Endo-1,4-β-xylanase (E.C.3.2.1.8) is a family of glycoside hydrolase. It is capable of hydrolyzing the backbone of substituted xylan polymers into fragments of random size. Due to this ability, xylanase can serve the degradation of lignocellulose, and facilitate the application of xylan. Cell-surface display of enzymes is one of the most attractive applications in yeast. It is a promising utilization in constructing the whole-cell biocatalyst of xylanase. For this purpose, a cDNA sequence of endo-1,4-β-xylanase B (XylB) from Aspergillus niger BCC14405 was optimized and synthesized according to the codon bias of Saccharomyces cerevisiae. The genes encoding galactokinase (GAL1) promoter, α-mating ... More
Endo-1,4-β-xylanase (E.C.3.2.1.8) is a family of glycoside hydrolase. It is capable of hydrolyzing the backbone of substituted xylan polymers into fragments of random size. Due to this ability, xylanase can serve the degradation of lignocellulose, and facilitate the application of xylan. Cell-surface display of enzymes is one of the most attractive applications in yeast. It is a promising utilization in constructing the whole-cell biocatalyst of xylanase. For this purpose, a cDNA sequence of endo-1,4-β-xylanase B (XylB) from Aspergillus niger BCC14405 was optimized and synthesized according to the codon bias of Saccharomyces cerevisiae. The genes encoding galactokinase (GAL1) promoter, α-mating factor 1 (MFα1) pre-pro secretion signal, fully codon-optimized XylB, the 320 amino acids of C terminal of α-agglutinin, alcohol dehydrogenase (ADH1) terminator and kanMX cassette were amplified and cloned into YEplac181 to construct a cell-surface display vector called pGMAAK-XylB with α-agglutinin as an anchor. Then pGMAAK-XylB was transformed into S. cerevisiae. The results show XylB was immobilized and actively expressed on S. cerevisiae. Meanwhile, a secretion expression plasmid was also constructed using the above elements except α-agglutinin as a control strain in the study of characteristic of XylB. After an induction of 48 h by 2% galactose, the activity of displayed XylB reached 63 U/g dry-cell weight. The optimal pH of displayed XylB has changed from 5 to 6 and the optimal temperature has changed from 50 °C to 60 °C, comparing to the recombinant secretion XylB.
