API ZYM System Research Articles

Determination of key enzyme activities in commercial peptidase and lipase preparations from microbial or animal sources

The enzyme complement of a selection of commercial food-grade peptidase and lipase preparations was investigated. Each preparation was assayed for protein content, proteinase activity at pH 5.5 and 7.0 at 37 °C using azocasein and semi-quantitatively assayed for lipase, peptidase, proteinase, phosphatase and glycosidase activity by the API-ZYM system. Each peptidase preparation was also assayed for various endo-, carboxy-, amino- and di-peptidase activities at pH 5.5 and 7.0 at 37 °C, using chromogenic or fluorogenic substrates, while each lipase preparation was assayed for esterase and lipase activity at pH 7.0 at 37 °C using p-nitrophenol substrates. All enzyme preparations were found to contain enzyme activities in addition to their stated main activity. According to the API-ZYM system the peptidase preparations contained varying levels of lipase, proteinase, peptidase, phosphatase and glycosidase activity, with the lipase preparations containing lipase, phosphatase and glycosidase activity. Only two peptidase and two lipase preparations contained significant amounts of proteinase activity as measured by azocasein. The peptidase and lipase activities of the preparations appeared to be dependent upon source. Most peptidase preparations had significantly more activity at pH 7.0 than at 5.5.

PROPERTIES OF COMMERCIAL MICROBIAL PROTEINASE PREPARATIONS

ABSTRACT Twenty three commercial microbial proteinase preparations derived from various Bacillus or Aspergillus spp. or from Rhizomucor niveus were assessed for proteolytic activity on azocasein at pH 5.5 or 7.0, or specificity on sodium caseinate at pH 5.5 and semi-quantitatively assessed for esterase, lipase, trypsin, chymotrypsin, general aminopeptidase, phosphatase and glycosidase activities using the API-ZYM system. Selected preparations were further assayed for peptidase, esterase and lipase activities at pH 7.0. The proteolytic activity of the Bacillus preparations was greater at pH 7.0, while that of the Aspergillus and Rhizomucor preparations was greater at pH 5.5. All the Bacillus preparations contained one of two main proteolytic activities, thought to be either bacillolysin or subtilisin. Most of the Aspergillus preparations contained the same proteinase, thought to be aspergillopepsin I, but two preparations appeared to contain a different unidentified proteinase. The proteolytic specificity of the Rhizomucor preparation was different from the Bacillus or Aspergillus preparations; thought to be rhizopuspepsin. According to the results of the API-ZYM system, all preparations contained enzyme activities in addition to their main proteolytic activity, with the Aspergillus and Rhizomucor preparations containing the highest levels and widest range of activities. Generally preparations derived from Aspergillus contained the highest level of general, proline and endopeptidase activities, with the Bacillus preparations conspicuous by the absence of general and proline-specific peptidase activities, while the Rhizomucor niveus preparation contained little or no general or endopeptidase activity. Esterase activity was found in all of the preparations evaluated, with only two Aspergillus preparations containing lipase activity.

Malabsorption syndrome observed in the common octopus Octopus vulgaris infected with Aggregata octopiana (Protista: Apicomplexa).

Octopus vulgaris infected with Aggregata octopiana were collected from an open-water culture system in the Ría of Aldán (NW Spain). Digestive tract infection values were determined with the use of a Neubauer chamber by counting the number of A. octopiana sporocysts. After determining enzyme activity values by the colorimetric Api-Zym system Biomerieux, one representative enzyme of glycosidases, peptid hydrolases and phosphoric hydrolases showing high activity was spectrophotometrically analysed. The enzymes were maltase and leucine-aminopeptidase (LAP) involved in the absorption process, and acid phosphatase, a lysosomic enzyme, respectively. Enzymatic activity of maltase and LAP decreased significantly, with increased sporocyst counts. However, acid phosphatase activity increased with severity of infection, indicating the presence of degradative enzymes from phagocytic cells in the infected area. A detrimental effect on gastrointestinal function may result from a decrease or malfunction of absorption enzymes. The results suggest a malabsorption syndrome resulting from parasitic infection.

Quality control Lactobacillus strains for use with the API 50CH and API ZYM systems at 37 degrees C.

The API 50CH and API ZYM systems fulfil an important role in the polyphasic taxonomic identification of lactobacilli. When the API 50CH fermentation profile of the quality control Lactobacillus casei var. alactosus (Lb. paracasei subsp. paracasei) strain NCFB 206 was determined at 37 degrees C, it was found to differ from that determined at 30 degrees C by BioMéreiux SA (Montalieu Vercieu, France) and the National Collection of Food Bacteria (Aberdeen, Scotland). In addition, the API 50CH fermentation and API ZYM profiles of Lb. casei strain ATCC 334T determined at 37 degrees C differed from those determined at 30 degrees C by Lee and Simard (1984). Strains NCFB 206 and ATCC 334T were thus assumed to exhibit temperature-dependent variation in fermentation profile, a phenomenon recently described by Nigatu et al. (2000). In contrast, Lb. rhamnosus strain ATCC 243T did not exhibit temperature-dependent variation in fermentation profile. Moreover, the fermentation profile obtained at 37 degrees C differed in only one respect (positive beta-gentiobiose utilisation) from that published by Collins et al. (1989). In addition, Lactobacillus strain GG produced a stable and reproducible API ZYM profile at 37 degrees C, although some variation in the level of enzyme activity was evident. Thus, strain NCFB 206 was replaced by strain ATCC 243T as the quality control strain of choice for use with the API 50CH fermentation system, and Lactobacillus strain GG adopted for use as a quality control strain with the API ZYM system for strain identification of lactobacilli at 37 degrees C. The API 50CH and API ZYM profiles of the commercially important Lactobacillus strains NCFB 1748, GG, KLD, F19, and ACA-DC 212.3 were determined at 37 degrees C after anaerobic growth in MRS broth. The fermentation and enzyme profiles of strain NCFB 1748 were almost identical to those of Lb. crispatus ATCC 33820T, those of strain GG were found to be more similar to those of Lb. rhamnosus strain 243T than Lb. zeae strain ATCC 15820T, those of strain KLD were most similar to those of Lb. fermentum DSM 20052T, while those of strains F19 and ACA-DC 212.3 were similar to those of Lb. casei strain ATCC 334T.

Enzyme activities of Borrelia burgdorferi sensu lato.

Activities of 19 enzymes were tested by the API ZYM system in 13 strains of Borrelia burgdorferi sensu lato (B. burgdorferi sensu stricto, B. afzelii, B. garinii, B. lusitaniae, B. valaisiana) grown in liquid BSK-H medium supplemented with rabbit serum. All strains produced acid phosphatase, esterase (C4), esterase-lipase (C8), leucine arylamidase and naphthol-AS-BI-phosphohydrolase. Nine strains also produced alkaline phosphatase, and three strains produced alpha-glucosidase. The API ZYM system probably cannot be used for differentiation between B. burgdorferi sensu lato genomospecies.

Ecophysiology of soil bacteria in the vicinity of Henryk Arctowski Station, King George Island, Antarctica

Microbial abundance and diversity in soil samples from a ca. 0.2km2 area in Admiralty Bay, King George Island, were analysed by epifluorescence microscopy and plate counts. Total bacterial counts (TC) varied little compared to the elemental content of the organic matter (c.v.TC 51% vs. c.v.C,N,S 131, 171 and 204%, respectively). The number of copiotrophic colony forming bacteria (CFU) fluctuated widely (CVCFU 171%). These factors together with the highly variable time course of colony formation by bacterial populations from each sampling site, indicate the high diversity and mosaic character of the microbial communities in the area. Morphological, physiological and enzymatic features of 48 bacterial strains isolated on nutrient agar were determined through API 20NE and API ZYM systems and by classical tests. These tests confirmed the high physiological diversity within the CFU populations. Only 14 of the 48 isolates tested by API 20NE system were identifiable in the API database.

Growth, dye degradation and ligninolytic activity studies on Zimbabwean white rot fungi

White rot fungi were collected from Chirinda and Chimanimani hardwood forests in Zimbabwe and studied with respect to growth temperature optima and dye decolorization. Temperature optima were found to vary (between 25-37 degrees C) amongst the isolates. The isolates were screened for their ability to degrade the polymeric dyes; blue dextran and Poly R478 and the triphenylmethane dyes; cresol red, crystal violet and bromophenol blue. Semi-quantitative determination of the hydrolytic enzyme activities possessed by the white rot fungi was determined using the API ZYM system. Lignin peroxidase (LiP), manganese peroxidase (MnP) and laccase activities in the fungi were also determined. No LiP was detected in any of the isolates but all isolates showed manganese peroxidase and laccase activities. Time related decolorization studies and optimum pH determinations for Poly R478 degradation by the isolates were carried out in liquid cultures. The most significant rates of Poly R478 decolorization in liquid cultures were found with the following isolates: Trametes cingulata, Trametes versicolor, Trametes pocas, DSPM95 (a species to be identified), Datronia concentrica and Pycnoporus sanguineus.

Extracellular enzymatic activity of Malassezia spp. isolates.

Extracellular enzymatic activity of different species of Malassezia spp was evaluated. Thirty-three isolates of animal origin (dogs and cats) and stock culture samples were studied. Twenty isolates of M. pachydermatis, 8 of M. furfur, 2 of M. sympodialis and M. globosa and one of M. restricta, M. obtusa and M. slooffiae were examined. The enzymatic activity was investigated using Api Zym system. The enzymatic patterns showed light differences. Esterase lipase, Phosphatase acid and Naphtol-AS-BI-phosphohydrolase were produced in significant amounts from most isolates excepted for M. restricta, confirming the limited enzymatic activity of this species. Data obtained from the other new species described after the revision of the genus, appear to be quite homogeneous. Dixon's broth appeared to be a valid medium for the growth of all Malassezia spp.

Extracellular enzymatic activity in 11 Cryptococcus species.

The extracellular enzymatic activity of 36 strains of yeast belonging to 11 species of the genus Cryptococcus, has been investigated, using the API-ZYM (BioMérieux, France) commercial system, with the objective of determining the differences in the enzymatic profiles of the various species. The strains studied were: 9 of C. neoformans, 7 of C. albidus, 6 of C. laurentii, 5 of C. uniguttulatus, 3 of C. humicolus, and 1 each of C. ater, C. curvatus, C. dimennae, C. hungaricus, C. infirmo-miniatus and C. magnus. All the strains showed enzymatic activity with positivity to Phosphatase alkaline, Esterase lipase C8, Leucine arylamidase, Phosphatase acid and Naphthol-AS-BI-phosphohydrolase, and negativity to Lipase C14, Trypsin, Chemotrypsin, beta-galactosidase, beta-glucuronidase and alpha-manosidase. Variable enzymatic activity was shown to Esterase C4, Valine arylamidase, Cystine arylamidase, alpha-galactosidase, alpha-glucosidase, beta-glucosidase, N-acetyl-beta-glucosaminidase and alpha-fucosidase. This allowed 11 separate enzymatic patterns to be established. The species C. neoformans and C. laurentii each presented two distinct patterns; C. uniguttulatus, C. hungaricus and C. magnus shared the same pattern; C. albidus, C. ater, C. curvatus, C. dimennae, C. humicolus and C. infirmo-miniatus presented an individual enzymatic pattern. The results obtained suggest that the API-ZYM system could be useful for the identification of species of the genus Cryptococcus and for the differentiation of the enzymotypes for epidemiological purposes.

Phenotypic and genetic characterization of a selected set of Lactococcus lactis strains isolated from a starter-free farmhouse cheese

Eleven strains of lactococci isolated from a farmhouse starter-free cheese manufactured from raw cow's milk were analysed in detail for some technologically-related properties. Large phenotypic differences were encountered between the isolates, some of which could be of practical relevance. Several strains produced lactic acid at a rate and at a final concentration suitable for large-scale cheesemaking. The enzymatic capabilities assayed with the API ZYM system showed that all strains possess similar profiles with weak proteinase and moderate leucine-arylamidase and esterase-lipase activities. Interestingly, two related strains presented a strong β -galactosidase activity. Plasmid and chromosomal analyses indicated a high degree of diversity among wild strains and showed low homology with some well-known Lactococcus lactis strains. Under highly stringent conditions, only one plasmid from a single strain gave a clear positive hybridization signal with lactococcal-derived probes for the β -phospho- galactosidase and the proteinase genes. In general, wild strains produced more odorous compounds and in higher amounts than the reference strains.

Enzymatic analyses of house dust mite extracts from Dermatophagoides pteronyssinus and Dermatophagoides farinae (Acari: Pyroglyphidae) during different phases of culture growth.

The majority of clinically important allergens of Dermatophagoides pteronyssinus (Trouessart, 1897) and Dermatophagoides farinae Hughes, 1961 present enzymatic activity. The allergenic enzymes described include cysteine proteases in group 1 allergens, trypsins in group 3, amylases in group 4, and chymotrypsins in group 6. Apart from these, other possibly allergenic enzymes also have been identified. Therefore, enzymatic profiles were studied during the 3 growth periods of the mite population--latency phase, exponential growth phase, and death phase. The activity of 19 different enzymes was analyzed by means of the Api Zym system, a method that has been used to study both mite extracts and other allergenic materials. Our study has demonstrated that the extracts contain a large variety of enzymes. It has been observed that enzymatic activity is caused exclusively by mites because the control carried out on the culture medium was negative for all the enzymes studied. Generally, the levels of diverse enzymatic activity increased with the growth of the culture, and decreased later, in both species. However, proteases are the exception; they maintain a high level of activity during the death phase of the cultured mites. The ratio between trypsin and chymotrypsin activity can be used as an excellent tool for quality control parameters during obtention of allergenic mite extracts.

A new biotyping method for Streptococcus mutans with the API ZYM system

OBJECTIVE: To test a new system for the biotyping of Streptococcus mutans, based on the measurement of enzyme activity, and to investigate the relationship between biotype and in vitro susceptibility to seven clinically useful antibiotics. METHODS: In total, 160 oral isolates of S. mutans were classified into different biotypes with the APIZYM test for enzyme activity, excluding results that were positive or negative in >80% of the strains. The susceptibility of all 160 strains to amoxycillin, cefazolin, erythromycin, clindamycin, vancomycin, teicoplanin and imipenem was tested by dilution in a solid medium. Statistical analysis of susceptibility (mean minimum inhibitory concentrations (MICs)) was based on chi-squared tests. RESULTS: Eight different biotypes (1-8) were identified on the basis of three kinds of enzyme activity: valine aryl amidase, acid phosphatase and alpha-galactosidase. Biotype 5 was found to be the most common. The mean MIC values showed strains belonging to biotype 4 to be the most susceptible to amoxycillin, cefazolin and erythromycin, whereas biotype 1 was the least susceptible to teicoplanin. CONCLUSIONS: The proposed biotyping method, which is relatively fast and simple to perform, provided reproducible results, and may contribute to clinically effective treatment of S. mutans infections.

A study of the enzymatic profile of soil isolates of Nocardia asteroides.

In this study, using the API-ZYM system, we have reported the enzyme profile of 42 soil strains and 2 clinical strains of Nocardia asteroides isolated locally. Of the 19 enzymes tested, only 7 were demonstrable in over 90% of the soil isolates. These included alkaline phosphatase, esterase lipase, leucine arylamidase, acid phosphatase, phosphohydrolase, alpha-glucosidase and beta-glucosidase. In addition, beta-galactosidase activity was demonstrated in all the strains by the O-nitrophenyl-beta-D-galactopyranoside (ONPG) test. The enzymes which were not demonstrable in > 95% of the strains included valine arylamidase, cystine arylamidase, trypsin, chymotrypsin, alpha-galactosidase, beta-glucoronidase, N-acetyl-beta-glucosaminidase, alpha-mannosidase and alpha-fucosidase. With the exception of valine arylamidase, which was lacking in all but one isolate, the enzyme profiles of the soil isolates were comparable with the clinical isolates of N. asteroides reported in previous studies. The reasons for this difference in the two sets of isolates is not clear. The study reinforces the view that specific differences in the enzymatic profiles of Nocardia species could be used for their rapid identification. However, more extensive studies are needed to establish the reproducibility of this method. To the best of our knowledge, this is the first study of the enzymatic profile of soil isolates of N. asteroides originating from a single geographic region.

Characterization of anaerobic, gram-negative, nonpigmented, saccharolytic rods from subgingival sites in dogs.

We characterized 52 anaerobic, gram-negative, nonpigmented, saccharolytic rods that were isolated from healthy and diseased subgingival sites of 16 family-owned dogs with spontaneous, clinically diagnosed periodontitis. Phenotypic features were determined with use of standard biochemical methods, by enzymatic profiling with the API ZYM system, and by cellular fatty acid profiling. Genotypic characterization was performed by DNA-DNA hybridization. Four phenotypic groups, defined as the Bacteroides fragilis group, the Prevotella buccae-like rods, the Prevotella heparinolytica/Prevotella zoogleoformans-like rods, and the slimy bile-tolerant rods, designated the Bacteroides pyogenes/Bacteroides tectum group, were detected. P. buccae and the B. pyogenes/B. tectum group organisms were isolated significantly more often (P values, < .05 and < .005) from the diseased than the healthy subgingival sites. The phenotypically similar group of bile-tolerant organisms, including B. pyogenes and B. tectum, most likely constitutes a major component of the anaerobic, gram-negative, saccharolytic microflora in periodontal lesions in dogs, a flora different from that in humans.

Characteristics of an unusual anaerobic pigmented gram-negative rod isolated from normal and inflamed appendices.

During our studies of the bacterial etiology of appendicitis, we often isolated a previously undescribed anaerobic gram-negative rod. This organism resembled the Bacteroides fragilis group because it was resistant to bile and because of its special-potency-disk pattern (resistant to vancomycin, kanamycin, and colistin), but unlike the B. fragilis group, this bacterium produced brown pigment on media containing hemolysed blood. The cellular fatty acid pattern, with iso-C15:0 being the predominant acid, was most closely related to the fatty acid profile of Porphyromonas species; however, this organism differed from Porphyromonas species by being bile-resistant and by not producing butyrate as a metabolic endproduct. Enzymatic activities of 31 isolates were determined with use of the API ZYM system and Rosco diagnostic tablets. These profiles were different from those of Prevotella, Porphyromonas, and related species. This organism was isolated from 40% of appendiceal tissue samples; no obvious qualitative or quantitative difference in rates of isolation from patients with inflamed or normal appendices was observed.

Toxic and enzymatic activities of Vibrio vulnificus biotype 2 with respect to host specificity.

In this work, the enzymatic activities of selected strains of biotypes 1 and 2 of Vabrio vulnificus were analyzed by using conventional methods and the API ZYM system. The toxic activities of extracellular products (ECPs) were further evaluated by in vitro and in vivo experiments. The ECPs of both biotypes (i) showed high-level hydrolytic activities, (ii) displayed cytotoxicity for fish cell lines, and (iii) were lethal for eels. Exotoxins seem to be proteinaceous since heat treatment of ECP samples destroyed their toxicity. Only biotype 2 strains were virulent for cels, suggesting that host specificity must be related to differences in cell surface properties. Infectivity trials with other fish species also revealed that only biotype 2 strains were virulent.

Identification of new enzyme activities of several strains of Thermus species.

New thermostable enzyme activities of seven Thermus strains were compared using the API ZYM system. All the strains exhibited high levels of alpha- and beta-glycosidases, esterase (C4) and esterase-lipase (C8) activities intracellularly. Only T. thermophilus HB8 (ATCC 27634) showed alpha-glucosidase and esterase activities in the supernatant. According to the intensity of beta-galactosidase activity, Thermus strains were divided in three groups. Group 0, which showed a weak beta-galactosidase activity, included Thermus spp. ATCC 31674 (T351) and 27978 (X-1) as well as T. thermophilus ATCC 27634 (HB8). Group I which consisted of T. aquaticus ATCC 25104 (YT-1), ATCC 25105 (Y-VII-51B) and Thermus sp. ATCC 27737 (T2), had a specific activity of approximately 40.0 U mg-1 and galactose as inducer. T. aquaticus ATCC 31558 (group 2) was particularly effective for beta-galactosidase production (2840 U) with a specific activity of 98 U mg-1. For each strain, galactose (0.5%) was a better inducer of beta-galactosidase production than lactose (1%). The detection of beta-galactosidase activity was dependent on the derivative chromogenic substrates used (naphthyl or nitrophenol coupled to sugar). Oligosaccharides were synthesized from cellobiose, lactulose, maltose or lactose as substrates at high temperature in some strains of Thermus.

Enzymatic profiles of Prevotella, Porphyromonas, and Bacteroides species obtained with the API ZYM system and Rosco diagnostic tablets.

Enzymatic profiles of Prevotella, Porphyromonas, and Bacteroides species obtained with the API ZYM system and Rosco diagnostic tablets.

Bacterial digestive enzyme activity in the stomach and hepatopancreas of Meganyctiphanes norvegica (M. Sars, 1857)

Northern krill, Meganyctiphaunes norvegica were maintained in antibiotic-treated (“A”), and untreated (control, “C”) aquaria. Each 24 h (for 72 h) numbers of saprophytic (Colony Forming Units, CFU), chitinolytic, and total bacteria (Acridine Orange Direct Count, AODC), were determined in the stomachs and hepatopancreata of animals. Parallely, the activities of chitinase, N-acetyl-β- d-glucosaminidase (NAGase), protease, cellulase and laminarinase were measured, and bacterial and endogenous chitinases compared by Fast Protein Liquid Chromatography (FPLC). CFU in “C” group stomachs were stable, but varied widely in the “A” group. AODC did not vary to any great extent. Hepatopancreas CFU decreased by four orders in the “A” compared to the “C” group. In “C” hepatopancreata, AODC were slighly higher than in “A”. Numbers of chitinolytic bacteria fell steeply in the “A” group. Chitinase, NAGase, protease, and cellulase activities in the stomach were significantly lower (5% level) after 24 h in “A” than in “C”. Laminarinase activity was significantly reduced in both organs after 72 h. Hepatopancreas chitinase and protease activities showed significant reductions only after 24 h. FPLC elution profiles of NAGase from krill, and bacteria grown on a medium semi-selective for chitinolytic bacteria differed. A number of bacterial chitinase peaks were absent in the “A” group. The API Zym system showed reduced proteolytic and lipase (C14) activity over 72 h. Endogenous bacteria are considered commensal with respect to the enzymes discussed.

Incidence and morphological variability of nocardia pinensis in australian activated sludge plants

Organisms exhibiting the typical microscopic branching patterns of Nocardia pinensis were seen in samples from 18/69 foaming and 13/75 non-foaming Australian activated sludge plants covering a wide geographic and climatic area. Although considerable variations in their morphology were observed under the microscope, particularly with respect to their branching angles, branch lengths and interbranch distances, pure cultures obtained by micromanipulation from 27 of these plants were remarkably consistent in their morphological and physiological properties, corresponded to previously described isolates of Nocardia pinensis, and were easily differentiated from 25 control cultures of related organisms using the API ZYM system. N. pinensis is readily differentiated from other “nocardias” when activated sludge samples are examined microscopically and should be reported separately.