A gene encoding a polysaccharide-degrading enzyme was cloned from the genome of the bacterium Nocardiopsis halotolerans. Analysis of the amino acid sequence of the protein showed the presence of the catalytic domain of the endo-1,4-β-xylanases of the GH11 family. The gene was amplified by PCR and ligated into the pPic9m vector. A recombinant producer based on Pichia pastoria was obtained. The production of the enzyme, which we called NhX1, was carried out in a 10 L fermenter. Enzyme production was 10.4 g/L with an activity of 927 U/mL. Purification of NhX1 was carried out using Ni-NTA affinity chromatography. The purified enzyme catalyzed the hydrolysis of xylan but not other polysaccharides. Endo-1,4-β-xylanase NhX1 showed maximum activity and stability at pH 6.0–7.0. The enzyme showed high thermal stability, remaining active at 90 °C for 20 min. With beechwood xylan, the enzyme showed K_m 2.16 mg/mL and V_max 96.3 U/mg. The products of xylan hydrolysis under the action of NhX1 were xylobiose, xylotriose, xylopentaose, and xylohexaose. Endo-1,4-β-xylanase NhX1 effectively saccharified xylan-containing products used for the production of animal feed. The xylanase described herein is a thermostable enzyme with biotechnological potential produced in large quantities by P. pastoria.

We demonstrate for the first time the feasibility of using bacterial laccase as a biocatalyst for oxidizing ferulic acid (FA) to enable nucleophilic crosslinking of chitosan under neutral conditions, targeting future applications in medicine and the food industry. We have established optimal conditions achieving a high degree of chitosan crosslinking (up to 85%). Employing quantum-chemical modeling and spectral analysis techniques, it was determined that the predominant mechanism for the nucleophilic attack of chitosan amino groups on the phenoxyl radical – the oxidation product of FA – involves Michael addition, followed by radical recombination. This process results in the formation of crosslinks consisting of β-β’ coupled FA dimers while retaining the phenoxyl functionalities.

A study of bacterial xylanases from Streptomyces prunicolor, Streptomyces carpinensis, and Streptomyces venezuelae was conducted. The xylanase-encoding genes were cloned from bacterial genomes. Recombinant producers based on E. coli M15(Rep4) and the pQE-30 vector were obtained, and the expression and purification of the recombinant proteins were carried out. Electrophoretically homogeneous preparations of four xylanases from the GH10 and GH11 families were obtained. The xylanases had a near-neutral pH optimum and moderate thermal stability (the enzymes were stable at 70 °C). The xylanases were more stable over a pH range of 5.0 to 8.0. The properties of the xylanases obtained correspond to the properties of most xylanase-producing bacteria of the Streptomyces genus described in the literature. Xylanases from the GH10 family of S. carpinensis form xylose, xylobiose and xylotetraose as main products of xylan hydrolysis. The GH10 xylanase from S. venezuelae hydrolyzes xylan into xylose, xylobiose, xylotriose, xylotetraose and xylopentaose. The main products of xylan hydrolysis by GH11 xylanase from S. prunicolor are xylobiose, xylopentaose and a trace amount of xylose. All four xylanases effectively saccharify cereals, indicating the biotechnological potential of these enzymes.