非蛋白質(zhì)氨基酸的酶促合成及其作為中間體參與新藥合成的研究
發(fā)布時間：2019-05-14 15:05
【摘要】：O-乙酰絲氨酸（硫醇）裂解酶(O-acetylserine sulfhydrylase, OASS)是大腸桿菌硫酸鹽同化途徑及半胱氨酸合成中重要的生物酶之一，和絲氨酸乙酰轉(zhuǎn)移酶(Serineacetyltransferase, SAT)以酶復合體的形式存在，通常被稱為半胱氨酸合酶(Cysteinesynthase)。在微生物和植物細胞內(nèi)，OASS和SAT將環(huán)境中的無機硫還原成-2價的硫，經(jīng)二步酶催化途徑由L-絲氨酸合成L-半胱氨酸。首先以絲氨酸和乙酰輔酶A為底物，在SAT催化下，合成O-乙酰絲氨酸(O-acetylserine, OAS)和輔酶A；然后在OASS的催化下，通過硫化物將OAS轉(zhuǎn)化為半胱氨酸。通過替代第二步反應(yīng)中底物，OASS能夠利用各種親核試劑合成出非蛋白質(zhì)氨基酸。 非蛋白質(zhì)氨基酸是自然界存在的蛋白質(zhì)中發(fā)現(xiàn)的20種氨基酸以外的其他氨基酸，因其豐富了肽鏈的多樣性，為跨越20種天然氨基酸屏障，人工合成蛋白質(zhì)提供了基礎(chǔ)材料，是國內(nèi)外合成肽類、類肽類藥物的主要成分，也是合成手性藥物的極具吸引力的侯選基礎(chǔ)材料，是眾多新藥的關(guān)鍵中間體，在科研和醫(yī)藥領(lǐng)域用途廣泛。 本論文以大腸桿菌Escherichia coli (E. coli) DH5α基因組DNA為模板，PCR擴增得到大小為972bp的O-乙酰絲氨酸（硫醇）裂解酶A (OASS-A)基因cysK ORF區(qū)基因片段，將目的片段與表達載體pET-22b(+)相連，得到重組質(zhì)粒CysK-pET-22b(+)，轉(zhuǎn)化到表達菌中，篩選誘導表達條件，進行蛋白純化，獲得高濃度可溶的OASS-A重組蛋白。以O(shè)-乙酰絲氨酸(OAS)為底物，對得到的重組蛋白進行酶活性檢測，測得純化的重組蛋白的活性單位為750U/mg，酶的粗提液中重組蛋白的活性單位為400U/mg。以重組OASS-A作為酶催化劑，O-乙酰絲氨酸和苯硫酚作為底物，合成非蛋白質(zhì)氨基酸S-苯基-L-半胱氨酸(S-phenyl-L-cysteine, S-P-C)，通過高效液相色譜和核磁共振技術(shù)對產(chǎn)物結(jié)構(gòu)進行鑒定，得到高純度的S-P-C。建立了通過得到大量的活性穩(wěn)定的重組酶作為生物催化劑，高效率合成非蛋白質(zhì)氨基酸的有效模式。根據(jù)藥物拼接原理，，將S-P-C與活性化合物積雪草酸、脫氧膽酸的有效基團進行偶聯(lián)，對產(chǎn)物進行結(jié)構(gòu)鑒定，并初步檢測合成產(chǎn)物對癌細胞增殖影響初步檢測，MTT實驗結(jié)果顯示，合成的終產(chǎn)物均具有一定的增殖抑制作用。
[Abstract]:O-acetylserine (mercaptan) lyase (O-acetylserine sulfhydrylase, OASS) is one of the important biological enzymes in sulfate assimilation pathway and cysteine synthesis of E. coli, and serine acetyltransferase (Serineacetyltransferase, SAT) exists in the form of enzyme complex. Commonly known as cysteinase (Cysteinesynthase). In microorganisms and plant cells, OASS and SAT reduced inorganic sulfur in the environment to-2 valence sulfur, and synthesized L-cysteine from L-serine by two-step enzyme catalysis. O-acetylserine (OAS) and coenzyme A were synthesized with serine and acetylcoenzyme A as substrate under the catalysis of SAT, and then OAS was converted to cysteine by sulfides catalyzed by OASS. By replacing the substrate in the second step, OASS can synthesize non-protein amino acids from various nucleophilic reagents. Non-protein amino acids are 20 kinds of amino acids other than 20 kinds of amino acids found in proteins existing in nature. Because they enrich the diversity of peptide chains, they provide the basic materials for artificial synthesis of proteins across 20 kinds of natural amino acid barriers. It is the main component of synthetic peptides and peptides at home and abroad, and it is also an attractive candidate for chiral drugs. It is a key intermediate of many new drugs and is widely used in scientific research and medicine. In this paper, the cysK ORF gene fragment of O-acetylserine (mercaptol) lyase A (OASS-A) gene was amplified by PCR using E. coli Escherichia coli (E. coli) DH5 偽 genomic DNA as template, and the fragment of Oacetylserine (mercaptol) lyase A (OASS-A) gene was amplified by PCR. The target fragment was connected with the expression vector pET-22b (), and the recombinant plasmid CysK-pET-22b (), was transformed into the expressing strain. The induced expression conditions were screened and the protein was purified to obtain the high concentration soluble OASS-A recombinant protein. Using O-acetylserine (OAS) as substrate, the enzyme activity of the recombinant protein was detected. The activity unit of the purified recombinant protein was 750 U 路mg, and the active unit of the recombinant protein in the crude extract of the enzyme was 400 U 路mg 路L ~ (- 1). Using recombinant OASS-A as enzyme catalyst and O-acetylserine and phenylthiophenol as substrate, non-protein amino acid S-phenyl-L-cystine (S-P-C) was synthesized. The structure of the product was identified by high performance liquid chromatography (HPLC) and nuclear magnetic resonance (NMR). An effective model for the efficient synthesis of non-protein amino acids by obtaining a large number of active and stable recombinant enzymes as biocatalysts was established. According to the principle of drug splicing, S-P-C was coupled with the effective groups of active compounds asiatidic acid and deoxycholic acid, the structure of the product was identified, and the effect of the synthesized product on the proliferation of cancer cells was preliminarily detected. The results of MTT test showed that the synthesized end products had certain inhibitory effect on proliferation.
【學位授予單位】：遼寧師范大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R914
本文編號：2476805
[Abstract]:O-acetylserine (mercaptan) lyase (O-acetylserine sulfhydrylase, OASS) is one of the important biological enzymes in sulfate assimilation pathway and cysteine synthesis of E. coli, and serine acetyltransferase (Serineacetyltransferase, SAT) exists in the form of enzyme complex. Commonly known as cysteinase (Cysteinesynthase). In microorganisms and plant cells, OASS and SAT reduced inorganic sulfur in the environment to-2 valence sulfur, and synthesized L-cysteine from L-serine by two-step enzyme catalysis. O-acetylserine (OAS) and coenzyme A were synthesized with serine and acetylcoenzyme A as substrate under the catalysis of SAT, and then OAS was converted to cysteine by sulfides catalyzed by OASS. By replacing the substrate in the second step, OASS can synthesize non-protein amino acids from various nucleophilic reagents. Non-protein amino acids are 20 kinds of amino acids other than 20 kinds of amino acids found in proteins existing in nature. Because they enrich the diversity of peptide chains, they provide the basic materials for artificial synthesis of proteins across 20 kinds of natural amino acid barriers. It is the main component of synthetic peptides and peptides at home and abroad, and it is also an attractive candidate for chiral drugs. It is a key intermediate of many new drugs and is widely used in scientific research and medicine. In this paper, the cysK ORF gene fragment of O-acetylserine (mercaptol) lyase A (OASS-A) gene was amplified by PCR using E. coli Escherichia coli (E. coli) DH5 偽 genomic DNA as template, and the fragment of Oacetylserine (mercaptol) lyase A (OASS-A) gene was amplified by PCR. The target fragment was connected with the expression vector pET-22b (), and the recombinant plasmid CysK-pET-22b (), was transformed into the expressing strain. The induced expression conditions were screened and the protein was purified to obtain the high concentration soluble OASS-A recombinant protein. Using O-acetylserine (OAS) as substrate, the enzyme activity of the recombinant protein was detected. The activity unit of the purified recombinant protein was 750 U 路mg, and the active unit of the recombinant protein in the crude extract of the enzyme was 400 U 路mg 路L ~ (- 1). Using recombinant OASS-A as enzyme catalyst and O-acetylserine and phenylthiophenol as substrate, non-protein amino acid S-phenyl-L-cystine (S-P-C) was synthesized. The structure of the product was identified by high performance liquid chromatography (HPLC) and nuclear magnetic resonance (NMR). An effective model for the efficient synthesis of non-protein amino acids by obtaining a large number of active and stable recombinant enzymes as biocatalysts was established. According to the principle of drug splicing, S-P-C was coupled with the effective groups of active compounds asiatidic acid and deoxycholic acid, the structure of the product was identified, and the effect of the synthesized product on the proliferation of cancer cells was preliminarily detected. The results of MTT test showed that the synthesized end products had certain inhibitory effect on proliferation.
【學位授予單位】：遼寧師范大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R914
【參考文獻】
相關(guān)期刊論文 前2條
1 趙龍鉉;田明珠;金禮吉;賀興隆;沈平;張曉翠;楊君微;;積雪草酸衍生物的合成與表征及體外抗癌活性的研究[J];有機化學;2011年05期
2 Maria J Perez;Oscar Briz;;Bile-acid-induced cell injury and protection[J];World Journal of Gastroenterology;2009年14期
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