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Characterization of Cryptopygus antarcticus Endo-β-1,4-Glucanase from Bombyx mori Expression Systems

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Endo-β-1,4-glucanase (CaCel) from Antarctic springtail, Cryptopygus antarcticus, a cellulase with high activity at low temperature, shows potential industrial use. To obtain sufficient active cellulase for characterization, CaCel gene was expressed in Bombyx mori-baculovirus expression systems. Recombinant CaCel (rCaCel) has been expressed in Escherichia coli (Ec-CaCel) at temperatures below 10 °C, but the expression yield was low. Here, rCaCel with a silkworm secretion signal (Bm-CaCel) was successfully expressed and secreted into pupal hemolymph and purified to near 90 % purity by Ni-affinity chromatography. The yield and specific activity of rCaCel purified from B. mori were estimated at 31 mg/l and 43.2 U/mg, respectively, which is significantly higher than the CaCel yield obtained from E. coli (0.46 mg/l and 35.8 U/mg). The optimal pH and temperature for the rCaCels purified from E. coli and B. mori were 3.5 and 50 °C. Both rCaCels were active at a broad range of pH values and temperatures, and retained more than 30 % of their maximal activity at 0 °C. Oligosaccharide structural analysis revealed that Bm-CaCel contains elaborated N- and O-linked glycans, whereas Ec-CaCel contains putative O-linked glycans. Thermostability of Bm-CaCel from B. mori at 60 °C was higher than that from E. coli, probably due to glycosylation.

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Acknowledgments

We thank Young-Sik Seok of the Institute of Kangwon-do Agricultural Pure Stock, Korea for the provision and maintenance of silkworms. This work was supported by the Bio-industry Technology Development Program, Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea (No: 111062-03-1-HD110 and 111116–01–1-SB010) and partially by the National Fisheries Research and Development Institute (RP-2014-AQ-027).

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  1. Research and Development Department, Gyeongbuk Institute for Marine Bioindustry, Uljin, 767-813, Republic of Korea

    Sun Mee Hong & Ho Sun Sung

  2. Marine Biology and Living Resources Research Department, Korea Institute Ocean Sciences Technology, Ansan, 426-744, Republic of Korea

    Mee Hye Kang, Choong-Gon Kim & Youn-Ho Lee

  3. Aquaculture Research Team, Headquarters for Bioscience and Technology, National Fisheries Research and Development Institute, Busan, 619-902, Republic of Korea

    Dae-Jung Kim

  4. Laboratory of Silkworm Science, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, Fukuoka, 812-8581, Japan

    Jae Man Lee & Takahiro Kusakabe

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Hong, S.M., Sung, H.S., Kang, M.H. et al. Characterization of Cryptopygus antarcticus Endo-β-1,4-Glucanase from Bombyx mori Expression Systems. Mol Biotechnol 56, 878–889 (2014). https://doi.org/10.1007/s12033-014-9767-8

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