ABSTRACT
Collagen II (COLII), the most abundant protein in vertebrates, helps maintain the structural and functional integrity of cartilage. Delivery of COLII from animal sources could improve cartilage regeneration therapies. Here we show that COLII can be purified from the Capra ear cartilage, a commonly available bio-waste product, with a high yield. MALDI-MS/MS analysis evidenced post-translational modifications of the signature triplet, Glycine-Proline-Hydroxyproline (G-P-Hyp), in alpha chain of isolated COLII (COLIIA1). Additionally, thirty-two peptides containing 59 Hyp residues and a few G-X-Y triplets with positional alterations of Hyp in COLIIA1 are also identified. Furthermore, we show that an injectable hydrogel formulation containing the isolated COLII facilitates chondrogenic differentiation towards cartilage regeneration. These findings show that COLII can be isolated from Capra ear cartilage and that positional alteration of Hyp in its structural motif, as detected by newly developed mass spectrometric method, might be an early marker of cartilage disorder.
Subject(s)
Collagen Type II/chemistry , Collagen Type II/isolation & purification , Ear Cartilage/chemistry , Goats/metabolism , Hydroxyproline/analysis , Tandem Mass Spectrometry/methods , Amino Acid Sequence , Animals , Biomarkers/analysis , Biomarkers/metabolism , Cartilage Diseases/metabolism , Collagen Type II/pharmacology , Glycine/chemistry , Goats/anatomy & histology , Hydrogels/pharmacology , Hydroxyproline/metabolism , Mesenchymal Stem Cells/drug effects , Mesenchymal Stem Cells/metabolism , Peptides/chemistry , Proline/chemistry , Protein Conformation , Protein Processing, Post-TranslationalABSTRACT
The extracellular biosurfactant product secreted by a marine bacterium was concentrated and purified directly from the fermentation broth in a single step by ultrafiltration (UF) employing YM 30 kDa (UF-I) and Omega 10 kDa (UF-II) polyethersulfone membranes. The optimum operating pressure required for both membranes, UF-I and UF-II, were found to be 30 and 35 psi, respectively. The biosurfactant from the fermentation broth was recovered in higher amounts using UF-II (89%) than using UF-I (73%). An analysis of the critical micelle concentrations (CMC) of the recovered lipopeptides showed a lower CMC value of 15 mg L⻹ for the UF-II product, indicating higher degree of purity (83%) when compared to that of the UF-I product (78%). The ultrafiltered products were characterized using Fourier transformed infrared spectroscopy and matrix-assisted laser desorption ionization time of flight mass spectral analysis, which demonstrated the presence of two families of lipopeptides.
Subject(s)
Aquatic Organisms/chemistry , Lipopeptides/analysis , Lipopeptides/biosynthesis , Lipopeptides/metabolism , Peptides, Cyclic/analysis , Peptides, Cyclic/biosynthesis , Peptides, Cyclic/metabolism , Ultrafiltration/methods , Adsorption , Aquatic Organisms/metabolism , Fermentation , Mass Spectrometry/methods , Membranes, Artificial , Micelles , Polymers/chemistry , Polymers/isolation & purification , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Spectroscopy, Fourier Transform Infrared/methods , Sulfones/chemistry , Sulfones/isolation & purification , Surface-Active Agents/chemistry , Surface-Active Agents/isolation & purificationABSTRACT
A marine Bacillus strain produced biosurfactant during its growth in a defined glucose-containing medium. An efficient method for separation and purification of biosurfactant isoforms was developed and optimized in high-performance liquid chromatography (HPLC) by manipulating solvent gradient program and flow rates. Starting with an initial run time of 60 min, the final optimized method had a significantly reduced run time of 20 min. By using this method, all the surface-active isoforms (fractions A-D) were eluted within 12 min of elution with much shortened retention time of each component. The purity levels of the isoforms were enhanced using the optimized method as evident from their lower CMC values. Among the four surface-active fractions, antimicrobial action was solely displayed by HPLC fraction A. FTIR analysis revealed all the HPLC fractions to be lipopeptide in nature and MALDI-ToF mass spectral analysis showed that these belonged to the fengycin family containing C(15), C(16), and C(17) fengycins.
