Molecular Cloning, Expression And Characterizations Of A Novel Agarase From Marine Pseudoalteromonas Sp. CY24

With rapid development of glycobiology, some agaro-oligosaccharides are found to be of valuable medicinal usage, as anti-tumor, anti-infection, anti-oxidant et al. Most importantly, these agaro-oligosaccharides are reported to have the therapeutic potential in the development of drugs and healthy tonic foods, promising potent developmental prospect. Agaro-oligosaccharides are traditionally prepared by the chemical hydrolysis. This traditional metkod-failed to yield the degraded products with homogeneous molecular weights, resulting in difficulty in obtaining the enough amounts of oligosaccharides with high purity. In fact, the agar hydrolytic enzymes are theoretically of substrate-specificity and favor the preparation of homogenous oligosacchardes. Yet the agar hydrolytic enzymes discovered until now are found to be low in activity and lack of substrate-specificity, becoming one of the bottle-neck in the preparation of agar oligosaccharides. Therefore, agar hydrolytic enzymes with high activity and high specificity are required to be developed for the preparation of agar oligosaccharides, which surely show a wide range of applications.A marine bacteria harboring high agarase activity was isolated with mediums containing agar as the sole carbon and energy source from coastal waters of Qingdao. On the basis of several phenotypic characters and a phylogenetic analysis of the genes coding for the 16S rRNA, this strain was identified as Pseudoalteromonas sp. CY24. An optimum culture medium has been obtained with NaCl 2.5%; Casein 0.25%; MgSO4-7H2O 0.5%; KC1 0.1%; FeSO4-7H2O 0.002%; CaCl2 0.02%; NaH2PO40.06% and 0.25% of Agar through optimizations of the fermentation conditions. The optimum temperature and tune of cultivation were 25 癈 and 24h, respectively. Cultivated in the optimum conditions, the highest agarase activity was found 1.8U/ml which was 20 times higher than that under the original conditions.The agarase gene (agaA) in Pseudoalteromonas sp. CY24 was cloned successfully by shotgun method. Genomic DNA of CY24 was digested by HindIII and PstI respectively, and ligated into the corresponding site of the pBluescript II KS+ vector, the genomic library was constructed when transformed into E. coli DH5a. 9 positive clones with sunk zone and white color were screened out from the genomiclibrary using agar plate containing ampicillin, X-Gal and IPTG. One of the recombinant plasmid pA5 was sequenced, the result of sequence analysis indicated that pA5 harbored a 3.5kb Hindlll insert of Pseudoalteromonas sp. CY24 DNA and the agarase gene was localized to a 1.36kb ORF through subcloning. the amino acid sequence of agarase was deduced using DNATools. The primary translation product is predicted to be a protein composed of 453 amino acid residues, with a calculated Mr of 50.8kDal and pI6.07. Sequence analysis of agaA indicates that there is a Shine-Dalgarno sequence and the conservative -10 region and the -35 region within the promoter upstream from the initiation codon. A typical transcriptional termination signal which is 25 base pairs downstream from the stop codon TAG was found. A 9 bp palindrome in this region which has the capability for hairpin formation with a free energy of -1-2.9 kcal may cause transcription termination. The NH2 terminus of the protein may function as a signal peptide in the export of the extracellular p-agarase as predicted by SignalP. Processing by a signal peptidase could potentially occur between residues 23 and 24 to yield a mature protein. Comparison of the agaA amino acid sequence with other agarase amino acid sequences reported so far showed no significant similarity (not more than 40% identity and 75% similarity). The result of CLASTALW indicates that we have cloned a new agarase gene. The agaA have been submitted to GenBank and the accession number is AY 150179.Expression system of agaA was constructed with the pET-24a(+) vector and E.coli BL21(DE3) as the host, recombinant protein with C-terminal His-tag was induced by IPTG. The recombinant agarase was purified by Ni...