Enhancements Of The Endoglucanase Activity Through Homologous And Heterologous And Heterologous Domain Reconstruction As Well As The Studies On The Enzymatic Characteristic

Cellulase is a term for a group of enzymes able to translate cellulose to glucose, which mainly contains endo-1,4-β-glucanase (EC 3.2.1.4),1,4-β-D-cellobiohydrolase (EC 3.2.1.9) and 1,4-β-glucosidase (EC 3.2.1.21). These three enzymes cooperate to translate cellulose to glucose completely. Endoglucanases, especially from fungi, have drawn the most interest in the previous cellulase study. Recently, bacterial cellulases have taken on satisfactory application performance and economic value in detergent industry, because they show relative stability in neutral and basic conditions. However, the large scale industrialized application of these enzymes was restricted by the low activity and high cost price. Improving their activity through genetic engineering and protein engineering was considered efficient approach to cut down the cost price. This paper realized the homologous and heterologous domain reconstruction by using the genetic engineering means, and expressed in Escherichia coli (BL21) and Pichia pastoris (GS115) respectively.1. According to the sequence of endoglucanase gene (GenBank Accession No.: DQ782954) from Bacillus subtilis, The protein sequences showed that it contains three domains:cellulase domain (CD) is about 253aa, carbohydrate-binding module (CBD) is about 82aa, and linker peptide(CP) is about 55aa, which are usually structural and functional independent. Analyzing the sequences derived from the fungus Corticium rolfsii (Strain AHU9627) the carbohydrate-binding module (CBD) and the linker peptide(CP) were found and named FCBD. the primers and the gene coding the carbohydrate-binding module (CBM) and linker(FCBM) of the endoglucanase from the Corticium rolfsii (GenBank Accession No.:D49448) was synthesized were synthesized by Invitrogen (Shaihai,China). After that,the products of the PCR were purified, and then digested with corresponding restriction enzymes, whose sites were designed in primers. Plasmid pET32a was digested with Kpn I and Xho I for vector fragment. Corresponding products were ligated to the vector fragment. After ligation 12 h at 16℃, E. coli strain BL21 (DE3) was transformed and plated on LB containing ampicillin. The positive colonies were screened by the clony PCR reactions and digested with corresponding restriction enzymes, which were named pET32a/EG, pET32a/EG/FCBD respectively. Hydrolysis zone analysis All the strains could see the hydrolysis ring In addition to the control. The enzyme activity of pET32a/EG and pET32a/EG/FCBD was 94.38978 U/mL and 174.3079U/mL respectively. The SDS-PAGE showed that 1)the molecular weight of pET32a/EG/FCBD was about 81kDa; 2)the molecular weight of pET32a/EG was about 72kDa; Compared with the control, the powder of degradating cellulose of the fusion enzyme EG2 was double compared to that of EG1. The optimal pH, temperature, pH stability and heat stability of pET32a/EG and pET32a/EG/FCBD had little differentiates,but the optimal pH values of the two enzymes were 7.0 and 6.5. Overall from the results we can conclude that increaseing the FCBM is helpful to enhance the Activity of the enzyme as we had anticipated.2. Redesigned endoglucanases enhanced cellulase domain(FCBD) from the fungus Corticium rolfsii (Strain AHU9627), added the FCBD on the CD of the endoglucanase. From Bacillus subtilis. Which were ligated to expression vector pPIC9k.The reconstructed endoglucanases were named pPIC9k/EG/FCBD and pPIC9k/CD/FCBD. The recombinant plasmids pPIC9k/EG/FCBD and pPIC9k/CD/FCBD were constructed and transformed to Pichia pastoris GS115. The positive colonies were screened by the high-throughput screening. After that their characters were also discussed and compared with the EGs (which were redesigned by our laboratory) endoglucanases enhanced cellulase domain, added and deleted CBM, named EG2, EG3 and EG4 respectively. The optimal temperature of the EGs was 45℃,55℃,40℃,55℃,55℃and 40℃, where their enzymatic activities in P. pastoris cultivation supernatant reached 884,770,797,918,779 and 874U/mL. EG2 showed 24.9% enzymatic activity loss compared to natural endoglucanase. The assays of enzyme resistance to different temperature indicated that when the temperature arrived at 60℃, the activity had a higher drop except 9KEGF. The reason is that the own body conformation was affected. The optimal pH values of EGs had little differentiates. EG1 and EG2 have same optimum pH of 7.0. EG3, EG4 and 9KCDF have same ph of 5.6. but the optimum pH of 9KEGF is 6.5. There are not much difference in temperature stability. When the temperature is 70℃, EG1 and 9KEGF still can preserve about more than 70% of its highest activity. At the same temperature EG4 and 9KCDF can preserve about 60%. But the EG2 and EG3 can only preserve about 13.8% and 21%. All the EGs showed strong alkali tolerance. The pH ranges where the EGs can reach above of 80% of their highest activities were between 6.5-8.0. When the pH=10, The activity of EG2 has significant reduction, at the same time the others still can maintained above 80% of their highest activities.