标记基因或用于检测导入基因表达的报告基因也可包含于本发明的表达载体中。选择标记基因的实例包括但不限于潮霉素抗性基因、卡那霉素抗性基因和氨苄青霉素抗性基因。报告基因的实例包括但不限于β-葡糖醛酸酶(GUS)基因、氯霉素乙酰转移酶(CAT)基因、荧光素酶(LUC)基因和绿色荧光蛋白(GFP)基因。
根据本发明另一个优选的实施方式,为了分泌表达本发明的玉米赤霉稀酮水解酶突变体、或为了便于纯化所表达的玉米赤霉稀酮水解酶突变体,本发明的表达载体中可进一步包含附加序列。在这种情况下,本发明的玉米赤霉稀酮水解酶突变体以融合蛋白(与附加序列编码的蛋白或肽融合)的形式表达。所述附加序列的实例包括但不限于编码信号肽或前肽的核苷酸序列;以及编码His标签或GST标签的核苷酸序列。
四.本发明的转化细胞
本发明的转化细胞是导入本发明的表达载体的细胞,该细胞能生产本发明的玉米赤霉稀酮水解酶突变体。该转化细胞可以是原核细胞,也可以是真核细胞。从方便快捷的角度来看,优选为原核细胞。
根据本发明一个优选的实施方式,所述原核细胞为大肠杆菌细胞。具体而言,由于目前对大肠杆菌细胞生产重组蛋白的条件有着十分深入的研究,且大肠杆菌细胞在生产制备过程中成本较低,能够满足技术需求和产品市场化的需求,因而大肠杆菌细胞被认为是玉米赤霉稀酮水解酶突变体生产中较为优选的转化细胞。然而,只要能导入本发明的表达载体并生产本发明的玉米赤霉稀酮水解酶突变体,本发明并不限于此。
可根据转化细胞的类型适当选择将本发明的表达载体导入转化细胞并进行蛋白表达的方法。这些方法都是本领域技术人员已知的。
根据本发明一个优选的实施方式,可通过以下方法获得大肠杆菌转化细胞:采用电击转化的方法,将本发明的表达载体转化到大肠杆菌感受态细胞中;随后将菌体悬液涂布于平板上,并培养直至出现单个菌落。
五.本发明的试剂盒
本发明的试剂盒是包含本发明的玉米赤霉稀酮水解酶突变体、编码本发明的玉米赤霉稀酮水解酶突变体的DNA分子、本发明的表达载体以及本发明的转化细胞中的一种或多种的试剂盒。
所述试剂盒可包含容器和在该容器上或与该容器相关联的标签或包装说明书。合适的容器包括例如瓶、小瓶、注射器等。该容器可由多种材料形成,如玻璃或塑料。所述标签和包装说明书指示了该试剂盒的使用方法及用途。任选地,本发明的试剂盒还可另外包含一种或多种组分,所述组分选自于试管、反应缓冲液、PCR引物、dNTP、Taq聚合酶、逆转录酶、DNA酶、RNA酶抑制剂、DEPC水和无菌水,但不总限于此。
六.本发明的玉米赤霉稀酮水解酶突变体的制备方法
通过培养本发明的转化细胞,可生产本发明的玉米赤霉稀酮水解酶突变体。通过与信号肽融合的融合蛋白的形式表达本发明的玉米赤霉稀酮水解酶突变体以用于分泌,本发明的玉米赤霉稀酮水解酶突变体可在培养基中积累。或者,当本发明的玉米赤霉稀酮水解酶突变体存在于转化细胞内时,可通过超声破碎等方式将转化细胞裂解并通过离心等方式获得本发明的玉米赤霉稀酮水解酶突变体。
当使用诱导型启动子时,优选在培养期间进行诱导。尽管培养所述转化细胞的方法随细胞的类型而不同,但可以使用传统方法。
根据本发明一个优选的实施方式,培养大肠杆菌转化细胞的方法的实例如下所述:将大肠杆菌菌种接种于100mL LB液体培养基中,于37℃下过夜培养活化;以1:100的接种量将其转接于500mL新鲜LB液体培养基(含50μg/mL卡那霉素)中,在37℃、200rpm的条件下,继续培养至OD600为0.6~0.8;然后加入终浓度为1mM的IPTG,在20℃、120rpm条件下培养14h,诱导ZHD101突变体基因的表达。
当本发明的玉米赤霉稀酮水解酶突变体(分泌到培养基中时)处在存在于发酵液上清中的状态时,可被使用;也可通过浓缩所述发酵液上清使用所述玉米赤霉稀酮水解酶突变体。可纯化或部分纯化所述玉米赤霉稀酮水解酶突变体(分泌到培养基中时)。
利用蛋白纯化的一般方法,可实现纯化或部分纯化。例如,可以使用包括色谱(如离子交换或凝胶过滤)、硫酸铵盐析或有机溶剂沉析等技术。还可通过冻干、超滤膜和有机溶剂沉析等浓缩纯化后的酶。
根据本发明一个优选的实施方式,利用在本发明玉米赤霉稀酮水解酶突变体的C端添加的六聚组氨酸标签对其进行纯化回收。
七.利用本发明的玉米赤霉稀酮水解酶突变体催化水解ZEN的方法和用途
根据本发明的一些实施方式,利用本发明的玉米赤霉稀酮水解酶突变体来催化水解ZEN的方法可包括如下步骤:使由4850μL缓冲液(pH范围为5.5~8.0)、50μL ZEN底物(溶于乙腈中,浓度范围0~2mg/mL)及100μL酶液(保存于100mMTris缓冲液中,pH 8.0,浓度范围100~200μg/mL)组成的5mL反应体系在37℃恒温水浴下反应10min。反应结束后在沸水中煮沸1min终止反应,反应产物通过HPLC进行检测。
在本发明一些优选的实施方式中,本发明的玉米赤霉稀酮水解酶突变体可用于在玉米深加工、燃料乙醇生产、氨基酸生产、有机酸生产、淀粉/淀粉糖加工、粮油食品加工、饲料生产中催化水解ZEN。
在本发明一些更为优选的实施方式中,所述氨基酸生产为谷氨酸生产和/或赖氨酸生产;所述有机酸生产为柠檬酸生产。
实施例
下文将结合具体实施例对本发明作进一步说明,但本发明并不限于以下实施例。下述实施例中,除非特别说明,所用试剂、培养基均为市售商品,所用方法均为常规方法。例如,所有的基因操作可按Molecular Cloning(Cold Spring Harbor Laboratory Press(1989))所记载的进行。
实施例1、ZHD101突变体的制备纯化
一、ZHD101突变体编码基因与表达载体的构建
将野生型玉米赤霉稀酮水解酶的编码序列(SEQ ID NO.2)替换为大肠杆菌偏爱密码子组成的DNA序列(SEQ ID NO.17),并引入相应突变,得到如SEQ ID NO.4、SEQ ID NO.6、SEQ ID NO.8、SEQ ID NO.10、SEQ ID NO.12、SEQ ID NO.14和SEQ ID NO.16所示的核苷酸序列,在上述序列5’端添加Nde I酶切位点CATATG,3’端顺次添加编码六聚组氨酸标签的DNA序列CACCATCACCACCATCAC(SEQ ID NO.18)、终止密码子TAA及Xho I酶切位点CTCGAG,化学合成所得的DNA序列(Invitrogen公司,美国)。组氨酸标签6×His添加于所编码的蛋白序列C端末尾以利于后续纯化步骤。所得DNA序列利用Nde I和Xho I双酶切并纯化后,与同样进行双酶切的表达载体pET30a(+)(含卡那霉素抗性基因)过夜连接,随后转入大肠杆菌感受态细胞BL21(DE3)(Takara公司,大连)中,即获得表达载体pET30a-ZHD101M1、pET30a-ZHD101M2、pET30a-ZHD101M3、pET30a-ZHD101M1M2、pET30a-ZHD101M1M3、pET30a-ZHD101M2M3和pET30a-ZHD101M1M2M3。
同时,作为对照,还采用相同方法制备了表达野生型玉米赤霉稀酮水解酶的表达载体pET30a-ZHD101。
经测序,表达载体pET30a-ZHD101、pET30a-ZHD101M1、pET30a-ZHD101M2、pET30a-ZHD101M3、pET30a-ZHD101M1M2、pET30a-ZHD101M1M3、pET30a-ZHD101M2M3和pET30a-ZHD101M1M2M3中均插入了正确的目的DNA片段。
二、转化细胞的获得
将步骤一获得的表达载体pET30a-ZHD101、pET30a-ZHD101M1、pET30a-ZHD101M2、pET30a-ZHD101M3、pET30a-ZHD101M1M2、pET30a-ZHD101M1M3、pET30a-ZHD101M2M3和pET30a-ZHD101M1M2M3转化大肠杆菌BL21(DE3)感受态细胞(Takara公司,大连),得到相应的转化大肠杆菌细胞E.coli BL21(DE3)/pET30a-ZHD101、E.coli BL21(DE3)/pET30a-ZHD101M1、E.coli BL21(DE3)/pET30a-ZHD101M2、E.coli BL21(DE3)/pET30a-ZHD101M3、E.coli BL21(DE3)/pET30a-ZHD101M1M2、E.coli BL21(DE3)/pET30a-ZHD101M1M3、E.coliBL21(DE3)/pET30a-ZHD101M2M3和E.coli BL21(DE3)/pET30a-ZHD101M1M2M3。
将得到的大肠杆菌转化细胞在含卡那霉素(50μg/mL)的LB平板上划线,37℃下过夜培养,再挑取生长良好的的单菌落接种于100mL LB液体培养基(含50μg/mL卡那霉素)中,在37℃、200rpm培养7h以上,以1:100的接种量进行步骤三的实验。
三、ZHD101突变体的获得
1、酶突变体的表达
将步骤二获得的大肠杆菌转化细胞以1:100的接种量转接于500mL新鲜LB液体培养基(含50μg/mL卡那霉素)中,在37℃、200rpm的条件下,继续培养至OD600为0.6~0.8。然后加入终浓度为1mM的IPTG,在20℃、120rpm条件下培养14h,诱导ZHD101突变体基因的表达。
诱导表达结束后,将所有的500mL菌液在4℃、10,000rpm(11,000g)的条件下离心10min,离心得到的菌体沉淀用Tris-HCl缓冲液重悬(100mM Tris,pH 8.0)。重悬液用超声破碎方法提取可溶蛋白(超声时间4s,间隔时间6s,60%功率,20min)。将裂解液在4℃、10,000rpm(11,000g)条件下离心10min,取上清即为含目的蛋白的粗酶液;再将含目的蛋白的粗酶液在4℃、10,000rpm(11,000g)条件下离心10min,进一步去除超声破碎带来的细胞杂质。
2、酶突变体的纯化
将含目的蛋白的粗酶液上样至Ni-NTA柱,通过镍离子作为亲和离子,利用不同咪唑浓度梯度洗脱目的蛋白。由于ZHD101及其突变体在254nm有特定吸收峰,故在纯化过程中,用254nm检测蛋白峰可以有效防止杂蛋白干扰。具体步骤如下:
首先,使用结合缓冲液A(100mM Tris,pH 8.0,含500mM NaCl和20mM咪唑)预平衡柱床;随后,使用结合缓冲液B(100mM Tris,pH 8.0,含500mM NaCl和50mM咪唑)洗脱杂蛋白,直至洗脱液在254nm下的吸光度与缓冲液B基本相同。使用洗脱缓冲液(100mM Tris,pH8.0,含500mM NaCl和250mM咪唑)收集目的蛋白,并用10kDa Ultra-0.5超滤离心管(Millipore)浓缩目的蛋白,并进行两次脱盐,移除目的蛋白中的咪唑和NaCl组分。纯化后的目的蛋白保存于Tris-HCl缓冲液中(100mM Tris,pH 8.0),并置于4℃冷藏保存。
目的蛋白的分子量在25kDa左右,其纯度通过SDS-PAGE(5%积层胶,12%分离胶)检验,结果如图2所示(以ZHD101和ZHD101M1为例,其它突变体的SDS-PAGE结果类似)。根据SDS-PAGE结果,在25kDa附近可以观测到明显的蛋白条带,收集的蛋白纯度大于90%。纯化后得到的目的蛋白浓度通过Bradford法并使用牛血清蛋白作为标准试剂测得,测定时所使用的吸光度为595nm。在此纯化条件下,500mL发酵液能得到约25mg目的蛋白。
实施例2、ZHD101突变体的催化活性检测
通过HPLC(LC-20AT,岛津公司)分析实施例1的步骤三中获得的目的蛋白,从而测定酶突变体的催化活性和动力学参数。HPLC图谱如图3所示(示例性示出ZHD101M3的实验结果)。
具体而言,所选用的色谱柱为Hypersil C18反相柱(依利特,5μm,4.6mM×250mM)。反应体系配比为:4850μL乙酸钠(pH 5.5)、50μL底物(溶于乙腈中,浓度呈梯度变化,最高浓度为2mg/mL)、100μL酶液(保存于100mM Tris缓冲液中,pH 8.0,浓度100μg/mL),在37℃恒温水浴下反应10min。反应结束后在沸水中煮沸1min终止反应。
HPLC的流动相配比(v/v)为:60%乙腈,40%三氟乙酸水溶液(0.1%,v/v),流速1.0mL/min,检测波长为254nm,柱温箱恒定为30℃。ZHD101、ZHD101M1、ZHD101M2、ZHD101M3、ZHD101M1M2、ZHD101M1M3、ZHD101M2M3以及ZHD101M1M2M3均在保留时间tR=6.4min处出现ZEN色谱吸收峰,在保留时间tR=3.3min处出现裂解产物色谱吸收峰。酶催化动力学参数(Vmax、KM、Vmax/KM)通过Lineweaver-Burk法将实验数据拟合为经典的米氏方程(Michaelis-Menten equation)得到。
在酸性条件(pH 5.5)下,本发明的玉米赤霉稀酮水解酶ZHD101突变体ZHD101M1对ZEN水解的比活为1.12U/mg,与对照ZHD101相比(0.635U/mg),比活提高了76.4%;突变体ZHD101M2对ZEN水解的比活为1.75U/mg,与对照ZHD101相比,比活提高了175.6%;突变体ZHD101M3对ZEN水解的比活为1.32U/mg,与对照ZHD101相比,比活提高了107.9%;突变体ZHD101M1M2对ZEN水解的比活为1.79U/mg,与对照ZHD101相比,比活提高了181.9%;突变体ZHD101M1M3对ZEN水解的比活为1.35U/mg,与对照ZHD101相比,比活提高了112.6%;突变体ZHD101M2M3对ZEN水解的比活为1.81U/mg,与对照ZHD101相比,比活提高了185.0%;突变体ZHD101M1M2M3对ZEN水解的比活为1.78U/mg,与对照ZHD101相比,比活提高了180.3%。可见,相对于一个位点的突变,将多个位点的突变结合可以进一步提高突变体的比活。
同时,在酸性条件(pH 5.5)下,本发明的玉米赤霉稀酮水解酶ZHD101突变体ZHD101M1对ZEN水解的催化效率(kcat/KM)为14417.2s-1·M-1,与对照ZHD101相比(10574.4s-1·M-1),催化效率提高了36.3%;突变体ZHD101M2对ZEN水解的催化效率为10430.8s-1·M-1,与对照ZHD101催化效率相当;突变体ZHD101M3对ZEN水解的催化效率为13640.8s-1·M-1,与对照ZHD101相比,催化效率提高了29.0%;突变体ZHD101M1M2对ZEN水解的催化效率为14259.7s-1·M-1,与对照ZHD101相比,催化效率提高了34.9%;突变体ZHD101M1M3对ZEN水解的催化效率为14823.1s-1·M-1,与对照ZHD101相比,催化效率提高了40.2%;突变体ZHD101M2M3对ZEN水解的催化效率为11942.8s-1·M-1,与对照ZHD101相比,催化效率提高了12.9%;突变体ZHD101M1M2M3对ZEN水解的催化效率为14597.5s-1·M-1,与对照ZHD101相比,催化效率提高了38.1%。
工业实用性
本发明的玉米赤霉稀酮水解酶突变体为进一步提高酶活和实现酸性条件下的工业应用奠定了基础。
序列表
<110> 吉林中粮生化有限公司 中粮营养健康研究院有限公司 清华大学
<120> 玉米赤霉烯酮水解酶ZHD101突变体及利用该突变体水解玉米赤霉烯酮的方法
<160> 18
<170> SIPOSequenceListing 1.0
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<211> 264
<212> PRT
<213> 粉红粘帚霉菌(Gliocladium roseum)
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Ala Lys Ala Pro Pro Glu Thr Tyr Thr Glu Val Thr Ala Gln Lys Leu
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Ala Ser Tyr Val Ile Ser Val Leu Asp Ala Leu Asp Ile Lys His Ala
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Thr Val Trp Gly Cys Ser Ser Gly Ala Ser Thr Val Val Ala Leu Leu
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Leu Gly Tyr Pro Asp Arg Ile Arg Asn Ala Met Cys His Glu Leu Pro
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Thr Lys Leu Leu Asp His Leu Ser Asn Thr Ala Val Leu Glu Asp Glu
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His Lys Asn Tyr Pro Val Trp Ala Arg Gly Tyr Pro Arg Thr Ile Pro
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Asp Trp Thr Val Gly Ala Ala Thr Pro Thr Glu Ser Phe Phe Asp Asn
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Met His Phe Pro Tyr Val Ser His Pro Asp Val Phe Ala Lys Tyr Val
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<210> 2
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<212> DNA
<213> 粉红粘帚霉菌(Gliocladium roseum)
<400> 2
atgcgcactc gcagcacaat ctcgaccccg aatggcatca cctggtacta tgagcaggag 60
ggtactggac ccgacgttgt cctcgtcccc gatggcctcg gagaatgcca gatgtttgac 120
agctccgtgt cgcaaattgc tgcccaaggc tttcgggtca ccacgtttga catgcccgga 180
atgtcccggt ctgcgaaggc accacccgag acctacactg aggtcacggc ccagaagctg 240
gcttcctatg tcatctccgt cctggatgct cttgacatca agcacgctac tgtctggggc 300
tgcagctcag gagcttccac cgtcgtggcg ctgttgctcg gttaccccga caggatacgc 360
aacgccatgt gccacgaact gccaacaaag ctactggacc acctttcaaa caccgctgtg 420
ctcgaagacg aggaaatctc aaagatcctg gccaatgtaa tgttgaacga cgtgtctgga 480
ggctcggagg cgtggcaagc catgggggac gaggtgcacg cgagactgca caagaactac 540
ccggtttggg ctcgaggata ccctcgcact attcctccct cagctccggt taaggatctg 600
gaggctctgc gtgggaagcc cctggactgg actgtcggcg ctgcgacacc aaccgagtct 660
ttctttgaca acattgttac cgctaccaag gctggtgtca acattgggtt gcttccaggg 720
atgcatttcc cttatgtttc ccacccggac gttttcgcta aatatgttgt ggaaactacg 780
cagaagcatc tt 792
<210> 3
<211> 264
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Met Arg Thr Arg Ser Thr Ile Ser Thr Pro Asn Gly Ile Thr Trp Tyr
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245 250 255
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260
<210> 4
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<213> Artificial Sequence(人工序列 M1突变体核苷酸序列)
<400> 4
atgcgtacgc gttctactat cagcacccct aacggtatca cctggtacta cgagcaagag 60
ggcaccggtc cagacgtggt tctggtccca gatggtctgg gtgaatgtca gatgttcgac 120
agctctgtga gccagatcgc tgctcagggt ttccgtgtta ctaccttcga catgccaggt 180
atgtcccgta gcgcaaaagc accgccggaa acttacaccg aggtgaccgc acagaaactg 240
gcttcctacg tcatctccgt cctggacgct ctggatatca aacacgctac cgtgtggggt 300
tgctcttccg gcgcttctac tgtggtagca ctgctgctgg gttatccgga tcgtatccgt 360
aacgcaatgt gccatgaact gccgactaaa ctgctggacc acctgtctaa cacggccgtt 420
ctggaagacg aagaaatctc caagattctg gccaacgtga tgctgaacaa agtgagcggc 480
ggctctgaag cgtggcaggc aatgggtgac gaagtacacg cacgcctgca taagaactac 540
cctgtttggg cgcgcggcta tccgcgcact attccgccgt ctgcgccggt taaagatctg 600
gaagccctgc gcggtaaacc gctggattgg accgtaggcg cggcgacccc gaccgaaagc 660
ttctttgata atattgttac cgcgaccaaa gcgggcgtaa acattggcct gctgccgggc 720
atgcacttcc cgtatgtttc ccacccggat gtttttgcca aatacgttgt agaaaccacg 780
cagaaacatc tg 792
<210> 5
<211> 264
<212> PRT
<213> Artificial Sequence(人工序列 M2突变体氨基酸序列)
<400> 5
Met Arg Thr Arg Ser Thr Ile Ser Thr Pro Asn Gly Ile Thr Trp Tyr
1 5 10 15
Tyr Glu Gln Glu Gly Thr Gly Pro Asp Val Val Leu Val Pro Asp Gly
20 25 30
Leu Gly Glu Cys Gln Met Phe Asp Ser Ser Val Ser Gln Ile Ala Ala
35 40 45
Gln Gly Phe Arg Val Thr Thr Phe Asp Met Pro Gly Met Ser Arg Ser
50 55 60
Ala Lys Ala Pro Pro Glu Thr Tyr Thr Glu Val Thr Ala Gln Lys Leu
65 70 75 80
Ala Ser Tyr Val Ile Ser Val Leu Asp Ala Leu Asp Ile Lys His Ala
85 90 95
Thr Val Trp Gly Cys Ser Ser Gly Ala Ser Thr Val Val Ala Leu Leu
100 105 110
Leu Gly Tyr Pro Asp Arg Ile Arg Asn Ala Met Cys His Glu Leu Pro
115 120 125
Thr Lys Leu Leu Lys His Leu Ser Asn Thr Ala Val Leu Glu Asp Glu
130 135 140
Glu Ile Ser Lys Ile Leu Ala Asn Val Met Leu Asn Asp Val Ser Gly
145 150 155 160
Gly Ser Glu Ala Trp Gln Ala Met Gly Asp Glu Val His Ala Arg Leu
165 170 175
His Lys Asn Tyr Pro Val Trp Ala Arg Gly Tyr Pro Arg Thr Ile Pro
180 185 190
Pro Ser Ala Pro Val Lys Asp Leu Glu Ala Leu Arg Gly Lys Pro Leu
195 200 205
Asp Trp Thr Val Gly Ala Ala Thr Pro Thr Glu Ser Phe Phe Asp Asn
210 215 220
Ile Val Thr Ala Thr Lys Ala Gly Val Asn Ile Gly Leu Leu Pro Gly
225 230 235 240
Met His Phe Pro Tyr Val Ser His Pro Asp Val Phe Ala Lys Tyr Val
245 250 255
Val Glu Thr Thr Gln Lys His Leu
260
<210> 6
<211> 792
<212> DNA
<213> Artificial Sequence(人工序列 M2突变体核苷酸序列)
<400> 6
atgcgtacgc gttctactat cagcacccct aacggtatca cctggtacta cgagcaagag 60
ggcaccggtc cagacgtggt tctggtccca gatggtctgg gtgaatgtca gatgttcgac 120
agctctgtga gccagatcgc tgctcagggt ttccgtgtta ctaccttcga catgccaggt 180
atgtcccgta gcgcaaaagc accgccggaa acttacaccg aggtgaccgc acagaaactg 240
gcttcctacg tcatctccgt cctggacgct ctggatatca aacacgctac cgtgtggggt 300
tgctcttccg gcgcttctac tgtggtagca ctgctgctgg gttatccgga tcgtatccgt 360
aacgcaatgt gccatgaact gccgactaaa ctgctgaaac acctgtctaa cacggccgtt 420
ctggaagacg aagaaatctc caagattctg gccaacgtga tgctgaacga cgtgagcggc 480
ggctctgaag cgtggcaggc aatgggtgac gaagtacacg cacgcctgca taagaactac 540
cctgtttggg cgcgcggcta tccgcgcact attccgccgt ctgcgccggt taaagatctg 600
gaagccctgc gcggtaaacc gctggattgg accgtaggcg cggcgacccc gaccgaaagc 660
ttctttgata atattgttac cgcgaccaaa gcgggcgtaa acattggcct gctgccgggc 720
atgcacttcc cgtatgtttc ccacccggat gtttttgcca aatacgttgt agaaaccacg 780
cagaaacatc tg 792
<210> 7
<211> 264
<212> PRT
<213> Artificial Sequence(人工序列 M3突变体氨基酸序列)
<400> 7
Met Arg Thr Arg Ser Thr Ile Ser Thr Pro Asn Gly Ile Thr Trp Tyr
1 5 10 15
Tyr Glu Gln Glu Gly Thr Gly Pro Asp Val Val Leu Val Pro Asp Gly
20 25 30
Leu Gly Glu Cys Gln Met Phe Asp Ser Ser Val Ser Gln Ile Ala Ala
35 40 45
Gln Gly Phe Arg Val Thr Thr Phe Asp Met Pro Gly Met Ser Arg Ser
50 55 60
Ala Lys Ala Pro Pro Glu Thr Tyr Thr Glu Val Thr Ala Gln Lys Leu
65 70 75 80
Ala Ser Tyr Val Ile Ser Val Leu Asp Ala Leu Asp Ile Lys His Ala
85 90 95
Thr Val Trp Gly Cys Ser Ser Gly Ala Ser Thr Val Val Ala Leu Leu
100 105 110
Leu Gly Tyr Pro Asp Arg Ile Arg Asn Ala Met Cys His Glu Leu Pro
115 120 125
Thr Lys Leu Leu Asp His Leu Ser Asn Thr Ala Val Leu Glu Asp Glu
130 135 140
Glu Ile Ser Lys Ile Leu Ala Asn Val Met Leu Asn Asp Val Ser Gly
145 150 155 160
Gly Ser Glu Ala Trp Gln Ala Met Gly Asp Lys Val His Ala Arg Leu
165 170 175
His Lys Asn Tyr Pro Val Trp Ala Arg Gly Tyr Pro Arg Thr Ile Pro
180 185 190
Pro Ser Ala Pro Val Lys Asp Leu Glu Ala Leu Arg Gly Lys Pro Leu
195 200 205
Asp Trp Thr Val Gly Ala Ala Thr Pro Thr Glu Ser Phe Phe Asp Asn
210 215 220
Ile Val Thr Ala Thr Lys Ala Gly Val Asn Ile Gly Leu Leu Pro Gly
225 230 235 240
Met His Phe Pro Tyr Val Ser His Pro Asp Val Phe Ala Lys Tyr Val
245 250 255
Val Glu Thr Thr Gln Lys His Leu
260
<210> 8
<211> 792
<212> DNA
<213> Artificial Sequence(人工序列 M3突变体核苷酸序列)
<400> 8
atgcgtacgc gttctactat cagcacccct aacggtatca cctggtacta cgagcaagag 60
ggcaccggtc cagacgtggt tctggtccca gatggtctgg gtgaatgtca gatgttcgac 120
agctctgtga gccagatcgc tgctcagggt ttccgtgtta ctaccttcga catgccaggt 180
atgtcccgta gcgcaaaagc accgccggaa acttacaccg aggtgaccgc acagaaactg 240
gcttcctacg tcatctccgt cctggacgct ctggatatca aacacgctac cgtgtggggt 300
tgctcttccg gcgcttctac tgtggtagca ctgctgctgg gttatccgga tcgtatccgt 360
aacgcaatgt gccatgaact gccgactaaa ctgctggacc acctgtctaa cacggccgtt 420
ctggaagacg aagaaatctc caagattctg gccaacgtga tgctgaacga cgtgagcggc 480
ggctctgaag cgtggcaggc aatgggtgac aaagtacacg cacgcctgca taagaactac 540
cctgtttggg cgcgcggcta tccgcgcact attccgccgt ctgcgccggt taaagatctg 600
gaagccctgc gcggtaaacc gctggattgg accgtaggcg cggcgacccc gaccgaaagc 660
ttctttgata atattgttac cgcgaccaaa gcgggcgtaa acattggcct gctgccgggc 720
atgcacttcc cgtatgtttc ccacccggat gtttttgcca aatacgttgt agaaaccacg 780
cagaaacatc tg 792
<210> 9
<211> 264
<212> PRT
<213> Artificial Sequence(人工序列 M1M2突变体氨基酸序列)
<400> 9
Met Arg Thr Arg Ser Thr Ile Ser Thr Pro Asn Gly Ile Thr Trp Tyr
1 5 10 15
Tyr Glu Gln Glu Gly Thr Gly Pro Asp Val Val Leu Val Pro Asp Gly
20 25 30
Leu Gly Glu Cys Gln Met Phe Asp Ser Ser Val Ser Gln Ile Ala Ala
35 40 45
Gln Gly Phe Arg Val Thr Thr Phe Asp Met Pro Gly Met Ser Arg Ser
50 55 60
Ala Lys Ala Pro Pro Glu Thr Tyr Thr Glu Val Thr Ala Gln Lys Leu
65 70 75 80
Ala Ser Tyr Val Ile Ser Val Leu Asp Ala Leu Asp Ile Lys His Ala
85 90 95
Thr Val Trp Gly Cys Ser Ser Gly Ala Ser Thr Val Val Ala Leu Leu
100 105 110
Leu Gly Tyr Pro Asp Arg Ile Arg Asn Ala Met Cys His Glu Leu Pro
115 120 125
Thr Lys Leu Leu Lys His Leu Ser Asn Thr Ala Val Leu Glu Asp Glu
130 135 140
Glu Ile Ser Lys Ile Leu Ala Asn Val Met Leu Asn Lys Val Ser Gly
145 150 155 160
Gly Ser Glu Ala Trp Gln Ala Met Gly Asp Glu Val His Ala Arg Leu
165 170 175
His Lys Asn Tyr Pro Val Trp Ala Arg Gly Tyr Pro Arg Thr Ile Pro
180 185 190
Pro Ser Ala Pro Val Lys Asp Leu Glu Ala Leu Arg Gly Lys Pro Leu
195 200 205
Asp Trp Thr Val Gly Ala Ala Thr Pro Thr Glu Ser Phe Phe Asp Asn
210 215 220
Ile Val Thr Ala Thr Lys Ala Gly Val Asn Ile Gly Leu Leu Pro Gly
225 230 235 240
Met His Phe Pro Tyr Val Ser His Pro Asp Val Phe Ala Lys Tyr Val
245 250 255
Val Glu Thr Thr Gln Lys His Leu
260
<210> 10
<211> 792
<212> DNA
<213> Artificial Sequence(人工序列 M1M2突变体核苷酸序列)
<400> 10
atgcgtacgc gttctactat cagcacccct aacggtatca cctggtacta cgagcaagag 60
ggcaccggtc cagacgtggt tctggtccca gatggtctgg gtgaatgtca gatgttcgac 120
agctctgtga gccagatcgc tgctcagggt ttccgtgtta ctaccttcga catgccaggt 180
atgtcccgta gcgcaaaagc accgccggaa acttacaccg aggtgaccgc acagaaactg 240
gcttcctacg tcatctccgt cctggacgct ctggatatca aacacgctac cgtgtggggt 300
tgctcttccg gcgcttctac tgtggtagca ctgctgctgg gttatccgga tcgtatccgt 360
aacgcaatgt gccatgaact gccgactaaa ctgctgaaac acctgtctaa cacggccgtt 420
ctggaagacg aagaaatctc caagattctg gccaacgtga tgctgaacaa agtgagcggc 480
ggctctgaag cgtggcaggc aatgggtgac gaagtacacg cacgcctgca taagaactac 540
cctgtttggg cgcgcggcta tccgcgcact attccgccgt ctgcgccggt taaagatctg 600
gaagccctgc gcggtaaacc gctggattgg accgtaggcg cggcgacccc gaccgaaagc 660
ttctttgata atattgttac cgcgaccaaa gcgggcgtaa acattggcct gctgccgggc 720
atgcacttcc cgtatgtttc ccacccggat gtttttgcca aatacgttgt agaaaccacg 780
cagaaacatc tg 792
<210> 11
<211> 264
<212> PRT
<213> Artificial Sequence(人工序列 M1M3突变体氨基酸序列)
<400> 11
Met Arg Thr Arg Ser Thr Ile Ser Thr Pro Asn Gly Ile Thr Trp Tyr
1 5 10 15
Tyr Glu Gln Glu Gly Thr Gly Pro Asp Val Val Leu Val Pro Asp Gly
20 25 30
Leu Gly Glu Cys Gln Met Phe Asp Ser Ser Val Ser Gln Ile Ala Ala
35 40 45
Gln Gly Phe Arg Val Thr Thr Phe Asp Met Pro Gly Met Ser Arg Ser
50 55 60
Ala Lys Ala Pro Pro Glu Thr Tyr Thr Glu Val Thr Ala Gln Lys Leu
65 70 75 80
Ala Ser Tyr Val Ile Ser Val Leu Asp Ala Leu Asp Ile Lys His Ala
85 90 95
Thr Val Trp Gly Cys Ser Ser Gly Ala Ser Thr Val Val Ala Leu Leu
100 105 110
Leu Gly Tyr Pro Asp Arg Ile Arg Asn Ala Met Cys His Glu Leu Pro
115 120 125
Thr Lys Leu Leu Asp His Leu Ser Asn Thr Ala Val Leu Glu Asp Glu
130 135 140
Glu Ile Ser Lys Ile Leu Ala Asn Val Met Leu Asn Lys Val Ser Gly
145 150 155 160
Gly Ser Glu Ala Trp Gln Ala Met Gly Asp Lys Val His Ala Arg Leu
165 170 175
His Lys Asn Tyr Pro Val Trp Ala Arg Gly Tyr Pro Arg Thr Ile Pro
180 185 190
Pro Ser Ala Pro Val Lys Asp Leu Glu Ala Leu Arg Gly Lys Pro Leu
195 200 205
Asp Trp Thr Val Gly Ala Ala Thr Pro Thr Glu Ser Phe Phe Asp Asn
210 215 220
Ile Val Thr Ala Thr Lys Ala Gly Val Asn Ile Gly Leu Leu Pro Gly
225 230 235 240
Met His Phe Pro Tyr Val Ser His Pro Asp Val Phe Ala Lys Tyr Val
245 250 255
Val Glu Thr Thr Gln Lys His Leu
260
<210> 12
<211> 792
<212> DNA
<213> Artificial Sequence(人工序列 M1M3突变体核苷酸序列)
<400> 12
atgcgtacgc gttctactat cagcacccct aacggtatca cctggtacta cgagcaagag 60
ggcaccggtc cagacgtggt tctggtccca gatggtctgg gtgaatgtca gatgttcgac 120
agctctgtga gccagatcgc tgctcagggt ttccgtgtta ctaccttcga catgccaggt 180
atgtcccgta gcgcaaaagc accgccggaa acttacaccg aggtgaccgc acagaaactg 240
gcttcctacg tcatctccgt cctggacgct ctggatatca aacacgctac cgtgtggggt 300
tgctcttccg gcgcttctac tgtggtagca ctgctgctgg gttatccgga tcgtatccgt 360
aacgcaatgt gccatgaact gccgactaaa ctgctggacc acctgtctaa cacggccgtt 420
ctggaagacg aagaaatctc caagattctg gccaacgtga tgctgaacaa agtgagcggc 480
ggctctgaag cgtggcaggc aatgggtgac aaagtacacg cacgcctgca taagaactac 540
cctgtttggg cgcgcggcta tccgcgcact attccgccgt ctgcgccggt taaagatctg 600
gaagccctgc gcggtaaacc gctggattgg accgtaggcg cggcgacccc gaccgaaagc 660
ttctttgata atattgttac cgcgaccaaa gcgggcgtaa acattggcct gctgccgggc 720
atgcacttcc cgtatgtttc ccacccggat gtttttgcca aatacgttgt agaaaccacg 780
cagaaacatc tg 792
<210> 13
<211> 264
<212> PRT
<213> Artificial Sequence(人工序列 M2M3突变体氨基酸序列)
<400> 13
Met Arg Thr Arg Ser Thr Ile Ser Thr Pro Asn Gly Ile Thr Trp Tyr
1 5 10 15
Tyr Glu Gln Glu Gly Thr Gly Pro Asp Val Val Leu Val Pro Asp Gly
20 25 30
Leu Gly Glu Cys Gln Met Phe Asp Ser Ser Val Ser Gln Ile Ala Ala
35 40 45
Gln Gly Phe Arg Val Thr Thr Phe Asp Met Pro Gly Met Ser Arg Ser
50 55 60
Ala Lys Ala Pro Pro Glu Thr Tyr Thr Glu Val Thr Ala Gln Lys Leu
65 70 75 80
Ala Ser Tyr Val Ile Ser Val Leu Asp Ala Leu Asp Ile Lys His Ala
85 90 95
Thr Val Trp Gly Cys Ser Ser Gly Ala Ser Thr Val Val Ala Leu Leu
100 105 110
Leu Gly Tyr Pro Asp Arg Ile Arg Asn Ala Met Cys His Glu Leu Pro
115 120 125
Thr Lys Leu Leu Lys His Leu Ser Asn Thr Ala Val Leu Glu Asp Glu
130 135 140
Glu Ile Ser Lys Ile Leu Ala Asn Val Met Leu Asn Asp Val Ser Gly
145 150 155 160
Gly Ser Glu Ala Trp Gln Ala Met Gly Asp Lys Val His Ala Arg Leu
165 170 175
His Lys Asn Tyr Pro Val Trp Ala Arg Gly Tyr Pro Arg Thr Ile Pro
180 185 190
Pro Ser Ala Pro Val Lys Asp Leu Glu Ala Leu Arg Gly Lys Pro Leu
195 200 205
Asp Trp Thr Val Gly Ala Ala Thr Pro Thr Glu Ser Phe Phe Asp Asn
210 215 220
Ile Val Thr Ala Thr Lys Ala Gly Val Asn Ile Gly Leu Leu Pro Gly
225 230 235 240
Met His Phe Pro Tyr Val Ser His Pro Asp Val Phe Ala Lys Tyr Val
245 250 255
Val Glu Thr Thr Gln Lys His Leu
260
<210> 14
<211> 792
<212> DNA
<213> Artificial Sequence(人工序列 M2M3突变体核苷酸序列)
<400> 14
atgcgtacgc gttctactat cagcacccct aacggtatca cctggtacta cgagcaagag 60
ggcaccggtc cagacgtggt tctggtccca gatggtctgg gtgaatgtca gatgttcgac 120
agctctgtga gccagatcgc tgctcagggt ttccgtgtta ctaccttcga catgccaggt 180
atgtcccgta gcgcaaaagc accgccggaa acttacaccg aggtgaccgc acagaaactg 240
gcttcctacg tcatctccgt cctggacgct ctggatatca aacacgctac cgtgtggggt 300
tgctcttccg gcgcttctac tgtggtagca ctgctgctgg gttatccgga tcgtatccgt 360
aacgcaatgt gccatgaact gccgactaaa ctgctgaaac acctgtctaa cacggccgtt 420
ctggaagacg aagaaatctc caagattctg gccaacgtga tgctgaacga cgtgagcggc 480
ggctctgaag cgtggcaggc aatgggtgac aaagtacacg cacgcctgca taagaactac 540
cctgtttggg cgcgcggcta tccgcgcact attccgccgt ctgcgccggt taaagatctg 600
gaagccctgc gcggtaaacc gctggattgg accgtaggcg cggcgacccc gaccgaaagc 660
ttctttgata atattgttac cgcgaccaaa gcgggcgtaa acattggcct gctgccgggc 720
atgcacttcc cgtatgtttc ccacccggat gtttttgcca aatacgttgt agaaaccacg 780
cagaaacatc tg 792
<210> 15
<211> 264
<212> PRT
<213> Artificial Sequence(人工序列 M1M2M3突变体氨基酸序列)
<400> 15
Met Arg Thr Arg Ser Thr Ile Ser Thr Pro Asn Gly Ile Thr Trp Tyr
1 5 10 15
Tyr Glu Gln Glu Gly Thr Gly Pro Asp Val Val Leu Val Pro Asp Gly
20 25 30
Leu Gly Glu Cys Gln Met Phe Asp Ser Ser Val Ser Gln Ile Ala Ala
35 40 45
Gln Gly Phe Arg Val Thr Thr Phe Asp Met Pro Gly Met Ser Arg Ser
50 55 60
Ala Lys Ala Pro Pro Glu Thr Tyr Thr Glu Val Thr Ala Gln Lys Leu
65 70 75 80
Ala Ser Tyr Val Ile Ser Val Leu Asp Ala Leu Asp Ile Lys His Ala
85 90 95
Thr Val Trp Gly Cys Ser Ser Gly Ala Ser Thr Val Val Ala Leu Leu
100 105 110
Leu Gly Tyr Pro Asp Arg Ile Arg Asn Ala Met Cys His Glu Leu Pro
115 120 125
Thr Lys Leu Leu Lys His Leu Ser Asn Thr Ala Val Leu Glu Asp Glu
130 135 140
Glu Ile Ser Lys Ile Leu Ala Asn Val Met Leu Asn Lys Val Ser Gly
145 150 155 160
Gly Ser Glu Ala Trp Gln Ala Met Gly Asp Lys Val His Ala Arg Leu
165 170 175
His Lys Asn Tyr Pro Val Trp Ala Arg Gly Tyr Pro Arg Thr Ile Pro
180 185 190
Pro Ser Ala Pro Val Lys Asp Leu Glu Ala Leu Arg Gly Lys Pro Leu
195 200 205
Asp Trp Thr Val Gly Ala Ala Thr Pro Thr Glu Ser Phe Phe Asp Asn
210 215 220
Ile Val Thr Ala Thr Lys Ala Gly Val Asn Ile Gly Leu Leu Pro Gly
225 230 235 240
Met His Phe Pro Tyr Val Ser His Pro Asp Val Phe Ala Lys Tyr Val
245 250 255
Val Glu Thr Thr Gln Lys His Leu
260
<210> 16
<211> 792
<212> DNA
<213> Artificial Sequence(人工序列 M1M2M3突变体核苷酸序列)
<400> 16
atgcgtacgc gttctactat cagcacccct aacggtatca cctggtacta cgagcaagag 60
ggcaccggtc cagacgtggt tctggtccca gatggtctgg gtgaatgtca gatgttcgac 120
agctctgtga gccagatcgc tgctcagggt ttccgtgtta ctaccttcga catgccaggt 180
atgtcccgta gcgcaaaagc accgccggaa acttacaccg aggtgaccgc acagaaactg 240
gcttcctacg tcatctccgt cctggacgct ctggatatca aacacgctac cgtgtggggt 300
tgctcttccg gcgcttctac tgtggtagca ctgctgctgg gttatccgga tcgtatccgt 360
aacgcaatgt gccatgaact gccgactaaa ctgctgaaac acctgtctaa cacggccgtt 420
ctggaagacg aagaaatctc caagattctg gccaacgtga tgctgaacaa agtgagcggc 480
ggctctgaag cgtggcaggc aatgggtgac aaagtacacg cacgcctgca taagaactac 540
cctgtttggg cgcgcggcta tccgcgcact attccgccgt ctgcgccggt taaagatctg 600
gaagccctgc gcggtaaacc gctggattgg accgtaggcg cggcgacccc gaccgaaagc 660
ttctttgata atattgttac cgcgaccaaa gcgggcgtaa acattggcct gctgccgggc 720
atgcacttcc cgtatgtttc ccacccggat gtttttgcca aatacgttgt agaaaccacg 780
cagaaacatc tg 792
<210> 17
<211> 792
<212> DNA
<213> Artificial Sequence(人工序列 大肠杆菌偏爱密码子)
<400> 17
atgcgtacgc gttctactat cagcacccct aacggtatca cctggtacta cgagcaagag 60
ggcaccggtc cagacgtggt tctggtccca gatggtctgg gtgaatgtca gatgttcgac 120
agctctgtga gccagatcgc tgctcagggt ttccgtgtta ctaccttcga catgccaggt 180
atgtcccgta gcgcaaaagc accgccggaa acttacaccg aggtgaccgc acagaaactg 240
gcttcctacg tcatctccgt cctggacgct ctggatatca aacacgctac cgtgtggggt 300
tgctcttccg gcgcttctac tgtggtagca ctgctgctgg gttatccgga tcgtatccgt 360
aacgcaatgt gccatgaact gccgactaaa ctgctggacc acctgtctaa cacggccgtt 420
ctggaagacg aagaaatctc caagattctg gccaacgtga tgctgaacga cgtgagcggc 480
ggctctgaag cgtggcaggc aatgggtgac gaagtacacg cacgcctgca taagaactac 540
cctgtttggg cgcgcggcta tccgcgcact attccgccgt ctgcgccggt taaagatctg 600
gaagccctgc gcggtaaacc gctggattgg accgtaggcg cggcgacccc gaccgaaagc 660
ttctttgata atattgttac cgcgaccaaa gcgggcgtaa acattggcct gctgccgggc 720
atgcacttcc cgtatgtttc ccacccggat gtttttgcca aatacgttgt agaaaccacg 780
cagaaacatc tg 792
<210> 18
<211> 18
<212> DNA
<213> Artificial Sequence(人工序列 六聚组氨酸标签)
<400> 18
caccatcacc accatcac 18
Claims (17)
1.一种玉米赤霉稀酮水解酶ZHD101突变体,其特征在于,所述ZHD101突变体在如SEQID NO. 1所示的野生型ZHD101氨基酸序列中引入了如下突变中的一种或多种:
第157位的D突变为K;
第133位的D突变为K;以及
第171位的E突变为K。
2.如权利要求1所述的ZHD101突变体,其中,所述ZHD101突变体的C端还具有六聚组氨酸标签。
3.编码如权利要求1或2所述的ZHD101突变体的DNA分子。
4. 如权利要求3所述的DNA分子,其中,所述DNA分子的核苷酸序列如SEQ ID NO. 4、SEQ ID NO. 6、SEQ ID NO. 8、SEQ ID NO. 10、SEQ ID NO. 12、SEQ ID NO. 14和SEQ IDNO. 16中任一者所示。
5.包含如权利要求3或4所述的DNA分子的表达载体。
6.如权利要求5所述的表达载体,其中,所用表达载体为pET30a(+)。
7.导入如权利要求5或6所述的表达载体的转化细胞。
8.如权利要求7所述的转化细胞,其中,所述转化细胞为原核细胞。
9.如权利要求8所述的转化细胞,其中,所述原核细胞为大肠杆菌细胞。
10.包含选自于如下物质中的一种或多种的试剂盒:
如权利要求1或2所述的ZHD101突变体;
如权利要求3或4所述的DNA分子;
如权利要求5或6所述的表达载体;以及
如权利要求7-9中任一项所述的转化细胞。
11. 制备如权利要求1或2所述的ZHD101突变体的方法,所述方法包括如下步骤:
在培养基中培养如权利要求7-9中任一项所述的转化细胞;以及
收集所述ZHD101突变体。
12.一种降解玉米赤霉烯酮的方法,其特征在于,使用如权利要求1或2所述的ZHD101突变体;如权利要求3或4所述的DNA分子;如权利要求5或6所述的表达载体;如权利要求7-9中任一项所述的转化细胞;和/或如权利要求10所述的试剂盒。
13.如权利要求1或2所述的ZHD101突变体;如权利要求3或4所述的DNA分子;如权利要求5或6所述的表达载体;如权利要求7-9中任一项所述的转化细胞;和/或如权利要求10所述的试剂盒在降解玉米赤霉烯酮中的用途。
14.如权利要求13所述的用途,其中,所述用途为玉米深加工、燃料乙醇生产、有机酸生产、淀粉/淀粉糖加工、粮油食品加工、饲料生产中的用途。
15.如权利要求13所述的用途,其中,所述用途为氨基酸生产中的用途。
16.如权利要求14所述的用途,其中,所述有机酸生产为柠檬酸生产。
17.如权利要求15所述的用途,其中,所述氨基酸生产为谷氨酸生产和/或赖氨酸生产。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201711180443.3A CN109825484B (zh) | 2017-11-23 | 2017-11-23 | 玉米赤霉烯酮水解酶zhd101突变体及利用该突变体水解玉米赤霉烯酮的方法 |
Applications Claiming Priority (1)
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