ISSN 00036838, Applied Biochemistry and Microbiology, 2010, Vol. 46, No. 7, pp. 697–705. © Pleiades Publishing, Inc., 2010. Original Russian Text © G.A. Kovalenko, L.V. Perminova, E.I. Chernyak, L.I. Sapunova, 2009, published in Biotekhnologiya, 2009, No. 5, pp. 63–73.
Investigation on Macrokinetics of Heterogeneous Process of Monosaccharide Isomerization using NonGrowing Cells of a Glucoisomerase Producer Arthrobacter nicotianae Immobilized inside SiO2Xerogel G. A. Kovalenkoa, b, L. V. Perminovaa, E. I. Chernyakc, and L. I. Sapunovad a
Institute of Catalysis, Russian Academy of Sciences, Siberian Branch, Novosibirsk, 630090 Russia email:
[email protected] b Novosibirsk State University, Novosibirsk, 630090 Russia c Institute of Organic Chemistry, Russian Academy of Sciences, Siberian Branch, Novosibirsk, 630090 Russia d Institute of Microbiology, National Academy of Sciences of Belarus, Minsk, 220141 Belarus Received September 2, 2008
Abstract—Macrokinetic peculiarites of heterogeneous process of monosaccharide (glucose/fructose) isomerization using biocatalysts prepared by incorporation of nongrowing cells of a glucose isomerasepro ducing strain Arthobacter nicotianae inside SiO2xerogel have been investigated. It was shown that the process proceeds in kinetic regime without diffusion limitation and biocatalyst activities at 60 and 75°C were 19 and 32 U/g, respectively. Time of equilibrium in the reaction of monosaccharide isomerization was a function of starting (“triggering”) glucose isomerase activity in a unit of reaction volume. When the activity exceeds 10 U/ml, equilibrium equimolar mixture of glucose and fructose was produced within a few hours. It was established that a continuous process carried out in a plugflow packedbed reactor is more efficient than a batch process accompanied with recycling, first of all, to significant improvement of operation stability of the designed biocatalysts. Under model conditions of industrial heterogeneous process of producing glucose fructose syrups, the halflife time of inactivation of the biocatalysts was more than 500 h at (65 ± 5)°C. Key words: Arthobacter nicotianae cells immobilized inside SiO2xerogel, glucose/fructose isomerization, a plugflow packedbed reactor DOI: 10.1134/S0003683810070045
Industrial technology of the manufacture of glu cosefructose syrups (GFS), starting from hydrolysis of the starch of renewable resources (corn, wheat, potato) and ending with the production of commercial products (treacle, syrups) with the content of dry sub stances at 71%, are described enough in the modern scientific–technical literature [1–4]. Heterogeneous biocatalysts for conducting the final step, isomeriza tion of glucose syrup, their properties and methods to the preparation are given in review literature [1–5]. The structure and mechanism of active component of heterogeneous biocatalysts, glucose isomerase enzyme, are studied in detail [5–7]. Let us focus on some features of the of process of glucose isomeriza tion into GFS. It is known that the enzyme reaction of glucose isomerization is equilibrium and the rate of glucose isomerization is equal to the rate of fructose isomer ization. The product of the reaction is the equimolar mixture of glucose and fructose, close (within the error of analysis) to the following content: 50% fructose and 50% glucose. In the case when glucose–fructose syrup
is a commercial product, corresponding technological requirements apply to it. For the first time these requirements were developed by Cargill Co., and the composition of commercial GFS42 (Isoclear 42 trade mark) satisfied the following requirements: not less than 42% fructose, 52% glucose, and 6% oligosac charides [8]. However, for their use in soft drinks, such as Coca and PepsiCola, with low value of pH, syrups with a higher content of fructose are required. As a result, next GFS55 standard on the composition of syrups was developed: not less than 55% fructose, 40% glucose, and 5% other. In this case, the sweetness of such syrups completely corresponds to sugar syrup of the same concentration, and the cost is at 10–20% lower than that of sugar, since, as was mentioned above, the raw material of its production is starch sus pension, obtained from plant vegetable renewable resources. As a rule, GFS55 is prepared by enriching the GFS42 with fructose by chromatography. The interesting solution of the given problem is suggested in work [9], where the reactor, called a simulated mov ing bed reactor (SMBR), is described, with the selec
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tion of reagent streams, in which reaction and separa tion of isomers (glucose and fructose) by adsorption on cationexchange membrane aare combined in a technological chain, which leads to a little shift of equilibrium of the reaction and enriching of syrups with fructose. Numerous scientific publications of recent years are devoted to the rational design of glucose isomerase enzyme (GI) by the search or selection of correspond ing “wild” strains of microorganisms [10, 11] or prep aration of recombinant strainproducers [12]. The purpose of the given investigations is primarily the increase of the thermostability of an enzyme and bio catalysts, prepared on its basis and active at 85–90°C, since it is known that at given temperatures the equi librium of the reaction shifts to the formation of fructose and it becomes possible to prepare syrups with a higher content of desired sugar (higher than 50%) [10]. The other approach in the change of the properties of glucose isomerase is the shift of pHoptimum to the acidic range, closer to 5–6. The use of acidresistant glucose isomerase would allow to combine the steps of starch saccharafication by glucoamylase (pH ~5–6) and isomerization by GI glucoamylase (pH ~7–8) and eliminate the intermediate steps of adjusting pH by addition of corresponding reagents (alkali). The syr ups, obtained by onestep technology, contain ~50% fructose and less than 1.5% of unknown oligosaccha rides [11]. Since GI is mainly an intracellular enzyme and a multiplestep procedure is required for its isolation and purification, immobilization is economically the most reasonable way for manufacture of the industrial biocatalysts. This makes it possible to use the enzyme multiple times, substantially reducing its expense. In recent years in the world, nearly 1500 t of biocatalysts with glucose isomerase activity were produced for the preparation of glucose–fructose syrups, the net pro duction of which is more than 10 million t [1]. On the market of biocatalysts five main companies are pre sented (table). For the preparation of heterogeneous biocatalysts isolated enzyme (CPC, GENENCOR, and UOP), homogenates of cells (NOVOZYMES), and whole cells (NAGASE) are used. The immobilization is per formed by adsorption and/or crosslinking by glutaric dialdehyde. From the data in the table, it is seen that biocatalysts, obtained by immobilization of isolated enzyme, is only by 1.4–4.0 times more active than bio catalysts, prepared on the basis of less active (by orders) nongrowing cells of microorganisms (see table), that is determined, probably, by the presence of diffusion restrictions. Despite the fact that the manu facture of GFS is the most largescale commercial heterogeneous biocatalytic process, the investigations on the development of novel effective biocatalysts are still carried out quite intensively at the present time. In scientific works published after 2000, novel polymer
and composite materials for the immobilization of glucose isomerase, are investigated, for instance, n trimethylaminopolystyrene particles [13], silk fibroin modified by glutaric dialdehyde [14], chitosane [15], polyacrylamide + carragenane/alginate [16], and phosphorylated polyvinyl alcohol + alginate [17]. For the industrial introduction of newly developed biocatalyst having commercial attractiveness, it is required, that the time of its halfinactivation under the conditions of isomerization of 40–47 wt% glucose syrup at 55–60°C was 80–150 days (1900–3600 h) and the total productivity was 12–20 t of GFS (by the dry substance) per 1 kg of catalyst [1]. In industry the process of production of GFS is performed in a column reactor of 1.5 m diameter and the height of 5 m with the updown flow of substrate solution at the rate of 1.4–6 volumes of biocatalyst bed per 1 h (see table [1]). This reactor is plugflow packedbed one, and major part of papers published after 2000, is devoted to the mathematical simulating of this type of reactor [18–20]. In design of reactors of new generations, the main attention is focused on the intensification of mass transfer of the substrate (liquid phase) to immobilized enzyme (solid) in order to over come diffusion limitations, and also on the elimina tion of stagnant zones and jet streams inside biocata lyst bed. Interesting by the principle of work are the vortex reactors, in particular the tangential flow two impinging streams reactor(TFTISR) [21–23], and Taylor–Poiseuille vortex flow reactor (VFR) [24, 25]. Vortex reactors (immersed and rotaryinertial) were developed by the authors of this article specially for diffusioncontrolled processes with the participation of heterogeneous biocatalysts, prepared on the basis of immobilized enzymes (in particular, glucoamylase) [26–28]. For the glucose isomerization, hollow fiber reactors with recirculation [29, 30] and down flow jet loop reactor (DJR) [31] are designed. In the works [32, 33], we described the screening of 85 strains of microorganisms, including those isolated from natural sources (soil), producing glucose isomerase. For the most active Arthrobacter nicotianae strainproducer of glucose isomerase, the biocatalytic properties were studied both in suspension and an immobilized state [33, 34]. It was discovered, that A. nicotianae have a weak ability to adhere on the sur face of the solid inorganic carriers; therefore, for the preparation of heterogeneous biocatalysts on the basis of A. nicotianae, a novel approach of immobilization was developed by the inclusion of microbial biomass into the silicate matrix, silica xerogel. The purpose of this work was to investigate the macrokinetics of isomerization of monosaccharides (glucose/fructose) with the participation of heteroge neous biocatalysts, prepared by the inclusion of non growing cells of the A. nicotianae strainproducer of glucose isomerase to the silica xerogel, and also to study the properties (activity, stability) of the effect of
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Characteristics of commercial heterogeneous biocatalysts used for the isomerization of glucose in GFS [1]
Company–pro Trade mark ducer
Method of immobilization
Initial volume Time of half Activity of inactivation in rate of the sub GI at 60°C, the conditions of strate injection, U/g the reaction the volume of the of biocatalyst layer of catalyst/h (60°C), h
CPC (ENZYME Gzyme BIOSYSTEMS) G994 GENENCOR Int.
NAGASE
NOVOZYMES A/S (NOVO NORDISK) UNIVERSAL OIL PROD UCTS (UOP)
Adsorption of the enzyme from Streptomyces olivochromogenes on the anion–exchange polymer Adsorption of purified enzyme Spezyme from S. ribiginosus on the com plex carrier, which contains DEAEcellulose, polystyrene, and titan dioxide Sweetase The binding of thermopro cessed S. phaeochromogenes cells with anion–exchange resin, granulation Sweetzyme The processing of homogenate S, Q, T obtained after the destruction of S. murinus and Bacillus coagulans cells, GA, extrusion Ketomax Binding of an enzyme from 100 Streptomyces olivochromogenes with GA with ceramics alumi num oxide coated by polyeth yleneimine polycondensate
Produc tion, kg of GFS/kg of biocatalyst
–
–
6
–
500–700
–
3.9
–
160–250
1200
1.4
4000
150–350
1800
2.1
1000–4000
5.2
–
1000
Note: “—” is the absence of the data in literature.
the process operational modes (periodic or continu ous regimes) on conversion of the monosaccharides (fructose). EXPERIMENTAL A. nicotianae strainproducer of glucose isomerase, deposited in the collection of nonpathogenic organ isms in the Institute of Microbiology, National Acad emy of Sciences of Belarus (Minsk), was used. The bacteria were grown under the conditions described in the work [34]. After the end of the cultivation, the microbial biomass was separated by centrifugation (~10000 g, 15 min, the humidity of the biomass was 75–80%) and used for the preparation of heteroge neous biocatalyst with glucose isomerase activity. The specific activity of A. nicotianae cells in suspension at 60 and 75°C (0.02 M phosphate buffer, pH 7.8) was, on average, 150 and 550 U/g of dry cells, respectively. The method of the preparation of the heteroge neous biocatalysts was the following. As a main inor ganic component, silicawas used as hydrogel, which was obtained by the solgel method [35] by the reac tion of sodium silicate (liquid glass) with ammonium nitrate at a temperature of 70°C, pH 7.5, and the rate of precipitation of 300 g SiO2/l/h. The humidity of hydrogel was 80–90%. Biocatalysts were prepared by the thorough mixing of the microbial biomass, silica APPLIED BIOCHEMISTRY AND MICROBIOLOGY
hydrogel and cobalt (II) hydroxocompounds (insolu ble cobalt hydroxocompounds were precipitated from the cobalt nitrate solutions by the ammonia solu tion at 20–22°C, and then washed with distilled water) in the following amounts, wt% of the dry substances: microbial biomass—10–15, SiO2—45–70, and CoxOy—20–40. The obtained humid mixture was dried at a temperature not higher than 50°C. When dry, from hydrogel of silica and biomass, xerogel with included cells of microorganisms was formed. The obtained dry mixture was mechanically grinded, pressed under excess pressure of 150 atm and fraction ated to obtain granules with the size from 0.2 to 4 mm. The maximal activity of GI of prepared biocatalysts at 60 and 75°C was 19 and 32 U/g, respectively. For the comparative investigations, Sweetzyme (Sigma, USA) heterogeneous biocatalyst was used, activity of which was 150 and 350 U/g of biocatalyst at 60 and 75°C, respectively. Measurement of glucose isomerase activity of A. nic otianae suspension and actinobacteria cells, included inside SiO2 xerogel, was performed at 60 and 75°C in the reaction medium, containing 0.02 M phosphate buffer, pH 7.8, and 1 mM Mg2+ as a sulfate. For the determination of activity of prepared heterogeneous biocatalyst, Co2+ ions were not added into the medium. For the determination of activity of Sweet zyme, the concentration of Co2+ was 1 mM in the
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reaction medium. Fructose, glucose, and also GFS of various carbohydrate compositions were used at a con centration of 2–3 M. The content of the product of the enzyme reaction (glucose) in the substrate solution (fructose) was varied from 5 to 30%. The rate of the reaction, equal to 1 μmol/min, corresponded to the unit of enzyme activity (U). The specific activity was expressed in μmol/min/g of dry cells or in μmol/min/g of biocatalyst (U/g), respectively, for the suspended and immobilized cells. Analysis of monosaccharides in the reaction medium was performed by HPLC under the condi tions described in the work [34], and also by the glu cose oxidase method (GO method) [36]. Since the last one is the most rapid and accurate, the test of the activity of obtained biocatalysts was performed using this method, and the experimental error did not exceed 15%. The isomerization reaction of monosaccharides with the participation of suspension of A. nicotianae cells (homogeneous conditions) was performed in a jacketed thermostatic reactor at intensive stirring with a magnetic stirrer at 60 and 75°C in periodical mode. After achievement of stationary state in the reaction medium, bacteria cells were separated by centrifuga tion (~10000 g, 15 min), and then resuspended in the reaction medium and the next reaction cycle was car ried out. Reaction of isomerization of monosaccharides with the participation of prepared biocatalyst in study and Sweetzyme (heterogeneous conditions) was carried out both in periodical and continuous modes in packedbed (1) differential gradientless and (2) plug flow reactors, respectively. The reaction equipment for the periodical mode consisted of a thermostat, a col umn packedbed reactor (by volume 10–15 cm3) with the bed of biocatalyst (by weight 0.1–0.2 g) thermo stated at 60 and 75°C, and a peristaltic pump, provid ing the circulation of the substrate solution through the fixed bed of prepared biocatalyst in study and Sweetzyme biocatalysts with the flow rate of 0.7 and 35 ml/min. The period of one reaction cycle was from 2 to 8 h, after that biocatalysts were washed with dis tilled water and 0.02 M phosphate buffer, pH 7.8. Used for the continuous mode were a column plug flow packedbed reactor filled by the biocatalyst (4.6 g, 10 cm3). Through the bed 45 wt% monosaccha ride solution, containing 1 mM MgSO4 was pumped down by a peristaltic pump with the rate of 0.03 ml/min. For improvement of the hydrodynamic properties of the fixed bed of biocatalyst, inert glass particles with the diameter of 2 mm were introduced inside the bed. The process was carried out at 65 ± 5°C. Periodically (once per day) the samples were taken on the outlet of the reactor and analyzed on the content of glucose and/or fructose. Heterogeneous biocatalytic process was character ized by the following parameters: (1) activity of GI of
biocatalyst, U/g; (2) conversion of substrate (x), cal C0 – Ct , %, where C0 is the culated by the formula x = C0 initial concentration of the monosaccharide, M, and Ct is the current concentration of monosaccharide, M; contact time (τ), equal to τ = volume of catalyst, cm3/flow rate, ml/h; and (4) “triggering” activity, which was determined as observed initial activity of GI in the unit of total volume of the reaction medium (U/ml) in the reactor set up including the joints between all its parts (reactor, mixer, peristaltic pump). RESULTS AND DISCUSSION Under heterogeneous conditions and periodic mode the influence of diffusion on the observed rate of monosaccharides isomerization was examined. The effect of following parameters (1) the flow circulation rate of reaction media through the bed of the biocata lyst (ω) and (2) the size of the granules, on the reaction rate, was studied. The maximal rate of isomerization reaction was observed at ω ~ 0.3–0.7 ml/min, then it reduced and was almost constant in the wide range of the flow rates from 2 to 60 ml/min (Fig. 1a). This phe nomenon is related to the fact that, at the low values of flow rate, the packedbed reactor operated in the mode of ideal displacement; in this case with the increase of the contact time (τ), the substrate conver sion and the observed rate reaction increased. With the increase of the flow rate, the mode of the operation of the reactor obviously changes and, at the high values of ω, the reactor operated for the mode of the ideal mix ing; the observed reaction rate does not depend on the flow rate (see Fig. 1a). From the obtained data, it can be concluded that the diffusion of the substrate (fruc tose) from the solution to the surface of the granule of the biocatalyst (external diffusion) did not limit the isomerization of monosaccharides. With the grinding of the granules, the observed activity of the biocatalyst did not increase substantially (see Fig. 1b). Consequently, the diffusion of the sub strate inside the granules of the biocatalyst (internal diffusion) did not play a substantial role in the isomer ization of monosaccharides with the participation of immobilized A. nicotianae cells. So, biocatalysts, prepared by the inclusion of A. nicotianae biomass inside the silica xerogel, work in the kinetical regime, and the mass transfer of substrate (glucose/fructose) to immobilized cells does not limit the rate of isomerization of monosaccharides. Prepared biocatalyst in study and Sweetzyme bio catalysts were studied in the periodical mode of per forming the isomerization of fructose in circulation setup described above. Upon investigation of kinetics of the isomerization reaction, it was found that there were two parts on the curve of dependence of conver sion on the duration of reaction performed in periodic mode (Fig. 2). Initially the reaction rates were maxi
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(b) Activity of biocatalyst, U/g
Activity of biocatalyst, U/g
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20 40 50 60 30 10 Flow rate of the substrate solution through the stationary layer of biocatalyst, ml/min
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 The size of the grains of biocatalyst, mm
Fig. 1. Dependence of the activity of the prepared biocatalyst from (a) the flow rate of the substrate through the fixed bedof bio catalyst and (b) the diameter of its granules.
(a)
(b) Conversion of substrate, %
Conversion of substrate, %
50 40 30 20
2 1
10
50 2 40 30 1
20 10
4 6 2 The period of the reaction cycle, h
4 6 2 The period of the reaction cycle, h
8
Fig. 2. The dependence of the substrate conversion on the duration of isomerization of fructose (3M) in periodic mode at 75°C with the participation of heterogeneous (a) prepared biocatalyst in study and (b) Sweetzyme biocatalysts at various “triggering” activity: (a) curves 1 and 2 are 6.7 and 11.3 U/ml, respectively; (b) curves 1 and 2 are 4.0 and 22.8 U/ml, respectively.
mal (dashed lines on Fig.2), than the reaction rates became close to zero (plateau on Fig.2). In those cases where “triggering” GI activity was not large (less then 10 U/ml), the reaching the plateau in kinetic curve was longer, and the rate of substrate conversion did not exceed 1 h (see Fig. 2a, curves 1, 2 and Fig. 2b, curve 1). In those cases where “triggering” activity of GI in the reaction medium was high (~20 U/ml), the equilib rium in the reaction of isomerization of monosaccha rides was achieved quite quickly (at 2–4 h) and the equilibrium mixture of glucose and fructose was equimolar (x = 50 ± 3%) (see Fig. 2b, curve 2). Comparative investigations, performed in periodi cal mode under homogeneous conditions with the par ticipation of suspended nongrowing cells, lead to an analogous result. At fructose isomerization, the complete conversion of substrate with the formation of equilibrium equimolar mixture of hydrocarbons (x = 48 ± 2%) was APPLIED BIOCHEMISTRY AND MICROBIOLOGY
observed within 2–4 h in those cases when the initial activity of GI in reaction medium was high (≥20 U/ml) (Fig. 3, curve 2). In those cases when the initial activity of GI in the reaction medium was rela tively low (500 h at 65 ± 5°C. The increase of the stability and activity of GI of newly developed heterogeneous biocatalysts was the subject of further researches. ACKNOWLEDGEMENTS We grateful to I.S. Andreeva and V.M. Lenskaya for qualified help in the area of microbiological investiga tions. This work was supported by Integration Project 4.4 of the Siberian Branch, Russian Academy of Sci ences, and the Foundation for Basic Research, National Academy of Sciences of Belarus, project no. B07CO011. REFERENCES 1. Bucholz, K., Kasche, V., and Bornscheuer, U.T., Bio catalysts and Enzyme Technology, Weinheim: Wiley VCH, 2005. 2. Bommarius, A.S. and Reibel, B.R., Biocatalysis, Wein heim: WileyVCH, 2004. 3. Toshiaki, K., J. Appl. Glycosci., 2003, vol. 50, pp. 55–60.
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