Studies On Purification And Characteri Zation Of Cholesterol Esterase From Acinetobacter Sp.

Cholesterol esterase(CHE;EC 3.1.1.13) is an important steroidal esterase, it is widely used in food processing and medical assay and paper industry.In present study,several cholesterol esterase-producing bacterial strains were isolated,and one of them with highiest cholesterol esterase acticity which was identified as Acinetobacter sp.,was further studied.The objective of this work is to obtain an appropriate source of cholesterol esterases for industrial and medicinal needs.Nine bacterial strains that express high level of inducible extracellular cholesterol esterase(CHE) were isolated from carnivore feces.One of these strains,named CHE4-1,belonging to the genus Acinetobacter sp., was found to produce the highest level of cholesterol esterase.No reports about the cholesterol esterase(CHE) from Acinetobacter sp.and the bacteria secreting cholesterol esterase(CHE) from carnivore feces have been reported by far.The growth and fermentation have been studied and the strategy of the control of sub-fermentation have been advanced,Fermentation process is optimized.The optimum culture medium is constituted as:cholesterol oleate 0.2%,TritonX-100 0.15%,peptone0.05%,NaNO₃ 0.008%,boracic acid 0.0075%,CaCl₂ 0.03%, MgSO₄·7H₂O 0.0125%,FeSO₄.7H₂0 0.0005%,KH₂PO₄ 0.037%.Fermentation condition for the strain was optimized as intitative pH 6.9,volume ofsubstrate 30ml, 30℃,cultivit for 48h.Emzyme activity reach at 950U/L. CHE from strain CHE4-1 was purified from the culture supematant by ultrafiltration followed with DEAE-Sepharose FF chromatography and Phenyl-Sepharose CL-4B chromatography,and then by Superdex-50 gel filtration.The specifiv activity of pure products reached 7.15U/mg,and the yield of enzyme activity is 27.02%,with a 9.67 purification factor.The purified cholesterol esterase migrated as a single protein band on reduced SDS-PAGE.The purity analyzed by HPLC is 98.22%. SDS-PAGE electrophoresis and gel filtration chromatography of CHE.showed that the purified enzyme was a monomer with a molecular weight of about 6.5kDa,and exhibited maximum absorption at 280 nm.It is the smallest cholesterol esterase among those reported by far.The kinetic property of cholesterol esterase from Acinetobacter sp.had been investgated.The enzyme was found to be stable from 4.0-10.0,and be stable from 0℃-55℃,and with optimum activity at pH7.0 and 45℃;The Km value for hydrolyzation of cholesterol oleate by this enzyme was 5.36×10^-4mol/L,the Km value for hydrolyzation of cholesterol linoleate by this enzyme was 1.75×10^-3mol/L.The enzyme showed higher vitality to both long-chain cholesterol ester and short-chain cholesterol ester.The studies of the effects of some metal ion and organic compounds on the enzyme activity also indicated its potential as a clinical diagnostic reagent.