Amin Karmali

Structure of amidase from Pseudomonas aeruginosa showing a trapped acyl transfer reaction intermediate state

Microbial amidases belong to the thiol nitrilases family and have potential biotechnological applications in chemical and pharmaceutical industries as well as in bioremediation. The amidase from Pseudomonas aeruginosa isa6 × 38-kDa enzyme that catalyzes the hydrolysis of a small range of short aliphatic amides. The hereby reported high resolution crystallographic structure shows that each amidase monomer is formed by a globular four-layer αββα sandwich domain with an additional 81-residue long C-terminal segment. This wraps arm-in-arm with a homologous C-terminal chain of another monomer, producing a strongly packed dimer. In the crystal, the biological active homo-hexameric amidase is built grouping three such dimers around a crystallographic 3-fold axis. The structure also elucidates the structural basis for the enzyme activity, with the nitrilases catalytic triad at the bottom of a 13-Å deep, funnel-shaped pocket, accessible from the solvent through a narrow neck with 3-Å diameter. An acyl transfer intermediate, resulting from the purification protocol, was found bound to the amidase nucleophilic agent, Cys166. These results suggest that some pocket defining residues should undergo conformational shifts to allow substrates and products to access and leave the catalytic pocket, for turnover to occur.

Substitutions of Thr-103-Ile and Trp-138-Gly in amidase from Pseudomonas aeruginosa are responsible for altered kinetic properties and enzyme instability

Pseudomonas aeruginosa Ph1 is a mutant strain derived from strain AI3. The strain AI3 is able to use acetanilide as a carbon source through a mutation (T103I) in the amiE gene that encodes an aliphatic amidase (EC 3.5.1.4). The mutations in the amiE gene have been identified (Thr103Ile and Trp138Gly) by direct sequencing of PCR-amplified mutant gene from strain Ph1 and confirmed by sequencing the cloned PCR-amplified gene. Site-directed mutagenesis was used to alter the wild-type amidase gene at position 138 for Gly. The wild-type and mutant amidase genes (W138G, T103I-W138G, and T103I) were cloned into an expression vector and these enzymes were purified by affinity chromatography on epoxy-activated Sepharose 6B-acetamide/phenylacetamide followed by gel filtration chromatography. Altered amidases revealed several differences in kinetic properties, namely, in substrate specificity, sensitivity to urea, optimum pH, and enzyme stability, compared with the wild-type enzyme. The W138G enzyme acted on acetamide, acrylamide, phenylacetamide, and p-nitrophenylacetamide, whereas the double mutant (W138G and T103I) amidase acted only on p-nitrophenylacetamide and phenylacetamide. On the other hand, the T103I enzyme acted on p-nitroacetanilide and acetamide. The heat stability of altered enzymes revealed that they were less thermostable than the wild-type enzyme, as the mutant (W138G and W138G-T103I) enzymes exhibited t1/2 values of 7.0 and 1.5 min at 55°C, respectively. The double substitution T103I and W138G on the amidase molecule was responsible for increased instabiliby due to a conformational change in the enzyme molecule as detected by monoclonal antibodies. This conformational change in altered amidase did not alter its Mr value and monoclonal antibodies reacted differently with the active and inactive T103I-W138G amidase.

Production, purification and characterisation of polysaccharides from Pleurotus ostreatus with antitumour activity

BACKGROUND Mushroom polysaccharides play an important role in functional foods because they exhibit biological modulator properties such as antitumour, antiviral and antibacterial activities. The present study involved the production, purification and characterisation of intracellular and extracellular free and protein-bound polysaccharides from Pleurotus ostreatus and the investigation of their growth-inhibitory effect on human carcinoma cell lines. RESULTS Several fermentation parameters were obtained: batch polysaccharide productivities of 0.013 ± 8.12 × 10⁻⁵ and 0.037 ± 0.0005 g L⁻¹ day⁻¹ for intracellular and extracellular polysaccharides respectively, a maximum biomass concentration of 9.35 ± 0.18 g L⁻¹ , P(max) = 0.935 ± 0.018 g L⁻¹ day⁻¹, µ(max) = 0.218 ± 0.02 day⁻¹, Y(EP/X) = 0.040 ± 0.0015 g g⁻¹ and Y(IP/X) = 0.014 ± 0.0003 g g⁻¹ . Some polysaccharides exhibited superoxide dismutase (SOD)-like activity of 50-200 units. Fourier transform infrared analysis of the polysaccharides revealed absorption bands characteristic of such biological macromolecules. Cytotoxicity assays showed that both intracellular and extracellular polysaccharides exhibited antitumour activity towards several tested human carcinoma cell lines in a dose-dependent manner. CONCLUSION The polysaccharides of P. ostreatus exhibited high SOD-like activity, which strongly supports their biological effect on tumour cell lines. The extracellular polysaccharides presented the highest antitumour activity towards the RL95 carcinoma cell line and should be further investigated as an antitumour agent.

Glucose 1- and 2-oxidases from fungal strains : isolation and production of monoclonal antibodies

Monoclonal antibodies (Mabs) against purified glucose 2-oxidase (EC 1.1.3.10) from Coriolus versicolor were raised by hybridoma technology using Sp2/0 myeloma cells as a fusion partner. Hybrid growth was observed in 42% of culture wells and 30% of these (i.e. 30 culture wells) contained anti-glucose 2-oxidase activity. Three positive wells containing hybrid cell lines were selected and cloned twice by the limiting dilution method and two hybridoma clones (E1A5 and E1A6) secreting Mabs were selected at random for purification and characterisation purposes. Both cell lines secreted Mabs of IgM class which were purified by gel filtration chromatography on a Sephacryl S-200 column with a final recovery of 80% and a purification factor of 16. The purified preparations were apparently homogeneous on native PAGE running with a M(r) of 950 kDa. Mabs were highly specific for glucose 2-oxidase as determined by Western blotting. These Mabs also crossreacted with glucose 1- and 2-oxidases from other fungal sources (Phanerochaeta chrysosporium, Penicillium amagasakiense and Aspergillus niger) as determined by Western blotting and by ELISA. Both glucose 1- and 2-oxidases from C. versicolor, P. chrysosporium, P. amagasakiense and A. niger were purified by hydrophobic interaction chromatography on Sepharose 4B-triazine dye with a recovery of enzyme activity in the range 85-92%. Purified preparations of glucose oxidases from fungal strains were apparently homogeneous on native PAGE. Glucose 2-oxidases were more hydrophobic than glucose 1-oxidases as determined by their chomatographic behaviour on Sepharose 4B-Cibacron Red G-E which could be used to study their roles in lignin biodegradation.

Substitution of Glu-59 by Val in amidase from Pseudomonas aeruginosa results in a catalytically inactive enzyme

A mutant strain, KLAM59, of Pseudomonas aeruginosa has been isolated that synthesizes a catalytically inactive amidase. The mutation in the amidase gene has been identified (Glu59Val) by direct sequencing of PCR-amplified mutant gene and confirmed by sequencing the cloned PCR-amplified gene. The wild-type and altered amidase genes were cloned into an expression vector and both enzymes were purified by affinity chromatography on epoxy-activated Sepharose 6B-acetamide followed by gel filtration chromatography. The mutant enzyme was catalytically inactive, and it was detected in column fractions by monoclonal antibodies previously raised against the wild-type enzyme using an ELISA sandwich method. The recombinant wild-type and mutant enzymes were purified with a final recovery of enzyme in the range of 70–80%. The wild-type and mutant enzymes behaved differently on the affinity column as shown by their elution profiles. The molecular weights of the purified wild-type and mutant amidases were found to be 210,000 and 78,000 Dalton, respectively, by gel filtration chromatography. On the other hand, the mutant enzyme ran as a single protein band on SDS-PAGE and native PAGE with a Mr of 38,000 and 78,000 Dalton, respectively. These data suggest that the substitution Glu59Val was responsible for the dimeric structure of the mutant enzyme as opposed to the hexameric form of the wild-type enzyme. Therefore, the Glu59 seems to be a critical residue in the maintenance of the native quaternary structure of amidase.

Immobilized metal affinity chromatography of monoclonal immunoglobulin M against mutant amidase from Pseudomonas aeruginosa.

The chromatographic behavior of monoclonal antibodies (MAbs) of immunoglobulin (Ig) M class against mutant (T103I) amidase from Pseudomonas aeruginosa was investigated on immobilized metal chelates. The effect of ligand concentration, the length of spacer arm, and the nature of metal ion were investigated in immobilized metal affinity chromatography (IMAC). The MAbs against mutant amidase adsorbed to Cu(II), Ni(II), Zn(II), Co(II), and Ca(II)-iminodiacetic acid (IDA) agarose columns. The increase in ligand concentration (epichlorohydrin: 30–60 and 1,4-butanediol-diglycidyl ether: 16–36) resulted in higher adsorption to IgM into immobilized metal chelates. The length of spacer arm was found to affect protein adsorption, as longer spacer arm (i.e., 1,4-butanediol-diglycidyl ether) increased protein adsorption of immobilized metal chelates. The adsorption of IgM onto immobilized metal chelates was pH dependent because an increase in the binding of IgM was observed as the pH varied from 6.0 to 8.0. The adsorption of IgM to immobilized metal chelates was the result of coordination of histidine residues to metal chelates that are available in the third constant domain of heavy chain (CH3) of immunoglobulins, as the presence of imidazole (5 mM) in the equilibration buffer abolished the adsorption of IgM to the column. The combination of tailor-made stationary phases for IMAC and a correct design of the adsorption parameters permitted to devise a one-step purification procedure for IgM. Culture supernatants containing IgM against mutant amidase (T103I) were purified either by IMAC on EPI-60-IDA-Co (II) column or by gel filtration chromatography on Sephacryl S-300HR. The specific content of IgM and final recovery of antibody activity exhibited similar values for both purification schemes. The purified preparations of IgM obtained by both schemes were apparently homogeneous on native polyacrylamide gel electrophoresis with a Mr of 851,000 Da. The results presented in this work strongly suggest that one-step purification of IgM by IMAC is a cost-effective and process-compatible alternative to other types of chromatography.

A high throughput colorimetric assay of β-1,3-D-glucans by Congo red dye.

Mushroom strains contain complex nutritional biomolecules with a wide spectrum of therapeutic and prophylactic properties. Among these compounds, β-d-glucans play an important role in immuno-modulating and anti-tumor activities. The present work involves a novel colorimetric assay method for β-1,3-d-glucans with a triple helix tertiary structure by using Congo red. The specific interaction that occurs between Congo red and β-1,3-d-glucan was detected by bathochromic shift from 488 to 516 nm (>20 nm) in UV-Vis spectrophotometer. A micro- and high throughput method based on a 96-well microtiter plate was devised which presents several advantages over the published methods since it requires only 1.51 μg of polysaccharides in samples, greater sensitivity, speed, assay of many samples and very cheap. β-D-Glucans of several mushrooms (i.e., Coriolus versicolor, Ganoderma lucidum, Pleurotus ostreatus, Ganoderma carnosum, Hericium erinaceus, Lentinula edodes, Inonotus obliquus, Auricularia auricular, Polyporus umbellatus, Cordyseps sinensis, Agaricus blazei, Poria cocos) were isolated by using a sequence of several extractions with cold and boiling water, acidic and alkaline conditions and quantified by this microtiter plate method. FTIR spectroscopy was used to study the structural features of β-1,3-D-glucans in these mushroom samples as well as the specific interaction of these polysaccharides with Congo red. The effect of NaOH on triple helix conformation of β-1,3-D-glucans was investigated in several mushroom species.

Biosensor for acrylamide based on an ion-selective electrode using whole cells of Pseudomonas aeruginosa containing amidase activity.

The aim of this work was to develop a biosensor for toxic amides using whole cells of Pseudomonas. aeruginosa containing amidase activity, which catalyses the hydrolysis of amides such as acrylamide producing ammonia and the corresponding organic acid. Whole cells immobilized in several types of membrane in the presence of glutaraldehyde and an ammonium ion-selective electrode, were used for biosensor development. This biosensor exhibited a linear response in the range of 0.1–4.0×10−3 M of acrylamide, a detection limit of 4.48×10−5 M acrylamide, a response time of 55 s, a sensitivity of 58.99 mV mM−1 of acrylamide and a maximum t1/2 of 54 days. The selectivity of this biosensor towards other amides was investigated, which revealed that it cross-reacted with acetamide and formamide, but no activity was detected with phenylacetamide, p-nitrophenylacetamide and acetanilide. It was successfully used for quantification of acrylamide in real industrial effluents and recovery experiments were carried out which revealed an average substrate recovery of 93.3%. The biosensor is cheap since whole cells of P. aeruginosa can be used as source of amidase activity.

Characterization of monoclonal antibodies against altered (T103I) amidase from Pseudomonas aeruginosa

Monoclonal antibodies (MAbs) against mutant (T103I) amidase from Pseudomonas aeruginosa were raised by hybridoma technology. To select MAbs suitable for immunoaffinity chromatography, hybridoma clones secreting polyol-responsive MAbs (PR-MAbs) were screened that bind antigen tightly but release under mild and nondenaturing elution conditions. It was found that about 10% of enzyme-linked immunosorbent assay (ELISA)-positive hybridoma produce these MAbs as their ag-ab complex can be disrupted by propylene glycol in the presence of a suitable salt. Two of these hybridoma clones (F6G7 and E2A6) secreting PR-MAbs against mutant amidase were selected for optimization of experimental conditions for elution of amidase by using ELISA elution assay. These hybridoma cell lines secreted MAbs of IgM class that were purified in a single step by gel filtration chromatography, which revealed a single protein band on native polyacrylamide gel electrophoresis (PAGE). Specificity studies of this MAb revealed that it recognized specifically a common epitope on mutant and wild-type amidases as determined by direct ELISA. This MAb exhibited a higher affinity for denatured forms of wild-type and mutant amidases than for native forms as revealed by affinity constants (K), suggesting that it recognizes a cryptic epitope on an amidase molecule. Furthermore, MAb E2A6 inhibited about 60% of wild-type amidase activity, whereas it activated about 60% of mutant amidase (T103I) activity. The data presented in this work suggest that this MAb acts as a very useful probe to detect conformational changes in native and denatured amidases as well as to differentiate wild-type and mutant (T103I) amidases.

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

Amidases (acylamide amidohydrolase EC 3.5.1.4) from mutant strains (i.e., B6, AI3, 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.