Dextran Enzyme Production Strain' Mutation And Expression Cloning Research

Dextranase can hydrolyze the a-1,6 glucosidic bonds within water-soluble polysaccharides,so this enzyme can widely be applied in suger industry and so on.In recent years,the dextranse also has been applied in the field of the inhibiting the biofilm formation,the production of blood substitutes.At present,many papers have reported that the existing dextranases were mainly came from fungi,comparing with the dextranase from bacterial,the fermentation period of fungi was very long and also has certain security hidden danger.The novel atmospheric and room temperature plasma(ARTP)method was used to breed high-yielding mutations of Arthrobacter KQ11.Mutagenesis produced two mutations4-1 and 4-13,which increased enzyme activity by 19%and 30%,respectively.Dents on the cell envelope were observed under scanning electron microscopy(SEM).The optimal temperature and pH of the mutant strains were 45 ?,pH 6.0(4-1)and 50 ?,pH 6.0(4-13).Through amino acid alignment,several nucleotides of the mutant strains were found to have changed,crystal structure suggested that the amino acid residue of 472 was located at random-coil,this may be associated with the improvement of the dextranase activity.In order to further improve the dextranase production of the mutant strain 4-13.The medium of mutant strains 4-13 was optimized.The optimal cultivate medium and fermentation conditions of the mutant strain was obtained through single factor experiment,PB experiments,the steepest climbing experiment and central composite experiment.Upon the optimal enzyme production conditions of the wild and mutant strains,the dextranase activity of 4-13 was 50%higher than that of the wild strain.The dextranase gene from the mutant strain 4-13 was cloned and expressed using Escherichia coli DH5a competent cells.Next,the recombinant dextran-hydrolysis enzyme was purified and its properties were characterized.The optimal temperature and pH were determined to be 60 ? and 6.5,respectively.The purified dextran-hydrolysis enzyme was activated by 5 mM or 10 mM K+,NH4+,Mg2+,and Co2+,while enzyme activity was inhibited by Cu2+ or Fe3+.High-performance liquid chromatography(HPLC)results suggested that the final hydrolysis products were glucose,maltose,maltotriose,and maltotetraose.Dextranase,which can hydrolyze the a-1,6 glucosidic bonds within water-soluble polysaccharides,could effectively inhibit biofilm formation.Through scanning electron microscopy(SEM)and confocal laser scanning microscopy(CLSM)observations,it was evident the dextranase inhibited biofilm formation and effectively removed previously formed biofilm.