Generation and properties of ascorbic acid-overproducing transgenic tobacco cells expressing sense RNA for <Emphasis Type="SmallCaps">l</Emphasis>-galactono-1,4-lactone dehydrogenase

l-Galactono-1,4-lactone dehydrogenase (GalLDH; EC 1.3.2.3) is the last enzyme in the putative l-ascorbic acid (AsA) biosynthetic pathway of plants. Here, we show for the first time that the overexpression of GalLDH can increase AsA content in tobacco (Nicotiana tabacum L.) BY-2 cells. To see the effect, we analyzed the properties of these AsA-overproducing transgenic cell lines, especially in relation to AsA content of cells, cell division, senescence and resistance to oxidative stress. The mitotic index in AsA-overproducing cells was higher than in wild-type cells. Moreover, the browning of these cells was markedly restrained, and the proportion of dead cells was reduced, especially in the later period of culture. These AsA-overproducing cells also acquired resistance to paraquat (methyl viologen), which produces active oxygen species. These results contribute to the previous insights about AsA and raise the possibility of the generation of plants that have resistance to environmental stresses by increasing their AsA content.     

Syntheses of dopa glycosides using glucosidases

Syntheses of l-dopa 1a glucoside 10a,b and dl-dopa 1b glycosides 10–18 with d-glucose 2, d-galactose 3, d-mannose 4, d-fructose 5, d-arabinose 6, lactose 7, d-sorbitol 8 and d-mannitol 9 were carried out using amyloglucosidase from Rhizopus mold, β-glucosidase isolated from sweet almond and immobilized β-glucosidase. Invariably, l-dopa and dl-dopa gave low to good yields of glycosides 10–18 at 12–49% range and only mono glycosylated products were detected through glycosylation/arylation at the third or fourth OH positions of l-dopa 1a and dl-dopa 1b. Amyloglucosidase showed selectivity with d-mannose 4 to give 4-O-C1β and d-sorbitol 8 to give 4-O-C6-O-arylated product. β-Glucosidase exhibited selectivity with d-mannose 4 to give 4-O-C1β and lactose 7 to give 4-O-C1β product. Immobilized β-glucosidase did not show any selectivity. Antioxidant and angiotensin converting enzyme inhibition (ACE) activities of the glycosides were evaluated glycosides, out of which l-3-hydroxy-4-O-(β-d-galactopyranosyl-(1′→4)β-d-glucopyranosyl) phenylalanine 16 at 0.9 ± 0.05 mM and dl-3-hydroxy-4-O-(β-d-glucopyranosyl) phenylalanine 11b,c at 0.98 ± 0.05 mM showed the best IC₅₀ values for antioxidant activity and dl-3-hydroxy-4-O-(6-d-sorbitol)phenylalanine 17 at 0.56 ± 0.03 mM, l-dopa-d-glucoside 10a,b at 1.1 ± 0.06 mM and dl-3-hydroxy-4-O-(d-glucopyranosyl)phenylalanine 11a-d at 1.2 ± 0.06 mM exhibited the best IC₅₀ values for ACE inhibition. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

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.     

Regulation of metabolite production by precursors and elicitors in liquid cultures of <Emphasis Type="Italic">Hypericum perforatum</Emphasis>

Four precursors (l-phenylalanine, l-tryptophan, cinnamic acid and emodin) and one signal elicitor (methyl jasmonate, MeJA) were added to liquid cultures of Hypericum perforatum L. to study their effect on production of hyperforin and hypericins (pseudohypericin and hypericin). The addition of l-phenylalanine (75 to 100 mg l⁻¹) enhanced production of hypericins, but hyperforin levels were decreased. Hypericin, pseudohypericin and hyperforin concentrations were all decreased when l-tryptophan (25 to 100 mg l⁻¹) was added to the medium. However, addition of l-tryptophan (50 mg l⁻¹) with MeJA (100 μM) stimulated hyperforin production significantly (1.81-fold) and resulted in an increased biomass. Cinnamic acid (25, 50 mg l⁻¹) and emodin (1.0 to 10.0 mg l⁻¹) each enhanced hyperforin accumulation in H. perforatum, but did not affect accumulation of hypericins.     

Biosynthesis,processing, and extracellular release of α-l-fucosidase in lymphoid cell lines of different genetic origins

In humans, the quantity of α-l-fucosidase in serum is determined by heredity. The mechanism controlling levels of the enzyme in serum is unknown. Lymphoid cell lines derived from individuals with either low, intermediate, or high α-l-fucosidase in serum were established. Steady-state levels of intracellular and extracellular α-l-fucosidase as well as rates of synthesis and secretion of enzyme overlapped among the cell lines. Thus,_vivo} serum phenotypes were not expressed in this system. No appreciable differences in the qualitative processing of newly made α-l-fucosidase were observed among these lymphoid cell lines. Cells pulse-labeled with³⁵S-methionine from 0.25 to 2 hr had an intracellular form of enzyme with aM _r=58,000. Cells pulsed for 1.5 hr and chased for 21 hr with unlabeled methionine had an intracellular form ofM _r=60,000 and an extracellular form ofM _r=62,000. All three enzyme forms were glycoproteins with a common polypeptide chain ofM _r=52,000 but with different carbohydrate moieties. No evidence for a high molecular mass precursor form of α-l-fucosidase was found. Fucosidosis is a rare, inherited disease in which α-l-fucosidase activity in tissues and body fluids is low or absent. The mutations for fucosidosis and the serum polymorphism map separately. Lymphoid cells from two siblings with fucosidosis had 8-fold to 341-fold less intracellular α-l-fucosidase protein with 11-fold to 56-fold lower specific activities than control cells. Residual mutant enzyme was a glycoprotein with a polypeptide chain virtually the same size (M _r=52,000) as control enzyme. However, residual mutant enzyme was hypoglycosylated and hypersecreted as compared to control enzyme. This research was supported by National Institutes of Health Grant DK 32161.     

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.     

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.     

Characterization of <Emphasis Type="SmallCaps">d</Emphasis>-tagatose-3-epimerase from <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis> that converts <Emphasis Type="SmallCaps">d</Emphasis>-fructose into <Emphasis Type="SmallCaps">d-</Emphasis>psicose

A non-characterized gene, previously proposed as the d-tagatose-3-epimerase gene from Rhodobacter sphaeroides, was cloned and expressed in Escherichia coli. Its molecular mass was estimated to be 64 kDa with two identical subunits. The enzyme specificity was highest with d-fructose and decreased for other substrates in the order: d-tagatose, d-psicose, d-ribulose, d-xylulose and d-sorbose. Its activity was maximal at pH 9 and 40°C while being enhanced by Mn²⁺. At pH 9 and 40°C, 118 g d-psicose l⁻¹ was produced from 700 g d-fructose l⁻¹ after 3 h. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Continuous 2-keto-<Emphasis Type="SmallCaps">l</Emphasis>-gulonic acid fermentation from <Emphasis Type="SmallCaps">l</Emphasis>-sorbose by <Emphasis Type="Italic">Ketogulonigenium vulgare</Emphasis> DSM 4025

A single-stage continuous fermentation process for the production of 2-keto-l-gulonic acid (2KGA) from l-sorbose using Ketogulonigenium vulgare DSM 4025 was developed. The chemostat culture with the dilution rate that was calculated based on the relationship between the 2KGA production rate and the 2KGA concentration was feasible for production with high concentration of 2KGA. In this system, 112.2 g/L of 2KGA on the average was continuously produced from 114 g/L of l-sorbose. A steady state of the fermentation was maintained for the duration of more than 110 h. The dilution rate was kept in the range of 0.035 and 0.043 h⁻¹, and the 2KGA productivity was 3.90 to 4.80 g/L/h. The average molar conversion yield of 2KGA from l-sorbose was 91.3%. Under the optimal conditions, l-sorbose concentration was kept at 0 g/L. Meanwhile, the dissolved oxygen level was changing in response to the dilution rate and 2KGA concentration. In the dissolved oxygen (DO) range of 16% to 58%, it was revealed that the relationship between DO and D possessed high degree of positive correlation under the l-sorbose limiting condition (complete consumption of l-sorbose). Increasing D closer to the critical value for washing out point of the continuous fermentation, DO value tended to be gradually increased up to 58%. In conclusion, an efficient and reproducible continuous fermentation process for 2KGA production by K. vulgare DSM 4025 could be developed using a medium containing baker’s yeast without using a second helper microorganism.     

Purification and characterization of anl-amino-acid oxidase fromChlamydomonas reinhardtii

Anl-amino-acid oxidase (EC 1.4.3.1) that catalyzes the oxidative deamination of twelvel-amino acids has been purified 21-fold and with 14% yield to electrophoretic homogeneity fromChlamydomonas reinhardtii cells by ammonium-sulfate fractionation, gel filtration through Sephacryl and Superose, anion-exchange chromatography and preparative electrophoresis in polyacrylamide gels. The native enzyme is a protein of 470 kDa and consists of eight identical or similarsized subunits of 60 kDa each. Optimum pH and temperature were 8.2 and 55° C, respectively, with a Q₁₀ (45–55° C) of 1.7 and an activation energy of 45 kJ · mol^–1. Its absorption spectrum showed, in the visible region, maxima at 360 and 444 nm, characteristic of a flavoprotein with a calculated flavin content of 7.7 mol FAD per mol of native enzyme. ApparentK _m values of the twelvel-amino acids which can act as substrates ofl-amino-acid oxidase ranged between 31 M for phenylalanine and 176 M for methionine. The effect of several specific group reagents, chelating agents and bivalent cations on enzyme activity has also been studied.This work was supported by Grant 780-CO2-01 from CICYT, Spain. The skillful secretarial assistance of C. Santos and I. Molina is gratefully acknowledged.     

Production,partial purification and characterization of α-<Emphasis Type="SmallCaps">l</Emphasis>-rhamnosidase from <Emphasis Type="Italic">Penicillium ulaiense</Emphasis>

Penicillium ulaiense is a post-harvest pathogenic fungus that attacks citrus fruits. The objective of this work was to study this microorganism as an α-l-rhamnosidase producer and to characterize it from P. ulaiense. The enzyme under study is used for different applications in food and beverage industries. α-l-Rhamnosidase was produced in a stirred-batch reactor using rhamnose as the main carbon source. The kinetic parameters for the growth of the fungi and for the enzyme production were calculated from the experimental values. A method for partial purification, including (NH₄)₂SO₄ precipitation, incubation at pH 12 and DEAE-sepharose chromatography yielded an enzyme with very low β-glucosidase activity. The pH and temperature optima were 5.0 and 60°C, respectively. The Michaelis–Menten constants for the hydrolysis of p-nitrophenyl-α-l-rhamnoside were V _max = 26 ± 4 IU ml⁻¹ and K _m  = 11 ± 2 mM. The enzyme showed good thermostability up to 60°C and good operational stability in white wine. Co²⁺ affected positively the activity; EDTA, Mn²⁺, Mg²⁺, dithiotreitol and Cu²⁺ reduced the activity by different amounts, and Hg²⁺ completely inhibited the enzyme. The enzyme showed more activity on p-nitrophenyl-α-l-rhamnoside than on naringin. According to these results, this enzyme has potential for use in the food and pharmacy industries since P. ulaiense does not produce mycotoxins.     

Molecular Cloning,Sequencing, and Expression of a <Emphasis Type="SmallCaps">l</Emphasis>-Glutamine <Emphasis Type="SmallCaps">d</Emphasis>-Fructose 6-Phosphate Amidotransferase Gene from <Emphasis Type="Italic">Volvariella volvacea</Emphasis>

Using 3′-RACE and 5′-RACE, we have cloned and sequenced the genomic gene and complete cDNA encoding l-glutamine d-fructose 6-phosphate amidotransferase (GFAT) from the edible straw mushroom, Volvariella volvacea. Gfat contains five introns, and encodes a predicted protein of 697 amino acids that is homologous to other reported GFAT sequences. Southern hybridization indicated that a single gfat gene locus exists in the V. volvacea genome. Recombinant native V. volvacea GFAT enzyme, over-expressed using Escherichia coli and partially purified, had an estimated molecular mass of 306 kDa and consisted of four equal-sized subunits of 77 kD. Reciprocal plots revealed K _m values of 0.55 and 0.75 mM for fructose 6-phosphate and l-glutamine, respectively. V. volvacea GFAT activity was inhibited by the end-product of the hexosamine pathway, UDP-GlcNAc, and by the glutamine analogues N ³-(4-methoxyfumaroyl)-l-2,3-diaminopropanoic acid and 2-amino-2-deoxy-d-glucitol-6-phosphate.     

Simultaneous utilization of D-cellobiose, D-glucose, and D-xylose by recombinant Corynebacterium glutamicum under oxygen-deprived conditions

Corynebacterium glutamicum R was metabolically engineered to broaden its sugar utilization range to d-xylose and d-cellobiose contained in lignocellulose hydrolysates. The resultant recombinants expressed Escherichia coli xylA and xylB genes, encoding d-xylose isomerase and xylulokinase, respectively, for d-xylose utilization and expressed C. glutamicum R bglF ^317A and bglA genes, encoding phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) β-glucoside-specific enzyme IIBCA component and phospho-β-glucosidase, respectively, for d-cellobiose utilization. The genes were fused to the non-essential genomic regions distributed around the C. glutamicum R chromosome and were under the control of their respective constitutive promoter trc and tac that permitted their expression even in the presence of d-glucose. The enzyme activities of resulting recombinants increased with the increase in the number of respective integrated genes. Maximal sugar utilization was realized with strain X5C1 harboring five xylA–xylB clusters and one bglF ^317A –bglA cluster. In both d-cellobiose and d-xylose utilization, the sugar consumption rates by genomic DNA-integrated strain were faster than those by plasmid-bearing strain, respectively. In mineral medium containing 40 g l⁻¹ d-glucose, 20 g l⁻¹ d-xylose, and 10 g l⁻¹ d-cellobiose, strain X5C1 simultaneously and completely consumed these sugars within 12 h and produced predominantly lactic and succinic acids under growth-arrested conditions.     

N-acetyl-l-glutamate in brain: Assay,levels, and regional and subcellular distribution

N-Acetyl-L-glutamate (NAG), the activator of mitochondrial carbamoyl phosphate synthetase (CPS), is demonstrated by several methods, including a new HPLC assay, in the brain of mammals and of chicken. The brain levels of NAG are 200–300 times lower than the levels of N-acetyl-l-aspartate (NAA), and are similar to the levels of NAG in rat liver. The NAG levels in chicken liver are very low. Although NAG is mitochondrial in the liver, it is cytosolic in brain. Using enzyme activity and immuno assays we did not detect CPS in brain (detection limit, 12.5 g/g brain), excluding that brain NAG is involved in citrullinogenesis. The regional distribution of brain NAG differs from that of NAA and resembles that of N-acetyl-l-aspartyl-l-glutamate (NAAG), suggesting that NAG and NAAG are related. NAG might be involved in the modulation of NAAG degradation.Special issue dedicated to Dr. Santiago Grisolía     

Enhanced synthesis of l - threo -3,4-dihydroxyphenylserine by high-density whole-cell biocatalyst of recombinant l -threonine aldolase from Streptomyces avelmitilis

l-threo-3,4-Dihydroxyphenylserine (DOPS) is a chiral unnatural β-hydroxy amino acid used for the treatment of Parkinson disease. We developed a continuous bioconversion system for DOPS production that uses whole-cell biocatalyst of recombinant Escherichia coli expressing l-threonine aldolase (l-TA) genes cloned from Streptomyces avelmitilis MA-4680. Maximum conversion rates were observed at 2 M glycine, 145 mM 3,4-dihydroxybenzaldehyde, 0.75% Triton-X, 5 g E. coli cells/l, pH 6.5 and 10°C. In the optimized condition, overall productivity was 8 g/l, which represents 40 times the synthesis yield possible with no optimization of conditions.     

Acetyl-l-carnitine influences the fluidity of brain microsomes and of liposomes made of rat brain microsomal lipid extracts

The fluorescence anisotropy (r) of diphenylhexatriene (DPH) was measured in different preparations (bovine spinal cord phosphatidylserine liposomes, rat brain microsomes, liposomes made with rat brain microsomal lipid having different phospholipid:cholesterol ratios) at temperatures ranging from 10° to 55°C. Phosphatidylserine liposomes exhibited an exponential relationship of rversus temperature, whereas the relationship shown by microsomes and liposomes prepared with microsomal lipid extracts was a linear one. The removal of protein and high phospholipid:cholesterol ratios decreased the slope of the lines (fluidity increased), although the intercept was unaffected. This means that differences were better appreciated at high temperatures and were well evident at 37°C. Acetyl-l-carnitine decreased r in rat brain microsomes and in liposomes made with microsomal lipids with different phospholipid:cholesterol ratios. The fluidifying effect of acetyl-l-carnitine was mild but statistically significant and could explain, at least in part, the data reported in the literature of acetyl-l-carnitine acting on some parameters affected by ageing. Besides, acetyl-l-carnitine seemed to oppose the changes of viscosity due to lipid peroxidation, which has been reported to increase in ageing and dementia.l-carnitine shares the properties of its acetyl ester, but only in part.Abbreviations DPH diphenylhexatriene - HEPES 4-(2-hydroxyethyl-l-piperazineethansulfonic) acid - r fluorescence anisotropy - SHB sucrose-HEPES-buffer (0.32 M sucrose, 2 mM HEPES, pH 7.0)     

The alternative<Emphasis Type="SmallCaps"> d</Emphasis>-galactose degrading pathway of<Emphasis Type="Italic"> Aspergillus nidulans</Emphasis> proceeds via<Emphasis Type="SmallCaps"> l</Emphasis>-sorbose

The catabolism of d-galactose in yeast depends on the enzymes of the Leloir pathway. In contrast, Aspergillus nidulans mutants in galactokinase (galE) can still grow on d-galactose in the presence of ammonium—but not nitrate—ions as nitrogen source. A. nidulans galE mutants transiently accumulate high (400 mM) intracellular concentrations of galactitol, indicating that the alternative d-galactose degrading pathway may proceed via this intermediate. The enzyme degrading galactitol was identified as l-arabitol dehydrogenase, because an A. nidulans loss-of-function mutant in this enzyme (araA1) did not show NAD⁺-dependent galactitol dehydrogenase activity, still accumulated galactitol but was unable to catabolize it thereafter, and a double galE/araA1 mutant was unable to grow on d-galactose or galactitol. The product of galactitol oxidation was identified as l-sorbose, which is a substrate for hexokinase, as evidenced by a loss of l-sorbose phosphorylating activity in an A. nidulans hexokinase (frA1) mutant. l-Sorbose catabolism involves a hexokinase step, indicated by the inability of the frA1 mutant to grow on galactitol or l-sorbose, and by the fact that a galE/frA1 double mutant of A. nidulans was unable to grow on d-galactose. The results therefore provide evidence for an alternative pathway of d-galactose catabolism in A. nidulans that involves reduction of the d-galactose to galactitol and NAD⁺-dependent oxidation of galactitol by l-arabitol dehydrogenase to l-sorbose.     

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.     

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.     

Synthesis and application of dipeptides; current status and perspectives

The functions and applications of l-α-dipeptides (dipeptides) have been poorly studied compared with proteins or amino acids. Only a few dipeptides, such as aspartame (l-aspartyl-l-phenylalanine methyl ester) and l-alanyl-l-glutamine (Ala-Gln), are commercially used. This can be attributed to the lack of an efficient process for dipeptide production though various chemical or chemoenzymatic method have been reported. Recently, however, novel methods have arisen for dipeptide synthesis including a nonribosomal peptide-synthetase-based method and an l-amino acid α-ligase-based method, both of which enable dipeptides to be produced through fermentative processes. Since it has been revealed that some dipeptides have unique physiological functions, the progress in production methods will undoubtedly accelerate the applications of dipeptides in many fields. In this review, the functions and applications of dipeptides, mainly in commercial use, and methods for dipeptide production including already proven processes as well as newly developed ones are summarized. As aspartame and Ala-Gln are produced using different industrial processes, the manufacturing processes of these two dipeptides are compared to clarify the characteristics of each procedure.