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標題: Streptoverticillium kentuckense CCRC 12429生產之麩醯基轉移純化、生化特性及應用
Purification, biochemical character and application of transglutaminase from Streptoverticillium kentuckense CCRC 12429
作者: 葉雅玟
Yeh, Yea-Wen
關鍵字: transglutaminase;麩醯基轉移;TGase;Streptoverticillium;purification;純化
出版社: 畜產學系
麩醯基轉移(Transglutaminase, TGase)可催化蛋白質分子間或分子內共價鍵的形成,進而改善食品蛋白質的功能性。傳統上,TGase的來源多來自動物組織,但基於經濟上的考量,必須去尋求更便利及經濟的生產來源。本試驗主要的目的即是從10株Streptoverticillium菌株間篩選出最佳的菌株以供生產TGase之用,其中所生產之TGase再經純化並分析其生化特性,以了解其最適作用條件。另外再將TGase添加入肌原纖維中以探討其催化作用,並應用於雞肉肉漿中測定其膠體流變性,以了解其應用時的適當使用量及作用條件。
試驗結果顯示在生產TGase菌株篩選方面,Streptoverticillium kentuckense CCRC 12429能於培養期間之第60小時產生最高的TGase活性-1.924unit/mL,且遠較其它菌株花費時間少;居於時間及產量的考量,本試驗則選定Streptoverticillium kentuckens CCRC 12429作為生產TGase的菌株。另外,在Streptoverticillium kentuckense CCRC 12429生長特性與TGase活性相關性上則發現TGase活性與蛋白質濃度、培養液pH變化、乾物質重及總生菌數於培養前期皆會隨時間的進行,而呈現上升的現象;但至培養後期則TGase活性與蛋白質濃度、培養液pH變化、乾物質重及總生菌數等皆會隨著培養時間的進行,而呈現下降的現象。
Streptoverticillium kentuckense CCRC 12429所生產之粗TGase以55-75﹪硫酸銨沈澱後純化倍率可提高5.19倍,產率為32﹪;再以CM-cellulose陽離子交換色層分析進一步純化可得純化倍率4.81,產率24﹪。
Streptoverticillium kentuckense CCRC 12429 所生產之TGase在pH 7.0時有最佳的pH安定性,其最適反應溫度為45℃,在45℃下的熱失活速率為1.55 ×10-4 sec-1;金屬離子Zn2+、Hg2+會抑制TGase活性,Ba2+、Fe2+、Fe3+、Mn2+及Mg2+雖會對TGase活性造成部分抑制,但仍保有80﹪以上的活性。Ca2+的添加不會對TGase活性造成影響。硫醇試劑-NEM(N-ethylamaleimide)會對TGase活性造成抑制,而還原劑則可促進TGase活性。
Streptoverticillium kentuckense CCRC 12429 TGase對雞胸肉肌原纖維蛋白質之催化作用則可藉由觀察蛋白質電泳而得知,結果顯示37℃作用時,添加粗TGase 0.1unit且須反應30min以上或於10℃作用,則須添加粗TGase 0.3unit且反應1小時以上,才能於電泳圖膠柱之上端觀察到大分子的聚合物。
Streptoverticillium kentuckense CCRC 12429 TGase對雞胸肉肉漿膠體流變性的影響結果則可發現在37 ℃的條件下,作用時間為60 min 時,添加0.5及1.0 ﹪的粗TGase粉末或0.05、0.1及0.3 ﹪初步純化的TGase粉末皆可有效地改善雞胸肉肉漿膠體強度、破斷強度及膠體硬度。但10 ℃的處理則可發現改善效果並不顯著。

Transglutaminase can catalyze the formation of covalent bond between inter- or intra- molecules of protein to improve the function of protein in food. Traditionally, most sources of TGase come from animal organs, but by the economics, it is necessary to look for more convenient and economical method for production. The purpose of this research is to select the most suitable strain from ten Streptoverticillium strains to produce TGase, then purify raw TGase and analyze its biochemical characteristics to obtain the optimal conditions. In addition, the catalysis of chicken myofibril polymerization is carried out as TGase addition. Finally, the chicken paste is treated by TGase to determine the rheological properties of gel to understand the levels and optimal acting conditions.
The results showed that a suitable strain for producing TGase is Streptoverticillium kentuckense CCRC 12429 because the highest TGase activity-1.924 unit/mL was obtained at the 60th hour of culture period, and spend less time than the other bacteria strains. In addition, the correlation between TGase activity and protein concentration, pH of medium, dry matter weight, and total bacterial counts increased as time increasing in the former incubation period, but decreased in the later period,.
After precipitation with 55~75% ammonium sulfate of the crude TGase of Streptoverticillium kentuckense CCRC 12429 producing, the purified multiples increased 5.19 times, and the yield was 32% . Furthermore the samples was purified with CM-cellulose cation-exchange chromatography eleuate multiples increased 4.81 times and obtained 24% yield.
The crude TGase from Streptoverticillium kentuckense CCRC 12429 had the highest pH stability at pH 7.0. The optimal reactive temperature was at 45℃, and thermal inactivative rate was 1.55×10-4 sec-1 at 45℃. The metal ions, Zn2+and Hg2+ inhibited the activity of TGase. Although Ba2+, Fe2+, Fe3+, Mn2+, and Mg2+ inhibited partial activity of TGase, and TGase activity still had 80 %. The adddition of Mg2+ did not affect the activity of TGase. Thiol reagent-NEM (N-ethylamaleimide) inhibited the activity of TGase, but reductant increased the activity of TGase.
The catalysis of Streptoverticillium kentuckense CCRC 12429 TGase on the myofibril proteins of chicken breast meat was analyzed by observing protein electrophoresis. The results showed that the polymers of large molecular were found in the top of SDS-PAGE electrophoretogram when 0.1 unit crude TGase incubated at 37℃ for over 30 min or 0.3 unit crude TGase incubated at 10℃ for over 1hr.
In rheological properties of gel, significantly effective improvement for the gel strength, breaking strength and gel hardness of chicken breast meat paste when 0.5 % and 1.0 % crude TGase powder or 0.05 %, 0.1 %, 0.3 % purified TGase powder incubated at 37℃, but no significant effective improvement when incubated at 10℃.
Appears in Collections:動物科學系

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