Expression of the melA gene from Rhizobium etli CFN42 in Escherichia coli and characterization of the encoded tyrosinase

The gene melA from the nitrogen-fixing bacterium Rhizobium etli CFN42 was amplified using PCR, cloned in the expression vector pTtrc99A to obtain plasmid pTrcmelA, and transformed into E. coli strain W3110. The resulting recombinant strain W3110/pTrcmelA synthesized a dark pigment when growing in solid or liquid media containing L-tyrosine and copper. This pigment was identified as melanin by comparing it with analytical grade melanin using a spectrophotometric assay. Melanin was synthesized when recombinant E. coli cells were incubated at 30 degrees C;. however, at 37 degrees C significantly less polymer was produced. The recombinant tyrosinase expressed intracellularly in E. coli was purified 40-fold with a 25% yield from a cell extract by ammonium sulfate precipitation and ion exchange chromatography. With the partially purified tyrosinase, the K. for L-dopa and L-tyrosine were determined as 2.44 and 0.19 mM, respectively. Temperature and pH for maximum activity were 50 degrees C and 6.5-7.5, respectively. Activation energy for thermal inactivation (50.77 kJ/mol; using L-dopa as substrate at pH 7) and half-life values indicate a higher thermal stability of R. etli tyrosinase in comparison with mushroom tyrosinase. Interestingly, for a bacterial tyrosinase, MelA showed an unusually higher activity for L-tyrosine than for L-dopa. (c) 2005 Elsevier Inc. All rights reserved.