Screening Of A Bacterium Capable Of Converting Agar To Oligosaccharides And Separation And Purification Of Agarase

In recent years, research and development of functional food which contain algal polysaccharide and oligosaccharide has drawn more and more attention.Agaro-oligosaccharide belongs to the kind of functional algal oligosaccharide.Research has shown that agaro-oligosaccharide possess many important physiological functions, such as anti-inflammatory, antioxidant, bacteriostasis, adsorption and moisturizing, skin-whitening, inhibitating the formation of melanin, anti-aging and so on. Preparation of agaro-oligosaccharide using biodegradation method is a popular eco-friendly technology nowadays. However, there are still problems that prevents the utilization of this technology, for instance, few sources of agarase and lower activity. Industrialization of agaro-oligosaccharide is somewhat impeded due to the reasons listed above. Therefore, it is important to screen the high yield agarase and also to scale up the production of agarase.To screen the high yield agarase strain, a gram-negative bacterial strain HJPHYXJ^-1 which is capable of transforming agar to neoagaro-oligosaccharide is isolated. Basic Local Alignment Search Tool（BLAST） search of HJPHYXJ^-1’s 16 S rDNA sequence on GenBank suggested that the similarity between this strain and Vibrio natriegens can reached up to 99%. In addition, the morphological,physiological and biochemical characteristics of HJPHYXJ^-1 have also shown high similarity to Vibrio natriegens. Therefore, HJPHYXJ^-1 was identified to be Vibrio natriegens. The Genbank serial number and Preservation number is KR181948 and CCTCC M 2015244, respectively.A single factor orthogonal design method has been adopted to optimize the agarase producing condition. The optimum condition to produce agarase is: 10% inoculums size, 8 hours for inoculation of age, fermentation at 30 oC and fermentation pH at 6.5.The fermentation broth has the highest agarase activity after the cultivation of 42 hours. The amount of reducing sugar in the hydrolysates was optimized from 149.00mg×L^-1to 714.00 mg×L^-1.The strain was cultivated under the optimum condition. The crude enzyme can beobtained after refrigerated centrifugation, ammonium sulfate precipitation, dialysis,and vacuum freeze drying from the fermentation broth. Agarase was purified up to14.36 times with a recovery of 4.4% by DEAE-Sepharose Fast Flow and Sephacryl S^-100 HR chromatography. The molecular weight of agarase is around 33.8 kD.Agarase is capable of converting agar to neoagaro oligosaccharides,indicating that it is β-agarase.Analysis of agarase properties showed that: the optimal reaction temperature was45 oC, and the optimal reaction pH was 8.5. The Michaelis constant and the maximum velocity was Km=12.24 mg×mL^-1and Vmax=0.051 mmol×（L×min）^-1, respectively. There are differences between the enzymatic hydrolysates with different digestion time. The results of HPLC suggested that the metabolite of enzymatic degradation was neoagaro-oligosaccharides. The molecular weight was between 342 and 1962. The longer the digestion time was, the lower the proportion of macromolecular substances was accounted for. The only two substances left were Neoagarobiose and neoagarotetraose after enzymolysising for 24 hours. The major component was neoagarotetraose.Based on the original strain Vibrio natriegens HJPHYXJ^-1, the experiment was designed by ultraviolet mutagenesis and screening. The enzyme activity was improved 151.85 U × m L^-1. The enzyme activity of agarase increased 46.30%compared to previous result.