極地微生物酶資源開(kāi)發(fā)研究進(jìn)展

王偉 姚從禹3 孫晶晶 郝建華

研究進(jìn)展

極地微生物酶資源開(kāi)發(fā)研究進(jìn)展

王偉1,2姚從禹1,2,3孫晶晶1,2郝建華1,2

(1中國(guó)水產(chǎn)科學(xué)研究院黃海水產(chǎn)研究所, 農(nóng)業(yè)農(nóng)村部極地漁業(yè)開(kāi)發(fā)重點(diǎn)實(shí)驗(yàn)室, 山東 青島 266071;2青島海洋科學(xué)與技術(shù)試點(diǎn)國(guó)家實(shí)驗(yàn)室海洋藥物與生物制品功能實(shí)驗(yàn)室, 山東 青島 266235;3上海海洋大學(xué)食品學(xué)院, 上海 201306)

兩極地區(qū)的微生物在極端環(huán)境中演化出能夠耐受低溫、高鹽等特殊性質(zhì)的酶。極地微生物的酶有潛在的應(yīng)用價(jià)值, 是重要的生物資源。極地微生物在各種復(fù)雜的極地環(huán)境中形成了高度多樣性, 蘊(yùn)含著可供開(kāi)發(fā)利用的大量新酶資源。近年來(lái)國(guó)內(nèi)外持續(xù)發(fā)現(xiàn)大量的極地微生物新酶, 說(shuō)明極地微生物已成為新酶的重要來(lái)源。本文簡(jiǎn)述了近5年來(lái)極地微生物新酶開(kāi)發(fā)的研究進(jìn)展, 分類(lèi)列舉了有較好商用前景的蛋白酶、脂肪酶/酯酶、糖類(lèi)降解酶等重要工業(yè)酶的篩選和性質(zhì)等研究簡(jiǎn)況。

極地微生物 酶資源開(kāi)發(fā) 蛋白酶 脂肪酶 酯酶 糖類(lèi)降解酶

0 引言

極地具有獨(dú)特的地理、氣候及環(huán)境特征, 如低溫、寡營(yíng)養(yǎng)、高pH、高鹽、極端光照條件等。極地微生物通過(guò)演化出一系列特定結(jié)構(gòu)和功能, 適應(yīng)了上述極端環(huán)境。具有在低溫下高效催化的低溫酶是極地微生物的重要適應(yīng)特征。

極地是一個(gè)微生物資源庫(kù), 其中的極端微生物能夠產(chǎn)生新型生物活性物質(zhì)(如酶、多糖、多肽等)[1]。極地微生物的多樣性與活性物質(zhì)研究已成為現(xiàn)代微生物學(xué)的研究熱點(diǎn)。極地微生物也是酶資源的重要來(lái)源, 極地微生物酶的研究從20世紀(jì)90年代開(kāi)始興起, 以蛋白酶、脂肪酶、淀粉酶等產(chǎn)業(yè)價(jià)值高的酶類(lèi)作為主要研究對(duì)象。極地微生物的低溫酶與耐鹽酶等結(jié)構(gòu)和功能新穎的極端酶憑借其獨(dú)特的催化作用大大拓寬了微生物酶的應(yīng)用范圍, 也給酶工程的研究帶來(lái)了新的思路和方向。本文對(duì)極地微生物來(lái)源的蛋白酶、脂肪酶/酯酶、糖類(lèi)降解酶等近5年來(lái)的研究進(jìn)展進(jìn)行綜述, 以期對(duì)極地微生物資源的綜合利用提供參考。

1 產(chǎn)酶的極地微生物篩選

雖然宏基因組文庫(kù)或數(shù)據(jù)挖掘等新方法已經(jīng)用于極地微生物新酶篩選, 但以經(jīng)典的分離培養(yǎng)法對(duì)菌株開(kāi)展酶活性篩選仍是近5年的主流方法(表1)。這些研究多選擇工業(yè)應(yīng)用潛力較大的水解酶(蛋白酶、脂肪酶、糖類(lèi)降解酶等)進(jìn)行篩選, 主要以平板透明圈法初步確定各種酶的活性。這些研究都能從來(lái)源多樣的極地環(huán)境樣品中篩選出多種產(chǎn)酶菌株, 說(shuō)明極地微生物酶資源豐富, 有待繼續(xù)廣泛深入地研究。

表1 近年從極地環(huán)境樣品中分離篩選產(chǎn)酶微生物簡(jiǎn)況

2 蛋白酶

蛋白酶是一類(lèi)催化蛋白質(zhì)或肽類(lèi)的肽鍵水解的酶類(lèi), 其種類(lèi)多樣, 在眾多行業(yè)及科研中廣泛應(yīng)用, 成為具有重要商業(yè)價(jià)值的工業(yè)酶。21世紀(jì)初報(bào)道了大量產(chǎn)適冷蛋白酶的極地微生物, 近年來(lái)極地微生物蛋白酶研究熱度不減。從3個(gè)南極淡水湖中分離出細(xì)菌18種63株, 真菌1種8株, 它們?cè)?℃均有蛋白酶活力[17]; Kim和Choi[18]研究了溫度對(duì)四株有生產(chǎn)低溫蛋白酶潛力的南極細(xì)菌的影響。

從表2可見(jiàn)近年從極地微生物中獲得的蛋白酶幾乎都是低溫酶; 從南極篩選的蛋白酶較多, 且主要為工業(yè)應(yīng)用最多的絲氨酸蛋白酶。來(lái)自真菌的酶少于細(xì)菌的酶, 說(shuō)明細(xì)菌是極地蛋白酶的主要來(lái)源。這些研究的目標(biāo)是獲得有潛在工業(yè)應(yīng)用價(jià)值(如洗滌劑和乳制品加工)的低溫酶, 工作主要集中在酶純化、性質(zhì)及應(yīng)用潛力, 對(duì)酶的空間結(jié)構(gòu)、催化機(jī)理等理論研究相對(duì)較少。以功能宏基因組篩選北極王灣海底沉積蛋白酶[31], 發(fā)現(xiàn)的新型中性金屬蛋白酶潛在應(yīng)用價(jià)值不高, 但證明了宏基因組法篩選蛋白酶的潛力。

3 脂肪酶/酯酶

脂肪酶/酯酶是能夠催化脂肪酸甘油酯水解和合成的酶, 在食品、制藥、洗滌、能源等工業(yè)中應(yīng)用廣泛。近年在南北極地區(qū)的微生物中獲得了大量脂肪酶/酯酶(表3)。

表2 近年新發(fā)現(xiàn)的極地微生物蛋白酶簡(jiǎn)況

近年發(fā)現(xiàn)的極地微生物脂肪酶/酯酶種類(lèi)多樣(表3), 主要是來(lái)自細(xì)菌的低溫酶。這些酶多數(shù)具有有機(jī)溶劑耐受能力, 展現(xiàn)出在不同領(lǐng)域的應(yīng)用價(jià)值。從表3可見(jiàn)宏基因組篩選已經(jīng)成為極地微生物新型酯酶篩選的主流方法, 用該方法獲得的酯酶數(shù)量已經(jīng)超過(guò)了傳統(tǒng)菌株分離的方法。一般情況下在極地微生物中篩選獲得低溫酶, 但也有可能獲得熱穩(wěn)定性理想的高溫酶。表3篩選到的脂肪酶/酯酶都是堿性或中性酶, 無(wú)酸性酶, 這可能和研究目標(biāo)多選擇細(xì)菌有關(guān), 也和對(duì)酸性脂肪酶/酯酶的應(yīng)用需求沒(méi)有堿性酶高有關(guān)。今后如果更關(guān)注極地真菌脂肪酶/酯酶, 可能會(huì)發(fā)現(xiàn)新的酸性酶。

4 糖類(lèi)降解酶

近年來(lái)從極地微生物中分離的糖類(lèi)降解酶種類(lèi)眾多, 性質(zhì)獨(dú)特。從北極斯匹次卑爾根島王灣分離的海洋細(xì)菌多具有褐藻膠、果膠、淀粉、木聚糖或羧甲基纖維素的低溫降解酶[49]。這也說(shuō)明了極地微生物糖類(lèi)降解酶資源開(kāi)發(fā)的良好前景。

表3 近年新發(fā)現(xiàn)的極地微生物脂肪酶/酯酶簡(jiǎn)況

續(xù)表3

*僅使用一種底物, 未作比較

4.1 水解葡萄糖單元的糖苷鍵的酶

淀粉酶能將淀粉轉(zhuǎn)化為低分子量的糖如葡萄糖、麥芽糖或寡糖。該酶在淀粉工業(yè)中使用廣泛, 在酶制劑市場(chǎng)占有很大份額。α-淀粉酶是內(nèi)切酶, 用于紡織品、紙張、食品、生物燃料、洗滌劑和制藥工業(yè)的多種生物加工工藝[50]。南極喬治王島海水細(xì)菌的α-淀粉酶(屬于糖苷水解酶GH13家族)最適條件20℃、pH 8.0, 是已知的α-淀粉酶的最低最適反應(yīng)溫度[50]。南極深海沉積細(xì)菌α-淀粉酶Amy172的最適條件50℃、pH 10[51]。南極真菌的耐熱α-淀粉酶, 最適條件70℃、pH 6。這是首次在適冷真菌中發(fā)現(xiàn)耐熱淀粉酶[52]。南極喬治王島土壤真菌新型適冷葡萄糖淀粉酶最適條件30℃、pH 6, 活性不依賴(lài)Ca2+[53]。北極王灣沉積物細(xì)菌α-淀粉酶Amy3的最適條件25℃、pH 8.5, 具有良好的低溫催化及嗜鹽性[54]。該菌株還具有兩種α葡萄糖苷酶: GH13家族的Pagl最適條件30℃、pH 8, 最適底物麥芽糖, 耐受葡萄糖[55]; GH97家族的PspAG 97A最適條件30℃、pH 7.5, 可水解α-1,2/1,4/1,6糖苷鍵[56]。北極斯匹次卑爾根島海水細(xì)菌普魯蘭酶(GH13家族)最適條件35℃、pH 6—7, 只水解α-1,6糖苷鍵[57]。

南極喬治王島土壤細(xì)菌β-葡糖苷酶EaBgl1A屬于GH1家族, 最適條件30℃、pH 7, 對(duì)葡萄糖的耐受性好于幾種商用酶[58], 晶體結(jié)構(gòu)解析表明其適應(yīng)低溫的主要機(jī)制是形成四聚體[59]。北極王灣表層海水細(xì)菌的新型內(nèi)切β-1,4-葡糖苷酶屬于GH10家族, 低溫耐鹽, 纖維素酶活性明顯而木聚糖酶活性很低[60]。從北極洋中脊熱液口宏基因組序列中得到了嗜熱纖維素酶, 最適條件100℃、pH 5.7, 其在85℃活性穩(wěn)定, 在GH9家族纖維素酶中熱穩(wěn)定性最好[61]。

4.2 卡拉膠酶

卡拉膠是一種硫酸化的紅藻多糖, 由半乳糖和3,6-脫水半乳糖通過(guò)α-1,3和β-1,4糖苷鍵交聯(lián)形成?？ɡz酶能降解卡拉膠產(chǎn)生水溶性好、生物活性高的卡拉膠寡糖, 在食品工業(yè)及醫(yī)藥等領(lǐng)域具有良好的應(yīng)用前景。潘愛(ài)紅等[62]優(yōu)化了南極普里茲灣沉積物細(xì)菌卡拉膠酶的產(chǎn)量。胡秋實(shí)等[63]從北極海水中篩選出四株高產(chǎn)卡拉膠酶的假交替單胞菌菌株, 酶的最適反應(yīng)溫度均為20℃。林歡等[64]從南極海藻分離一株高產(chǎn)卡拉膠酶的細(xì)菌, 優(yōu)化產(chǎn)酶條件, 確定酶最適條件為37℃、pH 7。

4.3 瓊膠酶

瓊脂(瓊膠)也是紅藻多糖, 由不同形式的半乳糖作為單糖單元構(gòu)成。瓊膠酶能降解瓊膠產(chǎn)生有活性的瓊寡糖。南極普里茲灣沉積物細(xì)菌NJ21有三種外切型β瓊膠酶, 產(chǎn)物均為新瓊二糖。GH42家族的瓊膠酶Aga1161最適條件40℃、pH 8, 40℃活性不穩(wěn)定[65]; Aga3463屬于GH86家族, 最適條件50℃、pH 7, 50℃活性不穩(wěn)定[66]; 瓊膠酶Aga3311也屬于GH42家族, 最適條件35℃、pH 7[67]。分離自東南極近岸海冰硅藻的細(xì)菌β瓊膠酶屬于GH16家族, 最適條件40℃、pH 7, 50℃不穩(wěn)定[68]。

4.4 β-半乳糖苷酶

β-半乳糖苷酶有兩種活性: 水解乳糖/低聚半乳糖; 連接半乳糖生成低聚半乳糖。該酶在乳制品工業(yè)中應(yīng)用廣泛。東南極湖泊嗜鹽古菌的半乳糖苷酶是GH42家族的單體酶, 比較其野生酶和六個(gè)突變酶的低溫催化參數(shù)及空間結(jié)構(gòu)模型, 說(shuō)明一個(gè)氨基酸突變就能使低溫催化能力明顯改變[69]。南極喬治王島土壤細(xì)菌β-半乳糖苷酶屬于GH2家族, 最適條件28℃、pH7, 低溫下具有水解和聚合兩種酶活[70]。該酶晶體結(jié)構(gòu)解析發(fā)現(xiàn)其適應(yīng)低溫的主要因素是二聚體酶分子表面增加了溶劑的可及性[71]。來(lái)自北極加拿大海盆海冰冰芯的海單胞菌有兩個(gè)半乳糖苷酶: BGAL584-1為不耐熱的低溫酶, 最適條件30℃、pH 7[72]; 而MaBGA最適條件60℃、pH 6[73]。兩個(gè)酶分別屬于GH2和GH42家族。北極王灣沉積物細(xì)菌低溫半乳糖苷酶為同源四聚體, 最適條件45℃、pH 7—8, 活性在45℃不穩(wěn)定[74]。

4.5 木聚糖酶

木聚糖是木糖以β-1,4糖苷鍵形成的多糖, 帶有阿拉伯糖和葡萄糖醛酸的側(cè)鏈。木聚糖酶可以將木聚糖降解為木糖及木寡糖, 廣泛應(yīng)用于造紙、紡織、食品、飼料等行業(yè)。從南極喬治王島海綿分離的真菌低溫木聚糖酶屬于GH10家族, 最適條件50℃、pH 6, 最適底物為阿拉伯木聚糖, 活性在35℃不穩(wěn)定, 是已知的最不耐熱的真菌木聚糖酶[75-76]。南極喬治王島海水細(xì)菌的GH10家族木聚糖酶最適條件35℃、pH 7—9, 以定向進(jìn)化和隨機(jī)突變結(jié)合的策略?xún)?yōu)化了該酶的熱穩(wěn)定性[77]。從北極洋中脊熱液口宏基因組序列中篩選到GH10家族的高溫木聚糖酶, 最適條件80℃、pH 5.6, 其降解纖維素的的活性高于木聚糖[78]。

4.6 褐藻酸裂解酶

褐藻酸是甘露糖醛酸和古洛糖醛酸以多種排列方式組成的線(xiàn)性共聚物。褐藻酸裂解酶能將褐藻酸降解成具有多種生物活性的寡糖。

東升[79]從北極海帶中分離了21株產(chǎn)褐藻酸裂解酶的細(xì)菌, 酶的最適溫度在20—50℃。高楊[80]從南大洋沉積物分離了10株能降解褐藻酸的真菌, 并表達(dá)了曲霉22-5的褐藻酸裂解酶Aly-i7, 其最適條件50℃、pH 7, 最適底物聚古洛糖醛酸, 裂解褐藻酸的產(chǎn)物為二糖。前述產(chǎn)瓊膠酶的南極細(xì)菌NJ21的褐藻酸裂解酶Al163屬于多糖裂解酶6家族(PL6)的內(nèi)切酶, 最適條件40℃、pH 7, 最適底物聚古洛糖醛酸[81]。從北極洋中脊熱液口宏基因組序列中獲得的褐藻酸裂解酶屬于PL7家族, 最適條件65℃、pH 6, 最適底物聚甘露糖醛酸, 該酶是已知最耐熱的褐藻酸裂解酶[82]。

4.7 果膠酶

果膠是植物細(xì)胞壁的組分, 是部分甲酯化的α-1,4-D-聚半乳糖醛酸。果膠酶在食品、紡織和造紙行業(yè)應(yīng)用廣泛, 可分為聚半乳糖醛酸酶、裂解酶和果膠甲酯酶三類(lèi)[83]。南極阿斯曼山細(xì)菌低溫堿性果膠裂解酶屬于PL6家族, 最適條件30℃、pH 10, 40℃活性不穩(wěn)定, 最適底物為聚半乳糖醛酸[84]。南極喬治王島土壤真菌聚半乳糖醛酸酶(GH28家族)在15℃、pH 3時(shí)活性明顯高于商用酶[83]。

5 其他酶

5.1 磷酸酶

磷酸酶能水解蛋白、核苷酸、生物堿等底物中的磷酸酯鍵, 在分子生物學(xué)、免疫學(xué)等領(lǐng)域應(yīng)用廣泛[85]。南極喬治王島土壤酵母磷酸酶最適條件47℃、pH 9.5, 其低溫活性較高而在47℃活性不穩(wěn)定[86]。喬治王島近岸表層海水細(xì)菌磷酸酶最適條件20—22℃、pH 7, 在48℃活性不穩(wěn)定, Mg2+能提高其活性和熱穩(wěn)定性[87]。

5.2 超氧化物歧化酶

超氧化物歧化酶(superoxide dismutase, SOD)能降解強(qiáng)氧化劑超氧陰離子自由基, 在制藥、化妝品等行業(yè)有應(yīng)用前景。南極海冰細(xì)菌SOD最適條件30℃、pH 8.0, 50℃活性不穩(wěn)定, 屬于Fe-SOD亞類(lèi)[88]。南極海冰酵母的Fe-SOD在pH 1.0—9.0和50℃的穩(wěn)定性都較好[89]。南極利文斯頓島曲霉有兩種Cu/Zn-SOD, 低溫誘導(dǎo)酶產(chǎn)量增加[90]。

6 展望

上文列舉了近年來(lái)極地微生物酶資源開(kāi)發(fā)簡(jiǎn)況, 主要關(guān)注了文獻(xiàn)報(bào)道較多、應(yīng)用價(jià)值較高的幾類(lèi)酶。報(bào)道的新酶以熱穩(wěn)定性較低的低溫酶為主, 個(gè)別酶熱穩(wěn)定性較好, 這些低溫酶大多有耐鹽能力。值得注意的是高溫酶的報(bào)道, 挪威生命科學(xué)大學(xué)Eijsink等從北極揚(yáng)馬延島北部的揚(yáng)馬延熱液口發(fā)現(xiàn)了三個(gè)高溫酶: 木聚糖酶[78]、褐藻酸裂解酶[82]和纖維素酶[61], 都具有理想的應(yīng)用前景。說(shuō)明極地的高溫環(huán)境雖然罕見(jiàn), 但也蘊(yùn)藏著獨(dú)特的酶資源。該研究將酶的底物(預(yù)處理的歐洲云杉木屑)在70℃的海底沉積中放置1年, 再回收測(cè)宏基因組獲取新酶基因。這種原位誘導(dǎo)篩選的方式很少用于極地微生物的新酶篩選, 有望成為將來(lái)常見(jiàn)的高效篩選策略。

目前傳統(tǒng)的平板培養(yǎng)法篩選菌株的新酶仍是國(guó)內(nèi)外最常用的方法。由于其簡(jiǎn)便易操作, 預(yù)計(jì)將來(lái)也不會(huì)被宏基因組等非培養(yǎng)方法替代。宏基因組作為重要的非培養(yǎng)篩選方法, 能克服眾多極地菌株無(wú)法培養(yǎng)的困難, 直接在基因水平發(fā)現(xiàn)新酶, 其基于功能[31,39-40,46]和基于序列[47,61,78,82]的篩選策略已經(jīng)在極地微生物資源開(kāi)發(fā)中獲得了眾多新酶, 今后必將成為常規(guī)的新酶篩選方法。此外, 從極地菌株的全基因組序列中發(fā)掘新酶也成為了常見(jiàn)的方法[23, 42, 44-45, 51, 53, 58, 60, 68-69, 72, 81, 83-84, 87]。隨著微生物全基因組測(cè)序費(fèi)用的下降和公開(kāi)的極地微生物全基因組數(shù)據(jù)的迅速增長(zhǎng), 可以推測(cè)從全基因組數(shù)據(jù)中獲取新酶基因的方法將逐漸替代傳統(tǒng)的酶基因克隆方法, 如基因組文庫(kù)篩選或簡(jiǎn)并引物PCR擴(kuò)增法。

多數(shù)極地微生物新酶篩選工作的應(yīng)用目標(biāo)明確, 獲得了大量有潛在工業(yè)應(yīng)用價(jià)值的酶, 不過(guò)未見(jiàn)真正轉(zhuǎn)化為商用酶制劑的報(bào)道, 可能受限于酶性質(zhì)、酶制劑開(kāi)發(fā)周期以及廠(chǎng)商對(duì)產(chǎn)品來(lái)源保密。

在極地微生物酶的研究?jī)?nèi)容上, 篩選、基因克隆表達(dá)和酶的純化與性質(zhì)研究是主流, 酶催化機(jī)理和晶體結(jié)構(gòu)解析等更深入的研究較少, 蛋白質(zhì)工程改造工作也不多。酶分子改造能進(jìn)一步提高酶的工作效能, 而理論研究是蛋白質(zhì)工程的重要指導(dǎo), 可以預(yù)期今后會(huì)有更多極地微生物酶理論研究及分子改造工作。

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A REVIEW OF NOVEL POLAR MICROBIAL ENZYMES FOR INDUSTRIAL APPLICATIONS

Wang Wei1,2, Yao Congyu1,2,3, Sun Jingjing1,2, Hao Jianhua1,2

(1Key Laboratory of Sustainable Development of Polar Fishery, Ministry of Agriculture and Rural Affairs, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China;2Laboratory for Marine Drugs and Bioproducts, Pilot National Laboratory for Marine Science and Technology, Qingdao 266235, China;3College of Food Science and Technology, Shanghai Ocean University, Shanghai 201306, China)

Special challenges for microorganisms in cold ecosystems include reduced enzymatic reaction rates, limited bioavailability of nutrients, and frequent extremes in pH and salinity. To thrive successfully in low temperature environments, psychrophiles have evolved a complex range of structural and functional adaptations. Psychrophiles produce cold-active enzymes, which can be up to ten times more active at low and moderate temperatures compared with their mesophilic homologues. The enzymes of polar microorganisms are shaped by their adaptations to the permanently low temperatures. In addition, strongly differing environments, such as permafrost, glaciers and sea ice, have contributed to additional functional diversity. Microorganisms that thrive in the polar zones are a vast reservoir of cold-adapted enzymes. These enzymes could be beneficial in many industrial applications. Research using polar microorganisms to find new bioproducts has been mainly focused on enzymes that can be used in a range of industrial processes. The biotechnological value of cold-adapted enzymes stems from their high turnover (kcat) at low to moderate temperatures and their high thermolability at elevated temperatures. In recent years, a large number of new polar microbial enzymes have been continuously discovered, indicating that polar microbes have become an important source of novel enzymes. This review describes the research progress of new microbial enzymes over the past five years, and focuses on the discovery of important industrial enzymes, such as protease, lipase/esterase and carbohydrate-degrading enzymes, with good commercial prospects.

polar microorganism, exploitation of enzyme resource, protease, lipase, esterase, carbohydrate- degrading enzymes

2019年7月收到來(lái)稿, 2019年11月收到修改稿

中國(guó)工程院戰(zhàn)略研究(2018-ZD-08)、農(nóng)業(yè)農(nóng)村部極地漁業(yè)開(kāi)發(fā)重點(diǎn)實(shí)驗(yàn)室開(kāi)放課題(2019OPF02)和中國(guó)水產(chǎn)科學(xué)研究院基本科研業(yè)務(wù)費(fèi)(2020TD67)資助

王偉, 男, 1980年生。博士, 副研究員, 主要從事極地海洋微生物資源開(kāi)發(fā)研究。E-mail: weiwang@ysfri.ac.cn

郝建華, E-mail: haojh@ysfri.ac.cn

10. 13679/j.jdyj.20190039