Bioprospecção de microrganismos produtores de celulases e otimização de sua produção enzimática

Abstract

Cellulose is the most abundant biopolymer on the planet, composed of glucose monomers linked by β-1,4-glycosidic bonds. Its degradation is carried out by enzymes known as cellulases, capable of hydrolyzing lignocellulosic material and releasing glucose. These enzymes can be obtained from various microorganisms, including fungi, bacteria, and actinobacteria. Although most research focuses on fungi due to their high productivity, bacteria also have great potential as cellulase producers, although they still receive less attention. In this context, this work sought to contribute to the study of cellulolytic bacteria through bioprospecting in different environmental samples: wood, decaying wood, bamboo, decaying bamboo, and leaf litter, cultivated in a selective medium containing carboxymethyl cellulose (CMC). For the isolates obtained, two preliminary tests were performed: (1) an assay in Congo red-stained CMC medium to calculate the enzymatic index (EI) and (2) degradation of filter paper strips after 10 days of culture. From these tests, five promising isolates were selected for further evaluation of total cellulolytic and endoglucanase enzyme activity. Subsequently, the two best-performing isolates underwent optimization of enzyme and biomass production using a 23 factorial design, which evaluated the effects of temperature (35 °C and 40 °C), pH (6 and 7), and the substrate used (CMC and treated corncob). Finally, molecular identification of the isolates was performed by 16S rRNA gene sequencing. A total of 40 microorganisms were obtained, of which isolates BA1A and BD6 stood out. These presented enzymatic indices of 4.07 and 7.45, respectively, in medium containing CMC. In the degradation of filter paper strips, isolate BD6 showed remarkable performance, reaching 100 % degradation in just 7 days. In the enzymatic activity analyses, BA1A presented values of 1.4796 ± 0.1686 U mg⁻1 (total cellulolytic) and 1.4585 ± 0.0714 U mg⁻1 (endoglucanases), while BD6 presented 1.4472 ± 0.1894 U mg⁻1 and 0.5203 ± 0.0101 U mg⁻1 , respectively. Optimization showed that the ideal conditions varied according to the microorganism, the substrate, and the evaluated response. It was observed that situations that increased total protein production did not always correspond to an increase in specific cellulolytic activity, indicating that some of the proteins were not involved in cellulose degradation. During molecular identification, isolate BA1A could not be sequenced, but its morphology suggests it is an actinobacterium. Isolate BD6, although not distinguished to the species level by 16S rRNA, was identified as belonging to the Enterobacteriaceae family, possibly Leclercia sp. or Enterobacter sp. Thus, this study contributes to expanding knowledge about cellulolytic bacteria and highlights the need for future investigations to clarify structural aspects, optimal conditions for enzymatic activity, and more precise molecular identification of the isolates.