Heterologous expression and characterization of <Emphasis Type="Italic">Bacillus coagulans</Emphasis><Emphasis Type="SmallCaps">l</Emphasis>-arabinose isomerase

Bacillus coagulans has been of great commercial interest over the past decade owing to its strong ability of producing optical pure l-lactic acid from both hexose and pentose sugars including l-arabinose with high yield, titer and productivity under thermophilic conditions. The l-arabinose isomerase (L-AI) from Bacillus coagulans was heterologously over-expressed in Escherichia coli. The open reading frame of the L-AI has 1,422 nucleotides encoding a protein with 474 amino acid residues. The recombinant L-AI was purified to homogeneity by one-step His-tag affinity chromatography. The molecular mass of the enzyme was estimated to be 56 kDa by SDS-PAGE. The enzyme was most active at 70°C and pH 7.0. The metal ion Mn²⁺ was shown to be the best activator for enzymatic activity and thermostability. The enzyme showed higher activity at acidic pH than at alkaline pH. The kinetic studies showed that the K _m, V _max and k _cat/K _m for the conversion of l-arabinose were 106 mM, 84 U/mg and 34.5 mM⁻¹min⁻¹, respectively. The equilibrium ratio of l-arabinose to l-ribulose was 78:22 under optimal conditions. l-ribulose (97 g/L) was obtained from 500 g/l of l-arabinose catalyzed by the enzyme (8.3 U/mL) under the optimal conditions within 1.5 h, giving at a substrate conversion of 19.4% and a production rate of 65 g L⁻¹ h⁻¹.     

Cloning and characterization of a novel <Emphasis Type="SmallCaps">l</Emphasis>-arabinose isomerase from <Emphasis Type="Italic">Bacillus licheniformis</Emphasis>

Based on analysis of the genome sequence of Bacillus licheniformis ATCC 14580, an isomerase-encoding gene (araA) was proposed as an l-arabinose isomerase (L-AI). The identified araA gene was cloned from B. licheniformis and overexpressed in Escherichia coli. DNA sequence analysis revealed an open reading frame of 1,422 bp, capable of encoding a polypeptide of 474 amino acid residues with a calculated isoelectric point of pH 4.8 and a molecular mass of 53,500 Da. The gene was overexpressed in E. coli, and the protein was purified as an active soluble form using Ni–NTA chromatography. The molecular mass of the purified enzyme was estimated to be ~53 kDa by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and 113 kDa by gel filtration chromatography, suggesting that the enzyme is a homodimer. The enzyme required a divalent metal ion, either Mn²⁺or Co²⁺, for enzymatic activity. The enzyme had an optimal pH and temperature of 7.5 and 50°C, respectively, with a k _cat of 12,455 min⁻¹ and a k _cat/K _m of 34 min⁻¹ mM⁻¹ for l-arabinose, respectively. Although L-AIs have been characterized from several other sources, B. licheniformis L-AI is distinguished from other L-AIs by its wide pH range, high substrate specificity, and catalytic efficiency for l-arabinose, making B. licheniformis L-AI the ideal choice for industrial applications, including enzymatic synthesis of l-ribulose. This work describes one of the most catalytically efficient L-AIs characterized thus far.     

Characterization of a thermostable endo-1,5-α-<Emphasis Type="SmallCaps">l</Emphasis>-arabinanase from <Emphasis Type="Italic">Caldicellulorsiruptor saccharolyticus</Emphasis>

A recombinant putative glycoside hydrolase from Caldicellulosiruptor saccharolyticus was purified with a specific activity of 12 U mg⁻¹ by heat treatment and His-Trap affinity chromatography, and identified as a single 56 kDa band upon SDS-PAGE. The native enzyme is a dimer with a molecular mass of 112 kDa as determined by gel filtration. The enzyme exhibited its highest activity when debranched arabinan (1,5-α-l-arabinan) was used as the substrate, demonstrating that the enzyme was an endo-1,5-α-l-arabinanase. The K _m, k _cat, and k _cat/K _m values were 18 mg ml⁻¹, 50 s⁻¹, and a 2.8 mg ml⁻¹ s⁻¹, respectively. Maximum enzyme activity was at pH 6.5 and 75°C. The half-lives of the enzyme at 65, 70 and 75°C were 2440, 254 and 93 h, respectively, indicating that it is the most thermostable of the known endo-1,5-α-l-arabinanases.     

Cloning,sequencing, overexpression and characterization of <Emphasis Type="SmallCaps">l</Emphasis>-rhamnose isomerase from <Emphasis Type="Italic">Bacillus pallidus</Emphasis> Y25 for rare sugar production

The l-rhamnose isomerase gene (L -rhi) encoding for l-rhamnose isomerase (l-RhI) from Bacillus pallidus Y25, a facultative thermophilic bacterium, was cloned and overexpressed in Escherichia coli with a cooperation of the 6×His sequence at a C-terminal of the protein. The open reading frame of L -rhi consisted of 1,236 nucleotides encoding 412 amino acid residues with a calculated molecular mass of 47,636 Da, showing a good agreement with the native enzyme. Mass-produced l-RhI was achieved in a large quantity (470 mg/l broth) as a soluble protein. The recombinant enzyme was purified to homogeneity by a single step purification using a Ni-NTA affinity column chromatography. The purified recombinant l-RhI exhibited maximum activity at 65°C (pH 7.0) under assay conditions, while 90% of the initial enzyme activity could be retained after incubation at 60°C for 60 min. The apparent affinity (K _m) and catalytic efficiency (k _cat/K _m) for l-rhamnose (at 65°C) were 4.89 mM and 8.36 × 10⁵ M⁻¹ min⁻¹, respectively. The enzyme demonstrated relatively low levels of amino acid sequence similarity (42 and 12%), higher thermostability, and different substrate specificity to those of E. coli and Pseudomonas stutzeri, respectively. The enzyme has a good catalyzing activity at 50°C, for d-allose, l-mannose, d-ribulose, and l-talose from d-psicose, l-fructose, d-ribose and l-tagatose with a conversion yield of 35, 25, 16 and 10%, respectively, without a contamination of by-products. These findings indicated that the recombinant l-RhI from B. pallidus is appropriate for use as a new source of rare sugar producing enzyme on a mass scale production.     

Purification and characterization of dihydroorotase fromPseudomonas putida

Dihydroorotase was purified to homogeneity fromPseudomonas putida. The relative molecular mass of the native enzyme was 82 kDa and the enzyme consisted of two identical subunits with a relative molecular mass of 41 kDa. The enzyme only hydrolyzed dihydro-l-orotate and its methyl ester, and the reactions were reversible. The apparentK _m andV _max values for dihydro-l-orotate hydrolysis (at pH 7.4) were 0.081 mM and 18 μmol min⁻¹ mg⁻¹, respectively; and those forN-carbamoyl-dl-aspartate (at pH 6.0) were 2.2 mM and 68 μmol min⁻¹ mg⁻¹, respectively. The enzyme was inhibited by metal ion chelators and activated by Zn²⁺. However, excessive Zn²⁺ was inhibitory. The enzyme was inhibited by sulfhydryl reagents, and competitively inhibited byN-carbamoylamino acids such asN-carbamoylglycine, with aK _i value of 2.7 mM. The enzyme was also inhibited noncompetitively by pyrimidine-metabolism intermediates such as dihydrouracil and orotate, with aK _i value of 3.4 and 0.75 mM, respectively, suggesting that the enzyme activity is regulated by pyrimidine-metabolism intermediates and that dihydroorotase plays a role in the control of pyrimidine biosynthesis.     

The dipeptidyl carboxypeptidase of <Emphasis Type="Italic">Escherichia coli</Emphasis> novablue: overproduction and molecular characterization of the recombinant enzyme

To express Escherichia coli novablue dipeptidyl carboxypeptidase (EcDCP), the gene was amplified by PCR and cloned into the expression plasmid pQE-31 to yield pQE-EcDCP. His₆-tagged EcDCP (His₆-EcDCP) was over-expressed in E. coli M15 (pQE-EcDCP) as a soluble and active form under 0.05 mM IPTG induction at 26°C for 12 h. The recombinant enzyme was purified to homogeneity by Ni²⁺-NTA resin and had a molecular mass of approximately 75 kDa. The temperature and pH optima for His₆-EcDCP were 37°C and 7.0, respectively. In the presence of 200 mM NaCl, His₆-EcDCP was stimulated by 1.5 fold. The K _M and k _cat values of the enzyme for N-benzoyl-l-glycyl-l-histidyl-l-leucine were 1.83 mM and 168.3 s⁻¹, respectively. His₆-EcDCP activity was dramatically inhibited by 10 mM EDTA, 0.25 mM 1.10-phenanthroline, and 2.5 mM DEPC, but it was not affected by Ser, Asp, Lys, and Trp protease inhibitors. Analysis of His₆-EcDCP by circular dichroism revealed that the secondary structures of the enzyme in 30 mM universal buffer (pH 7.0) were 17% α-helix, 35% β-sheet and 47% random coil. Mid point of thermal transition was calculated to be 55°C for the recombinant enzyme.     

Isolation and characterization of two serine proteases from metagenomic libraries of the Gobi and Death Valley deserts

Whole-genome sequence analysis of Bacillus halodurans ATCC BAA-125 revealed an isomerase gene (rhaA) encoding an l-rhamnose isomerase (l-RhI). The identified l -RhI gene was cloned from B. halodurans and over-expressed in Escherichia coli. DNA sequence analysis revealed an open reading frame of 1,257 bp capable of encoding a polypeptide of 418 amino acid residues with a molecular mass of 48,178 Da. The molecular mass of the purified enzyme was estimated to be ∼48 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 121 kDa by gel filtration chromatography, suggesting that the enzyme is a homodimer. The enzyme had an optimal pH and temperature of 7 and 70°C, respectively, with a k _cat of 8,971 min⁻¹ and a k _cat/K _m of 17 min⁻¹ mM⁻¹ for l-rhamnose. Although l-RhIs have been characterized from several other sources, B. halodurans l-RhI is distinguished from other l-RhIs by its high temperature optimum (70°C) with high thermal stability of showing 100% activity for 10 h at 60°C. The half-life of the enzyme was more than 900 min and ∼25 min at 60°C and 70°C, respectively, making B. halodurans l-RhI a good choice for industrial applications. This work describes one of the most thermostable l-RhI characterized thus far.     

Substrate specificity of a recombinant ribose-5-phosphate isomerase from <Emphasis Type="Italic">Streptococcus pneumoniae</Emphasis> and its application in the production of <Emphasis Type="SmallCaps">l</Emphasis>-lyxose and <Emphasis Type="SmallCaps">l</Emphasis>-tagatose

A putative ribose-5-phosphate isomerase (RpiB) from Streptococcus pneumoniae was purified with a specific activity of 26.7 U mg⁻¹ by Hi-Trap Q HP anion exchange and Sephacryl S-300 HR 16/60 gel filtration chromatographies. The native enzyme existed as a 96-kDa tetramer with activity maxima at pH 7.5 and 35°C. The RpiB exhibited isomerization activity with l-lyxose, l-talose, d-gulose, d-ribose, l-mannose, d-allose, l-xylulose, l-tagatose, d-sorbose, d-ribulose, l-fructose, and d-psicose and exhibited particularly high activity with l-form monosaccharides such as l-lyxose, l-xylulose, l-talose, and l-tagatose. With l-xylulose (500 g l⁻¹) and l-talose (500 g l⁻¹) substrates, the optimum concentrations of RpiB were 300 and 600 U ml⁻¹, respectively. The enzyme converted 500 g l⁻¹ l-xylulose to 350 g l⁻¹ l-lyxose after 3 h, and yielded 450 g l⁻¹ l-tagatose from 500 g l⁻¹ l-talose after 5 h. These results suggest that RpiB from S. pneumoniae can be employed as a potential producer of l-form monosaccharides.     

Isolation of <Emphasis Type="Italic">Aspergillus oryzae</Emphasis> mutants for heterologous protein production from a double proteinase gene disruptant

The recombinant Pichia pastoris harboring an improved methionine adenosyltransferase (MAT) shuffled gene was employed to biosynthesize S-adenosyl-l-methionine (SAM). Two l-methionine (l-Met) addition strategies were used to supply the precursor: the batch addition strategy (l-Met was added separately at three time points) and the continuous feeding strategies (l-Met was fed continuously at the rate of 0.1, 0.2, and 0.5 g l⁻¹ h⁻¹, respectively). SAM accumulation, l-Met conversion rate, and SAM productivity with the continuous feeding strategies were all improved over the batch addition strategy, which reached 8.46 ± 0.31 g l⁻¹, 41.7 ± 1.4%, and 0.18 ± 0.01 g l⁻¹ h⁻¹ with the best continuous feeding strategy (0.2 g l⁻¹ h⁻¹), respectively. The bottleneck for SAM production with the low l-Met feeding rate (0.1 g L⁻¹ h⁻¹) was the insufficient l-Met supply. The analysis of the key enzyme activities indicated that the tricarboxylic acid cycle and glycolytic pathway were reduced with the increasing l-Met feeding rate, which decreased the adenosine triphosphate (ATP) synthesis. The MAT activity also decreased as the l-Met feeding rate rose. The reduced ATP synthesis and MAT activity were probably the reason for the low SAM accumulation when the l-Met feeding rate reached 0.5 g l⁻¹ h⁻¹.     

Molecular characterization of phenylalanine ammonia lyase gene from <Emphasis Type="Italic">Cistanche deserticola</Emphasis>

We cloned the gene, CdPAL1, from Cistanche deserticola callus using RACE PCR with degenerate primers that were designed based on a multiple sequence alignment of known PAL genes from other plant species. The gene shows high homology to other known PAL genes registered in GenBank. The recombinant protein exhibited Michaelis–Menten kinetics with a K _m of 0.1013 mM, V _max of 4.858 μmol min⁻¹, K _cat of 3.36 S⁻¹, and K _cat/K _m is 33,168 M⁻¹ S⁻¹. The enzyme had an optimal pH of 8.5 and an activation energy of 38.92 kJ mol⁻¹ when l-Phenylalanine was used as a substrate; l-tyrosine cannot be used as substrate for this protein. The optimal temperature was 55°C, and the thermal stability results showed that, after a treatment at 70°C for 20 min, the protein retained 87% activity, while a treatment at 75°C for 20 min resulted in a loss of over 85% of the enzyme activity. Treatment with heavy metal ions (Hg²⁺, Pb²⁺, and Zn²⁺) showed remarkable inhibitory effects. Among the intermediates from the lignin (cinnamyl alcohol, cinnamyl aldehyde, coniferyl aldehyde, coniferyl alcohol), phenylpropanoid (cinnamic acid, coumaric acid, caffeic acid, and chlorogenic acid) and phenylethanoid (tyrosol and salidroside) biosynthetic pathways, only cinnamic acid showed strong inhibitory effects against CdPAL1 activity with a K _i of 8 μM. Competitive inhibitor AIP exhibited potent inhibition with K _i = 0.056 μM.     

Characterisation of <Emphasis Type="SmallCaps">l</Emphasis>-alanine and glycine absorption across the gut of an ancient vertebrate

This study utilised an in vitro technique to characterise absorption of two amino acids across the intestinal epithelium of Pacific hagfish, Eptatretus stoutii. Uptake of l-alanine and glycine conformed to Michaelis–Menten kinetics. An uptake affinity (K _m; substrate concentration required to attain a 50% uptake saturation) of 7.0 mM and an uptake capacity (J _max) of 83 nmol cm⁻² h⁻¹ were described for l-alanine. The K _m and J _max for glycine were 2.2 mM and 11.9 nmol cm⁻² h⁻¹, respectively. Evidence suggested that the pathways of l-alanine and glycine absorption were shared, and sodium dependent. Further analysis indicated that glycine uptake was independent of luminal pH and proline, but a component of uptake was significantly impaired by 100-fold excesses of threonine or asparagine. The presence of a short-term (24 h) exposure to waterborne glycine, similar in nature to that which may be expected to occur when feeding inside an animal carcass, had no significant impact on gastrointestinal glycine uptake. This may indicate a lack of cross talk between absorptive epithelia. These results are the first published data to describe gastrointestinal uptake of an organic nutrient in the oldest extant vertebrate and may provide potential insight into the evolution of nutrient transport systems.     

A family GH51 α-<Emphasis Type="SmallCaps">l</Emphasis>-arabinofuranosidase from <Emphasis Type="BoldItalic">Pleurotus ostreatus</Emphasis>: identification,recombinant expression and characterization

An α-l-arabinofuranosidase produced by Pleurotus ostreatus (PoAbf) during solid state fermentation on tomato pomace was identified and the corresponding gene and cDNA were cloned and sequenced. Molecular analysis showed that the poabf gene carries 26 exons interrupted by 25 introns and has an open reading frame encoding a protein of 646 amino acid residues, including a signal peptide of 20 amino acid residues. The amino acid sequence similar to the other α-l-arabinofuranosidases indicated that the enzyme encoded by poabf can be classified as a family 51 glycoside hydrolase. Heterologous recombinant expression of PoAbf was carried out in the yeasts Pichia pastoris and Kluyveromyces lactis achieving the highest production level of the secreted enzyme (180 mg L⁻¹) in the former host. rPoAbf produced in P. pastoris was purified and characterized. It is a glycosylated monomer with a molecular weight of 81,500 Da in denaturing conditions. Mass spectral analyses led to the localization of a single O-glycosylation site at the level of Ser160. The enzyme is highly specific for α-l-arabinofuranosyl linkages and when assayed with p-nitrophenyl α-l-arabinofuranoside it follows Michaelis–Menten kinetics with a K _M of 0.64 mM and a k _cat of 3,010 min⁻¹. The optimum pH is 5 and the optimal temperature 40°C. It is worth noting that the enzyme shows a very high stability in a broad range of pH. The more durable activity showed by rPoAbf in comparison to the other α-l-arabinofuranosidases enhances its potential for biotechnological applications and increases interest in elucidating the molecular bases of its peculiar properties.     

Biotechnological production of <Emphasis Type="SmallCaps">l</Emphasis>-ribose from <Emphasis Type="SmallCaps">l</Emphasis>-arabinose

l-Ribose is a rare and expensive sugar that can be used as a precursor for the production of l-nucleoside analogues, which are used as antiviral drugs. In this work, we describe a novel way of producing l-ribose from the readily available raw material l-arabinose. This was achieved by introducing l-ribose isomerase activity into l-ribulokinase-deficient Escherichia coli UP1110 and Lactobacillus plantarum BPT197 strains. The process for l-ribose production by resting cells was investigated. The initial l-ribose production rates at 39°C and pH 8 were 0.46 ± 0.01 g g⁻¹ h⁻¹ (1.84 ± 0.03 g l⁻¹ h⁻¹) and 0.27 ± 0.01 g g⁻¹ h⁻¹ (1.91 ± 0.1 g l⁻¹ h⁻¹) for E. coli and for L. plantarum, respectively. Conversions were around 20% at their highest in the experiments. Also partially purified protein precipitates having both l-arabinose isomerase and l-ribose isomerase activity were successfully used for converting l-arabinose to l-ribose.     

Characterization of NADP<Superscript>+</Superscript>-specific <Emphasis Type="SmallCaps">l</Emphasis>-rhamnose dehydrogenase from the thermoacidophilic Archaeon <Emphasis Type="Italic">Thermoplasma acidophilum</Emphasis>

Thermoplasma acidophilum utilizes l-rhamnose as a sole carbon source. To determine the metabolic pathway of l-rhamnose in Archaea, we identified and characterized l-rhamnose dehydrogenase (RhaD) in T. acidophilum. Ta0747P gene, which encodes the putative T. acidophilum RhaD (Ta_RhaD) enzyme belonging to the short-chain dehydrogenase/reductase family, was expressed in E. coli as an active enzyme catalyzing the oxidation of l-rhamnose to l-rhamnono-1,4-lactone. Analysis of catalytic properties revealed that Ta_RhaD oxidized l-rhamnose, l-lyxose, and l-mannose using only NADP⁺ as a cofactor, which is different from NAD⁺/NADP⁺-specific bacterial RhaDs and NAD⁺-specific eukaryal RhaDs. Ta_RhaD showed the highest activity toward l-rhamnose at 60 °C and pH 7. The K _m and k _cat values were 0.46 mM, 1,341.3 min⁻¹ for l-rhamnose and 0.1 mM, 1,027.2 min⁻¹ for NADP⁺, respectively. Phylogenetic analysis indicated that branched lineages of archaeal RhaD are quite distinct from those of Bacteria and Eukarya. This is the first report on the identification and characterization of NADP⁺-specific RhaD.     

Characterization of a tryptophan 6-halogenase from <Emphasis Type="Italic">Streptomyces toxytricini</Emphasis>

Tryptophan (Trp) halogenases are found in various bacteria and play an important role in natural product biosynthesis. Analysis of the genome of Streptomyces toxytricini NRRL 15443 revealed an ORF, stth, encoding a putative Trp halogenase within a non-ribosomal peptide synthetase gene cluster. This gene was cloned into pET28a and functionally overexpressed in Escherichia coli. The enzyme halogenated both l- and d-Trp to yield the corresponding 6-chlorinated derivatives. The optimum activity was at 40°C and pH 6 giving k _cat /K _M value of STTH of 72,000 min⁻¹ M⁻¹. The enzyme also used bromide to yield 6-bromo-Trp.     

Purification and Biochemical Characterization of Indole-3-acetyl-aspartic Acid Synthetase from Immature Seeds of Pea (Pisum sativum)

Indole-3-acetic acid (IAA) amidosynthetases catalyzing the ATP-dependent conjugation of IAA and amino acids play an important role in the maintenance of auxin homeostasis in plant cells. A new amidosynthetase, indole-3-acetic acid:l-aspartic acid ligase (IAA-Asp synthetase) involved in IAA-amino acid biosynthesis, was isolated via a biochemical approach from immature seeds of the pea (Pisum sativum L). The enzyme was purified to homogeneity by a three-step procedure, involving PEG 6000 fractionation, DEAE-Sephacel anion-exchange chromatography, and preparative PAGE, and characterized as a 70-kDa monomeric protein by analytical gel filtration and SDS-PAGE. Rabbit antiserum against recombinant AtGH3.5 cross-reacted with the pea IAA-Asp synthetase, and a single immunoreactive polypeptide band was observed at 70 kDa. The purified enzyme had an apparent isoelectric point at pH 4.7, the highest activity at pH 8.2, preferred Mg²⁺ as a cofactor, and was strongly activated by reducing agents. Similar to known recombinant GH3 enzymes, an IAA-Asp synthetase from pea catalyzes the conjugation of phytohormone acyl substrates to amino acids. The enzyme had the highest synthesizing activity on IAA, followed by 1-NAA, SA, 2,4-D, and IBA, whereas activities on l-Trp, IPA, PAA, (±)JA, and 2-NAA were not significant or not detected. Of 14 amino acids tested, the enzyme had the highest activity on Asp and lower activity on Ala and Lys. Glutamate was found to be a very poor substrate and no conjugating activity was observed on the rest of the amino acids. Steady-state kinetic analysis indicated that IAA and aspartate were preferred substrates for the pea IAA-Asp synthetase. The enzyme exhibited both higher affinities for IAA and Asp (K _m = 0.2 and 2.5 mM, respectively) and catalytic efficiencies (k _cat/K _m = 682,608.7 and 5080 s⁻¹ M⁻¹, respectively) compared with other auxins and amino acids examined. This study describes the first amidosynthetase isolated and purified from plant tissue and provides the foundation for future genetic approaches to explain the role of IAA-Asp in Pisum sativum physiology.     

A novel <Emphasis Type="SmallCaps">l</Emphasis>-aspartate dehydrogenase from the mesophilic bacterium <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> PAO1: molecular characterization and application for <Emphasis Type="SmallCaps">l</Emphasis>-aspartate production

l-aspartate dehydrogenase (EC 1.4.1.21; l-AspDH) is a rare member of amino acid dehydrogenase superfamily and so far, two thermophilic enzymes have been reported. In our study, an ORF PA3505 encoding for a putative l-AspDH in the mesophilic bacterium Pseudomonas aeruginosa PAO1 was identified, cloned, and overexpressed in Escherichia coli. The homogeneously purified enzyme (PaeAspDH) was a dimeric protein with a molecular mass of about 28 kDa exhibiting a very high specific activity for l-aspartate (l-Asp) and oxaloacetate (OAA) of 127 and 147 U mg⁻¹, respectively. The enzyme was capable of utilizing both nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP) as coenzyme. PaeAspDH showed a T _m value of 48°C for 20 min that was improved to approximately 60°C by the addition of 0.4 M NaCl or 30% glycerol. The apparent K _m values for OAA, NADH, and ammonia were 2.12, 0.045, and 10.1 mM, respectively; comparable results were observed with NADPH. The l-Asp production system B consisting of PaeAspDH, Bacillus subtilis malate dehydrogenase and E. coli fumarase, achieved a high level of l-Asp production (625 mM) from fumarate in fed-batch process with a molar conversion yield of 89.4%. Furthermore, the fermentative production system C released 33 mM of l-Asp after 50 h by using succinate as carbon source. This study represented an extensive characterization of the mesophilic AspDH and its potential applicability for efficient and attractive production of l-Asp. Our novel production systems are also hopeful for developing the new processes for other compounds production.     

Characterization of a mannose-6-phosphate isomerase from Geobacillus thermodenitrificans that converts monosaccharides

A recombinant mannose-6-phosphate isomerase from Geobacillus thermodenitrificans (GTMpi) isomerizes aldose substrates possessing hydroxyl groups oriented in the same direction at the C2 and C3 positions such as the d- and l-forms of ribose, lyxose, talose, mannose, and allose. The activity of GTMpi for d-lyxose isomerization was optimal at pH 7.0, 70°C and 1 mM Co²⁺. Under these conditions, the k _cat and K _m values were 74,300 s⁻¹ and 390 mM for d-lyxose and 28,800 s⁻¹ and 470 mM for l-ribose, respectively. The half-lives of the enzyme at 60, 65, and 70°C were 388, 73, and 27 h, respectively. GTMpi catalyzed the conversion of d-lyxose to d-xylulose with a 38% conversion yield after 3 h, and converted l-ribose to l-ribulose with a 29% conversion yield.     

Partial purification and characterization of pectinmethylesterase from ripening guava (<Emphasis Type="Italic">Psidium guajava</Emphasis> L.) fruits

Employing the techniques of (NH₄)₂SO₄ fractionation, ion exchange chromatography on DEAE cellulose and gel filtration through Sephadex G-100, pectinmethylesterase (EC 3.1.1.11) was purified from guava (Psidium guajava L.) fruits var. Hisar Safeda harvested at turning stage of maturity to 129-fold with 28% recovery. Molecular weight as determined by gel filtration was found to be 51 kDa and the enzyme preparation exhibited the same molecular weight under native (Native-PAGE) and denaturating conditions (SDS-PAGE) indicating that the enzyme was a monomer. With pectin as the substrate, it exhibited the Michaelis Menten kinetics with K _m value of 3.1 g l⁻¹. The enzyme was found to be stimulated by Ca⁺⁺ and Na⁺ and inhibited competitively by d-galacturonic acid with K _i value of 1.97 mM. The enzyme was completely inactivated by iodine while with diethyl pyrocarbonate and N-acetylimidazole, the enzyme was inhibited up to the extent of 56 and 45%, respectively. However, DTNB had no inhibitory effect whatsoever precluding the participation of any –SH group in the active centre. It is tentatively proposed that the enzyme has tyrosine and histidine residues at its active centre.