Skip to main content Accessibility help

Expression of β-agarase I DagA in prokaryotic cell and its activity identification

  • Zhou Yan-Sheng (a1), Wang Bao-Li (a1) (a2) and Qu Dong (a3)


The DagA gene and DagA(▽), which is a DagA gene encoding sequence without signal peptide, were cloned from genome DNA of Pseudoalteromonas atlantica 19262 by polymerase chain reaction (PCR). After ligation with pET21 vector, DagA and DagA(▽) were respectively expressed in Escherichia coli ER2566 using molecular chaperones DsbC and FkpA. A strain of ER2566-pET21a-DagA(▽)-DsbC was screened as a highly effective expressing system in the form of an inclusion body that had the target protein with up to 60% total bacterial protein. DagA protein was renatured and purified by dissolving it in 8 mol/l of urea, using Ni-NTA resin affinity chromatography and refolding using the urea gradient method. DagA with a molecular weight of ~30.8 kDa was identified by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and had the ability to digest agarose. In a pH range of 4.8–6.8, DagA maintained a bioactivity greater than 60%, with 5.8 being the optimum pH, and it exhibited activity at temperatures from 37°C to 60°C, with 55°C being the optimum temperature.


Corresponding author

*Corresponding author. E-mail:


Hide All
Bibb, MJ, Jones, GH, Joseph, R, Buttner, MJ and Ward, JM (1987) The agarase gene (DagA) of Streptomyces coelicolor A3 (2): Affinity purification and characterization of the cloned gene product. Journal of General Microbiology 133(8): 20892096.
Dong, JH, Hashikawa, S, Konishi, T, Tamaru, Y and Araki, T (2006) Cloning of the novel gene encoding β-agarase C from a marine bacterium, Vibrio sp. strain PO-303, and characterization of the gene product. Applied and Environmental Microbiology 72(9): 63996401.
Groleau, D and Yaphe, W (1977) Enzymatic hydrolysis of agar: Purification and characterization of beta neoagarotetraose hydrolase from Pseudomonas atlantica. Canadian Journal of Microbiology 23(6): 672697.
Hosoda, A, Sakal, M and Kanazawa, S (2003) Isolation and characterization of agar-degrading Paenibacillus spp. associated with the rhizosphere of spinach. Bioscience Biotechnology and Biochemistry 67(5): 10481055.
Kendall, K and Cullum, J (1984) Cloning and expression of an extracellular-agarase from Streptomyces coelicolor A3 (2) in Streptomyces lividans 66. Gene 29(3): 315321.
Ma, CP and Shi, C (2008) Advances in research of agarase. Microbiology 35(1): 109111 (in Chinese with English abstract).
Sugano, Y, Matsumoto, T, Kodama, H and Noma, M (1993) Cloning and sequencing of agar, a unique agarase 0107 gene from a marine bacterium, Vibrio sp. strain JT0107. Applied and Environmental Microbiology 59(11): 37503756.
Wang, GH and Zhang, M (2006) Research advances on agarase. Marine Science Bulletin 25(3): 7279. (in Chinese with English abstract).
Young, KS, Bhattacharjee, SS and Yaphe, W (1978) Enzyme cleavage of the alpha linkages in agarose to yield agarooligosaccharides. Carbohydrate Research 66: 207212.
Zhang, ST, Shi, J, Zhao, J, Qi, YF and Guo, AG (2006) Expression soluble and functional snake venom fibrinolytic enzyme fibrolase via the co-expression of DsbC in Escherichia coli. Protein & Peptide Letters 13(6): 559563.
Zhao, H, Zheng, WL and Ma, WL (2003) The effects of signal peptide on efficiency of foreign protein secretion. Journal of Biology 20(5): 13. (in Chinese with English abstract).
Zhang, Z and Huang, HL (2002) Escherichia coli disulfide-forming related proteins: structures, functions and their application in gene engineering for expressing heterologous proteins in Escherichia coli. Chinese Journal of Biotechnology 18(3): 261266. (in Chinese with English abstract).
Zhang, WW and Sun, L (2007) Cloning, characterization, and molecular application of a beta-agarase gene from Vibrio sp. strain V134. Applied and Environmental Microbiology 73(9): 28252831.
Zheng, B and Zhan, XM (2005) Signal peptide sequencing and its application to protein expression. Letters in Biotechnology 16(3): 296298. (in Chinese with English abstract).



Full text views

Total number of HTML views: 0
Total number of PDF views: 0 *
Loading metrics...

Abstract views

Total abstract views: 0 *
Loading metrics...

* Views captured on Cambridge Core between <date>. This data will be updated every 24 hours.

Usage data cannot currently be displayed