title: Design of aqueous two-phase systems for purification of hyaluronic acid produced by metabolically engineered Lactococcus lactis.
abstract: Hyaluronic acid has a wide range of biomedical applications and its commercial value is highly dependent on its purity and molecular weight. This study highlights the utility of aqueous two-phase separation as a primary recovery step for hyaluronic acid and for removal of major protein impurities from fermentation broths. Metabolically engineered cultures of a lactate dehydrogenase mutant strain of Lactococcus lactis (L. lactis NZ9020) were used to produce high-molecular-weight hyaluronic acid. The cell-free fermentation broth was partially purified using a polyethylene glycol/potassium phosphate system, resulting in nearly 100% recovery of hyaluronic acid in the salt-rich bottom phase in all the aqueous two-phase separation experiments. These experiments were optimized for maximum removal of protein impurities in the polyethylene glycol rich top phase. The removal of protein impurities resulted in substantial reduction of membrane fouling in the subsequent diafiltration process, carried out with a 300 kDa polyether sulfone membrane. This step resulted in considerable purification of hyaluronic acid, without any loss in recovery and molecular weight. Diafiltration was followed by an adsorption step to remove minor impurities and achieve nearly 100% purity. The final hyaluronic acid product was characterized by Fourier-transform IR and NMR spectroscopy, confirming its purity.
authors.affiliation: Department of Biotechnology, Manipal Institute of Technology, Manipal, Karnataka, India.
authors.forename: Vivek
authors.initials: V
authors.lastname: Rajendran
authors.affiliation: Department of Biotechnology, Bhupat and Jyoti Mehta School of Biosciences, Indian Institute of Technology Madras, Chennai, India.
authors.forename: Kirubhakaran
authors.initials: K
authors.lastname: Puvendran
authors.affiliation: Department of Biotechnology, Manipal Institute of Technology, Manipal, Karnataka, India.
authors.forename: Bharath Raja
authors.initials: BR
authors.lastname: Guru
authors.affiliation: Department of Biotechnology, Bhupat and Jyoti Mehta School of Biosciences, Indian Institute of Technology Madras, Chennai, India.
authors.forename: Guhan
authors.initials: G
authors.lastname: Jayaraman
mesh_terms: D000327:Adsorption; D019149:Bioreactors; D005591:Chemical Fractionation / Q000379:methods*; D005285:Fermentation; D006820:Hyaluronic Acid / Q000737:chemistry*; D007770:L-Lactate Dehydrogenase / Q000235:genetics; D013294:Lactococcus lactis / Q000235:genetics / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008970:Molecular Weight; D009154:Mutation; D010710:Phosphates / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011092:Polyethylene Glycols / Q000737:chemistry; D011108:Polymers / Q000737:chemistry; D017680:Potassium Compounds / Q000737:chemistry; D011506:Proteins / Q000737:chemistry; D017550:Spectroscopy, Fourier Transform Infrared; D013450:Sulfones / Q000737:chemistry
chemical_list: D010710:Phosphates; D011108:Polymers; D017680:Potassium Compounds; D011506:Proteins; D013450:Sulfones; C022840:polyether sulfone; D011092:Polyethylene Glycols; D006820:Hyaluronic Acid; C013216:potassium phosphate; D007770:L-Lactate Dehydrogenase
keywords: Aqueous two-phase Systems; Diafiltration; Hyaluronic acid; Lactococcus lactis