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

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Abstract

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, β-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.

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Funding

The authors would like to acknowledge the financial support provided by Fundação de Amparo à Pesquisa do Estado de São Paulo-FAPESP (process 2017/14629-9).

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Authors and Affiliations

  1. Instituto de Biociências Letras e Ciências Exatas (Ibilce), Câmpus São José do Rio Preto, Universidade Estadual Paulista (Unesp), São Paulo, Brazil

    Waldir Eduardo Simioni Pereira, Ronivaldo Rodrigues da Silva, Maurício Boscolo, Eleni Gomes & Roberto da Silva

  2. Instituto Federal de Educação, Ciência e Tecnologia de São Paulo campus Catanduva, Catanduva, São Paulo, Brazil

    Gabriela Salvador de Amo

  3. Universidade Federal de Mato Grosso do Sul, Campo Grande, Mato Grosso do Sul, Brazil

    Roberto Ruller

  4. Laboratório Nacional de Computação Científica, Petrópolis, Rio de Janeiro, Brazil

    Luciano Takeshi Kishi

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  1. Waldir Eduardo Simioni Pereira
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  2. Ronivaldo Rodrigues da Silva
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  3. Gabriela Salvador de Amo
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  4. Roberto Ruller
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  7. Eleni Gomes
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  8. Roberto da Silva
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Correspondence to Roberto da Silva.

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Pereira, W.E.S., da Silva, R.R., de Amo, G.S. et al. A Collagenolytic Aspartic Protease from Thermomucor indicae-seudaticae Expressed in Escherichia coli and Pichia pastoris. Appl Biochem Biotechnol 191, 1258–1270 (2020). https://doi.org/10.1007/s12010-020-03292-z

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  • DOI: https://doi.org/10.1007/s12010-020-03292-z

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