Identification, characterization, and cloning of a novel aminoacylase, L-pipecolic acid acylase from Pseudomonas species.

L-Pipecolic acid is utilized as a vital component of specific chemical compounds, such as immunosuppressive drugs, anticancer reagents, and anesthetic reagents. We isolated and characterized a novel L-aminoacylase, N-acetyl-L-pipecolic acid-specific aminoacylase (LpipACY), from Pseudomonas sp. AK2. The subunit molecular mass of LpipACY was 45 kDa and was assumed to be a homooctamer in solution. The enzyme exhibited high substrate specificity toward N-acetyl-L-pipecolic acid and a high activity for N-acetyl-L-pipecolic acid and N-acetyl-L-proline. This enzyme was stable at a high temperature (60°C for 10 min) and under an alkaline pH (6.0-11.5). The N-terminal and internal amino acid sequences of the purified enzyme were STTANTLILRNG and IMASGGV, respectively. These sequences are highly consistent with those of uncharacterized proteins from Pseudomonas species, such as amidohydrolase and peptidase. We also cloned and overexpressed the gene coding LpipACY in Escherichia coli. Moreover, the recombinant LpipACY exhibited properties similar to native enzyme. Our results suggest that LpipACY is a potential enzyme for the enzymatic synthesis of L-pipecolic acid. This study provides the first description of the enzymatic characterization of L-pipecolic acid specific amino acid acylase.

Abundance of Lactobacillus plantarum Strains with Beneficial Attributes in Blackberries (Rubus sp.), Fresh Figs (Ficus carica), and Prickly Pears (Opuntia ficus-indica) Grown and Harvested in Algeria.

This first study performed on traditional fruits consumed in North Africa reveals their richness in microorganisms with beneficial attributes like cholesterol lowering capabilities. Blackberries (Rubus sp.), fresh figs (Ficus carica), and prickly pears (Opuntia ficus-indica) are fruits largely and traditionally consumed in Kabylia, a beautiful northern Algerian region. Here, 85 lactic acid bacteria (LAB)-isolates were isolated and identified by MALDI-TOF mass spectrometry. The identified species belong to Lactobacillus and Leuconostoc genera. These 85 LAB-isolates were then assessed for their capabilities to grow under conditions mimicking the gastrointestinal tract, and the resulting data were statistically treated with principal component analysis (PCA). After which, only 26 LAB-isolates were selected and characterized for their genetic relatedness using random amplified polymorphic DNA (RAPD) method. Following the genetic relatedness assessment, only 10 LAB-strains, among which nine Lactobacillus plantarum and one Lactobacillus paracasei were studied for their pathoproperties and some probiotic features. Interestingly, all of these 10 LAB-strains were devoid of adverse effects, but capable to adhere to human epithelial colorectal adenocarcinoma Caco-2 cells. Of note, these 10 LAB-strains exhibited an important in vitro hypocholesteromia effect, in strain-dependent manner. Moreover, the Lactobacillus strains exhibited a high bile salt hydrolase (BSH) activity which was correlated with expression of bsh2, bsh3 and bsh4 genes.

Identification and characterization of a novel (+)-γ-lactamase from Microbacterium hydrocarbonoxydans.

2-Azabicyclo[2.2.1]hept-5-en-3-one (γ-lactam) is an important precursor of many carbocyclic nucleoside analogs and pharmaceuticals. (-)-γ-Lactam has attracted much attention because of its role as an intermediate of antiviral drugs such as abacavir and carbovir. (+)-γ-Lactamase can be used for the kinetic resolution of γ-lactam to obtain (-)-γ-lactam. In this study, a novel (+)-γ-lactamase (Mh33H4-5540) was discovered from the gene library of Microbacterium hydrocarbonoxydans based on a colorimetric high-throughput screening method and it could be used to enantioselectively catalyze the bioresolution of racemic γ-lactam with high enantiomeric excess (ee) (>99 %) and yield (>49 %). An unexpected finding was that Mh33H4-5540 was unrelated to other known γ-lactamases (5.7, 4.8, 7.2, and 5.4 % similarities in amino sequence with (+)-γ-lactamase from Comamonas acidovorans, Bradyrhizobium japonicum, Aeropyrum pernix, and Sulfolobus solfataricus, respectively) but rather related to isochorismatases. The homolog analysis of Mh33H4-5540 revealed that it was similar in structure with bacterial isochorismatases (an isochorismatase from Pseudomonas putida (PDB number 4H17) and a putative isochorismatase from Oleispira antarctica (PDB number 3LQY)). Thus, Mh33H4-5540 represented another type of (+)-γ-lactamase. Mh33H4-5540 was overexpressed in E. coli Rosetta (DE3), purified to homogeneity and functionally characterized. The enzyme displayed optimal activity at 25 °C and pH 8.0. The activity showed a 5.5-fold increase in the presence of 0.5 M Ni2+ or Co2+. Mh33H4-5540 displayed much higher (+)-γ-lactamase activity than any other biochemically characterized (+)-γ-lactamases. Overall, we discovered a novel (+)-γ-lactamase Mh33H4-5540 which displayed the highest activity. It could be a promising candidate of biocatalyst for industrial applications of highly valuable chiral pharmaceutical chemicals.

Improved efficiency of asymmetric hydrolysis of 3-substituted glutaric acid diamides with an engineered amidase.

AIMS: To improve the efficiency of asymmetric hydrolysis of 3-(4-chlorophenyl) glutaric acid diamide (CGD) using a recombinant Comamonas sp. KNK3-7 amidase (CoAM) produced in Escherichia coli. METHODS AND RESULTS: The CoAM gene was cloned, sequenced and found to comprise 1512 bp, encoding a polypeptide of 54 054 Da. CoAM-transformed E. coli were able to perform R-selective hydrolysis of CGD; however, complete conversion of 166·2 mmol l(-1) CGD in 28 h could not be obtained. We attempted to optimize the reactivity of CoAM by mutating single amino acids in the substrate-binding domain. Notably, the methionine-substituted L146M mutant enzyme showed increased reactivity, completing the conversion of 166·2 mmol l(-1) CGD in just 4 h. The Km value for L146M was lower than that of CoAM. CONCLUSIONS: We succeeded in creating the L146M mutant of CoAM with increased substrate affinity and found that this was the best mutant for the hydrolysis of CGD. SIGNIFICANCE AND IMPACT OF THE STUDY: Increasing the efficiency of hydrolysis of 3-substituted glutaric acid diamides is useful to improve the synthesis of optically active 3-substituted gamma-aminobutyric acid. This is the first report of efficient hydrolysis of CGD using amidase mutant-producing E. coli cells.

[Purification and characterization of a halophilic urethanase from Klebsiella pneumoniae].

Ethyl carbamate (EC) is a carcinogenic substance in many fermented foods. Enzymatic removal of ethyl carbamate from fermented foods is an important way to eliminate its potential health damage to consumers. To study the enzymatic properties of an ethyl carbamate hydrolase (urethanase) from Klebsiella pneumoniae, a strain isolated from murine somach, we purified the enzyme using ammonium sulfate precipitation, ion exchange chromatography and gel filtration chromatography. The molecular mass of this enzyme was estimated to be 55 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Its K(m) was 74 mmol/L when EC was used as the substrate. Moreover, its optimal reaction temperature was 55 degrees C, and the optimum pH was 7.0. The activity was enhanced by ethylene diamine tetraacetic acid (EDTA) and dithiothreitol (DTT), but strongly inhibited by Cu2+ and Zn2+. The enzyme was halophilic and tolerant to low concentration of ethanol. Therefore, it has the potential to remove EC from fermented foods.

Structural and functional characterization of ochratoxinase, a novel mycotoxin-degrading enzyme.

Ochratoxin, with ochratoxin A as the dominant form, is one of the five major mycotoxins most harmful to humans and animals. It is produced by Aspergillus and Penicillium species and occurs in a wide range of agricultural products. Detoxification of contaminated food is a challenging health issue. In the present paper we report the identification, characterization and crystal structure (at 2.2 Å) of a novel microbial ochratoxinase from Aspergillus niger. A putative amidase gene encoding a 480 amino acid polypeptide was cloned and homologously expressed in A. niger. The recombinant protein is N-terminally truncated, thermostable, has optimal activity at pH ~6 and 66°C, and is more efficient in ochratoxin A hydrolysis than carboxypeptidase A and Y, the two previously known enzymes capable of degrading this mycotoxin. The subunit of the homo-octameric enzyme folds into a two-domain structure characteristic of a metal dependent amidohydrolase, with a twisted TIM (triosephosphateisomerase)-barrel and a smaller ß-sandwich domain. The active site contains an aspartate residue for acid-base catalysis, and a carboxylated lysine and four histidine residues for binding of a binuclear metal centre.

Azobenzene switch with a long-lived cis-state to photocontrol the enzyme activity of a histone deacetylase-like amidohydrolase.

The control of enzymes by use of an external stimulus such as light enables the temporal and spatial regulation of defined chemical reactions in a highly precise manner. In this work we investigated and characterized the reversible photocontrol of a bacterial histone deacetylase-like amidohydrolase (HDAH) from Bordetella/Alcaligenes strain FB188, which holds great potential to control deacetylation reactions of a broad spectrum of substrates in biotechnological and biomedical applications. Several HDAH variants with a single surface accessible cysteine close to the active site were developed and covalently modified by a monofunctional azobenzene-based photoswitch [4-phenylazomaleinanil (4-PAM)]. The enzymatic activity of three HDAH variants (M30C, S20C and M150C) were shown to be controlled by light. The thermal cis-to-trans relaxation of azobenzene conjugated to HDAH was up to 50-fold retarded compared to unbound 4-PAM allowing light pulse switching rather than continuing irradiation to maintain the thermodynamically less stable cis-state of covalently attached 4-PAM.

Cross-functionalities of Bacillus deacetylases involved in bacillithiol biosynthesis and bacillithiol-S-conjugate detoxification pathways.

BshB, a key enzyme in bacillithiol biosynthesis, hydrolyses the acetyl group from N-acetylglucosamine malate to generate glucosamine malate. In Bacillus anthracis, BA1557 has been identified as the N-acetylglucosamine malate deacetylase (BshB); however, a high content of bacillithiol (~70%) was still observed in the B. anthracis ∆BA1557 strain. Genomic analysis led to the proposal that another deacetylase could exhibit cross-functionality in bacillithiol biosynthesis. In the present study, BA1557, its paralogue BA3888 and orthologous Bacillus cereus enzymes BC1534 and BC3461 have been characterized for their deacetylase activity towards N-acetylglucosamine malate, thus providing biochemical evidence for this proposal. In addition, the involvement of deacetylase enzymes is also expected in bacillithiol-detoxifying pathways through formation of S-mercapturic adducts. The kinetic analysis of bacillithiol-S-bimane conjugate favours the involvement of BA3888 as the B. anthracis bacillithiol-S-conjugate amidase (Bca). The high degree of specificity of this group of enzymes for its physiological substrate, along with their similar pH-activity profile and Zn²âº-dependent catalytic acid-base reaction provides further evidence for their cross-functionalities.

Purification and use of E. coli peptide deformylase for peptide deprotection in chemoenzymatic peptide synthesis.

Peptide deformylases (PDFs) catalyze the removal of the formyl group from the N-terminal methionine residue in nascent polypeptide chains in prokaryotes. Its deformylation activity makes PDF an attractive candidate for the biocatalytic deprotection of formylated peptides that are used in chemoenzymatic peptide synthesis. For this application it is essential to use PDF preparations that are free of contamination by peptidases that can cleave internal peptide bonds. Therefore, different purification methods were attempted and an industrially applicable purification procedure was developed based on a single anion-exchange chromatography step of an engineered PDF variant that was equipped with an anionic octaglutamate tag. The deformylation activity and stability of the engineered enzyme were similar to those of the wild-type PDF. This purification method furnished a PDF preparation with a 1500-fold decreased level of contamination by amidases and peptidases as compared to cell-free extract. It was shown that the enzyme could be used for deprotection of a formylated dipeptide that was prepared by thermolysin-mediated coupling.

Purification studies on a thermo-active amidase of Geobacillus pallidus BTP-5x MTCC 9225 isolated from thermal springs of Tatapani (Himachal Pradesh).

An intracellular aliphatic amide degrading inducible thermo-active amidase produced by Geobacillus pallidus BTP-5x MTCC 9225 was purified to apparent homogeneity using anion exchange and gel filtration chromatography, giving a yield of 6.7 % and a specific activity of 30.49 units mg(-1). The purified protein migrated as a single band of estimated molecular mass of 158 kDa (homo-tetramer) in 8 % polyacrylamide gel electrophoresis and ∼38.5 kDa in 12 % sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Optima of pH and temperature varied widely in broad pH range (pH 6-9) and temperature range (45-70 °C). The purified amidase was stable up to 6 h at 50 °C, with a t (1/2) of 7 h at 55 °C. The multimeric nature of the holozyme (tetramer) contributed to protection of the enzyme against thermal denaturation. The enzyme showed resistance to metal chelating agents (EDTA, 8-hydroxyquinoline, and sodium azide), explaining its non-metallic nature, and is strongly inhibited by thiol reagents that means cysteine is involved in catalysis. The amidase of G. pallidus BTP-5x preferentially hydrolyzed only small aliphatic amides and has a narrow substrate spectrum. The K (M) value for acrylamide is 10.54 mM, V (max) 45.19 µmol(-1) min(-1) mg(-1) protein, and k (cat) 4.29 min(-1). The sequence of amino acids of the purified enzyme MRHGDISSSHDTVGI appears similar to thermophilic amidases. Sequence analysis of the amidase gene showed that the enzyme is 347 amino-acid-long with a molecular weight of 38.4 kDa (as observed in SDS-PAGE), theoretical pI 5.38, and show strong similarity to thermostable amidases, possessing unique restriction sites.

Discovery of structurally-diverse inhibitor scaffolds by high-throughput screening of a fragment library with dimethylarginine dimethylaminohydrolase.

Potent and selective inhibitors of the enzyme dimethylarginine dimethylaminohydrolase (DDAH) are useful as molecular probes to better understand cellular regulation of nitric oxide. Inhibitors are also potential therapeutic agents for treatment of pathological states associated with the inappropriate overproduction of nitric oxide, such as septic shock, selected types of cancer, and other conditions. Inhibitors with structures dissimilar to substrate may overcome limitations inherent to substrate analogs. Therefore, to identify structurally-diverse inhibitor scaffolds, high-throughput screening (HTS) of a 4000-member library of fragment-sized molecules was completed using the Pseudomonas aeruginosa DDAH and human DDAH-1 isoforms. Use of a substrate concentration equal to its K(M) value during the primary screen allowed for the detection of inhibitors with different modes of inhibition. A series of validation tests were designed and implemented in the identification of four inhibitors of human DDAH-1 that were unknown prior to the screen. Two inhibitors share a 4-halopyridine scaffold and act as quiescent affinity labels that selectively and covalently modify the active-site Cys residue. Two inhibitors are benzimidazole-like compounds that reversibly and competitively inhibit human DDAH-1 with Ligand Efficiency values ≥0.3 kcal/mol/heavy (non-hydrogen) atom, indicating their suitability for further development. Both inhibitor scaffolds have available sites to derivatize for further optimization. Therefore, use of this fragment-based HTS approach is demonstrated to successfully identify two novel scaffolds for development of DDAH-1 inhibitors.

Purification, characterization, and primary structure of a novel N-acyl-D-amino acid amidohydrolase from Microbacterium natoriense TNJL143-2.

A novel N-acyl-D-amino acid amidohydrolase (DAA) was purified from the cells of a novel species of the genus Microbacterium. The purified enzyme, termed AcyM, was a monomeric protein with an apparent molecular weight of 56,000. It acted on N-acylated hydrophobic D-amino acids with the highest preference for N-acetyl-D-phenylalanine (NADF). Optimum temperature and pH for the hydrolysis of NADF were 45°C and pH 8.5, respectively. The k(cat) and K(m) values for NADF were 41 s⁻¹ and 2.5 mM at 37°C and pH 8.0, although the enzyme activity was inhibited by high concentrations of NADF. Although many known DAAs are inhibited by 1 mM EDTA, AcyM displayed a 65% level of its full activity even in the presence of 20 mM EDTA. Based on partial amino acid sequences of the purified enzyme, the full-length AcyM gene was cloned and sequenced. It encoded a protein of 495 amino acids with a relatively low sequence similarity to a DAA from Alcaligenes faecalis DA1 (termed AFD), a binuclear zinc enzyme of the α/ß-barrel amidohydrolase superfamily. The unique cysteine residue that serves as a ligand to the active-site zinc ions in AFD and other DAAs was not conserved in AcyM and was replaced by alanine. AcyM was the most closely related to a DAA of Gluconobacter oxydans (termed Gox1177) and phylogenetically distant from AFD and all other DAAs that have been biochemically characterized thus far. AcyM, along with Gox1177, appears to represent a new phylogenetic subcluster of DAAs.

Cloning, expression, characterization and application of atcA, atcB and atcC from Pseudomonas sp. for the production of L-cysteine.

An isolate of a Pseudomonas sp. uses the L-NCC (N-carbamoyl-L-cysteine) pathway to convert DL-2-amino-Δ(2)-thiazoline-4-carboxylic acid (DL-ATC) to L-cysteine. Genes encoding ATC racemase (AtcA), L-ATC hydrolase (AtcB) and L-NCC amidohydrolase (AtcC), involved in this pathway, were cloned from the Pseudomonas sp. and expressed in Escherichia coli BL21 via pET-28a(+). The resulting enzymes were purified, their functions identified, and their biochemical properties are described. In vitro catalysis experiments, using these enzymes, revealed that the bioconversion rate of L-cysteine from DL-ATC in the presence of AtcA was more efficient than in the absence of AtcA. This is the first report describing simultaneous cloning and expression of atcA, atcB and atcC and characterization of their enzymes for L-cysteine production from DL-ATC via the L-NCC pathway, enabling the complete L-NCC pathway to be elucidated.

Kinetic analyses and inhibition studies reveal novel features in peptide deformylase 1 from Trypanosoma cruzi.

In eubacteria and eukaryotic organelles N-terminal methionine excision requires the sequential action of two activities, a peptide deformylase (PDF), which systematically removes the N-formyl group present on all nascent polypeptides and methionine aminopeptidase (MAP), which exscinds methionine specifically and depends on the previous removal of the N-formyl group. In Trypanosoma cruzi two genes encoding bacterial PDF homologues have been identified and referred to as TcPDF-1 and TcPDF-2. Here we report the biochemical characterization of a truncated soluble version of TcPDF-1 lacking the hydrophobic N-terminal domain that is active with the bacterial PDF substrate formyl-methionyl-alanyl-serine but, in contrast to other PDFs, is not inhibited by actinonin. The enzyme is strongly activated by Cu(2+) and inhibited by Ni(2+). Our results show that T. cruzi PDF exhibits unique features thus providing a new avenue for the design of potential inhibitors for use in the treatment of diseases caused by trypanosomatid parasites.

Mass spectrometric characterization of human N-acylethanolamine-hydrolyzing acid amidase.

N-Acylethanolamine-hydrolyzing acid amidase (NAAA) is a lysosomal enzyme that primarily degrades palmitoylethanolamine (PEA), a lipid amide that inhibits inflammatory responses. We developed a HEK293 cell line stably expressing the NAAA pro-enzyme (zymogen) and a single step chromatographic purification of the protein from the media. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry MALDI-TOF MS analysis of the zymogen (47.7 kDa) treated with peptide-N-glycosidase F (PNGase F) identified 4 glycosylation sites, and acid cleavage of the zymogen into α- and ß-subunits (14.6 and 33.3 kDa) activated the enzyme. Size exclusion chromatography estimated the mass of the active enzyme as 45 ± 3 kDa, suggesting formation of an α/ß heterodimer. MALDI-TOF MS fingerprinting covered more than 80% of the amino acid sequence, including the N-terminal peptides, and evidence for the lack of a disulfide bond between subunits. The significance of the cysteine residues was established by their selective alkylation resulting in almost complete loss of activity. The purified enzyme was kinetically characterized with PEA and a novel fluorogenic substrate, N-(4-methyl coumarin) palmitamide (PAMCA). The production of sufficient quantities of NAAA and a high throughput assay could be useful in discovering novel inhibitors and determining the structure and function of this enzyme.

Cloning and expression of the FR901379 acylase gene from Streptomyces sp. no. 6907.

FR901379 acylase, an enzyme that catalyzes the hydrolysis of the palmitoyl moiety of the antifungal lipopeptide FR901379, was purified from the culture broth of Streptomyces sp. no. 6907 (FERM BP-5809), revealing the 80 kDa, two-subunit heterodimeric protein characteristic of the ß-lactam acylase family. Using oligodeoxyribonucleotide primers constructed on the basis of the N-terminal amino acid sequence of each purified subunit, the gene was identified from a cosmid library of Streptomyces sp. no. 6907 DNA. The deduced 775 amino acid sequence corresponded to a single polypeptide chain containing two subunits, and it shared 41.7% identity with aculeacin A acylase from Actinoplanes utahensis NRRL12052. FR901379 acylase activity was found to be 250-fold higher in the recombinant Streptomyces lividans 1326 carrying the cloned gene than in the original Streptomyces sp. no. 6907 strain.

Kinetics and inhibition of nicotinamidase from Mycobacterium tuberculosis.

Nicotinamidase/pyrazinamidase (PncA) is involved in the NAD+ salvage pathway of Mycobacterium tuberculosis and other bacteria. In addition to hydrolyzing nicotinamide into nicotinic acid, PncA also hydrolyzes the prodrug pyrazinamide to generate the active form of the drug, pyrazinoic acid, which is an essential component of the multidrug treatment of TB. A coupled enzymatic activity assay has been developed for PncA that allows for the spectroscopic observation of enzyme activity. The enzyme activity was essentially pH-independent under the conditions tested; however, the measurement of the pH dependence of iodoacetamide alkylation revealed a pK value of 6.6 for the active site cysteine. Solvent deuterium kinetic isotope effects revealed an inverse value for kcat of 0.64, reconfirming the involvement of a thiol group in the mechanism. A mechanism is proposed for PncA catalysis that is similar to the mechanisms proposed for members of the nitrilase superfamily, in which nucleophilic attack by the active site cysteine generates a tetrahedral intermediate that collapses with the loss of ammonia and subsequent hydrolysis of the thioester bond by water completes the cycle. An inhibitor screen identified the competitive inhibitor 3-pyridine carboxaldehyde with a Ki of 290 nM. Additionally, pyrazinecarbonitrile was found to be an irreversible inactivator of PncA, with a kinact/KI of 975 M(−1) s(−1).

Conjugates of plumbagin and phenyl-2-amino-1-thioglucoside inhibit MshB, a deacetylase involved in the biosynthesis of mycothiol.

N-Acetylglucosaminylinositol (GlcNAc-Ins)-deacetylase (MshB) and mycothiol-S-conjugate amidase (Mca), structurally related amidases present in mycobacteria and other Actinomycetes, are involved in the biosynthesis of mycothiol and in the detoxification of xenobiotics as their mycothiol-S-conjugates, respectively. With substrate analogs of GlcNAc-Ins, MshB showed a marked preference for inositol as the aglycon present in GlcNAc-Ins. The inhibition of MshB and Mca by 10 thioglycosides, 7 cyclohexyl-2-deoxy-2-C-alkylglucosides, and 4 redox cyclers was evaluated. The latter contained plumbagin tethered via 2 to 5 methylene carbons and an amide linkage to phenyl-2-deoxy-2-amino-1-thio-alpha-d-glucopyranoside. These proved to be the most potent amongst the 21 compounds tested as inhibitors of MshB. Their inhibitory potency varied with the length of the spacer, with the compound with longest spacer being the most effective.

First report of an antifungal amidase from Peltophorum pterocarpum. [corrected].

A 60 kDa antifungal amidase was purified from Peltophorum pterocarpum [corrected] seeds using an isolation procedure that entailed ion-exchange chromatography on Q-Sepharose, ion-exchange chromatography on DEAE-cellulose and FPLC-gel filtration on Superdex 75. Unlike most other antifungal proteins isolated previously, it was adsorbed on Q-Sepharose and DEAE-cellulose. The isolated protein, designated as peltopterin, exhibited an N-terminal amino acid sequence closely resembling those of amidases. It exhibited amidase activity and digested iodoacetamide with an optimum pH and temperature at pH 9 and 50 degrees C, respectively. It also hydrolyzed acrylamide and urea. It impeded mycelial growth in Rhizotonia solani with an IC(50) of 0.65 microm. Chitin deposition at hyphal tips in R. solani was observed by staining with Congo red after incubation with peltopterin. Its antifungal activity was stable throughout pH 0-14 and 25-100 degrees C. It potently inhibited HIV-1 reverse transcriptase with an IC(50) of 27 nm.

Purification, characterization, molecular cloning, and expression of a new aminoacylase from Streptomyces mobaraensis that can hydrolyze N-(middle/long)-chain-fatty-acyl-L-amino acids as well as N-short-chain-acyl-L-amino acids.

We report here on the purification, characterization, molecular cloning, and expression of a new aminoacylase, initially isolated from the supernatant of Streptomyces mobaraensis (Sm-AA). Purified wild-type Sm-AA was found to be a monomeric protein with a molecular mass of 55 kDa. The cloned gene of Sm-AA contained an ORF of 1,383 bp, encoding a polypeptide of 460 amino acids. A BLAST search revealed that Sm-AA belongs to the peptidase M20 family, with identities to a hypothetical protein from Streptomyces pristinaespiralis, a putative peptidase from Streptomyces avermitilis, peptidase M20 from Frankia sp., succinyl-diaminopimelate desuccinylase from Hemophilus influenzae, and aminoacylase-1 from porcine kidney at 89, 88, 67, 29, and 25% respectively. The Sm-AA gene was subcloned into an expression vector, pSH19, and was expressed in Streptomyces lividans TK24. The amount of the recombinant Sm-AA expressed in the S. lividans cells was approximately 42-fold higher than that of Sm-AA found in the supernatant of S. mobaraensis. Sm-AA showed high hydrolytic activity towards various N-acetyl-L-amino acids and N-(middle/long)-chain-fatty-acyl-L-amino acids, with a preference for the acyl derivatives of L-Met, L-Ala, L-Cys, etc. with an optimum pH and temperature for reaction of about 7.5 and 50 degrees Celsius (at pH 7.5).