Hydroxylation of L-tyrosine to 3,4-dihydroxyphenylalanine (L-DOPA) by immobilized tyrosinase in the presence of ascorbic acid (AH2), which reduces DOPA-quinone to L-DOPA, is characterized by low reaction yields that are mainly caused by the suicide inactivation of tyrosinase by L-DOPA and AH2. The main aim of this work was to compare processes with native and immobilized tyrosinase to identify the conditions that limit suicide inactivation and produce substrate conversions to L-DOPA of above 50% using HPLC analysis. It was shown that immobilized tyrosinase does not suffer from partitioning and diffusion effects, allowing a direct comparison of the reactions performed with both forms of the enzyme. In typical processes, additional aeration was applied and boron ions to produce the L-DOPA and AH2 complex and hydroxylamine to close the cycle of enzyme active center transformations. It was shown that the commonly used pH 9 buffer increased enzyme stability, with concomitant reduced reactivity of 76%, and that under these conditions, the maximal substrate conversion was approximately 25 (native) to 30% (immobilized enzyme). To increase reaction yield, the pH of the reaction mixture was reduced to 8 and 7, producing L-DOPA yields of approximately 95% (native enzyme) and 70% (immobilized). A three-fold increase in the bound enzyme load achieved 95% conversion in two successive runs, but in the third one, tyrosinase lost its activity due to strong suicide inactivation caused by L-DOPA processing. In this case, the cost of the immobilized enzyme preparation is not overcome by its reuse over time, and native tyrosinase may be more economically feasible for a single use in L-DOPA production. The practical importance of the obtained results is that highly efficient hydroxylation of monophenols by tyrosinase can be obtained by selecting the proper reaction pH and is a compromise between complexation and enzyme reactivity.

The main goal of this study was to select micro-and ultrafiltration membranes that can be used for the purification of mushroom tyrosinase, replacing salting-out dual-step processes followed by centrifugations. In experiments, a raw extract from white mushrooms was used with high level of ballast proteins and brownish impurities. Four microfiltration membranes for the removal of undesired high molecular weight compounds were screened and that made of nitrocellulose was selected due to high recovery of enzymatic activity. Then diafiltration and concentration on the membrane made of polyethersulphone (300 kDa) was selected to recover 8% of proteins and 58% of tyrosinase activity with five-to seven purification fold, 10% of proteases, and 8% of brown impurities. It was shown that tyrosinase can be pre-purified by selected membranes yielding the enzyme quality at least comparable to that after double salting-out method but in one device. In both cases, subsequent purification by ion-exchange chromatography slightly increased purification degree of the enzyme and brown impurity removal. The surplus of membrane pre-purification is substantially higher thermal stability of the enzyme, enlarged after the chromatographic step, due to very low content of proteolytic enzymes.