Studies on the aminopeptidase test for the distinction of gram-negative from gram-positive bacteria

Summary The aminopeptidase test was performed with representatives of gram-negative, gram-positive, and gram-variable bacteria. All gram-negative bacteria tested gave a positive test reaction with L-alanine-4-nitroanilide as test substrate. Representatives of the coryneform bacteria and some streptococci showed aminopeptidase activity after prolonged reaction times. A correlation between aminopeptidase activity and distinct interpeptide bridge composition in the peptidoglycan of many strains was demonstrated. The influence of growth conditions on aminopeptidase activity of intact bacterial cells is shown.     

Bacterial Colonization and Ectoenzymatic Activity in Phytoplankton-Derived Model Particles: Cleavage of Peptides and Uptake of Amino Acids

Abstract Phytoplankton-derived model particles were created in laboratory from a mixture of autoclaved diatom cultures. These particles were colonized by a marine bacterial community and incubated in rolling tanks in order to examine the relationship between aminopeptidase activity and leucine uptake. Bacteria inhabiting particles and ambient water were characterized for abundance, biovolume, aminopeptidase activity, leucine uptake, and growth rate. Particles were a less favorable habitat than ambient water for bacterial growth since growth rates of particle-attached bacteria were similar or even lower than those of free-living bacteria. During the first ∼100 h of the particle decomposition process, there were not statistically significant differences in the aminopeptidase activity:leucine uptake ratio between attached and free-living bacteria. From ∼100 h to ∼200 h, this ratio was higher for attached bacteria than for free-living bacteria. This indicates an uncoupling of aminopeptidase activity and leucine uptake. During this period, attached and free-living bacteria showed similar hydrolytic activities on a cell-specific basis. In the free-living bacterial community, variations in aminopeptidase activity per cell were associated with variations in leucine uptake per cell and growth rates. However, in the attached bacterial community, when leucine uptake and growth rates decreased, aminopeptidase activity remained constant. Thus, after ∼100 h, particle-attached bacteria were not taking advantage of their high aminopeptidase activity; consequently the hydrolysed amino acids were released into the ambient water, supporting the growth of free-living bacteria. These results demonstrate that over the particle decomposition process, the relationship between hydrolysis and uptake of the protein fraction shows different patterns of variation for attached and free-living bacterial communities. However, in our experiments, this uncoupling was not based on a hyperproduction of enzymes by attached bacteria, but on lower uptake rates when compared to the free-living bacteria. Received: 4 February 1997; Accepted: 9 May 1997     

An Aneurinibacillus sp. strain AM-1 produces a proline-specific aminopeptidase useful for collagen degradation

AIMS: We have been for a species of thermophilic bacteria that can effectively decompose collagen and collagen peptides that tend to be hard-to-degrade proteins because of their high content of proline residues. This study focused upon the enzymatic degradation of prolyl peptides by thermophilic bacteria. METHODS AND RESULTS: A strain, AM-1, producing a proline-specific aminopeptidase was isolated using a medium containing gelatin that was taken from soil samples collected at Arima Hot Spring located near Kobe, Japan. The strain showed the strongest level of hydrolysing activity toward prolyl-p-nitroanilide, and the activity proved to be thermostable. Phylogenetic analysis based on 16S rDNA sequences revealed that the isolated strain AM-1 was closest to Aneurinibacillus thermoaerophilus DSM10154T in its characteristics. Analysis of the purified proline-specific aminopeptidase suggested that the enzyme is an aminopeptidase containing metal that includes important disulphide bond(s). The strain AM-1 aminopeptidase has more similarities with leucyl aminopeptidases, but its activity level differs greatly with prolyl peptides. CONCLUSIONS: The proline-specific aminopeptidase from strain AM-1 is the first from the genus Aneurinibacillus and may be a new type of aminopeptidase for hydrolysing prolyl peptide. This enzyme also contributed to the degradation of collagen when used in combination with another collagenolytic protease. SIGNIFICANCE AND IMPACT OF THE STUDY: The proline-specific aminopeptidase obtained from strain AM-1 may be used in the treatment of wastewater containing collagen that is encountered in the meat industries, and for decreasing bitter peptides in milk products.     

Experimental study of Microcystis-associated and free-living cyanobacteria

In the experiment with water from the hypereutrophic Lake Frederiksborg Slotso (Denmark) sampled during the autumn peak of Microcystis growth, the quantity and production of free-living and cyanobacteria-associated heterotrophic bacteria were determined, as well as the extracellular enzymatic (aminopeptidase) activity. The functional diversities of associated and free-living bacterial communities were additionally compared using BIOLOG GN microplates to reveal the possible export of Microcystis-attached bacteria into ambient water. It has been shown that the cell size, production values, and growth rates of associated bacteria were less than those of free-living bacteria. At the same time, the potential aminopeptidase activity of associated bacteria was always higher than that of free-living bacteria. The experimental results have shown significant compositional differences in the structure of bacterial communities from different habitats.     

Enhanced instrumental sensitivity and selectivity for aminopeptidase profiling

Laser-excited fluorimetry has been applied to the identification of bacteria and fungus. The instrumental sensitivity and selectivity of the aminopeptidase profiling method has been enhanced by the use of laser excitation in conjunction with improved spectral and temporal background rejection. The linear dynamic range for the aminopeptidase technique has been increased by achieving a reduced lower limit of detection of the fluorescent tag, beta-naphthylamine. Standard aminopeptidase methodology only provides a linear dynamic range of 1.5 orders of magnitude. The laser-based method expanded the range to three orders of magnitude allowing the inherent specificity of aminopeptidase enzymes within the pathogen to be observed. The enhanced linear dynamic range was observed in profiles of Agrobacterium tumefaciens rubi and Phytophthora megasperma var. sojae.     

Experimental study of Microcystis-associated and free-living bacteria

In the experiment with water from the hypereutrophic Lake Frederiksborg Slotso (Denmark) sampled during the autumn peak of Microcystis growth, the quantity and production of free-living and cyanobacteria-associated heterotrophic bacteria were determined, as well as the extracellular enzymatic (aminopeptidase) activity. The functional diversities of associated and free-living bacterial communities were additionally compared using BIOLOG GN microplates to reveal the possible export of Microcystis-attached bacteria into ambient water. It has been shown that the cell size, production values, and growth rates of associated bacteria were less than those of free-living bacteria. At the same time, the potential aminopeptidase activity of associated bacteria was always higher than that of free-living bacteria. The experimental results have shown significant compositional differences in the structure of bacterial communities from different habitats.     

Antibacterial and proteolytic activity in venom from the endoparasitic wasp Pimpla hypochondriaca (Hymenoptera: Ichneumonidae)

Venom from the endoparasitic wasp, Pimpla hypochondriaca, is composed of a mixture of high and low molecular weight proteins, possesses phenoloxidase activity, has immunosuppressive properties, and induces paralysis in several insect species. In the present study we demonstrate that P. hypochondriaca venom also contains antibacterial and proteolytic activity. Antibacterial activity was detected against the Gram-negative bacteria Escherichia coli and Xanthamonas campestris but not against Pseudomonas syringae nor against two Gram-positive bacteria, Bacillus cereus and Bacillus subtilis. Endopeptidase and aminopeptidase activity in venom was detected using the synthetic fluorogenic substrates N-t-BOC-Phe-Ser-Arg-AMC, Arg-AMC and Leu-Arg. The aminopeptidase activity towards Arg-AMC was sensitive to amastatin (70% inhibition), an aminopeptidase inhibitor. Angiotensin-converting enzyme (ACE)-like enzyme activity was detected, by reverse-phase HPLC using the synthetic tripeptide Hip-His-Leu as a substrate. This activity was sensitive to captopril, an ACE inhibitor (IC(50) 3.8 x 10(-8) M). Using an antiserum raised against recombinant Drosophila melanogaster ACE-like enzyme, (rAnce), Western blot analysis revealed an immunoreactive protein, with a molecular weight estimate of 74 kDa, in P. hypochondriaca venom. The possibility that the endopeptidase, aminopeptidase and ACE are involved in the processing of peptide precursors in the venom sac is discussed.     

Release pattern of aminopeptidase from Biomphalaria glabrata hemocytes subjected to high-level bacterial challenge

Specimens of Biomphalaria glabrata, NIH albino strain, challenged with 2 μl of heat-killed Bacillus megaterium at a concentration of 9 × 10⁹ bacteria/ml, showed significant elevation of serum aminopeptidase activity level at 2 hr postchallenge when compared with the serum activity levels of the enzyme in snails of the other experimental groups at four time intervals postchallenge. It was found that challenge with a heavy dosage of heat-killed bacteria did not cause a shift in the highest level of enzyme activity from 2 hr postchallenge. Also, the activity of aminopeptidase was higher at 2 hr postchallenge with a greater dosage of heat-killed bacteria.     

Aminopeptidase Activity in Marine Chroococcoid Cyanobacteria

Synechococci are important primary producers in the ocean and can also utilize some components of the dissolved organic matter (DOM). The readily utilizable DOM in seawater is mainly polymeric (e.g., protein, polysaccharide) or phosphorylated and requires hydrolysis prior to uptake. We examined whether synechococci express ectoenzymes to hydrolyze DOM components and considered the possible significance of ectohydrolases for Synechococcus ecology and organic matter cycling in the sea. Five strains of non-nitrogen-fixing synechococci in axenic cultures were tested for enzyme activities with fluorogenic substrates. All strains show ectocellular aminopeptidase activity, but other enzymes were undetectable. The aminopeptidase level was in the range determined for five marine heterotrophic bacterial isolates tested for comparison. Aminopeptidase was not secreted into the medium; the majority (74%; tested in WH 7803) was cell surface bound, and a small fraction was periplasmic. The periplasmic activity was not released by cold osmotic shock of WH 7803. Phenylmethylsulfonyl fluoride and EDTA, inhibitors of serine and metalloproteases, strongly or completely inhibited WH 7803 aminopeptidase. The enzyme seemed constitutive; per-cell activity did not change during incubations in unenriched seawater, bovine serum albumin, or nitrate-replete mineral medium. In natural planktonic assemblages in the Southern California Bight, aminopeptidase activity was correlated with Synechococcus abundance as well as the abundance of other bacteria. Ectocellular aminopeptidase may be common in marine synechococci and play roles in their nitrogen nutrition, particularly in low-nitrate and low-light environments. Since synechococci are much less abundant than heterotrophic bacteria in seawater, the impact of Synechococcus aminopeptidase on proteolysis in the sea is likely to be episodic and restricted to specialized microenvironments.     

Aminopeptidase and phosphatase activities in basins of Lake Hiidenvesi dominated by cyanobacteria and in laboratory grown Anabaena

1. Extracellular enzyme activities were examined in freshwater basins representing a transition from hypertrophy to mesotrophy and in axenic cyanobacterial cultures to evaluate the ecological role of extracellular enzyme activities of cyanobacteria.
2. Aminopeptidase activity was related to the trophic status of the lake basins. The activity was highest in the most eutrophic basin and decreased in the less nutrient-rich basins. Cyanobacteria were the most important autotrophic organisms and aminopeptidase activity was positively associated with cyanobacterial biomass.
3. In an axenic Anabaena batch culture, nitrogenase activity was several orders of magnitude higher than leucine aminopeptidase activity. Nitrate did not have an effect on aminopeptidase activity or growth, but significantly reduced the rate of nitrogen fixation. A high phosphorus concentration at the beginning of the Anabaena batch-culture experiment resulted in reduced phosphatase activity.
4. In Lake Hiidenvesi, aminopeptidase activity probably originated mostly from attached bacteria and less so from cyanobacteria.     

Ultrasound Treatment for Harvesting an Aminopeptidase from Lactic Acid Bacteria and Quantitation of the Enzyme by Enzyme-Linked Immunosorbent Assays

Ultrasound treatment of Lactococcus lactis subsp. cremoris AM2 was optimized to release a maximum amount of intracellular aminopeptidase without modifying the antigenicity of the enzyme. The cells were sonicated three times for 30 s at 23 W. Antibodies produced against the aminopeptidase purified from L. lactis subsp. cremoris AM2 enabled us to use immunoblotting to detect the enzyme in the lysates of all of the lactococci tested but not in the lysates of Leuconostoc strains, lactobacilli, and Streptococcus salivarus subsp. thermophilus. A sandwich enzyme-linked immunosorbent assay (ELISA) was developed to quantify the purified aminopeptidase; the detection limit was 4 ng/ml. The aminopeptidase in the supernatant obtained after the ultrasound treatment of strain AM2 cells was detected with the ELISA starting with a total protein concentration of 200 ng/ml. The proportion of equivalent purified aminopeptidase in the supernatant of L. lactis subsp. cremoris AM2 was about 2% of the total protein. Similarly, the aminopeptidase was quantified in different lactococci; the percentages varied between 0.16 and 2%, depending on the strain. The aminopeptidase content in a mixture of several lactic bacteria was also determined with the sandwich ELISA.     

Synthesis of 2-arylbenzothiazole derivatives and their application in bacterial detection

A series of 2-arylbenzothiazole derivatives have been prepared as fluorogenic enzyme substrates in order to detect aminopeptidase, esterase, phosphatase and β-galactosidase activity in clinically important Gram-negative and Gram-positive bacteria. Substrates were incorporated into an agar-based culture medium and this allowed growth of intensely fluorescent bacterial colonies based on hydrolysis by specific enzymes. Substrate 20 targeted l-alanine aminopeptidase activity and was hydrolysed exclusively by a range of Gram-negative bacteria and inhibited the growth of a range of Gram-positive bacteria. Substrate 19a targeted β-alanyl aminopeptidase activity and generated fluorescent colonies of selected Gram-negative species including Pseudomonas aeruginosa. Substrate 21b targeted C8-esterase activity and resulted in strongly fluorescent colonies of selected species known to harbour such enzyme activity (e.g., Salmonella and Pseudomonas). Most Gram-negative species produced colonies with an intense blue fluorescence due to hydrolysis of phosphatase substrates 24a–c and substrate 24c was also hydrolysed by strains of Staphylococcus aureus. Compounds 26b and 26c targeted β-galactosidase activity and generated strongly fluorescent colonies with coliform bacteria that produced this enzyme (e.g., Escherichia coli).     

Decomposition of protein compounds in an eutrophic lake: spatial and temporal distribution of exopeptidase and endopeptidase activities in various size fractions

Activities of exopeptidases and endopeptidases in various size fractions and the possible regulation of these enzymes in response to the changes in substrate concentrations were studied. The endopeptidase activities were determined using a protein substrate labelled with a fluorochrome. Among the enzyme activities studied only leucine aminopeptidase and endopeptidase activities were recorded. The protein compounds did not seem to cleave by synergistic endo and exoenzyme mechanisms. The inhibitory effect of ethylenediamine tetraacetate (EDTA) and phenylmethylsulfonile fluoride (PMSF) suggested a high proportion of metallo-proteases and serine-proteases. The inhibitory profile at 1 and 5 m indicated that planktonic organisms probably produced the same type of proteases, whereas enzymes present in the 0–1.2 and 1.2–100 μm fractions were not similar. The mean percentages of aminopeptidase activity at 1, 5 and 14 m in the dissolved fraction were 12.5, 12.7 and 18.4%. This enzyme activity was low in the 0.2–1.2 μm fraction (12.1% at 1 m, 13.3% at 5 m and 19.1%, at 14 m) compared to that measured in the 1.2–100 μm fraction, whereas the average percentages of endopeptidase activities in this fraction were 50.9% at 1 m, 50.1% at 5 m and 53.5% at 14 m. The bacteria attached to particles had a higher specific activity than free-living bacteria. It was 11.7 times higher than the enzyme activity associated with larger free-living bacteria and 112.3 times higher than the activity of small free-living bacteria. In this study, the specific activity of the aminopeptidase (activity in the fraction 0.2–100 μm per number of bacteria) was correlated with Chl a at 1 m (r = 0.65, P <0.01), 5 m (r = 0.78, P <0.001) and 14 m (r = 0.96, P <0.001). The production of protein compounds by the phytoplankton could therefore regulate aminopeptidase activity. This revised version was published online in July 2006 with corrections to the Cover Date.     

Leucine aminopeptidase from Arabidopsis thaliana. Molecular evidence for a phylogenetically conserved enzyme of protein turnover in higher plants.

Leucine aminopeptidases are exopeptidases which are presumably involved in the processing and regular turnover of intracellular proteins; however, their precise function in cellular metabolism remains to be established. Towards this goal, a full-length complementary DNA encoding a plant leucine aminopeptidase was isolated from a cDNA library of Arabidopsis thaliana and sequenced. The nucleotide sequence showed 49.5% identity to the Escherichia coli xerB-encoded leucine aminopeptidase. Sequence analysis revealed that the cDNA encodes a polypeptide of 520 amino acids with a calculated molecular mass of 54,506 Da. The C-terminal part (amino acids 200-520) of the deduced amino acid sequence showed 43.8% sequence identity to the xerB-encoded leucine aminopeptidase and 42.6% sequence identity to the amino acid sequence of bovine lens leucine aminopeptidase (EC 3.4.11.1). No sequence similarity (not even over short sequence elements) was observed with any other known peptidase or proteinase sequence. The cDNA was expressed as a fusion protein from the lacZ promoter in E. coli. Enzymatic analysis proved that the cloned cDNA encoded an active leucine aminopeptidase. The properties of this enzyme, including metal requirements, inhibitor sensitivity, pH optimum and the remarkable temperature stability, are very similar to those reported for leucine aminopeptidases from other tissues. Amino acids involved in metal and substrate binding in bovine lens aminopeptidase are completely conserved in the plant enzyme as well as in the XerB protein. Our results show that leucine aminopeptidases form a superfamily of highly conserved enzymes, spanning the evolutionary period from the bacteria to animals and higher plants. This is the first aminopeptidase cloned from a plant.     

Novel chromogenic aminopeptidase substrates for the detection and identification of clinically important microorganisms

A series of amino acid derivatives 8–10, 42 and 43 have been prepared as chromogenic enzyme substrates in order to detect aminopeptidase activity in clinically important Gram-negative and Gram-positive bacteria. Enzymatic hydrolysis liberates the amino acid moiety and either a 4-aminophenol or a 4-dialkylaminoaniline derivative which undergoes oxidative coupling with 1-naphthol or a substituted 1-naphthol giving an indophenol dye. Substrates and 1-naphthols were incorporated into an agar-based culture medium and this allowed growth of intensely coloured bacterial colonies based on hydrolysis by specific enzymes. Red/pink coloured colonies were produced by the substrates 8–10 and blue coloured colonies were formed by the substrates 42 and 43. The l-alanyl aminopeptidase substrates 8 targeted l-alanyl aminopeptidase activity and gave coloured colonies with a range of Gram-negative bacteria. Substrates 9 targeted β-alanyl aminopeptidase activity and generated coloured colonies with selected Gram-negative species including Pseudomonas aeruginosa. Three substrates for l-pyroglutamyl acid aminopeptidase (10a, 10c and 43) were hydrolysed by enterococci and Streptococcus pyogenes to generate coloured colonies. Two yeasts were also included in the study, but they did not produce coloured colonies with any of the substrates examined.     

Aminopeptidase N from Streptococcus salivarius subsp. thermophilus NCDO 573: purification and properties

A 96 kDa aminopeptidase was purified from Streptococcus salivarius subsp. thermophilus NCDO 573. The enzyme had similar properties to aminopeptidases isolated from lactococci and lactobacilli and showed a high degree of N -terminal amino acid sequence homology to aminopeptidase N from Lactococcus lactis subsp. cremoris. It catalysed the hydrolysis of a range of aminoacyl 4-nitroanilides and 7-amido-4-methylcoumarin derivatives, dipeptides, tripeptides and oligopeptides. In common with aminopeptidases from other lactic acid bacteria, the enzyme from Strep. salivarius subsp. thermophilus showed highest activity with lysyl derivatives but was also very active with arginyl and leucyl derivatives. Relative activity with alanyl, phenylalanyl, tyrosyl, seryl and valyl derivatives was considerably lower and with glycyl, glutamyl and prolyl derivatives almost negligible. The aminopeptidase also catalysed the hydrolysis of dipeptides and tripeptides but mostly at rates much less than that with L-lysyl-4-nitroanilide and oligopeptides. The enzyme catalysed the successive hydrolysis of various amino acid residues from the N -terminus of several oligopeptides but it was unable to cleave peptide bonds on the N -terminal side of a proline residue.     

Experimentally induced elevation of aminopeptidase activity in hemolymph cells of the American oyster,Crassostrea virginica

The levels of aminopeptidase activity in the serum and hemolymph cells of the American oyster, Crassostrea virginica, have been determined to be 2.89 ± 2.25 and 0.62 ± 0.39 Sigma units/ml, respectively. Low levels of aminopeptidase activity have been demonstrated cytochemically within cytoplasmic granules, i.e., secondary phagosomes, of circulating hemolymph cells. Exposure of whole hemolymph to heat-killed Bacillus megaterium or to sterile sea water results in a significant increase in cellular aminopeptidase activity, with the level of activity being the highest in cells that had been exposed to bacteria. The level of aminopeptidase activity in serum is unaltered in similarly challenged whole hemolymph. It is concluded that aminopeptidase synthesized in cells during phagocytosis or as a result of stimulation by exposure to sea water is not released into the serum but is retained intracellularly. Our studies suggest that it is the intracellular and not the serum aminopeptidases whith are of primary importance in the degradation of B. megaterium.     

Experimental evidence for interactions between bacterial peptidase and alkaline phosphatase activity in the Baltic Sea

From the observed pattern of aminopeptidase and alkaline phosphatase activities in the Baltic Sea, the question arose whether there is an interaction between the activities of both enzymes. In experiments with 0.8 m filtered seawater, the effects of commercial alkaline phosphatase on bacterial aminopeptidase, the effects of commercial peptidase on bacterial alkaline phosphatase activity (APA), and the effects of proteins, carbohydrates and inorganic nutrients on the activities of both enzymes were investigated.Addition of commercial alkaline phosphatase stimulated bacterial aminopeptidase activity and, similarly, the addition of commercial peptidase increased the APA in bacteria. The proteins, albumin and casein, stimulated aminopeptidase activity and APA simultaneously. Experiments using ammonium and glucose suggested that stimulation of APA by peptidase could be mediated by nitrogen and carbon availability. There were also some indications that stimulation of aminopeptidase activity by alkaline phosphatase functioned by catalysing phosphate release from organic phosphorus compounds.     

Selective isolation of bacteria with dipeptidyl aminopeptidase type I activity from the sheep rumen

Five-hundred-and-six fresh isolates of rumen bacteria were tested for their ability to hydrolyse the synthetic substrate for dipeptidyl aminopeptidase type I, GlyArg-4-methoxy-2-naphthylamide (GlyArg-MNA), using a gel overlay technique. Twelve positive isolates were small Gram-negative rods which resembled Bacteroides ruminicola in their biochemical and morphological properties. SDS-PAGE of whole cell extracts indicated that two were similar to B. ruminicola strain B14, six resembled B. ruminicola strain M384, and four were similar to B. ruminicola GA33. All hydrolysed GlyArg-MNA, Ala2 and Ala5, and showed no activity against Leu-MNA. Ala3 and Ala2, but no Ala4, was produced from Ala5. The different groups had different, distinctive activity profiles. The two remaining positive isolates were Lactobacillus spp. with an exceptionally high Leu-MNA activity. It was concluded that, although different strains may only be distantly related, B. ruminicola forms the most important group of bacteria in the rumen to possess a dipeptidyl aminopeptidase type I activity.     

2-Arylbenzothiazole, benzoxazole and benzimidazole derivatives as fluorogenic substrates for the detection of nitroreductase and aminopeptidase activity in clinically important bacteria

A series of 2-(2-nitrophenyl)benzothiazole 7, 2-(2-nitrophenyl)benzoxazole 10 and 2-(2-nitrophenyl)benzimidazole 13 derivatives have been synthesised and assessed as indicators of nitroreductase activity across a range of clinically important Gram negative and Gram positive bacteria. The majority of Gram negative bacteria produced strongly fluorescent colonies with substrates 7 and 10 whereas fluorescence production in Gram positive bacteria was less widespread. The l-alanine 16 and 19 and β-alanine 21 and 23 derivatives have been prepared from 2-(2-aminophenyl)benzothiazole 14 and 2-(2-aminophenyl)benzoxazole 17. These four compounds have been evaluated as indicators of aminopeptidase activity. The growth of Gram positive bacteria was generally inhibited by these substrates but fluorescent colonies were produced with the majority of Gram negative bacteria tested.