Fusion of Carbohydrate Binding Modules to Bifunctional Cellulase to Enhance Binding Affinity and Cellulolytic Activity

Bifunctional cellulase (glycoside hydrolase 5, GH5) from Bacillus sp. D04 having both endo- and exoglucanase activities was fused with two types of carbohydrate binding modules (CBMs). CBM3 from Bacillus sp. D04 and CBM9 from Thermotoga maritima Xyn10A were added to GH5 to hydrolyze microcrystalline cellulose (Avicel) as well as water-soluble cellulose (carboxymethyl cellulose, CMC). The optimum temperature of GH5 was 50oC, while it increased to 60oC for the fusion GH5-CBM3 and GH5-CBM9, indicating that addition of CBM increased the thermostability of the enzyme. Addition of CBM3 and CBM9 enhanced the GH5 affinity (K_M), for which K_M decreased from 104 to 33.9 ~ 35.1 mg/mL for CMC, and from 115 to 55.5 ~ 80.3 mg/mL for Avicel, respectively. The catalytic efficiency (k_cat/K_M) also increased from 4.80 to 5.36 ~ 6.46 (mL/mg)/sec for CMC, and from 1.77 to 2.40 ~ 4.45 (mL/mg)/sec for Avicel, respectively, by addition of CBM3 and CBM9.     

Identification of a nodD-dependent locus in the Rhizobium strain NGR234 activated by phenolic factors secreted by soybeans and other legumes

Transfer of the strain NGR234nodD 1 gene into the narrow host range R. trifolii strain ANU843 on either a 6.7-kb HindIII or 17-kb XhoI fragment broadens the host range of this bacterium to include the tropical legumes Vigna unguiculata, Glycine ussuriensis, Leucaena leucocephala, and siratro (Macroptilium atropurpureum). Contrary to previous data (Bassam et al. 1986), mutagenesis of the 17-kb XhoI fragment with a mini-Mu lac transposon (Mu dII1734) showed that a functional nodD 1 gene was essential for extended host range. Gene expression studies using both Mu dII1734 fusions and a promoter-cloning vector indicated that several loci, including the nodD 1 gene, are constitutively expressed. No evidence was found for regulation of the strain NGR234 nodD 1 gene by its product. Another locus nod-81, was induced only in the presence of exudates from various plant species, including soybean (Glycine max). Whereas the expression of nod-81 was dependent on the presence of a functional nodD 1 gene product, a regulatory nod-box DNA sequence was not detected 5' to this gene by using available oligonucleotide hybridization probes. The nod-81 locus was induced by genistein, daidzein, naringenin, and coumestrol from both cotyledon and root tissue of freshly germinated soybean seedlings. A broad spectrum of commercially available phenolic compounds stimulated induction of the nod-81 locus, including some that antagonize nod gene induction in other Rhizobium species. The nodD 1 gene product from strain NGR234 was shown to determine the spectrum of compounds that induce nod-81 expression.     

Mycelium of fungi isolated from mouldy foods inhibits Staphylococcus aureus including MRSA – A rationale for the re-introduction of mycotherapy?

Fungal mycelium capable of producing antibacterial agents was isolated from samples of apple, beetroot, lemon and orange; the mycelium of all isolates produced penicillin, while the apple and beetroot samples also produced the antibacterial mycotoxin patulin. The known penicillin-producing fungi were shown to produce penicillin, but not patulin. The mycelial discs of all of fruit and vegetable isolates, as well as the two known penicillin producing fungi, inhibited Staphylococcus aureus, and mycelium of all isolates inhibited MRSA, in contrast, only one of the two known penicillin-producers did so. The results are discussed in relation to the possibility of using the mycelium of Penicillium species in mycotherapy.     

Silver staining DNA in polyacrylamide gels

This protocol describes a simple silver staining method used to visualize DNA fragments and other organic molecules with unsurpassed detail following traditional polyacrylamide gel electrophoresis (PAGE). Sensitivity rivals radioisotopic methods and DNA in the picogram range can be reliably detected. The described protocol is fast (approximately 1 h) and is implemented using readily available chemicals and materials. To achieve the sensitivity and visual clarity expected, quality reagents and clean handling are important. The updated protocol described here is based on the widely used method of Bassam et al. (1991), but provides improved image contrast and less risk of staining artefacts.     

Identification, cloning, and expression of the Scytalidium acidophilum XYL1 gene encoding for an acidophilic xylanase

We cloned XYL1, a Scytalidium acidophilum gene encoding for an acidophilic family 11 xylanase. The XYL1p protein was expressed in Pichia pastoris using the pPICZalphaA expression plasmid. The secreted protein was purified by TAXI affinity column chromatography. The purified XYL1p showed an optimum activity at pH 3.2 and 56 degrees C. The Michaelis-Menten constants were determined.     

Electro-Optical Imaging Microscopy of Dye-Doped Artificial Lipidic Membranes

Artificial lipidic bilayers are widely used as a model for the lipid matrix in biological cell membranes. We use the Pockels electro-optical effect to investigate the properties of an artificial lipidic membrane doped with nonlinear molecules in the outer layer. We report here what is believed to be the first electro-optical Pockels signal and image from such a membrane. The electro-optical dephasing distribution within the membrane is imaged and the signal is shown to be linear as a function of the applied voltage. A theoretical analysis taking into account the statistical orientation distribution of the inserted dye molecules allows us to estimate the doped membrane nonlinearity. Ongoing extensions of this work to living cell membranes are discussed.     

Central antalgic activity of resveratrol

A single dose of resveratrol (25 μg/10μl) was injected directly into the right lateral cerebral ventricle (icv) of Wistar rats via an implanted cannula in order to study the analgesic properties of the compound. A control group of rats received 10 μl NaCl 0.9%. The lengthening of the time to reaction to painful stimuli was assessed in the radiant heat tail-flick latency time test. In this study, the response to painful stimuli of the animals treated with resveratrol had a bimodal profile with hypoalgesia or hyperalgesia. In the selected experimental conditions, resveratrol had a definite analgesic effect; the increase in time to reaction ranged from 100-120% (8 rats) to 600-700% (9 rats). In this experiment resveratrol exerts evident central antalgic effects in the majority of rats, which are related to the individual level of excitation and vigilance at baseline. Antinociceptive induced by resveratrol icv injection was maximal at 4-10 min and lasted no longer than 15 min. The effect of resveratrol to produce analgesia after a single icv injection may be interesting for preventing chronic pain.     

Purification and sequencing of cyanogen bromide fragments from ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum

As a part of the goal to determine the total sequence of Rhodospirillum rubrum ribulosebisphosphate carboxylase/oxygenase, the cyanogen bromide fragments were fractionated and sequenced (or partially sequenced). Twelve of the anticipated 14 peptides were obtained in highly purified form. The other two peptides were located, respectively, within a trytophanyl cleavage product (which overlapped with four CNBr fragments) and within an active-site peptide characterized earlier (which overlapped with three CNBr fragments). These overlaps coupled with amino and carboxyl terminal sequence information of the intact subunit and the availability of the sequence of the corresponding enzyme from higher plants permitted alignment of all fragments. Eight CNBr peptides were sequenced completely; four of the CNBr peptides consisted of more than 80 residues and were only partially sequenced as permitted by direct Edman degradation. Of the approximate 475 residues per subunit, 339 were placed in sequence. The lack of extensive conservation of primary structure between R. rubrum and higher plant carboxylases permits the tentative identifications of those regions likely to be functionally important.