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Research background. Although there are many studies of the bioimprinting of lipases, there is no study comparing the strategies of bioimprinting prior to immobilization (pre-immobilization) and bioimprinting after immobilization (post-immobilization). Likewise, there is no study that compares bioimprinting of lipases immobilized from a pure lipase preparation and lipases immobilized from a crude extract. We therefore investigated these strategies, using the metagenomic lipase LipC12. Experimental approach. We immobilized LipC12 covalently on the commercial support Immobead 150P and treated it with various bioimprinting agents, either pre-immobilization or post-immobilization. We also compared immobilization from a pure LipC12 preparation and immobilization from a crude cell-free extract. Results and conclusions. The best improvements in triolein-hydrolyzing-activity in n-hexane, compared to a non-bioimprinted control, were obtained with post-immobilization bioimprinting, using oleic acid dissolved in t-butanol: a 12-fold improvement for immobilization from a pure LipC12 preparation and an almost 14-fold improvement for immobilization from the crude cell-free extract. This bioimprinting agent also gave a 3.5-fold increase in activity for the synthesis of ethyl oleate in n-hexane, this result being obtained for pre-immobilization bioimprinting and immobilization from the cell-free extract. Novelty and scientific contribution. This study is the first to compare pre-immobilization and post-immobilization bioimprinting strategies, as well as bioimprinting of enzymes immobilized from both pure enzyme preparations and crude cell-free extracts. These results encourage further investigation into bioimprinting strategies.

Bioimprinting was performed against ovalbumin (OVA) to confer its binding cavities for kwakhurin (Kwa), an isoflavonoid, produced solely by Pueraria candollei var. mirifica (P. candollei). The characterization of bioimprinted-OVA (biOVA), evaluated by an enzyme-linked immunosorbent assay (ELISA), revealed that it functioned as a specific receptor for Kwa. Using biOVA, two systems, i.e., an indirect competitive ELISA (icELISA) and the even simpler and more rapid competitive enzyme-linked bioimprinted-protein assay (cELBIA), were developed as novel techniques for the quantitative analysis of Kwa in P. candollei and its related products. The two analysis methods were found to have limits of detection (LOD) of 4.0 and 2.5 µg/mL, respectively. The high reliability of the developed icELISA and cELBIA using biOVA was also demonstrated by various validation analyses. Subsequently, bioimprinting was performed using eight other proteins to investigate them as candidate scaffolds for the generation of binding cavities for Kwa. Interestingly, two bioimprinted-IgG monoclonal antibodies (biMAbs) recognized Kwa, but their original binding affinity to hapten was lost. That is, the MAbs obtained a new binding ability to Kwa in exchange for their original binding affinity, raising the possibility that biMAb could be alternatively used as a probe for the quantitative analysis of Kwa as well as biOVA. This is the first report of small molecules recognition by MAbs used as proteins for bioimprinting.

A strategy to design imprinted proteins (IPs) able to detect a glycoprotein (ovalbumin, OVA) was proposed. Glucose oxidase (GOx) was used as a matrix for obtaining the binding cavities with high specificity towards the template protein. The effective method to purify the obtained IPs from the template molecules was developed based on a combination of dialysis and gel filtration. HRP was chosen as a model template to discover the optimal production conditions, and the optimal template concentration (100 µg⋅L−1) was chosen. The obtained imprinted proteins were characterized by the high adsorption selectivity towards the target protein (the imprinting factor towards OVA and HRP is 4.7). The developed method was transferred to the synthesis of the anti-OVA IPs. The binding properties of these IPs were estimated using the OVA conjugates with low- (FITC) and high- (HRP) molecular weight label molecules. The ability of the synthesized anti-OVA IPs as recognition elements in immunoassay was studied. Under the optimized experimental conditions, the proposed imprinted proteins exhibited a good linear response to OVA in the concentration range of 10–2000 ng⋅mL−1 with a detection limit of 6 ng⋅mL−1. The obtained recognition elements were tested for OVA determination in real samples of chicken egg white, and a sample of OVA-free cake spiked by OVA.

The technology of bioimprinting combined with adsorption immobilization by resin of Aspergillus niger lipase was studied for enhancing the catalytic esterification performance detected by model reaction (enzymatic synthesis of sucrose-6-acetate) of lipase. The immobilization yield of lipase on resin was 95.7 ± 1.9% after optimized adsorption conditions. Under the optimal reaction conditions, the esterification conversion and 6-hydroxy esterification conversion of model reaction catalyzed by immobilized lipase were 72.6 ± 0.4% and 83.6 ± 0.3%. The bioimprinting of A. Niger lipase was studied and the imprinting conditions were optimized based on the immobilization technique. When imprinted immobilized lipase was applied to the model reaction, the esterification conversion and 6-hydroxy esterification conversion were reached 82.4 ± 0.1% and 82.5 ± 2.5%, respectively. The sucrose-6-acetate yield was 73.31 ± 2.2% by internal standard method. The results indicate that based on enzyme immobilization combined with bioimprinting technology could improve the esterification effect with basically unchanged esterification selectivity.

This review considers the issues of molecular imprinting with the participation of proteins. Works in the field of bioimprinting published over the past five years and devoted to the determination of biomolecules and the enhancement of enzymatic activity are analyzed. Attention is focused on the imprinting of proteins as a method of modifying the molecular structure of proteins due to the formation of binding sites in the presence of substrates (proteins with molecular imprints or imprinted proteins). It is shown that the imprinting of proteins is promising for solving analytical problems. The ambiguous interpretation of the term bioimprinting in solving various problems is discussed.

Candida rugosa lipase (CRL) and Candida antarctica lipase A (CALA) with improved activity and selectivity were prepared for use in organic solvent media. CRL bioimprinted with fatty acids exhibited eightfold enhanced transesterification activity in hexane. Combination of bioimprinting and coating with lecithin or with immobilization did not improve the activity further. CALA was immobilized with and without bioimprinting, none of which improved the activity. All modified lipases were tested for selective ethanolysis of fish oil to concentrate omega-3 polyunsaturated fatty acids (PUFA). None of the preparations, except the immobilized ones catalysed ethanolysis. Immobilized CRL-catalyzed ethanolysis giving 27% (v/v) ethyl esters (EE) in 48 h, of which 43 mol% was oleic acid but no PUFA was detected in the EE fraction. Fatty acid selectivity of CALA was significantly improved by immobilization combined with bioimprinting, resulting in 5.5-fold lower omega-3 PUFA in EE.

Bioimprinting and immobilization of Candida antarctica lipase A (CALA) was optimized by response surface methodology to improve its selectivity for concentration of omega-3 polyunsaturated fatty acids (PUFA) by lipase-catalyzed ethanolysis of salmon oil obtained from salmon by-products. Enzyme-to-support ratio, oleic acid concentration, and immobilization time were the factors investigated. Total omega-3 PUFA lost to the ethyl esters (EE) fraction was monitored in relation to the varying levels of the factors. Results showed that the second-order polynomial model generated adequately fitted the experimental data. The most critical factor was the enzyme-to-support ratio, which led to decreased recovery of omega-3 PUFA in the glycerides fraction with increasing levels of enzyme amount. Optimum conditions were selected as follows: enzyme-to-support ratio, 0.2 (w/w); oleic acid concentration, 50 mM; immobilization time, 85 min. The total omega-3 PUFA lost to the EE fraction through ethanolysis of salmon oil decreased from 3.76 to 1.47 (mol%) under the optimum conditions.

Tannase has been extensively applied to synthesize gallic acid esters. Bioimprinting technique can evidently enhance transesterification-catalyzing performance of tannase. In order to promote the practical utilization of the modified tannase, a few enzymatic characteristics of the enzyme and its kinetic and thermodynamics properties in synthesis of propyl gallate by transesterification in anhydrous medium have been studied. The investigations of pH and temperature found that the imprinted tannase holds an optimum activity at pH 5.0 and 40 °C. On the other hand, the bioimprinting technique has a profound enhancing effect on the adapted tannase in substrate affinity and thermostability. The kinetic and thermodynamic analyses showed that the modified tannase has a longer half-time of 1,710 h at 40 °C; the kinetic constants, the activation energy of reversible thermal inactivation, and the activation energy of irreversible thermal inactivation, respectively, are 0.054 mM, 17.35 kJ mol −1 , and 85.54 kJ mol −1 with tannic acid as a substrate at 40 °C; the free energy of Gibbs (Δ G ) and enthalpy (Δ H ) were found to be 97.1 and 82.9 kJ mol -1 separately under the same conditions.

Geotrichum sp. lipase modified with a combined method composed of crosslinking and bioimprinting was employed to selectively hydrolyze waste fish oil for enrichment of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in glycerides. Crosslinked polymerization by monomer (polyethylene glycol 400 dimethyl acrylate), crosslinker (trimethylolpropane trimethylacrylate), and photoinitiator (benzoin methyl ether) coupled to bioimprinting using palmitic acid as imprint molecule, resulted in much more effective enzyme preparation used in aqueous hydrolysis reaction. Since the crosslinked polymerization modification maintained bioimprinted property and gave good dispersion of enzyme in reaction mixture, the crosslinked bioimprinted enzyme exhibited higher hydrolysis temperature, enhanced specific activity, shorter hydrolysis time, and better operational stability compared to free lipase. Crude fish oil was treated at 45 °C with this crosslinked bioimprinted lipase for 8 h, and 46% hydrolysis degree resulted in the production of glycerides containing 41% of EPA and DHA (EPA+DHA), achieving 85.7% recovery of initial EPA and DHA. The results suggested that bioimprinted enzymes did not lose their induced property in aqueous environment when prepared according to the described crosslinking-bioimprinting method. It could also be seen that the crosslinked bioimprinted lipase was effective in producing glycerides that contained a higher concentration of polyunsaturated fatty acid with better yield.

Pozadina istraživanja. Iako su objavljene mnoge studije biološkog označavanja lipaza, nijedna ne uspoređuje strategiju biološkog označavanja prije imobilizacije s onom nakon imobilizacije. Također, ne postoji istraživanje koje uspoređuje biološko označavanje lipaza imobiliziranih iz preparata čiste lipaze i lipaza imobiliziranih iz sirovog ekstrakta. Stoga smo istražili ove strategije koristeći metagenomsku lipazu LipC12. Eksperimentalni pristup. LipC12 smo kovalentno imobilizirali na komercijalnu podlogu Immobead 150P i tretirali je različitim agensima, prije ili nakon imobilizacije. Također smo usporedili imobilizaciju iz čistog preparata LipC12 i imobilizaciju iz sirovog ekstrakta bez stanica. Rezultati i zaključci. Najveći porast aktivnosti hidrolize trioleina u n-heksanu, u usporedbi s kontrolnim uzorkom bez biološkog označavanja, postignut je biološkim označavanjem nakon imobilizacije, korištenjem otopine oleinske kiseline u t-butanolu: 12 puta veća aktivnost pri imobilizaciji iz čistog preparata LipC12 i gotovo 14 puta veća aktivnost pri imobilizaciji iz sirovog ekstrakta bez stanica. Ovo sredstvo za biološko označavanje također je povećalo aktivnost pri sintezi etil-oleata u n-heksanu 3,5 puta, što je postignuto biološkim označavanjem prije imoblizacije i imobilizacijom iz ekstrakta bez stanica. Novina i znanstveni doprinos. Ovo je prvo istraživanje koje uspoređuje strategije biološkog označavanja prije i nakon imobilizacije, kao i biološkog označavanja enzima imobiliziranih iz preparata čistog enzima i sirovih ekstrakata bez stanica. Dobiveni rezultati potiču daljnja istraživanja strategija biološkog označavanja.