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

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

Abstract

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

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.

Figures/Tables 15

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

Fig.2 PCR electrophoresis result of amplified agarase gene aga2

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

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

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

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

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

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

Fig.9 Effects of different reagents on Aga2 activity

Fig. 10 Effects of NaCl concentration on Aga2 activity

Fig. 11 Linewaeaver-Burk diagram of Aga2

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

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

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

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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