ABSTRACT
Teixobactin, a cyclic-peptide antibiotic, destroys the cell walls of Gram-positive bacteria. It is therefore difficult for bacteria such as meticillin-resistant Staphylococcus aureus to develop resistance to it. Many scholars have studied the structure-activity relationship (SAR) of teixobactin. After reviewing the reports related to the discovery, structure, total synthesis and SAR of teixobactin, we found that the total synthesis of teixobactin was very difficult. The N-terminal methyl modification, ester bond and allo-End were unnecessary for the activity, the configuration of amino acids at the positions 1, 4, 5 and 8 could greatly influence the antibacterial activity, and the substitution of the amino acids at the 3, 4, 9 and 10 positions by Lys could retain the antibacterial activity.
ABSTRACT
A new method for the determination of peptide antibiotics in sediment from aquaculture environment by high performance liquid chromatography-tandem mass spectrometry was developed. The target analytes in sediments were ultrasonically extracted twice with citrate buffer solution and methol mixture (3∶ 4, V/ V), followed by complexation with 0. 5 g of Na2 EDTA, purification with 5 mL of methyl isobutyl ketone, and clean-up with HLB-SPE column. The analytes were separated on a MGII C18 column by gradient elution with 0. 1% formaic acid-0. 1% formaic acid acetonitrile as mobile phase, detected in multiple reaction monitoring (MRM) with electrospray ionization (ESI) under positive ion mode, and quantified by external standard method. The calibration curves were linear (R2 >0. 999) over a concentration range of 10 -10000μg / L for colistin and bacitracin and 4-4000 μg / L for virginiamycin M1 . The limits of detection (S / N = 3) were 5 μg / kg for colistin and bacitracin and 2 μg / kg for virginiamycin M1 . The limits of quantification (S / N=10) was 10 μg / kg for colistin and bacitracin and 4 μg / kg for virginiamycin M1 . At three spiked levels, the recoveries ranged from 79. 7% to 91. 6% (RSD=1. 9% -10. 8% ), showing high sensitivity, good reproducibility and wide applicability.
ABSTRACT
A total of 112 soil samples were taken from differents areas of district D.I.Khan and Kohat (KPK) Pakistan and screened for production of antibiotics against the Micrococcus luteus and Staphylococcus aureus. Widest zone of inhibition (18mm) was produced by microorganism isolated from saline soil. The strain was later identified as Bacillus GU057 by standard biochemical assays. Maximum activity (18mm inhibition zone) was observed against Staphylococcus aureus after 48 hours of incubation at pH 8 and 4% concentration of glucose. The antibiotic was identified by autobiography as bacitracin. The Bacillus strain GU057 was confirmed as good peptide antibiotic producer and can effectively be indulged as biocontrol agent.
Subject(s)
Anti-Bacterial Agents/isolation & purification , Bacillus/isolation & purification , Bacitracin/analysis , Bacitracin/isolation & purification , Glucose/analysis , Micrococcus luteus/isolation & purification , Saltpetre Soils/analysis , Staphylococcus aureus/isolation & purification , Methods , Process Optimization , Reference Standards , Soil Microbiology , MethodsABSTRACT
Objective:To screen and clone a carrier molecule for the expression of small bioactive peptides at high levels. Methods: A carrier molecule, PaP3.30, was screened out from the genome of Pseudomonas aeruginosa phage PaP3 and its gene was cloned by PCR method and inserted into pQE 32 expression plasmid, this recombinant plasmid was named pQE PaP30. The peptide antibiotics hPAB ? gene was then inserted into pQE PaP30 and induced to express the fusion protein in Escherichia coli . The ability of PaP3.30 to express other bioactive peptides was evaluated by fusing 6 different origins, varies in sizes and isoelectric points selected peptides to it. Results: After fused to PaP3.30, the peptide antibiotics hPAB ? could express as fusion protein above 30% of total bacterial proteins. Six selected peptides were also expressed by the level of 35%～44% total bacterial proteins when fused to carrier molecule, PaP3.30. Conclusion: The new carrier molecular, PaP3.30, is versatile in the expression of small bioactive peptides.
ABSTRACT
Objectives: To design the mutants of peptide antibiotics hPAB ? based on its molecular structure. Methods: The three dimension structure of hPAB ? was constructed by protein homology modeling method. The mutant molecules were designed and generated by PCR and inserted into pQE CP4 expression plasmid. The recombinant plasmids were identified by PCR and DNA sequencing and then transformed into Escherichia coli JM109 to express target fusion proteins. Results:Peptide hPAB ? shows one ? helical and three ? sheet in its structure. Its ? helical regions seem play a key role in the formation of active oligomer. Aside from positioning Thr 7 and Lys 10 into contact positions, the orientation of the ? helix is conserved about the oligome core, forming a ridge around it. Additionally, the dipoles of the helices would overlap to create a positively charged region near the core. These dipoles may be offset, however, by the presence of Asp 4 at the base of the helix. Two mutant molecules, hPAB ? 38 and hPAB ? 34, were designed by deleting N or/and C terminal 2～5 amino acid residues based on hPAB ? structure. The recombinant plasmids containing the mutants gene can express interest fusion proteins in E. coli JM109 successfully. Conclusions: Design, cloning and expression of the mutants of peptide antibiotics hPAB ? lay down the foundation for screening of the mutant of shorter peptide chain and having high or same antimicrobial activity.
ABSTRACT
Objective: To screen the best genetic engineering bacterium for the production of peptide antibiotic hPAB-? and evaluate its fermentation level in bottle. Methods:After analysis of the interest fusion protein expression levels of 8 recombinant bacteria containing 1-8 copies of human peptide antibiotic hPAB-? expressing plasmid respectively,2-5 copies expressing bacteria were chosen for the further study of their bacteria yield,expression forms of the target protein, dissolution of the inclusion bodies and the efficiency of fusion protein purification by affinity chromatography, then the best engineering bacterium with the certain copies of interest peptide expressing plasmid was screened out and its optimal fermentation parameters in bottle were also studied. Results:The recombinant bacterium transformed by 3 copies of interest peptide expressing plasmid was the best candidate for its bacteria yield (3.153 g/L) and fusion protein expression level (27.7%) were the highest among 1-8 copies candidates. The inclusion bodies of 3 copies target fusion protein could be easily dissolved by 8 mol/L urea and captured by Ni-NTA column. The elution of the fusion protein could be directly cleaved to monomer by adding 2 mol/L hydroxylamine, adjusting pH to 9.0 and incubating at 45℃ for 2 h. The optimal fermentation conditions of the selected recombinant bacteria were: culture the organisms with modified M9-CAA media at 37℃ and 160 r/min to (A 600 )≈2.5, then add IPTG to the final concentration 100 ?mol/L to induce the expression of target fusion protein for 5 h. Conclusion:The engineering bacterium containing 3 copies interest peptide recombinant expressing plasmid is the best candidate for the production of peptide antibiotic hPAB-?,and its fermentation parameters are confirmed.
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Objective To reconstruct the new engineered bacteria expressing hPAB-? triploids so as to improve the outputs of recombinant human peptide antibiotic ?. Methods The recombinant plasmid pQE31-hPAB-?(3) was transformed into E. coli. M15 to screen the new engineered bacteria expressing hPAB-? triploids. The stabilities of phPAB-?(3)/M15 were observed in continuous cultures. The expression levels of the fusion peptides of interest and the bacterial yields of the new engineered bacteria phPAB-?(3)/M15 were compared with that of phPAB-?(3)/JM109 in different fermentation scales. Results Genetic stability of the recombinant plasmid and phPAB-?(3)/M15 was 100 after 10 passages. Take bacterial yields into account, the new engineered bacteria phPAB-?(3)/M15 was better than phPAB-?(3)/JM109 at the similar expression levels of the target proteins by “t” test analysis (P
ABSTRACT
Objective To construct the recombinant plasmid with a human peptide antibiotic hPAB-? gene and to make it expressed in E. coli. Methods To replace the CNBr cleavage site in plasmid pFAST-hPAB-? (CNBr), a pair of primers containing the hydroxylamine cleavage site were designed, and the amplified PCR fragments were cloned into pFAST-HTa plasmid to produce pFAST-hPAB-? (HA), which was then transformed into E. coli DH10B. The constructed plasmid was identified by Ehe Ⅰ/Hind Ⅲ digestion and direct DNA sequencing. An Ehe Ⅰ/Hind Ⅲ digested fragment from pFAST-hPAB-? (HA) was subcloned into pQE32-CP to construct pQE32-CP-hPAB-?, which was transformed into E. coli JM109. The bacteria containing the expression plasmid were induced to express the fusion protein by IPTG. SDS-PAGE was carried out to analyze the molecular weight, expression quantity and expression form of the target fusion protein. After captured by Ni-NTA affinity column, the fusion protein was subjected to hydroxylamine cleavage analysis. Results An expected 230bp fragment was obtained by digesting pFAST-hPAB-? with Ehe Ⅰ/Hind Ⅲ. After this fragment was cloned into pQE32-CP, the recombinant plasmid was confirmed to contain the correct target sequence by DNA sequencing. The recombinant plasmid pQE32-CP-hPAB-? could express a desired protein with a relative molecular weight about 27kD, and its expression level reached 43 percent of the total bacterial proteins. The inclusion bodies were lysed by 8mol/L urea, and the fusion protein could then be captured by Ni-NTA column and cleaved by 2mol/L hydroxylamine at pH9.0. Conclusion The recombinant plasmid pQE32-CP-hPAB-? has been successfully constructed, and it can express the desired hPAB-? fusion protein in E. coli JM109 at high level. These results provide the foundation for future research.
ABSTRACT
Bacitracin was more growth-inhibitory to Neurospora crassa on a minimal magnesium medium than on a normal magnesium-medium. Both magnesium and manganese were able to counteract the growth inhibition. The antifungal activity of bacitracin was potentiated by zinc. Potassium could not counteract the growth inhibition by this antibiotic. The mycelial magnesium levels were low in bacitracin-inhibited cultures.