Pseudomonas sp.TKU008 所生產幾丁質酶之純化與定性

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题名:	Pseudomonas sp.TKU008 所生產幾丁質酶之純化與定性
其它题名:	Purification and characterization of a chitinase from pseudomonas sp.TKU008
作者:	林伯勳;Lin, Po-hsun
贡献者:	淡江大學生命科學研究所碩士班王三郎;Wang, San-lang
关键词:	幾丁質酶;蝦蟹殼粉;Pseudomonas sp.;chitinase:shrimp crab shell powder
日期:	2009
上传时间:	2010-01-11 02:28:54 (UTC+8)
摘要:	本研究以蝦蟹殼粉（SCSP）為 Pseudomonas sp.TKU008 生產蛋白酶與幾丁質酶之唯一碳/氮源，於含有 1% SCSP、0.05% MgSO4．7H2O、0.1% K2HPO4（pH7）之液態培養基搖瓶培養 4 天可得較高酵素活性，將所得發酵上清液進行酵素之純化分離及定性。首先將所得發酵上清液經硫酸銨沉澱濃縮、透析去除鹽類，再經過 DEAE-Sepharose、Phenyl Sepharose 及 Sephacryl S-100 管柱層析等分離步驟，純化出一種幾丁質酶，經 SDS-PAGE 測其分子量為 40 kDa。純化後幾丁質酶之活性回收率為 9%，純化倍數為 24.1倍，比活性為 0.175 U/mg。以懸浮態幾丁質為基質測得幾丁質酶最適pH值、最適反應溫度、pH安定性及熱安定性分別為 pH7、50℃、pH6～8 及 <50℃；幾丁質酶活性受 Cu2+、Mn2+所抑制。由幾丁質酶的液相層析串連式質譜分析得到胺基酸序列片段，序列比對有 26％序列接近 Bacillus cereus 的 chitinase B（GenBank accession number gi 10944304）。 The protease and chitinase producing strain, Pseudomonas sp.TKU008, was isolated from the soil in Taiwan. The shrimp and crab shell powder was used as sole carbon / nitrogen source. The optimized condition for protease and chitinase production were found when the culture was shaken at 30℃ for 4 days in 100mL of medium contain 1% shrimp and crab shell powder （SCSP）, 0.1% K2HPO4 and 0.05% MgSO4．7H2O （pH7）.One chitinase was purified by chromatography procedures of DEAE-sepharose CL-6B, Phenyl Sepharose and Sephacryl S-100. The molecular weight of the chitinase was 40k Da estimated by SDS–PAGE and gel-filtration, respectively. The optimum pH, optimum temperature, pH stability, and thermal stability of the chitinase were pH7, 50°C, pH6～8, and <50°C, respectively. The chitinase was inhibited completely by Mn2+, and Cu2+. The results of peptide mass mapping showed that three tryptic peptides of the chitinase were identical to a chitinase B from Bacillus cereus（GenBank accession number gi 10944304） with 26% sequence coverage.
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