Caulobacter crescentus: Superexpressão homóloga do gene xynA1 e aplicação do extrato enzimático enriquecido com a Xilanase I em panificação

Abstract

Exploration of microbial diversity along with genetic engineering allows the use and optimization of enzyme sources for use in the biotechnology industry. Analysis of the sequenced genome of Caulobacter crescentus demonstrates that it has numerous genes induced by agroindustrial residues and biomass, which encode enzymes used in industrial processes, including cellulases, xylanases and five different β-xylosidases. These enzymes are part of a growing demand in various industrial sectors, such as baking, paper clarification, fabric layering, animal feed, among others. In the present work, the mutant C. crescentus strain that overexpresses the xynA1 gene (CCNA_02894) encoding a bacterial endo-1-β xylanase (EC: 3.2.1.8) was created. The xynA1 gene was subcloned into the pAS22 vector, which features an xylose-inducible promoter that replaced the original xynA1 gene promoter, leading to an increase of more than 20 copies of the cloned gene product per bacterial cell. The conditional mutant obtained was named BS-xynA1 and grown in minimal medium M2 supplemented with xylose 0.3% (w/v) addition of different agro-indurstrial residues. The highest extracellular xylanase activity was 17.22 U mL-1 and specific activity 278.64 U mg-1 after 18 hours of bacterial growth in the presence of 0.2% (w / v) corn straw. Aliquots of crude enzyme extract with this high specific activity were used in the different tests applicability of biotechnological processes in regional flour industry baking laboratory. In baking tests, enzymatic addition reduced beating time by 9% and increased bread height by 18.2% compared to control, in addition to improving alveolar structure formation. In alveograph tests the enzymatic addition generated an increase of the following parameters compared to the controls: 18.8% increase in mass extensibility; 8.3% increase in mass deformation energy, showing an increase in mass force; and 5.2% increase in elasticity; In addition, the enzymatic addition also led to a 6.0% reduction in toughness and a 20.6% reduction in the toughness/elasticity ratio in relation to the control, demonstrating the decrease in mass stiffness by enzyme action. With the results of the analysis it was concluded that the enzyme extract containing xylanase I has applicability as a processing aid in the bakery industry because of its ability to improve the rheological characteristics of the dough, increasing the bread volume and improving the alveolar structure of the crumb. To our knowledge to date, this is the first time that xylanase I produced by homologous expression in C. crescentus has been successfully used in the bakery industry. This was made possible by the unpublished strain C. crescentus BS-xynA1.