Sistemas aquosos bifásicos uma ferramenta sustentável para a extração de ácido clavulânico a partir de diferentes fontes

O ácido clavulânico (AC) é um inibidor de β-lactamases que tem vindo a ser largamente utilizado na área médica. Embora seja de extrema importância, o desenvolvimento de processos alternativos de produção e purificação é ainda insignificante, sendo fundamental o estudo de técnicas de extração mais biocompatíveis, como os Sistemas Aquosos Bifásicos (SABs). Assim, este trabalho objetivou o estudo de Sistemas Aquosos Bifásicos baseados em polímeros como uma ferramenta alternativa para a extração de AC. Foram testados dois SPAB compostos por Polietileno Glicol (PEG) com massa molecular (M) de 4000 g/mol e Poliacrilato de Sódio de 8000 g/mol, nos quais foi alterado o eletrólito indutor da formação de fases, em particular, sulfato de sódio (Na2SO4,) e cloreto de sódio (NaCl). Ademais, este trabalho visou também avaliar a eficiência de extração do AC, bem como compreender o efeito dos contaminantes no processo de migração. Para tal, foi avaliada a extração do AC a partir de três fontes distintas: solução pura (99,9%); solução comercial (60%); diretamente a partir do sobrenadante de um meio fermentando de Streptomyces clavuligerus. Os resultados obtidos demonstraram que independentemente da fonte inicial do AC, ambos os SABs poliméricos promoveram uma partição preferencial do AC para a fase rica em PEG, sendo o coeficiente de partição maior nos sistemas com Na2SO4 do que com NaCl. Após identificar a grande capacidade de partição de AC, o SAB com PEG/NaPA/Na2SO4 foi também utilizado para avaliar a partição de proteínas presente no meio fermentado, sendo também obtida uma preferencial partição destas para a fase rica em PEG. Assim, apesar da baixa capacidade de purificação de AC frente a proteínas contaminantes, os SABs estudados demonstraram que podem ser uma técnica alternativa sustentável e bastante econômica para uma etapa inicial de clarificação/concentração de bioprodutos a partir de caldos fermentados

Sustainable lysis of Bacillus subtilis biomass to recover the biopharmaceutical L-asparaginase

The first-line biopharmaceutical used to treat Acute lymphoblastic leukemia (ALL), Oncaspar, is based on the enzyme L-asparaginase (ASNase), and has annual sales of ca. USD $100 million. In addition to other sources, genetically modified Bacillus subtilis is regarded as one of the most promising hosts for the ASNase production. The Aliivibrio fischeri ASNase type II, which has anti-tumour activity due its higher specific affinity for L-asparagine, expressed in B. subtillis is located in the periplasm. Therefore, cell lysis is required for the ASNase recovery. Nevertheless, typical cell lysis approaches, e.g. chemical methods with surfactants lead to some biocompatibility concerns and the need of extra purification steps. To overcome this drawback, in this work, ultrasound sonication (USS) conditions were studied to develop a greener and more biocompatible method for ASNase recovery from B. subtilis cell lysis. The USS cell lysis was optimized regarding the amplitude of USS pulse, number of lysis cycles and mass of cells/volume of solvent ratio. The identification and quantification of ASNase and major impurities present in the cell extract after lysis were investigated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and size exclusion high-performance liquid chromatography (SE-HPLC). ASNase activity was determined by monitoring the hydrolysis of the substrate, L-asparagine. The results obtained show that the ideal conditions for B. subtilis cell lysis are an amplitude of USS pulse of 60%, 40 cycles of lysis and 10 mL of phosphatebuffered saline (PBS) per 1 g of cells. Overall, an optimized sustainable B. subtilis cell lysis method was developed, avoiding the use of surfactants and with low energy consumption.publishe

Supported ionic liquid materials for L-asparaginase bioconjugation

Since the average life expectancy is increasing, several fatal diseases usually related to aging, such as cancer, heart and neurological diseases have become predominant. Biopharmaceuticals, namely nucleic-acid-based products, antibodies, recombinant proteins and enzymes are fundamental to overcome these age-related diseases. Actually, the gold standard enzyme for the treatment of acute chronic lymphoblastic leukemia (ALL) is L-asparaginase (ASNase). Hence, the reusability of this high-priced drug enables the cost reduction of treatments, which allows its routinely use by a widespread population. In this work, functionalized nanomaterials, namely supported ionic liquid materials (SILs) based on silica, formerly described in the literature for the separation of natural compounds from vegetable biomass, were studied as a cost effective support for ASNase immobilization and reuse. Commercial ASNase was used for preliminary tests. Several experimental immobilization conditions, such as pH, contact time, ASNase concentration and SILs recyclability were assessed and optimized, regarding the immobilized ASNase activity, assessed by Nessler reaction, which quantifies the amount of ammonium released after the enzymatic reaction with L-asparagine and immobilization yield. In fact, ASNase immobilization onto the SILs was successfully achieved with an immobilized ASNase activity ranging from 0.6 to 0.9 U of enzyme per mg of SILs under the optimum immobilization conditions. Moreover, all SILs allowed 5 cycles of reaction, while keeping more than 75% of initial ASNase activity. Through the envisioned immobilization strategy, process costs will be considerably reduced, which can lead to a wider use of ASNase in diverse fields of application.publishe

L-Asparaginase

L-Asparaginase (ASNase, EC 3.5.1.1) is a tetrameric aminohydrolase enzyme that catalyses the hydrolysis of the amino acid L-Asparagine into ammonia and L-aspartic acid. ASNase is present in different organisms such as bacteria, fungi, plant tissues and algae. ASNase is used in the pharmaceutical field as an anticancer drug for the treatment of acute lymphoblastic leukemia (ALL) and other malignant diseases such as Hodgkin’s disease. In the food sector, ASNase is used to prevent the formation of acrylamide, a toxic compound formed when starch-rich foods are cooked at temperatures above 100 °C. ASNase can also be used as a biosensor for the detection of L-asparagine levels.publishe

Reusability of L-asparaginase immobilized on silica-based supported ionic liquids

L-asparaginase (ASNase) is an aminohydrolase enzyme used as an anticancer drug, e.g. in the treatment of acute lymphoblastic leukemia, in acrylamide reduction and in biosensing. Nevertheless, its low stability and thermolability, and susceptibility to proteases, hinder its application in the health and food industries. Hence, the improvement of its properties through efficient immobilization methods is in high demand. Thus, this work aims the development of silica-based supported ionic liquids (SILs) for the ASNase immobilization to improve its stability and enable its reusability. While activated silica with no ILs only kept total initial ASNase activity during the first cycle of reaction, SILs allowed 5 cycles of reaction, keeping 82% of initial ASNase activity, reinforcing their potential as alternative enzymatic supports.publishe

Recent strategies and applications for l-asparaginase confinement

l-asparaginase (ASNase, EC 3.5.1.1) is an aminohydrolase enzyme with important uses in the therapeutic/pharmaceutical and food industries. Its main applications are as an anticancer drug, mostly for acute lymphoblastic leukaemia (ALL) treatment, and in acrylamide reduction when starch-rich foods are cooked at temperatures above 100 °C. Its use as a biosensor for asparagine in both industries has also been reported. However, there are certain challenges associated with ASNase applications. Depending on the ASNase source, the major challenges of its pharmaceutical application are the hypersensitivity reactions that it causes in ALL patients and its short half-life and fast plasma clearance in the blood system by native proteases. In addition, ASNase is generally unstable and it is a thermolabile enzyme, which also hinders its application in the food sector. These drawbacks have been overcome by the ASNase confinement in different (nano)materials through distinct techniques, such as physical adsorption, covalent attachment and entrapment. Overall, this review describes the most recent strategies reported for ASNase confinement in numerous (nano)materials, highlighting its improved properties, especially specificity, half-life enhancement and thermal and operational stability improvement, allowing its reuse, increased proteolysis resistance and immunogenicity elimination. The most recent applications of confined ASNase in nanomaterials are reviewed for the first time, simultaneously providing prospects in the described fields of application.publishe

Purification of antileukemic drugs through silica-based supported ionic liquids

L-asparaginase (LA) is an enzyme used as a biopharmaceutical for the treatment of acute lymphoblastic leukemia. LA can be produced via fermentation and its purification usually comprises several steps including precipitation, liquid-liquid extraction and chromatography techniques. Among these, ion exchange chromatography, which is often preceded by precipitation with salts as a first pre-chromatographic step, is the most used. However, theses common strategies for protein purification result in low yields and purity, requiring long processing times, while leading to a consequent increase of the process costs. Therefore, the demand for new cost-effective production/purification processes play now a priority role. This work aims the development of cost-effective technologies to purify LA from the complex fermentation medium from Bacillus Subtillis. Silica-based supported ionic liquids (SILs) are investigated as cost-effective purification materials for the target enzyme. The concentration of the extract from the fermentation, material/ extract from fermentation ratio and contact time effects in the purity and yield of LA were optimized. With this strategy, process costs, energy consumed, and waste generated, may be significantly decreased, which may lead to this biopharmaceutical price decrease and wider application.publishe

L-asparaginase-based biosensors

L-asparaginase (ASNase) is an aminohydrolase enzyme widely used in the pharmaceutical and food industries. Although currently its main applications are focused on the treatment of lymphoproliferative disorders such as acute lymphoblastic leukemia (ALL) and acrylamide reduction in starch-rich foods cooked at temperatures above 100 ºC, its use as a biosensor in the detection and monitoring of L-asparagine levels is of high relevance. ASNase-based biosensors are a promising and innovative technology, mostly based on colorimetric detection since the mechanism of action of ASNase is the catalysis of the L-asparagine hydrolysis, which releases L-aspartic acid and ammonium ions, promoting a medium pH value change followed by color variation. ASNase biosensing systems prove their potential for L-asparagine monitoring in ALL patients, along with L-asparagine concentration analysis in foods, due to their simplicity and fast response.publishe

Purification of antileukemic drugs through silica-based supported ionic liquids

L-asparaginase (LA) is an enzyme used as a biopharmaceutical for the treatment of acute lymphoblastic leukemia. LA can be produced via fermentation and its purification usually comprises ion exchange chromatography, which is often preceded by precipitation with salts as a first pre-chromatographic step. However, this purification strategy result in low yields and purity, requires long processing times, while leading to a consequent increase of the process costs. Therefore, the demand for new cost-effective purification processes play now a priority role. In this work silica-based supported ionic liquids (SILs) are investigated as an alternative technology to purify LA from the complex fermentation medium from Bacillus subtillis. The concentration of the extract from the fermentation, material/ extract from fermentation ratio and contact time effects in the purity and yield of LA were optimized. With this strategy, process costs, energy consumed, and waste generated, may be significantly decreased, which may lead to this biopharmaceutical price decrease and wider application.publishe

Enhanced enzyme reuse through the bioconjugation of L-asparaginase and silica-based supported ionic liquid-like phase materials

L-asparaginase (ASNase) is an amidohydrolase that can be used as a biopharmaceutical, as an agent for acrylamide reduction, and as an active molecule for L-asparagine detection. However, its free form displays some limitations, such as the enzyme’s single use and low stability. Hence, immobilization is one of the most effective tools for enzyme recovery and reuse. Silica is a promising material due to its low-cost, biological compatibility, and tunable physicochemical characteristics if properly functionalized. Ionic liquids (ILs) are designer compounds that allow the tailoring of their physicochemical properties for a given task. If properly designed, bioconjugates combine the features of the selected ILs with those of the support used, enabling the simple recovery and reuse of the enzyme. In this work, silica-based supported ionic liquid-like phase (SSILLP) materials with quaternary ammoniums and chloride as the counterion were studied as novel supports for ASNase immobilization since it has been reported that ammonium ILs have beneficial effects on enzyme stability. SSILLP materials were characterized by elemental analysis and zeta potential. The immobilization process was studied and the pH effect, enzyme/support ratio, and contact time were optimized regarding the ASNase enzymatic activity. ASNase–SSILLP bioconjugates were characterized by ATR-FTIR. The bioconjugates displayed promising potential since [Si][N3444]Cl, [Si][N3666]Cl, and [Si][N3888]Cl recovered more than 92% of the initial ASNase activity under the optimized immobilization conditions (pH 8, 6 × 10−3 mg of ASNase per mg of SSILLP material, and 60 min). The ASNase–SSILLP bioconjugates showed more enhanced enzyme reuse than reported for other materials and immobilization methods, allowing five cycles of reaction while keeping more than 75% of the initial immobilized ASNase activity. According to molecular docking studies, the main interactions established between ASNase and SSILLP materials correspond to hydrophobic interactions. Overall, it is here demonstrated that SSILLP materials are efficient supports for ASNase, paving the way for their use in the pharmaceutical and food industries.publishe