Expression and characterization of cold-adapted xylanase Xyl-L in Pichia pastoris for xylooligosaccharide (XOS) preparation.

Background: Xylan, the second most abundant polysaccharide in plant biomass, requires endoxylanases for its hydrolysis into xylooligosaccharides (XOS). Xylanases have been widely used in industries such as animal feed, bakery, juice production, and paper pulp. Recently, XOS have gained attention for their health benefits, including improved digestion, reduced cholesterol, and antioxidant effects. The cold-adapted GH10 xylanase of Antarctic origin Xyl-L was previously expressed in Escherichia coli, showing promising low-temperature activity. However, Pichia pastoris is currently a preferred host for industrial xylanase production due to its ability to express complex proteins and secrete them into the culture medium. This study explored the expression of Xyl-L in P. pastoris and evaluated its potential for XOS production using common flours as substrates, aiming for applications in the food and nutraceutical industry.

Results: Comparison between AOX1 ( ${\text{P}}_{AOX1}$ ) and GAP ( ${\text{P}}_{\mathrm{GAP}}$ ) promoters for recombinant Xyl-L production in P. pastoris showed that the ${\text{P}}_{AOX1}$ promoter resulted in higher activity per wet-cell weight. Co-transforming ${\text{P}}_{AOX1}$ -Xyl strains with plasmids encoding genes aiding in protein folding (HAC1 or PDI1) did not enhance Xyl-L catalytic activity compared to the parental ${\text{P}}_{AOX1}$ strain. Thus, ${\text{P}}_{AOX1}$ -Xyl was cultivated in 3 L bioreactors in fed-batch cultures; it is presumed that the enzyme is produced with glycosylations within its structure, given its migration within the SDS-PAGE gels. The produced Xyl-L was purified from the culture supernatant, resulting in peak xylanase activity after 90 h, with specific activity of 5.10 ± 0.21 U/mg, at pH 7.5 and ${25}^{\circ }$ C, using beechwood xylan. It also showed a Km of 3.5 mg/mL and a kcat of 9.16 ${\text{s}}^{-1}$ . Xyl-L maintained over 80% of relative activity between pH 5.6 $-$ 8.6 and $37-{44}^{\circ }$ C, and was activated by ${\text{CaCl}}_2$ and ${\text{MgCl}}_2$ , but inhibited by ${\text{MnCl}}_2$ . Xyl-L was tested using several flours (whole wheat, rye, oatmeal and all-purpose) as substrates, where XOS with a polymerization degree (DP) of 2 were obtained from each substrate, whole wheat flour generated XOS with DP 3, and XOS with DP 2, 3 and 4 were produced when beechwood xylan was used as substrate.

Conclusions: The xylanase Xyl-L was successfully expressed in P. pastoris and proved to be able to degrade various flour substrates, producing XOS with DP ranging from 2 to 4, indicating its potential applications in the nutraceutical and food industries. Further studies must be performed to optimize its production in bioreactors.