Design And Expression Of Hybrid Gene Encoding Thermostable Endoglucanase With High Activity And Property Of Hybrid Enzyme

The production and thermostability of endoglucanase are critical to its application in industry. In this study, a high-activity endoglucanase gene fragment from rumen fungus and a thermostabilizing domain from thermophilic fungus were ligated together using P. rhizinflata 2301 and S. racemosum BCC18080 as parents. Then the hybrid gene was expressed in E. coli and the silkworm pupae, respectively. The biochemical properties of the hybrid enzyme were assessed and a rat feeding trial was conducted to evaluate the effects of the hybrid enzyme on nutrient digestibility. The overall objectives of this study were to produce thermostable endoglucanase with high activity and to meet production requirements for feed additives.1.Expression of Trichoderma reesei endo-β-glucanaseⅡin silkworm, Bombyx mori L. by using BmNPV/Bac-to-Bac expression systemThe silkworm, Bombyx mori, was used to produce recombinant endo-β-glucanaseⅡ(rEGⅡ). The EGⅡgene (egl2) was cloned from the cellulolytic fungus Trichoderma reesei QM9414 and inserted into B. mori nucleopolyhedrovirus (BmNPV) genome using BmNPV/Bac-to-Bac expression vector. For expression of rEGⅡ, both the BmN cells and B. mori larvae were infected with the recombinant virus. The putative rEGⅡyield was about 386μg per larva and the enzyme activity of the purified rEGⅡwas approx 352 U/mg of rEGⅡ. The optimal activity of this purified protein was observed at 55℃and pH 4, respectively. It is inferred that the mass production of recombinant cellulases, like rEGⅡ, is feasible by the BmNPV/Bac-to-Bac expression system in silkworms. This exploratory experiment paves the way for further expression of hybrid endoglucanase in silkworm bioreactor.2.The molecular design and synthesis of hybrid endoglucanase CelcdT'C' geneA carboxymethyl-cellulase clone isolated from the cDNA library of the ruminal fungus, P. rhizinflata 2301 encoded a polypeptide containing a cellulase catalytic domain Celcd. The Celcd was flanked by a 28-amino acid linker peptide at the N-terminus and linked to a dockerin domain by a 7-amino acid linker at the C-terminus. Celcd plus the linker peptides at both termini was designated as CelcdN'C'. Deletion of the N-terminal flanking linker of CelcdN'C'(the resulting protein was designated CelcdC') did not alter the enzymatic function of the catalytic domain. However, the thermal stability of CelcdC was dramatically reduced. It was deduced that the N-terminal flanking linker of Celcd pay important role in the stabilization of the enzyme protein. Both CelcdN'C' and CelcdC' had a specific activity of 344.9 and 334.5 units of enzyme activity/mg protein against carboxyl methyl cellulose (CMC), respectively. The CelcdC' was finally chosen as the gene material encoding high activity.Endoglucanase is a major cellulolytic enzyme produced by S. racemosum (BCC18080) belonging to the Zygomycota subdivision. The optimal temperature for the purified enzyme was at 70℃, the highest among the family 45 endoglucanase from subdivision Zygomycotina. Search of NCBI-BLASTP revealed that the amino acid sequence deduced from the cellulose binding domain and the linker domain of BCC18080 endoglucanase was exactly the fragment we required.In orde to ligate these two gene materials together, we entrusted GenScript NanJing Co., Ltd to synthesize the complete encoding sequence of CelcdT'C' and make sure that the T'fragment was at the N-terminus and the CelcdC fragment at the C-terminus. Meanwhile, BamHI and HindⅢrestriction sites were designed at the N/C-terminus for the executive experiments, respectively. The CelcdT'C' gene was cloned into pUC57-simple vector by EcoRV, and stored at-20℃.3.The purification and expression of hybrid CelcdT'C' and its parental endoglucanase CelcdN'C' in Escherichia coliThe hybrid endoglucanase CelcdT'C' gene and its parental CelcdN'C'gene were subcloned into pET-30a(+) expression vector, and then transformed into E. coli BL21, respectively. The positive expression clones were thus obtained. For the bacterial liquid at 4 h post-induction with IPTG, the clearing zones could be seen on CMC-Na agar plate after congo red staining, showing that both ECelcdT'C' and ECelcdN'C' were biologically active. The molecular weight of ECelcdT'C' and ECelcdN'C' were 61 kDa and 54 kDa, respectively, consitent with the deduced data.The enzyme activities of the purified CelcdT'C' and CelcdN'C' were 11.12 and 16.36 U/ml, respectively, determined by the glucose standard curve method. The optimum temperature of both purified proteins was at 50℃. The parental CelcdN'C' had higher activity at 30-40℃. When incubated at 70-90℃, the hybrid endoglucanase CelcdT'C' was slightly less active than CelcdN'C', but much more thermostable. Both CelcdT'C' and CelcdN'C' had optimal pH of 5.0, with the hybrid cellulase CelcdT'C' being more active than CelcdN'C' at pH 7.0, while the activity of CelcdN'C' was a lilttle higher under weak acid condition.4.The expression of hybrid CelcdT'C' and its parental endoglucanase CelcdN'C' in silkworm pupa and study on the effect of feeding ratThe recombinant baculovirus harboring CelcdT'C' and CelcdN'C' gene was successfully construced by using BmNPV/Bac-to-Bac expression vector. For expression of the recombinant CelcdT'C' and CelcdN'C', both the BmN cells and silkworm pupa were infected with the recombinant virus. The expressed proteins were only detected in the cells but not in the supernatant. The enzyme activities of the crude enzymes were approx 463.4 and 477.9 U/mg for CelcdT'C' and CelcdN'C', respectively. The optimal activities of crude enzymes were observed at 60 and 50℃.In order to assess the effect of recombinant endoglucanases expressed in the silkworm pupae, rat was used in a feeding trial based on one-way factorial design. The experimental rats were divided into 4 groups:basal diet (negative control), basal diet added with 2.0% nomal silkworm pupa powder (positive control),2.0% of silkworm pupa containing endoglucanase CelcdN'C' (parental enzyme) or 2.0% of silkworm pupa powder containing CelcdT'C'(hybrid enzyme). The trial lasted for 27 d. Addition of 2.0% silkworm pupa powder did not affect palatability of feed. The digestibilities of dry matter, crude protein and crude fiber were significantly enhanced by inclusion of 2.0% silkworm pupae (P<0.05), compared to the negative control. The digestibility of nutrients in hybrid enzyme group was higher than the parental enzyme group, and the rats fed on hybrid enzyme tended to grow faster than other groups. From the results obtained in this study, it is indicated that it is feasible to efficiently produce the hybrid CelcdN'C'gene coding for thermostable endoglucanase with high activity by using BmNPV/Bac-to-Bac expression system. The spatial structure of the hybrid protein is excellent without damage. Furthermore, the hybrid enzyme could maintain higher cellulase activity than its parental endoglucanase CelcdN'C'during pelleting process.