טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentShuster-Ben-Yosef Vered
SubjectIsolation and Characterization of a Novel Bacterial
Tyrosinase
DepartmentDepartment of Biotechnology and Food Engineering
Supervisor Professor Ayelet Fishman
Full Thesis textFull thesis text - English Version


Abstract

Tyrosinases are copper-containing enzymes which are widely distributed throughout the phylogenetic scale, from bacteria to humans. Molecular oxygen is used by tyrosinases to catalyze two different enzymatic reactions: (i) the ortho-hydroxylation of monophenols to ortho-diphenols (monophenolase activity) and (ii) the oxidation of ortho-diphenols to ortho-quinones (diphenolase activity). The diphenolase activity is much faster than the monophenolase activity, limiting the use of tyrosinases for diphenol synthesis. There is a vast amount of literature available on the mechanism and biochemical nature of tyrosinases, however the information on the catalytic residues involved in catalysis is yet limited.

The overall goal of this research was to obtain a tyrosinase with an improved monophenolase/diphenolase activity ratio. In order to reach the desired enzymatic property the project was divided into three parts. The aim of the first part was to discover and isolate a tyrosinase producing bacteria using functional based screening from soil samples. Subsequently, to purify and characterize the tyrosinase. The aim of the third part was to use various protein engineering approaches to obtain an enzyme variant with altered activity and specificity.

A novel tyrosinase-producing bacterium was isolated on selective media from a soil sample and identified as Bacillus megaterium using 16S rDNA analysis. The extracellular enzyme was over-expressed in Escherichia coli BL21, was purified using an affinity column, and characterized biochemically. The enzymatic activity was enhanced in the presence of 10-50% water-miscible organic solvents as ethanol, methanol, 2-propanol and dimethyl sulfoxide.

Colorimetric assays were developed for high-throughput screening of enzyme libraries for enhanced monophenolase activity on tyrosine and phenol. The diphenolase activity of the best variants was examined using L-DOPA and catechol on a larger scale. This work highlights two new positions, R209 and N205, which influence tyrosinase activity and selectivity. By replacing Arg209 with His209, Phe209 or Ile209 the monophenolase/diphenolase activity ratio with tyrosine/L-DOPA was improved. In-silico modeling of R209H and R209F using the newly reported crystal structure of wild type tyrosinase of B. megaterium, suggested that the histidine and phenylalanine side-chain obstruct the entrance to the active site thus interfering with L-DOPA binding to CuB. Modeling of R209I and N205D suggested that a change in the electrostatic potential near CuB influences the enzyme selectivity.

Our results support the hypothesis that monophenols and diphenols bind differently in the active site of tyrosinase. In addition, this research emphasizes the benefits of combining random and rational design to get altered enzyme performance.