Purification and characterization of a novel thermo-active amidase from Geobacillus subterraneus RL-2a

A thermostable amidase produced by Geobacillus subterraneus RL-2a was purified to homogeneity, with a yield of 9.54 % and a specific activity of 48.66 U mg^?1. The molecular weight of the native enzyme was estimated to be 111 kDa. The amidase of G. subterraneus RL-2a is constitutive in nature, active at a broad range of pH (4.5–11.5) and temperature (40–90 °C) and has a half-life of 5 h and 54 min at 70 °C. Inhibition of enzyme activity was observed in the presence of metal ions, such as Co²⁺, Hg²⁺, Cu²⁺, Ni²⁺, and thiol reagents. The presence of mid-chain aliphatic and amino acid amides enhances the enzymatic activity. The acyl transferase activity was detected with propionamide, butyramide and nicotinamide. The enzyme showed moderate stability toward toluene, carbon tetrachloride, benzene, ethylene glycol except acetone, ethanol, butanol, propanol and dimethyl sulfoxide. The K _m and V _max of the purified amidase with nicotinamide were 6.02 ± 0.56 mM and 132.6 ± 4.4 μmol min^?1 mg^?1 protein by analyzing Michaelis–Menten kinetics. The results of MALDI-TOF analysis indicated that this amidase has homology with the amidase of Geobacillus sp. C56-T3 (gi|297530427). It is the first reported wide-spectrum thermostable amidase from a thermophilic G. subterraneus.     

Nitrilase superfamily aryl acylamidase from the halotolerant mangrove Streptomyces sp. 211726

A novel nitrilase superfamily amidase gene, designated azl13, was cloned from Streptomyces sp. 211726. Bioinformatic and biochemical analysis indicated that Azl13 belongs to a new subfamily in branch 13 of the nitrilase superfamily. His₆-Azl13 was expressed in Escherichia coli BL21(DE3) and had the expected molecular mass of 31 kDa, and the enzymatic activity was best at 40 °C, pH 8.0. His₆-Azl13 had amidase, aryl acylamidase, and acyl transferase activities, and it displayed an unusually wide substrate spectrum. His₆-Azl13 was most active on 4-guanidinobutyramide, which is probably its natural substrate, moderately active on short-chain aliphatic amides and weakly active hydrolyzing aromatic and heterocyclic amides. His₆-Azl13 also catalyzed acyl transfer to hydroxylamine from acetamide or the herbicide propanil. The substrate spectrum differs from that of the Pseudomonas amidase RamA, probably reflecting high salinity adaptation. The broad substrate spectrum of Azl13 is potentially useful for chemical synthesis and biodegradation.     

Optimization of arylacetonitrilase production from Alcaligenes sp. MTCC 10675 and its application in mandelic acid synthesis

Alcaligenes sp. MTCC 10675 has been isolated from soil sample using enrichment method and has nitrilase catalytic system which is highly specific for the hydrolysis of arylaliphatic nitriles. Optimization of culture conditions using response surface methodology and inducer-mediated approach enhanced arylacetonitrilase production significantly (2.4-fold). Isobutyronitrile acted as an effective inducer for the induction of arylacetonitrilase, and it is highly specific for arylacetonitriles (phenyl acetonitrile and mandelonitrile). Arylacetonitrilase has no effect on its relative velocity (V _r) up to 20 mM substrate (mandelonitrile) concentration and at 30 mM mandelonitrile, 23.4 % degree of inhibition (I _d) was recorded. Half life of arylacetonitrilase of Alcaligenes sp. MTCC 10675 was 27.5 h at 25 °C. Hg²⁺, Ag⁺, Pb³⁺, and Co²⁺ were strong inhibitor of arylacetonitrilase activity which resulted into 100 %, 91 %, 84 %, and 83 % inhibition, respectively. Polar protic solvent (dichloromethane, dimethylsulphooxide, and n-butanol) reduce arylacetonitrilase activity up to 80–94 % at 10 % concentration. Alcaligenes sp. MTCC 10675 has higher biocatalytic activity, i.e., 3.9 gg^-1 dcw, which is highest in comparison to till reported organism. Arylacetonitrilase-mediated hydrolysis of racemic mandelonitrile resulted into R-(-) mandelic acid with 99.0 % enantiomeric excess (e.e.)     

First Thermostable Endo-β-1,4-Glucanase from Newly Isolated Xanthomonas sp. EC102

A novel gene encoding thermostable endoglucanase was identified in Xanthomonas sp. EC102 from soil. The gene had 1,458 base pairs of open reading frame, which encode a 52-kDa protein of 486 amino acid residues. Sequence of the amino acid residues was similar with the endoglucanase from Xanthomonas campestris pv. campestris ATCC33913 (GenBank Accession No. NP_638867.1) (94 % identity). The endoglucanase was overexpressed in Escherichia coli BL21 and purified. Temperature for the highest enzymatic activity was 70 °C and pH optima was pH 5.5. The specific activity of the endoglucanase toward carboxymethylcellulose (CMC) was approximately 2 μmol min^?1 mg^?1, V _max for CMC was 1.44 μmol mg^?1 min^?1, and K _m values was 25.6 mg mL^?1. The EC102 endoglucanase was stable at temperatures up to 60 °C, and it was activated by 0.1 mM of Mn²⁺ and Co²⁺. This is the first report about thermostable endoglucanase from Xanthomonas sp.     

Heterologous overexpression of Vigna radiata epoxide hydrolase in Escherichia coli and its catalytic performance in enantioconvergent hydrolysis of p-nitrostyrene oxide into (R)-p-nitrophenyl glycol

Two native epoxide hydrolases (EHs) were previously discovered from mung bean powder (Vigna radiata), both of which can catalyze the enantioconvergent hydrolysis of p-nitrostyrene oxide (pNSO). In this study, the encoding gene of VrEH1 was successfully cloned from the cDNA of V. radiata by RT-PCR and rapid amplification of cDNA ends (RACE) technologies. High homologies were found to two putative EHs originated from Glycine max (80 %) and Medicago truncatula (79 %). The vreh1 gene constructed in pET28a(+) vector was then heterologously overexpressed in Escherichia coli BL21(DE3), and the encoded protein was purified to homogeneity by nickel affinity chromatography. It was shown that VrEH1 has an optimum activity at 45 °C and is very thermostable with an inactivation energy of 468 kJ mol^-1. The enzyme has no apparent requirement of metal ions for activity, and its activity was strongly inhibited by 1 mM of Ni²⁺, Cu²⁺, Fe²⁺, or Co²⁺. By adding 0.1 % Triton X-100, the enzyme activity could be significantly increased up to 340 %. VrEH1 shows an unusual ability of enantioconvergent catalysis for the hydrolysis of racemic pNSO, affording (R)-p-nitrophenyl glycol (pNPG). It displays opposite regioselectivity toward (S)-pNSO (83 % to C_α) in contrast to (R)-pNSO (87 % to C_β). The K _M and k _cat of VrEH1 were determined to be 1.4 mM and 0.42 s^-1 for (R)-pNSO and 5.5 mM and 6.2 s^-1 for (S)-pNSO. This thermostable recombinant VrEH1 with enantioconvergency is considered to be a promising biocatalyst for the highly productive preparation of enantiopure vicinal diols and also a good model for understanding the mechanism of EH stereoselectivity.     

Cloning and characterization of a novel amidase from <Emphasis Type="Italic">Paracoccus</Emphasis> sp. M-1, showing aryl acylamidase and acyl transferase activities

A novel amidase gene, designated pamh, was cloned from Paracoccus sp. M-1. Site-directed mutagenesis and bioinformatic analysis showed that the PamH protein belonged to the amidase signature enzyme family. PamH was expressed in Escherichia coli, purified, and characterized. The molecular mass of PamH was determined to be 52 kDa with an isoelectric point of 5.13. PamH displayed its highest enzymatic activity at 45°C and at pH 8.0 and was stable within a pH range of 5.0–10.0. The PamH enzyme exhibited amidase activity, aryl acylamidase activity, and acyl transferase activity, allowing it to function across a very broad substrate spectrum. PamH was highly active on aromatic and short-chain aliphatic amides (benzamide and propionamide), moderately active on amino acid amides, and possessed weak urease activity. Of the anilides examined, only propanil was a good substrate for PamH. For propanil, the k _cat and K _m were 2.8 s^?1 and 158 μM, respectively, and the catalytic efficiency value (k _cat/K _m) was 0.018 μM^?1 s^?1. In addition, PamH was able to catalyze the acyl transfer reaction to hydroxylamine for both amide and anilide substrates, including acetamide, propanil, and 4-nitroacetanilide; the highest reaction rate was shown with isobutyramide. These characteristics make PamH an excellent candidate for environmental remediation and an important enzyme for the biosynthesis of novel amides.     

Purification and characterization of a thermostable λ-carrageenase from a hot spring bacterium,Bacillus sp.

Purpose of work The purpose of this study is to report a thermostable λ-carrageenase that can degrade λ-carrageenan yielding neo-λ-carrabiose at 75 °C. A thermophilic strain Lc50-1 producing λ-carrageenase was isolated from a hot spring in Indonesia and identified as a Bacillus sp. The λ-carrageenase, Cga-L50, with an apparent molecular weight of 37 kDa and a specific activity of 105 U/mg was purified from the culture supernatant. The optimum pH and temperature of Cga-L50 were 8.0 and 75 °C, respectively. The enzyme was stable from pH 6–9 and retained ~50 % activity after holding at 85 °C for 10 min. Significant activation of Cga-L50 was observed with K⁺, Ca²⁺, Co²⁺, and Na⁺; whereas, the enzyme activity was inhibited by Sr²⁺, Mn²⁺, Fe²⁺, Cu²⁺,Cd²⁺, Mg²⁺, and EDTA. Cga-L50 is an endo-type λ-carrageenase that hydrolyzes β-1,4-linkages of λ-carrageenan, yielding neo-λ-carrabiose as the main product. This study is the first to present evidence of thermostable λ-carrageenase from hot spring bacteria.     

Characterization and application of a newly synthesized 2-deoxyribose-5-phosphate aldolase

A codon-optimized 2-deoxyribose-5-phosphate aldolase (DERA) gene was newly synthesized and expressed in Escherichia coli to investigate its biochemical properties and applications in synthesis of statin intermediates. The expressed DERA was purified and characterized using 2-deoxyribose-5-phosphate as the substrate. The specific activity of recombinant DERA was 1.8 U/mg. The optimum pH and temperature for DERA activity were pH 7.0 and 35 °C, respectively. The recombinant DERA was stable at pH 4.0–7.0 and at temperatures below 50 °C. The enzyme activity was inhibited by 1 mM of Ni²⁺, Ba²⁺ and Fe²⁺. The apparent K _m and V _max values of purified enzyme for 2-deoxyribose-5-phosphate were 0.038 mM and 2.9 μmol min^?1 mg^?1, for 2-deoxyribose were 0.033 mM and 2.59 μmol min^?1 mg^?1, respectively, which revealed that the enzyme had similar catalytic efficiency towards phosphorylated and non-phosphorylated substrates. To synthesize statin intermediates, the bioconversion process for production of (3R, 5S)-6-chloro-2,4,6-trideoxyhexose from chloroacetaldehyde and acetaldehyde by the recombinant DERA was developed and a conversion of 94.4 % was achieved. This recombinant DERA could be a potential candidate for application in production of (3R, 5S)-6-chloro-2,4,6-trideoxyhexose.     

Purification and biochemical characterization of a new alkali-stable laccase from Trametes sp. isolated in Tunisia: role of the enzyme in olive mill waste water treatment

A white-rot basidiomycete, isolated from decayed acacia wood (from Northwest of Tunisia) and identified as Trametes sp, was selected in a broad plate screening because of its ability to decolorize and dephenolize olive oil mill wastewater (OMW) efficiently. The major laccase was purified and characterized as a monomeric protein with apparent molecular mass of 61 kDa (SDS-PAGE). It exhibits high enzyme activity over broad pH and temperature ranges with optimum activity at pH 4.0 and a temperature of 60 °C. The purified laccase is stable at alkaline pH values. The enzyme retained 50 % of its activity after 90 min of incubation at 55 °C. Using ABTS, this laccase presented K _m and V _max values of 0.05 mM and 212.73 μmoL min^?1 mg^?1, respectively. It has shown a degrading activity towards a variety of phenolic compounds. The purified laccase was partially inhibited by Fe²⁺, Zn²⁺, Cd²⁺ and Mn²⁺, while Cu²⁺ acted as inducer. EDTA (10 mM) and NaN₃ (10 mM) were found to completely inhibit its activity. 73 % OMW was dephenolized after 315 min incubation at 30 °C with 2 U mL^?1 of laccase and 2 mM HBT.     

Expression,purification, and characterization of recombinant mangrove glutamine synthetase

To expand our knowledge about the relationship of nitrogen use efficiency and glutamine synthetase (GS) activity in the mangrove plant, a cytosolic GS gene from Avicennia marina has been heterologously expressed in and purified from Escherichia coli. Synthesis of the mangrove GS enzyme in E. coli was demonstrated by functional genetic complementation of a GS deficient mutant. The subunit molecular mass of GSI was ~40 kDa. Optimal conditions for biosynthetic activity were found to be 35 °C at pH 7.5. The Mg²⁺-dependent biosynthetic activity was strongly inhibited by Ni²⁺, Zn²⁺, and Al³⁺, whereas was enhanced by Co²⁺. The apparent K _m values of AmGLN1 for the substrates in the biosynthetic assay were 3.15 mM for glutamate, and 2.54 mM for ATP, 2.80 mM for NH₄ ⁺ respectively. The low affinity kinetics of AmGLN1 apparently participates in glutamine synthesis under the ammonium excess conditions.     

Influence of metal ions on bioremediation activity of protocatechuate 3,4-dioxygenase from Stenotrophomonas maltophilia KB2

The aim of this paper was to describe the effect of various metal ions on the activity of protocatechuate 3,4-dioxygenase from Stenotrophomonas maltophilia KB2. We also compared activity of different dioxygenases isolated from this strain, in the presence of metal ions, after induction by various aromatic compounds. S. maltophilia KB2 degraded 13 mM 3,4-dihydroxybenzoate, 10 mM benzoic acid and 12 mM phenol within 24 h of incubation. In the presence of dihydroxybenzoate and benzoate, the activity of protocatechuate 3,4-dioxygenase and catechol 1,2-dioxygenase was observed. Although Fe³⁺, Cu²⁺, Zn²⁺, Co²⁺, Al³⁺, Cd²⁺, Ni²⁺ and Mn²⁺ ions caused 20–80 % inhibition of protocatechuate 3,4-dioxygenase activity, the above-mentioned metal ions (with the exception of Ni²⁺) inhibited catechol 1,2-dioxygenase to a lesser extent or even activate the enzyme. Retaining activity of at least one of three dioxygenases from strain KB2 in the presence of metal ions makes it an ideal bacterium for bioremediation of contaminated areas.     

Characterization of the co-purified invertase and β-glucosidase of a multifunctional extract from Aspergillus terreus

The filamentous fungus Aspergillus terreus secretes both invertase and β-glucosidase when grown under submerged fermentation containing rye flour as the carbon source. The aim of this study was to characterize the co-purified fraction, especially the invertase activity. An invertase and a β-glucosidase were co-purified by two chromatographic steps, and the isolated enzymatic fraction was 139-fold enriched in invertase activity. SDS-PAGE analysis of the co-purified enzymes suggests that the protein fraction with invertase activity was heterodimeric, with subunits of 47 and 27 kDa. Maximal invertase activity, which was determined by response surface methodology, occurred in pH and temperature ranges of 4.0–6.0 and 55–65 °C, respectively. The invertase in co-purified enzymes was stable for 1 h at pH 3.0–10.0 and maintained full activity for up to 1 h at 55 °C when diluted in water. Invertase activity was stimulated by 1 mM concentrations of Mn²⁺ (161 %), Co²⁺ (68 %) and Mg²⁺ (61 %) and was inhibited by Al³⁺, Ag⁺, Fe²⁺ and Fe³⁺. In addition to sucrose, the co-purified enzymes hydrolyzed cellobiose, inulin and raffinose, and the apparent affinities for sucrose and cellobiose were quite similar (K_M = 22 mM). However, in the presence of Mn²⁺, the apparent affinity and V_max for sucrose hydrolysis increased approximately 2- and 2.9-fold, respectively, while for cellobiose, a 2.6-fold increase in V_max was observed, but the apparent affinity decreased 5.5-fold. Thus, it is possible to propose an application of this multifunctional extract containing both invertase and β-glucosidase to degrade plant biomass, thus increasing the concentration of monosaccharides obtained from sucrose and cellobiose.     

Cloning, expression and characterization of a novel esterase from Bacillus pumilus

The gene encoding esterase (CE1) from Bacillus pumilus ARA with a calculated molecular weight of 28.4 kDa was cloned, sequenced and efficiently expressed in Escherichia coli. The open reading frame of 747 nucleotides encoded a protein, which was classified as a carboxylesterase with an identity of 87 % to esterase from Bacillus subtilis 168. Recombinant CE1 was purified in a single step to electrophoretic homogeneity by IMAC (Ni²⁺). The enzyme displayed maximum activity toward p-nitrophenyl (pNP) acetate at 37–40 °C and pH?6.5–7.0. It was stable in the pH range from 6.5 to 8.0, and at temperature from 25 to 40 °C. Among four p-nitrophenyl esters tested, the best substrate was pNP acetate with K _m and k _cat values of 0.33 mM and 4.07 s^?1, respectively. Amounts of 2 mM Ca²⁺ and Co²⁺ significantly increased the esterase activity to 190 and 121 %, respectively. These results suggest that CE1 has very attractive applications of increasing feed digestibility in animal nutrition in this moderate temperature range.     

Production of ginsenosides Rg1 and Rh1 by hydrolyzing the outer glycoside at the C-6 position in protopanaxatriol-type ginsenosides using β-glucosidase from Pyrococcus furiosus

The specific activity of a recombinant β-glucosidase from Pyrococcus furiosus for protopanaxatriol (PPT)-type ginsenosides followed the order Rf > R₁ > Re > R₂ > Rg₂, which were converted to Rh₁, Rg₁, Rg₁, Rh₁, and Rh₁, respectively. No activity was observed with Rg₁ and Rh₁. Thus, P. furiosus β-glucosidase hydrolyzed the outer glycoside at the C-6 position in PPT-type ginsenosides whereas the enzyme did not hydrolyze the inner glucoside at the C-6 position and the glucoside at the C-20 position. The activity for Rf was optimal at 95 °C, pH 5.5, 5 mM ginsenoside, and 32 U enzyme l^?1. Under these conditions, P. furiosus β-glucosidase completely converted from R₁ to Rg₁ after 10 h, with a productivity of 0.4 g l^?1 h^?1 and completely converted Rf to Rh₁ after 1.2 h, with a productivity of 2.74 g l^?1 h^?1.     

Cloning, expression and characterization of a thermostable exo-β-D-glucosaminidase from the hyperthermophilic archaeon Pyrococcus horikoshii

An exo-β-d-glucosaminidase gene (PH0511) was cloned from the hyperthermophilic archaeon, Pyrococcus horikoshii, and expressed in Escherichia coli. The purified protein showed a strong exo-β-d-glucosaminidase activity by TLC analysis. DTT (50 mM) had little effect on its homodimeric structure during SDS-PAGE. The enzyme was optimally active at 90°C (over 20 min) and pH 6. It had a half-life of 9 h at 90°C and is the most thermostable glucosaminidase described up to now. The activity was not inhibited by ethanol, 2-propanol, DMSO, PEG-400, denaturing agents SDS (5%, w/v), urea, guanidine hydrochloride (5 M) and Mg²⁺, Mn²⁺, Co²⁺, Ca²⁺, Sr²⁺, Ni²⁺ (at up to 10 mM).     

Neutrophilic, nitrate-dependent, Fe(II) oxidation by a Dechloromonas species

A species of Dechloromonas, strain UWNR4, was isolated from a nitrate-reducing, enrichment culture obtained from Wisconsin River (USA) sediments. This strain was characterized for anaerobic oxidation of both aqueous and chelated Fe(II) coupled to nitrate reduction at circumneutral pH. Dechloromonas sp. UWNR4 was incubated in anoxic batch reactors in a defined medium containing 4.5–5 mM NO₃ ^?, 6 mM Fe²⁺ and 1–1.8 mM acetate. Strain UWNR4 efficiently oxidized Fe²⁺ with 90 % oxidation of Fe²⁺ after 3 days of incubation. However, oxidation of Fe²⁺ resulted in Fe(III)-hydroxide-encrusted cells and loss of metabolic activity, suggested by inability of the cells to utilize further additions of acetate. In similar experiments with chelated iron (Fe(II)-EDTA), encrusted cells were not produced and further additions of acetate and Fe(II)-EDTA could be oxidized. Although members of the genus Dechloromonas are primarily known as perchlorate and nitrate reducers, our findings suggest that some species could be members of microbial communities influencing iron redox cycling in anoxic, freshwater sediments. Our work using Fe(II)-EDTA also demonstrates that Fe(II) oxidation was microbially catalyzed rather than a result of abiotic oxidation by biogenic NO₂ ^?.     

Treatment of simulated wastewater containing toxic amides by immobilized <Emphasis Type="Italic">Rhodococcus rhodochrous</Emphasis> NHB-2 using a highly compact 5-stage plug flow reactor

Biodegradation of toxic amides by immobilized Rhodococcus rhodochrous NHB-2 has been studied to generate data for future development of reactors for the treatment of simulated wastewater containing various toxic amides. The whole resting cells were immobilized in different matrices like agar, polyacrylamide and alginate. Agar gel beads were selected for the treatment of simulated wastewater containing 100mM each acetamide, propionamide, and 10mM of acrylamide and packed in a highly compact five-stage plug flow reactor. The immobilized bacterium worked well in a broad pH range from 5 to 10, with an optimum at 8.7. The apparent K _m-value for the turnover of acetamide for the resting cells was determined to be around 40mM at pH 8.5 and 55°C, whereas the K _m-value of the purified amidase was predicted to be about 20 mM. This organism exhibited greater turnover of aliphatic amides as compared to aromatic amides. Although these cells showed maximal amide-degrading activity at 55°C, simulated wastewater treatment was carried out at 45°C, because of the greater stability of the amidase activity at that temperature. Of note, indices for overall temperature stability, based on the temperature dependence of apparent first order kinetic temperature denaturation constants, were determined to be –7.9±1.1×10^–4, and –13.7±1.3×10^–4, –14.5±0.7×10^–4, and –13.7±0.8×10^–4°Cmin, for free cells and cells immobilized in alginate, agar and polyacrylamide respectively. After 250min the reactor showed maximum degradation of acetamide, propionamide and acrylamide of about 97, 100 and 90%, respectively by using 883 enzyme activity units per reactor stage. The results of this investigation showed that R. rhodochrous NHB-2 expressing thermostable amidase could be used for the efficient treatment of wastewater containing toxic amides. Therefore, we suggest that this microbe has a very high potential for the detoxification of toxic amides from industrial effluents and other wastewaters.     

Cloning, Expression and Characterization of a Trehalose Synthase Gene From Rhodococcus opacus

Trehalose is a unique disaccharide capable of protecting proteins against environmental stress. A novel trehalose synthase (TreS) gene from Rhodococcus opacus was cloned and expressed in Escherichia coli Top10 and BL21 (DE3) pLysS, respectively. The recombinant TreS showed a molecular mass of 79 kDa. Thin layer chromatography (TLC) result suggested that this enzyme had the ability to catalyze the mutual conversion of maltose and trehalose. Moreover, high-performance liquid chromatography (HPLC) result suggested that glucose appeared as a byproduct with a conversion rate of 12 %. The purified recombinant enzyme had an optimum temperature of 25 °C and pH optimum around 7.0. Kinetic analysis revealed that the K _m for trehalose was around 98 mM, which was a little higher than that of maltose. The preferred substrate of TreS was maltose according to the analysis of k _cat/K _m. Both 1 and 10 mM of Hg²⁺, Cu²⁺ and Al³⁺ could inhibit the TreS activity, while only 1 mM of Ca²⁺ and Mn²⁺ could increase its activity. Five amino acid residues, Asp²⁴⁴, Glu²⁸⁶, Asp³⁵⁴, His¹⁴⁷ and His³⁵³, were shown to be conserved in R. opacus TreS, which were also important for α-amylase family enzyme catalysis.     

An extremely thermostable amylopullulanase from Staphylothermus marinus displays both pullulan- and cyclodextrin-degrading activities

A gene encoding an amylopullulanase of the glycosyl hydrolase (GH) family 57 from Staphylothermus marinus (SMApu) was heterologously expressed in Escherichia coli. SMApu consisted of 639 amino acids with a molecular mass of 75.3 kDa. It only showed maximal amino acid identity of 17.1 % with that of Pyrococcus furiosus amylopullulanase in all identified amylases. Not like previously reported amylopullulanases, SMApu has no signal peptide but contains a continuous GH57N_Apu domain. It had the highest catalytic efficiency toward pullulan (k _cat/K _m , 342.34 s^?1?mL?mg^?1) and was extremely thermostable with maximal pullulan-degrading activity (42.1 U/mg) at 105 °C and pH?5.0 and a half-life of 50 min at 100 °C. Its activity increased to 116 % in the presence of 5 mM CaCl₂. SMApu could also degrade cyclodextrins, which are resistant to the other amylopullulanases. The initial hydrolytic products from pullulan, γ-CD, and 6-O-maltooligosyl-β-CD were [6)-α-d-Glcp-(1?→?4)-α-d-Glcp-(1?→?4)-α-d-Glcp-(1→]_n, maltooctaose, and single maltooligosaccharide plus β-CD, respectively. The final hydrolytic products from above-mentioned substrates were maltose and glucose. These results confirm that SMApu is a novel amylopullulanase of the family GH57 possessing the cyclodextrin-degrading activity of cyclomaltodextrinase.     

Isolation of identical nitrilase genes from multiple bacterial strains and real-time PCR detection of the genes from soils provides evidence of horizontal gene transfer

Bacterial enzymes capable of nitrile hydrolysis have significant industrial potential. Microbacterium sp. AJ115, Rhodococcus erythropolis AJ270 and AJ300 were isolated from the same location in England and harbour identical nitrile hydratase/amidase gene clusters. Strain AJ270 has been well studied due to its nitrile hydratase and amidase activity. R. erythropolis ITCBP was isolated from Denmark and carries a very similar nitrile hydratase/amidase gene cluster. In this study, an identical nitrilase gene (nit1) was isolated from the four strains, and the nitrilase from strain AJ270 cloned and expressed in Escherichia coli. Analysis of the recombinant nitrilase has shown it to be functional with activity demonstrated towards phenylacetonitrile. A real-time PCR TaqMan^® assay was developed that allowed nit1 detection directly from soil enrichment cultures without DNA extraction, with nit1 detected in all samples tested. Real-time PCR screening of isolates from these soils resulted in the isolation of nit1 and also very similar nitrilase gene nit2 from a number of Burkholderia sp. The genes nit1 and nit2 have also been detected in many bacteria of different genera but are unstable in these isolates. It is likely that the genes were acquired by horizontal gene transfer and may be widespread in the environment.