Lorena Cardoso Cintra

Molecular cloning, expression and insulin reduction activity of a thioredoxin 1 homologue (TRX1) from the pathogenic fungus Paracoccidioides lutzii

The dimorphic fungi Paracoccidioides spp. are the etiological agents of paracoccidioidomycosis (PCM), a prevalent systemic mycosis in Latin America. The Paracoccidioides lutzii response to oxidative stress is largely unexplored. Thioredoxins (TRX) are involved in the regulation of the redox environment in the cell, responding to oxidative stress in several organisms. In this study, we describe the isolation and characterization of a cDNA encoding a thioredoxin 1 from yeast cells from P. lutzii. The cDNA codes for a 12kDa protein containing the characteristic thioredoxin active site. The thioredoxin 1 gene was expressed in Escherichia coli and the isolated thioredoxin 1 recombinant protein as the native PlTRX1 from yeast cells showed insulin reduction activity in vitro. We also showed by semi-quantitative RT-PCR analysis that the expression of thioredoxin 1 gene was induced in response to H2O2 and may exert an antioxidant activity in vivo. Our results suggest that the thioredoxin 1 may play an important role in controlling the redox status in P. lutzii which may contribute to this organisms virulence.

Characterization of a recombinant xylose tolerant β-xylosidase from Humicola grisea var. thermoidea and its use in sugarcane bagasse hydrolysis

One full-length β-xylosidase gene (hxylA) was identified from the Humicola grisea var. thermoidea genome and the cDNA was successfully expressed by Pichia pastoris SMD1168. An optimization of enzyme production was carried out, and methanol was found to be the most important parameter. The purified enzyme was characterized and showed the optimal conditions for the highest activity at pH 7.0 and 50°C, being thermostable by maintaining 41% of its activity after 12h incubated at 50°C. HXYLA is a bifunctional enzyme; it showed both β-xylosidase and α-arabinfuranosidase activities. The Km and Vmax values were 1.3mM and 39.1U/mg, respectively, against 4-nitrophenyl β-xylopyranoside. HXYLA showed a relatively strong tolerance to xylose with high Ki value of 603mM, with the xylose being a non-competitive inhibitor. HXYLA was successfully used simultaneously and sequentially with an endo-xylanase for analysis of synergism in the degradation of commercial xylans. Furthermore, commercial cellulases supplementation with HXYLA during sugarcane bagasse hydrolysis increased hydrolysis in 29%. HXYLA is distinguished from other β-xylosidases by the attractive characteristics for industrial applications such as thermostability, high tolerance xylose and saccharification of biomass by convert xylan into fementable monosaccharides and improve cellulose hydrolysis.

Improvement of bread making quality by supplementation with a recombinant xylanase produced by Pichia pastoris

Xylanases (EC 3.2.1.8) are hydrolytic enzymes, which randomly cleave the β-1,4-linked xylose residues from xylan. The synthetic gene xynBS27 from Streptomyces sp. S27 was successfully cloned and expressed in Pichia pastoris. The full-length gene consists of 729 bp and encodes 243 amino acids including 51 residues of a putative signal peptide. This enzyme was purified in two steps and was shown to have a molecular weight of 20 kDa. The purified r-XynBS27 was active against beechwood xylan and oat spelt xylan as expected for GH 11 family. The optimum pH and temperature values for the enzyme were 6.0 and 75 °C, respectively. The Km and Vmax were 12.38 mg/mL and 13.68 μmol min/mg, respectively. The r-XynBS27 showed high xylose tolerance and was inhibited by some metal ions and by SDS. r-XynBS27 was employed as an additive in the bread making process. A decrease in firmness, stiffness and consistency, and improvements in specific volume and reducing sugar content were recorded.