CN110358745A - 4-木糖醇脱氢酶突变体及其用途 - Google Patents
4-木糖醇脱氢酶突变体及其用途 Download PDFInfo
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- CN110358745A CN110358745A CN201910777021.7A CN201910777021A CN110358745A CN 110358745 A CN110358745 A CN 110358745A CN 201910777021 A CN201910777021 A CN 201910777021A CN 110358745 A CN110358745 A CN 110358745A
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- xylitol dehydrogenase
- xylitol
- dehydrogenase enzyme
- enzyme mutant
- xylulose
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Abstract
本发明公开了一种4‑木糖醇脱氢酶突变体及其用途。该4‑木糖醇脱氢酶突变体的氨基酸序列是SEQ ID NO:1所示的氨基酸序列的第41位亮氨酸突变为甲硫氨酸、第179位的异亮氨酸突变为苯丙氨酸的氨基酸序列。该4‑木糖醇脱氢酶突变体基因,其核苷酸序列如SEQ ID NO:4所示。将该基因导入大肠杆菌获得含有该基因的基因工程菌,实现重组4‑木糖醇脱氢酶的制备,本发明提供的4‑木糖醇脱氢酶突变体热稳定性大幅度提高,可用于高效转化木糖醇制备L‑木酮糖。
Description
技术领域
本发明属于生物工程技术领域,具体涉及一种4-木糖醇脱氢酶突变体及其用途。
背景技术
根据国际糖协会(ISRS)对稀有糖的定义:稀有糖是“在自然界存在但含量极少的一类单糖及其衍生物”。稀有糖虽然在自然界中含量少,但因具有潜在的生物活性和低毒性使其在膳食、保健、医药等领域发挥着重要作用。随着生活品质的提高,人们对膳食和健康的要求越来越高,对食品安全性和药物低毒性越来越关注。L-木酮糖(L-xylulose)是存在于多种生物体代谢途径的戊酮糖,其在自然界中浓度很低,是一种稀有糖。口服L-木酮糖可以抑制肠道蔗糖酶和麦芽糖酶活性,能有效地抑制餐后血糖升高,是降血糖食品的理想活性成分,L-木酮糖用于肿瘤治疗时能显著抑制恶性肿瘤细胞。此外,L-木酮糖可以作为其他稀有糖生产的前体物质。例如,L-木酮糖可以通过L-鼠李糖异构酶转化生产L-木糖和L-来苏糖。
根据Izumoring理论可以通过多种途径获得L-木酮糖。例如L-木糖和L-来苏糖通过醛糖异构酶的异构作用转化为L-木酮糖;L-核酮糖通过D-塔格糖3-差向异构酶作用于C-3位置羟基,通过差向异构作用转化为L-木酮糖;L-***糖醇和木糖醇通过氧化还原酶的氧化作用转化为L-木酮糖。其中L-木糖、L-来苏糖、L-核酮糖和L-***糖醇在自然界中含量极少,难以分离纯化且价格极其昂贵,无疑会提高L-木酮糖的生产成本。而木糖醇作为目前在商业领域应用最为广泛的稀有糖之一,已经实现工业化生产,价格相对低廉,易于获得。以木糖醇为底物,使得L-木酮糖的生产成本大大降低。
生物转化生产L-木酮糖,主要是通过菌体静息细胞催化氧化底物木糖醇。通常先培养菌体达到一定的浓度,通过过滤或离心方法收集并且洗涤培养的菌体细胞,再将这些细胞悬浮在含底物(主要为木糖醇)的反应体系中,在一定温度、pH和振荡条件下进行转化生产。该法反应条件温和,环境友好,成本较低且产品纯度高,易于实现规模化生产,基于长远考虑,生物转化法是最具潜力的,也是L-木酮糖生产技术的主要研究方向。微生物转化法生产L-木酮糖的研究最早见于1985年,美国康奈尔大学Doten等人在研究细菌性植物病原菌噬夏孢欧文氏菌(Erwinia uredovora,现更名为菠萝泛菌(Pantoea ananatis))的戊糖醇代谢过程中分离到1株能以木糖醇为唯一碳源的自发突变株(Doten et al.,1985),该突变菌株检测到具有4-木糖醇脱氢酶(XDH)和L-木酮糖激酶的活性,这种新型4-木糖醇脱氢酶,木糖醇为底物KM达到48Mm,可以催化NADH为辅酶的L-木酮糖还原反应。随后,他们通过转位子突变诱变筛选得到木糖醇阴性突变体突变株,该突变株可以合成4-木糖醇脱氢酶,同时缺乏L-木酮糖激酶活性。突变体可以转化木糖醇生成L-木酮糖,而不能进一步磷酸化L-木酮糖,进入磷酸戊糖途径(Doten et al.,1985)。2006年芬兰赫尔辛基大学Aarnikunnas等人成功将菠萝泛菌中的4-木糖醇脱氢酶基因分离并在大肠杆菌(E.coli)中进行了表达,这是首次细菌来源的4-木糖醇脱氢酶基因克隆并表达成功。通过对4-木糖醇脱氢酶影响因素的研究表明,XDH受到镁离子调节,其最适p H在碱性范围(Aarnikunnas etal.,2006)。通过响应面法优化重组大肠杆菌的静息细胞转化木糖醇生成L-木酮糖的影响因素,其在生物反应器中以250g/L木糖醇为底物时,L-木酮糖的生产力达到1.09g/(gh)(Usvalampiet al.,2009)。
1991年日本香川大学稀有糖研究中心Izumori小组以D-山梨糖醇为唯一碳源从土壤中分离筛选得到1株能将塔罗糖醇氧化为阿洛酮糖的产碱杆菌(Alcaligenes),该菌株体内多元醇脱氢酶具有转化木糖醇生产L-木酮糖的能力(Khan et al.,1991)。2007年日本香川大学稀有糖研究中心同一小组从土壤中分离得到的1株兼性嗜热菌,利用该菌株的静息细胞可以将木糖醇转化生成L-木酮糖,后将其鉴定为苍白芽胞杆菌(Bacillus pallidus)(Poonperm etal.,2007)。该菌株静息细胞在40℃下,甘氨酸-NaOH缓冲液(pH 10)中活性最高,作用于2%木糖醇时转化率达到最高为85%。2010年,Takata等人将苍白芽胞杆菌体内4-木糖醇脱氢酶基因在大肠杆菌体内成功表达。将重组大肠杆菌静息细胞置于pH 11.0甘氨酸-氢氧化钠缓冲溶液,以5%木糖醇为底物生产L-木酮糖,24h后木糖醇转化率达到35%(Takata et al.,2010)。
迄今为止,国内有关L-木酮糖生产方面的研究非常少,仅有两篇文献报道。2014年张玉宝等人从土壤中分离筛选得到1株可氧化木糖醇转化生产L-木酮糖的菌株ZN-14,经鉴定为巨大芽孢杆菌(Bacillus megaterium)(张玉宝等.,2014)。利用该菌株的静息细胞以木糖醇溶液(20g/L,pH 9.0)为底物在37℃转化24h,转化率达到26.62%,酶活力为2.6U/mL。2019年,朱雯惠等人将Pantoea ananatis来源的4-木糖醇脱氢酶基因在重组枯草芽孢杆菌中表达发酵和反应条件优化(朱雯惠等.,2019),并通过静息细胞反应得到最优的反应条件:底物质量浓度20g/L,缓冲溶液为甘氨酸-NaOH溶液(pH 10.0),反应温度为45℃。以上条件下得到的最终酶活为5.183U/L,与LB培养基相比提高231.2%,转化率达17.74%。
到目前为止,仅有以上提到的四种野生型(菠萝泛菌、产碱杆菌、苍白芽胞杆菌、巨大芽孢杆菌)不同微生物来源的4-木糖醇脱氢酶被报道,而且这些XDH热稳定性普遍较差,且活性较低导致L-木酮糖的产率低,限制了其工业应用范围。通常而言,在稀有糖工业化生产过程中,需要较高的操作温度。这是因为较高的反应温度可以带来很多的优势:提高反应效率(较高的反应速率和较低的扩散限制),降低溶液的粘度,增加反应的稳定性,获得更高的产量(增加底物和产物的溶解度,促使反应平衡向吸热反应移动),和减少微生物污染等。利用蛋白定向进化技术对野生型4-木糖醇脱氢酶进行改造,以获得活性高且热稳定性好、适合工业应用的4-木糖醇脱氢酶突变体,将是生物法制备L-木酮糖产业的关键。
参考文献
Doten R.C.,Mortlock R.P.Characterization of xylitol-utilizing mutantsof erwinia uredovora.Bacteriology,1985,161(2):529-533
Doten R.C.,Mortlock R.P..Production of D-and L-xylulose by mutants ofKlebsiella pneumoniae and Erwina uredovoru.Appl.Environ.Microbial.,1985,(49):158-162
Doten R.C.,Mortlock R.P..Inducible xylitol dehydrogenases in entericbacteria.Bacteriol.1985,162(2):845
Aarnikunnas J.S.,Pihlajaniemi A.,Palva A.,Leisola M.,A..Cloningand expression of a xylitol 4-dehydrogenase gene from Pantoea ananatis.ApplEnviron Microbiol,2006,(72):368–377
Usvalampi A.,Kiviharju K.,Leisola M.,M..Factors affecting theproduction of L-xylulose by resting cells of recombinant Escherichia coli.Ind.Microbiol.Biotechnol.2009,(36):1323-1330
Khan A.R.,Tokunaga H.,Yoshida K.,Izumori K..Conversion of xylitol toL-xylulose by Alcaligenes sp.701B-Cells.Fermention Bioengineering,1991,72(6):488-490
Poonperm W.,Takata G.,Morimoto K.,Granstrom T.G.,IzumoriK..Production of L-xylulose from xylitol by a newly isolated strain ofBacillus pallidus Y25and characterization of its relevant enzyme xylitoldehydrogenase.Enzyme and Microbial Technology.2007,(40):1206-1212
Takata G,Poonperm W,Morimoto K,et al.Cloning and overexpression ofthe xylitol dehydrogenase gene from Bacillus pallidus and its application toL-xylulose production.Journal of the Agricultural Chemical Society ofJapan.2010,74(9):1 807-1 813.
张玉宝,赵祥颖,杨丽萍,等.一株产L-木酮糖菌株的分离筛选及鉴定.食品科学,2014,35(1):199-203.
朱雯惠,孟青,江波,张涛.重组枯草芽孢杆菌表达4-木糖醇脱氢酶的发酵和反应条件优化.食品与发酵工业.2019,45(9):21-28
发明内容
本发明人经前期研究挖掘到了来源于克雷伯氏菌(Klebsiella oxytoca)的4-木糖醇脱氢酶尽管具有高活性,归因于低热稳定性而得不到良好的催化效果。因此需利用定点突变开发一种具有改良的热稳定性4-木糖醇脱氢酶突变体(GenBank:STR65190.1),提高其在催化生产L-木酮糖中的效率。
为实现上述目的,本发明利用分子对接建立底物(木糖醇)与酶(4-木糖醇脱氢酶)的复合物模型,对底物和酶结合的结构机理进行分析,选定可能影响4-木糖醇脱氢酶对木糖醇热稳定性的关键残基,通过定点突变得到了4-木糖醇脱氢酶突变体。该4-木糖醇脱氢酶突变体的氨基酸序列是SEQ ID NO:1所示的氨基酸序列的第41位亮氨酸突变为甲硫氨酸、第179位的异亮氨酸突变为苯丙氨酸的氨基酸序列。
本发明的另一目的是提供该4-木糖醇脱氢酶突变体的制备方法。
本发明的又一目的是提供该4-木糖醇脱氢酶突变体的用途。
本发明的目的可以通过以下技术方案实现:
4-木糖醇脱氢酶突变体基因,其野生型基因来源于克雷伯氏菌(GenBank:STR65190.1),经过定点突变获得该4-木糖醇脱氢酶突变体的基因,其核苷酸序列如SEQ IDNO:4所示。
一种4-木糖醇脱氢酶突变体,氨基酸序列如SEQ ID NO:2所示。
包含所述的4-木糖醇脱氢酶突变体基因的重组载体。其可通过本领域常规方法将本发明的4-木糖醇脱氢酶基因的核苷酸序列连接于各种载体上构建而成,重组质粒选自pET-22b(+)、pET-3a(+)、pET-3d(+)、pET-14b(+)、pET-15b(+)、pET-16b(+)、pET-17b(+)、pET-19b(+)、pET-20b(+)、pET-21a(+)、pET-23a(+)、pET-23b(+)、pET-24a(+)、pET-25b(+)、pET-26b(+)、pET-27b(+)、pET-28a(+)、pET-29a(+),pQE2、pQE9、pQE30、pQE3 1、pRSET-A、pRSET-B、pRSET-C、pGEX-5X-l、pGEX-6p-l、pGEX-6p-2、pBV220、pTrc99A、pTwin1、pEZZ18、pKK232-18、pBR322、pUC-18或pUC-19。更优选,上述重组质粒是pET-28a(+)。同时,为了在枯草芽孢杆菌中表达,优选地,可以使用的重组质粒选自pWB980、pHT43、pBE2、pMUTIN4、pUB110、pE194、pMA5、pMK3、pMK4、pHT304、pHY300PLK、pBest502、pDG1363、pSG1154、pAX01、pSAS144、pDL、pDG148-stu、pDG641、pUCX05-bgaB、pHT01、pUB110、pTZ4、pC194、φ1或φ105。更优选,上述重组质粒是pMA5。
一种生产所述的4-木糖醇脱氢酶突变体的基因工程菌,所述基因工程菌包含本发明所述的4-木糖醇脱氢酶突变体基因或本发明所述的重组载体。
所述的基因工程菌的宿主细胞包括原核细胞、酵母或真核细胞;优选所述原核细胞为大肠杆菌(E.coli)细胞或枯草芽孢杆菌。更优选的,所述宿主细胞为大肠杆菌BL21(DE3)细胞。
本发明所述的4-木糖醇脱氢酶突变体基因、所述的重组载体、所述的基因工程菌在制备L-木酮糖中的应用。
一种4-木糖醇脱氢酶突变体的制备方法,包括如下步骤:培养本发明所述的基因工程菌,获得重组表达的4-木糖醇脱氢酶突变体。
本发明的4-木糖醇脱氢酶突变体在转化木糖醇制备L-木酮糖中的用途。
催化反应条件,底物浓度30g/l,反应时间6小时,温度45℃。
有益效果
本发明的4-木糖醇脱氢酶(XDH)突变体与现有技术的4-木糖醇脱氢酶相比,在保持原有的高催化活性下还具有更优良的热稳定性,这两点对较高温度条件下催化木糖醇生产L-木酮糖是优良的性能,在工业生产中可大大降低催化剂的用量和缩短反应时间,从而降低生产成本。根据本发明的实施例,突变体XDH在45℃保温2小时后,任保留了80%的酶活。在使用重组基因工程菌酶转化木糖醇糖生产L-木酮糖实验中,底物浓度30g/l时,XDH突变体的转化率为88.7%,相比提高了近2倍,酶活高且热稳性好,催化效率高。
由此生产的L-木酮糖能够有效地用作食品或药物的研究。
附图说明
图1是显示在本发明实施方案条件下NADH标准曲线。
图2是显示在本发明实施方案条件下温度对于XDH及其突变体活性影响。
图3是显示在本发明实施方案条件下XDH及其突变体的热稳定性。
具体实施方式
下文中,将参考具体实施例对本发明进行更详细地描述。这些实施例仅仅是为了例证的目的,而非意欲限制本发明的范围。
实施例1:基因工程菌的建立
根据NCBI收录的4-木糖醇脱氢酶基因XDH(GenBank:STR65190.1),商业合成该4-木糖醇脱氢酶基因片段,以该基因片段为模版,通过PCR扩增扩展该片段(片段两侧加NdeI和BamHI限制性内切酶片段)其核苷酸序列如SEQ ID NO.3所示。并利用NdeI和BamHI限制性内切酶位点将基因***pET-28a质粒中,从而产生重组质粒pET-28a-XDH。通过常规转化方法,将该重组质粒转化到大肠杆菌BL21(DE3)中建立4-木糖醇脱氢酶基因程菌。将转化得到的含有野生型XDH基因的重组菌E.coli BL21(DE3)/pET-28a-XDH保藏在-80℃的超低温冰箱中。其中PCR扩增野生型XDH基因的引物为:
上游引物为:5'-CGCGGCAGCCATATGATGATTGAACCTGTGGCCTG-3'(SEQ ID NO.5)
下游引物为:5'-CTCGAATTCGGATCCTTACTTATCAGCTTTAATAA-3'(SEQ ID NO.6)
实施例2:定点突变
定点突变的引物采用Primer premier 5.0设计,引物设计的原则为:正反向扩增引物5’端包含15-21bp反向互补区域,各引物非互补区域长度至少为15bp,所需引入的突变包含在互补区域内。突变引物见表1。
表1突变引物
定点突变以pET-28a-XDH或pET-28a-XDH(L41M)重组质粒为模板,利用PrimerStarMix进行全质粒扩增,按照表2设置反应体系,扩增产物经过Dpn I酶消化去除PCR反应体系中的模板,然后在重组酶催化下将5’端和3’端进行同源重组,完成质粒的环化。最后将环化的扩增产物转入大肠杆菌BL21(DE3)宿主菌,并涂布在含卡那霉素的平板上,置于37℃培养箱中过夜培养。
表2定点突变体系
PCR程序为:95℃预变性300s,98℃变性10s,65℃退火15s,72℃延伸300s,反应30个循环后,再72℃延伸5min,最后4℃保温。PCR反应结束后,用0.8%的琼脂糖凝胶电泳检测PCR产物。然向每个PCR管中加入1μl Dpn I轻轻混匀后置37℃金属浴2h,之后将消化好的扩增产物按照表3中的配方进行重组反应。
表3重组反应体系
次日从平板中挑选含有突变质粒的重组大肠杆菌BL21三株,将上述重组菌从平板分别接种到装有5mL含有相应抗性的液体LB培养基(LB(g/L):蛋白胨10,氯化钠10,酵母提取物5)的50mL的摇管中,加入相应抗性,在摇床上37℃恒温培养12h,转速200rpm。培养结束后抽提质粒,送金斯瑞公司进行测序。最后将测序结果与野生型酶蛋白核酸序列进行比对,确定突变是否成功。
按照上述方法先进行单个位点突变得到重组表达菌E.coli BL21(DE3)/pET-28a-XDH(L41M),再以进过一次突变后的重组质粒pET-28a-XDH(L41M)为模板进行二次突变,最后得到了测序正确的包含XDH突变体的双位点突变重组表达菌E.coli BL21(DE3)/pET-28a-XDH(L41M+I179F)。
实施例3:重组大肠杆菌发酵培养
将实施例1、实施例2所得的重组菌E.coli BL21(DE3)/pET-28a-XDH、E.coli BL21(DE3)/pET-28a-XDH(L41M+I179F)分别接种至装有5mL含卡那霉素(50μg/mL)的LB培养基中,于37℃,200rpm的摇床中振荡培养8小时。取1ml菌体培养液转接至装有50mL TB发酵培养基((g/L):蛋白胨15,酵母提取物25,氯化钠10,含卡那霉素50μg/mL)的250ml摇瓶中,于37℃,200rpm振荡培养3小时,再于25-30℃,200rpm发酵培养12-16小时后,低温离心(8000rpm,10min,4℃)收集菌体,并用磷酸缓冲液(pH7.5,100mmol/L)清洗两次,分散于1ml同样的预冷的缓冲液中,于冰水中进行超声破碎。离心(8 000rpm,30min,4℃),弃菌体碎片,得到野生型XDH及其突变体粗酶液。
实施例4:XDH的纯化
为了纯化上述XDH及其突变体。采用GE公司的AKTA prime层析***,使用5mL的HisTrapHP镍柱亲和层析。层析柱用pH 8.5,0.5M NaCl,20mM咪唑,20mM磷酸钠缓冲(缓冲液A)预平衡,洗脱缓冲液为pH 8.5,0.5M NaCl,0.5M咪唑,20mM磷酸缓冲(缓冲液B),采用0%-100%缓冲B梯度洗脱,总洗脱时间为30min,然后将收集的活性蛋白过SephacrylS-300凝胶柱进一步脱盐去除杂蛋白,经280nm紫外检测仪监测,在高数值时收集目的蛋白。纯化后的酶进行冷冻干燥,得到冻干粉用于测定蛋白浓度和活性。
实施例5:XDH的酶活测定
XDH催化木糖醇生成L-木酮糖的氧化反应需要NAD+作为辅酶并生成NADH,通过测量单位时间内反应体系在340nm处吸光度的变化量来计算XDH的酶活力。在EP管中设置测酶活的反应体系:1.5M的木糖醇溶液100μL,2mM的NAD+溶液(使用100mM磷酸钠缓冲液新鲜配制,pH=8.5)100μL,10mMβ-巯基乙醇100μL,500mM的磷酸钠缓冲液(pH 8.5)400μL,dd H2O200μL;对照组为1.5M的木糖醇溶液0μL,2mM的NAD+溶液100μL,10mMβ-巯基乙醇100μL,磷酸钠缓冲液(pH 8.5)400μL,dd H2O 300μL。体系中添加β-巯基乙醇作为还原剂,具有防止二硫键相互交联的作用,能维持酶的活性状态。
使用移液器将上述反应液充分混匀后,转移至96孔酶标板,向每个孔里加入200μl混匀后的反应液,分别设置3组200μl的平行反应体系。待酶标仪在30℃保温5分钟后加入等量的纯酶液激活反应,立即检测波长340nm处检测吸光值变化,如公式(1)所:
酶的比活力计算公式如下:
其中V-反应体系总体积(200μL);D-稀释倍数;v-酶液体积(5μL);C-蛋白浓度(mg/mL);ε为NADH的摩尔消光系数(光程为1cm时,ε=6.220mM-1cm-1),此处采用NADH标准曲线方程(如图1)的斜率来校正,精确称量一定量的NADH,稀释不同倍数配置成不同浓度的NADH溶液,测定其在340nm处的吸光值,拟合线性方程,横坐标为NADH浓度,纵坐标为340nm波长下的吸光值。以每分钟生成1μmol NADH所需的酶量为一个活力单位(U)。蛋白定量采用TaKaRaBCA试剂盒测定。
实验结果,野生型XDH催化转化木糖醇的比活力为92.6U/mg冻干粉,而突变体XDH的酶比活力达到90.5U/mg冻干粉,相比野生型XDH的高活性只有略微下降。
实施例6:温度对XDH突变体活性的影响
(1)温度对野生型XDH及其突变体活力的影响
在不同温度条件下(25℃-60℃)按照实施例5所述方法测定实施例4中纯化后的XDH以木糖醇为底物的氧化活力,反应体系的缓冲液pH值为9.0。以最高的酶活力为100%,其对应的温度为最适温度,绘制XDH的温度-相对活力曲线。结果(如图2)显示,野生型XDH的最适温度为45℃,当反应温度超过50℃时酶活迅速下降,而XDH突变体的最适温度为50℃。
(2)野生型XDH及其突变体的热稳定性研究
将实施例4中纯化后的野生型XDH及其突变体酶分别置于45℃和50℃水浴条件下保温热处理,每隔一段时间取样测定剩余酶活。以初始的酶活力为最高酶活100%,绘制XDH的突变体的温度-相对活力曲线。结果(如图3)显示,野生型XDH在45℃保温2小时后酶活下降到60%以下,而其突变体在45℃保温2小时后,任保留了80%的酶活。
实施例7:重组基因工程菌酶转化木糖醇糖生产L-木酮糖
(1)两组静息细胞转化实验:1mL的反应体系中,加入1mL的用甘氨酸-NAOH缓冲液(50mM,pH 9.0)溶解的30g/L的木糖醇,按照实施例3所述发酵培养得到的50mL重组菌E.coliBL21(DE3)/pET-28a-XDH发酵液离心收集的菌体,45℃保温6h,然后煮沸10min以终止酶反应。
1mL的反应体系中,加入1mL的用甘氨酸-NAOH缓冲液(50mM,pH 9.0)溶解的30g/L的木糖醇,按照实施例3所述发酵培养得到的10mL重组菌E.coli BL21(DE3)/pET-28a-XDH(L41M+I179F)发酵液离心收集的菌体,45℃保温6h,然后煮沸10min以终止酶反应。
(2)用半胱氨酸咔唑比色法检测L-木酮糖的生成量。转化液经适当稀释后,移取1mL转化液,加入0.2mL 15g/L的半胱氨酸盐酸盐溶液,6mL体积分数为70%的硫酸溶液,摇匀后立即加入0.2mL 1.2g/L咔唑酒精溶液,70℃水浴保温10min,冷却10min。以空白调零,在560nm处测定吸光度。通过标准曲线计算生产的木酮糖含量。
反应6h后,含野生型XDH基因的重组菌催化生产L-木酮糖的转化率是45.2%,而突变体XDH的转化率为88.7%,相比提高了近2倍,由此表明,在较高温度下突变体XDH的反应催化活性要显著高于野生型XDH。
序列表
<110> 苏州科宁多元醇有限公司
<120> 4-木糖醇脱氢酶突变体及其用途
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Claims (8)
1.一种4-木糖醇脱氢酶突变体,其特征在于:所述4-木糖醇脱氢酶突变体的氨基酸序列是SEQ ID NO:1所示的氨基酸序列的第41位亮氨酸突变为甲硫氨酸、第179位的异亮氨酸突变为苯丙氨酸的氨基酸序列。
2.一种4-木糖醇脱氢酶突变体基因,其特征在于:所述4-木糖醇脱氢酶突变体基因的核苷酸序列如SEQ ID NO:4所示。
3.一种包含权利要求2所述的4-木糖醇脱氢酶突变体基因的重组载体。
4.一种生产权利要求1所述的4-木糖醇脱氢酶突变体的基因工程菌,其特征在于:所述基因工程菌包含权利要求2所述的4-木糖醇脱氢酶突变体基因或权利要求3所述的重组载体。
5.根据权利要求4所述的基因工程菌,其特征在于:所述基因工程菌的宿主细胞为大肠杆菌BL21(DE3)细胞。
6.权利要求2 所述的4-木糖醇脱氢酶突变体基因、权利要求3所述的重组载体或权利要求4- 5 中任一项所述的基因工程菌在制备4-木糖醇脱氢酶中的应用。
7.一种4-木糖醇脱氢酶突变体的制备方法,其特征在于:包括如下步骤:培养权利要求4或5所述的基因工程菌,获得重组表达的4-木糖醇脱氢酶突变体。
8.权利要求1所述的4-木糖醇脱氢酶突变体在转化木糖醇制备L-木酮糖中的用途。
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