Naslov
Optimization of aldolase immobilization on mesocellular silica foam

Autori
Skendrović, Dino ; Vrsalović Presečki, Ana

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, neobjavljeni rad, znanstveni

Skup
CHISA 2022.

Mjesto i datum
Prag, Češka Republika, 21.08.2022. - 25.08.2022

Vrsta sudjelovanja
Poster

Vrsta recenzije
Podatak o recenziji nije dostupan

Ključne riječi
Aldolase ; Immobilization ; Mesoporous silica

Sažetak
Due to the pharmaceutical industry’s need for sustainable and cost-effective production of chiral drug intermediates, biocatalytic processes offer a valuable alternative to conventional chemical synthesis.1 A notable example of such a chiral intermediate is the statin precursor, which can be synthesized in a single step by double aldol addition using the enzyme deoxyribose-phosphate aldolase (DERA) (Figure 1). Statins themselves are a class of drugs used for lowering levels of cholesterol in blood. Although the use of biocatalysis instead of chemical synthesis overcomes the problem of chirality, it is still necessary to deal with the instability of DERA in the presence of aldehyde substrates and reaction intermediate.2 Immobilization of the enzyme is one of the ways to overcome this obstacle. Covalent immobilization on a solid support can provide additional stability and in some cases even increase activity due to conformational changes of the bound enzyme. In this work, mesocellular silica foam (MCF), a type of mesoporous silica, was used as a solid support synthesized by the sol-gel method. As shown in Figure 2, MCF was then functionalized with (3-aminopropyl)trimethoxysilane (APTMS), a widely used agent for functionalization. Glutaraldehyde and succinic anhydride were used in three different concentrations to activate the functionalized support. After each activation and subsequent enzyme immobilization on the activated MCF, the immobilization efficiency, enzyme activity and stability in the aldol addition reaction were measured. The immobilization efficiency, activity and stability were defined as follows: Immobilization was performed by mixing enzyme and phosphate buffer solution together with the activated support for 1.5 h. The immobilization efficiency was calculated by measuring the protein content of the supernatant using the Bradford protein assay. Activity was measured in the batch reactor and 1 U of DERA activity was defined as the amount of enzyme required to form 1 µmol of product per minute. To measure the free enzyme activity, the reaction solution contained 100 µL of aldehyde solution and 100 µL of enzyme solution (3 mg/mL enzyme, 100 mM acetaldehyde, 50 mM chloroacetaldehyde). During the first 3 min of the reaction, samples were taken at regular intervals and analysed by HPLC to quantify the product concentration. To measure the activity of the immobilized enzyme, 20 mg of the activated MCF with immobilized enzyme was mixed with 100 µL of buffer and 100 µL of aldehyde solution. The sampling procedure is the same as for the free enzyme, but samples were taken during the first 30 minutes. After 30 minutes, the support was washed 3x with buffer and used for stability measurement by mixing it again with 100 µL buffer and 100 µL aldehyde solution, and sampling for activity measurement was performed for 5 minutes. Once the optimal activating agent was confirmed, the influence of different pH and temperature values during the immobilization process on the subsequent activity of the immobilized enzyme and immobilization efficiency was measured.

Izvorni jezik
Engleski

Znanstvena područja
Kemijsko inženjerstvo

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