Publications

genetic manipulation of bacillus amyloliquefaciens.	application of modern gene technology to strain improvement of the industrially important bacterium bacillus amyloliquefaciens is reported. several different plasmid constructions carrying the alpha-amylase gene (amye) from b. amyloliquefaciens were amplified in this species either extrachromosomally or intrachromosomally. the amye gene cloned on a pub110-derived high copy plasmid pkth10 directed the highest yields both in rich laboratory medium and in crude industrial medium. the alpha-amylase ...	1991	1367238
molecular cloning in lactobacillus helveticus by plasmid psa3::pva797 co-integrate formation and conjugal transfer.	a gene encoding beta-glucanase activity from bacillus amyloliquefaciens was subcloned in both orientations into plasmid shuttle vector psa3. in only one orientation could a co-integrate be generated with the conjugative plasmid pva797. the plasmid co-integrate was conjugated into lactobacillus helveticus strain cnrz450, where it was stably maintained without antibiotic selection and exhibited beta-glucanase activity. this method of introducing cloned dna into thermophilic lactobacilli will facil ...	1991	1367540
pathway and stability of protein folding.	we describe an experimental approach to the problem of protein folding and stability which measures interaction energies and maps structures of intermediates and transition states during the folding pathway. the strategy is based on two steps. first, protein engineering is used to remove interactions that stabilize defined positions in barnase, the rnase from bacillus amyloliquefaciens. the consequent changes in stability are measured from the changes in free energy of unfolding of the protein. ...	1991	1678536
levansucrase: a tool to study protein secretion in bacillus subtilis.	the bacillus amyloliquefaciens levansucrase gene (sacb[bamp]) was engineered in such a way that a heterologous gene could be inserted between the second and third codon of the mature levansucrase. extracellular levansucrase activity was detected only when the heterologous protein was secreted into the growth medium. a positive selection system to isolate suppressors of signal sequence mutants in bacillus subtilis has been developed based on the secretion of levansucrase.	1991	1784817
sequence regions of bacilli metalloproteinases that can affect enzyme thermostability.	by a computer analysis of the five bacilli metalloprotease sequences it was found that mesophilic bacillus amyloliquefaciens and b. subtilis proteases had lost two ca(2+)-binding sites due to the substitutions asp----ser 57, asp----thr 59, asp----pro 200, in comparison with the thermostable b. thermoproteolyticus thermolysin and b. stearothermophilus protease, which conserved three ca(2+)-binding sites, and b. cereus protease with the intermediate thermostability, which had presumably lost only ...	1991	1812491
optimization of bacillus alpha-amylase production by saccharomyces cerevisiae.	production of bacillus amyloliquefaciens alpha-amylase by saccharomyces cerevisiae using the multicopy plasmid paah5 and ways of improving the yields of secreted enzyme were studied. in standard non-buffered medium, alpha-amylase was rapidly inactivated but stabilization of the ph at 6 led to stable accumulation of alpha-amylase in the culture medium. removal of 1100 bp of the upstream sequence of the adh1 promoter present on paah5 resulted in delayed but increased alpha-amylase production: 29-f ...	1991	1872026
effect of signal sequence alterations on export of levansucrase in bacillus subtilis.	a series of alterations in the bacillus amyloliquefaciens levansucrase signal peptide were made by in vitro mutagenesis, and their effect on the secretion of levansucrase in bacillus subtilis was studied. some of the alterations resulted in a completely defective signal peptide. these included the removal of positively charged residues from the n-terminus and disruption of the hydrophobic core of the signal peptide either by introducing a charged residue or by deleting five or more amino acids. ...	1991	1898923
barnase toxin: a new chimeric toxin composed of pseudomonas exotoxin a and barnase.	we have constructed a chimeric toxin composed of pseudomonas exotoxin a (pe) and the extracellular ribonuclease of bacillus amyloliquefaciens, barnase. the chimeric protein, termed pe-bar, reacted with both anti-pe and anti-barnase antisera and had both adp ribosylation and ribonuclease activities. the chimeric toxin was cytotoxic to the murine fibroblast cell line l929 and to a murine hybridoma resistant to pe. a mutant form of pe-bar lacking adp-ribosylating activity was still cytotoxic to l92 ...	1991	1900455
use of alkaline phosphatase fusions to study protein secretion in bacillus subtilis.	we have constructed a vector designed to facilitate the study of protein secretion in bacillus subtilis. this vector is based on a translational fusion between the expression elements and signal sequence of bacillus amyloliquefaciens alkaline protease and the mature coding sequence for escherichia coli alkaline phosphatase (phoa). we show that export of alkaline phosphatase from b. subtilis depends on a functional signal sequence and that alkaline phosphatase activity depends upon secretion. the ...	1991	1901054
optimization of the signal-sequence cleavage site for secretion from bacillus subtilis of a 34-amino acid fragment of human parathyroid hormone.	we have effected the secretion from bacillus subtilis of a 34-amino acid (aa) fragment of human parathyroid hormone (pth,1-34), using a bacillus amyloliquefaciens neutral protease signal sequence. the secretion efficiency depended on the aa sequence near the signal-sequence cleavage site. we constructed a series of gene fusions encoding different pairs of aa between the signal sequence and pth,1-34. there was a correlation between those polypeptides which were efficiently secreted and the potent ...	1991	1908402
accurate measurements of coupling constants from two-dimensional nuclear magnetic resonance spectra of proteins and determination of phi-angles.	a new and simple method to measure 3jhnh alpha coupling constants of proteins by adding and subtracting traces from corresponding two-dimensional nuclear overhauser enhanced spectroscopy and two-dimensional correlated spectroscopy cross peaks after scaling is proposed. the optimal scaling for the addition and the subtraction of the two traces is obtained by minimizing an error function. the method was proven to give accurate and precise measurements of coupling constants when tested with a serie ...	1991	2005622
hybrid bacillus (1-3,1-4)-beta-glucanases: engineering thermostable enzymes by construction of hybrid genes.	hybrid (1-3,1-4)-beta-glucanase genes were constructed by extension of overlapping segments of the (1-3,1-4)-beta-glucanase genes from bacillus amyloliquefaciens and b. macerans generated by the polymerase chain reaction (pcr). four hybrid genes were expressed in escherichia coli cells. the mature hybrid enzymes contain a 16, 36, 78, or 152 amino acid n-terminal sequence derived from b. amyloliquefaciens (1-3,1-4)-beta-glucanase followed by a c-terminal segment derived from b. macerans (1-3,1-4) ...	1991	2005860
aromatic-aromatic interactions and protein stability. investigation by double-mutant cycles.	the side-chains of phenylalanine and tyrosine residues in proteins are frequently found to be involved in pairwise interactions. these occur both within repeating elements of secondary structure and in tertiary and quaternary interactions. it has been suggested that they are important in protein folding and stability, and non-bonded potential energy calculations indicate that a typical aromatic-aromatic interaction has an energy of between -1 and -2 kcal/mol and contributes between -0.6 and -1.3 ...	1991	2010920
crystal structure of a barnase-d(gpc) complex at 1.9 a resolution.	the ribonuclease excreted by bacillus amyloliquefaciens, barnase, was co-crystallized with the deoxy-dinucleotide d(gpc). the crystal structure was determined by molecular replacement from a model of free barnase previously derived by mauguen et al. refinement was carried out using data to 1.9 a resolution. the final model, which has a crystallographic r factor of 22%, includes 869 protein atoms, 38 atoms from d(gpc), a sulfate ion and 73 water molecules. only minor differences from free barnase ...	1991	2023257
overexpression, purification and crystallization of bamhi endonuclease.	the type ii restriction endonuclease bamhi has been expressed in e. coli, producing 100-fold more enzyme than the wild type bacillus amyloliquefaciens h strain. this high yield has facilitated purification to homogeneity of large amounts of the enzyme, along with its crystallization in a form which diffracts to at least 1.9 a in x-ray analysis.	1991	2030964
m.h2i, a multispecific 5c-dna methyltransferase encoded by bacillus amyloliquefaciens phage h2.	bacillus amyloliquefaciens phage h2 codes for a multispecific cytosine-5-dna- methyltransferase (mtase), m.h2i, which methylates ggcc, gcngc and [sequence: see text] target sequences. the gene coding for m.h2i was cloned in escherichia coli and its nucleotide (nt) sequence was determined. it consists of 1509 bp, corresponding to a protein of 503 amino acids (aa) with a calculated mr of 57,166. a comparison of the aa sequence of m.h2i with those of the multispecific mtases encoded by bacillus sub ...	1991	2055471
co-expression of a saccharomyces diastaticus glucoamylase-encoding gene and a bacillus amyloliquefaciens alpha-amylase-encoding gene in saccharomyces cerevisiae.	a glucoamylase-encoding gene (sta2) from saccharomyces diastaticus and an alpha-amylase-encoding gene (amy) from bacillus amyloliquefaciens were cloned separately into a yeast-integrating shuttle vector (yip5), generating recombinant plasmids psp1 and psp2, respectively. the sta2 and amy genes were jointly cloned into yip5, generating plasmid psp3. subsequently, the dominant selectable marker aph1, encoding resistance to geneticin g418 (gtr), was cloned into psp3, resulting in psp4. for enhanced ...	1991	2055483
inactivation and stabilization of stabilisins in neat organic solvents.	the stability of the serine proteases from bacillus amyloliquefaciens (subtillisin bpn') and bacillus licheniformis (subtilisin carlsberg) was investigated in various anhydrous solvents at 45 degrees c. the half-life of subtilisin bpn' in dimethyl-formamide dramatically depends on the ph of the aqueous solutions from which the enzyme was lyophilized, increasing from 48 min to 20 h when the ph is raised from 6.0 to 7.9. both subtilisins exhibited substantial inactivation during multihour incubati ...	1991	18600863
simple structured model for alpha-amylase synthesis by bacillus amyloliquefaciens.	a predictive, simple, structured model describing the synthesis of alpha-amylase by bacillus amyloliquefaciens was formulated. three key intracellular processes were identified (i.e, translation, and excretion) along with two key intracellular components (i.e., mrna and the intracellular form of the alpha-amylase enzyme). nearly all the model parameters were estimated by means of performing independent experiments, primarily fed-batch experiments. the model was shown to predict transient system ...	1991	18600872
[cloning and gene expression of bacillus cereus neutral proteinase in bacillus subtilis cells].	the neutral proteinase gene of bacillus cereus was cloned. its restriction map and the direction of transcription was determined. it was shown that the neutral proteinase gene could be expressed in bacillus cells. the thermostability of the product coded by the neutral proteinase gene and its natural analogue was explored. the obtained data indicate that the neutral proteinase of bacillus cereus is closely related to the enzyme of bacillus amyloliquefaciens by these parameters. it was found that ...	1992	1339956
identification of glutamic acid 105 at the active site of bacillus amyloliquefaciens 1,3-1,4-beta-d-glucan 4-glucanohydrolase using epoxide-based inhibitors.	bacillus amyloliquefaciens 1,3-1,4-beta-d-glucan 4-glucanohydrolase (ec 3.2.1.73) was modified by the mechanism-based, affinity-labeling reagent [14c](3,4)-epoxybutyl beta-d-cellobioside. following partial inactivation a completely inactivated enzyme preparation containing 1.1 mol of covalently bound inhibitor/mol of protein was obtained by chromatography on a cellulosic matrix. the inactivated enzyme was digested with endoproteinase glu-c and radioactive peptides purified by reversed-phase high ...	1992	1360982
processing of the prepropeptide portions of the bacillus amyloliquefaciens neutral protease fused to bacillus subtilis alpha-amylase and human growth hormone during secretion in bacillus subtilis.	a set of nested 3'-terminal deletions of the prepropeptide of the bacillus amyloliquefaciens neutral protease gene was constructed. alpha-amylase and human growth hormone were secreted using these truncated genes in bacillus subtilis. the level of the secreted alpha-amylase varied with the region for the truncated prepropeptide contained in the fusion gene but was independent of its length. even though length of the prepropeptide varied, the mobilities of secreted alpha-amylases were the same as ...	1992	1367948
effect of immobilisation on the production of alpha-amylase by an industrial strain of bacillus amyloliquefaciens.	the effect of immobilisation of an industrial strain of bacillus amyloliquefaciens in calcium alginate beads on production of alpha-amylase was investigated using lactose-based media in shake flasks and in a 0.3 dm3 glass fermenter. although the microorganism was a good alpha-amylase producer in batch cultures of free cells, it was unable to produce the enzyme for extended periods either in repeated batch cultures, or in continuous cultivation. in each case, parallel tests with cells immobilised ...	1992	1368003
secretion of correctly processed and folded pancreatic secretory trypsin inhibitor by bacillus subtilis.	we constructed a plasmid, designated pnpp126, containing a dna sequence encoding a fusion protein composed of bacillus amyloliquefaciens neutral protease prepeptide (signal peptide) and human pancreatic secretory trypsin inhibitor (hpsti), where the mature hpsti is accurately fused to the 3'-terminal of the prepeptide coding region. it was observed that the strain bacillus subtilis mt600 harboring pnpp126 could secrete a trypsin inhibitory activity into the culture medium. the n-terminal amino a ...	1992	1368060
expression of bacillus amyloliquefaciens amylase and vibrio alginolyticus protease a fusion genes.	previously we reported [deane, s. m., maharaj, r., robb, f. t. & woods, d. r. (1987) journal of general microbiology 133, 2295-2302] that the production of a vibrio alginolyticus sds-resistant alkaline serine protease (pro a) cloned in escherichia coli was characterized by a 12 h delay between the synthesis of an inactive precursor and secretion of active pro a. replacement of the v. alginolyticus promoter region by the alpha-amylase promoter region from bacillus amyloliquefaciens resulted in th ...	1992	1373436
positive theta-angles in proteins by nuclear magnetic resonance spectroscopy.	non-glycine residues with positive theta-angles have been identified in four proteins, barley serine proteinase inhibitor ci-2, bacterial ribonuclease (barnase) of bacillus amyloliquefaciens, hen egg white lysozyme and a basic protein from barley seed (barwin) by use of nuclear magnetic resonance spectroscopy. by accurate measurements of the coupling constant (3)jhnhalpha and integration of the nuclear overhauser hn-halpha cross peak, positive theta-angles could be determined reliably to 60 degr ...	1992	1392567
dna sequence and analysis of a cryptic 4.2-kb plasmid from the filamentous cyanobacterium, plectonema sp. strain pcc 6402.	the 4194-bp plasmid, prf1, from plectonema sp. strain pcc 6402 was completely sequenced and analyzed. seven potential open reading frames were identified. the predicted amino acid sequence of open reading frame c (orf c) had identities of 34, 29, and 25% with rep b from the staphylococcus aureus plasmid, pub110; rep from the bacillus amyloliquefaciens plasmid, pftb14; and protein a from the s. aureus plasmid, pc194, respectively. a 75-amino-acid region conserved in these proteins (rep b, rep, an ...	1992	1409974
synthesis and secretion of an erwinia chrysanthemi pectate lyase in saccharomyces cerevisiae regulated by different combinations of bacterial and yeast promoter and signal sequences.	nine different expression-secretion cassettes, comprising novel combinations of yeast and bacterial gene promoters and secretion signal sequences, were constructed and evaluated. a pectate lyase-encoding gene (pele) from erwinia chrysanthemi was inserted between each one of these expression-secretion cassettes and a yeast gene terminator, generating recombinant yeast-integrating shuttle plasmids pams1 through pams9. these yip5-derived plasmids were transformed and stably integrated into the geno ...	1992	1427097
regulation of the bamhi restriction-modification system by a small intergenic open reading frame, bamhic, in both escherichia coli and bacillus subtilis.	bamhi, from bacillus amyloliquefaciens h, is a type ii restriction-modification system recognizing and cleaving the sequence g--gatcc. the bamhi restriction-modification system contains divergently transcribed endonuclease and methylase genes along with a small open reading frame oriented in the direction of the endonuclease gene. the small open reading frame has been designated bamhic (for bamhi controlling element). it acts as both a positive activator of endonuclease expression and a negative ...	1992	1429443
microbial degradation of shrimp-shell waste.	a total of 40 strains of bacteria were isolated and tested for their potentiality to degrade chitin and utilize shrimp-shell waste for the production of chitinase. the activity ratio, percentage of weight loss and enzyme activity were determined for cultures exhibiting highest chitinolytic activities. the most active organisms were identified as alcaligenes denitrificans, bacillus amyloliquefaciens, b. megaterium and b. subtilis. the potentiality of the first two organisms to degrade chitin was ...	1992	1512701
a positive selection vector for cloning high molecular weight dna by the bacteriophage p1 system: improved cloning efficacy.	the bacteriophage p1 cloning system can package and propagate dna inserts that are up to 95 kilobases. clones are maintained in escherichia coli by a low-copy replicon in the p1 cloning vector and can be amplified by inducing a second replicon in the vector with isopropyl beta-d-thiogalactopyranoside. to overcome the necessity of screening clones for dna inserts, we have developed a p1 vector with a positive selection system that is based on the properties of the sacb gene from bacillus amyloliq ...	1992	1549564
translocation mediated by domain ii of pseudomonas exotoxin a: transport of barnase into the cytosol.	pseudomonas exotoxin a (pe) is a protein toxin composed of three structural domains. functional analysis of pe has revealed that domain i is the cell-binding domain and that domain iii functions in adp ribosylation. domain ii was originally designated as the translocation domain, mediating the transfer of domain iii to the cytosol, because mutations in this domain result in toxin molecules with normal cell-binding and adp-ribosylation activities but which are not cytotoxic. however, the results ...	1992	1567815
modular expression and secretion vectors for bacillus subtilis.	a modular vector system has been developed for the extracellular production of heterologous proteins in bacillus subtilis. this modular vector system consists of four secretion vectors which are based upon the genes encoding the bacillus amyloliquefaciens extracellular alkaline protease, neutral protease, barnase and levansucrase. the modular vectors contain compatible restriction sites downstream from the signal peptide-coding region. three reporter proteins (staphylococcal protein a, levansucr ...	1992	1587474
extensive comparison of the substrate preferences of two subtilisins as determined with peptide substrates which are based on the principle of intramolecular quenching.	subtilisins are serine endopeptidases with an extended binding cleft comprising at least eight binding subsites. interestingly, subsites distant from the scissile bond play a dominant role in determining the specificity of the enzymes. the development of internally quenched fluorogenic substrates, which allow polypeptides of more than 11 amino acids to be inserted between the donor and the acceptor, has rendered it possible to perform a highly systematic mapping of the individual subsites of the ...	1992	1627543
expression of the erwinia carotovora polygalacturonase-encoding gene in bacillus subtilis: role of signal peptide fusions on production of a heterologous protein.	the peha gene encoding an endopolygalacturonase (pectinase) of erwinia carotovora subsp. carotovora has been cloned previously [saarilahti et al., mol. microbiol. 4 (1990) 1037-1044]. we expressed peha in bacillus subtilis using a secretion vector based on the promoter and signal sequence of the alpha-amylase (amy)-encoding gene, amye, from bacillus amyloliquefaciens. to test whether the location of the junction between the secretion vector and peha affects the protein yield, we made four differ ...	1992	1628841
[isolation and analysis of protease-deficient mutants of bacillus amyloliquefaciens].	four types of protease negative mutants of bacillus amyloliquefaciens a50 were selected after four stages of step-by-step uv light mutagenesis. edta, and pmsf were used as inhibitors of protease activity to characterize the protease negative mutants with regard to the protease type. the electrophoretic patterns of proteases from culture medium of b. amyloliquefaciens protease deficient mutants were studied. the proinsulin stability in the culture medium of different mutants was analysed. proteas ...	1992	1639263
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
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
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
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
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
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
[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
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
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
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
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
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
[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
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
[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
fructan accumulation and sucrose metabolism in transgenic maize endosperm expressing a bacillus amyloliquefaciens sacb gene.	over 40,000 species of plants accumulate fructan, [beta]-2-1- and [beta]-2-6-linked polymers of fructose as a storage reserve. due to their high fructose content, several commercial applications for fructans have been proposed. however, plants that accumulate these polymers are not agronomically suited for large-scale cultivation or processing. this study describes the transformation of a bacillus amyloliquefaciens sacb gene into maize (zea mays l.) callus by particle bombardment. tissue-specifi ...	1996	12226187
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