Publications
| Title | Abstract | Year Filter | PMID(sorted ascending) Filter |
|---|
| secretion of escherichia coli beta-lactamase from bacillus subtilis by the aid of alpha-amylase signal sequence. | we describe a secretion vector system for introducing foreign genes into bacillus subtilis. we constructed secretion vectors from the plasmid pub110 and the promoter and signal sequence region of the alpha-amylase gene from bacillus amyloliquefaciens. foreign structural genes can be inserted into the various vectors after the signal sequence region of the alpha-amylase gene. demonstrating secretion of a foreign gene product from bacillus, we here report that the escherichia coli beta-lactamase g ... | 1982 | 6182566 |
| molecular cloning of alpha-amylase gene from bacillus amyloliquefaciens and its expression in b. subtilis. | the gene coding for alpha-amylase from bacillus amyloliquefaciens was isolated by direct shotgun cloning using b. subtilis as a host. the genome of b. amyloliquefaciens was partially digested with the restriction endonuclease mboi and 2- to 5-kb fragments were isolated and joined to plasmid pub110. competent b. subtilis amylase-negative cells were transformed with the hybrid plasmids and kanamycin-resistant transformants were screened for the production of alpha-amylase. one of the transformants ... | 1982 | 6183169 |
| amino acid sequence of alpha-amylase from bacillus amyloliquefaciens deduced from the nucleotide sequence of the cloned gene. | we have isolated by molecular cloning the gene coding for the alpha-amylase (1,4-alpha-d-glucan glucanohydrolase, ec 3.2.1.1.) from bacillus amyloliquefaciens and determined its complete nucleotide sequence. the gene cloned in the plasmid pub110 using bacillus subtilis as a host, was contained in a 2.3-kilobase insert. starting from an atg initiator codon, an open reading frame comprising a total of 514 amino acids (1542 base pairs) was found within the cloned dna fragment. the gene region encod ... | 1983 | 6185474 |
| increased production of alpha-amylase by bacillus amyloliquefaciens in the presence of glycine. | the production of alpha-amylase by bacillus amyloliquefaciens increased by a factor of 300 when glycine was added to a chemically defined simple medium at the early-logarithmic phase of growth. glycine was not metabolized to a significant extent under the conditions used, but it considerably prevented the lowering of the ph of the culture. | 1983 | 6193759 |
| in vivo transcription initiation and termination sites of an alpha-amylase gene from bacillus amyloliquefaciens cloned in bacillus subtilis. | the alpha-amylase gene, originally isolated by molecular cloning from chromosomal dna of bacillus amyloliquefaciens, is efficiently expressed from its own promoter in a bacillus subtilis host when present in the multicopy plasmid vector pub110. the flanking regions of this gene were sequenced and the ends of the in vivo-generated messenger rna were mapped by the s1 procedure. outside the coding sequence, the mrna for alpha-amylase contains about 30 nucleotides at the 5' end and 51 nucleotides at ... | 1984 | 6210229 |
| bacillus subtilis-phage phi 1 overcomes host-controlled restriction by producing bamnx inhibitor protein. | bacillus amyloliquefaciens n produces two restriction enzymes, bamni and bamnx. subtilis-phage phi 1 is strongly restricted by bamnx. we isolated phi 1 rh, a mutant of phage phi 1, which overcame the bamnx-restriction by producing inhibitor. this inhibitor inactivated bamnx specifically and reversibly. the inhibitor directly interacted with bamnx and the inactivation might be the result of formation of a binary complex. the inhibitory activity was sensitive to treatment with trypsin. the molecul ... | 1980 | 6255284 |
| the specificity of the bacillus amyloliquefaciens intracellular serine protease: a comparison with the specificity of secretory subtilisins. | 1981 | 6258997 | |
| dna cloning in bacillus subtilis. iii. efficiency of random-segment cloning and insertional inactivation vectors. | random segments of bacillus amyloliquefaciens and yeast saccharomyces cerevisiae dna were used to determine two parameters pertinent to cloning in bacillus subtilis, the yield of hybrids and the mean size of cloned segments. 10(3) to 10(4) hybrids/micrograms of dna segments were obtained. hybrids represented 11--18% of transformants. mean m. wt. of cloned dna segments was about 1 x 10(6), substantially lower than 3 x 10(6) found for donor dnas after digestion with restriction endonucleases. we h ... | 1980 | 6260584 |
| chemical synthesis and molecular cloning of a stop oligonucleotide encoding an uga translation terminator in all three reading frames. | we have chemically synthesized an oligonucleotide 5'd(tgattgattga)3' 3'd(actaactaact)5' that encodes the translation termination codon tga in all three reading frames. after ligation of appropriate restriction endonuclease linkers to the ends, the double-stranded oligonucleotide (stop-oligonucleotide) was joined to the plasmid pbr322 between the ecori and bamhi, or hindiii and bamhi sites, and the hybrid plasmids were transformed into escherichia coli hb101. four different constructions were obt ... | 1983 | 6313480 |
| the characteristics of liver glucose-6-phosphatase in the envelope of isolated nuclei and microsomes are identical. | we have compared the characteristics of glucose-6-phosphatase (ec 3.1.3.9) in the envelope of purified nuclei and microsomes from rat liver. the latency of mannose-6-p hydrolysis, permeability to edta, and susceptibility of the enzyme to protease-mediated inactivation all indicated that the permeability barrier defined by the envelope in situ is significantly disrupted in isolated nuclei (i.e. in vitro). latency of mannose-6-p hydrolysis was demonstrated to provide a quantitative measure of the ... | 1983 | 6313670 |
| [isolation of the specific endonuclease bamh1 in the multicyclic cultivation of bacillus amyloliquefaciens]. | the present article deals with the study of the multicyclic process of the cultivation of b. amyloliquefaciens in a liquid medium prepared from natural raw materials and with obtaining restrictase bam h1 from the biomass with different microbiological characteristics. the selected medium has been found to be suitable for the realization of the multicyclic cultivation process on an industrial scale. the tendency towards a drop in the level of nonspecific nucleases simultaneously with a decrease i ... | 1983 | 6314712 |
| construction of a bacillus subtilis cloning vehicle with heterologous dna sequence. | a cloning vehicle, pftb91, for the bacillus subtilis host was constructed with dna fragments heterologous to the host chromosome. it consists of three dna fragments: (i) chromosomal dna of bacillus amyloliquefaciens which complements the leua and ilvc mutations in b. subtilis; (ii) a b. amyloliquefaciens plasmid dna that supplies an autonomously replicating function; and (iii) a hindiii fragment of staphylococcus aureus plasmid ptp5 that carries gene tetr, conferring the tetr phenotype. it has s ... | 1983 | 6315540 |
| cloning, sequencing, and secretion of bacillus amyloliquefaciens subtilisin in bacillus subtilis. | the subtilisin gene from b. amyloliquefaciens has been cloned and expressed under its own promoter on a high copy plasmid, pbs42, in bacillus subtilis i-168 (marburg strain). greater than 95 percent of the expressed protease activity is secreted, and the activity is sensitive to inhibition by phenylmethylsulfonyl fluoride as expected for subtilisin. bacillus subtilis transformants carrying the bacillus amyloliquefaciens subtilisin gene in pbs42 (called ps4) secreted large amounts of a protein no ... | 1983 | 6316278 |
| an active-site carboxyl group in liquefying alpha-amylase: specific chemical modification. | bacillus amyloliquefaciens alpha-amylase activity is ph-dependent and the plot log (vmax/km) versus ph implicated a carboxyl group of aspartic acid/glutamic acid at the active site. chemical modification of alpha-amylase with edc confirmed this view. further, analysis of inactivation kinetics showed that modification of a single carboxyl group led to complete loss of the enzymic activity. | 1984 | 6332649 |
| isolation and the 5'-end nucleotide sequence of bacillus licheniformis alpha-amylase gene. | we have isolated and determined the 5'-end nucleotide sequence of the alpha-amylase gene from bacillus licheniformis atcc 14580. the alpha-amylase produced by this strain is thermostable and of liquefying type. the gene was originally cloned in a bacteriophage lambda 1059 vector. a subclone containing a 5.3 x 10(3)-base insert in pbr322 was further characterized. the nucleotide sequence coding for the 5' end of the structural gene together with the sequence coding for the upstream control region ... | 1984 | 6334606 |
| distribution of heterogeneous and homologous plasmids in bacillus spp. | a total of 75 strains (including 5 reference strains) of bacillus amyloliquefaciens, b. cereus, b. circulans, b. licheniformis, b. megaterium, b. pumilus, b. sphaericus, b. subtilis, and b. thuringiensis and 36 species-unidentified bacillus strains were surveyed for plasmids by cesium chloride-ethidium bromide equilibrium centrifugation of cell lysates in a study of antibiotic resistance in host cells. of the 111 strains, 13 (including 3 reference strains) were found to harbor plasmids, and 5 of ... | 1983 | 6410988 |
| replacement of the bacillus subtilis subtilisin structural gene with an in vitro-derived deletion mutation. | the entire subtilisin structural gene from bacillus subtilis i168 has been cloned, and its nucleotide sequence has been determined. when expressed on a high-copy-number shuttle vector, a fivefold increase in serine protease activity was observed. the dna sequence of the gene is 80% homologous to the bacillus amyloliquefaciens subtilisin structural gene, and the translated mature coding sequence is 85% homologous to the published protein sequence of subtilisin bpn'. the chloramphenicol resistance ... | 1984 | 6427178 |
| nucleotide sequence of the 5' region of the bacillus licheniformis alpha-amylase gene: comparison with the b. amyloliquefaciens gene. | the dna sequence of the 5' region of the bacillus licheniformis alpha-amylase gene is reported. comparison of the inferred amino acid sequence of the b. licheniformis alpha-amylase gene with that of the bacillus amyloliquefaciens gene shows that whereas the amino acid sequences of the mature proteins have considerable homology, the sequences for the signal peptides are distinct. | 1984 | 6609154 |
| [specific features of intracellular serine proteinase of bacillus amyloliquefaciens on native and denatured protein substrates]. | the effects of intracellular serine proteinase from b. amyloliquefaciens and of extracellular subtilisin bpn' on native and denaturated protein substrates were compared. the substrate hydrolysis by the enzymes was determined by a method initiating possible participation of intracellular serine protinease in intracellular protein degradation. this approach consists in a prolonged treatment of the products obtained after proteolysis with carboxypeptidase a with a subsequent amino acid assay. it wa ... | 1982 | 6753953 |
| [immunologic comparison of intracellular and extracellular serine proteases from bacillus amyloliquefaciens and some other proteases]. | antibodies against intracellular serine protease and extracellular subtilisin bpn' were raised in rabbits. using these antibodies and antisera against subtilisin carlsberg and thermitase (serine protease from thermoactinomyces vulgaris), it was shown that the proteases of the subtilisin family possess a pronounced immunological variability. immunological studies demonstrated that the vegetative and sporulating b. amyloliquefaciens cells contain no long-lived protein precursor of intracellular se ... | 1981 | 6791708 |
| beta-1.3.-1.4-glucanase in spore-forming microorganisms. vi. genetic instability of beta-glucanase production in a high-producer strain of bacillus amyloliquefaciens grown in a chemostat. | a bacillus amyloliquefaciens strain high-producing for beta-1.3-1.4-glucanase has gradually lost the ability to produce this enzyme during long-time continuous cultivation, independent of the culture conditions. mutant strains isolated after long-term cultivation exhibited changed behaviour concerning extracellular enzyme formation and sporulation. by agarose gel electrophoresis of alkaline dna extracts isolated form original and mutant strains we demonstrate that the observed pleiotropic phenom ... | 1982 | 7123998 |
| [characteristics of hybrid genes coding functionally-active secretory metalloproteinases from bacilli]. | a set of different hybrid genes encoding functionally active enzymes was obtained by homologous recombination between the fragments of related bacillus amyloliquefaciens and bacillus brevis metalloprotease genes cloned in plasmid vector in tandem orientation. the nucleotide sequences of hybrid genes were analyzed. it was demonstrated that the presence even of short homologous regions is sufficient for effective recombination in bacillus cells. | 1995 | 7476942 |
| the role of glu-60 in the specificity of the recombinant ribonuclease from bacillus amyloliquefaciens (barnase) towards dinucleotides, poly(a) and rna. | a computer model of the complex between g2'p5'g and barnase, the recombinant ribonuclease of bacillus amyloliquefaciens, was constructed, based on the known structure of the complex rnaase t1.g2'p5'g. this model suggests that the conserved residue glu-60 plays an important role in the specificity of barnase for guanosine. a barnase mutant was therefore made in which glu-60 was replaced by gln. this mutation increases the km for the dinucleotides gpc and gpa, by a factor of 10, but does not chang ... | 1994 | 7516656 |
| probing enzymic transition state hydrophobicities. | hydrophobic interactions are important in numerous biological processes; however, the nature and extent of hydrophobic interactions in nonaqueous enzymology remain poorly defined. we have estimated the free energies of enzyme--substrate hydrophobic interactions for a model reaction catalyzed by subtilisin bpn'(from bacillus amyloliquefaciens) in various solvents. transition state stabilization of subtilisin in water has contributions from both ground state destabilization of hydrophobic substrat ... | 1995 | 7547973 |
| bacillus amyloliquefaciens possesses a second type i signal peptidase with extensive sequence similarity to other bacillus spases. | a second sips2(ba) gene was pcr cloned from bacillus amyloliquefaciens. the deduced aa sequence is similar to those of the spases of b. subtilis, b. amyloliquefaciens, and b. licheniformis and the domain structure of the gene has been preserved. a low level of monocistronic gene transcription could be shown using northern analysis. the sips2(ba) gene was mapped to a region downstream of an e. coli frua gene homologue and shown to express a 21 kda protein in escherichia coli. | 1995 | 7578273 |
| hybrid bacillus amyloliquefaciens x bacillus licheniformis alpha-amylases. construction, properties and sequence determinants. | a series of 33 single and mosaic hybrid alpha-amylases was constructed from the genes amyba or amyli, encoding the alpha-amylases from bacillus amyloliquefaciens (amyba) and bacillus licheniformis (amyli). the hybrid proteins, consisting of the entire alpha-amylase sequence with a variable portion of amyba or amyli origin, were characterized in order to find enzymes with new properties (thermostability, temperature and ph optima, and substrate specificity), and to localize the amino acid sequenc ... | 1995 | 7607219 |
| unfolding simulations of the 85-102 beta-hairpin of barnase. | molecular dynamics simulations are used to investigate the unfolding reaction of an isolated beta-hairpin formed by residues 85 to 102 of barnase, a ribonuclease from bacillus amyloliquefaciens. this peptide was considered following evidence from experimental studies that it may act as an initiation site for barnase folding by adopting a native-like conformation early during the folding process. three successive molecular dynamics simulations of about 300 ps each were carried out for an all-atom ... | 1995 | 7650741 |
| [primary structure of the intracellular serine proteinase from bacillus amyloliquefaciens. iii. amino acid sequence of peptides obtained by hydrolysis with a glu,asp-specific proteinase. reconstruction of the entire amino acid sequence of the proteinase]. | glu,asp-specified protease hydrolysate of intracellular serine proteinase (isp) was separated by ion-exchange chromatography on a sulphocationite resin followed by hplc to yield 30 individual peptides. their sequences, spanning to 243 amino acid residues, were determined by the manual edman procedure. four overlapping fragments were reconstructed by comparing their sequences with those of tryptic and chymotryptic peptides. to arrange these fragments in the proteinase polypeptide chain and to rec ... | 1994 | 7695649 |
| a calorimetric study of the thermal stability of barstar and its interaction with barnase. | the temperature-induced unfolding of single, double, and triple mutants of barstar, the specific intracellular protein inhibitor of barnase from bacillus amyloliquefaciens, has been studied by high-sensitivity differential scanning calorimetry. the thermal unfolding of barstar mutants, where at least one of the two cysteine residues in the molecule had been replaced by alanine, follows a two-state mechanism at neutral and alkaline ph. the unfolding enthalpy and heat capacity changes are slightly ... | 1995 | 7711042 |
| influence of ca2+ on conformation and stability of three bacterial hybrid glucanases. | the three hybrid glucanases (1-12)amy x mac(13-214), (1-12)amy x des-tyr13mac(14-214); (1-16)amy x mac(17-214) are composed of short n-terminal segments of 12 or 16 amino acid residues derived from the bacillus amyloliquefaciens glucanase (amy) and of residues 13-214, 14-214 and 17-214, respectively, derived from the bacillus macerans enzyme (mac). the three proteins have similar conformational features as shown by the similar characteristics of their cd spectra in the far- and near-ultraviolet ... | 1995 | 7758469 |
| one-step enzymatic hydrolysis of starch using a recombinant strain of saccharomyces cerevisiae producing alpha-amylase, glucoamylase and pullulanase. | a recombinant strain of saccharomyces cerevisiae was constructed that contained the genes encoding a bacterial alpha-amylase (amy1), a yeast glucoamylase (sta2) and a bacterial pullulanase (pula). the bacillus amyloliquefaciens alpha-amylase and s. cerevisiae var. diastaticus glucoamylase genes were expressed in s. cerevisiae using their native promoters and the encoded enzymes secreted under direction of their native leader sequences. in contrast, the klebsiella pneumoniae pullulanase gene was ... | 1995 | 7766088 |
| modifications to the adh1 promoter of saccharomyces cerevisiae for efficient production of heterologous proteins. | the promoter of alcohol dehydrogenase i of the yeast saccharomyces cerevisiae was studied using bacillus amyloliquefaciens alpha-amylase as a marker protein. on glucose, activity of the original adh1 promoter decreases during late exponential, ethanol production growth phase. when 1100 bp (from -414 bp to -1500 bp) of the upstream sequence are deleted, activity increases into the late ethanol consumption phase but the promoter becomes active only after ethanol production growth phase (ruohonen e ... | 1995 | 7766401 |
| mechanism of solvent-induced thermal stabilization of alpha-amylase from bacillus amyloliquefaciens. | the transition temperature of irreversible thermal inactivation of alpha-amylase from bacillus amyloliquefaciens was estimated to be 60 degrees c. at this temperature, the enzyme inactivation followed first-order kinetics, having a half-life (t 1/2) of 12 min with a rate constant (k) of 0.06 min-1. conformational change was a prerequisite for this thermal inactivation. this is governed by stepwise temperature-dependent phenomena. among the solvent stabilizers tested, the enzyme was thermally sta ... | 1995 | 7782159 |
| dissociation constants and thermal stability of complexes of bacillus intermedius rnase and the protein inhibitor of bacillus amyloliquefaciens rnase. | binase, the extracellular ribonuclease of bacillus intermedius, is inhibited by barstar, the natural protein inhibitor of the homologous rnase, barnase, of b. intermedius. the dissociation constants of the binase complexes with barstar and its double cys40,82ala mutant are about 10(-12) m, only 5 to 43 times higher than those of the barnase-barstar complex. as with barnase, the denaturation temperature of binase is raised dramatically in the complex. calorimetric studies of the formation and sta ... | 1995 | 7789535 |
| an improved system for ribonuclease ba expression. | the extracellular ribonuclease from bacillus amyloliquefaciens (barnase, rnase ba) is a well-characterized enzyme extensively used in structure-function studies. a new system for efficient expression and purification of barnase has been developed. the strong regulated expression cassette with the pr promoter of lambda phage and the cooperative expression of barnase and barstar under its control have been applied to expression of these proteins in escherichia coli. the expression cassette contain ... | 1994 | 7858423 |
| stability and function: two constraints in the evolution of barstar and other proteins. | barstar is the intracellular inhibitor of barnase, an extracellular rnase of bacillus amyloliquefaciens. the dissociation constant of the barnase-barstar complex is 10(-14) m with an association rate constant between barnase and barstar of 3.7 x 10(8) s-1 m-1. the rapid association arises in part from the clustering of four acidic residues (asp35, asp39, glu76 and glu80) on the barnase-binding surface of barstar. the negatively charged barnase-binding surface of barstar effectively 'steers' the ... | 1994 | 7866746 |
| activation and cytotoxicity of 2-alpha-aminoacyl prodrugs of methotrexate. | in an effort to improve the selectivity of the anticancer drug methotrexate (mtx), a series of potential prodrugs in which the 2-amino group was acylated with various alpha-amino acids (as well as l-pyroglutamic acid) was synthesized. such derivatives are anticipated to be hydrolysed to mtx by appropriate aminopeptidases localized (over-expressed naturally or targeted as anti-tumor antibody conjugates) in the vicinity of the tumor. the l-leucyl, l-valyl, l-isoleucyl, d-alanyl and l-pyroglutamyl ... | 1995 | 7872963 |
| crystal structure of calcium-depleted bacillus licheniformis alpha-amylase at 2.2 a resolution. | the three-dimensional structure of the calcium-free form of bacillus licheniformis alpha-amylase (bla) has been determined by multiple isomorphous replacement in a crystal of space group p4(3)2(1)2 (a = b = 119.6 a, c = 85.4 a). the structure was refined using restrained crystallographic refinement to an r-factor of 0.177 for 28,147 independent reflections with intensities fobs > 0 at 2.2 a resolution, with root mean square deviations of 0.008 a and 1.4 degrees from ideal bond lengths and bond a ... | 1995 | 7877175 |
| characterization of the pcp gene of pseudomonas fluorescens and of its product, pyrrolidone carboxyl peptidase (pcp). | the gene pcp, encoding pyrrolidone carboxyl peptidase (pcp), from pseudomonas fluorescens mfo was cloned and its nucleotide sequence was determined. this sequence contains a unique open reading frame (pcp) coding for a polypeptide of 213 amino acids (m(r) 22,441) which has significant homology to the pcps from streptococcus pyogenes, bacillus subtilis, and bacillus amyloliquefaciens. comparison of the four pcp sequences revealed two highly conserved motifs which may be involved in the active sit ... | 1994 | 7909543 |
| the gene amye(tv1) codes for a nonglucogenic alpha-amylase from thermoactinomyces vulgaris 94-2a in bacillus subtilis. | we isolated the gene amye(tv1) from thermoactinomyces vulgaris 94-2a encoding a nonglucogenic alpha-amylase (amytv1). a chromosomal dna fragment of 2,247 bp contained an open reading frame of 483 codons, which was expressed in escherichia coli and bacillus subtilis. the deduced amino acid sequence of the amytv1 protein was confirmed by sequencing of several peptides derived from the enzyme isolated from a t. vulgaris 94-2a culture. the amino acid sequence was aligned with several known alpha-amy ... | 1994 | 7944369 |
| microcalorimetric determination of the thermostability of three hybrid (1-3,1-4)-beta-glucanases. | thermodynamic parameters of the three hybrid (1-3,1-4)-beta-glucanases h(a12-m), h(a12-m) delta y13, and h(a16-m) composed of short n-terminal regions derived from the bacillus amyloliquefaciens enzyme and a c-terminal region of the homologous bacillus macerans enzyme were determined in 2 mm sodium cacodylate ph 6.0, 1.5m guanidine hydrochloride, containing 1 mm cacl2 or 1 mm edta. melting of h(a12-m) delta y13 and h(a16-m) in the presence of calcium ions is characterized by two subtransitions; ... | 1994 | 7946082 |
| [primary structure of an intracellular serine proteinase from bacillus amyloliquefaciens. ii. amino acid sequence of peptides in a chymotrypsin hydrolysate]. | chymotrypsin hydrolyzate of the intracellular serine protease was separated by ion-exchange chromatography on a sulphocationite resin followed by hplc to yield fifty one individual peptides. their sequences, corresponding in total to 381 amino acid residues, were determined by the manual edman procedure. | 1994 | 8003042 |
| effect of modifying histidine residues on the action of bacillus amyloliquefaciens and barley-malt alpha-amylases. | modification of porcine pancreatic alpha-amylase (ppa) and taka-amylase a(taa) with diethyl pyrocarbonate (dep) causes activation of the release of p-nitrophenol from p-nitrophenol alpha-maltoside (g2pnp), and a decrease in amylase activity (hydrolysis of alpha-1,4 glucosidic bonds in starch). among the possible sites of modification, attention focuses on three histidine residues present around the active site of alpha-amylases of many different origins. in ppa these are his 101, his 201, and hi ... | 1994 | 8004636 |
| female sterile tobacco plants are produced by stigma-specific cell ablation. | we identified a tobacco stigma-specific gene, designated stig1. the stig1 gene is developmentally regulated and expressed specifically in the stigmatic secretory zone. we used a chimeric stig1-gus gene to show that the stigma-specific stig1 gene expression pattern is controlled primarily at the transcriptional level. we constructed a stigma-specific cytotoxic gene by fusing the stig1 gene 5' regulatory region with the coding sequence of the bacillus amyloliquefaciens barnase gene, to assess the ... | 1994 | 8039494 |
| [isolation of intracellular inhibitors of bacterial rnases on a column with immobilized bacillus intermedius rnase]. | intracellular inhibitors of rnases from bacillus amyloliquefaciens and bac. intermedius were isolated using affinity chromatography on covalently immobilized rnase from bac. intermedius. the inhibitor of rnase from bac. amyloliquefaciens was isolated from cells of e. coli hb 101 and purified to homogeneity. proteins with molecular weights of 70, 36 and 20 kd possessing an inhibitory activity were found in the extract obtained from frozen cells of bac. intermedius. | 1994 | 8047536 |
| [barnase mutant ser57ala: preparation and properties]. | barnase, an extracellular ribonuclease produced by bacillus amyloliquefaciens, belongs to a family of small microbial ribonucleases with similar structure and properties. these enzymes hydrolyze phosphodiester bonds on the 3' side of guanosine nucleotides in rna. the guanylic specificity of barnase is more pronounced in the hydrolysis of dinucleotides or cyclonucleotide phosphates as substrates than in the hydrolysis of rna or polynucleotides. to have an insight into the molecular basis of this ... | 1994 | 8052251 |
| effect of alteration of charged residues at the n termini of signal peptides on protein export in bacillus subtilis. | the role of positively charged residues at the n termini of signal peptides in protein export has been studied in bacillus subtilis. bacillus signal peptides (alkaline protease [apr] and neutral protease [npr] from bacillus amyloliquefaciens) were altered and fused to mature levansucrase (lvs). the effects of the various alterations on the export of lvs in b. subtilis were determined. the replacement of positively charged residues with neutral residues in both apr and npr signal peptides resulte ... | 1994 | 8083171 |
| pyroglutamyl peptidase gene from bacillus amyloliquefaciens: cloning, sequencing, expression, and crystallization of the expressed enzyme. | the pyroglutamyl peptidase [ec 3.4.11.8] gene from bacillus amyloliquefaciens was cloned and expressed in escherichia coli dh1. the transformant of e. coli dh1 harboring plasmid pbpg 1 with a 2.1 kb chromosomal dna fragment showed 80-fold higher activity than b. amyloliquefaciens. the nucleotide sequence of a 0.9 kb fragment that contains the promoter and the mature protein coding region was determined by the dideoxy chain-termination method. an open reading frame of 648 bp starting with an atg ... | 1993 | 8095933 |
| a calorimetric study of the thermal stability of barnase and its interaction with 3'gmp. | we have used high-sensitivity differential scanning calorimetry to characterize the thermal stability of barnase from bacillus amyloliquefaciens in the ph range 2.0-5.0. the energetics of the interaction between barnase and its inhibitor 3'gmp have been studied by isothermal titration calorimetry in the temperature range 15-30 degrees c. scanning calorimetry experiments were also made with the protein in the presence of various concentrations of 3'gmp at ph 4.5. a novel, simple procedure is prop ... | 1994 | 8142395 |
| different effects of n-glycosylation on the thermostability of highly homologous bacterial (1,3-1,4)-beta-glucanases secreted from yeast. | genes encoding bacillus amyloliquefaciens (1,3-1,4)-beta-glucanase (amy), b. macerans (1,3-1,4)-beta-glucanase (mac), and a series of hybrid enzymes containing n-terminal sequence segments of different length derived from amy with the remaining c-terminal segment derived from mac, were expressed in saccharomyces cerevisiae. the cells secreted active enzyme into the medium. while the quantity of n-glycan linked to the different enzymes was similar, pronounced differences in thermotolerance were o ... | 1994 | 8162185 |
| identification of active site carboxylic residues in bacillus licheniformis 1,3-1,4-beta-d-glucan 4-glucanohydrolase by site-directed mutagenesis. | active site residues of 1,3-1,4-beta-d-glucan 4-glucanohydrolase (ec 3.2.1.73) from bacillus licheniformis have been identified by site-directed mutagenesis. previous work revealed that glu-134 was essential for enzymatic activity, and it was proposed as the catalytic nucleophile by affinity labeling of the highly homologous bacillus amyloliquefaciens enzyme. to search for the general acid catalyst, the asp and glu residues conserved among the bacillus isozymes have been mutated to asn and gln, ... | 1994 | 8182059 |
| [cloning of the gene for extracellular bacillus circulans rnaase]. | the gene for extracellular low molecular weight ribonuclease of bacillus circulans bcf 247 was cloned. the strain was isolated from permafrost deposits of the kolyma lowland. the gene for the ribonuclease from bacillus intermedius (binase) was used as a specific probe. the cloning succeeded only in the e. coli strain producing the inhibitor of ribonuclease form bacillus amyloliquefaciens. selected clones secreted the active ribonuclease into the growth media. deletion derivatives of the parental ... | 1994 | 8183279 |
| bacillus mojavensis sp. nov., distinguishable from bacillus subtilis by sexual isolation, divergence in dna sequence, and differences in fatty acid composition. | a number of bacillus strains isolated from desert soil samples were shown to belong to a previously unidentified species, for which we propose the name bacillus mojavensis. the type strain is ro-h-1 (= nrrl b-14698). on the basis of restriction digest data, b. mojavensis is most closely related to bacillus amyloliquefaciens, bacillus atrophaeus, and bacillus subtilis. so far, b. mojavensis can be distinguished from b. subtilis only by differences in whole-cell fatty acid composition, divergence ... | 1994 | 8186089 |
| crystallization and preliminary x-ray analysis of restriction endonuclease bamhi-dna complex. | restriction endonuclease bamhi from bacillus amyloliquefaciens has been co-crystallized with a 12 bp dna fragment that encompasses its recognition site. the co-crystals diffract to at least 1.95 a resolution and belong to space group p2(1)2(1)2(1). the unit cell parameters are a = 108.8 a, b = 81.9 a, c = 68.8 a, consistent with one complex in the crystallographic asymmetric unit. the direction of the dna appears to be along the b axis. in order to achieve end to end stacking of dna, the complex ... | 1994 | 8201623 |
| crystallization and preliminary x-ray investigation of barstar, the intracellular inhibitor of barnase. | crystals of barstar, the intracellular inhibitor of the extracellular ribonuclease produced by bacillus amyloliquefaciens (barnase), were obtained through vapor phase equilibration using the hanging drop technique. three crystal forms have been characterized. forms i and ii, crystallized either in potassium phosphate or sodium citrate, are tetragonal; they exhibit a superstructure along the c-axis. form iii crystals, suitable for a high resolution structure determination, were grown from 55-65% ... | 1993 | 8272429 |
| equilibrium unfolding studies of barstar: evidence for an alternative conformation which resembles a molten globule. | the folding of the small protein barstar, which is the intracellular inhibitor to barnase in bacillus amyloliquefaciens, has been studied by equilibrium unfolding methods. barstar is shown to exist in two conformations: the a form, which exists at ph values lower than 4, and the n state, which exists at ph values above 5. the transition between the a form and the n state is completely reversible. uv absorbance spectroscopy, fluorescence spectroscopy, and circular dichroism spectroscopy were used ... | 1994 | 8286327 |
| regional sequence homologies in starch-degrading enzymes. | the enzymatic hydrolysis of starch, consisting of linear (amylose) and branched (amylopectin) glucose polymers, is catalyzed by alpha-, beta- and glucoamylases (gamma-amylases), cyclodextrinases, alpha-glucosidases, and debranching enzymes. saccharomyces cerevisiae cannot utilize starch. our laboratory has previously co-expressed the bacillus amyloliquefaciens alpha-amylase (amy) and the saccharomyces diastaticus glucoamylase (sta2) genes in s. cerevisiae. a gene encoding a debranching enzyme (p ... | 1993 | 8299155 |
| the roles of signal peptide and mature protein in rnase (barnase) export from bacillus subtilis. | barnase, an extracellular rnase from bacillus amyloliquefaciens is secreted post-translationally from b. subtilis. the rate of secretion of barnase from b. subtilis was improved by replacement of the barnase signal peptide with a heterologous signal peptide. however, the barnase signal peptide exported escherichia coli alkaline phosphatase faster than mature barnase. heat shock of b. subtilis cells did not significantly alter the export of barnase using the barnase signal peptide. the slow rate ... | 1993 | 8316212 |
| increase of specificity of rnase from bacillus amyloliquefaciens (barnase) by substitution of glu for ser57 using site-directed mutagenesis. | bacterial ribonucleases from bacillus amyloliquefaciens and bacillus intermedius show the specificity towards the nature of a nucleoside at the o3' end of the phosphodiester bond to be split in the preference order g > a >> u > c in the cleavage reactions of polynucleotides. it follows from the x-ray data that the substrate guanosine base is bound at the active site of these rnases in the same manner as for high-specificity guanylic rnases. we supposed that the difference in specificity for the ... | 1993 | 8344276 |
| folding of subtilisin bpn': role of the pro-sequence. | subtilisin bpn' is an extracellular serine protease from bacillus amyloliquefaciens that requires an n-terminal 77 amino acid pro-sequence for correct folding of the catalytic domain. we have expressed an inactive, stable pro-subtilisin variant in escherichia coli and show that it has structural properties similar to native subtilisin in terms of its near- and far-uv circular dichroism spectra, its compactness, and its capacity to bind calcium ions stoichiometrically. unlike subtilisin, the pro- ... | 1993 | 8377204 |
| determinants for the enhanced thermostability of hybrid (1-3,1-4)-beta-glucanases. | hybrid (1-3,1-4)-beta-glucanases which contain an n-terminal region derived from the bacillus amyloliquefaciens enzyme and a c-terminal region of the closely related b. macerans enzyme may exhibit a thermostability superior to both parental enzymes. a systematic series of hybrid enzymes were constructed in order to delineate the amino acid residues that affect protein stability. hybrid enzymes with between one and four of the n-terminal residues for the mature b. amyloliquefaciens (1-3,1-4)-beta ... | 1993 | 8404902 |
| identification of the barstar binding site of barnase by nmr spectroscopy and hydrogen-deuterium exchange. | the extracellular ribonuclease from bacillus amyloliquifaciens, barnase, forms a tightly-bound one-to-one complex with its intracellular inhibitor barstar. the barstar binding site on barnase was characterized by comparing the differences in the chemical shift and hydrogen-deuterium exchange rates between free and bound barnase. chemical shift assignments of barnase in the complex with barstar were determined from 3d noesy-hmqc and tocsy-hmqc spectra of a complex that had been prepared with unif ... | 1993 | 8405399 |
| folding of subtilisin bpn': characterization of a folding intermediate. | subtilisin bpn', an extracellular serine protease from bacillus amyloliquefaciens, requires a 77 amino acid pro-sequence for correct folding in vivo. we report the observation of a metastable folding intermediate during the refolding of wild-type and a proteolytically inactive mutant subtilisin bpn' that lack the pro-sequence. the addition of the pro-sequence as a separate polypeptide chain results in the folding of the intermediate to the native state. the intermediate state of subtilisin is st ... | 1993 | 8418836 |
| expression and secretion of bacillus amyloliquefaciens alpha-amylase by using the yeast pheromone alpha-factor promoter and leader sequence in saccharomyces cerevisiae. | replacement of the regulatory and secretory signals of the alpha-amylase gene (amy) from bacillus amylolique-faciens with the complete yeast pheromone alpha-factor prepro region (mf alpha 1p) resulted in increased levels of extracellular alpha-amylase production in saccharomyces cerevisiae. however, the removal of the (glu-ala)2 peptide from the mf alpha 1 spacer region (lys-arg-glu-ala-glu-ala) yielded decreased levels of extracellular alpha-amylase. | 1993 | 8476297 |
| [comparison of the heat stability and structure close homologs--bacillus amyloliquefaciens ribonuclease and bacillus intermedius 7p ribonuclease]. | parameters of heat denaturation and intrinsic fluorescence of barnase and its close homologue, binase, in the ph region 2-6 have been determined. barnase heat denaturation (ph 2.8-5.5) proceeds according to the "all-or-none" principle. barnase denaturation temperature is lower than that of binase and this difference increases from 2.5 degrees c at ph 5 to 7 degrees c at ph 3. enthalpy values of barnase and binase denaturation coincide only at ph 4.5-5.5, but as the ph decreases the barnase denat ... | 1993 | 8487771 |
| interaction of barnase with its polypeptide inhibitor barstar studied by protein engineering. | barnase, an extracellular ribonuclease of bacillus amyloliquefaciens, forms a very tight complex with its intracellular polypeptide inhibitor barstar. at ph 8, the values for the rate constants k1 (association) and k-1 (dissociation) are 6.0 x 10(8) s-1 m-1 and 8.0 x 10(-6) s-1, respectively. the value of ki, the dissociation constant of barstar and barnase, calculated from the ratio k-1/k1 is 1.3 x 10(-14) m, which corresponds to a delta g of -18.9 kcal/mol at 25 degrees c. the dissociation con ... | 1993 | 8494892 |
| directed mutagenesis and barnase-barstar recognition. | directed mutagenesis has been applied to the cloned genes of barnase and barstar, the extracellular ribonuclease of bacillus amyloliquefaciens and its intracellular inhibitor, to locate residues involved in the mutual recognition of these two proteins. arg59 and his102 of barnase and asp35 and asp39 of barstar have been so identified. with both cys40 and cys82 mutated to alanines, barstar is still produced in high yield and is functional both in vitro and in vivo. methods devised for determining ... | 1993 | 8507637 |
| photoreactivation in the genus bacillus. | photoreactivation of ultraviolet radiation-induced dna damage was examined in exponential-phase cells of six mesophilic species of the genus bacillus. under the experimental conditions used, it was observed that the laboratory strains b. cereus strain t and b. thuringiensis var. thuringiensis strain nrrl-b4039 exhibited strong photoreactivation (86-fold and 70-fold respectively). bacillus licheniformis strain atcc 8480 exhibited moderate (15-fold) photoreactivation. weak photoreactivation was ob ... | 1995 | 8528007 |
| [biosynthetic regulation of extracellular ribonucleases in native strains of bacilli and in recombinant strains of escherichia coli]. | regulation of the biosynthesis of extracellular ribonucleases of bacillus amyloliquefaciens h2 (barnase), bacillus intermedius 7p (binase), and bacillus pumilus kmm 62 (rnase bp) was studied in their native strains and recombinant escherichia coli strains. recombinant plasmids were obtained that contained genes encoding barnase, binase, and rnase bp under the control of their own regulatory sequences (plasmids pmt415, pml5, and pml61), genes encoding barnase and binase under the control of the t ... | 1995 | 8538511 |
| catalysis of amide proton exchange by the molecular chaperones groel and secb. | hydrogen-deuterium exchange of 39 amide protons of bacillus amyloliquefaciens ribonuclease (barnase) was analyzed by two-dimensional nuclear magnetic resonance in the presence of micromolar concentrations of the molecular chaperones groel and secb. both chaperones bound to native barnase under physiological conditions and catalyzed exchange of deeply buried amide protons with solvent. such exchange required complete unfolding of barnase, which occurred in the complex with the chaperones. subsequ ... | 1996 | 8571125 |
| subtilisin bpn' variants: increased hydrolytic activity on surface-bound substrates via decreased surface activity. | site-directed mutagenesis and random mutagenesis were used to produce variants of subtilisin bpn' (bacillus amyloliquefaciens) protease with variable surface adsorption properties. protease adsorption and peptide hydrolysis rate were measured for these variants using a model substrate consisting of a peptide covalently bound to a surface. while most variants adsorb at a level very similar to that of native bpn', several variants were identified which adsorb either more or less. for surface-bound ... | 1996 | 8605150 |
| protein-protein interaction: a genetic selection for compensating mutations at the barnase-barstar interface. | barnase and barstar are trivial names of the extracellular rnase and its intracellular inhibitor produced by bacillus amyloliquefaciens. inhibition involves the formation of a very tight one-to-one complex of the two proteins. with the crystallographic solution of the structure of the barnase-barstar complex and the development of methods for measuring the free energy of binding, the pair can be used to study protein-protein recognition in detail. in this report, we describe the isolation of sup ... | 1996 | 8637875 |
| studies on the activity of barnase toxins in vitro and in vivo. | pseudomonas exotoxin a (pe) is a protein toxin composed of three structural domains which are responsible for cell binding (domain ia, amino acids 1-252), translocation into the cytosol (domain ii, amino acids 253-364) and adp-ribosylation activity (domain iii, amino acids 405-613). we have previously described (prior, t. i., fitzgerald, d. j., and pastan, i. (1992) biochem. 31, 3555-3559) a molecule composed of amino acids 1-412 of pe and the extracellular ribonuclease of bacillus amyloliquefac ... | 1996 | 8741987 |
| experimental and theoretical study of electrostatic effects on the isoelectric ph and the pka of the catalytic residue his-102 of the recombinant ribonuclease from bacillus amyloliquefaciens (barnase). | barnase, the guanine specific ribonuclease of bacillus amyloliquefaciens, was subjected to mutations in order to alter the electrostatic properties of the enzyme. ser-85 was mutated into glu with the goal to introduce an extra charge in the neighborhood of his-102. a double mutation (ser-85-glu and asp-86-asn) was introduced with the same purpose but without altering the global charge of the enzyme. a similar set of mutations was made using asp at position 85. for all mutants the pi was determin ... | 1996 | 8778784 |
| direct selection of cloned dna in bacillus subtilis based on sucrose-induced lethality. | expression of the bacillus subtilis or bacillus amyloliquefaciens sacb gene in the presence of sucrose is lethal for a variety of bacteria. sucrose-induced lethality can be used to select for inactivation of sacb by insertion of heterologous dna in sensitive bacteria. this procedure has not been applicable to b. subtilis heretofore because expression of wild-type sacb is not detrimental to b. subtilis. the w29 mutation in the b. amyloliquefaciens sacb gene interferes with processing of the levan ... | 1996 | 8899981 |
| individual amino acids in the n-terminal loop region determine the thermostability and unfolding characteristics of bacterial glucanases. | thermostability and unfolding behavior of the wild-type (1,3-1,4)-beta-glucanases from bacillus macerans (mac) and bacillus amyloliquefaciens (amy) and of two hybrid enzymes h(a12-m) delta f14 and h(a12-m) delta y13f14a were studied by spectroscopic and microcalorimetric measurements. h(a12-m) delta f14 is constructed by the fusion of 12 n-terminal amino acids of amy with amino acids 13-214 of mac, and by deletion of f14. in h(a12-m) delta y13f14a, the n-terminal region of mac is exchanged again ... | 1996 | 8931144 |
| active-site titration of serine proteases using a fluoride ion selective electrode and sulfonyl fluoride inhibitors. | we report a general procedure for the determination of active enzyme concentrations for serine proteases. the method relies on the measurement of fluoride ion released from sulfonyl fluorides upon reaction with the active-site serine using an ion selective electrode. the results have been independently confirmed by amino acid analyses of subtilisins and by spectrofluorometric and spectrophotometric titrations. the minimal enzyme concentration detectable is 1-10 microm protease. the method is ins ... | 1996 | 8937565 |
| bacillus subtilis can modulate its capacity and specificity for protein secretion through temporally controlled expression of the sips gene for signal peptidase i. | bacillus subtilis contains three chromosomally encoded type i signal peptidases (sips, sipt and sipu), which remove signal peptides from secretory precursor proteins. in the present study the biological function of sips and the regulation of its synthesis were analysed. unlike the type i signal peptidase of escherichia coli, sips was essential neither for protein secretion nor viability of the cell. however, in the absence of sips the rate of processing of several preproteins was reduced, and fo ... | 1996 | 8951809 |
| relationship between thermal stability, degradation rate and expression yield of barnase variants in the periplasm of escherichia coli. | an advantage of exporting a recombinant protein to the periplasm of escherichia coli is decreased proteolysis in the periplasm compared with that in the cytoplasm. however, protein degradation in the periplasm also occurs. it has been widely accepted that the thermodynamic stability of a protein is an important factor for protein degradation in the cytoplasm of e.coli. to investigate the effect of the thermodynamic stability of an exported protein on the extent of proteolysis in the periplasm, b ... | 1996 | 9010933 |
| mechanical stability of compact modules of barnase. | globular proteins are composed of structural elements such as secondary structures and modules. modules are compact segments consisting of 10-40 contiguous amino acid residues and are often encoded by exons. therefore, the view that the modular organization of proteins is a result of exon-shuffling or -fusion is given support. secondary structures such as alpha-helix and beta-sheet are stabilized by hydrogen bonds and are thus considered to be stable, structural elements of a globular domain. si ... | 1997 | 9094422 |
| [effect of culture medium components on the accumulation of extracellular bacillary ribonucleases in culture fluid of recombinant strains of escherichia coli]. | the effect of the concentrations of peptone, yeast extract, and inorganic phosphate on the expression of genes of extracellular ribonucleases from bacillus intermedius 7p (binase) and bacillus amyloliquefaciens h2 (barnase) was studied in escherichia coli cells transformed with plasmids containing the structural genes of binase or barnase under the control of their own or synthetic regulatory region or the structural binase gene under the control of the regulatory regions of the genes of barnase ... | 1996 | 9102546 |
| nephrotoxic effects of bacterial ribonucleases in the isolated perfused rat kidney. | alterations of the renal function in the isolated perfused rat kidney system after application of two bacterial rnases, bacillus intermedius rnase (binase) and ribonuclease produced by bacillus amyloliquefaciens (barnase), were investigated with two different treatment regimens in comparison with catalytically inactive derivates of the enzymes, photooxidated at the active site his101 binase and inactive mutant his102gln barnase. for the in vitro approach the test enzymes were dissolved in the pe ... | 1997 | 9160109 |
| the role of glu73 of barnase in catalysis and the binding of barstar. | barnase, a small extracellular ribonuclease from bacillus amyloliquefaciens and its intracellular inhibitor barstar have co-evolved to bind tightly and rapidly. barnase has also evolved to be catalytically active. the active site of barnase and its binding site for barstar use the same subset of amino acids. the exception is glu73 (the general base in catalysis), which although located at the centre of the binding site, is separated by three ordered water molecules from barstar. we examined in t ... | 1997 | 9231905 |
| secretion of authentic 20-kda human growth hormone (20k hgh) in escherichia coli and properties of the purified product. | using bacillus amyloliquefaciens neutral protease gene (npr), we have constructed a secretion system of 20-kda human growth hormone (20k hgh) in e. coli. the secretion-signal region from npr was modified inserting a fragment coding a 2lys-5leu cluster. in this system we found that co-expression of glutathione reductase remarkably increased accumulation level of 20k hgh in periplasm and confirmed that secreted 20k hgh was correctly processed. the recombinant 20k hgh was highly purified and subjec ... | 1997 | 9232032 |
| rapid, sensitive, microbial detection by gene amplification using restriction endonuclease target sequences. | the use of primers synthesized to eight class ii restriction endonuclease target sequences, from haemophilus parainfluenzae, escherichia coli, staphylococcus aureus, salmonella infantis, rhodobacter sphaeroides, klebsiella pneumoniae, bacillus amyloliquefaciens and proteus vulgaris for single and multiplex pcr identification of the organisms is discussed. results indicate that the method is sensitive and specific enough to detect single cells and attogram amounts of target dna. it has also been ... | 1997 | 9281417 |
| [riboflavin biosynthetic genes in bacillus amyloliquefaciens: primary structure, organization and regulation of activity]. | 1997 | 9297088 | |
| bacillus subtilis contains four closely related type i signal peptidases with overlapping substrate specificities. constitutive and temporally controlled expression of different sip genes. | most biological membranes contain one or two type i signal peptidases for the removal of signal peptides from secretory precursor proteins. in this respect, the gram-positive bacterium bacillus subtilis seems to be exceptional, because it contains at least four chromosomally-encoded type i signal peptidases, denoted sips, sipt, sipu, and sipv. here, we report the identification of the sipt and sipv genes, and the functional characterization of sipt, sipu, and sipv. the four signal peptidases hav ... | 1997 | 9325333 |
| dsc studies of the conformational stability of barstar wild-type. | the temperature induced unfolding of barstar wild-type of bacillus amyloliquefaciens (90 residues) has been characterized by differential scanning microcalorimetry. the process has been found to be reversible in the ph range from 6.4 to 8.3 in the absence of oxygen. it has been clearly shown by a ratio of delta hvh/delta hcal near 1 that denaturation follows a two-state mechanism. for comparison, the c82a mutant was also studied. this mutant exhibits similar reversibility, but has a slightly low ... | 1997 | 9336842 |
| isolation and purification of two novel streptomycete rnase inhibitors, sai14 and sai20, and cloning, sequencing, and expression in escherichia coli of the gene coding for sai14. | two new rnase inhibitors, sai14 (mr, approximately 14,000) and sai20 (mr, approximately 20,000), were isolated and purified from a streptomyces aureofaciens strain. the gene sai14, coding for sai14 protein, was cloned and expressed in escherichia coli. the alignment of the deduced amino acid sequence of sai14 with that of barstar, the rnase inhibitor from bacillus amyloliquefaciens, showed significant similarity between them, especially in the region which contains most of the residues involved ... | 1998 | 9515932 |
| discrepancies between the nmr and x-ray structures of uncomplexed barstar: analysis suggests that packing densities of protein structures determined by nmr are unreliable. | the crystal structure of the c82a mutant of barstar, the intracellular inhibitor of the bacillus amyloliquefaciens ribonuclease barnase, has been solved to a resolution of 2.8 a. the molecule crystallizes in the space group i41 with a dimer in the asymmetric unit. an identical barstar dimer is also found in the crystal structure of the barnase-barstar complex. this structure of uncomplexed barstar is compared to the structure of barstar bound to barnase and also to the structure of barstar solve ... | 1998 | 9578582 |
| enzymatic properties of a novel liquefying alpha-amylase from an alkaliphilic bacillus isolate and entire nucleotide and amino acid sequences. | a novel liquefying alpha-amylase (lamy) was found in cultures of an alkaliphilic bacillus isolate, ksm-1378. the specific activity of purified lamy was approximately 5,000 u mg of protein-1, a value two- to fivefold greater between ph 5 and 10 than that of an industrial, thermostable bacillus licheniformis enzyme. the enzyme had a ph optimum of 8.0 to 8.5 and displayed maximum activity at 55 degreesc. the molecular mass deduced from sodium dodecyl sulfate-polyacrylamide gel electrophoresis was a ... | 1998 | 9726872 |
| recognition of rnase sa by the inhibitor barstar: structure of the complex at 1.7 a resolution. | we report the 1.7 a resolution structure of rnase sa complexed with the polypeptide inhibitor barstar. the crystals are in the hexagonal space group p65 with unit-cell dimensions a = b = 56.9, c = 135.8 a and the asymmetric unit contains one molecule of the complex. rnase sa is an extracellular microbial ribonuclease produced by streptomyces aureofaciens. barstar is the natural inhibitor of barnase, the ribonuclease of bacillus amyloliquefaciens. it inhibits rnase sa and barnase in a similar man ... | 1998 | 9757110 |
| crystallization and preliminary x-ray investigation of the complex of rnase sa with wild-type barstar. | rnase sa, an extracellular ribonuclease produced by streptomyces aureofaciens, is inhibited by barstar, the natural protein inhibitor of barnase, the ribonuclease of bacillus amyloliquefaciens. the complex of rnase sa with wild-type barstar was crystallized by hanging-drop vapour diffusion. it was shown by sodium dodecyl sulfate polyacrylamide gel electrophoresis that rnase sa and barstar are present in equimolar proportions in the crystals. the crystals are in the hexagonal space group p65 with ... | 1998 | 9761909 |
| conformational analysis of gpa and gpap in aqueous solution by molecular dynamics and statistical methods. | barnase, an extracellular endoribonuclease from bacillus amyloliquefaciens, hydrolyses single-stranded rna. its very low catalytic activity toward gpn dinucleotides, where n stands for any nucleoside, is markedly increased when a phosphate is added to the 3'-end, as in gpnp. here we investigate the conformational properties of gpa and gpap in solution, in order to determine whether differences in these properties may be related to the changes in enzymatic activity. two independent 1.3 ns molecul ... | 1998 | 9790845 |
| the unusually slow unfolding rate causes the high stability of pyrrolidone carboxyl peptidase from a hyperthermophile, pyrococcus furiosus: equilibrium and kinetic studies of guanidine hydrochloride-induced unfolding and refolding. | to elucidate the energetic features of the anomalously high-level stabilization of a hyperthermophile pyrrolidone carboxyl peptidase (pfpcp) from a hyperthermophilic archaeon, pyrococcus furiosus, equilibrium and kinetic studies of the guanidine hydrochloride (guhcl)-induced unfolding and refolding were carried out with cd measurements at 220 nm in comparison with those from the mesophile homologue (bapcp) from bacillus amyloliquefaciens. the mutant protein of pfpcp substituted with ser at both ... | 1998 | 9860869 |
| refinement and structural analysis of barnase at 1.5 a resolution. | the structure of bacillus amyloliquefaciens ribonuclease (barnase), an extracellular 110-residue enzyme initially solved at 2.0 a resolution, has been refined at 1.5 a using synchrotron radiation and an imaging-plate scanner. refinement with anisotropic atomic displacement parameters resulted in increased accuracy of the structure. the final model has a crystallographic r factor of 11.5% and an rfree of 17.4%. the three independent molecules in the asymmetric unit, referred to as a, b and c, all ... | 1999 | 10089345 |
| preliminary x-ray crystallographic analysis of a novel phytase from a bacillus amyloliquefaciens strain. | a novel bacterial phytase from a bacillus amyloliquefaciens strain was crystallized using the hanging-drop vapour-diffusion method. the amino-acid sequence of the enzyme does not show any homology to those of other known phytases or phosphatases, with the exception of a phytase from bacillus subtilis. the enzyme exhibits a thermal stability which is strongly dependent on calcium ions. high-quality single crystals of the enzyme in the absence of calcium ions were obtained using a precipitant solu ... | 1999 | 10089471 |
| from detergent additive to semisynthetic peroxidase-simplified and up-scaled synthesis of seleno-subtilisin | a simplified and up-scaled synthesis of the semisynthetic peroxidase seleno-subtilisin was developed. highly purified to technical grade subtilisin preparations from bacillus licheniformis and bacillus amyloliquefaciens were applied as starting materials. activation of ser 221 with phenylmethanesulfonyl fluoride, nucleophilic substitution by sodium hydrogen selenide, and oxidation to the seleninic acid with hydrogen peroxide completed the chemical active-site modification. the reactions were acc ... | 1998 | 10099399 |
| effect of temperature on the saccharide composition obtained after alpha-amylolysis of starch | the hydrolysis of starch to low-molecular-weight products (normally characterised by their dextrose equivalent (de), which is directly related to the number-average molecular mass) was studied at different temperatures. amylopectin potato starch, lacking amylose, was selected because of its low tendency towards retrogradation at lower temperatures. bacillus licheniformis alpha-amylase was added to 10% [w/w] gelatinised starch solutions. the hydrolysis experiments were done at 50, 70, and 90 degr ... | 1999 | 10099614 |
| thermodynamic stability of a cold-active alpha-amylase from the antarctic bacterium alteromonas haloplanctis. | the thermal stability of the cold-active alpha-amylase (aha) secreted by the antarctic bacterium alteromonas haloplanctis has been investigated by intrinsic fluorescence, circular dichroism, and differential scanning calorimetry. it was found that this heat-labile enzyme is the largest known multidomain protein exhibiting a reversible two-state unfolding, as demonstrated by the recovery of deltahcal values after consecutive calorimetric transitions, a deltahcal/deltaheff ratio close to unity, an ... | 1999 | 10194383 |