The Analysis Of A Bacteria Produced Hyaluronic Acid And The Clonging And Expression Of Its Hyalurondiase Gene

At first ,we observed the modaility of the strain GXH-1 which is kept in our lab and can produce hyaluronic acid .The optimum condition for HA yield is pH6.5,after fermentation at 37 C for 48hr the HA was about 4.12g per litre of fermentation liquor. A sequence about 1500bp 16SrDNA fragment was amplified from total DNA of the strain GXH-1 by PCR .The analysis result show the 16SrDNA of the strain share 100% homologywith the 16SrDNA sequence of type strain Streptococcus equi subsp zooepidemicus (gi|28849227|dbj|AB104843).After genetic background analysis ,we presume that an open read fragment about 2.6kb is the hyalurondiase of the Streptococcus equi,because of the high homogeneity between the ORF and some known hyalurondiase of the other strains. The ORF were amplified from totalDNA of GXH~1 by PCR .The analysis result show the sequence share 99% homology with an ORF in the Streptococcus equi.The ORF was inserted into expression vector pSE-380, then transformed into E.coli JM109 and E.coli BL21 . The SDS-PAGE showed that the relative molecular weights of the hyl expression products were about 97.5 kD and the expression in E.coli BL21 is much higher than in E.coli JM109. The Morgan-Elson reaction was used to assays the activity of hyalurondiase. Replacement plasmid for homologous recombination was constructed by inserting amp gene into hyalurondiase gene. We plan to get the homologous recombination of hyl gene in Streptococcus equi chromosomes achived by using nature or denatured linearized DNA fragments, and then the hyalurondiase deficient strain was obtained .