Identification And Breeding Of Agarase Degrading Bacterial And Its Fermentation Profucts

Agar is Generally Recognized as safe (GRAS) food additive. Agar-derived oligo-saccharides including agaro-oligosaccharides and neoagaro-oligosaccharides are a new class of marine functional oligosaccharides. Besides the basic activities of common oligo-saccharides, agar-derived oligosaccharides have many other biological activities, such as antioxidative, antiviral, anti-inflammatory and anti-caries. So far, the only commercially available agarase is the β-agarase Ⅱ from Pseudomonas atlantica T6c. However, the high cost of the β-agarase greatly impeded the development and application of the agarase and agar-derived oligosaccharides. So the study of agarase is desirable.In this study, an agarose-degrading bacterium was isolated from marine environment through screening. In order to provide a theoretical support on the research of agar-derived oligosaccharides and lays a foundation for its production and application, identification, growth conditions optimization, mutation breeding and producing agar-derived oligo-saccharides from Pseudoalteromonas sp. fmb-A18were proceed, respectively. The main results of this study are as follows:1. Screening and identification of an agarose-degrading bacterium from seawater. The fmb-A17strain which has an agarase activity of4.5U/mL was isolated from the seawater of the coach of Lianyungang, using artificial seawater media. This strain was preliminary identified as Pseudoalteromonas by morphological, physiological and biochemical tests. The16S rDNA gene (1147-bp) of fmb-A17was amplified by PCR. After a homology search on Genbank, a neighbor-joining phylogenetic tree was constructed using software ClustalX1.83and MEGA4. The results indicated that the16S rDNA gene sequence of fmb-A17showed the highest homology (99%) with that of Pseudoalteromonas sp. BSs20060. In the phylogenetic tree, strain fmb-A17and P. tunitaca were in the same branch. The result further indicated that strain fmb-A17belonged to Pseudoalteromonas.2. Mutation breeding of fmb-A17. The methods to breed the strain were chemical and physical mutagenesis of nitrosoguanidine (NTG), ehylmethanesulfonate (EMS), lithium chloride (LiCl) and low energy ion beam of N+. The optimized mutation conditions of NTG, EMS, LiCl and low energy N+are10μg/ml,0.07mol/mL,1%and10kev, respectively. Using to these protocols, we obtained a mutant stain which named finb-A18. It has an agarase activity of7.38U/mL and a well genetic stability.3. Optimization of growth conditions of fmb-A18. Using one-factor-at-a-time design, Plackett-Burman design and response surface methodology (RSM), we obtained the following results.28℃,180rpm, loading volume50mL/250mL, inoculation volume10%, fermentation at24h with0.1%maltose,0.2%agar,0.1%yeast extract,0.1%ammonium citrate,0.5%MgSO4·7H2O,1mmol/L KC1,100μmol/L K2HPO4,100μmol/L CaCl2and1μmol/L FeSO47H2O for one-factor-at-a-time design;0.24%yeast extract,0.2%maltose and1Mmol/L KC1for Plackett Burman design (PB). The optimized media compositions including1.706g/L maltose,1.174g/L yeast extract,1.021mmol/L KC1,40g/L NaCl,0.5μmol/L FeSO4,100μmol/L K2HPO4,1g/L Ammonium Citrate Tribasic,5g/L MgSO4and2.4g/L agar. The agarase activity of fmb-A18was about2-fold (15.044U/mL) higher than that of in origin medium.4. Agar-derived oligosaccharides produced by Pseudoalteromonas sp. fmb-A18. The crude agar-derived oligosaccharides were firstly obtained from the fermentation of Pseudoalteromonas sp. fmb-A18centrifugation. Then, using ethanol precipitation, macroporous resin NAK-Ⅱ, chromatography with DEAE-Sephcel, HiTrapTM esalting and Sephadex G15, we obtained purified agar-derived oligosaccharides which have only one peak tested by HPLC. The optimized reaction system for agar-derived oligosaccharides production was cultured at60h with agarose0.4%, yeast extract20g/L, Ammonium Citrate Tribasic8g/L, MgSO42g/L, NaCl6g/L. On this condition, the yield of agar-derived oligo-saccharides was about5mg/mL. NAK-II resin was selected from7different resins for its highest oligosaccharides recovery and deproteinization ratios. When treated with optimized conditions (flow rate of lmL/min and concentration of5mg/mL crude agar-derived oligo-saccharides solution for dynamic absorption and pH10.0,40℃and adsorption time of18h for static absorption), the deproteinization and oligosaccharide recovery ratios of NAK-II resin were80%and65%, respectively. After purified by chromatography with DEAE Sephcel, HiTrapTM esalting and Sephadex G15, we obtained oligosaccharide fraction GA-1which has a purity of91.36%. GA-1was identified as one kind of oligosaccharides with four sugar units using HPLC.