Heterologous expression of a coagulant protease produced by Thermomucor indicae-seudaticae N31 in Escherichia coli and Pichia pastoris

Proteases are enzymes whose function is the hydrolysis of proteins and peptides, generating products of various sizes. They are produced by the most diverse living organisms and have a wide spectrum of applications. The objective of this work was to promote the heterologous expression of a coagulant protease produced by Thermomucor indicae-seudaticae in Escherichia coli and Pichia pastoris through the identification of the gene encoding the enzyme. For this, a comparison was made between the genome of T. indicae-seudaticae and Rhizomucor pusillus used as reference. The vectors pET 28a (+) and pPICZαA were synthesized containing the gene of interest for transformation in E. coli and P. pastoris respectively. Competent cells from five different strains of E. coli were transformed and submitted to the expression test at 20 ° C and 37 ° C for 24 hours, analyzed both the soluble and the insoluble fraction, where they apparently presented the expression with the highest amount in insoluble fraction. P. pastoris cells were transformed and submitted to expression for 72 hours. The crude enzyme extract of the expression product generated was biochemically characterized. The recombinant enzyme produced by E. coli presented maximum activity of 250.89 U mL-1 using caseine as substrate under conditions of pH 5.0 and temperature of 50 ° C, but with stability after 24 hours only at pH 6.0 and 6.5, thus demonstrating to be sensitive at alkaline pH. While the recombinant enzyme produced by P. pastoris presented maximum activity of 57.82 U mL-1 under conditions of pH 5.0 and temperature of 60 ° C. In addition, the enzyme produced by P. pastoris presented better thermostability when compared to that produced by E. coli and both enzymes had strong inhibition of the activity by pepstatin A, confirming that it belongs to the class of aspartic proteases and also by the mercury ion, which confirms the presence of disulfide bonds in the structure of the enzyme. (AU)