The Construction Of Metagenomic Library From The Sediment Of The Zhongshan Station, Antarctica And The Clone, Expression And Analysis Of The Alkaline Protease ACPRO001

In this study,the metagenomic DNA were exrtracted and purified from a sediment sample core from ZSS.The samples ZSS was collected from the coast near the China Zhongshan Station.and the site ZSS was the sediment within the treated sewage effluent to the sea.The size of the DNA was more than 48Kb.Than the size was adjusted to 30-45Kb and was used to constract the metagenomic cosmid library.The library contained about 6500 clones.The length of the library covered 228Mb and provided more information about the microbial assemblages.Atfer screening the metagenomic library wih the sreening tacties of protease activity,we had got a positive clone which code number was 1146.This clone produced protease to hydrolyze defatted-milk on the special plates.The analysis indicated that this clone foreign DNA fragment contained an integrated ORF which coded 425 amino acids.The GC containing was 50.9%.The foreign DNA fragment contained an integrated alkaline portease gene after analyzed by BLAST software.The portein Mw was 44.833kD.A DNA sequence containing the DNA region encoding the protease gene was amplified by PCR and cloned into E.coli expression vector pET-His,rersulting in an experssion plasmid His-ACPRO001.The enzyme data had proved the enzyme reaction optimum pH was 9.0 and the optimum teperature was 60℃.The enzyme had a good heat-tolerance at the temperature of 50℃.The new gene was named ACPRO001 and registered in the Genebank database.The registered number was FM163400.In order to get a improvement on the activity,stability and the optimal pH of the protease,we used the Error-prone rolling circle amplification method to do a directed evolution of the protease.Date to now,we had got 800clones,but none of them showed better characters.We are still working on it.