Screening Of High Yield Strain For Pullulan Production By Irradiated Protoplasts From Aureobasidium Pullulans

Pullulan is a water-soluble extracelluler polysaccharide produced aerobically by growing a yeast like fungus Aureobasidium pullulans. It is an unbranched homopolysaccharide which consists of maltotriose units withα-1,6 glucosidic bonds. Pullulan have a wide range of commercial and industrial applications in many fields like environment polluted disposal, food science, and health care.Since the strain of Aureobasidium Pullulans N3.837 has low polysaccharide production rate and high levels of melanin production, it is still unfeasible to the application of this strain in Pullulan industrialized production. By using UV-irradiated protoplast process, we obtained 2 mutants with high yield polysaccharide production and deficiency in melanin formation. Fermentation ability and the relationship between cell morphology, the polysaccharide-producing capacity and pH, were also investigated.Present work established the optimium conditions for protoplasts preparation by using enzymatic hydrolysis and regeneration of protoplasts from Aureobasidium Pullulans, respectively. The strain was cultured about 20 h and then pretreated in liquid basal medium containing 1% Gly for 30min. Taken 25mmol /L Tris-HCl, pH 6.0 containing 0.5 mol /L KCl as high osmotic pressure liquid, eliminated cell wal1 by combined enzyme solution of 0.2% snailase, 0.2% cellulase and 0.2 % lysozyme(dissolved by 0.1 mol /L Na2SO4,0.8 mol /L KCl,25mmol /L Tris-HCl ,pH 6.0) at 28℃,pH6.0,for 1 h .The formation rate of the protoplast was 97.14%,and the regeneration rate reached 11.1%.The ultraviolet radiation (15W, distance of 30cm) was used to treat protoplast prepared in the optimum condition and 28 mutants were obtained. After measured the concentration of total sugar, and the yield of pullulan, two strains, designated as KR13 and KR28 which exhibit high polysaccharide yield and excellent properties were obtained. It is found that their transformation rates of sucrose are 29.28% and 26.92%, which is more than 3 times of that of the original strain.We further studied the fermentation process of KR13 and KR28 and found the fermentation time was reduced from 72h to 65h. And it is certificated that pullulan elaboration was occured with the yeast-like morphology of A. pullulans. The pullulan elaboration phase is mainly on the mid and later time of its steady period, i.e., between 40 to 70hours. Results described above pave the way to Pullulan industrialized production by fermentation.