where is maltase producedespn conference usa football teams 2023

; Visualization, T.V., K.E., A.M., K.P. According to annotations provided at the MycoCosm website [26], the genome of Blastobotrys (Arxula) adeninivorans [2] encodes 185 carbohydrate-active enzymes, including 88 glycoside hydrolases (GHs) assigned to different families. HHS Vulnerability Disclosure, Help A remote but significant sequence homology between glycoside hydrolase clan GH-H and family GH31. Fragments of sequence alignment of six maltases. However, as maltase-isomaltases also have a Thr at that position (Figure 1, upper panel; [12,16]), based on the amino acid signature, BaAG2 may also be a promiscuous enzyme with a wide substrate spectrum like O. polymorpha MAL1. Considering that BaAG2 had a remarkable activity on glycogen, the enzyme may contribute to glycogen catabolism in B. adeninivorans. SDs of at least two independent experiments with two replicates at each condition are shown by error bars. Several GHs of yeasts and filamentous fungi exhibit transglycosylating activity. All authors have read and agreed to the published version of the manuscript. At high maltose concentrations, BaAG2 exhibited transglycosylating ability producing potentially prebiotic di- and trisaccharides. In the BaAG2 protein, Asp216 was predicted as a nucleophile, Glu274 as an acid-base catalyst and Asp348 as a stabilizer (Figure 2). and T.A. Specifically: The small intestine is divided into 3 parts. We confirmed this by using the SignalP program (see Materials and Methods). BaAG2 was competitively inhibited by a diabetes drug acarbose (Ki = 0.8 M) and Tris (Ki = 70.5 M). ; Validation, T.V., K.E. This is demonstrated by the fact that a 2-deoxy analog is an effective competitive inhibitor (Ki = 10mM). Genome diversity and evolution in the budding yeasts (Saccharomycotina), Kelly C.T., Moriarty M.E., Fogarty W.M. Language links are at the top of the page across from the title. Most of the digestive enzymes in the small intestine are secreted by the pancreas and enter the small intestine via the pancreatic duct. Cloning and characterization of a, Alame T., Liiv L. Glucose repression of maltase and methanol-oxidizing enzymes in the methylotrophic yeast, Marn D., Linde D., Lobato M.F. Interestingly, this enzyme did not hydrolyze sucrose and had a quite low activity on pNPG. [17] Elimination of specific hydroxyl groups on the glucopyranose moiety does not eliminate catalysis.[17]. Saccharomyces cerevisiae maltase MAL62, studied for comparison, had only minimal ability to hydrolyze these polymers, and its transglycosylating activity was about three times lower compared to BaAG2. Isomaltose and isomaltose-like sugars are indicated by dark blue bars. The https:// ensures that you are connecting to the Purification and biochemical characterization of an -glucosidase from, da Silva T.M., Michelin M., de Lima Damsio A.R., Maller A., Almeida F.B.D.R., Ruller R., Ward R.J., Rosa J.C., Jorge J.A., Terenzi H.F., et al. 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]. For example, the four isomaltases of S. cerevisiae had all low thermostability. In the current work, we expressed heterologously in Escherichia coli and biochemically characterized the -glucosidase BaAG2 of B. adeninivorans encoded by rna_ARAD1D20130g. In current work, one of them, BaAG2, was produced in E. coli and characterized in detail. positions with residues of weakly similar properties. Wilson W.A., Roach P.J., Montero M., Baroja-Fernndez E., Muoz F.J., Eydallin G., Viale A.M., Pozueta-Romero J. The MAL62-containing plasmid pURI-ScMAL62Cter [12] was used to produce the S. cerevisiae maltase protein that was analyzed as a reference. kristen_lemke. BaAG2 and ScMAL62 had no activity on dextrans. Incubation for 30 min at 50 C totally inactivated the enzyme (Figure 4). For example, the -glucosidase of S. cerevisiae produced isomelezitose from sucrose when the substrate concentration was high [21]. Differential scanning fluorimetry (DSF) was used to evaluate the thermostability of BaAG2 in the presence and absence of ligands. WebThe enzyme is present in yeast, plants, and bacteria and is considered to be produced by cells of the mucous membrane in the lining of the intestinal wall in humans and other 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. [(accessed on 27 November 2019)]; Teste M.A., Marie Franois J., Parrou J.L. [4] Without lactase, lactose intolerant people pass the lactose undigested to the colon[5] where bacteria break it down, creating carbon dioxide and that leads to bloating and flatulence. Hydrolysis of 1 mM pNPG was measured at varied temperatures from 20 to 65 C. Presence of Tris raised the Tm value by 5.8 C, and of glucose by 4.4 C. [20], Mature human lactase consists of a single 160-kDa polypeptide chain that localizes to the brush border membrane of intestinal epithelial cells. Isolation and study of -glucosidases of the most basal lineages to Saccharomycotina, for example Lipomyces starkeyi, should verify the correctness of this hypothesis. Our study showed that BaAG2 is a maltase. SD, standard deviation. For example, its Vmax on maltose was 7.5 times higher compared to S. cerevisiae maltase MAL62 [23], and over two times higher compared to maltase-isomaltase of O. polymorpha [16]. Pancreatic lipase works with the help of the salts from bile secreted by the liver and the gallbladder. The site is secure. Reaction mixtures were spotted on TLC plates alongside with reference sugars (M): Glc (30 mM glucose); Mal (10 mM maltose); MalTri (10 mM maltotriose); DP4 (10 mM maltotetraose). WebMaltase is found in plants, bacteria, yeast, humans, and other vertebrates. For preliminary assay of substrate specificity of BaAG2, the enzyme was reacted with 100 mM concentration of various potential substrates: maltose, sucrose, maltotriose, isomaltose, melezitose, maltulose, turanose, palatinose or -MG. At fixed time points, aliquots were withdrawn, combined with three volumes of 200 mM Tris buffer (pH 8.3) and subsequently heated at 96C for 5 min to stop the reaction. In contrast to yeast maltases, BaAG2 showed exo-hydrolytic activity on starch, amylose, amylopectin and glycogen. The cloning procedure was conducted with recombinant Pfu polymerase (Thermo Fisher Scientific, Waltham, MA, USA). This work was funded by the Estonian Research Council (grant number PUT1050) to T.A. 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. 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). The cells were incubated on Greiner 96-well flat-bottom transparent polystyrene microplates (Greiner Bio-One, Frickenhausen, Germany) in 200 l under agitation for 24 h at 37 C. Cc, Clustal consensus. Table S1 also includes two B. adeninivorans enzymes that have been experimentally studied: a secreted invertase AINV belonging to GH31 family [10] and a secreted glucoamylase [9] of GH15 family. The MAL62 of S. cerevisiae that was assayed alongside could not hydrolyze MOS longer than maltotetraose (DP4) (Figure S4). Clustal Omega for making accurate alignments of many protein sequences. The small intestine. Location of Val216 in the structure is marked with a red circle. Arxula adeninivorans) belongs to a basal clade of Saccharomycotina subphylum and diverged in the evolution of fungi long before Saccharomyces [1,2,3,4,5]. Tamura K., Nei M., Kumar S. Prospects for inferring very large phylogenies by using the neighbor-joining method. 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]. Digestion of Lactase also catalyzes the conversion of phlorizin to phloretin and glucose. ; Resources, T.A. Hedges S.B., Marin J., Suleski M., Paymer M., Kumar S. Tree of life reveals clock-like speciation and diversification. On the other hand, -glucosidases of X. dendrorhous and A. niveus preferred polysaccharides such as starch, amylopectin and glycogen, and their kcat values on maltose were respectively 25 and 28 times lower compared to the value of BaAG2 [43,44]. 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). The oligonucleotide primers Ba20130g_PURICter_Fw (5 TAACTTTAAGAAGGAGATATACATATGGTTCTAGGATTTTTCAAAAAG 3) and Ba20130g_PURICter_Rev (5 GCTATTAATGATGATGATGATGATGGATTTCATAGATGACTGCCTCCA 3), designed according to the gene sequence of rna_ARAD1D20130g, were applied to amplify a 1789 bp fragment that represented the coding sequence of BaAG2 [12]. "Lactose-Free Milk, Low-Fat Cheese, and More Dairy Breakthroughs", "Asked: How do dairies make lactose-free milk? Phaffia rhodozyma) has been studied in detail, and synthesis of tri- and tetrasaccharides with -1,6 linkages was detected. Schrdinger L.L.C. [2] Technology to produce lactose-free milk, ice cream, and yogurt was developed by the USDA Agricultural Research Service in 1985. Multigene phylogenetic analysis of the Trichomonascus, Wickerhamiella and Zygoascus yeast clades, and the proposal of, Kunze G., Gaillardin C., Czernicka M., Durrens P., Martin T., Ber E., Gabaldn T., Cruz J.A., Talla E., Marck C., et al. Uitdehaag J.C.M., Mosi R., Kalk K.H., Van der Veen B.A., Dijkhuizen L., Withers S.G., Dijkstra B.W. S. cerevisiae has two types of -glucosidasesmaltases (EC 3.2.1.20) and isomaltases (EC 3.2.1.10)that differ for substrate specificity. What organ produces Maltase? The article processing charge was covered by University of Tartu Feasibility Fund grant PLTMRARENG13 to T.V. ; Writingreview and editing, T.A., T.V., K.E. Maltase is an enzyme produced by the cells lining the small intestine that breaks down the disaccharide maltose. Isomaltose and isomaltose-like substrates palatinose and -methylglucoside were not hydrolyzed (Figure 5). 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. Highlights: catalytic nucleophile (turquoise), acid-base catalyst (green), a transition state stabilizer (yellow) and a residue crucial for substrate specificity (red). Affinity of BaAG2 for maltose was slightly higher than for sucrose. After cooling the samples on ice, residual activity of the enzyme was determined according to the hydrolysis of 1 mM pNPG at 30 C. HPLC analysis was performed similarly as in [65]. The jejunum, the duodenum, and the ilium. It is located in the brush border of the small intestine of humans and other mammals. Other carbohydrates pass undigested into the large intestine, where they are digested by intestinal bacteria. Made in: Small intestine. Correia K., Yu S.M., Mahadevan R. AYbRAH: A curated ortholog database for yeasts and fungi spanning 600 million years of evolution. The maximum composite likelihood model [71] with 1000 bootstrap replicates was applied. BaAG2 was overexpressed in E. coli with the His6-tag in its C-terminus that enabled purification of the protein using Ni2+-affinity chromatography. Two independent experiments with two parallel measurements were conducted. [16] The enzyme is liberated from the -galactosyl moiety upon equatorial nucleophilic attack by water, which produces D-galactose. According to our assay, BaAG2 could hydrolyze universal substrates (pNPG and sucrose), maltose and maltose-like sugars such as turanose, maltotriose, melezitose and maltulose. 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 maltase has been characterized from C. albicans and four maltase-isomaltases from Scheffersomyces stipitis . 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. B. adeninivorans has been engineered for butanol production, applied in kits for the detection of hormones and dioxine in water as well as for manufacturing of tannase and cutinases [7]. Fructose, maltose, sucrose, isomaltose, palatinose, turanose, maltotriose, panose and melezitose were used as reference sugars. At pH 7.5, the activity was 81% from the maximum, and at pH 8.5, it was decreased to 52%. The maltase MAL62 of S. cerevisiae was used as a reference. These enzymes enter the small intestine in response to the hormone cholecystokinin, which is produced in response to the presence of nutrients. [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. 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. 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 studied the kinetics of the hydrolysis of pNPG, maltose, sucrose, maltotriose, melezitose, maltulose and turanose to calculate Km, Vmax, kcat and catalytic efficiency (kcat/Km) values for these substrates. Commercial amylolytic enzymes amyloglycosidase (glucoamylase) from Aspergillus niger, and -amylase from Aspergillus oryzae and S. cerevisiae MAL62 were used as reference enzymes.

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