Effects of exogenous ammonia or free amino acids on proteolytic activity and protein breakdown products in Streptococcus bovis,Prevotella albensis,and Butyrivibrio fibrisolvens

We studied in details the ammonia or free amino acids (AA) effects on proteolytic activity of three ruminal bacteria: enzymatic activities and protein breakdown products were measured at the end of the exponential growth phase. In Streptococcus bovis the simultaneous uptake of ammonia and probably small peptides induced a decline in total proteolytic activity as a result of changes in endopeptidasic activities. With free AA, the tendency for the endopeptidasic activities to specialise was more evident and the total proteolytic activity decreased too. In Prevotella albensis, the inhibition of proteolysis with free AA was linked to the disappearance of free endopeptidases, to the specialization of cell-associated endopeptidases and to the decrease in exopeptidases. The decrease of proteolysis in P. albensis when ammonia was added was more difficult to interpret. With ammonia or AA Butyrivibro fibrisolvens developed a distinct behavior of those expressed by the other species: the increase of the total proteolytic activity could be explained by a better balance of the endopeptidases expressed. It then clearly appeared that the expression of the proteolytic activities are linked to the nature and/or to the quantity of the nitrogen source. This leads each species to adopt its own nutritional strategy in order to adapt to the environmental conditions of the ruminal ecosystem. Received: 2 July 2001 / Accepted: 28 September 2001     

Proteolytic Activity of Streptococcus bovis Cultured Alone or Associated with Prevotella albensis,on two kinds of Protein Substrates: Casein or Pea Proteins

This work aims at studying in detail, the interaction between two major bacterial species involved in the ruminal proteolysis, Streptococcus bovis and Prevotella albensis. We chose to investigate this interaction by examining their behaviour both alone in monoculture and together in co-culture on two kinds of protein substrates. Comparison of the behaviour of S. bovis and P. albensis in monoculture, in terms of growth and total proteolytic activity with protein as the sole source of nitrogen, showed that S. bovis grew more rapidly and developed a higher total proteolytic activity. A higher proportion of short peptides was generated at the end of the exponential growth phase on pea protein medium whereas peptide accumulation did not appear on casein medium for both species; peptides of casein origin would be used more by species in monoculture than those of pea origin. S. bovis predominated in number in both co-cultures but the balance between species was greater on pea proteins (S. bovis: 64% and P. albensis: 36%) than on casein (S. bovis: 86% and P. albensis: 14%). At the same time, the decrease of the proteolytic activity was smaller on pea protein medium than that with casein and the use of proteolysis products was facilitated; peptides liberated by pea protein hydrolysis accumulated less than those obtained from casein. Moreover, the diversity of the endopeptidases expressed increased on pea proteins and the exopeptidase activities remained rather constant whereas they were highly stimulated on casein medium. All the results obtained in co-culture on pea proteins allowed us to suggest that a greater synergism occurred between the two species for the breakdown of proteins.     

Proteolytic Activities of the Starch-Fermenting Ruminal Bacterium, Streptococcus bovis

The objective of this study was to characterize the extracellular proteolytic activity of Streptococcus bovis. Strains KEG, JB1, NCFB 2476, and K11.21.09.6C produced very similar large molecular weight (160–200 kDa) extracellular proteases that were specifically inhibited by PMSF, a serine protease inhibitor. Further experiments with S. bovis KEG indicated that cultures grown with casein as the sole added N source produced the greatest level of proteolytic activity, and the level of proteolytic activity was independent of growth rate. Clarified ruminal fluid (CRF) decreased proteolytic activity by 54% compared with cultures grown with casein alone, and addition of exogenous peptides and carbohydrates (CHO) to the CRF further reduced the level of proteolytic activity by 44% and 52%, respectively. These results suggested that the proteolytic activity of S. bovis KEG was modulated by available N source and that the proteolytic activity was present for reasons other than providing N for growth. The role of S. bovis in ruminal proteolysis requires further definition, but phenotypic similarity among some ruminal strains would suggest a common niche in ruminal proteolysis. The uniformity of proteolytic activities could make S. bovis a prime candidate for manipulation in ruminal proteolysis control strategies. Received: 12 January 1999 / Accepted: 19 May 1999     

Digestion of Barley, Maize, and Wheat by Selected Species of Ruminal Bacteria

Differences in the digestion of barley, maize, and wheat by three major ruminal starch-digesting bacterial species, Streptococcus bovis 26, Ruminobacter amylophilus 50, and Butyrivibrio fibrisolvens A38, were characterized. The rate of starch digestion in all cereal species was greater for S. bovis 26 than for R. amylophilus 50 or B. fibrisolvens A38. Starch digestion by S. bovis 26 was greater in wheat than in barley or maize, whereas starch digestion by R. amylophilus 50 was greater in barley than in maize or wheat. B. fibrisolvens A38 digested the starch in barley and maize to a similar extent but was virtually unable to digest the starch in wheat. The higher ammonia concentration in cultures of B. fibrisolvens A38 when grown on wheat than when grown on barley or maize suggests that B. fibrisolvens A38 utilized wheat protein rather than starch. Scanning electron microscopy revealed that B. fibrisolvens A38 initially colonized cell wall material, while S. bovis 26 randomly colonized the endosperm and R. amylophilus 50 preferentially colonized starch granules. There was subsequent colonization but only superficial digestion of wheat starch granules by B. fibrisolvens A38. Variation in the association between starch and protein within the endosperm of cereal grains contributes to the differential effectiveness with which amylolytic species can utilize cereal starch.     

Diversity of Extracellular Proteolytic Activities Among Prevotella Species from the Rumen

The current research was aimed at comparing proteolytic activities among ruminal Prevotella spp. Growth rates of Prevotella sp. 2202, Prevotella ruminicola D31d, P. brevis GA33, P. albensis M384, and P. bryantii B₁4 varied with N source, and no one N source produced the fastest growth in all species. Proteolytic activity was greatest with casein compared with peptides, AA, and NH₄Cl in all species. Proteolytic activity of Prevotella sp. 2202, P. brevis GA33, and P. bryantii B₁4 was modulated by N source. With gelatin co-polymerized SDS-PAGE, the extracellular activities of the Prevotella spp. showed wide variation in number, size, and type of proteases. Prevotella sp. 2202 and P. albensis M384 produced metalloproteases of low molecular weight (40 kDa). P. ruminicola D31d produced one cysteine protease (100–200 kDa) and two metalloproteases (90–100 kDa). P. brevis GA33 generated a diffuse clearing zone (95–160 kDa) containing serine, cysteine, and metalloproteases. P. bryantii B₁4 produced a metalloprotease greater than 200 kDa in size. The molecular sizes provided are estimations and served only to differentiate among the bacterial species in this study. Large variations in proteolytic activities among species and the known genetic diversity of the Prevotella taxon suggested that targeting this bacterial assemblage for genetic manipulation in order to alter the bacterial impact on ruminal protein degradation would be difficult. Received: 12 January 1999 / Accepted: 19 May 1999     

Influence of Dipeptidyl Peptidase Inhibitors on Growth,Peptidase Activity,and Ammonia Production by Ruminal Microorganisms

The aim was to investigate known and potential new inhibitiors of dipeptidyl peptidases (DPP) for their effects on ruminal microorganisms. Gly-Phe diazomethylketone (GPD), Ala-Ala chloromethylketone (AAC), benserazide (DL-serine 2-(2,3,4- trihydroxybenzyl) hydrazide), and diprotin A (Ile-Pro-Ile) inhibited DPP activities of Prevotella albensis, P. ruminicola, P. bryantii, P. brevis, and mixed ruminal microorganisms, though incompletely and, except for diprotin A, without absolute specificity for any of the peptidases. Leucine aminopeptidase activity of Streptococcus bovis was also inhibited by GPD and benserazide. The inhibitors had no effect on the growth of the bacteria, except for GPD, which inhibited growth of P. albensis when only peptides were available for growth. Benserazide had some inhibitory effects on the growth of Megasphaera elsdenii and Prevotella spp., even in the absence of peptides. The predatory activity of ciliate protozoa on bacteria was unaffected by DPP inhibitors. Ammonia production from casein by mixed ruminal microorganisms was inhibited significantly (P < 0.05) by AAC (29% inhibition) and benserazide (33%). It was concluded that DPP inhibitors can influence the rate of NH₃ production in the rumen and may form the basis for developing protein-sparing feed additives for ruminants.     

The Ability of “Low G + C Gram-Positive” Ruminal Bacteria to Resist Monensin and Counteract Potassium Depletion

Gram-negative ruminal bacteria with an outer membrane are generally more resistant to the feed additive, monensin, than Gram-positive species, but some bacteria can adapt and increase their resistance. 16S rRNA sequencing indicates that a variety of ruminal bacteria are found in the “low G + C Gram-positive group,” but some of these bacteria are monensin resistant and were previously described as Gram-negative species (e.g., Selenomonas ruminantium and Megasphaera elsdenii). The activity of monensin can be assayed by its ability to cause potassium loss, and results indicated that the amount of monensin needed to catalyze half maximal potassium depletion (K_d) from low G + C gram-positive ruminal bacteria varied by as much as 130-fold. The K_d values for Butyrivibrio fibrisolvens 49, Streptococcus bovis JB1, Clostridium aminophilum F, S. ruminantium HD4, and M. elsdenii B159 were 10, 65, 100, 1020, and 1330 nM monensin, respectively. B. fibrisolvens was very sensitive to monensin, and it did not adapt. S. bovis and C. aminophilum cultures that were transferred repeatedly with sub-lethal doses of monensin had higher K_d values than unadapted cultures, but the K_d was always less than 800 nM. S. ruminantium and M. elsdenii cells were highly resistant (K_d > 1000 nM), and this resistance could be explained by the ability of these low G + C Gram-positive bacteria to synthesize outer membranes. Received: 14 May 1999 / Accepted: 24 June 1999     

Effects of vanillin, quillaja saponin, and essential oils on in vitro fermentation and protein-degrading microorganisms of the rumen

This study investigated the effects of vanillin on methanogenesis and rumen fermentation, and the responses of ruminal protein-degrading bacteria to vanillin (at concentrations of 0, 0.76 and 1.52 g/L), essential oils (clove oil, 1 g/L; origanum oil, 0.50 g/L, and peppermint oil, 1 g/L), and quillaja saponin (at concentration of 0 and 6 g/L) in vitro. Methane production, degradabilities of feed substrate, and ammonia concentration decreased linearly with increasing doses of vanillin. Concentration of total volatile fatty acids also decreased, whereas proportion of butyrate tended to increase linearly with increasing doses of vanillin. Protozoa population decreased, but abundances of Ruminococcus flavefaciens, Prevotella bryantii, Butyrivibrio fibrisolvens, Prevotella ruminicola, Clostridium aminophilum, and Ruminobacter amylophilus increased with increasing doses of vanillin. Origanum and clove oils resulted in lower ammonia concentrations compared to control and peppermint oil. All the tested essential oils decreased abundances of protozoa, Selenomonas ruminantium, R. amylophilus, P. ruminicola and P. bryantii, with the largest decrease resulted from origanum oil followed by clove oil and peppermint oil. The abundances of Megasphaera elsdenii, C. aminophilum, and Clostridium sticklandii were deceased by origanum oil while that of B. fibrisolvens was lowered by both origanum and clove oils. Saponin decreased ammonia concentration and protozoal population, but increased the abundances of S. ruminantium, R. amylophilus, P. ruminicola, and P. bryantii, though the magnitude was small (less than one log unit). The results suggest that reduction of ammonia production by vanillin and saponin may not be caused by direct inhibition of major known proteolytic bacteria, and essential oils can have different inhibitory effects on different proteolytic bacteria, resulting in varying reduction in ammonia production.     

Effect of the Microbial Feed Additive Saccharomyces cerevisiae CNCM I-1077 on Protein and Peptide Degrading Activities of Rumen Bacteria Grown In Vitro

We investigated the potential of the ruminant feed additive Saccharomyces cerevisiae CNCM I-1077 on protein and peptide degrading activities of the rumen bacterial species Prevotella albensis M384, Streptococcus bovis 20480, and Butyrivibrio fibrisolvens 3071 grown in vitro. Alive or heat-killed yeast cells were added to bacterial cultures in a complex casein–glucose medium. After incubation of the cultures at 39°C under O₂-free CO₂, peptidase activities were determined in the absence or in the presence of yeasts. Protease activities were detected after PAGE in gelatin-copolymerized gels. In co-incubations of bacteria and live S. cerevisiae I-1077, proteinase activities were reduced compared to the activities in the bacterial monocultures. Measurement of peptidase activities and microbial enumerations in the co-incubations suggested that live yeasts and bacteria interacted in a competitive way, leading to a decrease in peptidase activities. The mechanism responsible for such an effect could be mainly a competition for substrate utilization, but the release of small competitive peptides by the yeast cells is also likely to be implicated.     

Antimicrobial activity of Brazilian propolis extracts against rumen bacteria in vitro

The antimicrobial activity of three Brazilian propolis extracts was evaluated on bacterial strains representing major rumen functional groups. The extracts were prepared using different concentrations of propolis and alcohol, resulting in different phenolic compositions. The propolis extracts inhibited the growth of Fibrobacter succinogenes S85, Ruminococcus flavefaciens FD-1, Ruminococcus albus 7, Butyrivibrio fibrisolvens D1, Prevotella albensis M384, Peptostreptococcus sp. D1, Clostridium aminophilum F and Streptococcus bovis Pearl11, while R. albus 20, Prevotella bryantii B₁4 and Ruminobacter amylophilus H18 were resistant to all the extracts. The inhibited strains showed also different sensitivity to propolis; the hyper-ammonia-producing bacteria (C. aminophilum F and Peptostreptococcus sp. D1) being the most sensitive. Inhibition of hyper-ammonia-producing bacteria by propolis would be beneficial to the animal. The extract containing the lowest amount of phenolic compounds (LLOS C3) showed the lowest antimicrobial activity against all the bacteria. The major phenolic compounds identified in the propolis extracts (naringenin, chrysin, caffeic acid, p-coumaric acid and Artepillin C) were also evaluated on four sensitive strains. Only naringenin showed inhibitory effect against all strains, suggesting that naringenin is one of the components participating to the antibacterial activity of propolis.     

Regulation of avilamycin biosynthesis in Streptomyces viridochromogenes: effects of glucose, ammonium ion, and inorganic phosphate

Effects of glucose, ammonium ions and phosphate on avilamycin biosynthesis in Streptomyces viridochromogenes AS4.126 were investigated. Twenty grams per liter of glucose, 10 mmol/L ammonium ions, and 10 mmol/L phosphate in the basal medium stimulated avilamycin biosynthesis. When the concentrations of glucose, ammonium ions, and phosphate in the basal medium exceeded 20 g/L, 10 mmol/L, and 10 mmol/L, respectively, avilamycin biosynthesis greatly decreased. When 20 g/L glucose was added at 32 h, avilamycin yield decreased by 70.2%. Avilamycin biosynthesis hardly continued when 2-deoxy-glucose was added into the basal medium at 32 h. There was little influence on avilamycin biosynthesis with the addition of the 3-methyl-glucose (20 g/L) at 32 h. In the presence of excess (NH₄)₂SO₄ (20 mmol/L), the activities of valine dehydrogenase and glucose-6-phosphate dehydrogenase were depressed 47.7 and 58.3%, respectively, of that of the control at 48 h. The activity of succinate dehydrogenase increased 49.5% compared to the control at 48 h. The intracellular adenosine triphosphate level and 6-phosphate glucose content of S. viridochromogenes were 128 and 129%, respectively, of that of the control at 48 h, with the addition of the 40 mmol/L of KH₂PO₄. As a result, high concentrations of glucose, ammonium ions, and inorganic phosphate all led to the absence of the precursors for avilamycin biosynthesis and affected antibiotic synthesis.     

Cloning and expression of an amylase gene from Streptococcus bovis in Escherichia coli

An amylase gene was identified in a Streptococcus bovis 033 gtWESB genomic library. Using a starch overlay and a Congo red-iodine staining procedure, amylase positive clones could be identified by zones of clearing. Ten amylase positive clones were identified using this procedure. The clone chosen for further study, SBA105, contained an insert of approximately 7.5 kb. The insert was mapped, and subcloning localized the amylase gene to a region of approximately 3.1 kb. Cloning of the 3.1 kb amylase fragment into pUC18 in both orientations revealed that the amylase gene was transcribed from its own promoter. Amylase activity was expressed by the Escherichia coli subclones and was found to be largely associated with the cytoplasmic fraction. Southern hybridization of genomic DNA from the amylolytic strains, S. bovis 033, S. bovis 077, Butyrivibrio fibrisolvens 194 and 195 revealed a single hybridizing band in S. bovis 033 DNA only. This indicates that the amylase gene from S. bovis may differ from the amylases of these other amylolytic bacteria.     

Amino acid abundance and proteolytic potential in North American soils

Studies of nitrogen (N) cycling have traditionally focused on N mineralization as the primary process limiting plant assimilation of N. Recent evidence has shown that plants may assimilate amino acids (AAs) directly, circumventing the mineralization pathway. However, the general abundance of soil AAs and their relative importance in plant N uptake remains unclear in most ecosystems. We compared the concentrations and potential production rates of AAs and NH₄ ⁺, as well as the edaphic factors that influence AA dynamics, in 84 soils across the United States. Across all sites, NH₄ ⁺ and AA-N comprised similar proportions of the total bioavailable N pool (~20%), with NO₃ ⁻ being the dominant form of extractable N everywhere but in tundra and boreal forest soils. Potential rates of AA production were at least comparable to those of NH₄ ⁺ production in all ecosystems, particularly in semi-arid grasslands, where AA production rates were six times greater than for NH₄ ⁺ (P < 0.01). Potential rates of proteolytic enzyme activity were greatest in bacteria-dominated soils with low NH₄ ⁺ concentrations, including many grassland soils. Based on research performed under standardized laboratory conditions, our continental-scale analyses suggest that soil AA and NH₄ ⁺ concentrations are similar in most soils and that AAs may contribute to plant and microbial N demand in most ecosystems, particularly in ecosystems with N-poor soils.     

Alternative schemes of butyrate production in Butyrivibrio fibrisolvens and their relationship to acetate utilization, lactate production, and phylogeny

Butyrivibrio fibrisolvens strains D1 and A38 produced little lactate, but strain 49 converted as much as 75% of its glucose to lactate. Strain 49 had tenfold more lactate dehydrogenase activity than strains D1 or A38, this activity was stimulated by fructose 1,6-bisphosphate, and had a pH optimum of 6.25. A role for fructose 1,6-bisphosphate or pH regulation of lactate production in strain 49 was, however, contradicted by the observations that very low concentrations (< 0.2 mM) of fructose 1,6-bisphosphate gave maximal activity, and continuous cultures did not produce additional lactate when the pH was decreased. The lactate production of strain 49 was clearly inhibited by the presence of acetate in the growth medium. When strain 49 was supplemented with as little as 5 mM acetate, lactate production decreased dramatically, and most of the glucose was converted to butyrate. Strain 49 did not possess butyrate kinase activity, but it had a butyryl-CoA/acetate CoA transferase that converted butyryl-CoA directly to butyrate, using acetate as an acceptor. The transferase had a low affinity for acetate (K _m of 5 mM), and this characteristic explained the acetate stimulation of growth and butyrate formation. Strains D1 and A38 had butyrate kinase but not butyryl-CoA/acetate CoA transferase, and it appeared that this difference could explain the lack of acetate stimulation and lactate production. Based on these results, it is unlikely that B. fibrisolvens would ever contribute significantly to the pool of ruminal lactate. Since relatives of strain 49 (strains Nor37, PI-7, VV1, and OB156, based on 16S rRNA sequence analysis) all had the same method of butyrate production, it appeared that butyryl-CoA/acetate CoA transferase might be a phylogenetic characteristic. We obtained a culture of strain B835 (NCDO 2398) that produced large amounts of lactate and had butyryl-CoA/acetate CoA transferase activity, but this strain had previously been grouped with strains A38 and D1 based on 16S rRNA sequence analysis. Our strain B835 had a 16S rRNA sequence unique from the one currently deposited in GenBank, and had high sequence similarity with strains 49 and Nor37 rather than with strains A38 or D1. Received: 3 December 1998 / Accepted: 18 February 1999     

Development of the CELLOP optimisation model for plant cell cultivation

A mathematical computer-aided model CELLOP was constructed in which the desirability functions in a three-dimensional experimental design can be used to find the optimal growing conditions for plant cells. CELLOP is intended for the optimisation of 3 to 6 physical, chemical, or biological variables in the cultivation conditions of plant cell cultures. The model was used to optimise the culturing conditions (calcium, inorganic nitrogen, and sucrose concentrations) for coumarin-producing, spontaneously embryogenic cell lines of angelica Angelica archangelica L. subsp. archangelica and hogs fennel Peucedanum palustre (L.) Moench. For A. archangelica the overall optimum concentrations were 0.47 mM Ca²⁺, 5.06 mM NO₃ ⁻, 0.40 mM NH₄ ⁺, and 96.25 mM sucrose. The dry mass was 24.7 % higher and the coumarin content 40.5 % higher than those achieved in the standard 75 % Gamborg B5 medium. For A. archangelica the highest embryogenic activity was reached in the media containing 1.25 mM Ca²⁺. In the case of P. palustre the overall optimum concentrations were 1.60 mM Ca²⁺, 2.84 mM NO³ ₋, 0.23 mM NH₄ ⁺, and 85.10 mM sucrose. For P. palustre the dry mass production increased by 61.8 % and the coumarin content by 58.1 % compared to the values achieved in the Gamborg B5 medium. For P. palustre the highest embryogenic activity was reached in the presence of 50.0 mM NO₃ ⁻ and 4.01 mM NH₄ ⁺.     

不同磷、硫及二氧化碳浓度对标志链带藻生长和碳水化合物积累的影响

【目的】为了研究不同磷、硫及二氧化碳浓度对标志链带藻(Desmodesmus insignis)生长与碳水化合物积累的影响,本实验以改良BG11培养基为基础,设计了8种不同初始K_2HPO_4浓度、8种不同初始MgSO_4浓度及4种二氧化碳浓度培养标志链带藻。【方法】采用干重法和苯酚-硫酸法分别测定其生物质浓度与总碳水化合物的含量。【结果】实验结果显示,在高磷浓度(0.460 mmol/L)下生物量达到最高为6.37 g/L,磷浓度为0.230 mmol/L (对照组)时总碳水化合物含量及单位体积产率达到最高,分别为45.40%(%干重)和0.20 g/(L·d)。不同初始MgSO_4浓度实验结果显示,高硫浓度有利于标志链带藻生长及碳水化合物的积累,生物量、总碳水化合物含量及单位体积产率分别在硫浓度为1.217 mmol/L、0.609 mmol/L和1.824 mmol/L时达到最高,分别为7.02 g/L、51.6%(%干重)及0.26 g/(L·d)。当二氧化碳浓度为3%(V/V)时,标志链带藻生物量、总碳水化合物含量及单位体积产率均达到最高,分别为6.81 g/L、44.03%和0.20 g/(L·d)。【结论】因此,磷浓度为0.230 mmol/L、硫浓度为1.824 mmol/L和二氧化碳浓度为3%时最有利于标志链带藻生长及碳水化合物的积累。     

Regioselective hydroxylation of cholecalciferol,cholesterol and other sterol derivatives by steroid C25 dehydrogenase

Steroid C25 dehydrogenase (S25DH) from Sterolibacterium denitrificans Chol-1S is a molybdenum oxidoreductase belonging to the so-called ethylbenzene dehydrogenase (EBDH)-like subclass of DMSO reductases capable of the regioselective hydroxylation of cholesterol or cholecalciferol to 25-hydroxy products. Both products are important biologically active molecules: 25-hydroxycholesterol is responsible for a complex regulatory function in the immunological system, while 25-hydroxycholecalciferol (calcifediol) is the activated form of vitamin D₃ used in the treatment of rickets and other calcium disorders. Studies revealed that the optimal enzymatic synthesis proceeds in fed-batch reactors under anaerobic conditions, with 6–9 % (w/v) 2-hydroxypropyl-β-cyclodextrin as a solubilizer and 1.25–5 % (v/v) 2-methoxyethanol as an organic co-solvent, both adjusted to the substrate type, and 8–15 mM K₃[Fe(CN)₆] as an electron acceptor. Such thorough optimization of the reaction conditions resulted in high product concentrations: 0.8 g/L for 25-hydroxycholesterol, 1.4 g/L for calcifediol and 2.2 g/L for 25-hydroxy-3-ketosterols. Although the purification protocol yields approximately 2.3 mg of pure S25DH from 30 g of wet cell mass (specific activity of 14 nmol min⁻¹ mg⁻¹), the non-purified crude extract or enzyme preparation can be readily used for the regioselective hydroxylation of both cholesterol and cholecalciferol. On the other hand, pure S25DH can be efficiently immobilized either on powder or a monolithic silica support functionalized with an organic linker providing NH₂ groups for enzyme covalent binding. Although such immobilization reduced the enzyme initial activity more than twofold it extended S25DH catalytic lifetime under working conditions at least 3.5 times.

     

Enzymes of ammonium assimilation inStreptomyces avermitilis

Glutamine synthetase (GS), glutamate synthase (GOGAT), glutamate dehydrogenase (GDH), alanine dehydrogenase (ADH) and alanine aminotransferase (GPT) were detected in the cell-free homogenate ofStreptomyces avermitilis grown in a defined medium containing ammonium sulfate as the only nitrogen source. At an initial NH₄ ⁺ concentration of 7.5 mmol/L, high activities of GS, GOGAT and GDH were found while that of ADH was low. The ADH activity was markedly increased at initially millimolar NH₄ ⁺ concentrations. In some characteristics of its NH₄ ⁺-assimilating system (e.g. control of some enzyme activities, the NADPH specificity of GOGAT, the presence of alanine aminotransferase),S. avermitilis differs from other known streptomycetes.     

Deoxyribonuclease Activity in Selenomonas ruminantium, Streptococcus bovis, and Bacteroides ovatus

Six Selenomonas ruminantium strains (132c, JW13, SRK1, 179f, 5521c1, and 5934e), Streptococcus bovis JB1, and Bacteroides ovatus V975 were examined for nuclease activity as well as the ability to utilize nucleic acids, ribose, and 2-deoxyribose. Nuclease activity was detected in sonicated cells and culture supernatants for all bacteria except S. ruminantium JW13 and 179f sonicated cells. S. ruminantium strains were able to utilize several deoxyribonucleosides, while S. bovis JB1 and B. ovatus V975 showed little or no growth on all deoxyribonucleosides. When S. ruminantium strains 5934e, 132c, JW13, and SRK1 were incubated in medium that contained 15 mm ribose, the major end products were acetate, propionate, and lactate. S. ruminantium 5521c1 and S. bovis JB1 did not grow on ribose, and none of the S. ruminantium strains or S. bovis JB1 grew on 15 mm 2-deoxyribose. In contrast, B. ovatus V975 was able to grow on ribose and 2-deoxyribose. In conclusion, all S. ruminantium strains, S. bovis JB1, and B. ovatus V975 had nuclease activity. However, not all bacteria were able to utilize deoxyribonucleosides, ribose, or 2-deoxyribose. Received: 9 February 2000 / Accepted: 27 March 2000     

不同氮源及其浓度对标志链带藻合成淀粉和油脂的影响北大核心CSCD

【目的】以标志链带藻(Desmodesmus insignis)为实验材料,研究不同氮源及其浓度对该藻生长、总脂和淀粉(碳水化合物)含量的影响,为该藻在生物能源方面的应用提供一定的理论依据。【方法】以硝酸钠、碳酸氢铵或尿素为氮源,5个氮浓度(3、6、9、12和18 mmol/L)的BG-11培养基培养标志链带藻,采用干重法测定生物质浓度、重量法测定总脂、苯酚-硫酸法测定、总碳水化合物和淀粉的含量。【结果】标志链带藻在3种氮源下均能很好的生长。最高油脂含量出现在3 mmol/L硝酸钠实验组,达到32.61%(d.w)。当18 mmol/L碳酸氢铵作为氮源时,总碳水化合物与淀粉的含量以及产率都达到最高,分别为56.54%(d.w)和55.33%(d.w)、0.24和0.23 g/(L·d)。以尿素为氮源时,其生物质浓度和各组分含量与其它氮源实验组差别不大,均有利于该藻的生长及各生化组分含量的积累。【结论】以该藻种生产生物能源的成本等综合考虑,以18 mmol/L碳酸氢铵和尿素为氮源培养标志链带藻最优。