Screening For Laccase-Producing Strain And Preliminary Identification Of Fusant F49

Pleurous eryngii, alternate name almond abalone mushroom. The fungus is rich of nutrition and the texture is crisp and tender. Pleurous eryngii has a lot of highly active oxidase.The ability of decompose lignin is strong. They are widely used in degradation of lignin in microorganism and treatment of phenol waste water which is from paper industry. Laccase is a copper-bearing polyphenol oxidase, which are the highest in Pleurotus eryngii enzyme activity.Pleurotus ostreatus is one of the most abundant fungi species in China, high yield is its biggest feature. Through 30 years development, Pleurotus ostreatus had the biggest production output in edible fungi variety and became the most general edible fungi in China. In 2003, total output of Pleurotus ostreatus reached 2.73 million tons(fresh) in China, accounted for 26.3% of the total output of edible fungi.Protoplast fusion technology is one of the most significant technology in microorganism genetic breeding. It can overcome the breeding barrier, such as cell walls and mating system in conventional breeding method and it is a fact that species, genera and distant hybireds can hybird. Inactivated parental strain protoplasts fusion method reduce a very complicated work about finding a stable genetic marker and the posibilitity of losing advanced character of parents, and it can reduce the growth of parent strain without selective medium. Thus it will enhance the efficiency of selection.Screening for laccase-producing strain with high yield were studied by using the inactivated parental strain protoplasts electrofusion method in this paper. The optimal condition of preparing of Pleurous eryngii protoplast was that 6-day-old mycelia, when subjected to 1.5% Lywallzyme solution in 0.6 mol/L annitol at 30oC for 3.0 hours, with yield of protoplasts was 2.95×107/mL; The optimal condition of preparing of Pleurotus ostreatus protoplast was that 5-day-old mycelia, when subjected to 1.5% Lywallzyme solution in 0.6 mol/L annitol at 30 oC for 2.5 hours, with yield of protoplasts was 2.78×107/mL. The regeneration medium with the following composition: potato 200 g, glucose 20 g, yeast extract 2 g, KH2PO4 3 g, MgSO4·7H2O 1.5 g, VB10.1 g, VB60.1 g, agar 20 g, 0.6 mol/L annitol, pH6.5. Two layer plates of the above medium is beneficial to regeneration. The protoplast regeneration rate were 0.68% and 3.84%.With UV method, Pleurous eryngii was handled in 15W 20min, lethality rate were 100%; with lethality method, Pleurotus ostreatus was handled in 65 oC 30min, lethality rate were 100%. The ratio of Pleurous eryngii and Pleurotus ostreatus were mixed by 1∶1 and fused in electrofusion instrument. The optimal alternating electric field frequency and intensity were 1 MHz,250 V/cm and fusion pulse intensity was 5.5 kv/cm. The fusion rate was 3.2×10-3.A strain with characters of both parents was picked out and the enzyme activity of laccase and TTC-dehydrogenase respectively increased by 20.6% and 25.3%. The fusion strain heredity stability were tested and found out respectively to be 92.0% and 92.7%. The fusant was identified from its colony character, morphological state, antagonistic effect. Esterase isozyme zymograms was determined for further identification. The fusant had four isozyme bands, same with the Pleurous eryngii parental strain and had three isozyme bands, same with the Pleurotus ostreatus parental strain. However, The fusant had three isozyme bands which the parental strains did not have, exhibitding own characteristics.