L. V. Perminova

Immobilization of a recombinant strain producing glucose isomerase inside SiO2-xerogel and properties of prepared biocatalysts

An original method of immobilization of non-growing microorganism cells inside xerogel of silicium dioxide containing insoluble hydroxyl compounds of cobalt(II) has been developed. A recombinant strain producing glucose isomerase has been constructed on the basis of Escherichia coli with the use of a gene of Arthrobacter nicotianae. It was revealed that glucose isomerase activity and stability of biocatalysts prepared on the basis of the recombinant E. coli strain was 3–5 times greater compared with the biocatalysts prepared with the use of the donor strain A. nicotianae. Under conditions of continuous hydrolysis of 3 M fructose at 62–65°C in a fixed bed reactor, time of half-inactivation of the biocatalysts prepared from the recombinant strain and A. nicotianae was ∼60 and ∼25 days, respectively.

Synthesis of catalytic filamentous carbon on a nickel/graphite catalyst and a study of the resulting carbon-carbon composite materials in microbial fuel cells

The carbon-carbon composite materials obtained via the synthesis of catalytic filamentous carbon (CFC) on a Ni/graphite supported catalyst in the process of the pyrolysis of C3–C4 alkanes in the presence of hydrogen were systematically studied. The effects of the following conditions on the catalytic activity expressed as the yield of carbon (g CFC)/(g Ni) and on the character of CFC synthesis on graphite rods were studied: procedures for supporting Ni(II) compounds (impregnation and homogeneous precipitation), the concentrations of impregnating compouds (nickel nitrate, urea, and ethyl alcohol) in solution, graphite treatment (oxidation) conditions before supporting Ni(II) compounds, and the pyrolysis temperature of C3–C4 alkanes in the range of 400–600°C. Optimum conditions for preparing CFC/graphite composite materials, which are promising for use as electrodes in microbial fuel cells (MFCs), were chosen. The electrochemical characteristics of an MFC designed with the use of a CFC/graphite electrode (anode) and Gluconobacter oxydans glycerol-oxidizing bacteria were studied. The morphology of the surfaces of graphite, synthesized CFC, and also bacterial cells adhered to the anode was studied by scanning electron microscopy.

Catalytical properties of Arthrobacter nicotianae cells, a producer of glucose isomerase, immobilized inside xerogel of silicium dioxide

Arthrobacter nicotianae cells, producers of glucose isomerase, were immobilized inside xerogel of silicium dioxide, and properties of the resulted heterogeneous biocatalysts were investigated in the process of isomerization of monosaccharide (glucose and fructose). The glucose isomerase activity of the resulted biocatalysts was shown to be 10 U/g, on average, taking into account the loss of the activity upon the immobilization, which amounted to 50% of the cell activity in suspension. The rate of the fructose isomerization increased linearly in the range of 55–80°C with the temperature coefficient 1.3. The biocatalysts were stable in this range; they were rapidly inactivated, however, at increasing temperature. The half-pife time of inactivation was six to seven h and five min or less at 80 and 85°C, respectively. The half-pife time of inactivation of heterogeneous biocatalysts was 50–90 h in the periodic process of isomerization of 2 M monosaccharides at 60°C in the presence of the immobilized Arthrobacter nicotianae cells.

Glucose isomerase activity in suspensions of Arthrobacter nicotianae cells and adsorption immobilization of the microorganisms on inorganic carriers

Kinetics of monosaccharide isomerization has been studied in suspensions of intact, non-growing Arthrobacter nicotianae cells. Under the conditions of the study, glucose and fructose were isomerized at the same maximum rate of 700 μmol/min per 1 g dried cells, which increased with temperature (the dependence was linear at 60–80°C). The proposed means of adsorption immobilization of A. nicotianae cells involve inorganic carriers differing in macrostructure, chemical nature, and surface characteristics. Biocatalysts obtained by adsorbing the cells of A. nicotianae on carbon-containing foamed ceramics in the coarse of submerged cultivation were relatively stable and retained original activity (catalysis of monosaccharide isomerization) throughout 14 h of use at 70°C. Maximum glucose isomerase activity (2 μmol/min per 1 g) was observed with biocatalysts prepared by adsorption of non-growing A. nicotianae cells to the macroporous carbon-mineral carrier Sapropel and subsequent drying of the cell suspension together with the carrier.

Carbon-in-silica matrices for the preparation of heterogeneous biocatalysts: The synthesis of carbon nanofibers on a Ni/SiO2 catalyst and the characterization of the resulting adsorbents for the immobilization of thermostable lipase

Comparative studies of nanocarbons and nanocarbon-in-silica adsorbents for the immobilization of enzymes, for example, thermostable lipase from Thermomyces lanuginosus, were performed. Carbon nanotubes (CNTs) with different diameters, specific surface areas, and concentrations of surface carboxy groups were studied as the nanocarbon adsorbents. The nanocarbon-in-silica adsorbents were prepared by the synthesis of carbon nanofibers (CNFs) in SiO2 xerogel in the course of the pyrolysis of C3-C4 alkanes on Ni catalysts; their physicochemical and textural characteristics were studied by thermal analysis, scanning electron microscopy, and nitrogen porosimetry. It was found that carbon nanofibers of different diameters were synthesized in the bulk of a silica matrix only at Ni contents higher than 1–1.5%. The CNFs-in-silica supports were nanoporous: the mean pore diameter and the specific surface area were ∼10 nm and 250–300 m2/g, respectively. The heterogeneous biocatalysts prepared by the adsorption of thermostable lipase on the CNTs and CNFs-in-silica supports were investigated in the reaction of triglyceride (tributyrine) hydrolysis; the physicochemical properties of biocatalysts and their enzymatic activity and stability were studied depending on the hydrophobicity-hydrophilicity of the support/matrix.

Heterogeneous biocatalytic processes of vegetable oil interesterification to biodiesel

The heterogeneous biocatalytic interesterification of vegetable oils to ethyl esters of fatty acids is studied. Interesterification with ethanol or ethyl acetate is performed using biocatalysts obtained by incorporating cell lysates of the rE. coli/lip recombinant strain that produces thermostable Thermomyces lanuginosus lipase, into silica xerogel. The interesterification of vegetable oils is performed in both a batch stirred-tank reactor and a continuous reactor with a fixed bed of the prepared biocatalyst. It is shown that ethyl acetate is the best acylating reagent for the interesterification of vegetable oil triglycerides to ethyl esters of fatty acids. Unlike ethanol, ethyl acetate does not inactivate the biocatalyst irreversibly. Under the investigated conditions, the biocatalyst’s half-inactivation time was 720 hours at 40°C.

Preparation and characterization of supports with a synthesized layer of catalytic filamentous carbon: III. Synthesis of carbon nanofibers on nickel supported onto aluminum oxide

The synthesis of catalytic filamentous carbon (CFC) on catalysts prepared by supporting Ni2+ compounds onto the surface of various alumina modifications (macroporous α-Al2O3 and mesoporous ϑ-Al2O3 and δ-Al2O3) using two procedures (impregnation and homogeneous precipitation) was studied. The texture characteristics (specific surface area and pore structure) of the parent supports and adsorbents with a CFC layer were compared. The effect of the supporting procedure on the surface morphology of Ni/Al2O3 catalysts and the synthesized CFC layer was studied by scanning electron microscopy. It was found that the carbon yield on a macroporous catalyst prepared by homogeneous precipitation was higher than that on a catalyst prepared by impregnation by a factor of ∼2. The CFC layer exhibited a mesoporous structure because of a chaotic interlacing of carbon nanofibers, and the synthesis of CFC on macroporous supports resulted in the formation of a bidisperse pore structure of the adsorbent. Active and stable heterogeneous biocatalysts were prepared by the adsorptive immobilization of enzymatically active substances (glucoamylase and nongrowing baker’s yeast cells) on CFC.

Immobilized glucoamylase: A biocatalyst of dextrin hydrolysis

Heterogeneous biocatalysts of starch saccharification based on glucoamylase and carbon-containing carriers were obtained, and their biocatalytic properties in the enzymatic hydrolysis of corn dextrins were studied. It was shown that the morphology of the surface carbon layer of carriers markedly affected the properties of biocatalysts. Glucoamylase immobilized by adsorption on the surface of carriers covered with a layer of catalytic filamentous or pyrolytic carbon had the maximum enzymatic activity and stability, whereas biocatalysts prepared on the basis of carriers that had no carbon layer or were covered with graphite-like surface carbon had a low activity and stability.

Carbon-silica composite matrices for preparing heterogeneous biocatalysts with glucose isomerase activity

Comparative studies have been carried out on the preparation of carbon-silica composite matrices for heterogeneous biocatalysts with glucose isomerase activity. Carbon nanotubes and nanofibers have been included inside SiO2 xerogel. The enzymatic activity and operating stability of the biocatalysts have been investigated. Bacterial cells of a recombinant glucose isomerase producer strain have been used as the enzymatically active component of the biocatalysts. The steady-state activity of the biocatalysts subjected to “dry” cross linking with a glutaraldehyde solution (0.1–1%) is 1.5 times higher than the activity of the biocatalysts containing no nanocarbon. The initial and steady-state glucose-isomerase activities of the biocatalysts at 70°C are ∼520–540 and ∼150–160 μmol min−1 g−1, respectively. The half-inactivation time of the biocatalysts under continuous monosaccharide (glucose, fructose) isomerization conditions is up to ∼1500 h.

Catalytic properties of lipase adsorbed on nanocarbon-containing mesoporous silica in esterification and transesterification reactions

Nanocarbon-containing mesoporous silica covered with a varying amounts of nanostructured carbon of different morphologies were used as supports to immobilize Thermomyces lanuginosus lipase. The catalytic properties of the prepared biocatalysts were studied in both the transesterification of vegetable (linseed) oil in the presence of ethyl acetate and the esterification of the fatty acid (capric C10:0) in the presence of secondary (isopropyl or isoamyl) alcohols. The physico-chemical characteristics, such as the amount of adsorbed lipase, its specific activity, and the dependence of the activity and stability of the prepared biocatalysts on the support type were evaluated. The Michaelis-Menten kinetics was studied in the esterification of capric acid with isoamyl alcohol. The prepared biocatalysts were shown to retain up to 90% activity for >1000 h in the synthesis of isoamyl caprate. The half-time of the biocatalysts inactivation in the transesterification of linseed oil was found to be more than 700 h at 40°C.