Production of benzoylformic acid from phenylglycine by Saccharomycopsis lipolytica.

Microbial production of benzoylformic acid (BF), which can be used as a substrate of enzymatic synthesis of (R)-(-)-mandelic acid, was investigated. Among 145 strains of yeasts and actinomycetes, Saccharomycopsis lipolytica (IAM 4964) was the best producer of BF from DL-phenylglycine (DL-PG). Culture conditions for BF production by the organism were optimized. When 0.2% fructose as a carbon source and 0.7% Bacto-tryptone as a nitrogen source were used in the presence of 4% DL-PG, 14.5 mg/ml of BF was produced (about 37% molar yield) in 4 days of cultivation. BF was synthesized from the L-form of PG, but not from the D-form. The BF was isolated from culture broth in a crystalline form and physicochemically identified.


Bacillus stearothermophilus glyceraldehyde-3-phosphate dehydrogenase as a source of angiotensin-converting enzyme inhibitors.

The more potent inhibitory activity against angiotensin-converting enzyme (ACE) was excised from a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) preparation of Bacillus stearothermophilus by heating at 120 degrees C in 1 M AcOH-20 mM HCl, as compared with GAPDH preparations of yeast and pig. Sufficient excision of B. stearothermophilus ACE inhibitors required a longer proteolysis time of 60 min. Two inhibitors were then purified by gel-permeation and reverse-phase chromatographies. One of the B. stearothermophilus ACE inhibitors BG-1, was the GAPDH peptide 68-77 (Gly-Lys-Glu-Ile-Ile-Val-Lys-Ala-Glu-Arg, IC50: 32 microM). Another inhibitor, BG-2 (Gly-Lys-Met-Val-Lys-Val-Val-Ser-Trp-Tyr, IC50: 6 microM), correspond to GAPDH peptide 304-313. These sequences were quite different from those of vertebrate GAPDH peptides and the venom peptide family with ACE inhibitory activity. BG-2 was found to be a non-competitive type inhibitor, differing from many natural peptide inhibitors. Thus, B. stearothermophilus GAPDH seemed to be a good source of new type ACE inhibitors, in addition to the advantages due to its thermophilic property.


Biochemical mechanisms of C-P bond formation of bialaphos: use of gene manipulation for the analysis of the C-P bond formation step.

One of the three C-P bond formation steps, defined as step 5 in the bialaphos (BA) biosynthetic pathway, was analyzed using a new BA non-producing mutant NP71. The mutant was derived from a BA producer by gene replacement of an unidentified region next to the gene responsible for the step 5 deficiency of the mutant NP213, obtained by conventional mutation procedures. Biochemical analysis of these two mutants indicated that NP71 was defective in the formation of carboxyphosphonoenolpyruvate (CPEP), while NP213 lacked the enzyme CPEP phosphonomutase, which catalyzed the intramolecular rearrangement of CPEP.


Utilization and stability of vitamins in serum-containing and serum-free media in CHO cell culture.

The concentrations of four vitamins, ascorbic acid, nicotinamide, choline and thiamine were evaluated in the culture supernatant of Chinese hamster ovary (CHO) cells. The media used were alpha-modified Eagle's minimum essential medium (MEM-alpha) supplemented with 10% fetal calf serum, and a 1:1 mixture of Ham's F12 and Dulbecco's modified Eagle's medium (DME/F12), containing neither serum nor protein. The reference experiment without cells revealed instability of ascorbic acid and thiamine. Moreover, a significant amount of each vitamin decreased in the culture supernatant. The possibility of growth limitation by vitamin depletion is strongly suggested.


Structural analysis of repetitive DNA sequences in the goat growth hormone gene region.

Two clones that contain goat growth hormone (gGH) genes were isolated from goat genomic library using goat growth hormone cDNA as a probe. One clone CgGH contained gGH1 gene, and another clone, EgGH, contained gGH2 and gGH3 genes. The clone EgGH contained 491 bp inserted sequence, just upstream of the gGH3 gene, which was not present in the clone CgGH. From the sequencing data of the flanking regions of these gGH genes, short interspersed repetitive sequences (SINES) were found. Four SINES's existed in the CgGH clone and eight SINES's existed in the EgGH clone. The number of the repetitive DNA sequences in the goat genome was estimated about 10(2) copies from the analysis of the reassociation rate. As the number of gGH genes (gGH1, gGH2, and gGH3) per genome was different among individual goats, DNA fragments containing gGH1 gene, and that containing gGH2 and gGH3 genes, were estimated to be allelic on the goat chromosome.


The synthesis of new xylosyloligosaccharides by transxylosylation with Aspergillus niger beta-xylosidase.

The synthesis of xylosyloligosaccharides from beta-(1----4)-xylobiose in the presence of D-mannose by transxylosylation with beta-xylosidase from Aspergillus niger IFO 6662 was studied. At first, the transxylosylation products were separated by charcoal column chromatography in 5% ethanol elution into three peaks, P-1, P-2, and P-3. They were further separated on thin layer chromatography, and their three products, saccharides O-4, O-5, and O-N were purified by preparative paper chromatography. Saccharides O-4 and O-5 were identified as O-beta-D-xylopyranosyl-(1----4)-D- mannopyranose and O-beta-D-xylopyranosyl-(1----6)-D-mannopyranose, respectively, by measurement of the degree of polymerization, specific rotation, acid hydrolysis, and methylation analysis. By 1H-NMR spectroscopy in addition to these analyses, saccharide O-N was identified as O-beta-D-xylopyranosyl-(1----1')-beta-D-xylopyranose, a new nonreducing xylobiose.


Streptococcal antitumor protein: expression in Escherichia coli cells and properties of the recombinant protein.

Streptococcal antitumor protein (SAGP) was produced by transformed E. coli JM103 carrying the SAGP gene downstream from the tac promoter. The purified recombinant SAGP had the same N-terminal amino acid sequence as that of the native SAGP isolated from Streptococcus pyogenes Su cells. Gel filtration analysis showed that the recombinant SAGP was a dimer, while the native SAGP was a tetramer. When the antitumor activity was tested against sarcoma 180 cells, the IC50 of the recombinant SAGP was 0.3 microgram/ml, about a quarter as active as the native SAGP. These results suggest that the quaternary structure of SAGP is important for the antitumor activity.


New types of glycolipids from the surface lipids of Nicotiana umbratica.

The surface lipids of Nicotiana umbratica contained several varieties of glycolipids. Three types of glucose esters and five types of sucrose esters were isolated and identified from this species. These glycolipids contained short-chain fatty acids such as acetic, methylpropionic, methylbutyric, 3-methylpentanoic, 4-methylpentanoic and methylhexanoic acids. The acetylated positions of each compound were determined by 2-D NMR, using the HMBC technique. The structures of the glucose esters were 1,6-di-O-acetyl-2,3-4-tri-O-acyl-alpha-D-glucopyranose (I), 6-O-acetyl-2,3,4-tri-O-acyl-alpha-D-glucopyranose (II) and 2,3,4-tri-O-acyl-alpha, beta-D-glucopyranose (III). The structures of the sucrose esters were 6-O-acetyl-2,3,4-tri-O-acyl-alpha-D-glucopyranosyl-1,3-di-O-acetyl-beta- D- fructofuranoside (IV), 2,3,4-tri-O-acyl-alpha-D-glucopyranosyl-1,3-di-O-acetyl-beta-D-fructofur anoside (V), 6-O-acetyl-2,4-di-O-acyl-alpha-D-glucopyranosyl-3-O-acetyl-beta-D- fructofuranoside (VI), 2,3,4-tri-O-acyl-alpha-D-glucopyranosyl-3-O-acetyl-beta-D-fructofuranosi de 2,3,4-tri-O-acyl-alpha-D-glucopyranosyl-3-O-acetyl-beta-D-fructofuranosi de (VII) and 2,3,4-tri-O-acyl-alpha-D-glucopyranosyl-beta-D-fructofuranoside (VIII). Among them, I, IV, V and VI were isolated for the first time from plants of the family Solanaceae. These newly isolated glycolipids were inhibitory against barnyardgrass growth.


Inhibition of prolyl endopeptidase by synthetic peptide fragments of human beta-casein.

It has been suggested that peptide inhibitors of prolyl endopeptidase (PEP) may act as anti-amnestic agents. In the hope of finding PEP inhibitors in milk proteins, we synthesized a total of 37 human beta-casein peptide fragments containing proline residues. It was found that the peptides with PEP inhibition activity in vitro were located in the region of amino acid residues 49-59 of human beta-casein. The most potent inhibitor was Ile-Tyr-Pro-Phe-Val-Glu-Pro-Ile (IC50 = 8 microM).


Synthesis and antimicrobial activity of 2'-deoxypuromycin.

2'-Deoxypuromycin (2) was synthesized to learn the effect of the 2'-hydroxyl group on the biological activity. Acylated xylose 3 was condensed with silylated 6-chloropurine to give beta-D-xylofuranosyl-6-chloropurine derivative 4, whose 6-dimethylamination, 2'-deoxygenation and deprotection afforded 2'-deoxy-beta-D-xylofuranosyl purine analog 7. The latter was converted to 2'-deoxypuromycin (2) in 8 steps. 2'-Deoxy analog 2 showed only weak antimicrobial activity compared with that of puromycin (1).


Expression and secretion of bovine beta-lactoglobulin in Saccharomyces cerevisiae.

A bovine beta-lactoglobulin (beta-LG) was expressed in Saccharomyces cerevisiae carrying bovine pre-beta-LG cDNA and secreted into its growth medium. The expression plasmid was constructed by inserting the whole coding region of the cDNA encoding pre-beta-LG between the promoter and terminator of the yeast glyceraldehyde 3-phosphate dehydrogenase gene of pYG100, a yeast expression vector. In the supernatant of the yeast growth medium, beta-LG with a native conformation was detected by sandwich ELISA, and its amount was estimated to be 1.1 mg/l. A Western-immunoblotting analysis revealed that beta-LG secrected in the growth medium had the same mobility as that of authentic bovine beta-LG. The N-terminal sequence was also identical with that of authentic mature bovine beta-LG.


Solid-phase synthesis and crystallization of [Asn22, Gln25, Asn26]-A-chain-[Asn49, Glu50]-B-chain-monellin, an analogue of the sweet protein monellin.

The sweet protein monellin consists of two noncovalently associated polypeptide chains, the A chain of 44 amino acid residues and the B chain of 50 residues. The [Asn22, Gln25, Asn26]-A chain, an anologue of the A chain, was synthesized by the stepwise Fmoc-solid-phase method in an overall yield of 13.4%. The synthetic A chain analogue was combined with the [Asn49, Glu50]-B chain, which was left over from a previous work, to give [Asn22, Gln25, Asn26]-A-chain-[Asn49,Glu50]-B-chain-monellin in a yield of 26.2%. This synthetic monellin analogue was approximately 550 times sweeter than sucrose. Changing the carboxyl groups of Asp22, Glu25, and Asp26 of the A chain to amide groups significantly decreased the sweetness potency. Crystallization was performed by a vapor diffusion method.


Molecular cloning of cDNA encoding a human heat-shock protein whose expression is induced by adenovirus type 12 E1A in HeLa cells.

We have identified by differential plaque hybridization, human cDNA clones encoding a member of a heat-shock protein family (hsp 90 alpha) in the cDNA library of Adenovirus Type 12 E1A transfected HeLa cells. The complete nucleotide sequence of one of the clones (pHB76-114A) was identified. The sequence of 2912 base pairs had a single reading frame with a coding potential for an 84,672-Da protein. The amino acid sequence was highly homologous, but not identical, to that of the human hsp 90 alpha gene isolated from human peripheral blood lymphocytes [M. Yamazaki, K. Akaogi, T. Miwa, T. Imai, E. Soeda and K. Yokoyama, Nucleic Acids Res., 17, 7108 1989)]. This cDNA hybridized with RNA species which increased 5- to 20-fold upon heat shock and more than 5-fold in the differentiation stage of human Tera 2 cells.


Action pattern of Streptomyces griseus chitinase on partially N-acetylated chitosan.

Oligosaccharides produced during the course of the hydrolysis of 25% N-acetylated chitosan by Streptomyces griseus chitinase were fractionated by CM-Sephadex C-25 and Toyopearl HW-40F column chromatographies. Sugar compositions and sequences of main oligosaccharides were identified by N-acetylation, exo-splitting with beta-GlcNAcase and beta-GlcNase, and nitrous acid degradation. In addition to N-acetylated saccharides, GlcNAc, (GlcNAc)2, and (GlcNAc)3, hetero-chitooligosaccharides such as GlcN.GlcNAc, GlcN.GlcNAc.GlcNAc, GlcN.GlcN.GlcNAc, GlcN.GlcNAc.GlcNAc.GlcNAc, GlcNAc.GlcN.GlcNAc.GlcNAc, GlcN.GlcNAc.GlcN.GlcNAc, and GlcN.GlcN.GlcNAc.GlcNAc were identified. These results indicate that Streptomyces griseus chitinase specifically cleaves the N-acetyl-beta-D-glucosaminidic linkages in partially N-acetylated chitosan.


Purification and characterization of an antihypertensive compound from Lactobacillus casei.

Antihypertensive compounds were purified from an extract of autologous Lactobacillus casei cell lysates. The most effective compounds were polysaccharide-glycopeptide complexes, found in the cell wall. The average molecular weight was estimated as 180,000 from gel filtration using Sephacryl S-300. The polysaccharide moiety of the complexes consisted of glucose, rhamnose, and galactose, whereas the glycopeptide moiety consisted of N-acetylglucosamine, N-acetylmuramic acid, asparagine, glutamine, alanine, and lysine. The varieties of the components of these moieties were constant and independent of complex molecular size. When these complexes were orally administered to spontaneously hypertensive rats (SHR) and renal hypertensive rats (RHR) at doses of 1 mg/kg-body weight, systolic blood pressure (SBP) decreased by 10-20 mmHg 6 to 12 hr after administration without any change in heart rate. Appreciable hypotensive activity was lost by treating the complexes with hydrofluoric acid, which hydrolytically cleaves the phosphodiester bond between the polysaccharide and glycopeptide moiety.


Nucleotide sequence of the gene coding for the BanIII DNA methyltransferase in Bacillus aneurinolyticus.

The gene coding for the ATCGAT specific BanIII DNA methyltransferase (M-BanIII) of Bacillus aneurinolyticus was cloned and its nucleotides sequenced. The coding region was assigned on the nucleotide sequence on the basis of the N-terminal amino acid sequence and molecular weight of the enzyme. The M-BanIII gene coded for a protein of 580 amino acid residues (MW 66,344). Comparison with other methylases indicated that the M-BanIII sequence contained a segment of tetra-amino acids, NPPY, characteristic of N6-adenine methylases. In addition, some homologous regions were found in the sequences of type II adenine methylases PaeR7I(CTCGAG), TaqI(TCGA) and PstI(CTGCAG), containing TCGA within the recognition sequences.


Extracellular production of human tumor necrosis factor-alpha by Escherichia coli using a chemically-synthesized gene.

A DNA fragment of approximately 490 base pairs encoding human TNF was chemically synthesized and expressed within Escherichia coli cells. Furthermore, extracellular production of human TNF and several N-terminal deletion mutants of TNF was attempted using the excretion vector pEAP8. The TNF mutant with two N-terminal amino acids deleted (N delta 2-TNF) was efficiently excreted into the culture medium by E. coli carrying the plasmid pEXTNF3. In this clone, the signal peptide was correctly processed during the excretion. The E. coli-excreted N delta 2-TNF had higher antitumor activity than wild-type TNF or N delta 2-TNF produced intracellularly by E. coli.


Structures of sugar chains of water-soluble glycoproteins in developing castor bean cotyledons.

The structures of sugar chains from the water-soluble glycoproteins in developing castor beans have been identified. The structural analyses were done by a fluorescence method combined with exoglycosidase digestions and 1H-NMR spectroscopy. The identified structures fell into two categories; one was an oligomannose-type, the other xylomannose-type or xylose-containing type. Among these oligosaccharides, Man3Fuc1Xyl1GlcNAc2 (M3FX; 38%) and Man6GlcNAc2 (M6B; 22%) were the major structures. The higher mannose-content oligosaccharides (Man8-7GlcNAc2) were only 4.1%, and the further-modified structures (GNM3FX, M2FX) than M3FX were 22% of the total.


The complete amino acid sequence of antiviral protein from the seeds of pokeweed (Phytolacca americana).

The complete amino acid sequence of antiviral protein from the seeds of pokeweed (Phytolacca americana) has been determined. This has been done by the sequence analysis of peptides derived by enzymatic digestion with trypsin, pepsin, and lysylendopeptidase, as well as by chemical cleavage with cyanogen bromide. The protein consists of 261 amino acid residues containing two disulfide bonds and has a calculated molecular mass of 29167 Da. The two disulfide bonds connect Cys-34 to Cys-258 and Cys-84 to Cys-105. Comparison of this sequence with the sequence of the ricin A-chain shows that there are identical residues at 76 positions in the two molecules (30% identity), having an extended region of highly conserved sequence at positions 170-183 (IQMXSEAARFXYIE). In contrast, the internal regions at positions 77-119 and 141-169 and the C-terminal 15 amino acid residues are less homologous in the two proteins.


Survey of neutral aminopeptidases in bovine, porcine, and chicken skeletal muscles.

A survey of the total aminopeptidase activity of bovine, porcine, and chicken skeletal muscles at neutral pH was done, using the beta-naphthylamide derivatives of nine amino acids, DEAE-cellulose column chromatography of the muscle extract found at least four types of aminopeptidases in porcine and chicken muscles. Aminopeptidase B and aminopeptidase C were commonly recognized in bovine, porcine, and chickens muscles. Hydrolase H was recognized in porcine and chicken muscles. Aminopeptidase nC and hydrolase H had high activity against almost all substrates. The substrate specificities of both enzymes were fairly compatible with the pattern of free amino acids which increased during the storage of bovine, porcine, and chicken meats [Agric. Biol. Chem. 52, 2323 (1988)], indicating that aminopeptidase C and hydrolase H are responsible for the increment of free amino acids during aging of these muscles.


New pyrrolobenzodiazepine antibiotics, RK-1441A and B. II. Isolation and structure.

Antibacteriophage antibiotics, RK-1441A and B, related to neothramycin were isolated from the culture broth of Streptomyces sp. and their structures were deduced from spectroscopic analyses. The structure of RK-1441A was 8,11-dihydroxy-3,7-dimethoxy-5-oxo-1H-pyrrolo[2,1-c:1,4]benzodiazepine. RK-1441B is a tautomeric mixture at C-3 of the structure, 3,8,11-trihydroxy-7-methoxy-5-oxo-1H-pyrrolo[2,1-c:1,4]benzodiazepine.


Stereoselective total synthesis of wheat flour ceramide dihexoside.

A plant glycosphingolipid, O-(beta-D-mannopyranosyl)-(1----4)-O-(beta-D-glucopyranosyl)-(1----1)-(2 S,3S,4R)-4-hydroxy-N-tetracosanoylsphinganine 1, and the stereoisomer, O-(alpha-D-mannopyranosyl)-(1----4)-O-(beta-d-glucopyranosyl)-(1----1)-( 2S,3S,4R)-4-hydroxy-N-tetracosanoylsphinganine 6, were synthesized in a stereo- and regio-controlled way.


Toxicity of dimethyl sulfoxide as a solvent in bioassay system with HeLa cells evaluated colorimetrically with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide.

The toxicity of dimethyl sulfoxide (Me2SO) was examined in HeLa cells cultured at 37 degrees C for up to 72 hr. The growth of the cells was measured by a colorimetric method with the use of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), which gave good correlation between the cell number and the color development from the reduction of MTT under suitable conditions. When the initial number of cells was 3 x 10(4)/ml, Me2SO at 1% or less had no apparent effect on proliferation for up to 48 hr of incubation, but in longer incubations, cell growth was repressed. When the initial number of cells was 3 x 10(5)/ml, the effect of Me2SO was similar.


Complete amino acid sequence of luffin-a, a ribosome-inactivating protein from the seeds of sponge gourd (Luffa cylindrica).

The complete amino acid sequence of luffin-a has been determined. Twenty-two peptides were isolated from the tryptic digest of luffin-a and sequenced employing the DABITC/PITC double coupling method. Overlaping of these peptides was achieved by analyzing the chymotryptic peptides or CNBr-fragments of luffin-a and their S. aureus V8 protease peptides. Luffin-a consists of 248 amino acid residues and its relative molecular mass is calculated to be 27,021 Da, excluding the attached sugar chains reasoned to be present at each Asn residue of positions 28, 33, 77, 84, 206, and 227. A comparison with the sequence of ricin A-chain showed 33% sequence identity indicating that these proteins are homologous.


Promotive and inhibitory effects of raw starch adsorbable fragments from pancreatic alpha-amylase on enzymatic digestions of raw starch.

The enzymatically inactive but raw-starch-adsorbable peptide fragments designated as Gp-pan P and Gp-pan I were obtained from a tryptic digest of heat-inactivated hog pancreatic alpha-amylase. These two glycopeptide fragments were purified with Sephadex G-75, DEAE-Sephadex A-50, and HPLC and were found to be homogeneous on disc gel electrophoresis. Gp-pan P and I had molecular weights of 20,000 and 30,000 with SDS-PAGE, carbohydrate contents of 10% and 7%, N-terminal amino acids Gly-Trp and Ala-Val, and C-terminal amino acids Gly-Arg and Ile-Lys. Gp-pan P had promotive but Gp-pan I inhibitory effects on raw starch digestion by Aspergillus awamori var. kawachi glucoamylase I and Bacillus subtilis 65 alpha-amylase.


Purification and characterization of superoxide dismutase from Aerobacter aerogenes.

Superoxide dismutase (SOD) was purified from Aerobacter aerogenes IFO 3317 to an electrophoretically homogeneous state and partially characterized. SOD was purified by ammonium sulfate fractionation, column chromatography on DEAE-Sephadex A-50, gel filtration on Sephadex G-100, phenyl-Toyopearl 650 M hydrophobic chromatography, and hydroxyapatite adsorption chromatography. The molecular weight and subunit molecular weight of the purified enzyme were estimated to be 45,000 and 22,000, respectively. The isoelectric point of the enzyme was about pH 4.0. The purified enzyme remained stable at pH 7.0-11.0, 25 degrees C for 36 hrs, but was rapidly inactivated below pH 7.0. SOD was stable up to 35 degrees C at pH 7.0, but was inactivated at temperatures above that. The absorption maximum in the visible range was found at 360 nm, and the enzyme was insensitive to cyanide and fluoride, and sensitive to hydrogen peroxide and azide. These results suggest that the enzyme is an iron-containing SOD. The amino acid composition and the N-terminal sequence of the first 15 amino acids of the enzyme exhibited close homology with the other iron-containing SODs.


Relationship between the limited proteolysis of glycinin and its conformation.

The relationship between the limited proteolysis and conformation of native glycinin was studied by the following methods: analyses of the proteolytic digestion of chemically modified glycinin, circular dichroism (CD) measurements, a secondary structure prediction from the amino acid sequence and a hydropathy index analysis. The locations of tryptic fragments of glycinin were confirmed from the N-terminal amino acid sequences of the fragments. T fragments and P fragments were located at the N-terminal and central area of the acidic polypeptide chains, respectively. One of the cleavage sites was the Arg residue of around the 100th from the N-terminal side. The regions digested by limited trypsinolysis were presumed to be flexible, hydrophilic and near the surface of the molecule by prediction methods from the amino acid sequence. The other regions were predicted to be compact and beta-sheet in nature, almost 40-50% of the amino acid residues being predicted as beta-sheet from the amino acid sequence and from CD data.


Establishment and characterization of monoclonal antibodies against bovine ephemeral fever virus.

We established fourteen monoclonal antibodies (MAbs) reactive to bovine ephemeral fever virus YHL strain, and characterized six representatives including three IgG1s (YG3/4, YG5/8, and YG6/7) and three IgMs (YM4/9, YM2/6, and YM6/8). Among them, YG3/4 and YM4/9 gave especially strong reactivities to the virus. YM4/9 reacted specifically with a 43K antigen of the virus, corresponding to the matrix protein 1. The other MAbs reacted most strongly with the 43K antigen, but also reacted with unknown 23K and 21K antigens. By a simultaneous two-site method using YM4/9 and YG3/4, it was possible to detect 10(4.10)TCID50/ml of the virus, in the presence of serum.


Primary characterization of curved DNA segments cloned from Streptomyces DNA with extremely high G/C-contents.

Until recently, it was assumed that any short DNA segment could be regarded as a straight rod. Many instances, however, have been reported in which the helical axis was curved. In this study, to gain a general insight into the structural features of sequence-directed DNA curvatures, we constructed a set of plasmids carrying curved DNA segments, which were randomly cloned from Streptomyces total DNA with extremely high G/C-contents. The results of primary characterization of these curved DNA segments are presented. Although the cloned DNA segments had high G/C-contents, in all cases, several homopolymeric [dA] and [dT] stretches were found to be clustered around the centres of the determined nucleotide sequences. Furthermore, the clustered [dA] and [dT] stretches were located nearly in phase with the DNA helical screw. One of the DNA segments characterized in this study appeared to be derived from the giant linear plasmid, SCP1, and another from the circular fertility plasmid, SCP2.


Analysis by deletion and site-directed mutagenesis of promoters of the cell wall protein gene operon in Bacillus brevis 47.

The cell wall protein gene operon of Bacillus brevis 47 has multiple and tandem promoters. The precise locations of the two major promoters (P2 and P3) of the operon were determined by deletion analysis. This together with results of oligonucleotide-directed mutagenesis of the promoter regions confirmed that the -35 and -10 sequences of the two promoters proposed previously are essential for their promoter activity. A G + C-rich sequence upstream of the -35 region of P2 and an A-rich sequence upstream of the -35 region of P3 facilitate the transcription from P2 and P3, respectively.


The nucleotide sequences of Bacillus stearothermophilus ribosomal protein S12 and S7 genes: comparison with the str operon of Escherichia coli.

The primary structure of the ribosomal protein S7 from Bacillus stearothermophilus (BstS7) was analyzed by a combination of amino acid and DNA sequence analysis. Peptide sequence information was derived from tryptic peptides of BstS7 by manual Edman degradation. The nucleotide sequence of the 1.5-kb SalI fragment from B. stearothermophilus genome, cloned by the Escherichia coli S12 gene (rpsL) as a hybridization probe, was determined. Comparison of deduced amino acid sequence with the corresponding sequences of E. coli ribosomal proteins showed that this fragment contains the genes encoding S12, S7, and the N-terminus of the elongation factor G. Thus, the organization of this gene cluster is same as that in the str operon of E. coli. The amino acid sequence of B. stearothermophilus S12 (BstS12) deduced from the nucleotide sequence information agrees with the published amino acid sequence of BstS12 [M. Kimura and J. Kimura, FEBS Lett., 210, 91 (1987)]. The deduced sequence of B. stearothermophilus S7 (BstS7), together with sequence information of tryptic peptides, showed that protein S7 consists of 155 amino acid residues with a calculated molecular weight of 17933 and has 56% amino acid identity with the E. coli S7 (EcoS7).


Xylose (glucose) isomerase gene from the thermophile Clostridium thermohydrosulfuricum; cloning, sequencing, and expression in Escherichia coli.

The xylose isomerase gene from the thermophile Clostridium thermohydrosulfuricum has been cloned, using a fragment of the Bacillus subtilis gene as a probe. The complete nucleotide sequence of the gene was analyzed. C. thermohydrosulfuricum is the most thermophilic organism from which a xylose isomerase gene has been cloned and characterized. Comparison with amino acid sequences from other xylose isomerases showed that amino acids involved in substrate binding and isomerization are well conserved. Purification of the enzyme produced in E. coli was done by heating a cell-free extract at 85 degrees C for 10 min, giving a 20-fold purified enzyme. The native enzyme is a homomeric tetramer with a molecular weight of 200,000.


Complete amino acid sequence of luffin-b, a ribosome-inactivating protein from sponge gourd (Luffa cylindrica) seeds.

The complete amino acid sequence of luffin-b has been determined. All the twenty-seven tryptic peptides were isolated by reverse-phase HPLC from the tryptic digests of intact luffin-b and one of its CNBr fragments (CB4), and sequenced using the DABITC/PITC double coupling method. The overlap of these peptides was achieved by analyzing the CNBr fragments and their chymotryptic peptides. Luffin-b consists of 250 amino acid residues with a relative molecular mass of 27,275 Da. Investigation for glycosylation sites indicated that Asn at positions 2, 78, and 85 might carry sugars. Sequence comparison with luffin-a showed that amino acid substitution occurred in 55 positions. Luffin-b contains three glycosylation sites instead of the six sites in luffin-a, of which two were found to be conserved.


Purification and some properties of soybean saponin hydrolase from Aspergillus oryzae KO-2.

We had investigated the enzymatic hydrolysis of soybean saponins and selected soybean saponin hydrolase from Aspergillus oryzae KO-2. We attempted purification of this enzyme for further characterization. This enzyme was purified 1500-fold using ammonium sulfate fractionation and Sephadex G-200 gel filtrations. The enzyme was electrophoretically homogeneous and a glycoprotein by PAS staining. By gel filtration, the molecular weight of enzyme was 158,000 and SDS-PAGE showed the enzyme to have a tetrameric structure composed of heterogeneous subunits of 35,000 and 45,000. The enzyme activity was stable at temperatures below 40 degrees C and stable from pH 5.0 to 8.0. The optimum pH was pH 4.5 to 5.0 and the optimum temperature was 50 degrees C. The Km and Vmax for soyasaponin I were 0.48 mM and 9.8 mumol/hr mg protein, respectively. After hydrolysis with the enzyme, soyasapogenol B and alpha-L-rhamnopyranosyl (1----2)-beta-D-galactopyranosyl (1----2)-D-glucuronopyranoside were released from soyasaponin I.


Structure activity relationship of inhibitors specific for prolyl endopeptidase.

Structural requirements of N-blocked L-proline derivatives as specific inhibitors for prolyl endopeptidase were investigated using a series of substrate analogs. Replacement of L-proline by its D-isomer remarkably reduced the inhibition. Introduction of a sulfur atom in proline and/or in the penultimate pyrrolidine rings significantly increased the inhibition, but the introduction of oxygen rather diminished the activity. A peptide linkage (acid-amide bond) between the proline and the pyrrolidine ring was also required to keep the inhibitory activity. A benzyloxycarbonyl group was most effective as an N-blocked component of the inhibitors.


Isolation and sequencing of a gene, C-ADE1, and its use for a host-vector system in Candida maltosa with two genetic markers.

The host-vector systems of an n-alkane-assimilating-yeast, Candida maltosa, that we previously constructed consisted of a vector replicating with an ARS region of this yeast, and C. maltosa strains J288 (leu2) or CH1 (his5) as hosts. Since each of these hosts has a single genetic marker, we have developed a new host-vector system using two genetic markers. By UV irradiation of strain CH1, an adenine auxotrophic mutant, CHA1, forming red colonies was isolated. A DNA fragment complementing this deficiency was isolated from the C. maltosa genome. Since the DNA fragment also complemented the ade1 mutation of S. cerevisiae, we termed a gene contained in this DNA fragment C-ADE1. The nucleotides of C-ADE1 were sequenced. The deduced amino acid sequence (291 residues) had 65.6% homology with that of ADE1 of S. cerevisiae (306 residues). Having the cloned C-ADE1 DNA, we improved the host-vector system of C. maltosa.


Prothoracicotropic hormone of the silkworm, Bombyx mori: amino acid sequence and dimeric structure.

Prothoracicotropic hormone (PTTH) of the silkworm, Bombyx mori, was purified and its primary structure determined for the most part. From sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified PTTH under non-reducing and reducing conditions, and the amino acid sequence and composition analyses of the carboxamidomethylated PTTH, we concluded that Bombyx PTTH has a dimeric structure consisting of two identical, or nearly identical subunits, held together by disulfide bond(s). There exist subunit variants that differ by deletion of only a few amino acid residues at the N-terminus, and possibly at the C-terminus also, giving rise to a high heterogeneity in the PTTH molecule. The amino acid sequence up to the 104th residue from the N-terminus of the longest subunit, except for the 41st residue, was determined by sequence analysis of fragment peptides produced by lysyl endopeptidase, chymotrypsin and V8 protease digestions, leaving only a short C-terminal sequence undetermined. Bombyx PTTH is expected to contain a carbohydrate chain bound to an asparagine at position 41, deduced from the cDNA sequence.


Composition and structure of "group B saponin" in soybean seed.

The composition of "group B saponin" in soybean seed was analyzed by HPLC, and six kinds of "group B saponin," named Ba, Bb, Bb', Bc, Bd and Be according to elution order from HPLC, were detected. Of these saponins, Ba, Bb, Bb' and Bc were identified with soyasaponin V, I, III and II, respectively. Bd and Be were novel saponins possessing soyasapogenol E as the aglycone and the same sugar chain as Ba and Bb, respectively. These saponins were very unstable in the isolated state and had a tendency to form Ba and Bb, respectively. From these results, Bd and Be are presumed to be the precursors of Ba and Bb in soybean seed.


Cloning and characterization of the pectate lyase III gene of Erwinia carotovora Er.

A pectate lyase gene III (pel III) of Erwinia carotovora Er was cloned. The gene was expressed independently of a vector promoter in both E. carotovora Er and Escherichia coli. The pel III product was largely excreted in the culture medium of E. carotovora Er, while the product was only exported to the periplasmic space and was not excreted in the medium of E. coli. Nucleotide sequence analysis of pel III disclosed an open reading frame of 1,122 bp encoding a protein of 374 amino acids. The deduced amino acid sequence contained the N-terminal 30 amino acid sequence from the purified pectate lyase III (PL III) indicating the presence of a 22-amino-acid signal peptide. A putative ribosome-binding site was found to be 9 bp upstream of the start codon. The location of pel III was about 5.6 kb downstream of pel I. The PL III showed 80% homology in the amino acid sequence with the PL I of E. carotovora Er.


The glucoamylase cDNA from Aspergillus oryzae: its cloning, nucleotide sequence, and expression in Saccharomyces cerevisiae.

A cDNA for Aspergillus oryzae glucoamylase was cloned, using oligodeoxyribonucleotide probes derived from amino sequences of peptide fragments of the enzyme. The glucoamylase cDNA, when introduced into Saccharomyces cerevisiae, directed the secretion of active glucoamylase into the culture medium. The complete nucleotide sequence of the cDNA contained an open reading frame encoding 612 amino acid residues. Comparative studies with other fungal glucoamylases showed homologies of 67% with A. niger and 30% with Rhizopus oryzae of the deduced amino acid sequences. In the five conserved regions reported in other fungal glucoamylases, the levels of homologies between those regions of A. oryzae and A. niger enzymes were much higher (78-94%). A. oryzae glucoamylase contained no peptide region abundant in threonine and serine residue (TS-region), like that proposed to adsorb onto raw starch in A. awamori var. kawachii glucoamylase.


Purification and characterization of tissue plasminogen activator secreted by human embryonic lung diploid fibroblasts, IMR-90 cells.

The anti-urokinase-IgG-resistant plasminogen activator secreted by human embryonic lung diploid fibroblasts, IMR-90 cells (ATCC, CCL186) was purified to homogeneity from serum-free conditioned medium by a four-step procedure. The fibroblast plasminogen activator was identified as tissue plasminogen activator (t-PA) by the N-terminal sequence of the purified material and the complete amino acid sequence deduced from its complementary DNA (cDNA). The apparent molecular weight was the range of 64,000 to 68,000 by SDS-PAGE and was in the range of 69,000 to 72,000 by gel filtration. The fibroblast t-PA showed a stricter substrate specificity than urokinase in enzymatic hydrolysis of various chromogenic substrates. Compared to urokinase, the fibrobrast t-PA was more stable by heating at 95 degrees C for five min and was stable from pH 5 to 10. The fibrorast t-PA had a higher affinity for fibrin than urokinase.


Cloning and expression of cDNA coding for the major house dust mite allergen Der f II in Escherichia coli.

A cDNA library corresponding to mite protein was screened using anti-Der f II, a major allergen from the house dust mite Dermatophagoides farinae, antibody. Three possible clones were obtained that contained cDNA fragments coding for Der f II, and the nucleotide sequences of the fragments were determined. There were minor differences observed affecting the deduced amino acid sequence among the three cDNA fragments. The amino acid sequence of the purified native Der f II protein could be analyzed to 45 residues from the N-terminus. As a result of comparison, all the three cDNA fragments code for a mature protein with a derived molecular weight of about 14,000. The amino acid sequence was not homologous to any known protein sequences and it contained six cysteine residues and no N-glycosylation sites.


Cloning and expression of the sarcosine oxidase gene from Bacillus sp. NS-129 in Escherichia coli.

The gene coding for a thermostable sarcosine oxidase (EC 1.5.3.1) was isolated from Bacillus sp. NS-129. The primary structure of sarcosine oxidase deduced from the nucleotide sequence was a protein composed of 387 amino acids with molecular weight 42,955. When the sarcosine oxidase was overproduced to about 35% of soluble protein in E. coli under the control of a lac promoter, the sarcosine oxidase activity of the crude extract was increased 3-fold by the addition of FAD. This indicates that most of the enzyme is accumulated in an active form, a flavinless aporotein, in the cell.


Structures and activity of angiotensin-converting enzyme inhibitors in an alpha-zein hydrolysate.

Peptides that inhibit angiotensin-converting enzyme (ACE) were isolated from alpha-zein hydrolysate prepared with thermolysin. Their chemical structures were identified by Edman degradation and fast-atom bombardment mass spectrometry. Most of them were found to be tripeptides such as Leu-Arg-Pro, Leu-Ser-Pro, and Leu-Gln-Pro, having IC50 values of 0.27, 1.7, and 1.9 microM, respectively. These peptides were synthesized by a solid phase procedure and had similar ACE inhibitory activities as the isolated inhibitors. The hypotensive activity of Leu-Arg-Pro on spontaneously hypertensive rats was also investigated, with the result that the the blood pressure decreased by 15 mmHg after a 30 mg/kg intravenous injection.


N-acetyl-D-glucosamine-asparagine structure in ribosome-inactivating proteins from the seeds of Luffa cylindrica and Phytolacca americana.

Glycosylation-site-containing peptides were isolated from the proteolytic digests of luffin-a, luffin-b, PAP-S and CNBr-fragments of PAP-S by reverse-phase HPLC, and their amino acid compositions and sequences were analyzed. Six peptides were obtained from luffin-a, and three each from luffin-b and PAP-S. All of these peptides were negative toward the phenol-H2SO4 reaction and gave only N-acetyl-D-glucosamine in gas chromatography after methanolysis and reacetylation. Amounts of N-acetyl-D-glucosamine in these peptides were determined as D-glucosamine to be approximately one mol per peptide by an amino acid analyzer after HCl hydrolysis. Based on these results we concluded that Asn residues at positions of 28, 33, 77, 84, 206, and 227 in luffin-a, of 2, 78, and 85 in luffin-b, and of 10, 44, and 255 in PAP-S were glycosylated with only GlcNAc, and contained one residue per site.


Cloning, sequencing, and characterization of the intracellular invertase gene from Zymomonas mobilis.

The structural gene for the intracellular invertase E1 of Zymomonas mobilis strain Z6C was cloned in a 2.25-kb DNA fragment on pUSH11, and expressed in Escherichia coli HB101. The enzyme produced by the E. coli carrying pUSH11 was purified about 1,122 fold to homogenicity with a yield of 4%. The molecular weight and substrate specificity of the enzyme were identical with those of the intracellular invertase E1 from Z. mobilis. The nucleotides of the cloned DNA were sequenced; they included an open reading frame of 1,536 bp, coding for a protein with a molecular weight of 58,728. The N-terminal amino acid sequence predicted was identical with the sequence of the first 20 N-terminal amino acid residues of the protein obtained by Edman degradation. Comparison of the predicted amino acid sequence of E1 protein with those of the four other known beta-D-fructofuranosidases from Escherichia coli, Bacillus subtilis, and Saccharomyces cerevisiae indicated a stronger homology in the N-terminal portion than in the C-terminal portion.


Purification and some properties of protease I having transfer action from Streptomyces griseus var. alcalophilus.

Streptomyces griseus var. alcalophilus was selected because it secreted a unique protease (protease I) that catalyzed the transfer reaction forming the hydroxamic acids of various amino acids. Protease I was purified to the electrophoretically homogeneous state and an activity of more than 125-fold that of the culture broth. The molecular weight of the enzyme was estimated to be 25,000 by gel filtration. The enzyme was most active in neutral pH for the transfer reaction forming phenylalanine hydroxamic acid, although for the hydrolytic reaction with casein as substrate it was most active in alkaline pH. The enzyme was inhibited by diisopropylfluorophosphate. Protease I catalyzed the transfer reaction synthesizing the hydroxamic acids of hydrophobic, acidic, basic, and small aliphatic amino acids such as Phe, Tyr, Leu, Asp, Glu, Arg, Lys, Ala, and Gly. These results indicate that protease I has broad donor specificity. It is also considered that protease I is a unique enzyme with transfer activity, and distinct from the alkalophilic proteinase reported previously [Yamamoto et al., Agric. Biol. Chem., 38, 37 (1974)].


Nucleotide sequence of celC307 encoding endoglucanase C307 of Clostridium sp. strain F1.

The celC gene, encoding endoglucanase C, of Clostridium thermocellum was recently sequenced while a promoter region was not identified. In this study, the nucleotide sequence of celC307 of Clostridium sp. strain F1 was identified and compared with that of the C. thermocellum gene. The open reading frame was composed of 1029 nucleotides and the deduced amino acid sequence corresponded to a protein of a molecular weight of 40,905. We identified promoter sequences (TGGACA and TATAAT) at a position about 150 nucleotides upstream of the initiation codon. Six substitutions were found in the coding region, 3 leading to amino acid replacements. Five substitutions and 1 deletion of a nucleotide were found in the region upstream of the initiation codon, 1 present at the promoter sequence. Overproduction of endoglucanase C307 (EGC307) in Escherichia coli strongly inhibited the cell growth of the host strain. Around 50% of EGC307 produced in E. coli was detected in the periplasmic fraction. The N-terminal amino acid sequence suggested that this protein was exported into the periplasm without processing of a signal peptide.


Production and localization of enzymes on soft gel cultivation.

Production and localization of glutaminase and leucine aminopeptidase (LAP) in soft gel cultivation were compared with those in koji and liquid cultivations. The enzymes were detected only in the whole-mycelial-mat fraction by soft gel cultivation, but in both intracellular and extracellular fractions by the other two methods. The enzyme species of glutaminase and LAP in soft gel cultivation were analyzed by ion exchange and gel filtration column chromatographies. Three species of glutaminase and four (or five) species of LAP were formed in the whole-mycelial-mat fraction. The intracellular and extracellular fractions of the koji and liquid cultivations contained different species of enzymes.


Purification and primary structure of C-1027-AG, a selective antagonist of antitumor antibiotic C-1027, from Streptomyces globisporus.

C-1027-AG, a selective antagonist of antitumor antibiotic C-1027, was isolated by column chromatography on DEAE-cellulose, butyl-Toyopearl and Sephadex G-50 from a culture filtrate of Streptomyces globisporus. The amino acid sequence of purified C-1027-AG was determined with a protein sequencer on the basis of fragment peptides obtained by enzymatic hydrolysis with lysylendopeptidase, V8 protease, endopeptidase AspN and chymotrypsin, after performic acid oxidation. C-1027-AG is shown to consist of a single polypeptide chain cross-linked by two disulfide bonds, and to contain a total of 110 amino acid residues with alanine and glycine as its amino- and carboxyl-termini, respectively; its molecular weight was calculated to be 10,500 daltons. The primary structure of C-1027-AG is indicated to be identical to the protein moiety of C-1027, and is highly homologous to the sequences of antitumor proteins obtained from other Streptomyces species.


Purification, characterization, and amino acid sequence of foxtail millet trypsin inhibitor III.

One of the major trypsin inhibitors of foxtail millet, Setaria italica, was purified from a seed extract to an electrophoretically homogeneous protein by methods including chromatofocusing and affinity chromatography. This inhibitor (FMTI-III) was shown to be specific and single-headed for trypsin. The molecular weight and the amino acid composition together with the above nature were identical with those of another major trypsin inhibitor (FMTI-II) previously purified from foxtail millet grain. Sequence analysis of FMTI-III indicated that the protein contains 67 amino acid residues, the sequence of which is the same as that of FMTI-II except for the replacement of the C-terminal glutamine by glutamic acid. This single amino acid substitution had no effect on inhibitor-enzyme association.


Characterization and structure of the cellulase gene of Bacillus subtilis BSE616.

The Bacillus subtilis carboxymethyl cellulase (CMCase) gene originally cloned on a 3.2-kb PstI DNA fragment has been localized in a 1.5-kb Sau3AI fragment by a series of subclonings into plasmid pUC19. During the process the promoter region and Shine-Dalgarno (SD) sequence were deleted, but the 1.5-kb insert was shown to direct the synthesis of CMCase in scherichia coli to a high level, probably with the aid of lac promoter. We analyzed the complete nucleotide sequence of the CMCase gene. The CMCase gene is 1500-bp long, encoding a polypeptide of 499 amino acids and a stop codon. The putative "-35" region (TAGACA), "-10" region (TACAAT), and ribosome binding site (RBS) (AAGGAGG) have also been identified in the 5' flanking region. Comparison of the nucleotide sequence to three other published endo-beta-1,4-glucanase genes of B. subtilis strains shows that these sequences share very strong homology. It seems that the cellulase genes have been derived from a common ancestor by spontaneous mutation. The probability of carboxy-terminal processing of the CMCase protein is also discussed.


Further study on the 1,4-alpha-transglucosylation of rubusoside, a sweet steviol-bisglucoside from Rubus suavissimus.

Rubusoside (the beta-D-glucosyl ester of 13-O-beta-D-glucosyl-steviol), which is the major sweet principle of leaves of Rubus suavissimus S. Lee, was subjected to 1,4-alpha-transglucosylation by the cyclodextringlucanotransferase-starch system (the CGTase system). The tri- and tetra-glucosylated products were isolated together with the mono- and di-glucosylated products, which had already been isolated. A prominent increase in intensity of the sweetness was observed for the compounds which were di- and tri-glucosylated at the 13-O-glucosyl moiety. This result further substantiated the structure-sweetness relationship for 1,4-alpha-glucosylated compounds of steviol-glycosides reported previously. For protection of the 19-COO-glucosyl moiety against glucosylation by the CGTase system, the 4-hydroxyl group of the 19-COO-glucosyl moiety was beta-galactosylated by the beta-galactosidase-lactose system. This galactosylated compound was subjected to a regio-selective glucosylation of the 13-O-glucosyl moiety by the CGTase system, which was followed by enzymic elimination of the galactosyl group to furnish an exclusive preparation of the improved sweeteners just mentioned.


Cloning and nucleotide sequences of the complementary and genomic DNAs for the alkaline protease from Acremonium chrysogenum.

Complementary DNA encoding Ac. chrysogenum alkaline protease (Alp) was isolated from the Ac. chrysogenum ATCC11550 cDNA library by express-blot assay. The genomic DNAs encoding Ac. chrysogenum Alp were isolated from the Ac. chrysogenum genomic DNA library using the cloned cDNA as a probe. The 3150 nucleotides of the gene were sequenced. The prepro-Alp consists of 402 amino acids and two intervening sequences are found within the coding region. The amino acid sequence of Ac. chrysogenum Alp has 57% homology to that of Aspergillus oryzae Alp. The entire cDNA, encoding Ac. chrysogenum Alp, when introducing into the yeast Saccharomyces cerevisiae, directed the secretion of enzymatically active Alp into the culture medium.


Purification and properties of purine nucleoside phosphorylase from Brevibacterium acetylicum ATCC 954.

Purine nucleoside phosphorylase of Brevibacterium acetylicum ATCC 954, which catalyzes the production of ribavirin (1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide), a potent antiviral agent, from purine nucleoside and 1,2,4-triazole-3-carboxamide in a high yield, was purified 49-fold. This enzyme had a molecular weight of 31,000 and was a monomer. The isoelectric point of the enzyme was 4.7. The optimal temperature and pH of inosine phosphorolyzing reaction catalyzed by the enzyme was around 8.5 and 70 degrees C, respectively. The Michaelis constants for inosine, guanosine, and ribavirin were 1.43 mM, 2.44 mM and 2.08 mM, respectively, at 40 degrees C. This enzyme appeared to be a SH enzyme because it was inactivated by SH reagents, p-chloromercuribenzoate and N-ethylmaleimide, and HgCl2. In addition, this enzyme was completely inactivated by AgNO3 and was slightly inhibited by CuSO4. It showed nucleoside-phosphorolyzing activity toward inosine, 2'-deoxyinosine, 2',3'-dideoxyinosine, guanosine, 2'-deoxyguanosine, and xanthosine. However, adenosine and its derivatives could not be phosphorolyzed. This enzyme could not also phosphorolyze various 5'-mononucleotides. According to the amino terminal sequence analysis, the twenty residues from the amino terminal end of this enzyme were identified as follows: MTVNWNETRS-FLECKMQAKPE.


Solid-phase synthesis of crystalline monellin, a sweet protein.

The sweet protein, monellin, consists of two noncovalently associated polypeptide chains, the A chain of 44 amino acid residues and the B chain of 50 residues. The B chain was synthesized by the stepwise Fmoc solid-phase method in an overall yield of 6.2%. The synthetic B chain was combined with the synthetic A chain, which was left over from a previous work, to give monellin in a yield of 25.7%. The synthetic monellin was approximately 4000 times as sweet as sucrose, while the previously synthesized [Asn49, Glu50] B-chain monellin was also approximately 4000 times as sweet as sucrose. Exchanging Glu49 and Asn50 in the B chain did not affect the sweetness potency. Crystallization was performed by a vapor diffusion method.


Enhanced production of antibiotics by Pecilomyces lilacinus under alkaline conditions associated with morphological change.

The production of the group of peptide antibiotics called No. 1907 by Paecilomyces lilacinus was stabilized and enhanced by mono-spore isolation followed by strain improvement, and the production seemed to be related to a morphological change of the strain. Unstable and a low productivity (below 10 micrograms/ml) of the antibiotics No. 1907 by P. lilacinus was greatly improved up to 140 micrograms/ml by successive mono-spore isolation along with mutagenization. Alkaline conditions caused by adding Na2CO3, K2CO3, NaHCO3 or KHCO3 were essential to obtain high production of the antibiotics following simultaneous morphological change of the cells from filamentous mycelia to round arthroconidia. Resting cell experiments showed that a significant amount of antibiotics (213 micrograms/ml) was synthesized by the filamentous cells along with their morphological change to arthroconidia in the presence of glucose under the alkaline conditions.


Identification of Mycoplasma hyopneumoniae with a DNA probe.

From a genomic library of Mycoplasma hyopneumoniae a 1.3 kb DNA fragment was cloned which showed specific Southern hybridization and dot hybridization with the type strain of several porcine and bovine Mycoplasma species. This probe selectively recognized M. hyopneumoniae sequences in purified DNA or in broth-grown organisms. The 35S-labelled probe could detect as little as 100 pg of DNA or 10(5) colour changing units. This is a possible alternative diagnostic procedure for enzootic pneumonia of pigs.


Lysosomal enzymes produced by immobilized Tetrahymena thermophila.

The ciliated protozoon Tetrahymena thermophila was immobilized for production of secreted lysosomal enzymes in two ways. Cells entrapped in solid Ca-alginate spheres survived but were unable to grow and multiply. However, when encapsulated in hollow Ca-alginate spheres Tetrahymena multiplied well, reaching 0.9 x 10(7) cells/ml. These immobilized cells secreted large amounts of lysosomal enzymes when the medium was changed daily. This system was transferred to a reactor scale using a conical bubble column reactor for semicontinuous cultivation of the encapsulated cells. Under these conditions alpha-glucosidase, beta-glucosidase, beta-hexosaminidase and acid phosphatase were produced for at least 4 weeks. The hollow spheres were stable for 3 months and contained living and secreting Tetrahymena cells during this time. Immobilized T. thermophila cells can thus serve as a good source for production of commercially interesting enzymes.


High-performance liquid chromatographic measurement of tryptophan in blood, tissues, urine, and foodstuffs with electrochemical and fluorometric detections.

A rapid and convenient measurement of tryptophan in whole blood, serum, liver, brain, urine, and alkaline hydrolysates of proteins and foodstuff was done by high-performance liquid chromatography. The sample preparation was simply homogenized or mixed in a 5% trichloroacetic acid solution and a sample of the supernatant was injected onto a column after filtration with a 0.45-micron filter. The method used a Chemcosorb 5-ODS-H column (particle size, 5 microns, 150 x 4.6 mm i.d.) eluted with 20 mM potassium dihydrogen phosphate (pH adjusted to 3.7 by the addition of phosphoric acid) containing 1 g/l of sodium heptane sulfonate and 3 mg/l EDTA.2Na-acetonitrile (93:7, v/v) at a flow rate of 1.5 ml/min. The tryptophan contents in whole blood, serum, liver, and brain were electrochemically estimated at +1000 mV vs. Ag/AgCl, the detection limit being 0.2 pmol (40.84 pg) at a signal-to-noise ratio of 5:1. The tryptophan contents in urine, proteins, and foodstuff were fluorometrically estimated with an excitation wavelength of 280 nm and with an emission wavelength of 340 nm, the detection limit being 20 pmol (4.08 ng) at a signal-to-noise ratio of 5:1. Tryptophan was eluted at about 10.5 min. The total analysis time was about 12 min.


Cloning and nucleotide sequences of the AccI restriction-modification genes in Acinetobacter calcoaceticus.

The genes of the AccI restriction-modification system specific for GT(A/C) (G/T)AC were cloned from the chromosomal DNA of Acinetobacter calcoaceticus, and their nucleotides sequenced. The restriction and modification genes coded for polypeptides with calculated molecular weights of 42,494 and 63,078, respectively. Both the enzymes were coded by the same DNA strand and the restriction gene was upstream of the methylase gene, separated by 2 bp. The restriction gene was significantly expressed in E. coli cells, so that the AccI restriction endonuclease could be purified to homogeneity. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration indicated that the catalytically active form of the endonuclease was tetrameric. Sequence comparison with related enzymes indicated that AccI methylase contained a segment of tetra-amino acids, NPPY, characteristic of N6-adenine methylases. In addition, some homologous regions were found in the sequence of HincII methylase specific for GT(C/T) (A/G)AC.


Signal peptide of the colicin E2 lysis protein causes host cell death.

Colicin E2 is encoded by the SOS-inducible colicin operon of plasmid ColE2-P9. Highly induced colicin E2 is excreted from the E. coli host cell, accompanied by quasi-lysis and cell death. These three events are all directed by the colicin lysis protein that comprises the signal peptide and a small mature lipoprotein. We constructed a mutant lysis gene encoding its complete signal peptide with addition of two external amino acids at the C-terminus. By using this mutant peptide as a reference, the wild type lysis signal peptide was identified and shown to remain quite stable after the cleavage. This mutant peptide also mimics the cleaved-off signal peptide avoiding the influence of the mature lysis lipoprotein, and suggested that cell death by the lysis protein, but neither colicin excretion nor quasi-lysis, is decided by the nature of its signal region.


Molecular cloning and analysis of nucleotide sequence of the Bacillus subtilis lysA gene region using B. subtilis phage vectors and a multi-copy plasmid, pUB110.

A 3.8-kb EcoRI-fragment containing the lysA gene [diaminopimelate (DAP) decarboxylase] of Bacillus subtilis has been cloned into B. subtilis phage phi 105 and its nucleotides sequenced. The nucleotide sequence of a 3,762 bp stretch contained three open reading frames (ORF1, ORF2, and ORF3) in one orientation and another open reading frame (ORF4) in the opposite orientation. ORF2 coded for the lysA gene based on the complementation of a B. subtilis lys auxotroph and on the fact that the predicted amino acid sequence (440 amino acids with a molecular weight of 48,876) of ORF2 shared a 29.7%, 38.3%, and 32.9% identity with the sequences of Escherichia coli, Corynebacterium glutamicum and Pseudomonas aeruginosa lysA genes, respectively. ORF1, ORF3, and ORF4 did not correspond to E. coli lysR. Based on the comparison of the B. subtilis lysA sequence with a sequence of the DAP-decarboxylase gene cloned into pUB110 (Yamamoto et al., Nucleic Acids Res., 17, 10105 (1989], it was found that the lys gene in the plasmid was fused with the dnaN gene in its COOH-terminal region.


Analysing lymphokine-receptor interactions of IL-1 and IL-2 by recombinant-DNA technology.

The development of lymphokines as pharmaceutical agents is at the forefront of current biotechnological research. Lymphokines exert their regulatory action on cellular function through interaction with cell-surface receptors. An understanding of the biochemical nature of these molecular interactions should facilitate the design of small peptide analog pharmaceuticals which mimic lymphokines or their receptors.


Enzymatic properties of dipeptidyl carboxypeptidase from Bacillus pumilus.

Enzymatic properties of dipeptidyl carboxypeptidase (DCP) from Bacillus pumilus were investigated. The enzyme was more active on tri- and tetrapeptides than angiotensin-converting enzyme (ACE) from rabbit lung. The presence of chloride ion is essential for the hydrolysis. The Km value of angiotensin I for the enzyme was 0.119 x 10(-3) M. The enzyme was not inhibited by the mammalian ACE inhibitors lisinopril and enalaprilat. The enzyme is readily inhibited by EDTA but restored by Co2+, Mn2+, and Zn2+. Therefore, it seems to be a zinc-metallo protease.


Cloning and sequencing of a cyclodextrin glucanotransferase gene from Bacillus ohbensis and its expression in Escherichia coli.

A cyclodextrin glucanotransferase (CGTase) gene of Bacillus ohbensis was cloned in Escherichia coli and the nucleotide sequence was determined. A single open reading frame (2112 bp) with a TTG codon as an initiator was identified that encodes a typical signal peptide of 29 amino acids followed by the mature enzyme (675 amino acids), of which the partial amino acid sequences of the N-terminal region and some lysyl-endopeptidase fragments were determined by Edman degradation. The CGTase gene was expressed in E. coli under control of the lac promoter only when the upstream region containing a long inverted repeat structure (located at -108 to -67 bp from the initiation codon) was deleted. Substitution of an ATG codon for the initiation TTG triplet doubled the expression of the CGTase gene in E. coli. Enzyme preparations purified from the culture supernatant of B. ohbensis and from the periplasmic fraction of the E. coli transformant exhibited the same molecular weight (Mr) and enzymatic properties as follows: Mr, 80,000; optimum pH for activity, 5.0 (and a suboptimum at 10.0); stability between pH 6.5 and 10.0; optimum temperature for activity, 55 degrees C; and stability below 45 degrees C. The yields of the products from starch as the substrate were 25% for beta- and 5% for gamma-cyclodextrin.


The amino acid sequence of lysozyme from kalij pheasant (Lophura leucomelana) egg-white.

The amino acid sequence of kalij pheasant lysozyme has been analyzed. From the comparison of the tryptic peptide pattern of kalij pheasant lysozyme and maps from other bird lysozymes followed by the sequencing of tryptic peptides, the amino acid sequence of kalij pheasant was found to be: KVYGRCELAAAMKRLGLDNYRGYSLGNWVCAAKYESNFNTHATNRNTDGSTDYGIL- QINSRWWCNDGKTPGSRNLCHIPCSALLSSDITASVNCAKKIVSDGNGMNAW- VAWRNRCKGTDVSVWTRGCRL. This sequence had 9 amino acid substitutions compared with hen egg-white lysozyme. Two of these substitutions, positions 34 and 121, were newly detected in phasianid birds. The protein genealogy of phasianid bird lysozymes showed some discordance with the morphological classification of these birds.


The amino acid sequence of reeves' pheasant (Syrmaticus reevesii) lysozyme.

The amino acid sequence of reeves' pheasant lysozyme was analyzed. Carboxymethylated lysozyme was digested with trypsin and resulting peptides were analyzed using the DABITC/PITC double coupling manual Edman method. The established amino acid sequence had seven substitutions, Tyr3, Leu15, His41, His77, Ser79, Arg102, and Asn121, compared with hen egg-white lysozyme. Ser79 was the first found in a bird lysozyme. A substitution in the active site was found in position 102 which has been considered to participate in the substrate binding at subsites A-C.


Purification and some properties of a thermostable metal proteinase produced by Thermomicrobium sp. KN-22 strain.

An extreme thermophile that produces a heat-stable proteinase was isolated from hot-spring water and classified as Thermomicrobium sp. KN-22 (growth temperature, 50-83 degrees C; and optimum growth temperature, 70 degrees C). The proteinase was purified from the culture broth of this strain by fractionation with ammonium sulfate, chromatography on columns of DEAE-cellulose and CM-Sepharose CL-6B, and HPLC on TSKgel CM-5PW. The purified enzyme gave a single band on SDS-polyacrylamide gel electrophoresis and a single peak after HPLC (yield 8.8%). The enzyme had maximum activity at pH 8.5 and at 75 degrees C and it was stable up to 60 degrees C. The molecular weight of the enzyme was 35,000 by SDS-PAGE. Since the enzymatic activity was completely inhibited by EDTA, o-phenanthroline, and phosphoramidon, it appears that the enzyme is a metal proteinase.


Characterization of the leader peptide of an endo-type cellulase produced by an alkalophilic Streptomyces strain.

An endo-type semi-alkaline cellulase (CMCase I) produced by an alkalophilic Streptomyces strain has an extraordinarily long leader peptide of about 70 amino acids (aa), which can be grouped into four distinct regions, an NH2-terminal region (13 aa), an Arg-cluster region (13 aa), a hydrophobic region (23 aa), and an Ala/Pro-repeat region (12 aa). For identification of the function of each part of the leader peptide for secretion of the enzyme, mutation in the leader peptide were generated by site-directed mutagenesis using the cloned gene, and the mutant genes were expressed in a cellulase-negative mutant strain, Streptomyces lividans HN1. Although all the alterations in the leader peptide, except for one case, decreased secretion to various extents, in S. lividans, the following conclusions were obtained. Comparison of the intra- and extra-cellular enzyme activities of the mutants suggested that the Arg-cluster region was essential in secretion of the cellulase. Deletion of 8 amino acids rich in threonine and proline in the NH2-terminal region enhanced the secretion to a small extent. Deletion of the Ala/Pro repeat region had almost no effect on secretion.


Evidence that more than one gene encodes n-alkane-inducible cytochrome P-450s in Candida maltosa, found by two-step gene disruption.

An n-alkane-assimilating yeast, Candida maltosa, has a diploid genome. Probed with the previously isolated gene of n-alkane-inducible cytochrome P-450 (P-450alk), its allelic gene had been isolated, its nucleotides sequenced, and the interallelic divergence examined. Using one of the allelic genes, we disrupted the two alleles of the cytochrome P-450alk gene by a two-step gene disruption system. Surprisingly, the disruptant still assimilated n-alkane and contained n-alkane-inducible cytochrome P-450. This result indicates that, other than the disrupted two alleles, there is at least one other gene that encodes an n-alkane-inducible cytochrome P-450.


Purification and properties of aminopeptidase C from chicken skeletal muscle.

Aminopeptidase C was purified from fresh chicken skeletal muscle by ammonium sulfate fractionation, and by successive chromatography on DEAE-cellulose, Ultrogel AcA 34, DEAE-cellulose again, and an alanine AH-Sepharose 4B affinity column twice. The purified enzyme migrated as a single band by SDS-PAGE. Aminopeptidase C was purified about 300-fold over the crude extract with a yield of 0.6%. The molecular weight of this enzyme was found to be 185,000 by gel filtration in a Sepharose 6B column and 92,000 by SDS-PAGE. The optimum pH for the hydrolysis of L-leucine beta-naphthylamide was 6.0-7.0, the enzyme being stable in the range of pH 6.5-8.0. The activity of this enzyme was strongly inhibited by EDTA and puromycin, and was high against the beta-naphthylamide derivatives of Lys, Leu, Ala and Met. The enzyme was more active towards tri- and tetrapeptides than towards dipeptides.


Purification and some properties of sucrose phosphorylase from Leuconostoc mesenteroides.

Sucrose phosphorylase (EC 2.4.1.7) was purified to homogeneity from Leuconostoc mesenteroides cells with a specific activity of 173.8 units per mg protein by ammonium sulfate fractionation, anion exchange HPLC on TSKgel DEAE-5PW, and hydrophobic HPLC on TSKgel Ether-5PW. The purified enzyme was an acidic protein having an isoelectric point of pH 4.6 and s0(20),W of 4.34S. The molecular weight of this enzyme was estimated to be 56,400 by sedimentation equilibrium, 55,000 by SDS-polyacrylamide gel electrophoresis, and HPLC gel filtration on TSKgel G3000SW, suggesting that the enzyme is a monomeric protein. With regard to molecular weight, amino acid composition, and N-terminal amino acid sequence of 30 residues, this enzyme is close to the glucosyltransferase A of Streptococcus mutans.


Efficient production of Taka-amylase A by Trichoderma viride.

An efficient heterologous protein production system was developed in Trichoderma viride, a very efficient cellulase producer. An expression vector containing the Taka-amylase A gene from Aspergillus oryzae, which was fused to the strong promoter and signal peptide sequence of the cellobiohydrolase 1 gene (cbh1) of T. viride, and the hygromycin B resistance gene was used to transform protoplasts of T. viride. Using hygromycin B resistance, a frequency of 3 transformants per microgram DNA on average was obtained. One transformant showed highly elevated alpha-amylase production, 1.0 g/l, which was shown to be under the control of the cbh1 gene promoter. Analysis of the chromosomal DNA of the transformant showed the integration of more than one copy of the vector.


Solid-phase synthesis of crystalline [Ser41] B-chain monellin, an analogue of the sweet protein monellin.

The sweet protein monellin consists of two noncovalently associated polypeptide chains, the A chain of 44 amino acid residues and the B chain of 50 residues. Since blocking the free SH group of Cys41 in the B chain or treating the adjacent Met42 with CNBr removed its sweetness, this part of the molecule has been suggested to be essential for the sweetness. The [Ser41] B chain, an analogue of the B chain, was synthesized by the stepwise Fmoc solid-phase method in an overall yield of 1.9%. The synthetic B chain analogue was combined with the A chain, which was left over from a previous work, to give [Ser41] B-chain monellin in a yield of 31.0%. This synthetic monellin analogue was 2000 times as sweet as sucrose. Changing the Cys41 residue to the Ser residue significantly decreased the sweetness potency and stability of the molecule in solution. Crystallization was carried out by a vapor diffusion method.


Purifications and properties of orotidine-phosphorolyzing enzyme and purine nucleoside phosphorylase from Erwinia carotovora AJ 2992.

An orotidine-phosphorolyzing enzyme and a purine nucleoside phosphorylase (PNPase) of Erwinia carotovora AJ 2992, which is a potent producer of ribavirin (1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide), an antiviral agent, from orotidine and 1,2,4-triazole-3-carboxamide (TCA), were purified 23-fold and 103-fold, respectively. At each purification step, the orotidine-phosphorolyzing enzyme was always co-purified with an uridine phosphorylase (UPase) and its activity could not be separated from that of the UPase after it showed as a single band on SDS-polyacrylamide gel electrophoresis. These results suggest that this enzyme may be identical with UPase. The purified enzyme had a molecular weight of 68,000 +/- 2,000, and seemed to be a dimer. The optimal temperatures and pH values were 60 degrees C and 6.0 for orotidine phosphorolysis, and 70 degrees C and 7.0 for uridine phosphorolysis. The Michaelis constants for uridine and orotidine were 0.75 mM and 10.87 mM, respectively, at 40 degrees C. The PNPase of E. carotovora AJ 2992 had a molecular weight of 58,000 +/- 2,000 and seemed to be a dimer. The Michaelis constants for inosine and guanosine were 1.92 mM and 1.85 mM, respectively, at 40 degrees C. The PNPase was completely inactivated by p-chloromercuribenzoate.


A DNA transformation-competent Arabidopsis genomic library in Agrobacterium.

We have constructed a nuclear genomic library from the cruciferous plant Arabidopsis thaliana ecotype Columbia in a cosmid vector, pLZO3, and a host organism, Agrobacterium tumefaciens AGL1, which can directly DNA-transform the parent organism, Arabidopsis. The broad host range cosmid pLZO3 carries a gentamicin acetyltransferase gene as bacterial selective marker and tandem, chimeric neomycin and streptomycin phosphotransferase genes as plant selective markers. Agrobacterium AGL1 carries the hypervirulent, attenuated tumor-inducing plasmid pTiBo542 from which T-region DNA sequences have been precisely deleted, allowing optimal DNA transformation of many dicotyledonous plants. Agrobacterium AGL1 also carries an insertion mutation in its recA general recombination gene, which stabilizes the recombinant plasmids. The Arabidopsis genomic library consists of some 21,600 clones gridded onto 96-well microtiter dishes and, if random, carries at least three genomic equivalents. When probed for the presence of several Arabidopsis low copy-number genes, the genomic library seems representative. As with the unicellular organisms Escherichia coli and Saccharomyces cerevisiae, this DNA transformation competent genomic library should expedite gene isolation, by gene rescue, in multicellular organisms like Arabidopsis.


Commercial levels of chymosin production by Aspergillus.

We have increased the production of bovine chymosin in Aspergillus niger var. awamori to more than one gram per liter of secreted authentic enzyme by combining a mutagenesis protocol with a novel robotic screening program. Analysis of the superior chymosin producing strains indicated that they have enhanced capabilities to secrete extracellular proteins.


Cloning and nucleotide sequence of the KHS killer gene of Saccharomyces cerevisiae.

A 5.3-kbp fragment of the KHS gene was cloned from a genomic bank of Saccharomyces cerevisiae No. 115 constructed with an E. coli as the host and YEp13 as the vector. A non-killer yeast strain was transformed to a killer strain with the multi-copy vector containing the KHS gene, and the transformant could secrete 3-4 times more killer toxin into culture media than the donor, strain No. 115. The KHS toxin was purified 80-fold from the culture filtrate by gel filtration and column chromatography. The nucleotide sequence of a 2.8-kbp fragment of the KHS DNA that was enough for the expression of the killer activity was identified, and we found an open reading frame consisted of 2124 bp. Comparison of the open reading frame and N-terminal amino acid sequence of purified KHS toxin, suggested that the presumed peptide from the KHS gene might be processed between 36Gln and 37Ala before secretion.


Purification and properties of a xylanase from Cellvibrio gilvus that hydrolyzes p-nitrophenyl cellooligosaccharides.

An enzyme component that hydrolyzes pNP-G2 but not CMC has been isolated from a culture broth of Cellvibrio gilvus by a multi-step procedure involving Butyl-Toyopearl, DEAE-Toyopearl, and CM-Toyopearl chromatographies. The purified enzyme gave a single protein band on native, SDS-, and IEF-PAGE. The enzyme had a molecular weight of 40,000, an isoelectric point of 5.0, an optimum pH of 6.5, and an optimum temperature of 55 degrees C. It was stable from pH 4.0 to 9.0 at 37 degrees C for 1 hr and below 50 degrees C for 30 min. It hydrolyzed agluconic bonds not only of pNP-G2 but also of pNP-G3, pNP-G4, and pNP-G5. Cellooligosaccharides with D.P. of 3 to 5 were not hydrolyzed at all. Instead, the enzyme hydrolyzed xylan 4 times as fast as pNP-G2. Both HgCl2 and p-chloromercuribenzoic acid inhibited the two activities completely. Xylan inhibited the hydrolysis of pNP-G2 competitively. From these results, the purified enzyme was considered to be a unique xylanase that hydrolyzed the agluconic bonds of pNP-Gn.


Purification and characterization of a novel lyase from Cellulomonas sp. that degrades Fusarium and Gibberella acidic polysaccharides.

A Cellulomonas sp. isolated from soil produced a novel lyase that degraded the acidic polysaccharide of Fusarium sp. M7-1 with the formation of mannose and O-beta-D-mannopyranosyl-(1----2)-D-mannose. DEAE-Toyopearl 650M column chromatography showed three lyase activity peaks (fractions I, II, and III). The major fraction was purified to homogeneity by polyacrylamide gel electrophoresis analysis, and its molecular weight was 74,000. The optimum pH was 6.5 to 8.0 and the stable pH range was 6.0 to 8.0. The purified enzyme did not degrade glucuronic or galacturonic acid-containing polysaccharides such as chondroitin, hyaluronic acid, pectin, or pectic acid. However, the purified enzyme specifically degraded various Fusarium and Gibberella acidic polysaccharides, and unsaturated sugars were produced with the release of mannose and O-beta-D-mannopyranosyl-(1----2)-D-mannose. These results suggest that the acidic polysaccharides derived from Fusarium and Gibberella have similar structures.


Primary structures of N-linked oligosaccharides of momordin-a, a ribosome-inactivating protein from Momordica charantia seeds.

The structures of the N-linked oligosaccharides of momordin-a, which is a ribosome-inactivating protein from the seeds of Momordica charantia, were analyzed. First, the N-linked oligosaccharides of this glycoprotein were liberated by hydrazinolysis. After N-acetylation, the reducing ends of the oligosaccharides were coupled with 2-aminopyridine and the pyridylamino (PA-) derivatives were purified by gel filtration and high performance liquid chromatography (HPLC) on an ODS-silica column. Three kinds of oligosaccharide fractions were separated by HPLC. The structure of each oligosaccharide isolated was analyzed by a combination of sugar component analysis, exoglycosidase digestion, another kind of HPLC using an amide-silica column, and 500-MHz 1H NMR spectroscopy. The structures of two main oligosaccharides were established to be: [Formula; see text] and [Formula; see text]. These two oligosaccharides were the first examples having xylose (or fucose) but no alpha-mannosyl linkage among the N-linked oligosaccharides of glycoproteins from both animal and plant origins.


Transfer reaction catalyzed by exo-beta-1,4-galactanase from Bacillus subtilis.

A transfer reaction catalyzed by an exo-beta-1,4-galactanase from Bacillus subtilis was studied. The enzyme had a broad acceptor specificity and transferred galactobiosyl residues to acceptors such as various alcohols, including hydroxy benzenes and saccharides. Transfer products of glycerol formed by the enzyme were compared with those formed by Escherichia coli beta-galactosidase and by Penicillium citrinum endo-galactanase. E. coli enzyme transferred 90% of galactose residues to the primary hydroxyl groups of glycerol and P. citrinum endo-enzyme transferred 80% of saccharide residues to the secondary hydroxyl group. The B. subtilis exo-galactanase was less specific than the other two enzymes and formed two products (1-DG and 2-DG) with a 2-DG/1-DG ratio of about 2. The structures of the saccharides were examined by 13C-nuclear magnetic resonance analysis and by enzymatic hydrolysis. 1-DG and 2-DG were elucidated to be O-beta-D-galactosyl-(1----4)-O-beta-D-galactosyl-(1----1)-glycerol and O-beta-D-galactosyl-(1----4)-O-beta-D-galactosyl-(1----2)-glycerol, respectively. The efficiency of the transfer reaction was measured at various concentrations of glycerol using galactotriose as a donor. About 40-75% of galactobiosyl residues were transferred at an acceptor concentration range of 20-100 mg/ml.


Dye-sensitized photooxidation of neutral protease from Bacillus subtilis var. amylosacchariticus: assignment of histidine residue oxidized.

The neutral protease of Bacillus subtilis var. amylosacchariticus was photooxidized in the presence of methylene blue, by which treatment the enzyme was rapidly inactivated. The inactive enzyme was digested with endoproteinase Asp-N, the resultant peptides were separated by HPLC, and their amino acid sequences were compared with those obtained from the unmodified enzyme. Of four peptides that contained histidine residues, only the recovery of one peptide was found to be decreased by the photooxidation with the appearance of a new peptide. Comparisons of amino acid compositions and sequences between these two peptides showed that the latter peptide lacked His228 of the former one, indicating that His228 was photooxidized. This result suggests that His228 is involved in the catalytic reaction of the neutral protease or interaction with substrates.


Synthesis of the trisaccharide-protein conjugate of the phenolic glycolipid of Mycobacterium tuberculosis for the serodiagnosis of tuberculosis.

The trisaccharide segment of the phenolic glycolipid (PGL) of Mycobacterium tuberculosis, 2-O-methyl-3-O-[3-O-(2,3,4-tri-O-methyl-alpha-L-fucopyranosyl)-alpha-L- rhamnopyranosyl]-alpha-L-rhamnopyranose, was synthesized in the form of the p-(2-methoxycarbonylethyl)phenyl glycoside by a stepwise condensation. 2,4-Di-O-benzyl-3-O-acetyl-alpha-L-rhamnopyranosyl chloride was coupled to p-(2-methoxycarbonylethyl)phenyl 4-O-benzyl-2-O-methyl-alpha-L-rhamnopyranoside in the presence of silver triflate, and 2,3,4-tri-O-methyl-alpha-L-rhamnopyranosyl chloride was then coupled to the deacetylated disaccharide by the same procedure. The trisaccharide was deblocked and coupled to BSA, giving the neoglycoconjugate TB-NT-P-BSA. TB-NT-P-BSA showed its possibility as a useful tool for the serodiagnosis of tuberculosis.


Purification and properties of a novel surface-active agent- and alkaline-resistant protease from Bacillus sp. Y.

In the course of a search for an alkaline stable protease for industrial use, an alkaline protease (protease BYA) was isolated from an alkalophilic Bacillus sp. Y, and its properties were characterized. Its optimum pH was pH 10.0-12.5, when casein was used as a substrate. In addition to the stability of protease BYA at pH 6.5-13.0, it was also very stable towards various surface-active agents, such as sodium dodecyl sulfate and sodium linear alkylbenzene sulfonate. Protease BYA was most active at 70 degrees C. The isoelectric point (pI) of protease BYA was about 10.1. Protease BYA was characterized as a serine protease because of its sensitivity to phenylmethanesulfonyl fluoride and diisopropyl fluorophosphate. The protease seems to be related to proteases of the subtilisin family, such as subtilisin BPN', subtilisin Carlsberg, and No. 221 protease.


Gene encoding a putative zinc finger protein in Synechocystis PCC6803.

A 5.5-kb HindIII fragment of Synechocystis PCC6803 containing a liverwort (ORF316) homolog encoding a putative zinc finger protein was cloned. Nucleotide sequence analysis showed that the homology of the amino acid sequence deduced from the ORF326 of Synechocystis PCC6803 with the counterparts of a liverwort and tobacco was 50% and 46%, respectively. Synechocystis ORF326 also showed 38% homology with the dedB gene in Escherichia coli. The gene organization of the region in these species of organisms was quite different. This suggests that the Synechocystis ORF326 and liverwort ORF316 genes may be related to a common regulatory gene, but not photosynthetic gene characteristic to chloroplasts.


Cloning, nucleotide sequencing, and expression in Escherichia coli of a lipase and its activator genes from Pseudomonas sp. KWI-56.

A lipase gene (lip) and its activator gene (act) on a 2.9 kb BglII-EcoRI fragment from Pseudomonas sp. KWI-56 were cloned in Escherichia coli using pUC19 as a vector plasmid. From the sequencing results, the open reading frames of the lip and the act were found to contain 1092 and 1032 nucleotides, respectively. The act existed downstream of the lip with the same orientation. When the lip was expressed in E. coli using the lac promoter on the pUC plasmid vector, the lipase activity of E. coli carrying both the lip and the act was 200-fold greater than that carrying only the lip. This result suggested the act was important in the expression of the lip in E. coli.


Cloning and nucleotide sequence of thermostable lipase gene from Pseudomonas fluorescens SIK W1.

A gene coding for a thermostable lipase of Pseudomonas fluorescens SIK W1 was cloned into Escherichia coli JM83 by inserting Sau3AI-generated DNA fragments into the BamHI site of pUC19. Twenty colonies with esterase activity on the tributyrin agar plate were isolated by screening the constructed Pseudomonas fluorescens genomic library. Only one out of the esterase positive 20 colonies had lipase activity on the agar plate containing olive oil and Rhodamine-B. The complete nucleotide sequence of the lipase gene was identified. The lipase gene consists of an open reading frame, 1347bp long, commencing with an ATG start codon encoding a polypeptide of 449 amino acid residues and a TGA stop codon. Comparison of this lipase amino acid sequence with those from another organisms sequenced to data showed the presence of the short homologous region Gly-X-Ser-X-Gly.


Use of a triple protease-deficient mutant of Bacillus subtilis as a host for secretion of a B. subtilis cellulase and TEM beta-lactamase.

The mature portion of the TEM beta-lactamase (BLA) gene (bla) derived from pBR322 was fused with the promoter and signal region of Bacillus subtilis cellulase (BSC) gene (bsc), and the productivity was compared with that of the cloned native bsc gene, using a wild-type B. subtilis strain and a strain deficient in three proteases (i.e., extracellular serine protease, extracellular neutral protease, and the major intracellular serine protease) as hosts. The effects of the sen, sacQ, and prtR genes, carried on the same plasmids, were tested as for the productivities of BSC and BLA. The production of BSC was increased 9-fold by using the combination of the triple-protease deficient strain and the sacQ gene, compared with that by using the wild-type strain as a host, and no degradation of BSC was observed in the triple-protease deficient strain. On the other hand, though the production of the BSC-BLA fusion protein increased 2.5-fold in the triple-protease deficient strain, the BLA activity was decreased after the cell reached the stationary phase of growth, possibly due to some proteolysis. These observations show different sensitivity of secretary proteins to cellular proteases, and suggest that BLA is decomposed by remaining minor proteases.


A rapid isolation of the unknown 5'-flanking sequence of human CENP-B cDNA with polymerase chain reactions.

We rapidly and efficiently isolated the 5'-region of cDNA encoding the N-terminal region of human centromere antigen B (CENP-B) including an ATG methionine codon by polymerase chain reactions (PCR). The unknown 5'-flanking sequence of the cDNA was amplified using an adaptor-sequence ligated to the 5' end as a universal primer sequence. To locate the target fragments, we did an additional PCR with another set of two internal primers using samples of the size-fractionated products as templates, rather than using the conventional hybridization procedure. This approach can further be applied to the analysis of other unknown flanking sequences of cDNA or genomic DNA.


Antitumor activity of some polysaccharides isolated from a Chinese mushroom, "huangmo", the fruiting body of Hohenbuehelia serotina.

Polysaccharides were extracted from a Chinese mushroom, "Huangmo" with hot water (FI), 1% ammonium oxalate solution (FII), and 5% sodium hydroxide (FIII) and 20 more polysaccharides were isolated by ethanol precipitation, ion-exchange chromatography, gel filtration, and affinity chromatography. Of the six polysaccharides obtained, FI0, FA-1, FA-2, FII-2, FIII-1-b, and FIII-2-b were selected on the basis of the results of the antitumor activity test (the Sarcoma 180/mice, i.p. method). Six fractions which showed relatively high activities were examined: FI0, a mixture of galactose-containing heteroglycan and beta-D-glucan; FA-1 and -2, beta-D-glucan with galactose-containing hetero-glycan and protein; FII-2, beta-D-glucan; and FIII-1-b and FIII-2-b, (1----6)-beta-D-glucosyl branched (1----3)-beta-D-glucan.


Chemical modification of neutral protease from Bacillus subtilis var. amylosacchariticus: assignment of tyrosyl residues iodinated.

The neutral protease of Bacillus subtilis var. amylosacchariticus (B. amylosacchariticus) was iodinated with a 25-fold molar excess of iodine at pH 9.4 for 3 min at 0 degree C, by which treatment the proteolytic activity toward casein was markedly reduced, while the hydrolytic activity toward an N-blocked peptide substrate was rather increased. The modified enzyme was digested with Staphylococcus aureus V8 protease at pH 8.0 and the amino acid sequences of resultant peptides were compared with those obtained from the native enzyme. One of the peptides was found to have an amino acid sequence of Thr-Ala-Asn-Leu-Ile-Tyr-Glu, which corresponds to residue Nos. 153-159 of the enzyme, where Tyr-158 was identified to be mono-iodotyrosine. The other two peptides were those containing Tyr-21 which was mono- and di-iodinated, respectively. Referring to nitration experiments on the neutral protease and the active site structure of thermolysin, it was concluded that the iodination of Tyr-158 is mainly responsible for the activity changes of B. amylosacchariticus neutral protease.


Isolation and characterization of the alkaline protease gene of Aspergillus oryzae.

The genomic DNA for the alkaline protease (Alp) of the fungus Aspergillus oryzae was isolated using synthetic oligonucleotides as hydridization probes, and the complete nucleotide sequence was identified. The Alp gene is 1374 nucleotides long and contains three introns, one of which is in the pro region and two in the mature coding region. Sequences related to the TATA box (TATAAAT) and the CAAT box (CCAAAT) were found in the 5'-noncoding region. Primer extension analysis showed that three transcriptional start points are present.


Purification and characterization of beta-N-acetylhexosaminidase from Trichoderma harzianum.

beta-N-Acetylhexosaminidase was produced by Trichoderma harzianum cultivated with chitin as the growth substrate. The enzyme was purified 13.2-fold to homogeneity by ultrafiltration and sequential chromatography on SP-Toyopearl and Sephacryl S-200. The molecular weight of the enzyme was estimated to be about 150,000 by gel filtration. The pH and temperature optima were 4.0-5.5 and 50 degrees C, respectively. The enzyme hydrolyzed N-acetylchitooligosaccharides at the non-reducing ends to release GlcNAc monomer. The enzyme showed a strict substrate specificity to the sugar chains in complex carbohydrates, hydrolyzing only the linkage of GlcNAc beta 1-3Gal, but not hydrolyzing the other linkages such as GalNAc beta 1-3Gal and GlcNAc beta 1-2Man.


Characterization of Pseudomonas fluorescens carboxylesterase: cloning and expression of the esterase gene in Escherichia coli.

The Pseudomonas fluorescens gene (estB) that encodes a novel esterase (esterase II) was cloned into Escherichia coli JM83. DNA sequencing found a single open reading frame of 654 nucleotides. The open reading frame was confirmed by N-terminal amino acid sequence analysis of the esterase protein. A potential Shine-Dalgarno sequence is followed by the coding sequence of the estB gene. The amino acid sequence deduced from the nucleotide sequence contains the consensus active site sequence, G-X-S-X-G, of serine esterases. The enzyme expressed in an E. coli clone was purified by ion-exchange chromatography and gel filtration. Homogeneity of the purified enzyme was confirmed using SDS-polyacrylamide gel electrophoresis. The native enzyme exists as a dimer consisting of two identical subunits, each with a molecular weight of 23,000. The results of the experiments for identifying substrate specificity and the inhibitor studies suggest that this enzyme is a carboxylesterase (EC 3.1.1.1) and a serine residue is present at the active site of the esterase, as in the esterases of animal tissues.


Purification and characterization of rice peroxidases.

Four peroxidase components, named RP-2, 4, 6, and 7, were isolated from rice (Oryza sativa L.) green leaves. Isoelectric focusing indicated that each preparation was homogeneous. The molecular weights of RP-2, 4, 6, and 7 estimated by SDS-PAGE were 48,000, 48,000, 40,000, and 39,500, and their isoelectric points were 5.4, 8.1, 9.3, and 9.2, respectively. The activity of every preparation was maximum around pH 5.0. Antisera against these purified enzymes were raised in rabbits. Ouchterlony double diffusion tests with these antisera suggested that RP-6 and 7 were immunochemically identical and RP-2 and 4 were identical in parts and that RP-6 and 7 were quite different from RP-2 and 4. Analysis of the N-terminal amino acid sequences also showed that these peroxidase components were classified into two groups. The polymerase chain reaction showed that RP-2 and/or RP-4 contained an active central region, which is homologous to other plant peroxidases.


A lectin from the mushroom Pholiota aurivella.

A lectin was isolated from the fruiting bodies of Pholiota aurivella by preparative PAGE and named PAA (Pholiota aurivella agglutinin). PAA was a polymeric protein of more than several hundred kDa consisting of 18-kDa subunits. The amino acids were sequenced from the N-terminus of the lectin; they were YSVTTPNSVKGGTNQG. PAA agglutinated human erythrocytes regardless of blood type equally and Pronase treatment of erythrocytes increased the sensitivity to agglutination by the lectin. In a hemaggluting inhibition assay, none of the mono-and oligosaccharides used affected agglutination by the lectin. Among glycoproteins, asialofetuin was the best inhibitor. This is first isolated and characterized lectin from the family Strophariaceae.


Utilization of trans-activated T4 uvsY regulatory signal for a high yield of cloned gene products.

We established an efficient system for a high-level production of foreign gene products in Escherichia coli using a trans-activated gene expression under the control of a phage T4 regulatory signal cloned in a plasmid by T4 phage infection. The transcriptional and translational signal of the T4 uvsY gene cloned in a plasmid was fused translationally with the coding region of the lacZ gene. When E. coli cells carrying the uvsY-lacZ plasmid were infected with cytosine-substituting T4 phage at a multiplicity of infection of 5, the amount of beta-galactosidase increased about 2-fold (trans-activation) over that without phage infection. We examined conditions for the high-level production of a trans-activated gene product. We found that a large number of T4-infected cells in a lysis-inhibition state could be obtained by a low multiplicity of infection with cytosine-substituting T4 phage. Thus it is now possible to attain a high yield of the trans-activated gene products. We discuss the advantage of the trans-activated T4 uvsY regulatory signal for production of foreign products.


A novel expression system for production of a labile protein in Escherichia coli by infection with cytosin-substituting T4 phage.

A novel expression system was developed for the high level production of a labile protein in Escherichia coli. The regulatory signal of bacteriophage T4 uvsY gene was fused in frame with the coding region of human ventricular myosin alkali light chain (VLC1) gene. Expression from the regulatory signal was enhanced and continued in a lysis-inhibition state by infection with a cytosine-substituting T4 phage mutant. VLC1 protein was produced at a low level without infection because of its instability in the cells. Although the productivity was partly improved in a lon-deficient mutant without infection, it was improved about 100-fold with T4 phage infection. T4 phage produces protease inhibitor(s) (pin gene product) against proteases of host cell including the lon gene product (protease La).


Synthesis of 5-keto-5-oxime derivatives of milbemycins and their activities against microfilariae.

Starting from milbemycin D (1), milbemycin A4 (2) and milbemycin A3 (3), a series of 5-keto-5-oxime derivatives were synthesized by selective oximation at the alpha,beta-conjugated carbonyl function of the 5-ketomilbemycins (4-6). The activities of the synthesized compounds were studied in dogs naturally infested with microfilariae of Dirofilaria immitis. The 5-keto-5-oximes of milbemycin D (7), A4 (8) and A3 (9) had quite high efficacy to control the microfilariae and more potency than their parents, while the 5-O-acyl oximes (11-15) also exhibited high activity.


Structures of new cyclic peptides in young unshiu (Citrus unshiu Marcov.), orange (Citrus sinensis Osbeck.) and amanatsu (Citrus natsudaidai) peelings.

Four cyclic peptides were isolated from young unshiu (unripe fruit), orange and amanatsu peelings, and their structures were established on the basis of FAB-MS (CID method) and 2D-NMR spectroscopic data, and by chemical evidence. They were each found to consist of seven or eight amino acids.


Enzymic production of sweet stevioside derivatives: transglucosylation by glucosidases.

For the purpose of improving sweetness and a further study on the structure-sweetness relationship of steviol glycosides, transglycosylation of stevioside by a variety of commercial glucosidases was investigated. It was revealed that two alpha-glucosidases gave glucosylated products. Transglucosylation of stevioside by Pullulanase and pullulan exclusively afforded three products, 13-O-[beta-maltotriosyl-(1----2)-beta-D-glucosyl]-19-O-beta-D-g luc osyl- steviol (1), 13-O-[beta-maltosyl-(1----2)-beta-D-glucosyl]-19-O-beta-D-glucosyl- steviol (2) and 13-O-beta-sophorosyl-19-O-beta-maltotriosyl-steviol (3). All of these products have already been obtained by trans-alpha-1,4-glucosylation of stevioside by the cyclodextrin glucanotransferase starch system, and 1 and 2 have been proven to be tasty and potent sweeteners. Transglucosylation of stevioside by Biozyme L and maltose afforded three new products, 4, 5 and 6, the structures of these compounds being elucidated as 13-O-beta-sophorosyl-19-O-beta-isomaltosyl-steviol (4), 13-O-[beta-isomaltosyl(1----2)-beta-D-glucosyl]-19-O-beta-D-glucosyl- steviol (5) and 13-O-[beta-nigerosyl-(1----2)-beta-D-glucosyl]-19-O-beta-D- glucosyl-steviol (6). A significantly high quality of taste was evaluated for 4.