Abstract:A P.pastoris strain for efficient endo-β-1,3-glucanase expression was constructed to hydrolyze curdlan and produce curdlan oligosaccarides. Firstly, error-prone PCR technology was implemented on endo-β-1,3-glucanase gene from Trichoderma harzianum to construct a mutation library of BGN13.1a. High-throughput screening technology and Congo red plate screening were then employed to obtain high expression P. pastoris strains. Finally, TLC and MALDI-TOF MS were used to analyze the curdlan hydrolysate and their enzymatic properties. The results showed that the activity of endo-β-1,3-glucanase from the successfully screened mutant P. pastoris K827 was improved by 25% compared with that of the original enzyme. The expressed endo-β-1,3-glucanase hydrolyzed curdlan to produce oligosaccharides at 1.492 g/L with a polymerization degree of 2~10. The optimal reaction temperature and pH for endo-β-1,3-glucanase were 50 ℃ and pH 5.5, respectively. The pH stability and temperature stability of mutant K827 were significantly improved compared with the original enzyme. This study could provide experimental basis for the industrialized production and application of endo-β-1,3-glucanase.
