Molecular and computational approaches to characterize thermostable laccase gene from two xerophytic plant species.

Laccases are blue multicopper oxidases that carry out single electron transfers in the oxidation of phenols to quinones. In plants, they confer structural stability to the cell wall. Thermostable laccases were identified in xerophytes Cereus pterogonus and Opuntia vulgaris that could be used in biotechnology and industrial processes. Polyclonal anti-laccase antibodies were generated against purified laccase enzyme isoforms capable of 98-99% inhibition of the catalytic activity. Antibodies raised against lower molecular weight isoforms inhibited 70% of the catalytic activity of higher molecular forms. Only 20% inhibition was noted when assayed in reverse. A partial gene sequence of thermostable xerophytic laccase comprising 712 and 880 bp was identified employing cDNA as template. The nucleotide sequence was submitted to GenBank. The gene sequence was in silico translated into protein sequence and a 3-D structure was predicted using I-Tasser and Genesilico online servers that justified the experimental observations. Anti-laccase antibodies and nucleotide gene sequence of this thermostable plant laccase can be utilized for predicting laccase antigenic sequences and for cloning and expression of the thermostable eukaryotic laccase.

Characterization of xerophytic thermophilic laccase exhibiting metal ion-dependent dye decolorization potential.

Five laccase enzyme isoforms were isolated and purified to homogeneity from the cladodes of xerophytic Cereus pterogonus and Opuntia vulgaris plant species. Catalytic activity of all isoforms was enhanced 40 % by 1 mM Cu(2+) and 1 mM Mn(2+), whereas the activity was inhibited 100 % by 10 mM Fe(2+). Enzyme was found stable in 4 M urea and exhibited inactivity of 50 % in 8 M urea concentration. Ethylenediaminetetraacetic acid and cysteine-HCl were able to completely inhibit the enzyme activity at 1 mM and 100 µM, respectively. Preheated enzyme samples showed enhanced and stable catalytic activity in the presence of divalent cations over a period of 30 min compared with controls. In the presence of metal ions (1 mM Cu(2+) and 1 mM Mn(2+)), the preheated enzyme forms (60-90 °C) achieved 97 % of Malachite green and 98.75 % of Indigo blue (both at 2 %, w/v) dye decolorization in 12 h.

Biochemical and biophysical characterization of purified thermophilic xylanase isoforms in Cereus pterogonus plant spp.

Two thermostable xylanase isoforms T60 and T80 were purified to homogeneity from the cladodes of the xerophytic Cereus pterogonus plant species. After three consecutive purification steps, the specific activity of T60 and T80 isoforms were found to be 178.6 and 216.2 U mg⁻¹ respectively. The molecular mass of both isoforms was determined to be 80 kDa. The optimum temperature for T60 and T80 xylanase isoforms were 60 and 80 °C respectively. The pH was 5.0 for both isoforms. The presence of divalent metal ions (10 mM Co²âº) showed stimulatory effects of both catalytic activities, where as in the presence of Hg²âº, Cd²âº, Cu²âº showed inhibitory effect on these activities at all concentrations studied. The thermodynamic analysis of xylanase activity using denaturation kinetics and the presence divalent cations at 30-100 °C, showed lower ΔH, ΔS, and ΔG values at all the temperatures investigated. The melting temperature of purified T80 xylanase isoform as determined by TG/DTA analysis and it showed the unfolding temperature was 80 °C. The g value and hyperfine (A) value purified xylanase T80 isoform was 2.017 and 10.80 respectively. Immunoblot analysis with antiserum raised against the purified T80 xylanase isoforms revealed single immunolgically related polypeptides of 80 kDa, identical with the polypeptide band produced on SDS-PAGE. The results of double immunodiffusion against the T80 isoforms showed a single precipitin line indicating that the serum used was specific to these xylanase isoforms. The kinetic and thermodynamic properties suggested that xylanase from C. pterogonus may have a potential usage in various industries.

Thermophilic laccase from xerophyte species Opuntia vulgaris.

Two laccase temperature isoforms capable of oxidizing phenolic compounds to quinones were isolated and purified to homogeneity from the cladodes of the xerophyte species Opuntia vulgaris. These catalytically active proteins exhibit apparent molecular masses of 137 and 90 kDa. Under reducing conditions, both isoforms yielded a subunit molecular mass of 43 kDa, suggesting that the enzyme is a multimer of the 43 kDa subunit. The 137 kDa isoform when heated at 80°C for 3 min generated three polypeptide bands on activity stained polyacrylamide gels exhibiting 137, 90 and 43 kDa molecular forms. All isoforms of the enzyme exhibited an optimum pH of 10 when 2,6-dimethoxyphenol was used as a substrate. The optimum temperature of the 137 kDa enzyme form was noted to be 80°C and that of the 90 kDa enzyme form was 70°C. Denaturation kinetics of both the laccase isoforms carried out at their respective optimum temperatures for 30 min exhibited enzyme activity in excess of their t(1/2) values throughout the assay period. The K(m) for the 137 kDa form was determined to be 2.2 ± 0.3 mm and the V(max) was 2.8 ± 0.2 IU/mL. These high temperature stable laccase isoforms having alkaline pH optima can find significant industrial use.

Purification and characterization of thermophilic xylanase isolated from the xerophytic-Cereus pterogonus sp.

A thermo stable xylanase was purified and characterized from the cladodes of Cereus pterogonus plant species. The enzyme was purified to homogeneity by ammonium sulfate (80%) fractionation, ion exchange and size exclusion chromatography. The enzyme showed a final specific activity of 216.2 U/mg and the molecular mass of the protein was 80 KDa. The optimum pH and temperature for xylanase activity were 5.0 and 80 °C, respectively. With oat spelt xylan as a substrate the enzyme yielded a Km value of 2.24 mg/mL and a Vmax of 5.8 µmol min(-1) mg(-1). In the presence of metal ions (1 mM) such as Co(2+),Mn(2+), Ni(2+), Ca(2+) and Fe(3+) the activity of the enzyme increased, where as strong inhibition of the enzyme activity was observed with the use of Hg(2+), Cd(2+), Cu(2+), while partial inhibition was noted with Zn(2+) and Mg(2+). The substrate specificity of the xylanase yielded maximum activity with oat spelt xylan.