Purification, characterization, and substrate specificity of a glucoamylase with steroidal saponin-rhamnosidase activity from Curvularia lunata

Abstract

It has been previously reported that a glucoamylase from Curvularia lunata is able to hydrolyze the terminal 1,2-linked rhamnosyl residues of sugar chains at C-3 position of steroidal saponins. In this work, the enzyme was isolated and identified after isolation and purification by column chromatography including gel filtration and ion-exchange chromatography. Analysis of protein fragments by MALDI-TOF/TOF proteomics Analyzer indicated the enzyme to be 1,4-alpha-D-glucan glucohydrolase EC 3.2.1.3, GA and had considerable homology with the glucoamylase from Aspergillus oryzae. We first found that the glucoamylase was produced from C. lunata and was able to hydrolyze the terminal rhamnosyl of steroidal saponins. The enzyme had the general character of glucoamylase, which hydrolyze starch. It had a molecular mass of 66 kDa and was optimally active at 50 degrees C, pH 4, and specific activity of 12.34 U mg of total protein(-1) under the conditions, using diosgenin-3-O-alpha-L-rhamnopyranosyl(1-->4)-[alpha-L-rhamnopyranosyl (1-->2)]-beta-D-glucopyranoside (compound II) as the substrate. Furthermore, four kinds of commercial glucoamylases from Aspergillus niger were investigated in this work, and they had the similar activity in hydrolyzing terminal rhamnosyl residues of steroidal saponin.

Production of diosgenin from yellow ginger (Dioscorea zingiberensis C. H. Wright) saponins by commercial cellulase

“…A glucoamylase (from Curvularia lunata) which is capable of hydrolyzing the terminal 1, 2-linked rhamnosyl residues of sugar chains at C-3 in steroidal saponins had a molecular mass of 66 kDa (Feng et al 2007a). …”

“…3.2.1.X), may display new activities in producing diosgenin. To date, very few reports are available in the open literature focusing on the enzymatic hydrolysis of steroidal saponins to less glucosyl saponins (such as diosgenin) using enzymes with non-specific action (Feng et al 2007a). On the other hand, most previous reports investigate the biotransformation of a single steroidal saponin (Feng et al 2005;He et al 2006a;He et al 2006b), thus, the conditions of experiments, such as temperature, pH, as well as the ability to hydrolyze are to some extent different from those in practical use when an extract of saponin is used as the substrate.…”

Evaluation and characterization of new α-L-rhamnosidase-producing yeast strains

“…The purified enzyme having the optimum pH in the neutral pH range is more suitable for the enhancement of wine aroma (Yadav et al, 2012). The α-L-rhamnosidases reported from Curvularia lunata (Feng et al, 2007), Aspergillus niger (Puri and Kalra, 2005) and Aspergillus kawachii (Koseki et al, 2008) have same optimum pH i.e., 4. Yanai and Sato (2000) reported that the enzyme from yeast Pichia angusta showed an optimum activity at pH 6.…”

“…The optimum temperature obtained was similar to the α-L-rhamnosidases from Bacillus sp. GL1 (Hashimoto et al, 1999), Lactobacillus plantarum NCC 245 (Avila et al, 2009), Aspergillus flavus (Scaroni et al, 2002), Curvularia lunata (Feng et al, 2007), Aspergillus niger (Puri and Kalra, 2005) and Aspergillus kawachii (Koseki et al, 2008). Enzymes from Pichia angusta (Yanai and Sato, 2000), Aspergillus kawachii, Penicillium aureatiogriseum and Trichoderma longibrachiatu (Scaroni et al, 2002) were optimally active at 40°C and 60°C.…”

Aspergillus niger DLFCC-90 Rhamnoside Hydrolase, a New Type of Flavonoid Glycoside Hydrolase

“…The novel enzyme was classified in glycoside-hydrolase (GH) family 13. ␣ -L-Rhamnosidase (EC 3.2.1.40) cleaves terminal ␣-L-rhamnose from a large number of natural products, such as flavonoids, saponins, and many other natural glycosides, and this enzyme has been used in the food industry for eliminating the naringin bitterness of citrus juices (4,20,25,26), removing hesperidin crystals from orange juices and releasing 7-O-␤-D-glucoside-hesperetin, an important precursor in sweetener production (17,24), and enhancing wine aromas (7,6,22). In addition, the deglycosylation of flavonoids in vitro by ␣-L-rhamnosidase has ushered in a new era of drug development (5,8,19,27).…”