Identification Of Alkaline Protease-producing Strain SD11 And Purification And Studying Of Characteristics Of Enzyme

Alkaline protease that as an important protein hydrolase has a wide range of uses in lots of industrial production, such as washing, food and leather industries. Strain SD11 with high alkaline protease-producing ability was screened from the wetland of Qinhuangdao sea area and then was selected for the following study in this paper. Identification of strain, optimization of fermentation conditions, purification and enzymatic properties of alkaline protease were studied to provide theoretical basis for the practical application.Morphological, physiological and biochemical experiments and 16 S r DNA molecular taxonomy were used to identify strain SD11. It was a gram-negative bacteria. The colony were round, yellow, rod-shaped with smooth edges, not-atheletic. It could hydrolyze gelatin, starch and produce hydrogen sulfide, while could not produce indole. The results of methyl red and V-P tests were negative. Clustal X and Mega software were used to analyze the 16 S r DNA sequence of strain. Then the phylogenetic tree of strain SD11 was built. Thus strain SD11 was indentified may be Acinetobacter lwoffii according to the experiment results and the analysis. The strain of Acinetobacter lwoffii was firstly found to produce alkaline protease in the study.The optimal culture medium and fermentation conditions of strain SD11 were studied using single-factor method, taking the enzyme activities as the test index. The results showed that starch medium was optimum fermentation medium. The other optimal fermentation conditions were as below: fermentation time 54 h, initial p H 8.5, temperature 30℃ and the inoculum size 3%.The relatively pure alkaline protease was obtained after the ammonium sulfate precipitation, dialysis desalination and Sephacryl S-200 gel chromatograp of fermentation broth of strain SD11. The final specific activity of enzyme was 1.485 U/μg with the purified ratio of 2.559 times compared to the initial one of 0.580 U/μg. The recovery rate was 27.16%.Optimum temperature and p H of alkaline protease were 40℃ and 9, respectively. The stability range of the enzyme were 20-50℃ and p H 7.5-10.5. Therefore the alkaline protease of strain SD11 had great thermal and p H stability. The enzyme activities were inhibited by Mg2+, Ca2+ and Al3+, while activated evidently by Mn2+ and Cu2+. Then obvious inhibition effects occurred with PMSF, which indicated that the enzyme was belonged to the serine protease. The enzyme was also inhibited by EDTA and mercaptoethanol partly. The alkaline protease was worthy of research and application because of its favorable thermal and p H stability.