Characterization of a multi-tolerant tannin acyl hydrolase II from Aspergillus carbonarius produced under solid-state fermentation

Background Tannases are enzymes with biotechnological potential produced mainly by microorganisms as filamentous fungi. In this context, the production and characterization of a multi-tolerant tannase from Aspergillus carbonarius is described. Results The filamentous fungus A. carbonarius produced high levels of tannase when cultivated under solid-state fermentation using green tea leaves as substrate/carbon source and tap water at a 1:1 ratio as the moisture agent for 72 h at 30°C. Two tannase activity peaks were obtained during the purification step using DEAE-Cellulose. The second peak (peak II) was purified 11-fold with 14% recovery from a Sepharose CL-6B chromatographic column. The tannase from peak II (tannase II) was characterized as a heterodimeric glycoprotein of 134.89 kDa, estimated through gel filtration, with subunits of 65 kDa and 100 kDa, estimated through SDS-PAGE, and 48% carbohydrate content. The optimal temperature and pH for tannase II activity was 60°C and 5.0, respectively. The enzyme was fully stable at temperatures ranging from 20-60°C for 120 min, and the half-life (T1/2) at 75°C was 62 min. The activation energy was 28.93 kJ/mol. After incubation at pH 5.0 for 60 min, 75% of the enzyme activity was maintained. However, enzyme activity was increased in the presence of AgNO3 and it was tolerant to solvents and detergents. Tannase II exhibited a better affinity for methyl gallate (Km = 1.42 mM) rather than for tannic acid (Km = 2.2 mM). Conclusion A. carbonarius tannase presented interesting properties as, for example, multi-tolerance, which highlight its potential for future application.

Statistical optimization for tannase production by Aspergillus tubingensis in solid-state fermentation using tea stalks

Background A sequential statistical strategy was used to optimize tannase production from Aspergillus tubingensis using tea stalks by solid-state fermentation. Results First, using a Plackett-Burman design, inoculum size and incubation time (among seven tested variables) were identified as the most significant factors for tannase yield. The effects of significant variables were further evaluated through a single steepest ascent experiment and central composite design with response surface analysis. Under optimal conditions, the experimental value of 84.24 units per gram of dry substrate (U/gds) closely matched the predicted value of 87.26 U/gds. Conclusions The result of the statistical approach was 2.09 times higher than the basal medium (40.22 U/gds). The results were fitted onto a second-order polynomial model with a correlation coefficient (R²) of 0.9340, which implied an adequate credibility of the model.

Genetic analysis of violacein biosynthesis by Chromobacterium violaceum

Chromobacterium violaceum presents a distinctive phenotypic characteristic, the production of a deep violet pigment named violacein. Although the physiological function of this pigment is not well understood, the sequencing of the genome of this bacterium has given some insight into the mechanisms and control of violacein production. It was found that erythrose-4-phosphate (E4P), a precursor to aromatic amino acid biosynthesis, is produced by the non-oxidative portion of the hexose monophosphate pathway, since it lacks 6-phosphogluconate dehydrogenase. All genes leading from E4P plus phosphoenolpyruvate to tryptophan are present in the genome. Nevertheless, these genes are not organized in an operon, as in E. coli, indicating that other mechanisms are involved in expression. The sequencing data also indicated the presence and organization of an operon for violacein biosynthesis. Three of the four gene products of this operon presented similarity with nucleotide-dependent monooxygenases and one with a limiting enzyme polyketide synthase. As previously suggested, genes encoding proteins involved in quorum sensing control by N-hexanoyl-homoserine-lactone, an autoinducer signal molecule, are present in the bacterial genome. These data should help guide strategies to increase violacein biosynthesis, a potentially useful molecule