Amidase Research Articles

Protective efficacy of Alum adjuvanted Amidase protein vaccine against Staphylococcus aureus infection in multiple mouse models.

Staphylococcus aureus is an opportunistic pathogen of humans. No commercial vaccine is available to combat S. aureus infections. In this study, we have investigated the protective immune response generated by S. aureus non-covalently associated cell wall surface protein N-acetylmuramoyl-L-alanine amidase (AM) in combination with Alum (Al) and heat-killed S. aureus (hkSA) using murine models. BALB/c mice were immunized with increasing concentrations of AM antigen or hkSA to determine their optimum concentration for vaccination. Fifty micrograms of AM and hkSA each were found to generate maximum anti-AM IgG antibody production. BALB/c mice were immunized next with 50µg of AM, 50µg of hKSA and 1mg Al vaccine formulation. Vaccine efficacy was validated by challenging immunized BALB/c mice with S. aureus Newman and three clinical methicillin-resistant S. aureus strains. AM-hkSA-Al-immunized mice generated high anti-AM IgG antibody response with IgG1 and IgG2b as the predominant immunoglobulin subtypes. Increased survival (60%-90%) with decreased clinical disease symptoms was observed in the vaccinated BALB/c mice group. A significantly lower bacterial load and decreased kidney abscess formation was observed following the challenge with S. aureus in the vaccinated BALB/c mice group. Furthermore, the efficacy of AM-hkSA-Al vaccine was also validated using C57 BL/6 and Swiss albino mice. Using murine infection models, we have demonstrated that AM-hkSA-Al vaccine would be effective in preventing S. aureus infections. AM-hkSA-Al vaccine elicited strong immune response and may be considered for future vaccine design against S. aureus infections.

A novel flow cytometry assay based on bacteriophage-derived proteins for Staphylococcus detection in blood

Bloodstream infections (BSIs) are considered a major cause of death worldwide. Staphylococcus spp. are one of the most BSIs prevalent bacteria, classified as high priority due to the increasing multidrug resistant strains. Thus, a fast, specific and sensitive method for detection of these pathogens is of extreme importance. In this study, we have designed a novel assay for detection of Staphylococcus in blood culture samples, which combines the advantages of a phage endolysin cell wall binding domain (CBD) as a specific probe with the accuracy and high-throughput of flow cytometry techniques. In order to select the biorecognition molecule, three different truncations of the C-terminus of Staphylococcus phage endolysin E-LM12, namely the amidase (AMI), SH3 and amidase+SH3 (AMI_SH3) were cloned fused with a green fluorescent protein. From these, a higher binding efficiency to Staphylococcus cells was observed for AMI_SH3, indicating that the amidase domain possibly contributes to a more efficient binding of the SH3 domain. The novel phage endolysin-based flow cytometry assay provided highly reliable and specific detection of 1–5 CFU of Staphylococcus in 10 mL of spiked blood, after 16 hours of enrichment culture. Overall, the method developed herein presents advantages over the standard BSIs diagnostic methods, potentially contributing to an early and effective treatment of BSIs.

Structure and mechanism of the amidase from Geobacilus pallidus

Structure and mechanism of the amidase from <i>Geobacilus pallidus</i>

Diagnostic value of Streptococcus pneumoniae autolysin rLytA for community acquired pneumonia

Objective To obtain the recombinant protein LytA (rLytA) of Streptococcus pneumoniae strain (ATCC49619) through prokaryotic expression system and to investigate their diagnostic value for patients with community acquired pneumonia (CAP). Methods The specific primers were designed according to LytA gene sequence of Streptococcus pneumoniae M66 strain recorded in Genbank. The recombinant plasmid pET32a(+ )/LytA was constructed and transformed into BL21(DE3) to express LytA.The expressed protein LytA was purified by electroeluting of bag filter. Serum IgM of anti-LytA accordingly of patients with CAP were detected by ELISA. The results were evaluated by Chi-square test. Results The recombinant protein LytA was expressed and purified successfully with a relative molecular weight of 56 000. The IgM antibodies level of anti-LytA was significantly higher than the healthy control group (P=0.000). Diagnostic sensibility and specificity of LytA-IgM were 27.8% and 100.0%, while sensibility and specificity of sputum culture were 19.4% and 72.2%, respectively. The sensibility of LytA-IgM was equal to sputum culture(χ2=0.693, P=0.405), but the specificity was higher than it(χ2=14.316 P=0.000). Conclusions A rLytA-ELISA assay maybe has clinical value for diagnosis of pneumococcal infections. It is more rapid and objective than the culture method.(Chin J Lab Med, 2017, 40: 212-215) Key words: Streptococcus pneumoniae; Pneumonia; Community-acquired infections; N-acetylmuramoyl-L-alanine amidase; Serologic tests; Enzyme-linked immunosorbent assay

Identification and Application of Enantiocomplementary Lactamases for Vince Lactam Derivatives

AbstractFour enzymes showing hydrolytic activity on derivatives of 2‐azabicyclo[2.2.1]hept‐5‐en‐3‐one (Vince lactam) were successfully identified through analysis of protein crystal structure and amino acid sequence alignments. Enantiocomplementary activities were observed on Vince lactam and its saturated analog 2‐azabicyclo[2.2.1]heptan‐3‐one with non‐heme chloroperoxidase (CPO‐T) from Streptomyces aureofaciens, cyclic imide hydrolase (CIH) from Pseudomonas putida, polyamidase (NfpolyA) from Nocardia farcinica, and amidase (AMI) from Rhodococcus globerulus, and perfect kinetic resolution was achieved (E&gt;200). Computational analysis of amide bond resonance stabilization in lactams correlated well with the overall reactivity pattern of the lactams as a function of ring size and strain. The biocatalysts cloned and investigated in this study could be of interest for the synthesis of enantiopure carbocyclic nucleoside analogues.

Contribution of the Staphylococcus aureus Atl AM and GL Murein Hydrolase Activities in Cell Division, Autolysis, and Biofilm Formation

The most prominent murein hydrolase of Staphylococcus aureus, AtlA, is a bifunctional enzyme that undergoes proteolytic cleavage to yield two catalytically active proteins, an amidase (AM) and a glucosaminidase (GL). Although the bifunctional nature of AtlA has long been recognized, most studies have focused on the combined functions of this protein in cell wall metabolism and biofilm development. In this study, we generated mutant derivatives of the clinical S. aureus isolate, UAMS-1, in which one or both of the AM and GL domains of AtlA have been deleted. Examination of these strains revealed that each mutant exhibited growth rates comparable to the parental strain, but showed clumping phenotypes and lysis profiles that were distinct from the parental strain and each other, suggesting distinct roles in cell wall metabolism. Given the known function of autolysis in the release of genomic DNA for use as a biofilm matrix molecule, we also tested the mutants in biofilm assays and found both AM and GL necessary for biofilm development. Furthermore, the use of enzymatically inactive point mutations revealed that both AM and GL must be catalytically active for S. aureus to form a biofilm. The results of this study provide insight into the relative contributions of AM and GL in S. aureus and demonstrate the contribution of Atl-mediated lysis in biofilm development.

Phylogeny of the Staphylococcal Major Autolysin and Its Use in Genus and Species Typing

The major staphylococcal autolysin Atl is an important player in cell separation and daughter cell formation. In this study, we investigated the amino acid sequences of Atl proteins derived from 15 staphylococcal and 1 macrococcal species representatives. The overall organization of the bifunctional precursor protein consisting of the signal peptide, a propeptide (PP), the amidase (AM), six repeat sequences (R(1) to R(6)), and the glucosaminidase (GL) was highly conserved in all of the species. The most-conserved domains were the enzyme domains AM and GL; the least-conserved regions were the PP and R regions. An Atl-based phylogenetic tree for the various species representatives correlated well with the corresponding 16S rRNA-based tree and also perfectly matched the phylogenetic trees based on core genome analysis. The phylogenetic distance analysis of 18 AtlA proteins of various Staphylococcus aureus strains and 15 AtlE proteins of S. epidermidis revealed that both species representatives formed a relatively homogeneous cluster. Two S. epidermidis strains, M23864:W1 and VCU116, were identified by Atl typing that clustered far more distantly and belonged to either S. caprae and S. capitis or a new subspecies. Here we show that Atl typing is a useful tool for staphylococcal genus and species typing by using either the highly conserved AM domain or the less-conserved PP domain.

Erratum: First report of an antifungal amidase from Peltophorum ptercoarpum

Erratum: First report of an antifungal amidase from <i>Peltophorum ptercoarpum</i>

Crystallization, diffraction data collection and preliminary crystallographic analysis of hexagonal crystals ofPseudomonas aeruginosaamidase

The aliphatic amidase (acylamide amidohydrolase; EC 3.5.1.4) from Pseudomonas aeruginosa is a hexameric enzyme composed of six identical subunits with a molecular weight of approximately 38 kDa. Since microbial amidases are very important enzymes in industrial biocatalysis, the structural characterization of this enzyme will help in the design of novel catalytic activities of commercial interest. The present study reports the successful crystallization of the wild-type amidase from P. aeruginosa. Native crystals were obtained and a complete data set was collected at 1.4 A resolution, although the crystals showed diffraction to 1.25 A resolution. The crystals were found to belong to space group P6(3)22, with unit-cell parameters a = b = 102.60, c = 151.71 A, and contain one molecule in the asymmetric unit.

Production of glycosylated thermostable penicillin G amidase in

Production of glycosylated thermostable penicillin G amidase in

Evidence that cysteine-166 is the active-site nucleophile of Pseudomonas aeruginosa amidase: crystallization and preliminary X-ray diffraction analysis of the enzyme

Wild-type and site-specific mutants C166S and C166A (Cys-166-->Ser and Cys-166-->Ala respectively) of the amidase (acylamide amidohydrolase, EC 3.5.1.4) from Pseudomonas aeruginosa were expressed in Escherichia coli by using the vector pKK223-3. Both mutant proteins were catalytically inactive but showed complete cross-reactivity with polyclonal antiserum raised against the wild-type enzyme, as well as CD spectra identical with that of the wild-type enzyme, which were indicative of correct folding. Cys-166 is therefore implicated as the active-site nucleophile. Titration of free thiol groups with 5,5'-dithiobis-(2-nitrobenzoic acid) indicated that Cys-166 is not a rapidly reacting residue. Crystals of both wild-type and C166S amidase grew with identical, rhombohedral morphology; X-ray diffraction analysis established the unit cell dimensions (a=b=c=84 A; alpha=beta=gamma=75 degrees) and space group (R3 or R32). These results imply a quaternary structure of six subunits, with most probably 32 symmetry; the existence of a hexameric structure was supported by molecular mass determinations based on gel filtration and electrophoretic mobility.

Evidence that cysteine-166 is the active-site nucleophile of Pseudomonas aeruginosa amidase: crystallization and preliminary X-ray diffraction analysis of the enzyme

Wild-type and site-specific mutants C166S and C166A (Cys-166 → Ser and Cys-166 → Ala respectively) of the amidase (acylamide amidohydrolase, EC 3.5.1.4) from Pseudomonas aeruginosa were expressed in Escherichia coli by using the vector pKK223-3. Both mutant proteins were catalytically inactive but showed complete cross-reactivity with polyclonal antiserum raised against the wild-type enzyme, as well as CD spectra identical with that of the wild-type enzyme, which were indicative of correct folding. Cys-166 is therefore implicated as the active-site nucleophile. Titration of free thiol groups with 5,5ʹ-dithiobis-(2-nitrobenzoic acid) indicated that Cys-166 is not a rapidly reacting residue. Crystals of both wild-type and C166S amidase grew with identical, rhombohedral morphology; X-ray diffraction analysis established the unit cell dimensions (a = b = c = 84 Å; α = β = γ = 75 °) and space group (R3 or R32). These results imply a quaternary structure of six subunits, with most probably 32 symmetry; the existence of a hexameric structure was supported by molecular mass determinations based on gel filtration and electrophoretic mobility.

Proteolytic processing of penicillin amidase from Alcaligenes faecalis cloned in E. coli yields several active forms

Of four enzymatically active forms of Alcaligenes faecalis penicillin amidase (EC 3.5.1.11) observed in sonicated cells, two (PA5.5 and PA5.3; subscript denotes pI) could be isolated and purified in two steps from the cells destroyed by osmotic shock. Active enzyme was only found in the periplasm. PA5.5 converts further to PA5.3 which differs in the molecular mass of the A-chain. The origin of these differences is a conversion of the N-terminal Gln to pyrolideno-carboxilic acid and a loss of three amino acids from the C-terminus of the A-chain. PA5.3 had higher specific activity (10–30%) for the hydrolysis of benzylpenicillin and 6-nitro-3-phenylacetamidobenzoic acid than PA5.5. © Rapid Science Ltd. 1998

Zymographic characterization of Staphylococcus aureus cell wall.

Susceptibilities of several preparations of Staphylococcus aureus cells to various peptidoglycan hydrolases with known bond specificity were analyzed by zymography. The substrates were intact S. aureus cells, cells boiled in the presence of SDS and cells treated with trichloroacetic acid after treatment with boiling SDS solution (TCA-cells). Twofold dilutions of lysostaphin (LS), lysozyme (LZ), S. aureus 51 kDa glucosaminidase (GL) or S. aureus 62 kDa amidase (AM) were electrophoresed, and the minimal enzyme dose showing a visible bacteriolytic band was defined as MBD (minimal bacteriolytic dose). Under the same experimental conditions, this method gave reproducible results. As the substrate for zymogram, TCA-cells were the most sensitive to LS, LZ and AM, whereas the three substrate were equally sensitive to GL. A zymographic analysis of methicillin-resistant S. aureus treated with methicillin together with previous studies suggest that this method can be used for the preliminary characterization of S. aureus cell wall peptidoglycan.

Identification of endo-beta-N-acetylglucosaminidase and N-acetylmuramyl-L-alanine amidase as cluster-dispersing enzymes in Staphylococcus aureus.

Two proteins which are capable of dispersing cell clusters of Staphylococcus aureus have been purified from a S. aureus FDA209P culture supernatant. Both of them were found to have bacteriolytic activity. From the elution profile of column chromatography and Western blot (immunoblot) analysis, one of them was identified as a 51-kDa endo-beta-N-acetylglucosaminidase (GL). The other was a 62-kDa protein on the basis of sodium dodecyl sulfate gel electrophoresis. Analysis of the peptidoglycan fragments following treatment with the 62-kDa protein indicated that this protein is an N-acetylmuramyl-L-alanine amidase (AM). In vitro studies of cluster dispersion activities using S. aureus mutant strains Lyt66 or S. aureus Wood46 grown as clusters demonstrated that these two enzymes act synergistically to disperse clusters into single cells. Antiserum against the 51-kDa GL cross-reacted with the 62-kDa AM, and S. aureus FDA209P grown in the presence of anti-51-kDa-GL immunoglobulin G induced giant clusters. Clusters induced by anti-51-kDa GL and by Cibacron blue F3G-A were dispersed by coincubation with the 51-kDa GL and the 62-kDa AM. Western blot analysis demonstrated that the 51-kDa GL and the 62-kDa AM were missing in culture supernatants of S. aureus Lyt66, Wood46, and RUSAL2 (Tn551 autolysin-defective mutant), which grow in clusters. These results strongly suggest that the 51-kDa GL and 62-kDa AM are involved in cell separation of daughter cells after cell division.

Purification and characterisation of a microbial l-carnitine amidase

Purification and characterisation of a microbial l-carnitine amidase

Purification, properties and heterologous expression of formamidase from Methylophilus methylotrophus

Summary: The obligately methylotrophic bacterium Methylophilus methylotrophus readily uses formamide as a source of nitrogen for growth. Physiological investigations using batch, fed-batch and continuous cultures indicated that the organism contains a discrete formamidase (formamide amidohydrolase; EC 3.5.1.49), in addition to the previously characterized aliphatic amidase (acylamide amidohydrolase; EC 3.5.1.4) which is specific to short-chain aliphatic amides such as acetamide, propionamide and acrylamide. Formamidase synthesis was induced by formamide and acetamide, and repressed by ammonia. The enzyme was purified using anion-exchange and gel-filtration FPLC and shown to exhibit a narrow substrate specificity (high activity with formamide, little activity with short-chain aliphatic amides, no activity with urea). SDS-PAGE and gel-filtration FPLC showed that the enzyme comprises a single type of subunit and probably exists as a homodimer. A 3·2 kbp Pstl restriction fragment of chromosomal DNA from M. methylotrophus was cloned in pUC19 and expressed in Escherichia coli JM109. The purified gene product exhibited essentially identical properties to those of the M. methylotrophus formamidase in terms of its chromatographic behaviour, native M r (123 000), subunit M r (51 000), K cat (58 cf. 64 s−1), K m (1·6 cf. 2·1 mM), substrate specificity and N-terminal amino acid sequence (MKTIV-).

A modified and improved assay for sperm amidase activity.

An assay for sperm amidase activity has been modified so that many samples can be measured rapidly, making it more suitable for use in a clinical setting. The modified assay gave the same results (P greater than 0.05) as its lengthier parent assay (Kennedy et al, 1989). The measured enzyme activity was proportional to the number of sperm assayed over the range tested (3.5 x 10(5) to 2.1 x 10(6) sperm). The within-assay coefficient of variation was 7.8 +/- 1.8% (average +/- SE, n = 12), and the between-assay coefficient of variation was 7.2 +/- 1.2% (n = 3). The sensitivity was 7.6 microIU/well.

One-step affinity purification of amidase from mutant strains of Pseudomonas aeruginosa

Amidases (acylamide amido hydrolase EC 3.5.1.4) from mutant strains ( i.e., B6, AI103, AIU1N, OUCH 4 and L10) of Pseudomonas aeruginosa were purified in one-step by ligand affinity chromatography using Epoxy-activated Sepharose 4B-acetamide. The yields of the purified enzymes were about 90% for all mutant strains with purification factors of about 10 and were apparently homogeneous when analysed by SDS-PAGE and native PAGE. The protein bands on native PAGE coincided with the stained band of enzyme activity for all amidase preparations. Affinity columns had a maximum binding capacity of 0.5 mg amidase protein/ml of sedimented gel and could be regenerated and reused several times without any loss of binding capacity and resolution. Affinity gels containing either semicarbazide or urea were also found useful for the isolation of amidase. The differences in substrate specificity of these amidases reported previously were also observed in the elution behaviour of these enzymes from the affinity columns.

Purification and properties of an acylamide amidohydrolase (E. C. 3.5.1.4) with a wide activity spectrum from Brevibacterium sp. R 312

AbstractAn amidase with a wide activity spectrum was purified from the Brevibacterium sp. R 312 and studied. The purification was performed by precipitating the proteins of the supernatant fraction obtained after centrifugation of sonicated cells. The resulting proteins were submitted to a gel filtration step followed by DEAE‐Sephadex ion exchange chromatography and then by another gel filtration process. The purified amidase had a molecular weight of 180,000 and was composed of four subunits of the same molecular weight (43,000 + 2,000). Its isoelectric point was 3.5. This enzyme was able to hydrolyze a large number of amides into their corresponding organic acids and it also possessed an acyl transferase activity.