CN109182319A - 一种苏氨酸脱氨酶突变体及其制备方法和应用 - Google Patents

一种苏氨酸脱氨酶突变体及其制备方法和应用 Download PDF

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CN109182319A
CN109182319A CN201810947764.XA CN201810947764A CN109182319A CN 109182319 A CN109182319 A CN 109182319A CN 201810947764 A CN201810947764 A CN 201810947764A CN 109182319 A CN109182319 A CN 109182319A
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林建平
王莹
朱力
乔沛
薛海龙
李国四
吴绵斌
杨立荣
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Zhejiang University ZJU
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Abstract

本发明公开了一种苏氨酸脱氨酶突变体及其制备方法和应用，该突变体是将如SEQ ID NO.1所示氨基酸序列的14位、323位、344位、449位和510位氨基酸进行单点突变或多点突变获得的。与野生型苏氨酸脱氨酶相比，本发明苏氨酸脱氨酶具有极高的稳定性，可以大大提高工业化生产中苏氨酸脱氨酶的使用寿命。其中，G323D/F510L/T344A突变体应用于L‑2‑氨基丁酸的生产，可以使L‑苏氨酸转化率达到99％，NAD+添加量降为0.04g/L，这为L‑2‑氨基丁酸的大规模生产提供了良好的技术支持。

Description

一种苏氨酸脱氨酶突变体及其制备方法和应用
技术领域
本发明涉及苏氨酸脱氨酶突变体，和产苏氨酸脱氨酶基因工程菌及其构建方法和应用，属于基因工程领域。
背景技术
苏氨酸脱氨酶(Threonine deaminase，简称TD，EC 4.3.1.19)，催化L-苏氨酸脱氨生成α-酮丁酸，是异亮氨酸合成代谢途径的关键步骤。α-酮丁酸是生产L-2-氨基丁酸、异亮氨酸、D-2-羟基丁酸以及正丙醇的重要原材料，尤其是L-2-氨基丁酸，作为治疗癫痫的手性药物左乙拉西坦和布瓦西坦，抗结核药物盐酸乙胺丁醇，以及内皮素抗因子、连氮丝菌素类抗生素的重要前体，受到广泛的关注。
目前，已经有苏氨酸脱氨酶应用于L-2-氨基丁酸合成的报道，但其稳定性较差，限制了生物催化剂的使用寿命，限制了催化效率，也增加了工业生产中的成本。因此，提高苏氨酸脱氨酶的热稳定性，并通过基因工程技术构建高表达的基因工程菌在L-2-氨基丁酸的制备中具有重要意义。
发明内容
本发明的目的是提供一种苏氨酸脱氨酶突变体、编码基因、重组载体、基因工程菌以及在催化L-苏氨酸制备L-2-氨基丁酸中的应用。
本发明采用的技术方案是：
本发明提供一种苏氨酸脱氨酶突变体，所述突变体是将SEQ ID NO.1所示氨基酸序列第14位、323位、344位、449位和510位进行单、双、三或四点突变而成。
进一步，所述单突变是将第14位丙氨酸突为苏氨酸(A14T)、第323位甘氨酸突变为天冬氨酸(G323D)、第344位苏氨酸突变为丙氨酸(T344A)、第449位精氨酸突变为半胱氨酸(R449C)、第510位脯氨酸突变为亮氨酸(F510L)；所述双点突变为A14T/G323D、G323D/T344A、G323D/R449C、G323D/F510L；所述三点突变为G323D/F510L/T344A、G323D/F510L/R449C；所述四点突变为G323D/F510L/T344A/R449C。
更进一步，所述突变体氨基酸序列为下列之一：SEQ ID.2，SEQ ID.3，SEQ ID.4，SEQ ID.5，SEQ ID.6，SEQ ID.7，SEQ ID.8，SEQ ID.9，SEQ ID.10，SEQ ID.11，SEQ ID.12和SEQ ID.13。
本发明还涉及一种编码所述苏氨酸脱氨酶突变体的基因。
本发明还涉及一种携带所述基因的重组载体。
本发明还涉及一种由所述重组载体转化制备的重组工程菌。
本发明提供一种所述苏氨酸脱氨酶突变体在催化L-苏氨酸制备L-2-氨基丁酸中的应用。
与现有技术相比，本发明有益效果主要体现在：
本发明构建了一种高效表达苏氨酸脱氨酶突变体的重组大肠杆菌，提高了苏氨酸脱氨酶的热稳定性；该菌株可以用于L-2-氨基丁酸的生产，使脱氢酶法合成L-2-氨基丁酸的转化率达到99％以上，且NAD+的添加量低至0.04g/L，有利于实现脱氢酶法生产L-2-氨基丁酸的工业化。
附图说明
图1ilvA基因片段核酸电泳图，左侧泳道为DNA Marker，右侧泳道为ilvA基因PCR扩增产物。
图2野生型菌SDS-PAGE电泳图，泳道1为破碎液沉淀中蛋白，泳道2为破碎液上清中蛋白，泳道3为全细胞蛋白。
图3 E.coli K12来源的TD与热稳定蛋白的多重序列对比结果。
图4野生型苏氨酸脱氨酶和突变体G323D/F510L/T344A的最适反应温度。
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仅限于此：
实施例1：苏氨酸脱氨酶重组菌的构建
选取Escherichia coli K12来源的苏氨酸脱氨酶，利用PCR技术，以E.coli K12基因组为模板，以ilvA-F:5’-GGAATTCCATATGGCTGACTCGCAACCCCT G-3’和ilvA-R:5’-CCGCTCGAGTTAACCCGCCAAAAAGAAC-3’为引物，对苏氨酸脱氨酶进行扩增，并在其5’端和3’端分别引入Nde I和Xho I限制酶切位点。PCR反应体系(50μL)为：5×PrimeSTAR GXL buffer 10μL，PrimeSTAR GXL DNA Polymerase 1μL，2.5mM dNTPs 4μL，模板DNA 1μL，上下游引物各1μL，无菌水32μL。PCR反应条件为94℃预变性5min；94℃变性30s，55℃退火30s，68℃延伸2min，循环30次；68℃延伸10min。以1％琼脂糖凝胶电泳验证并回收PCR扩增产物，结果扩增到与SEQ ID NO.14序列大小相符的ilvA基因片段(图1中泳道1、2)。
将纯化后的PCR产物利用pEASY-Blunt Simple Cloning Kit连接到pEASY-BluntSimple载体上，将重组质粒转入到E.coli DH5α感受态细胞中，通过AmpR和KanR两种抗性的筛选板筛选阳性转化子。挑取阳性转化子以5mL LB培养基培养，提取质粒进行测序。将测序结果与数据库序列进行比对，确保序列克隆正确。将提取的重组质粒pEASY-Blunt Simple/ilvA及pET-28a(+)分别用Nde I和Xho I酶切。将酶切产物进行核酸电泳，取pEASY-BluntSimple/ilvA重组质粒酶切后约为1,500bp的片段以及pET-28a(+)酶切产物中约5,300bp的片段进行胶回收，将回收片段用T4连接酶25℃连接20min。将酶连产物转化到E.coli BL21(DE3)感受态细胞中：将20μL连接产物加入100μL刚刚解冻的大肠杆菌BL21(DE3)感受态细胞中，冰浴30min，42℃下热击45s，冰浴10min，然后加入250μL灭菌的LB培养基，在37℃，220r/min的摇床中培养1小时。取预培养后的细胞，将EP管在3000×g下离心30min，用移液枪移去清液，然后将沉淀的细胞轻轻吸起，使用涂布法完全转移到含有50μg/mL卡那霉素的LB琼脂平板上，37℃下培养10～12h。挑取阳性转化子进行菌落PCR，并提取质粒进行序列测定，得到正确构建的重组质粒pET28a(+)-ilvA。含有pET28a(+)-ilvA的阳性克隆为正确构建的重组E.coli BL21(DE3)/pET28a(+)-ilvA。
实施例2：苏氨酸脱氨酶基因的定向进化突变
将实施例1中的重组大肠杆菌BL21(DE3)/pET28a(+)-ilvA提取质粒，以质粒pET28a(+)-ilvA为模板，以Primers-F：5’-CGCGGATCCATGGCTGACTCG A-3’，Primers-R：5’-CCCAAGCTTAACCCGCCAAAAAGAAC-3’为引物，以加入二价锰离子和二价镁离子的方式向聚合酶链式反应中引入复制错误。定向进化的PCR反应体系为：5mM MnCl2 1μL，Taq聚合酶0.5μL，10×buffer 10μL，25mM MgCl2 14μL，dNTP Mixture 4μL，上下引物各1μL,模板DNA(50ng/μL)0.5μL和无菌水18μL。定向进化的PCR反应条件：94℃5min；94℃30s，55℃30s，72℃3min，30个循环；72℃10min。
纯化PCR产物，使用限制性核酸内切酶BamH I和Hind III在37℃下处理3小时。将“双酶切”处理后的PCR产物使用核酸电泳纯化，割胶回收含有突变位点的较小片段。将纯化后的突变片段连接到同样方法“双酶切”后得到的pET-28a(+)空质粒上，用T4连接酶于25℃连接20min。将连接产物全部用热转化导入事先制备好的大肠杆菌BL21(DE3)感受态细胞中。
构建突变库后，从突变库中随机挑取单菌落进行蛋白质热稳定性检测实验。将突变体库(重组子转化平板)的单菌落以灭菌牙签挑入96浅孔细胞培养板中，37℃，220r/min培养8小时培养板中每个孔径内均装有500μL LB液体培养基(含有50μg/mL卡那霉素)。吸取浅孔细胞培养板中的菌液(菌种)50μL，转接入含有600μL LB液体培养基(含有50μg/mL卡那霉素)的96深孔细胞培养板中，37℃，220r/min孵育约2小时，至OD600＝0.6～0.8时，向每个孔径中加入10μL 6mM的IPTG，28℃，220r/min诱导培养10小时。将诱导表达的细胞板4000×g，离心10min，去上清。向每个孔径中加入600μL 0.1M Tris-HCl(pH 7.5)，并重悬细胞。将50μL菌液分别置于在50℃水浴和4℃冰浴中90min，再分别向其中加入200μL 0.1M L-苏氨酸溶液，30℃反应30min。取50μL反应液与200μL 2,4-二硝基苯肼(2mM)37℃反应20min，再加入500μL 1M NaOH终止反应并溶解生成的腙。测定反应液在380nm下的吸收值。通过比对突变子与野生菌热处理后的吸收值大小，将吸收值大于对照组菌株的突变株选出。同时，剔除初始酶活降低超过50％的突变株。从相应的种子培养板中吸取菌液提取质粒进行测序，并进行50ml摇瓶培养和诱导，分离纯化后测定其比酶活、热稳定性和动力学参数。
筛选得到三株稳定性有所提高的突变株，分别对这三株突变株进行培养并提取质粒，送北京擎科新业生物技术有限公司测序，测序结果显示三株突变体均只有一个氨基酸发生突变，故将筛选到的这三株单突变体苏氨酸脱氨酶分别命名为A14T(其对应所述SEQID NO.1氨基酸序列第14位丙氨酸突变为苏氨酸)，T344A(其对应所述SEQ ID NO.1氨基酸序列第344位苏氨酸突变为丙氨酸)，R449C(其对应所述SEQ ID NO.1氨基酸序列第449位精氨酸突变为半胱氨酸)。
实施例3：大肠杆菌K12苏氨酸脱氨酶基因多重序列比对与计算机模拟突变
通过BLAST检索发现苏氨酸脱氨酶属于Trp-synth-beta II superfamily(accession ID:cd01562)。并找到了四个不同菌种来源的热稳定苏氨酸脱氨酶。它们分别为Deinococcus-Thermus(accession ID:WP_058978819.1),Chloroflexus sp.MS-G(accession ID:WP_031459137.1),Hydrogenophilales bacterium(accession ID:OGU19679.1)和Pseudomonas aeruginosa(accession ID:NP_250017.1)。根据氨基酸序列比对结果(如图3)，找到9个可能的氨基酸残基，分别为：Val131、Ala114、Phe157、Ala193、Glu240、Val281、Gly323、Ser443、Phe510。并通过计算机模拟突变分别计算突变株Val131Phe、Ala114Pro、Phe157Tyr、Ala193Ile、Glu240Asp、Val281Thr、Gly323Arg、Ser443Arg、Phe510Leu的“加权突变能量”，选择数值最小的突变类型进行突变实验。结果显示Ala114Pro,Glu240Asp,Gly323Arg,Ser443Arg以及Phe510Leu的能量数值最小，故选定114、240、323、443和510位分别进行定点饱和突变，对突变株进行热稳定筛选。
将实施例1中的重组大肠杆菌BL21(DE3)/pET28a(+)-ilvA提取质粒，以质粒pET28a(+)-ilvA为模板，根据亲本序列设计五对定点饱和突变的突变引物。
A114X-F:5’-CAACCGCCACCNNKGACATCAAAGTCGACGCGGTGCGCG-3’，
A114X-R:5’-GACTTTGATGTCMNNGGTGGCGGTTGGCATAACGATCAG-3’；
E240X-F:5’-GGCTATTTGCTNNKGGCGTAGCGGTAAAACGCATCGGTG-3’，
E240X-R:5’-ACCGCTACGCCMNNAGCAAATAGCCCTACGCGCGGCAGA-3’；
G323R-F:5’-TGAACTTCCACNNKCTGCGCTACGTCTCAGAACGCTGCGA-3’，
G323R-R:5’-ACGTAGCGCAGMNNGTGGAAGTTCACGTTGGCACCGGA-3’；
S443X-F:5’-AATTCCCGGAANNKCCGGGCGCGCTGCTGCGCTTCCTCAA-3’，
S443X-R:5’-AGCGCGCCCGGMNNTTCCGGGAATTCGAAGCTGTAGAGG-3’；
F510X-F:5’-CGGCGTTCAGGNNKTTTTTGGCGGGTTAAGCTTGCGGCCG-3’，
F510X-R:5’-CCCGCCAAAAAMNNCCTGAACGCCGGGTTATTGGTTTCG-3’。
利用PCR技术进行定点饱和突变。以EasyPfu DNA Polymerase进行PCR扩增(94℃5min；94℃30s，55℃30s，72℃3.5min，30个循环；72℃10min)。PCR产物经0.8％琼脂糖凝胶电泳验证后，结果扩增到与目的载体大小相符的基因片段。PCR产物经Dpn I酶切模板后，转入大肠杆菌BL21(DE3)感受态细胞(具体过程见实施例1)，感受态细胞在LB固体培养基(含卡那霉素50μg/mL)培养过夜，挑取单克隆进行培养并筛选热稳定突变株(具体过程见实施例2)。
筛选到两株突变株，其热稳定性都有显著提高。分别对这两株突变株进行培养并提取质粒，送北京擎科新业生物技术有限公司测序，测序结果显示这两株突变体均只有一个氨基酸发生突变，故将筛选到的这两株单突变体苏氨酸脱氨酶分别命名为G323D(其对应所述SEQ ID NO.1氨基酸序列第323位甘氨酸突变为天冬氨酸)，F510L(其对应所述SEQ IDNO.1氨基酸序列第510位脯氨酸突变为亮氨酸)。
实施例4：苏氨酸脱氨酶两点突变体的构建
以实施例3中获得单点突变体G323D编码基因为模板，以引入A14T、T344A、R449C、F510L突变点为模板进行突变，根据亲本序列设计定点突变的引物，引物为A14T-F:5’-CTCCGGAAGGTACTGAATATTTAAGAGCAGTGCT GCGCGCGCCGG-3’和A14T-R:5’-CTTAAATATTCAGTACCTTCCGGAGCAC CGGACAGGGGTTGCGAG-3’；T344A-F:5’-TGTTGGCGGTGGCCATTCCGGA AGAAAAAGGCAGCTTCCTCAAAT-3’和T344A-R:5’-TCTTCCGGAATGGCCACCGCCAACAACGCTTCACGCTGTTCGCCC-3’；R449C-F:5’-GCGCGCTGCT GTGTTTCCTCAACACGCTGGGTACGTACTGGAACA-3’和R449C-R:5’-GTGT TGAGGAAACACAGCAGCGCGCCCGGTGATTCCGGGAATTCG-3’；F510L-F:5’-CGGCGTTCAGGTTATTTTTGGCGGGTTAAGCTTGCGGCCGCACTC-3’和F510L-R:5’-CCCGCCAA AAATAACCTGAACGCCGGGTTATTGGTTTCGTCG TGG-3’(下划线标注处为突变碱基)。以EasyPfu DNA Polymerase进行PCR扩增(94℃5min；94℃30s，55℃30s，72℃3.5min，30个循环；72℃10min)。PCR产物经0.8％琼脂糖凝胶电泳验证后，结果扩增到与目的载体大小相符的基因片段。PCR产物经Dpn I酶切模板后，转入大肠杆菌BL21(DE3)感受态细胞(具体过程见实施例1)，感受态细胞在LB固体培养基(含卡那霉素50μg/mL)培养过夜，挑单克隆进行菌落PCR，验证为阳性克隆的菌落接种于LB液体培养基(含卡那霉素50μg/mL)中培养8h后，提取质粒，送北京擎科新业生物技术有限公司测序，测序结果显示正确，构建的双突变体苏氨酸脱氨酶命名为A14T/G323G、G323D/T344A、G323D/R449C、G323D/F510L。
实施例5：苏氨酸脱氨酶三点突变体的构建
以实施例4中获得的两点突变体G323D/F510L编码基因为模板，以引入T344A、R449C突变点为模板进行突变，根据亲本序列设计定点突变的引物，引物为T344A-F:5’-TGTTGGCGGTGGCCATTC CGGAAGAAAAAGGCAGCTTC CTCAAAT-3’和T344A-R:5’-TCTTCCGGAATGG CCACCGCCAACAACGCTTC ACGCTGTTCGCCC-3’；R449C-F:5’-GCGCGCTGCTGTGTTTCCTCAACACGCTGGGTACGTACTGGAACA-3’和R449C-R:5’-GTGTTGAGGAAACACAGCAGCGCGCCCGGTGATTCCGGGAATTCG-3’(下划线为突变碱基)。以EasyPfu DNA Polymerase进行PCR扩增(94℃5min；94℃30s，55℃30s，72℃3.5min，30个循环；72℃10min)。PCR产物经0.8％琼脂糖凝胶电泳验证后，结果扩增到与目的载体大小相符的基因片段。PCR产物经DpnI酶切模板后，转入大肠杆菌BL21(DE3)感受态细胞(具体过程见实施例1)，感受态细胞在LB固体培养基(含卡那霉素50μg/mL)培养过夜，挑单克隆进行菌落PCR，验证为阳性克隆的菌落接种于LB液体培养基(含卡那霉素50μg/mL)中培养8h后，提取质粒，送北京擎科新业生物技术有限公司测序，测序结果显示正确，构建的双突变体苏氨酸脱氨酶命名为G323D/F510L/T344A、G323D/F510L/R449C。
实施例6：苏氨酸脱氨酶四点突变体的构建
以实施例5中获得的三点突变体G323D/F510L/T344A的编码基因为模板，以引入R449C突变点为模板进行突变，根据亲本序列设计定点突变的引物，引物为R449C-F:5’-GCGCGCTGCTGTGTTTCCTCAACA CGCTGGGTACGTACT GGAACA-3’和R449C-R:5’-GTGTTGAGGAAACACAG CAGCGCGCCCGGTG ATTCCGGGAATTCG-3’(下划线为突变碱基)。以EasyPfu DNA Polymerase进行PCR扩增(94℃5min；94℃30s，55℃30s，72℃3.5min，30个循环；72℃10min)。PCR产物经0.8％琼脂糖凝胶电泳验证后，结果扩增到与目的载体大小相符的基因片段。PCR产物经Dpn I酶切模板后，转入大肠杆菌BL21(DE3)感受态细胞(具体过程见实施例1)，感受态细胞在LB固体培养基(含卡那霉素50μg/mL)培养过夜，挑单克隆进行菌落PCR，验证为阳性克隆的菌落接种于LB液体培养基(含卡那霉素50μg/mL)中培养8h后，提取质粒，送北京擎科新业生物技术有限公司测序，测序结果显示正确，构建的双突变体苏氨酸脱氨酶命名为G323D/F510L/T344A/R449C。
实施例7：野生型和突变型苏氨酸脱氨酶的诱导表达和纯化
将实施例1～6构建的野生型基因工程菌和突变型基因工程菌分别接种至含50μg/mL卡那霉素的50mL LB培养基中(1％蛋白胨，0.5％酵母提取物，1％氯化钠，pH 7.0)，37℃、220r/min振荡培养到OD600＝0.6～0.8时，加入终浓度0.1mM IPTG，28℃、220r/min振荡培养12h。4℃、9000×g离心10min收集菌体，生理盐水冲洗三次后，按每克菌体5mL的比例加入0.1M Tris-HCl缓冲液(pH 7.5)，冰预冷超声破胞，4℃，12000×g离心20min，取上清液与沉淀分别进行SDS-PAGE电泳分析，得到一条分子量约为51kDa的蛋白条带(见图2)。
破胞上清液即为粗酶液，经镍柱纯化，收集得到纯酶液，分别为wt-TD、A14T、G323D、T344A、R449C、F510L、A14T/G323D、G323D/T344A、G323D/R449C、G323D/F510L、G323D/F510L/T344A、G323D/F510L/R449C、G323D/F510L/T344A/R449C。
实施例8：野生型和突变型苏氨酸脱氨酶的酶活测定
取实施例7中野生型苏氨酸脱氨酶wt-TD，实施例7中单点突变体苏氨酸脱氨酶A14T、G323D、T344A、R449C、F510L，实施例7中双点突变体苏氨酸脱氨酶A14T/G323D、G323D/T344A、G323D/R449C、G323D/F510L，实施例7中三点突变体苏氨酸脱氨酶G323D/F510L/T344A、G323D/F510L/R449C，实施例7中四点突变体苏氨酸脱氨酶G323D/F510L/T344A/R449C纯酶液，测定该重组菌及其突变体对L-苏氨酸的酶活。
酶活测定方法：0.1M Tris-HCl(pH 7.5)，20μM PLP，2μg/mL纯化蛋白和10mM L-苏氨酸测定酶活。将酶预混液(含PLP)及底物分别加入酶标版，用酶标仪30℃保温5min，后将底物与酶预混液混合至终体积为200μL，起始反应。酶标仪每间隔10s采集一个数据，描述反应产物α-酮丁酸的变化情况，得到反应曲线。以初始3min内(线性范围，R^2>0.98)反应曲线的斜率为反应初始速率。
酶活单位定义：30℃，pH 7.5条件下，每分钟产生1μmolα-酮丁酸所需的酶蛋白量，即为一个苏氨酸脱氨酶活力单位，以U表示。苏氨酸脱氨酶比酶活以酶mg酶蛋白所含有的酶活力单位表示(U/mg)，结果见表1。突变株G323D/F510L/T344A的比酶活相较于野生型苏氨酸脱氨酶比酶活略有降低。
表1苏氨酸脱氨酶野生型及其突变体酶活力
实施例9：野生型和突变型苏氨酸脱氨酶的动力学常数测定
取实施例7中野生型苏氨酸脱氨酶wt-TD，实施例7中单点突变体苏氨酸脱氨酶A14T、G323D、T344A、R449C、F510L，实施例7中双点突变体苏氨酸脱氨酶A14T/G323D、G323D/T344A、G323D/R449C、G323D/F510L，实施例7中三点突变体苏氨酸脱氨酶G323D/F510L/T344A、G323D/F510L/R449C，实施例7中四点突变体苏氨酸脱氨酶G323D/F510L/T344A/R449C纯酶液，测定该重组菌及其突变体对L-苏氨酸的酶动力学参数。
测定方法：0.1M Tris-HCl(pH 7.5)，20μM PLP，2μg/mL纯化蛋白和0～100mM L-苏氨酸测定酶活。将酶预混液(含PLP)及底物分别加入酶标版，用酶标仪30℃保温5min，后将底物与酶预混液混合至终体积为200μL，起始反应。酶标仪每间隔10s采集一个数据，描述反应产物α-酮丁酸的变化情况，得到反应曲线。以初始3min内(线性范围，R^2>0.98)反应曲线的斜率为反应初始速率。
表2苏氨酸脱氨酶及其突变体酶动力学常数
实施例10：温度对野生型和突变型苏氨酸脱氨酶酶活和热稳定性影响测定
取实施例7中野生型苏氨酸脱氨酶wt-TD，实施例7中单点突变体苏氨酸脱氨酶A14T、G323D、T344A、R449C、F510L，实施例7中双点突变体苏氨酸脱氨酶A14T/G323D、G323D/T344A、G323D/R449C、G323D/F510L，实施例7中三点突变体苏氨酸脱氨酶G323D/F510L/T344A、G323D/F510L/R449C，实施例7中四点突变体苏氨酸脱氨酶G323D/F510L/T344A/R449C纯酶液，测定该重组菌及其突变体的热稳定参数。
酶在42℃下半衰期t1/2的测定：将一定量的酶置于42℃下保温，每间隔一定的时间取出等量的酶并测定其剩余酶活，从而确定处理后酶活损失一半所需的时间。突变株在42℃下的半衰期相较于野生型苏氨酸脱氨酶都有所延长，其中突变体G323D/F510l/T344A较野生型的半衰期提高了20倍。
T50 15(酶在该温度下热处理15min后，剩余原始酶活的一半)测定方法：取酶液在不同温度下(30～75℃)处理15min后，迅速冰浴，并测定其剩余酶活，从而确定T50 15。突变株在的T50 15相较于野生型苏氨酸脱氨酶的都有所提高，其中突变体G323D/F510l/T344A较野生型的T50 15提高了14℃。
纯化得到的野生型和突变株G323D/F510L/T344A在30～75℃下，按照上述检测酶活方法检测，结果参见图4。突变体G323D/F510l/T344A的最适反应温度较野生型苏氨酸脱氨酶的最适反应温度提高了20℃。
表3野生型和突变型苏氨酸脱氨酶热稳定性参数
实施例11：将本发明制得的苏氨酸脱氨酶突变体用于生产L-2-氨基丁酸
具体过程可以如下：
以L-苏氨酸、甲酸胺为底物，以NAD+为助剂，以0.1M Tris-HCl(pH 7.5)缓冲液为反应介质、加入酶源构成反应体系，在30～40℃、100r/min条件下反应，反应过程中以10％甲酸溶液调节溶液pH至7.5。
底物L-苏氨酸浓度为0.5～2M，甲酸胺浓度为0.5～2M，酶源用量为400～7000U/L，所述NAD+质量用量为0.03g/L～0.08g/L。
反应体系中酶源按如下步骤制备：将苏氨酸脱氨酶突变体重组菌(如上述实例中获得的G323D/F510L/T344A)、亮氨酸脱氢酶重组菌[Shinji N,et al.,Journal ofFermentaton&Bioengineering,1990,69(4):199-203]和甲酸脱氢酶重组菌[Jiang W,etal.,Applied and Environmental Microbiology,2017,83:1-12]接种至含终浓度50μg/mL卡那霉素的50mL LB液体培养基中，37℃、220r/min条件下培养至OD600＝0.6～0.8，添加终浓度0.1M IPTG，37℃、220r/min诱导培养10h，获得诱导培养液，将诱导培养液离心收集湿菌体，用0.1M Tris-HCl缓冲液(pH 7.5)润洗并重悬，超声破碎，置于50℃中水浴0～90min，4℃、12000×g离心30min后离心获得的上清液即为酶源。
上述反应中以L-苏氨酸为底物，反应体系中加入苏氨酸脱氨酶突变体将L-苏氨酸转化为α-酮丁酸，加入亮氨酸脱氢酶和甲酸脱氢酶，亮氨酸脱氢酶使α-酮丁酸转化为L-2-氨基丁酸同时消耗NADH，甲酸脱氢酶实现NADH的再生。
与野生型苏氨酸脱氨酶相比，本发明的苏氨酸脱氨酶具有极高的稳定性，可以大大提高工业化生产中苏氨酸脱氨酶的使用寿命。其中，G323D/F510L/T344A突变体应用于L-2-氨基丁酸的生产，可以使L-苏氨酸转化率达到99％，NAD+添加量只需0.04g/L，这为L-2-氨基丁酸的大规模生产提供了良好的技术支持。
序列表
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Leu Leu His Gly Ala Asn Phe Asp Glu Ala Lys Ala Lys Ala Ile Glu
130 135 140
Leu Ser Gln Gln Gln Gly Phe Thr Trp Val Pro Pro Phe Asp His Pro
145 150 155 160
Met Val Ile Ala Gly Gln Gly Thr Leu Ala Leu Glu Leu Leu Gln Gln
165 170 175
Asp Ala His Leu Asp Arg Val Phe Val Pro Val Gly Gly Gly Gly Leu
180 185 190
Ala Ala Gly Val Ala Val Leu Ile Lys Gln Leu Met Pro Gln Ile Lys
195 200 205
Val Ile Ala Val Glu Ala Glu Asp Ser Ala Cys Leu Lys Ala Ala Leu
210 215 220
Asp Ala Gly His Pro Val Asp Leu Pro Arg Val Gly Leu Phe Ala Glu
225 230 235 240
Gly Val Ala Val Lys Arg Ile Gly Asp Glu Thr Phe Arg Leu Cys Gln
245 250 255
Glu Tyr Leu Asp Asp Ile Ile Thr Val Asp Ser Asp Ala Ile Cys Ala
260 265 270
Ala Met Lys Asp Leu Phe Glu Asp Val Arg Ala Val Ala Glu Pro Ser
275 280 285
Gly Ala Leu Ala Leu Ala Gly Met Lys Lys Tyr Ile Ala Leu His Asn
290 295 300
Ile Arg Gly Glu Arg Leu Ala His Ile Leu Ser Gly Ala Asn Val Asn
305 310 315 320
Phe His Asp Leu Arg Tyr Val Ser Glu Arg Cys Glu Leu Gly Glu Gln
325 330 335
Arg Glu Ala Leu Leu Ala Val Thr Ile Pro Glu Glu Lys Gly Ser Phe
340 345 350
Leu Lys Phe Cys Gln Leu Leu Gly Gly Arg Ser Val Thr Glu Phe Asn
355 360 365
Tyr Arg Phe Ala Asp Ala Lys Asn Ala Cys Ile Phe Val Gly Val Arg
370 375 380
Leu Ser Arg Gly Leu Glu Glu Arg Lys Glu Ile Leu Gln Met Leu Asn
385 390 395 400
Asp Gly Gly Tyr Ser Val Val Asp Leu Ser Asp Asp Glu Met Ala Lys
405 410 415
Leu His Val Arg Tyr Met Val Gly Gly Arg Pro Ser His Pro Leu Gln
420 425 430
Glu Arg Leu Tyr Ser Phe Glu Phe Pro Glu Ser Pro Gly Ala Leu Leu
435 440 445
Arg Phe Leu Asn Thr Leu Gly Thr Tyr Trp Asn Ile Ser Leu Phe His
450 455 460
Tyr Arg Ser His Gly Thr Asp Tyr Gly Arg Val Leu Ala Ala Phe Glu
465 470 475 480
Leu Gly Asp His Glu Pro Asp Phe Glu Thr Arg Leu Asn Glu Leu Gly
485 490 495
Tyr Asp Cys His Asp Glu Thr Asn Asn Pro Ala Phe Arg Phe Phe Leu
500 505 510
Ala Gly
<210> 4
<211> 514
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 4
Met Ala Asp Ser Gln Pro Leu Ser Gly Ala Pro Glu Gly Ala Glu Tyr
1 5 10 15
Leu Arg Ala Val Leu Arg Ala Pro Val Tyr Glu Ala Ala Gln Val Thr
20 25 30
Pro Leu Gln Lys Met Glu Lys Leu Ser Ser Arg Leu Asp Asn Val Ile
35 40 45
Leu Val Lys Arg Glu Asp Arg Gln Pro Val His Ser Phe Lys Leu Arg
50 55 60
Gly Ala Tyr Ala Met Met Ala Gly Leu Thr Glu Glu Gln Lys Ala His
65 70 75 80
Gly Val Ile Thr Ala Ser Ala Gly Asn His Ala Gln Gly Val Ala Phe
85 90 95
Ser Ser Ala Arg Leu Gly Val Lys Ala Leu Ile Val Met Pro Thr Ala
100 105 110
Thr Ala Asp Ile Lys Val Asp Ala Val Arg Gly Phe Gly Gly Glu Val
115 120 125
Leu Leu His Gly Ala Asn Phe Asp Glu Ala Lys Ala Lys Ala Ile Glu
130 135 140
Leu Ser Gln Gln Gln Gly Phe Thr Trp Val Pro Pro Phe Asp His Pro
145 150 155 160
Met Val Ile Ala Gly Gln Gly Thr Leu Ala Leu Glu Leu Leu Gln Gln
165 170 175
Asp Ala His Leu Asp Arg Val Phe Val Pro Val Gly Gly Gly Gly Leu
180 185 190
Ala Ala Gly Val Ala Val Leu Ile Lys Gln Leu Met Pro Gln Ile Lys
195 200 205
Val Ile Ala Val Glu Ala Glu Asp Ser Ala Cys Leu Lys Ala Ala Leu
210 215 220
Asp Ala Gly His Pro Val Asp Leu Pro Arg Val Gly Leu Phe Ala Glu
225 230 235 240
Gly Val Ala Val Lys Arg Ile Gly Asp Glu Thr Phe Arg Leu Cys Gln
245 250 255
Glu Tyr Leu Asp Asp Ile Ile Thr Val Asp Ser Asp Ala Ile Cys Ala
260 265 270
Ala Met Lys Asp Leu Phe Glu Asp Val Arg Ala Val Ala Glu Pro Ser
275 280 285
Gly Ala Leu Ala Leu Ala Gly Met Lys Lys Tyr Ile Ala Leu His Asn
290 295 300
Ile Arg Gly Glu Arg Leu Ala His Ile Leu Ser Gly Ala Asn Val Asn
305 310 315 320
Phe His Gly Leu Arg Tyr Val Ser Glu Arg Cys Glu Leu Gly Glu Gln
325 330 335
Arg Glu Ala Leu Leu Ala Val Ala Ile Pro Glu Glu Lys Gly Ser Phe
340 345 350
Leu Lys Phe Cys Gln Leu Leu Gly Gly Arg Ser Val Thr Glu Phe Asn
355 360 365
Tyr Arg Phe Ala Asp Ala Lys Asn Ala Cys Ile Phe Val Gly Val Arg
370 375 380
Leu Ser Arg Gly Leu Glu Glu Arg Lys Glu Ile Leu Gln Met Leu Asn
385 390 395 400
Asp Gly Gly Tyr Ser Val Val Asp Leu Ser Asp Asp Glu Met Ala Lys
405 410 415
Leu His Val Arg Tyr Met Val Gly Gly Arg Pro Ser His Pro Leu Gln
420 425 430
Glu Arg Leu Tyr Ser Phe Glu Phe Pro Glu Ser Pro Gly Ala Leu Leu
435 440 445
Arg Phe Leu Asn Thr Leu Gly Thr Tyr Trp Asn Ile Ser Leu Phe His
450 455 460
Tyr Arg Ser His Gly Thr Asp Tyr Gly Arg Val Leu Ala Ala Phe Glu
465 470 475 480
Leu Gly Asp His Glu Pro Asp Phe Glu Thr Arg Leu Asn Glu Leu Gly
485 490 495
Tyr Asp Cys His Asp Glu Thr Asn Asn Pro Ala Phe Arg Phe Phe Leu
500 505 510
Ala Gly
<210> 5
<211> 514
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 5
Met Ala Asp Ser Gln Pro Leu Ser Gly Ala Pro Glu Gly Ala Glu Tyr
1 5 10 15
Leu Arg Ala Val Leu Arg Ala Pro Val Tyr Glu Ala Ala Gln Val Thr
20 25 30
Pro Leu Gln Lys Met Glu Lys Leu Ser Ser Arg Leu Asp Asn Val Ile
35 40 45
Leu Val Lys Arg Glu Asp Arg Gln Pro Val His Ser Phe Lys Leu Arg
50 55 60
Gly Ala Tyr Ala Met Met Ala Gly Leu Thr Glu Glu Gln Lys Ala His
65 70 75 80
Gly Val Ile Thr Ala Ser Ala Gly Asn His Ala Gln Gly Val Ala Phe
85 90 95
Ser Ser Ala Arg Leu Gly Val Lys Ala Leu Ile Val Met Pro Thr Ala
100 105 110
Thr Ala Asp Ile Lys Val Asp Ala Val Arg Gly Phe Gly Gly Glu Val
115 120 125
Leu Leu His Gly Ala Asn Phe Asp Glu Ala Lys Ala Lys Ala Ile Glu
130 135 140
Leu Ser Gln Gln Gln Gly Phe Thr Trp Val Pro Pro Phe Asp His Pro
145 150 155 160
Met Val Ile Ala Gly Gln Gly Thr Leu Ala Leu Glu Leu Leu Gln Gln
165 170 175
Asp Ala His Leu Asp Arg Val Phe Val Pro Val Gly Gly Gly Gly Leu
180 185 190
Ala Ala Gly Val Ala Val Leu Ile Lys Gln Leu Met Pro Gln Ile Lys
195 200 205
Val Ile Ala Val Glu Ala Glu Asp Ser Ala Cys Leu Lys Ala Ala Leu
210 215 220
Asp Ala Gly His Pro Val Asp Leu Pro Arg Val Gly Leu Phe Ala Glu
225 230 235 240
Gly Val Ala Val Lys Arg Ile Gly Asp Glu Thr Phe Arg Leu Cys Gln
245 250 255
Glu Tyr Leu Asp Asp Ile Ile Thr Val Asp Ser Asp Ala Ile Cys Ala
260 265 270
Ala Met Lys Asp Leu Phe Glu Asp Val Arg Ala Val Ala Glu Pro Ser
275 280 285
Gly Ala Leu Ala Leu Ala Gly Met Lys Lys Tyr Ile Ala Leu His Asn
290 295 300
Ile Arg Gly Glu Arg Leu Ala His Ile Leu Ser Gly Ala Asn Val Asn
305 310 315 320
Phe His Gly Leu Arg Tyr Val Ser Glu Arg Cys Glu Leu Gly Glu Gln
325 330 335
Arg Glu Ala Leu Leu Ala Val Thr Ile Pro Glu Glu Lys Gly Ser Phe
340 345 350
Leu Lys Phe Cys Gln Leu Leu Gly Gly Arg Ser Val Thr Glu Phe Asn
355 360 365
Tyr Arg Phe Ala Asp Ala Lys Asn Ala Cys Ile Phe Val Gly Val Arg
370 375 380
Leu Ser Arg Gly Leu Glu Glu Arg Lys Glu Ile Leu Gln Met Leu Asn
385 390 395 400
Asp Gly Gly Tyr Ser Val Val Asp Leu Ser Asp Asp Glu Met Ala Lys
405 410 415
Leu His Val Arg Tyr Met Val Gly Gly Arg Pro Ser His Pro Leu Gln
420 425 430
Glu Arg Leu Tyr Ser Phe Glu Phe Pro Glu Ser Pro Gly Ala Leu Leu
435 440 445
Cys Phe Leu Asn Thr Leu Gly Thr Tyr Trp Asn Ile Ser Leu Phe His
450 455 460
Tyr Arg Ser His Gly Thr Asp Tyr Gly Arg Val Leu Ala Ala Phe Glu
465 470 475 480
Leu Gly Asp His Glu Pro Asp Phe Glu Thr Arg Leu Asn Glu Leu Gly
485 490 495
Tyr Asp Cys His Asp Glu Thr Asn Asn Pro Ala Phe Arg Phe Phe Leu
500 505 510
Ala Gly
<210> 6
<211> 514
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 6
Met Ala Asp Ser Gln Pro Leu Ser Gly Ala Pro Glu Gly Ala Glu Tyr
1 5 10 15
Leu Arg Ala Val Leu Arg Ala Pro Val Tyr Glu Ala Ala Gln Val Thr
20 25 30
Pro Leu Gln Lys Met Glu Lys Leu Ser Ser Arg Leu Asp Asn Val Ile
35 40 45
Leu Val Lys Arg Glu Asp Arg Gln Pro Val His Ser Phe Lys Leu Arg
50 55 60
Gly Ala Tyr Ala Met Met Ala Gly Leu Thr Glu Glu Gln Lys Ala His
65 70 75 80
Gly Val Ile Thr Ala Ser Ala Gly Asn His Ala Gln Gly Val Ala Phe
85 90 95
Ser Ser Ala Arg Leu Gly Val Lys Ala Leu Ile Val Met Pro Thr Ala
100 105 110
Thr Ala Asp Ile Lys Val Asp Ala Val Arg Gly Phe Gly Gly Glu Val
115 120 125
Leu Leu His Gly Ala Asn Phe Asp Glu Ala Lys Ala Lys Ala Ile Glu
130 135 140
Leu Ser Gln Gln Gln Gly Phe Thr Trp Val Pro Pro Phe Asp His Pro
145 150 155 160
Met Val Ile Ala Gly Gln Gly Thr Leu Ala Leu Glu Leu Leu Gln Gln
165 170 175
Asp Ala His Leu Asp Arg Val Phe Val Pro Val Gly Gly Gly Gly Leu
180 185 190
Ala Ala Gly Val Ala Val Leu Ile Lys Gln Leu Met Pro Gln Ile Lys
195 200 205
Val Ile Ala Val Glu Ala Glu Asp Ser Ala Cys Leu Lys Ala Ala Leu
210 215 220
Asp Ala Gly His Pro Val Asp Leu Pro Arg Val Gly Leu Phe Ala Glu
225 230 235 240
Gly Val Ala Val Lys Arg Ile Gly Asp Glu Thr Phe Arg Leu Cys Gln
245 250 255
Glu Tyr Leu Asp Asp Ile Ile Thr Val Asp Ser Asp Ala Ile Cys Ala
260 265 270
Ala Met Lys Asp Leu Phe Glu Asp Val Arg Ala Val Ala Glu Pro Ser
275 280 285
Gly Ala Leu Ala Leu Ala Gly Met Lys Lys Tyr Ile Ala Leu His Asn
290 295 300
Ile Arg Gly Glu Arg Leu Ala His Ile Leu Ser Gly Ala Asn Val Asn
305 310 315 320
Phe His Gly Leu Arg Tyr Val Ser Glu Arg Cys Glu Leu Gly Glu Gln
325 330 335
Arg Glu Ala Leu Leu Ala Val Thr Ile Pro Glu Glu Lys Gly Ser Phe
340 345 350
Leu Lys Phe Cys Gln Leu Leu Gly Gly Arg Ser Val Thr Glu Phe Asn
355 360 365
Tyr Arg Phe Ala Asp Ala Lys Asn Ala Cys Ile Phe Val Gly Val Arg
370 375 380
Leu Ser Arg Gly Leu Glu Glu Arg Lys Glu Ile Leu Gln Met Leu Asn
385 390 395 400
Asp Gly Gly Tyr Ser Val Val Asp Leu Ser Asp Asp Glu Met Ala Lys
405 410 415
Leu His Val Arg Tyr Met Val Gly Gly Arg Pro Ser His Pro Leu Gln
420 425 430
Glu Arg Leu Tyr Ser Phe Glu Phe Pro Glu Ser Pro Gly Ala Leu Leu
435 440 445
Arg Phe Leu Asn Thr Leu Gly Thr Tyr Trp Asn Ile Ser Leu Phe His
450 455 460
Tyr Arg Ser His Gly Thr Asp Tyr Gly Arg Val Leu Ala Ala Phe Glu
465 470 475 480
Leu Gly Asp His Glu Pro Asp Phe Glu Thr Arg Leu Asn Glu Leu Gly
485 490 495
Tyr Asp Cys His Asp Glu Thr Asn Asn Pro Ala Phe Arg Leu Phe Leu
500 505 510
Ala Gly
<210> 7
<211> 514
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 7
Met Ala Asp Ser Gln Pro Leu Ser Gly Ala Pro Glu Gly Thr Glu Tyr
1 5 10 15
Leu Arg Ala Val Leu Arg Ala Pro Val Tyr Glu Ala Ala Gln Val Thr
20 25 30
Pro Leu Gln Lys Met Glu Lys Leu Ser Ser Arg Leu Asp Asn Val Ile
35 40 45
Leu Val Lys Arg Glu Asp Arg Gln Pro Val His Ser Phe Lys Leu Arg
50 55 60
Gly Ala Tyr Ala Met Met Ala Gly Leu Thr Glu Glu Gln Lys Ala His
65 70 75 80
Gly Val Ile Thr Ala Ser Ala Gly Asn His Ala Gln Gly Val Ala Phe
85 90 95
Ser Ser Ala Arg Leu Gly Val Lys Ala Leu Ile Val Met Pro Thr Ala
100 105 110
Thr Ala Asp Ile Lys Val Asp Ala Val Arg Gly Phe Gly Gly Glu Val
115 120 125
Leu Leu His Gly Ala Asn Phe Asp Glu Ala Lys Ala Lys Ala Ile Glu
130 135 140
Leu Ser Gln Gln Gln Gly Phe Thr Trp Val Pro Pro Phe Asp His Pro
145 150 155 160
Met Val Ile Ala Gly Gln Gly Thr Leu Ala Leu Glu Leu Leu Gln Gln
165 170 175
Asp Ala His Leu Asp Arg Val Phe Val Pro Val Gly Gly Gly Gly Leu
180 185 190
Ala Ala Gly Val Ala Val Leu Ile Lys Gln Leu Met Pro Gln Ile Lys
195 200 205
Val Ile Ala Val Glu Ala Glu Asp Ser Ala Cys Leu Lys Ala Ala Leu
210 215 220
Asp Ala Gly His Pro Val Asp Leu Pro Arg Val Gly Leu Phe Ala Glu
225 230 235 240
Gly Val Ala Val Lys Arg Ile Gly Asp Glu Thr Phe Arg Leu Cys Gln
245 250 255
Glu Tyr Leu Asp Asp Ile Ile Thr Val Asp Ser Asp Ala Ile Cys Ala
260 265 270
Ala Met Lys Asp Leu Phe Glu Asp Val Arg Ala Val Ala Glu Pro Ser
275 280 285
Gly Ala Leu Ala Leu Ala Gly Met Lys Lys Tyr Ile Ala Leu His Asn
290 295 300
Ile Arg Gly Glu Arg Leu Ala His Ile Leu Ser Gly Ala Asn Val Asn
305 310 315 320
Phe His Asp Leu Arg Tyr Val Ser Glu Arg Cys Glu Leu Gly Glu Gln
325 330 335
Arg Glu Ala Leu Leu Ala Val Thr Ile Pro Glu Glu Lys Gly Ser Phe
340 345 350
Leu Lys Phe Cys Gln Leu Leu Gly Gly Arg Ser Val Thr Glu Phe Asn
355 360 365
Tyr Arg Phe Ala Asp Ala Lys Asn Ala Cys Ile Phe Val Gly Val Arg
370 375 380
Leu Ser Arg Gly Leu Glu Glu Arg Lys Glu Ile Leu Gln Met Leu Asn
385 390 395 400
Asp Gly Gly Tyr Ser Val Val Asp Leu Ser Asp Asp Glu Met Ala Lys
405 410 415
Leu His Val Arg Tyr Met Val Gly Gly Arg Pro Ser His Pro Leu Gln
420 425 430
Glu Arg Leu Tyr Ser Phe Glu Phe Pro Glu Ser Pro Gly Ala Leu Leu
435 440 445
Arg Phe Leu Asn Thr Leu Gly Thr Tyr Trp Asn Ile Ser Leu Phe His
450 455 460
Tyr Arg Ser His Gly Thr Asp Tyr Gly Arg Val Leu Ala Ala Phe Glu
465 470 475 480
Leu Gly Asp His Glu Pro Asp Phe Glu Thr Arg Leu Asn Glu Leu Gly
485 490 495
Tyr Asp Cys His Asp Glu Thr Asn Asn Pro Ala Phe Arg Phe Phe Leu
500 505 510
Ala Gly
<210> 8
<211> 514
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 8
Met Ala Asp Ser Gln Pro Leu Ser Gly Ala Pro Glu Gly Ala Glu Tyr
1 5 10 15
Leu Arg Ala Val Leu Arg Ala Pro Val Tyr Glu Ala Ala Gln Val Thr
20 25 30
Pro Leu Gln Lys Met Glu Lys Leu Ser Ser Arg Leu Asp Asn Val Ile
35 40 45
Leu Val Lys Arg Glu Asp Arg Gln Pro Val His Ser Phe Lys Leu Arg
50 55 60
Gly Ala Tyr Ala Met Met Ala Gly Leu Thr Glu Glu Gln Lys Ala His
65 70 75 80
Gly Val Ile Thr Ala Ser Ala Gly Asn His Ala Gln Gly Val Ala Phe
85 90 95
Ser Ser Ala Arg Leu Gly Val Lys Ala Leu Ile Val Met Pro Thr Ala
100 105 110
Thr Ala Asp Ile Lys Val Asp Ala Val Arg Gly Phe Gly Gly Glu Val
115 120 125
Leu Leu His Gly Ala Asn Phe Asp Glu Ala Lys Ala Lys Ala Ile Glu
130 135 140
Leu Ser Gln Gln Gln Gly Phe Thr Trp Val Pro Pro Phe Asp His Pro
145 150 155 160
Met Val Ile Ala Gly Gln Gly Thr Leu Ala Leu Glu Leu Leu Gln Gln
165 170 175
Asp Ala His Leu Asp Arg Val Phe Val Pro Val Gly Gly Gly Gly Leu
180 185 190
Ala Ala Gly Val Ala Val Leu Ile Lys Gln Leu Met Pro Gln Ile Lys
195 200 205
Val Ile Ala Val Glu Ala Glu Asp Ser Ala Cys Leu Lys Ala Ala Leu
210 215 220
Asp Ala Gly His Pro Val Asp Leu Pro Arg Val Gly Leu Phe Ala Glu
225 230 235 240
Gly Val Ala Val Lys Arg Ile Gly Asp Glu Thr Phe Arg Leu Cys Gln
245 250 255
Glu Tyr Leu Asp Asp Ile Ile Thr Val Asp Ser Asp Ala Ile Cys Ala
260 265 270
Ala Met Lys Asp Leu Phe Glu Asp Val Arg Ala Val Ala Glu Pro Ser
275 280 285
Gly Ala Leu Ala Leu Ala Gly Met Lys Lys Tyr Ile Ala Leu His Asn
290 295 300
Ile Arg Gly Glu Arg Leu Ala His Ile Leu Ser Gly Ala Asn Val Asn
305 310 315 320
Phe His Asp Leu Arg Tyr Val Ser Glu Arg Cys Glu Leu Gly Glu Gln
325 330 335
Arg Glu Ala Leu Leu Ala Val Ala Ile Pro Glu Glu Lys Gly Ser Phe
340 345 350
Leu Lys Phe Cys Gln Leu Leu Gly Gly Arg Ser Val Thr Glu Phe Asn
355 360 365
Tyr Arg Phe Ala Asp Ala Lys Asn Ala Cys Ile Phe Val Gly Val Arg
370 375 380
Leu Ser Arg Gly Leu Glu Glu Arg Lys Glu Ile Leu Gln Met Leu Asn
385 390 395 400
Asp Gly Gly Tyr Ser Val Val Asp Leu Ser Asp Asp Glu Met Ala Lys
405 410 415
Leu His Val Arg Tyr Met Val Gly Gly Arg Pro Ser His Pro Leu Gln
420 425 430
Glu Arg Leu Tyr Ser Phe Glu Phe Pro Glu Ser Pro Gly Ala Leu Leu
435 440 445
Arg Phe Leu Asn Thr Leu Gly Thr Tyr Trp Asn Ile Ser Leu Phe His
450 455 460
Tyr Arg Ser His Gly Thr Asp Tyr Gly Arg Val Leu Ala Ala Phe Glu
465 470 475 480
Leu Gly Asp His Glu Pro Asp Phe Glu Thr Arg Leu Asn Glu Leu Gly
485 490 495
Tyr Asp Cys His Asp Glu Thr Asn Asn Pro Ala Phe Arg Phe Phe Leu
500 505 510
Ala Gly
<210> 9
<211> 514
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 9
Met Ala Asp Ser Gln Pro Leu Ser Gly Ala Pro Glu Gly Ala Glu Tyr
1 5 10 15
Leu Arg Ala Val Leu Arg Ala Pro Val Tyr Glu Ala Ala Gln Val Thr
20 25 30
Pro Leu Gln Lys Met Glu Lys Leu Ser Ser Arg Leu Asp Asn Val Ile
35 40 45
Leu Val Lys Arg Glu Asp Arg Gln Pro Val His Ser Phe Lys Leu Arg
50 55 60
Gly Ala Tyr Ala Met Met Ala Gly Leu Thr Glu Glu Gln Lys Ala His
65 70 75 80
Gly Val Ile Thr Ala Ser Ala Gly Asn His Ala Gln Gly Val Ala Phe
85 90 95
Ser Ser Ala Arg Leu Gly Val Lys Ala Leu Ile Val Met Pro Thr Ala
100 105 110
Thr Ala Asp Ile Lys Val Asp Ala Val Arg Gly Phe Gly Gly Glu Val
115 120 125
Leu Leu His Gly Ala Asn Phe Asp Glu Ala Lys Ala Lys Ala Ile Glu
130 135 140
Leu Ser Gln Gln Gln Gly Phe Thr Trp Val Pro Pro Phe Asp His Pro
145 150 155 160
Met Val Ile Ala Gly Gln Gly Thr Leu Ala Leu Glu Leu Leu Gln Gln
165 170 175
Asp Ala His Leu Asp Arg Val Phe Val Pro Val Gly Gly Gly Gly Leu
180 185 190
Ala Ala Gly Val Ala Val Leu Ile Lys Gln Leu Met Pro Gln Ile Lys
195 200 205
Val Ile Ala Val Glu Ala Glu Asp Ser Ala Cys Leu Lys Ala Ala Leu
210 215 220
Asp Ala Gly His Pro Val Asp Leu Pro Arg Val Gly Leu Phe Ala Glu
225 230 235 240
Gly Val Ala Val Lys Arg Ile Gly Asp Glu Thr Phe Arg Leu Cys Gln
245 250 255
Glu Tyr Leu Asp Asp Ile Ile Thr Val Asp Ser Asp Ala Ile Cys Ala
260 265 270
Ala Met Lys Asp Leu Phe Glu Asp Val Arg Ala Val Ala Glu Pro Ser
275 280 285
Gly Ala Leu Ala Leu Ala Gly Met Lys Lys Tyr Ile Ala Leu His Asn
290 295 300
Ile Arg Gly Glu Arg Leu Ala His Ile Leu Ser Gly Ala Asn Val Asn
305 310 315 320
Phe His Asp Leu Arg Tyr Val Ser Glu Arg Cys Glu Leu Gly Glu Gln
325 330 335
Arg Glu Ala Leu Leu Ala Val Thr Ile Pro Glu Glu Lys Gly Ser Phe
340 345 350
Leu Lys Phe Cys Gln Leu Leu Gly Gly Arg Ser Val Thr Glu Phe Asn
355 360 365
Tyr Arg Phe Ala Asp Ala Lys Asn Ala Cys Ile Phe Val Gly Val Arg
370 375 380
Leu Ser Arg Gly Leu Glu Glu Arg Lys Glu Ile Leu Gln Met Leu Asn
385 390 395 400
Asp Gly Gly Tyr Ser Val Val Asp Leu Ser Asp Asp Glu Met Ala Lys
405 410 415
Leu His Val Arg Tyr Met Val Gly Gly Arg Pro Ser His Pro Leu Gln
420 425 430
Glu Arg Leu Tyr Ser Phe Glu Phe Pro Glu Ser Pro Gly Ala Leu Leu
435 440 445
Cys Phe Leu Asn Thr Leu Gly Thr Tyr Trp Asn Ile Ser Leu Phe His
450 455 460
Tyr Arg Ser His Gly Thr Asp Tyr Gly Arg Val Leu Ala Ala Phe Glu
465 470 475 480
Leu Gly Asp His Glu Pro Asp Phe Glu Thr Arg Leu Asn Glu Leu Gly
485 490 495
Tyr Asp Cys His Asp Glu Thr Asn Asn Pro Ala Phe Arg Phe Phe Leu
500 505 510
Ala Gly
<210> 10
<211> 514
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 10
Met Ala Asp Ser Gln Pro Leu Ser Gly Ala Pro Glu Gly Ala Glu Tyr
1 5 10 15
Leu Arg Ala Val Leu Arg Ala Pro Val Tyr Glu Ala Ala Gln Val Thr
20 25 30
Pro Leu Gln Lys Met Glu Lys Leu Ser Ser Arg Leu Asp Asn Val Ile
35 40 45
Leu Val Lys Arg Glu Asp Arg Gln Pro Val His Ser Phe Lys Leu Arg
50 55 60
Gly Ala Tyr Ala Met Met Ala Gly Leu Thr Glu Glu Gln Lys Ala His
65 70 75 80
Gly Val Ile Thr Ala Ser Ala Gly Asn His Ala Gln Gly Val Ala Phe
85 90 95
Ser Ser Ala Arg Leu Gly Val Lys Ala Leu Ile Val Met Pro Thr Ala
100 105 110
Thr Ala Asp Ile Lys Val Asp Ala Val Arg Gly Phe Gly Gly Glu Val
115 120 125
Leu Leu His Gly Ala Asn Phe Asp Glu Ala Lys Ala Lys Ala Ile Glu
130 135 140
Leu Ser Gln Gln Gln Gly Phe Thr Trp Val Pro Pro Phe Asp His Pro
145 150 155 160
Met Val Ile Ala Gly Gln Gly Thr Leu Ala Leu Glu Leu Leu Gln Gln
165 170 175
Asp Ala His Leu Asp Arg Val Phe Val Pro Val Gly Gly Gly Gly Leu
180 185 190
Ala Ala Gly Val Ala Val Leu Ile Lys Gln Leu Met Pro Gln Ile Lys
195 200 205
Val Ile Ala Val Glu Ala Glu Asp Ser Ala Cys Leu Lys Ala Ala Leu
210 215 220
Asp Ala Gly His Pro Val Asp Leu Pro Arg Val Gly Leu Phe Ala Glu
225 230 235 240
Gly Val Ala Val Lys Arg Ile Gly Asp Glu Thr Phe Arg Leu Cys Gln
245 250 255
Glu Tyr Leu Asp Asp Ile Ile Thr Val Asp Ser Asp Ala Ile Cys Ala
260 265 270
Ala Met Lys Asp Leu Phe Glu Asp Val Arg Ala Val Ala Glu Pro Ser
275 280 285
Gly Ala Leu Ala Leu Ala Gly Met Lys Lys Tyr Ile Ala Leu His Asn
290 295 300
Ile Arg Gly Glu Arg Leu Ala His Ile Leu Ser Gly Ala Asn Val Asn
305 310 315 320
Phe His Asp Leu Arg Tyr Val Ser Glu Arg Cys Glu Leu Gly Glu Gln
325 330 335
Arg Glu Ala Leu Leu Ala Val Thr Ile Pro Glu Glu Lys Gly Ser Phe
340 345 350
Leu Lys Phe Cys Gln Leu Leu Gly Gly Arg Ser Val Thr Glu Phe Asn
355 360 365
Tyr Arg Phe Ala Asp Ala Lys Asn Ala Cys Ile Phe Val Gly Val Arg
370 375 380
Leu Ser Arg Gly Leu Glu Glu Arg Lys Glu Ile Leu Gln Met Leu Asn
385 390 395 400
Asp Gly Gly Tyr Ser Val Val Asp Leu Ser Asp Asp Glu Met Ala Lys
405 410 415
Leu His Val Arg Tyr Met Val Gly Gly Arg Pro Ser His Pro Leu Gln
420 425 430
Glu Arg Leu Tyr Ser Phe Glu Phe Pro Glu Ser Pro Gly Ala Leu Leu
435 440 445
Arg Phe Leu Asn Thr Leu Gly Thr Tyr Trp Asn Ile Ser Leu Phe His
450 455 460
Tyr Arg Ser His Gly Thr Asp Tyr Gly Arg Val Leu Ala Ala Phe Glu
465 470 475 480
Leu Gly Asp His Glu Pro Asp Phe Glu Thr Arg Leu Asn Glu Leu Gly
485 490 495
Tyr Asp Cys His Asp Glu Thr Asn Asn Pro Ala Phe Arg Leu Phe Leu
500 505 510
Ala Gly
<210> 11
<211> 514
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 11
Met Ala Asp Ser Gln Pro Leu Ser Gly Ala Pro Glu Gly Ala Glu Tyr
1 5 10 15
Leu Arg Ala Val Leu Arg Ala Pro Val Tyr Glu Ala Ala Gln Val Thr
20 25 30
Pro Leu Gln Lys Met Glu Lys Leu Ser Ser Arg Leu Asp Asn Val Ile
35 40 45
Leu Val Lys Arg Glu Asp Arg Gln Pro Val His Ser Phe Lys Leu Arg
50 55 60
Gly Ala Tyr Ala Met Met Ala Gly Leu Thr Glu Glu Gln Lys Ala His
65 70 75 80
Gly Val Ile Thr Ala Ser Ala Gly Asn His Ala Gln Gly Val Ala Phe
85 90 95
Ser Ser Ala Arg Leu Gly Val Lys Ala Leu Ile Val Met Pro Thr Ala
100 105 110
Thr Ala Asp Ile Lys Val Asp Ala Val Arg Gly Phe Gly Gly Glu Val
115 120 125
Leu Leu His Gly Ala Asn Phe Asp Glu Ala Lys Ala Lys Ala Ile Glu
130 135 140
Leu Ser Gln Gln Gln Gly Phe Thr Trp Val Pro Pro Phe Asp His Pro
145 150 155 160
Met Val Ile Ala Gly Gln Gly Thr Leu Ala Leu Glu Leu Leu Gln Gln
165 170 175
Asp Ala His Leu Asp Arg Val Phe Val Pro Val Gly Gly Gly Gly Leu
180 185 190
Ala Ala Gly Val Ala Val Leu Ile Lys Gln Leu Met Pro Gln Ile Lys
195 200 205
Val Ile Ala Val Glu Ala Glu Asp Ser Ala Cys Leu Lys Ala Ala Leu
210 215 220
Asp Ala Gly His Pro Val Asp Leu Pro Arg Val Gly Leu Phe Ala Glu
225 230 235 240
Gly Val Ala Val Lys Arg Ile Gly Asp Glu Thr Phe Arg Leu Cys Gln
245 250 255
Glu Tyr Leu Asp Asp Ile Ile Thr Val Asp Ser Asp Ala Ile Cys Ala
260 265 270
Ala Met Lys Asp Leu Phe Glu Asp Val Arg Ala Val Ala Glu Pro Ser
275 280 285
Gly Ala Leu Ala Leu Ala Gly Met Lys Lys Tyr Ile Ala Leu His Asn
290 295 300
Ile Arg Gly Glu Arg Leu Ala His Ile Leu Ser Gly Ala Asn Val Asn
305 310 315 320
Phe His Asp Leu Arg Tyr Val Ser Glu Arg Cys Glu Leu Gly Glu Gln
325 330 335
Arg Glu Ala Leu Leu Ala Val Ala Ile Pro Glu Glu Lys Gly Ser Phe
340 345 350
Leu Lys Phe Cys Gln Leu Leu Gly Gly Arg Ser Val Thr Glu Phe Asn
355 360 365
Tyr Arg Phe Ala Asp Ala Lys Asn Ala Cys Ile Phe Val Gly Val Arg
370 375 380
Leu Ser Arg Gly Leu Glu Glu Arg Lys Glu Ile Leu Gln Met Leu Asn
385 390 395 400
Asp Gly Gly Tyr Ser Val Val Asp Leu Ser Asp Asp Glu Met Ala Lys
405 410 415
Leu His Val Arg Tyr Met Val Gly Gly Arg Pro Ser His Pro Leu Gln
420 425 430
Glu Arg Leu Tyr Ser Phe Glu Phe Pro Glu Ser Pro Gly Ala Leu Leu
435 440 445
Arg Phe Leu Asn Thr Leu Gly Thr Tyr Trp Asn Ile Ser Leu Phe His
450 455 460
Tyr Arg Ser His Gly Thr Asp Tyr Gly Arg Val Leu Ala Ala Phe Glu
465 470 475 480
Leu Gly Asp His Glu Pro Asp Phe Glu Thr Arg Leu Asn Glu Leu Gly
485 490 495
Tyr Asp Cys His Asp Glu Thr Asn Asn Pro Ala Phe Arg Leu Phe Leu
500 505 510
Ala Gly
<210> 12
<211> 514
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 12
Met Ala Asp Ser Gln Pro Leu Ser Gly Ala Pro Glu Gly Ala Glu Tyr
1 5 10 15
Leu Arg Ala Val Leu Arg Ala Pro Val Tyr Glu Ala Ala Gln Val Thr
20 25 30
Pro Leu Gln Lys Met Glu Lys Leu Ser Ser Arg Leu Asp Asn Val Ile
35 40 45
Leu Val Lys Arg Glu Asp Arg Gln Pro Val His Ser Phe Lys Leu Arg
50 55 60
Gly Ala Tyr Ala Met Met Ala Gly Leu Thr Glu Glu Gln Lys Ala His
65 70 75 80
Gly Val Ile Thr Ala Ser Ala Gly Asn His Ala Gln Gly Val Ala Phe
85 90 95
Ser Ser Ala Arg Leu Gly Val Lys Ala Leu Ile Val Met Pro Thr Ala
100 105 110
Thr Ala Asp Ile Lys Val Asp Ala Val Arg Gly Phe Gly Gly Glu Val
115 120 125
Leu Leu His Gly Ala Asn Phe Asp Glu Ala Lys Ala Lys Ala Ile Glu
130 135 140
Leu Ser Gln Gln Gln Gly Phe Thr Trp Val Pro Pro Phe Asp His Pro
145 150 155 160
Met Val Ile Ala Gly Gln Gly Thr Leu Ala Leu Glu Leu Leu Gln Gln
165 170 175
Asp Ala His Leu Asp Arg Val Phe Val Pro Val Gly Gly Gly Gly Leu
180 185 190
Ala Ala Gly Val Ala Val Leu Ile Lys Gln Leu Met Pro Gln Ile Lys
195 200 205
Val Ile Ala Val Glu Ala Glu Asp Ser Ala Cys Leu Lys Ala Ala Leu
210 215 220
Asp Ala Gly His Pro Val Asp Leu Pro Arg Val Gly Leu Phe Ala Glu
225 230 235 240
Gly Val Ala Val Lys Arg Ile Gly Asp Glu Thr Phe Arg Leu Cys Gln
245 250 255
Glu Tyr Leu Asp Asp Ile Ile Thr Val Asp Ser Asp Ala Ile Cys Ala
260 265 270
Ala Met Lys Asp Leu Phe Glu Asp Val Arg Ala Val Ala Glu Pro Ser
275 280 285
Gly Ala Leu Ala Leu Ala Gly Met Lys Lys Tyr Ile Ala Leu His Asn
290 295 300
Ile Arg Gly Glu Arg Leu Ala His Ile Leu Ser Gly Ala Asn Val Asn
305 310 315 320
Phe His Asp Leu Arg Tyr Val Ser Glu Arg Cys Glu Leu Gly Glu Gln
325 330 335
Arg Glu Ala Leu Leu Ala Val Thr Ile Pro Glu Glu Lys Gly Ser Phe
340 345 350
Leu Lys Phe Cys Gln Leu Leu Gly Gly Arg Ser Val Thr Glu Phe Asn
355 360 365
Tyr Arg Phe Ala Asp Ala Lys Asn Ala Cys Ile Phe Val Gly Val Arg
370 375 380
Leu Ser Arg Gly Leu Glu Glu Arg Lys Glu Ile Leu Gln Met Leu Asn
385 390 395 400
Asp Gly Gly Tyr Ser Val Val Asp Leu Ser Asp Asp Glu Met Ala Lys
405 410 415
Leu His Val Arg Tyr Met Val Gly Gly Arg Pro Ser His Pro Leu Gln
420 425 430
Glu Arg Leu Tyr Ser Phe Glu Phe Pro Glu Ser Pro Gly Ala Leu Leu
435 440 445
Cys Phe Leu Asn Thr Leu Gly Thr Tyr Trp Asn Ile Ser Leu Phe His
450 455 460
Tyr Arg Ser His Gly Thr Asp Tyr Gly Arg Val Leu Ala Ala Phe Glu
465 470 475 480
Leu Gly Asp His Glu Pro Asp Phe Glu Thr Arg Leu Asn Glu Leu Gly
485 490 495
Tyr Asp Cys His Asp Glu Thr Asn Asn Pro Ala Phe Arg Leu Phe Leu
500 505 510
Ala Gly
<210> 13
<211> 514
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 13
Met Ala Asp Ser Gln Pro Leu Ser Gly Ala Pro Glu Gly Ala Glu Tyr
1 5 10 15
Leu Arg Ala Val Leu Arg Ala Pro Val Tyr Glu Ala Ala Gln Val Thr
20 25 30
Pro Leu Gln Lys Met Glu Lys Leu Ser Ser Arg Leu Asp Asn Val Ile
35 40 45
Leu Val Lys Arg Glu Asp Arg Gln Pro Val His Ser Phe Lys Leu Arg
50 55 60
Gly Ala Tyr Ala Met Met Ala Gly Leu Thr Glu Glu Gln Lys Ala His
65 70 75 80
Gly Val Ile Thr Ala Ser Ala Gly Asn His Ala Gln Gly Val Ala Phe
85 90 95
Ser Ser Ala Arg Leu Gly Val Lys Ala Leu Ile Val Met Pro Thr Ala
100 105 110
Thr Ala Asp Ile Lys Val Asp Ala Val Arg Gly Phe Gly Gly Glu Val
115 120 125
Leu Leu His Gly Ala Asn Phe Asp Glu Ala Lys Ala Lys Ala Ile Glu
130 135 140
Leu Ser Gln Gln Gln Gly Phe Thr Trp Val Pro Pro Phe Asp His Pro
145 150 155 160
Met Val Ile Ala Gly Gln Gly Thr Leu Ala Leu Glu Leu Leu Gln Gln
165 170 175
Asp Ala His Leu Asp Arg Val Phe Val Pro Val Gly Gly Gly Gly Leu
180 185 190
Ala Ala Gly Val Ala Val Leu Ile Lys Gln Leu Met Pro Gln Ile Lys
195 200 205
Val Ile Ala Val Glu Ala Glu Asp Ser Ala Cys Leu Lys Ala Ala Leu
210 215 220
Asp Ala Gly His Pro Val Asp Leu Pro Arg Val Gly Leu Phe Ala Glu
225 230 235 240
Gly Val Ala Val Lys Arg Ile Gly Asp Glu Thr Phe Arg Leu Cys Gln
245 250 255
Glu Tyr Leu Asp Asp Ile Ile Thr Val Asp Ser Asp Ala Ile Cys Ala
260 265 270
Ala Met Lys Asp Leu Phe Glu Asp Val Arg Ala Val Ala Glu Pro Ser
275 280 285
Gly Ala Leu Ala Leu Ala Gly Met Lys Lys Tyr Ile Ala Leu His Asn
290 295 300
Ile Arg Gly Glu Arg Leu Ala His Ile Leu Ser Gly Ala Asn Val Asn
305 310 315 320
Phe His Asp Leu Arg Tyr Val Ser Glu Arg Cys Glu Leu Gly Glu Gln
325 330 335
Arg Glu Ala Leu Leu Ala Val Ala Ile Pro Glu Glu Lys Gly Ser Phe
340 345 350
Leu Lys Phe Cys Gln Leu Leu Gly Gly Arg Ser Val Thr Glu Phe Asn
355 360 365
Tyr Arg Phe Ala Asp Ala Lys Asn Ala Cys Ile Phe Val Gly Val Arg
370 375 380
Leu Ser Arg Gly Leu Glu Glu Arg Lys Glu Ile Leu Gln Met Leu Asn
385 390 395 400
Asp Gly Gly Tyr Ser Val Val Asp Leu Ser Asp Asp Glu Met Ala Lys
405 410 415
Leu His Val Arg Tyr Met Val Gly Gly Arg Pro Ser His Pro Leu Gln
420 425 430
Glu Arg Leu Tyr Ser Phe Glu Phe Pro Glu Ser Pro Gly Ala Leu Leu
435 440 445
Cys Phe Leu Asn Thr Leu Gly Thr Tyr Trp Asn Ile Ser Leu Phe His
450 455 460
Tyr Arg Ser His Gly Thr Asp Tyr Gly Arg Val Leu Ala Ala Phe Glu
465 470 475 480
Leu Gly Asp His Glu Pro Asp Phe Glu Thr Arg Leu Asn Glu Leu Gly
485 490 495
Tyr Asp Cys His Asp Glu Thr Asn Asn Pro Ala Phe Arg Leu Phe Leu
500 505 510
Ala Gly

Claims (7)

1.一种苏氨酸脱氨酶突变体，其特征在于，所述苏氨酸脱氨酶突变体是将如SEQ IDNO.1所示氨基酸序列的第14位、第323位、第344位、第449位和第510位的氨基酸进行单、双、三点或四点突变获得。
2.如权利要求1所述的苏氨酸脱氨酶突变体，其特征在于，所述的突变具体为其第14位丙氨酸突变为苏氨酸、第323位甘氨酸突变为天冬氨酸、第344位苏氨酸突变为丙氨酸、第449位精氨酸突变为半胱氨酸、第510位脯氨酸突变为亮氨酸。
3.如权利要求1所述的苏氨酸脱氨酶突变体，其特征在于，其氨基酸序列为下列之一：SEQ ID.2，SEQ ID.3，SEQ ID.4，SEQ ID.5，SEQ ID.6，SEQ ID.7，SEQ ID.8，SEQ ID.9，SEQID.10，SEQ ID.11，SEQ ID.12和SEQ ID.13。
4.一种可编码如权利要求3所述的苏氨酸脱氨酶突变体的基因。
5.一种如权利要求4所述的苏氨酸脱氨酶突变体编码基因构建的重组载体。
6.一种如权利要求5所述重组载体转化制备的重组工程菌。
7.如权利要求1所述的苏氨酸脱氨酶突变体在生产L-2-氨基丁酸的用途。
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