Enzymatic production of fully deacetylated chitooligosaccharides and their neuroprotective and anti-inflammatory properties

Abstract

Among several commercial enzymes screened for chitosanolytic activity, Neutrase 0.8L (a protease from Bacillus amyloliquefaciens) was selected in order to obtain a product enriched in deacetylated chitooligosaccharides (COS). The hydrolysis of different chitosans with this enzyme was followed by size exclusion chromatography (SEC-ELSD), mass spectrometry (ESI-Q-TOF), and high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Neutrase 0.8L converted 10 g/L of various chitosans into mostly deacetylated oligosaccharides, yielding approximately 2.5 g/L of chitobiose, 4.5 g/L of chitotriose and 3 g/L of chitotetraose. We found out that the neutral protease was not responsible of the chitosanolytic activity in the extract, whilst it could participate in the deacetylating process. The synthesized COS were tested in vitro for their neuroprotective (towards human SH-S5Y5 neurons) and anti-inflammatory (in RAW macrophages) activities, and compared with other functional ingredients, namely fructooligosaccharides.

Tailored Enzymatic Synthesis of Chitooligosaccharides with Different Deacetylation Degrees and Their Anti-Inflammatory Activity

“…The highest effect was obtained using 250 ng/well ( Figure 5). It is worth noting that fdCOS exhibited a negligible anti-inflammatory effect at concentrations of 100 and 500 ng/well after a 2-h incubation, but this effect increased significantly at 6 h. This relates well with our preliminary results with fdCOS [18]. In contrast, paCOS (except for 100 ng/well) and faCOS displayed a more stable effect between 2 and 6 h. These results could be indicating a critical role of the acetamido group of COS in their properties.…”

“…Based on previous work [18], we selected the commercial proteolytic preparation Neutrase 0.8L and a chitosan (CHIT600) with a high DD (>90%) to scale up the production of fully deacetylated COS (fdCOS). The reaction was carried out over 24 h with 1% (w/v) chitosan at pH 5.0 and 50 • C. After this time, the enzyme and the remaining chitosan were eliminated by ultrafiltration with a 10 kDa membrane.…”

“…The size of COS (defined by the degree of polymerization, For the hydrolysis of chitosan, chitosanases (EC 3.2.1.132) catalyze specifically the cleavage of β (1→4) glycosidic linkages between GlcN moieties [29][30][31][32]. However, other enzymes such as pectinases [33], cellulases [34], and proteases [35] also display chitosanolytic activity yielding COS. We have recently reported that a proteolytic preparation from Bacillus amyloliquefaciens (Neutrase 0.8L) is able to produce a mixture of COS that is highly enriched in fdCOS [18].…”

“…Although the anti-inflammatory activity of chitosan oligosaccharides is well Among the methodologies to perform the partial hydrolysis of chitin/chitosan into COS, the use of chitinolytic/chitosanolytic enzymes offer some advantages over physical, chemical, or electrochemical depolymerization [17]. Glycosidic enzymes require mild reaction conditions (moderate temperature and slightly acidic pH), display high efficiency, and allow for control of the composition of the final product on the basis of enzyme specificity [4,[18][19][20][21][22], which can also be altered by protein engineering techniques [23,24]. The enzymatic strategies are more environmentally friendly and generate less waste than the chemical methods [3,25,26].…”

Application of Gelatin Decorated with Allura Red as Resonance Rayleigh Scattering Sensor to Detect Chito-Oligosaccharides

“…COS captured much attention from researchers on account of its good dissolvability [2] and a higher degree of deacetylation [3]. To our knowledge, COS has been proven to not only have antioxidant activity [4], and antimicrobial activity [5] but also have anti-tumor activity [6,7] and anti-inflammatory properties [8,9]. Therefore, COS was widely used in many fields, such as cosmeceutical products [10], nutraceuticals [11], pharmaceuticals [3], and core-shell nanoparticles [12].…”