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Isolation of at-amylase on crosslinked starch

Wim A. C. Somers,* Peter H. M. Koenen,* Hennie J. Rozie,+,* Jaap Visser,’ Frank M. Rombouts* and Klaas van ‘t Riet*
*Departments of Food and Bioprocess Engineering, ‘Molecular Genetics of Industrial Microorganisms and ‘Food Chemistry and Food Microbiology, Wageningen Agricultural University, Wageningen, The Netherlands
The isolation and purification starch powder stability of the enzyme, adsorption-desorption processes. resistance degraded, conditions process rohydrin of a-amylase from an industrial enzyme sample is described The adsorbent using crosslinked the

as an affinity adsorbent. process

The process

was studied with regard to the stability of the adsorbent, capacity and stability of the adsorbents during the repeated process by an increase during

and the capacity of the adsorbent for the enzyme. to evaluate the binding kinetics of the matrix was improved because This effect was accompanied levels adsorption

was used in a repeated in continuous the diffusion

The adsorption yielding

of the matri.x decreased. but no decline

of the capacity of the adsorbent The matri.x itself was slow~ly under the experimental IO-40 kg oj with epichlo-

for the enzyme,

levels up to 0.5 mg protein per mg adsorbent. was observed

of the adsorption

90 cycles

used. The lifetime of the matri.x was estimated to be 140 repeated can be obtained the adsorbent with I kg of adsorbent. is cheap and easy to prepare,

runs. Approximately

pure a-amylase because

This may yield an economically

attractive purification

as it consists of starch crosslinked


Crosslinked starch; cx-amylase; affinity chromatography; downstream processing

The introduction of affinity chromatography in an early stage of the downstream process can be an attractive method to isolate proteins on a large scale. l-6 The major drawback for the application of affinity chromatography in an early phase of the isolation process (i.e., directly on the fermentation broth) is twofold: Packed-bed chromatography can easily lead to clogging and fouling of the column and adsorbents often are very expensive. The first problem can be overcome by using continuous adsorption systems with affinity ligands coupled to o en ;P and porous matrices or with soluble affinity adsorbents.43 -” The majorit of the former studies use affinity ultrafiltration techniques.L1z The second problem can be overcome by the development of an affinity adsorbent that is cheap or that can be

Address reprint requests to Dr. Wim A. C. Somers at his present address. TN0 Nutrition and Food Research, Dept. of Biochemistry and Physical Chemistry, P.O. Box 360, 3700 AJ Zeist, The Netherlands Received 30 November 1992; accepted 6 May 1994

used for an extended period of time. An affinity adsorbent with these properties was designed for the isolation and purification of endo-polygalacturonase.7.‘3 The interaction between the enzyme and the adsorbent is based on the binding of the enzyme with alginate, the substrate analogue for the natural substrate pectate.14 For the isolation of cx-amylase, an affinity adsorbent based on enzyme-substrate interactions was also developed. l5 Starch crosslinked with epichlorohydrin is stable against biodegradation by a-amylase and has a good binding capacity for the enzyme.‘5-‘7 Adsorption and desorption conditions for this system depend on several parameters. Adsorption is preferably conducted at 4°C. At this temperature the adsorption levels are high, whereas breakdown of the adsorbent by the enzyme is minimal. I8 In addition there appears to be a relation between the adsorption characteristics and the activity optimum of bacterial a-amylases. Maximum adsorption is found at pH 5.0-6.0, the optimal pH for catalytic activity. It was shown that the interaction between the matrix and the enzyme is biospecific. This also allows recovery of the enzyme from the matrix using a competitive eluent-for instance, a limit dextrin fraction.” A major obstruction for the use of the powders in an

Enzyme and Microbial Technology 17:56-62, 1995 0 Elsevier Science Inc., 1995 655 Avenue of the Americas, New York, NY 10010

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pH 6. A total of 0. The powder obtained in this way had a maximum particle size of 0.6 ml epichlorohydrin (Merck.48 m) provided with a cooling mantle and with a glass filter at the bottom (cutoff 9O.‘s One unit was defined as the amount of enzyme that released 1 kmol of reducing end groups per minute at pH 6. adsorption capacity. pH I I .5 ml of desorption buffer. a heat-stable a-amylase from Bacillus licheniformis. ethanol (twice). After each step samples were taken to determine the enzyme activity. Breda. When used. Subsequently. was an enzyme preparation of Gist-brocades N. D-ghCOSe was used as the standard.08 mm.5 ml of an enzyme solution (50 . protein content.986) was a product of AVEBE (Veendam. After incubation the enzyme was eluted from the Enzyme Microb. 20.0 (temperature = 4°C. pH 6.0. The adsorption reaction was stopped after 100 min and the supematant was pumped out of the column. isolation process is the very low reaction velocity of the the capacities of the adsorption reaction.03 g of crosslinked starch was suspended in I . 0. lo-’ mol g-i) offer a good potential for the isolation of the enzyme.. The amount of adsorbed enzyme was found by measuring the decrease of enzyme activity in the liquid phase at equilibrium. The adsorbent has therefore been characterized with respect to its kinetic behavior. and acetone (twice).000 D) and specific activity (220 U mg . which was swollen in 100 ITIM sodium acetate at pH 6. no. Technol. Somers et al. vol.510. FRG).0 for I h.d.0-m] reaction vessel with a cap.0 . The change in the liquid volume was < IO’% in these experiments. The reaction mixture was shaken for 4 h in an orbital incubator (Gallenkamp) at 200 ‘pm (temperature = 45°C). C. IO’ U I-‘).85 ml 5 M sodium hydroxide were added.’ ). Merck). I@1000 . IO3 U I _ ’ ). The reaction was stopped by adding 200 ml 77~ (vol/vol) acetic acid. and usefulness in a repeated adsorptiondesorption process. ” Although crosslinked products under equilibrium conditions (5. 45 min). (Delft. Subsequently. The crosslinking reaction was also performed using a ground powder. 50 . and starch breakdown. Units can be converted to moles using the values for molecular weight (60. Desorption was accomplished in the adsorption buffer by the addition of 20% (volivol) glycerol (hereafter called desorption buffer). and 30. Then 20 PI of the supematant was taken and diluted to assay for enzyme activity. IO min) and the process was started again. The reaction velocity was followed with time. taking samples (20 ~1). ‘) was suspended in a Perspex column (i. The Netherlands).Isolation of cr-amylase on crosslinked starch: W.7 (temperature = 4°C. The reaction conditions for crosslinking were the same as described above.0 (temperature = 4°C.08 mm and was used for binding studies. Two milligrams of powder were suspended in I5 ml ethanol (p. The crosslinked powder was washed with 100 ml of ethanol. At the start of the experiment a-amylase (Maxamyl. Adsorption kinetics Determination of engme protein content activit_v and a-Amylase activity was determined by an automated neocuproin test using an autoanalyzer (Skalar.04 g of crosslinked starch was put in a 2. The column (I2 x 800 mm) was filled with the adsorbent.. Desorption kinetics Preparation of crosslinked potato starch powder A total of 25 g of drum-dried starch powder was suspended in I53 ml of ethanol.a. Repeated adsorption-desorption experiments Particle size reduction A total of 25 g of crosslinked starch was ground in a Retsch grinding device with a sieve size of 0. the average particle size increased (though <50’%) as a result of swelling in aqueous media. The samples were analyzed for reducing end groups by means of an automated neocuproin test using an autoanalyzer (Skalar). Then 1.’ ) was pumped into the column (temperature = 4°C). The reaction mixture was filtered over a glass filter (G2. The Netherlands) as described previously. 0. To this end the starch powder was ground in a Retsch grinder with a sieve size of 0. isotherms Adsorption isotherms were determined by incubating 0. FRG). Adsorption Materials and methods Materials Maxamyl. The sampling was done as described in “Adsorption kinetics. January 57 . The enzyme was desorbed by the addition of 100 mM glycine-sodium hydroxide. Particle size distributions were also determined by measuring chromatograph> Gel filtration experiments were carried out at room temperature. The drum-dried potato starch powder (Paselli WA 4. Gel filtration Powder size distribution Particle size distributions were determined with a Malvem 3300 particle sizer.08 mm.9 ml distilled water. 17.0.05 m: height 0. An activated matrix was obtained after incubation of 160 g of adsorbent in 800 ml 100 mM sodium acetate.‘). pH 6. 30.0 and 30°C. For sampling the tubes were centrifuged in an Eppendorf centrifuge to precipitate the powder (about 5 s). 13. with Maxamyl (50 .140 km).I9 It was the objective of this study to improve the adsorption characteristics of crosslinked starch.‘” Usually. The upper detection limit in this system was 564 km. A. lx Protein content was determined by using the method of Lowry et trl. The Netherlands). 5 min). 1995.1 g of crosslinked starch powder in 5 ml 100 mM sodium acetate. ref. the matrix was washed in cold 100 mM sodium acetate. 103-1000 IO’U I-‘) was added to the vessel at t = 0.” The total change in the liquid volume was ~10% in these experiments. Subsequently. The powder was air-dried. water (twice). The tube was placed in a rotating incubator. pH 6. no substantial binding of o-amylase was observed in the first 6 h of the reaction. Darmstadt. the diameter of a population of approximately 200 particles using a binocular microscope with a measuring device.03-0. Mainz. Crosslinked starch (24-33 g I -. Starch breakdown Starch powders were assayed for biodegradability as described previously” by incubating appropriate amounts of crosslinked starch with cu-amylase (50 . I O3 U I . IO’ U I . ‘*.V. biodegradation by o-amylase. Schott. the matrix was regenerated for adsorption by washing with 100 mM sodium acetate. with varying amounts of a-amylase (O-5000 .

The fast desorption and the partial adsorption at short reaction times indicate that the adsorption had only taken place at the particle outer surface. ferritin (450 kD).1 isotherm KS (103 u I-‘) 13.7 2 5. 10P” m*/s-’ was assumed for the diffusion of cw-amylase in the matrix.1 buffer starch 58 Enzyme Microb. resulting in higher amounts of soluble oligosaccharides in the experiments (Table 1). In the concentration range of enzyme relevant for the data presented in Table 1. The affinity of the enzyme toward the ground-crosslinked starch was decreased when compared with the crosslinked starch.6 2.42 Desorption 40°C % 1. diffusion was completed in a time on the order of 1 to 10 min. Although there was scatter in the adsorption data.) together with the calculated adsorption isotherms for these matrices and for the crosslinked starch starting product. bacitracine (1.32 0. The matrices could be stabilized during desorption by using glycerol18 or by applying a pH shift to pH 11 . Independent of the Table 1 Crosslinked starch and ground-crosslinked oligosaccharides starch: Adsorption isotherms and biodegradation measured by amount of soluble Biodegradation Adsorption Adsorption Matrix Crosslinked starch Ground-crosslinked q. c q = KS + c Table 1 shows the adsorption isotherms and the calculated capacities and Langmuir constants for the crosslinked starch and the ground-crosslinked starch. pH 6. this resulted in adsorption levels that were a factor of 1.5-5 higher than the values obtained for the matrix at the start of the experiment. kD). The adsorption levels predicted by the adsorption isotherm (see Table 1) were not reached within this time span. Figure 3 shows adsorption data for the matrices (M. The capacity of the ground-crosslinked starch was about 50% larger than the crosslinked starch. Repeated adsorption and desorption reactions: Development of adsorption characteristics Repeated adsorption and desorption reactions were carried out with the matrices using different enzyme concentrations in the adsorption experiment. vol. It was found that the desorption was complete in a few minutes. and sodium azide (63 D) were used. tyrosine (777 D). (10” U kg-‘) 6. When the first sample was taken after 1 min. During the experiment the adsorption levels increased for all three enzyme concentrations (see Figure 2a and b).0 g I--’ 0. with the addition of 20% (vol/vol) glycerol. ovalbumin (43 kD).6 ml min. The breakdown of the groundcrosslinked starch by the enzyme was larger at all conditions examined. Technol. it was clear that the adsorption characteristics of the three matrices were very different from that of the same Adsorption Figure kinetics la shows the adsorption of a Maxamyl sample (50 * lo3 U l.3 * 2.9 1. increased binding of the adsorbate was observed. Figure 2 shows the development of the adsorption process for two starting concentrations of enzyme [matrices M2 and M. January .. As a result of exposing the matrix to the enzyme. When a diffusion coefficient of 1. The fast desorption rate was also found for powders that were loaded by prolonged incubation (12 h) in the adsorption reaction.9 5. 90%-100% of the enzyme activity was already present in the supernatant. M.‘) to crosslinked starch and the groundcrosslinked starch. with 20% (vol/vol) glycerol at 70°C. The powders were used extensively for 50 to 100 runs.Papers matrix at 70°C by repeated incubation (three times) of the adsorbent in 300 ml sodium acetate buffer. lysozyme (25 kD).1 32.58 1. Only lo%-15% of the enzyme activity was adsorbed by the crosslinked power (Figure la).. Eventually. bovine serum albumin (BSA) (68 kD).06 buffer 40°C % 2. whereas the ground-crosslinked starch adsorbed 80% of the enzyme activity.1 g I-’ 0. dextran blue (MW + 450 kD). where the enzyme activity was reduced.3 g I-’ 0.O.. shown in Table I. 17.22 4°C % 0.‘). Ol$osaccharides may act as competitive inhibitors. and M. Subsequently.15 0.4 initial concentration of enzyme.8 9.. This can explain the apparent lower affinity constant K.0. The adsorption velocity was accelerated as a result of the particle size reduction. a fixed amount of approximately 40 * lo3 U lP ’ was absorbed in the first 10 min (Figure lb). l9 The desorption of the enzyme from the crosslinked starch could be accomplished by suspending a loaded powder in 100 mM sodium acetate. a Fourier analysis showed that for the fully loaded particles. (Table 2)]. the adsorbent was used in gelfiltration chromatography in the same column (flow 0. This value was larger than the experimentally found values..0.0 . this would result in lower adsorption levels at equilibrium.. Desorption kinetics Results Adsorption Adsorption equation: isotherms equilibria are described with the Langmuir 4m . chymotrypsinogen (25 kD).4 ? 0. As standards. 1995. pH 6.6% 1.

’ was obtained. wash solution. temperature = 4°C. a value of 145 .000 29.~ = 260. variation of initial enzyme concentration.. A. A.). 0. = 120.o = 950-l 100. lo3 U 1-l at the start of the experiment. buffer: 100 KIM sodium acetate. (a) c~. 0.” Temperature = 4°C lb) cb. adsorbent: 20 g I-‘.------q a* I 30 45 60 . bCalculated from experimental results. The adsorption capacity of the products..8 12.3 27.000 (cao) (U I-‘) No..A 75 30 45 60 t:mlni 0 15 t imln) Figure 1 (a) Adsorption kinetics of a-amylase on crosslinked starch.3 . and methods.7 End (g I-‘) 15. cb. vol. C. was 13. 20 g I -‘. Using these capacity values for an estimation of K. whereas K. 0. ground-crosslinked starch. was between 9. n . temperature = 4°C 20 30 40 50 60 0 20 40 60 80 100 cyclenumber 1-j cycle number I-) Figure 2 Repeated adsorption of a-amylase on crosslinked starch: enzyme concentrations of various 3oo I 250 3 200 150 .000 (U g ‘1 Ml M. 1995. January ..2 13. adsorbent. lo7 U kg-‘.~ = 225 lo3 U I ’ (matrix M. pH 6. 15-25 times larger than at the start of the experiment (see Table 1).4 * 10’ U kg-’ and 16. co. desorption solution. V. solution after adsorption.(t) (103 U/I 1 of wamylase on crosslinked starch: W. regeneration solution. estimated from Figure 3. -+--_-. Technol. .000 950. c. lo3 mol I-‘. As a result of these changes in the binding characteristics the performance of the matrices still improved after 60-90 59 Enzyme Microb. M3 “Obtained from adsorption isotherm. matrix in the first adsorption cycle.. Crosslinked starch. buffer. 0.6. 7 a l c. pi-l 6.0 = 50.).~ = 50. L-. 1O3 U 1. Somers et al. As a result of the process the affinity of the enzyme for the matrix was decreased and the capacity of the support was enlarged compared with the starting material (Table I). lo3 U I ~~‘. c~. n . 103-250 . O------Y 40 30-m. Starting solution.---AF\ 7 u 20 10 0 7 0 "V.9 Breakdown 37 59 53 (%I Theoreticala 1900 5000 6000 (U g Loading ‘) Experimental” 3000 14. Buffers: see “Materials Table 2 starch Repeated adsorption and desorption of cr-amylase to crosslinked starch powder: Adsorption characteristics of crosslinked Adsorbent Concentration Enzyme 50. 6i .\ '\ -.Isolation c.0.000 225. lo3 U I -’ (matrix M. 100 mM sodium acetate. IO3 U IV’. (b) Adsorption kinetics of a-amylase on ground-crosslinked starch. a.0. lo3 U I ‘. 17. '00 I" a_ b l x&_ . 0. Start (g I-‘) 24 33.

characterized by the corresponding equilibrium line.’ under the experimental conditions assuming mass transfer equilibrium to be reached. equilibrium data matrices M. and 0. showing that as a result of the incubation with the enzyme.64. KS = 200. 17. pH 6. for the third experiment. This resulted in increased loading values (q) for the matrix in every run (see Figure 5). resulting in increasing adsorption levels.). was approximately the same. matrix. Isi9 Independent of the bulk liquid concentration. Table 7). in that case the cycle times would have had to be considerably longer than for the case described above. The equilibrium. respectively). For two of the three experiments. pH 6. Crosslinked starch (starting product. an average breakdown of 0. lysozyme (14. BSA (68 kD). Adsorbent concentration.0. further breakdown did not result in a net increase in attainable binding sites.. the adsorbent could be used for at least 140 cycles at ch. The fractionation range of crosslinked starch increased when the matrix was exposed to a-amylase. Data for the last 10 runs of the experiments are collected in Table 3. and chymotrypsinogen (25 kD) were more retarded on an enzymeexposed column consisting of crosslinked starch (Kd = 0.4 kD).17. January became available for the enzyme. shifted as a result of the adsorbent degradation. However. Technol. 0. M. IO' U kg-‘. temperature = 4°C.c (1000 * lo3 U 1-l) at proceeding matrix biodegradation.‘9 The main 60 .34. there was no change in specificity in the concentration range examined. the total amount of enzyme bound in each following run decreased. Conclusions In previous papers the development of an affinity adsorbent for bacterial cr-amylases was reported. 0. which were exposed under different conditions (enzyme concentration and number of cycles). 20 g I-‘. Recovery and specificity Recovery of enzyme activity and protein in the repeated process was between 92% and 100% in all experiments. lo3 U I-‘) was used in every run. respectively.7 on a protein basis was achieved.‘5*‘8. However.l103U/L) enzyme concentration IlO U/L) Figure 3 Adsorption isotherms of a-amylase on crosslinked starch. Figure 5 shows the adsorption isotherm of Figure 3 with the shifting equilibrium lines for one concentration of cb. and ferritin (450 kD) were not retarded by either of the materials under the elution conditions chosen. When the whole particle was attainable for the enzyme. = 13.)V. and 0.Cl. temperature = 4°C Figure 4 Biodegradation of crosslinked starch as function of enzyme concentration. This also agrees with the conclusion that in the first adsorption experiments the enzyme was adsorbed only in the outer region of the particle. When a linear relationship between the cycle number and the biodegradation and a capacity q.c + 1000 . The adsorption process was characterized by a shifting equilibrium line for adsorption. It appears that the breakdown of the adsorbent did not follow the same trend as the changes in adsorption characteristics. 0. buffer. as described in Materials and methods]. and M.5 kg pure or-amylase was obtained with 1 kg adsorbent.Papers q (106U/kgl I16C breakdown 025 020 (g/L) T L 120 015 010 005 0 : 0 100 200 300 400 5 ‘0 500 1000 1500 2000 2500 3000 c. For the experiment shown in Figure 2a the actual breakdown of the matrix was observed.15. -. A purification factor of approximately 1.. Calculated adsorption isotherms. 100 mrv sodium acetate. which is in agreement with the results obtained earlier. q. the matrix degradation still proceeded. ---. It can be reasoned that the capacity of the three matrices.7 . 1995. The increase in the adsorption level could be explained by assuming that the matrix became more porous.16 g lP ’ per cycle was found. When breakdown was independent of the enzyme concentration over a large range and the whole capacity of the support (cb. the matrix became more accessible to larger molecules. = 13. cycles as the levels of adsorption and desorption still increased (see Figures 2a and b). where no large increase in adsorption levels was observed. respectively) than on a column of freshly prepared crosslinked starch (Kd = 0. The fact that the matrix became more porous because of interaction with the enzyme was confirmed by a comparison of the gel-filtration properties of a crosslinked starch before the first adsorption cycle and an adsorbent that had been exposed to the enzyme [measurement of the distribution factor Kd (Kd = (V.. In the initial part of the experiment binding sites Enzyme Microb. approximately 40 kg of enzyme could finally be obtained with 1 kg adsorbent. vol.05.34 g 1-l was found (Table 2). 0. Bacitracine (1440 D). With this process approximately 9.42. lo3 U I-‘. breakdown of the matrix appeared to be independent of the enzyme concentration in solution (Figure 4). In cycles 20-60. In addition.A.c = 1000 . Ovalbumin (43 kD). buffer: 100 mM sodium acetate. IO3 U I.0.-V..0. lo7 U kg-’ were assumed. M.

04 0.4 0. The kinetics of the complex formation and the equilibrium values finally reached were determined by the competition of the enzyme for the adsorbent and the soluble oligomen.‘) capacity of the adsorbent (U kg .5 0.I-- -1 80 40 0 0 500 1000 1500 2000 2500 3000 Cb IlO~Ull) Figure 5 Equilibrium lines for adsorption of a-amylase crosslinked starch. 2. Fraction Enzyme solution Adsorption (2) Wash step (3) Desorption (4) Regeneration (51 Total (2 + 3 + 4 + 5) 103 u I_’ 50 5 2 40 2 49 g I--’ 0. Although the matrix was degraded during the process. in the initial stage.5 1oaug 136 103 110 225 100 ’ 132 50 50 235 50 q(106U/kg) .25 0.. Proc.0700. 17. This increase in porosity was confirmed by comparing two adsorbents with respect to their gel-filtration properties. g I_’ 7. However.Isolation Table 3 Repeated adsorption and desorption of a-amylase 1~‘) and specific activity (U g-‘) of subfractions Matrix M. (higher affinity) than the one obtained from the equilibrium data.. E. Affinity chromatography: Its application to industrial scale processes. January 61 .17 0. Technol. New developments in downstream processing. the adsorption process was characterized by a lower value for K. Biotechnol. Therefore. on incubated with the enzyme. 103u 225 35 17 140 18 210 I ’ g I -’ 1. As a result of this interaction the matrix became more accessible to the enzyme. 31-66. This effect is explained by two phenomena that affect the binding of cr-amylase to this kind of products. C. Somers et al. M.) Langmuir constant (U 1~ ’ ) molecular weight (D) concentration of the enzyme on the adsorbent at equilibrium (U kg. 3-12 Hill. However. Further improvement could be achieved by enzyme-adsorbent interaction.38 0. 31-38 Lowe.25 1. c~. J Biotechnol. the adsorption of a-amylase to the adsorbent was accompanied by some biodegradation. even then the matrix appeared to be accessible only to a limited extent. Adv.1 0. when the equilibrium conditions of the system were studied it was found that the capacity of the adsorbent for the protein was much larger than initially expected from the kinetic experiments. References I 2 3 Janson.7 0.04 0. This can be explained by the fact that partial enzymic degradation of the matrix resulted in a more porous structure that allowed an increased penetration of the protein into the adsorbent. Trends Biorechnol..‘) distribution factor ( . protein content (g Matrix M. resulting in still increasing adsorption levels after 90 cycles. J.6 loa u g-1 133 70 70 230 70 IO3 u 1~ ’ 1020 600 100 340 30 1070 Matrix M. A. When the average particle size of the crosslinked starch was decreased by a grinding step a product was obtained with considerably better adsorption characteristics for the enzyme. matrix M.~ = 1000. 1984. and Hirtenstein. A. resulting in improved adsorption characteristics.‘) time (s) elution volume (I) volume liquid phase (I) void volume (1) pore volume (1) Acknowledgment These investigations were supported in part by the Netherlands Technology Foundation (STW). 1984. WLM 49. the amount of binding sites became 1530-fold enhanced in the continuous adsorption-desorption process. Furthermore. lo3 U I-‘. This can be attributed to the larger surface area that became available for binding. grant no. Enzyme Microb. R. C. the prepared crosslinked products consisted of relatively large particles in which most of the binding sites were not directly available for the enzyme.3 7. D. 1983. As shown. 1. where biodegradation had not fully evolved. the loss of material was fully made up for by an improved mass transfer and binding characteristics. which made it only suitable as a low-capacity affinity system for analytic purposes. First.5 0.35 103u go’ of cx-amylase on crosslinked starch: W. problem in the application of these adsorbents in the isolation of the enzyme was the very slow adsorption kinetics of this system.5 5. 1995. resulting in a larger fractionation range for the adsorbent that was Nomenclature ‘K K: M 9 9m t “. vol. on crosslinked starch powder: Enzyme activity (U I ‘1. Large-scale affinity purification--State of the art and future prospects. 1. C.6 0. “1 “0 “P bulk liquid concentration at equilibrium (U I.3 1.

B. Jr. Bonte.. On the interaction of o-amylase with crosslinked starch: Evaluation of process conditions. 997-1006 Lowry. J..Papers 4 Luong. Protein measurement with the Folin phenol reagent.. M. Visser. H. Purification of various pectic enzymes on crosslinked polyuronides. Rombouts. ed. Biol. 265-275 5 6 14 7 15 8 9 16 17 18 19 20 62 Enzyme Microb. R.. 1290-1293 Somers. F. Affinity cross-flow filtration for purifying biomolecules. K.. J. J. eds. Appl.. Visser. 281-286 Clonis. Continuous affinity-recycle extraction: A novel protein separation technique. Enzyme Microb. 1 It& 116 Rozie. 24. Eng. L. F. M. Amsterdam. Gordon. K. 5.. 5... W. 99-l 14. C. W. Developments in downstream processing of (poly)saccharide degrading enzymes. K. Rozie. M. 40. W. and Nivard. M. 283. Large-scale affinity chromatography. 323-330 Mattiasson. 35. T..X4-566 Mattiasson. Pungor E.. I. 1987. J. Biotechnol. 5. and Male. eds.56%563 Rombouts. Rombouts. Farr. and Van ‘t Riet. 1986. Rombouts.. Recent developments in downstream processing based on affinity interactions.. Study of the crosslinking reaction between epichlorohydrin and starch. Visser. 5. C. Bioltechnolog?. and Cooney. 1921 Kuniak. In: Affinity Chromatography and Related Techniques (Gribnau. 181-195 Somers. 255-260 Rozie. F.. Amsterdam. A. Technol.. M. and Pilnik. and Randall. Somers. M. Visser... A.. H. British patent no. 1989. H.. N.. H. J.. Isolation and purification of endo-polygalacturonase by affinity chromatography in a fluidized bed reactor. H. In: Proceedings of the 4th European Congress on Biotechnology (Neijssel.). K. J. 195 I. D. N. Bonte. In: Membrane Separations in Biotechnology (Courtney. NY. 604-608 Male. F. and Van ‘t Riet. Chem. 1991. 15. and Luong. W. Nguyen. 0. H. K. H.. F.. B. Chem. vol. G. J. Bioeng. L. B. Rozie. Visser. B7-B19 Luong. 1991. A.. I. K. Somers. 0. 1982.. and Van ‘t Riet. Van der Meer. F. Isolation and purification of endopolygalacturonase by affinity chromatography in a fluidized bed reactor. I. Van ‘t Riet. Technol. C. 13. W. N.. Chromatogr. Ch.q?: 1987. R. K. B. F. A. M. I. Bonte. Polym. Y. J. J. 1995.. Marcel Dekker. A. Elsevier Scientific. 13. 1987. Adsorption and desorption characteristics of bacterial o-amylases on cross-linked potato starch. W. Trends Biotechnol. Afeyan. L. M. 1989. M. and Visser.. A. W. J. H. and Luyben. J. Nguyen. Nguyen.). K. M.. K. T. F. F. Bioltechnolo. Biochem. Visser. T. B.. R. T. H. K.. Rozie. J. 349-365 Dreyfus. Crosslinked potato starch as an affinity adsorbent for bacterial a-amylase. 166. J. I. J. Ultrafiltration affinity purification-A process for large-scale biospecific separations.). Isolation of urokinase by affinity ultrafiltration. 11. L. M. R. and Male. W. and Marcbessault. B. H. and Ramstorp. Bioltechnology 1987.. T. and Rombouts. and Van ‘t Riet. Geraeds. Biotechnol. Rombouts. 199-222 Somers. 1987. I. Rosebrough. J. Biotechnol.. Van ‘t Riet. A. Carbohydr. 193. 1990. Visser.. C. Sttirke 1972. Elsevier Scientific Publishing. Ultrafiltration affinity purification-Isolation of concanavalin A from seeds of Canavalia ensiformis. 1984. January . R. A. 17. and Ling.. 1991. L. H. 87-93 Somers. Rombouts. L..