Production of fructosyl transferase by Aspergillus oryzae CFR 202 in solid-state fermentation using agricultural by-products.

Fructosyl transferase (FTase) production by Aspergillus oryzae CFR 202 was carried out by solid-state fermentation (SSF), using various agricultural by-products like cereal bran, corn products, sugarcane bagasse,cassava bagasse (tippi) and by-products of coffee and tea processing. The FTase produced was used for the production of fructo-oligosaccharides (FOS), using 60% sucrose as substrate. Among the cereal bran used, rice bran and wheat bran were good substrates for FTase production by A. oryzae CFR 202. Among the various corn products used, corn germ supported maximum FTase production, whereas among the by-products of coffee and tea processing used, spent coffee and spent tea were good substrates, with supplementation of yeast extract and complete synthetic media. FTase had maximum activity at 60 degrees C and pH 6.0. FTase was stable up to 40 degrees C and in the pH range 5.0-7.0. Maximum FOS production was obtained with FTase after 8 h of reaction with 60% sucrose. FTase produced by SSF using wheat bran was purified 107-fold by ammonium sulphate precipitation (30-80%), DEAE cellulose chromatography and Sephadex G-200 chromatography. The molecular mass of the purified FTase was 116.3 kDa by SDS-PAGE. This study indicates the potential for the use of agricultural by-products for the efficient production of FTase enzyme by A. oryzae CFR 202 in SSF, thereby resulting in value addition of those by-products.

Transgenic potato (Solanum tuberosum) tubers synthesize the full spectrum of inulin molecules naturally occurring in globe artichoke (Cynara scolymus) roots.

The ability to synthesize high molecular weight inulin was transferred to potato plants via constitutive expression of the 1-SST (sucrose:sucrose 1-fructosyltransferase) and the 1-FFT (fructan: fructan 1-fructosyltransferase) genes of globe artichoke (Cynara scolymus). The fructan pattern of tubers from transgenic potato plants represents the full spectrum of inulin molecules present in artichoke roots as shown by high-performance anion exchange chromatography, as well as size exclusion chromatography. These results demonstrate in planta that the enzymes sucrose:sucrose 1-fructosyltransferase and fructan:fructan 1-fructosyltransferase are sufficient to synthesize inulin molecules of all chain lengths naturally occurring in a given plant species. Inulin made up 5% of the dry weight of transgenic tubers, and a low level of fructan production also was observed in fully expanded leaves. Although inulin accumulation did not influence the sucrose concentration in leaves or tubers, a reduction in starch content occurred in transgenic tubers, indicating that inulin synthesis did not increase the storage capacity of the tubers.

Transgenic potato tubers accumulate high levels of 1-kestose and nystose: functional identification of a sucrose sucrose 1-fructosyltransferase of artichoke (Cynara scolymus) blossom discs.

By screening a cDNA library of artichoke (Cynara scolymus) blossom discs for fructosyltransferases, we isolated a clone designated Cy21. The deduced amino acid sequence shows homology to acid beta-fructosyl hydrolases and to the sucrose-fructan 6-fructosyltransferase (6-SFT) of barley. Transiently expressed in Nicotiana tabacum protoplasts, the Cy21 gene-product synthesized 1-kestose, indicating that Cy21 codes for a sucrose sucrose 1-fructosyltransferase (1-SST). The enzyme worked at physiologically relevant sucrose concentrations (25 mM sucrose). In the protoplast system, 1-kestose seemed to be the only fructan product of the 1-SST. The enzyme activity was not affected by pyridoxal-HCl, an inhibitor of both the beta-fructosyl hydrolase and the fructosyltransferase activity of invertases. The fructosyltransferase activity of the Cy21 gene-product, however, could be inhibited by Zn2+, Ag+ and Cu2+ ions. In artichoke plants the Cy21 transcript was highly abundant in primary roots and blossom discs. Transgenic potato tubers expressing Cy21 contain high levels of 1-kestose along with nystose and traces of fructosyl-nystose, supporting the conclusion that the Cy21 clone encodes a sucrose sucrose 1-fructosyltransferase.

The contribution of the cell wall to a transmembrane calcium gradient could play a key role in Bacillus subtilis protein secretion.

A weak Ca(2+)-binding site (Ka = 0.8 x 10(3) M-1, at pH 7) was identified in the mature part of levansucrase. An amino acid substitution (Thr-236-->Ile) in this site alters simultaneously the affinity for calcium, the folding transition and the efficiency of the secretion process of levansucrase. Moreover, the ability of the Bacillus subtilis cell wall to concentrate calcium ions present in the culture medium was studied. We confirm the results of Beveridge and Murray who showed that the concentration factor is about 100 to 120 times. This property preserves a high concentration of Ca2+ (> 2 mM) on the external side of the cytoplasmic membrane, even in the absence of further Ca2+ supplementation in the growth medium. Such local conditions allow the spontaneous unfolding-folding transition of levansucrase en route for secretion. Since several exocellular proteins of B. subtilis are calcium-binding proteins, we propose that the high concentration of calcium ion in the microenvironment of the cell wall may play a key role in the ultimate step of their secretion process.