Molecular modification of cellobiohydrolase gene cbh1 from Trichoderma reesei

【Objective 】Molecular modification of cellobiohydrolase gene cbh1 from T. reesei was carried out to improve cellulase activity in order to provide scientific reference for commercial production of cellulase. 【Method 】Cellobiohydrolase gene cbh1 from T. reesei was digested with DNase I, and the recycled fragments about 100 bp were linked with T4 DNA ligase randomly. By using the connection product as templates, the cbh1 sequence was amplified by PCR method. The modified cbh1 gene was transferred into T. reesei protoplast to conduct homologous recombination with original cbh1. Mutant strains with high cellulase activity were screened based on filter paper enzyme activity.cbh1 gene sequence alignment was performed in the NCBI. 【Result 】The cloned gene fragment was 637 bp in length and shared over 88% of homology with the other isolates of T. reesei, so it was identified as cbh1 gene fragment of T. reesei. After digestion of DNase I for 15 min, linking with T4 DNA ligase and PCR amplification, the modified cbh1 gene was obtained and transferred into T. reesei protoplast to construct mutant library. According to filter paper enzyme activity, mutant strain named E2-1was screened from cbh1 mutant library. The filter paper activity of E2-1cellulase was 1.7518 times higher than original strain. Sequence alignment revealed that, compared with original strain,14 bases of cbh1 distributing on 9 loci from mutant strain E2-1were changed, which included 5 bases substitution mutation, 8 bases deletion mutation and 1 base insertional mutation. 【Conclusion 】The modified cbh1 gene could be homologously recombined with chromosomal DNA of T. reesei, which resulted in improving the filter paper enzyme activity of mutant strain cellulase.