Primary capture of cyclodextrin glycosyltransferase derived from Bacillus cereus by aqueous two phase system

Separation and Purification Technology 81 (2011) 318–324

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Primary capture of cyclodextrin glycosyltransferase derived from Bacillus cereus by aqueous two phase system Hui Suan Ng a, Chin Ping Tan b, Soo Kien Chen c, Mohd Noriznan Mokhtar a, Arbakariya Ariff d, Tau Chuan Ling e,⇑ a

Department of Process and Food Engineering, Faculty of Engineering, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia Department of Food Technology, Faculty of Food Science and Technology, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia Department of Physics, Faculty of Science, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia d Department of Bioprocess Technology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia e Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia b c

a r t i c l e

i n f o

Article history: Received 17 May 2011 Received in revised form 26 July 2011 Accepted 27 July 2011 Available online 4 August 2011 Keywords: Aqueous two phase system Cyclodextrin glycosyltransferase Cyclodextrin Enzyme Purification

a b s t r a c t In this works, the polymer–salt aqueous two phase system (ATPS) which is polyethylene–glycol (PEG) with sodium citrate was constructed to purify the enzyme CGTase from fermentation broth. Impacts of parameters such as phase composition, tie-line length (TLL), volume ratio (VR), crude sample loading, pH and the addition of sodium chloride (NaCl) on the partition behavior of cyclodextrin glycosyltransferase (CGTase) were investigated. The study exhibited that the optimum system for the purification of the enzyme CGTase was achieved on the 19.0% PEG and 11.5% citrate system with TLL of 38.89% (w/w), VR of 2.0, with additional 4% (w/w) NaCl and 20% crude load at pH 7.0. CGTase from Bacillus sp. was partially purified by the ATPS up to 16.3-fold with a yield of 70%. Ó 2011 Elsevier B.V. All rights reserved.

1. Introduction Cyclodextrin glycosyltransferase (CGTase; E.C. 2.4.1.19) is an extracellular enzyme [1] that involved in the starch bioconversion in which it catalyzes the cyclization (transglycosylation) reaction in the conversion of starch into cyclodextrins (CDs) [2–4]. CGTase is classified as a hydrolytic enzyme which able to perform both intramolecular and intermolecular transglycosylation reactions such as cyclization, coupling and disproportionation of oligosaccharides. In the starch bioconversion, starch is being converted into a mixture of non-reducing cyclic structures of glucose residues which known as cyclodextrins (CDs) in different ratios via the cyclization reaction. Besides, CGTase also possesses a weak hydrolytic activity in which water acts as the glycosyl acceptor [5–7]. CGTase is produced by a variety of bacteria and among the bacteria; species of Bacillus is reported as the most common producers of this enzyme. In addition, Bacillus species was among the bacteria that produce most industrial enzymes which are widely involved in various industries ranging from pharmaceutical to even textile industries. Besides, Klebsiella, Micrococcus, Clostridium, Corynebacterium, Pseudomonas, Thermoanaerobacter, Thermoanaerobacterium

⇑ Corresponding author. Tel.: +60 3 79674354; fax: +60 3 79674178. E-mail address: [email protected] (T.C. Ling). 1383-5866/$ - see front matter Ó 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.seppur.2011.07.039

are other bacteria species that reported to be involved in the production of the enzyme CGTase [8–11]. Apart from that, CGTase was also recently reported to be produced by thermophilic Archae [8]. In this study, the species Bacillus cereus was found to produce the enzyme CGTase and being purified by ATPS purification method. The strain of B. cereus were isolated from rotten potatoes and was identified using 16S rDNA bacteria identification analysis. There are many studies reported on the purification methods employed to harvest enzyme CGTase from the fermentation broth. The common purification methods used to purify enzyme CGTase as reported includes starch adsorption, column chromatographyfollowed by gel filtration and ion exchange chromatography [6,11 ,12]. Nevertheless, these purification methods have their own drawbacks so far. Drawback with the starch column is that enzyme CGTase will react with the starch to form cyclodextrin during the elution and this reduces the recovery of the purified enzymes as additional steps to remove the CDs produced is required. Previous studies revealed that loss of enzyme reported even in multiple steps ion exchange chromatography during the purification processes [11]. In previous study, Chang et al. have successfully produced and separated cyclodextrin homologues (CDs) from CGTase by using PEG/salt and PEG/ dextran ATPS [13]. In the study, the CGTase was partitioned to the PEG-rich phase where the bioconversion took place there. However, there are no studies reported on the purification of CGTase enzyme by employing the ATPS [14] partitioning at present. Hence in this

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work, attempts on applying ATPS partitioning for the purification of enzyme CGTase from the fermentation broth was investigated. Apart from that, the factors affecting the partition behavior of the enzyme CGTase such as the molecular weight of the polymer, TLL, VR, pH, addition of NaCl was also investigated in order to achieve the optimum condition for the enzyme purification. Sodium citrate was selected in this study to construct the ATPS as it has lower eutrophication potential which is more biodegradable compared to other salts environmentally. [15]. 2. Materials and methods 2.1. Materials Cyclodextrins (CDs) were purchased from Sigma Chemical Co., MO, USA. Polyethylene glycol (PEG) of average molecular weight 6000, 8000 and 10,000 g/mol were obtained from Fluka Co. (USA). Phenolpthalein, Sodium citrate and citric acid were purchased from Merck (Darmstadt, Germany). Bicinchoninic acid solution was obtained from Sigma Aldrich (St. Loius, USA). All other chemicals used in this study were of analytical grade. 2.2. Bacterial strain and growth conditions for CGTase production B. cereus was isolated from rotten potatoes. A loopful of culture was transferred from the stock culture to a culture plate. The culture plate was incubated at 37 °C for 48 h. Then, the single colonies formed was transferred to a 50 ml of medium containing 1% (w/v) sago starch, 0.5% (w/v) peptone, 0.5% (w/v) yeast extract, 0.009% (w/v) MgSO4, 0.1% K2HPO4 and 1% Na2CO3. The culture was allowed to grow aerobically at 37 °C with continuous agitation at 250 rpm for 18 h. This was then applied as inoculum. A 10 ml of inoculum was then transferred into a 250 ml Erlenmeyer flask consisting 100 ml of the same medium mentioned previously and incubated at 37 °C at 250 rpm for 30 h with continuous shaking [2]. The crude enzyme was harvested after 15 min of centrifugation at 4000g as supernatant and was directly used for further studies. 2.3. CGTase activity assay The cyclizing activity of CGTase (standard enzyme assay) which is the b-CD production rate was measured spectrophotometrically at 550 nm according to phenolphthalein method as described in previous studies with modifications [16,17]. The procedure of CGTase activity assay has been described previously [16,17]. 2.4. Bicinchoninic acid assay (BCA assay) The total protein concentration was determined by bicinchoninic acid assay [18], as to minimize the interference of PEG and salts in the enzyme samples. The assay was performed in a microtiter plate where 50 ll of the enzyme sample was added with 200 ll of the working reagent and subsequently incubated at 37 °C for 30 min. The color intensity was measured at 562 nm. A set of blank solutions was prepared which containing the same amount of particular diluted phase solutions without the enzyme samples as a comparison to the enzyme samples for each system. 2.5. Binodal construction In order to introduce a productive and environmental friendly system for CGTase purification, sodium citrate salt which is biodegradable and non-toxic was selected against other common salts used in ATPS which gave similar partitioning behavior as described in other studies [13,19]. Binodal line is the boundary of the phase

separation where two or more phases formed beyond the curve but only one phase exists below the curve. Binodal curve of PEG 6000, 8000 and 10,000 and sodium citrate salt were constructed using turbidometric titration according to Albertsson [20]. Mixtures of PEG standard solutions and salt of known concentrations were titrated by the addition of diluents until the turbid solutions turned clear as shown in Table 1. 2.6. TLL Compositions of the two phase-forming components are in equilibrium if the two different points fall on the same TLL. The TLL can be determined by using the Eq. (1)

TLL ¼

qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi DP 2 þ DC 2

ð1Þ

where, DP is the difference between PEG concentration and DC is the difference between citrate concentrations. Concentrations of PEG and salts were evaluated by refractive index and conductivity, respectively [14]. A standard salt conductivity curve in a range of salt concentrations had been constructed in order determine the concentration of salts in the particular phaseforming mixture. PEG concentration was then estimated by subtracting the refractive index value contributed by salts [14]. 2.7. Partition experiments ATPS were prepared from PEG stock solutions (50% w/v) of different molecular weight (PEG 6000, 8000 and 10,000) and sodium citrate stock solutions (40% w/v). CGTase showed bottom phase preference in higher molecular weight as described in other studies [13]. As such, these three PEGs were chosen for further investigation. Seven different pHs (ranging from 5 to 8) of the citrate stocks were being adjusted appropriately by the addition of citric acid stock solutions (40% w/v). The 15 ml centrifuge tubes were used to prepare the phase systems. By calculations, known masses of PEG stock solution, citrate stock solution at definite pH and 20% (w/w) crude samples were weighed into the particular graduated centrifuge tubes. Subsequently, the systems were being added with appropriate distilled water in order to obtain a final mass of 10 g system. The feed solutions were stirred thoroughly by a vortex mixer and then centrifuged at 4000 rpm for 10 min for complete phase separations. Eventually, volumes of both phases formed were measured and samples from coexisting phases were pipetted out for further analysis by enzyme assay and protein assay. Final

Table 1 System composition for construction of PEG/citrate binodal curves. The system compositions used to construct the PEG/citrate binodal curves were shown. The system compositions were achieved by weighing equivalent amount of PEG and citrate stock solutions in to the system. [Citrate] (%, w/w)

[PEG] (%, w/w)

2.5 6.5 7.0 7.5 8.0 8.5 11.0 12.5 13.5 15.0 17.5 20.0 22.5 24.5

45.0 30.0 27.5 25.0 22.5 20.0 17.5 15.0 12.5 10.0 7.5 5.0 2.5 1.0

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system composition constructed for the investigations of the purification of CGTase at different molecular weight of PEG were shown as Table 2. 2.8. Partition coefficient

2.13. Purification fold The purification fold (PFT) was evaluated as the ratio of the CGTase specific activity in the top phase to the initial CGTase activity in the crude extract and determined by using Eq. (7)

The partition coefficient (K) of the CGTase was determined using the Eq. (2)

PFT ¼ SA of phase sample=SA of crude extract

K ¼ AT =AB

2.14. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) analysis

ð2Þ

where AT and AB are the CGTase cyclizing activities in units/ml in the top phase and bottom phase, respectively. 2.9. Yield of CGTase Yield of CGTase in top phase was evaluated as percent yield (YT) on weight basis according to the Eq. (3)

Y T ð%Þ ¼ 100C T V T =C I V I

ð3Þ

where, VT and VI are the volumes of the top phase and the volume of crude samples subjected to extraction, respectively. CT and CI are the concentrations of CGTase in the top phase and the crude extract, respectively. 2.10. Selectivity Selectivity (S) was defined as the ratio of the CGTase partition coefficient (Ke) to the protein partition coefficient (Kp) [14] using the Eq. (4)

S ¼ K e =K p

ð4Þ

2.11. Specific activity of CGTase The specific activity (SA) was calculated as the ratio between the enzyme activity (U) in the phase sample and the total protein concentration (mg) [14] according to Eq. (5)

SA ðU=mgÞ ¼ Enzyme activity ðUÞ=½Protein ðmgÞ

ð5Þ

2.12. Volume ratio (VR) VR was defined as the ratio of volume in the top phase (VT) to that in the bottom phase (VB) [14] and calculated by using Eq. (6)

V R ¼ V T =V B

ð6Þ

Table 2 System composition of PEG/citrate ATPS for the partition experiments. The system compositions of PEG/citrate ATPS used in the partition experiments were exhibited in terms of percentage of the concentrations (%, w/w). PEG molecular weight

Total system PEG/citrate

6000

16.5/10.5 17.5/11.0 19.0/11.5 20.5/12.0 21.5/12.5

8000

16.5/10.5 17.5/11.0 19.0/11.5 21.5/12.5 22.5/13.0

10,000

16.5/11.0 17.0/12.0 17.5/13.0 18.0/14.0 18.5/15.0

ð7Þ

The molecular weight of the CGTase was evaluated by SDS– PAGE using an electrophoresis unit (Bio-Rad). The acrylamide gel consisting of a 12% of resolving gel and a stacking gel of 4.5% was used. The phase samples obtained from the top phase were usually in very dilute conditions and were thus being concentrated and precipitated by 10% trichloroacetic acid (TCA) solution. The precipitation step was carried out to remove the salts which would affect the electrophoresis present in the phase samples. Besides, concentration of protein samples by using 7 ml protein concentrators (Pierce) and desalting column (Pierce) were carried out in order to obtain more concentrated samples for the electrophoresis process. The procedure has been described in a previous publication [14]. 3. Results and discussion 3.1. Effect of PEG molecular weight and TLL on CGTase partitioning The phase diagrams for PEG-citrate system with comparable TLLs were shown in Fig. 1a–c. Three phase diagrams with different molecular weight of PEG (i.e. 6000, 8000 and 10,000) were constructed, with increasing TLL while VR = 1.0 and pH = 7.0 were kept constant. Table 3 showed the partition coefficient, K for CGTase, and the purification fold of CGTase. It was shown that the K values exceeded one in almost all the systems prepared and these implied that CGTase showed top phase preference in which the CGTase were mostly partitioned to PEG-rich top phase. There is no obvious trend on the values of partition coefficient with the elevating TLL. For the system PEG6000/citrate, the K values and the purification fold were relatively constant for different TLL while the K values and purification fold exhibited by PEG10,000/citrate were relatively low compared to the other two systems investigated. Hence, the partitioning condition of PEG8000/citrate at long TLL of 38.89% (w/w) was selected for further experiments. The results revealed that the PEG molecular weight had slight effects on the partitioning behavior of the CGTase. As the PEG mass increases, the surface hydrophobicity increases in the PEG-rich top phase due to the reduction of the hydroxyl groups for the higher chain length PEG compared to the lower chain length PEG [21]. Besides, the increase in the PEG chain length also led to the increase of excluded volume, in which there were lesser space to accommodate protein in PEG-rich top phase. Hence, the K decrease with increasing PEG molecular weight in common due to the excluded volume effect [22]. On the other hand, most proteins would partition into salt-rich bottom phase in the PEG/salt system in general driven by the salting out effect [23,24]. According to the data obtained, the best system for CGTase was achieved in the PEG8000/citrate system at TLL of 38.89% (w/w) with K of 2.91 and purification fold of 5.32. It was suggested that at this point, the surface hydrophobicity property of PEG and the salting out force had achieved a great equilibrium which in turn led to optimum partitioning of CGTase. The CGTase partitioning into the salt rich bottom phase reduced with an increase in the molecular weight of PEG from 6000 to 8000 as shown by the

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Fig. 1. Phase diagram for PEG and sodium citrate. The bimodal curves for (a) PEG 6000, (b) PEG 8000 and (c) PEG 10,000 were plotted against different concentrations of sodium citrate. Tie-lines in different length were exhibited and the composition of ATPS with phase ratio of approximately 1 was labeled as (j) in each of the tie-line.

Table 3 Influence of the PEG molecular weight and TLL on the partitioning of CGTase. The influence of PEG molecular weight and concentration on the partitioning behaviour of CGTase were exhibited. The partition coefficient, K and purification fold were calculated by Eqs. (2) and (7) respectively. PEG molecular weight

Tie-line length (TLL)

K

Purification fold

29.77 33.13 36.99 42.16 43.07

1.27 1.08 0.98 1.09 1.26

4.29 3.00 3.08 3.56 3.04

8000

28.7 34.27 38.89 45.6 50.3

2.06 1.99 2.91 1.28 1.22

4.46 2.57 5.32 2.94 3.86

10,000

33.88 39.07 44.15 48.34 52.52

1.49 1.44 1.55 1.55 1.41

3.92 1.35 1.56 1.35 1.29

6000

increase in K-values. PEG 6000 or even lower molecular weight of PEG may not be a good option for the system as the low molecular weight of the polymer may draw all proteins to the top phase which caused poor separation and low purification factors of the samples. With increasing of TLL, more CGTase partitioned into top phase as there was a decrease in the relative free volume in the salt-rich bottom phase (Table 3). At longer TLL, the partition coefficient of CGTase seems to be constant as the longer tie-line

with higher concentration of citrate salt had led to the saltingout effect on CGTase. Hence, PEG8000/citrate system with TLL 38.89% (w/w) was chosen for further studies.

3.2. Effect of phase VR on CGTase partitioning Fig. 2 showed the effect of VR on CGTase partitioning. The partitioning of CGTase exhibited significant results at phase VR of 2.0 where the purification fold, K and selectivity of the CGTase were optimum at this VR. As the VR increased at the same TLL, these three factors were decreased gradually. In general, phase VR does not seem to be able to significantly give impact to the purification factor by decreasing the phase VR as the large variation of protein partition coefficient along same TLL remained constant [25,26]. The trend for the effect of increasing VR towards the CGTase partitioning were not significant obvious as exhibited in Fig. 2. However, it was suggested that as the VR increases, the protein partition coefficient increases as more target protein had been partitioned into polymer rich top phase as described by other authors [24,25]. Lower phase volume ratios may lead to precipitation at interphase when the limit of solubility of CGTase in the PEG phase had reached. As such, loss of CGTase by precipitation could be minimized at higher volume ratios. However, higher VR also had a drawback in which it leads to the dilution of CGTase in concentrations [14]. As we can observe in this study, the partition coefficient was maximal at VR of 2 with maximum purification factor of 9.3 and selectivity of 2.97 and therefore this VR was selected for further experiments.

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Fig. 2. Influence of the VR on the partitioning of CGTase. The partition behavior of CGTase at different volume ratios was exhibited. The volume ratios were manipulated from 0.13 to 3 along the 38.89% (w/w) TLL. The purification fold (), partition coefficient (j) and selectivity (N) of CGTase were evaluated and plotted against different volume ratios.

Table 4 Influence of the crude load on the partitioning of CGTase. The influence of crude load on the partitioning behaviour of CGTase was investigated. Different masses of loaded crudes were experimented varied from 10% to 40%. Crude load (% w/w)

Partition coefficient, K

Yield (%)

10 20 30 40

2.29 3.19 2.73 2.66

63.21 70.53 67.25 66.62

in Table 4. It was shown that the partition coefficients and yields of CGTase were almost similar for four different crude loads manipulated with the maximum K (3.19) and yield (70.53%) exhibited at the crude load of 20%. Increasing loaded mass into the ATPS may decrease the phase VR and thus alter the composition of the particular system. Besides, the components contained in the crude load may also change the characteristics of ATPS causing the particular system no longer considered the best for the partitioning of CGTase. These phenomenons may be caused by the losses of CGTase by precipitation along the ATPS partitioning process.

3.3. Effect of crude load on CGTase partitioning

3.4. Effect of pH on CGTase partitioning

The loaded crude in the enzyme partitioning had played a major role in ATPS as it not only may change the phase VR but also the loaded mass may affect the partitioning behavior of the target protein. The effect of crude load on CGTase partitioning was concluded

The pH had exhibited some impacts towards the partitioning behavior of CGTase in ATPS. In theory, negatively charged proteins would partition into the PEG-rich top phase while positively charged proteins would partition into the salt-rich lower phase

Fig. 3. The influence of pH on the partitioning of CGTase. The effect of the pH on the partitioning behavior of CGTase was exhibited. The pHs of the ATPS were varied from 5 to 8. The partition coefficient () and the selectivity (j) of CGTase were evaluated and plotted at different pH.

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Fig. 4. The influence of addition of NaCl on the partitioning of CGTase. The partitioning behavior of CGTase in addition of NaCl were investigated with PEG 8000/sodium citrate, TLL 38.89% at pH 7 and VR of 2. The partition coefficient and purification fold were exhibited.

[23,27]. In this study, the partitioning behavior of CGTase had been studied under different pH ranging from pH 5 to 8. The effect of pH on partition coefficient and selectivity of CGTase in ATPS was illustrated in Fig. 3. Based on the results obtained, K of CGTase increased with the increased of pH from 5 to 7 as more and more of the CGTase being partitioned into the PEG-rich top phase. CGTase, which had an isoelectric point, pI at about 6.9 [6], exhibited the optimum partitioning at pH 7 as shown in Fig. 3. At pH 7, CGTase was slightly negatively charged and as such more CGTase would be partition into the PEG phase theoretically. The partition coefficient and selectivity of CGTase were low at acidic conditions such as at pH 5 and 5.5 as at this pH, the CGTase, which was acting as positively charged protein at these point may prefer the salt-rich bottom phase instead of the PEG phase. As a result, pH 7 was proved to be the optimum pH for the ATPS of CGTase and thus being selected for further experiment.

3.5. Effect of addition of NaCl salt on CGTase partitioning The addition of neutral salts to ATPS may well alter the partitioning behavior of CGTase by changing the selectivity and yield of the enzyme. An electrical potential difference between two phases occurred as a result of the changes in salt type and concentration as the ions of added salt distributed unequally between the two phases [20,24]. The electrical potential difference that produced may affect the partitioning of the charged biomaterials in ATPS. In this study, the effect of addition of neutral salt NaCl on the partitioning of CGTase had been investigated. According to the results obtained as shown in Fig. 4, the optimum partitioning conditions for CGTase was reached with the addition of 4% (w/w) NaCl. At this optimum point, CGTase exhibited highest partition coefficient of 11.7, as more CGTase being partitioned to top phase due to the chemical potential of solutes alteration exerted by the salt. It also can be seen that at this point, the purification fold of the CGTase was the highest (16.3). This showed that addition of 4% (w/w) had brought about positive impact towards the ATPS of CGTase. As compared with the previous studies published by other researchers on the purification of CGTase using other conventional multiple steps purification method such as starch adsorption (purification fold of 43 with yield of 50%), ion exchange chromatography (purification fold of 23.1 and yield of 80.6%), ammonium sulfate

Fig. 5. SDS–PAGE analysis on the recovery of CGTase. The purity of the partitioned CGTase was evaluated by 12% SDS–PAGE analysis. Lanes 1 and 3: standard protein markers with molecular weight of 7–175 kDa; lane 2: crude enzyme CGTase; lane 4: ATPS top phase.

precipitation and affinity chromatography followed by gel filtration (purification fold of 157 and yield of 30%), ATPS was proved to be the less complicated and economic method to partially purify the enzyme CGTase even the purity and the recovery of CGTase obtained was not as high as to other conventional methods [6,11,28]. 3.6. CGTase recovery Optimum purification of CGTase was achieved by ATPS comprised of PEG8000, 38.89% (w/w) TLL with VR of 2, addition of 4% (w/w) NaCl and 20% crude load at pH7.0. Subsequently, the purity of the CGTase purified was evaluated using 12% SDS–PAGE. Fig. 5 illustrated the SDS–PAGE profile of the crude enzyme, standard protein markers and the purified enzyme. Lanes 1 and 3 was the standard protein marker. It was shown that crude enzyme (lane 2) exhibited a dense intensity of bands in which it indicated that there are large quantities of impurities and protein contaminants in the crude extract. Meanwhile, as can be seen from the Fig. 5, the enzyme extracted from the top phase of the system (lane 4) shows a small quantity of bands with a distinct band fell around the molecular weight of 55 kDa. The sample (lane 4) indicated highest enzyme specific activity after optimization of ATPS and it

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has been reported that the molecular weight of CGTase from Bacillus sp. generally falls in the range of 33–103 kDa [6]. The other fainter bands detected are probably the impurities which are not completely purified. Hence, the aqueous two phase system applied in this study could be used as a partial purification step for the enzyme CGTase. 4. Conclusion The direct primary capture of CGTase enzyme from fermentation broth using ATPS has been investigated. The CGTase was found in the top phase of PEG8000/citrate system while the impurities were partitioned to the bottom phase of ATPS. PEG molecular weight, VR, pH and addition of neutral salts to ATPS were shown to have impact on the partitioning of CGTase in ATPS. It was demonstrated that PEG8000/citrate with 38.89% (w/w) TLL, VR of 2.0, with additional 4% (w/w) NaCl and 20% crude load at pH 7.0 was the optimum conditions for the recovery of CGTase. A high purification fold of 16.3 and a yield of 70% were achieved under this optimized condition. The results proved that ATPS could be utilized as the potential primary step in the CGTase purification owing to its low cost and environmental friendly nature. Acknowledgements This study was supported by Research University Grant 9199681 from the Universiti Putra Malaysia and ESciencefund from the MOSTI, Malaysia. References [1] F.G. Priest, Extracellular enzyme synthesis in the genus Bacillus, Bacteriol. Rev. 41 (1977) 711–753. [2] X. Cao, Z. Jin, X. Wang, F. Chen, A novel cyclodextrin glycosyltransferase from an alkalophilic Bacillus species: purification and characterization, Food Res. Int. 38 (2005) 309–314. [3] N. Doukyu, H. Kuwahara, R. Aono, Isolation of Paenibacillus illinoisensis that produces cyclodextrin glucanotransferase resistant to organic solvents, Biosci. Biotechnol. Biochem. 67 (2003) 334–340. [4] D. Penninga, B. Strokopytov, H.J. Rozeboom, C.L. Lawson, B.W. Dijkstra, J. Bergsma, L. Dijkhuizen, Site-directed mutations in tyrosine 195 of cyclodextrin glycosyltransferase from Bacillus circulans strain 251 affect activity and product specificity, Biochemistry 34 (1995) 3368–3376. [5] A.R. Hedges, Industrial applications of cyclodextrins, Chem. Rev. 98 (1998) 2035–2044. [6] R.F. Martins, R. Hatti-Kaul, A new cyclodextrin glycosyltransferase from an alkaliphilic Bacillus agaradhaerens isolate: purification and characterisation, Enzyme Microb. Tech. 30 (2002) 116–124. [7] G. Schmid, Cyclodextrin glycosyltransferase production: yield enhancement by overexpression of cloned genes, Trends Biotechnol. 7 (1989) 244–248. [8] A. Biwer, G. Antranikian, E. Heinzle, Enzymatic production of cyclodextrins, Appl. Microbiol. Biot. 59 (2002) 609–617.

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