This study shows the research on the depolymerisation of insect and crab chitosans using novel enzymes. Enzyme preparations containing recombinant chitinase Chi 418 from Trichoderma harzianum, chitinase Chi 403, and chitosanase Chi 402 from Myceliophthora thermophila, all belonging to the family GH18 of glycosyl hydrolases, were used to depolymerise a biopolymer, resulting in a range of chitosans with average molecular weights (M_w) of 6–21 kDa. The depolymerised chitosans obtained from crustaceans and insects were studied, and their antibacterial and antifungal properties were evaluated. The results proved the significance of the chitosan’s origin, showing the potential of Hermetia illucens as a new source of low molecular weight chitosan with an improved biological activity.

Polysaccharide processing by means of low-temperature Electron Beam Plasma (EBP) is a promising alternative to the time-consuming and environmentally hazardous chemical hydrolysis in oligosaccharide production. The present paper considers mechanisms of the EBP-stimulated destruction of crab shell chitin, cellulose sulfate, and microcrystalline cellulose, as well as characterization of the produced oligosaccharides. The polysaccharide powders were treated in oxygen EBP for 1–20 min at 40 °C in a mixing reactor placed in the zone of the EBP generation. The chemical structure and molecular mass of the oligosaccharides were analyzed by size exclusion and the reversed phase chromatography, FTIR-spectroscopy, XRD-, and NMR-techniques. The EBP action on original polysaccharides reduces their crystallinity index and polymerization degree. Water-soluble products with lower molecular weight chitooligosaccharides (weight-average molecular mass, M_w = 1000–2000 Da and polydispersity index 2.2) and cellulose oligosaccharides with polymerization degrees 3–10 were obtained. The ¹H-NMR analysis revealed 25–40% deacetylation of the EBP-treated chitin and FTIR-spectroscopy detected an increase of carbonyl- and carboxyl-groups in the oligosaccharides produced. Possible reactions of β-1,4-glycosidic bonds’ destruction due to active oxygen species and high-energy electrons are given.

Microorganisms are a significant cause of damage to cultural heritage, including paintings. Currently, the palette of antiseptics that are used in painting has narrowed considerably. This is due to the increased demands placed on such substances. It was shown that low molecular weight chitosan (LMWC) obtained from the king crab (Paralithodes camtschaticus) exhibits high activity against dominant fungi-destructors of paintings in the State Tretyakov Gallery. Nevertheless, the increasing market demand for chitosan has prompted the exploration of alternative sources. Insects, notably the bioconverter black soldier fly (Hermetia illucens), stand out as one of the most cultivated options. This study investigates the effectiveness of chitosan, isolated from H. illucens by a novel method developed by authors, in inhibiting fungi that damage tempera paintings. The activity of 33 and 39 kDa chitosans from H. illucens is comparable to the most active chitosans previously studied from P. camtschaticus. However, there are characteristic differences between these compounds, as shown by the results of FTIR spectroscopy, which may affect their consumer properties when used in paint materials. Our studies suggest that LMWC from H. illucens is a promising material that can expand the range of antiseptics used in painting. Graphical Abstract

The depolymerization of chitosan using chitinolytic enzymes is one of the most promising approaches for the production of bioactive soluble chitooligosaccharides (COS) due to its high specificity, environmental safety, mild reaction conditions, and potential for development. However, the comparative efficacy of bacterial chitinases and chitosanases in terms of yield, solubility, and antimicrobial activity of produced COS remains understudied. In this work, chitinase (73 kDa) and chitosanase (40 kDa) from the strain Bacillus thuringiensis B-387 (Bt-387) were purified using various chromatographic techniques and compared by their action on chitosan (DD 85%). The molecular mass and structure of generated COS was determined using TLC, LC-ESI-MS, HP-SEC, and C¹³-NMR techniques. Chitosanase converted the polymer more rapidly to short COS (GlcN₂-GlcN₄), than chitinase, and was more specific in its action on mixed bonds between GlcN and GlcNAc. Chitosanase needed a noticeably shorter incubation time and enzyme–substrate ratio than chitinase for production of larger oligomeric molecules (Mw 2.4–66.5 and 15.4–77.7 kDa, respectively) during controlled depolymerization of chitosan. Moreover, chitosanase-generated oligomers demonstrate better solubility and a higher antifungal activity in vitro against the tested plant pathogenic fungi. These features, as well as the high enzyme production and its simplified purification protocol, make chitosanase B-387 more suitable for the production of antifungal chitooligomers than chitinase.

Chitooligosaccharides are widely used, which provides considerable interest in their biosynthesis. Enzymatic synthesis of hexa-N-acetylchitohexaose and penta-N-acetylchitopentaose using N-acetylglucoseaminyl transferase possessing the unique properties from rhizobial bacteria Rhizobium sp. GRH2 and M. loti has been performed in E. coli cells. Culturing of E. coli expressing the appropriate recombinant enzymes in the presence of N-acetylglucosamine as a substrate resulted in the synthesis of significant amounts of the desired chitooligosaccharides via high performance liquid chromatography and mass-spectrometry confirmed their conformity to standards.