Study On Breeding Of An Agarase-producing Strain And Preparation By Enzymatic Hydrolysis And Analysis Of Red Algae Oligosaccharides

Red algae breeding industry is developing rapidly in Chinese coasts. Output of red algae is growing quickly. Polysaccharides which are rich in red algae have good biological activities and important value for development. But for its high molecular weight and viscosity, it is difficult to have effect on the body cells, which restricts the application of polysaccharides. The decrease in the molecular weight and viscosity of the red alga polysacc haride is the key to the development and utilization of red algal polysaccharides. Developing an effectively agarase has profound meaning to the high value use of polysaccharide of red algae. Thus, this thesis studies the screening and identification of agarase-producing strains, enzyme production characteristics of the strain, purification, characterization of agarase, extraction and degradation of polysaccharides, and the structure function relationship of its product.1. An effectively agarase-producing strain was obtained through tablet screening method and rescreening method from the red algae collected from coastal areas of Qingdao. The strain was identified as Pseudoalteromonas according to the morphological and molecular biological identification and was named Pseudoalteromonas QJ97. The Fermentation conditions of the strain QJ97 were optimized using single factor experiments and response surface methodology(RSM) design. The optimized media agar, lactose, NH4NO3, K2HPO4 and NaCl were 3.96 g/L, 4.0 g/L,2.0 g/L,1.5 g/L and 11.27 g/L, respectively. The optimum cultivation conditions:temperature 31℃, inoculum size 2%, medium volume 30 mL/100 mL, initial pH value 8.0, and rotation speed 160 r/min. Enzyme activity of the crude enzyme made by fermentation was 625.93 U/mL, a 1.02-fold increase, under these conditions. The growth curve and agarase production curve showed the maximum biomass and enzyme activity were obtained at 28 h and 36 h respectively.2. Electrophoretic purity grade agarase was obtained from the fermentation broth by acetone precipitation, Q-sepharose fast flow ion exchange chromatography and Sephadex G-75 gel filtration chromatography. Agarase was purified 11.18-fold, with a specific activity of 23922.00 U/mg. The purified agarase exhibited a single band on SDS-PAGE with a molecular mass of 35 kDa. Its molecular mass is different from those previously reported. The characteristics of agarase were studied. The result showed the optimal reaction temperature and pH were 40℃ and 6.0 respectively and agarase had a strong tolerance for NaCL Ca2+, Mg2+, Ba2+ and K+ had little impact on enzyme activity. Zn2+, Cu2+, Mn2+ and Fe3+ had inhibitory effect on the activity. The low concentration of Co2+ had no influence on the activity, while the high concentration strongly inhibited the enzyme acticity. The low concentration of SDS could inactive agarase. Agarase for EDTA was relatively stable. The Km and Vmax values of agarase were 5.361 g/L and 0.244 g/L/min respectively. The agarase was highly specific for substrate, and it could degrade agar, not sodium alginate, carrageenan, chitosan, carboxymethyl cellulose, gelatin and soluble starch.3. Extraction conditions of polysaccharide from Porpyra haitanensis, Gracilaria lemaneiformis and Gelidium amansii were optimized by single factor test. The optimal so lid-liquid ratios were 1:40,130 and 130, respectively. The optimal extraction time was 4 h. The optimal enzymatic hydrolysis conditions of these polysaccharides were determined as 4%,40℃,6.0,2 h for enzyme dosage, hydrolysis temperature, pH and hydrolysis time. Conditions of ethanol precipitation were optimized. Optimal concentration ratios of these polysaccharides were 4,5,5, the optimum pH was 8, and it was advisable the the ethanol multiples was 4.4. Composition of polysaccharide from Porpyra haitanensis, Gracilaria lemaneiformis and Gelidium amansii was analysed by high efficiency liquid chromatography. The result showed that the main component of PHP, GLP and GAP was galactose, and contents were 93%,78.7%,79.3%, respectively. Each graded components of hydrolysates were analyzed by electrospray ionization mass spectrometry, and the structure-activity relationship was discussed combined with the antioxidant experiment and antimicrobial activity. The results showed that the hydroxyl radicals scavenging ability of red algal polysaccharides and its hydro lysate had a significant dose effect relationship with concentration. The influencing factors were researched through the vertical comparation of graded components from the same kind of raw material and the crosswise comparation of components with best biological activity from different raw material. The result showed that hydroxyl radicals scavenging activities decreased with reduction of the sulfate group and the degree of polymerization, and it also might be affected by the composition of some special groups and component. The degradation products with degree of polymerization of 8-16 had a strong ability of scavenging hydroxyl radicals. The red algae polysaccharides had no inhibitory effect on the test strains, while some graded components showed significant inhibitory effect The structure-antibacterial activity relationship was surveyed through the determination of minimum inhibitory concentration, and the result showed that the degree of polymerization and sulfate radical content had influence on the antibacterial activity, especially the degree of polymerization and the molecular weight. The degradation products with degree of polymerization of 8-16 might have better bacteriostasis effect.