CN105154457B - 一种来源于丁香假单胞菌的山梨醇脱氢酶基因及其应用 - Google Patents
一种来源于丁香假单胞菌的山梨醇脱氢酶基因及其应用 Download PDFInfo
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- CN105154457B CN105154457B CN201510601825.3A CN201510601825A CN105154457B CN 105154457 B CN105154457 B CN 105154457B CN 201510601825 A CN201510601825 A CN 201510601825A CN 105154457 B CN105154457 B CN 105154457B
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- sorbitol dehydrogenase
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Abstract
本发明公开了一种来源于丁香假单胞菌的山梨醇脱氢酶基因及其应用，基因核酸序列如SEQ ID NO：1所示，通过构建重组载体并在大肠杆菌中克隆表达，该基因编码的山梨醇脱氢酶氨基酸序列如SEQ ID NO：2所示。山梨醇脱氢酶能高效催化多种重要多元醇与其对应的酮糖的转化，山梨醇脱氢酶对底物的转化率均大于99%，相较于传统依赖异构酶的生产方法，大大降低了后期分离、脱色成本，具有重要的工业应用价值。
Description
技术领域
本发明属于基因工程和酶工程领域，具体涉及一种来源于丁香假单胞菌的山梨醇脱氢酶基因及其应用。
背景技术
山梨醇脱氢酶（Sorbitol dehydrogenase, 简称SDH, EC 1.1.1.14）广泛存在于微生物、植物以及动物之中，最早由Breusch, F. L.等发现于猫肝脏中（1942），是生物体内多元醇途径中的关键酶。未经细胞利用的葡萄糖进入多元醇通路时，醛糖还原酶首先将其还原为山梨醇，同时氧化NADPH生成NADP+。继而，SDH利用NAD+作为氢受体氧化山梨醇C2位的羟基生成果糖，果糖通过己糖激酶的磷酸化作用形成6-磷酸-果糖返回糖酵解途径。目前，研究者已考察了数十种不同来源的山梨醇脱氢酶，研究方向涉及与植物的滞育关系、与二型糖尿病的相关性、多元醇的生物转化等等，然而直到2003年，才由Philippsen, A.、Pauly, T.A等人相继利用X光衍射和晶体拓扑学分析得到首个微生物来源及人来源的SDH晶体及亚结构，分别属于短链脱氢酶家族、中链脱氢酶家族。
SDH大多为多聚体酶，是由相同或相似的亚基组成的二聚体、三聚体或四聚体，单亚基的分子量为25~60 kDa，能不同程度的氧化D-山梨醇，L-艾杜糖醇，D-半乳糖醇等多元醇C2位的羟基生成相对应的酮糖。在酶法检测方面，SDH可以用于快速、精确检测山梨醇的含量。在食品及其他复合材料领域，D-山梨醇是一种重要的大吨位原料，而在临床上，D-山梨醇则是糖尿病监测的重要指标。从羊肝脏、Bacillus subtilis中分离的SDH由于具有广泛的底物特异性，很难区分样品中的木糖醇（结构与山梨醇类似）；Schneider等从Pseudomonas sp.分离出对木糖醇低活性（仅为山梨醇的2%）的SDH，但因产率低并不适合工业应用；而后，Ikuko Masuda等从Pseudomonas sp. KS-E1806克隆了一条SDH基因并构建基因工程菌用于山梨醇脱氢酶的高效生产，同时申请了欧洲、美国、日本的专利保护（专利号：EP1262551 (A3)、US2003022336 (A1)、JP2002355046 (A)）。此外，SDH在酶法生产D-果糖或D-塔格糖方面，也具有潜在的工业应用价值。
发明内容
本发明的目的是提供一种来源于丁香假单胞菌（Pseudomonas syringae）的山梨醇脱氢酶基因及其应用，该基因编码的山梨醇脱氢酶能催化多元醇与相应酮糖相互转化，至今未发现该山梨醇脱氢酶基因的相关研究的报道。
来源于丁香假单胞菌的山梨醇脱氢酶基因，其核苷酸序列如SEQ ID NO：1所示，该基因序列含有774 bp碱基。
所述的山梨醇脱氢酶基因编码的山梨醇脱氢酶，其氨基酸序列如SEQ ID NO：2所示，其包含257个氨基酸。
一种表达载体，它包含所述的山梨醇脱氢酶基因。
一种重组菌，其是通过利用所述的表达载体转化宿主细胞获得的。
用细菌DNA Kit (TIANGEN, China)提取丁香假单胞菌基因组，基于如SEQ ID NO：1所示的核酸序列设计引物，扩增的DNA片段克隆到pET-28a载体中，将所构建的重组质粒pET-28a-sdh转化到大肠杆菌 BL21中构建工程菌，通过适宜浓度的IPTG诱导基因工程菌高效表达山梨醇脱氢酶。
应当理解，本领域技术人员可根据本发明公开的氨基酸序列，在不影响其活性的前提下，取代、缺失和/或增加一个或几个氨基酸，得到所述蛋白的突变序列。因此，本发明的来源于丁香假单胞菌的山梨醇脱氢酶还包括由SEQ ID No：2 所示的氨基酸序列中经取代、缺失或添加一个或几个氨基酸且具有同等活性的由SEQ ID No：2所示的蛋白质衍生的蛋白质。例如通过在末端添加标签序列，如His-tag 或Strep-tag 而衍生的蛋白质。
通过Blast软件在线比较分析，发现来源于丁香假单胞菌的山梨醇脱氢酶基因的核苷酸序列与其它微生物的己知山梨醇脱氢酶基因的同源性差异较大。
本发明中，术语“山梨醇脱氢酶基因”还包括能编码具有与山梨醇脱氢酶相同功能的蛋白的SEQ ID NO：1的突变形式，所述突变类型包括：确实、无义、插入、错义。本领域技术人员能够理解，作为遗传密码简并性的结果，许多不同的多核苷酸能够编码相同的多肽。另外，应当理解，本领域技术人员能够使用常规的技术进行核苷酸取代，所述取代不会影响本发明中所使用的多核苷酸编码的多肽序列。另外，还可以使用本领域中的已知的方法对多核苷酸进行修饰，以增强本发明多核苷酸在体内的活性或者存活期。
一种含有山梨醇脱氢酶基因的重组表达载体，是将SEQ ID NO：1所示基因与表达载体pET-28a连接所构建的重组载体。
一种含有上述重组表达载体的宿主细胞宿主大肠杆菌BL21（Escherichia coli BL21）。
所述的来源于丁香假单胞菌的山梨醇脱氢酶基因在多元醇与其对应的酮糖的转化中的应用。
将山梨醇脱氢酶基因通过构建重组载体并在宿主细胞中克隆表达，获得山梨醇脱氢酶，催化多元醇与其对应的酮糖的转化。
所述多元醇与其对应的酮糖的转化，例如，山梨醇转化为D-果糖，D-半乳糖醇转化为D-塔格糖，L-艾杜糖醇转化为L-山梨糖等。
有益效果：
本发明基于生物信息学的分析方法和分子生物学技术所获得的核酸序列是一种高活性、高选择性的山梨醇脱氢酶基因，可将其与载体连接后转化至宿主细胞内生产山梨醇脱氢酶，该酶能高效催化多种重要多元醇与其对应的酮糖的转化。本发明首次将氨基酸序列如SEQ ID NO：2所示的山梨醇脱氢酶应用于D-山梨醇合成D-果糖，D-半乳糖醇合成D-塔格糖，L-艾杜糖醇合成L-山梨糖中，取得很好的效果，在添加NADH氧化酶用于辅酶原位再生的情况下，分别可以催化合成100 g/L D-果糖，50 g/L D-塔格糖及50 g/L L-山梨糖。氨基酸序列如SEQ ID NO：2所示的山梨醇脱氢酶对底物的转化率高（均大于99%），相较于传统依赖异构酶的生产方法，大大降低了后期分离、脱色成本，具有重要的工业应用价值。
附图说明
图1为山梨醇脱氢酶基因表达载体的构建图；
图2为山梨醇脱氢酶基因诱导表达后的SDS-PAGE分析图。
具体实施方式
本发明提供了编码具有山梨醇脱氢酶活性的多肽的多聚核苷酸分子，该核苷酸分子是从丁香假单胞菌中克隆得到的，具有SEQ ID NO：1的核苷酸序列，它编码257个氨基酸的多肽。
本发明还涉及一种重组载体，该载体包含本发明的核苷酸序列SEQ ID NO：1，以及包含重组质粒的宿主细胞。同时，本发明包括构建该重组质粒和宿主细胞的方法，以及用重组工程菌生产山梨醇脱氢酶的方法。
在本发明中，“山梨醇脱氢酶”是指具有山梨醇脱氢酶活性的SEQ ID NO：2序列的多肽。该术语还包括SEQ ID NO：2序列的变异体，包括（但不限于）若干个氨基酸的缺失，插入和／或取代，以及在C末端和／或N末端添加一个或数个氨基酸，也可以使不影响序列的修饰形式上的差异。
本发明的山梨醇脱氢酶基因全长序列或其片段通常可以用PCR扩增法，重组法，或人工合成法获得。
本发明中，可选用本领域己知的各种载体，如质粒，粘粒，噬菌体及反转录病毒等。
重组表达载体可以用本领域熟知的方法导入宿主细胞中，这些方法包括：氯化钙热激法，电转化法，PEG介导法，基因枪法等。
本发明中，术语“宿主细胞”包括原核细胞和真核细胞。常用的原核细胞如大肠杆菌等。常用的真核细胞如酵母细胞，或各种动植物细胞。
本发明的实施将采用本领域技术人员的能力范围之内的化学、分子生物学等领域的传统技术。另外，除非另有说明，在本文中，核酸以5’至3’的方向从左向右书写，氨基酸序列则以氨基端到羧基端的方向从左向右书写。
根据下述实施例，可以更好地理解本发明。然而，本领域的技术人员容易理解，实施例所描述的具体的物料配比、工艺条件及其结果仅用于说明本发明，而不应当也不会限制权利要求书中所详细描述的本发明。
实施例1 山梨醇脱氢酶基因的克隆
丁香假单胞菌购于中国普通微生物菌种保藏管理中心（CGMCC），培养基LB（g·L-1）：酵母提取物5 g，蛋白胨10 g，NaCl 10 g，补蒸馏水至1 L。
将丁香假单胞菌接种于5 mL LB液体培养基中，30℃培养至对数生长期，使用DNAKit (TIANGEN, China)提取丁香假单胞菌基因组。构建表达载体所用的引物加设酶切位点，引物序列如下：
上游引物（SDH-sense含EcoRⅠ）为：
5'- CGGAATTCAAACGACTTGAAGGTAAAAGCG -3'
下游引物（SDH -anti含XhoⅠ）为：
5'- CCGCTCGAGTCAGTTCATCCAGTTGCCACCA -3'
所有引物均由南京金斯瑞生物科技有限公司合成。基因的PCR条件： 94℃变性7min，按如下参数循环30次：94℃变性1 min，60℃退火60 s，72℃延伸1 min。最后72℃延伸10 min。PCR反应结束后取产物2 μL，然后在浓度为0.8%的琼脂糖凝胶中，进行电泳分析。经凝胶成像系统成像确认片段大小正确后，采用TaKaRa公司的DNA纯化回收试剂盒（TaKaRaAgarose Gel DNA Purification）回收目的片段用于重组表达载体pET-28a-sdh的构建。
实施例2 重组表达载体pET-28a-sdh的构建
用XhoⅠ及EcoRⅠ分别酶切pET-28a（购于Novagen默克中国）及所扩增含有两个酶切位点的目的基因（实施例1PCR扩增获得），分别胶回收已双酶切的目的片段和表达载体，将已双酶切的表达载体pET-28a与目的基因（SEQ ID NO：1所示的基因）用T4-DNA连接酶（购于TaKaRa公司）进行过夜连接，得到重组载体pET-28a-sdh；将10 μL的连接产物加入100 μL的大肠杆菌BL21感受态细胞中，冰上放置30 min，42℃热激90 s。冰上放置2 min。加入预热的0.45 mL SOC培养基（2% (W/V)蛋白胨，0.5% (W/V)酵母浸粉，0.05% (W/V) NaCl，2.5 mMKCl，10 mM MgCl2，20 mM 葡萄糖。）。220 rpm 37℃ 1 h。将200 μL菌液加入含有30 μg/mL的卡那霉素的LB平板上，37℃过夜培养12~16 h，得到重组菌E.coli BL21（含pET-28a-sdh）。构建图谱见图1。
实施例3 山梨醇脱氢酶基因在大肠杆菌BL21中的诱导表达
挑取重组菌E.coli BL21（含pET-28a-sdh）及对照菌E. coli BL21（含pET-28a）至含30 μg/mL的卡那霉素的LB液体培养基中，37℃振荡培养过夜。然后按2%接种量分别接种到新鲜含30 μg/mL的卡那霉素的LB液体培养基中，37℃培养至OD600约为0.6时，加入IPTG至终浓度0.2 mmol·L-1，16℃，220 rpm，诱导表达24 h后，离心(4℃，5000 rpm，15 min)，菌泥用100 mM Tris-HCl缓冲(pH 9.0)重悬，超声破碎细胞(功率300 W，超声3 s，间歇5 s，共5min)，离心(4℃，12000 rpm，15 min)。SDS-PAGE分析显示，重组菌E.coli BL21（含pET-28a-sdh）表达出一条分子量约27 kDa的蛋白(见图2泳道3)，对照菌相应位置并没有明显表达条带（见图2泳道2）。
上清酶活的测定：酶反应体系包括100 mM Tris-HCl缓冲(pH 9.0)，1 mM NAD+，50mM 山梨醇，30℃，340 nm处测定吸光值的上升。酶活定义为每分钟内生成1 μmol NADH所需要的酶量为一个酶活单位U。蛋白采用Brandford法进行测定。结果显示，对照菌E.coliBL21（含pET-28a）的比酶活为0，而重组菌E.coli BL21（含pET-28a-sdh）的比酶活为15 U/mg。
实施例4
取实施例3诱导表达后离心收集的菌泥用Tris-HCl缓冲(100 mmol·L-1， pH 9.0)洗涤两次，称取10 g（湿重）的大肠杆菌菌泥，悬浮于200 mL的pH 9.0 Tris-HCl缓冲中。超声处理细胞(功率300 W，超声3 s，间歇5 s，共5 min)，加入山梨醇100 g/L，NADH氧化酶300U，NAD+ 0.2 mmol/L，25℃，280 rpm，12 h。产物D-果糖的产量为98.4 g/L，产物的得率为：99.5%。
实施例5
取实施例3诱导表达后离心收集的菌泥用Tris-HCl缓冲(100 mmol·L-1， pH 9.0)洗涤两次，称取10 g（湿重）的大肠杆菌菌泥，悬浮于200 mL的pH 9.0 Tris-HCl缓冲中。超声处理细胞(功率300 W，超声3 s，间歇5 s，共5 min)，加入半乳糖醇50 g/L，NADH氧化酶300 U，NAD+ 0.2 mmol/L，25℃，280 rpm，12 h。产物D-塔格糖的产量为49.5 g/L，产物的得率为：99.1%。
实施例6
取实施例3诱导表达后离心收集的菌泥用Tris-HCl缓冲(100 mmol·L-1， pH 9.0)洗涤两次，称取10 g（湿重）的大肠杆菌菌泥，悬浮于200 mL的pH 9.0 Tris-HCl缓冲中。超声处理细胞(功率300 W，超声3 s，间歇5 s，共5 min)，加入L-艾杜糖醇50 g/L，NADH氧化酶300 U，NAD+ 0.2 mmol/L，25℃，280 rpm，12 h。产物L-山梨糖的产量为49.1 g/L，产物的得率为：99.0%。
产物的检测方法：
采用高效液相色谱(HPLC)测定D-山梨醇、D-果糖、D-半乳糖醇、D-塔格糖、L-艾杜糖醇及L-山梨糖浓度。高效液相色谱仪采用美国戴安(DIONEX)公司UltiMate3000，色谱柱为美国Bio-Rad公司Aminex HPX-87H column（300 x 7.8 mm）色谱柱；流动相为5 mM H2SO4；流速0.6 mL/min；柱温为65℃；采用示差检测器。
<110> 南京工业大学
<120> 一种来源于丁香假单胞菌的山梨醇脱氢酶基因及其应用
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Claims (2)
1.山梨醇脱氢酶在多元醇与其对应的酮糖的转化中的应用，所述山梨醇脱氢酶由来源于丁香假单胞菌的山梨醇脱氢酶基因编码获得，其氨基酸序列如SEQ ID NO：2所示，所述来源于丁香假单胞菌的山梨醇脱氢酶基因的核苷酸序列如SEQ ID NO：1所示，其特征在于，在NADH氧化酶和NAD+条件下，所述山梨醇脱氢酶能够催化山梨醇、D-半乳糖醇或L-艾杜糖醇与其对应的酮糖的转化。
2.根据权利要求1所述的应用，其特征在于：所述对应的酮糖为D-果糖、D-塔格糖和L-山梨糖。
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2023122805A1 (en) * | 2021-12-20 | 2023-06-29 | Vestaron Corporation | Sorbitol driven selection pressure method |
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