M.D. Busto

Location, kinetics and stability of cellulases induced in Trichoderma reesei cultures

Amorphous cellulose induced the synthesis of endoglucanase in Trichoderma reesei to a greater extent than did cellobiose, lactose, sucrose or other commercial celluloses. In contrast, none of these carbohydrates was able to induce significant levels of endoglucanase activity in Aspergillus niger or Pseudomonas pickettii. Both endoglucanase and β-glucosidase activities were found in cell wall, cell-free extracts and extramycelial fractions of Trichoderma reesei cultures grown on amorphous cellulose. When carboxymethyl cellulose was used as substrate, Km and Vmax values of 1.32% (w/v) and 405.5 μmol glucose ml−1 h−1 for carboxymethyl cellulase were obtained Additionally, the activity was maximum over a pH range of 4.5–5.5, declining sharply beyond 5.5. The optimum temperature was between 50 and 70°C, with maximum activity at 60°C. The half-life of the enzyme appeared to be 9.4 h at 55°C and 4.3 h at 60°C.

Effect of immobilization on the stability of bacterial and fungal β-d-glucosidase

Abstract The thermal and proteolytic stability of free and immobilized β- d -glucosidase, isolated from Pseudomonas pickettii and Aspergillus niger , were determined. The optimal temperatures of soluble and entrapped β-glucosidase extracted from P. pickettii were 40 and 50°C, respectively. In contrast, the optimal temperature of enzyme isolated from A. niger remained unaltered (60°C). Free and immobilized A. niger β-glucosidase showed an unusual discontinuity around 40°C in the Arrhenius plot, suggesting that the enzyme could exist in two (or more) interconvertible forms with different activation energies. The polymeric network influenced the reactivity of both fungal and bacterial β-glucosidases since their E a values changed with respect to their soluble counterparts. Nevertheless, both the thermal stability and the resistance to proteolysis were apparently related to the origin (bacterial or fungal) and location (intracellular or exocellular) of the enzyme. The half-lives of soluble and immobilized β-glucosidases at six different temperatures were also calculated. The properties assayed were compared critically with those reported by other authors.

Optimisation of β-glucosidase entrapment in alginate and polyacrylamide gels

Abstract Immobilisation of β-glucosidase, isolated from Aspergillus niger, by entrapment in both calcium alginate and polyacrylamide gels was studied. A retention of 66% of initial activity was observed in the alginate beads prepared with 3% (w/v) alginate, 0.2 m CaCl 2 and 1 h of treatment. The maximum β-glucosidase activity in polyacrylamide gels (∼55%) was achieved in gels prepared with 20% acrylamide and 1.2% of crosslinking agent (bisacrylamide). β-Glucosidase immobilised in alginate gel did not follow pure Michaelis kinetics, exhibiting substrate inhibition. The K m of this enzyme was larger than that of the free β-glucosidase, suggesting that the alginate network limited the permeation rate of substrate and product. However, β-glucosidase entrapped in polyacrylamide gel showed a similar K m value to that of native enzyme. The pH value for maximum activity of free and immobilised enzymes was 4.0. The pH-activity curves were coincident, except at very low pH values where the enzyme trapped in alginate was more stable.

Studies of microbial β-d-glucosidase immobilized in alginate gel beads

About 50–60% β-d-glucosidase (isolated from Pseudomonas pickettii and Aspergillus niger) was immobilized in calcium alginate gel beads by entrapment, retaining 15–26% of original activity. The pH values for maximum activitiy of the immobilized bacterial and fungal enzymes appeared to be 5.0 and 3.0 respectively, very similar to those of the free enzymes. The pH-activity curves were coincident, except at very low pH values where the fungal immobilized enzymes was more stable. n nBacterial β-glucosidase did not follow pure Michaelis kinetics, exhibiting substrate inhibition and showing similar Km values for immobilized and soluble enzymes. The Km of the immobilized fungal enzyme was larger than that of the free enzyme, suggesting that the alginate network limited the permeation rate of substrate and product. The Vmax values of immobilized β-glucosidases from A. niger and P. pickettii were 14.7 and 29.5 smaller than those of the native enzymes, respectively.

Stabilisation of cellulases by cross-linking with glutaraldehyde and soil humates

Abstract β-Glucosidase and carboxymethyl cellulase were induced in and extracted from Aspergillus niger and Trichoderma reesei cultures and cross-linked with glutaraldehyde or complexed with a soil polyphenolic structure (humates). The concentration of the bifunctional reagent (2·5 or 5·0%) determined the activity of the immobilised β-glucosidase while the time and temperature of reaction had no significant influence. Although the catalytic activity of the cross-linked cellulases was 85% smaller than that of the free counterparts, they resisted thermal inactivation to a greater extent. When cellulases were bound to soil humates, their activity was much higher than that of the free enzymes. Furthermore, their thermal stability was enhanced by their complexation to the colloids. In contrast, when cellulases were bound to humates in the presence of glutaraldehyde, the cross-linked humate-enzyme complexes obtained showed a partial deactivation (β-glucosidase 10·4% and CMCase 37·1%). The thermostability of the immobilised CMCase was reduced (9·1%) but the thermostability of the β-glucosidase was increased (36·9%) with respect to its soluble counterpart at 60°C.

Induction of β-glucosidase in fungal and soil bacterial cultures

Abstract The production of β-glucosidase by soil enrichment cultures grown on cellobiose and carboxymethylcellulose and several experimental conditions were investigated in this work. The highest rates of induced enzyme activity were found in the soil cultures in which 0.4% cellobiose at pH 8.0 was used as the sole C and energy source. A bacterial strain, producing significant amounts of enzyme associated with the cell extracts and which was identified as Pseudomonas pickettii , was isolated from these soil cultures. In a comparative way, induction and location of β-glucosidase in Aspergillus niger cultures (using 0.4% cellobiose as C substrate) were also studied. In contrast with the results obtained with the bacterial cultures, only low levels of activity were detected when no N source was used. However, an appreciable amount of enzyme was assayed when 0.2% peptone was included in the culture medium with more than 70% of the total activity located in the extramycelial fraction.