Research On High-efficiency Expression Of Bacterial Urease In Prokaryotic Expression System

With the increasing demand for infrastructure,concrete materials widely used in modern engineering construction are prone to local damage such as cracks during long-term service,which can seriously endanger the safety of building structures and people's lives and property.Cracks repairment by using microbially induced calcite precipitation(MICP)has drawn attentions because of its environmentally favorable and durability in recent years.However,the urease activity of urease-producing wild strains and genetically engineered recombinant strains that have been obtained is still generally low,which severely restricts the development of microbial-induced calcium precipitation to repair concrete cracks.Therefore,how to efficiently express urease and improve urease activity has become an urgent problem to be solved.Based on this,this study intends to construct recombinant expression strains of urease gene clusters from different sources using molecular biological means,and plan to screen and obtain recombinant urease strains with high urease activity.The main works of the study are summarized as follows:(1)Ure ABC derived from Bacillus subtilis is heterologously expressed in E.coli.Using E.coli BL21(DE3)as the bottom bacterium,the Ure ABC gene cluster of urease from B.subtilis was successfully expressed.The recombinant urease was analyzed by SDS-PAGE and Nano LC-ESI-MS/MS analysis,which proved that the Ure A,Ure B and Ure C subunits of the target protein were successfully expressed,but Ure B and Ure C subunits mostly existed in the form of insoluble inclusion bodies.The urease activity of BL21/p ET32 a Ure ABC recombinant strain was 1.244±0.001 mmol/(L·min).(2)Ure ABC derived from B.subtilis was homologously expressed in B.subtilis AS1.The target genes Ure ABC and Ure ABC(His-tag)were subcloned into the expression vector p HT254 by homologous recombination,and the resulting recombinant strains AS1/p HT254 Ure ABC and AS1/p HT254 Ure ABC(His-tag)were successfully obtained by electroporation of B.subtilis AS1 competent method.Our results showed that Ure ABC was mostly soluble urease complex.And the urease activity of the recombinant strain AS1/p HT254 Ure ABC reached 2.555±0.001 mmol/(L·min),which is 2.8 times higher than that of the AS1/p HT254 Ure ABC(His-tag)recombinant strain containing histidine tag.This result indicated that the histidine tag of the constructed recombinant bacterium had a negative effect on the urease ABC.The formation and folding of the radical group and its physiological catalysis and function may have a certain degree of influence.(3)To investigate the high-efficiency expression of urease derived from Sporosarcina pasteurii with auxiliary protein in B.subtilis AS1 and the effect of urease gene coding sequence on the activity and function of urease.Amplify the urease gene cluster Ure ABCEFGD derived from S.pasteurii and subcloned into the vector p HT254 with strong promoter using homologous recombination strategy.The two expression vectors p HT254 Ure ABCEFGD(His-tag)and p HT254 Ure ABCEFGD were constructed with and without His-tag respectively.In addition,in order to study the effect of urease gene sequence on catalytic activity,we adopted the same strategy to construct expression vectors p HT254 Ure ABCDEFG(His-tag)and p HT254 Ure ABCDEFG with adjusted Ure D position in the gene cluster.The above recombinant expression vectors were electrotransformed into B.subtilis AS1,and four recombinant urease expression strains were obtained by screening.Among them,the highest urease activity of the AS1/p HT254 Ure ABCEFGD strain is 44.51±0.002 mmol/(L·min),which is 6.01 times higher than the histidine tag-containing AS1/p HT254 Ure ABCEFGD(His-tag)recombinant strain.Most importantly,the best activity of strain AS1/p HT254 Ure ABCEFGD was 1.91-fold higher than that of the well-known S.pasteurii strain as the highest urea decomposition ability.However,the urease catalytic activity of the recombinant strain obtained after adjusting the coding sequence of the urease gene was not detected owing to these polypepetide chains misfolded after regulation the coding sequence of urease family.In summary,we obtained the recombinant strain of AS1/p HT254 Ure ABCEFGD with a urease activity 1.91 times higher than the wild bacteria by comparing the enzyme activity of different urease resources expressed in prokaryotic expression system.It means that the AS1/p HT254 Ure ABCEFGD strain has potential application value in the repair of concrete cracks.Our study also laid the foundation for further research to develop the high-efficiency engineering strains of urease,which is suitable for industrial applications.