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Cyclodextrin glycosyltransferase variants experience different modes of product inhibition
Accepted Manuscript Title: Cyclodextrin glycosyltransferase variants experience different modes of product inhibition Author: Caiming Li Qi Xu Zhengbiao Gu Shuangdi Chen Jing Wu Yan Hong Li Cheng Zhaofeng Li PII: DOI: Reference:
S1381-1177(16)30160-6 http://dx.doi.org/doi:10.1016/j.molcatb.2016.08.016 MOLCAB 3422
To appear in:
Journal of Molecular Catalysis B: Enzymatic
Received date: Revised date: Accepted date:
28-4-2016 23-8-2016 23-8-2016
Please cite this article as: Caiming Li, Qi Xu, Zhengbiao Gu, Shuangdi Chen, Jing Wu, Yan Hong, Li Cheng, Zhaofeng Li, Cyclodextrin glycosyltransferase variants experience different modes of product inhibition, Journal of Molecular Catalysis B: Enzymatic http://dx.doi.org/10.1016/j.molcatb.2016.08.016 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Cyclodextrin glycosyltransferase variants experience different modes of product inhibition Caiming Li a,b,c, Qi Xu b, Zhengbiao Gu a,b,c, Shuangdi Chen b, Jing Wu a,d, Yan Hong a,b,c, Li Cheng a,b,c, Zhaofeng Li a,b,c* a
State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi,
Jiangsu 214122, People’s Republic of China b
School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu 214122,
People’s Republic of China c
Collaborative Innovation Center for Food Safety and Quality Control, Jiangnan
University, Wuxi, Jiangsu 214122, People’s Republic of China d
School of Biotechnology and Key Laboratory of Industrial Biotechnology Ministry of
Education, Jiangnan University, Wuxi, Jiangsu 214122, People’s Republic of China
*
Corresponding author at: School of Food Science and Technology, Jiangnan University,
Wuxi, Jiangsu 214122, People’s Republic of China. Tel./fax: +86 510 85329237. E-mail address: [email protected] (Z. Li).
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GRAPHICAL ABSTRACT
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Highlights:
he-, -, and -cyclization reactions of the α-CGTase from Paenibacillus macerans JFB05-01 experience identical modes of product inhibition, as do those of the β-CGTase from Bacillus circulans STB01.
Each cyclodextrin is the most potent inhibitor of its own production.
Product inhibition of the -CGTase from P. macerans JFB05-01 is competitive.
Product inhibition of the -CGTase from B. circulans STB01 is mixed-type.
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ABSTRACT Cyclodextrin glycosyltransferase (CGTase) can be used for the industrial production of cyclodextrins. However, product inhibition by cyclodextrins largely restrains the cyclization activities of CGTase and severely limits the application of cyclodextrins. In this paper, the kinetic mechanisms of the three kinds of cyclization reaction were studied, and the product inhibition modes of two CGTases from different sources were compared. The results confirm that the synthesis of each cyclodextrin is substantially inhibited by the corresponding cyclodextrin. Meanwhile, product inhibition studies indicate competitive inhibition for α-CGTase and a mixed pattern for β-CGTase. This demonstrates that the inhibition type is not decided by the kinds of cyclodextrins or the varieties of cyclization reactions, but by the structure of the CGTase.
Keywords: Cyclodextrin glycosyltransferase; Product inhibition; Kinetic study; Cyclodextrin; Cyclization reaction
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1. Introduction
Cyclodextrins, which contain unique hydrophobic cavities, can form inclusion complexes with small hydrophobic molecules [1-3], leading to their wide utility in the food, pharmaceutical, cosmetics and many other industries. At present, cyclodextrins are commonly produced by cyclodextrin glycosyltransferase (CGTase, EC 2.4.1.19), which can catalyze an intramolecular transglycosylation reaction and convert starch or starch derivatives into a mixture of three types of cyclodextrins (α-, β- and γ- cyclodextrins consisting of 6, 7, or 8 glucose units, respectively) [4-6]. Therefore, CGTase has great importance for the production of cyclodextrins [7]. However, product inhibition, which lowers the yield of cyclodextrins, is an obstacle to the large-scale utilization of cyclodextrins [8]. Product inhibition is a phenomenon in which the cyclization activity of CGTase is severely inhibited by the enzyme reaction products, particularly the cyclodextrins. It has been reported that cyclodextrins cause product inhibition by interfering with catalysis in the active sites and that maltose binding site function and -cyclization activity decline 80% in the presence of 10 mg/mL -cyclodextrin [9]. Various attempts have been made to solve this product inhibition problem in recent years, including enzyme immobilization and ultrafiltration [10-12]. These efforts have increased the cyclodextrin yield but have simultaneously increased the cost of industrial cyclodextrin production. Among the attempted methods, three effective strategies have been the most popular: adding organic solvents to the reaction system, changing the 5
CGTase protein structure (site-directed mutation) and removing the product as it is formed. Although many organic solvents can simultaneously increase the cyclodextrin specificity and the overall cyclodextrin yield [13, 14], the cost of separation techniques and the toxicity of the solvents (toluene, acetone, etc.) limit the large-scale application of cyclodextrins, especially in the food industry [15]. To avoid these drawbacks, it is important to look for more environmentally friendly and less costly solutions, such as protein structure modification by genetic engineering. Hence, the rational modification of CGTase to decrease the level of product inhibition is vital [16]. Before the ideal CGTase can be designed, the mechanism or mechanisms by which cyclodextrins inhibit the cyclization activity of CGTase must be understood. Although the mechanisms by which oligosaccharides (glucose, maltodextrin, maltotriose, etc.) inhibit the activity of CGTases have been studied [17], product inhibition of CGTase has not be adequately settled because little attention has been paid to cyclodextrins, which are major parts of the enzyme reaction, and useful information cannot be obtained solely by studying oligosaccharides. Crucial reports regarding the inhibition by acarbose have shown that acarbose is a much more powerful inhibitor than cyclodextrins [18, 19]. The mutation A230V decreases this inhibition 6700-fold, but at a significant cost to catalytic efficiency and stability [20], illustrating that mutations that overcome small molecule (including cyclodextrins) inhibition might also sacrifice catalytic capability and enzyme stability. Reports focusing on product inhibition by cyclodextrins have also had some limitations. Product inhibition of a single cyclization 6
reaction (mainly focusing on -cyclization activity) of CGTase has been studied [18]. However, scant attention has been paid to studying all three kinds of cyclization reactions (α-, - and γ-cyclizations) together and comparing them to understand their inhibition mechanisms simultaneously. Another important thing is that only CGTases from a single source, rather than from different sources, have been explored when investigating enzyme kinetics and cyclization characteristics. In this paper, product inhibition was thoroughly investigated to provide guidance for the design of CGTases that overcome or weaken the product inhibition caused by cyclodextrins. Furthermore, the kinetic parameters and inhibition patterns of two different CGTases from different sources were compared, revealing the influence of cyclodextrins on variant CGTases and the effect of cyclodextrins on different cyclization reactions.
2. Materials and methods
2.1. Production and purification of CGTases
Production of the two wild type CGTases used in this study, the α-CGTase from Paenibacillus macerans JFB05-01, which predominantly produces α-CD, and the β-CGTase from Bacillus circulans STB01, which predominantly produces β-CD, was performed as previously reported [21, 22]. Purification of these two CGTases was conducted according to the methods of Z. Li et al. [21] and C. Li et al. [22]. Protein 7
concentrations were determined using the Bradford method (Bio-Rad Laboratories, Hercules, CA) with bovine serum albumin as the standard.
2.2. Enzyme assays
All assays were performed by incubating 0.1 mL of appropriately diluted purified CGTase with 0.9 mL of 1% (w/v) maltodextrin (DE 5; Roquette Frères, Lestrem, France) in 10 mM phosphate buffer (pH 6.5) at 50 °C for 10 min. The amounts of α-, β-, and γ-cyclodextrin formed during these assays were determined using the methyl orange [23], phenolphthalein [24], and bromocresol green [25] methods, respectively. A comparison of these well-established colorimetric assays with HPLC assays, conducted in our laboratory, has shown the colorimetric assays to have sufficient sensitivity and reproducibility for use in enzyme kinetics (data not shown). Therefore, we have used these methods in our previous work in this area [26–28]. One unit of each activity was defined as the amount of enzyme that produced 1 μmol of the corresponding cyclodextrin per min. Each value represents the mean of three independent measurements and the deviation from the mean was <5%.
2.3. Optimization of substrate concentration and enzyme dilution ratio
Given the different optimal substrate concentrations and distinct initial velocities of 8
α-, β- and γ-cyclization activity [29], the impacts of substrate inhibition and enzyme activities were eliminated by using different concentrations of enzyme and different substrate concentrations ranges during kinetic studies. For α-CGTase (α-cyclization activity, 40 units/mL), the initial rate of α-cyclodextrin production was greater than those of the β-cyclization and γ-cyclization activities, so a higher enzyme dilution (1:200) was used to determine the kinetics of α-cyclodextrin production than was used to determine β-cyclization (1:50) and γ-cyclization (1:25) activities. For similar reasons, the dilution ratios used to determine the kinetics of the α-, β- and γ-cyclization activities of β-CGTase (β-cyclization activity, 30 units/mL) were 1:25, 1:100, 1:25, respectively. By this means, not only could we make the two enzymes have nearly equal activity in each cyclization reaction, we could also ensure the assays were conducted in the linear range. After diluting the enzyme to the appropriate concentrations, substrate solutions ranging from 1 mg/mL to 40 mg/mL in 10 mM phosphate buffer (pH 6.5) were prepared.
2.4. Determination of product inhibition modes
To determine the modes of product inhibition for the two CGTases, α-, β- or γ-cyclodextrin (Sigma-Aldrich Co. LLC.) was mixed with a series of substrate solutions at different concentrations (from 0.5 mg/mL to 10 mg/mL for -cyclization, and 0.5 9
mg/mL to 20 mg/mL for - and -cyclization ), then the corresponding cyclization activities were measured. Fitting of kinetic parameters was achieved using Origin 9.0 software (OriginLab Corporation, USA). The classification of the inhibition modes was determined from the kinetic parameters.
2.5. Analysis of kinetic parameters
Inhibition constants were determined by using the following equations. For competitive inhibition, Ki values were determined using Eq. (1): v=
Vmax [S] [I] Km(1 + ) + [S] Ki
(1)
For the linear mixed inhibition, Ki and Ki’ values were determined using Eq. (2): v
Vmax[S] [I] [I] Km(1 ) [S](1 ) Ki Ki'
(2)
In Eq. (1) and Eq. (2), v is the reaction rate, Vmax is the maximum reaction rate, Km is the Michaelis constant, Ki is the competitive inhibition constant, Ki’ is the uncompetitive inhibition constant, [S] and [I] are the concentrations of substrate and inhibitor. The Km and kcat values were precisely determined by applying 10 substrate concentrations ranging from 0.75 mg/mL to 10 mg/mL. The Ki values were calculated as described in section 2.4. All experiments were performed at least in triplicate.
2.6. Sequence alignments 10
The amino acid sequences of the CGTases were retrieved from the NCBI (http://www.ncbi.nlm.nih.gov) database. Multiple sequence alignment was carried out using the CLC Sequence Viewer (version 6.8).
3. Results
3.1. Substrate inhibition by maltodextrin (DE 5)
The influence of the maltodextrin (substrate) concentration on the rates of cyclodextrin production was studied so that a maltodextrin concentration free from substrate inhibition could be used in the kinetic studies using the two CGTases. The results showed that the α-CGTase and β-CGTase had similar optimal substrate concentrations when investigating the same type of cyclization reaction (data not shown). Interestingly, the initial α-cyclodextrin production velocity obviously decreased when the substrate concentration was above 10 mg/mL, whereas the -cyclodextrin and γ-cyclodextrin production velocities were only slightly reduced when the substrate concentration exceeded 20 mg/mL. The critical substrate concentrations to avoid substrate inhibition were 10 mg/mL for the α-cyclization reaction and 20 mg/mL for both the - and γ-cyclization reactions. The kinetic data from both of the CGTases could be well fitted with Michaelis-Menten kinetics when using maltodextrin (DE 5) as the substrate. The kinetic 11
parameters kcat and Km are presented in Table 1. The catalytic efficiencies (kcat/Km) of the three production reactions were quite different. The β-CGTase from B. circulans STB01 showed strong -cyclization activity (kcat/Km = 295.6 mL·mg-1·s-1), medium α-cyclization activity and weak γ-cyclization activity. In contrast, the α-CGTase from P. macerans JFB05-01 showed stronger α-cyclization activity (kcat/Km = 265.7 mL·mg-1·s-1) than -cyclization activity, and weak γ-cyclization activity.
3.2. Inhibition by cyclodextrins on α-cyclization reaction
To study the influence of α-, - and γ-cyclodextrins on the α-cyclization reaction, α-cyclization activity was measured using substrate solutions containing different concentrations of substrate (maltodextrin) and varying concentrations of α-, - or γ-cyclodextrin. The resulting data (Fig. 1) show that the α-cyclization activities of the two CGTases decrease drastically in the presence of certain amount of cyclodextrins. The feature that distinguishes one CGTase from another is the inhibition mode. The cyclodextrins inhibited α-CGTase via competitive inhibition (Fig. 1A, C and E), while they inhibited -CGTase via mixed-type inhibition, which is a combination of competitive inhibition and uncompetitive inhibition (Fig. 1B, D and F).
3.3. Inhibition of β- and γ-cyclization activities by cyclodextrins
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A comprehensive understanding of product inhibition in the production of cyclodextrins requires an investigation of the modes of product inhibition for the - and γ-cyclization reactions, which are not necessarily identical to those of the α-cyclization reaction. Therefore, cyclodextrin inhibition of the - and γ-cyclization activities of the two CGTases were studied by adding different concentrations of cyclodextrins to the substrate solutions, as done above for the α-cyclization reaction. The resulting data (Fig. 2 and Fig. 3) revealed that cyclodextrins inhibit the - and γ-cyclization reactions of α-CGTase via competitive inhibition, while they inhibit the and γ-cyclization reactions of -CGTase via mixed-type inhibition. These results demonstrated that inhibition of the - and γ-cyclization reactions of the two enzymes are identical to the inhibition modes of the α-cyclization reaction. The conclusion drawn from these results is that the CGTases themselves (enzyme structure) determines the mode of inhibition, not the kinds of cyclodextrins or the variety of the cyclization reactions. Initial analysis of the resulting data in Fig. 2 and Fig. 3 also revealed that the inhibition of the different cyclization reactions by the three kinds of cyclodextrin (α, , and γ) were different. The - and γ-cyclization activities of both enzymes were strongly suppressed by -cyclodextrin and γ-cyclodextrin, whereas α-cyclodextrin was a weaker inhibitor. The inhibition of γ-cyclization by α-cyclodextrin was so weak that it was even weaker than the substrate inhibition noted for maltodextrin. The α-cyclization activities of both enzymes were more potently inhibited by α-cyclodextrin than by - or 13
γ-cyclodextrin. To more accurately understand the relative inhibition of the different cyclization activities by the different cyclodextrins, Ki values were determined.
3.4. Kinetic inhibition parameters
The inhibition constants for α-, - and γ-cyclodextrin in each of the cyclization reactions of both the two CGTases are shown in Table 2. The Ki values for α-cyclodextrin in the α-cyclization reactions (Kiα) were the smallest among the Ki values in the α-cyclization reactions of α-CGTase and -CGTase. Thus, α-cyclodextrin was a stronger inhibitor of the α-cyclization reaction than the other two cyclodextrins. For the -cyclization reaction of the two enzymes, the Ki values showed that -cyclodextrin was the most potent inhibitor of -cyclization activity. Similarly, γ-cyclodextrin was the most potent inhibitor of the γ-cyclization reaction. Therefore, the data show that each cyclodextrin had the biggest impact on its corresponding cyclization reaction.
4. Discussion
There has been some dispute concerning which cyclodextrin is the strongest inhibitor of cyclization activities. Kim et al. [30] and K. C. P. Lee et al. [31] once reported that the enzymatic synthesis of each cyclodextrin was immensely inhibited by the corresponding cyclodextrin. Gawande and Patkar [32] also came to the same 14
conclusion when investigating α-cyclodextrin production by the cyclodextrin glycosyltransferase from Klebsiella pneumonia AS 22*. Nonetheless, other researchers drew conflicting conclusions and thought that γ-cyclodextrin was probably the strongest inhibitor among the three cyclodextrins, regardless of the type of cyclization reaction. Gastón et al. [33] found that γ-cyclodextrin (compared with α- and β-cyclodextrins) had the smallest Ki value for -cyclization by the CGTase from B. circulans DF 9R, meaning that γ-cyclodextrin was the strongest inhibitor. Matioli et al. [29] found that γ-cyclodextrin was a much stronger inhibitor than -cyclodextrin when examining the -cyclization activity of the CGTase from B. firmus strain 37. The results of the present study are consistent with those of Lee et al., Kim et al., and Gawande and Patkar, and inconsistent with those of Gastón et al. and Matioli et al. Considering these results together, we suggest that CGTases from different strains have different affinities for γ-cyclodextrin. Enzymes from a few strains, such as those studied by Gastón et al. and Matioli et al., exhibit an extraordinary affinity for γ-cyclodextrin that makes it the most potent inhibitor of all cyclization reactions. Enzymes from other strains, such as the ones studied here, have a more balanced affinity for γ-cyclodextrin that makes γ-cyclodextrin a less potent inhibitor of α- and β-cyclization. More detailed study of sequence differences among these enzymes may provide the basis for future site-directed mutagenesis studies designed to minimize product inhibition. Different modes of product inhibition by cyclodextrins have been observed in studies employing different CGTase substrates. Inhibition of the -cyclization reaction 15
of the CGTase from B. circulans strain 251 by -cyclodextrin when using partially hydrolyzed potato starch (DP 50) as the substrate was reported to be mixed-type [34]. Inhibition of the -cyclization reaction of the CGTase from B. circulans DF 9R by α-, βand γ-cyclodextrins when using maltodextrin as the substrate was reported to be competitive [33]. Leemhuis et al. [18] also found that inhibition of the CGTase from Thermoanaerobacterium thermosulfurigenes was competitive when investigating the disproportion reaction. Our previous kinetic analysis of this CGTase found that it has equal catalytic efficiency when using either soluble starch or maltodextrin (DE 5) as the substrate (data not shown). The use of maltodextrin reduces the operational difficulty of the experiment because solutions of maltodextrin (DE 5) are much easier to prepare than solutions of soluble starch. Thus, we used maltodextrin (DE 5) as the substrate in the kinetic constant measurements in this study. Inhibition of the β-cyclization reaction of the CGTase from B. circulans STB01 was shown to be mixed-type, and inhibition of the CGTase from P. macerans JFB05-01 was shown to be competitive. Taken together, these results suggest that CGTases can be grouped into those that exhibit competitive product inhibition and those that exhibit mixed-type product inhibition. They further suggest that each enzyme will display the same mode of product inhibition for each of its cyclization activities, as we saw with the CGTases from P. macerans JFB05-01 and B. circulans STB01. Competitive product inhibition, such as that seen for all the cyclization activities of the α-CGTase from P. macerans JFB05-01, suggests that inhibition results from the 16
product binding to a single binding site most likely the active site. Mixed-type product inhibition, such as that seen for all the cyclization activities of the β-CGTase from B. circulans STB01, suggests that this inhibition results from product binding to two different sites with equal affinity; one that precludes substrate binding (most likely the active site), and one that does not. Previous researchers have also suggested that two different binding sites, including the active site, are involved in product inhibition of CGTases showing mixed-type inhibition. For example, Penninga et al. [34] reported that maltose binding site 2 (MBS2) plays an important role in the cyclization reaction and that β-cyclodextrin bound in MBS2 might block the groove leading starch to the active site. In support of this, the mutation Y633A drastically reduced product inhibition. Gastón et al. found that the CGTase from B. circulans DF 9R, which displays competitive product inhibition, also has a Tyr residue at the site equivalent to residue 633. These results suggest that the presence of a tyrosine at this position reduces the affinity of the MBS2 site for the cyclodextrin products, and may be a signature residue for enzymes displaying competitive product inhibition. However, an alignment of the MBS2 sites from the nine sequences for which the mode of product inhibition has been established (Fig. 4) shows that many CGTases have a tyrosine residue at position 633, including B. circulans STB01, which exhibits mixed-type product inhibition. This observation suggests two possibilities: (1) the mixed-type product inhibition of B. circulans STB01 does not involve the MBS2 region or (2) binding of cyclization products to the MBS2 region is more complex than previously thought. We think the 17
second possibility is more likely to be correct. In summary, current evidence suggests that the active site and the MBS2 region are likely closely related to product inhibition. Our analysis suggests a way to develop CGTases that exhibit reduced levels of product inhibition that is based upon protein structure. For CGTases displaying mixed inhibition, if the structure of the MBS2 region is made similar to those of CGTases displaying competitive inhibition, the affinity of the MBS2 site for cyclodextrin may be decreased, weakening product inhibition to some degree. For CGTases exhibiting competitive product inhibition, mutations at or near the active site would be required to relieve product inhibition. In this case, compensatory mutations may be needed to minimize the impairment of enzyme activity and protein stability [20, 35]. Further investigations, particularly those involving analysis of the three-dimensional structures of CGTases and site-directed mutagenesis, need to be done in the future.
5. Conclusions We have comprehensively studied the kinetics of product inhibition for three kinds of cyclization reactions of two CGTases from different sources. Our results have demonstrated that the production of each type of cyclodextrin was most strongly inhibited by the corresponding cyclodextrin, because each cyclodextrin displayed the lowest Ki value for the cyclization reaction that produces it. Moreover, we found that cyclodextrins exhibit competitive inhibition with the CGTase from P. macerans 18
JFB05-01 and mixed-type inhibition with the CGTase from B. circulans STB01.
Acknowledgements This work received financial support from the Natural Science Foundation of Jiangsu Province (BK20150146), the Fok Ying-Tong Education Foundation, China (No. 131069), the China Postdoctoral Science Foundation Funded Project (2015M570406), the National Natural Science Foundation of China (No. 31101228), and the Jiangsu province “Collaborative Innovation Center for Food Safety and Quality Control” industry development program.
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FIGURE CAPTIONS
Fig. 1. Diagrams of the α-cyclization activity of α-CGTase (A, C, E) and β-CGTase (B, D, F) with α-, β- and γ-cyclodextrin as inhibitors. Cyclodextrins concentrations were: α-cyclodextrin (A, B, (■) 0 mg/mL, (●) 1 mg/mL, (▲) 2 mg/mL); β-cyclodextrin (C, D, (■) 0 mg/mL, (●) 1 mg/mL, (▲) 2 mg/mL); γ-cyclodextrin (E, F, (■) 0 mg/mL, (●) 3 mg/mL, (▲) 6 mg/mL). A series of maltodextrin (DE 5) solutions with different concentrations under 10 mg/mL were used as substrate and incubated at 50 oC for 10 min in 10 mM phosphate buffer, pH 6.5. Each value represents the mean of three independent measurements and the deviation from the mean is <5%.
Fig. 2. Diagrams of the β-cyclization activity of α-CGTase (A, C, E) and β-CGTase (B, D, F) with α-, β- and γ-cyclodextrin as inhibitors. Cyclodextrins concentrations were: α-cyclodextrin (A, B, (■) 0 mg/mL, (●) 10 mg/mL, (▲) 20 mg/mL); β-cyclodextrin (C, D, (■) 0 mg/mL, (●) 1 mg/mL, (▲) 2 mg/mL) and γ-cyclodextrin (E, F, (■) 0 mg/mL, (●) 3 mg/mL, (▲) 6 mg/mL). A series of maltodextrin (DE 5) solutions with different concentrations under 20 mg/mL were used as the substrate and incubated at 50 oC for 10 min in 10 mM phosphate buffer, pH 6.5. Each value represents the mean of three independent measurements and the deviation from the mean is <5%.
Fig. 3. Diagrams of the γ-cyclization activity of α-CGTase (A, C, E) and β-CGTase (B, 23
D, F) with α-, β- and γ-cyclodextrin as inhibitors. Cyclodextrins concentrations were: α-cyclodextrin (A, B, (■) 0 mg/mL, (●) 20 mg/mL, (▲) 40 mg/mL); β-cyclodextrin (C, D, (■) 0 mg/mL, (●) 2 mg/mL, (▲) 4 mg/mL) and γ-cyclodextrin (E, F, (■) 0 mg/mL, (●) 0.25 mg/mL, (▲) 0.5 mg/mL). A series of maltodextrin (DE 5) solutions with different concentrations under 20 mg/mL were used as substrate and incubated at 50 oC for 10 min in 10 mM phosphate buffer, pH 6.5. Each value represents the mean of three independent measurements and the deviation from the mean is <5%.
Fig. 4. Sequence alignment of the MBS2 regions of the nine CGTases for which product inhibition data are available. The sequence alignment was performed using the CLC Sequence Viewer (version 6.8).
24
TABLES Table 1 The kinetic parameters of cyclization reactions of the two CGTases at 50 oC. α-CGTase
β-CGTase
Reaction
kcat (s-1)
Km (mg/mL)
kcat/Km
kcat (s-1)
Km (mg/mL)
kcat/Km
α-cyclization
302.8 ± 11.3
1.1 ± 0.03
265.7
150.4 ± 5.2
1.7 ± 0.06
87.6
β-cyclization
205.3 ± 8.2
8.5 ± 0.3
24.2
768.0 ± 20.6
2.6 ± 0.1
295.6
γ-cyclization
24.5 ± 2.3
8.3 ± 0.4
3.0
64.7 ± 5.8
2.9 ± 0.1
22.2
25
Table 2 The inhibition constants of cyclodextrins in three different cyclization reactions at 50 o
C. Reaction
inhibition constant
α-CGTase
β-CGTase
α-cyclization
Kiαa
0.13 ± 0.01
0.17 ± 0.01
Kiβa
0.75 ± 0.03
0.85 ± 0.07
Kiγa
0.29 ± 0.02
1.69 ± 0.12
K’iαb
--
14.82 ± 0.32
K’iβb
--
2.31 ± 0.08
K’iγb
--
2.57 ± 0.09
Kiαa
12.97 ± 0.89
12.55 ± 0.71
Kiβa
0.41 ± 0.02
0.34 ± 0.01
Kiγa
0.48 ± 0.03
0.55 ± 0.01
K’iαb
--
25.58 ± 0.41
K’iβb
--
1.74 ± 0.05
K’iγb
--
0.86 ± 0.07
Kiαa
40.61 ± 1.84
14.56 ± 0.37
Kiβa
3.86 ± 0.12
2.85 ± 0.09
Kiγa
0.16 ± 0.01
0.19 ± 0.03
K’iαb
--
8.84 ± 0.43
K’iβb
--
3.94 ± 0.37
K’iγb
--
4.32 ± 0.35
β-cyclization
γ-cyclization
a
Kiα, Kiβ, Kiγ stand for the competitive inhibition constants for inhibition by α-, β- and
γ-cyclodextrin, respectively. b
K’iα, K’iβ, K’iγ stand for the noncompetitive inhibition constants for inhibition by α-, β-
and γ-cyclodextrin, respectively. 26
27
28
29
30
S1381-1177(16)30160-6 http://dx.doi.org/doi:10.1016/j.molcatb.2016.08.016 MOLCAB 3422
To appear in:
Journal of Molecular Catalysis B: Enzymatic
Received date: Revised date: Accepted date:
28-4-2016 23-8-2016 23-8-2016
Please cite this article as: Caiming Li, Qi Xu, Zhengbiao Gu, Shuangdi Chen, Jing Wu, Yan Hong, Li Cheng, Zhaofeng Li, Cyclodextrin glycosyltransferase variants experience different modes of product inhibition, Journal of Molecular Catalysis B: Enzymatic http://dx.doi.org/10.1016/j.molcatb.2016.08.016 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Cyclodextrin glycosyltransferase variants experience different modes of product inhibition Caiming Li a,b,c, Qi Xu b, Zhengbiao Gu a,b,c, Shuangdi Chen b, Jing Wu a,d, Yan Hong a,b,c, Li Cheng a,b,c, Zhaofeng Li a,b,c* a
State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi,
Jiangsu 214122, People’s Republic of China b
School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu 214122,
People’s Republic of China c
Collaborative Innovation Center for Food Safety and Quality Control, Jiangnan
University, Wuxi, Jiangsu 214122, People’s Republic of China d
School of Biotechnology and Key Laboratory of Industrial Biotechnology Ministry of
Education, Jiangnan University, Wuxi, Jiangsu 214122, People’s Republic of China
*
Corresponding author at: School of Food Science and Technology, Jiangnan University,
Wuxi, Jiangsu 214122, People’s Republic of China. Tel./fax: +86 510 85329237. E-mail address: [email protected] (Z. Li).
1
GRAPHICAL ABSTRACT
2
Highlights:
he-, -, and -cyclization reactions of the α-CGTase from Paenibacillus macerans JFB05-01 experience identical modes of product inhibition, as do those of the β-CGTase from Bacillus circulans STB01.
Each cyclodextrin is the most potent inhibitor of its own production.
Product inhibition of the -CGTase from P. macerans JFB05-01 is competitive.
Product inhibition of the -CGTase from B. circulans STB01 is mixed-type.
3
ABSTRACT Cyclodextrin glycosyltransferase (CGTase) can be used for the industrial production of cyclodextrins. However, product inhibition by cyclodextrins largely restrains the cyclization activities of CGTase and severely limits the application of cyclodextrins. In this paper, the kinetic mechanisms of the three kinds of cyclization reaction were studied, and the product inhibition modes of two CGTases from different sources were compared. The results confirm that the synthesis of each cyclodextrin is substantially inhibited by the corresponding cyclodextrin. Meanwhile, product inhibition studies indicate competitive inhibition for α-CGTase and a mixed pattern for β-CGTase. This demonstrates that the inhibition type is not decided by the kinds of cyclodextrins or the varieties of cyclization reactions, but by the structure of the CGTase.
Keywords: Cyclodextrin glycosyltransferase; Product inhibition; Kinetic study; Cyclodextrin; Cyclization reaction
4
1. Introduction
Cyclodextrins, which contain unique hydrophobic cavities, can form inclusion complexes with small hydrophobic molecules [1-3], leading to their wide utility in the food, pharmaceutical, cosmetics and many other industries. At present, cyclodextrins are commonly produced by cyclodextrin glycosyltransferase (CGTase, EC 2.4.1.19), which can catalyze an intramolecular transglycosylation reaction and convert starch or starch derivatives into a mixture of three types of cyclodextrins (α-, β- and γ- cyclodextrins consisting of 6, 7, or 8 glucose units, respectively) [4-6]. Therefore, CGTase has great importance for the production of cyclodextrins [7]. However, product inhibition, which lowers the yield of cyclodextrins, is an obstacle to the large-scale utilization of cyclodextrins [8]. Product inhibition is a phenomenon in which the cyclization activity of CGTase is severely inhibited by the enzyme reaction products, particularly the cyclodextrins. It has been reported that cyclodextrins cause product inhibition by interfering with catalysis in the active sites and that maltose binding site function and -cyclization activity decline 80% in the presence of 10 mg/mL -cyclodextrin [9]. Various attempts have been made to solve this product inhibition problem in recent years, including enzyme immobilization and ultrafiltration [10-12]. These efforts have increased the cyclodextrin yield but have simultaneously increased the cost of industrial cyclodextrin production. Among the attempted methods, three effective strategies have been the most popular: adding organic solvents to the reaction system, changing the 5
CGTase protein structure (site-directed mutation) and removing the product as it is formed. Although many organic solvents can simultaneously increase the cyclodextrin specificity and the overall cyclodextrin yield [13, 14], the cost of separation techniques and the toxicity of the solvents (toluene, acetone, etc.) limit the large-scale application of cyclodextrins, especially in the food industry [15]. To avoid these drawbacks, it is important to look for more environmentally friendly and less costly solutions, such as protein structure modification by genetic engineering. Hence, the rational modification of CGTase to decrease the level of product inhibition is vital [16]. Before the ideal CGTase can be designed, the mechanism or mechanisms by which cyclodextrins inhibit the cyclization activity of CGTase must be understood. Although the mechanisms by which oligosaccharides (glucose, maltodextrin, maltotriose, etc.) inhibit the activity of CGTases have been studied [17], product inhibition of CGTase has not be adequately settled because little attention has been paid to cyclodextrins, which are major parts of the enzyme reaction, and useful information cannot be obtained solely by studying oligosaccharides. Crucial reports regarding the inhibition by acarbose have shown that acarbose is a much more powerful inhibitor than cyclodextrins [18, 19]. The mutation A230V decreases this inhibition 6700-fold, but at a significant cost to catalytic efficiency and stability [20], illustrating that mutations that overcome small molecule (including cyclodextrins) inhibition might also sacrifice catalytic capability and enzyme stability. Reports focusing on product inhibition by cyclodextrins have also had some limitations. Product inhibition of a single cyclization 6
reaction (mainly focusing on -cyclization activity) of CGTase has been studied [18]. However, scant attention has been paid to studying all three kinds of cyclization reactions (α-, - and γ-cyclizations) together and comparing them to understand their inhibition mechanisms simultaneously. Another important thing is that only CGTases from a single source, rather than from different sources, have been explored when investigating enzyme kinetics and cyclization characteristics. In this paper, product inhibition was thoroughly investigated to provide guidance for the design of CGTases that overcome or weaken the product inhibition caused by cyclodextrins. Furthermore, the kinetic parameters and inhibition patterns of two different CGTases from different sources were compared, revealing the influence of cyclodextrins on variant CGTases and the effect of cyclodextrins on different cyclization reactions.
2. Materials and methods
2.1. Production and purification of CGTases
Production of the two wild type CGTases used in this study, the α-CGTase from Paenibacillus macerans JFB05-01, which predominantly produces α-CD, and the β-CGTase from Bacillus circulans STB01, which predominantly produces β-CD, was performed as previously reported [21, 22]. Purification of these two CGTases was conducted according to the methods of Z. Li et al. [21] and C. Li et al. [22]. Protein 7
concentrations were determined using the Bradford method (Bio-Rad Laboratories, Hercules, CA) with bovine serum albumin as the standard.
2.2. Enzyme assays
All assays were performed by incubating 0.1 mL of appropriately diluted purified CGTase with 0.9 mL of 1% (w/v) maltodextrin (DE 5; Roquette Frères, Lestrem, France) in 10 mM phosphate buffer (pH 6.5) at 50 °C for 10 min. The amounts of α-, β-, and γ-cyclodextrin formed during these assays were determined using the methyl orange [23], phenolphthalein [24], and bromocresol green [25] methods, respectively. A comparison of these well-established colorimetric assays with HPLC assays, conducted in our laboratory, has shown the colorimetric assays to have sufficient sensitivity and reproducibility for use in enzyme kinetics (data not shown). Therefore, we have used these methods in our previous work in this area [26–28]. One unit of each activity was defined as the amount of enzyme that produced 1 μmol of the corresponding cyclodextrin per min. Each value represents the mean of three independent measurements and the deviation from the mean was <5%.
2.3. Optimization of substrate concentration and enzyme dilution ratio
Given the different optimal substrate concentrations and distinct initial velocities of 8
α-, β- and γ-cyclization activity [29], the impacts of substrate inhibition and enzyme activities were eliminated by using different concentrations of enzyme and different substrate concentrations ranges during kinetic studies. For α-CGTase (α-cyclization activity, 40 units/mL), the initial rate of α-cyclodextrin production was greater than those of the β-cyclization and γ-cyclization activities, so a higher enzyme dilution (1:200) was used to determine the kinetics of α-cyclodextrin production than was used to determine β-cyclization (1:50) and γ-cyclization (1:25) activities. For similar reasons, the dilution ratios used to determine the kinetics of the α-, β- and γ-cyclization activities of β-CGTase (β-cyclization activity, 30 units/mL) were 1:25, 1:100, 1:25, respectively. By this means, not only could we make the two enzymes have nearly equal activity in each cyclization reaction, we could also ensure the assays were conducted in the linear range. After diluting the enzyme to the appropriate concentrations, substrate solutions ranging from 1 mg/mL to 40 mg/mL in 10 mM phosphate buffer (pH 6.5) were prepared.
2.4. Determination of product inhibition modes
To determine the modes of product inhibition for the two CGTases, α-, β- or γ-cyclodextrin (Sigma-Aldrich Co. LLC.) was mixed with a series of substrate solutions at different concentrations (from 0.5 mg/mL to 10 mg/mL for -cyclization, and 0.5 9
mg/mL to 20 mg/mL for - and -cyclization ), then the corresponding cyclization activities were measured. Fitting of kinetic parameters was achieved using Origin 9.0 software (OriginLab Corporation, USA). The classification of the inhibition modes was determined from the kinetic parameters.
2.5. Analysis of kinetic parameters
Inhibition constants were determined by using the following equations. For competitive inhibition, Ki values were determined using Eq. (1): v=
Vmax [S] [I] Km(1 + ) + [S] Ki
(1)
For the linear mixed inhibition, Ki and Ki’ values were determined using Eq. (2): v
Vmax[S] [I] [I] Km(1 ) [S](1 ) Ki Ki'
(2)
In Eq. (1) and Eq. (2), v is the reaction rate, Vmax is the maximum reaction rate, Km is the Michaelis constant, Ki is the competitive inhibition constant, Ki’ is the uncompetitive inhibition constant, [S] and [I] are the concentrations of substrate and inhibitor. The Km and kcat values were precisely determined by applying 10 substrate concentrations ranging from 0.75 mg/mL to 10 mg/mL. The Ki values were calculated as described in section 2.4. All experiments were performed at least in triplicate.
2.6. Sequence alignments 10
The amino acid sequences of the CGTases were retrieved from the NCBI (http://www.ncbi.nlm.nih.gov) database. Multiple sequence alignment was carried out using the CLC Sequence Viewer (version 6.8).
3. Results
3.1. Substrate inhibition by maltodextrin (DE 5)
The influence of the maltodextrin (substrate) concentration on the rates of cyclodextrin production was studied so that a maltodextrin concentration free from substrate inhibition could be used in the kinetic studies using the two CGTases. The results showed that the α-CGTase and β-CGTase had similar optimal substrate concentrations when investigating the same type of cyclization reaction (data not shown). Interestingly, the initial α-cyclodextrin production velocity obviously decreased when the substrate concentration was above 10 mg/mL, whereas the -cyclodextrin and γ-cyclodextrin production velocities were only slightly reduced when the substrate concentration exceeded 20 mg/mL. The critical substrate concentrations to avoid substrate inhibition were 10 mg/mL for the α-cyclization reaction and 20 mg/mL for both the - and γ-cyclization reactions. The kinetic data from both of the CGTases could be well fitted with Michaelis-Menten kinetics when using maltodextrin (DE 5) as the substrate. The kinetic 11
parameters kcat and Km are presented in Table 1. The catalytic efficiencies (kcat/Km) of the three production reactions were quite different. The β-CGTase from B. circulans STB01 showed strong -cyclization activity (kcat/Km = 295.6 mL·mg-1·s-1), medium α-cyclization activity and weak γ-cyclization activity. In contrast, the α-CGTase from P. macerans JFB05-01 showed stronger α-cyclization activity (kcat/Km = 265.7 mL·mg-1·s-1) than -cyclization activity, and weak γ-cyclization activity.
3.2. Inhibition by cyclodextrins on α-cyclization reaction
To study the influence of α-, - and γ-cyclodextrins on the α-cyclization reaction, α-cyclization activity was measured using substrate solutions containing different concentrations of substrate (maltodextrin) and varying concentrations of α-, - or γ-cyclodextrin. The resulting data (Fig. 1) show that the α-cyclization activities of the two CGTases decrease drastically in the presence of certain amount of cyclodextrins. The feature that distinguishes one CGTase from another is the inhibition mode. The cyclodextrins inhibited α-CGTase via competitive inhibition (Fig. 1A, C and E), while they inhibited -CGTase via mixed-type inhibition, which is a combination of competitive inhibition and uncompetitive inhibition (Fig. 1B, D and F).
3.3. Inhibition of β- and γ-cyclization activities by cyclodextrins
12
A comprehensive understanding of product inhibition in the production of cyclodextrins requires an investigation of the modes of product inhibition for the - and γ-cyclization reactions, which are not necessarily identical to those of the α-cyclization reaction. Therefore, cyclodextrin inhibition of the - and γ-cyclization activities of the two CGTases were studied by adding different concentrations of cyclodextrins to the substrate solutions, as done above for the α-cyclization reaction. The resulting data (Fig. 2 and Fig. 3) revealed that cyclodextrins inhibit the - and γ-cyclization reactions of α-CGTase via competitive inhibition, while they inhibit the and γ-cyclization reactions of -CGTase via mixed-type inhibition. These results demonstrated that inhibition of the - and γ-cyclization reactions of the two enzymes are identical to the inhibition modes of the α-cyclization reaction. The conclusion drawn from these results is that the CGTases themselves (enzyme structure) determines the mode of inhibition, not the kinds of cyclodextrins or the variety of the cyclization reactions. Initial analysis of the resulting data in Fig. 2 and Fig. 3 also revealed that the inhibition of the different cyclization reactions by the three kinds of cyclodextrin (α, , and γ) were different. The - and γ-cyclization activities of both enzymes were strongly suppressed by -cyclodextrin and γ-cyclodextrin, whereas α-cyclodextrin was a weaker inhibitor. The inhibition of γ-cyclization by α-cyclodextrin was so weak that it was even weaker than the substrate inhibition noted for maltodextrin. The α-cyclization activities of both enzymes were more potently inhibited by α-cyclodextrin than by - or 13
γ-cyclodextrin. To more accurately understand the relative inhibition of the different cyclization activities by the different cyclodextrins, Ki values were determined.
3.4. Kinetic inhibition parameters
The inhibition constants for α-, - and γ-cyclodextrin in each of the cyclization reactions of both the two CGTases are shown in Table 2. The Ki values for α-cyclodextrin in the α-cyclization reactions (Kiα) were the smallest among the Ki values in the α-cyclization reactions of α-CGTase and -CGTase. Thus, α-cyclodextrin was a stronger inhibitor of the α-cyclization reaction than the other two cyclodextrins. For the -cyclization reaction of the two enzymes, the Ki values showed that -cyclodextrin was the most potent inhibitor of -cyclization activity. Similarly, γ-cyclodextrin was the most potent inhibitor of the γ-cyclization reaction. Therefore, the data show that each cyclodextrin had the biggest impact on its corresponding cyclization reaction.
4. Discussion
There has been some dispute concerning which cyclodextrin is the strongest inhibitor of cyclization activities. Kim et al. [30] and K. C. P. Lee et al. [31] once reported that the enzymatic synthesis of each cyclodextrin was immensely inhibited by the corresponding cyclodextrin. Gawande and Patkar [32] also came to the same 14
conclusion when investigating α-cyclodextrin production by the cyclodextrin glycosyltransferase from Klebsiella pneumonia AS 22*. Nonetheless, other researchers drew conflicting conclusions and thought that γ-cyclodextrin was probably the strongest inhibitor among the three cyclodextrins, regardless of the type of cyclization reaction. Gastón et al. [33] found that γ-cyclodextrin (compared with α- and β-cyclodextrins) had the smallest Ki value for -cyclization by the CGTase from B. circulans DF 9R, meaning that γ-cyclodextrin was the strongest inhibitor. Matioli et al. [29] found that γ-cyclodextrin was a much stronger inhibitor than -cyclodextrin when examining the -cyclization activity of the CGTase from B. firmus strain 37. The results of the present study are consistent with those of Lee et al., Kim et al., and Gawande and Patkar, and inconsistent with those of Gastón et al. and Matioli et al. Considering these results together, we suggest that CGTases from different strains have different affinities for γ-cyclodextrin. Enzymes from a few strains, such as those studied by Gastón et al. and Matioli et al., exhibit an extraordinary affinity for γ-cyclodextrin that makes it the most potent inhibitor of all cyclization reactions. Enzymes from other strains, such as the ones studied here, have a more balanced affinity for γ-cyclodextrin that makes γ-cyclodextrin a less potent inhibitor of α- and β-cyclization. More detailed study of sequence differences among these enzymes may provide the basis for future site-directed mutagenesis studies designed to minimize product inhibition. Different modes of product inhibition by cyclodextrins have been observed in studies employing different CGTase substrates. Inhibition of the -cyclization reaction 15
of the CGTase from B. circulans strain 251 by -cyclodextrin when using partially hydrolyzed potato starch (DP 50) as the substrate was reported to be mixed-type [34]. Inhibition of the -cyclization reaction of the CGTase from B. circulans DF 9R by α-, βand γ-cyclodextrins when using maltodextrin as the substrate was reported to be competitive [33]. Leemhuis et al. [18] also found that inhibition of the CGTase from Thermoanaerobacterium thermosulfurigenes was competitive when investigating the disproportion reaction. Our previous kinetic analysis of this CGTase found that it has equal catalytic efficiency when using either soluble starch or maltodextrin (DE 5) as the substrate (data not shown). The use of maltodextrin reduces the operational difficulty of the experiment because solutions of maltodextrin (DE 5) are much easier to prepare than solutions of soluble starch. Thus, we used maltodextrin (DE 5) as the substrate in the kinetic constant measurements in this study. Inhibition of the β-cyclization reaction of the CGTase from B. circulans STB01 was shown to be mixed-type, and inhibition of the CGTase from P. macerans JFB05-01 was shown to be competitive. Taken together, these results suggest that CGTases can be grouped into those that exhibit competitive product inhibition and those that exhibit mixed-type product inhibition. They further suggest that each enzyme will display the same mode of product inhibition for each of its cyclization activities, as we saw with the CGTases from P. macerans JFB05-01 and B. circulans STB01. Competitive product inhibition, such as that seen for all the cyclization activities of the α-CGTase from P. macerans JFB05-01, suggests that inhibition results from the 16
product binding to a single binding site most likely the active site. Mixed-type product inhibition, such as that seen for all the cyclization activities of the β-CGTase from B. circulans STB01, suggests that this inhibition results from product binding to two different sites with equal affinity; one that precludes substrate binding (most likely the active site), and one that does not. Previous researchers have also suggested that two different binding sites, including the active site, are involved in product inhibition of CGTases showing mixed-type inhibition. For example, Penninga et al. [34] reported that maltose binding site 2 (MBS2) plays an important role in the cyclization reaction and that β-cyclodextrin bound in MBS2 might block the groove leading starch to the active site. In support of this, the mutation Y633A drastically reduced product inhibition. Gastón et al. found that the CGTase from B. circulans DF 9R, which displays competitive product inhibition, also has a Tyr residue at the site equivalent to residue 633. These results suggest that the presence of a tyrosine at this position reduces the affinity of the MBS2 site for the cyclodextrin products, and may be a signature residue for enzymes displaying competitive product inhibition. However, an alignment of the MBS2 sites from the nine sequences for which the mode of product inhibition has been established (Fig. 4) shows that many CGTases have a tyrosine residue at position 633, including B. circulans STB01, which exhibits mixed-type product inhibition. This observation suggests two possibilities: (1) the mixed-type product inhibition of B. circulans STB01 does not involve the MBS2 region or (2) binding of cyclization products to the MBS2 region is more complex than previously thought. We think the 17
second possibility is more likely to be correct. In summary, current evidence suggests that the active site and the MBS2 region are likely closely related to product inhibition. Our analysis suggests a way to develop CGTases that exhibit reduced levels of product inhibition that is based upon protein structure. For CGTases displaying mixed inhibition, if the structure of the MBS2 region is made similar to those of CGTases displaying competitive inhibition, the affinity of the MBS2 site for cyclodextrin may be decreased, weakening product inhibition to some degree. For CGTases exhibiting competitive product inhibition, mutations at or near the active site would be required to relieve product inhibition. In this case, compensatory mutations may be needed to minimize the impairment of enzyme activity and protein stability [20, 35]. Further investigations, particularly those involving analysis of the three-dimensional structures of CGTases and site-directed mutagenesis, need to be done in the future.
5. Conclusions We have comprehensively studied the kinetics of product inhibition for three kinds of cyclization reactions of two CGTases from different sources. Our results have demonstrated that the production of each type of cyclodextrin was most strongly inhibited by the corresponding cyclodextrin, because each cyclodextrin displayed the lowest Ki value for the cyclization reaction that produces it. Moreover, we found that cyclodextrins exhibit competitive inhibition with the CGTase from P. macerans 18
JFB05-01 and mixed-type inhibition with the CGTase from B. circulans STB01.
Acknowledgements This work received financial support from the Natural Science Foundation of Jiangsu Province (BK20150146), the Fok Ying-Tong Education Foundation, China (No. 131069), the China Postdoctoral Science Foundation Funded Project (2015M570406), the National Natural Science Foundation of China (No. 31101228), and the Jiangsu province “Collaborative Innovation Center for Food Safety and Quality Control” industry development program.
19
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FIGURE CAPTIONS
Fig. 1. Diagrams of the α-cyclization activity of α-CGTase (A, C, E) and β-CGTase (B, D, F) with α-, β- and γ-cyclodextrin as inhibitors. Cyclodextrins concentrations were: α-cyclodextrin (A, B, (■) 0 mg/mL, (●) 1 mg/mL, (▲) 2 mg/mL); β-cyclodextrin (C, D, (■) 0 mg/mL, (●) 1 mg/mL, (▲) 2 mg/mL); γ-cyclodextrin (E, F, (■) 0 mg/mL, (●) 3 mg/mL, (▲) 6 mg/mL). A series of maltodextrin (DE 5) solutions with different concentrations under 10 mg/mL were used as substrate and incubated at 50 oC for 10 min in 10 mM phosphate buffer, pH 6.5. Each value represents the mean of three independent measurements and the deviation from the mean is <5%.
Fig. 2. Diagrams of the β-cyclization activity of α-CGTase (A, C, E) and β-CGTase (B, D, F) with α-, β- and γ-cyclodextrin as inhibitors. Cyclodextrins concentrations were: α-cyclodextrin (A, B, (■) 0 mg/mL, (●) 10 mg/mL, (▲) 20 mg/mL); β-cyclodextrin (C, D, (■) 0 mg/mL, (●) 1 mg/mL, (▲) 2 mg/mL) and γ-cyclodextrin (E, F, (■) 0 mg/mL, (●) 3 mg/mL, (▲) 6 mg/mL). A series of maltodextrin (DE 5) solutions with different concentrations under 20 mg/mL were used as the substrate and incubated at 50 oC for 10 min in 10 mM phosphate buffer, pH 6.5. Each value represents the mean of three independent measurements and the deviation from the mean is <5%.
Fig. 3. Diagrams of the γ-cyclization activity of α-CGTase (A, C, E) and β-CGTase (B, 23
D, F) with α-, β- and γ-cyclodextrin as inhibitors. Cyclodextrins concentrations were: α-cyclodextrin (A, B, (■) 0 mg/mL, (●) 20 mg/mL, (▲) 40 mg/mL); β-cyclodextrin (C, D, (■) 0 mg/mL, (●) 2 mg/mL, (▲) 4 mg/mL) and γ-cyclodextrin (E, F, (■) 0 mg/mL, (●) 0.25 mg/mL, (▲) 0.5 mg/mL). A series of maltodextrin (DE 5) solutions with different concentrations under 20 mg/mL were used as substrate and incubated at 50 oC for 10 min in 10 mM phosphate buffer, pH 6.5. Each value represents the mean of three independent measurements and the deviation from the mean is <5%.
Fig. 4. Sequence alignment of the MBS2 regions of the nine CGTases for which product inhibition data are available. The sequence alignment was performed using the CLC Sequence Viewer (version 6.8).
24
TABLES Table 1 The kinetic parameters of cyclization reactions of the two CGTases at 50 oC. α-CGTase
β-CGTase
Reaction
kcat (s-1)
Km (mg/mL)
kcat/Km
kcat (s-1)
Km (mg/mL)
kcat/Km
α-cyclization
302.8 ± 11.3
1.1 ± 0.03
265.7
150.4 ± 5.2
1.7 ± 0.06
87.6
β-cyclization
205.3 ± 8.2
8.5 ± 0.3
24.2
768.0 ± 20.6
2.6 ± 0.1
295.6
γ-cyclization
24.5 ± 2.3
8.3 ± 0.4
3.0
64.7 ± 5.8
2.9 ± 0.1
22.2
25
Table 2 The inhibition constants of cyclodextrins in three different cyclization reactions at 50 o
C. Reaction
inhibition constant
α-CGTase
β-CGTase
α-cyclization
Kiαa
0.13 ± 0.01
0.17 ± 0.01
Kiβa
0.75 ± 0.03
0.85 ± 0.07
Kiγa
0.29 ± 0.02
1.69 ± 0.12
K’iαb
--
14.82 ± 0.32
K’iβb
--
2.31 ± 0.08
K’iγb
--
2.57 ± 0.09
Kiαa
12.97 ± 0.89
12.55 ± 0.71
Kiβa
0.41 ± 0.02
0.34 ± 0.01
Kiγa
0.48 ± 0.03
0.55 ± 0.01
K’iαb
--
25.58 ± 0.41
K’iβb
--
1.74 ± 0.05
K’iγb
--
0.86 ± 0.07
Kiαa
40.61 ± 1.84
14.56 ± 0.37
Kiβa
3.86 ± 0.12
2.85 ± 0.09
Kiγa
0.16 ± 0.01
0.19 ± 0.03
K’iαb
--
8.84 ± 0.43
K’iβb
--
3.94 ± 0.37
K’iγb
--
4.32 ± 0.35
β-cyclization
γ-cyclization
a
Kiα, Kiβ, Kiγ stand for the competitive inhibition constants for inhibition by α-, β- and
γ-cyclodextrin, respectively. b
K’iα, K’iβ, K’iγ stand for the noncompetitive inhibition constants for inhibition by α-, β-
and γ-cyclodextrin, respectively. 26
27
28
29
30