vuzbiochemiИзвестия вузов. Прикладная химия и биотехнологияProceedings of Universities. Applied Chemistry and Biotechnology2227-29252500-1558ИРНИТУ10.21285/2227-2925-2023-13-3-359-369DADDCOvuzbiochemi-1070Research ArticleФИЗИКО-ХИМИЧЕСКАЯ БИОЛОГИЯPHYSICOCHEMICAL BIOLOGYКаталитические свойства и иммобилизация ферментных препаратов, содержащих липазу галоалкалотолерантных бактерий Pseudomonas peli и Bacillus aequororisCatalytic properties and immobilization of enzyme preparations containing lipase of the haloalkalotolerant bacteria Pseudomonas peli and Bacillus aequororishttps://orcid.org/0000-0003-1870-1369МаксимоваЮ. Г.MaksimovaYu. G.

Максимова Юлия Геннадьевна - доктор биологических наук, заведующая лабораторией ИЭГМ УрО РАН – филиал Пермского федерального исследовательского центра УрО РАН; профессор, Пермский ГНИУ.

614081, Пермь, ул. Голева, 13; 614068, Пермь, ул. Букирева, 15

Yuliya G. Maksimova - Dr. Sci. (Biology), Head of the Laboratory, Institute of Ecology and genetics of Microorganisms, Perm FRC; Professor, Perm SU.

13, Golev St., Perm, 614081; 15, Bukirev St., Perm, 614068

yul_max@mail.ruhttps://orcid.org/0000-0002-0670-3829ПьянковаЕ. В.PyankovaE. V.

Пьянкова Екатерина Валерьевна - студент, Пермский ГНИУ; инженер, ИЭГМ УрО РАН – филиал Пермского федерального исследовательского центра УрО РАН,

614068, Пермь, ул. Букирева, 15; 614081, Пермь, ул. Голева, 13

Ekaterina V. Pyankova - Student, Perm SU; Engineer, Institute of Ecology and genetics of Microorganisms, Perm FRC,

15, Bukirev St., Perm, 614068; 13, Golev St., Perm, 614081

19katya991@rambler.ruhttps://orcid.org/0000-0002-6990-1902ЕлисееваА. Д.EliseevaA. D.

Елисеева Анна Дмитриевна – студент.

614068, Пермь, ул. Букирева, 15

Anna D. Eliseeva - Student,

15, Bukirev St., Perm, 614068

liamrik@list.ruhttps://orcid.org/0000-0002-6322-5755ЩеткоВ. А.ShchetkoV. A.

Щетко Виталий Анатольевич - кандидат биологических наук, начальник отдела «Научно-производственный центр биотехнологий».

220141, Минск, ул. Академика Купревича, 2

Vitaly A. Shchetko - Cand. Sci. (Biology), Head of the Department «Research and Production Center for Biotechnology».

2, Ak. Kuprevich St., Minsk, 220141

vental@yandex.ruhttps://orcid.org/0000-0003-2591-3351МаксимовА. Ю.MaksimovA. Yu.

Максимов Александр Юрьевич - кандидат биологических наук, старший научный сотрудник, ИЭГМ УрО РАН – филиал Пермского федерального исследовательского центра УрО РАН; доцент, Пермский ГНИУ.

614081, Пермь, ул. Голева, 13; 614068, Пермь, ул. Букирева, 15

Alexander Yu. Maksimov - Cand. Sci. (Biology), Senior Researcher, Institute of Ecology and genetics of Microorganisms, Perm FRC; Associate Professor, Perm SU.

13, Golev St., Perm, 614081; 15, Bukirev St., Perm, 614068

almaks1@mail.ruИнститут экологии и генетики микроорганизмов УрО РАН – филиал Пермского федерального исследовательского центра УрО РАН; Пермский государственный национальный исследовательский университетРоссияInstitute of Ecology and genetics of Microorganisms, Perm Federal Research Center; Perm State UniversityRussian FederationПермский государственный национальный исследовательский университетРоссияInstitute of Ecology and genetics of Microorganisms, Perm Federal Research Center; Perm State UniversityRussian FederationПермский государственный национальный исследовательский университетРоссияPerm State UniversityRussian FederationИнститут микробиологии НАН БеларусиРоссияInstitute of Microbiology of the National Academy of Sciences of BelarusRussian Federation202330092023133359369Copyright © Максимова Ю.Г., Пьянкова Е.В., Елисеева А.Д., Щетко В.А., Максимов А.Ю., 20232023Максимова Ю.Г., Пьянкова Е.В., Елисеева А.Д., Щетко В.А., Максимов А.Ю.Maksimova Y.G., Pyankova E.V., Eliseeva A.D., Shchetko V.A., Maksimov A.Y.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://vuzbiochemi.elpub.ru/jour/article/view/1070

Данная статья посвящена исследованию каталитических свойств и иммобилизации ферментных препаратов, которые содержат липазу галоалкалотолерантных бактерий Pseudomonas peli и Bacillus aequororis. Из галоалкалотолерантных бактерий Pseudomonas peli 3-Т и Bacillus aequororis 5-ДБ выделена липаза и иммобилизована на карбоксиметилцеллюлозе, активированном хитозане и кормовых дрожжах. Определена рН-зависимость активности и термостабильность нативного фермента, сохранение активности при иммобилизации и высушивании иммобилизованного препарата. Установлено, что активность липазы из обоих источников повышается с увеличением щелочности реакционной среды, причем у липазы P. peli 3-Т в кислой среде активность отсутствовала, у B. aequororis 5-ДБ при рН 6–7 составляла не более 20% от максимальной. Выделенная липаза обладает достаточно высокой термостабильностью. Так, липаза P. peli 3-Т при воздействии температуры 60 и 70 °С в течение 1 ч полностью сохраняет исходную активность, а в течение 15 мин воздействие температуры 80 и 90 °С приводит к снижению активности на 73 и 83% соответственно. Из изученных носителей для иммобилизации фермента наиболее перспективны активированный хитозан и кормовые дрожжи. При высушивании иммобилизованного препарата липазы на активированном хитозане сохраняется 3 и 46% активности нативного фермента из P. peli 3-Т и B. aequororis 5-ДБ соответственно, на кормовых дрожжах – 2 и 64% соответственно.

In this work, we investigate the catalytic properties and immobilization of enzyme preparations containing lipase of haloalkalotolerant bacteria Pseudomonas peli and Bacillus aequororis. Lipase was isolated from the P. peli 3-T and B. aequororis 5-DB followed by its immobilization on either carboxymethylcellulose, activated chitosan or fodder yeast. The pH-dependence of native enzyme activity and thermostability, as well as the residual activity upon immobilization and drying of immobilized product, were determined. The lipase activity from both sources enhances with increasing alkalinity of the reaction medium. Specifically, P. peli 3-T lipase exhibited no activity in an acidic medium, and B. aequororis 5-DB lipase exhibited around 20% of maximum activity at a pH value of 6–7. The isolated lipase has a rather high thermostability; thus, P. peli 3-T lipase fully retains its initial activity upon heating to 60°C and 70°C for 1 h. Moreover, 15 min exposure to temperatures of 80°C and 90°C leads to an activity decrease of 73% and 83%, respectively. Activated chitosan and fodder yeast are the most promising of the studied excipients for enzyme immobilization. Drying of the immobilized lipase product on the activated chitosan showed retention of 3% and 46% of the native enzyme activity derived from P. peli 3-T and B. aequororis 5-DB, respectively. In the case of fodder yeast, these values were 2% and 64%, respectively.

иммобилизованные ферментылипазагалотолерантные бактерииалкалотолерантные бактериитермостабильность ферментаimmobilized enzymeslipasehalotolerant bacteriaalkalotolerant bacteriaenzyme thermostabilityРабота выполнена при финансовой поддержке Правительства Пермского края в рамках научного проекта № С-26/507 и частично в рамках темы «Поиск и селекция новых перспективных микроорганизмов для целей биотехнологии. Создание иммунохимических диагностических систем», номер государственной регистрации НИОКТР 122010800029-1The work was supported financially by the government of the Perm Territory within the framework of scientific project no. С-26/507 and partly within the framework of the topic “Search and selection of new promising microorganisms for biotechnology purposes. Creation of immunochemical diagnostic systems”, state registration number NIOKTR 122010800029-1ReferencesБезбородов А.М., Загустина Н.А. Липазы в реакциях катализа в органическом синтезе (обзор) // Прикладная биохимия и микробиология. 2014. Т. 50. N 4. С. 347–373. https://doi.org/10.7868/S0555109914040187. EDN: QJJFEO.Bezborodov A.M., Zagustina N.A. Lipases in catalytic reactions of organic chemistry. Prikladnaya biokhimiya i mikrobiologiya = Applied Biochemistry and Microbiology. 2014;50(4):347-373. (In Russian). https://doi.org/10.7868/S0555109914040187. EDN: QJJFEO.Costa-Silva T.A., Carvalho A.K.F., Souza C.R.F., De Castro H.F., Bachmann L., Said S., et al. Enhancement lipase activity via immobilization onto chitosan beads used as seed particles during fluidized bed drying: application in butyl butyrate production // Applied Catalysis A: General. 2021. Vol. 622. P. 118217. https://doi.org/10.1016/j.apcata.2021.118217.Costa-Silva T.A., Carvalho A.K.F., Souza C.R.F., De Castro H.F., Bachmann L., Said S., et al. Enhancement lipase activity via immobilization onto chitosan beads used as seed particles during fluidized bed drying: application in butyl butyrate production. Applied Catalysis A: General. 2021;622:118217. https://doi.org/10.1016/j.apcata.2021.118217.Щербинин С. Экзогенная липаза снижает стоимость корма // Корма и кормопроизводство. 2018. N 4. С. 55–57.Shcherbinin S. Exogenous lipase reduces feed cost. Korma i kormoproizvodstvo. 2018;(4):55-57. (In Russian).Fujinami S., Fujisawa M. Industrial applications of alkaliphiles and their enzymes – past, present and future // Environmental Technology. 2010. Vol. 31, no. 8-9. P. 845–856. https://doi.org/10.1080/09593331003762807.Fujinami S., Fujisawa M. Industrial applications of alkaliphiles and their enzymes – past, present and future. Environmental Technology. 2010;31(8-9):845-856. https://doi.org/10.1080/09593331003762807.Oren A. Industrialandenvironmental applicationsof halophilic microorganisms // Environmental Technology. 2010. Vol. 31, no. 8-9. P. 825–834. https://doi.org/10.1080/09593330903370026.Oren A. Industrial and environmental applications of halophilic microorganisms. Environmental Technology. 2010;31(8-9):825-834. https://doi.org/10.1080/09593330903370026.Шилова А.В., Максимов А.Ю., Максимова Ю.Г. Выделение и идентификация алкалотолерантных бактерий с гидролитической активностью из содового шламохранилища // Микробиология. 2021. Т. 90. N 2. С. 155–165. https://doi.org/10.31857/S0026365621020130. EDN: KLWPKV.Shilova A.V., Maksimov A.Y., Maksimova Y.G. Isolation and identification of alkalitolerant bacteria with hydrolytic activity from a soda sludge storage. Mikrobiologiya = Microbiology. 2021;90(2):155-165. (In Russian). https://doi.org/10.31857/S0026365621020130. EDN: KLWPKV.Максимова Ю.Г., Шилова А.В., Егорова В.В., Щетко В.А., Максимов А.Ю. Физиолого-биохимическая характеристика и биотехнологический потенциал гидролитических галоалкалотолерантных бактерий содового шламохранилища // Известия РАН. Серия биологическая. 2023. N 2. C. 149–159. https://doi.org/10.31857/S1026347022700068. EDN: HANYNK.Maksimova Yu.G., Shilova A.V., Egorova V.V., Shchetko V.A., Maksimov A.Yu. Physiological and biochemical characteristics, and biotechnological potential of hydrolitic haloalkalitolerant bacteria of soda sludge storage. Izvestiya RAN. Seriya biologicheskaya = Proceedings of the Russian Academy of Sciences. Biological Series. 2023;(2):149-159. (In Russian). https://doi.org/10.31857/S1026347022700068. EDN: HANYNK.Xie J., Zhang Y., Simpson B. Food enzymes immobilization: novel carriers, techniques and applications // Current Opinion in Food Science. 2022. Vol. 43. P. 27–35. https://doi.org/10.1016/j.cofs.2021.09.004.Xie J., Zhang Y., Simpson B. Food enzymes immobilization: novel carriers, techniques and applications. Current Opinion in Food Science. 2022;43:27-35. https://doi.org/10.1016/j.cofs.2021.09.004.Fernandez-Lafuente R. Stabilization of multimeric enzymes: strategies to prevent subunit dissociation // Enzyme and Microbial Technology. 2009. Vol. 45, no. 6-7. P. 405–418. https://doi.org/10.1016/j.enzmictec.2009.08.009.Fernandez-Lafuente R. Stabilization of multimeric enzymes: strategies to prevent subunit dissociation. Enzyme and Microbial Technology. 2009;45(6-7):405-418. https://doi.org/10.1016/j.enzmictec.2009.08.009.Remonatto D., Miotti R.H., Monti R., Bassan J.C., de Paula A.V. Applications of immobilized lipases in enzymatic reactors: a review // Process Biochemistry. 2022. Vol. 114. P. 1–20. https://doi.org/10.1016/j.procbio.2022.01.004.Remonatto D., Miotti R.H., Monti R., Bassan J.C., de Paula A.V. Applications of immobilized lipases in enzymatic reactors: a review. Process Biochemistry. 2022;114:1-20. https://doi.org/10.1016/j.procbio.2022.01.004.Sampaio C.S., Angelotti J.A.F., Fernandez-Lafuente R., Hirata D.B. Lipase immobilization via crosslinked enzyme aggregates: problems and prospects – a review // International Journal of Biological Macromolecules. 2022. Vol. 215. P. 434–449. https://doi.org/10.1016/j.ijbiomac.2022.06.139.Sampaio C.S., Angelotti J.A.F., Fernandez-Lafuente R., Hirata D.B. Lipase immobilization via crosslinked enzyme aggregates: problems and prospects – a review. International Journal of Biological Macromolecules. 2022;215:434-449. https://doi.org/10.1016/j.ijbiomac.2022.06.139.Quayson E., Amoah J., Hama S., Kondo A., Ogino C. Immobilized lipases for biodiesel production: current and future greening opportunities // Renewable and Sustainable Energy Reviews. 2020. Vol. 134. P. 110355. https://doi.org/10.1016/j.rser.2020.110355.Quayson E., Amoah J., Hama S., Kondo A., Ogino C. Immobilized lipases for biodiesel production: current and future greening opportunities. Renewable and Sustainable Energy Reviews. 2020;134:110355. https://doi.org/10.1016/j.rser.2020.110355.Manan F.M.A., Attan N., Zakaria Z., Mahat N.A., Wahab R.A. Insight into the Rhizomucor miehei lipase supported on chitosan-chitin nanowhiskers assisted esterification of eugenol to eugenyl benzoate // Journal of Biotechnology. 2018. Vol. 280. P. 19–30. https://doi.org/10.1016/j.jbiotec.2018.05.015.Manan F.M.A., Attan N., Zakaria Z., Mahat N.A., Wahab R.A. Insight into the Rhizomucor miehei lipase supported on chitosan-chitin nanowhiskers assisted esterification of eugenol to eugenyl benzoate. Journal of Biotechnology. 2018;280:19-30. https://doi.org/10.1016/j.jbiotec.2018.05.015.Alves dos Santos L., Alnoch R.C., Soares G.A., Mitchell D.A., Krieger N. Immobilization of Pseudomonas fluorescens lipase on chitosan crosslinked with polyaldehyde starch for kinetic resolution of sec-alcohols // Process Biochemistry. 2022. Vol. 122. P. 238–247. https://doi.org/10.1016/j.procbio.2022.10.014.Alves dos Santos L., Alnoch R.C., Soares G.A., Mitchell D.A., Krieger N. Immobilization of Pseudomonas fluorescens lipase on chitosan crosslinked with polyaldehyde starch for kinetic resolution of sec-alcohols. Process Biochemistry. 2022;122:238-247. https://doi.org/10.1016/j.procbio.2022.10.014.Costa-Silva T.A., Carvalho A.K.F., Souza C.R.F., Freitas L., De Castro H.F., Oliveira W.P. Highly effective Candida rugosa lipase immobilization on renewable carriers: Integrated drying and immobilization process to improve enzyme performance // Chemical Engineering Research and Design. 2022. Vol. 183. P. 41–55. https://doi.org/10.1016/j.cherd.2022.04.026.Costa-Silva T.A., Carvalho A.K.F., Souza C.R.F., Freitas L., De Castro H.F., Oliveira W.P. Highly effective Candida rugosa lipase immobilization on renewable carriers: Integrated drying and immobilization process to improve enzyme performance. Chemical Engineering Research and Design. 2022;183:41-55. https://doi.org/10.1016/j.cherd.2022.04.026.Pourkhanali K., Khayati G., Mizani F., Raouf F. Characterization of free and immobilized lipase from Penicillium sp. onto three modified bentonites: a comparative study // Journal of Biotechnology. 2022. Vol. 344. P. 57–69. https://doi.org/10.1016/j.jbiotec.2021.12.013.Pourkhanali K., Khayati G., Mizani F., Raouf F. Characterization of free and immobilized lipase from Penicillium sp. onto three modified bentonites: a comparative study. Journal of Biotechnology. 2022;344:57-69. https://doi.org/10.1016/j.jbiotec.2021.12.013.Tan Z., Bilal M., Li X., Ju F., Teng Y., Iqbal H.M.N. Nanomaterial-immobilized lipases for sustainable recovery of biodiesel – a review // Fuel. 2022. Vol. 316. P. 123429. https://doi.org/10.1016/j.fuel.2022.123429.Tan Z., Bilal M., Li X., Ju F., Teng Y., Iqbal H.M.N. Nanomaterial-immobilized lipases for sustainable recovery of biodiesel – a review. Fuel. 2022;316:123429. https://doi.org/10.1016/j.fuel.2022.123429.Kovalenko G., Perminova L., Beklemishev A. Heterogeneous biocatalytical esterification by recombinant Thermomyces lanuginosus lipase immobilized on macroporous carbon aerogel // Catalysis Today. 2021. Vol. 379. P. 36–41. https://doi.org/10.1016/j.cattod.2020.11.018.Kovalenko G., Perminova L., Beklemishev A. Heterogeneous biocatalytical esterification by recombinant Thermomyces lanuginosus lipase immobilized on macroporous carbon aerogel. Catalysis Today. 2021;379:36-41. https://doi.org/10.1016/j.cattod.2020.11.018.Moguei M.R.S., Habibi Z., Shahedi M., Yousefi M., Alimoradi A., Mobini S., et al. Immobilization of Thermomyces lanuginosus lipase through isocyanidebased multi component reaction on multi-walled carbon nanotube: application for kinetic resolution of rac-ibuprofen // Biotechnology Reports. 2022. Vol. 35. P. e00759. https://doi.org/10.1016/j.btre.2022.e00759.Moguei M.R.S., Habibi Z., Shahedi M., Yousefi M., Alimoradi A., Mobini S., et al. Immobilization of Thermomyces lanuginosus lipase through isocyanidebased multi component reaction on multi-walled carbon nanotube: application for kinetic resolution of racibuprofen. Biotechnology Reports. 2022;35:e00759. https://doi.org/10.1016/j.btre.2022.e00759.Sun Y., Guo M., Hu S., Fang X., Jin Z., Wu R. Nanosurface-immobilized lipase and its degradation of phthalate wastewater // Molecular Catalysis. 2022. Vol. 529. P. 112522. https://doi.org/10.1016/j.mcat.2022.112522.Sun Y., Guo M., Hu S., Fang X., Jin Z., Wu R. Nanosurface-immobilized lipase and its degradation of phthalate wastewater. Molecular Catalysis. 2022;529:112522. https://doi.org/10.1016/j.mcat.2022.112522.Badoei-dalfard A., Tahami A., Karami Z. Lipase immobilization on glutaraldehyde activated graphene oxide/chitosan/cellulose acetate electrospun nanofibrous membranes and its application on the synthesis of benzyl acetate // Colloids and Surfaces B: Biointerfaces. 2022. Vol. 209. P. 112151. https://doi.org/10.1016/j.colsurfb.2021.112151.Badoei-dalfard A., Tahami A., Karami Z. Lipase immobilization on glutaraldehyde activated graphene oxide/ chitosan/cellulose acetate electrospun nanofibrous membranes and its application on the synthesis of benzyl acetate. Colloids and Surfaces B: Biointerfaces. 2022;209:112151. https://doi.org/10.1016/j.colsurfb.2021.112151.Ghide M.K., Li K., Wang J., Abdulmalek S.A., Yan Y. Immobilization of Rhizomucor miehei lipase on magnetic multiwalled carbon nanotubes towards the synthesis of structured lipids rich in sn-2 palmitic acid and sn-1,3 oleic acid (OPO) for infant formula use // Food Chemistry. 2022. Vol. 390. P. 133171. https://doi.org/10.1016/j.foodchem.2022.133171.Ghide M.K., Li K., Wang J., Abdulmalek S.A., Yan Y. Immobilization of Rhizomucor miehei lipase on magnetic multiwalled carbon nanotubes towards the synthesis of structured lipids rich in sn-2 palmitic acid and sn-1,3 oleic acid (OPO) for infant formula use. Food Chemistry. 2022;390:133171. https://doi.org/10.1016/j.foodchem.2022.133171.Yu D., Li Z., Zhou X., Wang W., Wang L., Liu T., et al. Study on the modification of magnetic graphene oxide and the effect of immobilized lipase // International Journal of Biological Macromolecules. 2022. Vol. 216. P. 498–509. https://doi.org/10.1016/j.ijbiomac.2022.06.203.Yu D., Li Z., Zhou X., Wang W., Wang L., Liu T., et al. Study on the modification of magnetic graphene oxide and the effect of immobilized lipase. International Journal of Biological Macromolecules. 2022;216:498509. https://doi.org/10.1016/j.ijbiomac.2022.06.203.Bilal M., Iqbal H.M.N. Naturally-derived biopolymers: potential platforms for enzyme immobilization // International Journal of Biological Macromolecules. 2019. Vol. 130. P. 462–482. https://doi.org/10.1016/j.ijbiomac.2019.02.152.Bilal M., Iqbal H.M.N. Naturally-derived biopolymers: potential platforms for enzyme immobilization. International Journal of Biological Macromolecules. 2019;130:462-482. https://doi.org/10.1016/j.ijbiomac.2019.02.152.Sharma A., Thatai K.S., Kuthiala T., Singh G., Arya S.K. Employment of polysaccharides in enzyme immobilization // Reactive and Functional Polymers. 2021. Vol. 167. P. 105005. https://doi.org/10.1016/j.reactfunctpolym.2021.105005.Sharma A., Thatai K.S., Kuthiala T., Singh G., Arya S.K. Employment of polysaccharides in enzyme immobilization. Reactive and Functional Polymers. 2021;167:105005. https://doi.org/10.1016/j.reactfunctpolym.2021.105005 .Vassiliadi E., Aridas A., Schmitt V., Xenakis A., Zoumpanioti M. (Hydroxypropyl)methyl cellulose-chitosan film as a matrix for lipase immobilization: operational and morphological study // Molecular Catalysis. 2022. Vol. 522. P. 112252. https://doi.org/10.1016/j.mcat.2022.112252.Vassiliadi E., Aridas A., Schmitt V., Xenakis A., Zoumpanioti M. (Hydroxypropyl)methyl cellulose-chitosan film as a matrix for lipase immobilization: operational and morphological study. Molecular Catalysis. 2022;522:112252. https://doi.org/10.1016/j.mcat.2022.112252 .Холназаров Б.А., Тураев Х.Х., Назаров Ю.Э. Синтез гидрогелей на основе биоматериалов крахмала и натрий-карбоксиметилцеллюлозы // Universum: химия и биология. 2020. N 10. C. 57–60.Kholnazarov B.A., Turaev H.H., Nazarov Yu.E. Synthesis of hydrogels based on starch biomaterials and sodium-carboxymethylcellulose. Universum: khimiya i biologiya. 2020;(10):57-60. (In Russian).

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