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|Title: ||Bacillus sp. YJ6 聚木醣酶之純化與特性分析|
Purification and Characterization of Xylanase from Bacillus sp. YJ6
|Authors: ||Yen-I Chiang|
|Contributors: ||NTOU:Department of Food Science|
|Keywords: ||Bacillus sp. YJ6;聚木醣酶;純化;生化特性|
Bacillus sp. YJ6;xylanase;purification;bio-properties
|Issue Date: ||2011-06-22T08:46:55Z
|Abstract: ||本研究目的為純化 Bacillus sp. YJ6 聚木醣酶 (xylanase) 並探討其生化特性。菌株接種於培養基後，置於 25oC、150 rpm 振盪培養，第四天可以得到最高聚木醣酶活性為 2.26 U/mL。收集胞外酵素液，經硫酸銨分劃、透析後，以 CM-Sepharose Fast Flow 管柱層析及 sephacryl S-100 HR 管柱層析可得到具有活性之聚木醣酶，其比活性為 1435.98 U/mg，回收率為 3.46%，純化倍率為 678.1 倍。經 SDS-PAGE 電泳分析呈現單一色帶，其分子量為 19 kDa。純化之聚木醣酶最適反應 pH 值為 pH 6.0，在 pH 5.0-9.0 間有較佳安定性，最適反應溫度為 50oC，在 40oC 以下安定性較佳，其活性會受到金屬離子 Hg2+、Cu2+、Fe2+ 及抑制劑 PMSF、TPCK、NEM、Leupeptin 抑制，而金屬離子 K+、Na+、Co2+、Mg2+ 及還原劑 b-mercaptoethanol、glutathione 則會提高其活性。此聚木醣酶僅對櫸木、樺木及燕麥聚木醣有活性，推論此酵素為一內切型聚木醣酶；N 端定序前八個胺基酸序列為 ASTDYWQN，設計引子經聚合酶連鎖反應得到基因全長，此序列具有642 個核甘酸，與其他菌株之聚木醣酶基因序列比較其相似度為 95% 左右，顯示該菌為一新穎的菌株。|
The cells of Bacillus sp. YJ6 were removed by passing through a 0.45 µm membrane after 4 days incubation at 25oC, which had the highest xylanase activity (2.26 U/mL). The xylanase was purified to electrophorectical homogeneity by precipitating at 40-60% saturation of ammonium sulphate, passing through CM-Sepharose FF and Sephacryl S-100 HR chromatographs. About 3.5% of xylanase was recovered and 678.1 purification fold were obtained. The purified xylanase was with a molecular mass of 19 kDa and specific activity of 1435.98 U/mg. It had an optimal pH and temperature at 6.0 and 50oC, respectively, and was stable at pH 5.0-9.0 and below 40oC. This xylanase was inhibited by Cu2+, Fe3+, Hg2+, PMSF, TPCK, NEM and Leupeptin, but activated by K+, Na+, Co2+, Mg2+, b-mercaptoethanol and glutathione. According to substrate specificity, the purified xylanase had high specificity to beechwood, birchwood and oat spelts xylans. The N-terminal sequence of xylanase was ASTDYWQN and used for desiging the primers to amply nucleotide sequence by PCR. This xylanase DNA consisted of 642 nucleotides, encoded 213 amino acid residues and exhibited upward 95% homology compared with 7 strains of Bacillus by BLAST system in NCBI database.
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