Yoji Tsukada

Molecular Cloning and Identification of N-Acyl-D-glucosamine 2-Epimerase from Porcine Kidney as a Renin-binding Protein

N-Acetylneuraminic acid (NeuAc) is an important molecule in biological recognition systems. NeuAc is known to be biosynthesized either from UDP-N-acetyl-D-glucosamine by an action of UDP-N-acetyl-D-glucosamine 2-epimerase or from N-acetyl-D-glucosamine by N-acyl-D-glucosamine 2-epimerase (GlcNAc 2-epimerase). However, the physiological function of the GlcNAc 2-epimerase in NeuAc biosynthesis has not been fully evaluated. To clarify the role of GlcNAc 2-epimerase in NeuAc biosynthesis, the enzyme and its gene were isolated from porcine kidney cortex. Escherichia coli cells transformed with the gene expressed the GlcNAc 2-epimerase having the same properties as those of the GlcNAc 2-epimerase from porcine kidney. Sequence analysis indicated that the gene was capable of synthesizing a 46.5-kDa protein (402 amino acids) with a conserved leucine zipper motif. Homology search for the cloned gene revealed that the GlcNAc 2-epimerase was identical with renin-binding protein (RnBP) in porcine kidney (Inoue, H., Fukui, K., Takahashi, S., and Miyake, Y. (1990) J. Biol. Chem. 265, 6556-6561) (identity: 99.6% in nucleotide sequence, 99.0% in amino acid sequence). That GlcNAc 2-epimerase is a RnBP was confirmed by its ability to bind porcine kidney renin and mask its protease activity. These findings provide unequivocal evidence that the enzyme GlcNAc 2-epimerase is a RnBP.

Simple and large-scale production of N-acetylneuraminic acid from N-acetyl-d-glucosamine and pyruvate using N-acyl-d-glucosamine 2-epimerase and N-acetylneuraminate lyase

N-Acetylneuraminate lyase and N-acyl-D-glucosamine 2-epimerase had been cloned and overexpressed in Escherichia coli. Simultaneous use of these two enzymes and feeding of appropriate amounts of pyruvate to the reaction mixture made possible the high conversion of N-acetylneuraminic acid (Neu5Ac) from N-acetyl-D-glucosamine (GlcNAc) with a 77% conversion rate on a molar basis. As a result, 29 kg of Neu5Ac was obtained from 27 kg of GlcNAc. The product was recovered by direct crystallization, and verified as identical to authentic Neu5Ac.

Production of microbial neuraminidases induced by colominic acid

Abstract Microorganisms capable of producing neuraminidases were sought by using culture media containing colominic acid, a homopolymer of N -acetylneuraminic acid, as a sole source of carbon. Among 1000 microorganisms screened, neuraminidases with strong activity and wide specificity were obtained from the culture filtrate of Sporotrichum schenckii, Penicillium urticae, Streptomyces sp., and an unidentified bacterium B 3831-1.

Why is sialic acid attracting interest now? complete enzymatic synthesis of sialic acid with N-acylglucosamine 2-epimerase

N-Acetylneuraminic acid (NeuAc), the representative of the family of sialic acids, is an important molecule in biological recognition systems. Currently, NeuAc-based novel pharmaceutical agents and diagnostic reagents for influenza viruses are highly required in medical fields, and larger amounts of NeuAc are in demand worldwide. NeuAc had been prepared either from colominic acid (a homopolymer of NeuAc) produced by fermentation or from natural sources such as edible birds nests, milk or eggs. However, the drawbacks of such conventional methods make them unsuitable for large-scale production of NeuAc. Recently, the N-acylglucosamine 2-epimerase (AGE) gene from porcine kidney was cloned in E. coli, and a strain with a high AGE expression level was constructed for practical applications, which enabled the complete enzymatic synthesis of NeuAc with a high conversion rate from the substrates, namely, N-acetylglucosamine and pyruvate. In addition, NeuAc of highest purity could be produced economically via its direct crystallization from the reaction mixture without any column purification processes. Such a simple procedure promises to be applicable to the mass production of sialic acid at the lowest cost.

Sulfated colominic acid: an antiviral agent that inhibits the human immunodeficiency virus type 1 in vitro

Colominic acid is a homopolymer of N-acetylneuraminic acid (NANA), which has an alpha-2,8 ketosidic linkage between its polymer units. In this study, colominic acids were sulfated under different conditions and their antiviral activities against human immunodeficiency virus type 1 (HIV-1) were examined. Sulfated colominic acids, containing 6-12% sulfur, blocked the expression of HIV-1 antigen in MT-4 cells or C8166 cells following exposure to MOLT-4/HTLV-IIIB or HIV-1[GUN-1]. The compounds inhibited syncytium formation upon co-cultivation of MOLT-4 cells (clone 8) with MOLT-4/HTLV-IIIB cells and abolished the production of HIV-1 p24 antigen in culture medium of peripheral blood lymphocytes (PBLs). HIV-1 reverse transcriptase (RT) activity was not directly affected by the drugs. The compounds did not prolong activated partial thromboplastin time (APTT) at 10 and 1.0 microgram/ml, suggesting that they may not have appreciable side effects in vivo. These agents were still able to block the expression of HIV-1 antigen even when the cells were infected with HIV-1 in RPMI-1640 medium containing high percentages of fetal calf serum (FCS). These properties may be therapeutically advantageous if these compounds were considered for possible clinical use.

Molecular cloning of the N-acetylneuraminate lyase gene in Escherichia coli K-12

SummaryThe gene for N-acetylneuraminate lyase [N-acetylneuraminate pyruvate-lyase; NPL] of Escherichia coli C600 was cloned onto pBR322 as a 9.8 kilobase HindIII fragment of chromosomal DNA and the hybrid plasmid was designated pMK2. The gene in the hybrid plasmid was subcloned in pBR322 as a 1.2 kilobase HindIII-EcoRI fragment and the resultant hybrid plasmid was designated pMK6. NPL activity level was increased more than 5-fold in the pMK6-bearing strain compared with that of the wild type, when the cells were grown on a medium containing inducer (N-acetylneuraminate: NANA). The transformants harbouring pMK6 also showed higher activity even in the absence of inducer. The NPL produced by pMK6-bearing cells was structurally and immunologically the same as that purified from E. coli C600.

Excretion of Ultraviolet-Absorbing Materials by Zygosaccharomyces soja Mutant: Part I. Accumulation of 5′-Nucleotides in the Culture Fluid of Zygosaccharomyces soja Mutant during GrowthPart II. Excretion of 5′-Nucleotides by Autodegradation of RNA from Intact Cells of Zygosaccharomyces soja MutantPart III. Accumulation of 5′-Nucleotides by Autodegradation of RNA in Cell-free Preparation of Zygosaccharomyces soja Mutant

Zygosaccharomyces soja mutant obtained from cycloheximide treatment accumulated several kinds of nucleotides together with riboflavin in the minimal glucose salt medium. Accumulation of nucleotides took place in parallel with the propagation of the microorganism after inoculation. That the nucleotides accumulated were identical to 5′-CMP, 5′-AMP, 5′-UMP, 5′-GMP and UDP, ADP, GDP respectively was verified using chemical and enzymic procedures. It was assumed that the excretion of these nucleotides was mainly carried out by the action of polynucleotide phosphorylase.It was also surmised that the excretion of AMP at the early stage of incubation meant leakage from de novo synthesis.

On the Character of Riboflavin Producing Mutant of Zygosaccharomyces soja

Riboflavin producing mutant of Zygosaccharomyces soja* was obtained by a treatment with cycloheximide. This mutant actively utilized various sugars and excreted riboflavin to the culture medium in a concentration of 30 to 40 μg per ml. Aerobic condition was prefered to sustain the growth of mutant and glucose catabolism was altered from alcohol fermentation in case of mother strain to respiration in mutant. This paper presents data obtained from morphological and physiological investigations.

Characteristics of TCA Cycle of Cycloheximide-resistant Mutant of Zygosaccharomyces soja

Glucose catabolism in Zygosaccharomyces soja was carried out. through fermentation, whereas riboflavin producing mutant obtained by cycloheximicie treatment was found to utilize oxidative mechanisms. For instance, growth of mutant was sustained in minimal medium containing TCA cycle intermediates as the sole carbon source, whereas the mother strain could not grow on such culture media. Furthermore, malonate inhibited the oxidation of succinate with resting cells of mutant. As these results support the existence of TCA cycle in mutant, various enzyme activities relating to TCA cycle were investigated by the use of cell-free extracts of both strains. Aconitase, α-ketoglutaric decarboxylase, succinic dehydrogenase, fumarase, isocitritase and glyoxylic reductase were not detected in mother strain. These data indicated that both the TCA cycle and glyoxylate cycle did not operate in mother strain. On the other hand, all enzyme activities relating to TCA cycle and glyoxylate cycle were verified in mutant. This f...