Heterologous expression of the antifungal ß-chitin binding protein CBP24 from bacillus thuringiensis and its synergistic action with bacterial chitinases.

The genome sequence analysis of Bacillus thuringiensis serovar konkukian S4 has shown to contain two chitinases (Chi74, Chi39) and two chitin-binding proteins (CBP50 and CBP24). The Chi74, Chi39 and CBP50 have been characterized previously. The chitin-binding protein CBP24 was cloned and heterologously expressed in Escherichia coli. The recombinant protein was purified using a Ni-NTA purification system. The purified protein was used to study its substrate binding activity using crystalline chitin variants as substrates. The Bmax and Kd values have shown that it preferably binds to ß-type of the crystalline chitin at a range of pH with peak activity between 5.5-7.5. To elucidate the role of CBP24 in the chitin degradation system of S4, the purified chitinases Chi74, Chi39 along with the ChiA from Serratia proteamcualans were used in different combinations with the CBP24 and chitinolytic activity was assayed. It was shown that the CBP24 acts synergistically with chitin degradation activity of bacterial chitinases non-specifically. Moreover, the CBP24 has shown antifungal activity against plant pathogenic fungi Fusarium oxysporum and Rhizoctonia solani. The present study will lead us to develop a technology for environmental friendly conversion of chitin to valuable products.

Thermodynamic properties of the ß-glucosidase from Thermotoga maritima extend the upper limit of thermophilicity.

Enzymes from thermophilic organisms are believed to be strong candidates for industrial applications due to their ability to withstand temperature-induced enzyme inactivation. The present study demonstrated molecular cloning, over-expression, purification and characterization of ß-glucosidase from Thermotoga maritima. The bglA gene with a capacity to encode a 51 kDa enzyme was heterologously expressed in E. coli M15. The enzyme was produced @130 mgL(-1) in LB media and @440 mgL(-1) in Dubos salt medium accounting 40-47 % of total cellular soluble proteins when lactose was used as an inducer. The enzyme showed a peak activity between pH and temperature range of 5.0-7.0 and 80-100 °C, respectively. The activity was fairly stable up to 140 °C. The turnover rate (kcat) of the enzyme was 187.1±20 s(-1), whereas the Km and Vmax values were 0.56 mM and 238±2.4 IU mg(-1) protein, respectively. The enzyme was shown to have half-life of 136, 71 and 12.6 h at 80, 90 and 100 °C, respectively. Thermodynamics parameters including melting temperature (130 °C), activation energy for inactivation (36.92 kJmole(-1)), enthalpy (33.73 kJmole(-1)), Gibb's free energy (127.96 kJmole(-1)) and entropy (-246.46 Jmole(-1)K(-1)) have shown that the enzyme have enhanced hydrophobic interactions to prevent its thermal unfolding. These features endorse the industrial applications of the enzyme.

Plasma homocysteine and DNA damage profiles in normal and obese subjects in the Pakistani population.

Dependence of plasma total homocysteine (tHcy) and DNA damage profiles on melanodialdehyde (MDA), oxidative stress, liver function tests (LFT), and lipids was studied in non-obese and obese subjects in the Pakistani population. Development of obesity is influenced by both genetic, biochemical and environmental factors. Plasma homocysteine (Hcy) and DNA damage profiles play a pivotal role in its progression. We studied 160 obesity patients and 160 lean subjects. Leukocytes were evaluated for DNA damage by comet assay and blood plasma for biochemical properties using commercial kits. Plasma Hcy level and DNA damage were strongly correlated with triglycerides (P < 0.000), LDL-cholesterol (P < 0.001), systolic blood pressure (P < 0.001), cholesterol (P < 0.004), MDA (P < 0.004) and total oxygen stress (P < 0.004) in obese individuals. Both Hyc and DNA damage were negatively associated with total anti-oxidant response and globulin. Both Hcy profile and DNA damage may have a role in the endothelium damage even in the normal range and are related to triglycerides, ALT, MDA, TOS, HDL- and LDL-cholesterol in the Pakistani population.

Double mutant of Aspergillus oryzae for improved production of L-dopa (3,4-dihydroxyphenyl-L-alanine) from L-tyrosine.

Aspergillus oryzae mutant strain UV-7 was further improved for the production of L-dopa from L-tyrosine using chemical mutation. Different putative mutant strains of the organism were tested for the production of L-dopa in triplicate shake-flask cultures. Among these putative mutants, the strain designated SI-12 gave a maximal production of L-dopa (444+/-14 mg of L-dopa/g of cells). The regulation of L-dopa from different carbon source solutions [initial substrate concentration (S(0))=30 g/l] by the mutant culture was investigated. At an initial pH (pH(0)) of 5.0 and a temperature (T) of 30 degrees C, 100% of sugars were utilized for product and cell mass formation, corresponding to final L-dopa product yield of 189+/-8 mg/g of substrate utilized and maximum volumetric and specific productivities of 145+/-5 mg/h per litre and 155+/-8 mg/h per g of cells respectively. There was up to 3-fold enhancement in product formation rate. This enhancement is, to our knowledge, the highest reported in the literature. To explain the kinetic mechanism of L-dopa formation and its thermal inactivation, the thermodynamic parameters were determined with the application of the Arrhenius model. Activation enthalpy and entropy for product formation, in the case of the mutant derivative, were 40 kJ/mol and 0.076 kJ.mol(-1).K(-1) for its production and 116 kJ/mol and 0.590 kJ.mol(-1).K(-1) for thermal inactivation respectively. The respective values for product formation and product de-activation were lower than the respective values for the parental culture. Therefore the mutant strain was thermodynamically more resistant to thermal denaturation during the product-formation process.