Protein sequences were aligned using Clustal Omega [72] to calculate identity values of the proteins. Chi Z., Ni X., Yao S. Cloning and overexpression of a maltase gene from. 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. We then visualized the S. cerevisiae IMA1 structure in complex with isomaltose (PDB: 3AXH) [29] using PyMol [30] in order to display all these amino acids (Figure 1, lower panel). Samples were withdrawn at fixed intervals, heated at 95 C to stop the reaction and analysed on TLC and by HPLC. Unfortunately, the sequence data of this protein is not available. official website and that any information you provide is encrypted According to the information present in the CBS-KNAW culture collection [37], B. adeninivorans CBS 8244 used in current work assimilates following -glucosidic sugars: maltose, sucrose, melezitose, trehalose and -MG. Of those, melezitose is a maltose-like sugar, and -MG (a synthetic analogue of isomaltose [38]) is an isomaltose-like sugar. SDs of at least two independent experiments with two replicates at each condition are shown by error bars. We tested panose hydrolysis by BaAG2 and conclude that it accumulated in transglycosylation reaction since it was not hydrolyzed by the enzyme even during extended (22 h) reaction time (Figure S3). 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). contact this location, Window Classics-Sarasota A maltase has been characterized from C. albicans [41] and four maltase-isomaltases from Scheffersomyces stipitis [12]. 4141 S Tamiami Trl Ste 23 Liiv L., Prn P., Alame T. Cloning of maltase gene from a methylotrophic yeast, Geber A., Williamson P.R., Rex J.H., Sweeney E.C., Bennett J.E. The most important brush border enzymes are dextrinase and glucoamylase, which further break down oligosaccharides. The small intestine is where most chemical digestion in the human body takes place. Which of the following reaction is catalysed by CH 18 quiz. Sarasota, FL34231 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. Pancreatic lipase works with the help of the salts from bile secreted by the liver and the gallbladder. 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. WebWhere produced Substrate Broken down into; Mouth: Salivary amylase: Salivary glands: Starch: Maltose: Small intestine - duodenum: Pancreatic amylase: Pancreas: Starch: 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. Gutirrez-Alonso P., Gimeno-Prez M., Ramrez-Escudero M., Plou F.J., Sanz-Aparicio J., Fernndez-Lobato M. Molecular characterization and heterologous expression of a. Chiba S., Murata M., Matsusaka K., Shimomura T. A new trisaccharide, 6F--D-glucosyl-sucrose, synthesized by transglucosylation reaction of brewers yeast -glucosidase. [(accessed on 27 November 2019)]; Teste M.A., Marie Franois J., Parrou J.L. Hydrolysis of the polymers was evaluated according to the release of glucose and by TLC analysis of reaction products. The site is secure. WebMaltase is found in plants, bacteria, yeast, humans, and other vertebrates. The article processing charge was covered by University of Tartu Feasibility Fund grant PLTMRARENG13 to T.V. It is located in the brush border of the small intestine of humans and other mammals. 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]. The MAL62-containing plasmid pURI-ScMAL62Cter [12] was used to produce the S. cerevisiae maltase protein that was analyzed as a reference. Preprolactase, the primary translation product, has a single polypeptide primary structure consisting of 1927 amino acids. SDs of two to three replicates are shown by error bars. Fragments of sequence alignment of six maltases. The initial velocity data analysis with enzyme kinetics module of the SigmaPlot (Systat Software, San Jose, CA, USA) yielded kinetic parameters (Km, Vmax) for the enzyme. Regulation of glycogen metabolism in yeast and bacteria. 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. The three major classes of nutrients that undergo digestion are proteins, lipids (fats), and carbohydrates. By 24 h of reaction with 500 mM maltose, the amount of transglycosylation products was 12.6% of total sugars in the reaction mixture, while the respective value for the MAL62 protein was about three times less4.5% (see Table S3). contact this location, Window Classics-Miami The genes encoding two putative -glucosidases designated as AG1 and AG2 were revealed in genome of B. adeninivorans. 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. Both types of enzymes hydrolyze sucrose and a synthetic substrate p-nitrophenyl--d-glucopyranoside (pNPG) [15,16,23,24]. Plants. Similar mode of melezitose hydrolysis was earlier shown for the maltase-isomaltase of O. polymorpha [16]. In the phylogram of -glucosidases from non-conventional yeasts, all eight Lipomyces enzymes clustered with those of B. adeninivorans [12]. 22.10C: Digestive Processes of the Small Intestine and T.A. Tamura K., Nei M., Kumar S. Prospects for inferring very large phylogenies by using the neighbor-joining method. The theoretical Mw value of 67,901 Da for the kcat calculation was computed in ExPASy Proteomics Server (http://expasy.org). The composition and linkage types of the tested substrates are indicated. 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]. Visnapuu T., Mardo K., Mosoarca C., Zamfir A.D., Vigants A., Alame T. Levansucrases from, Ernits K., Eek P., Lukk T., Visnapuu T., Alame T. First crystal structure of an endo-levanaseThe BT1760 from a human gut commensal. In BaAG2, a Thr is present at position of Val216 and therefore we predicted that this enzyme is most probably a maltase. Some properties of two forms of alpha-glucosidase from, Alame T., Viigand K., Pnograjeva K. Utilization of -glucosidic disaccharides by. Digestive System Test Review Flashcards | Quizlet Considering that BaAG2 had a remarkable activity on glycogen, the enzyme may contribute to glycogen catabolism in B. adeninivorans. The ability of B. adeninivorans to grow on sugars was assayed as in [12]. The pH optimum of BaAG2 was in a moderate acidic region as in the case of other yeast maltases [23,24]. Most of the digestive enzymes in the small intestine are secreted by the pancreas and enter the small intestine via the pancreatic duct. Saitou N., Nei M. The neighbor-joining method: A new method for reconstructing phylogenetic trees. 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]. 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. Identification, soluble expression, and characterization of a novel endo-inulinase from. Hedges S.B., Marin J., Suleski M., Paymer M., Kumar S. Tree of life reveals clock-like speciation and diversification. According to Hasegawa et al., the MalT protein of A. oryzae that has 51% of sequence identity to BaAG2 (Table S2) exhibited pNPG-hydrolyzing activity, its expression was induced when grown on maltose, and thereby the MalT was defined by the authors as a maltase [36]. BaAG2 content in reaction mixtures ranged from 0.02 to 3.5 g/mL. Thermostability of BaAG2 in the presence and absence of indicated ligands. Other carbohydrates pass undigested into the large intestine, where they are digested by intestinal bacteria. The maximum composite likelihood model [71] with 1000 bootstrap replicates was applied. Lipids (fats) are degraded into fatty acids and glycerol. According to literature data, thermolability has been reported for some other yeast -glucosidases. Concentration of released glucose was measured as described above and 3 l of the samples were analysed on TLC. 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. In the primers, the nucleotides annealing with the pURI3Cter vector [62] are shown in bold and those annealing with the BaAG2 gene sequence are shown in italics. A remote but significant sequence homology between glycoside hydrolase clan GH-H and family GH31. Accessibility StatementFor more information contact us atinfo@libretexts.org. contact this location, Window Classics-Pembroke Park 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. The effect of temperature on activity (left panel) and stability (right panel) of BaAG2. 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. SD values of three to five replicates on each substrate are indicated. This enzyme certainly has a biotechnological potentialit produced 3.6 times more transglycosylation products than the S. cerevisiae -glucosidase studied at the same conditions [17,20]. 8600 Rockville Pike ; Formal Analysis, T.V., A.M., K.P. Thus, an Aspergillus enzyme with high transglycosylating activity was reported to produce panose and isomaltose from maltose [18,59,60]. We showed that MOS of DP3 (see Table 1), DP4, DP5, DP6 and DP7 served as substrates for BaAG2 (Figure S4). Cc, Clustal consensus. [15], Substrate modification studies have demonstrated that the 3-OH and 2-OH moieties on the galactopyranose ring are essential for enzymatic recognition and hydrolysis. It has been shown that the S. cerevisiae maltase also produced panose from maltose, yet transglycosylating activity of the Saccharomyces enzyme was more than three times smaller compared to the X. dendrorhous enzyme. The .gov means its official. Presence of acarbose (the strongest inhibitor of BaAG2) increased the Tm of BaAG2 by 11.4 C. Incubation for 30 min at 50 C totally inactivated the enzyme (Figure 4). The purified enzyme was unstable if diluted therefore 5 g/L bovine serum albumin (BSA) was added to the dilution buffer as a protein stabilizer to retain its full catalytic activity. It is essential to the Bethesda, MD 20894, Web Policies an extracellular -glucosidase from L. starkeyi was biochemically characterized [51]. Genes potentially encoding for either maltases, isomaltases or maltase-isomaltases were recently discovered in the genomes of many non-conventional yeasts [12]. Maltase - Enzyme, Structure, Deficiency, and FAQs At first, the cultures were incubated for 2 h at 37 C followed by 20-h incubation at 22 C. Studier F.W., Moffatt B.A. Lactase also catalyzes the conversion of phlorizin to phloretin and glucose. 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. and T.V. and T.V. The genome of B. adeninivorans encodes two putative -glucosidases. 24850 Old 41 Ste 7 Figure 8 and Table S3 show that already within 2 h at 250 mM (85.6 g/L) maltose concentration, BaAG2 produced maltotriose (4.2 g/L) and panose, -d-Glc-(16)--d-Glc-(14)-d-Glc (1.6 g/L), in addition to a maltose hydrolysis productglucose. Aminopeptidase and dipeptidase free the end amino acid products. Prof. V. Passoth (SLU, Uppsala, Sweden). In the BaAG2 protein, Asp216 was predicted as a nucleophile, Glu274 as an acid-base catalyst and Asp348 as a stabilizer (Figure 2). 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]. The calculated molecular weight of the protein was 67.9 kDa and a prominent band of respective size was detected by electrophoresis of the lysate produced from induced E. coli cells overexpressing the AG2 gene (Figure S1). The BaAG2 protein deduced from the gene is 580 aa long. Other sets by this creator. The yeast strain was grown on solid YPD medium (20 g/L peptone, 20 g/L glucose, 10 g/L yeast extract, 20 g/L agar) at 30 C 24 h for harvesting the cells for genomic DNA extraction. Unraveling the function of glycosyltransferases in. Polysaccharide concentration in the reaction mixture was 5 g/L, and 20 g/mL of the enzyme was used. The activities (moles of glucose released per minute per mg of protein; U/mg) were calculated from initial velocities of the reaction. BaAG2 was overexpressed in E. coli with the His6-tag in its C-terminus that enabled purification of the protein using Ni2+-affinity chromatography. 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]. [21], Lactase is encoded by a single genetic locus on chromosome 2. Casa-Villegas M., Marn-Navarro J., Polaina J. Synthesis of isomaltooligosaccharides by, Kato N., Suyama S., Shirokane M., Kato M., Kobayashi T., Tsukagoshi N. Novel -glucosidase from. [2] Technology to produce lactose-free milk, ice cream, and yogurt was developed by the USDA Agricultural Research Service in 1985. All tested compounds inhibited pNPG hydrolysis competitively, and the most powerful inhibitors of BaAG2 were acarbose, Tris and glucose. [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. 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. Inclusion in an NLM database does not imply endorsement of, or agreement with, Specifically: The small intestine is divided into 3 parts. Sequence identity of BaAG2 with other maltases was only moderate being the highest (51%) with the maltase MalT of Aspergillus oryzae. The reaction samples were analyzed using TLC. Thermostable extracellular -amylase and -glucosidase of, Egeter O., Bruckner R. Characterization of a genetic locus essential for maltose-maltotriose utilization in, Schnert S., Buder T., Dahl M.K. However, yeasts synthesize glycogen as a reserve polysaccharide [58]. [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. 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. Digestive enzymes - The digestive system - AQA Synergy The small intestine. The LibreTexts libraries arePowered by NICE CXone Expertand are supported by the Department of Education Open Textbook Pilot Project, the UC Davis Office of the Provost, the UC Davis Library, the California State University Affordable Learning Solutions Program, and Merlot. At least two independent experiments were performed with two technical replicates. At least three independent measurements for each substrate and concentration were made. To confirm this, we conducted a differential scanning fluorimetry (DSF) assay of BaAG2 in the presence and absence of competing inhibitors as in [16,47]. In silico assay of -glucosidases of non-conventional yeasts [12] identified a putative -glucosidase protein AG1 of Lipomyces starkeyi as the closest homologue (50% identity) of BaAG2. Mcilvaine T.C. For example, -glucosidase of a yeast X. dendrorhous (syn. Several GHs of yeasts and filamentous fungi exhibit transglycosylating activity. The amino acids of these proteins corresponding to Val216 of ScIMA1 are shown inside a red frame as this position is considered of key importance in selective substrate binding [28]. [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. Genetic variation of the repeated MAL loci in natural populations of, Reinders A., Ward J.M. Stingele F., Newell J.W., Neeser J.R. It is thought to be synthesized by cells of the mucous membrane lining the intestinal wall. Janeek ., Gabriko M. Remarkable evolutionary relatedness among the enzymes and proteins from the -amylase family. The https:// ensures that you are connecting to the In the genomes of most yeasts addressed in Viigand et al. government site. For the analysis of products of polysaccharide degradation, 3 l of the reaction mixtures were spotted. Hydrolysis of polysaccharides (5 g/L) by BaAG2, ScMAL62 and amyloglycosidase of A. niger (AG) and -amylase of A. oryzae (-AM). The experiment was based on [16,47,65] with above-mentioned modifications. HHS Vulnerability Disclosure, Help Indeed, if respective Thr was substituted with Val in O. polymorpha maltase-isomaltase, utilization of maltose-like sugars was severely hampered [16]. [25] Both mutations, CT at position -13910 and G A at position -22018, have been independently linked to lactase persistence. 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. Maltase Definition & Meaning - Merriam-Webster Carboxypeptidase, a pancreatic brush border enzyme, splits one amino acid at a time. 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 E. coli DH5 (Thermo Fisher Scientific, Waltham, MA, USA) was used for DNA cloning and plasmid production. A.M., K.V., K.P., K.E. 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. Surprisingly, we detected the ability of BaAG2 to hydrolyze polysaccharides that is a rather exceptional feature among maltases. Acts on: Complex Carbohydrates. 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). Maltase - Wikipedia At high maltose concentrations, BaAG2 exhibited transglycosylating ability producing potentially prebiotic di- and trisaccharides. kcat and kcat/Km were calculated from these data. MycoCosm portal: Gearing up for 1000 fungal genomes. 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. The resulting supernatants were syringe-filtered (pore size 0.45 m) and loaded onto an IMAC HisTrap FF column coupled with an KTAprime plus chromatography system (GE Healthcare, Uppsala, Sweden). Hydrolysis of 1 mM pNPG was measured at varied temperatures from 20 to 65 C. The intracellular maltase (MAL1) protein of Schizosaccharomyces pombe, with 43.2% of sequence identity to BaAG2 hydrolyzed pNPG and maltose, but had also activity on soluble starch, and some activity on sucrose [33]. Catalytic constant of -glucosidase of A. niger on maltose was 144 1/s (2.6 times lower than BaAG2), but the affinity towards maltose was very high (0.75 mM) [45]. 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. Dextrans were hydrolyzed only by the amyloglycosidase, and the release of glucose was minimal. Figure 9 shows the phylogram of selected yeast species and A. oryzae based on sequence analysis of D1/D2 domains of large subunit ribosomal RNA to illustrate the evolutionary relationships between the species. One of those, the rna_ARAD1D20130g-encoded protein (BaAG2; 581 aa) was overexpressed in Escherichia coli, purified and characterized. The pURI3-BaAG2Cter and pURI-ScMAL62Cter containing the BaAG2 gene or MAL62 gene, respectively, were electroporated into E. coli BL21 (DE3) for heterologous expression. Km, Vmax, kcat and kcat/Km values of hydrolysis of pNPG and sugars by BaAG2. the contents by NLM or the National Institutes of Health. The Ki values were calculated using enzyme kinetics module of the SigmaPlot (Systat Software, San Jose, CA, USA). and K.P. A negative control containing 20 g/mL BSA instead of the enzyme was incubated alongside. Schrdinger L.L.C. [19] The prodomain has been shown to act as an intramolecular chaperone in the ER, preventing trypsin cleavage and allowing LPH to adopt the necessary 3-D structure to be transported to the Golgi apparatus. Highlights: catalytic nucleophile (turquoise), acid-base catalyst (green), a transition state stabilizer (yellow) and a residue crucial for substrate specificity (red). Extracellular maltase of, Kumara H.M., De Cort S., Verachtert H. Localization and characterization of alpha-glucosidase activity in. { "22.10A:_Anatomy_of_the_Small_Intestine" : "property get [Map MindTouch.Deki.Logic.ExtensionProcessorQueryProvider+<>c__DisplayClass228_0.