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

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

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