MOLECULAR CLONING OF HYALURONAN SYNTHASE GENE AND PRODUCTION OF HYALURONC ACID IN BACILLUS SUBTILIS

Background and Aim:Hyaluronic acid (HA) plays important roles in human tissue system, thus it is highly desirable for various applications, such as in medical, clinic and cosmetic fields. Expressing hyaluronan synthase alone could make B. subtilis produce HA. The hasA gene from Streptococcus equisimilis (sehasA), which encodes the enzyme hyaluronan synthase, has been expressed in Bacillus subtilis, resulting in the production of hyaluronic acid. In addition, the B. subtilis-derived material was shown to be secreted and of high quality, comparable to commercially available sources of HA.Methods:The sehasA gene encoding hyaluronan synthase was artificially synthesized with codon preference of B. subtilis with the restriction sites HindIII/BamHI. The B. subtilis WASD was used as the host for HA synthesis and E. coli DH5α was used for cloning of the shuttle plasmid. B. subtilis cells were made competent by the method of Anagnostopoulos and Spizizen. For the expression of HA synthase in B. subtilis to produce HA, MMG medium was employed. Presence of HA in broth was demonstrated by a modified cetyltrimethylammonium bromide (CTAB) turbidity method.Results:Codon analysis showed that the rare codon numbers in sehasA for host B. subtilis dropped to zero. Electrophoresis of digested Recombinant shuttle plasmid, showed a 1290 bp band of Hyaluronan synthase gene. Presence of HA in broth was demonsted by CTAB method.Conclusion:B. subtilis has proven to be a superior expression host for producing HA. Analysis by CTAB turbidity and FTIR have revealed that HA produced in B. subtilis.

Keywords:

hyaluronan synthase , Hyaluronic acid , Bacillus subtilis