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 and isolation of <Emphasis Type="SmallCaps">L</Emphasis>-to-<Emphasis Type="SmallCaps">D</Emphasis>-amino-acid-residue isomerase from platypus venom

Summary. Platypus venom contains an isomerase that reversibly interconverts the second amino-acid residue in some peptides between the L-form and the D-form. The enzyme acts on the natriuretic peptides OvCNPa and OvCNPb, and on the defensin-like peptides DLP-2 and DLP-4, but it does not act on DLP-1. While the isomerization of DLP-2 to DLP-4 is inhibited by the amino-peptidase inhibitor amastatin, it is not affected by the leucine amino-peptidase inhibitor bestatin. The enzyme, that is only present in minute quantities in an extract of the venom gland, is thermally stable up to 55 °C, and it was found by anion-exchange chromatography to be acidic. Isolation of the isomerase was carried out by combined ion-exchange chromatography and reverse-phase high performance liquid chromatography (HPLC).     

Purification and characterization of <Emphasis Type="SmallCaps">l</Emphasis>-arabinose isomerase from <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> producing <Emphasis Type="SmallCaps">d</Emphasis>-tagatose

l-arabinose isomerase (EC5.3.1.4. AI) mediates the isomerization of d-galactose into d-tagatose as well as the conversion of l-arabinose into l-ribulose. The AI from Lactobacillus plantarum SK-2 was purified to an apparent homogeneity giving a single band on SDS–PAGE with a molecular mass of 59.6 kDa. Optimum activity was observed at 50°C and pH 7.0. The enzyme was stable at 50°C for 2 h and held between pH 4.5 and 8.5 for 1 h. AI activity was stimulated by Mn²⁺, Fe³⁺, Fe²⁺, Ca²⁺ and inhibited by Cu²⁺, Ag⁺, Hg²⁺, Pb²⁺. d-galactose and l-arabinose as substrates were isomerized with high activity. l-arabitol was the strongest competitive inhibitor of AI. The apparent Michaelis–Menten constant (K _m), for galactose, was 119 mM. The first ten N-terminal amino acids of the enzyme were determined as MLSVPDYEFW, which is identical to L. plantarum (Q88S84). Using the purified AI, 390 mg tagatose could be converted from 1,000 mg galactose in 96 h, and this production corresponds to a 39% equilibrium.     

Anthraquinone polyamines: novel channel blockers of <Emphasis Type="Italic">N</Emphasis>-methyl-<Emphasis Type="SmallCaps">d</Emphasis>-aspartate receptors

Summary. Polyamines, in particular spermine, as well as some natural and synthetic polyamine derivatives have been found to be blockers of N-methyl-d-aspartate receptors. We developed novel, polyamine-based channel blockers to analyze the structure of NMDA receptors. Anthraquinone polyamines block NMDA receptors with some selectivity compared to other glutamate receptors. Results using mutant NR1 and NR2 subunits identified amino acid residues that influence blockade by anthraquinone polyamines. The head group (anthraquinone) may be positioned at the selectivity filter/narrowest constriction of the channel and the polyamine tail penetrates this constriction into the inner vestibule below the level of the selectivity filter. The results are consistent with other work showing that NR1 (Asn616) and NR2B (Asn616), but not NR2B (Asn615), make the narrowest constriction of NMDA channel, and that the M3 segments from the two subunits, which form the outer vestibule, are likely staggered relative to each other in the vertical axis of the channel.     

Alterations in <Emphasis Type="SmallCaps">D</Emphasis>-amino acid levels in the brains of mice and rats after the administration of <Emphasis Type="SmallCaps">D</Emphasis>-amino acids

Summary. To mutant ddY/DAO⁻ mice lacking D-amino-acid oxidase activity and normal ddY/DAO⁺ mice, five D-amino acids (D-Asp, D-Ser, D-Ala, D-Leu and D-Pro) were orally administered for two weeks, and the D-amino acid levels were examined in seven brain regions. The levels of D-Asp markedly increased in the pituitary and pineal glands in both strains. In the ddY/DAO⁺ mice, the levels of the other D-amino acids did not significantly change in most of the brain regions. While in the ddY/DAO⁻ mice the levels of D-Ser significantly increased in most of the brain regions except for the cerebrum and hippocampus. The levels of D-Ala and D-Leu increased in all regions but the levels of D-Pro did not significantly change. The same five D-amino acids were intravenously injected into Wistar rats and the D-amino acid levels in their brains were examined for 60 min after the administration. The levels of D-Asp markedly increased in the pineal gland 3 min after the administration, while the levels of D-Ser, D-Ala, and D-Pro increased both in the pineal and pituitary glands, the levels of D-Leu increased in all brain regions. These results are useful for the elucidation of the origins and regulation of D-amino acids in the mammalian body.     

Methods for syntheses of <Emphasis Type="Italic">N</Emphasis>-methyl-<Emphasis Type="Italic">DL</Emphasis>-aspartic acid derivatives

Summary. A novel practical method for the synthesis of N-methyl-DL-aspartic acid 1 (NMA) and new syntheses for N-methyl-aspartic acid derivatives are described. NMA 1, the natural amino acid was synthesized by Michael addition of methylamine to dimethyl fumarate 5. Fumaric or maleic acid mono-ester and -amide were regioselectively transformed into beta-substituted aspartic acid derivatives. In the cases of maleamic 11a or fumaramic esters 11b, the α-amide derivative 13 was formed, but hydrolysis of the product provided N-methyl-DL-asparagine 9 via base catalyzed ring closure to DL-α-methylamino-succinimide 4, followed by selective ring opening. Efficient methods were developed for the preparation of NMA-α-amide 13 from unprotected NMA via sulphinamide anhydride 15 and aspartic anhydride 3 intermediate products. NMA diamide 16 was prepared from NMA dimethyl ester 6 and methylamino-succinimide 4 by ammonolysis. Temperature-dependent side reactions of methylamino-succinimide 4 led to diazocinone 18, resulted from self-condensation of methylamino-succinimide via nucleophyl ring opening and the subsequent ring-transformation.     

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.     

NMDA Receptor Regulation by <Emphasis Type="SmallCaps">D</Emphasis>-serine: New Findings and Perspectives

The N-methyl-d-aspartate (NMDA) receptors play key roles in excitatory neurotransmission and are involved in several important processes, including learning, behavior, and synaptic plasticity. The regulation of NMDA receptor neurotransmission has been extensively studied, but many important questions still remain unsolved. One of the most debated aspects of the NMDA receptor regulation relates to the identity, role, and cellular origin of the NMDA coagonist(s). In addition to glutamate, the NMDA receptor activity was believed to be regulated by the coagonist glycine. More recently, d-serine has also been proposed to play a role as a key coagonist for NMDA receptor activity and neurotoxicity. A surprising unique biosynthetic pathway for d-serine has been demonstrated, indicating the conservation of d-amino acid metabolism in mammals. d-Serine was originally shown to be exclusively made in astrocytes, indicating a possible role as a gliotransmitter. Nevertheless, recent data indicate that d-serine has a neuronal origin as well, which raises several new questions on d-serine disposition. In this review, I discuss recent advances in the field and propose a novel model of d-serine signaling that includes a bidirectional flow of d-serine between astrocytes and neurons. This review is dedicated to the memory of Dr. Marcos Wolosker.     

Glutarate and <Emphasis Type="Italic">N</Emphasis>-acetyl-<Emphasis Type="SmallCaps">l</Emphasis>-glutamate buffers for cell-free synthesis of selectively <Superscript>15</Superscript>N-labelled proteins

Cell-free protein synthesis provides rapid and economical access to selectively ¹⁵N-labelled proteins, greatly facilitating the assignment of ¹⁵N-HSQC spectra. While the best yields are usually obtained with buffers containing high concentrations of potassium l-glutamate, preparation of selectively ¹⁵N-Glu labelled samples requires non-standard conditions. Among many compounds tested to replace the l-Glu buffer, potassium N-acetyl-l-glutamate and potassium glutarate were found to perform best, delivering high yields for all proteins tested, with preserved selectivity of ¹⁵N-Glu labelling. Assessment of amino-transferase activity by combinatorial ¹⁵N-labelling revealed that glutarate and N-acetyl-l-glutamate suppress the transfer of the ¹⁵N-α-amino groups between amino acids less well than the conventional l-Glu buffer. On balance, the glutarate buffer appears most suitable for the preparation of samples containing ¹⁵N-l-Glu while the conventional l-Glu buffer is advantageous for all other samples. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

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.     

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.     

Arabino-Galactan Proteins from <Emphasis Type="Italic">Pistacia lentiscus</Emphasis> var. <Emphasis Type="Italic">chia</Emphasis>: isolation,characterization and biological function

Summary. Arabino-Galactan Proteins (AGPs) were isolated from Chios mastic gum (CMG) by using a buffer containing 0.1 M NaCl, 20 mM Tris–HCl, pH 7.5. Protein analytical methods, combined with specific procedures for carbohydrate characterization, indicated the presence of highly glycosylated protein backbone. In particular, staining by Yariv reagent of the electrophoretically separated molecules revealed the existence of arabinose and galactose and such a modification is characteristic for AGPs. After experiments involving extensive dialysis of the isolated extracts against water and atomic absorption, there was evidence of the existence of zinc ions that are probably covalently bound to the AGPs. By using anion-exchange chromatography, capillary electrophoresis, colorimetric methods and GC-MS, it was found that the extracts were separated into three major populations (A, B, and C), which were consistent with their respective negative charge content namely, uronic acid. The characterization of neutral sugars that was investigated with GC-MS showed the existence of arabinose and galactose in different amounts for each group. Experiments concerning the inhibition of growth of Helicobacter pylori in the presence of AGPs, as is shown for other CMG constituents, showed that the extracts of at least 1.4 g CMG affected the viability of the bacterium. There is no evidence as to whether the AGPs provoke abnormal morphologies of H. pylori, as is reported for the total CMG, or for O-glycans that possess terminal α1, 4-linked N-acetylglucosamine and are expressed in the human gastric mucosa; this has to be further investigated. Authors’ address: Theodora Choli-Papadopoulou, Laboratory of Biochemistry, School of Chemistry, Aristotle University of Thessaloniki, TK 54124 Thessaloniki, Greece     

Engineering of an <Emphasis Type="SmallCaps">l</Emphasis>-arabinose metabolic pathway in <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

Corynebacterium glutamicum was metabolically engineered to broaden its substrate utilization range to include the pentose sugar l-arabinose, a product of the degradation of lignocellulosic biomass. The resultant CRA1 recombinant strain expressed the Escherichia coli genes araA, araB, and araD encoding l-arabinose isomerase, l-ribulokinase, and l-ribulose-5-phosphate 4-epimerase, respectively, under the control of a constitutive promoter. Unlike the wild-type strain, CRA1 was able to grow on mineral salts medium containing l-arabinose as the sole carbon and energy source. The three cloned genes were expressed to the same levels whether cells were cultured in the presence of d-glucose or l-arabinose. Under oxygen deprivation and with l-arabinose as the sole carbon and energy source, strain CRA1 carbon flow was redirected to produce up to 40, 37, and 11%, respectively, of the theoretical yields of succinic, lactic, and acetic acids. Using a sugar mixture containing 5% d-glucose and 1% l-arabinose under oxygen deprivation, CRA1 cells metabolized l-arabinose at a constant rate, resulting in combined organic acids yield based on the amount of sugar mixture consumed after d-glucose depletion (83%) that was comparable to that before d-glucose depletion (89%). Strain CRA1 is, therefore, able to utilize l-arabinose as a substrate for organic acid production even in the presence of d-glucose.     

<Emphasis Type="SmallCaps">l</Emphasis>-Homoarginine suppresses exocrine pancreas in rats

Summary.  Previously, we found that guanidinated casein, a l-homoarginine-containing protein, was a more potent stimulator of pancreatic enzyme secretion than intact casein in rats. In this study, we examined secretory response and adaptation of the exocrine pancreas to the administration of free l-homoarginine in normal and bile-pancreatic juice (BPJ)-diverted rats. An intraperitoneal injection of l-homoarginine (10 mg/rats) produced immediate and transient reduction in pancreatic secretion in BPJ-diverted rats, but not in normal rats. The BPJ-diverted rats were fed with either a 25% casein, 45% casein, or 45% casein diet supplemented with l-homoarginine (19 g/kg diet) for 4 days. Feeding of a diet containing l-homoarginine inhibited the pancreatic adaptation induced by the high-protein diet. These results indicate that l-homoarginine has an inhibitory effect on the secretion and production of exocrine pancreatic enzyme in BPJ-diverted rats, and l-homoarginine may have an antagonistic effect on CCK receptors. Received July 1, 2002 Accepted August 28, 2002 Published online December 20, 2002 Authors' address: Dr. Hiroshi Hara, Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Kita-9, Nishi-9, Kita-ku, Sapporo 060-8589, Japan E-mail: hara@chem.agr.hokudai.ac.jp     

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.     

Enzymatic production of <Emphasis Type="SmallCaps">l</Emphasis>-amino acids from the corresponding <Emphasis Type="SmallCaps">dl</Emphasis>-5-substituted hydantoins by <Emphasis Type="Italic">Bacillus fordii</Emphasis> MH602

Bacillus fordii MH602 was newly screened from soil at 45 °C and exhibited high activities of hydantoinase and carbamoylase, efficiently yielding l-amino acids including phenylalanine, phenylglycine and tryptophan with the bioconversion yield of 60–100% from the corresponding dl-5-substituted hydantoins. Hydantoinase activity was found to be cell-associated and inducible. The optimal inducer was dl-5-methylhydantoin with concentration of 0.014 mol L⁻¹ and added to the fermentation medium in the exponential phase of growth. In the production of optically pure amino acids from dl-5-benylhydantoin, the optimal temperature and pH of this reaction were 45–50 °C and 7.5 respectively. The hydantoinase was non-stereoselective, while carmbamoylase was l-selective. The hydantoinase activity was not subject to substrate inhibition, or product inhibition by ammonia. In addition, The activities of both enzymes from crude extract of the strain were thermostable; the hydantoinase and carbamoylase retained about 90% and 60% activity after 6 h at 50 °C, respectively. Since reaction at higher temperature is advantageous for enhancement of solubility and for racemization of dl-5-substituted hydantoins, the relative paucity of l-selective hydantoinase systems, together with the high level of hydantoinase and carbamoylase activity and unusual substrate selectivity of the strain MH602, suggest that it has significant potential applications.     

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.     

Preparation of tritiated oostatic peptides for study of radioactivity incorporation in flesh fly <Emphasis Type="Italic">Neobellieria bullata</Emphasis>

Summary. A series of insect oostatic peptides containing 3,4-dehydroproline in the C-terminal part or inside of the peptide chain was synthesized and tritiated by addition of ³H₂ to double bond of 3,4-dehydroproline residue. ³H-label was introduced also into tyrosine residue of oostatic tetra- and pentapeptides by isotopic exchange of benzyl β-hydrogens. In this way, three types of tritiated peptides were prepared, different in the radiolabeled amino acid position: [³H] Tyr-Asp-Pro-Ala-OH, H-Tyr-Asp-[³H] Pro-Ala-OH, [³H] Tyr-Asp-Pro-Ala-Pro-OH, H-Tyr-Asp-[³H] Pro-Ala-Pro-OH, H-Tyr-Asp-Pro-Ala-[³H] Pro-OH, H-Tyr-Asp-Pro-Ala-Pro₅-[³H] Pro-OH and H-Asp-[³H] Pro-OH. These peptides made possible a highly sensitive comparative study on radioactivity incorporation into head and ovaries of the flesh fly Neobellieria bullata, which revealed this process to proceed differently. The reasons of the found differences are discussed.     

<Emphasis Type="SmallCaps">d</Emphasis>-Psicose production from <Emphasis Type="SmallCaps">d</Emphasis>-fructose using an isolated strain, <Emphasis Type="Italic">Sinorhizobium</Emphasis> sp.

Sinorhizobium sp., which can convert d-fructose into d-psicose, was isolated from soil. The optimal pH, temperature, and cell concentration for d-psicose production with the isolated strain were 8.5, 40°C, and 60 mg/ml, respectively. The toluene-treated cells showed 2.5- and 4.8-fold increases in the d-psicose concentration and productivity compared with untreated washed cells. Under the optimal conditions, the toluene-treated cells produced 37 g d-psicose/l from 70% (w/v) (3.9 M) d-fructose after 15 h.     

Alanine racemase from the green alga <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis>

Summary. Chlamydomonas reinhardtii, a unicellular green microalga, could grow to a stationary phase having optical density of 2.0–2.5 at 750 nm in Tris-acetate-phosphate (TAP) medium containing 0.1% D-alanine. D-alanine has no inhibitory effect on growth and induced alanine racemase activity 130-fold more than without D-alanine in the green alga. Although C. reinhardtii cultured in the TAP medium showed alanine racemase activity, the content of free D-alanine was only 0.14%. The enzyme was partially purified by ammonium sulfate fractionation followed by three kinds of liquid chromatography using DEAE Toyopearl, Phenyl Sepharose, and TSK G3000 SWXL columns. The specific activity for L-alanine of the partially purified alanine racemase was 3.8 μmol/min/mg. The molecular weight of the enzyme was determined to be approximately 72,000 by gel filtration. The enzyme showed a maximum activity at 45 °C and pH 8.4 and requires pyridoxal 5′-phosphate as a coenzyme.