Characterization of a Glycoside Hydrolase Family 31 α-Glucosidase Involved in Starch Utilization inPodospora anserina

Abstract

For Podospora anserina, several studies of cellulolytic enzymes have been established, but characteristics of amylolytic enzymes are not well understood. When P. anserina grew in starch as carbon source, it accumulated glucose, nigerose, and maltose in the culture supernatant. At the same time, the fungus secreted α-glucosidase (PAG). PAG was purified from the culture supernatant, and was found to convert soluble starch to nigerose and maltose. The recombinant enzyme with C-terminal His-tag (rPAG) was produced with Pichia pastoris. Most rPAG produced under standard conditions lost its affinity for nickel-chelating resin, but the affinity was improved by the use of a buffered medium (pH 8.0) supplemented with casamino acid and a reduction of the cultivation time. rPAG suffered limited proteolysis at the same site as the original PAG. A site-directed mutagenesis study indicated that proteolysis had no effect on enzyme characteristics. A kinetic study indicated that the PAG possessed significant transglycosylation activity.

Efficient synthesis of α‐galactosyl oligosaccharides using a mutant Bacteroides thetaiotaomicron retaining α‐galactosidase (BtGH97b)

“…This result implied that the trisaccharide has three 17 anomeric protons with α, α, and β anomeric configurations, respectively, and is the non-reducing 18 sugar, β-maltosyl α-D-galactopyranoside [α-D-Galp-(1↔1)-β-D-Glcp-(4←1)α-D-Glcp]. 19 The formation of two saccharides from cellobiose was confirmed by HPLC. ESI-MS 20 revealed that the major product was a trisaccharide (m/z 527.16 for C18H32O16 + Na + ) and another 21 minor product was a tetrasaccharide (m/z 689.21 for C24H42O21 + Na + ).…”

“…Using glucose as the 18 acceptor generated one major and three minor products. Electrospray ionization-mass 19 spectrometry (ESI-MS) revealed that the major product (m/z 365.10 for C12H22O11 + Na + ) and one 20 minor product (m/z 365.11 for C12H22O11 + Na + ) were disaccharides, and the other two minor 21 saccharides (m/z 527.16 for C18H32O16 + Na + ) were trisaccharides. The trisaccharides are likely 22 formed from further reactions of the disaccharide products.…”

“…The initial 18 velocities of fluoride ion release from various concentrations of α-GalF as the sole substrate were 19 measured. The plots of velocities versus [α-GalF] exhibited standard Michaelis-Menten behavior 20 in each condition (Fig.…”

“…of 0.20 Å for 641 Cα atoms, indicating that the mutation had no 18 noticeable effect on the three-dimensional structure of the protein. The mutation of Asp415 to Gly 19 made a cavity that was sufficient to accommodate the formate or azide ion. A calcium ion, 20 characteristic of GH97 enzymes, and Gal-Lac were located at the active site (Fig.…”

“…We performed kinetic analyses of the transfer 17 reactions using lactose as an acceptor molecule. This is because we are interested in making α- 18 galactosyl carbohydrates that have uses in therapeutic processes, such as the α-gal epitope and 19 globosyl-Gb2/Gb3, although the transfer reaction resulted in the formation of a non-reducing 20 trisaccharide. The kinetic study suggests that the low reactivity of the glycosyl-azide intermediate 21 is involved in the limited transfer reaction in the azide-rescued reaction.…”