CN116286763A - 一种热稳定性提高的胱硫醚β-合成酶突变体及应用 - Google Patents
一种热稳定性提高的胱硫醚β-合成酶突变体及应用 Download PDFInfo
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Abstract
本发明公开了一种热稳定性提高的胱硫醚β‑合成酶突变体及应用,以氨基酸序列如SEQ ID NO.1所示的酶为亲本,将亲本第232位或第240位突变。一种热稳定性提高的胱硫醚β‑合成酶突变体显著提高了胱硫醚β‑合成酶的热稳定性和催化活性。
Description
技术领域
本发明属于酶工程技术领域,具体涉及一种热稳定性提高的胱硫醚β-合成酶突变体及应用。
背景技术
同型半胱氨酸(homocysteine,Hcy)亦称高半胱氨酸,是甲硫氨酸代谢过程中产生的一种含硫氨基酸。研究表明,Hcy水平与心脑血管疾病密切相关,是心脑血管疾病发病的一个重要危险因子,尤其是冠状动脉粥样硬化症和心肌梗死的重要危险指标,其浓度的升高程度与疾病的危险性成正比。血液中增高的Hcy因为刺激血管壁引起动脉血管的损伤,导致炎症和管壁的斑块形成,最终引起心脏血流受阻。另外,Hcy升高还可引起神经管畸形等出生缺陷类疾病。因此,人体Hcy浓度的测定对于心脑血管疾病的检测具有重要的临床意义。
血清中Hcy的正常参考值为5-15μmol/L,如果同型半胱氨酸值超过15μmol/L,则表明出现了同型半胱氨酸血症。同型半胱氨酸主要的检测方法包括高效液相色谱法(HPLC)、荧光偏振免疫分析、酶联免疫分析法以及毛细管电泳法,这些检测方法均存在测定程序复杂、耗时、样品需要预处理以及测试仪器价格昂贵等弊端。目前,临床上检测Hcy主要使用酶学检测法(循环酶法),可用于全自动生化分析仪。循环酶法测定Hcy的主要原理如下:样品中的Hcy在胱硫醚β-合成酶(CBS)的催化下和丝氨酸反应生成L-胱硫醚,L-胱硫醚被胱硫醚-β-裂解酶(CBL)分解成Hcy和丙酮酸,Hcy可再循环进入第一步反应,增强整个级联反应的信号,随后丙酮酸在乳酸脱氢酶(LDH)的催化下和还原型辅酶Ⅰ(NADH)生成乳酸和氧化型辅酶Ⅰ(NAD+),因此可以通过测量NADH在340nm波长下吸光值的变化反映样品中游离Hcy的浓度。循环酶法检测血浆Hcy具有测定结果稳定、操作简单快速,特异性高等优点。
胱硫醚β-合成酶(CBS)是循环酶法测定Hcy反应中的第一个酶,可以从不同来源的生物中制备获得,目前用于体外诊断的CBS主要来源于酿酒酵母(Saccharomycescerevisiae)。CBS的制备方法主要是重组大肠杆菌发酵。例如,王笃强等在大肠杆菌中表达了来自酿酒酵母的CBS和CBL基因,纯化获得了纯度达到90%的重组蛋白,重组CBS的单位酶活为15U/mg,CBL的单位酶活为72U/mg(中国生物工程杂志,2017,37(2):81-87)。王利群等构建了表达酵母CBS的重组大肠杆菌,纯化的重组CBS的单位酶活为22U/mg(中国医药工业杂志,2013,44(7):663-668)。然而,与循环酶法测定Hcy体系中其余两个酶胱硫醚-β-裂解酶(CBL)和乳酸脱氢酶(LDH)相比,CBS的热稳定性和催化活性均较低,导致生产的Hcy诊断试剂长期保存稳定性较差。因此,通过理性设计和酶工程研发高热稳定性和高活性的CBS具有重要的意义。
发明内容
本发明的目的是提供一种热稳定性提高的胱硫醚β-合成酶突变体及应用,显著提高了胱硫醚β-合成酶的热稳定性和催化活性。
本发明公开了一种热稳定性提高的胱硫醚β-合成酶突变体,以氨基酸序列如SEQID NO.1所示的酶为亲本,将亲本第232位或第240位突变。
本发明还公开了上述的一种热稳定性提高的胱硫醚β-合成酶突变体,将亲本第232位的天冬酰胺取代为脯氨酸;或者将第240位的天冬氨酸取代为色氨酸。
本发明还公开了编码上述的一种热稳定性提高的胱硫醚β-合成酶突变体的基因。
本发明还公开了含有上述基因的重组质粒。
本发明还公开了表达上述的突变体,或含有上述述重组质粒的宿主细胞。
本发明还公开了上述的一种热稳定性提高的胱硫醚β-合成酶突变体,或上述的基因,或上述的重组质粒,或上述的宿主细胞在生产同型半胱氨酸检测剂中的应用。
本发明的有益效果是:该稳定性提高的胱硫醚β-合成酶突变体催化效率提高了1.3倍,在55℃下孵育30min,突变酶残余酶活力为83%,比野生型酶残余酶活提高约4倍。显著提高了胱硫醚β-合成酶的热稳定性和催化活性,解决了胱硫醚β-合成酶热稳定性差的问题,为其在临床诊断试剂中的应用奠定了基础。
附图说明
图1:大肠杆菌重组表达野生型胱硫醚β-合成酶(CBS)的SDS-PAGE电泳图。其中,M表示蛋白分子量标准;1表示诱导后菌体总蛋白;2表示破碎后菌体上清液;3表示破碎后菌体沉淀;4表示镍柱亲和层析穿透液;5表示纯化的重组胱硫醚β-合成酶。
图2:基于计算机辅助半理性设计的突变氨基酸残基分析及突变体筛选结果,其中:A为候选氨基酸残基虚拟饱和突变后的最小自由能热图;B为野生型CBS与突变体蛋白在55℃下孵育30min后的残余酶活。
图3:纯化的CBS突变体N232P蛋白的SDS-PAGE电泳图。其中,M表示蛋白分子量标准;1表示诱导后菌体总蛋白;2表示诱导前菌体总蛋白;3表示破碎后菌体上清液;4表示破碎后菌体沉淀;5表示破碎后菌体上清液(使用0.22μm膜过滤);6表示镍柱亲和层析穿透液;7表示纯化的重组CBS突变体N232P蛋白。
图4:纯化的CBS突变体D240W蛋白的SDS-PAGE电泳图。其中,M表示蛋白分子量标准;1表示诱导后菌体总蛋白;2表示诱导前菌体总蛋白;3表示破碎后菌体上清液;4表示破碎后菌体沉淀;5表示破碎后菌体上清液(使用0.22μm膜过滤);6表示镍柱亲和层析穿透液;7表示纯化的CBS突变体D240W蛋白。
图5:胱硫醚β-合成酶(CBS)突变体N232P与野生型酶的热稳定性比较图。
图6:胱硫醚β-合成酶(CBS)突变体N232P与野生型酶的pH耐受性比较图。
具体实施方式
下面结合附图和具体实施方式对本发明进行详细说明。
本发明公开了一种热稳定性提高的胱硫醚β-合成酶突变体,以氨基酸序列如SEQID NO.1所示的酶为亲本,将亲本第232位或第240位突变。
亲本的编码基因的核苷酸序列如SEQ ID NO.2所示。
其一种实施方式中,,将亲本第232位的天冬酰胺取代为脯氨酸;或者将第240位的天冬氨酸取代为色氨酸。
本发明还公开了一种热稳定性提高的胱硫醚β-合成酶突变体的基因。在一种实施方式中,所述基因的核苷酸序列如SEQ ID NO.3或SEQ ID NO.4所示。
本发明还公开了含有上述基因的重组质粒。
在一种实施方式中,所述重组质粒的表达载体包括但不限于pET系列、pGEX系列或pACYC系列等。
在一种实施方式中,以pET-28a(+)为载体。
本发明还公开了上述的突变体,或上述重组质粒的宿主细胞。
在一种实施方式中,以大肠杆菌(E.coli)为宿主,表达所述胱硫醚β-合成酶突变体。
本发明还公开了上述的一种热稳定性提高的胱硫醚β-合成酶突变体,或上述基因,或上述重组质粒,或上述宿主细胞在生产同型半胱氨酸检测剂中的应用。
该一种热稳定性提高的胱硫醚β-合成酶突变体的制备方法,包含如下:
在线获取来源于酿酒酵母的胱硫醚β-合成酶晶体结构(PDB ID:6C2H),利用计算机辅助设计获得热稳定性提高的模拟突变体。
使用定点突变引物以表达野生型CBS的质粒为模板扩增获得目标突变体。
具体如下:
将含有重组质粒的宿主细胞单克隆接种至LB培养基(5mL)中,37℃培养12-16h得到种子液。
将种子液以1%接种量接种至500mL的LB培养基中,30℃培养至生物量OD600达到0.6-0.8,然后加入诱导剂在25℃下继续培养12-16h,离心收集菌体;
将收集的细胞在4℃条件下进行超声破碎,破碎液于4℃12000rpm的条件下离心20-30min,得到粗酶液;
将离心收集的粗酶液进行镍柱亲和层析和分子筛过滤脱盐,获得胱硫醚β-合成酶纯酶。
本发明通过对链球菌来源的磷酸甘油氧化酶进行理性分子改造,分别对其109、112、150、245、248、249、451、456等多个位点进行定点突变并筛选,最终获得热稳定性与催化活性均提高的磷酸氧化酶突变体。与野生型胱硫醚β-合成酶(CBS)的比对验证过程如下:
实施例1
重组野生型胱硫醚β-合成酶(CBS)的表达纯化和活性测定:
在大肠杆菌中表达来源于酿酒酵母(Saccharomyces cerevisiae)的胱硫醚β-合成酶(CBS)。所需试剂材料如下:大肠杆菌宿主菌DH5α、BL21(DE3)和表达质粒pET-28a(+)均为本实验室保存,也可购买得到;镍亲和层析填料(Ni Sepharose 6Fast Flow)和分子筛填料(Sephadex G-25)购自GE公司,根据蛋白纯化要求进行装柱;其余试剂均为国产或进口分析纯。
从在线数据库中(获取来源于酿酒酵母(Saccharomyces cerevisiae)的胱硫醚β-合成酶(CBS)的蛋白质序列(PDB ID:6C2H),氨基酸序列如SEQ ID NO.1序列所示。根据大肠杆菌密码子偏爱性推导出CBS基因序列(核苷酸序列如SEQ ID NO.2序列所示)并人工合成。通过酶切位点NdeⅠ和NotⅠ将合成的CBS基因连接到表达载体pET-28a(+),构建重组质粒pET-28a-CBS。将重组质粒转化至大肠杆菌BL21(DE3)得到表达野生型CBS的重组大肠杆菌。
将重组大肠杆菌划线于固体LB平板(含有50μg/mL的卡那霉素)上,37℃培养过夜得到单克隆。然后挑取单克隆于液体LB培养基(5mL)中37℃震荡培养过夜。将培养的种子液按照1%接种量接种于500mL LB培养基中,30℃培养至生物量OD600达到0.8-1.0,然后加入终浓度为0.3mM的诱导剂IPTG(异丙基-β-D-硫代半乳糖苷)在25℃下诱导12-16h,4℃离心收集菌体。将收集的菌体按照1g湿菌加入10mL破碎液悬浮,然后在冰水浴中进行超声破碎(功率400W,超声5s,间隙8s)30min,然后12000rpm离心20-30min收集上清液。上清液上镍亲和层析柱,使用平衡缓冲液(PBS,含20mM咪唑)洗脱杂蛋白,最后使用洗脱缓冲液(含有300mM咪唑)洗脱目标蛋白。洗脱的蛋白液再经分子筛脱盐后收集纯化的CBS。SDS-PAGE检测纯化蛋白的纯度超过95%,结果如图1所示。其中,M表示蛋白分子量标准;1表示诱导后菌体总蛋白;2表示破碎后菌体上清液;3表示破碎后菌体沉淀;4表示镍柱亲和层析穿透液;5表示纯化的重组胱硫醚β-合成酶。
酶活性检测体系为200μL,缓冲液为Tris-HCl(100mmol/L,pH8.0)、L-丝氨酸(5mmol/L)、同型半胱氨酸(10mmol/L)、NADH(1mmol/L)、乳酸脱氢酶(2KU/mL)、胱硫醚β-裂解酶(50U/mL)。将以上反应液混匀后加到96孔板上,在酶标仪中37℃孵育5min,然后在酶标板每个孔中加入2μl稀释至0.2mg/mL的待测CBS酶液反应10min,连续记录反应液在340nm下的吸光度,以不加待测CBS酶液的反应液为空白对照。其中,NADH在340nm的摩尔消光系数为6.22mM-1cm-1。CBS酶活性测定中所需的乳酸脱氢酶(LDH)和胱硫醚β-裂解酶(CBL)购买于北京索莱宝科技有限公司。酶活性定义:在pH 8.0,温度37℃条件下,每分钟催化产生1μmolL-胱硫醚所需要的酶量定义为1个单位(U)。在以上条件下测定纯化的野生型CBS比活性为43U/mg。
实施例2
胱硫醚β-合成酶(CBS)突变体库构建:从在线数据库中获取来源于酿酒酵母的胱硫醚β-合成酶(CBS)的晶体结构(PDB ID:6C2H),借助蛋白质分析软件FoldX进行计算机辅助设计,将CBS结构中柔性部位的氨基酸残基进行虚拟饱和突变,计算突变后的去折叠自由能变化(ΔΔG),选择ΔΔG<-1.0kcal/mol的氨基酸位点进行突变。候选氨基酸位点虚拟饱和突变后的最小自由能热图如附图2A所示。突变位点的虚拟筛选结果如下:Asp213Arg/Pro、Asn232Met/Pro、Asp240Met/Phe/Trp/Tyr、Glu302Met。
设计定点突变引物以表达野生型CBS基因的质粒pET-28a-CBS为模板PCR扩增获得以上9个目标突变体。PCR程序如下:预变性96℃5min;变性98℃15s,退火56℃30s,延伸72℃60s,30个循环;延伸72℃5min。上述PCR产物使用DpnI在37℃酶切2h,酶切后产物转化至大肠杆菌DH5α感受态细胞获得阳性克隆,挑取3个单克隆进行测序,测序正确后获得不同CBS突变体,然后使用质粒提取试剂盒提取9个目标突变体的重组质粒备用。PCR扩增CBS突变体的引物序列如表1所示。
表1定点突变所用引物
实施例3
胱硫醚β-合成酶(CBS)突变体的表达和筛选:
将实施例2中获得的胱硫醚β-合成酶(CBS)突变体质粒转化至大肠杆菌BL21(DE3)中用CBS突变体表达。具体步骤如下:
(1)表达CBS突变体的菌种制备。将筛选的大肠杆菌阳性菌株在LB固体培养基(50μg/mL卡那霉素)上划线培养至长出单克隆,挑取单克隆接种于LB液体培养基中,在摇床中37℃培养12-16h获得种子液。
(2)突变CBS诱导表达和热稳定性筛选。9种CBS突变酶的诱导表达条件和野生型CBS的表达条件一致(参见实施例1)。CBS突变体诱导培养后,4℃离心收集菌体后加入破碎液悬浮(1g湿菌/10mL破碎液),然后进行超声破碎(功率400W,超声5s,间隙8s)30min,破碎液12000rpm离心20-30min收集上清液。
分别测定野生型CBS和9个CBS突变体破碎上清液的酶活性,将未热处理的每种酶的活性设定为100%。同时,将野生型和9个突变体CBS上清液在55℃下处理30min,测定热处理后酶残余活性。计算热处理后酶残余活性与该酶未处理时的酶活性比值,即为该酶热处理后酶活性残余率,结果如图2B所示。野生型CBS在55℃下处理30min后残余酶活为21%,而CBS突变体N232P、D240W和D240Y的热稳定性显著提高,55℃下处理30min后残余酶活分别为83%、91%和34%。然后选择热稳定性大幅度提高的2个CBS突变体N232P、D240W进行表达纯化,SDS-PAGE检测纯化蛋白的纯度达到95%,结果如图3和图4所示。其中,M表示蛋白分子量标准;1表示诱导后菌体总蛋白;2表示诱导前菌体总蛋白;3表示破碎后菌体上清液;4表示破碎后菌体沉淀;5表示破碎后菌体上清液(使用0.22μm膜过滤);6表示镍柱亲和层析穿透液;7表示纯化的CBS突变体蛋白。
接下来,测定了纯化后CBS突变体N232P、D240W的比活性,CBS活性测定条件参见实施例1。结果表明CBS突变体N232P的比活性为56U/mg,突变体D240W的比活性为28U/mg。与野生型CBS活性(43U/mg)相比,突变体N232P的比活性提高了1.3倍,但是突变体D240W的比活性降低了1.5倍。突变体N232P的热稳定性和催化活性均得到了较大幅度的提高,突变体D240W的热稳定性具有大幅度提高,但是酶比活性下降明显。因此,接下来对CBS突变体N232P的酶学性质进行了进一步详细分析。
实施例4
CBS突变体N232P酶学性质分析:
将纯化的野生型CBS和突变体N232P蛋白使用分子筛进行缓冲液(100mM磷酸盐缓冲液,pH7.4)替换,测定脱盐后蛋白浓度并调整浓度为1mg/mL。然后在不同温度下孵育30min,然后测定酶活性,计算不同温度处理后残余酶活性。以未处理的野生型CBS和突变体N232P的活性分别作为100%,计算不同温度处理后酶残余活性,结果如图5所示。与野生型CBS相比,突变体N232P在30℃至65℃范围内热稳定性显著提高。野生型CBS在50℃条件下孵育30min后活性降低至68%,55℃条件下活性仅残余21%,60℃孵育30min后酶活性全部丢失。而N232P突变体在50℃条件下孵育30min后活性残留还超过90%,55℃条件下活性还残余83%,60℃孵育30min后酶活性仍保存57%。以上结果表明与野生型CBS相比,N232P突变体热稳定性得到大幅度提高。
(2)CBS突变体N232P的pH稳定性分析:
将野生型CBS和突变体N232P蛋白使用分子筛进行缓冲液(100mM磷酸盐缓冲液,pH7.4)替换,测定脱盐后蛋白浓度并调整浓度为5mg/mL。然后配置不同pH缓冲液(100mM),缓冲液如下:乙酸-乙酸钠缓冲液(pH4-6),磷酸盐缓冲液(pH6.5-7.4),Tris-HCl缓冲液(pH8-10)。使用不同pH缓冲液将5mg/mL的野生型和突变体N232P蛋白稀释成0.5mg/mL,然后在4℃下孵育12h,测定野生型CBS和突变体N232P的酶活性。分别以野生型CBS和突变体N232P的最高酶活性为100%,计算不同pH条件处理后的酶残余活性,结果如图6所示。结果表明N232P突变体在酸性溶液中(pH4-7)的稳定性显著高于野生型CBS,在pH 6的条件下N232P突变体活性残余83%,但野生型CBS活性仅残余63%。
SEQ ID NO.1:
MFSNKTRQDSIQKMQQEELDLLIIGGGITGAGVAVQAAASGIKTGLIEMQDFAE
GTSSRSTKLVHGGIRYLKTFDVEVVADTVGERAVVQGIAPHIPKPDPMLLPIYE
DEGATTFNMFSVKVAMDLYDKLANVTGTKYENYTLTPEEVLEREPFLKKEGL
KGAGVYLDFRNNDARLVIDNIKKAAEDGAYLVSKMKAVGFLYEGDQIVGVKA
RDLLTDEVIEIKAKLVINTSGPWVDKVRNLNFTRPVSPKMRPTKGIHLVVDAK
KLPVPQPTYFDTGKQDGRMVFAIPRENKTYFGTTDTDYQGDFTDPKVTQEDV
DYLLDVINHRYPEANITLADIEASWAGLRPLLIGNSGSDYNGGDNGSISDKSFN
KVVDTVSEYKENKVSRAEVEDVLNHLENSRDEKAPSTISRGSSLEREPDGLLT
LSGGKITDYRKMAEGALRLIRQLLKEEYGIETKEIDSKKYQISGGNFDPTKLEE
TVTELAKEGVAAGLEEEDATYIADFYGTNARRIFELAKEMAPYPGLSLAESAR
LRYGLEEEMVLAPGDYLIRRTNHLLFERDQLDEIKQPVIDAIAEYFGWTEEEKAQQTKRLEALIAESDLRELKGEK。
SEQ ID NO.2:
ATGTTCTCCAACAAGACTCGCCAGGACTCTATTCAGAAGATGCAGCAGGAG
GAACTGGATCTGCTGATTATCGGTGGTGGTATCACTGGTGCAGGTGTAGCAG
TGCAGGCAGCTGCTAGCGGTATCAAAACCGGCCTGATTGAGATGCAGGATT
TCGCTGAAGGTACCTCCTCCCGTTCCACCAAACTGGTTCATGGTGGTATCCG
TTATCTGAAAACCTTCGACGTTGAGGTGGTTGCGGACACCGTTGGCGAACG
TGCTGTTGTACAGGGTATCGCTCCGCACATCCCGAAACCGGACCCGATGCT
GCTGCCGATCTACGAGGACGAAGGTGCGACGACCTTCAACATGTTCAGCGT
GAAGGTAGCTATGGATCTGTACGACAAACTGGCCAACGTAACTGGTACCAA
ATACGAAAACTACACCCTGACCCCTGAAGAAGTTCTGGAACGTGAACCGTT
TCTGAAGAAGGAAGGCCTGAAAGGTGCAGGTGTGTATCTGGATTTCCGCAA
CAACGATGCGCGTCTGGTGATTGACAACATCAAAAAAGCAGCGGAAGACG
GCGCTTACCTGGTCTCTAAAATGAAAGCAGTTGGTTTCCTGTACGAAGGTG
ACCAGATCGTGGGTGTGAAGGCACGTGACCTGCTGACGGATGAAGTGATC
GAGATCAAAGCCAAACTGGTCATCAACACTTCTGGCCCGTGGGTGGACAA
AGTTCGCAACCTGAATTTCACCCGTCCGGTGTCTCCGAAAATGCGTCCGAC
CAAAGGCATCCACCTGGTTGTTGACGCGAAAAAACTGCCGGTACCGCAGC
CGACCTACTTCGACACGGGCAAACAGGACGGCCGCATGGTGTTTGCAATCC
CGCGTGAAAACAAAACCTACTTCGGTACCACTGACACCGACTACCAGGGT
GATTTCACGGACCCGAAAGTTACTCAAGAGGACGTAGACTACCTGCTGGAT
GTTATCAATCATCGCTATCCGGAAGCTAACATCACTCTGGCTGACATCGAAG
CTTCTTGGGCTGGCCTGCGTCCGCTGCTGATTGGTAACTCCGGCTCTGACTA
CAACGGTGGTGATAATGGTTCTATTTCCGACAAATCTTTCAACAAAGTTGTG
GACACTGTGTCTGAATACAAAGAAAACAAAGTTTCCCGCGCGGAAGTGGA
AGATGTACTGAACCACCTGGAAAACAGCCGTGATGAAAAAGCCCCTAGCA
CGATCAGCCGCGGTAGCTCTCTGGAACGTGAACCGGATGGTCTGCTGACCC
TGTCCGGTGGTAAAATCACTGATTATCGTAAAATGGCGGAAGGTGCTCTGC
GCCTGATCCGTCAGCTGCTGAAAGAGGAATATGGTATCGAAACTAAAGAAA
TCGACTCCAAAAAATACCAGATCAGCGGTGGCAACTTTGACCCGACTAAAC
TGGAAGAAACTGTAACCGAGCTGGCGAAAGAAGGCGTTGCTGCGGGTCTG
GAAGAAGAAGACGCGACGTACATCGCTGATTTCTACGGTACTAACGCGCGC
CGCATCTTTGAACTGGCGAAGGAGATGGCTCCATATCCAGGCCTGAGCCTG
GCAGAAAGCGCTCGTCTGCGCTACGGCCTGGAGGAAGAAATGGTTCTGGC
TCCGGGCGACTACCTGATCCGTCGTACTAACCACCTGCTGTTTGAGCGTGA
CCAGCTGGACGAAATCAAACAGCCGGTTATCGATGCGATCGCAGAATACTT
CGGCTGGACTGAGGAGGAAAAAGCTCAGCAGACTAAACGTCTGGAGGCTCTGATCGCGGAGAGCGATCTGCGCGAACTGAAAGGTGAAAAA。
SEQ ID NO.3:
ATGTTCTCCAACAAGACTCGCCAGGACTCTATTCAGAAGATGCAGCAGGAG
GAACTGGATCTGCTGATTATCGGTGGTGGTATCACTGGTGCAGGTGTAGCAG
TGCAGGCAGCTGCTAGCGGTATCAAAACCGGCCTGATTGAGATGCAGGATT
TCGCTGAAGGTACCTCCTCCCGTTCCACCAAACTGGTTCATGGTGGTATCCG
TTATCTGAAAACCTTCGACGTTGAGGTGGTTGCGGACACCGTTGGCGAACG
TGCTGTTGTACAGGGTATCGCTCCGCACATCCCGAAACCGGACCCGATGCT
GCTGCCGATCTACGAGGACGAAGGTGCGACGACCTTCAACATGTTCAGCGT
GAAGGTAGCTATGGATCTGTACGACAAACTGGCCAACGTAACTGGTACCAA
ATACGAAAACTACACCCTGACCCCTGAAGAAGTTCTGGAACGTGAACCGTT
TCTGAAGAAGGAAGGCCTGAAAGGTGCAGGTGTGTATCTGGATTTCCGCAA
CAACGATGCGCGTCTGGTGATTGACAACATCAAAAAAGCAGCGGAAGACG
GCGCTTACCTGGTCTCTAAAATGAAAGCAGTTGGTTTCCTGTACGAAGGTG
ACCAGATCGTGGGTGTGAAGGCACGTGACCTGCTGACGGATGAAGTGATC
GAGATCAAAGCCAAACTGGTCATCAACACTTCTGGCCCGTGGGTGGACAA
AGTTCGCAACCTGAATTTCACCCGTCCGGTGCCGCCGAAAATGCGTCCGAC
CAAAGGCATCCACCTGGTTGTTGACGCGAAAAAACTGCCGGTACCGCAGC
CGACCTACTTCGACACGGGCAAACAGGACGGCCGCATGGTGTTTGCAATCC
CGCGTGAAAACAAAACCTACTTCGGTACCACTGACACCGACTACCAGGGT
GATTTCACGGACCCGAAAGTTACTCAAGAGGACGTAGACTACCTGCTGGAT
GTTATCAATCATCGCTATCCGGAAGCTAACATCACTCTGGCTGACATCGAAG
CTTCTTGGGCTGGCCTGCGTCCGCTGCTGATTGGTAACTCCGGCTCTGACTA
CAACGGTGGTGATAATGGTTCTATTTCCGACAAATCTTTCAACAAAGTTGTG
GACACTGTGTCTGAATACAAAGAAAACAAAGTTTCCCGCGCGGAAGTGGA
AGATGTACTGAACCACCTGGAAAACAGCCGTGATGAAAAAGCCCCTAGCA
CGATCAGCCGCGGTAGCTCTCTGGAACGTGAACCGGATGGTCTGCTGACCC
TGTCCGGTGGTAAAATCACTGATTATCGTAAAATGGCGGAAGGTGCTCTGC
GCCTGATCCGTCAGCTGCTGAAAGAGGAATATGGTATCGAAACTAAAGAAA
TCGACTCCAAAAAATACCAGATCAGCGGTGGCAACTTTGACCCGACTAAAC
TGGAAGAAACTGTAACCGAGCTGGCGAAAGAAGGCGTTGCTGCGGGTCTG
GAAGAAGAAGACGCGACGTACATCGCTGATTTCTACGGTACTAACGCGCGC
CGCATCTTTGAACTGGCGAAGGAGATGGCTCCATATCCAGGCCTGAGCCTG
GCAGAAAGCGCTCGTCTGCGCTACGGCCTGGAGGAAGAAATGGTTCTGGC
TCCGGGCGACTACCTGATCCGTCGTACTAACCACCTGCTGTTTGAGCGTGA
CCAGCTGGACGAAATCAAACAGCCGGTTATCGATGCGATCGCAGAATACTT
CGGCTGGACTGAGGAGGAAAAAGCTCAGCAGACTAAACGTCTGGAGGCTCTGATCGCGGAGAGCGATCTGCGCGAACTGAAAGGTGAAAAA。
SEQ ID NO.4:
ATGTTCTCCAACAAGACTCGCCAGGACTCTATTCAGAAGATGCAGCAGGAG
GAACTGGATCTGCTGATTATCGGTGGTGGTATCACTGGTGCAGGTGTAGCAG
TGCAGGCAGCTGCTAGCGGTATCAAAACCGGCCTGATTGAGATGCAGGATT
TCGCTGAAGGTACCTCCTCCCGTTCCACCAAACTGGTTCATGGTGGTATCCG
TTATCTGAAAACCTTCGACGTTGAGGTGGTTGCGGACACCGTTGGCGAACG
TGCTGTTGTACAGGGTATCGCTCCGCACATCCCGAAACCGGACCCGATGCT
GCTGCCGATCTACGAGGACGAAGGTGCGACGACCTTCAACATGTTCAGCGT
GAAGGTAGCTATGGATCTGTACGACAAACTGGCCAACGTAACTGGTACCAA
ATACGAAAACTACACCCTGACCCCTGAAGAAGTTCTGGAACGTGAACCGTT
TCTGAAGAAGGAAGGCCTGAAAGGTGCAGGTGTGTATCTGGATTTCCGCAA
CAACGATGCGCGTCTGGTGATTGACAACATCAAAAAAGCAGCGGAAGACG
GCGCTTACCTGGTCTCTAAAATGAAAGCAGTTGGTTTCCTGTACGAAGGTG
ACCAGATCGTGGGTGTGAAGGCACGTGACCTGCTGACGGATGAAGTGATC
GAGATCAAAGCCAAACTGGTCATCAACACTTCTGGCCCGTGGGTGGACAA
AGTTCGCAACCTGAATTTCACCCGTCCGGTGTCTCCGAAAATGCGTCCGAC
CAAAGGCATCCACCTGGTTGTTGACGCGAAAAAACTGCCGGTACCGCAGC
CGACCTACTTCGACACGGGCAAACAGGACGGCCGCATGGTGTTTGCAATCC
CGCGTGAAAACAAAACCTACTTCGGTACCACTGACACCGACTACCAGGGT
GATTTCACGGACCCGAAAGTTACTCAAGAGGACGTAGACTACCTGCTGGAT
GTTATCAATCATCGCTATCCGGAAGCTAACATCACTCTGGCTGACATCGAAG
CTTCTTGGGCTGGCCTGCGTCCGCTGCTGATTGGTAACTCCGGCTCTGACTA
CAACGGTGGTGATAATGGTTCTATTTCCGACAAATCTTTCAACAAAGTTGTG
GACACTGTGTCTGAATACAAAGAAAACAAAGTTTCCCGCGCGGAAGTGGA
AGATGTACTGAACCACCTGGAAAACAGCCGTGATGAAAAAGCCCCTAGCA
CGATCAGCCGCGGTAGCTCTCTGGAACGTGAACCGGATGGTCTGCTGACCC
TGTCCGGTGGTAAAATCACTGATTATCGTAAAATGGCGGAAGGTGCTCTGC
GCCTGATCCGTCAGCTGCTGAAAGAGATGTATGGTATCGAAACTAAAGAAA
TCGACTCCAAAAAATACCAGATCAGCGGTGGCAACTTTGACCCGACTAAAC
TGGAAGAAACTGTAACCGAGCTGGCGAAAGAAGGCGTTGCTGCGGGTCTG
GAAGAAGAAGACGCGACGTACATCGCTGATTTCTACGGTACTAACGCGCGC
CGCATCTTTGAACTGGCGAAGGAGATGGCTCCATATCCAGGCCTGAGCCTG
GCAGAAAGCGCTCGTCTGCGCTACGGCCTGGAGGAAGAAATGGTTCTGGC
TCCGGGCGACTACCTGATCCGTCGTACTAACCACCTGCTGTTTGAGCGTGA
CCAGCTGGACGAAATCAAACAGCCGGTTATCGATGCGATCGCAGAATACTT
CGGCTGGACTGAGGAGGAAAAAGCTCAGCAGACTAAACGTCTGGAGGCTC
TGATCGCGGAGAGCGATCTGCGCGAACTGAAAGGTGAAAAA。
Claims (6)
1.一种热稳定性提高的胱硫醚β-合成酶突变体,其特征在于,以氨基酸序列如SEQ IDNO.1所示的酶为亲本,将亲本第232位或第240位突变。
2.如权利要求1所述的一种热稳定性提高的胱硫醚β-合成酶突变体,其特征在于,将亲本第232位的天冬酰胺取代为脯氨酸;或者将第240位的天冬氨酸取代为色氨酸。
3.编码权利要求1或2所述的一种热稳定性提高的胱硫醚β-合成酶突变体的基因。
4.含有权利要求3所述基因的重组质粒。
5.表达权利要求1或2所述的突变体,或含有权利要求4所述重组质粒的宿主细胞。
6.如权利要求1或2所述的一种热稳定性提高的胱硫醚β-合成酶突变体,或权利要求3所述的基因,或权利要求4所述的重组质粒,或权利要求5所述的宿主细胞在生产同型半胱氨酸检测剂中的应用。
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