Molecular cloning and characterization of a novel cold-active beta-1,4-D-mannanase from the Antarctic springtail, Cryptopygus antarcticus.
A cDNA encoding a beta-1,4-d-mannanase (CaMan) was identified among the expressed sequence tags (ESTs) of the Antarctic springtail, Cryptopygus antarcticus. The open reading frame consisted of 1149 bp encoding 382 amino acids with a putative signal peptide. Amino acid sequence comparison with other mannanases indicated that CaMan likely belongs to subfamily 10 of the glycoside hydrolase family 5, together with mollusc beta-mannanases. CaMan shows typical features of a cold-active enzyme: it has a high frequency of polar residues such as Asn, Gln, and Ser, and a low frequency of hydrophobic residues as well as a low ratio of Arg/(Arg+Lys) compared to the mesophilic beta-mannanases. When CaMan was fused with the thioredoxin gene in pET-32a(+), expressed in E. coli Rosetta-gami (DE3), and purified after thrombin treatment, catalytically active enzyme was obtained. CaMan has high specific activity (416.3 U/mg) toward locust bean gum at an optimal temperature of 30 degrees C and an optimal pH of 3.5. Its optimal temperature is the lowest among those of the known mannanases and the optimal pH is also the lowest except those of fungi. Even at 0-5 degrees C, this enzyme retained 20-40% of its maximum activity. Divalent metal ions such as Ca(2+), Mg(2+), Cu(2+), and Zn(2+) enhanced the enzyme activity, but Mn(2+), Hg(2+), and Ag(+) inhibited activity. This study represents the first record of a beta-mannanase from an arthropod and provides a new source of carbohydrate hydrolysis enzyme with novel characteristics.
Song
Jung Min
JM
Marine Resources Research Division, Korea Ocean Research and Development Institute, Ansan P.O. Box 29, Seoul 425-600, Republic of Korea.
Nam
Ki-Woong
KW
Kang
Sung Gyun
SG
Kim
Choong-Gon
CG
Kwon
Suk-Tae
ST
Lee
Youn-Ho
YH
D000595:Amino Acid Sequence; D000818:Animals; D000864:Antarctic Regions; D001181:Arthropods / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D003080:Cold Temperature; D005786:Gene Expression Regulation; D008969:Molecular Sequence Data; D044902:beta-Mannosidase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*
D044902:beta-Mannosidase