Process for the preparation of an antimicrobial peptide
The present disclosure relates to a process for preparation of a peptide of sequence ID No. 1 having a formula C 231 H 354 N 66 O 83 from an endophytic bacterium Paenibacillus peoriae IBSD35 isolated from the stem of Millettia pachycarpa . The genome size of Paenibacillus peoriae IBSD35 is 5.8 Mb, 45.6% GC content. The bioactive peptide designated as Peoriaerin IBSD35 of SEQ ID No. 1 was purified from the fermentation broth of P. peoriae strain IBSD35 and characterized with ESI (nano-spray)-TOF MS/MS spectrum. The peptide sequences were generated using MASCOT, MS-MS ion search. It has 54 amino acids residue and protein size is 5383.747 Da. The peptide of sequence ID No. 1 prepared by the present process exhibited broad spectrum of antibacterial and antifungal activity. It is highly resistant and stable to enzyme, detergent and environmental factors. It has the potential for drug development against AMR pathogens. It can also be used as food preservatives.
1 . A process for preparation of a peptide of sequence ID No. 1 having a formula C 231 H 354 N 66 O 83 , wherein the process comprises the steps of:
(i) selecting plant Millettia pachycarpa harboring microbial endophytes;
(ii) isolating endophyte Paenibacillus peoriae with surface sterilization technique;
(iii) culturing Paenibacillus peoriae on a growth medium at a temperature in a range of 35-40° C. for 5-7 days to obtain cells;
(iv) harvesting the cells by centrifugation for 10-20 minutes at a speed of 3000-4000 rpm and at a temperature of 4° € 4° C. and obtaining a supernatant;
(v) filtering the supernatant through a 0.2 μm ultrafiltration membrane to obtain a clarified supernatant;
(vi) subjecting the clarified supernatant obtained in step (v) with 70% ammonium sulphate precipitation and incubating for a period of 3-5 hours at room temperature to obtain a mixture;
(vii) centrifuging the mixture obtained in step (vi) at a speed of 4000 rpm for 30-40 minutes at a temperature of 4° C. to collect pellets, followed by dissolving the pellets in distilled water;
(viii) dialyzing the dissolved pellets obtained in step (vii) against sodium phosphate buffer twice for a period of 12 hours to obtain a solution;
(ix) purifying the solution obtained in step (viii) using column chromatography with DEAE-Cellulose and eluting with 100-700 mM NaCl followed by dialyses to obtain a dialysate sample;
(x) analyzing the dialysate sample of step (ix) with RP-HPLC to obtain an antimicrobial peptide (AMP) peak; and
(xi) analyzing the AMP peak with 12% Tricine SDS-PAGE and sequencing with LC-MS.
2 . The process as claimed in claim 1 , wherein the isolated endophyte is Paenibacillus peoriae IBSD35.
3 . The process as claimed in claim 1 , wherein the growth medium is Luria Bertani, or Brain Heart Infusion medium.
4 . The process as claimed in claim 1 , wherein the antimicrobial peptide has a molecular weight of 5383.747 Dalton.
5 . The process as claimed in claim 1 , wherein the antimicrobial peptide forms alpha helices and has at least 13 residues on a hydrophobic surface.
6 . The process as claimed in claim 1 , wherein the antimicrobial peptide is thermostable at a temperature in a range of 20 to 121° C. and at a pH in a range of 4-9.
7 . The process as claimed in claim 1 , wherein the antimicrobial peptide has a strong and broad spectrum of antimicrobial activity against the gram positive, gram negative and fungal anti-microbial resistant pathogens selected from the group consisting of Candida albicans ATCC 10231, Escherichia coli ATCC 25922, Klebsiella pneumoniae ATCC 4352, Salmonella typhimurium ATCC 14028, Candida tropicalis ATCC 750, and Staphylococcus aureus ATCC 25923 and acts as an antimicrobial agent against animal, poultry feeds, food preservatives, plant pathogens and for therapeutic purposes.
8 . The process as claimed in claim 1 , wherein the activity of antimicrobial peptide is in a range of 1818.18 to 275862.06 AU/mg and has a microbial inhibition count of 0.0365 μg μl−1.