A novel lipase-catalyzed method for preparing ELR-based bioconjugates

Abstract

Herein we present a novel one-pot method for the chemical modification of elastin-like recombinamers (ELRs) in a mild and efficient manner involving enzymatic catalysis with Candida antarctica lipase B. The introduction of different functionalities into such ELRs could open up new possibilities for the development of advanced biomaterials for regenerative medicine and, specifically, for controlled drug delivery given their additional ability to respond to stimuli other than pH or temperature, such as glucose concentration or electromagnetic radiation. Candida antarctica lipase B immobilized on a macroporous acrylic resin (Novozym 435) was used to enzymatically couple different aminated substrates to a recombinamer containing carboxylic groups along its amino acid chain by way of an amidation reaction. A preliminary study of the kinetics of this amidation in response to different reaction conditions, such as solvent, temperature or reagent ratio, was carried out using a phenylazobenzene derivative (azo-NH 2 ) as a model. The optimal amidation conditions were used to couple other amine reagents, such as phenylboronic acid (FB-NH 2 ) or polyethylene glycol (PEG-NH 2 ), thus allowing us to obtain photoresponsive, glucose-responsive or PEGylated ELRs that could potentially be useful as sensors in devices for controlled drug delivery.

Novozym 435: the “perfect” lipase immobilized biocatalyst?

“…388 Another paper shows that elastin-like recombinamers containing carboxylic groups may be modified by introducing different aminated compounds (aminophenylazobenzene, amino phenylboronic acid or aminopolyethylene glycol), using N435 as a catalyst, to produce photoresponsive, glucose-responsive or PEGylated elastin-like recombinamers. 389 N435 was also used as a catalyst for the transesterification/amidation of ethyl dihydroferulate. 390 Arachidonoyl ethanolamide was produced by amidation of arachidonic acid with ethanolamine catalyzed by N435 (95.6% yield), showing that the enzyme has an excellent chemoselectivity, even being an esterase.…”

Lipase-catalyzed solvent-free synthesis of monoglycerides from biodiesel-derived crude glycerol: Optimized using response surface methodology

“…This immobilized enzyme is prone to enzyme shedding caused by surfactant molecules (such as monoglycerides, and diglycerides) and high temperatures, resulting in enzyme deactivation [ 38 ]. Novozym 435 exhibits activity over a wide temperature range (20–110 °C), although it is typically employed within the optimal range of 30∼60 °C [ 49 ]. Raising the temperature enhances the pro-glycerolysis reaction catalyzed by Novozym 435 and reduces the time needed for the reaction to reach equilibrium.…”

“…However, beyond a certain temperature, protein thermal denaturation occurs, leading to a sharp decline in reaction rate [ 58 ]. Novozym 435, which is capable of catalyzing at 110 °C [ 49 ], does not undergo denaturation at 50–70 °C and significantly enhances the yield of monoglyceride. It is evident that the interaction between enzyme dosage and temperature has a significant effect on the monoglyceride content.…”

“In-situ” formation of elastin-like recombinamer hydrogels with tunable viscoelasticity through efficient one-pot process

“…Overall, the activation of carboxylic groups of glutamic or aspartic acid to conjugate with amine groups of another amino acid through the carbodiimide-mediated amidation reaction is particularly attractive for biological applications. Up to now, the amidation reaction using the ELR-bearing carboxylic groups has not been addressed except for the work that examined the chemical modification with p -phenylazoaniline (azo-NH 2 ), 4[(2 amino)carbamoyl]-phenylboronic acid (FB–NH 2 ), polyethyleneglycol (PEG-NH 2 ) and 1-(β-hydroxyethyl)-3,3-dimethyl-6′-nitrospiro-(indoline-2,2′[2 H -]benzopyran) (Sp-OH), but under anhydrous conditions in organic solvent to prevent the hydrolysis process [ [41] , [42] , [43] ]. Indeed, the chemical modification of carboxyl-containing macromolecules usually involves toxic organic solvents, as well as slow, complex and low-yield processes.…”

“…Indeed, the chemical modification of carboxyl-containing macromolecules usually involves toxic organic solvents, as well as slow, complex and low-yield processes. This is associated with low coupling ratios even with reagent excess, due to the complexity of the molecule to be modified [ [41] , [42] , [43] , [44] ]. Apart from these studies, to the authors' knowledge, neither the chemical modification of these ELRs through amidation of the carboxylic groups, in aqueous solutions, nor their subsequent application to the formation of chemical hydrogels, have been addressed.…”