The Construction Of Microbe Transformant Catalyzing The Synthesis Of Glyoxylic Acid

Glyoxyic acid is an fine chemical with diversified applications in perfumery, flavour, pharmaceutical, and agrochemical industries. Typical commercial production of glyoxylic acid employs either oxidation chemistry or electrochemical means, which produce waste streams containing various side-products and heavy metals. Increased public concern coupled with mounting government regulations surrounding the production of toxic wastes has provided impetus for a search for an alternative, cost effective, yet environmentally acceptable method of glyoxylic acid production. One such potential route lies in microbially-mediated enzymatic catalysis involving the oxidation of glycolic acid by glycolate oxidase and catalase. The goal of this work was to construct a strain in a genetic engineering way to catalyze the production of glyoxylate. It included the cloning and expression of spinach glycolate oxidase in E.coli, the expression of Helicobacterpylori catalase in E. coli, and the coexpression of the two enzymes in one host cell. First, the cDNA coding spinach glycolate oxidase (GO) was amplified by RT-PCR using the total RNA of spinach leaves as the template, and then cloned into cloning vector pMD18-T. After the DNA sequence was determined, the go gene was subcloned into E.coli expression vectors pBV220, pET-22b(+), pTIG-Trx and pThioHisC. SDS-PAGE analysis revealed that the recombinant E.coli BL21(DE3)(pTIG-Trx-GO) and E.coli BL21 (DE3)(pET-22b(+)-GO) expressed the predicted 38kDa glycolate oxidase, and the enzyme activity of E.coli BL21 (DE3)(pET-22b(+)-GO) (42U/g cells)was much higher than that of E.coli BL21 (DE3)(pTIG-Trx-GO) (6.3U/g cells). The enzyme activity ofE.coli BL21 (DE3)(pET-22b(+)-GO) was also a little higher than E.coli BL21 (DE3)(pKS20+), which was a gift from Prof. Ylva Lindqvist. Later on, we expressed the Helicobacter pylori catalase in E.coli, and the enzyme activity ofE.coli BL21 (DE3)(pET-22b(+)-CAT) was 500-600U/mg cells. Finally two methods were employed to express the tow enzymes in one E.coli cell. The first way is to transform E.coli BL21 (DE3)(pKS20+) with plasmid pET-22b(+)-CAT, and the second way is to subclone the genes of the two enzymes into one vector. By now the coexpression of glycolate oxidase and catalase has not succeeded.