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High molecular weight cyclic K K-1,4-glucan (referred to as cycloamylose) exhibited an artificial chaperone property toward three enzymes in different categories. The inclusion properties of cycloamylose effectively accommodated detergents, which keep the chemically denatured enzymes from aggregation, and promoted proper protein folding. Chemically denatured citrate synthase was refolded and completely recovered it's enzymatic activity after dilution with polyoxyethylenesorbitan buffer followed by cycloamylose treatment. The refolding was completed within 2 h, and the activity of the refolded citrate synthase was quite stable. Cycloamylose also promoted the refolding of denatured carbonic anhydrase B and denatured lysozyme of a reduced form. ß

The amylomaltase gene of the thermophilic bacterium Thermus aquaticus ATCC 33923 was cloned and sequenced. The open reading frame of this gene consisted of 1,503 nucleotides and encoded a polypeptide that was 500 amino acids long and had a calculated molecular mass of 57,221 Da. The deduced amino acid sequence of the amylomaltase exhibited a high level of homology with the amino acid sequence of potato disproportionating enzyme (D-enzyme) (41%) but a low level of homology with the amino acid sequence of theEscherichia coli amylomaltase (19%). The amylomaltase gene was overexpressed in E. coli, and the enzyme was purified. This enzyme exhibited maximum activity at 75°C in a 10-min reaction with maltotriose and was stable at temperatures up to 85°C. When the enzyme acted on amylose, it catalyzed an intramolecular transglycosylation (cyclization) reaction which produced cyclic α-1,4-glucan (cycloamylose), like potato D-enzyme. The yield of cycloamylose produced from synthetic amylose with an average molecular mass of 110 kDa was 84%. However, the minimum degree of polymerization (DP) of the cycloamylose produced by T. aquaticus enzyme was 22, whereas the minimum DP of the cycloamylose produced by potato D-enzyme was 17. The T. aquaticus enzyme also catalyzed intermolecular transglycosylation of maltooligosaccharides. A detailed analysis of the activity of T. aquaticus ATCC 33923 amylomaltase with maltooligosaccharides indicated that the catalytic properties of this enzyme differ from those of E. coliamylomaltase and the plant D-enzyme.

The thermostability of potato type L ␣-glucan phosphorylase (EC 2.4.1.1) was enhanced by random and site-directed mutagenesis. We obtained three single-residue mutations-Phe393Leu (F39L), Asn1353Ser (N135S), and Thr7063Ile (T706I)-by random mutagenesis. Although the wild-type enzyme was completely inactivated, these mutant enzymes retained their activity even after heat treatment at 60°C for 2 h. Combinations of these mutations were introduced by site-directed mutagenesis. The simultaneous mutation of two (F39L/N135S, F39L/T706I, and N135S/T706I) or three (F39L/N135S/T706I) residues further increased the thermostability of the enzyme, indicating that the effect of the replacement of the residues was cumulative. The triple-mutant enzyme, F39L/N135S/T706I, retained 50% of its original activity after heat treatment at 65°C for 20 min. Further analysis indicated that enzymes with a F39L or T706I mutation were resistant to possible proteolytic degradation.␣-Glucan phosphorylase (EC 2.4.1.1) catalyzes the reversible phosphorolysis of ␣-1,4 glucan and is widely distributed in microorganisms, plants, and animals. All known ␣-glucan phosphorylases require pyridoxal 5Ј-phosphate (PLP) for activity and seem to share a similar catalytic mechanism (17). Although all ␣-glucan phosphorylases belong to a large highly homologous group that includes glycogen phosphorylases from bacteria, yeast, and animals, starch phosphorylase from plants, and maltodextrin phosphorylases of bacteria, these enzymes from distinct origins are known to differ in their substrate preference and their mode of regulation (14,22).The wide distribution of ␣-glucan phosphorylase suggests that this enzyme plays an important role in the cellular metabolism of reserve polysaccharides: starch and glycogen. ␣-glucan phosphorylase is also potentially useful for the production of glucose 1-phosphate (G-1-P) and for the synthesis of linear amylose and other glucose polymers with various structures (6,23). To obtain ␣-glucan phosphorylase suitable for industrial applications, we previously reported the properties and primary structures of thermostable ␣-glucan phosphorylases from Bacillus stearothermophilus (19) and Thermus aquaticus (18). In these studies, we found that bacterial ␣-glucan phosphorylases appeared to have higher K m values of malto-oligosaccharides such as maltotetraose as the primer for glucan synthesis compared to plant ␣-glucan phosphorylases. To engineer useful ␣-glucan phosphorylase for various applications, the potato type L ␣-glucan phosphorylase gene was subjected to random mutagenesis by error-prone PCR, and thermostable variant enzymes were screened. Based on the results of random mutagenesis, we focused on specific amino acid replacements and obtained double and triple mutants by site-directed mutagenesis. We describe here the enhanced thermostability of potato type L ␣-glucan phosphorylase. Possible antiproteolytic degradation of the mutant enzymes is also described. MATERIALS AND METHODS Materials.Bacto tryptone and Bacto yeast extract ...

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The structural flexibility and conformational variety of the ions in room-temperature ionic liquids (RTILs) have significant effects on their physicochemical properties. To begin a systematic study of the thermodynamic properties of nonaromatic RTILs, 1-methyl-1-butylpiperidinium bis(fluorosulfonyl)amide ([Pip1,4][FSA]) was selected as the first sample. In addition to the rotational flexibility of the alkyl group, the [Pip1,4](+) cation has characteristic ring-flipping flexibility, which is very different from the behavior of the well-studied imidazolium-based cations. Calorimetry investigations using laboratory-made high-sensitivity calorimeters and Raman spectroscopy revealed that [Pip1,4][FSA] has two crystalline phases, Cryst-α and Cryst-β, and that every phase change is linked to conformational changes of both the cation and anion. Each phase change is also governed by very slow dynamics. The phase changes from supercooled liquid to Cryst-α and from Cryst-α to Cryst-β, which were observed only during heating, are not in fact phase transitions but structural relaxations. Notably, the temperatures of these structural relaxations exhibited heating rate dependences, from which the activation energy of the ring-flipping was estimated to be 38.8 kJ/mol. It is thought that this phenomenon is due to the associated conformational changes of the constituent ions in viscous surroundings.

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We studied the crystallization process of 1-butyl-3-methylimidazolium bromide ([C(4)mim]Br) using measurements of supersensitive scanning calorimetry, free induction decay (FID) signals of (1)H NMR, and direct observation. These three methods provided consistent, complementary results, which showed extremely slow dynamics at crystallization. This sample does not crystallize during the cooling process, loses mobility, and changes to a coagulated state, which is not the thermodynamic glass state. The FID signals and direct observation in the heating process indicate that the coagulated sample liquefies just before crystallization. The crystallization of [C(4)mim]Br does not occur from specialized locations such as the surface or wall of the sample tube but randomly in the liquid. The calorimetric measurements show that it takes 150 min for approximately 3 mg of this sample to crystallize perfectly. Conformational changes of the butyl group continue for approximately 330 min after crystallization. Such slow dynamics are thought to be due to the cooperative linking of crystallization and complex conformational changes in dense fields with high viscosity.