UDP-N-Acetylglucosamine 4-epimerase (EC 5.1.3.7) was purified from a cell extract of Bacillus subtilis by protamine sulfate treatment, ammonium sulfate fractionation, and column chromatographies on DEAE-Sephadex, hydroxylapatite, and Sephadex G-150. The purified enzyme was homogeneous on disc gel electrophoresis. The molecular weight of the enzyme was estimated to be about 62, 000 by gel filtration. The enzyme had an optimal pH in the range of 7.0 to 9.0, and was active at 30-35°C in the absence of added NAD and more active at 40-45°C in the presence of NAD. The enzyme activity was highly stimulated by small amounts of NAD. The estimated Km values were 4.4mM for UDP-N-acetylglucosamine and 0.13mM for NAD. No UDP-glucose 4-epimerase activity was found in the purified enzyme. The enzyme was inhibited by high concentrations of glucosamine and inhibition by glucosamine was competitive with UDP-N-acetylglucosamine. The inhibition constant was 37.0mM. N-Acetylglucosamine did not inhibit the enzyme.
Infant gut-associated bifidobacteria possess species-specific enzymatic sets to assimilate human milk oligosaccharides, and lacto-N-biosidase (LNBase) is a key enzyme that degrades lacto-N-tetraose (Galβ1–3GlcNAcβ1–3Galβ1–4Glc), the main component of human milk oligosaccharides, to lacto-N-biose I (Galβ1–3GlcNAc) and lactose. We have previously identified LNBase activity in Bifidobacterium bifidum and some strains of Bifidobacterium longum subsp. longum (B. longum). Subsequently, we isolated a glycoside hydrolase family 20 (GH20) LNBase from B. bifidum; however, the genome of the LNBase+ strain of B. longum contains no GH20 LNBase homolog. Here, we reveal that locus tags BLLJ_1505 and BLLJ_1506 constitute LNBase from B. longum JCM1217. The gene products, designated LnbX and LnbY, respectively, showed no sequence similarity to previously characterized proteins. The purified enzyme, which consisted of LnbX only, hydrolyzed via a retaining mechanism the GlcNAcβ1–3Gal linkage in lacto-N-tetraose, lacto-N-fucopentaose I (Fucα1–2Galβ1–3GlcNAcβ1–3Galβ1–4Glc), and sialyllacto-N-tetraose a (Neu5Acα2–3Galβ1–3GlcNAcβ1–3Galβ1–4Gal); the latter two are not hydrolyzed by GH20 LNBase. Among the chromogenic substrates examined, the enzyme acted on p-nitrophenyl (pNP)-β-lacto-N-bioside I (Galβ1–3GlcNAcβ-pNP) and GalNAcβ1–3GlcNAcβ-pNP. GalNAcβ1–3GlcNAcβ linkage has been found in O-mannosyl glycans of α-dystroglycan. Therefore, the enzyme may serve as a new tool for examining glycan structures. In vitro refolding experiments revealed that LnbY and metal ions (Ca2+ and Mg2+) are required for proper folding of LnbX. The LnbX and LnbY homologs have been found only in B. bifidum, B. longum, and a few gut microbes, suggesting that the proteins have evolved in specialized niches.Background: Phenotypically lacto-N-biosidase-positive Bifidobacterium longum JCM1217 does not possess a gene homologous to previously identified lacto-N-biosidase.Results: Hypothetical proteins BLLJ_1505 and BLLJ_1506 encode lacto-N-biosidase and its designated chaperone, respectively.Conclusion: The enzyme showed unique and unexpected substrate specificity.Significance: The enzyme is important for understanding how B. longum consumes human milk oligosaccharides and also may serve as a new tool in glycobiology. Infant gut-associated bifidobacteria possess species-specific enzymatic sets to assimilate human milk oligosaccharides, and lacto-N-biosidase (LNBase) is a key enzyme that degrades lacto-N-tetraose (Galβ1–3GlcNAcβ1–3Galβ1–4Glc), the main component of human milk oligosaccharides, to lacto-N-biose I (Galβ1–3GlcNAc) and lactose. We have previously identified LNBase activity in Bifidobacterium bifidum and some strains of Bifidobacterium longum subsp. longum (B. longum). Subsequently, we isolated a glycoside hydrolase family 20 (GH20) LNBase from B. bifidum; however, the genome of the LNBase+ strain of B. longum contains no GH20 LNBase homolog. Here, we reveal that locus tags BLLJ_1505 and BLLJ_1506 constitute LNBase from B. longum JCM1217. The gene products, designated LnbX and LnbY, respectively, showed no sequence similarity to previously characterized proteins. The purified enzyme, which consisted of LnbX only, hydrolyzed via a retaining mechanism the GlcNAcβ1–3Gal linkage in lacto-N-tetraose, lacto-N-fucopentaose I (Fucα1–2Galβ1–3GlcNAcβ1–3Galβ1–4Glc), and sialyllacto-N-tetraose a (Neu5Acα2–3Galβ1–3GlcNAcβ1–3Galβ1–4Gal); the latter two are not hydrolyzed by GH20 LNBase. Among the chromogenic substrates examined, the enzyme acted on p-nitrophenyl (pNP)-β-lacto-N-bioside I (Galβ1–3GlcNAcβ-pNP) and GalNAcβ1–3GlcNAcβ-pNP. GalNAcβ1–3GlcNAcβ linkage has been found in O-mannosyl glycans of α-dystroglycan. Therefore, the enzyme may serve as a new tool for examining glycan structures. In vitro refolding experiments revealed that LnbY and metal ions (Ca2+ and Mg2+) are required for proper folding of LnbX. The LnbX and LnbY homologs have been found only in B. bifidum, B. longum, and a few gut microbes, suggesting that the proteins have evolved in specialized niches. Background: Phenotypically lacto-N-biosidase-positive Bifidobacterium longum JCM1217 does not possess a gene homologous to previously identified lacto-N-biosidase. Results: Hypothetical proteins BLLJ_1505 and BLLJ_1506 encode lacto-N-biosidase and its designated chaperone, respectively. Conclusion: The enzyme showed unique and unexpected substrate specificity. Significance: The enzyme is important for understanding how B. longum consumes human milk oligosaccharides and also may serve as a new tool in glycobiology.
A novel alpha-galactosidase gene (aga2) was cloned from Bifidobacterium breve 203. It contained an ORF of 2226-bp nucleotides encoding 741 amino acids with a calculated molecular mass of 81.5 kDa. The recombinant enzyme Aga2 was heterogeneously expressed, purified and characterized. Regarding substrate specificity for hydrolysis, Aga2 was highly active towards p-nitrophenyl-alpha-d-galactopyranoside (pNPG). The Km value for pNPG was estimated to be 0.27 mM and for melibiose it was estimated to be 4.3 mM. Aga2 was capable of catalyzing transglycosylation as well as hydrolysis. The enzyme synthesized a trisaccharide (Gal-alpha-1, 4-Gal-alpha-1, 6-Glc) using melibiose as a substrate. It was a new oligosaccharide produced by glycosidase and contained Gal-alpha-1,4 linkage, a novel galactosidic link formed by microbial alpha-galactosidase. In the presence of pNPG as a donor, Aga2 was able to catalyze glycosyl transfer to various acceptors including monosaccharides, disaccharides and sugar alcohols.
Two types of α-L-fucosidase (F-I and F-II), that differ in substrate specificity, were produced in the culture fluid by Bacillus circulans isolated from soil when the bacterium was cultivated on medium containing porcine gastric mucin. F-I was able to cleave the α-(1→2), α-(1→3) and α-(1→4)-L-fucosidic linkages in various oligosaccharides and glyco proteins, but not p-nitrophenyl α-L-fucoside, as previously reported [Y. Tsuji et al. (1990) J. Biochem. 107, 324-330]. F-II was purified from the culture fluid obtained with glucose medium by ammonium sulfate fractionation and various subsequent column chromatogra phies. The purified enzyme was found to be homogeneous on PAGE and its molecular weight was estimated to be approximately 250,000. The maximal activity was observed between pH 6.0 to 7.0, the stable pH range being 6.0 to 8.5. The enzyme specifically cleaved a L-fucosidic bonds in low molecular weight substrates. The enzyme cleaved not only p-nitrophenyl α-L-fucOSide, but also 2′-fucosyllactose and 3-fucosyllactose. The enzyme was also able to act on the α-(1→6)-L-fucosidase linkages to N-acetyiglucosamine in 6-O-α L-fucopyranosyl-N-acetylglucosamlne, and bi- and tetrα-antennary oligosaceharides derived from porcine pancreatic lipase, which were not hydrolyzed by F-I.
An increasing number of projects in neuroscience require statistical analysis of high-dimensional data, as, for instance, in the prediction of behavior from neural firing or in the operation of artificial devices from brain recordings in brain-machine interfaces. Although prevalent, classical linear analysis techniques are often numerically fragile in high dimensions due to irrelevant, redundant, and noisy information. We developed a robust Bayesian linear regression algorithm that automatically detects relevant features and excludes irrelevant ones, all in a computationally efficient manner. In comparison with standard linear methods, the new Bayesian method regularizes against overfitting, is computationally efficient (unlike previously proposed variational linear regression methods, is suitable for data sets with large numbers of samples and a very high number of input dimensions) and is easy to use, thus demonstrating its potential as a drop-in replacement for other linear regression techniques. We evaluate our technique on synthetic data sets and on several neurophysiological data sets. For these neurophysiological data sets we address the question of whether EMG data collected from arm movements of monkeys can be faithfully reconstructed from neural activity in motor cortices. Results demonstrate the success of our newly developed method, in comparison with other approaches in the literature, and, from the neurophysiological point of view, confirms recent findings on the organization of the motor cortex. Finally, an incremental, real-time version of our algorithm demonstrates the suitability of our approach for real-time interfaces between brains and machines.
