Mutation Breeding,Enzyme Producting Optimizated And Enzymatic Properties Initial Research Of Cellulase Producing Strain Penicillium Oxalicum HP7-1

Lignocellulose is the most widely distributed and abundant renewable organic resources on the earth.At present,the cellulose resources were wasted because they have not been effectively used.Finding a degradation of cellulose into the useful product is the current hot topic.Cellulase has the ability to hydrolyze cellulose into reducing sugar,it is fermentated by microorganisms into fuel ethanol which can replace the oil-vehicle and provide a good raw material to solve such as the energy crisis.The purpose of this study is to find a strain which can product high cellulase and play an important role in the fuel ethanol produces.In this study,we slected the wild type strain HP7-1 as the original strain,using three times 60Co-y ray irradiation and two times ethyl methane sulphonate(EMS)composited with ultraviolet(UV)irradiation.At last,a slected mutant strain EU2106 which producted highest filter enzyme.The wild type strain HP7-1 and the mutant strain EU2106 were initial identified as Penicillium sp.based on the microscopical features and the internal transcribed spacers(ITS).The genetic stability experiment of mutant strain EU2106 showed that the enzyme production of this strain was stable.The filter paper enzyme(FPase)activity is from 2.74 U/mL to 6.87 U/mL when the liquid cultivated conditions of the mutant EU2106 were optimized.This represented about 2.5 fold interement as compared with the activity obtained under the basal conditions.The optimized medium composed of(g/L)KH2PO44,(NH4)2SO4,CaCl2 0.6,MgS04.7H20 0.6,Tween-80 2 mL,0.1 mL of trace elements,wheat bran 50,Avicel 10,pH 5.5.Incubation temperature is 28?,the noculum is 10/mL and sixth day is the time of optimal enzyme production.Determined different substrates enzyme activities of the selected mutants which were inoculated in the screen liquid medium and the results showed that the mutant strain EU2106(in addition to ?-glucosidase)produced the hightest activities of Fpase(6.79 U/mL),CMCase(11.64 U/mL),hydrochloric acid pretreatment bagassease(7.64 U/mL),bagasse-pulpase(10.20 U/mL),Avicelase(2.20 U/mL)and xylanase(40.72 U/mL),respectively,these levels were 3.3,1.4,2.2,2.4,3.2 and 1.4 fold higher than those in the parent strain.The HPLC analysis showed that the product from bagasse pulp hydrolyzed by the mutant strain EU2106 were cellobiose,glucose and xylose,the glucose is solely.In substrate specificity testing,the mutant strain EU2106 crude enzyme showed strong affinity towords bagasse pulp,peracetic acid pretreatment sugarcane bagasse,and steam explosion pretreatment.The cellulase from the mutant EU2106 showed that the optimal pH were 4.5,3.5 and 5.0,the optimal temperature were 55°C,45 °C and 70 °C towards the filter paper,CMC and p-NPG.In this study,we successfully screened a mutant strain EU2106,which the FPase activity was significantly increased.It provides a good basis material to others cellulase research,meanwhile the mutant strain can play an important application value in the fuel ethanol products.