Purification And Enzymatic Character Analysis Of β-1,4-endoglucanase From Pheretima

β-1,4- endoglucanase, also called Cx enzyme or CMC enzyme, belongs to the cellulases. It can degrade the macromolecule cellulose into cellobiose, and very important in the process of the hydrolysis of celluloses by cellulases.As the cellulases can solve the problem of agriculture, resources and environmental pollution, the research of cellulases are getting more and more extensive. Besides the cellulases which come from the mycetes, bacteria and microbes, the cellulin also become to the investigative focuses.This paper take Pheretima as the research object, extracted endo-cellulases from its intestinal tract, purified by DEAE-Sepharos Fast Flow anion exchange chromatography and SephadexG-75 gel filtration, one eluting peak had activity of β-1,4- endoglucanase were obtained, and it was a single band on the SDS-PAGE, the molecular weight of β-1,4-endoglucanase was 45kD, the purify multiple was 11 and recovery rate was 26.5%. Enzymology analysis demonstrated the enzyme had optimal temperaturei of 55℃, optimal pH of 6.0, was stabile within 40℃-60℃ and pH5.5-8.0.Km and Vmax of the enzyme was 0.289mg/ml 0.446U/ml · min repectively using Hanes-Woolf graphic method.In order to obtain gene sequences of endolucanase from Pheretima, after searching endoglucanase sequences refered on ncbi which has high coefficient of relationship with Pheretima, just as Mastotermes darwinensis,Biomphalaria glabrata and reticulitermes speratus,primer was designed on the basis of amino acid conservatism and a 340bp or so length fragment was got via RT-PCR. Consideration of both results of blastx and amino acid conservation, although the coefficient of relationship of this fragment with endoglucanase genes derived from other class was low, not yet gain endoglucanase gene's conserved sequence of Pheretima, so need to improve system or other routes cloned this cDNA.A β-glucosidase component from Pheretima was separated and purified, and the...