Magnetic core-shell cellulose system for the oriented immobilization of a recombinant ß-galactosidase with a protein tag.

The objective of this study was to immobilize a recombinant ß-galactosidase (Gal) tagged with a cellulose-binding domain (CBD) onto a magnetic core-shell (CS) cellulose system. After 30 min of reaction, 4 U/capsule were immobilized (CS@Gal), resulting in levels of yield and efficiency exceeding 80 %. The optimal temperature for ß-galactosidase-CBD activity increased from 40 to 50 °C following oriented immobilization. The inhibitory effect of galactose decreased in the enzyme reactions catalyzed by CS@Gal, and Mg2+ increased the immobilized enzyme activity by 40 % in the magnetic CS cellulose system. The relative enzyme activity of the CS@Gal was 20 % higher than that of the soluble enzyme activity after 20 min at 50 °C. The CS support and CS@Gal capsules exhibited an average size of 8 ± 1 mm, with the structure of the shell (alginate-pectin-cellulose) enveloping and isolating the magnetic core. The immobilized ß-galactosidase-CBD within the magnetic CS cellulose system retained ∼80 % of its capacity to hydrolyze lactose from skim milk after 10 reuse cycles. This study unveils a novel and promising support for the oriented immobilization of recombinant ß-galactosidase using a magnetic CS system and a CBD tag. This support facilitates ß-galactosidase reuse and efficient separation, consequently enhancing the catalytic properties of the enzyme.

Lactose hydrolysis in packed-and fluidized-bed reactors using a recombinant ß-galactosidase immobilized on magnetic core-shell capsules.

The objective of this study was to develop a bioprocess for lactose hydrolysis in diverse dairy matrices, specifically skim milk and cheese whey, utilizing column reactors employing a core-shell enzymatic system featuring ß-galactosidase fused to a Cellulose Binding Domain (CBD) tag (ß-galactosidase-CBD). The effectiveness of reactor configurations, including ball columns and toothed columns operating in packed and fluidized-bed modes, was evaluated for catalyzing lactose hydrolysis in both skim milk and cheese whey. In a closed system, these reactors achieved lactose hydrolysis rates of approximately 50% within 5 h under all evaluated conditions. Considering the scale of the bioprocess, the developed enzymatic system was capable of continuously hydrolyzing 9.6 L of skim milk while maintaining relative hydrolysis levels of approximately 50%. The biocatalyst, created by immobilizing ß-galactosidase-CBD on magnetic core-shell capsules, exhibited exceptional operational stability, and the proposed bioprocess employing these column reactors showcases the potential for scalability.

A systematic review about affinity tags for one-step purification and immobilization of recombinant proteins: integrated bioprocesses aiming both economic and environmental sustainability.

The present study reviewed and discussed the promising affinity tags for one-step purification and immobilization of recombinant proteins. The approach used to structure this systematic review was The Preferred Reporting Items for Systematic Review and Meta-analysis (PRISMA) methodology. The Scopus and Web of Science databases were used to perform the bibliographic survey by which 267 articles were selected. After the inclusion/exclusion criteria and the screening process, from 25 chosen documents, we identified 7 types of tags used in the last 10 years, carbohydrate-binding module tag (CBM), polyhistidine (His-tag), elastin-like polypeptides (ELPs), silaffin-3-derived pentalysine cluster (Sil3k tag), N-acetylmuramidase (AcmA tag), modified haloalkane dehalogenase (HaloTag®), and aldehyde from a lipase polypeptide (Aldehyde tag). The most used bacterial host for expressing the targeted protein was Escherichia coli and the most used expression vector was pET-28a. The results demonstrated two main immobilization and purification methods: the use of supports and the use of self-aggregating tags without the need of support, depending on the tag used. Besides, the chosen terminal for cloning the tag proved to be very important once it could alter enzyme activity. In conclusion, the best tag for protein one-step purification and immobilization was CBM tag, due to the eco-friendly supports that can be provided from industry wastes, the fast immobilization with high specificity, and the reduced cost of the process.

Application of cellulosic materials as supports for single-step purification and immobilization of a recombinant ß-galactosidase via cellulose-binding domain.

This study aimed to develop single-step purification and immobilization processes on cellulosic supports of ß-galactosidase from Kluyveromyces sp. combined with the Cellulose-Binding Domain (CBD) tag. After 15 min of immobilization, with an enzymatic load of 150 U/gsupport, expressed activity values reached 106.88 (microcrystalline cellulose), 115.03 (alkaline nanocellulose), and 108.47 IU/g (acid nanocellulose). The derivatives produced were less sensitive to the presence of galactose in comparison with the soluble purified enzyme. Among the cations assessed (Na+, K+, Mg2+, and Ca2+), magnesium provided the highest increase in the enzymatic activity of ß-galactosidases immobilized on cellulosic supports. Supports and derivatives showed no cytotoxic effect on the investigated cell cultures (HepG2 and Vero). Derivatives showed high operational stability in the hydrolysis of milk lactose and retained from 53 to 64% of their hydrolysis capacity after 40 reuse cycles. This study obtained biocatalyzers with promising characteristics for application in the food industry. Biocatalyzers were obtained through a low-cost one-step sustainable bioprocess of purification and immobilization of a ß-galactosidase on cellulose via CBD.

One-step purification of a recombinant beta-galactosidase using magnetic cellulose as a support: Rapid immobilization and high thermal stability.

For the first time, this work reported the one-step purification and targeted immobilization process of a ß-galactosidase (Gal) with the Cellulose Binding Domain (CBD) tag, by binding it to different magnetic cellulose supports. The process efficiency after ß-galactosidase-CBD immobilization on magnetic cellulose-based supports showed values of approximately 90% for all evaluated enzymatic loads. Compared with free Gal, derivatives showed affinity values between ß-galactosidase and the substrate 1.2 × higher in the lactose hydrolysis of milk. ß-Galactosidase-CBD's oriented immobilization process on supports increased the thermal stability of the immobilized enzyme by up to 7 × . After 15 cycles of reuse, both enzyme preparations showed a relative hydrolysis percentage of 50% of lactose in milk. The oriented immobilization process developed for purifying recombinant proteins containing the CBD tag enabled the execution of both steps simultaneously and quickly and the obtention of ß-galactosidases with promising catalytic characteristics for application in the food and pharmaceutical industries.

Synthesis of magnetic nanoparticles functionalized with histidine and nickel to immobilize His-tagged enzymes using ß-galactosidase as a model.

The aim of this study was to synthesize iron magnetic nanoparticles functionalized with histidine and nickel (Fe3O4-His-Ni) to be used as support materials for oriented immobilization of His-tagged recombinant enzymes of high molecular weight, using ß-galactosidase as a model. The texture, morphology, magnetism, thermal stability, pH and temperature reaction conditions, and the kinetic parameters of the biocatalyst obtained were assessed. In addition, the operational stability of the biocatalyst in the lactose hydrolysis of cheese whey and skim milk by batch processes was also assessed. The load of 600 Uenzyme/gsupport showed the highest recovered activity value (~50%). After the immobilization process, the recombinant ß-galactosidase (HisGal) showed increased substrate affinity and greater thermal stability (~50×) compared to the free enzyme. The immobilized ß-galactosidase was employed in batch processes for lactose hydrolysis of skim milk and cheese whey, resulting in hydrolysis rates higher than 50% after 15 cycles of reuse. The support used was obtained in the present study without modifying chemical agents. The support easily recovered from the reaction medium due to its magnetic characteristics. The iron nanoparticles functionalized with histidine and nickel were efficient in the oriented immobilization of the recombinant ß-galactosidase, showing its potential application in other high-molecular-weight enzymes.

Production of beta-galactosidase fused to a cellulose-binding domain for application in sustainable industrial processes.

This study aimed to produce and characterize a recombinant Kluyveromyces sp. ß-galactosidase fused to a cellulose-binding domain (CBD) for industrial application. In expression assays, the highest enzymatic activities occurred after 48 h induction on Escherichia coli C41(DE3) strain at 20 °C in Terrific Broth (TB) culture medium, using isopropyl ß-d-1-thiogalactopyranoside (IPTG) 0.5 mM (108.77 U/mL) or lactose 5 g/L (93.10 U/mL) as inducers. Cultures at bioreactor scale indicated that higher product yield values in relation to biomass (2000 U/g) and productivity (0.72 U/mL.h) were obtained in culture media containing higher protein concentration. The recombinant enzyme showed high binding affinity to nanocellulose, reaching both immobilization yield and efficiency values of approximately 70% at pH 7.0 after 10 min reaction. The results of the present study pointed out a strategy for recombinant ß-galactosidase-CBD production and immobilization, aiming toward the application in sustainable industrial processes using low-cost inputs.

Effect of by-products from the dairy industry as alternative inducers of recombinant ß-galactosidase expression.

OBJECTIVE: The aim of the present study was to evaluate the efficiency of lactose derived from cheese whey and cheese whey permeate as inducer of recombinant Kluyveromyces sp. ß-galactosidase enzyme produced in Escherichia coli. Two E. coli strains, BL21(DE3) and Rosetta (DE3), were used in order to produce the recombinant enzyme. Samples were evaluated for enzyme activity, total protein content, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis after induction with isopropyl-ß-D-1-thiogalactoside (IPTG) (0.05 and 1 mM) and lactose, cheese whey, and cheese whey permeate solutions (1, 10, and 20 g/L lactose) at shake-flask cultivation, and whey permeate solution (10 g/L lactose) at bioreactor scale. RESULTS: The highest specific activities obtained with IPTG as inducer (0.05 mM) after 9 h of induction, were 23 and 33 U/mgprotein with BL21(DE3) and Rosetta(DE3) strains, respectively. Inductions performed with lactose and cheese whey permeate (10 and 20 g/L lactose) showed the highest specific activities at the evaluated hours, exhibiting better results than those obtained with IPTG. Specific activity of recombinant ß-galactosidase using whey permeate (10 g/L lactose) showed values of approximately 46 U/mgprotein after 24-h induction at shake-flask study, and approximately 26 U/mgprotein after 16-h induction at bench bioreactor. CONCLUSIONS: The induction with cheese whey permeate was more efficient for recombinant ß-galactosidase expression than the other inducers tested, and thus, represents an alternative form to reduce costs in recombinant protein production.

Evaluation of the Ability of Miltefosine Associated with Topical GM-CSF in Modulating the Immune Response of Patients with Cutaneous Leishmaniasis.

Cutaneous leishmaniasis (CL) due to L. braziliensis is associated with an exaggerated inflammatory response and tissue damage. Miltefosine is more effective than pentavalent antimony (Sbv) in the treatment of CL, and here, we evaluate the ability of Sbv, miltefosine, and GM-CSF administered intravenously, orally, or topically, respectively, to modify the immune response. Patients were treated with miltefosine plus GM-CSF, miltefosine plus placebo, or Sbv. Mononuclear cells were stimulated with soluble Leishmania antigen (SLA) on day 0 and day 15 of therapy, and cytokine levels were determined in supernatants by ELISA. The lymphocyte proliferation and oxidative burst were evaluated by flow cytometry, and the degree of infection and Leishmania killing by optical microscopy. Proliferation of CD4+ T cells were enhanced in patients using miltefosine and in CD8+ T cells when GM-CSF was associated. Enhancement in the oxidative burst occurred in the miltefosine plus GM-CSF group on day 15 of therapy. Moreover, the number of L. braziliensis in infected monocytes on day 15 as well as the percentage of infected cells was lower after 48- and 72-hour culture in cells from patients treated with miltefosine plus GM-CSF. In addition to the ability of miltefosine to kill Leishmania, the changes in the immune response caused by miltefosine and GM-CSF may increase the cure rate of CL patients using these drugs.

Microbial ß-Galactosidases of industrial importance: Computational studies on the effects of point mutations on the lactose hydrolysis reaction.

Hydrolysis efficiency of ß-galactosidases is affected due to a strong inhibition by galactose, hampering the complete lactose hydrolysis. One alternative to reduce this inhibition is to perform mutations in the enzyme's active site. The aim of this study was to evaluate the effect of point mutations on the active site of different microbial ß-galactosidases, using computational techniques. The enzymes of Aspergillus niger (AnßGal), Aspergillus oryzae (AoßGal), Bacillus circulans (BcßGal), Bifidobacterium bifidum (BbßGal), and Kluyveromyces lactis (KlßGal) were used. The mutations were carried out in all residues that were up to 4.5 Å from the galactose/lactose molecules and binding energy was computed. The mutants Tyr96Ala (AnßGal), Asn140Ala and Asn199Ala (AoßGal), Arg111Ala and Glu355Ala (BcßGal), Arg122Ala and Phe358Ala (BbßGal), Tyr523Ala, Phe620Ala, and Trp582Ala (KlßGal) had the best results, with higher effect on galactose binding energy and lower effect on lactose affinity. To maximize enzyme reactions by reducing galactose affinity, double mutations were proposed for BcßGal, BbßGal, and KlßGal. The double mutations in BcßGal and BbßGal caused the highest reduction in galactose affinity, while no satisfactory results were observed to KlßGal. Using computational tools, mutants that reduced galactose affinity without significantly affecting lactose binding were proposed. The mutations proposed can be used to reduce the negative feedback process, improving the catalytic characteristics of ß-galactosidases and rendering them promising for industrial applications.

Production of recombinant human annexin V by fed-batch cultivation.

BACKGROUND: Annexin V, a 35.8 kDa intracellular protein, is a Ca⁺²-dependent phospholipid binding protein with high affinity to phosphatidylserine (PS), which is a well-known hallmark of apoptosis. Annexin V is a sensitive probe for PS exposure upon the cell membrane, and used for detection of apoptotic cells both in vivo and in vitro. Large-scale production of recombinant human annexin V is worth optimization, because of its wide use in nuclear medicine, radiolabeled with (99m)Tc, for the evaluation of cancer chemotherapy treatments, and its use in identification of apoptotic cells in histologic studies. Here we describe the high-yield production of a tag-free version of human annexin V recombinant protein by linear fed-batch cultivation in a bioreactor. RESULTS: We cloned the human ANXA5 coding sequence into the pET-30a (+) expression vector and expressed rhANXA5 in batch and fed-batch cultures. Using E. coli BL21 (DE3) in a semi-defined medium at 37°C, pH 7 in fed-batch cultures, we obtained a 45-fold increase in biomass production, respective to shaker cultivations. We developed a single-step protocol for rhANXA5 purification using a strong anion-exchange column (MonoQ HR16/10). Using these procedures, we obtained 28.5 mg of homogeneous, nontagged and biologically functional human annexin V recombinant protein from 3 g wet weight of bacterial cells from bioreactor cultures. The identity and molecular mass of rhANXA5 was confirmed by mass spectrometry. Moreover, the purified rhANXA5 protein was functionally evaluated in a FITC-annexin V binding experiment and the results demonstrated that rhANXA5 detected apoptotic cells similarly to a commercial kit. CONCLUSIONS: We describe a new fed-batch method to produce recombinant human annexin V in large scale, which may expand the commercial utilities for rhANXAV to applications such as in vivo imaging studies.

Molecular cloning, expression in Escherichia coli and production of bioactive homogeneous recombinant human granulocyte and macrophage colony stimulating factor.

Human granulocyte and macrophage colony stimulating factor (hGM-CSF) is a glycoprotein that activates and enhances the differentiation and survival of neutrophils, eosinophils and macrophages, which play a key role in the innate immune response. Here we describe the construction of the hGM-CSF encoding gene, cloning, expression in Escherichia coli, purification of recombinant hGM-CSF, N-terminal amino acid sequencing, and biological activity assay using TF-1 cells. The results presented show that the combination of experimental strategies employed to obtain recombinant hGM-CSF can yield biologically active protein, and may be useful to scaling-up production of biosimilar protein.

Human granulocyte colony stimulating factor (hG-CSF): cloning, overexpression, purification and characterization.

BACKGROUND: Biopharmaceutical drugs are mainly recombinant proteins produced by biotechnological tools. The patents of many biopharmaceuticals have expired, and biosimilars are thus currently being developed. Human granulocyte colony stimulating factor (hG-CSF) is a hematopoietic cytokine that acts on cells of the neutrophil lineage causing proliferation and differentiation of committed precursor cells and activation of mature neutrophils. Recombinant hG-CSF has been produced in genetically engineered Escherichia coli (Filgrastim) and successfully used to treat cancer patients suffering from chemotherapy-induced neutropenia. Filgrastim is a 175 amino acid protein, containing an extra N-terminal methionine, which is needed for expression in E. coli. Here we describe a simple and low-cost process that is amenable to scaling-up for the production and purification of homogeneous and active recombinant hG-CSF expressed in E. coli cells. RESULTS: Here we describe cloning of the human granulocyte colony-stimulating factor coding DNA sequence, protein expression in E. coli BL21(DE3) host cells in the absence of isopropyl-beta-D-thiogalactopyranoside (IPTG) induction, efficient isolation and solubilization of inclusion bodies by a multi-step washing procedure, and a purification protocol using a single cationic exchange column. Characterization of homogeneous rhG-CSF by size exclusion and reverse phase chromatography showed similar yields to the standard. The immunoassay and N-terminal sequencing confirmed the identity of rhG-CSF. The biological activity assay, in vivo, showed an equivalent biological effect (109.4%) to the standard reference rhG-CSF. The homogeneous rhG-CSF protein yield was 3.2 mg of bioactive protein per liter of cell culture. CONCLUSION: The recombinant protein expression in the absence of IPTG induction is advantageous since cost is reduced, and the protein purification protocol using a single chromatographic step should reduce cost even further for large scale production. The physicochemical, immunological and biological analyses showed that this protocol can be useful to develop therapeutic bioproducts. In summary, the combination of different experimental strategies presented here allowed an efficient and cost-effective protocol for rhG-CSF production. These data may be of interest to biopharmaceutical companies interested in developing biosimilars and healthcare community.

Molecular cloning and sequence analysis of cDNAs encoding for Boophilus microplus, Haemaphysalis longicornis and Rhipicephalus appendiculatus actins.

The nucleotide and deduced amino acid sequences of the actins from ticks, Boophilus microplus, Haemaphysalis longicornis and Rhipicephalus appendiculatus, have been determined. Nucleotide sequence analysis showed open reading frames of 1128-nucleotide-long encoding proteins of 376 amino acids with a predicted molecular weight of 41.82 kDa each. Comparison between the nucleic acid and deduced amino acid sequences as well as structural and phylogenetic analyses of these genes confirmed the high similarity among actins from ticks in comparison to other species.

Docking and small angle X-ray scattering studies of purine nucleoside phosphorylase.

Docking simulations have been used to assess protein complexes with some success. Small angle X-ray scattering (SAXS) is a well-established technique to investigate protein spatial configuration. This work describes the integration of geometric docking with SAXS to investigate the quaternary structure of recombinant human purine nucleoside phosphorylase (PNP). This enzyme catalyzes the reversible phosphorolysis of N-ribosidic bonds of purine nucleosides and deoxynucleosides. A genetic deficiency due to mutations in the gene encoding for PNP causes gradual decrease in T-cell immunity. Inappropriate activation of T-cells has been implicated in several clinically relevant human conditions such as transplant rejection, rheumatoid arthritis, lupus, and T-cell lymphomas. PNP is therefore a target for inhibitor development aiming at T-cell immune response modulation and has been submitted to extensive structure-based drug design. The present analysis confirms the trimeric structure observed in the crystal. The potential application of the present procedure to other systems is discussed.

Expression and immunolocalization of a Boophilus microplus cathepsin L-like enzyme.

Efforts are being undertaken to control tick infestations that cause important economic losses. A cathepsin L-like endopeptidase of Boophilus microplus was expressed in Escherichia coli; the recombinant enzyme was capable of hydrolysing gelatin, tick vitellin and bovine haemoglobin. In this paper we focus on the expression and local of synthesis of this enzyme in the tick. RT-PCR experiments showed that this endopeptidase is transcribed in the gut of partially engorged tick females. In immunoblotting, polyclonal antibodies against the recombinant enzyme reacted with proteins of larvae older than 5 days, of fully and partially engorged female gut. In immunolocalization experiments the enzyme was localized in probable secretory cells of the gut. Based on our findings we postulate that BmCL1 may be involved in haemoglobin degradation in the B. microplus gut. This enzyme may be used as target for the control of this parasite.