Study On Over-expression Of Bacillus Subtilis Neutral Endoglucanase Gene In Pichia Pastories And Its Structural Domain Reconstruction

Endoglucanase is one of the most important cellulose enzymes,which cooperates with cellobihydrolase andβ-1,4-glucosidase to translate cellulose to glucose completely. Recently,bacterial cellulose enzyme preparation had shown satisfactory application performance and economic value.It is our objective to express a Bacillus subtilis endoglucanase gene in Pichia pastoris.Structural domain reconstruction was also performed to study the effectiveness of cellulase domain(CD) and carbohydrate-binding module(CBM) in the endoglucanase.1.According to the sequence of endoglucanase gene(GenBank Accession No.: DQ782954) from Bacillus subtilis,a pair of primers was designed and synthesized,and a high-fidelity polymerase was used for amplifying the mature endoglucanase expression gene.A 1.4 kb fragment,which does not contain signal peptide sequence obtained,and was ligated to expression vector pPIC9k.Recombinant plasmid pPIC9k-End was constructed and transformed to Pichia pastoris GS115.Three transformants named GS115-Endâ… ,GS115-Endâ…¦and GS115-Endâ…§were obtained through MD,MM plate screening and enzymatic activity test.The expression conditions containing the initial cell density,pH and methanol concentration were studied in shaking culture.Optimal endoglucanase production was observed when the initial cell density OD₆₀₀ was 5,and the optimal methanol concentration was 0.5%～1.0%.The endoglucanase production increased obviously when provided adequate aeration,whereas it did not seem to vary when cells were induced at pH 4～8.Under the above expression condition,the endoglucanase activities of the three transformants were 860.7,760.3 and 786.2 U/mL,13.5,11.9 and 12.3 times compared with the endoglucanase activity of the original strain(63.78 U/mL) respectively.The enzyme maintained over 80%of the original enzymatic activity after incubated at 65℃for 30 min,and the SDS-PAGE showed that the molecular weight was about 79.82 kD.2.Redesigned endoglucanases enhanced cellulase domain,added and deleted CBM were constructed and expressed in Pichia Pastoris respectively in this work.The activities of reconstructed endoglucanases at different temperatures and pH values were also assayed. The optimal temperature and pH of the all endoglucanases were 65℃and 6.0,where only EG-2 showed 24.9%enzymatic activity loss comparing to natural endoglucanase.EG-4, which had deleted CBM,showed endoglucanase activity rapid loss under low temperature condition,only reached 46.7%of its highest activity at 40℃.However,the other three can preserve most of their activities below the optimal temperature,which all showed about 80%of the highest activities at 40℃.This work suggested the natural endoglucanase activity mainly attribute to its intact topologic structure and the extending CBM play an important role in preserving the most of enzymatic activity at lower temperature optimum, which all represented significance on protein engineering of endoglucanase to improve its enzymatic activity and to redesign novel endoglucanase more proper for industrial application.