Isolation And Identification Of Agar-degrading Bacteria Associated With Red Seaweed And Studies On Agarases From Strain HQM9

Through a systematic isolatation of agar-degrading bacteria from Gracilaria and Gelidium,25 strains of agar-degrading bacteria can be obtained. Among them,16 typical strains are selected for molecular identification and biodiversity analysis and thirteen strains'genera can be identified. The identification results show that these bacteria are mainly seen in genera Agarivorans, Cellulophaga, Alteromonas, Sac char ophagus, Glaciecola, Vibrio. Furthermore, based on 16Sr DNA sequence analysis and phylogenic analysis, it is estimated that the strain WH0801 could be a new species of Agarivorans; the strain JL1 may be a new genera or new species of Alteromonadales and the strain HQM9 a potential new species of Flavobacteriaceae.The identification test of WH0801 is conducted in this paper. According to the sequence analysis of 16Sr DNA, strain WH0801 shares approximately 96.1% of sequence homology with Agarivorans albus MKT106T. Thus, it can be preliminarily concluded that WH0801 is one member of Agarivorans. By further comprehensive analyses, including the analysis of morphology, biochemistry and fat acid analysis as well as DNA hybrid results and agarases production, we can see that, WH0801 different from Agarivorans albus, the only published species of Agarivorans, is a new species of Agarivorans and can be named as Agarivorans gilvus according to its features, whose type strain is WH0801T (=CGMCC 1.10131T=NRRL B-59247T).In the study, a new bacterium which has agar-degrading capability and produces flavochrome is isolated, which can be named temporally as HQM9. The 16S rDNA sequence analysis reveals that this strain HQM9 belongs to Flavobacteriaceae, showing maximum sequence homology (about 95.19%) with the identified member of Aquimarina intermedia LMG 23204T.Moreover, the phylogenetic analysis of the 16S rDNA sequence shows that strain HQM9 forms a separate branch within the genera Flavobacteriaceae. Thus strain HQM9 may represent a potential novel species in the genera.This thesis also studies on fermentation conditions for HQM9 and nature of the crude enzyme solution.The result shows that optimal conditions for enzyme production of HQM9 are as follows:initial pH7.5, temperature 28℃,2.5% concentration of NaCl; carbon and nitrogen sources are agar and peptone respectively. And the agar plays an inductive role in the production of agar enzyme but the enzyme is not fully influenced by the agar base. The most suitable environment for crude agarase is temperature 40-45℃, pH 7.0, agarose concentration of 1.2%, and various metal ion resistance. Zymogram analysis shows that there are at least eight different kinds of agarases in the agent.After the genetic sequence analysis of HQM9, it can be inferred according to BLAST results that there are 28 possible agarase genes in HQM9,13 of which are of GH16 family while the rest can not be clearly identified. The biological information test of two out of the 13, aga16G and aga16Y, shows that aga16G has the maximum protein-sequence homology of 47% and aga16Y,87%. Based on the prior tests, the primers including restriction enzyme cutting sites and promoters are designed and synthesized. After amplification with PCR, the product is subcloned into a highly efficient eukaryotic expression vector pET24a, forming expression vector pET24a-aga16G/aga16Y, and transforms into E. coil BL21 (DE3). The E. coil BL21 (DE3) with recombinant vector is induced to express protein by IPTG, showing there are large protein expressions and there is agarase active in the fermentation supernate. All this demonstrates that both genes aga16G and aga16Yare new Agarase genes and the restructured Agarase after expression can be named as rAga16G and rAga16Y.Further electrophoresis of rAga16Y shows that the recombined protein rAga16Y exists mainly in the form of inclusion body. The author of this thesis further purifies the recombined protein rAga16Y by High-Affinity Ni-IDA Resin, and tests the enzyme after dialysis and Native-PAGE analysis and finally obtains the purified recombinant agarase enzyme rAga16Y. Preliminary enzyme studies are conducted on the purified rAga16Y. The results show that the optimal reaction conditions for enzyme production of rAga16Y are as follows:substrate (agarose) with concentration 1.5%, temperature 55℃, and pH 8.0. In addition, K+ plays an active role in the enzymatic reaction while many other metal ions (Zn2+,Mn2+,Cu2+,Pb2+ Co2+) will inhibit to a different extent the enzymatic reaction.