隨意突變表現於大腸桿菌之Streptococcus pyogenes玻尿酸合成酶企圖提高玻尿酸產量

Abstract

玻尿酸是一個重複β(1,3)-GlcNAc-β(1,4)- GlcA單元的線型多醣，可被發現於哺乳動物組織細胞外及細菌孢子內。玻尿酸由玻尿酸合成酶催化而成，其為具有二重功能把GlcNAc和GlcA二種醣的帶UDP先質接到延長中的玻尿酸長鏈。既然最近的醫藥級化妝品對於玻尿酸的需求甚殷，我們將Streptococcus pyogenes c203S之玻尿酸合成酶在大腸桿菌膜上表達來產生分泌的高分子量玻尿酸，並企圖使用隨意突變來增加其產量。

Hyaluronan (HA), a linear polysaccharide composed of β(1,3)-GlcNAc-β(1,4)- GlcA repeats, is found in the extracellular matrix of vertebrate tissues as well as the capsule of several pathogenic bacteria. HAs are synthesized by HA synthases (HASs), which are dual-action glycosyltransferases that catalyze the addition of two different sugars GlcA and GlcNAc from UDP-linked precursors to the growing HA chain. Since the recent medical and cosmetic uses of HA have largely increased its demand, as described in this invention, we have overexpressed spHAS from Gram positive Streptococcus pyogenes c203S on the membrane of E. coli to produce and secrete HA with high molecular weight into the media. Random mutagenesis by error-prone PCR was performed attempting to isolate the mutants with improved yield of HA.