Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/98014
標題: 發展大腸桿菌溫控表現載體生產表皮葡萄球菌脂肪酶與其脂肪酶之應用
Development of thermo-regulated expression vector for E. coli B strain to produce lipase of Staphylococcus epidermidis and its application
作者: 劉丞桓
Cheng-Huan Liu
關鍵字: 溫控表現載體;表皮葡萄球菌;脂肪酶;丁醇酯;Thermo-regulated vector;Staphylococcus epidermidis;Lipase;Butyl esters
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摘要: 
過去發展出來的溫控型lacIts表現質體只應用在大腸桿菌K-12菌株,本研究希望發展適合應用於重組蛋白質生產的大腸桿菌B菌株,將經過隨機突變的lacI基因,構築在由T7A1啟動子(PA1)驅動的lacZ的質體中,並將質體轉型至lacI和lacZ基因被剃除的大腸桿菌B菌株,進而建立篩選lacI突變體之基因庫,藉由LacZ蛋白質的表現與活性測試,分離出一個熱敏感之lacI(lacIts),分析後顯示為第42號氨基酸被取代的突變Met42Lys,因此將此突變命名為lacI42ts,為了測試lacI42ts對溫度調控的能力,將脂肪酶gehC建構於由lacI42ts / PA1表現的質體上,並於大腸桿菌B菌株中進行重組蛋白質的表現,由初步測試結果得知,進行熱誘導的菌株脂肪酶活性比未誘導時高出49倍,並進一步優化蛋白質表現條件,於優化後之蛋白質表現條下進行測試,菌株中重組蛋白質GehC總活性可增加47%,此結果說明熱調控表現載體於大腸桿菌B菌株中進行重組蛋白質生產是可行的。過去的研究顯示,於表皮葡萄球菌中發現脂肪酶(SeLip,GehC),研究同時顯示此脂肪酶可用於水溶液中進行酯化反應生成香味化合物,為了解此催化機制針對分子量為43kDa的重組GehC(rGehC; Lys303-Lys688)進行蛋白質結晶條件篩選和晶體學分析,蛋白質結晶篩選過程使用PEG 10000作為沉澱劑,並在293K下可得到rGehC的蛋白質晶體,晶體於77K環境下可收集到完整性99.9%的原生數據(native data),其晶體的解析度能達到1.9Å、Rmerge值為7.3%、晶體為斜方晶系,屬於P212121晶格群,單位晶格參數為a = 42.07,b = 59.31,c = 171.30 Å,α = β = γ = 90。溶劑含量計算分析得知此脂肪酶可能含有一個不對稱單位(asymmetric unit)。

The temperature sensitive lacIts plasmids were developed in the past. They were applicated in Escherichia coli K-12 strain for protein produce. This study will develop thermo-regulated vector to produce recombinant protein in E. coli B strain. The lacI gene in the plasmid bearing the T7A1 promoter (PA1)-driven lacZ was randomly mutated. The mutant library was then screened in Escherichia coli B strain deficient in lacI and lacZ. Based on the LacZ phenotype, one heat-sensitive lacI (lacIts) was isolated and it revealed a mutation with an amino acid substitution, Met42Lys (designated lacI42ts). To examine its performance, the lacI42ts/PA1-based plasmid was employed for expression of gehC (encoding lipase) in E. coli B strain. Consequently, the strain that received a thermal induction produced 49-fold more GehC in terms of activity than the uninduced level. The expression condition was further optimized, finally leading to a 47% increase in the GehC activity for the strain. Overall, it indicates that the thermo-regulated vector is useful for the recombinant protein production in E. coli B strain. The Staphylococcus epidermidis lipase (SeLip, GehC) can be used in flavour-compound production via esterification in aqueous solution. This study reports the crystallization and crystallographic analysis of recombinant GehC (rGehC; Lys303–Lys688) with a molecular weight of 43 kDa. rGehC was crystallized at 293 K using PEG 10 000 as a precipitant, and a 99.9% complete native data set was collected from a cooled crystal at 77 K to a resolution of 1.9 Å with an overall Rmerge value of 7.3%. The crystals were orthorhombic and belonged to space group P212121, with unit-cell parameters a = 42.07, b = 59.31, c = 171.30 Å , α = β = γ = 90. Solvent-content calculations suggest that there is likely to be one lipase subunit in the asymmetric unit.
URI: http://hdl.handle.net/11455/98014
Rights: 同意授權瀏覽/列印電子全文服務,2021-08-15起公開。
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