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Special Issue "Enzyme Immobilization and Its Applications"

A special issue of Molecules (ISSN 1420-3049). This special issue belongs to the section "Chemical Biology".

Deadline for manuscript submissions: 30 June 2019

Special Issue Editor

Guest Editor
Prof. Dr. Roberto Fernandez-Lafuente

Institute of catalysis and petrochemsitry-CSIC, Campus UAM-CSIC, C/ Marie Curie 2, Cantoblanco, 28049 Madrid, Spain
Website | E-Mail
Fax: +34 91 585 4760
Interests: enzyme immobilization; stabilization and purification; biocatalyst and biosensors design; bioprocesses optimization

Special Issue Information

Dear Colleagues,

After the great successes of the 2014 and 2016 Special Issues on “Enzyme Immobilization”, this year we will launch a new edition with a small modification, “"Enzyme Immobilization and Its Applications". Thus, together to special emphasis on the development of new enzyme immobilization supports and/or protocols and the use of genetic, the development of new techniques for immobilized enzyme characterization, chemical or physical modification to enhance the enzyme performance after immobilization, this year we widen the focus to the use of immobilized enzymes where immobilization may play a key role. Special interest will be on enzyme coimmobilization and cascade reaction, as well as the use of nanomaterials to modify large or insoluble substrates. Both review and research papers will be welcome. 

Prof. Dr. Roberto Fernandez-Lafuente
Guest Editor

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.

Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. Molecules is an international peer-reviewed open access semimonthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for publication in this open access journal is 1800 CHF (Swiss Francs). Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Keywords

  • enzyme stabilization by immobilization
  • modulation of enzyme properties by immobilization
  • multipoint covalent attachment
  • multisubunit immobilization
  • immobilized enzyme characterization
  • cascade reactions
  • nanoparticles
  • enzymes coimmobilization

Related Special Issues

Published Papers (8 papers)

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Research

Open AccessArticle
Novel Carbon/PEDOT/PSS-Based Screen-Printed Biosensors for Acetylcholine Neurotransmitter and Acetylcholinesterase Detection in Human Serum
Molecules 2019, 24(8), 1539; https://doi.org/10.3390/molecules24081539
Received: 28 March 2019 / Revised: 15 April 2019 / Accepted: 16 April 2019 / Published: 18 April 2019
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Abstract
New reliable and robust potentiometric ion-selective electrodes were fabricated using poly(3,4-ethylenedioxythiophene)/poly(styrenesulfonate) (PEDOT/PSS) as the solid contact between the sensing membrane and electrical substrate for an acetylcholine (ACh) bioassay. A film of PEDOT/PSS was deposited on a solid carbon screen-printed platform made from ceramic [...] Read more.
New reliable and robust potentiometric ion-selective electrodes were fabricated using poly(3,4-ethylenedioxythiophene)/poly(styrenesulfonate) (PEDOT/PSS) as the solid contact between the sensing membrane and electrical substrate for an acetylcholine (ACh) bioassay. A film of PEDOT/PSS was deposited on a solid carbon screen-printed platform made from ceramic substrate. The selective materials used in the ion-selective electrode (ISE) sensor membrane were acetylcholinium tetraphenylborate (ACh/TPB/PEDOT/PSS-ISE) (sensor I) and triacetyl-β-cyclodextrin (β-CD/PEDOT/PSS-ISE) (sensor II). The sensors revealed clear enhanced Nernstian response with a cationic slope 56.4 ± 0.6 and 55.3 ± 1.1 mV/decade toward (ACh+) ions over the dynamic linear range 1.0 × 10−6–1 × 10−3 and 2.0 × 10−6–1.0 × 10−3 M at pH 5 with limits of detection 2.0 × 10−7 and 3.2 × 10−7 M for sensors I and II, respectively. The selectivity behavior of both sensors was also tested and the sensors showed a significant high selectivity toward ACh+ over different common organic and inorganic cations. The stability of the potential response for the solid-contact (SC)/ISEs was evaluated using a chronopotentiometric method and compared with that of electrodes prepared without adding the solid-contact material (PEDOT/PSS). Enhanced accuracy, excellent repeatability, good reproducibility, potential stability, and high selectivity and sensitivity were introduced by these cost-effective sensors. The sensors were also used to measure the activity of acetylcholinesterase (AChE). A linear plot between the initial rate of the hydrolysis of ACh+ substrate and enzyme activity held 5.0 × 10−3–5.2 IU L−1 of AChE enzyme. Application to acetylcholine determination in human serum was done and the results were compared with the standard colorimetric method. Full article
(This article belongs to the Special Issue Enzyme Immobilization and Its Applications)
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Figure 1

Open AccessArticle
A Highly Efficient Indirect P. pastoris Surface Display Method Based on the CL7/Im7 Ultra-High-Affinity System
Molecules 2019, 24(8), 1483; https://doi.org/10.3390/molecules24081483
Received: 27 February 2019 / Revised: 3 April 2019 / Accepted: 10 April 2019 / Published: 15 April 2019
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Abstract
Cell surface display systems for immobilization of peptides and proteins on the surface of cells have various applications, such as vaccine generation, protein engineering, bio-conversion and bio-adsorption. Though plenty of methods have been established in terms of traditional yeast surface display systems, the [...] Read more.
Cell surface display systems for immobilization of peptides and proteins on the surface of cells have various applications, such as vaccine generation, protein engineering, bio-conversion and bio-adsorption. Though plenty of methods have been established in terms of traditional yeast surface display systems, the development of a universal display method with high efficiency remains a challenge. Here we report an indirect yeast surface display method by anchoring Im7 proteins on the surface of P. pastoris, achieving highly efficient display of target proteins, including fluorescence proteins (sfGFP and mCherry) or enzymes (human Arginase I), with a CL7 fusion tag through the ultra-high-affinity interaction between Im7 and CL7. This indirect P. pastoris surface display approach is highly efficient and provides a robust platform for displaying biomolecules. Full article
(This article belongs to the Special Issue Enzyme Immobilization and Its Applications)
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Figure 1

Open AccessArticle
Degradation of Proteins and Starch by Combined Immobilization of Protease, α-Amylase and β-Galactosidase on a Single Electrospun Nanofibrous Membrane
Molecules 2019, 24(3), 508; https://doi.org/10.3390/molecules24030508
Received: 13 December 2018 / Revised: 19 January 2019 / Accepted: 21 January 2019 / Published: 31 January 2019
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Abstract
Two commercially available enzymes, Dextrozyme (α-amylase) and Esperase (protease), were covalently immobilized on non-woven electrospun poly(styrene-co-maleic anhydride) nanofiber mats with partial retention of their catalytic activity. Immobilization was achieved for the enzymes on their own as well as in different combinations [...] Read more.
Two commercially available enzymes, Dextrozyme (α-amylase) and Esperase (protease), were covalently immobilized on non-woven electrospun poly(styrene-co-maleic anhydride) nanofiber mats with partial retention of their catalytic activity. Immobilization was achieved for the enzymes on their own as well as in different combinations with an additional enzyme, β-galactosidase, on the same non-woven nanofiber mat. This experiment yielded a universal method for immobilizing different combinations of enzymes with nanofibrous mats containing maleic anhydride (MAnh) residues in the polymer backbone. Full article
(This article belongs to the Special Issue Enzyme Immobilization and Its Applications)
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Open AccessArticle
CALB Immobilized onto Magnetic Nanoparticles for Efficient Kinetic Resolution of Racemic Secondary Alcohols: Long-Term Stability and Reusability
Molecules 2019, 24(3), 490; https://doi.org/10.3390/molecules24030490
Received: 25 December 2018 / Revised: 17 January 2019 / Accepted: 24 January 2019 / Published: 30 January 2019
PDF Full-text (6246 KB) | HTML Full-text | XML Full-text | Supplementary Files
Abstract
In this study, an immobilization strategy for magnetic cross-linking enzyme aggregates of lipase B from Candida antarctica (CALB) was developed and investigated. Magnetic particles were prepared by conventional co-precipitation. The magnetic nanoparticles were modified with 3-aminopropyltriethoxysilane (APTES) to obtain surface amino-functionalized magnetic nanoparticles [...] Read more.
In this study, an immobilization strategy for magnetic cross-linking enzyme aggregates of lipase B from Candida antarctica (CALB) was developed and investigated. Magnetic particles were prepared by conventional co-precipitation. The magnetic nanoparticles were modified with 3-aminopropyltriethoxysilane (APTES) to obtain surface amino-functionalized magnetic nanoparticles (APTES–Fe3O4) as immobilization materials. Glutaraldehyde was used as a crosslinker to covalently bind CALB to APTES–Fe3O4. The optimal conditions of immobilization of lipase and resolution of racemic 1-phenylethanol were investigated. Under optimal conditions, esters could be obtained with conversion of 50%, enantiomeric excess of product (eep) > 99%, enantiomeric excess of substrate (ees) > 99%, and enantiomeric ratio (E) > 1000. The magnetic CALB CLEAs were successfully used for enzymatic kinetic resolution of fifteen secondary alcohols. Compared with Novozym 435, the magnetic CALB CLEAs exhibited a better enantioselectivity for most substrates. The conversion was still greater than 49% after the magnetic CALB CLEAs had been reused 10 times in a 48 h reaction cycle; both ees and eep were close to 99%. Furthermore, there was little decrease in catalytic activity and enantioselectivity after being stored at −20 °C for 90 days. Full article
(This article belongs to the Special Issue Enzyme Immobilization and Its Applications)
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Open AccessArticle
Preparation of Immobilized Lipase Based on Hollow Mesoporous Silica Spheres and Its Application in Ester Synthesis
Molecules 2019, 24(3), 395; https://doi.org/10.3390/molecules24030395
Received: 28 December 2018 / Revised: 14 January 2019 / Accepted: 17 January 2019 / Published: 22 January 2019
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Abstract
In this study, Candida rugosa lipase (CRL) was immobilized into modified hollow mesoporous silica (HMSS) materials with different hydrophobicity. Among propyl-(C3), phenyl-(C6), octyl-(C8), and octadecyl-(C18) modified HMSS as well as native HMSS, taking advantage of [...] Read more.
In this study, Candida rugosa lipase (CRL) was immobilized into modified hollow mesoporous silica (HMSS) materials with different hydrophobicity. Among propyl-(C3), phenyl-(C6), octyl-(C8), and octadecyl-(C18) modified HMSS as well as native HMSS, taking advantage of more hydrophobic microenvironment, the HMSS-C18-CRL showed exceptional performance in enzymatic esterification reaction. Using the novel HMSS-C18 with immobilized CRL (HMSS-C18-CRL), we investigated the esterification of phytosterols with polyunsaturated fat acid (PUFA) in a solvent-free system for the production of phytosterols esters. Response surface methodology (RSM) was applied to model and optimize the reaction conditions, namely, the enzyme load (5–25%), reaction time (10–110 min), molar ratio of α-linolenic acid (ALA)/phytosterols (1:1–7:1) and represented by the letters E, T, and M respectively. Best-fitting models were successfully established by multiple regressions with backward elimination. The optimum production was achieved at 70 min for reaction time, 20% based on the weight of substrate for enzyme loading, and 5.6:1 for ALA/phytosterols molar ratio. Under optimized conditions, a conversion of about 90 ± 2% was achieved. These results indicated that HMSS-C18-CRL demonstrates to be a promising catalyst and can be potentially applied in the functional lipid production. Full article
(This article belongs to the Special Issue Enzyme Immobilization and Its Applications)
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Graphical abstract

Open AccessArticle
Further Stabilization of Alcalase Immobilized on Glyoxyl Supports: Amination Plus Modification with Glutaraldehyde
Molecules 2018, 23(12), 3188; https://doi.org/10.3390/molecules23123188
Received: 21 November 2018 / Revised: 29 November 2018 / Accepted: 30 November 2018 / Published: 3 December 2018
Cited by 2 | PDF Full-text (1870 KB) | HTML Full-text | XML Full-text
Abstract
Alcalase was immobilized on glyoxyl 4% CL agarose beads. This permitted to have Alcalase preparations with 50% activity retention versus Boc-l-alanine 4-nitrophenyl ester. However, the recovered activity versus casein was under 20% at 50 °C, as it may be expected from [...] Read more.
Alcalase was immobilized on glyoxyl 4% CL agarose beads. This permitted to have Alcalase preparations with 50% activity retention versus Boc-l-alanine 4-nitrophenyl ester. However, the recovered activity versus casein was under 20% at 50 °C, as it may be expected from the most likely area of the protein involved in the immobilization. The situation was different at 60 °C, where the activities of immobilized and free enzyme became similar. The chemical amination of the immobilized enzyme or the treatment of the enzyme with glutaraldehyde did not produce any significant stabilization (a factor of 2) with high costs in terms of activity. However, the modification with glutaraldehyde of the previously aminated enzyme permitted to give a jump in Alcalase stability (e.g., with most than 80% of enzyme activity retention for the modified enzyme and less than 30% for the just immobilized enzyme in stress inactivation at pH 7 or 9). This preparation could be used in the hydrolysis of casein at pH 9 even at 67 °C, retaining around 50% of the activity after 5 hydrolytic cycles when the just immobilized preparation was almost inactive after 3 cycles. The modified enzyme can be reused in hydrolysis of casein at 45 °C and pH 9 for 6 cycles (6 h) without any decrease in enzyme activity. Full article
(This article belongs to the Special Issue Enzyme Immobilization and Its Applications)
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Graphical abstract

Open AccessArticle
Production of Omegas-6 and 9 from the Hydrolysis of Açaí and Buriti Oils by Lipase Immobilized on a Hydrophobic Support
Molecules 2018, 23(11), 3015; https://doi.org/10.3390/molecules23113015
Received: 8 October 2018 / Revised: 16 November 2018 / Accepted: 16 November 2018 / Published: 18 November 2018
Cited by 1 | PDF Full-text (6516 KB) | HTML Full-text | XML Full-text | Supplementary Files
Abstract
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 [...] Read more.
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. Full article
(This article belongs to the Special Issue Enzyme Immobilization and Its Applications)
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Graphical abstract

Open AccessFeature PaperArticle
Evaluation of Strategies to Produce Highly Porous Cross-Linked Aggregates of Porcine Pancreas Lipase with Magnetic Properties
Molecules 2018, 23(11), 2993; https://doi.org/10.3390/molecules23112993
Received: 7 November 2018 / Revised: 13 November 2018 / Accepted: 14 November 2018 / Published: 16 November 2018
Cited by 1 | PDF Full-text (5837 KB) | HTML Full-text | XML Full-text | Supplementary Files
Abstract
The preparation of highly porous magnetic crosslinked aggregates (pm-CLEA) of porcine pancreas lipase (PPL) is reported. Some strategies to improve the volumetric activity of the immobilized biocatalyst were evaluated, such as treatment of PPL with enzyme surface-modifying agents (polyethyleneimine or dodecyl aldehyde), co-aggregation [...] Read more.
The preparation of highly porous magnetic crosslinked aggregates (pm-CLEA) of porcine pancreas lipase (PPL) is reported. Some strategies to improve the volumetric activity of the immobilized biocatalyst were evaluated, such as treatment of PPL with enzyme surface-modifying agents (polyethyleneimine or dodecyl aldehyde), co-aggregation with protein co-feeders (bovine serum albumin and/or soy protein), use of silica magnetic nanoparticles functionalized with amino groups (SMNPs) as separation aid, and starch as pore-making agent. The combination of enzyme surface modification with dodecyl aldehyde, co-aggregation with SMNPs and soy protein, in the presence of 0.8% starch (followed by hydrolysis of the starch with α-amylase), yielded CLEAs expressing high activity (immobilization yield around 100% and recovered activity around 80%), high effectiveness factor (approximately 65% of the equivalent free enzyme activity) and high stability at 40 °C and pH 8.0, i.e., PPL CLEAs co-aggregated with SMNPs/bovine serum albumin or SMNPs/soy protein retained 80% and 50% activity after 10 h incubation, respectively, while free PPL was fully inactivated after 2 h. Besides, highly porous magnetic CLEAs co-aggregated with soy protein and magnetic nanoparticles (pm-SP-CLEAs) showed good performance and reusability in the hydrolysis of tributyrin for five 4h-batches. Full article
(This article belongs to the Special Issue Enzyme Immobilization and Its Applications)
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Graphical abstract

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.

Title: Methods to Measure and Evaluate Biocatalyst Performance for Industrial Implementation
Authors: Mafalda Dias Gomes and John M Woodley
Affiliation: Department of Chemical and Biochemical Engineering, Technical University of Denmark (DTU), DK-2800 Kgs Lyngby, Denmark
Emails: ;
Abstract: Enzyme stability is one of the most important parameters to assess the potential of a given biocatalyst for industrial application. In general, the lower the value of the product, the more important is the stability. However, this is not sufficient, because in economic terms it is the biocatalyst yield (amount of product produced per amount of biocatalyst added) which is important. In the laboratory the total turnover is often used to describe this, but this assumes full knowledge of the enzyme. In reality in an industrial setting enzymes are often used as impure, containing other proteins and impurities. For a direct comparison of alternative biocatalysts, therefore great care should be taken concerning the format of the enzyme. This concern is also relevant when considering enzyme immobilization, one of the more established ways of increasing stability. One of the features of biocatalyst yield is that it is the product of both reaction rate and stability. Hence this is a further complication which must be considered prior to implementation. In this paper methods to evaluate alternative biocatalysts for application in chemical synthesis and production will be discussed, along with some of the important research topics which need to be addressed in the future.

Title: Control of protein distribution across solid porous carriers by engineering polypeptide tags in fluorescent proteins
Author: Fernando Lopez-Gallego
Affiliation 1: Heterogeneous biocatalysis laboratory. Instituto de Síntesis Química y Catálisis Homogenea (ISQCH-CSIC). University of Zaragoza. Facultad de Ciencias, Edificio  C/Pedro Cerbuna, n 12, 50009, Zaragoza (Spain)
Affiliation 2: ARAID foundation, Zaragoza (Spain)
Email: [email protected]

Title: Co-encpasulation of beta-galactosidase and glucose isomerasa for the one-pot synthesis of lacto-fructose syrup
Authors: Lorena WIlson and Andrés Illanes
Affiliation: School of Biochemical Engineering, Pontificia Universidad Católica de Valparaíso, Avenida Brasil 2085, Valparaíso, Chile
Email: [email protected]

Title: Fishing and hunting - Biocatalysts by smart immobilization from fermentation media
Authors: Evelin Sánta-Bell, Flóra Nagy, Gábor Hornyánszky, Csaba Paizs, Diána Balogh-Weiser*, László Poppe*
Affiliation: Department of Organic Chemistry and Technology, Budapest University of Technology and Economics, Budapest, Hungary
Email: [email protected]
Abstract: The ever increasing importance of biocatalytic processes require production of various types of biocatalysts as enzymes or even as whole-cells. The road from fermentation providing these biocatalysts to the final application involves many technological difficulties with serious cost commitments. Among downstream issues, isolation and purification of enzymes or conservation and stabilization of whole-cells have significant impacts. Rational development of smart enzyme and cell immobilization methods can contribute to the radical simplification or even elimination of classic purification and preserving techniques such as chromatographic purification or lyophilization. In addition to a brief overview of the existing methodologies, this article will show possible solutions for creating efficient biocatalysts by “smart” immobilization. Creation of nanostructured materials with multifunctional surface properties such as tag recognition and groups allowing covalent binding enables selective immobilization of a recombinant enzyme with affinity tag in one process. Encapsulation of cell lysates or whole cells – by sol-gel technique or by electrostatic nanofiber fabrication – within nanoporous networks in presence of proper nanomaterials (nanospheres, nanoclays etc.) as supports provides promising opportunity for direct immobilization and stabilization. Due to the simplified isolation these immobilization techniques can significantly improve the biocatalyst production for single enzyme or multienzyme biotransformations.

Title: The microenvironment of immobilized enzymes: methods of characterization and role in determining enzyme performance
Authors: 
Juan M. Bolivar and Bernd Nidetzky
Affiliation:
Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Graz, Austria
Email: 
[email protected]

Title: Preparation of Immobilized Lipase Based on Hollow Mesoporous Silica Beads and the Application on Enzymatic Hydrolysis and Esterification
Author:
Mingming Zheng
Affiliation:
Oil Crops Research Institute, Chinese Academy of Agricultural Sciences, Hubei Key Laboratory of Lipid Chemistry and Nutrition, Wuhan 430062, China
Email: 
[email protected]

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