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A highly active α-cyclodextrin preferring cyclomaltodextrinase from Geobacillus thermopakistaniensis
Carbohydrate Research 481 (2019) 1–8
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A highly active α-cyclodextrin preferring cyclomaltodextrinase from Geobacillus thermopakistaniensis
T
Iqra Aroob, Nasir Ahmad, Mehwish Aslam, Abeera Shaeer, Naeem Rashid* School of Biological Sciences, University of the Punjab, Quaid-e-Azam Campus, Lahore, 54590, Pakistan
ARTICLE INFO
ABSTRACT
Keywords: Cyclomaltodextrinase Cyclodextrin Acarbose Thermostable Cloning Geobacillus thermopakistaniensis
Cyclomaltodextrinases show diverse hydrolyzing and/or transglycosylation activities against cyclodextrins, starch and pullulan. A gene annotated as cyclomaltodextrinase from Geobacillus thermopakistaniensis was cloned and overexpressed in Escherichia coli. The gene product, CDaseGt, was purified and biochemically characterized. The recombinant enzyme exhibited highest activity with α-cyclodextrin at 55 °C and pH 6.0. Specific hydrolytic activities towards α-, β- and γ-cyclodextrin were 1200, 735 and 360 μmol min−1 mg−1, respectively. To the best of our knowledge, the activity against α-cyclodextrin is the highest among the reported enzymes. Next to cyclodextrins, pullulan was the most preferred substrate with a specific activity of 105 μmol min−1 mg−1. CDaseGt was capable of hydrolysis of maltotriose and acarbose as well as transglycosylation of their hydrolytic products. At 65 °C, there was no significant loss in enzyme activity even after overnight incubation. Activity of CDaseGt was not metal ions dependent, however, the presence of Mn2+ significantly enhanced the α-CDase activity. EDTA had no significant effect on the CDaseGt activity, however, it enhanced the thermostability of the enzyme. CDaseGt existed in monomeric as well as dimeric form in solution. Dimeric form is more active compared to the monomeric one. Equilibrium between the two forms seems to be concentration dependent.
1. Introduction Cyclomaltodextrinase (CDase, EC 3.2.1.54), maltogenic amylase (MAase, EC 3.2.1.133) and neopullulanase (NPase, EC 3.2.1.135) are three groups of enzymes which efficiently hydrolyze cyclodextrins (CDs), cyclic oligomers of glucose units linked through α-D-(1,4)glycosidic bonds [1]. These enzymes also hydrolyze starch and pullulan but at a slower rate. They contain nearly 130 residues at the N-terminus, speculated to be associated with the catalytic domain, and around 70 residues at the C-terminus, that are absent in the αamylases. It has been proposed that these three groups of enzymes should come under a single name, cyclodextrinase (CDases), since cyclodextrins are their most preferred substrates [1,2]. The recent advancement in whole genome sequencing has revealed the presence of these enzymes in various microorganisms. They have been characterized from several bacterial and archaeal sources, including Alicyclobacillus acidocaldarius [3], Anoxybacillus flavithermus [4], Bacillus coagulans [5], Bacillus clarkii 7364 [6], Bacillus macerans [7], Bacillus sphaericus [8,9], Bacillus stearothermophilus [10], Bacillus sp. A2-5a [11], Bacillus sp. I-5 [12], Clostridium thermohydrosulfuricum 39E [13], Flavobacterium sp. [14], Klebsiella oxytoca strain M5a1 [15], Lactobacillus plantarum [16], Massilia timonae [17], *
Paenibacillus sp. A11 [18], Thermococcus sp. strain B1001 [19], Thermococcus kodakarensis [20], and Thermus sp. [21]. Geobacillus thermopakistaniensis is a Gram-positive aerobic thermophile, whose complete genome sequence has been determined [22]. We are interested in glycosyl hydrolase family 13 (GH13) enzymes. We have previously identified a novel enzyme from this family capable of hydrolyzing both α-1,4- and α-1,6-linked glucose polymers under industrial conditions and without the requirement of any additional metal ions [23]. Analysis of the genome sequence of G. thermopakistaniensis has revealed the presence of a full range of starch-hydrolyzing enzymes belonging to family GH13. They are annotated as α-amylase (ESU72338), cyclomaltodextrinase (ESU72342), amylopullulanase (ESU72863), glycogen branching enzyme (ESU73465), and pullulanase (ESU73761). However, none of them has been characterized. Currently, GH13 family contains 75319 sequences in total, including 70975 from bacterial sources. However, only 823 (1%) of them have been characterized, among them only 18 (2%) are cyclodextrin hydrolyzing enzymes (2019, http://www.cazy.org/GH13.html). These enzymes have great commercial importance in food industry [24]. Since a very few number of cycolodextrin hydrolyzing enzymes have been characterized and a search for better enzymatic toolkit is a need of the industry, we carried out the current study with the aim to find out a novel cyclodextrinase. In the present manuscript, we have described cloning and
Corresponding author. E-mail addresses: [email protected], [email protected] (N. Rashid).
https://doi.org/10.1016/j.carres.2019.06.004 Received 6 April 2019; Received in revised form 30 May 2019; Accepted 6 June 2019 Available online 08 June 2019 0008-6215/ © 2019 Elsevier Ltd. All rights reserved.
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Table 1 Percent identity between amino acid sequence of CDaseGt and other CDases. Enzyme and Source
1 2 3 4 5 6 7 8 9 10 11 12 13
Cyclomalodextrinase (CDaseGt) (Geobacillus thermopakistaniensis) Amylase (Bacillus sp. WPD616) Maltogenic amylase (Thermus sp. IM6501) Maltogenic Amylase (Geobacillus sp. SK70) Maltogenic amylase (Geobacillus sp. Gh6) Maltogenic-amylase (Geobacillus thermoleovorans) Neopullulanase (Geobacillus stearothermophilus) Neopullulanase (Geobacillus stearothermophilus) Maltogenic amylase (Parageobacillus caldoxylosilyticus) Cyclomaltodextrinase (Anoxybacillus flavithermus) Maltogenic amylase (Bacillus thermoalkalophilus) Maltogenic Amylase (Geobacillus stearothermophilus) Cyclomaltodextrinase (Bacillus sp.)
Sequence Identity (%) 1
2
3
4
5
6
7
8
9
10
11
12
13
100
99.8 100
99.4 99.3 100
97.2 97.1 97.1 100
96.4 96.2 96.2 97.9 100
95.9 95.7 95.7 97.4 98.2 100
86.2 86 86.2 86.7 86.9 87.2 100
86.2 86 86.2 86.5 86.5 86.9 98.2 100
73.8 73.6 73.8 74.4 74.3 74.4 75.3 75.3 100
69.2 69 69.3 69 69 69.2 69.5 69.2 72.1 100
66.3 66.1 66.3 66.8 66.6 66.8 67.6 67.8 76.5 68 100
64.2 64 64.2 64.9 64.7 64.7 63.8 63.7 68.9 67.9 78.1 100
55.2 55.1 55.2 55.1 54.7 54.7 55.4 55.9 58.9 57.5 64.2 62.3 100
Uniprot accession numbers or references used were: 1, V6VEH6; 2, Q52PU5; 3, O69007; 4, [Ref. [33]]; 5, E0X988; 6, I6RE37; 7, Q9AIV2; 8, P38940; 9, C0LZ63; 10, Q5BLZ6; 11, Q68KL3; 12, Q45490; 13, O82982.
expression, in Escherichia coli, of the gene corresponding to ESU72342, encoding a cyclomaltodextrinase (CDaseGt) from G. thermopakistaniensis.
Thermostability experiments revealed that CDaseGt is quite stable at temperatures up to 65 °C. There was no significant loss in enzyme activity even after overnight incubation at 65 °C. Presence of EDTA enhanced the thermostability of CDaseGt. The half-life of CDaseGt was 90 min at 70 °C in the absence of EDTA which increased to 360 min in the presence of 10 mM EDTA (Fig. 3C). The enzyme activity decreased drastically when incubated at 75 °C in the presence or absence of EDTA.
2. Results 2.1. Gene cloning and sequence analysis Genome sequence of G. thermopakistaniensis contains an open reading frame (ESU72342) coding for cyclomaltodextrinase of the GH13 family. The gene consisted of 1767 nucleotides, encoding a polypeptide of 588 amino acid residues having a theoretical molecular mass of 68,157 Da and an isoelectric point of 5.58. Sequence comparison demonstrated that CDaseGt exhibited the highest homology of 99% (identity) with amylase from Bacillus sp. WPD616 [25] and maltogenic amylase from Thermus sp. (1GVI_A) [21] (Table 1). Sequence alignment of CDaseGt with the characterized counterparts belonging to GH13 family, identified seven conserved regions typical of the members of this family. Among these conserved regions, region I was identical in all the counterparts (Fig. 1). However, there are slight differences in the other conserved regions. Similar to all other members of GH-13 family, the three catalytic residues, Asp328, Glu357 and Asp424, were conserved in CDaseGt.
2.4. Substrate preference and analysis of hydrolysis products In order to determine the substrate preference, various cyclic and acyclic saccharides were tested at a final concentration of 0.5%. Cyclodextrins were preferred among the tested substrates. Among cyclodextrins, α-cyclodextrin was the most preferred substrate with a specific activity of 1200 μmol min−1 mg−1, followed by β-cyclodextrin and then γ-cyclodextrin. Among the acyclic substrates, pullulan was the most preferred substrate but with less than 10% activity to that of αcyclodextrin (Table 3). Analysis of the reaction end products of these substrates demonstrated that maltose was the major product while glucose the minor (Fig. 4). CDaseGt was capable of hydrolysis as well as transglycosylation of maltotriose and higher oligoschararides. Major reaction products of maltotriose were maltose and glucose, while minor were maltotetraose and maltopentaose. These higher oligosachararides could be detected even after 15 min of the reaction. Oligosachararides higher than maltopentaose could not be detected even after a reaction of 1200 min (Table 4). However, when glucose or maltose were used independently or in combination with each other, no hydrolytic or transglycosylation activity could be detected. It appears that hydrolysis is the major activity of CDaseGt, whereas transglycosylation is the minor.
2.2. Gene expression in E. coli and purification of recombinant CDaseGt Expression of the gene in E. coli resulted in the production of recombinant CDaseGt in high amounts, approximately 30% of the host proteins (Fig. 2). Analysis of the soluble and insoluble fractions, after cell lysis, demonstrated that recombinant CDaseGt was produced in soluble form which remained in soluble fraction after heat treatment at 65 °C for 30 min, while most of the host proteins were denatured and precipitated, which were removed by centrifugation. CDaseGt, in the supernatant after centrifugation, was purified to apparent homogeneity, on SDS-PAGE, by anion-exchange and hydrophobic interaction column chromatographies (Fig. 2). Purification yield of recombinant CDaseGt was 30% with a 12-fold purification (Table 2).
2.5. Kinetic parameters When we determined the kinetic parameters of the reaction catalysed by CDaseGt, highest reaction rate was found with α-cyclodextrin followed by β- and γ-cyclodextrin. However, affinity of the enzyme towards these substrates was in the reverse order. Km value towards γcyclodextrin was 0.4 mM followed by 0.6 and 0.8 towards β- and αcyclodextrin, respectively (Table 5).
2.3. Biochemical characteristics of CDaseGt Examination of the enzyme activity at various pH values revealed that CDaseGt exhibits a broad pH optima. A minor difference in activity was observed at pH 5 to 7. However, the highest activity was observed at pH 6.0 in phosphate buffer (Fig. 3A). When the enzyme activity was examined at various temperatures by keeping the pH constant at 6.0 in phosphate buffer, CDaseGt exhibited the highest activity at 55 °C. (Fig. 3B).
3. Discussion The recent progress in sequencing has piled up more than seventy five thousand protein sequences belonging to members of family GH13 and only 1% of them have been characterized. In order to find novel 2
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Fig. 1. Alignment of amino acid sequences of the seven conserved regions found in CDaseGt and the characterized counterparts from GH13 family. Amino acids conserved in all the sequences are shown in white with black background. The numbers in superscripts show the corresponding position in the particular sequence. Catalytic triad is marked by asterisk below the aligned sequences. Uniprot accession numbers used were, V6VEH6: cyclomaltodextrinase from Geobacillus thermopakistaniensis; Q52PU5: amylase from Bacillus sp. WPD616; O69007: maltogenic amylase from Thermus sp. IM6501; I6RE37: maltogenic-amylase from Geobacillus thermoleovorans; Q9AIV2: neopullulanase from Geobacillus stearothermophilus; P38940: neopullulanase from Geobacillus stearothermophilus; C0LZ63: maltogenic amylase from Parageobacillus caldoxylosilyticus; Q68KL3: maltogenic amylase from Bacillus thermoalkalophilus; Q45490: maltogenic amylase from Geobacillus stearothermophilus; Q5BLZ6: cyclomaltodextrinase from Anoxybacillus flavithermus; A7DWA8: maltogenic amylase from Bacillus sp. US149; O82982: cyclomaltodextrinase from Bacillus sp.; Q57482: neopullulanase from Bacillus sp. KSM-1876; O06988: intracellular maltogenic amylase from Bacillus subtilis (strain 168); Q9R9H8: intracellular maltogenic amylase from Bacillus subtilis; X5CPN2: alpha-amylase from Bacillus lehensis G1; Q59226: cyclomaltodextrinase from Bacillus sp.; B0FZ47: oligosaccharide-producing multifunctional N-amylase from Bacillus sp. ZW2531-1; Q08341: cyclomaltodextrinase from Lysinibacillus sphaericus; Q9WX32: cyclomaltodextrinase from Alicyclobacillus acidocaldarius[37,40-46]. Table 2 Summary of the steps involved in purification of recombinant CDaseGt. Purification step
Total protein (mg)
Total activity (U)
Specific activity (U/mg)
Purification- fold
Yield (%)
Cell lysate Heat treatment HiTrap QFF HiTrap Phenyl HP
39 9.7 5.0 0.95
3775 3220 2280 1140
97 322 456 1200
1 3.3 4.7 12.4
100 85.2 60.4 30.2
thermopakistaiensis. CDaseGt was produced in soluble form in E. coli and possessed hydrolytic as well as transglycosylation activity. In order to get a better understanding of structure-function relationship among CDase-family members, the primary structure of CDaseGt was compared with those of closely related and characterized counterparts. Sequence comparison revealed the presence of seven conserved regions, identified in members of this family [26,27], were completely preserved in CDaseGt. Similar to other CDases, CDaseGt contains the N-terminal domain reported to be involved in dimerization of two identical subunits. In the dimeric structure, the active site pocket is a narrow groove which allows thin cyclodextrins to fit into it [28,29]. Truncation of this Nterminal domain resulted in a change in substrate specificity from cyclodextrins to starches [30]. We therefore, analyzed the quaternary state of CDaseGt by gel filtration chromatography and found that it existed in monomeric as well as dimeric form, similar to the counterpart from Thermus sp. [21]. This was further confirmed by non-denaturing PAGE (Fig. 5). Addition of KCl to the enzyme from Thermus sp. is reported to shift the monomer/dimer equilibrium toward the monomer resulting in a decrease in cyclodextrinase activity [30]. However, these results were in contrast to the cyclomaltodextrinase from Thermococcus sp. CL1 whose cyclodextrinase activity was increased in the presence of 5 mM KCl [31]. This prompted us to examine the cyclodextrinase activity in the presence of various concentrations of KCl. Lower
Fig. 2. Coomassie brilliant blue stained SDS-PAGE demonstrating production and purification of recombinant CDaseGt. Lane 1, total lysate of host cells carrying expression vector pET-21a(+); lane 2, total lysate of host cells carrying pET-CDaseGt gene; lane 3, soluble fraction of the sample in lane 2; lane 4, soluble fraction after heat treatment to the sample in lane 3; lane 5, sample after HiTrap QFF column; lane 6, sample after HiTrap PhenylHP column; lane M, molecular weight marker. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
enzymes from this family, there is a dire need to characterize more proteins. In this study we have cloned and characterized CDaseGt, an αcyclodextrin preferring cyclomaltodextrinase, from G. 3
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Fig. 3. Effect of pH and temperature on CDaseGt activity. A) Effect of pH. CDaseGt activity was examined at various pH without altering the temperature. Following buffers were used: sodium acetate buffer pH 4.5–6.0 (circles) and sodium phosphate buffer pH 6.0–7.5 (squares). B) Effect of temperature. CDaseGt activity was examined at various temperatures keeping the pH constant at 6.0 in sodium phosphate buffer. C) Thermostability of CDaseGt. The protein was incubated at 65 (circles) and 70 °C (squares) for various intervals of time in the presence (filled) and absence (empty) of 10 mM EDTA. Residual activity was examined in 50 mM sodium phosphate buffer (pH 6.0) at 55 °C.
protein was quite stable at 65 °C with no significant loss in activity even after overnight incubation at this temperature. However, at 70 °C the activity decreased gradually with the passage of time. Addition of EDTA has been reported to increase the activity [12] and thermal stability of a maltogenic amylase from Thermus sp. [21]. We therefore, examined the effect of EDTA on the enzyme activity and found no significant change in activity. Furthermore, we examined the stability of CDaseGt in the presence of 10 mM EDTA at 70 °C and found that the thermostability increased remarkably. However, overnight incubation at 4 °C is a prerequisite for enhancement of thermostability. If we directly incubate the protein at 70 °C, after addition of EDTA, no increase in thermostability was observed. There was no significant effect of EDTA on the thermostabilty at 75 °C or higher temperatures. Enhancement of thermostability, in the presence of EDTA, may be attributed to possible changes in the structure of CDaseGt. Such structural changes, in the presence of EDTA, have been reported for an endoglucanase from Aspergillus aculeatus [32]. Addition of Ca2+ has also been reported to increase the hydrolytic activity of cyclomaltodextrinase from Bacillus sp. I-5 [12] and Massilia timonae [17]. Similarly, a maltogenic amylase from Geobacillus sp. SK70, which was more than 97% identical to CDaseGt in primary structure (Table 1), was stimulated in the presence of Mn2+, Zn2+ and Tween-20 [33]. Furthermore, the enzyme activity of a maltogenic amylase from Bacillus sp. WPD616, which is more than 99% identical to CDaseGt, was completely inactivated in the presence of
Table 3 Relative hydrolysis rates of various substrates. Substrate
Relative hydrolysis rate (%)a
α-cyclodextrin β-cyclodextrin γ-cyclodextrin Pullulan Dextrin Wheat starch Rice starch Potato starch Corn starch Glycogen Acarbose Amylose Amylopectin
100 61 30 09 04 03 02 01 01 01 02 04 <1
a Determined by measuring the total reducing sugars, liberated from hydrolysis of these substrates at 55 °C.
concentrations of KCl, up to 200 mM, had no significant effect on the activity, however, at 0.5 M KCl, the cyclodextrinase activity was nearly abolished. As the optimum reaction temperature of CDaseGt was 55 °C, we therefore examined the thermostabilty at higher temperatures. The 4
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Fig. 4. High-performance liquid chromatography peaks showing the hydrolysis products α-cyclodextrin by CDaseGt. Reaction was carried out at 55 °C and pH 6.0. Aliquots were taken at various intervals of time and, after filtration, 20 μL were analyzed by an Aminex HPX-42A column. G1, G2, G3 and G4 correspond to glucose, maltose, maltotriose and maltotetraose, respectively.
Fig. 5. Zymogram analysis of purified CDaseGt. Purified CDaseGt was fractionated by gel filtration column (Superdex 200 10/30 GL) and two peak fractions were analysed by nondenaturing PAGE. One half part of the gel (left panel) was stained with Coomassie brilliant blue and the other half (right panel) was analysed by activity staining. Lane 1, first peak eluted at 10.8 mL (dimer); lane 2, second peak eluted at 13.4 mL (monomer). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Table 4 Time course analysis of end products of maltotriose. Time (min)
0 15 30 60 120 1200
Product released (%) G7
G6
G5
G4
G3
G2
G1
Total
– – – – – –
– – – – – –
– – – – 1.9 1.4
– 0.7 1.7 2.9 4.3 10.6
100 84 80.6 71 60 8.7
– 10 11.7 14.8 20.8 45.9
– 3.9 4.6 6.9 10.4 31.1
100 100 100 100 100 100
: Not detected Table 5 Kinetic parameters of CDaseGt against cyclodextrins.
α-cyclodextrin β-cyclodextrin γ-cyclodextrin a
Vmax (μmol min−1 mg−1)
Km (mM)
kcat (s−1)a
kcat/Km
1200 735 360
0.8 0.6 0.4
2739 1678 821
3424 2797 2053
kcat values were calculated based on the dimeric nature of CDaseGt.
1 mM Zn2+, and activity was reduced to 63% and 52% in the presence of 1 and 10 mM Mn2+, respectively [25]. In contrast to these studies, presence or absence of Ca2+, Zn2+ or Tween-20 exhibited no significant effect on the enzyme activity of CDaseGt. However, the enzyme activity of CDaseGt was nearly two-fold in the presence of 5 mM Mn2+. Acarbose, a psuedotetrasacharide, is a well-known inhibitor of αamylases. Among the enzymes in the subfamily, however, maltogenic amylases from Bacillus stearothermophilus [10], Thermus sp. [21]. and Geobacillus thermoleovorans [26], and cyclomaltodextrinase from Bacillus sp. I-5 [12] were able to hydrolyze acarbose to glucose [21]. When we examined the effect of acarbose on the cyclodextrinase activity of CDaseGt, we found that more than 90% activity was abolished in the presence of equimolar acarbose. CDaseGt was also capable
Fig. 6. Thin layer chromatography demonstrating hydrolysis of acarbose by CDaseGt. Acarbose (1%) was incubated with CDaseGt (5 μg) at 55 °C and pH 6.0 for 16 h and hydrolysis products were analyzed on TLC. Lane 1: standards; lane 2, acarbose (control); lane 3, hydrolysis products of acarbose. Symbols are: G1, glucose; G2, maltose; G3, maltotriose; AC, acarbose; and PT, pseudotrisaccharide.
5
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Table 6 Comparison of biochemical properties and kinetic parameters of closely related CDases. Organism
Geobacillus thermopakistaniensis Bacillus sp. WPD616 Thermus sp. Geobacillus sp. SK70 Geobacillus sp. Gh6 Geobacillus thermoleovorans B.stearothermophilusIMA6503. Geobacillus caldoxylosilyticus Bacillus thermoalkalophilus Bacillus stearothermophilus Bacillus sp. I-5
Homology (%)
100 99.8 99 98 96 96 86 75 72 69.4 56.4
Temp(°C)
55 50 60 55 60 80 55 50 70 55 50
pH
6 6.0 6 6 6 5-9 6.0 7 8 6.0 7.0
Activity (U)
kcat (s-1)
Km (mM)
kcat/Km
Reference
α
β
γ
α
β
γ
α
β
γ
α
β
γ
1200 * * * * 1151.2 * * * * *
735 * 278 ** * 548.9 ** ** 161 ** 1629
360 * * * * * * * * * *
0.8 * * * 3.9 2.01 * * * * 1.23
0.6 * 0.263 * 2.3 1.87 * * 0.129 * 0.83
0.4 * * * 1.9 * * * * * 0.92
2009 * * * 500 2.78 * * * * *
1242 * 167 * 280.7 1.32 * * 166 * *
605 * * * 225.6 ** * * * * *
2575 * * * 128.2 1.38 * * * * *
2484 * 636 * 121 0.70 * * 1383 * *
1375 * * * 118 * * * * * *
This Study [24] [21,30] [33] [34] [26] [36] [38] [39] [10] [12,47]
* : data not available
of hydrolysis of acarbose to glucose and pseudotrisaccharide (Fig. 6). However, the hydrolysis rate of acarbose was nearly fifty-times lower than that of α-cyclodexrin. The cyclodextrin hydrolysing enzymes from Bacillus sp. WPD616 [25], Thermus sp. IM6501 [21], G. thermoleovorans [26], Geobacillus sp. SK70 [33], Geobacillus Gh6 [34] and G. thermopakistaniensis exhibit more than 95% identity, however, some of their characteristics vary greatly (Table 6). As the monomeric form of CDaseGt is quite less active, therefore this variation in kinetic parameters can be attributed to the probable difference in equilibrium between dimeric and monomeric forms. In conclusion, we have characterized a CDase from G. thermopakistaniensis which exhibits the highest ever reported hydrolytic activity against α-cyclodextrin. Recombinant CDaseGt exists in monomeric as well as dimeric form in solution. Dimeric form is more active and stable than the monomeric form. Presence of EDTA enhances the thermostabilty of CDaseGt. Studies on site-directed mutagenesis, shifting the monomeric-dimeric equilibrium towards the dimeric form, and the industrial applications of this enzyme are in progress.
chain reaction (PCR). For amplification of the gene, a set of forward ( 5′-CATATGAGGAAAGAAGCCATCCACCACCGCTCAACCG-3′) and reverse (5′-TTACCAGCTTTCGACCGCGTAAAG-3′) primers was synthesized commercially (Gene Link, Hawthorne, NY). An NdeI restriction site was introduced in the forward primer (underlined sequence). The PCR-amplified product was cloned in pTG19-T, and the resulting plasmid was named pTG-CDaseGt. E. coli DH5-α cells were transformed using pTG-CDaseGt. For cloning in the expression vector, recombinant plasmid pTG-CDaseGt was digested with NdeI and HindIII to liberate the CDaseGt gene, which was purified and subsequently cloned in pET-21a (+) expression vector by utilizing the same restriction sites. The resulting plasmid was named pET-CDaseGt. 4.4. Production in E. coli and purification of CDaseGt All procedures were performed at room temperature unless stated otherwise. E. coli BL21 CodonPlus (DE3)-RIL cells containing pETCDaseGt were grown in LB medium, containing ampicillin, at 37 °C until the optical density at 600 nm reached at 0.4. Gene expression was induced with 0.2 mM IPTG. After 4.5 h of induction, cells were harvested by centrifugation at 12,000×g for 10 min at 4 °C. The cell pellet was resuspended in 50 mM Tris-Cl (pH 8.0) and disrupted by sonication using the Bandelin SonoPlus HD 2070 sonication system (Bandelin Electronic, GmbH). Cell debris was removed by centrifugation at 20,000×g for 10 min at 4 °C. The supernatant, obtained after centrifugation, was heated at different temperatures (from 50 °C to 70 °C) to get an optimized temperature at which recombinant CDaseGt remains in soluble fraction. Denatured heat-labile proteins from the host cells were separated by centrifugation at 20,000×g for 20 min at 4 °C. The supernatant, after centrifugation, was applied to HiTrap QFF column using ÄKTA purifier system (GE Healthcare, NJ). Elution of the proteins, bound to the column, was done with a 0–1 M sodium chloride (in 50 mM Tris-Cl, pH 8.0) linear gradient. Fractions, after HiTrap QFF column, containing significant amount of CDaseGt were pooled and equilibrated to 1 M (final concentration) ammonium sulphate. Protein sample was then applied to HiTrap Phenyl HP column equilibrated with 1 M ammonium sulphate. Elution of the proteins, bound to the column, was done with a 1 to 0 M ammonium sulphate linear gradient with 50 mM Tris-Cl, pH 8.0.
4. Experimental 4.1. Reagents and chemicals The reagents and chemicals used in this study were purchased either from Sigma-Aldrich (St. Louis, MO) or Fisher Scientific (Leicestershire). The restriction endonucleases, T4 DNA ligase, DNA and protein size markers, Taq DNA polymerase, RNase, and deoxynucleoside triphosphates (dNTPs) were from Thermo Scientific (ThermoFisher Scientific, MD). Cyclodextrins, maltooligosaccharides, pullulan, starch, amylopectin and amylose were purchased from Sigma-Aldrich. 4.2. Strains, plasmids, and media Escherichia coli DH5-α cells and plasmid pTG19-T (Vivantis Technologies) were used for cloning and general DNA manipulations. E. coli BL21 CodonPlus (DE3)-RIL cells (Stratagene, La Jolla, CA) and pET21a(+) expression vector (Novagen, Madison, WI) were used for gene expression. E. coli strains were routinely grown in Luria-Bertani (LB) medium at 37 °C. Recombinant E. coli cells containing pET-21a(+) were selected on LB agar containing ampicillin (100 μg mL−1), whereas 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (40 μg mL−1) and isopropyl-β-D-galactopyranoside (1 mM) were added when blue/white screening of recombinant E. coli cells containing pTG19-T was required.
4.5. Enzyme activity assay The enzyme activity of recombinant CDaseGt was measured in terms of the amount of reducing sugars liberated upon incubation with the substrate. Maltose was used as the standard for reducing sugars. In a standard assay mixture, 125 μL of 1% (w/v) substrate in 50 mM sodium phosphate buffer (pH 6.0) was mixed with 125 μL of solution containing 0.3 μg CDaseGt and incubated at 55 °C for 2–20 min. The reaction was
4.3. Cloning of CDaseGt gene CDaseGt gene (accession # ESU72342) was amplified from the genomic DNA of G. thermopakistaniensis strain MAS1 by polymerase 6
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stopped by quenching in ice water, and the released reducing ends were determined by the dinitrosalicylic acid (DNS) method [35].
4.10. Zymogram analysis Zymogram analysis was carried out for the detection of activities of monomeric and dimeric forms separated by gel filtration chromatography. Starch (0.1%) was incorporated into the non-denaturing polyacrylamide gel. Both monomer and dimer were loaded in duplicate. The gel was cut into two halves. First half was stained with coommassie brilliant blue and the second half was incubated in 50 mM phosphate buffer for 30 min at 55 °C followed by staining with KI/I2 (1.75% KI and 0.312%I2) solution.
4.6. Effects of pH and temperature on enzyme activity Effect of pH on the enzyme activity of recombinant CDaseGt was examined at 55 °C using various buffers of 50 mM strength. The buffers used were sodium acetate (pH 4.0 to 6.0), and sodium phosphate (pH 6.0 to 7.5). The pH values were adjusted at room temperature. In order to examine the effect of temperature on the enzymatic activity, assay mixtures were prepared in 50 mM sodium phosphate buffer, pH 6.0. Reaction mixtures were incubated for a fixed interval of time at temperatures from 45 to 75 °C. For thermostability analysis, the purified enzyme was diluted in 50 mM sodium phosphate buffer of pH 6.0. Properly diluted enzyme (0.3 μg/μL) was incubated at 65, 70 and 75 °C for various intervals of time and activity assays were performed at 55 °C and pH 6.0 as described above. Effect of EDTA (0.2–10 mM) was determined on enzyme activity and thermostability of CDaseGt. Activity assays were performed in the presence and absence of EDTA at 55 °C and pH 6.0 as described above.
References [1] K.H. Park, T.J. Kim, T.K. Cheong, J.W. Kim, B.H. Oh, B. Svensson, Structure, specificity and function of cyclomaltodextrinase, a multispecific enzyme of the αamylase family, Biochim. Biophys. Acta 1478 (2000) 165–185 https://doi.org/10. 1016/S0167-4838(00)00041-8. [2] K.H. Park, Function and tertiary-and quaternary-structure of cyclodextrin-hydrolyzing enzymes (CDase), a group of multisubstrate specific enzymes belonging to the α-amylase family, J. Appl. Glycosci. 53 (2006) 35–44 https://doi.org/10.5458/ jag.53.35. [3] R. Hulsmann, F. Lurz, F. Scheffel, E. Schneider, Maltose and maltodextrin transport in the thermoacidophilic gram-positive bacterium Alicyclobacillus acidocaldarius is mediated by a high-affinity transport system that includes a maltose binding protein tolerant to low pH, J. Bacteriol. 182 (2000) 6292–6301 https://doi.org/10.1128/ JB.182.22.6292-6301.2000. [4] P. Turner, A. Labes, O.H. Fridjonsson, G.O. Hreggvidson, P. Schonheit, J.K. Kristjansson, O. Holst, E.N. Karlsson, Two novel cyclodextrin-degrading enzymes isolated from thermophilic bacteria have similar domain structures but differ in oligomeric state and activity profile, J. Biosci. Bioeng. 100 (2005) 380–390 https://doi.org/10.1263/jbb.100.380. [5] S. Kitahata, M. Taniguchi, S.D. Beltran, T. Sugimoto, S. Okada, Purification and some properties of cyclodextrinase from Bacillus coagulans, Agric. Biol. Chem. 47 (1983) 1441–1447 https://doi.org/10.1080/00021369.1983.10865801. [6] Y. Nakagawa, W. Saburi, M. Takada, Y. Hatada, K. Horikoshi, Gene cloning and enzymatic characteristics of a novel gamma-cyclodextrin-specific cyclodextrinase from alkalophilic Bacillus clarkii 7364, Biochim. Biophys. Acta 1784 (2008) 2004–2011 https://doi.org/0.1016/j.bbapap.2008.08.022. [7] J.A. DePinto, L.L. Campbell, Purification and properties of the amylase of Bacillus macerans, Biochemistry 7 (1968) 121–124 https://doi/pdf/10.1021/bi00841a015. [8] T. Oguma, M. Kikuchi, K. Mizusawa, Purification and some properties of cyclodextrin-hydrolyzing enzyme from Bacillus sphaericus, Biochim. Biophys. Acta 1036 (1990) 1–5 https://doi.org/10.1016/0304-4165(90)90205-B. [9] T. Oguma, A. Matsuyama, M. Kikuchi, E. Nakano, Cloning and sequence analysis of the cyclomaltodextrinase gene from Bacillus sphaericus and expression in Escherichia coli cells, Appl. Microbiol. Biotechnol. 39 (1993) 197–203 https://doi.org/10.1007/ BF00228606. [10] H.J. Cha, H.G. Yoon, Y.W. Kim, H.S. Lee, J.W. Kim, K.S. Kwon, B.H. OH, K.H. Park, Molecular and enzymatic characterization of a maltogenic amylase that hydrolyzes and transglycosylates acarbose, Eur. J. Biochem. 253 (1998) 251–262 https://doi. org/10.1046/j.1432-1327.1998.2530251. [11] K. Ohdan, T. Kuriki, H. Takata, S. Okada, Introduction of raw starch-binding domains into Bacillus subtilis α-amylase by fusion with the starch-binding domain of Bacillus cyclomaltodextrin glucanotransferase, Appl. Microbiol. Biotechnol. 53 (2000) 430–434 https://doi.org/10.1128/AEM.66.7.3058-3064.2000. [12] T.J. Kim, J.H. Shin, J.H. Oh, M.J. Kim, S.B. Lee, S. Ryu, K. Kwon, J.W. Kim, E.H. Choi, J.F. Robyt, K.H. Park, Analysis of the gene encoding cyclomaltodextrinase from alkalophilic Bacillus sp. I-5 and characterization of enzymatic properties, Arch. Biochem. Biophys. 353 (1998) 221–227 https://doi.org/10.1006/abbi. 1998.0639. [13] S.M. Podkovyrov, J.G. Zeikus, Structure of the gene encoding cyclomaltodextrinase from Clostridium thermohydrosulfuricum 39E and characterization of the enzyme purified from Escherichia coli, J. Bacteriol. 174 (1992) 5400–5405 https://doi.org/ 10.1128/jb.174.16.5400-5405.1992. [14] H. Bender, Purification and characterization of a cyclodextrin-degrading enzyme from Flavobacterium sp, Appl. Microbiol. Biotechnol. 39 (1993) 714–719 https:// doi.org/10.1007/BF00164455. [15] G. Fiedler, M. Pajatsch, A. Böck, Genetics of a novel starch utilisation pathway present in Klebsiella oxytoca, J. Mol. Biol. 256 (1996) 279–291 https://doi.org/10. 1006/jmbi.1996.0085. [16] M.U. Jang, H.J. Kang, C.K. Jeong, Y. Kang, J.E. Park, T.J. Kim, Functional expression and enzymatic characterization of Lactobacillus plantarum cyclomaltodextrinase catalyzing novel acarbose hydrolysis, J. Microbiol. 56 (2018) 113–118 https:doi.org/10.1007/s12275-018-7551-3. [17] F.C.D. Santos, I.P. Barbosa-Tessmann, Recombinant expression, purification, and characterization of a cyclodextrinase from Massilia timonae, Protein Expr. Purif 154 (2019) 74–84 https:doi.org/10.1016/j.pep.2018.08.013. [18] J. Kaulpiboon, P. Pongsawasdi, Expression of cyclodextrinase gene from Paenibacillus sp. A11 in Escherichia coli and characterization of the purified cyclodextrinase, J. Biochem. Mol. Biol. 37 (2004) 408–415. [19] Y. Hashimoto, T. Yamamoto, S. Fujiwara, M. Takagi, T. Imanaka, Extracellular synthesis, specific recognition, and intracellular degradation of cyclomaltodextrins
4.7. Substrate specificity and analysis of the hydrolysis products The substrate preference and relative hydrolysis rates of cyclodextrins and various polysaccharides, including pullulan, starch, amylose, amylopectin, and dextrin, were determined by incubating each of them at a final concentration of 0.5% (w/v) with recombinant CDaseGt. Substrate solutions were prepared in 50 mM sodium phosphate buffer (pH 6.0), and after the addition of purified enzyme (0.3 μg), incubated at 55 °C for 2–30 min. The hydrolysis rates (μmol reducing sugar min−1 mg−1) were measured every 2 min by the DNS method [35]. In order to identify the saccharides obtained as hydrolysis products, incubations were carried out under similar conditions for various time intervals. The products were then analyzed by high-performance liquid chromatography (HPLC) on an Aminex HPX-42A column (300 × 78 mm; Bio-Rad Laboratories, Inc., CA). Signals were detected with a differential refractive index detector (S3580; Sykam GmbH, Germany). Double-distilled deionized water was used as the mobile phase/solvent. During separation, the column temperature was maintained at 85 °C and that of the detector at 45 °C. In addition to hydrolytic activity, transglycosylation activity of CDaseGt was also evaluated by using glucose, maltose and maltotriose or the mixtures of any two of these. 4.8. Thin layer chromatography Thin layer chromatography (TLC) was used to analyse hydrolytic products of acarbose. After incubating acarbose and CDaseGt, at 55 °C and pH 6.0, for various intervals of time, the hydrolytic products were analyzed by TLC, using 0.2 mm silica gel-coated aluminium sheets (Kieselgel 60 F254; Merck). Aliquots (5 μL) of the reaction mixtures were spotted on TLC plate and chromatographed using n-butanol-diethyl ether-acetic acid-water (9:3:3:1 [v/v/v/v]) as mobile phase. Hydrolytic products were identified by spraying the plate with 5% v/v sulphuric acid in methanol followed by baking the TLC plate at 120 °C for 5 min. 4.9. Gel filtration chromatography Oligomeric nature of CDaseGt was determined by gel filtration chromatography using Superdex 200 10/300 GL column (GE Healthcare, NJ). The column was equilibrated with 50 mM Tris-Cl pH 8 containing 150 mM NaCl. Protein sample (prepared in the same buffer) was loaded and eluted at a flow rate of 0.3 mL/min. Eluted fractions were used for zymogram analysis. 7
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[20] [21]
[22] [23]
[24]
[25] [26]
[27] [28]
[29]
[30]
[31]
[32] [33]
[34] S. Nasrollahi, L. Golalizadeh, R.H. Sajedi, M. Taghdir, S.M. Asghari, M. Rassa, Substrate preference of a Geobacillus maltogenic amylase: a kinetic and thermodynamic analysis, Int. J. Biol. Macromol. 60 (2013) 1–9 https://doi.org/10.1016/j. ijbiomac.2013.04.063. [35] P. Bernfeld, Amylases, alpha and beta, Methods Enzymol. 1 (1955) 149–158 https://doi.org/10.1016/0076-6879(55)01021-5. [36] K.A. Cheong, T.J. Kim, J.W. Yoon, C.S. Park, T.S. Lee, Y. B Kim, K.H. Park, J.W. Kim, Catalytic activities of intracellular dimeric neopullulanase on cyclodextrin, acarbose and maltose, Biotechnol. Appl. Biochem. 35 (2002) 27–34 https:// www.ncbi.nlm.nih.gov/pubmed/11834127. [37] T. Kuriki, T. Imanaka, Pattern of action of Bacillus stearothermophilus neopullulanase on pullulan, J. Bacteriol. 171 (1989) 369–374 https://doi.org/10.1128/jb.171.1. 369-374.1989. [38] Y. Kolcuoğlu, A. Colak, O. Faiz, A. Belduz, Cloning, expression and characterization of highly thermo-and pH-stable maltogenic amylase from a thermophilic bacterium Geobacillus caldoxylosilyticus TK4, Process Biochem. 45 (2010) 821–828 https://doi. org/10.1016/j.procbio.2010.02.001. [39] K.A. Cheong, K.A. Cheong, S.Y. Tang, T.K. Cheong, H. Cha, J.W. Kim, K.H. Park, Thermostable and Alkalophilic Maltogenic Amylase of Bacillus Thermoalkalophilus ET2 in Monomer-Dimer Equilibrium vol. 23, (2005), pp. 79–87 https://doi.org/10. 1080/10242420500090094. [40] S.B. Mabrouk, E.B. Messaoud, D. Ayadi, S. Jemli, A. Roy, M. Mezghani, S. Bejar, Cloning and sequencing of an original gene encoding a maltogenic amylase from Bacillus sp. US149 strain and characterization of the recombinant activity, Mol. Biotechnol. 38 (2008) 211 https://doi.org/10.1007/s12033-007-9017-4. [41] K. Igarashi, K. Ara, K. Saeki, K. Ozaki, S. Kawai, S. Ito, Nucleotide sequence of the gene that encodes a neopullulanase from an alkalophilic Bacillus, Biosci. Biotechnol. Biochem. 56 (1992) 514–516 https://doi.org/10.1271/bbb.56.514. [42] J. H Shim, J.T. Park, J.S. Hong, K. W Kim, M.J. Kim, J.H. Auh, Y.W. Kim, C.S. Park, W. Boos, J.W. Kim, K.W. Park, Role of maltogenic amylase and pullulanase in maltodextrin and glycogen metabolism of Bacillus subtilis 168, J. Bacteriol. 191 (2009) 4835–4844 https://doi.org/10.1128/JB.00176-09. [43] H.Y. Cho, Y.M. Kim, T.J. Kim, H.S. Lee, D.Y. Kim, J.W. Kim, Y.W. Lee, S.B. Leed, K.H. Park, Molecular characterization of a dimeric intracellular maltogenic amylase of Bacillus subtilis SUH4-2, Biochim. Biophys. Acta 1478 (2000) 333–340 https:// doi.org/10.1016/S0167-4838(00)00037-6. [44] N.H.A. Manas, S. Pachelles, N.M. Mahadi, R.M. Illias, The characterisation of an alkali-stable maltogenic amylase from Bacillus lehensis G1 and improved maltooligosaccharide production by hydrolysis suppression, PLoS One 9 (2014) e106481 https://doi.org/10.1371/journal.pone.0106481. [45] F. Li, X. Zhu, Y. Li, H. Cao, Y. Zhang, Functional characterization of a special thermophilic multifunctional amylase OPMA-N and its N-terminal domain, Acta Biochim. Biophys. 43 (2011) 324–334 https://doi.org/10.1093/abbs/gmr013. [46] J. Matzke, A. Herrmann, E. Schneider, E.P. Bakker, Gene cloning, nucleotide sequence and biochemical properties of a cytoplasmic cyclomaltodextrinase (neopullulanase) from Alicyclobacillus acidocaldarius, reclassification of a group of enzymes, FEMS Microbiol. Lett. 183 (2000) 55–61 https://doi.org/10.1111/j.15746968.2000.tb08933.x. [47] M. J Kim, W.S. Park, H.S. Lee, T. J Kim, J.H. Shin, S.H. Yoo, T.K. Cheong, S. Ryu, J.C. Kim, J.W. Kim, T.W. Moon, J.F. Robyt, K.H. Park, Kinetics and inhibition of cyclomaltodextrinase from alkalophilic Bacillus sp. I-5, Arch. Biochem. Biophys. 373 (2000) 110–115 https://doi.org/10.1006/abbi.1999.1471.
by the hyperthermophilic archaeon Thermococcus sp. strain B1001, J. Bacteriol. 183 (2001) 5050–5057 https: doi.org/10.1128/JB.183.17.5050-5057.2001. Y. Sun, X. Lv, Z. Li, J. Wang, B. Jia, J. Liu, Recombinant cyclodextrinase from Thermococcus kodakarensis KOD1: expression, purification, and enzymatic characterization, Archaea 26 (2015) 1–8 https://doi.org/10.1155/2015/397924. T.J. Kim, M.J. Kim, B.C. Kim, J.C. Kim, T.K. Cheong, J.W. Kim, K.H. Park, Modes of action of acarbose hydrolysis and transglycosylation catalyzed by a thermostable maltogenic amylase, the gene for which was cloned from a Thermus strain, Appl. Environ. Microbiol. 65 (1999) 1644–1651. M.A. Siddiqui, N. Rashid, S. Ayyampalayam, W.B. Whitman, Draft genome sequence of Geobacillus thermopakistaniensis strain MAS1, Genome Announc. 2 (2014) e00559-14 https:doi.org/10.1128/genomeA.00559-14. N. Ahmad, N. Rashid, M.S. Haider, M. Akram, M. Akhtar, Novel maltotriose-hydrolyzing thermoacidophilic type III pullulan hydrolase from Thermococcus kodakarensis, Appl. Environ. Microbiol. 80 (2014) 1108–1115 https:doi.org/10.1128/ AEM.03139-13. M.J.E.C. van der Maarel, B. van der Veen, J.C.M. Uitdehaag, H. Leemhuis, L. Dijkhuizen, Properties and applications of starch-converting enzymes of the αamylase family, J. Biotechnol. 94 (2002) 137–155 https://doi.org/10.1016/S01681656(01)00407-2. B. Liu, Y. Wang, X. Zhang, Characterization of a recombinant maltogenic amylase from deep sea thermophilic Bacillus sp. WPD616, Enzym. Microb. Technol. 39 (2006) 805–810 https://doi.org/10.1016/j.enzmictec.2006.01.003. D. Mehta, T. Satyanarayana, Dimerization mediates thermo-adaptation, substrate affinity and transglycosylation in a highly thermostable maltogenic amylase of Geobacillus thermoleovorans, PLoS One 8 (2013) e73612 https://doi.org/10.1371/ journal.pone.0073612. Š. Janeček, How many conserved sequence regions are there in the α-amylase family? Biologia 57 (2002) 29–41 https://http://biologia.savba.sk/suppl_11/janecek. pdf. J.S. Kim, S.S. Cha, H.J. Kim, T.J. Kim, N.C. Ha, S.T. Oh, H.S. Cho, M.J. Cho, M.J. Kim, H.S. Lee, J.W. Kim, K.Y. Choi, K.H. Park, B.H. Oh, Crystal structure of a maltogenic amylase provides insights into a catalytic versatility, J. Biol. Chem. 274 (1999) 26279–26286 https://doi.org/10.1074/jbc.274.37.26279. H.S. Lee, M.S. Kim, H.S. Cho, J.I. Kim, T.J. Kim, J.H. Choi, C. Park, H.S. Lee, B.H. Oh, K.H. Park, Cyclomaltodextrinase, neopullulanase, and maltogenic amylase are nearly indistinguishable from each other, J. Biol. Chem. 277 (2002) 21891–21897 https://doi.org/10.1074/jbc.M201623200. T.J. Kim, V.D. Nguyen, H.S. Lee, M.J. Kim, H.Y. Cho, Y.W. Kim, T.W. Moon, C.S. Park, J.W. Kim, B.H. Oh, S.B. Lee, B. Svensson, K.H. Park, Modulation of the multisubstrate specificity of Thermus maltogenic amylase by truncation of the Nterminal domain and by a salt-induced shift of the monomer/dimer equilibrium, Biochemistry 40 (2001) 14182–14190 https://doi.org/10.1021/bi015531u. J.E. Lee, I.H. Kim, J.H. Jung, D.H. Seo, S.G. Kang, J.F. Holden, J. Cha, C.S. Park, Molecular cloning and enzymatic characterization of cyclomaltodextrinase from hyperthermophilic archaeon Thermococcus sp. CL1, J. Microbiol. Biotechnol. 23 (2013) 1060–1069 https://doi.org/10.4014/jmb.1302.02073. G.S. Naika, P.K. Tikudx, Influence of ethylenediaminetetraacetic Acid (EDTA) on the structural stability of endoglucanase from Aspergillus aculeatus, J. Agric. Food Chem. 59 (2011) 7341–7345 https://doi.org/10.1021/jf103889m. M.R. Sulong, T. Leow, R. Rahman, M. Basri, A.B. Salleh, Enhancing thermostability of maltogenic amylase from Geobacillus sp. SK70 by single amino acid substitution, Int. J. New Technol. Sci. Eng. 2 (2015) 20–41.
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A highly active α-cyclodextrin preferring cyclomaltodextrinase from Geobacillus thermopakistaniensis
T
Iqra Aroob, Nasir Ahmad, Mehwish Aslam, Abeera Shaeer, Naeem Rashid* School of Biological Sciences, University of the Punjab, Quaid-e-Azam Campus, Lahore, 54590, Pakistan
ARTICLE INFO
ABSTRACT
Keywords: Cyclomaltodextrinase Cyclodextrin Acarbose Thermostable Cloning Geobacillus thermopakistaniensis
Cyclomaltodextrinases show diverse hydrolyzing and/or transglycosylation activities against cyclodextrins, starch and pullulan. A gene annotated as cyclomaltodextrinase from Geobacillus thermopakistaniensis was cloned and overexpressed in Escherichia coli. The gene product, CDaseGt, was purified and biochemically characterized. The recombinant enzyme exhibited highest activity with α-cyclodextrin at 55 °C and pH 6.0. Specific hydrolytic activities towards α-, β- and γ-cyclodextrin were 1200, 735 and 360 μmol min−1 mg−1, respectively. To the best of our knowledge, the activity against α-cyclodextrin is the highest among the reported enzymes. Next to cyclodextrins, pullulan was the most preferred substrate with a specific activity of 105 μmol min−1 mg−1. CDaseGt was capable of hydrolysis of maltotriose and acarbose as well as transglycosylation of their hydrolytic products. At 65 °C, there was no significant loss in enzyme activity even after overnight incubation. Activity of CDaseGt was not metal ions dependent, however, the presence of Mn2+ significantly enhanced the α-CDase activity. EDTA had no significant effect on the CDaseGt activity, however, it enhanced the thermostability of the enzyme. CDaseGt existed in monomeric as well as dimeric form in solution. Dimeric form is more active compared to the monomeric one. Equilibrium between the two forms seems to be concentration dependent.
1. Introduction Cyclomaltodextrinase (CDase, EC 3.2.1.54), maltogenic amylase (MAase, EC 3.2.1.133) and neopullulanase (NPase, EC 3.2.1.135) are three groups of enzymes which efficiently hydrolyze cyclodextrins (CDs), cyclic oligomers of glucose units linked through α-D-(1,4)glycosidic bonds [1]. These enzymes also hydrolyze starch and pullulan but at a slower rate. They contain nearly 130 residues at the N-terminus, speculated to be associated with the catalytic domain, and around 70 residues at the C-terminus, that are absent in the αamylases. It has been proposed that these three groups of enzymes should come under a single name, cyclodextrinase (CDases), since cyclodextrins are their most preferred substrates [1,2]. The recent advancement in whole genome sequencing has revealed the presence of these enzymes in various microorganisms. They have been characterized from several bacterial and archaeal sources, including Alicyclobacillus acidocaldarius [3], Anoxybacillus flavithermus [4], Bacillus coagulans [5], Bacillus clarkii 7364 [6], Bacillus macerans [7], Bacillus sphaericus [8,9], Bacillus stearothermophilus [10], Bacillus sp. A2-5a [11], Bacillus sp. I-5 [12], Clostridium thermohydrosulfuricum 39E [13], Flavobacterium sp. [14], Klebsiella oxytoca strain M5a1 [15], Lactobacillus plantarum [16], Massilia timonae [17], *
Paenibacillus sp. A11 [18], Thermococcus sp. strain B1001 [19], Thermococcus kodakarensis [20], and Thermus sp. [21]. Geobacillus thermopakistaniensis is a Gram-positive aerobic thermophile, whose complete genome sequence has been determined [22]. We are interested in glycosyl hydrolase family 13 (GH13) enzymes. We have previously identified a novel enzyme from this family capable of hydrolyzing both α-1,4- and α-1,6-linked glucose polymers under industrial conditions and without the requirement of any additional metal ions [23]. Analysis of the genome sequence of G. thermopakistaniensis has revealed the presence of a full range of starch-hydrolyzing enzymes belonging to family GH13. They are annotated as α-amylase (ESU72338), cyclomaltodextrinase (ESU72342), amylopullulanase (ESU72863), glycogen branching enzyme (ESU73465), and pullulanase (ESU73761). However, none of them has been characterized. Currently, GH13 family contains 75319 sequences in total, including 70975 from bacterial sources. However, only 823 (1%) of them have been characterized, among them only 18 (2%) are cyclodextrin hydrolyzing enzymes (2019, http://www.cazy.org/GH13.html). These enzymes have great commercial importance in food industry [24]. Since a very few number of cycolodextrin hydrolyzing enzymes have been characterized and a search for better enzymatic toolkit is a need of the industry, we carried out the current study with the aim to find out a novel cyclodextrinase. In the present manuscript, we have described cloning and
Corresponding author. E-mail addresses: [email protected], [email protected] (N. Rashid).
https://doi.org/10.1016/j.carres.2019.06.004 Received 6 April 2019; Received in revised form 30 May 2019; Accepted 6 June 2019 Available online 08 June 2019 0008-6215/ © 2019 Elsevier Ltd. All rights reserved.
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Table 1 Percent identity between amino acid sequence of CDaseGt and other CDases. Enzyme and Source
1 2 3 4 5 6 7 8 9 10 11 12 13
Cyclomalodextrinase (CDaseGt) (Geobacillus thermopakistaniensis) Amylase (Bacillus sp. WPD616) Maltogenic amylase (Thermus sp. IM6501) Maltogenic Amylase (Geobacillus sp. SK70) Maltogenic amylase (Geobacillus sp. Gh6) Maltogenic-amylase (Geobacillus thermoleovorans) Neopullulanase (Geobacillus stearothermophilus) Neopullulanase (Geobacillus stearothermophilus) Maltogenic amylase (Parageobacillus caldoxylosilyticus) Cyclomaltodextrinase (Anoxybacillus flavithermus) Maltogenic amylase (Bacillus thermoalkalophilus) Maltogenic Amylase (Geobacillus stearothermophilus) Cyclomaltodextrinase (Bacillus sp.)
Sequence Identity (%) 1
2
3
4
5
6
7
8
9
10
11
12
13
100
99.8 100
99.4 99.3 100
97.2 97.1 97.1 100
96.4 96.2 96.2 97.9 100
95.9 95.7 95.7 97.4 98.2 100
86.2 86 86.2 86.7 86.9 87.2 100
86.2 86 86.2 86.5 86.5 86.9 98.2 100
73.8 73.6 73.8 74.4 74.3 74.4 75.3 75.3 100
69.2 69 69.3 69 69 69.2 69.5 69.2 72.1 100
66.3 66.1 66.3 66.8 66.6 66.8 67.6 67.8 76.5 68 100
64.2 64 64.2 64.9 64.7 64.7 63.8 63.7 68.9 67.9 78.1 100
55.2 55.1 55.2 55.1 54.7 54.7 55.4 55.9 58.9 57.5 64.2 62.3 100
Uniprot accession numbers or references used were: 1, V6VEH6; 2, Q52PU5; 3, O69007; 4, [Ref. [33]]; 5, E0X988; 6, I6RE37; 7, Q9AIV2; 8, P38940; 9, C0LZ63; 10, Q5BLZ6; 11, Q68KL3; 12, Q45490; 13, O82982.
expression, in Escherichia coli, of the gene corresponding to ESU72342, encoding a cyclomaltodextrinase (CDaseGt) from G. thermopakistaniensis.
Thermostability experiments revealed that CDaseGt is quite stable at temperatures up to 65 °C. There was no significant loss in enzyme activity even after overnight incubation at 65 °C. Presence of EDTA enhanced the thermostability of CDaseGt. The half-life of CDaseGt was 90 min at 70 °C in the absence of EDTA which increased to 360 min in the presence of 10 mM EDTA (Fig. 3C). The enzyme activity decreased drastically when incubated at 75 °C in the presence or absence of EDTA.
2. Results 2.1. Gene cloning and sequence analysis Genome sequence of G. thermopakistaniensis contains an open reading frame (ESU72342) coding for cyclomaltodextrinase of the GH13 family. The gene consisted of 1767 nucleotides, encoding a polypeptide of 588 amino acid residues having a theoretical molecular mass of 68,157 Da and an isoelectric point of 5.58. Sequence comparison demonstrated that CDaseGt exhibited the highest homology of 99% (identity) with amylase from Bacillus sp. WPD616 [25] and maltogenic amylase from Thermus sp. (1GVI_A) [21] (Table 1). Sequence alignment of CDaseGt with the characterized counterparts belonging to GH13 family, identified seven conserved regions typical of the members of this family. Among these conserved regions, region I was identical in all the counterparts (Fig. 1). However, there are slight differences in the other conserved regions. Similar to all other members of GH-13 family, the three catalytic residues, Asp328, Glu357 and Asp424, were conserved in CDaseGt.
2.4. Substrate preference and analysis of hydrolysis products In order to determine the substrate preference, various cyclic and acyclic saccharides were tested at a final concentration of 0.5%. Cyclodextrins were preferred among the tested substrates. Among cyclodextrins, α-cyclodextrin was the most preferred substrate with a specific activity of 1200 μmol min−1 mg−1, followed by β-cyclodextrin and then γ-cyclodextrin. Among the acyclic substrates, pullulan was the most preferred substrate but with less than 10% activity to that of αcyclodextrin (Table 3). Analysis of the reaction end products of these substrates demonstrated that maltose was the major product while glucose the minor (Fig. 4). CDaseGt was capable of hydrolysis as well as transglycosylation of maltotriose and higher oligoschararides. Major reaction products of maltotriose were maltose and glucose, while minor were maltotetraose and maltopentaose. These higher oligosachararides could be detected even after 15 min of the reaction. Oligosachararides higher than maltopentaose could not be detected even after a reaction of 1200 min (Table 4). However, when glucose or maltose were used independently or in combination with each other, no hydrolytic or transglycosylation activity could be detected. It appears that hydrolysis is the major activity of CDaseGt, whereas transglycosylation is the minor.
2.2. Gene expression in E. coli and purification of recombinant CDaseGt Expression of the gene in E. coli resulted in the production of recombinant CDaseGt in high amounts, approximately 30% of the host proteins (Fig. 2). Analysis of the soluble and insoluble fractions, after cell lysis, demonstrated that recombinant CDaseGt was produced in soluble form which remained in soluble fraction after heat treatment at 65 °C for 30 min, while most of the host proteins were denatured and precipitated, which were removed by centrifugation. CDaseGt, in the supernatant after centrifugation, was purified to apparent homogeneity, on SDS-PAGE, by anion-exchange and hydrophobic interaction column chromatographies (Fig. 2). Purification yield of recombinant CDaseGt was 30% with a 12-fold purification (Table 2).
2.5. Kinetic parameters When we determined the kinetic parameters of the reaction catalysed by CDaseGt, highest reaction rate was found with α-cyclodextrin followed by β- and γ-cyclodextrin. However, affinity of the enzyme towards these substrates was in the reverse order. Km value towards γcyclodextrin was 0.4 mM followed by 0.6 and 0.8 towards β- and αcyclodextrin, respectively (Table 5).
2.3. Biochemical characteristics of CDaseGt Examination of the enzyme activity at various pH values revealed that CDaseGt exhibits a broad pH optima. A minor difference in activity was observed at pH 5 to 7. However, the highest activity was observed at pH 6.0 in phosphate buffer (Fig. 3A). When the enzyme activity was examined at various temperatures by keeping the pH constant at 6.0 in phosphate buffer, CDaseGt exhibited the highest activity at 55 °C. (Fig. 3B).
3. Discussion The recent progress in sequencing has piled up more than seventy five thousand protein sequences belonging to members of family GH13 and only 1% of them have been characterized. In order to find novel 2
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Fig. 1. Alignment of amino acid sequences of the seven conserved regions found in CDaseGt and the characterized counterparts from GH13 family. Amino acids conserved in all the sequences are shown in white with black background. The numbers in superscripts show the corresponding position in the particular sequence. Catalytic triad is marked by asterisk below the aligned sequences. Uniprot accession numbers used were, V6VEH6: cyclomaltodextrinase from Geobacillus thermopakistaniensis; Q52PU5: amylase from Bacillus sp. WPD616; O69007: maltogenic amylase from Thermus sp. IM6501; I6RE37: maltogenic-amylase from Geobacillus thermoleovorans; Q9AIV2: neopullulanase from Geobacillus stearothermophilus; P38940: neopullulanase from Geobacillus stearothermophilus; C0LZ63: maltogenic amylase from Parageobacillus caldoxylosilyticus; Q68KL3: maltogenic amylase from Bacillus thermoalkalophilus; Q45490: maltogenic amylase from Geobacillus stearothermophilus; Q5BLZ6: cyclomaltodextrinase from Anoxybacillus flavithermus; A7DWA8: maltogenic amylase from Bacillus sp. US149; O82982: cyclomaltodextrinase from Bacillus sp.; Q57482: neopullulanase from Bacillus sp. KSM-1876; O06988: intracellular maltogenic amylase from Bacillus subtilis (strain 168); Q9R9H8: intracellular maltogenic amylase from Bacillus subtilis; X5CPN2: alpha-amylase from Bacillus lehensis G1; Q59226: cyclomaltodextrinase from Bacillus sp.; B0FZ47: oligosaccharide-producing multifunctional N-amylase from Bacillus sp. ZW2531-1; Q08341: cyclomaltodextrinase from Lysinibacillus sphaericus; Q9WX32: cyclomaltodextrinase from Alicyclobacillus acidocaldarius[37,40-46]. Table 2 Summary of the steps involved in purification of recombinant CDaseGt. Purification step
Total protein (mg)
Total activity (U)
Specific activity (U/mg)
Purification- fold
Yield (%)
Cell lysate Heat treatment HiTrap QFF HiTrap Phenyl HP
39 9.7 5.0 0.95
3775 3220 2280 1140
97 322 456 1200
1 3.3 4.7 12.4
100 85.2 60.4 30.2
thermopakistaiensis. CDaseGt was produced in soluble form in E. coli and possessed hydrolytic as well as transglycosylation activity. In order to get a better understanding of structure-function relationship among CDase-family members, the primary structure of CDaseGt was compared with those of closely related and characterized counterparts. Sequence comparison revealed the presence of seven conserved regions, identified in members of this family [26,27], were completely preserved in CDaseGt. Similar to other CDases, CDaseGt contains the N-terminal domain reported to be involved in dimerization of two identical subunits. In the dimeric structure, the active site pocket is a narrow groove which allows thin cyclodextrins to fit into it [28,29]. Truncation of this Nterminal domain resulted in a change in substrate specificity from cyclodextrins to starches [30]. We therefore, analyzed the quaternary state of CDaseGt by gel filtration chromatography and found that it existed in monomeric as well as dimeric form, similar to the counterpart from Thermus sp. [21]. This was further confirmed by non-denaturing PAGE (Fig. 5). Addition of KCl to the enzyme from Thermus sp. is reported to shift the monomer/dimer equilibrium toward the monomer resulting in a decrease in cyclodextrinase activity [30]. However, these results were in contrast to the cyclomaltodextrinase from Thermococcus sp. CL1 whose cyclodextrinase activity was increased in the presence of 5 mM KCl [31]. This prompted us to examine the cyclodextrinase activity in the presence of various concentrations of KCl. Lower
Fig. 2. Coomassie brilliant blue stained SDS-PAGE demonstrating production and purification of recombinant CDaseGt. Lane 1, total lysate of host cells carrying expression vector pET-21a(+); lane 2, total lysate of host cells carrying pET-CDaseGt gene; lane 3, soluble fraction of the sample in lane 2; lane 4, soluble fraction after heat treatment to the sample in lane 3; lane 5, sample after HiTrap QFF column; lane 6, sample after HiTrap PhenylHP column; lane M, molecular weight marker. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
enzymes from this family, there is a dire need to characterize more proteins. In this study we have cloned and characterized CDaseGt, an αcyclodextrin preferring cyclomaltodextrinase, from G. 3
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Fig. 3. Effect of pH and temperature on CDaseGt activity. A) Effect of pH. CDaseGt activity was examined at various pH without altering the temperature. Following buffers were used: sodium acetate buffer pH 4.5–6.0 (circles) and sodium phosphate buffer pH 6.0–7.5 (squares). B) Effect of temperature. CDaseGt activity was examined at various temperatures keeping the pH constant at 6.0 in sodium phosphate buffer. C) Thermostability of CDaseGt. The protein was incubated at 65 (circles) and 70 °C (squares) for various intervals of time in the presence (filled) and absence (empty) of 10 mM EDTA. Residual activity was examined in 50 mM sodium phosphate buffer (pH 6.0) at 55 °C.
protein was quite stable at 65 °C with no significant loss in activity even after overnight incubation at this temperature. However, at 70 °C the activity decreased gradually with the passage of time. Addition of EDTA has been reported to increase the activity [12] and thermal stability of a maltogenic amylase from Thermus sp. [21]. We therefore, examined the effect of EDTA on the enzyme activity and found no significant change in activity. Furthermore, we examined the stability of CDaseGt in the presence of 10 mM EDTA at 70 °C and found that the thermostability increased remarkably. However, overnight incubation at 4 °C is a prerequisite for enhancement of thermostability. If we directly incubate the protein at 70 °C, after addition of EDTA, no increase in thermostability was observed. There was no significant effect of EDTA on the thermostabilty at 75 °C or higher temperatures. Enhancement of thermostability, in the presence of EDTA, may be attributed to possible changes in the structure of CDaseGt. Such structural changes, in the presence of EDTA, have been reported for an endoglucanase from Aspergillus aculeatus [32]. Addition of Ca2+ has also been reported to increase the hydrolytic activity of cyclomaltodextrinase from Bacillus sp. I-5 [12] and Massilia timonae [17]. Similarly, a maltogenic amylase from Geobacillus sp. SK70, which was more than 97% identical to CDaseGt in primary structure (Table 1), was stimulated in the presence of Mn2+, Zn2+ and Tween-20 [33]. Furthermore, the enzyme activity of a maltogenic amylase from Bacillus sp. WPD616, which is more than 99% identical to CDaseGt, was completely inactivated in the presence of
Table 3 Relative hydrolysis rates of various substrates. Substrate
Relative hydrolysis rate (%)a
α-cyclodextrin β-cyclodextrin γ-cyclodextrin Pullulan Dextrin Wheat starch Rice starch Potato starch Corn starch Glycogen Acarbose Amylose Amylopectin
100 61 30 09 04 03 02 01 01 01 02 04 <1
a Determined by measuring the total reducing sugars, liberated from hydrolysis of these substrates at 55 °C.
concentrations of KCl, up to 200 mM, had no significant effect on the activity, however, at 0.5 M KCl, the cyclodextrinase activity was nearly abolished. As the optimum reaction temperature of CDaseGt was 55 °C, we therefore examined the thermostabilty at higher temperatures. The 4
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Fig. 4. High-performance liquid chromatography peaks showing the hydrolysis products α-cyclodextrin by CDaseGt. Reaction was carried out at 55 °C and pH 6.0. Aliquots were taken at various intervals of time and, after filtration, 20 μL were analyzed by an Aminex HPX-42A column. G1, G2, G3 and G4 correspond to glucose, maltose, maltotriose and maltotetraose, respectively.
Fig. 5. Zymogram analysis of purified CDaseGt. Purified CDaseGt was fractionated by gel filtration column (Superdex 200 10/30 GL) and two peak fractions were analysed by nondenaturing PAGE. One half part of the gel (left panel) was stained with Coomassie brilliant blue and the other half (right panel) was analysed by activity staining. Lane 1, first peak eluted at 10.8 mL (dimer); lane 2, second peak eluted at 13.4 mL (monomer). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Table 4 Time course analysis of end products of maltotriose. Time (min)
0 15 30 60 120 1200
Product released (%) G7
G6
G5
G4
G3
G2
G1
Total
– – – – – –
– – – – – –
– – – – 1.9 1.4
– 0.7 1.7 2.9 4.3 10.6
100 84 80.6 71 60 8.7
– 10 11.7 14.8 20.8 45.9
– 3.9 4.6 6.9 10.4 31.1
100 100 100 100 100 100
: Not detected Table 5 Kinetic parameters of CDaseGt against cyclodextrins.
α-cyclodextrin β-cyclodextrin γ-cyclodextrin a
Vmax (μmol min−1 mg−1)
Km (mM)
kcat (s−1)a
kcat/Km
1200 735 360
0.8 0.6 0.4
2739 1678 821
3424 2797 2053
kcat values were calculated based on the dimeric nature of CDaseGt.
1 mM Zn2+, and activity was reduced to 63% and 52% in the presence of 1 and 10 mM Mn2+, respectively [25]. In contrast to these studies, presence or absence of Ca2+, Zn2+ or Tween-20 exhibited no significant effect on the enzyme activity of CDaseGt. However, the enzyme activity of CDaseGt was nearly two-fold in the presence of 5 mM Mn2+. Acarbose, a psuedotetrasacharide, is a well-known inhibitor of αamylases. Among the enzymes in the subfamily, however, maltogenic amylases from Bacillus stearothermophilus [10], Thermus sp. [21]. and Geobacillus thermoleovorans [26], and cyclomaltodextrinase from Bacillus sp. I-5 [12] were able to hydrolyze acarbose to glucose [21]. When we examined the effect of acarbose on the cyclodextrinase activity of CDaseGt, we found that more than 90% activity was abolished in the presence of equimolar acarbose. CDaseGt was also capable
Fig. 6. Thin layer chromatography demonstrating hydrolysis of acarbose by CDaseGt. Acarbose (1%) was incubated with CDaseGt (5 μg) at 55 °C and pH 6.0 for 16 h and hydrolysis products were analyzed on TLC. Lane 1: standards; lane 2, acarbose (control); lane 3, hydrolysis products of acarbose. Symbols are: G1, glucose; G2, maltose; G3, maltotriose; AC, acarbose; and PT, pseudotrisaccharide.
5
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Table 6 Comparison of biochemical properties and kinetic parameters of closely related CDases. Organism
Geobacillus thermopakistaniensis Bacillus sp. WPD616 Thermus sp. Geobacillus sp. SK70 Geobacillus sp. Gh6 Geobacillus thermoleovorans B.stearothermophilusIMA6503. Geobacillus caldoxylosilyticus Bacillus thermoalkalophilus Bacillus stearothermophilus Bacillus sp. I-5
Homology (%)
100 99.8 99 98 96 96 86 75 72 69.4 56.4
Temp(°C)
55 50 60 55 60 80 55 50 70 55 50
pH
6 6.0 6 6 6 5-9 6.0 7 8 6.0 7.0
Activity (U)
kcat (s-1)
Km (mM)
kcat/Km
Reference
α
β
γ
α
β
γ
α
β
γ
α
β
γ
1200 * * * * 1151.2 * * * * *
735 * 278 ** * 548.9 ** ** 161 ** 1629
360 * * * * * * * * * *
0.8 * * * 3.9 2.01 * * * * 1.23
0.6 * 0.263 * 2.3 1.87 * * 0.129 * 0.83
0.4 * * * 1.9 * * * * * 0.92
2009 * * * 500 2.78 * * * * *
1242 * 167 * 280.7 1.32 * * 166 * *
605 * * * 225.6 ** * * * * *
2575 * * * 128.2 1.38 * * * * *
2484 * 636 * 121 0.70 * * 1383 * *
1375 * * * 118 * * * * * *
This Study [24] [21,30] [33] [34] [26] [36] [38] [39] [10] [12,47]
* : data not available
of hydrolysis of acarbose to glucose and pseudotrisaccharide (Fig. 6). However, the hydrolysis rate of acarbose was nearly fifty-times lower than that of α-cyclodexrin. The cyclodextrin hydrolysing enzymes from Bacillus sp. WPD616 [25], Thermus sp. IM6501 [21], G. thermoleovorans [26], Geobacillus sp. SK70 [33], Geobacillus Gh6 [34] and G. thermopakistaniensis exhibit more than 95% identity, however, some of their characteristics vary greatly (Table 6). As the monomeric form of CDaseGt is quite less active, therefore this variation in kinetic parameters can be attributed to the probable difference in equilibrium between dimeric and monomeric forms. In conclusion, we have characterized a CDase from G. thermopakistaniensis which exhibits the highest ever reported hydrolytic activity against α-cyclodextrin. Recombinant CDaseGt exists in monomeric as well as dimeric form in solution. Dimeric form is more active and stable than the monomeric form. Presence of EDTA enhances the thermostabilty of CDaseGt. Studies on site-directed mutagenesis, shifting the monomeric-dimeric equilibrium towards the dimeric form, and the industrial applications of this enzyme are in progress.
chain reaction (PCR). For amplification of the gene, a set of forward ( 5′-CATATGAGGAAAGAAGCCATCCACCACCGCTCAACCG-3′) and reverse (5′-TTACCAGCTTTCGACCGCGTAAAG-3′) primers was synthesized commercially (Gene Link, Hawthorne, NY). An NdeI restriction site was introduced in the forward primer (underlined sequence). The PCR-amplified product was cloned in pTG19-T, and the resulting plasmid was named pTG-CDaseGt. E. coli DH5-α cells were transformed using pTG-CDaseGt. For cloning in the expression vector, recombinant plasmid pTG-CDaseGt was digested with NdeI and HindIII to liberate the CDaseGt gene, which was purified and subsequently cloned in pET-21a (+) expression vector by utilizing the same restriction sites. The resulting plasmid was named pET-CDaseGt. 4.4. Production in E. coli and purification of CDaseGt All procedures were performed at room temperature unless stated otherwise. E. coli BL21 CodonPlus (DE3)-RIL cells containing pETCDaseGt were grown in LB medium, containing ampicillin, at 37 °C until the optical density at 600 nm reached at 0.4. Gene expression was induced with 0.2 mM IPTG. After 4.5 h of induction, cells were harvested by centrifugation at 12,000×g for 10 min at 4 °C. The cell pellet was resuspended in 50 mM Tris-Cl (pH 8.0) and disrupted by sonication using the Bandelin SonoPlus HD 2070 sonication system (Bandelin Electronic, GmbH). Cell debris was removed by centrifugation at 20,000×g for 10 min at 4 °C. The supernatant, obtained after centrifugation, was heated at different temperatures (from 50 °C to 70 °C) to get an optimized temperature at which recombinant CDaseGt remains in soluble fraction. Denatured heat-labile proteins from the host cells were separated by centrifugation at 20,000×g for 20 min at 4 °C. The supernatant, after centrifugation, was applied to HiTrap QFF column using ÄKTA purifier system (GE Healthcare, NJ). Elution of the proteins, bound to the column, was done with a 0–1 M sodium chloride (in 50 mM Tris-Cl, pH 8.0) linear gradient. Fractions, after HiTrap QFF column, containing significant amount of CDaseGt were pooled and equilibrated to 1 M (final concentration) ammonium sulphate. Protein sample was then applied to HiTrap Phenyl HP column equilibrated with 1 M ammonium sulphate. Elution of the proteins, bound to the column, was done with a 1 to 0 M ammonium sulphate linear gradient with 50 mM Tris-Cl, pH 8.0.
4. Experimental 4.1. Reagents and chemicals The reagents and chemicals used in this study were purchased either from Sigma-Aldrich (St. Louis, MO) or Fisher Scientific (Leicestershire). The restriction endonucleases, T4 DNA ligase, DNA and protein size markers, Taq DNA polymerase, RNase, and deoxynucleoside triphosphates (dNTPs) were from Thermo Scientific (ThermoFisher Scientific, MD). Cyclodextrins, maltooligosaccharides, pullulan, starch, amylopectin and amylose were purchased from Sigma-Aldrich. 4.2. Strains, plasmids, and media Escherichia coli DH5-α cells and plasmid pTG19-T (Vivantis Technologies) were used for cloning and general DNA manipulations. E. coli BL21 CodonPlus (DE3)-RIL cells (Stratagene, La Jolla, CA) and pET21a(+) expression vector (Novagen, Madison, WI) were used for gene expression. E. coli strains were routinely grown in Luria-Bertani (LB) medium at 37 °C. Recombinant E. coli cells containing pET-21a(+) were selected on LB agar containing ampicillin (100 μg mL−1), whereas 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (40 μg mL−1) and isopropyl-β-D-galactopyranoside (1 mM) were added when blue/white screening of recombinant E. coli cells containing pTG19-T was required.
4.5. Enzyme activity assay The enzyme activity of recombinant CDaseGt was measured in terms of the amount of reducing sugars liberated upon incubation with the substrate. Maltose was used as the standard for reducing sugars. In a standard assay mixture, 125 μL of 1% (w/v) substrate in 50 mM sodium phosphate buffer (pH 6.0) was mixed with 125 μL of solution containing 0.3 μg CDaseGt and incubated at 55 °C for 2–20 min. The reaction was
4.3. Cloning of CDaseGt gene CDaseGt gene (accession # ESU72342) was amplified from the genomic DNA of G. thermopakistaniensis strain MAS1 by polymerase 6
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stopped by quenching in ice water, and the released reducing ends were determined by the dinitrosalicylic acid (DNS) method [35].
4.10. Zymogram analysis Zymogram analysis was carried out for the detection of activities of monomeric and dimeric forms separated by gel filtration chromatography. Starch (0.1%) was incorporated into the non-denaturing polyacrylamide gel. Both monomer and dimer were loaded in duplicate. The gel was cut into two halves. First half was stained with coommassie brilliant blue and the second half was incubated in 50 mM phosphate buffer for 30 min at 55 °C followed by staining with KI/I2 (1.75% KI and 0.312%I2) solution.
4.6. Effects of pH and temperature on enzyme activity Effect of pH on the enzyme activity of recombinant CDaseGt was examined at 55 °C using various buffers of 50 mM strength. The buffers used were sodium acetate (pH 4.0 to 6.0), and sodium phosphate (pH 6.0 to 7.5). The pH values were adjusted at room temperature. In order to examine the effect of temperature on the enzymatic activity, assay mixtures were prepared in 50 mM sodium phosphate buffer, pH 6.0. Reaction mixtures were incubated for a fixed interval of time at temperatures from 45 to 75 °C. For thermostability analysis, the purified enzyme was diluted in 50 mM sodium phosphate buffer of pH 6.0. Properly diluted enzyme (0.3 μg/μL) was incubated at 65, 70 and 75 °C for various intervals of time and activity assays were performed at 55 °C and pH 6.0 as described above. Effect of EDTA (0.2–10 mM) was determined on enzyme activity and thermostability of CDaseGt. Activity assays were performed in the presence and absence of EDTA at 55 °C and pH 6.0 as described above.
References [1] K.H. Park, T.J. Kim, T.K. Cheong, J.W. Kim, B.H. Oh, B. Svensson, Structure, specificity and function of cyclomaltodextrinase, a multispecific enzyme of the αamylase family, Biochim. Biophys. Acta 1478 (2000) 165–185 https://doi.org/10. 1016/S0167-4838(00)00041-8. [2] K.H. Park, Function and tertiary-and quaternary-structure of cyclodextrin-hydrolyzing enzymes (CDase), a group of multisubstrate specific enzymes belonging to the α-amylase family, J. Appl. Glycosci. 53 (2006) 35–44 https://doi.org/10.5458/ jag.53.35. [3] R. Hulsmann, F. Lurz, F. Scheffel, E. Schneider, Maltose and maltodextrin transport in the thermoacidophilic gram-positive bacterium Alicyclobacillus acidocaldarius is mediated by a high-affinity transport system that includes a maltose binding protein tolerant to low pH, J. Bacteriol. 182 (2000) 6292–6301 https://doi.org/10.1128/ JB.182.22.6292-6301.2000. [4] P. Turner, A. Labes, O.H. Fridjonsson, G.O. Hreggvidson, P. Schonheit, J.K. Kristjansson, O. Holst, E.N. Karlsson, Two novel cyclodextrin-degrading enzymes isolated from thermophilic bacteria have similar domain structures but differ in oligomeric state and activity profile, J. Biosci. Bioeng. 100 (2005) 380–390 https://doi.org/10.1263/jbb.100.380. [5] S. Kitahata, M. Taniguchi, S.D. Beltran, T. Sugimoto, S. Okada, Purification and some properties of cyclodextrinase from Bacillus coagulans, Agric. Biol. Chem. 47 (1983) 1441–1447 https://doi.org/10.1080/00021369.1983.10865801. [6] Y. Nakagawa, W. Saburi, M. Takada, Y. Hatada, K. Horikoshi, Gene cloning and enzymatic characteristics of a novel gamma-cyclodextrin-specific cyclodextrinase from alkalophilic Bacillus clarkii 7364, Biochim. Biophys. Acta 1784 (2008) 2004–2011 https://doi.org/0.1016/j.bbapap.2008.08.022. [7] J.A. DePinto, L.L. Campbell, Purification and properties of the amylase of Bacillus macerans, Biochemistry 7 (1968) 121–124 https://doi/pdf/10.1021/bi00841a015. [8] T. Oguma, M. Kikuchi, K. Mizusawa, Purification and some properties of cyclodextrin-hydrolyzing enzyme from Bacillus sphaericus, Biochim. Biophys. Acta 1036 (1990) 1–5 https://doi.org/10.1016/0304-4165(90)90205-B. [9] T. Oguma, A. Matsuyama, M. Kikuchi, E. Nakano, Cloning and sequence analysis of the cyclomaltodextrinase gene from Bacillus sphaericus and expression in Escherichia coli cells, Appl. Microbiol. Biotechnol. 39 (1993) 197–203 https://doi.org/10.1007/ BF00228606. [10] H.J. Cha, H.G. Yoon, Y.W. Kim, H.S. Lee, J.W. Kim, K.S. Kwon, B.H. OH, K.H. Park, Molecular and enzymatic characterization of a maltogenic amylase that hydrolyzes and transglycosylates acarbose, Eur. J. Biochem. 253 (1998) 251–262 https://doi. org/10.1046/j.1432-1327.1998.2530251. [11] K. Ohdan, T. Kuriki, H. Takata, S. Okada, Introduction of raw starch-binding domains into Bacillus subtilis α-amylase by fusion with the starch-binding domain of Bacillus cyclomaltodextrin glucanotransferase, Appl. Microbiol. Biotechnol. 53 (2000) 430–434 https://doi.org/10.1128/AEM.66.7.3058-3064.2000. [12] T.J. Kim, J.H. Shin, J.H. Oh, M.J. Kim, S.B. Lee, S. Ryu, K. Kwon, J.W. Kim, E.H. Choi, J.F. Robyt, K.H. Park, Analysis of the gene encoding cyclomaltodextrinase from alkalophilic Bacillus sp. I-5 and characterization of enzymatic properties, Arch. Biochem. Biophys. 353 (1998) 221–227 https://doi.org/10.1006/abbi. 1998.0639. [13] S.M. Podkovyrov, J.G. Zeikus, Structure of the gene encoding cyclomaltodextrinase from Clostridium thermohydrosulfuricum 39E and characterization of the enzyme purified from Escherichia coli, J. Bacteriol. 174 (1992) 5400–5405 https://doi.org/ 10.1128/jb.174.16.5400-5405.1992. [14] H. Bender, Purification and characterization of a cyclodextrin-degrading enzyme from Flavobacterium sp, Appl. Microbiol. Biotechnol. 39 (1993) 714–719 https:// doi.org/10.1007/BF00164455. [15] G. Fiedler, M. Pajatsch, A. Böck, Genetics of a novel starch utilisation pathway present in Klebsiella oxytoca, J. Mol. Biol. 256 (1996) 279–291 https://doi.org/10. 1006/jmbi.1996.0085. [16] M.U. Jang, H.J. Kang, C.K. Jeong, Y. Kang, J.E. Park, T.J. Kim, Functional expression and enzymatic characterization of Lactobacillus plantarum cyclomaltodextrinase catalyzing novel acarbose hydrolysis, J. Microbiol. 56 (2018) 113–118 https:doi.org/10.1007/s12275-018-7551-3. [17] F.C.D. Santos, I.P. Barbosa-Tessmann, Recombinant expression, purification, and characterization of a cyclodextrinase from Massilia timonae, Protein Expr. Purif 154 (2019) 74–84 https:doi.org/10.1016/j.pep.2018.08.013. [18] J. Kaulpiboon, P. Pongsawasdi, Expression of cyclodextrinase gene from Paenibacillus sp. A11 in Escherichia coli and characterization of the purified cyclodextrinase, J. Biochem. Mol. Biol. 37 (2004) 408–415. [19] Y. Hashimoto, T. Yamamoto, S. Fujiwara, M. Takagi, T. Imanaka, Extracellular synthesis, specific recognition, and intracellular degradation of cyclomaltodextrins
4.7. Substrate specificity and analysis of the hydrolysis products The substrate preference and relative hydrolysis rates of cyclodextrins and various polysaccharides, including pullulan, starch, amylose, amylopectin, and dextrin, were determined by incubating each of them at a final concentration of 0.5% (w/v) with recombinant CDaseGt. Substrate solutions were prepared in 50 mM sodium phosphate buffer (pH 6.0), and after the addition of purified enzyme (0.3 μg), incubated at 55 °C for 2–30 min. The hydrolysis rates (μmol reducing sugar min−1 mg−1) were measured every 2 min by the DNS method [35]. In order to identify the saccharides obtained as hydrolysis products, incubations were carried out under similar conditions for various time intervals. The products were then analyzed by high-performance liquid chromatography (HPLC) on an Aminex HPX-42A column (300 × 78 mm; Bio-Rad Laboratories, Inc., CA). Signals were detected with a differential refractive index detector (S3580; Sykam GmbH, Germany). Double-distilled deionized water was used as the mobile phase/solvent. During separation, the column temperature was maintained at 85 °C and that of the detector at 45 °C. In addition to hydrolytic activity, transglycosylation activity of CDaseGt was also evaluated by using glucose, maltose and maltotriose or the mixtures of any two of these. 4.8. Thin layer chromatography Thin layer chromatography (TLC) was used to analyse hydrolytic products of acarbose. After incubating acarbose and CDaseGt, at 55 °C and pH 6.0, for various intervals of time, the hydrolytic products were analyzed by TLC, using 0.2 mm silica gel-coated aluminium sheets (Kieselgel 60 F254; Merck). Aliquots (5 μL) of the reaction mixtures were spotted on TLC plate and chromatographed using n-butanol-diethyl ether-acetic acid-water (9:3:3:1 [v/v/v/v]) as mobile phase. Hydrolytic products were identified by spraying the plate with 5% v/v sulphuric acid in methanol followed by baking the TLC plate at 120 °C for 5 min. 4.9. Gel filtration chromatography Oligomeric nature of CDaseGt was determined by gel filtration chromatography using Superdex 200 10/300 GL column (GE Healthcare, NJ). The column was equilibrated with 50 mM Tris-Cl pH 8 containing 150 mM NaCl. Protein sample (prepared in the same buffer) was loaded and eluted at a flow rate of 0.3 mL/min. Eluted fractions were used for zymogram analysis. 7
Carbohydrate Research 481 (2019) 1–8
I. Aroob, et al.
[20] [21]
[22] [23]
[24]
[25] [26]
[27] [28]
[29]
[30]
[31]
[32] [33]
[34] S. Nasrollahi, L. Golalizadeh, R.H. Sajedi, M. Taghdir, S.M. Asghari, M. Rassa, Substrate preference of a Geobacillus maltogenic amylase: a kinetic and thermodynamic analysis, Int. J. Biol. Macromol. 60 (2013) 1–9 https://doi.org/10.1016/j. ijbiomac.2013.04.063. [35] P. Bernfeld, Amylases, alpha and beta, Methods Enzymol. 1 (1955) 149–158 https://doi.org/10.1016/0076-6879(55)01021-5. [36] K.A. Cheong, T.J. Kim, J.W. Yoon, C.S. Park, T.S. Lee, Y. B Kim, K.H. Park, J.W. Kim, Catalytic activities of intracellular dimeric neopullulanase on cyclodextrin, acarbose and maltose, Biotechnol. Appl. Biochem. 35 (2002) 27–34 https:// www.ncbi.nlm.nih.gov/pubmed/11834127. [37] T. Kuriki, T. Imanaka, Pattern of action of Bacillus stearothermophilus neopullulanase on pullulan, J. Bacteriol. 171 (1989) 369–374 https://doi.org/10.1128/jb.171.1. 369-374.1989. [38] Y. Kolcuoğlu, A. Colak, O. Faiz, A. Belduz, Cloning, expression and characterization of highly thermo-and pH-stable maltogenic amylase from a thermophilic bacterium Geobacillus caldoxylosilyticus TK4, Process Biochem. 45 (2010) 821–828 https://doi. org/10.1016/j.procbio.2010.02.001. [39] K.A. Cheong, K.A. Cheong, S.Y. Tang, T.K. Cheong, H. Cha, J.W. Kim, K.H. Park, Thermostable and Alkalophilic Maltogenic Amylase of Bacillus Thermoalkalophilus ET2 in Monomer-Dimer Equilibrium vol. 23, (2005), pp. 79–87 https://doi.org/10. 1080/10242420500090094. [40] S.B. Mabrouk, E.B. Messaoud, D. Ayadi, S. Jemli, A. Roy, M. Mezghani, S. Bejar, Cloning and sequencing of an original gene encoding a maltogenic amylase from Bacillus sp. US149 strain and characterization of the recombinant activity, Mol. Biotechnol. 38 (2008) 211 https://doi.org/10.1007/s12033-007-9017-4. [41] K. Igarashi, K. Ara, K. Saeki, K. Ozaki, S. Kawai, S. Ito, Nucleotide sequence of the gene that encodes a neopullulanase from an alkalophilic Bacillus, Biosci. Biotechnol. Biochem. 56 (1992) 514–516 https://doi.org/10.1271/bbb.56.514. [42] J. H Shim, J.T. Park, J.S. Hong, K. W Kim, M.J. Kim, J.H. Auh, Y.W. Kim, C.S. Park, W. Boos, J.W. Kim, K.W. Park, Role of maltogenic amylase and pullulanase in maltodextrin and glycogen metabolism of Bacillus subtilis 168, J. Bacteriol. 191 (2009) 4835–4844 https://doi.org/10.1128/JB.00176-09. [43] H.Y. Cho, Y.M. Kim, T.J. Kim, H.S. Lee, D.Y. Kim, J.W. Kim, Y.W. Lee, S.B. Leed, K.H. Park, Molecular characterization of a dimeric intracellular maltogenic amylase of Bacillus subtilis SUH4-2, Biochim. Biophys. Acta 1478 (2000) 333–340 https:// doi.org/10.1016/S0167-4838(00)00037-6. [44] N.H.A. Manas, S. Pachelles, N.M. Mahadi, R.M. Illias, The characterisation of an alkali-stable maltogenic amylase from Bacillus lehensis G1 and improved maltooligosaccharide production by hydrolysis suppression, PLoS One 9 (2014) e106481 https://doi.org/10.1371/journal.pone.0106481. [45] F. Li, X. Zhu, Y. Li, H. Cao, Y. Zhang, Functional characterization of a special thermophilic multifunctional amylase OPMA-N and its N-terminal domain, Acta Biochim. Biophys. 43 (2011) 324–334 https://doi.org/10.1093/abbs/gmr013. [46] J. Matzke, A. Herrmann, E. Schneider, E.P. Bakker, Gene cloning, nucleotide sequence and biochemical properties of a cytoplasmic cyclomaltodextrinase (neopullulanase) from Alicyclobacillus acidocaldarius, reclassification of a group of enzymes, FEMS Microbiol. Lett. 183 (2000) 55–61 https://doi.org/10.1111/j.15746968.2000.tb08933.x. [47] M. J Kim, W.S. Park, H.S. Lee, T. J Kim, J.H. Shin, S.H. Yoo, T.K. Cheong, S. Ryu, J.C. Kim, J.W. Kim, T.W. Moon, J.F. Robyt, K.H. Park, Kinetics and inhibition of cyclomaltodextrinase from alkalophilic Bacillus sp. I-5, Arch. Biochem. Biophys. 373 (2000) 110–115 https://doi.org/10.1006/abbi.1999.1471.
by the hyperthermophilic archaeon Thermococcus sp. strain B1001, J. Bacteriol. 183 (2001) 5050–5057 https: doi.org/10.1128/JB.183.17.5050-5057.2001. Y. Sun, X. Lv, Z. Li, J. Wang, B. Jia, J. Liu, Recombinant cyclodextrinase from Thermococcus kodakarensis KOD1: expression, purification, and enzymatic characterization, Archaea 26 (2015) 1–8 https://doi.org/10.1155/2015/397924. T.J. Kim, M.J. Kim, B.C. Kim, J.C. Kim, T.K. Cheong, J.W. Kim, K.H. Park, Modes of action of acarbose hydrolysis and transglycosylation catalyzed by a thermostable maltogenic amylase, the gene for which was cloned from a Thermus strain, Appl. Environ. Microbiol. 65 (1999) 1644–1651. M.A. Siddiqui, N. Rashid, S. Ayyampalayam, W.B. Whitman, Draft genome sequence of Geobacillus thermopakistaniensis strain MAS1, Genome Announc. 2 (2014) e00559-14 https:doi.org/10.1128/genomeA.00559-14. N. Ahmad, N. Rashid, M.S. Haider, M. Akram, M. Akhtar, Novel maltotriose-hydrolyzing thermoacidophilic type III pullulan hydrolase from Thermococcus kodakarensis, Appl. Environ. Microbiol. 80 (2014) 1108–1115 https:doi.org/10.1128/ AEM.03139-13. M.J.E.C. van der Maarel, B. van der Veen, J.C.M. Uitdehaag, H. Leemhuis, L. Dijkhuizen, Properties and applications of starch-converting enzymes of the αamylase family, J. Biotechnol. 94 (2002) 137–155 https://doi.org/10.1016/S01681656(01)00407-2. B. Liu, Y. Wang, X. Zhang, Characterization of a recombinant maltogenic amylase from deep sea thermophilic Bacillus sp. WPD616, Enzym. Microb. Technol. 39 (2006) 805–810 https://doi.org/10.1016/j.enzmictec.2006.01.003. D. Mehta, T. Satyanarayana, Dimerization mediates thermo-adaptation, substrate affinity and transglycosylation in a highly thermostable maltogenic amylase of Geobacillus thermoleovorans, PLoS One 8 (2013) e73612 https://doi.org/10.1371/ journal.pone.0073612. Š. Janeček, How many conserved sequence regions are there in the α-amylase family? Biologia 57 (2002) 29–41 https://http://biologia.savba.sk/suppl_11/janecek. pdf. J.S. Kim, S.S. Cha, H.J. Kim, T.J. Kim, N.C. Ha, S.T. Oh, H.S. Cho, M.J. Cho, M.J. Kim, H.S. Lee, J.W. Kim, K.Y. Choi, K.H. Park, B.H. Oh, Crystal structure of a maltogenic amylase provides insights into a catalytic versatility, J. Biol. Chem. 274 (1999) 26279–26286 https://doi.org/10.1074/jbc.274.37.26279. H.S. Lee, M.S. Kim, H.S. Cho, J.I. Kim, T.J. Kim, J.H. Choi, C. Park, H.S. Lee, B.H. Oh, K.H. Park, Cyclomaltodextrinase, neopullulanase, and maltogenic amylase are nearly indistinguishable from each other, J. Biol. Chem. 277 (2002) 21891–21897 https://doi.org/10.1074/jbc.M201623200. T.J. Kim, V.D. Nguyen, H.S. Lee, M.J. Kim, H.Y. Cho, Y.W. Kim, T.W. Moon, C.S. Park, J.W. Kim, B.H. Oh, S.B. Lee, B. Svensson, K.H. Park, Modulation of the multisubstrate specificity of Thermus maltogenic amylase by truncation of the Nterminal domain and by a salt-induced shift of the monomer/dimer equilibrium, Biochemistry 40 (2001) 14182–14190 https://doi.org/10.1021/bi015531u. J.E. Lee, I.H. Kim, J.H. Jung, D.H. Seo, S.G. Kang, J.F. Holden, J. Cha, C.S. Park, Molecular cloning and enzymatic characterization of cyclomaltodextrinase from hyperthermophilic archaeon Thermococcus sp. CL1, J. Microbiol. Biotechnol. 23 (2013) 1060–1069 https://doi.org/10.4014/jmb.1302.02073. G.S. Naika, P.K. Tikudx, Influence of ethylenediaminetetraacetic Acid (EDTA) on the structural stability of endoglucanase from Aspergillus aculeatus, J. Agric. Food Chem. 59 (2011) 7341–7345 https://doi.org/10.1021/jf103889m. M.R. Sulong, T. Leow, R. Rahman, M. Basri, A.B. Salleh, Enhancing thermostability of maltogenic amylase from Geobacillus sp. SK70 by single amino acid substitution, Int. J. New Technol. Sci. Eng. 2 (2015) 20–41.
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