Abstract of PhD Thesis

Purification and characterisation of tyrosinase and laccase from Pseudomonas putida F6

Aoife McMahon (2006) - University College Dublin

This study is concerned with the purification and characterisation of oxidases namely tyrosinase and laccase from Pseudomonas putida F6. Relevant physico-chemical characteristics of the enzyme were elucidated. The biotechnological potential of tyrosinase, in crude cell extracts as well as the purified enzyme, as a biosensor for the detection of phenols was also examined.

A comparison of the spectrophotometric detection and quantification of a number of 4-substituted phenols by two sources of the enzyme tyrosinase (Agaricus bisporus (mushroom) versus Pseudomonas putida) is described here.  Incubation of either source of tyrosinase with selected 4-substituted phenols results in the formation of coloured products that absorb light maximally within a narrow wavelength range (400-423 nm).  The inclusion of the nucleophile 3-methyl-2-benzothiazolinone (MBTH) in the tyrosinase assay resulted in more intensely coloured products that also absorb light within a narrow wavelength range (440-475 nm).  The molar extinction coefficient of the reaction products in the tyrosinase and tyrosinase-MBTH assay differed dramatically with values of between 714-1580 M-1cm-1 and 14213-26563 M-1cm-1 respectively.  The addition of MBTH improved the sensitivity of the reaction between 1.3 and 100 fold, depending on the substrate and source of the enzyme. The limit of detection of 4-substituted phenols also varied according to substrate and the source of enzyme used in the assay.  The lowest detectable concentration of 4-substituted phenol was 2.5 mM 4-hydroxyphenoxyacetic acid in the presence of mushroom tyrosinase and MBTH and 2.5 mM 2-(4-hydroxyphenyl) ethanol in the presence of cell extract of Pseudomonas putida F6 and MBTH.

The tyrosinase from P. putida F6 was purified by anion exchange chromatography, size-exclusion chromatography and protein excision from a non-denaturing SDS-PAGE gel. Inclusion of the purified tyrosinase from P. putida F6 into the tyrosinase-MBTH assay designed to detect phenols did not decrease the limit of detection of several 4-substituted phenols. In fact the detection limit increased for 2-(4-hydroxyphenyl) ethanol with the purified tyrosinase compared to crude cell extracts of P. putida F6.

P. putida F6 tyrosinase is a monomer. The relative molecular mass (Mr) of the purified F6 tyrosinase was approximately 39,000 as determined by SDS-PAGE and approximately 36,000 as determined by size exclusion chromatography. This result suggests that the enzyme is monomeric. Copper chelating compounds, EDTA and bathocuprione had no effect on enzyme activity while sulphur containing compounds, sodium diethyldithiocarbamate and b-mercaptoethanol, were shown to be strong inhibitors of P. putida F6 tyrosinase. Magnesium was found to stimulate enzyme activity and interestingly copper, at a concentration of 0.1 mM and above, was found to inhibit tyrosinase activity.

Kinetic parameters (Vmax and Km) of the P. putida F6 tyrosinase on seven substrates were determined showing typical Michaelis-Menten type kinetics. Tyrosinase showed the highest affinity for L-tyrosine (Km = 0.23 mM) and the lowest for 3-fluorophenol (Km = 3.21 mM). Both regiochemistry and stereochemistry had an effect on the affinity of tyrosinase for its substrates with a lower Km observed for a-methyl-DL-tyrosine compared to a-methyl-L-tyrosine and 4-fluorophenol compared to 3-fluorophenol, respectively. Maximum activity, with tyrosine (monophenolase) and L-dopa (diphenolase), was observed at 30 °C and pH 7.0. The enzyme was stable over a broad range of temperatures and was most stable at 30 °C. Both the monophenolase and diphenolase activities were relatively stable across a broad range of pH values with maximum stability at pH 5.0 and 4.0 respectively.

The coexistence of two o-diphenol oxidising activities in the crude cell extract of P. putida F6 is also reported here. The purification of the second o-diphenol oxidising activity was carried out by anion exchange chromatography followed by size exclusion chromatography. The o-diphenol oxidising enzyme isolated in this study has activity towards the o-diphenol, L-dopa, and was capable of oxidising the laccase specific substrate syringaldazine, it also consumed oxygen in the presence of hydroquinone and was unable to oxidize the monophenol, tyrosine. These preliminary observations suggest that it is a laccase type enzyme.  The native form of the laccase enzyme from P. putida F6 is a monomer of approximately 59,000 as determined by size exclusion chromatography. The Km of the laccase for syringaldazine and L-dopa were 0.11 and 0.84 mM respectively. The maximum rate of activity for syringaldazine and L-dopa was 151 and 38 nmoles/min/mg respectively.   Maximum activity was observed at 30 °C and pH 7.0 for the oxidation of syringaldazine.  The enzyme was stable over a broad range of temperatures and was most stable at 30 °C. It was most stable at pH 7 with a narrow bell shaped curve over a range of pH values.  

In conclusion this study investigates the ability of a previously uncharacterised bacterial tyrosinase (unpurified and purified) to detect a range of 4-substituted phenols. Crude cell extracts of Pseudomonas putida F6 contained more than one o-diphenol oxidizing activity. Both the tyrosinase and laccase enzymes were purified and characterised.