Sel’skokhozyaistvennaya Biologiya [Agricultural Biology], 2018, Vol. 53, ¹ 6, p. 1274-1284.
(how to cite this article)

doi: 10.15389/agrobiology.2018.6.1274eng

UDC 636.087.8:573.6.086.83:577.15

Acknowledgements:
This work is a part of the State Program to improve the world research and education competitiveness of the Kazan (Volga Region) Federal University
Supported financially by Russian Science Foundation (science project No. 16-16-04062)

 

GENE CONSTRUCT-BASED SERINE PROTEASE OF Bacillus pumilus AS A FEED ADDITIVE FOR POULTRY FARMING

A.O. Koryagina, N.L. Rudakova, M.T. Lutfullin, G.F. Khadieva,
A.A. Toymentseva, A.M. Mardanova, M.R. Sharipova

Kazan (Volga region) Federal University, Institute of Fundamental Medicine and Biology, 18, Kremlyovskaya ul.,Kazan, Republic of Tatarstan,420008 Russia, e-mail tihonovaao93@gmail.com (✉ corresponding author), natatialrudakova@gmail.com, MarTLutfullin@kpfu.ru, GuFHadieva@kpfu.ru, tojmencevaaa@mail.ru, mardanovaayslu@mail.ru, marsharipova@gmail.com

ORCID:
Koryagina A.O. orcid.org/0000-0003-1859-8269
Toymentseva A.A. orcid.org/0000-0002-7397-9585
Rudakova N.L. orcid.org/0000-0000-3685-2016
Mardanova A.M. orcid.org/0000-0003-0448-5685
Lutfullin M.T. orcid.org/0000-0003-1985-480X
Sharipova M.R. orcid.org/0000-0001-6168-9145
Khadieva G.F. orcid.org/0000-0002-9281-3994
The authors declare no conflict of interests

Received September 19, 2018

 

Bacterial enzymes, in particular proteinases, as dietary additives in poultry farming, improve digestibility of feed nutrients and, as a consequence, make animal diets cheaper. This explains why bioadditives are being actively developed worldwide. Proteinàses break down proteins and reduce the negative effect of digestive inhibitors thus allow the costs for purchasing synthetic amino acids to be lower. Bacteria and microscopic fungi, including those with gene constructs developed to increase yield and improve properties of the expressed enzymes, may be producers. Bacterial serine proteinases have a high thermostability and are resistant to inhibitors of animal origin. In this paper, we report for the first time about the production of highly purified secreted subtilisin-like serine proteinase from Bacillus pumilus upon expression of the recombinant vector in B. subtilis strains and evaluate the main physicochemical and biological characteristics of the synthesized product. The goal of our study is to obtain, by using the expression system, the highly purified subtilisin-like serine proteinase from B. pumilus as a promising feed additive for the poultry industry. The substrate specificity of the produced serine proteinase, i.e. the depth of hydrolysis, corresponds to the specificity of subtilisins, the enzyme cleaves the bonds formed by the carboxyl groups of the hydrophobic amino acids leucine, phenylalanine and tyrosine, as well as a number of hydrophilic amino acids. An investigation of the effect of temperature and pH on serine proteinase activity showed that in the presence of calcium ions at a final concentration of 5 mM, the temperature optimum of the enzyme reached 50 °Ñ. The enzyme remaines stable in the pH range from 7 to 10. The proteinase activity was studied at various pH values to simulate the conditions of the gastrointestinal tract of chickens. In a weakly acidic medium (pH 5.5, goiter) proteinase completely retains its activity (100 %), at pH 2.9 (stomach) the enzyme activity decreases by 40 %, and upon transition again to alkaline conditions (pH 6.5-8.0, small intestine and large intestine), the enzyme restores activity up to the values exceeding control by 13 %. Thus, the enzyme can remain active throughout the whole digestive tract of broiler chicks. The proteinase activity was not inhibited by natural inhibitors, such as a trypsin inhibitor, which would also allow the enzyme to function in the gastrointestinal tract of chickens. In experiments on the effect of chicken bile from 0.01 % to 0.05 % for 1 hour at 40 °Ñ on the microbial proteinase, the enzyme completely preserved its activity. With an increase in the concentration of chicken bile to 1 %, the enzyme activity decreased by 10 %. To study the toxicity of proteinase, 1-day-old Cobb 500 broiler chickens were observed for 10 days. The dietary proteinase at 100 EU/kg concentration showed no toxicity, and all the indices of the poultry remained normal. We found that in the early period, during 0-10 days of growth when the chickens are fed with Start ration, a dosage of 5 EU/kg of proteinase is effective. In the late stages of poultry growth (21-42 days), the use the Finisher mixed feed supplemented with bacterial proteinase at a dose of 15 EU/kg is optimal. In both cases, the dietary proteinase increases poultry weight gain by 13.9 % and 7.9 %, and also improves feed conversion by 14 % and 8.5 %, respectively. Thus, the amount of the introduced enzyme must be adjusted depending on the age of birds and the feed composition. The main indicators of Cobb 500 broiler chickens’ growth when using recombinant proteinase allow us to conclude that this proteinase is promising as a feed additive.

Keywords: Bacillus pumilus, recombinant subtilisin-like serine proteinase, substrate specificity, stability, activity, effects of pH and temperature, fodder additive, broiler chickens, Cobb 500.

 

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