Cloning, Expression And Characterization Of A Novel β-Agarase Gene From Marine Catenovulum Sp.X3

Agarase are glycoside hydrolase and catalyse the hydrolysis of agarose. According to the cleavage pattern, agarases are classified intoα-agarase (E.C.3.2.1) andβ-agarase (E.C.3.2.1.81), most of reported agarases areβ-agarases.β-Agarases have potential applications in the food, cosmetic and medical industries for the production of neoagarooligosaccharides from agar. It has been proved that neoagarooligosaccharides has various special chemical properties and biological activities, such as anti-tumor, anti-infecion and anti-oxidation. Moreover agarases have potential applications in biology as tool enzyme. In this study, a newβ-agarase gene agaXa from Catenovulum sp.X3 was cloned and expressed in E. coli BL21 (DE3), and the recombinant AgaXa was purified and characterized. The AgaXa displays some excellent enzymatic properties, such as high activity and thermostable. It has potential applications in industry.The main contents are as follows:An agar-degrading bacterium, X3 was isolated from seawater of Shantou and classified as Catenovulum by the analysis of its morphology and 16S rRNA gene sequences, named Catenovulum sp.X3. An optimum culture condition was determinate as: culture time 35h, culture temperature 35℃, pH 8.0 and with 2.5% NaCl.A novelβ-agarase gene agaXa was obtained by construct the gene library of Catenovulum sp.X3. The agaXa consists of 1,590 bp and encoded a protein of 529 amino acid residues with isolectric point of pH 7.8 and a predicted molecular weight of 56.6 kDa. SignalP 3.0 Server predicted that the agarase AgaXa contain a 28 amino acids signal peptide. The amino acid sequence of AgaXa was used for BLAST search and conserved domain search in the database of the NCBI. The result showed that the AgaXa was 39% similar only to theβ-agarase AgaB from pseudoalteromonas sp. CY24, no other protein showed significant similarity with AgaXa.The agarase AgaXa with signal peptide and 6×histidine residue on the C-terminus was overexpressed in pET-32a(+)/E. coli BL21(DE3) system, and induced using 0.1 mM IPTG, 0.01% Sodium deoxycholate, growth in a rotatory shaker for 20 h at 25℃, the culture supernatant showed high extracellular agarase activity. Finally, AgaXa was purified from culture supernatant with a Ni-NTA agarose column. The purified AgaXa was analyzed by SDS-PAGE, the result showed a single band with a apparent molecular mass of 52 kDa.The AgaXa showed high activity when the temperature was between 37℃and 52℃(the optimum temperature was 52℃) and it was stable when the temperature was below 42℃. The optimum pH of AgaXa was pH 7.4 and stabled at a wide range of pH 5.0～9.0 at 4℃.The activity of AgaXa was strongly inhibited by metal ions (Cu²⁺, Fe³⁺, Mn²⁺ and Al³⁺) and SDS. Oppositely, AgaXa's activity was activated by adding 10 mM DTT,β-Met, and metal ions Na⁺,K⁺,Ca²⁺,Mg²⁺. The activity increased by 127% with 10 mM DTT. The kinetic parameters of AgaXa for agarose were calculated based on Lineweaver–Burke plots. The Km, Vmax, Kcat, and Kcat/Km of AgaXa using agarose as substrate were 10.5 mg/mL, 588.2 U/mg, 5.15×102 s^-1 and 4.9×106 s^-1 M^-1, respectively.Enzymatic products from agarose in the course-time were analyzed by thin layer chromatography (TLC) and MALDI-TOF. The result showed the end enzymatic products were DP6, DP8, DP10 and DP12. Finally, the AgaXa main products were further identified by 13C NMR. The resonances at about 97 and 93 ppm were found, which were assigned toαandβanomeric carbon respectively at the reducing end of neoagarooligosaccharides. No evidence of a signal at 90.72 ppm was detected, which was typically found at the reducing end of agarooligosaccharides. The result confirmed that AgaXa is aβ-agarase. The recombinant AgaXa used to recover the DNA from agarose gels. The effect of recovering the DNA from agarose gels by recombinant AgaXa was not below the agarose gels DNA purification kit.