Purification, characterization and clinical applications of therapeutic fungal enzymes

This book chapter presents an overview of therapeutic fungal enzymes and their developments in biopharmaceuticals for the treatment of several diseases, clinical applications and investigation. Enzymes are biocatalysts of many reactions with widespread use in the pharmaceutical industry and medicine. Due to their high specificity, greater affinity, and high catalytic efficiency, enzymes have been widely used for therapeutic purposes. More specifically, therapeutic enzymes are being used in the treatment of several diseases, such as leukemia, cancer, pancreatic disorders, etc. For instance, L-asparaginase, which presents antineoplastic properties, has been used for the treatment of leukemia, namely acute lymphoblastic leukemia. Nowadays, more than 50% of the enzymes are produced by fungal sources, including the therapeutic enzymes, due to the advantages of being an economically feasible and consistent process, since it has high yield and is easy for modification and optimization of new therapeutic products. In this book chapter, readers from academies, research institutes and industries will gain useful information and in-deep knowledge on the emerging therapeutic fungal enzymes, their purification processes, characterization and medical applications.publishe

Propranolol resolution using enantioselective biphasic systems

The commercialization of chiral drugs is an increasing concern in the pharmacological field since the differences in the pharmacological activities of enantiomers may result in serious problems in the treatment of diseases using racemates. The resolution of chiral drugs is important for the development of safer and more active pharmaceuticals. This work aims to develop an enantioseparation platform for the resolution of propranolol (R/S-PRP) resorting to esters of tartaric acid and chiral ionic liquids (CILs) as chiral selectors in biphasic systems. More specifically, the efficiency of enantioselective liquid–liquid extraction (ELLE) systems, both aqueous and non-aqueous biphasic systems, are here studied, aiming to do a direct comparison between these two types of systems for the resolution of R/S-PRP. Studies were carried to evaluate the proper phase forming components of ELLE, R/S-PRP:chiral selector ratio, the potential of CIL over esters of tartaric acid, and the most suitable alkyl chain length for the esters of tartaric acid. It was found that the selected organic phase formers of ELLE, 1,2-dichloroethane and ethyl acetate, greatly impact the potential of the enantiorecognition of the system. The most efficient biphasic system identified was composed of 1,2-dichloroethane- water, and dipentyl-L-tartrate and boric acid as chiral selectors, with a enantioselectivity of 2.54. This system was further employed for the resolution of R/S-PRP in centrifugal partition chromatography, to assess its scalability potential, being shown that it was possible to increase the purity of R-PRP from 59% to 75%.publishe

A selective journey: enantioselective biphasic systems for the resolution of propranolol

Enantiomers may have different biological properties, leading to complications when using racemates for the treatment of diseases. Considering the difficulty in the synthesis of pure enantiomers, the synthesis of racemates followed by their chiral resolution is deemed as a simpler and cheaper alternative. Enantioselective liquid-liquid extraction (ELLE) is a promising separation process. ELLE is composed of two immiscible phases that enable the optimization of enantioseparation through the addition of a chiral selector, such as chiral ionic liquids (CIL) or tartaric acid derivatives. Upon their introduction in ELLE, these chiral selectors may help increase the selectivity of the system, contributing to high performant extraction/separation approaches. In this work, CILs and tartaric acid derivatives were used in biphasic systems as chiral selectors, aiming to separate R/S-propranolol (R/S-PRP) enantiomers. The most promising system was applied in centrifugal partition chromatography to further improve the enantiomeric purification rates.publishe

Design of a liquid-liquid extraction platform for the resolution of chiral pharmaceuticals

Enantiomers have different pharmacological properties, which can hinder the treatment of pathologies using racemic drugs. Racemates represent around 90 % of the commercialized chiral drugs, raising concerns by the FDA (Food and Drug Administration of United States) and EMA (European Medicines Agency). Therefore, the commercialization of the therapeutically active isomer should be preferential. Obtaining the pure enantiomer relies on direct synthesis or resolution of the existing racemates. Resolution is often considered a simpler and cheaper alternative. Enantioselective liquid-liquid extraction (LLE) is a promising separation process that can be operated in a continuous mode. LLE are composed of two tunable immiscible phases that allow the optimization of enantioseparation by the addition of a chiral selector, which is responsible for the chiral recognition. If the two immiscible phases are composed mainly of water, then the system is an aqueous biphasic system (ABS). Since the majority component of ABS is water, they are considered green, economical and reliable systems. A major advantage of LLE is that it can comprise both enantiomeric recognition and solvent extraction on a single technique. Ionic liquids are alternative solvents with great structural diversity, allowing the design of task-specific solvents, including chiral ionic liquids (CILs). The introduction of CILs in LLE may contribute to high performant extraction/separation systems. Another promising class of green chiral selectors is the tartaric acid esters family which in conjugation with boric acid appear as promising adjuvants for the LLE systems. In this work, two different approaches were explored for the purification of propranolol enantiomers using LLE and ABS. In the first one, CILs and tartaric acid esters were used in LLE systems as chiral selectors, and in the second one, CILs and tartaric acid esters were used as chiral selectors in polymer-polymer-based ABS. The best outcome was scaled-up resorting to centrifugal partition chromatography (CPC).publishe

Improvements in the enzymatic degradation of textile dyes using ionic-liquid-based surfactants

The intensive use of water containing dyes by the textile industry, and consequently the contamination of soils and water, represents serious environmental concerns. Amongst the several processes applied in the treatment of textile effluents, biological-based processes, if designed to be cost-effective and ecofriendly, are promising alternatives to decolorize textile effluents. In this work we investigate and propose the novel use of ionic liquids (ILs) with surfactant characteristics to improve the degradation of the largely used and highly hydrophobic textile dye indigo carmine (IC) by laccase. An initial screening on the activity of laccase in aqueous solutions of twelve surfactant-based ILs from three different families, namely tetraalkylammonium- and imidazolium-based cationic surfactants and cholinium-based anionic surfactants, at different concentrations, was carried out. Significant improvements in the activity of laccase were observed with decyltrimethylammonium bromide, [N10111]Br, and 1-decyl-3-methylimidazolium chloride, [C10mim]Cl, at 75 mM (above the critical micellar concentration of each IL). These ILs were then investigated in aqueous solutions to simultaneously encapsulate laccase and IC for the in situ enzymatic biodegradation of the dye. The use of ILs remarkably increases the degradation rate of the dye and decolorization efficiency; a degradation efficiency of IC of 82% is attained in 0.5 h using aqueous solutions of [N10111]Br, whereas without IL only 6% of IC is degraded. Furthermore, 93% of the dye decolorization was achieved with [N10111]Br. The overall gathered results show that it is possible to significantly improve the degradation of hydrophobic dyes by enzymes using appropriate surfactant-based ILs, while foreseeing the use of the treated water by the same textile industries in new dyeing steps and thus contributing to a substantial decrease of the economic input and environmental footprint of these industries.publishe

Development of suported ionic liquids for the purification of antileukemic drugs

L-asparaginase (LA) is an antileukemic biopharmaceutical of current high-cost. LA is produced via fermentation and its purification usually comprises precipitation, liquid- liquid extraction and chromatography techniques [1]. This work aims to develop sustainable technologies to purify LA. Functionalized nanomaterials, namely supported ionic liquids (SILs), are used as cost-effective purification techniques for the target enzyme. Initially, the synthesis and modification of SILs was performed. Different SILs were obtained and used for the purification of LA. Commercial LA was used for the first purification tests, in order to understand the behavior of the enzyme in contact with the nanomaterial. Experimental conditions, such as pH, and material/LA ratio, contact time were optimized. LA activity was quantified by Nessler reaction [2]. The first results reveal a total adsorption of LA by the SILs with a recovered activity reaching 90% depending on the SILs functionalization/ treatment. The modified SILs are shown to be very promising nanomaterials for the purification of LA. The LA was easily attached to SILs by adsorption under mild conditions. SILs supports can be a real alternative for a single step immobilization/purification of LA.publishe

Separation of albumin from bovine serum applying ionic-liquid-based aqueous biphasic systems

In this work, the extraction and separation of bovine serum albumin (BSA) from its original matrix, i.e., bovine serum, was performed using a novel ionic-liquid-based aqueous biphasic system (IL-based ABS). To this end, imidazolium-, phosphonium-, and ammonium-based ILs, combined with the anions’ acetate, arginate and derived from Good Buffers, were synthesized, characterized, and applied in the development of ABS with K2HPO4/KH2PO4 buffer aqueous solutions at pH 7. Initial studies with commercial BSA revealed a preferential migration of the protein to the IL-rich phase, with extraction efficiencies of 100% obtained in a single-step. BSA recovery yields ranging between 64.0% and 84.9% were achieved, with the system comprising the IL tetrabutylammonium acetate leading to the maximum recovery yield. With this IL, BSA was directly extracted and separated from bovine serum using the respective ABS. Different serum dilutions were further investigated to improve the separation performance. Under the best identified conditions, BSA can be extracted from bovine serum with a recovery yield of 85.6% and a purity of 61.2%. Moreover, it is shown that the BSA secondary structure is maintained in the extraction process, i.e., after being extracted to the IL-rich phase. Overall, the new ABS herein proposed may be used as an alternative platform for the purification of BSA from serum samples and can be applied to other added-value proteins.publishe

Efficient extraction of the RuBisCO enzyme from spinach leaves using aqueous solutions of biocompatible ionic liquids

Ribulose-1,5-biphosphate carboxylase/oxygenase (RuBisCO) is the most abundant protein on the planet, being present in plants, algae and various species of bacteria, with application in the pharmaceutical, chemical, cosmetic and food industries. However, current extraction methods of RuBisCO do not allow high yields of extraction. Therefore, the development of an efficient and selective RuBisCOs’ extraction method is required. In this work, aqueous solutions of biocompatible ionic liquids (ILs), i.e., ILs derived from choline and analogues of glycine-betaine, were applied in the RuBisCO’s extraction from spinach leaves. Three commercial imidazolium-based ILs were also investigated for comparison purposes. To optimize RuBisCO’s extraction conditions, response surface methodology was applied. Under optimum extraction conditions, extraction yields of 10.92 and 10.57 mg of RuBisCO/g of biomass were obtained with the ILs cholinium acetate ([Ch][Ac]) and cholinium chloride ([Ch]Cl), respectively. Circular dichroism (CD) spectroscopy results show that the secondary structure of RuBisCO is better preserved in the IL solutions when compared to the commonly used extraction solvent. The obtained results indicate that cholinium-based ILs are a promising and viable alternative for the extraction of RuBisCO from vegetable biomass.publishe

In Campylobacter jejuni, a new type of chaperone receives heme from ferrochelatase

Funding Information: JZ is a recipient of the MSCA-IF-2019 Individual Fellowship H2020-WF-02-2019, 101003441. FS acknowledges support from the European Research Council (ERC) under the European Union’s Horizon 2020 Research and Innovation program (grant agreement 803768). This work was also financially supported by Fundação para a Ciência e Tecnologia (Portugal) through PTDC/BIA-BQM/28642/2017 grant (LS), the MOSTMICRO-ITQB R&D Unit (UIDB/04612/2020 and UIDP/04612/2020), and the LS4FUTURE Associated Laboratory (LA/P/0087/2020). Publisher Copyright: Copyright © 2023 Zamarreño Beas, Videira, Karavaeva, Lourenço, Almeida, Sousa and Saraiva.Intracellular heme formation and trafficking are fundamental processes in living organisms. Bacteria and archaea utilize three biogenesis pathways to produce iron protoporphyrin IX (heme b) that diverge after the formation of the common intermediate uroporphyrinogen III (uro’gen III). In this study, we identify and provide a detailed characterization of the enzymes involved in the transformation of uro’gen III into heme in Campylobacter jejuni, demonstrating that this bacterium utilizes the protoporphyrin-dependent (PPD) pathway. In general, limited knowledge exists regarding the mechanisms by which heme b reaches its target proteins after this final step. Specifically, the chaperones necessary for trafficking heme to prevent the cytotoxic effects associated with free heme remain largely unidentified. In C. jejuni, we identified a protein named CgdH2 that binds heme with a dissociation constant of 4.9 ± 1.0 µM, and this binding is impaired upon mutation of residues histidine 45 and 133. We demonstrate that C. jejuni CgdH2 establishes protein–protein interactions with ferrochelatase, suggesting its role in facilitating heme transfer from ferrochelatase to CgdH2. Furthermore, phylogenetic analysis reveals that C. jejuni CgdH2 is evolutionarily distinct from the currently known chaperones. Therefore, CgdH2 is the first protein identified as an acceptor of intracellularly formed heme, expanding our knowledge of the mechanisms underlying heme trafficking within bacterial cells.publishersversionpublishe