楊海霞,鄧建軍,2,張 建,3,趙廣華,*
(1.中國(guó)農(nóng)業(yè)大學(xué)食品科學(xué)與營(yíng)養(yǎng)工程學(xué)院,北京100083;2.西北大學(xué)食品科學(xué)與工程系,陜西西安710069;3.石河子大學(xué)食品學(xué)院,新疆石河子832000)
植物幾丁質(zhì)酶純化測(cè)定及應(yīng)用研究進(jìn)展
楊海霞1,鄧建軍1,2,張 建1,3,趙廣華1,*
(1.中國(guó)農(nóng)業(yè)大學(xué)食品科學(xué)與營(yíng)養(yǎng)工程學(xué)院,北京100083;2.西北大學(xué)食品科學(xué)與工程系,陜西西安710069;3.石河子大學(xué)食品學(xué)院,新疆石河子832000)
植物幾丁質(zhì)酶是植物體中能夠水解幾丁質(zhì)多聚體的一種致病性相關(guān)蛋白(Pathogenesis-related proteins)。近年來(lái)對(duì)于幾丁質(zhì)酶的研究報(bào)道中,大量新型的植物幾丁質(zhì)酶被分離純化,并建立了不同的酶活測(cè)定方法,在幾丁質(zhì)酶的結(jié)構(gòu)及分類方面也逐步有了系統(tǒng)的研究。從幾丁質(zhì)酶的結(jié)構(gòu)及分類,分離純化以及已建立的酶活測(cè)定方法等方面取得的新進(jìn)展進(jìn)行了綜述,并展望了幾丁質(zhì)酶在農(nóng)業(yè)、食品生產(chǎn)及藥用領(lǐng)域的應(yīng)用前景。
植物,幾丁質(zhì)酶,分類,純化,酶活測(cè)定
植物在受到致病性微生物侵害時(shí),會(huì)誘導(dǎo)表達(dá)大量不同基因編碼的蛋白質(zhì)——致病性相關(guān)蛋白(Pathogenesis-related proteins)來(lái)保護(hù)植株免受病原菌的侵害[1-4]。而在這些誘導(dǎo)的致病性相關(guān)蛋白質(zhì)中,幾丁質(zhì)酶(Chitinase,E.C.3.2.1.14)被證明參與了寄主植物與真菌病原菌的相互作用[5-7]。幾丁質(zhì)酶又稱殼多糖酶,能夠水解幾丁質(zhì)內(nèi)部的β-1,4-糖苷鍵,生產(chǎn)N-乙酰葡萄糖胺寡聚體。它廣泛存在于植物體中,人們已從植物的根、莖、葉、果實(shí)以及種子中分離純化得到多種幾丁質(zhì)酶[8-10],并且發(fā)現(xiàn)幾丁質(zhì)酶可以通過破壞菌絲頂端及其側(cè)壁抑制真菌生長(zhǎng),從而達(dá)到抗真菌感染的目的;還可以通過釋放寡糖誘導(dǎo)植物抗霉素的產(chǎn)生。抑菌實(shí)驗(yàn)結(jié)果表明當(dāng)幾丁質(zhì)酶與β-1,3-葡聚糖酶共同作用時(shí),抑菌效果會(huì)大大提高[7]。由于幾丁質(zhì)酶具有很強(qiáng)的抗真菌活性,近年來(lái),幾丁質(zhì)酶已經(jīng)成為工業(yè)、農(nóng)業(yè)、生物技術(shù)以及食品等領(lǐng)域研究的熱點(diǎn)。本文就植物來(lái)源的幾丁質(zhì)酶在結(jié)構(gòu)、分類、分離純化方法、酶活測(cè)定方法以及應(yīng)用前景等方面進(jìn)行了綜述,為今后植物源幾丁質(zhì)酶的開發(fā)和應(yīng)用提供技術(shù)參考。
近年來(lái),對(duì)多種植物的幾丁質(zhì)酶基因進(jìn)行了克隆,研究發(fā)現(xiàn)植物幾丁質(zhì)酶的核苷酸序列與所編碼的氨基酸序列在結(jié)構(gòu)上存在一些規(guī)律性的變化,植物幾丁質(zhì)酶前體一般都含有N-端信號(hào)區(qū)(Signal peptide)、催化區(qū)(Catalytic domain)和 C-端延伸區(qū)(C-terminal extension),有的在N-端信號(hào)區(qū)之后有富含半胱氨酸的幾丁質(zhì)結(jié)合區(qū)域(Chitin binding domain,CBD),它與主結(jié)構(gòu)區(qū)之間有一段可變交聯(lián)區(qū)
(Flexible hinge region)[11](如圖 1)。N-端信號(hào)區(qū)一般由20多個(gè)氨基酸組成,為疏水性,作用是調(diào)節(jié)酶的分泌作用,引導(dǎo)幾丁質(zhì)酶通過細(xì)胞膜運(yùn)輸?shù)絻?nèi)質(zhì)網(wǎng)后被信號(hào)肽酶裂解掉。有的胞內(nèi)幾丁質(zhì)酶是沒有N-端信號(hào)區(qū)的[12],不同幾丁質(zhì)酶的N-端信號(hào)區(qū)也是不同的。CBD是一段由40個(gè)氨基酸組成的富含半胱氨酸的高度保守結(jié)構(gòu)[3],6個(gè)半胱氨酸殘基位置基本固定,之間形成二硫鍵,可能是維持三維結(jié)構(gòu)和蛋白之間的相互作用??勺兘宦?lián)區(qū)富含甘氨酸和脯氨酸,不同的幾丁質(zhì)酶的交聯(lián)區(qū)長(zhǎng)度差異很大。催化區(qū)的作用是水解底物,序列對(duì)比顯示催化區(qū)有兩段高度保守的區(qū)域,其中SxGG是結(jié)合底物部位,DxxDxDxE是催化水解底物區(qū)域[13],需要說(shuō)明的是催化區(qū)的底物結(jié)合部位和CBD是兩個(gè)獨(dú)立的區(qū)域,研究表明CBD不能夠增加幾丁質(zhì)水解速率,只能夠增加幾丁質(zhì)酶的結(jié)合能力[14-15]。C-端延伸區(qū)的作用是用來(lái)引導(dǎo)幾丁質(zhì)酶進(jìn)入植物液泡[16]。
圖1 植物幾丁質(zhì)酶的結(jié)構(gòu)分類圖
幾丁質(zhì)酶依據(jù)結(jié)構(gòu)特點(diǎn)和氨基酸序列被分為以下幾類[17],如圖1所示:ClassⅠ在 N-端信號(hào)區(qū)后有幾丁質(zhì)交聯(lián)區(qū)(CBD)以及可變交聯(lián)區(qū),連接著催化區(qū),一般含有約300個(gè)氨基酸,此類幾丁質(zhì)酶呈堿性,大部分存在于液泡內(nèi);ClassⅡ的主要氨基酸序列和ClassⅠ有很強(qiáng)的同源性,只是缺少CBD和可變交聯(lián)區(qū),此類幾丁質(zhì)酶多數(shù)呈酸性,存在于胞質(zhì)外;ClassⅢ的結(jié)構(gòu)和前兩類植物幾丁質(zhì)酶的結(jié)構(gòu)很不相同,但是和細(xì)菌、真菌幾丁質(zhì)酶具有較高的同源性,同樣不存在CBD區(qū)[18],它是胞外水解酶,在酶活性方面和溶菌酶有較大的相似性;ClassⅣ和ClassⅠ具有序列同源性,也同樣含有CBD和保守的主要結(jié)構(gòu),ClassⅣ和ClassⅠ可用血清學(xué)方法來(lái)區(qū)分,ClassⅣ的成熟蛋白質(zhì)基本含有241~255個(gè)氨基酸,分子量比ClassⅠ略小是由于有部分片段缺失[3]。Melchers el從煙草分離到一種新的幾丁質(zhì)酶,此酶的氨基酸序列與幾種細(xì)菌的幾丁質(zhì)外切酶相似,但與已報(bào)道的植物幾丁質(zhì)酶不同,故將其定義為ClassesⅤ[10]。目前在煙草、蕁麻、甜菜、豆科植物以及裸子植物中均已發(fā)現(xiàn) ClassesⅤ幾丁質(zhì)酶[19-20]。ClassⅥ幾丁質(zhì)酶的N端序列含有兩個(gè)重復(fù)排列的可變交聯(lián)區(qū)域,而ClassⅦ則缺失CBD區(qū)域,與ClassⅣ有較高的同源性,已從小麥、棉花中提取得到此類酶[21-22]。
如果依據(jù)糖基水解酶系統(tǒng)分類,植物幾丁質(zhì)酶可以被分為兩個(gè)家族,即Family 18和Family 19。所有的真菌、動(dòng)物、細(xì)菌幾丁質(zhì)酶以及植物ClassⅢ、Ⅳ幾丁質(zhì)酶均為Family 18,植物 ClassⅠ、Ⅱ、Ⅴ、Ⅵ、Ⅶ為Family 19[23]。二者在氨基酸序列、三維結(jié)構(gòu)以及分子機(jī)制等方面的差異表明它們分別起源于不同的祖先[21]。
植物幾丁質(zhì)酶的分子量介于25~35kD之間。在以幾丁質(zhì)為底物時(shí),最適反應(yīng)pH一般低于7,其等電點(diǎn)介于3~10之間,植物幾丁質(zhì)酶有著較好的熱穩(wěn)定性(有的在60℃仍很穩(wěn)定),且抗蛋白酶降解[24]。
植物幾丁質(zhì)酶可以通過不同的方法分離純化,主要有硫酸銨鹽析、離子交換層析、疏水作用層析、凝膠過濾層析、幾丁質(zhì)-親和層析以及電泳等方法。分離純化過程最好在低溫(4℃)下進(jìn)行。幾丁質(zhì)酶的酶活會(huì)隨著純化步驟的增加有所降低。提取植物幾丁質(zhì)酶一般按如下方法進(jìn)行:將植物原料用適當(dāng)pH的緩沖液勻漿,攪拌后過濾,得到的濾液離心去除不溶性淀粉及雜質(zhì),上清液中加入適當(dāng)濃度的硫酸銨,使幾丁質(zhì)酶沉淀,靜置一段時(shí)間,離心取沉淀,用緩沖液重新溶解成溶液狀態(tài),透析去除鹽分等雜質(zhì),即得到幾丁質(zhì)酶的粗提液。純化植物幾丁質(zhì)酶的方法有很多,依據(jù)不同幾丁質(zhì)酶的生物化學(xué)性質(zhì)選擇合適的純化方法。Bloch and Burger利用幾丁質(zhì)與幾丁質(zhì)酶結(jié)合的原理,將酶粗提液過幾丁質(zhì)親和柱,收集洗脫液即為純化的幾丁質(zhì)酶[25]。Takeshi Watanabe等人通過離子交換層析和凝膠過濾層析對(duì)幾丁質(zhì)酶進(jìn)行純化[26],純化過程中要選擇適當(dāng)?shù)膒H、緩沖液、洗脫鹽濃度、流速等條件,也可利用電泳(Polyacrylamide Gel Electrophoresis)的方法制備得到純化的幾丁質(zhì)酶[27]。
植物幾丁質(zhì)酶主要是水解幾丁質(zhì)多聚體中的β-1,4-糖苷鍵,幾丁質(zhì)酶的水解活性主要有三種方式,即內(nèi)切幾丁質(zhì)酶(Endochitinase)、外切幾丁質(zhì)酶(Exochitinase)以及 N-乙酰氨基己糖苷酶(GlcNAcase)[13,27]。內(nèi)切幾丁質(zhì)酶水解位點(diǎn)為同聚物任意中間位點(diǎn)的β-1,4-糖苷鍵,生成可溶的、低分子量的 N-乙酰氨基葡糖多聚體,如:chitotetraose、chitotriose以及diacetylchitobiose,其作用底物大多為四糖以上的低聚糖,而且酶與底物親和性也隨著底物聚合度提高而增大[28];外切幾丁質(zhì)酶則作用于非還原端口依次切下單糖,它生成的產(chǎn)物只有diacetylchitobiose,沒有單糖或低聚糖;N-乙酰氨基己糖苷酶利用外切形式將diacetylchitobiose和幾丁質(zhì)多聚體裂解成N-乙酰氨基葡糖單體[13]。離體實(shí)驗(yàn)表明,幾丁質(zhì)酶的活性底物還有其它物質(zhì);有的幾丁質(zhì)酶具有溶菌酶活性可作用于細(xì)菌肽聚糖、N-乙酰胞壁酸和N-乙酰葡萄糖之間的β-1,4-糖苷鍵;有的還能催化轉(zhuǎn)糖基反應(yīng)[29]。一般幾丁質(zhì)酶的內(nèi)切作用和外切作用是同時(shí)進(jìn)行的[12]。從許多植物包括大麥、玉米、煙草、馬鈴薯、大豆和大米中提取的幾丁質(zhì)酶都屬于內(nèi)切幾丁質(zhì)酶,主要產(chǎn)物為低聚糖[30]。
目前,關(guān)于檢測(cè)幾丁質(zhì)酶酶活的方法報(bào)道很多。最常用的方法是用膠體幾丁質(zhì)(Colloidal chitin)做底物,通過檢測(cè)酶解后的產(chǎn)物還原糖進(jìn)行酶活測(cè)定。膠體幾丁質(zhì)制備方法如Imanaka T等人所述[31],選擇合適的酶解條件進(jìn)行酶解反應(yīng)后,生成產(chǎn)物還原糖利用Imato和Yagishita的方法測(cè)定,1U等于釋放1μg 還原糖所需的酶量[32]。Nitoda T.K.等人發(fā)現(xiàn)用膠狀幾丁質(zhì)的顆粒均一大小的粉末作底物,靈敏度比用膠狀幾丁質(zhì)上清液高4倍[33]。幾丁質(zhì)的可溶性衍生物乙二醇幾丁質(zhì)(Glycolchitin)以及脫乙酰殼聚糖(Chitosan)被用作十分重要的測(cè)定酶活底物,酶解后通過紫外吸光值的測(cè)定反應(yīng)酶活[34]。還有報(bào)道認(rèn)為用膠狀幾丁質(zhì)做底物測(cè)定還原糖的含量只測(cè)出外切幾丁質(zhì)酶的活性,如要測(cè)內(nèi)切幾丁質(zhì)酶活性還需加入蝸牛腸酶酶解,再測(cè)定還原糖含量[35]。
為了更好地檢測(cè)溶液中幾丁質(zhì)酶的酶活,生色測(cè)定方法被建立并逐漸成熟起來(lái)。底物N-acetyl-D- glucosaminide(GlcNAc)、β - D - N,N′-diacetylchitobiose[(GlcNAc)2]以 及 β-D-N,N′,N″-triacetylchitotrios[(GlcNAc)3]可用生色基團(tuán)p-nitrophenyl(pNP)分別標(biāo)記為 pNP-GlcNAc,pNP-(GlcNAc)2以及 pNP-(GlcNAc)3。內(nèi)切幾丁質(zhì)酶、外切幾丁質(zhì)酶以及N-乙酰氨基己糖苷酶分別酶解pNP-(GlcNAc)3、pNP-(GlcNAc)2以及 pNP-GlcNAc后釋放pNP,通過熒光測(cè)定反應(yīng)酶活。Nagel R.等人提出利用pNP-GlcNAc作底物比固體幾丁質(zhì)靈敏,幾丁質(zhì)酶對(duì)pNP-GlcNAc的水解速率是固體幾丁質(zhì)的三倍,1U等于釋放1nmol pNP所用的酶量[36]。同樣,底物被4-methylumbelliferyl(4-MU)修飾的測(cè)定酶活的方法也被建立起來(lái)。4-MU-(GlcNAc)3、4-MU-(GlcNAc)2和4-MU-GlcNAc酶解后可釋放4-MU,通過熒光(激發(fā)波355nm,發(fā)射波460nm)分別測(cè)定反應(yīng)內(nèi)切幾丁質(zhì)酶、外切幾丁質(zhì)酶以及N-乙酰氨基己糖苷酶的酶活,1U即釋放1nmol 4-MU所用的酶量。
有些學(xué)者利用染料修飾膠狀幾丁質(zhì),利用酶解釋放的染料具有特異吸光值來(lái)測(cè)定幾丁質(zhì)酶的活性。早在1964年Hackman和Goldberg就提出用有色幾丁質(zhì)衍生物Chitin Azure作底物,酶解后通過550nm處測(cè)定吸光值反映幾丁質(zhì)酶酶活,1U酶活被定義為吸光值增加0.001所需的酶量[37]。M.Go′mez Ram?′rez提出了一種快速靈敏的方法,利用Remazol Brilliant Blue RR(RBB)染色的膠狀幾丁質(zhì)作底物,培養(yǎng)酶解后通過在595nm下測(cè)定吸光值反映幾丁質(zhì)酶的活性[38]。Ostazin Brilliant Red用作標(biāo)記幾丁質(zhì)底物,酶解后釋放的被染料標(biāo)記的GlcNAc在530nm測(cè) 定 吸 光 值[39]。Mojtaba Mamarabadi將 底 物 用Remazol Brilliant Violet(RBV)修飾,酶解后在550nm下測(cè)定吸光值反映酶活[40]。
近些年來(lái),人們從幾丁質(zhì)酶的酶學(xué)特性、抗菌機(jī)理以及分子克隆方面已經(jīng)有了系統(tǒng)的研究,但是對(duì)于幾丁質(zhì)酶防御反應(yīng)的機(jī)理還有待闡明;此外,目前學(xué)者的興趣主要集中于尋找或重組有更好動(dòng)力學(xué)特性的新型幾丁質(zhì)酶,以及對(duì)幾丁質(zhì)酶活性位點(diǎn)殘基的識(shí)別[27]。由于植物幾丁質(zhì)酶的重要作用,其在食品及藥物方面有著廣泛的應(yīng)用。
由于幾丁質(zhì)酶具有良好的抑菌活性[7],所以在食品生產(chǎn)中,幾丁質(zhì)酶與其它抗微生物酶(葡萄糖氧化酶和乳過氧化物酶)或與傳統(tǒng)的防腐措施(如山梨酸鹽、乙醇、溫度和低pH)聯(lián)合使用,可以提高食品微生物的安全性,在糕點(diǎn)或飲料等食品中適當(dāng)添加,可防止微生物的繁殖,起到防腐作用。
另外,幾丁質(zhì)對(duì)動(dòng)植物而言是一種較難吸收利用的氮素來(lái)源,幾丁質(zhì)酶可將食品中的幾丁質(zhì)酶解,作用后所產(chǎn)生的N-乙酰幾丁寡糖和殼聚幾丁寡糖都具有清爽的甜味,吸濕性和保濕性好,而且在水中溶解度比單糖低,有助于調(diào)整食品的水活性,增進(jìn)保水性,兼具調(diào)味和改良食品質(zhì)構(gòu)的功能。
幾丁質(zhì)酶的降解產(chǎn)物寡聚糖、葡糖胺以及N-乙酰氨基葡糖在醫(yī)藥及食品方面有著廣闊的應(yīng)用前景,我國(guó)漁業(yè)食品工業(yè)生產(chǎn)中的廢棄物中含有大量幾丁質(zhì),而傳統(tǒng)工藝用酸水解蝦殼等生產(chǎn)幾丁寡糖成本昂貴且對(duì)容器腐蝕性大,利用幾丁質(zhì)酶水解法制備其降解產(chǎn)物,不僅能徹底有效的處理廢殼,減少對(duì)環(huán)境的污染,還可以變廢為寶,使降解產(chǎn)物得以利用,這種環(huán)保、經(jīng)濟(jì)、快速的方法無(wú)疑為企業(yè)開拓一條新的途徑。
由于幾丁質(zhì)酶具有顯著的抗菌特性[7],近年來(lái),通過轉(zhuǎn)基因技術(shù),將水稻、大麥、煙草等幾丁質(zhì)酶基因轉(zhuǎn)入水稻、番茄等植物中用于病蟲害防治,效果十分顯著,不僅獲得了高效而廣譜的抗性,還克服了轉(zhuǎn)基因植物的表達(dá)水平低的問題,應(yīng)用前景十分廣闊。
總之,無(wú)論從幾丁質(zhì)資源的開發(fā)利用、減輕污染和保護(hù)環(huán)境的角度來(lái)看,還是幾丁質(zhì)原料及幾丁質(zhì)酶在農(nóng)業(yè)、工業(yè)、食品及藥物中的重要作用,充分研究幾丁質(zhì)酶,利用幾丁質(zhì)酶的特性開發(fā)新的應(yīng)用前景將會(huì)給社會(huì)以及人類帶來(lái)巨大幫助。
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Review on purification,enzyme assay and application of plant chitinases
YANG Hai-xia1,DENG Jian-jun1,2,ZHANG Jian1,3,ZHAO Guang-h(huán)ua1,*
(1.College of Food Science and Nutritional Engineering,China Agricultural University,Beijing 100083,China;2.Department of Food Science and Engineering,Northwest University,Xi’an 710069,China;3.Food College,Shihezi University,Shihezi 832003,China)
Plant chitinases which hydrolyze the chitin is one of pathogenesis-related proteins and can be induced in resistance of plants to fungal pathogens.Thus,plant chitinases has a wide range of application as a antisepticise material.Recently,chitinases from different plants have been purified and their enzymatic activities have been assayed with varied methods.The structures and classifications,different methods on enzymatic activity assay and purification were summarized.Moreover,applications of chitinases such as in food and medicine field were prospected.
plant;chitinases;classification;purification;enzymatic activity assay
TS201.2+5
A
1002-0306(2011)06-0431-05
2010-05-13 *通訊聯(lián)系人
楊海霞(1984-),女,博士研究生,研究方向:蛋白質(zhì)化學(xué)與營(yíng)養(yǎng)。