Enzymatic hyaluronan

Such enzymatic catalyzed polycondensations have allowed the synthesis of a number of natural polysaccharides, but has also allowed the production of nonnatural polysaccharides such as cellulose-xylan hybrids and functionalized hyaluronan, chondroitin sulfate, and chondroitin. Such work illustrates the ever-narrowing bridge between natural and synthetic polymers and polymer syntheses. 

Heparin/heparan, hyaluronan, and chondroitin are three prevalent glycosaminoglycans. Vertebrates use glycosaminoglycans in structural, recognition, adhesion, and signaling roles. Chemical synthesis of naturally occurring polysaccharides is considered to be impractical. Most polysaccharides, especially those from bacteria origins, are obtained by purification from natural sources or from cell culture, enzymatic approaches have been increasingly applied to obtain some structures. 

Kobayashi S, Morii H, Itoh R, Kimura S, Ohmae M. Enzymatic polymerization to artificial hyaluronan a novel method to synthesize a glycosaminoglycan using a transition state analogue monomer. J. Am. Chem. Soc. 2001 123 11825-11826. 

Animal venoms usually possess hyaluronidase activity [17]. The enzymatic properties, including hyaluronidase, of snake venoms have been extensively studied by Tan et al. [18]. Snake hyaluronidase acts on hyaluronan, chondroitin, and chondroitin-4- and -6-sulfate, producing various oligosaccharides, mainly tetrasaccharides [1]. 

Figure 9.15 Enzymatic polymerization of various monomers (series 1) to synthetic hyaluronan and its derivatives (series 2). (Reproduced with permission from [231]. Copyright (2005) American Chemical Society).

The proposed mechanism for enzymatic hydrolysis of hyaluronan involves nucleophilic attack of water at the anomeric carbon of the d-G1cNAc moiety via an oxazolinium ion intermediate (see Figure 9.16b). A water molecule can nucle-ophilically attack the oxazolinium anomeric carbon atom to open the oxazolinium ring, resulting in the formation of the hydrolysis products of a shortened HA molecule (see Figure 9.16c). 

Figure 1.2 Regulation of hyaluronan amount and chain length by expression of a specific HAS protein. Biochemical characterizations of the vertebrate HAS enzymes expressed in mammalian cell culture have revealed similarities and differences between the respective mammalian hyaluronan synthase enzymes. The differences are depicted in this cartoon. HASl produces small amounts of high-molar-mass hyaluronan. HAS2 produces significantly more high-molar-mass hyaluronan. HAS3 is the most active of the hyaluronan synthases, yet produces low-molar-mass hyaluronan chains. The physiological significance of these differences in enzymatic activity is not yet known [33].

Itano N, Sawai T, Yoshida M, Lenas P, Yamada Y, Imagawa M, Shinomura T, Hamaguchi M, Yoshida Y, Ohnuki Y, Miyauchi S, Spicer AP, McDonald JA, Kimata K. Three isoforms of mammahan hyaluronan synthases have distinct enzymatic properties. J Biol Chem 1999 274 25085-25092. 

Scheme 23.5 Enzymatic synthesis of hyaluronan hydrogel via an oxidative coupling.

Oligosaccharides derived from enzymatic cleavage of hyaluronan chondroHin sulfate, etc. Direct UV detection D A=200-232 nm C Fused silica (72 cm x 50 im i.d.) E 40 mmol 1 phosphate, 40 mmol 1 SDS, 10 mmol I borate, pH 9 V 15 kV Study of digests of connective tissues diagnosis of arthritis... 

Copolymers may also be produced using combinations of chemically modified mono- or disaccharides and enzymatic condensation. Such a route has been proposed to prepare an artificial hyaluronan, GlucA-/3-(1 3) GlucNAc oxazoline monomer reacting with hyaluronidase (324). 

Dvorakova, J., Kucera, L., Kucera, J., Svik, K., Foglarova, M., Muthny, T., Pravda, M., Nemcova, M., Velebny, V., Kubala, L., 2014. Chondrogenic differentiation of mesenchymal stem cells in a hydrogel system based on an enzymatically crosshnked tyramine derivative of hyaluronan. J. Biomed. Mater. Res. A 102, 3523—3530. 

The first method is a relatively simple and short process [5] that involves multiple water extractions of grounded rooster combs, precipitation from combined extracts of HA with acetic acid, treatment with base, enzymatic hydrolysis of the protein, ultrafiltration, ethanol precipitation and final dissolution of the precipitate hyaluronan in water. 

Then a solution of papain (200ml of 0.1% solution of papain per 1kg of material) is added. Enzymatic hydrolysis of proteins is carried out for 20-40h. Every 6-8 h the pH must be adjusted to 6-7 (to optimize enzyme activity). The HA mixture is purified by ultrafiltration and precipitation with 96% ethanol. The final product, sodium hyaluronate, is dried by lyophilization and the precipitate is then dissolved in a water-alcohol mixture containing 40% ethanol. The final purification of hyaluronan is performed by membrane filtration of a solution containing 30-50% ethanol. 

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