CN108048440A - 一种耐高温葡萄糖异构酶突变体及其应用 - Google Patents
一种耐高温葡萄糖异构酶突变体及其应用 Download PDFInfo
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- CN108048440A CN108048440A CN201810007649.4A CN201810007649A CN108048440A CN 108048440 A CN108048440 A CN 108048440A CN 201810007649 A CN201810007649 A CN 201810007649A CN 108048440 A CN108048440 A CN 108048440A
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Abstract
本发明公开了一种耐高温葡萄糖异构酶突变体及其在催化D‑葡萄糖异构化制备D‑果糖中的应用,突变体是将SEQ ID NO.1所示氨基酸序列的第89位、第136位、第352位中的一个或多个进行单突变或多突变获得的。本发明实现高转化率葡萄糖异构酶基因的高效表达,酶活为7.23U/mg。本发明中所述ToGI突变体的最适反应温度为110℃,该催化剂在100℃高温下催化400g/L D‑葡萄糖制备的高果糖浆中,D‑果糖浓度高达61.3%。
Description
(一)技术领域
本发明涉及一种葡萄糖异构酶突变体,具体地说涉及利用基因突变技术制备的具备超高反应温度的葡萄糖异构酶突变体的方法,以及该突变体在超高温下能稳定的异构化葡萄糖生产超高D-果糖浓度高果糖浆的应用。
(二)背景技术
葡萄糖异构酶(glucose isomerase,简称GI,EC 5.3.1.5),在体外主要用于催化D-葡萄糖异构化生成D-果糖,是工业上利用生物转化法制备高果糖浆的关键酶。根据GI的一级结构,可以将其分为两类,即I类和II类酶。与I类GI相比,II类GI的肽链N端含有多余的40-50个氨基酸残基(Deng H.etal.,Bioprocess and Biosystems Engineering,37:1211-1219,2014)。
高果糖浆(high fructose corn syrup,简称HFCS)是葡萄糖和果糖的混合物,是一种重要的甜味剂。高果糖浆具有溶解度高、化学和热稳定性好、渗透压大、吸潮性和保湿性强、与其它添加剂混合不影响食品风味等优势。根据其果糖含量的不同,高果糖浆主要有3种产品:HFCS-42、HFCS-55和HFCS-90。其中,HFCS-55的甜度优于蔗糖,是市场上的主流产品。但是,目前广泛采用的葡萄糖异构酶生物转化工艺无法一步制得F55型高果糖浆,需要将HFCS-42浓缩再与HFCS-90勾兑才能制得HFCS-55(MoellerS.M.et al.,Journal of theAmerican College of Nutrition,28:619-26,2009)。
GI介入的D-葡萄糖异构化过程是一个热力学平衡反应,随着异构化温度的升高,会促进异构化反应向果糖方向进行。目前,商业上用于生产高果糖浆的GI主要来源于Bacillus coagulans、Streptomyces murinus和Streptomyces rubiginosis等野生菌(DicosimoR.et al.,Chemical Society Reviews,42:6437-6474,2013)。由于上述这些GI的耐热性一般,只能在60-65℃的异构化温度稳定地进行催化反应,果糖转化率仅42-45%。因此,如果能在高温如100℃或更高的异构化温度下催化,所生成的具有高于55%D-果糖浓度的高果糖浆将有助于减少后续富集和勾兑的成本,对推进高果糖浆的生产技术变革具有重要意义。
目前,已有一些耐热GI的报道,例如Thermotoga maritima和Thermusthermophiles等,其最适温度分别达到105℃和95℃,但是,这些酶并没有制成酶制剂成功投放于市场。鉴于此背景,本发明提出将现有GI进行定点突变,并通过基因工程技术构建高表达的基因工程菌用于在高温下生产高果糖浆,对于填补缺乏耐高温酶的市场空白具有重大意义。
(三)发明内容
本发明所要解决的技术问题是提供一种具备超高反应温度的葡萄糖异构酶突变体,以及该突变体在超高温下能稳定异构化葡萄糖生产超高D-果糖浓度高果糖浆的应用。
本发明采用的技术方案是:
本发明提供一种耐高温葡萄糖异构酶突变体,所述突变体是将SEQ ID NO.1所示氨基酸序列的第89位、第136位、第352位中的一个或多个进行单突变或多突变获得的。
进一步,本发明所述突变体是将第89位、第136位、第352位中的一位、两位或者三位氨基酸突变为丙氨酸、丝氨酸、谷氨酰胺、甲硫氨酸或亮氨酸。
更进一步,所述突变体为下列之一:(1)将SEQ ID NO.1所示氨基酸序列的第89位苏氨酸(T)突变为谷氨酰胺(Q);(2)将SEQ IDNO.1所示氨基酸序列第89位苏氨酸(T)突变为谷氨酰胺(Q)、第136位色氨酸(W)突变为丝氨酸(S);(3)将SEQ ID NO.1所示氨基酸序列第89位苏氨酸(T)突变为谷氨酰胺(Q)、第136位色氨酸(W)突变为丝氨酸(S),同时第352位苏氨酸(T)突变为亮氨酸(L)。
编码本发明突变体的基因序列也属于本发明要求保护的范围。
本发明还提供一种生产耐高温葡萄糖异构酶突变体的方法:根据ToGI基因,设计定点突变的突变引物,以携带GI的克隆载体为模板进行定点突变构建突变体,以质粒pET28b或能表达该酶的载体为表达载体,将重组质粒转化Escherichia coliBL21(DE3)细胞或能表达该酶的宿主细胞,挑选验证后的阳性单克隆进行发酵培养。
本发明还涉及一种由所述耐高温葡萄糖异构酶突变体编码基因构建的重组载体,以及由所述重组载体转化获得的重组基因工程菌。
本发明还涉及一种耐高温葡萄糖异构酶突变体在催化D-葡萄糖异构化制备D-果糖中的应用,所述应用为:以含耐高温葡萄糖异构酶突变体基因的工程菌经发酵培养获得的湿菌体为酶源,以D-葡萄糖为底物,以锰盐为助剂,以50mM的Na2HPO4/NaH2PO4缓冲液(pH8.0)为反应介质构成反应体系,在50-110℃(优选100-110℃,最优选100℃),150r/min条件下反应,待反应结束,将反应液分离纯化,获得D-果糖;所述反应体系中,底物初始浓度为50-500g/L(优选400g/L),湿菌体的用量为10-50g/L(优选25g/L),所述锰盐终浓度为5-25mM(优选20mM)。
进一步,所述湿菌体按如下方法制备:构建含有所述耐高温葡萄糖异构酶突变体基因的重组载体,将所述重组载体转化至E.coli中,获得的重组基因工程菌进行诱导表达,取培养液分离得到湿菌体细胞。具体为:将含耐高温葡萄糖异构酶突变体基因的工程菌接种至含50μg/mL卡那霉素的LB液体培养基,在37℃、150r/min培养10h,获得种子液;将种子液以2%(v/v)接种量接种至新鲜的含有终浓度50μg/mL卡那霉素的LB培养基中,于37℃、150r/min培养OD600至0.6-0.8,再向培养液中加入终浓度为1mM的IPTG,于28℃诱导10h,8000r/min离心10min,弃去上清液,收集湿菌体;所述LB培养基组成:胰蛋白胨10g/L,酵母粉5g/L,NaCl 10g/L,溶剂为水,pH值自然。
与现有技术相比,本发明有益效果主要体现在:本发明提供了一种新的耐高温葡萄糖异构酶突变体,该突变体具有超级最适反应温度110℃,比原始酶ToGI提高了15℃,属于文献报道的最高水平。使用本突变体生产高果糖浆,在50-110℃、150r/min的转化条件下,ToGI突变体酶所生产的高果糖浆的转化率高达61.3%,高于原始酶和其他突变酶的转化效果,同时也是文献报道的最高水平。该ToGI突变体解决了长期困扰现有酶无法在高温生产高果糖浆的技术难题,具有显著的技术进步。
(四)附图说明
图1为ToGI突变体的最适温度示意图;
图2为含重组ToGI突变体的E.coli生产高果糖浆的应用示意图。
(五)具体实施方式
下面结合具体实施例对本发明进行进一步描述,但本发明的保护范围并不仅限于此:
实施例1:ToGI单位点突变体的构建与筛选
1、突变体构建
根据ToGI亲本序列(氨基酸序列为SEQ ID NO.1所示,核苷酸序列为SEQ ID NO.2所示)设计定点突变的突变引物,利用快速PCR技术,以重组载体pET28b/ToGI为模板,对第89位引入单突变,引物为:
正向引物CCGATGGTTNNNGCTAACCTGTTC(下划线为突变碱基)
反向引物CAGGTTAGCNNNAACCATCGGAACTTTC(下划线为突变碱基)
PCR反应体系:2×Phanta Max Buffer(含Mg2+)25μL,dNTPs 10mM,正向引物2μL,反向引物2μL,模板DNA 1μL,Phanta Max Super-Fidelity DNA Polymerase 50U,加入ddH2O至50μL。
PCR扩增条件为95℃ 3min;(95℃ 15s,50℃ 15s,61℃ 6.5min)30循环;72℃5min。
2、突变体转化表达
PCR产物转化E.coliBL21(DE3)感受态细胞,挑单克隆于含50μg/mL卡那霉素的LB液体培养基中,37℃培养过夜。利用半胱氨酸咔唑法显色法对突变体进行初筛,具体如下:将经菌落PCR鉴定后的含突变阳性菌株接种于5mL含50μg/mL卡那霉素的LB液体培养基试管中,于37℃、150r/min摇床培养至OD600约为0.6-0.8,再向培养物中加入IPTG至终浓度1mM,28℃诱导培养过夜12h,离心收集菌体于1.5mL离心管。
3、突变体筛选
配制反应混合液终浓度组成为:50mM Tris-HCl缓冲液(pH 7.0)、1mM Co2+、10mMMg2+、200mM葡萄糖。反应混合液85℃保温3min,快速吸取1mL加入含有菌体的1.5mL离心管中,振荡器振荡混匀后在85℃、500r/min反应10min,冰浴3min停止反应。以半胱氨酸-咔唑显色法筛选突变体,在1.5mL离心管中进行,反应体包括反应液167μL、半胱氨酸盐酸盐33μL、70%浓硫酸1000μL、咔唑酒精33μL。60℃下保温10min后观察颜色变化,以野生型葡萄糖异构酶产生菌株E.coli BL21(DE3)/pET28b/ToGI(SEQ ID NO.2所示基因转入E.coli BL21(DE3)制成)作为对照,颜色比野生型深的突变株进行酶活测定。
4、突变体酶活测定
采用超声破碎方法对湿菌体进行超声破碎,取1g步骤2的湿菌体,用20mL Tris-HCl缓冲液(pH 7.0)悬浮,在39W条件下超声破碎20min,制备获得无细胞抽提液(即超声破碎后的混悬液),离心,收集上清液,取1mL上清液用于反应。反应体系终浓度组成:Tris-HCl缓冲液(pH 7.0)、1mM Co2+和10mM Mg2+、200mM D-葡萄糖,共5mL体系。反应条件:于85℃、150r/min条件下反应20min,冰浴10min终止反应,8000r/min离心10min,取20μL上清液;采用HPLC检测D-葡萄糖和D-果糖的浓度。分析柱为Hypersil NH2柱(250×4.6mm,5μm)(依利特分析仪器有限公司,大连,中国)。Waters 2414示差折光检测器,Waters 1525泵,Waters717进样器。酶活定义:100℃和pH 7.0下,每分钟将D-葡萄糖异构化生成1μmol D-果糖所需酶量定义为一个酶活单位(U)。
该实施例的结果为:对115株重组转化菌初筛,筛选出5株酶活提高的突变株,再对其进行酶活测定,具体结果见表1。经分析确定,其余110株重组菌酶活保持不变或下降的原因是第89位苏氨酸(T)突变为A、S、Q、M和L外的其他氨基酸。
表1单点突变重组菌的酶活测定
将酶活提高最多的突变体ToGI-T89Q记为ToGI-1,获得重组菌E.coli BL21(DE3)/pET28b/ToGI-1。
实施例2:葡萄糖异构酶二位点突变体的构建与筛选
根据实施例1构建的单突变体ToGI-1序列设计定点突变的突变引物,利用快速PCR技术,以重组载体pET28b/ToGI-1为模板,对第136位引入单突变,引物为:
正向引物CGTTGTTNNNCCGGGTCGTG(下划线为突变碱基)
反向引物GACCCGGNNNAACAACGTAGATTTC(下划线为突变碱基)
PCR反应体系:2×Phanta Max Buffer(含Mg2+)25μL,dNTPs 10mM,正向引物2μL,反向引物2μL,模板DNA 1μL,Phanta Max Super-Fidelity DNA Polymerase 50U,加入ddH2O至50μL。
PCR扩增条件为95℃ 3min;(95℃ 15s,50℃ 15s,62℃ 6.5min)30循环;72℃5min。
PCR产物转化E.coliBL21(DE3)感受态细胞,挑单克隆于含50μg/mL卡那霉素的LB液体培养基中,37℃培养过夜。利用半胱氨酸咔唑法显色法对突变体进行初筛(同实施例1)。
采用超声破碎方法对湿菌体进行超声破碎,并进行酶活测定(同实施例1)。
该实施例的结果为:对97株重组转化菌初筛,筛选出5株酶活提高的突变株,再对其进行酶活测定,具体结果见表2。经分析确定,其余92株重组菌酶活保持不变或下降的原因是第136位苏氨酸(T)突变为A、S、Q、M和L外的其他氨基酸。
表2双点突变重组菌的酶活测定
将酶活提高最多的突变体ToGI-T89Q-W136S记为ToGI-2,获得重组菌E.coli BL21(DE3)/pET28b/ToGI-2。
实施例3:葡萄糖异构酶三位点突变体的构建与筛选
根据实施例2构建的突变体ToGI-2序列设计定点突变的突变引物,利用快速PCR技术,以重组载体pET28b/ToGI-2为模板,对第352位引入单突变,引物为:
正向引物CGTGCTNNNGCTCTGAAAG(下划线为突变碱基)
反向引物CAGAGCNNNAGCACGTTCAC(下划线为突变碱基)
PCR反应体系:2×Phanta Max Buffer(含Mg2+)25μL,dNTPs 10mM,正向引物2μL,反向引物2μL,模板DNA 1μL,Phanta Max Super-Fidelity DNA Polymerase 50U,加入ddH2O至50μL。
PCR扩增条件为95℃ 3min;(95℃ 15s,50℃ 15s,60℃ 6.5min)30循环;72℃5min。
PCR产物转化E.coliBL21(DE3)感受态细胞,挑单克隆于含50μg/mL卡那霉素的LB液体培养基中,37℃培养过夜。利用半胱氨酸咔唑法显色法对突变体进行初筛(同实施例1)。
采用超声破碎方法对湿菌体进行超声破碎,并进行酶活测定(同实施例1)。
该实施例的结果为:对104株重组转化菌初筛,筛选出5株酶活提高的突变株,再对其进行酶活测定,具体结果见表3。经分析确定,其余99株重组菌酶活保持不变或下降的原因是第352位苏氨酸(T)突变为A、S、Q、M和L外的其他氨基酸。
表3双点突变重组菌的酶活测定
将酶活提高最多的突变体ToGI-T89Q-W136S-T352L记为ToGI-3,获得重组菌E.coli BL21(DE3)/pET28b/ToGI-3。
实施例4:重组大肠杆菌发酵产酶
分别将实施例1、2和3的重组菌E.coliBL21(DE3)/pET28b/ToGI-1、E.coli BL21(DE3)/pET28b/ToGI-2、E.coliBL21(DE3)/pET28b/ToGI-3接种至含终浓度50μg/mL卡那霉素的LB液体培养基,在37℃、150r/min培养OD600约0.6-0.8,获得种子液;将种子液以2%(v/v)接种量接种至新鲜的含有终浓度50μg/mL卡那霉素的LB液体培养基中,于37℃、150r/min培养OD600至0.6-0.8,再向培养液中加入终浓度为0.1mM的IPTG,于28℃下诱导表达10h后,4℃、8000r/min离心10min,弃去上清液,用0.85%的生理盐水清洗两遍湿菌体,并收集湿菌体,备用。
实施例5:突变重组葡萄糖异构酶的纯化
收集实施例4制备的各重组菌的湿菌体在39W条件下超声破碎20min后,将破碎混合液离心,取上清液,在75℃热处理15min,然后在4℃、8000r/min离心10min,弃去沉淀,收集上清液使用nickel-NTA琼脂糖凝胶柱进行纯化,用平衡缓冲液(20mM磷酸盐缓冲液,300mM NaCl,20mM咪唑,pH 8.0)平衡层析柱,再使用洗脱液(50mM磷酸盐缓冲液,300mMNaCl,500mM咪唑,pH 8.0)进行洗脱,根据紫外检测器的信号响应,收集相应的洗脱液,即为各自纯酶液。
实施例6:纯化酶的最适温度
将实施例5中的纯酶液作为转化用酶,测定酶的最适反应温度。具体操作如下:50mM Tris-HCl缓冲液(pH 7.0)中,加入200mM D-葡萄糖、1mM Co2+、10mM Mg2+和1mL纯酶液,体系共5mL。分别于不同转化温度:60、70、80、90、100、110、120℃测定葡萄糖异构酶的活力(方法同实施例1,高于100℃的测定条件需要使用油浴)。由图1可知,ToGI-3的最适反应温度是110℃,比原始酶ToGI提高了15℃(Jiaet al,Enzyme Microbial Technology,99:1-8),同时也是文献报道的最高值。
实施例7:单、双和三突变重组菌全细胞制备高果糖浆。
按实施例4的方法,获得重组菌E.coliBL21(DE3)/pET28b/ToGI-1、E.coliBL21(DE3)/pET28b/ToGI-2和E.coliBL21(DE3)/pET28b/ToGI-3湿菌体作为生物催化剂,以D-葡萄糖为底物,生物转化制备高果糖浆。100mL催化体系包括:以50mM Na2HPO4/NaH2PO4缓冲液(pH 8.0)为反应介质,终浓度400g/L D-葡萄糖、终浓度20mM Mn2+、终浓度25g/L湿菌体。于100℃、150r/min,反应8h。每隔1h取1mL反应液,离心,0.22μm膜过滤后用HPLC检测D-果糖的浓度。由图2可知,E.coliBL21(DE3)/pET28b/ToGI-3在2h底物转化率达61.3%,高于原始酶和其他突变酶的转化率,同时也为文献报道的最高水平。
序列表
<110> 浙江工业大学
<120> 一种耐高温葡萄糖异构酶突变体及其应用
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Claims (8)
1.一种耐高温葡萄糖异构酶突变体,其特征在于所述突变体是将SEQ ID NO.1所示氨基酸序列的第89位、第136位、第352位中的一个或多个进行单突变或多突变获得的。
2.如权利要求1所述耐高温葡萄糖异构酶突变体,其特征在于所述突变体是将第89位、第136位、第352位的氨基酸突变为丙氨酸、丝氨酸、谷氨酰胺、甲硫氨酸或亮氨酸中的一种。
3.如权利要求1所述耐高温葡萄糖异构酶突变体,其特征在于所述突变体为下列之一:(1)将SEQ ID NO.1所示氨基酸序列的第89位苏氨酸突变为谷氨酰胺;(2)将SEQ ID NO.1所示氨基酸序列第89位的苏氨酸突变为谷氨酰胺、第136位色氨酸突变为丝氨酸,同时第352位苏氨酸突变为亮氨酸。
4.一种权利要求1所述耐高温葡萄糖异构酶突变体在催化D-葡萄糖异构化制备D-果糖中的应用。
5.如权利要求4所述的应用,其特征在于所述应用的方法为:以含耐高温葡萄糖异构酶突变体基因的重组菌经发酵培养获得的湿菌体为酶源,以D-葡萄糖为底物,以锰盐为助剂,以pH 8.0、50mM的Na2HPO4/NaH2PO4缓冲液为反应介质构成反应体系,在50-110℃、150r/min条件下反应,待反应结束,将反应液分离纯化,获得D-果糖。
6.如权利要求5所述的应用,其特征在于所述反应体系中,底物初始浓度为50-500g/L,湿菌体的用量为10-50g/L,所述锰盐终浓度为5-25mM。
7.如权利要求5所述的应用,其特征在于所述反应温度为100-110℃。
8.如权利要求5所述的应用,其特征在于所述湿菌体按如下方法制备:将含耐高温葡萄糖异构酶突变体基因的工程菌接种至含50μg/mL卡那霉素的LB液体培养基,在37℃、150r/min培养10h,获得种子液;将种子液以体积浓度2%接种量接种至新鲜的含有终浓度50μg/mL卡那霉素的LB培养基中,于37℃、150r/min培养OD600至0.6-0.8,再向培养液中加入终浓度为1mM的IPTG,于28℃诱导10h,8000r/min离心10min,弃去上清液,收集湿菌体;所述LB培养基组成:胰蛋白胨10g/L,酵母粉5g/L,NaCl 10g/L,溶剂为水,pH值自然。
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