Molecular cloning of a copper-dependent laccase from the dye-decolorizing strain Stenotrophomonas maltophilia AAP56

Abstract

Aims: To clone the gene encoding the enzyme with laccase activity expressed by Stenotrophomonas maltophilia AAP56 and to construct an insertional mutation in that gene to determine its effect on dye decolourization and laccase activity in this strain. Methods and Results: Comparative genomics of Sten. maltophilia strains K279a and R551‐3 revealed copA (coding for putative multicopper oxidase) as a candidate gene to encode an enzyme with laccase activity. Stenotrophomonas maltophilia AAP56 copA was amplified by degenerated PCR and cloned. A copA mutant strain, named Stemur, was constructed by homologous recombination. The comparison of wild‐type and mutant strains revealed that CopA shows laccase activity, and it is involved in copper resistance and in vitro dye decolorization. On the contrary, the mutation in copA did not affect the in vivo dye removal by Sten. maltophilia. Conclusions: Stenotrophomonas maltophilia AAP56 shows different mechanisms for dye decolorization. The gene encoding the laccase has been identified, and it has been shown that it is involved in the in vitro decolorization. Significance and Impact of the Study: Stenotrophomonas maltophilia is a micro‐organism of interest in different biotechnological processes including dye removal. This is the first report to address the molecular mechanism of this capacity, what will contribute to further improvements in the process.

Laccase versus Laccase-Like Multi-Copper Oxidase: A Comparative Study of Similar Enzymes with Diverse Substrate Spectra

“…In the case of the E. coli cuprous oxidase CueO it was suggested that the methionines are involved in binding cuprous oxide atoms which are oxidized by the enzyme [25]. For other enzymes it was suggested that copper binding at methionine-rich regions results in a conformational change required for the entry and oxidation of organic substrates [26], [27]. The question of whether these MCOs can be considered LMCOs or constitute a separate class of MCOs remains still open and may be solved by more closely at their substrate spectrum.…”

Characterization of yellow bacterial laccase SmLac/role of redox mediators in azo dye decolorization

“…Previous results with Syringaldazine as substrate also showed that phosphate buffer pH 7.0 is the optimal for SmLac . These results and other media studies, show that phosphate buffer is a convenient one for SmLac activity towards a wide range of substrates …”

“…Laccase was obtained from a copper induced 24 h culture of S. maltophilia AAP56 by sonication of harvested bacterial cells . The crude extract obtained was concentrated approximately 3‐fold in two steps: ammonium sulphate precipitation (80%) and ultrafiltration.…”

“…Purification and biochemical characterization of SmLac Laccase was obtained from a copper induced 24 h culture of S. maltophilia AAP56 by sonication of harvested bacterial cells. 15 The crude extract obtained was concentrated approximately 3-fold in two steps: ammonium sulphate precipitation (80%) and ultrafiltration. The strong yellow colour characterizing the enzymatic extract was eliminated by the gel filtration chromatography step, the purification rate was determined as 21% from crude laccase enzyme.…”

Combination of photoreactor and packed bed bioreactor for the removal of ethyl violet from wastewater

“…R. pickettii is capable of degrading 2,4,6-trichlorophenol and chlorobenzene Hatta et al, 2012). S. maltophilia exhibits laccase activity and therefore it is able to degrade dye compounds (Galai et al, 2011). Stenotrophomonas sp.…”