Β-glucosidase b from microbacterium sp. Bg28 as a biofilm control agent In food processing environment

About one in ten people contract a foodborne illness within a year. Children under the age of five are the most affected, with 125,000 deaths each year. Many of the foodborne illness outbreaks can be linked to the presence of biofilms in the food industry, and Salmonella enteritidis is an extremely important foodborne pathogen that thrives in these conditions. It has been shown that biofilms can be resistant to physical and chemical treatments used in cleaning and disinfection procedures in food processing. The problem with using more aggressive disinfectants is that they often violate food safety regulations. The use of enzymes which degrade biofilm matrix structural components should facilitate current disinfection procedures and not compromise food safety. In this study, the anti-biofilm activity of recombinantly expressed β-glucosidase B and its potential use as a protective agent to control Salmonella biofilm formation is investigated. The putative target of this enzyme is cellulose, the structural component of the Salmonella biofilm matrix. β-Glucosidase B deriving from the environmental strain Microbacterium sp. BG28 was heterologously expressed in Escherichia coli and successfully purified by affinity chromatography. The anti-biofilm activity of the enzyme was evaluated in in vitro assays using various clinical isolates of S. enteritidis. The toxicity of the enzyme was studied in Caenorhabditis elegans. β-glucosidase B effectively inhibited the formation of Salmonella biofilms grown in a temperature range of 8°C to 37°C, achieving 50% inhibition at concentrations of 100μg/ml. Biochemical characterization showed that the optimal pH activity of the enzyme is between 6 and 7, with the highest activity observed at temperatures between 37°C and 47°C. The absence of toxicity and other presented results indicate that beta-glucosidase B can be used in biofilm control in the food industry.Book of abstracts: International Conference of Biochemists and Molecular Biologists in Bosnia and Herzegovina - ABMBBIH May, 202

In silico pre-selection of β-glucosidase gene for heterologous recombinant expression

Biofilms are ubiquitous in nature, and the food industry is vulnerable to the risks posed by biofilm formation. Not only do they interfere with the food production process, but they also pose a public health threat. However, complete elimination of biofilms on food and food contact surfaces cannot be achieved by conventional methods (cleaning and disinfection) alone. New biofilm control strategies must be developed to prevent its formation and/or persistence. Novel approaches may be based on enzymes that depolymerize components of the biofilm matrix, making bacterial cells accessible to antimicrobial agents. Environmental microorganisms are an inexhaustible source of new enzymes. In Salmonella Enteritidis and Escherichia coli, known foodborne pathogens, cellulose is an important component of the biofilm matrix, so our isolates from untapped environments were tested for cellulolytic activity. Of the more than 70 isolates examined, isolate BG28 was selected as the most promising. Its genome was sequenced, annotated, and it was identified as Gram-positive Microbacterium sp. Genome mining revealed the presence of four complete genes for different β-glucosidases, one of three enzyme types of cellulase complexes. To select the best candidate for heterologous expression DeepTMHMM, ProtParam, and SoluProt were used to predict the presence/absence of signal peptide and transmembrane domains, instability index, aliphatic index, hydrophilicity, and soluble expression in E. coli. Based on the prediction results, the gene annotated as β-glucosidase B was selected for recombinant expression. In addition, I-TASSER was used to model the tertiary structure of the selected enzyme. The β-glucosidase B was recombinantly expressed, purified, and tested for its anti-biofilm activity. It was active and showed a 50% inhibitory effect on S. Enteritidis and E. coli biofilm formation at a concentration of 100 μg/ml. To further evaluate this in silico approach in the preselection of candidate enzymes for recombinant expression and purification, we will use it to identify other enzymes of the cellulase complex.Book of abstract: 4th Belgrade Bioinformatics Conference, June 19-23, 202

Optimization of the heterologous expression of banana glucanase in Escherichia coli (vol 77, pg 43, 2012)

For the heterologous production of a banana glucanase in Escherichia coli, its gene (GenBank GQ268963) was cloned into a pGEX-4T expression vector as a fusion protein with glutathione-S-transferase (GST). BL21 cells transformed with the GST-Mus a 5 construct were employed for production of the protein induced by 1 mM isopropyl-beta-D-thiogalactopyranoside (IPTG). The conditions for protein expression were optimized by varying the temperature (25, 30 and 37 C) and duration of protein expression (3, 6 and 12 h). The level of protein production was analyzed by densitometry of the sodium dodecyl sulfate-polyacrylamide gel (SDS-PAG) after electrophoretic resolution of the respective cell lysates. The optimal protein expression for downstream processing was obtained after 12 h of cell growth at 25 degrees C upon addition of IPTG. Recombinant GST-Mus a 5 purified by glutathione affinity chromatography revealed a molecular mass of about 60 kDa. The IgE and IgG reactivity of the rGST-Mus a 5 was confirmed by dot blot analysis with sera of individual patients from subjects with banana allergy and polyclonal rabbit antibodies against banana extract, respectively. The purified recombinant glucanase is a potential candidate for banana allergy diagnosis.Za potrebe proizvodnje u Escherichia coli gen glukanaze iz banane (GenBank GQ268963) je ukloniran u ekspresioni vektor pGEX-4T sa glutation-S-transferazom (GST). Proizvodnja ovog proteina u ćelijama je indukovana 1 mM izopropil-β-D-tiogalaktopiranozidom (IPTG). Uslovi za ekspresiju proteina su optimizovani variranjem temperature (25, 30 i 37°C) i dužine trajanja proteinske sinteze (3, 6 i 12 h). Nivo proizvodnje proteina je analiziran denzitometrijom SDS-PA gela nakon elektroforetskog razdvajanja ćelijskih lizata. Optimalna proizvodnja proteina za njegovo dalje procesovanje je dobijena gajenjem ćelija nakon dodatka IPTG na 25°C tokom 12 h. Rekombinantni GST-Mus a 5 prečišćen afinitetnom hromatografijom sa glutationom pokazuje molekulsku masu od 60 kDa. IgE i IgG reaktivnost izolovane glukanaze potvrđena je u 'dot blot' sa pojedinačnim serumima osoba alergičnih na bananu, i sa poliklonskim zečijim antitelima na ekstrakt banane, redom. Prečišćena rekombinantna glukanaza je potencijalan kandidat za dijagnozu alergije na bananu

Optimizacija heterologe proizvodnje glukanaze iz banane u E. coli

For the heterologous production of a banana glucanase in Escherichia coli, its gene (GenBank GQ268963) was cloned into a pG EX-4T expression vector as a fusion protein with glutathione-S-transferase (GST). BL21 cells transformed with the GST-Mus a 5 con struct were employed for production of the protein induced by 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG). The conditions for protein expression were optimized by varying the temperature (25, 30 and 37°C) and duration of protein expression (3, 6 and 12 h). The level of protein production was analyzed by densitometry of the sodium dodecyl sulfate-polyacrylamide gel (SDS-PAG) after electrophoretic resolution of the respective cell lysates. The optimal protein expression for downstream processing was obtained after 12 h of cell growth at 25°C upon addition of IPTG. Recombinant GST-Mus a 5 purified by glutathione affinity chromatography revealed a molecular mass of a bout 60 kDa. The IgE and IgG reactivity of the rGST-Mus a 5 was confirmed by dot blot an analysis with sera of individual patients from subjects with banana allergy and polyclonal rabbit antibodies against banana extract, respectively. The purified recombinant glucanase is a potential candidate for banana allergy diagnosis.Za potrebe proizvodnje u Escherichia coli gen glukanaze iz banane (GenBank GQ268963) je ukloniran u ekspresioni vektor pGEX-4T sa glutation-S-transferazom (GST). Proizvodnja ovog proteina u ćelijama je indukovana 1 mM izopropil-β-D-tiogalaktopiranozidom (IPTG). Uslovi za ekspresiju proteina su optimizovani variranjem temperature (25, 30 i 37°C) i dužine trajanja proteinske sinteze (3, 6 i 12 h). Nivo proizvodnje proteina je analiziran denzitometrijom SDS-PA gela nakon elektroforetskog razdvajanja ćelijskih lizata. Optimalna proizvodnja proteina za njegovo dalje procesovanje je dobijena gajenjem ćelija nakon dodatka IPTG na 25°C tokom 12 h. Rekombinantni GST-Mus a 5 prečišćen afinitetnom hromatografijom sa glutationom pokazuje molekulsku masu od 60 kDa. IgE i IgG reaktivnost izolovane glukanaze potvrđena je u 'dot blot' sa pojedinačnim serumima osoba alergičnih na bananu, i sa poliklonskim zečijim antitelima na ekstrakt banane, redom. Prečišćena rekombinantna glukanaza je potencijalan kandidat za dijagnozu alergije na bananu

Allergenic potency of kiwi fruit during fruit development

Food allergies, including kiwi fruit allergy, have been the subject of extensive research in the last few years. The aim of this study was to examine a possible relationship between the developmental stage of kiwi fruit and its allergenic potency. The protein and allergen patterns of kiwi fruit extracts in September, October, November and December fruit in the period from 2000-2002 were analysed. One of the factors that may contribute to the difficulties in proposing well-defined and standardized fruit extracts should also be the time of fruit harvesting. In this particular case, when the kiwi fruit was edible throughout November and December, we showed discrepancies in allergen content and potencies both in qualitative and quantitative terms. Two major allergens of kiwi fruit, Act c 1 and Act c 2, mainly accounted for the highest allergenic potential of November kiwi extract in vivo and in vitro. Not only the content of major allergens, but also the ratio of different proteins and even isoforms of the same allergen (Act c 2) change with fruit ripening. These findings should be taken into account during preparation of extracts for allergy diagnosis

Izolovanje i karakterizacija 68 kD alergena iz ekstrakta kućnih grinja

House dust mites (HDM) represent a major source of allergens, contributing to the increasing incidence of type I hypersensitivity disease worldwide. Over 30 different IgE-binding proteins from the HDM extract were detected. Although group 1 and 2 have been identified as major allergens, due to the safety and efficacy of allergy diagnosis and immunotherapy, there is a need to carefully evaluate the clinical relevance of other allergens present in the HDM extract. In regard to this, a high molecular mass allergen of about 68 kD was purified from the HDM extract using a combination of gel permeation chromatography and reversed-phase chromatography. The IgG and IgE reactivity of the purified protein were preserved during the purification process, as confirmed by Western blot analysis with polyclonal rabbit antibodies and dot blot analysis with a pool of sera from subjects with house dust mite allergy, respectively. In addition, the IgE reactivity was confirmed using ELISA testing with nine patient sera. The biological potency of the 68 kD allergen was confirmed by skin prick testing in five allergic subjects, suggesting that the high molecular mass allergen is a good candidate for component-resolved diagnosis of house dust mite allergy and eventual therapeutic treatment.Grinje iz kućne prašine predstavljaju jedan od glavnih izvora alergena koji su u značajnoj meri doprineli porastu prvog tipa preosetljivosti. Preko 30 IgE-vezujućih proteina iz kućne prašine je detektovano do danas. Alergeni grupe 1 i 2 označeni su kao glavni alergeni kućne prašine. Međutim, da bi se poboljšala sigurnost i efikasnost dijagnoze i terapije alergijskih oboljenja izazvanih grinjama iz kućne prašine, neophodno je odrediti klinički značaj svih alergena iz ovog alergenskog izvora. U ovom radu izolovan je alergen visoke molekulske mase od 68 kD iz ekstrakta kućne prašine kombinovanjem gel-permeacione hromatografije i reversno-fazne hromatografije. IgG i IgE reaktivnost prečišćenog proteina je proverena u 'Western blot'-u i 'dot blot'-u sa poliklonskim zečijim antitelima na ekstrakt kućne prašine i 'pool'-om seruma osoba alergičnih na kućnu prašinu, redom. 64 % pacijenata je pokazalo IgE reaktivnost na prečišćeni protein u ELISA testu. Biološka reaktivnost prečišćenog alergena je potvrđena u kožnim probama na pet pacijenata, ukazujući da je prečišćen alergen dobar kandidat za dijagnozu alergije na kućnu prašinu pojedinačnim komponentama i eventualni terapeutski tretman

Izolovanje i karakterizacija 68 kD alergena iz ekstrakta kućnih grinja

House dust mites (HDM) represent a major source of allergens, contributing to the increasing incidence of type I hypersensitivity disease worldwide. Over 30 different IgE-binding proteins from the HDM extract were detected. Although group 1 and 2 have been identified as major allergens, due to the safety and efficacy of allergy diagnosis and immunotherapy, there is a need to carefully evaluate the clinical relevance of other allergens present in the HDM extract. In regard to this, a high molecular mass allergen of about 68 kD was purified from the HDM extract using a combination of gel permeation chromatography and reversed-phase chromatography. The IgG and IgE reactivity of the purified protein were preserved during the purification process, as confirmed by Western blot analysis with polyclonal rabbit antibodies and dot blot analysis with a pool of sera from subjects with house dust mite allergy, respectively. In addition, the IgE reactivity was confirmed using ELISA testing with nine patient sera. The biological potency of the 68 kD allergen was confirmed by skin prick testing in five allergic subjects, suggesting that the high molecular mass allergen is a good candidate for component-resolved diagnosis of house dust mite allergy and eventual therapeutic treatment.Grinje iz kućne prašine predstavljaju jedan od glavnih izvora alergena koji su u značajnoj meri doprineli porastu prvog tipa preosetljivosti. Preko 30 IgE-vezujućih proteina iz kućne prašine je detektovano do danas. Alergeni grupe 1 i 2 označeni su kao glavni alergeni kućne prašine. Međutim, da bi se poboljšala sigurnost i efikasnost dijagnoze i terapije alergijskih oboljenja izazvanih grinjama iz kućne prašine, neophodno je odrediti klinički značaj svih alergena iz ovog alergenskog izvora. U ovom radu izolovan je alergen visoke molekulske mase od 68 kD iz ekstrakta kućne prašine kombinovanjem gel-permeacione hromatografije i reversno-fazne hromatografije. IgG i IgE reaktivnost prečišćenog proteina je proverena u 'Western blot'-u i 'dot blot'-u sa poliklonskim zečijim antitelima na ekstrakt kućne prašine i 'pool'-om seruma osoba alergičnih na kućnu prašinu, redom. 64 % pacijenata je pokazalo IgE reaktivnost na prečišćeni protein u ELISA testu. Biološka reaktivnost prečišćenog alergena je potvrđena u kožnim probama na pet pacijenata, ukazujući da je prečišćen alergen dobar kandidat za dijagnozu alergije na kućnu prašinu pojedinačnim komponentama i eventualni terapeutski tretman

Rana alergijska reakcija na metilprednizolon sa tolerancijom drugih kortikosteroida

Introduction. In spite of the wide usage of corticosteroids for the treatment of a plethora of diseases, sometimes they can induce immediate hypersensitivity reactions, which are however uncommon. Case Outline. We report a case of immediate allergic reaction induced by intravenous methylprednisolone given before operation for surgical repair of an arm contracture as a sequel of burns, which the child had tolerated a month before. Six weeks later the patient repeated the anaphylactic reaction during skin testing to methylprednisolone. In addition, basophile activation test with methylprednisolone (BAT) was positive. Conclusion. This case report describes a patient who experienced intraoperative anaphylaxis and anaphylactic reaction induced by skin testing. This is the first report on induction of both anaphylactic reactions by methylprednisolone in the same child. Clinical findings, positive BAT and positive skin tests with methylprednisolone imply that the child developed type-I hypersensitivity. The lack of cross-reactivity with other corticosteroids emphasizes that the reactions were caused by the steroid molecule.Uvod. Uprkos širokoj primeni kortikosteroida u lečenju od različitih bolesti, oni ponekad mogu izazvati ranu alergijsku reakciju. Prikaz bolesnika. Kod dvanaestogodišnjeg dečaka došlo je do rane alergijske reakcije izazvane intravenskom primenom metilprednizolona neposredno pre hirurške intervencije, tačnije, korekcije kontrakture šake koja se javila kao komplikacija opekotine. Mesec dana pre pojave alergijske reakcije dete je primalo metilprednizolon i dobro ga podnosilo. Šest nedelja posle operacije ponovo se javila anafilaktička reakcija tokom kožnog testiranja metilprednizolonom. Primenjen je i test aktivacije bazofila (BAT) ovim lekom, čiji je nalaz bio pozitivan. Zaključak. Ovo je prvi prikaz dve vrste anafilaktičke reakcije izazvane metilprednizolonom kod iste osobe. Klinička slika, pozitivni nalaz BAT i pozitivne kožne probe na metilprednizolon pokazuju da se kod deteta razvio prvi tip hipersenzitivne reakcije. Nedostatak unakrsne reaktivnosti s ostalim kortikosteroidima ukazuje na to da je alergijska reakcija izazvana steroidnim molekulom

Digestibilnost alergoida polena pelina u simuliranim uslovima gastrointestinalnog trakta

Chemically modified allergens (allergoids) have found use in both traditional and novel forms of immunotherapy of allergic disorders. Novel forms of immunotherapy include local allergen delivery, via the gastrointestinal tract. This study conveys the gastrointestinal stability of three types of mugwort pollen allergoids under simulated conditions of the gut. Allergoids of the pollen extract of Artemisia vulgaris were obtained by means of potassium cyanate, succinic and maleic anhydride. Gastrointestinal tract conditions (saliva, and gastric fluid) were simulated in accordance with the EU Pharmacopoeia. The biochemical and immunochemical properties of the derivatives following exposure to different conditions were monitored by determining the number of residual amino groups with 2,4,6-trinitrobenzenesulfonic acid, SDS PAGE, immunoblotting and inhibition of mugwort-specific IgE. Exposure to saliva fluid for 2 min did not influence the biochemical and immunochemical properties of the derivatives. In the very acidic conditions of the simulated gastric fluid, the degree of demaleylation and desuccinylation, even after 4 h exposure, was low, ranging from 10 to 30 %. The digestion patterns with pepsin proceeded rapidly in both the unmodified and modified samples. In all four cases, a highly resistant IgE-binding protein the Mwof which was about 28-35 kD, was present. Within the physiological conditions, no new IgE binding epitopes were revealed, as demonstrated by immunoblot and CAP inhibition of the mugwort specific IgE binding. An important conclusion of this study is the stability of the modified derivatives in the gastrointestinal tract of patients, within physiological conditions. The means that they are suitable for use in much higher concentrations in local forms of immunotherapy than unmodified ones.U ovom radu su prikazani rezultati ispitivanja stabilnosti tri tipa alergoida polena pelina u simuliranom želudačnom soku. Koristeći kalijum-cijanat anhidrid ćilibarne i anhidrid maleinske kiseline, napravljeni su alergoidi polena pelina (Artemisia vulgaris). Saliva i želudačni sok su simulirani na osnovu evropske farmakopeje. Biohemijske i imunohemijske osobine derivata posle izlaganja različitim uslovima, praćene su: određivanjem broja slobodnih amino grupa u reakciji sa TNBS, SDS PAG elektroforezom, imunoblotom i određivanjem pelin-specifičnog imunoglobulina E (IgE). Izlaganje salivi u trajanju od 2 minuta ne utiče na biohemijske i imunohemijske osobine derivata. U kiseloj sredini želudačnog soka ne dolazi do značajnog demaleilovawa i desukcinilovanja. Čak i posle četvoročasovnog izlaganja, taj procenat je u opsegu 10-30 %. Alergoidi pelina se trenutno digestuju pepsinom, sa izuzetkom visoko rezistentne proteinske trake molekulske mase 28-35 kD, koja odgovara važnom IgE-vezujućem proteinu polena pelina. Imunoblotom i CAP-inhibicijom je pokazano da, u okviru fizioloških uslova, ne dolazi do stvaranja novih IgE-vezujućih epitopa. Hemijska stabilnost modifikovanih derivata u simuliranim uslovima želudačnog soka omogućuje da se tokom imunoterapije mogu primenjivati veće doze alergoida nego nemodifikovanog ekstrakta polena pelina

GLYCOSIDE HYDROLASES FROM FRESHWATER FISH GILL MICROBIOTA AS BIOFILM INHIBITORS FOR ENHANCED FOOD SAFETY

The formation of biofilms by foodborne pathogens is a constant challenge in the food industry, leading to an increased risk of contamination and compromising food safety. Many of the chemicals commonly used for sanitation in the food industry are unable to remove biofilms, are harmful to surfaces and can be toxic. The effectiveness of disinfectants can be improved using enzymes that specifically target biofilm components such as exopolysaccharides, extracellular DNA, or proteins. In this study we investigated the potential of glycoside hydrolases originating from the gill microbiota of freshwater fish to control biofilm formation in the most common foodborne pathogens. We demonstrated that β-glucosidase from Microbacterium sp. BG28 (BglB-BG28) effectively inhibits cellulose-rich biofilms formed by Salmonella enteritidis, S. typhimurium, S. infantis, and Escherichia coli. When these bacteria were cultivated overnight with 200 μL/mL enzyme, up to 80% less biofilm was formed. By fluorescence microscopy, we visualised the inhibition of biofilms on plastic, glass and aluminium, materials commonly used in the food industry. When used as a pre-treatment, BglB-BG28 increased the bactericidal efficacy of Oxicid®S, a commercially available surface disinfectant. Its effectiveness at temperatures up to 50 °C and in a pH range from 4 to 8 together with compatibility with non-ionic detergents and high tolerance to sodium chloride and glucose give BglB-BG28 advantages in harsh and diverse industrial environments. Importantly, no toxicity to Caenorhabditis elegans was observed at enzyme concentrations of up to 1 mg/ml. Overall, these results demonstrate the suitability of the β-glucosidase BglB-BG28 for the formulation of a novel enzyme-based disinfectant to be used in food processing facilities.Book of abstract: From biotechnology to human and planetary health XIII congress of microbiologists of Serbia with international participation Mikromed regio 5, ums series 24: 4th – 6th april 2024, Mona Plaza hotel, Belgrade, Serbi