André Moreni Lopes

Liquid-liquid extraction of commercial and biosynthesized nisin by aqueous two-phase micellar systems.

Nisin is a natural additive for conservation of food, and can also be used as a therapeutic agent. Nisin inhibits the outgrowth of spores, the growth of a variety of Gram-positive and Gram-negative bacteria. In this paper we present a potentially scalable and cost-effective way to purify commercial and biosynthesized in bioreactor nisin, including simultaneously removal of impurities and contaminants, increasing nisin activity. Aqueous two-phase micellar systems (ATPMS) are considered promising for bioseparation and purification purposes. Triton X-114 was chosen as the as phase-forming surfactant because it is relatively mild to proteins and it also forms two coexisting phases within a convenient temperature range. Nisin activity was determined by the agar diffusion assay utilizing Lactobacillus sake as a sensitive indicator microorganism. Results indicated that nisin partitions preferentially to the micelle rich-phase, despite the surfactant concentration tested, and its antimicrobial activity increases. The successful implementation of this peptide partitioning, from a suspension containing other compounds, represents an important step towards developing a separation method for nisin, and more generally, for other biomolecules of interest.

LPS removal from an E. coli fermentation broth using aqueous two-phase micellar system

In biotechnology, endotoxin (LPS) removal from recombinant proteins is a critical and challenging step in the preparation of injectable therapeutics, as endotoxin is a natural component of bacterial expression systems widely used to manufacture therapeutic proteins. The viability of large‐scale industrial production of recombinant biomolecules of pharmaceutical interest significantly depends on the separation and purification techniques used. The aim of this work was to evaluate the use of aqueous two‐phase micellar system (ATPMS) for endotoxin removal from preparations containing recombinant proteins of pharmaceutical interest, such as green fluorescent protein (GFPuv). Partition assays were carried out initially using pure LPS, and afterwards in the presence of E. coli cell lysate. The ATPMS technology proved to be effective in GFPuv recovery, preferentially into the micelle‐poor phase (KGFPuv < 1.00), and LPS removal into the micelle‐rich phase (%REMLPS > 98.00%). Therefore, this system can be exploited as the first step for purification in biotechnology processes for removal of higher LPS concentrations.

Therapeutic l-asparaginase: upstream, downstream and beyond

Abstract l-asparaginase (l-asparagine amino hydrolase, E.C.3.5.1.1) is an enzyme clinically accepted as an antitumor agent to treat acute lymphoblastic leukemia and lymphosarcoma. It catalyzes l-asparagine (Asn) hydrolysis to l-aspartate and ammonia, and Asn effective depletion results in cytotoxicity to leukemic cells. Microbial l-asparaginase (ASNase) production has attracted considerable attention owing to its cost effectiveness and eco-friendliness. The focus of this review is to provide a thorough review on microbial ASNase production, with special emphasis to microbial producers, conditions of enzyme production, protein engineering, downstream processes, biochemical characteristics, enzyme stability, bioavailability, toxicity and allergy potential. Some issues are also highlighted that will have to be addressed to achieve better therapeutic results and less side effects of ASNase use in cancer treatment: (a) search for new sources of this enzyme to increase its availability as a drug; (b) production of new ASNases with improved pharmacodynamics, pharmacokinetics and toxicological profiles, and (c) improvement of ASNase production by recombinant microorganisms. In this regard, rational protein engineering, directed mutagenesis, metabolic flux analysis and optimization of purification protocols are expected to play a paramount role in the near future.

Stability, purification, and applications of bromelain: A review.

Bromelain is a cysteine protease found in pineapple tissue. Because of its anti‐inflammatory and anti‐cancer activities, as well as its ability to induce apoptotic cell death, bromelain has proved useful in several therapeutic areas. The market for this protease is growing, and several studies exploring various properties of this molecule have been reported. This review aims to compile this data, and summarize the main findings on bromelain in the literature to date. The physicochemical properties and stability of bromelain under different conditions are discussed. Several studies on the purification of bromelain from crude extracts using a wide range of techniques such as liquid–liquid extractions by aqueous two‐phase system, ultrafiltration, precipitation, and chromatography, have been reported. Finally, the various applications of bromelain are presented. This review therefore covers the main properties of bromelain, aiming to provide an up‐to‐date compilation of the data reported on this enzyme.

Can affinity interactions influence the partitioning of glucose-6-phosphate dehydrogenase in two-phase aqueous micellar systems?

In this work, we provide an investigation of the role and strength of affinity interactions on the partitioning of the glucose-6-phosphate dehydrogenase in aqueous two-phase micellar systems. These systems are constituted of micellar surfactant solutions and offer both hydrophobic and hydrophilic environments, providing selectivity to biomolecules. We studied G6PD partitioning in systems composed of the nonionic surfactants, separately, in the presence and absence of affinity ligands. We observed that G6PD partitions to the micelle-poor phase, owing to the strength of excluded-volume interactions in these systems that drive the protein to the micelle-poor phase, where there is more free volume available.

Extraction of Natural Red Colorants From the Fermented Broth of Penicillium Purpurogenum Using Aqueous Two-Phase Polymer Systems

Safety concerns related to the increasing and widespread application of synthetic coloring agents have increased the demand for natural colorants. Fungi have been employed in the production of novel and safer colorants. In order to obtain the colorants from fermented broth, suitable extraction systems must be developed. Aqueous two‐phase polymer systems (ATPPS) offer a favorable chemical environment and provide a promising alternative for extracting and solubilizing these molecules. The aim of this study was to investigate the partitioning of red colorants from the fermented broth of Penicillium purpurogenum using an ATPPS composed of poly(ethylene glycol) (PEG) and sodium polyacrylate (NaPA). Red colorants partitioned preferentially to the top (PEG‐rich phase). In systems composed of PEG 6,000 g/mol/NaPA 8,000 g/mol, optimum colorant partition coefficient (KC) was obtained in the presence of NaCl 0.1 M (KC = 10.30) while the PEG 10,000 g/mol/NaPA 8,000 g/mol system in the presence of Na2SO4 0.5 M showed the highest KC (14.78). For both polymers, the mass balance (%MB) and yield in the PEG phase (%ηTOP) were close to 100 and 79%, respectively. The protein selectivity in all conditions evaluated ranged from 2.0–3.0, which shows a suitable separation of the red colorants and proteins present in the fermented broth. The results suggest that the partitioning of the red colorants is dependent on both the PEG molecular size and salt type. Furthermore, the results obtained support the potential application of ATPPS as the first step of a purification process to recover colorants from fermented broth of microorganisms.

Evaluation of xylanases from Aspergillus niger and Trichoderma sp. on dough rheological properties

Although starch is the main polysaccharide used in the fermentation of bread dough, wheat flour also contain some non-starch polysaccharides such as pentosans or hemicelluloses, which may contribute up to 3% of the total polysaccharide content of the flour. Despite being present in relatively low amounts, pentosans and hemicelluloses play an important role in dough rheology and bread properties. The aim of this work is to understand how the xylanases from Aspergillus niger and Trichoderma sp. influence dough rheology, such as elasticity, extensibility, strength and stability. When the extensograph parameters such as extensibility (E) and elasticity (R) were determined, it was possible to note that all dosages of xylanase from A. niger were capable of decreasing the dough elasticity in 21%. Depending on the dosage, the xylanase from Trichoderma sp. can decrease dough stability in the mixing and, consequently, the mixing time during the process. An increased dosage of Trichoderma xylanase decreased the elasticity in 32% and increased the extensibility by 8% following 45 min. It was also observed that raising dosages of Trichoderma xylanase in flour content affected the dough rheology more significantly than raising dosages of A. niger xylanase .

Heterologous expression and purification of active L-asparaginase I of Saccharomyces cerevisiae in Escherichia coli host

l‐asparaginase (ASNase) is a biopharmaceutical widely used to treat child leukemia. However, it presents some side effects, and in order to provide an alternative biopharmaceutical, in this work, the genes encoding ASNase from Saccharomyces cerevisiae (Sc_ASNaseI and Sc_ASNaseII) were cloned in the prokaryotic expression system Escherichia coli. In the 93 different expression conditions tested, the Sc_ASNaseII protein was always obtained as an insoluble and inactive form. However, the Sc_ASNaseI (His)6‐tagged recombinant protein was produced in large amounts in the soluble fraction of the protein extract. Affinity chromatography was performed on a Fast Protein Liquid Chromatography (FPLC) system using Ni2+‐charged, HiTrap Immobilized Metal ion Affinity Chromatography (IMAC) FF in order to purify active Sc_ASNaseI recombinant protein. The results suggest that the strategy for the expression and purification of this potential new biopharmaceutical protein with lower side effects was efficient since high amounts of soluble Sc_ASNaseI with high specific activity (110.1 ± 0.3 IU mg−1) were obtained. In addition, the use of FPLC‐IMAC proved to be an efficient tool in the purification of this enzyme, since a good recovery (40.50 ± 0.01%) was achieved with a purification factor of 17‐fold.

ADSORPTION OF ENDOTOXINS ON IDA-Ca+2 BY ION METAL AFFINITY CHROMATOGRAPHY

A method was established for the determination of deoxynivalenol (vomitoxin) in grain and its products based on solid-phase extraction coupled with high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). The sample was firstly extracted by acetonitrile-water (84:16, v/v). The extract was then cleaned-up by an HLB solid phase extraction cartridge. The separation was carried out on a Phenomenex Kinetex C18 column (100 mm × 4.6 mm, 2.6 μm) with a gradient elution using 0.3 per mille ammonia solution-acetonitrile as mobile phases. The analysis of deoxynivalenol was performed under electrospray negative ionization mode. The limit of detection (LOD, S/N =3) and the limit of quantification (LOQ, S / N =10) were 20 μg/kg and 50 μg / kg, respectively. A good linearity (r ＞ 0.99) was achieved for the target compound over the range of 20 - 1000 μg/L. The recoveries at the three spiked levels (50, 100, 500 μg/kg) in the blank matrices such as flour, barley, soybean, rice, cornmeal, cassava and wheat, were varied from 75.6% to 111.0% with the relative standard deviations no more than 13.0%. The method is accurate, efficient, sensitive and practical. The cost of pretreatment is obviously reduced by replacing immunoaffinity columns and Mycosep columns with HLB columns which have the same purification effect.