Thermophilic Alkaline-Protease-Producing Bacterium - Tepidimonas taiwanensis sp.nov. from Southern Taiwan

Abstract

During screening for protease-producing bacteria, a strain designated Ⅰ1-1T was collected from a Sih-Chong-Si hot spring at Pingtung, located in the southern Taiwan. This strain formed light-yellow pigmented colonies. Cells of this organism were Gram-negative rods motile by means of a single polar flagellum. The sizes of rods were approximately 0.4-0.5 μm in diameter and 0.8-2.0 μm in length. Strain Ⅰ1-1T grew at temperatures ranging from 35 to 60℃, pH values between 6.0 and 10, and NaCl concentrations between 0-0.5%. Optimal growth condition was around 55℃, pH 7-8 and 0% NaCl. Comparison of the 16S rRNA gene sequence similarity of strainⅠ1-1T with those collected in public databases reveals that strain Ⅰ1-1T belonged to the comamonadaceae within Burkholderiales group of the β-Proteobacteria. According to the results from the phylogentic analysis, the 16S rDNA sequence similarty of strain Ⅰ1-1T clustered together with genus Tepimonas which contained species, On the basis of DNA-DNA similarity data, physiological and biochemical characteristics of API, Biolog, antibiotic susceptibility, fatty acid compositions, whole cell protein profile comparisons, as well as G+C content, the organism belonged to the protease genus Tepimonas and represented a novel species within this genus. We propose to classify Ⅰ1-1T strain as Tepidimonas taiwanesis sp.nov. The 16S rDNA sequence of strain Ⅰ1-1T clustered together with T. ignava(97.2﹪similarity)and T. aquatica.(96.8﹪similarity). Whole-genome DNA-DNA hybridization experiment was performed between strain Ⅰ1-1T and two type strains of it's nearest phylogenetic neighbors, T. ignava and T. aquatica strain. The binding levels of strain Ⅰ1-1T twords T. ignava and T. aquatica were 38.3﹪and 40.8﹪, respectively. The whole-cell protein profile of strain Ⅰ1-1T was compared with that of T. ignava(92.1﹪similarity) and T.aquatica (89.8﹪similarity). The G+C content of it's DNA is 68.1mol﹪. The predominant cellular fatty acids of strain Ⅰ1-1T were 16: 0 ( about 41﹪), 18:1 ω7 c ( about 13﹪), and summed feature 3a (16:1ω or 15:0 iso 2OH or both) (about 26﹪). A zymogram analysis of the strain Ⅰ1-1T in the alkaline medium reveales the presence of protease activity near the molecular weights of 32, 60, and 75 kDa. The condition of protease activity from the strain I1-1T in this research were assayed, and the optimal temperature was at 55℃, and optimal pH was at 8.

本研究之菌種來自南臺灣屏東地區四重溪溫泉水中之一株具分解蛋白能力的好氧性嗜熱桿菌,名為Ⅰ1-1T 菌株。Ⅰ1-1T 菌株經培養後菌落顏色呈現半透明淡黃色,菌體形態大小為0.4-0.5μm x 0.8-2.0μm之革蘭氏陰性菌,具單鞭毛運動能力。Ⅰ1-1T 菌株生長溫度範圍為35-60 ℃( 最適55℃),生長pH值範圍為6-10(最適pH7-8)‚生長鹽度範圍為0-0.5%的NaCl。Ⅰ1-1T 菌株經由16S rDNA 序列分析與資料庫比對,Ⅰ1-1T 菌株屬於β-Proteobacteria 綱、Burkholderiable 目、Comamonadaceae 科。經由系統分類學分析,確認Ⅰ1-1T 菌株與Tepidimonas 菌屬中的菌種之16S rDNA 序列相似度,再經由DNA-DNA 雜合反應、生化分析之API 系統、Biolog 系統、抗生素感受性、脂肪酸組成、全細胞蛋白質電泳、G+C content分析比對結果, Ⅰ1-1T 菌株屬於Tepidimonas 菌屬中的另一新種,因此命名為Tepidimonas taiwanesis。 Ⅰ1-1T 菌株與Tepidimonas 菌屬中之二菌種T. ignava 及T. aquatica 之16S rDNA 序列相似度分別為97.2﹪及96.8﹪,而Ⅰ1-1T 菌株與T. ignava 及T. aquatica DNA-DNA 雜合結果,其相似度分別為38.3﹪、40.8﹪。另全細胞蛋白質電泳結果Ⅰ1-1T 菌株與T. ignava 、T. aquatica 之序列相似度分別為92.1﹪、89.8﹪。此外, Ⅰ1-1T 菌株脂肪酸成分以16:0 ( 約41﹪)、18:1 ω7 c ( 約13﹪)和summed feature 3a (16:1ω or 15:0 iso 2OH or both) ( 約26﹪)為主。以Ⅰ1-1T菌株之胞外粗酵素液進行SDS-PAGE電泳分離，證明Ⅰ1-1T菌株具有在鹼性下生產蛋白酶的能力，其蛋白酶分子量約為32、60與75 kDa。本研究菌株粗酵素液的蛋白酶活性最適反應溫度為55℃ ，而最適作用pH值為8之鹼性條件。