篩選角蛋白酶生產菌及其酵素性質研究

Isolation and characterization of keratinase from microorganisms

沈潔瑩(Chieh-Yin Sheng)

指導教授 ： 謝寶全 ； 鄭作林

國立屏東科技大學/農學院/食品科學系所/碩士(2007年)

https://doi.org/10.6346/NPUST.2007.00039

摘要

本實驗利用羽毛聚集培養方式，從養雞場附近土壤中篩選出可分泌角蛋白酶之菌株，由南投某養雞場的雞糞堆肥中篩選到一株具有生產高角蛋白酶活性之菌株，該菌為革蘭氏陰性之短桿菌，經BIOLOG鑑定系統判定歸屬為綠膿桿菌Pseudomonas aeruginosa。此菌株於三角瓶中以羽毛粉為唯一碳源的培養基進行2天振盪培養，可在其培養液中獲得胞外的角蛋白酶活性，其中以2%的羽毛粉添加具有最佳的角蛋白酶之誘導活性，但是額外醣類添加反而會抑制酵素活性的生成。此酵素的最適反應溫度及酸鹼值分別為60℃及pH 6，粗酵素液在4℃冷藏條件下經20天儲存仍維持有一定的活性，酵素在中性偏鹼的pH環境下具有較佳安定性。粗酵素液經硫酸銨沉澱劃分可回收角蛋白酶，再先後經DEAE陰離子交換樹脂、Sephacryl S-100HR膠體過濾管柱層析純化，最後純化倍率為33.1倍，回收率2.9%，該酵素以azo-keratin為受質，求得其酵素動力學 Km為35 mg/mL，Vmax為714.3 μg/min。純化後之酵素在SDS-PAGE電泳片34.5 kDa處呈現有唯一的蛋白質色帶，另以Sephacryl S-100HR膠體過濾管柱分析其原態酵素的分子量則為64.6 kDa，由此可知Pseudomonas aeruginosa所分泌之角蛋白酶是一種二單元體結構之蛋白質。

關鍵字

角蛋白酶 ； 羽毛 ； 綠膿桿菌

並列摘要

This study was to screen high keratinase producing microorganisms from the soil sample around poultry farm in the whole feather enrichment medium. We have isolated a Gram-negative, rod.shaped bacterial strain from compost sample of the poultry farm in Nantou. The isolate is able to produce high amount keratinase when cultured in medium containing feather and was identified as Pseudomonas aruginnosa by BIOLOG system. This bacterium secreted a high activity extracellular keratinase when cultured on feather meal medium. Enzyme production was best at 2 days of incubation in shaking flask. The maximum enzyme activity was induced by a medium containing 2% feather meal and repressed by extra source of carbohydrates. Optimum temperature and pH of keratinase were 60℃ and pH 6.0, respectively. The enzyme was stable at 4℃ for 20 days and exhibited a better pH stability range from neutral to alkaline condition. The crude keratinase produced by Pseudomonas aruginnosa was punified by ammonium sulfate precipitation, DEAE anion-exchange chromatography, and Sephacryl S-100HR gel filtration chromatography. After the purification; the purification fold was 33.1 and the yield was 2.9%. The Km and Vmax of keratinase were 35 mg/mL and 714.3 μg/min, respectively. The purified enzyme had a molecular weight mass of 34.5 kDa by SDS-PAGE analyze and 64.6 kDa by gel filtration chromatography. The result revealed that keratinase enzyme from Pseudomonas aeruginosa might be a dimmer.

並列關鍵字

keratinase ； feather meal ； Pseudomonas aeruginosa

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