Recombination And Expression Of Novel Endo-β-glucanase Gene From Penicillium Echinulatum In Trichoderma Reesei

As the major cellulase component, exo-β-glucanase has great value in textile industry and pulp bleaching industry. However, the endo-β-glucanase produced by Trichoderma reesei only applies to acidic system, which limits its further applications in above-mentioned areas. In this study, recombinant DNA technology is adopted to constructing the novel endo-β-glucanase producing strain. This study plays an important role in promoting industrial applications of cellulaseIn this study, a novel endo-β-glucanase (EGL1) secreted by mutant Penicillium echinulatum was optimized according to the codon bias of Trichoderma reesei, obtaining the new gene Eglln. The length of Eglln is 1175 bp, encoding 387 amino acid residues. The codon-optimized gene(Eglln) was inserted between Trichoderma reesei strong promoter Pcbh1 (including secreting signal peptide sequence) and terminator, further ligated to pCAMBIA1300 vector to construct a recombination plasmid pCB-PET with hygromycin B resistance marker.The plasmid pCB-PET was transformed into the conidium of T. reesei ZU-02 via an optimized Agrobacterium tumefaciens mediated transformation. 226 positive transformants were screened by hygromycin B, and 5 fast growth transformants were obtained by further rescreening on the culture plates using carboxymethylcellulose as sole carbon source. The chromosomal DNA from 5 recombinant transformants were extracted separately after subculture for 10 generations. It was further confirmed that the Eglln gene had been integrated into the chromosomal DNA of T. reesei by PCR detection. An obvious protein band (approximately 41 kDa) corresponding to the expressing product of target gene Eglln was detected in fermentation broth of recombinant T. reesei by SDS-PAGE which proved that the Eglln gene had extracellularly expressed successfully.Enzyme production by the 5 recombinant transformants was performed in shaking flasks. After 48 h fermentation at 30℃ under agitation (200 r/min), endo-β-glucanase activity in the culture broth was measured at pH 8.0,60 ℃. The activity produced by transformants reached 382.6 U/ml, which was 22.5-fold as high as that of the host strain. The batch fermentation experiment of the transformant T1 was carried out in a 2 m3 fermenter. The recombinant endo-β-glucanase activity reached 1070 U/ml after 96 h fermentation, which was 2.8-fold as high as that under the shaking flask condition.The study on enzymatic properties showed that the recombinant EGL1N exhibited high thermostability. It was stable below 70℃ and its optimal temperature for reaction was 60 ℃. The enzyme also presented good hydrolytic activity and stability over a broad pH range (5.0-10.5) and an optimum pH value of 8.0. The CMCase activity was strongly stimulated when Ca2+ was employed. The CMCase activity was completely inhibited by Hg2+ and an effective inhibition was also observed with Mg2+ 、Al3+、Zn3+, Cu2+ and Fe3+. These characteristics of the cellulase will be of great importance to modern industry.This study showed that the optimized Penicillium echinulatum endo-β-glucanase gene had been successfully transformed and expressed in T. reesei. The research result acts as a significant role in the directed evolution of cellulase producing strains and related industrial applications as well.