Identification Of A Marine Bacterium HQM9T And Studies On Its Agarase

Agarases are the hydrolytic enzymes that degrade agar into oligo-saccharides. As research on carbohydrate biology goes further in recent years, people discovered that agaro-oligosaccharides, derived from agarose, have plenty of important biological activities, such as anti-inflammation, anti-tumor and antioxidant, etc, so agarase can be widely used in food, feed, cosmetics and medical industry. The traditional way to produce agaro-oligosaccharides is through acid hydrolysis of agar with some problem such as the hydrolysis product is not homogenous and the hydrolyzing reaction is uncontrollable. Enzymatic degradation of polysaccharides is better than the acid hydrolysis method with high specificity and mild conditions. In former study, our laboratory isolated an agar-degrading bacterium HQM9T. This article mainly centered on the identification, optimization of the condition of agarse production and cloning and expression of the agarase genes.The strain HQM9T was isolated from the surface of Gelidium grow on the coast of weihai with strong agar-degrading ability. After growth on2216E plate for3-5d, the strain could liquefy the plate. Strain HQM9T had MK-6as the major menaquinone and C15:0iso, C17:0iso3OH, C16:1w7c/15iso2OH, C16.03OH C16:0, C17:02OH, C14:0, C13:1AT12-13, C13:0iso as the major fatty acids. The major polar lipids were Phosphatidylethanolamine and an unknown Lipid. The DNA (G+C) content was33.20mol%Phylogenetic analysis based on the16S rRNA gene sequences showed that strain HQM9T was a member of the Bacteroidetes, Flavobacteria, Flavobacteriaceae, had the most sequence similarities to the Aquimarina agarilytica ZCI with97.09%similatarity. So, strain HQM9T was considered to represent a novel species in the Aquimarina, for which the name Aquimarina agarivorans sp.nov. was proposed. The GenBank accession number for the16S rRNA gene sequence of is Aquimarina agarivorans HQM9T is HQ593612.In this article,we researched the optimal conditions for HQM9T to produce agarase, former one-factor-at-a-time methodology suggested that the optimum NaCl content, pH, additional carbon source and nitrogen source to produce agarase was NaCL25‰, pH8.0, agar and peptone. Based on these results, we designed orthogonal experiment (L1837) to optimize the factors effected the agarase production, such as the content of agar, peptone, yeast extract powder, bottle filling quantity, inoculum size, temperature for fermentation and time for fermentation. We confirmed the optimal culture medium was:agar0.5%, peptone0.6%, yeast extract powder0.08%, NaCL2.5%, pH8.0and the culture condition was settled as50ml in250ml conical flask,28℃, inoculated as1.0%. After incubate at150rpm/min for72h, the highest enzyme activity was got as35U/mL, twice more than the enzyme activity before optimization. The genetic sequence analysis of HQM9T, based on BLAST results (KEGG, Swissprot, COG, etc) infers that there are36possible agarase genes,19of which is of GH16family, the others did not belong to the unknown GH families, the length of agrase genes was different from309bp-5217bp, reflected the novelty of strain HQM9T and its agarase gene.Collected the crude enzyme of HQM9T in the culture supernatant after fermentation, from the result of Native-PAGE and enzymatic activity stain after that, we dicovered that there were at least seven type of agarase in the culture supernatant.These agarase stripe was sent to conduct mass spectrum identification, according to the mass spectrum identification and the sequence online virtual enzyme digestion, we determined that the seven agarse stripe was expressed by agal6A, agal6l, aga16L, aga16S, aga16AB, aga16AD and agaAJ, the molecular weight of agarase was96.68kDa,128.19kDa,72.91kDa,98.88kDa,44.35kDa,57.61kDa and73.98kDa, respectively.Then we used pET prokaryotic expression system to express these agarase genes, these genes was cloned into a highly efficient eukaryotic expression vector pET-24a(+), and transformed into E.coil BL21(DE3). The E. coil BL21(DE3) with recombinant vector was induced by1PTG to express peotein, the SDS-PAGE showing there was only gene aga16AD had large protein expressions and showd agarase active in the fermentation supernate, while the other genes did not have any additional stripe compared to the control that did not induced by IPTG. Then, we purified the recombined protein by High-Affinity Ni-IDA Resin and SDS-PAGE showed only one stripe, preliminary enzyme studies were conducted on the purified protein. The optimum substrate concentration, temperature and pH for the protein was agarose1.0%,50℃and pH6. As for the different metals, K+, Mg2+and Na+barely had any influence on the enzyme, Ca2+could slightly enhance the activity while Zn2+, Mn2+Cu2+, Co2+and Pb2+could strongly inhibit the activity. Then we used the pMAL protein fusion expression system to express the other genes, SDS-PAGE showed after induced the there were large protein stripe that had the same molecular weight with our former estimation. However, the agarase activity detection suggested that the expressed protein did not have agarase activity. The reason maybe the recombined protein expressed in periplasmic space did not fold correctly, form the wrong spatial structure and could not combine with the substrate-agar as to lose the activity to degrade agar.