The yeast Yarrowia lipolytica is a hemiascomycete and represents a homogeneous phylogenetic group with physiological and ecological diversity [1]. It is a non-conventional yeast, often used in research and isdistantly related to Candida glabrata, Kluyveromyces lactis and Debaryomyces hansenii. Strains of Y. lipolytica can produce significant amounts of intra- or extra-cellular metabolites including vitamins, lipases, storage lipids, citric acid and pyruvic acid and can be used for biodegradation of various wastes (e.g., olive-mill waters and raw glycerol) [2-6]. The 3,4-dihydroxy phenyl L-alanine (L-dopa) is a drug used for Parkinson's disease, and is capable of changing the enzymes of energy metabolism of myocardium following neurogenic injury. The process of bioconversion of L-tyrosine to L-dopa in microorganisms is generally slow, but is accelerated by a small amount of L-dopa in the broth [7]. L-dopa has also been produced with Erwinia herbicola cells carrying a mutant transcriptional regulator TyrR from pyrocatechol and DL-serine [8,9]. It can also be produced using L-tyrosine as a substrate, tyrosinase as a biocatalyst and L-ascorbate as the reducing agent [10,11]. The general reaction is:
Tyrosinases (monophenol, o-diphenol:oxygen oxidoreductase, EC 1.14.18.1) belong to a larger group of type-3 copper proteins, which include catecholoxidases and oxygen-carrier haemocyanins [12]. Tyrosinases are involved in the melanin pathway and are responsible for the first steps of melanin synthesis from L-tyrosine, leading to the formation of L-dopaquinone and L-dopachrome [13]. Tyrosinases catalyse the o-hydroxylation of monophenols (cresolase activity or "monophenolase") and the ensuing oxidation of molecular oxygen. Subsequently, the o-quinones undergo non-enzymatic reactions with various nucleophiles, producing intermediates [14]. The immobilization of tyrosinases on solid supports can increase enzyme stability [15-19], protect tyrosinase from inactivation by reaction with quinones, (preserving them from proteolysis) [20], improve thermal stability of fungal tyrosinases [21], and increase activity in comparison to soluble enzymes [22].
Diatomite (2:1 clay mineral) is a naturally occurring, soft, chalk-like sedimentary rock that is easily crumbled into a fine, off-white powder which has K+ in the interlayer. This powder has an abrasive feeling similar to pumice and is light-weight due to its porosity. By adding diatomite into the reaction, an increased substrate uptake and enzyme production rate with concomitant L-dopa production could result. We have previously reported the effect of cresoquinone and vermiculite on the microbial transformation of L-tyrosine to L-dopa by Aspergillus oryzae [23,24]. In the present study, different concentrations of diatomite were added into the reaction mixture to achieve a high performance transformation of L-tyrosine to L-dopa.
A. oryzae is an organism typically used for L-dopa production. The easy handling, rapid growth rate and environmentally friendly nature of alternative yeasts such as Y. lipolytica have created an interest in their use for fermentation. Because tyrosinases are intracellular enzymes, pre-grown cells harvested from fermented broth were used for the microbiological transformation of L-tyrosine to L-dopa.
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