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 ALDHTt 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 ALDHTt led to optimum enzymatic working conditions of 50 °C, and a pH of 8. ALDHTt possesses dual enzymatic activity, with the ability to act as a dehydrogenase and an esterase. ALDHTt 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, ALDHTt 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.

Enzyme Immobilization on Metal Organic Frameworks: the Effect of Buffer on the Stability of the Support

“…These MOFs were then used to encapsulate two dehydrogenase enzymes, the thermophilic ALDH Tt and LDH, from rabbit muscle. Fe-BTC has previously been used for the immobilization of enzymes, including ADH, Lip, GOx, and Lac, reporting improved catalytic performance in terms of thermal and pH stability. , The use of Fe-BTC as a support allows for a rapid and facile in-situ immobilization process for the development of an immobilized enzyme, with times as short as 1 h. Here, we confirm the successful immobilization of ALDH Tt , a thermophilic enzyme that can be used at temperatures up to 50 °C with a range of aldehyde substrates including hexanal and terephthalaldehyde to produce the respective carboxylic acids using NAD + as a cofactor. Three concentrations of enzyme were examined0.47, 1.80, and 2.34 mg/mL (in aqueous solution 2).…”

“… 11 Encapsulation of ALDH Tt in Fe-BTC was confirmed by SDS-PAGE ( Figure S7 ). 13 , 60 The presence of the 59 kDa ALDH Tt 36 was confirmed in the analyzed Fe-BTC samples, while no protein was detected in the supernatant.…”

“…Aldehyde dehydrogenase from Thermus thermophilus (ALDH Tt ) was expressed in E. coli BL21­(DE3) and subsequently purified as previously reported …”

“…Aldehyde dehydrogenase from Thermus thermophilus (ALDH Tt ) was expressed in E. coli BL21(DE3) and subsequently purified as previously reported. 36 In-Situ Immobilization of ALDH Tt in Fe-BTC. Enzyme immobilization in Fe-BTC was carried out as above with the addition of the appropriate amount of enzyme to solution 2.…”

“…Sodium acetate was identified as a suitable buffer for use at an acidic pH as significantly less degradation of the support was observed. The most stable MOFs—Fe-BTC, Cu-TMA, and ZIF-zni—were successfully used to encapsulate two dehydrogenase enzymes—aldehyde dehydrogenase (ALDH Tt ) 36 and lactate dehydrogenase (LDH). Additionally, we describe the screening of a range of polymers to enhance the stability of MOFs in buffer.…”