Promising Industrial Enzymes

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

Deadline for manuscript submissions: closed (31 December 2022) | Viewed by 22417

Special Issue Editors


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Guest Editor
Department of Chemical, Biological and Environmental Engineering, EE, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain
Interests: biochemical engineering; bioprocess engineering, production of recombinant proteins in the cell factory Pichia pastoris; monitoring modeling and control of bioprocesses and applied biocatalysis (lipases)

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Guest Editor
Department of Chemical, Biological and Environmental Engineering, EE, Universitat Autònoma de Barcelona, Bellaterra, 08193 Barcelona, Spain
Interests: The main research interests are focused on industrial biotechnology, especially on applied biocatalysis, bioprocess development, multi-enzymatic systems, bioreactor design and enzyme immobilization

Special Issue Information

Dear Colleagues,

The industrial applications of enzymes have been increasing in the last decade, becoming a real cost-effective alternative as a substitute for chemical processes. It represents a more environmentally sustainable approach, greener than the traditional chemical industry and in agreement with the fundamentals of circular economy. In fact, the number of enzymes with industrial applications is increasing, including not only the classical proteases, amylases or lipases.

The present Special Issue is focused on recent research and reviews about how different approaches have managed to improve enzymes for industrial applications. Research topics may include: i) examples of enhanced enzymes by directed evolution, enzyme engineering and metagenomics; ii) advances in bioprocess engineering and cell factories to increase the heterologous production of enzymes; iii) bioprocess scaling-up to industrial levels;  iv) novel approaches to improve the stability and reutilization of the industrial enzymes; v) medium engineering as the used of supercritical fluids, ionic liquids, deep eutectic solvents and novel reaction medium; vi) multi-enzymatic systems development, including cofactor regeneration; vii) process intensification and successful examples of new industrial enzymatic applications.

Prof. Dr. Francisco Valero
Dra. Marina Guillén Montalbán
Guest Editors

Manuscript Submission Information

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Keywords

  • industrial enzymes
  • direct evolution
  • metagenomics
  • enzyme engineering
  • heterologous production
  • bioprocess engineering
  • scale-up
  • immobilization
  • industrial biocatalysis
  • multi-enzymatic synthesis
  • cofactor regeneration
  • bioreactor design
  • medium engineering
  • process intensification
  • bioeconomy

Published Papers (9 papers)

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Research

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7 pages, 888 KiB  
Article
Cyanate Degradation in Different Matrices Using Heat-Purified Enzymes
by Chia-Jung Hsieh and Chi-Yang Yu
Catalysts 2023, 13(1), 76; https://doi.org/10.3390/catal13010076 - 30 Dec 2022
Cited by 1 | Viewed by 1122
Abstract
A green and low-cost removal method for cyanate, a toxic byproduct from the treatment of cyanide, is still needed. Cyanase converts cyanate to CO2 and NH3, but its industrial practicality is limited because the reaction requires HCO3 as [...] Read more.
A green and low-cost removal method for cyanate, a toxic byproduct from the treatment of cyanide, is still needed. Cyanase converts cyanate to CO2 and NH3, but its industrial practicality is limited because the reaction requires HCO3 as a substrate. In this study, we used carbonic anhydrase from Sulfurihydrogenibium azorense (SazCA) to provide HCO3 for cyanase from Thermomyces lanuginosus (TlCyn); both TlCyn and SazCA were purified by one-step heating without prior cell lysis. The heat treatment resulted in higher activities of both enzymes than the conventional two-step process. From a 50 mL-culture, the highest total activity of 147 U and 47,174 WAU was obtained from 5 min of heating at 60 and 80 °C for TlCyn and SazCA, respectively. The coupled enzymatic system was used to degrade cyanate in three different matrices: 50 mM Tris-HCl (pH 8), industrial wastewater, and artificial wastewater. In the industrial wastewater, with the addition of 0.75 WAU (Wilbur-Anderson unit) of SazCA, cyanate degradation using 0.5 mM NaHCO3 was similar to that using 3 mM NaHCO3, indicating an 83% reduction in NaHCO3. We have demonstrated that the dependence on HCO3 of cyanate degradation can be effectively alleviated by using low-cost heat-purified TlCyn and SazCA; the industrial practicality of the coupled enzymatic system is therefore improved. Full article
(This article belongs to the Special Issue Promising Industrial Enzymes)
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13 pages, 1712 KiB  
Article
The Stability of Dimeric D-amino Acid Oxidase from Porcine Kidney Strongly Depends on the Buffer Nature and Concentration
by Diego Carballares, Javier Rocha-Martin and Roberto Fernandez-Lafuente
Catalysts 2022, 12(9), 1009; https://doi.org/10.3390/catal12091009 - 06 Sep 2022
Cited by 1 | Viewed by 1230
Abstract
The first step of the inactivation of the enzyme D-amino acid oxidase (DAAO) from porcine kidney at pH 5 and 7 is the enzyme subunit dissociation, while FAD dissociation has not a relevant role. At pH 9, both dissociation phenomena affect the [...] Read more.
The first step of the inactivation of the enzyme D-amino acid oxidase (DAAO) from porcine kidney at pH 5 and 7 is the enzyme subunit dissociation, while FAD dissociation has not a relevant role. At pH 9, both dissociation phenomena affect the enzyme stability. A strong effect of the buffer nature and concentration on enzyme stability was found, mainly at pH 7 and 9 (it was possible at the same temperature to have the enzyme fully inactivated in 5 mM of Hepes while maintaining 100% in 5 mM of glycine). The effect of the concentration of buffer on enzyme stability depended on the buffer: at pH 5, the acetate buffer had no clear effect, while Tris, Hepes and glycine (at pH 7) and carbonate (at pH 9) decreased enzyme stability when increasing their concentrations; phosphate concentration had the opposite effect. The presence of 250 mM of NaCl usually increased enzyme stability, but this did not occur in all cases. The effects were usually more significant when using low concentrations of DAAO and were not reverted upon adding exogenous FAD. However, when using an immobilized DAAO biocatalyst which presented enzyme subunits attached to the support, where dissociation was not possible, this effect of the buffer nature on enzyme stability almost disappeared. This suggested that the buffers were somehow altering the association/dissociation equilibrium of the enzyme. Full article
(This article belongs to the Special Issue Promising Industrial Enzymes)
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15 pages, 901 KiB  
Article
Constitutive Expression in Komagataella phaffii of Mature Rhizopus oryzae Lipase Jointly with Its Truncated Prosequence Improves Production and the Biocatalyst Operational Stability
by Josu López-Fernández, Maria Dolors Benaiges and Francisco Valero
Catalysts 2021, 11(10), 1192; https://doi.org/10.3390/catal11101192 - 30 Sep 2021
Cited by 6 | Viewed by 1662
Abstract
Rhizopus oryzae lipase (ROL) containing 28 C-terminal amino acids of the prosequence fused to the N-terminal mature sequence in ROL (proROL) was successfully expressed in the methylotrophic yeast Komagataella phaffii (Pichia pastoris) under the constitutive glyceraldehyde-3-phosphate dehydrogenase promoter (PGAP). [...] Read more.
Rhizopus oryzae lipase (ROL) containing 28 C-terminal amino acids of the prosequence fused to the N-terminal mature sequence in ROL (proROL) was successfully expressed in the methylotrophic yeast Komagataella phaffii (Pichia pastoris) under the constitutive glyceraldehyde-3-phosphate dehydrogenase promoter (PGAP). Although the sequence encoding the mature lipase (rROL) was also transformed, no clones were obtained after three transformation cycles, which highlights the importance of the truncated prosequence to obtain viable transformed clones. Batch cultures of the K. phaffii strain constitutively expressing proROL scarcely influenced growth rate and exhibited a final activity and volumetric productivity more than six times higher than those obtained with proROL from K. phaffii under the methanol-inducible alcohol oxidase 1 promoter (PAOX1). The previous differences were less marked in fed-batch cultures. N-terminal analysis confirmed the presence of the 28 amino acids in proROL. In addition, immobilized proROL exhibited increased tolerance of organic solvents and an operational stability 0.25 and 3 times higher than that of immobilized rROL in biodiesel and ethyl butyrate production, respectively. Therefore, the truncated prosequence enables constitutive proROL production, boosts bioprocess performance and provides a more stable biocatalyst in two reactions in which lipases are mostly used at industrial level, esterification (ethyl butyrate) and transesterification (biodiesel). Full article
(This article belongs to the Special Issue Promising Industrial Enzymes)
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14 pages, 1641 KiB  
Article
Chiral Synthesis of 3-Amino-1-phenylbutane by a Multi-Enzymatic Cascade System
by Natàlia Alcover, Gregorio Álvaro and Marina Guillén
Catalysts 2021, 11(8), 973; https://doi.org/10.3390/catal11080973 - 14 Aug 2021
Cited by 2 | Viewed by 2354
Abstract
Asymmetric synthesis of chiral amines from prochiral ketones using transaminases is an attractive biocatalytic strategy. Nevertheless, it is hampered by its unfavorable thermodynamic equilibrium. In the present work, an insitu by-product removal strategy was applied for the synthesis of 3-amino-1-phenylbutane (3-APB) by coupling [...] Read more.
Asymmetric synthesis of chiral amines from prochiral ketones using transaminases is an attractive biocatalytic strategy. Nevertheless, it is hampered by its unfavorable thermodynamic equilibrium. In the present work, an insitu by-product removal strategy was applied for the synthesis of 3-amino-1-phenylbutane (3-APB) by coupling a transaminase with a pyruvate decarboxylase (PDC), which does not require the use of any expensive additional cofactor. Using this strategy, the pyruvate obtained in the transamination reaction is transformed by PDC into acetaldehyde and CO2 which are of high volatility. Two different transaminases from Chromobacterium violaceum (CviTA) and Vibrio fluvialis (VflTA) were characterized to find out the appropriate pH conditions. In both cases, the addition of PDC dramatically enhanced 3-APB synthesis. Afterwards, different reaction conditions were tested to improve reaction conversion and yield. It was concluded that 30 °C and a 20-fold alanine excess lead to the best process metrics. Under the mentioned conditions, yields higher than 60% were reached with nearly 90% selectivity using both CviTA and VflTA. Moreover, high stereoselectivity for (S)-3-APB was obtained and ee of around 90% was achieved in both cases. For the first time, the asymmetric synthesis of 3-APB using PDC as by-product removal system using CviTA is reported. Full article
(This article belongs to the Special Issue Promising Industrial Enzymes)
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21 pages, 3313 KiB  
Article
Immobilization of the Peroxygenase from Agrocybe aegerita. The Effect of the Immobilization pH on the Features of an Ionically Exchanged Dimeric Peroxygenase
by Diego Carballares, Roberto Morellon-Sterling, Xiaomin Xu, Frank Hollmann and Roberto Fernandez-Lafuente
Catalysts 2021, 11(5), 560; https://doi.org/10.3390/catal11050560 - 28 Apr 2021
Cited by 14 | Viewed by 2785
Abstract
This paper outlines the immobilization of the recombinant dimeric unspecific peroxygenase from Agrocybe aegerita (rAaeUPO). The enzyme was quite stable (remaining unaltered its activity after 35 h at 47 °C and pH 7.0). Phosphate destabilized the enzyme, while glycerol stabilized it. [...] Read more.
This paper outlines the immobilization of the recombinant dimeric unspecific peroxygenase from Agrocybe aegerita (rAaeUPO). The enzyme was quite stable (remaining unaltered its activity after 35 h at 47 °C and pH 7.0). Phosphate destabilized the enzyme, while glycerol stabilized it. The enzyme was not immobilized on glyoxyl-agarose supports, while it was immobilized albeit in inactive form on vinyl-sulfone-activated supports. rAaeUPO immobilization on glutaraldehyde pre-activated supports gave almost quantitative immobilization yield and retained some activity, but the biocatalyst was very unstable. Its immobilization via anion exchange on PEI supports also produced good immobilization yields, but the rAaeUPO stability dropped. However, using aminated agarose, the enzyme retained stability and activity. The stability of the immobilized enzyme strongly depended on the immobilization pH, being much less stable when rAaeUPO was adsorbed at pH 9.0 than when it was immobilized at pH 7.0 or pH 5.0 (residual activity was almost 0 for the former and 80% for the other preparations), presenting stability very similar to that of the free enzyme. This is a very clear example of how the immobilization pH greatly affects the final biocatalyst performance. Full article
(This article belongs to the Special Issue Promising Industrial Enzymes)
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14 pages, 3222 KiB  
Article
The β-Fructofuranosidase from Rhodotorula dairenensis: Molecular Cloning, Heterologous Expression, and Evaluation of Its Transferase Activity
by María Gimeno-Pérez, Zoran Merdzo, Eva Castillo-Rosa, Carlos Martín de Hijas and María Fernández-Lobato
Catalysts 2021, 11(4), 476; https://doi.org/10.3390/catal11040476 - 07 Apr 2021
Cited by 4 | Viewed by 2885
Abstract
The β-fructofuranosidase from the yeast Rhodotorula dairenensis (RdINV) produces a mixture of potential prebiotic fructooligosaccharides (FOS) of the levan-, inulin- and neo-FOS series by transfructosylation of sucrose. In this work, the gene responsible for this activity was characterized and its functionality proved in [...] Read more.
The β-fructofuranosidase from the yeast Rhodotorula dairenensis (RdINV) produces a mixture of potential prebiotic fructooligosaccharides (FOS) of the levan-, inulin- and neo-FOS series by transfructosylation of sucrose. In this work, the gene responsible for this activity was characterized and its functionality proved in Pichia pastoris. The amino acid sequence of the new protein contained most of the characteristic elements of β-fructofuranosidases included in the family 32 of the glycosyl hydrolases (GH32). The heterologous yeast produced a protein of about 170 kDa, where N-linked and O-linked carbohydrates constituted about 15% and 38% of the total protein mass, respectively. Biochemical and kinetic properties of the heterologous protein were similar to the native enzyme, including its ability to produce prebiotic sugars. The maximum concentration of FOS obtained was 82.2 g/L, of which 6-kestose represented about 59% (w/w) of the total products synthesized. The potential of RdINV to fructosylate 19 hydroxylated compounds was also explored, of which eight sugars and four alditols were modified. The flexibility to recognize diverse fructosyl acceptors makes this protein valuable to produce novel glycosyl-compounds with potential applications in food and pharmaceutical industries. Full article
(This article belongs to the Special Issue Promising Industrial Enzymes)
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Review

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16 pages, 1640 KiB  
Review
Recent Progress in the Mechanism and Engineering of α/β Hydrolases for Chiral Chemical Production
by Mingzhe Qiu, Sheng Dong, Qiu Cui, Yingang Feng and Jinsong Xuan
Catalysts 2023, 13(2), 288; https://doi.org/10.3390/catal13020288 - 27 Jan 2023
Cited by 1 | Viewed by 1363
Abstract
Chiral compounds are valuable industrial products and intermediates, and the production of chemicals with high enantiopurity is one of the major objects in asymmetric catalysis. Compared with traditional chemical synthesis, enzymatic synthesis can produce chiral molecules under sustainable conditions which are much greener, [...] Read more.
Chiral compounds are valuable industrial products and intermediates, and the production of chemicals with high enantiopurity is one of the major objects in asymmetric catalysis. Compared with traditional chemical synthesis, enzymatic synthesis can produce chiral molecules under sustainable conditions which are much greener, more economical, and more environmentally friendly. The superfamily of α/β hydrolases includes a lot of diverse enzymes showing excellent chemo-, regio-, and enantio-selectivity in asymmetric synthesis and many of them are biocatalysts in industry. This review outlines the current knowledge of the structures and reaction mechanism of α/β hydrolases and summarizes the screening and protein engineering efforts to develop biocatalysts for chiral chemicals production in recent years. Other strategies such as whole-cell catalysis and protein immobilization to improve the performance of α/β hydrolases are also discussed. The progress in biocatalyst development based on α/β hydrolases will promote the biosynthesis of chiral compounds, thus contributing to the green and sustainable development of the chemical and pharmaceutical industry. Full article
(This article belongs to the Special Issue Promising Industrial Enzymes)
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16 pages, 726 KiB  
Review
Anticancer Asparaginases: Perspectives in Using Filamentous Fungi as Cell Factories
by Pedro Henrique Dias Garcia, Tales Alexandre Costa-Silva, Martí Morera Gómez, Fabiano Jares Contesini, Paula Renata Bueno Campos Canella and Patrícia de Oliveira Carvalho
Catalysts 2023, 13(1), 200; https://doi.org/10.3390/catal13010200 - 15 Jan 2023
Cited by 2 | Viewed by 2247
Abstract
The enzyme L-asparaginase (L-asparagine amidohydrolase) catalyzes the breakdown of L-asparagine into aspartate and ammonia, which leads to an anti-neoplastic activity stemming from its capacity to deplete L-asparagine concentrations in the bloodstream, and it is therefore used in cases of acute lymphoblastic leukemia (ALL) [...] Read more.
The enzyme L-asparaginase (L-asparagine amidohydrolase) catalyzes the breakdown of L-asparagine into aspartate and ammonia, which leads to an anti-neoplastic activity stemming from its capacity to deplete L-asparagine concentrations in the bloodstream, and it is therefore used in cases of acute lymphoblastic leukemia (ALL) to inhibit malignant cell growth. Nowadays, this anti-cancer enzyme, largely produced by Escherichia coli, is well established on the market. However, E. coli L-asparaginase therapy has side effects such as anaphylaxis, coagulation abnormality, low plasma half-life, hepatotoxicity, pancreatitis, protease action, hyperglycemia, and cerebral dysfunction. This review provides a perspective on the use of filamentous fungi as alternative cell factories for L-asparaginase production. Filamentous fungi, such as various Aspergillus species, have superior protein secretion capacity compared to yeast and bacteria and studies show their potential for the future production of proteins with humanized N-linked glycans. This article explores the past and present applications of this important enzyme and discusses the prospects for using filamentous fungi to produce safe eukaryotic asparaginases with high production yields. Full article
(This article belongs to the Special Issue Promising Industrial Enzymes)
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24 pages, 1616 KiB  
Review
Recent Advances in Lipases and Their Applications in the Food and Nutraceutical Industry
by Ana Laura Reyes-Reyes, Francisco Valero Barranco and Georgina Sandoval
Catalysts 2022, 12(9), 960; https://doi.org/10.3390/catal12090960 - 29 Aug 2022
Cited by 13 | Viewed by 5295
Abstract
Lipases are efficient enzymes with promising applications in the nutraceutical and food industry, as they can offer high yields, pure products under achievable reaction conditions, and are an environmentally friendly option. This review addresses the production of high-value-added compounds such as fatty acid [...] Read more.
Lipases are efficient enzymes with promising applications in the nutraceutical and food industry, as they can offer high yields, pure products under achievable reaction conditions, and are an environmentally friendly option. This review addresses the production of high-value-added compounds such as fatty acid esters, with the potential to be used as flavoring agents or antioxidant and antimicrobial agents, as well as structured lipids that offer specific functional properties that do not exist in nature, with important applications in different food products, and pharmaceuticals. In addition, the most recent successful cases of reactions with lipases to produce modified compounds for food and nutraceuticals are reported. Full article
(This article belongs to the Special Issue Promising Industrial Enzymes)
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