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PubMed2005

[High level expression of PNGase F in Escherichia coli and its bioactivities].

In order to obtain active recombinant PNGase F in Escherichia coli, a prokaryotic expression vector pET28a/PNGase F was constructed. Amplification of PNGase F was obtained using PCR technique employing suitable primers designed according to the PNGase F gene sequence from Flavobacterium nmeningosepticum. The expression of PNGase F gene in LB medium or M9 medium led to the formation of inclusion body and soluble protein, respectively. The refolding of the denatured inclusion body was successful by gradual dilution. Further purification of the refolded protein and soluble crude extract from M9 medium with Ni2+ -NTA argarose resulted a 90% purified PNGase F. The purified protein catalyzed the complete and intact cleavage of N-linked oligosaccharides from various glycoproteins. The efficiency of this cleavage was affected by the substrate status in the reaction system. In summary, we have developed an enzyme production system where PNGase F was over-expressed in recombinant E. coli. This system can provide more than 15 mg/L purified active PNGase F. This purified active PNGase F can be used as tools in analyzing the oligosaccharide structure.

Bacterial Proteins↗

PubMed1991

[Production of hydroxy and oxo fatty acids by microorganisms as a model of adipocere formation].

Microbial synthesis of hydroxy and oxo fatty acids was studied as one of the model of experimental adipocere formation. Conversion of various fatty acids into 10-hydroxy and 10-oxo fatty acids by Micrococcus luteus was also studied. Fatty acids possessing cis-9-unsaturated forms were converted into 10-hydroxy and 10-oxo fatty acids. On the other hand, enoic acids possessing trans-9-unsaturated form or the ones which do not have double bond at the 9 -carbon position were inactive as substrates. 10-Hydroxypalmitic and 10-hydroxystearic acids were converted into the corresponding 10-oxo fatty acids but the 10-oxo fatty acids were inactive as substrates. To study the mechanism of the formation of 10-hydroxy and 10-oxo fatty acids, the crude enzyme preparation from Flavobacterium meningosepticum solubilized by sonication was used. The mechanism of hydration and dehydrogenation was proved by gas chromatography-mass spectrometry of 10-hydroxy and 10-oxo fatty acids produced from oleic acid in the presence of D2O or H218O. These results indicate that oleic acid is hydrated to 10-hydroxystearic acid at first and then, the latter compound is dehydrogenated to 10-oxostearic acid.

Fatty Acids↗

PubMed1990

Purification and characterization of a novel heparinase.

A unique heparinase was isolated from a recently discovered Gram-negative soil bacterium. The enzyme (heparinase III) was purified by hydroxylapatite chromatography, chromatofocusing, and gel permeation chromatography. The enrichment was 48x, and the specific activity of catalytically pure heparinase was 127 IU/mg of protein. Similar to the heparinase I from Flavobacterium heparinum, heparinase III also degrades heparin to mainly disaccharide fragments. It is specific for heparin and also breaks down heparan sulfate, but not hyaluronic acid and chondroitin sulfate. Heparinase III, however, differs markedly from heparinase I in several other aspects: it has a higher molecular mass (94 versus 43 kDa), pI (9.2 versus 8.5), its Km and kcat are different, and it has a higher energy of activation (15.6 versus 6.3 kcal/mol). Optimal activity was also found at higher pH (7.6 versus 6.5) and temperature (45 versus 37 degrees C). Furthermore, the amino acid composition of heparinase III is quite different from that of heparinase I.

Amino Acids↗

PubMed1990

Cloning and expression of peptide-N4-(N-acetyl-beta-D-glucosaminyl)asparagine amidase F in Escherichia coli.

The peptide-N4-(N-acetyl-beta-D-glucosaminyl) asparagine amidase F (PNGase F) gene from Flavobacterium meningosepticum was cloned into a high copy number Escherichia coli plasmid. Levels of PNGase F activity produced in cultures of the recombinant strain were up to 100-fold higher than those obtained in cultures of F. meningosepticum. The complete PNGase F gene sequence was determined. Comparison of the predicted amino acid sequence of pre-PNGase F to the N-terminal sequence of the native mature enzyme indicates that the protein is synthesized with a 40-amino acid signal sequence that is removed during secretion in F. meningosepticum. The recombinant PNGase F produced in E. coli is a mixture of products comprised predominantly of two proteins with molecular masses of 36.3 and 36.6 kDa. These proteins have a higher apparent molecular mass than the 34.7-kDa native enzyme. N-terminal amino acid sequencing demonstrated that these higher molecular mass products result from cleavage of the pre-PNGase F in E. coli upstream of the native N terminus. The PNGase F gene was engineered to encode a preenzyme that was processed in E. coli to give an N terminus identical to that of the native enzyme. Purified preparations of this form of recombinant PNGase F were shown to be suitable for glycoprotein analyses since they possess no detectable endo-beta-N-acetylglucosaminidase F, exoglycosidase, or protease activity.

Amidohydrolases↗

PubMed1989

[Bacteriologic investigation of the hospital air-conditioning water].

We have isolated a pathogenic strain of LDB from the water of the hospital cooling-water storage tank for air-conditioning system. At the same time, other bacteria were also isolated with Pseudomonas and Flavobacterium being predominant, most of these organisms in the water of air-conditioning system were resistant to antibacterial drugs. A proposal to improve architectural design and to sterilize air-conditioning water was suggested.

Air Conditioning↗

PubMed1989

The fokI restriction-modification system. I. Organization and nucleotide sequences of the restriction and modification genes.

A DNA fragment that carried the genes coding for FokI endonuclease and methylase was cloned from the chromosomal DNA of Flavobacterium okeanokoites, and the coding regions were assigned to the nucleotide sequence by deletion analysis. The methylase gene was 1,941 base pairs (bp) long, corresponding to a protein of 647 amino acid residues (Mr = 75,622), and the endonuclease gene was 1,749 bp long, corresponding to a protein of 583 amino acid residues (Mr = 66,216). The assignment of the methylase gene was further confirmed by analysis of the N-terminal amino acid sequence. The endonuclease gene was downstream from the methylase gene in the same orientation, separated by 69 bp. The promoter site, which could be recognized by Escherichia coli RNA polymerase, was upstream from the methylase gene, and the sequences adhering to the ribosome-binding sequence were identified in front of the respective genes. Analysis of the gene products expressed in E. coli cells by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the molecular weights of both enzymes coincided well with the values estimated from the nucleotide sequences, and that the monomeric forms were catalytically active. No significant similarity was found between the sequences of the two enzymes. Sequence comparison with other related enzymes indicated that FokI methylase contained two copies of a segment of tetra-amino acids which is characteristic of adenine-specific methylase.

Amino Acid Sequence↗

PubMed1986

Transfer of glycerol by Endo-beta-N-acetylglucosaminidase F to oligosaccharides during chitobiose core cleavage.

N-Linked oligosaccharides, when hydrolyzed by glycerol-containing preparations of endo-beta-N-acetylglucosaminidase (Endo) F from Flavobacterium meningosepticum were found to have glycerol attached to their reducing ends. The absence of a reducing end was confirmed by high-field 1H NMR spectroscopy, and the incorporated glycerol was verified through mass spectrometry and collisionally activated decomposition fast atom bombardment/mass spectrometry/mass spectrometry techniques. Periodate oxidation of [1(3)-14C]glycerol-labeled oligosaccharides indicated glycerol was glycosidically linked via its 1(3) carbon to the C1 of the reducing end N-acetylglucosamine. In a second, less favored reaction, the glycerol glycoside was hydrolyzed by Endo F using water as the terminal nucleophile, thus regenerating the N-acetylglucosamine reducing end. Glycerol could be removed from Endo F preparations without affecting enzyme stability, and chitobiosyl core hydrolysis in its absence provided intact oligosaccharides with normal N-acetylglucosamine reducing ends. The incorporation of labeled glycerol may provide a useful method for monitoring of Endo F release of oligosaccharides.

Acetylglucosamine↗

PubMedDate not supplied

[Coagulation of bacteria by the action of montmorrillonite].

Conditions were studied for the coagulation of aggregation-stable Bacillus subtilis, Flavobacterium rigens and Escherichia coli cell dispersions. Their aggregative stability decreased when montmorillonite, a clay mineral, was added to a weakly acid medium or in the presence of Al3+ ions. Bacterial heterocoagulation under the action of montmorillonite can be used as a universal strategy for biomass isolation from aqueous dispersion media.

Bacillus subtilis↗

PubMed1988

Differentiation of some gram-negative glucose nonfermenting bacteria using miniaturized carbon sources assimilation tests.

In water and soil the gram-negative nonfermenting bacteria play an important role in the biological mineralization process. To improve the methods for species differentiation of these heterogenous bacterial group, a total of 481 reference strains of gram-negative glucose nonfermenting bacteria belonging to the genera Pseudomonas, Alcaligenes, Bordetella, Agrobacterium, Moraxella, Acinetobacter, Flavobacterium and some CDC groups have been investigated for their ability to utilize 42 different carbon substrates with the help of a standardized and automated micromethod. Most species showed a typical pattern of carbon utilization and hence could be differentiated from each other within their genera. As already has been demonstrated by more clinical significant Pseudomonas species, this method proves to be a useful alternative to existing methods of differentiation, especially with representatives of the families Pseudomonadaceae and Alcaligenaceae.

Acinetobacter↗

PubMed1988

Studies on fermented corn flour food poisoning in rural areas of China. II. Isolation and identification of causal microorganisms.

Using potato dextrose agar medium, 40 strains of microorganisms were isolated from leftover fermented corn flour samples involved in outbreaks of food poisoning. All strains produced powerful toxins which caused the same intoxication to mice, dogs, and monkeys as the leftover food samples. On the basis of results obtained from the morphology of this bacteria and its colony, from biochemical tests, and from the G-C mole percentage in DNA, the bacteria was identified as Flavobacterium farinofermentans nov. sp. (Meng, Z. and Wang, D.).

Agglutination Tests↗

PubMed1988

Bacterial species detected in some bottled mineral waters sold in Italy.

A total of 51 samples from 32 different kinds of mineral water were examined to investigate their microbial facies in view of a possible relationship with ionic composition. No difference was found between mineral and oligomineral waters. The genera most represented were Pseudomonas, Flavobacterium and Alcaligenes; only rarely could members of these genera be assigned precisely to a defined species, because many strains showed intermediate patterns. The authors suggest widening the microbiological examination of mineral waters and improving ecological and taxonomic knowledge of the autochthonous species in all processing phases in order to evaluate not only their compliance with the law, but also the variations imposed on microflora by different conditions related to container materials, time and temperature of storage.

Alcaligenes↗

PubMed1985

Isolation and characterization of moderately halophilic nonmotile rods from different saline habitats.

A total of 736 strains were isolated from samples taken from three different saline habitats: solar saltern, saline soils and the sea, near Alicante (Spain). For a further study 60 moderately halophilic nonmotile rods were selected and studied for 57 phenotypic characteristics. The highest proportion of moderately halophilic nonmotile rods were isolated from saline soils and in media with 10 or 20% salts, being very scarce in sea water samples. All were Gram-negative rods and were included in two groups: 33 oxidase positive strains could be assigned to the genus Flavobacterium and 24 oxidase negative strains to the genus Acinetobacter.

Acinetobacter↗

PubMed1986

Requirements of cleavage of high mannose oligosaccharides in glycoproteins by peptide N-glycosidase F.

Peptide N-glycosidase from Flavobacterium meningosepticum cleaves complex as well as neutral glycoproteins (Plummer, T.H., Jr., Elder, J.H., Alexander, S., Phelan, A.W., and Tarentino, A.L. (1984) J. Biol. Chem. 259, 10700-10704). Examples of neutral glycoprotein substrates include ribonuclease B (one high mannose oligosaccharide chain) and yeast external invertase (nine chains/invertase subunit). The rate of deglycosylation by the glycosidase was greatly enhanced if the glycoprotein substrate was denatured prior to enzyme treatment, from a low of 11-fold for external invertase to a high of 844-fold for ribonuclease B. Peptide N-glycosidase F was unable to cleave the asparaginyl-N-acetylglucosamine bond in endo-beta-N-acetylglucosaminidase H-modified external invertase or ribonuclease B, although that in similarly modified glycopeptide substrate was cleaved. Ribonuclease B was digested sequentially with various exoglycosidases to produce an oligosaccharide chain of varied length. Using the resulting forms of ribonuclease B as substrates for peptide N-glycosidase F, the minimum oligosaccharide chain for cleavage was the di-N-acetyl-chitobiosyl core unit.

Amidohydrolases↗

PubMed1985

[Qualitative and quantitative determination of bacterial populations in an aquatic environment. 7. Development of bacterial growth on raw materials exposed to potable water].

Refined steel plates coated with different materials that contained available organic compounds led to a microbial growth on the surface. Even plastics and bitumen which were used in the sphere of drinking water showed after an exposure time of three months up to 192 ml slime per square meter. The number of viable bacteria within the Aufwuchs was in the range of 10(7) cfu/ml. The production of slime increased with time. The relation of carbohydrate and protein content significantly changed from 2 at the beginning to 30 after 12 months of incubation the bitumen coating test plates. This indicates an increase synthesis of carbohydrate containing extracellular polymeric substances during the late phase of growth. The bacteria isolated from the Aufwuchs mainly belonged to the genera Pseudomonas, Flavobacterium, Acinetobacter, Caulobacter, sheated bacteria and other gramnegative physiologically nonreactiv roads. During exposure of the plates the relation changed within the bacterial communities of the main groups. Comparing the bacteria communities of inlet and outflow water it became evident that the later one was influenced by bacteria of the Aufwuchs.

Bacteria↗

PubMed1977

Oxidase positive rods from cases of suspected gonorrhoea. A comparison of conventional, gas chromatographic and genetic methods of identification.

Genito-urethral specimens from 3260 women and 1170 men, with ailments suggestive of gonorrhoea, were examined for growth of oxidase positive rodshaped bacteria, as well as of gonococci. Moraxella osloensis was identified in 26 cases (0.64 per cent of women and 0.43 per cent of men). Three patients harboured phenylalanine negative (or weakly reacting) and tryptophan deaminase negative M. phenylpyrouvica and, in three cases, a Flavobacterium species was detected. Among six oropharyngeal specimens from patients suspected of gonorrhoea, two yielded growth of oxidase positive rods, Kingella kingae and Neisseria elongata, respectively, N. gonorrhoeae was isolated from 537 patients, i.e., 12.1 per cent of all cases. The isolates of oxidase positive rods were in most cases completely identified by streptomycin resistance transformation. On this basis, the diagnostic reliability of some morphological and cultural-biochemical tests and gas chromatography was examined. Gas chromatographic analysis of fatty acid and alcohol composition of whole cells proved distinctive of species defined genetically, irrespective of confusing behaviour of some strains in other tests.

Bacteriological Techniques↗

PubMed1982

Studies on pectolytic bacteria in water and bottom sediments of two lakes of different trophy.

Pectolytic bacteria were found in greater numbers in the eutrophic than in the mesotrophic lake. However, higher pectolytic activity was exhibited by the bacteria derived from the mesotrophic lake. In the eutrophic lake higher endo- and exo-PG activity was found in the benthic than in the panktonic bacteria, whereas in the mesotrophic one higher endo-PG activity was exhibited by the planktonic and higher exo-PG activity by the benthic bacteria. The polygalacturonases synthesized by the bacteria of the genera Achromobacter, Bacillus, Pseudomonas, of the group Flavobacterium - Cytophaga and of the family Enterobacteriaceae were active in the pH range 6.0-9.0. The majority of tested strains showed maximum activity at a temperature of 30 degrees C.

Bacillus↗

PubMed1984

Demonstration of peptide:N-glycosidase F activity in endo-beta-N-acetylglucosaminidase F preparations.

Endo-beta-N-acetylglucosaminidase F preparations from Flavobacterium meningosepticum have been found to contain peptide:N-glycosidase activity. Only the second activity, designated as peptide:N-glycosidase F, readily cleaves the beta-aspartylglycosylamine linkage of a fetuin triantennary complex glycopeptide, as shown by the isolation of the corresponding carbohydrate-free peptide containing aspartic acid and of an intact oligosaccharide with a di-N-acetylchitobiosyl moiety at the reducing end. Both activities in the mixture will hydrolyze a high mannose octaglycopeptide from ovalbumin, with the type of product formed being influenced by pH. At pH 4.0, only the endo-beta-N-acetylglucosaminidase F activity is functional, releasing octapeptide-GlcNAc and oligosaccharide-GlcNAc. At pH 9.3, the predominant cleavage is by peptide:N-glycosidase F at the glycosylamine bond, releasing octapeptide and oligosaccharide-GlcNAc-GlcNAc. This latter oligosaccharide is then hydrolyzed by endo-beta-N-acetylglucosaminidase F to oligosaccharide-GlcNAc plus GlcNAc.

Acetylglucosaminidase↗

PubMed1984

Chitynolytic bacteria in water and bottom sediments of two lakes of different trophy.

Chitynolytic bacteria were found in greater numbers in the eutrophic lake Jeziorak than in the mesotrophic lake Jasne. In both lakes higher chitynolytic activity was found in the planktonic than in the benthic bacteria. The capacity of chitin hydrolysis was found in bacteria of the genera Achromobacter, Bacillus, Nocardia, Pseudomonas, of the Flavobacterium-Cytophaga group and of the Enterobacteriaceae family. The chitinases synthesized by the studied bacteria were active in the pH range 5.0--8.0. The majority of the strains tested showed maximum activity at pH 5.0 or 6.0.

Alcaligenes↗