ИЗУЧЕНИЕ ВЛИЯНИЯ ТЕМПЕРАТУРЫ И PH НА СИНТЕЗ ГИДРОЛИТИЧЕСКИХ ФЕРМЕНТОВ МИКРООРГАНИЗМАМИ

Тремасова Анна Михайловна; Ганиев Ильнур Махмутович; Мухаммадиев Ринат Салаватович; Мусин Рифкат Расимович; Ерошин Артур Игоревич; Сафарова Энже Ришатовна

CC BY

7

2

i Надоели баннеры? Вы всегда можете отключить рекламу.

Журнал

Ветеринарный врач

2024

ВАК

Область наук

Ветеринарные науки

Ключевые слова

микроорганизмы / продуценты / ферменты / активность / кислотность среды / температура культивирования / microorganisms / producers / enzymes / activity / acidity of the medium / cultivation temperature

Аннотация научной статьи по ветеринарным наукам, автор научной работы — Тремасова Анна Михайловна, Ганиев Ильнур Махмутович, Мухаммадиев Ринат Салаватович, Мусин Рифкат Расимович, Ерошин Артур Игоревич

Использование гидролитических ферментов является активно развивающимся направлением в кормлении сельскохозяйственных птиц и животных. Целью исследований явилось изучение влияния температуры культивирования штаммов и кислотность питательной среды на уровень продукции гидролитических ферментов продуцентами. Для проведения исследования использованы микроорганизмы (Bacillus subtilis VY-04, Bacillus subtilis RS-15-58 и Trichoderma reesei АM-16-71), выделенные нами из природных субстратов, и отобранные в ходе трехэтапного скрининга продуцентов гидролитических ферментов, как наиболее активные и перспективные продуценты амилаз, протеаз, ксиланаз, целлюлаз и липаз. Влияние температуры на регуляцию продукции гидролитических ферментов изучали при культивировании продуцентов в диапазоне температур от 25 °С до 45 °С в зависимости от биологических объектов; влияние начальной величины рН жидкой питательной среды определяли в диапазоне значений рН от 3,0 ед до 9,0 ед. Различные величины рН среды устанавливали добавлением 1 М раствора соляной кислоты или гидроксида натрия. Количественное определение активности гидролитических ферментов осуществляли колориметрическим и спектрофотометрическим методами. В результате проведённых исследований установлены оптимальные условия культивирования штаммов B. subtilis VY-04, B. subtilis RS-15-58 и Т. reesei АM-16-71, обеспечивающие максимальный биосинтез гидролитических ферментов. Установлено, что наиболее высокая протеазная и амилазная активность у B. subtilis VY-04 отмечена при температуре культивирования от 37,0 °С до 38,0 °С, pH питательной среды от 7,0 ед до 8,0 ед. Наиболее выраженная липазная активность у B. subtilis RS-15-58 обнаружена при температуре культивирования от 35,5 °С до 36,5 °С, pH среды от 6,5 ед до 7,5 ед. Максимальные значения ксиланазной и целлюлазной активности у Т. reesei АM-16-71 определены в диапазоне температур культивирования от 29,0 °С до 31,0 °С, pH среды от 4,0 ед до 6,0 ед.

i Надоели баннеры? Вы всегда можете отключить рекламу.

STUDY OF THE EFFECT OF TEMPERATURE AND PH ON THE SYNTHESIS OF HYDROLYTIC ENZYMES BY MICROORGANISMS

The use of hydrolytic enzymes is an actively developing area in the feeding of poultry and farm animals. The aim of the present research was to study the effect of the temperature of cultivation of strains and the acidity of the nutrient medium on the level of production of hydrolytic enzymes by producers. To conduct the study, we used microorganisms (Bacillus subtilis VY-04, Bacillus subtilis RS-15-58 and Trichoderma reesei AM-16-71), which were isolated from natural substrates and selected during a three-stage screening of producers of hydrolytic enzymes as the most active and promising producers of amylases, proteases, xylanases, cellulases and lipases. The effect of temperature on the regulation of hydrolytic enzyme production was studied during the cultivation of producers in the temperature range from 25 ° C to 45 ° C, depending on biological objects; the effect of the initial pH value of the liquid nutrient medium was determined in the pH range 3.0 9.0. Various pH values of the medium were determined by adding 1 М solution of hydrochloric acid or sodium hydroxide. Quantitative determination of the activity of hydrolytic enzymes was carried out by colorimetric and spectrophotometric methods. As a result of the conducted studies, optimal conditions for the cultivation of B. subtilis VY-04, B. subtilis RS-15-58 and T. reesei AM-16-71 strains were established, ensuring maximum biosynthesis of hydrolytic enzymes. It was found that the highest protease and amylase activity in B. subtilis VY-04 was observed at cultivation temperatures from 37.0 °C to 38.0 °C, pH of the nutrient medium from 7.0 to 8.0. The most pronounced lipase activity in B. subtilis RS-15-58 was found at culture temperatures from 35.5 °C to 36.5 °C, pH of the medium from 6.5 to 7.5. The maximum values of xylanase and cellulase activity in T. reesei AM-16-71 were determined in the range of cultivation temperatures from 29.0 ° C to 31.0 ° C, pH of the medium from 4.0 to 6.0.

Текст научной работы на тему «ИЗУЧЕНИЕ ВЛИЯНИЯ ТЕМПЕРАТУРЫ И PH НА СИНТЕЗ ГИДРОЛИТИЧЕСКИХ ФЕРМЕНТОВ МИКРООРГАНИЗМАМИ»

�� . 2024. � 2. �. 73 � 79

�he Veterinarian. 2024; (2): 73 � 79

��

�� 579.222: 577.152.3

DOI: 10.33632/1998-698�_2024_2_73

�� PH ��

�� , �� , anuta.tremasova@yandex.ru

�� , �� , ilnurgm-vgora@mail.ru

�� , �� , tanirtashir@mail.ru

�� , �� , musinrifkat@mail.ru

�� , erosartur@yandex.ru

�� , enn.kvant@mail.ru

�� , �� , ��, ��

��, �� : �� .

��. �� . �� . �� (Bacillus subtilis VY-04, Bacillus subtilis RS-15-58 � Trichoderma reesei �M-16-71), �� , � �� , �� , ��, ��, �� . �� 25 �� 45 �� ; �� 3,0 �� 9,0 ��. �� 1 � �� . �� . � �� B. subtilis VY-04, B. subtilis RS-15-58 � �. reesei �M-16-71, �� . ��, �� B. subtilis VY-04 �� 37,0 �� 38,0 ��, pH �� 7,0 �� 8,0 ��. �� B. subtilis RS-15-58 �� 35,5 �� 36,5 ��, pH �� 6,5 �� 7,5 ��. �� . reesei �M-16-71 �� 29,0 �� 31,0 ��, pH �� 4,0 �� 6,0 ��.

�� : ��, ��, ��, ��, �� , ��

�� : �� .�., �� .�., �� .�., �� .�., �� .�., �� .�. �� // �� . 2024. � 2 �. 73-79. DOI: 10.33632/1998-698�_2024_2_73

STUDY OF THE EFFECT OF TEMPERATURE AND PH ON THE SYNTHESIS OF HYDROLYTIC ENZYMES BY MICROORGANISMS

Anna M. Tremasova, doctor of biological sciences, anuta.tremasova@yandex.ru

Ilnur M. Ganiev, candidate of biological sciences, ilnurgm-vgora@mail.ru

Rinat S. Mukhammadiev, candidate of biological sciences, tanirtashir@mail.ru

Rifkat R. Musin, candidate of veterinary sciences, musinrifkat@mail.ru

Artur I. Eroshin, erosartur@yandex.ru

Ange R. Safarova, enn.kvant@mail.ru

Federal Center for Toxicological, Radiation and Biological Safety, Kazan, Russian Federation

�orresponding author: Anna M. Tremasova.

Abstract. The use of hydrolytic enzymes is an actively developing area in the feeding of poultry and farm animals. The aim of the present research was to study the effect of the temperature of cultivation of strains and the acidity of the nutrient medium on the level of production of hydrolytic enzymes by producers. To conduct the study, we used microorganisms (Bacillus subtilis VY-04, Bacillus subtilis RS-15-58 and Trichoderma reesei AM-16-71), which were isolated from natural substrates and selected during a three-stage screening of producers of hydrolytic enzymes as the most active and promising producers of amylases, proteases, xylanases, cellulases and lipases. The effect of temperature on the regulation of hydrolytic enzyme production was studied during the cultivation of producers in the temperature range from 25 � C to 45 � C, depending on biological objects; the effect of the initial pH value of the liquid nutrient medium was determined in the pH range 3.0 - 9.0. Various pH values of the medium were determined by adding 1 � solution of hydrochloric acid or sodium hydroxide. Quantitative determination of the activity of hydrolytic enzymes was carried out by colorimetric and spectrophotometric methods. As a result of the conducted studies, optimal conditions for the cultivation of B. subtilis VY-04, B. subtilis RS-15-58 and T. reesei AM-16-71 strains were established, ensuring maximum biosynthesis of hydrolytic enzymes. It was found that the highest protease and amylase activity in B. subtilis VY-04 was observed at cultivation temperatures from 37.0 �C to 38.0 �C, pH of the nutrient medium from 7.0 to 8.0. The most pronounced lipase activity in B. subtilis RS-15-58 was found at culture temperatures from 35.5 �C to 36.5 �C, pH of the medium from 6.5 to 7.5. The maximum values of xylanase and cellulase activity in T. reesei AM-16-71 were determined in the range of cultivation temperatures from 29.0 � C to 31.0 � C, pH of the medium from 4.0 to 6.0.

Keywords: microorganisms, producers, enzymes, activity, acidity of the medium, cultivation temperature

��. �� - �� . �� . �� , �� , � �� .

�� . �� , �� , �� , �� -�� [1, 2]. �� . �� . �� -�� , �� , �� [3].

�� , ��, ��, �� [4]. �� (�� ) � �� , �� , � �� [5]. �� (�� ), �� . � �� , �� .

�� .

�� . �� (B. subtilis VY-04, B. subtilis RS-15-58 � �. reesei �M-16-71), �� , �� , �� , ��, �� [6].

�� , �� (�/��3):

1) ��, �� 120,0; �� 15,0; �� 15,0; �� 15,0; ��3 - 2,5; MgSO4?7�2� - 2,5; MnSO4?5H2O � 0,05 (pH �� 6,9 �� 7,1 ��);

2) ��, �� 30,0; �� 2,0; �� 1,0; (NH4)2SO4 � 1,0; CaCl2 � 0,04, MgSO4?7H2O � 0,04; �� 80 � 5,0 (pH �� 7,0 �� 7,5 ��);

3) ��, �� 40,0; �� 40,0; �� 40,0; �� 10,0; KH2PO4 � (NH4)2SO4 � 1,0; CaCl2?2�2� � 0,04; MgSO4?7H2O � 0,6 (pH �� 6,7 �� 6,9 ��).

�� 25 �� 45 �� ; �� 3,0 �� 9,0 ��. �� 1 � �� .

�� . �� (10000 g, � �� 10 �� 4 ��) � �� . �� Millex (�� 33 ��, �� 0,45 ��, �� ) (�� Merck Millipore�, ��).

�� (�� , ��), �� Watman �1 (�� , ��), �� (�� , ��), �� (�� , ��) � �� -80 (�� , ��).

�� , �� 1 �� .

�� , �� 1 � �� , �� 30 % �� 50 % �� .

�� 1 � �� . �� , �� 1 �� 1 �� . �� 260 �� 280 �� [4].

�� Microsoft Office Excel 2013 (�Windows�, ��) �� -�� (M�m). �� t-�� . �� 95 % �� (p?0,05).

�� . �� , �� , �� (�� ) �� . �� , �� [7]. � �� , ��, ��, �� B. subtilis VY-04, B. subtilis RS-15-58 � �. reesei �M-16-71 �� . �� B. subtilis VY-04. �� (�� ) �� B. subtilis VY-04 �� 1.

�� 1 � �� B. subtilis VY-04

� �

�) �� ; �) ��

�� B. subtilis VY-04 ��, �� 35,0 �� 40,0 ��. �� B. subtilis VY-04 �� 37,0 �� 38,0 ��. �� . �� 39,0 �� 40,0 �� 36,0 �� 35,0 �� .

�� pH �� B. subtilis VY-04 �� , �� pH �� 4,0 �� 9,0 ��. �� 7,0 �� 8,0 ��. �� pH �� 7,5 �� (96,83�4,51) ��/��3 � (101,04�3,96) ��/��3, ��. �� 9,0 �� 11,9 % � �� 6,0 %. �� 6,0 �� 4,0 �� (�� 4,0 �� 2 �� 5 ��). �� , �� pH �� 7,0 �� Bacillus [8].

�� B. subtilis RS-15-58 (��. 2).

�� 2 � �� B. subtilis RS-15-58

� �

�) �� ; �) ��

�� 2, �� 35,0 �� 40,0 ��. �� 35,5 �� 36,5 ��. �� 37,0 �� 40,0 �� 35,0 �� . �� H �� B. subtilis RS-15-58 �� , �� pH �� 4,0 �� 9,0 ��. �� 6,5 �� 7,5 ��. �� 8,0 �� 9,0 ��. �� 4,0 �� 6,0 �� , �� 7,0 �� (�� 4 �� ). �� . reesei �M-16-71 (�� 3 � 4).

�� 3 � �� . reesei �M-16-71

� �

�) �� ; �) ��

�� 4 � �� . reesei �M-16-71

� �

�) �� ; �) ��

�� (��. 3 � 4), �� . reesei �M-16-71 �� 27,0 �� 32,0 ��. �� 30,0 �� 31,0 ��, � �� 29,0 �� 30,0 ��. �� 32,0 �� 35,0 �� . �� pH �� , �� , �� pH �� 4,0 �� 9,0 ��. �� : �� 5,0 �� 6,0 ��, �� 4,0 �� 5,0 ��. �� (�� 3,0 �� 4,0 ��) � �� (�� 7,0 �� 9,0 ��) �� . �� , �� pH �� 7,0 �� Trichoderma [9]. �� , �� , ��, ��, �� B. subtilis VY-04, B. subtilis RS-15-58 � �. reesei �M-16-71 �� , �� .

��. � �� B. subtilis VY-04, B. subtilis RS-15-58 � �. reesei �M-16-71, �� . ��, �� B. subtilis VY-04 �� 37,0 �� 38,0 ��, pH �� 7,0 �� 8,0 ��. �� B. subtilis RS-15-58 �� 35,5 �� 36,5 ��, pH �� 6,5 �� 7,5 ��. �� . reesei �M-16-71 �� 29,0 �� 31,0 ��, pH �� 4,0 �� 6,0 ��.

��

1. ��, �.�. �� Bacillus subtilis / �.�. ��, �.�. ��, �.�. ��, �.�. ��, �.�. �� // �� . � 2019. � � 3. � �. 19-23.

2. �� / �.�. ��, �.�. ��, �.�. �� // �� . � 2011. � � 1. � �. 4-6.

3. ��, �.�. �� 101 �� -�� / �.�. ��, �.�. ��, �.�. �� // �� . � 2020. � � 1. � �. 48-53.

4. ��, �.�. �� : �� / �.�. ��. � �� : ��, 2005. � 608 �.

5. ��, �.�. �� / �.�. ��, �.�. ��, �.�. ��, �.�. ��, �.�. �� // �� . � 2023. � � 1. � �. 26-31.

6. ��, ��.�. �� - �� / ��.�. ��, �.�. ��, �.�. ��, �.�. ��, �.�. �� // �� : �� -�� , ��-��, 23�24 �� 2023 ��. �� XXV. � ��-�� : �� , 2023. � �. 820-823.

7. ��, �.�. �� : �� / �. �. ��. � �� : ��-�� , 2015. � 19 �.

8. ��, �.�. �� Bacillus licheniformis � �� ?�� / �.�. ��, �.�. ��, �.�. ��, �.�. ��i, �.�. ��i, �.�. ��, �.�. �� // �� . � 2002. � �. 38, � 5. � �. 502�508.

9. Burlacu, A. Microbial xylanase: a review / A. Burlacu, P. Cornea, F. Israel-Roming // Scientific Bulletin. Series F. Biotechnologies. � 2016. � Vol. 20. �P. 335�342.

References

1. Mukhammadiev, R.S. Enzymatic activity of xylanases and cellulases of probiotic strains of Bacillus subtilis / R.S. Mukhammadiev, R.S. Mukhammadiev, L.R. Valiullin, V.V. Biryulya, E.V. Skvortsov // Veterinarian. � 2019. � No. 3. � pp. 19-23.

2. The state of intestinal microbiocenosis of piglets with mycotoxicosis against the background of probiotic use / T.A. Shumilova, L.E. Matrosova, F.G. Akhmetov // Veterinarian. � 2011. � No. 1. � pp. 4-6.

3. Krotova, N.Yu. Compound feed with the enzyme preparation Akstra KHAR 101 in the cultivation of broiler chickens / N.Yu. Krotova, A.Yu. Lavrentiev, V.S. Sherne // Veterinarian. � 2020. � No. 1. � pp. 48-53.

4. Netrusov, A.I. Practicum on microbiology: textbook / A.I. Netrusov. � Moscow : Akademiya, 2005. � 608 p.

5. Musin, R.R. Evaluation of the effectiveness of lactic acid bacteria in harvesting haylage from alfalfa / R.R. Musin, E.V. Skvortsov, A.M. Tremasova, P.V. Bykova, A.I. Eroshin // Veterinarian. � 2023. � No. 1. � pp. 26-31.

6. Mukhammadiev, Rin.S. Search for new strains of microorganisms producing a complex of hydrolytic enzymes / Rin.S. Mukhammadiev, R.R. Musin, I.M. Ganiev, V.Yu. Titova, A.M. Tremasova // Topical issues of improving the technology of production and processing of agricultural products : Materials of the international scientific and practical conference, Yoshkar-Ola, March 23-24, 2023. Volume Issue XXV. � Yoshkar-Ola : Mari State University, 2023. � pp. 820-823.

7. Astashkina, A.P. Preparation of nutrient media and cultivation of microorganisms : methodological guidelines / A. P. Astashkina. Tomsk : Publishing House of Tomsk Polytechnic University, 2015. � 19 p.

8. Tsurikova, N.V. Obtaining an active strain of Bacillus licheniformis � a producer of thermostable ?�amylase / N.V. Tsurikova, L.I. Nefedova, E.V. Kostyleva, V.I. Zvenigorodsky, V.G. Yasinovsky, T.A. Voikova, A.P. Sinitsyn // Applied Biochemistry and microbiology. - 2002. � Vol. 38, No. 5. � pp. 502-508.

9. Burlacu, A. Microbial xylanase: a review / A. Burlacu, P. Cornea, F. Israel-Roming // Scientific Bulletin. Series F. Biotechnologies. � 2016. � Vol. 20. �P. 335�342.

�� .

�� /�� , �� .

All authors have made an equivalent contribution to the preparation of the publication.

The authors declare that there is no conflict of interest.

�� / accepted for publication 22.01.2024;

� �� .�., �� .�., �� .�., �� .�., �� .�., �� .�. 2024

i Надоели баннеры? Вы всегда можете отключить рекламу.

ИЗУЧЕНИЕ ВЛИЯНИЯ ТЕМПЕРАТУРЫ И PH НА СИНТЕЗ ГИДРОЛИТИЧЕСКИХ ФЕРМЕНТОВ МИКРООРГАНИЗМАМИ Текст научной статьи по специальности «Ветеринарные науки»

STUDY OF THE EFFECT OF TEMPERATURE AND PH ON THE SYNTHESIS OF HYDROLYTIC ENZYMES BY MICROORGANISMS

Текст научной работы на тему «ИЗУЧЕНИЕ ВЛИЯНИЯ ТЕМПЕРАТУРЫ И PH НА СИНТЕЗ ГИДРОЛИТИЧЕСКИХ ФЕРМЕНТОВ МИКРООРГАНИЗМАМИ»