Enzyme preparations witchused in animal breeding are unstable. Instability is due to a partial or complete inactivation of the enzymes in the gastrointestinal tract under the influence of a strongly acidic environment, inhibitors and proteases. Increasing the effectiveness of the use of exogenous enzyme preparations is possible by the creation of stabilized forms of biopreparations. To do this, use the principles and methods of engineering enzymology. The aim of the research is to determine the optimal conditions for the immobilization of the enzyme preparation of protosubtilin G3X (proteolytic spectrum of the action) by the adsorption method. Conduct a comparative evaluation of the properties of native and immobilized biocatalysts on the conditions in vitro and in vivo.
Proteolytic activity was determined by Anson's method. The amount of protein on the carrier was evaluated by reducing its concentration in the reaction mixture, measured with Lowry O.H. et al .. The activity of the immobilized enzyme was expressed as a percentage of the activity of the native enzyme. The pH solutions were measured on the potentiometer pH- 340. For immobilization weused enzyme preparation of protosubtilin G3X with an activity of 70 units / g, as a carrier we used zeolite.
The immobilization procedure consisted of mixing the buffer solution of the enzyme with the carrier. During research and study of the influence of the ionic strength of the solution and pH on the adsorption process it was established that the catalytic activity of the obtained preparation falls in the buffers: phosphate, citrate, borate, acetate. Moreover, with an increase in the ionic strength of the solution, regardless of its composition, the enzyme activity of preparation was reduced. Optimal for immobilization was a 0.1 M phosphate buffer solution with pH in the range of 7.0–7.4. Protosubtilin G3X lost 80 % of the initial activity during immobilization in water.
When determining the capacity of a carrier, it is found that 1 g of zeolite adsorbs 29.8 mg of protein. The maximum specific activity of the enzyme (0.16 U / mg protein) is appeared at the optimum load of the carrier 22.5 mg protein per 1 g zeolite and corresponded to 85.7 % of the activity of the native protosubtilin G3X. Consequently, the optimal conditions for the immobilization of protosubtilin G3X on zeolite are: 0.1 M phosphate buffer solution with pH 7.0–7.4, temperature 20–25 ºС, carrier capacity 22.5 mg / g, duration of the process 2 years.
In experiments in vitro studied the dependence of the catalytic activity of the pH value in the range of 1.5 to 8.0. After 1 hour of incubation in the buffer solutions, it was found that the optimal value of pH for both forms of preparations coincides (pH 7.2). With pH-inactivation of native and immobilized forms of protosubtilin G3X, the loss of catalytic activity of the modified preparation was significantly less than the native. Moreover, a significant expansion of the pH profile in the acidic zone was observed for the immobilized enzyme. If the native enzyme retained 20 % of the original activity at pH 5.0 and irreversibly inactivated at pH 4.5–4.8; the immobilized enzyme retained 42 % of the activity at pH 4.0.
We studied the proteolytic activity of digestive enzymes in different parts of the gastrointestinal tract of broilers: goitre (pH 4.5–5.8), glandular stomach (pH 3.6–4.7), duodenum (pH 5.7–6.2). The proteolytic activity of the contents of goiter in the groups of chickens witch received native and immobilized protosubtilin G3X, was the same or higher, than in the control groups (p <0.01). The value of the proteolytic activity of the contents of the glandular stomach in the group of broiler chickens witch received the native enzyme sharply decreased almost to the level of the value in the control group. same value in the group witch received the immobilized enzyme was higher by 30 % (p <0.05). There was also an increase of the proteolytic activity in the chyme of the duodenum in the experimental groups of chicks (p <0.05); however, the catalytic activity in the group witch received the immobilized enzyme, was higher by 12.8 %.
It should be noted that in conditions in vivo, in contrast to similar conditions in vitro, was observed partial reactivation of the catalytic properties of the native protosubtilin G3X after exposure to the strongly acid medium of the glandular stomach, which is obviously related to the biological environment of the protein molecule.
To show the intensity of metabolic processes witch provide growth and development of broiler, we use the integral indicator as productivity. Feeding of immobilized protosubtilin G3X to chickens positively influenced the weight gain and helped to reduce feed costs. At the end of the experiment, the weight of the chicks receiving the native enzyme was higher by 9.0 % (p> 0.1) and immobilized by 16.0 % (p <0.05) compared to the control group. The feed costs for 1 kg of gain in both experimental groups were the same (2.40 kg) and less than in the control group by 5.8 %.
Key words: enzyme, immobilization, native enzyme, zeolite, adsorption, proteolytic activity, pH, buffer solution.
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