Isolation, cloning and characterization of a tyrosinase with improved activity in organic solvents from Bacillus megaterium.
A tyrosinase-expressing bacterium was isolated from soil, and extracellular enzymatic activity was induced by the presence of tyrosine and CuSO(4). 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 x 10(3) and 32.9 x 10(3) (s(-1) x M(-1)). The maximum activity for both monophenolase and diphenolase was observed at 50 degrees 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.
Shuster
Vered
V
Department of Biotechnology and Food Engineering, Technion-Israel Institute of Technology, Haifa, Israel.
Fishman
Ayelet
A
D000595:Amino Acid Sequence; D001410:Bacillus megaterium / Q000201:enzymology* / Q000302:isolation & purification; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004275:DNA, Ribosomal / Q000737:chemistry / Q000235:genetics; D020536:Enzyme Activators / Q000494:pharmacology; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007980:Levodopa / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D014442:Monophenol Monooxygenase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D009930:Organic Chemicals / Q000494:pharmacology; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D012988:Soil Microbiology; D012997:Solvents; D013696:Temperature; D014443:Tyrosine / Q000378:metabolism
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D020536:Enzyme Activators; D009930:Organic Chemicals; D012336:RNA, Ribosomal, 16S; D012997:Solvents; D014443:Tyrosine; D007980:Levodopa; D014442:Monophenol Monooxygenase