(560ak) Preparation of a New Enzymatic Biocatalyst Via the Taguchi Method: Application to Lipase a from Candida Artarctica Immobilized Onto Cashew Apple Bagasse
AIChE Annual Meeting
2019
2019 AIChE Annual Meeting
Catalysis and Reaction Engineering Division
Poster Session: Catalysis and Reaction Engineering (CRE) Division
Wednesday, November 13, 2019 - 3:30pm to 5:00pm
Due to their high stability, high substrate specificity, high region, chemo and
stereoselectivity, lipases are the most commonly used enzymes in biocatalysis. Among
lipases, lipase A from Candida antarctica (CALA) is a very attractive biocatalyst, once
it presents high thermostability, selectivity for trans fatty acids, stability in the acid pH
range and high chemo-selectivity in relation to the amine groups. However, once lipases
in their free form are soluble in water, it is necessary to immobilize them to improve their
biocatalytic properties and enable their recovery and reuse easier. Among the methods
for enzyme immobilization, the chemical ones allow to form covalent bonds are between
the lipases and the support, resulting in rigidity and improved enzymatic stability. An
alternative support for enzyme immobilization is cashew bagasse in natura, which is rich
in hydroxyl groups, allowing multipoint covalent bonds. Activating the support with
divinylsulfone (DVS) allows such types of bonds to be obtained on supports containing
on their surface a very broad range of amino, thiol or hydroxyl groups, such as cashew
bagasse in natura. In this study, CALA was immobilized onto cashew bagasse in natura
activated with DVS and optimal conditions (DVS concentration: 1, 3, 5 and 7.5; buffer
molarity: 5, 25, 100 and 350 mM; pH: 3, 5, 7 and 12.5; temperature: 4, 15, 25 and 30 °C
and time of activation: 0,5, 3, 12 and 24 hours) for the immobilization procedure was
determined. For this communication, an advanced experimental design by the Taguchi
method with a standard orthogonal matrix L16’ (the ‘L†and “16†represent the Latin
square and the number of experiments, respectively) was used to examine five factors at
four levels in order to maximize the derivative activity. For the preparation of the support,
cashew bagasse in natura was washed three times with water, dried at 60 °C for 24h and
milled in a hammer mill to obtain an average particle size of less than 0.177mm. The
support was then activated as follows:1.0g of cashew bagasse in natura was added to 20
mL of 350 mM sodium carbonate solution, at neutral pH, containing 4.5 M divinyl
sulfone. The resulting solution was kept under constant stirring for 30 minutes at 15 °C;
the support was then washed with distilled water and stored at 6 °C. For the
immobilization procedure, 1.0 mg of enzyme per g of support was used in the presence
of 0.01% Triton-X and 5 mM sodium phosphate buffer and pH 7.0, for 24 h and 25 °C,
under continuous agitation. After immobilization, the biocatalysts were incubated in 100
mM bicarbonate buffer at pH 10.0 (1:10 w/v) at 25 °C for 24 h, then in 1 M EDA pH 10.0
also for 24 h at 4 °C. Finally, the biocatalysts were washed with distilled water, vacuum
dried and stored at 4 °C. Then, the hydrolytic activity of soluble and immobilized CALA
was performed, as well as the protein concentration was determined. Once the
experimental design was finished, the optimal conditions was found to be: DVS
concentration: 3, buffer molarity: 3 mM, pH: 3; temperature: 30 °C and time of activation:
12 hours, obtaining a derivative activity of 4.31 U/g.