Preparation, Purification And Characterization Of Agrase From Paenibacillus Sp.

Strain Paenibacillus sp. WL was isolated from sea waters of Zhejiang coast. Forscreening high-activity agarase producing strains, Paenibacillus sp. WL was mutated byUV treatment, γ-irradiation and implanting low-energy N+ion consecutively. After UVtreatment, a strain WL-68with agarase activity of26.7U/mL,12.2%higher than that ofthe original strain was selected. Atter γ-irradiation, a mutant WL-68-46with agaraseactivity of31.5U/mL,25.0%higher than that of WL-68was selected. Finally, a mutantstrain Paenibacillus sp. WL-68-46-15was screened out after implanting low-energy N+ion, which produced agarase with the activity of47.3U/mL,53.6%higher than strainWL-68-46and108%higher than the original strain. Strain WL-68-46was marked asWL-15. Property of producing agarase with high activity of WL-15was stable after5generation transfer inoculation. Afterward, the concentrations of the main componentsin fermentation medium were optimized to improve agarase activity. When theconcentration of agar, NaNO3and NaCl were2.5g/L,6.0g/L and15.0g/L respectively,the agarase activity could reach53.3U/mL,17.9%higher than that of pre-optimization.Agarase produced by the mutant strain Paenibacillus sp. WL-15was purified byammonium sulfate precipitate, DEAE-ion column chromatography and gel-filtrationchromatography consecutively. Supernatant was obtained by centrifuging culture broth（8000r/min for20min） below4℃. Precipitate was collected with solid ammoniumsulfate saturation of20%-80%. Precipitate obtained by centrifugation （10000r/min for30min） was dissolved in20mmol/L sodium phosphate buffer （pH7.5）（buffer A）.Insoluble substance was discarded by centrifugation （10000r/min for20min）.Supernatant was dialyzed against the same buffer for2d. The dialysate wasconcentrated and then applied onto a DEAE-anion column equilibrated with buffer A.Active fraction was collected during a linear gradient elution of0to1.0mol/L NaClcontained in buffer A and concentrated. Concentrates was loaded onto a SephacrylS-100column equilibrated with buffer A. Active fractions were collected and lyophilized as the final preparation of purified enzyme. The agarase was purified11.9fold with a yield of5.1%, giving a single bind on SDS-PAGE with relative molecularmass of30kDa. Activity of the agarase was detected from pH4to9, and the resultshowed that the optimal agar-hydrolysis pH value was6. It was stable at thetemperature range of30-50℃and with the optimal agar-hydrolysis temperature of40℃.Na⁺, K⁺, Ca²⁺, Fe²⁺, Al³⁺, Mg²⁺, SDS, and EDTA could active the reaction of hydrolysisof agar by agarases. However, Cu²⁺and Mn²⁺inhabited its activity. Kmand Vmaxvaluesof agarase was3.41mg/mL and444.3U/（mL·min）, respectively. The results of substratespecificity detection of agarase towards different substrates indicated that it was highlyagar specific, and had only a little activity towards carrageenan. This agarase will be apowerful tool in the preparation of agar-oligosaccharide.