Kazuo Nakahama

Cloning and expression of a cDNA encoding a novel human neurotrophic factor

A cDNA encoding a novel human neurotrophic factor (designated nerve growth factor‐2; NGF‐2) was cloned from a human glioma cDNA library using a synthetic DNA corresponding to human nerve growth factor (NGF). The cloned cDNA encodes a polypeptide composed of 257 amino acid residues including a prepro‐sequence of 138 residues and a mature region of 119 residues. The amino acid sequence of human NGF‐2 exhibits 58% similarity with that of human NGF. Conditioned medium of COS‐7 cells transfected with an expression plasmid for human NGF‐2 cDNA supported the survival of sensory neurons isolated from dorsal root ganglia of embryonic chicks. A 1.5 kb of NGF‐2 mRNA can be detected from an early development stage in rat brain, by Northern blotting analysis.

Asparaginase and Glutaminase Activities of Micro-organisms

Summary: l-Asparaginase and l-glutaminase activities were detected in many microorganisms and the distribution of these activities was found to be related to the classification of micro-organisms. Among 464 bacteria, the activities occurred in many Gram-negative bacteria and in a few Gram-positive bacteria. Most members of the family Enterobacteri-aceae possessed l-asparaginase. l-Asparaginase and l-glutaminase occurred together in a large proportion of pseudomonads. Among Gram-positive bacteria many strains of Bacillus pumilus showed strong l-asparaginase activity. Amidase activities were also observed in several strains in other families. l-Asparaginase activity was not detected in culture filtrates of 261 strains of species of the genera Streptomyces and Nocardia, but l-asparaginase and l-glutaminase were detected when these organisms were sonicated. The amidase activities in culture filtrates of 4158 fungal strains were tested. All the strains of Fusarium species formed l-asparaginase. Organisms of the genera Hypomyces and Nectria, which are regarded as the perfect stage of the genus Fusarium, also formed l-asparaginase. Several Penicillium species formed l-asparaginase. Two organisms of the family Moniliaceae formed l-glutaminase together with l-asparaginase, and a fewascomycetous fungi formed l-asparaginase or l-glutaminase. Among 1326 yeasts, l-asparaginase or l-glutaminase occurred frequently in certain serological groups of yeasts: VI (Hansenula) group, Cryptococcus group and Rhodotorula group. Many strains of Sporobolomyces species also showed l-asparaginase activity. Several strains of Cryptococcus and Rhodotorula group possessed l-glutaminase and l-asparaginase. l-Glutaminase alone was formed in many strains of Candida scottii and Cryptococcus albidus, both of which are related to Basidiomycetes.

Differences between Saccharomyces cerevisiae and Bacillus subtilis in secretion of human lysozyme.

Saccharomyces cerevisiae secreted human lysozyme in the medium as an active form when the signal peptides of chicken lysozyme and a chicken lysozyme-Aspergillus awamori glucoamylase hybrid were used, whereas it did not synthesize any human lysozyme protein by using the signal peptide of A. awamori glucoamylase. The secreted lysozyme was easily purified and crystallized. On the other hand, Bacillus subtilis secreted an inactive human lysozyme, which seemed to have incorrect disulfide bonds, with the signal peptide of amylase and its mutants. The free energy changes for the membrane translocation of the signal peptides are related to the secretion of human lysozyme in S. cerevisiae, but not in B. subtilis. These results indicate that differences exist between S. cerevisiae and B. subtilis in the secretion of human lysozyme.

Production, purification and characterization of biologically active recombinant human nerve growth factor

The human NGF gene was isolated and inserted downstream from murine leukemia virus LTR in a plasmid having dihydrofolate reductase cDNA. The expression plasmid was introduced into CHO cells. Selection of the transformants for the resistance to methotrexate gave a CHO cell line which produced human NGF at a level of 4 mg/L in the culture medium. The recombinant human NGF was purified to near homogeneity from the culture supernatant. The NH2-terminal amino acid sequence, the COOH-terminal amino acid (Ala), and the amino acid composition of the human NGF were identical to those deduced from the nucleotide sequence of the human NGF gene. The recombinant human NGF was composed of 120 amino acid residues. Three disulfide linkages were determined to be Cys15-Cys80, Cys-58-Cys108, and Cys68-Cys110; the locations were identical to those in the mouse 2.5S NGF molecule. The specific biological activity of the recombinant human NGF was comparable with that of authentic mouse 2.5S NGF as determined by stimulation of neurite outgrowth from PC12 cells.

Formation of L-Asparaginase by Fusarium Species

SUMMARY: L-Asparaginase was formed in the culture filtrates of a number of Fusarium species, as well as in those of ascomycetous fungi having a Fusarium imperfect state, such as species of Hypomyces and Nectria. Species of Gibberella, though having a Fusarium state, formed little L-asparaginase. The distribution of the ability to form the enzyme was related to taxonomic position.

Kinetic Resolution of an Indan Derivative Using Bacillus sp.SUI-12 : Synthesis of a Key Intermediate of the Melatonin Receptor Agonist TAK-375

The chiral indan derivative (S)-2 (2-[(8S)-1,6,7,8-tetrahydro-2H-indeno[5,4-b]furan-8-yl]ethyl-amine) was synthesized by enzyme-catalyzed asymmetric hydrolysis of the racemic acetamide 1 (N-[2-(1,6,7,8-tetrahydro-2H-indeno[5,4-b]furan-8-yl)ethyl]acetamide). The reaction was carried out using Bacillus sp. SUI-12 screened for the ability to hydrolyze 1 to give (S)-2 with high enantioselectivity. In a scaled-up experiment, a low reaction rate was observed. However, by changing the culture medium and the reaction conditions, it became possible to run the reaction to 40% conversion on a 10-g or more scale, obtaining (S)-2 at >;99% enantiomeric excess (ee). The (S)-2 obtained was available for the synthesis of the melatonin receptor agonist TAK-375 (N-[2-[(8S)-1,6,7,8-tetrahydro-2H-indeno[5,4-b]furan-8-yl]ethyl]propanamide).

Increased expression of hepatitis B virus surface antigen gene in Escherichia coli by IS1 insertion

SummaryWe selected faster growing colonies of Escherichia coli harbouring an expression plasmid for hepatitis B virus surface antigen (HBsAg) gene after mutagenesis. Among these colonies, three were found to produce an increased level of HBsAg as a consequence of alteration of the plasmid. Analysis of this plasmid showed that an insertion sequence, IS1, was inserted into a middle region of the HBsAg gene (codon for Pro 127) to generate a termination codon 20 bp downstream from the junction site between the HBsAg gene and the left end of IS1. Insertion of a chemically synthesized termination codon into the same region of the HBsAg gene also increased the expression of the HBsAg gene. These results suggest that HBsAg lacking the COOH-terminal region is produced at a high level because it does not inhibit the growth of the host.

Kinetic Resolutions of Indan Derivatives Using Bacteria

Racemic indan derivatives have been resolved by the hydrolysis of amide bonds using Corynebacterium ammoniagenes IFO12612 to produce (S)-amine and (R)-amides. In the kinetic resolution of 1 (N-[2-(6-methoxy-indan-1-yl)ethyl]acetamide), it was possible to run the reaction to 44% conversion on a 10-g scale, obtaining (S)-amine 4 ((S)-2-(6-methoxy-indan-1-yl)ethylamine) at >99% enantiomeric excess (ee) and (R)-1 at 98% ee.

A promoter for Bacillus subtilis expression vector

A 117-bp EcoRI-PstI fragment with strong promoter activity (P1 promoter) was cloned from Bacillus subtilis chromosomal DNA and sequenced. The P1 promoter was shown to contain a putative -35 region (TTTACT) and -10 region (TAGATT), and promotes expression of cloned human interleukin-2 (IL-2) and human interferon-gamma (IFN-gamma) genes in B. subtilis.

Microbial synthesis of three metabolites of a tachykinin receptor antagonist, TAK-637.

The compound TAK-637 ((aR,9R)-7-[3,5-bis(trifluoromethyl)benzyl]-8,9,10,11-tetrahydro-9-methyl-5-(4-methylphenyl)-7H-[1,4]diazocino[2,1-g][1,7]naphthyridine-6,13-dione), a tachykinin receptor antagonist, has been shown to be converted into three metabolites in rats and guinea pigs. It was difficult to isolate the metabolites from rats and guinea pigs administered TAK-637 and elucidate the structures. A total of 100 actinomycete strains were screened for the ability to convert TAK-637 into its metabolites. Three strains, Streptomyces subrutilus IFO13388, Streptomyces tanashiensis subsp. cephalomyceticus IFO13929 and Streptomyces lavenduligriseus IFO13405, were found to convert TAK-637 into the metabolites consistent with the metabolites formed in rats and guinea pigs as determined by HPLC analyses. The metabolites were synthesized by microbial conversion using the actinomycetes. The structures of the metabolites were elucidated by spectral analyses. It was found that the methyl group at the C(5)-phenyl group of TAK-637 was hydroxylated and the resulting alcohol was converted to carboxylic acid via aldehyde. One of the metabolites (hydroxylated TAK-637) was obtained using a 200-l fermentor in a large-scale cultivation to evaluate its biological activity.