Shukun Yu

α-1,4-Glucan lyases producing 1,5-anhydro-d-fructose from starch and glycogen have sequence similarity to α-glucosidases

Abstract In the past few years a novel enzyme α-1,4-glucan lyase (EC 4.2.2.13), which releases 1,5-anhydrofructose from starch and glycogen, has been cloned and characterized from red algae and fungi. Accumulated evidence indicates that the lytic degradation of starch and glycogen also occurs in other organisms. The present review focuses on the biochemical and molecular aspects of eight known α-1,4-glucan lyases and their genes from red algae and fungi. While the amino acid sequence identity is 75–80% among the α-1,4-glucan lyases from each of the taxonomic groups, the identity between the algal and fungal α-1,4-glucan lyases is only 25–28%. Notably database searches disclosed that the α-1,4-glucan lyases have a clear identity of 23–28% with α-glucosidases of glycoside hydrolase family 31, thus for the first time linking enzymes from the class of hydrolases with that of lyases. The alignment of lyases and α-glucosidases revealed seven well-conserved regions, three of which have been reported to be involved in catalysis and substrate binding in α-glucosidases. The shared substrate and inhibitor specificity and sequence similarity of α-1,4-glucan lyases with α-glucosidases suggest that related structural elements are involved in the two different catalytic mechanisms.

Methods for the assay of 1,5-anhydro-d-fructose and α-1,4-glucan lyase

Abstract 1,5-Anhydro- d - arabino -hex-2-ulose (1,5-anhydro- d -fructose, 1,5AnFru), produced by α-1,4-glucan lyase (EC 4.2.2.13) acting on starch, glycogen, or related d -glucose oligo- and polysaccharides as substrate, reacts with alkaline 3,5-dinitrosalicylic acid reagent (DNS) at room temperature (22 °C) within 10 min. The absorbance of the reaction mixture at 550 nm at the end of the reaction was proportional to a 1,5AnFru content in the range of 0.5 to 16 μmol (80 μg to 2.6 mg) mL −1 . 1,5AnFru determined by this colorimetric, one test tube one reagent method, was in good agreement with that found by 1 H-NMR spectroscopy and HPLC. The DNS method is also specific as other reducing sugars, such as glucose, maltose maltosaccharides, starch and glycogen do not give a colour with DNS at 22 °C; therefore, they do not interfere in the determination. The DNS method is applicable to lyase assay for both cell-free extracts and purified enzyme. Methods for reducing sugar analyses, based on the reduction of ferric and cupric ions, were examined for 1,5AnFru and they proved to be quantitative but in contrast to the DNS method, they were not specific. Instead of assaying 1,5AnFru, the activity of α-1,4-glucan lyase was analysed enzymatically by quantifying glucose or 4-nitrophenol released using maltose and 4-nitrophenyl α-maltopentaoside as substrate, respectively.

Physico-chemical Characterization of Floridean Starch of Red Algae

The current work reports on isolation and physico-chemical characterization of floridean starch from three species of agarophytic macro red algae. As determined by 1H-NMR spectroscopy, the average chain length and degree of branching frequency of this starch were 18 and 4.8, respectively. According to its amylopectin chain length distribution obtained by Dionex analysis, the crystalline polymorph of floridean starch from the red alga Gracilariopsis lemaneiformis was deduced to be C-type and this was further supported from its X-ray crystallographic pattern. Enzymatic analysis of its glucose 6-phosphate content showed that floridean starch had a low level of covalently linked phosphate (1 nmol per milligram starch) and this was further confirmed by 31P-NMR. The absorbance peak of floridean starch with iodine occurred at 527—530 nm and the blue value was low (0.1), indicating the absence of amylose, which was confirmed by differential scanning calorimetry (DSC). Floridean starch exhibited low gelatinization temperature, low viscosity, high clarity and little or no retrogradation upon repetitive freeze-thaw cycles, as studied by DSC and rapid viscosity analysis (RVA). These results are discussed in light of the functional properties and the structure of floridean starch.

1,5-Anhydro-D-fructose; a versatile chiral building block: biochemistry and chemistry.

There is a steadily increasing need to expand sustainable resources, and carbohydrates are anticipated to play an important role in this respect, both for bulk and fine chemical preparation. The enzyme alpha-(1-->4)-glucan lyase degrades starch to 1,5-anhydro-D-fructose. This compound, which has three different functional properties, a prochiral center together with a permanent pyran ring, renders it a potential chiral building block for the synthesis of valuable and potentially biologically active compounds. 1,5-Anhydro-D-fructose is found in natural materials as a degradation product of alpha-(1-->4)-glucans. The occurrence of lyases and the metabolism of 1,5-anhydro-D-fructose are reviewed in the biological part of this article. In the chemical part, the elucidated structure of 1,5-anhydro-D-fructose will be presented together with simple stereoselective conversions into hydroxy/amino 1,5-anhydro hexitols and a nojirimycin analogue. Synthesis of 6-O-acylated derivatives of 1,5-anhydro-D-fructose substituted with long fatty acid residues is carried out using commercially available enzymes. Those reactions lead to compounds with potential emulsifying properties. The use of protected derivatives of 1,5-anhydro-D-fructose for the synthesis of natural products is likewise reviewed. The potential utilization of this chemical building block is far from being exhausted. Since 1,5-anhydro-D-fructose now is accessible in larger amounts through a simple-enzyme catalyzed degradation of starch by alpha-(1-->4)-glucan lyase, the application of 1,5-anhydro-D-fructose may be considered a valuable contribution to the utilization of carbohydrates as the most abundant resource of sustainable raw materials.

Efficient purification, characterization and partial amino acid sequencing of two α-1,4-glucan lyases from fungi

alpha-1,4-Glucan lyases from the fungi Morchella costata and M. vulgaris were purified by affinity chromatography on beta-cyclodextrin-sepharose, followed by ion exchange and gel filtration. The purified enzymes produced 1,5-anhydro-D-fructose from glucose oligomers and polymers with alpha-1,4-glucosidic linkages, such as maltose, maltosaccharides, amylopectin, and glycogen. The lyases were basically inactive towards glucans linked through alpha-1,1, alpha-1,3 or alpha-1,6 linkages. For both enzymes the molecular mass was around 121,000 Da as determined by matrix-assisted laser desorption mass spectrometry. The pI for the lyases from M. costata and M. vulgaris was 4.5 and 4.4, respectively. The lyases exhibited an optimal pH range of pH 5.5 to pH 7.5 with maximal activity at pH 6.5. Optimal temperature was between 37 degrees C and 48 degrees C for the two lyases, depending on the substrates. The lyases were examined with 12 inhibitors to starch hydrolases and it was found that they were inhibited by the -SH group blocking agent PCMB and the following sugars and their analogues: glucose, maltitol, maltose, 1-deoxynojirimycin and acarbose. Partial amino acid sequences accounting for about 35% of the lyase polypeptides were determined. In the overlapping region of the sequences, the two lyases showed 91% identity. The two lyases also cross-reacted immunologically.

A group of α-1,4-glucan lyases and their genes from the red alga Gracilariopsis lemaneiformis: purification, cloning, and heterologous expression

We present here the first report of a group of alpha-1,4-glucan lyases (EC 4.2.2.13) and their genes. The lyases produce 1, 5-anhydro-D-fructose from starch and related oligomers and polymers. The enzymes were isolated from the red alga Gracilariopsis lemaneiformis from the Pacific coasts of China and USA, and the Atlantic Coast of Venezuela. Three lyase isozymes (GLq1, GLq2 and GLq3) from the Chinese subspecies, two lyase isozymes (GLs1 and GLs2) from the USA subspecies and one lyase (GLa1) from the Venezuelan subspecies were identified and investigated. GLq1, GLq3, GLs1 and GLa1 were purified and partially sequenced. Based on the amino acid sequences obtained, three lyase genes or their cDNAs (GLq1, GLq2 and GLs1) were cloned and completely sequenced and two other genes (GLq3 and GLs2) were partially sequenced. The coding sequences of the lyase genes GLq1, GLq2 and GLs1 are 3267, 3276 and 3279 bp, encoding lyases of 1088, 1091 and 1092 amino acids, respectively. The deduced molecular masses of the mature lyases from the coding sequences are 117030, 117667 and 117790 Da, respectively, close to those determined by mass spectrometry using purified lyases. The amino acid sequence identity is more than 70% among the six algal lyase isozymes. The algal GLq1 gene was expressed in Pichia pastoris and Aspergillus niger, and the expression product was identical to the wild-type enzyme.

Structure of 1,5-Anhydro-D-Fructose: X-ray Analysis of Crystalline Acetylated Dimeric Forms

Abstract Acetylation of 1,5-anhydro-D-fructose under acidic conditions gave two crystalline acetylated dimeric forms, which by X-ray analysis were shown to be diastereomeric spiroketals formed between C-2 and C-2/C-3. The structures of the compounds differed only at the configuration at C-2. Acetylation or benzoylation of 1,5-anhydro-D-fructose in pyridine yielded 3,6-di-O-acetyl-1,5-anhydro-4-deoxy-D-glycero-hex-3-enos-2-ulopyra-nos or crystalline 1,5-anhydro-3,6-di-O-benzoyl-4-deoxy-D-glycero-hex-3-enos-2-ulo-pyranose. 1. Presented at the 9th European Carbohydrate Symposium, Utrecht, Netherlands, July 6-1 1, 1997, Poster A138.

1,5-anhydro-D-fructose and its derivatives: biosynthesis, preparation and potential medical applications.

1,5-Anhydro-D-fructose (AF) was first found in fungi and red algae. It is produced by the degradation of glycogen, starch and maltosaccharides with α-1,4-glucan lyase (EC 4.2.2.13). In vivo, AF is metabolized to 1,5-anhydro-D-glucitol (AG), ascopyrone P (APP), microthecin and other derivatives via the anhydrofructose pathway. The genes coding for the enzymes in this pathway have been cloned, enabling the large-scale production of AF and related products in a cell-free reactor. The possible applications of these products in medicine have been evaluated using both in vitro and in vivo systems. Thus AF is a useful anticariogenic agent as it inhibits the growth of the oral pathogen Streptococcus mutans, impairing the production of plaque-forming polysaccharides and lactic acid. AF also shows anti-inflammatory and anticancer effects. AG is used as a diabetic marker for glycemic control. AG also stimulates insulin secretion in insulinoma cell lines. in vivo, APP has been shown to lengthen the life span of cancer-afflicted mice. It interferes with tumor growth and metastasis by its cidal effects on fast multiplying cells. Microthecin inhibits the growth of the human pathogen Pseudomonas aeruginosa PAO1, particularly under anaerobic conditions. The pharmaceutical usefulness of the other AF metabolites 1,5-anhydro-D-mannitol,1-deoxymannojirimycin, haliclonol, 5-epipentenomycin I, bissetone, palythazine, isopalythazine, and clavulazine remains to be investigated. In this review AF and its metabolites as the bioactive natural products for their pharmaceutical potentials are discussed.

1,5-Anhydro-d-fructose: regioselective acylation with fatty acids

Abstract Regioselective acylation of 1,5-anhydro- d -fructose was performed with dodecanoic acid to give 1,5-anhydro-6- O -dodecanoyl- d -fructose, chemically in 50% yield and enzymatically in quantitative yield. Quantitative conversions were also obtained using hexadecanoic and octadecanoic acids as acyl donors.

α-1,4-Glucan lyase-producing endophyte of Gracilariopsis sp. (Rhodophyta) from China

Purification of α-1,4-glucan lyase from red algae and fungi has previously been reported. The α-glucan lyase converts α-glucans to 1,5- anhydro-d-fructose. In this study, an endophyte was detected between the algal cells of Gracilariopsis sp., but not penetrating the cell walls. Histological staining was consistent with the endophyte being fungal and immunohistochemistry revealed that it possessed an α-1,4-glucan lyase differing from that of the host. The endophyte α-glucan lyase was recognized by antisera against both fungal and red algal α-glucan lyases, whereas the α-glucan lyase found in the algal tissue was recognized only by the antisera against algal α-glucan lyase. The lyases occur independently in the host and in the endophyte, and were sometimes detected simultaneously. The endophyte was only observed in Gracilariopsis sp. collected in China; no endophyte could be detected in Gracilariopsis sp. from California or in Gracilaria chilensis.