A Collagenolytic Aspartic Protease from Thermomucor indicae-seudaticae Expressed in Escherichia coli and Pichia pastoris.

Proteases are produced by the most diverse microorganisms and have a wide spectrum of applications. However, the use of wild microorganisms, mainly fungi, for enzyme production has some drawbacks. They are subject to physiological instability due to metabolic adaptations, causing complications and impairments in the production process. Thus, the objective of this work was to promote the heterologous expression of a collagenolytic aspartic protease (ProTiN31) from Thermomucor indicae seudaticae in Escherichia coli and Pichia pastoris. The pET_28a (+) and pPICZαA vectors were synthesized containing the gene of the enzyme and transformed into E. coli and P. pastoris, respectively. The recombinant enzymes produced by E. coli and P. pastoris showed maximum activity at pH 5.0 and 50 °C, and pH 5.0 and 60 °C, respectively. The enzyme produced by P. pastoris showed better thermostability when compared to that produced by E. coli. Both enzymes were stable at pH 6.0 and 6.5 for 24 h at 4 °C, and sensitive to pepstatin A, ß-mercaptoethanol, and Hg2+. Comparing the commercial collagen hydrolysate (Artrogen duo/Brazil) and gelatin degradation using protease from P. pastoris, they showed similar peptide profiles. There are its potential applications in a wide array of industrial sectors that use collagenolytic enzymes.

Heterologous expression, purification and biochemical characterization of a new xylanase from Myceliophthora heterothallica F.2.1.4.

Myceliophthora heterothallica is a thermophilic fungus potentially relevant for the production of enzymes involved in the degradation of plant biomass. A xylanase encoding gene of this species was identified by means of RT-PCR using primers designed based on a xylanase coding sequence (GH11) of the fungus M. thermophila. The obtained gene was ligated to the vector pET28a(+) and the construct was transformed into Escherichia coli cells. The recombinant xylanase (r-ec-XylMh) was heterologously expressed, and the highest activity was observed at 55 °C and pH 6. The enzyme stability was greater than 70% between pH 4.5 and 9.5 and the inclusion of glycerol (50%) resulted in a significant increase in thermostability. Under these conditions, the enzyme retained more than 50% residual activity when incubated at 65 °C for 1 h, and approximately 30% activity when incubated at 70 °C for the same period. The tested cations did not increase xylanolytic activity, and the enzyme indicated significant tolerance to several phenolic compounds after 24 h, as well as high specificity for xylan, with no activity for other substrates such as CMC (carboxymethylcellulose), Avicel, pNPX (p-nitrophenyl-ß-D-xylopyranoside) and pNPA (p-nitrophenyl-α-L-arabinofuranoside), and is thus, of potential relevance in pulp bleaching.