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ПРИМЕНЕНИЕ СВЕРХСШИТЫХ ПОЛИМЕРОВ В КАЧЕСТВЕ НОСИТЕЛЕЙ ГЕТЕРОГЕННЫХ БИОКАТАЛИЗАТОРОВ ДЛЯ РЕАКЦИИ ГИДРОЛИЗА ИНУЛИНА
И.В. Шкутина, Н.В. Мироненко, В.Ф. Селеменев
Ирина Викторовна Шкутина (ORCID 0000-0001-6733-0627)*
Кафедра общей и медицинской химии им. проф. В.В. Хорунжего, Санкт-Петербургский государственный педиатрический медицинский университет, ул. Литовская, 2, Санкт-Петербург, Российская Федерация, 194100
Е-mail: [email protected]*
Наталья Владимировна Мироненко (ORCID 0000-0002-3049-6647)
Кафедра естественнонаучных дисциплин, кафедра аналитической химии, Воронежский государственный университет, Университетская пл., 1, Воронеж, Российская Федерация, 394006 Е-mail: [email protected]
Владимир Федорович Селеменев (ORCID 0000-0002-5061-2588)
Кафедра аналитической химии, Воронежский государственный университет, Университетская пл., 1, Воронеж, Российская Федерация, 394006
В данной работе проведено исследование адсорбционной иммобилизации инули-назы на сверхсшитых макропористых сорбентах на основе стирола и дивинилбензола: низкоосновном анионообменнике А100, высокоосновном анионообменнике Ä500R, сильнокислотном катионообменнике C100H. Рассмотрено влияние продолжительности процесса сорбции, значений концентраций ионов водорода и белка в растворе на количество иммобилизованного фермента и активность полученных гетерогенных биокатализаторов. Выявлено, что адсорбция достигает своего максимального значения в среднем через 4 ч при рН 4,7-5,0 для рассматриваемых сорбентов. Активность полученных гетерогенных биокатализаторов составляет 64,8-83,5% от активности свободной инулиназы. Хотя активность инулиназы при адсорбции на используемых в работе полимерных носителях снижается, интегральное количество полученного продукта будет выше, чем для нативного катализатора. Изучены изотермы адсорбции инулиназы на сверхсшитых полимерах. Стабильность полученных гетерогенных биокатализаторов объясняется высокой сорбционной способностью сверхсшитых сорбентов. Выявлено, что иммобилизованная инулиназа может применяться в течение 8-11 последовательных циклов реакции гидролиза субстрата. Используя адсорбционную теорию БЭТ, проведен анализ изотерм сорбции фермента и рассчитаны равновесные параметры сорбции: предельное количество сорбированной инулиназы, константы, характеризующие взаимодействие сорбат-сор-бент и сорбат-сорбат. Среди рассматриваемых носителей наиболее высокое значение величины константы сорбционногоравновесия (KL=4,25±0.04л/ммоль) соответствует биокатализатору инулиназа-катионообменник, величина сорбционной емкости которого также наибольшая. Полученные данные представляют интерес для оценки эффективности использования сорбентов в качестве носителей инулиназы в последующих технологических процессах переработки инулинсодержащего сырья.
Ключевые слова: сверхсшитые полимеры, инулиназа, иммобилизация, каталитическая активность, гетерогенный биокатализатор
Для цитирования:
Шкутина И.В., Мироненко Н.В., Селеменев В.Ф. Применение сверхсшитых полимеров в качестве носителей гетерогенных биокатализаторов для реакции гидролиза инулина. Изв. вузов. Химия и хим. технология. 2022. Т. 65. Вып. 8. С. 48-54. DOI: 10.6060/ivkkt.20226508.6559.
For citation:
Shkutina I.V., Mironenko N.V., Selemenev V.F. Application of super-crosslinked polymers as carriers of heterogeneous biocatalysts for inulin hydrolysis reaction. ChemChemTech [Izv. Vyssh. Uchebn. Zaved. Khim. Khim. Tekhnol.]. 2022. V. 65. N 8. P. 48-54. DOI: 10.6060/ivkkt.20226508.6559.
APPLICATION OF SUPER-CROSSLINKED POLYMERS AS CARRIERS OF HETEROGENEOUS BIOCATALYSTS FOR INULIN HYDROLYSIS REACTION
I.V. Shkutina, N.V. Mironenko, V.F. Selemenev
Irina V. Shkutina (ORCID 0000-0001-6733-0627)*
Department of General and Medical Chemistry named after Prof. V.V.Khorunzhy, St. Petersburg State Pediatric
Medical University, Litovskaya st., 2, St. Petersburg, 194100, Russia
E-mail: [email protected]*
Natalia V. Mironenko (ORCID 0000-0002-3049-6647)
Department of Natural Sciences, Department of Analytical Chemistry, Voronezh State University, Universi-
tetskaya sq., 1, Voronezh, 394006, Russia
E-mail: [email protected]
Vladimir F. Selemenev (ORCID 0000-0002-5061-2588)
Department of Analytical Chemistry, Voronezh State University, Universitetskaya sq., 1, Voronezh, 394006,
Russia
In this work the study of adsorption immobilization of inulinase on super-crosslinked macroporous sorbents based on styrene and divinylbenzene: low-base anion exchanger A100, highbase anion exchanger A500R, strong acid cation exchanger C100H was carried out. The influence of the duration of the sorption process, the value of the concentration of hydrogen ions and the concentration of protein in solution on the amount of immobilized enzyme and the activity of the obtained heterogeneous biocatalysts is considered. It was revealed that adsorption reaches its maximum value on average after 4 h at pH of 4.7-5.0 for the sorbents under consideration. The activity of the obtained heterogeneous biocatalysts is 64.8-83.5% of the activity offree inulinase. Although the activity of inulinase during adsorption on the polymer carriers used in the work decreases, the integral amount of the resulting product will be higher than for the native catalyst. The isotherms of inulinase adsorption on super-crosslinked polymers have been studied. The high stability of the obtained heterogeneous biocatalysts is explained by the high sorption capacity of super-crosslinked sorbents. It was revealed that immobilized inulinase can be used during 8-11 consecutive cycles of the substrate hydrolysis reaction. Using the BET adsorption theory, the enzyme sorption isotherms were analyzed and the equilibrium sorption parameters were calculated: the maximum amount of sorbed inulinase, the constants characterizing the sorbate-sorbent and sorbate-sorbate interaction. Among the considered carriers, the highest value of the sorption equilibrium constant (KL=4.25±0.04 l/mmol) corresponds to the inulinase-cation exchanger biocatalyst, the sorption capacity of which is also the largest. The data obtained is of interest for evaluating the effectiveness of the use of sorbents as inulinase carriers in subsequent technological operations ofprocessing of inulin-containing raw materials.
Key words: super-crosslinked polymers, inulinase, immobilization, catalytic activity, heterogeneous biocatalyst
INTRODUCTION
Inulinase (2.1-P-D-fructan-fructanohydrolase, E.C. 3.2.1.7) is widely used in technological processes to obtain fructose from vegetable raw materials and makes it possible to convert inulin into fructose in one stage [1, 2]. Fructose has a twice lower glycemic index, and the assimilation of fructose occurs without the participation of insulin. Due to its properties, syrups with a high fructose content obtained by enzymatic hydrolysis of extracts of inulin-containing plants can be used in the confectionery industry, in therapeutic nutrition as a sweetener for diabetic patients [3, 4].
However, the use of native enzymes has a number of limitations: the impossibility of reuse, low thermal stability, the complexity of separating the biocatalyst and the final product. The effectiveness of enzymatic processes can be improved with the help of immobilized drugs [5-8].
The results obtained during the immobilization of enzymes on super-crosslinked polymer sorbents are of continued interest [9,10]. Previously, we investigated the immobilization of inulinase on sorbents of the "Styrosorb" type [11, 12].
Despite the fact that a large number of enzyme preparations immobilized on different carriers have been already described, it should be noted that the choice of the sorbent and the method of binding the protein to the carrier remains largely empirical. The most important factors on which the adsorption immobilization of enzymes depends are determined, on the one hand, by the surface properties of carriers: their chemical structure, hydrophilicity or hydrophobicity, porosity, swelling, on the other hand - the structure of enzymes, the size of macromolecules, chemical properties.
Super-crosslinked sorbents based on styrene with divinylbenzene have a uniquely high sorption capacity in relation to organic substances in aqueous or airborne media. They have found wide application in large-scale sorption processes in the chemical, food, pharmaceutical industries, to solve a number of environmental problems [13].
The purpose of this work was to study the conditions of adsorption immobilization of inulinase on super-crosslinked ion-exchangers.
EXPERIMENTAL PART
The object of the study was Aspergillus niger (Sigma Aldrich) inulinase, inulin (Spofa) was used as a substrate. The sorbents were ion exchangers (Puro-lite): a weakly basic anion exchanger A100 containing mainly tertiary amino groups; a highly basic anion exchanger A500R; a strong acid cation exchanger C100H with sulfogroups. These carriers belong to the group of super-crosslinked polymer materials based on styrene and divinylbenzene with a highly developed specific surface area and macroporous structure [14]. To prepare for the experiment, the sorbents were previously conditioned [15].
Kinetic experiments were carried out under static conditions with continuous mixing of the solution by the limited volume method. To do this, the air-dry sorbent suspensions weighing 1.0000 ±0.0002 g were placed in conical flasks with a lapped stopper of 1000 ml, and filled with an inulinase solution with a concentration of 2-10"2 mmol/l. Inulinase solutions were prepared on the basis of an acetate buffer. At certain intervals, 1.0 ml samples were taken and the concentration of the substance was determined by the spectrophotometric method. The total amount of protein in native enzyme preparations was determined spectrophotometrically using the Lowry method, in immobilized enzymes - by the modified Lowry method. The process was considered to be completed if the substance content in the solution did not change for some time. The absorption spectra of solutions
were recorded on a Shumadzu UV-2401 spectrophotometer, the spectral width of the slit is 0.5 nm, the scanning step is 0.5 nm, the scanning speed corresponded to the Slow mode.
The method of variable concentrations was used to obtain sorption isotherms. Sorbent suspensions (1.0000 ± 0.0002 g) were brought into contact with solutions of various concentrations ((0.25-2.5)-10-2 mmol/l), based on 0.1mol/l acetate buffer pH 4.7. Immobilization was carried out during for 4 h under static conditions at a temperature of 20.0 ± 1.5 °C with periodic stirring. The amount of substance in the sorbent phase was calculated from the difference in the concentrations of the initial and equilibrium solutions. Protein desorption into buffer solutions was no more than 2%.
Experiments to determine the activity of enzyme preparations were conducted in a thermostatically controlled reactor in which the incubated liquid was stirred using a magnetic stirrer. The catalytic activity was measured with the help of the spectrophotometry method by the Selivanov reaction using resor-cinol [16]. For a unit of inulinase activity, such an amount of an enzyme was taken that catalyzes the hydrolysis of inulin for 1 min with the formation of 1 ^mol of fructose.
Inulinase activity was calculated by the formula:
A = , (1)
180bt
where A is the catalytic activity per mg of protein; a -the amount of fructose, mmol; b - the amount of enzyme in the reaction mixture, mg/ml of hydrolysate; t - the hydrolysis time, min; 180 - the molecular mass of fructose.
The standard deviation of the obtained results did not exceed 0.01.
RESULTS AND DISCUSSION
Kinetic sorption curves were obtained to determine the time to achieve equilibrium in the sorbent enzyme solution system. The equilibrium value of in-ulinase adsorption for the studied sorbents is reached on average within 250 min (Fig. 1).
To use polymer carriers in real technological processes, an important requirement is not only a high sorption capacity of the sorbent relative to the sorbate, but also a short time period to achieve equilibrium in the system. Considering the size of the inulinase (radius - 12.6 nm, height of the molecule - 6.4 nm) [17], it is possible to note good kinetic properties of the sorbents used.
The conducted studies have shown that the highest value of inulinase sorption is observed in the pH range 4.5-5.2 (Fig. 2), at this value heterogeneous
biocatalysts showed the highest value of catalytic activity. The activity of inulinase preparations immobilized on the exchanger C100H at pH 4.7 is 83.5%, on A500R -78.4%, on A100 - 64.8% of the activity of the native enzyme (2500 U/g). It can be assumed that the active centers of inulinase after immobilization become sterically less accessible to the high-molecular substrate - inulin. On the other hand, adsorption immobilization stabilizes the inulinase molecule, primarily the mobility of the tertiary structure which is responsible for the formation of the enzyme-substrate complex, thus protecting the protein from the effects of denaturing factors, pH in particular. Despite the decrease in the maximum reaction rate of inulin hydrolysis and catalytic activity compared to free inulinase, a significant advantage is the possibility of reuse of a heterogeneous biocatalyst.
Fig. 1. Kinetic curves of inulinase sorption on C100H (1), A500R (2), A100 (3). Q - the amount of sorbed inulinase, mmol/g; t - is the
duration of the process, min Рис. 1. Кинетические кривые сорбции инулиназы на C100H (1), A500R (2), А100 (3). Q - количество сорбированной инулиназы, ммоль/г; t - продолжительность процесса, мин
Fig. 2. Dependence of the amount of sorbed inulinase (Q, mmol/g) on C 100H (1), A 500R (2), A 100 (3) on the pH of the
equilibrium solution Рис. 2. Зависимость количества сорбированной инулиназы (Q, ммоль/г) на C100H (1), A500R (2), А100 (3) от рН равновесного раствора
The isotherms of inulinase sorption on the carriers under consideration, obtained at pH 4.7, are shown in Fig. 3. In the region of low concentrations at sorption isotherms, we observe an almost linear dependence and the subsequent appearance of a plateau, which corresponds to the formation of a monomolecu-lar sorbate layer. Further, immobilization occurs due to the interaction of inulinase molecules with each other, which leads to the formation of polymolecular layers on the surface of the sorbent.
Fig. 3. Isotherms of inulinase sorption at C100H (1), A500R (2), and 100 (3) at pH 4.7. C - equilibrium protein concentration in
solution, mmol/l Рис. 3. Изотермы сорбции инулиназы на C100H (1), A500R (2), А100 (3) при рН 4,7. С - равновесная концентрация белка в растворе, ммоль/л
Hydrophobic interactions and emerging hydrogen bonds between molecules, leading to the formation of supramolecular complexes, probably play a decisive role in the binding of inulinase to the carrier. The three-dimensional structure of super-stitched polystyrene used as carriers is a polymer mesh with a developed inner surface (up to 1500 m2/g) and a certain mobility capable of forming structured phases in such a polymer matrix. This assumption has been noted during the sorption of a number of organic substances [ 18-20]. The presence of reactive functional groups also makes it possible to implement electrostatic interactions to ensure stronger carrier - enzyme bonds.
During immobilization, the catalytic activity of a heterogeneous biocatalyst was controlled for each value of the enzyme concentration. It was proved that despite the decreasing of the inulinase activity during immobilization, the total amount of the resulting product will be higher than that of the native enzyme.
The study of the mechanism of formation of the enzyme-carrier complex is necessary for the targeted creation of immobilized enzyme preparations. It is proved that enzymes are able to form supramolecular
complexes when their microenvironment changes. This phenomenon must be taken into account when selecting conditions and methods of immobilization, since the association-dissociation processes are one of the key mechanisms for regulating the catalytic activity of the biocatalyst [21]. Theoretical processing of sorption isotherms allows us to draw a conclusion that the nature of the interaction in the sorbate-sorbent, sorb-ate-sorbate, sorbate-solvent system can be used to optimize immobilization processes.
To describe quantitatively the isotherms of in-ulinase sorption by super-crosslinked sorbents, the BET model was used [22] (Table). The amount of immobilized enzyme was calculated by the formula:
Q =_KLce_, (2)
^ (1 - Kscs )(1 - KsCe + KLce ) where Q-10"3 is the amount of sorbed protein, mmol/g; QoT 10-3 - the maximum amount of sorbed protein (the amount of the monolayer protein to be sorbed), mmol/g; ce-10"2 - the equilibrium concentration of the solution, mmol/l; Kl - the sorption equilibrium constant characterizing the intensity of the sorption process, l/mmol; Ks - the sorption equilibrium constant for the polymolecular layer, l/mmol.
The values of the sorption equilibrium constants included in the BET isotherm equation were calculated using the linearized equation:
1
^ kl - к8л
Q(1 - Ksce )
Q Kl
c„ + ■
Q» Kl
(3)
The maximum quantity of the immobilized enzyme that composes the monomolecular layer was determined from equations (4,5):
0„ =
1
b ■ K,
Kl = \ + KS • b
(4)
(5)
The sorption equilibrium constant for the polymolecular layer (Ks) was determined from the maximum authentic value of the line approximation
ce -ce/ Q(1-Ksc,). (6)
Table
Values of sorption parameters calculated using BET equations
Таблица. Значения сорбционных параметров,
Sorbent Kl, l/mmol Q^-10'3, mmol/g Ks, l/mmol R2
А 100 3.58±0.05 0.42±0.01 0.72±0.06 0.95
A500R 4.05±0.02 0.60±0.03 0.85±0.01 0.97
C100H 4.25±0.04 0.65±0.02 0.92±0,02 0.96
The calculated values of sorption parameters are presented in Table. It follows from the data obtained that the maximum value of the sorption equilibrium constant (Kl) has the cation exchanger C100H, which indicates a greater affinity of the carrier to the enzyme, compared to the anion exchangers studied. Since the values of Kl for all carriers are higher than Ks, it is possible to assume a greater role of the sorbent-sorbate interaction, compared to the formation of complexes of protein molecules. The obtained values of the constants are consistent with the values of the constants Kl and Ks for organic substances during sorption by carriers [23, 24].
The study of the cyclical action of the obtained heterogeneous biocatalysts was carried out. To do this, the immobilized enzyme (100 mg) was placed in test tubes with an inulin solution (10 ml; 1 mmol/l) and hydrolysis was performed, the substrate was removed every hour. It was revealed that a heterogeneous bi-ocatalyst based on the sorbent C100H can be used for an average of 11 cycles (Fig. 4), A500R - 9 cycles, A100 - 8 cycles, which indicates a sufficiently high stability of the obtained preparations.
90 80 70 60 50 40 30 20 10 0
А, %
1 2 3 4 5 6 7
9 10 11 n
Fig. 4. The multiplicity of the use of immobilized inulinase in the reaction of hydrolysaimilin. A- the activity (% of the maximum);
n - the number of reaction cycles Рис. 4. Кратность использования иммобилизованной инули-назы в реакции гидролиза инулина. А - активность (% от максимальной); n - количество реакционных циклов
Despite the decrease in the maximum reaction rate of inulin hydrolysis and catalytic activity compared to free inulinase, a significant advantage is the possibility of reuse of a heterogeneous biocatalyst.
The results obtained during the experiment confirm the relevance and prospects of further investigation of biocatalysts based on super-crosslinked sorbents.
CONCLUSION
The paper considers the regularities of adsorption immobilization of inulinase on macroporous su-per-crosslinked ion exchangers. Optimal conditions for the immobilization process were established: time - 4 h, pH - 4.7, sorbate concentration - (2-2.5)-10-2 mmol/l. The catalytic activity of heterogeneous biocatalysts is 64.8-83.5% of the activity of the native enzyme. One of the advantages of immobilized enzymes is the possibility of their reuse. It has been established that immobilized inulinase can be used during 8-11 cycles in the inulin hydrolysis reaction.
It is noted that inulinase sorption isotherms on the carriers under consideration have a polymolecular character. The values of sorption parameters for prediction and description of polymolecular sorption on carriers are calculated using the BET equations. The data obtained in this work confirm the expediency of
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using super-crosslinked ion exchangers C100H and A500R for the immobilization of inulinase.
ACKNOWLEDGMENTS
The work was carried out with the support of the Ministry of Science and Higher Education of the Russian Federation as part of the state assignment to universities in the field of scientific activity for 20202022, project FZGU-2020-0044.
The authors declare the absence a conflict of interest warranting disclosure in this article.
Работа выполнена при поддержке Министерства науки и высшего образования Российской Федерации в рамках государственного задания вузам в сфере научной деятельности на 20202022 гг., проект ФЗГУ-2020-0044.
Авторы заявляют об отсутствии конфликта интересов, требующего раскрытия в данной статье.
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Поступила в редакцию (Received) 06.12.2021 Принята к опубликованию (Accepted) 25.05.2022
