Maltooligosaccharides (MOS) are homooligosaccharides that consist of 3–10 glucose molecules linked by α-1,4 glycosidic bonds. As they have physiological functions, they are commonly used as ingredients in nutritional products and functional foods. Many researchers have investigated the potential applications of MOS and their derivatives in the pharmaceutical industry. In this review, we summarized the properties and methods of fabricating MOS and their derivatives, including sulfated and non-sulfated alkylMOS. For preparing MOS, different enzymatic strategies have been proposed by various researchers, using α-amylases, maltooligosaccharide-forming amylases, or glycosyltransferases as effective biocatalysts. Many researchers have focused on using immobilized biocatalysts and downstream processes for MOS production. This review also provides an overview of the current challenges and future trends of MOS production.

α-Nitrophenyl derivatives of glycosides are convenient substrates used to detect and characterize α-N-acetylgalactosaminidase. A new procedure combining chemical and biocatalytic steps was developed to prepare 4-nitrophenyl-2-acetamido-2-deoxy-α-D-galactopyranoside (4NP-α-GalNAc). The α-anomer was prepared through chemical synthesis of an anomeric mixture followed by selective removal of the β-anomer using specific enzymatic hydrolysis. Fungal β-N-acetylhexosaminidase (Hex) from Penicillium oxalicum CCF 1959 served this purpose owing to its high chemo-and regioselectivity towards the β-anomeric N-acetylgalactosamine (GalNAc) derivative. The kinetic measurements of the hydrolytic reaction showed that the enzyme was not inhibited by the substrate or reaction products. The immobilization of Hex in lens-shaped polyvinyl alcohol hydrogel capsules provided a biocatalyst with very good storage and operational stability. The immobilized Hex retained 97% of the initial activity after ten repeated uses and 90% of the initial activity after 18 months of storage at 4 °C. Immobilization inactivated 65% of the enzyme activity. However, the effectiveness factor and kinetic and mass transfer phenomena approached unity indicating negligible mass transfer limitations.

The application of immobilized enzymes often plays a key role in successfully implementing an economically feasible biocatalytic process at an industrial scale. Designing an immobilized biocatalyst involves solving several tasks, from the selection of the carrier and immobilization method to the characterization of the kinetic properties of the immobilized enzyme. In this study, we focused on the kinetic properties of free and immobilized ß-N-acetylhexosaminidase (Hex), a promising enzyme for application in the field of biotechnology, especially for the synthesis of bioactive carbohydrates. Hex was immobilized via covalent binding in methacrylate particles. The effect of immobilizing Hex from Penicillium oxalicum into porous particles on kinetic properties was investigated, and mathematical and experimental modeling showed that the kinetic behavior of the enzyme was significantly influenced by diffusion in the particles. Along with the study on kinetics, a simple method was developed to investigate the reversible inhibition of the immobilized enzyme in a continuous-flow system. The method is suitable for application in cases where a chromogenic substrate is used, and here it was applied to demonstrate the inhibitory effects of N-acetyl-glucosaminyl thiazoline (NAG-thiazoline) and O-(2-Acetamido-2-deoxy-D-glucopyranosylidene)amino N-phenyl carbamate ((Z)-PugNAc) on Hex.

β-N-acetylhexosaminidases have great potential in applied biocatalysis owing to their ability to act on a wide range of natural and modified substrates. In this work, β-N-acetylhexosaminidases from four Penicillium crustosum strains (PcHex) were studied. The production strains showed the highest enzymatic activity in the culture medium after 11–14 days of cultivation. The specific activity of the isolated and purified PcHex of hydrolysis of 4-nitrophenyl-N-acetyl-β-D-galactopyranoside was 15–20 U/mg protein. All isolated β-N-acetylhexosaminidases showed similar pH–activity profiles, with the optimum pH being 4.0–5.0 and the optimum temperature being 40 °C–50 °C. Apart from standard substrates, two synthetic substrates (5-bromo-4-chloro-3-indolyl-N-acetyl-β-D-galactoside and 2-chloro-4-nitrophenyl-β-D-galactopyranoside) were tested and successfully hydrolyzed using β-N-acetylhexosaminidase preparations. Maximum activity toward the fluorogenic substrate 4-methylumbelliferyl-N-acetyl-β-D-galactopyranoside (4MU-β-GalNAc) was observed for enzyme preparations from PcHex1 (15.38 U/mg protein) isolated from swab samples of books in the Slovak National Museum in Martin (Slovak Republic) and stored in our laboratory. The same enzyme preparation was used for the selective hydrolysis of β-anomer of 4MU-GalNAc from an anomeric mixture of 4MU-α/β-GalNAc. Thus, pure α-anomer (with the total yield being 90%) was separated from the mixture, which suggests the application potential of these enzymes.