Abstract
The effect of five culture variables on the molecular weight properties of hyaluronic acid (HA) produced by Streptococcus zooepidemicus was studied in batch culture with a complex medium containing glucose and 10 g of yeast extract per liter. Neither the culture pH (pH 6.3 to 8.0) nor the agitation speed (300 to 1,000 rpm) affected the weight-average molecular weight (M(infw)) of HA under anaerobic conditions at 37(deg)C when 20 g of glucose per liter was used initially. M(infw) was in the narrow range of 1.5 x 10(sup6) to 2.3 x 10(sup6), and polydispersity (P) was between 1.8 and 2.5. When S. zooepidemicus was grown at lower temperatures or with aeration, higher-molecular-weight polymer and increased yields were observed. The polydispersity, however, remained unaffected. Anaerobically, the mean M(infw) (based on three samples taken within 4 h of glucose exhaustion) was (2.40 (plusmn) 0.10) x 10(sup6) and (1.90 (plusmn) 0.05) x 10(sup6) at 32 and 40(deg)C respectively. Aeration of the culture at 1 vol/vol/min produced HA with mean M(infw) of (2.65 (plusmn) 0.05) x 10(sup6) compared with (2.10 (plusmn) 0.10) x 10(sup6) under equivalent anaerobic conditions. The initial glucose concentration had the most pronounced effect on polymer characteristics. Increasing this concentration from 20 to 40 g/liter produced HA with mean M(infw) of (3.1 (plusmn) 0.1) x 10(sup6) at 1-vol/vol/min aeration. The molecular weight of HA also exhibited time dependency, with smaller chains (M(infw), ca. 2.5 x 10(sup6)) detected early in the culture time course, rising to a maximum (M(infw), 3.2 x 10(sup6)) in the late exponential phase of growth. The mean polydispersity was also greater (2.7 (plusmn) 0.1) under these conditions. Replicate experiments performed under conditions resulting in the lowest (40(deg)C, anaerobic) and highest (40 g of glucose per liter, 1-vol/vol/min aeration)-M(infw) polymer demonstrated excellent experimental reproducibility.
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- DeAngelis P. L., Papaconstantinou J., Weigel P. H. Molecular cloning, identification, and sequence of the hyaluronan synthase gene from group A Streptococcus pyogenes. J Biol Chem. 1993 Sep 15;268(26):19181–19184. [PubMed] [Google Scholar]
- DeAngelis P. L., Weigel P. H. Immunochemical confirmation of the primary structure of streptococcal hyaluronan synthase and synthesis of high molecular weight product by the recombinant enzyme. Biochemistry. 1994 Aug 9;33(31):9033–9039. doi: 10.1021/bi00197a001. [DOI] [PubMed] [Google Scholar]
- Dougherty B. A., van de Rijn I. Molecular characterization of a locus required for hyaluronic acid capsule production in group A streptococci. J Exp Med. 1992 May 1;175(5):1291–1299. doi: 10.1084/jem.175.5.1291. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kvam C., Granese D., Flaibani A., Zanetti F., Paoletti S. Purification and characterization of hyaluronan from synovial fluid. Anal Biochem. 1993 May 15;211(1):44–49. doi: 10.1006/abio.1993.1230. [DOI] [PubMed] [Google Scholar]
- Lansing M., Lellig S., Mausolf A., Martini I., Crescenzi F., O'Regan M., Prehm P. Hyaluronate synthase: cloning and sequencing of the gene from Streptococcus sp. Biochem J. 1993 Jan 1;289(Pt 1):179–184. doi: 10.1042/bj2890179. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mausolf A., Jungmann J., Robenek H., Prehm P. Shedding of hyaluronate synthase from streptococci. Biochem J. 1990 Apr 1;267(1):191–196. doi: 10.1042/bj2670191. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Motohashi N., Nakamichi Y., Mori I., Nishikawa H., Umemoto J. Analysis by high-performance gel permeation chromatography of hyaluronic acid in animal skins and rabbit synovial fluid. J Chromatogr. 1988 Jan 8;435(2):335–342. doi: 10.1016/s0021-9673(01)82193-2. [DOI] [PubMed] [Google Scholar]
- O'Regan M., Martini I., Crescenzi F., De Luca C., Lansing M. Molecular mechanisms and genetics of hyaluronan biosynthesis. Int J Biol Macromol. 1994 Dec;16(6):283–286. doi: 10.1016/0141-8130(94)90056-6. [DOI] [PubMed] [Google Scholar]
- Shimada E., Matsumura G. Viscosity and molecular weight of hyaluronic acids. J Biochem. 1975 Sep;78(3):513–517. doi: 10.1093/oxfordjournals.jbchem.a130935. [DOI] [PubMed] [Google Scholar]
- van de Rijn I. Streptococcal hyaluronic acid: proposed mechanisms of degradation and loss of synthesis during stationary phase. J Bacteriol. 1983 Dec;156(3):1059–1065. doi: 10.1128/jb.156.3.1059-1065.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]