Hydrogels in pharmaceutical formulations.

Phylogenetic surveys of soil ecosystems have shown that the number of prokaryotic species found in a single sample exceeds that of known cultured prokaryotes. Soil metagenomics, which comprises isolation of soil DNA and the production and screening of clone libraries, can provide a cultivation-independent assessment of the largely untapped genetic reservoir of soil microbial communities. This approach has already led to the identification of novel biomolecules. However, owing to the complexity and heterogeneity of the biotic and abiotic components of soil ecosystems, the construction and screening of soil-based libraries is difficult and challenging. This review describes how to construct complex libraries from soil samples, and how to use these libraries to unravel functions of soil microbial communities.

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The metagenomics of soil

Form and functionality of starch

Lipid vesicles and other colloids as drug carriers on the skin.

http://imb.savba.sk/~janecek/Papers/PDF/BBA_01.pdf

Relationship of sequence and structure to specificity in the alpha-amylase family of enzymes.

Analysis of errors in medical rapid prototyping models.

It has been estimated that less than 1% of the microorganisms in nature can be cultivated by conventional techniques. Thus, the classical approach of isolating enzymes from pure cultures allows the analysis of only a subset of the total naturally occurring microbiota in environmental samples enriched in microorganisms. To isolate useful microbial enzymes from uncultured soil microorganisms, a metagenome was isolated from soil samples, and a metagenomic library was constructed by using the pUC19 vector. The library was screened for amylase activity, and one clone from among approximately 30,000 recombinant Escherichia coli clones showed amylase activity. Sequencing of the clone revealed a novel amylolytic enzyme expressed from a novel gene. The putative amylase gene (amyM) was overexpressed and purified for characterization. Optimal conditions for the enzyme activity of the AmyM protein were 42°C and pH 9.0; Ca2+ stabilized the activity. The amylase hydrolyzed soluble starch and cyclodextrins to produce high levels of maltose and hydrolyzed pullulan to panose. The enzyme showed a high transglycosylation activity, making α-(1, 4) linkages exclusively. The hydrolysis and transglycosylation properties of AmyM suggest that it has novel characteristics and can be regarded as an intermediate type of maltogenic amylase, α-amylase, and 4-α-glucanotransferase.

Characterization of a Novel Amylolytic Enzyme Encoded by a Gene from a Soil-Derived Metagenomic Library

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.

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

Porcine pancreatic alpha-amylase activity on native starch granules is more accurately described as a function of surface area of the granules rather than of substrate concentration. The apparent K(m) of alpha-amylolysis of native starch from potato, maize, and rice expressed as a function of substrate concentration was largest for potato with a single value of V(max). However, the ratio of the slope of a Lineweaver-Burk plot to that of rice for enzymatic hydrolysis of native potato and maize starch were 7.78 and 2.58, respectively, which were very close to the ratio of surface area per mass of the two starch granules to that of rice. Therefore, the reciprocal of initial velocity was a linear function of the reciprocal of surface area for each starch granule. Surface area was calculated assuming the starch granules were spherical. The values obtained by this calculation were in good agreement with the value obtained by the photomicrographic method. By comparing enzymatic digestion of native maize granules to that of rice granules, it was concluded that the presence of pores in maize granules appeared to significantly affect overall rate of digestion after sufficient reaction time, but not at the very initial stage of hydrolysis.

Myo-Jeong Kim

Papers

Analysis of errors in medical rapid prototyping models.

Characterization of a Novel Amylolytic Enzyme Encoded by a Gene from a Soil-Derived Metagenomic Library

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

Porcine Pancreatic α-Amylase Hydrolysis of Native Starch Granules as a Function of Granule Surface Area

Targeted and sustained delivery of hydrocortisone to normal and stratum corneum-removed skin without enhanced skin absorption using a liposome gel