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2.
Biochem Pharmacol ; 182: 114230, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32979352

RESUMO

L-asparaginase (ASNase) from Escherichia coli (EcAII) is used in the treatment of acute lymphoblastic leukaemia (ALL). EcAII activity in vivo has been described to be influenced by the human lysosomal proteases asparaginyl endopeptidase (AEP) and cathepsin B (CTSB); these hydrolases cleave and could expose epitopes associated with the immune response against EcAII. In this work, we show that ASNase resistance to CTSB and/or AEP influences the formation of anti-ASNase antibodies, one of the main causes of hypersensitivity reactions in patients. Error-prone polymerase chain reaction was used to produce variants of EcAII more resistant to proteolytic cleavage by AEP and CTSB. The variants with enzymatic activity and cytotoxicity levels equivalent to or better than EcAII WT were submitted to in vivo assays. Only one of the mutants presented increased serum half-life, so resistance to these proteases is not the only feature involved in EcAII stability in vivo. Our results showed alteration of the phenotypic profile of B cells isolated after animal treatment with different protease-resistant proteoforms. Furthermore, mice that were exposed to the protease-resistant proteoforms presented lower anti-asparaginase antibodies production in vivo. Our data suggest that modulating resistance to lysosomal proteases can result in less immunogenic protein drugs.

3.
World J Microbiol Biotechnol ; 35(8): 114, 2019 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-31332537

RESUMO

N-acetyl-D-glucosamine (GlcNAc) is an important amino-monosaccharide with great potential for biotechnological applications. It has traditionally been produced by the chemical hydrolysis of chitin, despite certain industrial and environmental drawbacks, including acidic wastes, low yields and high costs. Therefore, enzymatic production has gained attention as a promising environmentally-friendly alternative to the chemical processes. In this study we demonstrate the GlcNAc bioproduction from colloidal α-chitin using an enzyme cocktail containing endochitinases and exochitinases (chitobiosidases and N-acetyl-glucosaminidases). The enzyme cocktail was extracted after fermentation in a bioreactor by Aeromonas caviae CHZ306, a chitinolytic marine bacterium with great potential for chitinase production. Hydrolysis parameters were studied in terms of temperature, pH, enzyme and substrate concentration, and reaction time, achieving over 90% GlcNAc yield within 6 h. The use of colloidal α-chitin as substrate showed a substantial improvement of GlcNAc yields, when compared with ß-chitin and α-chitin polymorphs. Such result is directly related to a significant decrease in crystallinity and viscosity from natural α-chitin, providing the chitinase with greater accessibility to the depolymerized chains. This study provides valuable information on the GlcNAc bioproduction from chitin using an enzymatic approach, addressing the key points for its production, including the enzyme cocktail composition and the substrate structures.


Assuntos
Acetilglucosamina/biossíntese , Aeromonas caviae/enzimologia , Quitina/metabolismo , Quitinases/metabolismo , Meios de Cultura/química , Concentração de Íons de Hidrogênio , Hidrólise , Espectroscopia de Ressonância Magnética , Peso Molecular , Temperatura , Viscosidade , Difração de Raios X
4.
AAPS PharmSciTech ; 20(6): 251, 2019 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-31300911

RESUMO

Polymersomes are versatile nanostructures for protein delivery with hydrophilic core suitable for large biomolecule encapsulation and protective stable corona. Nonetheless, pharmaceutical products based on polymersomes are not available in the market, yet. Here, using commercially available copolymers, we investigated the encapsulation of the active pharmaceutical ingredient (API) L-asparaginase, an enzyme used to treat acute lymphoblastic leukemia, in polymersomes through a quality-by-design (QbD) approach. This allows for streamlining of processes required for improved bioavailability and pharmaceutical activity. Polymersomes were prepared by bottom-up (temperature switch) and top-down (film hydration) methods employing the diblock copolymers poly(ethylene oxide)-poly(lactic acid) (PEG45-PLA69, PEG114-PLA153, and PEG114-PLA180) and the triblock Pluronic® L-121 (poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide), PEG5-PPO68-PEG5). Quality Target Product Profile (QTPP), Critical Quality Attributes (CQAs), Critical Process Parameters (CPPs), and the risk assessment were discussed for the early phase of polymersome development. An Ishikawa diagram was elaborated focusing on analytical methods, raw materials, and processes for polymersome preparation and L-asparaginase encapsulation. PEG-PLA resulted in diluted polymersomes systems. Nonetheless, a much higher yield of Pluronic® L-121 polymersomes of 200 nm were produced by temperature switch, reaching 5% encapsulation efficiency. Based on these results, a risk estimation matrix was created for an initial risk assessment, which can help in the future development of other polymersome systems with biological APIs nanoencapsulated.


Assuntos
Antineoplásicos/síntese química , Asparaginase/síntese química , Nanoestruturas/química , Poloxâmero/síntese química , Polietilenoglicóis/síntese química , Antineoplásicos/farmacocinética , Asparaginase/farmacocinética , Interações Hidrofóbicas e Hidrofílicas , Poloxâmero/farmacocinética , Polietilenoglicóis/farmacocinética , Propilenoglicóis/síntese química , Propilenoglicóis/farmacocinética
5.
Int Braz J Urol ; 45(3): 435-448, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31038864

RESUMO

OBJECTIVES: Prostate cancer is the most common and fatal cancer amongst Brazilian males. The quality of prostate cancer care in Brazil was systematically reviewed and compared to United Kingdom (UK) National Institute for Health and Care Excellence (NICE) guidelines, which are considered an international benchmark in care, to determine any treatment gaps in Brazilian practice. MATERIALS AND METHODS: A systematic review of Brazilian and UK literature was undertaken. Additionally, quality of life scores was measured using a FACT-P questionnaire of 36 prostate cancer patients attending the Farmácia Universitária da Universidade de São Paulo (FARMUSP). These scores were compared against NICE care measures for patient safety, clinical effi cacy and quality of life indicators determined by either quantitative or qualitative methods. Key fi ndings: The quality of prostate cancer care in Brazil was considered good when compared to NICE guidelines. However, FACT-P data strongly indicated a poor understanding of treatment received by Brazilian patients and that their mental health needs were not being met. CONCLUSIONS: NICE quality statements that address the holistic needs of patients should be implemented into Brazilian outpatient care plans. Addressing the non-medical concerns of patients may improve quality of life and can be easily rolled-out through existing Brazilian pharmacy services at no fi nancial cost to the Brazilian Unifi ed Health System (SUS).


Assuntos
Assistência Ambulatorial/normas , Assistência Farmacêutica/normas , Neoplasias da Próstata/tratamento farmacológico , Garantia da Qualidade dos Cuidados de Saúde/métodos , Qualidade de Vida , Brasil , Lista de Checagem/normas , Humanos , Masculino , Padrões de Referência , Inquéritos e Questionários/normas , Reino Unido
6.
Appl Microbiol Biotechnol ; 103(13): 5161-5166, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31104099

RESUMO

L-asparaginase is an enzyme produced by microorganisms, plants, and animals, which is used clinically for the treatment for acute lymphoblastic leukemia (ALL) and, in the food industry, to control acrylamide formation in baked foods. The purpose of this review was to evaluate the available literature regarding microbial sources of L-asparaginase, culture media used to achieve maximum enzyme expression in microbial fermentations, and assay methods employed to assess L-asparaginase activity. Studies were gathered by searching PubMed, and Web of Science databases before January 22, 2018, with no time restrictions. The articles were evaluated according to the source of L-asparaginase being studied, the nitrogen source in the culture medium, the type of sample, and the method employed to evaluate L-asparaginase activity. Bacterial L-asparaginase appeared to be the most commonly studied source of the enzyme and, most often, the enzyme activity was assayed from crude protein extracts using the Nessler method, which is an indirect measurement of asparaginase activity that determines the concentration of ammonia generated after the action of the enzyme on the substrate, L-asparagine. However, ammonia is also generated throughout microbial fermentations and this endogenous ammonia will also reduce the Nessler reagent if crude microbial extracts are used to determine total L-asparaginase activity. We suggest that current estimates of L-asparaginase activity reported in the literature may be overestimated when Nessler reagent is used, since we were unable to find a single study that made reference to the possible inference of fermentation derived ammonia.


Assuntos
Asparaginase/metabolismo , Bactérias/enzimologia , Bioensaio/normas , Amônia/metabolismo , Asparagina/metabolismo , Bioensaio/métodos , Meios de Cultura , Fermentação
7.
Prep Biochem Biotechnol ; 49(7): 679-685, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30990115

RESUMO

L-Asparaginase (L-ASNase) is an important enzyme used to treat acute lymphoblastic leukemia, recombinantly produced in a prokaryotic expression system. Exploration of alternatives production systems like as extracellular expression in microorganisms generally recognized as safe (such as Pichia pastoris Glycoswitch®) could be advantageous, in particular, if this system is able to produce homogeneous glycosylation. Here, we evaluated extracellular expression into Glycoswitch® using two different strains constructions containing the asnB gene coding for Erwinia chrysanthemi L-ASNase (with and without His-tag), in order to find the best system for producing the extracellular and biologically active protein. When the His-tag was absent, both cell expression and protein secretion processes were considerably improved. Three-dimensional modeling of the protein suggests that additional structures (His-tag) could adversely affect native conformation and folding from L-ASNase and therefore the expression and cell secretion of this enzyme.


Assuntos
Asparaginase/genética , Clonagem Molecular/métodos , Pectobacterium chrysanthemi/enzimologia , Pectobacterium chrysanthemi/genética , Asparaginase/química , Expressão Gênica , Genes Bacterianos , Glicosilação , Modelos Moleculares , Pectobacterium chrysanthemi/química , Pichia/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética
8.
Biologicals ; 59: 47-55, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30871932

RESUMO

Acute lymphoblastic leukemia (ALL) is a type of cancer with a high incidence in children. The enzyme l-asparaginase (ASNase) constitutes a key element in the treatment of this disease. Four formulations of ASNase from a bacterial source are currently available. However, these formulations are characterized by their high immunogenicity, resulting in the inactivation of the drug, as well as in the occurrence of hypersensitivity reactions in a large number of patients. In this work, we performed an immunoinformatic analysis in order to clarify structural aspects of the immunogenicity of the asparaginase from Escherichia coli and Erwinia carotovora. For this purpose, we performed the prediction of immunogenic and allergenic epitopes in the structure of asparaginases by using the relative frequency of immunogenic peptides for the eight alleles most frequently distributed worldwide. This study showed that there are no significant differences in the level of immunogenicity between the two enzymes, while asparaginase from E. coli presented a higher relative frequency of allergenic epitopes. These results are consistent with previously published reports. However, from a structural point of view, to the best of our knowledge, this is the first report describing the structural determinants that contribute to the hypersensitivity response to this treatment.


Assuntos
Asparaginase/imunologia , Proteínas de Bactérias/imunologia , Epitopos/imunologia , Escherichia coli/enzimologia , Pectobacterium carotovorum/enzimologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/imunologia , Sequência de Aminoácidos , Asparaginase/efeitos adversos , Asparaginase/uso terapêutico , Proteínas de Bactérias/química , Proteínas de Bactérias/uso terapêutico , Criança , Simulação por Computador , Epitopos/química , Escherichia coli/genética , Humanos , Hipersensibilidade/etiologia , Hipersensibilidade/imunologia , Pectobacterium carotovorum/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Conformação Proteica , Homologia de Sequência de Aminoácidos , Especificidade da Espécie
9.
Mater Sci Eng C Mater Biol Appl ; 98: 524-534, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30813054

RESUMO

l-Asparaginase (ASNase) is an amidohydrolase used as a chemotherapeutic agent for the treatment of acute lymphoblastic leukemia (ALL). The nanoencapsulation of this enzyme is strategic to avoid its immediate immunogenic effects that lead to a decrease in the enzyme half-life. In this work, ASNase-containing nanoparticles (NPs) were prepared by double emulsification, through an ultrasonic sonicator or an Ultra-Turrax, using two copolymers of 50:50 (w/w) poly (lactic-co-glycolic acid) (PLGA) with different ranges of molecular weight (24-38 kDa and 30-60 kDa) and varying the concentration of polyvinyl alcohol (PVA) as a stabilizer (0.5, 1.0, 1.5 and 2.0%) as well as the emulsification time (30 and 60 s). Using 24-38 kDa PLGA and 1.0% PVA, we obtained by cavitation NPs with hydrodynamic diameter of 384 nm, polydispersity index of 0.143 and Zeta potential of -16.4 mV, whose ASNase encapsulation efficiency was as high as 87 ±â€¯2%. The encapsulated enzyme showed an activity 22% higher than that of the free enzyme, and no conformational changes were detected by circular dichroism. The enzyme release from NPs entrapped in dialysis bags (500 kDa molecular weight cut-off) allowed selecting a controlled system able to release about 60% of the enzyme within 14 days, for which the Korsmeyer-Peppas model provided the best correlation (R2 = 0.966).


Assuntos
Asparaginase/metabolismo , Nanosferas/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , Animais , Emulsões/química , Estabilidade Enzimática , Enzimas Imobilizadas/metabolismo , Hemólise , Hidrodinâmica , Nanosferas/ultraestrutura , Ovinos
10.
Artigo em Inglês | MEDLINE | ID: mdl-30800657

RESUMO

L-Asparaginase (ASNase) is used in the treatment of acute lymphoblastic leukemia, being produced and commercialized only from bacterial sources. Alternative Saccharomyces cerevisiae ASNase II coded by the ASP3 gene was biosynthesized by recombinant Pichia pastoris MUT s under the control of the AOX1 promoter, using different cultivation strategies. In particular, we applied multistage fed-batch cultivation divided in four distinct phases to produce ASNase II and determine the fermentation parameters, namely specific growth rate, biomass yield, and enzyme activity. Cultivation of recombinant P. pastoris under favorable conditions in a modified defined medium ensured a dry biomass concentration of 31 gdcw.L-1 during glycerol batch phase, corresponding to a biomass yield of 0.77 gdcw.g glycerol - 1 and a specific growth rate of 0.21 h-1. After 12 h of glycerol feeding under limiting conditions, cell concentration achieved 65 gdcw.L-1 while ethanol concentration was very low. During the phase of methanol induction, biomass concentration achieved 91 gdcw.L-1, periplasmic specific enzyme activity 37.1 U.g dcw - 1 , volumetric enzyme activity 3,315 U.L-1, overall enzyme volumetric productivity 31 U.L-1.h-1, while the specific growth rate fell to 0.039 h-1. Our results showed that the best strategy employed for the ASNase II production was using glycerol fed-batch phase with pseudo exponential feeding plus induction with continuous methanol feeding.

11.
Food Chem ; 280: 175-186, 2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-30642484

RESUMO

Utilization of marine algae has increased considerably over the past decades, since biodiversity within brown, red and green marine algae offers possibilities of finding a variety of bioactive compounds. Marine algae are rich sources of dietary fibre. The remarkable positive effects of seaweed dietary fibre on human body are related to their prebiotic activity over the gastrointestinal tract (GIT) microbiota. However, dietary modulation of microorganisms present in GIT can be influenced by different factors such as type and source of the dietary fibre, their molecular weight, type of extraction and purification methods employed, composition and modification of polysaccharide and oligosaccharide. This review will demonstrate evidence that polysaccharides and oligosaccharides from marine algae can be used as prebiotics, emphasizing their use in human health, their application as food and other possible applications. Furthermore, an important approach of microbial enzymes employment during extraction, modification or production of those prebiotics is highlighted.


Assuntos
Clorófitas/metabolismo , Oligossacarídeos/metabolismo , Polissacarídeos/metabolismo , Prebióticos/análise , Alga Marinha/metabolismo , Microbioma Gastrointestinal/efeitos dos fármacos , Humanos , Oligossacarídeos/química , Oligossacarídeos/farmacologia , Feófitas/metabolismo , Polissacarídeos/química , Polissacarídeos/farmacologia , Rodófitas/metabolismo
12.
Nat Prod Res ; : 1-5, 2018 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-30499338

RESUMO

This work aimed at evaluating the prebiotic potential of the aqueous extract and crude polysaccharides from Agave sisalana boles by an in vitro screening. Crude polysaccharides were obtained from the aqueous bole extract by precipitation with acetone and resuspension in water. The liquid extract and the polysaccharide solution were then spray dried and submitted to thermal analysis and quantification of metabolites. Prebiotic activity was checked on probiotic strains belonging to the Lactobacillus genus using inulin, fructo-oligosaccharides, fructose and glucose as positive controls. The powder of A. sisalana bole extract, which has recently been identified as a rich source of inulin, exhibited higher potential of fermentation compared with crude polysaccharides.

13.
Braz. j. microbiol ; 49(4): 856-864, Oct.-Dec. 2018. graf
Artigo em Inglês | LILACS | ID: biblio-974294

RESUMO

ABSTRACT The growth of yeasts in culture media can be affected by many factors. For example, methanol can be metabolized by other pathways to produce ethanol, which acts as an inhibitor of the heterologous protein production pathway; oxygen concentration can generate aerobic or anaerobic environments and affects the fermentation rate; and temperature affects the central carbon metabolism and stress response protein folding. The main goal of this study was determine the implication of free fatty acids on the production of heterologous proteins in different culture conditions in cultures of Pichia pastoris. We evaluated cell viability using propidium iodide by flow cytometry and thiobarbituric acid reactive substances to measure cell membrane damage. The results indicate that the use of low temperatures and low methanol concentrations favors the decrease in lipid peroxidation in the transition phase from glycerol to methanol. In addition, a temperature of 14 ºC + 1%M provided the most stable viability. By contrast, the temperature of 18 ºC + 1.5%M favored the production of a higher antibody fragment concentration. In summary, these results demonstrate that the decrease in lipid peroxidation is related to an increased production of free fatty acids.


Assuntos
Pichia/metabolismo , Ácidos Graxos não Esterificados/metabolismo , Pichia/crescimento & desenvolvimento , Pichia/genética , Temperatura , Proteínas Recombinantes/genética , Meios de Cultura/metabolismo , Meios de Cultura/química , Metanol/metabolismo , Fermentação , Glicerol/metabolismo
14.
Anal Bioanal Chem ; 410(27): 6985-6990, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30155702

RESUMO

L-asparaginase or ASNase (L-asparagine aminohydrolase, E.C.3.5.1.1) is an enzyme clinically accepted as an antitumor agent to treat acute lymphoblastic leukemia (ALL) and lymphosarcoma through the depletion of L-asparagine (L-Asn) resulting in cytotoxicity to leukemic cells. ASNase is also important in the food industry, preventing acrylamide formation in processed foods. Several quantification techniques have been developed and used for the measurement of the ASNase activity, but standard pharmaceutical quality control methods were hardly reported, and in general, no official quality control guidelines were defined. To overcome this lack of information and to demonstrate the advantages and limitations, this work properly compares the traditional colorimetric methods (Nessler; L-aspartic acid ß-hydroxamate (AHA); and indooxine) and the high-performance liquid chromatography (HPLC) method. A comparison of the methods using pure ASNase shows that the colorimetric methods both overestimate (Nessler) and underestimate (AHA and indooxine) the ASNase activity when compared to the values obtained with HPLC, considered the most precise method as this method monitors both substrate consumption and product formation, allowing for overall mass-balance. Correlation and critical analysis of each method relative to the HPLC method were carried out, resulting in a demonstration that it is crucial to select a proper method for the quantification of ASNase activity, allowing bioequivalence studies and individualized monitoring of different ASNase preparations. Graphical abstract ᅟ.


Assuntos
Asparaginase/metabolismo , Colorimetria/métodos , Ensaios Enzimáticos/métodos , Asparaginase/análise , Asparagina/análogos & derivados , Asparagina/metabolismo , Ácido Aspártico/metabolismo , Cromatografia Líquida de Alta Pressão/métodos , Humanos
15.
Eur J Pharm Biopharm ; 131: 92-98, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30053482

RESUMO

Osmolytes are small organic molecules accumulated by cells in response to environmental stresses. They are represented by amino acids, sugars, polyols, tertiary sulphonium and quaternary ammonium compounds. These molecules present a protective behaviour and favour the equilibrium of macromolecules towards the native form, preventing denaturation and promoting the folding of unfolded proteins. Protein formulations due to their biological character require greater care during the manufacturing process, shelf-life and administration of the drug, as variations in these factors may result in denaturation, inactivation and/or protein aggregation. These drawbacks can be surpassed using osmolytes as excipients in protein formulations as stabilisers, bulking agents and even buffers. A number of 133 biologics, including vaccines and immunoglobulins, approved by the U.S. Food and Drug Administration (FDA) between 1998 and 2017 were analysed in this work in order to identify the most used group of osmolyte molecules. A deep insight into their role in protein formulations was discussed and compared to data in the literature. The advantages and disadvantages of their use in specific formulations were also extensively discussed here. In conclusion, investigation into the role of osmolytes in each formulation is essential for understanding their effect and provides a background to be used when selecting the best osmolyte to fit a specific formulation without excluding the patient needs.


Assuntos
Biofarmácia , Composição de Medicamentos , Aminoácidos/química , Animais , Excipientes , Humanos
16.
Braz J Microbiol ; 49 Suppl 1: 119-127, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-29858140

RESUMO

Nowadays, it is necessary to search for different high-scale production strategies to produce recombinant proteins of economic interest. Only a few microorganisms are industrially relevant for recombinant protein production: methylotrophic yeasts are known to use methanol efficiently as the sole carbon and energy source. Pichia pastoris is a methylotrophic yeast characterized as being an economical, fast and effective system for heterologous protein expression. Many factors can affect both the product and the production, including the promoter, carbon source, pH, production volume, temperature, and many others; but to control all of them most of the time is difficult and this depends on the initial selection of each variable. Therefore, this review focuses on the selection of the best promoter in the recombination process, considering different inductors, and the temperature as a culture medium variable in methylotrophic Pichia pastoris yeast. The goal is to understand the effects associated with different factors that influence its cell metabolism and to reach the construction of an expression system that fulfills the requirements of the yeast, presenting an optimal growth and development in batch, fed-batch or continuous cultures, and at the same time improve its yield in heterologous protein production.


Assuntos
Carbono/metabolismo , Pichia/genética , Pichia/metabolismo , Regiões Promotoras Genéticas , Proteínas Recombinantes/biossíntese , Microbiologia Industrial , Pichia/crescimento & desenvolvimento , Proteínas Recombinantes/genética , Temperatura
17.
Nanomaterials (Basel) ; 8(6)2018 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-29861449

RESUMO

Polymersomes (PL), vesicles formed by self-assembly of amphiphilic block copolymers, have been described as promising nanosystems for drug delivery, especially of biomolecules. The film hydration method (FH) is widely used for PL preparation, however, it often requires long hydration times and commonly results in broad size distribution. In this work, we describe the challenges of the self-assembly of poly (ethylene glycol)-poly(lactic acid) (PEG-PLA) into PL by FH exploring different hydrophilic volume fraction (f) values of this copolymer, stirring times, temperatures and post-FH steps in an attempt to reduce broad size distribution of the nanostructures. We demonstrate that, alongside f value, the methods employed for hydration and post-film steps influence the PEG-PLA self-assembly into PL. With initial FH, we found high PDI values (>0.4). However, post-hydration centrifugation significantly reduced PDI to 0.280. Moreover, extrusion at higher concentrations resulted in further improvement of the monodispersity of the samples and narrow size distribution. For PL prepared at concentration of 0.1% (m/v), extrusion resulted in the narrower size distributions corresponding to PDI values of 0.345, 0.144 and 0.081 for PEG45-PLA69, PEG114-PLA153 and PEG114-PLA180, respectively. Additionally, we demonstrated that copolymers with smaller f resulted in larger PL and, therefore, higher encapsulation efficiency (EE%) for proteins, since larger vesicles enclose larger aqueous volumes.

18.
Fungal Biol ; 122(5): 302-309, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29665956

RESUMO

Laccase production in saline conditions is still poorly studied. The aim of the present study was to investigate the production of laccase in two different types of bioreactors by the marine-derived basidiomycete Peniophora sp. CBMAI 1063. The highest laccase activity and productivity were obtained in the Stirred Tank (ST) bioreactor, while the highest biomass concentration in Air-lift (AL) bioreactor. The main laccase produced was purified by ion exchange and size exclusion chromatography and appeared to be monomeric with molecular weight of approximately 55 kDa. The optimum oxidation activity was obtained at pH 5.0. The thermal stability of the enzyme ranged from 30 to 50 °C (120 min). The Far-UV Circular Dichroism revealed the presence of high ß-sheet and low α-helical conformation in the protein structure. Additional experiments carried out in flask scale showed that the marine-derived fungus was able to produce laccase only in the presence of artificial seawater and copper sulfate. Results from the present study confirmed the fungal adaptation to marine conditions and its potential for being used in saline environments and/or processes.


Assuntos
Organismos Aquáticos/metabolismo , Basidiomycota/metabolismo , Reatores Biológicos/microbiologia , Meios de Cultura/química , Lacase/metabolismo , Solução Salina/metabolismo , Organismos Aquáticos/crescimento & desenvolvimento , Basidiomycota/crescimento & desenvolvimento , Cromatografia em Gel , Cromatografia por Troca Iônica , Dicroísmo Circular , Sulfato de Cobre/metabolismo , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Lacase/química , Lacase/isolamento & purificação , Peso Molecular , Oxirredução , Estrutura Secundária de Proteína , Temperatura
19.
Braz J Microbiol ; 49(4): 856-864, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29705163

RESUMO

The growth of yeasts in culture media can be affected by many factors. For example, methanol can be metabolized by other pathways to produce ethanol, which acts as an inhibitor of the heterologous protein production pathway; oxygen concentration can generate aerobic or anaerobic environments and affects the fermentation rate; and temperature affects the central carbon metabolism and stress response protein folding. The main goal of this study was determine the implication of free fatty acids on the production of heterologous proteins in different culture conditions in cultures of Pichia pastoris. We evaluated cell viability using propidium iodide by flow cytometry and thiobarbituric acid reactive substances to measure cell membrane damage. The results indicate that the use of low temperatures and low methanol concentrations favors the decrease in lipid peroxidation in the transition phase from glycerol to methanol. In addition, a temperature of 14°C+1%M provided the most stable viability. By contrast, the temperature of 18°C+1.5%M favored the production of a higher antibody fragment concentration. In summary, these results demonstrate that the decrease in lipid peroxidation is related to an increased production of free fatty acids.


Assuntos
Ácidos Graxos não Esterificados/metabolismo , Pichia/metabolismo , Meios de Cultura/química , Meios de Cultura/metabolismo , Fermentação , Glicerol/metabolismo , Metanol/metabolismo , Pichia/genética , Pichia/crescimento & desenvolvimento , Proteínas Recombinantes/genética , Temperatura
20.
Methods Mol Biol ; 1674: 183-191, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-28921437

RESUMO

The bacterial expression of glycoproteins has experienced significant progress in recent years, particularly in regard to the production of conjugate vaccines against pathogens. In this case, a protein carrier conjugated with glycosides is used to produce intense stimulation of the immune system. Glycoconjugate vaccines account for 35% of the global vaccine market, and consequently, several biotechnological companies have developed products for the purification of glycosylated proteins to attain homogeneity. In this chapter we present a general process for glycoprotein production in Escherichia coli and a practice method for purification of glycosylated proteins, using affinity chromatography.


Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Glicoconjugados/metabolismo , Glicoproteínas/metabolismo , Vacinas Conjugadas/metabolismo , Cromatografia de Afinidade/métodos , Glicosilação
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