Breeding and Efficiency Evaluation of a High-Yielding Cellobiohydrolase Strain

Objective: Energy and environmental issues are one of the most severe challenges for human survival and development in the 21st century. Therefore, the global interest in developing lignocellulosic biomass resources is growing, and cellobiohydrolase is one of the key enzymes for converting cellulose into sugars. Based on this, we want to breed strains with high cellobiohydrolase production. Methods: The strains with high production of cellobiohydrolase were screened by Congo red medium firstly. Then, strains producing transparent circle were screened again by enzyme activity assay, so as to screen out the strain with high production of cellobiohydrolase (with Trichoderma reesei as control). Observed the growth state of the strain and identified the strain. Atmospheric and Room Temperature Plasma mutagenesis was used to mutagenize the strain, and selected dominant mutants in order to further improve the enzyme activity. Finally, the enzymatic property analysis was carried out to evaluate the stability of cellobiohydrolase. Results: 12 strains were obtained through the primary screening by Congo red staining. After fermentation and re-screening, a strain with high enzyme activity was obtained, which was H9, and the maximum enzyme activity was 32.4 IU/mL. The colony of the strain nearly round, thick and fluffy, with a slight bulge in the middle, it was light gray flocculent in the initial stage of culture, and turns light green in the later stage; It was identified as Cladosporium. In this study, 36 positive mutants were screened, of which H9-18 mutant strain had the highest enzyme activity of 43.2 IU/mL, which was 33.33% higher than that before mutation. The results of the enzymatic property analysis showed that the cellobiohydrolase produced by the H9-18 strain selected in this study showed highest enzyme activity of 43.2 IU/mL when the temperature was 50 ℃. And the highest enzyme activity was 44.1 IU/mL when the pH was 4.8. Fe2+ had the strongest promoting effect on the enzyme activity of 38.67%, while Na+ had the most obvious inhibitory effect of 34.03%. Conclusion: This experiment finally obtained a strain H9-18 with high cellobiohydrolase production efficiency and stable enzyme activity, which further laid a theoretical and experimental foundation for the efficient utilization of lignocellulosic biomass resources.