Researches About Three Carrageenases From Pseudoalteromonas Carrageenovora ASY5

Carrageenase,mainly secreted by bacteria,could be used as a powerful tool to prepare specific carrageenan oligosaccharides for further study on biological activity-structure relationship and industrial exploitation.The result from Genome sequence of Pseudoalteromonas carrageenovora ASY5 indicated the existence of a lot of carrageenase genes,in addition to two identified genes.In this study,two ?-carrageenase genes were cloned from marine bacterium P.carrageenovora ASY5 into expression vector pET-28 a and expressed in Escherichia coli BL21(DE3).Besides,the potential application of a new ?-carrageenase from P.carrageenovora ASY5 was evaluated,and the optimal culture medium and conditions of ?-carrageenase production in shaking flask fermentation on the P.carrageenovora ASY5 were carried out by single factor experiment.The results were as follwes:(1)A ?-carrageenase gene car 1 was cloned from bacterium P.carrageenovora ASY5 into expression vector pET-28 a and expressed in Escherichia coli BL21(DE3).The ?-carrageenase gene contained 1194 bp full-length gene sequence and encoded 398 aa residues of the protein with a molecular weight of 48 kDa in SDS-PAGE analysis.Based on domain structure analysis,the ?-carrageenase belongs to glycohydrolase family.Then,the characterization of the recombinant ?-carrageenase was studied and the results showed that ?-carrageenan could specifically be degraded by this enzyme.The results also indicated that the optimal temperature and pH of the enzyme was 55°C and pH 9.0,respectively.And it is demonstrated that the recombinant ?-carrageenase retained more than 95.0% of the maximum enzyme activity after incubation for l h at 40°C.The recombinant ?-carrageenase retained more than 60.0% of the maximum enzyme activity after incubation for l h below 45°C.The recombinant ?-carrageenase retained more then 80.0% of the maximum enzyme activity after incubation for 30 min over a range of pH 8.0-10.0.The enzyme could hydrolyze ?-carrageenan into ?-carrabiose and ?-carratetraose,which were characterized by ESI-MS.(2)This study was carried out to clone the car 30 gene in P.carrageenovora ASY5 and provide a basic foundation for the study of car 30 genetic characteristics.The homology analysis found that the amino acid sequence of car 30 gene share the highest 100% identity with the protein from Pseudoalteromonas sp.H103(WP 058549724.1).The secondary structure of car 30 was mainly composed of random coil,?-helix and ?-extended.The tertiary structure analysis showed that the structure of the ?-carrageenase from P.carrageenovora ASY5 is nearly spherical and the enzyme approximately consist of ?-structure.There is a core structure of hydrophobic protein on it's surface and it has the typical ?/? domain structure of hydrolase.In conclusion,the ?-carrageenase from P.carrageenovora ASY5 has the same characteristics of the enzyme in GH16 family.The results indicated that the optimal temperature and pH of the enzyme LJ 30 was 45°C and pH 6.5,respectively.And it was demonstrated that the recombinant ?-carrageenase retained more than 50.0% of the maximum enzyme activity after incubation for 30 min at 50°C.The recombinant ?-carrageenase retained more than 50.0% of the maximum enzyme activity after incubation for 60 min over a range of pH 6.5-7.5.(3)A novel ?-carrageenase-producing strain was screened from mangroves and authenticated as P.carrageenovora ASY5 in our laboratory.The potential application of this new strain was evaluated.Medium compositions and culturing conditions in shaking flask fermentation were first optimized by single-factor experiment.?-Carrageenase activity increased from 0.34 U/m L to 1.08 U/m L after test optimization.Optimal fermentation conditions were 20°C,pH 7.0,incubation time of 40 h,15 g/L NaCl,1.5%(w/v)yeast extract as nitrogen source,and 0.9%(w/v)?-carrageenan as carbon source.Then,the crude ?-carrageenan was characterized.The optimum temperature and pH of ?-carrageenase were 40°C and 8.0,respectively.The enzymatic activity at 35-40°C for 45 min retained more than 40% of the maximum activity.The structure of oligosaccharides derived from ?-carrageenan was detected using electrospray ionization mass spectrometry(ESI-MS).?-Carrageenase degraded ?-carrageenan,yielding disaccharides and tetrasaccharides as main products.In addition,The optimum temperature and p H of ?-carrageenase induced by ?-carrageenan from P.carrageenovora ASY5 were 50°C and pH 9.0,respectively.The enzyme activity maintained more than 90% of the maximum activity when incubated at 40-55°C for 45 min.The structure of oligosaccharides derived from ?-carrageenan was detected using electrospray ionization mass spectrometry(ESI-MS).?-Carrageenase degraded ?-carrageenan,yielding ?-carrabiose as main products.