␤-Lactamases are enzymes responsible for the hydrolysis of ␤-lactam antibiotics, being produced by several pathogenic bacteria. Clavulanic acid is a commercially and clinically important ␤-lactamase inhibitor, its extraction being possible by the application of aqueous two-phase system. In this study, clavulanic acid stability was investigated at different molar mass PEG (400, 1 000 and 20 000 g mol −1) and at different citrate concentrations (5 and 20%) PEG/citrate aqueous-two phase systems (ATPS), under different pH values (4.0-8.0). Clavulanic acid extraction was also evaluated. Low citrate concentration and PEG 20 000 (g mol −1) ATPS, at pH 6.0, were shown to be the systems that presented the highest clavulanic acid stability. Based on this, a factorial design 2 2 was used to evaluate CA extraction, being PEG (20, 25 and 30%) and citrate (5, 10 and 15%) concentrations the parameters evaluated. Clavulanic acid was extracted into the PEG-rich phase (133.66 mg L −1), the highest values of the partition coefficient and yield being K = 5.92, Y = 103.53%, respectively The ATPS was not only effective on clavulanic acid extraction, but also its degradation was minimal. These results clearly indicate that ATPS can be successfully applied as a first step for the purification of clavulanic acid.

This work aimed to compare the production of collagenolytic proteases produced by M. subtilissimus UCP1262 in submerged fermentation (SF) and solidstate fermentation (SSF) as well as extracting in aqueous two-phase system (ATPS).Collagenolytic protease production was performed in using MS-2 culture medium (SF) and soybean bran as substrate (SSF). Subsequently, the fermented liquid from both fermentations were used for the extraction of enzyme by ATPS, it was verified the influence of different variables from a factorial design 2 3 . In SSF the highest protease and collagenolytic activities were achieved with 362.66 U/mL and 179.81 U/mL, respectively.When compared with SF (26.33 and 18.70 U/mL) higher values were obtained in the activities. The protease partitioning from SF and SSF in ATPS showed a similar profile showing higher affinity for the polymer rich phase. The highest value for the response variable purification factor (3.49) was obtained in the system using SSF. Thus, SSF shows promise as a bioprocess for extracellular production of collagenolytic proteases, using of soybean bran as substrate had used sustainable raw material, aiming application this possible enzyme in the treatment of burns and postoperative scarring.