Primary recovery of hyaluronic acid produced in Streptococcus equi subsp. zooepidemicus using PEG–citrate aqueous two-phase systems

Abstract

Given its biocompatibility, rheological, and physiological properties, hyaluronic acid (HA) has become a biomaterial of increasing interest with multiple applications in medicine and cosmetics. In recent decades, microbial fermentations have become an important source for the industrial production of HA. However, due to its final applications, microbial HA must undergo critical and long purification processes to ensure clinical and cosmetic grade purity. Aqueous two-phase systems (ATPS) have proven to be an efficient technique for the primary recovery of high-value biomolecules. Nevertheless, their implementation in HA downstream processing has been practically unexplored. In this work, polyethylene glycol (PEG)–citrate ATPS were used for the first time for the primary recovery of HA produced with an engineered strain of Streptococcus equi subsp. zooepidemicus. The effects of PEG molecular weight (MW), tie-line length (TLL), volume ratio (VR), and sample load on HA recovery and purity were studied with a clarified fermentation broth as feed material. HA was recovered in the salt-rich bottom phase, and its recovery increased when a PEG MW of 8000 g mol−1 was used. Lower VR values (0.38) favoured HA recovery, whereas purity was enhanced by a high VR (3.50). Meanwhile, sample load had a negative impact on both recovery and purity. The ATPS with the best performance was PEG 8000 g mol−1, TLL 43% (w/w), and VR 3.50, showing 79.4% HA recovery and 74.5% purity. This study demonstrated for the first time the potential of PEG–citrate ATPS as an effective primary recovery strategy for the downstream process of microbial HA.

Production, characterization and techno-economic evaluation of Aspergillus fusant l-asparaginase

“…Santos et al ( 2018 ) reported extraction of 87.94% and 20.09-fold purified periplasmic l -ASNase from Escherichia coli with ATPS using PEG/Citrate system under optimized conditions. Primary (one-step) purification of other microbial products by PEG/Citrate aqueous two-phase system has also been reported by Flores-Gatica et al ( 2021 ) who recovered hyaluronic acid with 79.4% recovery and 74.5-fold purification factor. However, pre-purification of crude extract by ammonium sulfate precipitation before ATPS application significantly improved purification by 173.8-fold.…”

Recovery Techniques Enabling Circular Chemistry from Wastewater

“…Diverse examples of successful polymer/salt systems for the recovery of biopolymers can be found in the literature. For example, Gatica et al [208] employed a PEG/citrate ATPS to extract microbial, high molecular weight hyaluronic acid. This polysaccharide was mostly extracted in the citrate-rich bottom phase (80%), and it could be recovered with a purity level of 75%.…”

Primary recovery strategies of low‐molecular‐weight toxins from Crotalus molossus nigrescens and Crotalus atrox using aqueous two‐phase and three‐phase partition systems

“…9 ATPS are formed when two water-soluble molecules (e.g., two polymers or a polymer and salt) are mixed above a certain critical concentration, when phase separation takes place. 9,10 This separation method has been used extensively for the separation of a wide type of biomolecules such as enzymes, 11 proteins, 12 peptides, 13 biopolymers, 14 antibodies, 15 and low-molecular-weight products, 16 among others molecules. Although several molecules from different sources have been recovered using ATPS, its use for snake venom fractionation has been poorly explored.…”