耐低溫Serratia屬菌株β1,4-半乳糖??之定性分析與基因選殖

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Title:	耐低溫Serratia屬菌株β1,4-半乳糖??之定性分析與基因選殖
Other Titles:	Characterization and cloning of the β1,4-galactosidase from psychrotrophic Serratia isolate strain D04
Authors:	蘇郁欣 Su, Yu-Hsin
Contributors:	羅能文 Lo, Neng-Wen 東海大學畜產與生物科技學系
Keywords:	耐低溫菌;半乳糖?;?;ONPG;基因選殖 β1;ONPG;Gene cloning;Psychrotrophs;4-galactosidase
Date:	2005
Issue Date:	2011-06-14T03:16:28Z (UTC)
Abstract:	在4℃篩選之耐低溫分離菌株D04，依其16S rRNA序列親源分析推測此菌株為Serratia屬細菌的成員；菌株之型態及生理特性亦支持16S rRNA序列分析結果。對耐低溫Serratia屬菌株D04之β1,4-半乳糖??做生化特性分析，研究結果顯示此酵素最適作用溫度為55℃，溫度升高至75℃則完全失去活性；在pH 7.0環境下則擁有最佳活性，鹼性環境下酵素較不穩定，而在酸性環境下酵素活性變化並不顯著。帶有酵素之細胞震碎液於55℃下，測得水解ONPG之Km值為1.61 mM，最大反應速率Vmax為6.50μmol/min/mg protein；水解PNPG之Km值則為1.49 mM及Vmax值11.01μmol/min/mg protein。對D04菌株之β1,4-半乳糖??做基因選殖所得之核?酸序列進行開放讀架分析，得知序列共有3,051個核?酸，所轉譯成之β1,4-半乳糖??蛋白質與Erwinia carotovora subsp. atroseptica菌最高約有66﹪之相同度。依據糖?水解?特有之功能區胺基酸序列之分析，此酵素與糖?水解?家族之第二家族（Family 2）最為接近。利用基因重組技術所得之D04菌株β1,4-半乳糖??經Ni-NTA親和吸著膠體純化後，可得24.3倍高於細菌萃取液總蛋白質中原酵素含量的純β1,4-半乳糖??，且經純化過之重組蛋白酵素仍保有分解X-gal的能力。 Psychrotrophic strain D04 isolated at 4℃ was subjected to the phylogenetic analysis of the 16S rRNA gene sequence. The result showed that the isolated strain is a member of the genus Serratia. This assignment is consistent with the observation from its morphology and physiological characteristics. Characterization of the β1,4-galactosidase from this psychrotrophic Serratia indicated that the enzyme had an optimal activity at 55℃ and was completely inactive at 75℃. The enzyme also had highest activity at pH 7.0 and was labile to high pH. However, its enzyme activity was not noticeably decreased at low pH values. The observed Km of the enzyme in cell lysate at 55℃ was 1.610 mM and 1.486 mM, and the Vmax was 6.502 μmol/min/mg and 11.013 μmol/min/mg, respectively with ONPG and PNPG as substrate. When the nucleotide sequence obtained by gene cloning for the isolate D04 was subjected to open reading frame analysis, it was determined that the coding sequence of the β1,4-galactosidase gene of isolate D04 was 3051 bp long and encoded a 1016 amino-acid polypeptide, which shared about 66% identity with Erwinia carotovora subsp. Atroseptica. According to the amino acid sequence alignment with glycosyl hydrolases specific domain, the cloned enzyme was classified most likely to the family 2. The recombinantβ1,4-galactosidase purified by Ni-NTA agarose resulted in a 24.3 fold enrichment from total protein in cell lysate. Furthermore, the purified recombinant enzyme retained the capability of hydrolyzing X-gal.
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