Rapid purification of subtilisin YaB and its protein-engineered mutant enzymes and evaluation of their feasibility as meat tenderizers.

Abstract

Subtilisin YaB為鹼性枯草桿菌YaB (alkalophilic Bacillus YaB) 所分泌生產，此酵素由於對側鏈殘基較小之胺基酸基質特異性高，因此被認為是極具潛力的肉品嫩化酵素。目前已有學者利用蛋白工程改造本酵素的受質特異性，希望能改造出更優良的肉品嫩化劑。本研究以Bacillus DB104操作pEX600A表現鹼性彈性蛋白(wild-type) 及四種定點突變酵素 (G124A、G124V、G159A、G159S)，並評估其實際應用在肉品嫩化時的效果。 這些酵素在工業化大量生產時，需要快速簡便的純化方法。本研究中利用酵素等電點偏鹼性 (pI=10.6) 之特性，以QM-cellulose chromatography ("SEPRASORB" kit) 來純化酵素，最理想條件為先利用超過濾方式濃縮，再以 pH 9.0 之緩衝液系統進行 QM-cellulose SEPRASORB純化。純化倍數可達42 倍，酵素活性 559.7 U/ml。較傳統純化程序有令人滿意的改進，而此系統所得的部份純化酵素已足以利用來探討肉品應用性。 在探討此等突變酵素應用於肉品之條件方面，結果顯示突變酵素之特性無論在pH穩定性、耐熱性、最適反應pH及最適反應溫度並沒有重大改變。由實驗結果我們建議，在一般肉品或調理肉品之 pH或中、低溫 (0 ~ 40℃) 的情況下不論野生或突變酵素均可應用之，而加熱至55℃以上即可停止其反應。 在肉品嫩化效果方面，以這五種野生及突變酵素及市面上常用的肉品嫩化劑papain與bromelain以及elastase與collagenase，共同進行評估。觀察酵素對彈性蛋白、膠原蛋白、酪蛋白及肌肉纖維蛋白質這四種基質的特異性。優良的肉品嫩化酵素應對彈性蛋白及膠原蛋白等組締組織蛋白具高特異性，而對酪蛋白及肌肉纖維蛋白特異性低。結果發現鹼性彈性蛋白較符合優良肉品嫩化劑的特性，而突變酵素基質特異性更優於野生酵素，其中以G159A之水解特性表現最佳。 進一步以酵素直接應用在牛肉之嫩化，觀察經酵素分解後的牛肉。結果顯示鹼性彈性蛋白YaB及其定點突變酵素在肌肉纖維分解上較弱，而其膠原蛋白質的分解能力則優於bromelain且與其餘商業酵素接近。証實鹼性彈性蛋白YaB及其定點突變酵素較一般商業肉品嫩化酵素有更優良之肉品嫩化的特性。並且突變酵素的結果均較wild-type 有提升，顯示以蛋白工程手法所獲得的突變酵素，其基質特異性已被修飾得更適於肉品嫩化的應用。

Subtilisin YaB produced by alkalophilic Bacillus YaB has substrate specificity for small aliphatic amino acids and thus promising as a potential meat tenderizer. Recently, the substrate specificity of the enzyme has been improved by protein-engineering technology. In this study, alkaline elastase (wild-type) and four site-directed mutant enzymes G124A, G124V, G159A and G159S expressed by harboring pEX600A-based vectors in Bacillus DB104 are evaluated for their feasibility as meat tenderizers. To apply these enzymes in industrial, a simple and quick purification method is highly demanded. Here we use ultrafiltration and QM-cellulose SEPRASORB with pH9.0 buffer system to simplify the purification step. The purification fold and specific activity for the purified subtilisin YaB were 42, 559.67 U/ml. It is better than traditional method. This partial purified enzyme from the system we developed can be applied in meat industry directly. The characteristics of these mutant enzymes show no significant change on the properties such as pH stability, thermostability, optimal pH and optimal temperature. From the result, we suggest the wild-type and mutant enzymes can be applied under board pH range from 5-12, temperature range from 0-40℃, simply heating above 55℃ can stop the enzyme activity. To evaluate the properties as meat tenderizer, wild-type, four mutant enzymes, papain, bromelain, elastase and collagenase are tested for their substrate specificity in elastin, collagen, casein and myofibrillar proteins. An ideal meat tenderizer should have high specificity toward elastin and collagen, and low specificity toward casein and myofibrillar protein. The results indicate that wild and mutant alkaline elastases have better properties as meat tenderizer than other commercial meat tenderizers. G159A is the best among all the wild and mutant enzymes. The study in applying the enzyme on beef meat shows that elastase YaB and mutant enzyme are weak in hydrolyzing myofibrillar protein and no significant difference on hydrolyzing collagen. This result implicates that alkaline elastase YaB and its mutant enzymes are better meat tenderizers than commercial meat tenderizers. Mutant enzymes are better than wild-type. The results confirm that mutant enzymes are modified as better meat tenderizers.