Purification,Properties And Prospects Of Urease From The Bacillus Atrophaeus C89 Associated With Sponge Dysidea Avara

As the biological enzyme with a crystal structure obtained at the earliest time,urease has been separated from plants,soil and animal gastrointestinal tracts.However,alkalescent microbial urease separated from marine environments was rarely reported.In the research,a strain of urease-producing microorganism Bacillus atrophaeus C89 was separated from Dysidea avara in the South China Sea.The marine microbial urease was finally obtained through multiple steps of separation and purification such as ultrasonication,ammonium sulfate chromatography,Superdex 200gel filtration chromatography and Hi Trap DEAE FF anion exchange chromatography.As measured,the specific enzyme activity was 1,306.54U/mg;the purification fold was 7.13;the yield was 15.66%.According to Bacillus atrophaeus C89 genome information and SDS-PAGE experiments,the urease was composed of three subunits includingα,βandγwith sizes of 61 KDa,14 KDa and 10 KDa respectively.Native-Page experiment showed that the molecular weight of urease was about 260 KDa.Through Swiss-Model homologous modeling,it was predicted that the urease could form（αβγ）₃ trimer.The optimum pH of this marine microbial urease was 8.2;The activity was kept over 80%at pH 8.0～8.6.The optimum pH of plant jack bean urease was 7.4;the enzyme activity was kept over 80%at pH 7.0～7.6.Hence,this marine microbial urease has higher alkali resistance than that of plant jack bean urease.The optimum salinity of the marine microbial urease was 15%;the activity was kept over 80%within the salinity range of 0～25%.The optimum salinity of plant jack bean urease was 5%;the remnant enzyme activity was about 70%when the salinity reached 25%.In comparison with the plant jack bean urease,the marine microbial urease has better resistance to high salinity concentrations.The optimum temperature of the marine microbial urease was 40℃;under the temperature of 45℃,the remnant specific enzyme activity was kept over80%.The optimum temperature of plant jack bean urease was 35℃;under the temperature of 40℃,the remnant specific enzyme activity exceeded80%.Thus,the marine microbial urease has higher temperature resistance than the jack bean urease sourced from lands.Based on the inhibition experiments of heavy metal ions(Hg²⁺,Cu²⁺,Pb²⁺and Cd²⁺)in the artificial seawater system,significant positive correlation existed between concentrations of four types of heavy metal ions and the urease inhibition rate.The inhibition of each heavy metal ions on urease activity obeyed the order of Hg²⁺>Cu²⁺>Cd²⁺>Pb²⁺;the combined inhibition of mutiple heavy metal ions had different synergistic or antagonistic effects,Hg²⁺,Cu²⁺and Cd²⁺showed synergistic effects,while Pb²⁺demonstrated certain antagonistic effect.