豬毛篩選菌Bacillus cereus H10角蛋白酶及蛋白酶之純化、特性與應用之研究

Abstract

本研究冀由豬毛上篩選一株具高角蛋白及蛋白水解能力的菌株，確認菌株生產酵素的最佳活性表現條件及其基本特性，應用於水解台灣大宗的事業廢棄物 - 豬毛，以取代目前的焚燒處理造成環境污染及能源耗費，同時探討其用於飼料添加之營養價值。研究中先篩選出 32 株具豬毛水解能力之菌株，以呈色法測定各菌株水解偶氮基酪蛋白 (azocasein) 及偶氮基角蛋白 (azokeratin) 之能力，藉統計分析決定試驗菌株後，利用變性梯度膠體電泳法 (denaturing gradient gel electrophoresis, DGGE) 及定序確認菌株身分為 Bacillus cereus H10。 由菌數、pH 值、蛋白分解酶比活性、角蛋白分解酶比活性及可溶性蛋白量決定菌株酵素表現最佳天數為培養第三日，接著利用離子交換樹脂管柱及快速蛋白質液相層析系統純化得到一種蛋白酶及角蛋白酶，分子量分別約為 102.3 及 44.8 kDa。針對溫度及 pH 值兩項因子對酵素活性表現的探討中可發現，角蛋白酶在 50∼90 °C 及 pH 5.0 的條件下表現良好活性，然而，蛋白酶則在 50 °C 及 pH 6.0~8.0 有最佳的活性表現，為了使蛋白酶及角蛋白酶同時有最佳的活性表現，利用反應曲面法 (response surface methodology, RSM) 及序列二次規劃法 (sequential quadratic programming, SQP) 尋求最佳酵素活性表現，為 pH 7.57及59 °C 時可得到最高的蛋白酶活性及角蛋白酶活性。在生化特性分析部分，利用蛋白質抑制劑確認該菌株生產蛋白酶及角蛋白酶皆屬於金屬型蛋白酶 (metalloprotease)。在變性劑、有機溶劑及還原劑中皆可維持穩定活性。添加二硫蘇糖醇 (DTT) 和巰基乙醇 (β-mercaptoethanol) 後蛋白分解活性皆降低，而角蛋白分解活性，在 β-mercaptoethanol 的處理後有些微的增加；但 DTT 的添加則降低其活性表現。Ca2+ 和Zn2+ 的添加，皆使蛋白和角蛋白分解活性降低，而添加 Fe3+ 則提升角蛋白酶的活性表現。耐熱性測試中，蛋白分解活性在 59、70 或 80 °C 作用 120 分鐘，仍維持 30~40% 活性，而角蛋白分解活性在三種溫度作用 120 分鐘皆可維持 95% 活性。 就 B. cereus H10 角蛋白酶之應用面進行探討，由褐藻酸鈉及氯化鈣膠囊化之酵素，蛋白分解活性在 59 °C 下有提高，70 °C 無明顯改變，80 °C 作用 120 分鐘則有降低的情況；角蛋白分解活性在三種溫度無差異。將膠囊化酵素重複試驗六次後，蛋白分解活性仍可維持 40~60%，而角蛋白分解活性則可維持 80%。若同時比較不同來源之蛋白質水解時，以豬毛粉水解效果最佳 (70%)，與大豆粕水解情形相似，豬毛及羽毛粉較差 (30%)。與 Proteinase K 相比，B. cereus H10 生產酵素的水解效果亦高出約兩倍左右。分析其水解產物之胺基酸組成量，B. cereus H10 酵素處理組皆顯著高於未添加酵素處理組。若利用必需胺基酸計算其化學積分，以酪胺酸和苯基丙胺酸為第一限制胺基酸，離胺酸是第二限制胺基酸，異白胺酸是第三限制胺基酸。當考量豬隻及家禽之理想蛋白質組成，幾乎所有必需胺基酸皆可符合需求，因此，未來若要供飼料應用，可搭配較高量離胺酸的飼料以符合動物生長所需。

Keratin, which is increasingly accumulating in the environment mainly in the form of feathers and hair, becomes a part of solid waste management. However, keratin is difficult to degrade due to its highly rigid structure rendered by extensive disulfide bonds and cross-linkages. There is a demand for developing alternatives for recycling of such wastes. Thus, the purpose of this study was to purify and characterize a keratinase secreted by a high keratinolytic bacterium isolated from hog hair. In addition, the potential for utilization of the hydrolyzates of hog hair and hog hair meal as ingredients in animal feed was also evaluated. Isolation results indicated that a bacterium designated H10 from hog hair showed high keratinolytic and proteolytic activities. Further identified by PCR-DGGE and 16S rRNA sequencing, H10 was placed in a cluster with Bacillus cereus. The unique of this strain is comprised two types of extra-cellular proteins: proteolytic and keratinolytic. After purification of these enzymes secreted by B. cereus H10 using ion exchange chromatography, the specific activity of the finally purified enzyme for proteolysis and keratinolysis was 29080 and 6391.25 U/mg, respectively. The molecular mass of keratinase and protease were 45.4 kDa and 102.3 kDa, respectively. The enzyme sample possessed two proteases which displayed a very different active temperature and pH range for proteolysis and keratinolysis. Response surface methodology (RSM) combining with sequential quadratic programming (SQP) helps in evaluating the effect factors and in building models. Thus, RSM with SQP was used in the following work to develop a prediction model for the optimal pH and temperature for the enzyme sample from B. cereus H10. Optimization results indicated that the optimal pH and temperature were at pH 7.57 and 59 °C. The results on the biochemical properties showed that both proteases belonged to the metalloproteases and were stable toward solvents, detergents and reduced agents. Among the metal ions, calcium and zinc ion showed an inhibition for enzyme activities, but the keratinase was activated by ferric ion. On the keratinolytic activity, the enzyme displayed stability at 59, 70 and 80 °C, with more than 95% of its initial activity up to 2hr, whereas, the proteolytic activity only remained 30~40% activity. Evaluating the possible applications of this enzyme, immobilized enzymes could stand higher temperature than free enzymes on both activities. After 6 times usages, the immobilized enzymes displayed stability with more than 80% of its initial activity, whereas, the proteolytic activity also remained 40~60% activity. Testing the protease activity on various protein substrates indicated this enzyme sample could easily hydrolyze hog hair meal which showed no significant difference with hydrolyzing soy bean meal. However, it displayed a relatively low digestibility for feather meal and hog hair. Further comparison with proteinase K, the hydrolyzation of hog hair by B. cereus H10 enzyme was 2-fold higher than that of proteinase K. Nutrition improvement test proved that hydrolyzates of hog hair and hog hair meal might have potential for utilization as ingredients in animal feed. In conclusion, this enzyme sample is very effective in hog hair degradation and shows the high thermal stability, presenting potential use for biotechnological processes involving keratin hydrolysis. To the best of our knowledge, this is the first report on isolation and purification of keratinase and protease from hog hair and cooperation of these two enzymes resulting in the effective degradation of keratin.