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

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 degrees 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.

Automated summary

The thermophilic ALDH(Tt) has been found to possess dual enzymatic activity and broad substrate specificity, showing activity for a range of aldehydes and ester substrates. However, it may exhibit differing levels of activity towards specific substrates with para-substituted benzaldehydes being efficiently processed while ortho-substituted ones show no catalytic activity.