Stabilization of Hydroxynitrile Lyases from Two Variants of Passion Fruit, Passiflora edulis Sims and Passiflora edulis Forma flavicarpa, by C-Terminal Truncation.

Because the synthesis of chiral compounds generally requires a broad range of substrate specificity and stable enzymes, screening for better enzymes and/or improvement of enzyme properties through molecular approaches is necessary for sustainable industrial development. Herein, the discovery of unique hydroxynitrile lyases (HNLs) from two species of passion fruits, Passiflora edulis forma flavicarpa (yellow passion fruit, PeHNL-Ny) and Passiflora edulis Sims (purple passion fruit, PeHNL-Np), isolated and purified from passion fruit leaves is reported. These are the smallest HNLs (comprising 121 amino acids). Amino acid sequences of both enzymes are 99 % identical; there is a difference of one amino acid in a consensus sequence. PeHNL-Np has an Ala residue at position 107 and is nonglycosylated at Asn105. Because it was confirmed that natural and glycosylated PeHNL-Ny showed superior thermostability, pH stability, and organic tolerance to that of PeHNL-Np, it has been speculated that protein engineering around the only glycosylation site, Asn105, located at the C-terminal region of PeHNL-Ny, might contribute to the stabilization of PeHNL. Therefore, the focus is on improved stability of the nonglycosylated PeHNL by truncating its C-terminal region. The C-terminal-truncated PeHNLΔ107 was obtained by truncating 15 amino acids from the C terminus followed by expression in Escherichia coli. PeHNLΔ107 expressed in E. coli was not glycosylated, and showed improved thermostability, solvent stability, and reusability similar to that of the wild-type glycosylated form of PeHNL expressed in Pichia pastoris. These data reveal that the lack of the high-flexibility region at the C terminus of PeHNL might be a possible reason for improving the stability of PeHNL.

Purification and characterization of a hydroxynitrile lyase from Amygdalus pedunculata Pall.

Hydroxynitrile lyases (HNLs) are widely used in the asymmetric synthesis of cyanohydrins which are organic compounds used in the production of fine chemicals and pharmaceuticals, because these enzymes exhibit high catalytic efficiency and are very economical. In the present study, seeds of A. pedunculata Pall were identified as new potential source of HNLs. The HNL from A. pedunculata Pall (APHNL) was purified 138 fold and 4.20% yield with a specific activity of 661 U/mg. SDS-PAGE result showed the enzyme to be present as a monomer and the relative molecular mass determined by MALDI-TOF MS was 61 kDa. APHNL owned highest activity at pH 6.0 and at 60 °C temperature, showing activity up to 80 °C and stable up to 60 °C. APHNL has a Km of 0.5 mM, Vmax of 665.9 µmol mg-1 min-1, Kcat of 676.5 s-1 and Kcat/Km of 1353 s-1 mM-1 using mandelonitrile as substrate. Syntheses of (R)-mandelonitrile and (R)-2-Hydroxy-2-(3-phenoxy-phenyl)-acetonitrile were carried out using APHNL and molar conversion of (R)-mandelonitrile and (R)-2-Hydroxy-2-(3-phenoxy-phenyl)-acetonitrile were 90% and 98% with 94% and 93% ee, respectively. These results indicated that APHNL was an excellent biocatalyst and has very high potential for synthesis of enantiopure cyanohydrins.

Structural and functional insights into the reaction specificity of catalase-related hydroperoxide lyase: A shift from lyase activity to allene oxide synthase by site-directed mutagenesis.

Two highly identical fusion proteins, an allene oxide synthase-lipoxygenase (AOS-LOX) and a hydroperoxide lyase-lipoxygenase (HPL-LOX), were identified in the soft coral Capnella imbricata. Both enzymes initially catalyze the formation of 8R-hydroperoxy-eicosatetraenoic acid (8R-HpETE) from arachidonic acid by the C-terminal lipoxygenase (LOX) domain. Despite the fact that the defined catalytically important residues of N-terminal catalase-related allene oxide synthase (cAOS) domain are also conserved in C. imbricata hydroperoxide lyase (cHPL), their reaction specificities differ. In the present study, we tested which of the amino acid substitutions around the active site of cHPL are responsible for a control in the reaction specificity. The possible candidates were determined via comparative sequence and structural analysis of the substrate channel and the heme region of coral cAOSs and C. imbricata cHPL. The amino acid replacements in cHPL-R56G, ME59-60LK, P65A, F150L, YS176-177NL, I357V, and SSSAGE155-160PVKEGD-with the corresponding residues of cAOS were conducted by site-directed mutagenesis. Although all these mutations influenced the catalytic efficiency of cHPL, only F150L and YS176-177NL substitutions caused a shift in the reaction specificity from HPL to AOS. The docking analysis of P. homomalla cAOS with 8R-HpETE substrate revealed that the Leu150 of cAOS interacts with the C5-C6 double bond and the Leu177 with the hydrophobic tail of 8R-HpETE. We propose that the corresponding residues in cHPL, Phe150 and Ser177, are involved in a proper coordination of the epoxy allylic radical intermediate necessary for aldehyde formation in the hydroperoxide lyase reaction.

Recombinant overexpression of dihydroneopterin aldolase catalyst potentially regulates folate-biofortification.

We aim to investigate the prospects of increased production of folate through the overexpression of heterologous dihydroneopterin aldolase catalyst. The gene encoding aldolase catalyst was cloned into an expression vector and the induced recombinant protein was purified through metal-affinity chromatography which appeared at 14 kDa position on polyacrylamide-gel. Remarkably, a periodic increase in the extracellular and intracellular folic acid concentration was observed at 4 h growth of induced recombinant DHNA samples than control in a pH-dependent manner. Maximum folate concentration was observed with at least twofold increase in induced recombinant samples at pH8.0 compared to the significant decline at 6 h growth. Consistently, heterologous overexpression of bacterial aldolase through Agrobacterium-mediated genetic transformation of tobacco led to more than 2.5-fold increase in the folate concentration in the transgenic leaves than control tissues. These data are veritable inspecting metabolic flux in both bacterial and plant systems, thus providing directions for future research on folate agri-fortification.

Characterization of Two Recombinant 3-Hexulose-6-Phosphate Synthases from the Halotolerant Obligate Methanotroph Methylomicrobium alcaliphilum 20Z.

Two key enzymes of the ribulose monophosphate (RuMP) cycle for formaldehyde fixation, 3-hexulose-6-phosphate synthase (HPS) and 6-phospho-3-hexulose isomerase (PHI), in the aerobic halotolerant methanotroph Methylomicrobium alcaliphilum 20Z are encoded by the genes hps and phi and the fused gene hps-phi. The recombinant enzymes HPS-His6, PHI-His6, and the two-domain protein HPS-PHI were obtained by heterologous expression in Escherichia coli and purified by affinity chromatography. PHI-His6, HPS-His6 (2 × 20 kDa), and the fused protein HPS-PHI (2 × 40 kDa) catalyzed formation of fructose 6-phosphate from formaldehyde and ribulose-5-phosphate with activities of 172 and 22 U/mg, respectively. As judged from the kcat/Km ratio, HPS-His6 had higher catalytic efficiency but lower affinity to formaldehyde compared to HPS-PHI. AMP and ADP were powerful inhibitors of both HPS and HPS-PHI activities. The two-domain HPS-PHI did not show isomerase activity, but the sequences corresponding to its HPS and PHI regions, when expressed separately, were found to produce active enzymes. Inactivation of the hps-phi fused gene did not affect the growth rate of the mutant strain. Analysis of annotated genomes revealed the separately located genes hps and phi in all the RuMP pathway methylotrophs, whereas the hps-phi fused gene occurred only in several methanotrophs and was absent in methylotrophs not growing under methane. The significance of these tandems in adaptation and biotechnological potential of methylotrophs is discussed.

First characterization of extremely halophilic 2-deoxy-D-ribose-5-phosphate aldolase.

2-Deoxy-d-ribose-5-phosphate aldolase (DERA) catalyzes the aldol reaction between two aldehydes and is thought to be a potential biocatalyst for the production of a variety of stereo-specific materials. A gene encoding DERA from the extreme halophilic archaeon, Haloarcula japonica, was overexpressed in Escherichia coli. The gene product was successfully purified, using procedures based on the protein's halophilicity, and characterized. The expressed enzyme was stable in a buffer containing 2 M NaCl and exhibited high thermostability, retaining more than 90% of its activity after heating at 70 °C for 10 min. The enzyme was also tolerant to high concentrations of organic solvents, such as acetonitrile and dimethylsulfoxide. Moreover, H. japonica DERA was highly resistant to a high concentration of acetaldehyde and retained about 35% of its initial activity after 5-h' exposure to 300 mM acetaldehyde at 25 °C, the conditions under which E. coli DERA is completely inactivated. The enzyme exhibited much higher activity at 25 °C than the previously characterized hyperthermophilic DERAs (Sakuraba et al., 2007). Our results suggest that the extremely halophilic DERA has high potential to serve as a biocatalyst in organic syntheses. This is the first description of the biochemical characterization of a halophilic DERA.

Discovery and molecular and biocatalytic properties of hydroxynitrile lyase from an invasive millipede, Chamberlinius hualienensis.

Hydroxynitrile lyase (HNL) catalyzes the degradation of cyanohydrins and causes the release of hydrogen cyanide (cyanogenesis). HNL can enantioselectively produce cyanohydrins, which are valuable building blocks for the synthesis of fine chemicals and pharmaceuticals, and is used as an important biocatalyst in industrial biotechnology. Currently, HNLs are isolated from plants and bacteria. Because industrial biotechnology requires more efficient and stable enzymes for sustainable development, we must continuously explore other potential enzyme sources for the desired HNLs. Despite the abundance of cyanogenic millipedes in the world, there has been no precise study of the HNLs from these arthropods. Here we report the isolation of HNL from the cyanide-emitting invasive millipede Chamberlinius hualienensis, along with its molecular properties and application in biocatalysis. The purified enzyme displays a very high specific activity in the synthesis of mandelonitrile. It is a glycosylated homodimer protein and shows no apparent sequence identity or homology with proteins in the known databases. It shows biocatalytic activity for the condensation of various aromatic aldehydes with potassium cyanide to produce cyanohydrins and has high stability over a wide range of temperatures and pH values. It catalyzes the synthesis of (R)-mandelonitrile from benzaldehyde with a 99% enantiomeric excess, without using any organic solvents. Arthropod fauna comprise 80% of terrestrial animals. We propose that these animals can be valuable resources for exploring not only HNLs but also diverse, efficient, and stable biocatalysts in industrial biotechnology.

The crystal structure of D-threonine aldolase from Alcaligenes xylosoxidans provides insight into a metal ion assisted PLP-dependent mechanism.

Threonine aldolases catalyze the pyridoxal phosphate (PLP) dependent cleavage of threonine into glycine and acetaldehyde and play a major role in the degradation of this amino acid. In nature, L- as well as D-specific enzymes have been identified, but the exact physiological function of D-threonine aldolases (DTAs) is still largely unknown. Both types of enantio-complementary enzymes have a considerable potential in biocatalysis for the stereospecific synthesis of various ß-hydroxy amino acids, which are valuable building blocks for the production of pharmaceuticals. While several structures of L-threonine aldolases (LTAs) have already been determined, no structure of a DTA is available to date. Here, we report on the determination of the crystal structure of the DTA from Alcaligenes xylosoxidans (AxDTA) at 1.5 Å resolution. Our results underline the close relationship of DTAs and alanine racemases and allow the identification of a metal binding site close to the PLP-cofactor in the active site of the enzyme which is consistent with the previous observation that divalent cations are essential for DTA activity. Modeling of AxDTA substrate complexes provides a rationale for this metal dependence and indicates that binding of the ß-hydroxy group of the substrate to the metal ion very likely activates this group and facilitates its deprotonation by His193. An equivalent involvement of a metal ion has been implicated in the mechanism of a serine dehydratase, which harbors a metal ion binding site in the vicinity of the PLP cofactor at the same position as in DTA. The structure of AxDTA is completely different to available structures of LTAs. The enantio-complementarity of DTAs and LTAs can be explained by an approximate mirror symmetry of crucial active site residues relative to the PLP-cofactor.

Purification and simultaneous immobilization of Arabidopsis thaliana hydroxynitrile lyase using a family 2 carbohydrate-binding module.

Tedious, time- and labor-intensive protein purification and immobilization procedures still represent a major bottleneck limiting the widespread application of enzymes in synthetic chemistry and industry. We here exemplify a simple strategy for the direct site-specific immobilization of proteins from crude cell extracts by fusion of a family 2 carbohydrate-binding module (CBM) derived from the exoglucanase/xylanase Cex from Cellulomonas fimi to a target enzyme. By employing a tripartite fusion protein consisting of the CBM, a flavin-based fluorescent protein (FbFP), and the Arabidopsis thaliana hydroxynitrile lyase (AtHNL), binding to cellulosic carrier materials can easily be monitored via FbFP fluorescence. Adsorption properties (kinetics and quantities) were studied for commercially available Avicel PH-101 and regenerated amorphous cellulose (RAC) derived from Avicel. The resulting immobilizates showed similar activities as the wild-type enzyme but displayed increased stability in the weakly acidic pH range. Finally, Avicel, RAC and cellulose acetate (CA) preparations were used for the synthesis of (R)-mandelonitrile in micro-aqueous methyl tert-butyl ether (MTBE) demonstrating the applicability and stability of the immobilizates for biotransformations in both aqueous and organic reaction systems.

Crosslinked enzyme aggregates of hydroxynitrile lyase partially purified from Prunus dulcis seeds and its application for the synthesis of enantiopure cyanohydrins.

Hydroxynitrile lyases are powerful catalysts in the synthesis of enantiopure cyanohydrins which are key synthons in the preparations of a variety of important chemicals. The response surface methodology including three-factor and three-level Box-Behnken design was applied to optimize immobilization of hydroxynitrile lyase purified partially from Prunus dulcis seeds as crosslinked enzyme aggregates (PdHNL-CLEAs). The quadratic model was developed for predicting the response and its adequacy was validated with the analysis of variance test. The optimized immobilization parameters were initial glutaraldehyde concentration, ammonium sulfate saturation concentration, and crosslinking time, and the response was relative activity of PdHNL-CLEA. The optimal conditions were determined as initial glutaraldehyde concentration of 25% w/v, ammonium sulfate saturation concentration of 43% w/v, and crosslinking time of 18 h. The preparations of PdHNL-CLEA were examined for the synthesis of (R)-mandelonitrile, (R)-2-chloromandelonitrile, (R)-3,4-dihydroxymandelonitrile, (R)-2-hydroxy-4-phenyl butyronitrile, (R)-4-bromomandelonitrile, (R)-4-fluoromandelonitrile, and (R)-4-nitromandelonitrile from their corresponding aldehydes and hydrocyanic acid. After 96-h reaction time, the yield-enantiomeric excess values (%) were 100-99, 100-21, 100-99, 83-91, 100-99, 100-72, and 100-14%, respectively, for (R)-mandelonitrile, (R)-2-chloromandelonitrile, (R)-3,4-dihydroxymandelonitrile, (R)-2-hydroxy-4-phenyl butyronitrile, (R)-4-bromomandelonitrile, (R)-4-fluoromandelonitrile, and (R)-4-nitromandelonitrile. The results show that PdHNL-CLEA offers a promising potential for the preparation of enantiopure (R)-mandelonitrile, (R)-3,4-dihydroxymandelonitrile, (R)-2-hydroxy-4-phenyl butyronitrile, and (R)-4-bromomandelonitrile with a high yield and enantiopurity.

Molecular cloning, characterization and expression of a gene encoding phosphoketolase from Termitomyces clypeatus.

A phosphoketolase (pk) gene from the fungus Termitomyces clypeatus (TC) was cloned and partially characterized. Oligonucleotide primers specific for the phosphoketolase gene (pk) were designed from the regions of homologies found in the primary structure of the enzyme from other fungal sources related to TC, using multiple sequence alignment technique. The cDNA of partial lengths were amplified, cloned and sequenced in three parts by 3' and 5' RACE and RT-PCR using these oligonucleotide primers. The full length ds cDNA was constructed next by joining these three partial cDNA sequences having appropriate overlapping regions using Overlap Extension PCR technique. The constructed full length cDNA exhibited an open reading frame of 2487 bases and 5' and 3' UTRs. The deduced amino acid sequence, which is of 828 amino acids, when analyzed with NCBI BLAST, showed high similarities with the phosphoketolase enzyme (Pk) superfamily with expected domains. The part of the TC genomic DNA comprising of the pk gene was also amplified, cloned and sequenced and was found to contain two introns of 68 and 74 bases that interrupt the pk reading frame. The coding region of pk cDNA was subcloned in pKM260 expression vector in correct frame and the protein was expressed in Escherichia coli BL21 (DE3) transformed with this recombinant expression plasmid. The recombinant protein purified by His-tag affinity chromatography indicated the presence of a protein of the expected size. In vivo expression studies of the gene in presence of different carbon sources indicated synthesis of Pk specific mRNA, as expected. Phylogenetic studies revealed a common ancestry of the fungal and bacterial Pk. The TC is known to secrete several industrially important enzymes involved in carbohydrate metabolism. However, the presence of Pk, a key enzyme in pentose metabolism, has not been demonstrated conclusively in this organism. Cloning, sequencing and expression study of this gene establishes the functioning of this gene in T. clypeatus. The Pk from TC is a new source for commercial exploitation.

Isolation, expression, and characterization of a hydroperoxide lyase gene from cucumber.

A full-length cDNA coding for hydroperoxide lyase (CsHPL) was isolated from cucumber fruits of No. 26 (Southern China type) and No.14-1 (Northern China type), which differed significantly in fruit flavor. The deduced amino acid sequences of CsHPL from both lines show the same and significant similarity to known plant HPLs and contain typical conserved domains of HPLs. The recombinant CsHPL was confirmed to have 9/13-HPL enzymatic activity. Gene expression levels of CsHPL were measured in different organs, especially in fruits of different development stages of both lines. The HPL activities of fruit were identified basing on the catalytic action of crude enzyme extracts incubating with 13-HPOD (13-hydroperoxy-(9Z,12E)-octadecadienoic acid) and 13-HPOD + 9-HPOD (9-hydroperoxy-(10E,12Z)-octadecadienoic acid), and volatile reaction products were analyzed by GC-MS (gas chromatography-mass spectrometry). CsHPL gene expression in No. 26 fruit occurred earlier than that of total HPL enzyme activity and 13-HPL enzyme activity, and that in No. 14-1 fruit was consistent with total HPL enzyme activity and 9-HPL enzyme activity. 13-HPL enzyme activities decreased significantly and the 9-HPL enzyme activities increased significantly with fruit ripening in both lines, which accounted for the higher content of C6 aldehydes at 0-6 day post-anthesis (dpa) and higher content of C9 aldehydes at 9-12 dpa.

Utilization of D-ribitol by Lactobacillus casei BL23 requires a mannose-type phosphotransferase system and three catabolic enzymes.

Lactobacillus casei strains 64H and BL23, but not ATCC 334, are able to ferment D-ribitol (also called D-adonitol). However, a BL23-derived ptsI mutant lacking enzyme I of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) was not able to utilize this pentitol, suggesting that strain BL23 transports and phosphorylates D-ribitol via a PTS. We identified an 11-kb region in the genome sequence of L. casei strain BL23 (LCABL_29160 to LCABL_29270) which is absent from strain ATCC 334 and which contains the genes for a GlpR/IolR-like repressor, the four components of a mannose-type PTS, and six metabolic enzymes potentially involved in D-ribitol metabolism. Deletion of the gene encoding the EIIB component of the presumed ribitol PTS indeed prevented D-ribitol fermentation. In addition, we overexpressed the six catabolic genes, purified the encoded enzymes, and determined the activities of four of them. They encode a D-ribitol-5-phosphate (D-ribitol-5-P) 2-dehydrogenase, a D-ribulose-5-P 3-epimerase, a D-ribose-5-P isomerase, and a D-xylulose-5-P phosphoketolase. In the first catabolic step, the protein D-ribitol-5-P 2-dehydrogenase uses NAD(+) to oxidize D-ribitol-5-P formed during PTS-catalyzed transport to D-ribulose-5-P, which, in turn, is converted to D-xylulose-5-P by the enzyme D-ribulose-5-P 3-epimerase. Finally, the resulting D-xylulose-5-P is split by D-xylulose-5-P phosphoketolase in an inorganic phosphate-requiring reaction into acetylphosphate and the glycolytic intermediate D-glyceraldehyde-3-P. The three remaining enzymes, one of which was identified as D-ribose-5-P-isomerase, probably catalyze an alternative ribitol degradation pathway, which might be functional in L. casei strain 64H but not in BL23, because one of the BL23 genes carries a frameshift mutation.

Structural characterization of hydroperoxide lyase in dodecyl maltoside by using circular dichroism.

Fatty acid hydroperoxide lyase (HPL) is a membrane protein, member of the lipoxygenase pathway, which holds a central role in plant defense. Green bell pepper fatty acid hydroperoxide lyase, overexpressed in Escherichia coli, was purified and solubilized in two different non ionic detergents, Triton X-100 and dodecyl maltoside (DM). DM is considered to be more useful compared to Triton X-100, as it allows characterization of the protein with spectroscopic techniques, for which Triton X-100 was inapplicable. Circular dichroism demonstrated that HPL's secondary structure in DM consists of 13.53 % α-helix, 32.73 % ß-sheet, 21.76 % turn and 31.13 % unordered.

S-selective hydroxynitrile lyase from a plant Baliospermum montanum: molecular characterization of recombinant enzyme.

A novel S-hydroxynitrile lyase (HNL) was purified from leaves of a plant, Baliospermum montanum, by ammonium sulfate fractionation and column chromatographies. Full-length cDNA and genomic DNA were cloned and sequenced. The latter contained two introns and one ORF encoding a 263-residue protein (subunit: 29.5 kDa). The hnl gene was expressed in Escherichia coli and the enzyme was characterized including detailed kinetic studies of 20 substrates for (S)-cyanohydrin synthesis. The enzyme exhibited the highest specific activity (178 U/mg), k(cat) (98/s) and k(cat)/K(m) ratio for piperonal. k(cat)/K(m) ratio for aromatic aldehydes was much larger than those of aliphatic aldehydes and ketones. It was strongly inhibited by AgNO3, PMSF, phenol and methyl ethyl ketone, showed an optimum at pH 5, while having activity at range of 4-6.5. It exhibited stability at wide pH range 2.4-11, the highest activity at 20 °C, being active at 0-65 °C. The enzyme showed variations in residues involved in substrate pocket and substrate entrance channel compared to other S-selective HNLs, based on a model was built. C-terminal short truncations provided more enzyme production. Gel filtration revealed a 60-65 kDa molecular mass for this non-FAD enzyme and its C-terminally truncated forms using three buffer compositions, indicating dimeric structures.

Characterization of a new (R)-hydroxynitrile lyase from the Japanese apricot Prunus mume and cDNA cloning and secretory expression of one of the isozymes in Pichia pastoris.

PmHNL, a hydroxynitrile lyase from Japanese apricot ume (Prunus mume) seed was purified to homogeneity by ammonium sulfate fractionation and chromatographic steps. The purified enzyme was a monomer with molecular mass of 58 kDa. It was a flavoprotein similar to other hydroxynitrile lyases of the Rosaceae family. It was active over a broad temperature, and pH range. The N-terminal amino acid sequence (20 amino acids) was identical with that of the enzyme from almond (Prunus dulcis). Based on the N-terminal sequence of the purified enzyme and the conserved amino acid sequences of the enzymes from Pr. dulcis, inverse PCR method was used for cloning of a putative PmHNL (PmHNL2) gene from a Pr. mume seedling. Then the cDNA for the enzyme was cloned. The deduced amino acid sequence was found to be highly similar (95%) to that of an enzyme from Pr. serotina, isozyme 2. The recombinant Pichia pastoris transformed with the PmHNL2 gene secreted an active enzyme in glycosylated form.

Preliminary X-ray crystallographic analysis of the D-xylulose 5-phosphate phosphoketolase from Lactococcus lactis.

Phosphoketolases are thiamine diphosphate-dependent enzymes which play a central role in the pentose-phosphate pathway of heterofermentative lactic acid bacteria. They belong to the family of aldehyde-lyases and in the presence of phosphate ion cleave the carbon-carbon bond of the specific substrate D-xylulose 5-phosphate (or D-fructose 6-phosphate) to give acetyl phosphate and D-glyceraldehyde 3-phosphate (or D-erythrose 4-phosphate). Structural information about phosphoketolases is particularly important in order to fully understand their mechanism as well as the steric course of phosphoketolase-catalyzed reactions. Here, the purification, preliminary crystallization and crystallographic characterization of D-xylulose 5-phosphate phosphoketolase from Lactococcus lactis are reported. The presence of thiamine diphosphate during purification was essential for the enzymatic activity of the purified protein. The crystals belonged to the monoclinic space group P2(1). Diffraction data were obtained to a resolution of 2.2 A.

Expression and characterization of a novel deoxyribose 5-phosphate aldolase from Paenibacillus sp. EA001.

A novel deoC gene was identified from Paenibacillus sp. EA001 isolated from soil. The gene had an open reading frame (ORF) of 663 base pairs encoding 220 amino acids with a molecular mass of 24.5 kDa. The amino acid sequence was 79 % identical to that of deoxyribose 5-phosphate aldolase (DERA) from Geobacillus sp. Y412MC10. The deoC gene encoding DERA was cloned into expression vector and the protein was expressed in Escherichia coli. The recombinant DERA was purified by using Ni-NTA affinity chromatography and characterized. The optimum temperature and pH for DERA were 50 degrees C and 6.0, respectively. The specific activity for deoxyribose 5-phosphate (DR5P), substrate, was 62 micronmol/min/mg. The Km value for DR5P was determined to be 145 mM with the Kcat value of 3.2 times 10(2 /sec) from Lineweaver-Burk plots. The EA001 DERA showed stability toward a high concentration of acetaldehyde (100 mM).

Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of 2-keto-3-deoxy-6-phosphogluconate aldolase from Zymomonas mobilis ZM4.

Zymomonas mobilis ZM4 is an organism optimized for ethanol production which uses the Entner-Doudoroff (ED) pathway for the breakdown of glucose. The key enzyme in this process is 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase, which produces glyceraldehyde 3-phosphate and pyruvate. In order to provide a molecular background for the KDPG aldolase from this ethanologenic organism (zmKDPG aldolase), the ZMO0997 gene of Z. mobilis ZM4 coding for zmKDPG aldolase was cloned and expressed and the purified protein was crystallized from 25%(w/v) polyethylene glycol 3350 and 0.1 M bis-tris pH 5.5. Diffraction data were collected to 1.8 A resolution using synchrotron radiation. The crystal belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 63.7, b = 83.0, c = 117.2 A. A trimeric zmKDPG aldolase molecule was present in the asymmetric unit, resulting in a crystal volume per unit protein weight of 2.40 A(3) Da(-1) and a solvent content of 48%.

Stability of hydroperoxide lyase activity from Amaranthus tricolor (Amaranthus mangostanus L.) leaves: influence of selected additives.

BACKGROUND: Hydroperoxide lyase (HPL) has potential value for the flavour additive industry. Currently, the production and application of HPL suffer from stability problems. The objective of this study was to investigate the stabilisation of HPL preparation from Amaranthus tricolor leaves by the addition of selected chemical additives. RESULTS: Amaranthus tricolor leaves were identified as a particularly rich source of 13-HPL activity. The addition of 100 g L(-1) sucrose and trehalose to microsomal HPL prior to lyophilisation could retain nearly 100% enzymatic activity, compared to only 20% for the lyophilised control. The lyophilised microsomal HPL containing sucrose maintained full activity for even 40 days storage at -20 degrees C. For HPL solution, glycerol was effective for long-term stability at -20 degrees C. Moreover, poyols (sucrose and trehalose) and amino acid (glycine) enhanced the thermostability of HPL, while KCl and polyol mannitol decreased the thermostability of HPL. CONCLUSION: The flavour-producing enzyme HPL, found in the leaves of Amaranthus tricolor, was stabilised by the addition of chemical additives.