Gene Engineered Strain's Construction And Characteristics' Studies Of α-amylse Gene From Thermotoga Maritime

The gene α-amyA from Thermotoga maritima was analyzed by using DNA analysisprograms. Some factors were found to affect the expression of α-amyA in E. coli. Thecomplete gene encoding extracellular α-amylase was amplified from the genomic DNA ofThermotoga maritima by polymerase chain reaction. The recombinant plasmid pET-amyAwas constructed by inserting the amplified segment into the expression vector pET-20(b). Thesignal peptide of amyA gene bound of unoptimal codon was cut off to form amyAⅠ. Therecombinant plasmid pET-amyAⅠand pHsh-amyAⅠwas obtained by inserting the amyAⅠinto the vector pET-20(b) and pHsh. The complete argU gene was amplified from the genomeof E. coli by polymerase chain reaction and was inserted into the plasmid pET-amyAⅠtoform pET-R-amyAⅠ.The recombinant plasmids pET-amyA, pET-amyAⅠ, pHsh-amyAⅠ, pET-R-amyAⅠwere transformed into the E.coli JM109(DE3) respectively , The recombinant plasmidspET-amyAⅠ, pHsh-amyAⅠwere transformed into the E.coli BL21-CodonPlus(DE3)-RIL.The recombinant strains produced amylase activities of 1658.0 U/ ml, 6721.7 U/ml, 7420.0U/mL, 8904.5 U/ml, 13867.7 U/ml, 14860.7 U/ml through induction, respectively. Incomparison, enzyme activity produced by E.coli BL21-CodonPlus(DE3)-RIL harbouringpHsh-amyAⅠwas 8.97 times higher than that by E.coli JM109(DE3) harbouring pET-amyAat their optimized induction conditions.The recombinant protein was purified by the heat treatment and immobilized metalaffinity chromatography, purified enzyme presented as a single protein band on SDS-PAGEwith molecular weight of 63.11 kD. The optimum activity of a α-amyA was found to be at pH6.5~7.0 and 85~90℃. In a pH range of 5.0~9.0, the enzyme was stable, and had a half-life of17 h at 90℃. The apparent Michaelis constant of the α-amyA was 1.5459 g/L for amylum,and Vmax was 23.430 mmol/min.mg protein.