TWI654304B - 具提升酶活性的溶菌酶 - Google Patents
具提升酶活性的溶菌酶Info
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- TWI654304B TWI654304B TW106140742A TW106140742A TWI654304B TW I654304 B TWI654304 B TW I654304B TW 106140742 A TW106140742 A TW 106140742A TW 106140742 A TW106140742 A TW 106140742A TW I654304 B TWI654304 B TW I654304B
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- lysozyme
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- C—CHEMISTRY; METALLURGY
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2462—Lysozyme (3.2.1.17)
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- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01017—Lysozyme (3.2.1.17)
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Abstract
本案係關於一種具提升酶活性的溶菌酶,其胺基酸序列係為將序列編號2第166個位置的組胺酸突變成賴胺酸的序列。
Description
本案係關於一種溶菌酶,尤指一種具提升酶活性的溶菌酶。
溶菌酶(Lysozyme, EC 3.2.1.17)是一種作用於微生物細胞壁的水解酶,又稱為胞壁質酶。它能有效地水解細菌細胞壁的肽聚糖(peptidoglycan),其機制主要是透過水解N-乙醯胞壁酸(N-acetylmuramic acid)和N-乙醯葡萄糖胺(N-acetylglucosamine)之間的b-1,4糖苷鍵,使肽聚糖骨架結構斷裂後造成細胞壁破裂, 最終導致細菌溶解。溶菌酶廣泛存在於自然界中,像是哺乳動物的眼淚、唾液、鼻涕、組織及鳥類和家禽的蛋清中。依其不同來源,溶菌酶可分為六大類:分別為雞型(C-type)、鵝型(G-type)、無脊椎動物型(I-type)、噬菌體型(Phage-type)、植物型及細菌型。溶菌酶是一種無毒、且對人和哺乳動物無副作用的蛋白質,因其具有溶菌的特性,近年來也已被廣泛地應用於不同工業上。它在乳製品工業中可當作天然防腐劑,例如在巴氏殺菌奶添加溶菌酶能有效地延長其保存期。在食品應用上,可延長水產品及肉食品的儲存時間。而在動物飼料工業上能提高動物的生產性能。研究結果顯示,在仔豬飼料中額外添加溶菌酶可以提高動物對飼料的轉換率及降低腹瀉率,改善仔豬健康並減少抗生素的使用。
近年來由於全球抗生素藥物的濫用,越來越多的細菌發展成抗藥性的菌株,許多國家已開始禁止在畜禽飼料中添加抗生素,因此科學家們正努力尋找能夠替代傳統抗生素的方案,而使得溶菌酶的研究受到越來越廣泛的重視。目前許多研究不論是從大自然中篩選新基因或是改造現有的酶,都企圖想要得到能滿足各種工業應用的溶菌酶。在許多改造酶的策略中,根據酶蛋白結構分析,進而邏輯性地設計突變點是改造酶的主要方法之一。而在此策略中提升酶蛋白的活性也是改良工業酶的一大重點,酶活性愈高就代表成本的下降以及利潤的提高,也較有利於工業應用。
本案即欲藉由邏輯性地設計突變來提升溶菌酶的活性,進而增加其在工業上的應用價值。
本案之目的在於改造現有溶菌酶,利用結構分析及點突變技術,以有效提升溶菌酶的活性,進而增加溶菌酶的工業應用價值。
為達上述目的,本案之一較廣義實施態樣為提供一種溶菌酶,其胺基酸序列係為將序列編號2第166個位置或與序列編號2具有80%以上序列相似度的序列中對應位置的組胺酸突變成賴胺酸的序列。
在一實施例中,編碼該序列編號2的基因係從美洲鴕鳥(Rhea americana)的蛋清中所分離出來的Rham基因。
在一實施例中,該溶菌酶係為一鵝型溶菌酶。
在一實施例中,該溶菌酶的胺基酸序列如序列編號5所示。
本案之另一較廣義實施態樣為提供一種編碼前述溶菌酶之核酸分子。
本案之又一較廣義實施態樣為提供一種重組質體,其係包含前述核酸分子。
體現本案特徵與優點的一些典型實施例將在後段的說明中詳細敘述。應理解的是本案能夠在不同的態樣上具有各種的變化,其皆不脫離本案的範圍,且其中的說明及圖式在本質上係當作說明之用,而非用以限制本案。
本案的溶菌酶基因Rham是從美洲鴕鳥(Rhea americana,又名三趾鴕鳥)的蛋清(egg white)中所分離出來的,其蛋白特性為高耐熱性及耐酸性,屬於鵝型溶菌酶。近期發現它可以大量地表達在工業上常用的畢赤酵母(
Pichia pastoris)中,具有潛在的工業應用價值。為了深入研究以及進一步地改造溶菌酶Rham,本案分析其蛋白結構後針對其活性區域具有關鍵特性的四十多個胺基酸來做修改。第1圖顯示溶菌酶Rham的蛋白立體結構圖。根據蛋白結構重疊比對的結果,Rham蛋白序列第166個位置的組胺酸(H166)位於蛋白立體結構中的活性區內,故被選擇作為進行點突變(site-directed mutagenesis)的位置之一,且發現針對H166來進行點突變能提升溶菌酶活性。其它的突變沒有增強活性的效果,在此即不再贅述。
以下將詳述本案改造溶菌酶之方法及其所得到之改良溶菌酶。
第2圖顯示野生型溶菌酶的核苷酸序列以及胺基酸序列。如第2圖所示,野生型溶菌酶Rham基因包含558個鹼基(含終止密碼子,核苷酸序列以序列編號1標示),且編碼185個胺基酸(胺基酸序列以序列編號2標示)。首先,將Rham基因利用
EcoRI以及
NotI限制酶切位構築到pPICZaA載體中,再將該重組質體轉形入一勝任細胞(competent cell)中,形成一野生型表達載體。
為了提高溶菌酶Rham之活性,本案利用點突變技術以野生溶菌酶基因做為模版進行聚合酶連鎖反應(polymerase chain reaction, PCR)步驟,當中所用的突變引子列於第3圖,其引子序列以序列編號3標示。原始的模板DNA則利用
DpnI移除掉。接著把突變質體送入大腸桿菌勝任細胞中以抗生素(Zeocin)作初步篩選,並藉由DNA定序步驟確認成功突變基因。在此,本案構築了一個突變株H166K,其中H166K意指為溶菌酶Rham第166個位置的胺基酸由組胺酸(histidine)突變成賴胺酸(lysine)。第4圖即顯示H166K突變型溶菌酶的核苷酸序列以及胺基酸序列,其中核苷酸序列以序列編號4標示,胺基酸序列以序列編號5標示。
之後在畢赤酵母中表現溶菌酶重組基因。先將溶菌酶之野生型及突變型的重組基因利用
PmeI 把DNA質體進行線性化之後,藉由電轉步驟將DNA送入畢赤酵母內。接著將菌液塗於含有100 µg/ml Zeocin抗生素的YPD培養基於30
oC下進行培養兩天。再挑選菌落並接種到5 ml YPD於30
oC下培養,再次接種到50 ml BMGY中於30
oC下培養至隔天。接著,將培養基換成含有0.5%甲醇的 20 ml BMMY來誘導蛋白表達,同樣培養於30
oC下。每隔24小時取樣並補充0.5%甲醇。在經過4天的蛋白質誘導表現之後,將菌液以3500 rpm離心並收集上清液,進而檢測溶菌酶活性。
溶菌酶的活性測試方式是參考先前文獻方法再經過修改而成。在本案中是以溶壁微球菌(
Micrococcus lysodeikticus)為底物,並利用濁度法來測定溶菌酶活性。首先將溶壁微球菌粉末以pH 6.2的磷酸二氫鉀緩衝液(66 mM)配製成一定濃度(A
450=0.6~0.7),接著取2.5 ml配製好的溶壁微球菌底物加入0.1 ml適當稀釋的溶菌酶蛋白,迅速混合後偵測1分鐘內OD
450吸光值的變化(25
oC)。而 1 unit 的定義為25
oC、波長450mm下吸光度降低0.001為1個酶活力單位。
第5圖顯示野生型及H166K突變型溶菌酶的活性分析。如第5圖所示, H166K突變型溶菌酶之相對比活性為野生型溶菌酶的191%,其活性明顯地高於野生型溶菌酶。此外,本案也將野生型及H166K突變型基因進行了50L模擬工業生產的醱酵試驗,而在50L醱酵的結果中,H166K突變型蛋白的活性也是明顯地高於野生型。
此外,酵素在不同物種間通常存在一些變異,但仍具有相同功能,且彼此間大多具有80%以上的胺基酸序列相似度,顯見要保有酵素功能,亦可容許有部分胺基酸序列的變異。換言之,本案改造之溶菌酶序列當不僅限於將序列編號2第166個位置的組胺酸突變成賴胺酸的序列,亦可包含將與序列編號2具有80%以上序列相似度的序列中對應位置的組胺酸突變成賴胺酸的序列。
綜上所述,為了增加溶菌酶Rham的活性來提升其工業應用價值,本案根據其結構分析挑選位在活性區附近的第166個位置的組胺酸(histidine)進行改造,利用點突變技術突變為賴胺酸(lysine)。根據溶菌酶活性分析結果可知,本案所改造的H166K突變型蛋白的活性明顯高於野生型蛋白。因此,本案成功且大大地提升溶菌酶Rham的活性,進而降低其生產成本並增加此溶菌酶的工業應用價值。
縱使本發明已由上述實施例詳細敘述而可由熟悉本技藝人士任施匠思而為諸般修飾,然皆不脫如附申請專利範圍所欲保護者。
無
第1圖顯示溶菌酶Rham的蛋白立體結構圖。 第2圖顯示野生型溶菌酶的核苷酸序列以及胺基酸序列。 第3圖顯示點突變技術所採用的引子序列。 第4圖顯示H166K突變型溶菌酶的核苷酸序列以及胺基酸序列。 第5圖顯示野生型及H166K突變型溶菌酶的活性分析。
Claims (6)
- 一種溶菌酶,其胺基酸序列係為將序列編號2第166個位置的組胺酸突變成賴胺酸的序列。
- 如申請專利範圍第1項所述之溶菌酶,其中編碼該序列編號2的基因係從美洲鴕鳥(Rhea americana)的蛋清中所分離出來的Rham基因。
- 如申請專利範圍第1項所述之溶菌酶,其中該溶菌酶係為一鵝型溶菌酶。
- 如申請專利範圍第1項所述之溶菌酶,其中該溶菌酶的胺基酸序列如序列編號5所示。
- 一種編碼如申請專利範圍第1項所述之溶菌酶之核酸分子。
- 一種重組質體,其係包含如申請專利範圍第5項所述之核酸分子。
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Pooart J et al., Biosci Biotechnol Biochem. 2004 Jan;68(1):159-69. |
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