Immobilization of β‐galactosidase in collodion microcapsules

Paine, Michelle; Carbonell, Ruben G.

doi:10.1002/bit.260170414

Cited by 8 publications

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“…Lactases from bacterial (Hustad e t al. 1973a,b; Ostergaard and Martiny 1973;Paine and Carlsonell 1975;Reagan et al 1974;Wondolowski and Woychik 1974), fungal ; Coughlin and Charles 1976;Coughlin et al 1974;Hasselberger e t al. 1974;Hyrkas et al 1976; Okos and Harper 1974;Okos et al 1978; Olson and Stanley 1973;Paruchuri 1976;Pitcher et al 1976;Weetall et al 1974a,b;Wierzbicki et al 1973;Woychik and Wondolowski 1972;, and yeast (Dahlquist et al 1973;Kilara et al 1977; Pastore e t 1976;Woychik et al 1974) sources have previously been immobilized on a number of support materials and used in the hydrolysis of lactose present in different solutions.…”

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confidence: 99%

Use of Immobilized Lactase in Processing Cheese Whey Ultrafiltrate

Roodpeyma

Hill

Amundson

1983

J Food Process Engineering

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Enzymatic hydrolysis of lactose in cottage cheese whey ultrafiltrate was investigated. Lactase of A. niger was immobilized on an alumina‐silica catalyst support by the linking agent tolylene‐2, 4‐diisocyanate. The resulting immobilized enzyme preparation had an activity of 3 standard international units of lactase per gram at pH 4 and 37 °C. The optimum pH and temperature for hydrolysis of lactose by immobilized lactase were 3.5 and 50°C, respectively. Immobilization of the enzyme resulted in reductions of 1.1 pH units and 15°C in optimum pH and temperature, respectively. The two constants of the simple Michaelis‐Menten rate expression were obtained from Lineweaver‐Burk plots of the initial reaction rate data obtained at 37°C. Estimated values for Vmax and the apparent Km were 7.8 (μmoles/min‐g) and 0.26 (M), respectively. Inhibition by the product galactose was measured by studying the hydrolysis reaction in a batch reactor. The inhibition constant Ki was estimated from batch reactor data to be 0.005 and 0.053 (M) at 35 and 50°C, respectively. Activation energies of 8.1 and 6.4 (kcal/gmole) were obtained for the immobilized and soluble enzyme reactions, respectively. The behavior of the batch reactor as measured in terms of a plot of conversion versus time was essentially the same for both conventional and deionized whey ultrafiltrate.

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Section: Introductionmentioning

confidence: 99%

Use of Immobilized Lactase in Processing Cheese Whey Ultrafiltrate

Roodpeyma

Hill

Amundson

1983

J Food Process Engineering

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A new method for cell immobilization

Petre

Noël

Thomas

1978

Biotech & Bioengineering

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INTRODUCTIONOwing to the high cost of enzyme purification there is a growing interest devoted to the immobilization of whole microorganisms. Several papers have dealt with the immobilization of cells by entrapment inside polyacrylamide gels,'-'' agar and polymers of cellulose tria~etate.'~ Microorganisms have also been immobilized by absorption and then crosslinked within a collagen film'5*'" or by a direct linkage on zirconium hydroxide.'? An elegant method was recently described" in which concanavalin A was specifically studied for selective binding of yeast and bacteria by bioaffnity.A new method of cell immobilization is described in this paper using Escherichia coli as an example. The &plactosidase activity is measured as a function of a number of parameters during the immobilization process. P-Galactosidase has been immobilized previously by several methods.The behavior of a packed bed containing immobilized cells is described and discussed. MATERIAL AND METHODS SonicationA O.O5M, pH 7, 0.1N NaCl tris-HC1 buffer solution containing 2.5% (wlv) of lyophilized bacteria was sonicated with a 100 W sonicator (B 12-Branson, Sonic Power Co.). The suspension was thermostated at 4°C during the process. The sonication took place for 15 sec of each 75 sec period. The same process was used with immobilized bacteria. One hundred-seventy-five mg of particles were induced into 50 ml of the same buffer solution. Measurement of Enzyme ActivityThe enzyme activity was measured in a Tris-HC1 buffer solution (0.05 M , pH 7, 0.1 N NaCI) containing 5 x 10-3M ONPG at a temperature of 25°C. The kinetics was measured by the increase of the optical density at 410 nm owing t o ONP production. With immobilized systems the solution flowed continuously through a glass cuvet in a double beam spectrophotometer (Beckman DBGT). Cell ImmobilizationCells were immobilized within membranes and porous particles. The procedure for membranes was: 1.25 ml of a pH 6.8, 0.ON phosphate buffer sdution, containing 6 rng ml-' glutaraldehyde, 60 mg ml-I bovine albumin (Sigma chemical Corp.), and 10 mg ml-' lyophilized E. coli K 10 were spread on a plane gkss surface. After drying for 2 hr, the film produced was rinsed in distilled water.The procedure for porous particles was: the solution described above was frozen slowly at -30°C for 4 hr and then the temperature was raised to 4°C. An insduble sponge-like structure was obtained. The insoluble phase was rinsed with a 10 mg ml-' glycine solution and with distilled water. The sponge was ground and then lyophilized. The preparation is stable when stored below 0°C.Biotechnology and Bioengineering Vol. XX, Pp. 127-134 (1978) Packed BedPorous particles containing enzyme were stirred at 25°C in a 0.05M. 0. IN NaCI, pH 7 Tns-buffer solution for 2 hr and then introduced into a column 1.5 cm in diam and 20 cm in length (Whatman). The packed-bed and flowing solutions were both thermostated at 25°C. RESULTS Properties of Immobilized BacteriaThe activity yield as a function of glutaraldehyde concentration during t...

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Ion exchange immobilization of changed β‐galactosidase fusions for lactose hydrolysis

Heng¹,

Glatz²

1994

Biotech & Bioengineering

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The use of charged peptides fused to enzymes for immobilization onto ion-exchange membranes was explored for the enzyme x-galactosidase. The additional charged peptides, containing 1, 5, 11, and 16 aspartates, fused to x-galactosidase, for the most part did not interfere with the kinetic behavior for lactose hydrolysis. There was a 2-fold decline in V(m) for the 16-aspartate fusion, but the others were quite similar to the wild type enzyme (BGWT). BGWT and the fusions all retained approximately 50% of their activities when adsorbed onto ion-exchange membranes. In contrast to BGWT, the enhanced binding strength of the 11 aspartate fusion provided the ability to hydrolyze whey permeate at 0.3 M ionic strength without enzyme leakage, and to immobilize the enzyme directly from diluted cell extract with 83% purity.

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Immobilization of β‐galactosidase in collodion microcapsules

Cited by 8 publications

References 1 publication

Use of Immobilized Lactase in Processing Cheese Whey Ultrafiltrate

Use of Immobilized Lactase in Processing Cheese Whey Ultrafiltrate

A new method for cell immobilization

Ion exchange immobilization of changed β‐galactosidase fusions for lactose hydrolysis

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