| Carrageenans consist of linear chains of galactopyranose residues in the D-configuration linked by alternatingα(1→3) andβ(1→4) linkages, with sulfate-ester groups commonly present. On the basis of the level and position of sulfate substitution, they are commercially classified into three major types asκ-,ι- andλ-carrageenan (with 1, 2 and 3 sulfate groups, respectively). Recently, some carrabiose oligosaccharides are found to be of valuable pharmacological activities, such as antitumor, antiviral, anticoagulant and immunomodulate activities, in the development of drugs. Especially,λ-carrabiose oligosaccharides that contain 1,4-linked D-galactose 2,6-disulphate units and highly sulfated groups, are considered to have better biological activities and may be exploited as new drugs.Carrageenases, which are glycoside hydrolases that cleave the internal (1→4) linkages of carrageenans yielding products of the neo-carrabiose series, display a strict substrate specificity namelyκ-,ι-, andλ-carrageenases. Up to now,κ- andι-carrageenases have been widely studied, however, there was only oneλ-carrageenase reported. Therefore, the discovery of novelλ-carrageenases with high activity has become the focus of research and development of new drugs.In the previous study, a marine bacterium Cellulophaga sp. QY201 was isolated from Grateloupia livida and it could produceκ-,ι- andλ-carrageenases. The optimal medium forλ-carrageenase production was as follows (%, w/v): 3% NaCl, 0.3% MgSO4·7H2O, 0.02% CaCl2, 0.01% KCl, 0.002% FeSO4, 0.3% Casein, 0.15% NaH2PO4, 0.1% NaH2PO4, 0.25%λ-carrageenan and the initial pH 7.0. The optimal cultivation conditions forλ-carrageenase production were 500mL flasks containing 150mL medium, shaking cultivation at 120rpm and 28℃for 36 hours. Theλ-carrageenase activity could reach 3.6U/mL when Cellulophaga sp. QY201 was incubated under the optimal cultivation conditions, which was 2.5 times higher than that under the original conditions. Theλ-carrageenase of Cellulophaga sp. QY201 was extracted by (NH4)2SO4 precipitation and purified by ion-exchange chromatography and Superdex 75 gel filtration chromatography. This procedure resulted in 40-fold purification of theλ-carrageenase from the crude culture supernatant with a 3% recovery. The purified enzyme gave a specific activity of 8 U/mg and a single band on SDS-PAGE with a molecular mass of 38.0 kD. Size exclusion chromatography indicated that this enzyme existed as a monomer in solution.The optimal temperature and pH for hydrolysis ofλ-carrageenan by theλ-carrageenase were 30℃and 7.5, respectively; the enzyme was stable below 30℃and over a range of pH 6.0-8.5. Na+, K+, Cu2+ and Mg2+ enhanced the enzyme activity significantly, whereas Ba2+, Mn2+, (NH4)2SO4, Ni2+ and EDTA decreased the enzyme activity. The value of Km was 4.926×10-6 mol/L forλ-carrageenan. Analysis of the substrate specificity showed thatλ-carrageenase had high activity onλ-carrageenan and little activity onκ-carrageenan,ι-carrageenan and agar. The pattern ofλ-carrageenan hydrolysis showed that the enzyme was an endo-typeλ-carrageenase, and the main final products have been purified and characterized to be neo-λ-carratetraose (DP4) and neo-λ-carradiaose(DP2) using MALDI-TOF-MS.In conclusion, aλ-carrageenase was purified from Cellulophaga sp. QY201 and the molecular weight and degradation products of this enzyme indicated that it was a novel glycoside hydrolase. It will not only enrich the diversity ofλ-carrageenases, but also provide an opportunity for investigating the structure-function relationship of carrageenases. Furthermore, its high activity and product specificity will enable this enzyme to be applied in many different fields, e.g., medicine, agriculture and food technology.
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