N-Terminal Domain Truncation and Domain Insertion-Based

Article Cite This: J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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N‑Terminal Domain Truncation and Domain Insertion-Based Engineering of a Novel Thermostable Type I Pullulanase from Geobacillus thermocatenulatus Lingmeng Li,† Fengying Dong,† Lin Lin,‡,§ Dannong He,§ Wei Wei,*,† and Dongzhi Wei*,†

J. Agric. Food Chem. Downloaded from pubs.acs.org by UNIV OF SOUTH DAKOTA on 09/17/18. For personal use only.

†

State Key Laboratory of Bioreactor Engineering, Newworld Institute of Biotechnology, East China University of Science and Technology, Shanghai 200237, People’s Republic of China ‡ Shanghai University of Medicine and Health Sciences, Shanghai 200093, People’s Republic of China § Research Laboratory for Functional Nanomaterial, National Engineering Research Center for Nanotechnology, Shanghai 200241, People’s Republic of China S Supporting Information *

ABSTRACT: A novel thermostable type I pullulanase gene (pulGT) from Geobacillus thermocatenulatus DSMZ730 was cloned. It has an open reading frame of 2154 bp encoding 718 amino acids. G. thermocatenulatus pullulanase (PulGT) was found to be optimally active at pH 6.5 and 70 °C. It exhibited stable activity in the pH range of 5.5−7.0. PulGT lacked three domains (CBM41 domain, X25 domain, and X45 domain) compared with the pullulanase from Bacillus acidopullulyticus (2WAN). Different N-terminally domain truncated (730T) or spliced (730T-U1 and 730T-U2) mutants were constructed. Truncating the N-terminal 85 amino acids decreased the Km value and did not change its optimum pH, an advantageous biochemical property in some applications. Compared with 2WAN, PulGT can be used directly for maize starch saccharification without adjusting the pH, which reduces cost and improves efficiency. KEYWORDS: Geobacillus thermocatenulatus, pullulanase, domain truncation and insertion, maize starch

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INTRODUCTION Pullulanase (EC 3.2.1.41), a well-known starch-debranching enzyme, has been utilized to catalyze the hydrolysis of α-1,4and α-1,6-glycosidic linkages in amylopectin, pullulan, and the α-and β-limit dextrin of amylopectin. Based on the specificity of action and the hydrolysis products, pullulanases are subdivided into type I and II pullulanases, and the pullulan hydrolases are divided into three subgroups: type I, II, and III. Type I pullulanases specifically hydrolyze α-1,6-glycosidic linkages in starch, pullulan, and other polysaccharides to produce maltotriose and linear oligosaccharides.1 Type II pullulanases, also known as amylopullulanases/α-amylasepullulanases, cleave not only α-1,6-glucosidic linkages in pullulan but also α-1,4- and α-1,6-linkages in starch and related oligosaccharides.2 Pullulan hydrolase type I (neopullulanase) attacks α-1,4-glycosidic linkages in pullulan to release panose.3 Pullulan hydrolase type II, also called isopullulanase, hydrolyzes α-1,4-glycosidic linkages in pullulan, forming isopanose.4 Pullulan hydrolase type III is an enzyme capable of cleaving α-1,4- as well as α-1,6-glycosidic linkages in pullulan to produce a mixture of maltose, maltotriose, and panose.5 As a major debranching enzyme, pullulanase can hydrolyze branch points, greatly reduce the demand for glucoamylase, and significantly improve the utilization of starch glucosidase when used in conjunction with glucoamylase.6 Low-carbohydrate “light beer” can be produced by a combination of pullulanase and a fungal amylase or glucoamylase in the wheat fermentation industry.7 Researchers have used high-gravity maize mashes as raw materials to produce ethanol and obtained the highest possible utilization © XXXX American Chemical Society

of starch in the raw material along with a high yield of available sugars; simultaneously, they have achieved maintenance of high-quality spirit by adding pullulanase for deep enzymatic degradation.8 Pullulanase can be used not only for liquefaction and saccharification, but also as an antistaling agent when producing bread and other baked products, preparing resistant starch, and producing panose- and isopanose-containing syrups.9 However, the high temperatures (60−65 °C) required for saccharification necessitate the use of thermostable enzymes for catalysis. Thermostable pullulanase exhibits great potential for application in the starch saccharification process. Many pullulanases from different species have been discussed, such as those from BacillusBacillus sp., Klebsiella sp., fungi, yeasts, and higher plants.10 However, low yields and high production cost limit their commercial application. To overcome this bottleneck, cloning pullulanase genes from wild strains and expressing them in heterologous hosts have been confirmed as a possible approach.11 Among the reported pullulanases, most are type II pullulanases and mesophilic type I pullulanases. A few thermophilic type I pullulanases have been investigated, such as Thermotoga maritima MSB8,12 Anoxybacillus sp. LM18−11,13 Fervidobacterium pennavorans Ven5,14 Bacillus sp. AN-7,15 Thermus thermophilus HB27,16 and Thermotoga neapolitana.17 Thermophilic bacteria that likely produce thermostable enzymes have been studied for their Received: June 27, 2018 Revised: September 3, 2018 Accepted: September 5, 2018

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DOI: 10.1021/acs.jafc.8b03331 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry potential industrial applications.18 Enzymes from Geobacillus sp. have been classified as thermophilic and are suitable for industrial use. These include amylopullulanase from Geobacillus thermoleovorans NP33,19 type I pullulanase from Geobacillus thermoleovorans US105,20 and type II pullulanase from Bacillus stearothermophilus G-82.21 However, there have been no reports about pullulanases from G. thermocatenulatus. In recent years, protein engineering and genetic modifications offer alternative options for developing new enzymes with industrial application potential.22 Protein engineering strategies, such as site-directed mutagenesis and directed evolution are used to create potential excellent pullulanases.23 However, these strategies have certain limitations. Site-directed mutagenesis should be based on the crystal structure of the protein, while directed evolution requires a random mutant library for high-throughput screening.24 Protein domains often correspond to structural domains, which are self-stabilizing and fold independent of the rest of the protein chain. They may occur independently or as part of complex multidomain protein architectures that evolve by domain truncation and insertion or recombination. Some previous studies have shown that truncation and insertion of unstable pullulanase domains or peptides can enhance the thermostability and improve pullulanase expression level and activity. It is generally known that pullulanase from Bacillus acidopullulyticus (2WAN) has many ideal biochemical properties (mainly heat resistance, acid resistance, and specific enzyme activity) for application in starch saccharification, and has been used in industrial production.25 Previous studies on the structure and functions of the 2WAN mature protein have shown that it is composed of 921 amino acids, and the structure indeed forms an uncommon domain in which the Nterminal CBM41 domain is disordered/partially absent incrystal but the X45a-X25-X45b-CBM48-GH13 multidomain architecture is completely clear.26 In recent years, many researchers have attempted to improve thermostability and catalytic efficiency by altering the structure of pullulanase and have achieved some success. For example, Wang et al. found that disorder prediction-based truncation would be helpful to efficiently improve enzyme activity and catalytic efficiency.27 The N-terminal deletion construct of amylopullulanase exhibits similar optimum pH and temperature as that of the wild-type enzyme but exhibits enhanced thermostability, hydrolytic action, and specific activity to pullulan.28 Previous research has also suggested that the N-terminal domains of Bacillus deramificans pullulanase negatively affect the secretion and stability of the enzyme but positively impact its activity by enhancing its substrate affinity.29 In this study, a novel thermostable type I pullulanase was cloned and expressed, and its enzymatic properties were determined. Based on protein engineering, the N-terminal domain truncation (85 amino acids) and insertion (CBM41X45a-X25-X45b and X45a-X25-X45b from 2WAN) variants of PulGT were constructed. Finally, PulGT and 2WAN (commonly used commercial pullulanase from B. acidopullulyticus) were applied in saccharification experiments using maize starch as a substrate. Compared with 2WAN, PulGT could be used directly for maize starch saccharification without adjusting the pH, suggesting its potential application in the starch industry.

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Information (SI) Table S1. Strain DSMZ730 (named G. thermocatenulatus DSMZ730) was purchased from Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH (DSMZ730). Escherichia coli DH5α (Invitrogen) and plasmid pMD19-T (TaKaRa) were used for gene cloning and sequencing. E. coli BL21 (DE3) and plasmid pET-28a(+) were used for gene expression. LA Taq polymerase, T4 DNA ligase, restriction endonucleases, and a DNA marker were purchased from TaKaRa (Otsu, Japan). A protein marker was purchased from MBI Fermentas (Vilnius, Lithuania). Isopropyl-β-D-thiogalactopyranoside (IPTG), ampicillin, and kanamycin were purchased from Amresco (Shanghai Genebase Co., Ltd., China). DNA Mini Kit and Plasmid Mini Prepare Kit were purchased from Axygen Biosciences (Union City, CA, U.S.A.). The substrates (pullulan, soluble starch and amylose) and markers (glucose, maltose, and maltotriose) were purchased from Sigma Chemical Company (St. Louis, MO, U.S.A.). Other chemicals were obtained commercially and were of reagent grade. The α-amylase (40000 U/mL) and pullulanase (industrial enzyme, pullulanase from B. acidopullulyticus; 2000 U/mL) were purchased from Novozymes (Bagsvaerd, Denmark). DNA Manipulation and Sequence Analysis. DNA manipulation was carried out following standard procedures. G. thermocatenulatus DSMZ730 was harvested after overnight growth in medium 1 culture (peptone 0.5%, meat extract 0.3%, Mn2+ 0.001‰, pH 7.0) at 60 °C, and DNA extraction was performed using a DNA Mini Kit (Axygen). Based on the information on Geobacillus pullulanase in GenBank, PCR primers T730-U/T730-D (SI Table S2) for the pullulanase gene sequence were designed. The PCR thermocycling conditions were as follows: predenaturation at 94 °C for 4 min, followed by 30 cycles at 94 °C for 30 s, 58 °C for 30 s, and 72 °C for 2 min 15 s, with a final extension step at 72 °C for 10 min. The target fragment with the approximate length was cloned into the pMD19-T simple vector (named pMD-pulGT). Then, E. coli DH5α competent cells were transformed with the pMD-pulGT. The nucleotide sequences and predicted amino acid sequences were analyzed by the BLAST program (NCBI). The open reading frame (ORF) was predicted using the NCBI ORF Finder tool (http://www. ncbi.nlm.nih.gov/gorf/gorf.html). The signal peptide was predicted by the SignalP 4.0 server (http://www.cbs.dtu.dk/services/SignalP/). ExPASy proteomics server (http://web.expasy.org/compute_pi/) was used to predict the MW and pI of the enzyme, and the SWISSMODEL server (http://swissmodel.expasy.org/SWISS-MODEL. html) was used to predict its three-dimensional structure. The models were visualized and analyzed using PDB Viewer, and the figures were constructed using Pymol. Gene Expression of Pullulanase in E. coli. Using the primers PulGT-U/PulGT-D (SI Table S2), the amplified product was digested with restriction endonucleases (Sac I/Xho I) and ligated into the pET28a(+) vector that had been digested with the same restriction endonucleases (SI Figure S1). The recombinant vector was transformed into E. coli DH5α competent cells, and then positive recombinant colonies were screened and their identity was verified by polymerase chain reaction (PCR) and sequencing. The recombinant plasmid pET-28a-pulGT was transformed into E. coli BL21 (DE3). Optimization of Induction Conditions and Purification of Pullulanase-Producing Recombinants. Pullulanase activity of recombinant colonies was analyzed after overnight incubation at 37 °C in Luria−Bertani medium (LB medium) supplemented with kanamycin (50 μg/mL). Colonies were then transferred to shake flasks for amplification. IPTG was added to induce the expression of pullulanase in the recombinant vector when the optical density at 600 nm (OD600) reached 0.6. Initial induction conditions were as follows: IPTG concentration: 0.1 mM, temperature: 20 °C, working time: 24 h. Different induction times (0, 4, 12, 16, 20, 24, 28, and 32 h) and temperatures (20, 30, and 37 °C) were tested to investigate the effect of induction time and temperature on recombinant protein expression. The results were confirmed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and enzyme analysis. The recombinant protein contained a His-Tag, therefore we purified

MATERIALS AND METHODS

Bacterial Strains, Plasmids, and Chemicals. The bacterial strains and plasmids used in this study are listed in Supporting B


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Journal of Agricultural and Food Chemistry Table 1. Pairwise Similarity between the Type I Pullulanase Amino Acid Sequencesa similarity value (%) for pullulanase sequence from pullulanase sequence from

G. thermocatenulatus DSMZ730

G. thermoleovorans US105

G. kaustophilus DSMZ7263

Anoxybacillus sp. LM18-11

Bacillus sp. CICIM 263

F. pennavorans Ven5

B. acidopullulyticus

G. thermocatenulatus DSMZ730 G. thermoleovorans US10520 G. kaustophilus DSMZ726332 Anoxybacillus sp. LM18-1113 Bacillus sp. CICIM 26337 F. pennavorans Ven514 B. acidopullulyticus26

100

95

94

55

46

41

39

100

99

56

46

42

40

100

55

46

41

39

100

47

39

40

100

40

40

100

41 100

a

The sequences are from the following sources: G. thermocatenulatus DSMZ730 (this study; GenBank accession no. KT899327); G. thermoleovorans US105 (GenBank accession no. AJ315595); G. kaustophilus DSMZ7263 (GenBank accession no. KT899327); Anoxybacillus sp. LM18-11 (GenBank accession no. HQ844266); Bacillus sp. CICIM 263 (GenBank accession no. KT897705); F. pennavorans Ven5 (GenBank accession no. AF096862); B. acidopullulyticus. stability of PulGT, the enzyme was incubated in sodium phosphate buffer (pH 6.5) at different temperatures (60 and 70 °C) for various time intervals. The enzyme reaction system without any incubation was used as a control (100%). Different metal ions (1, 5, and 10 mM) and chemical reagents were added to study their effects on PulGT in the standard assay medium. The enzyme reaction mixtures were incubated at 70 °C for 20 min in sodium phosphate buffer (pH 6.5). The enzyme reaction system without any incubation was used as a control (100%). The enzymatic properties and thermal stability of mutants were measured in each optimum catalysis environment. Substrate Specificity and Hydrolysis Product Analysis. The ability of the purified enzyme to hydrolyze various carbohydrates was examined at 70 °C and pH 6.5 in 0.1 M sodium phosphate buffer. The carbohydrates tested included pullulan, amylose, soluble starch, amylopectin, glycogen, α-cyclodextrin, and γ-cyclodextrin at a concentration of 1% (w/v). The enzyme reaction mixtures containing different substrates were incubated at 70 °C for 3 h. Silica gel plates were used for thin-layer chromatography (TLC) analysis. Glucose, maltose, and maltotriose were used as the standards. Pullulanase (about 0.5 U) was incubated with 1% (w/v) pullulan substrate at 70 °C for 3 h, and the resulting products of this reaction were spotinoculated onto TLC sheets. The plate was placed in a chamber containing a solvent system of acetic acid/n-butanol/water (1:2:1) for a while and then was withdrawn. After the plate was dried, reducing sugars were detected using sulfuric acid solution (containing 0.3% N1-naphthyl-ethylenediamine and 5% H2SO4 in methanol) at 110 °C for 10 min. High-performance liquid chromatography (HPLC) was used to analyze the hydrolysis products of different substrates by about 1 U of enzyme. A Waters analysis column (Waters Sugar Pak1 column, WAT085188) with a refractive index detector was used for HPLC. A double-distilled water solvent system was used as the mobile phase at a flow rate of 0.5 mL/min. The temperatures of the column and refractive index detector were 75 and 35 °C, respectively. The sample quantity was 10 μL each time. Kinetic Parameters of the Recombinant PulGT and Mutants. The kinetic parameters of mutants were measured in each optimal catalysis environment. The DNS method was used to measure the kinetic parameters Km and Vmax of PulGT (730T and 730T-U2) in pH 6.5 sodium phosphate buffer at 70 °C (65 °C). Different substrate concentrations of pullulan (2, 4, 6, 8, and 10 mg/mL) were reacted with the standard reaction system. The reaction solution was sampled every 2 min and measured. According to the initial velocity values (the amount of maltotriose generated per minute) and substrate concentrations, nonlinear regression curves were plotted.11 Subsequently, the Km and Vmax values were calculated from the fitting formulas, respectively, as indicated in the graph (SI Figure S2).

the target protein by a Ni-NTA column. The mutant enzyme was induced under the optimum PulGT induction conditions. Design and Construction of N-Terminal Truncation and Insertion Mutant Proteins. The primers in SI Table S2 were designed for constructing the N-terminal truncation and insertion variants. The recombinant plasmids pET-28a-pulGT and pET-28a2WAN (constructed in our lab) were used as templates for PCR. The PCR products (85 amino acids truncated from the N-terminal of PulGT) using Truncated-PulGT-U/Truncated-PulGT-D and plasmid pET-28a(+) were digested by restriction endonucleases at the Sac I/ Xho I sites and then ligated to obtain the respliced plasmid pET-28a730T. Thereafter, the PCR products (CBM41-X45a-X25-X45b/X45aX25-X45b) and pET-28a-730T were digested by the restriction endonucleases at the Nde I/Sac I sites and then ligated to obtain the respliced plasmids pET-28a-730TU1 and pET-28a-730TU2, respectively (SI Figure S1). The plasmids containing the correct mutated genes, as confirmed by sequencing, were finally transformed into E. coli BL21 (DE3) to express the pullulanase mutants. Enzymatic Property Assays. Enzyme activity was determined by measuring the enzymatic release of reducing sugar from pullulan, and the 3,5-dinitrosalicylic acid (DNS) method was adopted to measure the activity of PulGT in pullulan hydrolysis. In this study, 250 μL of 1% (w/v) pullulan solution was mixed with 175 μL of 100 mM sodium phosphate buffer (pH 6.5) and preincubated in a water bath at 70 °C. A suitably diluted enzyme sample (75 μL) was added to the preheated solution. Furthermore, 500 μL of the mixture was incubated at 70 °C. After 20 min, 750 μL of DNS was added to terminate the reaction. The reaction was boiled in a water bath for 5 min, and then the solution was cooled on ice immediately. The reaction system with no enzyme sample with DNS reagent was set as a control. Enzyme activity was expressed as an average of U (mg protein). One U is defined as the amount of enzyme that released 1 μmol of reducing sugar (equivalent to glucose) per minute under specified assay conditions. All pullulanase activities were converted to the entire fermentation volume (volumetric activity U or mg/mL medium). Enzymatic properties’ graphs were drawn to analyze data by the statistical tool Origin 8.5. All values are expressed as the mean ± standard deviation (SD; repeat SD, ≤ 5%) of three independent experiments. The optimum pH of PulGT enzyme activity was determined by measuring its activity in different buffer systems with pH ranging from 4.0 to 9.0. To assess pH stability, the enzyme was incubated in different buffers for 60 min at 4 °C. The buffer system was as follows: 100 mM citrate buffer (pH 4.0−6.0), 100 mM sodium phosphate buffer (pH 6.5−7.0), and 50 mM Tris-HCl buffer (pH 8.0−9.0). The optimum temperature of PulGT enzyme activity was determined by measuring its activity at 40−80 °C (pH 6.5). To evaluate the thermal C


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Journal of Agricultural and Food Chemistry

Figure 1. Protein sequence and structure of PulGT. (a) Sequence alignment of the conserved region of PulGT and other type I pullulanases from different bacterial sources. The sequences of the following strains were obtained from GenBank: G. thermocatenulatus; Geobacillus kaustophilus GKpp1; G. stearothermophilus; Anoxybacillus sp. LM18−11; G. thermoleovorans US105; and B. acidopullulyticus 2WAN. Regions I, II, III, and IV are underlined. The conserved sequence (YNWGYNP) and the catalytic site (Asp411, Glu440, Asp528) in all type I pullulanases is boxed. The numbering refers to the amino acid position in each sequence. The structures are denoted as follows: ϒ, YNWGYDP conserved sequence and the catalytic site. (b) The whole three-dimensional structure. The three-dimensional structure of PulGT was predicted by the SWISS-MODEL server. (c) The catalytic domain of (β/α)8 structure. The catalytic triad: Asp411, Glu440, and Asp528 residues are marked in green. Saccharification of Maize Starch. Maize starch (10 g/100 mL) was stirred with a magnetic stirrer and gelatinized at 100 °C for 10 min. After the temperature dropped to 90 °C, heat-stable α-amylase (0.18 U) was added to the gelatinized liquid for liquefaction for 30 min. Then, the temperature was decreased to 65 °C, and the saccharification process used the same total activity of 2WAN and purified PulGT (0.24 U) for reaction in 30 min. The reaction products were boiled immediately for 10 min and analyzed by HPLC.

glycoside hydrolase family enzymes (GH13) were also identified in PulGT (Figure 1a). On account of these commonalities, we confirmed that PulGT is a type I pullulanase. Based on the sequence alignment in the conserved region of PulGT and type I pullulanase from other bacterial sources, amino acid sequence conservation in the following regions associated with catalysis and protein stabilization was determined: the catalytic site (Asp411, Glu440, Asp528), the putative Ca2+-binding site (Asp280, Glu286, Glu306), and the active site (Tyr297, Asn298, His345, Arg409, Asp411, Leu412, Glu440, Trp442, Asp470, Arg473, His527, Asp528, Asn529, Asn583, Tyr585). The G. thermocatenulatus DSMZ730 has not been reported in literature previously. The PDB Viewer was used to visualize the protein structure of PulGT and the three-dimensional structure was predicted by SWISS-MODEL server (Figure 1b,c). In the starch industry, amylase and pullulanase are utilized in the starch conversion reaction together. Since this complex reaction occurs in a heated environment, a series of thermophilic type I pullulanases have been researched for application in starch saccharification.6 Therefore, selecting new thermophilic bacterial strains is a prerequisite and effective solution for obtaining new pullulanases, and it is important for maximal production in order to get better yields in the starch industry. Geobacillus species are well-known for their ability to secrete thermophilic enzymes. There are few reports on pullulanases from Geobacillus species,20,32 but there are no reports on pullulanases isolated from G. thermocatenulatus, including wild-type or recombinant strains. Construction and Expression of Recombinant Plasmids. The pullulanase gene (pulGT) from G. thermocatenulatus was cloned and successfully expressed in E. coli. The expression vector pET-28a(+) was used to construct and express target proteins in this study. The induction temperature had some effect on PulGT production. Compared with the lower temperature conditions (20 °C), more protein formed inclusion bodies at 30 or 37 °C. (SI Figure S3a). These results are consistent with earlier reports that low temperatures are more conducive for folding to the correct threedimensional structure. We also measured the pullulanase

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RESULTS AND DISCUSSION Pullulanase Gene Cloning and Sequence Analysis. Through PCR amplification, a 2154 bp DNA fragment encoding a polypeptide of 718 amino acids was cloned and sequenced. The GenBank accession number of pulGT is KY613502. The G+C content (%) of pulGT is 58.64%. The mature protein contains a single catalytic domain of the AmyAC family. The molecular weight of PulGT was estimated to be 80.37 kDa, and the pI value was calculated to be 5.53 by the ExPASy compute pI/Mw program algorithm. In addition to other details reported regarding type I pullulanase (Table 1), PulGT from G. thermocatenulatus DSMZ730 showed low sequence homology. Homology analysis revealed that PulGT showed highest identity (95%) with pullulanase from G. thermoleovorans US105, and it had only 39% homology with pullulanase AF096862 from B. acidopullulyticus (commonly used commercial pullulanase) (Table 1). Although the overall similarity value was very low, a highly conserved region consisting of 7 amino acids (YNWGYNP) is found in all type I pullulanases. Visual inspection of the alignment revealed conserved amino acids in regions associated with catalysis and protein stabilization. These 7 amino acids (YNWGYNP) from type I pullulanase have been reported to bind with the substrate and/or exert catalytic activity toward α-1,6-glycosidic linkages.30 The same conserved sequence is present in PulGT at amino acids 292− 298. It has been reported that regions I, II, III, and IV are highly conserved in the enzymes belonging to the GH family 13 α-amylase superfamily.31 Meanwhile, four conserved regions (region I, II, III, and IV) that are common to the D


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Figure 2. Enzymatic properties of purified PulGT. Optimum temperature (a), thermostability (b), optimum pH (c), and pH stability (d) of the purified PulGT from G. thermocatenulatus. The optimum temperature was determined by assaying enzyme activity at different temperatures (40−80 °C) in 100 mM sodium phosphate buffer (pH 6.5). Thermal denaturing half-lives of the recombinant pullulanase were determined by measuring residual activities after the enzyme was treated at 60 °C (■) or 70 °C (●) in 100 mM sodium phosphate buffer (pH 6.5) for different times. Effect of pH on PulGT activity at 70 °C in buffers ranging from pH 4.0−9.0. After incubation at 4 °C for 60 min in buffers ranging from pH 4.5−9.0, residual activity was measured at pH 6.5. Each value represents the mean and SD of triplicate experiments.

pullulan, which is obviously higher than those of the pullulanases from B. thermoleovorans US105 (3.7 U/mg, 75 °C, pH 6.0),33 T. neapolitana (28.7 U/mg, 80 °C, pH 6.5),17 Geobacillus kaustophilus (64.75 U/mg, 65 °C, pH 6.0),32 and Paenibacillus barengoltzii CAU904 (68.3 U/mg, 50 °C, pH 5.5).34 These results showed that optimizing the expression conditions and selecting appropriate purification methods could result in higher specific activity. Enzymatic Properties of the Purified Recombinant Pullulanase. Pullulan was chosen as the substrate to measure PulGT activity at a range of temperatures and pH values (Figure 2). Maximum enzyme activity was observed at the optimum temperature of 70 °C (Figure 2a). PulGT had a high optimum temperature of 70 °C, which is apparently higher than that of pullulanases from P. barengoltzii CAU904 (50 °C),34 B. deramificans (60 °C),23c and Anoxybacillus sp. LM18−11 (60 °C),35 and also higher than that of B. acidopullulyticus pullulanase (60 °C), which is widely used in the starch industry.36 The industrial application of pullulanase requires thermostability at about 60 °C, and PulGT exhibited a preferable thermal stability at 60 °C. The residual activity of PulGT retained more than 50% after it was incubated at pH 6.5 for 3 h at 60 °C and about 46.8% after incubation for 2 h at 70 °C. (Figure 2b). The temperature properties of pullulanase indicated that PulGT has remarkable potential for industrial application. In addition, we compared the pH properties of several thermostable pullulanases, as shown in Table 2. Purified PulGT exhibited an optimum pH of 6.5 in sodium phosphate buffer, which was similar to that of pullulanases from G. kaustophilus,31 Bacillus sp. CICIM 263,37 T.

activity in different induction conditions and found that the highest PulGT activity was induced at 20 °C SI Figure S3b). Through analyzing SDS-PAGE data, the fermentation time was regarded as another remarkable parameter that influences the expression level of PulGT. The yield of PulGT protein increased with extended incubation time, within a certain range (SI Figure S3c, lines 1 to 5). At the same time, pullulanase activity increased with increased expression of recombinant protein. Cells were cultured for over 20 h, and significant accumulation of recombinant proteins was not detected (SI Figure S3c, lines 5 to 8). Thus, the culture time of 20 h was chosen to harvest recombinant strains (BL21-pET-28a-pulGT). The empty pET-28a(+) plasmids in E. coli BL21 (DE3) and recombinant strains lacking the inducer (IPTG) were cultured in the same conditions as controls. From our results, we concluded that the optimal induction conditions were 0.1 mM IPTG at 20 °C for 20 h (SI Figure S3d), where recombinant pullulanase (PulGT) activity and expression level reached about 17.25 U/mL and 0.35 mg/mL, respectively. Purification of Recombinant Protein. The recombinant protein (PulGT) contained a His-tag, and hence Ni-NTA was used for protein purification. Approximately 2.85 mg PulGT was obtained and the recovery rate was 63.62% by Ni-NTA affinity chromatography. The specific activity improved from 49.28 to 192.56 U/mg. As shown in SI Figure S3e, SDS-PAGE analysis determined that a single band with a molecular mass of approximately 80 kDa was purified. Ni-NTA affinity chromatography was an appropriate method to purify this protein SI Table S3). The purified PulGT exhibited the high specific activity of 192.56 U/mg (70 °C, pH 6.5) toward E


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Journal of Agricultural and Food Chemistry Table 2. Comparison of Type I Pullulanases from Different Sources source

Topt (°C)

pHopt

relative activity >80%a

residual activity >70%b

B. acidopullulyticus25

60

5.0

4.5−5.5

G. kaustophilus32

65

6.0

6.0

G. thermocatenulatus US10520 Anoxybacillus sp. LM18-1135 T. maritima MSB812 Bacillus sp. CICIM 26337 T. thermophilus HB2716 G. thermocatenulatus

75

6.0

NA

3.0−8.0 (4 °C 1 h) 5.0−9.0(65 °C 1 h) NA

60

6.0

5.5−6.5

NA

95 70

6.0 6.5

NA 5.5−9.0(>70%)

70

6.5

6.5−7.0

70

6.5

5.0−7.0

T. neapolitana17

80

6.5

5.0−7.0

6.0−8.0 6.0−8.5 (60 °C 1 h) 6.0−8.0 (70 °C 1 h) 5.0−7.0 (4 °C 1 h) NA

Table 3. Effect of Various Metal Ions and Chemical Reagents on PulGT Activity relative activity (%)c metal-ion compounds none CaCl2 MgCl2 CuCl2 CoCl2 ZnSO4 reagents none EDTA TritonX-100 DTT α-cyclodextrin urea

a

The pullulanase from different sources retained more than 80% relative activity at corresponding pH. bThe pullulanase from different sources retained more than 70% residual activity at corresponding pH. The comments in brackets are measurement conditions of pH stability.

1 mM

5 mM

10 mM

100.0 100.0 100.0 182.52 ± 0.042 34.03 ± 0.006 2.55 ± 0.009 151.18 ± 0.028 180.62 ± 0.139 108.39 ± 0.181 0.00 ± 0.003 1.67 ± 0.001 1.11 ± 0.012 2.64 ± 0.005 0.00 ± 0.006 0.60 ± 0.003 0.00 ± 0.002 0.00 ± 0.002 0.79 ± 0.005 concentration relative activity (%)c 5 mM 0.1% 1% 10 mM 1% 2M

100 0.00 ± 0.001 117.01 ± 0.003 130.67 ± 0.015 235.87 ± 0.019 0.46 ± 0.010 0.00 ± 0.011

c The data represent the mean of three experimental repeats with SD of ±5%.

The hydrolysates of enzyme reactions were detected by TLC and HPLC. To facilitate identification, we used known sugar solutions (maltose and maltotriose) as standards (Figure 3d). TLC and HPLC results indicated that the hydrolyzed product from pullulan was maltotriose, not panose or isopanose (Figure 3b,c). The substrate and product profiles (analyzed by TLC and HPLC) indicated that PulGT specifically attacks α-1,6linkages of branched oligosaccharides. In summary, PulGT from G. thermocatenulatus is a novel thermostable temperature type I pullulanase and has high potential for industrial applications. According to the nonlinear regression curves, the kinetic parameters (Km and Vmax) of PulGT toward pullulan were measured to be 22.4 mg/mL and 0.079 g/(L·min), respectively. Design, Construction, and Purification of Truncated or Spliced Enzymes. Computational analysis of the 2WAN sequence revealed that it comprises 921 amino acids with a predicted molecular mass of 102.1 kDa. A three-dimensional model (Figure 4a,c) of 2WAN showed that this protein consisted of six domains characteristic of type I pullulanases of the GH13 family. The characteristic domains were recognized as a CBM41 domain, an X25 domain, an X45 domain, a CBM48 domain, and an amylase catalytic domain (Figure 4c).26 It is said that these N-terminal domains can bring pullulanases to the vicinity of starch substrates, allowing the substrate-binding sites in the AmyC domain to readily splice the substrate and hydrolyze α-1,6-glycosidic bonds. In order to obtain a higher yield and improve the application of this enzyme, protein engineering based on domain splicing was carried out in our study. Furthermore, previous research showed that the N-terminal domain (CBM41) of B. deramificans was disordered and the domain structure could not be solved entirely, and truncation could improve its thermal stability.29 We tried to provide a foundation for the properties of domains and domain evolution for further studies, because PulGT contains 85 amino acids of an uncharacterized domain at its N-terminal region (Figure 4b). Schematic representations of pullulanase and truncated enzyme variants are shown in Figure 4c.

thermophilus HB27,16 and T. neapolitana.17 Furthermore, PulGT retained superior enzyme activity (over 80% activity) at pH 5.0−7.0 (Figure 2c), indicating that it has a wider range of pH adaptability than pullulanase from G. kaustophilus,32 Anoxybacillus sp. LM18-11,35 and T. thermophilus HB27.16 Compared with pullulanase from B. acidopullulyticus, PulGT showed higher activity in near-neutral pH. PulGT still retained more than 70% residual activity after incubation for 60 min at pH 5.0−7.0 (Figure 2d). These enzymatic properties are favorable for industrial use. The effects of different metal ions and chemical reagents on the enzyme were examined in pH 6.5 citrate buffers at 70 °C for 20 min. It has been reported that Ca2+ not only activates pullulanase activity17 but also enhances their thermostability.37 NCBI Conserved Domain Search (CDSearch) analysis predicted that three calcium-binding sites exist in PulGT: D280, E286, and E306. In the present study, the presence of Ca2+ affected enzyme activity. Except for Mg2+ and lower concentrations of Ca2+, other metal ions showed significant inhibitory effects. Furthermore, enzyme activity was greatly inhibited by ethylene diamine tetraacetic acid (EDTA), α-cyclodextrin, and urea, but activated by Triton X100 and dithiothreitol (DTT) at certain concentrations (Table 3). Substrate Specificity, Hydrolysis Properties, and Kinetic Parameters of PulGT. Consistent with previous reports, PulGT showed broad substrate specificity for various polysaccharides that have α-1,6-glycosidic bonds in their structures, such as pullulan, amylopectin, and soluble starch.17,31,38 The hydrolytic specificity of PulGT toward various substrates was determined. PulGT displayed highest specific activity to pullulan followed by soluble starch (34.66%). The hydrolytic activity of amylopectin was only 18.88%, but it exhibited no activity toward amylose, glycogen, or γ-cyclodextrin (Figure 3a). Hence, our results revealed that PulGT can hydrolyze pullulan, soluble starch, and amylopectin, all of which contain α-1,6-glycosidic linkages, but it has no activity toward α-1,4-glycosidic linkages, suggesting that PulGT belongs to type I pullulanase. F


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Figure 3. Substrate specificity and analysis of hydrolyzed products. (a) Relative activity on different substrates. (b) TLC analysis of hydrolysis products with pullulan as the substrate. Lane G2: maltose; Lane G3: maltotriose; Lane 1: pullulan with 1 U PulGT for 60 min; Lane 2: pullulan with 5 U PulGT for 60 min. (c) HPLC analysis of pullulan hydrolysates. (d) HPLC analysis of maltose and maltotriose standards.

Figure 4. Protein structure and diagrammatic sketch of wild-type and mutant proteins. (a) The three-dimensional structure of 2WAN (Protein Data Bank, PDB). (b) The three-dimensional structure of PulGT as predicted by the SWISS-MODEL server. (c) Schematic representation of pullulanase and enzyme variants. Bars: green, CBM41 domain; yellow, X45a domain; blue, X25 domain; orange, X45b domain; dark gray, CBM48 domain; purple (2WAN)/pink (PulGT), GH13 superfamily catalytic domain; light gray, the N-terminal 85 amino acids of PulGT. 730T is the mutant protein lacking the N-terminal 85 amino acids. 730T-U1 is the N-terminus of 730T spliced with CBM41 and X45-X25 domains. 730T-U2 is the Nterminus of 730T spliced with X45-X25 domains. G


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Figure 5. Enzymatic properties of pullulanase mutants. Optimum temperature (a), optimum pH (b), thermostability (c) and (d) of the purified mutants. Line: black (■), PulGT; red (●), 730T; blue (▲), 730T-U2. Enzyme activity was assayed at different temperatures (40−80 °C) in 100 mM sodium phosphate buffer (pH 6.5). Effect of pH on PulGT (730T and 730T-U2) activity at 70 °C (65 °C) in buffers ranging from pH 5.0−8.0. Thermostability of the mutant pullulanases was determined by measuring residual activity after the enzyme was treated at 60 °C (c) or 70 °C (d) in 100 mM sodium phosphate buffer (pH 6.5) for different durations. Each value represents the mean and SD of triplicate experiments.

activity after incubation for 3 h at 60 °C (Figure 5c), but the thermal stabilities of 730T and 730T-U2 declined rapidly at 65 °C (Figure 5d). Splicing the X45-X25 domain did not influence protein expression, but it negatively affected thermal stability. To further evaluate the effect of truncation or combination at the N-terminus on substrate affinity and catalytic efficiency of the enzyme, kinetic analysis of the mutant was conducted with pullulan as the substrate under each optimum condition. As shown in Table 4, the Km values of 730T and 730T-U2

The genes encoding the N-terminal truncated or spliced pullulanases (730T, 730T-U1, and 730T-U2) were cloned into the vector pET-28a(+) and expressed in E. coli BL21 (DE3). The expression was attained by incubation with 0.1 mM IPTG at 20 °C for 20 h. The ExPASy compute MW program algorithm estimated the molecular weights of 730T, 730T-U1, and 730T-U2 as 71.1, 103.7, and 91.2 kDa, respectively. As shown in SI Figure S4, the experimental data were consistent with the theoretical prediction. We found that PulGT could exhibit almost entirely soluble expression, but inclusion bodies were readily formed after truncating 85 amino acids from its Nterminus (SI Figure S4b). This indicated that the N-terminal 85 amino acids of PulGT were responsible for ensuring secretory protein folding and maintaining its stability. Experiments showed that the protein expression levels of 730T-U1 were obviously lower than those of PulGT, and the relative expression amount of 730T-U2 compared with that of PulGT was not significantly different. A recent study found that proteins with high molecular weights and complicated structures have a high tendency to form inclusion bodies and have a low secretion ratio.39 These results agree with our experimental results. We did not perform enzymatic characterization of 730T-U2, which formed a large number of inclusion bodies, leading to difficulties in purification. The optimum temperature of 730T and 730T-U2 was 65 °C, which was below the optimum temperature of the wild-type protein, but the relative activity of 730T was higher than that of PulGT at 60 °C (Figure 5a). The optimum pH of the mutant proteins did not significantly change at the optimum temperature (Figure 5b). The variants could retain more than 80% of residual

Table 4. Protein Properties and Kinetic Parameters of the Wild-Type Pullulanase and the Truncated or Spliced Mutants enzyme

amino acids

molecular weight (kDa)

Km (mg/mL)

Vm (g/(L·min))

PulGT 730T 730T-U2

718 633 823

80.37 71.1 91.2

22.4 17.9 32.9

0.079 0.051 0.087

were 17.9 and 32.9 mg/mL, respectively, which are 0.81- and 1.41-fold higher than that of the PulGT enzyme. 730T exhibited lower Km value than the wild-type enzyme. Thus, N-terminal truncation of pullulanase may make the substrate more accessible to the active center to improve catalytic efficiency. On the other hand, the Km value of 730T-U2 was higher than that of the wild-type enzyme, suggesting a decline in catalytic efficiency. N-terminal insertion also reduced the affinity of enzymes and substrates with higher Km values, possibly because of the high molecular weight domain. In addition, H


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Figure 6. TLC and HPLC analysis of maize starch fermentation product. (a) TLC analysis of hydrolysis products of 2WAN with pullulan as the substrate at pH 6.0. Lane G1: glucose; Lane G3: maltotriose. Pullulan with 1 U 2WAN for 0 min (lane 1), 20 min (lane 2), 40 min (lane 3), and 60 min (lane 4). (b) TLC analysis of hydrolysis products of PulGT with pullulan as the substrate. Lane G1: glucose; Lane G3: maltotriose. Pullulan with 1 U PulGT for 0 min (lane 1), 20 min (lane 2), 40 min (lane 3), and 60 min (lane 4). (c) HPLC analysis of corn starch fermentation product. Red line: PulGT; Blue line: 2WAN. The coordinate axis of time and signal peak has been staggered, respectively.

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truncated or spliced mutants had higher relative activities at 65 °C, which would be more useful for industrial applications. The wide pH range did not change, suggesting the possibility of application of these enzymes in other areas. Analysis of Maize Starch Saccharification Product. The hydrolysis products when 2WAN and PulGT hydrolyzed pullulan for 0, 20, 40, or 60 min at 65 °C in pH 6.0 were analyzed by TLC. We found that PulGT could completely hydrolyze pullulan to maltotriose in 60 min, whereas intermediate products were generated after 20 and 40 min of the reaction (Figure 6b). However, 2WAN, which has low enzyme activity at pH 6.0, could hardly react with pullulan (Figure 6a). Thus, we used PulGT and 2WAN for maize starch saccharification at pH 6.0, which is the natural pH of liquefied maize starch at 65 °C. The HPLC spectrum of the saccharification products is shown in Figure 6c. Based on the peak area, we concluded that PulGT could hydrolyze more dextrin to produce more polysaccharide, maltotriose, maltose, and glucose without adjusting the pH value to 4.5. When maize starch is fermented to produce syrup or resistant starch, the natural pH is about 6.0 after high-temperature-resistant αamylase is used for liquefaction. 2WAN exhibits a greater loss of enzyme activity under this pH condition.8,25 2WAN requires a pH change to 4.5−5.5 after saccharification, thereby increasing energy consumption and hydrolysis time. However, PulGT, with a high activity at pH 6.0, could directly be applied to saccharification without adjusting the pH value. Therefore, its industrial use can reduce cost, improve efficiency, and shorten the time required for hydrolyzing maize starch.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.8b03331. Bacterial strains, plasmids and proteins used in this study, primers used to prepare truncated pullulanase genes, purification of PulGT by Ni-NTA affinity chromatography, plasmid profile of wild type and mutant pullulanases, nonlinear regression curves of wild type and mutants, SDS-PAGE analysis showing recombinant PulGT protein production in E. coli, and SDS-PAGE analysis of wild-type and mutant pullulanase protein expression in E. coli (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*Tel.: +86-21-64251803. Fax: +86-21-64251803. E-mail: [email protected]. *Tel.: +86-21-64252078. Fax: +86-21-64252078. E-mail: [email protected]. ORCID

Lingmeng Li: 0000-0002-6630-6788 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was financially supported by the National Natural Science Foundation of China (No. C31570795), the Shanghai International Science and Technology Cooperation I


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Project (No. 14520720500), the Minhang District Leading Talent Project (No. 201541), and the Shanghai Talent Development Project (No. 201531).

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N-Terminal Domain Truncation and Domain Insertion-Based

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