Directed Evolution Of An Endoglucanase Gene From Thermotoga Maritima

The endoglucanase obtained from Thermotoga maritima which is one of the thermophilic bacteria, exhibited excellent thermostablity with considerable potential in industrial applications. In this thesis, gene cloning and expression were studied to get recombinant expressed strain. To improve the thermostability of endoglucanase, Error-prone PCR technology was employed in the study. Several muntants with higher enzyme activity were obtained through high throughput screening. Furthermore, the enzymatic properties of recombinant endoglucanase were investigated. The main research contents and results are shown as following:1,The endoglucanase gene was cloned with primers designed according to the analysis of position of the restriction enzyme of the endoglucanase gene and multiple cloning sites of vector pET-20b.The sequencing result showed that its nucleotide magnitude was 759 bp.2,The obtained DNA fragment was inserted into the vector pET-20b with both Nde I-Xho I double digests, then transformed into E.coli BL21(DE3). The endoglucanase activity of E.coli pET-20b-Cel12B was 22. 9U/L in optimality condition.3,Error-prone PCR was used for leading into random mutation.With improving experiments condition, the PCR system was determined as: 7 mM MgCl2,0.15 mM MnCl2, 0.2 mM dATP, 0.2 mM dGTP,1 mM dCTP,1 mM dTTP and 5 U of Taq DNA polymerase, with lower annealing temperature and without hot start.4,Five mutants with higher enzyme activity were obtained from several rounds of random mutation.Base sequence has transformed leading to change amino acid sequence which mainly caused improving enzyme activity. Most of sequences were intended to be changed between A and T or C and T. The mutant enzyme activity was 2 to 3 times higher than original when using CMC as substrate to determine the enzyme activity.5,The suitable pH and temperature for enzyme activity of endoglucanase were 6.5 and 90℃, respectively. The endoglucanase has a good stability when pH was 6.0-7.0. The remained enzyme activity maintained at 90% compared to the unkept one after heating at 90℃for 2 hours.