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The high-specificity self-assembling nature of DNA makes the molecule a candidate for the template for the construction of molecular devices. In order to construct a functional device, the template must be positioned onto a predetermined site on a substrate to allow external connections, and the components must be properly aligned onto the template. A key factor is the high yield of binding, especially when the device consists of many components. Such high yield requires that the bases of the template DNA be exposed so that its counterpart can interact freely, and the template be stretched to avoid folding or coiling that hampers the interaction. We have developed electrokinetic DNA manipulation method, by which a double-stranded DNA is stretched and immobilized bridging over an electrode pair, with the molecular ends anchored while the middle part is left free to interact with foreign molecules. However, double-stranded DNA has a closed structure and the bases inside are not easily accessible. To have a template DNA with accessible bases, two methods are developed in this paper; one being the direct stretching of single-stranded DNA, and the other being the use of a recombination protein to make the bases of double-stranded DNA accessible. The former has an advantage in its simplicity, and the latter in its mechanical stability. We expect that these stretch-and-positioned DNA with accessible base-pairs will lead to the high-yield molecular construction. [ABSTRACT FROM AUTHOR]

The enzyme β-mannanase (EC 3.2.1.78) cleaves mannans of hemicellulose fraction of plant cell walls, which are resistant to the gastrointestinal tract of animals and poultry. One of the important directions of the use of β-mannanases is the production of mannose-containing hydrolysates from vegetable raw materials. The products of mannans hydrolysis - mannose and manno-oligosaccharides - exhibit bifidogenic and immunostimulatory effects. As a result of genetic modification of the strain B. subtilis performed by us there has been received an active producer of β- mannanase. The encoding part of the gene of the β-mannanase B. subtilis 168 was amplified by PCR and cloned into the vector pET28b (+) which contains hexahistidine sequence (6His-Tag), and then after induced gene expression in E. coli cells, the recombinant protein was purified by affinity chromatography on a Ni2+-NTA agarose. The purified protein has a molecular weight of 40 kDa and a specific activity of 774 U/mg, the highest activity of β-mannanase was observed at 40° C and pH 7.0. The investigation of pH and temperature stability of the enzyme showed that at pH 7.0 and a temperature of 30° C β-mannanase maintained about 70% activity during the 6 hours of incubation. The maximum degree of enzymatic hydrolysis of the galactomannan of locust bean gum, being 85%, was observed at the optimum temperature and pH and with the enzyme dosage of 9 U/g of substrate for 3 h. The identification of hydrolysis products showed the presence of mannose, mannotriose, mannotetrose and mannopentose in the derived hydrolysates that indirectly indicates the affiliation of β- mannanase B. subtilis to the enzymes of endo-effect. [ABSTRACT FROM AUTHOR]