Properties of mouse α-galactosidase

α-Galactosidase has been examined in various murine tissues using the substrate 4-methylumbelliferyl-α-galactoside. Mouse liver appears to contain a single major form of the enzyme, as judged by chromatography and electrophoresis. The enzyme was purified 467-fold with a yield of about 40% by a method involving chromatography on Concanavalin A-Sepharose. It has maximal activity at pH 4.2, a K_m value of 1.4 mM, an energy of activation of 16 400 cal/mol, and a molecular weight of 150 000 at pH 5.2. It is inhibited at high concentrations of myoinositol and appears to contain N-acetylneuraminic acid. In these characteristics it resembles human α-galactosidase A.The enzyme from various tissues differs in electrophoretic mobility. After treatment with neuraminidase, however, the enzyme from all tissues comigrates as a single band of activity. By this criterion the α-galactosidase of liver is most heavily sialylated and that from kidney the least. As estimated by gel filtration, the enzyme from liver and kidney exists as species of molecular weight 320 000, 150 000 and 70 000, depending upon pH and ionic strength. This appears to be the result of aggregation of the enzyme, since the forms are interconvertible and under some conditions a single molecular weight species is observed. The liver enzyme is primarily lysosomal, while the kidney enzyme is distributed approximately equally between lysosomal and microsomal fractions.     

The purification and immobilization of Penicillium notatum β-galactosidase

Penicillium notatum No. 1 as a producer of β-galactosidase was cultivated in a 5–1 fermenter. Various methods of protein isolation and concentration from the culture fluid were optimized. Then the conditions of β-galactosidase purification using an affinity chromatographic technique were established. The purified enzyme was immobilized on a controlled porous glass (CPG). The optimum temperature and pH values of the native and immobilized forms of β-galactosidase were determined as 50°C and 30–50°C as well as pH 3 and pH 3–5, respectively.     

A comparison of different methods of β-galactosidase immobilization

β-Galactosidase was immobilized in a range of supports showing suitable physico-chemical characteristics for use in fluidized bed reactors. Uncoated porous glass, alginate and κ-carrageenan beads and chromosorb-W were used as carriers. The intrinsic kinetic constants (V_max and K_M) and coupling parameters for the immobilization were calculated. The highest immobilized protein percentages and activity yields were obtained when β-galactosidase was attached through its amine groups to aldehyde-glass. The final choice of derivative for use in fluidized bed reactors should be based not only on the enzymatic activity shown by the derivatives but also on the hydrodynamic behaviour of the supports.     

Optimization of β-galactosidase extraction from Kluyveromyces marxianus

The influence of several parameters, such as temperature, pH, and concentration of buffer and solvent, on the release of β-galactosidase from Kluyveromyces marxianus cells was studied. In optimal conditions (37°C, pH 9.5–10.5) greater than 90% of the intracellular β-galactosidase activity was released into 0.1-0.5 phosphate buffer after 1.5-2.0 h treatment with 1% chloroform. The described method is simple, effective, relatively fast, and selective.     

Purification of β-acetylglucosaminase and β-galactosidase from ram testis

1. The presence of beta-galactosidase (EC 3.2.1.23) in an acetic acid extract of ram testis is reported. Some properties of the crude enzyme preparation were studied. 2. The purification of beta-acetylglucosaminase (EC 3.2.1.30) and of beta-galactosidase from the ram-testis extract by ammonium sulphate precipitation and chromatography on a CM-cellulose column is described. 3. The final purifications of the separated enzymes achieved were for the beta-acetylglucosaminase 35 times and for the beta-galactosidase 99 times. 4. The possibility of using DEAE-cellulose and Sephadex G-200 to purify the enzymes was investigated.     

The kinetics of β-galactosidase attached to porous cellulose sheets

The properties of β-galactosidase attached to cellulose and DEAE-cellulose sheets arc described. Those insoluble enzyme derivatives obey the Michael-Menten relationship but, the measured kinetic parameters are very dependent on the flow conditions. The results of long-term stability tests are given.     

Immobilization of β-galactosidase and other enzymes onto p-amino-carbanilated cellulose derivatives

β-Galactosidase and other enzymes were immobilized on p-amino-carbanilated derivatives of cellulose and methylol cellulose using the diazo method and through glutaraldehyde. The optimum conditions for coupling cellulose tri-(p-amino-carbanilate) (CTAC) to β-galactosidase were established. The diazo coupling method with CTAC gave greater activity than with glutaraldehyde when coupled to β-galactosidase (Escherichia coli). The stability of the CTAC–β-galactosidase system was examined. The disubstituted p-amino-carbanilate derivative (CDAC) gave a lower activity, whereas the methylol analog (MCTAC) gave slightly greater activity. The CTAC was also used to immobilize glucose oxidase, trypsin, pepsin, and papain.     

Study of the deactivation of β-galactosidase entrapped in alginate-carrageenan gels

The stability of β-galactosidase entrapped in Ca-alginate–K-κ-carrageenan gels under operation conditions was studied. The thermal deactivation of the immobilised enzyme and the biocatalyst protein loss due to gel swelling were taken into account in the mass balance of the enzymatic reaction rate expression.

Time-temperature effect was the most important factor in the biocatalyst deactivation reaction. However, results showed that the enzyme entrapped in gels was partially lost by gel swelling, which was a source of error in predicting results in continuous processes. The enzyme loss determined in this work showed a non-linear behaviour and it depended on mixing conditions of the reactor.

Values of protein loss were used in the modelling of a fixed-bed reactor with similar flow conditions to reduce the error in predicting the operation conditions to maintain a constant conversion.

For reaction conditions similar to those analysed in this work, the β-galactosidase was well entrapped in alginate-carrageenan matrices.     

Structural classification of αββ and ββα supersecondary structure units in proteins

We present a fully automatic structural classification of supersecondary structure units, consisting of two hydrogen-bonded β strands, preceded or followed by an α helix. The classification is performed on the spatial arrangement of the secondary structure elements, irrespective of the length and conformation of the intervening loops. The similarity of the arrangements is estimated by a structure alignment procedure that uses as similarity measure the root mean square deviation of superimposed backbone atoms. Applied to a set of 141 well-resolved nonhomologous protein structures, the classification yields 11 families of recurrent arrangements. In addition, fragments that are structurally intermediate between the families are found; they reveal the continuity of the classification. The analysis of the families shows that the α helix and β hairpin axes can adopt virtually all relative orientations, with, however, some preferable orientations; moreover, according to the orientation, preferences in the left/right handedness of the α–β connection are observed. These preferences can be explained by favorable side by side packing of the α helix and the β hairpin, local interactions in the region of the α–β connection or stabilizing environments in the parent protein. Furthermore, fold recognition procedures and structure prediction algorithms coupled to database-derived potentials suggest that the preferable nature of these arrangements does not imply their intrinsic stability. They usually accommodate a large number of sequences, of which only a subset is predicted to stabilize the motif. The motifs predicted as stable could correspond to nuclei formed at the very beginning of the folding process. Proteins 30:193–212, 1998. © 1998 Wiley-Liss, Inc.     

Extraction of β-galactosidase fused protein a in aqueous two-phase systems

A method for the purification of proteins hybridized with β-galactosidase and produced in Escherichia coli is suggested. The method is based on the dominating properties of the β-galactosidase part of the molecule that are utilized for extraction in a poly(ethylene glycol) 4000/potassium phosphate aqueous two-phase system. The purification of the hybrid protein Staphylococcal protein A-Escherichia coli-β-galactosidase (SpA-βgal) produced in Escherichia coli is described. The partitioning of the cell debris and SpA-βgal depended on the distance to the critical point, i.e., the length of the tie line. A poly(ethylene glycol) top phase and an interface free from cell debris were obtained for a composition close to the binodial with a relatively short tie line. At this composition no Spa-βgal partitioned to the interface. When the length of the tie line was increased, more of the SpA-βgal was caught by the interface. The partitioning of SpA-βgal to the top phase was also affected by the salts present during the extraction. The utilization of SpA-βgal for affinity extraction has been investigated. Experiments with SpA-βgal and fluorescence-labeled human IgG(hIgG-F) in a poly(ethylene glycol) 4000/potassium phosphate aqueous two-phase system showed that the complex SpA-βgal-hlgG-F was partitioned to the interface, probably as a precipitate.     

Temperature and pH dependence of mold α-galactosidase

The effect of temperature and pH on kinetic behavior of α-galactosidase of Mortierella vinacea was investigated on the hydrolysis of p-nitrophenyl-α-D -galactopyranoside (PNPG). A very unusual kinetic behavior was observed for the soluble α-galactosidase i.e., substrate inhibition diminished gradually with increasing temperature or near the neutral pH range, and the kinetics approached the ordinary Michaelis-Menten (MM) type. On the other hand, with decreasing temperature or in acidic pH range, substrate inhibition was accelerated. Therefore, Arrhenius plots based on the initial reaction rate did not give straight lines. Furthermore, the slope in the Arrhenius plot changed with substrate concentration, which would make the determination of a characteristic value using conventional methods meaningless. However, the Arrhenius plots of individual kinetic parameters in the rate equation resulted in straight lines in the temperature range 15 to 50°C. From this, the drastic change in kinetic behavior could be explained in connection with the temperature and pH dependence of kinetic parameters in the model. For mold pellets (whole-cell enzyme), however, the influence of temperature and pH was less apparent than that of soluble enzyme because of the limitation in intraparticle diffusion. By using the rate equation that was determined for soluble enzyme and the theoretically derived effectiveness factor, the overall reaction rate for mold pellets at various temperature and pH could be predicted to some extent.     

The phospho-β-galactosidase and synaptotagmin predictions

Two bona fide consensus predictions of secondary and tertiary structure in a protein family, made and announced before experimental structures were known, are evaluated in light of the subsequently determined experimental structures. The first, for phospho-β-galactosidase, identified the core strands of an 8-fold α–β barrel, and identified the 8-fold α–β barrel itself, which was found in the subsequently determined experimental structure to be the core folding domain. The second, for synaptotagmin, identified seven out of eight β-strands in the structure correctly, missing only a noncore strand. Three preferred “topologies” were selected from several hundred thousand possible topologies of these seven predicted strands using a rule-based analysis. The subsequently determined experimental structure showed that these seven strands in synaptotagmin adopt one of the three preferred topologies. We were unable, however, to identify the correct topology from among these three topologies. © 1995 Wiley-Liss, Inc.     

Kinetic studies of mold α-galactosidase on PNPG hydrolysis

The kinetic properties of α-galactosidase of Mortierella vinacea were investigated in detail using PNPG (p-nitrophenyl-α-D -galactopyranoside) as a substrate. Consequently, the enzyme was markedly inhibited not only by the substrate, but also by the galactose hydrolized. The initial rate of reaction at sufficiently high substrate concentrations, however, did not fall to zero and did approach a finite value. Galactose behaved as a mixed inhibitor and was neither totally competitive nor totally noncompetitive. A rate equation was obtained from a generalized equation derived from a kinetic model which took both the inhibitions into consideration. The constants used in the equation were appropriately estimated. The calculated rate agreed fairly well with the observed initial rate. Moreover, the PNPG hydrolysis progressing in a batch system was found to be approximately representable by simple first order kinetics in which the rate constant was dependent on the initial substrate concentration.     

Hydrolysis of lactose in whey permeate by immobilized β-galactosidase from Penicillium notatum

A new low-cost β-galactosidase (lactase) preparation for whey permeate saccharification was developed and characterized. A biocatalyst with a lactase activity of 10 U/mg, a low transgalactosylase activity and a protein content of 0.22 mg protein/mg was obtained from a fermenter culture of the fungus Penicillium notatum. Factors influencing the enzymatic hydrolysis of lactose, such as reaction time, pH, temperature and enzyme and substrate concentration were standardized to maximize sugar yield from whey permeate. Thus, a 98.1% conversion of 5% lactose in whey permeate to sweet (glucose-galactose) syrup was reached in 48 h using 650 β-galactosidase units/g hydrolyzed substrate. After the immobilization of the acid β-galactosidase from Penicillium notatum on silanized porous glass modified by glutaraldehyde binding, more than 90% of the activity was retained. The marked shifts in the pH value (from 4.0 to 5.0) and optimum temperatures (from 50°C to 60°C) of the solid-phase enzyme were observed and discussed. The immobilized preparation showed high catalytic activity and stability at wider pH and temperature ranges than those of the free enzyme, and under the best operating conditions (lactose, 5%; β-galactosidase, 610–650 U/g lactose; pH 5.0; temperature 55°C), a high efficiency of lactose saccharification (84–88%) in whey permeate was achieved when lactolysis was performed both in a batch process and in a recycling packed-bed bioreactor. It seems that the promising results obtained during the assays performed on a laboratory scale make this immobilizate a new and very viable preparation of β-galactosidase for application in the processing of whey and whey permeates.     

Purification and enzymatic properties of α-galactosidase from Penicillium janthinellum

α-Galactosidase (E.C.3.2.1.22) from Penicillium janthinellum was purified by precipitation and fractionation with ammonium sulphate, cold acetone or ethanol, calcium phosphate gel, and column chromatographies on Sephadex G-100 and G-200. The enzyme was purified about 110.39-fold when Sephadex G-100 was used. α-Galactosidase exhibited the optimum pH and temperature at 4.5 and 60°C, respectively. The optimum enzyme stability was obtained at pH 3.5 for 24 h (at room temperature). The enzyme was found to be thermostable below 65°C up to 40 minutes and was gradually inactivated by increasing the temperature above this degree. The MICHAELIS constant was 0.55 mM for p-nitrophenyl-α-D-galactoside. The α-galactosidase activity was strongly inhibited by Hg⁺⁺ and slightly activated by Mn⁺⁺. The results show the possibility of producing a thermostable enzyme from a low-priced agricultural product, for instance, lupine.     

Studies on the 5α-androstane-3β, 17β-diol-6α-hydroxylase and on the 5α-androstane-3β, 17β-diol-7α-hydroxylase in anterior hypophysis of male rats.

In anterior pituitaries from male rats, it appeared that 5α-androstane-3β, 17β-diol was quickly metabolized into 5α-androstane-3β,6α-17β-triol and 5α-androstane-3β,7α, 17β-triol by action of 6α- and 7α-hydroxylases. Hydroxysteroid hydroxylases were located in endoplasmic reticulum and were dependent on NADPH⁺. Their optimum pH was 8.0, optima temperature, 37°C, and their apparent Km was 2.7 μM. Hydroxylative reactions were not reversible and not modified by gonadectomy. Hydroxylation seemed an efficient control of the pituitary level of 5α-andros-tane-3β, 17β-diol.