Bacillus cereus TKU027所生產幾丁質酶、幾丁聚醣酶及蛋白酶之純化、定性與應用

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Title:	Bacillus cereus TKU027所生產幾丁質酶、幾丁聚醣酶及蛋白酶之純化、定性與應用
Other Titles:	Purification and characterization of a chitinase, a chitosanase and a protease from Bacillus cereus TKU027 and their applications Bacillus cereus TKU027所生產幾丁質酶幾丁聚醣酶及蛋白酶之純化定性與應用
Authors:	劉金佩;Liu, Chin-Pei
Contributors:	淡江大學化學學系碩士班王三郎;Wang, San-Lang
Keywords:	仙人掌桿菌;幾丁質酶;幾丁聚醣酶;蛋白酶;Bacillus cereus;chitinase;chitosanase;protease
Date:	2011
Issue Date:	2011-12-28 18:08:10 (UTC+8)
Abstract:	菌株TKU027 係以蝦頭粉為唯一碳/氮源，篩選自台灣彰化土壤之幾丁質酶、幾丁聚醣酶及蛋白酶生產菌，經鑑定為 Bacillus cereus。幾丁質酶與蛋白酶較適生產條件為，分別於含有 1% 蝦頭粉末、0.1% K2HPO4、0.05% MgSO4．7H2O 之50 mL和100 mL 液態培養基（pH 6），於 37℃ 搖瓶（150 rpm）培養 2 天。將所得發酵液經離心、硫酸銨沉澱，以及DEAE-Sepharose與Sephacryl S-100 層析等步驟，純化出幾丁質酶CHI與幾丁聚醣酶CHS，經SDS-PAGE分別測定分子量為 65 kDa 及 63 kDa。CHI及CHS之最適反應pH、最適反應溫度、pH安定性、熱安定性分別為: pH 6、50℃、pH 5 – 8、< 40℃ 和 pH 6、60℃、pH 3 – 10、< 50℃。CHI 活性會受 Mn2+ 及PMSF 所抑制；CHS 活性則會受 Cu2+、Mn2+ 及EDTA 所抑制。利用B. cereus TKU027 較適培養條件，將所得發酵上清液添加，分別可促進L. paracasei TKU012生長達 175% 、L. paracasei 12193 生長達144% 與 L. kefir 14011 生長達 132% 之影響。另外，利用製備自上清液的粗酵素液，水解水溶性幾丁聚醣後所得之寡醣，亦可促進 L. paracasei 12193 生長達 324% 與 L. kefir 14011 生長達 174% 之影響。此外利用PCR-DGGE技術，探討添加 B. cereus TKU027 與蝦頭粉，於淡水紅樹林土壤之菌相變化與生物降解，並於培養 5 週有最高總糖量（3511 µg/g soil）及還原糖量（1442 µg/g soil），提高總生菌數至 6 × 107 CFU/g soil。 The chitinase, chitosanase and protease-producing strain TKU027 was isolated from soil in Changhua of Taiwan with shrimp head powder as the sole carbon/nitrogen source and identified as Bacillus cereus. The optimized culture conditions for chitinase and protease production were found to be shaken at 37℃ for 2 days in 50 mL and 100 mL of medium containing 1% shrimp head powder (SHP) , 0.1% K2HPO4 and 0.05% MgSO4．7H2O (pH 6). A chitinase CHI and a chitosanase CHS were purified from the culture supernatant by ammonium sulfate precipitation, DEAE-Sepharose and Sephacryl S-100. The molecular masses of CHI and CHS determined by SDS-PAGE were approximately 65 and 63 kDa, respectively. The optimum pH, optimum temperature, pH stability, and thermal stability of CHI and CHS were pH 6, 50℃, pH 5–8, <40℃ and pH 6, 60℃, pH 3–10, <50℃, respectively. The chitinase activity was inhibited by Mn2+ and PMSF. The chitosanase activity was inhibited by Cu2+, Mn2+ and EDTA. The culture supernatant obtained from B. cereus TKU027 in the optimized culture conditions enhanced the growth of L. paracasei TKU012 most obviously up to 175%, followed by L. paracasei 12193 up to 144% and L. kefir 14011 up to 132%. The crude enzyme from B. cereus TKU027 culture supernatant hydrolyzed water soluble chitosan to produce N-acetyl chitooligosaccharides. The N-acetyl chitooligosaccharides also exhibited activity of enhancing growth for L. paracasei 12193 up to 324% and L. kefir 14011 up to 174%. In addition, B. cereus TKU027 and SHP were added in soil respectively to investigate the biodegradation of SHP and change of bacteria flora by PCR-DGGE analysis. The highest reducing sugar（1442 µg/g soil）, total sugar（3511 µg/g soil）and total viable cell counts（6 × 107 CFU/g soil）were found at the 5th week incubation with B. cereus TKU027 and SHP in Tamsui mangrove river soil.
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