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

doi:10.13560/j.cnki.biotech.bull.1985.2021-1617

摘要/Abstract

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¹³C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90 μmol/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Ni²⁺、Ca²⁺、Ba²⁺、K⁺、Mg²⁺、Zn²⁺、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。

关键词: Paraglaciecola hydrolytica, 琼胶酶, 异源表达, 酶学性质, 琼胶寡糖

Abstract:

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. ¹³C 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 90 μmol/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 Ni²⁺，Ca²⁺，Ba²⁺，K⁺，Mg²⁺，Zn²⁺，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 industrial application prospects.

Key words: Paraglaciecola hydrolytica, agarase, heterologous expression, enzymatic property, agarooligosaccharide

王小桃, 邹杭, 吴怡, 向省维, 吕华, 刘超兰, 林家富, 王欣荣, 褚以文, 宋涛. Paraglaciecola hydrolytica中新型β-琼胶酶Aga2的异源表达及酶学性质分析[J]. 生物技术通报, 2022, 38(11): 258-268.

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. Heterologous Expression and Enzymatic Properties Analysis of Novel β-agarase Aga2 from Paraglaciecola hydrolytica[J]. Biotechnology Bulletin, 2022, 38(11): 258-268.

图/表 15

图1 最大似然法构建琼胶酶的进化树

Fig. 1 Phylogenetic tree analysis of Aga2 using max-likelihood method

图2 琼胶酶基因aga2的PCR扩增电泳图

Fig.2 PCR electrophoresis result of amplified agarase gene aga2

图3 琼胶酶Aga2的总蛋白电泳图 M：蛋白 marker；C：空载对照；上样量为 5-10 μg

Fig. 3 SDS-PAGE analysis of agarases Aga2 M：Protein marker. C：Empty control；the loading amount is 5-10 μg

图4 IPTG浓度、诱导温度和诱导时间对Aga2的影响 M：蛋白marker；A：1-7：表示IPTG的浓度为0 μmol/L、30 μmol/L、60 μmol/L、90 μmol/L、120 μmol/L、150 μmol/L、180 μmol/L时的蛋白样品；C：1-5：诱导温度为16℃、20℃、24℃、28℃、32℃时的蛋白样品；E：1-6：诱导时间为0 h、3 h、6 h、9 h、12 h、15 h时的蛋白样品

Fig.4 Effects of IPTG，temperature and induction time concentration on Aga2 M：Protein marker. A：1-7：protein samples when the concentration of IPTG is 0，30，60，90，120，150和 180 μmol/L，respectively. C：1-5：protein samples when the induction temperatures is 16℃，20℃，24℃，28℃，and 32℃. E：1-6：protein samples when the induction times is 0，3，6，9，12 and 15 h

图5 镍金属亲和层析纯化后的Aga2蛋白电泳图 M：蛋白marker；Aga2：纯化的Aga2；上样量为1-5 μg

Fig. 5 SDS-PAGE analysis of purified Aga2 after Ni affi-nity chromatography M：Protein marker. Aga2：Purified Aga2. The loading amount is 1-5 μg

图6 Aga2针对不同底物的酶活

Fig 6 Aga2’s enzyme activities against different substrates CMC-Na：Carboxymethylcellulose sodium

图7 琼胶酶Aga2最适温度（A）和耐热性分析（B）

Fig. 7 Optimal temperature（A）and hot tolerance（B）analysis of Aga2

图8 Aga2最适pH（A）和pH耐受性（B）分析

Fig. 8 Optimal pH（A）and pH tolerance（B）analysis of Aga2

图9 不同试剂对Aga2活性的影响

Fig.9 Effects of different reagents on Aga2 activity

图10 NaCl浓度对琼胶酶活性的影响

Fig. 10 Effects of NaCl concentration on Aga2 activity

图11 Aga2的Linewaeaver-Burk图

Fig. 11 Linewaeaver-Burk diagram of Aga2

图12 Aga2降解琼胶生成底物的碳核磁共振分析 G和A：D-半乳糖和3,6-L-内醚半乳糖；r和nr：还原端和非还原端；α和β：α碳原子和β碳原子

Fig.12 CNMR analysis of agar hydrolysis product catalyz-ed by Aga2 G and A：D-galactose and 3,6-Anhydro-L-galactose；r and nr：reducing and non-reducing ends；α and β：α carbon atoms and β carbon atoms

图13 琼胶酶 Aga2降解琼脂糖不同时间点的TLC图 NA8：新琼八糖标准品；NA6：新琼六糖标准品；NA4：新琼四糖标准品；NA2表示新琼二糖标准品

Fig. 13 TLC analysis of hydrolysis product by Aga2 during different time points NA8：Neoagarooctaose standard；NA6：neoagarohexaose standard；NA4：neoagarotetraose standard；NA2：neoagarobiose standard

图14 Aga2降解琼胶生成寡糖的MALDI-TOF-MS结果示意图 NA6：新琼六糖标准品；NA4：新琼四糖标准品

Fig. 14 MALDI-TOF-MS analysis of oligosaccharides from agar hydrolysis catalyzed by Aga2 NA6：Neoagarohexaose standard；NA4：neoagarotetraose standard

图15 Aga2降解不同长度寡糖的TLC结果分析 1-4：Aga2分别和新琼二糖、新琼四糖、新琼六糖、新琼八糖混合反应的产物；5-8分别为：新琼二糖标准品、新琼四糖标准品、新琼六糖标准品、新琼八糖标准品

Fig. 15 TLC analysis of different oligosaccharide hydroly-sis product catalyzed by Aga2 1-4：Mixed reaction products of Aga2 and neoagarobiose，Aga2 and neoagarote-traose，Aga2 and neoagarohexaose，Aga2 and neoagarooctaose，respectively. 5-8：Neoagarobiose standard，neoagarotetraose standard，neoagarohexaose standard，and neoagarooctaose standard，respectively

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