Mutation Of The Strain Highly Producing Laccase From Coriolrs Versicolor Protoplasts

In order to obtain most of protoplasts from Coriolrs Versicolor 1.028 ,the initial strain,we tested from three aspects: enzyme and their compounding,age of mycelial, osmotic stabilization, taking into account the yield of protoplast.At last ,we finded the optimal conditions, which can be described: at 32℃, 1% Snailase+1% cellulase dissolved by mannitol, age of mycelial 6 days.Considering ratio of regeneration of protoplast, mortality and ratio of positive mutation ,we treated the protoplast with U.V. at mortality60%.A mutant with high laccase activity was screened,naming Cv₃₀-9 .The laccase activity of the mutant kept stable after 8 generations.The RAPD revealed that the initial strain and the mutant contained differential bands,which reflected the genetic difference at genome. The variety of electrophoresis chart,including the increase , absence of the sum of bands and the change of colour of bands, reflected the change at genome, frequency of variety is 40.00%⁸1.82%.After single factor designing experiment,the optimum fermentation conditions of Cv₃₀-9 were obtained,which is as followedxoncentration of the carbon resource 3.0g/L, concentration of the nitrogen resource 18mmol/l,the initial PH of liquid medium 2.5,25mL/250ml flask.The PAGE revealed that kind and quantity of enzyme contained difference,which proved that Cv₃₀-9 was got from the chang of the initial strain at genome furthermore.The chang of laccase activity of the mutant and the initial strain is acute at different reaction temperature,their optimum reaction temperature is almost same.At the aspect the stability of heat,they are stable between 20 ℃ 45 ℃ ,their half-activity temperature are 70℃ and 60℃ respectively,they are all heat-resistant.Laccase of the initial strain and the mutant have their optimum activity at pH4.0 and pH4.5. The initial strain has better stability in the range pH2.5⁵.0,but the mutant in the range pH2.5⁶.0.