Lactobacillus delbrueckii CIDCA 133 fermented milk modulates inflammation and gut microbiota to alleviate acute colitis.

Lactobacillus delbrueckii subsp. lactis CIDCA 133 is a health-promoting bacterium that can alleviate gut inflammation and improve the epithelial barrier in a mouse model of mucositis. Despite these beneficial effects, the protective potential of this strain in other inflammation models, such as inflammatory bowel disease, remains unexplored. Herein, we examined for the first time the efficacy of Lactobacillus delbrueckii CIDCA 133 incorporated into a fermented milk formulation in the recovery of inflammation, epithelial damage, and restoration of gut microbiota in mice with dextran sulfate sodium-induced colitis. Oral administration of Lactobacillus delbrueckii CIDCA 133 fermented milk relieved colitis by decreasing levels of inflammatory factors (myeloperoxidase, N-acetyl-ß-D-glucosaminidase, toll-like receptor 2, nuclear factor-κB, interleukins 10 and 6, and tumor necrosis factor), secretory immunoglobulin A levels, and intestinal paracellular permeability. This immunobiotic also modulated the expression of tight junction proteins (zonulin and occludin) and the activation of short-chain fatty acids-related receptors (G-protein coupled receptors 43 and 109A). Colonic protection was effectively associated with acetate production and restoration of gut microbiota composition. Treatment with Lactobacillus delbrueckii CIDCA 133 fermented milk increased the abundance of Firmicutes members (Lactobacillus genus) while decreasing the abundance of Proteobacteria (Helicobacter genus) and Bacteroidetes members (Bacteroides genus). These promising outcomes influenced the mice's mucosal healing, colon length, body weight, and disease activity index, demonstrating that this immunobiotic could be explored as an alternative approach for managing inflammatory bowel disease.

Purification and partial physical-chemical characterization of a new bovine trypsin proteoform (zeta-trypsin).

Recent advancements in enzyme research have unveiled a new proteoform of bovine trypsin, expanding our understanding of this well-characterized enzyme. While generally similar to other trypsins, this novel proteoform comprises three polypeptide chains, marking a significant difference in activity, kinetic properties, and conformational stability. Compared with the already known bovine trypsin proteoforms, the results showed a lower: activity, kcat and kcat.KM-1 and protein 'foldedness' ratio for the new proteoform. Molecular autolysis, a common feature in trypsin and chymotrypsin, has been explored through comparative physical chemistry properties with other proteoforms. This new proteoform of trypsin not only enriches the existing enzyme repertoire but also promises to shed light on the intricate physiological pathway for enzyme inactivation. Our results suggest that the new trypsin proteoform is one of the likely final pathways for enzyme inactivation in a physiological environment. This discovery opens up new avenues for further research into the functional implications of this new trypsin proteoform.

Association of Fructo-oligosaccharides and Arginine Improves Severity of Mucositis and Modulate the Intestinal Microbiota.

Mucositis is defined as inflammatory and ulcerative lesions along of the gastrointestinal tract that leads to the imbalance of the intestinal microbiota. The use of compounds with action on the integrity of the intestinal epithelium and their microbiota may be a beneficial alternative for the prevention and/or treatment of mucositis. So, the aim of this study was to evaluate the effectiveness of the association of fructo-oligosaccharides (FOS) and arginine on intestinal damage in experimental mucositis. BALB/c mice were randomized into five groups: CTL (without mucositis + saline), MUC (mucositis + saline), MUC + FOS (mucositis + supplementation with FOS-1st until 10th day), MUC + ARG (mucositis + supplementation with arginine-1st until 10th day), and MUC + FOS + ARG (mucositis + supplementation with FOS and arginine-1st until 10th day). On the 7th day, mucositis was induced with an intraperitoneal injection of 300 mg/kg 5-fluorouracil (5-FU), and after 72 h, the animals were euthanized. The results showed that association of FOS and arginine reduced weight loss and oxidative stress (P < 0.05) and maintained intestinal permeability and histological score at physiological levels. The supplementation with FOS and arginine also increased the number of goblet cells, collagen area, and GPR41 and GPR43 gene expression (P < 0.05). Besides these, the association of FOS and arginine modulated intestinal microbiota, leading to an increase in the abundance of the genera Bacteroides, Anaerostipes, and Lactobacillus (P < 0.05) in relation to increased concentration of propionate and acetate. In conclusion, the present results show that the association of FOS and arginine could be important adjuvants in the prevention of intestinal mucositis probably due to modulated intestinal microbiota.

Treatment with Distinct Antibiotic Classes Causes Different Pulmonary Outcomes on Allergic Airway Inflammation Associated with Modulation of Symbiotic Microbiota.

Background: Asthma is a chronic pulmonary disease that affects about 300 million people worldwide. Previous studies have associated antimicrobial use with allergies, but the real impact of antibiotics on asthma is still elusive. We investigated the potential impact of amoxicillin (Amox), trimethoprim/sulfamethoxazole (TMP/SMX), and metronidazole (Metro) in a murine model of OVA-induced allergic airway inflammation. Methods: BALB/c mice received three cycles of 7 days of antibiotics in drinking water followed by 7 days washout and were sensitized i.p. with OVA/Alum at days 0 and 14. After the end of the last antibiotic washout, the mice were challenged with aerosolized OVA. Pulmonary parameters were evaluated, and serum, BAL, and feces were collected for analysis. Results: Amox- and TMP/SMX-treated animals displayed more severe allergic airway inflammation parameters with increased airway hyperresponsiveness, reduced lung alveolar volume, and increased levels in BAL of IL-4 and IL-6. In contrast, Metro-treated mice showed preserved FEV-50, decreased lung inflammation, and higher levels of butyrate and propionate in their feces. Metro treatment was associated with increased OVA-specific IgA in serum. BAL microbiota was abundant in allergic groups but not in nonallergic controls with the Amox-treated group displaying the increased frequency of Proteobacteria, while Metro and TMP/SMX showed increased levels of Firmicutes. In the gut, we observed the enrichment of Akkermansia muciniphila associated with reduced airway inflammation phenotype in the Metro group, even after the recovery period. Conclusion: Our data suggest that different antibiotic treatments may impact the course of experimental allergic airway inflammation in diverse ways by several mechanisms, including modulation of short-chain fat acids production by intestinal microbiota.

Evaluation of biological activities, structural and conformational properties of bovine beta- and alpha-trypsin isoforms in aqueous-organic media.

The study of the biological activity of trypsin isoforms in aqueous-organic media is of great interest to various fields of knowledge and biochemistry applications. Thus enzymatic, structural, and energetic properties of bovine ß- and α-trypsin isoforms were compared in aqueous-organic media using 30 mg of each isoform. The results showed that the changes induced on the structure and activity of the same trypsin isoform occur at different concentrations. Better results for activity (ionic strength of 0.11 mol·L-1, at 37 °C and pH 8.0) were found in 0-40% of ethanolic media in which the activity for ß-trypsin was about 60% higher than ɑ-trypsin. The ethanolic system does not cause significant changes in the level of secondary structure but the ß-trypsin isoform undergoes a major rearrangement. The use of until 60% (v/v) ethanol showed that ß-trypsin presents a denaturation process 17% more cooperative. The organic solvent causes redistribution in the supramolecular arrangement of both isoforms: all concentrations used induced the ß-trypsin molecules to rearrange into agglomerates. The ɑ-trypsin rearranges into agglomerates up to 60% (v/v) of ethanol and aggregates at 80% (v/v) of ethanol. Both isoforms keep the enzymatic activity up to 60% (v/v) of ethanol.

Association of casein micelle size and enzymatic curd strength and dry matter curd yield / Associação do tamanho das micelas de caseína com a força e produção de massa da coalhada

ABSTRACT: The aim of the present study was to explore the association between milk protein content and casein micelle size and to examine the effects of casein micelle size on enzymatic curd strength and dry matter curd yield using reduced laboratory-scale cheese production. In this research, 140 bulk tank milk samples were collected at dairy farms. The traits were analyzed using two linear models, including only fixed effects. Smaller micelles were associated with higher κ-casein and lower αs-casein contents. The casein micellar size (in the absence of the αs-casein and κ-casein effects) did not affect the enzymatic curd strength; however, smaller casein micelles combined with higher fat, lactose, casein and κ-casein contents exhibited a favorable effect on the dry matter curd yield. Overall, results of the present study provide new insights into the importance of casein micelle size for optimizing cheese production.

RESUMO: Este trabalho foi desenvolvido com o objetivo de investigar a associação da composição proteica do leite com o tamanho das micelas de caseína, e o efeito do TMCN sobre a firmeza do coágulo enzimático e da produção de massa seca do coágulo produzido em escala reduzida. Foram coletadas 140 amostras de leite cru de diferentes fazendas. Os dados foram analisados usando dois modelos lineares, incluindo somente efeitos fixos. Menores micelas de caseína foram associadas com maior conteúdo de k-caseína e menor conteúdo de αs-caseína. O tamanho das micelas de caseína sem o efeito da αs-caseína e k-caseína não apresentou efeito sobre a firmeza do coágulo, porém apresentou efeito significatico sobre a produção de massa seca do coágulo. Esses resultados demonstram a importância do tamanho das micelas de caseína para otimizar a produção de queijo.

Determination of structural and thermodynamic parameters of bovine α-trypsin isoform in aqueous-organic media.

The α-trypsin isoform is a globular protein that belongs to serine-protease family and has a polypeptide chain of 223 amino acid residues, six disulfide bridges and two domains with similar structures. The effects of aqueous-organic solvent (ethanol) in different concentration on the α-trypsin structure have been investigated by spectroscopic techniques and thermodynamic data analysis. The results from spectroscopic measurements, including far-UV Circular Dichroism, UV-vis absorption spectroscopy, intrinsic tryptophan fluorescence and dynamic light scattering (DLS) suggest the formation of partially folded states, instead of aggregate states, at high ethanol concentration (>60% v/v ethanol), with little loss of secondary structure, but with significant tertiary structure changes. The thermodynamic data (Tm and ΔH) suggest a loosening of intramolecular weak interactions, which reflects in a flexibility increase such that the catalytic capacity can be increased or decreased according to the ethanol concentration into the system. Overall results we suggest that in range of 0-60% v/v ethanol/buffer, α-trypsin undergoes reversible multimerization phenomena with catalytic activity. However from 60% v/v ethanol/buffer, population of folded partially states with less catalytic activity are predominant.

Oral butyrate reduces oxidative stress in atherosclerotic lesion sites by a mechanism involving NADPH oxidase down-regulation in endothelial cells.

Butyrate is a 4-carbon fatty acid that has antiinflammatory and antioxidative properties. It has been demonstrated that butyrate is able to reduce atherosclerotic development in animal models by reducing inflammatory factors. However, the contribution of its antioxidative effects of butyrate on atherogenesis has not yet been studied. We investigated the influence of butyrate on oxidative status, reactive oxygen species (ROS) release and oxidative enzymes (NADPH oxidase and iNOS) in atherosclerotic lesions of ApoE(-/-) mice and in oxLDL-stimulated peritoneal macrophages and endothelial cells (EA.hy926). The lesion area in aorta was reduced while in the aortic valve, although lesion area was unaltered, superoxide production and protein nitrosylation were reduced in butyrate-supplemented mice. Peritoneal macrophages from the butyrate group presented a lower free radical release after zymosan stimulus. When endothelial cells were pretreated with butyrate before oxLDL stimulus, the CCL-2 and superoxide ion productions and NADPH oxidase subunit p22phox were reduced. In macrophage cultures, in addition to a reduction in ROS release, nitric oxide and iNOS expression were down-regulated. The data suggest that one mechanism related to the effect of butyrate on atherosclerotic development is the reduction of oxidative stress in the lesion site. The reduction of oxidative stress related to NADPH oxidase and iNOS expression levels associated to butyrate supplementation attenuates endothelium dysfunction and macrophage migration and activation in the lesion site.

Antigenicity, Immunogenicity and Protective Efficacy of Three Proteins Expressed in the Promastigote and Amastigote Stages of Leishmania infantum against Visceral Leishmaniasis.

In the present study, two Leishmania infantum hypothetical proteins present in the amastigote stage, LiHyp1 and LiHyp6, were combined with a promastigote protein, IgE-dependent histamine-releasing factor (HRF); to compose a polyproteins vaccine to be evaluated against L. infantum infection. Also, the antigenicity of the three proteins was analyzed, and their use for the serodiagnosis of canine visceral leishmaniasis (CVL) was evaluated. The LiHyp1, LiHyp6, and HRF DNA coding sequences were cloned in prokaryotic expression vectors and the recombinant proteins were purified. When employed in ELISA assays, all proteins were recognized by sera from visceral leishmaniasis (VL) dogs, and presented no cross-reactivity with either sera from dogs vaccinated with a Brazilian commercial vaccine, or sera of Trypanosoma cruzi-infected or Ehrlichia canis-infected animals. In addition, the antigens were not recognized by antibodies from non-infected animals living in endemic or non-endemic areas for leishmaniasis. The immunogenicity and protective efficacy of the three proteins administered in the presence of saponin, individually or in combination (composing a polyproteins vaccine), were evaluated in a VL murine model: BALB/c mice infected with L. infantum. Spleen cells from mice inoculated with the individual proteins or with the polyproteins vaccine plus saponin showed a protein-specific production of IFN-γ, IL-12, and GM-CSF after an in vitro stimulation, which was maintained after infection. These animals presented significant reductions in the parasite burden in different evaluated organs, when compared to mice inoculated with saline or saponin. The decrease in parasite burden was associated with an IL-12-dependent production of IFN-γ against parasite total extracts (produced mainly by CD4+ T cells), correlated to the induction of parasite proteins-driven NO production. Mice inoculated with the recombinant protein-based vaccines showed also high levels of parasite-specific IgG2a antibodies. The polyproteins vaccine administration induced a more pronounced Th1 response before and after challenge infection than individual vaccines, which was correlated to a higher control of parasite dissemination to internal organs.

Evaluation of adjuvant activity of fractions derived from Agaricus blazei, when in association with the recombinant LiHyp1 protein, to protect against visceral leishmaniasis.

The development of effective prophylactic strategies to prevent leishmaniasis has become a high priority. No less important than the choice of an antigen, the association of an appropriate adjuvant is necessary to achieve a successful vaccination, as the majority of the tested antigens contain limited immunogenic properties, and need to be supplemented with immune response adjuvants in order to boost their immunogenicity. However, few effective adjuvants that can be used against leishmaniasis exist on the market today; therefore, it is possible to speculate that the research aiming to identify new adjuvants could be considered relevant. Recently, Agaricus blazei extracts have proved to be useful in enhancing the immune response to DNA vaccines against some diseases. This was based on the Th1 adjuvant activity of the polysaccharide-rich fractions from this mushroom. In this context, the present study evaluated purified fractions derived from Agaricus blazei as Th1 adjuvants through in vitro assays of their immune stimulation of spleen cells derived from naive BALB/c mice. Two of the tested six fractions (namely F2 and F4) were characterized as polysaccharide-rich fractions, and were able to induce high levels of IFN-γ, and low levels of IL-4 and IL-10 in the spleen cells. The efficacy of adjuvant action against L. infantum was evaluated in BALB/c mice, with these fractions being administered together with a recombinant antigen, LiHyp1, which was previously evaluated as a vaccine candidate, associated with saponin, against visceral leishmaniasis (VL). The associations between LiHyp1/F2 and LiHyp1/F4 were able to induce an in vivo Th1 response, which was primed by high levels of IFN-γ, IL-12, and GM-CSF, by low levels of IL-4 and IL-10; as well as by a predominance of IgG2a antibodies in the vaccinated animals. After infection, the immune profile was maintained, and the vaccines proved to be effective against L. infantum. The immune stimulatory effects in the BALB/c mice proved to be similar when comparing the F2 and F4 fractions with a known Th1 adjuvant (saponin), though animals vaccinated with saponin did present a slight to moderate inflammatory edema on their hind footpads. In conclusion, the F2 and F4 fractions appear to induce a Th1-type immune response and, in this context, they could be evaluated in association with other protective antigens against Leishmania, as well as in other disease models.

Gamma trypsin: purification and physicochemical characterization of a novel bovine trypsin isoform.

A novel bovine trypsin isoform was purified from commercial sample by ion exchange chromatography by Sephadex SP C50®. New isoform contains in addition of loss of N-terminus hexapeptide (as found in parent molecule ß-trypsin) an intra-chain split between Lys-155 and Ser-156. The novel enzyme denominate γ-trypsin showed similar properties with α-trypsin isoform in polypeptide number chain (two chain), molecular masses (23,312 Da), secondary structure, hydrodynamic radius and others. In spite of enzymatic and structural similarities of both isoforms, γ-trypsin preferably has a lower rate formation from ß-trypsin, a lower surface charge, but the γ-trypsin has a higher thermal stability than α-trypsin. Due to obtaining facility of purification of bovine trypsin isoforms from commercial font, and properties described above, this enzyme becomes an interesting alternative for the food industry, detergent and biocatalysis research.

Antagonistic activity expressed by Shigella sonnei: identification of a putative new bacteriocin.

Bacteriocins are antibacterial, proteinaceous substances that mediate microbial dynamics. Bacteriocin production is a highly disseminated property among all major lineages of bacteria, including Shigella. In this paper, we addressed the purification and characterisation of a bacteriocin produced by a Shigella sonnei strain (SS9) isolated from a child with acute diarrhoea. The substance was purified through ammonium-sulphate precipitation and sequential steps of chromatography. The intracellular fraction obtained at 75% ammonium sulphate maintained activity following exposure to pH values from 1-11 and storage at -80ºC for more than two years and was inactivated by high temperatures and proteases. The molecular mass of the purified bacteriocin was determined by mass spectrometry to be 18.56 kDa. The N-terminal sequence of the bacteriocin did not match any other antibacterial proteins described. A putative new bacteriocin produced by S. sonnei has been detected. This bacteriocin may represent a newly described protein or a previously described protein with a newly detected function. Considering that SS9 expresses antagonism against other diarrhoeagenic bacteria, the bacteriocin may contribute to S. sonnei virulence and is potentially applicable to either preventing or controlling diarrhoeal disease.

Antagonistic activity expressed by Shigella sonnei: identification of a putative new bacteriocin

Bacteriocins are antibacterial, proteinaceous substances that mediate microbial dynamics. Bacteriocin production is a highly disseminated property among all major lineages of bacteria, including Shigella. In this paper, we addressed the purification and characterisation of a bacteriocin produced by a Shigella sonnei strain (SS9) isolated from a child with acute diarrhoea. The substance was purified through ammonium-sulphate precipitation and sequential steps of chromatography. The intracellular fraction obtained at 75% ammonium sulphate maintained activity following exposure to pH values from 1-11 and storage at -80ºC for more than two years and was inactivated by high temperatures and proteases. The molecular mass of the purified bacteriocin was determined by mass spectrometry to be 18.56 kDa. The N-terminal sequence of the bacteriocin did not match any other antibacterial proteins described. A putative new bacteriocin produced by S. sonnei has been detected. This bacteriocin may represent a newly described protein or a previously described protein with a newly detected function. Considering that SS9 expresses antagonism against other diarrhoeagenic bacteria, the bacteriocin may contribute to S. sonnei virulence and is potentially applicable to either preventing or controlling diarrhoeal disease.

Activation of Leishmania spp. leishporin: evidence that dissociation of an inhibitor not only improves its lipid-binding efficiency but also endows it with the ability to form pores.

We have previously shown that various species of Leishmania produce a lytic activity, which, in Leishmania amazonensis, is mediated by a pore-forming cytolysin, called leishporin. It is toxic for macrophages in vitro and optimally active at pH 5.0 to 5.5 and at 37 °C, suggesting that it might be active inside phagolysosomes. Leishporin from both L. amazonensis (a-leishporin) and Leishmania guyanensis (g-leishporin) can be activated by proteases, suggesting either a limited proteolysis of an inactive precursor or a proteolytic degradation of a non-covalently linked inhibitor. Here, we show that both a- and g-leishporin are also activated in dissociating conditions, indicating the second hypothesis as the correct one. In fact, we further demonstrated that inactive leishporin is non-covalently associated with an inhibitor, possibly more than one oligopeptide that, when removed, renders leishporin hemolytically active. This activation was shown to be the result of both the improvement of leishporin's ability to bind to phospholipids and the emergence of its pore-forming ability. In vitro results demonstrate that leishporin can be released by the parasites, as they evolve in axenic cultures, in an inactive form that can be activated. These results are compatible with our hypothesis that leishporin can be activated in the protease-rich, low pH, and dissociating environment of parasitophorous vacuoles, leading to disruption of both vacuoles and plasma membranes with the release of amastigotes.

Purification of a membrane-bound trypsin-like enzyme from the gut of the velvetbean caterpillar (Anticarsia gemmatalis Hübner) / Purificação de uma enzima "tipo tripsina" não-solúvel do intestino da lagarta da soja (Anticarsia gemmatalis Hübner)

Disruption of protein digestion in insects by specific endoprotease inhibitors is being regarded as an alternative to conventional insecticides for pest control. To optimize the effectiveness of this strategy, the understanding of the endoprotease diversity of the target insect is crucial. In this sense, a membrane-bound trypsin-like enzyme from the gut of Anticarsia gemmatalis fifth-instar larvae was purified. Non-soluble fraction of the gut extract was solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and subjected to a p-aminobenzamidine affinity chromatography followed by anion-exchange chromatography. The yield of the purified enzyme was 11% with a purification factor of 143 and a final specific activity of 18.6 µM min.-1 mg-1 protein using N-α-benzoyl-L- Arg-p-nitroanilide (L-BApNA) as substrate. The purified sample showed a single band with proteolytic activity active and apparent molecular mass of 25 kDa on SDS-PAGE. Molecular mass determined by MALDI-TOF mass spectrometry was 28,632 ± 26 Da. Although the low recovery and the difficulties in purifying large enzyme amounts limited its further characterization, the results contribute for the understanding of the proteases present on A. gemmatalis gut, which are potential targets for natural or specifically designed protease inhibitors.

Comprometer a digestão de proteínas dos insetos pelo uso de inibidores específicos de endoproteases tem sido amplamente estudado como um método de controle de pragas alternativo ao uso dos inseticidas convencionais. No processo de otimização desta estratégia, o conhecimento da diversidade das endoproteases do inseto alvo torna-se crucial. Neste sentido, uma enzima "tipo-tripsina" ligada à membrana obtida do intestino de larvas do 5° instar de A. gemmatalis foi purificada. A fração insolúvel do extrato do intestino foi solubilizada com 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) e submetida à uma cromatografia de afinidade em uma coluna de p-aminobenzamidina, seguida por uma cromatografia de troca-aniônica. O rendimento da enzima purificada foi de 11% com fator de purificação de 143 e uma atividade específica final de 18.6 µM min.-1 mg-1 de proteína usando N-α-benzoyl-L- Arg-p-nitroanilide (L-BApNA) como substrato. Após a separação da amostra purificada por SDS-PAGE e incubação subsequente com caseína, uma única banda ativa com massa molecular aparente de 25 kDa foi observada. A massa molecular determinada por espectrometria de massa (MALDI-TOF) foi de 28,632 ± 26 Da. O baixo rendimento e as dificuldades em purificar grandes quantidades da enzima limitaram caracterização complementar. A observação desta enzima, no entanto, é mais uma etapa no processo de conhecer as endoproteases presentes no intestino de A. gemmatalis, alvos potenciais de inibidores de proteases naturais ou especificamente projetados.

Purification and partial characterization of proteinase inhibitors of equine seminal plasma.

The aims of the study were: 1/ to isolate and identify equine seminal plasma proteinase inhibitors, 2/ to evaluate their inhibitory potential, and 3/ to test a correlation between protein concentration in seminal plasma supernatant (obtained after precipitation with 36% ammonium sulfate) and stallion sexual maturity. Seminal plasma proteins obtained from six stallions were chromatographed in a Superose 12 (FPLC system) column followed by C(18) HPLC reverse-phase. Inhibition of trypsin amidase activity was evaluated in the collected fractions. Active proteins with a molecular mass of 6.3-7.0 KDa were identified using mass spectrometry. The older stallions showed a reduction in total seminal plasma protein concentration, but had similar concentrations of proteinase inhibitors (0.28+/-0.10 mg/ml) in seminal plasma supernatant. Different proteinase inhibitor isoforms were found in semen of all stallions which suggests that the isoforms may be used as biomarkers of individual animals.

Improved purification process of β- and α-trypsin isoforms by ion-exchange chromatography

The purpose of this work was to improve the separation and yield of pure β- and α-trypsin isoforms by ion-exchange chromatography and to characterize some physical-chemical properties of these isoforms. Purification of trypsin isoforms was performed by ion-exchange chromatography in 0.1 mol/L tris-HC buffer, pH 7.10 at 4ºC. The sample loading, salt concentration, flow rate and pH of mobile phase were varied to determine their effects on the resolution of the separation. The resolution was optimized mainly between β- and α-trypsin. Pure isoforms were obtained by chromatographying 100 mg of commercial trypsin during seven days, yielding 51 mg of high purity β-trypsin and 13 mg of α-trypsin partially pure, with small amounts of contaminating of ψ-trypsin. Thus, time and resolution of purification were optimized yielding large amounts of pure active enzymes that are useful for several research areas and biotechnology.

O propósito deste trabalho foi melhorar a separação e o rendimento das isoformas puras β- e α-tripsina por meio de cromatografia de troca iônica e caracterizar algumas propriedades físico-químicas dessas isoformas. A purificação de isoformas de tripsina foi realizada em SE Sephadex, com tampão tris-HCl, pH 7,10 a 4ºC. A quantidade de amostra, a concentração salina, o fluxo e o pH da fase móvel foram variados para determinar o efeito sobre a resolução da separação. A resolução foi otimizada principalmente entre β- e α-tripsina, utilizando o pH 7,10 a 4ºC. Isoformas puras foram obtidas a partir de 100 mg de tripsina comercial bovina depois de sete dias de cromatografia, fornecendo 51,0 mg de β-tripsina totalmente pura e 13,0 mg de α-tripsina parcialmente pura, com quantidades pequenas de contaminação por ψ-Tripsina. Assim, tempo e resolução da purificação foram otimizados redendo grandes quantidades de enzimas puras e ativas que são úteis em várias áreas de pesquisa e ciências biotecnológicas.

Actinomycetemcomitin: a new bacteriocin produced by Aggregatibacter (Actinobacillus) actinomycetemcomitans.

Aggregatibacter (Actinobacillus) actinomycetemcomitans P(7-20) strain isolated from a periodontally diseased patient has produced a bacteriocin (named as actinomycetemcomitin) that is active against Peptostreptococcus anaerobius ATCC 27337. Actinomycetemcomitin was produced during exponential and stationary growth phases, and its amount decreased until it disappeared during the decline growth phase. It was purified by ammonium sulphate precipitation (30-60% saturation), and further by FPLC (mono-Q ionic exchange and Phenyl Superose hydrophobic interaction) and HPLC (C-18 reversed-phase). This bacteriocin loses its activity after incubation at a pH below 7.0 or above 8.0, following heating for 30 min at 45 degrees C, and after treatment with proteolytic enzymes such as trypsin, alpha-chymotrypsin, and papain. Actinomycetemcomitin has a molecular mass of 20.3 KDa and it represents a new bacteriocin from A. actinomycetemcomitans.