3.3.2.1 Solvent Effects
The effect of organic solvents as a process parameter on the bioreduction should be tested, if a catalyst is to consider for use in an industrial approach. Low wa-ter-solubility of most ene reductase substrates, in combination with sub-strate/product inhibition, presents a considerable challenge, often resulting in low substrate loadings and significant amounts of waste water.[22] Addition of organic co-solvents and use of biphasic reaction systems therefore offer signifi-cant advantages, often increasing the biocatalyst performance in synthetic chemistry.[234,235] It is important to keep in mind that the reaction system used here consists of two enzymes, the ene reductase and a glucose dehydrogenase that belongs to the recycling system, which supplies the consumed hydride from a sacrificial agent. The following results are therefore specific to the reac-tion system.
It could be determined that water-miscible solvents lead to diminished activi-ties when 10% (v/v) is exceeded (Figure 25). The most destructive effect on ac-tivity shows the panel of TsER variants with small polar solvents like C1 to C3
alcohols, dimethyl sulfoxide or acetonitrile, common co-solvents for organic molecules in biotransformation. Other ene reductases have likewise shown a similar intolerance against such co-solvents,[81,84,220] but no clear trend is
appar-ent.[65,236–240] In contrast, the combination of TsER variant and the glucose
dehy-drogenase (GDH-60 from EVOCATAL) tolerates up to 20% (v/v) of various water-immiscible solvents without any loss in activity for the production of 40b. Im-portantly, already a small volumetric amount of organic solvent (5-10% (v/v)) increased 40b productivity up to 2-fold, enabling full conversion of the sub-strate. Encouraged by this result, the non-active substrates 49a and 52a were tested in the presence of 10% tert-butyl methyl ether (MTBE). No conversion was observed, excluding the possibility that they are not converted due to their low solubility.
Figure 25. Effect of organic solvents to the biocatalyst. Addition of organic solvents increased formation of 40b. Full conversion was reached with toluene, n-pentane and MTBE with either of the variants. Using 20% (v/v) organic solvent has a positive effect in most cases, and n-pentane is even tolerated up to 40% (v/v). Grey line indicates the conversion rate without (w.o.) organic solvent (C25D/I67T 43%, C25G/I67T 48%). MTBE, methyl tert-butyl ether; MeOH, methanol, 2-PrOH, 2-propanol; DIPE, diisopropylether; EtOAc, ethyl acetate; EtOH, ethanol; DMSO, di-methyl sulfoxide; MeCN, acetonitrile.
Addition of n-pentane, MTBE, diisopropyl ether (DIPE) or toluene proved to be particularly effective. The low water miscibility of n-pentane (39 mg·l−1)[241] even allowed reactions with 40% (v/v) and the recovery of a clean product by simple phase separation. The findings that immiscible solvents are more compatible and enhanced conversion levels confirm observations made for other OYE wild types.[81,220,242] Immiscible solvents provide a substrate and product sink for the
organic compound, keeping concentrations of reactants in the aqueous phase
low.[243–246] This is beneficial for enzyme catalysis, because such a reaction setup
often circumvents inhibition due to high product concentrations in the aqueous phase.
It was noted that the enantiopurity of 33b increased up to 30% upon addition of an organic phase. Interestingly, the effect was only observed for the wild type and TsER C25D based variants (Table 4). The beneficial effect of a biphasic sys-tem on enantiopurity was previously described for compounds prone to race-mization, like 33b and 22b.[65]
Table 4. Solvent effect on conversion and ee of 33a and 40a. For 40b the ee values were always
>99%. All reactions were done in triplicates; standard deviation for conversion is ± 5%. The reac-tions were stopped after 24 h.
wt C25D/I67C C25D/I67T C25D/I67V C25G C25G/I67C C25G/I67T C25G/I67V
33b
conv%
ee/%
conv/%
ee/%
conv%
ee/%
conv%
ee/%
conv%
ee/%
conv/%
ee/%
conv/%
ee/%
conv%
ee/%
without solvent 99 79R
99 66R
91 57R
99 69R
90 87R
>99 84R
99 81R
96 85R with 10%(v/v)
MTBE
99 96R
99 71R
91 88R
99 88R
90 88R
>99 82R
99 80R
96 84R
40b
conv% conv/% conv% conv% conv% conv/% conv/% conv%
without solvent 40 58 43 60 38 45 48 37
with 10%(v/v)
MTBE 91 88 88 96 39 42 90 37
In addition, pH variation,[191] choice of nicotinamide recycling system, as well as the enzyme concentration or the presence of oxygen[210] are also described to have a small but notable effect on the enantiopurity of ene reductase reactions.
While all these observations might be explained by the solvent effect on stereo-selectivity and the formation of different solute-solvent clusters at various reac-tion condireac-tions,[247] it was surprising to see such a significant difference between the two variant groups.
3.3.2.2 Temperature Effects
Enzymes with high thermal stability are of particular interest to biotechnology and basic research.[248] Increased stability and life-times enables more flexibility in process design, use in new reactions and exploration of basic research ques-tions without the limitation of a narrow temperature window.
Before investigating the thermal stability of the whole reaction system, the indi-vidual thermal stability of both enzymes, glucose dehydrogenase and ene re-ductase in the reaction system, used during this thesis, was assessed. The recy-cling system based on GDH-60 from EVOCATAL was found to be active up to 50 °C (Table 5). The engineered GDH from Bacillus subtilis (BsGDH E170K/Q252L)[59] showed still 60% specific activity at 70 °C under the reaction conditions which were used in this thesis. Thus, the investigation of tempera-ture differences was performed with the BsGDH.
Table 5. Spectrophotometric determination of temperature dependency of used glucose dehy-drogenases. The engineered BsGDH shows highest activity at temperatures between 40-60 °C.
Activity is reduced by 50% at 30 and 70 °C, whereas the GDH-060 from EVOCATAL shows no activity at 60 and 70 °C. Therefore, BsGDH was used analysing the temperature dependency of the bioreduction. The temperature range of the spectrophotometer is 0-70 °C.
Temperature 30 °C 40 °C 50 °C 60 °C 70 °C BsGDH activity [U/mL] 32.80 57.78 61.70 58.94 38.31
relative activity 0.53 0.93 1.00 0.95 0.62 GDH-60 (EVOCATAL) activity [U/mL] 23.05 41.42 46.78 0.00 0.00 relative activity 0.49 0.88 1.00 0.00 0.00
The thermal stability of variant C25G/I67T and C25D/I67T was assessed by in-cubating the purified enzymes in buffer at temperatures between 4 and 70 °C for 14.5 h prior to a reaction at 30 °C (Figure 26A). Both TsER enzymes retain full activity up to an incubation temperature of 60 °C. At 70 °C incubation, C25D/I67T lost 3% and C25G/I67T 14% of its initial activity. This thermal stabil-ity is comparable to that of the wild type.[179] Notable is the 1.06-fold activity enhancement for C25G/I67T after incubation at 30 °C compared to no
pre-incubation, while the activation effect might be masked for C25D/I67T, since full conversion was reached.
Figure 26. Thermostability assessment of the ene reductase reaction system. A) Residual activity at screening conditions of TsER C25G/I67T and C25D/I67T after 14.5 h incubation at different temperatures. Temperature-time dependent formation of 22b with the reaction system contain-ing a TsER variant and the engineered BsGDH[59] at temperatures between 30 and 85 °C. C) C25G/I67T, D) C25D/I67T, B) Enantioselectivity of 22b in the presence of TsER decreases rapidly with higher temperatures, while in absence of an ene reductase and recycling system, the enan-tioselectivity is stable up to 60 °C but at 65 °C and above, 22b racemizes and an ee of 8% is ob-served.
After ensuring thermal stability of both enzymes, the reaction system was test-ed for levodione (22b) production at reaction temperatures between 30 °C and 85 °C. Formation of 22b completes in 150 min under screening conditions. In-creasing the temperature accelerated the reaction, now reaching full conversion in less than 20 min (C25D/I67T) or 40 min (C25G/I67T) at 55 °C (Figure 26C, D).
Productivity is not affected up to 65 °C, whereas both variants start losing activ-ity at 70 °C and above. In general, working above 75 °C reduced the lifetime of
the reaction system below 20 min. Not only the enzymes, but also the nicotina-mide cofactor (NADPH/NADP+) is labile at higher temperatures. The half-life of dissolved NADPH at 70 °C and above is less than 10 min.[249,250] Therefore, it is impossible to distinguish between cofactor or enzyme degradation as source for lost activity at high temperatures. This might be a general challenge for pro-cesses with oxidoreductases at higher reaction temperatures and long reaction times. The enzyme can be engineered to be more stable,[51,251,252] but the stability of the cofactor cannot be altered unless another molecule is used. Consequently, as long as thermolabile nicotinamide cofactors are used, increased reaction temperatures may negatively impact yields for reaction times longer than a few hours, despite the increased reaction rate and enhances solubility of organic molecules at higher temperatures.
It is known that (R)-levodione (22b) racemizes in buffered aqueous solution approximately 3% ee per hour at ambient temperatures.[218,253] It was expected, that the effect would be more dominant at increased temperatures and could be found that incubation of (R)-22b in aqueous buffer without ene reductase at 65 °C and above for one hour results in a loss of enantiopurity yielding an ee of 8%. The reaction system used here produces (R)-22b with an ee of 92% at 30 °C.
When increasing the temperature, the enantiopurity drops by 50% (Figure 26B).
At 70 °C, 37% ee is still observed, indicating that the reduction is faster than the racemization. Thus, an increased reaction temperature seems only beneficial for non-racemizable or achiral compounds.
3.3.2.3 Divalent Metal Impacts and pH Effects
The glucose based recycling system is irreversible due to the hydrolytic cleav-age of gluconolactone to gluconic acid, benefitting NADPH production but also leading to acidification of the reaction. Therefore investigations for the effect of pH on the reaction system were performed and it was found to work well in the
range between pH 6-8 (Figure 27A and C). The C25D/I67T variant is less affect-ed by a change in pH and still shows production of 22b without loss of efficien-cy at pH 9.
Figure 27. Effect of pH and divalent metal salts on productivity. A) Effect of pH on 22b-production with the reaction system consisting of C25D/I67T or C25G/I67T and GDH-60. B) Divalent metal chloride salts marginally affect productivity of 17b with stoichiometric amounts of NADPH (light grey). When the recycling system is used instead (GDH-60/glucose), higher productivities are achieved and the addition of Co2+ becomes slightly beneficial. C) pH depend-ency of the two used recycling systems, consisting glucose dehydrogenase, glucose and NADP+.
Remarkably, 40-60% formation of 22b was still observed at a pH of 5 and 10, especially considering that NADPH/NADP+ rapidly degrade at pH values be-low 7.[254] The buffer capacity is 100 mM, ten-times higher than the amount of possible acid equivalents under screening conditions. On this scale, acidification of the reaction mixture was never observed. When aiming for higher substrate loadings (>100 mM), gluconic acid equivalents exceed the buffer capacity and process engineering is needed to compensate for the pH drop.
In view of the fact that enzymes and classical catalysis are more and more com-bined in one-pot reactions,[255,256] the effect of divalent metal salts on the TsER variant panel and on the reaction system was tested. Water soluble chloride salts of divalent metals (Figure 27B) had no significant effect on productivity of
17b. The metal salts had a minimal positive effect when the GDH-60 based re-cycling system was used, where the minimal improvement of Co2+ became sig-nificant and an overall improvement of 0.2 fold was observed.