Mechanical stability and diffusional resistance of a polymeric gel used for biocatalyst immobilization.

The mechanical strength of gelatin gels insolubilized by crosslinking with formaldehyde was measured at various gelatin percentages and formaldehyde-to-gelatin ratios. This property was shown to be related to the characteristic sponge-like structure of the insolubilized gelatin gel, a structure that unexpectedly is also responsible for the resistance to substrate and product diffusion. A comparison between immobilizates of invertase and invertase-active yeast cells prepared with different gelatin concentrations showed that the enzyme, in contrast to cells, is deeply involved in the gel insolubilization process. The catalytic behavior of agar, kappa-carrageenan, alginate, and gelatin immobilizates was compared under the same conditions of cell loading.

Enzyme immobilization by means of ultrafiltration techniques.

Unstirred, plane membrane, ultrafiltration cells have been used as enzymatic reactor units. Because of the concentration polarization phenomena which take place in the system, at steady-state the enzyme is confined (dynamically immobilized) within an extremely narrow region upstream the ultrafiltration membrane. Correspondingly its concentration attains fairly high values. Kinetic studies have been therefore performed under quite unusual experimental conditions in order to better approximate local enzyme concentration levels in immobilized enzyme systems. Studies have been also carried out on the kinetics of enzyme deactivation in the continuous presence of substrate and reaction products. Once the enzyme concentration profile is completely developed, further injection into the system of suitable amounts of an inert proteic macromolecule (albumin polymers) gives rise to the formation of a gel layer onto the ultrafiltration membrane within which the enzyme is entrapped (statically immobilized). The effect of this immobilization technique has been studied as far as the kinetics of the main reaction, the substrate mass transfer resistances and the enzyme stability are concerned. The rejective properties of such gel layers towards enzymatic molecules have been exploited in producing multilayer, multi-enzymatic reactors.

Theoretical and experimental analysis of a soluble enzyme membrane reactor.

Recently enzyme immobilization techniques have been proposed that are mainly founded on the formation of an enzyme-gel layer onto the active surface of an ultrafiltration membrane within an unstirred ultrafiltration cell. If the membrane molecular-weight cutoff is less than the enzyme molecular weight and hence such as to completely prevent enzyme permeation (once the enzyme solution has been charged into the test cell and pressure applied to the system), a time progressive increase in enzyme concentration takes place at the upstream membrane surface that can eventually lead to gelation and hence to enzyme immobilization. However, depending on the total enzyme amount fed, the maximum enzyme concentration achieved in the unsteady state could be less than the gelation level. In this situation, no immobilization occurs and the enzyme still remains in the soluble form although it is practically confined within a limited region immediately upstream the membrane and at fairly high concentrations. In this paper, the experimental conditions that allow gelling to occur are discussed together with a theoretical analysis of the soluble enzyme membrane reactor which is obtained when no gelling takes place. Such a system could be usefully employed in performing kinetic analyses at high enzyme concentration levels that are still in the soluble form.

Kinetic behaviour of acid phosphatase-albumin co-polymers in homogeneous phase and under gel-immobilized conditions.

1. An analysis of the kinetic behaviour of immobilized acid phosphatase (EC 3.1.3.2) layers, gelled on the active surface of an ultrafiltration membrane, was carried out. 2. Two possible forms of such immobilized-enzyme systems were dealt with, namely enzyme-polyalbumin co-gelation through an ultrafiltration process, and enzyme co-polymerization to the same albumin polymers and subsequent gelation. 3. A preliminary analysis was also performed on both the corresponding homogeneous-phase (soluble systems to provide reference kinetics. 4. The main conclusions drawn are: (i) the enzyme-albumin co-polymers show a decrease in specific activity compared with the corresponding free enzyme in both soluble and immobilized forms; (ii) in the homogeneous phase a slight increase in the apparent Michaelis constant was measured for the co-polymerized enzyme compared with the free one, which suggests a decrease in affinity towards substrate; (iii) the activation energy in the immobilized phase is halved, compared with that in the homogeneous phase, which indicates that the combined mass-transfer/reaction step is rate-controlling.

Preparation and properties of co-reticulated invertase supported by an ultrafiltration membrane.

Yeast invertase was co-reticulated with glutaraldehyde to bovine serum albumin to give a soluble bound enzyme that was immobilized as a tightly adhering layer on the active surface of an ultrafiltration membrane. The Michaelis constant and stability of this immobilized-enzyme system are compared with those of the enzyme either in the native form or immobilized as a dynamically formed gel layer on an ultrafiltration membrane, as previously described by us [Drioli, Gianfreda, Palescandolo & Scardi (1975) Biotechnol, Bioeng, 17, 1365-1367].

Inhibition of amylases from different origins by albumins from the wheat kernel.

The amylase activity of water extracts from 18 insect species, from 23 marine species and from 17 different species of birds and mammals was determined quantitatively. The inhibition of amylase in these extracts by three albumin fractions from the mature wheat kernel, which had been separated according to their molecular weights (60 000, 24 000 and 12 500 D), was determined as well. The inhibition activity of the three albumin fractions toward amylases extracted from a number of cereal species or from immature and germinating wheat kernel was also tested. The extracts from insects that are destructive of wheat grain and stored wheat products showed much higher amylase activities as compared to the other insect species that do not attack wheat and wheat products. On the basis of the effectiveness with which the three albumin fractions inhibit their activities, the amylase preparations tested were divided into susceptible, partially susceptible and resistent. Susceptible amylases, inhibited by any of the three albumin fractions, were found mainly in insects that attack wheat and in marine species. Partially susceptible amylases, inhibited by only one or two of the three albumin fractions, were present in a few avain and mammalian species including man. Resistent amylases were largely distributed in cereal, avian and mammalian species as well as in insect species that do not usually attack wheat grain or wheat flour products. At no stage of development, wheat alpha-amylase was inhibited by the albumin fractions from the mature kernel. The 12 500 dalton albumin fraction was the most effective in inhibiting insect amylases, but it was inactive toward avian and mammalian amylases. The 24 000 dalton albumin fraction was the most effective in inhibiting amylases from marine avian and mammalian species and inhibited as much as 33 amylases over 66 different amylases tested. It is suggested that protein inhibitors of amylase contributed to natural selection of polyploid wheats by giving some insect resistence to such wheats, even though some insect species were able to overcome this biochemical defense toa large degree by producing higher amylase activities.

Effect of pH, ionic strength and univalent inorganic ions on the reconstitution of aspartate aminotransferase.

1. The effect of pH change on the reconstitution of aspartate aminotransferase (EC 2.6.1.1), i.e. the reactivation of the apoenzyme with coenzyme (pyridoxal phosphate and pyridoxamine phosphate), was studied in the pH range 4.2-8.9 by using three buffer systems at concentrations ranging from 0.025 to 0.1m. 2. Although the profile of the reconstitution rate-pH curve in the range pH5.2-6.8 (covered by sodium cacodylate-HCl buffer) reflects the influence of the H(+) concentration on the reconstitution process, the profile of the curve in the pH ranges 4.2-5.6 and 7.2-8.25 (covered respectively by sodium acetate-acetic acid and Tris-HCl buffers) appears to be influenced by the ionic strength of the buffer. 3. The reconstitution is also influenced by univalent inorganic ions such as halide ions and, to a lesser extent, alkali metal ions, which are known to alter the water structure.

The influence of chloride and other univalent inorganic anions on the visible-absorption spectrum of aspartate aminotransferase.

1. The influence of Cl(-), Br(-), NO(3) (-) and F(-) ions on the visible-absorption spectrum of deionized aspartate aminotransferase was investigated. 2. Except for F(-), these anions caused an increase of the extinction at 430mmu with a concomitant decrease of that at 362mmu. 3. The affinity constants for Cl(-) and NO(3) (-) ions were calculated by a procedure based on the assumption that the anion stabilizes the protonated form of the enzyme chromophore (lambda(max.) 430mmu). 4. The true pK of the chromophore of the enzyme was found to be 5.25.