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

• Published in: Cells (Basel, Switzerland) Vol. 10; no. 12; p. 3535
• Main Authors: Shortall, Kim, Durack, Edel, Magner, Edmond, Soulimane, Tewfik
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 14-12-2021; MDPI
• Subjects: Acetic acid; aldehyde dehydrogenase; Aldehyde Dehydrogenase - chemistry; Aldehyde Dehydrogenase - isolation & purification; Aldehyde Dehydrogenase - metabolism; Aldehydes; biochemical characterisation; Biosynthesis; Carboxylic acids; Chromatography; Cytochrome; Dehydrogenases; E coli; Enzymatic activity; Enzymes; Esterase; esterase activity; Esterases - metabolism; Esters; heat treatment purification; Heat treatments; Hexanal; Kinetics; Molecular Weight; NAD; NAD - metabolism; Oxidation; p-Nitrophenyl butyrate; p-Nitrophenylacetic acid; Proteins; Recombinant Proteins - isolation & purification; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Substrate Specificity; thermophilic enzyme; Thermus thermophilus; Thermus thermophilus - enzymology; heat treatment purification; aldehyde dehydrogenase; thermophilic enzyme; biochemical characterisation; substrate specificity; esterase activity
• ISSN: 2073-4409; 2073-4409
• DOI: 10.3390/cells10123535

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
-nitrophenyl esters as substrates. The ALDH
from
was recombinantly expressed in
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
led to optimum enzymatic working conditions of 50 °C, and a pH of 8. ALDH
possesses dual enzymatic activity, with the ability to act as a dehydrogenase and an esterase. ALDH
possesses broad substrate specificity, displaying activity for a range of aldehydes, most notably hexanal and the synthetic dialdehyde, terephthalaldehyde. Interestingly,
-substituted benzaldehydes could be processed efficiently, but
-substitution resulted in no catalytic activity. Similarly, ALDH
displayed activity for two different esterase substrates,
-nitrophenyl acetate and
-nitrophenyl butyrate, but with activities of 22.9% and 8.9%, respectively, compared to the activity towards hexanal.