Ingrid Persson

Fungal cellulolytic enzyme production: A review

Abstract Cellulolytic enzyme production from fungi has been reviewed. Enzyme concentrations, enzyme productivities and enzyme yield have been compared for soluble substrates, purified cellulose and lignocellulosic substrates in relation to the estimated technical-economical goals of 20 filter paper units (FPU) ml−1 and 200 FPU litre−1 h−1. This can be achieved with Trichoderma reesei mutants in fed-batch cultures. The relative contributions from strain development, nature of substrate, substrate concentration and cultivation conditions on the improvements in enzyme production with Trichoderma species were found to be of the same order of magnitude.

Semicontinuous cellulase production in an aqueous two-phase system with Trichoderma reesei rutgers C30

Cellulases [see 1,4(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] from Trichoderma reesei, Rutgers C30, can be semicontinuously produced in an aqueous two-phase system composed of dextran and poly(ethylene glycol) using Solka Floc BW 200 as substrate. When substrate was intermittently added along with fresh top phase, which replaced the withdrawn top phase containing the produced enzymes, a yield of 1740 U endo-β-d-glucanase/g cellulose and 59.3 FPU/g cellulose was extracted with the top phase. Without fresh substrate added, a yield of 3920 U endo-β-d-glucanase/g cellulose and 127.7 FPU/g cellulose was extracted after five runs.

Influence of cultivation conditions on the production of cellulolytic enzymes withTrichoderma reesei Rutgers C30 in aqueous two-phase systems

Cellulolytic enzyme production in aqueous two-phase systems withTrichoderma reesei Rutgers C30 has been investigated. The influ ence of different phase systems, as well as addition of media compo nents and substrate on enzyme production have been studied. Extractive enzyme production in fed-batch cultivations was per formed in a phase system composed of PEG 8000 5%-Dextran T500 7% with 1% Solka-Floc BW 200 as substrate. The cellulolytic enzyme system was intermittently withdrawn with the top phase. Addition of media components every 24 h and cellulose every 72 h gave an aver age enzyme activity in the withdrawn top phase of 2.2 FPU/mL dur ing 170 h cultivation. The corresponding productivity was 18 FPU/lh. The productivity was increased to 24 FPU/l.h when media compo nents and cellulose were added every 72 h. The average enzyme con centration was then 1.6 FPU/mL. The results are discussed in relation to methods for cellulolytic enzyme production involving immobiliza tion and cell recycling.

Semicontinuous production of cellulolytic enzymes withTrichoderma reesei Rutgers C30 in an aqueous two-phase system

Cellulolytic enzyme production was studied in an aqueous twophase system, PEG 8000 5%-Dextran 7%, withTrichoderma reesei Rutgers C30 in a 7L fermentor. In batch cultivations, an average of 2.5 filter paper units (FPU)/mL were obtained in the top phase. In cultiva tions in regular media without polymers, the same enzyme concen tration was obtained. The enzyme yield was 205 FPU/g cellulose in the phase system, and 259 FPU/g cellulose in the regular medium. An extractive fed-batch cultivation was maintained in the aqueous twophase system for 360 h. The enzyme containing top phase was with drawn after phase separation. New cellulose substrate and nutrients were added with the new top phase. The enzyme extraction was started after 120 h of cultivation, and was repeated every 72 h. The total substrate concentration was 40 g/L. A maximum enzyme concentration of 4.8 FPU/mL was obtained in the withdrawn cell-free top phase. The enzyme yield was 148 FPU/g cellulose.

Production of β-glucosidase withAspergillus phoenicis QM329 in aqueous two-phase systems

The production of β-glucosidase withAspergillus phoenicis QM 329 was studied in two different aqueous two-phase systems: polyethylene glycol (PEG) 1550 7.5%/Dextran T2000 9.5% and PEG 8000 4%/polyvinyl alcohol (PVA) 14000 8%. The enzyme concentrations in the top phase of the phase systems were 3.4 IU/ml and 3.2 IU/ml, respectively, compared with 2.0 IU/ml obtained in a regular medium. The total amount of β-glucosidase obtained in the phase systems was 265 IU and 176 IU, respectively, compared with 200 IU in a regular medium.

In situ immobilized β-glucosidase fromAspergillus phoenicis QM 329

Aspergillus phoenicis QM 329 was found to grow in the shape of beads in shake flasks and in an air-lift fermentor. Initial culture pH, pH profile during cultivation, carbon source, inoculum size and fermentor configuration influenced the retainment of the β-glucosidase activty in the mycelium. Glucose and soluble starch produced beads with the highest activity and the best stability. Glucose-derived beads were more homogeneous in size and shape than the starch-derived beads. These beads kept their integrity for 10 d at 50° C. After 48 h hydrolysis of 50 g/l cellobiose 75% of the initial enzyme activity remained. The beads could be air-dried and alcohol-sterilized with only minor loss of activity. The size of the beads could be controlled by varying the size of the conidia inoculum. In an air-lift fermentor with a working volume of 1500 ml,900 ml beads with an average diameter of 2 mm (estimated to 37–45,000 beads) were produced in less than 3 d with glucose as carbon source. The beads held a β-glucosidase activity of 0.140 IU/bead determined with the pNP assay.

Continuous cellobiose hydrolysis using self-immobilized β-glucosidase fromAspergillus phoenicis QM 329 in a fluidized-bed reactor

Aspergillus phoenicis QM 329 was grown in the shape of beads in shake flasks and in an air-lift fermentor. The production of β-glucosidase started when the carbon source, glucose, was consumed. The β-glucosidase activity was retained in the beads at a pH below 6.0. The influence of bead diameter on enzyme activity and the pH and temperature optima for cellobiose hydrolysis has been studied. The enzyme-containing beads were used in a fluidized-bed reactor for continuous cellobiose hydrolysis, and a productivity of 2.0 g/L-h at a substrate conversion of 76% was obtained. The self-immobilized β- glucosidase is a stable and reusable enzyme with a half-life of 700 h when operating at 50°C and pH 4.8.