CN111662886A - 一种l-脯氨酸4-羟化酶及其应用 - Google Patents
一种l-脯氨酸4-羟化酶及其应用 Download PDFInfo
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- CN111662886A CN111662886A CN202010382954.9A CN202010382954A CN111662886A CN 111662886 A CN111662886 A CN 111662886A CN 202010382954 A CN202010382954 A CN 202010382954A CN 111662886 A CN111662886 A CN 111662886A
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- leu
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- amino acid
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- Enzymes And Modification Thereof (AREA)
Abstract
本发明涉及一种具有高催化活性的L‑脯氨酸4‑羟化酶,其氨基酸序列在SEQ ID NO.1所示序列中具有如下突变中的一种或多种:第92位氨基酸由谷氨酰胺突变为丝氨酸、苏氨酸、半胱氨酸、赖氨酸、天冬氨酸或谷氨酸;第94位氨基酸由赖氨酸突变为丝氨酸、半胱氨酸、组氨酸或天冬氨酸;第115位氨基酸由色氨酸突变为谷氨酰胺、赖氨酸或谷氨酸;第217位氨基酸由丝氨酸突变为苏氨酸、半胱氨酸、谷氨酰胺、精氨酸、组氨酸或天冬氨酸;第226位氨基酸由精氨酸突变为苏氨酸、酪氨酸、天冬氨酸或谷氨酸。本发明还涉及上述酶的应用。
Description
技术领域
本发明属于生物催化领域,具体涉及一种L-脯氨酸4-羟化酶及其在合成反式-4-羟基-L-脯氨酸中的应用。
背景技术
L-羟脯氨酸(L-hydroxyproline,Hyp)由L-脯氨酸羟化酶(L-prolinehydroxylase)对L-脯氨酸羟基化修饰而产生的亚氨基酸。由于L-脯氨酸羟基化过程中羟基的添加位置不同,形成了4种同分异构体—反式-4-羟脯氨酸、反式-3-羟脯氨酸、顺式-3-羟脯氨酸和顺式-4-羟脯氨酸。其中,自然界中反式-4-羟脯氨酸(trans-4-hydroxy-L-proline,trans-Hyp)普遍存在于动植物体内,其以糖蛋白的形式存在植物的细胞壁蛋白质中,以多肽的形式存在动物的胶原蛋白(如明胶、骨胶)中。它在生物体的一些生理和病理过程中都发挥有关键的作用,可直接通过动物的骨胶水解得到,作为医药合成中间体、食品添加剂和化妆品抗氧化剂而广泛应用于食品、化妆品、医药、化工等多个领域。在医药方面上,由于L-羟脯氨酸有着2个手性中心,故其药理性质多变,衍生物众多,能够作为一些药物合成的前体物,也能作为原料用于合成新一代的药物,如新一代培南抗生素、抗肿瘤抗高血压药物、新型胃药等。羟脯氨酸的衍生物N-乙酰羟脯氨酸是治疗结缔组织损伤、风湿性关节炎等疾病的良好药物。羟脯氨酸还可以促进伤口、骨骼的愈合;调节脂肪的乳化降低体脂而达到减肥的功效。
国内外羟脯氨酸的生产方法主要有3种:水解法、微生物转化法(发酵法)和化学合成法。由于水解法和化学合成方法对环境污染严重,目前,国外生产L-羟脯氨酸主要靠生物转化法。而我国起步较晚且技术落后,羟脯氨酸严重依赖进口。致使每年都有大量的动物骨胶出口到国外,部分再高价返销国内,因而探索一种高效、经济、环境友好的羟脯氨酸制备方法具有重要的研究意义。微生物转化法就是利用微生物细胞内脯氨酸羟化酶的催化作用进行合成反应。该酶是一种依赖α-酮戊二酸的双加氧酶,需要在α-酮戊二酸、抗坏血酸、DTT的共基质以及Fe2+为共因子的条件下,进行反应。该反应中以游离的L-脯氨酸作为底物,通过脯氨酸-4-羟化酶将游离态的L-脯氨酸羟化为反式-4-羟脯氨酸。
目前,使用最多的即为指孢囊菌RH1所产生的脯氨酸-4-羟化酶,但该酶的催化活性较低,产率不高,严重限制了其在工业上的应用,有待进一步改良优化。
发明内容
本发明的目的是提供一种具有高催化活性的脯氨酸-4-羟化酶。
本发明采用如下技术方案:
一种L-脯氨酸4-羟化酶,来源于蜡样芽孢杆菌,其氨基酸序列如SEQ ID NO.1所示。
编码氨基酸序列如SEQ ID NO.1所示的L-脯氨酸4-羟化酶的DNA,其碱基序列(bp4h)如SEQ ID NO.2所示。
一种L-脯氨酸4-羟化酶,其氨基酸序列在SEQ ID NO.1所示序列中具有如下突变中的一种或多种:
(1)SEQ ID NO.1中第92位氨基酸由谷氨酰胺突变为丝氨酸(Q92S)、苏氨酸(Q92T)、半胱氨酸(Q92C)、赖氨酸(Q92K)、天冬氨酸(Q92D)或谷氨酸(Q92E);
(2)SEQ ID NO.1中第94位氨基酸由赖氨酸突变为丝氨酸(K94S)、半胱氨酸(K94C)、组氨酸(K94H)或天冬氨酸(K94D);
(3)SEQ ID NO.1中第115位氨基酸由色氨酸突变为谷氨酰胺(W115Q)、赖氨酸(W115K)或谷氨酸(W115E);
(4)SEQ ID NO.1中第217位氨基酸由丝氨酸突变为苏氨酸(S217T)、半胱氨酸(S217C)、谷氨酰胺(S217Q)、精氨酸(S217R)、组氨酸(S217H)或天冬氨酸(S217D);
(5)SEQ ID NO.1中第226位氨基酸由精氨酸突变为苏氨酸(R226T)、酪氨酸(R226Y)、天冬氨酸(R226D)或谷氨酸(R226E)。
作为优选的,一种L-脯氨酸4-羟化酶,其氨基酸序列在SEQ ID NO.1所示序列中第92位氨基酸由谷氨酰胺突变为丝氨酸(Q92S),其氨基酸序列如SEQ ID NO.3所示。
作为优选的,一种L-脯氨酸4-羟化酶,其氨基酸序列在SEQ ID NO.1所示序列中第217位氨基酸由丝氨酸突变为组氨酸(S217H),其氨基酸序列如SEQ ID NO.4所示。
一种编码上述的L-脯氨酸4-羟化酶的DNA。
一种上述DNA的基因工程菌。
一种利用上述L-脯氨酸4-羟化酶在合成反式-4-羟基-L-脯氨酸中的应用。
本发明的有益效果在于:本发明对蜡样芽孢杆菌(Bacillus cereus)HBU-AI中的脯氨酸-4-羟化酶的关键氨基酸位点进行了研究。本发明首先通过同源建模获得了 BP4H的三维模型,再结合该酶家族保守性序列分析和分子对接模拟结果,找到了5个位于催化核心区域的氨基酸位点,即Gln92、Lys94、Trp115、Ser217和Arg226,其中预测Gln92、Lys94、Trp115与底物或产物通过氢键和盐键结合,Ser217、Arg226与辅底物α-酮戊二酸结合。
实验中采用定点半饱和突变技术,用极性氨基酸替换的方式对这5个氨基酸位点作用进行考察。通过建库、测序,得到了部分极性氨基酸替换或代换的突变株,共计23株。对它们进行活性验证,结果显示,大部分突变株都能保持>20%的野生酶活性,其中Q92S和S217H能保持85%以上的野生酶活性,这说明Gln92、Lys94、Trp115、Ser217和Arg226 5个氨基酸对酶的活性影响很大,更进一步证实了模型的准确性,验证了保守序列分析和分子对接的结果,最后再通过定点半饱和突变技术对该酶进行改造,筛选具有高催化活性的L-脯氨酸-4-羟化酶,筛选得到的优良转化酶对羟基化催化反应的进一步放大具有重要的实用价值和现实意义。
附图说明
图1为BP4H酶结构模型与4q5o的三维结构叠合。
图2为BP4H酶结构模型的Ramachandran。
图3为结合Fe2+的保守2-His-1-羧酸酯面三联体。
图4为结合α-酮戊二酸的保守位点RXS motifs。
图5为分子对接结果。
图6为BP4H酶定点突变菌株的相对活性。
具体实施方式
以下对本文的详细内容和实施方式进行具体说明。如无特殊说明,本文中所用术语的含义与本领域技术人员一般理解的含义相同,但如有冲突,则以本文中的定义为准。
本文中,所涉及的数值一般指重量或重量百分比,除非特殊说明。
来源于蜡样芽孢杆菌的L-脯氨酸4-羟化酶是已报道的具有较高羟基化活性的酶,它可以催化L-脯氨酸形成反式4-羟基-L-脯氨酸,其基因bp4h的碱基序列如SEQ ID NO.2所示,共有819个碱基。氨基酸序列如SEQ ID NO.1所示,共有273个氨基酸。
本发明得到的L-脯氨酸4-羟化酶的突变体是基于上述bp4h基因产生的突变体。
以下实施例用于说明本发明,下述实施例中所使用的实验方法如无特殊说明,均为常规方法。
下述实施例中所用的材料、试剂等, 如无特殊说明,均可从商业途径得到。
下面结合具体实施例对本文作进一步说明,但本文并不限于以下实施例。
在下述实施例中使用到的培养基、转化以及培养方法总结如下:
培养基:氯化钠10g/L、酵母粉5g/L、胰蛋白胨10g/L(纯水配置),pH7.0。混匀分装,灭菌待用(121℃,20min)。
LB固体培养基:在LB液体培养基的基础上,加1.5%的琼脂,灭菌待用。
含氨苄(Amp)的平板:将灭菌后的LB固体培养基加热融化,待冷却至60℃时,加入千分之一含量的氨苄青霉素(50mg/mL)使培养基内氨苄浓度为50μg/mL。混匀后倒入平板,冷却后待用。
转化及培养:在超净台中取2.5μL重组环化产物,加到50μL大肠杆菌DH5a感受态细胞中,轻弹管壁混匀,冰上放置30min。42℃热激90s,冰水浴5min。加入650μL 的LB培养基,于37℃、150rpm条件下振荡培养1h。然后取100μL菌液均匀涂布在含有氨苄的平板上。平板倒置,37℃培养过夜。第二天从平板上随机挑取单菌落若干,接种于含氨苄抗生素的LB液体培养基(试管,3mL)中,在37℃、150rpm条件下振荡培养8h,观察培养液是否浑浊。取2μL浑浊菌液进行PCR验证,将验证阳性的菌株取600μL菌液送测序公司测序。
实施例1 同源建模及评价
利用SWISS-MODEL同源建模服务器(https://www.swissmodel.expasy.org/)对来源于蜡样芽孢杆菌HBU-AI的脯氨酸羟化酶(BP4H)进行同源建模,具体方法的步骤如下:输入目标序列,搜寻合适的模板,再选择合适的模板进行同源建模。P4H结构研究的实验结果较少,选择与目标序列同源性较高的四氢嘧啶羟化酶(PDB:4q5o)作为模板,使用自动模式(Automated Mode)进行建模。采用Procheck程序的Ramachandran plot以及verify3D模型结构进行打分,评价三维结构与其一级序列的兼容性。
结果表明:将BP4H蛋白序列输入到SWISS-MODEL 同源建模服务器进行序列比对发现,BP4H和模板蛋白的序列同一性在30%~36%之间,其中,四氢嘧啶羟化酶(PDB:4q5o)与BP4H蛋白的同源性最高,为36.02%。因此,选用四氢嘧啶羟化酶(PDB:4q5o)进行同源建模获取其三维结构,经过能量优化和模型评估后得到了合理的三维结构模型。ProteinsSuperimposing显示BP4H模型与四氢嘧啶羟化酶(PDB:4q5o)的RMSD 值为 0.64 Å,表明两者具有十分相似的三维架构(见图1)。使用 Procheck 程序分析模型主链和侧链的立体化学构型的合理性(见图2)结果表明 BP4H模型的绝大多数 φ、 ψ 二面角均在合理范围内,其中85.6%的残基在核心区域,11.9%的残基在允许区域,仅有0.9%的残基在不允许区域内。运用 verify3D 方法对该模拟结构进行打分,结果显示95.35%的残基 3D-1D 兼容性分值>0.2,表明该模型一级序列与三级结构的相容性较好。综合 Procheck 和 verify3D 方法的评价结果,表明通过同源建模所得到的 BP4H 的三维结构合理可信。
实施例2序列保守性分析与分子对接
序列同源性比对检索发现,脯氨酸羟化酶基因与四氢嘧啶羟化酶基因(ectD)具有较高的同源性,同属于Fe2+与α-酮戊二酸依赖性的双加氧酶超家族,都存在结合 Fe2+的保守 2-His-1-羧酸酯面三联体(见图3)和结合α-酮戊二酸的保守位点RXS motifs(见图4)。非铁红素和α-酮戊二酸依赖性的双加氧酶家族的酶促功能主要取决于高度活泼的铁离子。Proteins Superimposing揭示了在脯氨酸羟化酶BP4H的活性中心,氨基酸残基 His109、Asp111和 His215的功能基团(2个咪唑基团和1个羧基基团)与 Fe2+结合形成所谓的 2-His-1-羧酸酯面三联体。在所有非铁红素和α-酮戊二酸依赖性的双加氧酶家族的酶中,共底物α-酮戊二酸结合于酶蛋白活性中心底部,并通过其 1-羧基基团和 2-氧代基团参与协调亚铁离子与酶的结合,其5-羧基基团则通过与精氨酸或赖氨酸的侧链碱性基团形成的盐桥及与氨基酸残基的羟基形成氢键而稳定结合。在脯氨酸羟化酶BP4H的活性中心,氨基酸残基Ser217的醇羟基可能与α-酮戊二酸的5-羧基基团相互作用,且该位点严格保守;氨基酸残基Arg226的侧链碱性基团与α-酮戊二酸的5-羧基基团形成盐桥,将α-酮戊二酸固定在酶的活性中心。
分子对接结果(见图5)发现,脯氨酸位于Gln92、Lys94与Trp115等3个极性氨基酸残基与Fe2+组成的活性口袋中,并预测Lys94与L-脯氨酸的羧基形成盐桥,Gln92、Trp115分别与L-脯氨酸的羧基和α-亚氨基形成氢键。因此,根据序列保守性分析和分子对接结果,我们选择Gln92、Lys94、Trp115、Ser217和Arg226五个氨基酸位点进行定点半饱和突变。
实施例3 BP4H酶定点半饱和突变
向质粒引入单碱基定点突变,只需要设计一对引物将质粒进行反向PCR扩增即可,所设计引物序列见下表1,利用定点突变试剂盒Mut Express@ Ⅱ Fast Mutagenesis Kit和针对每个关键氨基酸位点设计的编码所有极性氨基酸的简并引物,对含有BP4H基因的质粒进行PCR扩增,将扩增产物进行琼脂糖凝胶电泳检测,最后我们成功获得了含突变后BP4H基因的质粒,且条带清晰。
表1 引物序列
注:n代表四种核苷酸;v代表A/C/G;k代表G/T,n、v、k编码了几乎全部极性氨基酸,适合我们来验证这五个氨基酸位点的可取代性。
实施例4 BP4H酶定点突变结果统计
将得到的PCR产物,经DpnI酶消化、回收、环化,并转入大肠杆菌感受态DH5a中,随机挑选菌株进行BP4H基因测序,测序后的BP4H定点半饱和突变结果统计见表2。
表2 BP4H酶定点突变结果统计
实施例5 突变菌株的HPLC活性验证
从验证正确的菌株中取50μL菌液接种于装有1mL LB液体培养基(含千分之一氨苄)的24孔板内,37℃、150rpm条件下振荡培养2h后,依次加入1μL的IPTG和3.9μL的L-脯氨酸,转25℃、150rpm振荡培养24h。
待测样品的衍生化,从24孔板中各取200μL菌液至1.5mL离心管中,9000r/min离心2min,取上清液50μL于新的1.5mL离心管中,再加入200μL四硼酸钠水溶液、410μL纯水、320μL乙腈、20μL FMOC-Cl乙腈溶液(0.38mol/L),混匀后放置反应10min,用0.22μm的微孔滤膜过滤到液相瓶中,进行高效液相色谱分析。
转化产物的液相色谱分析方法如下:色谱柱为Agilent Extend C-18(4.6mm×250mm,5μm,Agilent公司),流动相A为0.05%三氟乙酸水溶液,B为0.05%三氟乙酸乙腈溶液,检测波长263nm,进样量5μL,梯度洗脱条件:0~5min,30% B;5~8min,30%~40% B;8~15min,40% B;15~20 min,40%~50% B;20~25 min,50% B;25~30min,50%~90% B;30~38min,90% B;38~41min,90%~30% B;41~45 min,30% B。
结果:将得到的上述突变菌株进行活性验证,结果如表3。
表3 BP4H酶定点突变菌株活性验证结果
从表3中可以看出,这5个氨基酸位点经突变后,其活性均受到了较大影响,这说明Gln92、Lys94、Trp115、Ser217和Arg226 5个氨基酸位点确实如序列分析和分子对接预测的那样,位于酶催化的活性中心。
从突变菌株的相对活性结果(图4)来看,大部分突变菌株能保持>20%的野生酶活性,其中,部分氨基酸位点,如Gln92、Ser217,其突变菌株Q92S和S217H能保持85%以上的野生酶活性,这说明极性氨基酸之间的互换确实能保持其野生酶活性。这说明Gln92、Lys94、Trp115、Ser217和Arg226 5个氨基酸对酶的活性影响很大,更进一步证实了模型的准确性,验证了保守序列分析和分子对接的结果。这为我们接下来以同源建模为基础,通过定点突变提高BP4H酶活性,提供了有价值的理论基础。
以上公开的本发明优选实施例只是用于帮助阐述本发明。优选实施例并没有详尽叙述所有的细节,也不限制该发明仅为所述的具体实施方式。显然,根据本说明书的内容,可作很多的修改和变化。本说明书选取并具体描述这些实施例,是为了更好地解释本发明的原理和实际应用,从而使所属技术领域技术人员能很好地理解和利用本发明。本发明仅受权利要求书及其全部范围和等效物的限制。
SEQUENCE LISTING
<110> 河北博伦特药业有限公司
<120> 一种L-脯氨酸4-羟化酶及其应用
<130> 4
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 272
<212> PRT
<213> Bacillus cereus
<400> 1
Met Leu Thr Pro Thr Glu Leu Lys Gln Tyr Arg Glu Ala Gly Tyr Leu
1 5 10 15
Leu Ile Glu Asp Gly Leu Gly Pro Arg Glu Val Asp Cys Leu Arg Arg
20 25 30
Ala Ala Ala Ala Leu Tyr Ala Gln Asp Ser Pro Asp Arg Thr Leu Glu
35 40 45
Lys Asp Gly Arg Thr Val Arg Ala Val His Gly Cys His Arg Arg Asp
50 55 60
Pro Val Cys Arg Asp Leu Val Arg His Pro Arg Leu Leu Gly Pro Ala
65 70 75 80
Met Gln Ile Leu Ser Gly Asp Val Tyr Val His Gln Phe Lys Ile Asn
85 90 95
Ala Lys Ala Pro Met Thr Gly Asp Val Trp Pro Trp His Gln Asp Tyr
100 105 110
Ile Phe Trp Ala Arg Glu Asp Gly Met Asp Arg Pro His Val Val Asn
115 120 125
Val Ala Val Leu Leu Asp Glu Ala Thr His Leu Asn Gly Pro Leu Leu
130 135 140
Phe Val Pro Gly Thr His Glu Leu Gly Leu Ile Asp Val Glu Arg Arg
145 150 155 160
Ala Pro Ala Gly Asp Gly Asp Ala Gln Trp Leu Pro Gln Leu Ser Ala
165 170 175
Asp Leu Asp Tyr Ala Ile Asp Ala Asp Leu Leu Ala Arg Leu Thr Ala
180 185 190
Gly Arg Gly Ile Glu Ser Ala Thr Gly Pro Ala Gly Ser Ile Leu Leu
195 200 205
Phe Asp Ser Arg Ile Val His Gly Ser Gly Thr Asn Met Ser Pro His
210 215 220
Pro Arg Gly Val Val Leu Val Thr Tyr Asn Arg Thr Asp Asn Ala Leu
225 230 235 240
Pro Ala Gln Ala Ala Pro Arg Pro Glu Phe Leu Ala Ala Arg Asp Ala
245 250 255
Thr Pro Leu Val Pro Leu Pro Ala Gly Phe Ala Leu Ala Gln Pro Val
260 265 270
<210> 2
<211> 819
<212> DNA
<213> Bacillus cereus
<400> 2
atgctgaccc cgacggagct caagcagtac cgcgaggcgg gctatctgct catcgaggac 60
ggcctcggcc cgcgggaggt cgactgcctg cgccgggcgg cggcggccct ctacgcgcag 120
gactcgccgg accgcacgct ggagaaggac ggccgcaccg tgcgcgcggt ccacggctgc 180
caccggcgcg acccggtctg ccgcgacctg gtccgccacc cgcgcctgct gggcccggcg 240
atgcagatcc tgtccggcga cgtgtacgtc caccagttca agatcaacgc gaaggccccg 300
atgaccggcg atgtctggcc gtggcaccag gactacatct tctgggcccg agaggacggc 360
atggaccgtc cgcacgtggt caacgtcgcg gtcctgctcg acgaggccac ccacctcaac 420
gggccgctgt tgttcgtgcc gggcacccac gagctgggcc tcatcgacgt ggagcgccgc 480
gcgccggccg gcgacggcga cgcgcagtgg ctgccgcagc tcagcgccga cctcgactac 540
gccatcgacg ccgacctgct ggcccggctg acggccgggc ggggcatcga gtcggccacc 600
ggcccggcgg gctcgatcct gctgttcgac tcccggatcg tgcacggctc gggcacgaac 660
atgtcgccgc acccgcgcgg cgtcgtcctg gtcacctaca accgcaccga caacgccctg 720
ccggcgcagg ccgctccgcg cccggagttc ctggccgccc gcgacgccac cccgctggtg 780
ccgctgcccg cgggcttcgc gctggcccag cccgtctag 819
<210> 3
<211> 272
<212> PRT
<213> 人工合成
<400> 3
Met Leu Thr Pro Thr Glu Leu Lys Gln Tyr Arg Glu Ala Gly Tyr Leu
1 5 10 15
Leu Ile Glu Asp Gly Leu Gly Pro Arg Glu Val Asp Cys Leu Arg Arg
20 25 30
Ala Ala Ala Ala Leu Tyr Ala Gln Asp Ser Pro Asp Arg Thr Leu Glu
35 40 45
Lys Asp Gly Arg Thr Val Arg Ala Val His Gly Cys His Arg Arg Asp
50 55 60
Pro Val Cys Arg Asp Leu Val Arg His Pro Arg Leu Leu Gly Pro Ala
65 70 75 80
Met Gln Ile Leu Ser Gly Asp Val Tyr Val His Ser Phe Lys Ile Asn
85 90 95
Ala Lys Ala Pro Met Thr Gly Asp Val Trp Pro Trp His Gln Asp Tyr
100 105 110
Ile Phe Trp Ala Arg Glu Asp Gly Met Asp Arg Pro His Val Val Asn
115 120 125
Val Ala Val Leu Leu Asp Glu Ala Thr His Leu Asn Gly Pro Leu Leu
130 135 140
Phe Val Pro Gly Thr His Glu Leu Gly Leu Ile Asp Val Glu Arg Arg
145 150 155 160
Ala Pro Ala Gly Asp Gly Asp Ala Gln Trp Leu Pro Gln Leu Ser Ala
165 170 175
Asp Leu Asp Tyr Ala Ile Asp Ala Asp Leu Leu Ala Arg Leu Thr Ala
180 185 190
Gly Arg Gly Ile Glu Ser Ala Thr Gly Pro Ala Gly Ser Ile Leu Leu
195 200 205
Phe Asp Ser Arg Ile Val His Gly Ser Gly Thr Asn Met Ser Pro His
210 215 220
Pro Arg Gly Val Val Leu Val Thr Tyr Asn Arg Thr Asp Asn Ala Leu
225 230 235 240
Pro Ala Gln Ala Ala Pro Arg Pro Glu Phe Leu Ala Ala Arg Asp Ala
245 250 255
Thr Pro Leu Val Pro Leu Pro Ala Gly Phe Ala Leu Ala Gln Pro Val
260 265 270
<210> 4
<211> 272
<212> PRT
<213> 人工合成
<400> 4
Met Leu Thr Pro Thr Glu Leu Lys Gln Tyr Arg Glu Ala Gly Tyr Leu
1 5 10 15
Leu Ile Glu Asp Gly Leu Gly Pro Arg Glu Val Asp Cys Leu Arg Arg
20 25 30
Ala Ala Ala Ala Leu Tyr Ala Gln Asp Ser Pro Asp Arg Thr Leu Glu
35 40 45
Lys Asp Gly Arg Thr Val Arg Ala Val His Gly Cys His Arg Arg Asp
50 55 60
Pro Val Cys Arg Asp Leu Val Arg His Pro Arg Leu Leu Gly Pro Ala
65 70 75 80
Met Gln Ile Leu Ser Gly Asp Val Tyr Val His Gln Phe Lys Ile Asn
85 90 95
Ala Lys Ala Pro Met Thr Gly Asp Val Trp Pro Trp His Gln Asp Tyr
100 105 110
Ile Phe Trp Ala Arg Glu Asp Gly Met Asp Arg Pro His Val Val Asn
115 120 125
Val Ala Val Leu Leu Asp Glu Ala Thr His Leu Asn Gly Pro Leu Leu
130 135 140
Phe Val Pro Gly Thr His Glu Leu Gly Leu Ile Asp Val Glu Arg Arg
145 150 155 160
Ala Pro Ala Gly Asp Gly Asp Ala Gln Trp Leu Pro Gln Leu Ser Ala
165 170 175
Asp Leu Asp Tyr Ala Ile Asp Ala Asp Leu Leu Ala Arg Leu Thr Ala
180 185 190
Gly Arg Gly Ile Glu Ser Ala Thr Gly Pro Ala Gly Ser Ile Leu Leu
195 200 205
Phe Asp Ser Arg Ile Val His Gly His Gly Thr Asn Met Ser Pro His
210 215 220
Pro Arg Gly Val Val Leu Val Thr Tyr Asn Arg Thr Asp Asn Ala Leu
225 230 235 240
Pro Ala Gln Ala Ala Pro Arg Pro Glu Phe Leu Ala Ala Arg Asp Ala
245 250 255
Thr Pro Leu Val Pro Leu Pro Ala Gly Phe Ala Leu Ala Gln Pro Val
260 265 270
Claims (6)
1.一种L-脯氨酸4-羟化酶,其特征在于,其氨基酸序列在SEQ ID NO.1所示序列中具有如下突变中的一种或多种:
(1)SEQ ID NO.1中第92位氨基酸由谷氨酰胺突变为丝氨酸、苏氨酸、半胱氨酸、赖氨酸、天冬氨酸或谷氨酸;
(2)SEQ ID NO.1中第94位氨基酸由赖氨酸突变为丝氨酸、半胱氨酸、组氨酸或天冬氨酸;
(3)SEQ ID NO.1中第115位氨基酸由色氨酸突变为谷氨酰胺、赖氨酸或谷氨酸;
(4)SEQ ID NO.1中第217位氨基酸由丝氨酸突变为苏氨酸、半胱氨酸、谷氨酰胺、精氨酸、组氨酸或天冬氨酸;
(5)SEQ ID NO.1中第226位氨基酸由精氨酸突变为苏氨酸、酪氨酸、天冬氨酸或谷氨酸。
2.一种L-脯氨酸4-羟化酶,其特征在于,其氨基酸序列在SEQ ID NO.1所示序列中第92位氨基酸由谷氨酰胺突变为丝氨酸。
3.一种L-脯氨酸4-羟化酶,其特征在于,其氨基酸序列在SEQ ID NO.1所示序列中第217位氨基酸由丝氨酸突变为组氨酸。
4.一种编码如权利要求1~3任一项所述的L-脯氨酸4-羟化酶的DNA。
5.一种包含权利要求4所述DNA的基因工程菌。
6.一种利用如权利要求1~3任一项所述的L-脯氨酸4-羟化酶在合成反式-4-羟基-L-脯氨酸中的应用。
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WO2010111622A2 (en) * | 2009-03-27 | 2010-09-30 | Codexis, Inc. | Amidases and methods of their use |
CN109207459A (zh) * | 2018-11-23 | 2019-01-15 | 福州大学 | 一种定点突变改造热稳定性提高的琼胶酶突变体 |
CN110846288A (zh) * | 2019-11-27 | 2020-02-28 | 浙江华睿生物技术有限公司 | 一种谷胱甘肽双功能酶突变体及其应用 |
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WO2010111622A2 (en) * | 2009-03-27 | 2010-09-30 | Codexis, Inc. | Amidases and methods of their use |
CN109207459A (zh) * | 2018-11-23 | 2019-01-15 | 福州大学 | 一种定点突变改造热稳定性提高的琼胶酶突变体 |
CN110846288A (zh) * | 2019-11-27 | 2020-02-28 | 浙江华睿生物技术有限公司 | 一种谷胱甘肽双功能酶突变体及其应用 |
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