where is maltase produced

We expected that binding of the substrates or competitive inhibitors of the enzyme should increase its thermostability. [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. [21], Lactase is encoded by a single genetic locus on chromosome 2. Acarbose, palatinose, Tris and glucose (strong inhibitors of pNPG hydrolysis by BaAG2) and fructose that inhibited the reaction only weakly (see Table 2) were used as ligands. -Glucosidases have been popular objects to study protein evolution and phylogenesis [13,14,15,16], but they also have a biotechnological value. Every temperature point was assayed in triplicate. Isomaltose and isomaltose-like substrates palatinose and -methylglucoside were not hydrolyzed (Figure 5). Digestion of Presence of Tris raised the Tm value by 5.8 C, and of glucose by 4.4 C. Viigand K., Pnograjeva K., Visnapuu T., Alame T. Genome mining of non-conventional yeasts: Search and analysis of MAL clusters and proteins. WebLactase (EC 3.2.1.108) is an enzyme produced by many organisms. [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. [20], Mature human lactase consists of a single 160-kDa polypeptide chain that localizes to the brush border membrane of intestinal epithelial cells. 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]. It was produced by both enzymes, but its concentration was certainly higher in the case of BaAG2its concentration in the 72-h reaction sample was 5.2 g/L (Table S3). contact this location, Window Classics-West Palm Beach 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]. E. coli DH5 (Thermo Fisher Scientific, Waltham, MA, USA) was used for DNA cloning and plasmid production. Sequence identity of BaAG2 with other maltases was only moderate being the highest (51%) with the maltase MalT of Aspergillus oryzae. Lactase also catalyzes the conversion of phlorizin to phloretin and glucose. After cooling the samples on ice, residual activity of the enzyme was determined according to the hydrolysis of 1 mM pNPG at 30 C. Where is lactase produced? Chi Z., Ni X., Yao S. Cloning and overexpression of a maltase gene from. CH 18 quiz. 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. Ccontrol sample without the enzyme but containing 5 g/L bovine serum albumin (BSA) incubated at the same conditions for 74/96 h. Glucose release was quantified enzymatically. Of those, the IMA1 was most stable, and IMA5 the least stable [55]. The above-mentioned enzyme of L. starkeyi had equally high activity on maltose and isomaltose, but it also hydrolyzed maltotriose, isomaltotriose, panose, amylopectin and starch. One of those, the rna_ARAD1D20130g-encoded protein (BaAG2; 581 aa) was overexpressed in Escherichia coli, purified and characterized. 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. Transglycosylation of maltose by BaAG2 and ScMAL62. 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 Our assay confirmed that five above-mentioned -glucosidic sugars were indeed all assimilated. 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. Janeek ., Gabriko M. Remarkable evolutionary relatedness among the enzymes and proteins from the -amylase family. Inclusion in an NLM database does not imply endorsement of, or agreement with, Some carbohydrates are degraded into simple sugars, or monosaccharides (e.g., glucose, galactose) and are absorbed by the small intestine. Preprolactase, the primary translation product, has a single polypeptide primary structure consisting of 1927 amino acids. Other carbohydrates pass undigested into the large intestine, where they are digested by intestinal bacteria. SDs of two to three HPLC measurements at each time point were up to 20%. 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. Thr217 is located next to the catalytic nucleophile Asp216 in BaAG2 (Figure 2). A highly active endo-inulinase of B. adeninivorans was cloned and recently characterized [11]. We hypothesize that the ability to degrade malto-oligosaccharides and -glycosidic polysaccharides may be characteristic for the maltases of yeasts with deep phylogeny. Glucose and many other monosaccharides are also assimilated. the contents by NLM or the National Institutes of Health. The complete genome of. Further purification steps were conducted as described in [63]. [8][9] The U.S. Food and Drug Administration has not independently evaluated these products. 24850 Old 41 Ste 7 Isolation and study of -glucosidases of the most basal lineages to Saccharomycotina, for example Lipomyces starkeyi, should verify the correctness of this hypothesis. ; Investigation, T.V. and K.E. 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]. After we noticed that BaAG2 could hydrolyze soluble starch with the release of glucose, we performed a more detailed assay testing the hydrolysis of a set of polymeric -glucans: amylose and amylopectin from potato, glycogen from oysters and dextrans of Mw 20 and 110 kDa. 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 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. Hydrolysis of the polymers was evaluated according to the release of glucose and by TLC analysis of reaction products. and T.A. Literature mining revealed Tris as a competitive inhibitor of Bacillus brevis maltase with the Ki of 14.5 mM [56]. The effect of temperature on activity (left panel) and stability (right panel) of BaAG2. The same marker sugars were used in all assays but are marked only on TLC plate of glycogen degradation. Studies have linked the occurrence of lactase persistence to two different single-nucleotide polymorphisms about 14 and 22 kilobases upstream of the 5-end of the LPH gene. Two independent experiments with two parallel measurements were conducted. CBS Database. Surprisingly, we detected the ability of BaAG2 to hydrolyze polysaccharides that is a rather exceptional feature among maltases. 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. In the phylogram of -glucosidases from non-conventional yeasts, all eight Lipomyces enzymes clustered with those of B. adeninivorans [12]. Federal government websites often end in .gov or .mil. SD, standard deviation. At least two independent experiments were performed with two technical replicates. In the human enzyme, the lactase activity has been connected to Glu-1749, while Glu-1273 is the site of phlorizin hydrolase function. BaAG2 is the first maltase characterized from B. adeninivorans. Interestingly, isomelezitose was hydrolyzed by BaAG2 with release of palatinose (Figure S3). 2 terms. Assay of reaction course indicated that exo-hydrolysis occurreda glucose residue was stripped off from the oligomer. [7] Its primary commercial use, in supplements and products such as those from Lacteeze and Lactaid, is to break down lactose in milk to make it suitable for people with lactose intolerance. In Kelly et al. 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. The Saccharomycotina clades according to [50] are designated by background coloring. [24] This mutation has allowed almost half of the world's population to metabolize lactose without symptoms. 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. ; supervision, T.A. Marking below the sequence alignment is according to Clustal consensus showing conservation: * positions with fully conserved residue; : positions with residues of strongly similar properties; . Specifically, it catalyzes the hydrolysis of the -1,4-glucosidic linkages Identification, soluble expression, and characterization of a novel endo-inulinase from. ", "Lactose intolerance - Symptoms and causes", "Innovative Products for Food Industries: The Lactaid Story", "Agency Response Letter GRAS Notice No. [1] Lactase can be purchased as a food supplement, and is added to milk to produce "lactose-free" milk products. According to literature data, thermolability has been reported for some other yeast -glucosidases. The bootstrap values (1000 replicates) are shown at the nodes. The theoretical Mw value of 67,901 Da for the kcat calculation was computed in ExPASy Proteomics Server (http://expasy.org). For example, the four isomaltases of S. cerevisiae had all low thermostability. At pH 7.5, the activity was 81% from the maximum, and at pH 8.5, it was decreased to 52%. 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. 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]. [26], The lactase promoter is 150 base pairs long and is located upstream of the site of transcription initiation. All authors have read and agreed to the published version of the manuscript. As a library, NLM provides access to scientific literature. contact this location. 2781 Vista Pkwy N Ste K-8 [26] Cdx-2, HNF-1, and GATA have been identified as transcription factors. Ki values and inhibition mode for BaAG2 inhibitors of pNPG hydrolysis. CC LICENSED CONTENT, SPECIFIC ATTRIBUTION. 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. 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. Action: Starch to a disaccharide. Lactase supplements are sometimes used to treat lactose intolerance. Several GHs of yeasts and filamentous fungi exhibit transglycosylating activity. On the phylogram (Figure 9), B. adeninivorans clusters with Lipomyces starkeyi. The BaAG2 protein deduced from the gene is 580 aa long. West Palm Beach, FL33411 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). Thus, BaAG2 may have a biotechnological value. Maltase is an enzyme produced by the cells lining the small intestine that breaks down the disaccharide maltose. The activity measured after incubation at 10 C was taken as 100%. bruxellensis and Bacillus brevis maltases, BaAG2 was much more sensitive to Tris. Figure 4 shows that at temperature over 50 C, the activity of the enzyme rapidly declined. small intestine Students also viewed. Functional characterization of the -glucoside transporter Sut1p from, Dujon B.A., Louis E.J. Location of Val216 in the structure is marked with a red circle. In contrast, the maltase of Schizosaccharomyces pombe prefers maltose to sucrose [33]. [16] The enzyme is liberated from the -galactosyl moiety upon equatorial nucleophilic attack by water, which produces D-galactose. Hydrolysis of pNPG was assayed as in [16,40] according to the release of p-nitrophenol. 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. 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. At least three independent measurements for each substrate and concentration were made. At pH 4.5 and 4.4, the respective values were 93 and 15%. [26] Studies of hypolactasia onset have demonstrated that despite polymorphisms, little difference exists in lactase expression in infants, showing that the mutations become increasingly relevant during development. The activities (moles of glucose released per minute per mg of protein; U/mg) were calculated from initial velocities of the reaction. 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]. BaAG2 was competitively inhibited by a diabetes drug acarbose (Ki = 0.8 M) and Tris (Ki = 70.5 M). A remote but significant sequence homology between glycoside hydrolase clan GH-H and family GH31. The identity matrix of these proteins is presented in Supplementary Materials (Table S2). Our study showed that BaAG2 is a maltase. Thus, an Aspergillus enzyme with high transglycosylating activity was reported to produce panose and isomaltose from maltose [18,59,60]. 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]. The so-produced glucose is either utilized by the body or can be stored in the liver as glycogen (or called animal starch). 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. Maltase. H11 -glucosidase to 51% with Aspergillus oryzae maltase MalT (Table S2). Considering -glucosidases of yeasts, they have mostly been studied in S. cerevisiae as these enzymes are crucial in baking and brewing [22]. The maltase MAL62 of S. cerevisiae was used as a reference. Genomic DNA of B. adeninivorans was extracted using PowerMax Soil DNA Isolation Kit (MoBio Laboratories, Carlsbad, CA, USA) and the standard protocol by manufacturer. 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. 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]. and T.V. In contrast, maltotriose was hydrolysed by BaAG2 (Figure 5, Table 1), and due to that, its content decreased at prolonged transglycosylation reaction (Figure 8, Table S3). Gabriko M. Evolutionary history of eukaryotic -glucosidases from the -amylase family. Universal substrates are indicated by green, maltose and maltose-like sugars by yellow, and isomaltose and isomaltose-like sugars by blue bars. 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. For kinetic analysis, initial rates of p-nitrophenol or glucose release from substrates were measured at four to seven concentrations ranging from 0.13.0 mM for pNPG and 2.5250 mM for di- and trisaccharides. 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. At desired time points (2 h, 24 h, 74/96 h) aliquots were withdrawn and heated to stop the reaction. 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. Thermal stability assay of BaAG2 indicated that the enzyme was rather thermolabile: if kept for 30 min at temperatures above 37 C, its catalytic activity reduced significantly (Figure 4, right panel). The maximum composite likelihood model [71] with 1000 bootstrap replicates was applied. WebMaltase is found in plants, bacteria, yeast, humans, and other vertebrates. [(accessed on 27 November 2019)]; Teste M.A., Marie Franois J., Parrou J.L. SD, standard deviation. Webmaltase: [noun] an enzyme that catalyzes the hydrolysis of maltose to glucose. Small intestine. Produced In Site of Release pH Level; Carbohydrate Digestion: Salivary amylase: Salivary glands: Mount: Neutral: Pancreatic amylase: Pancreas: Small intestine: To produce the BaAG2 protein with a C-terminal His6-tag, an expression plasmid pURI3-AG2Cter was constructed by cloning the BaAG2 gene into pURI3-Cter vector similarly as in [63]. The respective extinction coefficients of BaAG2 [133,855 1/(M cm)] and ScMAL62 [148,990 1/(M cm)] were computed at ExPASy Proteomics Server (http://expasy.org). BaAG2 was proven to be a maltasehydrolysing -1,4 and -1,3 but not -1,6 linkages in glucose-containing substrates. The composition and linkage types of the tested substrates are indicated. ; Visualization, T.V., K.E., A.M., K.P. 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. 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]. The https:// ensures that you are connecting to the contact this location, Window Classics-Pembroke Park Amino acid signature of yeast -glucosidases, including B. adeninivorans AG2 (upper panel) and their designation on the three-dimensional (3D) structure of S. cerevisiae isomaltase IMA1 in complex with isomaltose (RCSB Protein Data Bank, PDB: 3AXH [29]) (lower panel). WebMade in: Salivary Gland. ; funding acquisition, T.A and T.V. 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. Clustal Omega for making accurate alignments of many protein sequences. Miami, FL33155 BaAG2 and ScMAL62 had no activity on dextrans. The overall reaction that lactase catalyzes is as follows: The catalytic mechanism of D-lactose hydrolysis retains the substrate anomeric configuration in the products. These enzymes enter the small intestine in response to the hormone cholecystokinin, which is produced in response to the presence of nutrients. When mining the genome of B. adeninivorans [2] for the genes related to maltose hydrolysis, we found two genes encoding intracellular GH13 family proteins. Tris also considerably increased thermostability of BaAG2 in a DSF assay (Figure 6). Samples from the reaction mixtures were withdrawn at designated time points, heated to terminate the reaction and analyzed for sugar composition by HPLC as described in Materials and Methods, paragraphs 4.5. and 4.6. However, these putative -glucosidases of L. starkeyi have not been cloned for protein expression and characterization. Phaffia rhodozyma) has been studied in detail, and synthesis of tri- and tetrasaccharides with -1,6 linkages was detected. 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. This work was funded by the Estonian Research Council (grant number PUT1050) to T.A. Some carbohydrates, such as cellulose, are not digested at all, despite being made of multiple glucose units. Atypically for yeast maltases, a low but clearly recordable exo-hydrolytic activity on amylose, amylopectin and glycogen was detected. Unraveling the function of glycosyltransferases in. Error bars are representing standard deviations (SD) of two independent experiments with two replicates. [26] The sequence is highly conserved in mammals, suggesting that critical cis-transcriptional regulators are located nearby. Many -glucosidases can transglycosylate and produce short oligosaccharides, especially at high concentration of the substrate [17,18,19,20,21]. Proteolytic enzymes, including trypsin and chymotrypsin, are secreted by the pancreas and cleave proteins into smaller peptides. and K.P. government site. Legal. Blastobotrys (Arxula) adeninivorans LS3 (CBS 8244) was kindly provided by Assoc. 20 g/mL of the enzyme (BaAG2 or ScMAL62) was incubated in 100 mM K-phosphate buffer (pH 6.5) with 0.2 g/L Na-azide and 250 mM or 500 mM maltose at 30 C up to 72 h. The samples with BaAG2 also contained 5 g/L BSA. Pancreatic lipase works with the help of the salts from bile secreted by the liver and the gallbladder. Language links are at the top of the page across from the title. 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. Reconstruction of ancestral metabolic enzymes reveals molecular mechanisms underlying evolutionary innovation through gene duplication. Made in: Small intestine. In most of the world's population, lactase transcription is down-regulated after weaning, resulting in diminished lactase expression in the small intestine,[22] which causes the common symptoms of adult-type hypolactasia, or lactose intolerance. In current work, one of them, BaAG2, was produced in E. coli and characterized in detail. ; Resources, T.A. The pH optimum of BaAG2 was in moderately acidic regionfrom 5.5 to 6.5 (Figure S2). The Tm of BaAG2 without a ligand was similar to that of maltase-isomaltase MAL1 of O. polymorpha51 C [16]. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. 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). 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]. FOIA The commercial enzymes hydrolyzed amylose, amylopectin and glycogen rapidly and with the expected pattern of products (Figure 7). and T.V. Maltases degrade maltose and maltose-like sugars, i.e., maltotriose, turanose and maltulose, but cannot degrade isomaltose and isomaltose-like sugars (-1,6 linkages) such as palatinose. A buffer solution for colorimetric comparison. [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. It is located in the brush border of the small intestine of humans and other mammals. Activity of BaAG2 on 100 mM sugars and 1 mM pNPG. [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. 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. Bacterial cells were grown overnight in LB-Amp medium at 37 C and diluted 100 times in autoinduction medium. The Ki values were calculated using enzyme kinetics module of the SigmaPlot (Systat Software, San Jose, CA, USA). Interestingly, BaAG2 was strongly and competitively inhibited not only by acarbose, a diabetes drug and a well-known inhibitor of -glucosidases, but also by Tris. Substrate specificity of -glucosidases can be in silico predicted based on so-called amino acid signaturea set of amino acids that locate in the vicinity of the substrate-binding pocket [12,15,27,28]. The protein was predicted as intracellularno secretion signal was detected by the SignalP program v. 5.0 [39]. The PCR product was cloned into a pJET vector from CloneJET PCR Cloning Kit (Thermo Fisher Scientific, Waltham, MA, USA), yielding pJET-BaAG2. Voordeckers K., Brown C.A., Vanneste K., van der Zande E., Voet A., Maere S., Verstrepen K.J. [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. ; Validation, T.V., K.E. Not typical for yeast maltases, BaAG2 had exo-hydrolytic activity on amylose, amylopectin and glycogen. [16] The removal of the D-glucose leaving group may be facilitated by Mg-dependent acid catalysis. The ability of B. adeninivorans to grow on sugars was assayed as in [12]. 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. Some other enzymes of B. adeninivorans have also been investigated, including alcohol dehydrogenase [8], extracellular glucoamylase [9] and invertase [10]. [12]. A maltase has been characterized from C. albicans [41] and four maltase-isomaltases from Scheffersomyces stipitis [12]. Compared to Br. 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. By 72 h of reaction, the maltotriose content was decreased and panose content increased to 2.6 g/L (Figure 8, Table S3). 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. 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. 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. 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. 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. * For the structure and linkage type of the substrates, see Figure 5. To date no additional data on MalT protein is available. The small intestine is where most chemical digestion takes place. 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. 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. In the case of ScMAL62, no hydrolysis of amylose was detected, and hydrolysis of glycogen and amylopectin became detectable only by 72 h of the reaction (Figure 7).
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