Exploring and exploiting starch-modifying amylomaltases from thermophiles

Starch is a staple food present in water-insoluble granules in many economically important crops. It is composed of two glucose polymers: the linear α-1,4-linked amylose and amylopectin with a backbone of α-1,4-glycosidic bonds and α-1,6-linked side chains. To dissolve starch completely in water it needs to be heated; when it cools down too much the starch solution forms a thermo-irreversible gel. Amylomaltases (EC 2.4.1.25) are enzymes that transfer a segment of an α-1,4-D-glucan to a new 4-position in an acceptor, which may be glucose or another α-1,4-D-glucan. Acting upon starch, amylomaltases can produce cycloamylose or a thermoreversible starch gel, both of which are of commercial interest.

Database mining and transcriptional analysis of genes encoding inulin-modifying enzymes of Aspergillus niger

As a soil fungus, Aspergillus niger can metabolize a wide variety of carbon sources, employing sets of enzymes able to degrade plant-derived polysaccharides. In this study the genome sequence of A. niger strain CBS 513.88 was surveyed, to analyse the gene/enzyme network involved in utilization of the plant storage polymer inulin, and of sucrose, the substrate for inulin synthesis in plants. In addition to three known activities, encoded by the genes suc1 (invertase activity; designated sucA), inuE (exo-inulinase activity) and inuA/inuB (endo-inulinase activity), two new putative invertase-like proteins were identified. These two putative proteins lack N-terminal signal sequences and therefore are expected to be intracellular enzymes. One of these two genes, designated sucB, is expressed at a low level, and its expression is up-regulated when A. niger is grown on sucrose- or inulin-containing media. Transcriptional analysis of the genes encoding the sucrose- (sucA) and inulin-hydrolysing enzymes (inuA and inuE) indicated that they are similarly regulated and all strongly induced on sucrose and inulin. Analysis of a creA mutant strain of A. niger revealed that expression of the extracellular inulinolytic enzymes is under control of the catabolite repressor CreA. Expression of the inulinolytic enzymes was not induced by fructose, not even in the creA background, indicating that fructose did not act as an inducer. Evidence is provided that sucrose, or a sucrose-derived intermediate, but not fructose, acts as an inducer for the expression of inulinolytic genes in A. niger

Paenibacillus granivorans sp. nov., a new Paenibacillus Species which Degrades Native Potato Starch Granules

From a native potato starch-degrading enrichment culture, strain A30 had been isolated and had tentatively been identified as a member of the Bacillus firmus/lentus group. In this paper the isolate A30 is further characterized using phylogentic analysis of the 16S rDNA and determination of a number of additional phenotypic characteristics. These data are compared to those of Paenibacillus amylolyticus, P. chibensis, and P. thiaminolyticus. It is concluded that strain A30 is a new Paenibacillus species, for which the name Paenibacillus granivorans is suggested.

Kinetic properties of an inulosucrase from Lactobacillus reuteri 121

Inulosucrases catalyze transfer of a fructose moiety from sucrose to a water molecule (hydrolysis) or to an acceptor molecule (transferase), yielding inulin. Bacterial inulin production is rare and a biochemical analysis of inulosucrase enzymes has not been reported. Here we report biochemical characteristics of a purified recombinant inulosucrase enzyme from Lactobacillus reuteri. It displayed Michaelis-Menten type of kinetics with substrate inhibition for the hydrolysis reaction. Kinetics of the transferase reaction is best described by the Hill equation, not reported before for these enzymes. A C-terminal deletion of 100 amino acids did not appear to affect enzyme activity or product formation. This truncated form of the enzyme was used for biochemical characterization.

The Unique Branching Patterns of Deinococcus Glycogen Branching Enzymes Are Determined by Their N-Terminal Domains

Glycogen branching enzymes (GBE) or 1,4-α-glucan branching enzymes (EC 2.4.1.18) introduce α-1,6 branching points in α-glucans, e.g., glycogen. To identify structural features in GBEs that determine their branching pattern specificity, the Deinococcus geothermalis and Deinococcus radiodurans GBE (GBEDg and GBEDr, respectively) were characterized. Compared to other GBEs described to date, these Deinococcus GBEs display unique branching patterns, both transferring relatively short side chains. In spite of their high amino acid sequence similarity (88%) the D. geothermalis enzyme had highest activity on amylose while the D. radiodurans enzyme preferred amylopectin. The side chain distributions of the products were clearly different: GBEDg transferred a larger number of smaller side chains; specifically, DP5 chains corresponded to 10% of the total amount of transferred chains, versus 6.5% for GBEDr. GH13-type GBEs are composed of a central (β/α) barrel catalytic domain and an N-terminal and a C-terminal domain. Characterization of hybrid Deinococcus GBEs revealed that the N2 modules of the N domains largely determined substrate specificity and the product branching pattern. The N2 module has recently been annotated as a carbohydrate binding module (CBM48). It appears likely that the distance between the sugar binding subsites in the active site and the CBM48 subdomain determines the average lengths of side chains transferred.

Kartläggning och testning av läs- och skrivsvårigheter hos vuxna med svenska som andraspråk

Denna uppsats behandlar kartläggning och testning av läs- och skriv-svårigheter hos vuxenstuderande som har svenska som andraspråk. Arbetet tar sin utgångspunkt i den fonologiska förklaringsmodellen av dyslexi/specifika läs- och skrivsvårigheter, vilken framhåller att svårigheterna med att läsa och skriva bottnar i en svaghet i det fonologiska systemet. Om man inte har möjlighet att testa en person på modersmålet, vilket annars utgör den mest rättvisande metoden för bedömning av läs- och skrivsvårigheter, kan man enligt vissa forskare ändå använda fonologiska tester på majoritetsspråket, förutsatt att personen vistats i målspråkslandet under en tid. Forskare framhåller också vikten av att vid bedömningen väga in kringliggande faktorer avseende social och kulturell bakgrund, samt hur situationen kring läsning och skrivning hittills sett ut, innefattande läs- och skriv-inlärningen på modersmålet. I denna studie sker, med hjälp av kartläggningssamtal och tester av den fonologiska förmågan, en bedömning av fyra kvinnors läs- och skrivsvårigheter. Undersökningen visar att det går att få fram en djupare och mer tillförlitlig information om vilka läs- och skrivsvårigheterna är och möjlig orsak till dessa, om man kombinerar fonologiska test med ingående kartläggningssamtal. Att enbart använda sig av en av metoderna kan göra att man vid bedömningen missar information som är viktigt för förståelsen av svårigheterna. Studien visar också på vikten av en tidig identifiering av specifika läs- och skrivsvårigheter. Det finns ett tydligt samband mellan dessa svårigheter och en negativ självbild, något som kan leda till passivitet, uppgivenhet och undvikande strategier. Att få en förklaring till svårigheterna och adekvat stöttning, innebär för många en upprättelse och ny motivation

Detection of methanogenic archaea in seawater particles and the digestive tract of a marine fish species

A methanogen-specific nested PCR approach was used to detect methanogenic archaea in seawater particles of the North Sea and the feces and the digestive tract of flounder (Platichthys flesus), a fish found in the North Sea. A number of 16S rDNA sequences with 97.6-99.5% similarity to Methanococcoides methylutens were found in the seawater particles as well as the digestive tract and fecal samples. (C) 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved

Properties of the Glucan Branching Enzyme of the Hyperthermophilic Bacterium Aquifex aeolicus

Glucan branching enzymes are responsible for the synthesis of α(1→6) glycosidic bonds in glycogen and amylopectin. The glucan branching enzyme of the hyperthermophile Aquifex aeolicus is the most thermoactive and thermostable glucan branching enzyme described. The gene encoding this glucan branching enzyme was overexpressed in E. coli and purified using γ-cyclodextrin affinity chromatography. Subsequently, the enzyme was subjected to a biochemical characterization. The optimum temperature for activity was 80°C, and the enzyme was stable up to 90°C. Its thermostability may be explained by the relatively high number of aromatic amino acid residues present, in combination with a relatively low number of glutamine/asparagine residues. The Km for amylose was 4 µM and the Vmax was 4.9 U/mg of protein (at optimal pH and temperature). The side-chain distribution of the branched glucan formed from amylose was determined.