Screening And Mutagenesis Of Cellulase-producing Microorganisms And Heterologous Expression Of ?-glucosidase Gene

With the increasingly prominent energy crisis and environmental pollution,the world's energy structure is changing from fossil energy to renewable energy.Fuel ethanol is considered to be the best fuel to replace and save petroleum fuels,especially cellulosic bioethanol has become a research hotspot in industrialized utilization technology.The conversion of biomass resources into biofuels or high value-added products is very attractive,and cellulase enzymes play a key role in this process.Microbes can not only produce cellulase,but also effectively degrade straw to protect the environment.Therefore,screening high-yield composite cellulase and efficient straw degradation microorganisms is the primary task.In this study,we isolate and identify straw-degrading bacteria with high cellulase production capacity,optimize the conditions for producing enzymes,and determine the best conditions for cellulase hydrolysis of the strains and their ability to tolerate ionic liquids.At the same time,the wild strain was mutagenized by ⁶⁰Co-?rays,and a mutant strain with high cellulase production was obtained,and its?-glucosidase was expressed heterologously.The saccharification rate of ionic liquid pretreatment and cellulase hydrolysis of straw was studied.Through collecting soil samples,separation and screening,17 strains with higher cellulase activity were obtained.After morphological and molecular biological identification,they were respectively named Penicillium oxalicum G6-2B?Penicillium oxalicum SB16?Penicillium oxalicum SB12?Penicillium oxalicum F3-7?Penicillium oxalicum Y31?Penicillium oxalicum G1-1?Aspergillus terreus L39?Aspergillus fumigatus H11?Aspergillus flavus G1-4?Aspergillus aculeatus G1-3?Trichoderma erinaceus L3?Trichoderma erinaceus L4?Trichoderma hamatum C2?Trichoderma hamatum R?Trichoderma harzianum L5?Fusarium Axysporum DZ4?Talaromyces wortmannii L9.The strain A.aculeatus G1-3 with efficient straw degradation ability was selected carry out the optimization of enzyme production conditions under the optimal fermentation conditions,the three enzyme activities of A.aculeatus G1-3 were Fpase(0.165 U/m L)and CMCase(1.303 U/m L),?-glucosidase(1.943 U/m L),respectively.Mutation breeding of strain A.aculeatus G1-3 by ⁶⁰Co-?rays has resulted in many dominant strains.The cellulase produced by the mutant strain A.aculeatus P6 are:?-glucosidase(7.023 U/m L),CMCase(1.543 U/m L)and FPase(0.094 U/m L),respectively.The cellulase enzymes of mutant strain A.aculeatus C51 were?-glucosidase(3.443 U/m L),CMCase(0.618 U/m L)and FPase(0.134 U/m L),respectively.Compared with of the wild strain A.aculeatus G1-3,the cellulase activity of the mutant strain changed significantly and cellulase remained stable for 10generations.The optimum p H value and temperature of cellulase hydrolysis were determined.The results showed that the optimum p H value of A.aculeatus G1-3 cellulase was 5.0,the hydrolysis temperature was 65?,The optimum p H value and hydrolysis temperature of cellulase of mutant strain A.aculeatus P6 and A.aculeatus C51 were basically consistent with those of wild strain.The results show that when lonic liquids tolerance of cellulase was determined.It was found that when the ionic liquid concentration of[EMIM]CH₃COOH was 2.5%(w/v),the mutant strain P6 achieved a high enzymatic activity with?-glucosidase(95.1%),CMCase(98.9%)and FPase(85.9%).When the concentration of[EMIM]CH₃COOH was increased to15%(w/v),the A.aculeatus P6 maintained the enzymatic activity with?-glucosidase(63.1%),CMCase(50.6%)and FPase(53.1%).The activity of the three celluloses is maintained above50%.Using plasmid p PIC9K and Pichia pastoris as heterologous expression vectors,the?-glucosidase protein of A.aculeatus P6 was heterologously expressed.Verification by agarose gel electrophoresis and colony PCR showed that?-glucosidase protein was successfully expressed in Pichia pastoris and the enzyme activity could reach(2.352 U/m L).After untreated corn straw hydrolyzed by cellulase of wild strain A.aculeatus G1-3,mutant strain A.aculeatus P6 and A.aculeatus C51 for 72 h,the yields of reducing sugar were 4.121mg/m L,9.415 mg/m L,8.206 mg/m L,respectively.The corn straw pretreated with ionic liquid([EMIM]CH₃COOH)was hydrolyzed by cellulase for 72 h,and the yield of A.aculeatus G1-3reducing sugar was 19.434 mg/m L.The reducing sugar yields of A.aculeatus P6 and A.aculeatus C51 were 21.224 mg/m L and 20.072 mg/m L,respectively.Therefore,the yield of reducing sugar in corn straw pretreated with[EMIM]CH₃COOH synergistic hydrolyzed by strain cellulase was significantly higher than that in the untreated corn straw.In this study,we obtained a high-producing cellulase strain using straw as a carbon source.The advantage mutant strain obtained by mutagenesis treatment also obtained cellulase that can tolerate a certain concentration of ionic liquid,and successfully expressed?-glucosidase heterologously.At the same time,the saccharification rate of cellulase hydrolyzed straw by the ionic liquid pretreatment synergistic strain was studied,which provided an effective way for straw degradation to prepare reducing sugar.