Immobilization of lactase from Kluyveromyces lactis greatly reduces the inhibition promoted by glucose. full hydrolysis of lactose in milk (original) (raw)
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Applied Biochemistry and Biotechnology, 2010
β-D-galactosidase (EC 3.2.1.23) from Kluyveromyces marxianus YW-1, an isolate from whey, has been studied in terms of cell disruption to liberate the useful enzyme. The enzyme produced in a bioreactor on a wheat bran medium has been successfully immobilized with a view to developing a commercially usable technology for lactose hydrolysis in the food industry. Three chemical and three physical methods of cell disruption were tested and a method of grinding with river sand was found to give highest enzyme activity (720 U). The enzyme was covalently immobilized on gelatin. Immobilized enzyme had optimum pH and temperature of 7.0 and 40°C, respectively and was found to give 49% hydrolysis of lactose in milk after 4 h of incubation. The immobilized enzyme was used for eight hydrolysis batches without appreciable loss in activity. The retention of high catalytic activity compared with the losses experienced with several previously reported immobilized versions of the enzyme is significant. The method of immobilization is simple, effective, and can be used for the immobilization of other enzymes.
Enzyme and Microbial Technology, 2000
The kinetic model of the hydrolysis of lactose with a -galactosidase from Kluyveromyces fragilis immobilized on a commercial silica-alumina (KA-3, from Südchemie) has been determined. A wide experimental range of the main variables has been employed: temperature, concentrations of substrate, and products and concentration of enzyme. The runs were performed in a complex buffer with the salt composition of milk. The effect of pH and temperature on the stability and the activity of the enzyme have been studied. The optimum pH for the enzyme activity was, approximately, seven. The immobilized enzyme was more stable than the free one at acidic pH, but more instable at basic pH. The maximum temperature used for the hydrolysis runs performed to select the kinetic model was 40°C, so inactivation of the enzyme during the kinetic runs has been avoided. Agitation, concentration of enzyme in the solid and particle size were selected to ensure that the overall rate was that of the chemical reaction. Eleven kinetic models were proposed to fit experimental data, from first order to more complex ones, such as those taking into account inhibition by one of the compounds involved in the hydrolysis reaction. Applying statistical and physical criteria, a Michaelis-Menten model with a competitive inhibition by galactose has been selected. The model is able to fit the experimental data correctly in the wide experimental range studied. Finally, the model obtained is compared to the one selected in a previous work for the hydrolysis of lactose with the free enzyme.
Journal of Food Biochemistry, 2001
A potentially low cost P-galactosidase was prepared as a crude permeabilized cell mass of the yeast Kluyveromyces lactis that had been grown in ultrafiltered cheese whey. The enzyme preparation was immobilized in alginate beads. Milk lactose hydrolysis rates were 25 % higher in manganese alginate beads than in calcium alginate beads. The immobilized biocatalyst lost activity when stored in calcium chloride solution, however, storage in 5 mM DTT or 50% glycerol allowed the biocatalyst to be recycled at least 5 times without any loss of activity.
Lactose hydrolysis by immobilized β-galactosidase: the effect of the supports and the kinetics
Catalysis Today, 2003
The kinetic behaviour of -galactosidase from Kluyveromices marxianus (Saccharomyces) lactis, immobilized on different oxides supports, such as alumina, silica, and silicated alumina has been studied. We observed a strong dependence of the immobilized enzyme activities on the chemical nature and physical structure of the supports. In particular, when the particle sizes of the supports are increased, the enzymatic activity strongly decreases.
Operational stability and kinetics of lactose hydrolysis by β- galactosidase from
2016
The kinetic modeling of the lactose hydrolysis and the operational stability of the enzyme β-galactosidase of Kluyveromyces fragilis was determined using as substrate skimmed powdered milk, reconstituted in a way to supply two lactose concentrations: 5% and 10% (w/v). For the operational stability both lactose concentrations were studied in the presence and in the absence of the buffer pH 6.5. Every 40 minutes the residual activity was determined by the method of the initial velocities. The experimental results showed that the buffer leads to enzyme inactivation. However, for lactose solutions without buffer, the residual activity only declines 15% in 6 hours and later it decreases abruptly. In the kinetic modeling the hydrolysis reaction was led to 40 o C/7h, being used enzyme concentrations equivalent to 1500 and 8100 LAU/L. The adjusted model allowed us to conclude that it is necessary to use the quantity of 3450 LAU/L of the enzyme to obtain the hydrolysis from 70% to 80% of the milk lactose in two to three hours of reaction.
Hydrolysis of whey lactose by immobilized β-Galactosidase
Brazilian Archives of Biology and Technology, 2008
Hydrolysis of whey lactose to glucose and galactose by immobilized galactosidase comes as an alternative to enlarge the possibilities of commercial use of this feedstock. To be applied at industrial scale, the process should be performed continuously .This work aimed to study the hydrolysis of whey lactose by an immobilized enzyme reactor. b-Galactosidase from Aspergillus oryzae was immobilized on silica and activity and stability were evaluated. The best immobilization results were attained by using glutaraldehyde as support's activator and enzyme stabilizer. The optimized enzyme proportion for immobilization was 15-20 mg g-1 of support. Treatments of whey were performed (microfiltration, thermal treatment and ultrafiltration), seeking the elimination of sludge, and the effects on operating the fixed bed reactor were evaluated. Ultrafiltration was the best treatment towards a proper substrate solution for feeding the reactor.
Enzyme and Microbial Technology, 2002
We study the enzymatic hydrolysis of lactose by a commercial enzyme from a selected strain of Kluyveromyces fragilis. The variables analyzed were: temperature (25-40 • C), enzyme concentration (0.1-3.0 g l −1 ), lactose concentration (0.0278-0.208 M), and initial galactose concentration (0.0347 M). On the basis of the data analyzed, both published and in the present work, we propose a Michaelis-Menten kinetic model with inhibition by the product (galactose), which reveals that the substrate (lactose) and the product (galactose) present similar affinity for the active site of the enzyme.
Applied Microbiology and Biotechnology, 2002
The enzyme β-galactosidase was purified from a cold-adapted organism isolated from Antarctica. The organism was identified as a psychrotrophic Pseudoalteromonas sp. The enzyme was purified with high yields by a rapid purification scheme involving extraction in an aqueous two-phase system followed by hydrophobic interaction chromatography and ultrafiltration. The β-galactosidase was optimally active at pH 9 and at 26°C when assayed with o-nitrophenyl-β-D-galactopyranoside as substrate for 2 min. The enzyme activity was highly sensitive to temperature above 30°C and was undetectable at 40°C. The cations Na + , K + , Mg 2+ and Mn 2+ activated the enzyme while Ca 2+ , Hg 2+ , Cu 2+ and Zn 2+ inhibited activity. The shelf life of the pure enzyme at 4°C was significantly enhanced in the presence of 0.1% (w/v) polyethyleneimine. The pure β-galactosidase was also evaluated for lactose hydrolysis. More than 50% lactose hydrolysis was achieved in 8 h in buffer at an enzyme concentration of 1 U/ml, and was increased to 70% in the presence of 0.1% (w/v) polyethyleneimine. The extent of lactose hydrolysis was 40-50% in milk. The enzyme could be immobilized to Sepharose via different chemistries with 60-70% retention of activity. The immobilized enzyme was more stable and its ability to hydrolyze lactose was similar to that of the soluble enzyme.
Journal of Molecular Catalysis B: Enzymatic, 1998
. The covalent immobilization of b-galactosidase from KluyÕeromyces lactis b-gal on to two different porous carriers, CPC-silica and agarose, is reported. CPC-silica was silanizated and activated with glutaraldehyde. The activation of agarose Ž . via a cyanylating agent CDAP was optimized. Gel-bound protein and gel-bound activity were both measured directly, Ž . allowing the determination of apparent specific activities S.A. . Higher amounts of b-gal were immobilized on the activated Ž y1 . CPC-silica maximum capacity, 23 mg ml of packed support than on the CDAP-activated agarose. For the lower enzyme Ž y1 . loading assayed 12.6 mg ml packed support , 100% of the enzyme was immobilized but only 34% of its activity was Ž y1 expressed. This inactivation during immobilization was confirmed by the S.A. values 22-29 EU mg for the CPC-deriva-y1 . Ž . tives and 80 EU mg for soluble b-gal . The K 3.4 mM for the CDAP-derivative with ONPG as substrate was higher app Ž . than the K value for soluble b-gal 2 mM . When the enzyme loading was increased five-fold, the K increased M app four-fold, to 13 mM. The V values for the CPC-derivatives were remarkably lower than the V for soluble app max b-galactosidase. CDAP-derivatives showed better thermal stabilities than CPC-derivatives but neither of them enhanced the stability of the soluble enzyme. When stored at 48C, the activity of both derivatives remained stable for at least 2 months. Ž . Both derivatives displayed high percentages of lactose conversion 90% in packed bed mini-reactors. Glucose production was 3.3-fold higher for the CPC-derivative than for the CDAP-derivative, as a consequence of the higher flow rates achieved. q 1998 Elsevier Science B.V. All rights reserved.