Purification Of Alkaline Protease Produced By Acinetobacter Lwoffii And Study Of Its Hydrolysis Characteristics And Structure

Alkaline protease is a kind of important proteolytic enzyme,which is widely used in many industries,such as laundry,food and leather industry.A strain highly producing alkaline protease was screened in our laboratory,which was identified as Acinetobacter lwoffii.On the basis of previous studies,the separation and purification of alkaline protease produced by Acinetobacter lwoffii was studied in this paper.The amino acid sequence was analyzed through biological software.And the optimum conditions of the alkaline protease hydrolyzing soybean protein isolate were optimized.The alkaline protease produced by Acinetobacter lwoffii in the fermentation broth was isolated by ammonium sulfate precipitation,dialysis,CM Sepharose Fast Flow ion-exchange column chromatography and Sephacryl S-200 gel column chromatography.The specific activity of the enzyme was increased from 568.46 U/mg to 1494.79 U/mg.The purification efficiency was 2.63 times,and the recovery rate was up to 27.16%.The amino acid sequence of alkaline protease was carried out and analyzed through biological software.The alkaline protease contained 248 amino acid residues,which was non-secretory protein.The hydrophilic region of the alkaline protease was relatively larger,so the alkaline protease was belong to the hydrophilic protein.The protein was once transmembrane protein by alpha helix.The secondary structures were alpha helix 42.34%,beta chain 16.13%,beta angle 9.68%,and irregular curl 31.85%.The binding sites of alkaline protease were also analyzed,and the homology modeling of 3D structure was carried out.Alkaline protease was applied to hydrolyze soybean protein isolate.The optimum hydrolysis conditions were investigated.Single factors of enzyme dosage,time,temperature,pH and substrate concentration were analyzed.Then the response surface methodology(RMS)was used to optimize the latter three factors.The optimum hydrolysis conditions were determined as follows: 3.85% substrate concentration,enzyme 6000 U/g,43.99?,pH 8.93,hydrolysis time 2 h.The hydrolysis degree could reach 14.41%.