Immobilisation and stabilisation of β-galactosidase from Kluyveromyces lactis using a glyoxyl support

β-galactosidase from Kluyveromyces lactis was covalently immobilised on a Glyoxyl Sepharose (GS) support by multi-point attachment. The enzyme immobilisation process was very efficient; the supports immobilised almost all the protein responsible for the catalytic activity in a short period of time, retaining approximately 82% of the activity in the case of the optimal immobilised preparations. Stability of the GS derivatives varied as a function of enzyme-support incubation time. The optimal immobilised preparation was produced after 2 h of incubation with the support at alkaline pH. This derivative, obtained by multi-point covalent attachment, was 100-fold more stable at pH 7 and 50 °C than the cyanogen bromide Sepharose derivative obtained by a one-point covalent immobilisation method. Stabilisation was also observed under a wide range of experimental conditions. This method allowed the immobilisation of 9000 IU enzyme g^?1 of support, resulting in highly active and stable derivatives suitable for industrial processes.     

Performance comparison of free and immobilised chicken liver esterase inhibited by four different pesticides

BACKGROUND: In enzyme inhibition‐based biosensors for the detection of organophosphate and carbamate pesticides, the biological element is the immobilised esterase which is inhibited selectively by the pesticides. The free chicken liver esterase, which possesses comparable inhibition response to the pesticides as AChE, has been reported. However, the responses to the pesticides are different between the free and immobilised enzyme due to the diffusion limitation. Therefore, in this study four typical pesticides were selected to compare the ability of free and (ion exchange) immobilised chicken enzyme for pesticide detection and reasons for the difference were investigated. RESULTS: For dichlorvos and malathion at a concentration of 0.1 mg L^?1, the inhibition of the immobilised enzyme was 26.98% and 48.72%, respectively, higher than that for the free enzyme, while the percentage inhibition of free and immobilised enzyme differed very little for trichlorfon at 0.1 mg L^?1. In the meantime, carbaryl at a concentration of 2.5 mg L^?1 showed a 17.72% inhibition for immobilised enzyme which was 13.64% higher than that for the free enzyme. The Michaelis constant of immobilised enzyme was lower than that of the free one and suitable pH values for the free and immobilised enzyme were 7.5 and 8.0, respectively. CONCLUSIONS: The sensitivity of chicken liver esterase to the pesticide inhibition could be improved by immobilisation. Smaller K_m and a lower pH for the micro‐environment of immobilised enzyme should result in higher percentage inhibition compared with that of the free enzyme. Copyright © 2008 Society of Chemical Industry     

Characteristics of immobilised β‐glucosidase and its effect on bound volatile compounds in orange juice

In this study, bound volatile compounds were isolated and extracted with Amberlite XAD‐2 resin and then hydrolysed by free or immobilised β‐glucosidase. The released bound volatiles were analysed by GC‐MS. In addition, the optimisation of immobilisation method on sodium alginate and the characteristics of immobilised β‐glucosidase were studied. The results showed that crosslinking‐entrapment was the best method. The optimal conditions of this method were as follows: sodium alginate concentration 3.5%, glutaraldehyde concentration 1%, crosslinking time 3 h, immobilisation time 2 h and CaCl₂ concentration 3%. The optimum temperature for β‐glucosidase (65 °C) was decreased by 10 °C after immobilisation, while the optimum pH values for free and immobilised β‐glucosidase were both at pH 5.0. The K_m values of free and immobilised β‐glucosidase were 14.89 and 0.59 m , respectively. In total, thirteen and six bound volatile compounds were detected in orange juice hydrolysed by free and immobilised β‐glucosidase, including benzenic compounds, terpenic compounds, hydroxy esters, C₁₃‐norisoprenoids and alcohols.     

Peroxidase activity of germinating Sorghum bicolor grains: effect of some cations

We investigated some properties of the major ionic peroxidase (POD) from germinating Sorghum bicolor var Fara Fara. Peroxidase activity increased eightfold during the first 36 h of germination and declined thereafter to four times the value in the dry grain after 144 h. Gel filtration followed by ion exchange chromatography resolved the major peroxidase into two forms. The predominant POD had an optimum temperature of 55 °C, two pH optima at 5.0 and 7.0, with a sharp decline in activity below pH 4.5, an apparent activation energy of 463 kJ mol^?1, an apparent molecular weight of 63.1 kDa and was relatively heat‐stable up to 70 °C. The enzyme was strongly inhibited by dithiothreitol and sodium metabisulphite. Calcium chloride and magnesium chloride below 2 mM enhanced POD activity without any adverse effect on germination, while ammonium chloride and ferric chloride inhibited the enzymatic activity of both forms. At 5 mM , magnesium chloride inhibited POD activity by 50% with only a 14% reduction in germination, while calcium chloride achieved the same effect at 10 mM . These results are pertinent to controlling the undesirable activity of peroxidase in a typical malting or brewing process. © 2002 Society of Chemical Industry     

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.     

Dephosphorylation of phytate compounds by means of acid phosphatase from Aspergillus niger

A non-purified preparation of intracellular acid phosphatase (EC 3.1.3.2) from a waste mycelium of Aspergillus niger was utilised for dephosphorylation of phytate compounds present in food and feed ingredients. The enzymic hydrolysis of p-nitrophenylphosphate was used for assaying acid phosphatase activity, expressed in standard units (u). The hydrolysis of phytate phosphorus in wheat bran, soya bean meal and fully formulated feedstuffs for broilers (Galus galus; ‘Cornish’ × White Rock') was carried out at 40°C, a pH value of 4.5 and an enzyme dosage ranging from 12 to 30 u g^?1. Complete dephosphorylation of soya bean protein isolates was performed at 60°C, a pH value of 4.5 and an enzyme dosage of 12 u g^?1. In the gastrointestinal tract of broilers the in-vivo dephosphorylation of phytates present in feed was observed when the preparation of acid phosphatase was added to the diet.     

REVISED CONFORMATIONAL STABILITY PARAMETERS FOR BETA-LACTOGLOBULIN ISOTHERMAL DENATURATION BY UREA

Isothermal unfolding studies for beta‐lactoglobulin (β‐Lg) usually apply a denaturation model suitable for a single submit protein. A method is described for correcting such results in retrospect. This leads to accurate estimates for the Gibbsfree energy of stabilization of native β‐Lg aimer. The denaturation of β‐Lg aimer is described by a dissociation coupled unfolding (DCU) process; Dimer → monomer → unfolded state. The Gibbs free energy change for DCU (ΔG°_DCU) was 80 kJ mol^?1 at pH 7 and 97 kJ mol^?1 for β‐Lg at pH 2.6. With the addition of a range of salts (pH 6.85) then ΔG°_DCU increased to 105 kJ mol^?1. The literature is replete with denaturation studies employing β‐Lg as the principal protein. In many instances, it would be prudent to reevaluate stability data for β‐Lg in line with techniques outlined here.     

Immobilised yeast grape must deacidification in a recycle fixed bed reactor

Maloalcoholic fermentation (MAF) of grape must by Schizosaccharomyces pombe immobilised in calcium‐alginate double‐layer beads (ProMalic^®) was studied in Erlenmeyer flasks and in a total recycle fixed‐bed reactor operating in batch mode. The reaction is pseudo‐first order with respect to l ‐malic acid and under similar conditions deacidification is faster in the recycle reactor. This was attributed to mass transfer limitations which were confirmed in the recycle reactor by studying the influence of yeast load on the rate of MAF. Mass transfer limitations are also responsible for the lower activation energy of fermentation with the immobilised yeast (67 ± 9 kJ mol^?1) in comparison with the free cells (126 ± 19 kJ mol^?1). Alcoholic fermentation and MAF were performed simultaneously, both in the recycle reactor and in the industrial trials, confirming the efficacy of immobilised S. pombe to reduce grape must acidity without interfering with the main fermentation. Altogether, the present results are useful for the scale‐up of a recycle reactor to process large volumes of grape must.     

Hydrolysis of sucrose by invertase entrapped in polyvinyl alcohol hydrogel capsules

Invertase was immobilised into polyvinyl alcohol capsules (LentiKats^®). The enzyme retained 86% of its original activity after the immobilisation process. Kinetic constants, pH and temperature profiles, and stability were determined, for both free and immobilised enzyme. Entrapped invertase was used for 45 repeated batch hydrolyses and no decrease in enzymatic activity was observed. The immobilised enzyme retained 78% of its initial activity after 8 months of storage.     

SARCOPLASMIC RETICULUM Ca2+-ATPase ACTIVITY IN COD (GADUS MORHUA) MUSCLE MEASURED IN CRUDE HOMOGENATES

An assay for sarcoplasmic reticulum Ca²⁺‐ATPase in an unfractionated homogenate from cod(Gadus morhua) muscle has been established. Specificity of the assay was demonstrated by the Ca²⁺ dependence of the enzyme and the effect of a specific inhibitor, thapsigargin. Stimulation and inhibition of enzyme activity was observed at low and high Ca²⁺ concentration, respectively. Half the maximal activity was obtained at 0.2 μM free calcium and no activity could be detected above 20 mM free Ca²⁺. Thapsigargin inhibited 92% of enzyme activity. Sarcoplasmic reticulum Ca²⁺‐ATPase showed maximal activity around pH 7 and 25C. A nonlinear Arrhenius plot was found with pronounced changes in the slope in the temperature interval 6–15C. The activation energies obtained from the approximately linear parts above 15C and below 6C were 25 kJ mot^?1 and 172 kJ mol^?1, respectively.     

Galacto-oligosaccharide synthesis using chemically modified β-galactosidase from Aspergillus oryzae immobilised onto macroporous amino resin

The goal of this study was to establish an efficient immobilisation protocol for β-galactosidase from Aspergillus oryzae onto the polystyrenic macroporous resin Purolite^® A-109 for better utilisation of its transglactosylation activity and application in galacto-oligosaccharide (GOS) synthesis. This was achieved by improving simple ionic adsorption by carboxyl group activation on the enzyme surface with carbodiimide, enabling covalent immobilisation. This yielded significantly increased operational stability, assayed as GOS synthesis, in a batch reactor, and even more prominently, in a fluidised bed reactor (73% activity retained after 10 cycles). The immobilised enzyme showed two very beneficial advantages over the free enzyme for future applications: higher affinity towards catalysing transgalactosylation than towards hydrolysis and shift of pH optimum towards more acidic conditions. GOS synthesis performed under the optimum conditions obtained (400 g L⁻¹ lactose, pH 4.5, 50 °C) yielded 87 g L⁻¹ and 100 g L⁻¹ for batch and fluidised bed reactors, respectively.     

Production of soya milk containing low flatulence‐causing oligosaccharides in a packed bed reactor using immobilised α‐galactosidase

Peanut α‐galactosidase was immobilised in calcium alginate beads and used to hydrolyse the flatulence‐causing oligosaccharides, raffinose and stachyose, in soya milk in batch and in packed bed reactor with recycle. The immobilised enzyme exhibited a slightly lower activity than the free enzyme. The activity yield of immobilised α‐galactosidase was 75.1% and the immobilisation yield was 82.6%. Batch hydrolysis using immobilised enzyme at 55 °C resulted in 96% reduction in the oligosaccharides after 12 h. For the continuous process, a packed bed reactor with recycle was used. More than 98% of the oligosaccharides were hydrolysed after 6 h of reaction at 55 °C. The immobilised enzyme also proved to be stable up to three repeated hydrolysis reactions.     

Acid phosphatase in the tubers of Dioscorea species and its purification from the white yam (D. rotundata poir)

Acid phosphatase activity was determined in 15 cultivars from four species of yam. A 12-fold purification of the enzyme from Dioscorea rotundata (cv. chikakwondo) gave a homogeneous preparation as demonstrated by polyacrylamide gel electrophoresis. This enzyme preparation has an apparent molecular weight of 115 000 ±2000 and an optimum activity at a pH of 5·20 and a temperature of 50°C. The K_m of the enzyme is 3·81 mM with disodium p-nitrophenylphosphate (p-NNP) as a substrate. The energy of activation, heat of activation, energy of inactivation and heat of inactivation are 7·0, 6·4, 4·41 and 4·34 kcal M^?1, respectively. Although it has very little activity with most organic phosphoric acid esters, it is significantly inhibited by Ca²⁺, Hg²⁺ and EDTA and activated by Mg²⁺. The enzyme has a half-life of 50,17 or 13 days, respectively, when stored at 6-8°C, 0°C or room temperature (29±2°C).     

Urea and Heat Unfolding of Cold-Adapted Atlantic Cod(Gadusmorhua)Trypsin and Bovine Trypsin

The reversible unfolding reactions for phenylmethylsulphonyl fluoride (PMSF)-modified trypins from Atlantic cod (cod PMS-trypsin) and cattle (bovine PMS-trypsin) were monitored by fluorescence spectrophotometry as a function of urea concentration and temperature. For urea unfolding at 25°C, the free energy change at zero concentration of urea (ΔG(H₂O)) for cod PMS-trypsin was 11(±4·4) kJ mol⁻¹ compared with 18(±1·14) kJ mol⁻¹ for bovine PMS-trypsin, while the mid-point concentration for urea unfolding curve ([urea]_1/2) was 3·0(±0·57) M and 4·1(±0·16) M, respectively. From studies of enzyme heat unfolding, the mid point temperature of the thermal unfolding curve ( T _m ) was 46(±1·4)°C for cod PMS-trypsin compared with 57(±2)°C for bovine PMS-trypsin. The standard free energy change (Δ G° ) for reversible thermal unfolding of cod PMS-trypsin was 9(±1) kJ mol⁻¹ compared with 19(±1) kJ mol⁻¹ for bovine PMS-trypsin. Values for the enthalpy (Δ H _m ), entropy (Δ S _m ) and heat capacity (Δ C _p ) for heat unfolding are compared. Results from urea and thermal unfolding studies show that cod PMS-trypsin has a significantly lower conformational stability than bovine PMS-trypsin.     

Purification and immobilisation of inulinase from Aspergillus candidus for producing fructose

A high‐inulinase‐producing strain of Aspergillus candidus (10 units cm⁻³ of medium) for producing fructose from inulin has been identified. The extracellular inulinase from this fungi was purified 56‐fold by ammonium sulphate fractionation, DEAE cellulose and Sephadex G‐150 column chromatography. Invertase to inulinase ratio of 1.8 in culture filtrate was reduced to 0.14 in the purified preparation. The pH and temperature optima were 5.5 and 45°C, respectively. The molecular weight of inulinase was determined as 54±4 kDa. K_m of inulinase with inulin as a substrate was 3.8 mmole dm ⁻³. The purified preparation produced only fructose as the product of inulin, indicating that inulinase has primarily exo‐inulinase activity. Inulinase was immobilised on chitin and casein using glutaraldehyde as a linking agent and on cellulose using FeCl₃‐HCl as a metal chelation agent. Maximum immobilisation of 45.8% was achieved on cellulose. All three immobilised preparations had a higher temperature optima of 55°C. The inulinases immobilised on cellulose and casein were stable at pH 5–7. The cellulose‐immobilised preparation was more stable than the other two preparations after heating for 1 h at 55°C. © 1999 Society of Chemical Industry     

Properties of polyphenol oxidase in mango (Mangifera indica) kernel

Polyphenol oxidase (PPO) activity of filtered extract of ground mango kernel suspension (400 g litre⁻¹) was studied spectrophotometrically at 420 nm using catechol as substrate. The enzyme was most active at pH 6·0 and 25°C. Activity was reduced by 50% at pH values of 5·0 and 7·1, and also at temperatures of 14°C and 30°C. The calculated activation energy and the Michaelis constant (K_m) were 21·4 kcal mol⁻¹ °C⁻¹ and 24·6 mM , respectively. The V_max value was 2·14 units g⁻¹ mango kernel. The time to heat inactivate PPO decreased rapidly to < 10 min with increasing temperature of ⩾ 70°C at 50% activity. © 1998 SCI.     

Characterization of Sultaniye grape (Vitis vinifera L. cv. Sultana) polyphenol oxidase

Polyphenol oxidase (PPO) was extracted from Sultaniye grapes grown in Turkey, and its characteristics in terms of pH and temperature optima, thermal inactivation, kinetic parameters and potency of some PPO inhibitors were studied. Optimum pH and temperature for grape PPO were found to be 3.4 and 30 °C, using catechol as substrate. K_m and V_max values were found to be 44.5 ± 5.47 mm and 0.695 ± 0.0353 OD₄₁₀ min^?1, respectively. Four inhibitors were tested in this study and the most potent inhibitor was sodium metabisulphite, followed by ascorbic acid. From the thermal inactivation studies in the range of 65–80 °C, the half‐life values of the enzyme ranged between 2.6 and 49.5 min. Activation energy (E_a) and Z values were calculated to be 208.5 kJ mol^?1 (r² = 0.9544) and 10.95 °C (r² = 0.9517), respectively.     

Purification and properties of polyphenol oxidase in head lettuce (Lactuca sativa)

Polyphenol oxidase (EC 1.10.3.1) in head lettuce (Lactuca sativa L) was purified by ammonium sulphate fractionation, ion exchange chromatography and gel filtration. The enzyme was found to be homogeneous by polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be about 56 000 amu by Sephadex G-100 gel filtration. The purified enzyme quickly oxidised chlorogenic acid (5-caffeoyl quinic acid) and (—)-epicatechin. The K^m values for the enzyme, using chlorogenic acid (pH 4·5, 30°C) and (—)-epicatechin (pH 7·0, 30°C) as substrate, were 0·67 mM and 0·91 mM, respectively. The optimal pH of chlorogenic acid oxidase and (—)-epicatechin oxidase activities were 4·5 and 7·8, respectively, and both activities were stable in the pH range 6–8 at 5°C for 20 h. Potassium cyanide and sodium diethyldithiocarbamate markedly inhibited both activities of the purified enzyme. The inhibitory effect of metallic ions such as Ca²⁺, Mn²⁺, Co²⁺ and Ni²⁺ for chlorogenic acid oxidase activity was stronger than that for (—)-epicatechin oxidase activity.     

Protein stability function relations: native β‐lactoglobulin sulphhydryl–disulphide exchange with PDS

Intermolecular sulphhydryl–disulphide exchange with β‐lactoglobulin dimer occurs when this dissociates to form monomers exposing two SH groups. This notion is re‐evaluated in the light of recent structural data suggesting that the degree of SH group exposure in β‐lactoglobulin is unaffected by dissociation. β‐Lactoglobulin was treated with 2,2′‐dipyridyl disulphide (PDS). The rate of sulphhydryl–disulphide exchange was measured at sub‐denaturation temperatures of 25–60 ° C. Parallel studies were conducted by reacting PDS with reduced glutathione (GSH). The SH group of GSH was up to 31 000 times more reactive than β‐lactoglobulin. At pH 7 the reaction activation enthalpy (ΔH^#) and entropy (ΔS^#) was 26 kJ mol⁻¹ and −100 J mol⁻¹ K⁻¹ respectively for GSH. For β‐lactoglobulin, ΔH^# was 157.2 kJ mol⁻¹ and ΔS^# was 254 J mol⁻¹ K⁻¹. At pH 2.6, ΔH^# was 14.4 kJ mol⁻¹ and ΔS^# was −213 J mol⁻¹ K⁻¹ for GSH. The corresponding results for β‐lactoglobulin were 20.3 kJ mol⁻¹ and −147 J mol⁻¹ K⁻¹. These and other thermodynamic results are discussed in terms of the effects of β‐lactoglobulin conformational structure and stability on SH group reactivity. For native β‐lactoglobulin at neutral pH, intermolecular sulphhydryl–disulphide exchange appears to involve the dissociated monomer. SH group activation probably arises from the lower structural stability of the monomer relative to the dimer. At pH 2.6 the mechanism of SH–disulphide exchange does not require protein dissociation and probably involves breathing motions or localised changes in protein structure. © 2000 Society of Chemical Industry     

In-vitro and in-vivo dephosphorylation of rapeseed meal by means of phytate-degrading enzymes derived from Aspergillus Niger

A meal of ‘double low’ rapeseed (var ‘Jantar’) was subjected to phytate hydrolysis using enzyme preparations derived from a mycelium of Aspergillus niger which contained phytase (EC 3.1.3.8) and acid phosphatase (EC 3.1.3.2) activities. The complete conversion of myo-inositol hexa- and pentaphosphates present in rapeseed meal to lower phosphate esters of myo-inositol was accomplished at 40°C, a pH value of 4.5, phytase dosage (in phytase units (PhytU)) 0.1 PhytU g^?1 accompanied by acid phosphatase activity 37.1 units g^?1, in 1 h. Under these conditions, complete dephosphorylation was observed in 4 h. Decreasing the pH value to 3.0 caused a rise in the amount of inorganic phosphorus released, while increasing to 5.5 resulted in substantial reduction in the reaction rate. Purification of phytase to a specific activity 0.375 PhytU mg^?1 of protein exhibited a negative influence upon the yield of rapeseed dephosphorylation. The substitution of calcium phosphate for a preparation of phytase in feed containing rapeseed meal did not cause significant differences in the body weight gain or in tibia mineralisation of broilers (Gains galus, ‘Astra B’).