Pepsin immobilized on inorganic supports for the continuous coagulation of skim milk.

The milk-clotting enzyme pepsin was immobilized onto beads of alumina, titania, glass, stainless steel, iron oxide, and Teflon for treating skim milk in a fluidized-bed reactor. Two covalent attachment procedures using silanized supports and glutaraldehyde and two adsorption procedures were evaluated. The three best catalysts were titania and glass, using the covalent attachment procedure, and alumina, using the adsorption procedure at pH 1.2. The pepsin adsorbed on alumina catalyst has commercial potential compared to the previously used glass catalyst. Attempts to increase the stability of pepsin adsorbed on alumina by cross-linking with glutaraldehyde were unsuccessful owing to the low pH necessary for optimum pepsin adsorption; Desorption of pepsin from alumina during reactor operation was determined. Regeneration of spent catalysts was only partially successful.     

Production of glycerol by immobilizedPichia farinosa

Summary Cells of the osmophilic yeastPichia farinosa were immobilized in sintered glass Raschig rings for the production of glycerol. The kinetics of production were observed under different conditions in batch, fed-batch and semicontinuous fermentations in fixed-bed column reactors and compared with those of free cells. 2.6 × 10⁹ cells/g sintered glass were adsorbed. The glycerol productivity amounted to 8.1 g/l per day. The highest concentration reached in batch culture was 86 g/l with immobilized cells. Fermentations using immobilized cells were accelerated compared to fermentations using free cells and maximum yield and productivity were reached at lower initial sugar concentrations. Using scanning electron microscopy it was observed that the shape of the cells was related to the sugar concentration in the medium. The experiments show thatP. farinosa produces glycerol with a high and constant productivity over long periods of time.     

Ethanol production by immobilized cells of Zymomonas mobilis

Summary Columnar reactors containing immobilized cells of Zymomonas mobilis were utilized for the continuous production of ethanol from glucose. Two different immobilization strategies were investigated. In one case, cells were entrapped in borosilicate glass fiber pads, while in the other, cells were immobilized via flocculation. The reactors were operated in both the fixed-bed and expanded-bed manner. Ethanol productivities as high as 132 g/l·h were achieved. Data obtained from studies employing 5.0 and 10.0% glucose concentrations are presented. Problems encountered during the operation of the continuous, immobilized cell reactors are discussed.Operated by Union Carbide Corporation under contract W-7405-eng-26 with the U.S. Department of Energy.     

Characterization and immobilization of E. coli (ATCC-26) Beta-galactosidase

The β-galactosidase from Escherichia coli ATTCC-26 was partially purified and characterized. It was found to be comparable to galactosidases from other E. coli strains in stability, pH and temperature maxima, and activity requirements, but it had a more favorable ratio of activity toward lactose versus synthetic substrates. The galactosidase was immobilized on porous glass beads by three covalent bonding methods. Kinetic data for the free and bound enzymes were determined using natural and synthetic substrates. Activity characteristics of the free and immobilized enzymes were comparable, however, the bound forms were less stable to heat.     

Multiple immobilized enzyme reactors: determination of pyruvate and phosphoenolpyruvate concentrations using immobilized lactate dehydrogenase and pyruvate kinase

Lactate dehydrogenase (LDH) and pyruvate kinase (PK), immobilized on solid glass beads by diazotization, were used in packed bed reactors to analyze for both pyruvate (PYR) and phosphoenolpyruvate (PEP) through the disappearance of β-nicotinamide adenine dinucleotide (NADH) monitored spectrophotometrically at 340 nm. Packed bed reactors containing PK and/or LDH were also capable of monitoring continuously varying concentrations of adenosine-5′-diphosphate (ADP), PEP, and PYR. The immobilized enzymes (～40 μg/g glass) retained about 4% of the activity of the soluble enzymes. Preparations of immobilized LDH and PK exhibited enhanced stability when maintained in the presence of β-mercaptoethanol and NADH or EDTA, respectively, and were shown to regain 75% of the original activity after four months storage at 4°C.     

Removal of skim milk lactose by fermentation using free and immobilized Kluyveromyces marxianus cells

Kluyveromyces marxianus CBS 6164 cells, free or immobilized in Ca-alginate (2%) beads, are able to consume more than 99% of the skim milk lactose in anaerobic conditions. In batches at 30 °C, the lactose consumption after 3.5 h of skim milk fermentation by 30 and 50 g free K. marxianus cells per liter was around 99 and 99.6% respectively, with an approximate conversion of lactose to ethanol and CO₂ of 80%. The immobilized cells, easy to handle and showing a faster and easier separation from the fermented medium compared to the free ones, were used in more than 23 batches (cycles of re-use) without losing their activity.     

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.     

Open tubular heterogeneous enzyme reactors: preparation and kinetic behavior

Tubes with immobilized enzymes on the inner wall, called open tubular heterogeneous enzyme reactors, were prepared by binding enzymes either directly to the tube inside surface or to a layer of a porous matrix attached to the inner wall. Kinetic studies of the hydrolysis of N-benzoyl-L -arginine ethylester as a model reaction indicated that the reaction was kinetically controlled in reactors with surface bound trypsin and the kinetic parameters were evaluated by conventional methods. On the other hand, substrate diffusion in both the porous matrix and the bulk substrate solution strongly affected the rate of reaction in porous layer trypsin reactors. The highest overall rates of reaction were obtained when the reaction was bulk diffusion controlled and the measured rates were in agreement with those calculated from expressions derived from heat transfer theory. The design of reactors for the limiting cases of kinetic and bulk diffusion controlled reaction as well as a method for the determination of substrate diffusivity are outlined.     

Evaluation of the effectiveness factor along immobilized enzyme fixed-bed reactors: Design of a reactor with naringinase covalently immobilized into glycophase-coated porous glass

A design equation is presented for packed-bed reactors containing immobilized enzymes in spherical porous particles with internal diffusion effects and obeying reversible one-intermediate Michaelis-Menten kinetics. The equation is also able to explain irreversible and competitive product inhibition kinetics. It allows the axial substrate profiles to be calculated and the dependence of the effectiveness factor along the reactor length to be continuously evaluated. The design equation was applied to explain the behavior of naringinase immobilized in Glycophase-coated porous glass operating in a packed-bed reactor and hydrolyzing both p-nitrophenyl-alpha-L-rhamnoside and naringin. The theoretically predicted results were found to fit well with experimentally measured values.     

Sulphydryl oxidase: Properties and applications

Sulphydryl oxidase has been isolated and purified from bovine milk and partially characterized. The enzyme is obtained from the whey fraction produced by chymosin (rennin) treatment of skim milk. Several procedures have been developed for its isolation, all of which use to advantage its concentration-dependent aggregate size. This enzyme exhibits a much broader substrate specificity than that shown by other ‘aerobic oxidases’ presently characterized which catalyse the formation of disulphides from thiols. Using molecular oxygen as an electron acceptor in a twoelectron reduction to hydrogen peroxide, the enzyme catalyses oxidation of cysteine and its analogues, some volatile thiols, peptides, and also thiols in proteins.Vis-a-vis protein-disulphide oxidoreductase and protein-disulphide isomerase, this enzyme does not catalyse thiol-disulphide interchange and thus functions only in de novo formation of disulphides. Enzyme-bound iron and most likely a free sulphydryl group are essential for catalytic activity; however, the carbohydrate moiety of this glycoprotein is probably not required. The broad substrate specificity suggests several potential industrial uses for the enzyme, including flavour modification in the food industry or synthesis of disulphides in pharmaceuticals. Consequently, immobilized forms were developed and characterized. Reactors containing sulphydryl oxidase covalently immobilized on porous glass and silica have been examined for their efficacy in eliminating the cooked flavour in ultra-high temperature sterilized milk. Both the degree of oxidation and cooked flavour were correlated with a normalized residence time in the reactor.     

Hydrolysis of lactose by immobilized microorganisms.

Cells of Lactobacillus bulgaricus, Escherichia coli, and Kluyveromyces (Saccharomyces) lactis immobilized in polyacrylamide gel beads retained 27 to 61% of the beta-galactosidase activity of intact cells. Optimum temperature and pH and thermostability of these microbial beta-galactosidases were negligibly affected by the immobilization. Km values of beta-galactosidase in immobilized cells of L. bulgaricus, E. coli, and K. lactis toward lactose were 4.2, 5.4, and 30 mM, respectively. Neither inhibition nor activation of beta-galactosidase in immobilized L. bulgaricus and E. coli appeared in the presence of galactose, but remarkable inhibition by galactose was detected in the case of the enzyme of immobilized K. lactis. Glucose inhibited noncompetitively the activity of three species of immobilized microbial cells. These kinetic properties were almost the same as those of free beta-galactosidase extracted from individual microorganisms. The activity of immobilized K. lactis was fairly stable during repeated runs, but those of E. coli and L. bulgaricus decreased gradually. These immobilized microbial cells, when introduced into skim milk, demonstrated high activity for converting lactose to monosaccharides. The flavor of skim milk was hardly affected by treatment with these immobilized cells, although the degree of sweetness was raised considerably.     

Hydrolysis of lactose by immobilized microorganisms.

Cells of Lactobacillus bulgaricus, Escherichia coli, and Kluyveromyces (Saccharomyces) lactis immobilized in polyacrylamide gel beads retained 27 to 61% of the beta-galactosidase activity of intact cells. Optimum temperature and pH and thermostability of these microbial beta-galactosidases were negligibly affected by the immobilization. Km values of beta-galactosidase in immobilized cells of L. bulgaricus, E. coli, and K. lactis toward lactose were 4.2, 5.4, and 30 mM, respectively. Neither inhibition nor activation of beta-galactosidase in immobilized L. bulgaricus and E. coli appeared in the presence of galactose, but remarkable inhibition by galactose was detected in the case of the enzyme of immobilized K. lactis. Glucose inhibited noncompetitively the activity of three species of immobilized microbial cells. These kinetic properties were almost the same as those of free beta-galactosidase extracted from individual microorganisms. The activity of immobilized K. lactis was fairly stable during repeated runs, but those of E. coli and L. bulgaricus decreased gradually. These immobilized microbial cells, when introduced into skim milk, demonstrated high activity for converting lactose to monosaccharides. The flavor of skim milk was hardly affected by treatment with these immobilized cells, although the degree of sweetness was raised considerably.     

Microwave‐assisted covalent immobilization of enzymes on inorganic surfaces

Enzymes on carriers can be easily recycled or used in fixed bed reactors. The immobilization often results in an improved stability. Depending on the support used and the method of coupling, this is a time‐consuming process. While the wide applicability of microwaves (MWs) within organic synthesis is known since the 1980s, proteins (including enzymes) are generally considered as too sensitive toward MW irradiation. In this article, MW methods were investigated to improve the processing speed of covalent enzyme immobilization on inorganic supports. Herein two laccases from Trametes versicolor and Myceliophthora thermophilia (Novozyme 51003®) and the glucose oxidase from Aspergillus niger were immobilized onto samples of ceramic honeycomb and porous glass (TRISOPERL® 1000 AMINO). The enzymes showed different sensitivity to MW irradiation, but all were suitable for MW‐assisted immobilization. Subsequent stability tests were conducted to compare conventional immobilization methods with those with MW irradiation. The glucose oxidase provided the best results. For all cases, a successful MW irradiation assisted covalent enzyme immobilization on solid support was obtained with a total 20‐fold reduction of the time necessary.     

Evaluation of intrinsic immobilized kinetics in hollow fiber reactor systems.

Immobilized cell and enzyme hollow fiber reactors have been developed for a variety of biochemical and biomedical applications. Reported mathematical models for predicting substrate conversion in these reactors have been limited in accuracy because of the use of free-solution kinetic parameters. This paper describes a method for determining the intrinsic kinetics of enzymes immobilized in hollow fiber reactor systems using a mathematical model for diffusion and reaction in porous media and an optimization procedure to fit intrinsic kinetic parameters to experimental data. Two enzymes, a thermophilic beta-galactosidase that exhibits product inhibition and L-lysine alpha-oxidase, were used in the analysis. The intrinsic kinetic parameters show that immobilization enhanced the activity of the beta-galactosidase while decreasing the activity of L-lysine alpha-oxidase. Both immobilized enzymes had higher Km values than did the soluble enzyme, indicating less affinity for the substrate. These results are used to illustrate the significant improvement in the ability to predict substrate conversion in hollow fiber reactors.     

Regeneration of NADH with immobilized systems of alanine dehydrogenase and hydrogen dehydrogenase

Summary An alternative approach to the regeneration of coenzymes using immobilized hydrogen dehydrogenase (hydrogenase) is described. Hydrogenase isolated from Alcaligenes Eutrophus was immobilized to porous glass particles and used in combination with alanine dehydrogenase for formation of alanine, while the NADH consumed was regenerated by molecular hydrogen. Different physical arrangements of the two enzymes were compared. Alanine was conveniently assayed with a specially designed enzyme thermistor method.     

Protective Effect of Administration of Skim Milk on Exogenous and Endogenous Infection in Mice

In order to minimize the denaturation of proteins in milk, normal cow's milk was pasteurized at 61 C for 20 min. The protective effects of the thus prepared skim milk (low-heat skim milk) on exogenous and endogenous infection were examined as compared with conventional skim milk which was pasteurized at 121 C for 2 sec. The antibody titers to Listeria monocytogenes and Escherichia coli of low-heat skim milk were almost equal to that of raw milk, while no antibody was detected in the conventional skim milk. When mice were given low-heat skim milk or conventional skim milk, the incidence of the translocation of orally inoculated Listeria monocytogenes to the spleen was lower in the low-heat skim milk group than that in the conventional skim milk group. The life span of 7 Gy X-ray irradiated mice given low-heat skim milk was significantly prolonged in comparison to that of mice given conventional skim milk. However, there were no differences in the number of bacteria in the feces or IgA production by Peyer's patch cells between the two groups. These results suggest that antibodies in low-heat skim milk, which still have reactivity to exogenous or indigenous bacteria, may contribute to the protective effects against bacterial infection.     

A new fluorogenic substrate for chymotrypsin.

The action of pepsin on a dilute solution of skim milk produces a sigmoidal increase in the turbidity of the solution. The time course of the increase depends on the amount of pepsin present. Turbidity changes can be measured in a recording spectrophotometer, providing an automated form of the milk clotting assay, which will easily determine 20 ng of pepsin.     

A novel thermostable lipase from Basidiomycete <Emphasis Type="Italic">Bjerkandera adusta </Emphasis>R59: characterisation and esterification studies

Microbial lipases are widely diversified in their enzymatic properties and substrate specificities, which make them very attractive for industrial application. Partially purified lipase from Bjerkandera adusta R59 was immobilized on controlled porous glass (CPG) and its properties were compared with those of the free enzyme. The free and immobilized lipases showed optimal activities at 45 and 50°C, respectively. Both enzyme forms were highly thermostable up to 60°C. The enzymes were stable at pH from 6.0 to 9.0 and their optimal pH for activity was 7.0. The free lipase was more thermostable in n-hexane than in aqueous environment. Both lipase preparations had good stabilities in non-polar solvents and were capable of hydrolysing a variety of synthetic and natural fats. Non-immobilized lipase activity was inhibited by disulphide bond reagents, serine and thiol inhibitors, while EDTA and eserine had no effect on enzyme activity. All anionic detergents tested in experiments inhibited lipase activity. The free lipase showed good stability in the presence of commercial detergents at laundry pH and temperatures. Applications of free and immobilized lipases for esterification were also presented.     

Immobilization of enzymes in porous supports: Effects of support-enzyme solution contacting

Proteins have been immobilized in porous support particles held in a fixed-bed reactor through which protein solution is continuously circulated. Changing the recirculation flow rate alters the observed immobilization kinetics and the maximum enzyme loading which can be achieved for glucose oxidase and glucoamylase on carbodiimide-treated activated carbon and for glucoamylase immobilized on CNBr-Sepharose 4B. Direct microscopic examination of FITC-labelled protein in sectioned Sepharose particles and indirect activity-loading studies with activated carbon-enzyme conjugates all indicate that immobilized enzyme is increasingly localized near the outer surface of the support particles at larger recirculation flow rates. Restricted diffusion of enzymes may be implicated in this phenomenon. These contacting effects may be significant considerations in the scaleup of processes for protein impregnation in porous supports, since apparent activity and stability of the final preparation depend on internal protein distribution.     

Conversion of D-sorbit to L-sorbose by cells of Acetobacter suboxydans immobilized in sintered glass

A simple method for immobilizing cells of Acetobacter suboxydans by adsorption on inorganic sintered glass carriers is described. The immobilized cell preparations exhibited 100% of the initial activity when converting d-sorbit to l-sorbose. This sintered glass was used in a fixed-bed loop reactor with a working volume of 0.2 l for semicontinuous and continuous experiments. A prolonged working life span was achieved, which could possibly satisfy requirements for scaled-up operations.