Purification and Characterization of Cycloisomaltooligosaccharide Glucanotransferase and Cloning of cit from Bacillus circulans U-155

Abstract

The enzyme cycloisomaltooligosaccharide glucanotransferase (CITase) was isolated from Bacillus circulans U-155, which had a yield of 25.8%, and purified to homogeneity. The M_r was approximately 98,000, similar to that for all known CITases. Specific activity of the purified enzyme was 2.11 U/mg protein, with maximal activity at approximately pH 6.0. The enzyme was stable at pH 4.5 up to pH 9.0 and at temperatures up to 50°C. The main product of the initial enzyme reaction was cycloisomalto-heptaose. The cit gene has a 2,895-bp open reading frame and encodes CITase in B. circulans U-155. We cloned this gene into a recombinant plasmid pCI811 and expressed it in Escherichia coli. A comparison between DNA sequence data from the transformant and the N-terminal amino acid sequence of the purified enzyme from B. circulans U-155 suggested that CITase was translated as a secretory precursor with a 30-amino-acid signal peptide. The mature enzyme contained 934 residues with a predicted molecular mass of 103.93 kDa. The enzyme activity in the transformants was approximately 3.0 mU/mL, similar to that of the purified enzyme secreted by B. circulans U-155. The enzyme also showed 72 and 67% identity with CITase from Paenibacillus sp. 598K and B. circulans T-3040, respectively. These results suggest that the enzyme isolated from B. circulans U-155 shares a greater similarity with CITase expressed by Paenibacillus sp. 598K than B. circulans T-3040.

Abbreviations

CI, cyclodextran (cycloisomaltooligosaccharide); CITase, cycloisomaltooligosaccharide glucanotransferase; CI-7, cycloisomalto-heptaose; CI-8, cycloisomalto-octaose; X-Gal, 5-bromo-4-chloro-3-indolyl β-D-galactopyranoside; IPTG, isopropyl β-Dthiogalactopyranoside; PCR, polymerase chain reaction; ORF, open reading frame; SD-sequence, Shine-Dalgarno sequence; CD, cyclodextrin.

INTRODUCTION

Cyclodextrans (cycloisomaltooligosaccharides, CIs) are cyclic glucose oligomers that consist of several D-glucose molecules linked by α-1,6 glycosidic linkages. The synthesis of these oligosaccharides from dextran is catalyzed by CI glucanotransferase (CITase, EC 2.4.1.248).^{1) 2)} Although several bacteria produce CIs from dextran, only 2 enzymes have been reported, that catalyze the intramolecular transglycosylation reaction required to produce CIs from dextran, namely, CITase from Bacillus circulans T-3040²⁾ and Paenibacillus sp. 598K.³⁾

As CIs can form inclusion complexes with hydrophobic substances, they are thought to be novel solubilizers and stabilizers.⁴⁾ They are also thought to be cariostatic reagents due to their ability to inhibit activity of glucan synthase from Leuconostoc mesenteroides and Streptococcus mutans.⁵⁾ It has been shown that cycloisomalto-heptaose (CI-7) has the strongest inhibitory activity.

CITase from B. circulans T-3040 produces cycloisomalto-octaose (CI-8) as its main product. In contrast, CITase from Paenibacillus sp. 598K mainly produces CI-7.^{2) 3)} To facilitate further research into CIs, we aimed to identify strains that produced CITases similar to Paenibacillus sp. 598K. Therefore, we screened for strains that produced CITase that synthesized CI-7 as its main product. As a result, we identified strain U-155, of the soil bacterium B. circulans, which accumulated CI-7 in culture. We have previously reported information regarding B. circulans U-155.^{6) 7)}

In this report, we describe the purification and characterization of CITase from B. circulans U-155, the cloning of CITase-encoding gene cit from B. circulans U-155 into an Escherichia coli plasmid vector (pUC118), and the expression of cit in E. coli.

MATERIALS AND METHODS

Reagents. Dextran-40 was purchased from Meito Sangyo Co. (Tokyo, Japan). The series of isomaltooligosaccharides that were used as standards were purchased from two separate vendors: isomaltose to isomaltoheptaose was purchased from Seikagaku Kogyo Co. (Tokyo, Japan) and isomaltooctaose to isomaltodecaose was obtained from Funakoshi Co. (Tokyo, Japan).

Strains and growth conditions. Strain U-155, which was isolated from soil and identified as B. circulans, was used for this study. It was cultured according to a method described previously.¹⁾ E. coli XL1-Blue MRF′ Δ(mcrA)183 Δ(mcrCB-hsdSMR-mrr)173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac [F′ proAB lacl^q ZΔM15Tn10 (Tet′)] was used as the host strain for routine transformation. Luria-Bertani (LB) broth and agar plates containing 40 µg/mL 5-bromo-4-chloro-3-indolyl β-D-galactopyranoside (X-Gal) and 47.2 µg/mL isopropyl β-D-thiogalactopyranoside (IPTG; X-Gal plates) were used for screening E. coli. Ampicillin (Sigma Chemical Co., Tokyo, Japan) was routinely added up to a final concentration of 50 μg/mL, when necessary.

Enzyme assay. CITase activity was assayed as described previously, except that 100 mM acetate buffer (pH 6.0) was used.²⁾ One unit of the enzyme was defined as the amount of enzyme that produced 1 μmol of CIs per min under the above conditions.

Purification of the CITase from B. circulans U-155. CITase was purified as described previously, with some modifications as follows.²⁾ Sixteen liters of the culture broth was centrifuged at 10,000×G for 20 min to remove the cells. The supernatant was then concentrated to approximately 200 mL using a hollow fiber membrane with a molecular weight cut-off of 6,000 Da and ultrafiltration using an Amicon PM-10 membrane, and the concentrate was used as a crude extract. Ammonium sulfate was added to a final concentration of 1.0 M. The enzyme was subjected to HPLC (Tosoh Co., Tokyo, Japan) using a preparative TSKgel Phenyl 5PW column (21.5 mm×150 mm; Tosoh Co.). Elution conditions were as follows: solvent, 10 mM EDTA-100 mM phosphate buffer (pH 7.0); elution, a linear gradient of ammonium sulfate (1.0 to 0.0 M); flow rate, 4 mL/min; and fraction volume, 8 mL/fraction. The enzyme was eluted with approximately 0.1 M of ammonium sulfate. The active fractions (total 40 mL) were concentrated to 1.0 mL by ultrafiltration using the Amicon PM-10 membrane. The concentrate was diluted 20 times with 1 mM EDTA-10 mM phosphate buffer (pH 7.0) and subjected to HPLC using a TSKgel DEAE 5PW column (7.5 mm × 75 mm, Tosoh Co.) equilibrated with the same buffer. The enzyme was eluted with a linear gradient of NaCl (0.0 to 0.4 M) at a flow rate of 1 mL/min. The active fractions (total 8 mL) were concentrated to 0.9 mL by ultrafiltration using Centriprep (Amicon Co., Bedford, USA). A portion (0.3 mL) of the concentrate was subjected to molecular sieve HPLC using a TSKgel G3000 SW column (7.6 mm × 600 mm × 2, Tosoh Co.) and eluted with 200 mM NaCl-10 mM EDTA-100 mM phosphate buffer (pH 7.0) at a flow rate of 1 mL/min. The enzyme was eluted as a single peak. The active fractions were combined (total 11.9 mL).

General analytical methods. Protein concentration was determined using the Bradford assay,⁸⁾ followed by SDS-PAGE as per the method described by Laemmli.⁹⁾ Reducing power in the reaction mixture was measured using the Somogyi-Nelson method.¹⁰⁾

Determination of the N-terminal amino acid sequences of CITase. The N-terminal sequence of purified CITase from B. circulans U-155 was determined by repetitive Edman degradation in a pulse liquid protein sequencer (model 473A; Applied Biosystems, Foster City, USA).

DNA manipulation. Unless otherwise indicated, DNA manipulations were performed essentially as described by Maniatis et al.¹¹⁾ For DNA sequence analysis, overlapping deletions of regions that were to be sequenced were constructed using the Deletion Kit (Takara Shuzo Co., Ltd., Kyoto, Japan), as described previously.¹²⁾ Sequencing was performed with an automated DNA sequencer (model 370A; Applied Biosystems) using DNA Sequencing System/Taq Polymerase Kit (Applied Biosystems).

Nucleotide sequence accession number. The nucleotide sequence reported in this paper has been submitted to the DDBJ, EMBL and GenBank nucleotide sequence databases under the accession number D88360.

RESULTS

Purification and characterization of CITase from B. circulans U-155.

CITase isolated from B. circulans U-155, which had a 25.8% yield, was purified to homogeneity (Table 1). The enzyme was eluted as a symmetrical single peak by molecular sieve HPLC and it migrated as a single band on SDS-PAGE (Fig. 1). The M_r was approximately 98,000, similar to that of all known CITases. Specific activity of the purified enzyme was 2.11 U/mg protein (Table 1). Although this value is far lower than that of hydrolases such as dextranase, it is almost same as that of T-3040 CITase (2.16 U/mg).²⁾

Table. 1.

Summary of purification steps for the Bacillus circulans U-155-derived CITase.

Fig. 1.

SDS-PAGE of purified CITase from Bacillus circulans U-155.

Approximately 10 μg protein was treated with 2.5% 2-mercaptoethanol at 100°C for 3 min, prior to loading the gel. After the gel-run, the gel was stained with Coomassie Brilliant Blue R250. Lane 1, marker proteins; lane 2, purified enzyme.

As shown in Table 2, the enzyme showed maximal activity at approximately pH 6.0 and was stable at pH 4.5 through pH 9.0. Thermostability tests between 10 and 70°C in 0.1 M phosphate buffer (pH 6.0) for 15 min showed that the enzyme was stable at temperatures up to 50°C. This value was the same as that for CITase from Paenibacillus sp. 598K and 10°C higher than that of CITase from B. circulans T-3040.

Table. 2.

Comparison of properties of CITases from Bacillus circulans U-155, Paenibacillus sp. 598K and Bacillus circulans T-3040.

^a) The characteristics presented in the table were from literature³⁾ and the molecular weight was calculated from the mature Paenibacillus sp. 598K CITase amino acid sequence information. ^b) The characteristics presented in table were from literature^{2) 13)} and the molecular weight was calculated from the mature Bacillus circulans T-3040 CITase amino acid sequence information. ^c) The molecular weight was calculated using amino acid sequence information of mature CITase from Bacillus circulans U-155.

The time course of the cyclization reaction was investigated with a reaction mixture containing 1 mL of 4% substrate solution, 200 μL of 100 mM acetate buffer (pH 5.5), 780 μL distilled water, and 20 μL of the enzyme (2.71 U/mL). Aliquots of the reaction mixture were withdrawn at regular intervals. As shown in Fig. 2, CITase from B. circulans U-155 produced 3 CIs (CI-7, CI-8 and CI-9) from dextran. However, the ratio of CI-7, CI-8 and CI-9 produced by CITase from strain U-155 was different from that produced by CITase from strain T-3040. At the initial stage of the reaction, the main product of CITase from B. circulans U-155 was CI-7, although CI-8 was the main product at the final stage. The total yields of CI-7, CI-8 and CI-9 of CITase from B. circulans U-155 were almost the same as those from B. circulans T-3040-derived CITase (data not shown). In addition, CITase from B. circulans U-155 did not act on amylopectin and pullulan, both of which contain α-1,6 glycosidic linkages at their branch points. In summary, these results show that the main product of CITase from B. circulans U-155 at the initial stage of the reaction is CI-7 and that the thermostability of B.circulans U-155-derived CITase is 10°C higher than that of B. circulans T-derived CITase. Therefore, the properties of B. circulans U-155-derived CITase are more similar to those of Paenibacillus sp. 598K-derived CITase than to those of B. circulans T-3040-derived CITase.

Fig. 2.

Time course of cyclization reaction of CITases from Bacillus circulans U-155 and T-3040.

One milliliter of a 4.0% dextran-40 solution, 0.2 mL of 100 mM acetate buffer (pH 6.0 for B. circulans U-155-derived CITase and pH 5.5 for B. circulans T-3040-derived CITase), 0.78 mL distilled water and 0.02 mL of the respective CITases (2.71 U/mL) were mixed and incubated for 10 h at 40°C. One hundred-microliter aliquots of the reaction mixtures were withdrawn and boiled for 5 min. After centrifugation, the supernatants were analyzed by HPLC. (A) Time course of CI-7, (B) Time course of CI-8, (C) Time course of CI-9. Symbols: B. circulans U-155 (■), B. circulans T-3040 (□).

Cloning, DNA sequencing, amino acid sequence homology of B. circulans U-155-derived CITase and expression of B. circulans U-155 cit in E. coli.

The CITase-encoding gene, cit, was cloned by the shotgun method using X-Gal plates containing 0.5% blue dextran (Blue dextran plates) as described previously.¹³⁾

One in approximately 3,000 transformants showed a positive signal. Plasmid DNA was isolated from such a clone and designated as pCI811, which had a Sau3AI fragment insert of approximately 4 kb.

An XL1-Blue MRF′ strain carrying pCI811 was incubated aerobically in LB broth at 37°C for 16 h, sonicated crude lysates were prepared, and CITase activity was determined. Enzyme activity from the strain harboring pCI811 was approximately 3 mU/mL, similar to that of the enzyme purified from B. circulans U-155 culture.

Examination of the nucleotide sequence showed a 2,895-bp open reading frame (ORF, data not shown). When this was compared with the N-terminal amino acid sequence of the purified CITase, we found that the latter was consistent with the deduced amino acid sequence from the ORF, starting at position 31. Therefore this enzyme was confirmed to be a typical extracellular enzyme with a signal peptide consisting of 30 amino acid residues, while the mature enzyme contained 934 residues. This is similar to that of B. circulans T-3040-derived cit, with a predicted molecular mass of 103.930 kDa. The molecular mass of the deduced amino acid sequence corresponded with the result from SDS-PAGE.

A potential ribosome-binding site was found beginning at nucleotide 11 with a Shine-Dalgarno sequence, separated by 7 bp from the putative ATG translation start codon at nucleotide 1 (data not shown). The downstream end of the ORF was followed by a possible transcription termination signal, which had a potential stem structure at nucleotides 2,952‒2,966 (data not shown).

The deduced amino acid sequence of CITase from B. circulans U-155 is presented in Fig. 3, together with those of other known CITases. The mature B. circulans U-155 CITase sequence showed about 72% identity with that of Paenibacillus sp. 598K and 67% with that of B. circulans T-3040.^{3) 13)} Asp145, Asp270 and Glu342, which are thought to be catalytically important residues in CITases, were also conserved in this CITase.^{3) 14)} On the other hand, a comparison of the R1 region (from Tyr404 to Tyr492), which is thought to contribute to the preference for CI-8 production, showed that the similarity of B. circulans U-155-derived CITase R1 to Paenibacillus sp. 598K-derived CITase R1 (87%) was far higher than that with B. circulans T-3040-derived CITase R1 (60%).¹⁵⁾ The amino acid sequence of B. circulans U-155-derived CITase was similar to that of dextran hydrolases, but the similarity was low. Comparison of B. circulans U-155-derived CITase with the representative dextranase from Streptococcus mutans showed 28% similarity and 18% identity, while CITase differed from the dextranases with respect to the presence of the R1 region (Tyr404‒Tyr492), as reported previously by Aoki et al.¹⁶⁾

Fig. 3.

Alignment of the amino acid sequences of the CITases from Bacillus circulans U-155, Paenibacillus sp. 598K and B. circulans T-3040.

The primary amino acid sequence of the CITases were obtained from GenBank (IDs D88360 and D61382) for B. circulans U-155 and T-3040, and from DDBJ (ID AB685169) for Paenibacillus sp. 598K, respectively. The numbers begin from the N-terminal amino acid of the mature CITase. Amino acid residues that are identical between all CITases are indicated in gray. Amino acid residues that are identical between two CITases are shown as bold. The solid arrow shows cleavage sites for the signal peptidases. The open arrows show catalytic amino acid residues.

DISCUSSION

So far, CITase has been isolated from just 3 strains including the one described in this study: B. circulans T-3040, Paenibacillus sp. 598K and B. circulans U-155. Although these enzymes can produce CIs from dextran, the ratios of the 3 CIs, CI-7, CI-8 and CI-9, produced by these strains differ. B. circulans T-3040-derived CITase mainly produces CI-8.²⁾ On the other hand, Paenibacillus sp. 598K-derived CITase mainly produces CI-7.³⁾ B. circulans U-155-derived CITase also produces mainly CI-7 at the initial stage of the reaction. The properties of B. circulans U-155-derived CITase are more similar to those of Paenibacillus sp. 598K-derived CITase than to those of B. circulans T-3040-derived CITase. In fact, when we compared the deduced amino acid sequences of these 3 CITases, we found that the similarity of B. circulans U-155-derived CITase to Paenibacillus sp. 598K-derived CITase (72%) was higher than to B. circulans T-3040 (67%). Funane et al. divided the amino acid sequence of CITase into several regions and investigated the role of each region by monitoring the activity of their deletion-mutants. They reported that the R1 region (Tyr404‒Tyr492) contributes to the preference for CI-8 production.¹⁵⁾ The similarity of B. circulans U-155-derived CITase R1 region to Paenibacillus sp. 598K-derived CITase R1 region (87%) was high, whereas similarity to B. circulans T-3040 CITase R1 region (60%) was low. This large difference in similarity between R1 regions was reflected in the different main products.

We have previously isolated several strains of bacteria capable of producing CIs from dextran. However, they are either of the B. circulans T-3040 type or the Paenibacillus sp. 598K type, although recently Funane et al. reported a new strain of CI-producing bacteria that mainly produces CI-10, CI-11 and CI-12.¹⁷⁾ We could not detect larger CIs than those from Funane’s report since our detective system was adapted to oligosaccharides.

Cyclodextrin (CD) glucanotransferases (CGTases) produce 3 kinds of CDs (α-CD, β-CD and γ-CD) as main products and are classified into 3 types. One mainly produces α-CD from starch, an example is B. macerans-derived CGTase.¹⁸⁾ The second type mainly produces β-CD from starch, an example is B. megaterium-derived CGTase.¹⁹⁾ The third type mainly produces γ-CD and CGTase isolated from the alkalophilic B. clarkii is an example of this type.²⁰⁾ Given that 3 types of CGTases have been identified so far, it is likely that a third type of CITase, one that mainly produces CI-9, may also be found in the future.

ACKNOWLEDGMENTS

The authors extend their deepest gratitude to Miss T. Kurokawa for her devoted technical assistance and to Mr. K. Tobe for his skilled preparation of CIs. The authors would also like to extend their thanks to Dr. K. Funane for her critical discussion of the manuscript.

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