Journal of Molecular Catalysis B: Enzymatic 84 (2012) 121–127 Contents lists available at SciVerse ScienceDirect Journal of Molecular Catalysis B: Enzymatic jo u rn al hom epa ge: www.elsevier.com/locate/molcatb Improving process conditions of hydroxytyrosol synthesis by toluene-4-monooxygenase Moran Brouk, Ayelet Fishman ∗ Department of Biotechnology and Food Engineering, Technion – Israel Institute of Technology, Haifa 32000, Israel a r t i c l e i n f o Article history: Available online 22 May 2012 Key words: Hydroxytyrosol Boric acid gel Toluene-4-monooxygenase Scale-up Adsorption a b s t r a c t Toluene-4-monooxygenase from Pseudomonas mendocina KR1 was recently engineered for the synthesis of hydroxytyrosol, a potent antioxidant. Following a 190-fold improvement in the enzyme activity by protein engineering means, improving the process conditions of this biocatalytic route was under taken for developing a liter-scale bioprocess. The growth stage was improved by selection of a rich media and harvesting the cells at the end of the logarithmic stage. The biotransformation stage was optimized by evaluating substrate concentration, cell density, and different operational modes. It was found that although reusing the cells in successive batch modes is feasible, their activity is dramatically decreased after the first use. In comparison, the activity of the cells following subsequent substrate addition in a fed batch mode was only slightly decreased. Furthermore, a better yield was obtained by extending the duration of the biotransformation stage, rather than adding more substrate. An overall concentration of 133 mg/L HTyr, corresponding to a volumetric productivity of 54 mg/L/h and a yield of 48% was achieved by a batch mode using 2 mM substrate. This is an order of magnitude improvement compared with the enzyme productivity before the process optimization. The use of beads conjugated with phenylboronic acid residues for adsorbing the product from the biotransformation bulk was evaluated. Though the recovery yield and purity were shown to be oppositely dependent, an average recovery procedure led to 2-fold purification of HTyr resulting in 84% purity with 70% recovery yield. © 2012 Elsevier B.V. All rights reserved. 1. Introduction There is an increasing interest in the use and application of bio- catalytic processes at an industrial scale [1–4]. Hence, as the scale of the biocatalytic process increases, different considerations become more significant, and the process is required to overcome several limitations; mainly, low solubility of the substrate, toxicity of either substrate and/or products, low enzyme productivity, and metabolic diversity which leads to undesired by-products or further degra- dation of the desired molecules. For overcoming such weaknesses, efforts can be carried out from the point of view of either the bio- catalyst (exploiting protein engineering to adjust the biocatalysts to a specific process) or the process (exploiting different biochem- ical tools for adjusting the process in a way that optimizes the biocatalyst activity) [1,4]. The biocatalytic process described in the present study is the whole cell biotransformation of 2-phenylethanol (PEA) to form the substituted catechol hydroxytyrosol (HTyr). HTyr, a commer- cially valuable antioxidant, is naturally present in olives and has ∗ Corresponding author. Tel.: +972 4 829 5898; fax: +972 4 829 3399. E-mail address: afishman@tx.technion.ac.il (A. Fishman). been shown to be beneficial in preventing various diseases, such as diabetes, atherosclerosis and cancer [5–7]. Recently, we reported the designing of toluene 4-monooxygenase (T4MO) variants for the biosynthesis of HTyr by whole (resting) cell biotransformation [8–10]. T4MO, the biocatalyst chosen to perform this reaction, is an O 2 -dependent multicomponent monooxygenase which requires NADH as a cofactor [11]. Hence, performing the reaction with a whole cell system enables NADH regeneration saving the tedious and costly process of protein isolation and purification. Moreover, the formation of undesired byproducts or further product degrada- tion is minimized by the heterologous expression of the enzyme in Escherichia coli TG1 cells [11]. Earlier attempts to enhance the production of HTyr from PEA focused on the biocatalyst itself, by exploiting protein engineering to improve its catalytic activity [8–10]. These attempts lead to the discovery of a variant, TmoA I100A/E214G/D285Q, which exhibited an initial oxidation rate of 4.4 ± 0.3 nmol/min/mg protein, which is 190-fold faster than the rate obtained by wild-type [10]. How- ever, the productivity achieved by the engineered biocatalysts is not yet ideal nor economically feasible for industrial applications. Hence, with the aim of enhancing HTyr production using recombi- nant E. coli cells, we changed the focus towards the process itself, to optimize the biocatalyst activity. 1381-1177/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.molcatb.2012.05.010