Characterization and biotechnological application of protease from thermophilic Thermomonas haemolytica.

In this study, it was aimed to determine the ability to produce protease enzyme of Thermomonas haemolytica isolated from geothermal Nenehatun hot spring in Turkey and utilization of this enzyme in the detergent industry to remove protein stains. The protease-producing strains were screened from hot springs, and a potential strain was identified as T. haemolytica according to morphological, physiological and biochemical characteristics and sequence of 16S rRNA gene. Maximum protease activity was observed at 55 °C and pH 9.0 at 72 h of incubation. Activity was very stable between 50 and 65 °C and pH 8.0-10.0, respectively. The enzyme activity was significantly inhibited by PMSF and partly inhibited by EDTA, EGTA, SDS, and urea. Some divalent metal ions such as Ca2+, Mg2+, and Mn2+ increased the enzyme activity, while Zn2+ and Cu2+ decreased. Michaelis-Menten constant (Km) and maximum velocity (Vmax) values were calculated by Lineweaver-Burk plot as 125 EU/ml and 1262 mg/ml, respectively. The biochemical characterization of the protease obtained from T. haemolytica was performed and applied on the blood and grass-stained fabrics with detergent to evaluate the stain removal performance of the enzyme. It was observed that the application of detergent with enzyme was more effective than the detergent without enzyme to clean up the stained fabrics. This is the first report of characterization of the protease of T. haemolytica. According to results obtained from this study, this new strain is a promising candidate for industrial applications in production of detergent.

Characterization of a novel cold-active xylanase from Luteimonas species.

Biotechnological application of xylanolytic enzymes is normally hindered by their temperature-dependent catalytic property. To satisfy the industrial demands, xylanases that can perform catalysis under cold condition are attracting attention. In this study, the biochemical properties of a predicted xylanase (laXynA) encoded in the genome of marine bacterium Luteimonas abyssi XH031T were characterized. Structure modeling and structure-based sequence alignment indicated that laXynA belongs to the glycoside hydrolase family 10, and it is 20-26% identical to other characterized cold-active xylanases in the same family. Recombinant laXynA was successfully produced in Escherichia coli system by autoinduction and purified by Ni-affinity chromatography. The isolated enzyme showed an optimum temperature of 30 °C toward beechwood xylan and retained important percentage of optimal activity at low temperatures (64, 55, and 29% at 10, 5, and 0 °C, respectively). A remarkable characteristic of laXynA was extreme halophilicity as demonstrated by fourfold enhancement on xylanase activity at 0.5 M NaCl and by maintaining nearly 100% activity at 4 M NaCl. Thin layer chromatography analysis demonstrated that laXynA is an endo xylanase. This study is the first to report the over-expression and characterization of a cold-active xylanase from Luteimonas species. The enzymatic property revealed the cold-active nature of laXynA. The enzyme is a promising candidate in saline food processing application.

Crystal structures of a bacterial dipeptidyl peptidase IV reveal a novel substrate recognition mechanism distinct from that of mammalian orthologues.

Dipeptidyl peptidase IV (DPP IV, DPP4, or DAP IV) preferentially cleaves substrate peptides with Pro or Ala at the P1 position. The substrate recognition mechanism has been fully elucidated for mammalian DPP IV by crystal structure analyses but not for bacterial orthologues. Here, we report the crystal structures of a bacterial DPP IV (PmDAP IV) in its free form and in complexes with two kinds of dipeptides as well as with a non-peptidyl inhibitor at 1.90 to 2.47 Å resolution. Acyl-enzyme intermediates were observed for the dipeptide complexes of PmDAP IV, whereas tetrahedral intermediates were reported for the oligopeptide complexes of mammalian DPP IVs. This variation reflects the different structural environments of the active site Arg residues, which are involved in the recognition of a substrate carbonyl group, of mammalian and bacterial enzymes. A phylogenetic analysis revealed that PmDAP IV is a closer relative of dipeptidyl peptidases 8 and 9 (DPP8 and DPP9, DPP IV-family enzymes) than DPP IV. These results provide new insights into the substrate recognition mechanism of bacterial DAP IVs and may assist in the development of selective inhibitors for DAP IVs from pathogenic asaccharolytic bacteria, which utilise proteins or peptides as an energy source.

Periplasmic form of dipeptidyl aminopeptidase IV from Pseudoxanthomonas mexicana WO24: purification, kinetic characterization, crystallization and X-ray crystallographic analysis.

Dipeptidyl aminopeptidase IV (DAP IV or DPP IV) from Pseudoxanthomonas mexicana WO24 (PmDAP IV) preferentially cleaves substrate peptides with Pro or Ala at the P1 position [NH2-P2-P1(Pro/Ala)-P1'-P2'…]. For crystallographic studies, the periplasmic form of PmDAP IV was overproduced in Escherichia coli, purified and crystallized in complex with the tripeptide Lys-Pro-Tyr using the hanging-drop vapour-diffusion method. Kinetic parameters of the purified enzyme against a synthetic substrate were also determined. X-ray diffraction data to 1.90 Šresolution were collected from a triclinic crystal form belonging to space group P1, with unit-cell parameters a = 88.66, b = 104.49, c = 112.84 Å, α = 67.42, ß = 68.83, γ = 65.46°. Initial phases were determined by the molecular-replacement method using Stenotrophomonas maltophilia DPP IV (PDB entry 2ecf) as a template and refinement of the structure is in progress.

LaaA, a novel high-active alkalophilic alpha-amylase from deep-sea bacterium Luteimonas abyssi XH031(T).

Alpha-amylase is a kind of broadly used industrial enzymes, most of which have been exploited from terrestrial organism. Comparatively, alpha-amylase from marine environment was largely undeveloped. In this study, a novel alkalophilic alpha-amylase with high activity, Luteimonas abyssi alpha-amylase (LaaA), was cloned from deep-sea bacterium L. abyssi XH031(T) and expressed in Escherichia coli BL21. The gene has a length of 1428bp and encodes 475 amino acids with a 35-residue signal peptide. The specific activity of LaaA reached 8881U/mg at the optimum pH 9.0, which is obvious higher than other reported alpha-amylase. This enzyme can remain active at pH levels ranging from 6.0 to 11.0 and temperatures below 45°C, retaining high activity even at low temperatures (almost 38% residual activity at 10°C). In addition, 1mM Na(+), K(+), and Mn(2+) enhanced the activity of LaaA. To investigate the function of potential active sites, R227G, D229K, E256Q/H, H327V and D328V mutants were generated, and the results suggested that Arg227, Asp229, Glu256 and Asp328 were total conserved and essential for the activity of alpha-amylase LaaA. This study shows that the alpha-amylase LaaA is an alkali-tolerant and high-active amylase with strong potential for use in detergent industry.

phoD Alkaline Phosphatase Gene Diversity in Soil.

Phosphatase enzymes are responsible for much of the recycling of organic phosphorus in soils. The PhoD alkaline phosphatase takes part in this process by hydrolyzing a range of organic phosphoesters. We analyzed the taxonomic and environmental distribution of phoD genes using whole-genome and metagenome databases. phoD alkaline phosphatase was found to be spread across 20 bacterial phyla and was ubiquitous in the environment, with the greatest abundance in soil. To study the great diversity of phoD, we developed a new set of primers which targets phoD genes in soil. The primer set was validated by 454 sequencing of six soils collected from two continents with different climates and soil properties and was compared to previously published primers. Up to 685 different phoD operational taxonomic units were found in each soil, which was 7 times higher than with previously published primers. The new primers amplified sequences belonging to 13 phyla, including 71 families. The most prevalent phoD genes identified in these soils were affiliated with the orders Actinomycetales (13 to 35%), Bacillales (1 to 29%), Gloeobacterales (1 to 18%), Rhizobiales (18 to 27%), and Pseudomonadales (0 to 22%). The primers also amplified phoD genes from additional orders, including Burkholderiales, Caulobacterales, Deinococcales, Planctomycetales, and Xanthomonadales, which represented the major differences in phoD composition between samples, highlighting the singularity of each community. Additionally, the phoD bacterial community structure was strongly related to soil pH, which varied between 4.2 and 6.8. These primers reveal the diversity of phoD in soil and represent a valuable tool for the study of phoD alkaline phosphatase in environmental samples.

Crystallization and preliminary X-ray crystallographic studies of dipeptidyl aminopeptidase BII from Pseudoxanthomonas mexicana WO24.

Dipeptidyl aminopeptidase BII from Pseudoxanthomonas mexicana WO24 (DAP BII) is able to cleave a variety of dipeptides from the amino-terminus of substrate peptides. For crystallographic studies, DAP BII was overproduced in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. X-ray diffraction data to 2.3 Å resolution were collected using an orthorhombic crystal form belonging to space group P2(1)2(1)2(1), with unit-cell parameters a = 76.55, b = 130.86, c = 170.87 Å. Structural analysis by the multi-wavelength anomalous diffraction method is in progress.

A unique enzyme of acetic acid bacteria, PQQ-dependent alcohol dehydrogenase, is also present in Frateuria aurantia.

A membrane-bound, pyrroloquinoline quinone (PQQ)-dependent alcohol dehydrogenase (ADH) was purified from Frateuria aurantia LMG 1558(T). Although F. aurantia belongs to a group of γ-Proteobacteria, the characteristics of its PQQ-ADH were similar to the enzyme characteristics of the typical high-acetic acid-resistant bacterium Gluconacetobacter europaeus from the group of α-Proteobacteria. The PQQ-dependent ADH was solubilized from the membranes and purified after anionic, cationic, and affinity chromatography with specific activity of 117 U/mg. The purified enzyme was estimated to be composed of two subunits of ca. 72 and 45 kDa, as judged by SDS-polyacrylamide gel electrophoresis. The purified enzyme had maximum activity at pH 4.5 and showed the highest substrate specificity to ethanol, isoamyl alcohol, 1-butanol, and 1-propanol. The deduced sequences of cloned genes adhA and adhB encoding subunits I and II of PQQ-ADH showed 80 % amino acid (AA) identity to AdhA and 68 % AA identity to AdhB of Ga. europaeus V3 (LMG 18494). Because of the high similarity between genes encoding subunits I and II of PQQ-ADH and its homologous genes found in a distantly related taxonomic group of acetic acid bacteria, the results suggest the possibility of horizontal gene transfer between these two groups of genera.

Promiscuous esterase activities of the C-C hydrolases from Dyella ginsengisoli.

A C-C hydrolase gene (bphD(LA-4)) from strain Dyella ginsengisoli LA-4 was cloned and expressed in Escherichia coli BL21 (DE3). BphD(LA-4) together with another hydrolase MfphA(LA-4), which derived from the same strain, possessed esterase activities. p-Nitrophenyl butyrate was the best substrate for both enzymes. BphD(LA-4) had high catalytic efficiency to p-nitrophenyl benzoate, whereas MfphA(LA-4) had no activity. Homology modeling and docking studies demonstrated that the proper hydrogen bond interaction was important for the reactivity of specific substrate.

Characterization of a novel ginsenoside-hydrolyzing α-L-arabinofuranosidase, AbfA, from Rhodanobacter ginsenosidimutans Gsoil 3054T.

The gene encoding an α-L-arabinofuranosidase that could biotransform ginsenoside Rc {3-O-[ß-D-glucopyranosyl-(1-2)-ß-D-glucopyranosyl]-20-O-[α-L-arabinofuranosyl-(1-6)-ß-D-glucopyranosyl]-20(S)-protopanaxadiol} to ginsenoside Rd {3-O-[ß-D-glucopyranosyl-(1-2)-ß-D-glucopyranosyl]-20-O-ß-D-glucopyranosyl-20(S)-protopanaxadiol} was cloned from a soil bacterium, Rhodanobacter ginsenosidimutans strain Gsoil 3054(T), and the recombinant enzyme was characterized. The enzyme (AbfA) hydrolyzed the arabinofuranosyl moiety from ginsenoside Rc and was classified as a family 51 glycoside hydrolase based on amino acid sequence analysis. Recombinant AbfA expressed in Escherichia coli hydrolyzed non-reducing arabinofuranoside moieties with apparent K (m) values of 0.53 ± 0.07 and 0.30 ± 0.07 mM and V (max) values of 27.1 ± 1.7 and 49.6 ± 4.1 µmol min(-1) mg(-1) of protein for p-nitrophenyl-α-L-arabinofuranoside and ginsenoside Rc, respectively. The enzyme exhibited preferential substrate specificity of the exo-type mode of action towards polyarabinosides or oligoarabinosides. AbfA demonstrated substrate-specific activity for the bioconversion of ginsenosides, as it hydrolyzed only arabinofuranoside moieties from ginsenoside Rc and its derivatives, and not other sugar groups. These results are the first report of a glycoside hydrolase family 51 α-L-arabinofuranosidase that can transform ginsenoside Rc to Rd.

Nitroreductase activity of ferredoxin reductase BphA4 from Dyella ginsengisoli LA-4 by catalytic and structural properties analysis.

Ferredoxin reductase BphA4 was well known as a component of biphenyl dioxygenase. However, there was little information about whether it could utilize nonphysiological oxidants as electron acceptors. In the present study, we reported the novel nitroreductase activity of BphA4(LA)₋4. The homology model of ferredoxin reductase BphA4 from Dyella ginsengisoli LA-4 was constructed. According to the alignment of three-dimensional structures, it was supposed that BphA4(LA)₋4 could function as nitroreductase. Recombinant His-tagged BphA4(LA)₋4 was purified with a molecular mass of 49.6 ± 1 kDa. Biochemical characterization of purified BphA4(LA)₋4 possessed the nitroreductase activity with the optimal temperature 50°C and pH 8.0. The substrate spectrum and kinetics indicated BphA4(LA)₋4 could reduce several nitroaromatics with different apparent K(m) values: m-dinitrobenzene (560 µM), o-dinitrobenzene (1,060 µM), o-nitroaniline (1,570 µM), m-nitrobenzoic acid (1,300 µM) and m-nitrophenol (67 µM). The nitroreductase activity was further explained by docking studies, which was indicated that Arg 288 should play an important role in binding nitroaromatics. Moreover, there existed a good linear correlation between lnK(m) and calculated binding energy.

Symbiotic adaptation of bacteria in the gut of Reticulitermes speratus: low endo-beta-1,4-glucanase activity.

The termite is a good model of symbiosis between microbes and hosts and possesses an effective cellulose digestive system. Oxygen-tolerant bacteria, such as Dyella sp., Chryseobacterium sp., and Bacillus sp., were isolated from Reticulitermes speratus gut. Notably, the endo-beta-1,4-glucanase (EG) activity of all 16 strains of isolated bacteria was low. Due to the combined activity of EG from the termites and their symbiotic protozoa, the bacteria might not be compelled to express EG. This observation demonstrates how well intestinal bacteria have assimilated themselves into the efficient cellulose digestive systems of termites.

Enzyme-substrate interaction and characterization of a 2,3-dihydroxybiphenyl 1,2-dioxygenase from Dyella ginsengisoli LA-4.

A bphC gene (915 bp) encoding 2,3-dihydroxybiphenyl 1,2-dioxygenase (BphC) was amplified by PCR from Dyella ginsengisoli LA-4, which was heterologously expressed in Escherichia coli. The purified His-Tag BphC was able to catalyze the meta-cleavage reaction of the dihydroxylated aromatic rings. According to the specificity constant (K(cat)/K(m)) of BphC_LA-4, the specificity of BphC_LA-4 was determined in the following order: 2,3-dihydroxybiphenyl>3-methylcatechol>catechol>4-chlorocatechol>4-methylcatechol. The experimental data were consistent with the prediction of enzyme-substrate complexes. The highest specific activity of BphC_LA-4 was 118.3 U mg(-1) for 2,3-dihydroxybiphenyl.

Despite having a common P1 Leu, eglin C inhibits alpha-lytic proteinase a million-fold more strongly than does turkey ovomucoid third domain.

Results of the inhibition of alpha-lytic proteinase by two standard mechanism serine proteinase inhibitors, turkey ovomucoid third domain (OMTKY3) and eglin C, and many of their variants are presented. Despite similarities, including an identical P1 residue (Leu) in their primary contact regions, OMTKY3 and eglin C have vastly different association equilibrium constants toward alpha-lytic proteinase, with Ka values of 1.8 x 10(3) and 1.2 x 10(9) M(-1), respectively. Although 12 of the 13 serine proteinases tested in our laboratory for inhibition by OMTKY3 and eglin C are more strongly inhibited by the latter, the million-fold difference observed here with alpha-lytic proteinase is the largest we have seen. The million-fold stronger inhibition by eglin C is retained when the Ka values of the P1 Gly, Ala, Ser, and Ile variants of OMTKY3 and eglin C are compared. Despite the small size of the S1 pocket in alpha-lytic proteinase, interscaffolding additivity for OMTKY3 and eglin C holds well for the four P1 residues tested here. To better understand this difference, we measured Ka values for other OMTKY3 variants, including some that had residues elsewhere in their contact region that corresponded to those of eglin C. Assuming intrascaffolding additivity and using the Ka values obtained for OMTKY3 variants, we designed an OMTKY3-based inhibitor of alpha-lytic proteinase that was predicted to inhibit 10,000-fold more strongly than wild-type OMTKY3. This variant (K13A/P14E/L18A/R21T/N36D OMTKY3) was prepared, and its Ka value was measured against alpha-lytic proteinase. The measured Ka value was in excellent agreement with the predicted one (1.1 x 10(7) and 2.0 x 10(7) M(-1), respectively). Computational protein docking results are consistent with the view that the backbone conformation of eglin C is not significantly altered in the complex with alpha-lytic proteinase. They also show that the strong binding for eglin C correlates well with more favorable atomic contact energy and desolvation energy contributions as compared to OMTKY3.

Isolation and characterization of a new extracellular bacteriolytic endopeptidase of Lysobacter sp. XL1.

The previously unstudied bacteriolytic enzyme L(4) was isolated from the culture liquid of the bacterium Lysobacter sp. XL1 in electrophoretically homogeneous state. The enzyme L(4) is a diaminopimelinoyl-alanine endopeptidase relative to peptidoglycan of Lysobacter sp. XL1. The enzyme is an alkaline protein of approximately 21 kD. The N-terminal amino acid sequence of the enzyme has been determined - A V V N G V N Y V Gx T T A ... The maximal activity of the enzyme was observed in 0.05 M Tris-HCl at pH 8.0 and 50-55 degrees C. The half-inactivation temperature of the enzyme is 52 degrees C. The endopeptidase L(4) is not a metalloenzyme since it is not affected by EDTA. The enzyme is inhibited by p-chloromercuribenzoic acid by 72% and by phenylmethylsulfonyl fluoride by 43%, which indicates the involvement of serine and thiol groups in its functioning.

The role of clp-regulated factors in antagonism against Magnaporthe poae and biological control of summer patch disease of Kentucky bluegrass by Lysobacter enzymogenes C3.

A global regulator was previously identified in Lysobacter enzymogenes C3, which when mutated, resulted in strains that were greatly reduced in the expression of traits associated with fungal antagonism and devoid of biocontrol activity towards bipolaris leaf-spot of tall fescue and pythium damping-off of sugarbeet. A clp gene homologue belonging to the crp gene family was found to globally regulate enzyme production, antimicrobial activity, and biological control activity expressed by Lysobacter enzymogenes C3 (Kobayashi et al. 2005). Here, we report on the expansion of the biocontrol range of L. enzymogenes C3 to summer patch disease caused by Magnaporthe poae. The clp- mutant strain 5E4 was reduced in its ability to suppress summer patch disease compared with the wild-type strain C3 and was completely devoid of antifungal activity towards M. poae. Furthermore, cell suspensions of 5E4 were incapable of colonizing M. poae mycelium in a manner that was distinct for C3. Strain C3 demonstrated biosurfactant activity in cell suspensions and culture filtrates that was associated with absorption into the mycelium during the colonization process, whereas 5E4 did not. These results describe a novel interaction between bacteria and fungi that intimates a pathogenic relationship.

[Effect of the proteolytic enzymes of Bacillus licheniformis and the lysoamidase of Lysobacter sp. XL1 on Proteus vulgaris and Proteus mirabilis].

Preparations of culture liquid of three Bacullus licheniformis strains (S, 103, and 60.4) and the enzymatic preparation lysoamidase from culture liquid of Lysobacter sp. strain XL1 actively lysed preliminarily autoclaved cells of gram-negative bacteria Proteus vulgaris and P. mirabilis. Living Proteus cells treated with these enzymatic preparations were lysed during their subsequent autoclaving. Inoculation of enzyme-treated Proteus cells, taken either separately or in combination with one another and polymyxin B, into a rich medium led to cell repair and restoration of viability of culture.

Dyella koreensis sp. nov., a beta-glucosidase-producing bacterium.

A bacterial strain (designated BB4(T)), which has beta-glucosidase activity, was isolated from soil around the roots of bamboo plants. Cells were Gram-negative, aerobic, non-motile and straight-rod-shaped. Phylogenetic analysis of 16S rRNA gene sequences revealed a clear affiliation with members of the family 'Xanthomonadaceae'. The 16S rRNA gene sequence of strain BB4(T) showed the following sequence similarities: 97.7% to Dyella japonica XD53(T), 97.1% to Frateuria aurantia LMG 1558(T), 96.2% to Fulvimonas soli LMG 19981(T), 94.3% to Rhodanobacter lindaniclasticus RP5575(T) and <90% to other members of the 'Gammaproteobacteria'. The G+C content of the genomic DNA was 63.8 mol%. The major fatty acids were branched forms, especially large proportions of iso-C(15:0), iso-C(17:0) and iso-C(17:1)omega9c, similar to the profile of the genus Dyella. The results of DNA-DNA hybridization with D. japonica XD53(T) and Frateuria aurantia LMG 1558(T), in combination with phenotypic characteristics and 16S rRNA gene sequence analysis, demonstrated that strain BB4(T) should be classified as a novel Dyella species. The name Dyella koreensis sp. nov. is proposed, with strain BB4(T) (=KCTC 12359(T)=NBRC 100831(T)) as the type strain.

A clp gene homologue belonging to the Crp gene family globally regulates lytic enzyme production, antimicrobial activity, and biological control activity expressed by Lysobacter enzymogenes strain C3.

Lysobacter enzymogenes strain C3, a biological control agent for plant diseases, produces multiple extracellular hydrolytic enzymes and displays antimicrobial activity against various fungal and oomycetous species. However, little is known about the regulation of these enzymes or their roles in antimicrobial activity and biocontrol. A study was undertaken to identify mutants of strain C3 affected in extracellular enzyme production and to evaluate their biocontrol efficacy. A single mini-Tn5-lacZ(1)-cat transposon mutant of L. enzymogenes strain C3 that was globally affected in a variety of phenotypes was isolated. In this mutant, 5E4, the activities of several extracellular lytic enzymes, gliding motility, and in vitro antimicrobial activity were reduced. Characterization of 5E4 indicated that the transposon inserted in a clp gene homologue belonging to the Crp gene family of regulators. Immediately downstream was a second open reading frame similar to that encoding acetyltransferases belonging to the Gcn5-related N-acetyltransferase superfamily, which reverse transcription-PCR confirmed was cotranscribed with clp. Chromosomal deletion mutants with mutations in clp and between clp and the acetyltransferase gene verified the 5E4 mutant phenotype. The clp gene was chromosomally inserted in mutant 5E4, resulting in complemented strain P1. All mutant phenotypes were restored in P1, although the gliding motility was observed to be excessive compared with that of the wild-type strain. clp mutant strains were significantly affected in biological control of pythium damping-off of sugar beet and bipolaris leaf spot of tall fescue, which was partially or fully restored in the complemented strain P1. These results indicate that clp is a global regulatory gene that controls biocontrol traits expressed by L. enzymogenes C3.

[Mechanism of action of the extracellular bacteriolytic enzymes of Lysobacter sp. on gram-positive bacteria: role of the cell wall anionic polymers of the target bacteria]. / Rol' anionnykh polimerov kletochnykh stenok grampolozhitel'nykh bakterii-mishenei v mekhanizme deistviia na nikh vnekletochnykh bakterioliticheskikh fermentov Lysobacter sp.

The study of the extracellular bacteriolytic enzymes of Lysobacter sp. showed that they can efficiently hydrolyze the peptidoglycan of gram-positive bacteria provided that there is an electrostatic interaction of these enzymes with the cell wall anionic polymers, teichoic and teichuronic acids in particular. The hydrolytic action of bacteriolytic enzymes on the cell wall largely depends on the negative charge of teichoic and teichuronic acids, rather than on their chemical composition.