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

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.

Integration of a Nucleoside Triphosphate Regeneration System in the One‐pot Synthesis of UDP‐sugars and Hyaluronic Acid

“…[12] Setting the reaction parameters for a high UDP-GlcNAc/UDP-GlcA ratio secures high activity of PmHAS 1À 703 as demonstrated in our previous studies. [12,13] Moreover, we hypothesize that modulation of PmHAS 1À 703 activity directs the amount of HA chains by de novo synthesis and thus finally the HA MW. With an MW of 1.54 MDa, a low dispersity of 1.02, and a final HA concentration of 1.4 g/L, enzymatic synthesis can compete with results from bacterial fermentation.…”

“…[10,11] In our previous study, we established an in vitro one-pot synthesis to produce high molecular weight (HMW) HA. [12] The enzymatic cascade contains six enzymes: AtGlcAK, AtUSP (both from Arabidopsis thaliana), BlNahK (Bifidobacterium longum), SzGlmU (Streptococcus zooepidemicus), PmPpA, and PmHAS 1À 703 (both from Pasteurella multocida). We could show that the MW of HA could be controlled by the key factors pH and the MgCl 2 concentration because these factors influence the syntheses of UDP-GlcA and UDP-GlcNAc.…”

“…With an MW of 1.54 MDa, a low dispersity of 1.02, and a final HA concentration of 1.4 g/L, enzymatic synthesis can compete with results from bacterial fermentation. [12,14] However, in the current enzymatic batch synthesis, a stoichiometric amount of ATP (20 mM) is needed, which is costly and inefficient for large-scale processes. [15] Also, high ATP and ADP concentrations can reduce the activity of kinases and other enzymes as we have found out with AtGlcAK.…”

“…[15] Also, high ATP and ADP concentrations can reduce the activity of kinases and other enzymes as we have found out with AtGlcAK. [12] Applying an ATP regeneration system reduces starting ATP concentration, which reduces cost and avoids limitations. [15] Several kinases were used to regenerate nucleoside triphosphates (NTPs) such as acetate kinase, carbamate kinase, creatine kinase, pyruvate kinase (PK), and PPK.…”

Biocatalysis: “A Jack of all Trades...”

“…In contrast, enzymatic systems often provide a single-step selective process, yielding directly a “deprotected” product. This has been nicely demonstrated in the one-pot enzyme cascade synthesis of high molecular weight hyaluronic acid, which is largely used in cosmetic applications [ 4 ]. This elegant procedure, an alternative to the use of animal tissues, combines two enzyme modules that in situ generate the respective precursors UDP–GlcA and UDP–GlcNAc using the hyaluronan synthase from Pasteurella multocida ( Pm HAS).…”