Analysis of Transglucosylation Products of Aspergillus niger α-Glucosidase that Catalyzes the Formation of α-1,2- and α-1,3-Linked Oligosaccharides.

According to whole-genome sequencing, Aspergillus niger produces multiple enzymes of glycoside hydrolases (GH) 31. Here we focus on a GH31 α-glucosidase, AgdB, from A. niger . AgdB has also previously been reported as being expressed in the yeast species, Pichia pastoris ; while the recombinant enzyme (rAgdB) has been shown to catalyze tranglycosylation via a complex mechanism. We constructed an expression system for A. niger AgdB using Aspergillus nidulans . To better elucidate the complicated mechanism employed by AgdB for transglucosylation, we also established a method to quantify glucosidic linkages in the transglucosylation products using 2D NMR spectroscopy. Results from the enzyme activity analysis indicated that the optimum temperature was 65 °C and optimum pH range was 6.0-7.0. Further, the NMR results showed that when maltose or maltopentaose served as the substrate, α-1,2-, α-1,3-, and small amount of α-1,1-ß-linked oligosaccharides are present throughout the transglucosylation products of AgdB. These results suggest that AgdB is an α-glucosidase that serves as a transglucosylase capable of effectively producing oligosaccharides with α-1,2-, α-1,3-glucosidic linkages.

A Novel α-Glucosidase of the Glycoside Hydrolase Family 31 from Aspergillus sojae.

We characterized an α-glucosidase belonging to the glycoside hydrolase family 31 from Aspergillus sojae. The α-glucosidase gene was cloned using the whole genome sequence of A. sojae, and the recombinant enzyme was expressed in Aspergillus nidulans. The enzyme was purified using affinity chromatography. The enzyme showed an optimum pH of 5.5 and was stable between pH 6.0 and 10.0. The optimum temperature was approximately 55 °C. The enzyme was stable up to 50 °C, but lost its activity at 70 °C. The enzyme acted on a broad range of maltooligosaccharides and isomaltooligosaccharides, soluble starch, and dextran, and released glucose from these substrates. When maltose was used as substrate, the enzyme catalyzed transglucosylation to produce oligosaccharides consisting of α-1,6-glucosidic linkages as the major products. The transglucosylation pattern with maltopentaose was also analyzed, indicating that the enzyme mainly produced oligosaccharides with molecular weights higher than that of maltopentaose and containing continuous α-1,6-glucosidic linkages. These results demonstrate that the enzyme is a novel α-glucosidase that acts on both maltooligosaccharides and isomaltooligosaccharides, and efficiently produces oligosaccharides containing continuous α-1,6-glucosidic linkages.

Effect of purification method of ß-chitin from squid pen on the properties of ß-chitin nanofibers.

The relationship between purification methods of ß-chitin from squid pen and the physicochemical properties of ß-chitin nanofibers (NFs) were investigated. Two types of ß-chitin were prepared, with ß-chitin (a→b) subjected to acid treatment for decalcification and then base treatment for deproteinization, while ß-chitin (b→a) was treated in the opposite order. These ß-chitins were disintegrated into NFs using wet pulverization. The ß-chitin (b→a) NF dispersion has higher transmittance and viscosity than the ß-chitin (a→b) NF dispersion. For the first time, we succeeded in obtaining 3D images of the ß-chitin NF dispersion in water by using quick-freeze deep-etch replication with high-angle annular dark field scanning transmission electron microscopy. The ß-chitin (b→a) NF dispersion has a denser and more uniform 3D network structure than the ß-chitin (a→b) NF dispersion. Widths of the ß-chitin (a→b) and (b→a) NFs were approximately 8-25 and 3-10nm, respectively.