生薑蛋白酶之製備與生化特性之探討

Abstract

生薑 (Zingiber officinale roscoe) 源自於東南亞地區，可當作香料、藥品及化妝品等。生薑中含有生薑蛋白酶，使用在肉類嫩化上，但應用在凝乳之研究上甚少，因此本實驗藉由萃取及純化生薑蛋白酶，探討其之生化特性。 生薑榨汁後可取得薑汁，經過濾後分為兩組，一組添加0.2% L-抗壞血酸，另一組則不添加。薑汁於4℃離心，過濾後濾液為新鮮薑汁。在新鮮薑汁中緩慢加入3倍 (w/v) 冰丙酮，經由攪拌後於4℃離心，移除上清液，可得丙酮沉澱之生薑蛋白酶。丙酮沉澱之生薑蛋白酶有兩組，一組由新鮮薑汁進行丙酮沉澱為FA，另ㄧ組由添加0.2% L-抗壞血酸之新鮮薑汁進行丙酮沉澱為VA。將丙酮沉澱之生薑蛋白酶溶在 50 mM MES buffer (pH 6.0)，於4℃離心，取上清液置入DEAE FF陰離子交換管柱進行純化。 無添加L-抗壞血酸之新鮮薑汁，凝乳活性與蛋白質分解活性於4℃下只能維持24小時；當添加0.2% L-抗壞血酸，於4℃貯存6天期間可以維持凝乳活性與蛋白質分解活性。新鮮薑汁及丙酮沉澱之生薑蛋白酶溶液在-80℃貯存約兩個月期間，可維持凝乳活性與蛋白質分解活性。丙酮沉澱之生薑蛋白酶溶液進行DEAE FF陰離子交換管柱進行純化，有兩個蛋白質分劃P1及P2。凝乳比活性與蛋白質分解比活性方面，P1會顯著大於P2 (P＜0.05)。半胱胺酸蛋白酶抑制劑E64、leupeptin及iodoacetic acid對FA-P1、VA-P1、FA-P2與VA-P2均有顯著性之抑制效果 (P＜0.05)，尤其iodoacetic acid抑制效果最好，因此可推測P1及P2屬於半胱胺酸蛋白酶。丙酮沉澱之生薑蛋白酶經純化收集蛋白質分劃P1及P2，P1在分子量為82 KDa具有蛋白質分解活性，P2在分子量為62 KDa及82 KDa具有蛋白質分解活性，均會分解α、β及κ-酪蛋白。 綜合上述，由實驗結果可得知生薑蛋白酶之生化特性，日後可提供於製作凝乳製品之參考，增加多元化乳製品開發及提高附加價值。

Ginger rhizome (Zingiber officinale roscoe), originated from Southeast Asia, has been used as ingredient for the making of spices, herb medicine and cosmetics. It contains the ginger proteases and has the proteolytic activity. Ginger proteases are found in the use in meat tendering, but rarely used in milk clotting before. The purpose of this study was to purify ginger proteases, and to conduct research for its biochemical characteristics. Ginger rhizome was crushed in a juicer and some juice was obtained. The juice was filterd and divided into two parts, one was with 0.2% L-ascorbic acid, and the other one without L-ascorbic acid. The ginger juice was centrifuged at 4℃, and then filterd to obtain the fresh ginger juice. The fresh ginger juice was then slowly added 3 parts (w/v) of cold acetone. The solution was stirred and precipitated by centrifugation at 4℃. The supernatant was removed, and the acetone precipitate contained ginger proteases. The acetone precipitate from the fresh ginger juice was FA, and the acetone precipitate from the fresh ginger juice with 0.2% L-ascorbic acid was VA. The acetone precipitate was dissolved in 0.05 M MES buffer (pH 6.0) and purified by FPLC on a DEAE FF ion-exchange chromatography column. Controlling at 4℃, the milk clotting activity (MCA) and the proteolytic activity (PA) were maintained for 24 hours. MCA and PA of the fresh ginger juice with 0.2% L-ascorbic acid were stored stably for six days at 4℃.Controlling at -80℃ for two months, MCA and PA of the fresh ginger juice and acetone precipitate were stable. The ginger proteases of the acetone precipitate were separated on a DEAE FF ion-exchange chromatography column into two fractions, P1 and P2. The specific activity of MCA and PA of P1 was found higher than P2 (P＜0.05). The protease activity of FA-P1, VA-P1, FA-P2 and VA-P2 were significantly inhibited by E-64, leupeptin and iodoacetic acid (P＜0.05), especially in iodoacetic acid. P1 and P2 were inhibited by E-64, leupeptin and iodoacetic acid which belonged to cysteine protease inhibitors so they might be cysteine protease. The protein of P1 that had protetolytic activity was 82 KDa molecular weight. The proteins of P2 that had protetolytic activity were 62 and 82 KDa molecular weight. P1 and P2 could digested α, β and κ-casein. The data described above provides the biochemical characteristics of ginger proteases. It will provide useful information for making milk curd products in Taiwan and increase diversity and additional value of milk products.