Immobilization of dextranase from Chaetomium erraticum

Journal of Biotechnology 131 (2007) 440–447

Immobilization of dextranase from Chaetomium erraticum Frank Alwin Erhardt, Hans-Joachim J¨ordening ∗ Institute for Technical Chemistry, Department for Carbohydrate Technology, Technical University Braunschweig, Hans-Sommer-Straße 10, D-38106 Braunschweig, Germany Received 16 February 2007; accepted 25 July 2007

Abstract In order to facilitate the Co-Immobilization of dextransucrase and dextranase, various techniques for the immobilization of industrial endodextranase from Chaetomium erraticum (Novozymes A/S) were researched. Adsorption isotherms at various pH-values have been determined for bentonite (Montmorillonite), hydroxyapatite and Streamline DEAE. Using bentonite and hydroxyapatite, highest activity loads (12,000 U g−1 ; 2900 U g−1 , respectively) can be achieved without a significant change of the apparent Michaelis–Menten constant KM . For successful adsorption, enzyme to bentonite ratios greater than 0.4 (w/w) have to be used as lower ratios lead to 90% enzyme inactivation due to bentonite contact. In addition, covalent linkage using the activated oxiran carriers Eupergit C and Eupergit C250L as well as linkage with aminopropyl silica via metaperiodate activation of glycosyl moiety of dextranase are discussed. This is also the first report probing the structure of a matrix containing dextranase by use of substrate species with different molecular weights. From this we can observe a relationship between the porosity of Eupergit and dextran dependent activity. For the reactor concept using Co-Immobilisates, hydroxyapatite will be preferred to Eupergit because of its higher specific activity and dispersity. © 2007 Elsevier B.V. All rights reserved. Keywords: Dextranase; Immobilization; Bentonite; Hydroxyapatite; Eupergit

1. Introduction Dextranase (1,6-␣-d-glucan-6-glucanohydrolase, EC 3.2.1.11) is an important industrial enzyme and has traditionally found its main application in the sugar industry, where it renders the technical processing of alternated sugar beet possible (Zimmer, 1999). In addition there is a range of speciality applications like the preparation of clinical low molecular weight dextran (Novak and Stoycos, 1961) or the coupling of dextranase to tumor cell specific antibodies followed by administration of cytotoxic dextran-conjugate (Hansen, 1998). Recently dextranase has gained much interest in the directed synthesis of isomaltooligosaccharides (IMOs), which have been shown to exhibit prebiotic effects (Goulas et al., 2004a,b; Kubik et al., 2004; Paul et al., 1989; Thitaram et al., 2005). Generally the hydrolysis of dextran by dextranase could yield IMOs. Yet a sustainable process would utilize sucrose rather than dextran, because the latter compound is a microbial or enzymatic product

∗

Corresponding author. Tel.: +49 531 234 4784; fax: +49 531 391 7263. E-mail address: [email protected] (H.-J. J¨ordening).

0168-1656/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.jbiotec.2007.07.946

of sucrose conversion; moreover dextran hydrolysis generates about 20% of unwanted branched oligomers (Paul et al., 1989). Thus, for enzymatic synthesis two enzymes must be present, dextransucrase for the conversion of sucrose to dextran and dextranase for dextran breakdown (Goulas et al., 2004b; Kubik et al., 2004). Fig. 1 outlines the process flow sheet for the technical isomaltose production using the dextransucrase dsrS only (Ergezinger, 2006). We aim to design a biocatalyst for IMO synthesis, which contains both enzymes in immobilized form and by that delivers higher IMO yields. It is essential to restrict the free access of dextranase to dextransucrase dsrS as it is known that the dsrS enzyme becomes rapidly destabilized when exposed to dextranolytic activity (Berensmeier et al., 2004). As far as work groups operate with soluble dextranase and soluble/immobilized dextransucrase, they consistently report on a pronounced decrease of dsrS activity. Within 10 repetitive batches half of the initial activity has been lost (Kubik et al., 2004), other studies observe an activity decrease of 10% during the first 400 min of operation in a membrane reactor (Goulas et al., 2004a). In order to restrict the free access of dextranase to dextransucrase, we decided to immobilize dextranase and co-entrap this

F.A. Erhardt, H.-J. J¨ordening / Journal of Biotechnology 131 (2007) 440–447

441

Fig. 1. Process flow sheet of a multiphase fluidized bed reaction adsorber (MFBRA) for the integrated synthesis and adsorption of isomaltose using dextransucrase (Ergezinger, 2006).

immobilisate and the dsrS into a hydrogel. For the first step we looked at the immobilization of dextranase, which should yield a highly active carrier and should suit our overall reactor concept. For our studies we used an industrial dextranase from Novozymes A/S, because its KM is low as compared to dextranases from Amano or Asa. Adsorption of dextranase onto bentonite according to the protocol of Madhu and Prabhu (1985) brought deviant and inadequate results, thus we also studied several other immobilization methods. There are several boundary conditions we were confined to: the works of Reischwitz et al. (1995) suggest that the immobilization of dextransucrase for technical operation ought to be performed by entrapment in alginate. Hence particle size of dextranase immobilisate must fall far below the size of alginate beads, because it has to be co-entrapped within the alginate beads (Erhardt et al., 2005). For increased stability of the dsrS enzyme, we work at pH 5.4 (Berensmeier et al., 2004; Kaboli and Reilly, 1980). Regarding the hydrodynamics of our reactor system (fluidised bed reactor) the density of dextranase immobilisate should ideally be well above 1.2 g cm−3 . 2. Materials and methods Unless otherwise stated all materials and chemicals are purchased from Sigma–Aldrich, Munich, Germany. For adsorption experiments a rotating device (MBT-TR 28 Rotator, 10 rpm) is used. Activity of immobilisates is measured using a thermocycler (HLC HTMR-133), where moderate mixing means 600 rpm and turbulent mixing 1200 rpm. 2.1. Enzymes Enzyme “Dextranase Plus L” is kindly provided by Novozymes A/S, Denmark. The enzyme is a fungal endo-dextranase

obtained from submerged culture of a strain of Chaetomium erraticum. The enzyme solution does not contain significant amounts of impurities as can be seen from protein and activity profiles of SEC runs (BioSep-SEC-S2000, Phenomenex) as well as from SDS-PAGEs. Enzyme preparations are subjected to intensive ultrafiltration to ensure complete buffer exchange >99.9% (MWCO 10.000; Vivaspin 20, Sartorius Germany). One Unit of dextranase activity is defined as the amount of enzyme that liberates reducing sugars equivalent to 1 ␮mol maltose per minute at pH 5.4 and 30 ◦ C. The substrate solution is comprised of 30 g L−1 dextran (MW: 425–575 kDa) in 0.1 M acetate buffer at pH 5.4 and reducing sugars are determined according to Sumner and Howell (1935) using dinitrosalicylic acid solution. For protein measurements of enzyme solutions or supernatants of centrifuged suspensions, the Bradford method is used (Bradford, 1976). 2.2. Bentonite adsorption experiments Prior to adsorption, bentonite (Montmorillonite) is subjected to hydrogen peroxide conditioning and to NaCl or CaCl2 incubation as reported by Nowikow (1995). After that 2 mL of 2 g L−1 bentonite solution is contacted with dextranase solution overnight at 4 ◦ C in a rotating device at 10 rpm. The ratio of bentonite to dextranase ranges from 20:1 to 1:1.5 on a mass basis. After the incubation, samples of the suspension and the supernatant are withdrawn and protein concentration and activity are determined; an incubated dextranase solution serves as a control. Suspensions are then washed five times using 50 mM sodium acetate pH 5.4 for at least 20 min at 4 ◦ C at 10 rpm. Generally enzyme bleeding in the last washing step was still significant. To correct for possible enzyme detachment, the activity of the supernatant is subtracted from the suspension activity. All experiments are carried out in triplicate and 50 mM acetate

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(pH 4.0–5.4), 50 mM phosphate (pH 7.0: sodium bentonite) and 50 mM imidazole (pH 7.0: calcium bentonite), respectively serve as buffer. During the washing procedure triplets are combined. 2.3. Further adsorption experiments Protein adsorption isotherms of hydroxyapatite (Type I, Sigma–Aldrich; 26.8 g L−1 ) follow the procedure described above. The initial protein concentration varies from 0.1 to 3 g L−1 . As an anion exchanger Streamline DEAE (GE Healthcare) is equilibrated with 500 mM NaCl in 50 mM sodium acetate at pH 5.4. The carriers are then washed with the buffer (pH 4.2: 20 mM sodium acetate; pH 6.3 and pH 8.1: 20 mM sodium phosphate). For adsorption 2 mL 4% (v/v) Streamline are contacted with 3 g L−1 dextranase solution at 4 ◦ C in a rotating device at 10 rpm overnight. Batches are washed five times using 50 mM sodium acetate pH 5.4.

2.6. Alginate immobilization Preparation of alginate beads (1.1% (w/v), dp = 2 mm) is done by standard ionotropic gelation through a syringe as described by Berensmeier et al. (2004). By modifying the protocol of Patel et al. (1967), the immobilization of dextranase onto alginate using Woodward’s reagent K (N-ethyl-5-phenylisoxazolium3 sulfonat) is realized. One gram of alginate beads is treated with 10 mL 200 mM Woodward’s reagent K in 20 mM borate buffer pH 9.0 for 2 h at room temperature in a rotating device (10 rpm). After that, beads are washed 5 times with 25 mM calcium acetate buffer pH 5.4 and the activated alginate beads are contacted with 5340 Units dextranase in 20 mM calcium acetate pH 4.8 or 20 mM borate pH 8.0 for 2 h at room temperature, with or without 1 M CaCl2 . Alternatively, a linker could be integrated before enzyme loading (incubation with 100 mM 6-aminohexanoic acid; anew activation step). 2.7. Definition of applied models and equations

2.4. Immobilization on Eupergit® Eupergit® C or Eupergit® C250L (20 mg each) is suspended in 2 mL dextranase solution at different pH-values. Dextranase solution is comprised of 440–740 Units in 20 mM buffer including 1 M NaCl (pH 4.0: sodium acetate, 440 U; pH 6.4: sodium phosphate, 740 U; pH 8.8: borate, 730 U). Incubation takes place at 4 ◦ C for 96 h in a rotating device (10 rpm). The carriers are washed using 1 M NaCl until no activity could be detected in the supernatant. After the last wash step, the carriers are resuspended twice in 25 mM sodium acetate pH 5.4. 2.5. Immobilization on aminopropyl silica via activated carbohydrate moiety Aminopropyl silica is prepared according to Angeletti et al. (1989) and washed with 5% (v/v) ethanol before use. The carrier is then conditioned with the buffer. For the carrier activation, a protocol of Monsan (1977) is modified, for glycoenzyme activation the protocol of Hsiao and Royer (1979) is followed. The carrier is activated using 5% (v/v) glutardialdehyde in 50 mM phosphate buffer at pH 8.5 for 2 h at 30 ◦ C. The glycoenzyme is activated using 20 mM sodium periodate at pH 4.3 for 1 h at 4 ◦ C in the dark. For the formation of a stable glycoenzyme, the partly oxidized dextranase is incubated with 50 mM 1,6-diaminohexane in 10 mM sodium phosphate at pH 8.0 for 2 h at 4 ◦ C. The enzyme derivative is subjected to ultrafiltration (99.3% buffer exchange), so that 350 Units enzyme preparations in 20 mM sodium acetate pH 4.0, in 20 mM sodium phosphate pH 6.4 and in 20 mM borate pH 8.8 are available; all preparations contain 1 M NaCl too. The enzyme solution is added to the activated carrier (20 mg) and incubated at 4 ◦ C overnight. Sampling during the entire immobilization procedure is achieved by adding 2 volumes 500 mM 1,6-diamino hexane to 1 volume sample. By doing so, the activated enzyme should remain stable until activity is tested. The washing procedure is analogous to Eupergit® washings.

Unless otherwise stated, error bars shown in the plotted data are based on sample size n = 3 and a confidence band p = 95% (Student t-distribution). The activity yields are determined by the division of the immobilisate activity by the putative amount of adsorbed dextranase (see Eq. (1)). y% =

Aimmob. × 100 Ain − Aeq

(1)

For the computation of catalytic efficiencies, the ratio of the specific activities of the soluble dextranase and the immobilized dextranase is formed (Eq. (2)). c% =

U mg−1 dextranase

−1 U g−1 support /(mgdextranase gsupport )

× 100

(2)

3. Results and discussion 3.1. Bentonite adsorption To characterize the adsorption of dextranase onto bentonite, adsorption isotherms at different pH-values are presented in Fig. 2. Generally, at low protein concentrations adsorption increases linearly and eventually levels off, so the adsorption isotherms can be modelled by Langmuir. Based on Table 1, isotherms can be clustered according to their pH-values: a pH greater than 5.4 results in monolayer adsorption capacities close to 0.2 g g−1 . Within this band it is rather insignificant, whether a Na- or Ca-bentonite is used. However, the bentonite form is crucial when we look at the monolayer capacity at adsorption of pH 4.0: the homoionic sodium bentonite surpasses the load of Ca-bentonite by a factor of 5. The interlattice space between bentonite crystals is dependent on the intercalated ion species, so that Na-bentonite lattices are more distant compared to those found in Ca-bentonite (Nowikow, 1995). In solution the delaminated Na-bentonite exhibits roughly four times the surface of a Ca-bentonite (Schramm and Kwak, 1982), thus more protein can be bound

F.A. Erhardt, H.-J. J¨ordening / Journal of Biotechnology 131 (2007) 440–447

Fig. 2. pH-dependent adsorption isotherms of dextranase on bentonites. Filled black symbols: Na-bentonite. Open grey symbols: Ca-bentonite. Isotherms modelled by Langmuir. (),(): pH 4.0; (): pH 4.8; (䊉),(): pH 5.4; (),(): pH 7.0.

and higher loads achieved. This effect has been observed by other work groups to different extents (Hamzehi and Pflug, 1981; McLaren et al., 1958). As for the maximum adsorption capacity of 0.8 g g−1 this agrees well with the work of other groups (Buttersack et al., 1995; Morgan and Corke, 1975; Nowikow, 1995). From our data we can further derive the pHdependence of dextranase adsorption. Mapping the dextranase on a 2D-PAGE, the isoelectric point can be estimated to be 5.1 (data not shown), thus a pH lower than this confers an overall positive net charge to the enzyme, so that adsorption onto the cation-exchanger bentonite is facilitated. Although this behaviour can be expected, there exist exceptions from this mechanism: Hamzehi and Pflug (1981) report on the highest adsorption load of ␣-Amylase onto bentonite at the pI of their enzyme. Fig. 3 displays the activity adsorption isotherm both in equilibrium at pH 4.0 and after the washing steps at pH 5.4. The equilibrium isotherm takes account of a possible inactivation of dextranase upon bentonite contact, so the equilibrium load is calculated by the subtraction of the activity of suspension from the supernatant. The activity loss observed is due to enzyme inactivation and not due to diffusional limitation, since the activity of a different dextranase is fully conserved upon bentonite contact. Table 1 Bentonite adsorption isotherms aL (g−1 g)

KL aL −1 (g g−1 )

R2

Sodium bentonite pH 4.0 56.8 pH 4.5 28.9 pH 4.8 5.16 pH 5.4 0.77 pH 7.0 0.08

68.9 49.8 12.7 3.34 0.30

0.82 0.58 0.41 0.23 0.27

0.891 0.862 0.909 0.937 0.997

Calcium bentonite pH 4.0 8.70 pH 5.4 0.386 pH 7.0 0.256

61.1 2.27 2.99

0.14 0.17 0.09

0.890 0.954 0.963

KL (1)

443

Fig. 3. Activity adsorption isotherms of dextranase on Na-bentonite. Black solid line: Equilibrium isotherm at pH 4.0, Langmuir model [KL = 55.5; aL = 0.59]. Grey dashed line: Isotherm after intensive washing at pH 5.4, Freundlich model [KF = 2.76; n = 3.80], and abscissa of equilibrium isotherm adopted.

The inactivation as listed in Table 2 results from the activity difference between the blind control and the suspension. The maximum activity load in equilibrium is 94,000 U g−1 and after washing steps 12,000 U g−1 . Low enzyme to bentonite ratios cause almost a complete loss of dextranase activity in the fluid phase of suspension as well as in the adsorbed state. Madhu and Prabhu (1985) report 50–100% activity yield upon dextranase adsorption onto bentonite with enzyme to carrier ratios being 20–400 times below our lowest ratio (mass base). We could not confirm these results with dextranase from C. erraticum, as extensive enzyme inactivation occurred at ratios lower than 0.4. When the amount of applied enzyme increases, the yield of immobilization improves and the inactivation declines. This is in agreement with other protein adsorption studies onto bentonite where the portion of active adsorbed enzyme rises at high enzyme concentrations (Bajpai and Sachdeva, 2001; Buttersack et al., 1995; Garwood et al., 1983; Morgan and Corke, 1975; Ross, 1983). The Freundlich shape of adsorption isotherm after washing may be due to the concentration-dependent inactivation of dextranase. Morgan and Corke (1975) as well as Garwood et al. (1983) provided evidence for cooperative effects upon enzyme adsorption onto bentonite. The adsorbed dextranase is further characterized by the determination of the apparent Michaelis–Menten constants (Fig. 4). The apparent KM -values of soluble and adsorbed enzymes do not differ significantly (6.51 and 7.1 g L−1 ). The mean particle size of suspended bentonite is determined by laser diffraction to be 1.8 ␮m (x50 ) and the average pore diameter amounts to 0.6 ␮m (mercury porosimetry). The high dispersity, comparatively large pores and the delaminated structure of bentonite permit kinetics without significantly effecting diffusion. The maximum activity of the pH-profile of adsorbed dextranase remains unchanged at pH 5.4. In the case of our reactor system, the application of a dextranase immobilisate based on bentonite is ruled out by its detrimental inactivating potency towards dextransucrase, which we will report on in a separate paper.

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Table 2 Dextranase inactivation and yields of immobilization Equilibrium (U g−1 )

Dextranase inactivation upon adsorption at pH 4.0 (% of Ainitial )

Yield % in equilibrium at pH 4.0 Aads /(Ainitial − Aequil )

Yield % after pH-shift to pH 5.4 Aads /(Ainitial − Aequil )

318 61.6 1.41 0.10 0.09 0.05 0.06

44 44 69 90 90 92 91

13.5 34 30 10 10 8 8

2.9 4.1 3.8 1.1 0.7 0.5 0.6

(2940 U g−1 as compared to 540 U g−1 , both after washing). If dextranase is adsorbed at pH 4.1 and hydroxyapatite is washed several times with buffer at pH 5.4, we achieve the highest activity yield so far (28%). The results suggest that adsorption below the pI features a conformation, which supports the protein binding in an active and firm state. The kinetics of dextranase immobilisate can be seen in Fig. 4. As with bentonite, the apparent KM -values of enzyme adsorbed onto hydroxyapatite do not differ significantly from soluble enzyme (5.8 versus 6.5 g L−1 ). 3.3. Adsorption onto Streamline DEAE

Fig. 4. Michaelis–Menten kinetics of soluble and adsorbed dextranase (normalized to vmax ). Apparent KM -values: () KM,sol = (6.5 ± 0.3) g L−1 , (䊉) KM,bentonite = (7.1 ± 0.4) g L−1 and () KM,hydroxyapatite = (5.8 ± 0.7). R2 > 0.999.

3.2. Hydroxyapatite adsorption In addition to bentonite we investigated hydroxyapatite, which also has a high density and dispersity due to its small particle size (5.6 ␮m). Table 3 reflects the Langmuir parameters obtained by non-linear regression of protein adsorption isotherms of dextranase. The equilibrium load q* with 40 mg g−1 at pH 5.4 surpasses the load achieved at pH 4.1, here q* is 32 mg g−1 . The KL parameter of protein and activity isotherms at pH 5.4 lays 2–3 fold above the distribution coefficient at pH 4.1, which also points at the protein adsorption being favored at pH 5.4. Activity loads in equilibrium as well as after washing steps both reveal that the adsorption at pH 4.1 yields an immobilisate with higher activity Table 3 Protein and activity adsorption isotherms of hydroxyapatite adsorption Protein

KL (1)

aL (g−1 g)

KL aL −1 (g g−1 )

R2

HA pH 5.4 HA pH 4.0

6.91 2.63

175 81.4

0.0395 0.0323

0.992 0.992

Activity HA pH 5.4, eq HA pH 5.4, a.w. HA pH 4.1, eq HA pH 4.1, a.w.

KL (1) 7.44 2.10 3.34 0.65

aL (U−1 mg) 1.16 3.89 0.37 0.22

KL aL −1 (U g−1 ) 6417 539 9027 2941

eq: equilibrium; a.w.: after washing steps at pH 5.4.

I2 0.936 0.979 0.994 0.995

For anion exchange resins, we tested Dowex 2×8 and Streamline DEAE. Initial experiments showed that the dextranase adsorption works better with Streamline DEAE, because equilibrium loads of DEAE exceeded Dowex loads 10-fold at pH 6.3. The primary focus was to see, whether DEAE supports would facilitate higher activity yields, thus the particle size of 100–300 ␮m has been accepted. Table 4 displays activity loads of Streamline adsorbers in equilibrium and after washing steps. The specific loads as well as the activity yields are optimal at pH 6.3, the immobilisate exerts an activity of 93 U mL−1 and 4.5% of adsorbed dextranase are eventually found on the carrier. The load of the carrier is suitable for our process, however, the particle size allows only dextransucrase prepared in alginate beads with dp > 1.0 mm, which may result in diffusion limitation of this enzyme (Berensmeier et al., 2004). In general the adsorption experiments exhibited low activity yields. In addition the immobilized dextranase had to be washed very thoroughly. Although enzyme bleeding was not very substantial, it can become a problem in a continuous reactor system. To overcome both problems, we researched the covalent immobilization of dextranase onto commercial Eupergit® supports, activated aminopropyl silica (involves activation of glycoenzyme) and activated alginate. Table 4 Streamline DEAE adsorption at different pH-values

pH 4.2 pH 6.3 pH 8.1

Equilibrium load (U mL−1 )

Specific load at pH 5.4 (U mL−1 )

Activity yield (%)

1026 2061 1868

25 93 54

2.4 4.5 2.9

F.A. Erhardt, H.-J. J¨ordening / Journal of Biotechnology 131 (2007) 440–447

Fig. 5. Activity of dextranase covalently bound onto Eupergit® at different pH-values using two different substrate sizes at turbulent mixing: solid bars without pattern refer to 7.5 kDa dextran, bars with sparse pattern to 500 kDa species. Protein loads at pH 4.0 (black)/pH6.4 (grey)/pH 8.8: Eupergit C = 16/8.9/8.4 mg g−1 ; Eupergit C250L = 11/5.2/4.6 mg g−1 .

Eupergit® belongs to the group of macroporous carriers (dp = 150 ␮m), thus the experiments extend into kinetic aspects of the final enzyme immobilisates. The pH upon Eupergit® immobilization, the substrate sizes (both Fig. 5) and the external mass transfer (Table 5) are studied. Fig. 5 shows that the activity of immobilisate strongly depends on the substrate in use. The averaged pH activities add up to 741 U g−1 (Eupergit® C) and 509 U g−1 (Eupergit® C250L), respectively. However, using the high molecular weight dextran as a substrate, Eupergit® C250L has an activity clearly higher than Eupergit® C. This can be attributed to the pore size of both supports: the works of de Segura et al. (2004) measure the pores to be 10 nm (Eup. C) and 180–300 nm (Eup. C250L), respectively. Given that the hydrodynamic diameter of 7.5 kDa dextran is 1.5–2.7 nm (Lebrun and Junter, 1993; Pluen et al., 1999) and of 500 kDa dextran 15.3 nm (Lebrun and Junter, 1993), it becomes clear that reduced activity of Eupergit® C with a 500 kDa dextran is due to a strong internal diffusion limitation and partly due to total size exclusion. Although the internal difTable 5 Activity of Eupergit and aminopropyl silica immobilisates using 500 kDa dextran under different mixing conditions Activity (U g−1 ), turb. mixing

Catalytic efficiency (%)

12 10 12

16 27 33

Eupergit C250L pH 4.0 25 pH 6.4 23 pH 8.8 30

93 72 86

18 28 41

Aminopropyl silica pH 4.0 16 pH 6.4 14 pH 8.8 12

14 13 13

n.a. n.a. n.a.

Eupergit C pH 4.0 pH 6.4 pH 8.8

7.3 5.7 5.5

fusion in Eupergit® C250L is less distinct, this support suffers from an external mass transfer limitation (Table 5). At pH 4.0 both matrices bind approximately twice as much protein as at other pH-values, however the activities of immobilisates at pH 4.0 do not differ from the activities at other pH-values. This can be explained by the fact that incubation at pH 4.0 inactivates dextranase by 38%. Higher protein loads at Eupergit® C can be attributed to the twofold higher content of oxiran groups of this Eupergit® (de Segura et al., 2004). The dextranase immobilisate is needed for our reactor concept involving dextransucrase, which produces dextran in a bimodal way (Tsuchiya et al., 1955), so that Eupergit® C250L would be required for the hydrolysis of high molecular weight dextran (>1 MDa). However, as mass transfer within alginate is facilitated only by diffusion, external mass transfer limitations could then be expected. 3.5. Immobilization using activated aminopropyl silica and activated dextranase

3.4. Immobilization using Eupergit® supports

Activity (U g−1 ), mod. mixing

445

Catalytic efficiency refers to 7.5 kDa activity tests under turbulent mixing.

The carbohydrate spines of glycoenzymes may hinder the protein from approaching the binding groups on the surface of the solid support. Thus, functionalized glycoenzyme-derivatives can be prepared and used for immobilization (Hsiao and Royer, 1979). We prepared a hexamethylenediamine adduct, which was covalently attached onto an activated aminopropyl silica gel (glutardialdehyde activation, (Monsan, 1977)). Table 5 shows the activity of silica-bound dextranase; protein and activity equilibrium loads could not be determined due to interference of sampling with analytics. Similar to Eupergit® immobilization, the pH does not significantly influence the activities obtained, at pH 4.0 the highest activity is achieved (55 U g−1 ). We have not detected external mass transfer limitation (data not shown), and in comparison with Eupergit® the internal diffusion limitations are rather low (using 7.5 kDa substrate: activity increase by factor 3.6 ± 0.5). 3.6. Immobilization via activated alginate The last immobilization approaches are derived from our overall reactor concept. We evaluate a possible generation of a bilayer alginate catalyst with entrapped dextransucrase inside and coupled dextranase on the outer shell. Alginate is activated with Woodward’s reagent K and an optional C-6 spacer can be included before dextranase is added at different pH-values. Immobilization by this procedure has not turned out satisfactory as all activities obtained are between 2.2 U g−1 without spacer and 3.4 U g−1 with spacer. Kirstein et al. (1986) investigated the binding of glucose dehydrogenase onto differently activated supports and found the immobilization onto alginate activated by Woodward’s reagent problematic too (no yields/activities given). When low and high molecular weight dextran species are used as substrates, activity loads remain unchanged. This suggests that the enzyme binding takes place on the outer shell of alginate. Furthermore high calcium chloride concentrations (1 M) during immobilization reduce the achieved activity by 40%.

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4. Conclusions In our studies we investigated the immobilization of industrial dextranase, which should deliver a highly active adsorbent compatible with the boundary conditions of our reactor concept, involving the use of entrapped dextransucrase in a fluidised bed reactor at pH 5.4. From adsorption experiments, the bentonite carriers exhibit high monolayer protein and activity loads at pH 4.0 (q* = 0.8 g g−1 ; amax = 94,000 U g−1 ) and after pH-shift to 5.4 (amax = 12,000 U g−1 ), however dextranase to bentonite ratios above 0.4 must be used to avoid extensive enzyme inactivation. Yet follow-up experiments indicate detrimental effects of bentonite in connection with the entrapped dextransucrase, thus bentonite cannot be used for co-immobilization. Other selected carriers used are hydroxyapatite (q* = 0.032 g g−1 ; amax = 2900 U g−1 ) and Streamline DEAE (q* = 0.009 g mL−1 ; amax = 93 U mL−1 ). In general, immobilization yields are low and in case of DEAE the rather high particle size is problematic. For the covalent attachment, porous Eupergit® differs only moderately in activity using low molecular weight substrate (Eupergit® C: 741 U g−1 ; Eupergit® C250L: 509 U g−1 ). However, macroporous Eupergit® C250L reveals enhanced activity for high molecular weight substrates (83 U g−1 versus 11 U g−1 Eupergit® C), which increases the susceptibility to external mass transfer limitations. Activation of the carbohydrate moiety of dextranase by metaperiodate results in active silica gels of 49 U g−1 , whereas the activation of alginate via Woodward’s reagent K exhibits extremely low activities in the range of 2–3 U g−1 . In future, we will focus on the realization of CoImmobilisates of dextranase and dextransucrase based on our investigated carriers. Acknowledgements The authors would like to thank the German Research Foundation DFG that supported this project in the form of a grant to the collaborative research centre SFB 578 “From gene to product”. We are also grateful to Novozyme A/S, Denmark, for supplying dextranase. References Angeletti, E., Canepa, C., Martinetti, M., Venturello, P., 1989. Amino groups immobilized on silica gel: an efficient and reusable heterogeneous catalyst for the Knoevenagel condensation. J. Chem. Soc. Perkin Trans. 1, 105–107. Bajpai, A.K., Sachdeva, R., 2001. Immobilization of diastase onto bentonite clay surfaces. Coll. Polym. Sci. 280, 892–899. Berensmeier, S., Ergezinger, M., Bohnet, M., Buchholz, K., 2004. Design of immobilised dextransucrase for fluidised bed application. J. Biotechnol. 114 (3), 255–267. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of proteins utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. Buttersack, C., Nowikow, K., Schaper, A., Buchholz, K., 1995. Gewinnung von Enzymen aus Zuckerr¨uben. Zuckerindustrie 119 (4), 284–291. de Segura, A.G., Alcalde, M., Yates, M., Rojas-Cervantes, M.L., L´opez-Cort´es, N., Ballesteros, A., Plou, F.J., 2004. Immobilization of dextransucrase from leuconostoc mesenteroides nrrl b-512f on Eupergit C supports. Biotechnol. Prog. 20 (5), 1414–1420.

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