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Applied Biochemistry and Microbiology (v.43, #1)
Transformation of steroids by actinobacteria: A review by M. V. Donova (pp. 1-14).
Development of pharmaceutical industry is currently aimed at introducing biotechnological processes on a large-scale and thereby replacing multiple-stage chemical syntheses. Actinobacteria are efficient biocatalysts of many processes involving steroid bioconversion, which hold considerable importance for the synthesis of hormonal drugs. The potential to catalyze the conversion of a broad spectrum of steroid substrates makes it possible to expect efficient utilization of these microorganisms in development of new technologies of manufacturing steroid pharmaceutical substances. The review is a first attempt to systematize data on the potential of actinobacteria to catalyze diverse reactions of steroid transformation (such as hydroxylation, introduction and reduction of double bonds, oxidation of steroid alcohols, reduction of ketones, side chain de-esterification and degradation, etc.), with emphasis on processes of practical biotechnological importance and progress in steroid bioconversion over the last ten years.
Oxygen can be replaced by artificial electron acceptors in reactions catalyzed by alcohol oxidase by G. P. Shumakovich; S. V. Shleev; O. V. Morozova; M. V. Gonchar; A. I. Yaropolov (pp. 15-20).
For the first time, spectrometric and electrochemical studies demonstrated the possibility of using artificial electron acceptors in reactions catalyzed by alcohol oxidase. We report kinetic parameters of homogenous catalytic oxidation of formaldehyde by organic redox compounds belonging to different structural classes (toluidine blue, methylene blue, 2,6-dichlorophenolindo-phenol, and p-benzoquinone) and replacing dioxygen in these reactions. p-Benzoquinone, having the highest redox potential, proved to be the most efficient artificial electron acceptor of all compounds studied.
Regulation of phenoloxidase activity with sulfur dioxide as the base of production of high-quality instant green tea by N. T. Omiadze; N. I. Mchedlishvili; N. G. Pruidze; L. K. Gulua; M. O. Abutidze; K. Sh. Akhvlediani; E. G. Kvesitadze; V. R. Aplakov (pp. 21-24).
The possibility to regulate phenoloxidase activity with sulfur dioxide was studied. It was found that this compound is a potent inhibitor of phenoloxidase of the reversible and mixed type. The inhibitory effect of sulfur dioxide on phenoloxidase provided grounds for a new biotechnological approach to the production of instant green tea. This approach allows increasing the yield of the extractive and the proportion of phenolics in the extractive, thereby improving the organoleptic quality of the product.
Luminol oxidation catalyzed by royal palm leaf peroxidase by I. S. Alpeeva; I. Yu. Sakharov (pp. 25-28).
We optimized the conditions for oxidation of luminol by hydrogen peroxide in the presence of peroxidase (EC 1.11.1.7) from royal palm leaves (Roystonea regia). The pH range (8.3–8.6) corresponding to maximum chemiluminescence was similar for palm tree peroxidase and horseradish peroxidase. Variations in the concentration of the Tris buffer were accompanied by changes in chemiluminescence. Note that maximum chemiluminescence was observed in the 30 mM Tris solution. The detection limit of the enzyme assay during luminol oxidation by hydrogen peroxide was 1 pM. The specific feature of palm tree peroxidase was the generation of a long-term chemiluminescent signal. In combination with the data on the high stability of palm tree peroxidase, our results indicate that this enzyme is promising for its use in analytical studies.
Simultaneous co-immobilization of glucose oxidase and catalase in their substrates by G. Ozyilmaz; S. S. Tukel (pp. 29-35).
Glucose oxidase (GOD) and catalase (CAT) were simultaneously co-immobilized onto magnesium silicate (Florisil®) by covalent coupling. Glucose was added in immobilization mixture and hydrogen peroxide, which is the substrate of CAT, was produced in coupling mixture during immobilization time. Therefore, co-immobilization of GOD and CAT was carried out in the presence of both their substrates: glucose and hydrogen peroxide, respectively. The effect of glucose concentration in immobilization mixture on activities of GOD and CAT of co-immobilized samples were investigated. Maximum GOD and CAT activities were determined for samples co-immobilized in the presence of 15 and 20 mM glucose, respectively. Co-immobilization of GOD and CAT in the presence of their substrates highly improved the activity and reusability of both enzymes.
Transglycosylation of L-ascorbic acid by A. A. Markosyan; L. A. Abelyan; M. O. Adamyan; Zh. I. Akopyan; V. A. Abelyan (pp. 36-40).
Cyclodextrin glucanotransferases (CGTs, EC 2.4.1.19) from mesophilic, thermophilic, and halophilic bacteria and maltase (EC 3.2.1.20) from the yeast Saccharomyces cerevisiae were used for transglycosylation of ascorbic acid with starch, maltodextrin, γ-cyclodextrin, and maltose. These compounds served as donors of glucosyl residues. CGT from thermophilic strains was shown to be the most potent in this respect (the degree of transglycosylation was as high as 60%).
The effect of liposomes on the growth and sensitivity of Mycobacterium smegmatis to isoniazide by O. A. Troshkina; E. G. Salina; G. M. Sorokoumova; A. S. Kaprelyants; A. A. Selishcheva (pp. 41-46).
The effects of the liposome form of isoniazide (IN) and liposomes without IN on the growth of Mycobacterium smegmatis were studied. Fluorescent assay demonstrated that the fraction of liposomes that interacted with M. smegmatis amounted to 1–3%. It was shown that the IN efficiency in a liposomal form decreased depending on the liposome composition and concentration as compared with the IN in water solution. A preincubation of mycobacteria with liposomes led to a decrease in their sensitivity to IN. An analogous effect was observed when incubating M. smegmatis with oleic acid. It was postulated that the relative resistance of M. smegmatis to the antibiotic when using lipids as a carbon substrate appeared due to a change in the agent’s metabolism and should be taken into account when testing in vitro the liposomal forms of antibiotics.
Adaptation of aerobic methylobacteria to dichloromethane degradation by M. L. Torgonskaya; Yu. E. Firsova; N. V. Doronina; Yu. A. Trotsenko (pp. 47-51).
A shortening of the lag phase in dichloromethane (DCM) consumption was observed in the methylobacteria Methylopila helvetica DM6 and Albibacter methylovorans DM10 after prior growth on methanol with the presence of 1.5% NaCl. Neither heat nor acid stress accelerated methylobacterium adaptation to DCM consumption. Sodium azide (1 mM) and potassium cyanide (1 mM) inhibited consumption of DCM by these degraders but not by transconjugants Methylobacterium extorquens AM1, expressing DCM dehalogenase but unable to grow on DCM. This indicates that the degrader strains possess energy-dependent systems of transport of DCM or chloride anions produced during DCM dehalogenation. Inducible proteins were found in the membrane fraction of A. methylovorans DM10 cells adapted to DCM and elevated NaCl concentration.
Features of the toxic action of 2,4,6-trinitrotoluene on Escherichia coli K12 by B. M. Kurinenko; N. A. Denivarova; R. E. Davydov; G. Yu. Yakovleva (pp. 52-56).
2,4,6-Trinitrotoluene present in a culture of Escherichia coli K12, at a concentration of 200 mg/l, caused a decrease in the total cell population and the population of colony-forming units, increased permeability of the external lipoprotein envelope, and increased the refractive index of cells. The shape of some cells changed from rod-like to coccoid, and cell size decreased. The specific rate of glucose consumption and the content of NADH (NADPH) in cells decreased. The changes of these morphological and physiological features were reversible, tending to normalize after reduction of 2,4,6-trinitrotoluene concentration in the course of cultivation.
Temperature cycling to improve the ethanol production with solid state simultaneous saccharification and fermentation by H. Z. Chen; J. Xu; Z. H. Li (pp. 57-60).
Simultaneous saccharification and fermentation (SSF) widely used in submerged state could be effective in solid state. Solid state SSF was first compared with solid-state separate hydrolysis and fermentation on ethanol production. Ethanol yield using solid-state separate hydrolysis and fermentation (SHF) in 5 days was only half of that in solid state SSF in 3 days. In solid state SSF, the ethanol concentration using temperature cycling (10 h at 37°C followed by 15 min at 42°C) reached 5.2% which was 2 times higher than that observed at 37°C within 72 h.
A comparative characterization of fungal melanin and the humin-like substances synthesized by Cerrena maxima 0275 by O. V. Koroleva; N. A. Kulikova; T. N. Alekseeva; E. V. Stepanova; V. N. Davidchik; E. Yu. Belyaeva; E. A. Tsvetkova (pp. 61-67).
Comparative studies of fungal melanin and two preparations of the high-molecular-weight humin-like substances formed during a solid-phase cultivation of the basidiomycete Cerrena maxima 0275 for 45 and 70 days were performed. The fungal melanin from Aspergillus niger and the humin-like substances synthesized by the basidiomycete C. maxima 0275 are similar in their physicochemical properties (elemental composition and behavior in acids and alkalis) and auxin-like activities. However, these biopolymers differ, essentially, at the structural level. According to IR spectroscopy data, the obtained humin-like substances display a higher similarity to natural humic acids and are more diverse in their functional groups compared with fungal melanins. Presumably, this is connected with the fact that laccase is involved in formation of humin-like substances; moreover, this enzyme is involved not only in the synthesis of these polymers, but also in their modification and degradation.
The correlation between the synthesis of β-carotene and zygote formation by Blakeslea trispora heterothallic strains by N. V. Kalinina; V. M. Tereshina; A. S. Memorskaya; E. P. Feofilova (pp. 68-72).
It was demonstrated for the first time that the level of carotenogenesis by the heterothallic Blakeslea trispora strains intensively forming zygospores decreased under conditions of a surface cocultivation during their sexual interaction as compared with the strains grown separately. On the contrary, carotenogenesis was stimulated during a sexual interaction of the strains incapable of forming zygotes. In a submerged culture, the zygote-forming pairs of strains synthesized a considerably larger amount of trisporic acids but a smaller amount of carotenoids than the strains not forming zygospores. The discovered inverse dependence between zygote formation and carotenogenesis allowed us to suggest the inability to form zygotes as a criterion for selecting carotenogenic strain pairs.
Galactosylated derivatives of low-molecular-weight chitosan: Obtaining and properties by A. V. Il’ina; V. P. Varlamov (pp. 73-77).
Chitosan, a binary heteropolysaccharide consisting of 2-acetamide-2-deoxy-β-D-glucopyranose and 2-amino-2-deoxy-β-D-glucopyranose residues linked in different proportions via β-glycosidic bonds. The presence of a primary amino group in the chitosan structure allows for the synthesis of various derivatives. The procedure of obtaining activated N-hydroxysuccinimide esters with the use of lactobionic acid was applied to obtain galactosylated derivatives of low-molecular-weight chitosan with a substitution degree varying from 8 to 23%. The properties of these derivatives (viscosity, solubility, and biodegradability) were studied. These derivatives are well soluble at pH values greater than the acidity constant of amino groups of chitosan (6.5). Broadening the pH range towards increase and the presence of galactose residues allows these derivatives to be used in working with biological objects.
A highly branched storage polyglucan in the thermoacidophilic red microalga Galdieria maxima cells by I. N. Stadnichuk; L. R. Semenova; G. P. Smirnova; A. I. Usov (pp. 78-83).
The thermoacidophilic red alga Galdieria maxima is capable of heterotrophic growth. The content of carbohydrates in G. maxima grown heterotrophically increases by a factor of 4, reaching as much as 60% of cell dry weight. The increase in the level of carbohydrates in cells is due to accumulation of a storage α-glycan. According to a specific cleavage to glucose catalyzed by amyloglucosidase and the high positive specific optical rotation characteristic of polyglucans, this polysaccharide can be classified as a floridean starch. The data of 1H NMR spectroscopy and the results of methylation showed that the average length of the unbranched regions of the polysaccharide molecule is six to seven glucose residues. The degree of branching of the starch molecule of G. maxima is greater than that of storage polysaccharides of other red algae, glycogens of yeast, and phytoglycogens of cyanobacteria.
Polysaccharides of cell cultures of Silene vulgaris by E. A. Günter; Yu. S. Ovodov (pp. 84-90).
Callus and suspension cultures of campion (Silene vulgaris) produced pectin polysaccharides, similar in structure to the polysaccharides of intact plants. The major components of the pectins were D-galacturonic acid, galactose, arabinose, and rhamnose residues. The maximum content of pectins was found in callus. The monosaccharide composition of arabinogalactans isolated from cells and a culture medium of callus cultures were similar, with the ratio between arabinose and galactose of 1: (2.3–6.5) being retained. The arabinogalactans from the cells and culture medium of the suspension cultures also had a similar structure, and the arabinose to galactose ratio was 1: (1.5–1.8). In contrast to the callus cultures, the suspension cultures produced arabinogalactans with an increased content of arabinose residues and a decreased content of galactose residues. The greatest content of arabinogalactan was detected in the culture medium of the suspension cultures.
The effect of intercultivar substitution of wheat Triticum aestivum L. chromosomes on lipoxygenase activity and its correlation with the technological properties of flour by V. A. Trufanov; M. D. Permyakova; T. A. Pshenichnikova; M. F. Ermakova; V. A. Davydov; A. V. Permyakov; E. V. Berezovskaya (pp. 91-97).
The effects of intercultivar substitution of individual chromosome pairs (except for 1B, 6D, and 7A) in the wheat cultivars Saratovskaya 29 and Janetzkis Probat, differing in quality, on specific lipoxygenase activity, the grain yield per spike, and the main technological properties of flour and dough were studied. It was demonstrated that the substitution of individual chromosomes of the recipient cultivar Saratovskaya 29 with the homologous chromosomes of the donor cultivar Janetzkis Probat caused significant changes in lipoxygenase activity and several other quality characteristics. The correlations between the lipoxygenase activity and the parameters of physical dough properties were determined. Three molecular forms of lipoxygenase (Lpx-1, Lpx-2, and Lpx-3), differing in the value of surface charge and enzymatic activity, were detected by native PAGE.
Use of monoclonal antibodies against horse immunoglobulin in an enzyme immunoassay of bacterial toxins and toxoids by M. A. Burkin; I. A. Gal’vidis; I. V. Yakovleva; V. V. Sviridov (pp. 98-101).
Immunization of BALB/c mice by horse antiserum against diphtheria made it possible to obtain IgG1 monoclonal antibodies (MoAbs) 2B7E4 specific for light chains of horse immunoglobulin (Ig). Unlike commercial preparations of anti-horse immunoglobulin antibodies, which are specific for the whole Ig molecule or its Fc-fragment, the peroxidase (HRP) conjugate of the MoAb, 2B7E4-HRP did not interact with human, mouse, rabbit, and sheep Igs, or horse albumin. The conjugate obtained was used with MoAbs against bacterial toxins and commercial horse antitoxins, as a universal reagent in sandwich enzyme immunoassay (ELISA) for bacterial toxins and toxoids. The detection sensitivity of diphtheria toxin/toxoid equaled 0.0005 Lf/ml; tetanus toxin and toxoid were detected with sensitivities of 20 LD50/ml and 0.005 UI/ml, respectively. A similar sandwich ELISA for botulinum toxoids (group measurement) allowed types A, B, and E to be detected at 0.02, 0.002, and 0.001 UI/ml, respectively; selective measurement was only possible in the case of type E toxoid (0.001 UI/ml).
On the possibility of oral treatment with protein solutions by I. L. Valuev; L. K. Starosel’tseva; L. I. Valuev; G. A. Sytov; M. V. Ul’yanova; L. V. Vanchugova; Yu. A. Talyzenkov; N. A. Plate (pp. 102-104).
Experiments on animals showed that native proteins may diffuse into the blood flow after oral administration of diluted protein solutions. An in vitro study led us to hypothesize that treatment with diluted solutions is accompanied by a decrease in the rate of protein proteolysis and accelerated protein diffusion through the intestinal mucosa.
An improved method of photometric determination of cyclodextrin glucanotransferase activity by N. G. Usanov; E. A. Gil’vanova; P. A. Elizar’ev; E. A. Prutsakova; A. I. Melent’ev (pp. 105-110).
The method of spectrophotometric determination of cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19) activity with the use of phenolphthalein as a colored reagent has been improved. This technique includes an enzymatic reaction at 40°C for 60 min in 2% starch, with subsequent supplementation of the reaction mixture (0.5 ml) with the phenolphthalein reagent (3.0 ml) prepared in 0.1 M potassium carbonate buffer (pH 11.0) according to a special procedure, and measurement of the optical density of the obtained mixture at 553 nm. The activity was calculated using the exponential growth equation that connects a drop in the optical density and the degree of dilution of the enzyme. The described technique is suitable for working in a sufficiently broad range of specific activity of β-CGTase and does not require precise adjustment of the degree of dilution of solutions analyzed.