Expression of Alginate lyase Gene in E.coli and Characterization of the Enzyme

Abstract:

The algl gene(GenBank accession number: GU585575) encoding alginate lyase(ALGL) from Bacillus cereus F1-5-10 was cloned into the vector pET-30a and expressed in Escherichia coli BL21 in this study.The optimum expression conditions of algl in E.coli was study and listed as follows: the concentration of IPTG 0.4 mmol/L,expression time 4 h,and expression temperature 32 ℃.Under the optimum conditions,the activity of recombinant enzyme achieved at 11.7 U/mg.Then,the recombinant enzyme was purified and the characteristics were determined.It was found that(1) the molecular weight was approximately 45 000;(2)the optimum pH and temperature were 6.6 and 35 ℃,respectively;(3)the activity was stimulated by 1.0 mmol/L Ca2+ and Mg2+;(4)the Km and Vmax values for alginate were 1.89 mg/mL and 15.01 U/mL,respectively.