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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 ...19846427178
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.19846609154
dna hybridization procedure to detect pseudorabies virus dna in swine tissue.a dna hybridization technique was developed to detect the presence of pseudorabies virus (prv) dna. p nick translated probes of high specific activity were prepared from transformed escherichia coli plasmids into which bacillus amyloliquefaciens h (bam h1) restriction fragments of prv dna had been inserted. swine cellular dna and tissue culture prv dna were digested with bam h1, separated by agarosegel electrophoresis, transferred onto nitrocellulose paper, hybridized to the radioactive probes, ...19852408522
[limited proteolysis of human leukocyte interferon-alpha2 and localization of the antigenic determinant binding the monoclonal antibodies].large peptide fragments of human leucocyte interferon-alpha 2 (inf-alpha 2) were obtained by limited proteolysis with trypsin, pepsin, thermolysine and bacillus amyloliquefaciens intracellular serine proteinase. the ability of the fragments to bind murine monoclonal antibodies nk2 raised against inf-alpha 2 was studied by the immunoblotting technique. the region of sequence 110-149 is the most sensitive to proteolytic attack, being probably exposed on the surface of the inf-alpha 2 molecule. inf ...19852415171
sigma 29-like protein is a common sporulation-specific element in bacteria of the genus bacillus.a monoclonal antibody specific for an antigenic determinant on the bacillus subtilis sporulation-induced sigma factor sigma 29 reacted with proteins similar in size to sigma 29 in extracts of sporulating bacillus licheniformis, bacillus amyloliquifaciens, bacillus cereus, bacillus natto, and bacillus pumilus but not in extracts prepared from vegetatively growing cultures of these bacteria. these results indicate that rna polymerase modifications, initially described for b. subtilis, are likely t ...19852415506
complete nucleotide sequence of a gene coding for heat- and ph-stable alpha-amylase of bacillus licheniformis: comparison of the amino acid sequences of three bacterial liquefying alpha-amylases deduced from the dna sequences.the gene coding for the heat-stable and ph-stable alpha-amylase of bacillus licheniformis 584 (atcc 27811) was cloned in escherichia coli and the nucleotide sequence of a dna fragment of 1,948 base pairs containing the entire amylase gene was determined. as inferred from the dna sequence, the b. licheniformis alpha-amylase had a signal peptide of 29 amino acid residues and the mature enzyme comprised 483 amino acid residues, giving a molecular weight of 55,200. the amino acid sequence of b. lich ...19852418011
[mapping and cloning of the gene coding for beta-glucanase from various bacilli].beta-glucanase gene from bacillus subtilis 168 has been mapped by bacteriophage pbs1 transduction technique between saca and pura genes. the stimulating effect of pleiotropic mutations pap, amyb and sacuh on beta-glucanase production in bacillus subtilis and bacillus amyloliquefaciens has been described. beta-glucanase gene from bacillus amyloliquefaciens has been cloned ona charon 4a vector. expression of the gene in e. coli cells depended on the orientation of the cloned dna on a pbr322 vector ...19853842743
an active center tryptophan residue in liquefying alpha-amylase from bacillus amyloliquefaciens.liquefying alpha-amylase from bacillus amyloliquefaciens was inactivated on treatment with n-bromosuccinamide. preincubation of the enzyme with either of the substrate, or competitive inhibitor provided significant protection against inactivation. the relationship between activity loss and the number of tryptophan residues modified, as well as presence of substrate/inhibitor in the reaction mixture, demonstrated that only one of three modifiable tryptophan residues is at or near the active cente ...19853872124
active site studies on bacillus amyloliquefaciens alpha-amylase (i).modification of liquefying alpha-amylase by diethylpyrocarbonate or its photo-oxidation in the presence of rose bengal caused rapid loss of enzyme activity. the photo-oxidation followed pseudo-first-order kinetics giving maximal value at ph 8.0. the photo-oxidized enzyme showed a characteristic increase in absorbance at 250 nm which was directly proportional to the extent of inactivation. diethylpyrocarbonate at low concentration at ph 6.0 and 30 degrees c completely inactivated alpha-amylase. i ...19853872408
chemical modification of liquefying alpha-amylase: role of tyrosine residues at its active center.liquefying alpha-amylase from bacillus amyloliquefaciens was inactivated by treatment with tetranitromethane and n-acetylimidazole. the loss of activity occurred with modification of five tyrosine residues. preincubation of the enzyme with either the substrate or the competitive inhibitor at saturating levels provided complete protection against inactivation. however, the presence of substrate/inhibitor in the reaction mixture protected only two of the five modifiable tyrosine residues, suggesti ...19853875315
[synthesis and processing in escherichia coli of human leukocyte interferon linked with the signal sequence of bacillus amyloliquefaciens alpha-amylase]. 19853896711
[expression of the alpha-amylase gene of bacillus amyloliquefaciens in saccharomyces cerevisiae yeasts]. 19853912145
transcription and translation of foreign genes in bacillus subtilis by the aid of a secretion vector.expression levels of bacillus amyloliquefaciens alpha-amylase, escherichia coli tem-beta-lactamase, and semliki forest virus glycoprotein e1 genes were compared in bacillus subtilis. all three model genes were expressed by using a secretion vector, constructed by joining the b. amyloliquefaciens alpha-amylase promoter and signal sequence with plasmid pub110 (i. palva, m. sarvas, p. lehtovaara, m. sibakov, and l.kääriäinen, proc. natl. acad. sci. u.s.a. 79:5582-5586, 1982). when transformed b. su ...19853920200
secretion of semliki forest virus membrane glycoprotein e1 from bacillus subtilis.the gene coding for the semliki forest virus (sfv) membrane protein e1 was joined to a secretion vector containing the promoter and signal sequence regions of the alpha-amylase gene from bacillus amyloliquefaciens. to facilitate secretion, the regions coding for the n-terminal signal peptide (the 6k protein) and the c-terminal hydrophobic transmembrane domain of the e1 gene were deleted. after transformation into b. subtilis, e1 was shown by immunoblotting to be expressed at a low level (about 0 ...19853920841
nucleotide sequence of the bacillus stearothermophilus alpha-amylase gene.the nucleotide sequence of the bacillus stearothermophilus alpha-amylase gene and its flanking regions was determined. an open reading frame was found, comprising a total of 1,647 base pairs (549 amino acids) and starting from a gug codon as methionine. it was shown by nh2-terminal amino acid sequence analysis that the extracellular amylase consisted of 515 amino acid residues, which corresponded to a molecular weight of 58,779. thus the nh2-terminal portion of the gene encodes 34 amino acid res ...19853924897
export of alpha-amylase by bacillus amyloliquefaciens requires proton motive force.the secretion of protein directly into the extracellular medium by bacillus amyloliquefaciens, a gram-positive bacterium, was shown to be dependent on proton motive force. when the electrochemical membrane potential gradient of protons was dissipated either by uncouplers or by valinomycin in combination with k+, a precursor form of alpha-amylase accumulated on the cellular membrane. the proton motive force could be dissipated without altering the intracellular level of atp, indicating that the o ...19852997128
bacteriophage spo2-mediated plasmid transduction in transpositional mutagenesis within the genus bacillus.a single copy of the streptococcus faecalis transposon tn917, located in the bacillus subtilis chromosome, was able to transpose onto the spo2 cos plasmid ppl1017, which codes for chloramphenicol resistance and contains the bacteriophage phi 105 immunity region. selection for ppl1017::tn917 chimeras was performed by spo2-mediated plasmid transduction of transposon-borne resistance to macrolide-lincosamide-streptogramin b antibiotics (mlsr). the transposition of tn917 onto plasmid ppl1017 occurre ...19852999078
cloning, sequencing and transcription of an inactivated copy of bacillus amyloliquefaciens extracellular ribonuclease (barnase).the gene for bacillus amyloliquefaciens extracellular rnase (barnase) has been cloned in an inactive form in escherichia coli following insertional mutagenesis by transposon tn917. the nucleotide (nt) sequence of the gene was determined and the deduced amino acid (aa) sequence found to correspond almost precisely to the previously determined sequence. an open reading frame (orf) of 72 codons precedes the mature sequence. the probable translation start site is 46 or 47 codons before the n-termina ...19853007290
the dependence of protein release from bacillus amyloliquefaciens on the growth phase in batch culture.for the recovery of intracellular material from bacteria it is often necessary to disrupt the cells. much work has been done on the kinetics of protein release in beadmills,(1) homogenizers,(2) and by ultrasonication.(3) in this paper we report how the growth phase of bacillus amyloliquefaciens grown in batch culture affects the rate of protein release by ball milling, ultrasonication, and autolysis. we further suggest that autolysis is a feasible method for disrupting bacillus.198518553751
protoplast transformation in coryneform bacteria and introduction of an alpha-amylase gene from bacillus amyloliquefaciens into brevibacterium lactofermentum.[this corrects the article on p. 638 in vol. 51.].198616347093
protoplast transformation in coryneform bacteria and introduction of an alpha-amylase gene from bacillus amyloliquefaciens into brevibacterium lactofermentum.the goal of this study was to investigate the likelihood of developing useful transformation systems for coryneform bacteria. two species of coryneform bacteria, brevibacterium lactofermentum and corynebacterium lilium, were transformed with chimeras constructed from pub110 and a cryptic coryneform plasmid (pgx1901). c. lilium protoplasts were also efficiently transfected with phage cs1 dna. high transformation and transfection frequencies were obtained after only 2 min of lysozyme treatment of ...19863008649
synthesis of ompa protein of escherichia coli k12 in bacillus subtilis.we have inserted a c-terminally truncated gene of the major outer membrane protein ompa of escherichia coli downstream from the promoter and signal sequence of the secretory alpha-amylase of bacillus amyloliquefaciens in a secretion vector of bacillus subtilis. b. subtilis transformed with the hybrid plasmid synthesized a protein that was immunologically identified as ompa. all the protein was present in the particulate fraction. the size of the protein compared to the peptide synthesized in vit ...19863016148
use of plasmid ptv1 in transposon mutagenesis and gene cloning in bacillus amyloliquefaciens.the plasmid ptv1, constructed in bacillus subtilis as a tool for insertional mutagenesis by the transposon tn917, has been transferred to bacillus amyloliquefaciens by transduction with the phage pbs1. insertional mutants containing tn917 were observed in the new host. southern blot analysis of such mutants indicated no preference for insertion sites. the copy numbers of ptv1 in b. amyloliquefaciens and b. subtilis were found to be 1.4 and 14, respectively; the plasmid is less stable against los ...19863016781
detection of latent pseudorabies virus in porcine tissue, using a dna hybridization dot-blot assay.a dna-hybridization dot-blot technique was developed to detect the presence of pseudorabies virus (prv) dna in porcine tissue. seven 32p-nick translated probes of high specific activity were prepared from transformed escherichia coli plasmids into which bacillus amyloliquefaciens h (bam hi) restriction fragments of prv-dna had been inserted. samples of dna that had been extracted from porcine tissue or from prv grown in tissue culture were transferred to nitrocellulose paper, using a microsample ...19863024534
cloning and sequencing of a gene from bacillus amyloliquefaciens that complements mutations of the sporulation gene spoiid in bacillus subtilis.a segment of dna from bacillus amyloliquefaciens, which complemented a mutant sporulation gene, spoiid68, in bacillus subtilis, was cloned into a derivative of the temperate bacteriophage phi 105. the segment of dna included an entire structural gene and complemented the mutation spoiid298, in addition to spoiid68, in b. subtilis. the nucleotide sequence of the gene from b. amyloliquefaciens was determined and compared with that of the b. subtilis gene; 74% homology was found in the coding regio ...19863114424
the effect of the inverted repeat structure on the production of the cloned bacillus amyloliquefaciens alpha-amylase.an inverted repeat structure from bacillus natto preceding the bacillus subtilis alpha-amylase gene has been suggested to be responsible for the enhancement of alpha-amylase production [yamazaki et al. (1983) j. bacteriol. 156, 327-337]. a similar inverted repeat region has also been found upstream from the bacillus amyloliquefaciens alpha-amylase gene and shown to function as a transcription termination signal of an upstream operon of 2.2 x 10(3) bases (2.2 kb) (kallio et al., following paper i ...19863488213
isolation and characterization of a 2.2-kb operon preceding the alpha-amylase gene of bacillus amyloliquefaciens.a dna region of 2.8 x 10(3) base pairs (2.8 kb) upstream of the bacillus amyloliquefaciens alpha-amylase gene has been isolated. this dna gave rise to a 2.2-kb transcript. the 3' end of the transcript was mapped with s1 nuclease and shown to terminate 49 base pairs upstream of the -35 region of the alpha-amylase promoter. in b. subtilis minicells this 2.2-kb transcript coded for three different polypeptides, thus indicating a polycistronic operon-type structure. the location and the order of the ...19863488214
substrate binding nodes of bacillus amyloliquefaciens alpha-amylase. 19863491781
in vivo formation and stability of engineered disulfide bonds in subtilisin.computer modeling suggested that a disulfide bond could be built into bacillus amyloliquefaciens subtilisin between positions 22 (wild-type, thr) and 87 (ser) or between positions 24 (ser) and 87 (ser). single cysteines were introduced into this cysteine-free protease at positions 22, 24, or 87 by site-directed mutagenesis of the cloned subtilisin gene. the corresponding double-cysteine mutants were constructed, and recombinant plasmids were expressed in bacillus subtilis. double-cysteine mutant ...19863516996
secretion and autoproteolytic maturation of subtilisin.the sequence of the cloned bacillus amyloliquefaciens subtilisin gene suggested that this secreted serine protease is produced as a larger precursor, designated preprosubtilisin [wells, j. a., ferrari, e., henner, d. j., estell, d. a. & chen, e. y. (1983) nucleic acids res. 11, 7911-7925]. biochemical evidence presented here shows that a subtilisin precursor is produced in bacillus subtilis hosts. the precursor is first localized in the cell membrane, reaching a steady-state level of approximate ...19863517850
site-directed mutagenesis and the role of the oxyanion hole in subtilisin.oligonucleotide-directed mutagenesis was used to investigate the nature of transition state stabilization in the catalytic mechanism of the serine protease, subtilisin bpn'. the gene for this extracellular enzyme from bacillus amyloliquefaciens has been cloned and expressed in bacillus subtilis. in the transition state complex, the carbonyl group of the peptide bond to be hydrolyzed is believed to adopt a tetrahedral configuration rather than the ground-state planar configuration. crystallograph ...19863520553
proteases of enhanced stability: characterization of a thermostable variant of subtilisin.a procedure has been developed for the isolation and identification of mutants in the bacterial serine protease subtilisin that exhibit enhanced thermal stability. the cloned subtilisin bpn' gene from bacillus amyloliquefaciens was treated with bisulfite, a chemical mutagen that deaminates cytosine to uracil in single-stranded dna. strains containing the cloned, mutagenized subtilisin gene which produced subtilisin with enhanced thermal stability were selected by a simple plate assay procedure w ...19863329733
expression of the staphylococcal protein a gene in bacillus subtilis by gene fusions utilizing the promoter from a bacillus amyloliquefaciens alpha-amylase gene.gene fusions of dna sequences encoding protein a from staphylococcus aureus (spa) with expression elements from an alpha-amylase gene from bacillus amyloliquefaciens (amyebamp) directed the synthesis and efficient secretion of protein a in bacillus subtilis. the fusions were established on multicopy pub110-based plasmid vectors, in contrast to the intact spa gene, which could not be stably established on plasmids in b. subtilis. some of the resulting b. subtilis strains secreted protein a at lev ...19863081489
secretion of a heterologous protein from bacillus subtilis with the aid of protease signal sequences.secretion vectors based on the genes from bacillus amyloliquefaciens p for alkaline protease (aprbamp) and neutral protease (nprbamp) were constructed. with both aprbamp and nprbamp, a unique restriction site was introduced 3' of the predicted signal coding region by using the technique of oligonucleotide-directed mutagenesis. the new sites enabled us to fuse a heterologous gene to the expression and secretion elements. we used the protein a gene (spa) from staphylococcus aureus as a heterologou ...19863081490
two tandemly located promoters, artificially constructed, are active in a bacillus subtilis alpha-amylase secretion vector.an 85 bp dna fragment, the nucleotide sequence of which had 84% homology with the sequence for the promoter, ribosome binding site and nh2-terminal five amino acids of the bacillus amyloliquefaciens alpha-amylase gene, was chemically synthesized. in order to analyze the promoter activity of a bacillus subtilis alpha-amylase secretion vector, the fragment was inserted between the promoter and signal peptide-coding region of bacillus subtilis alpha-amylase gene. both promoters, tandemly repeated, ...19863086305
the beta-glucanase gene from bacillus amyloliquefaciens shows extensive homology with that of bacillus subtilis.the nucleotide sequence of a 1583-bp dna fragment containing gene bg1a for endo-beta-1,3-1,4-glucanase (ec 3.2.1.73) of bacillus amyloliquefaciens strain be20/78, a high producer of secreted enzymes, has been determined. the gene bg1a comprises an open reading frame (orf) of 717 bp (= 239 codons) starting with atg at 469 up to the translation stop codon taa at 1188. upstream from the translation initiation codon atg, the ribosome-binding sequence 5'-aaaaaaggggg-3' and two putative bgla promoters ...19863106158
tunicamycin-resistant mutants of bacillus amyloliquefaciens are deficient in amylase, protease and penicillinase synthesis and have altered sensitivity to antibiotics and autolysis.mutants of bacillus amyloliquefaciens resistant to at least 10 micrograms/ml of tunicamycin were isolated and shown to be pleiotropic. the mutants were more resistant to streptomycin, chloramphenicol, kanamycin and neomycin than was the parent strain but less resistant to penicillin g and tetracycline. they were more autolytic, presumably due to an altered cell wall. the mutants produced reduced levels of amylase, penicillinase and both metal and serine protease besides having an enhanced sporul ...19862424885
secretion of human serum albumin from bacillus subtilis.we have fused the structural gene (hsa) for human serum albumin (hsa) to the expression elements and signal sequence coding region of each of two genes from bacillus amyloliquefaciens p, an alpha-amylase gene (amybamp) and a neutral protease gene (nprbamp). bacillus subtilis strains harboring either of these gene fusions synthesized a protein with the antigenic characteristics and size (68 kilodaltons) of hsa. results from pulse-labeling studies indicated that the bacterially produced hsa was se ...19873110129
stimulation by hyphopichia burtonii and bacillus amyloliquefaciens of aflatoxin production by aspergillus flavus in irradiated maize and rice grains.aspergillus flavus was grown on maize and rice extract agars and on irradiated viable cracked maize and rice grains, either in pure culture or in dual culture with wild strains of either hyphopichia burtonii or bacillus amyloliquefaciens. aflatoxin production by a. flavus and its growth and interactions with the other microorganisms were studied at three water activities (aw) (0.98, 0.95, and 0.90) and two temperatures (25 and 16 degrees c). both h. burtonii and b. amyloliquefaciens markedly sti ...19873111368
improvement in the alkaline stability of subtilisin using an efficient random mutagenesis and screening procedure.an efficient random mutagenesis procedure coupled to a replica plate screen facilitated the isolation of mutant subtilisins from bacillus amyloliquefaciens that had altered autolytic stability under alkaline conditions. out of about 4000 clones screened, approximately 70 produced subtilisins with reduced stability (negatives). two clones produced a more stable subtilisin (positives) and were identified as having a single mutation, either ile107val or lys213arg (the wild-type amino acid is follow ...19873334089
use of lambda exonuclease for efficient oligonucleotide-mediated site-directed deletion and point mutation of double-stranded dna.a novel approach to oligonucleotide-mediated, site-directed in vitro mutagenesis is described that allows for the efficient generation of sequence modifications on double-stranded substrates without the need for subcloning into special vectors. site-directed deletions as well as point mutations were introduced into the genes encoding human tissue plasminogen activator (tpa) and the bacillus amyloliquefaciens alpha-amylase gene using lambda exonuclease to enzymatically degrade dna 5' to 3' in ord ...19872954801
expression of bacillus amyloliquefaciens extracellular ribonuclease (barnase) in escherichia coli following an inactivating mutation.an inactivated gene for bacillus amyloliquefaciens extracellular ribonuclease (barnase) has previously been cloned and sequenced following transposon mutagenesis. the intact gene could not be assembled in escherichia coli and is presumed to be lethal. therefore, we introduced specific mutations into the barnase gene to prevent its lethal effect. a gln-73 mutant gene was stable in e. coli but only produced low amounts of barnase antigen. mutants containing asp, gln or arg, instead of his-102, at ...19873297926
recruitment of substrate-specificity properties from one enzyme into a related one by protein engineering.the bacillus licheniformis and bacillus amyloliquefaciens subtilisins differ by 31% in protein sequence and by factors of greater than 60 in catalytic efficiency, kcat/km, toward various substrates. despite large differences in sequence and substrate specificity for these serine proteases, only two amino acid substitutions (residues 156 and 217) occur within 4 a (contact distance) of modeled substrates, and a third substitution (residue 169) is within 7 a. the three b. licheniformis substitution ...19873299378
engineering enzyme specificity by "substrate-assisted catalysis".a novel approach to engineering enzyme specificity is presented in which a catalytic group from an enzyme is first removed by site-directed mutagenesis causing inactivation. activity is then partially restored by substrates containing the missing catalytic functional group. replacement of the catalytic his with ala in the bacillus amyloliquefaciens subtilisin gene (the mutant is designated his64ala) by site-directed mutagenesis reduces the catalytic efficiency (kcat/km) by a factor of a million ...19873299704
electrostatic effects on modification of charged groups in the active site cleft of subtilisin by protein engineering.the dielectric constant in the active site cleft of subtilisin from bacillus amyloliquefaciens has been probed by mutating charged residues on the rim and measuring the effect on the pka value of the active site histidine (his64) by kinetics. mutation of a negatively charged surface residue, which is 12 to 13 a from his64, to an uncharged one asp----ser99) lowers the pka of the histidine by up to 0.4 unit at low ionic strength (0.005 to 0.01 m). this corresponds to an apparent dielectric constan ...19873302273
[exonuclease iii from bacillus amyloliquefaciens].bacillus amyloliquefaciens cells were found to contain an exonuclease which catalyzes the sequential hydrolysis of mononucleotides from the 3'-termini of duplex dna. the enzyme was purified to homogeneity and its molecular weight (as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate) is 29,000. the exonuclease possesses an additional catalytic activity, i.e., 3'-5' exonuclease specific for the rna strand in an rna--dna hybrid duplex (rnase h activity). in ...19873311177
prediction of electrostatic effects of engineering of protein charges.accurate prediction of electrostatic effects on catalytic activity is an essential component of protein design. site-directed mutagenesis of charged groups in subtilisin of bacillus amyloliquefaciens has provided experimental measurements of electrostatic interactions which may be used to test such theoretical methods. the pka of the histidine of the active site has been perturbed by +0.08 to -1.0 units by modifying one or two residues. electrostatic effects in proteins can be modelled by the al ...19873313059
[determination of inhibition sites during hydrolysis of polydeoxyribonucleotides by exonucleases iii from bacillus amyloliquefaciens and escherichia coli].the influence of the primary structure of polydeoxyribonucleotides on the rate of hydrolysis with exonuclease iii from bacillus amyloliquefaciens and escherichia coli was investigated. the substrates used were synthetic oligodeoxyribonucleotides and pbr 322 dna fragments labeled with 32p at the 5'-termini of one of the chains. according to the data from polyacrylamide gel electrophoresis performed under denaturing conditions, the hydrolysis of these substrates by unsaturating concentrations of b ...19873315009
[design of a hybrid gene coding for the leader sequence of bacillus amyloliquefaciens alpha-amylase and for human proinsulin].the chemically synthesized structure gene of human proinsulin was cloned in e. coli on the secretory vector containing regulatory elements of the bacillus amyloliquefaciens alpha-amylase gene. the proinsulin gene was inserted by the ecori site located immediately after the dna area encoding the alpha-amylase signal peptide. the e. coli cells transformed by such a plasmid produced hybrid protein consisting of the alpha-amylase signal peptide, five amino acid residues after the gene mating and hum ...19873326519
h2, a temperate bacteriophage isolated from bacillus amyloliquefaciens strain h.bacillus amyloliquefaciens strain h is lysogenic for a large temperate phage we call h2. h2 has a polyhedral head 85 nm in diameter and a tail of about 17 x 434 nm. h2 lysogenizes bacillus subtilis between the tyra and metb genes, and gives specialized transduction of metb and, at lower frequencies, of ilvd and ilva. the phage carries a thymidylate synthase gene and converts thymine auxotrophs of b. subtilis to prototrophy. the h2 genome is a linear dna molecule about 129 kb in length. dna extra ...19873449602
molecular structure of the replication origin of a bacillus amyloliquefaciens plasmid pftb14.the structure of a 1.5-kb dna sequence that is necessary and sufficient for the replication of an 8.2-kb cryptic plasmid, pftb14, isolated from a strain of bacillus amyloliquefaciens has been characterized. the 1.5-kb dna sequence contains an open reading frame, rep, stretching for 1017 bp, a promoter region for rep expression, and a possible replication origin for the plasmid upstream of the promoter. the rep product is trans-active and essential for plasmid replication. the predicted rep prote ...19873481020
efficient secretion of bacillus amyloliquefaciens alpha-amylase by [corrected] its own signal peptide from saccharomyces cerevisiae host cells [corrected].the expression and secretion of bacillus amyloliquefaciens alpha-amylase was studied in yeast saccharomyces cerevisiae. the bacillus promoter was removed by bal 31 digestion and three forms of the alpha-amylase gene were constructed: the bacillus signal sequence was either complete (yep alpha a1), partial (yep alpha a2) or missing (yep alpha a3). secretion of alpha-amylase into the culture medium was obtained with the complete signal sequence only. the secreted alpha-amylase was glycosylated and ...19872830166
expression of a bacillus alpha-amylase gene in yeast.a recombinant plasmid, psr11.3, containing the alpha-amylase gene (amy) of bacillus amyloliquefaciens was characterized and expressed in bacillus subtilis. a 2.3 kilobase bamhi-bglii fragment carrying amy was cloned into pbr322 (pel322) and in both orientations into a multi-copy escherichia coli-yeast shuttle vector yep13 (pam13) and expressed in e. coli hb101 and various saccharomyces stains. we report on the successful secretion of an active bacterial enzyme in yeast without using yeast promot ...19882843299
why is one bacillus alpha-amylase more resistant against irreversible thermoinactivation than another?half-lives of bacillus alpha-amylases at 90 degrees c and ph 6.5 greatly increase in the series from bacillus amyloliquefaciens to bacillus stearothermophilus to bacillus licheniformis, e.g. the difference in thermostability between the first and the third enzymes exceeds 2 orders of magnitude. this stabilization is achieved by lowering the rate constant of monomolecular conformational scrambling, which is the cause of irreversible thermoinactivation of b. amyloliquefaciens and b. stearothermoph ...19883125174
quantification of alpha-amylase mrna in bacillus subtilis by nucleic acid sandwich hybridization.a novel hybridization test for quantification of mrna from bacterial cells lysed directly in the culture medium was developed and optimized. the method uses two adjacent probes from a dna fragment of interest in a sandwich hybridization. an unlabeled probe is immobilized on a solid support to capture homologous nucleic acids in the test solution. hybrid detection is performed with the other, labeled, probe, which can bind to the filter only as a sample-mediated hybrid. we used the bacillus amylo ...19883129270
genome homology and host range of some sp beta-related bacteriophages of bacillus subtilis and bacillus amyloliquefaciens.temperate bacillus subtilis bacteriophages sb beta, phi 3t and spr and b. amyloliquefaciens phage h2 were compared with respect to dna-dna homology by southern blot analysis to each other and to members of the genus bacillus. the results show that h2 is a distantly related member of the group iii b. subtilis phages. detectable homology to group iii phages could be found in the dna of several other bacillus species, including b. natto and b. amyloliquefaciens, demonstrating the widespread occurre ...19883133451
[use of the thermoinducible temperate phage phi 105cts139 for cloning in bacillus subtilis].the gene for alpha-amylase from bacillus amyloliquefaciens having a foreign promoter providing gene expression in logarithmic growth phase and the cat gene of plasmid pc194 (ac fragment) were inserted into thermoinducible prophage phi 105 cts139. possibility of amylolytic activity enhancement was studied after thermoinduction. when ac fragment and random psti restricts of phage dna were ligated and used to transform bacillus subtilis 1a289 (phi 105 cts139) the amy+ cmr transformants were obtaine ...19883139998
engineering thermostability in subtilisin bpn' by in vitro mutagenesis.a procedure has been developed for the isolation and identification of mutants of the bacterial serine protease, subtilisin, which exhibit enhanced thermostability. the cloned subtilisin bpn' gene from bacillus amyloliquefaciens was treated with a variety of chemical mutagens to introduce random mutations in the coding sequence. strains containing the cloned, mutagenized subtilisin gene which produced subtilisin with enhanced thermostability were selected by a simple plate assay procedure, which ...19883145814
stabilization of protein structure by interaction of alpha-helix dipole with a charged side chain.the alpha-helix in proteins has a dipole moment resulting from the alignment of dipoles of the peptide bond which can perturb the pkas of ionizing groups. one of the two histidine residues (his18) in barnase, the small ribonuclease from bacillus amyloliquefaciens, is located at the negatively charged end (c-terminal) of an alpha-helix. from nmr titrations of wild-type and engineered mutants we find that the pka of his18 is 7.9 in wild-type enzyme, 1.6 units above the value in the urea-denatured ...19883173493
[primary structure of intracellular serine proteinase from bacillus amyloliquefaciens. i. isolation of the enzyme and amino acid sequence of peptides of tryptic hydrolysate].method of isolation of intracellular serine protease was modified. gramicidin s-sepharose cl-4b with a higher content of the ligand, synthesized through a modified procedure, was used as an affinity sorbent which simplified the purification and led to the pure enzyme with high specific activity and 90% yield. trypsin hydrolyzate of the protease was separated by ion-exchange chromatography on a sulphocationite resin followed by paper chromatography and paper electrophoresis to yield twenty-five i ...19883190767
mechanisms of irreversible thermal inactivation of bacillus alpha-amylases.molecular mechanisms of irreversible thermal inactivation of two bacterial alpha-amylases, from the mesophile bacillus amyloliquefaciens and from the thermophile bacillus stearothermophilus, have been elucidated in the ph range of relevance to enzymatic catalysis. at ph 5.0, 6.5, and 8.0, b. amyloliquefaciens alpha-amylase irreversibly inactivates due to a monomolecular conformational process, formation of incorrect (scrambled) structures which subsequently undergo aggregation. at the last ph, t ...19883257756
[mutations in the alpha-amylase gene of bacillus amyloliquefaciens, leading to a decrease in the temperature of protein inactivation].alpha-amylase genes of bacillus amyloliquefaciens, coding proteins with reduced thermostability, had been obtained as a result of hydroxylamine mutagenesis. temperature, ph and starch concentration dependences of two mutant alpha-amylases were investigated. the synthesis of the alpha-amylases by several b. subtilis strains with different levels of extracellular proteases was also studied. the mutation containing fragments were localized and the structures of the mutations were determined. it was ...19883265469
dissecting the catalytic triad of a serine protease.serine proteases are present in virtually all organisms and function both inside and outside the cell; they exist as two families, the 'trypsin-like' and the 'subtilisin-like', that have independently evolved a similar catalytic device characterized by the ser, his, asp triad, an oxyanion binding site, and possibly other determinants that stabilize the transition state (fig. 1). for bacillus amyloliquefaciens subtilisin, these functional elements impart a total rate enhancement of at least 10(9) ...19883282170
the three-dimensional structure of bacillus amyloliquefaciens subtilisin at 1.8 a and an analysis of the structural consequences of peroxide inactivation.the three-dimensional structure of the subtilisin from bacillus amyloliquefaciens (bas) has been refined to 1.8 a using the amino acid sequence deduced from the dna coding sequence. the structure is essentially the same as the previously reported structures of subtilisin bpn' (wright, c.s., alden, r.a., and kraut, j. (1969) nature 221, 235-242) and novo (drenth, j., hol, w. g. j., jansonius, j. n., and koekoek, r. (1972) eur. j. biochem. 26, 177-181) determined in different crystal forms, at 2.5 ...19883286644
barnase and barstar. expression of its cloned inhibitor permits expression of a cloned ribonuclease.barnase is the extracellular ribonuclease of bacillus amyloliquefaciens and barstar its specific intracellular inhibitor. the gene for barstar has now been cloned and sequenced. when the wild-type gene for barnase is reconstructed from its previously cloned parts on the same plasmid as the barstar gene, the lethal effect of its expression is suppressed. a plasmid has been devised which directs the secretion of 100 mg per active barnase liter by escherichia coli and another which provides large ( ...19883050134
[mutations in escherichia coli pmp and htpr genes stabilize the products of foreign gene expression].the half lives of mrna for escherichia coli chloramphenicol-acetyltransferase, bacillus amyloliquefaciens alpha-amylase and human leucocyte interferon were measured in e. coli cells by molecular rna.dna hybridization. the effect of mutation in pnp gene, coding polynucleotide phosphorylase, on the stability of these mrna was studied. the half life of interferon mrna increases from 25 to 90 s in the pnp mutant, resulting in an increase of interferon accumulation. the stability of interferon in e. ...19883054497
cloning of the bamhi methyl transferase gene from bacillus amyloliquefaciens.we wish to report the initial characterization of a recombinant clone containing the bamhi methylase gene. genomic chromosomal dna purified from bacillus amyloliquefaciens was partially cleaved with hindiii, fractionated by size, and cloned into psp64. plasmid dna from this library was challenged with bamhi endonuclease and transformed into escherichia coli hb101. a recombinant plasmid pbamm6.5 and a subclone pbamm2.5 were shown to contain the bamhi methylase gene based on three independent obse ...19883063644
expression of a cloned beta-glucanase gene from bacillus amyloliquefaciens in an escherichia coli rela strain after plasmid amplification.amino acid starvation of cells of the escherichia coli rela strain, cp79, which cannot accumulate guanosine tetraphosphate (ppgpp) in response to amino acid limitation, increased the peg1 plasmid content about 5- to 7-fold in comparison with exponentially growing cells (peg1:pbr322 with an insertion of bacillus amyloliquefaciens dna coding for beta-glucanase). in contrast, no peg1 amplification occurred in e. coli cp78, the stringently controlled counterpart, after amino acid starvation. in orde ...19883071739
contribution of hydrophobic interactions to protein stability.a major factor in the folding of proteins is the burying of hydrophobic side chains. a specific example is the packing of alpha-helices on beta-sheets by interdigitation of nonpolar side chains. the contributions of these interactions to the energetics of protein stability may be measured by simple protein engineering experiments. we have used site-directed mutagenesis to truncate hydrophobic side chains at an alpha-helix/beta-sheet interface in the small ribonuclease from bacillus amyloliquefac ...19883386721
[two-codon mutagenesis of alpha-amylase gene of bacillus amyloliquefaciens].the oligonucleotide encoding bam hi recognition site having the structure pcgggatc had been inserted into the recognition sites mspi of the b. amyloliquefaciens alpha-amylase gene, which was cloned in ptg29b plasmid. the alpha-amylase gene had no bamhi sites before mutagenesis. the set of pnsbamhi plasmids with bamhi site at four different positions was obtained. it was shown that all the mutant alpha-amylases possess different specific activities. one of the mutant proteins possesses reduced th ...19882464738
kinetics of alpha-amylase synthesis from bacillus amyloliquefaciens.the microbial production of alpha-amylase from bacillus amyloliquefaciens was investigated. the microorganism was grown using media containing glucose or maltose at 37 degrees c and under aerobic conditions in a 16-l fermentor. the alpha-amylase synthesis from maltose was not found to be inducible but was found to be subject to catabolite repression. the maltose uptake rate was observed to be the rate-limiting step compared to the conversion rate of maltose to glucose by intracellular alpha-gluc ...198818584616
fed-batch fermentation for the production of alpha-amylase by bacillus amyloliquefaciens.fed-batch cultures were performed to maximize the alpha-amylase activity in a bioreactor. kinetic equations containing a catabolite repression effect were used to model the enzyme formation from bacillus amyloliquefaciens. fed-batch culture experiments were performed using maltose to implement the optimal feeding strategy. optimal fed-batch culture based on sequential parameter estimation was performed successfully using off-line analysis while the fermentation was in progress. the enzyme activi ...198818584627
effect of yeast extract on alpha-amylase synthesis by bacillus amyloliquefaciens. 198918587980
stabilization of restriction endonuclease bam hi by cross-linking reagents.bacillus amyloliquefaciens h produces a restriction endonuclease enzyme bamhl which is heat labile even at low temperatures. studies were conducted to enhance thermal stability of bamhl using cross-linking reagents, namely, glutaraldehyde, dimethyl adipimidate (dma), dimethyl suberimidate (dms), and dimethyl 3,3'-dithiobispropionimidate (dtbp). reaction with glutaraldehyde did not result in a preparation with enhanced thermal stability. however, the dma-, dms-, and dtbp-cross-linked preparations ...198918587865
cell growth and alpha-amylase production characteristics of bacillus amyloliquefaciens.growth and alpha-amylase production characteristics of bacillus amyloliquefaciens strain f (atcc 23350) in batch cultures are examined using glucose or maltose as the carbon source. while the cell growth is rapid when glucose is used as the carbon source, higher cell mass, higher total and specific enzyme activities, and higher enzyme production rates are obtained when maltose is used as the carbon source. the overall specific enzyme activity decreases with an increase in the initial concentrati ...198918587901
cell growth and alpha-amylase production characteristics of bacillus amyloliquefaciens. 198918588140
on the mechanism of growth of cells (bacillus amyloliquefaciens) in the mixed aqueous two-phase system.the growth of bacillus amyloliquefaciens in the aqueous two-phase system, made up of polyethylene glycol, dextran, and water, was investigated. generally, bacillus partitions in the dextran phase, but the magnitude of the separation depends largely on the overall composition of polymers in the phase system. the kinetics of growth of bacillus amyloliquefaciens was studied in the polyethylene glycol-rich continuous phase, dextran-rich dispersed phase, and in the mixed phase. from the kinetic data ...19892472777
expression of bovine pancreatic ribonuclease a coded by a synthetic gene in bacillus subtilis.two different hybrid genes were constructed which fuse the bacillus amyloliquefaciens alkaline protease gene (apr[bamp]) promoter and signal peptide coding region to a synthetic bpr gene coding for the mature bovine pancreatic rnase a. the first gene fusion (apr-bpr1) contained the apr[bamp] signal peptide coding region fused to mature bpr through a linker coded 3-amino acid region and retained the signal processing site ala-ala of the alkaline protease. the second fusion (apr-bpr2) joined the e ...19892501158
production of pneumolysin, a pneumococcal toxin, in bacillus subtilis.we have expressed the pneumolysin gene of streptococcus pneumoniae in bacillus subtilis, both from its own promoter and as a fusion protein. the level of expression of pneumolysin from its own promoter was low. the protein produced was hemolytically active. a higher level of expression (about 10 micrograms/ml of culture) was achieved when either one of two c-terminal fragments (corresponding to amino acids 265-471 and 55-471, respectively) or the entire coding part of the pneumolysin gene were f ...19892502471
protein engineering of disulfide bonds in subtilisin bpn'.five single-disulfide mutants were studied in subtilisin bpn', a cysteine-free, secreted serine protease from bacillus amyloliquefaciens. the disulfides were engineered between residues 26-232, 29-119, 36-210, 41-80, and 148-243. these bonds connected a variety of secondary structural elements, located in buried or exposed positions at least 10 a from the catalytic ser-221, and linked residues that were separated by 39 up to 206 amino acids. all disulfide bonds formed in the enzyme when the expr ...19892504281
[cloning the alpha-amylase gene of bacillus amyloliquefaciens in cyanobacteria cells].the recombinant plasmids of piah4amy series were constructed containing the alpha-amylase gene of bacillus amyloliquefaciens a50 with its own promoter and leading sequence within an integrative vector plasmid piah4 (cmr) for cyanobacterium anacystis nidulans r2. at anacystis nidulans transformation the hybrid plasmids integrate into cyanobacterium chromosome with high efficiency and all cmr transformants produce alpha-amylase. expression of bacillar alpha-amylase gene in cyanobacterium cells is ...19892515431
transformation of bacillus amyloliquefaciens by electroporation.a method for transformation of whole bacillus amyloliquefaciens cells by electroporation was developed. the procedure is as efficient as the protoplast transformation method, resulting in up to 10(5) transformants/micrograms plasmid dna, but requires less effort and time. cells for electroporation were grown to late exponential phase in a rich medium supplemented with 0.25 m sucrose, washed with and resuspended in 0.25 m sucrose, 1 mm hepes, 1 mm mgcl2, 10% (v/v) glycerol, ph 7.0, at 3-5 x 10(10 ...19892599355
kinetic characterization of the recombinant ribonuclease from bacillus amyloliquefaciens (barnase) and investigation of key residues in catalysis by site-directed mutagenesis.barnase, the ribonuclease from bacillus amyloliquefaciens, has been cloned and expressed in escherichia coli [hartley, r. w. (1988) j. mol. biol. 202, 913-915], thus enabling the overproduction and site-directed mutagenesis of one of the smallest enzymes (mr equals 12,382). as barnase is also composed of just a single polypeptide chain with no disulfide bridges and has a reversible folding transition, it affords a fine system for studying protein folding and design. we show here that the recombi ...19892665810
synthesis and secretion of bacterial alpha-amylase by the yeast saccharomyces cerevisiae.alpha-amylase from bacillus amyloliquefaciens, synthesized in yeast saccharomyces cerevisiae without substitution of the signal sequence, is efficiently secreted from yeast cells: 60-70% of the overall amount of the enzyme is found in the culture fluid. in contrast to many yeast secretory proteins, which accumulate in the periplasmic space and in the cell wall, intracellular alpha-amylase is localized mainly in the cytoplasm. obviously, transfer across the cell wall is not a rate-limiting step i ...19892666166
hybrid bacillus endo-(1-3,1-4)-beta-glucanases: construction of recombinant genes and molecular properties of the gene products.hybrid beta-glucanase genes were constructed by the reciprocal exchange of the two halves of the isolated beta-glucanase genes from bacillus amyloliquefaciens and b. macerans. the beta-glucanase hybrid enzyme 1 (h1) contains the 107 amino-terminal residues of mature b. amyloliquefaciens beta-glucanase and the 107 carboxyl-terminal amino acid residues of b. macerans beta-glucanase. the reciprocal beta-glucanase hybrid enzyme 2 (h2) consists of the 105 amino-terminal residues from the b. macerans ...19892673278
barnase and barstar: two small proteins to fold and fit together.barnase and barstar are the extracellular ribonuclease and its intracellular inhibitor produced by bacillus amyloliquefaciens. both are small single-chain proteins and thus are suitable for application to the study of how a protein's sequence directs its fold. barnase has neither disulfide bonds nor non-peptide components and unfolds reversibly in what closely approximates a two-state reaction. the genes for both these proteins have been cloned in e. coli. expression of barstar is necessary to c ...19892696173
investigation into the nature of a bacillus promoter cloned into a promoter-probe plasmid.the alpha-amylase-coding gene (amy) of bacillus amyloliquefaciens ncp1 was cloned into the bacillus subtilis promoter probe vector ppl603b.1, using a bglii digest of chromosomal dna. the resulting plasmid, pvc102, was shown to have a bglii site within the insert. it was determined that this was the result of the fortuitous co-cloning of 2.88-kb and 0.92-kb bglii fragments separated in ncp1 dna by approx. 3 kb. unexpectedly, this co-cloning was readily repeated. subcloning showed that while the 2 ...19892806909
amino acid residues stabilizing a bacillus alpha-amylase against irreversible thermoinactivation.the alpha-amylase of bacillus licheniformis (bla) is stable and active at high temperature. more than 80% of its activity is retained after heat treatment at 90 degrees c for 30 min, and the optimum temperature for its activity is 80-85 degrees c. in contrast, the alpha-amylase of bacillus amyloliquefaciens (baa), the amino acid sequence of which shows 80% homology with that of bla, is rapidly inactivated at 90 degrees c. various chimeric genes were constructed from the structural genes for the ...19892808401
capping and alpha-helix stability.the first and last four residues of alpha-helices differ from the rest by not being able to make the intrehelical hydrogen bonds between the backbone greater than c=o groups of one turn and the greater than nh groups of the next. physico-chemical arguments and statistical analysis suggest that there is a preference for certain residues at the c and n termini (the c- and n-caps) that can fulfil the hydrogen bonding requirements. we have tested this hypothesis by constructing a series of mutations ...19892812029
translation and processing of bacillus amyloliquefaciens extracellular rnase.bacillus amyloliquefaciens extracellular rnase has been previously cloned and expressed in bacillus subtilis. site-specific mutagenesis experiments have identified codon -39 as the start site of translation. we have determined the primary signal peptide cleavage site of preprobarnase and propose a pathway for the conversion of probarnase to mature barnase.19892914867
production and secretion of pertussis toxin subunits in bacillus subtilis.pertussis toxin (pt) is a major component of today's acellular whooping cough vaccines. the use of acellular vaccines is predicted to increase sharply in the near future. there is therefore a need to produce pt in a way that makes its purification as easy as possible. our approach was to express all five pt subunits individually in bacillus subtilis. we have used vectors containing the promoter and signal sequences of the alpha-amylase gene of bacillus amyloliquefaciens followed by an insert enc ...19902110091
bacillus amyloliquefaciens alpha-amylase signal sequence fused in frame with human proinsulin is properly processed by bacillus subtilis cells.the plasmid pbins1, containing the promoter, sd and leader peptide sequences of bacillus amyloliquefaciens alpha-amylase gene and 267 bp long sequence coding for human proinsulin directs the efficient synthesis of hybrid preproinsulin, as well as quantitative secretion of proinsulin outside of protease-deficient bacillus subtilis aj73 cells. the recombinant proinsulin has been isolated from the culture medium and its n-terminal sequence shown to be identical with that of natural human prohormone ...19902112381
excretion into the culture medium of a bacillus beta-glucanase after overproduction in escherichia coli.the beta-glucanase gene (bgl) from bacillus amyloliquefaciens was expressed in e. coli csh 55 under the control of the pr promoter of phage lambda that is repressed by the thermosensitive repressor c1857. production of beta-glucanase was drastically stimulated by a temperature shift to 42 degrees c. this overexpression of the bgl gene (about 20% of the total cellular protein) led to an almost complete excretion of the otherwise periplasmic protein into the extracellular medium, beta-glucanase ac ...19902114118
efficiency of transcriptional terminators in bacillus subtilis.to promote more efficient synthesis of heterologous gene products in a bacillus subtilis host, we have developed a system for rapidly testing the effect of a putative terminator on in vivo gene expression. terminator structures from the bacillus amyloliquefaciens amye gene, the bacillus licheniformis penp gene, the b. subtilis bgls gene, and the bacillus thuringiensis cry gene were subcloned and inserted into a vector in such a way as to disrupt expression of the cat-86 gene. comparisons are mad ...19902123811
secretion of the alpha-galactosidase from cyamopsis tetragonoloba (guar) by bacillus subtilis.a fusion of dna sequences encoding the spo2 promoter, the alpha-amylase signal sequence from bacillus amyloliquefaciens, and the mature part of the alpha-galactosidase from cyamopsis tetragonoloba (guar) was constructed on a bacillus subtilis multicopy vector. bacillus cells of the protease-deficient strain db104 harboring this vector produced and secreted the plant enzyme alpha-galactosidase up to levels of 1,700 u/liter. a growth medium suppressing the residual proteolytic activity of strain d ...19902160224
the complete sequence of the bacillus amyloliquefaciens strain h, cellular bamhi methylase gene. 19902235511
isolation and characterization of levansucrase-encoding gene from bacillus amyloliquefaciens.the gene encoding levansucrase (lvs) from bacillus amyloliquefaciens (sacb[bamp]) was isolated, sequenced and expressed in bacillus subtilis. analysis of the nucleotide sequence of sacb[bamp] reveals extensive homology with that of the b. subtilis lvs-encoding gene in the promoter and coding region. the sacb[bamp] gene cloned in a multicopy plasmid is induced by sucrose in b. subtilis.19902265762
an investigation of the cause of the eosinophilia-myalgia syndrome associated with tryptophan use.the eosinophilia-myalgia syndrome is a newly recognized illness that has been associated with the consumption of tryptophan products. it is not known whether the cause is related to the tryptophan itself or to chemical constituents introduced by the manufacturing process.19902370887
the complete sequence of the bacillus amyloliquefaciens proviral h2, bamhi methylase gene. 19902374727
cloning and expression of alpha-amylase from bacillus amyloliquefaciens in a stable plasmid vector in lactobacillus plantarum.lactobacillus plantarum is used in a wide range of agricultural and food fermentations. in this paper we report the introduction of alpha-amylase into the organism from bacillus amyloliquefaciens on a stable recombinant plasmid. the genetically manipulated organism grew on mrsb medium supplemented with starch and it may be a prototype for the development of lactobacilli able to use an increased range of substrates in commercial fermentations.19901366859
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