Special Issue "Immobilization of Enzymes"

A special issue of Catalysts (ISSN 2073-4344). This special issue belongs to the section "Biocatalysis".

Deadline for manuscript submissions: 30 September 2019.

Special Issue Editors

Guest Editor
Dr. Immacolata Serra E-Mail
University of Milano, Department of Food, Environmental and Nutritional Sciences (DeFENS), Milano, Italy
Interests: biocatalysis, heterologous protein expression, enzyme immobilization, enzyme engineering, multi-enzymatic processes, acylases, ketoreductases, nucleoside phosphorylases, fermentations
Guest Editor
Prof. Daniela Ubiali E-Mail
University of Pavia, Department of Drug Sciences, Pavia, Italy
Interests: biocatalysis, enzyme immobilization, nucleoside phosphorylases, lipases, omega-transaminases, biorefinery, medicinal chemistry

Special Issue Information

Dear Colleagues,

Enzymes are extremely versatile and powerful catalysts used in several fields (fine chemicals, pharmaceuticals, food, plastics, cosmetics, biofuels); moreover, they often represent an environmentally friendly alternative to conventional chemical catalysts. However, the competitiveness of biocatalyzed processes with respect to the “conventional” chemical ones is often hampered by the poor long-term operational stability of the biocatalysts and their difficult recovery and re-use. These drawbacks can generally be overcome by enzyme immobilization that, by definition, produces “an enzyme confined in a defined region of space with retention of catalytic activity, increased stability and which can be used repeatedly and continuously”. Thus, it is not surprising that enzyme immobilization is frequently considered as a pre-requisite to the development of an enzyme as a biocatalyst for synthetic applications.

Many efforts have been devoted over the years to the development of various immobilization techniques and materials. Nevertheless, the optimal immobilization technique often needs to be tailored for each enzyme to fit both the peculiar enzyme features and the final process. Combining information derived from protein sequence, 3-D structure, reaction mechanism and process features with data on physical/chemical properties of the carrier is pivotal to develop ad hoc immobilization strategies.

For this Special Issue, we welcome contributions from all aspects of enzyme immobilization, may they be related to fundamental science or practical applications, that can be outlined by the following keywords:

  • Enzyme immobilization techniques
  • Enzyme co-immobilization for cascade reactions
  • Rational design of immobilization
  • Improved enzyme properties via immobilization
  • Novel supports for enzyme immobilization
  • Enzyme modification to improve immobilization on solid support
  • Biotransformations catalyzed by immobilized enzymes

Dr. Immacolata Serra
Prof. Daniela Ubiali
Guest Editors

Manuscript Submission Information

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Keywords

  • Enzyme immobilization techniques
  • Enzyme co-immobilization for cascade reactions
  • Rational design of immobilization
  • Improved enzyme properties via immobilization
  • Novel supports for enzyme immobilization
  • Enzyme modification to improve immobilization on solid support
  • Biotransformations catalyzed by immobilized enzymes

Published Papers (12 papers)

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Research

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Open AccessArticle
Enzyme Immobilization for Solid-Phase Catalysis
Catalysts 2019, 9(9), 732; https://doi.org/10.3390/catal9090732 - 29 Aug 2019
Abstract
The covalent immobilization of an enzyme to a solid support can broaden its applicability in various workflows. Immobilized enzymes facilitate catalyst re-use, adaptability to automation or high-throughput applications and removal of the enzyme without heat inactivation or reaction purification. In this report, we [...] Read more.
The covalent immobilization of an enzyme to a solid support can broaden its applicability in various workflows. Immobilized enzymes facilitate catalyst re-use, adaptability to automation or high-throughput applications and removal of the enzyme without heat inactivation or reaction purification. In this report, we demonstrate a step-by-step procedure to carry out the bio-orthogonal immobilization of DNA modifying enzymes employing the self-labelling activity of the SNAP-tag to covalently conjugate the enzyme of interest to the solid support. We also demonstrate how modifying the surface functionality of the support can improve the activity of the immobilized enzyme. Finally, the utility of immobilized DNA-modifying enzymes is depicted through sequential processing of genomic DNA libraries for Illumina next-generation sequencing (NGS), resulting in improved read coverage across AT-rich sequences. Full article
(This article belongs to the Special Issue Immobilization of Enzymes)
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Open AccessArticle
Two-Step Production of Neofructo-Oligosaccharides Using Immobilized Heterologous Aspergillus terreus 1F-Fructosyltransferase Expressed in Kluyveromyces lactis and Native Xanthophyllomyces dendrorhous G6-Fructosyltransferase
Catalysts 2019, 9(8), 673; https://doi.org/10.3390/catal9080673 - 07 Aug 2019
Abstract
Fructo-oligosaccharides (FOS) are prebiotic low-calorie sweeteners that are synthesized by the transfer of fructose units from sucrose by enzymes known as fructosyltransferases. If these enzymes generate β-(2,6) glycosidic bonds, the resulting oligosaccharides belong to the neoseries (neoFOS). Here, we characterized the properties of [...] Read more.
Fructo-oligosaccharides (FOS) are prebiotic low-calorie sweeteners that are synthesized by the transfer of fructose units from sucrose by enzymes known as fructosyltransferases. If these enzymes generate β-(2,6) glycosidic bonds, the resulting oligosaccharides belong to the neoseries (neoFOS). Here, we characterized the properties of three different fructosyltransferases using a design of experiments approach based on response surface methodology with a D-optimal design. The reaction time, pH, temperature, and substrate concentration were used as parameters to predict three responses: The total enzyme activity, the concentration of neoFOS and the neoFOS yield relative to the initial concentration of sucrose. We also conducted immobilization studies to establish a cascade reaction for neoFOS production with two different fructosyltransferases, achieving a total FOS yield of 47.02 ± 3.02%. The resulting FOS mixture included 53.07 ± 1.66 mM neonystose (neo-GF3) and 20.8 ± 1.91 mM neo-GF4. Full article
(This article belongs to the Special Issue Immobilization of Enzymes)
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Open AccessArticle
Preparation of Cross-Linked Enzyme Aggregates (CLEAs) of an Inulosucrase Mutant for the Enzymatic Synthesis of Inulin-Type Fructooligosaccharides
Catalysts 2019, 9(8), 641; https://doi.org/10.3390/catal9080641 - 27 Jul 2019
Abstract
Fructooligosaccharides are well-known carbohydrate molecules that exhibit good probiotic activity and are widely used as sweeteners. Inulin-type fructooligosaccharides (IFOs) can be synthesized from sucrose using inulosucrase. In this study, cross-linked enzyme aggregates (CLEAs) of Lactobacillus reuteri 121 inulosucrase (R483A-LrInu) were prepared and used [...] Read more.
Fructooligosaccharides are well-known carbohydrate molecules that exhibit good probiotic activity and are widely used as sweeteners. Inulin-type fructooligosaccharides (IFOs) can be synthesized from sucrose using inulosucrase. In this study, cross-linked enzyme aggregates (CLEAs) of Lactobacillus reuteri 121 inulosucrase (R483A-LrInu) were prepared and used as a biocatalyst for IFOs production. Under optimum conditions, R483A-LrInu CLEAs retained 42% of original inulosucrase activity. Biochemical characterization demonstrated that the optimum pH of inulosucrase changed from 5 to 4 after immobilization, while the optimum temperature was unchanged. Furthermore, the pH stability and thermostability of the R483A-LrInu CLEAs was significantly improved. IFOs product characterization indicated that the product specificity of the enzyme was impacted by CLEA generation, producing a narrower range of IFOs than the soluble enzyme. In addition, the R483A-LrInu CLEAs showed operational stability in the batch synthesis of IFOs. Full article
(This article belongs to the Special Issue Immobilization of Enzymes)
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Open AccessArticle
Combined Cross-Linked Enzyme Aggregates of Monoamine Oxidase and Putrescine Oxidase as a Bifunctional Biocatalyst for Determination of Biogenic Amines in Foods
Catalysts 2019, 9(7), 579; https://doi.org/10.3390/catal9070579 - 30 Jun 2019
Abstract
In order to determine total biogenic amines in fermented foods, the combined cross-linked enzyme aggregates of a monoamine oxidase and a putrescine oxidase (combi-CLEAs) and the cross-linked enzyme aggregates (CLEAs) of the fused enzyme of two amine oxidases (MonoAmine Putrescien Oxidase, MAPO) were [...] Read more.
In order to determine total biogenic amines in fermented foods, the combined cross-linked enzyme aggregates of a monoamine oxidase and a putrescine oxidase (combi-CLEAs) and the cross-linked enzyme aggregates (CLEAs) of the fused enzyme of two amine oxidases (MonoAmine Putrescien Oxidase, MAPO) were prepared. The effects of various parameters were examined to optimize the CLEAs formation. Biochemical characterization and stability of free and the CLEAs enzymes were performed. Through optimization of the CLEAs formation condition, the combi-CLEAs and the CLEAs-MAPO were prepared with 82% and 78% of residual activities relative to the activities of the subjected enzymes were in a preparative scale. The optimal pH for tyramine-activities of the CLEAs enzymes were shifted to relatively basic pH, leading to synchronization of the optimal performances of combi-CLEAs over pH for tyramine and putrescine. In addition, thermostability of the CLEAs enzymes were improved with almost double half-lives at 65 °C in comparison to the free enzymes. The catalytic efficiencies of combi-CLEAs for tyramine, histamine and putrescine were reduced by 41%, 56%, and 31%, respectively, and the inhibition potency by the substrate was reduced by two-fold in comparison of the mixed free enzymes. In conclusion, combi-CLEAs are a promising catalyst with the improved stability and the same optimum pH for dual activities in enzymatic determination of biogenic amines in foods. Full article
(This article belongs to the Special Issue Immobilization of Enzymes)
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Open AccessArticle
Fabrication and Optimization of a Lipase Immobilized Enzymatic Membrane Bioreactor based on Polysulfone Gradient-Pore Hollow Fiber Membrane
Catalysts 2019, 9(6), 495; https://doi.org/10.3390/catal9060495 - 28 May 2019
Abstract
Enzymatic membrane bioreactors (EMBRs) possess the characteristic of combining catalysis with separation, and therefore have promising application potentials. In order to achieve a high-performance EMBR, membrane property, as well as operating parameters, should give special cause for concerns. In this work, an EMBR [...] Read more.
Enzymatic membrane bioreactors (EMBRs) possess the characteristic of combining catalysis with separation, and therefore have promising application potentials. In order to achieve a high-performance EMBR, membrane property, as well as operating parameters, should give special cause for concerns. In this work, an EMBR based on hollow fiber polysulfone microfiltration membranes with radial gradient pore structure was fabricated and enzyme immobilization was achieved through pressure-driven filtration. Lipase from Candida rugosa was used for immobilization and EMBR performance was studied with the enzymatic hydrolysis of glycerol triacetate as a model reaction. The influences of membrane pore diameter, substrate feed direction as well as operational parameters of operation pressure, substrate concentration, and temperature on the EMBR activity were investigated with the production of hydrolysates kinetically fitted. The complete EMBR system showed the highest activity of 1.07 × 104 U⋅g−1. The results in this work indicate future efforts for improvement in EMBR. Full article
(This article belongs to the Special Issue Immobilization of Enzymes)
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Open AccessArticle
Preparation of Crosslinked Enzyme Aggregates of a Thermostable Cyclodextrin Glucosyltransferase from Thermoanaerobacter sp. Critical Effect of the Crosslinking Agent
Catalysts 2019, 9(2), 120; https://doi.org/10.3390/catal9020120 - 30 Jan 2019
Cited by 4
Abstract
Crosslinked enzyme aggregates (CLEAs) of a thermostable cyclodextrin glucosyltransferase (CGTase) from Thermoanaerobacter sp. have been prepared for the production of cyclodextrins (CDs). Different parameters in the precipitation (nature and concentration of precipitant) and crosslinking steps (time of reaction with cross-linker, nature and concentration [...] Read more.
Crosslinked enzyme aggregates (CLEAs) of a thermostable cyclodextrin glucosyltransferase (CGTase) from Thermoanaerobacter sp. have been prepared for the production of cyclodextrins (CDs). Different parameters in the precipitation (nature and concentration of precipitant) and crosslinking steps (time of reaction with cross-linker, nature and concentration of the crosslinker) were evaluated on the production of CLEAs of CGTase. Among the seven studied precipitants, acetone with a 75% (v/v) concentration produced the aggregates of CGTase with higher activity, which retained 97% of the initial activity. Concerning the cross-linker (glutaraldehyde, starch–aldehyde, and pectin–aldehyde), starch–aldehyde produced the most active CLEAs. The use of bovine serum albumin as co-feeder decreased the expressed activity. Addition of polyethylenimine at the end of cross-linking step prevented the leakage of the enzyme and the subsequent Schiff’s bases reduction with sodium borohydride permitted to maintain 24% of the initial activity even with the large dextrin as substrate. The optimal conditions for the immobilization process required were defined as 75% (v/v) acetone as precipitation reagent for 1 h at 20 °C, 20 mM starch–aldehyde as crosslinking reagent for 2 h at 20 °C, treatment with 1 mg/mL of polyethylenimine for 5 min, reduction with 1 mg/mL of sodium borohydride. The CLEAs of CGTase were active catalyst (similarly to the free enzyme) in the production of cyclodextrins at 50 °C and pH 6.0 for 6 h reaction, maintaining intact their structures. Besides this, after five cycles of 3 h the total cyclodextrin yield was 80% of the initial value (first batch, with around 45% CD yield). Full article
(This article belongs to the Special Issue Immobilization of Enzymes)
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Open AccessArticle
Immobilization of Arylmalonate Decarboxylase
Catalysts 2018, 8(12), 603; https://doi.org/10.3390/catal8120603 - 02 Dec 2018
Cited by 1
Abstract
Arylmalonate decarboxylase (AMD) is a monomeric enzyme of only 26 kDa. A recombinant AMDase from Bordetella bronchiseptica was expressed in Escherichia coli and the enzyme was immobilized using different techniques: entrapment in polyvinyl alcohol (PVA) gel (LentiKats®), covalent binding onto magnetic [...] Read more.
Arylmalonate decarboxylase (AMD) is a monomeric enzyme of only 26 kDa. A recombinant AMDase from Bordetella bronchiseptica was expressed in Escherichia coli and the enzyme was immobilized using different techniques: entrapment in polyvinyl alcohol (PVA) gel (LentiKats®), covalent binding onto magnetic microparticles (MMP, PERLOZA s.r.o., Lovosice, Czech Republic) and double-immobilization (MMP-LentiKats®) using the previous two methods. The double-immobilized AMDase was stable in 8 repeated biocatalytic reactions. This combined immobilization technique has the potential to be applied to different small proteins. Full article
(This article belongs to the Special Issue Immobilization of Enzymes)
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Open AccessArticle
Rice Husk as an Inexpensive Renewable Immobilization Carrier for Biocatalysts Employed in the Food, Cosmetic and Polymer Sectors
Catalysts 2018, 8(10), 471; https://doi.org/10.3390/catal8100471 - 19 Oct 2018
Cited by 4
Abstract
The high cost and environmental impact of fossil-based organic carriers represent a critical bottleneck to their use in large-scale industrial processes. The present study demonstrates the applicability of rice husk as inexpensive renewable carrier for the immobilization of enzymes applicable sectors where the [...] Read more.
The high cost and environmental impact of fossil-based organic carriers represent a critical bottleneck to their use in large-scale industrial processes. The present study demonstrates the applicability of rice husk as inexpensive renewable carrier for the immobilization of enzymes applicable sectors where the covalent anchorage of the protein is a pre-requisite for preventing protein contamination while assuring the recyclability. Rice husk was oxidized and then functionalized with a di-amino spacer. The morphological characterization shed light on the properties that affect the functionalization processes. Lipase B from Candida antarctica (CaLB) and two commercial asparaginases were immobilized covalently achieving higher immobilization yield than previously reported. All enzymes were immobilized also on commercial epoxy methacrylic resins and the CaLB immobilized on rice husk demonstrated a higher efficiency in the solvent-free polycondensation of dimethylitaconate. CaLB on rice husk appears particularly suitable for applications in highly viscous processes because of the unusual combination of its low density and remarkable mechanical robustness. In the case of the two asparaginases, the biocatalyst immobilized on rice husk performed in aqueous solution at least as efficiently as the enzyme immobilized on methacrylic resins, although the rice husk loaded a lower amount of protein. Full article
(This article belongs to the Special Issue Immobilization of Enzymes)
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Open AccessArticle
Preparation of a Flower-Like Immobilized D-Psicose 3-Epimerase with Enhanced Catalytic Performance
Catalysts 2018, 8(10), 468; https://doi.org/10.3390/catal8100468 - 18 Oct 2018
Cited by 1
Abstract
In this present study, we proposed a smart biomineralization method for creating hybrid organic–inorganic nanoflowers using a Co2+-dependent enzyme (D-psicose 3-epimerase; DPEase) as the organic component and cobalt phosphate as the inorganic component. The prepared nanoflowers have many separated [...] Read more.
In this present study, we proposed a smart biomineralization method for creating hybrid organic–inorganic nanoflowers using a Co2+-dependent enzyme (D-psicose 3-epimerase; DPEase) as the organic component and cobalt phosphate as the inorganic component. The prepared nanoflowers have many separated petals that have a nanometer size. Under optimum conditions (60 °C and pH of 8.5), the nanoflower can display its maximum activity (36.2 U/mg), which is about 7.2-fold higher than free DPEase. Furthermore, the immobilized DPEase presents enhanced pH and thermal stabilities. The DPEase-nanoflower maintained about 90% of its activity after six reaction cycles, highlighting its excellent reusability. Full article
(This article belongs to the Special Issue Immobilization of Enzymes)
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Open AccessArticle
Stabilization of Enzymes by Multipoint Covalent Attachment on Aldehyde-Supports: 2-Picoline Borane as an Alternative Reducing Agent
Catalysts 2018, 8(8), 333; https://doi.org/10.3390/catal8080333 - 15 Aug 2018
Cited by 4
Abstract
Enzyme immobilization by multipoint covalent attachment on supports activated with aliphatic aldehyde groups (e.g., glyoxyl agarose) has proven to be an excellent immobilization technique for enzyme stabilization. Borohydride reduction of immobilized enzymes is necessary to convert enzyme–support linkages into stable secondary amino groups [...] Read more.
Enzyme immobilization by multipoint covalent attachment on supports activated with aliphatic aldehyde groups (e.g., glyoxyl agarose) has proven to be an excellent immobilization technique for enzyme stabilization. Borohydride reduction of immobilized enzymes is necessary to convert enzyme–support linkages into stable secondary amino groups and to convert the remaining aldehyde groups on the support into hydroxy groups. However, the use of borohydride can adversely affect the structure–activity of some immobilized enzymes. For this reason, 2-picoline borane is proposed here as an alternative milder reducing agent, especially, for those enzymes sensitive to borohydride reduction. The immobilization-stabilization parameters of five enzymes from different sources and nature (from monomeric to multimeric enzymes) were compared with those obtained by conventional methodology. The most interesting results were obtained for bacterial (R)-mandelate dehydrogenase (ManDH). Immobilized ManDH reduced with borohydride almost completely lost its catalytic activity (1.5% of expressed activity). In contrast, using 2-picoline borane and blocking the remaining aldehyde groups on the support with glycine allowed for a conjugate with a significant activity of 19.5%. This improved biocatalyst was 357-fold more stable than the soluble enzyme at 50 °C and pH 7. The results show that this alternative methodology can lead to more stable and active biocatalysts. Full article
(This article belongs to the Special Issue Immobilization of Enzymes)
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Review

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Open AccessReview
Surface Display—An Alternative to Classic Enzyme Immobilization
Catalysts 2019, 9(9), 728; https://doi.org/10.3390/catal9090728 - 28 Aug 2019
Abstract
Enzyme immobilization to solid matrices often presents a challenge due to protein conformation sensitivity, desired enzyme purity, and requirements for the particular carrier properties and immobilization technique. Surface display of enzymes at the cell walls of microorganisms presents an alternative that has been [...] Read more.
Enzyme immobilization to solid matrices often presents a challenge due to protein conformation sensitivity, desired enzyme purity, and requirements for the particular carrier properties and immobilization technique. Surface display of enzymes at the cell walls of microorganisms presents an alternative that has been the focus of many research groups worldwide in different fields, such as biotechnology, energetics, pharmacology, medicine, and food technology. The range of systems by which a heterologous protein can be displayed at the cell surface allows the appropriate one to be found for almost every case. However, the efficiency of display systems is still quite low. The most frequently used yeast for the surface display of proteins is Saccharomyces cerevisiae. However, apart from its many advantages, Saccharomyces cerevisiae has some disadvantages, such as low robustness in industrial applications, hyperglycosylation of some heterologous proteins, and relatively low efficiency of surface display. Thus, in the recent years the display systems for alternative yeast hosts with better performances including Pichia pastoris, Hansenula polymorpha, Blastobotrys adeninivorans, Yarrowia lipolytica, Kluyveromyces marxianus, and others have been developed. Different strategies of surface display aimed to increase the amount of displayed protein, including new anchoring systems and new yeast hosts are reviewed in this paper. Full article
(This article belongs to the Special Issue Immobilization of Enzymes)
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Open AccessFeature PaperReview
Dextran Aldehyde in Biocatalysis: More Than a Mere Immobilization System
Catalysts 2019, 9(7), 622; https://doi.org/10.3390/catal9070622 - 21 Jul 2019
Cited by 1
Abstract
Dextran aldehyde (dexOx), resulting from the periodate oxidative cleavage of 1,2-diol moiety inside dextran, is a polymer that is very useful in many areas, including as a macromolecular carrier for drug delivery and other biomedical applications. In particular, it has been widely used [...] Read more.
Dextran aldehyde (dexOx), resulting from the periodate oxidative cleavage of 1,2-diol moiety inside dextran, is a polymer that is very useful in many areas, including as a macromolecular carrier for drug delivery and other biomedical applications. In particular, it has been widely used for chemical engineering of enzymes, with the aim of designing better biocatalysts that possess improved catalytic properties, making them more stable and/or active for different catalytic reactions. This polymer possesses a very flexible hydrophilic structure, which becomes inert after chemical reduction; therefore, dexOx comes to be highly versatile in a biocatalyst design. This paper presents an overview of the multiple applications of dexOx in applied biocatalysis, e.g., to modulate the adsorption of biomolecules on carrier surfaces in affinity chromatography and biosensors design, to serve as a spacer arm between a ligand and the support in biomacromolecule immobilization procedures or to generate artificial microenvironments around the enzyme molecules or to stabilize multimeric enzymes by intersubunit crosslinking, among many other applications. Full article
(This article belongs to the Special Issue Immobilization of Enzymes)
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Planned Papers

The below list represents only planned manuscripts. Some of these manuscripts have not been received by the Editorial Office yet. Papers submitted to MDPI journals are subject to peer-review.

Collagenase activity of bromelain immobilized on gold nanoparticles

Adrianne Brito 1, Marcelo Y. Icimoto 2 and Iseli Nantes-Cardoso 1,*

1   Federal University of ABC, Santo André SP, Brazil

2   The Federal University of São Paulo, São Paulo, SP, Brazil

*   Correspondence: [email protected] and [email protected] ;

Abstract: Bromelain (Bro) is a multiprotein complex extract (from the pineapple plant [Ananas comosus]) composed of at least eight cysteine-proteases with broad applicability in medicine and industry. The stability of proteases is considered a key factor, obtained, for example, by immobilization in nanoparticles (NPs). The present study described the synthesis and characterization of gold nanoparticles (GNPs) produced with the use of Bro as reducing and stabilizing agents. The synthesis results in active Bro immobilized on GNPs (BroGNPs) with a gain of thermal stability and aging resistance. Consistent with the structural changes observed by circular dichroism analysis, the association with GNPs had effects on enzyme activity. The activity assays were done on type I collagen membrane and characterized by UV-Vis spectroscopy, fluorescence spectroscopy after reaction with OPA (O-phthaldialdehyde), scanning electron microscopy, scanning confocal microscopy by laser and high-performance liquid chromatography. The interaction of BroGNPs with these membranes promoted their partial digestion, but in a differentiated way relative to the free enzyme and papain. Another differential observed for BroGNPs was the significant increase in the sensitivity to inhibition by E64 (protease inhibitor), indicating a gain of cysteine ​​protease activity. The improvement of stability and gain of specific activity using nanotechnology contributes to the broadening and improvement of Bro applications.

Keywords: Bromelain; gold nanoparticles; collagen

Surface display - an alternative to classic enzyme immobilization

Mateja Lozančić, Amir Sk. Hossain, Vladimir Mrša, Renata Teparić

Laboratory of Biochemistry, Faculty of Food Technology and Biotechnology, University of Zagreb

Abstract: Enzyme immobilization to solid matrices often presents a challenge due to protein conformation sensitivity, desired enzyme purity and requirements for the particular carrier properties and immobilization technique. Surface display of enzymes at the cell walls of microorganisms presents an alternative that reached the focus of many research groups worldwide in different fields of biotechnology, energetics, pharmacology, medicine and food technology. The range of systems by which a heterologous protein can be displayed at the cell surface allows finding the one appropriate for almost every case. However, the efficiency of display systems is still quite low. The most frequently used yeast for surface display of proteins is S. cerevisiae. However, besides many advantages S. cerevisiae has some disadvantages as well, such as low robustness in industrial applications, hyperglycosylation of some heterologous proteins and relatively low efficiency of surface display. Thus, in the recent years the display systems for alternative yeast hosts with better performances including Pichia pastoris, Hansenula polymorpha, Arxula adeninivorans, Yarrowia lipolytica, Kluyveromyces lactis, and others have been developed. Different strategies of surface display aimed to increase the amount of displayed protein, including new anchoring systems and new yeast hosts are reviewed in this paper.

Keywords: surface display; genetic immobilization; yeast cell wall; cell wall proteins

Increased Stability of Oligopeptidases immobilized in Gold Nanoparticles

Marcelo Y. Icimoto1*, Adrianne Brito2, Marcos P. C. Ramos1 ,Vitor Oliveira1 and Iseli Nantes-Cardoso2

1   The Federal University of São Paulo, São Paulo, SP, Brazil

2   Federal University of ABC, Santo André SP, Brazil

*   Correspondence: [email protected];

Abstract: The related metallopeptidases Thimet Oligopeptidase (THOP, E.C.3.4.24.25) and Neurolysin (NEL, E.C.3.4.24.26) belongs the zinc endopeptidase M13 family. Numerous in vitro studies indicate that these peptidases can participate in the catalysis/processing of bioactive peptides such as angiotensins and bradykinin. Some in vivo works demonstrate the use of THOP and NEL inhibitors potentiating the bradykinin-induced hypotension, indicating a role for both peptidases in the metabolism of bradykinin in the circulation. Efforts have been conducted in order to Biotechnologically use both proteases to regulate blood pressure and stroke in mice, but the low plasmatic stability of recombinant enzymes suggests that this aspect is a limiting step. In order to improve the catalytic function and stability of both proteases, here we showed, for the first time, the increased stability and gain of specific activity using nanotechnology, by immobilizing recombinant THOP and NEL in Gold Nanoparticles (GNPs). We demonstrate the importance of cysteine residues contributing to both GNP formation and stabilization by site-directed mutagenesis, activity-based and structural assays using fluorogenic substrates and circular dichroism. The generated GNP-THOP and GNP-NEL were also characterized using UV-Vis spectroscopy, atomic force microscopy and scanning electron microscopy. Altogether, our data suggests a potential use of oligopeptidases immobilized in gold nanoparticles as therapies related to pressure regulation and stroke, also contributing to better comprehend the physiological and pathological roles of both proteases.

Keywords: THOP; NEL; Gold Nanoparticles; Oligopeptidases

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