References

vavilov

Вавиловский журнал генетики и селекции

Vavilov Journal of Genetics and Breeding

2500-3259

Institute of Cytology and Genetics of Siberian Branch of the RAS

10.18699/VJ21.015

vavilov-2925

Research Article

БИОТЕХНОЛОГИЯ

BIOTECHNOLOGY

Продукция субтилизиновых протеаз в бактериях и дрожжах

Production of subtilisin proteases in bacteria and yeast

Розанов

А. С.

Rozanov

A. S.

лаборатория молекулярных биотехнологий

Новосибирск

Laboratory of Molecular Biotechnologies

Novosibirsk

rozanov@bionet.nsc.ru

https://orcid.org/0000-0001-5604-5601

Шеховцов

С. В.

Shekhovtsov

S. V.

лаборатория молекулярных биотехнологий

Новосибирск

Laboratory of Molecular Biotechnologies

Novosibirsk

Богачева

Н. В.

Bogacheva

N. V.

лаборатория молекулярных биотехнологий

Новосибирск

Laboratory of Molecular Biotechnologies

Novosibirsk

https://orcid.org/0000-0003-2658-7906

Першина

Е. Г.

Pershina

E. G.

лаборатория молекулярных биотехнологий

Новосибирск

Laboratory of Molecular Biotechnologies

Novosibirsk

Ряполова

А. В.

Ryapolova

A. V.

с. Малобыково, Белгородская область

Malobykovo village, Belgorod region

Бытяк

Д. С.

Bytyak

D. S.

с. Малобыково, Белгородская область

Malobykovo village, Belgorod region

https://orcid.org/0000-0002-3524-0456

Пельтек

С. Е.

Peltek

S. E.

лаборатория молекулярных биотехнологий

Новосибирск

Laboratory of Molecular Biotechnologies

Novosibirsk

Курчатовский геномный центр Института цитологии и генетики Сибирского отделения Российской академии наук; Федеральный исследовательский центр Институт цитологии и генетики Сибирского отделения Российской академии наукРоссияKurchatov Genomic Center of the Institute of Cytology and Genetics of Siberian Branch of the Russian Academy of Sciences; Institute of Cytology and Genetics of Siberian Branch of the Russian Academy of SciencesRussian Federation

Инновационный центр «Бирюч-НТ»РоссияInnovation Centre “Biruch-NT”Russian Federation

2021

15032021

251125134

2021

Розанов А.С., Шеховцов С.В., Богачева Н.В., Першина Е.Г., Ряполова А.В., Бытяк Д.С., Пельтек С.Е.

Rozanov A.S., Shekhovtsov S.V., Bogacheva N.V., Pershina E.G., Ryapolova A.V., Bytyak D.S., Peltek S.E.

This work is licensed under a Creative Commons Attribution 4.0 License.

https://vavilov.elpub.ru/jour/article/view/2925

В настоящей работе мы рассматриваем прогресс в изучении и модификации субтилизиновых протеаз. Несмотря на длительное время применения микробиальных протеаз и значительное число работ, посвященных их исследованию, поиск новых генов протеаз, создание продуцентов и развитие методов их применения остаются актуальными, о чем говорит высокий уровень цитирования публикаций, описывающих протеазы и их продуценты. На данный класс ферментов приходится максимальный объем производства промышленных белков в мире, что объясняет большой интерес к нему. Это говорит о чрезвычайно высокой важности получения собственных технологий их производства. В статье представлены сведения о классификации субтилизинов, истории их открытия и дальнейших работ по оптимизации их свойств. Дан обзор классов субтилизиновых протеаз и родственных им ферментов. Проанализированы проблемы поиска и отбора субтилаз из природных штаммов различных микроорганизмов, пути и особенности их модификации и используемые при этом методы генетической инженерии. Детально изучены методы оптимизации продукции промышленных субтилаз у различных штаммов, касающихся важнейших аспектов культивирования: состава среды, времени культивирования, влияния температуры и pH. Приводятся результаты последних исследований по техникам культивирования – глубинному и твердофазному культивированию. На основании рассмотренных литературных данных можно заключить, что в настоящее время практически не применяются нативные, т. е. обнаруженные в природе ферменты, в связи с решающими преимуществами, предоставляемыми белками, модифицированными при помощи генной инженерии и обладающими улучшенными свойствами: термостабильностью, общей устойчивостью к детергентам и специфической – к различным окислителям, высокой активностью в разных диапазонах температур, независимостью от ионов, стабильностью в отсутствие кальция и т. д. Большинство субтилизиновых протеаз синтезируется в штаммах-продуцентах, относящихся к разным видам рода Bacillus. В то же время ведутся работы по адаптации синтеза этих ферментов в других микроорганизмах, в частности дрожжей Pichia pastoris.

In this review, we discuss the progress in the study and modif ication of subtilisin proteases. Despite longstanding applications of microbial proteases and a large number of research papers, the search for new protease genes, the construction of producer strains, and the development of methods for their practical application are still relevant and important, judging by the number of citations of the research articles on proteases and their microbial producers. This enzyme class represents the largest share of the industrial production of proteins worldwide. This situation can explain the high level of interest in these enzymes and points to the high importance of designing domestic technologies for their manufacture. The review covers subtilisin classif ication, the history of their discovery, and subsequent research on the optimization of their properties. An overview of the classes of subtilisin proteases and related enzymes is provided too. There is a discussion about the problems with the search for (and selection of) subtilases from natural strains of various microorganisms, approaches to (and specifics of) their modif ication, as well as the relevant genetic engineering techniques. Details are provided on the methods for expression optimization of industrial subtilases of various strains: the details of the most important parameters of cultivation, i. e., composition of the media, culture duration, and the inf luence of temperature and pH. Also presented are the results of the latest studies on cultivation techniques: submerged and solid-state fermentation. From the literature data reviewed, we can conclude that native enzymes (i. e., those obtained from natural sources) currently hardly have any practical applications because of the decisive advantages of the enzymes modified by genetic engineering and having better properties: e. g., thermal stability, general resistance to detergents and specif ic resistance to various oxidants, high activity in various temperature ranges, independence from metal ions, and stability in the absence of calcium. The vast majority of subtilisin proteases are expressed in producer strains belonging to different species of the genus Bacillus. Meanwhile, there is an effort to adapt the expression of these enzymes to other microbes, in particular species of the yeast Pichia pastoris.

субтилизинсубтилазапротеазащелочная сериновая протеазаPichia pastorisBacillus subtilisбиотехнологиягенетическая инженериякультивирование

subtilisinsubtilaseproteasealkaline serine proteasePichia pastorisBacillus subtilisbiotechnologygenetic engineeringcultivation

The study was supported by the budget project No. 0259-2019-0005. This work was done within the framework of State Assignment Kurchatov Genomic Center of the Institute of Cytology and Genetics of Siberian Branch of the Russian Academy of Sciences (075-15-2019-1662).

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The authors declare that there are no conflicts of interest present.