BaAG2 efficiently hydrolyzed maltose and maltose-like sugars, but could not hydrolyze isomaltose, palatinose nor -MG that are specific substrates for yeast isomaltases (Figure 5, Table 1). Yamamoto K., Miyake H., Kusunoki M., Osaki S. Crystal structures of isomaltase from, Yamamoto K., Nakayama A., Yamamoto Y., Tabata S. Val216 decides the substrate specificity of -glucosidase in, Yamamoto K., Miyake H., Kusunoki M., Osaki S. Steric hindrance by 2 amino acid residues determines the substrate specificity of isomaltase from. However, transport and intracellular hydrolysis of -glucosidic sugars have also been investigated in Ogataea polymorpha [25] and Schizosaccharomyces pombe [33,49]. We also acknowledge previous National Science Foundation support under grant numbers 1246120, 1525057, and 1413739. The hormone secretin also causes bicarbonate to be released into the small intestine from the pancreas to neutralize the potentially harmful acid coming from the stomach. The melting temperature (Tm) values calculated from the DSF data are presented in Figure 6. [24] This mutation has allowed almost half of the world's population to metabolize lactose without symptoms. The effect of temperature on activity (left panel) and stability (right panel) of BaAG2. The scale bar shows the number of base substitutions per site. Notably, a new transglycosylation product, isomaltose, emerged. Most of the digestive enzymes in the small intestine are secreted by the pancreas and enter the small intestine via the pancreatic duct. Table 1 shows that natural sugars, maltose and sucrose (-d-Glc-(12)--d-Fru) were hydrolyzed by BaAG2 with the highest catalytic efficiency (kcat/Km). In the current work, we routinely used the buffer with pH of 6.5 to characterize substrate specificity, kinetics and other properties of the enzyme. Digestive System Test Review Flashcards | Quizlet WebWhere is maltase produced? Regulation of glycogen metabolism in yeast and bacteria. We also discovered a very strong inhibition of BaAG2 by Tris with the Ki of 70.5 M (Table 2). Mcilvaine T.C. Figure 4 shows that at temperature over 50 C, the activity of the enzyme rapidly declined. ; Writingreview and editing, T.A., T.V., K.E. ; Writingoriginal draft preparation, T.A. and T.V. Maltose - an overview | ScienceDirect Topics [20], Mature human lactase consists of a single 160-kDa polypeptide chain that localizes to the brush border membrane of intestinal epithelial cells. an extracellular -glucosidase from L. starkeyi was biochemically characterized [51]. When testing the effect of different buffers during the experiments, we noticed that the activity of BaAG2 was lost in Tris-HCl buffer. Viigand K., Visnapuu T., Mardo K., Aasamets A., Alame T. Maltase protein of, Fernndez-Arrojo L., Marn D., Gmez De Segura A., Linde D., Alcalde M., Gutirrez-Alonso P., Ghazi I., Plou F.J.J., Fernndez-Lobato M., Ballesteros A. small intestine Students also viewed. SD, standard deviation. Degradation of glucose polymers, i.e., amylopectin-free amylose from potato, amylopectin from potato, glycogen from oysters and dextrans of Mw 20 kDa and 110 kDa, was assayed in K-phosphate buffer (100 mM, pH 6.5) containing 0.2 g/L Na-azide. Curiel J.A., de Las Rivas B., Mancheo J.M., Muoz R. The pURI family of expression vectors: A versatile set of ligation independent cloning plasmids for producing recombinant His-fusion proteins. [26] The sequence is highly conserved in mammals, suggesting that critical cis-transcriptional regulators are located nearby. The E. coli lysate exhibited catalytic activity of 1 mM pNPG hydrolysis at 30 C (71 U/mg), which after purification of the protein increased 5.8 times, reaching 411.5 U/mg. Preprolactase, the primary translation product, has a single polypeptide primary structure consisting of 1927 amino acids. As shown in Figure 4 (left panel), catalytic activity of BaAG2 was the highest at 45 C being 24% higher than activity measured at 30 Cthe temperature we routinely used for enzyme activity assay. 100 mM K-phosphate buffer (pH 6.5) was used and reactions were conducted at 30 C if not stated otherwise. We have earlier shown that O. polymorpha maltase-isomaltase MAL1 could use maltotriose and maltotetraose as a substrate, while malto-oligosaccharides (MOS) of higher degree of polymerization (DP) were not hydrolyzed [16]. and T.A. The genes potentially encoding -glucosidases in the genomes of non-conventional yeasts were analysed in Viigand et al. Interestingly, this enzyme did not hydrolyze sucrose and had a quite low activity on pNPG. Other carbohydrates pass undigested into the large intestine, where they are digested by intestinal bacteria. International Journal of Molecular Sciences, http://creativecommons.org/licenses/by/4.0/, https://www.mdpi.com/1422-0067/21/1/297/s1, http://www.wi.knaw.nl/Collections/DefaultInfo.aspx?Page=Home. Medium for transformant selection contained ampicillin (Amp, 150 mg/L) for plasmid preservation. The GH13 enzymes use an Asp (D) as a nucleophile and a Glu (E) as an acid-base catalyst [31]. Proteins were quantified by measuring the absorbance at 280 nm. Needleman R.B., Marmur J., Federoff H.J., Eccleshall T.R., Buchferer B. Purification and characterization of an -glucosidase from, Krakenate R.P., Glemzha A.A. Wilson W.A., Roach P.J., Montero M., Baroja-Fernndez E., Muoz F.J., Eydallin G., Viale A.M., Pozueta-Romero J. West Palm Beach, FL33411 contact this location, Window Classics-Tampa Shen X.-X., Zhou X., Kominek J., Kurtzman C.P., Hittinger C.T., Rokas A. Reconstructing the backbone of the Saccharomycotina yeast phylogeny using genome-scale data. Beta [12], the -glucosidase genes resided in maltose utilization (MAL) clusters, whereas no MAL clusters were detected in B. adeninivorans. Maltase | Exercise.com The pH optimum of BaAG2 was in moderately acidic regionfrom 5.5 to 6.5 (Figure S2). In the phylogram of -glucosidases from non-conventional yeasts, all eight Lipomyces enzymes clustered with those of B. adeninivorans [12]. Saitou N., Nei M. The neighbor-joining method: A new method for reconstructing phylogenetic trees. Universal substrates are indicated by green, maltose and maltose-like sugars by yellow, and isomaltose and isomaltose-like sugars by blue bars. 2781 Vista Pkwy N Ste K-8 Unfortunately, the sequence data of this protein is not available. Presence of acarbose (the strongest inhibitor of BaAG2) increased the Tm of BaAG2 by 11.4 C. Hydrolysis of pNPG was assayed as in [16,40] according to the release of p-nitrophenol. From amylopectin (5 g/L), 0.1 g/L of glucose was released by 24 h, and 0.3 g/L by 72 h of the reaction. The activity measured after incubation at 10 C was taken as 100%. Importantly, at high maltose concentrations, BaAG2 exhibited transglycosylating ability producing potentially prebiotic di- and trisaccharides: isomaltose, panose and maltotriose. [23], Some population segments exhibit lactase persistence resulting from a mutation that is postulated to have occurred 5,00010,000 years ago, coinciding with the rise of cattle domestication. 20 g/mL of the enzyme was reacted with 250 mM or 500 mM of maltose. At pH 4.5 and 4.4, the respective values were 93 and 15%. Non-Conventional Yeasts: From Basic Research to Application. SDs of two to three replicates are shown by error bars. Which of the following reaction is catalysed by WebLactase (EC 3.2.1.108) is an enzyme produced by many organisms. S. cerevisiae maltase MAL62 assayed for comparison had only minimal ability towards these polymers and its transglycosylating activity was much lower. Brush border enzymes take over from there. [26], The lactase promoter is 150 base pairs long and is located upstream of the site of transcription initiation. See Materials and Methods, paragraphs 4.3 and 4.6 for details. FOIA Fragments of sequence alignment of six maltases. [26] Cdx-2, HNF-1, and GATA have been identified as transcription factors. Ojima T., Saburi W., Yamamoto T., Kudo T. Characterization of, Hasegawa S., Takizawa M., Suyama H., Shintani T., Gomi K. Characterization and expression analysis of a maltose-utilizing (. Aside from these two GH13 proteins, three putative extracellular -glucosidases of GH31 family were detected in B. adeninivorans genome (see Table S1 of Supplementary Materials). Genome of an early-diverged yeast Blastobotrys (Arxula) adeninivorans (Ba) encodes 88 glycoside hydrolases (GHs) including two -glucosidases of GH13 family. Location of Val216 in the structure is marked with a red circle. Notably, in addition to maltotriose and panose, isomaltose was also detected among the transglycosylation products produced from maltose by BaAG2 (Figure 8, Table S3). -Glucosidases have been popular objects to study protein evolution and phylogenesis [13,14,15,16], but they also have a biotechnological value. The activities (moles of glucose released per minute per mg of protein; U/mg) were calculated from initial velocities of the reaction. [25] Both mutations, CT at position -13910 and G A at position -22018, have been independently linked to lactase persistence. Protein sequences were aligned using Clustal Omega [72] to calculate identity values of the proteins. One of those, the rna_ARAD1D20130g-encoded protein (BaAG2; 581 aa) was overexpressed in Escherichia coli, purified and characterized. At desired time points (2 h, 24 h, 74/96 h) aliquots were withdrawn and heated to stop the reaction. To assay the hydrolysis of malto-oligosaccharides (DP 47), panose and melezitose by BaAG2 and ScMAL62, 50 mM of the sugar was reacted with 2.6 g/mL of the enzyme in K-phosphate buffer (100 mM, pH 6.5) containing 0.1 g/L of Na-azide and samples were withdrawn at fixed time points. HPLC analysis was performed similarly as in [65]. We assume that the ability to hydrolyze MOS and to cleave polymeric -glucans, at least to some extent, may be characteristic to maltases of early-diverged yeasts. Additionally, BaAG2 had a high affinity and activity towards a synthetic substrate pNPGthe Km for pNPG was 0.76 mM and the Vmax over 750 U/mg. From this aspect, BaAG2 differs from the maltases of S. cerevisiae [23,24] and Candida albicans [41], and also from the maltase-isomaltase of O. polymorpha [16,40,42], for which affinity for sucrose is about twice higher than for maltose. On the phylogram (Figure 9), B. adeninivorans clusters with Lipomyces starkeyi. The genome of B. adeninivorans was sequenced in 2014 [2]. Pancreatic lipase works with the help of the salts from bile secreted by the liver and the gallbladder. The Tm of BaAG2 without a ligand was similar to that of maltase-isomaltase MAL1 of O. polymorpha51 C [16]. 24850 Old 41 Ste 7 The concentration of BaAG2 was used with 2 M and for ligands: 100 mM fructose, 50 mM palatinose, 50 mM glucose, 5 mM Tris and 5 mM acarbose. BaAG2 was competitively inhibited also by isomaltose-like sugars and a hydrolysis productglucose. [17] The 3-hydroxy group is involved in initial binding to the substrate while the 2- group is not necessary for recognition but needed in subsequent steps. Our study showed that BaAG2 is a maltase. [15] While the details of the mechanism are uncertain, the stereochemical retention is achieved off a double displacement reaction. Janeek ., Gabriko M. Remarkable evolutionary relatedness among the enzymes and proteins from the -amylase family. Further purification steps were conducted as described in [63]. According to literature data, thermolability has been reported for some other yeast -glucosidases. 22.10C: Digestive Processes of the Small Intestine is shared under a CC BY-SA license and was authored, remixed, and/or curated by LibreTexts. This work was funded by the Estonian Research Council (grant number PUT1050) to T.A. Purified proteins were maintained in 100 mM K-phosphate buffer (pH 6.5) at 4 C. In the genomes of most yeasts addressed in Viigand et al. In current work, one of them, BaAG2, was produced in E. coli and characterized in detail. Atypically for yeast maltases, a low but clearly recordable exo-hydrolytic activity on amylose, amylopectin and glycogen was detected. Samples were withdrawn at fixed intervals, heated at 95 C to stop the reaction and analysed on TLC and by HPLC. Highlights: catalytic nucleophile (turquoise), acid-base catalyst (green), a transition state stabilizer (yellow) and a residue crucial for substrate specificity (red). Thermostability of the enzyme was not highafter keeping the enzyme at 45 C for 30 min, the enzymes activity dropped to 46% from the initial. The genome of B. adeninivorans encodes two putative -glucosidases. BaAG2, Blastobotrys adeninivorans AG2 (580 aa); SpMal1, Schizosaccharomyces pombe Mal1 (579 aa, {"type":"entrez-protein","attrs":{"text":"NP_595063.1","term_id":"19111855","term_text":"NP_595063.1"}}NP_595063.1) [33]; ScMAL62, Saccharomyces cerevisiae MAL62 (584 aa, {"type":"entrez-protein","attrs":{"text":"P07265","term_id":"126716","term_text":"P07265"}}P07265) [23]; GsAG, Geobacillus stearothermophilus exo--1,4-glucosidase (555 aa, {"type":"entrez-protein","attrs":{"text":"BAA12704.1","term_id":"1321625","term_text":"BAA12704.1"}}BAA12704.1) [34]; HaAG, Halomonas sp. [18] The signal sequence is cleaved in the endoplasmic reticulum, and the resulting 215-kDa pro-LPH is sent to the Golgi apparatus, where it is heavily glycosylated and proteolytically processed to its mature form. Department of Genetics, Institute of Molecular and Cell Biology, University of Tartu, Riia 23, 51010 Tartu, Estonia; Received 2019 Nov 28; Accepted 2019 Dec 30. The reaction samples were analyzed using TLC. Thin layer chromatography (TLC) analysis showed that the glycosidic bond of turanose moiety of melezitose was hydrolyzed first, yielding sucrose and glucose as products (Figure S3). We showed that maltose, other maltose-like substrates (maltulose, turanose, maltotriose, melezitose, malto-oligosaccharides of DP 47) and sucrose were hydrolyzed by BaAG2, whereas isomaltose and isomaltose-like substrates (palatinose, -methylglucoside) were not, confirming that BaAG2 is a maltase. The Saccharomycotina clades according to [50] are designated by background coloring. The cloning procedure was conducted with recombinant Pfu polymerase (Thermo Fisher Scientific, Waltham, MA, USA). ; funding acquisition, T.A and T.V. We would like to emphasize that the activity of BaAG2 (Table 1) was higher compared to some of other studied maltases of yeasts and filamentous fungi. Initial velocity data were plotted against the temperature to reveal the temperature optimum. The theoretical Mw value of 67,901 Da for the kcat calculation was computed in ExPASy Proteomics Server (http://expasy.org). Some carbohydrates, such as cellulose, are not digested at all, despite being made of multiple glucose units. The temperature optimum for BaAG2 was between 4050 C, with maximum activity (530 U/mg) achieved at 45 C. Not typical for yeast maltases, BaAG2 had exo-hydrolytic activity on amylose, amylopectin and glycogen. 22.10C: Digestive Processes of the Small Intestine contact this location, Window Classics-Sarasota Specifically, it catalyzes the hydrolysis of the -1,4-glucosidic linkages Naumov G.I., Naumova E.S., Michels C.A. Compared to Br. As BaAG2 is an intracellular enzyme, and this yeast possesses a secreted glucoamylase [9], the maltase BaAG2 has most probably no role in starch degradation. Maltase is Chapter 17 Example We confirmed that BaAG2 is a maltase with a considerable transglycosylating activity. Action: Starch to a disaccharide. National Library of Medicine and transmitted securely. X-ray structures along the reaction pathway of cyclodextrin glycosyltransferase elucidate catalysis in the -amylase family. At pH 7.5, the activity was 81% from the maximum, and at pH 8.5, it was decreased to 52%. Cc, Clustal consensus. Hydrolysis of 1 mM pNPG was measured at varied temperatures from 20 to 65 C. Identification and enzymatic characterization of the maltose-inducible -glucosidase MalL (sucrase-isomaltase-maltase) of, Rolfsmeier M., Blum P. Purification and characterization of a maltase from the extremely thermophilic crenarchaeote, Deng X., Petitjean M., Teste M.A., Kooli W., Tranier S., Franois J.M., Parrou J.L. The activity was the highest with amylopectin, and the lowest with amylose (Figure 7). Similarities and differences in the biochemical and enzymological properties of the four isomaltases from, McWethy S.J., Hartman P.A. Every temperature point was assayed in triplicate. Sievers F., Higgins D.G. The pH optimum of the O. polymorpha maltase is 6.06.5 [40], of S. cerevisiae maltase 6.56.8 [23,24] and of Schizosaccharomyces pombe maltase 6.0 [33].
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