Characterization of novel α-galactosidase in glycohydrolase family 97 from Bacteroides thetaiotaomicron and its immobilization for industrial application.

Bacteroides thetaiotaomicron (B. thetaiotaomicron), which resides in the human intestinal tract, has a number of carbohydrate enzymes, including glycoside hydrolase (GH) family 97. Only a few GH 97 enzymes have been characterized to date. In this study, a novel α-galactosidase (Bt_3294) was cloned from B. thetaiotaomicron, expressed in Escherichia coli, and purified using affinity chromatography. This novel enzyme showed optimal activity at 60 °C and pH 7.0. Enzyme activity was reduced by 94.4% and 95.7% in the presence of 5 mM Ca2+ and Fe2+, respectively. It is interesting that Bt_3294 specifically hydrolyzed shorter α-galactosyl oligosaccharides, such as melibiose and raffinose. The D-values of Bt_3294 at 40 °C and 50 °C were about 107 and 6 min, respectively. After immobilization of Bt_3294, the D-values at 40 °C and 50 °C were about 37.6 and 29.7 times higher than those of the free enzyme, respectively. As a practical application, the immobilized Bt_3294 was used to hydrolyze raffinose family oligosaccharides (RFOs) in soy milk, decreasing the RFOs by 98.9%.

Development and Application of Cyclodextrin Hydrolyzing Mutant Enzyme Which Hydrolyzes ß- and γ-CD Selectively.

Cyclodextrins (CDs) are produced from starch by cyclodextrin glucanotransferase (CGTase), which has cyclization activity. Specifically, α-CD is an important biomolecule, as it is a molecular carrier and soluble dietary fiber used in the food industry. Upon inspection of the conserved regions of the glycoside hydrolase (GH) 13 family amylases, the amino acids K232 and H233 of CGTase were identified as playing an important role in enzyme reaction specificity. A novel CD hydrolyzing enzyme, cyclodextrin glycosyl transferase (CGTase)-alpha, was developed using site-directed mutagenesis at these positions. Action pattern analysis using various substrates revealed that CGTase-alpha was able to hydrolyze ß- and γ-CD, but not α-CD. This selective CD hydrolyzing property was employed to purify α-CD from a CD mixture solution. The α-CD that remained after treatment with CGTase-alpha and exotype glucoamylase was purified using hydrophobic interaction chromatography with 99% purity.

Enzymatic Synthesis of a Novel Kaempferol-3-O-ß-d-glucopyranosyl-(1→4)-O-α-d-glucopyranoside Using Cyclodextrin Glucanotransferase and Its Inhibitory Effects on Aldose Reductase, Inflammation, and Oxidative Stress.

Kaempferol-3-O-ß-d-glucopyranoside (astragalin, AS), a major flavonoid that exists in various plants, exerts antioxidant, antitumor, anti-human immunodeficiency virus (HIV), and anti-inflammatory effects. However, the low water solubility of AS limits its use. In this study, we used cyclodextrin glucanotransferase (CGTase) with maltose (G2) as a donor molecule to enzymatically modify AS to improve its water solubility and physiochemical properties. We isolated the glycosylated astragalin (G1-AS) and identified the structure of G1-AS as kaempferol-3-O-ß-d-glucopyranosyl-(1→4)-O-α-d-glucopyranoside, where one glucose residue was transferred to AS. G1-AS retained the antioxidative activity of the original AS compound; however, the solubility of G1-AS was 65-fold higher than that of AS. In addition, G1-AS showed enhanced anti-inflammatory effects and aldose reductase inhibitory activity compared to AS when applied to rat lenses.

Characterization of novel thermophilic alpha-glucosidase from Bifidobacterium longum.

In this study, the gene encoding α-glucosidase from Bifidobacterium longum subsp. longum JCM1217 (BLAG) was cloned and expressed in Escherichia coli. The amino acid sequence alignment demonstrated that BLAG belongs to glycoside hydrolase (GH) family 13. The optimal temperature for enzyme activity was 75°C; about 80% of the catalytic activity was lost at 50°C, which is very unusual for enzymes from the Bifidobacterium genus. In the presence of 5mM of Co2+ and Ca2+, enzyme activity was reduced to 47% and 48%, respectively. Furthermore, BLAG lost catalytic activity following the addition of 5mM of Fe2+ ion. The BLAG enzyme was able to hydrolyze α-1,2, α-1,3, α-1,4, and α-1,6 glycosidic O-linkages and liberated glucose from the non-reducing end of substrates. The kinetic study revealed that among the maltooligosaccharides, BLAG showed the highest kcat/Km value to maltotriose (G3), and had relatively low kcat/Km values on long-chain maltooligosaccharides. This is the first report describing the production of a thermophilic α-glucosidase from the Bifidobacterium genus.

Characterization and Application of BiLA, a Psychrophilic α-Amylase from Bifidobacterium longum.

In this study, a novel α-amylase was cloned from Bifidobacterium longum and named BiLA. The enzyme exhibited optimal activity at 20 °C and a pH value of 5.0. Kinetic analysis using various carbohydrate substrates revealed that BiLA had the highest k(cat/)K(m) value for amylose. Interestingly, analysis of the enzymatic reaction products demonstrated that BiLA specifically catalyzed the hydrolysis of oligosaccharides and starches up to G5 from the nonreducing ends. To determine whether BiLA can be used to generate slowly digestible starch (SDS), starch was treated with BiLA, and the kinetic parameters were analyzed using porcine pancreatic α-amylase (PPA) and amyloglucosidase (AMG). Compared to normal starch, BiLA-treated starch showed lower k(cat)/K(m) values with PPA and AMG, suggesting that BiLA is a potential candidate for the production of SDS.

Characterization of a Novel Maltose-Forming α-Amylase from Lactobacillus plantarum subsp. plantarum ST-III.

A novel maltose (G2)-forming α-amylase from Lactobacillus plantarum subsp. plantarum ST-III was expressed in Escherichia coli and characterized. Analysis of conserved amino acid sequence alignments showed that L. plantarum maltose-producing α-amylase (LpMA) belongs to glycoside hydrolase family 13. The recombinant enzyme (LpMA) was a novel G2-producing α-amylase. The properties of purified LpMA were investigated following enzyme purification. LpMA exhibited optimal activity at 30 °C and pH 3.0. It produced only G2 from the hydrolysis of various substrates, including maltotriose (G3), maltopentaose (G5), maltosyl ß-cyclodextrin (G2-ß-CD), amylose, amylopectin, and starch. However, LpMA was unable to hydrolyze cyclodextrins. Reaction pattern analysis using 4-nitrophenyl-α-d-maltopentaoside (pNPG5) demonstrated that LpMA hydrolyzed pNPG5 from the nonreducing end, indicating that LpMA is an exotype α-amylase. Kinetic analysis revealed that LpMA had the highest catalytic efficiency (kcat/Km ratio) toward G2-ß-CD. Compared with ß-amylase, a well-known G2-producing enzyme, LpMA produced G2 more efficiently from liquefied corn starch due to its ability to hydrolyze G3.