CN108034649B - 一种葡萄糖异构酶突变体及其应用 - Google Patents
一种葡萄糖异构酶突变体及其应用 Download PDFInfo
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- CN108034649B CN108034649B CN201810007946.9A CN201810007946A CN108034649B CN 108034649 B CN108034649 B CN 108034649B CN 201810007946 A CN201810007946 A CN 201810007946A CN 108034649 B CN108034649 B CN 108034649B
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Abstract
本发明公开了一种葡萄糖异构酶突变体及催化D‑葡萄糖异构化制备D‑果糖中的应用,本发明由Thermus oshimai GI(ToGI)的基因序列为模板,经三位点的定点突变后,ToGI突变体以质粒pET‑28b或能表达该酶的载体为表达载体,以Escherichia coli BL21(DE3)或能表达该酶的菌株为表达宿主,实现高转化率葡萄糖异构酶基因的高效表达,酶活为8.45U/mg。本发明中所述ToGI突变体具备优良热稳定性,于100℃保温7d,酶活保持不变。运用固定化技术,该催化剂在100℃条件下连续催化400g/L D‑葡萄糖生成D‑果糖的15个批次中,转化率均大于60%。
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获得的具有超级高温热稳定性的葡萄糖异构酶突变体的方法,以及该固定化葡萄糖异构酶突变体在超高温下连续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D-葡萄糖和D-果糖组成的混合物,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.,Journalof the American 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,主要原因是该耐高温酶在高温的热稳定性没有达到工业化生产的要求。鉴于此背景,本发明提出将现有GI进行定点突变以提高其高温热稳定性,并通过基因工程技术构建高表达的基因工程菌,经固定化后用于在高温下连续化、低成本化生产高果糖浆,对于填补缺乏耐高温GI酶制剂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葡萄糖异构酶突变体,提高其高温热稳定性,经固定化后用于在高温下连续化、低成本化生产高果糖浆。
本发明采用的技术方案为:
本发明提供一种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216位、第228位、第345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216位、第228位、第345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216位谷氨酸(E)突变为丝氨酸(S);(2)将SEQ ID NO.1所示氨基酸序列第216位谷氨酸(E)突变为丝氨酸(S)、第228位缬氨酸(V)突变为亮氨酸(L);(3)将SEQ ID NO.1所示氨基酸序列第216位谷氨酸(E)突变为丝氨酸(S)、第228位缬氨酸(V)突变为亮氨酸(L),同时第345位脯氨酸(P)突变为谷氨酰胺(Q)。
编码本发明的ToGI突变体的基因序列也属于本发明要求保护的范围。
本发明ToGI突变体的制备方法: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ToGI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。
本发明提供一种所述含葡萄糖异构酶突变体在催化D-葡萄糖异构化制备D-果糖中应用,所述应用的方法为:以含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或湿菌体经固定化得到的固定化颗粒为催化剂,以D-葡萄糖为底物,以锰盐为助剂,以50mMNa2HPO4-NaH2PO4缓冲液(pH 6.5-7.5)为反应介质,在200r/min,85℃下进行反应,反应完全后,将反应液分离纯化,获得D-果糖;所述底物初始浓度为50-500g/L缓冲液(优选400g/L),催化剂用量为10-50g/L缓冲液(优选40g/L),锰盐终浓度为5-25mM(优选8mM)。
本发明所述湿菌体按如下方法制备:构建含有所述GI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,取培养液分离得到含ToGI突变酶的菌体细胞。具体为:将含GI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。
进一步,所述固定化颗粒按如下方法制备:将含GI突变体基因的重组菌经发酵培养获得的湿菌体用pH值6.5-7.5的缓冲液制成菌悬液;向菌悬液中添加硅藻土,搅拌混匀,再加入聚乙烯亚胺(PEI)室温(优选25℃、100r/min)搅拌絮凝1-2h,然后加入三羟甲基磷(THP),在0-30℃(优选20-25℃)、100r/min搅拌交联1-2h后,抽滤,滤饼用蒸馏水洗涤后用轴向挤压机挤压成长条状,室温风干后,粉碎成粒(优选粒径为0.5-2mm),得到所述固定化颗粒;所述硅藻土与菌悬液中湿菌体重量比为0.01-0.1:1,所述聚乙烯亚胺以体积浓度5%聚乙烯亚胺水溶液的形式加入,所述聚乙烯亚胺水溶液体积用量以菌悬液中湿菌体重量计为2mL/6g,所述THP以30%(v/v)三羟甲基磷水溶液的形式加入,所述THP水溶液体积用量以菌悬液中湿菌体重量计为0.25mL/6g。
本发明所述30%(v/v)THP水溶液按如下方法制备:取15g四羟甲基氯化磷溶于90mL去离子水,3.4g的氢氧化钾溶于10mL去离子水,室温25℃、100r/min条件下将两者缓慢混合,制得30%(v/v)THP水溶液,现用现配,四羟甲基氯化磷和氢氧化钾按摩尔比1:0.995。
当本发明以固定化颗粒为催化剂时,反应结束后,将反应液离心,沉淀用50mMNa2HPO4-NaH2PO4缓冲液(pH 6.5-7.5)洗涤后重复利用,上清分离纯化,获得D-果糖。
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ToGI异构酶突变体。本发明中所述ToGI突变体具有超级高温热稳定性,于100℃保温7d,酶活保持不变,该结果为文献报道最高值。运用改良的固定化技术,经交联剂THP固定的催化剂具有优良重复使用率,于100℃条件下连续催化D-葡萄糖生成D-果糖的15个批次中,转化率均大于60%,该固定化催化剂具备在高温生物催化生产高果糖浆的工业化应用潜力。
(四)附图说明
图1为ToGI突变体的于100℃孵育时的热稳定性示意图;
图2为固定化产品重复使用批次示意图。
(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216位引入单突变,引物为:
正向引物GAACCCANNNTTCGCTCACG(下划线为突变碱基)
反向引物GAGCGAANNNTGGGTTCAGAC(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,57℃ 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20mLTris-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,Waters 717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235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230株重组菌酶活保持不变或下降的原因是第216位谷氨酸(E)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-E216S记为ToGI-I,获得重组菌E.coli BL21(DE3)/pET28b/ToGI-I。
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-I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-I为模板,对第228位引入单突变,引物为:
正向引物GAACTTCNNNCACGCTGTTG(下划线为突变碱基)
反向引物CAGCGTGNNNGAAGTTCAGAC(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,57℃ 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170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165株重组菌酶活保持不变或下降的原因是第228位缬氨酸(V)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-E216S-V228L记为ToGI-II,获得重组菌E.coliBL21(DE3)/pET28b/ToGI-II。
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-II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-II为模板,对第345位引入单突变,引物为:
正向引物GCTGGGTNNNTACTCTCGTG(下划线为突变碱基)
反向引物GAGAGTANNNACCCAGCAGAC(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103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98株重组菌酶活保持不变或下降的原因是第345位脯氨酸(P)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-E216S-V228L-P345Q记为ToGI-III,获得重组菌E.coli BL21(DE3)/pET28b/ToGI-III。
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.coli BL21(DE3)/pET28b/ToGI-I、E.coli BL21(DE3)/pET28b/ToGI-II、E.coli BL21(DE3)/pET28b/ToGI-III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℃诱导表达10h,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,收集相应的洗脱液为纯酶液。
实施例6:纯化酶的高温热稳定性
将实施例5中的纯酶液作为转化用酶,测定纯化酶的热稳定性。具体操作如下:将酶液中加入20mM Mn2+,于100℃保温,并在0、1、2、3、4、6、7d取1mL酶液样品,测定残余酶活,将初始酶活力定义为100%。由图1可知,ToGI-III在孵育7d后活力仍能保维持初始酶活的100%,其高温热稳定性优于ToGI、ToGI-I和ToGI-II,同时也属文献报道的最高水平。
实施例7:以THP为交联剂固定单、双和三突变重组菌细胞
制备30%(v/v)THP水溶液:取15g的四羟甲基氯化磷(浓度80%)溶于90mL去离子水,3.4g的氢氧化钾溶于10mL去离子水,室温25℃、100r/min条件下将两者缓慢混合,制得30%(v/v)THP水溶液,现用现配,四羟甲基氯化磷和氢氧化钾按摩尔比1:0.995。
将实施例4制备的重组ToGI-I、ToGI-II和ToGI-III细胞各6g,分别用50mL磷酸盐(Na2HPO4-NaH2PO4)缓冲液(pH 7.0)悬浮,加入0.3g的硅藻土(545),适当搅拌。加入2mL 5%(v/v)PEI水溶液在25℃、100r/min条件下絮凝,再加入0.25mL体积浓度30%THP水溶液,在25℃、100r/min条件下交联反应2h。然后抽滤,滤饼用蒸馏水洗涤后用轴向挤压机挤压成长条状,室温风干后,粉碎成粒(优选粒径为0.5-2mm),得含耐高温ToGI突变体的固定化颗粒。
实施例8:三种固定化颗粒异构化制备D-果糖的重复使用率
以实施例7中的三种固定化颗粒为生物催化剂,以D-葡萄糖为底物,生物转化制备D-果糖。20mL催化体系包括:以50mM磷酸盐缓冲液(pH 7.0)为反应介质,终浓度400g/L D-葡萄糖、终浓度8mM Mn2+、终浓度40g/L ToGI-III固定化颗粒。于100℃、200r/min,异构化2h。反应液于4℃离心,少量上清液经0.22μm膜过滤后用HPLC检测D-果糖浓度;收集ToGI-III固定化颗粒经缓冲液洗涤进行下一批转化。由图2可知,突变体ToGI-III细胞经THP固定化后,生物转化15批次的D-果糖转化率均在60%以上,效果优于原始酶ToGI和突变酶ToGI-I和ToGI-II,同时也为文献报道的最高水平。
序列表
<110> 浙江工业大学
<120> 一种葡萄糖异构酶突变体及其应用
<160> 2
<170> SIPOSequenceListing 1.0
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Met Tyr Glu Pro Lys Pro Glu His Lys Phe Thr Phe Gly Leu Trp Thr
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Tyr Ala Leu Arg Lys Ser Leu Glu Thr Met Asp Leu Gly Ala Glu Leu
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Gly Ala Glu Ile Tyr Val Val Trp Pro Gly Arg Glu Gly Ala Glu Val
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Glu Ala Thr Gly Lys Ser Arg Arg Val Trp Gly Trp Val Arg Glu Ala
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Leu Asn Phe Met Ala Ala Tyr Ala Glu Asp Gln Gly Tyr Gly Tyr Arg
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Phe Ala Leu Glu Pro Lys Pro Asn Glu Pro Arg Gly Asp Ile Tyr Phe
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Ala Thr Val Gly Ser Phe Leu Ala Phe Ile Tyr Thr Leu Asp Gln Pro
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Glu Arg Phe Gly Leu Asn Pro Glu Phe Ala His Glu Thr Met Ala Gly
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atgtacgaac cgaaaccgga acacaaattc accttcggtc tgtggaccgt tggtaacgtt 60
ggtcgtgacc cgttcggtga cgctgttcgt gaaaaactgg acccggttta cgttgttcac 120
aaactggctg aactgggtgt ttacggtatc aacctgcacg acgaagacct gatcccgcgt 180
ggtaccccgc cggctgaacg tgaccgtata gttcgtaggt tccgtaaagc tctcgaagaa 240
accggtctga aagttccgat ggttaccgct aacctgttct ctgacccggc gttcaaagac 300
ggtgcgttca cctctccgga cccgtgggtt cgtgcttacg ctctgcgtaa atctctggaa 360
accatggacc tgggtgctga actgggtgct gaaatctacg ttgtttggcc gggtcgtgaa 420
ggtgctgaag ttgaagctac cggtaaatct cgtcgtgttt ggggttgggt tcgtgaagct 480
ctgaacttca tggctgctta cgctgaagac cagggttacg gttaccgttt cgctctggaa 540
ccgaaaccga acgaaccgcg tggtgacatc tacttcgcta ccgttggttc tttcctggct 600
ttcatctaca ccctcgacca gccagaaagg ttcggtctga acccagaatt cgctcacgaa 660
accatggctg gtctgaactt cgttcacgct gttgctcagg ttctggacgc tggtaaactg 720
ttccacatcg acctgaacga ccagcgtatg tctcgtttcg accaggacct gcgtttcggt 780
tctgaaaacc tgaaagctgc tttcttcctg gttgacctgc tggaatcttc tggttaccag 840
ggtccgcgtc acttcgacgc tcacgctctg cgtaccgaag acgaagaagg tgtttgggct 900
ttcgctcgtg gttgcatgcg tacctacctg atcttcaaag aaaaggcgca ggcgttccgt 960
gaagacccag aagttcgttc tctgctggaa gaatactacg gtgaagaccc gcaggctctg 1020
ggtctgctgg gtccgtactc tcgtgaacgt gctaccgctc tgaaagaagt tgctctgccg 1080
ctggaagcta aacgtcgtcg tggttacgct ctggaacgtc tggaccagct ggttgttgaa 1140
cacctgctgg gtgttcgtgg tcaccaccac caccaccac 1179
Claims (7)
1.一种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216位谷氨酸突变为丝氨酸;(2)将SEQ ID NO.1所示氨基酸序列第216位谷氨酸突变为丝氨酸、第228位缬氨酸突变为亮氨酸;(3)将SEQ ID NO.1所示氨基酸序列第216位谷氨酸突变为丝氨酸、第228位缬氨酸突变为亮氨酸,同时第345位脯氨酸突变为谷氨酰胺。
2.一种权利要求1所述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。
3.如权利要求2所述的应用,其特征在于所述应用的方法为:以含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或湿菌体经固定化得到的固定化颗粒为催化剂,以D-葡萄糖为底物,以锰盐为助剂,以pH 6.5-7.5、50 mM Na2HPO4/NaH2PO4缓冲液为反应介质构成反应体系,在85oC、200 r/min条件下反应,待反应结束,将反应液分离纯化,获得D-果糖。
4.如权利要求3所述的应用,其特征在于所述反应体系中,底物初始浓度为50-500 g/L,湿菌体的用量为10-50 g/L,所述锰盐终浓度为5-25 mM。
5.如权利要求3所述的应用,其特征在于所述湿菌体按如下方法制备:将含葡萄糖异构酶突变体基因的工程菌接种至含50 μg/mL卡那霉素的LB液体培养基,在37oC、150 r/min培养10 h,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50 μg/mL卡那霉素的LB培养基中,于37oC、150 r/min培养OD600至0.6-0.8,再向培养液中加入终浓度为1 mM的IPTG,于28oC诱导10 h,8000 r/min离心10 min,弃去上清液,收集湿菌体;所述LB培养基组成:胰蛋白胨10 g/L,酵母粉5 g/L,NaCl 10 g/L,溶剂为水,pH值自然。
6.如权利要求3所述的应用,其特征在于所述固定化颗粒按如下方法制备:将含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用pH值6.5-7.5的缓冲液制成菌悬液;向菌悬液中添加硅藻土,搅拌混匀,再加入聚乙烯亚胺室温搅拌絮凝1-2 h,然后加入三羟甲基磷,在0-30oC、100 r/min搅拌交联1-2 h后,抽滤,滤饼用蒸馏水洗涤后用轴向挤压机挤压成长条状,室温风干后,粉碎成粒,得到所述固定化颗粒;所述硅藻土与菌悬液中湿菌体重量比为0.01-0.1:1,所述聚乙烯亚胺以体积浓度5%聚乙烯亚胺水溶液的形式加入,所述聚乙烯亚胺水溶液体积用量以菌悬液中湿菌体重量计为2mL/6g,所述三羟甲基磷以体积浓度30%三羟甲基磷水溶液的形式加入,所述三羟甲基磷水溶液体积用量以菌悬液中湿菌体重量计为0.25 mL/6g。
7.如权利要求6所述的应用,其特征在于所述体积浓度30%三羟甲基磷水溶液按如下方法制备:取15 g的四羟甲基氯化磷溶于90 mL去离子水,3.4 g的氢氧化钾溶于10 mL去离子水,室温25oC、100 r/min条件下将两者缓慢混合,制得体积浓度30%THP水溶液,现用现配,四羟甲基氯化磷和氢氧化钾按摩尔比1:0.995。
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