Overproduction, purification, and characterization of extracellular lipoxygenase of Pseudomonas aeruginosa in Escherichia coli

Lipoxygenase (LOX; EC 1.13.11.12,) is an enzyme that is widely used in food industry to improve aroma, rheological, or baking properties of foods. In this study, we described the expression and characterization of Pseudomonas aeruginosa LOX in Escherichia coli. The recombinant LOX was successfully expressed and secreted by E. coli using its endogenous signal peptide. When induced with 1 mM isopropyl β-d-1-thiogalactopyranoside (final concentration) at 20 °C for 47 h, the titer of the recombinant enzyme reached 3.89 U/mL. In order to characterize the catalytic properties, the recombinant LOX was purified to homogeneity on Q High Performance and Mono Q5/50GL sequentially. The molecular weight of the LOX was estimated as 70 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The K _m and V _max of the recombinant enzyme were 48.9 μM and 0.226 μmol/min, respectively. The purified enzyme exhibited a maximum activity at 25 °C and pH 7.5. High-performance liquid chromatography analysis of the linoleic acid hydroperoxides produced by recombinant LOX revealed that the LOX from P. aeruginosa falls into linoleic acid 13(S)-LOX. To the best of our knowledge, this is the first report on the overexpression of extracellular LOX in microorganisms, and the achieved LOX yield is the highest ever reported.     

Characterization and preliminary mutation analysis of a thermostable alanine racemase from Thermoanaerobacter tengcongensis MB4

A thermostable alanine racemase from Thermoanaerobacter tengcongensis MB4 was successfully expressed in Escherichia coli and characterized. The full-length gene MBalr2 (1164 bp) encodes 388 amino acid residues including 6 out of 8 highly conserved amino acid residues at the entryway to the active site of alanine racemase. Recombinant MBAlr2 and three mutants (S171A, H359Y and double mutation S171A/H359Y) of MBAlr2 were purified by His₆-tag affinity column and gel filtration chromatography. The purified protein MBAlr2 was a dimeric PLP-dependent enzyme with broad substrate specificity. The optimal racemization temperature and pH were 70–75 °C and 11.0, respectively. The kinetic parameters K _m and V _max of MBAlr2 at 70 °C, determined by HPLC, were 20.16 mM and 1414 μmol min^?1 for l-alanine, and 9.95 mM and 702.6 μmol min^?1 for d-alanine, respectively. Enzymatic assays showed that the activity of both mutants (S171A and H359Y) was lost, but the activity of mutant S171A/H359Y was recovered to 69.8 % of wild type, which suggested that residues Ser171 and His359 might be the important residues for catalytic mechanisms of MBAlr2.     

A valine-resistant mutant ofArabidopsis thaliana displays an acetolactate synthase with altered feedback control

A valine-resistant mutant line, VAL-2, ofArabidopsis thaliana (L.) Heynh. was identified by screening M 2 populations of ethylmethane-sulfonate-mutagenized seeds. The resistance was found to be due to a single, dominant, nuclear gene mutation. Assay of acetolactate synthase (ALS) indicated that the valine resistance in this mutant is caused by decreased sensitivity of ALS to the branched-chain amino acids, valine, leucine andisoleucine. A two fold decrease in apparentK _m value for pyruvate of the mutant ALS enzyme was detected compared with that of the wild type. The sensitivity of the ALS enzyme to sulfonylurea, imidazolinone and triazolopyrimidine herbicides was not altered in the mutant. At the plant growth level the mutant was also resistant to valine plus leucine, but was sensitive to leucine orisoleucine alone. The mutant gene,var1, maps, or is very closely linked, toCSR1, the gene encoding acetolactate synthase inArabidopsis.Abbreviations ALS acetolactate synthase - BCAA branched-chain amino acid - CS chlorsulfuron - IM imidazolinone - SU sulfonylurea - TP triazolopyrimidine We thank Dr. George W. Haughn for providing Arabidopsis lines MSU12, MSU15, MSU21, MSU22 and MSU23. This work was supported by a Research Grant from the Natural Sciences and Engineering Research Council of Canada to J.K., K.W. is grateful for a University of Saskatchewan Graduate Scholarship.     

Extracellular lipase from <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> JCM5962(T): Isolation,identification, and characterization

The study was done to isolate, identify, and characterize a good lipolytic strain from soil. Lipolytic strain isolation was done using tributyrin agar medium. The biochemical testing and 16S rRNA gene sequencing analysis was done for identification. The enzyme was purified using ammonium sulfate precipitation and column chromatography. Results have shown a novel high lipolytic strain of P. aeruginosa JCM5962(T), isolated from soil of sugarcane field. The 16S rRNA sequence analysis confirmed the strain as P. aeruginosa JCM5962(T); further, the sequence was submitted to Genbank (KX946966.1). The isolate produced an extracellular lipase which was purified as single band of 31 kDa. Maximum lipase activity was observed at 50 °C and pH 8.0. Activity was enhanced in the presence of cobalt and benzene solvent, whereas mercury, sodium dodecyl sulfate, and chloroform inhibited it. The enzyme’s marked stability and activity at high temperature, alkaline pH and organic solvents suggest that this can be effectively used in a variety of applications in industries and as biotechnological tools.     

Cloning of Intron-Removed Enolase Gene and Expression,Purification, Kinetic Characterization of the Enzyme from Theileria annulata

Tropical theileriosis is a disease caused by infection with an apicomplexan parasite, Theileria annulata, and giving rise to huge economic losses. In recent years, parasite resistance has been reported against the most effective antitheilerial drug used for the treatment of this disease. This emphasizes the need for alternative methods of treatment. Enolase is a key glycolytic enzyme and can be selected as a macromolecular target of therapy of tropical theileriosis. In this study, an intron sequence present in T. annulata enolase gene was removed by PCR-directed mutagenesis, and the gene was first cloned into pGEM-T Easy vector and then subcloned into pLATE31 vector, and expressed in Escherichia coli cells. The enzyme was purified by affinity chromatography using Ni–NTA agarose column. Steady-state kinetic parameters of the enzyme were determined using GraFit 3.0. High quantities (~65 mg/l of culture) of pure recombinant T. annulata enolase have been obtained in a higly purified form (>95 %). Homodimer form of purified protein was determined from the molecular weights obtained from a single band on SDS-PAGE (48 kDa) and from size exclusion chromatography (93 kDa). Enzyme kinetic measurements using 2-PGA as substrate gave a specific activity of ~40 U/mg, K _m: 106 μM, k_cat: 37 s^?1, and k _cat/K _m: 3.5 × 10⁵ M^?1 s^?1. These values have been determined for the first time from this parasite enzyme, and availability of large quantities of enolase enzyme will facilitate further kinetic and structural characterization toward design of new antitheilerial drugs.     

Purification and kinetic characterization of polygalacturonase - 3 from palmyrah palm (Borassus flabellifer L)

Polygalacturonase-3 was isolated and purified to homogeneity from palmyrah palm (Borassus flabellifer L.) fruit using Con A-Sepharose affinity column. The purified enzyme migrated as a single band on native and SDS–polyacrylamide gel electrophoresis. The molecular mass of the purified enzyme was estimated to be 66 kDa by size elution chromatography. Optimum polygalacturonase activity as a function of pH and temperature was determined using polygalacturonic acid as substrate. Optimum pH and temperature values ranged between the pH?4.0–5.0 and temperature 30–40 °C. At the optimum pH and temperature, the K_m and V_max values were determined by Lineweaver–Burk method. The value K_m (0.33 mM) reveals that polygalacturonase has significant reactivity towards polygalacturonic acid. The enzyme showed varied responses towards divalent and monovalent metal ions. Ca²⁺ activated the polygalacturonase-3 enzyme protein. Both teepol and cetyltrimethylammonium bromide inhibited polygalacturonase-3 activity by 44 %, while 2-mercaptoethanol stimulated the enzyme marginally.     

Herbicide Resistance in Datura innoxia: Kinetic Characterization of Acetolactate Synthase from Wild-Type and Sulfonylurea-Resistant Cell Variants

Acetolactate synthase (ALS, EC 4. 1.3. 18), the first enzyme in the biosynthesis of branched-chain amino acids, was isolated from wild-type and sulfonylurea-resistant Datura innoxia cell variants and characterized. Apparent K_m values of the ALS for pyruvate from three sulfonylurea-resistant variants (CSR2, CSR6, and CSR10) were manyfold greater than that of the wild type. The inhibition of wild-type and herbicide-resistant ALS activity by chlorsulfuron (CS), a sulfonylurea herbicide, and l-leucine (l-Leu), one of the feedback inhibitors of the enzyme, was examined. ALS from two CS-resistant variants exhibited severalfold greater resistance to CS than did the wild-type enzyme. Inhibition of ALS by l-Leu fitted a partially competitive pattern most closely. It is proposed that the herbicide resistance mutation accentuated the partial inhibition characteristics of ALS by l-Leu. ALS from one of the two CS-resistant variants (CSR6) had a K_i for l-Leu an order of magnitude greater than that of the wild-type enzyme. The alterations in kinetic properties observed in the ALS from sulfonylurea-resistant variants are discussed in relation to the possible evolutionary significance of the herbicide binding site of this enzyme, the physiological effects of such biochemical alterations, and their practical utility in genetic studies.     

Expression and Functional Characterization of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> Recombinant <Emphasis Type="SmallCaps">l</Emphasis>.Asparaginase

Recombinant _l.asparaginase, L.ASNase, from Pseudomonas aeruginosa was purified using nickel affinity chromatography. The affinity purified L.ASNase exhibited a protein band with a molecular weight of 72.4 kDa on a native polyacrylamide gel and 36.276 kDa using SDS–PAGE. The activity of the purified L.ASNase was enhanced by Mg²⁺ and inhibited by Zn²⁺ at a concentration of 5 mM. The specificity of the recombinant L.ASNase towards different substrates was examined, and it was found that the enzyme showed the highest activity towards l.asparagine. Moreover, the enzyme showed lower activity towards other substrates such as _L.glutamine, urea and acrylamide. The in vitro hemolysis assay revealed that the purified L.ASNase did not show hemolysis effect on blood erythrocytes. Serum and trypsin half-life of L.ASNase suggested that the recombinant L.ASNase retained 50% of its initial activity after 90 and 60 min incubation period in serum and trypsin separately.     

Cloning and sequence analysis of the heat-stable acrylamidase from a newly isolated thermophilic bacterium, Geobacillus thermoglucosidasius AUT-01

A thermophilic bacterium capable of degrading acrylamide, AUT-01, was isolated from soil collected from a hot spring area in Montana, USA. The thermophilic strain grew with 0.2 % glucose as the sole carbon source and 1.4 mM acrylamide as the sole nitrogen source. The isolate AUT-01 was identified as Geobacillus thermoglucosidasius based on 16S rDNA sequence. An enzyme from the strain capable of transforming acrylamide to acrylic acid was purified by a series of chromatographic columns. The molecular weight of the enzyme was estimated to be 38 kDa by SDS-PAGE. The enzyme activity had pH and temperature optima of 6.2 and 70 ºC, respectively. The influence of different metals and amino acids on the ability of the purified protein to transform acrylamide to acrylic acid was evaluated. The gene from G. thermoglucosidasius encoding the acrylamidase was cloned, sequenced, and compared to aliphatic amidases from other bacterial strains. The G. thermoglucosidasius gene, amiE, encoded a 38 kDa, monomeric, heat-stable amidase that catalysed the cleavage of carbon–nitrogen bonds in acrylamide. Comparison of the amino acid sequence to other bacterial amidases revealed 99 and 82 % similarity to the amino acid sequences of Bacillus stearothermophilus and Pseudomonas aeruginosa, respectively.     

Physico-kinetic and functional features of a novel β-glucosidase isolated from milk thistle (Silybum marianum Gaertn.) flower petals

A highly abundant β-glucosidase from petals of Silybum marianum has been purified and characterized for its physico-kinetic properties. The 135 kDa enzyme was a homodimer with subunit molecular mass of 67.6 kDa. The characteristic catalytic properties of the enzyme included acidic pH optimum (5.5), meso-thermostability, and β-linked substrate specificity with preference for gluco-conjugate but a marked (>50 %) activity with D-fuco-conjugates and considerable (~16 %) activity towards D-galacto-conjugates. The enzyme showed high affinity for p-nitrophenyl glucoside (pNPG) with K_m and V_max values of 0.25 mM and 5.35 μkat.mg^?1 enzyme protein. Thus, the enzyme had a very high (292,000 M^?1.s^?1) catalytic efficiency (K_cat/K_m). Thermal catalytic optimum of enzyme was 40 °C with activation energy value 8.26 kCal.Mol^?1. The enzyme showed significant insensitivity to D-gluconic acid lactone inhibition (57 % at 5 mM) with an apparent K_i 3.8 mM. The transglucosylating ability of enzyme was noticed for glucosylation of geraniol and withaferin-A with pNPG as glucosyl donor but cellobiose did not serve as the glycosyl donor. Partial proteomics of the enzyme revealed two peptide fragment sequences, VTPSNEVH and KRSEESNF. These motifs showed significant matching/sequence conservation with some other glycohydrolases. The novelties of purified enzyme hold potential to expand a library of catalytically characteristic members of the hydrolase family from plants for use in biotransformation applications.     

Promising properties of a formate dehydrogenase from a methanol-assimilating yeast Ogataea parapolymorpha DL-1 in His-tagged form

The cDNA gene coding for formate dehydrogenase (FDH) from Ogataea parapolymorpha DL-1 was cloned and expressed in Escherichia coli. The recombinant enzyme was purified by nickel affinity chromatography and was characterized as a homodimer composed of two identical subunits with approximately 40 kDa in each monomer. The enzyme showed wide pH optimum of catalytic activity from pH 6.0 to 7.0. It had relatively high optimum temperature at 65 °C and retained 93, 88, 83, and 71 % of its initial activity after 4 h of exposure at 40, 50, 55, and 60 °C, respectively, suggesting that this enzyme had promising thermal stability. In addition, the enzyme was characterized to have significant tolerance ability to organic solvents such as dimethyl sulfoxide, n-butanol, and n-hexane. The Michaelis–Menten constant (K _m), turnover number (k _cat), and catalytic efficiency (k _cat/K _m) values of the enzyme for the substrate sodium formate were estimated to be 0.82 mM, 2.32 s^?1, and 2.83 mM^?1 s^?1, respectively. The K _m for NAD⁺ was 83 μM. Due to its wide pH optimum, promising thermostability, and high organic solvent tolerance, O. parapolymorpha FDH may be a good NADH regeneration catalyst candidate.     

A novel nitrilase from Rhodobacter sphaeroides LHS-305: cloning, heterologous expression and biochemical characterization

In this study, a novel nitrilase gene from Rhodobacter sphaeroides was cloned and overexpressed in Escherichia coli. The open reading frame of the nitrilase gene includes 969 base pairs, which encodes a putative polypeptide of 322 amino acid residues. The molecular weight of the purified native nitrilase was about 560 kDa determined by size exclusion chromatography. This nitrilase showed one single band on SDS-PAGE with a molecular weight of 40 kDa. This suggested that the native nitrilase consisted of 14 subunits with identical size. The optimal pH and temperature of the purified enzyme were 7.0 and 40 °C, respectively. The kinetic parameters V _max and K _m toward 3-cyanopyridine were 77.5 μmol min^?1 mg^?1 and 73.1 mmol/l, respectively. The enzyme can easily convert aliphatic nitrile and aromatic nitriles to their corresponding acids. Furthermore, this enzyme demonstrated regioselectivity in hydrolysis of aliphatic dinitriles. This specific characteristic makes this nitrilase have a great potential for commercial production of various cyanocarboxylic acids by hydrolyzing readily available dinitriles.     

Directed evolution of GH43 β-xylosidase XylBH43 thermal stability and L186 saturation mutagenesis

Directed evolution of β-xylosidase XylBH43 using a single round of gene shuffling identified three mutations, R45K, M69P, and L186Y, that affect thermal stability parameter K _t ^0.5 by ?1.8 ± 0.1, 1.7 ± 0.3, and 3.2 ± 0.4 °C, respectively. In addition, a cluster of four mutations near hairpin loop-D83 improved K _t ^0.5 by ~3 °C; none of the individual amino acid changes measurably affect K _t ^0.5 . Saturation mutagenesis of L186 identified the variant L186K as having the most improved K _t ^0.5 value, by 8.1 ± 0.3 °C. The L186Y mutation was found to be additive, resulting in K _t ^0.5 increasing by up to 8.8 ± 0.3 °C when several beneficial mutations were combined. While k _cat of xylobiose and 4-nitrophenyl-β-d-xylopyranoside were found to be depressed from 8 to 83 % in the thermally improved mutants, K _m, K _ss (substrate inhibition), and K _i (product inhibition) values generally increased, resulting in lessened substrate and xylose inhibition.     

Molecular Basis of Imidazolinone Herbicide Resistance in Arabidopsis thaliana var Columbia

Acetolactate synthase (ALS), the first enzyme in the biosynthetic pathway of leucine, isoleucine, and valine, is inhibited by imidazolinone herbicides. To understand the molecular basis of imidazolinone resistance, we isolated the ALS gene from an imazapyr-resistant mutant GH90 of Arabidopsis thaliana. DNA sequence analysis of the mutant ALS gene demonstrated a single-point mutation from G to A at nucleotide 1958 of the ALS-coding sequence. This would result in Ser to Asn substitution at residue 653 near the carboxyl terminal of the matured ALS. The mutant ALS gene was introduced into tobacco using Agrobacterium-mediated transformation. Imidazolinone-resistant growth of transformed calli and leaves of transgenic plants was 100-fold greater than that of nontransformed control plants. The relative levels of imidazolinone-resistant ALS activity correlated with the amount of herbicide-resistant growth in the leaves of transgenic plants. Southern hybridization analysis confirmed the existence of transferred ALS gene in the transformant showing high imazapyr resistance. The results demonstrate that the mutant ALS gene confers resistance to imidazolinone herbicides. This is the first report, to our knowledge, of the molecular basis of imidazolinone resistance in plants.     

Isolation and mutation of recombinant EPSP synthase from pathogenic bacteria Pseudomonas aeruginosa

The Pseudomonas aeruginosa aroA gene encodes an enzyme called 5-enol-pyruvylshikimate-3-phosphate (EPSP) synthase, which has been shown as the primary target of the herbicide glyphosate. We have cloned this gene and constructed a system for the high level expression of a recombinant form of this enzyme by amplifying the aroA gene from the P. aeruginosa genomic DNA and subcloning into a vector suitable for expression in Escherichia coli. The resulting plasmid, pTrcPA, produced the EPSP synthase in large quantities which has been purified to homogeneity. Furthermore, the site-directed mutants of P. aeruginosa ESPS synthase have been constructed in order to compare in vitro glyphosate sensitivity between the wild-type and the mutant enzymes. The k_cat and K_m values for substrates in both forward and reverse reactions were obtained from both wild-type and mutant EPSP synthases.     

Characterization of mutation-induced changes in the maize (Zea mays L.) ADH1-1S1108 alcohol dehydrogenase

The homodimeric alcohol dehydrogenase gene product of maize (Zea mays L.)Adh1-1S1108 mutation was purified and compared with the parentalAdh1-1S enzyme. The mutant alcohol dehydrogenase activity had pH optima and substrate specificity similar to those of the parental enzyme, but exhibited somewhat increased and decreasedK _mvalues for acetaldehyde and NADH, respectively. The mutant enzyme was also markedly less stable than the enzyme from parental tissues to temperatures as low as 50°C. Sequence analysis of a polymerase chain reaction (PCR)-generated cDNA clone revealed a G-to-C mutation at position 406 and a C-to-T mutation at position 974. These would result in residue 103 of each protein subunit being changed from an alanine to a proline and residue 292 being changed from an alanine to a valine. Whether one or both of these changes in primary sequence is responsible for the altered substrate affinities and stability is not yet understood.     

Cloning,purification and characterization of novel Cu-containing nitrite reductase from the <Emphasis Type="Italic">Bacillus firmus</Emphasis> GY-49

Nitrite is generated from the nitrogen cycle and its accumulation is harmful to environment and it can be reduced to nitric oxid by nitrite reductase. A novel gene from Bacillus firmus GY-49 is identified as a nirK gene encoding Cu-containing nitrite reductase by genome sequence. The full-length protein included a putative signal peptide of 26 amino acids and shown 72.73% similarity with other Cu-containing nitrite reductase whose function was verified. The 993-bp fragment encoding the mature peptide of NirK was cloned into pET-28a (+) vector and overexpressed as an active protein of 36.41 kDa in the E.coli system. The purified enzyme was green in the oxidized state and displayed double gentle peaks at 456 and 608 nm. The specific activity of purified enzyme was 98.4 U/mg toward sodium nitrite around pH 6.5 and 35 °C. The K _m and K _cat of NirK on sodium nitrite were 0.27 mM and 0.36?×?10³ s^?1, respectively. Finally, homology model analysis of NirK indicated that the enzyme was a homotrimer structure and well conserved in Cu-binding sites for enzymatic functions. This is a first report for nitrite reductase from Bacillus firmus, which augment the acquaintance of nitrite reductase.     

Cloning of a Family 11 Xylanase Gene from Bacillus amyloliquefaciens CH51 Isolated from Cheonggukjang

Bacillus amyloliquefaciens CH51, an isolate from cheonggukjang, Korean fermented soyfood, secretes several enzymes into culture medium. A gene encoding 19 kDa xylanase was cloned by PCR. Sequencing showed that the gene encoded a glycohydrolase family 11 xylanase and named xynA. xynAHis, xynA with additional codons for his-tag, was overexpressed in Escherichia coli BL21(DE3) using pET-26b(+). XynAHis was purified using HisTrap affinity column. K_m and V_max of XynAHis were 0.363 mg/ml and 701.1 μmol/min/mg, respectively with birchwood xylan as a substrate. The optimum pH and temperature were pH 4 and 25 °C, respectively. When xynA was introduced into Bacillus subtilis WB600, active XynA was secreted into culture medium.     

Purification and characterization of a chitinase from Serratia proteamaculans

A chitinase gene from Serratia proteamaculans 18A1 was cloned, sequenced, and expressed in Escherichia coli M15. Recombinant enzyme (ChiA) was purified by Ni-NTA affinity column chromatography. The ChiA gene contains an open reading frame (ORF), encoding an endochitinase with a deduced molecular weight 60 kDa and predicted isoelectric point of 6.35. Comparison of ChiA with other chitinases revealed a modular structure containing an N-terminal PKD-domain, a family 18 catalytic domain and a C-terminal putative chitin-binding domain. Turn over rate (K _cat) of the enzyme was determined using colloidal chitin (49.71 ± 1.15 S^?1) and crystalline β-chitin (17.20 ± 0.83 S^?1) as substrates. The purified enzyme was active over a broad range of pH (pH 4.5–9.0) and temperature (4–70°C) with a peak activity at pH 5.5 and 55°C. However, enzyme activity was found to be stable up to 45°C for longer incubation periods. Purified enzyme was shown to inhibit fungal spore germination and hyphal growth of pathogenic fungi Fusarium oxysporum and Aspergillus niger.     

Mechanism of Sulfonylurea Herbicide Resistance in the Broadleaf Weed, Kochia scoparia

Selection of kochia (Kochia scoparia) biotypes resistant to the sulfonylurea herbicide chlorsulfuron has occurred through the continued use of this herbicide in monoculture cereal-growing areas in the United States. The apparent sulfonylurea resistance observed in kochia was confirmed in greenhouse tests. Fresh and dry weight accumulation in the resistant kochia was 2- to >350-fold higher in the presence of four sulfonylurea herbicides as compared to the susceptible biotype. Acetolactate synthase (ALS) activity isolated from sulfonylurea-resistant kochia was less sensitive to inhibition by three classes of ALS-inhibiting herbicides, sulfonylureas, imidazolinones, and sulfonanilides. The decrease in ALS sensitivity to inhibition (as measured by the ratio of resistant I₅₀ to susceptible I₅₀) was 5- to 28-fold, 2- to 6-fold, and 20-fold for sulfonylurea herbicides, imidazolinone herbicides, and a sulfonanilide herbicide, respectively. No differences were observed in the ALS-specific activities or the rates of [¹⁴C]chlorsulfuron uptake, translocation, and metabolism between susceptible and resistant kochia biotypes. The K_m values for pyruvate using ALS from susceptible and resistant kochia were 2.13 and 1.74 mm, respectively. Based on these results, the mechanism of sulfonylurea resistance in this kochia biotype is due solely to a less sulfonylurea-sensitive ALS enzyme.