Purification And Characterization Of Pullulan From Aureobasidium Pullulans Protoplast By Laser Mutagenesis

Pullulan, an extracelluler polysaccharide produced by Aureobasidium Pullulans, in the process of growing ,which ferments with sucrose or soluble starch and so on. It is an unbranched homopolysaccharide which consists of maltotriose units with α-1,6 glucosidic bonds. There is extensive applying prospect for Pullulan in the domains of food packing, medicine manufacturing, keeping fruit and seafood fresh, etc, as for the good features of edibility, no poison, membranability and so on. It discussed the preparation of Aureobasidium Pullulans protoplast, the laser mutagenesis to select high-yield strain, the purification and characterization of the polysaccharides produced by Aureobasidium Pullulans and the exploration about the conditions of fermentation in the dissertation. Prepare the protoplast with the Aureobasidium Pullulans 6518 as the original strain. After the strain was cultured in liquid basal medium for 20 hours, Gly and high osmotic pressure liquid were added into the medium and the strain was pre-cultivated for 40 min. Later, the strain was washed by the high osmotic pressure liquid, centrifuged and precipitated for twice and then suspended. The suspension 1 mL diluted properly was spread in solid basal medium plate and cultured for 24 h. Afterwards, the colonies were counted as the number of living molds undigested by enzymes. Another suspension 2 mL was put in twice volume mixed enzymes ( snailuse, celluluse and lysozyme ) liquid and digested for certain time. Then the mixture was centrifuged and the sediment was washed by the high osmotic pressure liquid in order to eliminate the mixed enzymes. Protoplast, which was obtained after centrifuged again and discarded the supernatant, was suspended in 2 mL high osmotic pressure liquid. Protoplast suspension 1mL was diluted by aseptic water so as to destroy the protoplast and then spread in solid basal medium plate and cultured. After 24 h the colonies, which did not take off the cell wall, were counted as the number of living molds digested by enzymes. Another 1mL protoplast suspension diluted properly was cultured in regenerate medium combining agar gel screen technology for 48 h, the number of colonies counted was the sum of that of living molds, which did not take off the cell wall after digested by enzymes, and that of molds from regenerate protoplasts. The study explored the effects on protoplast preparation in the aspects about the concengtration of the enzyme mixed and the high osmotic pressure liquid, the time of the pretreatment and the digestion, respectively. Using the product of the formation rate and the regeneration rate of the protoplast as the value standard, the optimum preparation condition was confirmed eventually, which is the digestion by snailuse 0.2 %, cellulose 0.1 % and lysozyme 0.2 % ( 30 ℃, 15 min ) after it was cultured in Cit-NaCl high osmotic pressure liquid medium containing 1% Gly. The product of the formation rate and the regeneration rate of the protoplast prepared in this condition reached the supreme value, 11.7 %. He-Ne laser ( λ=632.8 nm ) with four kinds of power density ( 5 mW/cm2,10 mW/cm2,20 mW/cm2,40 mW/cm2 ) was used to treatprotoplast prepared in the optimum condition for 5 min,10 min,20 min and 30 min, respectively. In the same treatment time, lethal rate increased along with the rise of the power density. The lethal got to the highest value 99.8 % when it is treated in 40 mW/cm2 for 30 min. 16 Aureobasidium Pullulans strains from the living ones in every group after mutation were selected to ferment, 11 of which was positive mutation. The concentration of the polysaccharides was determined with improved phenol-sulphuric acid method. Finally, the mutant strain J208 produced the most polysaccharide, whose transformation rate of sucrose is 53.3 %, just 10.6 times as much as the original strain 6518. The extropolysaccharides produced by the mutant strain J208 fermentation was precipitated and dried by ethanol ( 95 % ) with twice volume as much as the polysaccharides. 2 g crude polysaccharides were obtained and then purified by Savage method for four times repeatedly so as to remove proteins. Continuously, they were excluded small molecular matters and inorganic salts and then refined by vacuum concentration, precipitation with ethanol ( 100 % ), washing with acetone and ether successively. After dried by P2O5, there are 1.48 g purified polysaccharides left. The percent of protein in crude polysaccharides reduced from 3.4 % to 0.6 % after purification, which was determined by ameliorated Lowry method on the basis of bovine serum albumin ( BSA ). Purified polysaccharides were dissolved into water till get to the concentration 1.0 % and then were assayed by Sephadex G-75 Gel-filtration ( 1.5 cm×120 cm ). The elusion fraction was collected and then froze anddried to receive white powder 1.17 g. The polysaccharides powder was made up to 1 mg/mL liquid and scanned in full absorption spectrum with UV/VIS spectroscopy. The liquid did not have feature peaks in 260 nm or 280 nm, indicating that the sample was polysaccharides purification without proteins, nucleic acid or compound polysaccharides binging proteins. The feature peaks proved the purified polysaccharides, which were analyzed by means of Infrared Spectra with standard pullulans, were composed of dextroside linked by α-glycoside bond. Moreover, that the feature peaks between the polysaccharides and the standard pullulans are nearly the same can also contribute to the conclusion. When the purified polysaccharides were analyzed by means of 13C NMR, all the 18 C-formants can be discriminated and classified, which proved that the molecule is pullulans as for it is a linear one consisted of maltoses linked by α-1,6 glucosidic bonds. The result analyzed with circular dichroism ( CD ) spectra suggested that the configure of pullulans in water was randomly coiled conformation. Comparing the variant J208 with the original strain 6518, there were remarkable differences in the process of fermentation rather than in the shape of cells and colonies. The most important divergence is that the output of polysaccharides produced by mutant strain J208 in basal medium increased notably, whose transformation rate of sucrose is 53.3 %, just 10.6 times as much as the original strain 6518. Secondly, the change of pH on mutant strain J208 was less than that of the original strain 6518, and the...