Characterization of Cryptopygus antarcticus endo-β-1,4-glucanase from Bombyx mori expression systems.
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.
Research and Development Department, Gyeongbuk Institute for Marine Bioindustry, Uljin, 767-813, Republic of Korea, hongsunmee@gimb.or.kr.
Sun Mee
SM
Hong
Ho Sun
HS
Sung
Mee Hye
MH
Kang
Choong-Gon
CG
Kim
Youn-Ho
YH
Lee
Dae-Jung
DJ
Kim
Jae Man
JM
Lee
Takahiro
T
Kusakabe
D000595:Amino Acid Sequence; D000818:Animals; D001181:Arthropods / Q000201:enzymology*; D012831:Bombyx / Q000378:metabolism*; D002480:Cellulase / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D015870:Gene Expression; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D037121:Plant Lectins / Q000378:metabolism; D011134:Polysaccharides / Q000737:chemistry; D011679:Pupa / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D013696:Temperature
D037121:Plant Lectins; D011134:Polysaccharides; D011994:Recombinant Proteins; D002480:Cellulase