極地微生物酶資源開發研究進展

王偉 姚從禹3 孫晶晶 郝建華

研究進展

極地微生物酶資源開發研究進展

王偉1,2姚從禹1,2,3孫晶晶1,2郝建華1,2

(1中國水產科學研究院黃海水產研究所, 農業農村部極地漁業開發重點實驗室, 山東 青島 266071;2青島海洋科學與技術試點國家實驗室海洋藥物與生物制品功能實驗室, 山東 青島 266235;3上海海洋大學食品學院, 上海 201306)

兩極地區的微生物在極端環境中演化出能夠耐受低溫、高鹽等特殊性質的酶。極地微生物的酶有潛在的應用價值, 是重要的生物資源。極地微生物在各種復雜的極地環境中形成了高度多樣性, 蘊含著可供開發利用的大量新酶資源。近年來國內外持續發現大量的極地微生物新酶, 說明極地微生物已成為新酶的重要來源。本文簡述了近5年來極地微生物新酶開發的研究進展, 分類列舉了有較好商用前景的蛋白酶、脂肪酶/酯酶、糖類降解酶等重要工業酶的篩選和性質等研究簡況。

極地微生物 酶資源開發 蛋白酶 脂肪酶 酯酶 糖類降解酶

0 引言

極地具有獨特的地理、氣候及環境特征, 如低溫、寡營養、高pH、高鹽、極端光照條件等。極地微生物通過演化出一系列特定結構和功能, 適應了上述極端環境。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特征。

極地是一個微生物資源庫, 其中的極端微生物能夠產生新型生物活性物質(如酶、多糖、多肽等)[1]。極地微生物的多樣性與活性物質研究已成為現代微生物學的研究熱點。極地微生物也是酶資源的重要來源, 極地微生物酶的研究從20世紀90年代開始興起, 以蛋白酶、脂肪酶、淀粉酶等產業價值高的酶類作為主要研究對象。極地微生物的低溫酶與耐鹽酶等結構和功能新穎的極端酶憑借其獨特的催化作用大大拓寬了微生物酶的應用范圍, 也給酶工程的研究帶來了新的思路和方向。本文對極地微生物來源的蛋白酶、脂肪酶/酯酶、糖類降解酶等近5年來的研究進展進行綜述, 以期對極地微生物資源的綜合利用提供參考。

1 產酶的極地微生物篩選

雖然宏基因組文庫或數據挖掘等新方法已經用于極地微生物新酶篩選, 但以經典的分離培養法對菌株開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應用潛力較大的水解酶(蛋白酶、脂肪酶、糖類降解酶等)進行篩選, 主要以平板透明圈法初步確定各種酶的活性。這些研究都能從來源多樣的極地環境樣品中篩選出多種產酶菌株, 說明極地微生物酶資源豐富, 有待繼續廣泛深入地研究。

表1 近年從極地環境樣品中分離篩選產酶微生物簡況

2 蛋白酶

蛋白酶是一類催化蛋白質或肽類的肽鍵水解的酶類, 其種類多樣, 在眾多行業及科研中廣泛應用, 成為具有重要商業價值的工業酶。21世紀初報道了大量產適冷蛋白酶的極地微生物, 近年來極地微生物蛋白酶研究熱度不減。從3個南極淡水湖中分離出細菌18種63株, 真菌1種8株, 它們在4℃均有蛋白酶活力[17]; Kim和Choi[18]研究了溫度對四株有生產低溫蛋白酶潛力的南極細菌的影響。

從表2可見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, 且主要為工業應用最多的絲氨酸蛋白酶。來自真菌的酶少于細菌的酶, 說明細菌是極地蛋白酶的主要來源。這些研究的目標是獲得有潛在工業應用價值(如洗滌劑和乳制品加工)的低溫酶, 工作主要集中在酶純化、性質及應用潛力, 對酶的空間結構、催化機理等理論研究相對較少。以功能宏基因組篩選北極王灣海底沉積蛋白酶[31], 發現的新型中性金屬蛋白酶潛在應用價值不高, 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、能源等工業中應用廣泛。近年在南北極地區的微生物中獲得了大量脂肪酶/酯酶(表3)。

表2 近年新發現的極地微生物蛋白酶簡況

近年發現的極地微生物脂肪酶/酯酶種類多樣(表3), 主要是來自細菌的低溫酶。這些酶多數具有有機溶劑耐受能力, 展現出在不同領域的應用價值。從表3可見宏基因組篩選已經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量已經超過了傳統菌株分離的方法。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, 無酸性酶, 這可能和研究目標多選擇細菌有關, 也和對酸性脂肪酶/酯酶的應用需求沒有堿性酶高有關。今后如果更關注極地真菌脂肪酶/酯酶, 可能會發現新的酸性酶。

4 糖類降解酶

近年來從極地微生物中分離的糖類降解酶種類眾多, 性質獨特。從北極斯匹次卑爾根島王灣分離的海洋細菌多具有褐藻膠、果膠、淀粉、木聚糖或羧甲基纖維素的低溫降解酶[49]。這也說明了極地微生物糖類降解酶資源開發的良好前景。

表3 近年新發現的極地微生物脂肪酶/酯酶簡況

續表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。該酶在淀粉工業中使用廣泛, 在酶制劑市場占有很大份額。α-淀粉酶是內切酶, 用于紡織品、紙張、食品、生物燃料、洗滌劑和制藥工業的多種生物加工工藝[50]。南極喬治王島海水細菌的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溫度[50]。南極深海沉積細菌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現耐熱淀粉酶[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Ca2+[53]。北極王灣沉積物細菌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菌β-葡糖苷酶EaBgl1A屬于GH1家族, 最適條件30℃、pH 7, 對葡萄糖的耐受性好于幾種商用酶[58], 晶體結構解析表明其適應低溫的主要機制是形成四聚體[59]。北極王灣表層海水細菌的新型內切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℃活性穩定, 在GH9家族纖維素酶中熱穩定性最好[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α-1,3和β-1,4糖苷鍵交聯形成。卡拉膠酶能降解卡拉膠產生水溶性好、生物活性高的卡拉膠寡糖, 在食品工業及醫藥等領域具有良好的應用前景。潘愛紅等[62]優化了南極普里茲灣沉積物細菌卡拉膠酶的產量。胡秋實等[63]從北極海水中篩選出四株高產卡拉膠酶的假交替單胞菌菌株, 酶的最適反應溫度均為20℃。林歡等[64]從南極海藻分離一株高產卡拉膠酶的細菌, 優化產酶條件, 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成。瓊膠酶能降解瓊膠產生有活性的瓊寡糖。南極普里茲灣沉積物細菌NJ21有三種外切型β瓊膠酶, 產物均為新瓊二糖。GH42家族的瓊膠酶Aga1161最適條件40℃、pH 8, 40℃活性不穩定[65]; Aga3463屬于GH86家族, 最適條件50℃、pH 7, 50℃活性不穩定[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菌β瓊膠酶屬于GH16家族, 最適條件40℃、pH 7, 50℃不穩定[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。該酶在乳制品工業中應用廣泛。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突變酶的低溫催化參數及空間結構模型, 說明一個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]。該酶晶體結構解析發現其適應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]。來自北極加拿大海盆海冰冰芯的海單胞菌有兩個半乳糖苷酶: 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]。兩個酶分別屬于GH2和GH42家族。北極王灣沉積物細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鏈。木聚糖酶可以將木聚糖降解為木糖及木寡糖, 廣泛應用于造紙、紡織、食品、飼料等行業。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菌的GH10家族木聚糖酶最適條件35℃、pH 7—9, 以定向進化和隨機突變結合的策略優化了該酶的熱穩定性[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株產褐藻酸裂解酶的細菌, 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, 裂解褐藻酸的產物為二糖。前述產瓊膠酶的南極細菌NJ21的褐藻酸裂解酶Al163屬于多糖裂解酶6家族(PL6)的內切酶, 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、紡織和造紙行業應用廣泛, 可分為聚半乳糖醛酸酶、裂解酶和果膠甲酯酶三類[83]。南極阿斯曼山細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、免疫學等領域應用廣泛[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℃活性不穩定[86]。喬治王島近岸表層海水細菌磷酸酶最適條件20—22℃、pH 7, 在48℃活性不穩定, Mg2+能提高其活性和熱穩定性[87]。

5.2 超氧化物歧化酶

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

6 展望

上文列舉了近年來極地微生物酶資源開發簡況, 主要關注了文獻報道較多、應用價值較高的幾類酶。報道的新酶以熱穩定性較低的低溫酶為主, 個別酶熱穩定性較好, 這些低溫酶大多有耐鹽能力。值得注意的是高溫酶的報道, 挪威生命科學大學Eijsink等從北極揚馬延島北部的揚馬延熱液口發現了三個高溫酶: 木聚糖酶[78]、褐藻酸裂解酶[82]和纖維素酶[61], 都具有理想的應用前景。說明極地的高溫環境雖然罕見, 但也蘊藏著獨特的酶資源。該研究將酶的底物(預處理的歐洲云杉木屑)在70℃的海底沉積中放置1年, 再回收測宏基因組獲取新酶基因。這種原位誘導篩選的方式很少用于極地微生物的新酶篩選, 有望成為將來常見的高效篩選策略。

目前傳統的平板培養法篩選菌株的新酶仍是國內外最常用的方法。由于其簡便易操作, 預計將來也不會被宏基因組等非培養方法替代。宏基因組作為重要的非培養篩選方法, 能克服眾多極地菌株無法培養的困難, 直接在基因水平發現新酶, 其基于功能[31,39-40,46]和基于序列[47,61,78,82]的篩選策略已經在極地微生物資源開發中獲得了眾多新酶, 今后必將成為常規的新酶篩選方法。此外, 從極地菌株的全基因組序列中發掘新酶也成為了常見的方法[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序費用的下降和公開的極地微生物全基因組數據的迅速增長, 可以推測從全基因組數據中獲取新酶基因的方法將逐漸替代傳統的酶基因克隆方法, 如基因組文庫篩選或簡并引物PCR擴增法。

多數極地微生物新酶篩選工作的應用目標明確, 獲得了大量有潛在工業應用價值的酶, 不過未見真正轉化為商用酶制劑的報道, 可能受限于酶性質、酶制劑開發周期以及廠商對產品來源保密。

在極地微生物酶的研究內容上, 篩選、基因克隆表達和酶的純化與性質研究是主流, 酶催化機理和晶體結構解析等更深入的研究較少, 蛋白質工程改造工作也不多。酶分子改造能進一步提高酶的工作效能, 而理論研究是蛋白質工程的重要指導, 可以預期今后會有更多極地微生物酶理論研究及分子改造工作。

1 DE Pascale D, De Santi C, Fu J, et al. The microbial diversity of Polar environments is a fertile ground for bioprospecting[J]. Marine Genomics, 2012, 8: 15-22.

2 丁新彪, 叢柏林, 張揚, 等. 南極普里茲灣及鄰近海域沉積物微生物多樣性與生理生化研究[J]. 海洋科學進展, 2014, 32(2): 209-218.

3 董寧. 東南極格羅夫山土壤微生物多樣性及其可培養細菌的產酶和抗菌活性篩選[D]. 青島: 中國海洋大學, 2014.

4 郝文惠. 大西洋與南海深海沉積物真菌多樣性分析及極地產低溫酶菌種的篩選[D]. 廈門: 廈門大學, 2014.

5 張麗珉, 趙琳, 叢柏林. 南極羅斯海區域可培養微生物分離鑒定及產低溫酶能力初步篩選[J]. 海洋學報, 2018, 40(8): 152-164.

6 LEE Y M, JUNG Y J, HONG S G, et al. Diversity and physiological characteristics of culturable bacteria from marine sediments of Ross Sea, Antarctica[J]. The Korean Journal of Microbiology, 2014, 50(2): 119-127.

7 TOMOVA I, STOILOVA-DISHEVA M, VASILEVA-TONKOVA E. Characterization of heavy metals resistant heterotrophic bacteria from soils in the Windmill Islands region, Wilkes Land, East Antarctica[J]. Polish Polar Research, 2014, 35(4): 593-607.

8 TSUJI M. Genetic diversity of yeasts from East Ongul Island, East Antarctica and their extracellular enzymes secretion[J]. Polar Biology, 2018, 41(2): 249-258.

9 BARAHONA S, YUIVAR Y, SOCIAS G, et al. Identification and characterization of yeasts isolated from sedimentary rocks of Union Glacier at the Antarctica[J]. Extremophiles, 2016, 20(4): 479-491.

10 LEE Y M, KIM E H, LEE H K, et al. Biodiversity and physiological characteristics of Antarctic and Arctic lichens-associated bacteria[J]. World Journal of Microbiology and Biotechnology, 2014, 30(10): 2711-2721.

11 SINGH P, SINGH S M, ROY U. Taxonomic characterization and the bio-potential of bacteria isolated from glacier ice cores in the High Arctic[J]. Journal of Basic Microbiology, 2016, 56(3): 275-285.

12 SINGH P, SINGH S M, DHAKEPHALKAR P. Diversity, cold active enzymes and adaptation strategies of bacteria inhabiting glacier cryoconite holes of High Arctic[J]. Extremophiles, 2014, 18(2): 229-242.

13 SINGH P, ROY U, TSUJI M. Characterisation of yeast and filamentous fungi from Br?ggerbreen glaciers, Svalbard[J]. Polar Record, 2016, 52(4): 442-449.

14 Salam S, Lekshmi S, Silvester R, et al. Effect of environmental factors on growth and enzyme production of cold adapted bacteria from water and sediment of Kongsjord, Ny-Alesund, Arctic[J]. Journal of Environmental Biology, 2017, 38(4): 579-585.

15 DE SANTI C, ALTERMARK B, DE PASCALE D, et al. Bioprospecting around Arctic Islands: Marine bacteria as rich source of biocatalysts[J]. Journal of Basic Microbiology, 2016, 56(3): 238-253.

16 張良. 北極海洋沉積物細菌群落結構及其胞外水解酶研究[D]. 青島: 國家海洋局第一海洋研究所, 2018.

17 MATSUI M, KAWAMATA A, KOSUGI M, et al. Diversity of proteolytic microbes isolated from Antarctic freshwater lakes and characteristics of their cold-active proteases[J]. Polar Science, 2017, 13: 82-90.

18 KIM H D, CHOI J I. Effect of temperature on growth rate and protease activity of Antarctic microorganisms[J]. Korean Journal of Microbiology and Biotechnology, 2014, 42(3): 293-296.

19 Pereira J Q, Lopes F C, Petry M V, et al. Isolation of three novel Antarctic psychrotolerant feather-degrading bacteria and partial puri?cation of keratinolytic enzyme fromsp. A03[J]. International Biodeterioration and Biodegradation, 2014, 88: 1-7.

20 Pereira J Q, Ambrosini A, Passaglia L M P, et al. A new cold-adapted serine peptidase from Antarcticsp. A03: Insights about enzyme activity at low temperatures[J]. International Journal of Biological Macromolecules, 2017, 103: 854-862.

21 PARK H J, LEE C W, KIM D, et al. Crystal structure of a cold-active protease (Pro21717) from the psychrophilic bacterium,PAMC 21717, at 1.4 ? resolution: Structural adaptations to cold and functional analysis of a laundry detergent enzyme[J]. PLoS One, 2018, 13(2): e0191740.

22 da Silva Nascimento T C E, de Sena A R, Gomes J E G, et al. Extracellular serine proteases bysp. L1-4B isolated from Antarctica: Overproduction using cactus pear extract with response surface methodology[J]. Biocatalysis and Agricultural Biotechnology, 2015, 4(4): 737-744.

23 LYLLOFF J E, HANSEN L B S, JEPSEN M, et al. Genomic and exoproteomic analyses of cold- and alkaline-adapted bacteria reveal an abundance of secreted subtilisin-like proteases[J]. Microbial Biotechnology, 2016, 9(2): 245-256.

24 Alias N, AHMAD MAZIAN M, Salleh A B, et al. Molecular cloning and optimization for high level expression of cold-adapted serine protease from Antarctic yeastPI12[J]. Enzyme Research, 2014, 2014: 1-20.

25 KIM H D, KIM S M, CHOI J I. Purification, characterization, and cloning of a cold-adapted protease from Antarctic[J]. Journal of Microbiology and Biotechnology, 2018, 28(3): 448-453.

26 LARIO L D, CHAUD L, ALMEIDA M G, et al. Production, purification, and characterization of an extracellular acid protease from the marine Antarctic yeastL7[J]. Fungal Biology, 2015, 119(11): 1129-1136.

27 GAO B, HE L, WEI D Z, et al. Identification and magnetic immobilization of a pyrophilous aspartic protease from Antarctic psychrophilic fungus[J]. Journal of Food Biochemistry, 2018, 42(6): e12691.

28 SANTOS A F, PIRES F, JESUS H E, et al. Detection of proteases fromandisolated from Antarctic soil[J]. Annals of the Brazilian Academy of Sciences, 2015, 87(1): 109-119.

29 PARK H J, LEE Y M, KIM S, et al. Identification of proteolytic bacteria from the Arctic Chukchi Sea expedition cruise and characterization of cold-active proteases[J]. Journal of Microbiology, 2014, 52(10): 825-833.

30 Qoura F, Kassab E, Rei?e S, et al. Characterization of a new, recombinant thermo-active subtilisin-like serine protease derived from[J]. Journal of Molecular Catalysis B: Enzymatic, 2015, 116: 16-23.

31 王光龍. 利用功能宏基因組技術對北極、大西洋海底沉積物中的新型蛋白酶、酯酶進行篩選、鑒定和性質研究[D]. 濟南: 山東大學, 2014.

32 郝文惠, 王凡羽, 郭玉, 等. 南極深海沉積物中產低溫脂肪酶菌株的篩選與基因克隆[J]. 應用海洋學學報, 2014, 33(3): 306-311.

33 Maharana A K, SINGH S M. A cold and organic solvent tolerant lipase produced by Antarctic strainsp. Y-23[J]. Journal of Basic Microbiology, 2018, 58(4):331-342.

34 WI A R, JEON S J, KIM S, et al. Characterization and a point mutational approach of a psychrophilic lipase from an arctic bacterium,[J]. Biotechnology Letters, 2014, 36(6): 1295-1302.

35 ZHANG Y, JI F L, WANG J Y, et al. Purification and characterization of a novel organic solvent-tolerant and cold-adapted lipase fromsp. ZY124[J]. Extremophiles, 2018, 22(2): 287-300.

36 楊文娟. 極端環境細菌脂肪酶資源挖掘及其在生物制藥領域的應用探索[D]. 武漢: 華中科技大學, 2017.

37 PARK S H, KIM S J, PARK S, et al. Characterization of organic solvent-tolerant lipolytic enzyme fromisolated from the Antarctic Ocean[J]. Applied Biochemistry and Biotechnology, 2019, 187(3): 1046-1060.

38 Wicka M, Wanarska M, Krajewska E, et al. Cloning, expression, and biochemical characterization of a cold-active GDSL-esterase of asp. S9 isolated from Spitsbergen island soil[J]. Acta Biochimica Polonica, 2016, 63(1): 117-125.

39 PETROVSKAYA L E, NOVOTOTSKAYA-VLASOVA K A, SPIRINA E V, et al. Expression and characterization of a new esterase with GCSAG motif from a permafrost metagenomic library[J]. FEMS Microbiology Ecology, 2016, 92(5): fiw046.

40 DE SANTI C, ALTERMARK B, PIERECHOD M M, et al. Characterization of a cold-active and salt tolerant esterase identified by functional screening of Arctic metagenomic libraries[J]. BMC Biochemistry, 2016, 17: 1.

41 Castilla A, Panizza P, Rodríguez D, et al. A novel thermophilic and halophilic esterase fromsp. R02, the first member of a new lipase family (Family XVII)[J]. Enzyme and Microbial Technology, 2017, 98: 86-95.

42 HONG D K, JANG S H, LEE C W. Gene cloning and characterization of a psychrophilic phthalate esterase with organic solvent tolerance from an Arctic bacteriumPAMC 26605[J]. Journal of Molecular Catalysis B: Enzymatic, 2016, 133(Suppl.1): 337-345.

43 KASHIF A, TRAN L H, JANG S H, et al. Roles of active-site aromatic residues in cold adaption ofesterase EstSP1[J]. ACS Omega, 2017, 2(12): 8760-8769.

44 De Santi C, Tedesco P, Ambrosino L, et al. A new alkaliphilic cold-active esterase from the psychrophilic marine bacteriumsp.: Functional and structural studies and biotechnological potential[J]. Applied Biochemistry and Biotechnology, 2014, 172(6): 3054-3068.

45 鄧盾, 張云, 孫愛君, 等. 一個新穎南極微生物酯酶EST112-2的功能鑒定和在手性叔醇(S)-芳樟醇制備中的應用[J]. 有機化學, 2018, 38(5): 1185-1192.

46 TCHIGVINTSEV A, TRAN H, POPOVIC A, et al. The environment shapes microbial enzymes: five cold-active and salt-resistant carboxylesterases from marine metagenomes[J]. Applied Microbiology and Biotechnology, 2015, 99(5): 2165-2178.

47 DE SANTI C, WILLASSEN N P, WILLIAMSON A. Biochemical characterization of a family 15 carbohydrate esterase from a bacterial marine arctic metagenome[J]. PLoS One, 2016, 11(7): e0159345.

48 DE SANTI C, GANI O A, HELLAND R, et al. Structural insight into a CE15 esterase from the marine bacterial metagenome[J]. Scientific Reports, 2017, 7: 17278.

49 JAIN A, KRISHNAN K P. A glimpse of the diversity of complex polysaccharide-degrading culturable bacteria from Kongsfjorden, Arctic Ocean[J]. Annals of Microbiology, 2017, 67(2): 203-214.

50 Sanchez A, Ravanal M C, Andrews B A, et al. Heterologous expression and biochemical characterization of a novel cold-active α-amylase from the Antarctic bacteriasp. 2-3[J]. Protein Expression and Purification, 2019, 155: 78-85.

51 潘愛紅, 李江, 谷曉倩, 等. 南極菌sp. A211-5產堿性α-淀粉酶Amy172的克隆表達及酶學性質研究[J]. 化學與生物工程, 2019, 36(5): 36-42.

52 GAO B, MAO Y, ZHANG L, et al. A novel saccharifying α-amylase of Antarctic psychrotolerant fungi: Gene cloning, functional expression, and characterization[J]. Starch, 2016, 68(1/2): 20-28.

53 CARRASCO M, ALCAíNO J, CIFUENTES V, et al. Purification and characterization of a novel cold adapted fungal glucoamylase[J]. Microbial Cell Factories, 2017, 16: 75.

54 薛毅, 王梅, 方澤民, 等. 低溫、嗜鹽α-淀粉酶Amy3的克隆、表達及重組酶性質[J]. 微生物學報, 2018, 58(2): 336-345.

55 LI W, XUE Y, LI J, et al. A cold-adapted and glucose-stimulated type II α-glucosidase from a deep-sea bacteriumsp. K8[J]. Biotechnology Letters, 2016, 38(2): 345-349.

56 LI W, FAN H, HE C, et al. PspAG97A: a halophilic α-glucoside hydrolase with wide substrate specificity from glycoside hydrolase family 97[J]. Journal of Microbiology and Biotechnology, 2016, 26(11): 1933-1942.

57 Elleuche S, Qoura F M, Lorenz U, et al. Cloning, expression and characterization of the recombinant cold-active type-I pullulanase from[J]. Journal of Molecular Catalysis B: Enzymatic, 2015, 116: 70-77.

58 Crespim E, Zanphorlin L M, de Souza F H M, et al. A novel cold-adapted and glucose-tolerant GH1 β-glucosidase fromB7[J]. International Journal of Biological Macromolecules, 2016, 82: 375-380.

59 Zanphorlin L M, de Giuseppe P O, Honorato R V, et al. Oligomerization as a strategy for cold adaptation: Structure and dynamics of the GH1 β-glucosidase fromB7[J]. Scientific Reports, 2016, 6: 23776.

60 ZHAO F, CAO H, ZHAO L, et al. A novel subfamily endo-β-1,4-glucanases in glycoside hydrolase family 10[J]. Applied and Environmental Microbiology, 2019, 85(18): e01029-19.

61 STEPNOV A A, FREDRIKSEN L, STEEN I H, et al. Identification and characterization of a hyperthermophilic GH9 cellulase from the Arctic Mid-Ocean Ridge vent field[J]. PLoS One, 2019, 14(9): e0222216.

62 潘愛紅, 李江, 王蕾, 等. 南極交替單胞菌R11-5產卡拉膠酶的發酵條件優化[J]. 微生物學通報, 2018, 45(9): 2022-2034.

63 胡秋實, 蘇忠亮, 李江. 兩種極端環境微生物產卡拉膠酶的研究[J]. 化學與生物工程, 2014, 31(5): 17-20.

64 林歡, 李然, 王瑞玉, 等. 南極低溫降解卡拉膠菌株的篩選、鑒定、產酶條件及酶學性質的初步研究[J]. 化學與生物工程, 2018, 35(5): 47-52.

65 LI J, SHA Y. Expression and enzymatic characterization of a cold-adapted β-agarase from Antarctic bacteriumsp. NJ21[J]. Chinese Journal of Oceanology and Limnology, 2015, 33(2): 319-327.

66 LI J, XIE M S, GAO Y. Identification and biochemical characterization of a novel exo-type β-agarase Aga3463 from an Antarcticsp. strain[J]. International Journal of Biological Macromolecules, 2019, 129: 162-170.

67 劉秀萌, 李江, 侯旭光, 等. 南極菌產瓊膠酶aga3311的表達、性質及其降解特性[J]. 微生物學報, 2016, 56(9): 1468-1476.

68 HAN Z, ZHANG Y, YANG J. Biochemical characterization of a new β-agarase from[J]. International Journal of Molecular Sciences, 2019, 20(9): 2143.

69 LAYE V J, KARAN R, KIM J M, et al. Key amino acid residues conferring enhanced enzyme activity at cold temperatures in an Antarctic polyextremophilic β-galactosidase[J]. Proceedings of the National Academy of Sciences of the United States of America, 2017, 114(47): 12530-12535.

70 Pawlak-Szukalska A, Wanarska M, Popinigis A T, et al. A novel cold-active β-D-galactosidase with transglycosylation activity from the Antarcticsp. 32cB – Gene cloning, puri?cation and characterization[J]. Process Biochemistry, 2014, 49(12): 2122–2133.

71 RUTKIEWICZ M, BUJACZ A, BUJACZ G. Structural features of cold-adapted dimeric GH2β-D-galactosidase fromsp. 32cB[J]. Biochimica et Biophysica Acta-Proteins and Proteomics, 2019, 1867(9): 776-786.

72 孫茜. 北極海單胞菌基因組學分析及其β-半乳糖苷酶基因的功能研究[D]. 上海: 華東理工大學, 2015.

73 DING H T, ZENG Q, ZHOU L L, et al. Biochemical and structural insights into a novel thermostable β-1, 3-galactosidase fromsp. BSi20414[J]. Marine Drugs, 2017, 15(1): 13.

74 Alikunju A P, Joy S, Salam J A, et al. Functional characterization of a new cold-adapted β-galactosidase from an arctic fjord sediment bacteriaMCC 3423[J]. Catalysis Letters, 2018, 148(10): 3223-3235.

75 DEL-CID A, UBILLA P, RAVANAL M C, et al. Cold-active xylanase produced by fungi associated with Antarctic marine sponges[J]. Applied Biochemistry and Biotechnology, 2014, 172(1): 524-532.

76 Gil-Durán C, Ravanal M C, Ubilla P, et al. Heterologous expression, puri?cation and characterization of a highly thermolabile endoxylanase from the Antarctic fungussp.[J]. Fungal Biology, 2018, 122(9): 875-882.

77 ACEVEDO J P, REETZ M T, ASENJO J A, et al. One-step combined focused epPCR and saturation mutagenesis for thermostability evolution of a new cold-active xylanase[J]. Enzyme and Microbial Technology, 2017, 100: 60-70.

78 FREDRIKSEN L, STOKKE R, JENSEN M S, et al. Discovery of a thermostable GH10 xylanase with broad substrate specificity from the arctic mid-ocean ridge vent system[J]. Applied and Environmental Microbiology, 2019, 85(6): e02970-18.

79 東升. 北極褐藻酸裂解酶分泌菌株的多樣性分析和褐藻酸裂解酶的成熟與催化機制研究[D]. 濟南: 山東大學, 2014.

80 高楊. 南極真菌產褐藻膠裂解酶Aly-i7的基因克隆、表達和酶學性質研究[D]. 青島: 青島科技大學, 2018.

81 XIE M S, LI J, HE P Q, et al. Expression and characterization of a bifunctional alginate lyase named Al163 from the Antarctic bacteriumsp. NJ-21[J]. Journal of Oceanology and Limnology, 2018, 36(4): 1304-1314.

82 VUORISTO K S, FREDRIKSEN L, OFTEBRO M, et al. Production, characterization, and application of an alginate lyase, AMOR_PL7A, from hot vents in the arctic mid-ocean ridge[J]. Journal of Agricultural and Food Chemistry, 2019, 67(10): 2936-2945.

83 Carrasco M, Rozas J M, Alcaíno J, et al. Pectinase secreted by psychrotolerant fungi: identification, molecular characterization and heterologous expression of a cold-active polygalacturonase fromsp.[J]. Microbial Cell Factories, 2019, 18: 45.

84 TANG Y M, WU P, JIANG S J, et al. A new cold-active and alkaline pectate lyase from Antarctic bacterium with high catalytic efficiency[J]. Applied Microbiology and Biotechnology, 2019, 103(13): 5231-5241.

85 Nalini P, Ellaiah P, Prabhakar T, et al. Microbial alkaline phosphatases in bioprocessing[J]. International Journal of Current Microbiology and Applied Sciences, 2015, 4(3): 384-396.

86 Yuivar Y, Barahona S, Alcaíno J, et al. Biochemical and thermodynamical characterization of glucose oxidase, invertase, and alkaline phosphatase secreted by Antarctic yeasts[J]. Frontiers in Molecular Biosciences, 2017, 4: 86.

87 Pellizza L A, Smal C, Ithuralde R E, et al. Structural and functional characterization of a cold-adapted stand-alone TPM domain reveals a relationship between dynamics and phosphatase activity[J]. The FEBS Journal, 2016, 283(23): 4370-4385.

88 WANG Q F, WANG Y F, HOU Y H, et al. Cloning, expression and biochemical characterization of recombinant superoxide dismutase from Antarctic psychrophilic bacteriumsp. ANT506[J]. Journal of Basic Microbiology, 2016, 56(7): 753-761.

89 KAN G F, WEN H, WANG X F, et al. Cloning and characterization of iron-superoxide dismutase in Antarctic yeast strainAN5[J]. Journal of Basic Microbiology, 2017, 57(8): 680-690.

90 ABRASHEV R, FELLER G, KOSTADINOVA N, et al. Production, purification, and characterization of a novel cold-active superoxide dismutase from the Antarctic strain363[J]. Fungal Biology, 2016, 120(5): 679-689.

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月收到來稿, 2019年11月收到修改稿

中國工程院戰略研究(2018-ZD-08)、農業農村部極地漁業開發重點實驗室開放課題(2019OPF02)和中國水產科學研究院基本科研業務費(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發研究。E-mail: weiwang@ysfri.ac.cn

郝建華, E-mail: haojh@ysfri.ac.cn

10. 13679/j.jdyj.20190039