Bacillus amyloliquefaciens之角蛋白酶之研究

Abstract

羽毛由角蛋白﹝keratin﹞構成，成分中80%以上為胺基酸，角蛋白不易分解，一般工業上使用之方法有將羽毛磨成粉末作為飼料添加物，或以強酸強鹼，處理廢棄羽毛，以羽毛粉作為添加物有分解速度緩慢、應用範圍少之缺點，以強酸強鹼方式處理羽毛則使成分受破壞因而再利用效率降低之缺點，因此開始有多篇研究針對以生物降解羽毛，使羽毛在自然狀態下分解，並保有其胺基酸結構，提供再利用價值，而部分研究亦指出分解羽毛之酵素角蛋白酶﹝keratinase﹞，除降解羽毛外，尚有其他功能，因此實驗先由環境分離菌株，接著針對菌株產生之酵素活性及其基因功能等進行探討，提供酵素開發之參考。 研究中環境篩選所得Bacillus cereus及Bacillus amyloliquefaciens將其訂為Bacillus cereus Ker103及Bacillus amyloliquefaciens Ker103，以直接培養獲得粗萃蛋白質及大腸桿菌表現系統，兩種方式獲得分解羽毛之酵素，比較酵素生產情況，並測試酵素於不同環境條件下，酵素之活性及穩定性，依據生產方式不同，獲得之酵素純度及數量亦有所差異，此兩菌所產生之羽毛分解酵素均對環境具有良好耐受性及穩定性，在pH 3.0-10.0條件下均保有活性，溫度介於20-70℃之間酵素皆可作用，其中Bacillus cereus Ker103之粗萃酵素在pH 8.0及50℃下活性最佳，Bacillus amyloliquefaciens Ker103粗萃酵素則在pH 7.0及40℃下有最佳活性，以PCR方式選殖酵素基因，Bacillus cereus Ker103分解羽毛之基因vpr，蛋白大小為99 kDa，Bacillus amyloliquefaciens Ker103分解羽毛基因為kerk，分子量為28.8 kDa，從蛋白質變性電泳結果判斷，此兩菌產生之羽毛分解酵素為胞外酵素，其中Vpr會形成內含體而不溶於水，因此不易純化，因此僅以直接培養獲得粗萃酵素，而kerk則以大腸桿菌BL21轉殖株培養於LB液態培養基中，以IPTG誘導，並使用6X His-tag進行純化， kerk純化之效益良好，較易生產純度較高之酵素，經純化後之kerk其最佳酸鹼反應條件為pH 9，在含高濃度鹽類環境中易失活，添加介面活性劑成分PEG-3350及Tween 20具提升酵素活性之情形，因此未來若要進行大量生產，Bacillus amyloliquefciens Ker103之KerK具有開發價值，亦可作為產品中額外添加成分，可再進行進一步探討。

Feather is composed of keratin, with more than 80% amino acid. According to research, keratin is difficult to degrade. Strong acid and basic chemical solvent is used on industry, which will be easily degrade feather. Some factory boil and grind feather into powder, and use it as additive fodder. But there should be more usage by feather. Since 1950, more research study about degrading feather by biodegradation. It is not only a better way to degrade feather but also maintains ingredient and function of feather. And some research also found that feather-degrading enzyme with other function. So we isolated bacterial strain from environment and have cloned a functional gene of keratinase. Then we purified the enzyme to study feature of the enzyme. In our study, after 16s rRNA sequencing, isolated strains were confirmed as Bacillus cereus and Bacillus amyloliquefaciens, and identified as Bacillus cereus Ker103 and Bacillus amyloliquefaciens Ker103. We produced enzyme by directly culture and Escherichia coli BL21 expression system, then analyzed enzyme activity and stability. Between these two methods, purity, productivity and activity was different. Enzymes produced from these two strains are stable and tolerated in different environment. Under pH 3.0-10.0 and 20-70℃, the two enzymes keep their activity and stability. Crude enzyme produced from Bacillus cereus Ker103 indicated the best activities are under incubation condition pH 8.0 and 50℃. The crude enzyme produced from Bacillus amyloliquefaciens Ker103 show the best activity under pH 7.0 and 40℃. We use PCR to detect and clone the enzyme gene. Bacillus cereus Ker103 includes enzyme gene, vpr, with predicted molecular weight 98.9 kDa and Bacillus amyloliquefaciens Ker103 includes enzyme gene, kerk, with predicted molecular weight 28.8 kDa. From the result of SDS-PAGE of these two cultural supernatant, indicated that these two protein are extracellular enzymes. Among these two enzymes, Vpr formed inclusion body, which made it difficult to produce and purify. So we especially try to purify KerK protein by Escherichia coli BL21 expression system to improve its purity. Then we detected enzyme activity, we found that purified enzyme show the best activity under pH 9.0, easily get lost activity under high salt concentration condition. The addition of surfactant Tween 20 and PEG-3350 improve the enzyme activity. We concluded that KerK showed its potential on large scale production and application.