Production and characterization of extracellular lignin peroxidase from immobilized Phanerochaete chrysosporium in a 10-l bioreactor

Abstract

Lignin peroxidase was produced by nylon-web immobilized Phanerochaete chrysosporium in carbon-limited medium in a modified Biostat ® E bioreactor. Mixing took place only with air and/or oxygen flow. During the growth phase, glucose ( 2 g l −1 ) was consumed in 26 to 28 h, almost twice as fast as under comparable conditions in 250-ml Erlenmeyer shake cultures. Veratryl alcohol was employed as an enzyme production activator. The production of the extracellular lignin peroxidase began approximately 70 h after inoculation, immediately after the addition of more glucose ( 1 g l −1 ). In semicontinuous production, a maximum activity of 730 U l −1 was reached after 130 h, and the bioreactor produced a total of ∼6200 U of lignin peroxidase during the first 5.5 days. After fermentation for a total of 330 h, the biocatalyst was stored for 1 week at 5°C, after which it was still capable of producing high ligninase activities in 400-ml repeated batch shake cultures, with an estimated total production of at least 5000 U per week for at least 1 month. With isoelectric focusing, a total of 14 peroxidases ( eight lignin peroxidases and two Mn ( II)- dependent peroxidases) were found from culture supernatants concentrated by laminar-flow ultrafiltration 13- to 21-fold. Enzymes of 40 000, 43 000, and 45 000 molecular weight were identified by gel electrophoresis. The isoelectric point varied from 4.00 to 4.95, and some homology was observed between P. chrysosporium peroxidases but not between P. chrysosporium and Pleurotus ostreatus ligninolytic enzymes.