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1.
A colorimetric enzyme assay using Escherichia coli to determine nutritionally available lysine in biological materials 总被引:2,自引:1,他引:1
A novel bacterial method is described for determining nutritionally available lysine in protein foods with a lysine auxotroph of Escherichia coli (strain M2626). Lysine-dependent synthesis of the induced enzyme bT-galactosidase is determined by a colorimetric method. With this approach sensitivity is increased ca 100-fold and assay time decreased to ca 2 h. The improved procedure was applied to the assay of lysine present as the free amino acid or in small peptides, and after enzymic pre-digestion in vitro with a mixture of pronase and intestinal peptidases, to pure proteins and a variety of feed meals and rice cultivars. In addition, heat treatment of complex samples was shown to lower their content of available lysine, as judged by the decreased nutritional response of the E.coli strain. 相似文献
2.
A 4.0 kb fragment from a plasmid genomic DNA library of the marine bacterium Alteromonas haloplanktis ATCC 19855 was found in the presence of Na+ to complement the dagA gene of Escherichia coli. We have completely sequenced this fragment and the position of the Na(+)-linked D-alanine glycine permease gene (dagA) on the fragment has been determined by complementation. The predicted carrier protein consists of 542 amino acid residues (M(r) 58,955). Its hydropathy profile suggests it is composed of eight transmembrane segments with a long hydrophilic region between segments six and seven. Significant similarity has been found between this Na(+)-linked permease and the Na+/proline permeases of E. coli and Salmonella typhimurium and the human and rabbit intestinal Na+/glucose cotransporters. 相似文献
3.
David A. Frisch Andrew M. Tommey Burle G. Gengenbach David A. Somers 《Molecular & general genetics : MGG》1991,228(1-2):287-293
Summary Dihydrodipicolinate synthase (DHPS; EC 4.2.1.52) is the first committed enzyme in the lysine branch of the aspartate-derived amino acid biosynthesis pathway and is common to bacteria and plants. Due to feedback inhibition by lysine, DHPS serves in a regulatory role for this pathway in plant metabolism. To elucidate the molecular genetic characteristics of DHPS, we isolated a putative full-length cDNA clone for maize DHPS by direct genetic selection in an Escherichia coli dapA
–auxotroph. The maize DHPS activity expressed in the complemented E. coli auxotroph showed the lysine inhibition characteristics of purified maize DHPS, indicating that the cDNA encoded sequences for both the catalytic function and regulatory properties of the enzyme. The N-terminal amino acid sequence of purified maize DHPS was determined by direct sequencing and showed homology to a sequence within the cDNA, indicating that the clone contained the entire coding region for a mature polypeptide of 326 amino acids plus a 54 amino acid transit peptide sequence. The molecular weight of 35854, predicted from the deduced amino acid sequence, was similar to the 38 000 Mr determined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) for the purified enzyme from maize. DHPS mRNAs complementary to the cDNA were detected in RNA isolated from developing maize endosperm and embryo tissues. Southern blots indicated the presence of more than one genomic sequence homologous to DHPS per haploid maize genome. 相似文献
4.
Isolation of Escherichia coli synthesized recombinant eukaryotic proteins that contain epsilon-N-acetyllysine.
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B. N. Violand M. R. Schlittler C. Q. Lawson J. F. Kane N. R. Siegel C. E. Smith E. W. Kolodziej K. L. Duffin 《Protein science : a publication of the Protein Society》1994,3(7):1089-1097
Recombinant porcine (rpST) and bovine somatotropins (rbST) synthesized in Escherichia coli contain the amino acid, epsilon-N-acetyllysine. This amino acid was initially discovered in place of the normal lysine144 in a modified reversed-phase HPLC (RP-HPLC) species of rpST. Mass spectrometry and amino acid sequencing of a tryptic peptide isolated from this RP-HPLC purified protein were used to identify this altered residue as epsilon-N-acetyllysine. Ion-exchange chromatography was utilized to prepare low isoelectric point (pI) forms of rpST and rbST, which are enriched in epsilon-N-acetyllysine. Electrospray mass spectrometry demonstrated that the majority of the protein in these low pI fractions contained species 42 Da larger than normal. Immobilized pH gradient electrophoresis (IPG) of the ion-exchange purified low pI proteins was used to isolate several monoacetylated species of rpST and rbST. The location of the acetylated lysine in each IPG-purified protein was determined by tryptic peptide mapping and amino acid sequencing of the altered tryptic peptides. Amino acid analyses of enzymatic digests of rpST and rbST were also used to confirm the presence of epsilon-N-acetyllysine in these recombinant proteins. These data demonstrate that a significant portion of rpST and rbST produced in E. coli contain this unusual amino acid. 相似文献
5.
The ability of Escherichia coli K-12 4212 to utilize a variety of oligopeptides as sources of required amino acids was examined. Triornithine-resistant mutants of this strain were oligopeptide permease deficient (Opp-) as judged by their inability to utilize (Lys)3 and (Lys)4 as sources of lysine and their resistance to the toxic tripeptide (Val)3. These same mutants were able to grow when Met-Met-Met, Met-Gly-Met, Met-Gly-Gly, Gly-Met-Gly, Gly-Gly-Met, Gly-Met-Met, Met-Met-Gly, or Leu-Leu-Leu were supplied in place of the requisite amino acid. The system mediating the uptake of these peptides, herein designated Opr I, was not able to transport N-alpha-acetylated peptides, nor the tetrapeptides Met-Gly-Met-Met, Met-Met-Gly-Met, or Met-Met-Met-Gly. Competition experiments indicated that trimethionine and trileucine enter E. coli K-12 via either Opp or Opr I. Analogous results were found using the methionine, leucine-requiring auxotroph E. coli B163. It appears that more than one oligopeptide transport system exists in E. coli and that the system mediating peptide uptake is complex. 相似文献
6.
The complete amino acid sequence of the inorganic pyrophosphatase from thermophilic bacterium PS-3 was determined by automated Edman analysis of the intact protein and of peptides derived from digests obtained with lysylendopeptidase, Staphylococcus aureus strain V8 protease, and arginylendopeptidase. The monomer peptide chain comprises 164 amino acid residues and has a calculated molecular weight of 18,792. The sequence is identical at about 46% of the amino acid positions with that of the Escherichia coli enzymes. 相似文献
7.
8.
A multiple amino acid auxotroph and a wild type of Escherichia coli K12 were used to study the effects of near minimum growth temperatures on the binding, transport, and cellular incorporation of selected amino acids. Both strains of the bacterium showed the same minimum growth temperature (8 degrees C) when previously grown at 15 degrees C. At 8 degrees C and above, the auxotroph exhibited an overall greater ability to bind and transport amino acids than did the wild type. Below the minimum growth temperature, transport and cellular incorporation including respiration ((uptake) were significantly lower for either organism. The NEU and HEPPEL osmotic shock treatment indicated the removal of the specific histidine-binding protein and the ability to bind histidine was not recovered by further incubation below 8 degrees C. At 8 degrees C and above, the cells recovered their ability to bind histidine within one hour. The evidence presented indicates a direct relationship between the auxotroph's minimum growth temperature and its ability to bind amino acids, specifically methionine. 相似文献
9.
Cloning and sequencing of the Thermoanaerobacterium saccharolyticum B6A-RI apu gene and purification and characterization of the amylopullulanase from Escherichia coli. 总被引:4,自引:2,他引:2
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The amylopullulanase gene (apu) of the thermophilic anaerobic bacterium Thermoanaerobacterium saccharolyticum B6A-RI was cloned into Escherichia coli. The complete nucleotide sequence of the gene was determined. It encoded a protein consisting of 1,288 amino acids with a signal peptide of 35 amino acids. The enzyme purified from E. coli was a monomer with an M(r) of 142,000 +/- 2,000 and had same the catalytic and thermal characteristics as the native glycoprotein from T. saccharolyticum B6A. Linear alignment and the hydrophobic cluster analysis were used to compare this amylopullulanase with other amylolytic enzymes. Both methods revealed strictly conserved amino acid residues among these enzymes, and it is proposed that Asp-594, Asp-700, and Glu-623 are a putative catalytic triad of the T. saccharolyticum B6A-RI amylopullulanase. 相似文献
10.
Cloning and sequencing of pel gene responsible for CMCase activity from Erwinia chrysanthemi PY35 总被引:1,自引:0,他引:1
The phytopathogenic bacterium Erwinia chrysanthemi secretes multiple isozymes of plant cell wall disrupting enzymes such as pectate lyase and endoglucanase. We cloned genomic DNA from Erwinia chrysanthemi PY35. One of the E. coli XL1-Blue clones contained a 5.1-kb BamHI fragment and hydrolyzed carboxymethyl cellulose and polygalacturonic acid. By subsequent subcloning, we obtained a 2.9-kb fragment (pPY100) that contained the pel gene responsible for CMCase and pectate lyase activities. The pel gene had an open reading frame (ORF) of 1,278 bp encoding 425 amino acids with a signal peptide of 25 amino acids. Since the deduced amino acid sequence of this protein was very similar to that of PelL of E. chrysanthemi EC16, we concluded that it belonged to the pectate lyase family EC 4.2.2.2, and we designated it PelL1. Sequencing showed that the PeIL1 protein contains 400 amino acids and has a calculated pI of 7.15 and a molecular mass of 42,925 Da. The molecular mass of PelL1 protein expressed in E. coli XL1-Blue, as analyzed by SDS-PAGE, appeared to be 43 kDa. The optimum pH for its enzymatic activity was 9, and the optimum temperature was about 40 decreased C. 相似文献
11.
12.
Vesela I. Chalova Irene B. Zabala-Díaz Cassie L. Woodward Steven C. Ricke 《World journal of microbiology & biotechnology》2008,24(3):353-359
As an essential amino acid, lysine is an important component of animal and human diets and its bioavailability can depend
on a variety of factors. Therefore, an accurate pre-determination of bioavailable lysine in foods and feeds is important.
In this study a whole cell fluorescent biosensor for the quantification of lysine in protein sources was constructed. A gene
encoding for green fluorescent protein (GFPmut3) was introduced into an E. coli lysine auxotroph genome as a part of a mini-Tn5-Km transposon. The location of the transposon was determined and the growth
kinetics of the newly constructed biosensor were examined. The transposon disrupted the ybhM gene, which encodes for the synthesis of a protein with an unknown function. No effect of the transposon’s location in the
genome or the expression of gfp on bacterial growth rates was observed. Based on the fluorescence emitted by GFPmut3, a standard curve after 6-h growth of
the strain was generated. A correlation coefficient of 0.95 was observed when the fluorescence method was compared to the
conventional optical density (OD) growth-based lysine assay. Using the newly developed lysine fluorescent whole cell sensor
we determined the total lysine in casein acid hydrolyzate (7.13 ± 0.34%). When lysine added to 12 μg/ml and 30 μg/ml of casein
acid hydrolyzate was quantified, recoveries of 97 ± 1.65% and 103 ± 4.66% respectively were detected. The results suggest
that the microbial assay using GFP fluorescence represents a promising alternative method for the potential estimation of
lysine in protein sources. 相似文献
13.
Characterization of a protein inhibitor of extracellular proteases produced by Erwinia chrysanthemi 总被引:10,自引:0,他引:10
Erwinia chrysanthemi, a phytopathogenic bacterium, produces a protease inhibitor which is a low-molecular-weight, heat-stable protein. In addition to its action on the three E. chrysanthemi extracellular proteases A, B and C, it also strongly inhibits the 50 kD extracellular protease of Serratia marcescens. Its structural gene (inh) was subcloned and expressed in Escherichia coli, in which it encodes an active inhibitor which was purified. The nucleotide sequence of the inh gene shows an open reading frame of 114 condons. The N-terminal amino acid sequence of the purified inhibitor was also determined. It indicated the existence of an amino-terminal signal peptide absent from the mature protein. The inhibitor is entirely periplasmic in E. chrysanthemi and partially periplasmic in E. coli. 相似文献
14.
AGITATION DURING INCUBATION REDUCES THE TIME REQUIRED FOR A LYSINE MICROBIOLOGICAL GROWTH ASSAY USING AN ESCHERICHIA COLI AUXOTROPHIC MUTANT 总被引:2,自引:0,他引:2
X. LI A.M. ERICKSON S.C. RICKE 《Journal of Rapid Methods and Automation in Microbiology》2000,8(2):83-94
Microbiological assays involving Escherichia coli lysine auxotrophs must be optimized to facilitate routine use. Our objectives in this study were to characterize growth of an auxotrophic E. coli lysine mutant (American Type Culture Collection strain #23812) and examine the effect of agitation on E. coli mutant growth. A defined minimal salts basal medium was used and supplemented with various lysine concentrations. The E. coli lysine auxotroph responded to increasing lysine concentration with increasing optical density. When maximum optical density (MOD) was determined for the auxotroph, a linear increase was obtained as lysine concentrations were increased (R2 ± 0.96) for both agitation and static cultures. Growth rates were not significantly (p > 0.05) affected by lysine concentrations, cultural conditions or their combined effect. However, growth with agitation significantly (p < 0.05) reduced the assay time by shortening the lag phase and causing stationary phase to occur earlier. The values of R2 (± 0.96) relatively remained constant over the range while the bacterial population were in the stationary phase. In conclusion, the lysine growth assay using the E. coli lysine auxotroph can be made more rapid by agitating the culture during incubation. 相似文献
15.
Growth responses of lysine auxotrophic mutants of Escherichia coli have been used as a measurement of bioavailable lysine in protein sources and animal feeds. Sterilizing feed samples by autoclaving to eliminate non-specific background growth of indigenous feed micro-organisms prior to conducting the bacterial assay may introduce chemical and physical alterations to the feeds, influencing the estimation of available feed lysine. In this study, an antibiotic- and antifungal-supplemented medium was constructed to support growth of an E. coli lysine auxotroph assay organism, and was tested for its ability to repress indigenous bacterial and fungal growth in feed samples. To determine which antibiotics to include, an ampicillin-sensitive E. coli lysine mutant strain (ATCC no. 23812) was screened for antibiotic resistance and transformed with a plasmid carrying an ampicillin resistance gene. Maximum optical density quantitative response of the E. coli auxotroph to lysine was not altered by the antibiotic medium amendments (ampicillin, novobiocin and cycloheximide). Indigenous microfloral growth in a variety of typical animal feeds was suppressed in the presence of the antistatic agents. The estimated lysine recovery was 91.6% and 98.1% when the medium was used in an assay of available lysine in a lysine-supplemented feed. This indicates that the antibiotic-amended basal medium can be used for the E. coli-determined lysine availability of a variety of animal feeds without prior sterilization of the feed sources. 相似文献
16.
Molecular cloning of bile acid 7-dehydroxylase from Eubacterium sp. strain VPI 12708. 总被引:8,自引:8,他引:0
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Eubacterium sp. strain VPI 12708 is a human intestinal bacterium which contains an inducible bile acid 7-dehydroxylase. Two-dimensional polyacrylamide gel electrophoresis showed that at least four new polypeptides were synthesized after exposure of growing cells to sodium cholate. One of these, of molecular weight 27,000 (PP-27), was implicated in 7-dehydroxylase catalysis. PP-27 was purified to greater than 95% homogeneity by DEAE-cellulose chromatography, high-pressure liquid chromatographic gel filtration, high-pressure liquid chromatography-DEAE chromatography, and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The first 33 amino acid residues of the N terminus of PP-27 were determined with a gas-phase sequencer, and a corresponding mixed oligonucleotide (17-mer) was synthesized. Southern blot analysis of EcoRI total digests of chromosomal DNA showed a 2.2-kilobase fragment which hybridized to the 32P-labeled 17-mer. This fragment was enriched for by size fractionation of an EcoRI total digest of genomic DNA, ligated into the bacterial plasmid pUC8, and used to transform Escherichia coli HB101. Transformants containing the putative 7-dehydroxylase gene were detected with the 32P-labeled 17-mer by colony hybridization techniques. The insert was 2.2 kilobases in length and contained the first 290 bases of the PP-27 gene. Preliminary nucleic acid sequence data correlate with the amino acid sequence. The entire gene was cloned on a 1,150-base-pair TaqI fragment. Western blot analysis of E. coli strains containing these plasmids indicated that PP-27 is expressed in E. coli but is not regulated by bile acids under the conditions used. 相似文献
17.
The argF gene encoding ornithine carbamoyl-transferase (OTCase; EC2.1.3.3) has been cloned from Corynebacterium glutamicum by transforming the Escherichia coli arginine auxotroph with the genomic DNA library. The cloned DNA also complements the E. coli argG mutant, suggesting a clustered organization of the genes in the genome. We have determined the DNA sequence of the minimal fragment complementing the E. coli argF mutant. The coding region of the cloned gene is 957 nucleotides long with a deduced molecular mass of about 35 kDa polypeptide. The enzyme activity and size of the expressed protein in the E. coli auxotroph carrying the argF gene revealed that the cloned gene indeed codes for OTCase. Analysis of the amino acid sequence of the predicted protein revealed a strong similarity to the corresponding protein of other bacteria. 相似文献
18.
Two overlapping clones encoding for a ribonuclease from six-day-old larvae of the insect Ceratitis capitata (Cc-RNase) have been isolated by immunoscreening a cDNA library and by 5' RACE. The sequence of the Cc-RNase cDNA contains an open reading frame of 414 nucleotides encoding for a precursor protein of 138 amino acids long with a putative signal peptide consisting of 19 amino acids. The calculated M(r) of the mature protein was found to be 13.7 kDa. Multiple alignments of the deduced amino acid Cc-RNase sequence with other ribonucleases revealed an approximate 25% average identity. Despite the low percentage of identity, histidine and lysine residues which are essential for its catalytic activity, were found to be completely conserved. Furthermore, expression of the clone in E. coli resulted in the production of a recombinant product that showed strong immunoreactivity with anti-RNase specific antibodies. These results support the hypothesis that the identified clone encodes for a protein which is a new member of the RNase superfamily. 相似文献
19.
AIMS: To examine the utility of an Escherichia coli green fluorescent protein (GFP) containing biosensor for quantification of bioavailable lysine in selected feed samples under nonsterile conditions and to estimate the background fluorescence of analyzed feed samples and evaluate the risk of confounding GFP emission from the lysine assay organism. METHODS AND RESULTS: Escherichia coli lysine auxotroph GFP based biosensor was used to determine the percentage of bioavailable lysine in two samples of soybean-, cottonseed-, and meat and bone meal under nonsterile conditions. The fluorescence emitted by GFP was successfully measured using a spectrofluorimeter to monitor bacterial growth response to protein-derived lysine and lysine containing small peptides. The autofluorescence of analyzed feed samples at different concentrations could also be estimated. CONCLUSIONS: When feed protein concentrations are decreased, autofluorescence interference can be avoided. SIGNIFICANCE: The E. coli lysine auxotroph GFP-based biosensor can successfully be used for the determination of bioavailable lysine in these selected animal feed proteins under nonsterile conditions. IMPACT OF THE STUDY: E. coli GFP biosensor for lysine has potential for routine application in animal feeds. 相似文献
20.
We have studied the mechanisms by which amino acid starvation of Escherichia coli induces resistance against the lytic and bactericidal effects of penicillin. Starvation of E. coli strain W7 of the amino acids lysine or methionine resulted in the rapid development of resistance to autolytic cell wall degradation, which may be effectively triggered in growing bacteria by a number of chemical or physical treatments. The mechanism of this effect in the amino acid-starved cells involved the production of a murein relatively resistant to the hydrolytic action of crude murein hydrolase extracts prepared from normally growing E. coli. Resistance to the autolysins was not due to the covalently linked lipoprotein. Resistance to murein hydrolase developed most rapidly and most extensively in the portion of cell wall synthesized after the onset of amino acid starvation. Lysozymes digests of the autolysin-resistant murein synthesized during the first 10 min of lysine starvation yielded (in addition to the characteristic degradation products) a high-molecular-weight material that was absent from the lysozyme-digests of control cell wall preparations. It is proposed that inhibition of protein synthesis causes a rapid modification of murein structure at the cell wall growth zone in such a manner that attachment of murein hydrolase molecules is inhibited. The mechanism may involve some aspects of the relaxed control system since protection against penicillin-induced lysis developed much slower in amino acid-starved relaxed controlled (relA) cells than in isogenic stringently controlled (relA+) bacteria. 相似文献