Special Issue "Enzyme Immobilization 2016"
Deadline for manuscript submissions: closed (1 December 2016)
Prof. Dr. Roberto Fernandez-Lafuente
After the success of the first Special Issue edited by Molecules in 2014, I am very happy to announce the launching of a second issue on this topic. The immobilization of enzymes and proteins is a seemingly ancient research area that still attracts great interest. The initial interest in enzyme immobilization was motivated by a desire to simplify the reuse of these expensive biocatalysts. Nowadays, the research in this area has focused in its application to overcome many enzyme limitations, for example improving enzyme stability, activity, selectivity or specificity, reducing inhibition problems, and even coupling immobilization and purification.
Two years after the publication of the first issue in May 2014, where many interesting immobilization protocols were reported, still the control of the orientation of the enzyme on the support surface and of the intensity of the support-enzyme interactions is not properly solved. Coupling of genetic and chemical tools has been utilized to produce enzymes that may be better immobilized on tailor made supports. Each day more researches couple immobilization to any other strategy for enzyme stabilization and enhancement of their properties.
Thus, the efforts to design strategies involving the coupled use of immobilization with microbiological (e.g., the use of thermophilic enzymes), chemical or genetic modifications are of special interest. Enzymes are co-immobilized to catalyze cascade reactions; however, this process may produce additional complications, no always co-immobilization will be recommended. This Special Issue invites submissions (i.e., research or review papers) discussing the design of new immobilization protocols, especially when the control of the enzyme orientation is intended by design of the support, or modification of the enzyme. The use of the immobilized enzymes to take advantage of the catalytic improved properties will be also suitable for this second special issue. Papers related to the modification of immobilized enzymes, or to the modification of the support surface after enzyme immobilization, are also welcome.
Dr. Roberto Fernandez-Lafuente
Manuscript Submission Information
Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. All papers will be peer-reviewed. Accepted papers will be published continuously in the journal (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as short communications are invited. For planned papers, a title and short abstract (about 100 words) can be sent to the Editorial Office for announcement on this website.
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- controlled enzyme immobilization and co-immobilization
- heterofunctional supports
- improved enzyme properties via immobilization
- chemical or genetic modification and immobilization
- immobilization of enzymes from thermophilic microorganisms
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.
Type of the paper: article
Title: Immobilization of glycoside hydrolases families GH1, GH13 and GH70: state of art and perspectives
Authors: Natália G. Graebin, Jéssie da N. Schöffer, Diandra de Andrades, Manuela P. Klein, Plinho F. Hertz, Marco A. Z. Ayub, Rafael C. Rodrigues *
Affiliation: Biotechnology, Bioprocess, and Biocatalysis Group, Food Science and Technology Institute, Federal University of Rio Grande do Sul, Av. Bento Gonçalves 9500, PO Box 15090, ZC 91501-970 Porto Alegre, RS, Brazil
Abstract: Glycoside hydrolases (GH) are a widespread group of enzymes that hydrolyzes the glycosidic bond between two carbohydrates or between a carbohydrate and a non-carbohydrate moiety. Due to the increasing interest for industrial application of these enzymes, immobilization of GH is an important feature to improve activity, stability, and possible reuses in batch reaction or in continuous processes. Thus, in this review we will focus in the immobilization of enzymes from specific GH families. Firstly, a brief introduction of enzyme immobilization methods will be presented, discussing some advantages and drawbacks. Following, it will be reviewed the studies of enzyme immobilization from families GH1, GH13 and GH70, focusing in the enzymes β-glucosidase, β-galactosidase, α-amylase, cyclodextrin glycosyltransferase and dextransucrase. In each case, the immobilization protocols will be evaluated considering their pros and cons. Finally, the perspectives for new immobilization methods, as well as industrial applications will be briefly presented.
Keywords: enzyme immobilization; glycoside hydrolases; β-glucosidase; β-galactosidase; amylase; dextransucrase.
Type of the paper: article
Tentative title: Site-Specific Immobilization Of Dehalogenase St2570 Mediated By Formylglycine-Generating Enzyme And Applications Of The Process In Batch And Semi-Continuous Flow Reactors
Authors: Hui Jian, Yingwu Wang*, Yan Bai, Rong Li, Renjun Gao*
Affiliations: Key Laboratory for Molecular Enzymology and Engineering, the Ministry of Education, School of Life Science, Jilin University, Changchun 130012, China
Abstract: Formylglycine-generating enzymes can selectively recognise and oxidise cysteine residues within the sulphatase sub motif at the terminus of proteins to form aldehyde-bearing formylglycine (FGly) residues; these residues can form covalent bonds with amino functionalised fluoresceins or supports through the Schiff base reaction. In this experiment, aldehyde-tagged proteins were prepared using a previously designed set of the pET28a plasmid system. The C-terminal aldehyde-tagged protein (ST2570CQ) exhibits significant enzymological properties, such as generation of new free aldehyde groups, high level of protein expression and improved enzyme activity. The immobilization of ST2570CQ on SBA15 modified with 2% APTES ethanol solution (V/V) was performed under the following optimal conditions: 2 mg of ST2570CQ per 10 mg of SBA15-NH2 was incubated in 50 mM PB buffer (pH 6.5) at 10 °C for more than 3 h. The immobilized ST2570CQ shows threefold higher thermal stability, 1.2-fold higher catalytic ability and improved operational stability than free ST2570. The immobilized ST2570CQ retains 60% of its original activity after seven cycles of batch operation and 100% of its original activity after 10 cycles of reuse in the semi-continuous flow reactor. These results provide a basis for industrial-scale production of immobilized ST2570.
Key words: Immobilization; Dehalogenase; Formylglycine-generating enzyme; Sulfolobus tokodaii
Type of the paper: article
Tentative title: Reversible Immobilization Of Lipases On Heterofunctional Octyl-Amino Agarose Beads Prevents Enzyme Desorption
Authors: Nazzoly Rueda 1,2,+, Tiago L. Albuquerquea 3,+, Rocio Bartolome-Cabrero 1, Laura Fernandez-Lopez 1, Rodrigo Torres 2,4, Claudia Ortiz 5, Cleiton S. dos Santos 1,3, Oveimar Barbosa 6, Roberto Fernandez-Lafuente 1,*
Affiliations: 1 Departamento de Biocatalisis. ICP-CSIC, Campus UAM-CSIC Madrid. Spain.
2 Escuela de Química, Grupo de investigación en Bioquímica y Microbiología (GIBIM), Edificio Camilo Torres 210, Universidad Industrial de Santander, Bucaramanga, Colombia.
3 Departamento de Engenharia Química, Universidade Federal Do Ceará, Campus Do Pici, CEP 60455-760, Fortaleza, CE, Brazil.
4 Current address: Laboratorio de Biotecnología, Instituto Colombiano del Petróleo-Ecopetrol, Piedecuesta, Bucaramanga, Colombia.
5 Escuela de Microbiología, Universidad Industrial de Santander, Bucaramanga, Colombia
6 Departamento de Química, Facultad de Ciencias. Universidad del Tolima, Ibagué, Colombia
+ Both authors have evenly participated in this research
Abstract: Two different heterofunctional octyl-amino supports have been prepared using ethylenediamine and hexylendiamine (OCEDA and OCHDA) and utilized to immobilize five lipases (lipases A (CALA) and B (CALB ) from Candida antarctica, lipases from Thermomyces lanuginosus (TLL), from Rhizomucor miehei (RML) and from Candida rugosa (CRL) and the phospholipase Lecitase Ultra (LU). Using pH 5 and 50 mM sodium acetate, the immobilizations proceeded via interfacial activation on the octyl layer, after some ionic bridges were established. These supports did not release enzyme at Triton X-100 concentrations that released all enzyme from the octyl support. The activities of the immobilized enzymes were usually slightly higher using the new supports than the octyl ones (that, except for CALB, produced significant enzyme hyperactivation). Thermal and solvent stabilities of LU and TLL were significantly improved compared to the OC counterparts, while in the other enzymes the stability used to decrease (depending on the pH value). As a general rule, OCEDA had lower negative effects immobilized enzyme stability than OCHDA. While in solvent inactivation the enzyme molecules remained attached to the support using the new supports and not using monofunctional octyl supports, in thermal inactivation this only occurred in certain cases.
Type of the paper: Article
Title: Stabilization of CALB Immobilized on Octyl Agarose by Physical Intermolecular Crosslinking with PEI. Prevention of the Enzyme desorption
Authors: Sara Peirce 1,2, Veymar G. Tacias-Pascacio 1,3, Maria Elena Russo 4, Antonio Marzocchella 2, José J. Virgen-Ortíz 1,* and Roberto Fernandez-Lafuente 1,*
Affiliation: 1 Departamento de Biocatálisis. Instituto de Catálisis-CSIC, Campus UAM-CSIC Madrid, Spain
2 Dipartimento di Ingegneria Chimica, dei Materiali e della Produzione Industriale. Universita' degli Studi di Napoli Federico II, Italy
3 Unidad de Investigación y Desarrollo en Alimentos. Instituto Tecnológico de Veracruz, Calzada Miguel A. de Quevedo 2779, 91897 Veracruz, Mexico
4 Istituto di Ricerche sulla Combustione–Consiglio Nazionale delle Ricerche, Napoli, Italy
E-Mails: firstname.lastname@example.org (J.J.V.-O.); email@example.com (R.F.-L.)
Abstract: Lipase B from Candida antarctica (CALB) was immobilized on octyl agarose (OC) and physically modified with PEI in order to confer a strong ion exchanger to the enzyme and that way, enable the immobilization of other enzymes over it. The enzyme activity was fully maintained during the coating and the thermal stability was marginally improved. The enzyme release from the support by incubation in the non-ionic detergent Triton X-100 was more difficult after the PEI-coating suggesting that some intermolecular physical crosslinking had been achieved making more difficult this desorption. Thermal stability was marginally improved, but the stability of the OCCALB-PEI was significantly better than that of OCCALB during inactivation in mixtures of aqueous buffer and organic cosolvents. SDS-PAGE analysis of the inactivated biocatalyst showed the OCCALB released some enzyme to the medium during inactivation, and this was partially prevented by coating with PEI. This effect was obtained without preventing the possibility of re-use of the support by incubation in 2% ionic detergents. That way, this modified CALB not only has a strong anion exchange nature, keeping the activity, but also improves its stability under diverse reaction conditions without losing the reversibility of the immobilization.
Keywords: reversible immobilization, interfacial adsorption, PEI modification, enzyme stabilization, enzyme physical intermolecular crosslinking
Type of the paper: Commentary
Title: Solid-state metalloproteins—An alternative to immobilisation
Author: Trevor D. Rapson
Affiliation: CSIRO, Black Mountain, Canberra, ACT, 2601, Australia
Abstract: Immobilisation is often the key step required to use an enzyme in an industrial processes. Ideally immobilisation will stabilise the enzyme without compromising its functional properties. While great progress has been made in this area, immobilisation continues to remain a bottleneck in the commercialisation process. This commentary outlines an alternative approach to immobilisation which uses a recombinant silk protein into which metal active sites can be incorporated to produce solid‐state materials which mimic metalloprotein function. These solid15 state materials are remarkably stable at a range of temperatures and different solvent conditions. I discuss the genesis of this approach and highlight areas where such solid‐state materials could find application.
Keywords: biocatalysis; industrial biotechnology; silk; biosensors; de novo engineering
Type of the paper: Article
Title: Tailoring the Spacer Arm for Covalent Immobilization of Candida antarctica Lipase B–Thermal Stabilization by Bisepoxide-activated Aminoalkyl Resins in Continuous-flow Reactors
Authors: Emese Abaházi 1, Dávid Lestál 1, Zoltán Boros 2 and László Poppe 1,2,*
Affiliation: 1 Budapest University of Technology and Economics
2 SynBiocat LLC
Abstract: An efficient and easy-to-perform method was developed for immobilization of CaLB on mesoporous aminoalkyl polymer supports by bisepoxide activation. Polyacrylate resins (100–300 µm; ~50 nm pores) with different aminoalkyl functional groups (ethylamine: EA and hexylamine: HA) were modified with bisepoxides differing in the length, rigidity and hydrophobicity of the units linking the two epoxy functions. After immobilization, the different CaLB preparations were evaluated using the lipase-catalyzed kinetic resolution (KR) of racemic 1-phenylethanol (rac-1) in batch mode and in continuous-flow reactor as well. Catalytic activity, enantiomer selectivity, recyclability, the mechanical and long-term stability of CaLB immobilized on the various supports were tested. The most active CaLB preparation (on HA-resin activated with 1,6-hexanediol diglycidyl ether-HDGE) retained 90% of its initial activity after 13 consecutive reaction cycles or after 12 month of storage at 4 °C. The specific rate (rflow), enantiomer selectivity (E) and enantiomeric excess (ee) achievable with the best immobilized CaLB preparations were studied as a function of temperature in kinetic resolution of rac-1 performed in continuous-flow packed-bed bioreactors. The optimum temperature of the most active HA-HDGE CaLB in continuous-flow mode was 60 °C. Although CaLB immobilized on the glycerol diglycidyl ether (GDGE)-activated EA-resin was less active and less selective, a much higher optimum temperature (80 °C) was observed with this form in continuous-flow mode KR of rac-1.
Keywords: Lipase, immobilization, covalent attachment, kinetic resolution, thermal stabilization, continuous-flow reactor.
Type of the paper: Article
Title: Construction of an Immobilized Thermophilic Esterase on Epoxy Support for Poly(ε-caprolactone) Synthesis
Authors: Hui Ren 1, Zhen Xing 2, Jiebing Yang 2, Wei Jiang 3, Gang Zhang 3,*, Jun Tang 3,*,
Quanshun Li 2,*
Affiliation: 1 Department of Colorectal Surgery, the Second Hospital of Jilin University, Changchun 130041, China
2 Key Laboratory for Molecular Enzymology and Engineering of Ministry of Education, School of Life Sciences, Jilin University, Changchun 130012, China
3 Department of Polymer Science, College of Chemistry, Jilin University, Changchun 130012, China
E-Mails: firstname.lastname@example.org (Q.L.); email@example.com (J.T.); firstname.lastname@example.org (G.Z.)
Abstract: Developing an efficient immobilized enzyme is of great significance for improving the operational stability of enzymes in poly(ε-caprolactone) synthesis. In this paper, a thermophilic esterase AFEST from the archaeon Archaeoglobus fulgidus was successfully immobilized on the epoxy support Sepabeads EC-EP via covalent attachment, and the immobilized enzyme was then employed as a biocatalyst for poly(ε-caprolactone) synthesis. The enzyme loading on support was measured to be 72 mg/g support after the immobilization. Through the optimization of reaction conditions (enzyme concentration, temperature, reaction time and medium), poly(ε-caprolactone) was obtained with 100% monomer conversion and low number-average molecular weight (<1300 g/mol). Further, the immobilized enzyme exhibited excellent reusability, with monomer conversion values exceeding 75% during 15 batch reactions.
Keywords: thermophilic esterase; immobilization; epoxy support; ring-opening polymerization; poly(ε-caprolactone)
Type of the paper: Article
Title: Catalytic Oxidation of Phenolic Wastewater by Using Horseradish Peroxidase Immobilized on Graphene Oxide/Fe3O4
Authors: Qing Chang, Jia Huang, Yaobin Ding and Heqing Tang*
Affiliation: College of Resources and Environmental Science, South Central University for Nationalities, South-Central University for Nationalities, Wuhan 430074, China
Abstract: Graphene oxide/Fe3O4 (GO/Fe3O4) nanoparticles were synthesized by an ultrasonic-assisted reverse co-precipitation method, and then horseradish peroxidase (HRP) was covalently immobilized onto GO/Fe3O4 with 1-ethyl-3-(3-dimethyaminopropyl)carbodiimide (EDC) as a cross-linking agent. In order to enhance the phenol removal efficiency and prevent the inactivation of the enzyme, the polyethylene glycol with highly hydrophilicity was added in this reaction, because the adsorption capacity for the polymer by degradation was stronger than the HRP. The results showed that the immobilized enzyme removed over 95% of phenol from aqueous solution. The catalytic condition was extensively optimized among the range of pH, mass ratio of PEG/phenol as well as initial concentration of immobilized enzyme and H2O2. The HRP immobilized on GO/Fe3O4 composite could be easily separated under a magnetic field from the reaction solution and reused.
Keywords: graphene oxide; magnetite; phenolic wastewater; immobilized enzyme; catalytic oxidation