Oxygen-dependent desulphation of monomethyl sulphate by Agrobacterium sp. M3C.

Agrobacterium sp. M3C, previously isolated from canal-water for its ability to grow on monomethyl sulphate, degraded this ester with stoichiometric liberation of inorganic sulphate. In contrast with the biodegradation of monomethyl sulphate in Hyphomicrobium sp., and of other longer-chain alkyl sulphates in Pseudomonas spp., the pathway in Agrobacterium appeared not to involve a sulphatase enzyme capable of catalysing ester-bond hydrolysis. No such sulphatase was detectable under a range of conditions of bacterial culture, or using various methods for preparing cell-extracts, or different assay conditions. There was no incorporation of 18O-label from H2(18O) into the liberated inorganic sulphate. No methanol was detectable during biodegradation, and the organism was incapable of growth on methanol, and did not produce methanol dehydrogenase activity when grown on monomethyl sulphate. Tracer studies using mono[14C]-methyl sulphate indicated that formate serine and glycine were produced during the biodegradation. The presence of these amino acids, together with high activity of hydroxypyruvate reductase, indicated the operation of the serine pathway common in methylotrophs. Use of an oxygen electrode in conjunction with monomethyl[35S]sulphate showed that release of 35SO2(-4) was dependent on availability of O2, and that there was equimolar stoichiometry among monomethyl sulphate degraded, O2 consumed and 35SO2(-4) released. A proposed pathway for the degradation involved an initial mono-oxygenation to methanediol monosulphate with subsequent elimination of SO2(-4) and concomitant formation of formaldehyde. The pathway was compared with degradation mechanisms for other C1 compounds and for other sulphate esters.


Enzymes involved in anaerobic degradation of acetone by a denitrifying bacterium.

The pathway of anaerobic acetone degradation by the denitrifying bacterial strain BunN was studied by enzyme measurements in extracts of anaerobic acetone-grown cells. An ADP- and MgCl2-dependent decarboxylation of acetoacetate was detected which could not be found in cell-free extracts of acetate-grown cells. It is concluded that free acetoacetate is formed by ATP-dependent carboxylation of acetone. Acetoacetate was converted into its coenzyme A ester by succinyl-CoA: acetoacetate CoA transferase, and cleaved by a thiolase into acetyl-CoA. The acetyl residue was completely oxidized in the citric acid cycle. The ADP-dependent decarboxylation of acetoacetate was inhibited by EDTA, but not by avidin. High myokinase activities led to equilibrium amounts of ATP, ADP, and AMP in the reaction mixtures, and prevented determination of the decarboxylase reaction stoichiometry, therefore.


Reductive dechlorination of 1,2-dichloroethane and chloroethane by cell suspensions of methanogenic bacteria.

Concentrated cell suspensions of methanogenic bacteria reductively dechlorinated 1,2-dichloroethane via two reaction-mechanisms: a dihalo-elimination yielding ethylene and two hydrogenolysis reactions yielding chloroethane and ethane, consecutively. The transformation of chloroethane to ethane was inhibited by 1,2-dichloroethane. Stimulation of methanogenesis caused an increase in the amount of dechlorination products formed, whereas the opposite was found when methane formation was inhibited. Cells of Methanosarcina barkeri grown on H2/CO2 converted 1,2-dichloroethane and chloroethane at higher rates than acetate or methanol grown cells.


Degradation of 2,4,5-trichlorophenoxyacetic acid by a Nocardioides simplex culture.

A Nocardioides simplex strain 3E was isolated which totally dechlorinated 2,4,5-trichlorophenoxyacetic acid and was capable of its utilization as the sole source of carbon. The mechanism of 2,4,5-trichlorophenoxyacetic acid degradation by this strain was investigated. Chloroaromatic metabolites that occur in the lag, exponential and stationary growth phases of the strain Nocardioides simplex 3E were isolated and identified bases on a combination of TLC, GC-MS and HPLC data. Decomposition of 2,4,5-trichlorophenoxyacetic acid at the initial stage was shown to proceed by two pathways: via the splitting of the two-carbon fragment to yield 2,4,5-trichlorophenol and the reductive dechlorination to produce 2,4-dichlorophenoxyacetic acid. Hydrolytic dechlorination of 2,4,5-trichlorophenoxyacetic acid was found to yield dichlorohydroxyphenoxyacetic acid, thus pointing to the possible existence of a third branch at the initial stage of degradation of the xenobiotic. 2,4,5-Trichlorophenol and 2,4-dichlorophenoxyacetic acid produced during the metabolism of 2,4,5-trichlorophenoxyacetic acid and in experiments with resting cells are utilized by the strain Nocardioides simplex 3E as growth substrates.


Identification of metabolites from degradation of naphthalene by a Mycobacterium sp.

A Mycobacterium sp. isolated from oil-contaminated sediments was previously shown to mineralize 55% of the added naphthalene to carbon dioxide after 7 days of incubation. In this paper, we report the initial steps of the degradation of naphthalene by a Mycobacterium sp. as determined by isolation of metabolites and incorporation of oxygen from 18O2 into the metabolites. The results indicate that naphthalene is initially converted to cis- and trans-1,2-dihydroxy-1,2-dihydronaphthalene by dioxygenase and monooxygenase catalyzed reactions, respectively. The ratio of the cis to trans-naphthalene dihydrodiol isomers was approximately 25: 1. Thin layer and high pressure liquid chromatographic and mass spectrometric techniques indicated that besides the cis- and trans-1,2-dihydroxy-1,2-dihydronaphthalene, minor amounts of ring cleavage products salicylate and catechol were also formed. Thus the formation of both cis and trans-naphthalene dihydrodiols by the Mycobacterium sp. is unique. The down-stream reactions to ring cleavage products proceed through analogous dioxygenase reactions previously reported for the bacterial degradation of naphthalene.


Dechlorination of pentachlorophenol by membrane bound enzymes of Rhodococcus chlorophenolicus PCP-I.

Dechlorination (para-hydroxylation) of pentachlorophenol (PCP) and tetrachloro-para-hydroquinone (TeCH) and O-methylation of TeCH were demonstrated in cell extracts of Rhodococcus chlorophenolicus PCP-I. PCP para-hydroxylating activity was membrane bound, whereas TeCH dechlorinating enzyme was soluble. The PCP para-hydroxylating enzyme was solubilized by Triton X-100 and the requirement for both FAD and NADPH was shown. The dechlorinating activities were inducible in contrast to the constitutive TeCH O-methylating activity. The PCP para-hydroxylation was inhibited by its product TeCH, by anoxic conditions, and by different inhibitors of P450. Participation of this cytochrome in the PCP hydroxylation was confirmed by the appearance of a carbon monoxide dependent peak of absorbance at 457 nm in the membrane fraction prepared from PCP degrading cells.


Anaerobic degradation of sorbic acid by sulfate-reducing and fermenting bacteria: pentanone-2 and isopentanone-2 as byproducts.

Strictly anaerobic bacteria were enriched and isolated from freshwater sediment sources in the presence and absence of sulfate with sorbic acid as sole source of carbon and energy. Strain WoSo1, a Gram-negative vibrioid sulfate-reducing bacterium which was assigned to the species Desulfoarculus (formerly Desulfovibrio) baarsii oxidized sorbic acid completely to CO2 with concomitant stoichiometric reduction of sulfate to sulfide. This strain also oxidized a wide variety of fatty acids and other organic compounds. A Gram-negative rod-shaped fermenting bacterium, strain AmSo1, fermented sorbic acid stoichiometrically to about equal amounts of acetate and butyrate. At concentrations higher than 10 mM, sorbic acid fermentation led to the production of pentanone-2 and isopentanone-2 (3-methyl-2-butanone) as byproducts. Strain AmSo1 fermented also crotonate and 3-hydroxybutyrate to acetate and butyrate, and hexoses to acetate, ethanol, hydrogen, and formate. The guanine-plus-cytosine content of the DNA was 41.8 +/- 1.0 mol%. Sorbic acid at concentrations higher than 5 mM inhibited growth of this strain while strain WoSo1 tolerated sorbic acid up to 10 mM concentration.


Kinetics of aerobic biodegradation of benzene and toluene in sandy aquifer material.

Monod's equation adequately described aerobic biodegradation rates of benzene and toluene by the microbial population of a sandy aquifer when these compounds were initially present at concentrations lower than 100 mg/l each. Concentrations higher than 100 mg/l were inhibitory, and no benzene or toluene degradation was observed when these compounds were initially present at 250 mg/l each. The Monod coefficients were calculated as k = 8.3 g-benzene/g-cells/day and Ks = 12.2 mg/l for benzene, and k = 9.9 g-toluene/g-cells/day and Ks = 17.4 mg/l for toluene. Specific first-order coefficients would be 0.68 l/mg.day for benzene and 0.57 l.mg.day for toluene.


Evidence for two distinct phosphonate-degrading enzymes (C-P lyases) in Arthrobacter sp. GLP-1.

Arthrobacter sp. GLP-1 can utilize a wide range of organophosphonates as its sole source of phosphorus. The in-situ formation of sarcosine and methane from glyphosate and methanephosphonic acid respectively was studied. These two processes are differentially induced during phosphorus-deprivation. Methanephosphonic acid strongly inhibits glyphosate degradation (I50 10 microM), but glyphosate has very little effect on methane generation (I50 150 mM). The pattern of inhibition by other organophosphonates and organophosphonate analogues is also very different for the two systems. Degradation of glyphosate and methanephosphonic acid therefore represent distinct processes.


Use of antisense RNA to confer bacteriophage resistance in dairy starter cultures.

The strategy and implementation of a unique system for engineering bacteriophage resistant starter cultures of Lactococcus lactis employing antisense RNA is reviewed. As a necessary prerequisite for developing this system, we have cloned and sequenced a number of bacteriophage genes coding for minor and major structural proteins. In addition, we have also identified a series of genes whose function(s) is not known but their sequences appear to be conserved in a vast number of isolates. One of these latter sequences, designated gp51C, codes for a 51-kDa protein which is extremely charged and shares some homology with yeast translation initiation factor. Resistance to a broad class of isometric bacteriophages has been achieved by expression of an antisense RNA targeted against, for example, gp51C. In the best case, expression of the antisense gp51C RNA results is a greater than 99% reduction in the total number of plaque forming units. Additional antisense RNA constructs directed against other bacteriophage genes, including the major capsid protein, also appear effective at inhibiting infection from 40-55% suggesting that this approach may prove useful for engineering a set of truly isogenic strains to be used in a starter culture rotation plan.


Maintenance and killing efficiency of conditional lethal constructs in Pseudomonas putida.

Conditional lethal (suicidal) genetic constructs were designed and employed in strains of Pseudomonads as models for containment of genetically-engineered microbes that may be deliberately released into the environment. A strain of Pseudomonas putida was formed with a suicide vector designated pBAP24h that was constructed by cloning the host killing gene (hok) into the RSF1010 plasmid pVDtac24 and placing it under the control of the tac promoter. After hok induction in P. putida only 40% of surviving cells continued to bear the hok sequences within 4 h of induction; in contrast, 100% of the cells in uninduced controls bore hok. A few survivors that demonstrated resistance to hok-induced killing developed in P. putida, which may have been due to a mutation or physiological adaptation that rendered the membrane 'resistant' to hok. Conditional lethal strains of P. putida also were formed by inserting gef (a chromosomal homolog of hok) under the control of the tac promoter into the chromosome using a transposon. Constructs with chromosomal gef, as well as an RK2-derived plasmid construct containing gef, were only marginally more stable than the hok constructs; they were effective in killing P. putida when induced and within 2 h post-induction killing from either gef construct resulted in a 10(3)-10(5)-fold reduction in viable cell count compared to uninduced controls.


Ribosome-inactivating proteins from plants: present status and future prospects.

Plant ribosome-inactivating proteins (RIPs) are N-glycosidases which cleave the N-glycosidic bond of adenine in a specific ribosomal RNA sequence. Most commonly RIPs are single-chain proteins (type 1 RIPs), but some (type 2 RIPs) possess a galactose-specific lectin domain that binds to cell surfaces. The latter RIPs are potent toxins, the best known of which is ricin. RIPs have antiviral and abortifacient activities, and, in a widespread application, can also be linked to antibodies or ligands to form immunotoxins or conjugates specifically toxic to a given type of cell.


Simultaneous amplification and detection of specific DNA sequences.

We have enhanced the polymerase chain reaction (PCR) such that specific DNA sequences can be detected without opening the reaction tube. This enhancement requires the addition of ethidium bromide (EtBr) to a PCR. Since the fluorescence of EtBr increases in the presence of double-stranded (ds) DNA an increase in fluorescence in such a PCR indicates a positive amplification, which can be easily monitored externally. In fact, amplification can be continuously monitored in order to follow its progress. The ability to simultaneously amplify specific DNA sequences and detect the product of the amplification both simplifies and improves PCR and may facilitate its automation and more widespread use in the clinic or in other situations requiring high sample throughput.


Increased resistance to potato virus X and preservation of cultivar properties in transgenic potato under field conditions.

During the last three years we performed field trials to assess levels of resistance against potato virus X (PVX) and changes in intrinsic properties of the potato cultivars Bintje and Escort upon the introduction of the PVX coat protein (CP) gene. Analysis of leaf and tuber samples collected in the field at two week intervals revealed a stable expression of the PVX CP gene throughout the growing season. This resulted in a large decrease in PVX incidence among clonal progeny obtained from previously infected Bintje and Escort clones. Based on evaluation of 50 defined morphological characteristics, tuber yield and grading, 81.8% of the Escort and 17.9% of the Bintje derived transgenic clones proved to be true to type. Overall lightsprout morphology was a useful criterion for the early detection of deviant transgenic clones. Using the polymerase chain reaction (PCR) with convergent primers spanning transgenic sequences, true to type clones could be distinguished unambiguously from the corresponding untransformed cultivars. Clear distinctions between independent transgenic clones could be made by inverted PCR (IPCR) diagnosis revealing integration-specific border fragments. These results demonstrate the commercial feasibility of improving potato cultivars by selectively adding new traits while preserving intrinsic properties, and the possibility of unambiguously identifying independent transgenic cultivars.


Antigen responsive antibody-receptor kinase chimera.

We have constructed chimeric receptors, combining murine IgM and the cytoplasmic portion of human epidermal growth factor receptor (EGFR), with the aim of developing a novel immunosensor with antigen-dependent phosphorylation activity. When intact IgM was used, the chimeric receptor showed both antigen binding and protein tyrosine kinase activity, but the kinase activity was constitutive and independent of antigen binding. However, with IgM lacking the CH2 domain, the autophosphorylation activity increased with increasing concentrations of anti-IgM or hapten-BSA conjugate. Monovalent hapten could not induce phosphorylation but inhibited stimulation by hapten-conjugated BSA.


Cysteine to serine substitutions in basic fibroblast growth factor: effect on inclusion body formation and proteolytic susceptibility during in vitro refolding.

We have investigated the effect of cysteine to serine substitutions in human basic fibroblast growth factor (bFGF) on the formation of inclusion bodies in Escherichia coli. Using a temperature-sensitive expression, system, about 30% of human bFGF, which contains four cysteines at positions 26, 70, 88, and 93, is deposited into inclusion bodies. A single mutation at position 88 and a double mutation at positions 70 and 88 do not greatly alter the partition of bFGF into soluble and insoluble cell fractions. However, a single substitution of cysteine 70 by serine decreases the fraction of soluble bFGF significantly. When cysteines 26 and 93 (conserved among related growth factors) are replaced by serines, no soluble bFGF is formed in E. coli. Cysteine to serine substitutions also affect proteolytic susceptibility of bFGF during in vitro refolding from crude inclusion bodies. About 60% of human bFGF is lost to proteolytic degradation during in vitro refolding. Replacement of cysteines by serines increases the total recovery of bFGF, although more aggregates are formed during refolding. Ser-88-bFGF was expressed at the highest level, gave the highest soluble fraction in vivo, and exhibited the greatest fractional recovery and was recovered with the largest insoluble fraction after in vitro refolding. Thermal stability experiments at 42 degrees C and 70 degrees C revealed that cysteine to serine substitutions did not cause aggregation of the folded protein in vitro.


Monoclonal antibodies can protect L-asparaginase against inactivation by trypsin.

We show that a non-inhibitory monoclonal antibody (MAB) can be selected that provides substantial and sustained protection against proteolytic inactivation of L-asparaginase by trypsin. Of six non-inhibitory, high affinity, monoclonal antibodies to L-asparaginase, one afforded approximately 70% protection. Inactivation of L-asparaginase is associated with a single cleavage adjacent to lysine-29 that results in loss of an N-terminal fragment with a calculated MW of 2,647. The protective MAB prevented this trypsin cleavage. The products of gene fusions of "humanized" fragments of such antibodies and L-asparaginase could have increased clinical utility.


Comparison of the catalytic and inhibitory properties of Pachysolen tannophilus xylose reductase to rat lens aldose reductase.

The catalytic and inhibitory profiles of xylose reductase isolated from the yeast Pachysolen tannophilus (PTXR) are compared to those of aldose reductase (AR) obtained from rat lens. While both PTXR and rat lens AR are NADPH-specific enzymes and have an affinity for a variety of substrates such as D-xylose, D,L-glyceraldehyde, and 4-nitrobenzaldehyde, the enzymes differ in their substrate affinity profiles. Also, PTXR is not inhibited by standard inhibitors of AR thus supporting a hypothesis that this enzyme may not possess the inhibitor binding site found in rat lens AR.


Quantification of denitrification by strain T1 during anaerobic degradation of toluene.

Strain T1 is a denitrifying bacterium that is capable of toluene degradation under anaerobic conditions. During anaerobic growth on toluene, the specific growth rate of strain T1 was 0.14 h-1. Nitrite accumulated in the medium stoichiometrically with the depletion of nitrate. When nitrate was nearly depleted from the medium nitrite reduction and dinitrogen formation began. A non-kinetic model was formulated that was based on a hypothesis of non-simultaneous nitrate and nitrite reduction, independent of the concentrations of nitrate and nitrite. The model was verified experimentally over a wide range of conditions that included nitrate and nitrite limitation, toluene limitation, and various ratios of nitrate to nitrite. The model and its experimental verification demonstrated that strain T1 reduces nitrate and nitrite non-simultaneously, even if nitrite is initially present in the medium in addition to nitrate.


Stereoselective epoxidation of phenyl allyl ether by alkene-utilizing bacteria.

Eighteen newly isolated ethene- and propene-utilizing bacteria were screened for the ability to produce phenyl glycidyl ether, a common precursor for the synthesis of beta blockers, from phenyl allyl ether. These organisms included Aerococcus, Alcaligenes, Micrococcus and Staphylococcus spp. and a variety of Gram-negative, Gram-positive and Gram-variable mesophilic rods/coccobacilli not yet identified. The majority of ethene- and propene-grown cultures (14 strains) accumulated phenyl glycidyl ether (0.4-1.7 mM) as the sole oxidation product. The bioconversions with the three most promising ethene-utilizers (M26, M90C, M93A) were scaled-up to yield essentially optically pure (enantiomeric excess = 93%) S-(+)-phenyl glycidyl ether. This is currently under investigation for commercial production of optically pure beta blockers.


Batch and continuous ribonuclease production by immobilized Aspergillus clavatus cells in a bubble-column bioreactor.

Ribonuclease production using immobilized cells (IC) of Aspergillus clavatus has been studied under batch, repeated-batch and continuous fermentation conditions in a bubble-column bioreactor and compared with production by free cells. Immobilization was achieved by the method of cryostructurization in polyvinyl alcohol beads. The effect of various aeration rates in a column bioreactor has been investigated. Enzyme production by IC [42,000 units (U).1(-1)] during batch fermentations was comparable to that of a free-cell system. The specific productivity of IC was 8.5 times higher than that of free cells. In repeated batch fermentation at various aeration rates, successful reuse of IC was obtained, with comparable levels of enzyme production. Continuous ribonuclease production was achieved for 44 days at 1 vvm aeration and a dilution rate of 0.01 h-1 with high volumetric productivity (450 U.1-1.h-1) and yield.


The effect of Pluronic F-68 on hybridoma cells in continuous culture.

The comparative effects of Pluronic on cell growth of hybridomas were studied. The addition of 0.05% Pluronic decreased the steady-state cell number in continuous culture by about 12% compared to a non-supplemented culture. In short-term experiments, results demonstrated a gradual decrease in cell number with increasing concentration of Pluronic. Such growth inhibition was found to be a result of lowering the rate of DNA synthesis.


Optimization of protein-production by the baculovirus expression vector system in shake flasks.

Shake flasks were successfully employed for the cultivation of Spodoptera frugiperda (Sf-9) insect cells and for the production of beta-galactosidase, a recombinant model protein, utilizing the baculovirus expression vector system. The culture doubling time and maximal cell density were 20 h and 5 x 10(6) cells/ml respectively. The optimal liquid volumes for flasks rotating at 100 rpm were 25-40% of the flask total volume. Enzyme production (about 600 mg/l) was best at a multiplicity of infection of between 1 and 20 and at a cell density at time of infection of 0.7 x 10(6) cells/ml. At a rotation speed of 100 rpm, Pluronic F-68 had no effect on growth and enzyme production.


Antisense RNA directed against the major capsid protein of Lactococcus lactis subsp. cremoris bacteriophage 4-1 confers partial resistance to the host.

Antisense RNA targeted against the major capsid protein (MCP) of Lactococcus lactis subsp. cremoris bacteriophage F4-1 reduced bacteriophage replication by up to 50%. The region containing the mcp gene was oriented to transcribe the antisense strand using a L. lactis subsp. cremoris Wg2 promoter. The size of the mcp insert transcribed affected the level of bacteriophage inhibition and the greatest level of inhibition was achieved using a 301-bp fragment from the 5' end of the mcp. Antisense mcp RNA constructs were stable and did not alter the endogenous plasmid profile in the host, L. lactis subsp. cremoris F4-1. There were, however, some adverse effects on the host during the stationary phase as exhibited by a decline in cell density.


Cloning and heterologous expression of the penicillin biosynthetic gene cluster from penicillum chrysogenum.

A cosmid clone containing the putative penicillin biosynthetic gene cluster from Penicillium chrysogenum was used to transform the related filamentous fungi Neurospora crassa and Aspergillus niger, which do not produce beta-lactam antibiotics. Both of the transformed hosts contained intact P. chrysogenum DNA derived from the cosmid clone and produced authentic penicillin V. Assays of penicillin biosynthetic enzyme activity additionally demonstrated that they possessed delta-(L-alpha-amino-adipyl)-L-cysteinyl-D-valine synthetase (ACVS), isopenicillin N synthetase (IPNS) and acyl coenzyme A:6-aminopenicillanic acid acyltransferase (ACT) activity. The data suggests that genes encoding all the enzymes necessary for the biosynthesis of penicillin from amino acid precursors are closely linked in P. chrysogenum and constitute a gene cluster.


Selective determination of L-lysine with L-lysine epsilon-dehydrogenase.

Simple and sensitive spectrophotometric methods were developed for the selective determination of L-lysine. The assay methods were based on the spectrophotometric determination of NADH formed in the reaction catalyzed by L-lysine epsilon-dehydrogenase (Procedure A) or the formazan produced on coupling of the dehydrogenase reaction with the reduction of a tetrazolium salt (Procedure B). Procedure B was applicable to the direct determination of L-lysine in rabbit serum.


Beta-eliminative cleavage of the acidic polysaccharide of Fusarium sp. M7-1 by an enzyme preparation of Cellulomonas sp.

By digestion with an enzyme preparation derived from a culture filtrate of Cellulomonas sp., an unsaturated disaccharide was produced accompanied by the release of mannose and beta 1----2 mannobiose from the acidic oligomer mixture that was obtained from the acidic polysaccharide of Fusarium sp. M7-1 by acetolysis. The unsaturated disaccharide produced was isolated and identified as 4-deoxy-L-threo-hex-4-enopyranouronosyl alpha(1----2)-D-galactose. The same unsaturated sugar linked to the polysaccharide was also produced accompanied by the release of mannose and beta 1----2 mannobiose from the acidic polysaccharide by the enzyme digestion.


Construction of shuttle vector plasmid between Clostridium acetobutylicum and Escherichia coli.

A high copy number plasmid pCS86 (3.0 kb) was isolated from Clostridium acetobutylicum strain No. 86. For developing the vector plasmid for gene cloning of saccharolytic clostridia, two hybrid plasmids were constructed by joining pCS86 to the E. coli promoter probe vector pKK232-8 carrying the promoterless gene of chloramphenicol acetyltransferase (CAT). One of the hybrid plasmids designated as pTY10 replicated and expressed the CAT gene in E. coli, and transformed a plasmid-free strain, C. acetobutylicum strain No. 220, as a shuttle vector. This is the first shuttle vector constructed from C. acetobutylicum and E. coli plasmids. Reciprocal transformation was possible between E. coli and C. acetobutylicum, though deletion occurred in the sequence originated from pKK232-8. A deleted plasmid pTY101 only replicated in Clostridium, though it showed a high copy number.


Electroporation-transformation system for coryneform bacteria by auxotrophic complementation.

We evaluated electroporation as an alternative system for genetic exchange for one of the coryneform bacteria, Brevibacterium flavum MJ233. The maximum number of transformants, 6 x 10(4) cells, was obtained when cells were cultured with Penicillin G (1 U/ml) and harvested at the middle-log phase. Electroporation was done using 12.5 kV/cm of pulse field strength, 1 x 10(10) cells, and 1 microgram of plasmid DNA. Other coryneform bacteria, Brevibacterium lactofermentum ATCC 13869, Corynebacterium glutamicum ATCC 31830, and B. stationis IFO 12144 were also transformed by electroporation. Electroporation has the advantage that intact cells can be used as host cells without the need for protoplast formation and regeneration. Moreover, minimal medium can be used, so auxotrophic complementation of the transformants is possible.


Structure of rice-straw arabinoglucuronoxylan and specificity of Streptomyces xylanase toward the xylan.

Rice-straw treated with 5% ammonia water was hydrolyzed with the beta-xylanase of Streptomyces sp. E-86, and four kinds of hetero-oligosaccharides, two arabinoxylo- and two glucuronoxylo-oligosaccharides, were isolated from the hydrolysate by charcoal column, gel filtration, ion-exchange, and aminopropyl silica column chromatographies. The structures of the oligosaccharides were identified as 3(2)-alpha-L-arabinofuranosyl-xylobiose, 3(2)-alpha-L-arabinofuranosylxylotriose, 2(3)-4-O-methyl-alpha-D-glucuronosylxylotriose, and 2(3)-alpha-D-glucuronosylxylotriose by the analysis of component sugar, partial acid hydrolysis, methylation analysis etc. Based on the structures of above oligosaccharides, the structure of rice-straw arabinoglucuronoxylan and the specificity of the xylanase toward the xylan are discussed.


Purification and characterization of citrate synthase from Streptomyces hygroscopicus SF-1293 and comparison of its properties with those of 2-phosphinomethylmalic acid synthase.

To study the relationship between citrate synthase and 2-phosphinomethylmalic acid (PMM) synthase, which catalyzes a very similar reaction comparable to citrate formation in the biosynthesis of a herbicide, bialaphos, citrate synthase was purified from the mycelium of Streptomyces hygroscopicus SF-1293, a bialaphos-producing organism. The overall purification was 440-fold with a yield of 4.4% from cell-free extract. Based on comparison with PMM synthase, it has been concluded that citrate synthase of S. hygroscopicus is quite different from PMM synthase in several aspects such as enzymatic properties, amino acid composition. N-terminal amino acid sequence, and stereo-chemical reaction mechanism.


Structural characterization of the immunoactive and antiviral water-solubilized lignin in an extract of the culture medium of Lentinus edodes mycelia (LEM).

The active principle of EP3, a fraction from an extract of the culture medium of Lentinus edodes mycelia (LEM), which activates murine macrophages, causes proliferation of bone marrow cells, and inhibit the replication of Human Immunodeficiency Virus in vitro, was characterized as a water-solubilized lignin. The detailed structural feature of this water-solubilized lignin was investigated and shown to be a highly condensed and polycarboxylated lignin which is denatured and solubilized by Lentinus edodes from bagasse. The water-solubilized lignin itself was confirmed to have both immunological activities and the antiviral activity.


Identification of soybean protein components that modulate the action of insulin in vitro.

We have reported that a 37-kDa protein from a soybean isolate shows an affinity for bile acids and modulates insulin action on fat decomposition in vitro [Makino et al., Agric. Biol. Chem., 52, 803 (1988)]. In this study, the major components of the protein were identified as the acidic A1a and A2 subunits of glycinin, via amino-terminal sequence analyses of purified proteins and examination of their effects on fat decomposition in rat adipose cells. The most hydrophobic region of the subunits was found to be responsible for the bile acid-binding ability, and the binding region probably does not contribute to the insulin-modulating activity. These bile acid-binding and insulin-modulating properties were also noted in a 40-kDa protein from pea seeds, probably acidic subunits of legumin, suggesting that these characteristics may be common to legume proteins.


Limited proteolysis of silkworm antitrypsin by several proteinases.

Silkworm antitrypsin (sw-AT) isolated from larval hemolymph was limitedly digested by Achromobacter lysylendopeptidase, alpha-chymotrypsin, subtilisin BPN', subtilisin Carlsberg, papain, or Pseudomonas elastase. Each proteinase could cleave specific site(s) around the reactive site identified for the reaction of sw-AT and bovine trypsin. Among these proteinases, only subtilisin BPN' was inhibited by sw-AT, although weakly. By the cleavable amino acid sequence in sw-AT, it was suggested that whether or not these proteinases were inhibited by sw-AT did not solely depend on their substrate specificities. The susceptibility to the attack of proteinase should indicate that this region is exposed on the molecular surface. The amino acid sequence in the COOH-terminal region slightly away from the reactive site in sw-AT had homology with that in the corresponding region of the serine proteinase inhibitor (serpin) group.


Single site proteolysis in silkworm antitrypsin causes structural changes in behavior against denaturing reagents.

Silkworm antitrypsin (sw-AT), which was thought to belong to serpin family, changed its behavior against denaturation after chymotryptic cleavage of a single peptide bond (Tyr-Val) two amino acids away from the reactive site for trypsin (Lys-Val). This chymotrypsin-modified sw-AT became resistant to denaturation by heat, sodium dodecyl sulfate, or guanidine hydrochloride, and this characteristic was evident in its circular dichroism spectrum. The modified sw-AT was also indigestible by S. aureus V8 protease. These facts should indicate a structural change from a stressed, unstable state to a stable one accompanying the cleavage of the single peptide bond in sw-AT. The stabilizing factor was in part attributed to the interaction of a COOH-terminal fragment (5 kDa) and an NH2-terminal one (36 kDa) in modified sw-AT.


Structural analyses of sugar chains from ricin A-chain variant.

Two glycopeptides were isolated from a tryptic digest of ricin A-chain variant by gel filtration and reversed-phase HPLC. Their amino acid compositions and sequences showed that Asn-10 and Asn-236 are glycosylated. The sugar chains were liberated from these glycopeptides by hydrazynolysis, N-acetylated, and pyridylaminated. Component analysis and comparison of elution positions of the purified pyridylamino (PA-) sugar chains with those of authentic PA-sugar chains from the major ricin A-chain by reversed-phase HPLC and size fractionation HPLC suggested that both sugar chains linked to Asn-10 and Asn-236 of the ricin A-chain variant are M3FX, identical to that linked to Asn-10 of the major ricin A-chain.


Subunit structure of karatasin, the proteinase isolated from Bromelia plumieri (karatas).

Close to 15% of the karatasin proteinase activity in the fruit juice of Bromelia plumieri (karatas) is present outside dialysis Visking tubing in 7 days in 0.2 M acetate buffer (pH) 3.5 or 6.5) containing phenyl mercuric acetate. The small proteinase(s), distinct from the 85% activity in juice due to nondialysable karatasin with a reported Mr of 24,868, separates across Spectrapore (13 kDa) membranes but not across Spectrapore with 3.5 kDa average pore diameter. The dialyzed proteinase is named karatasin-D (K-D). Purified non-Dialysable karatasin can be dissociated to what seems to be K-D by incubation in a buffer solution, containing SDS and 2-mercaptoethanol with phenyl mercuric acetate, in dialysis experiments for 8 days at room temperature using Spectrapore 13 kDa tubing. Thus, native karatasin in B. plumieri fruit juice seem to be the result of association of 2 small molecular mass K-D subunits, linked together by disulfide bonds and electrostatic forces, in equilibrium with small amounts of free K-D molecules. The amino acid composition and partial sequence of karatasin up to the 14th position from the amino terminus have discrete analogies with papain and with stem bromelain.


Comparison of CD composition produced by chimeric CGTases.

Using 12 chimeric cyclomaltodextrin glucanotransferases (CGTases) constructed from two genes, the CGTase gene from the alkalophilic Bacillus sp. strain No. 38-2 (CGTase 38-2 gene) and the CGTase gene from the alkalophilic Bacillus sp. strain No. 17-1 (CGTase 17-1 gene), we compared the effect of those chimeric enzymes on cyclodextrin (CD) production, especially on the composition of the CDs produced. It was found that the N-terminal and the C-terminal segments were important for CD production. Chimeric enzymes that contained the N-terminal and the C-terminal segments derived from CGTase 38-2 produced large amounts of CD, and especially a higher proportion of alpha-CD than those of other chimeric enzymes.


Primary structure of rat monoamine oxidase A deduced from cDNA and its expression in rat tissues.

The cDNA for rat monoamine oxidase A (MAO-A) mRNA was isolated from a liver cDNA library in lambda gt11 as the cDNA species cross-hybridizing with rat monoamine oxidase B (MAO-B) cDNA. The primary structure of the protein, deduced from the nucleotide sequence, consisted of 526 amino acid residues and its molecular mass was calculated to be 59.6 kD. The amino acid sequence shows about 70% identity with that of rat MAO-B. Northern blot analysis showed that MAO-B mRNA was expressed in various rat tissues and the highest expression was observed in heart. The MAO-B mRNA was quite different from MAO-A mRNA in the tissue-specific expression and liver had the highest level of the mRNA.


Practical debittering using model peptides and related compounds.

In order to develop a practical debittering method for amino acids and peptides, several debittering methods were studied. The authors found that hooking acidic amino acids to and acetylation of bitter amino acids is very effective to remove the bitterness from their concentrated solution. For debittering by mixing with additives, skin milk and other peptide compounds were effective. Acidic amino acids were also effective to reduce the bitterness. Gelatinized starch was found to be useful for debittering because it takes bitter substances into its net structure.


Molecular and kinetic properties of Aspergillus ficuum inulinases.

The beta-fructosidases from the thermotolerant fungus, A. ficuum, were characterized. All were active as to inulin and sucrose hydrolysis with different specificity constants (kcat/Km). The enzymes were classified according to the ratio (alpha) of the specificity constants for inulin (I) and sucrose (S). The invertase showed an alpha value of lower than one, while the inulinases had alpha values of higher than one. The alpha ratio is proposed for the classification of beta-fructosidases into the inulinase (EC 3.2.1.7) and invertase (EC 3.2.1.26) groups. The amino acid composition, pH and temperature profiles, ultracentrifugation analysis results and effects of metal ions and effectors were studied. The data confirmed the preceding classification based on the I/S ratio, on the mode of action of inulinases (exo or endo) during inulin hydrolysis and on the molecular properties of the proteins.


The effects of metal ions on the DNA damage induced by hydrogen peroxide.

The effects of metal ions on DNA damage induced by hydrogen peroxide were investigated using two methods, agarose-gel electrophoretic analysis of supercoiled DNA and sequencing-gel analysis of single end-labeled DNA fragments of defined sequences. Hydrogen peroxide induced DNA damage when iron or copper ion was present. At least two classes of DNA damage were induced, one being direct DNA-strand cleavage, and the other being base modification labile to hot piperidine. The investigation of the damaged sites and the inhibitory effects of radical scavengers revealed that hydroxyl radical was the species which attacked DNA in the reaction of H2O2/Fe(II). On the other hand, two types of DNA damage were induced by H2O2/Cu(II). Type I damage was predominant and inhibited by potassium iodide, but type II was not. The sites of the base-modification induced by type I damage were similar to those by lipid peroxidation products and by ascorbate in the presence of Cu(II), suggesting the involvement of radical species other than free hydroxyl radical in the damaging reactions.


Purification and characterization of a novel fungal endo-beta-N-acetylglucosaminidase acting on complex oligosaccharides of glycoproteins.

A novel endo-beta-N-acetylglucosaminidase acting on complex type sugar chains of glycoproteins was found in the culture broth of a fungus isolated from soil and identified as Mucor hiemalis f. hiemalis on the basis of various characteristics. The endo-beta-N-acetylglucosaminidase, named Endo-M, was purified to almost homogeneity by polyacrylamide gel electrophoresis involving ammonium sulfate fractionation, and column chromatographies on DEAE-Sepharose CL-6B, Sephadex G-200, hydroxylapatite, TSK-gel HW-65F and Con A-Sepharose 4B. The molecular weight of the enzyme was estimated to be about 95,000 by gel chromatography. The optimum pH was found to be 6.0-6.5 and the enzyme was stable in the pH range of 7 to 8. The enzyme showed high activity on dansyl ovalbumin glycopeptide, and also could act on dansyl transferrin glycopeptide, and dansyl asialotransferrin glycopeptide containing biantennary complex type sugar chains. The Km value for dansyl asialotransferrin glycopeptide as the substrate was 2.0 x 10(-3) M. The enzyme released complex type sugar chains from intact asialotransferrin without the addition of any detergent and the liberated sugar chains were identified by chromatography on a Bio-Gel P-4 column, calibrated with markers of known structure, and 1H-NMR analysis.


Enzymatic synthesis of p-nitrophenyl alpha-maltoheptaoside by transglycosylation of maltohexaose-forming amylase.

An extracellular maltohexaose-forming amylase [EC 3.2.1.98] from Klebsiella pneumoniae mutant is a normal hydrolytic enzyme that hydrolyzes short-chain amylose (DP = 23) to give about 40% maltohexaose. Transglycosylation from maltoheptaose to the 4-position of p-nitrophenyl alpha-glucoside was efficiently induced through the use of maltohexaose-forming amylase in an aqueous methanol solution. The enzyme specifically produced p-nitrophenyl alpha-maltoheptaoside (13% of the p-nitrophenyl alpha-glucoside) from maltoheptaose as a donor and p-nitrophenyl alpha-glucoside as an acceptor. The yield of p-nitrophenyl alpha-maltoheptaoside depended on the concentration of methanol solvent, the pH, and temperature. Furthermore, the use of the aqueous methanol system in the reaction not only improved the solubility of p-nitrophenyl alpha-glucoside but also greatly increased the formation of p-nitrophenyl alpha-maltoheptaoside, which is a useful substrate for assay of human amylase in serum and urine.


Immunochemical analysis of the glucomannan from Candida utilis.

A glucomannan isolated from a Candida utilis mutant having a new chemotype was further studied by inhibition of the homologous precipitin reaction with oligosaccharides obtained from the glucomannan by partial acid hydrolysis and controlled acetolysis. Oligosaccharides having at least two consecutive alpha-(1----2)-linked mannose residues at the non-reducing end and gluco-manno-pentasaccharide were effective inhibitors. Thus, it appears that the glucomannan had two groups of antigenic determinants, one corresponding to the side chains of two, three, and four mannose units connected by alpha-(1----2)-linkage, and the other corresponding to a side chain composed of an O-alpha-D-glucopyranosyl-(1----6)-O-alpha-D-mannopyranosyl- (1----2)-O-alpha-D-mannopyranosyl-(1----2)-D-mannose unit. These results support a probable structure of repeating units for the glucomannan presented previously. The relative susceptibility of intersaccharidic linkages to acid hydrolysis in the mannan is discussed.


Deletion of D-helix in bovine pancreatic phospholipase A2.

D-Helix-deleted bovine pancreatic phospholipase A2 was designed using molecular mechanic calculations, and synthesized. Effects of the deletion on the enzymatic activity and on the structure were investigated. The enzymatic activity of the mutant protein was about 40% of that of the native one for a micellar substrate of 1,2-dioctanoyl-phosphatidylcholine. Although the Michaelis constant of the mutant enzyme was not changed, the catalytic constant was decreased. The dissociation constant of calcium ion was also changed. 1H NMR study revealed a slight conformation change around the active site of the mutant enzyme in addition to changes around the mutated region. The effect on the activity of the mutation seems to be due to the conformational changes around the active site.


Expression of the beta-cyclodextrin glucanotransferase gene of an alkalophilic Bacillus sp. #1011 in Escherichia coli cells and characterization of the synthesized enzyme.

To express efficiently the gene for extracellular beta-cyclodextrin glucanotransferase (beta-CGTase) of an alkalophilic Bacillus sp. #1011 using the E. coli promoters (tac, trp and PL promoters), three DNA fragments starting from the nucleotide positions +1, -18, and -48 of the translation initiation site of the gene were prepared and they were fused with the promoters. The maximum production of the enzyme, which was located mainly (90%) in the periplasm of the E. coli strain, was observed in the combination of the trp promoter and the beta-CGTase gene starting from the -48 nucleotide position in the presence of the inducer, IAA. The production of the enzyme was increased to 5.5 times that by the E. coli harboring the original plasmid and to approximately 3 times higher than the extracellular production of the enzyme by the parental Bacillus sp. #1011. The properties including the stability and optimum in the high pH range (pH 9) of the extracellular beta-CGTase from the alkalophilic Bacillus was conserved in the periplasmic enzymes of the E. coli cells.


Stereoselective synthesis of marine antibiotic (-)-malyngolide and its stereoisomers.

A convenient synthetic method for the marine antibiotic (-)-malyngolide and its stereoisomers was accomplished from a chiral alpha-alkoxyketone (4), which was readily available as a chiron. Chiral quaternary carbon synthons (5a) and (5b) as the key intermediates were constructed by the chelation controlled addition of Grignard reagent to 4. The diastereomeric mixture of 5a and 5b was readily transformed into a separable mixture of lactones (7a) and (7b), each of which could be easily separated by silica-gel column chromatography. (-)-Malyngolide and its three stereoisomers were obtained in optically pure form without the need for optical resolution.


Construction of chimeric insecticidal proteins between the 130-kDa and 135-kDa proteins of Bacillus thuringiensis subsp. aizawai for analysis of structure-function relationship.

Eight chimeric insecticidal protein (IP) genes were constructed between the 130-kDa and 135-kDa IP genes of Bacillus thuringiensis subsp. aizawai, and expressed in Escherichia coli JM103 cells. The characterization of the produced chimeric IPs indicated that the variable region (VR1) in the amino-terminal half of the IPs is responsible for the insecticidal activity against larvae of Spodoptera litura and Plutella xylostella. The carboxy-terminal half of VR1 was important for the formation of the 60-kDa active fragment in the gut juice of S. litura larvae. Also, combination of the other two variable regions (VR2 and VR3), which were in the central and carboxy-terminal portions of the IPs, appeared to be related to the solubility of the IPs in the gut juice.


Purification and characterization of two forms of maltotetraose-forming amylase from Pseudomonas stutzeri.

Pseudomonas stutzeri MO-19 produced two active forms of extracellular maltotetraose-forming amylase. Both forms, G4-1 and G4-2, were purified to electrophoretic homogeneity. The molecular masses of G4(-1) and G4(-2) were 57 kd and 46 kd by SDS-polyacrylamide gel electrophoresis, respectively. An identical N-terminal sequence up to 20 amino acid residues and similar amino acid compositions were obtained from both forms, but different C-terminal amino acids, leucine from G4(-1) and alanine from G4(-2), were released by carboxypeptidase Y. By in vitro incubation with a culture supernatant containing protease activity, G4(-1) was converted into G4(-2) without any loss of the amylase activity. It was concluded that G4(-2) was a product derived by the limited proteolysis of G4(-1), and that the proteolysis occurred in the C-terminal region of G4-1. G4-2 was more thermostable than G4(-1), and had a 20-fold higher Michaelis constant value for glycogen, which was 50 mg/ml against 2.3 mg/ml of G4(-1). G4(-1) adsorbed onto raw starch granules while G4(-2) did not.


A new depsipeptide antibiotic, variapeptin.

A culture similar to Streptomyces variabilis was found to produce a novel cyclic hexadepsipeptide antibiotic named variapeptin. Variapeptin is structurally related to azinothricin, A83586C, and citropeptin. The antibiotic was active against Gram-positive bacteria and showed cytotoxic activity against mammalian cells.


Cellulase production by Neurospora crassa: purification and characterization of cellulolytic enzymes.

In studies on cellulase production by the cell-1 mutant of Neurospora crassa, eight enzymes (three exoglucanases, four endoglucanases, and one beta-glucosidase) were identified and characterized by gel filtration, ion exchange chromatography, and chromatofocusing. After purification, each of the proteins ran as a single band in polyacrylamide gel electrophoresis, using both native and denaturing gels. The molecular weights of the proteins were found to be between 70,000 and 22,000 daltons, and all were glycosylated, with carbohydrate contents ranging between 5.6% and 36%.


Magnetic aqueous two-phase separation in preparative applications.

Magnetic aqueous two-phase separation is a new technique to speed up the separation of aqueous two-phase systems (Anal. Biochem. 1987, 167, 331-339). It is based on the addition of magnetically susceptible material (e.g. 1-micron iron oxide particles) which induces rapid phase separation when a mixed system is placed in a magnetic field. The technique has been applied to a number of two-phase systems. The time for phase separation was decreased by a factor of 5-240,000, with the largest improvement for systems containing high concentrations of protein and for systems with viscous or nearly isopycnic phases. An apparatus for preparative multistage extraction with magnetic separation was constructed and tested on glycolytic enzymes present in a yeast extract using a dextran/Cibacron blue-polyethylene glycol system. The presence of iron oxide particles did not adversely affect the extracted enzymes. An electromagnet-based apparatus for continuous phase separation on a larger scale was also designed. A phase system containing crude dextran and unpurified cell homogenate was effectively processed. The apparatus also allowed effective separation when the phase containing iron oxide particles was only a small fraction (4%) of the total phase system.


Expression and characterization of a tripartite fusion protein consisting of chimeric IgG-binding receptors and beta-galactosidase.

Using protein engineering, a tripartite fusion protein was constructed consisting of five IgG-binding regions of protein A from Staphylococcus aureus, two IgG-binding regions of protein G from Streptococcus strain G148 and beta-galactosidase from Escherichia coli. The resulting protein lacks the serum albumin binding regions of native protein G. The fusion protein, which is a tetramer of approximately 660 kDa, was designed as a tool for immunological assays taking advantage of its broad spectrum of antibody affinity. The gene was placed under control of two promoters, the PR promoter and the lac UV5 promoter and the expression from the two promoters was studied in a bioreactor. Induction of the PR promoter gave an intracellular product concentration corresponding to 20% of the cell dry weight. By utilizing the properties of beta-galactosidase, the protein was purified by extraction in an aqueous two-phase system. The fusion protein was not proteolytically degraded during the cultivation and purification steps. The biological activity of all three parts of the protein was demonstrated with a competitive ELISA.


A new depsipeptide antibiotic, citropeptin.

An actinomycete identified as Streptomyces flavidovirens was found to produce a new cyclic hexadepsipeptide antibiotic, designated citropeptin. Citropeptin showed antitumor activity against P388 murine leukemia.


Action pattern of Aeromonas hydrophila chitinase on partially N-acetylated chitosan.

Oligosaccharides from the digestion of 34% N-acetylated chitosan by Aeromonas hydrophila chitinase were separated by CM-Sephadex C-25 column chromatography. Sugar compositions and the sequences of main oligosaccharides were identified through their N-acetylation, their cleavage with exo-glycosidases, and their degradation with nitrous acid. Hetero-chitooligosaccharides such as GlcN.GlcNAc, GlcN.GlcNAc.GlcNAc, GlcNAc.GlcN.GlcNAc, and GlcNAc.GlcN.GlcNAc.GlcNAc, together with GlcNAc and (GlcNAc)2, were detected. The structure of GlcN.GlcNAc was confirmed by the analysis with proton and carbon NMR spectroscopy. These studies indicate that Aeromonas hydrophila chitinase is more specific toward the N-acetyl-beta-D-glucosaminidic bonds in partially N-acetylated chitosan.


Purification and some properties of beta-fructofuranosidase I from Arthrobacter sp. K-1.

Arthrobacter sp. K-1, isolated from soil, produces beta-fructofuranosidase. This enzyme preparation was separated into three fractions (I, II, and III) by butyl-Toyopearl 650 M column chromatography. The beta-fructofuranosidase I was purified to homogeneity by disc-electrophoresis after consecutive column chromatographies. The enzyme had a molecular weight of 52,000 by SDS-polyacrylamide gel electrophoresis and 51,000 by gel filtration with Ultrogel AcA 44, and an isoelectric point of 4.3. The enzyme was most active at pH 6.5-6.8 and at 55 degrees C and stable up to 45 degrees C at pH 6.5 for 30 min of incubation, and from pH 5.5 to 10.0 at 40 degrees C in 2 hr of incubation. This enzyme hydrolyzed sucrose, erlose, raffinose, fructosylxyloside, neokestose, and stachyose at the relative velocities of 100, 75, 61, 59, 54, and 11, but hardly hydrolyzed 1-kestose and nystose (less than 0.02).


Purification and some properties of endo-1,3-beta-D-xylanase from Pseudomonas sp. PT-5.

An endo-1,3-beta-D-xylanase (1,3-beta-D-xylan xylanohydrolase, EC 3.2.1.32) was purified from the culture fluid of Pseudomonas sp. PT-5 by ammonium sulfate fractionation, DEAE-Sepharose CL-6B, Toyopearl HW-50S, and Butyl-Toyopearl 650 M column chromatography. The purified enzyme gave a single band on polyacrylamide gel disc electrophoresis and its molecular weight was 35,000 by SDS-polyacrylamide gel electrophoresis. The enzyme was stable from pH 5.5 to 8.0 and had its maximum activity at pH 7.5. The enzyme rapidly reduced the viscosity of glycol beta-1,3-xylan solutions and produced xylose and xylooligosaccharides from seaweed beta-1,3-xylan. The enzyme activity was greatly inhibited by Hg2+, SDS, ethylenediamine tetraacetic acid (EDTA), and N-bromosuccinimide (NBS).


Protoplast transfection of Lactobacillus casei by phage PL-1 DNA.

Polyethylene glycol (PEG) mediated transfection of Lactobacillus casei ATCC 27092 protoplasts by phage PL-1 DNA was done. The protoplasts were obtained by treatment with purified PL-1 phage N-acetylmuramidase in the presence of citrate. Optimum conditions for transfection were 50% PEG 4,000, 15 micrograms protamine sulfate/ml, 0.15 M sucrose, and 10 mM MgSO4 in MR medium (pH 6.0). The extent of transfection was proportional to the amounts of DNA added, and the greatest efficiency of transfection after a 10-min incubation was about 3.3 x 10(5) PFU/micrograms DNA. The eclipse period of growth of progeny phages in the transfectants was 3 hr and the average burst size was 200.


Expression of recombinant bovine beta-lactoglobulin in Escherichia coli.

Bovine beta-lactoglobulin A was expressed in Escherichia coli in its mature form. The gene was constructed using a cDNA clone which coded for amino acid residues Leu-11 to Ile-162 and a synthetic oligonucleotide coding for the initial 10 amino acids preceded by a translational start. The met-beta-lactoglobulin was expressed using a tac promoter vector, pTTQ18, and accounted for approximately 15% of the total cellular protein. The recombinant met-beta-lactoglobulin migrated with the same molecular weight as native beta-lactoglobulin A on SDS-PAGE. The majority of the met-beta-lactoglobulin produced was found in an insoluble form but could be solubilized using guanidine-HCl. The renatured preparation was greater than 80% pure and migrated similarly to purified beta-lactoglobulin A under nondenaturing conditions.


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

The KHR gene cloned from a genomic library was on 4.7-kbp DNA fragment and was inserted into YCpG11 vector (KHR-YCp) and YEp vector (KHP-YEp). Transformants with KHR-YEp could secrete 3-4 times as much killer toxin into the media as the donor strain. The complete nucleotide sequence of the KHR gene was analyzed. It was found that the KHR gene consisted of 888 bp. It was suggested that this protein was processed before being secreted into the media, because its molecular mass presumed from the nucleotide sequence was larger than that of the mature killer toxin.


Purification and some properties of dipeptidyl carboxypeptidase from Bacillus pumilus.

An intracellular protease from a bacterium, Bacillus pumilus HL721, was purified about 5000-fold by chromatography with a Q-Sepharose Fast Flow column, TSK-gel HA-1000 glass column, and TSK-gel G3000SWXL column using Bz-Gly-Ala-Pro as a substrate. The enzyme was the most active at pH around 7.5 and stable from 4.5 and 8.0. The enzyme activity was inhibited by Cu2+, EDTA, N-ethylmaleimide, o-phenanthroline, and p-chloromercuribenzoic acid. The molecular weight of the enzyme was 155,000 by gel filtration. The enzyme removed dipeptide from the carboxyl end of some peptides used as substrates. From these results the enzyme seems to be a dipeptidyl carboxypeptidase.


A practical test for in vitro evaluation of photosensitization: assessment with hematoporphyrin derivative (HPD).

A simple procedure is described for the determination of the photosensitizing potency of drugs, using three leukemic cell lines, two of lymphocytic origin, L1210 and P388 and one of erythroid type, Friend-745. The procedure allows one to investigate several aspects of the photosensitization properties of tested compounds such as cellular localization and direct (trypan blue exclusion) or delayed (clonogenicity) photomediated toxicities. The method was assessed using crude hematoporphyrin derivative (HPD) as well as dihematoporphyrin ether (DHE) or commercially available Photofrin II. Results were compared to those obtained with normal cells, e.g spleen lymphocytes and erythropoietic stem cells (CFU-e), and discussed in the light of the relative response of normal versus transformed cells.


Soluble receptors for IL-1 and IL-4: biological activity and therapeutic potential.

Cytokine research has yielded a range of products which have now reached the stage of clinical trials and a plethora of novel therapeutics may be expected. Recently, cytokine receptors have also become an area of intensive research. Preliminary results indicate that recombinant soluble receptors can interfere with the biological functions of cytokines and thus may be appropriate for the treatment of certain pathological conditions where cytokine activity needs to be modulated.


Mechanism for the bitter tasting potency of peptides using O-aminoacyl sugars as model compounds.

In order to study the role of hydrophobicity in bitter peptides, several O-aminoacyl sugars, in which amino acids or peptides were attached to the 2- and 3-position of methyl alpha-D-glucopyranoside, were synthesized and sensory analyses were carried out. It was found that the bitterness increased as the hydrophobicity of compounds increased, implying that the bitterness receptor recognizes the hydrophobicity of bitter peptides. A structure for the bitterness receptor is also discussed.


Recovery of Clostridium thermosulfurogenes produced beta-amylase by (hydroxypropyl)methylcellulose partition.

A procedure for recovering Clostridium thermosulfurogenes produced beta-amylase from fermentation broth by partition was developed. The partition was achieved by addition of ammonium sulfate to an aqueous solution of the enzyme with (hydroxypropyl)methylcellulose. The beta-amylase-containing pellet formed upon centrifugation could be redissolved and the polymer recovered by a second salt addition. The process was not dependent on polymer/enzyme solution pH, but it was affected by temperature, polymer nominal molecular weight and loading, and fermentation carbon source. Unlike more traditional aqueous-phase partitions, such as poly(ethylene glycol)/dextran, the current approach appeared to be biospecific.


Immobilization of fatty acid synthetase from Mycobacterium smegmatis by radiation-induced polymerization.

Fatty acid synthetase of Type I from Mycobacterium smegmatis was immobilized by radiation-induced polymerization of 2-hydroxyethyl methacrylate (HEMA) in the presence of trimethylolpropane trimethacrylate (TMPTMA). The stability of immobilized synthetase toward low ionic strength increased in comparison with the free form, but the stabilities of immobilized preparations assessed by pH and temperature were identical to those of the free form. The apparent Km of immobilized enzyme for acetyl-CoA and malonyl-CoA were both 6 microM, essentially the same as those of the free form; acetyl-CoA, 5 microM and malonyl-CoA, 6 microM.


Continuous synthesis of a tripeptide by successive condensation and transesterification catalyzed by two immobilized proteinases in organic solvent.

The tripeptide Z-GlyPheLeuNH2 was continuously synthesized in a high yield from three amino acid derivatives, Z-Gly, PheOMe, and LeuNH2, by immobilized thermolysin (IMT) and immobilized alpha-chymotrypsin (IMC) in an organic solvent, ethyl acetate. The optimal conditions for the synthesis of Z-GlyPheOMe were established theoretically. The yield of Z-GlyPheOMe with IMT in ethyl acetate saturated with buffer was more than 88% after continuous synthesis for 116 hr. The optimal conditions for the synthesis of Z-GlyPheLeuNH2 from Z-GlyPheOMe and LeuNH2 by IMC through transesterification was established in batch reaction experiments. When the concentration of water in the reaction solution was 17-20 microliters/ml, the activity of IMC was highest. The equilibrium between the water concentration in the reaction solution and that in the resin used for enzyme immobilization depended on the resin and was not affected by the presence of the enzyme immobilized. Z-GlyPheLeuNH2 was synthesized from Z-GlyPheOMe and LeuNH2 with a yield of 100%, by continuous reaction for 160 hr. The reactor for synthesis of this tripeptide was efficient and stable because of the use of transesterification and the choice of an appropriate organic solvent. The series plug-flow reactor was successfully operated for 220 hr with a yield of more than 80%. The residual activity of IMT was 94% and that of IMC was 100%.


Lambda vectors for stable cloned gene expression.

The bacteriophage lambda offers a unique opportunity concurrently to minimize segregational instability in recombinant systems by chromosomal integration of the cloned gene and to achieve high cloned gene expression during an abortive lytic phase. Lysis leads approximately to a 100-fold amplification of the cloned gene. Cell lysis in the lytic state is blocked by a specific mutation (Sam), allowing the cell to maintain its integrity, and lambda DNA packaging is blocked by other mutations (Wam, Eam) that keep cloned genes open to transcription. In the presence of these mutations, extremely high levels of cloned beta-galactosidase (more than 15% of total cell protein) have been obtained during abortive lysis from vectors found to be essentially 100% stable for over 75 generations in the lysogenic phase.


Cloning and expression of the creatinase gene from Flavobacterium sp. U-188 in Escherichia coli.

The gene coding for creatinase (creatine amidinohydrolase, EC 3.5.3.3) was isolated from Flavobacterium sp. U-188. The primary structure of creatinase deduced from the nucleotide sequence showed a protein (molecular weight, 42, 651) composed of 378 amino acids. The creatinase gene was over-expressed in Escherichia coli under the control of the lac promoter and the amount of this enzyme was over 20% of the soluble protein in the cell.


Leucine dehydrogenase from Corynebacterium pseudodiphtheriticum: purification and characterization.

Leucine dehydrogenase [EC 1.4.1.9] was purified to homogeneity from Corynebacterium pseudodiphtheriticum ICR 2210. The enzyme consisted of a single polypeptide with a molecular weight of about 34,000. Stepwise Edman degradation provided the N-terminal sequence of the first 24 amino acids, and carboxypeptidase Y digestion provided the C-terminal sequence of the last 2 amino acids. Although the enzyme catalyzed the reversible deamination of various branched-chain L-amino acids, L-valine was the best substrate for oxidative deamination at pH 10.9 and the saturated concentration. The enzyme, however, had higher reactivity for L-leucine, and the kcat/Km value for L-leucine was higher than that for L-valine. The enzyme required NAD+ as a natural coenzyme. The NAD+ analogs 3-acetylpyridine-NAD+ and deamino-NAD+ were much better coenzymes than NAD+. The enzyme activity was significantly reduced by sulfhydryl reagents and pyridoxal 5'-phosphate. D-Enantiomers of the substrate amino acids competitively inhibited the oxidation of L-valine.


Chromosomal transformation in Saccharomyces cerevisiae with DNA isolated by pulse field gel electrophoresis.

We isolated and purified yeast chromosome DNA molecules using pulse field gel electrophoresis (PFG). The isolated DNA had nearly the same size as the native chromosomal DNA on PFG. We could directly transform Saccharomyces cerevisiae yeasts with it, and obtain transformants that were selected by complementation of several markers. They had new chromosome DNA bands observed on PFG. The new chromosome was very stable during mitosis and mating processes, and each of the three homologous chromosomes in the derivative zygotes of transformants was separated equally in daughter cells.


Solid-phase synthesis and crystallization of monellin, an intensely sweet protein.

Monellin, a sweet protein, consists of two noncovalently associated polypeptide chains, the A chain of 44 amino acid residues and the B chain of 50 residues. Two different primary structures have been reported for each of the A and B chains. The A and B chains corresponding to one of the reported monellin structures were synthesized by the stepwise solid-phase method using the Fmoc strategy in overall yields of 14.1% and 5.6%, respectively. The characterization of the synthetic peptides by HPLC, FAB-MS, amino acid analysis and sequencing fully supported the expected structures. The individual synthetic A and B chains were not sweet. Combination of the two chains, and subsequent HPLC purification gave monellin in a yield of 53.9%. The synthetic monellin had a distinct, lingering sweet taste (4000 times sweeter than sucrose) and was crystallized by a vapor diffusion method. The synthetic product was identical to natural monellin by HPLC, but not by tryptic mapping. These results indicate that the reported structure for monellin differs slightly from that of natural monellin.


Syntheses and biological activities of (+/-)-streptovitacin A and E-73.

(+/-)-Streptovitacin A (1) and its stereoisomers were synthesized by an aldol reaction of (+/-)-2,4-dimethyl-4-trimethylsiloxy-1-cyclohexanones (4b and 9) with 4-(2-oxoethyl)-2,6-piperidinedione (5). E-72 (2) was derived from synthetic 1. (+/-)-1 showed moderate growth inhibition against fungi and lettuce seeds.


Transformation of Lactobacillus helveticus subsp. jugurti with plasmid pLHR by electroporation.

Lactobacillus helveticus subsp. jugurti was transformed with plasmid, pLHR (8.5 kilobases), by electroporation. The plasmid, pLHR, consists of a cryptic plasmid, pLJ1, from L. helveticus subsp. jugurti, the Escherichia coli vector pBR329, and the erythromycin resistance gene of pAM beta 1 from Enterococcus faecalis. Maximum transformation efficiency of 1.3 x 10(4) transformants per microgram of DNA was obtained by exposure to a pulse with an exponential decay waveform at 4 kV/cm with 25 microF capacitance. The presence of glycine in the growth medium was essential for transformation. Plasmid DNA isolated from transformants had not undergone detectable rearrangements or deletions. In addition, it was found that L. helveticus subsp. jugurti has a restriction and modification system.


A new cDNA clone relating to larger molecular species of rat insulin-like growth factor-I mRNA.

A new cDNA clone for rat IGF-I mRNA was obtained. The cDNA contained a new base sequence as cDNA in the 3' region. The sequence coincided with a part of the sequence reported earlier by Shimatsu and Rotwein in exon 5 of the rat IGF-I gene. The presence of this cDNA proved the previous suggestion to be true that the size heterogeneity of IGF-I mRNA is primarily due to the heterogeneity of the 3'-untranslated region.


Molecular cloning, nucleotide sequence and expression in Escherichia coli of the beta-cyclodextrin glycosyltransferase gene from Bacillus circulans strain no. 8.

The beta-cyclodextrin glycosyltransferase (beta-CGTase) gene was isolated from a lambda-library prepared from Bacillus circulans strain no. 8. It was subcloned into plasmid pTZ and expressed by its endogenous regulatory sequences in Escherichia coli JM 103. The structural gene was sequenced and showed an open reading frame for a polypeptide of 718 amino acid residues. The recombinant beta-CGTase had the same enzymatic properties as the extracellular CGTase (684 amino acid residues, corresponding to a mol. wt. of 74416) produced by B. circulans strain no. 8. The amino acid sequence showed the highest homology (74.6% identical amino acids) with the CGTase of B. circulans strain F-2, which had been erroneously described as an amylase. The homology with the enzyme from the alkalophilic Bacillus sp. strain no. 1011 was 71.4%. The amino acid sequence derived will be used for elucidating the three-dimensional structure of the enzyme.


Synthesis and biological activities of griseofulvin analogs.

In order to elucidate the structure-activity relationship of griseofulvin (1), (+/-)-6-demethyl analog (3), 2-demethoxy-6-demethyldihydro analog (4), (+/-)-dechloro-6-ethyl analog (5), (+/-)-dechloro-6-epi-ethyl analog (6), (+/-)6-ethyl analog (7) and (+/-)-6-epi-ethyl analog (8) were synthesized by a Diels-Alder cycloaddition of alkylidene ketones (16, 17 18, 19 and 20) with modified 1,3-butadienes (21 or 22). Their biological activities were examined against fungi.


Complete amino acid sequence 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. Two different primary structures have been reported for each of these chains. The complete amino acid sequence of monellin was determined by a combination of FAB- and ESI-mass spectrometry, and by automatic Edman degradation.


Evidence of introduction by molecular cloning of artificial inverted sequence at the 5' terminus of the sense strand of rat insulin-like growth factor-I cDNA.

A cDNA of insulin-like growth factor (IGF)-I mRNA was obtained from a rat liver cDNA library. This cDNA contained a 5' terminal sequence of 48 base pairs complementary to a sequence in the 3' terminal region (an inverted repeat sequence) except for one nucleotide insertion in the 3' region. Base sequence analysis of this cDNA, northern blot analysis of rat liver mRNA using a probe specific to 5' region of cDNA, base sequence studies of the 5' upstream region of a clone of the rat IGF-I gene, and some other studies suggested strongly that the inverted repeat sequence of the IGF-I cDNA used was introduced artificially during preparation of the cDNA library.


A cis-acting locus needed for stable maintenance of the yeast plasmid pSB3.

The Zygosaccharomyces rouxii plasmid pSB3 is one of the yeast plasmids resembling 2-microns DNA. The presence of a cis-acting locus needed for stable maintenance of pSB3 has been expected by analogy with the functions encoded by other yeast plasmids such as 2-microns DNA and pSR1. We found such a locus and designated it STB-SB3. The STB-SB3 locus was delimited within 371 bp in a unique region of pSB3. This region is included in the plasmid region where no transcript was detected. No prominent feature of the nucleotide sequence was found in this region except for a direct repeat with 65% homology consisting of 27 bp and 29 bp. This is in clear contrast with the structure of the STB locus of 2-microns DNA which has five and a half tandem repeats consisting of a highly homologous 62 bp sequence. By analyzing the sites hypersensitive to nucleases using partially purified nuclei containing pSB3, the STB-SB3 locus was found not to be tightly folded into a nucleosome structure.


The amino acid sequence of Lady Amherst's pheasant (Chrysolophus amherstiae) and golden pheasant (Chrysolophus pictus) egg-white lysozymes.

The amino acids of Lady Amherst's pheasant and golden pheasant egg-white lysozymes have been sequenced. The carboxymethylated lysozymes were digested with trypsin followed by sequencing of the tryptic peptides. Lady Amherst's pheasant lysozyme proved to consist of 129 amino acid residues, and a relative molecular mass of 14,423 Da was calculated. This lysozyme had 6 amino acids substitutions when compared with hen egg-white lysozyme: Phe3 to Tyr, His15 to Leu, Gln41 to His, Asn77 to His, Gln 121 to Asn, and a newly found substitution of Ile124 to Thr. The amino acid sequence of golden pheasant lysozyme was identical to that of Lady Amherst's phesant lysozyme. The phylogenetic tree constructured by the comparison of amino acid sequences of phasianoid birds lysozymes revealed a minimum genetic distance between these pheasants and the turkey-peafowl group.


Enhancing effect of malondialdehyde modification on the mouse IgE response to protein antigens.

To investigate the immunochemical properties of proteins reacted with malondialdehyde (MDA), which is a major degradation product of lipid oxidation, the antibody responses of mice immunized with MDA-modified bovine serum albumin (BSA) were examined. The specific IgE response to MDA-BSA was 4 to 8 times higher than that to native BSA, suggesting that the specific IgE antibody response to MDA-modified protein was enhanced by MDA bound to the surface of the protein. The antibodies to MDA-BSA did not distinguish the modified protein from the native one, and the antibodies raised against the modified proteins were not specific for the MDA portion but were for the carrier proteins.


Molecular cloning and nucleotide sequence of the aminopeptidase T gene of Thermus aquaticus YT-1 and its high-level expression in Escherichia coli.

Aminopeptidase T (AP-T) is a metallo-dependent dimeric enzyme of Thermus aquaticus YT-1, an extremely thermophilic bacterium. We cloned the AP-T gene from T. aquaticus YT-1 into Escherichia coli using a synthetic oligonucleotide as a hybridization probe. The nucleotide sequence of the AP-T gene was found to encode 408 amino acid residues with GTG as a start codon. The molecular weight was calculated to be 44,820. The AP-T was overproduced in E. coli (about 5% of total soluble protein) when the start codon of the gene was changed from GTG to ATG, and the gene was downstream from the tac promoter. The AP-T expressed in E. coli was heat stable and easily purified by heat treatment (80 degrees C, 30 min). The N-terminal amino acid sequence of AP-T showed similarity with that of aminopeptidase II from Bacillus stearothermophilus.


Ren-2 as well as Ren-1 renin exists in the kidney and plasma of a two-renin gene mouse.

Ren-1 renin is synthesized in the kidney of every mouse. Ren-2 renin has been observed in the submandibular gland (SMG) of male mice carrying two renin genes. However, it is not known if Ren-2 renin is in the kidney and blood of the two-renin gene mice. In this study, a direct ELISA for Ren-2 renin (SMG renin) was established by a sandwich method. This ELISA could measure the Ren-2 active renin in the range from 1 to 100 ng and distinguish Ren-2 active from not only Ren-1 renin but also Ren-2 prorenin. By a combination of this assay system and conventional methods, the pro-form as well as the active form of Ren-2 renin was found in the kidney and plasma of male AKR mice carrying two-renin genes.


Stabilization of tumor antigens by chemical modification with diethyl pyrocarbonate.

C3H mice were immunized with MH134 tumors modified with two different chemicals, DEPC and TNBS, and the tumor-neutralizing activities of the spleen cells were examined after separating the cells by a Nylon-wool column, plastic dish, or treatment with specific antibodies (anti-mouse IgG and anti-mouse theta sera). The effector cells induced in mice by immunization with TNBS-treated tumors were T cells. In contrast, in the case of tumors modified with DEPC, macrophages were the major effector cells. The effects of chemical modification on the solubilization of cell surface proteins was also examined. DEPC inhibited solubilization of specific proteins from tumor cell surfaces, while TNBS did not have such an effect on the solubilization of proteins. These results indicate that the antitumor response in mice primed with DEPC-modified tumor is different from the case of TNBS-modified tumors, and that the immunological significance of chemical modification with DEPC is supposedly due to the stabilization of antigenic proteins on the tumor cell surface.


N-terminal extension of sweet peptides in relation to the structural features of peptide sweeteners.

Sweet aspartyl di- and tripeptide esters were extended toward the N-terminus in relation to the structural features of sweet peptides. The sweet peptides were designed on the basis of the receptor site model. It was found that an extension of the sweet aspartyl dipeptide esters by adding a small D-amino acid residue mostly gave sweet compounds (e.g., D-Ala-L-Asp-D-Ala-OMe), although this significantly decreased their sweetness potencies. Further extension at the N-terminus of the extended sweet tripeptide esters to yield the tetrapeptide esters resulted in a loss of the sweet taste. The N-terminal extension of sweet aspartyl tripeptide esters resulted in faintly sweet or nonsweet tetrapeptide esters. Interestingly, an analogous extension at the N-terminus of the sweet aminomalonyl dipeptide esters gave bitter compounds (e.g., D-Ala-DL-Ama-L-Phe-OMe). These results indicate that the receptor has a small space that can accomodate an additional small D-amino acid residue at the site facing the N-terminus of sweet aspartyl dipeptide esters.


FAB-MS/MS spectrometry in determining the primary structure of gamma-glutamyl-containing peptides.

Positive fast atom bombardment tandem mass spectrometry (FAB-MS/MS) was applied for peptide sequencing, particularly for determining the gamma glutamyl linkage involved in metal-binding peptides such as Cadystin (gamma EC)3G = Cadystin A and Cadystin (gamma EC)2G = Cadystin B (MW 771 and 539, respectively). The fragmentation patterns between the natural gamma glutamyl peptide and the synthetic alpha glutamyl one were clearly distinguishable. FAB-MS/MS was proved to be a good method for determining these peptides, since it needed no chemical degradation and only a small amount of the peptide was needed for determination.


Occurrence of free O-phosphoserine and O-phosphothreonine in porcine liver.

The occurrence of free O-phosphoserine and O-phosphothreonine in porcine liver is demonstrated. These amino acids were separated from the tissue extracts by anion- and cation-exchange chromatography and thin-layer chromatography, and were identified by gas chromatography with flame photometric detection and gas chromatography-mass spectrometry. The contents of O-phosphoserine and O-phosphothreonine in the liver were estimated to be 377 +/- 13 ng/g and 115 +/- 2 ng/g, respectively.


High-level secretion of a Rhizopus niveus aspartic proteinase in Saccharomyces cerevisiae.

The gene encoding an extracellular Rhizopus niveus aspartic proteinase I (RNAP-I) was introduced into Saccharomyces cerevisiae. The yeast cell carrying a plasmid containing the intact RNAP-I gene under the control of the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene promoter of S. cerevisiae did not synthesize RNAP-I at all. On the other hand, when the intron of the RNAP-I gene had been removed from the gene in the plasmid, the cell secreted RNAP-I with high efficiency. Processing of the pro-sequence occurred at the same region of the pro-enzyme during cultivation as observed in the culture of R. niveus. Moreover, the promoter and the terminator of the original RNAP-I gene were found to be weakly functional in the yeast cell with respect to expression of the intronless RNAP-I gene, although the initiation and termination sites were heterogeneous. The effects of vector-types on the extracellular production of RNAP-I were also investigated.


Purification of a membrane glycoprotein with an inositol-containing phospholipid anchor from Dictyostelium discoideum.

Large-scale purification of a Dictyostelium discoideum cell surface glycoprotein, which is anchored in the membrane via a glycosylphosphatidylinositol (GPI) moiety, is described. The purification protocol involved four steps: separation of crude cell membranes by low-speed centrifugation, delipidization of these membranes using acetone, extraction of the membrane proteins using the detergent Octyl beta-D-thioglucopyranoside (OTP), and purification of a specific membrane protein by monoclonal antibody immunoaffinity chromatography. The protein purified, PsA (prespore-specific antigen), is a developmentally regulated membrane glycoprotein found on a subset of cells from the cellular slime mould, D. discoideum. The protocol provides an efficient, economical, and technically simple way to purify GPI proteins in sufficient quantities for structural and functional studies. PsA was recovered at a yield of about 60%; with a purity of 97%, the extraction of 1 x 10(10) cells (1.1 g dry weight) yielded about 0.5 mg PsA glycoprotein. Techniques are described for growing kilogram quantities of D. discoideum cells in stainless steel trays at little cost. D. discoideum has considerable potential as a novel expression system for the production of foreign membrane-associated proteins. The purification strategy provides a means of purifying other GPI proteins, including those produced by protein engineering techniques.


A novel isolation method for hen egg yolk antibody, "IgY".

A method for isolation of egg yolk immunoglobulin, IgG, a livetin protein, was investigated. Several natural gums (carrageenan and xanthan gum) were found to be effective for removal of yolk lipoprotein as a precipitate. The effect was pronounced with lambda-carrageenan and the lipid content in the supernatant after removal of the resulting precipitate was less than 0.4% of that of egg yolk. IgY remained in this supernatant, with a yield of 86%, and was isolated by chromatography on a column of DEAE-Sephacel followed by salting-out with sodium sulfate. IgY thus isolated was almost pure (98%) and the yield was 70 to 100 mg per egg.


Identification and characterization of a thermolabile antigen (TLAa, enolase) in Saccharomyces cerevisiae.

Five kinds of proteins of Saccharomyces cerevisiae were recently purified to a homogeneous state and identified as yeast cell surface antigens (TLAa, TLAb, TLAc, TLAd, and TLAe), but their physiological functions remained uncertain. In this paper, TLAa was identified as a yeast enolase (EC 4.2.1.11) from the following evidence: (1) molecular weights and amino acid compositions of both proteins were similar, (2) the N-terminal 22 amino acid sequences of both proteins were the same (3), TLAa had enolase activity, and (4) the authentic yeast enolase gave a positive reaction with anti-TLAa serum in the Ouchterlony immunodiffusion test and the immunoblotting test. Two isoproteins of TLAa (enolase) were separated by non-denatured polyacrylamide gel electrophoresis and detected by immunoblotting with anti-TLAa serum. The contents of the two isoproteins varied depending on the following culture conditions: glucose starvation, growth in the presence of non-fermentable carbon sources, and growth in media containing sodium chloride and 2-deoxyglucose. The contents did not vary, however, with heat shock treatment or with growth in media containing sodium azide, tunicamycin, or sorbitol. These results showed that TLAa was a cytoplasmic antigen, and that its synthesis was regulated by some environmental stresses.


Structure of di-O-alpha-maltosyl cyclodextrins produced from alpha-maltosylfluoride and cyclodextrins.

The structures of di-O-alpha-maltosyl beta-cyclodextrins ((G2)2-beta-CDs), which were produced from alpha-maltosylfluoride (alpha-G2F) and cyclodextrin (CD) by the transfer action of debranching enzymes, were examined by the enzymic method using Bacillus subtilis saccharifying alpha-amylase (BSA). (G2)2-beta-CD was converted to (G1)2-beta-CD by treatment with glucoamylase before the examination. BSA completely hydrolyzed (G1)2-beta-CD to produce glucose, 6(3)-O-alpha-glucosylmaltotriose, and 6(3),6(5)-di-O-alpha-glucosyl maltopentaose. (G2)2-beta-CD was the mixture of 6A,6C-di-O-alpha-maltosyl beta-CD and 6A,6D-di-O-alpha-maltosyl beta-CD. The ratio of A,C/A,D in (G2)2-beta-CD synthesized with Pseudomonas isoamylase and Aerobacter pullulanase were 40:60-45:55 and 30:70, respectively. The content of 6A,6C-di-O-alpha-maltosyl gamma-CD in (G2)2-gamma-CD synthesized by isoamylase was about 35%.


Isolation of restriction-reduced mutants from Streptomyces.

Restriction-reduced mutants were isolated from Streptomyces rosa subsp. notoensis KA301 and S. tanashiensis strain Kala which produce the benzoisochromanequinone antibiotics nanaomycin and kalafungin, respectively. The mutants of S. rosa, which can be transformed with a multi-copy plasmid and in which the actinophage Pa16 can propagate, were selected. They were transformed with a single-copy plasmid propagated in S. lividans TK24, and with its modified plasmid propagated in the mutant at higher efficiency. The mutants of S. tanashiensis were selected by their capability to be transformed with a multi-copy plasmid. The efficiency of transformation with a single-copy plasmid propagated in S. lividans TK24 was low, but was much increased by heating the protoplasts at 42 degrees C for 15 min prior to the transformation. These mutants derived from both strains probably lack at least one of their restriction systems.


Cloning and expression of endo-beta-1,3-glucanase gene from Flavobacterium dormitator in Escherichia coli and characterization of the gene product.

The beta-1,3-glucanase (1,3-beta-D-glucan glucanohydrolase, EC 3.2.1.6) gene from Flavobacterium dormitator var. glucanolyticae was cloned into Escherichia coli C600 with a vector plasmid, pBR322. The E. coli cells carrying a recombinant plasmid, pKU beta G1 (8.2 kb), showed a high beta-1,3-glucanase activity and a lytic activity on viable yeast cells. These activities were found in the periplasmic space of E. coli clone cells. Southern hybridization analysis showed that the cloned gene was derived from F. dormitator chromosomal DNA. The gene products were purified from the periplasmic fraction of E. coli by ammonium sulfate fractionation and ion-exchange chromatography. The purified enzymes were demonstrated to be identical with a lytic endo-beta-1,3-glucanase II and a nonlytic endo-beta-1,3-glucanase I from F. dormitator from their enzymological and immunological properties. In the E. coli cells, endo-beta-1,3-glucanase I was also formed by a proteolytic digestion of endo-beta-1,3-glucanase II during the cultivation as in F. dormitator. Thus, the only endo-beta-1,3-glucanase II was coded for in the cloned gene.


Hyper-heterogeneity of the N-terminus of recombinant BSF-2/IL-6 produced in CHO and NIH3T3 cells.

Recombinant human BSF-2 (B cell stimulatory factor 2/Interleukin 6; IL-6) proteins were purified from CHO and NIH3T3 cell cultures and characterized. The lectin binding patterns suggested that the proteins have N-linked oligosaccharide(s) with tri-antennary structure of bisecting GlcNAc. Their N-termini were highly heterogeneous; at least five closely related N-termini were detected. This N-terminal heterogeneity was not generated in the cell culture because no processing activity was found in the culture medium.


Molecular cloning of the glucoamylase gene of Aspergillus shirousami and its expression in Aspergillus oryzae.

The glucoamylase enzyme (GAase) gene from Aspergillus shirousami was cloned and sequenced from genomic and cDNA libraries. The genomic gene was located in the 5.4 kb EcoRI fragment. The deduced amino acid sequence of GAase contained 639 amino acid residues with a relative molecular mass of approximately 68,000 daltons (non-glycosylated form). The genomic gene of A. shirousami GAase was introduced into Aspergillus oryzae. These transformants had increased GAase and raw starch degradation (RSD) activity in culture media and in rice-koji extracts. Analysis by Southern, Northern, SDS-PAGE, and Western blot techniques confirmed the foreign gene was correctly transcribed, translated, and expressed in A. oryzae.


Isolation and some properties of acid phosphatase-1(1) from tomato leaves.

An isozyme of acid phosphatase-1, acid phosphatase-1(1), was purified from the leaves of tomato (Lycopersicon esculentum) to homogeneity and characterized. The purified enzyme was homogeneous on polyacrylamide gel electrophoresis with or without sodium dodecyl sulfate. The gel filtration analysis showed that the native molecule had a relative molecular mass of about 61 kilodaltons (kDa). The relative molecular mass of the subunit on gel electrophoresis with sodium dodecyl sulfate was about 32 kDa, indicating that the native form of the enzyme was a homodimer. It was suggested by periodic acid-Schiff staining on the gel that the enzyme was a glycoprotein. The Km for p-nitrophenylphosphate was 2.9 x 10(-3) M. The enzyme had a pH optimum of 4.5 in 0.15 M potassium acetate buffer with p-nitrophenylphosphate as a substrate. This enzyme was activated by divalent metal ions, such as Zn2+, Mg2+, and Mn2+. The N-terminal amino acids were sequenced after the purified enzyme was treated with pyroglutamylpeptidase. It was suggested that the N-terminal amino acid was pyroglutamate.


Preparation of polyclonal antibodies to an anti-tumor (1----3)-beta-D-glucan, schizophyllan.

Antibodies against Schizophyllan (SPG) or SPG conjugated with bovine serum albumin (BSA) were raised in rabbits by multiple subcutaneous immunization. The titer of SPG-reactive antibodies in the antiserum to SPG-BSA conjugate was significantly higher than in the antiserum to SPG as assessed by enzyme-linked immunosorbent assay (ELISA). The SPG-reactive antibodies purified by affinity chromatography using a Protein A-Sepharose CL-4B column interacted with the SPG-BSA conjugate spotted on a nitrocellulose membrane filter in a dose-dependent manner, but the anti-SPG antibodies did not interact with BSA. Immunocytochemical staining has also shown that the anti-SPG antibodies reacted with SPG taken up by mouse peritoneal macrophages.


The enzymatic and molecular characteristics of Saccharomycopsis alpha-amylase secreted from Saccharomyces cerevisiae.

The Saccharomycopsis fibuligera alpha-amylase (Sfamy) gene was expressed in Saccharomyces cerevisiae. The highest productivity of Sfamy was 70 mg per liter of culture broth. We purified Sfamy from the culture broth and identified the NH2 terminal primary sequence. This sequence suggests that the Sfamy gene product is synthesized as a pre-pro-precursor, and the pro-sequence is cleaved after a Lys-Arg sequence with the calpain-like endopeptidase encode by the KEX2 gene, resulting in mature Sfamy protein composed of 468 amino acids. Furthermore, the enzyme Sfamy is a glycoprotein in which one N-linked sugar chain containing mannose residues is attached to the Asn residue at the 198 position. The Km and kcat values were 1.1 x 10(-4) M and 1.4 x 10(2) sec-1, respectively, using amylose (the degree of polymerization n = 18) as a substrate. Moreover, the secondary structure, the location of the secondary elements including alpha-helix, beta-sheet, and loop, and tertiary structure were predicted theoretically on the basis of the molecular structure of Aspergillus oryzae alpha-amylase. Taka-amylase A (TAA). These results indicate that Sfamy protein is composed of main (M) and C-terminal (C) domains. The molecular structure of M domain closely resembles that of TAA, but the C domain appears to be more compact than that of TAA because of deletions at three regions forming turns and one region forming alpha-helix.


Quantification of physical and cyto-physiological conditions for the electrofusion of Saccharomyces cerevisiae.

Various conditions for obtaining hybrids of the auxotrophic mutants SH1509 and SH1512 of Saccharomyces cerevisiae by electrofusion were investigated. An AC field of 400 Vp/cm and a DC field of 2 square pulses (7 kV/cm; 60 microsec each) at an interval of 0.5 sec were effective. Treatment with 0.2 (SH1509) or 1.0 mg/ml (SH1512) Zymolyase for 1 or 1.5 hr was essential. As to the molarity of the osmotic stabilizer (sorbitol), the hybrid yield peaked at 0.6 M. The presence of CaCl2 (up to 0.4 mM) or 0.1 mM CaCl2 with 0.1 mM MgCl2 enhanced the yield. The temperature of the spheroplast suspension during pulsations also affected the yield, the most suitable temperature being 28 degrees C.


Cloning and nucleotide sequence of an esterase gene from Pseudomonas fluorescens and expression of the gene in Escherichia coli.

A gene coding for a novel esterase of Pseudomonas fluorescens was cloned in this study. DNA sequencing showed that the open reading frame is comprised of 708 nucleotides. The coding sequence of the gene is preceded by a potential Shine-Dalgarno sequence and by a promoter-like structure. Following the stop codon a structure reminiscent of the E. coli rho-independent terminator is present. The enzyme expressed in an E. coli clone was mostly in the periplasmic space, released to the outside of the cell by osmotic shock and purified to homogeneity by QAE-Sephadex A-50 and DEAE-Sepharose columns. The native form of the enzyme consisted of two identical subunits, each with a molecular weight of 27,000. By studying the properties and substrate specificity, the enzyme was classified as an arylesterase (EC 3.1.1.2).


Thermostable S-alkylcysteine alpha, beta-lyase from a thermophile: purification and properties.

S-Alkylcysteine alpha, beta-lyase was found in a thermophile, Bacillus sp. 41A, which was newly isolated from soil, and purified to homogeneity from the cell extract. The enzyme has a molecular weight of about 76,000, and is composed of two subunits identical in molecular weight (39,000). The enzyme requires pyridoxal 5'-phosphate as a coenzyme, and catalyzes alpha, beta-elimination of S-methyl-L-cysteine and its analogs such as S-ethyl-L-cysteine, L-djenkolate, L-cystine, Se-methyl-L-selenocysteine, and O-methyl-DL-serine. However, S-methyl-D-cysteine, D-cystine, L-methionine, and L-norleucine were inert. The enzyme also catalyzes the beta-replacement reaction of S-methyl-L-cysteine with various thiols to yield the corresponding S-substituted cysteines. In addition to S-methyl-L-cysteine, Se-methyl-L-selenocysteine and O-methyl-DL-serine also serve as beta-substituent acceptors in the beta-replacement reaction. The enzyme is most active at 70 degrees C and stable at high temperatures. Automated Edman degradation provided the N-terminal sequence of the first 44 amino acids. The amino acid sequence in the vicinity of the lysyl residue to which pyridoxal 5'-phosphate is bound, was -Lys-His-Gln-Arg- by Edman degradation of the pyridoxyl peptide obtained by digestion with trypsin after reduction with sodium borohydride.


Isolation and partial characterization of three protein-synthesis inhibitory proteins from the seeds of Luffa cylindrica.

Three new proteins which inhibit protein synthesis in rabbit reticulocyte lysates were isolated from an extract of sponge gourd (Luffa cylindrica) seeds by chromatography on a AF-Blue Toyopearl column followed by FPLC with a Mono S column. These three protein-synthesis inhibitory proteins (PSIs) have molecular masses of 19 kDa, 15 kDa, and 9 kDa, and were designated 19K-PSI, 15K-PSI, and 9K-PSI, respectively. Although the 19K-PSI had no effect on protein synthesis in HeLa cells, its inhibitory activity on the cell-free protein synthesis was 340- and 83-fold stronger than those of ricin A-chain and luffin-a, respectively, probably due to hydrolyzing mRNA. The inhibitory activities of 15K- and 9K-PSIs on the cell-free protein synthesis were weaker than those of ricin A-chain and luffin-a. The 19K-PSI was a glycoprotein having an ordinary amino acid composition, three intramolecular disulfide bonds and a blocked N-terminal residue, while the 15K-PSI was extraordinarily rich in glycine and the 9K-PSI in arginine and glutamic acid (and/or glutamine). The amino acid composition of 19K-PSI was: Ser27Glx3Gly164Tyr7Lys9His6, and that of 9K-PSI was: Asx3Glx25Pro2Gly5Lys2His2Arg25Trp3.