Support Enzyme Loading Influences the Effect of Aldehyde Dextran Modification on the Specificity of Immobilized Ficin for Large Proteins.

It has been reported that the modification of immobilized glyoxyl-ficin with aldehyde dextran can promote steric hindrances that greatly reduce the activity of the immobilized protease against hemoglobin, while the protease still maintained a reasonable level of activity against casein. In this paper, we studied if this effect may be different depending on the amount of ficin loaded on the support. For this purpose, both the moderately loaded and the overloaded glyoxyl-ficin biocatalysts were prepared and modified with aldehyde dextran. While the moderately loaded biocatalyst had a significantly reduced activity, mainly against hemoglobin, the activity of the overloaded biocatalyst was almost maintained. This suggests that aldehyde dextran was able to modify areas of the moderately loaded enzyme that were not available when the enzyme was overloaded. This modification promoted a significant increase in biocatalyst stability for both biocatalysts, but the stability was higher for the overloaded biocatalyst (perhaps due to a combination of inter- and intramolecular crosslinking).

Tuning Immobilized Enzyme Features by Combining Solid-Phase Physicochemical Modification and Mineralization.

Lipase B from Candida antarctica (CALB) and lipase from Thermomyces lanuginosus (TLL) were immobilized on octyl agarose. Then, the biocatalysts were chemically modified using glutaraldehyde, trinitrobenzenesulfonic acid or ethylenediamine and carbodiimide, or physically coated with ionic polymers, such as polyethylenimine (PEI) and dextran sulfate. These produced alterations of the enzyme activities have, in most cases, negative effects with some substrates and positive with other ones (e.g., amination of immobilized TLL increases the activity versus p-nitro phenyl butyrate (p-NPB), reduces the activity with R-methyl mandate by half and maintains the activity with S-isomer). The modification with PEI increased the biocatalyst activity 8-fold versus R-methyl mandelate. Enzyme stability was also modified, usually showing an improvement (e.g., the modification of immobilized TLL with PEI or glutaraldehyde enabled to maintain more than 70% of the initial activity, while the unmodified enzyme maintained less than 50%). The immobilized enzymes were also mineralized by using phosphate metals (Zn2+, Co2+, Cu2+, Ni2+ or Mg2+), and this affected also the enzyme activity, specificity (e.g., immobilized TLL increased its activity after zinc mineralization versus triacetin, while decreased its activity versus all the other assayed substrates) and stability (e.g., the same modification increase the residual stability from almost 0 to more than 60%). Depending on the enzyme, a metal could be positively, neutrally or negatively affected for a specific feature. Finally, we analyzed if the chemical modification could, somehow, tune the effects of the mineralization. Effectively, the same mineralization could have very different effects on the same immobilized enzyme if it was previously submitted to different physicochemical modifications. The same mineralization could present different effects on the enzyme activity, specificity or stability, depending on the previous modification performed on the enzyme, showing that these previous enzyme modifications alter the effects of the mineralization on enzyme features. For example, TLL modified with glutaraldehyde and treated with zinc salts increased its activity using R-methyl mandelate, while almost maintaining its activity versus the other unaltered substrates, whereas the aminated TLL maintained its activity with both methyl mandelate isomers, while it decreased with p-NPB and triacetin. TLL was found to be easier to tune than CALB by the strategies used in this paper. In this way, the combination of chemical or physical modifications of enzymes before their mineralization increases the range of modification of features that the immobilized enzyme can experienced, enabling to enlarge the biocatalyst library.

Tuning Immobilized Commercial Lipase Preparations Features by Simple Treatment with Metallic Phosphate Salts.

Four commercial immobilized lipases biocatalysts have been submitted to modifications with different metal (zinc, cobalt or copper) phosphates to check the effects of this modification on enzyme features. The lipase preparations were Lipozyme®TL (TLL-IM) (lipase from Thermomyces lanuginose), Lipozyme®435 (L435) (lipase B from Candida antarctica), Lipozyme®RM (RML-IM), and LipuraSelect (LS-IM) (both from lipase from Rhizomucor miehei). The modifications greatly altered enzyme specificity, increasing the activity versus some substrates (e.g., TLL-IM modified with zinc phosphate in hydrolysis of triacetin) while decreasing the activity versus other substrates (the same preparation in activity versus R- or S- methyl mandelate). Enantiospecificity was also drastically altered after these modifications, e.g., LS-IM increased the activity versus the R isomer while decreasing the activity versus the S isomer when treated with copper phosphate. Regarding the enzyme stability, it was significantly improved using octyl-agarose-lipases. Using all these commercial biocatalysts, no significant positive effects were found; in fact, a decrease in enzyme stability was usually detected. The results point towards the possibility of a battery of biocatalysts, including many different metal phosphates and immobilization protocols, being a good opportunity to tune enzyme features, increasing the possibilities of having biocatalysts that may be suitable for a specific process.

Immobilization of Penicillin G Acylase on Vinyl Sulfone-Agarose: An Unexpected Effect of the Ionic Strength on the Performance of the Immobilization Process.

Penicillin G acylase (PGA) from Escherichia coli was immobilized on vinyl sulfone (VS) agarose. The immobilization of the enzyme failed at all pH values using 50 mM of buffer, while the progressive increase of ionic strength permitted its rapid immobilization under all studied pH values. This suggests that the moderate hydrophobicity of VS groups is enough to transform the VS-agarose in a heterofunctional support, that is, a support bearing hydrophobic features (able to adsorb the proteins) and chemical reactivity (able to give covalent bonds). Once PGA was immobilized on this support, the PGA immobilization on VS-agarose was optimized with the purpose of obtaining a stable and active biocatalyst, optimizing the immobilization, incubation and blocking steps characteristics of this immobilization protocol. Optimal conditions were immobilization in 1 M of sodium sulfate at pH 7.0, incubation at pH 10.0 for 3 h in the presence of glycerol and phenyl acetic acid, and final blocking with glycine or ethanolamine. This produced biocatalysts with stabilities similar to that of the glyoxyl-PGA (the most stable biocatalyst of this enzyme described in literature), although presenting just over 55% of the initially offered enzyme activity versus the 80% that is recovered using the glyoxyl-PGA. This heterofuncionality of agarose VS beads opens new possibilities for enzyme immobilization on this support.

Functionalization of Porous Cellulose with Glyoxyl Groups as a Carrier for Enzyme Immobilization and Stabilization.

The functionalization of the internal surface of macroporous carriers with glyoxyl groups has proven to highly stabilize a large variety of enzymes through multipoint covalent immobilization. In this work, we have translated the surface chemistry developed for the fabrication of glyoxyl-agarose carriers to macroporous cellulose (CEL). To that aim, CEL-based microbeads were functionalized with glyoxyl groups through a stepwise alkoxylation (or alkylation)/oxidation synthetic scheme. This functionalization sequence was analyzed by solid-state NMR, while the scanning electron miscroscopy of CEL microbeads reveals that the mild oxidation conditions negligibly affect the morphological properties of the material. Through the optimal functionalization protocol using rac-glycidol, we introduce up to 200 µmols of aldehyde groups per gram of wet CEL, a similar density to the one obtained for the benchmarked agarose-glyoxyl carrier. This novel CEL-based carrier succeeds to immobilize and stabilize industrially relevant enzymes such as d-amino acid oxidase from Trigonopsis variabilis and xylanases from Trichoderma reseei. Remarkably, the xylanases immobilized on the optimal CEL-based materials present a half-life time of 51 h at 60 °C and convert up to 90% of the xylan after four operation cycles for the synthesis of xylooligosaccharides.

Stabilization of multimeric sucrose synthase from Acidithiobacillus caldus via immobilization and post-immobilization techniques for synthesis of UDP-glucose.

Sucrose synthases (SuSys) have been attracting great interest in recent years in industrial biocatalysis. They can be used for the cost-effective production of uridine 5'-diphosphate glucose (UDP-glucose) or its in situ recycling if coupled to glycosyltransferases on the production of glycosides in the food, pharmaceutical, nutraceutical, and cosmetic industry. In this study, the homotetrameric SuSy from Acidithiobacillus caldus (SuSyAc) was immobilized-stabilized on agarose beads activated with either (i) glyoxyl groups, (ii) cyanogen bromide groups, or (iii) heterogeneously activated with both glyoxyl and positively charged amino groups. The multipoint covalent immobilization of SuSyAc on glyoxyl agarose at pH 10.0 under optimized conditions provided a significant stabilization factor at reaction conditions (pH 5.0 and 45 °C). However, this strategy did not stabilize the enzyme quaternary structure. Thus, a post-immobilization technique using functionalized polymers, such as polyethyleneimine (PEI) and dextran-aldehyde (dexCHO), was applied to cross-link all enzyme subunits. The coating of the optimal SuSyAc immobilized glyoxyl agarose with a bilayer of 25 kDa PEI and 25 kDa dexCHO completely stabilized the quaternary structure of the enzyme. Accordingly, the combination of immobilization and post-immobilization techniques led to a biocatalyst 340-fold more stable than the non-cross-linked biocatalyst, preserving 60% of its initial activity. This biocatalyst produced 256 mM of UDP-glucose in a single batch, accumulating 1 M after five reaction cycles. Therefore, this immobilized enzyme can be of great interest as a biocatalyst to synthesize UDP-glucose.

Stabilization of Immobilized Lipases by Intense Intramolecular Cross-Linking of Their Surfaces by Using Aldehyde-Dextran Polymers.

Immobilized enzymes have a very large region that is not in contact with the support surface and this region could be the target of new stabilization strategies. The chemical amination of these regions plus further cross-linking with aldehyde-dextran polymers is proposed here as a strategy to increase the stability of immobilized enzymes. Aldehyde-dextran is not able to react with single amino groups but it reacts very rapidly with polyaminated surfaces. Three lipases-from Thermomyces lanuginosus (TLL), Rhizomucor miehiei (RML), and Candida antarctica B (CALB)-were immobilized using interfacial adsorption on the hydrophobic octyl-Sepharose support, chemically aminated, and cross-linked. Catalytic activities remained higher than 70% with regard to unmodified conjugates. The increase in the amination degree of the lipases together with the increase in the density of aldehyde groups in the dextran-aldehyde polymer promoted a higher number of cross-links. The sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of those conjugates demonstrates the major role of the intramolecular cross-linking on the stabilization of the enzymes. The highest stabilization was achieved by the modified RML immobilized on octyl-Sepharose, which was 250-fold more stable than the unmodified conjugate. The TLL and the CALB were 40-fold and 4-fold more stable than the unmodified conjugate.

Production of Omegas-6 and 9 from the Hydrolysis of Açaí and Buriti Oils by Lipase Immobilized on a Hydrophobic Support.

This paper describes a bioprocess to obtain omegas-6 and 9 from the hydrolysis of Açaí (Euterpe oleracea Martius) and Buriti (Mauritia flexuosa) oils by lipases immobilized on octyl-sepharose. For this, oils and butters were initially selected as the carbon source which resulted in higher production of lipases in Beauveria bassiana and Fusarium oxysporum cultures. The carbon source that provided secretion of lipase by B. bassiana was Açaí oil, and for F. oxysporum, Bacuri butter. Lipases obtained under these conditions were immobilized on octyl-sepharose, and both, the derivatives and the crude extracts were biochemically characterized. It was observed that the immobilization promoted an increase of stability in B. bassiana and F. oxysporum lipase activities at the given temperatures and pH. In addition, the immobilization promoted hyperactivation of B. bassiana and F. oxysporum lipase activities being 23.5 and 11.0 higher than free enzyme, respectively. The hydrolysis of Açaí and Buriti oils by the derivatives was done in a biphasic (organic/aqueous) system, and the products were quantified in RP-HPLC. The results showed the potential of these immobilized lipases to obtain omegas-6 and 9 from Brazilian natural oils. This work may improve the enzymatic methodologies for obtaining foods and drugs enriched with fatty acids.

Modulation of the regioselectivity of Thermomyces lanuginosus lipase via biocatalyst engineering for the Ethanolysis of oil in fully anhydrous medium.

BACKGROUND: Enzymatic ethanolysis of oils (for example, high oleic sunflower oil containing 90% of oleic acid) may yield two different reaction products depending on the regioselectivity of the immobilized lipase biocatalyst. Some lipase biocatalysts exhibit a 1,3-regioselectivity and they produced 2 mols of fatty acid ethyl ester plus 1 mol of sn2-monoacylglycerol (2-MAG) per mol of triglyceride without the release of glycerol. Other lipase biocatalysts are completely non-regioselective releasing 3 mols of fatty acid ethyl ester and 1 mol of glycerol per mol of triglyceride. Lipase from Thermomyces lanuginosus (TLL) adsorbed on hydrophobic supports is a very interesting biocatalyst for the ethanolysis of oil. Modulation of TLL regioselectivity in anhydrous medium was intended via two strategies of TLL immobilization: a. - interfacial adsorption on different hydrophobic supports and b.- interfacial adsorption on a given hydrophobic support under different experimental conditions. RESULTS: Immobilization of TLL on supports containing divinylbenezene moieties yielded excellent 1,3-regioselective biocatalysts but immobilization of TLL on supports containing octadecyl groups yielded non-regioselective biocatalysts. On the other hand, TLL immobilized on Purolite C18 at pH 8.5 and 30 °C in the presence of traces of CTAB yielded a biocatalyst with a perfect 1,3-regioselectivity and a very interesting activity: 2.5 µmols of oil ethanolyzed per min per gram of immobilized derivative. This activity is 10-fold higher than the one of commercial Lipozyme TL IM. Immobilization of the same enzyme on the same support, but at pH 7.0 and 25 °C, led to a biocatalyst which can hydrolyze all ester bonds in TG backbone. CONCLUSIONS: Activity and regioselectivity of TLL in anhydrous media can be easily modulated via Biocatalysis Engineering producing very active immobilized derivatives able to catalyze the ethanolysis of triolein. When the biocatalyst was 1,3-regioselective a 33% of 2-monoolein was obtained and it may be a very interesting surfactant. When biocatalyst catalyzed the ethanolysis of the 3 positions during the reaction process, a 99% of ethyl oleate was obtained and it may be a very interesting drug-solvent and surfactant. The absence of acyl migrations under identical reaction conditions is clearly observed and hence the different activities and regioselectivities seem to be due to the different catalytic properties of different derivatives of TLL.

Emerging strategies and integrated systems microbiology technologies for biodiscovery of marine bioactive compounds.

Marine microorganisms continue to be a source of structurally and biologically novel compounds with potential use in the biotechnology industry. The unique physiochemical properties of the marine environment (such as pH, pressure, temperature, osmolarity) and uncommon functional groups (such as isonitrile, dichloroimine, isocyanate, and halogenated functional groups) are frequently found in marine metabolites. These facts have resulted in the production of bioactive substances with different properties than those found in terrestrial habitats. In fact, the marine environment contains a relatively untapped reservoir of bioactivity. Recent advances in genomics, metagenomics, proteomics, combinatorial biosynthesis, synthetic biology, screening methods, expression systems, bioinformatics, and the ever increasing availability of sequenced genomes provides us with more opportunities than ever in the discovery of novel bioactive compounds and biocatalysts. The combination of these advanced techniques with traditional techniques, together with the use of dereplication strategies to eliminate known compounds, provides a powerful tool in the discovery of novel marine bioactive compounds. This review outlines and discusses the emerging strategies for the biodiscovery of these bioactive compounds.

The effects of the chemical modification on immobilized lipase features are affected by the enzyme crowding in the support.

In this article, we have analyzed the interactions between enzyme crowding on a given support and its chemical modification (ethylenediamine modification via the carbodiimide route and picryl sulfonic (TNBS) modification of the primary amino groups) on the enzyme activity and stability. Lipase from Thermomyces lanuginosus (TLL) and lipase B from Candida antarctica (CALB) were immobilized on octyl-agarose beads at two very different enzyme loadings, one of them exceeding the capacity of the support, one well under this capacity. Chemical modifications of the highly loaded and lowly loaded biocatalysts gave very different results in terms of activity and stability, which could increase or decrease enzyme activity depending on the enzyme support loading. For example, both lowly loaded biocatalysts increased their activity after modification while the effect was the opposite for the highly loaded biocatalysts. Additionally, the modification with TNBS of highly loaded CALB biocatalyst increased its stability while decrease the activity.

Tailoring the specificity of ficin versus large hemoglobin and small casein by co-immobilizing inert proteins on the immobilized enzyme layer and further modification with aldehyde dextran.

Ficin has been immobilized at full loading on glyoxyl agarose beads. Then, ficin was blocked with 2,2'-dipyridyldisulfide. To be effective, the modification must be performed in the presence of 0.5 M urea, as the enzyme was not inhibited under standard conditions, very likely because the catalytic Cys was not fully exposed to the medium. Activity could be fully recovered by incubation with 1 M mercaptoethanol. This biocatalyst could hydrolyze hemoglobin and casein. The objective of this paper was to increase the enzyme specificity versus small proteins by generating steric hindrances to the access of large proteins. The step by step blocking via ionic exchange of the biocatalyst with aminated bovine serum albumin (BSA), aldehyde dextran and a second layer of aminated BSA produced a biocatalyst that maintained its activity versus small synthetic substrates, increased the biocatalyst stability, while reduced its activity to over 50 % versus casein. Interestingly, this treatment almost fully annulled the activity versus hemoglobin, more effectively at 37 °C than at 55 °C. The biocatalyst could be reused 5 times without changes in activity. The changes could be caused by steric hindrances, but it cannot be discarded some changes in enzyme sequence specificity caused by the modifications.

Adsorption features of reduced aminated supports modified with glutaraldehyde: Understanding the heterofunctional features of these supports.

Immobilization of enzymes on aminated supports using the glutaraldehyde chemistry may involve three different interactions, cationic, hydrophobic, and covalent interactions. To try to understand the impact this heterofunctionality, we study the physical adsorption of the beta-galactosidase from Aspergillus niger, on aminated supports (MANAE) and aminated supports with one (MANAE-GLU) or two molecules of glutaraldehyde (MANAE-GLU-GLU). To eliminate the chemical reactivity of the glutaraldehyde, the supports were reduced using sodium borohydride. After enzyme adsorption, the release of the enzyme from the supports using different NaCl concentrations, Triton X100, ionic detergents (SDS and CTAB), or different temperatures (4 °C to 55 °C) was studied. Using MANAE support, at 0.3 M NaCl almost all the immobilized enzyme was released. Using MANAE-GLU, 0.3 M, and 0.6 M NaCl similar results were obtained. However, incubation at 1 M or 2 M NaCl, many enzyme molecules were not released from the support. For the MANAE-GLU-GLU support, none of the tested concentrations of NaCl was sufficient to release all enzyme bound to the support. Only using high temperatures, 0.6 M NaCl, and 1 % CTAB or SDS, could the totality of the proteins be released from the support. The results shown in this paper confirm the heterofunctional character of aminated supports modified with glutaraldehyde.

Tuning almond lipase features by the buffer used during immobilization: The apparent biocatalysts stability depends on the immobilization and inactivation buffers and the substrate utilized.

The lipase from Prunus dulcis almonds was inactivated under different conditions. At pH 5 and 9, enzyme stability remained similar under the different studied buffers. However, when the inactivation was performed at pH 7, there were some clear differences on enzyme stability depending on the buffer used. The enzyme was more stable in Gly than when Tris was employed for inactivation. Then, the enzyme was immobilized on methacrylate beads coated with octadecyl groups at pH 7 in the presence of Gly, Tris, phosphate and HEPES. Its activity was assayed versus triacetin and S-methyl mandelate. The biocatalyst prepared in phosphate was more active versus S-methyl mandelate, while the other ones were more active versus triacetin. The immobilized enzyme stability at pH 7 depends on the buffer used for enzyme immobilization. The buffer used in the inactivation and the substrate used determined the activity. For example, glycine was the buffer that promoted the lowest or the highest stabilities depending on the substrate used to quantify the activities.

Green Enzymatic Synthesis of Geranyl Butyrate: Process Optimization and Mechanistic Insights.

Flavor esters are organic compounds widely used in the food industry to enhance the aroma and taste of products. However, most chemical processes for the production of these flavoring compounds use toxic organic solvents. Some organic solvents derived from petroleum can leave behind residual traces in food products, which may raise concerns about potential health risks and contamination. In this study, we employ Eversa Transform 2.0, a commercial lipase derived from the lipase from Thermomyces lanuginosus, to produce geranyl butyrate in aqueous media. The chemical process was optimized using the Taguchi method, and a conversion of 93% was obtained at the optimal reaction conditions of: 1:5 molar ratio (v/v), 15% biocatalyst load (w/w), at 50 °C, in 6 h. Classic (molecular dynamics) and quantum (density functional theory) simulations unveiled amino acid residues involved in the stabilization of the enzyme-substrate complex. Detailed QM/MM mechanistic studies identified the nucleophilic attack of the deacylation reaction as the rate-limiting step of the entire mechanism, which has a free energy barrier of 14.0 kcal/mol.

Glycosylation of polyphenolic compounds: Design of a self-sufficient biocatalyst by co-immobilization of a glycosyltransferase, a sucrose synthase and the cofactor UDP.

Glycosyltransferases catalyze the regioselective glycosylation of polyphenolic compounds, increasing their solubility without altering their antioxidant properties. Leloir-type glycosyltransferases require UDP-glucose as a cofactor to glycosylate a hydroxyl of the polyphenol, which is expensive and unstable. To simplify these processes for industrial implementation, the preparation of self-sufficient heterogeneous biocatalysts is needed. In this study, a glycosyltransferase and a sucrose synthase (as an UDP-regenerating enzyme) were co-immobilized onto porous agarose-based supports coated with polycationic polymers: polyethylenimine and polyallylamine. In addition, the UDP cofactor was strongly ionically adsorbed and co-immobilized with the enzymes, eliminating the need to add it separately. Thus, the optimal self-sufficient heterogeneous biocatalyst was able to catalyze the glycosylation of three polyphenolic compounds (piceid, phloretin and quercetin) with in situ regeneration of the UDP-glucose, allowing multiple consecutive reaction cycles without the addition of exogenous cofactor. A TTN value of 50 (theoretical maximum) was obtained in the reaction of piceid glycosylation, after 5 reaction cycles, using the self-sufficient biocatalyst based on an improved sucrose synthase variant. This result was 5-fold higher than the obtained using soluble cofactor and the co-immobilized enzymes, and much higher than those reported in the literature for similar processes.

Glutaraldehyde modification of lipases immobilized on octyl agarose beads: Roles of the support enzyme loading and chemical amination of the enzyme on the final enzyme features.

Lipase B from Candida antarctica (CALB) and lipase from Thermomyces lanuginosus (TLL) have been immobilized on octyl agarose at low loading and at a loading exceeding the maximum support capacity. Then, the enzymes have been treated with glutaraldehyde and inactivated at pH 7.0 in Tris-HCl, sodium phosphate and HEPES, giving different stabilities. Stabilization (depending on the buffer) of the highly loaded biocatalysts was found, very likely as a consequence of the detected intermolecular crosslinkings. This did not occur for the lowly loaded biocatalysts. Next, the enzymes were chemically aminated and then treated with glutaraldehyde. In the case of TLL, the intramolecular crosslinkings (visible by the apparent reduction of the protein size) increased enzyme stability of the lowly loaded biocatalysts, an effect that was further increased for the highly loaded biocatalysts due to intermolecular crosslinkings. Using CALB, the intramolecular crosslinkings were less intense, and the stabilization was lower, even though the intermolecular crosslinkings were quite intense for the highly loaded biocatalyst. The stabilization detected depended on the inactivation buffer. The interactions between enzyme loading and inactivating buffer on the effects of the chemical modifications suggest that the modification and inactivation studies must be performed under the target biocatalysts and conditions.

Biocatalytic production of biolubricants: Strategies, problems and future trends.

The increasing worries by the inadequate use of energy and the preservation of nature are promoting an increasing interest in the production of biolubricants. After discussing the necessity of producing biolubricants, this review focuses on the production of these interesting molecules through the use of lipases, discussing the different possibilities (esterification of free fatty acids, hydroesterification or transesterification of oils and fats, transesterification of biodiesel with more adequate alcohols, estolides production, modification of fatty acids). The utilization of discarded substrates has special interest due to the double positive ecological impact (e.g., oil distillated, overused oils). Pros and cons of all these possibilities, together with general considerations to optimize the different processes will be outlined. Some possibilities to overcome some of the problems detected in the production of these interesting compounds will be also discussed.

Oriented immobilization of antibodies onto sensing platforms - A critical review.

The immunosensor has been proven a versatile tool to detect various analytes, such as food contaminants, pathogenic bacteria, antibiotics and biomarkers related to cancer. To fabricate robust and reproducible immunosensors with high sensitivity, the covalent immobilization of immunoglobulins (IgGs) in a site-specific manner contributes to better performance. Instead of the random IgG orientations result from the direct yet non-selective immobilization techniques, this review for the first time introduces the advances of stepwise yet site-selective conjugation strategies to give better biosensing efficiency. Noncovalently adsorbing IgGs is the first but decisive step to interact specifically with the Fc fragment, then following covalent conjugate can fix this uniform and antigens-favorable orientation irreversibly. In this review, we first categorized this stepwise strategy into two parts based on the different noncovalent interactions, namely adhesive layer-mediated interaction onto homofunctional support and layer-free interaction onto heterofunctional support (which displays several different functionalities on its surface that are capable to interact with IgGs). Further, the influence of ligands characteristics (synthesis strategies, spacer requirements and matrices selection) on the heterofunctional support has also been discussed. Finally, conclusions and future perspectives for the real-world application of stepwise covalent conjugation are discussed. This review provides more insights into the fabrication of high-efficiency immunosensor, and special attention has been devoted to the well-orientation of full-length IgGs onto the sensing platform.

Chemical amination of immobilized enzymes for enzyme coimmobilization: Reuse of the most stable immobilized and modified enzyme.

Although Lecitase and the lipase from Thermomyces lanuginosus (TLL) could be coimmobilized on octyl-agarose, the stability of Lecitase was lower than that of TLL causing the user to discard active immobilized TLL when Lecitase was inactivated. Here, we propose the chemical amination of immobilized TLL to ionically exchange Lecitase on immobilized TLL, which should be released to the medium after its inactivation by incubation at high ionic strength. Using conditions where Lecitase was only adsorbed on immobilized TLL after its amination, the combibiocatalyst was produced. Unfortunately, the release of Lecitase was not possible using just high ionic strength solutions, and if detergent was added, TLL was also released from the support. This occurred when using 0.25 M ammonium sulfate, Lecitase did not immobilize on aminated TLL. That makes the use octyl-vinylsulfone supports necessary to irreversibly immobilize TLL, and after blocking with ethylendiamine, the immobilized TLL was aminated. Lecitase immobilized and released from this biocatalyst using 0.25 M ammonium sulfate and 0.1% Triton X-100. That way, a coimmobilized TLL and Lecitase biocatalyst could be produced, and after Lecitase inactivation, it could be released and the immobilized, aminated, and fully active TLL could be utilized to build a new combibiocatalyst.