Article ID Journal Published Year Pages File Type
18394 Enzyme and Microbial Technology 2006 8 Pages PDF
Abstract

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 °C; however, at 37 °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 Km for l-dopa and l-tyrosine were determined as 2.44 and 0.19 mM, respectively. Temperature and pH for maximum activity were 50 °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.

Related Topics
Physical Sciences and Engineering Chemical Engineering Bioengineering
Authors
, , , , , , , , ,