Cloning And Expression Of An Endoglucanase Gene From Bacillus Thermoliquefaciens

Bacillus thermoliquefaciens is one of thermophilic bacteria. The endoglucanase found in B. thermoliquefaciens has exhibited the excellent thermostablity and considerable potential in industrial applications. In order to achieve high-level expression of recombinant endoglucanase, the endoglucanase gene Cel5A from B. thermoliquefaciens-NL was cloned and expressed in Escherichia coli and Pichia pastoris. Further, the full length of the endoglucanase gene was designed and synthesized, then expressed in P. pastoris. The main results obtained are as followed:1. The endoglucanase gene of B. thermoliquefaciens-NL was cloned with primers designed according to the conserved regions of the endoglucanase genes in Genbank. Sequencing result showed that its nucleotide sequence was 1,500 bp, encoding a peptide of 499 amino acids, with a signal peptide of 29 amino acids. The deduced molecular weight of enzyme was 55.2kDa. Comparison to this sequence with endoglucanase genes of the other Bacillus sp, the nucleotide sequence and amino acid sequence identity were 99.8%and 99.6% for Bacillus Subtilis. PAP115(Z29076)99.73% and 99.4% for B. subtilis.CK-2(X67044)respectively. The Glu490 and Thr496 of this two endoglucanase gene were changed into Arg and Ala, respectively.2. The gene Cel5AⅠencoding an endoglucanase and Cel5AⅡencoding an endoglucanase without signal peptide from B. thermoliquefaciens-NL were subcloned into pET-20b, resulting in pET-20b-Cel5AⅠand pET-20b-Cel5AⅡ, and were expressed in E.coli BL21 CodonPlus (DE3)-RIL. The endoglucanase activities of E.coli BL21 CodonPlus (DE3)-RIL harbouring pET-20b-Cel5AⅠand pET-20b-Cel5AⅡwere 0.0238U/ml and 0.0242U/ml, respectively. The result of SDS-PAGE showed that the expression of recombinant? endoglucanase Cel5AⅡwas enhanced and most of the recombinant protein was inclusion bodies.3. P. pastoris GS115 harbouring pPICZαB-Cel5AⅡproduced the higher endoglucanase activity at 0.089U/ml. This was 3.7 fold higher than endoglucanase produced by E.coli JM109 harbouring pET-20b-Cel5AⅡ.4.?The secondary structure of the translation initiation region (TIR) is a crucial factor for translation initiation rate. Meanwhile, codon preference plays an important role. According to the two principles, the gene Cel5A was optimized. The full length of the endoglucanase gene Cel5AⅢwas constructed by overlap extension PCR. The recombinant gene Cel5AⅢconsisted of 1,427bp and have expressed in P. pastoris. The recombinant P. pastoris GS115 Cel5AⅢproduced the higher endoglucanase activity at 0.198U/ml. Which was 2.2 fold higher than the endoglucanase produced by GS115 Cel5AⅡ, This indicated that the constructed gene Cel5AⅢwith P. pastoris codon preferences can improve the endoglucanase's expression.