Characterization Of Tyrosinase Research Articles

Biochemical characterization with kinetic studies of melanogenic enzyme tyrosinase from white button mushroom, Agaricus bisporus

In Agaricus bisporus, color is a key determinant for marketability and consumer acceptability. However, postharvest browning has become a major concern, affecting the overall economics of the mushroom industry. In button mushrooms, the tyrosinase enzyme (E.C.1.14.18.1) is responsible for the browning reactions by catalyzing the conversion of monophenols and diphenols into quinones which polymerize to form melanin. Thus, the present study focused on the purification and characterization of tyrosinase from A. bisporus. This enzyme was purified with a final yield of 19.71% and 32.05 purification fold. The study of enzymatic activity over a temperature (5-45°C) and pH range (3-10) showed that the optimum temperature was 35°C with pH 7. The kinetic studies revealed that Km values were different for catechol (0.71 mM) and L-dopa (0.87 mM), which indicated a higher affinity of the enzyme for catechol. Inhibition studies showed that cinnamic acid is a non-competitive inhibitor while salicylic acid is a competitive inhibitor of tyrosinase. The molecular weight of the enzyme was found to be 43 kDa and different amide regions were reflected by the FTIR spectra of the enzyme. This study may provide valuable insights into the structure, biochemical properties, and inhibition of tyrosinase enzyme for controlling mushroom browning.

Discovery and characterization of tyrosinases from sea anemone pedal disc

The formation of underwater adhesion is a complicated physiological process and many different types of enzymes are found to be essential apart from structural proteins. Previous studies have shown that various tyrosinases were present in marine adhesives, but little information is available about the over-expression and enzymatic characterization of these enzymes. Specifically, this study first identified four significantly up-regulated tyrosinases in the pedal disc of Haliplanella luciae by means of multi-omics technology, and made preliminary bioinformatics predictions. Sequence alignment showed that the Tyr1_Hl contained six conserved His residues that bind to copper ions, of which a tyrosinase with diphenolase activity named as Tyr1_HlΔ, was expressed in Escherichia coli BL21 (DE3) cells and purified by affinity chromatography. Enzymatic characterization showed that the activity of Tyr1_HlΔ was Cu2+ dependent and maximum catalytic activities were in 20 mM Tris–HCl (pH 8.0) at 37 °C. In summary, we identified novel tyrosinases in the pedal disks of sea anemone for the first time and the Tyr1_HlΔ was successfully recombinant expressed. Our study will provide basis for future exploration of bio-adhesion mechanism and design of bio-adhesives derived from sea anemones.

Optimization and Characterization of Tyrosinases from Multi-enzyme Producing Fusarium solani and Fumago sp.

Tyrosinase is a copper-containing metalloprotein that catalyzes the oxidation of tyrosine, in particular, L-DOPA to L-Dopaquinone, which are precursors of brown pigments in some wounded eukaryotic tissues. The present study focused on screening, production and characterization of tyrosinase from multi-enzyme producing Fusarium solani B1 and Fumago sp. A total of 25 strains were isolated from rotting wood samples and screened for hydrolytic and oxidative multi-enzyme potentials using different polymeric substrates. The two most consistent strains: Fusarium solani B1 and Fumago sp. B13 were further evaluated for tyrosinase production. Some media cultural parameters and physiological conditions were optimized in order to maximize tyrosinase production. Incubation of Fumago sp. B13 and Fusarium solani B1 for 96 and 144 h in medium containing 2 % and 0.2 % ratios of Glucose and NaNO3 with pH 6 and 7, respectively, was most suitable for tyrosinase production. Characterization of the partially purified tyrosinase from Fumago sp. B13 and Fusarium solani B1 exhibited optimal activities at pH 6-7, 30 °C, and 1 mM Cu2+, respectively, thereby suggesting their potentials for novel biotechnological applications.

Purification and characterization of tyrosinase from walnut leaves (Juglans regia)

Polyphenol oxidase (PPO) is a type-3 copper enzyme catalyzing the oxidation of phenolic compounds to their quinone derivates, which are further converted to melanin, a ubiquitous pigment in living organisms. In this study a plant originated tyrosinase was isolated from walnut leaves (Juglans regia) and biochemically characterized. It was possible to isolate and purify the enzyme by means of an aqueous two-phase extraction method followed by chromatographic purification and identification. Interestingly, the enzyme showed a rather high monophenolase activity considering that the main part of plant PPOs with some exceptions solely possess diphenolase activity. The average molecular mass of 39,047Da (Asp101→Arg445) was determined very accurately by high resolution mass spectrometry. This proteolytically activated tyrosinase species was identified as a polyphenol oxidase corresponding to the known jrPPO1 sequence by peptide sequencing applying nanoUHPLC–ESI-MS/MS. The polypeptide backbone with sequence coverage of 96% was determined to start from Asp101 and not to exceed Arg445.

Isolation and Characterization of Novel Tyrosinase from Laceyella sacchari

We here describe the isolation and characterization of a tyrosinase from a newly isolated soil bacterium. 16S rDNA sequence analysis revealed that the bacterium most probably belongs to the species Laceyella sacchari (Ls) ( > 99.9 % identity). The tyrosinase extracellular enzymatic activity was induced in the presence of L-methionine and CuSO4. The crude enzyme was first purified by centrifugation followed by ammonium sulphate precipitation and ultrafiltration. After removal of a brown pigment, probably melanin, a purified enzyme was obtained by further separation of the crude protein mixture using size exclusion chromatography. Some 10.5 mg of pure tyrosinase (LsTyr) was isolated with a molecular mass of 30 910 Da, based on MALDI mass spectrometry. Together with the observed enzymatic activity, N-terminal chemical sequence analysis confirmed that the isolated enzyme is homologous to other tyrosinases. The kinetic parameters for the diphenol substrates L-DOPA and dopamine and for the monophenol substrate L-tyrosine were determined to be KM = 4.5 mM , 1.5 mM and 0.055 mM, and kcat/KM = 261.5 mM-1 s -1 , 30.6 mM-1 s-1 and 56.3 mM-1 s-1, respectively. Maximal activities of the purified enzyme were found to occur at pH 6.8.

Production and characterization of tyrosinase activity in Pycnoporus sanguineus CCT-4518 crude extract

Tyrosinase is an enzyme of industrial interest. The production and characterization of tyrosinase from P. sanguineus CCT-4518 were investigated. The selection of inductors, luminosity influence, inoculum size and type of culture medium on the production of tyrosinase and the effect of inhibitors on enzyme activity were performed. Optimum conditions for intracellular tyrosinase production was observed after 2 days using 0.15% L-tyrosine as inducer, in the presence of light, with inoculum size of 10 mycelium discs, using 2% malt extract broth medium, incubated at 30°C, and constant agitation of 150 rpm. Tyrosinase activity was completely inhibited by the addition of 6 mM salicylhydroxamic acid or phenylthiourea, however an inhibition of 4.15% was recorded by the addition of 0.1 mM sodium azide. No inhibition could be detected in case of 0.1 mM phenyl methanesulfonyl fluoride addition. Optimal conditions for intracellular tyrosinase activity using L-dopa as substrate were observed at pH 6.6 and 45°C. Thermal stability studies indicated that the enzyme is stable at 45°C for 15 minutes. Higher temperatures decreased tyrosinase activity. Enzyme production was confirmed by non-denaturing polyacrylamide gel electrophoresis and the protein profile was investigated by denaturing polyacrylamide gel electrophoresis.

Purification and Characterization of Tyrosinases from Streptomyces albus

The bacterium Streptomyces albus has so far never been investigated for tyrosinase activity. The studies presented in this communication show that this bacterium may be a future source for larger production of tyrosinase. The enzyme was purified starting with 5,600 ml of culture filtrate. The crude enzyme was first purified by centrifugation, followed by ammonium sulfate precipitation and ultrafiltration. Then, melanin was removed applying a Servacell DEAE 52 resin, using the batch technique. Thereafter, the crude enzyme was loaded on a SEC Sephacryl S-100 column and, after ultrafiltration, 1.17 mg of purified tyrosinase were obtained. The molecular mass of the purified enzyme was determined by MALDI mass spectrometry to be 30,096 Da which corresponds to the obtained results from SDS-PAGE. Using the diphenol L-DOPA and the monophenol L-tyrosine as substrates, the kinetic parameters for both substrates, Km = 7.8 mM and 0.5 mM and k(cat)/Km = 157 mM(-1) s(-1) and 23 mM(-1) s(-1), respectively, were determined. Maximal activities of the purified enzyme were recorded at pH 7.0. Long-term experiments with Streptomyces albus tyrosinase revealed that storage of the lyophilized enzyme sample at temperatures below zero turned out to be the best. For tyrosinase in buffer containing 20% glycerol, no loss of activity was observed at 4 degrees C and -60 degrees C.

Identification and Characterization of Tyrosinase from Streptomyces Albus by Mass Spectrometry

ABSTRACTThe bacterium Streptomyces albus has so far never been investigated for tyrosinase activity. The studies presented in this communication show that his bacterium is maybe a future source for larger production of tyrosinase. The enzyme was purified starting with 5 600 ml of culture filtrate. The crude enzyme was first purified by centrifugation, followed by ammonium sulphate precipitation and ultrafiltration. Then, melanin was removed applying a Servacell DEAE 52 resin, using the batch technique. Thereafter, the crude enzyme was loaded on a SEC Sephacryl S-100 column and after ultrafiltration 1.17 mg of purified tyrosinase were obtained. The molecular mass of the purified enzyme was determined by MALDI mass spectrometry to be 30 096 Da which corresponds to the obtained results from SDS-PAGE.Using diphenol L-DOPA and the monophenol L-tyrosine as substrates, the kinetic parameters for both substrates, Km= 7.8 mM and 0.5 mM and Kcat/Km= 157 mM−1s−1 and 23 mM−1s−1, respectively, were determined. Maximal activities of the purified enzyme were recorded at pH 7.0.Several isolated peptides were sequenced by MALDI MS/MS and a spectrum of the peptide with mass 1357.64 was characterized and allowing to deduce the sequence SDRQVTTGPFAYRHG. A spectrum of the peptide with molecular mass 1536.61 was characterized and deduced sequence is WVGGQMATGVSPN. Other spectrum of a peptide with molecular mass 1334.3 gave sequence DTDSGERTGHR. The identified amino acid sequences of the peptides showed very high similarity with database sequences for other tyrosinases from Streptomyces species.

Partial purification and characterization of tyrosinase extracted from mushroom (Agaricus bisporus)

Partial purification and characterization of tyrosinase extracted from mushroom (Agaricus bisporus)

Purification and characterization of tyrosinase from gill tissue of Portabella mushrooms

A group of tyrosinase isoforms with isoelectric points between 4.9 and 5.2 was isolated from gill tissue of Portabella mushrooms. Use of protease inhibitors was not able to increase the amount of latent forms significantly in crude extracts or to preserve latent tyrosinase activity during purification. The tyrosinase in gill tissue extracts showed latent activity above pH 5.5 and suppressed or displayed no latent activity below pH 5.5 when assayed in the presence of SDS. The purified isoforms showed monophenolase activity toward 4-hydroxyanisole but practically no activity toward tyrosine or tyramine. The purified isoforms showed greater activity toward catechol than either 4-methylcatechol, dopa, dopamine, chlorogenic acid, t-butylcatechol, or catechin. The Km for catechol was similar for the group of isolated isoforms (4.3 mM) compared to the isoforms in crude extracts (5.3 mM). Crude extracts showed several isoforms ranging from 50 to 230 kDa after partially denaturing SDS PAGE, while the purified isoforms showed molecular weights of 70 kDa.

Characterization of tyrosinase- and polyphenol esterase-catalyzed end products using selected phenolic substrates.

The oxidative end products that result from the biocatalysis of tyrosinase (PPO) and/or a polyphenol esterase (PPE) extract have been investigated simultaneously in model systems containing selected phenolic compounds as substrates. The spectrophotometric scanning of brown color, formed in the presence of both PPO and PPE, showed a decrease in the absorbance compared to that obtained with PPO only. Graphical analyses of the iterative spectra of oxidized phenolic end products by PPO confirmed the presence of, at least, three kinetically related absorbing species. HPLC analyses of the end products, obtained by the biocatalysis of PPE or PPO activity, indicated the presence of two main groups of compounds: colored ones of lambda(max) at 294-324 nm and colorless products of lambda(max) at 264-290 nm. PPE produced both compounds when selected substrates were used as substrates, whereas PPO produced only one type of oxidation product. However, when both enzymes were incubated together, the nature of the end products was similar to that obtained with PPE only.

Characterization of tyrosinase from the cap flesh of portabella mushrooms.

Tyrosinase, purified from the cap flesh tissue of portabella mushrooms, was characterized with regard to its physical and biochemical properties. A native molecular size of 41 kDa for the enzyme was obtained by size exclusion chromatography, whereas SDS-PAGE indicated that the enzyme contained a single subunit with a size of approximately 48 kDa under reduced and nonreduced conditions. The purified enzyme showed a single immunological cross-reacting protein after Western blotting when probed with antibodies against Agaricus bisporus tyrosinase. Isoelectric focusing demonstrated that the enzyme preparation, apparently homogeneous by electrophoresis, still contained three isoforms of pI 5.1, 5.2, and 5.3. The purified enzyme was able to oxidize a variety of mono-, di-, and triphenolic compounds. An apparent K(m) of 5 mM was obtained using catechol as the substrate, and an apparent K(m) of 9 mM was found using L-Dopa as a substrate. Ascorbic acid, kojic acid, tropolone, mercaptobenzothiazole, and salicylhydroxamic acid inhibited the enzyme severely at 100 microM.

Purification and properties of tyrosinases from Vibrio tyrosinaticus

Two tyrosinase isoenzymes (EC1. 10.3.1) have been highly purified from Vibrio tyrosinaticus . This represents the first characterization of tyrosinases from eubacteria. The enzymes have molecular weights of 41,000 and 38,500, closer to the tyrosinase of Neurospora crassa (33,000) than to the mammalian enzymes from mouse and hamster melanoma (55,000 and 70,000). The bacterial tyrosinases are also different from the mammalian enzymes in that the former possess basic isoelectric points. The Vibrio tyrosinases do not cross-react with antiserum against a hamster melanoma tyrosinase. The K m values for l -tyrosine are 3.1 m m for both isoenzymes while the K m value for l -DOPA is 36 m m for one and 67 m m for the other. The values for tyrosine are tenfold, and those for dopa about 100-fold, larger than the corresponding K m values for the tumor enzymes. Apoenzyme was prepared by treatment with diethyldithiocarbamate, and activity was partially or wholly restored by addition of Cu 2+ , Mn 3+ , Cd 2+ , or Fe 2+ . The one isoenzyme examined has some activity on d -tyrosine and very slight activity against d, l - m -tyrosine. No activity was detected with catechol or l -phenylalanine.