Preparation of lactose‐free milk by using salt‐fractionated almond (Amygdalus communis) β‐galactosidase

β‐galactosidase was isolated from almond (Amygdalus communis) extract by ammonium sulfate precipitation. Almond proteins precipitated by using ammonium sulfate and then dialysed exhibited 5.3‐fold purification of β‐galactosidase, and the yield of enzyme preparation was 96.5%. The partially purified β‐galactosidase exhibited pH and temperature optima at pH 5.5 and 50 °C, respectively. The enzyme was significantly stable against heat, pH, calcium and magnesium ions and D ‐galactose. The almond β‐galactosidase preparation exhibited over 89% activity even after 2 months storage at 4 °C. Hydrolysis of lactose in milk and whey was performed in a stirred batch process by using this enzyme preparation. These observations indicated that the hydrolysis of lactose increased continuously with time. The enzyme could hydrolyse 94% of lactose in buffer solution and whey whereas 90% of lactose hydrolysis was achieved in milk. The main aim of the present study was to prepare lactose‐free milk, which must be free from contamination, and the process should be inexpensive. Copyright © 2007 Society of Chemical Industry     

Optimisation of culture conditions and development of a novel fed‐batch strategy for high production of β‐galactosidase by Kluyveromyces lactis

The aim of this study was to enhance β‐galactosidase production by Kluyveromyces lactis CICC1773. Firstly, the optimum culture conditions were obtained by response surface methodology, and the maximum β‐galactosidase activity reached 20.6 U mL^?1, about two‐fold increase than that of the initial conditions (initial fermentation medium and conditions). To further improve β‐galactosidase production, a new fed‐batch strategy based on pH feedback control was developed successfully in a 7‐L fermenter, using 400 g L^?1 lactose as feeding medium. As a result, the β‐galactosidase activity and productivity reached up to 111.61 U mL^?1 and 5.31 U/(mL·h), enhanced by 15.3‐fold and 17.6‐fold superior than the results of initial conditions, respectively. To our knowledge, β‐galactosidase activity obtained was the highest value among the results reported by nonrecombinant strains. These results demonstrated that the new fed‐batch strategy based on optimum culture conditions could be automatic control easily and be conductive to further scale up for industrial fermentation.     

Evaluation of potential side activities of commercial enzyme preparations used in winemaking

Twenty‐one commercial enzyme preparations used in winemaking were characterised for the α‐L‐rhamnosidase, α‐L‐arabinosidase, β‐D‐xylosidase, ?β‐D‐galactosidase, β‐D‐glucosidase, esterase, protease, cinnamoyl esterase and laccase activities. A new rapid fluorimetric method to assay esterase activity was developed. Enzyme preparations differed for the level of each enzyme activity assayed rather than for the type of enzymatic activity detected. High levels of protease, glycosidase, esterase and cinnamoyl esterase activity were found among enzyme preparations for different technological applications. A drastic reduction in the level of cinnamoyl esterase was observed in commercial grape juice, pH 3.6, total acidity 5.3 g L^?1_, sugar 170 g L^?1. Protease activity was only weakly reduced, from 10 to 20%, in commercial grape juice. β‐glucosidase activity levels are reduced in the presence of increasing concentration of glucose but are still present at the higher glucose concentration (100 g L^?1). The extent of the reduction observed was dependent on the enzymatic preparations tested.     

Characterization of glucoamylase immobilized on magnetic nanoparticles

Magnetic support was prepared by precipitation from an alkaline solution of divalent and trivalent iron ions and subsequently was modified with 3‐aminopropyltriethoxysilane. FTIR analysis showed existence of a new Si–O–Fe bond in obtained particles. Scanning electronic microscopy images shows that the nanoparticles of all samples have particle size below 30 nm. Glucoamylase AMG 300L was immobilized onto the modified magnetic support using glutaraldehyde as a coupling agent. Obtained preparations had specific activity of 148 U/g of the support when measured at 55°C using maltose as substrate. The immobilized enzyme exhibited mass transfer limitation as reflected by a higher apparent K_m value and a lower energy of activation. The immobilization was almost completely terminated after 30 min of the reaction at 30°C. The highest immobilization yield of the enzyme was achieved at glutaraldehyde concentration of 10 mM. The immobilization did not influence considerably on optimum pH and temperature of substrate hydrolysis catalyzed by investigated enzyme (55°C, pH 4.5). Moreover, immobilized glucoamylase was easily separated from the reaction medium by an external magnetic field and retained about 60% of initial activity after nine repeated cycles of enzyme reaction followed by magnetic separation.     

Study of galacto‐oligosaccharide formation from lactose using Pectinex Ultra SP‐L

BACKGROUND: Galacto‐oligosaccharides (GOS) are synthesised from lactose by transglycosylation using β‐galactosidase (EC 3.2.1.23) and are recognised as prebiotics. The commercial enzyme preparation Pectinex Ultra SP‐L produced by Aspergillus aculeatus possesses β‐galactosidase activity; however, because its use has been directed towards the formation of 6′‐β‐galactosyl‐lactose, no data have been reported on the formation of other GOS. Since the composition of the oligosaccharide mixture obtained during lactose hydrolysis may affect the prebiotic properties, in this study the influence of various parameters (pH, temperature, time and enzyme and lactose concentrations) on the formation of GOS using Pectinex Ultra SP‐L was investigated. RESULTS: High‐performance anion exchange chromatography with pulsed amperometric detection (HPAEC‐PAD) analysis allowed the detection of disaccharides other than lactose, trisaccharides and minor amounts of higher‐molecular‐weight GOS. The main GOS formed were a trisaccharide identified as β‐D ‐Galp‐(1 → 6)‐Lac (6′‐β‐galactosyl‐lactose) and a disaccharide identified as β‐D ‐Galp‐(1 → 6)‐D ‐Gal (galactobiose). Other GOS detected were tentatively identified as β‐D ‐Galp‐(1 → 6)‐D ‐Glc (allolactose), β‐D ‐Galp‐(1 → 3)‐D ‐Glc and β‐D ‐Galp‐(1 → 3)‐Lac. Trisaccharide formation was favoured by a pH increase from 4.5 to 6.5, whereas the disaccharide content increased as the pH decreased, reaching a level of 11% at pH 4.5. 6′‐β‐Galactosyl‐lactose production increased gradually with increasing temperature, attaining a maximum value of 17% at 60 °C after 7 h, whereas disaccharide formation was optimal at 50 °C, reaching a level of 10% after 24 h. CONCLUSION: The results indicate that Pectinex Ultra SP‐L can be used to obtain GOS mixtures of different composition depending on the operating conditions. It has been shown for the first time that Pectinex Ultra SP‐L can be used for the selective formation of disaccharides. Copyright © 2008 Society of Chemical Industry     

A Modelling study on skimmed milk lactose hydrolysis and β‐galactosidase stability using three reactor types

The hydrolysis of lactose in skimmed milk and β‐galactosidase inactivation studies were carried out in three different devices – bioreactor, sonicator and homogeniser – to evaluate the performance of such reactors that have different operational systems. The experiments were carried out using β‐galactosidase produced from Kluyveromyces marxianus lactis. At the optimum process conditions obtained from the experiments performed in bioreactor, sonicator and homogeniser, 89%, 90% and 54% of lactose were hydrolysed and the enzyme lost its activity by 14%, 13% and 24%, respectively, at the end of the processing time of 30 min. The commercial milk lactose content (1 g/L lactose) was reached at 60 min for bioreactor and sonicator. After evaluation of the data, it was found that the kinetics of hydrolysis and enzyme inactivation could be represented by a first‐order kinetic model and a single‐step non‐first‐order enzyme inactivation kinetic model, respectively, for all process conditions applied. The activation energy for hydrolysis reaction and the enzymatic inactivation energy values were also calculated.     

The effect of thermal treatment on β‐glucosidase inactivation in vanilla bean (Vanilla planifolia Andrews)

The conditions for enzyme activity (pH and temperature) and kinetic parameters for the thermal inactivation of β‐glucosidase enzyme in vanilla beans have been investigated. The maximum enzyme activity was detected at pH 6.5 and 38 °C. The values obtained for V_max and K_m were 62.05 units and 2.07 mm, respectively. When hot water treatment (the most practical method of vanilla bean killing) was applied, β‐glucosidase treated at pH 6.0 and 60 °C for 3 min lost 51% of activity, while at 70 °C for 90 s the enzyme lost 60% of activity and at 80 °C for 30 s the enzyme lost 48% of its activity. When vanilla beans were cured in an oven at 60 °C for 36 to 48 h all β‐glucosidase activity was lost.     

Immobilisation of amylases on krill chitin

Krill chitin was used as a support for diastase. The enzyme was immobilised by simple adsorption or in the presence of 0.1% glutaraldehyde. Alternatively, the enzyme was supported on chitin previously activated with glutaraldehyde. The best results were achieved by the first procedure using chitin fractions of 22–52 mesh, obtained by demineralisation of raw dried krill offal with 22% HCl (1:10) for 2h at room temperature and deproteinisation with 28% KOH solution for 2h at 95°C. Supporting diastase without any treatment with glutaraldehyde did not reduce the enzyme activity. The optimum pH for binding of the diastase on chitin preparations was 6.2 in the presence of glutaraldehyde or 6.7 without the crosslinking agent. Immobilisation shifts the optimum pH for the activity of diastase by 0.5 units towards the acid side.     

Properties of Gel-Entrapped Glucoamylase

Glucoamylase from Aspergillus niger was immobilized by entrapping in glutaraldehyde crosslinked gelatin. Preparations were prepared in the form of beads and thin layers on activated glass, polyester and aluminium foils. Immobilization did not cause changes of pH and temperature optimum. However, it increased thermal stability additionally stabilized by the presence of a substrate. The most stable preparations acted without change in activity for 34 days at a temperature of 50°C. Obtained preparations hydrolyzed α-amylase liquefied starch to a DE value of about 97%. The preparations of glucoamylase immobilized in a thin layer of gelatin on the polyester and aluminium foil were used in a thin layer flow reactor for hydrolysis of 10% starch solution.     

Immobilized Glucose Isomerase on DEAE Cellulose Beads

Purified glucose isomerase from Actinoplanes missouriensis was immobilized on porous DEAE-cellulose beads by simple adsorption. The immobilized glucose isomerase retained over 70% of its original activity. The hinderance of immobilized enzyme activity due to pore diffusion and film diffusion was insignificant with the bead size at 35 mesh or smaller. The relative substrate flow rate can be kept at 0.04 cm/s or higher. The optimum pH of the imobilized enzyme did not change, however, the optimum pH range became broader. The broader pH profile indicated that immobilized enzymes are less sensitive to pH change in the substrate. The half life of the immobilized enzyme was at around 1,000 h at 60°C. Cobalt ions are not required for enzyme stability. The cost of using immobilized enzyme on DEAE cellulose beads should be less be than that of the whole cell immobilization system due primarily to the fact that DEAE-cellulose beads are reusable for immobilization as well as for enzyme purification.     

Immobilization of <Emphasis Type="BoldItalic">Aspergillus oryzae</Emphasis> β-Galactosidase onto Duolite A568 Resin via Simple Adsorption Mechanism

In this study, a rapid, simple and economic method of enzyme immobilization was developed to hydrolyze lactose. Duolite A568 resin was used for the immobilization of β-galactosidase via simple adsorption mechanism. The effects of immobilization parameters such as time, pH, and temperature were studied. Immobilization parameters for maximum enzyme activity were estimated at 35 °C temperature, pH 4.5, 5 mg/mL enzyme concentration, and approximately 60 min immobilization time. A significant amount of enzyme was immobilized with high catalytic activity. Enzyme immobilization procedure explained in this study slightly affected the enzyme kinetic. The value of Michaelis constant K _m for immobilized enzyme was significantly larger, indicating decreased affinity by the enzyme for its substrate. It was observed that both free and immobilized enzyme showed maximum activity at 65 °C reaction temperature. Immobilized β-galactosidase was significantly more active at all temperatures as compared to its free form. However, optimal pH of immobilized enzyme was slightly affected by immobilization procedure. The optimum pH of immobilized enzyme was shifted up 0.5 unit to a more alkaline value of 6.0 compared to the free enzyme.     

Immobilization of invertase on celite and on polyacrylamide by an absorption procedure

Invertase from Saccharomyces cerevisiae was immobilized on celite and on polyacrylamide by an absorption procedure. The properties of the immobilized invertase were characterized and compared with those of soluble invertase. The activity yield for immobilized invertase on celite and on polyacrylamide was 92% and 81% respectively. The optimum pH and temperature for both soluble and immobilized invertase activity were 4.6 and 60 °C respectively. The activity of immobilized invertase is stable in the range pH 4.0–6.5. The immobilized invertase was thermostable when incubated at temperatures ranging from 40 to 60 °C. Immobilized invertase had a high stability when stored at room temperature for 90 days and had an excellent operational stability when used 20 times repeatedly. The invertase immobilized on celite was more stable than invertase immobilized on polyacrylamide. The invertase preparations immobilised by absorption procedures exhibited marked stability towards temperature, pH changes and had high storage and operational stability, suggesting their excellent potential for use as supports. Copyright © 2003 Society of Chemical Industry     

Immobilization and characterization of naringinase for the hydrolysis of naringin

The degree of hydrolysis of naringin was investigated at various temperatures (40, 50, 60 °C), enzyme concentrations (0.01–0.30 mg ml⁻¹), and pH values (2.5–5.5) for naringinase enzyme. Naringinase was immobilized on celite by simple adsorption. Naringin content was determined by HPLC method. The degree of hydrolysis of naringin showed a linear increase up to an enzyme concentration of 0.2 mg ml⁻¹ that corresponds to 82% hydrolysis. The optimum values of pH for the hydrolysis of naringin were 4.0 for free and 3.5 for immobilized enzymes. Maximum enzyme activities were found to be 70 and 60 °C for free and immobilized enzymes, respectively. The values of K _m,app and V _max,app calculated were 1.22 mM and 0.45 μmol min⁻¹ mg enzyme⁻¹ for free and 2.16 mM and 0.3 μmol min⁻¹ mg enzyme⁻¹ for immobilized enzyme, respectively. The mathematical modelling was applied to the experimental data for hydrolysis of naringin as a function of time at 30, 40 and 50 °C. The increase in temperature from 30 to 50 °C increased the rate constant 3.09 times for free enzyme. However, the rate constants found for immobilized enzyme applications did not increase in a similar trend as a function of temperature. The retained activity of celite-adsorbed naringinase was found to be 83% at their optimum conditions. The retained activity of immobilized enzyme was followed up to the fifth run and was found to be almost unchanged after the third use at optimum reaction conditions (pH 3.5, 60 °C).     

Properties of <Emphasis Type="Italic">Aspergillus subolivaceus</Emphasis> free and immobilized dextranase

Aspergillus subolivaceus dextranase is immobilized on several carriers by entrapment and covalent binding with cross-linking. Dextranase immobilized on BSA with a cross-linking agent shows the highest activity and considerable immobilization yield (66.7%). The optimum pH of the immobilized enzyme is shifted to pH 6.0 as compared with the free enzyme (pH 5.5). The optimum temperature of the reaction is resulted at 60 °C for both free and immobilized enzyme. Thermal and pH stability are significantly improved by the immobilization process. The calculated K _m of the immobilized dextranase (14.24 mg mL⁻¹) is higher than that of the free dextranase (11.47 mg mL⁻¹), while V _max of the immobilized enzyme (2.80 U μg protein⁻¹) is lower than that of the free dextranase (11.75 U μg protein⁻¹). The immobilized enzyme was able to retain 76% of the initial catalytic activity after 5.0 cycles.     

Multiple forms of β‐galactosidase from mango (Mangifera indica L Alphonso) fruit pulp

Mango (Mangifera indica L cv Alphonso) was found to contain three isoforms (I, II and III) of β‐galactosidase which, upon purification on Sephadex G‐200, had relative abundances of 44, 38 and 18%, respectively. The total specific activity increased from 20 to 727 µmol l^?1 upon purification, representing a ～36‐fold increase with a recovery of 0.28 U U^?1. The optimal pH for activity and stability were in the ranges 3.6–4.3 and 4–6.2, respectively. The optimal temperature for β‐galactosidase activity was between 42 and 47 °C with T_m in the range 45–51 °C. The K_m for pNP‐β‐galactopyranoside was 0.98, 1.11 and 0.95 mM , and V_max was 0.56, 0.53 and 0.35 µmol pNP min^?1, respectively for isoforms I, II and III. Hg²⁺ caused strong inhibition, whereas galacturonic acid, galactose, xylose, fucose and mannose slightly inhibited the activity of β‐galactosidase isoforms. The apparent molecular weights by GPC were 78, 58 and 91 kDa for isoforms I, II and III, respectively. The ability of these isoforms to degrade the endogenous substrate (arabinogalactan) possibly suggests a role in pectin dissolution during tissue softening/fruit ripening. Copyright © 2004 Society of Chemical Industry     

Purification and characterization of an amylase from Opuntiaficus‐indica seeds

BACKGROUND: In Tunisia, prickly pear fruit grow spontaneously; it is consumed as fresh fruit, juice or jam. When the fruit is used for juice production, the seeds are discarded and go to waste. Our study aimed to extract biomolecules from seeds by producing value‐added products from the fruits. RESULTS: An amylase from Opuntia ficus‐indica seeds was extracted and purified to homogeneity. An increase in specific activity of 113‐fold was observed. The apparent molecular mass of the enzyme is 64 kDa. The optimum pH and temperature for enzyme activity were pH 5 and 60 °C, respectively. Under these conditions, the specific activity is 245.5 U mg^?1. The enzyme was activated by Co²⁺ and Mg²⁺ (relative activity 117% and 113% respectively) at lower ion concentrations. It was strongly inhibited by Mn²⁺ and Fe²⁺. Cu²⁺ inhibited totally the activity of this enzyme, but Ca²⁺ has an inhibitory effect which increases with ion concentration. CONCLUSION: The extracted enzyme belongs to the exo type of amylases and is classified as a β‐cyclodextrin glycosyltransferase since it generates mainly β‐cyclodextrin from starch. It exhibits high thermal stability and a broad range of pH stability, making it a promising prospect for industrial and food applications. Copyright © 2012 Society of Chemical Industry     

Stability and Activity of β-Galactosidase in Sonicated Cultures of Lactobacillus delbrueckii ssp. bulgaricus 11842 as Affected by Temperature and Ionic Environments

Stability of β‐galactosidase and related lactose hydrolyzing activity in sonicated cultures of Lactobacillus bulgaricus 11842 was investigated in the presence of Na⁺ or K⁺ ions. After sonication in Na⁺ or K⁺ buffers at various pH levels, cultures were held at various temperatures, before adding test lactose solutions. Hydrolysis was monitored by cryoscopy. Cultures sonicated in K⁺ buffer had higher activity and stability than those in Na⁺ buffer; both were highest at pH 6 and 7. Stability was unaffected at pH 6 and 7 below 56 °C. Holding at 61 °C for 60 min caused 70% activity loss with K⁺ ions at pH 6 and 7, while with Na⁺, activity loss was almost complete.     

Immobilization of β-galactosidase from Cicer arietinum (gram chicken bean) and its catalytic actions

β-Galactosidase (β-d-galactosidase galactohydrolase EC 3.2.1.23) isolated and purified from Cicer arietinum (gram chicken bean) was immobilized on two kinds of modified resin D202 with glutaraldehyde. Both the immobilized enzymes had high protein-binding capacity and high yield of enzyme activity. Kinetics results showed that the enzyme activity attained its maximum at 57°C, pH 6 for the immobilized β-galactosidase I and 52°C, pH 6 for the immobilized β-galactosidase II, respectively. The operational pH range was increased. Kinetic constants (K_m, V_max and E_a) for the free and bound enzymes, with ONPG as substrate, were studied. Results showed that K_m and V_max of immobilized enzymes were decreased while E_a of them was increased. The effects of some compounds and organic solvents for the free and immobilized enzymes were discussed. Inhibitory constants for raffinose, lactose and d-galactose, which were all reversible inhibitors of the enzymes, were also obtained.     

大豆β-淀粉酶、产气杆菌异淀粉酶在虾壳几丁质上的固定化及在麦芽糖生产中的应用

本文研究了大豆β—淀粉酶和产气杆菌异淀粉酶在同一载体上的固定化作用。以虾壳几丁质和壳聚糖为载体,证明几丁质更适于固定化异淀粉酶。用5％甲酸和50％乙酸预处理载体可增加固定化异淀粉酶的活性和稳定性,这可能是由于载体中壳聚糖的溶解造成的。经甲酸处理的几丁质先与4～6％pH7～9的戊二醛反应1小时,再同酶液(10mg/ml,pH6)反应2小时,并添加0.2mol/1Ca~#,将有利于固定化作用。用本方法得到的固定化酶具有高度活性和稳定性。固定化异淀粉酶最适pH由6.5变为5.0,而固定化β—淀粉酶最适pH变化不大。固定化酶在60℃稳定,在70℃5分钟失活。固定化酶在水中贮藏稳定,并具有良好的操作稳定性,使其在麦芽糖生产中具有潜在的应用价值。     

Lysyl oxidase from jumbo squid (Dosidicus gigas) muscle: purification and partial characterization

Lysyl oxidase (LOX; E.C.1.4.3.13) was purified from jumbo squid muscle (Dosidicus gigas) with 1900‐fold and yield 1.9%, and characterized for the first time. The purification procedure consisted of fractionation with urea and a combination of size‐exclusion and anion‐exchange chromatography. The enzyme had a molecular weight of 32 kDa, as estimated by SDS‐PAGE. Using a specific LOX substrate (1,5‐diaminopentane), its optimum activity was determined at pH 8.2 and 65 °C. Activation energy (E_a) of the enzyme was 69.94 kJ K^?1 mol^?1. The enzyme was strongly inhibited by β‐aminopropionitrile fumarate (BAPN), a specific LOX inhibitor. Moreover, purified LOX was able to work at different temperatures (20–90 °C) at pH 8.2. Although further research is needed, the results from this work suggest that based on LOX activity, this enzyme may be of practical use in preventing textural changes in jumbo squid during storage or processing.