Optimizing the BFD-catalyzed synthesis of 2-HPP in a resting cells-based bioprocess
Biotransformation is an efficient tool for the synthesis of different chiral α-hydroxy ketones. Bioconversion can be performed via growing cells, resting cells, partially purified or thoroughly purified enzymes. The industrial production of PAC is currently done using growing yeast cells in a process where the exogenous benzaldehyde is condensed with acetaldehyde using the carboligation activity of PDC enzyme [53]. A similar process was also examined applying growing P. putida cells for a fermentative 2-HPP synthesis in the presence of aldehyde substrates in this study. However, this strain did not have the ability to produce 2-HPP coupled to growth as expected. Since high concentrations of growth-inhibitory substrates could cause some toxicity problems in actively growing cells [54, 55]. The mandelate pathway also needs to be induced in the presence of mandelic acid and employing growing biocatalysts would not be particularly useful. Moreover, the microbial transformation of benzaldehyde and acetaldehyde to 2-HPP by P. putida growing cells may lead to the conversion of benzaldehyde to benzoate by benzaldehyde dehydrogenase in the mandelate pathway even prior to BFD induction [56]. So, it was envisioned that the bioconversion with P.putida in a resting cells process could be a better choice. Acyloin synthesis by resting cells is a mode of production in which there is no cell multiplication. Nevertheless, these cells are able to carry out the biotransformation using the enzymes and active cell metabolism [57]. To this end, biotransformation reactions were conducted utilizing P. putida whole resting cell biocatalysts as enzyme sources. Cells in Pseudomonas mineral medium (PMM) were supplemented with 3 g L− 1 ammonium mandelate and 1 g L− 1 yeast extract to completely induce BFD enzyme in cells. Cells were harvested in the middle of the exponential growth phase (OD600 0.9-1) by centrifugation, washed and resuspended in phosphate buffer, and then incubated aerobically at 30°C. Under these conditions, biomass duplication was prevented. So, the benzoin condensation reaction of P. putida resting cells was conducted in a batch synthesis in the presence of aldehydes without further processing. The reduction in the substrates concentration and the accumulation of 2-HPP in the reaction medium were monitored by frequent sample analysis by GC. Several parameters involved in reaction performance including substrate concentration, biomass concentration, reaction time, and pH were optimized to achieve the highest possible productivity and enantiomeric purity [15, 29, 39, 58].
Optimization Of Acetaldehyde Concentration
The cross condensation of benzaldehyde and acetaldehyde leads to the formation of S-2-HPP through carboligase activity of the BFD enzyme from P. putida. Acetaldehyde concentration and ratio of acetaldehyde to benzaldehyde concentration are key parameters affecting the biotransformation reaction and are prerequisites for the successful setup of a stable process catalyzed by BFD. Therefore, at a fixed benzaldehyde concentration of 50 mM, varied concentrations of acetaldehyde from 200 mM up to 1600 mM were examined. As shown in Fig. 1, increasing acetaldehyde concentration from 200 to 600 mM was concomitant with two-fold increase in the 2-HPP titer. High concentrations of acetaldehyde up to 600 mM can decrease the production of benzyl alcohol since acetaldehyde can deactivate oxidoreductase enzymes responsible for the conversion of benzaldehyde to benzyl alcohol [58]. However, further increase in acetaldehyde concentration negatively affected 2-HPP production probably due to irreversible inhibition of BFD by acetaldehyde [29, 59]. Besides, at acetaldehyde concentrations exceeding 600 mM, substrates were not used completely and more than 65 percent of benzaldehyde remained unconsumed. Park et al. [60] studied production of benzaldehyde by free and immobilized whole-cell benzoyl formate decarboxylase. They also investigated the effect of acetaldehyde added to the reactant mixture containing 100 mM (15 g L− 1) benzoylformate on the bio-products produced by a whole-cell enzyme. It was shown that a concentration of 2.73 g L− 1 2-HPP was produced in the presence of 800 mM acetaldehyde as a co-reactant. The amount of benzaldehyde and benzyl alcohol produced were 5.13 and 0.3 g L− 1, respectively, and TPP, the expensive coenzyme of BFD, had to be added to the reactant mixture.
Optimization Of Biotransformation Duration
The effect of biotransformation time on the production of 2-HPP was studied. 2-HPP titers after 2 and 3 hours were 0.7 g L− 1 and 1.2 g L− 1, respectively. However, lower 2-HPP were obtained over more prolonged time periods (Fig. 2). Therefore, biotransformation duration of 3 hours was considered as the optimum time for further experiments.
Optimization Of Biomass Concentration
The initial cell concentration is one of the most critical parameters in the biotransformation of benzaldehyde and acetaldehyde to 2-HPP. Hence, the biotransformation reaction was set up at varying biomass concentrations to find the optimum value. Figure 3 represents the impact of whole-cell concentration on 2-HPP formation.
This figure shows that maximum 2-HPP titers were achieved at OD600 20 which is equivalent to cell concentration of 0.033 g w.w/ml (0.006 g DCW/ml). The yield of 2-HPP is not correlated to the increase in biomass concentration and 2-HPP production was not improved in a linear pattern proportional to the increase in cell load. This is probably due to the degradation of 2-HPP by the cell metabolism. We also speculated that at high biomass concentrations, cells demand a higher oxygen level. Similar results were obtained with resting cells of Salinivibrio costicula GL6 [61], Halomonas elongata [62], and P. fluorescence strain BF13 [63]. Furthermore, unwanted benzaldehyde side reaction leading to the formation of benzyl alcohol as a by-product was promoted with cell load surplus. Therefore, it was decided to use a cell load of 0.033 g w.w/ml (0.006 g DCW/ml) in the following experiments to keep the by-product concentration at the lowest levels. Wilcocks and co-workers [18] demonstrated 2-HPP production from benzoylformate using acetaldehyde as the co-substrate with whole induced cells of P. putida for the first time. Although whole cells yielded less 2-HPP than crude cell extracts, a maximum 2-HPP concentration of 4.5 g L− 1 was achieved by 0.015 g DCW/ml of whole cells in the presence of 15 g L− 1 benzoylformate as the main substrate after 2 hours of reaction. Furthermore, benzaldehyde (4.2 g L− 1) and benzyl alcohol (0.29 g L− 1) were formed as by-products in this biotransformation condition. Thiamine PPi and magnesium chloride were also used for all the reaction conditions tested, including cell extracts and whole cells. Since the price of the α-ketoacid is usually higher than the price of aldehydes, this biotransformation reaction is relatively economical and cost-effective due to the use of inexpensive benzaldehyde instead of expensive and infrequent benzoylformate as the main substrate.
Optimization Of Benzaldehyde Concentration
Increasing benzaldehyde concentration may yield higher product titers. However, high benzaldehyde concentrations could be toxic to the cells. Therefore, finding optimum benzaldehyde concentration is vital. The results in Fig. 4 indicate that complete depletion of benzaldehyde did not occur in any of the reactions. Nevertheless, further experiments revealed that benzaldehyde was consumed entirely by increasing the reaction time up to 20 hours but it may not be economically justified to continue the process for such prolonged times. As shown in Fig. 4, maximum 2-HPP titer was observed at 20 mM benzaldehyde. Interestingly, the minimum accumulation of benzyl alcohol as the main by-product was measured at benzaldehyde concentrations of 10 and 20 mM (Fig. 4).
It has been demonstrated that the ratio of benzaldehyde/acetaldehyde is important to form 2-HPP in an effective cross acyloin reaction. Higher concentrations of acetaldehyde in presence of lower benzaldehyde concentration is needed to produce 2-HPP with high yield [25]. Demir et al. [39] reported that 35-fold excess of acetaldehyde to benzaldehyde resulted in the formation of more 2-HPP since it prevents benzoin formation from homocoupling of two benzaldehyde molecules catalyzed by BFD. Our results showed maximum 2-HPP production at benzaldehyde and acetaldehyde concentrations of 20 mM and 600 mM, respectively. Applying an excess of benzaldehyde (30–40 and 50 mM) in relation to optimal acetaldehyde (600 mM) would mean that full conversion to 2-HPP cannot be achieved. Furthermore, unwanted self-condensation of benzaldehyde leading to the formation of R-benzoin and benzyl alcohol formation is promoted with a benzaldehyde surplus. Therefore, the optimum acetaldehyde to benzaldehyde concentration ratio was considered as 30.
Optimization Of Ph
Figure 5 demonstrates the effect of pH on 2-HPP titer. Highest 2-HPP concentrations were obtained at pH 7. Lower pH values resulted in a sharp decrease in 2-HPP production and reached almost zero at pH values below 5. pH values higher than 7 also had a negative impact on 2-HPP production.
2-HPP production using immobilized P. putida cells
The synthesis of 2-HPP starting from benzaldehyde and acetaldehyde has been examined by covalently immobilized BFD on magnetic epoxy attached magnetic nanoparticles. The activity of the immobilized BFD was determined to be 53.0% related to the activity of the free enzyme. The immobilized biocatalyst retained 95% of its original activity after five reaction cycles [64].
It is proven in several investigations that both aldehydes, benzaldehyde and acetaldehyde, have an inactivating effect on the activity of PpBFD. However, the inactivating impact of benzaldehyde is much higher in comparison to acetaldehyde [65, 66]. On the other hand, it would be better to use higher acetaldehyde concentration to achieve higher reaction rates. So, the simplest approach to overcome these limitations would be the use of immobilized whole cells.
Immobilization of cells enables recovery and reuse of P. putida cells for several cycles; thereby reducing the costs associated with 2-HPP production. Another merit to this immobilization system is that 2-HPP is produced purely without any terminal by-product formation since 2-HPP accumulated in the beads can inhibit alcohol dehydrogenases responsible for the conversion of benzaldehyde to benzyl alcohol [60, 67]. Cells immobilized in CA-PVA produced 0.6 g L− 1 2-HPP in the first cycle and 0.44 g L− 1 in the second cycle using 20 mM benzaldehyde and 600 mM acetaldehyde with 200 beads after 10 hours (Fig. 6-a). Increasing the benzaldehyde concentration to 40 mM resulted in the production of 0.9 g L− 1 2-HPP in the first and second cycles after 5 hours (Fig. 6-b). However, 2-HPP production decreased to 0.6 g L− 1 and 0.3 g L− 1 in the third and fourth cycles, respectively (Fig. 6-b). Increasing the biomass load from 0.033 g w.w/ml to 0.05 g w.w/ml improved total 2-HPP production from 2.8 g L− 1 to 3.3 g L− 1 during successive cycles (Fig. 6-c). Although the residual activity of immobilized catalyst decreased gradually with the increasing number of reaction cycles, with the immobilized cells several biotransformation cycles can be repeated quickly without having to grow cells each time.