Cellulase-producing Actinomycetes With The Capacity Of High-temperature Tolerance And Characterization Of The Enzymatic Properties Of Its Cellulase

Cellulose is the world's most widely available renewable resource,and cellulase produced by microorganisms can be used to convert it into a small molecule that can be directly utilized.It is of great significance to solve problems such as environmental pollution and waste of crop resources.The purpose of this thesis is to screen out a strains capable of efficiently degrading cellulose with high temperature environment,study and analyze the optimization conditions of the enzyme-producing,separation and purification of single component cellulase,and study on the enzymatic properties of protease enzymatic properties of the cellulase produced by target strain.The results are as follows:?1?12 strains are isolated from compost in high temperature stage,they are compared by preliminary screening and second screening.Which used to screen the high temperature resistant strain A7 that efficiently degraded cellulose.After morphological identification of the strain and phylogenetic tree analysis of the gene,the strain is identified as Streptomyces sp.and used as the target strain for the next experiment.?2?Optimize the liquid fermentation medium composition and fermentation conditions of A7.The best enzyme production conditions are:the best carbon source is peanut straw,the carbon concentration is 4%,the inorganic nitrogen source is potassium nitrate,and the organic nitrogen source is peanut powder cake,the ratio of the two is 4:1,the total nitrogen concentration is 0.8%,the Ca Cl₂is 0.075%,the inoculum is 4%;the initial p H is 4,the temperature is 50?,and the incubation time is 96 h.Under these conditions,finally the strain produce?-Gase reach 165.07 U/m L,CMCase reach 156 U/m L,and FPase reache150 U/m L.?3?Single factor optimization experiment,central combination experiment and response surface analysis show that the culture temperature,fermentation time,C/N and inoculum in solid fermentation medium are the key factors affecting the production of enzyme by Streptomyces sp.A7.According to the response surface analysis,the enzyme production of CMCase increased to 110.76 U/m L,which is 1.18 times that of single factor optimization,and the enzyme production of?-Gase increased to 114.76 U/m L,which is 1.2 times that of single factor optimization.?4?A crude enzyme solution for liquid fermentation of the strain is subjected to ammonium sulfate precipitation and Sephadex G-75 chromatography to obtain a set of endoglucanase.SDS-PAGE shows a single band with a monomeric mass of approximately 23k Da.The enzymatic properties of the obtained pure protease are analyzed,and the optimum reaction p H is 5,and the stability is high in an acidic environment having a p H 3-6.The optimum temperature for the enzymatic reaction is 50?,and the thermal stability is higher than 60?.Low concentrations of K⁺,Ca²⁺and Na⁺promote the enzyme activity on the enzymatic reaction.Low concentration of Ba²⁺,Co²⁺and Mn²⁺inhibited the activity of the enzyme.Na N₃,DTT and EDTA have a strong inhibitory effect on enzyme activity.For cellulase substrate specificity,it has a higher affinity for CMC-Na and MCC.The michaelis constant Km value of this enzyme is 0.4 mg/m L,and the maximum reaction rate is 1.87min.m L/mg.