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. Comparison between AOX1 ( $$\hbox {P}_{AOX1}$$ ) and GAP ( $$\hbox {P}_{GAP}$$ ) promoters for recombinant Xyl-L production in P. pastoris showed that the $$\hbox {P}_{AOX1}$$ promoter resulted in higher activity per wet-cell weight. Co-transforming $$\hbox {P}_{AOX1}$$ -Xyl strains with plasmids encoding genes aiding in protein folding (HAC1 or PDI1) did not enhance Xyl-L catalytic activity compared to the parental $$\hbox {P}_{AOX1}$$ strain. Thus, $$\hbox {P}_{AOX1}$$ -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 $$\hbox {s}^{-1}$$ . Xyl-L maintained over 80% of relative activity between pH 5.6 $$-$$ 8.6 and $$37-44^{\circ }$$ C, and was activated by $$\hbox {CaCl}_2$$ and $$\hbox {MgCl}_2$$ , but inhibited by $$\hbox {MnCl}_2$$ . 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. 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.

The Atacama Desert is emerging as an unexpected source of microbial life and, thus, a source of bioactive compounds and novel enzymes. Baeyer–Villiger monooxygenases (BVMOs), a subclass of flavin-dependent monooxygenases (FPMOs), have gained attention as promising biocatalysts for the biosynthesis of industrially relevant molecules for a wide range of applications, such as pharmaceuticals and polymers, among others. BVMOs catalyze the oxidation of ketones and cyclic ketones to esters and lactones, respectively, by using molecular oxygen and NAD(P)H. BVMOs may also catalyze heteroatoms oxidation including sulfoxidations and N-oxidations. This work aims to search for novel BVMOs in the genomes of new bacterial strains isolated from the Atacama Desert. Bioinformatic analysis led to the identification of 10 putative BVMOs, where the monooxygenase named MO-G35A was selected. Genome context showed, downstream of the MO-G35A, a gene encoding for an enzyme from the short-chain dehydrogenase/reductase family, suggesting a closer redox loop between both enzymes. MO-G35A was successfully expressed in three Escherichia coli expression systems, where higher yields were achieved using the E. coli Shuffle T7 as host, suggesting that correct disulfide bond formation is necessary for correct folding. Enzyme characterization showed that it operates optimally at 35–38 °C, exhibiting a Km of 0.06 mM and a kcat of 0.15 s⁻¹ for bicyclo [3.2.0] hept-2-en-6-one (BHC). Furthermore, the study revealed high stability in the presence of organic solvents, making it suitable for applications in various industrial processes, especially when the substrates have poor solubility in aqueous solutions. These results highlight the robustness and adaptability of enzymes in extreme environments, making them valuable candidates for biotechnological applications.