hydrophobic interaction chromatography on octyl

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HYDROPHOBIC INTERACTION CHROMATOGRAPHY ON OCTYL SEPHAROSE—AN APPROACH FOR ONESTEP PLATFORM PURIFICATION OF CYCLODEXTRIN GLUCANOTRANSFERASES a

a

b

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Premalatha Shetty , Smitha Bhat , J. L. Iyer , Srikant Shenoy , b

J. S. Pai & K. Satyamoorthy

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Department of Biotechnology, Manipal Life Sciences Centre, Manipal University, Manipal, Karnataka, India b

Food and Fermentation Technology Division, Institute of Chemical Technology, University of Mumbai, Nathalal Parekh Marg, Matunga, Mumbai, Maharashtra, India Available online: 03 Oct 2011

To cite this article: Premalatha Shetty, Smitha Bhat, J. L. Iyer, Srikant Shenoy, J. S. Pai & K. Satyamoorthy (2011): HYDROPHOBIC INTERACTION CHROMATOGRAPHY ON OCTYL SEPHAROSE—AN APPROACH FOR ONE-STEP PLATFORM PURIFICATION OF CYCLODEXTRIN GLUCANOTRANSFERASES, Preparative Biochemistry and Biotechnology, 41:4, 350-364 To link to this article: http://dx.doi.org/10.1080/10826068.2010.548434

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Preparative Biochemistry & Biotechnology, 41:350–364, 2011 Copyright # Taylor & Francis Group, LLC ISSN: 1082-6068 print/1532-2297 online DOI: 10.1080/10826068.2010.548434


HYDROPHOBIC INTERACTION CHROMATOGRAPHY ON OCTYL SEPHAROSE—AN APPROACH FOR ONE-STEP PLATFORM PURIFICATION OF CYCLODEXTRIN GLUCANOTRANSFERASES

Premalatha Shetty,1 Smitha Bhat,1 J. L. Iyer,2 Srikant Shenoy,1 J. S. Pai,2 and K. Satyamoorthy1 1 Department of Biotechnology, Manipal Life Sciences Centre, Manipal University, Manipal, Karnataka, India 2 Food and Fermentation Technology Division, Institute of Chemical Technology, University of Mumbai, Nathalal Parekh Marg, Matunga, Mumbai, Maharashtra, India

& Cyclodextrin glucanotransferase (CGTase) from Bacillus circulans ATCC 21783 was concentrated by ultrafiltration and subsequently purified by hydrophobic interaction chromatography on Octyl Sepharose 4 fast flow. The matrix was able to bind selectively to the enzyme at a very low ammonium sulfate concentration of 0.67 M and enzyme desorption was performed by decreasing gradient of the salt. The overall recovery was 80% with 689-fold purity. CGTases derived from four soil isolates and Toruzyme, the commercial preparation of CGTase, also bound to Octyl Sepharose under similar conditions at 0.67 M and eluted at 0.55–0.5 M of ammonium sulfate. Octyl Sepharose chromatography can thus be used as a platform approach for purification of CGTases from various bacterial sources. Long stretches of sequence predominated by hydrophobic amino acids are reportedly present in the starch binding domains of CGTases. Starch binding experiments indicated the binding of the enzymes to the octyl matrix through these domains. Keywords alkalophilic Bacillus, cyclodextrin glucanotransferase, hydrophobic interaction chromatography, Octyl Sepharose, purification, starch binding domain

INTRODUCTION Cyclodextrin glucanotransferase (CGTase; EC 2.4.1.19) is an extracellular transglucosidase that hydrolyzes starch and related carbohydrates, resulting in formation of cyclodextrins (CDs). CDs, the nonreducing cyclic polymers of a-(1-4)-linked D-glucopyranose units, possess an interior cavity that is relatively apolar compared to water.[1,2] Guest molecules of appropriate size and shape can be included into this cavity. This ability of CDs to form Address correspondence to Premalatha Shetty, Department of Biotechnology, Manipal Life Sciences Centre, Manipal University, Manipal 576 104, Karnataka, India. E-mail: [email protected]


Purification of CGTases by Octyl Sepharose Chromatography

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inclusion complexes with a wide range of molecules has been exploited extensively in food, pharmaceutical, chemical, cosmetic, and agricultural industries.[3,4] The most common CDs comprised of six, seven, and eight glucose units are known as a-, b-, and c- CD, respectively. The CGTase enzyme can also transfer glycosyl residues from a donor to a suitable glycosyl acceptor, resulting in formation of various other functional oligosaccharides.[5] A wide variety of bacteria belonging to the genera Bacillus, Klebsiella, Corynebacter, and Thermoanaerbacterium produce CGTases.[4,6] Production and purification of this enzyme have hence been extensively studied. Majority of the purification protocols reported for this enzyme employ two or more column fractionations involving techniques such as adsorption onto starch, gel filtration, affinity and ion-exchange chromatography, etc.[7–10] Multistep purification is laborious and generally results in low yield. Single-step purification methods using affinity ligands have also been reported.[11–14] Preparation of affinity matrices using cyclodextrins as ligands is time-consuming and expensive. The present investigation employs hydrophobic interaction chromatography for purification of CGTase from various bacteria. Moreover, the matrix Octyl Sepharose used in this study is known to show differential selectivity.[15] Hydrophobic interaction chromatography is relatively a more economically viable process in comparison to affinity chromatography. Hence the chromatographic behavior of various CGTases on this matrix was studied as a possible approach for singlestep purification of this enzyme. The results indicate the possibility of the starch binding domain (SBD) of the enzyme being involved in binding to the matrix.

MATERIALS AND METHODS Materials Bacillus circulans (ATCC 21783) was procured from American Type Culture Collection Centre, Rockville, MD. Soluble starch (Merck, India), Toruzyme 3.0 L (Novozymes), ribonuclease (SRL, India), and lysozyme (SRL, India) were procured. The bovine serum albumin and standard b-cyclodextrin were from Sigma (St. Louis, MO). Octyl Sepharose 4 fast flow resin was purchased from Amersham Pharmacia Biotech, Uppsala, Sweden. Polyethersulfone membrane (molecular mass cutoff 10 kD) from Millipore, USA, was used. Molecular mass markers (PMW-M) were from Bangalore Genei, India. Chromatographic experiments were performed using Pharmacia Frac 100 fraction collector. Spectronic Genesys 5 was employed for spectrophotometric measurements.

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Production of CGTase The production medium contained (g=L): tapioca starch, 20.0; yeast extract, 15.0; magnesium sulfate, 0.2; dipotassium hydrogen phosphate, 1.0; and sodium carbonate 10.0. All other components except sodium carbonate were added to gelatinized starch and autoclaved. Sodium carbonate solution was autoclaved and added separately. Production was carried out as reported in our earlier publication.[16] The medium was dispensed in 50-mL aliquots in 500-mL Erlenmeyer flasks. Inoculum (1.0%; OD at 660 nm ¼ 0.1) was added to the flasks, which were incubated on a rotary shaker at 200 rpm, 30 C for 84 hr. The broth was centrifuged at 6,000 g for 20 min and the supernatant was assayed for CGTase activity by dextrinising assay. A cyclizing assay was performed to confirm formation of cyclodextrins.

Assay of Starch Dextrinizing Activity The method for measuring dextrinizing activity (ability of the enzyme to break down starch into dextrins) was modified.[7] This was considered an assay for CGTase activity since preliminary testing employing dinitrosalicylic acid showed that the enzyme action did not lead to formation of significant amount of reducing sugars. Substrate solution (0.25 mL), containing 1.5 mg of soluble starch in 0.05 M glycine=NaCl=NaOH buffer, pH 8.5, was incubated with 0.05 mL of suitably diluted enzyme at 50 C for 10 min. The reaction was stopped by addition of 1 mL of 0.5 M acetic acid. After addition of 0.5 mL of 0.02% iodine–0.2% potassium iodide reagent, the solution was diluted to 10 mL with distilled water. Absorbance was measured at 700 nm. One unit is defined as the amount of enzyme that can dextrinise 1 mg starch per minute at 50 C.

Estimation of b-CD The amount of b-CDs was measured based on the reduction in the color of phenolphthalein due to complexation with b-CD, as per the method of Kaneko et al.[17] Nine hundred and fifty microliters of 4% (w=v) soluble starch dissolved in 0.1 M phosphate buffer, pH 7.0, was mixed with 50 mL of the enzyme solution. The reaction mixture was incubated at 50 C for 30–60 min. The reaction was stopped by adding 3.5 mL of 0.03 M NaOH followed by addition of 0.5 mL of 0.04% phenolphthalein prepared in 0.01 M sodium carbonate solution. A550 nm was read after 15 min. b-CD concentrations ranging from 0 to 2 mg=mL were prepared for standardization. A calibration curve was constructed by plotting milligrams of b-CD against percent reduction in absorbance (y axis).


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Estimation of Protein Protein concentration was determined using Folin–Ciocalteau’s phenol reagent. The protein concentration in the eluted fractions of the chromatographic run was monitored at 280 nm. Purification of CGTase


Concentration by Ultrafiltration (UF) The cell-free broth was concentrated approximately five- to sixfold by ultrafiltration (UF) using a 10-kD membrane. Hydrophobic Interaction Chromatography Concentrated crude enzyme containing 0.67 M (NH4)2SO4 was applied to an Octyl Sepharose 4 fast flow column (9 mm 98 mm, 6 mL) preequilibrated with 0.67 M ammonium sulfate ((NH4)2SO4) in 0.1 M phosphate buffer, pH 7. The flow rate was maintained at 0.6 mL per minute and fractions of 3.0 mL were collected. After washing with three bed volumes of the buffer, the enzyme was eluted with a decreasing gradient of (NH4)2SO4. The fractions were analyzed for dextrinizing activity. Concentration of ammonium salt was estimated by Nesslerization. Molecular Mass Determination SDS-PAGE electrophoresis in 10% polyacrylamide gel was carried out as per the method of Laemmli.[18] Molecular mass markers in the range of 14–97.4 kD were used. Enzyme Binding Assays Starch Binding Assay The retentates obtained by UF were used for the study. UF retentate from B.circulans (ATCC 21783) was diluted with 0.1 M phosphate buffer, pH 7, to obtain various concentrations of CGTase ranging from 0.2 to 8 U=mL. Ammonium sulfate was added to a final concentration of 0.67 M. For control tubes, dilutions were prepared without (NH4)2SO4. Two milliliters from each dilution was incubated with 0.5 g starch (prewashed with the buffer with=without ammonium sulfate for test=control, respectively), for 30 min at 4 C with intermittent shaking (every 5 min). The suspension was centrifuged at 2000 rpm for 10 min at 4 C. The supernatant was assayed for dextrinizing activity to calculate the enzyme that remained unadsorbed. The starch pellet was subjected to washing with respective buffers at 4 C. The washing was discarded and the pellet was resuspended in

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2 mL of the phosphate buffer containing 4% soluble starch. The suspension was incubated at 50 C for 1 hr in a shaken water bath. The supernatant obtained after centrifugation was kept in boiling water bath for 4–5 min for inactivation of the enzyme which may have leached into solution. Amount of b-CD formed in this supernatant due to enzyme action was measured by analyzing appropriate volumes of the aliquots by phenolphthalein complexation. Starch binding assay was also performed by incubating 1 U each of the CGTases from Bacillus sp. M4, Bacillus sp. M1a, and Toruzyme with 0.5 g of the starch. Effect of Starch on Binding of the Enzyme to Octyl Sepharose in the Presence of (NH4)2SO4 Starch solution (1.2% w=v, 0.5 mL) in 0.1 M phosphate buffer, pH 7, containing 0.67 M (NH4)2SO4 and 0.9=1.2 units of CGTase was incubated with 0.2 mL Octyl Sepharose for 10 min at 4 C with intermittent shaking (every 2 min). Control experiment was performed in absence of starch. The suspension was centrifuged at 2000 rpm for 10 min at 4 C. The resin was washed with the buffer containing (NH4)2SO4 and the bound enzyme was desorbed using 1 mL of the buffer without (NH4)2SO4. The desorbate was subjected to phenolphthalein complexation method to measure the b-CDs produced by the desorbed enzyme. RESULTS AND DISCUSSION Bacillus circulans (ATCC 21783) is well documented as a CGTase producer. The cell-free broth containing extracellular CGTase from this organism was concentrated by UF. Retentate (100 U) containing 0.67 M (NH4)2SO4 was applied to the Octyl Sepharose 4 column. Enzyme adsorption was studied at different concentrations of the (NH4)2SO4 ranging from 0.4 M to 1.2 M. It was found that the enzyme bound to the matrix at a concentration of 0.6 M. The concentration of (NH4)2SO4 was further optimized to 0.67 M (around 16% saturation) for adsorption onto the matrix. As shown in Figure 1, the chromatographic profile of the proteins (A280 nm) showed that the majority of the contaminant proteins got eluted in the flow through and in the subsequent washings with the equilibrating buffer. Elution of the bound enzyme was carried out using decreasing gradient of the (NH4)2SO4. The enzyme elution began at around 0.55 M of the salt. A280 of the fractions exhibiting CGTase activity was insignificant, as can be seen from Figure 1, indicating a high degree of purification. The enzyme was purified approximately 689-fold with 80% yield and a specific activity of 2341 U=mg protein, as shown in Table 1. Octyl Sepharose is made of highly cross-linked agarose beads, which offer an excellent flow property to the medium. At a flow rate of 36 mL=h, hydrophobic interaction chromatography gave good fold purity



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FIGURE 1 Chromatographic profile of B. circulans CGTase purification using Octyl Sepharose. Fractions 1–6: flow through (unadsorbed fraction) and washings. Fractions 7 onward: eluant fractions. (Color figure available online.)

and yield, and hence can be used for processing large volumes. The enzyme was purified to homogeneity as the purified preparation showed a single band, and the molecular mass of the enzyme as exhibited by SDS PAGE was found to be around 80 kD (Figure 2). TABLE 1

Purification of CGTases by Hydrophobic Interaction on Octyl Sepharose

Source B. circulans (ATCC 21783)

Bacillus sp. M4

Bacillus sp. 1a

Toruzyme 3.0 L

a

Parametera

Crude

UF

HICb

Specific activityc Fold purity Yield (%) Specific activity Fold purity Yield (%) Specific activity Fold purity Yield (%) Specific activity Fold purity Yield (%)

3.4 0.24 1 100 4.3 0.21 1 100 0.11 0.01 1 100 71.82 2.92 1 100

6.3 0.29 1.85 91 2.2 9.2 0.45 2.14 91.7 1.6 0.23 0.03 2.1 93.8 5.4 101.9 7.05 1.42 90.1 3.6

2341 170 688.5 79.3 1.2 1988 107 462.33 81.85 2.5 40.4 4.1 367 83.7 3.3 1625 110 22.62 80.5 2.4

Mean of three determinations. Hydrophobic interaction chromatography. c Specific activity in U=mg. b


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FIGURE 2 SDS-PAGE of purified CGTases from different sources. The gel was stained with Coomassie brilliant blue. Lane 1, Bacillus circulans (ATCC 21783); lane 2, Toruzyme; lane 3, molecular mass protein markers; lane 4, Bacillus sp. M1a; lane 5, Bacillus sp. M4.

The ability of Octyl Sepharose matrix to bind to CGTase from various other bacteria was further tested. Four CGTase-yielding bacteria have been isolated from soil samples in our laboratory. All four strains were found to be alkalophilic. Bacillus coagulans (MTCC 6201) and B. circulans (MTCC 6200) have been deposited at MTCC, Chandigarh, India. The identification of the other two isolates, viz., Bacillus sp. M4 and Bacillus sp. M1a, to species level is ongoing. CGTases from these isolates (crude cell free broth) were subjected to the same purification process as described earlier. In all four cases, the CGTase enzymes behaved identically. The binding condition was in the presence of 0.67 M (NH4)2SO4 at pH 7 and elution at 0.5 M (NH4)2SO4. The commercial preparation of CGTase, i.e., Toruzyme, which is derived from a strain of Thermoanaerobacter, could also be purified using precisely the same hydrophobic interaction chromatography process. Of the four isolates, the isolates Bacillus sp. M4 (slender rods) and Bacillus sp. M1a (thick rods) produced the highest and lowest units, respectively (unpublished results). Bacillus sp. M4 was not able to grow at neutral pH. However, Bacillus sp. M1 was found to grow at neutral pH, albeit at a slow rate. The CGTases from these two distinct strains were further subjected to the same purification protocol as that for Bacillus circulans ATCC 21783. The UF retentate (100 U and 20 U, respectively, of CGTase from Bacillus sp. M4 and Bacillus M1a) was purified as described earlier. Results are shown



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in Table 1. The enzyme produced by Bacillus sp. M4 was purified to the specific activity of 1988 and recovered to an extent of 82% with an overall fold purity of 462. The Bacillus M1a CGTase was purified 367 times with a yield of around 84%. CGTase from Toruzyme 3.0 L, subjected to a similar purification protocol, was purified 23-fold with a recovery of 81%. Figure 2 shows the sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) results for the purified preparations of the enzymes. Purified CGTases from Toruzyme, Bacillus sp. M1a, and Bacillus sp. M4 showed protein bands with molecular mass of 76.5, 76, and 77.5 kD, respectively. Purification of CGTase by affinity chromatography using starch or CDs as ligands is extensively reported. Starch adsorption followed by immunoaffinity purification of cyclodextrin glycosyltransferase from Bacillus circulans A11 gave an overall purification fold of 155 at a recovery of 45%.[19] A purification protocol involving starch adsorption for CGTase from an alkalophilic Bacillus agaradhaerens isolate was reported to give 43-fold purity and a yield of 50%.[20] CGTase from Klebsiella pneumoniae AS-22A was purified 736 times with a recovery of 68% by chromatography on cornstarch followed by Sephacryl S 200 gel filtration.[21] Affinity chromatography using cyclodexrins as ligands has been reported. Using an /-CD (epoxy)Sepharose 6B column, Sian et al. reports an increase in purity to an extent of 2200-fold. However, the yield was only around 4%.[13,22] CGTase from B. coagulans was purified by Akimaru et al.[23] using a series of steps involving starch adsorption and four different chromatographic techniques, which included hydrophobic interaction chromatography with Phenyl Sepharose as one of the steps. Enzyme adsorbed to Phenyl Sepharose at 1 M (NH4)2SO4. The hydrophobic interaction chromatography step was able to purify the enzyme 1.9-fold with a yield of around 75%. A report on B. firmus CGTase wherein the UF concentrate was chromatographed using Phenyl Sepharose led to 8.9-fold purification and a percent recovery of 64.7.[24] In the present study, a recovery of 88% was achieved for the enzymes from all the three different sources with the fold purity ranging from 175 to 371 in the hydrophobic interaction chromatography step (specific activity of the purified enzyme:specific activity of UF retentate). As mentioned earlier, the octyl derivative is known to give differential selectivity. It appears that CGTases from various bacteria could thus be selectively purified using Octyl Sepharose. To investigate further, ribonuclease, bovine serum albumin, and lysozyme were loaded onto an Octyl Sepharose column under similar conditions. We found that all the three enzymes eluted unadsorbed. Thus, these studies indicate that hydrophobic interaction chromatography on Octyl Sepharose can serve as a common purification platform process for CGTases. In hydrophobic interaction chromatography, (NH4)2SO4 concentration of 0.8–1 M is generally used as a starting point for screening experiments.[15,24]

358 TABLE 2 Bacteria

P. Shetty et al. Stretch of Sequence Dominated by Hydrophobic Amino Acids in CGTases from Various


Source (NCBI Version=PUBMED Total Number Stretch of Sequence Rich in Number) of Amino Acids Hydrophobic Amino Acids

Domain

Bacillus sp. 1-1 (P31746.1=GI: 399220) Bacillus sp. B1018 (GI: 98164= PUBMED: 1689153) Bacillus sp. I-5 (AAR32682.1=GI: 39933006) Paenibacillus sp. JB-13 (ACB71089.1=GI: 172054128) Bacillus sp. 17-1 (PUBMED: 2534600) Paenibacillus macerans (P04830, GI: 461717) Bacillus sp. 38-2 (PUBMED: 2972812)

703

481–544 and 565–600

NA

713

440553 and 565–642

NA

712

440–640

NA

713

468–644

NA

713

451–644

613. . .709: SBD

714 712

440–530 and 539–600 and 631639 440–643

B. circulans strain 251 (1CXK_A= GI: 4930027)

686

413–526 and 574–617

Anaerobranca gottschalkii (CAH61550.1=GI: 54304042) Geobacillus stearothermophilus (P31797.1=GI: 399224) Alkalophilic Bacillus sp. 1011 (1UKQ_B=GI: 46015834)

721

514–606

438. . .522: Aamy C 614 . . . 710: SBD 437 . . . 521: Aamy_C 612. . .708: SBD 410. . .494: Aamy_C 586. . .682: SBD NA

711

440–593 and 610–640

NA

686

423–617

Thermoanaerobacterium thermosulfurigenes (1CIU_A=GI: 157830625)

683

415–537 and 571–613

411. . .494: Aamy_C 586. . .682: SBD 410. . .492: Aamy_C 582. . .681: SBD

Note. SBD, starch binding domain.

Hydrophobic ligands such as butyl, phenyl, and octyl and decyl agarose were screened for purification of alkaline protease by Adikane et al.[25] They report optimum results with phenyl agarose in a single-step operation, with 20-fold purity and 40% yield. Adsorption of this enzyme on octyl agarose was reported to be only 35–45% in the presence of 2 M (NH4)2SO4. However, in the present study, adsorption of CGTase on octyl sepharose occurred at a much lower salt concentration of 0.67 M. Cell membrane-bound enzymes are known to bind to hydrophobic matrices at concentrations below 0.8 M. In the present investigation, the enzyme adsorbed to the matrix at a relatively lower salt concentration. The results imply that all these CGTases must comprise a common structural feature that gets exposed in the presence of 0.67 M of (NH4)2SO4 enabling them to bind to an octyl matrix. Additionally, lower consumption of the salt is not only economic but also environmentally friendly.



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Addition of (NH4)2SO4 is known to unmask the hydrophobic patches on the surface of proteins. The CGTases may be having regions rich in hydrophobic amino acids at or closer to the surface that gets exposed to the environment, at such a low saturation level of (NH4)2SO4. CGTases in general are composed of five domains (A, B, C, D, and E). Domains A, B, and C are involved in the catalytic role of the enzyme. The primary structure of domain E contains a typical starch binding motif found in other starch-binding proteins.[26–30] The amino acid sequences of CGTases from various sources were retrieved from the NCBI database and were analyzed by the Kyte–Doolittle hydropathy plot at a window size of 19 (http://gcat. davidson.edu/rakarnik/kyte-doolittle.htm). Long stretches of the sequence exhibiting positive values (hydropathicity) were selected (Table 2). The CGTases from all the bacteria showed stretches of sequence close to the C-terminus dominated by hydrophobic amino acids. In case of CGTases derived from Bacillus sp. 17-1, Paenibacillus macerans, Bacillus sp. 38-2, Bacillus circulans strain 251, and alkalophilic Bacillus sp. 1011, the details of the Aamy_C and starch binding domain are available in the database. When the data were analyzed it was observed that the Aamy_C domains and starch binding domains contain stretches of sequence dominated by hydrophobic amino acids. The results of the Kyte–Doolittle plot for CGTase from Bacillus sp. 38-2 is given in Figure 3. The continuous stretch of sequence from

FIGURE 3 Kyte–Doolittle plot for the CGTase from Bacillus sp. 38-2; x axis indicates the amino acid number and y axis denotes the hydropathy index. Arrow indicates a hydropathic index of 0 on the x axis. (A) The protein sequence was analyzed by Kyte–Doolittle plot. The stretch of sequence predominated by hydrophobic amino acids (440–643) is shown in the inset (B).


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FIGURE 4 Effect of (NH4)2SO4 on the binding of CGTase from B. circulans (ATCC 21783) to starch granules. Starch, 0.5 g each, was incubated with increasing units of CGTase in the presence=absence of 0.67 M (NH4)2SO4. Experimental conditions are given in the text. Supernatant was assayed for dextrinizing activity to determine the unbound activity. Difference between the applied units and the unbound units is considered as bound units. Pellet containing the adsorbed enzyme was incubated with solubilized starch and the resultant b-CD was measured by phenolphthalein complexation method. Amount of b-CD formed is proportional to bound activity. (Color figure available online.)

440–643 contributing to predominantly positive hydrophathic index shown in the inset includes 96.4% of the Aamy_C domain (440..521 of 437–521) and a long stretch encompassing around one-third of the starch binding domain domain (612 . . . 643 of 612 . . . 708). The reports suggest that the starch binding domain of CGTases is an independent domain that appears to have been conserved through evolution and that it retains its starchbinding ability while maintaining its original conformation, even if separated from the other four domains.[26–28,30] The possibility of the enzyme binding to the octyl matrix via its Aamy_C domain or starch binding domain was further investigated. If the hypothesis is that (NH4)2SO4 at 0.67 M uncovers the Aamy_C domain, then there is a possibility that the activity of the enzyme could be affected (as Aamy_C domain is involved in the enzyme activity). To test this hypothesis, the enzyme activity was monitored in the presence and absence of (NH4)2SO4. For all three CGTases, activity remained unaltered. Purification techniques wherein enzyme binds to the matrix through its catalytic site often lead



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to significant loss in recovery. In this study, we were able to achieve a very good recovery of the enzyme of up to 88%. The focus was then shifted to find the involvement of starch binding domain in binding. Effects of starch binding studies were examined in the presence of (NH4)2SO4. Increasing concentrations of the enzyme (UF concentrate) from B. circulans were incubated with granular starch in the presence=absence of (NH4)2SO4. As shown in Figure 4, the supernatant (unbound fraction) collected in the presence of 0.67 M salt exhibited lower dextrinizing activity as compared to the unbound fraction obtained from the corresponding control tubes. It appears that increased binding of the enzyme had occurred in the presence of salt, leaving behind lower amount of the enzyme in supernatent. To confirm the increased binding of enzyme that had occurred in the presence of the salt, the enzyme-bound starch fractions were further incubated with solubilized starch. It was found that the starch fraction to which enzyme had bound in the presence of (NH4)2SO4 produced more b-CDs in comparison to the control tubes to which the same units of CGTase had been applied (Figure 4). Thus, it was confirmed that the presence of 0.67 M (NH4)2SO4 facilitated the binding of enzyme to starch. Starch binding study was also carried out for CGTases from Bacillus sp. M4, Bacillus sp. M1a, and Toruzyme. The results are summarized in Table 3. The presence of salt enhanced the binding of the enzyme to starch. It appears that the starch binding domain or a region in close vicinity to this domain gets exposed in the presence of the salt. If a region at or TABLE 3

Starch Binding Assay


Bacillus sp. M4

Bacillus sp. M1a

Incubation period (min)

(NH4)2SO4 (M)

Units unbound (U)

Units bounda(B)

b-CD formedb (m) g

10 30 10 30 10 30 10 30 10 30 10 30

0 0 0.67 0.67 0 0 0.67 0.67 0 0 0.67 0.67

0.34 0.01 0.24 0.01 0.22 0.02 neg 0.38 0.03 0.26 0.02 0.24 0.02 neg 0.37 0.02 0.29 0.02 0.23 0.01 0.05

0.66 0.76 0.78 1.0 0.62 0.74 0.76 1 0.63 0.71 0.77 0.95

2.21 0.08 2.56 0.11 2.72 0.19 3.29 0.3 2.05 0.09 2.38 0.08 2.85 0.17 3.44 0.21 2.2 0.07 2.39 0.13 2.68 0.21 3.32 0.08

Note. Experimental conditions are given in text. One unit of the ultrafiltrate was incubated with 0.5 g of the starch. For the incubation period of 10 min, intermittent shaking was performed every 2 min. Supernatent was assayed for dextrinising activity to determine the unadsorbed activity. Amount of b-CD formed by the bound enzyme was estimated by phenolphthalein complexation method. a B, Difference between the applied unit and the unbound units ¼ 1 – U. b Milligrams of b-CD formed=hr by bound enzyme at 50 C.

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TABLE 4

Binding of Enzyme to Octyl Sepharose in the Presence of Starch


Bacillus sp. M4


Bacillus sp. M1a

Units Applied

Starch (%)

Units Unbound

Units Desorbeda

b-CDb(mg)

0.9 1.2 0.9 1.2 0.9 1.2 0.9 1.2 0.9 1.2 0.9 1.2

0 0 1 1 0 0 1 1 0 0 1 1

Negligible Negligible 0.25 0.01 0.3 0.02 0.04 0.06 0.26 0.02 0.31 0.02 0.06 0.09 0.21 0.01 0.31 0.01

0.76 0.05 1.01 0.03 0.57 0.02 0.83 0.05 0.76 0.04 0.94 0.05 0.53 0.02 0.79 0.02 0.73 0.03 0.98 0.04 0.57 0.01 0.81 0.06

2.61 0.11 3.1 0.13 1.68 0.08 2.63 0.11 2.46 0.12 2.99 0.09 1.62 0.04 2.45 0.16 2.27 0.05 3.04 0.15 1.59 0.11 2.44 0.17

Note. Ultrafiltrate containing 0.9=1.2 units was incubated with 0.2 mL of the resin in phosphate buffer containing 0.67 M ammonium sulfate in the presence or absence of starch. The enzyme bound to the resin was desorbed and was analyzed for bound activity. Experimental conditions are given in text. a Dextrinizing activity in the desorbate, which reflects the bound activity. b Milligrams of b-CD formed=hr by desorbed enzyme at 50 C.

near the starch binding domain is involved in binding to the octyl matrix, then the presence of starch may reduce the binding of enzyme to the matrix as starch and the octyl matrix may compete for this binding site. In the experiment wherein the CGTase was allowed to bind to the octyl matrix in the presence of starch at 0.67 M (NH4)2SO4, decreased binding of the enzyme to the matrix was observed. These results are discussed in Table 4. The results indicated that the activity bound to the matrix was lower than that in the control tubes. Thus, it can be concluded that regions at or near the starch binding domain are perhaps involved in binding of the enzyme to the matrix. The potential of using microbial starch binding domains as immobilization tags has been reviewed by Rodrı´guez-Sanoja et al. (2005).[30] Studies involving recombinant DNA techniques have already been reported to understand the contribution of starch binding domain to the functioning of CGTases.[31,32] Deletion of starch binding domain should render the enzyme unable to adsorb onto the octyl resin. If proved, the starch binding domain reportedly being an independent domain can be employed as a tag for expression of fusion proteins, which can then be easily purified using octyl derivatized matrices. CONCLUSION Simplicity and efficiency of the method should allow hydrophobic interaction chromatography employing Octyl Sepharose 4 fast flow to be a readily


363

adapted process for the large scale purification of CGTases. The possibility of regeneration and reuse of the column and mechanical stability of the matrix make hydrophobic interaction chromatography a valuable approach to purify CGTase in a single chromatographic step. As in the present study all the six CGTases bound to the column at 0.67 M (NH4)2SO4 and could be eluted by decreasing the salt concentration to 0.5 M, it appears that hydrophobic interaction chromatography using an octyl matrix can be employed as a platform approach for purification of CGTases from various sources.


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