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

Paper ID Volume ID Publish Year Pages File Format Full-Text
18394 42721 2006 8 PDF Available
Title
Expression of the melA gene from Rhizobium etli CFN42 in Escherichia coli and characterization of the encoded tyrosinase
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.

Keywords
Rhizobium etli; Escherichia coli; melA gene; Tyrosinase; Melanin
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Expression of the melA gene from Rhizobium etli CFN42 in Escherichia coli and characterization of the encoded tyrosinase
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Publisher
Database: Elsevier - ScienceDirect
Journal: Enzyme and Microbial Technology - Volume 38, Issue 6, 1 April 2006, Pages 772–779
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us