Studies On Screening High-yield Polysaccharides And Laccase Strains From Pleurotus Eryngii Using Protoplast Technology

Two major types of important bioactive metabolites which were polysaccharides and laccase from P. eryngii had been attracted more and more attentions. Fungi polysaccharides as "biological effects modifier" had very strong nonspecific immunity enhancement effect and antitumor activity through various mechanisms. At present numerous fungi polysaccharides in clinical application had been used for the treatment of diseases, such as tumor, immune deficiency, virus infection diseases and so on. In addition, Fungi polysaccharides also had been developed for functional food additives as they had many other kinds of pharmacological activities such as antioxidative activities, hypoglycemic effect, anti-hyperlipidemic effect and so on. Fungi laccases had important roles on lignin degradation and had extensive substrates so that they had wide application in environmental protection, food, pulp, textile and other industrial fields.P. eryngii was a kind of rare edible and medicinal fungi which was introduced in the1990s from foreign country. It was also called "xingbaogu" because of its almond-like fragrance and abalone-like flesh. It was quite popular because it was rich in nutrition and delicious best among oyster mushrooms and had all kinds of health cares suggested by recent researches. However, the publications about two important bioactive metaoblites that were polysaccharides and laccase for P. eryngii were less available.Protoplast was a tool for genetic manipulation which had been successfully applied in varieties of edible and medicinal fungi. Protoplast clonal variation technology and protoplast UV mutagenesis technology were all based on protoplast and had been achieved numerous high-quality and high-quantity strains from edible and medicinal fungi. However, the two technologies had been so rarely applied to P. eryngii.Random amplified polymorphic DNA (RAPD) was a kind of molecular markers, had been used successfully in genetic and breeding fields such as germplasm identification, classification, phylogenesis, genetic diversity, genetic linkage map construction and gene location and cloning. However, application of RAPD in P. eryngii had been reported less.Based on the above conditions, firstly, high efficient preparation system of protoplast from P. eryngii was established through systematic optimization of influence factors during protoplast isolation in this paper. Secondly, high efficient regeneration system of protoplast was constructed according to the effect of all factors on regeneration rate. Again, screening systems of high-yield polysaccharides and laccase strains by protoplast clonal variation technology and UV mutagenesis technology were established in order to screen high-yield strains. Finally, RAPD technology system was constructed in order to identify the genetic difference between screened strains and parental strain.The results showed that the optimum culture days was6d, hydrolytic temperature, pH, substrate concentration, enzyme concentration and reaction time of lywallzyme was30℃,6.0,200mg/ml,1.5%and2.5hours, respectively. Under this condition, the yield of protoplast was up to6.34×107cells/ml.The best regeneration conditions for the protoplast were followed as:osmotic stabilizer and dilution agent were all0.6M mannitol, regeneration culture mode was single layer and spreading culture, regeneration culture medium was PPR culture medium. Under this condition, protoplast regeneration rate was0.76%.High-yield IPS, EPS and laccase strains screened by protoplast clonal variation technology accounted for35.7%,39.3%and50%among protoclones strains, respectively. Strain HF15, HF17, HM41showed the highest IPS, EPS content or laccase acitivy of127.8±3.2mg/g,324.3±8.3mg/1and5488±101U.min-1.ml-1which was more34.8%,42.1%and22.5%than parental strain, respectively.High-yield IPS, EPS and laccase strains screened by protoplast UV mutagenesis technology accounted for42.4%,39.4%and15.2%, respectively. Strain HU30-2, HU50-15, HU50-12showed the highest IPS, EPS content or laccase actitivy of114.1±2.7mg/g,360.9±8.0mg/l,5889+108U.ml-1.1-1, which was more20.4%,58.1%and31.4%than parental strain, respectively. Their yields or activities had genetic stability.The optimum conditions to measure laccase activity of P. eryngii using ortho-tolidine method were determined:the wavelength was620nm, temperature was30℃, pH was4.2, ortho-tolidine concentration was5mmol/l, reaction time was within3min. Under this condition, laccase activity was highest and98%than before screening.Genetic differences between high-yield strains and parental strain HB3were analyzed using RAPD technology and the results showed that genetic variation of strain HU50-15comparison with strain HB3was the biggest and HM41was followed. Genetic variation of strain HF15, HU30-12, HU50-12was smaller and variation of strain HF17didn’t occur.New methods were offered to screen high-yield polysaccharides and laccase strains from P. eryngii in this paper and it could not only provide high-yield polysaccharides and laccase strains for production but also broaden application fields of the protoplast clonal variation technology and UV mutagenesis technology and thus it had potential theoretical and realistic significance.