Characterization and molecular cloning of thermostable alpha-amylase from Streptomyces sp.To1

Summary A new thermophilic Streptomyces sp. TO1, isolated from Tunisian soil, produced a thermostable alpha-amylase and pullulanase. The gene encoding for the alpha-amylase activity was cloned into the multicopy cloning plasmid pLM1 using S. lividans ZX1 as host strain. The ZX1 / pLM1 strain has the same activity than the initial TO1 strain and about 25 fold higher activity than the ZX1 strain. This alpha-amylase has an optimum of pH and temperature at 6 and 70 °C respectively.     

Novel Method for the Synthesis of 2′ -Phosphorylated Oligonucleotides

We have developed a new method for the preparation of oligodeoxyribonucleotides and oligo(2′-O-methylribonucleotides) that contain a 2′-phosphorylated ribonucleoside residue, and optimized it to avoid 2′ -3′ -isomerization and chain cleavage. Structures of the 2′ -phosphorylated oligonucleotides were confirmed by MALDI-TOF MS and enzymatic digestion, and the stability of their duplexes with DNA and RNA was investigated. 2′-Phosphorylated oligonucleotides may be useful intermediates for the introduction of various chemical groups for a wide range of applications.     

Antioxidant enzymes activities and bilirubin level in adult rat treated with lead

Lead toxicity is closely related to its accumulation in several tissues and its interference with bioelements, whose role is critical for several biological processes. Recently, oxidative stress has been proposed as a possible mechanism involved in lead toxicity. This study was carried out to investigate the effect of dose-dependent lead exposure on haematological and oxidative stress parameters. Adult male 'Wistar' rats (150-200 g) were divided into three groups: group [Pb 5] and group [Pb 15] received respectively 5 mg Pb(2+) (n=16) and 15 mg Pb(2+)/kg b.w. (n=16) as lead acetate solution i.p. for a period of seven days. Group [T] (n= 16) served as control and received 15 mg Na(+)/kg b.w. as sodium acetate solution i.p. for the same period. All animals were sacrificed 24 h after the last injection. Blood superoxide dismutase (SOD) and blood glutathione peroxidase (GPx) activities and plasma bilirubin level were measured. Liver was quickly excised for the estimation of alteration in lipid peroxidation indices (MDA). Lead exposure induces, in both treated groups, a marked decline in haematocrit and haemoglobin levels (p<0.01) when compared to control. The results show also a significant decrease (p<0.01) in SOD activity, but only in group [Pb 15]. SOD activity did not decrease in group [Pb 5] in comparison with control (p>0.05). However, lead exposure caused a light increase in GPx activity in group [Pb 15], which remains non-significant (p>0.05) compared to control. Group [Pb 5] did not record significant changes in the activity of GPx. Lead exposure for a period of seven days resulted in a significant (p<0.05) increase in bilirubin level in group [Pb 15] compared to control. The bilirubin level from rats of group [Pb 5] did not reach a statistical significance. Changes in liver MDA content in lead-exposed rats from [Pb 5] and [Pb 15] groups did not reach a statistical (p<0.05) significance. It is concluded that lead induces oxidative stress in a dose-dependent manner. No dose-dependent response was observed in blood GPx activity and liver MDA content. These results could be due to the short duration of the treatment.     

Two detergent stable alkaline serine-proteases from Bacillus mojavensis A21: Purification,characterization and potential application as a laundry detergent additive

Two detergent stable alkaline serine-proteases (BM1 and BM2) from Bacillus mojavensis A21 were purified. The molecular weights of BM1 and BM2 enzymes determined by SDS–PAGE were approximately 29,000 Da and 15,500 Da, respectively. The optimum pH values of BM1 and BM2 proteases were shown to be 8.0–10.0 and 10.0, respectively. Both enzymes exhibited maximal activity at 60 °C, using casein as a substrate.     

Chicken feathers: a complex substrate for the co-production of α-amylase and proteases by <Emphasis Type="Italic">B. licheniformis</Emphasis> NH1

This study is concerned with the co-production of alkaline proteases and thermostable α-amylase by some feather-degrading Bacillus strains: B. mojavensis A21, B. licheniformis NH1, B. subtilis A26, B. amyloliquefaciens An6 and B. pumilus A1. All strains produced both enzymes, except B. pumilus A1, which did not exhibit amylolytic activity. The best enzyme co-production was obtained by the NH1 strain when chicken feathers were used as nitrogen and carbon sources in the fermentation medium. The higher co-production of both enzymes by B. licheniformis NH1 strain was achieved in the presence of 7.5 g/l chicken feathers and 1 g/l yeast extract. Strong catabolic repression on protease and α-amylase production was observed with glucose. Addition of 0.5% glucose to the feather medium suppressed enzyme production by B. licheniformis NH1. The growth of B. licheniformis NH1 using chicken feathers as nitrogen and carbon sources resulted in its complete degradation after 24 h of incubation at 37°C. However, maximum protease and amylase activities were attained after 30 h and 48 h, respectively. Proteolytic activity profiles of NH1 enzymatic preparation grown on chicken feather or casein-based medium are different. As far as we know, this is the first contribution towards the co-production of α-amylase and proteases using keratinous waste. Strain NH1 shows potential use for biotechnological processes involving keratin hydrolysis and industrial α-amylase and proteases co-production. Thus, the utilization of chicken feathers may result in a cost-effective process suitable for large-scale production.     

Silencing activity of 2'-O-methyl modified anti-MDR1 siRNAs with mismatches in the central part of the duplexes

The thermodynamic properties of siRNA duplexes are important for their silencing activity. siRNAs with high thermodynamic stability of both the central part of the duplex and in the whole, usually display low silencing activity. Destabilization of the central part of the siRNA duplex could increase its silencing activity. However, mismatches located in the central part of the duplex could substantially decrease the amount of RNAi efficacy, hindering active RISC formation and function. In this study, we examined the impact of duplex destabilization by nucleotide substitutions in the central part (7-10 nt counting from the 5'-end of the antisense strand) of the nuclease-resistant siRNA on its silencing activity.     

Head-to-head bis-hairpin polyamide minor groove binders and their conjugates with triplex-forming oligonucleotides: studies of interaction with target double-stranded DNA

Two hairpin hexa(N-methylpyrrole)carboxamide DNA minor groove binders (MGB) were linked together via their N-termini in head-to-head orientation. Complex formation between these bis-MGB conjugates and target DNA has been studied using DNase I footprinting, circular dichroism, thermal dissociation, and molecular modeling. DNase I footprint revealed binding of these conjugates to all the sites of 492 b.p. DNA fragment containing (A/T)(n)X(m)(A/T)(p) sequences, where n>3, p>3; m=1,2; X = A,T,G, or C. Binding affinity depended on the sequence context of the target. CD experiments and molecular modeling showed that oligo(N-methylpyrrole)carboxamide moieties in the complex form two short antiparallel hairpins rather than a long parallel head-to-head hairpin. Binding of bis-MGB also stabilized a target duplex thermodynamically. Sequence specificity of bis-MGB/DNA binding was validated using bis-conjugates of sequence-specific hairpin (N-methylpyrrole)/(N-methylimidazole) carboxamides. In order to increase the size of recognition sequence, the conjugates of bis-MGB with triplex-forming oligonucleotides (TFO) were synthesized and compared to TFO conjugated with single MGB hairpin unit. Bis-MGB-oligonucleotide conjugates also bind to two blocks of three and more A.T/T.A pairs similarly to bis-MGB alone, independently of the oligonucleotide moiety, but with lower affinity. However, the role of TFO in DNA recognition was demonstrated for mono-MGB-TFO conjugate where the binding was detected mainly in the area of the target sequence consisting of both MGB and TFO recognition sites. Basing on the molecular modeling, three-dimensional models of both target DNA/bis-MGB and target DNA/TFO-bis-MGB complexes were built, where bis-MGB forms two antiparallel hairpins. According to the second model, one MGB hairpin is in the minor groove of 5'-adjacent A/T sequence next to the triplex-forming region, whereas the other one occupies the minor groove of the TFO binding polypurine tract. All these data together give a key information for the construction of MGB-MGB and MGB-oligonucleotide conjugates possessing high specificity and affinity for the target double-stranded DNA.     

Hydrolytic efficiency of Penicillium occitanis cellulase: kinetic aspects

Summary The parameters controlling the activity of the hyper-cellulolytic mutant Pol 6 of Penicillium occitanis cellulase were studied with regard to its efficiency for the hydrolysis of esparto grass cellulose. The optimal operational hydrolysis parameters were pH 5.0, temperature 45–55°C and 32 enzyme units/g of substrate. The maximum conversion ratio to reducing sugars was 84%. The cellulase was thermally quite stable, its activity decreasing by 20% when held at 50°C for 48 h. The cellulase was subject to end-product inhibition, with filter paper activity decreasing by 30% in the presence of 5% glucose. The results generally indicate the high efficiency of P. occitanis cellulase. It compares well with that from other microorganisms such as Trichoderma reesei.     

The effect of methylation on DNA replication in Salmonella enterica serovar typhimurium

The DNA adenine methylase of Salmonella typhimurium methylates adenine at GATC sequences. Strains deficient in this methylase are not well transformed by methylated plasmids, but unmethylated plasmids transform them at high frequencies. Hemimethylated daughter molecules accumulate after the transformation of dam(-) strains with fully methylated plasmids, suggesting that hemimethylation prevents DNA replication. It will also be shown that plasmids isolated from dam(-) bacteria are hemimethylated by restriction enzyme digestion. These results may explain why newly formed daughter molecules are not substrates for immediate reinitiation of DNA replication in dam(-) bacteria.