Cloning And Enhancing Thermostability By Directed Evolution Of Endoglucanases Ⅰ Form T. Longibrachiatum

Cellulase is the key factor in the process of transformed lignocellulose into fuel. However, many factors restrict the application of cellulose in industry. For example, low specific activity of cellulase, which is the most important in those inhibitory factors. So the protein engineering research in cellulase improvement has become a hot topic in priority program, In this research, the thermostability of endoglucanases I from T. longibrachiatum improved by directed evolution including the random mutagenesis and DNA shuffling. The main results are as follows:Firstly, an endoglucanases I CDS with 1386 bps in length was cloned from the mRNA of T. longibrachiatum A002 by reverse transcription PCR. This CDS codes a hydrophilous protein in length of 461 amino acid with signal peptide, which has glycosylation sites and 48 KD molecular weights. Sequence analysis showed that:this gene has significant homology with many kinds of EG I of Trichoderma. Conserved domain analysis showed that this protein has two conserved domain:Glyco_hydro₇ and CBM₁. Tertiary structure prediction showed that the active centre was constituted by a β-jelly roll structure.Secondly, the EG I was expressed in Pichia pastoris by expression vector pPIC9K. CMCase activity was detected in culture supernate. A protein band was isolated in about 48 KD molecular weights through SDS-PAGE. The optimum temperature and optimum pH of the recombinant protein were 45 ℃ and 5.0 separately. After induced with methanol 168 h, the recombinant protein expression would be reach crest value which was 12.9 U/mL. The optimum concentration of methanol was 1.0%, the CMCase activity could be 15.5 U/mL.Thirdly, the eg I gene was random mutated by error-prone PCR and DNA shuffling in order to improve its thermostability by directed evolution. A mutational enzyme named Mut A19 was isolated from 5400 mutants. This mutant enzyme’s thermostability was enhanced compared with the wild type enzyme. On one hand, the optimum temperature of Mut A19 was 65 ℃, which was 20 ℃ higher than the wild type. On the other hand, Mut A19 was stabilized after thermal insulation 60 min in 65 ℃ simultaneously, the wild type enzyme was inactivated in the same stressful condition.Above all, in this research, an EG I CDS was cloned and expressed. An effective method for error-prone PCR and DNA shuffling was established. A mutant with enhanced thermostability was obtained by directed evolution.