The cloning procedure was conducted with recombinant Pfu polymerase (Thermo Fisher Scientific, Waltham, MA, USA). Produced In Site of Release pH Level; Carbohydrate Digestion: Salivary amylase: Salivary glands: Mount: Neutral: Pancreatic amylase: Pancreas: Small intestine: Pancreatic lipase works with the help of the salts from bile secreted by the liver and the gallbladder. H11 -glucosidase (538 aa, {"type":"entrez-protein","attrs":{"text":"BAL49684.1","term_id":"374428620","term_text":"BAL49684.1"}}BAL49684.1) [35]; AoMalT, Aspergillus oryzae maltase MalT (574 aa, {"type":"entrez-protein","attrs":{"text":"XP_001825184.1","term_id":"169781442","term_text":"XP_001825184.1"}}XP_001825184.1) [36]. At first, the cultures were incubated for 2 h at 37 C followed by 20-h incubation at 22 C. 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. Though the proteins aligned sufficiently well over the entire sequence, BaAG2 showed only moderate sequence identity with the other maltases ranging from 35% with Halomonas sp. The genes potentially encoding -glucosidases in the genomes of non-conventional yeasts were analysed in Viigand et al. 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. This is because the cellulose is made out of beta-glucose that makes the inter-monosaccharidal bindings different from the ones present in starch, which consists of alpha-glucose. In the human enzyme, the lactase activity has been connected to Glu-1749, while Glu-1273 is the site of phlorizin hydrolase function. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (, GUID:F7FE2AAD-6745-4A4F-A3F5-1B8CC927D301, {"type":"entrez-protein","attrs":{"text":"NP_595063.1","term_id":"19111855","term_text":"NP_595063.1"}}, {"type":"entrez-protein","attrs":{"text":"P07265","term_id":"126716","term_text":"P07265"}}, {"type":"entrez-protein","attrs":{"text":"BAA12704.1","term_id":"1321625","term_text":"BAA12704.1"}}, {"type":"entrez-protein","attrs":{"text":"BAL49684.1","term_id":"374428620","term_text":"BAL49684.1"}}, {"type":"entrez-protein","attrs":{"text":"XP_001825184.1","term_id":"169781442","term_text":"XP_001825184.1"}}, {"type":"entrez-protein","attrs":{"text":"Q9P8G8","term_id":"74665415","term_text":"Q9P8G8"}}, Kurtzman C.P., Robnett C.J. Maltase is an enzyme produced by the cells lining the small intestine that breaks down the disaccharide maltose. Universal substrates are indicated by green, maltose and maltose-like sugars by yellow, and isomaltose and isomaltose-like sugars by blue bars. government site. The upper panel of Figure 1 shows the amino acid signature of O. polymorpha maltase-isomaltase MAL1, S. cerevisiae maltase MAL62, isomaltase IMA1, and B. adeninivorans AG2. Transglycosylation of maltose was enhanced at 500 mM (171.2 g/L) concentration: up to 13.3 g/L of maltotriose was produced by 2 h and 10.4 g/L of panose by 72 h of reaction. Gene sequences from domains 1 and 2 (D1/D2) of large subunit ribosomal RNA were aligned to build a neighbor-joining phylogenetic tree [69] of yeasts using MEGA v. 7.0 [70]. See Materials and Methods, paragraphs 4.3 and 4.6 for details. For thermostability determination, BaAG2 was incubated in 100 mM K-phosphate buffer (pH 6.5) buffer containing 5 g/L BSA for 30 min at temperatures 10, 20, 30, 37, 45 and 50 C. Total amount of detected saccharides at each time point was equalled to 100%. To visualize hydrolysis and polymerization products, the TLC analysis was conducted as in [16] on Silica Gel 60 F254 plates with concentrating zone (Merck, Darmstadt, Germany). 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. At least three independent measurements for each substrate and concentration were made. The cells were harvested by centrifugation (2400 g, 10 min) at 4 C and stored at 20 C until further use. This is demonstrated by the fact that a 2-deoxy analog is an effective competitive inhibitor (Ki = 10mM). Location of Val216 in the structure is marked with a red circle. WebMaltose (or malt sugar) is an intermediate in the intestinal digestion (i.e., hydrolysis) of glycogen and starch, and is found in germinating grains (and other plants and Cells were disrupted by sonication with Ultrasonic Homogenizer (Cole-Parmer Instrument Company, Vernon Hills, IL, USA) in 100 mM K-phosphate buffer (pH 6.5) with the cOmplete, EDTA-free Protease Inhibitor Cocktail (Roche Diagnostics, Mannheim, Germany), and centrifuged 30 min at 2400 g at 4 C. At desired time points (2 h, 24 h, 74/96 h) aliquots were withdrawn and heated to stop the reaction. 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. After cooling the samples on ice, residual activity of the enzyme was determined according to the hydrolysis of 1 mM pNPG at 30 C. sharing sensitive information, make sure youre on a federal Growth of B. adeninivorans on sugars (supplemented at 2 g/L) evaluated by an optical density (OD) of the culture at 600 nm achieved by 24-h cultivation on a microplate at 37 C. The ATG start codon and the stop codon are underlined in the primer sequences. The PCR product was cloned into a pJET vector from CloneJET PCR Cloning Kit (Thermo Fisher Scientific, Waltham, MA, USA), yielding pJET-BaAG2. Therefore, BaAG2 should be considered as a maltase. WebWhen individuals have Acid Maltase Deficiency, maltase is either not produced, or produced in very low amounts; therefore it cannot be used properly in the stomach or [10], Lactase (or a similar form of -galactosidase) is also used to screen for blue white colonies in the multiple cloning sites of various plasmid vectors in Escherichia coli or other bacteria. Furthermore, after substitution of Val216 in S. cerevisiae IMA1 with Thr, the isomaltase IMA1 gained the ability to hydrolyze maltose [28,29]. Which of the following reaction is catalysed by Some carbohydrates are degraded into simple sugars, or monosaccharides (e.g., glucose, galactose) and are absorbed by the small intestine. Thus, some of them have a high transglycosylating activity thanks to which they produce prebiotic oligosaccharides and potential functional food ingredients such as panose, melezitose, isomelezitose and isomalto-oligosaccharides [17,18,19,20,21]. Therefore, we routinely performed enzymatic assays at 30 C since some reactions (e.g., transglycosylation and polysaccharide degradation assays) were conducted up to several days. [12], the -glucosidase genes resided in maltose utilization (MAL) clusters, whereas no MAL clusters were detected in B. adeninivorans. 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). The theoretical Mw value of 67,901 Da for the kcat calculation was computed in ExPASy Proteomics Server (http://expasy.org). Made in: Small intestine. The content of the released glucose was determined spectrophotometrically as in [16,47]. and K.E. X-ray structures along the reaction pathway of cyclodextrin glycosyltransferase elucidate catalysis in the -amylase family. Gabriko M. Evolutionary history of eukaryotic -glucosidases from the -amylase family. ; funding acquisition, T.A and T.V. It is essential to the complete digestion of whole milk; it breaks down lactose, a sugar which gives milk its sweetness. Presence of Tris raised the Tm value by 5.8 C, and of glucose by 4.4 C. or sucrose [16,24] (Table 1) whereas the opposite is true for maltases of bacteria and archaea [52,53,54]. Saitou N., Nei M. The neighbor-joining method: A new method for reconstructing phylogenetic trees. [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. Protein sequences of S. cerevisiae maltase MAL62 (UniProtKB: {"type":"entrez-protein","attrs":{"text":"P07265","term_id":"126716","term_text":"P07265"}}P07265), S. cerevisiae isomaltase IMA1 (UniProtKB: YGR287C), O. polymorpha maltase-isomaltase MAL1 (UniProtKB: {"type":"entrez-protein","attrs":{"text":"Q9P8G8","term_id":"74665415","term_text":"Q9P8G8"}}Q9P8G8) and B. adeninivorans AG2 were aligned using Clustal Omega [72] and amino acids corresponding to IMA1 signature positions determining the substrate specificity [12,15] were extracted from the alignment. Maltase Flashcards | Quizlet Incubation for 30 min at 50 C totally inactivated the enzyme (Figure 4). Based on the literature, only one -glucosidase of yeast and filamentous fungi has much higher kcat on maltose than that of BaAG2the extracellular -glucosidase of Schizosaccharomyces pombe (kcat = 709 1/s) [46]. These enzymes enter the small intestine in response to the hormone cholecystokinin, which is produced in response to the presence of nutrients. For example, the -glucosidase of S. cerevisiae produced isomelezitose from sucrose when the substrate concentration was high [21]. The bootstrap values (1000 replicates) are shown at the nodes. Ernits K., Viigand K., Visnapuu T., Pnograjeva K., Alame T. Thermostability measurement of an -glucosidase using a classical activity-based assay and a novel Thermofluor method. Based on phylogenetic analysis of orthologous proteins [4], this group diverged from S. cerevisiae lineage 200 to 400 million, and from the CTG clade 200 million years ago. Identification, soluble expression, and characterization of a novel endo-inulinase from. Some properties of two forms of alpha-glucosidase from, Alame T., Viigand K., Pnograjeva K. Utilization of -glucosidic disaccharides by. E. coli DH5 (Thermo Fisher Scientific, Waltham, MA, USA) was used for DNA cloning and plasmid production. Transformation of maltose into prebiotic isomaltooligosaccharides by a novel -glucosidase from. HHS Vulnerability Disclosure, Help The small intestine uses different enzymes and processes to digest proteins, lipids, and carbohydrates. Pembroke Park, FL33023 BaAG2 was competitively inhibited by a diabetes drug acarbose (Ki = 0.8 M) and Tris (Ki = 70.5 M). The same marker sugars were used in all assays but are marked only on TLC plate of glycogen degradation. H11 -glucosidase to 51% with Aspergillus oryzae maltase MalT (Table S2). [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. At least two independent experiments were performed with two technical replicates. Figure 4 shows that at temperature over 50 C, the activity of the enzyme rapidly declined. 2401 SW 32nd Ave The Co-evolution of Genes and Culture, Lactase persistence shows indication of association with Obesity, Galactose-1-phosphate uridylyltransferase, https://en.wikipedia.org/w/index.php?title=Lactase&oldid=1161883688, Pages using infobox enzyme with unknown parameters, Creative Commons Attribution-ShareAlike License 4.0, This page was last edited on 25 June 2023, at 16:21. Yeast cells grown overnight on BD Difco YNB medium (Thermo Fisher Scientific, Waltham, MA, USA) without amino acids containing 2 g/L glucose were used as inoculum. Isomaltose and isomaltose-like sugars are indicated by dark blue bars. Licensee MDPI, Basel, Switzerland. Chapter 17 Example Genes for eight intracellular -glucosidases (five maltases and three isomaltases) were predicted in the genome of L. starkeyi. The MAL62 of S. cerevisiae that was assayed alongside could not hydrolyze MOS longer than maltotetraose (DP4) (Figure S4). However, transport and intracellular hydrolysis of -glucosidic sugars have also been investigated in Ogataea polymorpha [25] and Schizosaccharomyces pombe [33,49]. Dextrans were hydrolyzed only by the amyloglycosidase, and the release of glucose was minimal. A maltase has been characterized from C. albicans [41] and four maltase-isomaltases from Scheffersomyces stipitis [12]. contact this location. lambicus), both extra- and intracellular maltases were inhibited not only by acarbose (Ki values between 28.5 and 57 M) but also by Tris (Ki values between 7.45 and 15.7 mM) [57]. A maltase has been characterized from C. albicans and four maltase-isomaltases from Scheffersomyces stipitis . [18] It can be divided into five domains: (i) a 19-amino-acid cleaved signal sequence; (ii) a large prosequence domain that is not present in mature lactase; (iii) the mature lactase segment; (iv) a membrane-spanning hydrophobic anchor; and (v) a short hydrophilic carboxyl terminus. The temperature optimum for BaAG2 was between 4050 C, with maximum activity (530 U/mg) achieved at 45 C. [(accessed on 19 May 2017)]; Janeek ., Svensson B., MacGregor E.A. SDs of two to three replicates are shown by error bars. It is located in the brush border of the small intestine of humans and other mammals. Action: Maltase to Glucose. Genomic DNA of B. adeninivorans was extracted using PowerMax Soil DNA Isolation Kit (MoBio Laboratories, Carlsbad, CA, USA) and the standard protocol by manufacturer. [17] Elimination of specific hydroxyl groups on the glucopyranose moiety does not eliminate catalysis.[17]. 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. Thus, B. adeninivorans grows on both maltose-like and isomaltose-like sugars, meaning that it should possess enzymes for the hydrolysis of both types of sugars. S. cerevisiae has two types of -glucosidasesmaltases (EC 3.2.1.20) and isomaltases (EC 3.2.1.10)that differ for substrate specificity. Isomaltose synthesis from maltose was also confirmed for ScMAL62, even though the content of it was only minimal (Figure 8, Table S3). Ki values and inhibition mode for BaAG2 inhibitors of pNPG hydrolysis. Where does maltase function in the body? - Studybuff.com SDs of two to three HPLC measurements at each time point were up to 20%. * For the structure and linkage type of the substrates, see Figure 5. 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]. Bile salts attach to triglycerides and help to emulsify them; this aids access by pancreatic lipase because the lipase is water-soluble, but the fatty triglycerides are hydrophobic and tend to orient toward each other and away from the watery intestinal surroundings. Wilson W.A., Roach P.J., Montero M., Baroja-Fernndez E., Muoz F.J., Eydallin G., Viale A.M., Pozueta-Romero J. The maltase MAL62 of S. cerevisiae was used as a reference. [22] It is expressed exclusively by mammalian small intestine enterocytes and in very low levels in the colon during fetal development. Studies of E. coli lactase have proposed that hydrolysis is initiated when a glutamate nucleophile on the enzyme attacks from the axial side of the galactosyl carbon in the -glycosidic bond. On the phylogram (Figure 9), B. adeninivorans clusters with Lipomyces starkeyi. [22] Humans are born with high levels of lactase expression. One of those, the rna_ARAD1D20130g-encoded protein (BaAG2; 581 aa) was overexpressed in Escherichia coli, purified and characterized. Carboxypeptidase, a pancreatic brush border enzyme, splits one amino acid at a time. In current work, one of them, BaAG2, was produced in E. coli and characterized in detail. 43 terms. Digestive System Test Review Flashcards | Quizlet Aminopeptidase and dipeptidase free the end amino acid products. Typically, yeast -glucosidases hydrolyze pNPG more rapidly than maltose. 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. Initial velocity data were plotted against the temperature to reveal the temperature optimum. In metabolism, the -glycosidic bond in D-lactose is hydrolyzed to form D-galactose and D-glucose, which can be absorbed through the intestinal walls and into the bloodstream. Maltose - an overview | ScienceDirect Topics The small intestine is where most chemical digestion in the human body takes place. Many -glucosidases can transglycosylate and produce short oligosaccharides, especially at high concentration of the substrate [17,18,19,20,21]. Almagro Armenteros J.J., Tsirigos K.D., Snderby C.K., Petersen T.N., Winther O., Brunak S., von Heijne G., Nielsen H. SignalP 5.0 improves signal peptide predictions using deep neural networks. and T.V. MycoCosm portal: Gearing up for 1000 fungal genomes. The melting temperature (Tm) values calculated from the DSF data are presented in Figure 6. Prof. V. Passoth (SLU, Uppsala, Sweden). In contrast, the maltase of Schizosaccharomyces pombe prefers maltose to sucrose [33]. By 72 h of reaction, the maltotriose content was decreased and panose content increased to 2.6 g/L (Figure 8, Table S3). Other brush border enzymes are maltase, sucrase, and lactase. SD values of three to five replicates on each substrate are indicated. Chi Z., Ni X., Yao S. Cloning and overexpression of a maltase gene from. Having seen that isomaltose and isomaltose-like sugars are not hydrolyzed by BaAG2 (Figure 5), we measured inhibition of pNPG hydrolysis reaction by these sugars as in [16]. Highlights: catalytic nucleophile (turquoise), acid-base catalyst (green), a transition state stabilizer (yellow) and a residue crucial for substrate specificity (red). and K.P. [1] Lactase can be purchased as a food supplement, and is added to milk to produce "lactose-free" milk products. Where Digestive Enzymes are Produced Flashcards | Quizlet [27] Developmentally regulated DNA-binding proteins may down-regulate transcription or destabilize mRNA transcripts, causing decreased LPH expression after weaning.[27]. The most important brush border enzymes are dextrinase and glucoamylase, which further break down oligosaccharides. We expected that binding of the substrates or competitive inhibitors of the enzyme should increase its thermostability. [26], The lactase promoter is 150 base pairs long and is located upstream of the site of transcription initiation. A simplified autoinduction medium as in [64] was used for protein overproduction: the LB-based medium (20 g/L tryptone, 5 g/L yeast extract, 5 g/L NaCl) was supplemented with 25 mM phosphate buffer (Na2HPO4/KH2PO4; pH 7.2) and 3 g/L glycerol to which filter-sterilized 0.25 g/L glucose and 1 g/L lactose were added. SDs of at least two independent experiments with two replicates at each condition are shown by error bars. However, BaAG2 exhibited moderate, but clearly detectable and recordable exo-hydrolysis of amylose, amylopectin and glycogen. Thus, an Aspergillus enzyme with high transglycosylating activity was reported to produce panose and isomaltose from maltose [18,59,60]. All authors have read and agreed to the published version of the manuscript. WebERT by Genzyme, Inc. using a recombinant human GAA produced in a Chinese Hamster Ovary (CHO) cell line, has shown moderate success in patients using a biweekly infusion The Saccharomycotina clades according to [50] are designated by background coloring. DNA Clean & Concentrator-5 Kit (Zymo Research, Irvine, CA, USA) was used for purification and concentration of PCR products. Conceptualization, T.A. In addition, maltotriose, maltulose, turanose (maltose-like sugars) as well as isomaltose and palatinose (an isomaltose-like sugar) were identified as new growth substrates for this yeast. Utilization of -glucosidic sugars by maltases and isomaltases has earlier been thoroughly studied in S. cerevisiae because metabolism of these sugars is crucial in brewing and baking [22,48].

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