Development of a Biosensor for the Detection of Acetoin during Wine Fermentation

Acetoin is most commonly used in food, flavor, cosmetics and chemical synthesis and is formed [...

( R,R )-Butane-2,3-diol dehydrogenase from Bacillus clausii DSM 8716 T : Cloning and expression of the bdhA -gene, and initial characterization of enzyme

The gene encoding a putative (R,R)-butane-2,3-diol dehydrogenase (bdhA) from Bacillus clausii DSM 8716T was isolated, sequenced and expressed in Escherichia coli. The amino acid sequence of the encoded protein is only distantly related to previously studied enzymes (identity 33–43%) and exhibited some uncharted peculiarities. An N-terminally StrepII-tagged enzyme variant was purified and initially characterized. The isolated enzyme catalyzed the (R)-specific oxidation of (R,R)- and meso-butane-2,3-diol to (R)- and (S)-acetoin with specific activities of 12 U/mg and 23 U/mg, respectively. Likewise, racemic acetoin was reduced with a specific activity of up to 115 U/mg yielding a mixture of (R,R)- and meso-butane-2,3-diol, while the enzyme reduced butane-2,3-dione (Vmax 74 U/mg) solely to (R,R)-butane-2,3-diol via (R)-acetoin. For these reactions only activity with the co-substrates NADH/NAD+ was observed. The enzyme accepted a selection of vicinal diketones, α-hydroxy ketones and vicinal diols as alternative substrates. Although the physiological function of the enzyme in B. clausii remains elusive, the data presented herein clearly demonstrates that the encoded enzyme is a genuine (R,R)-butane-2,3-diol dehydrogenase with potential for applications in biocatalysis and sensor development

Acetoin Biosensor Based on Acetoin Reductase-Modified Field-Effect Sensor Applied for Acetoin Detection in Beer Samples

Introduction In food industry, the use and dosing of flavoring substances is an important aspect during fabrication processes. Acetoin and diacetyl are flavoring agents which can be found in many food products, like yogurt, cheese, milk, wine and beer [1]. In beer, acetoin/diacetyl is a critical parameter for the maturation degree of the beverage during fabrication. They are formed during the first phase of beer fermentation and getting reduced during the later phase of fermentation and the maturation. Both can have a negative effect on the beer flavor, when they are above a certain concentration. Typical acetoin concentrations in beer are in the range of 10-50 µM. Measuring the acetoin/diacetyl concentration during the production can serve as a quality control and unnecessary maturation time could be avoided [2]. Until now, the quantitative determination of the acetoin/diacetyl concentration is mostly done “off-line” by gas chromatography. Therefore, a sample has to be sent to an external laboratory, which takes time till results are achieved, or the brewery needs an own laboratory, whereby enormous costs arise and trained staff is needed. Because of that, we recently developed a biosensor for the detection of acetoin based on an enzyme-modified electrolyte-insulator-semiconductor (EIS) sensor, which was characterized in buffer solutions [3]. In this work, the novel biosensor was applied for acetoin measurements in beer samples for the first time. Materials and Methods As sensor structures, capacitive Al-p-Si-SiO2-Ta2O5 field-effect sensors have been used, schematically shown in Fig. 1(a). The Ta2O5 transducer surface was modified with a novel acetoin reductase by means of cross-linking. The acetoin reductase (from B. clausii DSM 8716T) catalyzes the reduction of acetoin to 2,3-butanediol, while NADH serves as a cofactor [4]. The fabricated acetoin biosensors have been electrochemically characterized by capacitance-voltage- and constant-capacitance (ConCap) measurements in buffer solution (0.2 mM Tris-HCl) with varying acetoin concentration and beer samples, spiked with different amounts of acetoin. Results and Conclusions The acetoin sensitivity was investigated in beer solutions and the biosensors showed a linear behavior in the concentration range between 10 µM and 90 μM with a sensitivity of ca. 30 mV/dec, see Fig. 1(b). A detailed description of experiments and the obtained results will be presented and discussed. Figure 1: Schematic measurement set-up with the layer structure of the EIS sensor modified with acetoin reductase (a). Exemplary ConCap-measurement curve recorded in buffer-beer mixture (ratio 20:1) spiked with different acetoin concentrations, respectively (b). Acknowledgments The authors thank the ministry of innovation, science and research of the state of North-Rhine-Westphalia for financial support (funding program FH-Struktur 2016). References [1] P. Li, Y. Zhu, H. Shun, J. Fan, Q. Hu, Y. Cao, Development and validation of high-performance liquid chromatography method for the determination of diacetyl in beer using 4-nitro-o-phenylenediamine as the derivatization reagent, Journal of Agricultural and Food Chemistry 60 (2012) 3013–3019. doi: 10.1021/jf3007163. [2] A. D. Haukeli, S. Lie, Formation and removal of acetoin during yeast fermentation, Journal of the Institute of Brewing 81 (1975) 58–64. doi: 10.1002/j.2050-0416.1975.tb03662.x. [3] D. Molinnus, M. Muschallik, L. O. Gonzalez, J. Bongaerts, T. Wagner, T. Selmer, P. Siegert, M. Keusgen, M. J. Schöning, Development and characterization of a field-effect biosensor for the detection of acetoin, Biosensors and Bioelectronics 115 (2018) 1–6. doi: 10.1016/j.bios.2018.05.023. [4] L. Muschallik, D. Molinnus, J. Bongaerts, M. Pohl, T. Wagner, M. J. Schöning, P. Siegert, T. Selmer, (R,R)-Butane-2,3-diol dehydrogenase from Bacillus clausii DSM 8716T: Cloning and expression of the bdhA-gene, and initial characterization of enzyme, Journal of Biotechnology 258 (2017) 41– 50. doi: 10.1016/j.jbiotec.2017.07.020. Figure 1 <jats:p /

Synthesis of α-hydroxy ketones and vicinal diols with the Bacillus licheniformis DSM 13T butane-2,3-diol dehydrogenase

The enantioselective synthesis of α-hydroxy ketones and vicinal diols is an intriguing field because of the broad applicability of these molecules. Although, butandiol dehydrogenases are known to play a key role in the production of 2,3-butandiol, their potential as biocatalysts is still not well studied. Here, we investigate the biocatalytic properties of the meso-butanediol dehydrogenase from Bacillus licheniformis DSM 13T (BlBDH). The encoding gene was cloned with an N-terminal StrepII-tag and recombinantly overexpressed in E. coli. BlBDH is highly active towards several non-physiological diketones and α-hydroxyketones with varying aliphatic chain lengths or even containing phenyl moieties. By adjusting the reaction parameters in biotransformations the formation of either the α-hydroxyketone intermediate or the diol can be controlled

Capacitive Field‐Effect Biosensor Applied for the Detection of Acetoin in Alcoholic Beverages and Fermentation Broths

An acetoin biosensor based on a capacitive electrolyte–insulator–semiconductor (EIS) structure modified with the enzyme acetoin reductase, also known as butane-2,3-diol dehydrogenase (Bacillus clausii DSM 8716T), is applied for acetoin detection in beer, red wine, and fermentation broth samples for the first time. The EIS sensor consists of an Al/p-Si/SiO2/Ta2O5 layer structure with immobilized acetoin reductase on top of the Ta2O5 transducer layer by means of crosslinking via glutaraldehyde. The unmodified and enzyme-modified sensors are electrochemically characterized by means of leakage current, capacitance–voltage, and constant capacitance methods, respectively