Clone, Expression Of The GlyA Gene Isolated From Two Kind Of Strains And Enzyme Assay

L-serine is widely used in cosmetics, medicine and chemical industry.At present L-serine is expensive but the demand of L-serine is very large, only depend import.The fermentative production of L-serine directly from carbo-hydrates has not been established because the metaboic turnover of this amino acid is much quicker than that of other amino acids: L-serine is a key intermediate for the synthesis of several other amino acids, nucleic acids and phospholipids. Because of its advantages of cheao raw material,available raw material and high concentration of L-serine in the reaction solution, the enzymatic method becomes the most promising one for manufacturing L-serine.The enzyme serine hydroxymethyltransferase(SHMT), a pyridoxal-phosphate enzyme, has been shown to have activity toward a wide variety of substrates. One catalytic function of this enzyme is to reversibly convert glycine and formaldehyde to serine in the presence of a cofactor, tetrahydrofolate(THF).In this reseach, isolation of the complete coding sequence of the glyA gene from E.coli K12 and S.thermophilus AS1.2471 were obtained directly from the genomic DNAs by PCR amplification. The PCR fragments of two kind were inserted into pET-28a(+) respectively. The expression vector pET28-K12-glyA and pET28-AS1.2471-glyA were transformed into E.coli BL21 and induced by IPTG.. Identification via SDS-PAGE showed that SHMT expressed successfully. The purified protein was obtained by nickel affinity gel column chromatography.Activity of SHMT expressed by the glyA gene isolated from E.coli K12 and S.thermophilus AS1.2471 was measured. The result is that the activity of SHMT encoded by the glyA gene isolated from S.thermophilus is almost twice as high as which from E.coli. So SHMT encoded by the glyA gene isolated from S.thermophilus possesses efficiency of catalytic activity,which will have a better application for industry. Through the reseach of clone and expression of the glyA gene isolated from two kind of strains and enzyme assay,SHMT with higher activity was obtained. It is valuable for enzymatic production of L-serine.