CN113462669B - 酮泛解酸羟甲基转移酶突变体、编码基因及其应用 - Google Patents
酮泛解酸羟甲基转移酶突变体、编码基因及其应用 Download PDFInfo
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Abstract
发明涉及基因工程领域，具体涉及谷氨酸棒状杆菌来源的酮泛解酸羟甲基转移酶突变体及其编码基因及其在制备酮泛解酸和D‑泛酸中的应用。本发明通过借助计算机模拟技术，对底物通道及活性口袋周围关键氨基酸位点进行预测和突变，得到了一系列突变体。所述对酮泛解酸羟甲基转移酶突变体是是将SEQ ID NO:1所示氨基酸序列第18位、第20位、第21位、第24位、第25位、第26位、第27位、第49位进行定点突变获得的。最终得到的酮泛解酸羟甲基转移酶突变体中有酶活显著改变的突变体，该突变体相比于野生型酶在催化α‑酮异戊酸产生酮泛解酸时催化活力和底物耐受性大大提高。
Description
(一)技术领域
本发明属于生物技术领域，具体涉及一种来源于谷氨酸棒杆菌(Corynebacteriumglutamicum)的酮泛解酸羟甲基转移酶突变体及其编码基因、含有该突变体基因的重组载体以及酮泛解酸羟甲基转移酶突变体在生成酮泛解酸和制备D-泛酸中的应用。
(二)背景技术
酮泛解酸羟甲基转移酶(Ketolytic acid hydroxymethyltransferase,KPHMT,EC2.1.2.11)，是panB基因的表达产物，也是大肠杆菌W3110催化泛酸生物合成途径的关键步骤：底物α-酮异戊酸与5,10-亚甲基四氢叶酸在酮泛解酸羟甲基转移酶催化作用下反应生成酮泛解酸，该反应过程是可逆的。
D-泛酸，又称遍多酸或维生素B5，是一种水溶性维生素，D-泛酸的化学索引号(CAS)为79-83-4，相对分子质量为219.23500，相对密度为1.266g/cm3，其化学式为CH2OHC(CH3)2CHOHCONHCH2CH2COOH，由泛解酸和β-丙氨酸组成。有旋光性，仅D型([α]＝+37.5°)有生物活性。消旋泛酸具有吸湿性和静电吸附性；纯游离泛酸是一种淡黄色粘稠的油状物，具酸性，易溶于水和乙醇，不溶于苯和氯仿，泛酸在酸、碱、光及热等条件下都不稳定。泛酸是脂肪酸合成类固醇所必需的物质；也可参与类固醇紫质、褪黑激素和亚铁血红素的合成；还是体内柠檬酸循环、胆碱乙酰化、合成抗体等代谢所必需的中间物。泛酸在体内可作用于正常的上皮器官如神经、肾上腺、消化道及皮肤，提高动物对病原体的抵抗力。泛酸也可以增加谷胱甘肽的生物合成从而减缓细胞凋亡和损伤。泛酸及其衍生物还可以减轻抗生素等药物引起的毒副作用，参与多种营养成分的吸收和利用。
现有的D-泛酸生产方法是化学-酶法，异丁醛与甲醛在碱性、高温条件下羟醛缩合，再添加氢氰酸，在酸性条件下进行醇氰化反应形成氰醇；氰醇在酸性条件下通过水解环化得到DL-泛解酸内酯，DL-泛解酸内酯经L-泛解酸内酯水解酶水解，留下D-泛解酸内酯，产生的L-泛解酸再经化学内酯、外消旋转为DL-泛解酸内酯。得到的D-泛解酸内酯与β-氨基丙酸钙缩合直接制得D-泛酸钙。综合考虑D-泛酸现有生产方法的回收率和环境因素，以可再生的廉价基质用微生物发酵生产D-泛酸受到越来越多的关注。
大肠杆菌W3110中D-泛酸的生物合成途径(泛酸生物合成途径参见图1)需要四种酶的参与，包括酮泛解酸羟甲基转移酶(panB)，酮泛解酸还原酶(panE,Ketopantoatereductase,EC 1.1.1.169)，泛酸合成酶(panC,Pantothenate synthetase,EC 6.3.2.1)和L-天冬氨酸-α-脱羧酶(panD,Aspartate decarboxylase,EC 4.1.1.11)。第一步：5,10-亚甲基四氢叶酸和α-酮异戊酸生成酮泛解酸，该反应由panB基因编码酮的泛解酸羟甲基转移酶催化，在大肠杆菌、尼度曲霉和伤寒沙门氏菌中已有功能鉴定。第二步：由panE基因编码的酮泛解酸还原酶，还原酮泛解酸生成泛解酸；泛酸生物合成的最后一步是由panC编码的泛酸合成酶催化的，该酶催化泛解酸和β-丙氨酸(由panD编码的天冬氨酸脱羧酶催化L-天冬氨酸生成)缩合生成泛酸。Aileen Rubi和D.M.Downs认为由panB基因编码的酮泛解酸羟甲基转移酶催α-酮异戊酸与5,10-亚甲基四氢叶酸反应生成酮泛解酸，是合成D-泛酸的限速步骤(Elevated levels of ketopantoate hydroxymethyltransferase(PanB)lead toa physiologically significant coenzyme A elevation in Salmonella entericaserovar Typhimurium.[J].Journal of bacteriology,2002,184(10))，通过加强panB基因的表达或提高酮泛解酸羟甲基转移酶的催化活力可提高泛酸的产量。
自上个世纪90年代开始，一些著名的化学制药公司BASF、DSM和Degussa AG等开始关注D-泛酸发酵法合成。Miki Hiroshi等人通过运用紫外诱变和亚硝基胍诱变技术与特殊培养基筛选相结合进行高产D-泛解酸菌株的选育，筛选得到一株高产D-泛酸的基因工程菌，通过外源添加β-丙氨酸在经过72h补料发酵后D-泛酸产量达65.4g/L(Process forproducing D-pantoic acid and D-pantothenicacid or salts thereof:US 5932457[P].1999-08-03.)。Rogers R.Y ocum等人致力于构建高产D-泛酸的枯草芽孢杆菌(Bacillus subtilis)，通过解除panBCD、ilvBNC、panE和ilvD的调控来增强D-泛酸合成，构建过表达panBD的质粒，能有效促进D-泛酸积累(Microorganisms and processes forenhancedproduction of pantothenate.US 7291489[P].2007-11-06.)。显然，KPHMT(panB)作为催化泛酸生物合成中的第一步，也是关键一步，起着重要的影响。从1957年起，McIntosh等人首次从大肠杆菌中提取得到KPHMT粗酶，并于1993年由Carol等人纯化该蛋白，对其进行了晶体结构研究。2002年，Aileen Rubio等人发现在来源于鼠伤寒沙门(Salmonella enterica)的panB基因上游添加一个CG碱基对，使启动子的10和35个六聚体之间的间距达到了17bp的一致间距，导致泛操纵子转录增加。并且这种突变引起的panB过度表达，增加了泛酸和辅酶A合成的增加。2003年，Florian Schmitzberger等人对KPHMT的晶体结构进行了详细的比较分析，并对其结构同系物进行了鉴定，表明KPHMT的结构属于(β/α)8磷酸烯醇式丙酮酸/丙酮酸超家族(Comparative analysis of the Escherichiacoli ketopantoate hydroxymethyltransferase crystal structure confirms that itis a member of the(β/α)8phosphoenolpyruvate/pyruvate superfamily.[J].Journalof bacteriology,2003,185(14))。
总体而言，目前报道的关于发酵法生产D-泛酸存在的问题是产量偏低，生产成本较高，不能达到规模化工业生产的要求。并且对于panB的研究较少，因此panB是一个有力的突破点。
(三)发明内容
本发明的目的是提供一种谷氨酸棒杆菌(Corynebacterium glutamicum ATCC13032)来源的酮泛解酸羟甲基转移酶突变体、编码基因、含有该突变体基因的重组载体，以及酮泛解酸羟甲基转移酶突变体在制备酮泛解酸和制备D-泛酸中的应用。该突变体对5,10-亚甲基四氢叶酸和α-酮异戊酸催化生成酮泛解酸的催化活力较野生酶有提高，可有效提高终产物D-泛酸的产量，降低了生产成本。
本发明采用的技术方案是：
一种酮泛解酸羟甲基转移酶突变体，由SEQ ID NO:1所示氨基酸经单点突变获得，所述单点突变的位点为第20位、第24位、第25位、第26位、第27位、第83位、第109位、第123位、第189位、第222位、第254位；或者将SEQ ID NO:1所示氨基酸经双位点突变获得，所述双位点突变的位点为第21与24位、第24与27位进行双位点突变获得。
进一步，所述点突变为下列之一：第20位的赖氨酸突变为丙氨酸(K20A)；第24位谷氨酰胺突变为丙氨酸(Q24A)；第25位的赖氨酸突变为丙氨酸(K25A)；第26位缬氨酸突变为天冬酰胺(V26N)；第27位丝氨酸突变为丙氨酸(S27A)；第83位丝氨酸突变为精氨酸(T83R)；第109位异亮氨酸突变为亮氨酸(I109L)；第123位谷氨酸突变为丙氨酸(E123A)；第189位谷氨酸突变为丙氨酸(E189A)；第222位缬氨酸突变为天冬酰胺(V222N)；第254位丙氨酸突变为精氨酸(A254R)；第21位缬氨酸突变为谷氨酸与第24位谷氨酰胺突变为丙氨酸(V21E/Q24A)；第24位谷氨酰胺突变为丙氨酸与第27位丝氨酸突变为丙氨酸(Q24A/S27A)。
本发明还提供所述酮泛解酸羟甲基转移酶酶突变体的编码基因，含所述编码基因的重组载体以及由所述编码基因构建的工程菌，所述工程菌表达宿主通常为E.coli BL21(DE3)。
本发明所述重组酮泛解酸羟甲基转移酶突变体是通过对野生型酮泛解酸羟甲基转移酶进行多个氨基酸的突变，通过过表达panB，提高其对α-酮异戊酸的催化活力进而提高泛酸的产率。
所述突变体获得方法如下：首先将野生型酮泛解酸羟甲基转移酶编码基因(SEQID NO.2)与表达载体pET28a(+)连接，构建重组表达质粒。然后将panB基因转化至表达宿主E.coli BL21(DE3)中，得到含有酮泛解酸羟甲基转移酶基因的重组基因工程菌。以含有酮泛解酸羟甲基转移酶基因的重组表达质粒为模板，通过定点突变技术进行基因改造。对所得突变后的基因工程菌进行IPTG诱导培养，培养液与菌体分离后通过超声破碎得到酮泛解酸羟甲基转移酶突变体粗酶液。并将突变体酮泛解酸羟甲基转移酶与原始酮泛解酸羟甲基转移酶进行催化活力的比较，筛选得到催化性能优异的突变体。
本发明还涉及所述酮泛解酸羟甲基转移酶突变体在生物催化制备酮泛解酸和泛酸中的应用。
所得酶活有明显改变的酮泛解酸羟甲基转移酶突变体可与panE基因编码的酮泛解酸还原酶(KPR)和panC基因编码的泛酸合成酶(Ps)耦联，用于酶法生产泛酸。
本发明的酮泛解酸羟甲基转移酶突变体的氨基酸数量只有271个，且结构明确，因此本领域技术人员很容易获得其编码基因、包括这些基因的表达盒和质粒、以及包含该质粒的转化体。
这些基因、表达盒、质粒、转化体可以通过本领域技术人员所熟知的基因工程构建方式获得。
当做为生物催化剂用于生产时，本发明的酮泛解酸羟甲基转移酶突变体可以呈现酶的形式或者菌体的形式。所述酶的形式包括游离酶、固定化酶，包括纯化酶、粗酶、发酵液、载体固定的酶、细胞破碎物等：所述菌体的形式包括存活菌体细胞和死亡菌体细胞。
与现有技术相比，本发明的有益效果主要体现在：
本发明得到了一系列对酮泛解酸羟甲基转移酶酶活有明显改变的酮泛解酸羟甲基转移酶突变体，所得酮泛解酸羟甲基转移酶突变体可与panE基因编码的酮泛解酸还原酶(KPR)和panC编码的泛酸合成酶(Ps)耦联，用于酶法生产泛酸。相较野生型酮泛解酸羟甲基转移酶，本发明构建的酮泛解酸羟甲基转移酶突变体C.glu-panB-K20A，C.glu-panB-Q24A，C.glu-panB-K25A，C.glu-panB-V26N，C.glu-panB-S27A，C.glu-panB-T83R，C.glu-panB-I109L，C.glu-panB-E123A，C.glu-panB-E189A，C.glu-panB-V222N，C.glu-panB-A254R，C.glu-panB-V21E/Q24A，C.glu-panB-Q24A/S27A，通过与野生型酶比较，酶活有所提高，优于现有报道的酮泛解酸羟甲基转移酶。
(四)附图说明
图1为泛酸生物合成途径。
图2为实施例中1，2和3中的panB(C.glu)，panC(E.coli)和panE(E.coli)基因扩增片段和pET28a线性化载体的琼脂糖凝胶电泳图，其中1kb:DNA分子量marker，panB-pET28a(6.0kb)，panC-pET28a(6.0kb)，panE-pET28a(6.0kb)；panB-C.glu(813bp)，panC-E.coli(855bp)，panE-E.coli(912bp)。
图3为实施例中4的panB-Histag构建图。
图4为实施例中5中的panB(C.glu)诱导表达，超声破碎后上清的SDS-PAGE电泳图，其中M：蛋白质marker，panB-C.glu(28kDa)。
图5为实施例中5中的panC(E.coli)和panE(E.coli)诱导表达，超声破碎后上清的SDS-PAGE电泳图，其中M：蛋白质marker，panC-E.coli(32kDa)和panE-E.coli(34kDa)。图6为实施例中6中的panB-Histag(C.glu)诱导表达后纯酶液与粗酶液的SDS-PAGE电泳图，其中M：蛋白质marker，panB-Histag(28kDa)，panB-C.glu(28kDa)。
图7为实施例中7中的KPHMT(panB)蛋白浓度标准曲线图。
图8为实施例中8中的突变体C.glu-panB-muts构建的琼脂糖凝胶电泳图，其中1kb：DNA分子量marker，C.glu-panB-K20A，C.glu-panB-Q24A，C.glu-panB-K25A，C.glu-panB-V26N，C.glu-panB-S27A，C.glu-panB-T83R，C.glu-panB-I109L C.glu-panB-E123A，C.glu-panB-E189A，C.glu-panB-V222N，C.glu-panB-A254R，C.glu-panB-V21E/Q24A，C.glu-panB-Q24A/S27A(6.5kbp)。
图9为实施例中9中的底物α-酮异戊酸浓度标准曲线图。
图10为实施例中10中的泛解酸浓度标准曲线图。
图11为实施例中11中的泛酸浓度标准曲线图。
图12为实施例中12和13中的底物α-酮异戊酸的HPLC液相谱图。
图13为实施例12中的野生型酮泛解酸羟甲基转移酶以及突变体酶的酶活柱状图。
图14为实施例中13中的中间产物泛解酸的HPLC液相谱图。
图15为实施例中13中的产物泛酸的HPLC液相谱图。
(五)具体实施方式
下面结合具体实例对本发明做进一步详细地说明，但本发明并不限于以下实施例：
本发明中涉及到多种物质的添加量、含量及浓度，其中所述的百分含量，除特别说明外，皆指质量百分含量。
实施例1：野生型酮泛解酸羟甲基转移酶基因工程菌panB-CG的构建
将基因库中来源于Corynebacterium glutamicum ATCC 13032酮泛解酸羟甲基转移酶KPHMT的基因序列(GenBank登录号：BX927148.1)，通过全基因合成获得panB-CG质粒。设计表达引物1(tttgtttaactttaagaaggagatataccATGCCCATGTCAGGCATTGATGCAAAG)、引物2(tctcagtggtggtggtggtggtgctcgagAAAGGACTCCGCTTCGCCTGGGAAGGT)，利用 Max高保真DNA聚合酶进行PCR扩增，获得813bp的酮泛解酸羟甲基转移酶基因序列(氨基酸序列如SEQ ID NO.1所示，核苷酸序列SEQ ID NO.2所示)。以pET28a载体为模板，通过PCR扩增技术得到线性化载体。引物设计如下，引物3(ACCTTCCCAGGCGAAGCGGAGTCCTTTctcgagcaccaccaccaccaccactgaga)，引物4(CTTTGCATCAATGCCTGACATGGGCATggtatatctccttcttaaagttaaacaaa)，利用/> II One Step Cloning Kit一步无缝克隆试剂盒将酮泛解酸羟甲基转移酶基因片段与pET28a线性化载体片段(panB-CG基因扩增片段和pET28a线性化载体的琼脂糖凝胶电泳图参见图2)进行同源重组连接，构建表达载体pET28a-panB-CG。将构建的载体线性化，通过化学转化法将线性化的pET28a-panB-CG导入大肠杆菌(E.coli)BL21(DE3)中，获得野生型酮泛解酸羟甲基转移酶基因工程菌pET28a-panB-CG，记为野生型工程菌panB-CG。
实施例2：野生型酮泛解酸还原酶酶基因工程菌panE-EC的构建
将基因库中来源于Escherichia coli str.K-12substr，W3110酮泛解酸还原酶KPR的基因序列(GenBank登录号：BAE76205.1)，通过全基因合成获得panE-EC质粒。设计表达引物5(tttgtttaactttaagaaggagatataccATGAAAATTACCGTATTGGGATGCGGTGCC)、引物6(tctcagtggtggtggtggtggtgctcgagCTACCAGGGGCGAGGCAAACCAGTGCCGAT)，利用 Max高保真DNA聚合酶进行PCR扩增，获得912bp的酮泛解酸还原酶基因序列(核苷酸序列如SEQ IDNO.3所示，氨基酸序列SEQ ID NO.4所示)。以pET28a载体为模板，通过PCR扩增技术得到线性化载体。引物设计如下，引物7(ATCGGCACTGGTTTGCCTCGCCCCTGGTAGctcgagcaccaccaccaccaccactgaga)，引物8(GGCACCGCATCCCAATACGGTAATTTTCATggtatatctccttcttaaagttaaacaaa)，利用/> II One Step Cloning Kit一步无缝克隆试剂盒将酮泛解酸还原酶基因片段与pET28a质粒片段进行同源重组连接，构建表达载体pET28a-panE-EC。将构建的载体线性化(panE-EC基因扩增片段和pET28a线性化载体的琼脂糖凝胶电泳图参见图2)，通过化学转化法将线性化的pET28a-panE-EC导入大肠杆菌(E.coli)BL21(DE3)中，获得野生型酮泛解酸还原酶基因工程菌pET28a-panE-EC，记为野生型工程菌panE-EC。
实施例3：野生型泛酸合成酶基因工程菌panC-EC的构建
将基因库中来源于Escherichia coli str.K-12substr，W3110泛酸合成酶Ps的基因序列(GenBank登录号：BAE76042.1)，通过全基因合成获得panC-EC质粒。设计表达引物9(tttgtttaactttaagaaggagatataccATGTTAATTATCGAAACCCTGCCGCTGC)、引物10(tctcagtggtggtggtggtggtgctcgagTTACGCCAGCTCGACCATTTTGTTGTCGAT)，利用 Max高保真DNA聚合酶进行PCR扩增，获得855bp的泛酸合成酶基因序列(核苷酸序列如SEQ ID NO.5所示，氨基酸序列SEQ ID NO.6所示)。以pET28a载体为模板，通过PCR扩增技术得到线性化载体。引物设计如下，引物11(ATCGACAACAAAATGGTCGAGCTGGCGTAActcgagcaccaccaccaccaccactgaga)，引物12(GCAGCGGCAGGGTTTCGATAATTAACATggtatatctccttcttaaagttaaacaaa)，利用 II One Step Cloning Kit一步无缝克隆试剂盒将泛酸合成酶基因片段与pET28a质粒片段进行同源重组连接，构建表达载体pET28a-panC-EC。将构建的载体线性化(panC(E.coli)基因扩增片段和pET28a线性化载体的琼脂糖凝胶电泳图参见图2)，通过化学转化法将线性化的pET28a-panC-EC导入大肠杆菌(E.coli)BL21(DE3)中，获得野生型泛酸合成酶基因工程菌pET28a-panC-EC，记为野生型工程菌panC-EC。
实施例4：panB-His基因工程菌的构建
通过设计引物及同源臂，将6个组氨酸，其包含18个碱基(CATCATCATCACCACCAC)设计在panB目的基因的终止密码子TAA前，通过PCR扩增技术得到含有组氨酸标签的panB-CG目的基因片段及panB-CG大框架。再通过一步克隆将目的基因片段及大框架连接，后通过化学转化至BL21(DE3)感受态细胞中，测序结果正确后得到panB-His基因工程菌株。
具体步骤：先获取线性化质粒框架，以质粒panB-CG为模板，以panB-His-F和panB-His-R为引物，引物设计如下，引物13(CCAGGCGAAGCGGAGTCCTTTCACCACCACCATCATCATTAAAAGCTTG)，引物14(ATCAATGCCTGACATGGGCATGGTATATCTCCTTCTTAAAGTTAAACAA)通过PCR扩增获得长度为6000bp带有His-tag同源臂的片段，并采用水平电泳仪和核酸成像仪验证片段大小是否正确。之后采用PCR Cleanup试剂盒处理消化后的PCR产物，得到较为纯净的目的基因片段，并用Nano Drop测量核酸浓度。
为获得目的片段panB，需以panB-F/panB-R为引物，引物设计如下，引物15(ACTTTAAGAAGGAGATATACCATGCCCATGTCAGGCATTGATGCAAAGAAAATCCGC)，引物16(GGTGCTCGAGTGCGGCCGCAAGCTTTTAATGATGATGGTGGTGGTGAAAGGACTC)，同样采用PCR扩增技术获得。并以质粒panB-CG为模板。然后同样采用水平电泳仪和核酸成像仪验证PCR产物的片段大小是否正确，并对PCR产物进行Cleanup纯化，最后同样用Nano Drop测量核酸浓度。
PCR扩增获得panB-CG质粒框架和panB目的片段后，再采用诺唯赞公司的一步克隆试剂盒将质粒框架和目的片段无缝连接。连接好的产物及时转化到E.coli BL21(DE3)感受态中，涂布于含Kan抗性的LB固体平板中，倒置于37℃恒温培养箱中过夜培养。在超净台中用无菌小枪头从平板中挑取若干单菌落，放置于装有20μL超纯水的EP管中，沸水浴煮沸10min，12000rpm离心1min，吸取1μL上清液作为菌落PCR使用的模板，在质粒panB-CG上插入位点两端设计验证引物panB-VF/panB-VR，引物设计如下，引物17(TTCTGGAAAGCTCATCGCCGAT)，引物18(TTTAGAGGCCCCAAGGGGTTA)，PCR扩增后，采用水平电泳仪和核酸成像仪验证PCR产物的片段大小是否正确，条带大小与目的基因大小一致则视为阳性克隆，再从原平板挑取对应菌落接种于LB试管中，过夜培养，提取质粒后以panB-VF/panB-VR为测序引物送测序公司测序验证。
panB-Histag基因工程菌构建图参见图3。
实施例5：酮泛解酸羟甲基转移酶、酮泛解酸还原酶、泛酸合成酶的诱导表达
将保藏在-80℃冰箱里含有panB-His基因、panE-EC基因、panC-EC基因的三种基因工程菌分别用接种环在含有终浓度为50μg/mL Kan固体LB培养基平板上划线，倒置于37℃恒温培养箱中培养12h后，将平板中单菌落接种到含有终浓度为50μg/mL Kan抗性的5mL LB试管中，于37℃，180rpm恒温摇床培养10h后即为种子液，将种子液按照2％的接种量加入到含有100mL新鲜灭菌LB液体培养基的500mL锥形瓶中，再加入Kan母液至终浓度为50μg/mL，于28℃，180rpm培养至OD600值达到0.6-0.8时加入20μL IPTG母液至终浓度为0.1mM，于37℃，180rpm恒温振荡摇床中过夜培养。将收集的菌液于8000rpm，4℃下离心10min，即为含有目的基因的大肠杆菌湿菌体，置于-20℃冰箱内保存待用。
酮泛解酸羟甲基转移酶KPHMT(panB)、酮泛解酸还原酶KPR(panE)、泛酸合成酶Ps(panC)诱导表达后的SDS-PAGE图参见图4、5。
实施例6：KPHMT(panB)的制备及分离纯化
将收集到的湿菌体(100g/L)重悬于20mM pH8.0磷酸缓冲液中(PB)，置于4℃冰浴，按照30W，破1s停1s，超声破碎30min之后，再将细胞破碎液于4℃，10000rpm低温离心20min，即为酮泛解酸羟甲基转移酶粗酶液；
在实验室前期工作中，酮泛解酸羟甲基转移酶已携带组氨酸(6*His)标签。将获得的粗酶液于0.22μm滤膜除杂后，上清液用Nickel-NTA亲和层析柱(简称Ni柱)纯化蛋白，具体步骤：
(1)预平衡：用5倍柱体积的binding buffer平衡Ni柱，直至基线平衡；
(2)上样：样品以5mL/min的流速上样，使目标蛋白充分和Ni柱充分结合；
(3)除杂：以10mL/min的流速用6倍柱体积的Washing buffer洗脱杂蛋白，直至基线平衡；
(4)洗脱：以5mL/min的流速用Elution buffer洗脱目的蛋白并收集蛋白置于冰上。
(5)平衡：用5倍柱体积的binding buffer平衡Ni柱，直至基线平衡；
(6)保柱：用5倍柱体积含20％乙醇的超纯水清洗Ni柱后，置于4℃冰箱备用；
(7)脱盐：将目的蛋白倒入截留分子量(MWCO)为10kDa的透析袋中置于20mMpH8.0PB缓冲液中4℃过夜透析除盐(前4h每两小时更换一次缓冲液以加快透析速度，减少酶活损失)；
(8)将脱盐后的目的蛋白分装于10mL EP管中，置于-80℃超低温冰箱保存备用。
诱导表达后纯酶液与粗酶液的SDS-PAGE电泳图参见图6。
实施例7：SDS-PAGE蛋白电泳检测及蛋白浓度测定
(1)样品制备：将待测样品与4×Protein Loading Buffer按照3:1的体积比加到EP管中充分混合后，置于沸水浴中5～10min，备用；
(2)样品检测：将预制蛋白胶安装在电泳槽上，倒入电泳缓冲液并检查是否漏液，缓慢向上拔掉梳子，上样，Marker和样品的上样量为5μL。180V电泳大约35min后条带移动至蛋白胶底部结束电泳。
(3)染色及脱色：将蛋白胶取出放入染色脱色仪内，染色脱色10min后，将蛋白胶放入凝胶成像仪中，观察目的条带大小，确定目的蛋白是否成功表达。
采用BCA法测定蛋白浓度，用BCA(Bicinchoninic acid)试剂盒检测，其步骤如下：
(1)空板测量：在37℃，562nm下，将空96孔板在酶标仪上读取对应的吸光值作为背景对照，平行之间测量三次；
(2)标准曲线制备：分别向96孔板内加入20μL梯度浓度的蛋白标准液来配制蛋白标准曲线，如表1所示：
表1：蛋白标准溶液配制表
(3)工作液配制：工作液A和工作液B按照50:1的体积比配制BCA工作液，充分混匀，现配现用；
(4)蛋白含量测定：向酶标板各孔中加入200μL工作液，振荡30s，于37℃保温30min后，测定各孔在562nm处吸光值，纵坐标为测量值，对应蛋白含量为横坐标作图，即蛋白标准曲线y＝0.0475x+0.0039，R2>0.99，如图7所示。
(5)根据测得的吸光值，计算样品蛋白浓度为10.38807mg/mL。
(6)将含有酮泛解酸羟甲基转移酶(panB)的基因工程菌在2L大摇瓶中发酵培养，获得发酵液的菌体含量大约为3g/L。将湿菌体超声破碎、离心收集、过滤除杂后即可得到酮泛解酸羟甲基转移酶粗酶液，并利用AKTA蛋白分离纯化仪通过Ni柱亲和层析对粗酶液进行分离纯化，得到蛋白浓度约为10.39mg/mL的KPHMT纯酶液。
酮泛解酸羟甲基转移酶KPHMT(panB)蛋白标准曲线参见图7。
实施例8：定点突变构建C.glu-panB-muts
通过Swiss-Model对谷氨酸棒杆菌来源的panB进行3D模型的构建，并使用AutoDock分子对接软件将KPHMT(panB)的模型和底物小分子α-酮异戊酸和5,10-亚甲基四氢叶酸进行分子对接，选取panB基因上的关键位点进行突变。设计引物如下：
K20A
上游引物19：CATTTCCGCGAAGCTGCTGTAAACGGCCAGAAA
下游引物20：AGCAGCTTCGCGGAAATGACGGGTGCGGATTTT
Q24A
上游引物21：GCTAAAGTAAACGGCGCTAAAGTTTCGGTTCTC
下游引物22：AGCGCCGTTTACTTTAGCTTCGCGGAAATGACG
K25A
上游引物23：AAAGTAAACGGCCAGGCTGTTTCGGTTCTCACC
下游引物24：AGCCTGGCCGTTTACTTTAGCTTCGCGGAAATG
V26N
上游引物25：GTAAACGGCCAGAAAACCTCGGTTCTCACCAGC
下游引物26：GGTTTTCTGGCCGTTTACTTTAGCTTCGCGGAA
S27A
上游引物27：AACGGCCAGAAAGTTGCTGTTCTCACCAGCTAT
下游引物28：AGCAACTTTCTGGCCGTTTACTTTAGCTTCGCG
T83R
上游引物29：GCGGTGACGATCGCTCGCAAGCGTGCGCTTGTG
下游引物30：GCGAGCGATCGTCACCGCCTTGGCCAGCACAAT
I109L
上游引物31：GCGGTGGAGTCCGCGTTGCGGGTCATGCGTGAA
下游引物32：CAACGCGGACTCCACCGCCTGATTTGGGCTCAC
E123A
上游引物33：GCTGCGGTGAAGATCGCTGGTGGCGTGGAGATC
下游引物34：AGCGATCTTCACCGCAGCCGCACCCGTTTCACG
E189A
上游引物35：TTTGCGGTTGTGTTGGCTATGGTTCCAGCAGAG
下游引物36：AGCCAACACAACCGCAAACGCACCCGCCTGCTC
V222N
上游引物37：GATGGGCAGGTTTTGGCTTGGCAGGATGCCTTC
下游引物38：AGCCAAAACCTGCCCATCTGTGCCATTGCCGGC
A254R
上游引物39：CACGACGCCGCGCAGCGCTACATCGCCGATATC
下游引物40：GCGCTGCGCGGCGTCGTGCAAGGAATCGCCCAA
V21E/Q24A
上游引物41：GCTAAAGAGAACGGCGCTAAAGTTTCGGTTCTC
下游引物42：AGCGCCGTTCTCTTTAGCTTCGCGGAAATGACG
Q24A/S27A
上游引物43：AACGGCGCTAAAGTTGCTGTTCTCACCAGCTAT
下游引物44：AGCAACTTTAGCGCCGTTTACTTTAGCTTCGCG
以pET28a-panB-CG质粒为模板，通过PCR引入定点突变，PCR反应程序如下：95℃5min，95℃30s，58℃30s，72℃0.5min(2kbp/min)，重复30个循环；72℃继续延伸10min。将PCR产物用DpnI于37℃处理15min，灭活后转化至E.coli BL21(DE3)感受态细胞中，涂布于含终浓度50mg/L卡那抗性的LB固体平板上，37℃培养12h后，随机挑取单菌落进行测序，分别获得突变体C.glu-panB-K20A，C.glu-panB-Q24A，C.glu-panB-K25A，C.glu-panB-V26N，C.glu-panB-S27A，C.glu-panB-T83R，C.glu-panB-I109L C.glu-panB-E123A，C.glu-panB-E189A，C.glu-panB-V222N，C.glu-panB-A254R，C.glu-panB-V21E/Q24A，C.glu-panB-Q24A/S27A。
突变体C.glu-panB-muts构建的琼脂糖凝胶电泳图参见图8。
实施例9：α-酮异戊酸标准曲线图制作
称取α-酮异戊酸对照品0.05g(准确到0.001g)，置于一10mL容量瓶中，加水溶解并稀释至不同浓度(0.5，1，2，2.5，5g/L)，制成系列标准工作溶液。后使用0.22μm有机滤膜过膜，用于高效液相色谱(HPLC)检测。检测α-酮异戊酸的高效液相色谱操作条件如下所述。
A流动相成分：8mM H2SO4与超纯水，使用0.20μm微孔水系滤膜过膜并超生除气泡；
B色谱柱型号：Organic Acid Analysis Column Aminex HPX-87H Ion ExclusionColumn(300mm×7.8mm)
C参数设置：进样量：20μL，柱温：60℃，流速：0.6mL/min，检测波长：210nm。
α-酮异戊酸标准曲线图参见图9。
实施例10：泛解酸浓度标准曲线图制作
称取泛解酸对照品0.05g(准确到0.001g)，置于一10mL容量瓶中，加水溶解并稀释至不同浓度(0.5，1，2，2.5，5g/L)，制成系列标准工作溶液。后使用0.22μm有机滤膜过膜，用于高效液相色谱(HPLC)检测。检测α-酮异戊酸的高效液相色谱操作条件如下所述。
A流动相成分：95％超纯水、4.9％乙腈、0.1％磷酸，使用0.20μm微孔水系滤膜过膜并超生除气泡；
B色谱柱型号：ACQUITY UPLC BEH C18 column(100mm×2.1mm,1.7μm,Waters,UK)；
C参数设置：进样量：10μL，柱温：30℃，流速：0.9mL/min，检测波长：200nm。
泛解酸标准曲线图参见图10。
实施例11：D-泛酸浓度标准曲线图制作
称取D-泛酸钙对照品0.05g(准确到0.001g)，置于一10mL容量瓶中，加水溶解并稀释至不同浓度(0，0.5，1，2，2.5g/L)，制成系列标准工作溶液。后使用0.22μm有机滤膜过膜，用于高效液相色谱(HPLC)检测。检测α-酮异戊酸的高效液相色谱操作条件如下所述。
A流动相成分：95％超纯水、4.9％乙腈、0.1％磷酸，使用0.20μm微孔水系滤膜过膜并超生除气泡；
B色谱柱型号：ACQUITY UPLC BEH C18 column(100mm×2.1mm,1.7μm,Waters,UK)；
C参数设置：进样量：10μL，柱温：30℃，流速：0.9mL/min，检测波长：200nm。
D-泛酸标准曲线图参见图11。
实施例12：酮泛解酸羟甲基转移酶(panB)酶活的测定
酮戊酸羟甲基转移酶(panB)酶活的测定：以α-酮异戊酸(α-酮异戊酸HPLC检测图参见图12)为底物生成酮泛解酸。酶活测定体系为1.5mL：α-酮异戊酸终浓度为10mM，缓冲液为磷酸钾缓冲液(100mM，pH6.8)，反应前先将甲醛(终浓度5mM)与四氢叶酸(终浓度0.05mM)于37℃孵育15min来制备N5,N10-亚甲基四氢叶酸，后添加硫酸镁(终浓度5mM)，α-酮异戊酸和KPHMT纯酶液20μL(panB)，于37℃，600rpm反应15min制备酮泛解酸，后取反应液200μL于20μL HCl(6M)中终止反应。使用0.22μm有机滤膜过膜，用于高效液相色谱(HPLC)检测。高效液相色谱操作条件如下所述。
A流动相成分：8mM H2SO4与超纯水，使用0.20μm微孔水系滤膜过膜并超生除气泡；
B色谱柱型号：Organic Acid Analysis Column Aminex HPX-87H Ion ExclusionColumn(300mm×7.8mm)
C参数设置：进样量：20μL，柱温：60℃，流速：0.6mL/min，检测波长：210nm。
酶活单位定义:在pH6.8，温度37℃的条件下，每分钟催化底物α-酮异戊酸产生1微摩尔(μmol)酮泛解酸所需要的酶量定义为1个单位(U)。通过对多个突变体克隆筛选经测序发现，发现有些位点的氨基酸的替换会导致突变体的酶活力的显著变化。
野生型酮泛解酸羟甲基转移酶以及突变体酶的酶活检测结果见下表：
相较野生型酮泛解酸羟甲基转移酶，本发明构建的酮泛解酸羟甲基转移酶突变体C.glu-panB-K20A，C.glu-panB-Q24A，C.glu-panB-K25A，C.glu-panB-V26N，C.glu-panB-S27A，C.glu-panB-T83R，C.glu-panB-I109L，C.glu-panB-E123A，C.glu-panB-E189A，C.glu-panB-V222N，C.glu-panB-A254R，C.glu-panB-V21E/Q24A，C.glu-panB-Q24A/S27A，通过与野生型酶比较，酶活有所提高，分别提高了17％，42％，150％，25％，8％，25％，58％，17％，25％，58％，75％，58％，150％。
野生型酮泛解酸羟甲基转移酶以及突变体酶的酶活柱状图见图13。
实施例13：KPHMT(panB)，KPR(panE)与PS(panC)耦联制备泛酸
挑选平板上的单菌落，接种到含100mLLB培养基的500mL摇瓶中，培养基中含50ug/mL卡那霉素，37℃培养2h后，加入终浓度0.1mM IPTG，降温至28℃，培养过夜。5000rpm离心10min，弃上清，收集湿菌体。称取1g湿菌体，加入10mL 20mM磷酸盐缓冲液(pH8.0)，重悬菌体，后于冰浴上超声破碎，后得到粗酶液进行中间产物酮泛解酸、泛解酸(泛解酸HPLC检测图参见图14)以及终产物泛酸(泛酸HPLC检测图参见图15)的制备。酶活测定反应体系包含(以下浓度为终浓度)：反应前先在缓冲液为磷酸钾缓冲液(100mM，pH6.8)中加入100μL甲醛(终浓度5mM)与100μL四氢叶酸(终浓度0.2mM)于37℃孵育10min来制备5,10-亚甲基四氢叶酸，后加入100ml 5mM硫酸镁，100μLα-酮异戊酸(100mM)，20μL KPHMT(panB)，1h后加入50μLNADPH(0.1mM)，50μL KPR(panE)，100μLβ-丙氨酸(100mM)和50μL Ps(panC)，总体积为1.5mL。2h后取反应液200μL于20μL HCl(6M)中终止反应。使用0.22μm有机滤膜过膜，用于高效液相色谱(HPLC)检测。检测泛酸的高效液相色谱操作条件如下所述。
高效液相色谱操作条件：
A流动相成分：95％超纯水、4.9％乙腈、0.1％磷酸，使用0.20μm微孔水系滤膜过膜并超生除气泡；
B色谱柱型号：ACQUITY UPLC BEH C18 column(100mm×2.1mm,1.7μm,Waters，UK)；
C参数设置：进样量：10μL，柱温：30℃，流速：0.9mL/min，检测波长：200nm。
野生型酮泛解酸羟甲基转移酶以及突变体酶与KPR(panE)与PS(panC)耦联制备泛酸检测结果见下表：
相较野生型酮泛解酸羟甲基转移酶，本发明构建的酮泛解酸羟甲基转移酶突变体酶C.glu-panB-K20A，C.glu-panB-Q24A，C.glu-panB-K25A，C.glu-panB-V26N，C.glu-panB-S27A，C.glu-panB-T83R，C.glu-panB-109L，C.glu-panB-E123A，C.glu-panB-E189A，C.glu-panB-V222N，C.glu-panB-A254R，C.glu-panB-V21E/Q24A，C.glu-panB-Q24A/S27A，与KPR(panE)与PS(panC)耦联制备泛酸，泛酸产量有所提高，分别提高了7.1％，5.8％，6.6％，15.0％，11.3％，10.0％，7.9％，2.1％，4.2％，7.1％，19.3％，8.7％。
序列表
<110> 浙江工业大学
<120> 酮泛解酸羟甲基转移酶突变体、编码基因及其应用
<160> 10
<170> SIPOSequenceListing 1.0
<210> 1
<211> 271
<212> PRT
<213> 未知(Unknown)
<400> 1
Met Pro Met Ser Gly Ile Asp Ala Lys Lys Ile Arg Thr Arg His Phe
1 5 10 15
Arg Glu Ala Lys Val Asn Gly Gln Lys Val Ser Val Leu Thr Ser Tyr
20 25 30
Asp Ala Leu Ser Ala Arg Ile Phe Asp Glu Ala Gly Val Asp Met Leu
35 40 45
Leu Val Gly Asp Ser Ala Ala Asn Val Val Leu Gly Arg Asp Thr Thr
50 55 60
Leu Ser Ile Thr Leu Asp Glu Met Ile Val Leu Ala Lys Ala Val Thr
65 70 75 80
Ile Ala Thr Lys Arg Ala Leu Val Val Val Asp Leu Pro Phe Gly Thr
85 90 95
Tyr Glu Val Ser Pro Asn Gln Ala Val Glu Ser Ala Ile Arg Val Met
100 105 110
Arg Glu Thr Gly Ala Ala Ala Val Lys Ile Glu Gly Gly Val Glu Ile
115 120 125
Ala Gln Thr Ile Arg Arg Ile Val Asp Ala Gly Ile Pro Val Val Gly
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His Ile Gly Tyr Thr Pro Gln Ser Glu His Ser Leu Gly Gly His Val
145 150 155 160
Val Gln Gly Arg Gly Ala Ser Ser Gly Lys Leu Ile Ala Asp Ala Arg
165 170 175
Ala Leu Glu Gln Ala Gly Ala Phe Ala Val Val Leu Glu Met Val Pro
180 185 190
Ala Glu Ala Ala Arg Glu Val Thr Glu Asp Leu Ser Ile Thr Thr Ile
195 200 205
Gly Ile Gly Ala Gly Asn Gly Thr Asp Gly Gln Val Leu Val Trp Gln
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Asp Ala Phe Gly Leu Asn Arg Gly Lys Lys Pro Arg Phe Val Arg Glu
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Tyr Ala Thr Leu Gly Asp Ser Leu His Asp Ala Ala Gln Ala Tyr Ile
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Ala Asp Ile His Ala Gly Thr Phe Pro Gly Glu Ala Glu Ser Phe
260 265 270
<210> 2
<211> 816
<212> DNA
<213> 未知(Unknown)
<400> 2
atgcccatgt caggcattga tgcaaagaaa atccgcaccc gtcatttccg cgaagctaaa 60
gtaaacggcc agaaagtttc ggttctcacc agctatgatg cgctttcggc gcgcattttt 120
gatgaggctg gcgtcgatat gctccttgtt ggtgattccg ctgccaacgt tgtgctgggt 180
cgcgatacca ccttgtcgat caccttggat gagatgattg tgctggccaa ggcggtgacg 240
atcgctacga agcgtgcgct tgtggtggtt gatctgccgt ttggtaccta tgaggtgagc 300
ccaaatcagg cggtggagtc cgcgatccgg gtcatgcgtg aaacgggtgc ggctgcggtg 360
aagatcgagg gtggcgtgga gatcgcgcag acgattcgac gcattgttga tgctggaatt 420
ccggttgtcg gccacatcgg gtacaccccg cagtccgagc attccttggg cggccacgtg 480
gttcagggtc gtggcgcgag ttctggaaag ctcatcgccg atgcccgcgc gttggagcag 540
gcgggtgcgt ttgcggttgt gttggagatg gttccagcag aggcagcgcg cgaggttacc 600
gaggatcttt ccatcaccac tatcggaatc ggtgccggca atggcacaga tgggcaggtt 660
ttggtgtggc aggatgcctt cggcctcaac cgcggcaaga agccacgctt cgtccgcgag 720
tacgccacct tgggcgattc cttgcacgac gccgcgcagg cctacatcgc cgatatccac 780
gcgggtacct tcccaggcga agcggagtcc ttttaa 816
<210> 3
<211> 303
<212> PRT
<213> 未知(Unknown)
<400> 3
Met Lys Ile Thr Val Leu Gly Cys Gly Ala Leu Gly Gln Leu Trp Leu
1 5 10 15
Thr Ala Leu Cys Lys Gln Gly His Glu Val Gln Gly Trp Leu Arg Val
20 25 30
Pro Gln Pro Tyr Cys Ser Val Asn Leu Val Glu Thr Asp Gly Ser Ile
35 40 45
Phe Asn Glu Ser Leu Thr Ala Asn Asp Pro Asp Phe Leu Ala Thr Ser
50 55 60
Asp Leu Leu Leu Val Thr Leu Lys Ala Trp Gln Val Ser Asp Ala Val
65 70 75 80
Lys Ser Leu Ala Ser Thr Leu Pro Val Thr Thr Pro Ile Leu Leu Ile
85 90 95
His Asn Gly Met Gly Thr Ile Glu Glu Leu Gln Asn Ile Gln Gln Pro
100 105 110
Leu Leu Met Gly Thr Thr Thr His Ala Ala Arg Arg Asp Gly Asn Val
115 120 125
Ile Ile His Val Ala Asn Gly Ile Thr His Ile Gly Pro Ala Arg Gln
130 135 140
Gln Asp Gly Asp Tyr Ser Tyr Leu Ala Asp Ile Leu Gln Thr Val Leu
145 150 155 160
Pro Asp Val Ala Trp His Asn Asn Ile Arg Ala Glu Leu Trp Arg Lys
165 170 175
Leu Ala Val Asn Cys Val Ile Asn Pro Leu Thr Ala Ile Trp Asn Cys
180 185 190
Pro Asn Gly Glu Leu Arg His His Pro Gln Glu Ile Met Gln Ile Cys
195 200 205
Glu Glu Val Ala Ala Val Ile Glu Arg Glu Gly His His Thr Ser Ala
210 215 220
Glu Asp Leu Arg Asp Tyr Val Met Gln Val Ile Asp Ala Thr Ala Glu
225 230 235 240
Asn Ile Ser Ser Met Leu Gln Asp Ile Arg Ala Leu Arg His Thr Glu
245 250 255
Ile Asp Tyr Ile Asn Gly Phe Leu Leu Arg Arg Ala Arg Ala His Gly
260 265 270
Ile Ala Val Pro Glu Asn Thr Arg Leu Phe Glu Met Val Lys Arg Lys
275 280 285
Glu Ser Glu Tyr Glu Arg Ile Gly Thr Gly Leu Pro Arg Pro Trp
290 295 300
<210> 4
<211> 912
<212> DNA
<213> 未知(Unknown)
<400> 4
atgaaaatta ccgtattggg atgcggtgcc ttagggcaat tatggcttac agcactttgc 60
aaacagggtc atgaagttca gggctggctg cgcgtaccgc aaccttattg tagcgtgaat 120
ctggttgaga cagatggttc gatatttaac gaatcgctga ccgccaacga tcccgatttt 180
ctcgccacca gcgatctgct cctggtgacg ctgaaagcat ggcaggtttc cgatgccgtc 240
aaaagcctcg cgtccacact gcctgtaact acgccaatac tgttaattca caacggcatg 300
ggcaccatcg aagagttgca aaacattcag cagccattac tgatgggcac caccacccat 360
gcagcccgcc gcgacggcaa tgtcattatt catgtggcaa acggtatcac gcatattggc 420
ccggcacggc aacaggacgg ggattacagt tatctggcgg atattttgca aaccgtgttg 480
cctgacgttg cctggcataa caatattcgc gccgagctgt ggcgcaagct ggcagtcaac 540
tgcgtgatta atccactgac tgccatctgg aattgcccga acggtgaatt acgtcatcat 600
ccgcaagaaa ttatgcagat atgcgaagaa gtcgcggcgg tgatcgaacg cgaagggcat 660
catacttcag cagaagattt gcgtgattac gtgatgcagg tgattgatgc cacagcggaa 720
aatatctcgt cgatgttgca ggatatccgc gcgctgcgcc acactgaaat cgactatatc 780
aatggttttc tcttacgccg cgcccgcgcg catgggattg ccgtaccgga aaacacccgc 840
ctgtttgaaa tggtaaaaag aaaggagagt gaatatgagc gcatcggcac tggtttgcct 900
cgcccctggt ag 912
<210> 5
<211> 283
<212> PRT
<213> 未知(Unknown)
<400> 5
Met Leu Ile Ile Glu Thr Leu Pro Leu Leu Arg Gln Gln Ile Arg Arg
1 5 10 15
Leu Arg Met Glu Gly Lys Arg Val Ala Leu Val Pro Thr Met Gly Asn
20 25 30
Leu His Asp Gly His Met Lys Leu Val Asp Glu Ala Lys Ala Arg Ala
35 40 45
Asp Val Val Val Val Ser Ile Phe Val Asn Pro Met Gln Phe Asp Arg
50 55 60
Pro Glu Asp Leu Ala Arg Tyr Pro Arg Thr Leu Gln Glu Asp Cys Glu
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Lys Leu Asn Lys Arg Lys Val Asp Leu Val Phe Ala Pro Ser Val Lys
85 90 95
Glu Ile Tyr Pro Asn Gly Thr Glu Thr His Thr Tyr Val Asp Val Pro
100 105 110
Gly Leu Ser Thr Met Leu Glu Gly Ala Ser Arg Pro Gly His Phe Arg
115 120 125
Gly Val Ser Thr Ile Val Ser Lys Leu Phe Asn Leu Val Gln Pro Asp
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Ile Ala Cys Phe Gly Glu Lys Asp Phe Gln Gln Leu Ala Leu Ile Arg
145 150 155 160
Lys Met Val Ala Asp Met Gly Phe Asp Ile Glu Ile Val Gly Val Pro
165 170 175
Ile Met Arg Ala Lys Asp Gly Leu Ala Leu Ser Ser Arg Asn Gly Tyr
180 185 190
Leu Thr Ala Glu Gln Arg Lys Ile Ala Pro Gly Leu Tyr Lys Val Leu
195 200 205
Ser Ser Ile Ala Asp Lys Leu Gln Ala Gly Glu Arg Asp Leu Asp Glu
210 215 220
Ile Ile Thr Ile Ala Gly Gln Glu Leu Asn Glu Lys Gly Phe Arg Ala
225 230 235 240
Asp Asp Ile Gln Ile Arg Asp Ala Asp Thr Leu Leu Glu Val Ser Glu
245 250 255
Thr Ser Lys Arg Ala Val Ile Leu Val Ala Ala Trp Leu Gly Asp Ala
260 265 270
Arg Leu Ile Asp Asn Lys Met Val Glu Leu Ala
275 280
<210> 6
<211> 852
<212> DNA
<213> 未知(Unknown)
<400> 6
gtgttaatta tcgaaaccct gccgctgctg cgtcagcaaa ttcgccgcct gcgtatggaa 60
ggcaagcgcg tggcgctggt gcctaccatg ggtaacctgc acgatggcca tatgaagctg 120
gtcgacgaag ccaaagcccg cgccgatgtg gtcgtcgtca gtattttcgt taacccgatg 180
cagttcgacc gcccggaaga tctggctcgt tatccacgga ccttgcagga ggactgcgag 240
aagctaaaca aacgtaaagt ggatttagtt ttcgcccctt cggtaaaaga gatctacccg 300
aacggtactg aaacccacac ttacgttgac gttcctggcc tttcgaccat gctggaaggt 360
gccagccgtc cgggacattt tcgcggcgtt tcgactattg tcagcaagct gttcaacctg 420
gtccagccgg acatcgcctg cttcggtgaa aaagattttc agcaactggc gctgatccgc 480
aaaatggttg ccgatatggg cttcgatatt gagattgtcg gtgtgccaat tatgcgcgcc 540
aaagacggtc tggcgctaag ttcccgtaac ggttatctga cggcggaaca acgcaaaatt 600
gcgcctggtc tgtacaaagt tttaagttcg attgctgaca aattgcaggc tggggaacgg 660
gatctcgatg aaattattac cattgcgggg caagaactga atgaaaaagg cttccgcgcc 720
gatgatattc agattcgcga tgccgacaca ttgctggaag tttctgaaac cagcaaacgg 780
gcagtaattc tggtagccgc ctggcttggc gatgctcgcc tgatcgacaa caaaatggtc 840
gagctggcgt aa 852
<210> 7
<211> 56
<212> DNA
<213> 未知(Unknown)
<400> 7
tttgtttaac tttaagaagg agatatacca tgcccatgtc aggcattgat gcaaag 56
<210> 8
<211> 56
<212> DNA
<213> 未知(Unknown)
<400> 8
tctcagtggt ggtggtggtg gtgctcgaga aaggactccg cttcgcctgg gaaggt 56
<210> 9
<211> 56
<212> DNA
<213> 未知(Unknown)
<400> 9
accttcccag gcgaagcgga gtcctttctc gagcaccacc accaccacca ctgaga 56
<210> 10
<211> 51
<212> DNA
<213> 未知(Unknown)
<400> 10
ctttgcatca atgcctgaca tgggcatggt atatctcctt cttaaagtta a 51
Claims (5)
1.一种酮泛解酸羟甲基转移酶突变体，由SEQ ID NO:1所示氨基酸经单点突变获得，所述单点突变为：第25位的赖氨酸突变为丙氨酸。
2.编码权利要求1所述酮泛解酸羟甲基转移酶突变体的基因。
3.含有权利要求2所述编码基因的表达载体。
4.权利要求1所述酮泛解酸羟甲基转移酶突变体在生物催化制备酮泛解酸中的应用。
5.权利要求1所述酮泛解酸羟甲基转移酶突变体在生物催化制备泛酸中的应用。
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