title: Novel cellobiose 2-epimerases for the production of epilactose from milk ultrafiltrate containing lactose.
abstract: A selected number of enzymes have recently been assigned to the emerging class of cellobiose 2-epimerases (CE). All CE convert lactose to the rare sugar epilactose, which is regarded as a new prebiotic. Within this study, the gene products of 2 potential CE genes originating from the mesophilic bacteria Cellulosilyticum lentocellum and Dysgonomonas gadei were recombinantly produced in Escherichia coli and purified by chromatography. The enzymes have been identified as novel CE by sequence analysis and biochemical characterizations. The biochemical characterizations included the determination of the molecular weight, the substrate spectrum, and the kinetic parameters, as well as the pH and temperature profiles in buffer and food matrices. Both identified CE epimerize cellobiose and lactose into the C2 epimerization products glucosylmannose and epilactose, respectively. The epimerization activity for lactose was maximal at pH 8.0 or 7.5 and 40°C in defined buffer systems for the CE from C. lentocellum and the CE from D. gadei, respectively. In addition, biotransformations of the foodstuff milk ultrafiltrate containing lactose were demonstrated. The CE from D. gadei was produced in a stirred-tank reactor (12 L) and purified using an automatic system. Enzyme production and purification in this scale indicates that a future upscaling of CE production is possible. The bioconversions of lactose in milk ultrafiltrate were carried out either in a batch process or in a continuously operated enzyme membrane reactor (EMR) process. Both processes ran at an industrially relevant low temperature of 8°C to reduce undesirable microbial growth. The enzyme was reasonably active at the low process temperature because the CE originated from a mesophilic organism. An epilactose yield of 29.9% was achieved in the batch process within 28 h of operation time. In the continuous EMR process, the epilactose yield in the product stream was lower, at 18.5%. However, the enzyme productivity was approximately 6 times higher because the continuous epilactose formation was carried out for about 6 d without further addition of biocatalyst. Within this time, 24g of epilactose in 2.8 L of permeate were produced. The batch and the EMR process showed that the milk ultrafiltrate, which is a sidestream of the milk protein production, might be upgraded to a dairy product of higher value by the enzymatic in situ production of epilactose.
authors: Department of Biotechnology and Enzyme Science, University of Hohenheim, 70599 Stuttgart, Germany.
authors: Manuel
authors: M
authors: Krewinkel
authors: Department of Biotechnology and Enzyme Science, University of Hohenheim, 70599 Stuttgart, Germany.
authors: Jana
authors: J
authors: Kaiser
authors: Department of Biotechnology and Enzyme Science, University of Hohenheim, 70599 Stuttgart, Germany.
authors: Michael
authors: M
authors: Merz
authors: Department of Biotechnology and Enzyme Science, University of Hohenheim, 70599 Stuttgart, Germany.
authors: Eva
authors: E
authors: Rentschler
authors: Department of Biotechnology and Enzyme Science, University of Hohenheim, 70599 Stuttgart, Germany.
authors: Beatrice
authors: B
authors: Kuschel
authors: Department of Soft Matter Science and Dairy Technology, Institute of Food Science and Biotechnology, University of Hohenheim, 70599 Stuttgart, Germany.
authors: Jörg
authors: J
authors: Hinrichs
authors: Department of Biotechnology and Enzyme Science, University of Hohenheim, 70599 Stuttgart, Germany. Electronic address: Lutz.Fischer@uni-hohenheim.de.
authors: Lutz
authors: L
authors: Fischer
mesh_terms: D000818:Animals; D001419:Bacteria / Q000201:enzymology*; D002238:Carbohydrate Epimerases / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D007785:Lactose / Q000737:chemistry*; D008892:Milk / Q000737:chemistry*; D056692:Prebiotics; D013696:Temperature
chemical_list: D056692:Prebiotics; D002238:Carbohydrate Epimerases; D007785:Lactose
keywords: cellobiose 2-epimerase; epilactose; lactose; milk; prebiotic