Amylase

About: Amylase is a research topic. Over the lifetime, 14164 publications have been published within this topic receiving 296069 citations.

Modes of action of acarbose hydrolysis and transglycosylation catalyzed by a thermostable maltogenic amylase, the gene for which was cloned from a Thermus strain.

Abstract: Several maltogenic amylases (EC 3.2.1.-) and closely related enzymes were cloned from gram-positive bacteria, including Bacillus species (4, 13). The enzymes were different from typical amylases in that they (i) were not secreted outside the cell, (ii) preferred cyclodextrins to starch or pullulan as a substrate, and (iii) exhibited both transglycosylation and hydrolysis activities on various substrates. They hydrolyzed starch and β-cyclodextrin mainly to maltose and pullulan to panose. Many of these properties, if not all, were shared by some amylolytic enzymes, including neopullulanases (EC 3.2.1.135) and cyclomaltodextrinases (EC 3.2.1.54; CDases) (7, 10, 17, 20, 26). The action modes of two maltogenic amylases (4, 13) and a CDase (17) isolated from three different Bacillus species were investigated by time course experiments with soluble starch or maltotriose as a substrate. The enzymes transferred a sugar molecule (donor) released after the hydrolysis of an α-(1,4)-glycosidic linkage to a reducing end of another sugar molecule (acceptor) by forming an α-(1,6)-glycosidic linkage. The coupled transglycosylation and hydrolysis activities of these enzymes were used for the production of branched oligosaccharides (BOS) from liquefied starch (15, 23), giving a more efficient process than the traditional one (31). The maltogenic amylases from Bacillus licheniformis (BLMA [13]), Bacillus stearothermophilus (BSMA, [4]), and B. subtilis (unpublished data) could hydrolyze acarbose, an amylase inhibitor, at different levels of efficiency. Acarbose is a pseudotetrasaccharide that has a pseudosugar ring at the nonreducing end [4,5,6-trihydroxy-3-(hydroxymethyl)-2-cyclohexene-1-yl] linked to the nitrogen of 4-amino-4,6-dideoxy-d-glucopyranose (4-amino-4-deoxy-d-quinovo-pyranose), which is linked via an α-(1,4)-glycosidic linkage to maltose. The pseudotrisaccharide (PTS) resulting from the hydrolysis of acarbose by these enzymes was transferred to the C-6 of glucose forming isoacarbose. This indicated that the catalytic properties unique to maltogenic amylases are probably due to differences in the tertiary structures of the proteins. The primary structures of maltogenic amylases in four regions were well conserved, and their secondary structure was likely to constitute a (β/α)8-barrel domain as with other amylolytic enzymes (11, 12). The characterization of amylolytic enzymes that exhibit transglycosylation and/or cyclodextrin hydrolyzing activity at the level of protein structure and enzymatic properties would be quite useful for understanding catalytic activities and substrate binding patterns more precisely. In this paper, we report on the cloning and physicochemical properties of another maltogenic amylase of a Thermus strain (ThMA) that was capable of hydrolyzing acarbose and transferring PTS to various acceptors. The enzyme was isolated from a thermophilic gram-negative bacterium, Thermus strain IM6501, and was more stable at high temperatures than other maltogenic amylases. Studies of the transferring activity of the thermostable enzyme by using acarbose and methylation of the resulting transfer products revealed additional modes of transglycosylation. Transglycosylation of a donor sugar molecule (PTS) to an acceptor molecule by forming an α-(1,3)-glycosidic linkage was demonstrated for the first time by using acarbose and ThMA.

Purification and properties of the raw-starch-digesting glucoamylases from Corticium rolfsii

Abstract: Corticium rolfsii AHU 9627, isolated from a tomato stem, is one of the strongest producers of a raw-starch-digesting amylase. The amylase system secreted by C. rolfsii AHU 9627 consisted of five forms of glucoamylase (G1–G5) and a small amount of α-amylase. Among these amylases, G1, G2 and G3 were able to hydrolyze raw starch. Five forms of glucoamylase were separated from each other and purified to an electrophoretically homogeneous state. The molecular masses were: G1 78 kDa, G2 78 kDa, G3 79 kDa, G4 70 kDa, and G5 69 kDa. The isoelectric points were: G1 3.85, G2 3.90, G3 3.85, G4 4.0, and G5 4.1. These glucoamylases showed nearly identical characteristics except that G4 and G5 were unable to hydrolyze raw starch.

Banana starch breakdown in the human small intestine studied by electron microscopy.

Abstract: Objective To determine the origin of the poor digestibility of banana starch granules in the human small intestine. Design The subjects received the same experimental meal. Setting Nutrition Research Unit, Laennec Hospital, CHU, Nantes. Subjects Six healthy young subjects. Interventions The digestion of raw green banana flour in the upper part of the gut was studied by the intubation technique. After ingestion of 30 g banana flour mixed with a complex meal, ileal samples were continuously collected during 14 h. In order to determine the structural nature of this resistant starch, the dried ileal samples were observed with scanning and transmission electron microscopy. Transmission electron microscopy was performed after treatment with periodic acid-thiosemicarbazide-silver nitrate. Results Banana starch proved very resistant to in vivo amylase hydrolysis since 84% of the starch ingested reached the terminal ileum. The microscopic observations showed that raw banana flour contained irregularly shaped dense starch granules with smooth surfaces. After their passage through the small intestine, starch granules appeared exocorroded, with porous surfaces, and some exhibited several irregular pits, crevices or holes by which the enzymes had penetrated and hydrolysed the inner part. Cell walls closely associated with starch granules could have hindered enzyme access to starch. Conclusions Encapsulation could be partly responsible for the low digestibility of starch in banana flour, together with the intrinsic resistance of banana starch granules.