Effect of storage conditions on population variability and activity of Streptomyces recifensis var. lyticus

The results of the study on survival and variation of Streptomyces recifensis var. lyticus 2435 producing lytic enzymes are presented. The culture was maintained for 2.5 years under a layer of vaseline oil, at a temperature of -20 degrees C and in lyophilized state. It was shown that irrespective of the storage method strain 2435 preserved its viability. However, the most intensive growth was observed in the lyophilized cultures. During the storage the content of the productive colonies characteristic of the morphological type culture in the population decreased while the number of the low active variants increased. Lyophilization of the strain spores in the sucrose-gelatine medium provided insignificant morphological variation of the culture and preservation of the initial level of its lytic activity against a number of test-microbes except S. aureus and M. lysodeikticus. Storage of the culture under vaseline oil and at a temperature of -20 degrees C resulted in lowering of its lytic activity against all the test-microbes used. For long-term maintenance of Streptomyces recifensis var. lyticus 2435 the method of lyophilization in the sucrose-gelatine medium is recommended.     

Interrelation between the activity of the lytic enzyme producer Actinomyces recifensis var. lyticus 2435 and its population variability

Spontaneous variability of Actinomyces recifensis var. lyticus 2435 producing a complex of lytic enzymes was studied. A correlation between the strain activity and the content of the variants of the main morphological type in the population was shown. The carbon sources influenced the proportion of different type variants in the population. Strain 2435 was rather stable with respect to the level of the synthesis of yeast-like enzymes and showed a significant variation with respect to the level of the biosynthesis of the bacteriolytic complex. The population variation of strain 2435 with respect to the staphylolytic (synthesis of specific endopeptidases) and muramidase activity was most pronounced. The high activity levels of strain 2435 and wide lytic spectrum were provided by selection of the variants of the first morphological type with respect to the property of high staphylolytic activity.     

Isolation and identification of the enzyme complex in Streptomyces recifensis var. lyticus 2435 with beta-lactamase activity

Ability of an enzyme complex from Streptomyces recifensis var. lyticus 2435 to inactivate beta-lactam antibiotics was shown. Two lytic endopeptidases with beta-lactamase activity were isolated and identified as beta-lactamases of classes II and V according to the Richmond and Sykes classification system. The ability of the endopeptidases to hydrolyze the beta-lactam ring confirmed the absence of strict substrate specificity in them. Correlation between the capacity of the lytic endopeptidases for lysing staphylococcal cells and their capacity for inactivating beta-lactam antibiotics was observed.     

Enzymatic lysis of staphylococcal cells and isolation of cell walls

Procedures for lyzing staphylococcal cells with the use of ultrasound, lysozyme and a lytic enzyme complex of Actinomyces recifensis var. lyticus, 2435 were compared. The lysis level was estimated by two parameters: lower optical density and protein yield percentage. It was found that ultrasound provided rather high levels of cell destruction reaching 60-68 per cent. The use of lysozyme enabled to destroy 16 per cent of the cells. The enzyme complex of strain 2435 showed high lytic activity with respect to the tested culture. For destroying dense staphylococcal suspensions it appeared necessary to study the effect of preliminary treatment of the cells with various chemical substances on their liability to the effect of the enzyme complex. It was demonstrated that treatment of the cells with 0.01-0.1 M cystein HCl solutions, 0.01-0.02 M sodium dodecylsulfate solutions or 0.05-0.5 M sodium hydroxide solutions increased 2.6-4.7-fold the cell liability to enzymatic hydrolysis. The studies enabled to develop conditions providing complete lysis of 10-percent staphylococcal cell suspension within 5 to 15 minutes under the effect of the lytic enzyme complex of strain 2435. A procedure for isolating cell walls was developed.     

Enzymatic lysis of staphylococci in relation to their species and strain properties

The lysoenzyme preparation from Streptomyces recifensis subsp. lyticus 2435 had a marked lytic activity against staphylococci of different species, spectra and antibiotic sensitivity. Certain strain differences of the cells in the population could be easily eliminated with increasing the dose. The preparation is a complex of lytic enzymes with high antimicrobial activity. It was concluded that it could be considered as a potentially promising chemotherapeutic agent for treatment of staphylococcal infections.     

Rifampicin use for potency stabilization in Rif(r) mutants of Streptomyces recifensis subsp. lyticus, an organism producing lytic enzymes]

Rif(r) mutants 1P-92 and 2P-15 were isolated as a result of selection of Streptomyces recifensis subsp. lyticus, an organism producing lytic enzymes. The effect of rifampicin on the biosynthetic potency of the mutants was studied. When added to the medium for cultivation of Rif(r) mutants 1P-92 and 2P-15 in the optimal concentrations (7.5 and 10.0 mcg/ml respectively), the antibiotic showed stabilizing effect on their potency in successive subcultures and recovered the initial potency of the old laboratory strains. Preliminary cultivation of strain 2P-15 after its storage for 6 years at a temperature of -20 degrees C made it possible to increase the efficiency of the initial potency recovery in the analytical selection.     

Use of enzyme preparation from Streptomyces recifensis subsp. lyticus for isolation of membrane substances from staphylococcal cells

A procedure for isolating staphylococcal membranes including preprocessing of the cells with 0.1 M solution of cysteine hydrochloride and subsequent differential centrifugation was developed. The procedure is based on enzymatic lysis with an enzyme preparation from Streptomyces recifensis subsp. lyticus 2435. The membrane preparations had oxidase and dehydrogenase activity and were characterized by a high specific activity of the membrane-bound ATPase. Determination of the cytochrome differential spectra revealed the presence of cytochromes a, b and o in the membrane preparations.     

Use of a lysoenzyme preparation from Streptomyces recifensis subsp. lyticus 2435 for isolating DNA from staphylococcal cells

It was shown that the preparation 2435 from Streptomyces recifensis subsp. lyticus, including a complex of bacteriolytic and concomitant enzymes provided lysis of thick staphylococcal suspensions within 15 to 20 minutes under optimal conditions after preliminary treatment of the cells with 0.1 M cystein-HCl. A procedure was developed for isolating DNA from the cells of staphylococci and other microorganisms based on enzymatic lysis. In terms of major physicochemical properties, the preparations of DNA were not inferior to the preparations of DNA isolated by the classical Marmur technique with Kirbi's deproteinization and had transforming activity. The developed procedure for isolation of DNA with using the lysoenzyme preparation widened the possibilities of investigating the genetics of staphylococci and other microorganisms.     

Sporulation and regeneration efficiency of phosphobacteria (<Emphasis Type="Italic">Bacillus megaterium</Emphasis> var <Emphasis Type="Italic">phosphaticum</Emphasis>)

Sporulation in Bacillus megaterium var phosphaticum (PB — 1) was induced using modified nutrient media. This modified medium induced sporulation within 36 h. After spore induction the spores were kept under refrigerated (5°C) and room temperature (32°C) for five months and survival of spores was studied at 15 days intervals by plating them in nutrient agar medium. It was observed that there was not much variation in the storage temperature (5°C & 32°C). The spore cells of Bacillus megaterium var phosphaticum (PB — 1) were observed up to five months of storage under refrigerated (5°C) and room temperature (32°C). Regeneration of spore cells into vegetative cells was studied in tap water, rice gruel, nutrient broth, sterile lignite and sterile water at different concentrations of spore inoculum. The multiplication of sporulated Bacillus megaterium var phosphaticum culture was fast and reached its maximum (29.5 × 10⁸ cfu ml⁻¹) in nutrient broth containing 5 per cent inoculum level.     

Optimization of the medium and cultivation conditions of Penicillium roquefortii f39 producing the diketopiperazine alkaloid roquefortine

We optimized the medium for cultivation of Penicillium roquefortii f39, a producer of roquefortine. In this medium, the roquefortine yield increased 1.5-2-fold. The increase in roquefortine content was associated with high biomass yield, but not with an increase in biosynthetic activity of the mycelium. Direct correlation was found between extracellular roquefortine concentration and amount of the inoculum. The introduction of sucrose into the growth medium allowed us to increase the concentration of roquefortine during fermentation to 90 mg/l.     

Role of iron ions in the production of hemolysin by toxigenic and nontoxigenic Vibrio cholerae of different serogroups

The hemolytic activity of ctx- and ctx+ V. cholerae, serogroups eltor and O39, in a medium free of FeCl3 was studied. During the cultivation in this medium, the strains of both V. cholerae serogroups proved to be capable of lysing sheep red blood cells in the Graig test, irrespective of the presence of ctx genes. The cultivation of V. cholerae ctx+ strains of both serogroups under such conditions facilitated the production of hemolysin with the same spectrum of lytic activity as hemolysin produced by ctx- strains.     

Influence of antitumor preparations on the concentration of free radicals in cells of Fusarium bulbigenum var. blasticola fungus during primary and tumour-like secondary growth

The fungus Fusarium bulbigenum var. blasticola in which secondary tumor-like formations appear under certain conditions in aging was used as a new test system to examine the action of antitumor preparations. Free radicals in the primary mycelium and tumor-like formations without introduction of preparations (control samples) and after the introduction of preparation into the cultivation medium of the fungus have been studied by EPR spectroscopy. The EPR spectra of the fungus represent single, somewhat asymmetrical lines with a width of deltaH = 0.4 divided by 0.6 mT and g = 2.0036 +/- 0.006, which enabled one to assign the paramagnetic centers observed to melanine radicals. It was found that the concentration of free radicals in tumor-like formations is always higher than in the primary mycelium, which may be related to intensive metabolism in tumor-like formations. It has been established that several antitumor preparations (fluorouracil, hydrea, methotrexat, and vepezide) completely inhibit the growth of tumor-like formations. Another group of preparations (cyclophosphanum, dacarbazin, adriablastin, and vinblastin), on the contrary, stimulate their growth, which is accompanied by an increase in the concentration of free radicals in cells of the primary mycelium and tumor-like formations. The preparations of the third group (mercaptopurine, lanvis, and farmorubicin), despite the increased level of free radicals in cells, have a weak inhibitory effect. It has been shown that, in the concentration range studied, vitamins B2, B12, C, and PP stimulate the growth of tumor-like formations, and, when used in combination with antitumor preparations, enhance or reduce the inhibitory properties of these preparations.     

Observations consistent with autocrine stimulation of hybridoma cell growth and implications for large-scale antibody production

Existence of autocrine growth factors (aGFs) may influence the serum requirement for growth of hybridoma cells and thus significantly influence process economics. For the murine hybridoma cell line S3H5/2bA2, critical inoculum density (cID) and serum requirement for growth were inversely related for cultivation in both T flasks and spinner flasks. In spinner flasks, an inoculum density of 10⁶ cells/ml was necessary for the cells to grow in RPMI 1640 medium without serum supplement, and an inoculum density of 10³ cell/ml was necessary in RPMI 1640 medium with 10% serum. In T flasks, where the local cell density is higher than in spinner flasks, an inoculum density of 10⁶ cells/ml was necessary for the cells to grow in RPMI 1640 medium without serum supplement, and an inoculum density of 1 cell/ml was also necessary in RPMI 1640 medium with 10% serum. Further, immobilized cells at high local cell density could grow under conditions where cells in T flasks at corresponding overall cell density could not grow. The cells at high inoculum density were less sensitive to shear induced by mechanical agitation than the cells at low inoculum density. Taken together these observations support the existence of secreted aGF(s) by the hybridoma cell line used. Since the specific MAb production rate was independent of cultivation method and inoculum density, the existence of autocrine growth factors would suggest that the use of immobilized cells should improve the economics of MAb production.     

Effect of an introduced inoculum on soil microbial diversity

Abstract A laboratory study was carried out to evaluate the impact of the introduction of genetically modified microorganisms into soil, in terms of effect on the diversity of the indigenous microflora, and at the process level. The impact on microbial phenotypic diversity, and on soil denitrification of an inoculum of a lux -modified denitrifier, Pseudomonas fluorescens , was examined using two different soil types in re-packed soil microcosms. The effect on diversity was found to depend on the soil pore size class into which the modified inoculum was introduced. The introduction of lux -modified cells into the 15–30 μm pore neck size class caused a short-term reduction in the overall microbial diversity. There was no significant change in the diversity of the indigenous microbial community, however, when cells were introduced into the 40–60 μm pore class. Partial chloroform fumigation proved useful in differentiating cell populations with respect to pore location. No change in diversity was observed when dead cells (either heat killed or glutaraldehyde fixed) were introduced into either pore size class. At the process level, the effect on soil denitrification of introduction of lux -modified P. fluorescens was not significantly different from introduction of the equivalent inoculum of the parental wild-type, although denitrification was found to be dependent upon both soil structure and pore size location of the introduced inoculum.     

A mechanistic analysis of the inoculum requirement for the cultivation of mammalian cells on microcarriers

For the cultivation of mammalian cells on microcarriers a minimum inoculum concentration is required to initiate cell attachment and subsequent cell growth. A critical cell number model has been proposed to elucidate the mechanism of the inoculum requirement. In this model it was hypothesized that after inoculation a critical number of cells per microcarrier is required for normal growth to occur; failure to acquire enough cells will impede cell growth. This critical cell number model was expressed mathematically and used to simulate cell distribution and growth on microcarriers under different cultivation conditions. By comparing the simulated growth kinetics with the experimental results, the actual critical cell number per microcarrier was identified. The critical number could be reduced by employing an improved medium for the cultivation.     

Effect of the inoculum on beta-fructofuranosidase synthesis by Aspergillus awamori]

The influence of the age and amount of the inoculum on the beta-fructofuranosidase synthesis by Aspergillus awamori 16 was studied during submerged cultivation. The level of the synthesis increased significantly when the 36-hour mycelial inoculum was used.     

Role of gramicidin S in the sporulation of the R+ and R- variants of Bacillus brevis var. G.-B.]

The effect of gramicidin S added to the cultivation medium on sporulation of the gramicidin S-producing P+ variant and gramicidin S-nonproducing P- variant of Bacillus brevis var. G.-B. was studied. Gramicidin S added to the synthetic medium with glucose in an amount of 30 and 100 microgram/ml 4 and 7 hours after inoculation with the vegetative cells of R- variant had no effect on the growth of the culture but retarded its sporulation. When gramicidin S was added in an amount of 100 microgram/ml 4 hours after inoculation, the sporulation rate of R- variant strongly decreased, rohile sporulation was not suppressed as it was noted before with respect to R+ variant. Active stimulation of Bacillus brevis var. G.-B. sporulation was observed after addition of gramicidin S 13 hours after development of R+ and R- variants without the antibiotic biosynthesis. Synthesis of gramicidin S by R+ strain was suppressed by the specific inhibitor beta-phenyl-beta-alanine. The amount of gramicidin S added to the medium during the sporulation process of R+ and R- variants decreased. On addition of 30 microgram/ml of the antibiotic it was practically not detectable when the culture showed the greatest number of the spores. Therefore, gramicidin S added to the medium is probably adsorbed by the cells of Bac. brevis var. G.-B. and affects sporulation of R- and R+ variants thus accelerating or retarding this process depending on the cultivation conditions.     

杏鲍菇菌丝体水溶性多糖提取及培养条件优化

以马铃薯葡萄糖综合培养基(PDP)为基础培养基,采用正交试验法优化杏鲍菇菌丝体多糖发酵条件,对接种量、摇床转速和培养时间等因素对多糖含量的影响进行了研究。采用水提醇沉法提取多糖,苯酚—浓硫酸法进行多糖含量测定。结果表明,最佳培养条件:接种量为每瓶1块直径为1cm的菌块,转速为140r/min,培养时间为8d。此时杏鲍菇菌丝体多糖含量最高,为75.1mg/g。     

微载体高密度培养Vero细胞的研究

微载体是动物细胞高密度培养的有效手段。首先在硅化的方瓶中对Cytodex 1、Cy-todex 3、Biosilon、Bellco Glass Microcarrier、CT-1、CT-3、MC-1、CT-28种国产和进口微载体进行了比较和筛选。确定以Biosilon作为Vero细胞高密度培养的首选微载体。用500mlWheaton搅拌瓶探索影响Vero细胞高密度培养的条件,表明50～60mg/ml的微载体浓度、1～2×10⁶/ml的细胞接种密度、适当的通气(95％O_2+5％CO₂)对该细胞的高密度培养具有重要意义。在200ml培养体积的Wheaton搅拌瓶中,微载体浓度为50～60mg/ml,细胞接种密度为9.24×10⁵/ml,搅拌速度为65～85r/min,经25d培养,Vero细胞密度可达2.34×10⁷/ml,表明50～60mg/ml的微载体浓度对培养细胞没有毒性。接着在1.5L CelliGen生物反应器中进行培养,细胞接种密度为4.98×10⁵/ml,培养体积为1.2L,日灌流量从0.20L逐渐加大到3.65L,经22d连接灌流培养,最终细胞密度可达2.05×10⁷/ml。     

Effect of gramicidin C on the formation and germination of Bacillus brevis var. GB (P+-variant) spores]

The effect of gramicidin C added to the medium at various periods of cultivation in concentrations of 20, 40 and 100 gamma/ml on sporulation of P+-variant of Bac. brevis var. GB was studied. The most effective increase in the sporulation rate and percentage of the cells germinating into the spores was observed on addition of the antibiotic to the medium in amounts of 20 and 40 gamma/ml in 13 hours of the culture development. The amount of gramicidin C during sporulation decreased and partially passed into the spores which did not differ after germination from those of P+-variant grown on the synthetic medium with glucose and without preliminary addition of the antibiotic. Addition of gramicidin C in an amount of 100 gamma/ml at the end of the lag phase, i.e. 4 hours after the culture inoculation suppressed sporulation and had no effect on growth of the cells of its own producing organism.