Characterization of a fungal thermostable endoglucanase from Chinese Nong-flavor daqu by metatranscriptomic method

Chinese Nong-flavor (NF) daqu has been enriched with plenty of active enzymes by man-made environment for thousand years. Based on our previous metatranscriptomics, an endo-β-glucanase gene (NFEg16A), which showed high expression level in NF daqu, was directly obtained and expressed in Escherichia coli BL21 (DE3). NFEg16A shared the highest sequence identity of 87% with endo-1,3-1,4-β-glucanase from Paecilomyces thermophile. It was optimally active at pH 6.5 and 60 °C and highly stable (>75% residual activity) at pH 3-8 and temperature 30-90 °C. The activity of NFEg16A was strongly inhibited by 10 mM Fe³⁺ and Hg²⁺. Compared with endoglucanases with high similarities, NFEg16A was more stable at 70 °C and had higher half-lives of 3.4 h and 1.4 h at 80 °C and 90 °C, respectively. Its specific activity was 85.3 U/mg on barley β-glucan. Moreover, NFEg16A could efficiently hydrolyze substrate at high concentration of 15 mg/mL, and released glucose and cellobiose as its main end-products. Therefore, this work to some extent verified the important role of NFEg16A in NF daqu, and it would stimulate the acquisition of more enzymes from NF daqu to improve the baijiu quality in future. High thermostability of NFEg16A could also strengthen its potential applications in feed industry.