N-terminal truncation of a maleate cis–trans isomerase from Rhodococcus jostii RHA1 results in a highly active enzyme for the biocatalytic production of fumaric acid

• The truncation at N-terminus of a putative maleate isomerase from Rhodococcus jostii RHA1 resulted in three active mutant enzymes.
• Two hydrophilic and two hydrophobic amino acid clusters at the N-terminus of this isomerase are not needed for its activity.
• One of the mutants (MaiR-D48) possessed relatively high thermo-stability and a broad reaction pH range.
• The E. coli cells expressing MaiR-D48 catalyzed the isomerization of maleate to fumarate at 1 M substrate concentration.

As part of the project to develop an efficient biocatalytic process for the production of fumaric acid, a full-length putative maleate cis–trans isomerase gene from Rhodococcus jostii RHA1 was synthesized and expressed in Escherichia coli Rosetta2 (DE3) pLysS, but the protein was not soluble and showed no catalytic activity. Bioinformatics analysis of the protein sequence indicated that there were two hydrophilic and two hydrophobic amino acid clusters in an alternate arrangement at the N-terminus, and 50 extra amino acid residues at the N-terminus were not present in the known maleate cis–trans isomerases. The alternate hydrophilic and hydrophobic clusters at the N-terminus were thus truncated one by one to evaluate their effect on the gene expression and enzyme activity. Three mutants (MaiR-D41/42-304AA, MaiR-D48/49-304AA and MaiR-D52/53-304AA) without the hydrophilic and hydrophobic clusters were expressed as soluble protein with maleate cis–trans isomerase activity. Among them, MaiR-D48 was purified and its properties were studied. The purified enzyme had a temperature optimum of 40 °C and a wide pH range (5.0–9.0) with the optimum pH being 8.0. The whole cells of E. coli expressing MaiR-D48 catalyzed the isomerization of maleic acid to fumaric acid at 1 M substrate concentration, showing its potential for industrial use.

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