Experimental evidence for a 9-binding subsite of Bacillus licheniformis
thermostable
a
-amylase
Phuong Lan Tran
a, Jin-Sil Lee
a, Kwan-Hwa Park
a,b,⇑ aDepartment of Foodservice Management and Nutrition, Sangmyung University, Seoul 110-743, Republic of Korea
b
Department of Food Science and Biotechnology, Seoul National University, Seoul 151-742, Republic of Korea
a r t i c l e
i n f o
Article history:
Received 12 November 2013 Revised 17 December 2013 Accepted 27 December 2013 Available online 17 January 2014 Edited by Judit Ovádi
Keywords:
Bacillus licheniformis thermostablea -amylase
Kinetic parameter Subsite affinity
Linear maltooligosaccharides High-performance anion exchange chromatography
a b s t r a c t
The action pattern of Bacillus licheniformis thermostablea-amylase (BLA) was analyzed using a ser-ies of14C-labeled and non-labeled maltooligosaccharides from maltose (G2) to maltododecaose
(G12). Maltononaose (G9) was the preferred substrate, and yielded the smallest Km= 0.36 mM, the
highest kcat= 12.86 s1, and a kcat/Kmvalue of 35.72 s1mM1, producing maltotriose (G3) and
malto-hexaose (G6) as the major product pair. Maltooctaose (G8) was hydrolyzed into two pairs of prod-ucts: G3 and maltopentaose (G5), and G2 and G6 with cleavage frequencies of 0.45 and 0.30, respectively. Therefore, we propose a model with nine subsites: six in the terminal non-reducing end-binding site and three at the reducing end-binding site in the binding region of BLA. Ó 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
1. Introduction
a
-Amylase (1,4-a
-D-glucan glucanohydrolase, E.C. 3.2.1.1) isfound naturally in plants, animals, and microorganisms, and cata-lyzes the hydrolysis of
a
-1,4-glycosidic linkages in the interior of starch into fine dextrins and smaller-molecular-weight products[1,2]. The
a
-amylase from Bacillus licheniformis, a mesophilic bacte-rium predominantly found in temperate soil, is a highly thermosta-ble starch-degrading enzyme [3]. It has significant potential applications in the alcohol, sugar, and brewing industries, and more recently in the pharmaceutical industry [4–7]. In addition, the determination ofa
-amylase activity in human serum and urine has been widely used in clinical laboratories for the diagnosis of pancreatic disorders[8]. For a direct and automation-ready proce-dure for measuringa
-amylase activity in a clinical laboratory, ethylidene-pNP-G7 is used as a substrate in which G7 is the basic sugar unit fora
-amylase action. Since amylase is found in humans and other mammals, in addition to being an enzyme present in plant seeds, bacteria, and fungi, it is desirable to develop a substrate suitable for measuring the activity of a wide variety ofa
-amylases from various sources[9–11].a
-Amylase is known asa multi-attack enzyme that cleaves the
a
-1,4 linkage of glucan ran-domly and in multiple ways. Determination of the rate of polymer substrate hydrolysis (such as starch) may confuse the results of thea
-amylase reaction, since macro-substrate molecules are degraded by successive attacks by the enzyme[12]. Moreover, the rate of hydrolysis depends on the chain length of the substrate. Moreover, modifications of the substrate G7 at both the reducing and non-reducing ends may result in different affinities of the enzyme to the substrate. Therefore, an understanding of the action pattern and reaction mechanism ofa
-amylase would allow for a more complete description of starch hydrolysis. Human salivary amylase and porcine pancreatic amylase have six and five subsites, respec-tively[13,14], whereas other amylases, includinga
-amylase and maltogenic amylase, vary from seven to ten subsites. Kandra et al. [15,16] found G6 and higher maltooligosaccharides to be suitable hydrolytic substrates for Bacillus licheniformisa
-amylase, suggesting eight subsites: five glycone (5, 4, 3, 2, 1) and three aglycone (+1, +2, +3) binding sites [15]. However, many investigations ofa
-amylase have used substrates with a limited range [i.e., a degree of polymerization (DP) 3–7] with and without chromophore substitute ions[12,17,18], which were found to have too narrow a range of chain lengths to cover all possible subsites of 8–10.Therefore, this study investigated the action pattern and subsite affinity of B. licheniformis ATCC 27811 thermostable
0014-5793/$36.00 Ó 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.febslet.2013.12.032
⇑Corresponding author at: Department of Foodservice Management and Nutri-tion, Sangmyung University, Seoul 110-743, Republic of Korea. Fax: +82 2 781 7528.
E-mail address:parkkwanhwa@gmail.com(K.-H. Park).
a
-amylase (BLA)[19]on linear maltooligosaccharides using a wide range of DP 2–12 substrates with non- and 14C-labeledmaltooligosaccharides. 2. Materials and methods
2.1. Preparation of BLA
Escherichia coli strain MC1061 was used as a host for BLA expression. Recombinant E. coli harboring the BLA gene with six histidines at the N-terminus was cultured in Luria–Bertani (LB) medium containing ampicillin (100
l
g/ml) overnight at 37 °C with shaking. The enzyme BLA was purified using nickel (Ni2+)nitrilotri-acetic acid (NTA) resin (QIAGEN, Hilden, Germany) packed in a Poly-PrepÒchromatography column (Bio-Rad, Hercules, CA, USA),
as described by Park et al.[20]. The protein concentration was determined by the Bradford method[21], and the purity of the protein was confirmed by employing a 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) analysis according to Laemmli[22].
2.2. Substrate preparation
Maltooligosaccharides from G2 to G6 were purchased from Sigma Chemical (St. Louis, MO, USA), and G7 and G8 were kindly provided by Dr. Byung Cheol Min (Daesang Corporation, Korea). Maltononaose (G9) and maltoundecaose (G11), or maltodecaose (G10), and maltododecaose (G12), were produced by Thermotoga maritima maltosyltransferase (TmMT) transferring maltosyl units or disproportionating G7 and G8, respectively, to form a set of maltosyl transfer products[23]. The target substrates were isolated using 3MM (Whatman, Maidstone, Kent, UK) paper chromatogra-phy using acetonitrile/n-propanol alcohol/ethyl acetate/water (8.5:5:2:7, v/v/v/v) as the solvent. The purity of substrates was confirmed by thin-layer chromatography (TLC) analysis.
Maltooligosaccharides labeled with 10 mCi/mmol 14
C-D-glu-cose at their reducing end (G3⁄
to G10⁄
) were synthesized using
14C-D-glucose and b-CD and the Toruzyme coupling reaction
(Novozyme, Bagsvaerd, Denmark) at 80 °C for 30 min [24]. The reaction products were separated by paper chromatography, as de-scribed above. The labeled maltooligosaccharides were eluted and subjected to a second separation using isopropanol/ethyl acetate/ water (3:1:1, v/v/v) as the solvent, then eluted again in deionized water, concentrated using a SpeedVac, and then resuspended in deionized water to the desired concentration.
2.3. Thin layer chromatography (TLC) analysis
The TLC analysis was carried out using a Whatman K5F silica gel plate with isopropanol/ethyl acetate/water (3:1:1, v/v/v) as the sol-vent to check the purity of substrate products and to confirm the reaction mode of BLA on linear maltooligosaccharides.
14
C-labeled substrates from G3 to G10 were reacted with BLA. The TLC plate containing reaction products was developed twice and then covered with an imaging plate in a cassette for 10 h. The reducing-end radioactivity products were detected by scan-ning with a BAS2500 image analyzer (Fuji Film, Tokyo, Japan).
2.4. Analysis of oligosaccharides by high-performance anion-exchange chromatography (HPAEC)
The enzyme reaction was carried out at 70 °C using each substrate at concentrations from 0.2 to 2 mM for G6 to G12, and from 0.2 to 5 mM for G2 to G5, and samples were taken every minute in a double volume 0.1 N NaOH. The reacted samples were then analyzed using a HPAEC system (Dionex-300, Dionex,
Sunny-vale, CA, USA) with an electrochemical detector (ED40, Dionex), a CarboPac™ PA-1 anion-exchange column (250 4 mm, Dionex), and a guard column. The column was first equilibrated with 150 mM NaOH. The sample was eluted with varied gradients of 0–600 mM sodium acetate in 150 mM NaOH at a flow rate of 1 ml/min[20]. The resultant decrease in substrate concentration was determined as the initial velocity.
2.5. Kinetic analysis of the enzyme reaction and subsite binding affinity The reaction rate of BLA on a series of maltooligosaccharides was analyzed using the Michaelis constant, Km, and the turnover
number, kcat, as parameters for a Lineweaver–Burk plot.
The subsite affinities of BLA were determined using the kinetic parameters and the bond cleavage frequency data according to the methods described by Suganuma et al.[25]:
Aiþ1¼ RT ln ðkcat=KmÞnþ1e o Piþ1= Xn iþ1 Piþ1 ! =ðkcat=KmÞne o Pi= Xn i Pi ! " #
The sum of the subsite affinity (Ar+ Ar+1) for the subsite on either
side of the catalytic site was calculated as the kcat/Kmof the smallest
substrate using kintbased on the following equation:
ðkcat=KmÞn¼ 0:018kint X p exp X cov: i Ai=RT ! n;p
This kinetic method can be applied to either exo- or endo-amylases
[26].
2.6. Molecular modeling
The G9 ligand complex model was initially constructed by manual fitting of G9 ligand into the active site of BLA based on superposition of various lengths of carbohydrate ligands to alpha amylases. Initial complex models were subjected to energy minimization followed by 1 ps of molecular dynamics at 3008 K after equilibration. They were minimized to a maxi-mum derivative of 1.0 kcal per step using the Discover module in the Insight II software (Accelrys; San Diego, CA) with Amber force fields.
3. Results and discussion
3.1. Action and cleavage pattern of oligosaccharides by BLA
The cleavage distribution of BLA was analyzed quantitatively using HPAEC analysis and an image analyzer employing the non-labeled and 14C-labeled maltooligosaccharides from G2 to G12
and G3⁄
to G10⁄
, respectively (Table 1). As shown inFigs. 1 and 2, when maltooligosaccharides from G2 to G6 were reacted with the enzyme, the reaction exclusively produced the pair products G1 and glucann1as the major products. Glucose was released
from the reducing end of the substrates (Fig. 2). The BLA hydro-lyzed the G7 maltooligosaccharides into two product pairs—G2 & G5 and G1 & G6—with cleavage frequencies of 0.60 and 0.30, respectively. Similarly, the products of G8 hydrolysis by BLA were identified as two pairs: G3 & G5 and G2 & G6, with cleavage affin-ities of 0.45 and 0.30, respectively (Fig. 1). These findings indicated that binding sites 5 and 6 were favorable to substrates G8 and
G7 (Table 1,Fig. 1). G9 was converted into G3 and G6 as the main
pair product of hydrolysis, whereby the maltotriosyl moiety was cleaved from the reducing end of the substrates (Figs. 1 and 2). The production of G6 was significant from the substrates G7 to G9 whose arrangements were not as influenced by the filling of
the three aglycone subsites, clearly indicating that the substrate first filled five or six subsites of the non-reducing end.
The cleavage pattern of the enzyme reaction on substrates with higher DPs is in agreement with the results of Kandra et al.[15,16]. The reaction produces G5 as the main product from the
non-reduc-Fig. 1. HPAEC analysis of the BLA reaction with maltotetraose, maltopentaose, maltoheptaose, maltooctaose, and maltononaose.
G1 G2 G3 G4 G5 G6 G7 G8 G9 G10
A
B
G3 G4 G5 G6 G7 G8 G9 G10 G3 G4 G5 G6 G7 G8 G9 G10Fig. 2. TLC analysis of the BLA reaction with14
C-labeled maltooligosaccharides from maltotriose to maltodecaose. The chromatogram was visualized using naphthol-H2SO4(A) and by autoradiography (B).
Table 1
Cleavage distribution of maltooligosaccharides from maltotriose to maltodecaose by BLA.
Substrate So (mM) DP eo(M) 3 G.020G.980G 0.2 1.4 x 10-9 1.000 .000 * * * * * * * * 4 G.047G.010 .943G G 0.2 1.4 x 10-9 .003 .970 .000 5 G.001G.061G.008 .930G G 0.2 1.4 x 10-9 .130 .870 .000 .000 6 G G G.083G.016G.899G 0.2 2.2 x 10-10 .001 .001 .000 .100 .110 .790 .000 7 G.001G.001G.010G.019G0.701G.268G 0.2 2.2 x10-10 0.600 .012 .280 .000 .000 .000 8 G.001G.001G.032G.043G.515G.288G.120G 0.2 2.2 x 10-10 .450 .000 .005 .300 .200 .000 .000 0.2 2.2 x 10-10 9 G.001G.002G.035G.040G.047G.528G.225G.122G .004 .510 .260 .190 .000 .000 .000 .000 0.2 2.2 x 10-10 10 G.001 .001 .008 .030G G G G.070G.127G.401G.247 .115G G .009 .130 .370 .220 .190 .000 .000 .000 .000 (A) (B) (A) (B) (A) (B) (B) (A) (B) (A) (B) (A) (B) (A) (B) (A)
DP, degree of polymerization; so, substrate concentration; eo, enzyme concentration;⁄, reducing end.
(A) Calculation based on the results of HPAEC analysis. (B) Calculation based on the results of TLC and image analysis.
ing end of chloro-4-nitrophenyl-G6, -G7, and -G8, whereas 2-chloro-4-nitrophenyl-G3 was the main product cleaved from the reducing end of 2-chloro-4-nitrophenyl-G5, -G8, -G9, and -G10. Therefore, the cleavage frequency at the first and second subsites of the G4 substrate was similar, and the highest cleavage frequency occurred at the third subsite of G5[16]. However, in this study, cleavage at the first subsite from the reducing end exclusively dominated the reaction. The different pattern found here compared with other studies may have been due to the substrates having their reducing end modified by 2-choro-4-nitrophenyl. Re-search using the amylase from Thermoactinomyces vulgaris (TVAI) suggested that cleavage occurred most frequently at the second subsite (2) for the substrates G4 and G5 in a 15-min reaction
[12]. Conversely, we found in this study that BLA preferentially split the first glycosidic bond from the reducing end of the substrates labeled with14C-D-glucose (Fig. 2), producing cleavage frequencies of 0.97 and 0.87 for G4 and G5, respectively (Table 1). In addition, the time-series reaction analysis of G4 and G5 over a 60-min period showed that G2 was not produced in any significant amount compared with G1 and G3 (data not shown). Nitta et al.
[27]found that a methyl group significantly affected the binding of a substrate analog with Taka-amylase A. Therefore, the discrep-ancy in results might have been caused by the different structures of substrates with modifications at the reducing end. This alter-ation may affect short oligosaccharides more significantly due to difficulties in filling all the subsites of the enzyme.
As shown in the TLC analysis (Fig. 2), the substrates with DP > 8 produced G3 exclusively from the reducing end, indicating the presence of three binding subsites (+1, +2, +3).
3.2. Kinetic parameters of BLA
As shown in the product analysis using HPAEC (Fig. 1) the sub-strate concentration was sufficiently low and reaction products were analyzed at the initial reaction time to minimize other reac-tions such as transglycosylation and condensation. The Kmvalues
decreased with increasing chain length, whereas kcat increased
with a chain length up to DP 9 and became almost constant for
DP > 9 (Table 2) with no significant difference, since kcatis expected
to be constant when DP exceeds the number of subsites [26,28]. The enzyme had the smallest Km= 0.36 mM and highest kcat
result-ing in the highest catalytic efficiency, kcat/Km, for G9. These results
indicated that BLA catalyzes most of the hydrolysis of G9, which fills all nine binding subsites in the catalytic region. Therefore, the number of subsites of BLA is estimated to be nine. On the other hand, the rate of hydrolysis of
a
-amylase from B. licheniformis could not be compared with those in the study in which kinetic data were not available for the subsite mapping of the enzyme[15]. TVA II, a neopullulanase-like amylase from T. vulgaris R-47, and ThMA, a thermostable maltogenic amylase from Thermus spe-cies, have different patterns of kcat/Kmvalues: the highest kcat/Km
was of TVA II for G7 and of ThMA for G3[18,29].
3.3. Subsite affinities of BLA
The subsite affinities of BLA were calculated using the kinetic parameters and bond cleavage frequencies according to the meth-ods described by Suganuma et al. [25]. The sum of (A1+ A+1),
being adjacent to the central catalytic residue, was 3.80 kcal mol1. Subsite A
2 (3.89 kcal mol1) and A+2
(0.32 kcal mol1) of the non-reducing and reducing ends binding
sites, respectively, had a relatively larger positive affinity on both sides of the catalytic site. This enzyme had a dominant binding site at A5(0.48 kcal mol1) at the non-reducing end (Table 3). In
com-parison with other subsites in the catalytic region of BLA (Table 3), subsite A6had moderate affinity (0.2 kcal mol1). The proportion
of G6 produced from both G7 and G8 substrates reached 30%, while that from G9 was 51%, suggesting that A6retains significant
affinity. The subsite structure of
a
-amylase from various sources has been reported as 8–10[15,25,29,30], of which Taka-amylase A was suggested by Suganuma et al. to have nine subsites in the binding site[25]. Comparison with the subsite structures of other types of amylase revealed that they differ in the strength of their affinities to substrate. TVA II has eight subsites (5 to +3) [18], and ThMA has seven subsites (5 to +2)[29]. Based on the above findings of the action pattern and kinetic parameters, we propose a subsite structure model of BLA for nine subsites of the six non-reducing end binding sites (6, 5, 4, 3, 2, 1) and the three reducing end binding sites (+1, +2, +3) with a catalytic site between the sixth and seventh subsites. Furthermore, the relationship be-tween subsite binding affinity of the malto-oligomers with and without modification will provide further insight into the structure and substrate specificity of amylolytic enzymes, as well as provid-ing the possibility of novel substrate development.3.4. Molecular modeling
Molecular modeling with the G9 substrate showed a groove surface that extends from the active site to the putative subsite of 6 with a platform architecture; it becomes wide open beyond subsite +3 at the reducing end (Fig. 3).
Table 2
Kinetic data of BLA on maltose to maltododecaose.
Substrate Km(mM) kcat(s1) kcat/Km(s1mM1)
G2 113.88 ± 0.12 0.04 ± 0.01 0.35 103± 0.00 G3 6.68 ± 0.06 1.68 ± 0.15 0.25 ± 0.02 G4 5.42 ± 0.05 1.91 ± 0.16 0.35 ± 0.03 G5 5.13 ± 0.08 2.01 ± 0.14 0.39 ± 0.03 G6 1.56 ± 0.04 9.50 ± 0.14 6.09 ± 0.18 G7 0.69 ± 0.02 9.84 ± 0.17 14.26 ± 0.48 G8 0.39 ± 0.02 11.23 ± 0.16 28.80 ± 1.18 G9 0.36 ± 0.01 12.86 ± 0.10 35.72 ± 1.03 G10 0.37 ± 0.03 12.81 ± 0.08 34.62 ± 2.82 G11 0.39 ± 0.03 12.11 ± 0.10 31.05 ± 2.40 G12 0.40 ± 0.01 11.92 ± 0.14 29.80 ± 0.82 Table 3
Subsite affinities of BLA. Subsite (i)a
6 5 4 3 2 1 +1 +2 +3
Subsite affinity (Ai) A6 A5 A4 A3 A2 A1 A+1 A+2 A+3
Aivalue (kcal mol1)
(A) 0.20 0.48 0.02 0.18 3.89 3.80 0.32 0.25
(B) 0.13 0.51 0.06 0.18 3.88 3.80 0.36 0.24
(A) Calculation based on the results of HPAEC analysis. (B) Calculation based on the results of TLC and image analysis.
a
Acknowledgments
The authors thank Dr. Eui-Jeon Woo, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Korea, for molecular modeling and Ms. Myung-Ok Kyung, Seoul National University, for preparing 14C-labeled maltooligosaccharides. This study was
supported in part by the Basic Research Program through the Na-tional Research Foundation (2012R1A1A2005012) and in part by the Next Generation BioGreen21 Program (SSAC, No. PJ009086), Rural Development Administration, Republic of Korea.
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The catalytic residues of BLA are colored blue and the G9 substrate is shown as sticks with the corresponding subsite numbers.
