Bacillus licheniformis CCRC 14353和Bacillus licheniformis CCRC 11594之粗酵素對雞羽毛水解效果之評估

Abstract

壹、中文摘要 羽毛含90 %以上之角蛋白，而角蛋白因為含硫胺基酸與α螺旋之間複雜的交叉鍵結，使其結構間的鍵結相當穩固，不易為一般蛋白質水解酵素所破壞。本試驗利用Bacillus. licheniformis CCRC 14353和Bacillus. licheniformis CCRC 11594為試驗菌株，測試此二菌株之生長特性並進一步利用其分泌之粗酵素進行羽毛水解試驗，以評估其利用性。 實驗結果顯示在生長特性方面：B. licheniformis CCRC 14353和 B. licheniformis CCRC 11594之菌數於培養的第96小時達最高量，分別為8.94和9.06 log CFU / mL，隨後開始下降，而其酵素活性則隨著菌體的生長而上升，尤其在培養後第132小時達最佳酵素活性，兩者分別為2.14和1.74 unit / mL。培養後第24小時兩者之溶液pH值皆有些微下降之現象，培養至第96小時期間，pH隨培養時間的增加而上升，而培養150小時後其溶液pH分別為pH 7.21和7.29。SDS-PAGE顯示，兩者之粗酵素在30 kDa及31 kDa處有蛋白質之環帶。 CCRC 14353酵素活性之最適pH為pH 7及pH 11，分別為30.59及30.48 unit / g protein；而CCRC 11594之最適pH亦為pH 7及pH 11，酵素活性分別為45.3及42.9 unit / g protein。CCRC 14353酵素最適活性溫度為70℃，為35.12 unit / g protein；而CCRC 11594粗酵素之最適溫度60℃，為65.71 unit / g protein；CCRC 14353酵素對溫度之敏感度較CCRC 11594者為低。 CCRC 143534及CCRC 11594之粗酵素對羽毛水解144小時後，其溶液中之總氮含量皆有顯著的增加；CCRC 11594水解羽毛後之溶液中總氮量較CCRC 14353高；預先經高溫高壓處理之羽毛，水解效果較未處理者佳；pH 11對經高溫高壓處理之1 %羽毛水解效果具有更大之助益。CCRC 143534及CCRC 1159之粗酵素於pH 7溶液中水解羽毛，其溶液pH僅在水解後第24小時呈下降現象，之後隨著時間增加而上升，而於pH 11中作用時，則是隨水解時間的增加，而有下降之現象，但仍保持在鹼性狀態；而經高溫高壓處理過之組別在相同初始pH作用環境水解144小時過程中，其溶液中之pH值皆較未經處理組別之pH值高。兩者之粗酵素於pH 11中水解羽毛144小時之羽毛損失重量皆較pH 7者為高；經高溫高壓處理之羽毛於水解期間之損失重量較未經處理者高，且以1 %組別之羽毛損失重量最為明顯；CCRC 11594處理組別之羽毛損失重量較CCRC 14353者高。 羽毛表面在經過粗酵素分解處理後，皆較處理前有顯著被破壞之現象，尤以預先經高溫高壓處理之羽毛較易為酵素所分解，且以CCRC 11594之效果較CCRC 14353者佳。高溫高壓處理之羽毛經CCRC 14353及CCRC 11594粗酵素水解後，其外觀已看不到羽毛完整的結構，且羽毛溶液的濁度亦明顯升高。

捌、英文摘要 The Evaluation of Hydrolysis of Chicken Feather with Raw Enzyme from Bacillus licheniformis CCRC 14353 and Bacillus licheniformis CCRC 11594 Ting-Rou Chen Abstract Feathers composed of over 90% protein, the main component being keratin, a α-helix and the sulfide amino acid highly cross-linked interaction results in mechanical stability and resistance to proteolyses. The objective of the present study was to evaluate the efficiency of enzymes from Bacillus licheniformis CCRC 14353 and Bacillus licheniformis CCRC 11594 for hydrolyses of chicken feather. The results were as the following : the CFU of B. licheniformis CCRC 14353 and B. licheniformis CCRC 11594 reached the maximum value (8.94 and 9.06 log CFU/mL) for 96 hrs of incubation, respectively. The enzyme activity of the bacteria strains increased with increasing of growth curve, and reached the maximum values which were 1.74 and 2.14 unit/mL for 132 hrs of incubation, respectively. The pH of two bacteria strains were slightly decreased when incubated for 24 hrs, but rose later until incubated for 150 hrs, the pH were 7.21 and 7.29 when determined at the end time in this research, respectively. SDS-PAGE analysis of the raw enzyme from the two bacteria strains revealed that the 30 kDa and 31 kDa protein can be detected. The optimum pH of enzyme activity of CCRC 14353 was pH 7 and pH 11, and the enzyme activity was 30.59 and 30.48 units/g protein, respectively; The optimum pH of enzyme activity of CCRC 11594 was pH 7 and pH 11, and the enzyme activity was 45.3 and 42.9 units/g protein, respectively. The optimum temperatures of enzyme activity of CCRC 14353 and CCRC 11594 were 70 and 60℃, and the enzyme activity was 35.12 and 65.71 units/g protein, individually. After the raw enzymes of CCRC 143534 and CCRC 11594 hydrolyzed chicken feather for 144 hrs, the total nitrogen content was significantly increased in solution when compared to the initial （0 hr）; as well as the total nitrogen content of CCRC 11594 was higher than that of CCRC 14353. The hydrolyzing efficiency of chicken feather treated by high temperature and high pressure was higher than the control. The raw enzymes of CCRC 143534 and CCRC 11594 were used to hydrolyze chicken feather in pH 7 solution, the pH were decreased after hydrolyzing for 24 hrs, and then increased with time. However, the pH of the same treatments in pH 11 solution was decreased with time, and maintained an alkalinity condition during hydrolysis. The loss percentage of chicken feather treated by both raw enzymes in pH 11 solution for 144 hrs were higher than those in pH 7 solution. The loss percentage of chicken feather treated by high temperature and high pressure was higher than the samples without treatment, especially in 1 % feather lot; The loss percentage of chicken feather by raw enzyme of CCRC 11594 was significantly more than that of CCRC 14353. In SEM observation, some chicks and broken particles were found on the surface of chicken feather barbs treated by raw enzymes from the two bacteria strains. Moreover, a distinctly more checks and broken particles were observed when chicken feather per treated by high temperature and pressure then treated by raw enzymes from the two bacteria strains. The appearance of chicken feathers were broken down and resulted in a high turbidity solution when they were treated by raw enzymes from the two bacteria strains for 144 hrs. Overall, conclusion chicken feather treated by raw enzymes from CCRC 11594 had better hydrolysis efficiency than CCRC 14353. On the other hand, a synergism was found when chicken feather per-treated by high temperature and pressure then treated by raw enzymes.