葡萄糖酸桿菌之頭孢菌素醯化酶的基因選殖表現與比較

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题名:	葡萄糖酸桿菌之頭孢菌素醯化酶的基因選殖表現與比較 Cloning, Expression and Comparison of Cephalosporin Acylase Genes from Gluconobacter Species
作者:	何金玉
贡献者:	生物科技研究所
关键词:	頭孢菌素酰化酶葡萄糖酸桿菌 Cephalosporin acylase Gluconobacter Species
日期:	2024
上传时间:	2024-08-02 15:03:48 (UTC+8)
摘要:	頭孢氨芐(cephalexin)是應用最廣泛的 β-內酰胺類抗生素之一，具有廣效性抗感染性微生物的作用，但工業上以化學法生產頭孢氨芐存在許多問題，因此酵素法被認為是生產頭孢氨芐的優越方法。一步驟反應，減少污染。然而，由於酵素的特性導致產物水解，酵素法仍存在低生產率的挑戰。在本研究中，利用已知的基因體序列進行引子的設計，包括來自葡萄糖酸桿菌BCRC10683的α-氨基酸酯水解酶（AEH），以聚合酶鏈鎖反應(PCR)放大擴增來自不同葡萄糖酸桿菌物種的頭孢氨芐合成酶（也稱為頭孢氨芐合成酶，CSE），包括來自弗氏葡萄糖酸桿菌BCRC 10383 的戊二醯-7-氨基頭孢菌酸醯化酶（GL-7-ACA 醯化酶），以及來自葡萄糖酸桿菌BCRC 10384 的青黴素醯化酶。將來自葡萄糖酸桿菌BCRC 10683 的AEH 和來自葡萄糖酸桿菌BCRC 10683 的GL-7-ACA 醯化酶的基因選殖出來，將選殖的基因片段進行定序並和在GenBank中的序列比對，確認基因序列是正確的。接著將葡萄糖酸桿菌BCRC 10683的AEH基因進一步選殖到載體pET21b和pET28a，並送到大腸桿菌中大量表現AEH蛋白質（命名為GoAEH）。經由一步驟的Ni-NTA管柱純化後，純化的蛋白質溶液以苯基甘氨酸甲酯 (PGME)和7-氨基去乙醯氧基頭孢烷酸(7-ADCA) 進行頭孢氨芐的合成反應，並進行TLC和HPLC分析。結果顯示一步驟純化的GoAEH確實具有能催化PGME和7-ADCA縮合反應以合成頭孢氨芐的活性，這顯示AEH是葡萄糖酸桿菌中的CSE。根據演化樹和多重序列並列比對分析，葡萄糖酸桿菌屬中有多個與GoAEH具有很高的同源性的蛋白質，預計它們均具有相同的生產頭孢氨芐的催化能力，但需要從基因選殖、蛋白表現和活性測定等方面再進一步研究才能驗證它。 Cephalexin is one of the most widely used β-lactam antibiotics, which possesses a broad spectrum against infectious microorganisms, but the industrial production of cephalexin using chemical scheme suffers from many problems, so enzymatic methods are regarded as a superior method that can produce cephalexin in one-step reaction and cut down the pollution. However, the enzymatic method still has remaining challenges in low productivity due to hydrolysis of the product causing by the characteristic of the enzyme. In this study, the enzymes for cephalexin synthesis (also called cephalexin-synthesizing enzyme, CSE) from different Gluconobacter species have been amplified by PCR using the primers designed based on the genome sequences including α-amino acid ester hydrolase (AEH) from G. oxydans BCRC 10683, glutaryl-7-aminocephalosporanic acid acylase (GL-7-ACA acylase) from Gluconobacter frateurii BCRC 10383, and penicillin acylase from G. oxydans BCRC 10384. Both genes encoding AEH from G. oxydans BCRC 10683 and GL-7-ACA acylase from G. frateurii BCRC 10383 were successfully cloned and the cloned DNA fragments were sequenced and blast in GenBank to confirm to be correct. The AEH gene from G. oxydans BCRC 10683 was further subcloned into pET21b and pET28a in Escherichia coli to overexpress AEH (named GoAEH). After purification by one-step Ni-NTA column, the purified protein fractions were conducted cephalexin-synthesizing reactions from phenylglycine methyl ester (PGME) and 7-aminodeacetoxycephalosporanic acid (7-ADCA), and examined by TLC and HPLC analysis. The results showed that the one-step purified GoAEH did possess good activity to catalyze the condensing reaction of PGME and 7-ADCA to synthesize cephalexin, which suggested that AEH is the CSE in G. oxydans. There are several proteins have very high homology with GoAEH in Gluconobacter species according to the phylogeny and multiple sequence alignment analyses, which was expected that all of them have the same catalyzing ability to producing cephalexin, but needs to further study in gene cloning, protein expression and activity assay to validate it.
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