In vitro and in vivo studies have demonstrated the prebiotic potential of isomalto-oligosaccharides (IMO), comprising α-(1,6)-gluco-oligosaccharides and panose, which selectively stimulate the growth of probiotic bifidobacteria and lactobacilli. The protein machinery conferring the utilization of IMO by probiotics, however, remains vaguely described. We have used genomic, transcriptomic, enzymatic, and biophysical analyses to explore IMO utilization routes in probiotic lactobacilli and bifidobacteria as represented by Lactobacillus acidophilus NCFM and Bifidobacterium animalis subsp. lactis Bl-04, respectively. Utilization of IMO and malto-oligosaccharide (α-(1,4)-glucosides) appears to be linked both at the genetic and transcriptomic level in the acidophilus group lactobacilli as suggested by reverse transcriptase PCR (RT-PCR) and gene landscape analysis. Canonical intracellular GH13_31 glucan 1,6-α-glucosidases active on IMO longer than isomaltose occur widely in acidophilus group lactobacilli. Interestingly, however, isomaltose, isomaltulose and panose seem to be internalized through a phosphoenoyl pyruvate transferase system (PTS) and subsequently hydrolyzed by a GH4 6-phosphate-α-glucosidases based on whole genome microarray analysis. This sub-specificity within GH4 seems to be unique for lactobacilli mainly from the gut niche, as the sequences from this group segregate from characterized GH4 maltose-6-phosphate-α-glucosidases in other organisms. By comparison, IMO utilization in bifidobacteria is linked to soybean oligosaccharide utilization loci harboring GH36 α-galactosidases, GH13_31 oligo 1,6-α-glucosidases and a dual specificity ATP-binding cassette (ABC) transport system active in the uptake of both classes of α-(1,6)-glycosides. These data bring novel insight to advance our understanding of the basis of selectivity of IMO metabolism by important gut microbiome members.
Marie S. Møller, Darrell Cockburn, Jonas W. Nielsen, Johanne M. Jensen, Malene B. Vester-Christensen, Morten M. Nielsen, Joakim M. Andersen, Casper Wilkens, Julie Rannes, Per Hägglund, Anette Henriksen, Maher Abou Hachem, Martin Willemoës, Birte Svensson
Certain enzymes interact with polysaccharides at surface binding sites (SBSs) situated outside of their active sites. SBSs are not easily identified and their function has been discerned in relatively few cases. Starch degradation is a concerted action involving GH13 hydrolases. New insight into barley seed α-amylase 1 (AMY1) and limit dextrinase (LD) includes i) kinetics of bi-exponential amylopectin hydrolysis by AMY1, one reaction having low Km (8 µg/mL) and high kcat (57 s－1) and the other high Km (97 µg/mL) and low kcat (23 s－1). β-Cyclodextrin (β-CD) inhibits the first reaction by binding to an SBS (SBS2) on domain C with Kd = 70 µM, which for the SBS2 Y380A mutant increases to 1.4 mM. SBS2 thus has a role in the fast, high-affinity component of amylopectin degradation. ii) The N-terminal domain of LD, the debranching enzyme in germinating seeds, shows distant structural similarity with domains including CBM21 present in other proteins and involved in various molecular interactions, but no binding site identity. LD is controlled by barley limit dextrinase inhibitor (LDI) which belongs to the cereal-type inhibitor family and forms a tight 1:1 complex with LD. iii) LDI in turn is regulated by disulfide reduction mediated by the barley thioredoxin h (trxh) NADPH-dependent thioredoxin reductase (NTR) system. Based on the progress monitored by released free thiol groups from LDI and its failure to inhibit LD as elicited by trxh, the LDI inactivation is proposed to stem from loss of structural integrity due to reduction of all four disulfides.
Fungal cellobiose dehydrogenases (CDHs) are divided on the basis of amino acid sequence into class I (from basidiomycetes) and class II (from ascomycetes), which show quite different pH-dependence of the activity. Here, we mutated glutamine 734 (Q734) in the flavin domain of class I CDH from the basidiomycete Phanerochaetechrysosporium to the corresponding amino acid in class II CDHs (serine or threonine), and compared the kinetics of the mutant enzymes (Q734S and Q734T) and wild-type enzyme (WT). The two mutant enzymes showed almost identical absorption spectra, although that of WT was slightly different. When the steady-state kinetic parameters were compared at pH 4.0 and 7.0, WT showed the highest activity at both pH values. However, kcat/Km for Q734S was similar at both pHs, whereas kcat/Km for Q734T was 3.5 times higher at pH 4.0 than that at pH 7.0. As for the pH-dependence of the specific activity, Q734S did not have an apparent optimum pH in the pH range tested, whereas Q734T showed an acidic optimum pH profile compared with WT. These differences can be explained in terms of the effect of the side chain of the amino acid residue at position 734 on the reactivity of the flavin cofactor.
Deproteinized wheat starch (Act-WS) was prepared by twice digesting with actinase large granular wheat starch classified from wheat starch that had been extracted with 70% ethanol. A small amount of preoteinaceous component still remained in Act-WS. The effect of lysine (Lys), monosodium glutamate (GluNa), glycine (Gly) and alanine (Ala) on the thermal behavior of Act-WS, and the dispersion state of the swollen starch granules in the Act-WS paste was investigated. Consequently, Gly and Ala had little effect on the gelatinization temperature and viscosity, whereas GluNa and Lys markedly elevated these parameters. This increased viscosity with Lys contrasted the results for potato starch. Such charged amino acids as GluNa and Lys reduced the swelling and solubility, while Lys elevated the turbidity, median diameter and ratio of aggregates of the swollen granules. The increased viscosity was thus caused by forming aggregates with Lys, presumably through interacting with a starch-granule-associated protein.
Four types of corn starch (waxy corn starch (WC), normal corn starch (NC), high-amylose corn starch class 5 (HAC class 5) and high-amylose corn starch class 7 (HAC class 7)) were hydrolyzed with 1.5% hydrochloric acid and the resistant starch (RS) content was measured. The acid-hydrolyzed HAC class 5 and class 7 show significantly higher RS content. The change in RS content, X-ray crystallinity, molecular size distribution, thermal property and appearance of HAC class 7 after up to 100 h acid hydrolysis were analyzed. The RS content increased to 69.3 from 40.5% at 16 h and then decreased gradually, while crystallinity continued to increase during acid hydrolysis. The fractionation profiles indicated that with the decrease of the amylose fraction correlating well with the increase in RS content. Granules hydrolyzed for 24 h retained their natural shape, whereas after 100 h hydrolysis granules were damaged. The enzyme-digested residues of native and acid-hydrolyzed HAC class 7 were recovered. The crystalline regions of HAC class 7 acid-hydrolyzed for 24 h were only slightly digested by enzymatic treatment, whereas native and HAC class 7 acid-hydrolyzed for 100 h were extensively digested. Thermal analysis showed the transition peak in high temperature region increased with acid hydrolysis and this peak was also shown in enzyme-digested residues. Acid hydrolysis may influence RS content by two mechanisms: (1) moderate acid hydrolysis increases the crystalline regions and thus resistance to enzymatic digestion of the hydrolyzed granules and (2) excess acid hydrolysis damages granular structure and decreases resistance to enzymatic digestion.
Amylose can be synthesized with phosphorylase (EC 126.96.36.199) by using malto-oligosaccharides as primers. In this context, the glucose-1-phosphate (G-1-P) units are consumed through the polymerization process. In this study, we investigated the use of amylopectin as a primer for the synthesis of a novel starch. The obtained starch was characterized by dynamic light scattering, small-angle X-ray scattering (SAXS) and X-ray diffraction. The SAXS results were well represented by the star-polymer model scattering function. According to these results, we think that the amylopectin chain elongation was implied, and the transformation of crysralline type of amylopectin was observed by X-ray diffraction.
We developed an enzymatic colorimetric method for the quantification of α-D-mannose 1-phosphate by adding phosphomannomutase, mannose 6-phosphate isomerase and glucose 6-phosphate isomerase to a conventional glucose 6-phosphate assay using glucose 6-phosphate dehydrogenase. In this method, α-D-mannose 1-phosphate is converted into D-glucose 6-phosphate via D-mannose 6-phosphate and D-fructose 6-phosphate and the resultant D-glucose 6-phosphate is ultimately converted into 6-phosphogluconolactone under concomitant reduction of thio-NAD+ to thio-NADH, which can be quantified by its wavelength of 400 nm. This method is not altered by the presence of D-mannose, D-mannosamine, N-acetyl-D-mannosamine, L-mannose, β-1,4-mannobiose, α-1,2-mannobiose, methyl α-D-mannoside or dimethyl sulfoxide and it would be useful in studies involving enzymes such as phosphorylases belonging to glycoside hydrolase family 130, which release α-D-mannose 1-phosphate as the reaction product.
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