Lactose hydrolysis by β-galactosidase enzyme: optimization using response surface methodology (original) (raw)
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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.
Journal of Food Process Engineering, 2009
To overcome the permeability barrier and prepare whole cell biocatalysts with high activities, permeabilization of Kluyveromyces marxianus var. lactis NCIM 3566 in relation to b-galactosidase activity was optimized using cetyltrimethylammonium bromide (CTAB) as permeabilizing agent. Permeabilized whole cells can be advantageous over pure enzyme preparations in terms of cost-effectiveness and increased stability maintained by the intracellular environment. Response surface methodology (RSM) was applied to optimize concentration of CTAB, temperature and the treatment time for maximum permeabilization of yeast cells. The optimum operating conditions for permeabilization process to achieve maximum enzyme activity obtained by RSM were 0.06% (w/v) CTAB concentration, 28C temperature and process duration of 14 min. At these conditions of process variables, the maximum value of enzyme activity was found to be 1,334 IU/g. The permeabilized yeast cells were highly effective and resulted in 90.5% lactose hydrolysis in whey.
The enzymatic synthesis of galacto-oligosaccharides (GOS) from lactose was studied using commercial grade b-galactosidase (Biolacta FN5) from Bacillus circulans. The reaction was carried out under free enzyme condition varying initial lactose concentration (ILC: 55e525 g/L), enzyme concentration (0.05e1.575 g/L), temperature (30e50 C) and pH (5.0e6.0). Reaction mixture compositions were analyzed utilizing high performance liquid chromatography (HPLC). A maximum GOS formation of 39% (dry basis) was achieved at an ILC of 525 g/L converting 60% of the lactose fed. Tri-saccharides were the major types of GOS formed, accounting approximately 24%; whereas, tetra-saccharides and penta-saccharides account approximately 12% and 3%, respectively. Design correlation was developed in order to observe the quantitative effect of operating parameters on GOS yield. Further, based on MichaeliseMenten model, fourstep reaction pathways were considered for simplistic understanding of the kinetics. Apart from predicting the reaction mixture composition, the approach also provided kinetic parameters though simulation using COPASI 4.7 Ò. Excellent agreements were observed between simulated and experimental results.
Electron. J. Biotechnol, 2008
This paper investigates the production and optimization of β-galactosidase enzyme using synthetic medium by Kluyveromyces lactis NRRL Y-8279 in shake flask cultures. Among the different cell disintegration methods used, the highest specific activity was obtained when the cells were permeabilized using isoamyl alcohol. Response surface methodology was used to investigate the effects of four fermentation parameters (agitation speed, pH, initial substrate concentration and incubation time) on β-galactosidase enzyme production. Results of the statistical analysis showed that the fit of the model was good in all cases. Maximum specific enzyme activity of 4218.4 U g-1 was obtained at the optimum levels of process variables (pH 7.35, agitation speed 179.2 rpm, initial sugar concentration 24.9 g l-1 and incubation time 50.9 hrs). The response surface methodology was found to be useful in optimizing and determining the interactions among process variables in β-galactosidase enzyme production. The enzyme β-galactosidase (lactase, EC 3.2.1.23) catalyzes the hydrolysis of lactose to glucose and galactose. This enzyme is industrially important because it can be
Chemical Engineering and Processing: Process Intensification, 2009
Calcium alginate entrapped  galactosidase preparations were used for the hydrolysis of lactose from solution, milk and whey in batch processes as well as in continuous packed bed columns. The efficiency of columns, containing calcium alginate entrapped soluble and crosslinked concanavalin A complex of  galactosidase was examined at various flow rates at room temperature 32 • C, for the continuous hydrolysis of 0.1 M lactose for over 2 months. From the kinetic study the Michaelis constant (K m) were found to be 2.51 mM and 5.18 mM for the free and immobilized  galactosidase, respectively. The V max for the soluble enzyme and immobilized preparation was 4.8 × 10 −4 mol/min and 4.2 × 10 −4 mol/min, respectively. Furthermore, we found that entrapped crosslinked Con A- galactosidase was more efficient in the hydrolysis of lactose present in milk (77%) and whey (86%) in batch processes as compared to the entrapped soluble  galactosidase.
Optimization of β-galactosidase production by response surface methodology
β-galactosidase production is carried out using lactose or lactose rich substrates like whey. Tamarind seed powder has not been reported as a substrate for production of this enzyme before. In this study, statistical optimization of medium components for production of β-galactosidase, using tamarind seed powder, by Aspergillus terreus was attempted. Screening for the effects of eleven medium components on enzyme activity was carried out by Plackett-Burman design which showed that NH4(SO4)2, lactose and MgSO4 has significant (p<0.001) positive influence and pH, yeast extract, maltose and NaNO3 has significant negative influence. Optimal levels of positively influencing parameters were determined by ridge analysis and was found to be 2.97, 2.88 and 2.67 g/L of NH4(SO4)2, lactose and MgSO4 respectively. In the optimized medium, enzyme activity increased 2.8 folds in comparison with basal medium. Improved activity, being achieved by the use of a cheaper substrate, could reduce the co...
Indian Journal of Dairy Science
In the study, pH 6.5 and 40°C were selected on the basis of maximum β-galactosidase activity produced by L.helveticus MTCC 5463 (V3), L.rhamnosus (NK2) and L.casei (NK9) during optimizing the pH and temperatures using Response Surface Methodology (RSM). During evaluation of lactose hydrolysis using partially purified β-galactosidase enzyme, V3 showed highest reduction of lactose and maximum production of glucose and galactose at 2 % rate of β-galactosidase upto 12h of incubation. Partially purified β-galactosidase enzyme showed maximum lactose hydrolysis at 2% rate of addition in sterilized reconstituted skim milk for V3, NK2 & NK9 cultures up to 12h of incubation. Lactose hydrolysed milk which were prepared using partially purified enzyme were added @2% in sterilized reconstituted skim milk for 12h at 37°C. Based on sensory scores, B (Commercially available enzyme sample), A (Reconstituted sterilized skim milk), and C (Partially purified β-galactosidase produced from V3) showed highest sensory score and were more acceptable compared to D (NK2), E (NK9) and F (Market sample: Lactose free milk) after 12h of incubation at 37°C by adding 2% partially purified β-galactosidase enzyme in sterilized reconstituted skim milk.
Food Science and Technology, 2019
The cheese whey shows an organic nutrient charge that can be used to obtain metabolites of interest by biotechnology of microorganisms. Thus, fermentative processes for enzyme production, in particular beta-galactosidase becomes feasible. The enzyme plays an important role in the biotech food industry to obtain milk and dairy products with low lactose content for consumption by intolerant individuals. The objective of this work was to determine the enzyme activity of the concentrated beta-galactosidase (CBG) and the permeabilized cells (PC) both obtained from Saccharomyces fragilis OZ 275. The enzyme beta-galactosidase obtained from the fermentation of Saccharomyces fragilis OZ 275 in cheese whey was used to determine the optimal conditions for the hydrolysis of lactose solution at 1% (w/v). Response Surface Methodology (RSM) by Box-Behnken Design (BBD) was employed to determine beta-galactosidase activity for such factors pH, temperature and enzyme concentration suitable for the lactose hydrolysis. Based on the statistical analysis, the optimum operational conditions for maximizing lactose hydrolysis thus optimizing the enzyme activity for CBG were, temperature 30 °C, pH 6.0 and enzyme concentration 3% (v/v) and for PC was temperature 44 °C, pH 7.0 and enzyme concentration 4% (v/v).
Journal of Molecular Catalysis B: Enzymatic, 2013
This work studied the hydrolysis of lactose using -galactosidase from Aspergillus oryzae immobilized with a combination of adsorption and glutaraldehyde cross-linking onto the ion exchange resin Duolite A568 as a carrier. A central composite design (CCD) was used to study the effects of lactose concentration and feed flow rate on the average hydrolysis reaction rate and lactose conversion in a fixed bed reactor operating continuously with an upflow at a temperature of 35 ± 1 • C. The optimal conditions for the average hydrolysis reaction rate and the lactose conversion included a lactose concentration of 50 g/L and a feed flow rate of 6 mL/min. The average reaction rate and conversion reached 2074 U and 65%, respectively. The immobilized enzyme activity was maintained during the 30 days of operation in a fixed bed reactor with a 0.3 mL/min feed flow rate of a 50 g/L lactose solution at room temperature. Feed flows ranging from 0.6 to 12 mL/min were used to determine the distribution of residence times and the kinetics of the fixed bed reactor. A non-ideal flow pattern with the formation of a bypass flow in the fixed bed reactor was identified. The conditions used for the kinetics study included a lactose solution concentration of 50 g/L at pH 4.5 and a temperature of 35 ± 1 • C. Kinetic models using a PFR and axial dispersion methods were used to describe the lactose hydrolysis in the fixed bed reactor, thus accounting for the competitive inhibition by galactose. To increase the lactose conversion, experiments were performed for two fixed bed reactors in series, operating in continuous duty with upflow, with the optimal conditions determined using the CCD for a fixed bed reactor. The total conversion for the two reactors in series was 82%.
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.