Jean-Louis Leuba

β‐Galactosidase from Aspergillus niger

The enzyme beta-galactosidase (EC 3.2.1.23) from Aspergillus niger was purified and resolved into three multiple forms, using molecular sieving, ion-exchange, an hydrophobic chromatography. The isolated enzyme forms accounted for 83%, 8%, and 9% of the total beta-galactosidase activity, respectively. They were glycoproteins with estimated molecular weights of 124,000, 150,000 and 173,000, isoelectric points of about 4.6, and pH optima between 2.5 and 4.0. Amino acid and carbohydrate analyses showed that multiplicity was mainly due to dissimilar carbohydrate contents (about 12.5%, 20.5% and 29% neutral carbohydrates, respectively). The multiple form pattern might depend on the culture conditions. The beta-galactosidase forms were heat-stable up to about 60 degrees C. The Km values for lactose ranged from 85 mM to 125 mM, whereas those for the synthetic substrate o-nitrophenyl-beta-D-galactopyranoside were equal to about 2.4 mM. The V values obtained at 30 degrees C for lactose and o-nitrophenyl-beta-D-galactopyranoside were 104 units/mg enzyme protein and 121 units/mg enzyme protein, respectively (weighted averages for the three enzyme forms). The slight reactional dissimilarities between the three enzyme forms are unlikely to be physiologically relevant. The biological significance of A. niger beta-galactosidase multiplicity might be related to the observed differences in carbohydrate content, as suggested by recent reports on other microbial glycoprotein enzymes.

Chitinolytic Enzyme Activity of Penicillium janthinellum P9 in Bench-Top Bioreactor

The chitinolytic activity of Penicillium janthinellum P9 was studied in shaken cultures and in a 3-l bench-top bioreactor by varying culture conditions such as initial medium pH, growth temperature, stirrer speed and aeration site. In shaken flasks, the highest levels of enzyme activity (468 and 483 U·l−1) were obtained at a growth temperature of 24°C and at an initial medium pH of 4.0, respectively. In the bioreactor, both agitation and aeration significantly influenced the enzyme production: the highest level of enzyme activity (497 U·l−1) was obtained at an impeller speed of 500 rpm and an aeration rate of 1.5 vvm. Culturing P. janthinellum P9 under optimised conditions led to an increase in the enzyme activity of ca. 65% (686 U·l−1 as compared to the 415 U·l−1 obtained under the initial culture conditions).

Repeated-batch and continuous production of chitinolytic enzymes by Penicillium janthinellum immobilised on chemically-modified macroporous cellulose

Abstract Conidiospores of the fungus Penicillium janthinellum P9 were immobilised on polyurethane sponge and chemically-modified macroporous cellulose and cultivated for the production of chitinolytic enzymes in shaken culture, repeated-batch processes. The highest enzyme production (342 U l−1) was obtained using the macroporous cellulose BR-L01T(70) (Biomaterial Co. Ltd., Japan); with this carrier cell leakage was negligible. The immobilised mycelium of P. janthinellum P9 was then used for repeated-batch and continuous production of chitinolytic enzymes and N-acetyl-β- d -glucosamine in a fluidised-bed reactor (1.0 l working volume). While the repeated-batch cultivation resulted in almost the same enzyme activity (338 U l−1) obtained in shaken culture, with the continuous operation a steady enzyme production of about 450 U l−1 was reached and maintained for 168 h at a feed rate of 0.036 l h−1. At this feed rate, enzyme volumetric productivity and total N-acetyl-β- d -glucosamine production were 14.76 U l−1 h−1 and 15.9 g, respectively. Carrier colonisation and hyphal growth were followed by observations under the scanning electron microscope.

Inactivation of Mucor plumbeus by the combined actions of chitinase and high hydrostatic pressure.

Sporangiospores were treated with high hydrostatic pressure and/or fungal chitinase in order to study the inhibition of germination and growth of the food spoiling mold Mucor plumbeus. Total fungal inhibition was obtained either at 4.0 kbar or by 10 U/ml of chitinase from Penicillium janthinellum. A pretreatment with 1 U/ml of the same chitinase reduced the pressure necessary to obtain complete spore inhibition to 3 kbar.

Immobilization of proteinases on Chitosan

SummaryTrypsin, pronase and subtilisin were immobilized on chitosan by glutaraldehyde coupling. Significant retention of activity was observed when synthetic substrates as well as casein were used. The specific activities of the bound proteinases ranged from 38% to 79% of their initial specific activities. The pH-activity profile of trypsin was slightly shifted toward alkaline values, and its thermal stability was increased. Immobilized trypsin was found to be less sensitive to its natural inhibitors than the soluble enzyme.

Immobilization of the β-galactosidase fromAspergillus niger on chitosan

Crude or purifiedAspergillus niger β-galactosidase preparations were immobilized on chitosan (deacetylated chitin, activated with glutaraldehyde). The most active immobilized systems were obtained withcrude enzyme preparations. The immobilized enzyme hydrolyzed lactose in pure lactose solutions, ultrafiltrate whey permeates, or acid wheys at similar rates. The pH activity profiles and Km values of the chitosan-bound enzyme were not significantly altered on immobilization, and its stability on repetitive use up to 60°C was increased by reduction with NaBH4. After 8 weeks on discontinuous operation (8 h use per day), β-galactosidase-chitosan columns were found to retain about 90, 50, or 60% of their initial activities with lactose, ultrafiltrate permeate, or acid whey solutions, respectively. The efficiency of the β-galactosidase-chitosan conjugate appears to be comparable or greater than those of other described systems, and its stability should allow its use on an industrial scale. A preliminary report of this work has already been presented at the 4th International Enzyme Engineering Conference (September 25-30, 1977, Bad Neuenahr, FRG).