Directed Evolution Of Heparanase Gene And Preparation Of LWMH

Heparin is an important biochemical medicine,and its molecular weight ranges from 3000 to 37500Da,as a kind of glycosaminoglycan which is widely existed in animal organs,tissues(such as small intestinal mucosa,lungs,etc).it is inear chain macromolecule formed by six or eight monose,which is alternately linked with glucosamine sulfate and hyaluronic acid molecules,and it contains many different size of non-uniform components.Low molecular heparin(LWMH)ranging from 4000Da to 8000Da are components separated from ordinary heparin or fragments from heparin by degradated,so its structure is single.Although they are all able to be the preparation of antithrombotic drugs,but Compared with heparin,LWMH have small molecular weight,easy to be absorbed,high bioavailability,long half-life in the body and not easy side effects such as bleeding,thus it arouses people's great interest.At present,because of the low and unstable heparinase hydrolysis activity,LWMH yield is low,the price is very expensive.and limits its wide application in the field of pharmaceutical industry,therefore,through the appropriate method to improve the activity of heparinase hydrolysis and to improve LWMH production have an important significance in the field of scientific research.The research group have completed the cloning and recombinant of heparinase gene using E.coli expression system.The objective of my subject is regardding this heparinase gene as starting material for Directed Evolution,through error-prone PCR and high-throughput screening methods to get a strains with efficient expression of heparinase;And then,we do some research on optimization strains recombinant protein with purification and studing enzymatic properties;Finally,use efficient expression strains to catalyze heparin for the preparation of LMWN.The specific research results and methods as follows:Firstly,regarding recombinant plasmid pET-28-a(+)-Hpa I y as a template for error-prone PCR technology to build mutant library,useing high-throughput screening methods to get an efficient expression strains Hpa I 14.The enzyme activity increased by 57%compared with the original strain,the enzyme activity is as high as 366 U/L.Sequencing results shows that there are four bases mutated gene:T398C,A804C,C805A,T951A.This resulted some change:Ile194,Pro570Thr.And the protease crude enzyme liquid is purified by Ni2+ affinity chromatography.Then we study The purified heparinase enzymology properties,SDS-PAGE electrophoresis results showed that the purified protein molecular weight is 49 kDa Basically consistent with the theoretical value.discovered that the optimal temperature of heparinase is 30? and enzyme activity is greater than the original strains,enzyme activity heat stability is lower than the original strains,the Optimal pH is 7.5 and the enzyme activity is higher than the original strains,When the pH is between 7 to 8 enzyme activity is stable,outside of the range will induce larger influence on enzyme activity.Compared with eight different metal ions influence on heparinase,found the Ca2+ and Fe2+ to the activation of the enzyme has obvious effect,however,Mn2+,Ni2+,Zn2+,Cu2+ and Mg2+ have obvious inhibitory effect.Finally the restructured cells of efficiently express heparinase optimization strains can be used in catalyzing hydrolysis of heparin.the hydrolysis reaction is inhibited by the over 125?L concentration of substrate concentration.improving the reaction temperature within the range of 20?30? improve the reaction conversion rate,Within the scope of the pH 7?9 efficiency of catalytic reaction is relatively stable,degradation of heparin by catalytic of Recombinant cells requires more moderate pH conditions.Due to restructuring cells under high speed can be fully contacted with the substrate,therefore when high speed conditions,the cells catalytic efficiency tend to be stable and the efficiency is significantly higher.recombinant cells to the degradation of heparin is controllable,respectively in 2,4,6h reaction termination,with the extension of time,the degree of degradation of heparin has been improved,this further proved that useing of optimized recombinant cells to catalytic heparin for low molecular weight heparin is feasible.