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.

A review on Lactococcus lactis: from food to factory

“…cremoris as the former produces GABA while the latter does not [94]. Other examples of medicinal metabolites successfully synthesized by L. lactis includes hyaluronic acid, which is a carbohydrate polymer used in wound healing, dermatitis and cosmetic-based applications [67]. …”

Comparative Economic Analysis Between Endogenous and Recombinant Production of Hyaluronic Acid

“…Hyaluronic acid is a biopolymer utilized in different current sectors of biotechnology ( Papakonstantinou et al, 2012 ; Bukhari et al, 2018 ; Bowman et al, 2018 ), and decreasing its production costs is important. Multiple reports for improvements in the production ( Liu et al, 2011 ) and recovery/purification ( Cavalcanti et al, 2019 ; Sousa et al, 2009 ; Choi et al, 2014 ; Rajendran et al, 2016 ; Akdamar et al, 2009 ; Murado et al, 2012 ; Rangaswamy and Jain, 2008 ) exist. However, a theoretical framework is required in order to determine which options provide the best production costs and have the potential to be incorporated into real-life production.…”

“…On the other hand, HA downstream bioprocessing has been extensively tested. The vast majority of the methods involved rely on a series of precipitations, filtrations, liquid-liquid partitions or chromatographic operations ( Cavalcanti et al, 2019 ; Sousa et al, 2009 ; Choi et al, 2014 ; Rajendran et al, 2016 ; Akdamar et al, 2009 ; Murado et al, 2012 ; Rangaswamy and Jain, 2008 ), each of these with specific variations to reduce material consumption or increase recovery yields.…”

Isolation and Characterization of Polysaccharides from Oysters (Crassostrea gigas) with Anti-Tumor Activities Using an Aqueous Two-Phase System

“…The most commonly explored type of ATPS in recent years for separation of polysaccharide/proteins is the polymer–salt system. High-molecular-weight hyaluronic acid was purified with a polyethylene glycol/potassium phosphate system, and nearly all of the hyaluronic acid in the salt-rich bottom phase and its purity reached almost 100% [ 21 ]. Xing and Li purified the aloe from bovine serum albumin with a PEG6000/(NH 4 ) 2 SO 4 system, and partitioned the aloe polysaccharides in the bottom phase.…”