Paraglaciecola hydrolytica中新型β-琼胶酶Aga2的异源表达及酶学性质分析  

Heterologous Expression and Enzymatic Properties Analysis of Novelβ-agarase Aga2 from Paraglaciecola hydrolytica

作　　者：王小桃 邹杭 吴怡 向省维 吕华 刘超兰[1] 林家富[1] 王欣荣[1] 褚以文[1] 宋涛 WANG Xiao-tao;ZOU Hang;WU Yi;XIANG Shen-wei;LV Hua;LIU Chao-lan;LIN Jia-fu;WANG Xin-rong;CHU Yi-wen;SONG Tao(Antibiotics Research and Re-evaluation Key Laboratory of Sichuan Province,School of Pharmacy,Chengdu University,Chengdu 610106)

机构地区：[1]成都大学药学院抗生素研究与再评价四川省重点实验室,成都610106

出　　处：《生物技术通报》2022年第11期258-268,共11页Biotechnology Bulletin

基　　金：中国湖泊微生物多样性及资源调查(科技部科技基础工作专项2017FY100300);四川省科技项目(2020YJ0276);抗生素研究与再评价四川省重点实验室开放课题(ARRLKF19-01)。

摘　　要：琼胶寡糖具有抗氧化、抗肿瘤和调节肠道菌群等多种生物活性,而微生物来源的琼胶酶是酶法制备琼胶寡糖的重要工具酶。目前报道的琼胶酶数量较少,而具有优良酶学特性的琼胶酶数量更少,极大阻碍了酶法制备琼胶寡糖的工艺开发进程。因此有必要发掘更多微生物来源的新颖琼胶酶。从副居冰菌属Paraglaciecola hydrolytica细菌基因组中挖掘到一个新颖琼胶酶基因aga2,构建至表达载体pET28a(+),并在大肠杆菌BL21(DE3)中进行表达;通过镍金属亲和层析纯化蛋白并探究温度、pH、金属离子、NaCl浓度对Aga2活性的影响;采用^(13)C核磁共振、薄层色谱和基质辅助激光解吸飞行时间质谱分析酶解产物。Aga2与已知琼胶酶的最高相似度为53.7%。同时Aga2在IPTG(Isopropyl-beta-D-thiogalactopyranoside)浓度为90μmol/L,20℃下诱导9 h时,可溶性表达量最高。纯化的Aga2最适反应温度为50℃,且40℃孵育3 h后仍保持72.9%的相对酶活力,具有较好的温度稳定性。Aga2的最适pH为6.0,在不同pH(4-9)下放置5 h后,仍保持62.6%以上的相对酶活力,具有较好的pH稳定性。Aga2在NaCl浓度为2.5 mol/L时仍保持78%的相对酶活力,具有较强的盐耐受性。同时Aga2对Ni^(2+)、Ca^(2+)、Ba^(2+)、K^(+)、Mg^(2+)、Zn^(2+)、EDTA、DTT、Urea、SDS、TritionX-100有耐受性。薄层色谱结果表明,该酶属于内切型β琼胶酶,产物为新琼四糖和新琼六糖。Aga2具有温度和pH稳定性、盐耐受性和重金属离子耐受性,具有良好的工业应用前景。Agarooligosaccharides have various biological activities including anti-oxidant,anti-tumor and intestinal flora regulation,therefore microbe-sourced agarase is an importantone for enzymatic preparation of agarooligosaccharides.At present,the number of reported agarases is small,and the number of agarases with excellent enzymatic properties is very few,which seriously hinders the development of enzymatic preparation of agarooligosaccharides.Therefore,it is necessary to study more novel agarases from microorganism.A novel agarase gene aga2 was mined from Paraglaciecola hydrolytica,then constructed into the expression vector pET28a(+),and expressed in Escherichia coli BL21(DE3).The protein was purified by nickel ion affinity chromatography,and then the effects of temperature,pH,metal ions and NaCl concentration on the Aga2 activity were studied.^(13)C nuclear magnetic resonance,thin-layer chromatography and matrix-assisted laser desorption time-of-flight mass spectrometry were used to analyze the enzymatic hydrolysis products.The highest similarity between Aga2 and known agarase was 53.7%.The soluble expression of Aga2 was the highest when the concentration of IPTG(isopropyl-beta-D-thiogalactopyranoside)was 90μmol/L and induced at 20℃for 9 h.The optimal temperature for purified Aga2 was 50℃,and 72.9%enzyme activity remained after incubating at 40℃for 3 h,showing good temperature stability.The optimal pH of Aga2 was 6.0 and over 62.6%relative enzyme activity remained after 5 h at pH 4-9,thus had good pH stability.Aga2 still maintained 78%relative enzyme activity when the NaCl concentration was 2.5 mol/L,and had strong salt tolerance.At the same time,Aga2 was resistant to Ni^(2+),Ca^(2+),Ba^(2+),K^(+),Mg^(2+),Zn^(2+),EDTA,DTT,Urea,SDS,and TritonX-100.The results of thin-layer chromatography showed that the enzyme belonged to endo-β-agarase,and the products were new neoagarotetraose and neoagarohexaose.Aga2 has temperature stability and pH stability,NaCl tolerance and heavy metal ion tolerance,thus has good ind

关 键 词：Paraglaciecola hydrolytica 琼胶酶 异源表达 酶学性质 琼胶寡糖 

分 类 号：O636.1[理学—高分子化学] Q78[理学—化学]