Biotechnology Bulletin ›› 2022, Vol. 38 ›› Issue (11): 258-268.doi: 10.13560/j.cnki.biotech.bull.1985.2021-1617

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

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()   

  1. Antibiotics Research and Re-evaluation Key Laboratory of Sichuan Province,School of Pharmacy,Chengdu University,Chengdu 610106
  • Received:2021-12-30 Online:2022-11-26 Published:2022-12-01
  • Contact: SONG Tao E-mail:wangxiaotao@stu.cdu.edu.cn;songtao@cdu.edu.cn

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