Anti-polyHistidine monoclonal antibody, clone JKS-3 [HRP]

A synthetic cDNA-AmyA gene was cloned and successfully expressed in Pichia pastoris as a His-tagged enzyme under the methanol inducible AOX1 promoter. High level of extracellular amylase production of 72 U/mL was obtained after a 72h induction by methanol. As expected, the recombinant strain produced only the AmyA isoform since the host is a protease deficient strain. Besides, the purified r-AmyA showed a molecular mass of 54kDa, the same pH optimum equal to 5.6 but a higher thermoactivity of 60 degrees C against 50 degrees C for the native enzyme. Unlike AmyA which maintained 50% of its activity after a 10-min incubation at 60 degrees C, r-AmyA reached 45min. The higher thermoactivity and thermostability could be related to the N-glycosylation. The r-AmyA activity was enhanced by 46% and 45% respectively in the presence of 4mM Fe2+ and Mg2+ ions. This enzyme was more efficient in bread-making since such ions were reported to have a positive impact on the nutriment quality and the rheological characteristics of the wheat flour dough. The thermoactivity/thermostability as well as the iron and magnesium activations could also be ascribed to the presence of an additional C-terminal loop containing the His tag.

Phenylacetone monooxygenase (EC 1.14.13.92, PAMO) catalyzes oxidation of ketones with molecular oxygen and NADPH with the formation of esters. PAMO is a promising enzyme for biotechnological processes. In this work, we generated genetic constructs coding for PAMO from Thermobifida fusca, containing N- or C-terminal His(6)-tags (PAMO N and PAMO C, respectively), as well as PAMO L with the His(6)-tag attached to the enzyme C-terminus via a 19-a.a. spacer. All PAMO variants were expressed as catalytically active proteins in Escherichia coli BL21(DE3) cells; however, the expression level of PAMO N was 3 to 5 times higher than for the other two enzymes. The catalytic constants (k(cat)) of PAMO C and PAMO L were similar to that published for PAMO L produced in a different expression system; the catalytic constant for PAMO N was slightly lower (by 15%). The values of Michaelis constants with NADPH for all PAMO variants were in agreement within the published data for PAMO L (within the experimental error); however, the K-M for benzylacetone was several times higher. Thermal inactivation studies and differential scanning calorimetry demonstrated that the thermal stability of PAMO N was 3 to 4 times higher compared to that of the enzymes with the C-terminal His(6)-tag.