Expression And Application Of Heparinase Ⅰ AndⅢ And Gene Knockout Of E. Coli K5Waar

Low molecular weight heparin is an anticoagulant that is widely used in clinical treatment. Heparosan is an acidic polysaccharide natural product, bio synthesized as the polysaccharide capsule in Escherichia coli K5. It is the precursor polysaccharide in the biosynthesis of heparin and heparan sulfate, with an average molecular weight of10kDa to20kDa. Reaearch on heparosan to heparin through biological pathway is vital important.This study was aimed at preparation of low molecular weight heparin. To this end, Heparinase I and Heparinase III were expressed and purified. Then these two enzymes were used to depolymerize heparin and heparosan respectively. An enzymatic method was established to determine the heparosan content in the E. coli K5fermentation products based on Heparinase Ⅲ’s activity on heparosan. With the goal of enhancing E. coli K5heparosan production, the waaR gene which is the critical gene for the biosynthesis of outer core was removed using Red homologous recombination technique.Heparinase I was successfully expressed in engineering bacteria which contains HepA gene through induction of IPTG. The molecular weight of this protein is44.83kDa. Soluble fusion heparinase I was obtained after low temperature culturing and the help of molecular chaperones. The soluble fraction was purified by Ni-NTA affinity chromatography. The specific activity of the purified enzyme was10427.50U/mg, purification fold was6.47. Heparinase I was applied to depolymerize heparin, the depolymerizing effect is good. Heparinase Ⅲ was also successfully expressed in engineering bacteria contains HepC through induction of IPTG. The molecular weight was73.07kDa. Experiments were carried out to optimize the best conditions for enzyme production. After purification by Ni-NTA affinity chromatography, the specific activity of the enzyme was5372U/mg, purification fold was1.61. Heparinase Ⅲ can eliminate heparosan well.An enzymatic method was established to determine the content of heparosan in the sample based on Heparinase Ⅲ’s activity on heparosan. This method was convenient, simple to use and the reaction was gentle. Heparosan concentration can be calculated just through the standard formula by detection the absorbance of products.WaaR gene was successfully cloned using E. coli K5DNA as template by PCR. The sequence was99%similarity with the waaR of E. coli strain F632, two bases were different. Plasmid pKD46was transformed to E. coli K5to express λ Red recombinase. Primers including56bp homologous sequences of waaR in both ends were synthesized. Then PCR reaction was done to obtain targeting DNA which contains kanamycin resistance gene using pKD4as template. The targeting DNA was transformed to cells. It was found that more kanamycin resistant strains grow when incubation overnight than just2h. PCR reactions were done to confirm the genome structure of the mutants. It was found that the recombination structure was1607bp length, the homologous arms were existed, but the rest part of the waaR gene was totally replaced by selection marker. Comparing the capsule form on the cell surface of wide type and waaR mutant through TEM, a flocculent structure which is the characteristic of capsule was found in wide type, while the cell surface of waaR mutant looks smooth, no capsule was found.