Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/24544
DC FieldValueLanguage
dc.contributor.advisor劉登城zh_TW
dc.contributor.advisorMing-Tsao Chen , Ph. D.en_US
dc.contributor.advisor陳明造zh_TW
dc.contributor.author葉雅玟zh_TW
dc.contributor.authorYeh, Yea-Wenen_US
dc.date2000zh_TW
dc.date.accessioned2014-06-06T07:23:10Z-
dc.date.available2014-06-06T07:23:10Z-
dc.identifier.urihttp://hdl.handle.net/11455/24544-
dc.description.abstract中文摘要 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。zh_TW
dc.description.abstractAbstract 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℃.en_US
dc.description.tableofcontents目 錄 頁次 壹、中文摘要................................................ 1 貳、前言.................................................... 3 參、文獻檢討................................................ 4 一、麩醯基轉移的催化機制................................ 4 二、麩醯基轉移的分類.................................... 5 (一)分泌型TGase....................................... 5 (二)組織型TGase....................................... 6 (三)凝血因子十三...................................... 7 (四)血球TGase......................................... 9 (五)角質細胞TGase..................................... 9 (六)表皮型TGase....................................... 10 (七)微生物型TGase..................................... 10 1.由鏈黴菌 (Streptomyces)產生的TGase.................. 11 2.由Physarum polycephalum產生的TGase.................. 15 3.由Bacillus subtilis產生的TGase...................... 16 (八)其他來源的TGase................................... 17 三、TGase於食品加工上的應用............................... 19 (一)肉類製品.......................................... 20 (二)魚肉製品.......................................... 21 (三)乳製品............................................ 23 (四)植物性蛋白質製品.................................. 24 四、TGase的未來利用性..................................... 26 肆、材料與方法.............................................. 27 一、菌株之培養及特性的測定................................ 27 (一)菌株之培養及篩選.................................... 27 (二)乾物質重............................................ 30 (三) pH................................................. 30 (四)總生菌數............................................ 30 (五)蛋白質濃度.......................................... 31 (六) TGase活性.......................................... 32 二、TGase粗酵素液之製備及純化............................. 33 (一) TGase粗酵素液的製備................................ 33 (二) TGase的純化........................................ 33 (三)蛋白質SDS-PAGE電泳分析.............................. 34 (四)TGase生化特性的分析................................. 38 1.酸鹼度安定性......................................... 38 2.最適反應溫度......................................... 38 3金屬離子對TGase活性之影響............................. 39 4.抑制劑對TGase活性的影響.............................. 39 5.還原劑對TGase活性之影響.............................. 39 6.熱失活速率........................................... 40 三、S. kentuckense CCRC 12429所生產之TGase 對雞胸肉肌原纖維蛋 白質的作用.................................................. 41 (一)雞胸肉肌原纖維蛋白(myofibrillar protein)的製備...... 41 (二)肌原纖維蛋白質之萃取步驟............................ 41 (三)TGase與肌原纖維蛋白質之作用......................... 42 四、S. kentuckense CCRC 12429所生產之TGase對雞胸肉肉漿流變性的影響........................................................ 43 (一)雞胸肉肉漿的製作.................................... 43 (二)TGase的添加......................................... 43 (三)流變性的測定........................................ 44 伍、結果與討論.............................................. 45 一、菌株的選擇培養及特性.................................. 45 二、S. kentuckense CCRC 12429之生長特性及TGase活性........ 51 三、S. kentuckense CCRC 12429TGase粗酵素液之純化及其生化特 性...........................................................56 (一) TGase的純化........................................ 56 (二) TGase生化特性...................................... 60 1. pH對TGase活性的影響................................ 60 2. TGase之最適反應溫度................................ 62 3. S. kentuckense CCRC 12429TGase的熱失活速率......... 62 4.金屬離子對TGase活性的影響........................... 70 5.抑制劑對TGase活性的影響............................. 72 6.還原劑對TGase活性的影響............................. 72 四、S. kentuckense CCRC 12429 TGase對雞胸肉肌原纖維蛋白質的作用.......................................................... 75 五、S. kentuckense CCRC 12429TGase對雞胸肉肉漿膠體流變性的影響.......................................................... 87 陸、結 論.................................................. 97 柒、參考文獻................................................ 99 捌、英文摘要............................................... 112zh_TW
dc.language.isoen_USzh_TW
dc.publisher畜產學系zh_TW
dc.subjecttransglutaminaseen_US
dc.subject麩醯基轉移zh_TW
dc.subjectTGaseen_US
dc.subjectStreptoverticilliumen_US
dc.subjectpurificationen_US
dc.subject純化zh_TW
dc.titleStreptoverticillium kentuckense CCRC 12429生產之麩醯基轉移純化、生化特性及應用zh_TW
dc.titlePurification, biochemical character and application of transglutaminase from Streptoverticillium kentuckense CCRC 12429en_US
dc.typeThesis and Dissertationzh_TW
item.grantfulltextnone-
item.fulltextno fulltext-
item.cerifentitytypePublications-
item.languageiso639-1en_US-
item.openairecristypehttp://purl.org/coar/resource_type/c_18cf-
item.openairetypeThesis and Dissertation-
Appears in Collections:動物科學系
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