A new κ-carrageenase gene cgkS was cloned from marine bacterium Shewanella sp. Kz7 by using degenerate and site-finding PCR. The gene was comprised of an open reading frame of 1224bp, encoding 407 amino acid residues...A new κ-carrageenase gene cgkS was cloned from marine bacterium Shewanella sp. Kz7 by using degenerate and site-finding PCR. The gene was comprised of an open reading frame of 1224bp, encoding 407 amino acid residues, with a signal peptide of 24 residues. Based on the deduced amino acid sequence, the κ-carrageenase CgkS was classified into the Glycoside Hy- drolase family 16. The cgkS gene was expressed in Escherichia coli, and the recombinant enzyme was purified to homogeneity with a specific activity of 716.8 U mg-1 and a yield of 69%. Recombinant CgkS was most active at 45 ℃ and pH 8.0. It was stable at pH 6.0-9.0 and below 30℃. The enzyme did not require NaCl for activity, although its activity was enhanced by NaCI. CgkS degraded κ-carrageenan in an endo-fashion releasing tetrasaccharides and disaccharides as main hydrolysis products.展开更多
基金supported by the Key Technologies Research and Development Program of China(2013BA B01B02)National Science Foundation of China(310707 12)+1 种基金Special Fund for Marine Scientific Research in the Public Interest(201005024 and 201105027)National Hightech R&D Program of China(2011AA09070304)
文摘A new κ-carrageenase gene cgkS was cloned from marine bacterium Shewanella sp. Kz7 by using degenerate and site-finding PCR. The gene was comprised of an open reading frame of 1224bp, encoding 407 amino acid residues, with a signal peptide of 24 residues. Based on the deduced amino acid sequence, the κ-carrageenase CgkS was classified into the Glycoside Hy- drolase family 16. The cgkS gene was expressed in Escherichia coli, and the recombinant enzyme was purified to homogeneity with a specific activity of 716.8 U mg-1 and a yield of 69%. Recombinant CgkS was most active at 45 ℃ and pH 8.0. It was stable at pH 6.0-9.0 and below 30℃. The enzyme did not require NaCl for activity, although its activity was enhanced by NaCI. CgkS degraded κ-carrageenan in an endo-fashion releasing tetrasaccharides and disaccharides as main hydrolysis products.
