Isolation Of Alkaline Protease-produced Marine Bacteria And Cloning Of The Alkaline Protease Gene

Three alkaline protease-produced marine bacterial strains, named as 3B, 6CW and 15E, were isolated form samples of sea-water,sea-mud and seeweed. These isolates were characterized and identified as species of Pseudomonas, Microbacterium, and Brevibacterium respectively, based on their physiological and biochemical characteristics and analysis of 16S rDNA sequence.And, marine bacterium Brevibacterium oxydans secreting alkaline protease is firstly reported.Fermentation conditions of the alkaline protease secreting from Brevibacterium oxydans 15E were researched. In the condition that initial pH of culture medium was 9.0, culture-broth-quantum was about 20%, inoculum was 2%, speed of shake-culture was 200 r/min and culture time was 40 hours, the strain 15E grew well and total protease activity reached its peak. The casein medium adding 0.5% beef extract and 0.1% glucose was suitable for the growth of the organism and the production of the protease. The concentration of K⁺, Ca²⁺ and Mg²⁺ in the artificial-seawater was suitable for the growth and protease synthesis of strain 15E, while K⁺ was vital for the protease production.Strain 15E could secrete alkaline protease, whose molecular weight was 49.0 kDa, isoelectric point was 9.3 and Km value was 5.6× 10^-3 g/mL. The optimum condition for the protease activity was 60℃ and pH ranging from 7.0 to 9.0. Ions Ca²⁺,Mg²⁺ and K⁺ promoted the activity of the protease.The protease was tolerable to some denaturing agents such as SDS, Urea, Guanidine HC1. PMSF and AEBSF could greatly inhibit protease activity, which indicated the alkaline protease belonged to the type of serine protease; the enzyme could also be inhibited by EDTA, which showed that some metal ions played a role on the protein for catalytic activity.The result of cloning alkaline protease gene apr showed that the length of sequence in nucleotides is 1 357 nt, and the max ORF encoded 447 amino acid. Amino acid sequences of transmembrane were from 122th to 144th amino acid, and it was probably a precursor...