CN114854728B - 一种脯氨酸消旋酶及其制备和应用 - Google Patents
一种脯氨酸消旋酶及其制备和应用 Download PDFInfo
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- CN114854728B CN114854728B CN202210514276.6A CN202210514276A CN114854728B CN 114854728 B CN114854728 B CN 114854728B CN 202210514276 A CN202210514276 A CN 202210514276A CN 114854728 B CN114854728 B CN 114854728B
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Abstract
本发明公开了一种脯氨酸消旋酶的制备及其应用，该脯氨酸消旋酶的氨基酸序列如SEQIDNO.2所示，其最适pH为9.0；经pH6.0～12.0的缓冲液处理1h后，剩余酶活55％以上；最适温度为40℃；在30℃、37℃、40℃条件下耐受1h，仍保持100％以上酶活，50℃条件下耐受50min达到半衰期；在pH9.0、40℃条件下，该酶的Km和Vmax分别为0.06mmol/L和2.1610μmol/L/min，本发明制备的脯氨酸消旋酶具有较好的热稳定性和较宽的pH作用范围，且该酶已被确定为治疗恰加斯病的药物筛选靶点，其抑制剂的开发具有巨大的研究价值。
Description
技术领域
本发明涉及基因工程技术领域，具体涉及一种脯氨酸消旋酶的制备及其应用。
背景技术
脯氨酸消旋酶(Proline racemase，PRAC，EC5.1.1.4)是一种二聚体酶，可催化L-脯氨酸和D-脯氨酸相互转化，其催化机制为“双碱基催化”，由N端和C端的两个半胱氨酸残基共同完成，不需要辅因子或其它已知的辅酶，迄今为止仅在三种物种中描述过。最初于1968年由Cardinale和Abeles首次从梭菌属细菌Clostridium sticklandii中分离出来，研究发现PRAC可通过Stickland途径对梭菌属细菌的能量代谢进行调控，是梭菌属的主要能量来源，属于细菌性的PRAC。2000年Reina-San-Martin B等在人类致病性寄生虫克氏锥虫(Trypanosoma cruzi)中分离克隆得到第一个真核PRAC，研究发现该酶的分泌形式是一种有效的宿主B细胞有丝分裂原，可以帮助寄生虫逃避宿主的特异性免疫反应而在宿主中继续存活。最近，在超嗜热古菌(Hypert hermophilic archaeon)中发现了一个双功能脯氨酸消旋酶/羟脯氨酸差向异构酶，是新发现的第三种PRAC。由于该酶可调控梭菌属细菌的能量代谢以及克氏锥虫的免疫逃逸反应，因此被作为开发抗微生物剂和治疗恰加斯病的新靶标。
克氏锥虫是南美锥虫病(恰加斯病)的病原体，是大多数拉丁美洲国家的一个严重公共卫生问题，影响着数千万人。寄生虫病有两个主要阶段:急性期，在感染后不久出现；慢性期，在数年或数十年的沉默期之后，约有三分之一的感染者出现。由于这种长期无症状状态，恰加斯病通常被认为是一个“沉默的杀手”，损害了早期特异性诊断和治疗。其发病机制归因于低等级寄生虫会分泌PRAC，该酶会导致宿主免疫系统紊乱。到目前为止还没有开发出有效的疫苗，主要的治疗药物为杂环硝基化合物(硝呋替莫和苯硝唑)，但该类药物具有较大的毒副作用，对慢性患者非常不利。因此，为了获得更好的治疗手段，研究人员将寄生虫中的PRAC基因进行敲除，发现当PRAC基因被敲除后寄生虫不再存活。因此将其确定为针对恰加斯病的新化学疗法的靶标，其抑制剂的开发对该疾病的治疗具有重大的意义。
虽然最近也在其他病原体中发现了PRAC，但是目前对PRAC的研究较少，主要集中于PRAC对克氏锥虫细胞内循环的影响，以及这些酶在寄生虫与哺乳动物细胞间的相互作用。对于其抑制剂的研究较少，未得到对恰加斯病有效的治疗药物(抑制剂)。主要原因可能有两方面：(1)该酶仅在部分细菌和寄生虫中发现，不易获得，研究来源较少，因此未被广泛研究；(2)该酶的催化位点太小，不利于任何药物的设计，增加了抑制剂筛选和设计的难度。
发明内容
本发明的目的是提供一种脯氨酸消旋酶的制备及其应用，该脯氨酸消旋酶利用宏基因组学技术，直接从西黑冠长臂猿粪便微生物中获得，具有良好的热稳定性和较宽的pH作用范围，在恰加斯病药物筛选的靶标等方面具有潜在的应用价值。
为了达到上述目的，本发明提供了一种脯氨酸消旋酶，该脯氨酸消旋酶的氨基酸序列如SEQ ID NO.2所示。
本发明的另一目的是提供所述的脯氨酸消旋酶的编码基因，该脯氨酸消旋酶编码基因的核苷酸序列如SEQ ID NO.1所示。
本发明的另一目的是提供一种包含上述脯氨酸消旋酶编码基因的重组载体。
优选地，该重组载体为质粒pEASY-E2。
本发明的另一目的是提供一种包含上述编码基因的重组菌。
优选地，该重组菌为大肠杆菌BL21(DE3)。
本发明的另一目的是提供一种上述脯氨酸消旋酶的制备方法，该方法包含：
步骤1.将含有上述脯氨酸消旋酶编码基因的重组表达载体转化至宿主细胞得到重组菌株，培养该重组菌株，诱导重组脯氨酸消旋酶的表达；
步骤2.纯化所表达的脯氨酸消旋酶。
优选地，采用的载体为质粒pEASY-E2。
优选地，该制备方法采用的菌为大肠杆菌BL21(DE3)。
优选地，上述的脯氨酸消旋酶编码基因是以宏基因组DNA为模板并以核苷酸序列如SEQ ID NO.3和SEQ ID NO.4所示的引物进行PCR扩增所得。
本发明的另一目的是提供该脯氨酸消旋酶在抗寄生虫药物靶标中的应用，可应用于恰加斯病药物的靶标筛选。
本发明的一种脯氨酸消旋酶的制备及其应用，具有以下优点：
本发明利用宏基因组学技术直接从西黑冠长臂猿粪便微生物宏基因组中筛选PRAC基因，设计引物扩增PRAC基因片段，并在大肠杆菌中异源表达。宏基因组学避开了微生物分离培养的问题，直接对环境样品中所有微生物进行测序，极大地扩展了微生物资源的利用空间。
本发明的脯氨酸消旋酶的最适pH为9.0；经pH 6.0～12.0的缓冲液处理1h后，剩余酶活在55％以上；最适温度为40℃；在30℃、37℃、40℃条件下耐受1h，其仍保持100％以上酶活，在50℃条件下耐受1h，50min时达到半衰期；在pH＝9.0，温度40℃条件下，该酶的Km和Vmax分别为0.06mmol/L和2.1610μmol/L/min；除了Na+、Li+、K+和Mg2+对重组酶活性影响不大，其余金属离子对重组酶具有强烈的抑制作用；10mmol/L的DDT、乙酸和β-巯基乙醇对重组酶具有强烈的抑制作用，其余化学试剂对重组酶影响较小，可将其活性提高1％～20％。该酶活性随着NaCl浓度的提高而降低，在NaCl浓度为3.5～5M时，相对酶活低于10％，在0.5～5MNaCl中处理1h后酶活性维持在25％以上。以上性质表明，本发明制备的脯氨酸消旋酶具有热稳定性和较宽的pH作用范围，在新型抗寄生虫药物筛选的靶标等方面具有潜在的应用价值。
附图说明
图1为本发明脯氨酸消旋酶基因NCPrac2的PCR电泳结果图。
图2为本发明重组脯氨酸消旋酶NCPRAC2在大肠杆菌中诱导表达的SDS-PAGE结果图。
图3为本发明重组脯氨酸消旋酶的最适pH。
图4为本发明脯氨酸消旋酶的pH稳定性。
图5为本发明脯氨酸消旋酶的最适温度。
图6为本发明脯氨酸消旋酶的温度稳定性。
图7为本发明脯氨酸消旋酶NaCl的影响。
图8为本发明脯氨酸消旋酶NaCl的耐受性。
图9为本发明脯氨酸消旋酶对8种L-氨基酸的催化能力。
具体实施方式
下面将对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
所需的试验材料和试剂如下所述：
1、样品、菌株及载体：西黑冠长臂猿粪便微生物宏基因组DNA、表达载体pEASY-E2；BL21(DE3)购自北京擎科新业生物技术有限公司。
2、基因工程操作酶类、试剂盒及其它生化试剂：限制性内切酶、DNA聚合酶、连接酶购自TaKaRa公司，质粒提取试剂盒、胶回收纯化试剂盒为美国Omega公司；其他试剂均为分析纯。
3、LB培养基：Peptone 10g、Yeast extract 5g、NaCl 10g，加蒸馏水至1000mL，pH自然(约为7)。固体培养基在以上基础上加2.0％(w/v)琼脂。
说明：以下实验例中未作具体说明的分子生物学实验方法，均参照《分子克隆实验指南》(第三版)J.萨姆布鲁克一书中所列的具体方法进行，或者按照试剂盒和产品说明书进行。
实验例1脯氨酸消旋酶基因NCParc2的获得
(1)长臂猿粪便微生物宏基因组脯氨酸消旋酶基因的筛选
从已构建长臂猿粪便微生物文库中根据基因预测、功能注释和分泌蛋白预测分析结果，筛选注释结果为脯氨酸消旋酶的基因，从而得到脯氨酸消旋酶基因NCPrac2，该基因序列如SEQ ID NO.1所示。
(2)脯氨酸消旋酶基因NCPrac2的克隆
以NCPrac2-F/NCPrac2-R为引物对，以西黑冠长臂猿粪便微生物宏基因组DNA为模板进行PCR扩增。
PCR反应体系(20.0μL)：宏基因组DNA 0.5μL，PrimeSTAR Max 10μL，NCPrac2-F0.5μL，NCPrac2-R0.5μL，ddH2O补足20.0μL。
PCR反应参数为：98℃，10s；55℃，15s；72℃，90s；30个循环；72℃，10min；4℃，10min。
PCR结果得到目的基因NCPrac2，参见图1，其中，泳道“M”为DNA Marker；泳道“1”为NCPrac2 PCR扩增产物。
其中，所用到的引物序列如下(5’→3’):
NCPrac2-F(SEQ ID NO.3)：
TAAGAAGGAGATATACATATGGAATTGATGCATCTCAGCCACCTCGTTG；
NCPrac2-R(SEQ ID NO.4)：
GTGGTGGTGGTGGTGCTCGAGTTACGCCAGCCGGAAGCCATG。
实验例2脯氨酸消旋酶NCPRAC2的制备
将实施例1制备的脯氨酸消旋酶基因NCPrac2与质粒pEASY-E2连接得到重组表达载体pEASY-E2-NCPrac2，然后转化大肠杆菌BL21(DE3)获得重组大肠杆菌菌株BL21(DE3)/NCPrac2。取含有重组表达载体pEASY-E2-NCPrac2的大肠杆菌菌株BL21(DE3)/NCPrac2，以0.1％的接种量接种于LB(含100μg/mLAmp)培养液中，37℃，180rpm过夜培养。然后将此活化的菌液以1％接种量接种到新鲜的LB(含100μg/mLAmp)培养液中，37℃，180rpm培养约5～6h(OD600达到0.8～1.0)后，加入终浓度0.7mmol/L的IPTG诱导，于16℃、180rpm培养约16h。5000rpm离心10min，收集菌体。用适量的pH＝7.0的Tris-HCl缓冲液悬浮菌体后，于冰浴条件下超声波破碎菌体。以上胞内浓缩的初酶液经12,000rpm，4℃离心10min后，吸取上清并用Nickel-NTA Agarose纯化目的蛋白，得到脯氨酸消旋酶NCPRAC2，其氨基酸序列如SEQ IDNO.2所示。
对所述纯化目的蛋白进行SDS-PAGE分析，结果参见图2，图2是本发明实施例提供的在大肠杆菌中表达的重组脯氨酸消旋酶的SDS-PAGE分析，其中，泳道“M”为蛋白Marker；泳道“1”为仅含有pEASY-E2空载体的大肠杆菌诱导表达情况；泳道“2”为载体中带有脯氨酸消旋酶基因的大肠杆菌诱导表达情况。由图2可知，重组脯氨酸消旋酶在大肠杆菌中得到了表达，且获得较明显的蛋白条带。
实验例3脯氨酸消旋酶NCPRAC2的性质测定
重组脯氨酸消旋酶NCPRAC2的酶活采用消旋反应和氧化反应两步法进行测定。
其中，一个酶活力单位(U)定义为1min内催化生成1μmol D-Pro所需要的酶量。
(1)脯氨酸消旋酶NCPRAC2的最适pH和pH稳定性的测定
酶的最适pH测定：将实施例2纯化的脯氨酸消旋酶NCPRAC2在37℃，pH 3～pH 13的缓冲液中进行酶促反应。
酶的pH稳定性测定：将纯化的酶液置于pH＝3～13的缓冲液中，在37℃下处理1h后进行酶促反应，以未处理的酶液作为对照。
缓冲液为：20mmol/L伯瑞坦-罗宾森缓冲液(pH＝3～13)。以L-脯氨酸为底物，反应10min，测定纯化的脯氨酸消旋酶的酶学性质。
结果参见图3和图4，图3为本发明实施例提供的脯氨酸消旋酶最适pH，图4为本发明实施例提供的脯氨酸消旋酶的pH稳定性。由图3和图4可知，本发明提供的脯氨酸消旋酶的最适pH为9.0；经pH 6.0～12.0的缓冲液处理1h后，酶活剩余55％以上。
(2)脯氨酸消旋酶的最适温度及温度稳定性测定
酶的最适温度测定：在pH＝12.0，于0～70℃下进行酶促反应。
酶的温度稳定性测定：将同样酶量的酶液置于设定的温度(30、37、40、50℃)中处理1h，每隔10分钟在pH＝9.0及40℃下进行酶促反应，以未处理的酶液作为对照。
结果参见图5和图6，图5为本发明实施例提供的脯氨酸消旋酶的最适温度，图6为本发明实施例提供的脯氨酸消旋酶的温度稳定性。结果表明：脯氨酸消旋酶的最适温度为40℃，在30、37、40℃条件下保持稳定，在50℃条件下处理50min达到半衰期。
(3)脯氨酸消旋酶的NaCl影响及NaCl耐受性测定
酶的NaCl影响测定：在40℃，pH＝9.0，0.5～5M NaCl反应条件下进行酶促反应。
酶的NaCl稳定性测定：将同样酶量的酶液置于0.5～5M NaCl反应条件中处理1h，在pH＝9.0及40℃下进行酶促反应，以未处理的酶液作为对照。
结果参见图7、图8，图7为本发明实施例提供的脯氨酸消旋酶的NaCl影响，图8为本发明实施例提供的脯氨酸消旋酶的NaCl耐受。结果表明：该酶活性随着NaCl浓度的提高而降低，在NaCl浓度为3.5～5M时酶活性低于10％。在0.5～5MNaCl条件下处理1h后剩余25％以上的酶活性。
(4)重组脯氨酸消旋酶的动力学参数测定
动力学参数在pH＝9.0、温度40℃和一级反应时间下以不同浓度的L-脯氨酸为底物(0～50mM)进行测定，根据Lineweaver-Burk法计算出Km和Vmax值。经测定，在pH 9.0及温度40℃条件下，该酶的Km和Vmax分别为0.06mmol/L和2.1610μmol/L/min。
(5)不同金属离子及化学试剂对重组脯氨酸消旋酶活力影响测定
在酶促反应体系中加入化学试剂(终浓度为10mmol/L)，研究其对酶活的影响。在40℃，pH＝9.0条件下，测定酶活(同样条件下以未加金属离子及化学试剂的酶促反应作为对照)，结果参见表1。
表1金属离子和化学试剂对重组酶NCPRAC2活力的影响
表1表明，除了Na+、Li+、K+和Mg2+对重组酶活性影响不大，其余金属离子对重组酶具有强烈的抑制作用；10mmol/L的DDT、乙酸和β-巯基乙醇对重组酶具有强烈的抑制作用，其余化学试剂对重组酶影响较小，可将其活性提高1％-20％。
(6)重组脯氨酸消旋酶对不同底物降解的测定
40℃，pH＝9.0条件下，上述酶活性测定体系中加入相同浓度的不同底物：脯氨酸(Ala)、丝氨酸(Ser)、半胱氨酸(Cys)、脯氨酸(Pro)、酪氨酸(Tyr)、亮氨酸(Leu)、谷氨酸(Glu)、天冬氨酸(Asp)，测定酶活(以L-脯氨酸为底物的酶活作为对照)，结果表明重组脯氨酸消旋酶均不分解其它底物。
结果参见图9，重组脯氨酸消旋酶底物特异性较强，对其他L-氨基酸的催化活性均低于11％。
其中，本发明的脯氨酸消旋酶与其它生物来源的脯氨酸消旋酶酶学性质比较如下表所示：
注：
[1]A B-cell mitogen from a pathogenic trypanosome is a eukaryoticproline racemase.[J].Nature Medicine,2000,6(8):890-897.
[2]Amino Acid Racemization in Pseudomonas putida KT2440[J].Journal ofBacteriology,2013,195(22):5016-5024.
[3]Proline racemases are conserved mitogens:characterization of aTrypanosoma vivax proline racemase.[J].Molecular&Biochemical Parasitology,2009,165(2):170-179.
[4]Identification and Characterization of Bifunctional ProlineRacemase/Hydroxyproline Epimerase from Archaea:Discrimination of Substratesand Molecular Evolution[J].Plos One,2015,10.
[5]Biochemical characterization of proline racemases from the humanprotozoan parasite Trypanosoma cruzi and definition of putative proteinsignatures.[J].The Journal of biological chemistry,2003.
由表可知，本发明的脯氨酸消旋酶的最适pH为9.0，不同于已知脯氨酸消旋酶的最适pH，可被应用于更广的范围，且其Km远低于已知的脯氨酸消旋酶Km。
实验例4脯氨酸消旋酶NCPRAC2与已知的脯氨酸消旋酶蛋白结构对比
为进一步验证本发明的脯氨酸消旋酶NCPRAC2的结构差异，将脯氨酸消旋酶NCPRAC2与现有PDB蛋白结构数据库进行对比，结果发现，目前仅有5种脯氨酸消旋酶获得了晶体结构，而本发明的脯氨酸消旋酶NCPRAC2与已解析结构的嗜热球菌(Thermococcuslitoralis)来源的脯氨酸消旋酶相似性最高，为41.09％，与其它4个已知晶体结构的脯氨酸消旋酶的相似性在30％左右，说明本发明获得的脯氨酸消旋酶属于一种新型脯氨酸消旋酶，对丰富脯氨酸消旋酶资源以及抑制剂的开发具有较大的研究价值。
尽管本发明的内容已经通过上述优选实施例作了详细介绍，但应当认识到上述的描述不应被认为是对本发明的限制。在本领域技术人员阅读了上述内容后，对于本发明的多种修改和替代都将是显而易见的。因此，本发明的保护范围应由所附的权利要求来限定。
序列表
<110> 云南师范大学
<120> 一种脯氨酸消旋酶及其制备和应用
<141> 2023-10-24
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atgcatctca gccacctcgt tgaaacgatt gatacgcata ccgcgggcaa tcccacccgc 60
aacctcatcg ccggggttcc ccgcatcgag gggaaaacca tgcaggacaa gatggcctat 120
gccgaggagc atctggactg gatgcgcacc gccgtcatga tggaaccccg cgggcacagc 180
aacatgtccg gcacaatctg ggtggaaccg tgccatcccg aggccgacat gggcatcctg 240
ttcatcgacg cgggcgggca catgcccatg tgcgggcaca gcaccatcgg ctgcgtcacc 300
gccatgttgg aaagcggccg ggtgcccatt accggggaag tgaccgaagt gaacatcgat 360
accccagccg gactcgtgcg cacccgggcc accgtggaaa acggcagcgt gaccagcgtc 420
gcgttccgca acgtgccctc cttcctgttc tgctcgggga cggtcgaggt tcccggtata 480
ggggctgtcc cctttgatgt ggcttacggc ggcaacacgt atgccattgc cgacgccgcg 540
tattttcccg gccttgaact ccgttccggc caccgttccg ccatcgaaaa gaccgcgcag 600
gcattcggcg atgctgtgcg cgcggcggtg aagttccagc atcccctcca gccgttcatc 660
aatgtcatca cgcacgtcat gttctatacg aagccggatg atcccacagc cacttaccgc 720
aataccgtcg tttttctgcc tgactccctc gaccgttcac cctgcgggac gggcacctcc 780
gcccgtgtgg cctcgctgtt cgccaagggc gagctcggcc tgaacgaagc ttttgtccat 840
gaaagcgtca tcggcaccca gttccgggcg cgtatcgtcg agcccgcaat ggtcgggccg 900
tacaagggag gcatccctga agtatccggt tcggcttacg tgaccggcct gctcaaactg 960
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Arg Thr Ala Val Met Met Glu Pro Arg Gly His Ser Asn Met Ser Gly
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Phe Ile Asp Ala Gly Gly His Met Pro Met Cys Gly His Ser Thr Ile
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His Val Met Phe Tyr Thr Lys Pro Asp Asp Pro Thr Ala Thr Tyr Arg
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Claims (9)
1.一种脯氨酸消旋酶，其特征在于，编码该脯氨酸消旋酶的核苷酸序列如SEQ ID NO.1所示。
2.如权利要求1所述的脯氨酸消旋酶的编码基因，其特征在于，其核苷酸序列如SEQ IDNO.1所示。
3.一种包含如权利要求2所述的编码基因的重组载体。
4.根据权利要求3所述的重组载体，其特征在于，采用的载体为质粒 pEASY-E2。
5.一种包含如权利要求2所述的编码基因的重组菌。
6.根据权利要求5所述的重组菌，其特征在于，采用的菌为大肠杆菌BL21（DE3）。
7.一种如权利要求1所述的脯氨酸消旋酶的制备方法，其特征在于，包含以下步骤：
S1. 将含有如权利要求2所述的脯氨酸消旋酶编码基因的重组表达载体转化至宿主细胞得到重组菌株，培养该重组菌株，并诱导所述脯氨酸消旋酶基因表达；
S2. 纯化所表达的脯氨酸消旋酶，得到如权利要求1所述的脯氨酸消旋酶。
8.根据权利要求7所述的制备方法，其特征在于，采用的重组表达载体为质粒pEASY-E2；采用的菌为大肠杆菌BL21（DE3）。
9.如权利要求1所述的脯氨酸消旋酶在作为恰加斯病药物筛选靶标中的应用。
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Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001040449A2 (en) * | 1999-12-03 | 2001-06-07 | Institut Pasteur | Cloning of a gene encoding an amino acid racemase from trypanosoma cruzi, and uses thereof |
| BRPI0410975A (pt) * | 2003-05-30 | 2006-07-04 | Pasteur Institut | forma cristalina de prolina racemase de trypanosoma cruzi praca (tcpraca), composição, métodos para fabricar uma forma cristalina de tcpraca e para identificar uma substáncia que pode ligar-se à tcpraca e consequentemente afetar a atividade biológica de tcpraca, substáncia, dispositivo para realizar o método, método para projetar uma molécula que afeta a atividade biológica de tcpraca, e, molécula |
| CN1820070A (zh) * | 2003-02-11 | 2006-08-16 | 巴斯德研究院 | 消旋酶的鉴别和鉴定、蛋白质特征的确定、及用于检测 d －氨基酸和筛选能抑制消旋酶尤其是脯氨酸消旋酶活性的分子的测试 |
| EP2272510A1 (en) * | 2009-07-06 | 2011-01-12 | Institut Pasteur | Inhibitors of the proline racemase of trypanosoma cruzi |
| KR20170002235A (ko) * | 2015-06-29 | 2017-01-06 | 광운대학교 산학협력단 | 돌연변이 프롤린 라세미화 효소의 제조방법 |
| CN111032874A (zh) * | 2017-07-12 | 2020-04-17 | Cj第一制糖株式会社 | 表达活性d-脯氨酸还原酶的微生物以及生产活性d-脯氨酸还原酶的方法 |
| CN111041016A (zh) * | 2019-12-12 | 2020-04-21 | 云南师范大学 | 一种耐盐六磷酸海藻糖水解酶及其制备方法和应用 |
| CN113106082A (zh) * | 2021-05-27 | 2021-07-13 | 云南师范大学 | 动物粪便宏基因组来源的丙氨酸消旋酶及其制备和应用 |
| CN113661239A (zh) * | 2018-12-28 | 2021-11-16 | 催化剂生物科学公司 | 经修饰的尿激酶型纤溶酶原激活物多肽和使用方法 |
| CN115372290A (zh) * | 2022-04-11 | 2022-11-22 | 云南师范大学 | 一种快速测定脯氨酸消旋酶酶活的方法 |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2383358A1 (en) * | 1999-08-31 | 2001-03-08 | Board Of Regents, The University Of Texas System | Methods and compositions of a novel serine protease inhibitor |
| US20080138352A1 (en) * | 2003-02-11 | 2008-06-12 | Paola Minoprio | Identification and characterization of novel proline racemases and hydroxyproline-2-epimerases, uses thereof, and methods of identifying proline racemases and hydroxyproline-2-epimerases |
| US20120076815A1 (en) * | 2003-02-11 | 2012-03-29 | Institut Pasteur | Identification and characterization of racemases, definition of protein signatures, and a test for detecting d-amino acid and for screening molecules capable of inhibiting the activity of racemase, especially proline racemase |
| US20120058135A1 (en) * | 2010-09-07 | 2012-03-08 | Institut Pasteur | Identification and characterization of racemases, definition of protein signatures, and a test for detecting D-amino acid and for screening molecules capable of inhibiting the activity of racemase, especially proline racemase |
| US11613744B2 (en) * | 2018-12-28 | 2023-03-28 | Vertex Pharmaceuticals Incorporated | Modified urokinase-type plasminogen activator polypeptides and methods of use |
-
2022
- 2022-05-12 CN CN202210514276.6A patent/CN114854728B/zh active Active
Patent Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001040449A2 (en) * | 1999-12-03 | 2001-06-07 | Institut Pasteur | Cloning of a gene encoding an amino acid racemase from trypanosoma cruzi, and uses thereof |
| CN1820070A (zh) * | 2003-02-11 | 2006-08-16 | 巴斯德研究院 | 消旋酶的鉴别和鉴定、蛋白质特征的确定、及用于检测 d －氨基酸和筛选能抑制消旋酶尤其是脯氨酸消旋酶活性的分子的测试 |
| BRPI0410975A (pt) * | 2003-05-30 | 2006-07-04 | Pasteur Institut | forma cristalina de prolina racemase de trypanosoma cruzi praca (tcpraca), composição, métodos para fabricar uma forma cristalina de tcpraca e para identificar uma substáncia que pode ligar-se à tcpraca e consequentemente afetar a atividade biológica de tcpraca, substáncia, dispositivo para realizar o método, método para projetar uma molécula que afeta a atividade biológica de tcpraca, e, molécula |
| EP2272510A1 (en) * | 2009-07-06 | 2011-01-12 | Institut Pasteur | Inhibitors of the proline racemase of trypanosoma cruzi |
| KR20170002235A (ko) * | 2015-06-29 | 2017-01-06 | 광운대학교 산학협력단 | 돌연변이 프롤린 라세미화 효소의 제조방법 |
| CN111032874A (zh) * | 2017-07-12 | 2020-04-17 | Cj第一制糖株式会社 | 表达活性d-脯氨酸还原酶的微生物以及生产活性d-脯氨酸还原酶的方法 |
| CN113661239A (zh) * | 2018-12-28 | 2021-11-16 | 催化剂生物科学公司 | 经修饰的尿激酶型纤溶酶原激活物多肽和使用方法 |
| CN111041016A (zh) * | 2019-12-12 | 2020-04-21 | 云南师范大学 | 一种耐盐六磷酸海藻糖水解酶及其制备方法和应用 |
| CN113106082A (zh) * | 2021-05-27 | 2021-07-13 | 云南师范大学 | 动物粪便宏基因组来源的丙氨酸消旋酶及其制备和应用 |
| CN115372290A (zh) * | 2022-04-11 | 2022-11-22 | 云南师范大学 | 一种快速测定脯氨酸消旋酶酶活的方法 |
Non-Patent Citations (4)
| Title |
|---|
| Inhibition of Trypanosoma cruzi proline racemase affects host-parasite interactions and the outcome of in vitro infection;Coutinho L等;Mem Inst Oswaldo Cruz.;第1055-1062页 * |
| proline racemase family protein [Bilophila wadsworthia];Reina-San-Martin,B.等;Genbank Database;Accession No：WP_029434478.1 * |
| 寄生虫来源的外泌体及其功能研究进展;范晓斌;何兴;潘卫庆;;中国热带医学(第12期);第1-6页 * |
| 西黑冠长臂猿粪便微生物宏基因组来源的丙氨酸消旋酶 的异源表达及性质研究;杨金茹等;微生物学报;第1362-1378页 * |
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