[show abstract][hide abstract] ABSTRACT: A cell-free extract of Saccharomyces cerevisiae containing the angiotensin I-converting enzyme (ACE) inhibitory peptide was treated in a successive simulated gastric-intestinal bioreactor (step 1: amylase digestion, step 2: gastric fluid digestion, step 3: intestinal fluid digestion) to illustrate the absorption pattern of antihypertensive ACE inhibitory peptide, and the ACE inhibitory activities of each step were determined. Total ACE inhibitory activities of step 1, step 2, and step 3 were 55.96%, 80.09%, and 76.77%, respectively. The peptide sequence of each steps was analyzed by MS/MS spectrophotometry. Eleven kinds of representative peptide sequences were conserved in each step, and representative new peptides including RLPTESVPEPK were identified in step 3.
[show abstract][hide abstract] ABSTRACT: Recently, a cyclic AMP receptor protein homologue, GlxR, was reported to bind to the upstream regions of several genes involved in the regulation of diverse physiological processes in Corynebacterium glutamicum. However, the function of GlxR has not yet been explored in C. glutamicum in vivo using a glxR deletion mutant. Therefore, this study examines the role of GlxR as a repressor in glyoxylate bypass and carbon catabolite repression (CCR) using a deletion mutant. The disruption of glxR resulted in a severe growth defect, but growth was restored by complementation with the glxR and crp genes from C. glutamicum and Streptomyces coelicolor, respectively. The production of isocitrate lyase (ICL) and malate synthase (MS) was significantly increased in the glxR mutant. The specific activities of both enzymes were increased in the glxR mutant, regardless of the carbon source. In accordance, the promoter activities of ICL and MS using lacZ fusion were derepressed in the glxR mutant. In addition, the glxR mutant exhibited derepression of the gluA gene for glutamate uptake in the presence of glucose, thereby relieving CCR by glucose. These results indicate that GlxR plays an important role in CCR as well as in acetate metabolism.
[show abstract][hide abstract] ABSTRACT: Genome analysis of C. glutamicum ATCC 13032 has showed one putative adenylate cyclase gene, cyaB (cg0375) which encodes membrane protein belonging to class III adenylate cyclases. To characterize the function of cyaB, a deletion mutant was constructed, and the mutant showed decreased level of intracellular cyclic AMP compared to that of wild-type. Interestingly, the cyaB mutant displayed growth defect on acetate medium, and this effect was reversed by complementation with cyaB gene. Similarly, it showed growth defect on glucose-acetate mixture minimal medium, and the utilization of glucose was retarded in the presence of acetate. The deletion mutant retained the activity of glyoxylate bypass enzymes. Additionally, the mutant could grow on ethanol but not on propionate medium. The data obtained from this study suggests that adenylate cyclase plays an essential role in the acetate metabolism of C. glutamicum, even though detailed regulatory mechanisms involving cAMP are not yet clearly defined. The observation that glyoxylate bypass enzymes are derepressed in cyaB mutant indicates the involvement of cAMP in the repression of aceB and aceA.
Applied Microbiology and Biotechnology 08/2009; 85(4):1061-8. · 3.69 Impact Factor
[show abstract][hide abstract] ABSTRACT: Bacteria sense their population density and coordinate the expression of target genes, including virulence factors in Gram-negative bacteria, by the N-acylhomoserine lactones (AHLs)-dependent quorum-sensing (QS) mechanism. In contrast, several soil bacteria are able to interfere with QS by enzymatic degradation of AHLs, referred to as quorum quenching. A potent AHL-degrading enzyme, AiiA, of Bacillus thuringiensis has been reported to effectively attenuate the virulence of bacteria by quorum quenching. However, little is known about the role of AiiA in B. thuringiensis itself. In the present study, an aiiA-defective mutant was generated to investigate the role of AiiA in rhizosphere competence in the root system of pepper. The aiiA mutant showed no detectable AHL-degrading activity and was less effective for suppression of soft-rot symptom caused by Erwinia carotovora on the potato slice. On the pepper root, the survival rate of the aiiA mutant significantly decreased over time compared with that of wild type. Interestingly, viable cell count analysis revealed that the bacterial number and composition of E. carotovora were not different between treatments of wild type and the aiiA mutant, although root application of the aiiA mutant in pepper failed to protect the plant from root rot. These results provide evidence that AiiA can play an important role in rhizosphere competentce of B. thuringiensis and bacterial quorum quenching to Gram-negative bacteria without changing bacterial number or composition.
Journal of Microbiology and Biotechnology 10/2008; 18(9):1518-21. · 1.40 Impact Factor
[show abstract][hide abstract] ABSTRACT: The M37 lipase from Photobacterium lipolyticum shows an extremely low activation energy and strong activity at low temperatures, with optimum activity seen at 298 K and more than 75% of the optimum activity retained down to 278 K. Though the M37 lipase is most closely related to the filamentous fungal lipase, Rhizomucor miehei lipase (RML) at the primary structure level, their activity characteristics are completely different. In an effort to identify structural components of cold adaptation in lipases, we determined the crystal structure of the M37 lipase at 2.2 A resolution and compared it to that of nonadapted RML. Structural analysis revealed that M37 lipase adopted a folding pattern similar to that observed for other lipase structures. However, comparison with RML revealed that the region beneath the lid of the M37 lipase included a significant and unique cavity that would be occupied by a lid helix upon substrate binding. In addition, the oxyanion hole was much wider in M37 lipase than RML. We propose that these distinct structural characteristics of M37 lipase may facilitate the lateral movement of the helical lid and subsequent substrate hydrolysis, which might explain its low activation energy and high activity at low temperatures.
Proteins Structure Function and Bioinformatics 01/2008; 71(1):476-484. · 3.34 Impact Factor
[show abstract][hide abstract] ABSTRACT: In this review, we describe the phosphotransferase system (PTS) of Corynebacterium glutamicum and discuss genes for putative global carbon regulation associated with the PTS. C. glutamicum ATCC 13032 has PTS genes encoding the general phosphotransferases enzyme I, HPr and four enzyme II permeases, specific for glucose, fructose, sucrose and one yet unknown substrate. C. gluamicum has a peculiar sugar transport system involving fructose efflux after hydrolyzing sucrose transported via sucrose EII. Also, in addition to their primary PTS, fructose and glucose are each transported by a second transporter, glucose EII and a non-PTS permease, respectively. Interestingly, C. glutamicum does not show any preference for glucose, and thus co-metabolizes glucose with other sugars or organic acids. Studies on PTS-mediated sugar uptake and its related regulation in C. glutamicum are important because the production yield of lysine and cell growth are dependent on the PTS sugars used as substrates for fermentation. In many bacteria, the PTS is also involved in several regulatory processes. However, the detailed molecular mechanism of global carbon regulation associated with the PTS in this organism has not yet been revealed.
Journal of Molecular Microbiology and Biotechnology 02/2007; 12(1-2):43-50. · 1.68 Impact Factor
[show abstract][hide abstract] ABSTRACT: N-acylhomoserine lactones (AHLs) are conserved signal molecules that control diverse biological activities in quorum sensing system of Gram-negative bacteria. Recently, several soil bacteria were found to degrade AHLs, thereby interfering with the quorum sensing system. Previously, Rhodococcus erythropolis W2 was reported to degrade AHLs by both oxido-reductase and AHL-acylase. In the present study, two AHL-utilizing bacteria, strains LS31 and PI33, were isolated and identified as the genus Rhodococcus. They exhibited different AHL-utilization abilities: Rhodococcus sp. strain LS31 rapidly degraded a wide range of AHLs, including N-3-oxo-hexanoyl-l-homoserine lactone (OHHL), whereas Rhodococcus sp. strain PI33 showed relatively less activity towards 3-oxo substituents. Coculture of strain LS31 with Erwinia carotovora effectively reduced the amount of OHHL and pectate lyase activity, compared with coculture of strain PI33 with E. carotovora. A mass spectrometry analysis indicated that both strains hydrolyzed the lactone ring of AHL to generate acylhomoserine, suggesting that AHL-lactonases (AHLases) from the two Rhodococcus strains are involved in the degradation of AHL, in contrast to R. erythropolis W2. To the best of our knowledge, this is the first report on AHLases of Rhodococcus spp.
[show abstract][hide abstract] ABSTRACT: A Gram-positive, rod- or coccoid-shaped and N-hexanoyl-l-homoserine lactone-degrading bacterial strain, A2-4(T), was isolated from a soil in Korea, and its taxonomic position was investigated by using a polyphasic approach. Strain A2-4(T) grew optimally at pH 7.0-8.0 and 30 degrees C without NaCl. Phylogenetic analyses based on 16S rRNA gene sequences revealed that strain A2-4(T) is most closely related to members of the genus Nocardioides. Strain A2-4(T) possessed chemotaxonomic properties indicative of members of the genus Nocardioides; the cell-wall peptidoglycan type was based on ll-diaminopimelic acid, MK-8(H(4)) was the predominant menaquinone and iso-C(16 : 0) was the predominant fatty acid. The DNA G+C content was 72.1 mol%. The 16S rRNA gene sequence of strain A2-4(T) was 98.3-99.1 % similar to those of the type strains of Nocardioides simplex, Nocardioides aromaticivorans and Nocardioides nitrophenolicus and 93.8-96.3 % similar to those of the type strains of other Nocardioides species. Strain A2-4(T) could be distinguished from the three phylogenetic relatives, N. nitrophenolicus, N. aromaticivorans and N. simplex, by DNA-DNA relatedness (25-42 %) and by differences in some phenotypic characteristics. On the basis of the phenotypic, phylogenetic and genetic data, the strain represents a novel species of the genus Nocardioides, for which the name Nocardioides kongjuensis sp. nov. is proposed. The type strain is A2-4(T) (=KCTC 19054(T)=JCM 12609(T)).
International journal of systematic and evolutionary microbiology 09/2006; 56(Pt 8):1783-7. · 2.11 Impact Factor
[show abstract][hide abstract] ABSTRACT: In many Gram-negative bacteria, including a number of pathogens such as Pseudomonas aeruginosa and Erwinia carotovora, virulence factor production and biofilm formation are linked to the quorum-sensing systems that use diffusible N-acyl-L-homoserine lactones (AHLs) as intercellular messenger molecules. A number of organisms also contain genes coding for lactonases that hydrolyze AHLs into inactive products, thereby blocking the quorum-sensing systems. Consequently, these enzymes attract intense interest for the development of antiinfection therapies. However, the catalytic mechanism of AHL-lactonase is poorly understood and subject to controversy. We here report a 2.0-angstroms resolution structure of the AHL-lactonase from Bacillus thuringiensis and a 1.7-angstroms crystal structure of its complex with L-homoserine lactone. Despite limited sequence similarity, the enzyme shows remarkable structural similarities to glyoxalase II and RNase Z proteins, members of the metallo-beta-lactamase superfamily. We present experimental evidence that AHL-lactonase is a metalloenzyme containing two zinc ions involved in catalysis, and we propose a catalytic mechanism for bacterial metallo-AHL-lactonases.
Proceedings of the National Academy of Sciences 01/2006; 102(49):17606-11. · 9.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: Lipase L1 from Geobacillus stearothermophilus L1 contains an unusual extra domain, making a tight intramolecular interaction with the main catalytic domain through a Zn2+-binding coordination. To elucidate the role of the Zn2+, we disrupted the Zn2+-binding site by mutating the zinc-ligand residues (H87A, D61A/H87A, and D61A/H81A/H87A/D238A). The activity vs. temperature profiles of the mutant enzymes showed that the disruption of the Zn2+-binding site resulted in a notable decrease in the optimal temperature for maximal activity from 60 to 45-50 degrees C. The mutations also abolished the Zn2+-induced thermal stabilization. The wild-type enzyme revealed a 34.6-fold increase in stabilization with the addition of Zn2+ at 60 degrees C, whereas the mutant enzymes exhibited no response to Zn2+. Additional circular dichroism spectroscopy studies also confirmed the structural stabilizing role of Zn2+ on lipase L1 at elevated temperatures.
[show abstract][hide abstract] ABSTRACT: N-acylhomoserine lactones (AHLs) play an important role in regulating virulence factors in pathogenic bacteria. Recently, the enzymatic inactivation of AHLs, which can be used as antibacterial targets, has been identified in several soil bacteria. In this study, strain M664, identified as a Streptomyces sp., was found to secrete an AHL-degrading enzyme into a culture medium. The ahlM gene for AHL degradation from Streptomyces sp. strain M664 was cloned, expressed heterologously in Streptomyces lividans, and purified. The enzyme was found to be a heterodimeric protein with subunits of approximately 60 kDa and 23 kDa. A comparison of AhlM with known AHL-acylases, Ralstonia strain XJ12B AiiD and Pseudomonas aeruginosa PAO1 PvdQ, revealed 35% and 32% identities in the deduced amino acid sequences, respectively. However, AhlM was most similar to the cyclic lipopeptide acylase from Streptomyces sp. strain FERM BP-5809, exhibiting 93% identity. A mass spectrometry analysis demonstrated that AhlM hydrolyzed the amide bond of AHL, releasing homoserine lactone. AhlM exhibited a higher deacylation activity toward AHLs with long acyl chains rather than short acyl chains. Interestingly, AhlM was also found to be capable of degrading penicillin G by deacylation, showing that AhlM has a broad substrate specificity. The addition of AhlM to the growth medium reduced the accumulation of AHLs and decreased the production of virulence factors, including elastase, total protease, and LasA, in P. aeruginosa. Accordingly, these results suggest that AHL-acylase, AhlM could be effectively applied to the control of AHL-mediated pathogenicity.
Applied and Environmental Microbiology 06/2005; 71(5):2632-41. · 3.68 Impact Factor
[show abstract][hide abstract] ABSTRACT: Corynebacterium glutamicum ATCC 13032 has four enzyme II (EII) genes of the phosphotransferase system in its genome encoding transporters for sucrose, glucose, fructose, and an unidentified EII. To analyze the function of these EII genes, they were inactivated via homologous recombination and the resulting mutants characterized for sugar utilization. Whereas the sucrose EII was the only transport system for sucrose in C. glutamicum, fructose and glucose were each transported by a second transporter in addition to their corresponding EII. In addition, the ptsF ptsG double mutant carrying deletions in the EII genes for fructose and glucose accumulated fructose in the culture broth when growing on sucrose. As no fructokinase gene exists in the C. glutamicum genome, the fructokinase gene from Clostridium acetobutylicum was expressed in C. glutamicum and resulted in the direct phosphorylation of fructose without any fructose efflux. Accordingly, since fructokinase could direct fructose flux to the pentose phosphate pathway for the supply of NADPH, fructokinase expression may be a potential strategy for enhancing amino acid production.
[show abstract][hide abstract] ABSTRACT: The mature lipase LipA and its 56aa-truncated chaperone DeltaLipBhis (with 6xhis-tag) from Ralstonia sp. M1 were over-expressed in Escherichia coli BL21 under the control of T7 promoter with a high level of 70 and 12mg protein per gram of wet cells, respectively. The simply purified lipase LipA was effectively refolded by Ni-NTA purified chaperone DeltaLipBhis in molar ratio 1:1 at 4 degrees C for 24 hours in H2O. The in vitro refolded lipase LipA had an optimal activity in the temperature range of 50-55 degrees C and was stable up to 45 degrees C with more than 84% activity retention. The maximal activity was observed at pH 10.75 for hydrolysis of olive oil and found to be stable over alkaline pH range 8.0-10.5 with more than 52% activity retention. The enzyme was found to be highly resistant to many organic solvents especially induced by ethanolamine (remaining activity 137-334%), but inhibited by 1-butanol and acetonitrile (40-86%). Metal ions Cu2+, Sn2+, Mn2+, Mg2+, and Ca2+ stimulated the lipase slightly with increase in activity by up to 22%, whereas Zn2+ significantly inhibited the enzyme with the residual activity of 30-65% and Fe3+ to a lesser degree (activity retention of 77-86%). Tween 80, Tween 60, and Tween 40 induced the activation of the lipase LipA (222-330%) and 0.2-1% (w/v) of Triton X-100, X-45, and SDS increased the lipase activity by up to 52%. However, 5% (w/v) of Triton X-100, X-45, and SDS inhibited strongly the activity by 31-89%. The inhibitors including DEPC, EDTA, PMSF, and 2-mercaptoethanol (0.1-10mM) inhibited moderately the lipase with remaining activity of 57-105%. The lipase LipA hydrolyzed a wide range of triglycerides, but preferentially short length acyl chains (C4 and C6). In contrast to the triglycerides, medium length acyl chains (C8 and C14) of p-nitrophenyl (p-NP) esters were preferential substrates of this lipase. The enzyme preferentially catalyzed the hydrolysis of cottonseed oil (317%), cornoil (227%), palm oil (222%), and wheatgerm oil (210%) in comparison to olive oil (100%).
Protein Expression and Purification 02/2005; 39(1):97-106. · 1.43 Impact Factor
[show abstract][hide abstract] ABSTRACT: A Gram-negative, motile, non-spore-forming, pleomorphic and lipolytic bacterial strain, M37T, was isolated from an intertidal sediment of the Yellow Sea in Korea. This organism grew optimally at 25-28 degrees C and in the presence of 1-2 % NaCl. It did not grow without NaCl or in the presence of more than 6 % NaCl. Strain M37T was characterized chemotaxonomically by having Q-8 as the predominant respiratory lipoquinone and C(16 : 1)omega7c and/or iso-C(15 : 0) 2-OH and C(16 : 0) as the major fatty acids. The DNA G+C content was 47 mol%. Phylogenetic analyses based on 16S rRNA gene sequences placed strain M37T within the clade comprising Photobacterium species, forming a coherent cluster with the type strains of Photobacterium profundum and Photobacterium indicum (16S rRNA gene similarity levels of 97.5-98.0 %). The mean DNA-DNA relatedness levels between strain M37T and P. profundum JCM 10084T and P. indicum DSM 5151T were in the range 12-15 %. Similarities between 16S rRNA gene sequences of strain M37T and those of the type strains of the other Photobacterium species ranged from 93.9 % (with Photobacterium fischeri) to 96.2 % (with Photobacterium phosphoreum). On the basis of phenotypic properties and phylogenetic and genomic distinctiveness, strain M37T (=KCTC 10562BPT=DSM 16190T) should be placed in the genus Photobacterium as a novel species, for which the name Photobacterium lipolyticum sp. nov. is proposed.
International journal of systematic and evolutionary microbiology 02/2005; 55(Pt 1):335-9. · 2.11 Impact Factor
[show abstract][hide abstract] ABSTRACT: A sequence-based approach was used to retrieve functional lipases from microbial genome databases. Many novel genes assigned as putative lipases were tested using the criteria of the typical lipase sequence rule, based on a consensus sequence of a catalytic triad (Ser, Asp, His) and oxyanion hole sequence (HG). To obtain the lipase genes satisfying the sequence rule, PCR cloning was performed, while the lipase activities were tested using a tributyrin/tricaprylin plate and p-nitrophenyl caproate. Among nine putative lipases from four strains, five functional lipolytic proteins were obtained from Archaeoglobus fulgidus, Deinococcus radiodurans, and Agrobacterium tumefaciens. All five lipases exhibited a relatively low sequence similarity (less than 26.7%) with known lipases and turned out to belong to different lipase families. Accordingly, the current results indicate that the proposed strategic approach based on the microbial genome is an efficient and rapid method for finding novel and functional lipases.
[show abstract][hide abstract] ABSTRACT: Quorum sensing is a signalling mechanism that controls diverse biological functions, including virulence, via N-acylhomoserine lactone (AHL) signal molecules in Gram-negative bacteria. With the aim of isolating strains or enzymes capable of blocking quorum sensing by inactivating AHL, bacteria were screened for AHL degradation by their ability to utilize N-3-oxohexanoyl-L-homoserine lactone (OHHL) as the sole carbon source. Among four isolates, strain IBN110, identified as Arthrobacter sp., was found to grow rapidly on OHHL, and to degrade various AHLs with different lengths and acyl side-chain substitutions. Co-culture of Arthrobacter sp. IBN110 and the plant pathogen Erwinia carotovora significantly reduced both the AHL amount and pectate lyase activity in co-culture medium, suggesting the possibility of applying Arthrobacter sp. IBN110 in the control of AHL-producing pathogenic bacteria. The ahlD gene from Arthrobacter sp. IBN110 encoding the enzyme catalysing AHL degradation was cloned, and found to encode a protein of 273 amino acids. A mass spectrometry analysis showed that AhlD probably hydrolyses the lactone ring of N-3-hexanoyl-L-homoserine lactone, indicating that AhlD is an N-acylhomoserine lactonase (AHLase). A comparison of AhlD with other known AHL-degrading enzymes, Bacillus sp. 240B1 AiiA, a Bacillus thuringiensis subsp. kyushuensis AiiA homologue and Agrobacterium tumefaciens AttM, revealed 25, 26 and 21 % overall identities, respectively, in the deduced amino acid sequences. Although these identities were relatively low, the HXDH approximately H approximately D motif was conserved in all the AHLases, suggesting that this motif is essential for AHLase activity. From a genome database search based on the conserved motif, putative AhlD-like lactonase genes were found in several other bacteria, and AHL-degrading activities were observed in Klebsiella pneumoniae and Bacillus stearothermophilus. Furthermore, it was verified that ahlK, an ahlD homologue, encodes an AHL-degrading enzyme in K. pneumoniae. Accordingly, the current results suggest the possibility that AhlD-like AHLases could exist in many other micro-organisms.
[show abstract][hide abstract] ABSTRACT: An esterase-producing Bacillus megaterium strain (20-1) was isolated from a soil sample collected in South Korea. The cloned gene showed that the esterase 20-1 composed of 310 amino acids corresponding to a molecular mass (Mr) of 34,638. Based on the Mr and the protein sequence, the esterase 20-1 belonged to the H lipase/esterase group. The optimum temperature and pH of the purified His-tagged enzyme were 20–35°C and 8.0, respectively. The esterase 20-1 showed a ‘nonionic detergent-induced activation’ phenomenon, which was a detergent type- and concentration-dependent process. In comparison with the native enzyme, the Tween 80-treated enzyme had relatively a similar kcat value of 274s−1 but a very low Km value of 0.037mM for PNPC (C6), therefore, it showed a 14-fold increase in kcat/Km value.
[show abstract][hide abstract] ABSTRACT: Gram-negative bacteria can communicate with each other by N-acyl homoserine lactones (AHLs), which are quorum-sensing autoinducers. Recently, the aiiA gene (encoding an enzyme catalyzing the degradation of AHL) has been cloned from Bacillus sp. strain 240B1. During investigations in the course of the ongoing Bacillus thuringiensis subsp. morrisoni genome project, an aiiA homologue gene in the genome sequence was found. These results led to consideration of the possibility of the widespread existence of the gene in B. thuringiensis. aiiA homologue genes were found in 16 subspecies of B. thuringiensis, and their sequences were determined. Comparison of the Bacillus sp. strain 240B1 aiiA gene with the B. thuringiensis aiiA homologue genes showed high homologies of 89 to 95% and 90 to 96% in the nucleotide sequence and deduced amino acid sequence, respectively. Among the subspecies of B. thuringiensis having an aiiA gene, the subspecies aizawai, galleriae, kurstaki, kyushuensis, ostriniae, and subtoxicus were shown to degrade AHL. It was observed that recombinant Escherichia coli producing AiiA proteins also had AHL-degrading activity and could also attenuate the plant pathogenicity of Erwinia carotovora. These results indicate that insecticidal B. thuringiensis strains might have potential to compete with gram-negative bacteria in natural ecosystems by autoinducer-degrading activity.
Applied and Environmental Microbiology 09/2002; 68(8):3919-24. · 3.68 Impact Factor