Enzymatic Generation of Chitooligosaccharides from Chitosan Using Soluble and Immobilized Glycosyltransferase (Branchzyme)

Abstract

Chitooligosaccharides possessing remarkable biological properties can be obtained by enzymatic hydrolysis of chitin. In this work, the chitosanase activity of soluble and immobilized glycosyltransferase (Branchzyme) toward chitosan and biochemical characterization are described for the first time. This enzyme was found to be homotetrameric with a molecular weight of 256 kDa, an isoelectric point of 5.3, and an optimal temperature range of between 50 and 60 °C. It was covalently immobilized to glutaraldehyde-agarose with protein and activity immobilization yields of 67% and 17%, respectively. Immobilization improved enzyme stability, increasing its half-life 5-fold, and allowed enzyme reuse for at least 25 consecutive cycles. The chitosanase activity of Branchzyme on chitosan was similar for the soluble and immobilized forms. The reaction mixture was constituted by chitooligosaccharides with degrees of polymerization of between 2 and 20, with a higher concentration having degrees of polymerization of 3-8.

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

“…Based on previous works that report the presence of chitosanolytic activity in commercial enzyme preparations (Cabrera et al 2005, Montilla et al 2013, Pantaleone et al 1992, we screened the hydrolytic activity towards chitosan CHIT100 (100-300 kDa, DD ≥ 90%) of a series of commercial enzymes whose stated activity was pectinase, cellulase (endo-1,4-β-D-glucanase), xylanase or protease (Table 1). Using the DNS assay based on the detection of released reducing sugars, the two more active preparations were Rapidase TF (a pectinase from Aspergillus niger) and Neutrase 0.8L (a protease from Bacillus amyloliquefaciens), which showed a moderate activity towards chitosan (0.47 ± 0.03 and 0.54 ± 0.03 U per mL, respectively).…”

“…Mass spectrometry (MS) techniques, such as LC-MS/MS (Kim et al 2013) or MALDI-TOF (Montilla et al 2013), are of great potential to identify (or to discard) the formation of certain COS in the reactions, especially combined with NMR spectroscopy (Mahata et al 2014). Figure 3 illustrates the ESI-Q-TOF MS spectrum that resulted from the hydrolysis of chitosan CHIT600 with Neutrase 0.8L.…”

Bioproduction of Chitooligosaccharides: Present and Perspectives

“…This enzyme catalyzes the transfer of a segment of a 1,4-α- d -glucan chain to a primary hydroxyl group in a similar glucan chain to create 1,6-α-linkages, thereby increasing the number of branch points [131]. Interestingly, Branchzyme was active on chitosan and produced COS with DP 2-20, with a higher concentration having COS with DP 3–8 [132]. Recently, a family 46 chitosanase from S. coelicolor A3(2) was employed to degrade both a fully deacetylated chitosan and a 68% deacetylated chitosan for the production of a series of COS and to study in-depth the enzyme's mode of action [133].…”

Continuous production of chitooligosaccharides by an immobilized enzyme in a dual-reactor system

“…For this type of enzyme, covalent binding is preferred over simple adsorption because the substrate chitosan possesses an extraordinary ability partly due to the presence of amino groups to bind biomolecules, which may cause the desorption of the enzyme from the carrier. In fact, most of the strategies reported to immobilize chitosanases involve the formation of covalent bonds between the enzyme and the carrier, using silica gel [21], agar gel [22,23], agarose [24], polyacrylonitrile [25], DEAE-cellulose [26], amylose-coated magnetic nanoparticles [27] and even chitin itself [28,29]. Other methodologies, in particular alginate entrapment, have been also explored [30,31].…”