從新分離出的 Serratia spp.分析其分泌性蛋白酶特性

Characterization of Extracellular Proteases from Newly Isolated Serratia spp.

蛋白酶應用非常廣泛，可應用於醫藥、食品（釀造、烘焙、乳酪製造、阿斯巴甜合成）及工業上（清潔劑及皮革業等），故能產生分泌性鹼性蛋白酶（extracellular alkaline proteases） 的微生物是很有潛力應用於工業上，故我們著手於篩選此類的菌種。初步已篩選出三株耐鹼性且能產生高活性的分泌性蛋白酶細菌，將在37℃ 及pH 為7.5 的條件下生產分泌性蛋白酶活性最高的菌株做16SrRNA 序列菌種鑑定，得知其為Serratia spp. 。利用SDS-PAGE 及gelatin zymography 檢測得知此菌培養上清液有兩種蛋白水解帶，其相對應的分子量分別約為45 及50 kDa 。當加入絲胺酸蛋白酶抑制劑（serine protease inhibitors ）PMSF（2 mM） 或TPCK （1 mM） 時，蛋白酶活性喪失超過90% ，故其所產生的分泌性蛋白酶絕大部分屬於絲胺酸蛋白酶。

Proteases are enzymes of central importance due to having a primary position both inphysiological and commercial fields. They are widely used in food (brewing, baking, meat tenderization,dairy industry, synthesis of aspartame), detergent and leather industries. In this study, we screened thealkaline-tolerant bacteria that could produce the extracellular protease(s). Three strains that exhibitedclear zones on skim milk were isolated. One of these strains, which exhibited strongest protease activeityat 37℃ and pH 7.5, was identified as Serratia spp. by 16S rRNA sequence analysis. SDS-PAGE andgelatin zymography revealed that two bands with proteolytic activeity were observed from the culturemedium of Serratia spp. and molecular mass of the two proteolytic bands about 45 and 50 kDa wereestimated, respectively. Enzyme activity was lost > 90% when serine protease inhibitors PMSF (2 mM),or TPCK (1 mM) was added in the assay. These results suggest that most of proteases in supernatant ofSerratia spp. should be the serine proteases.