Factors influencing the operational stability of NADPH-dependent alcohol dehydrogenase and an NADH-dependent variant thereof in gas/solid reactors
Graphical abstract
Research highlights
▶ The order of magnitude of operational stability of LbADH and a variant thereof in the gas/solid bioreactor is governed by the thermostability of the dry protein, but not by that of the cofactor. ▶ Both in solid and in dry state, the proteins denature by partial unfolding with subsequent aggregation. ▶ Stability investigations of enzymes in dry state predict the level of operational stability in gas/solid reactions.
Introduction
Gas/solid biocatalysis represents an alternative to common liquid biocatalytic reaction systems, where the solid dry enzyme catalyzes conversion of gaseous substrates to gaseous products. The technology is used for production of volatile compounds like esters and alcohols [1]. It exhibits significant advantages, such as high productivity, pronounced stability of the immobilized biocatalyst [2] and simplified downstream processing [3]. The possibility to control thermodynamic parameters in a gas/solid system additionally allows adjusting the enzyme micro-environment, which is important for scientific purposes, such as studies of enzyme hydration or solvent influences [4].
It is generally accepted that dry enzymes are significantly more stable than the dissolved ones. However, progressive inactivation of alcohol dehydrogenases is observed under the gas/solid reactor conditions [5], [6], [7], [8]. Yang and Russell described the conversion of 3-methyl-2-buten-1-ol, catalyzed by immobilized yeast alcohol dehydrogenase in a continuous gas/solid reactor at 22–50 °C at different water activities and observed steady-state periods of 4–16 days prior to progressive inactivation [8]. Reduction of acetophenone with the solid immobilized wild type ADH from Lactobacillus brevis (LbADHwt) performed in a continuous gas/solid reactor at 40 °C and a water activity (aw) of 0.5 revealed a half-life between 1 and more than 40 days for the enzymatic activity depending on the immobilization conditions [7]. Up to now the mechanisms leading to enzyme inactivation in the solid state remain unclear.
To elucidate the effects of temperature on activity and stability, previous studies focused on the comparison of operational stabilities of ADHs derived from different thermophilic and mesophilic sources [9]. It was shown that high thermal stability in aqueous media was not necessarily correlated with higher stability under gas/solid reactor conditions [9]. However, the enzymes used in that study were from different organisms, showing significant sequence and structural differences. Engineered enzymes, differing only in few sequence positions were never comparatively studied in the gas/solid system.
Here we present a comparative study of the NADPH-dependent wild type ADH from L. brevis (LbADHwt) and the NADH-dependent variant thereof, LbADH G37D. Both are able to reduce ketones to the corresponding (R)-alcohols with high stereoselectivity [10], [11]. As can be deduced from structural data, both enzyme variants are homotetramers with a molecular weight of 107 kDa, they contain two Mg2+ binding sites and four active centres (one per subunit) [11], [12]. The variant was created to accept cheaper and more stable cofactor NADH [13], [14] with higher affinity than NADPH [11].
Activity and stability of the LbADHwt and the LbADH G37D were studied in aqueous solution, in the dry solid state and with respect to their operational stability in a gas/solid reactor (see principle in Fig. 1A). To identify the main factors causing enzyme inactivation in the gas/solid system, the reduction of acetophenone to (R)-1-phenylethanol with concomitant oxidation of 2-propanol to acetone for the cofactor regeneration was studied (Fig. 1B), while varying thermodynamic parameters, such as water activity, and acetophenone activity. Additionally, the thermostability of both enzyme variants and the cofactors was investigated in the solid state and in solution using tryptophan fluorescence spectroscopy and HPLC/MS, respectively.
Section snippets
Results and discussion
In case of substrate-coupled cofactor regeneration the cofactors for the biocatalytic reduction step are required in equimolar amounts with respect to the enzyme's active sites, here four per LbADH molecule. Therefore, the cofactor stability in the gas/solid reactor is as important as the stability of the respective enzyme. In aqueous solution the oxidized and the reduced cofactor molecules are able to freely diffuse between the binding site in the enzyme and the solution, enabling substrate
Conclusions
In this study, two single-amino-acid exchange enzyme variants, NADPH-dependent LbADHwt and NADH-dependent LbADH G37D were investigated with respect to their stability both in non-reactive and reactive systems. The aim was to elucidate factors that affect the operational stability in the gas/solid reactor and find parameters that may predict the performance in the gas/solid system.
It could be clearly demonstrated that in spite of a lower thermostability and increased number of degradation
Chemicals
Media components, salts and substrates for the enzymatic assays were purchased from Sigma–Aldrich. Q-Sepharose and G25 column filling beads were supplied by Pharmacia. High purity β-NADPH and β-NADH were ordered from Biomol GmbH.
Protein over expression and purification
LbADHwt and LbADH G37D were recombinantly expressed in the pET21a vector purified by ion-exchange chromatography technique as described by Niefind et al. [12]. Purified proteins were desalted on G25 material in 10 mM triethanolamine, 1 mM MgCl2 buffer (pH 7.5), dissolved
References (26)
- et al.
Journal of Molecular Biology
(2005) - et al.
Journal of Molecular Biology
(2003) - et al.
Biotechnology Advances
(2007) - et al.
Journal of Biological Chemistry
(1961) - et al.
Journal of Biological Chemistry
(1961) - et al.
The Journal of Biological Chemistry
(1963) Analytical Biochemistry
(1976)- et al.
Journal of Bioscience and Bioengineering
(2005) - et al.
Analytical Biochemistry
(1992) - et al.
Biocatalysis and Biotransformation
(2001)
Applied Microbiology and Biotechnology
Biotechnology and Bioengineering
Green Chemistry
Cited by (18)
Carboxylic acid reductases: Structure, catalytic requirements, and applications in biotechnology
2023, International Journal of Biological MacromoleculesStructural adaptation of thermostable carboxylic acid reductase from Mycobacterium phlei
2022, Molecular CatalysisOn the modelling and surface response analysis of a non-conventional wall-cooled solid/gas bioreactor with application in esterification
2022, Chemical Engineering JournalCitation Excerpt :Even when SG biocatalysis offers advantages over liquid-based biocatalytic systems, the design of the bioreactor is the main engineering challenge for its application at full scale. Its implementation in the industry has been hindered by a lack of understanding of the complex interaction between kinetics and transport phenomena in bioreactors.[11–17] Although mathematical modeling is essential to carry out the conceptual design or guide the experimental work in the SG bioreactor, as far as the authors know, there is not, today, a model developed for this technology with application in esterification.
A robust and stereocomplementary panel of ene-reductase variants for gram-scale asymmetric hydrogenation
2021, Molecular CatalysisCitation Excerpt :Not only the enzymes, but also the nicotinamide cofactor (NADPH/NADP+) is labile at higher temperatures. The half-life of dissolved NADPH at 70 °C and above is less than 10 min [46]. Therefore, it is impossible to distinguish between cofactor or enzyme degradation as source for lost activity at high temperatures.
Optimization for simultaneous enhancement of biobutanol and biohydrogen production
2021, International Journal of Hydrogen EnergyCitation Excerpt :A comparison of these outcomes with other reported literature reveals that C. saccharoperbutylacetonicum gives the maximum biobutanol production at 30 °C using TYA media [42–44]. The reason behind the decrease of biobutanol concentration from 6.5 g/L to 5.3 g/L at temperatures above 30 °C would be due to the denaturation of enzymes responsible for butanol formation, such as butanol dehydrogenase [41,45]. However, an in-depth study is needed to find out the reason for the decrease in biobutanol formation at a higher temperature, which is vice versa in case of biohydrogen.
Solid/gas biocatalysis for aroma production: An alternative process of white biotechnology
2020, Biochemical Engineering JournalCitation Excerpt :Experimentation at the laboratory level can be considered to be the starting point for the intrinsic kinetic evaluation of the biocatalyst [44,107,108,112–114], although it is not mandatory when kinetics cannot be decoupled from transport phenomena resistances, as observed in SG systems such as those presented in Table 4. In the case of the production of NACs, the reaction rate must first be determined experimentally in the SG bioreactor using a supported biocatalyst [98,103] or not using one [102,115]. Observations are thus obtained in the transient state, accounting for the catalytic stability of the biocatalyst.
- 1
Both authors contributed equally to this work.