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Enzyme Immobilization and Its Applications
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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: closed (31 July 2019) | Viewed by 74467

Special Issue Editor

Prof. Dr. Roberto Fernandez-Lafuente

grade E-Mail Website
Guest Editor
Institute of Catalysis and Petrochemsitry-CSIC, Campus UAM-CSIC, C/ Marie Curie 2, Cantoblanco, 28049 Madrid, Spain
Interests: biocatalysis; enzyme immobilization; enzyme stabilization; enzyme chemical modification; bioprocess optimization
Special Issues, Collections and Topics in MDPI journals

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

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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 (18 papers)

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Editorial

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4 pages, 186 KiB  
Editorial
Editorial for Special Issue: Enzyme Immobilization and Its Applications
by Roberto Fernandez-Lafuente
Molecules 2019, 24(24), 4619; https://doi.org/10.3390/molecules24244619 - 17 Dec 2019
Cited by 13 | Viewed by 2524
Abstract
Modern chemistry demands cleaner processes, for which more efficient catalysts are required [...] Full article
(This article belongs to the Special Issue Enzyme Immobilization and Its Applications)

Research

Jump to: Editorial, Review, Other

16 pages, 2143 KiB  
Article
Improving the Yields and Reaction Rate in the Ethanolysis of Soybean Oil by Using Mixtures of Lipase CLEAs
by Margarita Díaz Ramos, Letícia Passos Miranda, Roberto Fernandez-Lafuente, William Kopp and Paulo Waldir Tardioli
Molecules 2019, 24(23), 4392; https://doi.org/10.3390/molecules24234392 - 01 Dec 2019
Cited by 31 | Viewed by 3141
Abstract
Due to the heterogeneity of oils, the use of mixtures of lipases with different activity for a large number of glycerol-linked carboxylic acids that compose the substrate has been proposed as a better alternative than the use of one specific lipase preparation in [...] Read more.
Due to the heterogeneity of oils, the use of mixtures of lipases with different activity for a large number of glycerol-linked carboxylic acids that compose the substrate has been proposed as a better alternative than the use of one specific lipase preparation in the enzymatic synthesis of biodiesel. In this work, mixtures of lipases from different sources were evaluated in their soluble form in the ethanolysis of soybean oil. A mixture of lipases (50% of each lipase, in activity basis) from porcine pancreas (PPL) and Thermomyces lanuginosus lipase (TLL) gave the highest fatty acid ethyl ester (FAEE) yield (around 20 wt.%), while the individual lipases gave FAEE yields 100 and 5 times lower, respectively. These lipases were immobilized individually by the cross-linked enzyme aggregates (CLEAs) technique, yielding biocatalysts with 89 and 119% of expressed activity, respectively. A mixture of these CLEAs (also 50% of each lipase, in activity basis) gave 90.4 wt.% FAEE yield, while using separately CLEAs of PPL and TLL, the FAEE yields were 84.7 and 75.6 wt.%, respectively, under the same reaction conditions. The mixture of CLEAs could be reused (five cycles of 6 h) in the ethanolysis of soybean oil in a vortex flow-type reactor yielding an FAEE yield higher than 80% of that of the first batch. Full article
(This article belongs to the Special Issue Enzyme Immobilization and Its Applications)
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17 pages, 5010 KiB  
Article
Immobilization of Naringinase from Penicillium decumbens on Chitosan Microspheres for Debittering Grapefruit Juice
by Joanna Bodakowska-Boczniewicz and Zbigniew Garncarek
Molecules 2019, 24(23), 4234; https://doi.org/10.3390/molecules24234234 - 21 Nov 2019
Cited by 41 | Viewed by 3867
Abstract
Naringinase is an enzyme complex which exhibits α-l-rhamnosidase and β-d-glucosidase activity. This enzymatic complex catalyzes the hydrolysis of naringin (4′,5,7-trihydroxy flavanone 7-rhamnoglucoside), the main bittering component in grapefruit. Reduction of the level of this substance during the processing of [...] Read more.
Naringinase is an enzyme complex which exhibits α-l-rhamnosidase and β-d-glucosidase activity. This enzymatic complex catalyzes the hydrolysis of naringin (4′,5,7-trihydroxy flavanone 7-rhamnoglucoside), the main bittering component in grapefruit. Reduction of the level of this substance during the processing of juice has been the focus of many studies. The aim of the study was the immobilization of naringinase on chitosan microspheres activated with glutaraldehyde and, finally, the use of such immobilized enzyme for debittering grapefruit juice. The effect of naringinase concentration and characterization of the immobilized enzyme compared to the soluble enzyme were investigated. The maximum activity was observed at optimum pH 4.0 for both free and immobilized naringinase. However, the optimum temperature was shifted from 70 to 40 °C upon immobilization. The KM value of the immobilized naringinase was higher than that of soluble naringinase. The immobilization did not change the thermal stability of the enzyme. The immobilized naringinase had good operational stability. This preparation retained 88.1 ± 2.8% of its initial activity after ten runs of naringin hydrolysis from fresh grapefruit juice. The results indicate that naringinase immobilized on chitosan has potential applicability for debittering and improving the sensory properties of grapefruit juices. Full article
(This article belongs to the Special Issue Enzyme Immobilization and Its Applications)
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23 pages, 3612 KiB  
Article
“Fishing and Hunting”—Selective Immobilization of a Recombinant Phenylalanine Ammonia-Lyase from Fermentation Media
by Evelin Sánta-Bell, Zsófia Molnár, Andrea Varga, Flóra Nagy, Gábor Hornyánszky, Csaba Paizs, Diána Balogh-Weiser and László Poppe
Molecules 2019, 24(22), 4146; https://doi.org/10.3390/molecules24224146 - 15 Nov 2019
Cited by 14 | Viewed by 4066
Abstract
This article overviews the numerous immobilization methods available for various biocatalysts such as whole-cells, cell fragments, lysates or enzymes which do not require preliminary enzyme purification and introduces an advanced approach avoiding the costly and time consuming downstream processes required by immobilization of [...] Read more.
This article overviews the numerous immobilization methods available for various biocatalysts such as whole-cells, cell fragments, lysates or enzymes which do not require preliminary enzyme purification and introduces an advanced approach avoiding the costly and time consuming downstream processes required by immobilization of purified enzyme-based biocatalysts (such as enzyme purification by chromatographic methods and dialysis). Our approach is based on silica shell coated magnetic nanoparticles as solid carriers decorated with mixed functions having either coordinative binding ability (a metal ion complexed by a chelator anchored to the surface) or covalent bond-forming ability (an epoxide attached to the surface via a proper linker) enabling a single operation enrichment and immobilization of a recombinant phenylalanine ammonia-lyase from parsley fused to a polyhistidine affinity tag. Full article
(This article belongs to the Special Issue Enzyme Immobilization and Its Applications)
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17 pages, 3876 KiB  
Article
Co-Immobilization of Tri-Enzymes for the Conversion of Hydroxymethylfurfural to 2,5-Diformylfuran
by Zhuofu Wu, Linjuan Shi, Xiaoxiao Yu, Sitong Zhang and Guang Chen
Molecules 2019, 24(20), 3648; https://doi.org/10.3390/molecules24203648 - 10 Oct 2019
Cited by 23 | Viewed by 3302
Abstract
Acting as a “green” manufacturing route, the enzyme toolbox made up of galactose oxidase, catalase, and horseradish peroxidase can achieve a satisfactory yield of 2,5-diformylfuran derived from 30 mM hydroxymethylfurfural. However, as the concentration of hydroxymethylfurfural increases, the substrate causes oxidative damage to [...] Read more.
Acting as a “green” manufacturing route, the enzyme toolbox made up of galactose oxidase, catalase, and horseradish peroxidase can achieve a satisfactory yield of 2,5-diformylfuran derived from 30 mM hydroxymethylfurfural. However, as the concentration of hydroxymethylfurfural increases, the substrate causes oxidative damage to the activity of the tri-enzyme system, and the accumulated hydrogen peroxide produced by galactose oxidase causes tri-enzyme inactivation. The cost of tri-enzymes is also very high. These problems prevent the utilization of this enzyme toolbox in practice. To address this, galactose oxidase, catalase, and horseradish peroxidase were co-immobilized into Cu3(PO4)2 nanoflowers in this study. The resulting co-immobilized tri-enzymes possessed better tolerance towards the oxidative damage caused by hydroxymethylfurfural at high concentrations, as compared to free tri-enzymes. Moreover, the 2,5-diformylfuran yield of co-immobilized tri-enzymes (95.7 ± 2.7%) was 1.06 times higher than that of separately immobilized enzymes (90.4 ± 1.9%). This result could be attributed to the boosted protective effect provided by catalase to the activity of galactose oxidase, owing to the physical proximity between them on the same support. After 30 recycles, co-immobilized tri-enzymes still achieves 86% of the initial yield. Moreover, co-immobilized tri-enzymes show enhanced thermal stability compared with free tri-enzymes. This work paves the way for the production of 2,5-diformylfuran from hydroxymethylfurfural via co-immobilized tri-enzymes. Full article
(This article belongs to the Special Issue Enzyme Immobilization and Its Applications)
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10 pages, 1594 KiB  
Article
Insight into the Modification of Phosphatidylcholine with n-3 Polyunsaturated Fatty Acids-Rich Ethyl Esters by Immobilized MAS1 Lipase
by Xiumei Wang, Xiaoli Qin, Xiuting Li, Zexin Zhao, Bo Yang and Yonghua Wang
Molecules 2019, 24(19), 3528; https://doi.org/10.3390/molecules24193528 - 29 Sep 2019
Cited by 6 | Viewed by 2743
Abstract
This study reported the modification of phosphatidylcholine (PC) with n-3 polyunsaturated fatty acids (PUFA)-rich ethyl esters (EE) by immobilized MAS1 lipase-catalyzed transesterification in the solvent-free system. Effects of n-3 PUFA-rich EE/PC mass ratio, enzyme loading, reaction temperature, and water dosage on the incorporation [...] Read more.
This study reported the modification of phosphatidylcholine (PC) with n-3 polyunsaturated fatty acids (PUFA)-rich ethyl esters (EE) by immobilized MAS1 lipase-catalyzed transesterification in the solvent-free system. Effects of n-3 PUFA-rich EE/PC mass ratio, enzyme loading, reaction temperature, and water dosage on the incorporation of n-3 PUFA into PC were investigated, respectively. The results indicate that the maximum incorporation of n-3 PUFA into PC reached 33.5% (24 h) under the following conditions: n-3 PUFA-rich EE/PC mass ratio of 6:1, enzyme loading of 20%, reaction temperature of 55 °C, and water dosage of 1.0%. After 72 h of reaction, the incorporation of n-3 PUFA into PC was 43.55% and the composition of the reaction mixture was analyzed by 31P nuclear magnetic resonance (NMR). The results show that the reaction product consisted of 32.68% PC, 28.76% 1-diacyl-sn-glycero-3-lysophosphatidylcholine (sn-1 LPC), 4.90% 2-diacyl-sn-glycero-3-lysophosphatidylcholine (sn-2 LPC), and 33.60% sn-glycero-3-phosphatidylcholine (GPC). This study offers insight into the phospholipase activity of immobilized MAS1 lipase and suggests the extended applications of immobilized MAS1 lipase in the modification of phospholipids for industrial purpose. Full article
(This article belongs to the Special Issue Enzyme Immobilization and Its Applications)
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13 pages, 1854 KiB  
Article
Synthesis with Immobilized Lipases and Downstream Processing of Ascorbyl Palmitate
by Carolina Tufiño, Claudia Bernal, Carminna Ottone, Oscar Romero, Andrés Illanes and Lorena Wilson
Molecules 2019, 24(18), 3227; https://doi.org/10.3390/molecules24183227 - 05 Sep 2019
Cited by 23 | Viewed by 4317
Abstract
Ascorbyl palmitate is a fatty acid ester endowed with antioxidant properties, used as a food additive and cosmetic ingredient, which is presently produced by chemical synthesis. Ascorbyl palmitate was synthesized from ascorbic acid and palmitic acid with a Pseudomonas stutzeri lipase immobilized on [...] Read more.
Ascorbyl palmitate is a fatty acid ester endowed with antioxidant properties, used as a food additive and cosmetic ingredient, which is presently produced by chemical synthesis. Ascorbyl palmitate was synthesized from ascorbic acid and palmitic acid with a Pseudomonas stutzeri lipase immobilized on octyl silica, and also with the commercial immobilized lipase Novozym 435. The latter was selected for optimizing the reaction conditions because of its high reactivity and stability in the solvent 2-methyl-2-butanol used as reaction medium. The reaction of the synthesis was studied considering temperature and molar ratio of substrates as variables and synthesis yield as response parameter. The highest yield in the synthesis of ascorbyl palmitate was 81%, obtained at 55 °C and an ascorbic acid to palmitic acid molar ratio of 1:8, both variables having a strong effect on yield. The synthesized ascorbyl palmitate was purified to 94.4%, with a purification yield of 84.2%. The use of generally recognized as safe (GRAS) certified solvents with a polarity suitable for the solubilization of the compounds made the process a viable alternative for the synthesis and downstream processing of ascorbyl palmitate. Full article
(This article belongs to the Special Issue Enzyme Immobilization and Its Applications)
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17 pages, 3710 KiB  
Article
Selective Immobilization of Fluorescent Proteins for the Fabrication of Photoactive Materials
by Ana I. Benítez-Mateos, Ehsan Mehravar, Susana Velasco-Lozano, Radmila Tomovska, Luca Salassa and Fernando López-Gallego
Molecules 2019, 24(15), 2775; https://doi.org/10.3390/molecules24152775 - 30 Jul 2019
Cited by 5 | Viewed by 3211
Abstract
The immobilization of fluorescent proteins is a key technology enabling to fabricate a new generation of photoactive materials with potential technological applications. Herein we have exploited superfolder green (sGFP) and red (RFP) fluorescent proteins expressed with different polypeptide tags. We fused these fluorescent [...] Read more.
The immobilization of fluorescent proteins is a key technology enabling to fabricate a new generation of photoactive materials with potential technological applications. Herein we have exploited superfolder green (sGFP) and red (RFP) fluorescent proteins expressed with different polypeptide tags. We fused these fluorescent proteins to His-tags to immobilize them on graphene 3D hydrogels, and Cys-tags to immobilize them on porous microparticles activated with either epoxy or disulfide groups and with Lys-tags to immobilize them on upconverting nanoparticles functionalized with carboxylic groups. Genetically programming sGFP and RFP with Cys-tag and His-tag, respectively, allowed tuning the protein spatial organization either across the porous structure of two microbeads with different functional groups (agarose-based materials activated with metal chelates and epoxy-methacrylate materials) or across the surface of a single microbead functionalized with both metal-chelates and disulfide groups. By using different polypeptide tags, we can control the attachment chemistry but also the localization of the fluorescent proteins across the material surfaces. The resulting photoactive material formed by His-RFP immobilized on graphene hydrogels has been tested as pH indicator to measure pH changes in the alkaline region, although the immobilized fluorescent protein exhibited a narrower dynamic range to measure pH than the soluble fluorescent protein. Likewise, the immobilization of Lys-sGFP on alginate-coated upconverting nanoparticles enabled the infrared excitation of the fluorescent protein to be used as a green light emitter. These novel photoactive biomaterials open new avenues for innovative technological developments towards the fabrication of biosensors and photonic devices. Full article
(This article belongs to the Special Issue Enzyme Immobilization and Its Applications)
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14 pages, 2566 KiB  
Article
Novel Carbon/PEDOT/PSS-Based Screen-Printed Biosensors for Acetylcholine Neurotransmitter and Acetylcholinesterase Detection in Human Serum
by Nashwa H. Ashmawy, Abdulrahman A. Almehizia, Teraze A. Youssef, Abd El-Galil E. Amr, Mohamed A. Al-Omar and Ayman H. Kamel
Molecules 2019, 24(8), 1539; https://doi.org/10.3390/molecules24081539 - 18 Apr 2019
Cited by 32 | Viewed by 3953
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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14 pages, 2845 KiB  
Article
A Highly Efficient Indirect P. pastoris Surface Display Method Based on the CL7/Im7 Ultra-High-Affinity System
by Shuntang Li, Jie Qiao, Siyu Lin, Yi Liu and Lixin Ma
Molecules 2019, 24(8), 1483; https://doi.org/10.3390/molecules24081483 - 15 Apr 2019
Cited by 13 | Viewed by 4130
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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9 pages, 993 KiB  
Article
Degradation of Proteins and Starch by Combined Immobilization of Protease, α-Amylase and β-Galactosidase on a Single Electrospun Nanofibrous Membrane
by William J. Cloete, Stefan Hayward, Pieter Swart and Bert Klumperman
Molecules 2019, 24(3), 508; https://doi.org/10.3390/molecules24030508 - 31 Jan 2019
Cited by 11 | Viewed by 4406
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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17 pages, 6246 KiB  
Article
CALB Immobilized onto Magnetic Nanoparticles for Efficient Kinetic Resolution of Racemic Secondary Alcohols: Long-Term Stability and Reusability
by Xiu Xing, Jun-Qi Jia, Jing-Fan Zhang, Zi-Wen Zhou, Jun Li, Na Wang and Xiao-Qi Yu
Molecules 2019, 24(3), 490; https://doi.org/10.3390/molecules24030490 - 30 Jan 2019
Cited by 29 | Viewed by 4479
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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15 pages, 4475 KiB  
Article
Preparation of Immobilized Lipase Based on Hollow Mesoporous Silica Spheres and Its Application in Ester Synthesis
by Zhe Dong, Meng-Ying Jiang, Jie Shi, Ming-Ming Zheng and Feng-Hong Huang
Molecules 2019, 24(3), 395; https://doi.org/10.3390/molecules24030395 - 22 Jan 2019
Cited by 26 | Viewed by 4017
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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15 pages, 1870 KiB  
Article
Further Stabilization of Alcalase Immobilized on Glyoxyl Supports: Amination Plus Modification with Glutaraldehyde
by Fouzia Hussain, Sara Arana-Peña, Roberto Morellon-Sterling, Oveimar Barbosa, Sabrina Ait Braham, Shagufta Kamal and Roberto Fernandez-Lafuente
Molecules 2018, 23(12), 3188; https://doi.org/10.3390/molecules23123188 - 03 Dec 2018
Cited by 17 | Viewed by 4051
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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18 pages, 6595 KiB  
Article
Production of Omegas-6 and 9 from the Hydrolysis of Açaí and Buriti Oils by Lipase Immobilized on a Hydrophobic Support
by Malena Martínez Pérez, Enrico Cerioni Spiropulos Gonçalves, Jose Carlos Santos Salgado, Mariana de Souza Rocha, Paula Zaghetto de Almeida, Ana Claudia Vici, Juliana da Conceição Infante, Jose Manuel Guisán, Javier Rocha-Martin, Benevides Costa Pessela and Maria de Lourdes Teixeira de Moraes Polizeli
Molecules 2018, 23(11), 3015; https://doi.org/10.3390/molecules23113015 - 18 Nov 2018
Cited by 13 | Viewed by 5409
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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21 pages, 5837 KiB  
Article
Evaluation of Strategies to Produce Highly Porous Cross-Linked Aggregates of Porcine Pancreas Lipase with Magnetic Properties
by José Renato Guimarães, Raquel de Lima Camargo Giordano, Roberto Fernandez-Lafuente and Paulo Waldir Tardioli
Molecules 2018, 23(11), 2993; https://doi.org/10.3390/molecules23112993 - 16 Nov 2018
Cited by 45 | Viewed by 5167
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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Review

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24 pages, 4009 KiB  
Review
The Microenvironment in Immobilized Enzymes: Methods of Characterization and Its Role in Determining Enzyme Performance
by Juan M. Bolivar and Bernd Nidetzky
Molecules 2019, 24(19), 3460; https://doi.org/10.3390/molecules24193460 - 24 Sep 2019
Cited by 48 | Viewed by 5631
Abstract
The liquid milieu in which enzymes operate when they are immobilized in solid materials can be quite different from the milieu in bulk solution. Important differences are in the substrate and product concentration but also in pH and ionic strength. The internal milieu [...] Read more.
The liquid milieu in which enzymes operate when they are immobilized in solid materials can be quite different from the milieu in bulk solution. Important differences are in the substrate and product concentration but also in pH and ionic strength. The internal milieu for immobilized enzymes is affected by the chemical properties of the solid material and by the interplay of reaction and diffusion. Enzyme performance is influenced by the internal milieu in terms of catalytic rate (“activity”) and stability. Elucidation, through direct measurement of differences in the internal as compared to the bulk milieu is, therefore, fundamentally important in the mechanistic characterization of immobilized enzymes. The deepened understanding thus acquired is critical for the rational development of immobilized enzyme preparations with optimized properties. Herein we review approaches by opto-chemical sensing to determine the internal milieu of enzymes immobilized in porous particles. We describe analytical principles applied to immobilized enzymes and focus on the determination of pH and the O2 concentration. We show measurements of pH and [O2] with spatiotemporal resolution, using in operando analysis for immobilized preparations of industrially important enzymes. The effect of concentration gradients between solid particle and liquid bulk on enzyme performance is made evident and quantified. Besides its use in enzyme characterization, the method can be applied to the development of process control strategies. Full article
(This article belongs to the Special Issue Enzyme Immobilization and Its Applications)
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Other

11 pages, 363 KiB  
Concept Paper
Considerations when Measuring Biocatalyst Performance
by Mafalda Dias Gomes and John M. Woodley
Molecules 2019, 24(19), 3573; https://doi.org/10.3390/molecules24193573 - 03 Oct 2019
Cited by 43 | Viewed by 3990
Abstract
As biocatalysis matures, it becomes increasingly important to establish methods with which to measure biocatalyst performance. Such measurements are important to assess immobilization strategies, different operating modes, and reactor configurations, aside from comparing protein engineered variants and benchmarking against economic targets. While conventional [...] Read more.
As biocatalysis matures, it becomes increasingly important to establish methods with which to measure biocatalyst performance. Such measurements are important to assess immobilization strategies, different operating modes, and reactor configurations, aside from comparing protein engineered variants and benchmarking against economic targets. While conventional measurement techniques focus on a single performance metric (such as the total turnover number), here, it is argued that three metrics (achievable product concentration, productivity, and enzyme stability) are required for an accurate assessment of scalability. Full article
(This article belongs to the Special Issue Enzyme Immobilization and Its Applications)
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