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Enzyme Immobilization and Biocatalysis

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

Deadline for manuscript submissions: closed (20 December 2021) | Viewed by 37798

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Prof. Dr. Aniello Costantini

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Department of Chemical Engineering, Materials and Industrial Production DICMaPI, Università degli Studi di Napoli Federico II, Naples, Italy
Interests: nanostructured materials; sol–gel synthesis; enzyme immobilization; catalysis
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Dr. Valeria Califano

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Institute of Sciences and Technologies for Sustainable Energy and Mobility (STEMS), Italian National Research Council CNR, Naples, Italy
Interests: enzyme immobilization; biocatalysis; biofuels; mesoporous silica
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Dear Colleagues,

Enzymes are catalysts with outstanding properties. A great deal of eﬀort has been devoted to enzymatic conversion, since it offers many advantages over chemical catalysis, including sustainability, selectivity towards the products, and milder operational conditions. However, the cost of enzymes is one of the main obstacles to the large-scale commercialization of enzymatic processes. Furthermore, enzymes have several features that limit their use for industrial applications, such as inhibition by high concentrations of substrates and products, low stability, and lack of reusability. Enzyme immobilization often gives the possibility of improving these unsuitable characteristics. The immobilization of biocatalysts on/in a solid matrix can reduce the production cost by increasing efficiency, allowing reuse, and facilitating the separation of the products from the reaction mixture. Besides, enzyme immobilization often results in the stabilization of enzymes, sometimes increasing or modifying their activity. However, although a large number of immobilization protocols have been developed, the design of new protocols that make it possible to improve the enzyme properties is still an exciting goal.

This Special Issue aims to collect a series of articles that can help the progress in the field of enzymatic immobilization for industrial applications.

Prof. Dr. Aniello Costantini
Dr. Valeria Califano
Guest Editors

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Keywords

biocatalysis
enzyme immobilization

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Published Papers (12 papers)

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Editorial

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3 pages, 150 KiB

Open AccessEditorial

Enzyme Immobilization and Biocatalysis

by Valeria Califano and Aniello Costantini

Catalysts 2021, 11(7), 823; https://doi.org/10.3390/catal11070823 - 7 Jul 2021

Cited by 4 | Viewed by 2701

Abstract

Enzymes are catalysts with outstanding properties [...] Full article

(This article belongs to the Special Issue Enzyme Immobilization and Biocatalysis)

Research

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12 pages, 3107 KiB

Open AccessArticle

Enzymatic Production of Ecodiesel by Using a Commercial Lipase CALB, Immobilized by Physical Adsorption on Mesoporous Organosilica Materials

by Carlos Luna, Victoria Gascón-Pérez, Francisco J. López-Tenllado, Felipa M. Bautista, Cristóbal Verdugo-Escamilla, Laura Aguado-Deblas, Juan Calero, Antonio A. Romero, Diego Luna and Rafael Estévez

Catalysts 2021, 11(11), 1350; https://doi.org/10.3390/catal11111350 - 9 Nov 2021

Cited by 8 | Viewed by 2619

Abstract

The synthesis of two biocatalysts based on a commercial Candida antarctica lipase B, CALB enzyme (E), physically immobilized on two silica supports, was carried out. The first support was a periodic mesoporous organosilica (PMO) and the second one was a commercial silica modified with octyl groups (octyl-MS3030). The maximum enzyme load was 122 mg enzyme/g support on PMO and 288 mg enzyme/g support on octyl-MS3030. In addition, the biocatalytic efficiency was corroborated by two reaction tests based on the hydrolysis of p-nitrophenylacetate (p-NPA) and tributyrin (TB). The transesterification of sunflower oil with ethanol was carried out over the biocatalysts synthesized at the following reaction conditions: 6 mL sunflower oil, 1.75 mL EtOH, 30 °C, 25 μL NaOH 10 N and 300 rpm, attaining conversion values over 80% after 3 h of reaction time. According to the results obtained, we can confirm that these biocatalytic systems are viable candidates to develop, optimize and improve a new methodology to achieve the integration of glycerol in different monoacylglycerol molecules together with fatty acid ethyl esters (FAEE) molecules to obtain Ecodiesel. Full article

(This article belongs to the Special Issue Enzyme Immobilization and Biocatalysis)

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10 pages, 1839 KiB

Open AccessArticle

Immobilization of Fusarium solani Cutinase onto Magnetic Genipin-Crosslinked Chitosan Beads

by Zhanyong Wang, Tingting Su and Jingjing Zhao

Catalysts 2021, 11(10), 1158; https://doi.org/10.3390/catal11101158 - 26 Sep 2021

Cited by 6 | Viewed by 2395

Abstract

Genipin was used as a crosslinking agent to prepare magnetic genipin-crosslinked chitosan beads, which were then used as a carrier for immobilizing recombinant cutinase from Fusarium solani (FSC) to obtain immobilized FSC. The optimal temperature for the immobilized FSC was 55 °C, which was 5 °C higher than that of the free enzyme, whereas its optimal pH was increased from 8.0 to 9.0; this indicates that the immobilized FSC had improved pH and thermal stability. After repeated use for 10 cycles, the activity of the immobilized FSC remained at more than 50%; after being stored at 4 °C for 30 days, its activity was still approximately 88%. We also found that the Km of the immobilized FSC was higher than that of the free enzyme. These results indicate that the performance of FSC was improved after immobilization, which is an important basis for the subsequent application of FSC in industrial production. Full article

(This article belongs to the Special Issue Enzyme Immobilization and Biocatalysis)

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17 pages, 12618 KiB

Open AccessArticle

Engineering of a Novel, Magnetic, Bi-Functional, Enzymatic Nanobiocatalyst for the Highly Efficient Synthesis of Enantiopure (R)-3-quinuclidinol

by Qingman Li, Qihua Jiang, Pengcheng Gu, Lianju Ma and Yiwu Wang

Catalysts 2021, 11(9), 1126; https://doi.org/10.3390/catal11091126 - 18 Sep 2021

Cited by 2 | Viewed by 1996

Abstract

Ni²⁺-NTA-boosted magnetic porous silica nanoparticles (Ni@MSN) to serve as ideal support for bi-functional enzyme were fabricated for the first time. The versatility of this support was validated by one-step purification and immobilization of bi-functional enzyme MLG consisting of 3-Quinuclidinone reductase and glucose dehydrogenase, which can simultaneously catalyze both carbonyl reduction and cofactor regeneration, to fabricate an artificial bi-functional nanobiocatalyst (namely, MLG-Ni@MSN). The enzyme loading of 71.7 mg/g support and 92.7% immobilization efficiency were obtained. Moreover, the immobilized MLG showed wider pH and temperature tolerance and greater storage stability than free MLG under the same conditions. The nanosystem was employed as biocatalyst to accomplish the 3-quinuclidinone (70 g/L) to (R)-3-quinuclidinol biotransformation in 100% conversion yield with >99% selectivity within 6 h and simultaneous cofactor regeneration. Furthermore, the immobilized MLG retained up to 80.3% (carbonyl reduction) and 78.0% (cofactor regeneration) of the initial activity after being recycled eight times. In addition, the MLG-Ni@MSN system exhibited almost no enzyme leaching during biotransformation and recycling. Therefore, we have reason to believe that the Ni@MSN support gave great promise for constructing a new biocatalytic nanosystem with multifunctional enzymes to achieve some other complex bioconversions. Full article

(This article belongs to the Special Issue Enzyme Immobilization and Biocatalysis)

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9 pages, 3931 KiB

Open AccessArticle

Immobilization of Camel Liver Catalase on Nanosilver-Coated Cotton Fabric

by Omar A. M. Al-Bar, Reda M. El-Shishtawy and Saleh A. Mohamed

Catalysts 2021, 11(8), 900; https://doi.org/10.3390/catal11080900 - 26 Jul 2021

Cited by 11 | Viewed by 3149

Abstract

Nanoparticles have the advantage of a superior surface area to volume ratio, and thus such materials are useful for enzyme immobilization. A silver nanoparticle coated cotton fabric (AgNp-CF) is used to immobilize camel liver catalase in the present work. The effect of loading levels of AgNp inside cotton fabrics on the immobilization of catalase was investigated. The results revealed that a 6 mL loading level of AgNp precursor (silver nitrate, 2 mM) at pH 8 showed the maximum immobilization efficiency (76%). The morphological properties of the cotton fabric (CF), AgNp-CF and AgNp-CF-catalase were characterized by SEM. The reusability of the immobilized enzyme was tested over ten reuses to show a 67% retained function of its initial activity. Compared with the soluble enzyme’s working pH (6.5), a rather broader working pH (6.5–7.0) was observed for the immobilized catalase. Additionally, the optimum working temperature increased from 30 for the soluble enzyme to 40 °C for the immobilized one, indicating thermal stability. The free and immobilized catalase enzyme’s Km values were 22.5 and 25 mM H₂O₂, respectively, reflecting the enzyme’s effective properties. The inhibitory effect of metal ions on the enzyme activity was higher toward soluble catalase than the immobilized catalase. This work has developed a method for immobilizing catalase to be useful for several applications. Full article

(This article belongs to the Special Issue Enzyme Immobilization and Biocatalysis)

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13 pages, 2422 KiB

Open AccessArticle

Immobilization of Catalase on Chitosan/ZnO and Chitosan/ZnO/Fe₂O₃ Nanocomposites: A Comparative Study

by Reda M. El-Shishtawy, Nahed S. E. Ahmed and Yaaser Q. Almulaiky

Catalysts 2021, 11(7), 820; https://doi.org/10.3390/catal11070820 - 6 Jul 2021

Cited by 54 | Viewed by 3963

Abstract

The strong catalytic performance, eco-friendly reaction systems, and selectivity of enzyme-based biocatalysts are extremely interesting. Immobilization has been shown to be a good way to improve enzyme stability and recyclability. Chitosan-incorporated metal oxides, among other support matrices, are an intriguing class of support matrices for the immobilization of various enzymes. Herein, the cross-linked chitosan/zinc oxide nanocomposite (CS/ZnO) was synthesized and further improved by adding iron oxide (Fe₂O₃) nanoparticles. The final cross-linked CS/ZnO/Fe₂O₃ nanocomposite was used as an immobilized support for catalase and is characterized by SEM, EDS, and FTIR. The nanocomposite CS/ZnO/Fe₂O₃ enhanced the biocompatibility and immobilized system properties. CS/ZnO/Fe₂O₃ achieved a higher immobilization yield (84.32%) than CS/ZnO (37%). After 10 repeated cycles, the remaining immobilized catalase activity of CS/ZnO and CS/ZnO/Fe₂O₃ was 14% and 45%, respectively. After 60 days of storage at 4 °C, the remaining activity of immobilized enzyme onto CS/ZnO and CS/ZnO/Fe₂O₃ was found to be 32% and 47% of its initial activity. The optimum temperature was noticed to be broad at 25–30 °C for the immobilized enzyme and 25 °C for the free enzyme. Compared with the free enzyme optimum pH (7.0), the optimum pH for the immobilized enzyme was 7.5. The K_m and V_max values for the free and immobilized enzyme on CS/ZnO, and the immobilized enzyme on CS/ZnO/Fe₂O₃, were found to be 91.28, 225.17, and 221.59 mM, and 10.45, 15.87, and 19.92 µmole ml⁻¹, respectively. Catalase immobilization on CS/ZnO and CS/ZnO/Fe₂O₃ offers better stability than free catalase due to the enzyme’s half-life. The half-life of immobilized catalase on CS/ZnO/Fe₂O₃ was between 31.5 and 693.2 min. Full article

(This article belongs to the Special Issue Enzyme Immobilization and Biocatalysis)

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8 pages, 895 KiB

Open AccessArticle

Polymeric Nanoparticles Decorated with Monoclonal Antibodies: A New Immobilization Strategy for Increasing Lipase Activity

by Laura Chronopoulou, Viviana Couto Sayalero, Hassan Rahimi, Aurelia Rughetti and Cleofe Palocci

Catalysts 2021, 11(6), 744; https://doi.org/10.3390/catal11060744 - 17 Jun 2021

Cited by 3 | Viewed by 2452

Abstract

Recent advances in nanotechnology techniques enable the production of polymeric nanoparticles with specific morphologies and dimensions and, by tailoring their surfaces, one can manipulate their characteristics to suit specific applications. In this work we report an innovative approach for the immobilization of a commercial lipase from Candida rugosa (CRL) which employs nanostructured polymeric carriers conjugated with anti-lipase monoclonal antibodies (MoAbs). MoAbs were chemically conjugated on the surface of polymeric nanoparticles and used to selectively adsorb CRL molecules. Hydrolytic enzymatic assays evidenced that such immobilization technique afforded a significant enhancement of enzymatic activity in comparison to the free enzyme. Full article

(This article belongs to the Special Issue Enzyme Immobilization and Biocatalysis)

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20 pages, 3971 KiB

Open AccessArticle

Characterization of an Immobilized Amino Acid Racemase for Potential Application in Enantioselective Chromatographic Resolution Processes

by Isabel Harriehausen, Jonas Bollmann, Thiane Carneiro, Katja Bettenbrock and Andreas Seidel-Morgenstern

Catalysts 2021, 11(6), 726; https://doi.org/10.3390/catal11060726 - 11 Jun 2021

Cited by 5 | Viewed by 2427

Abstract

Enantioselective resolution processes can be improved by integration of racemization. Applying environmentally friendly enzymatic racemization under mild conditions is in particular attractive. Owing to the variety of enzymes and the progress in enzyme engineering, suitable racemases can be found for many chiral systems. An amino acid racemase (AAR) from P. putida KT2440 is capable of processing a broad spectrum of amino acids at fast conversion rates. The focus of this study is the evaluation of the potential of integrating AAR immobilized on Purolite ECR 8309 to racemize L- or D-methionine (Met) within an enantioselective chromatographic resolution process. Racemization rates were studied for different temperatures, pH values, and fractions of organic co-solvents. The long-term stability of the immobilized enzyme at operating and storage conditions was found to be excellent and recyclability using water with up to 5 vol% ethanol at 20 °C could be demonstrated. Packed as an enzymatic fixed bed reactor, the immobilized AAR can be coupled with different resolution processes; for instance, with chromatography or with preferential crystallization. The performance of coupling it with enantioselective chromatography is estimated quantitatively, exploiting parametrized sub-models. To indicate the large potential of the AAR, racemization rates are finally given for lysine, arginine, serine, glutamine, and asparagine. Full article

(This article belongs to the Special Issue Enzyme Immobilization and Biocatalysis)

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11 pages, 4180 KiB

Open AccessArticle

Alternative Routes for the Production of Natural 4-Vinylguaiacol from Sugar Beet Fiber Using Basidiomycetous Enzymes

by Thorben Günther, Lasse Schoppe, Franziska Ersoy and Ralf G. Berger

Catalysts 2021, 11(5), 631; https://doi.org/10.3390/catal11050631 - 14 May 2021

Cited by 3 | Viewed by 2491

Abstract

Traditional smoking generates not only the impact flavor compound 4-vinylguaiacol, but concurrently many unwanted and potent toxic compounds such as polycyclic aromatic hydrocarbons. Enzyme technology provides a solution without any side-product formation. A feruloyl esterase from Rhizoctonia solani (RspCAE) liberated ferulic acid from low-priced sugar beet fiber. Decarboxylation of ferulic acid to 4-vinylguaiacol was achieved by a second enzyme from Schizophyllum commune (ScoFAD). Both enzymes were covalently immobilized on agarose to enable reusability in a fixed-bed approach. The two enzyme cascades showed high conversion rates with yields of 0.8 and 0.95, respectively, and retained activity for nearly 80 h of continuous operation. The overall productivity of the model process with bed volumes of 300 µL and a substrate flow rate of 0.25 mL min⁻¹ was 3.98 mg 4-vinylguaiacol per hour. A cold online solid phase extraction using XAD4 was integrated into the bioprocess and provided high recovery rates during multiple elution steps. Attempting to facilitate the bioprocess, a fused gene coding for the two enzymes and a set of different linker lengths and properties was constructed and introduced into Komagataella phaffii. Longer and rigid linkers resulted in higher activity of the fusion protein with a maximum of 67 U L⁻¹. Full article

(This article belongs to the Special Issue Enzyme Immobilization and Biocatalysis)

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13 pages, 2717 KiB

Open AccessArticle

Laccase and Its Mutant Displayed on the Bacillus subtilis Spore Coat for Oxidation of Phenolic Compounds in Organic Solvents

by Silu Sheng and Edgardo T. Farinas

Catalysts 2021, 11(5), 606; https://doi.org/10.3390/catal11050606 - 10 May 2021

Cited by 9 | Viewed by 2182

Abstract

Enzymes displayed on the Bacillus subtilis spore coat have several features that are useful for biocatalysis. The enzyme is preimmobilized on an inert surface of the spore coat, which is due to the natural sporulation process. As a result, protein stability can be increased, and they are resistant to environmental changes. Next, they would not lyse under extreme conditions, such as in organic solvents. Furthermore, they can be easily removed from the reaction solution and reused. The laboratory evolved CotA laccase variant T480A-CotA was used to oxidize the following phenolic substrates: (+)-catechin, (−)-epicatechin, and sinapic acid. The kinetic parameters were determined and T480A-CotA had a greater V_max/K_m than wt-CotA for all substrates. The V_max/K_m for T480A-CotA was 4.1, 5.6, and 1.4-fold greater than wt-CotA for (+)-catechin, (−)-epicatechin, and sinapic acid, respectively. The activity of wt-CotA and T480A-CotA was measured at different concentrations from 0–70% in organic solvents (dimethyl sulfoxide, ethanol, methanol, and acetonitrile). The V_max for T480A-CotA was observed to be greater than the wt-CotA in all organic solvents. Finally, the T480A-CotA was recycled 7 times over a 23-h period and up to 60% activity for (+)-catechin remained. The product yield was up to 3.1-fold greater than the wild-type. Full article

(This article belongs to the Special Issue Enzyme Immobilization and Biocatalysis)

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13 pages, 3460 KiB

Open AccessArticle

Biocatalysis for Rare Ginsenoside Rh2 Production in High Level with Co-Immobilized UDP-Glycosyltransferase Bs-YjiC Mutant and Sucrose Synthase AtSuSy

by Jianlin Chu, Jiheng Yue, Song Qin, Yuqiang Li, Bin Wu and Bingfang He

Catalysts 2021, 11(1), 132; https://doi.org/10.3390/catal11010132 - 18 Jan 2021

Cited by 13 | Viewed by 3420

Abstract

Rare ginsenoside Rh2 exhibits diverse pharmacological effects. UDP-glycosyltransferase (UGT) catalyzed glycosylation of protopanaxadiol (PPD) has been of growing interest in recent years. UDP-glycosyltransferase Bs-YjiC coupling sucrose synthase in one-pot reaction was successfully applied to ginsenoside biosynthesis with UDP-glucose regeneration from sucrose and UDP, which formed a green and sustainable approach. In this study, the his-tagged UDP-glycosyltransferase Bs-YjiC mutant M315F and sucrose synthase AtSuSy were co-immobilized on heterofunctional supports. The affinity adsorption significantly improved the capacity of specific binding of the two recombinant enzymes, and the dual enzyme covalently cross-linked by the acetaldehyde groups significantly promoted the binding stability of the immobilized bienzyme, allowing higher substrate concentration by easing substrate inhibition for the coupled reaction. The dual enzyme amount used for ginsenoside Rh2 biosynthesis is Bs-YjiC-M315F: AtSuSy = 18 mU/mL: 25.2 mU/mL, a yield of 79.2% was achieved. The coimmobilized M315F/AtSuSy had good operational stability of repetitive usage for 10 cycles, and the yield of ginsenoside Rh2 was kept between 77.6% and 81.3%. The high titer of the ginsenoside Rh2 cumulatively reached up to 16.6 mM (10.3 g/L) using fed-batch technology, and the final yield was 83.2%. This study has established a green and sustainable approach for the production of ginsenoside Rh2 in a high level of titer, which provides promising candidates for natural drug research and development. Full article

(This article belongs to the Special Issue Enzyme Immobilization and Biocatalysis)

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29 pages, 8877 KiB

Open AccessFeature PaperEditor’s ChoiceReview

Lipase Immobilization in Mesoporous Silica Nanoparticles for Biofuel Production

by Aniello Costantini and Valeria Califano

Catalysts 2021, 11(5), 629; https://doi.org/10.3390/catal11050629 - 13 May 2021

Cited by 47 | Viewed by 5913

Abstract

Lipases are ubiquitous enzymes whose physiological role is the hydrolysis of triacylglycerol into fatty acids. They are the most studied and industrially interesting enzymes, thanks to their versatility to promote a plethora of reactions on a wide range of substrates. In fact, depending on the reaction conditions, they can also catalyze synthesis reactions, such as esterification, acidolysis and transesterification. The latter is particularly important for biodiesel production. Biodiesel can be produced from animal fats or vegetable oils and is considered as a biodegradable, non-toxic and renewable energy source. The use of lipases as industrial catalysts is subordinated to their immobilization on insoluble supports, to allow multiple uses and use in continuous processes, but also to stabilize the enzyme, intrinsically prone to denaturation with consequent loss of activity. Among the materials that can be used for lipase immobilization, mesoporous silica nanoparticles represent a good choice due to the combination of thermal and mechanical stability with controlled textural characteristics. Moreover, the presence of abundant surface hydroxyl groups allows for easy chemical surface functionalization. This latter aspect has the main importance since lipases have a high affinity with hydrophobic supports. The objective of this work is to provide an overview of the recent progress of lipase immobilization in mesoporous silica nanoparticles with a focus on biodiesel production. Full article

(This article belongs to the Special Issue Enzyme Immobilization and Biocatalysis)

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