来源于Wenyingzhuangia属海洋细菌的一种β-琼胶酶的克隆表达及性质研究
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摘要
琼胶是一种重要的海洋多糖,其寡糖被证实具有丰富的生理调节功能。该研究拟从微生物基因组出发,利用生物信息学及分子生物学技术发掘新型琼胶酶。以海洋细菌Wenyingzhuangia fucanilytica CZ1127~T为实验材料,从该菌的基因组中发掘出1条潜在的GH16家族β-琼胶酶编码基因序列aga16A,通过分子克隆、异源表达及分离纯化,证实其编码的蛋白Aga16A_Wf具有琼胶酶活力。酶学性质及作用方式分析表明,Aga16A_Wf的最适反应温度(50℃)高于琼胶的凝胶温度,且该酶展现出适冷性;Aga16A_Wf的动力学常数K_m为3.6 g/L,K_(cat)为287.88 s~(-1);该酶为内切型水解酶,最小作用底物为四糖,最小产物为二糖。Aga16A_Wf可作为高效制备琼胶寡糖的工具酶,该酶的获取为琼胶及琼胶寡糖的进一步研究与应用提供了良好支撑。
Agarose is an important marine polysaccharide and its oligosaccharides were considered to have a series of bioactive functions. A gene,aga16 A,encoding a putative GH16 amily β-agarase was recovered from the genome of Wenyingzhuangia funcanilytica CZ1127 T,and heterologously expressed and purified. The recombinant Aga16 A_Wf was confirmed to be an agarase with the optimum reaction temperature of 50 ℃,higher than the gelling temperature of agar. Besides,the enzyme was active under cold environment. Its kinetic constants Kmand Kcatwere3. 6 g/L and 287. 88 s-1,respectively. Aga16 A_Wf was an endo-acting enzyme,its smallest substrate and product were tetrasaccharide and disaccharide,respectively. Aga16 A_Wf could be served as a favorable tool to produce agar oligosaccharides efficiently. It is also useful for further studies on agar and its oligosaccharides.
Agarose is an important marine polysaccharide and its oligosaccharides were considered to have a series of bioactive functions. A gene,aga16 A,encoding a putative GH16 amily β-agarase was recovered from the genome of Wenyingzhuangia funcanilytica CZ1127 T,and heterologously expressed and purified. The recombinant Aga16 A_Wf was confirmed to be an agarase with the optimum reaction temperature of 50 ℃,higher than the gelling temperature of agar. Besides,the enzyme was active under cold environment. Its kinetic constants Kmand Kcatwere3. 6 g/L and 287. 88 s-1,respectively. Aga16 A_Wf was an endo-acting enzyme,its smallest substrate and product were tetrasaccharide and disaccharide,respectively. Aga16 A_Wf could be served as a favorable tool to produce agar oligosaccharides efficiently. It is also useful for further studies on agar and its oligosaccharides.
引文
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[25]LI Renkuan,CHEN Zeng,YING Xijuan,et al. A novel GH16 beta-agarase isolated from a marine bacterium,Microbulbifer sp. BN3 and its characterization and high-level expression in Pichia pastoris[J]. International Journal of Biological Macromolecules,2018,119:1 164-1 170.
[26]ZHANG Weiwei,SUN Li. Cloning,characterization,and molecular application of a beta-agarase gene from Vibrio sp. strain V134[J]. Applied and Environmental Microbiokogy,2007,73(9):2 825-2 831.
[27]OHTA Y,HATADA Y,NOGI Y,et al. Enzymatic properties and nucleotide and amino acid sequences of a thermostable beta-agarase from a novel species of deep-sea Microbulbifer[J]. Applied Microbiology and Biotechnology,2004,64(4):505-514.
[28]OHTA Y,NOGI Y,MIYAZAKI M,et al. Enzymatic properties and nucleotide and amino acid sequences of a thermostable beta-agarase from the novel marine isolate,JAMB-A94[J]. Bioscience Biotechnology and Biochemistry,2004,68(5):1 073-1 081.
[29]MEI Jianfeng,TANG Zhongxiu,YI Yu,et al. Purification and characterization of beta-agarase from Paenibacillus sp.[J]. Food Science and Biotechnology,2014,23(5):1 605-1 609.
[30]杨光.海洋来源琼胶酶及其基因的克隆与表达[D].福州:福州大学,2015.
[31]CHAUHAN P S,SAXENA A. Bacterial carrageenases:An overview of production and biotechnological applications[J]. 3 Biotech,2016,6(2):146.
[32]SCHEFER L,USOV I,MEZZENGA R. Anomalous stiffening and ion-induced coil-helix transition of carrageenans under monovalent salt conditions[J]. Biomacromolecules,2015,16(3):985-991.
[2]汪春牛,时杰,葛思思,等.卡拉胶与琼胶提取工艺的研究进展[J].海洋科学,2012,36(6):122-126.
[3]DUCKWORTH M,YAPHE W. The structure of agar:Part I. Fractionation of a complex mixture of polysaccharides[J]. Carbohydrate Research,1971,16(1):189-197.
[4]GIORDANO A,ANDREOTTI G,TRAMICE A,et al.Marine glycosyl hydrolases in the hydrolysis and synthesis of oligosaccharides[J]. Biotechnology Journal,2006,1(5):511-530.
[5]WU Shaochi,WEN Tuannan,PAN Chorngliang. Algal-oligosaccharide-lysates prepared by two bacterial agarases stepwise hydrolyzed and their anti-oxidative properties[J].Fisheries Science,2005,71(5):1 149-1 159.
[6]MAZUMDER S,GHOSAL P K,PUJOL C A,et al. Isolation,chemical investigation and antiviral activity of polysaccharides from Gracilaria corticata(Gracilariaceae,Rhodophyta)[J]. International Journal of Biological Macromolecules,2002,31(1-3):87-95.
[7]黄能平,阮灵伟,施泓. Flammeovirga sp. SJP92琼脂酶的分离纯化及其酶学性质[J].应用海洋学学报,2016,35(1):116-121.
[8]崔方元.嗜琼胶卵链菌比较基因组学研究及系列琼胶酶基因的克隆表达[D].青岛:中国海洋大学,2015.
[9]CHEN Feng,CHANG Yaoguang,DONG Shujun,et al.Wenyingzhuangia fucanilytica sp. nov.,a sulfated fucan utilizing bacterium isolated from shallow coastal seawater[J]. International Journal of Systematic and Evolutionary Microbiology,2016,66(9):3 270-3 275.
[10]CHANG Yaoguang,XUE Changhu,TANG Qingjuan,et al. Isolation and characterization of a sea cucumber fucoidan-utilizing marine bacterium[J]. Letters in Applied Microbiology,2010,50(3):301-307.
[11]YIN Yanbin,MAO Xizeng,YANG Jincai,et al. dbCAN:A web resource for automated carbohydrate-active enzyme annotation[J]. Nucleic Acids Research,2012,40(W1):W445-W451.
[12]PETERSEN T N,SREN B,HEIJNE G V,et al. Signal P 4. 0:Discriminating signal peptides from transmembrane regions[J]. Nature Methods,2011,8(10):785-786.
[13]WILKINS M R,GASTEIGER E,BAIROCH A,et al.Protein identification and analysis tools in the Ex PASy server[J]. Methods in Molecular Biology,1999,112:531-552.
[14]ALTSCHUL S F,MADDEN T L,SCHAFFER A A,et al. Gapped BLAST and PSI-BLAST:A new generation of protein database search programs[J]. Nucleic Acids Reseaech,1997,25(17):3 389-3 402.
[15]THOMPSON J D,GIBSON T J,FRDRIC P,et al. The CLUSTAL_X windows interface:Flexible strategies for multiple sequence alignment aided by quality analysis tools[J]. Nucleic Acids Research,1997,25(24):4 876-4 882.
[16]TAMURA K, STECHER G, PETERSON D, et al.MEGA6:Molecular evolutionary genetics analysis version6. 0[J]. Molecular Biology and Evolution,2013,30(12):2 725-2 729.
[17]LEVER M. A new reaction for colorimetric determination of carbohydrates[J]. Analytical Biochemistry,1972,47(1):273-279.
[18]张翠玉,薛长湖,于龙,等.基于p HBH法的岩藻聚糖硫酸酯酶酶活测定方法[J].中国食品学报,2013,13(7):200-206.
[19]ALLOUCH J,JAM M,HELBERT W,et al. The threedimensional structures of two beta-agarases[J]. Journal of Biological Chemistry,2003,278(47):47 171-47 180.
[20]史升耀,张燕霞,刘万庆,等.江蓠琼胶产率、物理性质和化学组成的季节变化[J].海洋与湖沼,1983,14(3):272-278.
[21]ALI A,EVA-MARA G D P,FABRA M J,et al. Agarose-based freeze-dried capsules prepared by the oil-induced biphasic hydrogel particle formation approach for the protection of sensitive probiotic bacteria[J]. Food Hydrocolioids,2019,87:487-496.
[22]CUI X,JIANG Yuechen,CHANG Liuyi,et al. Heterologous expression of an agarase gene in Bacillus subtilis,and characterization of the agarase[J]. International Journal of Biological Macromolecules,2018,120(Pt A):657-664.
[23]CUI Fangyuan,DONG Sujie,SHI Xiaochong,et al.Overexpression and characterization of a novel thermostable beta-Agarase YM01-3,from marine bacterium Catenovulum agarivorans YM01(T)[J]. Marine Drugs,2014,12(5):2 731-2 747.
[24]AN Ke,SHI Xiaochong,CUI Fangyuan,et al. Characterization and overexpression of a glycosyl hydrolase family16 beta-agarase YM01-1 from marine bacterium Catenovulum agarivorans YMO1(T)[J]. Protein Expression and Purification,2018,143:1-8.
[25]LI Renkuan,CHEN Zeng,YING Xijuan,et al. A novel GH16 beta-agarase isolated from a marine bacterium,Microbulbifer sp. BN3 and its characterization and high-level expression in Pichia pastoris[J]. International Journal of Biological Macromolecules,2018,119:1 164-1 170.
[26]ZHANG Weiwei,SUN Li. Cloning,characterization,and molecular application of a beta-agarase gene from Vibrio sp. strain V134[J]. Applied and Environmental Microbiokogy,2007,73(9):2 825-2 831.
[27]OHTA Y,HATADA Y,NOGI Y,et al. Enzymatic properties and nucleotide and amino acid sequences of a thermostable beta-agarase from a novel species of deep-sea Microbulbifer[J]. Applied Microbiology and Biotechnology,2004,64(4):505-514.
[28]OHTA Y,NOGI Y,MIYAZAKI M,et al. Enzymatic properties and nucleotide and amino acid sequences of a thermostable beta-agarase from the novel marine isolate,JAMB-A94[J]. Bioscience Biotechnology and Biochemistry,2004,68(5):1 073-1 081.
[29]MEI Jianfeng,TANG Zhongxiu,YI Yu,et al. Purification and characterization of beta-agarase from Paenibacillus sp.[J]. Food Science and Biotechnology,2014,23(5):1 605-1 609.
[30]杨光.海洋来源琼胶酶及其基因的克隆与表达[D].福州:福州大学,2015.
[31]CHAUHAN P S,SAXENA A. Bacterial carrageenases:An overview of production and biotechnological applications[J]. 3 Biotech,2016,6(2):146.
[32]SCHEFER L,USOV I,MEZZENGA R. Anomalous stiffening and ion-induced coil-helix transition of carrageenans under monovalent salt conditions[J]. Biomacromolecules,2015,16(3):985-991.
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