Pub Date : 2023-11-07DOI: 10.55959/msu0579-9384-2-2023-64-5-468-477
Galina V. Presnova, Tatiana V. Bulko, Victoria V. Shumyantseva, Maya Yu. Rubtsova
An electrochemical immunosensor based on screen-printed graphite electrodes has been developed for the determination of the antibiotic chloramphenicol in water and milk samples. It has been shown that the immobilization of chloramphenicol-specific antibodies in a liquid crystal layer of a membrane-like didodecyldimethylammonium bromide preserves the mobility and accessibility of active centers of antibodies, and the addition of gold nanoparticles improves electron transfer from the electrode surface to the redox centers of horseradish peroxidase used as a label. The limit of detection of chloramphenicol in water was 0.02 μg/L, in milk - 0.04 μg/L. The method can be used to determine residual amounts of chloramphenicol in animal products.
{"title":"IMMUNOSENSOR BASED ON SCREEN-PRINTED GRAPHITE ELECTRODES MODIFIED WITH GOLD NANOPARTICLES AND SYNTHETIC MEMBRANE-LIKE SUBSTANCE FOR THE DETERMINATION OF CHLORAMPHENICOL","authors":"Galina V. Presnova, Tatiana V. Bulko, Victoria V. Shumyantseva, Maya Yu. Rubtsova","doi":"10.55959/msu0579-9384-2-2023-64-5-468-477","DOIUrl":"https://doi.org/10.55959/msu0579-9384-2-2023-64-5-468-477","url":null,"abstract":"An electrochemical immunosensor based on screen-printed graphite electrodes has been developed for the determination of the antibiotic chloramphenicol in water and milk samples. It has been shown that the immobilization of chloramphenicol-specific antibodies in a liquid crystal layer of a membrane-like didodecyldimethylammonium bromide preserves the mobility and accessibility of active centers of antibodies, and the addition of gold nanoparticles improves electron transfer from the electrode surface to the redox centers of horseradish peroxidase used as a label. The limit of detection of chloramphenicol in water was 0.02 μg/L, in milk - 0.04 μg/L. The method can be used to determine residual amounts of chloramphenicol in animal products.","PeriodicalId":23660,"journal":{"name":"Vestnik Moskovskogo Universiteta Seriya 2 Khimiya","volume":"49 218","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2023-11-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"135540462","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-11-07DOI: 10.55959/msu0579-9384-2-2023-64-5-478-489
Ksenia A. Afanasyeva, Anastasia М. Gileva, Elena A. Markvicheva, Ulyana A. Budanova, Yurii L. Sebyakin
The work is devoted to the preparation of a carbohydrate derivative of lipotripeptide (N-lactitol-Gly)2-LysC16 of irregular structure with two terminal residues of D-galactose, a branching link based on aliphatic L-lysine and its carbohydrate-free analog with 1-pyrenbutanol as a fluorescent label in a hydrophobic fragment. The developed synthesis scheme includes universal approaches of peptide chemistry, as well as the stages of formation of an acyclic carbohydrate based on lactose in the hydrophilic domain of amphiphile. The compounds are designed to create compositions of vector BAS delivery systems with the ability to visualize the process of interaction with target cells.
{"title":"GLYCOLIPOTRIPEPTIDE (N-LACTITOL-Gly)2-LysС16 AND ITS FLUORESCENTLY LABELED ANALOGUE FOR VISUALIZATION OF VECTOR SYSTEMS FOR BIOLOGICALLY ACTIVE SUBSTANCES DELIVERY TO TARGET CELLS","authors":"Ksenia A. Afanasyeva, Anastasia М. Gileva, Elena A. Markvicheva, Ulyana A. Budanova, Yurii L. Sebyakin","doi":"10.55959/msu0579-9384-2-2023-64-5-478-489","DOIUrl":"https://doi.org/10.55959/msu0579-9384-2-2023-64-5-478-489","url":null,"abstract":"The work is devoted to the preparation of a carbohydrate derivative of lipotripeptide (N-lactitol-Gly)2-LysC16 of irregular structure with two terminal residues of D-galactose, a branching link based on aliphatic L-lysine and its carbohydrate-free analog with 1-pyrenbutanol as a fluorescent label in a hydrophobic fragment. The developed synthesis scheme includes universal approaches of peptide chemistry, as well as the stages of formation of an acyclic carbohydrate based on lactose in the hydrophilic domain of amphiphile. The compounds are designed to create compositions of vector BAS delivery systems with the ability to visualize the process of interaction with target cells.","PeriodicalId":23660,"journal":{"name":"Vestnik Moskovskogo Universiteta Seriya 2 Khimiya","volume":"5 18","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2023-11-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"135540483","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-11-07DOI: 10.55959/msu0579-9384-2-2023-64-5-490-499
Sergei Yu. Zaitsev
Recently, more attention has been paid to the study of the amino acid composition of gelatins, which is associated with the quality of the corresponding gels as intermediates for human and animal nutrition. In a brief review, a modification of the general method of acid extraction of collagens for the preparation of gelatins using enzymes (such as papain, actinidin, and others) is considered and the corresponding changes in the amino acid composition of gelatins are discussed. It is clear that there are changes in the content of glycine in gelatins from any collagens, but in all cases the content of glycine is about a third of the content of all amino acids (as in the original collagens). It is important that the content of imino acids (the sum of proline and hydroxyproline, which largely determines the properties of gels) in gelatins from any collagens with the use of all the studied enzymes is much higher than without them. In addition, the content of imino acids in gelatin from the bovine skin of cows with the use of any enzymes is significantly higher than in gelatins from the skin of pigs and fish. This holds true for other key “proteinogenic” amino acids as well. The reverse trend is observed only for a few amino acids: serine, threonine, tyrosine, phenylalanine, the content of which is low in gelatins from any collagens.
{"title":"CHANGES IN THE AMINO ACID COMPOSITION OF GELATIN AFTER TREATMENT OF BOVINE COLLAGEN WITH ENZYME PREPARATION","authors":"Sergei Yu. Zaitsev","doi":"10.55959/msu0579-9384-2-2023-64-5-490-499","DOIUrl":"https://doi.org/10.55959/msu0579-9384-2-2023-64-5-490-499","url":null,"abstract":"Recently, more attention has been paid to the study of the amino acid composition of gelatins, which is associated with the quality of the corresponding gels as intermediates for human and animal nutrition. In a brief review, a modification of the general method of acid extraction of collagens for the preparation of gelatins using enzymes (such as papain, actinidin, and others) is considered and the corresponding changes in the amino acid composition of gelatins are discussed. It is clear that there are changes in the content of glycine in gelatins from any collagens, but in all cases the content of glycine is about a third of the content of all amino acids (as in the original collagens). It is important that the content of imino acids (the sum of proline and hydroxyproline, which largely determines the properties of gels) in gelatins from any collagens with the use of all the studied enzymes is much higher than without them. In addition, the content of imino acids in gelatin from the bovine skin of cows with the use of any enzymes is significantly higher than in gelatins from the skin of pigs and fish. This holds true for other key “proteinogenic” amino acids as well. The reverse trend is observed only for a few amino acids: serine, threonine, tyrosine, phenylalanine, the content of which is low in gelatins from any collagens.","PeriodicalId":23660,"journal":{"name":"Vestnik Moskovskogo Universiteta Seriya 2 Khimiya","volume":"91 12","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2023-11-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"135539683","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-11-07DOI: 10.55959/msu0579-9384-2-2023-64-5-407-440
Maria S. Andrianova, Olga S. Panova, Alexey A. Titov, Natalia V. Komarova, Alexander E. Kuznetsov
The 2019 coronavirus disease (COVID-19) outbreak has become global. The unprecedented socioeconomic impact of the pandemic has highlighted the need to improve existing diagnostic methods and develop new methods to control the spread of the disease. Traditional technologies such as quantitative real-time polymerase chain reaction (qRT-PCR) have been considered the gold standard for testing for COVID-19 since the SARS-CoV-2 genome sequence was published. However, they are time-consuming, labor-intensive and do not guarantee the absence of false results. Electrochemical biosensors present alternative approaches to detect viral nucleic acids or viral antigens. High sensitivity, relatively low cost of sensors and equipment, convenient management, rapid analysis, and suitability for miniaturization may contribute to the development of point-of-care (POC) testing for COVID-19. The review examines and critically discusses modern electrochemical biosensors for SARS-CoV-2 detection and related technologies.
{"title":"ELECTROCHEMICAL BIOSENSORS FOR SARS-COV-2 DETECTION","authors":"Maria S. Andrianova, Olga S. Panova, Alexey A. Titov, Natalia V. Komarova, Alexander E. Kuznetsov","doi":"10.55959/msu0579-9384-2-2023-64-5-407-440","DOIUrl":"https://doi.org/10.55959/msu0579-9384-2-2023-64-5-407-440","url":null,"abstract":"The 2019 coronavirus disease (COVID-19) outbreak has become global. The unprecedented socioeconomic impact of the pandemic has highlighted the need to improve existing diagnostic methods and develop new methods to control the spread of the disease. Traditional technologies such as quantitative real-time polymerase chain reaction (qRT-PCR) have been considered the gold standard for testing for COVID-19 since the SARS-CoV-2 genome sequence was published. However, they are time-consuming, labor-intensive and do not guarantee the absence of false results. Electrochemical biosensors present alternative approaches to detect viral nucleic acids or viral antigens. High sensitivity, relatively low cost of sensors and equipment, convenient management, rapid analysis, and suitability for miniaturization may contribute to the development of point-of-care (POC) testing for COVID-19. The review examines and critically discusses modern electrochemical biosensors for SARS-CoV-2 detection and related technologies.","PeriodicalId":23660,"journal":{"name":"Vestnik Moskovskogo Universiteta Seriya 2 Khimiya","volume":"62 2","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2023-11-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"135539993","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-11-04DOI: 10.55959/msu0579-9384-2-2023-64-4-334-352
Nikolay V. Panin, Dorel T. Guranda, Irina V. Shapovalova, Vytas K. Švedas
The review considers the contribution of works carried out at the scientific school of Ilya Vasilievich Berezin to the study of the kinetics and thermodynamics of penicillin acylase-catalyzed reactions. Methods for determining the activity of penicillin acylases, the reversibility of the enzymatic hydrolysis of a number of penicillins, cephalosporins and related compounds, the influence of the β-lactam ring on the thermodynamics of the synthesis of new penicillins and cephalosporins by direct condensation as well as by acyl transfer, the issues of optimizing the conditions for enzymatic acyl transfer and the use of supersaturated reagent solutions are discussed. The role of chromogenic substrates in the study of penicillin acylase, the possibility of using the methods of titration of active sites of the enzyme and the creation of “smart” biocatalysts based on penicillin acylase due to the formation of conjugates with stimulus-sensitive polymers are considered.
{"title":"PENICILLIN ACYLASE: A RETROSPECTIVE OF STUDYING THE KINETICS AND THERMODYNAMICS OF PRACTICALLY SIGNIFICANT REACTIONS","authors":"Nikolay V. Panin, Dorel T. Guranda, Irina V. Shapovalova, Vytas K. Švedas","doi":"10.55959/msu0579-9384-2-2023-64-4-334-352","DOIUrl":"https://doi.org/10.55959/msu0579-9384-2-2023-64-4-334-352","url":null,"abstract":"The review considers the contribution of works carried out at the scientific school of Ilya Vasilievich Berezin to the study of the kinetics and thermodynamics of penicillin acylase-catalyzed reactions. Methods for determining the activity of penicillin acylases, the reversibility of the enzymatic hydrolysis of a number of penicillins, cephalosporins and related compounds, the influence of the β-lactam ring on the thermodynamics of the synthesis of new penicillins and cephalosporins by direct condensation as well as by acyl transfer, the issues of optimizing the conditions for enzymatic acyl transfer and the use of supersaturated reagent solutions are discussed. The role of chromogenic substrates in the study of penicillin acylase, the possibility of using the methods of titration of active sites of the enzyme and the creation of “smart” biocatalysts based on penicillin acylase due to the formation of conjugates with stimulus-sensitive polymers are considered.","PeriodicalId":23660,"journal":{"name":"Vestnik Moskovskogo Universiteta Seriya 2 Khimiya","volume":"33 6","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2023-11-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"135775660","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-11-04DOI: 10.55959/msu0579-9384-2-2023-64-4-289-311
Vladimir I. Tishkov, Anastasia A. Pometun, Svyatoslav S. Savin
NAD(P)+ -dependent formate dehydrogenase (EC 1.2.1.2, FDH) catalyzes the simple from chemical and biological point of view reaction of formate ion oxidation to carbon dioxide with corresponding reduction of NAD(P)+ to NAD(P) H. Advances in the life sciences have shown that this reaction plays an extremely important role in a wide variety of organisms. The areas and types of practical applications of FDH are also permanently expanding. In this review we considered the main stages in the development of understanding and knowledge about the role of formate dehydrogenase in living systems. Achievements in creation of highly effi cient catalysts based on FDH for classic biotechnology as well as for new areas are also considered. The importance of appropriate choice of the initial FDH for the creation of a biocatalyst with the required and prescribed properties with minimal costs is shown. The prospects for the use of FDH for the fixation of CO2 are discussed.
{"title":"FORMATE DEHYDROGENASE: FROM NAD(P)H REGENERATION TO THE TARGET IN PATHOGENS BIOFILMS, A COMPONENT OF HIGHLY EFFICIENT HYBRID BIOCATALYSTS AND CO2 FIXATION FROM THE ATMOSPHERE","authors":"Vladimir I. Tishkov, Anastasia A. Pometun, Svyatoslav S. Savin","doi":"10.55959/msu0579-9384-2-2023-64-4-289-311","DOIUrl":"https://doi.org/10.55959/msu0579-9384-2-2023-64-4-289-311","url":null,"abstract":"NAD(P)+ -dependent formate dehydrogenase (EC 1.2.1.2, FDH) catalyzes the simple from chemical and biological point of view reaction of formate ion oxidation to carbon dioxide with corresponding reduction of NAD(P)+ to NAD(P) H. Advances in the life sciences have shown that this reaction plays an extremely important role in a wide variety of organisms. The areas and types of practical applications of FDH are also permanently expanding. In this review we considered the main stages in the development of understanding and knowledge about the role of formate dehydrogenase in living systems. Achievements in creation of highly effi cient catalysts based on FDH for classic biotechnology as well as for new areas are also considered. The importance of appropriate choice of the initial FDH for the creation of a biocatalyst with the required and prescribed properties with minimal costs is shown. The prospects for the use of FDH for the fixation of CO2 are discussed.","PeriodicalId":23660,"journal":{"name":"Vestnik Moskovskogo Universiteta Seriya 2 Khimiya","volume":"13 7","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2023-11-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"135774553","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-11-04DOI: 10.55959/msu0579-9384-2-2023-64-4-312-333
Arkady P. Sinitsyn, Olga A. Sinitsyna, Ivan N. Zorov, Aleksandra M. Rozhkova
The review describes the history of the development of research on carbohydrasеs conducted at the Department of Chemical Enzymology from the mid-1970s to the present. The results concerning the study of the mechanism and kinetics of the processes of enzymatic conversion of cellulose and renewable plant raw materials under the action of a multi-enzyme cellulases complexes; the role of individual components of these complexes - basic (endoglucanases and cellobiohydrolases) and auxiliary enzymes (polysaccharide monooxygenase, β-glucosidase, xylanase) and their synergistic interaction. The features of using reactors of various designs for bioconversion of plant raw materials are described: periodic type, continuous column type, reactor for hydrolysis in a constant electric field, reactor with intensive mixing by ferromagnetic particles in magnetic field. The possibilities of increasing the reactivity of plant raw materials using various pretreatment methods, as well as the influence of the structural and physico-chemical properties of cellulose on the efficiency of its enzymatic conversion are discussed. Data on the creation of highly active strains of microscopic fungi-producers of cellulases and other carbohydrases using methods of induced mutagenesis - Trichoderma (Hypocrea), Penicillium (Talaromyces), Aspergillus, Chrysosporium (Myceliophtora) spp., as well as data on the composition of the enzyme complexes produced by them and the properties of the enzymes forming them are presented. It describes the creation of expression systems based on P. canescens and P. verruculosum and the production of recombinant producer strains with their help, which made it possible to obtain enzyme preparations (EP) that ensure high efficiency of bioconversion processes of plant raw materials, as well as to create producers of a wide range of carbohydrases for practical use in various fields of industry and agriculture. A number of industrially important EP obtained using the P. verruculosum expression system are currently being produced at the Agroferment LLC plant.
{"title":"CARBOHYDRASES – 50 YEARS OF RESEARCH AT THE DEPARTMENT OF CHEMICAL ENZYMOLOGY OF LOMONOSOV MOSCOW STATE UNIVERSITY, HISTORY AND PROSPECTS","authors":"Arkady P. Sinitsyn, Olga A. Sinitsyna, Ivan N. Zorov, Aleksandra M. Rozhkova","doi":"10.55959/msu0579-9384-2-2023-64-4-312-333","DOIUrl":"https://doi.org/10.55959/msu0579-9384-2-2023-64-4-312-333","url":null,"abstract":"The review describes the history of the development of research on carbohydrasеs conducted at the Department of Chemical Enzymology from the mid-1970s to the present. The results concerning the study of the mechanism and kinetics of the processes of enzymatic conversion of cellulose and renewable plant raw materials under the action of a multi-enzyme cellulases complexes; the role of individual components of these complexes - basic (endoglucanases and cellobiohydrolases) and auxiliary enzymes (polysaccharide monooxygenase, β-glucosidase, xylanase) and their synergistic interaction. The features of using reactors of various designs for bioconversion of plant raw materials are described: periodic type, continuous column type, reactor for hydrolysis in a constant electric field, reactor with intensive mixing by ferromagnetic particles in magnetic field. The possibilities of increasing the reactivity of plant raw materials using various pretreatment methods, as well as the influence of the structural and physico-chemical properties of cellulose on the efficiency of its enzymatic conversion are discussed. Data on the creation of highly active strains of microscopic fungi-producers of cellulases and other carbohydrases using methods of induced mutagenesis - Trichoderma (Hypocrea), Penicillium (Talaromyces), Aspergillus, Chrysosporium (Myceliophtora) spp., as well as data on the composition of the enzyme complexes produced by them and the properties of the enzymes forming them are presented. It describes the creation of expression systems based on P. canescens and P. verruculosum and the production of recombinant producer strains with their help, which made it possible to obtain enzyme preparations (EP) that ensure high efficiency of bioconversion processes of plant raw materials, as well as to create producers of a wide range of carbohydrases for practical use in various fields of industry and agriculture. A number of industrially important EP obtained using the P. verruculosum expression system are currently being produced at the Agroferment LLC plant.","PeriodicalId":23660,"journal":{"name":"Vestnik Moskovskogo Universiteta Seriya 2 Khimiya","volume":"30 5","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2023-11-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"135775503","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-11-04DOI: 10.55959/msu0579-9384-2-2023-64-3-377-390
Leonid A. Shaposhnikov, Svyatoslav S. Savin, Denis L. Atroshenko, Tatyana A. Chubar, Evgenii V. Pometun, Vladimir I. Tishkov, Anastasia A. Pometun
NAD(P)+ -dependent formate dehydrogenase (FDH, EC 1.2.1.2.) catalyzes the oxidation of formate ion with the coupled reduction of NAD(P)+ to NAD(P)H. Previously, in our laboratory, a genetic construct was obtained with the soyfdh2 gene encoding isoenzyme 2 of formate dehydrogenase from soybean Glycine max (SoyFDH). In this construct the nucleotide sequence encoding the signal peptide responsible for the transport of the pro-enzyme into the mitochondria of plant cells (the SoyFDH_L enzyme) was deleted. In this work, a second variant of SoyFDH_S was obtained, in which, compared to SoyFDH_L, the sequence at the N-terminus was reduced and changed to mimic the N-terminus sequence in FDH from Pseudomonas sp.101 bacteria. Next, a sequence of six histidine residues (His-tag) was added to the N-terminus of the long and short forms of SoyFDH. All four SoyFDH variants were expressed in E. coli BL21(DE3)CodonPlus, these enzymes were purified, their kinetic parameters were determined, and thermal stability was studied. In the case of SoyFDH_L, which is similar to the natural variant of the enzyme, both with and without His-tag, the expression level is two times higher compared to the truncated variant. The addition of His-tag to the N-terminus of enzymes reduces the level of expression. Changing the sequence of the N-terminus, as well as introducing the His-tag sequence to the N-terminus, does not significantly affect thermal stability of the enzymes at temperatures of 50–56 °C. However, due to the higher values of the activation enthalpy ΔH≠ of the thermal inactivation process, the shortened form at normal temperatures is 3 times more stable than the natural one. A comparison of the kinetic parameters of the two SoyFDH variants shows that the catalytic constants are the same, but the long version of SoyFDH_L has lower values KM HCOO– , and the short version has lower KM NAD+ values. The introduction of His-tag into the N-terminus of enzymes does not affect their kinetic parameters.
{"title":"ENGINEERING THE N-TERMINAL SEQUENCE OF GLYCINE MAX SOYBEAN FORMATE DEHYDROGENASE","authors":"Leonid A. Shaposhnikov, Svyatoslav S. Savin, Denis L. Atroshenko, Tatyana A. Chubar, Evgenii V. Pometun, Vladimir I. Tishkov, Anastasia A. Pometun","doi":"10.55959/msu0579-9384-2-2023-64-3-377-390","DOIUrl":"https://doi.org/10.55959/msu0579-9384-2-2023-64-3-377-390","url":null,"abstract":"NAD(P)+ -dependent formate dehydrogenase (FDH, EC 1.2.1.2.) catalyzes the oxidation of formate ion with the coupled reduction of NAD(P)+ to NAD(P)H. Previously, in our laboratory, a genetic construct was obtained with the soyfdh2 gene encoding isoenzyme 2 of formate dehydrogenase from soybean Glycine max (SoyFDH). In this construct the nucleotide sequence encoding the signal peptide responsible for the transport of the pro-enzyme into the mitochondria of plant cells (the SoyFDH_L enzyme) was deleted. In this work, a second variant of SoyFDH_S was obtained, in which, compared to SoyFDH_L, the sequence at the N-terminus was reduced and changed to mimic the N-terminus sequence in FDH from Pseudomonas sp.101 bacteria. Next, a sequence of six histidine residues (His-tag) was added to the N-terminus of the long and short forms of SoyFDH. All four SoyFDH variants were expressed in E. coli BL21(DE3)CodonPlus, these enzymes were purified, their kinetic parameters were determined, and thermal stability was studied. In the case of SoyFDH_L, which is similar to the natural variant of the enzyme, both with and without His-tag, the expression level is two times higher compared to the truncated variant. The addition of His-tag to the N-terminus of enzymes reduces the level of expression. Changing the sequence of the N-terminus, as well as introducing the His-tag sequence to the N-terminus, does not significantly affect thermal stability of the enzymes at temperatures of 50–56 °C. However, due to the higher values of the activation enthalpy ΔH≠ of the thermal inactivation process, the shortened form at normal temperatures is 3 times more stable than the natural one. A comparison of the kinetic parameters of the two SoyFDH variants shows that the catalytic constants are the same, but the long version of SoyFDH_L has lower values KM HCOO– , and the short version has lower KM NAD+ values. The introduction of His-tag into the N-terminus of enzymes does not affect their kinetic parameters.","PeriodicalId":23660,"journal":{"name":"Vestnik Moskovskogo Universiteta Seriya 2 Khimiya","volume":"20 6","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2023-11-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"135774944","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-11-04DOI: 10.55959/msu0579-9384-2-2023-64-4-391-400
Andrey V. Levashov, Vladimir V. Fedorchuk, Svyatoslav S. Savin, Vladimir I. Tishkov
As society develops, its relationship with science and influence on it becomes more and more significant. The ability to navigate the current trends in the development of society and science in particular is the most important factor in choosing new topics for scientific work and understanding the prospects for the development of scientific research. Therefore, training young professionals in this understanding (primarily in the field of natural sciences) is no less important aspect of higher education than the process of teaching fundamental and practical knowledge. This article discusses the development of relationships between society, chemistry and biotechnology (primarily applied enzymology) at different stages of human evolution. The article was written based on the materials of the introductory lecture of the section on biotechnology and applied enzymology as part of the general course “Chemical Foundations of Biological Processes”, read at the Faculty of Chemistry of Moscow State University named after M.V. Lomonosov, The features and aspects of the interaction of society, chemistry and biotechnology at different stages of the development of our world, when biotechnology has gone through the stages of development from “cave-memorable” (unconsciously natural) to “smart”, are considered. A great and important contribution to the writing of this article was made by the discussion of this problem at seminars with students of 3-6 courses of the Faculty of Chemistry of Moscow State University. The impact of changes in our society as a result of the SARS Cov-2 pandemic is also discussed separately.
{"title":"RELATIONSHIP BETWEEN SOCIETY, CHEMISTRY AND BIOTECHNOLOGY. SCIENTIFIC, ECONOMIC AND ETHICO-MORAL ASPECTS","authors":"Andrey V. Levashov, Vladimir V. Fedorchuk, Svyatoslav S. Savin, Vladimir I. Tishkov","doi":"10.55959/msu0579-9384-2-2023-64-4-391-400","DOIUrl":"https://doi.org/10.55959/msu0579-9384-2-2023-64-4-391-400","url":null,"abstract":"As society develops, its relationship with science and influence on it becomes more and more significant. The ability to navigate the current trends in the development of society and science in particular is the most important factor in choosing new topics for scientific work and understanding the prospects for the development of scientific research. Therefore, training young professionals in this understanding (primarily in the field of natural sciences) is no less important aspect of higher education than the process of teaching fundamental and practical knowledge. This article discusses the development of relationships between society, chemistry and biotechnology (primarily applied enzymology) at different stages of human evolution. The article was written based on the materials of the introductory lecture of the section on biotechnology and applied enzymology as part of the general course “Chemical Foundations of Biological Processes”, read at the Faculty of Chemistry of Moscow State University named after M.V. Lomonosov, The features and aspects of the interaction of society, chemistry and biotechnology at different stages of the development of our world, when biotechnology has gone through the stages of development from “cave-memorable” (unconsciously natural) to “smart”, are considered. A great and important contribution to the writing of this article was made by the discussion of this problem at seminars with students of 3-6 courses of the Faculty of Chemistry of Moscow State University. The impact of changes in our society as a result of the SARS Cov-2 pandemic is also discussed separately.","PeriodicalId":23660,"journal":{"name":"Vestnik Moskovskogo Universiteta Seriya 2 Khimiya","volume":"27 6","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2023-11-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"135775349","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-11-04DOI: 10.55959/msu0579-9384-2-2023-64-4-353-376
Ilya N. Kurochkin, Alexandra D. Vasilyeva, Evgeniy G. Evtushenko, Arkady V. Eremenko, Dmitry V. Pergushov, Larisa V. Sigolaeva
The paper addresses the possibilities of voltammetry, surface-enhanced Raman spectroscopy (SERS) and mass spectrometry in tandem with enzymatic catalysis. The basic principles of operation of electrochemical biosensors based on oxidases and dehydrogenases are described. It has been shown that biosensors using cyclic enzymatic reactions and substrate recycling have the best sensitivity. The variants of significant improvement of the analytical potential of biosensor analysis due to the use of polymers for effective modification of the electrode surface and non-destructive immobilization of enzymes are illustrated. The data demonstrating how the use of enzyme labels expands the range of bioanalytical applications of SERS are presented. The possibility of highly sensitive measurement of the activity of enzyme labels (peroxidase, alkaline phosphatase, β-galactosidase) using SERS, in fact, opens up a new universal platform for the development of methods for the determination of various antigens. By way of example of the most commonly used proteases, the main trends in the development of the methodology of proteomic studies by mass spectrometry, as well as the role of proteases in the design of mass spectrometric experiments, are considered.
{"title":"ENZYMES IN THE DEVELOPMENT OF PHYSICO-CHEMICAL METHODS FOR BIOMEDICAL RESEARCH","authors":"Ilya N. Kurochkin, Alexandra D. Vasilyeva, Evgeniy G. Evtushenko, Arkady V. Eremenko, Dmitry V. Pergushov, Larisa V. Sigolaeva","doi":"10.55959/msu0579-9384-2-2023-64-4-353-376","DOIUrl":"https://doi.org/10.55959/msu0579-9384-2-2023-64-4-353-376","url":null,"abstract":"The paper addresses the possibilities of voltammetry, surface-enhanced Raman spectroscopy (SERS) and mass spectrometry in tandem with enzymatic catalysis. The basic principles of operation of electrochemical biosensors based on oxidases and dehydrogenases are described. It has been shown that biosensors using cyclic enzymatic reactions and substrate recycling have the best sensitivity. The variants of significant improvement of the analytical potential of biosensor analysis due to the use of polymers for effective modification of the electrode surface and non-destructive immobilization of enzymes are illustrated. The data demonstrating how the use of enzyme labels expands the range of bioanalytical applications of SERS are presented. The possibility of highly sensitive measurement of the activity of enzyme labels (peroxidase, alkaline phosphatase, β-galactosidase) using SERS, in fact, opens up a new universal platform for the development of methods for the determination of various antigens. By way of example of the most commonly used proteases, the main trends in the development of the methodology of proteomic studies by mass spectrometry, as well as the role of proteases in the design of mass spectrometric experiments, are considered.","PeriodicalId":23660,"journal":{"name":"Vestnik Moskovskogo Universiteta Seriya 2 Khimiya","volume":"34 2","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2023-11-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"135775658","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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