Изучено содержание пяти фракций богатых энергией неорганических полифосфатов (полиР), АТФ и активность Н+-АТФазы плазматической мембраны у низкоактивного продуцента цефалоспорина С (цефС) Acremonium chrysogenum ATCC 11550 и селекционированного высокоактивного штамма 26/8 при выращивании гриба на глюкозе и комплексной среде, обеспечивающей активный синтез антибиотика. Показано, что на комплексной среде штамм 26/8 образует в 26 раз больше цефС, чем штамм АТСС 11550. При этом происходило резкое падение содержания в клетках АТФ и высокомолекулярных фракций полиР2, полиР3 и полиР5 с одновременным возрастанием низкомолекулярной фракции полиР1. Эти данные свидетельствуют об участии полиР как источников энергии, в синтезе цефС. Активность Н+-АТФазы мало изменялась как при низкой, так и высокой продукции цефС. Это подтверждает предположение, что у A. chrysogenum наряду с cefT, присутствуют и другие, альтернативные, транспортеры антибиотика. Полученные данные могут быть использованы при оптимизации процесса биосинтеза цефалоспорина С в промышленных масштабах.

Исследованы изменения в клеточной стенке Acremonium chrysogenum при интенсивном образовании цефалоспорина C. С помощью электронной микроскопии показано, что клеточная стенка клеток штамма ATCC 11550 (“дикий” тип) в процессе роста становилась более рыхлой и утолщалась. Клеточная стенка клеток штамма 26/8 (суперпродуцент цефалоспорина C) к стационарной фазе в значительной степени деградировала. Биохимический анализ показал, что указанные изменения сопровождались снижением содержания белков как ковалентно, так и нековалентно связанных с полисахаридами клеточных стенок обоих штаммов. В процессе роста наблюдалось увеличение чувствительности клеточных стенок штамма-суперпродуцента к действию литических ферментов хитиназы, ламинариназы, протеиназы К, трипсина и препарата литиказы; в случае “дикого” типа подобного увеличения выявлено не было. Полученные результаты свидетельствуют о нарушении структуры клеточных стенок A. chrysogenum, сопровождающем интенсивное образование антибиотика.

Разработана система трансформации гетерологичных генов методом агробактериального переноса в штаммы Acremonium chrysogenum дикого типа ATCC 11550, природного продуцента бета-лактамного антибиотика цефалоспорина С и полученного на его основе в результате многоступенчатого отбора штамма-суперпродуцента цефалоспорина С №26/8. Сконструированы вектора для агробактериальной трансформации A. chrysogenum, содержащие кассеты экспрессии генов устойчивости к антибиотикам генетицину (G418) и блеомицину (ZeocinTM) под контролем TEF1 промоторов Ashbya gossypii и Saccharomyces cerevisiae. Проведена сравнительная оценка методов агротрансформации при ко-культивировании клеток грибов и агробактерий на фильтрах и в “глубинных условиях”. Отобранные трансформанты A. chrysogenum, устойчивые к генетицину и блеомицину, охарактеризованы методами ПЦР и Саузерн-блоттинга.

Mutagenic (Ames tests) and genotoxic (SOS chromotest) activities of highly-efficient natural anthracycline monosaccharides possessing antitumor activity-daunorubicin (also known as daunomycin or rubomycin), doxorubicin (adriamycin), and carminomycin-were studied. At the same time, the hypothesis was tested that intercalation of the antibiotic moiety into the helix of cell DNA, which was mediated by the saccharide amino group, played a crucial role in genotoxicity of these anthracyclines. The hydrolysis products of these antibiotics (the corresponding aglycones) and aclacynomycin A (an anthracycline trisaccharide), as well as aclavinone (its derivative aglycone), were studied. All these compounds lacked the saccharide amino group necessary for intercalation. It was found that all anthracycline monosaccharides studied had a strong mutagenic effect on strain TA98 and a moderate effect on strain TA100 of Salmonella typhimurium. Aclacynomycin A was found to have no mutagenic effect on any strain. Lack of the glycoside amino group did not necessarily result in loss of mutagenic activity in the derivative aglycones of anthracycline monosaccharides: they exhibited moderate mutagenic activity in strain TA98 and low but significant activity in strain TA100. The S9 microsomal fraction did not alter the mutagenic activity of either anthracycline monosaccharides or their aglycones; however, it dramatically increased the mutagenic activity of aclavinone: correspondence between positive responses in Ames tests and the SOS chromotest was found. Apparently, the mutagenic activity of the substances studied in bacterial cells was mediated by inducing the SOS-repair process. If the compound contained the amino glycoside moiety, functional and structural precursors of the SOS response were formed via intercalation of the reagents into the DNA duplex; if the substance did not contain this moiety, the precursors were formed via ionic interaction.

Correlation between the dynamics of intracellular pH and antibiotic synthesis in Fusidium coccineum strains with different biosynthetic capacity was studied. At the beginning of the intensive antibiotic synthesis the intracellular pH in the low-active and highly-active strains was minimum and maximum respectively. In the highly active strain an increase in the intracellular pH of the cytoplasm after the addition of Tween-80 and Factor-d2 analogs to the growth medium was observed.

The paper presents the results of the studies carried out by the authors within 20 years on development of processes for production of beta-lactam antibiotics with using biocatalysis. The proposed general principles for the development of efficient biocatalytic technologies are discussed in regard to production of the key compounds and synthesis of beta-lactams. The paper includes 4 parts concerned with comparison of the biocatalytic and chemical processes for production of beta-lactam antibiotics, requirements to the quality of the biocatalysts used and criteria for estimation of the efficiency of the stage of the technological biocatalyst production. The criteria provided determination of the optimal biocatalyst for production of the key compounds in the synthesis of beta-lactams. A retrospective analysis of the biocatalytic processes for production of 6-amino-penicillanic acid is presented and the impact of the activity and the form of the biocatalyst on the process efficiency is substantiated. Various schemes of the enzymatic synthesis of beta-lactam antibiotics and the approaches to the improvement of the technological processes including those with the use of immobilized microbial cultures at the stage of the production of the initial biosynthetic antibiotics are described. The prospects for the improvement of the processes for production of drugs with the use of biocatalysis are indicated and the main trends of the research required for the large scale use of the immobilized enzymes, microbial cells, oligo-enzymatic and poly-enzymatic systems in transformation and synthesis of organic compounds are defined.

A new method and a device for enzymatic express assay of beta-lactam antibiotics are described. The principle of the device operation is based on the use of native enzymes specific to the antibiotics assayed. The method makes it possible to exclude the influence of the buffer capacity of the samples on the results of the assay. The data on the testing of the method and device with model solutions of benzylpenicillin are presented. The relative error of a single measurement does not exceed 2 per cent. The time of an assay is not more than 3 minutes. Recommendations on the use of the device in research studies and manufacture are presented.

A fermentation medium balanced by the main components was developed for Cryptococcus diffluens strains producing penicillin-V-acylases (PA). It was shown that the culture needed for production of the enzyme was inductor, which was phenoxyacetic acid (POAA). Additional introduction of ethanol to the medium provided an increase in production of PA by 36 per cent and the culture growth by 25 per cent. Introduction of one of the following substances to the medium with POAA and ethanol i.e. (CN3COO)2Ca, FeSO4, proline or asparagine provided an additional increase in the production level of PA by 24 to 94 per cent. The use of the medium varieties will permit one to isolate highly productive cells of the culture.

It was found possible to use organic sorbents and in particular carboxylic cationites for isolation of nisin from the fermentation broth filtrate and its purification. Nisin is known as a polypeptide antibiotic applied as a preservative. The sorbents were shown to have high exchange capacities by the isolated substance and mechanical strength and resistance. They also proved to be highly stable.

The results of developing a semiautomatic apparatus with oxygen detection for enzymatic control of glucose concentration are presented. The design of a glucose sensitive electrode is based on an oxygen probe and a membrane with immobilized glucose oxidase. Materials for the probe were chosen and the operating conditions for measuring temperature, pH, linear agitation velocity and other parameters were optimized. The semiautomatic analyzer was constructed and its main characteristics were studied. The results of the apparatus testing during biosynthesis of various antibiotics are presented. It was shown that the required glucose concentration in the cultivation medium was provided for any specific circumstances in relation to the carbohydrate source.

Methods for development of bioengineering systems of different types useful in synthesis and transformation of antibiotics are discussed. It was shown that in development of monoenzymatic biocatalysts on the basis of immobilized cells and in preparation of immobilized cultures producing secondary metabolites with enzymological engineering directed action on the cells could be provided which made it possible to establish highly efficient bioengineering systems. Various means for providing the directed action and method for estimation of the carrier-culture interation are proposed. The prospects of using the second generation biocatalysts in improvement of the processes for production of antibiotics are described.

Comparative investigation of lipogenesis in 2 initial and 4 mutant strains of Penicillium chrysogenum showed that there were no noticeable differences in the composition of the lipid fatty acids in these strains. Certain shifts in the ratio of definite lipid fatty acids in the mutant strain deficient by synthesis of lysine and isoleucine (increased contents of oleic acid) were revealed. A marked influence of the physiological conditions on lipogenesis in the mutant with multiple deficiency by amino acids, vitamins and nucleotides was observed.

A population of Escherichia coli strains producing penicillinacylase (PA) and differing in the level of their enzyme activity was studied. Their structural-functional analysis showed that selection according to the property of PA production yielded strains with aberrations in the processes of cell division. The population of microbial cells producing PA had a correlation between its morphological composition and enzyme activity, and the two characteristics depended on the conditions under which the strains were cultivated. The highest enzyme activity was exerted by normally dividing cells. A type of colonies optimal for stabilising the level of PA production was determined for the enzyme-producing strain. The structure of cell envelopes, their composition and permeability changed considerably as the ability to synthesize PA increased. The results allow one to specify further rational selection of strains superproducing the enzyme on the basis of changes in the membrane permeability.

The dynamics of cephalosporine C and desacetylcephalosporine C alterations in cultures of Acremonium chrysogenum 309A under different conditions was studied. It was shown expedient to determine the fermantation period by the data of HPLC. Genetic predisposition of the strain used to production of both cephalosporine C and desacetylcephalosporine C is discussed. With changing the cultivation procedure, medium active acidity and cultivation time it is possible to change the biosynthesis pathway.

A laboratory unit for production of calcium alginate gel granules with immobilized microorganisms is described. It provides sterile production of particles from tens micrometers to 2 mm in diameter. Expediency of using biocatalysts in the form of fine granules is exemplified with a number of immobilized microorganisms. Conditions for immobilizing the erythromycin producing organism by its incorporation into the calcium alginate gel were studied. Viability of the actinomycete in the gel was shown by consumption of the nutrients and biosynthesis of the antibiotic.

For using actinomyceteous extracellular aminoacylase in production of optically active amino acids it is necessary to have the fermentation broth with lowered contents of extracellular pigments whose accumulation dynamics in the process of the strain growth correlates with the dynamics of the enzyme biosynthesis. The results of the studies showed that it was possible to regulate biosynthesis of the aminoacylase and pigments in the direction of increasing the enzyme production and decreasing the pigment formation by using a medium of the respective composition and providing the respective high temperature and sufficient aeration for the strains.