Biocatalytic engineering was carried out by varying monotonically the binary CNTs-silica composition and, accordingly, the physicochemical characteristics of adsorbents developed for immobilization of recombinant T. lanuginosus lipase (rPichia/lip). The adsorbents based on composite carbon-silica materials (CCSMs) were produced by impregnating finely dispersed multi-walled carbon nanotubes with silica hydrosol followed by calcination in argon at 350°C; the mass ratio of the hydrophobic and the hydrophilic components varied over a wide range. Biocatalysts (BCs) for green low-temperature synthesis of various esters in a non-aqueous medium of organic solvents were prepared by adsorption of rPichia/lip with subsequent drying under ambient conditions. The characteristics of the CCSMs and BCs were characterized by thermogravimetry, nitrogen porosimetry and electron microscopy. The catalytic properties of BCs, such as enzymatic activity, substrate conversion and specificity, as well we their operational stability depending on the chemical composition of CCSMs were extensively studied in the esterification of saturated monocarboxylic acids (C4, C7, C18) and primary aliphatic alcohols (C2, C4, C16) in hexane at 20°C. It was found that the esterifying activity manyfold decreased with increasing the silica content primarily due to a decrease in adsorption ability of CCSMs toward rPichia/lip. The substrate specificity and operational stability of the lipase-active BCs did not greatly depend on the composition of CCSMs. Biocatalysts retained more than half of their initial esterifying activity after 10 reaction cycles.

Carbon–silica composite materials (CSCMs) containing different amounts of silica and carbon components are obtained using two silica precursors (silica sol and silane) and multiwalled carbon nanotubes (MWNTs). At the initial stage of obtaining CSCMs by method 1, a fine MWNT powder is subjected to impregnation by moisture capacity with silica sol; in accordance with method 2, MWNTs are treated with tetraethoxysilane and then subjected to hydrolysis and polycondensation. The silica (SiO2) content in the composites is varied in a range of 3–60 wt

Composite carbon–silica materials (CCSM), differing in the content of carbon and silica components, were obtained using two silicon dioxide precursors (silica sol and silane) and multi-walled carbon nanotubes (MWNTs). At the initial stage of obtaining CCSM by method 1, impregnation of finely dispersed MWCNT powder with silica sol was used, method 2 was carried out using treatment of MWCNTs with tetraethoxysilane followed by hydrolysis and polycondensation. The content of silica (SiO2) in the composites varied from 3 to 60 wt %. After drying and appropriate heat treatment at 250–350°C, the composite materials were studied by various physicochemical methods: nitrogen porosimetry, electron microscopy, X-ray fluorescence analysis, and synchronous thermal analysis. Significant differences in parameters were found depending on the chemical composition of CСSM, including textural characteristics. Thus, with an increase in the SiO2 content, the specific surface area of composite materials increased (by a factor of 2), and maxima were observed on the distribution curves over pore diameters (at 20–40 nm).The composite carbon–silica materials were tested as adsorbent for the preparation of heterogeneous biocatalysts (BC) for the low-temperature synthesis of esters; the active component of these BC was lipase immobilized exclusively on the carbon surface of nanotubes. With a decrease in the content of MWCNTs in the composite materials, the enzymatic activity and operational stability of biocatalysts, measured in the reaction of esterification of heptanoic acid (C7) with butanol (C4), decreased monotonically, reaching a 2–8-fold drop in activity at the maximum content of SiO2 (58 wt %).

Biocatalysis in both the homogeneous and heterogeneous versions is an independent interdisciplinary direction in scientific and practical research of single-stage conversions of feed reagents (substrates) to valuable marketable products involving, in most cases, a single enzyme as a catalyst. Single-enzyme biocatalytic processes exhibiting all specific features of enzymatic catalysis are a fairly competitive alternative to conventional chemical production. This review provides information on the results achieved by domestic research teams/laboratories that have been involved in extensive and efficient studies in the field of biocatalysis over the past decades and have practical developments protected by RF patents, which, under favorable circumstances, can be offered to commercial enterprises/companies for testing and use on a laboratory and/or pilot-scale with the prospects of industrial scale-up. In the review, special attention is given to targeted systematic studies of lipolytic enzymes (lipases), which have a unique ability to catalyze reactions in a medium of organic solvents, primarily esterification and transesterification reactions, which lead to the formation of valuable products of organic synthesis, such as esters. Lipases are active components of heterogeneous biocatalysts (BCs) synthesized by fixing (immobilizing) these enzymes on the surface of solid adsorbing supports. The review briefly describes the results of studies of domestic research teams and gives complete lists of their reports providing information on the methods of immobilization of target enzymes, the catalytic properties of the developed BCs (enzyme activity, substrate specificity, operational stability) and describing the conditions for biocatalytic processes involving heterogeneous BCs, such as the synthesis of acrylamide and various esters. Taking into account the average activity (А), which is similar to the activity measured at the half-inactivation time (t1/2), and the operational stability of BCs characterized by the t1/2 value, a fairly rough assessment of the productivity of BCs is conducted by calculating the amount of valuable product produced (in tons) per kilogram of BC.

The supramolecular aggregation processes occurring on metallic (aluminum and gold) surfaces in aqueous solutions of bovine serum albumin (BSA) during drying were studied using advanced scanning electron microscopy (SEM). The possible mechanism for the formation of amazing intricate fractal structures on metallic surfaces was proposed based on the analysis of SEM images, size distribution diagrams and EDX-scanning element distribution maps.

The processes of supramolecular aggregation occurring on carbon surfaces in aqueous solutions of bovine serum albumin (BSA) during drying were studied using modern scanning electron microscopy (SEM). The carbon materials studied were highly oriented pyrolytic graphite (HOPG) and glassy carbon (GC). Based on the analysis of SEM images and EDX-scanning element distribution maps, a possible mechanism for the formation of the observed intricate structures on the surface was proposed. The formation of fuzzy lacy structures resembling shadow replicas was explained by relatively strong hydrophobic–hydrophobic interactions of albumin molecules with carbon surfaces.

A study was made of the low-temperature synthesis of esters of heptanoic (enanthic, C7:0) acid and various diols using heterogeneous biocatalysts prepared by the adsorptive immobilization of the recombinant lipase rPichia/lip on macroporous carbon aerogel. The substrates were diols differing in length of the carbon skeleton (2 to 6 C atoms), position of the OH group, and isomerism of the carbon skeleton, namely, 1,2-ethanediol (ethylene glycol) and its oligomers (dimers and trimers), 1,2-propanediol (propylene glycol), 1,3‑propanediol, 1,4-butanediol, 1,6-hexanediol, and 2-ethyl-1,3-hexanediol. The esterification and synthesis of monoesters of heptanoic acid were carried out in batch reactors under very mild conditions (20 ± 2°C, 1 bar). The properties of the prepared biocatalysts, such as enzymatic activity, substrate specificity, and operational stability, were investigated depending on the structure of the diol molecule and the nature of the organic solvent (chloroform, hexane, acetone). It was found that C2–C4 short-chain diols irreversibly inhibited the immobilized lipase, and the biocatalyst was completely inactivated within 1–3 reaction cycles. The maximum activity (83 U/g) and the conversion of acid (94% in 24 h) were observed in the esterification of heptanoic acid with 1,6-hexanediol; under the studied conditions, the fraction of monoester was more than 99%. A correlation was found between the biocatalytic activity and the molecular length of symmetric diols: the esterification reaction rate increased with increasing distance between the terminal OH groups. Because chloroform inactivated the adsorbed rPichia/lip, the conditions for the reactivation of the biocatalysts were selected by replacing the reaction medium: the solvent by hexane and the diol by butanol.

Biocatalysts with lipase activity (BLAs) were prepared by adsorptive immobilization of recombinant lipase (r Pichia /lip) from thermophilic microscopic fungi Thermomyces lanuginosus produced by a genetically engineered strain of methylotrophic yeast Komagataella phafii ( Pichia pastoris ). Supports with different physicochemical properties were used as adsorbents: mesoporous hydrophilic silica (SiO 2 ) and macroporous hydrophobic carbon aerogel (MCA). The enzymatic activity, substrate specificity and operational stability of BLAs were studied in the esterification of saturated fatty acids with aliphatic alcohols differing in the number of carbon atoms in the molecule from 2 to 18. Matrices of relative activities were compiled for more than 60 pairs of substrates, an acid and an alcohol, by comparing the reaction rates of the esterification under identical conditions, which allowed us to reveal differences in the specificity of adsorbed lipase depending on the chemical nature of the support. It was found that for both types of biocatalysts, r Pichia /lip on SiO 2 (PLSi) and r Pichia /lip on MCA (PLC), the maximum reaction rate was observed under esterification of heptanoic acid (C 7 ) with butyl alcohol (C 4 ). Under the same conditions of the synthesis of esters (20 ± 2°C, 1 bar, a mixture of hexane and diethyl ether as an organic solvent), including the synthesis of butylheptanoate, r Pichia /lip adsorbed on silica showed an order of magnitude lower activity than lipase adsorbed on carbon aerogel. The catalytic constants, equal to 3.7 s –1 and 1.1 × 10 2 s –1 , respectively, differed by 30 times. It was found that esters of short chain fatty acids C 4 –C 7 and ethyl alcohol C 2 were synthesized 2–3 times faster using the hydrophobic PLC type than using the hydrophilic PLSi type of BLAs. At the same time, esters of high-molecular-weight acids С 9 , C 10 , С 18 and alcohols С 8 –С 16 with pronounced hydrophobicity were synthesized 1.5–2 times faster using of PLSi type BLAs. The operational stability of the biocatalysts was quite high: the prepared BLAs retained 82–99% of their initial activity after more than 30 reaction cycles, while the duration of each cycle to reach an acid conversion above 85% was several hours (4–6 h).

This work was devoted to the construction of recombinant strains Escherichia coli BL21 (DE3) and Pichia pastoris X33, which produce a 1,3-specific thermostable lipase from Thermomyces lanuginosus. The sequences of two lipase genes were optimized for expression in bacteria and methylotrophic yeasts, then synthesized and cloned in the corresponding expression vectors. As a result of genetic engineering manipulations, E. coli and P. pastoris strains were constructed that efficiently produced T. lanuginosus recombinant lipase. Recombinant E. coli clones accumulated lipase in the cytoplasm at a level of 30–40% of the total cellular protein. Recombinant P. pastoris clones secreted lipase into the culture medium at a concentration of at least 1 g/L. Lipases produced by the recombinant clones, designated as rE.coli/lip and rPichia/lip, contained a six-histidine sequence (-His6) in the C-terminal region. The resulting lipases were immobilized on/in solid inorganic supports in order to develop heterogeneous biocatalysts (HBs) for the enzymatic conversion of triglycerides and fatty acids. The rPichia/lip enzyme was adsorbed on mesoporous silica and macroporous carbon aerogel. The properties of the prepared HBs, their enzymatic activity, substrate specificity, and operational stability were studied in the reaction of esterification of fatty acids with aliphatic alcohols in organic solvents at 20 ± 2°C. It was found that immobilized lipases had a relatively wide substrate specificity, as well as high operational stability, and the prepared HBs almost completely retained their high esterifying activity for several tens of reaction cycles.

Heterogeneous biocatalysis is a part of biotechnology and it has commercial potential for industrial implementation, in particular the final stages of deep processing of renewable raw materials. The commercially attractive heterogeneous biocatalysts are prepared by immobilizing practically valuable enzymatic active substances onto solid inorganic supports. Heterogeneous biocatalytic processes of the target conversion of substrate into valuable market product are carried out in periodic or continuous modes using traditional batch and packed-bed reactors, as well as novel types of vortex reactors in accordance with the principles of green chemistry. Heterogeneous biocatalysts for the final stages of deep processing of vegetable raw materials such as starch and oils are described here. One of the biocatalysts is glucoamylase immobilized by adsorption on mesoporous carbon support Sibunit™ type. This glucoamylase-active biocatalyst is used at the stage of starch saccharification, i.e., hydrolysis of dextrin to treacle and glucose syrups used in food and confectionary industries. The second of the biocatalysts is recombinant T. lanuginosus lipase immobilized on mesoporous silica KSK™ type and macroporous carbon aerogel. These lipase-active biocatalysts can effectively compete with traditional organic synthesis catalysts, and they are used in low-temperature processes carried out in unconventional anhydrous media such as interesterification of vegetable oils’ triglycerides with ethyl acetate for producing ethyl esters of fatty acids (biodiesel and vitamin F) and esterification of fatty acids with aliphatic alcohols for synthesis of various esters used as fragrances, flavorings, odors, emollients, and nonionic surfactants in perfume and cosmetics industries. The prepared heterogeneous biocatalysts due to their high enzymatic activity and operational stability are promising for practical implementation.

The lipase-active heterogeneous biocatalysts were prepared by adsorptive immobilization of Thermomyces lanuginosus lipase (designated as rPichia/lip) produced by authoring recombinant Pichia pastoris strain. Biocatalysts of LipoCarb type were prepared by spontaneous adsorption of rPichia/lip on the authoring macroporous carbon aerogel. Biocatalysts of LipoSil type were prepared by forced adsorption of rPichia/lip on mesoporous silica. The prepared biocatalysts were studied carefully in the esterification of short-chain fatty acids (butyric C4, C5-C7) and aliphatic alcohols (ethanol C2, C3-C5, C8) in order to produce valuable fruity-smelling esters. Biocatalytic properties, such as enzymatic activity, substrates specificity, and operational stability, were found to depend on chemical nature of both the adsorbents and organic solvents, primarily on their polarity. When comparing the activity in the synthesis of n-butyl heptanoate, it was found that the LipoCarb biocatalysts demonstrated activity an order of magnitude higher than LipoSil type. The Michaelis-Menten kinetic constants differed by a factor of 30, e.g. 1.1.102 s(-1) and 3.7 s(-1), respectively. The operational stability of the lipase-active biocatalysts was sufficiently high; rPichia/lip adsorbed on carbon aerogel retained till 96% of the initial activity after 29 reaction cycles, whereas rPichia/lip adsorbed on silica completely retained the activity after 38 reaction cycles of esterification under optimal reaction conditions selected. The prepared biocatalysts of LipoCarb and LipoSil types were promising for the Green enzymatic synthesis of fragrance and aroma esters under very mild ambient conditions (20 +/- 2 degrees C, 1 bar).

Processes of the low-temperature enzymatic synthesis of esters in nonaqueous media of organic solvents with participation of heterogeneous biocatalysts synthesized by the adsorption immobilization of a recombinant lipase from Thermomyces lanuginosus (Pichia pastoris producer) on macroporous carbon aerogel were investigated. The esterification reaction was conducted using individual organic solvents of different polarity, such as nonpolar ones (lоgP > 2.5) – hexadecane, hexane and toluene, and polar solvents (lоgP < 1) – diethyl ether, tert-butanol and acetone, as well as their binary mixtures of different composition, for example, a mixture of hexane with diethyl ether having the composition 1 : (1/5÷3, vol. fr.). The effect of the solvent and co-solvent nature on the properties of synthesized biocatalysts, namely the enzymatic activity and operational stability, was studied in the batch esterification of heptanoic acid (C7:0) by alcohols, particularly the polar substrates – ethanol and n-butanol, or nonpolar alcohols – n-octanol and n-hexadecanol. Activity of the synthesized biocatalysts was found to depend to a great extent on polarity of both the solvents used in the reaction and the alcohol substrates. The maximum activity of 400 EA·g–1 was observed in a medium of nonpolar and nonviscous hexane (lоgP = 2.9), whereas the minimum activity of 4 EA·g–1 – in acetone (lоgP = 0.4); esterification of heptanoic acid in the presence of tert-butanol (lоgP = 0.6) virtually did not proceed. The activity of biocatalysts was shown to depend nonmonotonically on the lоgP value of individual solvent. In the binary mixtures of organic solvents, such as hexane/diethyl ether, biocatalytic activity linearly increased with an increase in lоgPmixture; in addition, biocatalysts exhibited the maximum operational stability.

Heterogeneous biocatalysts prepared by immobilizing recombinant lipases are promising for implementation in organic synthesis of various esters used as fragrances, emollients, emulsifiers, non-ionic surfactants, plasticizers, etc. For this research, the Thermomyces lanuginosus lipase produced by the authoring recombinant Pichia pastoris strain was adsorbed on the authoring macroporous carbon aerogel. The prepared biocatalysts were studied in the periodic processes of enzymatic esterification carried out in nonconventional anhydrous media of organic solvents at ambient conditions (22 +/- 2 degrees C, 1 bar). Biocatalytical activities depended strongly on both the selected pair of lipase substrates (acid and alcohol) involved in reaction and the organic solvent used for esterification. The reaction rates of fatty C7 acid esterification with double and triple bond C3 alcohols, such as allyl and propargyl alcohols, were found to be 2-4 times lower than with aliphatic propanol. Isomerism of an acid (not alcohol) molecule greatly affected the esterification rates. It was observed that isomers of C4 and C5 acids (isobutyric and iso-valeric) practically did not react with butanol. The esterification rates strongly depended on the polarity of the organic solvent characterized by the parameter logP. With an increase in the solvent polarity from non-polar hexane to polar acetone, the esterification rate decreased by two orders of magnitude. The prepared lipase-active biocatalysts had a sufficiently high operational stability under the chosen optimal reaction conditions; their stationary activity (300 +/- 100 U.g(-1)) did not change in periodic reaction cycles during several tens of hours.

The recombinant rPichia/lip lipase was immobilized by adsorbing the enzyme on aggregated carbon nanotubes (CNTs), unmodified, or doped with nitrogen (N-CNTs). Heterogeneous biocatalysts prepared in this way were investigated in the reaction of the low-temperature synthesis of esters proceeding in organic solvents at room temperature. We studied the effect of the texture and morphology of the CNTs, as well as the nitrogen concentration in doped N-CNTs, on the catalytic properties of immobilized lipase, such as activity, specificity, and stability. The activity of biocatalysts and the specific activity of the adsorbed enzyme increased by 1.5–1.6 times with an increase in the concentration of nitrogen introduced into the CNTs (2 and 5 wt % of nitrogen). When studying the specificity of the esterification of saturated fatty acids (heptanoic, stearic) with aliphatic alcohols (n-butanol, n-hexadecanol), the maximum rate was observed in the synthesis of n-butyl heptanoate. The prepared biocatalysts were highly stable in the batch process of the low-temperature synthesis of esters, while retaining at least 80–85% of the activity of the conditioned biocatalysts for 36 reaction cycles (720 h).

Immobilization of recombinant Thermomyces lanuginosus lipase (designated as rPichia/lip) was carried out by moisture capacity impregnation of mesoporous silica granules followed by drying, and forcible adsorption of enzyme occurred. Eventually prepared lipase-active heterogeneous biocatalysts were systematically studied for enzymatic esterification performed at ambient conditions (20 ± 2 °C, 1 bar) in unconventional anhydrous media of organic solvents such as hexane and diethyl ether. The saturated fatty acids differing in the number of carbon atoms (C2–C10, C18), and aliphatic alcohols differing in the structure of the molecules, namely both the number of carbon atoms (C2–12, C16), and the isomerism of the carbon skeleton (n- and iso-), and OH-group position (prim-, sec-, tert-) were studied as substrates for enzymatic esterification. The specificity of the heterogeneous enzymatic esterification was determined by comparing the reaction rates for various pairs of substrates; and the matrix of relative units of activities was composed. The immobilized on silica rPichia/lip was found to have sufficiently wide specificity toward saturated fatty acids and aliphatic alcohols. High reaction rates were measured in esterification of fatty acids and primary n- and iso-aliphatic alcohols possessing more than four carbon atoms in the molecules. The enanthic acid (heptanoic, C7:0) reacted with butanol (C4) with the highest rate; and the kinetic parameters such as Michaelis constant (KM) for acid and maximal reaction rate (Vmax) were determined under the studied conditions of esterification. Substrates containing aromatic residues did not participate in esterification. The lipase-active heterogeneous biocatalysts possessed considerably high operational stability, and the catalytic activity was completely retained for several tens of reaction cycles in a periodic batch process of low-temperature synthesis of various fatty acid esters.

The monograph consists of reviews prepared by specialists having scientific publications, theoretical knowledge and practical experience in research of immobilized cells of different microorganisms, plants and animals, which they conducted for the last decade. The basis of the reviews is composed by the scientific results of the authors and relevant data on the discussed topics, presented in the modern world literature. The monograph collected information about the characteristics of immobilized cells, various approaches used to their regulation, the possible long-term functioning and storage of such cells. It performs the prospects for application of immobilized cells in biomedicine, biodetection systems, synthetic processes of biologically active substances and in overcoming environmental problems. The monograph is intended for specialists in the field of biotechnology, heterogeneous catalysis, green chemistry, biochemistry, biophysics, ecology, cytology, biomedicine as well as for teachers and students of natural science and technological faculties of higher educational institutions, for anyone interested in new results of research on the properties of various cells and applied aspects of their possible use.

This work is a continuation of the studies devoted to the synthesis of nanostructured carbon (NSC) as a result of the pyrolysis of a mixture of H 2 + C 3 –C 4 alkanes on supported Ni catalysts. Mesoporous alumina (γ-Al 2 O 3 ) and titania (TiO 2 ), on which Ni(II) compounds are deposited by impregnation or homogeneous precipitation, are studied as carriers. Using the methods of thermogravimetric analysis and scanning electron microscopy, it is shown that the activity of Ni catalysts (carbon yield) and the morphology of synthesized NSC are largely determined by the chemical nature of the support. It is found that the synthesis of NSC in the form of carbon nanofibers with a pronounced filamentary structure proceeds only on a Ni catalyst supported on titanium dioxide. The mesoporous carbon–mineral supports obtained after catalytic pyrolysis were studied in the adsorptive immobilization of the enzyme such as Thermomyces lanuginosus lipase. The adsorption properties of the supports, as well as the enzymatic activity and stability of the prepared biocatalysts in the esterification of saturated fatty acids (capric, C10: 0) with aliphatic alcohols (isopentanol, C 5 ) in the non-aqueous media of organic solvents (hexane and diethyl ether) at ambient temperature, are studied. Biocatalysts prepared by lipase adsorption on NSC/TiO 2 show the maximum esterification activity of 100 EA/g, which is 20–45 times higher than the activity of lipase adsorbed on NSC/Al 2 O 3 .

Heterogeneous biocatalysts prepared by immobilizing a recombinant lipase from Thermomyces lanuginosus on mesoporous inorganic supports—silica (SiO2), alumina (Al2O3), and titania (TiO2)—are comparatively studied in the esterification of fatty acids with aliphatic alcohols. It is found that the T. lanuginosus lipase adsorbed on silica has the highest esterifying activity, while the lipase adsorbed on titania is completely inactivated. SiO2-based catalysts have high activity and stability in the esterification of saturated fatty acids containing 4–18 carbon atoms (C4–C18) with aliphatic alcohols (C5–C16) in organic solvents (hexane and diethyl ether). The catalysts operate in this reaction for several tens of reaction cycles (>40) without loss of activity. The recombinant rPichia/lip lipase immobilized on silica exhibits the most pronounced specificity for its first substrate, a fatty acid. For instance, the rate of synthesis for esters of low molecular weight acids (С4–С6) is three to four times slower than for the esters of acids with more than seven carbon atoms. The catalyst has a relatively broad specificity for the second substrate, an aliphatic alcohol. It is found that the ester of enanthic acid (C7:0) and butanol (C4) is synthesized at the maximum rate.