Expression Of Ureases From Different Sources In Prokaryotic System

Urease(EC 3.5.1.5)is the first nickel-containing metal enzyme discovered by humans,which can hydrolyze urea to produce ammonium and carbonate.At present,the most widely used urease-producing bacteria is mainly Sporosarcina pasteurii in the field of biomineralization.However,there are fewer reports about molecular modification of wild S.pasteurii till now.The screening of high-yielding S.pasteurii urease strains by nitrosoguanidine chemical mutagenesis is arduous.Therefore,it is of great significance to achieve high-efficiency expression of urease by combination protein engineering methods and molecular biology methods based on screening suitable urease gene cluster in different expression systems.In this study,our aim is to obtain recombinant engineered strains with high urease activity based on excavating of urease gene resources and compare the enzyme activity of urease gene expressed in different prokaryotic systems.The main results of this study are summarized as follows:(1)Heterologous expression of Bacillus megaterium urease gene cluster in Escherichia coli.After codon optimization,urease gene cluster ure ABCEFGD_B.mefrom B.megaterium was cloned to vector p ET28a,and the resulting expression vector p ET28a-ure ABCEFGD_B.mewas transformed into two different E.coli strains.Results of SDS-PAGE and Nano LC-ESI-MS/MS mass spectrometry analysis proved that seven subunits of urease were all expressed.The recombinant urease activity reached 304±15 U/L.(2)Heterologous expression of Morganella morganii urease gene cluster and its urea transporter gene I in E.coli.The urease gene cluster ure ABCEFGD_M.moand the urea transporter gene ure I_M.mowere amplified from the wild M.morganii genome and subcloned to plasmid p ET28a respectively.These two resulting expression vectors of p ET28a-ure ABCEFGD_M.moand p ET28a-ure ABCEFGDI_M.mowere transferred into different expression strain of E.coli.Results of SDS-PAGE and Nano LC-ESI-MS/MS mass spectrometry analysis showed that all seven subunits of M.morganii urease were expressed.Unfortunately,most of the protein in E.coli exists in the form of inclusion bodies.Among them,the recombinant strain BL21(DE3)/pET28a-ure ABCEFGDI_M.mocontaining the urea transporter gene has the highest urease activity,which reached 4500±240 U/L.(3)Co-expression of the urease gene cluster of M.morganii and urea transporter gene I in Bacillus.subtilis.Recombinant strains of B.subtilis AS1/p HT254-ure ABCEFGDI_M.mowere obtained to express target urease gene ure ABCEFGDI_M.moby using B.subtilis as the chassis organism.The results of SDS-PAGE and Nano LC-ESI-MS/MS mass spectrometry analysis of the recombinant protein showed that all seven subunits of the M.morganii urease gene cluster were achieved soluble expression.The best urease activity of the recombinant strain AS1/p HT254-ure ABCEFGDI_M.mois 8665±300 U/L,which is 1.93-fold higher than that of the same urease gene cluster in E.coli recombinant strain,and 4-fold higher than that of wild M.morganii urease activity.This above result shows that B.subtilis is a suitable prokaryotic expression system for urease expression.(4)Expression of Sporosarcina pasteurii urease gene cluster in B.subtilis.In order to modify the expression vector p HT254-ure ABCEFGD_S.pawith high bioactivity,its original promoter was replaced with p43 using overlap extension PCR strategy,and RBS sequence before ure F was added by whole plasmid PCR strategy.The above recombinant vector was electrotransformed into B.subtilis AS1.As expected,The urease activity of AS1/p HT254-ure ABCEFGD_S.pastrain reached 15880±370U/L.Results of field emission scanning electron microscopy and X-ray diffraction showed that the main ingredient of precipitation component was calcite,which suggesting that the crystal precipitation induced by microorganisms has potential application.