Carboxyamide Group Research Articles

Supramolecular assemblies of organogelators featuring benzimidazole and long‐chain pyridine dicarboxyamide

AbstractTwo organogelators, CO‐01 and CO‐03, featuring benzimidazole and long‐chain pyridine carboxyamide groups were prepared and their physical properties were fully explored. CO‐01 and CO‐03 are capable of transforming various organic solvents to organogels. The morphologic investigations of these organogels have shown that the supramolecular assemblies, in the forms of fibers and spheres, are readily generated from the aggregates of CO‐01 and CO‐03. The sol–gel interconversion can be readily achieved by tuning the gelator concentration and temperature. By manipulating the redox state of anthraquinone group in CO‐03, the gel–sol transition for the organogels of CO‐03 can be reversibly tuned by either chemical or electrochemical oxidation/reduction reactions. The presence of polarized imidazole and amide N–H groups allows anion‐induced gel collapse. The accompanying colorimetric and ratiometric fluorescent responses of gels CO‐01 and CO‐03 to F− and CN− render these organogelators being sensitive and selective anion probes.

Atomic view of cosolute-induced protein denaturation probed by NMR solvent paramagnetic relaxation enhancement

The cosolvent effect arises from the interaction of cosolute molecules with a protein and alters the equilibrium between native and unfolded states. Denaturants shift the equilibrium toward the latter, while osmolytes stabilize the former. The molecular mechanism whereby cosolutes perturb protein stability is still the subject of considerable debate. Probing the molecular details of the cosolvent effect is experimentally challenging as the interactions are very weak and transient, rendering them invisible to most conventional biophysical techniques. Here, we probe cosolute-protein interactions by means of NMR solvent paramagnetic relaxation enhancement together with a formalism we recently developed to quantitatively describe, at atomic resolution, the energetics and dynamics of cosolute-protein interactions in terms of a concentration normalized equilibrium average of the interspin distance, [Formula: see text], and an effective correlation time, τc The system studied is the metastable drkN SH3 domain, which exists in dynamic equilibrium between native and unfolded states, thereby permitting us to probe the interactions of cosolutes with both states simultaneously under the same conditions. Two paramagnetic cosolute denaturants were investigated, one neutral and the other negatively charged, differing in the presence of a carboxyamide group versus a carboxylate. Our results demonstrate that attractive cosolute-protein backbone interactions occur largely in the unfolded state and some loop regions in the native state, electrostatic interactions reduce the [Formula: see text] values, and temperature predominantly impacts interactions with the unfolded state. Thus, destabilization of the native state in this instance arises predominantly as a consequence of interactions of the cosolutes with the unfolded state.

بهبود خواص فیزیکی و رئولوژیکی خمیر نان با استفاده از دو آنزیم ترانس گلوتامیناز و آسپاراژیناز و پودر آب پنیر و اینولین

اثر افزودنی های مختلف (اینولین به عنوان پری بیوتیک، پودر آب پنیر، آنزیم آسپاراژیناز و ترانس گلوتامیناز) بر ویژگی‌های فارینوگرافی و اکستنسوگرافی دو نوع آرد (قوی و ضعیف) گندم با استفاده از طرح مورد مطالعه قرار گرفت. ویژگی‌های فارینوگرافی مانند درصد جذب آب، زمان گسترش، پایداری خمیر، درجه نرم شدگی خمیر، عدد کیفیت فارینوگراف و ویژگی‌های اکستنسوگرافی مانند مقدار انرژی، قابلیت کششی، مقاومت کششی و نسبت مقاومت کششی به قابلیت کششی تعیین شدند. تمام مدل‌های رگرسیون در سطح 01/0 و با ضریب تبیین (R2) 70/81 تا %67/98 معنی‌دار و قادر به پیش بینی ویژگی‌های خمیر فرموله شده بودند. نوع آرد و سطح اینولین اثر منفی زیادی بر درجه نرم شدگی خمیر و اثر مثبت بر زمان گسترش، پایداری، عدد کیفیت فارینوگراف، قابلیت کششی و مقاومت کششی نشان داد. درصد جذب آب با افزایش سطح اینولین، کاهش و با کاربرد آرد قوی، افزایش یافت. پودر آب پنیر اثر مشابه با اینولین بر پارامترهای فارینوگرافی و اکستنسوگرافی داشت. آنزیم آسپاراژیناز و ترانس گلوتامیناز به تنهایی اثر معنی‌دار بر ویژگی‌های فارینوگرافی نداشتند ولی اثر بر همکنش آنها بر کاهش درجه نرم شدگی خمیر معنی‌دار بود. تمام پارامترهای اکستنسوگراف به جز قابلیت کششی به طور قابل توجهی تحت تاثیر ترانس گلوتامیناز قرار گرفت. درمیان برهمکنش‌ها، برهمکنش بین آنزیم ترانس گلوتامیناز و نوع آرد مصرفی اثر مثبت قابل توجهی بر مقاومت کششی خمیر داشت.

The oxidation of alkylaryl sulfides and benzo[b]thiophenes by Escherichia coli cells expressing wild-type and engineered styrene monooxygenase from Pseudomonas putida CA-3

Nine different sulfur-containing compounds were biotransformed to the corresponding sulfoxides by Escherichia coli Bl21(DE3) cells expressing styrene monooxygenase (SMO) from Pseudomonas putida CA-3. Thioanisole was consumed at 83.3 μmoles min(-1) g cell dry weight(-1) resulting mainly in the formation of R-thioanisole sulfoxide with an enantiomeric excess (ee) value of 45 %. The rate of 2-methyl-, 2-chloro- and 2-bromo-thioanisole consumption was 2-fold lower than that of thioanisole. Surprisingly, the 2-methylthioanisole sulfoxide product had the opposite (S) configuration to that of the other 2-substituted thioanisole derivatives and had a higher ee value (84 %). The rate of oxidation of 4-substituted thioanisoles was higher than the corresponding 2-substituted substrates but the ee values of the products were consistently lower (10-23 %). The rate of benzo[b]thiophene and 2-methylbenzo[b]thiophene sulfoxidation was approximately 10-fold lower than that of thioanisole. The ee value of the benzo[b]thiophene sulfoxide could not be determined as the product racemized rapidly. E. coli cells expressing an engineered SMO (SMOeng R3-11) oxidised 2-substituted thioanisoles between 1.8- and 2.8-fold faster compared to cells expressing the wild-type enzyme. SMOeng R3-11 oxidised benzo[b]thiophene and 2-methylbenzo[b]thiophene 10.1 and 5.6 times faster that the wild-type enzyme. The stereospecificity of the reaction catalysed by SMOeng was unchanged from that of the wild type. Using the X-ray crystal structure of the P. putida S12 SMO, it was evident that the entrance of substrates into the SMO active site is limited by the binding pocket bottleneck formed by the side chains of Val-211 and Asn-46 carboxyamide group.

Unique Interaction Pattern for a Functionally Biased Ghrelin Receptor Agonist

Based on the conformationally constrained D-Trp-Phe-D-Trp (wFw) core of the prototype inverse agonist [D-Arg(1),D-Phe(5),D-Trp(7,9),Leu(11)]substance P, a series of novel, small, peptide-mimetic agonists for the ghrelin receptor were generated. By using various simple, ring-constrained spacers connecting the D-Trp-Phe-D-Trp motif with the important C-terminal carboxyamide group, 40 nm agonism potency was obtained and also in one case (wFw-Isn-NH(2), where Isn is isonipecotic acid) ~80% efficacy. However, in contrast to all previously reported ghrelin receptor agonists, the piperidine-constrained wFw-Isn-NH(2) was found to be a functionally biased agonist. Thus, wFw-Isn-NH(2) mediated potent and efficacious signaling through the Gα(q) and ERK1/2 signaling pathways, but in contrast to all previous ghrelin receptor agonists it did not signal through the serum response element, conceivably the Gα(12/13) pathway. The recognition pattern of wFw-Isn-NH(2) with the ghrelin receptor also differed significantly from that of all previously characterized unbiased agonists. Most importantly, wFw-Isn-NH(2) was not dependent on GluIII:09 (Glu3.33), which otherwise is an obligatory TM III anchor point residue for ghrelin agonists. Molecular modeling and docking experiments indicated that wFw-Isn-NH(2) binds in the classical agonist binding site between the extracellular segments of TMs III, VI, and VII, interacting closely with the aromatic cluster between TMs VI and VII, but that it does so in an opposite orientation as compared with, for example, the wFw peptide agonists. It is concluded that the novel peptide-mimetic ligand wFw-Isn-NH(2) is a biased ghrelin receptor agonist and that the selective signaling pattern presumably is due to its unique receptor recognition pattern lacking interaction with key residues especially in TM III.

Transglutaminase를 생산하는 Streptomyces platensis의 분자생물학적인 연구를 위한 접합 전달법 확립

식품단백질의 물성과 기능성을 개선시켜 산업적인 가치가 매우 높은 TGase를 생산하는 S. platensis YK-2의 분자 유전학적인 연구를 위해 형질전환방법을 확립하였으며 본 연구를 위해 도입된 방법은 대장균을 plasmid DNA의 공여체로, S. platensis의 포자를 수용체로 사용하는 접합전달법이다. S. platensis를 위한 최적 접합전달조건은 50 mM의 <TEX>$MgCl_2$</TEX>를 첨가한 MS배지에 열처리를 하지 않은 포자와 <TEX>$5{\times}10^7$</TEX>의 plasmid DNA 공여체인 E. coli를 사용하는 것이다. 또 본 연구를 통해 얻어진 접합전달체의 attB site에 대한 분석을 통해 S. platensis chromosome의 pirin 상동체를 코드하는 ORF내에 attB site가 단일위치로 존재하고 있으며 이미 밝혀진 다른 방선균유래 attB site의 염기서열에 대해 77.8%~96.3%의 상동성을 나타냈다. Streptomyces platensis YK-2, newly isolated from forest soil, produces transglutaminase (TGase), which catalyses an acyl transfer reaction between the primary grade amine and protein or <TEX>$\gamma$</TEX>-carboxyamide group of peptide bound glutamine residues. For a molecular genetic study of S. platensis, an effective transformation method was established by using a conjugal transfer of DNA from Escherichia coli to spores of actinomycetes. The highest transconjugation frequency of S. platensis was obtained on an MS medium containing 50 mM <TEX>$MgCl_2$</TEX>, using <TEX>$5{\times}10^7\;E$</TEX>. coli as a DNA donor and <TEX>$1{\times}10^8$</TEX> spores without heat treatment as a host. We also identified that S. platensis contains a single attB site within an ORF encoding a pirin-homolog, and that its attB site sequence shows high homology to that of S. logisporoflavus. In addition, it was confirmed by phenotypic analyses of exconjugants that the introduction of heterologous DNA into the attB site of the S. platensis chromosome does not affect its morphological differentiation and TGase production.

Simultaneous NMR assignment of backbone and side chain amides in large proteins with IS-TROSY

A new strategy for the simultaneous NMR assignment of both backbone and side chain amides in large proteins with isotopomer-selective transverse-relaxation-optimized spectroscopy (IS-TROSY) is reported. The method considers aspects of both the NMR sample preparation and the experimental design. First, the protein is dissolved in a buffer with 50%H2O/50%D2O in order to promote the population of semideuterated NHD isotopomers in side chain amides of Asn/Gln residues. Second, a 13C'-coupled 2D 15N-1H IS-TROSY spectrum provides a stereospecific distinction between the geminal protons in the E and Z configurations of the carboxyamide group. Third, a suite of IS-TROSY-based triple-resonance NMR experiments, e.g. 3D IS-TROSY-HNCA and 3D IS-TROSY-HNCACB, are designed to correlate aliphatic carbon atoms with backbone amides and, for Asn/Gln residues, at the same time with side chain amides. The NMR assignment procedure is similar to that for small proteins using conventional 3D HNCA/3D HNCACB spectra, in which, however, signals from NH2 groups are often very weak or even missing due to the use of broad-band proton decoupling schemes and NOE data have to be used as a remedy. For large proteins, the use of conventional TROSY experiments makes resonances of side chain amides not observable at all. The application of IS-TROSY experiments to the 35-kDa yeast cytosine deaminase has established a complete resonance assignment for the backbone and stereospecific assignment for side chain amides, which otherwise could not be achieved with existing NMR experiments. Thus, the development of IS-TROSY-based method provides new opportunities for the NMR study of important structural and biological roles of carboxyamides and side chain moieties of arginine and lysine residues in large proteins as well as amino moieties in nucleic acids.

A Substrate-Assisted Concerted Mechanism for Aminoacylation by a Class II Aminoacyl-tRNA Synthetase

Aminoacyl-tRNA synthetases (aaRS) join amino acids to their cognate transfer RNAs, establishing an essential coding relationship in translation. To investigate the mechanism of aminoacyl transfer in class II Escherichia coli histidyl-tRNA synthetase (HisRS), we devised a rapid quench assay. Under single turnover conditions with limiting tRNA, aminoacyl transfer proceeds at 18.8 s(-)(1), whereas in the steady state, the overall rate of aminoacylation is limited by amino acid activation to a rate of 3 s(-)(1). In vivo, this mechanism may serve to allow the size of amino acid pools and energy charge to control the rate of aminoacylation and thus protein synthesis. Aminoacyl transfer experiments using HisRS active site mutants and phosphorothioate-substituted adenylate showed that substitution of the nonbridging Sp oxygen of the adenylate decreased the transfer rate at least 10 000-fold, providing direct experimental evidence for the role of this group as a general base for the reaction. Other kinetic experiments revealed that the rate of aminoacyl transfer is independent of the interaction between the carboxyamide group of Gln127 and the alpha-carboxylate carbon, arguing against the formation of a tetrahedral intermediate during the aminoacyl transfer. These experiments support a substrate-assisted concerted mechanism for HisRS, a feature that may generalize to other aaRS, as well as the peptidyl transferase center.

One‐Pot Synthesis of Pyrazole‐5(3)‐carboxyamides

A convenient one‐pot synthesis of eight pyrazole‐5(3)‐carboxyamides from the reaction of the 5(3)‐trichloromethylpyrazoles with amines, in good yield, is reported. These reactions show that the trichloromethyl group is a convenient precursor to carboxyamide groups.

Biocompatibility studies of anionic collagen membranes with different degree of glutaraldehyde cross-linking

The work describes the biocompatibility and biodegradation studies of anionic collagen membranes casted form collagen gels collagen, that were selective hydrolyzed at the carboxyamide groups, as a function of the degree of cross-links induced by glutaraldehyde. Independently from the degree of cross-links, all membranes studied were characterized by a similar inflammatory response, inversely dependent on glutaraldehyde reaction time, that decreased from the time of the implant. Cell alterations, mineralization or contact necrosis were not observed in any of the membranes studied. Rates for membrane tissue biodegradation were directly related to glutaraldehyde reaction time, and ranged from 30 to periods longer than 60 days, associated with good biocompatibility. Although other properties must be considered, their use in the treatment of periodontal diseases, the biological behavior observed with the 8 h GA cross-linked membrane suggests that, anionic collagen membrane described in this work may be of potential use, not only in association with guided tissue regeneration technique for periodontal tissue reconstruction, but also in other collagen biomaterial applications where controlled biodegradability is required.

Hidrólise seletiva de carboxiamidas de resíduos de asparagina e glutamina em colágeno: preparação e caracterização de matrizes aniônicas para uso como biomateriais

This work describes the selective hydrolysis of carboxyamide groups of asparagine and glutamine of collagen matrices for the preparation of negatively charged collagen biomaterials. The reaction was performed in the presence of chloride and sulfate salts of alkaline and alkaline earth metals in aqueous dimethylsulfoxide solution and, selectively hydrolysis of carboxyamide groups of collagen matrices was promoted without cleavage of the peptide bond. The result is a new collagen material with controlled increase in negative charge content. Although triple helix secondary structure of tropocollagen was preserved, significative changes in thermal stabilities were observed in association with a new pattern of tropocollagen macromolecular association, particularly in respect microfibril assembly, thus providing at physiological pH a new type of collagen structure for biomaterial preparation, characterized by different charge and structural contents .

Complexing Behaviour of Vanadate Towards Thrombin

The vanadium(V)-thrombin complex was prepared in solution by the interaction of H2VO4 − with human thrombin. The interaction between vanadate and the protein was characterized by UV spectroscopy and titrations. The binding constant was calculated to be 104−−9 M-2. A model complex of vanadium with trypsin was also prepared and studied by UV difference spectra to compare the binding sites of trypsin with thrombin for vanadate. It is concluded that vanadium is binding as vanadate to carboxyamide groups of the glutamine residues.

Recognition of C-terminal amide groups by (serine) carboxypeptidase Y investigated by site-directed mutagenesis.

Serine carboxypeptidases have the ability to hydrolyze peptides as well as peptide amides. Previously, it has been demonstrated that Asn51 and Glu145 (in the protonated form) each donate a hydrogen bond to the alpha-carboxylate of peptide substrate. It is here demonstrated by characterization of carboxypeptidase Y derivatives, mutationally altered at positions 51 and 145, that the same groups are involved in the interaction with the C-terminal carboxyamide group of peptide amides. Asn51 donates a hydrogen bond to the C = O group of the substrate, and Glu145 (in the charged form) accepts one from the NH2 group of the substrate. Thus, the ionic state of Glu145 is different when peptides are hydrolyzed as compared with when peptide amides are hydrolyzed. This explains why Km for the hydrolysis of peptides increases with pH, whereas it remains constant for peptide amides. As a consequence, kcat/Km for the hydrolysis of peptide amides is higher than for the hydrolysis of peptides at pH > 8. At physiological pH, peptides and peptide amides are hydrolyzed with rates of the same order of magnitude; this is in accordance with reports describing that serine carboxypeptidases are involved in the degradation of biologically active peptide amides.

A theoretical study of glucosamine synthase. II. Combined quantum and molecular mechanics simulation of sulfhydryl attack on the carboxyamide group.

Continuing our theoretical studies of glucosamine synthase catalysis, we have carried out MNDO and ab initio calculations of the first stage of the reaction, which involves the attack of a cysteine thiol group from the enzyme active site on the side chain carboxyamide group of glutamine, producing ammonia and thioester. The reactants were modelled by methyl mercaptate and acetamide, respectively. For two considered mechanisms of the reaction the energy surfaces were evaluated. Mechanism I, proposed by Chmara et al. (1985) involves the nucleophilic attack of a deprotonated thiol group on the carbonyl carbon atom. Mechanism II, postulated in our previous work (Tempczyk et al. 1989), assumes the concerted binding of the mercaptate sulphur to the carbonyl carbon and the sulfhydryl hydrogen to the amide nitrogen with simultaneous breaking of the S-H bond. The energy surface of mechanism I shows no minimum on the approach of the mercaptide anion towards the carbonyl carbon, which is also consistent with ab initio calculations in a 4-31 G basis set. Therefore, mechanism I seems to be unlikely. The same analysis of mechanism II shows that it leads to the desired products: methyl thioacetate and ammonia. The presence of a sulfhydryl hydrogen causes apparent pyramidicity of the amido nitrogen and lengthening of the C-N bond in the transition state, making conditions for the release of the ammonia molecule. The MNDO calculated energy barrier of the reaction is 50.1 kcal/mol and the approximate 4-31 G ab initio barrier (at the MNDO geometries of the substrate complex and the transition state) is 63 kcal/mol.(ABSTRACT TRUNCATED AT 250 WORDS)

Subunit assembly and active site location in the structure of glutamate dehydrogenase.

The three-dimensional crystal structure of the NAD(+)-linked glutamate dehydrogenase from Clostridium symbiosum has been solved to 1.96 A resolution by a combination of isomorphous replacement and molecular averaging and refined to a conventional crystallographic R factor of 0.227. Each subunit in this multimeric enzyme is organised into two domains separated by a deep cleft. One domain directs the self-assembly of the molecule into a hexameric oligomer with 32 symmetry. The other domain is structurally similar to the classical dinucleotide binding fold but with the direction of one of the strands reversed. Difference Fourier analysis on the binary complex of the enzyme with NAD+ shows that the dinucleotide is bound in an extended conformation with the nicotinamide moiety deep in the cleft between the two domains. Hydrogen bonds between the carboxyamide group of the nicotinamide ring and the side chains of T209 and N240, residues conserved in all hexameric GDH sequences, provide a positive selection for the syn conformer of this ring. This results in a molecular arrangement in which the A face of the nicotinamide ring is buried against the enzyme surface and the B face is exposed, adjacent to a striking cluster of conserved residues including K89, K113, and K125. Modeling studies, correlated with chemical modification data, have implicated this region as the glutamate/2-oxoglutarate binding site and provide an explanation at the molecular level for the B type stereospecificity of the hydride transfer of GDH during the catalytic cycle.

The characterization of a membrane-bound protein carboxylmethylation system in brain

The membrane-bound component of the cerebral protein carboxylmethylation system, consisting of the membrane-bound enzyme protein carboxylmethyltransferase II (PCMT) and of selected membrane-bound methyl accepting proteins (MAP), is described. The cellular localization of this membrane-bound protein carboxylmethylation system is shown to include, in addition to nerve cell bodies and purified synaptosomes, astrocytes and oligodendroglia. The membrane-bound nature of the protein carboxylmethylation system was investigated and these studies revealed a tight association which exposure to several detergents could only partially solubilize. The membrane-bound PCMT could be shown to undergo activation after treatment with Na-deoxycholate and CHAPs, while after its detergent-induced solubilization PCMT activation was observed after Na-deoxycholate, Nonidet P-40 and Lubrol-P(X). Solubilization of the carboxylmethylation system in CHAPS appeared to be more effective at 0 degrees C than at 25 degrees C or 37 degrees C. Detergent treatment was shown to be deleterious to the MAPs as PCMT substrates, particularly when the exposure was extended to more than 1 h. These observations prompted exposure of the brain membranes and of their Lubrol-P(X) and Nonidet P-40 extracts to NH(4)OH, treatment which promotes the conversion of protein asparagine residues to atypical l-isoaspartate residues, recently shown (in synthetic peptides) to be the single most effective residue recognized for carboxylmethylation by PCMT. We found up to a 400% enhancement of the carboxylmethylation of solubilized membrane MAPs by the equally solubilized PCMT (which resisted the alkaline treatment virtually unscathed) after 90 min at 37 degrees C in 0.05 M NH(4)OH. However, when brain membrane Lubrol-P(x) extracts were first subjected to bis(I,I-trifluoroacetoxy)-iodobenzene, a reagent which converts the carboxyamide group of protein-bound asparagine to the corresponding primary amine, the amount of MAPs susceptible to be acted upon by 0.05 M NH(4)OH became greatly reduced. Finally, acidic slab gel electrophoresis of membrane-bound MAPs, carboxyl-[(3)H]-methylated by the membrane-bound PCMT, revealed the presence of about 12 radioactive protein bands, ranging in MW from under 20 KDa to about 90 KDa.

A mathematical description of the process of dehydrocyclization of polyamido acids

Modeling of two mechanisms of the imidization of polyamido acids has been carried out. The assumption has been made that the kinetic characteristics which are available in practice can be described by a set of parallel-sequential reactions, taking into account the different reactivity of structural conformations of the ortho carboxyamide groups.

Application of the Ammonia Gas-Sensing Electrode: Determination of Drugs Having a Carboxyamide Group by Decomposition with Acid

A simple potentiometric method for the determination of drugs having a carboxyamide group is described. Ethenzamide, niacinamide, pyrazinamide, or salicylamide was refluxed with 20% HC1 and the carboxyamide was hydrolyzed. The ammonia evolved at a pH >11 and was determined without separation from the decomposition solution using an ammonia gas-sensing electrode. A linear calibration plot was obtained with drugs in the range of 2 × 10–5-1 × 10–2M. This method was applied to the analysis of injection and powder-containing auxiliary compounds.

Functional group and substituent effects, both steric and electronic, upon the ultraviolet absorption spectra of some N-alkyl- and N,N-dialkyl-derivatives of some cinnamamides and benzalcyanoacetamides

Conjugative and steric constants for N-substituted carboxyamide groups have been derived which allow the calculation of the long wavelength absorption maxima of N-alkyl- and N,N-dialkyl-derivatives of cinnamamide and benzalcyanoacetamide. Deviations between calculated and observed values indicate that there may be steric interference between bulky N,N-dialkylcarboxyamide groups and the benzylic hydrogen atom.

Studies on acylase activity and microorganisms. XXIV. Properties of delta-ornithine acylase (5-N-acylornithine amidohydrolase).

In order to study the enzymatic properties of the δ-ornithine acylase in KT 801 (Pseudomonas sp.), experiments were carried out with the purified enzyme preparation. This enzyme has a pH optimum around 8.2 toward 5-N-benzoyl-L-ornithine. It is inhibited by Co2+, Zn2+, Fe3+, Cu2+, Hg2+, EDTA, and o-phenanthroline. The inactivation for EDTA is reversed by addition of Ca2+ or Mn2+ after dialysis. Km value toward 5-N-benzoyl-L-ornithine was calculated to be 1.8×10-4M. As a results of the investigation of substrate specificity, it was found that this enzyme hydrolyses not only L form of 5-N-benzoylornithine, but D form in the rate of about one sixth. The ratio of the activities toward L and D form remained constant all through the purification steps. These result strongly suggest that one enzyme has the both activities. Also, susceptible substrate to δ-ornithine acylase require absolutely the presence of a free carboxyl group, the presence of a free α-amino group is desirable for ready susceptibility of the substrate, but it is not an absolute requirement, and the susceptibility are considerably affected by carbon chain length between the carboxyamide group and the free carboxyl group. This enzyme shows no α-amino acylase activity. On the other hand, δ-ornithine acylase is quantitatively released when EDTA-lysozyme spheroplasts are made. From these result, it has been proposed that δ-ornithine acylase of KT 801 is at the cell surface.