Pichia Pastoris Lab Report
The recombinant hemicellulolytic enzymes: an endo-xylanase (HXYN2) and a -xylosidase (HXYLA), both from H. grisea (CARVALHO, W. R. 2008; Cintra et al. 2016) and an -L-arabinofuranosidase (ABF3) from Penicilium Penicillium purpurogenum (Ravanal et al. 2010) were used in the hydrolysis tests. These three enzymes were heterologously expressed and produced in Pichia pastoris.
The enzyme-producing clones were induced according to the instructions of the Pichia Expression Kit (Invitrogen, USA). Interactions between the recombinant enzymes (HXYN2, HXYLA and ABF3) and a commercial cellulase mixture (Accellerase 1500, Dupont, Rochester, NY, USA) were analyzed. The sugarcane bagasse (SCB) used in the hydrolysis experiments was pre-treated by steam …show more content…
pastoris was concentrated by ultrafiltration using a Vivaspin (GE Healthcare, UK) membrane filter with a 10 kDa molecular weight cut-off, and directly used for the enzymatic hydrolysis of SCB. The interaction between the three recombinant enzymes for SCB hydrolysis was performed with different concentrations of HXYN2, HXYLA and ABF3 in different ratios according to a central composite rotational design (CCRD) 23, including six axial points and six central points, totalizing 20 assays according to the table 1.
The factors influence was assessed by analyzing the main effects and interaction between the factors and was calculated using Statistica 8.0 software (StatSoft Inc. Tulsa, OK, USA). Student’s t-test was used to analyze the level of significance. The Pareto chart was constructed with this software and shows the values of the Student’s t-test for each component of the medium. It was considered as response variable the quantification of reducing sugars by DNS …show more content…
The hydrolysis was performed using ACR in a concentration of 5 FPU.g-1. The EH was performed following the standard conditions in sequential and in simultaneous reactions. After the hydrolysis, glucose concentration was determined using the glucose oxidase kit (Doles Reagents ®) following the manufacturer's guidelines. The results were compared by statistical analyses (Student’s t testy-test). Significant differences were obtained when p ≤
The enzyme-producing clones were induced according to the instructions of the Pichia Expression Kit (Invitrogen, USA). Interactions between the recombinant enzymes (HXYN2, HXYLA and ABF3) and a commercial cellulase mixture (Accellerase 1500, Dupont, Rochester, NY, USA) were analyzed. The sugarcane bagasse (SCB) used in the hydrolysis experiments was pre-treated by steam …show more content…
pastoris was concentrated by ultrafiltration using a Vivaspin (GE Healthcare, UK) membrane filter with a 10 kDa molecular weight cut-off, and directly used for the enzymatic hydrolysis of SCB. The interaction between the three recombinant enzymes for SCB hydrolysis was performed with different concentrations of HXYN2, HXYLA and ABF3 in different ratios according to a central composite rotational design (CCRD) 23, including six axial points and six central points, totalizing 20 assays according to the table 1.
The factors influence was assessed by analyzing the main effects and interaction between the factors and was calculated using Statistica 8.0 software (StatSoft Inc. Tulsa, OK, USA). Student’s t-test was used to analyze the level of significance. The Pareto chart was constructed with this software and shows the values of the Student’s t-test for each component of the medium. It was considered as response variable the quantification of reducing sugars by DNS …show more content…
The hydrolysis was performed using ACR in a concentration of 5 FPU.g-1. The EH was performed following the standard conditions in sequential and in simultaneous reactions. After the hydrolysis, glucose concentration was determined using the glucose oxidase kit (Doles Reagents ®) following the manufacturer's guidelines. The results were compared by statistical analyses (Student’s t testy-test). Significant differences were obtained when p ≤