Studies On Screening With Laser Mutagenesis And Optimization Of Solid Fermentationo Conditions Of Manganese Peroxidase High Producing Fungi

Lignin is a kind of inexpensive and affluent renewable resource on the earth.The biodegradation of lignin is the pivotal step in the circulation of carbon in nature.However, due to its extremely complicated and indefinite structure,such polymer is hard to be degraded. Consequently,serious environment pollution has been resulted in around the world. Traditional ways,both physical and chemical,can not resolve this problem effectively and thoroughly.In such conditions,biotechnology is an optimum choice for scientists.How to decompose and convert lignin into unharmful matter with catabolic enzymes produced by microorganism has become a new topic in scientific field,which strongly attracted scientists' eyes.In recent years,most studies related to lignin's catabolic enzymes have been focused on white-rot fungi and Phanerochaete chrysosporium has been regarded as mode fungus.The catabolic enzymes produced by Phanerochaete chrysosporium include lignin peroxidase(Lip) and manganese peroxidase(MnP) and MnP has a broad prospect in application,both in industry and agriculture.Yet,the MnP produced by natural strains exhibit low activity,which has been restrict its practical use in production.In such a case,finding a new way to improve strains' MnP activity is in urgent demand and it's the base of widely application.What's more, so far,most studies on fermentation of Phanerochaete chrysosporium have been concentrated on fluid fermentation,which is highly cost and has strict requirements of circumstances.Thus, making studies on Phanerochaete chrysosporium's solid fermentation is of practical importance.Four strains which could effectively degrade dye were separated from bark with brown or ivory white locus ferrugineus.One strain with the strongest MnP activity was selected by measuring enzyme activity with natrium lacticum,which is 8.59U/g.According to morphous characteristics,it was regarded as Phanerochaete chrysosporium.Orthogonal experiment was carried out to determine the optimum conditions of Phanerochaete chrysosporium protoplast production and regeneration.The results showed that the strains should be treated with 1.5mg/mL enzyme,at 30℃,for 2 hours.The enzyme was a mixture of glusulase and cellulase. They were dissolved with 0.4 mol/L amchlor-0.3%β-ME and 0.2mol/L phosphate buffer, according to S:C=5:4.Then,He-Ne laser(λ=632.8nm,output power was 12mw,the diameter of light spot was 1.5nm) irradiated protoplasts for different time in a distance of 30cm.7# strain with 14.28U/g MnP activity and better genetic stability was selected after analysis of esterase isozymogram,MnP activity and genetic stability.The solid fermentation medium should be added with 0.015mmol/g Mn²⁺ and 0.1%Tween-20 and the fermentation conditions were as follows:domestication time of strains was 5d,inoculation rate was 0.5%,temperature was 37℃and water ratio was 85%.Under such circumstances,the MnP activity of Phanerochaete chrysosporium hit 20.71U/g.The study showed that after laser mutagenesis and optimization of solid fermentation conditions,strains' MnP activity could be raised by 141.1%.