Characterization Of κ-carrageenase From Pseudoalteromonas Sp.AJ5-913 And Analysis For Its Hydrolyzed Products

This paper aims at screening aκ-carrageenase-producing bacterium with high enzyme activity, optimizing its culture conditions and medium components, mutagenizing the bacterium to obtain the mutant with higher activity, purifying theκ-carrageenase from the mutant cultural supernatant, studying the enzymatic properties, preparingκ-carrageenan-derived oligosaccharides fromκ-carrageenan using the enzyme, analyzing the composition and structure of the enzyme hydrolyzed products, providing the theoretical and technological supports for commercial production ofκ-carrageenase andκ-carrageenan oligosaccharides. By enrichment culture technique aκ-carrageenan-degrading bacterium AJ5, capable of utilizingκ-carrageenan as sole source of carbon and energy, was isolated from the intestine of holothurian Apostichopus japonicus. The strain was identified as the genus Pseudoalteromonas sp. according to its morphological and physiological characterization and 16S rRNA gene analysis. It was found that the strain had different physiological characteristics compared with the only one bacterium, P. carrageenovora, in this genus. The culture conditions and medium components for the bacterium have been standardized for the maximal productivity of the extracellularκ-carrageenase using the single factor and orthogonal tests. The optimal culture conditions was found as following:75 mL medium in 250 mL Erlenmeyer flask, shaking speed of 150 r/min, inoculum volume 7%, pH8.0 and temperature 28°C. The optimal medium components were observed as the following:κ-carrageenan 1 g/L, beef extract 2 g/L, NaCl20 g/L, K₂HPO₄·3H₂O 1 g/L, MgSO₄·7H₂O 0.5 g/L, MnCl₂·4H₂O 0.2 g/L, FePO₄·4H₂O 0.01 g/L. By using the complex mutagenesis of UV irradiation and Ethyl Methanesulphonate (EMS) treatment, the mutant Pseudoalteromonas sp.AJ5-913 withκ-carrageenase activity of 6.788 U/mL was obtained from Pseudoalteromonas sp.AJ5, which was 2.9-fold higher than that of the parent culture. An extracellularκ-carrageenase was purified from Pseudoalteromonas sp.AJ5-913 cultural supernatant by ammonium sulfate fractionation, gel filtration chromatography (Sephadex G-200) and cation-exchange chromatography (CM-cellulose 52). The purified enzyme yielded a single band on SDS-PAGE with the molecular mass of 35 kDa. The sequence of the 20 amino acids at the N-terminal of the enzyme was N-P-T-C-H-I-A-K-P-G-E-T-T-I-L-Q-E-C-R-S. Compared with known N-terminal amino acid residues ofκ-carrageenases, noκ-carrageenase with the same corresponding N-terminal amino acid sequence was observed, indicating that this protein might be a novelκ-carrageenase. The pI ofκ-carrageenase was 8.5 on isoelectric focusing. The optimum pH for the enzyme was 8.0 and its activity was stable in the pH range of 6.6–8.6. The optimum temperature was 55°C and the enzyme was stable at 28°C, but 95% of the activity was lost at 50°C-75°C for 30 min. The activity of the enzyme was optimum at the presence of 50 mmol/L NaCl. The enzyme activity was almost completely inhibited by Co²⁺, Cu²⁺ and Zn²⁺ at 1 mmol/L. Thisκ-carrageenase showed Michaelis-type kinetics when hydrolyzing κ-carrageenan, as calculated from Lineweaver plot, the apparent Km value was 9.8±0.2 mg/mL. The enzyme specifically hydrolyzedκ-carrageenan.Ï„- andλ-carrageenans and agarose were not hydrolyzed by thisκ-carrageenase. The composition and structure of main enzyme hydrolyzed products examined by ESI-TOF-MS and 13C-NMR wereκ-neocarrabiose, -tetraose, -hexaose, -octaose, and -decaose sulfates with 3-linkedβ-D-galactopyranose 4-sulfate as the reducing end, indicating theκ-carrageenase from Pseudoalteromonas sp. AJ5-913 specifically hydrolyzed theβ-1, 4 glycosidic linkage between 3,6-anhydro-D-galactose and D-galactose. The main enzyme hydrolyzed products of this enzyme were different from those ofκ-carrageenases from other bacterial strains. The anti-HSV-1 activities ofκ-neocarraoligosaccharides were determined.κ-Neocarraoligosaccharides (3.12-200μg/mL) could interfere absorption of HSV-1 to Vero cells and there was obvious relationship between the concentrations and the effects ofκ-neocarraoligosaccharides.