Gene Cloning, Expression And Halophilic Characteristics Of A Halophilic Amylase From Enterobacter Cloacae

A halophilic amylase gene in sequenced genome of Enterobacter cloacae which is a non-halophilic strain, was cloned from the total DNA of mixed culture microorganisms in the cow manure soil of dairy plant in Guangxi University, was cloned by PCR method. The halophilic amylase gene was expressed in E. coli JM109, and extracted total intracellular protein via cracking the strains’cell to detect amylase activity. Enzymatic characteristics showed that it was no enzyme activity when the substrate was soluble starch. Adding NaCl to the soluble starch, it had the enzyme activity. The results showed that it was a kind of halophilic amylase. Purification and enzyme properties of recombinant indicated that its maximum reaction temperature was50℃and pH value was6.0. When the NaCl concentration of the substrate reached4mol/L, it had the highest enzyme activity. It still maintained60%relative activity at the5.5mol/L salt concentration. It is the extreme halophilic enzyme, and the characteristics of hydrolysis product conform to alpha amylase.Analysis of amino acids, it showed that the acidic amino acid (aspartic acid and glutamic acid) content of the halophilic enzyme was15.8%, higher than some kind of non-halophilic amylase was10%. Compare and analysis of the halophilic and non-halophilic amylase’ amino acid sequence, selecting the amino acid sites where non-halophilic and halophilic enzymes were not the same amino acid to site-specific mutation and then studied the halophilic mechanism. The results showed that the Alanine(A), aspartic acid (D), serine (S), tryptophan (W), asparagine (N) can reduce the NaCl dependence of the enzyme. Meanwhile, selecting different mutation sites and different amino acid would have different effects on the enzyme activity.