Screening And Mutagenesis Of Cellulase-producing Strain S-1

Abstract:Cellulose is an important constituent of plant cell walls.It is degraded into glucose,which is easily used,by the hydrolysis of cellulase enzymes.The application of cellulase has a very important significance for solving the global energy and environmental crisis,and has now become a research hotspot in the world.In this study,strains with cellulase-producing ability were screened from soil samples containing decayed straw in Henan,and S-1 strain with high capacity for cellulase production was screened according to the production of transparent rings after Congo red staining,and the molecules were subjected to molecular analysis.Biological identification,using orthogonal experimental methods to optimize the enzyme production conditions;Strain S-1 as the starting strain,ion beam and plasma mutagenesis,at the same time establish cellulase activity high-throughput screening method,breeding The cellulase produces a high yield of mutant strains and optimizes the enzyme production conditions of the mutant strains.The result is as follows:Seven bacterial strains with ability of producing cellulase were selected from soil samples containing decayed straw in Henan Region.Of them,the ratio of transparent ring diameter and colony diameter?D/H?formed by bacterial strain S-1after being dyed with Congo red was highest,namely 2.33.bacterial strain S-1 was identified as Bacillus amyloliquefaciens after the phylogenetic tree was built according to 16S rRNA identification result.Orthogonal experiment was used for studying the impact of different factors?p H,temperature,C source,N source?on CMCase activity produced by fermentation of bacterial strain S-1 at different levels.Result indicates:in the factor level range set in the experiment,the optimum pH of bacterial strain is 5,the optimum temperature is40?,the optimum C source and N source contents are 15g/L.Factor sequence of affecting CMCase produced by fermentation of bacterial strain S-1 is:temperature<C source<pH<N source.The ion beam mutagenesis was carried out for selected bacterial strain S-1 and the irradiation doses were 1×10¹⁴,5×10¹⁴,1×10¹⁵,5×10¹⁵,1×10¹66 and 5×10¹⁶N⁺/cm².It was discovered,the larger the dose was,the higher the lethality rate was.When the irradiation dose of ion beam was 5×10¹⁶N⁺/cm²,the lethality rate was highest,up to100%.When the irradiation dose was 5×10¹⁴N⁺/cm²,the positive mutation rate was highest,namely 44%.When power was 3.2W and irradiation distance was 3mm,the plasma could be used for mutagenesis.When the irradiation time was 2,4,6,8,10,12,14,16,18 and20min,it was discovered that the longer the irradiation time was,the higher the lethality rate was.When the irradiation time of plasma was 20min,the lethality rate was highest,up to 98.50%.In the screening process of mutant strain produced by the mutagenesis of ion beam,the CMCase activity of mutant strain after mutagenesis was tanked as the preliminary screening indicator and CMCase activity of mutant strain was taken as the affination indicator.On this basis,20 mutant strains were screened.Of them,CMCase activity of mutant strain 308 was highest,it was increased by 84.4%after24h than that of original strain S-1.After the 10 generation,the CMCase activity of mutant strain 308,D36,W10 and D79 was stable,of which,the CMCase activity of mutant strain 308 was highest,16.83 U/ml.the CMCase activity of mutant strain D36was 14.88U/ml.The genetic stability test results indicate that CMCase produced by308,D36,W10 and D79 has genetic stability.The results of thallus and gemma dyed by methylene blue indicate that,the mycelial morphology of subcultured G10 original strain S-1 is similar with that of mutant strain 308,and both of them are of rod shape,but the spore morphology is different,the spores of original strain S-1 and mutant strain 308 are oval and spheroidal.The activities of intercellular CMCase and intracellular CMCase of original strain S-1,mutant strain 308 and D36 were determined using eliasa high flux method.Result indicates,the intracellular CMCase activity of bacterial strain before and after mutagenesis was lower than the intercellular CMCase activity.Of them,the intracellular CMCase activity of mutant strain 308 is only 5.94U/ml,and the intercellular CMCase activity is 16.69 U/ml;the intracellular CMCase activity of original strain S-1 is 6.68 U/ml and the intercellular CMCase activity is 11.11 U/ml.The results indicate that ion beam mutagenesis can enhance the yield of intercellular CMCase of bacterial strain.The growth curve of CMCase production curve of original strain S-1 and mutant strain 308 and D36 show that,compared with original strain S-1,the thallus growth of mutant strain 308 and D36 is faster and the thallus content is higher.CMCase output grows fast in the logarithmic phase of bacterial strain,reaches a maximum in the stable period,and maintains stable in a certain period while the CMCase output of mutant strain 308 and D36 is higher than that of original strain S-1.Single factor experiment was used for studying impacts of culture temperature,rotation sped on the CMCase produced by mutant strain 308,finally,the optimal culture temperature was determined as 40?and CMCase activity on this condition reached 17.37U/ml;the optimal rotation speed was 180r/min and CMCase activity on this condition reached 19.56 U/ml.