Bacillus mycoides TKU038所生產之幾丁聚醣酶之純化、定性與應用

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Title:	Bacillus mycoides TKU038所生產之幾丁聚醣酶之純化、定性與應用
Other Titles:	Purification, characterization, and application of a chitosanase from Bacillus mycoides TKU038
Authors:	陳威廷;Chen, Wei-Ting
Contributors:	淡江大學化學學系碩士班王三郎
Keywords:	烏賊軟骨;幾丁聚醣酶;幾丁寡醣;抗發炎;Bacillus mycoides;squid pen;chitosanase;chitooligomers;anti-inflammatory
Date:	2016
Issue Date:	2017-08-24 23:38:44 (UTC+8)
Abstract:	本篇研究目的在工業和廢棄處理當中來生產新穎幾丁聚醣酶。將幾丁殼素之廢棄物轉換成生物活性幾丁寡醣 (COS) ，幾丁聚醣酶是最具有潛力的應用之一。幾丁聚醣酶生產菌株篩選於台灣土壤，以0.5%烏賊軟骨粉為唯一碳/氮源，經鑑定為Bacillus mycoides。酵素從培養上清液經管住層析等步驟分離。胞外幾丁聚醣酶純化倍率為130倍與活性回收率35 %，測得分子量約為48 kDa。純化之幾丁聚醣酶最適反應溫度為50 °C，pH為6、10，熱安定性為30-50 °C，pH安定性為4-10。幾丁聚醣酶活性受到Cu2+, Ba2+, Zn2+, Fe2+, Mn2+, EDTA 及PMSF所抑制，其Km 及 Vmax分別為0.098 mg/mL 及 1.336 U/mL。 HPLC及MALDI-TOF MS之組合結果分析，酵素水解水溶性幾丁聚醣所獲得之幾丁寡醣，其TKU038包括具有多種聚合度 (DP) ，範圍3-9。TKU038之發酵上清液與幾丁寡醣混合物具有DPPH·自由基清除活性。高聚合度之幾丁寡醣 DPPH·自由基清除活性較低聚合度之幾丁寡醣強。而幾丁寡醣也有抗發炎之活性與促進L. paracasei TKU010 及L. paracasei TKU012之生長。TKU038對於幾丁寡醣之生產在SPP廢棄處理與工業上有潛在的應用。 The objectives of this investigation were to produce a novel chitosanase for application in industries and waste treatment. The conversion of chitinous biowaste into bioactive chitooligomers (COS) is one of the most promising applications of chitosanase. A chitosanase-producing strain was isolated form Taiwan soil and identified as Bacillus mycoides. The chitosanase was produced using 0.5% (w/v) squid pen powder (SPP) as the sole carbon/nitrogen source, and these enzymes was purified from the culture supernatant by column chromatography. Extracellular chitosanase was purified to 130-fold with a 35% yield, and its molecular mass was approximately 48 kDa. The purified chitosanase exhibited optimum activity at 50 °C, pH 6, 10 and was stable at 30-50 °C, pH 4-10. The chitosanase was significantly inhibited by Cu2+, Ba2+, Zn2+, Fe2+, Mn2+, EDTA and PMSF. The value of Km and Vmax for chitosanase were 0.098 mg/mL and 1.336 U/mL, respectively. A combination of the HPLC and MALDI-TOF MS results showed that the chitosan oligosaccharides obtained from the hydrolysis of water-soluble chitosan by TKU038 comprise oligomers with various degrees of polymerization (DP), ranging from 3 to 9. The TKU038 culture supernatant and COS mixture exhibited 2, 2-diphenyl-1-picrylhydrazyl (DPPH·) scavenging activities. The COS with high DP exhibited enhanced DPPH· radical scavenging compared with COS with low DP. The COS also had anti-inflammatory activity and enhanced the growth of L. paracasei TKU010 and L. paracasei TKU012. TKU038 has potential applications in SPP waste treatment and industries for COS production as a medical prebiotic.
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