Affinity Immobilization of Escherichia coli: Catalysis by Intact

Transcription

1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 1983, p /83/2384-5$2./ Copyright C 1983, American Society for Microbiology Vol. 45, No. 2 Affinity Immobilization of Escherichia coli: Catalysis by Intact and Permeable Cells Bound to Starch T. FERENCI Department of Microbiology, University of Sydney, New South Wales 26, Australia Received 13 July 1982/Accepted 3 September 1982 The binding of viable Escherichia coli cells to an immobilized ligand of a surface receptor for maltodextrins has recently been demonstrated (T. Ferenci and K. S. Lee, J. Mol. Biol. 16: , 1982). The interaction of bacteria and ligand immobilized in a chromatographic column was investigated over a wide range of applied cell densities, temperatures, eluant ph values, osmotic concentrations, and flow rates. Over 95% retention of bacteria applied to starch-sepharose was found at cell densities up to 19 per ml of matrix, between ph 5.5 and 8., between 8 and 55 C, in the presence of to.5 M NaCl, and at elution flow rates up to 37 column volumes per h. The catalytic capability and stability of affinity-immobilized cells was demonstrated with the cytoplasmic 3-galactosidase activity of starch-bound cells. Intact immobilized bacteria exhibited slowly increasing 1Bgalactosidase activity over several days with a plateau after 6 days. Bacteria made permeable by treatment with toluene were also bound to starch-sepharose but showed maximum 1-galactosidase activity within 1 day and exhibited no loss of enzyme activity in 8 days of continuous elution at ambient temperatures. Immobilized bacterial cells have a number of well-established applications as catalysts and have considerable potential in other areas of biotechnology (review in ref. 2). The best-studied means of immobilization involve entrapment of cells in a semipermeable polymeric matrix (2). An alternative means of bacterial immobilization that has not been investigated is the specific binding of cells onto matrices that contain a ligand recognized by bacterial surface receptors, referred to here as affinity immobilization. The potential advantage of such an approach is that the bacteria so bound are readily accessible to the medium surrounding the matrix. With entrapped cells, substrates and products have to gain access through polymeric gels surrounding the bacteria. Such bacteria would be particularly poor when applied to the production or conversion of extracellular macromolecules such as polysaccharides or proteins. Gram-negative bacteria like Escherichia coli have several surface proteins that have specific binding sites for extracellular ligands (6). One of these surface proteins is the lambda receptor, which is physiologically involved in the transport of maltodextrins across the outer membrane and has a binding site for linear oa-1-4 linked glucans (5). This receptor is responsible for the ability of E. coli cells to bind starch covalently linked to a Sepharose matrix, which in turn has permitted the affinity-chromatographic separation of E. coli populations that differ in surface-receptor characteristics (4). This study presents data on the factors affecting the interaction of bacteria with immobilized starch and on the feasibility of using affinityimmobilized bacteria as catalysts. As indicated below, the stable immobilization of E. coli through binding to starch is possible under a wide range of conditions. The catalytic capability of affinity-immobilized cells was tested by means of the,b-galactosidase activity of bound bacteria. Other means of immobilizing,b-galactosidase have been tested previously with purified enzymes and immobilized cells for the hydrolysis of lactose in milk or whey (11). MATERUILS AND METHODS Materials. Starch covalently coupled to oxiraneactivated Sepharose 6B (Pharmacia Fine Chemicals) was synthesized as previously described (4). The matrix used in these experiments contained 6 to 8 mg of starch in a 1-ml packed volume gel. IPTG (isopropyl P-D-thiogalactopyranoside) and ONPG (o-nitrophenyl-,-d-galactopyranoside) were from Sigma Chemical Co. The columns used in all experiments were Bio- Rad Laboratories polypropylene Econo-columns with 7-,um polyethylene bed supports; these columns do not retain bacteria (4). Bacterial strains and culture conditions. A K-12 strain of E. coli, HFr G6 (13), was used in the reported studies. In all cases when the binding of bacteria to starch-sepharose was measured, the bacteria were 384

2 VOL. 45, 1983 AFFINITY IMMOBILIZATION OF E. COLI 385 grown in the presence of.2% maltose on media previously described (13). This was necessary to achieve full induction of the lambda receptors (1). When cells were immobilized to assay for B-galactosidase activity, the growth media also included.2 mm IPTG to induce the enzyme (8). Cells were toluene treated as previously described (3). In all experiments reported, 1 ml of starch-sepharose was packed in columns and used to test the retention of bacteria. The columns and matrix were presterilized by elution with 1 column volumes of.1 M NaOH and then were equilibrated with minimal medium A (MMA, 8). The bacteria grown under the appropriate conditions were harvested through centrifugation and washed with MMA. Except in experiments where the applied cell density was varied, cells were applied in.1 ml of a suspension of 11 bacteria per ml of MMA. The numbers of bacteria eluted under various conditions were estimated by viable counts when low numbers of bacteria were assayed or were estimated routinely through turbidometric measurements when 19 bacteria were applied. There was no significant difference in absorbance at 58 nm between toluene-treated and untreated bacteria. In experiments involving elution over extended periods, elution buffers contained.2% (wt/vol) sodium azide to prevent growth or gross contamination in the column. RESULTS Immobilization of bacteria through starch binding. For affinity binding to be a useful immobilization technique, the retention of large numbers of bacteria in a small volume of matrix is necessary. E. coli cells were efficiently retained by columns loaded with over 19 bacteria per ml of matrix (Table 1). More than 95% of applied bacteria were bound at these densities. As previously demonstrated, this retention is starch dependent (4). At higher applied cell densities, even higher numbers were retained, although not necessarily in a starch-dependent manner. With 11 or more cells applied to a column, a thick layer of bacteria formed on top TABLE 1. Retention of E. coli cells at increasing applied densities' Bacteria Bacteria retained applied No. % 2.3 x x x x x x X x x x x x a Columns of starch-sepharose containing 1 ml of packed volume gel were equilibrated with MMA and loaded with.1 ml of suspension containing the given number of E. coli cells. The columns were eluted with 3 ml of MMA, and the numbers of bacteria eluted was determined. These experiments were performed at 22"C and at a flow rate of approximately 13 ml/h. TABLE 2. Effect of continuous elution on the retention of E. coli by starch-sepharosea Vol eluted Bacteria eluted (ml) (x16) a A column was prepared as described in Table 1 footnote a and was loaded with 3 x 19 bacteria in.1 ml of MMA. The column was continuously eluted with MMA at a flow rate of 1 ml/h at ambient temperatures. Fractions were taken, and the numbers of bacteria eluted were determined by plate counts. of the matrix and could not be recovered by eluting the column with soluble starch; bacteria bound to starch-sepharose in a receptor-dependent manner are eluted by soluble ligands of the lambda receptor (4). Presumably, cell-cell interactions are more likely than cell-ligand interactions at very high cell densities. Stability of bacterial binding to starch-sepharose. The stability of interaction between E. coli and starch-sepharose was investigated by subjecting columns containing immobilized bacteria to continuous elution. After the initial application of bacteria, when approximately 3 to 5% of bacteria passed through unadsorbed (Table 1), the washout of bacteria was followed by continuing the elution with 2 column volumes of MMA under nongrowth conditions. The numbers of bacteria eluted per column volume of eluant settled to approximately.1% of bacteria present in the column (Table 2). In total, 1.1% of retained bacteria were lost after elution with 2 column volumes of MMA. In considering the effect of flow rate on the loss of bacteria, the numbers of bacteria washed out were investigated at flow rates between 2.3 and 37 column volumes per h. Even at the fastest rate of elution tested, only 6% of applied bacteria eluted at the void volume of the column. At the highest rates, column stability and not bacterial elution became the limiting factor. Effect of ph and ionic strength on rentention by starch-sepharose. The binding of E. coli to columns of starch-sepharose was investigated in the ph range 4 to 9.5 (Fig. 1). Optimum retention was obtained in columns equilibrated and eluted at ph 6 to 8. Poorest retention was found at ph above 9, although considerable numbers

3 386 FERENCI 1 6 ai ph FIG. 1. Effect of ph on the binding of E. coli to starch-sepharose. Columns containing 1 ml of matrix were equilibrated by elution buffers at the appropriate ph containing.1 M phthalate (),.1 M phosphate (A), and.1 M Tris (E). Bacterial suspensions (2 x 19 bacteria) in the same buffers were applied and eluted with 3 ml of buffer at the same ph. The bacteria eluted were estimated turbidometrically. of bacteria were retained even at these ph values. Tris, phosphate, and phthalate buffers gave similar retention at equivalent ph values. Receptor-dependent binding was also followed in columns equilibrated with distilled water, 1-fold diluted MMA, and MMA supplemented with.1 M,.25 M, and.5 M NaCl. Under all these conditions, with 19 bacteria applied, less than 5% of the bacteria eluted at the void volume of the column with the given eluant. Hence, ionic composition had little effect on retention by starch. Effect of temperature on retention by starch- Sepharose. Columns submerged in a water bath equilibrated at to 65'C were loaded with approximately 19 bacteria and eluted with MMA at a flow rate of 1 ml/h. Retention by starch- Sepharose gave a broad temperature optimum and was over 95% effective between 8 and 55 C (Table 3). Even at 65 C, close to 95% of bacteria were initially retained, and continued elution with buffer at this temperature did not result in increased rates of washout over ambient temperatures (data not shown). Catalysis by affinity-immobilized E. cohl. The 3-galactosidase activity of bacteria bound to starch-sepharose was followed by continuous elution of columns with ONPG as the substrate and by photometric monitoring of the eluate for nitrophenol (8). When viable cells were immobilized and eluted with MMA containing 5 mm ONPG, the hydrolysis of this substrate could be immediately detected. However, the extent of hydrolysis increased over a number of days until it corresponded to near-total conversion of the APPL. ENVIRON. MICROBIOL. substrate (Fig. 2). This increase in activity was not due to the growth of E. coli or contaminants since these experiments were conducted with a buffer containing.2% sodium azide, and indeed, few viable cells were found in the column after 8 days of elution under these conditions. More likely, the initial rise in P-galactosidase activity was due to the increased permeability of cells, allowing increased access of substrate to enzyme; it is known that the rate of hydrolysis of ONPG by intact bacteria is limited by the rate of permeation into the cells (7). Consistent with this idea were the results of experiments with cells made permeable by prior treatment with toluene. Such cells after treatment exhibited under.1% viability but were as efficiently retained by starch-sepharose as viable cells. Toluene-treated cells also showed approximately 95% retention when applied to starch-sepharose, as determined from turbidity measurements of the eluates. When ONPG was passed through a column of toluene-treated cells, the rate of hydrolysis reached the maximum level without showing the slow rate of increase exhibited by viable cells (Fig. 2). The maximum extent of hydrolysis was close to that found for untreated cells after 5 days and corresponded to a rate of 25,umol of ONPG hydrolyzed per h per 19 cells. Since near-total conversion of substrate occurred under the conditions of Fig. 2, it was investigated whether even faster rates of ONPG hydrolysis could be observed at higher flow rates and temperatures. At an elution rate of 17 ml/h, increased rates of,b-galactosidase activity were found at temperatures up to 45 C; rates were up to 81,umol of ONPG hydrolyzed per h per 19 bacteria (Fig. 3). Continuous elution at temperatures above 45 C was not possible since the P-galactosidase activity was not stable; there was no drop in activity after 15 h of continuous elution at 45'C, whereas there was a 5% drop in activity in 18 h at 47 C and in 3.6 h at 5 C. Since there is no loss of retention by starch-sepharose TABLE 3. Effect of temperature on the retention of E. coli by starch-sepharosea Temp Bacteria retained (OC) (%) a Columns of starch-sepharose containing 1 ml of matrix were equilibrated by elution with MMA at the given temperatures. The columns were loaded with.1 ml of E. coli suspension containing 2.4 x 19 bacteria and were eluted with MMA at a flow rate of 1 ml/h at the given temperatures. The numbers of bacteria eluted in 2 ml of MMA were estimated turbidometrically.

4 VOL. 45, 1983 AFFINITY IMMOBILIZATION OF E. COLI z 25 l Time (days) FIG. 2. P-Galactosidase activity of E. coli bound to starch-sepharose. Columns containing 1 ml of matrix were loaded with 2 x 19 E. coli cells induced for P- galactosidase. The columns were eluted with MMA containing 5 mm ONPG and.2% sodium azide at ambient temperature and at a flow rate of 4.8 ml/h. The hydrolysis of ONPG was followed by monitoring the absorbance of the eluate at 42 nm. The cells used were either viable bacteria () or bacteria made permeable by treatment with toluene (). at these temperatures, it was likely that the loss of activity was due to denaturation of P-galactosidase, which in the pure state is inactivated at these temperatures (12). The specific activities of immobilized and nonimmobiized toluene-treated cells were compared. By using the 3-galactosidase assay of Miller (8), an activity of 26 pmol of ONPG hydrolyzed per h per 19 bacteria was found for cell suspensions. Immobilized bacteria, toluene treated in the same way and equilibrated with nonlimiting ONPG concentration and a flow rate of 17 mi/h, gave an activity of 33,umol per h per 19 bacteria. Hence, immobilization does not result in the loss of P-galactosidase activity. It is not clear why the immobilized activity is slightly higher; possibly extended elution in the column makes more cells permeable to substrate. DISCUSSION The data in this paper demonstrates that the affinity immobilization of bacteria for biotechnological applications is worthy of consideration as an alternative to established techniques such as entrapment or binding to ion-exchange resins (2). Although the stability of adsorbed as against entrapped bacteria has been questioned (2), the results with starch-bound bacteria suggest that stable catalysis by affinity-immobilized cells is possible for extended periods. Also, the minor effects on starch binding found over fairly broad ranges of ph, temperature, ionic strength, and elution rate suggest that affinity immobilization need not be adversely affected by fluctuations in operating conditions or by the adoption of particular conditions that optimize catalysis by a given enzyme. The use of starch as a ligand in affinity immobilization also suggests that such an approach need not be expensive. In the present study, for the sake of reproducibility and fast flow rates, the starch was chemically cross-linked to Sepharose. If such a process is to be adopted on a larger scale, the possible use of starch homopolymers that have reasonable flow properties would need to be investigated. As shown through the use of toluene-treated cells, affinity immobilization of nonviable but highly permeable cells may be of definite advantage in attempts to immobilize cytoplasmic enzymes inside cells. Maximum,B-galactosidase activity may be reached far sooner with permeable than with intact cells (Fig. 2) without any apparent sacrifice in stability. This study used E. coli for affinity immobilization since the receptor-dependent binding by this bacterium is well characterized (4, 5). The lamb gene coding for the receptor responsible is also well characterized (1). Since the transfer of this gene to Salmonella spp. has already been demonstrated (9), it is not difficult to regard the transfer of the ability to bind starch to any gramnegative bacterium as also feasible. A possible disadvantage to the use of starch as a ligand is that it is readily degraded by amylases from a wide range of sources. To protect the immobilized starch in these experiments, all E Temp (IC) FIG. 3. Effect of temperature on the hydrolysis of ONPG by affinity-immobilized bacteria. A column containing 1 ml of matrix was equilibrated with MMA at 2 C and was loaded with 19 E. coli cells previously treated with toluene. The column was then eluted with MMA containing 5 mm ONPG and.2% sodium azide. Elution was continued for 12 to 15 h until a steady rate of ONPG hydrolysis was attained. The temperature of the column was then increased and eluted until new steady-state rates of ONPG hydrolysis were attained at each temperature.

5 388 FERENCI long-term storage and use of starch-sepharose was done in the presence of sodium azide to prevent contamination by organisms that have extracellular amylases. Another possible disadvantage is that affinity immobilization on starch is not possible when the eluting medium contains soluble starch, maltose, or starch-derived maltodextrins since the bacteria themselves are eluted under these conditions (4). However, it should be pointed out that bacteria have surface receptors for other types of ligands (6), and these alternative surface receptors could be usefully investigated for potential application in affinity immobilization. ACKNOWLEDGMENTS I thank Peter Ernst and Kin-Sang Lee for excellent experimental contributions to this project. This work was financially supported by the Australian Research Grants Commission. LITERATURE CITED 1. Braun-Breton, C., and M. Hofnung In vivo and in vitro functional alterations of the bacteriophage lambda receptor in lamb missense mutants of Escherichia coli K- 12. J. Bacteriol. 148: Chibata, I., and T. Tosa Use of immobilized cells. Annu. Rev. Biophys. Bioeng. 1: Ferend, T., and H. L. Konberg The utilization of APPL. ENVIRON. MICROBIOL. fructose by Escherichia coli. Biochem. J. 132: Ferencl, T., and K.-S. Lee Directed evolution of the lambda receptor of Escherichia coli through affinity-chromatographic selection. J. Mol. Biol. 16: Ferencl, T., M. Schwentorat, S. Ulirich, and J. Vilmart Lambda receptor in the outer membrane of Escherichia coli as a binding protein for maltodextrins and starch polysaccharides. J. Bacteriol. 142: Kadner, R. J., and P. J. Bassford, Jr The role of the outer membrane in active transport, p In B. P. Rosen (ed.), Bacterial transport. Marcel Dekker, Inc., New York. 7. Kennedy, E. P The lactose permease system of Escherichia coli, p. 49. In J. R. Beckwith and D. Zipser (ed.), The lactose operon. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 8. Miller, J. H Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 9. Palva, E. T., P. Lluestr6m, and S. Harayama Cosmid cloning and transposon mutagenesis in Salmonella typhimurium. Mol. Gen. Genet. 181: Schwartz, M The adsorption of coliphage lambda to its host: effect of variations in the surface density of receptor and in phage-receptor affinity. J. Mol. Biol. 13: Thomas, D The future of enzyme technology. Trends Biochem. Sci. 4: Wadlenfels, K., and R. Weil P-Galactosidase, p In P. D. Boyer (ed.), The enzymes, vol. 7, 3rd ed. Academic Press, Inc., New York. 13. Wandersman, C., M. Schwartz, and T. Ferencd Escherichia coli mutants impaired in maltodextrin transport. J. Bacteriol. 14:1-13. Downloaded from on April 14, 219 by guest