Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/50762
標題: Bacillus sp. P-6 中蛋白之生產與性質分析
Production and characterization of the protease from Bacillus sp. P-6.
作者: Lay, Uei-An
賴威安
關鍵字: 蛋白質分解酵素
proteolytic enzyme
蛋白酉每
好氣桿菌
金屬蛋白酉每
protease
Bacillus sp.
metalloprotease
出版社: 食品科學系
摘要: A protease producing bacterium was isolated and identified as Bacillus sp. strain P-6. The optimal condition for protease production was found, when the culture was shaken at 37℃ with 100 rpm for 30 hours in 250 ml Hinton flask containing 50 ml of medium consisted of 0.5 % disodium succinate ( 6 H2O ) , 0.2 % casamino acid, 0.2 % yeast extract, 0.14 % NaH2PO4, 0.44 % K2HPO4, 0.01 % CaCl2 and 0.02 % MgSO4. Under such conditions, protease activity of 342 U/ml was attained. The protease was purified through ultrafiltration, ammonium sulfate fractionation, CM-Sepharose CL-6B ionic exchange chromatography, and Sephacryl S-200 gel filtration. A 9-fold purification of the enzyme over crude enzyme preparation and specific activity of 8066 U/mg was shown. And the recovery of activity was 14 %. The molecular weight of the enzyme was determined to be 35 kDa by SDS-PAGE. The N-terminal amino acid sequence of this protease is VTGTN(Y/V)VGTGIG , and showed a high degree of homology with other neutral proteases belonging to thermolysin superfamily . The protease was found to have a pH optimum at 7.0, a temperature optimum at 60℃ as acting on casein. The protease was stable at ~55℃and pH 8.0-10.0. Among several natural substrates determined, casein was the best substrate. Limited substrate specificity of the protease was observed while the product of casein digested by the protease was analysed with SDS-PAGE.The activity of protease was strongly inhibited by EDTA. The EDTA-inhibited protease was partial reactivated by Co2+, Ca2+, Mg2+, Fe+2.
篩選具蛋白酉每生產能力的菌株,經初步鑑定並命名該菌株為Bacillus sp. P-6。其最適生產條件為: 以250 ml Hinton錐形瓶盛裝50 ml 含有0.5 % 琥珀酸二鈉(含六分子水)、0.2 % Casamino acid、0.2 %酵母抽出物、0.14 % 磷酸二氫鈉、0.44 % 磷酸氫二鉀、0.01 %氯化鈣和0.02 % 硫酸鎂的培養基,於37℃ 100 rpm下,恆溫振盪培養30小時,可得較高蛋白酉每活性( 342 U/ml )的粗酵素液。 將粗酵素液經超過濾濃縮、硫酸銨區分、CM-Sepharose CL-6B陽離子交換層析及Sephacryl S-200膠體過濾層析,得到比活性為8066 U/mg的蛋白酉每,其純化倍數為9倍,而回收率為14 %。以SDS-PAGE測得此蛋白酉每的分子量為35 kDa。分析蛋白酉每之N-端月安基酸序列為VTGTN(Y/V)VGTGIG,顯示此蛋白酉每與熱溶酉每超家系的中性金屬蛋白酉每同源性較高。 此蛋白酉每的最適溫度為60℃,最適pH為7.0,對於蛋白質的水解以酪蛋白的活性最高。在55℃以下以及pH 8.0-10.0之間均可維持較高安定性。分析蛋白酉每催化之酪蛋白水解產物,推測此蛋白酉每具有限制性的基質專一性。蛋白酉每活性受到EDTA強烈的抑制作用。添加Co2+、Ca2+、Mg2+和Fe2+ 於EDTA抑制的蛋白則有部份復活化之效果。
URI: http://hdl.handle.net/11455/50762
Appears in Collections:食品暨應用生物科技學系

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