CN112359030B - 一种ω-转氨酶突变体及其应用 - Google Patents
一种ω-转氨酶突变体及其应用 Download PDFInfo
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- CN112359030B CN112359030B CN202011344825.7A CN202011344825A CN112359030B CN 112359030 B CN112359030 B CN 112359030B CN 202011344825 A CN202011344825 A CN 202011344825A CN 112359030 B CN112359030 B CN 112359030B
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- aminotransferase
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Abstract
本发明公开了一种ω‑转氨酶突变体,由来自土曲霉(Aspergillus terreus)的ω‑转氨酶突变所得,野生型ω‑转氨酶的氨基酸序列如SEQ ID No.2所示,所述ω‑转氨酶突变体的突变位点为:E133Q、D224K、D231A、E253A或D268K中的至少一种。本发明筛选得到的ω‑转氨酶突变体在热力学稳定性、酶活性方面明显优于野生酶。
Description
技术领域
本发明涉及生物技术领域,特别是涉及一种ω-转氨酶突变体及其应用。
背景技术
手性胺是手性药物的重要组成部分,是一类极其重要的精细化工和医药中间体。随着手性药物市场的不断扩大,手性胺的需求迅速增长。对手性胺类药物进行更加深层次的研究具有较大的商业经济效益和工业应用价值。目前,超过70%的药物都是手性胺及其衍生物,如神经类药物、心血管药物、抗高血压药物、抗感染药物及疫苗等的合成都是以手性胺作为中间体。
转氨酶是催化某基团从供体化合物转移到受体化合物上的一类酶,可以通过简单的催化剂里,可逆地将来源于氨基酸供体的氨基转移到氨基酸受体上。目前,在不对称合成手性胺类化合物及胺类化合物外消旋体拆分中,转氨酶都是关键的生物技术酶。由于其不对称催化合成手性胺的特点,转氨酶已成为工业上用于生产氨基酸、手性胺、氨基醇和氨基糖等重要农药或医药中间体的常用酶之一。来自于土曲霉属(Aspergillus terreus)的ω-转氨酶以酮类化合物为原料,通过立体选择性地转氨基作用,可以高效生产手性胺,催化氨基供体上的氨基转移到前手性的受体酮,得到手性胺和副产物酮,反应过程需要磷酸吡哆醛(pyridoxal phosphate,PLP)的参与,催化过程如下所示:
实验表明,ω-转氨酶野生型在40℃下的半衰期仅为6.9min,不利于应用到工业生产中,其热稳定性有待进一步提高。如CN105441404A、CN105950581A公开了利用定点突变技术对ω-转氨酶野生型进行改造,获得热稳定性进一步提高的ω-转氨酶突变体,使其更适合工业应用。
转氨酶在合成手性胺方面具有较好的应用前景,但由于野生型酶在底物特异性、稳定性、催化效率等方面存在诸多不足,目前满足工业应用需求的转氨酶仍较为有限。基于非理性、理性及半理性设计策略的蛋白质工程技术能有效改善转氨酶的应用性能,为手性胺的高效制备提供了可能。随着转氨酶蛋白结构和催化机制相关研究的深入,利用理性或半理性设计策略对转氨酶进行分子改造的研究备受关注。
在自然进化的过程中,大多数的蛋白质的表面氨基酸残基具有较大的不保守性,对酶的催化性能和热稳定性有重要的微调作用。蛋白质表面带点残基的等电点(isoelectric,pI)与蛋白质的等电点相差较大时,可以预估该氨基酸对蛋白质的结构稳定可能存在负面效果。改变蛋白质表面的相应带电残基可能会引起突变位点周围,甚至是蛋白质催化活性部位周围的静电场发生改变,从而达到影响酶的热稳定性以及催化性能。研究表明,优化蛋白质表面电荷是一种提高酶催化性能的有效策略,该方法通过对酶蛋白表面的电荷-电荷间相互作用能进行计算预测,将蛋白表面不利于酶催化性能的带电氨基酸残基替换为丙氨酸或者相反电荷的氨基酸残基以达到提高其热稳定性和催化性能的目的。Schweiker等(Schweiker KL,Zarrine-Afsar A,Davidson AR,etal.Computationaldesign of the Fyn SH3 domain with increased stability through optimization ofsurface chargecharge interactions[J].Protein Science.2007;16(12):2694-702.)使用TK-SA模型合理优化Fyn SH3结构域表面电荷间相互作用能,成功地提高了该蛋白的稳定性,最稳定突变体的热解折叠温度(Tm)比野生型高12.3℃。Zhang等(Zhang LJ,Tang XM,Cui DB,etal.A method to rationally increase protein stability based on thecharge-charge interaction,with application to lipase LipK107[J].ProteinScience.2014;23(1):110-6.)通过改进TK-SA模型算法,发布了一套新的设计算法-ETSS,运用ETSS算法预测位于脂肪酶Lipk107表面且对酶的稳定性至关重要4个残基(D113、D149、D213和D253),利用酸性或中性氨基酸对4个位点的氨基酸进行替换得到4个Lipk107突变体(D113A、D149K、D213A和D253A),研究结果显示,突变体D113A、突变体D149K、突变体D213A和突变体D253A在50℃下的半衰期(t1/2)分别是野生型的12倍、14倍、4.5倍和6倍,且突变体D253A的酶活力是野生型的1.2倍。Tu等(Tu T,Luo H,Meng K,etal.Improvement inthermostability of an Achaetomium sp.strain Xz8 endopolygalacturonase via theoptimization of charge-charge interactions[J].Applied and EnvironmentalMicrobiology.2015;81(19):6938-44.)利用ETSS程序计算了多聚半乳糖醛酸酶带电氨基酸i点和j之间的总相互作用能(Eij),推断出影响酶蛋白稳定性的9个残基,利用定点突变构建9个突变体,研究表明,突变体D244A和突变体D299R表现出较好的热稳定性;双突变体D244A/D299R的半失活温度(T50)和Tm分别比野生型提高17℃和10.2℃,其在50℃和55℃的t1/2分别比野生型延长8.4h和45min。
但是蛋白质结构具有极其复杂的特点,存在三级结构中的各个氨基酸均受到近端或远端氨基酸残基改变的影响,而这些影响无法进行精确地预知。通过ETSS算法能够预知突变氨基酸的位点,但是对于氨基酸突变之后对蛋白质整体的影响是未知的。通过yasara软件进行转氨酶单突变体动力学计算,预测不同温度下ω-转氨酶的氨基酸残基在突变前后的均方根偏差(RMSD)的差值,初步对突变体进行验证,从而达到理性设计的目的。
目前尚无通过酶蛋白表面的电荷-电荷间相互作用能计算以及yasara模拟计算均方根偏差(RMSD)的差值的方法来提高土曲霉属(Aspergillus terreus)ω-转氨酶热稳定性的相关研究报道。
发明内容
本发明基于一种酶蛋白表面电荷电荷相互作用能的计算预测的方法,通过该方法获得酶活、热稳定性进一步提高的土曲霉(Aspergillus terreus)ω-转氨酶突变体。
本发明利用一套新的表面电荷-电荷间相互作用能算法-ETSS,筛选与野生酶氨基酸序列中等电点处于劣势的氨基酸残基,作为优选突变氨基酸,进而利用定点突变技术进行改造。本发明利用蛋白质动力学计算软件yasara计算不同温度下带电氨基酸残基突变体的RMSD差值,筛选较野生酶氨基酸序列中RMSD值逆向变化的突变体,作为优选突变体,进而利用定点突变技术进行改造。
一种ω-转氨酶突变体,由来自土曲霉(Aspergillus terreus)的ω-转氨酶突变所得,野生型ω-转氨酶的氨基酸序列如SEQ ID No.2所示,所述ω-转氨酶突变体的突变位点为:E133Q、D224K、D231A、E253A或D268K中的至少一种。
本发明又提供了所述的ω-转氨酶突变体在催化(R)-(+)-α-甲基苄胺生成苯乙酮中的应用。相较于野生型酶,突变体酶在较高温条件下具有较好的热力学稳定性,更适合工业应用。
本发明又提供了编码所述ω-转氨酶突变体的基因。优选的,所述的基因,突变位点为E133Q、D224K、D231A、E253A或D268K的ω-转氨酶突变体的基因序列分别如SEQ IDNo.3~7。
本发明又提供了所述基因在催化(R)-(+)-α甲基苄胺生成苯乙酮中的应用。
本发明还提供了包含所述基因的重组表达质粒。
本发明还提供了包含所述重组表达质粒的基因工程菌。
本发明还提供了所述基因工程菌在催化(R)-(+)-α甲基苄胺生成苯乙酮中的应用。
与现有技术相比,本发明具有以下有益效果:
本发明方法基于技术成熟的遗传算法,以及TK-SA模型算法,在此基础上改进得到ETSS算法,结合ω-转氨酶表面电荷-电荷相互作用,确定需要突变的氨基酸残基位点,通过定点突变技术进行实验验证。通过yasara进行蛋白质动力学计算不同温度下氨基酸残基位点在突变前后的RMSF,初步分析出突变氨基酸对近端或远端带电氨基酸的影响,并用对应温度下的蛋白质均方根偏差(RMSD)进行计算验证。该方法可有效地提高正突变概率,提高实验效率及可行性,并筛选得到热力学稳定性、酶活性明显优于野生酶的突变酶。
附图说明
图1为ω-转氨酶中各带电残基的Eij值计算结果图。
图2为野生型和突变体的SDS-PAGE电泳分析结果图,其中,各泳道分别为M:protein marker;1:野生型(未纯化);2:野生型(纯化);3:E133Q;4:D224K;5:D231A;6:E253A;7:D268K。
图3为野生型和突变体的酶活力检测结果图。
图4为野生型和突变体的残余酶活力检测结果图。
图5为野生型和突变体的稳定性检测结果图,分别为野生型和突变体E133Q,D224K,D231A,E253A,D268K的T50 10。
图6为野生型和突变体的稳定性检测结果图,分别为野生型和突变体E133Q,D224K,D231A,E253A,D268K在40℃下的t1/2。
图7为野生型和突变体的MD模拟结果图,其值显示为MD模拟阶段(4-10ns)的RMSD值,其中,(a)313K和400K条件下野生型和突变体E133Q的RMSD差值;(b)313K和400K条件下野生型和突变体D224K的RMSD差值;(c)313K和400K条件下野生型和突变体D231A的RMSD差值;(d)313K和400K条件下野生型和突变体E253A的RMSD差值;(e)313K和400K条件下野生型和突变体D268K的RMSD差值。
图8为野生型和突变体的MD模拟结果图,其值分别显示为313K(a)和400K(b)条件下野生型和突变体E133Q,D224K,D231A,E253A,D268K部分氨基酸残基的RMSF差值。
具体实施方式
实施例1
(1)材料与试剂
(R)-ω-TA全基因由安徽通用生物公司合成,重组质粒pET-28a-ω-TA由本实验室保藏。PrimeSTAR Max DNA聚合酶购自TaKaRa公司;Dpn I购自Thermo Scientific公司;SanPrep柱式质粒DNA小量抽提试剂盒、异丙基-b-D-硫代半乳糖苷(IPTG)、硫酸卡那霉素(Kanamycin sulfate)、磷酸吡哆醛(pyrodoxal-5’-phosphate,PLP)、改良型Bradford蛋白浓度测定试剂盒购自生工生物工程(上海)股份有限公司;DNA Marker、Protein Marker、E.coli BL21(DE3)、E.coli DH5α、Ni-NTA层析介质购自北京全式金生物技术有限公司;丹磺酰氯(DNS-C1)购自Sigma公司(美国);γ-氨基丁酸标品(γ-aminobutyric acid)购自Fluka公司(瑞士);十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)凝胶制备试剂盒购于康为世纪生物科技有限公司;二甲基亚砜(DMSO)、丙酮酸、(R)-α-甲基苄胺购自阿拉丁生化科技股份有限公司
(2)突变位点的选择
在不考虑其他相互作用的前提下,利用Linux系统(Centos6.0版本)的ETSS程序计算野生型ω-转氨酶(wild-type)带电氨基酸残基i位点和j位点之间的总相互作用能(Eij),Eij值为正表示其对应残基对ω-转氨酶的整体稳定性呈负面作用,而Eij值为负表示其对应残基对ω-转氨酶的整体稳定性呈积极作用。理论上,蛋白质特定位置带电残基的电荷发生变化可能引起其表面电位的改变,从而提高蛋白质的稳定性。因此,利用定点突变技术将特定位置Eij值为正的氨基酸突变为简单的中性氨基酸(丙氨酸)和电荷相反的氨基酸,构建突变体。按照以下3种标准对所有带电氨基酸残基进行筛选:(1)Eij能量值(正值)高的残基;(2)远离ω-转氨酶活性中心的残基,(3)处于ω-转氨酶二级结构上的残基,优先选择处于loop区域与二级结构交叉位置。
野生型ω-转氨酶带电氨基酸残基i位点和.j位点之间的总Eij如图1所示,在ω-转氨酶中共发现95个带电氨基酸,其中49个残基的Eij值为正,46个残基的Eij值为负。按照3种标准对所有带电氨基酸残基进行筛选,筛选到的6个影响ω-转氨酶催化性能的关键氨基酸残基(E133,D224,D231,E253,E262和D268),随后,利用定点突变技术获得5个潜在突变体(E133Q,D224K,D231A,E253A,D268K)。
使用表1中的引物、以含有ω-转氨酶基因(基因序列如SEQ ID No.1所示,长度为978bp,从土曲霉Aspergillus terreus中克隆所得)的质粒为模板进行PCR扩增。在37℃条件下,PCR产物经Dpn I进行消化后,进行PCR产物纯化试剂盒进行纯化,之后采用化学转化法转入E.coli DH 5α感受态细胞中,1h后将复苏液涂布于含有终浓度为50μg/μL卡那霉素的LB固体平板得到定点突变文库。重组质粒送至安徽通用生物系统有限公司进行核苷酸序列测定,将测序正确的重组质粒转化入E.coli BL21(DE3)感受态细胞,以获取目标重组菌株。
表1 定点突变引物
引物名称 | 序列(5’-3’) |
E133Q-F | GAACTCGTCCGCAAGATATAGTGAACAACCT |
E133Q-R | GTTGTTCACTATATCTTGCGGACGAGTTCCT |
D224K-F | TTAGTCAAAAAAGGCGTCCTGTATACGCCAGAT |
D224K-R | TGGCGTATACAGGACGCCTTTTTTGACTAATAC |
D231A-F | TCCTGTATACGCCAGCTCGCGGGGTGCTGC |
D231A-R | TGCAGCACCCCGCGAGCTGGCGTATACAGG |
E253A-F | TTTGGAATAGCGGTGCGGGTTGAG |
E253A-R | TCAACCCGCACCGCTATTCCAAAG |
D268K-F | GCCTACCGGTGTAAAGAGATTTTCATGT |
D268K-R | CATGAAAATCTCTTTACACCGGTAGG |
备注:下划线表示突变位置。
(3)突变文库的建立与质粒的提取
取出-80℃保存的E.coli DH5α感受态细胞并将其放冰上解冻。5μL的消化产物加入到50μL E.coli DH5α感受态细胞中,用枪轻轻混匀,冰上静置30min。42℃水浴锅内热激90s,迅速将管放冰上冷却3~5min。每管中加入600μL预冷的LB培养基,37℃,180pm条件下复苏培养1h,使细菌恢复到正常生长状态。将菌液6000rpm离心2min,除去500μL上清液,取余下菌液混匀后,均匀地涂布于含有终浓度为50μg/mL Kan的LB固体培养基平板上。37℃培养箱中培养平板(正面向上)20-30min后,倒置平板,过夜培养。
随机挑取平板上的单菌落接种于5mL含50μg/mL Kan的LB培养基中,37℃,230rpm条件下培养至OD600值为0.8时,取1mL菌液送至杭州擎科梓熙生物技术有限公司进行核苷酸序列的测定,1mL菌液用于保存菌种,3mL菌液用于提取质粒,具体步骤参照质粒小量提取试剂盒(康为世纪)说明书。测序正确的菌种质粒用1%DNA琼脂糖凝胶电泳验证目的条带的大小和纯度后,5μL质粒被转入E.coli BL21(DE3)感受态细胞中,以获取目标重组菌株,剩余质粒放入-20℃冰箱保存备用。
(4)酶的表达与纯化
挑取野生型及突变体单菌落接种于5mL含有终浓度为50μg/mL Kan的LB液体培养基中,37℃、230rpm条件下摇床培养12h。菌液以2%的接种量(V/V)转接至200mL含有终浓度为50μg/mL Kan的LB液体培养基中,37℃、230rpm条件下继续培养2~3h。当OD600达到0.8时,加入终浓度为0.5mM的IPTG,并在25℃、150rpm条件下诱导蛋白表达。诱导20h后,在6000r/min,4℃条件下收集菌体。
菌体细胞用20mM的PBS缓冲液(pH 8.0)洗涤2次去除残留培养基后悬浮于50mL破胞缓冲液(50mM磷酸二氢钠,300mM氯化钠,20mM咪唑,pH 8.0)中。冰浴条件下对菌体细胞进行均质机破碎细胞。细胞破碎液在8000rpm,4℃条件下离心1h,收集得到的上清液即为含有ω-转氨酶的粗酶液。随后,采用Ni-NTA亲和层析法对粗酶液进行分离纯化,粗酶经上样、清洗、洗脱后得到纯酶液,操作步骤参照说明书进行。
(5)蛋白含量的测定
采用改良型Bradford蛋白浓度测定试剂盒建立蛋白含量标准曲线,测定纯酶的浓度,蛋白标准曲线的制备步骤参照说明书进行。采用SDS-PAGE方法(12%分离胶和5%浓缩胶)鉴定纯化后蛋白的分子量和纯度。
野生型和突变体的SDS-PAGE电泳图谱如图2所示。野生型和突变体的电泳条带位于同一水平线,分子量约为36kDa,与理论分子量(36.1kDa)相一致,为后续实验的进行奠定了基础。
(6)酶活力的测定
1)酶活力的测定
20μL纯酶与180μL底物溶液(10mM PLP,2.5mM(R)-α-MBA,2.5mM丙酮酸,0.25%DMSO,50mM PBS,pH=8.0)于25℃条件下反应3min,测定OD245丙酮酸的生成量,具体方法参考(S,/>M,Redestad E,et al.Rapid and Sensitive Kinetic Assay forCharacterization ofω-Transaminases[J].Anal Chem,2009,81:8244-8248.)酶活力(U)定义为在一定条件下,每分钟转氨酶催化底物丙酮酸和(R)-(+)-α-甲基苄胺发生转氨反应生成1μmoL苯乙酮所需酶量。
野生型和5个突变体的酶活力如图3所示,与ω-转氨酶野生型的酶活力相比,5个突变体的的酶活力显著提高,分别是野生型酶活力的2.80至3.26倍,说明对133、224、231、253、268五个位点进行合理的突变,ω-转氨酶的催化活性得到改善。
2)酶残余活力的测定
将纯化的野生型和突变体在40℃下孵育10min,孵育结束后,立即放冰上冷却10min。随后,20μL经热处理的酶液与180μL底物溶液(10mM PLP,2.5mM(R)-α-MBA,2.5mM丙酮酸,0.25%DMSO,50mM PBS,pH=8.0)于25℃条件下反应3min,测定野生型和突变体的残余活力。实验平行三次,以未经热处理得到的酶活力为100%,筛选出相对酶活力比野生型高的突变体。40℃下热处理10min后,野生型和5个突变体的残余活力如图4所示。突变体E133Q,D224K,D231A,E253A,D268K的残余活力百分比均比野生型高,分别是野生型的1.75倍、1.64倍、1.95倍2.17倍和2.06倍。
综上所述,利用优化蛋白表面电荷的方法,筛选到5个热稳定性和酶活力都提升的突变体E133Q,D224K,D231A,E253A,D268K。此外,133位点突变为谷氨酰胺之后,酶活力得到提升,说明133位点更加适合略带电负性的氨基酸。相反的,224位点和268位点突变为带正电的赖氨酸之后的酶活力升高,说明224和268两个位点适合带正电氨基酸,将其突变为中性氨基酸反而不能够很有效的提升酶活力。
(7)酶学参数的测定
用含0.01mM PLP的PBS缓冲液(50mM,pH=8.0)分别配制0、0.125、0.25、0.5、1.0、1.5、2.0、2.5、3.0不同浓度的(R)-α-MBA和丙酮酸底物溶液。采用酶活力测定的方法测定在不同浓度下ω-转氨酶野生型和突变体的酶活力。将不同底物以及不同底物浓度[S]下对应的反应速率V带入米氏方程V=Vmax×[S]/(Km+[S]),利用Origin 8.0软件进行非线性拟合,并计算野生型和突变体对应的酶动力学参数Km和Vmax;由公式kcat=Vmax/[E]([E]为酶的摩尔浓度),计算野生型和突变体对应的kcat和kcat/Km。
从表2可知,与野生型相比的动力学参数相比,五种突变体对氨基供体的的亲和力(Km α-MBA)上升,对氨基受体的亲和力(Km pyruvate)也上升(Km值越低,酶对底物的亲和力越强),从而五种突变体的催化效率均得到显著提升,导致突变体E133Q,D224K,D231A,E253A,D268K的kcat/Kmm pyruvate分别是野生型的1.13至1.69倍,kcat/Km α-MBA分别是野生型的1.60至3.12倍。
表2 野生型和突变体的动力学参数
(8)热稳定性的测定
1)T50 10的测定
T50 10是指纯酶在4~60℃下孵育10min后,酶残余活力降低到50%时所对应的温度。将纯化后的野生酶及其突变体分别在4~60℃下孵育10min,孵育结束后迅速放置冰上冷却10min,测定野生型及其突变体的残余活力。以温度为横坐标,以热处理后与未经热处理的酶活力比值为纵坐标,运用Origin 8.0软件作图,计算野生型和突变体的T50 10。
2)t1/2的测定
t1/2是指纯酶在40℃下孵育不同时间后,酶残余活力降低到50%时所对应的时间。将纯化后的野生型及其突变体分别在40℃下孵育0~80min,孵育结束后立即放冰上冷却10min,测定野生型及其突变体的残余活力。以时间为横坐标,以热处理后与未经热处理的酶活力比值为纵坐标,运用Origin 8.0软件作图,计算野生型和突变体在40℃下的t1/2。
3)Tm的测定
Tm是指纯酶达到最大反应速率所需要的最适温度。将20μL纯化后的野生型及其突变体与180μL底物溶液(0.25%DMSO,2.5mM(R)-α-MBA,2.5mM丙酮酸)混合后,迅速放入25~55℃的恒温混匀仪中(400r/min),3min后迅速放入100℃的沸水浴中变性10min,最后在冰上放置10min后,利用酶标仪检测OD245,运用Origin 8.0软件作图,计算野生型和突变体的Tm。
突变体E133Q,D224K,D231A,E253A和D268K的稳定性测定结果如图5、图6和表3所示。野生型的T50 10为38.5℃,突变体E133Q,D224K,D231A,E253A和D268K的T50 15分别为40.70至44.59℃,分别比野生型提高了2.2至6.09℃。突变体E133Q,D224K,D231A,E253A和D268K的t1/2分别为13.17至27.89min,分别是野生型t1/2(6.9min)的1.91至4.04倍。
表3 野生型和突变体的稳定性参数
名称 | T50 15(℃) | t1/2(min) |
WT-AT | 38.50±0.5 | 6.90±0.6 |
E133Q | 41.12±0.5 | 15.05±0.5 |
D224K | 44.59±0.5 | 27.89±0.3 |
D231A | 42.51±0.8 | 19.52±0.5 |
E253A | 40.70±0.5 | 13.17±0.5 |
D268K | 43.80±0.5 | 23.35±0.5 |
(9)分子动力学模拟
分子动力学(MD)模拟是分析蛋白质稳定性及其分子机制的有效方法。骨架原子的均方根偏差(RMSD)表示整个蛋白质的稳定性,与蛋白质的热稳定性呈负相关。单个残基的均方根涨落(RMSF)代表单个氨基酸残基在蛋白质结构中的稳定性,与蛋白质的热稳定性呈负相关。
以土曲霉属(Aspergillus terreus)ω-转氨酶野生型三维结构为模板,利用Pymol软件构建突变体三维结构。野生型和突变体三维结构经FoldX软件(version 3.0beta5.1)进行处理后,运用YASARA软件(版本16.4.6)的Amber 14力场对其在313K和400K下温度下进行10ns的分子动力学模拟。将PDB格式的野生型和突变体三维结构载入YASARA软件,结构经加氢处理后置于水密度为0.998mg/L以及边长为20埃的立方体中,钠离子和氯离子作为反离子使体系呈电中性。范德华力相互作用的截断距离为7.86埃,采用ParticleMesh Ewald(PME)方法计算长程静电相互作用。时间步长为2.5fs,模拟的三维结构构象每25ps保存一次,利用Visual Molecular Dynamics(VMD)软件对模拟轨迹进行可视化。
将野生型和突变体E133Q,D224K,D231A,E253A和D268K在313K和400K下分别进行时长为10ns的MD模拟,并且对两个模拟结果的值进行差值比较,对比结果如图7和图8所示。由图7可知,在整个MD模拟过程中,突变体E133Q,D224K,D231A,E253A和D268K的平均RMSD差值明显低于野生型,表明E133Q,D224K,D231A,E253A和D268K具有更高的稳定性。由图8可知,在313K和400K温度下,野生型蛋白中24位氨基酸区域的RMSF差值值高于突变体E133Q,D224K,D231A,E253A和D268K,52位氨基酸区域的RMSF差值值高于突变体E133Q,D224K,D231A,E253A和D268K,说明野生型ω-转氨酶的九种突变体能够产生远端氨基酸残基活跃性的影响,从而导致蛋白稳定性提高。
序列表
<110> 浙江科技学院
宁波酶赛生物工程有限公司
<120> 一种ω-转氨酶突变体及其应用
<160> 17
<170> SIPOSequenceListing 1.0
<210> 1
<211> 978
<212> DNA
<213> 土曲霉(Aspergillus terreus)
<400> 1
atggccagta tggataaggt ttttgcaggc tatgctgccc gtcaagcaat cttagaaagt 60
accgaaacta cgaacccgtt tgccaaagga attgcctggg tcgaagggga actcgttcct 120
ttagctgaag cacgcattcc actcctcgat cagggcttca tgcactccga tctgacctac 180
gacgtaccgt ctgtttggga tgggcgattt tttcgtttag atgatcatat tacacgcctg 240
gaagcaagct gcaccaagct gaggctgcgt ctacccttac cacgtgatca agttaaacaa 300
atcctggtgg aaatggtcgc aaaatctggt attcgggatg catttgttga attgatagtc 360
acccgcggtc ttaaaggggt gcgaggaact cgtccgcatg atatagtgaa caacctgtac 420
atgtttgtgc agccgtacgt gtgggttatg gagccggata tgcagcgcgt aggcggcagc 480
gcagtggtgg ctaggaccgt ccgccgggta ccaccgggcg ctattgatcc gaccgtcaag 540
aatcttcagt ggggtgatct tgttcgtgga atgtttgaag cggctgatcg tggcgcaaca 600
tatcccttcc ttaccgacgg cgatgcgcac ctgactgaag gatcgggttt taatatagta 660
ttagtcaaag atggcgtcct gtatacgcca gatcgcgggg tgctgcaggg agtgactcgc 720
aagtccgtta tcaacgctgc tgaagccttt ggaatagaag tgcgggttga gttcgttcca 780
gttgagctgg cctaccggtg tgacgagatt ttcatgtgca cgacggcggg tggcattatg 840
cctatcacaa cattggacgg tatgcctgta aatggtgggc aaattgggcc tattacgaaa 900
aaaatatggg acggttattg ggcgatgcat tatgacgccg cgtattcgtt cgagatcgac 960
tataatgaga gaaattag 978
<210> 2
<211> 325
<212> PRT
<213> 土曲霉(Aspergillus terreus)
<400> 2
Met Ala Ser Met Asp Lys Val Phe Ala Gly Tyr Ala Ala Arg Gln Ala
1 5 10 15
Ile Leu Glu Ser Thr Glu Thr Thr Asn Pro Phe Ala Lys Gly Ile Ala
20 25 30
Trp Val Glu Gly Glu Leu Val Pro Leu Ala Glu Ala Arg Ile Pro Leu
35 40 45
Leu Asp Gln Gly Phe Met His Ser Asp Leu Thr Tyr Asp Val Pro Ser
50 55 60
Val Trp Asp Gly Arg Phe Phe Arg Leu Asp Asp His Ile Thr Arg Leu
65 70 75 80
Glu Ala Ser Cys Thr Lys Leu Arg Leu Arg Leu Pro Leu Pro Arg Asp
85 90 95
Gln Val Lys Gln Ile Leu Val Glu Met Val Ala Lys Ser Gly Ile Arg
100 105 110
Asp Ala Phe Val Glu Leu Ile Val Thr Arg Gly Leu Lys Gly Val Arg
115 120 125
Gly Thr Arg Pro Glu Asp Ile Val Asn Asn Leu Tyr Met Phe Val Gln
130 135 140
Pro Tyr Val Trp Val Met Glu Pro Asp Met Gln Arg Val Gly Gly Ser
145 150 155 160
Ala Val Val Ala Arg Thr Val Arg Arg Val Pro Pro Gly Ala Ile Asp
165 170 175
Pro Thr Val Lys Asn Leu Gln Trp Gly Asp Leu Val Arg Gly Met Phe
180 185 190
Glu Ala Ala Asp Arg Gly Ala Thr Tyr Pro Phe Leu Thr Asp Gly Asp
195 200 205
Ala His Leu Thr Glu Gly Ser Gly Phe Asn Ile Val Leu Val Lys Asp
210 215 220
Gly Val Leu Tyr Thr Pro Asp Arg Gly Val Leu Gln Gly Val Thr Arg
225 230 235 240
Lys Ser Val Ile Asn Ala Ala Glu Ala Phe Gly Ile Glu Val Arg Val
245 250 255
Glu Phe Val Pro Val Glu Leu Ala Tyr Arg Cys Asp Glu Ile Phe Met
260 265 270
Cys Thr Thr Ala Gly Gly Ile Met Pro Ile Thr Thr Leu Asp Gly Met
275 280 285
Pro Val Asn Gly Gly Gln Ile Gly Pro Ile Thr Lys Lys Ile Trp Asp
290 295 300
Gly Tyr Trp Ala Met His Tyr Asp Ala Ala Tyr Ser Phe Glu Ile Asp
305 310 315 320
Tyr Asn Glu Arg Asn
325
<210> 3
<211> 978
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
atggccagta tggataaggt ttttgcaggc tatgctgccc gtcaagcaat cttagaaagt 60
accgaaacta cgaacccgtt tgccaaagga attgcctggg tcgaagggga actcgttcct 120
ttagctgaag cacgcattcc actcctcgat cagggcttca tgcactccga tctgacctac 180
gacgtaccgt ctgtttggga tgggcgattt tttcgtttag atgatcatat tacacgcctg 240
gaagcaagct gcaccaagct gaggctgcgt ctacccttac cacgtgatca agttaaacaa 300
atcctggtgg aaatggtcgc aaaatctggt attcgggatg catttgttga attgatagtc 360
acccgcggtc ttaaaggggt gcgaggaact cgtccgcgtg atatagtgaa caacctgtac 420
atgtttgtgc agccgtacgt gtgggttatg gagccggata tgcagcgcgt aggcggcagc 480
gcagtggtgg ctaggaccgt ccgccgggta ccaccgggcg ctattgatcc gaccgtcaag 540
aatcttcagt ggggtgatct tgttcgtgga atgtttgaag cggctgatcg tggcgcaaca 600
tatcccttcc ttaccgacgg cgatgcgcac ctgactgaag gatcgggttt taatatagta 660
ttagtcaaag atggcgtcct gtatacgcca gatcgcgggg tgctgcaggg agtgactcgc 720
aagtccgtta tcaacgctgc tgaagccttt ggaatagaag tgcgggttga gttcgttcca 780
gttgagctgg cctaccggtg tgacgagatt ttcatgtgca cgacggcggg tggcattatg 840
cctatcacaa cattggacgg tatgcctgta aatggtgggc aaattgggcc tattacgaaa 900
aaaatatggg acggttattg ggcgatgcat tatgacgccg cgtattcgtt cgagatcgac 960
tataatgaga gaaattag 978
<210> 4
<211> 978
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
atggccagta tggataaggt ttttgcaggc tatgctgccc gtcaagcaat cttagaaagt 60
accgaaacta cgaacccgtt tgccaaagga attgcctggg tcgaagggga actcgttcct 120
ttagctgaag cacgcattcc actcctcgat cagggcttca tgcactccga tctgacctac 180
gacgtaccgt ctgtttggga tgggcgattt tttcgtttag atgatcatat tacacgcctg 240
gaagcaagct gcaccaagct gaggctgcgt ctacccttac cacgtgatca agttaaacaa 300
atcctggtgg aaatggtcgc aaaatctggt attcgggatg catttgttga attgatagtc 360
acccgcggtc ttaaaggggt gcgaggaact cgtccggaag atatagtgaa caacctgtac 420
atgtttgtgc agccgtacgt gtgggttatg gagccggata tgcagcgcgt aggcggcagc 480
gcagtggtgg ctaggaccgt ccgccgggta ccaccgggcg ctattgatcc gaccgtcaag 540
aatcttcagt ggggtgatct tgttcgtgga atgtttgaag cggctgatcg tggcgcaaca 600
tatcccttcc ttaccgacgg cgatgcgcac ctgactgaag gatcgggttt taatatagta 660
ttagtcaaaa aaggcgtcct gtatacgcca gatcgcgggg tgctgcaggg agtgactcgc 720
aagtccgtta tcaacgctgc tgaagccttt ggaatagaag tgcgggttga gttcgttcca 780
gttgagctgg cctaccggtg tgacgagatt ttcatgtgca cgacggcggg tggcattatg 840
cctatcacaa cattggacgg tatgcctgta aatggtgggc aaattgggcc tattacgaaa 900
aaaatatggg acggttattg ggcgatgcat tatgacgccg cgtattcgtt cgagatcgac 960
tataatgaga gaaattag 978
<210> 5
<211> 978
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
atggccagta tggataaggt ttttgcaggc tatgctgccc gtcaagcaat cttagaaagt 60
accgaaacta cgaacccgtt tgccaaagga attgcctggg tcgaagggga actcgttcct 120
ttagctgaag cacgcattcc actcctcgat cagggcttca tgcactccga tctgacctac 180
gacgtaccgt ctgtttggga tgggcgattt tttcgtttag atgatcatat tacacgcctg 240
gaagcaagct gcaccaagct gaggctgcgt ctacccttac cacgtgatca agttaaacaa 300
atcctggtgg aaatggtcgc aaaatctggt attcgggatg catttgttga attgatagtc 360
acccgcggtc ttaaaggggt gcgaggaact cgtccggaag atatagtgaa caacctgtac 420
atgtttgtgc agccgtacgt gtgggttatg gagccggata tgcagcgcgt aggcggcagc 480
gcagtggtgg ctaggaccgt ccgccgggta ccaccgggcg ctattgatcc gaccgtcaag 540
aatcttcagt ggggtgatct tgttcgtgga atgtttgaag cggctgatcg tggcgcaaca 600
tatcccttcc ttaccgacgg cgatgcgcac ctgactgaag gatcgggttt taatatagta 660
ttagtcaaag atggcgtcct gtatacgcca gctcgcgggg tgctgcaggg agtgactcgc 720
aagtccgtta tcaacgctgc tgaagccttt ggaatagaag tgcgggttga gttcgttcca 780
gttgagctgg cctaccggtg tgacgagatt ttcatgtgca cgacggcggg tggcattatg 840
cctatcacaa cattggacgg tatgcctgta aatggtgggc aaattgggcc tattacgaaa 900
aaaatatggg acggttattg ggcgatgcat tatgacgccg cgtattcgtt cgagatcgac 960
tataatgaga gaaattag 978
<210> 6
<211> 978
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
atggccagta tggataaggt ttttgcaggc tatgctgccc gtcaagcaat cttagaaagt 60
accgaaacta cgaacccgtt tgccaaagga attgcctggg tcgaagggga actcgttcct 120
ttagctgaag cacgcattcc actcctcgat cagggcttca tgcactccga tctgacctac 180
gacgtaccgt ctgtttggga tgggcgattt tttcgtttag atgatcatat tacacgcctg 240
gaagcaagct gcaccaagct gaggctgcgt ctacccttac cacgtgatca agttaaacaa 300
atcctggtgg aaatggtcgc aaaatctggt attcgggatg catttgttga attgatagtc 360
acccgcggtc ttaaaggggt gcgaggaact cgtccggaag atatagtgaa caacctgtac 420
atgtttgtgc agccgtacgt gtgggttatg gagccggata tgcagcgcgt aggcggcagc 480
gcagtggtgg ctaggaccgt ccgccgggta ccaccgggcg ctattgatcc gaccgtcaag 540
aatcttcagt ggggtgatct tgttcgtgga atgtttgaag cggctgatcg tggcgcaaca 600
tatcccttcc ttaccgacgg cgatgcgcac ctgactgaag gatcgggttt taatatagta 660
ttagtcaaag atggcgtcct gtatacgcca gatcgcgggg tgctgcaggg agtgactcgc 720
aagtccgtta tcaacgctgc tgaagccttt ggaatagcgg tgcgggttga gttcgttcca 780
gttgagctgg cctaccggtg tgacgagatt ttcatgtgca cgacggcggg tggcattatg 840
cctatcacaa cattggacgg tatgcctgta aatggtgggc aaattgggcc tattacgaaa 900
aaaatatggg acggttattg ggcgatgcat tatgacgccg cgtattcgtt cgagatcgac 960
tataatgaga gaaattag 978
<210> 7
<211> 978
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
atggccagta tggataaggt ttttgcaggc tatgctgccc gtcaagcaat cttagaaagt 60
accgaaacta cgaacccgtt tgccaaagga attgcctggg tcgaagggga actcgttcct 120
ttagctgaag cacgcattcc actcctcgat cagggcttca tgcactccga tctgacctac 180
gacgtaccgt ctgtttggga tgggcgattt tttcgtttag atgatcatat tacacgcctg 240
gaagcaagct gcaccaagct gaggctgcgt ctacccttac cacgtgatca agttaaacaa 300
atcctggtgg aaatggtcgc aaaatctggt attcgggatg catttgttga attgatagtc 360
acccgcggtc ttaaaggggt gcgaggaact cgtccggaag atatagtgaa caacctgtac 420
atgtttgtgc agccgtacgt gtgggttatg gagccggata tgcagcgcgt aggcggcagc 480
gcagtggtgg ctaggaccgt ccgccgggta ccaccgggcg ctattgatcc gaccgtcaag 540
aatcttcagt ggggtgatct tgttcgtgga atgtttgaag cggctgatcg tggcgcaaca 600
tatcccttcc ttaccgacgg cgatgcgcac ctgactgaag gatcgggttt taatatagta 660
ttagtcaaag atggcgtcct gtatacgcca gatcgcgggg tgctgcaggg agtgactcgc 720
aagtccgtta tcaacgctgc tgaagccttt ggaatagaag tgcgggttga gttcgttcca 780
gttgagctgg cctaccggtg taaagagatt ttcatgtgca cgacggcggg tggcattatg 840
cctatcacaa cattggacgg tatgcctgta aatggtgggc aaattgggcc tattacgaaa 900
aaaatatggg acggttattg ggcgatgcat tatgacgccg cgtattcgtt cgagatcgac 960
tataatgaga gaaattag 978
<210> 8
<211> 31
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
gaactcgtcc gcaagatata gtgaacaacc t 31
<210> 9
<211> 31
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
gttgttcact atatcttgcg gacgagttcc t 31
<210> 10
<211> 33
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
ttagtcaaaa aaggcgtcct gtatacgcca gat 33
<210> 11
<211> 33
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
tggcgtatac aggacgcctt ttttgactaa tac 33
<210> 12
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
tcctgtatac gccagctcgc ggggtgctgc 30
<210> 13
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
tgcagcaccc cgcgagctgg cgtatacagg 30
<210> 14
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
tttggaatag cggtgcgggt tgag 24
<210> 15
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
tcaacccgca ccgctattcc aaag 24
<210> 16
<211> 28
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
gcctaccggt gtaaagagat tttcatgt 28
<210> 17
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
catgaaaatc tctttacacc ggtagg 26
Claims (7)
1.一种ω-转氨酶突变体,其特征在于,由来自土曲霉(Aspergillus terreus)的ω-转氨酶突变所得,野生型ω-转氨酶的氨基酸序列如SEQ ID No.2所示,所述ω-转氨酶突变体的突变位点为:D224K。
2.如权利要求1所述ω-转氨酶突变体在催化(R)-(+)-α甲基苄胺生成苯乙酮中的应用。
3.编码如权利要求1所述ω-转氨酶突变体的基因。
4.如权利要求3所述的基因,其特征在于,突变位点为D224K的ω-转氨酶突变体的基因序列如SEQ ID No.4所示。
5.包含如权利要求3所述基因的重组表达质粒。
6.包含如权利要求5所述重组表达质粒的基因工程菌。
7.如权利要求6所述基因工程菌在催化(R)-(+)-α甲基苄胺生成苯乙酮中的应用。
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