Selection of Optimal Conditions for Cultivation of Bacillus megaterium UCM B-5710 – Producer of Keratinase

Abstract

Every year the volume of production of poultry products all over the world is growing steadily. This contributes to a constant increase in the amount of by-products of poultry processing in the form of down and feather waste, which are dangerous for the environment due to the hard-to-degrade keratin protein and a large number of microbial pathogens. Therefore, the use of environmentally friendly methods for the destruction of keratin substrates due to keratinases of microorganisms is an urgent area of research. The aim of this work was to select the optimal cultivation conditions for the Bacillus megaterium strain UCM B-5710 to increase the activity of the keratinase synthesized by it. Methods. The culture was grown at 28°C, 201 rpm for 7 days on a basic nutrient medium containing defatted chicken feathers as the only source of carbon and nitrogen. The selection of optimal cultivation conditions was carried out according to the following parameters: temperature (21°C, 28°C, 42°C), stirring speed (201 rpm, 212 rpm), amount of inoculum (5%, 10%, 15% , 20%, 25%), the initial pH value of the nutrient medium (4.0–11.0), concentration of keratin-containing substrate (0.1%, 0.2%, 0.5%, 1.0%, 1.5%, 2.0%), additional carbon source (glucose, galactose, lactose, maltose, sucrose, mannitol, potato and corn starch, soluble starch, soybean meal) and nitrogen (NH4Cl, NH4NO3, (NH4)2SO4, NaNO3, urea, peptone, tryptone, yeast extract and soybean meal) at a concentration of 1%. Keratinase activity was assessed by the UV absorption at 280 nm of the hydrolysis products of keratin-containing raw materials. Protein was determined by the Lowry method. Results. The dynamics of the enzyme synthesis showed that the culture of B. megaterium UCM B-5710 exhibited the highest keratinase activity on the 3rd day, and complete splitting of feathers was observed on the 4–5th days. The selection of the concentration of the keratin-containing substrate showed that 0.5% is the optimal concentration. The study of the influence of the initial pH value of the nutrient medium indicates that the culture grew well at pH 6.0–7.0 and pH 9.0–11.0, but at pH 8.0 its growth was very weak. The culture exhibited the maximum keratinase activity at pH 10.0. In addition, at this pH value, complete splitting of feathers was visually observed. The influence of such a key factor as temperature on the growth and synthesis of the enzyme by B. megaterium UCM B-5710 culture demonstrated complete splitting of feathers already on the 2nd day of cultivation at 42°C, at 21°C the culture split feathers very poorly. The introduction of the inoculum into the composition of the nutrient medium in an amount of 15% of the volume of the medium and the mixing intensity of 212 rpm turned out to be optimal. Besides, it was shown that the introduction of an additional source of carbon or nitrogen had an ambiguous effect on the level of keratinase activity of B. megaterium UCM B-5710. Complete inhibition of enzyme synthesis was observed when ammonium sulfate was added to the nutrient medium, and partial inhibition was observed in the case of glucose, lactose, and maltose. Potato, corn, and soluble starch stimulated keratinase synthesis. The majority of inorganic nitrogen sources (ammonium chloride and nitrate) did not affect the synthesis of B. megaterium UCM B-5710 keratinase, while organic sources (urea, peptone, tryptone, yeast extract) increased the level of keratinase activity by 20–50%. However, the most effective result was obtained using soybean meal, the addition of which to the nutrient medium increased the keratinase activity by 2.5 times. Conclusions. As a result of the studies, the optimal conditions for cultivation of the B. megaterium UCM B-5710 strain were selected: the optimum temperature for the growth and development of the culture is 42°C, the initial pH value is 10.0, the stirring speed is 212 rpm and the amount of inoculum introduced is 15%, an additional source of carbon and nitrogen in the form of soybean meal at a concentration of 0.5%. This made it possible to increase the activity of keratinase by 4 times.

Substrate Specificity of Bacillus megaterium UСM B-5710 Keratinase

“…Keratinases are unique enzymes capable of breaking down the hard-to-split protein keratin. Th e studied culture of B. megaterium UCM B-5710, as we showed earlier [8], is an eff ective producer of keratinase. Th e study of the growth curve of this culture, the level of keratinase and proteolytic activity, protein, the content of free amino acids in the SCL in dynamics showed that keratinase synthesis began 6 hours aft er the start of cultivation, and the levels of the above indicators increased with increasing culture cultivation time.…”

“…In connection with the foregoing, studies aimed at fi nding highly active producers of keratinases and studying their ability to split various keratin-containing substrates are relevant. Previously [8], we have shown that the culture of Bacillus megaterium UCM B-5710 is an active producer of keratinase. Th e aim of this work was to study the substrate specifi city of B. megaterium UCM B-5710 keratinase.…”