Optimization of C. crescentus β-Xylosidases and Expression of xynB1–5 Genes in Response to Agro-Industrial Waste
- 8 Downloads
The effect of the response surface methodology was applied to the production of Caulobacter crescentus (strain NA1000) β-xylosidases using corn cob. The components of the medium that presented the greatest influence on the enzyme production were chosen for optimization including the concentration of the corn cob residue and temperature variation. Optimal concentrations were determined by a central composite rotational design and a combination of 3.5% (w/v) corn cob concentration and 27 °C temperature was found to be optimal. When C. crescentus was cultivated using the optimized conditions, a maximum activity of 393.36 U/mL of β-xylosidases was achieved in 24-h cultures with a yield of 95% in real test conditions compared to the predicted one. In parallel, there was an increase of 3.6 times in the production of intracellular xylanases when compared to cultures without statistical application. In the C. crescentus genome, 5 genes that encode β-xylosidases are present. In order to evaluate which of them would be induced in the optimized conditions, the quantitative expression (qPCR) of the xynB1–xynB5 genes was evaluated in the presence of 1 or 3.5% corn cob (w/v) and surprisingly, all showed a constitutive expression in relation to the control. Assays of Western Blot performed with a polyclonal antiserum against C. crescentus β-xylosidase II in the optimized condition also did not show mass variation of the referred protein. These data strongly suggest that post-transcriptional controls are operating in the induced condition to increase the activity of C. crescentus β-xylosidases I-V, but β-xylosidase II. To our knowledge, this is the first time that these data are reported in literature for a bacterial system.
Keywordsβ-Xylosidases Expression Experimental design Bacteria Agro-industrial wastes
J.M. Corrêa was a PNPD/CAPES scholar. E.L. Santos was a CNPq scholar. R.C.G. Simão was a productivity scholar at Fundação Araucária.
Compliance with Ethical Standards
Conflict of interest
The authors declare that they have no conflicts of interest.
- 6.Graciano, L., Corrêa, J.M., Gandra, R.F., Seixas, F.A.V., Kadowaki, M.K., Sampaio, S.C., da Conceição-Silva, Osaku, C.A., Simão, R.C.G.: The cloning, expression, purification, characterization and modeled structure of Caulobacter crescentus β-xylosidase I. World J. Microbiol. Biotechnol. 28, 2879–2888 (2012)CrossRefGoogle Scholar
- 8.Justo, P.I., Corrêa, J.M., Maller, A., Kadowaki, M.K., Da Conceição-Silva, J.L., Gandra, R.F., Simão, R.C.G.: Analysis of the xynB5 gene encoding a multifunctional GH3-BglX β-glucosidase-β-Xylosidases-α-Arabinosidase member in Caulobacter crescentus. Antonie Van Leeuwenhoek 108, 993–1007 (2015)CrossRefGoogle Scholar
- 11.Zhang, S., Xie, J., Zhao, L., Pei, J., Su, E., Xiao, W., Wang, Z.: Cloning, overexpression and characterization of a thermostable β-xylosidase from Thermotoga petrophila and cooperated transformation of ginsenoside extract to ginsenoside 20(S)-Rg3 with a β-glucosidase. Bio-organ. Chem. 85, 159–167 (2019)Google Scholar
- 12.Evinger, M., Agabian, N.: Envelope-associated nucleoid from Caulobacter crescentus stalked and swarmer. J. Bacteriol. 132, 294–301 (1977)Google Scholar
- 13.Johnson, R.C., Ely, B.: Isolation of spontaneously derived mutants of Caulobacter crescentus. Genetics 86, 25–32 (1977)Google Scholar
- 19.Sambrook, J., Fritsch, E.F., Maniatis, T.: Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, New York (1989)Google Scholar
- 20.Bustin, S.A., Benes, V., Garson, J.A., Hellemans, J., Huggett, J., Kubista, M., Mueller, R., Nolan, T., Pfaffl, M.W., Shipley, G.L., Vandesompele, J., Wittwer, C.T.: The Miqe Guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin. Chem. 55, 611–622 (2009)CrossRefGoogle Scholar
- 23.Vieria, F.G.N., Christ, D., Graciano, L., Corrêa, J.M., Kadowaki, M.K., da Conceição-Silva, J.L., Gandra, R.F., Maller, A., Polizeli, M.L.T.M., Simão, R.C.G.: Experimental design for optimization of β-xylosidase production by A. fumigatus isolated from the Atlantic Forest (Brazil). J. Adv. Biol. Biotechnol. 21(3), 1–16 (2019)CrossRefGoogle Scholar
- 25.Midorikawa, G.E.O., Correa, C.L., Noronha, E.F., Ferreira-Filho, E.X., Togawa, R.C., Costa, M.M.C., Silva-Junior, O.B., Grynberg, P., Miller, R.N.G.: Analysis of the transcriptome in Aspergillus tamarii during enzymatic degradation of sugarcane bagasse. Front. Bioeng. Biotechnol. 6(123), 1–17 (2018)Google Scholar