台灣嗜熱細菌Thermus taiwanensis WL-219含錳超氧歧化?之研究

Abstract

本研究室在過去幾年針對台灣地區的溫泉進行高溫微生物的採樣以及篩選，建立了台灣嗜熱菌種源庫。本研究以實驗室所收集的台灣嗜熱性細菌以原態聚丙烯醯氨膠片進行蛋白質電泳分析，配合超氧歧化?活性染色結果，發現分離菌株strain WL-219含有錳超氧歧化?，其活性區與已知嗜熱菌屬標準菌株不同。而且分離菌株strain WL-219菌株在生理特性的測試、脂肪酸組成以及16S rDNA序列比對結果顯示該菌株也與已知嗜熱菌屬的各標準菌株有相當差異，可能為台灣嗜熱菌屬之新種。建議將其命名為Thermus taiwanensis WL-219。 利用聚合?連鎖反應，以T. aquaticus與T. Thermophilus含錳超氧歧化?基因保留區域核酸序列自行設計引子，可以將嗜熱菌屬各標準菌株與Thermus taiwanensis WL-219之genomic DNA放大得到單一條帶，定序後與NCBI（National Center of Biotechnology Information）等基因庫所收錄的核酸序列進行比對，發現該片段與含錳超氧歧化?之核酸序列相似，經由序列相似度比較分析發現Thermus taiwanensis WL-219的含錳超氧歧化?核酸序列與T. thermophilus的序列相似度為92％，與T. aquaticus相似度為88％。將序列進行系統發生（phylogenic）分析，其結果與利用16S rDNA序列所得的結果相似，可以輔助菌種之分類。 為了研究嗜熱性細菌所生產的耐熱性酵素應用的可行性，本研究將所得到的Thermus taiwanensis WL-219含錳超氧歧化?基因片段選殖到蛋白質表現載體pET20b（＋）中，並送至大腸桿菌表現，結果雖然可以大量表現分子量接近（23kDa）的超氧歧化?蛋白質，但是僅有少量蛋白質為可溶性，可溶性蛋白質可以經由親和性層析純化，並具有超氧歧化?活性，酵素在80℃加熱20分鐘仍保有70％活性。

In the past few years, our laboratory had sampled and investigated the thermophilic microbes from many hot springs in Taiwan. Superoxide dismutase (SOD) activities of the thermophiles isolated from Taiwan were assayed by combining polyacrylamide gel electrophoresis and activity staining. One of our tested strains, strain WL-219, the electromorphs of manganese superoxide dismutase were different from all the other electromorphs of superoxide dismutase of Thermus type strains. The physiological characteristics, fatty acid composition and 16S rDNA gene sequence comparison showed that strain WL-219 might represent a new species of the genus Thermus. The name, Thermus taiwanensis WL-219 is suggested for this new species. By using PCR primer designed from the conserved sequences of sod gene of T. aquaticus and T. thermophilus, an internal fragment of sod gene from 10 Thermus type strains and 4 Thermus sp. isolates was amplified. The sequence of the sod fragment of T. taiwanensis WL-219 display 92% and 88% identity with the sequence of the type strains of T. aquaticus and T. thermophilus, respectively. The phylogenetic tree of partial sequences of sod gene similar to that of 16S rDNA sequences for Thermus type strains, and suggested that this gene sequence could be useful to differentiate closely related species. DNA fragment coding for manganese superoxide dismutase from T. taiwanensis WL-219 was cloned and overexpressed in Escherichia coli by use of the pET-20b(+) expression vector. The recombinant enzyme was efficiently purified from crude E. coli cell lysate by affinity chromatography. Seventy percent of emzymatic activity of recombinant Mn-SOD was retained after heated at 80℃ for 20 minutes.