Abstract
Background: β-1, 4-glucosidases play important roles in the degradation of lignocellulosic biomass. It helps generate glucose from cellobiose and oligosaccharides, which could enhance the productivity in biorefinery and bioconversion process for energy and chemicals. Discovering novel β-1, 4-glucosidases can provide broader possibilities and understanding. The purpose of this study was to find a new β-1, 4-glucosidase in Aspergillus fresenii by an efficient method based on the high throughput sequencing technique. Results: With the high throughput sequencing technique, a novel β-1, 4-glucosidase, named bgl T2, was cloned from Aspergillus fresenii , which was 2586 bp encoding 862 amino acid residues based on the sequencing analysis. Its amino acid sequence shared 91%, 80%, 80%, and 78% identity with the β-glucosidases of Aspergillus steynii IBT 23096 (XP_024702113.1), Aspergillus oryzae (5FJJ_A), Aspergillus aculeatus (P48825.1), and Aspergillus fumigatus A1163 (B0XPE1.1), respectively. The β-glucosidase bgl T2 gene was optimized according to the codon bias of Komagataella phaffii (≡ Pichia pastoris (nom. illeg.)) and synthetized. The optimized bgl T2 gene was inserted into plasmid pPICZαA, and transformed into K.phaffii X33 for its heterologous expression and enzymatic characters determination. The heterologous expressed β-glucosidase bgl T2 presented the highest activity at 55 °C and pH 5.5. When bgl T2 treated in citric acid- disodium hydrogen phosphate buffer (from pH 2.5 to pH 8.0) for one hour, the enzymatic activity was stable for pH 3.0 to pH 8.0 treatment, while the enzymatic activity dropped down to 22% with the pH 2.5 treatment. The thermostability half-life of bgl T2 was 9 min 36 s, 4 min 22 s, 117 s, and 68 s under 50 °C, 55 °C, 60 °C, 65 °C incubation, respectively. The Michaelis constant ( K m ) of bgl T2 was 0.0007 mol/L. The maximum rate of bgl T2 theoretical enzymatic reaction ( V max ) was 9×10 -8 mol/L/s. In a 5 L fermentation vessel, the recombinant K.phaffii X33 could yield a β-1, 4-glucosidase activity of 4.45 U/mL after 96 h methanol inducement. Conclusions: As the important enzyme to release glucose in the hydrolysis processes of cellulose, the novel bgl T2 provide a possibility to apply in bioenergy engineering, food processing, feed industry, and nutritional study, etc. This study also developed a path to obtain new enzymes depending on high throughput sequencing technique.
Targeted high-throughput sequencing technique for the molecular diagnosis of primary immunodeficiency disorders
