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標題: Characterization and Protein Engineering of D-hydantoinase from Agrobacterium Radiobacter and Mutational Analysis of the Catalytic Sites of D-hydantoinase from Bacillus Caldolyticus
Agrobacterium Radiobacter D-hydantoinase的改造和利用定點突變法分析 Bacillus Caldolyticus D-hydantoinase 的活性部位
作者: Perng, Renn-Yeuan
關鍵字: Agrobacterium Radiobacter D-hydantoinase;定點突變法
出版社: 分子生物研究所
D-Hydantoinase is an industrial enzyme used in the production of semisynthetic penicillins and cephalosporins. The dht gene of Agrobacterium radiobacter and Bacillus were prepared by ploymerase chain reaction. The genes were cloned into pQE30 expression vector and expressed in E. coli. D-Hydantoinase was tagged with six histidine residues and purified by metal affinity chromatography.A. radioacter D-hydantoinase was enhanced by of Mn2+ or Co2+ ions and inhibited by Cu2+ ion, while the activity of B. caldolyticus D-hydantoinase was only considerably enhanced by Mn2+ ion. Both A. radioacter D-hydantoinase and B. caldolyticus D-hydantoinase were resistance to H2O2 oxidation. Half-life of B. caldolyticus D-hydantoinase and A.radioacter D-hydantoinase were 40 and 2 days at 50℃ respectively. After incubation at 50℃ for 112 days, about 10% of B.caldolyticus D-hydantoinase activity were still retained in the reaction mixture containing Mn2+ ion.
Alignment of D-hydantoinase and dihydropyrimidinase sequences from diverse microorganisms and animals, three conserved cysteine residues were found in thermostable D-hydantoinases. To develop a thermostable A. radioacter D-hydantoinase, three mutant, Arg-47-Cys, Phe-223-Cys and Leu-429-Cys, were constructed by site-directed mutagenesis. The Phe-223-Cys mutant enzyme shows a better thermostability than wild-type D-hydantoinases and remaning 10% activity after incubation at 50℃ for 7 days.
Asp-56, Asp-392, Ser-148 and Cys-317 residue was highly conserved in fifteen functional releated D-hydantoinases and dihydropyrimidinases. Site-directed mutagenesis revealed that mutant enzymes Asp-56-Ala and Asp-392-Ala were remaining 0.44% and complete lose activity, respectively, while Ser-148-Ala and Cys-317-Ala still retained 54.5 and 71.2% activity. Asp-56-Ala mutant enzyme has the same specific activity as wild-type in the reaction mixture containing 38.4 mM Mn2+ ion. Furthermore, its Kd is increase 182-fold than wild-type. It revealed that Asp-56 is involved in metal binding and Asp-392 residue might be involved in enzyme catalysis.

D-Hydantoinase是工業上用來生產penecillin和cephalsporin類抗生素的酵素。利用PCR方法由Agrobacterium radiobacter和Bacillus caldolyticus選殖dht基因,將其構築到pQE30表現載體,並以E.coli進行dht基因的表現。利用金屬親和性層析法純化回收N端帶有六個histidine之D-Hydantoinase,進行活性分析。結果發現,Mn2+和Co2+離子會提高A. radiobacter D-hydantoinase的活性,Cu2+離子則會抑制其活性,而Mu2+離子則能使B.caldolyticus D-hydantoinase的活性大幅提高。A. radiobacter D-hydantoinase和B. caldolyticus D-hydantoinase對H2O2皆有很強的抗性。在50℃的水浴中,B. caldolyticus D-hydantoinase和A. caldolyticus D-hydantoinase之半衰期分別為40天和20天。在B, caldolyticus D-hydantoinase溶液中,添加100uM之Mu2+離子,置於50℃水浴中,保溫112天後,D-hydantoinase仍保有10%的活性。
將來自不同微生物或動物之D-hydantoinase胺基酸序列加以比對,發現有三個保守的cysteine,為嗜熱菌之D-hydantoinase所獨物具有的。為了嘗試改造A. radiobacter D-hydantoinase成為耐熱酵素,於是針對這三個cysteine,進行定點突變,產生了Arg-47-Cys、Phe-223-Cys和Leu-223-Cys三個變異D-hydantoinase。結果顯示Phe-223-Cys變異□於50℃水浴中,保溫7天後,仍殘留有10%活性,較野生型D-hydantoinase為佳。比對15個功能相似的D-hydantoinase及dihydropyrimidinase之胺基酸序列,以找出高度保守之胺基酸殘基,定點突變之結果顯示變異□Asp-56-Ala和Asp-392-Ala幾乎完全失去活性,而變異□Ser-148-Ala和Cys-317-Ala則分別殘存有54.5和71.2%之活性。Asp-56-Ala變異□在38.4mM Mn2+離子溶液中,比活性與野生型D-hydantoinase相近,而Kd值增大182倍,因此推測Asp-56殘基與Mn2+離子之結合有關,而Asp-392殘基可能與催化反應的進行有關。
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