Galacto-oligosaccharide production with immobilized ß-galactosidase in a packed-bed reactor vs. free ß-galactosidase in a batch reactor
Warmerdam, A. ; Benjamins, E. ; Leeuw de, T.F. ; Broekhuis, T.A. ; Boom, R.M. ; Janssen, A.E.M. - \ 2014
Food and Bioproducts Processing 92 (2014)4. - ISSN 0960-3085 - p. 383 - 392.
bacillus-circulans - lactose hydrolysis - eupergit-c - covalent immobilization - enzymatic-synthesis - enzymes - supports - temperature - milk - art
We report here that the usage of immobilized enzyme in a continuous packed bed reactor (PBR) can be a good alternative for GOS production instead of the traditional use of free enzyme in a batch reactor. The carbohydrate composition of the product of the PBR with immobilized enzyme was comparable to that of the batch reactor with free enzyme. The stability of the immobilized enzyme at a lactose concentration of 38% (w/v) and at 50¿C was very high: the half-life time of the immobilized enzyme was approximately 90 days. The enzymatic productivity of GOS production using immobilized enzyme in a PBR can be more than six times higher than that of GOS production with free enzyme in a batch reactor. Besides, when aiming for an equal volumetric productivity to the batch process in designing a PBR, the volume of the PBR can be much smaller than that of the batch reactor, depending on the enzyme dosage and the run time of a single batch
Kinetic Characterization of Galacto-Oligosaccharide (GOS) Synthesis by Three Commercially Important b-Galactosidases
Warmerdam, A. ; Zisopoulos, F.K. ; Boom, R.M. ; Janssen, A.E.M. - \ 2014
Biotechnology Progress 30 (2014)1. - ISSN 8756-7938 - p. 38 - 47.
bacillus-circulans - aspergillus-oryzae - lactose hydrolysis - kluyveromyces-lactis - enzymatic-synthesis - escherichia-coli - skim milk - purification - onpg
Many b-galactosidases show large differences in galacto-oligosaccharide (GOS) production and lactose hydrolysis. In this study, a kinetic model is developed in which the effect of lactose, glucose, galactose, and oligosaccharides on the oNPG converting activity of various b-galactosidases is quantified. The use of oNPG as a competing substrate to lactose yields more information than can be obtained by examining only the conversion of lactose itself. The reaction rate with lactose or oligosaccharides as substrate relative to that with water as acceptor is much higher for the b-galactosidase of Bacillus circulans than the bgalactosidases of Aspergillus oryzae and Kluyveromyces lactis. In addition, the bgalactosidase of B.circulans has a high reaction rate with galactose as acceptor, in contrast to those of A. oryzae and K. lactis. The latter two are strongly inhibited by galactose. These differences explain why b-galactosidase of B. circulans gives higher yields in GOS production than other b-galactosidases. Many of the reaction rate constants for the b-galactosidase isoforms of B. circulans increase with increasing molecular weight of the isoform. This indicates that the largest isoform b-gal-A is most active in GOS production. However, its hydrolysis rate is also much higher than that of the other isoforms, which results in a faster hydrolysis of oligosaccharides as well.
Effects of Carbohydrates on the oNPG Converting Activity of ß-Galactosidases
Warmerdam, A. ; Wang, J. ; Boom, R.M. ; Janssen, A.E.M. - \ 2013
Journal of Agricultural and Food Chemistry 61 (2013)26. - ISSN 0021-8561 - p. 6458 - 6464.
bacillus-circulans - physiological consequences - enzymatic-synthesis - aspergillus-oryzae - lactose hydrolysis - oligosaccharides - confinement
The effects of high concentrations of carbohydrates on the o-nitrophenyl ß-D-galactopyranoside (oNPG) converting activity of ß-galactosidase from Bacillus circulans are studied to get a better understanding of the enzyme behavior in concentrated and complicated systems in which enzymatic synthesis of galacto-oligosaccharides is usually performed. The components that were tested were glucose, galactose, lactose, sucrose, trehalose, raffinose, Vivinal GOS, dextran-6000, dextran-70 000, and sarcosine. Small carbohydrates act as acceptors in the reaction. This speeds up the limiting step, which is binding of the galactose residue with the acceptor and release of the product. Simultaneously, both inert and reacting additives seem to cause some molecular crowding, which results in a higher enzyme affinity for the substrate. The effect of molecular crowding on the enzyme activity is small compared to the effect of carbohydrates acting in the reactions as acceptors. The effects of reactants on ß-galactosidases from B. circulans, A. oryzae, and K. lactis are compared. KEYWORDS: galacto-oligosaccharides, GOS, ß-galactosidase, Bacillus circulans, enzyme activity, carbohydrates, crowding, galactosyl transfer, transgalactosylation, Aspergillus oryzae, Kluyveromyces lactis
Oligosaccharide synthesis by the hyperthermostable b-glucosidase from Pyrococcus furiosus: kinetics and modelling
Bruins, M.E. ; Strubel, M. ; Lieshout, J.F.T. van; Janssen, A.E.M. ; Boom, R.M. - \ 2003
Enzyme and Microbial Technology 33 (2003)1. - ISSN 0141-0229 - p. 3 - 11.
escherichia-coli - enzymatic-synthesis - bacillus-circulans - hydrolysis - galactosidase - lactose - temperature - disaccharides - glycosidases - glucoamylase
Oligosaccharides can be synthesised from monosaccharides or disaccharides, using glycosidases as a catalyst. To investigate the potential of this synthesis with beta-glycosidase from Pyrococcus furiosus we determined kinetic parameters for substrate conversion and product formation from cellobiose, lactose, glucose and galactose. The obtained parameters for initial rate measurements of disaccharide conversion were also used for the interpretation of experiments in time. The model for cellobiose gave a good description of the experiments. The enzyme was found to be uncompetitively inhibited by cellobiose and competitively inhibited by glucose. Lactose conversion however, could not be modelled satisfactorily; apparently additional reactions take place. Monosaccharide condensation also yielded oligosaccharides, but much slower. The use of a hyperthermostable, enzyme was found to be positive. More substrate could be dissolved at higher temperatures, which benefited all reactions.