Appl Biochem Biotechnol DOI 10.1007/s12010-015-1675-2
Single Amino Acid Substitution in the Pullulanase of Klebsiella variicola for Enhancing Thermostability and Catalytic Efficiency Guo Cui Mu 1 & Yao Nie 1,3 & Xiao Qing Mu 1,3 & Yan Xu 1,2,3 & Rong Xiao 4
Received: 23 January 2015 / Accepted: 19 May 2015 # Springer Science+Business Media New York 2015
Abstract Based on conserved sites and homology modeling analysis, the residue Phe581 in the Klebsiella variicola SHN-1 pullulanase was selected as the potential thermostabilityrelated site and its role on thermostability and activity was investigated by site-saturated mutagenesis. Compared with the wild-type pullulanase, the optimum temperature of the mutants including F581L, F581Q, F581R, F581T, F581V, and F581Y was increased from 53 to 56 °C, and correspondingly the half lives of these mutants at 55 °C were increased by 4.20, 3.70, 1.90, 7.16, 3.01, and 1.75 min, respectively. By modeling the structure of the pullulanase, formation of more hydrogen bonds by single-site substitution was supposed to be responsible for the improvement of thermostability. Of these mutants, furthermore, F581L and F581V exhibited higher values of Vmax and kcat/Km, compared with the wild-type enzyme. Therefore, the residue Phe581 was identified as an important site relevant to the activity and thermostability of the pullulanase of K. variicola, and by mutation at this single site, the mutated enzymes with enhanced thermostability and catalytic efficiency were achieved consequently. Electronic supplementary material The online version of this article (doi:10.1007/s12010-015-1675-2) contains supplementary material, which is available to authorized users.
* Yao Nie [email protected] * Yan Xu [email protected] 1
Key Laboratory of Industrial Biotechnology, Ministry of Education and School of Biotechnology, Jiangnan University, Wuxi 214122, China
2
State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, China
3
2011 Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University, Wuxi 214122, China
4
Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, NJ 08854, USA
Appl Biochem Biotechnol
Keywords Pullulanase . Site-saturated mutagenesis . Thermostability . Optimum temperature . Catalytic efficiency
Introduction Pullulanase (EC 3. 2. 1. 41) is known as an outstanding debranching enzyme and mostly utilized with other amylolytic enzymes involving α-amylase, β-amylase, and glucoamylase to produce glucose or maltose syrups from starch [1], where two enzymatic hydrolysis processes including liquefaction and saccharification are generally involved. In the saccharification process, glucoamylase mainly hydrolyzes α-1,4-glucosidic links and release glucose molecules from the non-reducing end of the dextrins [2], while pullulanase is usually applied to specifically cleave the α-1,6-glucosidic linkages in amylaceous polysaccharides. Therefore, the combination of glucoamylase and pullulanase has been widely adopted to produce glucose from starch raw materials with high efficiency. However, the saccharification reaction is usually carried out at the temperature ranging from 55 to 60 °C, and there is a requirement for the involved pullulanase to be active and stable around that temperature [3]. Pullulanases have been discovered from various microorganisms, such as Anaerobranca gottschalkii [4], Bacillus acidopullulyticus [5], Bacillus subtilis TU [6], Thermus aquaticus YT-1 [7], Desulfurococcus mucosus [8], and so on. These discovered enzymes have been further identified to perform diverse characteristics. Associating with the working conditions of enzymatic saccharification, only if pullulanase is adaptable in respect of resistance to higher temperature can it act together with glucoamylase to promote the efficiency of the mixed enzymatic saccharification. However, so far the available pullulanases with thermostability are yet limited to be suitable for the saccharification reaction and practical application [3]. To improve the availability of enzymes with application potential, site-directed mutagenesis has been widely adopted to engineer certain enzymes for desired properties based on the knowledge of structure-function relationships [9]. The α-amylase has been mutated with the half-life of 3.8-fold longer to the wild-type enzyme and its melting temperature was correspondingly increased 3.2 °C compared with the wild-type enzyme by structurebased rational design and site-directed mutagenesis [10]. The Aspergillus niger N25 phytase has been reported to undergo the site-directed mutagenesis by substitution of the residues of Ile44 and Thr252 with Glu and Arg, respectively. The obtained mutants exhibited enhanced thermostability and overall catalytic efficiency, compared to the wildtype enzyme [11]. Therefore, the approaches involving active sites analysis and subsequent site-directed mutagenesis should be very useful in development of enzymatic properties including catalytic activity and stability. Nevertheless, there are only few reports about improving thermostability and catalytic efficiency concurrently by single-site substitution. In previous work, the pullulanase from Klebsiella variicola SHN-1 (kvPulA) has been discovered and identified to posses the potential in practical application [12]. However, the thermostability and optimum catalytic temperature of the enzyme need to be further improved to meet the requirement of enzymatic saccharification process. For the pullulanase from Klebsiella aerogene, the residue of Phe562 would be the possible site to have a close relationship with its thermostability [13]. Based on sequence alignment and conserved sites
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analysis, in this investigation, the role of the residue in kvPulA corresponding to Phe562 in K. aerogene pullulanase was investigated by site-saturated mutagenesis, especially concerning its influence on thermostability and catalytic efficiency. Then the positive mutants were achieved with enhanced thermostability and catalytic efficiency, and the effect of involved residue substitutions at this site was further elucidated by homology modeling and molecular interaction analysis.
Materials and Methods Strains, Plasmids, and Materials The recombinant plasmid pET-28a-pul carrying the pullulanase-encoded gene kvpulA (GenBank accession no. JX087429) from K. variicola SHN-1 was constructed as described in the previous work [12]. The strains including E. coli JM109 and BL21were used as the recombinant hosts for the gene cloning and expression, respectively. PrimeSTAR® HS DNA Polymerase for PCR reaction and restriction endonuclease Dpn I was purchased from TAKARA Biotechnology (Dalian, China). Bradford Protein Assay Kit was obtained from TIANGEN Biotech Co., Ltd. (Beijing, China). The DNA primers for mutagenesis were synthesized in Sangon Co., Ltd. (Shanghai, China) and Plasmid Mini Kit was obtained from Omega bio-tek (Norcross, GA, USA). The polysaccharide of pullulan for determination of pullulanase activity was purchased from Tokyo Kasei Kogyo Co., Ltd. (Tokyo, Japan).
Site-Directed Mutagenesis Site-directed mutagenesis was performed using Megaprimer PCR of the whole recombinant plasmid bearing the pullulanase-encoded gene kvpulA [14]. The primers for site-saturated mutagenesis at the residue Phe581 were designed according to the principle of codon usage. The mutations were generated by using PrimeSTAR HS DNA polymerase with the recombinant plasmid pET-28a-pul as the template. The condition for PCR was as follows: 30 cycles at 98 °C for 10 s, 68 °C for 15 s, and 68 °C for 9 min 30 s. The PCR product was digested with DpnI, followed by transformation into E. coli JM109 for cloning. The plasmids containing correct mutated genes confirmed by sequencing were finally transformed into E. coli BL21 (DE3) for expression of the mutants of the pullulanase.
Expression of the Wild-Type Enzyme and the Mutants The colony of the recombinant E. coli BL21 (DE3) carrying kvpulA or mutated gene was grown in 4 ml Luria–Bertani (LB) liquid medium containing 50 μg ml−1 kanamycin at 37 °C and 200 rpm overnight. Then the culture was inoculated to 50 ml LB liquid medium containing 50 μg ml−1 kanamycin in a 250-ml shake flask and incubated at 37 °C and 200 rpm. When the culture turbidity (OD600 nm) reached to the level between 0.6 and 0.8, 0.4 mM IPTG was added to induce target protein expression. The expression of the enzyme and the mutants was performed at 25 °C for 20 h. After centrifugation at 4 °C and 10,000 rpm for 15 min, the resulted culture supernatants were collected for further research.
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Protein and Enzyme Activity Assay SDS-PAGE of the enzyme sample was performed on a 9 % (w/v) running gel. Protein concentration was determined by Bradford method with bovine serum albumin (BSA) as a standard [15]. Pullulanase activity was assayed by measuring the aldehyde groups released during incubation from a reaction mixture consisting of pullulan solution and the diluted enzyme solution. The enzyme solution was diluted with sodium acetate buffer (pH5.0) in advance. Then 100 mM sodium acetate buffer (pH5.0) containing 0.1 ml 1 % (w/v) pullulan was mixed with the diluted enzyme solution. The mixture was incubated at 50 °C for 30 min. Then, the amount of released aldehyde groups was assayed by dinitrosalicylic acid method. One unit of enzyme activity was defined as the amount of enzyme that causes the liberation of 1 μmol of aldehyde groups in 1 min under the reaction conditions [16].
Characterization of the Mutants The kinetic parameters (Km, Vmax, and kcat values) of the pullulanase were determined based on the method described previously [17], where different concentrations of pullulan (1, 2, 5, 10, and 15 g l−1) were adopted. The values of Vmax and Km were estimated by fitting the initial rate data to the Michaelis-Menten equation using nonlinear regression with GraphPad Prism software. To determine the optimum temperature, enzyme activity was measured across the temperature range from 50 to 65 °C. For each enzyme, the highest activity in the temperature range was taken as 100 % and the percentage of relative activity was calculated relative to the highest activity. The optimum pH of the mutants was determined by assaying the enzyme activity in sodium acetate buffer of different pH value ranging from 4.0 to 6.0. For each enzyme, the highest activity in the pH range was taken as 100 % and the percentage of relative activity was calculated relative to the highest activity. The half-lives (T1/2) of the enzymes at 55 °C were taken as an indicator of the thermostability. Enzymatic reaction solution was incubated at 55 °C and pH 5.0, and enzyme activity was measured after incubation at 55 °C for 5, 10, 15, 20, 25, and 30 min, respectively. The values of T1/2 at 55 °C were calculated according to the equations as shown below. lnðU t =U o Þ ¼ −kt T 1=2 ¼ ln2 = k T50 was defined as the temperature at which 50 % of the initial activity remained after incubation for 15 min and was calculated according to the above equations [18]. The residual activity was assessed in the temperature range from 35 to 55 °C with an interval of 5 °C.
Glucose Production by Pullulanase-Involved Mixed Enzymatic Saccharification The enzyme solutions comprising glucoamylase and pullulanase involving kvPulA, F581L, or F581V were added in the reactor vessel (dosage equal to 50 U of glucoamylase and 0.5 U of pullulanase per gram of dry maltodextrin, i.e., the ratio of glucoamylase to pullulanase was
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100:1). Dry maltodextrin (10 g) and sodium acetate buffer (pH 5.0) was added into the reactor vessel till the total weight of the slurry reached 50 g in a 250-ml batch reactor vessel, leading to the final concentration of dry maltodextrin 20 % (w/w). The saccharification procedure was carried out at 55 °C in a water bath, and a high-temperature-resistant magnetic stirrer was used for saccharification to obtain a constant stirring speed. Samples were taken at certain time intervals and then boiled for 5 min to inactivate enzymes for measuring glucose content (DX) by HPLC analysis. The DX value was defined as the percentage of glucose on a dry basis. The amount of glucose was analyzed by HPLC on a Sepax HP-Amino column (4.6×250 mm, 5 μm) with a Hitachi Chromaster 5450 refraction index detector and the mobile phase of acetonitrile/water (75:25, v/v) at a flow rate of 0.8 ml min−1.
Homology Modeling and Molecular Interaction Analysis Based on sequence identity of 98 % between the Klebsiella pneumoniae pullulanase and kvPulA, the protein structure of the K. pneumoniae pullulanase (PDB ID=2fhf) was used as the template for homology modeling to generate the model structures of the wild-type enzyme and the mutants of kvPulA with SWISS-MODEL. Multiple sequence alignment was conducted by EMBL-EBI (http://www.ebi.ac.uk/Tools/msa/). Structure comparison and analysis of the wild-type enzyme and the mutants was performed by the program of PyMOL.
Results and Discussion Thermostability-Related Site Selected for Saturated Mutagenesis The pullulanase from K. aerogene has been investigated for the structure-function relationships by random mutagenesis, and the residue of Phe562 was considered to be the possible site relating to its thermostability [13]. Based on sequence alignment and conserved sites analysis, 98.52 % identity was found between the K. aerogene pullulanase and kvPulA, and the residue Phe562 in K. aerogene pullulanase was identified to correspond to Phe581 in kvPulA. Therefore, the residue of Phe581 was taken into account to investigate its role on thermostability and catalytic activity. To understand the effect of other amino acid substitutions at the position 581 on thermostability and catalytic efficiency of the enzyme, saturated mutagenesis of kvPulA was carried out by replacing the residue of Phe581 with other 19 amino acid residues. The sequencingconfirmed mutant genes were cloned and expressed successfully in recombinant Escherichia coli.
Effects of Single Point Mutations on Optimum Temperature and Thermostability To investigate the effects of site-saturated mutagenesis on optimum temperature of enzymatic catalysis, the enzyme activities of the wild-type enzyme and the mutants were measured at temperature ranging from 50 to 65 °C, respectively. Compared with the wild-type pullulanase kvPulA, the optimum temperature of enzyme activity was increased from 53 to 56 °C for most of the mutants including F581A, F581C, F581E, F581G, F581H, F581L, F581Q, F581R, F581S, F581T, F581V, and F581Y, while the mutants F581K, F581M, and F581N exhibited the optimum temperature lower than that of kvPulA.
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Then the effect of site-saturated mutagenesis at Phe581 on thermostability of the enzyme was further evaluated under the incubation conditions of 55 °C and pH 5.0. After heat treatment for different periods from 5 to 30 min, the enzyme activities of kvPulA and its mutants were measured. As shown in Fig. 1, compared with kvPulA, the half-lives were increased by 0.65, 4.20, 3.70, 1.90, 7.16, 3.01, 5.12, and 1.75 min for the mutants of F581I, F581L, F581Q, F581R, F581T, F581V, F581W, and F581Y, respectively. In addition, the values of T50 of the mutants were measured by assessing the residual activity of the enzymes after incubation at temperature ranging from 35 to 55 °C for 15 min. As shown in Fig. 2, most of the mutants had a positive shift of T50 except for F581A and F581C. Integrating the above results, the mutants F581L, F581Q, F581R, F581T, and F581V were found to be more active and stable against increased temperature. To gain insight into the role of Phe581 in kvPulA, based on the protein structure of the K. pneumoniae pullulanase (PDB ID: 2fhf) which shares the sequence identity of 98 % with kvPulA, the model structures of the wide-type enzyme and the mutants were generated by homology modeling. In the structure of kvPulA (Fig. 1 in Electronic supplementary material), Phe581 was located by a loop structure, in which the region A comprising the sequence NWGYDP (residue 575–580) was highly conserved in type I pullulanase, and additionally Trp576 and Tyr578 in the conserved sequence would be important to act to the glucose residues in subsites −1 and −2 of substrate by van der Waals interactions [19]. Because loops are likely to accommodate a large variety of stabilizing mutations than the regions with higher rigidity without disrupting the threedimensional structure [20, 21], the stability of the loop close to Phe581 would be critical to the stability of the whole protein and also its affinity towards substrate. Compared with the wild-type enzyme, the mutations at Phe581 formed more hydrogen-bond interactions, such as F581Q, F581R, and F581T (Fig. 2 in Electronic supplementary material), resulting in enhanced stability of the loop structure [22, 23]. Furthermore, hydrophobic residues in the interior region of protein would also affect the protein stability [24]. As Leu and Val are more hydrophobic than Phe, therefore, the mutants F581L and F581V would be more stable due to hydrophobic interactions.
pH Profile of Thermostability-Enhanced Mutants Protein engineering by mutations generally introduces the change of structure and function of enzyme. For the thermostability-enhanced mutants involving site-directed mutagenesis at
Fig. 1 Half-lives of the wild-type kvPulA and its mutants at 55 °C
Appl Biochem Biotechnol
Fig. 2 Thermostabilities of the wild-type kvPulA and its mutants. For each enzyme, the activity without heat treatment was taken as 100 %
Phe581, the effects of these mutations on optimum pH value of enzyme activity were further investigated. As shown in Fig. 3, substitutions of Phe581 with the residues increasing the enzyme stability did not change the pH profile of the enzyme. As the requirement of pH condition of saccharification process, pullulanase should exhibit its maximum activity at pH 4.5–5.0. Therefore, thermostability-enhanced mutants with unchanged optimum pH value would be more suitable for the enzymatic saccharification process.
Catalytic Efficiency of Thermostability-Enhanced Mutants In addition to thermostability and optimum catalytic temperature and pH value, the effects of these mutations on enzyme activity and catalytic efficiency were further investigated. The kinetic parameters of the wild-type kvPulA and its mutants were determined under the enzyme activity assay conditions. Compared with the wild-type enzyme, as shown in Table 1, most of the mutants performed obviously increased kcat values and similar Km values, such as F581L
Fig. 3 Optimum pH values of the wild-type kvPulA and its mutants. Activity assay was carried out at 55 °C in buffers of various pH values. For each enzyme, the highest activity in the temperature range was taken as 100 %
Appl Biochem Biotechnol Table 1 Kinetic parameters of the wild-type kvPulA and the mutants of higher catalytic efficiency Enzyme
Km (mg ml−1)
Vmax (mg ml−1 s−1)
kcat (s−1)
kcat/Km (ml mg−1 s−1)
F581L
7.77±0.04
95.24±0.36
198.70±0.91
25.59±0.09
F581Q
6.12±0.02
43.97±0.28
167.80±0.74
27.44±0.09
F581R
11.86±0.05
68.13±0.39
120.40±0.68
10.15±0.03
F581T F581V
8.42±0.04 6.81±0.03
56.24±0.41 97.87±0.65
130.00±0.52 172.90±0.88
15.44±0.17 25.40±0.19
WT
6.57±0.03
34.31±0.18
105.20±0.43
16.02±0.76
and F581V, indicating that substitutions of Phe581 with these residues did not change much of substrate binding, while the activity and catalytic efficiency was obviously enhanced. As known, proper conformational flexibility of the substrate-binding site would be critical to the enzyme activity and affinity towards substrate, thus the replacement of Phe581 by other amino acid residues would affect the molecular interactions among the active sites and ligands in the functional domain [25]. The location of Phe581 in the functional domain further indicated that even the mutation around the catalytic center would have an influence on the catalytic activity of the enzyme [26, 27]. Although it has already been reported that enhancement of thermostability and other properties of certain enzyme could be achieved simultaneously [28], improvement of both thermostability and catalytic efficiency of the pullulanase by single-site substitution was not yet common. Consequently, the candidates of the pullulanase with high application potential were obtained by the strategy involving active site analysis and site-directed mutagenesis.
Glucose Production by Pullulanase-Involved Mixed Enzymatic Saccharification For the mutated pullulanases with enhanced thermostability and catalytic efficiency, F581L and F581V were further investigated for glucose production from maltodextrin hydrolysis,
Fig. 4 Glucose production from maltodextrin hydrolysis by the mixed enzymatic saccharification with the combination of glucoamylase and pullulanase involving the mutants and the wild-type enzyme. Dry maltodextrin was incubated with 50 U of glucoamylase and 0.5 U of pullulanase at 55 °C. The glucose content (DX) produced from mixed enzymatic saccharification of maltodextrin was measured by HPLC
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combined with glucoamylase in the mixed enzymatic saccharification. The values of glucose content (DX) during the pullulanase-involved saccharification process were measured for the mutants of F581L and F581V, with the wild-type enzyme as the negative control. As shown in Fig. 4, both of the mutants gave higher DX values than the wild-type pullulanase during the whole saccharification process. Additionally, the maximum DX values over 98 % were achieved for the mutants of F581L (98.30 %) and F581V (98.92 %), which was obviously higher than that of wild-type enzyme (97.58 %). Therefore, from the viewpoint of application, the mutated pullulanase, F581L and F581V, would be more feasible and promising for glucose production from starch saccharification.
Conclusion In this work, based on conserved sites and homology modeling analysis, the residue Phe581 in the K. variicola SHN-1 pullulanase was selected for site-saturated mutagenesis to investigate the effects of other amino acid residue substitutions at this position on the thermostability and catalytic efficiency of the enzyme. Some mutants from single-site substitutions of Phe581 were found to modulate the catalytic domain flexibility and thereby improve the thermostability and catalytic efficiency of the enzyme. Modeling structure analysis revealed that formation of hydrogen bonds generated from mutations would be responsible for the improvement of enzymatic properties. Therefore, the mutated enzyme with higher thermostability and catalytic efficiency was achieved concurrently and would be promising in application of enzymatic saccharification process. Acknowledgments Financial supports from the National Key Basic Research and Development Program of China (973 Program) (2011CB710800), the National High Technology Research and Development Program of China (863 Program) (2012AA022207), the National Natural Science Foundation of China (NSFC) (21376107 and 21336009), the 111 Project (111-2-06), the High-end Foreign Experts Recruitment Program (GDW20133200113), the Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions, and the Jiangsu province BCollaborative Innovation Center for Advanced Industrial Fermentation^ industry development program are greatly appreciated.
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Single Amino Acid Substitution in the Pullulanase of Klebsiella variicola for Enhancing Thermostability and Catalytic Efficiency Guo Cui Mu 1 & Yao Nie 1,3 & Xiao Qing Mu 1,3 & Yan Xu 1,2,3 & Rong Xiao 4
Received: 23 January 2015 / Accepted: 19 May 2015 # Springer Science+Business Media New York 2015
Abstract Based on conserved sites and homology modeling analysis, the residue Phe581 in the Klebsiella variicola SHN-1 pullulanase was selected as the potential thermostabilityrelated site and its role on thermostability and activity was investigated by site-saturated mutagenesis. Compared with the wild-type pullulanase, the optimum temperature of the mutants including F581L, F581Q, F581R, F581T, F581V, and F581Y was increased from 53 to 56 °C, and correspondingly the half lives of these mutants at 55 °C were increased by 4.20, 3.70, 1.90, 7.16, 3.01, and 1.75 min, respectively. By modeling the structure of the pullulanase, formation of more hydrogen bonds by single-site substitution was supposed to be responsible for the improvement of thermostability. Of these mutants, furthermore, F581L and F581V exhibited higher values of Vmax and kcat/Km, compared with the wild-type enzyme. Therefore, the residue Phe581 was identified as an important site relevant to the activity and thermostability of the pullulanase of K. variicola, and by mutation at this single site, the mutated enzymes with enhanced thermostability and catalytic efficiency were achieved consequently. Electronic supplementary material The online version of this article (doi:10.1007/s12010-015-1675-2) contains supplementary material, which is available to authorized users.
* Yao Nie [email protected] * Yan Xu [email protected] 1
Key Laboratory of Industrial Biotechnology, Ministry of Education and School of Biotechnology, Jiangnan University, Wuxi 214122, China
2
State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, China
3
2011 Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University, Wuxi 214122, China
4
Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, NJ 08854, USA
Appl Biochem Biotechnol
Keywords Pullulanase . Site-saturated mutagenesis . Thermostability . Optimum temperature . Catalytic efficiency
Introduction Pullulanase (EC 3. 2. 1. 41) is known as an outstanding debranching enzyme and mostly utilized with other amylolytic enzymes involving α-amylase, β-amylase, and glucoamylase to produce glucose or maltose syrups from starch [1], where two enzymatic hydrolysis processes including liquefaction and saccharification are generally involved. In the saccharification process, glucoamylase mainly hydrolyzes α-1,4-glucosidic links and release glucose molecules from the non-reducing end of the dextrins [2], while pullulanase is usually applied to specifically cleave the α-1,6-glucosidic linkages in amylaceous polysaccharides. Therefore, the combination of glucoamylase and pullulanase has been widely adopted to produce glucose from starch raw materials with high efficiency. However, the saccharification reaction is usually carried out at the temperature ranging from 55 to 60 °C, and there is a requirement for the involved pullulanase to be active and stable around that temperature [3]. Pullulanases have been discovered from various microorganisms, such as Anaerobranca gottschalkii [4], Bacillus acidopullulyticus [5], Bacillus subtilis TU [6], Thermus aquaticus YT-1 [7], Desulfurococcus mucosus [8], and so on. These discovered enzymes have been further identified to perform diverse characteristics. Associating with the working conditions of enzymatic saccharification, only if pullulanase is adaptable in respect of resistance to higher temperature can it act together with glucoamylase to promote the efficiency of the mixed enzymatic saccharification. However, so far the available pullulanases with thermostability are yet limited to be suitable for the saccharification reaction and practical application [3]. To improve the availability of enzymes with application potential, site-directed mutagenesis has been widely adopted to engineer certain enzymes for desired properties based on the knowledge of structure-function relationships [9]. The α-amylase has been mutated with the half-life of 3.8-fold longer to the wild-type enzyme and its melting temperature was correspondingly increased 3.2 °C compared with the wild-type enzyme by structurebased rational design and site-directed mutagenesis [10]. The Aspergillus niger N25 phytase has been reported to undergo the site-directed mutagenesis by substitution of the residues of Ile44 and Thr252 with Glu and Arg, respectively. The obtained mutants exhibited enhanced thermostability and overall catalytic efficiency, compared to the wildtype enzyme [11]. Therefore, the approaches involving active sites analysis and subsequent site-directed mutagenesis should be very useful in development of enzymatic properties including catalytic activity and stability. Nevertheless, there are only few reports about improving thermostability and catalytic efficiency concurrently by single-site substitution. In previous work, the pullulanase from Klebsiella variicola SHN-1 (kvPulA) has been discovered and identified to posses the potential in practical application [12]. However, the thermostability and optimum catalytic temperature of the enzyme need to be further improved to meet the requirement of enzymatic saccharification process. For the pullulanase from Klebsiella aerogene, the residue of Phe562 would be the possible site to have a close relationship with its thermostability [13]. Based on sequence alignment and conserved sites
Appl Biochem Biotechnol
analysis, in this investigation, the role of the residue in kvPulA corresponding to Phe562 in K. aerogene pullulanase was investigated by site-saturated mutagenesis, especially concerning its influence on thermostability and catalytic efficiency. Then the positive mutants were achieved with enhanced thermostability and catalytic efficiency, and the effect of involved residue substitutions at this site was further elucidated by homology modeling and molecular interaction analysis.
Materials and Methods Strains, Plasmids, and Materials The recombinant plasmid pET-28a-pul carrying the pullulanase-encoded gene kvpulA (GenBank accession no. JX087429) from K. variicola SHN-1 was constructed as described in the previous work [12]. The strains including E. coli JM109 and BL21were used as the recombinant hosts for the gene cloning and expression, respectively. PrimeSTAR® HS DNA Polymerase for PCR reaction and restriction endonuclease Dpn I was purchased from TAKARA Biotechnology (Dalian, China). Bradford Protein Assay Kit was obtained from TIANGEN Biotech Co., Ltd. (Beijing, China). The DNA primers for mutagenesis were synthesized in Sangon Co., Ltd. (Shanghai, China) and Plasmid Mini Kit was obtained from Omega bio-tek (Norcross, GA, USA). The polysaccharide of pullulan for determination of pullulanase activity was purchased from Tokyo Kasei Kogyo Co., Ltd. (Tokyo, Japan).
Site-Directed Mutagenesis Site-directed mutagenesis was performed using Megaprimer PCR of the whole recombinant plasmid bearing the pullulanase-encoded gene kvpulA [14]. The primers for site-saturated mutagenesis at the residue Phe581 were designed according to the principle of codon usage. The mutations were generated by using PrimeSTAR HS DNA polymerase with the recombinant plasmid pET-28a-pul as the template. The condition for PCR was as follows: 30 cycles at 98 °C for 10 s, 68 °C for 15 s, and 68 °C for 9 min 30 s. The PCR product was digested with DpnI, followed by transformation into E. coli JM109 for cloning. The plasmids containing correct mutated genes confirmed by sequencing were finally transformed into E. coli BL21 (DE3) for expression of the mutants of the pullulanase.
Expression of the Wild-Type Enzyme and the Mutants The colony of the recombinant E. coli BL21 (DE3) carrying kvpulA or mutated gene was grown in 4 ml Luria–Bertani (LB) liquid medium containing 50 μg ml−1 kanamycin at 37 °C and 200 rpm overnight. Then the culture was inoculated to 50 ml LB liquid medium containing 50 μg ml−1 kanamycin in a 250-ml shake flask and incubated at 37 °C and 200 rpm. When the culture turbidity (OD600 nm) reached to the level between 0.6 and 0.8, 0.4 mM IPTG was added to induce target protein expression. The expression of the enzyme and the mutants was performed at 25 °C for 20 h. After centrifugation at 4 °C and 10,000 rpm for 15 min, the resulted culture supernatants were collected for further research.
Appl Biochem Biotechnol
Protein and Enzyme Activity Assay SDS-PAGE of the enzyme sample was performed on a 9 % (w/v) running gel. Protein concentration was determined by Bradford method with bovine serum albumin (BSA) as a standard [15]. Pullulanase activity was assayed by measuring the aldehyde groups released during incubation from a reaction mixture consisting of pullulan solution and the diluted enzyme solution. The enzyme solution was diluted with sodium acetate buffer (pH5.0) in advance. Then 100 mM sodium acetate buffer (pH5.0) containing 0.1 ml 1 % (w/v) pullulan was mixed with the diluted enzyme solution. The mixture was incubated at 50 °C for 30 min. Then, the amount of released aldehyde groups was assayed by dinitrosalicylic acid method. One unit of enzyme activity was defined as the amount of enzyme that causes the liberation of 1 μmol of aldehyde groups in 1 min under the reaction conditions [16].
Characterization of the Mutants The kinetic parameters (Km, Vmax, and kcat values) of the pullulanase were determined based on the method described previously [17], where different concentrations of pullulan (1, 2, 5, 10, and 15 g l−1) were adopted. The values of Vmax and Km were estimated by fitting the initial rate data to the Michaelis-Menten equation using nonlinear regression with GraphPad Prism software. To determine the optimum temperature, enzyme activity was measured across the temperature range from 50 to 65 °C. For each enzyme, the highest activity in the temperature range was taken as 100 % and the percentage of relative activity was calculated relative to the highest activity. The optimum pH of the mutants was determined by assaying the enzyme activity in sodium acetate buffer of different pH value ranging from 4.0 to 6.0. For each enzyme, the highest activity in the pH range was taken as 100 % and the percentage of relative activity was calculated relative to the highest activity. The half-lives (T1/2) of the enzymes at 55 °C were taken as an indicator of the thermostability. Enzymatic reaction solution was incubated at 55 °C and pH 5.0, and enzyme activity was measured after incubation at 55 °C for 5, 10, 15, 20, 25, and 30 min, respectively. The values of T1/2 at 55 °C were calculated according to the equations as shown below. lnðU t =U o Þ ¼ −kt T 1=2 ¼ ln2 = k T50 was defined as the temperature at which 50 % of the initial activity remained after incubation for 15 min and was calculated according to the above equations [18]. The residual activity was assessed in the temperature range from 35 to 55 °C with an interval of 5 °C.
Glucose Production by Pullulanase-Involved Mixed Enzymatic Saccharification The enzyme solutions comprising glucoamylase and pullulanase involving kvPulA, F581L, or F581V were added in the reactor vessel (dosage equal to 50 U of glucoamylase and 0.5 U of pullulanase per gram of dry maltodextrin, i.e., the ratio of glucoamylase to pullulanase was
Appl Biochem Biotechnol
100:1). Dry maltodextrin (10 g) and sodium acetate buffer (pH 5.0) was added into the reactor vessel till the total weight of the slurry reached 50 g in a 250-ml batch reactor vessel, leading to the final concentration of dry maltodextrin 20 % (w/w). The saccharification procedure was carried out at 55 °C in a water bath, and a high-temperature-resistant magnetic stirrer was used for saccharification to obtain a constant stirring speed. Samples were taken at certain time intervals and then boiled for 5 min to inactivate enzymes for measuring glucose content (DX) by HPLC analysis. The DX value was defined as the percentage of glucose on a dry basis. The amount of glucose was analyzed by HPLC on a Sepax HP-Amino column (4.6×250 mm, 5 μm) with a Hitachi Chromaster 5450 refraction index detector and the mobile phase of acetonitrile/water (75:25, v/v) at a flow rate of 0.8 ml min−1.
Homology Modeling and Molecular Interaction Analysis Based on sequence identity of 98 % between the Klebsiella pneumoniae pullulanase and kvPulA, the protein structure of the K. pneumoniae pullulanase (PDB ID=2fhf) was used as the template for homology modeling to generate the model structures of the wild-type enzyme and the mutants of kvPulA with SWISS-MODEL. Multiple sequence alignment was conducted by EMBL-EBI (http://www.ebi.ac.uk/Tools/msa/). Structure comparison and analysis of the wild-type enzyme and the mutants was performed by the program of PyMOL.
Results and Discussion Thermostability-Related Site Selected for Saturated Mutagenesis The pullulanase from K. aerogene has been investigated for the structure-function relationships by random mutagenesis, and the residue of Phe562 was considered to be the possible site relating to its thermostability [13]. Based on sequence alignment and conserved sites analysis, 98.52 % identity was found between the K. aerogene pullulanase and kvPulA, and the residue Phe562 in K. aerogene pullulanase was identified to correspond to Phe581 in kvPulA. Therefore, the residue of Phe581 was taken into account to investigate its role on thermostability and catalytic activity. To understand the effect of other amino acid substitutions at the position 581 on thermostability and catalytic efficiency of the enzyme, saturated mutagenesis of kvPulA was carried out by replacing the residue of Phe581 with other 19 amino acid residues. The sequencingconfirmed mutant genes were cloned and expressed successfully in recombinant Escherichia coli.
Effects of Single Point Mutations on Optimum Temperature and Thermostability To investigate the effects of site-saturated mutagenesis on optimum temperature of enzymatic catalysis, the enzyme activities of the wild-type enzyme and the mutants were measured at temperature ranging from 50 to 65 °C, respectively. Compared with the wild-type pullulanase kvPulA, the optimum temperature of enzyme activity was increased from 53 to 56 °C for most of the mutants including F581A, F581C, F581E, F581G, F581H, F581L, F581Q, F581R, F581S, F581T, F581V, and F581Y, while the mutants F581K, F581M, and F581N exhibited the optimum temperature lower than that of kvPulA.
Appl Biochem Biotechnol
Then the effect of site-saturated mutagenesis at Phe581 on thermostability of the enzyme was further evaluated under the incubation conditions of 55 °C and pH 5.0. After heat treatment for different periods from 5 to 30 min, the enzyme activities of kvPulA and its mutants were measured. As shown in Fig. 1, compared with kvPulA, the half-lives were increased by 0.65, 4.20, 3.70, 1.90, 7.16, 3.01, 5.12, and 1.75 min for the mutants of F581I, F581L, F581Q, F581R, F581T, F581V, F581W, and F581Y, respectively. In addition, the values of T50 of the mutants were measured by assessing the residual activity of the enzymes after incubation at temperature ranging from 35 to 55 °C for 15 min. As shown in Fig. 2, most of the mutants had a positive shift of T50 except for F581A and F581C. Integrating the above results, the mutants F581L, F581Q, F581R, F581T, and F581V were found to be more active and stable against increased temperature. To gain insight into the role of Phe581 in kvPulA, based on the protein structure of the K. pneumoniae pullulanase (PDB ID: 2fhf) which shares the sequence identity of 98 % with kvPulA, the model structures of the wide-type enzyme and the mutants were generated by homology modeling. In the structure of kvPulA (Fig. 1 in Electronic supplementary material), Phe581 was located by a loop structure, in which the region A comprising the sequence NWGYDP (residue 575–580) was highly conserved in type I pullulanase, and additionally Trp576 and Tyr578 in the conserved sequence would be important to act to the glucose residues in subsites −1 and −2 of substrate by van der Waals interactions [19]. Because loops are likely to accommodate a large variety of stabilizing mutations than the regions with higher rigidity without disrupting the threedimensional structure [20, 21], the stability of the loop close to Phe581 would be critical to the stability of the whole protein and also its affinity towards substrate. Compared with the wild-type enzyme, the mutations at Phe581 formed more hydrogen-bond interactions, such as F581Q, F581R, and F581T (Fig. 2 in Electronic supplementary material), resulting in enhanced stability of the loop structure [22, 23]. Furthermore, hydrophobic residues in the interior region of protein would also affect the protein stability [24]. As Leu and Val are more hydrophobic than Phe, therefore, the mutants F581L and F581V would be more stable due to hydrophobic interactions.
pH Profile of Thermostability-Enhanced Mutants Protein engineering by mutations generally introduces the change of structure and function of enzyme. For the thermostability-enhanced mutants involving site-directed mutagenesis at
Fig. 1 Half-lives of the wild-type kvPulA and its mutants at 55 °C
Appl Biochem Biotechnol
Fig. 2 Thermostabilities of the wild-type kvPulA and its mutants. For each enzyme, the activity without heat treatment was taken as 100 %
Phe581, the effects of these mutations on optimum pH value of enzyme activity were further investigated. As shown in Fig. 3, substitutions of Phe581 with the residues increasing the enzyme stability did not change the pH profile of the enzyme. As the requirement of pH condition of saccharification process, pullulanase should exhibit its maximum activity at pH 4.5–5.0. Therefore, thermostability-enhanced mutants with unchanged optimum pH value would be more suitable for the enzymatic saccharification process.
Catalytic Efficiency of Thermostability-Enhanced Mutants In addition to thermostability and optimum catalytic temperature and pH value, the effects of these mutations on enzyme activity and catalytic efficiency were further investigated. The kinetic parameters of the wild-type kvPulA and its mutants were determined under the enzyme activity assay conditions. Compared with the wild-type enzyme, as shown in Table 1, most of the mutants performed obviously increased kcat values and similar Km values, such as F581L
Fig. 3 Optimum pH values of the wild-type kvPulA and its mutants. Activity assay was carried out at 55 °C in buffers of various pH values. For each enzyme, the highest activity in the temperature range was taken as 100 %
Appl Biochem Biotechnol Table 1 Kinetic parameters of the wild-type kvPulA and the mutants of higher catalytic efficiency Enzyme
Km (mg ml−1)
Vmax (mg ml−1 s−1)
kcat (s−1)
kcat/Km (ml mg−1 s−1)
F581L
7.77±0.04
95.24±0.36
198.70±0.91
25.59±0.09
F581Q
6.12±0.02
43.97±0.28
167.80±0.74
27.44±0.09
F581R
11.86±0.05
68.13±0.39
120.40±0.68
10.15±0.03
F581T F581V
8.42±0.04 6.81±0.03
56.24±0.41 97.87±0.65
130.00±0.52 172.90±0.88
15.44±0.17 25.40±0.19
WT
6.57±0.03
34.31±0.18
105.20±0.43
16.02±0.76
and F581V, indicating that substitutions of Phe581 with these residues did not change much of substrate binding, while the activity and catalytic efficiency was obviously enhanced. As known, proper conformational flexibility of the substrate-binding site would be critical to the enzyme activity and affinity towards substrate, thus the replacement of Phe581 by other amino acid residues would affect the molecular interactions among the active sites and ligands in the functional domain [25]. The location of Phe581 in the functional domain further indicated that even the mutation around the catalytic center would have an influence on the catalytic activity of the enzyme [26, 27]. Although it has already been reported that enhancement of thermostability and other properties of certain enzyme could be achieved simultaneously [28], improvement of both thermostability and catalytic efficiency of the pullulanase by single-site substitution was not yet common. Consequently, the candidates of the pullulanase with high application potential were obtained by the strategy involving active site analysis and site-directed mutagenesis.
Glucose Production by Pullulanase-Involved Mixed Enzymatic Saccharification For the mutated pullulanases with enhanced thermostability and catalytic efficiency, F581L and F581V were further investigated for glucose production from maltodextrin hydrolysis,
Fig. 4 Glucose production from maltodextrin hydrolysis by the mixed enzymatic saccharification with the combination of glucoamylase and pullulanase involving the mutants and the wild-type enzyme. Dry maltodextrin was incubated with 50 U of glucoamylase and 0.5 U of pullulanase at 55 °C. The glucose content (DX) produced from mixed enzymatic saccharification of maltodextrin was measured by HPLC
Appl Biochem Biotechnol
combined with glucoamylase in the mixed enzymatic saccharification. The values of glucose content (DX) during the pullulanase-involved saccharification process were measured for the mutants of F581L and F581V, with the wild-type enzyme as the negative control. As shown in Fig. 4, both of the mutants gave higher DX values than the wild-type pullulanase during the whole saccharification process. Additionally, the maximum DX values over 98 % were achieved for the mutants of F581L (98.30 %) and F581V (98.92 %), which was obviously higher than that of wild-type enzyme (97.58 %). Therefore, from the viewpoint of application, the mutated pullulanase, F581L and F581V, would be more feasible and promising for glucose production from starch saccharification.
Conclusion In this work, based on conserved sites and homology modeling analysis, the residue Phe581 in the K. variicola SHN-1 pullulanase was selected for site-saturated mutagenesis to investigate the effects of other amino acid residue substitutions at this position on the thermostability and catalytic efficiency of the enzyme. Some mutants from single-site substitutions of Phe581 were found to modulate the catalytic domain flexibility and thereby improve the thermostability and catalytic efficiency of the enzyme. Modeling structure analysis revealed that formation of hydrogen bonds generated from mutations would be responsible for the improvement of enzymatic properties. Therefore, the mutated enzyme with higher thermostability and catalytic efficiency was achieved concurrently and would be promising in application of enzymatic saccharification process. Acknowledgments Financial supports from the National Key Basic Research and Development Program of China (973 Program) (2011CB710800), the National High Technology Research and Development Program of China (863 Program) (2012AA022207), the National Natural Science Foundation of China (NSFC) (21376107 and 21336009), the 111 Project (111-2-06), the High-end Foreign Experts Recruitment Program (GDW20133200113), the Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions, and the Jiangsu province BCollaborative Innovation Center for Advanced Industrial Fermentation^ industry development program are greatly appreciated.
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