高溫古生菌Sulfolobus acidocaldarius DSM639 之重組蛋白Ketol-acid reductoisomerase之生化特性分析

Characterization of recombinant ketol-acid reductoisomerase from thermoacidophilic archaeon Sulfolobus acidocaldarius DSM639

江沂庭(Yi-ting Chiang)

指導教授 ： 黃雪莉

國立中央大學/理學院/生命科學系/碩士(2012年)

摘要

Sulfolobus acidocaldarius DSM639為一株嗜熱、嗜酸之古生菌，能生長於55-85 ˚C之溫泉與酸性環境(pH 2-3)，其適應極端環境之策略能對酵素和應用具研發潛力。本實驗先前研究中，經由蛋白質體學分析此菌之分泌蛋白體，鑑定到多種外泌的酵素，Ketol-acid reductoisomerase (KARI, EC 1.1.1.86)為其中之一，此酵素在細胞內參與三種胺基酸:纈胺酸、亮氨酸及異亮氨酸之生物合成路徑，另外也被專利保護下應用在工業發酵生成丁醇作為生質能源，因此本研究將此種酵素利用大腸桿菌大量表達，並進行生化特性分析以探討應用潛力；基因體序列分析本酵素乃以GTG為起始密碼子，因古生菌具有多種罕見起始密碼子，為了更進一步證實本酵素轉譯是由GTG開始，進行N端定序佐證之。結果依據核甘酸和N端定序結果推測KARI轉譯是由GTG開始。此外，KARI酵素活性測試發現重組酵素在65 ˚C、pH 7.3下有最高活性，經初步純化比活性值為3.5 μmole/min/mg，對菸鹼醯胺腺嘌呤二核苷酸磷酸(NADPH)之Km為14 μM。

關鍵字

重組蛋白 ； 高溫古生菌

並列摘要

Sulfolobus acidocaldarius DSM639 is a thermoacidophilic crenarchaeon which can grow in hot springs at 55-85 ˚C and acidic environments (pH 2-3). As we facing the extreme living environmental challenge now and in near future, the extreme adaptation genes, extremozymes and strategies from archaea may help us to adapt in hostile environments. In previous study, ketol-acid reductoisomerase (KARI, EC 1.1.1.86) was identified as one of numerous extra-cellular proteins of S. acidocaldarius DSM639 by LC/MS/MS. This enzyme not only participates in valine, leucine and isoleucine biosynthesis pathway, but also can be expressed in recombinant organism for bio-butanol production. In this study, KARI with GTG as start codon was overexpressed by recombinated E. coli system, and the biochemical properties of KARI were also explored. The results of N-terminal sequencing and DNA sequencing verified that KARI was expressed by using GTG as start codon in E. coli. It suggested that GTG can be used as start codon for protein expression in both archaeobacteria and E. coli. Furthermore, the KARI enzyme assay showed that optimal temperature and pH value of recombinant KARI was 65 ˚C and pH 7.3 in 100 mM HEPES-potassium containing 10 mM MgCl2 condition. The specific activity of purified enzyme was 3.5 μmole/min/mg, and the Km was 14 μM responding to co-substrate: NADPH.

並列關鍵字

ketol-acid reductoisomerase ； Sulfolobus

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