Recombinant Expression And Enzymatic Characterization Of Acetolactate Decarboxylase In Vitro

Acetoin is an important C-4 platform compound, also with its oxidation products diacetyl and reduced product 2,3- butanediol. They are widely used in food, pharmaceuticals, fuel, aerospace and other fields. In the fermentation pathway to produce these C-4 platform compounds in bacteria, acetolactate decarboxylase (ALDC) is the key enzyme which regulates thea-acetolactate metabolic flux, and it can decarboxylate the racemic a-acetolactate to generate a single chiral (R)-acetoin. Although ALDC has been discovered in diverse microorganisms, they are various in structure and enzymatic properties. Enterobacter aerogenes (E.a) and Enterobacter cloacae (E.c) were common bacteria converting pyruvate to 2,3-butanediol. However, Bacillus subtilis DL01 (B.s) isolated from offshore sludge exhibited extremely high production of acetoin (41.63 g/L) without byproduct diacetyl and 2,3-butanediol formations during the fermentation. In order to understand the relationship between the structure and the enzyme activity from different ALDCs, genes of ALDCs from the above three bacteria were cloned, recombinantly expressed and studied in vitro.In this study, genes of ALDCs from three different bacteria were cloned and transformed into E.coli BL21. ALDC proteins were induced by IPTG with a soluble expression at 37 ℃. Through ion exchange chromatography and gel filtration chromatography, the ALDC with high purity was obtained. The molecular weight of all three ALDCs estimated by SDS-PAGE were all about 30 kDa. Bioinformatics analysis showed, the amino acids sequence of ALDC-B.s only exhibits a similarity of 40% to those of ALDC-E.c and ALDC-E.a. Molecular docking reveals that ALDCs prefer to catalyse (S)-a-acetolactate directly rather than (R)-a-acetolactate. ALDC-E.c displays the smallest binding energy when binding to the substrate, while ALDC-B.s the greatest, suggesting a higher affinity between the substrate and ALDC-E.c than ALDC-B.s. Due to the inherent chirality of the ALDC’s catalysed reaction, circular dichroism (CD) spectra was used to measure the enzymatic activity of ALDCs. The results showed the specific activity of ALDC-B.s was 1853.26 U/mg, about 1.8-fold of ALDC-E.c and 2.2-fold of ALDC-E.a, respectively. The enzyme activity was different at the same pH, and ALDC was activated by part of the divalent metal ions. Kinetic parameters revealed that Km value of ALDC-B.s was 20.94 mM, while those of ALDC-E.c and ALDC-E.a are 12.20 mM and 14.83 mM, respectively, indicating a better substrate affinity of ALDC-E.c and ALDC-E.a when compared to ALDC-B.s. Furthermore, Kcat value of ALDC-B.s is 2.21 s-1, higher than 0.96 s-1 of ALDC-E.c and 0.81 s-1 of ALDC-E.a, showing a greater catalytic efficiency of ALDC-B.s. The kinetic experiment results are essentially consistent with our previous bioinformatic predictions.