title: Hydrolysis of lactose by beta-glycosidase CelB from hyperthermophilic archaeon Pyrococcus furiosus: comparison of hollow-fiber membrane and packed-bed immobilized enzyme reactors for continuous processing of ultrahigh temperature-treated skim milk.
abstract: Recombinant beta-glycosidase CelB from the hyperthermophilic archaeon Pyrococcusfuriosus was produced through expression of the plasmid-encoded gene in Escherichia coli. Bioreactor cultivations of E. coli in the presence of the inductor isopropyl-1-thio-beta-D-galactoside (0.1 mM) gave approx 100,000 U of enzyme activity/L of culture medium after 8 h of growth. A technical-grade enzyme for the hydrolysis of lactose was prepared by precipitating the mesophilic protein at 80 degrees C. A hollow-fiber membrane reactor was developed, and its performance during continuous processing of ultrahigh temperature-treated (UHT) skim milk at 70 degrees C was analyzed regarding long-term stability, productivity, and diffusional limitation thereof. CelB was covalently attached onto Eupergit C in yields of 80%, and a packed-bed immobilized enzyme reactor was used for the continuous hydrolysis of lactose in UHT skim milk at 70 degrees C. The packed-bed reactor was approximately 10-fold more stable and gave about the same productivity at 80% substrate conversion as the hollow-fiber reactor at 60% substrate conversion. The marked difference in the stability of free and immobilized CelB seems to reflect mainly binding of the soluble enzyme to the membrane surface of the hollow-fiber module. Under these bound conditions, CelB is essentially inactive. CelB is essentially inactive. Microbial contamination of the reactors did not occur during reaction times of up to 39 d, given that UHT skim milk and not pasteurized skim milk was used as the substrate.
authors.affiliation: Division of Biochemical Engineering, Institute of Food Technology, University of Agricultural Sciences, Vienna, Austria.
authors.forename: Barbara
authors.initials: B
authors.lastname: Splechtna
authors.forename: Inge
authors.initials: I
authors.lastname: Petzelbauer
authors.forename: Bernhard
authors.initials: B
authors.lastname: Kuhn
authors.forename: Klaus D
authors.initials: KD
authors.lastname: Kulbe
authors.forename: Bernd
authors.initials: B
authors.lastname: Nidetzky
mesh_terms: D000818:Animals; D019149:Bioreactors; D002480:Cellulase / Q000378:metabolism*; D006868:Hydrolysis; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D007700:Kinetics; D007785:Lactose / Q000378:metabolism*; D008892:Milk; D020027:Pyrococcus furiosus / Q000201:enzymology* / Q000254:growth & development
chemical_list: D007544:Isopropyl Thiogalactoside; C109965:CelB endoglucanase; D002480:Cellulase; D007785:Lactose