Immobilized enzyme reactors based on monoliths: Effect of pore size and enzyme loading on biocatalytic process

Abstract

Macroporous monolithic columns with different mean pore size (from 360 to 2020 nm) and appropriate flow-through properties were synthesized using free radical in situ copolymerization of glycidyl methacrylate, 2-hydroxyethyl methacrylate and ethylene dimethacrylate. In order to predict the composition of porogen mixture to generate the pores in the interested size interval, the Hildebrand theory was used. Ribonuclease A and its specific low- and macromolecular substrates cytidine-2',3'-cyclic monophosphate sodium salt and RNA were applied as model system. The effect of mean pore size of macroporous monoliths used for enzyme immobilization on molecular recognition and biocatalytic characteristics was examined. The monitoring of RNA degradation was performed using anion-exchange HPLC on monolithic CIM DEAE analytical column. The high efficiency of heterogeneous biocatalysts obtained comparatively to the catalytic reaction of RNA degradation in solution was demonstrated. Additionally, the series of six monolithic immobilized enzyme reactors with different amount of biocatalyst was prepared and studied regarding to the biocatalytic properties at recirculation mode at two experimental variants, e.g. (i) fixed range of concentrations of circulated substrate solutions, and (ii) fixed range of substrate/enzyme molar ratios.

Molecularly imprinted macroporous polymer monolithic layers for L‐phenylalanine recognition in complex biological fluids

“…Several works describing the application of Hildebrand's theory for the prediction of the compatibility of polymer and solvent(s) have been recently published. [34][35][36] According to Hildebrand's theory, the solubility of a polymer with solubility parameter δ 1 in a solvent with solubility parameter δ 2 is favored when (δ 1δ 2 ) 2 or (δ 1 -δ 2 ) 1/2 is minimized. [37][38][39] In turn, at the synthesis of macroporous materials, the deference in polymer and solvent(s) solubility parameters has to be significant to consider the solvent(s) as porogens.…”

Investigating Monoliths (Vinyl Azlactone-co-Ethylene Dimethacrylate) as a Support for Enzymes and Drugs, for Proteomics and Drug-Target Studies

“…The poly(VDM-co-EDMA) monoliths show an average backpressure of 30 bar/m at a flow rate of 1 μL/min of 100% ACN with a permeability of 5.0 × 10 −14 m 2 , calculated as described in Meller et al (2016), based on backpressure measurements (n = 17). The permeability of the poly(VDM-co-EDMA) monolith is in same range as other organic monoliths (Vlakh et al, 2013; Volokitina et al, 2017), commercial particle packed columns (Song et al, 2014), and silica based monoliths (Motokawa et al, 2002; Zhang et al, 2013). The performance of the produced trypsin IMERs was evaluated by comparing the peptide fingerprint region of an on-line IMER digestion (5 min, Figure 2B) to that of myoglobin digested in-solution (Figure 2A).…”

A strategy for screening trypsin inhibitors from traditional Chinese medicine based on a monolithic capillary immobilized enzyme reactor coupled with offline liquid chromatography and mass spectrometry

“…Trypsin solutions of different concentrations (0.2, 0.5, 0.8, and 1 mg/mL) were continuously pumped through the monolithic matrix for 2 h. The enzyme activity was highest at a concentration of 0.8 mg/mL, however, a lower enzyme activity appeared at a concentration of 1 mg/mL (Supporting Information Figure S1A). This could be explained by the fact that a too high enzyme density on the matrix surface resulted in steric hindrance‐related phenomena . Therefore, 0.8 mg/mL trypsin concentration was chosen for enzyme immobilization.…”

“…By prolonging the immobilization time to 8 h, trypsin activity did not increase, this even resulted in a small decrease (Supporting Information Figure S1B). This may also be attributed to an increase in the quantity of enzyme immobilized on the support, causing steric hindrance and reducing the catalytic efficiency . Therefore, an immobilization time of 6 h was chosen for the subsequent experiments.…”