Identification and Characterization of Tyrosinase from Streptomyces Albus by Mass Spectrometry

ABSTRACT

The 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, K_m= 7.8 mM and 0.5 mM and K_cat/K_m= 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.

Keywords:

• enzyme kinetics
• mass spectrometry
• tyrosinase
• Streptomyces albus