1
|
Zheng J, Zhang Q, Zhong N. Selective synthesis of triacylglycerols by the ADS-17-supported Candida antarctica lipase B through esterification of oleic acid and glycerol. JOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE 2025; 105:3931-3941. [PMID: 39835430 DOI: 10.1002/jsfa.14137] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/07/2024] [Revised: 12/02/2024] [Accepted: 01/07/2025] [Indexed: 01/22/2025]
Abstract
BACKGROUND Immobilized enzyme possessing both high activity and good selectivity is important in practice. In this study, Candida antarctica lipase B (CALB) was immobilized onto the macroporous resin ADS-17 for triacylglycerol (TAG) synthesis through esterification of oleic acid and glycerol. The reaction conditions were optimized by single-factor study and orthogonal test, and the reusability of the immobilized CALB (CALB@ADS-17) was evaluated. In addition, the mechanism of lipase immobilization was studied and the catalytic mechanism of CALB@ADS-17 was investigated. RESULTS Oleic acid conversion up to 99.20% and TAG content at 91.58 wt% could be obtained under optimal conditions. In addition, the CALB@ADS-17 retained 84.28% of its initial activity after 11 cycles of reuse. The mechanism of lipase immobilization was through hydrophobic adsorption. The relationship between temperature and oleic acid conversion was lnV0 = 6.3316 - 4.3321/T, and the activation energy (Ea) was 36.02 kJ mol-1. CALB@ADS-17 did not exhibit an obvious interfacial activation phenomenon. Its kinetic behavior can be described by the Michaelis-Menten model, whose kinetic parameters of vmax, kcat, Km, Ki, and kcat/Km were 0.01265 μmol L-1 s-1, 9310.72 s-1, 0.4907 mmol L-1, 3.997 mmol L-1, and 1.90 × 104 L mmol-1 s-1, respectively. CONCLUSION CALB@ADS-17 showed good esterification performance and exhibited good selectivity towards TAG generation. In addition, CALB@ADS-17 exhibited good reusability in esterification reactions and has potential in practical applications. © 2025 Society of Chemical Industry.
Collapse
Affiliation(s)
- Jiawei Zheng
- School of Food Science, Guangdong Pharmaceutical University, Zhongshan, China
| | - Qiangyue Zhang
- School of Food Science, Guangdong Pharmaceutical University, Zhongshan, China
| | - Nanjing Zhong
- School of Food Science, Guangdong Pharmaceutical University, Zhongshan, China
| |
Collapse
|
2
|
Dos Santos KM, de França Serpa J, de Castro Bizerra V, Melo RLF, Sousa Junior PGD, Santos Alexandre V, da Fonseca AM, Fechine PBA, Lomonaco D, Sousa Dos Santos JC, Martins de Souza MC. Enhanced Biodiesel Production with Eversa Transform 2.0 Lipase on Magnetic Nanoparticles. LANGMUIR : THE ACS JOURNAL OF SURFACES AND COLLOIDS 2024; 40:26835-26851. [PMID: 39591534 DOI: 10.1021/acs.langmuir.4c02542] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/28/2024]
Abstract
This research investigated the usefulness of magnetic iron oxide nanoparticles (Fe3O4) as a support to immobilize the lipase Eversa Transform 2.0 (ET 2.0) to obtain an active and stable biocatalyst, easily recoverable from the reaction medium for applications in the production of biodiesel. Biodiesel was an alternative fuel composed mainly of fatty acid esters with strong transesterification and esterification capabilities. The study focused on the esterification of oleic acid with ethanol to synthesize ethyl oleate. Magnetic nanoparticles were prepared by coprecipitation, then activated with glutaraldehyde and functionalized with γ-aminopropyltriethoxysilane (APTES). The optimal conditions for immobilizing ET 2.0 were pH 10, 25 mM sodium carbonate buffer, an enzymatic load of 200 U/g, and 1 h of contact time, obtaining 78% yield and enzymatic activity of 205.9 U/g. Postimmobilization evaluation showed that the immobilized enzyme performed better than its free form. Kinetic studies were conducted under these optimized conditions (2-96 h at 150 rpm and 37 °C). The biocatalyst was tested for the synthesis of ethyl oleate using oleic acid as the substrate and ethanol, achieving a conversion of 88.1%. Subsequent recirculation tests maintained approximately 80% conversion until the fourth cycle, confirming the sustainability of ester production. Molecular docking studies revealed that the binding affinity for the enzyme-docked oil composition was estimated at -5.8 kcal/mol, suggesting that the combination of the substrate and lipase was stable and suitable for esterification.
Collapse
Affiliation(s)
- Kaiany Moreira Dos Santos
- Instituto de Engenharia e Desenvolvimento Sustentável - IEDS, Campus das Auroras, Universidade da Integração Internacional da Lusofonia Afro-Brasileira - UNILAB, Rua José Franco de Oliveira, s/n - Zona Rural, Redenção 62790-970, CE, Brazil
| | - Juliana de França Serpa
- Instituto de Engenharia e Desenvolvimento Sustentável - IEDS, Campus das Auroras, Universidade da Integração Internacional da Lusofonia Afro-Brasileira - UNILAB, Rua José Franco de Oliveira, s/n - Zona Rural, Redenção 62790-970, CE, Brazil
| | - Viviane de Castro Bizerra
- Instituto de Engenharia e Desenvolvimento Sustentável - IEDS, Campus das Auroras, Universidade da Integração Internacional da Lusofonia Afro-Brasileira - UNILAB, Rua José Franco de Oliveira, s/n - Zona Rural, Redenção 62790-970, CE, Brazil
| | - Rafael Leandro Fernandes Melo
- Departamento de Engenharia Metalúrgica e de Materiais, Universidade Federal do Ceará-UFC, Campus do Pici, Bloco 729, Fortaleza CEP 60440-554, CE, Brazil
| | - Paulo Gonçalves de Sousa Junior
- Instituto de Engenharia e Desenvolvimento Sustentável - IEDS, Campus das Auroras, Universidade da Integração Internacional da Lusofonia Afro-Brasileira - UNILAB, Rua José Franco de Oliveira, s/n - Zona Rural, Redenção 62790-970, CE, Brazil
| | - Valdilane Santos Alexandre
- Instituto de Engenharia e Desenvolvimento Sustentável - IEDS, Campus das Auroras, Universidade da Integração Internacional da Lusofonia Afro-Brasileira - UNILAB, Rua José Franco de Oliveira, s/n - Zona Rural, Redenção 62790-970, CE, Brazil
| | - Aluísio Marques da Fonseca
- Instituto de Engenharia e Desenvolvimento Sustentável - IEDS, Campus das Auroras, Universidade da Integração Internacional da Lusofonia Afro-Brasileira - UNILAB, Rua José Franco de Oliveira, s/n - Zona Rural, Redenção 62790-970, CE, Brazil
| | - Pierre Basílio Almeida Fechine
- Departamento de Química Analítica e Físico-Química, Universidade Federal do Ceará - UFC, Campus do Pici, Bloco 940, Av. Humberto Monte, 2825, CEP 60455760 Fortaleza, CE, Brazil
| | - Diego Lomonaco
- Laboratório de Produtos e Tecnologia em Processos (LPT), Universidade Federal do Ceará-UFC, Fortaleza 60440-900, CE, Brasil
| | - José Cleiton Sousa Dos Santos
- Instituto de Engenharia e Desenvolvimento Sustentável - IEDS, Campus das Auroras, Universidade da Integração Internacional da Lusofonia Afro-Brasileira - UNILAB, Rua José Franco de Oliveira, s/n - Zona Rural, Redenção 62790-970, CE, Brazil
| | - Maria Cristiane Martins de Souza
- Instituto de Engenharia e Desenvolvimento Sustentável - IEDS, Campus das Auroras, Universidade da Integração Internacional da Lusofonia Afro-Brasileira - UNILAB, Rua José Franco de Oliveira, s/n - Zona Rural, Redenção 62790-970, CE, Brazil
| |
Collapse
|
3
|
Yingngam B, Makewilai L, Chaisawat S, Yingngam K, Chaiburi C, Khumsikiew J, Netthong R. Vibration-assisted Microbead Production: A New Frontier for Biocompatible Surfaces. MEDICAL APPLICATIONS FOR BIOCOMPATIBLE SURFACES AND COATINGS 2024:251-285. [DOI: 10.1039/9781837675555-00251] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/05/2025]
Abstract
The encapsulation of active pharmaceutical ingredients (APIs) in microbeads is an essential step in drug delivery; however, it is also inherently associated with the need to control particle size and drug release profiles. Nevertheless, most conventional methods of microencapsulation fail to provide consistent results. A new method called vibration-assisted microbead coating is a novel unified technique utilizing mechanical vibrations to enable the controlled, uniform coating of microbeads on APIs. This chapter discusses the technology of vibration-assisted encapsulation performed by the authors through microbead formation and the physical activity of coating APIs. This chapter focuses on achieving uniform control of the final coated surface of the API, microbead shape, size, and loading through vibration parameters. Additionally, this chapter discusses the biocompatibility and stability of the final coated surface. This new means of encapsulation has high potential for drug delivery. This method reduces most of the traditional challenges of encapsulation, if not eliminates them, and is more reliable. Based on the abovementioned findings, the authors propose the following main areas for their further work: optimisation of vibration parameters for various APIs, research into the long-term stability of the loading–release profile, and possible use of the technique in targeted drug delivery.
Collapse
Affiliation(s)
- B. Yingngam
- aFaculty of Pharmaceutical Sciences, Ubon Ratchathani University, Ubon Ratchathani, 34190, Thailand
| | - L. Makewilai
- bIntegrated Biopharmaceutical Research Cluster, Faculty of Pharmaceutical Sciences, Ubon Ratchathani University, Ubon Ratchathani, 34190, Thailand
| | - S. Chaisawat
- bIntegrated Biopharmaceutical Research Cluster, Faculty of Pharmaceutical Sciences, Ubon Ratchathani University, Ubon Ratchathani, 34190, Thailand
| | - K. Yingngam
- bIntegrated Biopharmaceutical Research Cluster, Faculty of Pharmaceutical Sciences, Ubon Ratchathani University, Ubon Ratchathani, 34190, Thailand
| | - C. Chaiburi
- cFaculty of Science and Digital Innovation, Thaksin University (Phattalung Campus), Pa Payom, Phattalung, 93210, Thailand
| | - J. Khumsikiew
- aFaculty of Pharmaceutical Sciences, Ubon Ratchathani University, Ubon Ratchathani, 34190, Thailand
| | - R. Netthong
- aFaculty of Pharmaceutical Sciences, Ubon Ratchathani University, Ubon Ratchathani, 34190, Thailand
| |
Collapse
|
4
|
Naim M, Mohammat MF, Mohd Ariff PNA, Uzir MH. Biocatalytic approach for the synthesis of chiral alcohols for the development of pharmaceutical intermediates and other industrial applications: A review. Enzyme Microb Technol 2024; 180:110483. [PMID: 39033578 DOI: 10.1016/j.enzmictec.2024.110483] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/15/2024] [Revised: 06/27/2024] [Accepted: 07/14/2024] [Indexed: 07/23/2024]
Abstract
Biocatalysis has emerged as a strong tool for the synthesis of active pharmaceutical ingredients (APIs). In the early twentieth century, whole cell biocatalysis was used to develop the first industrial biocatalytic processes, and the precise work of enzymes was unknown. Biocatalysis has evolved over the years into an essential tool for modern, cost-effective, and sustainable pharmaceutical manufacturing. Meanwhile, advances in directed evolution enable the rapid production of process-stable enzymes with broad substrate scope and high selectivity. Large-scale synthetic pathways incorporating biocatalytic critical steps towards >130 APIs of authorized pharmaceuticals and drug prospects are compared in terms of steps, reaction conditions, and scale with the corresponding chemical procedures. This review is designed on the functional group developed during the reaction forming alcohol functional groups. Some important biocatalyst sources, techniques, and challenges are described. A few APIs and their utilization in pharmaceutical drugs are explained here in this review. Biocatalysis has provided shorter, more efficient, and more sustainable alternative pathways toward existing small molecule APIs. Furthermore, non-pharmaceutical applications of biocatalysts are also mentioned and discussed. Finally, this review includes the future outlook and challenges of biocatalysis. In conclusion, Further research and development of promising enzymes are required before they can be used in industry.
Collapse
Affiliation(s)
- Mohd Naim
- School of Chemical Engineering, Engineering Campus, Universiti Sains Malaysia, Nibong Tebal, Pulau Pinang 14300, Malaysia.
| | - Mohd Fazli Mohammat
- Centre for Chemical Synthesis & Polymer Technology, Institute of Science (IoS), Kompleks Inspirasi, Universiti Teknologi MARA, Shah Alam, Selangor Darul Ehsan 40450, Malaysia.
| | - Putri Nur Arina Mohd Ariff
- Department of Nanopharmaceutical Sciences, Nagoya Institute of Technology, Gokiso, Showa-ku, Nagoya 466-8555, Japan.
| | - Mohamad Hekarl Uzir
- School of Chemical Engineering, Engineering Campus, Universiti Sains Malaysia, Nibong Tebal, Pulau Pinang 14300, Malaysia.
| |
Collapse
|
5
|
Alishah Aratboni H, Martinez M, Olvera C, Ayala M. Thermostabilization of a fungal laccase by entrapment in enzymatically synthesized levan nanoparticles. PLoS One 2024; 19:e0304242. [PMID: 39024280 PMCID: PMC11257323 DOI: 10.1371/journal.pone.0304242] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/04/2023] [Accepted: 05/08/2024] [Indexed: 07/20/2024] Open
Abstract
In this work, we present a comprehensive investigation of the entrapment of laccase, a biotechnologically relevant enzyme, into levan-based nanoparticles (LNPs). The entrapment of laccase was achieved concomitantly with the synthesis of LNP, catalyzed by a truncated version of a levansucrase from Leuconostoc mesenteroides. The study aimed to obtain a biocompatible nanomaterial, able to entrap functional laccase, and characterize its physicochemical, kinetic and thermal stability properties. The experimental findings demonstrated that a colloidal stable solution of spherically shaped LNP, with an average diameter of 68 nm, was obtained. An uniform particle size distribution was observed, according to the polydispersity index determined by DLS. When the LNPs synthesis was performed in the presence of laccase, biocatalytically active nanoparticles with a 1.25-fold larger diameter (85 nm) were obtained, and a maximum load of 243 μg laccase per g of nanoparticle was achieved. The catalytic efficiency was 972 and 103 (μM·min)-1, respectively, for free and entrapped laccase. A decrease in kcat values (from 7050 min-1 to 1823 min-1) and an increase in apparent Km (from 7.25 μM to 17.73 μM) was observed for entrapped laccase, compared to the free enzyme. The entrapped laccase exhibited improved thermal stability, retaining 40% activity after 1 h-incubation at 70°C, compared to complete inactivation of free laccase under the same conditions, thereby highlighting the potential of LNPs in preserving enzyme activity under elevated temperatures. The outcomes of this investigation significantly contribute to the field of nanobiotechnology by expanding the applications of laccase and presenting an innovative strategy for enhancing enzyme stability through the utilization of fructan-based nanoparticle entrapments.
Collapse
Affiliation(s)
- Hossein Alishah Aratboni
- Departamento de Ingeniería Celular y Biocatálisis, Instituto de Biotecnología UNAM, Cuernavaca, Morelos, Mexico
| | - Maura Martinez
- Departamento de Ingeniería Celular y Biocatálisis, Instituto de Biotecnología UNAM, Cuernavaca, Morelos, Mexico
| | - Clarita Olvera
- Departamento de Ingeniería Celular y Biocatálisis, Instituto de Biotecnología UNAM, Cuernavaca, Morelos, Mexico
| | - Marcela Ayala
- Departamento de Ingeniería Celular y Biocatálisis, Instituto de Biotecnología UNAM, Cuernavaca, Morelos, Mexico
| |
Collapse
|
6
|
Dokuzparmak E. Industrial Approach to Invertase Production from Fruit Waste for Enhanced Efficiency and Conservation. ACS OMEGA 2024; 9:26183-26194. [PMID: 38911758 PMCID: PMC11190939 DOI: 10.1021/acsomega.4c01732] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 02/22/2024] [Revised: 05/21/2024] [Accepted: 05/24/2024] [Indexed: 06/25/2024]
Abstract
This study investigates the commercial viability of repurposing fruit waste for enzyme production, specifically focusing on the invertase enzyme derived from Saccharomyces cerevisiae. By utilizing fruit pulp that incorporates mulberry, carob, Figure, and grape pulp as a nutrient source, it is observed that the culture medium containing carob pulp exhibits the highest invertase activity. Specifically, the invertase activity in this medium is approximately 2.5 times greater (12.90 U/mg protein) than that observed in the peptone medium (5.98 U/mg protein). The extract undergoes several purification steps, including ultrafiltration, ammonium sulfate precipitation, dialysis, and ion-exchange chromatography (purification ratio: 12.11 times, yield: 26.93%). The purified enzyme is immobilized using alginate beads, improving pH and thermal stability. The immobilized enzyme exhibits optimal activity between pH 3.50 and pH 7.00, thereby broadening the enzyme's high-activity pH range. The thermal stability of the immobilized invertase enzyme is significantly improved, especially at 65 °C. Activity studies in the presence of metal ions and certain chemicals have been conducted. The immobilized enzyme's activity increases by approximately 40% in the presence of Ca2+ and Mg2+, and the immobilized enzyme maintains its activity in the presence of detergents such as SDS, Tween-20, and organic solvents like ethanol and methanol. The potential for the reuse of immobilized invertase was investigated under standard assay conditions. After 20 cycles, the immobilized enzyme was found to retain 80% of its initial activity. Overall, the study establishes the commercial potential of fruit pulp, typically discarded in fruit juice production, as a valuable source for obtaining an invertase enzyme. Furthermore, this study also aims to develop a suitable purification process for invertase in the fruit juice industry. By harnessing fruit waste and implementing innovative enzyme production strategies, industries can enhance their efficiency, reduce their environmental footprint, and optimize resource utilization.
Collapse
Affiliation(s)
- Emre Dokuzparmak
- Ege University, Department
of Bioengineering, Faculty of Engineering, İzmir 35040, Turkey
| |
Collapse
|
7
|
Han Y, Jiang H, Huang C, Wu X, Ouyang Y, Chen H, Lan D, Wang Y, Zheng B, Xia J. Enzymatic interfacial conversion of acylglycerols in Pickering emulsions stabilized by hydrogel microparticles. J Colloid Interface Sci 2024; 661:228-236. [PMID: 38301461 DOI: 10.1016/j.jcis.2024.01.192] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/07/2023] [Revised: 01/04/2024] [Accepted: 01/26/2024] [Indexed: 02/03/2024]
Abstract
HYPOTHESIS A critical challenge in the enzymatic conversion of acylglycerols is the limited exposure of the enzyme dissolved in the aqueous solution to the hydrophobic substrate in the oil phase. Positioning the enzyme in a microenvironment with balanced hydrophobicity and hydrophilicity in Pickering emulsion will facilitate the acylglycerol-catalyzing reactions at the interface between the oil and liquid phases. EXPERIMENTS In this work, to overcome the challenge of biphasic catalysis, we report a method to immobilize enzymes in polyethylene glycol (PEG)-based hydrogel microparticles (HMPs) at the interface between the oil and water phases in Pickering emulsion to promote the enzymatic conversion of acylglycerols. FINDINGS 3 wt% of HMPs can stabilize the oil-in-water Pickering emulsion for at least 14 days and increase the viscosity of emulsions. Lipase-HMP conjugates showed significantly higher hydrolytic activity in Pickering emulsion; HMP-immobilized lipase SMG1 showed an activity about three times that of free lipase SMG1. Co-immobilization of a lipase and a fatty acid photodecarboxylase from Chlorella variabilis (CvFAP) in Pickering emulsion enables light-driven cascade conversion of triacylglycerols to hydrocarbons, transforming waste oil to renewable biofuels in a green and sustainable approach. HMPs stabilize the Pickering emulsion and promote interfacial biocatalysis in converting acylglycerols to renewable biofuels.
Collapse
Affiliation(s)
- Yongxu Han
- Department of Chemistry, The Chinese University of Hong Kong, Shatin, Hong Kong, China
| | - Hao Jiang
- Department of Chemistry, The Chinese University of Hong Kong, Shatin, Hong Kong, China
| | - Chen Huang
- Department of Chemistry, The Chinese University of Hong Kong, Shatin, Hong Kong, China
| | - Xue Wu
- Department of Chemistry, The Chinese University of Hong Kong, Shatin, Hong Kong, China
| | - Yinghan Ouyang
- Department of Chemistry, The Chinese University of Hong Kong, Shatin, Hong Kong, China
| | - Hongfei Chen
- Department of Chemistry, The Chinese University of Hong Kong, Shatin, Hong Kong, China
| | - Dongming Lan
- School of Food Science and Engineering, South China University of Technology, Guangzhou 510640, China
| | - Yonghua Wang
- School of Food Science and Engineering, South China University of Technology, Guangzhou 510640, China
| | - Bo Zheng
- Department of Chemistry, The Chinese University of Hong Kong, Shatin, Hong Kong, China
| | - Jiang Xia
- Department of Chemistry, The Chinese University of Hong Kong, Shatin, Hong Kong, China.
| |
Collapse
|
8
|
Scheibel DM, Gitsov IPI, Gitsov I. Enzymes in "Green" Synthetic Chemistry: Laccase and Lipase. Molecules 2024; 29:989. [PMID: 38474502 DOI: 10.3390/molecules29050989] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/30/2023] [Revised: 02/14/2024] [Accepted: 02/22/2024] [Indexed: 03/14/2024] Open
Abstract
Enzymes play an important role in numerous natural processes and are increasingly being utilized as environmentally friendly substitutes and alternatives to many common catalysts. Their essential advantages are high catalytic efficiency, substrate specificity, minimal formation of byproducts, and low energy demand. All of these benefits make enzymes highly desirable targets of academic research and industrial development. This review has the modest aim of briefly overviewing the classification, mechanism of action, basic kinetics and reaction condition effects that are common across all six enzyme classes. Special attention is devoted to immobilization strategies as the main tools to improve the resistance to environmental stress factors (temperature, pH and solvents) and prolong the catalytic lifecycle of these biocatalysts. The advantages and drawbacks of methods such as macromolecular crosslinking, solid scaffold carriers, entrapment, and surface modification (covalent and physical) are discussed and illustrated using numerous examples. Among the hundreds and possibly thousands of known and recently discovered enzymes, hydrolases and oxidoreductases are distinguished by their relative availability, stability, and wide use in synthetic applications, which include pharmaceutics, food and beverage treatments, environmental clean-up, and polymerizations. Two representatives of those groups-laccase (an oxidoreductase) and lipase (a hydrolase)-are discussed at length, including their structure, catalytic mechanism, and diverse usage. Objective representation of the current status and emerging trends are provided in the main conclusions.
Collapse
Affiliation(s)
- Dieter M Scheibel
- Department of Chemistry, State University of New York-ESF, Syracuse, NY 13210, USA
| | - Ioan Pavel Ivanov Gitsov
- Science and Technology, Medtronic Incorporated, 710 Medtronic Parkway, Minneapolis, MN 55432, USA
| | - Ivan Gitsov
- Department of Chemistry, State University of New York-ESF, Syracuse, NY 13210, USA
- The Michael M. Szwarc Polymer Research Institute, Syracuse, NY 13210, USA
- Biomedical and Chemical Engineering Department, Syracuse University, Syracuse, NY 13210, USA
- BioInspired Institute, Syracuse, NY 13210, USA
| |
Collapse
|
9
|
Melo RLF, Sales MB, de Castro Bizerra V, de Sousa Junior PG, Cavalcante ALG, Freire TM, Neto FS, Bilal M, Jesionowski T, Soares JM, Fechine PBA, Dos Santos JCS. Recent applications and future prospects of magnetic biocatalysts. Int J Biol Macromol 2023; 253:126709. [PMID: 37696372 DOI: 10.1016/j.ijbiomac.2023.126709] [Citation(s) in RCA: 15] [Impact Index Per Article: 7.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/10/2023] [Revised: 08/25/2023] [Accepted: 09/03/2023] [Indexed: 09/13/2023]
Abstract
Magnetic biocatalysts combine magnetic properties with the catalytic activity of enzymes, achieving easy recovery and reuse in biotechnological processes. Lipases immobilized by magnetic nanoparticles dominate. This review covers an advanced bibliometric analysis and an overview of the area, elucidating research advances. Using WoS, 34,949 publications were analyzed and refined to 450. The prominent journals, countries, institutions, and authors that published the most were identified. The most cited articles showed research hotspots. The analysis of the themes and keywords identified five clusters and showed that the main field of research is associated with obtaining biofuels derived from different types of sustainable vegetable oils. The overview of magnetic biocatalysts showed that these materials are also employed in biosensors, photothermal therapy, environmental remediation, and medical applications. The industry shows a significant interest, with the number of patents increasing. Future studies should focus on immobilizing new lipases in unique materials with magnetic profiles, aiming to improve the efficiency for various biotechnological applications.
Collapse
Affiliation(s)
- Rafael Leandro Fernandes Melo
- Departamento de Engenharia Metalúrgica e de Materiais, Universidade Federal do Ceará, Campus do Pici, Bloco 729, Fortaleza CEP 60440-554, CE, Brazil; Grupo de Química de Materiais Avançados (GQMat), Departamento de Química Analítica e Físico-Química, Universidade Federal do Ceará, Campus do Pici, Fortaleza CEP 60451-970, CE, Brazil
| | - Misael Bessa Sales
- Instituto de Engenharias e Desenvolvimento Sustentável, Universidade da Integração Internacional da Lusofonia Afro-Brasileira, Campus das Auroras, Redenção CEP 62790-970, CE, Brazil
| | - Viviane de Castro Bizerra
- Instituto de Engenharias e Desenvolvimento Sustentável, Universidade da Integração Internacional da Lusofonia Afro-Brasileira, Campus das Auroras, Redenção CEP 62790-970, CE, Brazil
| | - Paulo Gonçalves de Sousa Junior
- Departamento de Química Orgânica e Inorgânica, Centro de Ciências, Universidade Federal do Ceará, Campus Pici, Fortaleza CEP 60455-760, CE, Brazil
| | - Antônio Luthierre Gama Cavalcante
- Departamento de Química Analítica e Físico-Química, Centro de Ciências, Universidade Federal do Ceará, Campus Pici, Fortaleza CEP 60455-760, CE, Brazil
| | - Tiago Melo Freire
- Grupo de Química de Materiais Avançados (GQMat), Departamento de Química Analítica e Físico-Química, Universidade Federal do Ceará, Campus do Pici, Fortaleza CEP 60451-970, CE, Brazil; Departamento de Química Analítica e Físico-Química, Centro de Ciências, Universidade Federal do Ceará, Campus Pici, Fortaleza CEP 60455-760, CE, Brazil
| | - Francisco Simão Neto
- Departamento de Engenharia Química, Universidade Federal do Ceará, Campus do Pici, Bloco 709, Fortaleza CEP 60455-760, CE, Brazil
| | - Muhammad Bilal
- Department of Sanitary Engineering, Faculty of Civil and Environmental Engineering, Gdansk University of Technology, G. Narutowicza 11/12 Str., 80-233 Gdansk, Poland
| | - Teofil Jesionowski
- Institute of Chemical Technology and Engineering, Faculty of Chemical Technology, Poznan University of Technology, Berdychowo 4, PL-60965 Poznan, Poland
| | - João Maria Soares
- Departamento de Física, Universidade do Estado do Rio Grande do Norte, Campus Mossoró, Mossoró CEP 59610-090, RN, Brazil
| | - Pierre Basílio Almeida Fechine
- Grupo de Química de Materiais Avançados (GQMat), Departamento de Química Analítica e Físico-Química, Universidade Federal do Ceará, Campus do Pici, Fortaleza CEP 60451-970, CE, Brazil; Departamento de Química Analítica e Físico-Química, Centro de Ciências, Universidade Federal do Ceará, Campus Pici, Fortaleza CEP 60455-760, CE, Brazil
| | - José Cleiton Sousa Dos Santos
- Instituto de Engenharias e Desenvolvimento Sustentável, Universidade da Integração Internacional da Lusofonia Afro-Brasileira, Campus das Auroras, Redenção CEP 62790-970, CE, Brazil; Departamento de Engenharia Química, Universidade Federal do Ceará, Campus do Pici, Bloco 709, Fortaleza CEP 60455-760, CE, Brazil.
| |
Collapse
|
10
|
Patil PD, Salokhe S, Karvekar A, Suryavanshi P, Phirke AN, Tiwari MS, Nadar SS. Microfluidic based continuous enzyme immobilization: A comprehensive review. Int J Biol Macromol 2023; 253:127358. [PMID: 37827414 DOI: 10.1016/j.ijbiomac.2023.127358] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/14/2023] [Revised: 10/05/2023] [Accepted: 10/09/2023] [Indexed: 10/14/2023]
Abstract
Conventional techniques for enzyme immobilization suffer from suboptimal activity recovery due to insufficient enzyme loading and inadequate stability. Furthermore, these techniques are time-consuming and involve multiple steps which limit the applicability of immobilized enzymes. In contrast, the use of microfluidic devices for enzyme immobilization has garnered significant attention due to its ability to precisely control immobilization parameters, resulting in highly active immobilized enzymes. This approach offers several advantages, including reduced time and energy consumption, enhanced mass-heat transfer, and improved control over the mixing process. It maintains the superior structural configuration in immobilized form which ultimately affects the overall efficiency. The present review article comprehensively explains the design, construction, and various methods employed for enzyme immobilization using microfluidic devices. The immobilized enzymes prepared using these techniques demonstrated excellent catalytic activity, remarkable stability, and outstanding recyclability. Moreover, they have found applications in diverse areas such as biosensors, biotransformation, and bioremediation. The review article also discusses potential future developments and foresees significant challenges associated with enzyme immobilization using microfluidics, along with potential remedies. The development of this advanced technology not only paves the way for novel and innovative approaches to enzyme immobilization but also allows for the straightforward scalability of microfluidic-based techniques from an industrial standpoint.
Collapse
Affiliation(s)
- Pravin D Patil
- Department of Basic Science & Humanities, SVKM'S NMIMS Mukesh Patel School of Technology Management & Engineering, Mumbai, Maharashtra 400056, India
| | - Sakshi Salokhe
- Department of Biotechnology Engineering, Kolhapur Institute of Technology's College of Engineering, Kolhapur 416 234, India
| | - Aparna Karvekar
- Department of Biotechnology Engineering, Kolhapur Institute of Technology's College of Engineering, Kolhapur 416 234, India
| | - Prabhavati Suryavanshi
- Department of Biotechnology Engineering, Kolhapur Institute of Technology's College of Engineering, Kolhapur 416 234, India
| | - Ajay N Phirke
- Department of Basic Science & Humanities, SVKM'S NMIMS Mukesh Patel School of Technology Management & Engineering, Mumbai, Maharashtra 400056, India
| | - Manishkumar S Tiwari
- Department of Data Science, SVKM'S NMIMS Mukesh Patel School of Technology Management & Engineering, Mumbai, Maharashtra 400056, India
| | - Shamraja S Nadar
- Department of Chemical Engineering, Institute of Chemical Technology, Matunga (E), Mumbai 400019, India.
| |
Collapse
|
11
|
Yadav N, Chahar D, Bisht M, Venkatesu P. Assessing the compatibility of choline-based deep eutectic solvents for the structural stability and activity of cellulase: Enzyme sustain at high temperature. Int J Biol Macromol 2023; 249:125988. [PMID: 37499720 DOI: 10.1016/j.ijbiomac.2023.125988] [Citation(s) in RCA: 6] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/06/2023] [Revised: 07/13/2023] [Accepted: 07/23/2023] [Indexed: 07/29/2023]
Abstract
As a new generation of 'green solvents' deep eutectic solvents (DESs) represents a promising alternative to the conventional solvents. Their environmental-benign nature and designer properties promote their utility in biocatalysis. Enzymes are marginally stable when exposed to physical/chemical disturbances. One such enzyme is cellulase which is a propitious catalyst for the depolymerization of cellulose under mild conditions. Therefore, their stability is a prerequisite condition to match demands of biorefineries. To address this issue of low stability, activity and thermal denaturation of cellulase, there is a need to find a sustainable and suitable co-solvent that is biocompatible with enzymes ultimately to facilitate their application in bio-industries. In this regard, we synthesized three choline-based DESs, choline chloride (ChCl)-glycerol, ChCl-ethylene glycol and ChCl-lactic acid and employed them to analyze their suitability for cellulase. The present study systematically evaluates the influence of the mentioned DESs on stability, activity and thermal stability of cellulase with the help of various spectroscopic techniques. The spectroscopic analysis revealed that the structural stability and activity of the enzyme were improved in presence of ChCl-glycerol and ChCl-ethylene glycol. The thermal stability was also very well maintained in both the DESs. Interestingly, the relative activity of cellulase was >80 % even after incubation at 50 °C after 48 h for both the DESs. This activity preservation behaviour was more pronounced for ChCl-ethylene glycol than ChCl-glycerol. Moreover, temperature variations studies also reveal promising results by maintain conformational intactness. On the other side, ChCl-lactic acid showed a deleterious effect on the enzyme both structurally as well as thermally. The dynamic light scattering (DLS) analysis provides more specific information about the negative influence of ChCl-lactic acid towards cellulase native structure. This DES induces unavoidable alterations in the enzyme structure which leads to the unfolding of enzyme, ultimately, destabilizing it. Overall, our results present a physical insight into how the enzyme stability and activity depend on the nature of DES. Also, the findings will help to facilitate the development and application of DESs as biocatalytic process.
Collapse
Affiliation(s)
- Niketa Yadav
- Department of Chemistry, University of Delhi, Delhi 110 007, India
| | - Deepak Chahar
- Department of Chemistry, University of Delhi, Delhi 110 007, India
| | - Meena Bisht
- Department of Chemistry, Sri Venkateswara College, University of Delhi, Delhi 110 007, India
| | | |
Collapse
|
12
|
dos Santos LN, Perna RF, Vieira AC, de Almeida AF, Ferreira NR. Trends in the Use of Lipases: A Systematic Review and Bibliometric Analysis. Foods 2023; 12:3058. [PMID: 37628057 PMCID: PMC10453403 DOI: 10.3390/foods12163058] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/19/2023] [Revised: 07/27/2023] [Accepted: 07/28/2023] [Indexed: 08/27/2023] Open
Abstract
Scientific mapping using bibliometric data network analysis was applied to analyze research works related to lipases and their industrial applications, evaluating the current state of research, challenges, and opportunities in the use of these biocatalysts, based on the evaluation of a large number of publications on the topic, allowing a comprehensive systematic data analysis, which had not yet been conducted in relation to studies specifically covering lipases and their industrial applications. Thus, studies involving lipase enzymes published from 2018 to 2022 were accessed from the Web of Science database. The extracted records result in the analysis of terms of bibliographic compatibility among the articles, co-occurrence of keywords, and co-citation of journals using the VOSviewer algorithm in the construction of bibliometric maps. This systematic review analysis of 357 documents, including original and review articles, revealed studies inspired by lipase enzymes in the research period, showing that the development of research, together with different areas of knowledge, presents good results related to the applications of lipases, due to information synchronization. Furthermore, this review showed the main challenges in lipase applications regarding increased production and operational stability; establishing well-defined evaluation criteria, such as cultivation conditions, activity, biocatalyst stability, type of support and reactor; thermodynamic studies; reuse cycles; and it can assist in defining goals for the development of successful large-scale applications, showing several points for improvement of future studies on lipase enzymes.
Collapse
Affiliation(s)
- Lucely Nogueira dos Santos
- Postgraduate Program in Food Science and Technology, Institute of Technology, Federal University of Pará (UFPA), Belém 66075-110, Brazil;
| | - Rafael Firmani Perna
- Graduate Program in Chemical Engineering, Institute of Science and Technology, Federal University of Alfenas (UNIFAL-MG), Poços de Caldas 37715-400, Brazil; (R.F.P.); (A.C.V.)
| | - Ana Carolina Vieira
- Graduate Program in Chemical Engineering, Institute of Science and Technology, Federal University of Alfenas (UNIFAL-MG), Poços de Caldas 37715-400, Brazil; (R.F.P.); (A.C.V.)
| | - Alex Fernando de Almeida
- Engineering of Bioprocesses and Biotechnology, Federal University of Tocantins (UFT-TO), Gurupi 77402-970, Brazil;
| | - Nelson Rosa Ferreira
- Postgraduate Program in Food Science and Technology, Institute of Technology, Federal University of Pará (UFPA), Belém 66075-110, Brazil;
- Faculty of Food Engineering, Institute of Technology, Federal University of Pará (UFPA), Belém 66075-110, Brazil
| |
Collapse
|
13
|
Scuto FR, Ciarlantini C, Chiappini V, Pietrelli L, Piozzi A, Girelli AM. Design of a 3D Amino-Functionalized Rice Husk Ash Nano-Silica/Chitosan/Alginate Composite as Support for Laccase Immobilization. Polymers (Basel) 2023; 15:3127. [PMID: 37514516 PMCID: PMC10383677 DOI: 10.3390/polym15143127] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/11/2023] [Revised: 07/19/2023] [Accepted: 07/20/2023] [Indexed: 07/30/2023] Open
Abstract
Recycling of agro-industrial waste is one of the major issues addressed in recent years aimed at obtaining products with high added value as a future alternative to traditional ones in the per-spective of a bio-based and circular economy. One of the most produced wastes is rice husk and it is particularly interesting because it is very rich in silica, a material with a high intrinsic value. In the present study, a method to extract silica from rice husk ash (RHA) and to use it as a carrier for the immobilization of laccase from Trametes versicolor was developed. The obtained mesoporous nano-silica was characterized by X-ray diffraction (XRD), ATR-FTIR spectroscopy, Scanning Elec-tron Microscopy (SEM), and Energy Dispersive X-ray spectroscopy (EDS). A nano-silica purity of about 100% was found. Nano-silica was then introduced in a cross-linked chitosan/alginate scaffold to make it more easily recoverable after reuse. To favor laccase immobilization into the composite scaffold, functionalization of the nano-silica with (γ-aminopropyl) triethoxysilane (APTES) was performed. The APTES/RHA nano-silica/chitosan/alginate (ARCA) composite al-lowed to obtain under mild conditions (pH 7, room temperature, 1.5 h reaction time) a robust and easily reusable solid biocatalyst with 3.8 U/g of immobilized enzyme which maintained 50% of its activity after six reuses. The biocatalytic system, tested for syringic acid bioremediation, was able to totally oxidize the contaminant in 24 h.
Collapse
Affiliation(s)
- Francesca Romana Scuto
- Department of Chemistry, Sapienza University of Rome, P. le A. Moro 5, 00185 Rome, Italy
| | - Clarissa Ciarlantini
- Department of Chemistry, Sapienza University of Rome, P. le A. Moro 5, 00185 Rome, Italy
| | - Viviana Chiappini
- Department of Chemistry, Sapienza University of Rome, P. le A. Moro 5, 00185 Rome, Italy
| | - Loris Pietrelli
- DAFNE Department, Tuscia University, Via Santa Maria in Gradi 4, 01100 Viterbo, Italy
| | - Antonella Piozzi
- Department of Chemistry, Sapienza University of Rome, P. le A. Moro 5, 00185 Rome, Italy
| | - Anna M Girelli
- Department of Chemistry, Sapienza University of Rome, P. le A. Moro 5, 00185 Rome, Italy
| |
Collapse
|
14
|
Mineralization of Lipase from Thermomyces lanuginosus Immobilized on Methacrylate Beads Bearing Octadecyl Groups to Improve Enzyme Features. Catalysts 2022. [DOI: 10.3390/catal12121552] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/03/2022] Open
Abstract
Lipase from Thermomyces lanuginosus (TLL) has been immobilized on Purolite Lifetech® ECR8806F (viz. methacrylate macroporous resin containing octadecyl groups, designated as Purolite C18-TLL), and the enzyme performance has been compared to that of the enzyme immobilized on octyl-agarose, designated as agarose C8-TLL. The hydrolytic activity versus p-nitrophenol butyrate decreased significantly, and to a lower extent versus S-methyl mandelate (more than twofold), while versus triacetin and R-methyl mandelate, the enzyme activity was higher for the biocatalyst prepared using Purolite C18 (up to almost five-fold). Regarding the enzyme stability, Purolite C18-TLL was significantly more stable than the agarose C8-TLL. Next, the biocatalysts were mineralized using zinc, copper or cobalt phosphates. Mineralization increased the hydrolytic activity of Purolite C18-TLL versus triacetin and R-methyl mandelate, while this activity decreased very significantly versus the S-isomer, while the effects using agarose C8-TLL were more diverse (hydrolytic activity increase or decrease was dependent on the metal and substrate). The zinc salt treatment increased the stability of both biocatalysts, but with a lower impact for Purolite C18-TLL than for agarose-C8-TLL. On the contrary, the copper and cobalt salt treatments decreased enzyme stability, but more intensively using Purolite C18-TLL. The results show that even using enzymes immobilized following the same strategy, the differences in the enzyme conformation cause mineralization to have diverse effects on enzyme stability, hydrolytic activity, and specificity.
Collapse
|
15
|
Holyavka MG, Goncharova SS, Sorokin AV, Lavlinskaya MS, Redko YA, Faizullin DA, Baidamshina DR, Zuev YF, Kondratyev MS, Kayumov AR, Artyukhov VG. Novel Biocatalysts Based on Bromelain Immobilized on Functionalized Chitosans and Research on Their Structural Features. Polymers (Basel) 2022; 14:5110. [PMID: 36501516 PMCID: PMC9739615 DOI: 10.3390/polym14235110] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/03/2022] [Revised: 11/18/2022] [Accepted: 11/22/2022] [Indexed: 11/27/2022] Open
Abstract
Enzyme immobilization on various carriers represents an effective approach to improve their stability, reusability, and even change their catalytic properties. Here, we show the mechanism of interaction of cysteine protease bromelain with the water-soluble derivatives of chitosan-carboxymethylchitosan, N-(2-hydroxypropyl)-3-trimethylammonium chitosan, chitosan sulfate, and chitosan acetate-during immobilization and characterize the structural features and catalytic properties of obtained complexes. Chitosan sulfate and carboxymethylchitosan form the highest number of hydrogen bonds with bromelain in comparison with chitosan acetate and N-(2-hydroxypropyl)-3-trimethylammonium chitosan, leading to a higher yield of protein immobilization on chitosan sulfate and carboxymethylchitosan (up to 58 and 65%, respectively). In addition, all derivatives of chitosan studied in this work form hydrogen bonds with His158 located in the active site of bromelain (except N-(2-hydroxypropyl)-3-trimethylammonium chitosan), apparently explaining a significant decrease in the activity of biocatalysts. The N-(2-hydroxypropyl)-3-trimethylammonium chitosan displays only physical interactions with His158, thus possibly modulating the structure of the bromelain active site and leading to the hyperactivation of the enzyme, up to 208% of the total activity and 158% of the specific activity. The FTIR analysis revealed that interaction between N-(2-hydroxypropyl)-3-trimethylammonium chitosan and bromelain did not significantly change the enzyme structure. Perhaps this is due to the slowing down of aggregation and the autolysis processes during the complex formation of bromelain with a carrier, with a minimal modification of enzyme structure and its active site orientation.
Collapse
Affiliation(s)
- Marina G. Holyavka
- Biophysics and Biotechnology Department, Voronezh State University, 1 Universitetskaya Square, 394018 Voronezh, Russia
- Laboratory of Bioresource Potential of Coastal Area, Institute for Advanced Studies, Sevastopol State University, 33 Studencheskaya Street, 299053 Sevastopol, Russia
| | - Svetlana S. Goncharova
- Biophysics and Biotechnology Department, Voronezh State University, 1 Universitetskaya Square, 394018 Voronezh, Russia
| | - Andrey V. Sorokin
- Biophysics and Biotechnology Department, Voronezh State University, 1 Universitetskaya Square, 394018 Voronezh, Russia
- Laboratory of Bioresource Potential of Coastal Area, Institute for Advanced Studies, Sevastopol State University, 33 Studencheskaya Street, 299053 Sevastopol, Russia
- Metagenomics and Food Biotechnologies Laboratory, Voronezh State University of Engineering Technologies, 19 Revolutsii Avenue, 394036 Voronezh, Russia
| | - Maria S. Lavlinskaya
- Biophysics and Biotechnology Department, Voronezh State University, 1 Universitetskaya Square, 394018 Voronezh, Russia
- Laboratory of Bioresource Potential of Coastal Area, Institute for Advanced Studies, Sevastopol State University, 33 Studencheskaya Street, 299053 Sevastopol, Russia
- Metagenomics and Food Biotechnologies Laboratory, Voronezh State University of Engineering Technologies, 19 Revolutsii Avenue, 394036 Voronezh, Russia
| | - Yulia A. Redko
- Biophysics and Biotechnology Department, Voronezh State University, 1 Universitetskaya Square, 394018 Voronezh, Russia
| | - Dzhigangir A. Faizullin
- Kazan Institute of Biochemistry and Biophysics, FRC Kazan Scientific Center of the RAS, 2/31 Lobachevsky Street, 420111 Kazan, Russia
| | - Diana R. Baidamshina
- Institute of Fundamental Medicine and Biology, Kazan Federal University, 18 Kremlevskaya Street, 420008 Kazan, Russia
| | - Yuriy F. Zuev
- Kazan Institute of Biochemistry and Biophysics, FRC Kazan Scientific Center of the RAS, 2/31 Lobachevsky Street, 420111 Kazan, Russia
| | - Maxim S. Kondratyev
- Biophysics and Biotechnology Department, Voronezh State University, 1 Universitetskaya Square, 394018 Voronezh, Russia
- Laboratory of Structure and Dynamics of Biomolecular Systems, Institute of Cell Biophysics of the RAS, 3 Institutskaya Street, 142290 Pushchino, Russia
| | - Airat R. Kayumov
- Institute of Fundamental Medicine and Biology, Kazan Federal University, 18 Kremlevskaya Street, 420008 Kazan, Russia
| | - Valeriy G. Artyukhov
- Biophysics and Biotechnology Department, Voronezh State University, 1 Universitetskaya Square, 394018 Voronezh, Russia
| |
Collapse
|
16
|
Souza PMP, Carballares D, Gonçalves LRB, Fernandez-Lafuente R, Rodrigues S. Immobilization of Lipase B from Candida antarctica in Octyl-Vinyl Sulfone Agarose: Effect of the Enzyme-Support Interactions on Enzyme Activity, Specificity, Structure and Inactivation Pathway. Int J Mol Sci 2022; 23:ijms232214268. [PMID: 36430745 PMCID: PMC9697615 DOI: 10.3390/ijms232214268] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/06/2022] [Revised: 10/21/2022] [Accepted: 11/11/2022] [Indexed: 11/21/2022] Open
Abstract
Lipase B from Candida antarctica was immobilized on heterofunctional support octyl agarose activated with vinyl sulfone to prevent enzyme release under drastic conditions. Covalent attachment was established, but the blocking step using hexylamine, ethylenediamine or the amino acids glycine (Gly) and aspartic acid (Asp) altered the results. The activities were lower than those observed using the octyl biocatalyst, except when using ethylenediamine as blocking reagent and p-nitrophenol butyrate (pNPB) as substrate. The enzyme stability increased using these new biocatalysts at pH 7 and 9 using all blocking agents (much more significantly at pH 9), while it decreased at pH 5 except when using Gly as blocking agent. The stress inactivation of the biocatalysts decreased the enzyme activity versus three different substrates (pNPB, S-methyl mandelate and triacetin) in a relatively similar fashion. The tryptophane (Trp) fluorescence spectra were different for the biocatalysts, suggesting different enzyme conformations. However, the fluorescence spectra changes during the inactivation were not too different except for the biocatalyst blocked with Asp, suggesting that, except for this biocatalyst, the inactivation pathways may not be so different.
Collapse
Affiliation(s)
- Priscila M. P. Souza
- Departamento de Biocatálisis, ICP-CSIC, Campus UAM-CSIC, 28049 Madrid, Spain
- Food Engineering Department, Federal University of Ceará, Campus do Pici, Bloco 858, Fortaleza CEP 60440-900, CE, Brazil
| | - Diego Carballares
- Departamento de Biocatálisis, ICP-CSIC, Campus UAM-CSIC, 28049 Madrid, Spain
| | - Luciana R. B. Gonçalves
- Chemical Engineering Department, Federal University of Ceará, Campus do Pici, Bloco 709, Fortaleza CEP 60440-900, CE, Brazil
| | - Roberto Fernandez-Lafuente
- Departamento de Biocatálisis, ICP-CSIC, Campus UAM-CSIC, 28049 Madrid, Spain
- Center of Excellence in Bionanoscience Research, Member of the External Scientific Advisory Academics, King Abdulaziz University, Jeddah 21589, Saudi Arabia
- Correspondence: (R.F.-L.); (S.R.)
| | - Sueli Rodrigues
- Food Engineering Department, Federal University of Ceará, Campus do Pici, Bloco 858, Fortaleza CEP 60440-900, CE, Brazil
- Correspondence: (R.F.-L.); (S.R.)
| |
Collapse
|
17
|
Nano-fibrillated cellulose-based scaffolds for enzyme (co)-immobilization: Application to natural product glycosylation by Leloir glycosyltransferases. Int J Biol Macromol 2022; 222:217-227. [PMID: 36165869 DOI: 10.1016/j.ijbiomac.2022.09.160] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/25/2022] [Revised: 09/15/2022] [Accepted: 09/16/2022] [Indexed: 11/21/2022]
Abstract
Polysaccharide-based scaffolds are promising carriers for enzyme immobilization. Here, we demonstrate a porous scaffold prepared by direct-ink-writing 3D printing of an ink consisting of nanofibrillated cellulose, carboxymethyl cellulose and citric acid for immobilization application. Negative surface charge introduced by the components made the scaffold amenable for an affinity-like immobilization via the cationic protein module Zbasic2. Zbasic2 fusions of two sugar nucleotide-dependent glycosyltransferases (C-glycosyltransferase, Z-CGT; sucrose synthase, Z-SuSy) were immobilized individually, or co-immobilized, and applied to synthesize the natural C-glycoside nothofagin. The cascade reaction involved β-C-glycosylation of phloretin (10 mM, ~90 % conversion) from UDP-glucose, provided from sucrose and catalytic amounts of UDP (1.0 mM). Enzymes were co-immobilized at ~65 mg protein/g carrier to receive activities of 9.5 U/g (Z-CGT) and 4.5 U/g (Z-SuSy) in 22-33 % yield (protein) and an effectiveness of 23 % (Z-CGT) and 13 % (Z-SuSy). The scaffold-bound enzymes were recyclable for 5 consecutive reactions.
Collapse
|
18
|
Morellon-Sterling R, Bolivar JM, Fernandez-Lafuente R. Switch off/switch on of a cysteinyl protease as a way to preserve the active catalytic group by modification with a reversible covalent thiol modifier: Immobilization of ficin on vinyl-sulfone activated supports. Int J Biol Macromol 2022; 220:1155-1162. [PMID: 36037909 DOI: 10.1016/j.ijbiomac.2022.08.155] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/23/2022] [Revised: 08/11/2022] [Accepted: 08/23/2022] [Indexed: 11/05/2022]
Abstract
The immobilization of ficin (a cysteinyl proteases) on vinyl sulfone agarose produced its almost full inactivation. It was observed that the incubation of the free and immobilized enzyme in β-mercaptoethanol produced a 20 % of enzyme activity recovery, suggesting that the inactivation due to the immobilization could be a consequence of the modification of the catalytic Cys. To prevent the enzyme inactivation during the immobilization, switching off of ficin via Cys reaction with dipyridyl-disulfide was implemented, giving a reversible disulfide bond that produced a fully inactive enzyme. The switch on of ficin activity was implemented by incubation in 1 M β-mercaptoethanol. Using this strategy to immobilize the enzyme on vinyl sulfone agarose beads, the expressed activity of the immobilized ficin could be boosted up to 80 %. The immobilized enzyme presented a thermal stabilization similar to that obtained using ficin-glyoxyl-agarose beads. This procedure may be extended to many enzymes containing critical Cys, to permit their immobilization or chemical modification.
Collapse
Affiliation(s)
- Roberto Morellon-Sterling
- Departamento de Biocatálisis, ICP-CSIC, Marie Curie 2, Campus UAM-CSIC Cantoblanco, 28049 Madrid, Spain; Student of Departamento de Biología Molecular, Universidad Autónoma de Madrid, Darwin 2, Campus UAM-CSIC, Cantoblanco, 28049 Madrid, Spain
| | - Juan M Bolivar
- FQPIMA Group, Chemical and Materials Engineering Department, Faculty of Chemical Sciences, Complutense University of Madrid, Complutense Ave., Madrid 28040, Spain
| | - Roberto Fernandez-Lafuente
- Departamento de Biocatálisis, ICP-CSIC, Marie Curie 2, Campus UAM-CSIC Cantoblanco, 28049 Madrid, Spain; Center of Excellence in Bionanoscience Research, External Scientific Advisory Academics, King Abdulaziz University, Jeddah 21589, Saudi Arabia.
| |
Collapse
|
19
|
Sampaio CS, Angelotti JAF, Fernandez-Lafuente R, Hirata DB. Lipase immobilization via cross-linked enzyme aggregates: Problems and prospects - A review. Int J Biol Macromol 2022; 215:434-449. [PMID: 35752332 DOI: 10.1016/j.ijbiomac.2022.06.139] [Citation(s) in RCA: 41] [Impact Index Per Article: 13.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/04/2022] [Revised: 06/06/2022] [Accepted: 06/20/2022] [Indexed: 02/08/2023]
Abstract
In this review we have focused on the preparation of cross-linked enzyme aggregates (CLEAs) from lipases, as these are among the most used enzyme in bioprocesses. This immobilization method is considered very attractive due to preparation simplicity, non-use of supports and the possibility of using crude enzyme extracts. CLEAs provide lipase stabilization under extreme temperature or pH conditions or in the presence of organic solvents, in addition to preventing enzyme leaching in aqueous medium. However, it presents some problems in the preparation and limitations in their use. The problems in preparation refer mainly to the crosslinking step, and may be solved using an aminated feeder. The problems in handling have been tackled designing magnetic-CLEAs or trapping the CLEAs in particles with better mechanical properties, the substrate diffusion problems has been reduced by producing more porous-CLEAs, etc. The enzyme co-immobilization using combi-CLEAs is also a new tendency. Therefore, this review explores the CLEAs methodology aimed at lipase immobilization and its applications.
Collapse
Affiliation(s)
- Camila S Sampaio
- Postgraduate Program in Biotechnology, Federal University of Alfenas, 37130-001 Alfenas, MG, Brazil
| | - Joelise A F Angelotti
- Postgraduate Program in Biotechnology, Federal University of Alfenas, 37130-001 Alfenas, MG, Brazil
| | - Roberto Fernandez-Lafuente
- Department of Biocatalysis, ICP-CSIC, Campus UAM-CSIC, Cantoblanco, 28049 Madrid, Spain.; Center of Excellence in Bionanoscience Research, Member of The External Scientific Advisory Board, King Abdulaziz University, Jeddah, Saudi Arabia.
| | - Daniela B Hirata
- Postgraduate Program in Biotechnology, Federal University of Alfenas, 37130-001 Alfenas, MG, Brazil.
| |
Collapse
|
20
|
Chen X, Chen X, Zhu L, Liu W, Jiang L. Efficient production of inulo-oligosaccharides from inulin by exo- and endo-inulinase co-immobilized onto a self-assembling protein scaffold. Int J Biol Macromol 2022; 210:588-599. [PMID: 35513090 DOI: 10.1016/j.ijbiomac.2022.04.213] [Citation(s) in RCA: 12] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/28/2022] [Revised: 04/19/2022] [Accepted: 04/28/2022] [Indexed: 11/19/2022]
Abstract
Inulin can be hydrolyzed by inulinases to yield inulo-oligosaccharides (IOSs), which have great application potential in the food and nutraceutical industries. However, conventional enzymatic production of IOSs is limited by long hydrolysis times and poor thermo-stability of inulinases. Here, the self-assembling protein scaffold EutM was engineered to co-immobilize exo-inulinase (EXINU) and endo-inulinase (ENINU) for synergistic hydrolysis of inulin to produce IOSs with 3 to 5 monosaccharide units (DP3-5 IOSs). The immobilization of EXINU/ENINU onto the EutM scaffold resulted in an increase of catalytic efficiency, a 65% increase of the Vmax of ENINU, as well as an increase of thermo-stability, with 4.26-fold higher residual activity of EXINU after 22 h-incubation at 50 °C. After optimization, two efficient production protocols were obtained, in which the yield and productivity of DP3-5 IOSs reached 80.38% and 70.86 g·(L·h)-1, respectively, which were at a high level in similar studies. Overall, this study provides an attractive self-assembling protein platform for the co-immobilization of inulinases, as well as optimized bioprocesses with great promise for the industrial production of DP3-5 IOSs.
Collapse
Affiliation(s)
- Xinyi Chen
- College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing, Jiangsu 211816, China
| | - Xianhan Chen
- College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing, Jiangsu 211816, China
| | - Liying Zhu
- School of Chemistry and Molecular Engineering, Nanjing Tech University, Nanjing, Jiangsu, 210009, China
| | - Wei Liu
- College of Food Science and Light Industry, State Key Laboratory of Materials-Oriented Chemical Engineering, Nanjing Tech University, Nanjing, Jiangsu 211816, China..
| | - Ling Jiang
- College of Food Science and Light Industry, State Key Laboratory of Materials-Oriented Chemical Engineering, Nanjing Tech University, Nanjing, Jiangsu 211816, China..
| |
Collapse
|
21
|
Chen X, Chen X, Zhu L, Liu W, Jiang L. Programming an Orthogonal Self-Assembling Protein Cascade Based on Reactive Peptide-Protein Pairs for In Vitro Enzymatic Trehalose Production. JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY 2022; 70:4690-4700. [PMID: 35404598 DOI: 10.1021/acs.jafc.2c01118] [Citation(s) in RCA: 15] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/14/2023]
Abstract
Trehalose is an important rare sugar that protects biomolecules against environmental stress. We herein introduce a dual enzyme cascade strategy that regulates the proportion of cargos and scaffolds, to maximize the benefits of enzyme immobilization. Based upon the self-assembling properties of the shell protein (EutM) from the ethanolamine utilization (Eut) bacterial microcompartment, we implemented the catalytic synthesis of trehalose from soluble starch with the coimmobilization of α-amylase and trehalose synthase. This strategy improved enzymatic cascade activity and operational stability. The cascade system enabled the efficient production of trehalose with a yield of ∼3.44 g/(L U), 1.5 times that of the free system. Moreover, its activity was maintained over 12 h, while the free system was almost completely inactivated after 4 h, demonstrating significantly enhanced thermostability. In conclusion, an attractive self-assembly coimmobilization platform was developed, which provides an effective biological process for the enzymatic synthesis of trehalose in vitro.
Collapse
Affiliation(s)
- Xianhan Chen
- College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing, Jiangsu 211816, People's Republic of China
| | - Xinyi Chen
- College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing, Jiangsu 211816, People's Republic of China
| | - Liying Zhu
- School of Chemistry and Molecular Engineering, Nanjing Tech University, Nanjing, Jiangsu 210009, People's Republic of China
| | - Wei Liu
- College of Food Science and Light Industry, State Key Laboratory of Materials-Oriented Chemical Engineering, Nanjing Tech University, Nanjing, Jiangsu 211816, People's Republic of China
| | - Ling Jiang
- College of Food Science and Light Industry, State Key Laboratory of Materials-Oriented Chemical Engineering, Nanjing Tech University, Nanjing, Jiangsu 211816, People's Republic of China
| |
Collapse
|
22
|
Immobilization Techniques on Bioprocesses: Current Applications Regarding Enzymes, Microorganisms, and Essential Oils. FOOD BIOPROCESS TECH 2022. [DOI: 10.1007/s11947-022-02780-w] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/04/2022]
|