Unveiling antibiofilm potential: proteins from Priestia sp. targeting Staphylococcus aureus biofilm formation.

Staphylococcus aureus is the etiologic agent of many nosocomial infections, and its biofilm is frequently isolated from medical devices. Moreover, the dissemination of multidrug-resistant (MDR) strains from this pathogen, such as methicillin-resistant S. aureus (MRSA) strains, is a worldwide public health issue. The inhibition of biofilm formation can be used as a strategy to weaken bacterial resistance. Taking that into account, we analysed the ability of marine sponge-associated bacteria to produce antibiofilm molecules, and we found that marine Priestia sp., isolated from marine sponge Scopalina sp. collected on the Brazilian coast, secretes proteins that impair biofilm development from S. aureus. Partially purified proteins (PPP) secreted after 24 hours of bacterial growth promoted a 92% biofilm mass reduction and 4.0 µg/dL was the minimum concentration to significantly inhibit biofilm formation. This reduction was visually confirmed by light microscopy and Scanning Electron Microscopy (SEM). Furthermore, biochemical assays showed that the antibiofilm activity of PPP was reduced by ethylenediaminetetraacetic acid (EDTA) and 1,10 phenanthroline (PHEN), while it was stimulated by zinc ions, suggesting an active metallopeptidase in PPP. This result agrees with mass spectrometry (MS) identification, which indicated the presence of a metallopeptidase from the M28 family. Additionally, whole-genome sequencing analysis of Priestia sp. shows that gene ywad, a metallopeptidase-encoding gene, was present. Therefore, the results presented herein indicate that PPP secreted by the marine Priestia sp. can be explored as a potential antibiofilm agent and help to treat chronic infections.

Exploring Scaffold Hopping for Novel 2-(Quinolin-4-yloxy)acetamides with Enhanced Antimycobacterial Activity.

Utilizing a scaffold-hopping strategy from the drug candidate telacebec, a novel series of 2-(quinolin-4-yloxy)acetamides was synthesized and evaluated as inhibitors of Mycobacterium tuberculosis (Mtb) growth. These compounds demonstrated potent activity against drug-sensitive and multidrug-resistant strains (MIC ≤ 0.02 µM). Leading compounds were evaluated against a known qcrB resistant strain (T313A), and their loss in activity suggested that the cytochrome bc1 complex is the likely target. Additionally, these structures showed high selectivity regarding mammalian cells (selectivity index > 500) and stability across different aqueous media. Furthermore, some of the synthesized quinolines demonstrated aqueous solubility values that exceeded those of telacebec, while maintaining low rates of metabolism. Finally, a selected compound prevented Mtb growth by more than 1.7 log10 colony forming units in a macrophage model of tuberculosis (TB) infection. These findings validate the proposed design and introduce new 2-(quinolin-4-yloxy)acetamides with potential for development in TB drug discovery campaigns.

Metarhizium anisopliae E6 secretome reveals molecular players in host specificity and toxicity linked to cattle tick infection.

Although Metarhizium anisopliae is one of the most studied fungal biocontrol agents, its infection mechanism is far from being completely understood. Using multidimensional protein identification technology (MudPIT), we evaluated the differential secretome of M. anisopliae E6 induced by the host Rhipicephalus microplus cuticle. The proteomic result showed changes in the expression of 194 proteins after exposure to host cuticle, such as proteins involved in adhesion, penetration, stress and fungal defense. Further, we performed a comparative genomic distribution of differentially expressed proteins of the M. anisopliae secretome against another arthropod pathogen, using the Beauveria bassiana ARSEF2860 protein repertory. Among 47 analyzed protein families, thirty were overexpressed in the M. anisopliae E6 predicted genome compared to B. bassiana. An in vivo toxicity assay using a Galleria mellonella model confirmed that the M. anisopliae E6 secretome was more toxic in cattle tick infections compared to other secretomes, including B. bassiana with cattle ticks and M. anisopliae E6 with the insect Dysdereus peruvianus, which our proteomic results had also suggested. These results help explain molecular aspects associated with host infection specificity due to genetic differences and gene expression control at the protein level in arthropod-pathogenic fungi.

SARS-CoV-2 immune complex triggers human monocyte necroptosis.

We analyzed the ability of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) itself and SARS-CoV-2-IgG immune complexes to trigger human monocyte necroptosis. SARS-CoV-2 was able to induce monocyte necroptosis dependently of MLKL activation. Necroptosis-associated proteins (RIPK1, RIPK3 and MLKL) were involved in SARS-CoV-2N1 gene expression in monocytes. SARS-CoV-2 immune complexes promoted monocyte necroptosis in a RIPK3- and MLKL-dependent manner, and Syk tyrosine kinase was necessary for SARS-CoV-2 immune complex-induced monocyte necroptosis, indicating the involvement of Fcγ receptors on necroptosis. Finally, we provide evidence that elevated LDH levels as a marker of lytic cell death are associated with COVID-19 pathogenesis.

Identification of potential inhibitors of Mycobacterium tuberculosis shikimate kinase: molecular docking, in silico toxicity and in vitro experiments.

Tuberculosis (TB) is one of the main causes of death from a single pathological agent, Mycobacterium tuberculosis (Mtb). In addition, the emergence of drug-resistant TB strains has exacerbated even further the treatment outcome of TB patients. It is thus needed the search for new therapeutic strategies to improve the current treatment and to circumvent the resistance mechanisms of Mtb. The shikimate kinase (SK) is the fifth enzyme of the shikimate pathway, which is essential for the survival of Mtb. The shikimate pathway is absent in humans, thereby indicating SK as an attractive target for the development of anti-TB drugs. In this work, a combination of in silico and in vitro techniques was used to identify potential inhibitors for SK from Mtb (MtSK). All compounds of our in-house database (Centro de Pesquisas em Biologia Molecular e Funcional, CPBMF) were submitted to in silico toxicity analysis to evaluate the risk of hepatotoxicity. Docking experiments were performed to identify the potential inhibitors of MtSK according to the predicted binding energy. In vitro inhibitory activity of MtSK-catalyzed chemical reaction at a single compound concentration was assessed. Minimum inhibitory concentration values for in vitro growth of pan-sensitive Mtb H37Rv strain were also determined. The mixed approach implemented in this work was able to identify five compounds that inhibit both MtSK and the in vitro growth of Mtb.

Novel 4-aminoquinolines: Synthesis, inhibition of the Mycobacterium tuberculosis enoyl-acyl carrier protein reductase, antitubercular activity, SAR, and preclinical evaluation.

Herein a series of 4-aminoquinolines were synthesized in an attempt to optimize and study the structural features related to LABIO-17 biological activity, a Mycobacterium tuberculosis NADH-dependent enoyl-acyl carrier protein reductase (MtInhA) inhibitor previously identified by a virtual-ligand-screening approach. Structure-activity relationships led to novel submicromolar inhibitors of MtInhA and potent antitubercular agents. The lead compound is 87-fold more potent as enzymatic inhibitors and 32-fold more potent against M. tuberculosis H37Rv strain in comparison with LABIO-17. These molecules were also active against multidrug-resistant strains, devoid of apparent toxicity to mammalian cells and showed favorable in vitro ADME profiles. Additionally, these compounds were active in an intracellular model of tuberculosis (TB) infection, showed no genotoxicity signals, satisfactory absorption parameters and absence of in vivo acute toxicity. Finally, treatment with selected 4-aminoquinoline for two weeks produced bacteriostatic effect in a murine model of TB. Taken together, these findings indicate that this chemical class may furnish candidates for the future development of drug-sensitive and drug-resistant tuberculosis treatments.

Antitubercular Activity of Novel 2-(Quinoline-4-yloxy)acetamides with Improved Drug-Like Properties.

Using cycloalkyl and electron-donating groups to decrease the carbonyl electrophilicity, a novel series of 2-(quinoline-4-yloxy)acetamides was synthesized and evaluated as in vitro inhibitors of Mycobacterium tuberculosis (Mtb) growth. Structure-activity relationship studies led to selective and potent antitubercular agents with minimum inhibitory concentrations in the submicromolar range against drug-sensitive and drug-resistant Mtb strains. An evaluation of the activity of the lead compounds against a spontaneous qcrB mutant strain indicated that the structures targeted the cytochrome bc 1 complex. In addition, selected molecules inhibited Mtb growth in a macrophage model of tuberculosis infection. Furthermore, the leading compound was chemically stable depending on the context and showed good kinetic solubility, high permeability, and a low rate of in vitro metabolism. Finally, the pharmacokinetic profile of the compound was assessed after oral administration to mice. To the best of our knowledge, for the first time, a 2-(quinoline-4-yloxy)acetamide was obtained with a sufficient exposure, which may enable in vivo effectiveness and its further development as an antituberculosis drug candidate.

Viral isolation allows characterization of early samples of SARS-CoV-2 lineage B1.1.33 with unique mutations (S: H655Y and T63N) circulating in Southern Brazil in 2020.

Different approaches are in use to improve our knowledge about the causative agent of coronavirus disease (COVID-19). Cell culture-based methods are the better way to perform viral isolation, evaluate viral infectivity, and amplify the virus. Furthermore, next-generation sequencing (NGS) have been essential to analyze a complete genome and to describe new viral species and lineages that have arisen over time. Four naso-oropharyngeal swab samples, collected from April to July of 2020, were isolated and sequenced aiming to produce viral stocks and analyze the mutational profile of the found lineage. B.1.1.33 was the lineage detected in all sequences. Although the samples belong to the same lineage, it was possible to evaluate different mutations found including some that were first described in these sequences, like the S:H655Y and T63N. The results described here can help to elicit how the pandemic started to spread and how it has been evolving in south Brazil.

µProteInS-a proteogenomics pipeline for finding novel bacterial microproteins encoded by small ORFs.

SUMMARY: Genome annotation pipelines traditionally exclude open reading frames (ORFs) shorter than 100 codons to avoid false identifications. However, studies have been showing that these may encode functional microproteins with meaningful biological roles. We developed µProteInS, a proteogenomics pipeline that combines genomics, transcriptomics and proteomics to identify novel microproteins in bacteria. Our pipeline employs a model to filter out low confidence spectra, to avoid the need for manually inspecting Mass Spectrometry data. It also overcomes the shortcomings of traditional approaches that usually exclude overlapping genes, leaderless transcripts and non-conserved sequences, characteristics that are common among small ORFs (smORFs) and hamper their identification. AVAILABILITY AND IMPLEMENTATION: µProteInS is implemented in Python 3.8 within an Ubuntu 20.04 environment. It is an open-source software distributed under the GNU General Public License v3, available as a command-line tool. It can be downloaded at https://github.com/Eduardo-vsouza/uproteins and either installed from source or executed as a Docker image. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

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.

EPSP Synthase-Depleted Cells Are Aromatic Amino Acid Auxotrophs in Mycobacterium smegmatis.

The epidemiological importance of mycobacterial species is indisputable, and the necessity to find new molecules that can inhibit their growth is urgent. The shikimate pathway, required for the synthesis of important bacterial metabolites, represents a set of targets for inhibitors of Mycobacterium tuberculosis growth. The aroA-encoded 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) enzyme catalyzes the sixth step of the shikimate pathway. In this study, we combined gene disruption, gene knockdown, point mutations (D61W, R134A, E321N), and kinetic analysis to evaluate aroA gene essentiality and vulnerability of its protein product, EPSPS, from Mycolicibacterium (Mycobacterium) smegmatis (MsEPSPS). We demonstrate that aroA-deficient cells are auxotrophic for aromatic amino acids (AroAAs) and that the growth impairment observed for aroA-knockdown cells grown on defined medium can be rescued by AroAA supplementation. We also evaluated the essentiality of selected MsEPSPS residues in bacterial cells grown without AroAA supplementation. We found that the catalytic residues R134 and E321 are essential, while D61, presumably important for protein dynamics and suggested to have an indirect role in catalysis, is not essential under the growth conditions evaluated. We have also determined the catalytic efficiencies (Kcat/Km) of recombinant wild-type (WT) and mutated versions of MsEPSPS (D61W, R134A, E321N). Our results suggest that drug development efforts toward EPSPS inhibition may be ineffective if bacilli have access to external sources of AroAAs in the context of infection, which should be evaluated further. In the absence of AroAA supplementation, aroA from M. smegmatis is essential, its essentiality is dependent on MsEPSPS activity, and MsEPSPS is vulnerable. IMPORTANCE We found that cells from Mycobacterium smegmatis, a model organism safer and easier to study than the disease-causing mycobacterial species, when depleted of an enzyme from the shikimate pathway, are auxotrophic for the three aromatic amino acids (AroAAs) that serve as building blocks of cellular proteins: l-tryptophan, l-phenylalanine, and l-tyrosine. That supplementation with only AroAAs is sufficient to rescue viable cells with the shikimate pathway inactivated was unexpected, since this pathway produces an end product, chorismate, that is the starting compound of essential pathways other than the ones that produce AroAAs. The depleted enzyme, the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), catalyzes the sixth step of shikimate pathway. Depletion of this enzyme inside cells was performed by disrupting or silencing the EPSPS-encoding aroA gene. Finally, we evaluated the essentiality of specific residues from EPSPS that are important for its catalytic activity, determined with experiments of enzyme kinetics using recombinant EPSPS mutants.

Targeting thymidine phosphorylase inhibition in human colorectal cancer xenografts.

Human thymidine phosphorylase (hTP) is overexpressed in several solid tumors and is commonly associated with aggressiveness and unfavorable prognosis. 6-(((1,3-Dihydroxypropan-2-yl)amino)methyl)-5-iodopyrimidine-2,4(1H,3H)-dione (CPBMF-223) is a noncompetitive hTP inhibitor, which has been described as a tumor angiogenesis inhibitor. The present study investigated the effects of CPBMF-223 in a xenograft tumor induced by human colorectal carcinoma cells (HCT-116). Additionally, CPBMF-223 capacity to reduce cell migration, its toxicological profile, and pharmacokinetic characteristics, were also evaluated. The intraperitoneal treatment with CPBMF-223 markedly prevented the relative tumor growth with an efficacy similar to that observed for 5-fluorouracil. Interestingly, number of vessels were significantly decreased in the treated groups. Moreover, CPBMF-223 significantly reduced the migration of cell line HCT-116. In the Ames assay and in an acute oral toxicity test, the molecule did not alter any evaluated parameter. Using the zebrafish toxicity model, cardiac and locomotor parameters were slightly changed. Regarding the pharmacokinetics profile, CPBMF-223 showed clearance of 9.42 L/h/kg after intravenous administration, oral bioavailability of 13.5%, and a half-life of 0.75 h. Our findings shed new light on the role of hTP in colorectal cancer induced by HCT-116 cell in mice, pointing out CPBMF-223 as, hopefully, a promising drug candidate.

Expression of Bacillus amyloliquefaciens transglutaminase in recombinant E. coli under the control of a bicistronic plasmid system in DO-stat fed-batch bioreactor cultivations.

We studied the expression of Bacillus amyloliquefaciens transglutaminase cloned in Escherichia coli BL21(DE3)pLysS harboring the plasmid pBAD/3C/bTGase, a bicistronic expression system, in bioreactor cultivation. Batch and fed-batch controlled as DO-stat strategies were employed for the production of the recombinant enzyme. In 30 h-batch cultivations using Terrific broth (TB), 6 g/L of biomass and 3.12 U/mgprotein of transglutaminase activity were obtained. DO-stat fed-batch cultivations under the control of oxygen concentration (DO-stat) using TB as medium but fed with glucose allowed the increment in biomass formation (17.5 g/L) and enzyme activity (6.43 U/mgprotein). DO-stat fed-batch using mineral medium (M9) and fed with glucose under the same conditions produced even higher enzymatic activity (9.14 U/mgprotein). The pH effect was investigated, and the best enzymatic activity could be observed at pH 8. In all cultivations, the bicistronic system remained stable, with 100% of plasmid-bearing cells. These results show that E. coli bearing bicistronic plasmid constructs to express recombinant TGase could be cultivated in bioreactors under DO-stat fed-batch using mineral medium and it is a promising strategy in future optimizations to produce this important enzyme.

Ultrasound-Assisted Synthesis of 4-Alkoxy-2-methylquinolines: An Efficient Method toward Antitubercular Drug Candidates.

Tuberculosis (TB) has been described as a global health crisis since the second half of the 1990s. Mycobacterium tuberculosis (Mtb), the etiologic agent of TB in humans, is a very successful pathogen, being the main cause of death in the population among infectious agents. In 2019, it was estimated that around 10 million individuals were contaminated by this bacillus and about 1.2 million succumbed to the disease. In recent years, our research group has reported the design and synthesis of quinoline derivatives as drug candidates for the treatment of TB. These compounds have demonstrated potent and selective growth inhibition of drug-susceptible and drug-resistant Mtb strains. Herein, a new synthetic approach was established providing efficient and rapid access (15 min) to a series of 4-alkoxy-6-methoxy-2-methylquinolines using ultrasound energy. The new synthetic protocol provides a simple procedure utilizing an open vessel system that affords the target products at satisfactory yields (45-84%) and elevated purities (≥95%). The methodology allows the evaluation of a larger number of molecules in assays against the bacillus, facilitating the determination of the structure-activity relationship with a reduced environmental cost.

8-Mercaptoguanine-based inhibitors of Mycobacterium tuberculosis dihydroneopterin aldolase: synthesis, in vitro inhibition and docking studies.

The dihydroneopterin aldolase (DHNA, EC 4.1.2.25) activity of FolB protein is required for the conversion of 7,8-dihydroneopterin (DHNP) to 6-hydroxymethyl-7,8-dihydropterin (HP) and glycolaldehyde (GA) in the folate pathway. FolB protein from Mycobacterium tuberculosis (MtFolB) is essential for bacilli survival and represents an important molecular target for drug development. S8-functionalized 8-mercaptoguanine derivatives were synthesised and evaluated for inhibitory activity against MtFolB. The compounds showed IC50 values in the submicromolar range. The inhibition mode and inhibition constants were determined for compounds that exhibited the strongest inhibition. Additionally, molecular docking analyses were performed to suggest enzyme-inhibitor interactions and ligand conformations. To the best of our knowledge, this study describes the first class of MtFolB inhibitors.

Anti-tubercular profile of new selenium-menadione conjugates against Mycobacterium tuberculosis H37Rv (ATCC 27294) strain and multidrug-resistant clinical isolates.

Tuberculosis (TB) is one of the most fatal diseases and is responsible for the infection of millions of people around the world. Most recently, scientific frontiers have been engaged to develop new drugs that can overcome drug-resistant TB. Following this direction, using a designed scaffold based on the combination of two separate pharmacophoric groups, a series of menadione-derived selenoesters was developed with good yields. All products were evaluated for their in vitro activity against Mycobacterium tuberculosis H37Rv and attractive results were observed, especially for the compounds 8a, 8c and 8f (MICs 2.1, 8.0 and 8.1 µM, respectively). In addition, 8a, 8c and 8f demonstrated potent in vitro activity against multidrug-resistant clinical isolates (CDCT-16 and CDCT-27) with promising MIC values ranging from 0.8 to 3.1 µM. Importantly, compounds 8a and 8c were found to be non-toxic against the Vero cell line. The SI value of 8a (>23.8) was found to be comparable to that of isoniazid (>22.7), which suggests the possibility of carrying out advanced studies on this derivative. Therefore, these menadione-derived selenoesters obtained as hybrid compounds represent promising new anti-tubercular agents to overcome TB multidrug resistance.

Pentacyanoferrate(II) complex of pyridine-4- and pyrazine-2-hydroxamic acid as source of HNO: investigation of anti-tubercular and vasodilation activities.

A pharmacophore design approach, based on the coordination chemistry of an intimate molecular hybrid of active metabolites of pro-drugs, known to release active species upon enzymatic oxidative activation, is devised. This is exemplified by combining two anti-mycobacterial drugs: pyrazinamide (first line) and delamanid (third line) whose active metabolites are pyrazinoic acid (PyzCOOH) and likely nitroxyl (HNO (or NO.)), respectively. Aiming to generate those active species, a hybrid compound was envisaged by coordination of pyrazine-2-hydroxamic acid (PyzCONHOH) with a Na3[FeII(CN)5] moiety. The corresponding pentacyanoferrate(II) complex Na4[FeII(CN)5(PyzCONHO-)] was synthesized and characterized by several spectroscopic techniques, cyclic voltammetry, and DFT calculations. Chemical oxidation of this complex with H2O2 was shown to induce the release of the metabolite PyzCOOH, without the need of the Mycobacterium tuberculosis (Mtb) pyrazinamidase enzyme (PncA). Control experiments show that both H2O2- and N-coordinated pyrazine FeII species are required, ruling out a direct hydrolysis of the hydroxamic acid or an alternative oxidative route through chelation of a metal center by a hydroxamic group. The release of HNO was observed using EPR spectroscopy in the presence of a spin trapping agent. The devised iron metal complex of pyrazine-2-hydroxamic acid was found inactive against an actively growing/non-resistant Mtb strain; however, it showed a strong dose-dependent and reversible vasodilatory activity with mostly lesser toxic effects than the reference drug sodium nitroprussiate, unveiling thus a potential indication for acute or chronic cardiovascular pathology. This is a priori a further indirect evidence of HNO release from this metal complex, standing as a possible pharmacophore model for an alternative vasodilator drug.

Design, synthesis, and evaluation of new 2-(quinoline-4-yloxy)acetamide-based antituberculosis agents.

Using a classical molecular simplification approach, a series of 36 quinolines were synthesized and evaluated as in vitro inhibitors of Mycobacterium tuberculosis (M. tuberculosis) growth. Structure-activity relationship (SAR) studies leaded to potent antitubercular agents, with minimum inhibitory concentration (MIC) values as low as 0.3 µM against M. tuberculosis H37Rv reference strain. Furthermore, the lead compounds were active against multidrug-resistant strains, without cross-resistance with some first- and second-line drugs. Testing the molecules against a spontaneous mutant strain containing a single mutation in the qcrB gene (T313A) indicated that the synthesized quinolines targeted the cytochrome bc1 complex. In addition, leading compounds were devoid of apparent toxicity to HepG2 and Vero cells and showed moderate elimination rates in human liver S9 fractions. Finally, the selected structures inhibited M. tuberculosis growth in a macrophage model of tuberculosis infection. Taken together, these data indicate that this class of compounds may furnish candidates for the future development of antituberculosis drugs.

Cloning and expression of the Bacillus amyloliquefaciens transglutaminase gene in E. coli using a bicistronic vector construction.

Transglutaminases (TGases) are a class of transferases widely used in the food and biotechnology industries. In this work, we describe the production of recombinant Bacillus amyloliquefaciens TGase in Escherichia coli, obtaining the protein in its soluble and active form. In order to reduce TGase activity inside host cells and consequently its toxicity, we constructed a bicistronic plasmid containing the B. amyloliquefaciens TGase gene fused to the inhibitory Streptomyces caniferus prodomain. To make the enzyme active and avoid the need of prodomain removal in vitro, we also cloned the 3C protease gene into the same plasmid. After a fast single-step purification protocol, we obtained a partially purified recombinant TGase with 37 mU/mg protein activity, that crosslinked bovine serum albumin (BSA). This is the first report on the expression of B. amyloliquefaciens TGase in E. coli in its mature and active form.