Study of ALDH from Thermus thermophilus-Expression, Purification and Characterisation of the Non-Substrate Specific, Thermophilic Enzyme Displaying Both Dehydrogenase and Esterase Activity

Abstract

Aldehyde dehydrogenases (ALDH), found in all kingdoms of life, form a superfamily of enzymes that primarily catalyse the oxidation of aldehydes to form carboxylic acid products, while utilising the cofactor NAD(P)⁺. Some superfamily members can also act as esterases using p-nitrophenyl esters as substrates. The ALDH_Tt from Thermus thermophilus was recombinantly expressed in E. coli and purified to obtain high yields (approximately 15-20 mg/L) and purity utilising an efficient heat treatment step coupled with IMAC and gel filtration chromatography. The use of the heat treatment step proved critical, in its absence decreased yield of 40% was observed. Characterisation of the thermophilic ALDH_Tt led to optimum enzymatic working conditions of 50 °C, and a pH of 8. ALDH_Tt possesses dual enzymatic activity, with the ability to act as a dehydrogenase and an esterase. ALDH_Tt possesses broad substrate specificity, displaying activity for a range of aldehydes, most notably hexanal and the synthetic dialdehyde, terephthalaldehyde. Interestingly, para-substituted benzaldehydes could be processed efficiently, but ortho-substitution resulted in no catalytic activity. Similarly, ALDH_Tt displayed activity for two different esterase substrates, p-nitrophenyl acetate and p-nitrophenyl butyrate, but with activities of 22.9% and 8.9%, respectively, compared to the activity towards hexanal.

Keywords: aldehyde dehydrogenase; biochemical characterisation; esterase activity; heat treatment purification; substrate specificity; thermophilic enzyme.

Scheme 1

ALDH reaction mechanism highlighting ALDH_Tt monomeric and tetrameric structure (PDB ID: 6FJX) (a) dehydrogenase activity using cofactor, (b) esterase activity [14].

Figure 1

(a) SDS-PAGE of ALDH_Tt protein purification samples. Lane 1: PageRuler pre-stained protein ladder (ThermoFisher Scientific, Cork, Ireland), lane 2: E. coli BL21(DE3) cell lysate expressing ALDH_Tt, lane 3: Ni affinity chromatography 200 mM imidazole elution, lane 4: purified ALDH_Tt, lane 5: Ni affinity chromatography flow through. (b) Western blot of cell lysate expressing ALDH_Tt and purified protein following gel filtration chromatography. Lane 1: PageRuler pre-stained protein ladder (Thermofisher Scientific, Cork, Ireland), lane 2: E. coli BL21(DE3) cell lysate expressing ALDH_Tt, lane 3: purified ALDH_Tt.

Figure 2

Elution profile of ALDH_Tt via Ni-affinity chromatography and gel filtration chromatography. Absorbance was monitored at 280 nm. (a) Chromatogram displaying purification of ALDH_Tt via Ni-affinity chromatography using a step gradient of imidazole from 50–500 mM. E. coli host proteins are eluted from 10–50 mM imidazole in the first two peaks, while ALDH_Tt is eluted at 200 mM imidazole in the third peak. (b) Chromatogram displaying a Gaussian peak of the apparent homogenous ALDH_Tt with elution at approximately 70 min using gel filtration chromatography at a flow rate of 1 mL/min.

Figure 3

MALDI-TOF spectrum of recombinant ALDH_Tt utilising HCCA as the matrix, run in linear cationic 5–60 kDa mode, using 2500 shots.

Figure 4

ALDH_Tt enzymatic activity profiles utilising NAD⁺ as cofactor and hexanal as substrate (a) Plot of activity of ALDH_Tt as a function of temperature in the range 20–50 °C in 10 mM potassium phosphate pH 8. (b) Plot of activity of ALDH_Tt as a function of pH at 50 °C. Optimum conditions were determined as pH 8 at 50 °C.

Figure 5

ALDH_Tt substrate screening and specificity utilising a range of aliphatic, aromatic, and cyclic aldehydes. Specific activity is demonstrated at (a) 25 °C and (b) 50 °C, displaying the thermophilic nature and broad substrate scope of the enzyme.

Figure 6

Dehydrogenase and esterase activity of ALDH_Tt at 25 °C purified in the absence of β-mercaptoethanol (a) dehydrogenase activity using hexanal in the presence of acetone, (b) comparison of specific activity of dehydrogenase and esterase activity using different esters, (c) esterase activity using PNP-acetate in the presence of low concentrations of NAD⁺.