ENZYMATIC CATALYSIS BY PERMEABILIZED CELLS

ENZYMATIC CATALYSIS BY PERMEABILIZED CELLS

K. Q. Wilberg, T. L. M. Alves

PEQ/COPPE - Universidade Federal de Rio de Janeiro - Centro de Tecnologia

Ilha do Fundão - bloco G - C. P. 68502 - CEP 21945-970 - Rio de Janeiro, RJ – Brazil

(Received: June 11, 1997; Accepted: October 30, 1997)

Abstract - This paper presents an enzymatic process for sorbitol and gluconic acid production using cells of Zymomonas mobilis permeabilized with CTAB. Equimolar solutions of glucose and fructose (from 96.0 to 422.2 g/L) were used. In a batch reactor, conversions of 97% were attained after 15 to 20 hours of reaction. The effect of the initial concentration of the substrates was evaluated in experiments using 20% more and 20% less glucose than fructose. It was observed that the reaction performed with more fructose reached completion faster and with a higher value of conversion.

Keywords: Sorbitol, gluconic acid, Zymomonas mobilis, permeabilized cells, glucose-fructose oxidoreductase.

INTRODUCTION

The conventional process of sorbitol production consists of high pressure hydrogenation of dextrose syrup, while gluconic acid, or its salts, is produced by a process of fermentation of glucose solutions using Acetobacter suboxydans or Aspergillus niger. Alternatively, these two compounds can be synthesized by an enzymatic process catalyzed by the enzymes glucose-fructose oxidoredutase (GFOR) and gluconolactonase (GL) of Zymomonas mobilis (Zachariou and Scopes, 1986). GFOR reduces fructose to sorbitol and oxidizes glucose to gluconolactone using the NADP(H) cofactor as an electron transfer agent. This cofactor is tightly bound to the active site of the enzyme and is regenerated by the enzymatic reaction itself. In a sequential reaction, the gluconolactone is hydrolyzed to gluconic acid by the action of GL (2). The enzymatic system described is exclusive to the bacterium Zymomonas mobilis (Zachariou and Scopes, 1986) and may be used either as isolated enzymes or in permeabilized cells.

When cells are permeabilized with an appropriate detergent, their cytoplasmatic membrane becomes highly permeable, allowing small compounds such as metallic ions and cofactors to diffuse out of the cells while keeping the macromolecules inside. The enzymes retained by the cellular wall may be easily accessed by substrates (Chun and Rogers, 1988). As the NADP(H) cofactor of GFOR remains bound to the enzyme even after the permeabilization process, sorbitol and gluconic acid production is performed without the interference of parallel metabolic routes (Chun and Rogers, 1988).

The kinetic mechanism which describes a normal conversion of substrates is illustrated in Figure 1, which shows sorbitol accumulation in the medium and gluconate conversion to ethanol by the Entner-Doudoroff pathway. Using efficiently permeabilized cells, the route for ethanol production is blocked and both sorbitol and gluconate accumulate in the medium.

The present paper describes a batch process for sorbitol and gluconic acid production using cells of Zymomonas mobilis permeabilized with Cetyltrimethylammonium bromide (CTAB). The kinetics of the reaction is investigated for different equimolar solutions, as well as for different initial concentrations of each substrate.

EXPERIMENTAL

Microorganism and Growth Conditions

All experiments were performed with Zymomonas mobilis CP4 (ATCC 31821) grown at 30^oC in a medium containing 100 g/L glucose, 1 g/L (NH₄)₂SO₄, 1 g/L MgSO₄.7H₂O and 5 g/L yeast extract (Chun and Rogers, 1988). No phosphate salts were added in order to minimize the levels of phosphorylated intermediates in the cells that are necessary for ethanol production (Chun and Rogers, 1988).

Cell Permeabilization

Approximately 1g of cells collected at the late exponential phase were separated by centrifugation. A permeabilized solution prepared with CTAB 0.2% (w/v) in citrate buffer (pH = 6.2) was added to the cells, in a proportion of 0.033 to 0.05g of CTAB per gram of dried cells (Rehr et al., 1991). The suspension was homogenized and placed in a shaker at 300 rpm for 30 min (Gowda et al., 1988). The cells were centrifuged, washed with citrate buffer (pH = 6.2) and water (pH adjusted to 6.2) and resuspended in water (pH = 6.2).

Kinetic Studies

Kinetic studies were carried out using different concentrations of glucose and fructose mixtures and free permeabilized cells. Reactions were performed in a pH-Stat titrator (Mettler DL 21) with a working volume of 50mL. A temperature of 39^oC and pH of 6.2, optimal for the activity of GFOR (Zachariou and Scopes, 1986), were held constant by the use of a thermostatic bath and the controlled addition of a sodium hydroxide solution, respectively. This solution, of known concentration, was used to neutralize the gluconic acid.

Analytical Methods

Concentration of cells was determined as dry weight by measuring the absorvance of the culture medium at 340 nm with a spectrophotometer Shimadzu UV-2201. Glucose and fructose concentrations were determined as total reduction sugars by the Somogyi colorimetric method (Somogyi, 1951) with the same spectrophotometer. Sodium gluconate concentration was determined by HPLC (Waters Assoc.) using a Shodex S-801 column at room temperature. The mobile phase was water at a flow rate of 0.8 mL/min. Gluconate was detected by the differential refractometer CG 410.

Since the product formation reaction is equimolar, it is possible to determine the concentrations of all components during the batch by monitoring just one of them or from the amount of sodium hydroxide added.

RESULTS AND DISCUSSION

Figures 2a and 2b show the effect of permeabilization on Zymomonas mobilis cells. Photographs of cells, taken by transmission electron microscopy, before (Figure 2a) and after (Figure 2b) permeabilization show the distinction between the cytoplasmatic contents caused by intracellular compound leakage.

Figure 1: Mechanism of sorbitol and gluconic acid production by the GFOR and GL enzymes and gluconic acid conversion to ethanol by the Entner-Doudoroff pathway.

Figure 2a: Electron transmission microphotograph of Zymomonas mobilis bacterium before permeabilization.

Figure 2b: Electron transmission microphotograph of Zymomonas mobilis bacterium after permeabilization.

Kinetic studies were carried out at various initial substrate concentrations. The experimental results were calculated by using the sodium gluconate concentration measured by HPLC, as well as by adding NaOH. Owing to volume variation in the reactional system and mass loss by aliquot withdrawal, results were corrected and expressed in number of mols. Reactions were performed in equimolar solutions of glucose and fructose with concentration varying from 96.0 to 422.2 g/L of each sugar. The results of these experiments are presented in Figure 3.

Values of final conversion and specific rate for the first two hours of the preceding experiments are presented in Table 1. Initial cell concentration was measured for each experiment in order to compare the specific product formation rates for different experiments.

It is interesting to note that the values calculated by the amount of NaOH added were higher than the ones obtained by HPLC, indicating that gluconate is being consumed during the process, specially after 15 hours (see Figure 3). This observation suggests that not all the cells were properly permeabilized. As may be seen in Figure 2b, some cells present the same cytoplasmatic shade as the unpermeabilized ones.

Knowing that GFOR has higher affinity for glucose than for fructose (Zachariou and Scopes, 1986), experiments were performed in order to develop a kinetic model of this process. Initial substrate concentrations of 20% more and 20% less glucose than fructose were used in two preliminary tests. Table 2 presents the experimental conditions together with the results of the specific rates for the first two hours and final conversion, both calculated by NaOH consumption.

Figure 3: Sodium gluconate production using equimolar solutions of substrates. Results in terms of number of mols of sodium gluconate measured by HPLC (symbols) and of NaOH added (+ and line). The values given are the initial concentrations of each substrate and cells for each experiment.

Experiment Specific rate (g gluconate/(g cell.h)) Conversion (%) calculated by NaOH calculated by HPLC calculated by NaOH calculated by HPLC* 96.00 g/L 1.50 1.39 97.6 100.0 202.95 g/L 1.22 1.26 97.4 96.3 260.97 g/L 2.18 1.88 97.4 92.0 422.21 g/L 3.54 3.14 98.8 92.9

Experiment	Specific rate (g gluconate/(g cell.h))		Conversion (%)
	calculated by NaOH	calculated by HPLC	calculated by NaOH	calculated by HPLC*
96.00 g/L	1.50	1.39	97.6	100.0
202.95 g/L	1.22	1.26	97.4	96.3
260.97 g/L	2.18	1.88	97.4	92.0
422.21 g/L	3.54	3.14	98.8	92.9

Table 1: Values of specific rate and conversion calculated by the amount of NaOH added and by the HPLC measurement

* Using the highest value of concentration measured

Experiment Specific rate calculated by NaOH (g gluconate/(g cell . h)) Conversion calculated by NaOH (%) 1) F0 = 1.2.G0 G0 = 100.0 g/L 0.835 100.0 2) G0 = 1.2.F0 F0 = 98.4 g/L 0.886 93.3

Experiment	Specific rate calculated by NaOH (g gluconate/(g cell . h))	Conversion calculated by NaOH (%)
1) F0 = 1.2.G0 G0 = 100.0 g/L	0.835	100.0
2) G0 = 1.2.F0 F0 = 98.4 g/L	0.886	93.3

Table 2: Effect of initial concentrations of each substrate on specific rate and conversion of reaction

Figure 4: Sodium gluconate production using different initial concentrations of each substrate. Results in terms of number of mols of sodium gluconate measured by HPLC (symbols) and of NaOH added (+ and line). The values given are the initial concentrations of each substrate and cells for each experiment.

Conversions were calculated with respect to the limiting substrate of each experiment, i. e., in the first case glucose and in the second case fructose. Product formation results are presented in Figure 4.

Despite the fact that the curve slope in the first case is greater than it is in the second, specific rates of product formation were very similar in both tests because of the difference in cell concentrations (see Table 2). It can be seen in Figure 4 that the rate of the second test is significantly reduced after five hours of reaction, which explains the lower conversion attained. Since reaction is equimolar, the results show that an excess of fructose promotes higher conversion of substrates due to the greater affinity of GFOR for glucose than for fructose (Zachariou and Scopes, 1986).

CONCLUSIONS

Even for lower concentrations of cells, the conversion values obtained in this work are higher than those reported in the literature, where toluene was used as the permeabilizing agent (Chun and Rogers, 1988). Although CTAB was shown to be a better agent for permeabilization, gluconate consumption and microphotographs indicate that the permeabilization procedure has yet to be improved.

It can be concluded that the enzymatic reaction has not yet reached the saturating concentrations of the substrates. Experiments are still being conducted to establish this operational condition and to develop a kinetic model of the process.

The possibility of cell reutilization, the facility in acquiring and maintaining the enzymatic system of interest and the high conversions which can be obtained make the process performed with permeabilized cells a very attractive alternative for the synthesis of high-value products from cheap and abundant raw materials such as sucrose.

ACKNOWLEDGMENT

The financial support received from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPQ) is gratefully acknowledged.

NOMENCLATURE

CTAB Cetyltrimetilammonium-bromide

F0 Initial concentration of fructose, g/L

G0 Initial concentration of glucose, g/L

GFOR Glucose-fructose oxidoreductase

GL Gluconolactonase

X0 Initial concentration of cells, g/L

• Chun, U.H. and Rogers, P.L., The Simultaneous Production of Sorbitol from Fructose and Gluconic Acid from Glucose Using an Oxidoreductase of Zymomonas mobilis Appl. Microbiol. Biotechnol., 29, p. 19 (1988).
• Zachariou, M. and Scopes, R.K., Glucose-Fructose Oxidoreductase, a New Enzyme Isolated from Zymomonas mobilis that is Responsible for Sorbitol Production. J. Bacteriol., 167, No. 3, p. 863 (1986).
• Rehr, B.; Wilhelm, C. and Sahm, H., Production of Sorbitol and Gluconic Acid by Permeabilized Cells of Zymomonas mobilis Appl. Microbiol. Biotechnol., 35, p. 144 (1991).
• Gowda, L.R.; Joshi, M.S. and Bhat, S.G., In situ Assay of Intracellular Enzymes of Yeast (Kluyveromyces fragilis) by Digitonin Permeabilization of Cell Membrane. Anal. Biochem., 175, p. 531 (1988).
• Somogyi, M., Notes on Sugar Determination. J. Biol. Chem., 195, p. 19 (1951).

*

and R. Nobrega

*

To whom correspondence should be addressed.

Publication Dates

• Publication in this collection
  06 Oct 1998
• Date of issue
  Dec 1997

History

• Accepted
  30 Oct 1997
• Received
  11 June 1997