CN101824420B - 纳豆芽孢杆菌谷氨酸脱氢酶基因的突变基因及编码蛋白 - Google Patents
纳豆芽孢杆菌谷氨酸脱氢酶基因的突变基因及编码蛋白 Download PDFInfo
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Abstract
一种生物技术领域的纳豆芽孢杆菌谷氨酸脱氢酶基因的突变基因及编码蛋白；所述纳豆芽孢杆菌谷氨酸脱氢酶编码基因的突变基因的碱基序列如SEQ ID NO：3、SEQ ID NO：5、SEQID NO：7、SEQ ID NO：9、SEQ ID NO：11、SEQ ID NO：13或SEQ ID NO：15所示；所述纳豆芽孢杆菌谷氨酸脱氢酶编码基因的突变基因编码的蛋白的氨基酸序列如SEQ ID NO：4、SEQ IDNO：6、SEQ ID NO：8、SEQ ID NO：10、SEQ ID NO：12、SEQ ID NO：14或SEQ ID NO：16所示；本发明还涉及一种包含前述突变基因的重组载体。通过同源重组将该本发明的突变引入野生菌株后，可获得低产氨且发生长速度不变的基因工程菌，从而生产出无氨味纳豆。
Description
技术领域
本发明涉及一种生物技术领域的基因及编码蛋白，具体是一种纳豆芽孢杆菌谷氨酸脱氢酶基因的突变基因及编码蛋白。
背景技术
纳豆芽孢杆菌(Bacillus Subtilis Natto)是日本纳豆的发酵菌，发酵后能产生多种独特的保健功效成份，例如纳豆激酶、多聚谷氨酸、维生素K2等，纳豆激酶已被证实具有良好的溶解血栓功能，且无毒副作用。另外，纳豆还具有预防骨质疏松、抗氧化、抗菌、调节肠胃等生理预防和调节功能。除预防骨质疏松与大豆本身含有的异黄酮有关外，其他功能主要取决于发酵后产生的新物质。日本人将每年的7月10日定为纳豆节。但纳豆在中国市场却难觅身影，究其原因，主要是发酵过程中产氨，氨在产品中累积使其风味让国人难以接受。然而，当今世界，食疗越来越受到人们的认可和追捧，治未病的思想也开始成为各国政府对于国民健康的主导政策。因此作为一种功能性食品，如果能改良其风味，纳豆一定会有极大的市场需求。纳豆芽孢杆菌氨基酸代谢产氨主要是联合脱氨基作用，GDH为纳豆菌代谢产氨途径中的关键酶。
Shigeki Kada等在《Bioscience，Biotechnology and Biochemistry》(生物科学、生物技术和生物化学)2008年第72卷第7期1869～1876页发表了题为《Identification of Two MajorAmmonia-Releasing Reactions Involved in Secondary Natto Fermentation》(纳豆二次发酵过程中与氨还原释放相关的两个关键酶的鉴定)一文，认为GDH和尿素酶是产氨的关键酶，GDH编码基因敲除后氨的含量可降低50％左右，若将编码尿素酶的基因同时敲除，几乎可完全抑制产氨。BORIS R.BELITSKY等在《Journal of Bacterology》(细菌学杂志)1998年第180卷第23期6298～6305页发表了题为《Role and Regulation of Bacillus subtilis Glutamate DehydrogenaseGenes》(枯草芽孢杆菌谷氨酸脱氢酶基因的作用和调节)一文，文中研究结果表明，如果敲除编码GDH的基因，菌种的生长速度会下降，在普通的营养肉汤培养基中，代增时间由36min增加到52min。因此如果仅仅通过敲除GDH编码基因来达到减少产氨量的目的，会付出发酵时间延长及底物要求更高的代价。所以通过研究GDH的活性位点，利用定点突变技术获得不同GDH酶活的突变体，进而获得具有不同GDH酶活的发酵菌株，有可能达到在不影响发酵周期的前提 下，减少产氨量，改善纳豆风味。
纳豆芽孢杆菌GDH完全失活虽然可以降低产氨量，但同时延长了发酵周期，增加了对底物的要求，因此有必要对此酶进行结构改造。
经对现有技术的文献检索发现，尚未见与本发明的主题有关的报道。
发明内容
本发明的目的在于克服现有技术中的不足，提供一种纳豆芽孢杆菌谷氨酸脱氢酶基因的突变基因及编码蛋白。通过同源重组将该本发明的突变引入野生菌株后，可获得低产氨且发生长速度不变的基因工程菌，从而生产出无氨味纳豆。
本发明是通过以下的技术方案实现的，
本发明涉及一种纳豆芽孢杆菌谷氨酸脱氢酶编码基因的突变基因，该突变基因的碱基序列及其编码蛋白的氨基酸序列如SEQ ID NO：3、SEQ ID NO：5、SEQ ID NO：7、SEQ ID NO：9、SEQ ID NO：11、SEQ ID NO：13或SEQ ID NO：15所示。
本发明还涉及一种纳豆芽孢杆菌谷氨酸脱氢酶基因的突变基因的编码蛋白，所述蛋白的氨基酸序列如SEQ ID NO：4、SEQ ID NO：6、SEQ ID NO：8、SEQ ID NO：10、SEQ ID NO：12、SEQ ID NO：14或SEQ ID NO：16所示。
本发明还涉及一种重组载体，该重组载体包含前述的突变基因。
所述重组载体使用的载体为pET-28a。
本发明确定了纳豆芽孢杆菌的GDH的三维结构，利用SAMM分子模拟分析等软件分析了GDH的活性位点和突变方案，利用QuikChange定点突变试剂盒对活性位点进行定点突变，得到7个突变体。通过同源重组将该突变引入野生菌株后，可获得低产氨且发生长速度不变的基因工程菌，从而生产出无氨味纳豆。
本发明涉及的纳豆芽孢杆菌具体为纳豆芽孢杆菌(Bacillus Natto)SJTU 1-4 CGMCCNO：2801，已于2008年12月9日提交中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)保藏，该单位的地址为：北京市朝阳区大屯路，中国科学院微生物研究所，邮编，100101。
附图说明
图1模型min的拉式图检验结果；
图2谷氨酸脱氢酶的三维模型；
图3酶活性中心氢键结构的模拟；
图4氨基酸残基化学修饰对酶活性影响的分析；
图5突变质粒转化子菌落PCR产物电泳分析；
图6GDH表达的SDS-PAGE分析；
图7纯化蛋白的SDS-PAGE分析；
图8野生型和突变型谷氨酸脱氢酶活性的比较。
具体实施方式
以下对本发明的实施例作详细说明：本实施例在以本发明技术方案为前提下进行实施，给出了详细的实施方式和过程，但本发明的保护范围不限于下述的实施例。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等著的《分子克隆：实验室手册》(NewYork：Cold Spring Harbor Laboratory Press，1989)中所述的条件，或按照制造厂商所建议的条件。
实施例
步骤一，纳豆芽孢杆菌GDH的同源建模；首先在PDB中搜寻与GDH蛋白质(SEQ ID NO：2)序列的高度相似的蛋白质序列，在Swiss model上尝试性建模，经Prosa2003残基能量图分析后，最后选择了来自强烈炽热球菌的1GTM蛋白，嗜温球菌的1EUZ蛋白作为同源建模的模版，建模，利用SAMM分子模拟分析软件对得到的蛋白模型进行优化；具体过程如下：
利用NCBI的BLASTn对得到的纳豆芽孢杆菌GDH编码基因序列(SEQ ID NO：1)进行比对分析，使用ORF Finder模块搜寻序列中可能的开放式阅读框，并将所找到的一个含有422个氨基酸残基的蛋白质在BLASTp中进行比对分析；
在PDB中得到六段与该蛋白质序列的高度相似的蛋白质序列(相似度在50％左右)，分别为1GTM、1EUZ、1PZO、1HWY、1V9L和2RIR。以前五个蛋白质序列作为模版，在Swissmodel上得到蛋白模型model1，经Prosa2003残基能量图分析后，来自强烈炽热球菌的1GTM蛋白，嗜温球菌的1EUZ蛋白与model1的残基能量变化趋势最为接近。选取1EUZ、1GTM作为同源建模的模版，在Swiss model上再一次建模得到蛋白模型swiss；
Swiss Model和PredictProtein：在Swiss model中使用1GTM和1EUZ作为模版，按默认设置进行同源建模，在PredictProtein中，按默认设置对纳豆枯草芽孢杆菌GDH的二级结构特点进行逐一分析；
步骤二，纳豆芽孢杆菌GDH活性位点和定点突变方案的确定，利用SAMM分子模拟分析软件对蛋白模型进行能量最小化分析，并经分子动力学模拟后得到可能与底物谷氨酸(Glu)存 在氢键连接的氨基酸残基，对其中出现概率较高的几个氨基酸残基作突变模拟；具体如下：
ClustalX：利用ClustalX，以1GTM、1EUZ、1PZO、1HWY、1V9L、和2RIR作为比对对象，对纳豆枯草芽孢杆菌GDH进行多序列比对，得到四段高度一致性的氨基酸序列，分别为：80-82号残基，112-120号残基，195-200号残基，351-358号残基，该四段序列均为与底物谷氨酸空间距离十分接近 
的区域，酶活中心很有可能包含于其中；
SAMM：利用SAMM分子模拟分析软件对Swiss model自动生成GDH的蛋白模型利用PAMM94力场进行能量最小化，得到一个蛋白模型min。经Prosa2003残基能量分析后，模型min中几段GDH的保守序列的氨基酸残基的残基能量略小于蛋白模型swiss。经拉氏图检验，模型min中只有3个氨基酸残基的ψ， 
角在非核心区域内，如图1所示，图中较亮的十字形点为在非核心区域内的氨基酸残基；
在SAMM中固定蛋白骨架，利用ClustalX得到的信息，选择CHAM22力场，进行GDH与其辅酶NADP+以及底物谷氨酸的分子对接，得到该酶与底物谷氨酸和辅酶NADP+对接结果，如图2所示；
以2ns为步长，环境温度为310K，经分子动力学模拟后得到1000个分子模型，以时间t对模型能量E作图。模型的能量随着模拟时间增长而逐渐下降，并趋于稳定，表明该蛋白与底物和辅酶的作用模型是稳定而可信的。统计模型中与底物Glu存在氢键连接的氨基酸残基，如图3所示；
模拟结果表明Lys80，Lys116，Arg196，Thr200，Ser351这五个氨基酸残基与谷氨酸形成氢键连接的可能性较大，见表1，即意味着它们很有可能是酶活中心直接与底物发生连接或催化反应的位点，故选取它们进行突变模拟实验。Lys，Ser，Arg这三个氨基酸残基种类与谷氨酸形成氢键的概率最大，故对其进行特异性的蛋白质化学修饰研究，由于Thr没有特异性的修饰剂，故只进行定点突变模拟试验。利用SAMM软件中的定点突变模块，以突变后的蛋白ΔG变化最小，突变模型的分子动力学曲线与原蛋白模型的变化趋势一致，与底物的结合能变化尽可能大为原则，选出了五对突变体，分别为Lys80Ala，Lys116Gln，Arg196Phe，Thr200Ala，Ser351Ala，其ΔG变化值如表2。原酶与底物的结合能在7800J/mol左右，突变后基本在14000J/mol左右，证明这些位点的确对酶活有一定的作用。
表1氨基酸残基与底物形成氢键的概率模拟分析
| 氨基酸残基 | 概率(％) |
| Lys80 | 62 |
| Gly82 | 5 |
| Lys104 | 11 |
| Lys116 | 26 |
| Pro155 | 14 |
| Asp156 | 15 |
| Arg196 | 50 |
| Glu197 | 9 |
| Thr200 | 41 |
| Asn326 | 16 |
| Ser351 | 29 |
| Ser358 | 10 |
表2各突变方案的ΔG变化
| 突变方案 | ΔG(kJ/mol) |
| K80A | -0.02 |
| K116Q | -0.08 |
| R196F | -0.06 |
| T200V | -0.06 |
| S116A | -0.12 |
步骤三，纳豆芽孢杆菌GDH的纯化和化学修饰；提取纳豆芽孢杆菌发酵液中的粗酶液后，用硫酸铵分级沉淀、DE32离子交换层析、Sephacryl S-300凝胶层析和羟基磷灰石层析对GDH进行纯化。TNBS、PMSF和PG分别对纯化酶中氢键出现概率较高的赖氨酸残基、丝氨酸残基和精氨酸残基进行特异性化学修饰，并以底物保护后再进行修饰的效果作对照，对GDH的活性位点进行初步验证；具体如下：
1、纳豆枯草芽孢杆菌NADP+依赖型GDH的纯化(结果见表3)
在37℃下培养9.5小时后，用差速离心法(1800rpm和4800rpm)收集对数生长后期的菌种，以下所有纯化步骤均在常温下进行：
粗酶液的提取，将收集的菌体置于细胞裂解液中25℃裂解1小时，料液比(W/V)为1∶4；裂解液配方为0.05M Tris-HCl，pH 7.0，1mM EDTA，1mM DDT，100mM NaCl，1mg/L溶菌酶。裂解后超声处理20min，每次5s，工作与停息比为1∶4；8000rpm离心5min后收集上清液，即为粗酶液。
硫酸铵分级沉淀：在粗酶液中加入25.8g/mL的硫酸铵(40％饱和度)，在0～4℃静止过夜，10000rpm离心10min去沉淀。再在上清液中添加硫酸铵至38.1g/ml(65％饱和度)，0～4℃静 止过夜，12000rpm离心15min取沉淀，并溶于10ml缓冲液A(50mmol/L Tris-HCl缓冲液、pH8.0，内含1.0mmol/L EDTA、1.0mmol/L DDT)中，在4℃下保存。
DE32离子交换层析：将盐析后得到的粗酶液4.5mL在缓冲液A中透析6小时后用PEG20000透析脱水至3mL；DE32柱(2.6cm×50cm)层析条件为：上样量为3mL，采用0～0.6M NaCl线性梯度洗脱，流速为20mL/h，缓冲体系为pH8.0的缓冲液A。
Sephacryl S-300凝胶层析：将离子交换柱分离到的高酶活组分合并后经PEG20000透析浓缩至3ml；Sephacryl S-300凝胶柱(2.6cm×90cm)层析条件为：上样量为3mL，0.15M NaCl等梯度洗脱，流速为40mL/h，缓冲体系为pH8.0的缓冲液A。
羟基磷灰石层析：将凝胶柱分离到的高酶活组分合并后经PEG20000透析浓缩至1ml；羟基磷灰石柱(1.6cm×10cm)层析条件为：上样量为1mL，0～0.3M KCl线性梯度洗脱，流速为4mL/h，缓冲体系为pH8.0的磷酸缓冲液。
酶活检测，25℃下，取酶液与酶活测定液各1.5毫升测定GDH催化辅酶NADP+还原所引起的光吸收值在340nm波长处的变化。脱氨酶活力测定液配制：1mmol/L NADP，30mmol/L谷氨酸，缓冲液：pH8.050mmol/L Tris-HCl；每分钟催化1μmol NADP+还原所需的酶量定义为一个酶活力单位(μ)。所有测定都在Beckman DU800分光光度计上完成。对照为不含GDH的缓冲液A。
表3谷氨酸脱氢酶的纯化结果
| 体积(mL) | 活性 | 蛋白 | 单位活性 | 回收 | 纯化倍 | |
| 提取物 | 18 | 14.53 | 14.4 | 1.01 | 100 | 1 |
| 盐析 | 4.5 | 8.46 | 3.83 | 2.21 | 58.2 | 2.2 |
| DEAE-32 | 3 | 7.85 | 0.72 | 10.89 | 54.03 | 10.8 |
| Sephycral | 3 | 7.04 | 0.32 | 22.24 | 48.49 | 22.0 |
| HTP | 1 | 5.96 | 0.17 | 34.29 | 41.00 | 34.0 |
蛋白含量测定，280nm和260nm的吸收差法，用下面的经验公式算出蛋白质的浓度：蛋白质浓度＝1.45×A280-0.74×A260(mg/ml)。
蛋白质化学修饰实验
赖氨酸残基的修饰：将纯化后的蛋白在pH9.0的重碳酸缓冲液中透析12小时，每6小时换一次缓冲溶液，取50μL纯化后的酶溶液(0.1mg/mL，10U)，加入等体积的含有不同浓度TNBS溶液(0.05～1.5mM)。在黑暗中反应半小时后用5μL的4M HCl和10％SDS溶液中止 反应，取10μL按酶活性测定方法测定修饰后的酶活，其余部分用来测定每个蛋白分子中被TNBS修饰的赖氨酸残基数。方法为：用分光光度计测定反应体系在346nm处吸收值，三硝基苯赖氨酸的吸收系数14.6mM-1cm-1，对照为不含TNBS的重碳酸缓冲液。
精氨酸残基的修饰：将纯化后的蛋白在pH9.0的重碳酸缓冲液中透析12小时，每6小时换一次缓冲溶液，取50μL纯化后的酶溶液(0.1mg/mL，10U)，加入等体积的不同浓度PG溶液(0.05～5mM)。PG先溶于DMSO(DMSO对酶活没有影响)，再用重碳酸缓冲液定容。反应半小时后用5μL 4M HCl中止反应，用分光光度计测定反应体系在250nm处吸收值以确定每个蛋白分子中被修饰的精氨酸残基数，PG修饰的精氨酸在250nm处的吸收系数为11.3mM-1cm-1。然后将反应体系16000rpm超速离心，取上清按酶活性测定方法测定酶的残活，对照为重碳酸缓冲液。
丝氨酸残基的修饰：将纯化后的蛋白在pH9.0的重碳酸缓冲液中透析12小时，每6小时换一次缓冲溶液，取50μL纯化后的酶溶液(0.1mg/mL，10U)，加入等体积的不同浓度PMSF溶液(0.05～5mM)。PMSF先溶于DMSO，再用重碳酸缓冲液进行定容，反应半小时后测定残活，对照为重碳酸缓冲液。
结果表明：GDH对于TNBS、PMSF存在浓度依赖性的失活。经浓度为1.5mM的TNBS修饰后，GDH便完全失活，1mM的TNBS可以抑制约90％的酶活，如图4(A)所示。经浓度为0.2mM的PMSF修饰后，谷氨酸脱氢酶便完全失活。0.1mM的PMSF可以抑制约90％的酶活，如图4(C)所示。PG能造成小部分的酶活性丧失，1mM的PG可以抑制约17％的酶活力，更高的浓度也无法提升其对酶活性的抑制效果，如图4(B)所示。说明该酶的酶促反应中心存在对酶活性有贡献的赖氨酸和丝氨酸残基。
底物保护实验
所有底物保护实验均先在酶溶液中加入谷氨酸至浓度为0.1mol/L，再与不同的修饰剂(浓度至可使酶基本失活)反应，残活用酶活测定方法测定。纯酶以及修饰后的酶(半失活)的动力学系数Km，Kcat均通过测定底物谷氨酸含量为0.01～30mM时酶的活力计算得到，计算方法为LB法作图，使用Excel线性回归模块作图分析。
进行底物保护后进行TNBS修饰，GDH活性为原酶活性的98.7％。进行底物保护后进行PMSF修饰，GDH活性几乎不变。因此可以推测，酶活性中心必定含有Lys和Ser残基，并且其与酶的催化作用有重要的关联。而Arg残基的特异性抑制剂PG对于该酶的抑制效果不明显，经底物保护后，进行PG修饰酶活可恢复到原水平的97％，所以Arg残基并不在酶的活性位点上， 不参与底物结合与催化反应。根据先前的计算模拟分析，Arg残基处于酶活性中心的非核心区域，其可能通过与其他在酶活性中心氨基酸残基形成氢键作用以稳定酶活性中心结构，Arg196Phe，Arg196Glu两个模拟突变体对于底物的结合能都大幅上升。然而Arg残基并不直接参与到酶促反应中，对它进行化学修饰后，由于其侧链与PG相连，很有可能对酶的底物结合产生一定的空间位阻效应，使酶的活性部分下降。
步骤四，定点突变，针对三个有效活性位点设计突变扩增引物，利用QuikChange定点突变试剂盒对活性位点进行定点突变，得到单位点和多位点突变的突变体；具体步骤如下：
1、引物设计
根据测序得到的序列，结合遗传密码子表，针对Lys80、Lys116及Ser351三个活性位点，设计了3对突变引物：
Lys(AAA)80-Ala(GCA)，引物如下：
5’-GTGAAAACGTATCCCGCCTGCCGTTACCGACAGAG-3’
GCCCACTTTTGCATAGGGCGGAAAGCAATGGCTGTCTCAGT
CGGGTGAAAACGTATCCCTCCTTTCGTTACCGACAGAGTCA，
3’-CACTTTTGCATAGGGAGGACGGCAATGGCTGTCTC-5’；
Lys(AAA)116-Gln(CAA)，引物如下：
5’-GGATCACAAACAATTCCGCCTTGACCACCGCCATAT-3’
GAACCTAGTGTTTGTTAAGGCGGAAATGGTGGCGGTATACCT
CTTGGATCACAAACAATTCCGCCTTTACCACCGCCATATGGA，
3’-CCTAGTGTTTGTTAAGGCGGAACTGGTGGCGGTATA-5’；
Ser(TCA)351-Ala(GCT)
5’-AACTGTTACGCCACCGGCAGCTGCCAGCAGC-3’
TCTTTGACAATGCGGTGGCCGTCAACGGTCGTCGAGA
AGAAACTGTTACGCCACCGGGAGTTGCCAGCAGCTCT，
3’-TTGACAATGCGGTGGCCGTCGACGGTCGTCG-5’；
2、定点突变
以pMD19-T为克隆载体，经TA克隆得到纳豆芽孢杆菌GDH编码基因。利用Quikchange试剂盒对GDH进行基因定点突变。Quikchange试剂盒定点突变体系：
5μL 10×reaction Buffer
3μL(10～100ng)质粒DNA(pMD19-T/GDH)
2.5μL突变引物(100ng/μL)
1μL dNTP混合物
1.5μL QuikSolution
36μL dH20(使整个体系体积为50μl)
1μL QuikChange Lighting聚合酶；
3、PCR扩增
将混合物在95℃加热1min后，95℃变性20s，60℃退火10s，68℃延伸
4min，循环18轮，进行PCR。最后一轮循环结束后，于68℃保温5min，使反应产物扩增充分。PCR扩增后加2μL Dpn I限制性酶直接降解其中经甲基化修饰与半甲基化修饰的原质粒模板与质粒杂环，轻轻混匀，离心后，放在37℃ 5min。
4、大肠杆菌(DH5α)感受态细胞的制备(CaCl2法)：
将-70℃冰箱内冻存的DH5α宿主菌在冰上融化后，用接种环挑取菌液在LB平板上(无抗生素)上划线，37℃倒置培养过夜；
从平板上挑取DH5α单菌落，接入5mL灭菌液体LB培养基中，37℃，250rpm，振摇培养过夜；
将宿主菌以1％的转接量转接至100mL液体LB培养基中，37℃，250rpm，培养菌液至OD600＝0.4～0.6；
在无菌条件下将菌液转移至预冷的灭菌离心管中，4℃，5000rpm离心4min收集菌体；
用30mL的预冷灭菌CaCl2(0.1moL/L)重悬菌体，4℃过夜；
4℃下5000rpm离心4min，倒掉上清，收集菌体；
用9mL灭菌CaCl2(0.1moL/L)重悬菌体，加甘油至终浓度为20％；
取1.5mL离心管，按100μL/管分装，超低温保存。
5、DH5α感受态细胞转化
取分装的感受态细胞，在冰上融解；
加入2μLβ-Me，轻轻混匀，在冰上放2min；
加102μL质粒，轻轻旋转离心管混匀内容物，在冰上培养30min；
将离心管在42℃水浴中热击90s，然后快速转移到冰上，冷却静置2min，该过程不要摇动离心管；
加入灭菌LB培养液(无抗生素)500μL，混匀后在37℃下150rpm振荡培养60min，使菌体复苏；
吸取100～300μL离心管内容物，涂布在含有100ng/mL氨苄青霉素的LB固体培养基平板上，37℃过夜培养(＞16h)。
6、菌落PCR检测筛选突变质粒阳性转化子
挑选突变质粒转化子为模板，进行菌落PCR，筛选阳性转化子；PCR参数设置：94℃下加热5min后，94℃变性1min，57℃退火1min，72℃延伸1min，循环35轮，进行PCR。最后一轮循环结束后，于72℃下保温10min。用1％琼脂糖凝胶电泳检查扩增结果。引物如下：
P1：5′GCCTGCAAGAGTATGGTAAG3′和P2：5′GATAGTCCACAAGGTCCTCC3′；
电泳图如图5所示。图中各泳道分别为：
M：标准分子量(200bp)；
N：阴性对照；
泳道1～4：pMD19-T/GDH/K80A突变质粒转化子的扩增结果；
泳道5～8：pMD19-T/GDH/K116Q突变质粒转化子的扩增结果；
泳道9～13：pMD19-T/GDH/S351A突变质粒转化子的扩增结果。
7、DNA测序
将PCR扩增结果为阳性的转化子送上海桑尼生物科技有限公司测序，测序结果用VectorNTI软件进行比对分析，分析结果显示突变实验成功。单位点突变后，再以突变子为模板，利用相同的条件进行多位点突变。结果如下：
野生型GDH的碱基序列如SEQ ID NO：1所示，GDH的氨基酸序列如SEQ ID NO：2所示；
突变型GDH的碱基序列和氨基酸序列具体为：
(1)K80A，在DNA序列为238～240从AAA变为GCA(DNA的碱基序列为SEQ ID NO：3)，在氨基酸序列为第80位的赖氨酸变为丙氨酸(氨基酸序列为SEQ ID NO：4)；
(2)K116Q，在DNA序列为346～348从AAA变为CAA(DNA的碱基序列为SEQ IDNO：5)，在氨基酸序列为第116位的赖氨酸变为谷氨酰胺(氨基酸序列为SEQ ID NO：6)；
(3)S351A，在DNA序列为1051～1053从AGT变为GCT(DNA的碱基序列为SEQ IDNO：7)，在氨基酸序列为351位的丝氨酸变为丙氨酸(氨基酸序列为SEQ ID NO：8)；
(4)K80A-K116Q是指第80位的赖氨酸变为丙氨酸，同时第116位的赖氨酸变为谷氨酰胺；(DNA的碱基序列为SEQ ID NO：9，氨基酸序列为SEQ ID NO：10)；
(5)K80A-S351A是指第80位的赖氨酸变为丙氨酸，同时第351位的丝氨酸变为丙氨酸；(DNA的碱基序列为SEQ ID NO：11，氨基酸序列为SEQ ID NO：12)；
(6)K116-S351A是指第116位的赖氨酸变为谷氨酰胺，同时第351位的丝氨酸变为丙氨酸；(DNA的碱基序列为SEQ ID NO：13，氨基酸序列为SEQ ID NO：14)；
(7)K80A-K116Q-S351A是指第80位的赖氨酸变为丙氨酸，同时第116位的赖氨酸变为谷氨酰胺，第351位的丝氨酸变为丙氨酸；DNA的碱基序列为SEQ ID NO：15，氨基酸序列为SEQ ID NO：16)。
步骤五，基因表达和酶活检测；构建表达载体，将野生型基因和突变基因在大肠杆菌中表达，将得到的包涵体复性后测定GDH酶活，检测突变的有效性；具体步骤如下：
1、构建表达载体
设计带有酶切位点的引物：
F(含NheI酶切位点)CGTACGGCTAGCATGGCGGCCGATCGAAAC，
R(含EcoRI酶切位点)CGCTAGGAATTCCGATTGGCATTTCACTTT。
分别以野生型和突变型的pMD19-T/GDH为模板，用上述引物和pyrobest DNA聚合酶进行PCR扩增。用NheI和EcoRI对纯化后的PCR产物和原核表达载体pET28a分别进行双酶切。酶切9小时后对酶切产物进行纯化。在T4DNA连接酶的作用下将PCR产物和载体连接，转化DH5α，构建含目的基因GDH的原核表达载体。重组质粒经PCR和酶切鉴定后送上海桑尼生物科技有限公司测序。
2、诱导表达
将重组质粒pET28a-GDH转化表达宿主菌E.coli BL21(DE3)。挑取单菌落接种于含50μg/ml Kan的LB液体培养基，在37℃、180rpm振荡过夜培养后，按1％比例转接入新鲜 的Kan-LB，37℃摇床培养3小时后，加IPTG至终浓度为0.4mM，28℃，180rpm振荡培养7小时。
3、表达产物SDS-PAGE分析
取诱导表达菌液1mL，12000rpm离心1min，将细胞用50μL水重悬后，加入40μL的2×SDS上样缓冲液，混匀，再加入10μL的DTT，99℃干浴5min，取15μL进行SDS-PAGE电泳。电泳条件如下：
10mL 12％的分离胶：H2O 3.2mL，30％丙烯酰胺混合液4.0mL，1.5mol/lTris(pH8.8)2.6mL，10％SDS 0.1mL，10％过硫酸铵0.1mL，TEMED 4μL。
2ml 5％的积层胶：H2O 1.4mL，30％丙烯酰胺混合液0.33mL，1.0mol/lTris(pH6.8)0.25mL，10％SDS 0.02mL，10％过硫酸铵0.02mL，TEMED 2μL。
5×Tris甘氨酸电泳缓冲液：Tris 15.1g，甘氨酸94.0g，10％SDS 50mL，定容至1000mL。
脱色液：125mL乙醇，40mL冰醋酸，加水至500mL。
电压设定为80V，待染料前沿到达分离胶位置后，电压加至120V。染料前沿迁移至凝胶的底部时，停止电泳。将胶放入考马斯亮蓝快速染色液中，轻轻震荡30min；弃去染色液，将凝胶在水中漂洗数次，放入脱色液中，置于摇床轻轻震荡脱色；将脱色完全的凝胶在成像系统内拍照。
4、GDH纯化
超声破菌：诱导培养后的细菌在4℃以10000rpm离心5min，收集菌体。用0.01M磷酸盐缓冲液(PBS)洗菌体一遍，再用PBS重悬一次，置于冰浴中，超声破菌(350W，持续5S，间隔10S，共20次)。10000rpm离心10min后，分别将上清和沉淀进行12％SDS-PAGE分析。分析结果显示，超声破菌后，重组蛋白主要以包涵体的形式存在于沉淀中，如图6所示，图中各泳道分别为：
泳道1：诱导表达E.coli BL21(DE3)/pET28a；
泳道2、3：分别为诱导表达E.coli BL21(DE3)/pET28a/WT的上清和沉淀；
泳道4、5：分别为诱导表达E.coli BL21(DE3)/pET28a/K80A的上清和沉淀；
泳道6、7：分别为诱导表达E.coli BL21(DE3)/pET28a/K116Q的上清和沉淀；
泳道8、9：分别为诱导表达E.coli BL21(DE3)/pET28a/S351A的上清和沉淀；
包涵体的变性、纯化和复性
变性条件纯化所需缓冲液：
变性裂解/结合缓冲液B：8M尿素；0.1M NaH2P04；0.01M Tris-CI，pH8.0；
变性漂洗缓冲液C：8M尿素；0.1M NaH2P04；0.01M Tris-CI pH6.3；
变性洗脱缓冲液：
缓冲液D：8M尿素；0.1M NaH2P04；0.01M Tris-CI pH5.9；
缓冲液E：8M尿素；0.1M NaH2P04；0.01M Tris-CI pH4.5；
注意：缓冲液B、C、D、E应在使用前调至适宜pH值，以避免尿素解离。
(1)取100mL经适宜浓度IPTG诱导6h后的细菌培养物，8000×g离心5min，收集菌体，以PBS(pH＝7.2)洗涤菌体1次；
(2)将菌体重悬于1/10体积的PBS中，在冰上超声破碎至溶液不再粘稠；
(3)5，000×g离心15min；
(4)移去上清，将100mL培养物的沉淀重悬于10mL 1×结合缓冲液(含6M尿素)；
(5)冰浴孵育1小时，彻底溶解包涵体，离心去除不溶成分；
(6)将1mL 50％Ni-NTA His·Bind树脂悬液加到4mL细胞裂解液中，轻柔混匀(如旋转混合器200rpm)，室温结合15～60min。
(7)将裂解液与Ni-NTA His·Bind树脂的混合物小心加入下端封闭的空色谱柱中；
(8)除去柱下端封闭盖子，收集流出液，用于SDS-PAGE分析；
(9)以4ml缓冲液C漂洗杂蛋白2次。保存漂洗组分用于SDS-PAGE分析；
(10)以0.5mL缓冲液D洗脱目的蛋白4次，再以0.5mL缓冲液E洗4次，收集组分，并用SDS-PAGE分析。蛋白单体通常用缓冲液D即可洗脱，而多聚体、聚合物和含两个His标签的蛋白通常需由缓冲液E洗脱；
(11)在收集的蛋白洗脱液中，加105μL 20％的PEG4000、210μL 50mmol/L的氧化型谷胱甘肽和210μL 100mmol/L的还原型谷胱甘肽，室温静置约2h。转至处理好的透析袋中，用TE(pH＝7.2)透析2～3天，每天换液5～7次。纯化后蛋白的电泳图如图7所示，图中泳道M为标准蛋白，泳道1为野生型GDH，泳道2为GDH/K80A，泳道3为GDH/K116Q，泳道4为GDH/S351A，泳道5为未纯化的野生型GDH。
5、蛋白浓度及GDH酶活性测定
测定GDH氧化脱氨基作用的酶活性测定液为：0.5mmol/L NADP+，15mmol/L谷氨酸，25mmol/L Tris-Hcl，pH8.0。取酶液与酶活性测定液各1.5mL测定GDH催化辅酶NADP+还 原所引起的光吸收值在340nm波长处的变化。每分钟催化1μmol NADP+还原所需的酶量定义为一个酶活力单位(U)。酶活性＝ΔOD340×0.2×2/0.0486，蛋白浓度＝1.45×OD280-0.74×OD260，单位酶活性＝酶活性/蛋白浓度。酶活性测定结果如图8所示。
从图8可以看出，野生型GDH NADP+依赖酶活性为9.42U/mg protein，单点突变的K80A、K116Q和S351A酶活性分别为4.35U/mg protein，4.24U/mg protein和3.60U/mg protein，酶活性分别降至野生型的46.2％、45.0％和38.2％；K80A/K116Q、K80A/S351A、K116Q/S351A和K80A/K116Q/S351A的酶活性分别为1.77U/mg protein、2.03U/mg protein、1.62U/mgprotein和1.76U/mg protein，即双点突变和三点突变可使酶活性降低至18.8％、21.5％、17.2％和18.7％
由此，可以证明K80、K116、S351位于谷氨酸脱氢酶的活性中心。也证明本发明采用的突变方案是有效的，突变后的蛋白有稳定的结构，且表现出部分酶活性。
序列表
<110>上海交通大学
<120>纳豆芽孢杆菌谷氨酸脱氢酶基因的突变基因及编码蛋白
<130>01001
<160>16
<170>PatentIn version 3.3
<210>1
<211>1275
<212>DNA
<213>纳豆芽孢杆菌(Bacillus Subtillis Natto)
<220>
<221>CDS
<222>(1)..(1275)
<400>1
atg gcg gcc gat cga aac acc ggt cat aca gaa ggg gac aaa ctt gat 48
Met Ala Ala Asp Arg Asn Thr Gly His Thr Glu Gly Asp Lys Leu Asp
1 5 10 15
gta tta aaa tcg acc caa acc gta ata cat aag gct ctg gaa aaa ttg 96
Val Leu Lys Ser Thr Gln Thr Val Ile His Lys Ala Leu Glu Lys Leu
20 25 30
gga tat ccc gaa gag gta tac gaa ttg tta aaa gag ccg atg aga tta 144
Gly Tyr Pro Glu Glu Val Tyr Glu Leu Leu Lys Glu Pro Met Arg Leu
35 40 45
tta acg gta aaa ata cct gtt cgt atg gac gac ggt tca gtt aag att 192
Leu Thr Val Lys Ile Pro Val Arg Met Asp Asp Gly Ser Val Lys Ile
50 55 60
ttc aca gga tat cgt gcg cgg cac aat gac tct gtc ggt cca acg aaa 240
Phe Thr Gly Tyr Arg Ala Arg His Asn Asp Ser Val Gly Pro Thr Lys
65 70 75 80
ggc ggg ata cgt ttt cac ccg aat gta aca gaa aaa gag gtg aag gcg 288
Gly Gly Ile Arg Phe His Pro Asn Val Thr Glu Lys Glu Val Lys Ala
85 90 95
ctt tca att tgg atg agt tta aaa tgc ggc ata att gat ctt cca tat 336
Leu Ser Ile Trp Met Ser Leu Lys Cys Gly Ile Ile Asp Leu Pro Tyr
100 105 110
ggc ggt ggt aaa ggc gga att gtt tgt gat cca agg gat atg tcg ttt 384
Gly Gly Gly Lys Gly Gly Ile Val Cys Asp Pro Arg Asp Met Ser Phe
115 120 125
aga gag ctg gag cgt ctg agc aga ggg tat gtc aga gcg atc agc caa 432
Arg Glu Leu Glu Arg Leu Ser Arg Gly Tyr Val Arg Ala Ile Ser Gln
130 135 140
att gtc ggc ccg aca aaa gac gtg ccg gca ccg gat gta ttt aca aac 480
Ile Val Gly Pro Thr Lys Asp Val Pro Ala Pro Asp Val Phe Thr Asn
145 150 155 160
tca caa atc atg gct tgg atg atg gat gag tat tca aga att gat gaa 528
Ser Gln Ile Met Ala Trp Met Met Asp Glu Tyr Ser Arg Ile Asp Glu
165 170 175
ttt aat tcg cct gga ttt att aca ggc aaa ccg ctt gtg ctt ggc gga 576
Phe Asn Ser Pro Gly Phe Ile Thr Gly Lys Pro Leu Val Leu Gly Gly
180 185 190
tct cac ggg aga gaa tct gcg aca gca aat ggt gtt acc atc tgt att 624
Ser His Gly Arg Glu Ser Ala Thr Ala Asn Gly Val Thr Ile Cys Ile
195 200 205
aaa gaa gcg gct aag aag aga ggc atc gat att aaa ggt gcg cgt gtc 672
Lys Glu Ala Ala Lys Lys Arg Gly Ile Asp Ile Lys Gly Ala Arg Val
210 215 220
gtt gtc caa ggc ttc gga aac gcg gga agc tat ttg gca aaa ttt atg 720
Val Val Gln Gly Phe Gly Asn Ala Gly Ser Tyr Leu Ala Lys Phe Met
225 230 235 240
cat gat gcg ggg gca aaa gtt gtc ggc atc tca gat gcg tat ggc gga 768
His Asp Ala Gly Ala Lys Val Val Gly Ile Ser Asp Ala Tyr Gly Gly
245 250 255
ctt tat gat ccg gaa ggc ctt aat atc gat tgt tta ctc gac cga cgc 816
Leu Tyr Asp Pro Glu Gly Leu AsnIle Asp Cys Leu Leu Asp Arg Arg
260 265 270
gac agc ttc ggt acc gta aca aag ctt ttc aac gat acc att acc aac 864
Asp Ser Phe Gly Thr Val Thr Lys Leu Phe Asn Asp Thr Ile Thr Asn
275 280 285
caa gag ctg ctg gag ctg gat tgt gat att ctc gtt cct gct gcg att 912
Gln Glu Leu Leu Glu Leu Asp Cys Asp Ile Leu Val Pro Ala Ala Ile
290 295 300
gaa aat caa att aca gaa gaa aat gcc cat aat att cgg gct aaa att 960
Glu Asn Gln Ile Thr Glu Glu Asn Ala His Asn Ile Arg Ala Lys Ile
305 310 315 320
gtc gtt gaa gca gcg aac gga cca aca acg ctt gaa gga aca aag att 1008
Val Val Glu Ala Ala Asn Gly Pro Thr Thr Leu Glu Gly Thr Lys Ile
325 330 335
ctt tca gac cgg gac att ctg ctt gta cca gac gtg ctg gca agt gcc 1056
Leu Ser Asp Arg Asp Ile Leu Leu Val Pro Asp Val Leu Ala Ser Ala
340 345 350
ggt ggc gta aca gtt tct tat ttt gaa tgg gtt cag aat aac caa ggc 1104
Gly Gly Val Thr Val Ser Tyr Phe Glu Trp Val Gln Asn Asn Gln Gly
355 360 365
ttc tac tgg agt gaa gaa gag gta gaa gaa aaa tta gaa aaa atg atg 1152
Phe Tyr Trp Ser Glu Glu Glu Val Glu Glu Lys Leu Glu Lys Met Met
370 375 380
gtc aaa tca ttt aac aat att tat gaa atg gct aac aac cga aga att 1200
Val Lys Ser Phe Asn Asn Ile Tyr Glu Met Ala Asn Asn Arg Arg Ile
385 390 395 400
gac atg agg ctc gct gca tat atg gtc ggc gtt cgc aaa atg gct gaa 1248
Asp Met Arg Leu Ala Ala Tyr Met Val Gly Val Arg Lys Met Ala Glu
405 410 415
gct tcg cgt ttt aga ggc tgg ata taa 1275
Ala Ser Arg Phe Arg Gly Trp Ile
420
<210>2
<211>424
<212>PRT
<213>纳豆芽孢杆菌(Bacillus Subtillis Natto)
<400>2
Met Ala Ala Asp Arg Asn Thr Gly His Thr Glu Gly Asp Lys Leu Asp
1 5 10 15
Val Leu Lys Ser Thr Gln Thr Val Ile His Lys Ala Leu Glu Lys Leu
20 25 30
Gly Tyr Pro Glu Glu Val Tyr Glu Leu Leu Lys Glu Pro Met Arg Leu
35 40 45
Leu Thr Val Lys Ile Pro Val Arg Met Asp Asp Gly Ser Val Lys Ile
50 55 60
Phe Thr Gly Tyr Arg Ala Arg His Asn Asp Ser Val Gly Pro Thr Lys
65 70 75 80
Gly Gly Ile Arg Phe His Pro Asn Val Thr Glu Lys Glu Val Lys Ala
85 90 95
Leu Ser Ile Trp Met Ser Leu Lys Cys Gly Ile Ile Asp Leu Pro Tyr
100 105 110
Gly Gly Gly Lys Gly Gly Ile Val Cys Asp Pro Arg Asp Met Ser Phe
115 120 125
Arg Glu Leu Glu Arg Leu Ser Arg Gly Tyr Val Arg Ala Ile Ser Gln
130 135 140
Ile Val Gly Pro Thr Lys Asp Val Pro Ala Pro Asp Val Phe Thr Asn
145 150 155 160
Ser Gln Ile Met Ala Trp Met Met Asp Glu Tyr Ser Arg Ile Asp Glu
165 170 175
Phe Asn Ser Pro Gly Phe Ile Thr Gly Lys Pro Leu Val Leu Gly Gly
180 185 190
Ser His Gly Arg Glu Ser Ala Thr Ala Asn Gly Val Thr Ile Cys Ile
195 200 205
Lys Glu Ala Ala Lys Lys Arg Gly Ile Asp Ile Lys Gly Ala Arg Val
210 215 220
Val Val Gln Gly Phe Gly Asn Ala Gly Ser Tyr Leu Ala Lys Phe Met
225 230 235 240
His Asp Ala Gly Ala Lys Val Val Gly Ile Ser Asp Ala Tyr Gly Gly
245 250 255
Leu Tyr Asp Pro Glu Gly Leu Asn Ile Asp Cys Leu Leu Asp Arg Arg
260 265 270
Asp Ser Phe Gly Thr Val Thr Lys Leu Phe Asn Asp Thr Ile Thr Asn
275 280 285
Gln Glu Leu Leu Glu Leu Asp Cys Asp Ile Leu Val Pro Ala Ala Ile
290 295 300
Glu Asn Gln Ile Thr Glu Glu Asn Ala His Asn Ile Arg Ala Lys Ile
305 310 315 320
Val Val Glu Ala Ala Asn Gly Pro Thr Thr Leu Glu Gly Thr Lys Ile
325 330 335
Leu Ser Asp Arg Asp Ile Leu Leu Val Pro Asp Val Leu Ala Ser Ala
340 345 350
Gly Gly Val Thr Val Ser Tyr Phe Glu Trp Val Gln Asn Asn Gln Gly
355 360 365
Phe Tyr Trp Ser Glu Glu Glu Val Glu Glu Lys Leu Glu Lys Met Met
370 375 380
Val Lys Ser Phe Asn Asn Ile Tyr Glu Met Ala Asn Asn Arg Arg Ile
385 390 395 400
Asp Met Arg Leu Ala Ala Tyr Met Val Gly Val Arg Lys Met Ala Glu
405 410 415
Ala Ser Arg Phe Arg Gly Trp Ile
420
<210>3
<211>1275
<212>DNA
<213>纳豆芽孢杆菌(Bacillus Subtillis Natto)
<220>
<221>CDS
<222>(1)..(1275)
<400>3
atg gcg gcc gat cga aac acc ggt cat aca gaa ggg gac aaa ctt gat 48
Met Ala Ala Asp Arg Asn Thr Gly His Thr Glu Gly Asp Lys Leu Asp
1 5 10 15
gta tta aaa tcg acc caa acc gta ata cat aag gct ctg gaa aaa ttg 96
Val Leu Lys Ser Thr Gln Thr Val Ile His Lys Ala Leu Glu Lys Leu
20 25 30
gga tat ccc gaa gag gta tac gaa ttg tta aaa gag ccg atg aga tta 144
Gly Tyr Pro Glu Glu Val Tyr Glu Leu Leu Lys Glu Pro Met Arg Leu
35 40 45
tta acg gta aaa ata cct gtt cgt atg gac gac ggt tca gtt aag att 192
Leu Thr Val Lys Ile Pro Val Arg Met Asp Asp Gly Ser Val Lys Ile
50 55 60
ttc aca gga tat cgt gcg cgg cac aat gac tct gtc ggt cca acg gca 240
Phe Thr Gly Tyr Arg Ala Arg His Asn Asp Ser Val Gly Pro Thr Ala
65 70 75 80
ggc ggg ata cgt ttt cac ccg aat gta aca gaa aaa gag gtg aag gcg 288
Gly Gly Ile Arg Phe His Pro Asn Val Thr Glu Lys Glu Val Lys Ala
85 90 95
ctt tca att tgg atg agt tta aaa tgc ggc ata att gat ctt cca tat 336
Leu Ser Ile Trp Met Ser Leu Lys Cys Gly Ile Ile Asp Leu Pro Tyr
100 105 110
ggc ggt ggt aaa ggc gga att gtt tgt gat cca agg gat atg tcg ttt 384
Gly Gly Gly Lys Gly Gly Ile Val Cys Asp Pro Arg Asp Met Ser Phe
115 120 125
aga gag ctg gag cgt ctg agc aga ggg tat gtc aga gcg atc agc caa 432
Arg Glu Leu Glu Arg Leu Ser Arg Gly Tyr Val Arg Ala Ile Ser Gln
130 135 140
att gtc ggc ccg aca aaa gac gtg ccg gca ccg gat gta ttt aca aac 480
Ile Val Gly Pro Thr Lys Asp Val Pro Ala Pro Asp Val Phe Thr Asn
145 150 155 160
tca caa atc atg gct tgg atg atg gat gag tat tca aga att gat gaa 528
Ser Gln Ile Met Ala Trp Met Met Asp Glu Tyr Ser Arg Ile Asp Glu
165 170 175
ttt aat tcg cct gga ttt att aca ggc aaa ccg ctt gtg ctt ggc gga 576
Phe Asn Ser Pro Gly Phe Ile Thr Gly Lys Pro Leu Val Leu Gly Gly
180 185 190
tct cac ggg aga gaa tct gcg aca gca aat ggt gtt acc atc tgt att 624
Ser His Gly Arg Glu Ser Ala Thr Ala Asn Gly Val Thr Ile Cys Ile
195 200 205
aaa gaa gcg gct aag aag aga ggc atc gat att aaa ggt gcg cgt gtc 672
Lys Glu Ala Ala Lys Lys Arg Gly Ile Asp Ile Lys Gly Ala Arg Val
210 215 220
gtt gtc caa ggc ttc gga aac gcg gga agc tat ttg gca aaa ttt atg 720
Val Val Gln Gly Phe Gly Asn Ala Gly Ser Tyr Leu Ala Lys Phe Met
225 230 235 240
cat gat gcg ggg gca aaa gtt gtc ggc atc tca gat gcg tat ggc gga 768
His Asp Ala Gly Ala Lys Val Val Gly Ile Ser Asp Ala Tyr Gly Gly
245 250 255
ctt tat gat ccg gaa ggc ctt aat atc gat tgt tta ctc gac cga cgc 816
Leu Tyr Asp Pro Glu Gly Leu Asn Ile Asp Cys Leu Leu Asp Arg Arg
260 265 270
gac agc ttc ggt acc gta aca aag ctt ttc aac gat acc att acc aac 864
Asp Ser Phe Gly Thr Val Thr Lys Leu Phe Asn Asp Thr Ile Thr Asn
275 280 285
caa gag ctg ctg gag ctg gat tgt gat att ctc gtt cct gct gcg att 912
Gln Glu Leu Leu Glu Leu Asp Cys Asp Ile Leu Val Pro Ala Ala Ile
290 295 300
gaa aat caa att aca gaa gaa aat gcc cat aat att cgg gct aaa att 960
Glu Asn Gln Ile Thr Glu Glu Asn Ala His Asn Ile Arg Ala Lys Ile
305 310 315 320
gtc gtt gaa gca gcg aac gga cca aca acg ctt gaa gga aca aag att 1008
Val Val Glu Ala Ala Asn Gly Pro Thr Thr Leu Glu Gly Thr Lys Ile
325 330 335
ctt tca gac cgg gac att ctg ctt gta cca gac gtg ctg gca agt gcc 1056
Leu Ser Asp Arg Asp Ile Leu Leu Val Pro Asp Val Leu Ala Ser Ala
340 345 350
ggt ggc gta aca gtt tct tat ttt gaa tgg gtt cag aat aac caa ggc 1104
Gly Gly Val Thr Val Ser Tyr Phe Glu Trp Val Gln Asn Asn Gln Gly
355 360 365
ttc tac tgg agt gaa gaa gag gta gaa gaa aaa tta gaa aaa atg atg 1152
Phe Tyr Trp Ser Glu Glu Glu Val Glu Glu Lys Leu Glu Lys Met Met
370 375 380
gtc aaa tca ttt aac aat att tat gaa atg gct aac aac cga aga att 1200
Val Lys Ser Phe Asn Asn Ile Tyr Glu Met Ala Asn Asn Arg Arg Ile
385 390 395 400
gac atg agg ctc gct gca tat atg gtc ggc gtt cgc aaa atg gct gaa 1248
Asp Met Arg Leu Ala Ala Tyr Met Val Gly Val Arg Lys Met Ala Glu
405 410 415
gct tcg cgt ttt aga ggc tgg ata taa 1275
Ala Ser Arg Phe Arg Gly Trp Ile
420
<210>4
<211>424
<212>PRT
<213>纳豆芽孢杆菌(Bacillus Subtillis Natto)
<400>4
Met Ala Ala Asp Arg Asn Thr Gly His Thr Glu Gly Asp Lys Leu Asp
1 5 10 15
Val Leu Lys Ser Thr Gln Thr Val Ile His Lys Ala Leu Glu Lys Leu
20 25 30
Gly Tyr Pro Glu Glu Val Tyr Glu Leu Leu Lys Glu Pro Met Arg Leu
35 40 45
Leu Thr Val Lys Ile Pro Val Arg Met Asp Asp Gly Ser Val Lys Ile
50 55 60
Phe Thr Gly Tyr Arg Ala Arg His Asn Asp Ser Val Gly Pro Thr Ala
65 70 75 80
Gly Gly Ile Arg Phe His Pro Asn Val Thr Glu Lys Glu Val Lys Ala
85 90 95
Leu Ser Ile Trp Met Ser Leu Lys Cys Gly Ile Ile Asp Leu Pro Tyr
100 105 110
Gly Gly Gly Lys Gly Gly Ile Val Cys Asp Pro Arg Asp Met Ser Phe
115 120 125
Arg Glu Leu Glu Arg Leu Ser Arg Gly Tyr Val Arg Ala Ile Ser Gln
130 135 140
Ile Val Gly Pro Thr Lys Asp Val Pro Ala Pro Asp Val Phe Thr Asn
145 150 155 160
Ser Gln Ile Met Ala Trp Met Met Asp Glu Tyr Ser Arg Ile Asp Glu
165 170 175
Phe Asn Ser Pro Gly Phe Ile Thr Gly Lys Pro Leu Val Leu Gly Gly
180 185 190
Ser His Gly Arg Glu Ser Ala Thr Ala Asn Gly Val Thr Ile Cys Ile
195 200 205
Lys Glu Ala Ala Lys Lys Arg Gly Ile Asp Ile Lys Gly Ala Arg Val
210 215 220
Val Val Gln Gly Phe Gly Asn Ala Gly Ser Tyr Leu Ala Lys Phe Met
225 230 235 240
His Asp Ala Gly Ala Lys Val Val Gly Ile Ser Asp Ala Tyr Gly Gly
245 250 255
Leu Tyr Asp Pro Glu Gly Leu AsnIle Asp Cys Leu Leu Asp Arg Arg
260 265 270
Asp Ser Phe Gly Thr Val Thr Lys Leu Phe Asn Asp Thr Ile Thr Asn
275 280 285
Gln Glu Leu Leu Glu Leu Asp Cys Asp Ile Leu Val Pro Ala Ala Ile
290 295 300
Glu Asn Gln Ile Thr Glu Glu Asn Ala His Asn Ile Arg Ala Lys Ile
305 310 315 320
Val Val Glu Ala Ala Asn Gly Pro Thr Thr Leu Glu Gly Thr Lys Ile
325 330 335
Leu Ser Asp Arg Asp Ile Leu Leu Val Pro Asp Val Leu Ala Ser Ala
340 345 350
Gly Gly Val Thr Val Ser Tyr Phe Glu Trp Val Gln Asn Asn Gln Gly
355 360 365
Phe Tyr Trp Ser Glu Glu Glu Val Glu Glu Lys Leu Glu Lys Met Met
370 375 380
Val Lys Ser Phe Asn Asn Ile Tyr Glu Met Ala Asn Asn Arg Arg Ile
385 390 395 400
Asp Met Arg Leu Ala Ala Tyr Met Val Gly Val Arg Lys Met Ala Glu
405 410 415
Ala Ser Arg Phe Arg Gly Trp Ile
420
<210>5
<211>1275
<212>DNA
<213>纳豆芽孢杆菌(Bacillus Subtillis Natto)
<220>
<221>CDS
<222>(1)..(1275)
<400>5
atg gcg gcc gat cga aac acc ggt cat aca gaa ggg gac aaa ctt gat 48
Met Ala Ala Asp Arg Asn Thr Gly His Thr Glu Gly Asp Lys Leu Asp
1 5 10 15
gta tta aaa tcg acc caa acc gta ata cat aag gct ctg gaa aaa ttg 96
Val Leu Lys Ser Thr Gln Thr Val Ile His Lys Ala Leu Glu Lys Leu
20 25 30
gga tat ccc gaa gag gta tac gaa ttg tta aaa gag ccg atg aga tta 144
Gly Tyr Pro Glu Glu Val Tyr Glu Leu Leu Lys Glu Pro Met Arg Leu
35 40 45
tta acg gta aaa ata cct gtt cgt atg gac gac ggt tca gtt aag att 192
Leu Thr Val Lys Ile Pro Val Arg Met Asp Asp Gly Ser Val Lys Ile
50 55 60
ttc aca gga tat cgt gcg cgg cac aat gac tct gtc ggt cca acg aaa 240
Phe Thr Gly Tyr Arg Ala Arg His Asn Asp Ser Val Gly Pro Thr Lys
65 70 75 80
ggc ggg ata cgt ttt cac ccg aat gta aca gaa aaa gag gtg aag gcg 288
Gly Gly Ile Arg Phe His Pro Asn Val Thr Glu Lys Glu Val Lys Ala
85 90 95
ctt tca att tgg atg agt tta aaa tgc ggc ata att gat ctt cca tat 336
Leu Ser Ile Trp Met Ser Leu Lys Cys Gly Ile Ile Asp Leu Pro Tyr
100 105 110
ggc ggt ggt caa ggc gga att gtt tgt gat cca agg gat atg tcg ttt 384
Gly Gly Gly Gln Gly Gly Ile Val Cys Asp Pro Arg Asp Met Ser Phe
115 120 125
aga gag ctg gag cgt ctg agc aga ggg tat gtc aga gcg atc agc caa 432
Arg Glu Leu Glu Arg Leu Ser Arg Gly Tyr Val Arg Ala Ile Ser Gln
130 135 140
att gtc ggc ccg aca aaa gac gtg ccg gca ccg gat gta ttt aca aac 480
Ile Val Gly Pro Thr Lys Asp Val Pro Ala Pro Asp Val Phe Thr Asn
145 150 155 160
tca caa atc atg gct tgg atg atg gat gag tat tca aga att gat gaa 528
Ser Gln Ile Met Ala Trp Met Met Asp Glu Tyr Ser Arg Ile Asp Glu
165 170 175
ttt aat tcg cct gga ttt att aca ggc aaa ccg ctt gtg ctt ggc gga 576
Phe Asn Ser Pro Gly Phe Ile Thr Gly Lys Pro Leu Val Leu Gly Gly
180 185 190
tct cac ggg aga gaa tct gcg aca gca aat ggt gtt acc atc tgt att 624
Ser His Gly Arg Glu Ser Ala Thr Ala Asn Gly Val Thr Ile Cys Ile
195 200 205
aaa gaa gcg gct aag aag aga ggc atc gat att aaa ggt gcg cgt gtc 672
Lys Glu Ala Ala Lys Lys Arg Gly Ile Asp Ile Lys Gly Ala Arg Val
210 215 220
gtt gtc caa ggc ttc gga aac gcg gga agc tat ttg gca aaa ttt atg 720
Val Val Gln Gly Phe Gly Asn Ala Gly Ser Tyr Leu Ala Lys Phe Met
225 230 235 240
cat gat gcg ggg gca aaa gtt gtc ggc atc tca gat gcg tat ggc gga 768
His Asp Ala Gly Ala Lys Val Val Gly Ile Ser Asp Ala Tyr Gly Gly
245 250 255
ctt tat gat ccg gaa ggc ctt aat atc gat tgt tta ctc gac cga cgc 816
Leu Tyr Asp Pro Glu Gly Leu Asn Ile Asp Cys Leu Leu Asp Arg Arg
260 265 270
gac agc ttc ggt acc gta aca aag ctt ttc aac gat acc att acc aac 864
Asp Ser Phe Gly Thr Val Thr Lys Leu Phe Asn Asp Thr Ile Thr Asn
275 280 285
caa gag ctg ctg gag ctg gat tgt gat att ctc gtt cct gct gcg att 912
Gln Glu Leu Leu Glu Leu Asp Cys Asp Ile Leu Val Pro Ala Ala Ile
290 295 300
gaa aat caa att aca gaa gaa aat gcc cat aat att cgg gct aaa att 960
Glu Asn Gln Ile Thr Glu Glu Asn Ala His Asn Ile Arg Ala Lys Ile
305 310 315 320
gtc gtt gaa gca gcg aac gga cca aca acg ctt gaa gga aca aag att 1008
Val Val Glu Ala Ala Asn Gly Pro Thr Thr Leu Glu Gly Thr Lys Ile
325 330 335
ctt tca gac cgg gac att ctg ctt gta cca gac gtg ctg gca agt gcc 1056
Leu Ser Asp Arg Asp Ile Leu Leu Val Pro Asp Val Leu Ala Ser Ala
340 345 350
ggt ggc gta aca gtt tct tat ttt gaa tgg gtt cag aat aac caa ggc 1104
Gly Gly Val Thr Val Ser Tyr Phe Glu Trp Val Gln Asn Asn Gln Gly
355 360 365
ttc tac tgg agt gaa gaa gag gta gaa gaa aaa tta gaa aaa atg atg 1152
Phe Tyr Trp Ser Glu Glu Glu Val Glu Glu Lys Leu Glu Lys Met Met
370 375 380
gtc aaa tca ttt aac aat att tat gaa atg gct aac aac cga aga att 1200
Val Lys Ser Phe Asn Asn Ile Tyr Glu Met Ala Asn Asn Arg Arg Ile
385 390 395 400
gac atg agg ctc gct gca tat atg gtc ggc gtt cgc aaa atg gct gaa 1248
Asp Met Arg Leu Ala Ala Tyr Met Val Gly Val Arg Lys Met Ala Glu
405 410 415
gct tcg cgt ttt aga ggc tgg ata taa 1275
Ala Ser Arg Phe Arg Gly Trp Ile
420
<210>6
<211>424
<212>PRT
<213>纳豆芽孢杆菌(Bacillus Subtillis Natto)
<400>6
Met Ala Ala Asp Arg Asn Thr Gly His Thr Glu Gly Asp Lys Leu Asp
1 5 10 15
Val Leu Lys Ser Thr Gln Thr Val Ile His Lys Ala Leu Glu Lys Leu
20 25 30
Gly Tyr Pro Glu Glu Val Tyr Glu Leu Leu Lys Glu Pro Met Arg Leu
35 40 45
Leu Thr Val Lys Ile Pro Val Arg Met Asp Asp Gly Ser Val Lys Ile
50 55 60
Phe Thr Gly Tyr Arg Ala Arg His Asn Asp Ser Val Gly Pro Thr Lys
65 70 75 80
Gly Gly Ile Arg Phe His Pro Asn Val Thr Glu Lys Glu Val Lys Ala
85 90 95
Leu Ser Ile Trp Met Ser Leu Lys Cys Gly Ile Ile Asp Leu Pro Tyr
100 105 110
Gly Gly Gly Gln Gly Gly Ile Val Cys Asp Pro Arg Asp Met Ser Phe
115 120 125
Arg Glu Leu Glu Arg Leu Ser Arg Gly Tyr Val Arg Ala Ile Ser Gln
130 135 140
Ile Val Gly Pro Thr Lys Asp Val Pro Ala Pro Asp Val Phe Thr Asn
145 150 155 160
Ser Gln Ile Met Ala Trp Met Met Asp Glu Tyr Ser Arg Ile Asp Glu
165 170 175
Phe Asn Ser Pro Gly PheIle Thr Gly Lys Pro Leu Val Leu Gly Gly
180 185 190
Ser His Gly Arg Glu Ser Ala Thr Ala Asn Gly Val Thr Ile Cys Ile
195 200 205
Lys Glu Ala Ala Lys Lys Arg Gly Ile Asp Ile Lys Gly Ala Arg Val
210 215 220
Val Val Gln Gly Phe Gly Asn Ala Gly Ser Tyr Leu Ala Lys Phe Met
225 230 235 240
His Asp Ala Gly Ala Lys Val Val Gly Ile Ser Asp Ala Tyr Gly Gly
245 250 255
Leu Tyr Asp Pro Glu Gly Leu Asn Ile Asp Cys Leu Leu Asp Arg Arg
260 265 270
Asp Ser Phe Gly Thr Val Thr Lys Leu Phe Asn Asp Thr Ile Thr Asn
275 280 285
Gln Glu Leu Leu Glu Leu Asp Cys Asp Ile Leu Val Pro Ala Ala Ile
290 295 300
Glu Asn Gln Ile Thr Glu Glu Asn Ala His Asn Ile Arg Ala Lys Ile
305 310 315 320
Val Val Glu Ala Ala Asn Gly Pro Thr Thr Leu Glu Gly Thr Lys Ile
325 330 335
Leu Ser Asp Arg Asp Ile Leu Leu Val Pro Asp Val Leu Ala Ser Ala
340 345 350
Gly Gly Val Thr Val Ser Tyr Phe Glu Trp Val Gln Asn Asn Gln Gly
355 360 365
Phe Tyr Trp Ser Glu Glu Glu Val Glu Glu Lys Leu Glu Lys Met Met
370 375 380
Val Lys Ser Phe Asn Asn Ile Tyr Glu Met Ala Asn Asn Arg Arg Ile
385 390 395 400
Asp Met Arg Leu Ala Ala Tyr Met Val Gly Val Arg Lys Met Ala Glu
405 410 415
Ala Ser Arg Phe Arg Gly Trp Ile
420
<210>7
<211>1275
<212>DNA
<213>纳豆芽孢杆菌(Bacillus Subtillis Natto)
<220>
<221>CDS
<222>(1)..(1275)
<400>7
atg gcg gcc gat cga aac acc ggt cat aca gaa ggg gac aaa ctt gat 48
Met Ala Ala Asp Arg Asn Thr Gly His Thr Glu Gly Asp Lys Leu Asp
1 5 10 15
gta tta aaa tcg acc caa acc gta ata cat aag gct ctg gaa aaa ttg 96
Val Leu Lys Ser Thr Gln Thr Val Ile His Lys Ala Leu Glu Lys Leu
20 25 30
gga tat ccc gaa gag gta tac gaa ttg tta aaa gag ccg atg aga tta 144
Gly Tyr Pro Glu Glu Val Tyr Glu Leu Leu Lys Glu Pro Met Arg Leu
35 40 45
tta acg gta aaa ata cct gtt cgt atg gac gac ggt tca gtt aag att 192
Leu Thr Val Lys Ile Pro Val Arg Met Asp Asp Gly Ser Val Lys Ile
50 55 60
ttc aca gga tat cgt gcg cgg cac aat gac tct gtc ggt cca acg aaa 240
Phe Thr Gly Tyr Arg Ala Arg His Asn Asp Ser Val Gly Pro Thr Lys
65 70 75 80
ggc ggg ata cgt ttt cac ccg aat gta aca gaa aaa gag gtg aag gcg 288
Gly Gly Ile Arg Phe His Pro Asn Val Thr Glu Lys Glu Val Lys Ala
85 90 95
ctt tca att tgg atg agt tta aaa tgc ggc ata att gat ctt cca tat 336
Leu Ser Ile Trp Met Ser Leu Lys Cys Gly Ile Ile Asp Leu Pro Tyr
100 105 110
ggc ggt ggt aaa ggc gga att gtt tgt gat cca agg gat atg tcg ttt 384
Gly Gly Gly Lys Gly Gly Ile Val Cys Asp Pro Arg Asp Met Ser Phe
115 120 125
aga gag ctg gag cgt ctg agc aga ggg tat gtc aga gcg atc agc caa 432
Arg Glu Leu Glu Arg Leu Ser Arg Gly Tyr Val Arg Ala Ile Ser Gln
130 135 140
att gtc ggc ccg aca aaa gac gtg ccg gca ccg gat gta ttt aca aac 480
Ile Val Gly Pro Thr Lys Asp Val Pro Ala Pro Asp Val Phe Thr Asn
145 150 155 160
tca caa atc atg gct tgg atg atg gat gag tat tca aga att gat gaa 528
Ser Gln Ile Met Ala Trp Met Met Asp Glu Tyr Ser Arg Ile Asp Glu
165 170 175
ttt aat tcg cct gga ttt att aca ggc aaa ccg ctt gtg ctt ggc gga 576
Phe Asn Ser Pro Gly Phe Ile Thr Gly Lys Pro Leu Val Leu Gly Gly
180 185 190
tct cac ggg aga gaa tct gcg aca gca aat ggt gtt acc atc tgt att 624
Ser His Gly Arg Glu Ser Ala Thr Ala Asn Gly Val Thr Ile Cys Ile
195 200 205
aaa gaa gcg gct aag aag aga ggc atc gat att aaa ggt gcg cgt gtc 672
Lys Glu Ala Ala Lys Lys Arg Gly Ile Asp Ile Lys Gly Ala Arg Val
210 215 220
gtt gtc caa ggc ttc gga aac gcg gga agc tat ttg gca aaa ttt atg 720
Val Val Gln Gly Phe Gly Asn Ala Gly Ser Tyr Leu Ala Lys Phe Met
225 230 235 240
cat gat gcg ggg gca aaa gtt gtc ggc atc tca gat gcg tat ggc gga 768
His Asp Ala Gly Ala Lys Val Val Gly Ile Ser Asp Ala Tyr Gly Gly
245 250 255
ctt tat gat ccg gaa ggc ctt aat atc gat tgt tta ctc gac cga cgc 816
Leu Tyr Asp Pro Glu Gly Leu Asn Ile Asp Cys Leu Leu Asp Arg Arg
260 265 270
gac agc ttc ggt acc gta aca aag ctt ttc aac gat acc att acc aac 864
Asp Ser Phe Gly Thr Val Thr Lys Leu Phe Asn Asp Thr Ile Thr Asn
275 280 285
caa gag ctg ctg gag ctg gat tgt gat att ctc gtt cct gct gcg att 912
Gln Glu Leu Leu Glu Leu Asp Cys Asp Ile Leu Val Pro Ala Ala Ile
290 295 300
gaa aat caa att aca gaa gaa aat gcc cat aat att cgg gct aaa att 960
Glu Asn Gln Ile Thr Glu Glu Asn Ala His Asn Ile Arg Ala Lys Ile
305 310 315 320
gtc gtt gaa gca gcg aac gga cca aca acg ctt gaa gga aca aag att 1008
Val Val Glu Ala Ala Asn Gly Pro Thr Thr Leu Glu Gly Thr Lys Ile
325 330 335
ctt tca gac cgg gac att ctg ctt gta cca gac gtg ctg gca gct gcc 1056
Leu Ser Asp Arg Asp Ile Leu Leu Val Pro Asp Val Leu Ala Ala Ala
340 345 350
ggt ggc gta aca gtt tct tat ttt gaa tgg gtt cag aat aac caa ggc 1104
Gly Gly Val Thr Val Ser Tyr Phe Glu Trp Val Gln Asn Asn Gln Gly
355 360 365
ttc tac tgg agt gaa gaa gag gta gaa gaa aaa tta gaa aaa atg atg 1152
Phe Tyr Trp Ser Glu Glu Glu Val Glu Glu Lys Leu Glu Lys Met Met
370 375 380
gtc aaa tca ttt aac aat att tat gaa atg gct aac aac cga aga att 1200
Val Lys Ser Phe Asn Asn Ile Tyr Glu Met Ala Asn Asn Arg Arg Ile
385 390 395 400
gac atg agg ctc gct gca tat atg gtc ggc gtt cgc aaa atg gct gaa 1248
Asp Met Arg Leu Ala Ala Tyr Met Val Gly Val Arg Lys Met Ala Glu
405 410 415
gct tcg cgt ttt aga ggc tgg ata taa 1275
Ala Ser Arg Phe Arg Gly Trp Ile
420
<210>8
<211>424
<212>PRT
<213>纳豆芽孢杆菌(Bacillus Subtillis Natto)
<400>8
Met Ala Ala Asp Arg Asn Thr Gly His Thr Glu Gly Asp Lys Leu Asp
1 5 10 15
Val Leu Lys Ser Thr Gln Thr Val Ile His Lys Ala Leu Glu Lys Leu
20 25 30
Gly Tyr Pro Glu Glu Val Tyr Glu Leu Leu Lys Glu Pro Met Arg Leu
35 40 45
Leu Thr Val Lys Ile Pro Val Arg Met Asp Asp Gly Ser Val Lys Ile
50 55 60
Phe Thr Gly Tyr Arg Ala Arg His Asn Asp Ser Val Gly Pro Thr Lys
65 70 75 80
Gly Gly Ile Arg Phe His Pro Asn Val Thr Glu Lys Glu Val Lys Ala
85 90 95
Leu Ser Ile Trp Met Ser Leu Lys Cys Gly Ile Ile Asp Leu Pro Tyr
100 105 110
Gly Gly Gly Lys Gly Gly Ile Val Cys Asp Pro Arg Asp Met Ser Phe
115 120 125
Arg Glu Leu Glu Arg Leu Ser Arg Gly Tyr Val Arg Ala Ile Ser Gln
130 135 140
Ile Val Gly Pro Thr Lys Asp Val Pro Ala Pro Asp Val Phe Thr Asn
145 150 155 160
Ser Gln Ile Met Ala Trp Met Met Asp Glu Tyr Ser Arg Ile Asp Glu
165 170 175
Phe Asn Ser Pro Gly Phe Ile Thr Gly Lys Pro Leu Val Leu Gly Gly
180 185 190
Ser His Gly Arg Glu Ser Ala Thr Ala Asn Gly Val Thr Ile Cys Ile
195 200 205
Lys Glu Ala Ala Lys Lys Arg Gly Ile Asp Ile Lys Gly Ala Arg Val
210 215 220
Val Val Gln Gly Phe Gly Asn Ala Gly Ser Tyr Leu Ala Lys Phe Met
225 230 235 240
His Asp Ala Gly Ala Lys Val Val Gly Ile Ser Asp Ala Tyr Gly Gly
245 250 255
Leu Tyr Asp Pro Glu Gly Leu Asn Ile Asp Cys Leu Leu Asp Arg Arg
260 265 270
Asp Ser Phe Gly Thr Val Thr Lys Leu Phe Asn Asp Thr Ile Thr Asn
275 280 285
Gln Glu Leu Leu Glu Leu Asp Cys Asp Ile Leu Val Pro Ala Ala Ile
290 295 300
Glu Asn Gln Ile Thr Glu Glu Asn Ala His Asn Ile Arg Ala Lys Ile
305 310 315 320
Val Val Glu Ala Ala Asn Gly Pro Thr Thr Leu Glu Gly Thr Lys Ile
325 330 335
Leu Ser Asp Arg Asp Ile Leu Leu Val Pro Asp Val Leu Ala Ala Ala
340 345 350
Gly Gly Val Thr Val Ser Tyr Phe Glu Trp Val Gln Asn Asn Gln Gly
355 360 365
Phe Tyr Trp Ser Glu Glu Glu Val Glu Glu Lys Leu Glu Lys Met Met
370 375 380
Val Lys Ser Phe Asn Asn Ile Tyr Glu Met Ala Asn Asn Arg Arg Ile
385 390 395 400
Asp Met Arg Leu Ala Ala Tyr Met Val Gly Val Arg Lys Met Ala Glu
405 410 415
Ala Ser Arg Phe Arg Gly Trp Ile
420
<210>9
<211>1275
<212>DNA
<213>纳豆芽孢杆菌(Bacillus Subtillis Natto)
<220>
<221>CDS
<222>(1)..(1275)
<400>9
atg gcg gcc gat cga aac acc ggt cat aca gaa ggg gac aaa ctt gat 48
Met Ala Ala Asp Arg Asn Thr Gly His Thr Glu Gly Asp Lys Leu Asp
1 5 10 15
gta tta aaa tcg acc caa acc gta ata cat aag gct ctg gaa aaa ttg 96
Val Leu Lys Ser Thr Gln Thr Val Ile His Lys Ala Leu Glu Lys Leu
20 25 30
gga tat ccc gaa gag gta tac gaa ttg tta aaa gag ccg atg aga tta 144
Gly Tyr Pro Glu Glu Val Tyr Glu Leu Leu Lys Glu Pro Met Arg Leu
35 40 45
tta acg gta aaa ata cct gtt cgt atg gac gac ggt tca gtt aag att 192
Leu Thr Val Lys Ile Pro Val Arg Met Asp Asp Gly Ser Val Lys Ile
50 55 60
ttc aca gga tat cgt gcg cgg cac aat gac tct gtc ggt cca acg gca 240
Phe Thr Gly Tyr Arg Ala Arg His Asn Asp Ser Val Gly Pro Thr Ala
65 70 75 80
ggc ggg ata cgt ttt cac ccg aat gta aca gaa aaa gag gtg aag gcg 288
Gly Gly Ile Arg Phe His Pro Asn Val Thr Glu Lys Glu Val Lys Ala
85 90 95
ctt tca att tgg atg agt tta aaa tgc ggc ata att gat ctt cca tat 336
Leu Ser Ile Trp Met Ser Leu Lys Cys Gly Ile Ile Asp Leu Pro Tyr
100 105 110
ggc ggt ggt caa ggc gga att gtt tgt gat cca agg gat atg tcg ttt 384
Gly Gly Gly Gln Gly Gly Ile Val Cys Asp Pro Arg Asp Met Ser Phe
115 120 125
aga gag ctg gag cgt ctg agc aga ggg tat gtc aga gcg atc agc caa 432
Arg Glu Leu Glu Arg Leu Ser Arg Gly Tyr Val Arg Ala Ile Ser Gln
130 135 140
att gtc ggc ccg aca aaa gac gtg ccg gca ccg gat gta ttt aca aac 480
Ile Val Gly Pro Thr Lys Asp Val Pro Ala Pro Asp Val Phe Thr Asn
145 150 155 160
tca caa atc atg gct tgg atg atg gat gag tat tca aga att gat gaa 528
Ser Gln Ile Met Ala Trp Met Met Asp Glu Tyr Ser Arg Ile Asp Glu
165 170 175
ttt aat tcg cct gga ttt att aca ggc aaa ccg ctt gtg ctt ggc gga 576
Phe Asn Ser Pro Gly Phe Ile Thr Gly Lys Pro Leu Val Leu Gly Gly
180 185 190
tct cac ggg aga gaa tct gcg aca gca aat ggt gtt acc atc tgt att 624
Ser His Gly Arg Glu Ser Ala Thr Ala Asn Gly Val Thr Ile Cys Ile
195 200 205
aaa gaa gcg gct aag aag aga ggc atc gat att aaa ggt gcg cgt gtc 672
Lys Glu Ala Ala Lys Lys Arg Gly Ile Asp Ile Lys Gly Ala Arg Val
210 215 220
gtt gtc caa ggc ttc gga aac gcg gga agc tat ttg gca aaa ttt atg 720
Val Val Gln Gly Phe Gly Asn Ala Gly Ser Tyr Leu Ala Lys Phe Met
225 230 235 240
cat gat gcg ggg gca aaa gtt gtc ggc atc tca gat gcg tat ggc gga 768
His Asp Ala Gly Ala Lys Val Val Gly Ile Ser Asp Ala Tyr Gly Gly
245 250 255
ctt tat gat ccg gaa ggc ctt aat atc gat tgt tta ctc gac cga cgc 816
Leu Tyr Asp Pro Glu Gly Leu Asn Ile Asp Cys Leu Leu Asp Arg Arg
260 265 270
gac agc ttc ggt acc gta aca aag ctt ttc aac gat acc att acc aac 864
Asp Ser Phe Gly Thr Val Thr Lys Leu Phe Asn Asp Thr Ile Thr Asn
275 280 285
caa gag ctg ctg gag ctg gat tgt gat att ctc gtt cct gct gcg att 912
Gln Glu Leu Leu Glu Leu Asp Cys Asp Ile Leu Val Pro Ala Ala Ile
290 295 300
gaa aat caa att aca gaa gaa aat gcc cat aat att cgg gct aaa att 960
Glu Asn Gln Ile Thr Glu Glu Asn Ala His Asn Ile Arg Ala Lys Ile
305 310 315 320
gtc gtt gaa gca gcg aac gga cca aca acg ctt gaa gga aca aag att 1008
Val Val Glu Ala Ala Asn Gly Pro Thr Thr Leu Glu Gly Thr Lys Ile
325 330 335
ctt tca gac cgg gac att ctg ctt gta cca gac gtg ctg gca agt gcc 1056
Leu Ser Asp Arg Asp Ile Leu Leu Val Pro Asp Val Leu Ala Ser Ala
340 345 350
ggt ggc gta aca gtt tct tat ttt gaa tgg gtt cag aat aac caa ggc 1104
Gly Gly Val Thr Val Ser Tyr Phe Glu Trp Val Gln Asn Asn Gln Gly
355 360 365
ttc tac tgg agt gaa gaa gag gta gaa gaa aaa tta gaa aaa atg atg 1152
Phe Tyr Trp Ser Glu Glu Glu Val Glu Glu Lys Leu Glu Lys Met Met
370 375 380
gtc aaa tca ttt aac aat att tat gaa atg gct aac aac cga aga att 1200
Val Lys Ser Phe Asn Asn Ile Tyr Glu Met Ala Asn Asn Arg Arg Ile
385 390 395 400
gac atg agg ctc gct gca tat atg gtc ggc gtt cgc aaa atg gct gaa 1248
Asp Met Arg Leu Ala Ala Tyr Met Val Gly Val Arg Lys Met Ala Glu
405 410 415
gct tcg cgt ttt aga ggc tgg ata taa 1275
Ala Ser Arg Phe Arg Gly Trp Ile
420
<210>10
<211>424
<212>PRT
<213>纳豆芽孢杆菌(Bacillus Subtillis Natto)
<400>10
Met Ala Ala Asp Arg Asn Thr Gly His Thr Glu Gly Asp Lys Leu Asp
1 5 10 15
Val Leu Lys Ser Thr Gln Thr Val Ile His Lys Ala Leu Glu Lys Leu
20 25 30
Gly Tyr Pro Glu Glu Val Tyr Glu Leu Leu Lys Glu Pro Met Arg Leu
35 40 45
Leu Thr Val Lys Ile Pro Val Arg Met Asp Asp Gly Ser Val Lys Ile
50 55 60
Phe Thr Gly Tyr Arg Ala Arg His Asn Asp Ser Val Gly Pro Thr Ala
65 70 75 80
Gly Gly Ile Arg Phe His Pro Asn Val Thr Glu Lys Glu Val Lys Ala
85 90 95
Leu Ser Ile Trp Met Ser Leu Lys Cys Gly Ile Ile Asp Leu Pro Tyr
100 105 110
Gly Gly Gly Gln Gly Gly Ile Val Cys Asp Pro Arg Asp Met Ser Phe
115 120 125
Arg Glu Leu Glu Arg Leu Ser Arg Gly Tyr Val Arg Ala Ile Ser Gln
130 135 140
Ile Val Gly Pro Thr Lys Asp Val Pro Ala Pro Asp Val Phe Thr Asn
145 150 155 160
Ser Gln Ile Met Ala Trp Met Met Asp Glu Tyr Ser Arg Ile Asp Glu
165 170 175
Phe Asn Ser Pro Gly Phe Ile Thr Gly Lys Pro Leu Val Leu Gly Gly
180 185 190
Ser His Gly Arg Glu Ser Ala Thr Ala Asn Gly Val Thr Ile Cys Ile
195 200 205
Lys Glu Ala Ala Lys Lys Arg Gly Ile Asp Ile Lys Gly Ala Arg Val
210 215 220
Val Val Gln Gly Phe Gly Asn Ala Gly Ser Tyr Leu Ala Lys Phe Met
225 230 235 240
His Asp Ala Gly Ala Lys Val Val Gly Ile Ser Asp Ala Tyr Gly Gly
245 250 255
Leu Tyr Asp Pro Glu Gly Leu Asn Ile Asp Cys Leu Leu Asp Arg Arg
260 265 270
Asp Ser Phe Gly Thr Val Thr Lys Leu Phe Asn Asp Thr Ile Thr Asn
275 280 285
Gln Glu Leu Leu Glu Leu Asp Cys Asp Ile Leu Val Pro Ala Ala Ile
290 295 300
Glu Asn Gln Ile Thr Glu Glu Asn Ala His Asn Ile Arg Ala Lys Ile
305 310 315 320
Val Val Glu Ala Ala Asn Gly Pro Thr Thr Leu Glu Gly Thr Lys Ile
325 330 335
Leu Ser Asp Arg Asp Ile Leu Leu Val Pro Asp Val Leu Ala Ser Ala
340 345 350
Gly Gly Val Thr Val Ser Tyr Phe Glu Trp Val Gln Asn Asn Gln Gly
355 360 365
Phe Tyr Trp Ser Glu Glu Glu Val Glu Glu Lys Leu Glu Lys Met Met
370 375 380
Val Lys Ser Phe Asn Asn Ile Tyr Glu Met Ala Asn Asn Arg Arg Ile
385 390 395 400
Asp Met Arg Leu Ala Ala Tyr Met Val Gly Val Arg Lys Met Ala Glu
405 410 415
Ala Ser Arg Phe Arg Gly Trp Ile
420
<210>11
<211>1275
<212>DNA
<213>纳豆芽孢杆菌(Bacillus Subtillis Natto)
<220>
<221>CDS
<222>(1)..(1275)
<400>11
atg gcg gcc gat cga aac acc ggt cat aca gaa ggg gac aaa ctt gat 48
Met Ala Ala Asp Arg Asn Thr Gly His Thr Glu Gly Asp Lys Leu Asp
1 5 10 15
gta tta aaa tcg acc caa acc gta ata cat aag gct ctg gaa aaa ttg 96
Val Leu Lys Ser Thr Gln Thr ValIle His Lys Ala Leu Glu Lys Leu
20 25 30
gga tat ccc gaa gag gta tac gaa ttg tta aaa gag ccg atg aga tta 144
Gly Tyr Pro Glu Glu Val Tyr Glu Leu Leu Lys Glu Pro Met Arg Leu
35 40 45
tta acg gta aaa ata cct gtt cgt atg gac gac ggt tca gtt aag att 192
Leu Thr Val Lys Ile Pro Val Arg Met Asp Asp Gly Ser Val Lys Ile
50 55 60
ttc aca gga tat cgt gcg cgg cac aat gac tct gtc ggt cca acg gca 240
Phe Thr Gly Tyr Arg Ala Arg His Asn Asp Ser Val Gly Pro Thr Ala
65 70 75 80
ggc ggg ata cgt ttt cac ccg aat gta aca gaa aaa gag gtg aag gcg 288
Gly Gly Ile Arg Phe His Pro Asn Val Thr Glu Lys Glu Val Lys Ala
85 90 95
ctt tca att tgg atg agt tta aaa tgc ggc ata att gat ctt cca tat 336
Leu Ser Ile Trp Met Ser Leu Lys Cys Gly Ile Ile Asp Leu Pro Tyr
100 105 110
ggc ggt ggt aaa ggc gga att gtt tgt gat cca agg gat atg tcg ttt 384
Gly Gly Gly Lys Gly Gly Ile Val Cys Asp Pro Arg Asp Met Ser Phe
115 120 125
aga gag ctg gag cgt ctg agc aga ggg tat gtc aga gcg atc agc caa 432
Arg Glu Leu Glu Arg Leu Ser Arg Gly Tyr Val Arg Ala Ile Ser Gln
130 135 140
att gtc ggc ccg aca aaa gac gtg ccg gca ccg gat gta ttt aca aac 480
Ile Val Gly Pro Thr Lys Asp Val Pro Ala Pro Asp Val Phe Thr Asn
145 150 155 160
tca caa atc atg gct tgg atg atg gat gag tat tca aga att gat gaa 528
Ser Gln Ile Met Ala Trp Met Met Asp Glu Tyr Ser Arg Ile Asp Glu
165 170 175
ttt aat tcg cct gga ttt att aca ggc aaa ccg ctt gtg ctt ggc gga 576
Phe Asn Ser Pro Gly PheIle Thr Gly Lys Pro Leu Val Leu Gly Gly
180 185 190
tct cac ggg aga gaa tct gcg aca gca aat ggt gtt acc atc tgt att 624
Ser His Gly Arg Glu Ser Ala Thr Ala Asn Gly Val Thr Ile Cys Ile
195 200 205
aaa gaa gcg gct aag aag aga ggc atc gat att aaa ggt gcg cgt gtc 672
Lys Glu Ala Ala Lys Lys Arg Gly Ile Asp Ile Lys Gly Ala Arg Val
210 215 220
gtt gtc caa ggc ttc gga aac gcg gga agc tat ttg gca aaa ttt atg 720
Val Val Gln Gly Phe Gly Asn Ala Gly Ser Tyr Leu Ala Lys Phe Met
225 230 235 240
cat gat gcg ggg gca aaa gtt gtc ggc atc tca gat gcg tat ggc gga 768
His Asp Ala Gly Ala Lys Val Val Gly Ile Ser Asp Ala Tyr Gly Gly
245 250 255
ctt tat gat ccg gaa ggc ctt aat atc gat tgt tta ctc gac cga cgc 816
Leu Tyr Asp Pro Glu Gly Leu AsnIle Asp Cys Leu Leu Asp Arg Arg
260 265 270
gac agc ttc ggt acc gta aca aag ctt ttc aac gat acc att acc aac 864
Asp Ser Phe Gly Thr Val Thr Lys Leu Phe Asn Asp Thr Ile Thr Asn
275 280 285
caa gag ctg ctg gag ctg gat tgt gat att ctc gtt cct gct gcg att 912
Gln Glu Leu Leu Glu Leu Asp Cys AspIle Leu Val Pro Ala Ala Ile
290 295 300
gaa aat caa att aca gaa gaa aat gcc cat aat att cgg gct aaa att 960
Glu Asn Gln Ile Thr Glu Glu Asn Ala His Asn Ile Arg Ala Lys Ile
305 310 315 320
gtc gtt gaa gca gcg aac gga cca aca acg ctt gaa gga aca aag att 1008
Val Val Glu Ala Ala Asn Gly Pro Thr Thr Leu Glu Gly Thr Lys Ile
325 330 335
ctt tca gac cgg gac att ctg ctt gta cca gac gtg ctg gca gct gcc 1056
Leu Ser Asp Arg Asp Ile Leu Leu Val Pro Asp Val Leu Ala Ala Ala
340 345 350
ggt ggc gta aca gtt tct tat ttt gaa tgg gtt cag aat aac caa ggc 1104
Gly Gly Val Thr Val Ser Tyr Phe Glu Trp Val Gln Asn Asn Gln Gly
355 360 365
ttc tac tgg agt gaa gaa gag gta gaa gaa aaa tta gaa aaa atg atg 1152
Phe Tyr Trp Ser Glu Glu Glu Val Glu Glu Lys Leu Glu Lys Met Met
370 375 380
gtc aaa tca ttt aac aat att tat gaa atg gct aac aac cga aga att 1200
Val Lys Ser Phe Asn Asn Ile Tyr Glu Met Ala Asn Asn Arg Arg Ile
385 390 395 400
gac atg agg ctc gct gca tat atg gtc ggc gtt cgc aaa atg gct gaa 1248
Asp Met Arg Leu Ala Ala Tyr Met Val Gly Val Arg Lys Met Ala Glu
405 410 415
gct tcg cgt ttt aga ggc tgg ata taa 1275
Ala Ser Arg Phe Arg Gly Trp Ile
420
<210>12
<211>424
<212>PRT
<213>纳豆芽孢杆菌(Bacillus Subtillis Natto)
<400>12
Met Ala Ala Asp Arg Asn Thr Gly His Thr Glu Gly Asp Lys Leu Asp
1 5 10 15
Val Leu Lys Ser Thr Gln Thr Val Ile His Lys Ala Leu Glu Lys Leu
20 25 30
Gly Tyr Pro Glu Glu Val Tyr Glu Leu Leu Lys Glu Pro Met Arg Leu
35 40 45
Leu Thr Val Lys Ile Pro Val Arg Met Asp Asp Gly Ser Val Lys Ile
50 55 60
Phe Thr Gly Tyr Arg Ala Arg His Asn Asp Ser Val Gly Pro Thr Ala
65 70 75 80
Gly Gly Ile Arg Phe His Pro Asn Val Thr Glu Lys Glu Val Lys Ala
85 90 95
Leu Ser Ile Trp Met Ser Leu Lys Cys Gly Ile Ile Asp Leu Pro Tyr
100 105 110
Gly Gly Gly Lys Gly Gly Ile Val Cys Asp Pro Arg Asp Met Ser Phe
115 120 125
Arg Glu Leu Glu Arg Leu Ser Arg Gly Tyr Val Arg Ala Ile Ser Gln
130 135 140
Ile Val Gly Pro Thr Lys Asp Val Pro Ala Pro Asp Val Phe Thr Asn
145 150 155 160
Ser Gln Ile Met Ala Trp Met Met Asp Glu Tyr Ser Arg Ile Asp Glu
165 170 175
Phe Asn Ser Pro Gly Phe Ile Thr Gly Lys Pro Leu Val Leu Gly Gly
180 185 190
Ser His Gly Arg Glu Ser Ala Thr Ala Asn Gly Val Thr Ile Cys Ile
195 200 205
Lys Glu Ala Ala Lys Lys Arg Gly Ile Asp Ile Lys Gly Ala Arg Val
210 215 220
Val Val Gln Gly Phe Gly Asn Ala Gly Ser Tyr Leu Ala Lys Phe Met
225 230 235 240
His Asp Ala Gly Ala Lys Val Val Gly Ile Ser Asp Ala Tyr Gly Gly
245 250 255
Leu Tyr Asp Pro Glu Gly Leu Asn Ile Asp Cys Leu Leu Asp Arg Arg
260 265 270
Asp Ser Phe Gly Thr Val Thr Lys Leu Phe Asn Asp Thr Ile Thr Asn
275 280 285
Gln Glu Leu Leu Glu Leu Asp Cys Asp Ile Leu Val Pro Ala Ala Ile
290 295 300
Glu Asn Gln Ile Thr Glu Glu Asn Ala His Asn Ile Arg Ala Lys Ile
305 310 315 320
Val Val Glu Ala Ala Asn Gly Pro Thr Thr Leu Glu Gly Thr Lys Ile
325 330 335
Leu Ser Asp Arg Asp Ile Leu Leu Val Pro Asp Val Leu Ala Ala Ala
340 345 350
Gly Gly Val Thr Val Ser Tyr Phe Glu Trp Val Gln Asn Asn Gln Gly
355 360 365
Phe Tyr Trp Ser Glu Glu Glu Val Glu Glu Lys Leu Glu Lys Met Met
370 375 380
Val Lys Ser Phe Asn Asn Ile Tyr Glu Met Ala Asn Asn Arg Arg Ile
385 390 395 400
Asp Met Arg Leu Ala Ala Tyr Met Val Gly Val Arg Lys Met Ala Glu
405 410 415
Ala Ser Arg Phe Arg Gly Trp Ile
420
<210>13
<211>1275
<212>DNA
<213>纳豆芽孢杆菌(Bacillus Subtillis Natto)
<220>
<221>CDS
<222>(1)..(1275)
<400>13
atg gcg gcc gat cga aac acc ggt cat aca gaa ggg gac aaa ctt gat 48
Met Ala Ala Asp Arg Asn Thr Gly His Thr Glu Gly Asp Lys Leu Asp
1 5 10 15
gta tta aaa tcg acc caa acc gta ata cat aag gct ctg gaa aaa ttg 96
Val Leu Lys Ser Thr Gln Thr Val Ile His Lys Ala Leu Glu Lys Leu
20 25 30
gga tat ccc gaa gag gta tac gaa ttg tta aaa gag ccg atg aga tta 144
Gly Tyr Pro Glu Glu Val Tyr Glu Leu Leu Lys Glu Pro Met Arg Leu
35 40 45
tta acg gta aaa ata cct gtt cgt atg gac gac ggt tca gtt aag att 192
Leu Thr Val Lys Ile Pro Val Arg Met Asp Asp Gly Ser Val Lys Ile
50 55 60
ttc aca gga tat cgt gcg cgg cac aat gac tct gtc ggt cca acg aaa 240
Phe Thr Gly Tyr Arg Ala Arg His Asn Asp Ser Val Gly Pro Thr Lys
65 70 75 80
ggc ggg ata cgt ttt cac ccg aat gta aca gaa aaa gag gtg aag gcg 288
Gly Gly Ile Arg Phe His Pro Asn Val Thr Glu Lys Glu Val Lys Ala
85 90 95
ctt tca att tgg atg agt tta aaa tgc ggc ata att gat ctt cca tat 336
Leu Ser Ile Trp Met Ser Leu Lys Cys Gly Ile Ile Asp Leu Pro Tyr
100 105 110
ggc ggt ggt caa ggc gga att gtt tgt gat cca agg gat atg tcg ttt 384
Gly Gly Gly Gln Gly Gly Ile Val Cys Asp Pro Arg Asp Met Ser Phe
115 120 125
aga gag ctg gag cgt ctg agc aga ggg tat gtc aga gcg atc agc caa 432
Arg Glu Leu Glu Arg Leu Ser Arg Gly Tyr Val Arg Ala Ile Ser Gln
130 135 140
att gtc ggc ccg aca aaa gac gtg ccg gca ccg gat gta ttt aca aac 480
Ile Val Gly Pro Thr Lys Asp Val Pro Ala Pro Asp Val Phe Thr Asn
145 150 155 160
tca caa atc atg gct tgg atg atg gat gag tat tca aga att gat gaa 528
Ser Gln Ile Met Ala Trp Met Met Asp Glu Tyr Ser Arg Ile Asp Glu
165 170 175
ttt aat tcg cct gga ttt att aca ggc aaa ccg ctt gtg ctt ggc gga 576
Phe Asn Ser Pro Gly Phe Ile Thr Gly Lys Pro Leu Val Leu Gly Gly
180 185 190
tct cac ggg aga gaa tct gcg aca gca aat ggt gtt acc atc tgt att 624
Ser His Gly Arg Glu Ser Ala Thr Ala Asn Gly Val Thr Ile Cys Ile
195 200 205
aaa gaa gcg gct aag aag aga ggc atc gat att aaa ggt gcg cgt gtc 672
Lys Glu Ala Ala Lys Lys Arg Gly Ile Asp Ile Lys Gly Ala Arg Val
210 215 220
gtt gtc caa ggc ttc gga aac gcg gga agc tat ttg gca aaa ttt atg 720
Val Val Gln Gly Phe Gly Asn Ala Gly Ser Tyr Leu Ala Lys Phe Met
225 230 235 240
cat gat gcg ggg gca aaa gtt gtc ggc atc tca gat gcg tat ggc gga 768
His Asp Ala Gly Ala Lys Val Val Gly Ile Ser Asp Ala Tyr Gly Gly
245 250 255
ctt tat gat ccg gaa ggc ctt aat atc gat tgt tta ctc gac cga cgc 816
Leu Tyr Asp Pro Glu Gly Leu Asn Ile Asp Cys Leu Leu Asp Arg Arg
260 265 270
gac agc ttc ggt acc gta aca aag ctt ttc aac gat acc att acc aac 864
Asp Ser Phe Gly Thr Val Thr Lys Leu Phe Asn Asp Thr Ile Thr Asn
275 280 285
caa gag ctg ctg gag ctg gat tgt gat att ctc gtt cct gct gcg att 912
Gln Glu Leu Leu Glu Leu Asp Cys Asp Ile Leu Val Pro Ala Ala Ile
290 295 300
gaa aat caa att aca gaa gaa aat gcc cat aat att cgg gct aaa att 960
Glu Asn Gln Ile Thr Glu Glu Asn Ala His Asn Ile Arg Ala Lys Ile
305 310 315 320
gtc gtt gaa gca gcg aac gga cca aca acg ctt gaa gga aca aag att 1008
Val Val Glu Ala Ala Asn Gly Pro Thr Thr Leu Glu Gly Thr Lys Ile
325 330 335
ctt tca gac cgg gac att ctg ctt gta cca gac gtg ctg gca gct gcc 1056
Leu Ser Asp Arg Asp Ile Leu Leu Val Pro Asp Val Leu Ala Ala Ala
340 345 350
ggt ggc gta aca gtt tct tat ttt gaa tgg gtt cag aat aac caa ggc 1104
Gly Gly Val Thr Val Ser Tyr Phe Glu Trp Val Gln Asn Asn Gln Gly
355 360 365
ttc tac tgg agt gaa gaa gag gta gaa gaa aaa tta gaa aaa atg atg 1152
Phe Tyr Trp Ser Glu Glu Glu Val Glu Glu Lys Leu Glu Lys Met Met
370 375 380
gtc aaa tca ttt aac aat att tat gaa atg gct aac aac cga aga att 1200
Val Lys Ser Phe Asn Asn Ile Tyr Glu Met Ala Asn Asn Arg Arg Ile
385 390 395 400
gac atg agg ctc gct gca tat atg gtc ggc gtt cgc aaa atg gct gaa 1248
Asp Met Arg Leu Ala Ala Tyr Met Val Gly Val Arg Lys Met Ala Glu
405 410 415
gct tcg cgt ttt aga ggc tgg ata taa 1275
Ala Ser Arg Phe Arg Gly Trp Ile
420
<210>14
<211>424
<212>PRT
<213>纳豆芽孢杆菌(Bacillus Subtillis Natto)
<400>14
Met Ala Ala Asp Arg Asn Thr Gly His Thr Glu Gly Asp Lys Leu Asp
1 5 10 15
Val Leu Lys Ser Thr Gln Thr Val Ile His Lys Ala Leu Glu Lys Leu
20 25 30
Gly Tyr Pro Glu Glu Val Tyr Glu Leu Leu Lys Glu Pro Met Arg Leu
35 40 45
Leu Thr Val Lys Ile Pro Val Arg Met Asp Asp Gly Ser Val Lys Ile
50 55 60
Phe Thr Gly Tyr Arg Ala Arg His Asn Asp Ser Val Gly Pro Thr Lys
65 70 75 80
Gly Gly Ile Arg Phe His Pro Asn Val Thr Glu Lys Glu Val Lys Ala
85 90 95
Leu Ser Ile Trp Met Ser Leu Lys Cys Gly Ile Ile Asp Leu Pro Tyr
100 105 110
Gly Gly Gly Gln Gly Gly Ile Val Cys Asp Pro Arg Asp Met Ser Phe
115 120 125
Arg Glu Leu Glu Arg Leu Ser Arg Gly Tyr Val Arg Ala Ile Ser Gln
130 135 140
Ile Val Gly Pro Thr Lys Asp Val Pro Ala Pro Asp Val Phe Thr Asn
145 150 155 160
Ser Gln Ile Met Ala Trp Met Met Asp Glu Tyr Ser Arg Ile Asp Glu
165 170 175
Phe Asn Ser Pro Gly Phe Ile Thr Gly Lys Pro Leu Val Leu Gly Gly
180 185 190
Ser His Gly Arg Glu Ser Ala Thr Ala Asn Gly Val Thr Ile Cys Ile
195 200 205
Lys Glu Ala Ala Lys Lys Arg Gly Ile Asp Ile Lys Gly Ala Arg Val
210 215 220
Val Val Gln Gly Phe Gly Asn Ala Gly Ser Tyr Leu Ala Lys Phe Met
225 230 235 240
His Asp Ala Gly Ala Lys Val Val Gly Ile Ser Asp Ala Tyr Gly Gly
245 250 255
Leu Tyr Asp Pro Glu Gly Leu Asn Ile Asp Cys Leu Leu Asp Arg Arg
260 265 270
Asp Ser Phe Gly Thr Val Thr Lys Leu Phe Asn Asp Thr Ile Thr Asn
275 280 285
Gln Glu Leu Leu Glu Leu Asp Cys Asp Ile Leu Val Pro Ala Ala Ile
290 295 300
Glu Asn Gln Ile Thr Glu Glu Asn Ala His Asn Ile Arg Ala Lys Ile
305 310 315 320
Val Val Glu Ala Ala Asn Gly Pro Thr Thr Leu Glu Gly Thr Lys Ile
325 330 335
Leu Ser Asp Arg Asp Ile Leu Leu Val Pro Asp Val Leu Ala Ala Ala
340 345 350
Gly Gly Val Thr Val Ser Tyr Phe Glu Trp Val Gln Asn Asn Gln Gly
355 360 365
Phe Tyr Trp Ser Glu Glu Glu Val Glu Glu Lys Leu Glu Lys Met Met
370 375 380
Val Lys Ser Phe Asn Asn Ile Tyr Glu Met Ala Asn Asn Arg Arg Ile
385 390 395 400
Asp Met Arg Leu Ala Ala Tyr Met Val Gly Val Arg Lys Met Ala Glu
405 410 415
Ala Ser Arg Phe Arg Gly Trp Ile
420
<210>15
<211>1275
<212>DNA
<213>纳豆芽孢杆菌(Bacillus Subtillis Natto)
<220>
<221>CDS
<222>(1)..(1275)
<400>15
atg gcg gcc gat cga aac acc ggt cat aca gaa ggg gac aaa ctt gat 48
Met Ala Ala Asp Arg Asn Thr Gly His Thr Glu Gly Asp Lys Leu Asp
1 5 10 15
gta tta aaa tcg acc caa acc gta ata cat aag gct ctg gaa aaa ttg 96
Val Leu Lys Ser Thr Gln Thr ValIle His Lys Ala Leu Glu Lys Leu
20 25 30
gga tat ccc gaa gag gta tac gaa ttg tta aaa gag ccg atg aga tta 144
Gly Tyr Pro Glu Glu Val Tyr Glu Leu Leu Lys Glu Pro Met Arg Leu
35 40 45
tta acg gta aaa ata cct gtt cgt atg gac gac ggt tca gtt aag att 192
Leu Thr Val Lys Ile Pro Val Arg Met Asp Asp Gly Ser Val Lys Ile
50 55 60
ttc aca gga tat cgt gcg cgg cac aat gac tct gtc ggt cca acg gca 240
Phe Thr Gly Tyr Arg Ala Arg His Asn Asp Ser Val Gly Pro Thr Ala
65 70 75 80
ggc ggg ata cgt ttt cac ccg aat gta aca gaa aaa gag gtg aag gcg 288
Gly Gly Ile Arg Phe His Pro Asn Val Thr Glu Lys Glu Val Lys Ala
85 90 95
ctt tca att tgg atg agt tta aaa tgc ggc ata att gat ctt cca tat 336
Leu Ser Ile Trp Met Ser Leu Lys Cys Gly Ile Ile Asp Leu Pro Tyr
100 105 110
ggc ggt ggt caa ggc gga att gtt tgt gat cca agg gat atg tcg ttt 384
Gly Gly Gly Gln Gly Gly Ile Val Cys Asp Pro Arg Asp Met Ser Phe
115 120 125
aga gag ctg gag cgt ctg agc aga ggg tat gtc aga gcg atc agc caa 432
Arg Glu Leu Glu Arg Leu Ser Arg Gly Tyr Val Arg Ala Ile Ser Gln
130 135 140
att gtc ggc ccg aca aaa gac gtg ccg gca ccg gat gta ttt aca aac 480
Ile Val Gly Pro Thr Lys Asp Val Pro Ala Pro Asp Val Phe Thr Asn
145 150 155 160
tca caa atc atg gct tgg atg atg gat gag tat tca aga att gat gaa 528
Ser Gln Ile Met Ala Trp Met Met Asp Glu Tyr Ser Arg Ile Asp Glu
165 170 175
ttt aat tcg cct gga ttt att aca ggc aaa ccg ctt gtg ctt ggc gga 576
Phe Asn Ser Pro Gly Phe Ile Thr Gly Lys Pro Leu Val Leu Gly Gly
180 185 190
tct cac ggg aga gaa tct gcg aca gca aat ggt gtt acc atc tgt att 624
Ser His Gly Arg Glu Ser Ala Thr Ala Asn Gly Val Thr Ile Cys Ile
195 200 205
aaa gaa gcg gct aag aag aga ggc atc gat att aaa ggt gcg cgt gtc 672
Lys Glu Ala Ala Lys Lys Arg Gly Ile Asp Ile Lys Gly Ala Arg Val
210 215 220
gtt gtc caa ggc ttc gga aac gcg gga agc tat ttg gca aaa ttt atg 720
Val Val Gln Gly Phe Gly Asn Ala Gly Ser Tyr Leu Ala Lys Phe Met
225 230 235 240
cat gat gcg ggg gca aaa gtt gtc ggc atc tca gat gcg tat ggc gga 768
His Asp Ala Gly Ala Lys Val Val Gly Ile Ser Asp Ala Tyr Gly Gly
245 250 255
ctt tat gat ccg gaa ggc ctt aat atc gat tgt tta ctc gac cga cgc 816
Leu Tyr Asp Pro Glu Gly Leu Asn Ile Asp Cys Leu Leu Asp Arg Arg
260 265 270
gac agc ttc ggt acc gta aca aag ctt ttc aac gat acc att acc aac 864
Asp Ser Phe Gly Thr Val Thr Lys Leu Phe Asn Asp Thr Ile Thr Asn
275 280 285
caa gag ctg ctg gag ctg gat tgt gat att ctc gtt cct gct gcg att 912
Gln Glu Leu Leu Glu Leu Asp Cys Asp Ile Leu Val Pro Ala Ala Ile
290 295 300
gaa aat caa att aca gaa gaa aat gcc cat aat att cgg gct aaa att 960
Glu Asn Gln Ile Thr Glu Glu Asn Ala His Asn Ile Arg Ala Lys Ile
305 310 315 320
gtc gtt gaa gca gcg aac gga cca aca acg ctt gaa gga aca aag att 1008
Val Val Glu Ala Ala Asn Gly Pro Thr Thr Leu Glu Gly Thr Lys Ile
325 330 335
ctt tca gac cgg gac att ctg ctt gta cca gac gtg ctg gca gct gcc 1056
Leu Ser Asp Arg Asp Ile Leu Leu Val Pro Asp Val Leu Ala Ala Ala
340 345 350
ggt ggc gta aca gtt tct tat ttt gaa tgg gtt cag aat aac caa ggc 1104
Gly Gly Val Thr Val Ser Tyr Phe Glu Trp Val Gln Asn Asn Gln Gly
355 360 365
ttc tac tgg agt gaa gaa gag gta gaa gaa aaa tta gaa aaa atg atg 1152
Phe Tyr Trp Ser Glu Glu Glu Val Glu Glu Lys Leu Glu Lys Met Met
370 375 380
gtc aaa tca ttt aac aat att tat gaa atg gct aac aac cga aga att 1200
Val Lys Ser Phe Asn Asn Ile Tyr Glu Met Ala Asn Asn Arg Arg Ile
385 390 395 400
gac atg agg ctc gct gca tat atg gtc ggc gtt cgc aaa atg gct gaa 1248
Asp Met Arg Leu Ala Ala Tyr Met Val Gly Val Arg Lys Met Ala Glu
405 410 415
gct tcg cgt ttt aga ggc tgg ata taa 1275
Ala Ser Arg Phe Arg Gly Trp Ile
420
<210>16
<211>424
<212>PRT
<213>纳豆芽孢杆菌(Bacillus Subtillis Natto)
<400>16
Met Ala Ala Asp Arg Asn Thr Gly His Thr Glu Gly Asp Lys Leu Asp
1 5 10 15
Val Leu Lys Ser Thr Gln Thr Val Ile His Lys Ala Leu Glu Lys Leu
20 25 30
Gly Tyr Pro Glu Glu Val Tyr Glu Leu Leu Lys Glu Pro Met Arg Leu
35 40 45
Leu Thr Val Lys Ile Pro Val Arg Met Asp Asp Gly Ser Val Lys Ile
50 55 60
Phe Thr Gly Tyr Arg Ala Arg His Asn Asp Ser Val Gly Pro Thr Ala
65 70 75 80
Gly Gly Ile Arg Phe His Pro Asn Val Thr Glu Lys Glu Val Lys Ala
85 90 95
Leu Ser Ile Trp Met Ser Leu Lys Cys Gly Ile Ile Asp Leu Pro Tyr
100 105 110
Gly Gly Gly Gln Gly Gly Ile Val Cys Asp Pro Arg Asp Met Ser Phe
115 120 125
Arg Glu Leu Glu Arg Leu Ser Arg Gly Tyr Val Arg Ala Ile Ser Gln
130 135 140
Ile Val Gly Pro Thr Lys Asp Val Pro Ala Pro Asp Val Phe Thr Asn
145 150 155 160
Ser Gln Ile Met Ala Trp Met Met Asp Glu Tyr Ser Arg Ile Asp Glu
165 170 175
Phe Asn Ser Pro Gly Phe Ile Thr Gly Lys Pro Leu Val Leu Gly Gly
180 185 190
Ser His Gly Arg Glu Ser Ala Thr Ala Asn Gly Val Thr Ile Cys Ile
195 200 205
Lys Glu Ala Ala Lys Lys Arg Gly Ile Asp Ile Lys Gly Ala Arg Val
210 215 220
Val Val Gln Gly Phe Gly Asn Ala Gly Ser Tyr Leu Ala Lys Phe Met
225 230 235 240
His Asp Ala Gly Ala Lys Val Val Gly Ile Ser Asp Ala Tyr Gly Gly
245 250 255
Leu Tyr Asp Pro Glu Gly Leu Asn Ile Asp Cys Leu Leu Asp Arg Arg
260 265 270
Asp Ser Phe Gly Thr Val Thr Lys Leu Phe Asn Asp ThrIle Thr Asn
275 280 285
Gln Glu Leu Leu Glu Leu Asp Cys Asp Ile Leu Val Pro Ala Ala Ile
290 295 300
Glu Asn Gln Ile Thr Glu Glu Asn Ala His Asn Ile Arg Ala Lys Ile
305 310 315 320
Val Val Glu Ala Ala Asn Gly Pro Thr Thr Leu Glu Gly Thr Lys Ile
325 330 335
Leu Ser Asp Arg Asp Ile Leu Leu Val Pro Asp Val Leu Ala Ala Ala
340 345 350
Gly Gly Val Thr Val Ser Tyr Phe Glu Trp Val Gln Asn Asn Gln Gly
355 360 365
Phe Tyr Trp Ser Glu Glu Glu Val Glu Glu Lys Leu Glu Lys Met Met
370 375 380
Val Lys Ser Phe Asn Asn Ile Tyr Glu Met Ala Asn Asn Arg Arg Ile
385 390 395 400
Asp Met Arg Leu Ala Ala Tyr Met Val Gly Val Arg Lys Met Ala Glu
405 410 415
Ala Ser Arg Phe Arg Gly Trp Ile
420
Claims (3)
1.一种纳豆芽孢杆菌谷氨酸脱氢酶编码基因的突变基因，其特征在于，该突变基因的碱基序列如SEQ ID NO：3、SEQ ID NO：5、SEQ ID NO：7、SEQ ID NO：9、SEQ ID NO：11、SEQ ID NO：13或SEQ ID NO：15所示。
2.一种权利要求1所述的纳豆芽孢杆菌谷氨酸脱氢酶编码基因的突变基因编码的蛋白质，其特征在于，所述蛋白质的氨基酸序列如SEQ ID NO：4、SEQ ID NO：6、SEQ ID NO：8、SEQ ID NO：10、SEQ ID NO：12、SEQ ID NO：14或SEQ ID NO：16所示。
3.一种重组载体，其特征在于，所述重组载体包含权利要求1所述的突变基因，使用的载体为pET-28a。
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| CN105624265A (zh) * | 2014-10-30 | 2016-06-01 | 中国科学院青岛生物能源与过程研究所 | 一种利用表面展示谷氨酸脱氢酶的全细胞测定l-谷氨酸含量的方法 |
| CN105505846B (zh) * | 2016-01-07 | 2019-11-29 | 南京工业大学 | 表面展示谷氨酸脱氢酶的重组芽孢及其构建方法与应用 |
| CN110724726B (zh) * | 2019-10-31 | 2021-10-15 | 中国农业大学 | 一种鱼肉腐败菌氨基酸脱氨能力的检测方法 |
| CN112280760B (zh) * | 2020-11-26 | 2022-04-15 | 宿迁市江南大学产业技术研究院 | 一种谷氨酸脱氢酶突变体及其应用 |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1198777A (zh) * | 1995-10-06 | 1998-11-11 | 佛罗里达大学 | 有关谷氨酸脱氢酶α－和β－亚基的新多肽和多核苷酸及用法 |
| CN101578361A (zh) * | 2005-06-17 | 2009-11-11 | 米克罗比亚精密工程股份有限公司 | 改进的氨基酸和代谢物生物合成 |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1198777A (zh) * | 1995-10-06 | 1998-11-11 | 佛罗里达大学 | 有关谷氨酸脱氢酶α－和β－亚基的新多肽和多核苷酸及用法 |
| CN101578361A (zh) * | 2005-06-17 | 2009-11-11 | 米克罗比亚精密工程股份有限公司 | 改进的氨基酸和代谢物生物合成 |
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| Kada S等.Identification of two major ammonia-releasing reactions involved in secondary Natto fermentation.《Biosci.Biotechnol.Biochem.》.2008,第72卷(第7期),1869-1876. * |
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