The sterol esterase from the ascomycete Ophiostoma piceae was immobilized on novel polyacrylate-based epoxy-activated carriers (Dilbeads TM). Six supports with particle sizes between 120-165 m were prepared varying the composition of monomers, crosslinkers and porogens. Their surface areas and porosities were determined by N 2 adsorption and mercury intrusion porosimetry. The pore volumes ranged from 0.63 to 1.32 cm 3 /g, but only Dilbeads TM RS and NK had narrow pore size distributions (with maxima at 33.5 and 67.0 nm, respectively). The distribution of the enzyme in the support was studied by fluorescence confocal microscopy. The immobilized esterase on Dilbeads TM TA showed a significant pH and thermal stability and was assayed in the continuous hydrolysis of cholesteryl esters-present in the pulp industry process waters- .
This study deals with the potential of Pichia pastoris X-33 for the production of penicillin G acylase (PGA) from Achromobacter sp. CCM 4824. Synthetic gene matching the codon usage of P. pastoris was designed for intracellular and secretion-based production strategies and cloned into vectors pPICZ and pPICZα under the control of AOX1 promoter. The simple method was developed to screen Pichia transformants with the intracellularly produced enzyme. The positive correlation between acylase production and pga gene dosage for both expression systems was demonstrated in small scale experiments. In fed-batch bioreactor cultures of X-33/PENS2, an extracellular expression system, total PGA expressed from five copies reached 14,880 U/L of an active enzyme after 142 h; however, 60% of this amount retained in the cytosol. The maximum PGA production of 31,000 U/L was achieved intracellularly from nine integrated gene copies of X-33/PINS2 after 90 h under methanol induction. The results indicate that in both expression systems the production level of PGA is similar but there is a limitation in secretion efficiency.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.