Isolation, Cloning and Characterization of a Tyrosinase with Improved Activity in Organic Solvents from <i>Bacillus megaterium</i>

Abstract

A tyrosinase-expressing bacterium was isolated from soil, and extracellular enzymatic activity was induced by the presence of tyrosine and CuSO₄. Amplification of the 16S rDNA genes revealed a high similarity with Bacillus megaterium. The enzyme was over-expressed in Escherichia coli BL21 and purified using an affinity column. The tyrosinase was composed of 297 amino acids and was determined to be a monomer with a relative molecular mass of 31 kDa according to gel filtration. The K_m values for 3,4-dihydroxy-L-phenylalanine (L-DOPA) and L-tyrosine were 0.35 and 0.075 mM, respectively, and the k_cat/K_m values were 28.9·10³ and 32.9·10³ (s^–1·M^–1). The maximum activity for both monophenolase and diphenolase was observed at 50°C and pH 7.0. Enzymatic activity was enhanced in the presence of 10–50% water-miscible organic solvents, which included ethanol, methanol, 2-propanol and dimethyl sulfoxide (DMSO). The activity in 30% DMSO was 170% of the activity in water and the enantioselectivity towards L-DOPA decreased by 40%. The residual activity following an incubation period of 17 h in 0–70% methanol was constant. This newly isolated and characterized tyrosinase may have potential applications in organic synthesis due to its high activity and stability at typically denaturing conditions.

Characterization of Tyrosinase Enzyme From Native Bacillus Megaterium Sp. Strain M36

“…This analysis showed almost 34kDa bond of the enzyme ( Figure 5). This result was similar to the result of Shuster and Fishman (2009) who have demonstrated the tyrosinase from Bacillus megaterium to be almost 35kDa. …”

Determination of tyrosinase substrate-binding modes reveals mechanistic differences between type-3 copper proteins

“…A tyrosinase producing TyrBm was isolated from soil samples and the gene encoding for the tyrosinase was cloned into Escherichia coli BL21. The enzyme was expressed, purified and crystalized as previously described 7,24,25 . Briefly, E. coli cells were grown overnight at 37°C in 0.5 l of TB medium (tryptone 1.2%, yeast extract 2.4%, glycerol 0.4% (w/v) and potassium phosphate buffer 89 mM) and then harvested by centrifugation (8,000 g for 10 min at room temperature).…”

Characterization of oil-in-water emulsions stabilized by tyrosinase-crosslinked soy glycinin

“…TyrBm was isolated in our laboratory from soil samples, and the gene encoding for the tyrosinase was cloned into Escherichia coli BL21, expressed and purified as previously described, and the enzyme activity was determined on L-tyrosine, and L-dopa (Shuster & Fishman, 2009). In this work, the enzyme activity on caffeic acid was determined as well.…”

“…Chlorogenic acid has a very large side chain which might limit the substrate availability to the active site thus leading to a smaller activity rate. The reaction mixture in the presence of a phenolic reagent contained a strong pigment attributed to self-polymerization of the oxidized phenolic reagent and the formation of melanin (Goldfeder et al, 2012;Shuster & Fishman, 2009). In the control tubes, which were incubated under the same conditions with distilled water rather than enzyme, a low amount of pigment was observed, indicating that the reactive diphenols (caffeic acid and chlorogenic acid) spontaneously oxidized to quinones and further polymerized.…”

“…Tyrosinase from the soil bacteria Bacillus megaterium (TyrBm) was isolated and characterized in our lab and was investigated for its monophenolase and diphenolase activities (Goldfeder, Egozy, Shuster Ben-Yosef, Adir, & Fishman, 2012;Sendovski, Kanteev, Shuster Ben-Yosef, Adir, & Fishman, 2010;Shuster & Fishman, 2009). TyrBm is a "type-3-copper" enzyme, which contains two copper ions in its active site that are necessary for its activity.…”