Key Factors for A One-Pot Enzyme Cascade Synthesis of High Molecular Weight Hyaluronic Acid.

Gottschalk Gottschalk,Elling Elling,Kuballa Kuballa,Zaun Zaun,Eisele Eisele

doi:10.3390/ijms20225664

Gottschalk Gottschalk, Elling Elling + Show 3 more

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https://doi.org/10.3390/ijms20225664

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Abstract

In the last decades, interest in medical or cosmetic applications of hyaluronic acid (HA) has increased. Size and dispersity are key characteristics of biological function. In contrast to extraction from animal tissue or bacterial fermentation, enzymatic in vitro synthesis is the choice to produce defined HA. Here we present a one-pot enzyme cascade with six enzymes for the synthesis of HA from the cheap monosaccharides glucuronic acid (GlcA) and N-acetylglucosamine (GlcNAc). The combination of two enzyme modules, providing the precursors UDP–GlcA and UDP–GlcNAc, respectively, with hyaluronan synthase from Pasteurella multocida (PmHAS), was optimized to meet the kinetic requirements of PmHAS for high HA productivity and molecular weight. The Mg2+ concentration and the pH value were found as key factors. The HA product can be tailored by different conditions: 25 mM Mg2+ and 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES)-NaOH pH 8 result into an HA product with high Mw HA (1.55 MDa) and low dispersity (1.05). Whereas with 15 mM Mg2+ and HEPES–NaOH pH 8.5, we reached the highest HA concentration (2.7 g/L) with a yield of 86.3%. Our comprehensive data set lays the basis for larger scale enzymatic HA synthesis.

Highlights

Introduction and MotivationHyaluronic acid (HA) is a natural, non-sulfated, linear polymer consisting of repeating disaccharide units of [-3)GlcNAc(β1-4)glucuronic acid (GlcA)(β1-]n
We recently demonstrated the in vitro one-pot synthesis of hyaluronic acid (HA) from sucrose and GlcNAc with in situ regeneration of UDP–GlcA
We showed that substrate inhibition by UDP–GlcA and a high Km value for UDP–GlcNAc afford a favorable UDP–sugar ratio for the production of HA by PmHAS

Summary

Introduction

Hyaluronic acid (HA) is a natural, non-sulfated, linear polymer consisting of repeating disaccharide units of [-3)GlcNAc(β1-4)GlcA(β1-]n. In contrast to other glycosaminoglycans, HA is a polysaccharide with a size of up to 108 Da. Due to the anionic character, HA is able to bind large amounts of water, resulting in a viscoelastic gel [1,2,3]. HA forms are found in the extracellular matrix of connective tissues [3]. Because of the unique rheological behavior and the non-immunogenic feature, HA is widely used for medical and cosmetic applications such as drug/cosmetic agents, ophthalmic surgery, and tissue engineering [5,6,7,8,9,10]. With the increasing demand for HA, the market is expected to develop from USD 7.2 billion in 2016 to USD 15.5 billion in 2025 [11]

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