Production of XynX, a large multimodular protein of thermoanaerobacterium sp., by protease-deficient Bacillus subtilis strains [corrected].

XynX of Thermoanaerobacterium sp. [corrected] is a large, multimodular xylanase of 116 kDa. An Escherichia coli transformant carrying the entire xynX produced three active truncated xylanase species of 105, 85, and 64 kDa intracellularly. The Bacillus subtilis WB700 transformant with the xynX, a strain deficient in seven proteases including Vpr, secreted two active truncated xylanase species of 65 and 44 kDa. The B. subtilis WB800 transformant with xynX, a strain deficient in eight proteases including Vpr and WprA, secreted more active enzymes, 8.46 U ml(-1), mostly in the form of 105 and 85 kDa, than the WB700 transformant, 6.93 U ml(-1). This indicates that the additional deletion of wprA enabled the WB800 to secrete XynX in its intact form. B. subtilis WB800 produced more total enzyme activity than E. coli (1,692 ± 274 U vs. 141.9 ± 27.1 U), and, more importantly, secreted almost all the enzyme activity. The results suggest the potential use of B. subtilis WB800 as a host system for the production of large multimodular proteins.

Protection of Penaeus monodon against white spot syndrome virus by inactivated vaccine with herbal immunostimulants.

To improve the immune response in tiger shrimp Penaeus monodon against WSSV infection, juveniles (350 ± 10 mg) were vaccinated with formalin-inactivated WSSV and fed with herbal immunostimulants. The methanolic extracts of herbal immunostimulants such as Acalypha indica, Cynodon dactylon, Picrorrhiza kurrooa, Withania somnifera and Zingiber officinalis were incorporated in formulated diets at different concentrations; 250 (ED(1)), 500 (ED(2)), 1000 (ED(3)) and 2000 (ED(4)) mg kg(-1) of feed and fed for 60 days after vaccination. After 30 and 60 days intervals of feeding, the shrimps were challenged with WSSV, which were isolated and propagated from the infected crustaceans. The shrimps fed with control diets (C(1)) succumbed to death within 5 days after WSSV challenge, when no vaccination and immunostimulations were given. The other control groups (C(2) and C(3)) had slight improvements in all parameters including survival. The percentage survival was significantly (P < 0.05) increased to 30, 50 and 60% in the ED(2), ED(3) and ED(4) diets respectively after 60 days challenging. The better haematological, biochemical and immunological parameters were also found in the herbal extracts supplemented diets fed vaccinated shrimps. The present study revealed that the combined effect of immunostimulation and vaccination helped to boost the immune system against WSSV infection and hence this application can be adopted for shrimp culture.

The role of carbohydrate-binding module (CBM) repeat of a multimodular xylanase (XynX) from Clostridium thermocellum in cellulose and xylan binding.

A non-cellulosomal xylanase from Clostridium thermocellum, XynX, consists of a family-22 carbohydratebinding module (CBM22), a family-10 glycoside hydrolase (GH10) catalytic module, two family-9 carbohydrate-binding modules (CBM9-I and CBM9-II), and an S-layer homology (SLH) module. E. coli BL21(DE3) (pKM29), a transformant carrying xynX', produced several truncated forms of the enzyme. Among them, three major active species were purified by SDS-PAGE, activity staining, gel-slicing, and diffusion from the gel. The truncated xylanases were different from each other only in their C-terminal regions. In addition to the CBM22 and GH10 catalytic modules, XynX(1) had the CBM9-I and most of the CBM9-II, XynX(2) had the CBM9-I and about 40% of the CBM9-II, and XynX(3) had about 75% of the CBM9-I. The truncated xylanases showed higher binding capacities toward Avicel than those toward insoluble xylan. XynX(1) showed a higher affinity toward Avicel (70.5%) than XynX(2) (46.0%) and XynX(3) (42.1%); however, there were no significant differences in the affinities toward insoluble xylan. It is suggested that the CBM9 repeat, especially CBM9-II, of XynX plays a role in xylan degradation in nature by strengthening cellulose binding rather than by enhancing xylan binding.

A new approach for enhanced multiplication of arbuscular mycorrhizal fungi and isolation of ITS regions from Glomus deserticola and Laccaria fraterna.

Rootlets induced from the petiole base of L. purpureus, using IAA and kinetin was used for enhanced multiplication of arbuscular mycorrhizal (AM) fungus, G. deserticula. Using conserved short arbitrary oligonucleotides, as specific primers, we amplified the ITS-region, a molecular marker for fungal identification, from the genomic DNA extracted from cultured spores of G. deserticola, and genomic DNA extracted from the mycelium of L. fraterna. The capacity of fungal colonization and subsequent spore formation of G. deserticola, compared with the natural root system was evaluated. This technology would provide a simple way to multiply AM fungi and to produce spores without microbial contamination useful for further molecular characterization.

Use of sucrose-agar globule with root exudates for mass production of vesicular arbuscular mycorrhizal fungi.

A sucrose-agar globule (SAG) was newly introduced to increase production of the vesicular arbuscular mycorrhizal (VAM) fungal spores, Gigaspora gigantea and Glomus fasciculatum. An SAG inoculum and a sucrose-agar globule with root exudates (SAGE) inoculum were prepared, and their spore productions were compared with a soil inoculum. When the SAGE was used as the inoculum on sucrose-agar medium plates the number of spores was increased (35% more than the soil inoculum). After the soil inoculum and SAGE were inoculated on an experimental plant, Zingiber officinale, the percentage root colonization, number of VAM spores, and dry matter content were analyzed. It was observed that the SAGE showed a higher percentage of root colonization (about 10% more), and increases in the number of spores (about 26%) and dry matter (more than 13%) for the two VAM fungal spores than the soil inoculum. The results of this study suggested that the SAGE inoculum may be useful for the mass production of VAM fungi and also for the large scale production of VAM fungal fertilizer.