由 Bacillus cereus NTU-FC-11 (BCRC 17275) 生產可溶性豬血水解物及其相關酵素

Studies on the soluble porcine blood hydrolysate produced by Bacillus cereus NTU-FC-11 (BCRC 17275) and the related enzyme

林凱建(Kai-Chien Lin)

指導教授 ： 李敏雄

國立臺灣大學/生物資源暨農學院/農業化學研究所/碩士(2004年)

https://doi.org/10.6342/NTU.2004.01829

摘要

本研究自土壤中篩選得到一株豬血塊分解酵素生產菌Bacillus cereus NTU-FC-11 (BCRC 17275)。此菌株具有生產豬血塊分解酵素及酪蛋白酶分解酵素之能力。其最適生產條件為：以500 mL錐形瓶盛裝200 mL含有1 % 糙米基質，於27℃下、120 rpm，培養18小時，可得較高的豬血塊分解酵素活性 (194 U/mL) 及鹼性蛋白酶活性 (256 U/mL)。將上述培養條件獲得之培養液，以凍乾的豬血粉末當親和性吸附劑吸附後，再以10 mM Carbonate buffer pH 10.5將酵素洗出，洗出液經陰離子交換後，可得到比活性為4338 U/mg的豬血塊分解酵素。以SDS-PAGE測得此純化酵素之分子量為39.2 kDa。酵素性質分析結果顯示，此酵素屬於金屬蛋白酶，其最適反應溫度為50℃，最適反應pH值為9.0，在pH 3-9時安定性良好。另外，以豬血塊為基質與此菌株進行發酵生產後所得到的發酵液，離心去除菌體與沉澱物，上清液經分光光度儀全波長偵測，發現深褐色的豬血塊蛋白水解物，最大吸收波長為406 nm。經培養72小時後，對豬血塊蛋白水解物有最大的收率。水解物在pH 4、5和6時，會沉澱析出。利用此特性，可方便且有效率地回收豬血塊蛋白水解物。水解物的性質分析結果發現，其熱安定性很高，且富含鐵質 (由豬血塊之0.18 % 增加為0.35 %)，可作為鐵質補充劑。

關鍵字

鐵質 ； 豬血塊分解酵素

並列摘要

In this study, a strain of bacteria was isolated from soil can hydrolyze porcine blood. This strain possess not only good porcine blood fibrinolytic activity but also good caseinolytic activity. This strain was tentatively identified as Bacillus cereus. We designated this strain as Bacillus cereus NTU-FC-11. The optimal cultural condition of Bacillus cereus NTU-FC-11 for production was found, when the culture was shaken at 27℃ with 120 rpm for 18 hours in 500 mL flask containing 200 mL of medium consisted of 1.0 % brown rice. Under such conditions, procine blood fibrinolytic activity (194 U/mL) and alkaline protease activity (256 U/mL) was attained. The enzyme was purified through affinity adsorption of porcine blood powder and ultrafiltration. A 4338 U/mg of specific activity for procine blood fibrinolytic enzyme was shown. The molecular weight of the enzyme was determined to be 39.2 kDa by SDS-PAGE. The purified enzyme is considered as metalloprotease, its optima temperature and pH are 55℃and pH 9.0, respectively. It could remain 100% of activity at pH 3-9 for 1 hour. The fermented porcine blood hydrolysates was scanned the full wave-length by ultraviolet-visible spectrometry, and the maxima absorbance was observed at 406 nm. After 72 hours of incubation, the production of hydrolysates reach the maxima. Moreover, the hydrolysates become insoluble at the solutions of pH 4, 5 or 6. After adjusting to pH lower than 4 or higher than 6, the precipitates redissolve. The hydrolysates have good thermostability. The pH 5 precipitate can concentrate the iron content of porcine blood from 0.18 % to 0.35 %. By means of these characteristics, the hydrolysates could be recovered from the fermentation broth easily and effectively.

並列關鍵字

fibrinolytic activity ； iron

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