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PubMed1975

Bacillus subtilis N-acetylmuramic acid L-alanine amidase.

The N-acetymuramic acid L-alanine amidase from Bacillus subtilis (ATCC 6051) has been purified to homogeneity. It is a monomeric protein of molecular weight 50,000. The enzyme has a high affinity for homologous cell walls and once attached to a cell wall will hydrolyze the wall completely before initiating the hydrolysis of a new cell wall. The affinity of the enzyme for cell walls devoid of teichoic acid or for cell walls of Bacillus megaterium is much lower than that for B. subtilis cell walls. A second homogenous protein has been isolated from B. subtilis which specifically combines with the amidase in a 1:1 molar ratio and stimulates enzyme activity. This modifier protein has no intrinsic cell wall lytic activity. The binding of enzyme and modifier protein has a dissociation constant of 8.5 times 10-9 M in 0.1 M LiCl, pH 8.0, but the two proteins can be completely dissociated in 3 M LiCl at pH 8.0.

Alanine↗

PubMed1993

Two forms of acidic arginine amidases in human kidney.

Two forms of acidic arginine amidases were separated from human kidney extract using the techniques of basic ion exchange adsorption and elution as well as lima bean trypsin inhibitor (LBTI) and aprotinin affinity adsorptions and elutions. The enzymes were tentatively named acidic human renal arginine amidase-L (AHRAA-L, with affinity to an LBTI column) and -A (AHRAA-A, with affinity to an aprotinin column). Both enzymes showed a similar molecular mass of approximately 3.0 x 10(4) daltons, differing from that of human renal kallikrein (HRK, molecular mass of 4.8 x 10(4) daltons). The specific activity of AHRAA-L and -A were 106 and 680 nmol/min/A280 of Val-Leu-Arg-pNA amidolysis, respectively, and they were strongly inhibited by LBTI and human urinary trypsin inhibitor (UTI), while ethylenglycol-bis(beta-amino ethylether)-N,N,N',N'-tetraacetic acid (EGTA) showed a weak or no effect on both enzymes.

Esterases↗

PubMed1976

Demonstration of pyroglutamylpeptidase and amidase activities toward thyrotropin-releasing hormone in hamster hypothalamus extracts.

Using a radioimmunoassay method for thyrotropin-releasing hormone, the presence of thyrotropin-releasing hormone-metabolizing activity in various hamster tissues was demonstrated. While there was substantial activity degrading thyrotropin-releasing hormone in hypothalamus, there was a notable absence of such activity in pituitary. The enzymatic activity in the hypothalamus was shown to be soluble and separable into two fractions. Analysis of the metabolic products formed by the two enzymes indicated that one possessed an amidase activity (less than Glu-His-Pro-NH2 leads to less than Glu-His-Pro) and the other possessed pyroglutamylpeptidase activity (less than Glu-His-Pro-NH2 leads to less than Glu+His-Pro-NH2). Other peptides containing NH2-terminal pyroglutamic acid or COOH-terminal amide groups did not block the hydrolysis of thyrotropin-releasing hormone, suggesting that the enzymes were specific. Some inhibitors preferentially blocked the activity of one or the other enzymes. Of possible biological significance is the observation that thyroid-stimulating hormone inhibited the amidase activity while hydrocortisone inhibited the pyroglutamylpeptidase activity.

Amidohydrolases↗

PubMed1979

Enzyme immobilization techniques on poly(glycidyl methacrylate-co-ethylene dimethacrylate) carrier with penicillin amidase as model.

Two types of bead-form macroporous carriers based on glycidyl methacrylate with ethylene dimethacrylate copolymers were used for the immobilization of penicillin amidase either directly or after chemical modification. Direct binding through oxirane groups, which is equally efficient at pH 4.2 and 7, is relatively slow and brings about an activity loss at low enzyme concentrations. The most efficient immobilization was achieved on glutaraldehyde-activated amino carrier, irrespective of whether the amino groups were formed by ammonia or 1,6-diaminohexane treatment of the original oxirane carrier. Hydrazine treatment gave lower immobilization yields. The same is true of the azide method independent of the length of the spacer. Most enzyme activity was preserved by coupling the carbodiimide-activated enzyme to the carrier with alkyl or arylamino groups at the end of a longer substituent. Immobilization on diazo-modified carrier gave average results. Rapid immobilization by a lysine-modified phosgene-treated carrier resulted in an activity loss. It is suggested that multipoint and very tight attachment of the enzyme molecule to the matrix decreased the activity. The immobilized activity is quite stable in solution and very stable upon lyophilization with sucrose.

Amidohydrolases↗

PubMed1994

Crystallization and preliminary crystallographic analysis of peptide-N4-(N-acetyl-beta-D-glucosaminyl)asparagine amidase PNGase F.

PNGase F is an amidase that hydrolyzes the beta-aspartylglucosylamine bond of asparagine-linked glycopeptides and glycoproteins. Enzymatic activity of PNGase F requires the recognition of both the peptide and the carbohydrate moiety. Crystals of PNGase F were grown by sitting drop vapor diffusion methods at 10 degrees C. The precipitating buffer contains both polyethylene glycol 3350 and (NH4)2SO4 in sodium acetate buffer at pH 4.3. The crystals belong to the orthorhombic space group C222(1) with cell dimensions: a = 87.16 A, b = 125.10 A, c = 79.33 A and diffract to 1.8 A resolution.

Amidohydrolases↗

PubMed1995

Characterization of human serum N-acetylmuramyl-L-alanine amidase purified by affinity chromatography.

Human serum N-acetylmuramyl-L-alanine amidase was purified to homogeneity by a relatively short procedure including affinity chromatography. For this purpose, a specific adsorbent was prepared by coupling the main substrate of the enzyme, N-acetyl-muramyl-L-alanine-gamma-D-glutamyl-L-meso-2,6[3,4,5-3H] diaminopimelic acid, to a divinylsulfone agarose gel. The enzyme is unable to hydrolyze this muramylpeptide when it is attached as a ligand to the gel, whereas a high affinity is conserved. In addition to affinity chromatography, the presented purification scheme includes ion exchange chromatography on DEAE-Sepharose and molecular sieving on Superdex 200. The enzyme was purified 739-fold with a yield of 22.5%. One single band at 135 kDa was obtained on native gradient PAGE. Gradient PAGE in denaturing conditions gave one single band at 74 kDa, which was lowered to 64 kDa when the enzyme was denatured in nonreducing conditions. This suggests that the native enzyme is a dimer consisting of two subunits of identical molecular weight with only intramolecular disulfide bonds. Isoelectric focusing gave one single band at pI 5.0. Glycan detection before and after treatment with N-glycosidase F showed that the enzyme is a glycoprotein. Further analysis by lectin immuno detection on dot blots confirmed that the enzyme is an N-glycosylated protein of complex type with sialic acid, terminally linked alpha (2-->6) to galactose or N-acetylgalactosamine. The 15 amino acid N-terminal sequence was determined by microsequence analysis.

Acetylmuramyl-Alanyl-Isoglutamine↗

PubMed1994

Periplasmic aggregation limits the proteolytic maturation of the Escherichia coli penicillin G amidase precursor polypeptide.

The Escherichia coli penicillin G amidase (PGA), which is a key enzyme in the production of penicillin G derivatives is generated from a precursor polypeptide by an unusual internal maturation process. We observed the accumulation of the PGA precursor polypeptide in the insoluble material recovered after sonication of recombinant E. coli JM109 cells grown at 26 degrees C. The aggregated nature of the accumulated molecules was demonstrated using detergents and chaotropic agents in solubilization assays. The periplasmic location of the aggregates was shown by trypsin-accessibility experiments performed on the spheroplast fraction. Finally, we showed that addition of sucrose or glycerol in the medium strongly reduces this periplasmic aggregation and as a consequence PGA production is substantially increased. Thus, periplasmic aggregation of the PGA precursor polypeptide limits PGA production by recombinant E. coli and this limitation can be overcome by addition in the medium of a non-metabolizable sugar, such as sucrose, or of glycerol.

Biotechnology↗

PubMed1992

Peptide-N4-(N-acetylglucosaminyl)asparagine amidase (PNGase) activity could explain the occurrence of extracellular xylomannosides in a plant cell suspension.

We have previously isolated mannoside and xylomannoside oligosaccharides with one or two terminal reducing N-acetylglucosamine residues from the extracellular medium of white campion (Silene alba) suspension culture. We have now demonstrated the presence of peptide-N4-(N-acetylglucosaminyl)asparagine amidase (PNGase) activity in cell extracts as well in the culture medium that could explain the production of those compounds. An additional xylomannoside, (GlcNAc)Man3(Xyl)GlcNAc(Fuc)GlcNAc, was characterized, and 1H- and 13C-NMR assignments for the oligosaccharide Man3(Xyl)GlcNAc(Fuc)GlcNAc were obtained using homonuclear and heteronuclear spectroscopy (COSY).

Amidohydrolases↗

PubMed1995

Characterization of the peptide-N4-(N-acetylglucosaminyl) asparagine amidase (PNGase Se) from Silene alba cells.

The peptide-N4-(N-acetylglucosaminyl) asparagine amidase (PNGase Se) earlier described [Lhernould S., Karamanos Y., Bourgerie S., Strecker G., Julien R., Morvan H. (1992) Glycoconjugate J 9:191-97] was partially purified from cultured Silene alba cells using affinity chromatography. The enzyme is active between pH 3.0 and 6.5, and is stable in the presence of moderate concentrations of several other protein unfolding chemicals, but is readily inactivated by SDS. Although the enzyme cleaves the carbohydrate from a variety of animal and plant glycopeptides, it does not hydrolyse the carbohydrate from most of the corresponding unfolded glycoproteins in otherwise comparable conditions. The substrate specificity of this plant PNGase supports the hypothesis that this enzyme could be at the origin of the production of 'unconjugated N-glycans' in a suspension medium of cultured Silene alba cells.

Amidohydrolases↗

PubMed1990

Optimized deglycosylation of glycoproteins by peptide-N4-(N-acetyl-beta-glucosaminyl)-asparagine amidase from Flavobacterium meningosepticum.

Peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F(PNGase F) from Flavobacterium meningosepticum is a highly useful enzyme for the structural analysis of N (asparagine)-linked carbohydrate chains derived from glycoproteins. The enzyme was enriched using a published procedure [Tarentino AL, Gomez CM, Plummer TH, Jr (1984) Biochemistry 1985:4665-71; Tarentino AL, Plummer TH, Jr (1987) Methods Enzymol 138:770-78] and further purified by hydrophobic interaction HPLC on a weak hydrophobic TSK-Ether column from which it was eluted by a decreasing gradient of 1.7 M ammonium sulphate in 100 mM sodium phosphate, pH 7.0, containing 5 mM EDTA. To determine the optimal conditions for a complete deglycosylation of glycoproteins by PNGase F, experiments were performed with human alpha 1-acid glycoprotein, because the five complex type carbohydrate chains are quite resistant to enzymic hydrolysis. The influence of different detergents on the enzyme reaction was studied. Complete deglycosylation of human alpha 1-acid glycoprotein was achieved by the use of 60 mU/ml PNGase F in 0.25 M sodium phosphate buffer, pH 8.6, containing 0.2% (w/v) SDS, 20 mM mercaptoethanol and 0.5% Mega-10.

Amidohydrolases↗

PubMed1994

The expression of the penicillin G amidase gene of Escherichia coli by primer extension analysis.

Escherichia coli ATCC 11105 and JM109, transformed with a multicopy plasmid carrying the penicillin G amidase (PGA) gene, were grown at 26 degrees and 37 degrees C, in the presence or the absence of phenylacetic acid (PAA) or of glucose. A method based on primer extension was developed to quantify in vivo levels of PGA mRNAs. A unique transcription start site was found to be used in all the fermentation conditions tested. This site is located 28 nucleotides upstream of the initiation codon. Its utilization is subjected to catabolic repression and is induced by PAA. This site is used at 37 degrees C, but the PGA mRNA level in E. coli ATCC 11105 is lower at 37 degrees C than at 26 degrees C. Induction of the pga gene by PAA was found to be more efficient in the producer strain. Taking into account the amount of PGA mRNA present in the cells at 37 degrees C, one would expect the production of active PGA at this temperature. This is not the case. Thus, at 37 degrees C, expression is blocked at a step after transcription.

Base Sequence↗

PubMed1990

Antibody-penicillin-V-amidase conjugates kill antigen-positive tumor cells when combined with doxorubicin phenoxyacetamide.

The two monoclonal antibodies (mAb), L6 (anti-carcinoma), and 1F5 [anti-(B-cell-lymphoma)], were chemically linked to the enzyme penicillin-V amidase (PVA), which hydrolyzes phenoxyacetamides, to explore the potential of using mAb-enzyme conjugates for the localization of chemotherapeutic drugs at tumor cells. The phenoxyacetamide derivatives of doxorubicin and melphalan were prepared, yielding the less toxic amides, doxorubicin-N-p-hydroxyphenoxyacetamide (DPO) and melphalan-N-p-hydroxyphenoxyacetamide (MelPO). These were hydrolyzed by PVA to doxorubicin and melphalan respectively. In vitro studies with the L6-positive lung carcinoma cell line, H2981, and the 1F5-positive B-cell lymphoma line, Daudi, showed that DPO was 80-fold less toxic to H2981 cells and 20-fold less toxic to Daudi cells than doxorubicin, and its toxicity was substantially increased when the H2981 cells were pretreated with L6-PVA or the Daudi cells were pretreated with 1F5-PVA. The cytotoxic effect was antigen-specific, since only the binding mAb-enzyme conjugate increased the cytotoxicity of the prodrug. MelPO was more than 1000-fold less toxic than melphalan to H2981 cells and more than 100-fold less toxic than melphalan to Daudi cells. Pretreatment with the mAb-PVA conjugates did not enhance the toxicity of MelPO in either cell line, because PVA hydrolyzes the phenoxyacetamide bond of MelPO too slowly to generate a toxic level of melphalan.

Amidohydrolases↗

PubMed1991

Cell aggregates of Escherichia coli with benzylpenicillin amidase activity.

Intact cells Escherichia coli CCM 2843, exhibiting substantial benzylpenicillin amidase activity, were bound mutually with supporting waste microbial cells, native or treated, to obtain an inexpensive biocatalyst for the production of 6-aminopenicillanic acid (6-APA). The bond was effected by glutaraldehyde (GA) and Sedipur CL-930 (PEI), without any carrier. The optimal concentration of GA was 2%, that of PEI 1%. The optimal biocatalyst was obtained by immobilization of productive cells with their fragments at a mass ratio of 4:1. The cell aggregates were used for hydrolysis of potassium benzyl-penicillin at a concentration of 5% to 6-APA. After 25 repeated batch conversions the degree of conversion did not decrease; its average value was 96.4%.

Bacterial Adhesion↗

PubMed1988

Structural requirements of choline derivatives for 'conversion' of pneumococcal amidase. A new single-step procedure for purification of this autolysin.

Tertiary amines appear to be the minimal structure needed to convert in vitro the inactive form (E-form) of pneumococcal amidase to the catalytic active form (C-form). Diethylethanolamine was one of the compounds that converted the E-form, a finding that has been used successfully to develop an affinity chromatography system in DEAE-cellulose for the rapid and efficient purification of lytic enzymes of pneumococcus and its bacteriophages.

Amidohydrolases↗

PubMed1994

Perfusible and non-perfusible supports with monoclonal antibodies for bioaffinity chromatography of Escherichia coli penicillin amidase within its pH stability range.

Several monoclonal antibodies (mABs) have been prepared and immobilized for the biospecific isolation of penicillin amidase (PA) from Escherichia coli (EC 3.5.1.11), an enzyme without S-S bridges and a pH stability range of 4-9. During the immobilization the fluorescence emission maxima of the mABs were found to change from 336 nm to ca. 350 nm. Only one of these mABs was found to be suitable for preparative bioaffinity chromatography of PA within the pH stability range. This mAB was immobilized on different spherical supports (Eupergit C 250 L and Sepharose) and one perfusible support (Knauer Quick Disc) and used for analytical and preparative bioaffinity chromatography. Under isocratic conditions the plate height for the perfusible biospecific adsorbent was found to be an order of magnitude lower than for the other supports. The different forms of this proteolytically processed bacterial enzyme could not be separated, however, by the biospecific adsorbents. At the mAB density used in the adsorbents (10-30 microM), less than 30% of the theoretical binding capacity of the immobilized mABs could be used to adsorb the enzyme.

Animals↗

PubMed1995

Endo-N-acetyl-beta-D-glucosaminidase and peptide-N4-(N-acetyl-glucosaminyl) asparagine amidase activities during germination of Raphanus sativus.

Endo-N-acetyl-beta-D-glucosaminidase (ENGase, EC 3.2.1.96) and peptide-N4-(N-acetyl-beta-D-glucosaminyl) asparagine amidase (PNGase, EC 3.5.1.52) activities were monitored during germination and postgerminative development in Raphanus sativus. The PNGase activity was found in dry seeds and its level was constant during germination and postgermination. The ENGase activity was first detected about 18 hr after the start of imbibition (HAI) and displayed a maximum level at 36 HAI. After 36 HAI the production of both enzymes was constant until days 4-5. Both enzymes displayed substrate specificities corresponding to the potential glycoprotein substrates found in plants. They are in agreement (i) with the hypothesis that ENGase and PNGase are at the origin of the production of 'unconjugated N-glycans' and (ii) with the possibility that protein activity could be regulated by the removal of N-glycans.

Amidohydrolases↗

PubMed1986

Sequencing and heterologous expression of the gene encoding penicillin V amidase from Bacillus sphaericus.

The Bacillus sphaericus gene encoding penicillin V amidase, which catalyzes the hydrolysis of penicillin V, has been characterized. The entire nucleotide sequence of the coding region, as well as 5'- and 3'-flanking regions, was determined using an improved sequencing strategy. The deduced amino acid sequence suggests a protein consisting of 338 residues with an Mr of 37,500. The ATG initiator codon is preceded by a putative ribosome-binding site, typical for genes of Gram-positive origin. High expression of the gene was obtained in Escherichia coli using an inducible promoter, showing that the gene product is stable in this heterologous host.

Amidohydrolases↗

PubMed1991

The role of penicillin amidases in nature and in industry.

Penicillin amidase (PA) is the enzyme used commercially for the production of semisynthetic penicillins. During the past decade, a detailed picture of the structure and regulation of the gene encoding this enzyme has emerged, revealing a variety of interesting features that are unique among microorganisms. Clues to the biological role of this enzyme have been provided, as well as new strategies for the commercial production and utilization of PA.

Escherichia coli↗