Identification of a novel type I pullulanase from Fervidobacterium nodosum Rt17-B1, with high thermostability and suitable optimal pH

Journal Pre-proofs Identification of a novel type I pullulanase from Fervidobacterium nodosum Rt17-B1, with high thermostability and suitable optimal pH Yang Yang, Yingying Zhu, Joy Ujiroghene Obaroakpo, Shuwen Zhang, Jing Lu, Lan Yang, Dawei Ni, Xiaoyang Pang, Jiaping Lv PII: DOI: Reference:

S0141-8130(19)37464-1 https://doi.org/10.1016/j.ijbiomac.2019.10.112 BIOMAC 13613

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International Journal of Biological Macromolecules

Received Date: Revised Date: Accepted Date:

15 September 2019 9 October 2019 11 October 2019

Please cite this article as: Y. Yang, Y. Zhu, J.U. Obaroakpo, S. Zhang, J. Lu, L. Yang, D. Ni, X. Pang, J. Lv, Identification of a novel type I pullulanase from Fervidobacterium nodosum Rt17-B1, with high thermostability and suitable optimal pH, International Journal of Biological Macromolecules (2019), doi: https://doi.org/ 10.1016/j.ijbiomac.2019.10.112

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Identification of a novel type I pullulanase from Fervidobacterium nodosum Rt17-B1, with high thermostability and suitable optimal pH

Yang Yanga, Yingying Zhuc, JOY UJIROGHENE OBAROAKPOa, Shuwen Zhanga, Jing Lua, Lan Yanga, Dawei Nic, Xiaoyang Panga,b,*, Jiaping Lva,*

a

Institute of Food Science and Technology, Chinese Academy of Agricultural Science, Beijing

100193, China. b State

Key Laboratory of Dairy Biotechnology, Shanghai Engineering Research Center of Dairy

Biotechnology, Dairy Research Institute, Bright Dairy Company Ltd., Shanghai 200436, China c

State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu

214122, China.

* Corresponding authors: [email protected], Tel: (8610)62815542 [email protected], Tel: (8610)62819421

Abstract Pullulanase could be used in many industrial processes due to its ability to hydrolyze α-1,6-glucosidic linkage. During the use of high temperature conditions in industrial production, pullulanase requires high resistance of heat. In this study, a novel type I pullulanase from Fervidobacterium nodosum Rt17-B1 (FN-pullulanase) with a suitable optimal pH and thermostability was discovered. Sequence analysis of FN-pullulanase showed that the enzyme had the typical motif of type I pullulanase (YNWGYDP). The recombinant FN-pullulanase, expressed in Escherichia coli, was purified as a single band on SDS-PAGE with a molecular mass of about 95 kDa. The enzyme showed optimum activity at pH 5.0 and 80 °C, and its specific activity was 25.93 U/mg. FN-pullulanase also exhibited good pH stability and thermostability. More than 80% of its initial activity was retained after incubated on ice at pH 3.5-9.0 for 24 h. Its half-life at 65 °C was 115.5 h. The enzyme could completely convert pullulan to maltotriose, as well as hydrolyze soluble starch or amylopectin to maltose, maltotriose, maltotetraose, maltopentaose and maltohexaose (G2-G6). Generally, this study identified a novel FN-pullulanase with both high thermostability and suitable optimum pH, which had the potential to be used in starch conversion process. Keywords: Pullulanase; Thermostability; Heterologous expression; Characterization;

Introduction Because various products processed from starch can be widely used in a lot of subsequent industrial production, including food [1], nutraceutical [2], material [3], pharmaceutical [4] and detergent industries [5], starch is regarded as a major industrial raw material. Most starches used in industries contain about 75% to 85% of amylopectin [6]. Amylopectin is a highly-branched polysaccharide with α-1,4-glucosidic linkages in the glucan chain and α-1,6-glucosidic linkages at the branch points after every 20 to 30 glucose units [7]. So, there are approximately 2.5% to 4.25% of α-1,6-glucosidic bonds in starches for industrial use. Due to the inefficiency of glucoamylase, as well as the inability of α-amylase and β-amylase to hydrolyze α-1,6-glucosidic bonds, a kind of barrier to the complete hydrolysis of starches occurs [5]. Pullulanase, belonging to the direct debranching enzyme, catalyzes the hydrolysis of pullulan to maltotriose as a single production [8]. According to the catalytic specificity, pullulanase can be divided into type I pullulanase and type II pullulanase. Type I pullulanase (EC 3.2.1.41) can specifically hydrolyze α-1,6-glucosidic bonds [9], while type II pullulanase (EC 3.2.1.1/41), also known as amylopullulanase, can hydrolyze α-1,6-glucosidic bonds as well as α-1,4-glucosidic bonds [10]. Compared with isoamylase, pullulanase has many advantages, which makes pullulanase more suitable for saccharification process: pullulanase has greater temperature stability and more β-amylase compatible pH range [5]; pullulanase is able to hydrolyze substrates of shorter side chains, which makes maximum use of starch [11]; the activity of isoamylase might be inhibited by β-amylase, maltotriose and maltotetraose which exists in saccharification [5,12]. A number of studies had shown that in many industrial starch conversions processing by a combination of pullulanase and glucoamylase, there is a consequent increase in the maximum concentration of glucose by about 2% and a reduction in the consumption of glucoamylase by 60%. Furthermore, the saccharification time could also be shortened (from 80 h to 30 h), while the concentration of substrate in saccharification could be increased from 25% - 30% to 30% - 40% [6,13]. Similarly, the combined use of pullulanase and β-amylase increased maltose production by at least 20% [6,14]. Additionally, pullulanase had been widely used in the production of beer [5], resistant starch [15], high-amylose starch [16] and biodegradable packing materials [17], and pullulanase could also be used in cyclodextrins production [18], detergent [19], baking industry

[20] and plaque control agent [21]. There are diversified demands for the characteristics of pullulanase due to its wide applications. Starch saccharification is the primary application of pullulanase, and the conventional process of saccharification is ordinarily carried out at 55-65 °C and pH 4.5-5.5. Therefore, the use of pullulanase with thermostability and acid resistance is desirable [22]. A large number of pullulanases, especially from thermophilic and hyper-thermophilic microorganisms, have been characterized. Pullulanases from Bacillus acidopullulyticus [23], Fervidobacterium pennavorans Ven5 [24], Bacillus thermoleovorans US105 [25], Klebsiella aerogenes W70 [26,27], Caldicellulosiruptor saccharolyticus [28], Anaerobranca gottschalkii DSM 13577 [29], Thermotoga maritima MSB8 [30,31], Thermus thermophilus HB8 [32], Bacillus sp. CICIM 263 [33], Thermotoga neapolitana KCCM 41025 [34] and Bacillus flavocaldarius KP 1228 [35] have been studied at genetic levels. However during production, most of these identified type I pullulanases were unsuitable in starch conversion processes because only a few of them with optimal pH ranges of 4.5 – 5.5 also had good heat resistance. Therefore, it is necessary to invest in more researches to screen new enzymes with excellent properties or improve their properties by mutation. This study therefore aimed to identify a novel pullulanase from Fervidobacterium nodosum Rt17-B1, expressed in Escherichia coli and purified to electrophoresis homogeneity. Its enzymatic properties were also investigated in detail. To the best of our knowledge, pullulanase from a F. nodosum strain has never been previously reported. The characteristic thermostability and acid resistance of the identified pullulanase could make it a potential candidate in starch conversion.

2. Materials and methods 2.1 Cloning and expression of FN-pullulanase A thermostable type I pullulanase from Fervidobacterium pennavorans Ven5 was used as a template to search for putative pullulanase in National Center for Biotechnology Information (NCBI). A full-length nucleotide sequence from Fervidobacterium nodosum Rt17-B1 (accession number of NC_009718.1) encoding a putative pullulanase (accession number of WP_011994577.1) was obtained from GenBank database. The signal peptide of the putative pullulanase was

predicted by an online tool (http://www.cbs.dtu.dk/services/SignalP/). After removal of the signal peptide-encoding sequence, the gene was synthesized by BGI Genomics Technology Co., Ltd (Beijing, China) and cloned into pET-22b(+) at NdeI/XhoI sites, to yield pET-22b(+)-pulF. The recombinant plasmid for expression was transformed into E. coli BL21(DE3). The recombinant strain was inoculated in LB medium containing 100 μg/mL ampicillin, and then cultured at 37 °C and 200 rpm. When the OD600nm was reached (0.6-0.8), 1 mM final concentration of isopropyl-β-D-1-thiogalactopyranoside (IPTG) was added and continuously cultured at 28 °C and 200 rpm for 6 h. The cells were harvested by centrifugation for 10 min at 6000 rpm and 4°C, and resuspended in 50mM sodium phosphate buffer (pH 7.0) containing 100mM NaCl.

2.2 Purification of recombinant protein The suspended cells were disrupted on ice by sonication using a Vibra-CellTM 72405 Sonicator for 15min (1 s pulse and 3 s pulse off). Cell debris was subsequently removed by centrifugation for 15 min at 10000×g and 4 °C. The supernatant was collected as crude enzyme and filtered through a 0.45 μm filter. Then enzyme solution was loaded onto a column packed with Ni2+ Sepharose High Performance affinity resin (GE, Healthcare, Sweden) for immobilized metal affinity chromatography (IMAC). To pre-equilibrate the column and wash the unbound proteins, binding buffer (50 mM sodium phosphate buffer, 500 mM NaCl, pH 7.0) and washing buffer (50 mM sodium phosphate buffer, 500 mM NaCl, 100 mM imidazole, pH 7.0) were used respectively. The recombinant pullulanase was collected by elution buffer (50 mM sodium phosphate buffer, 500 mM NaCl, 500 mM imidazole, pH 7.0). The collection was dialyzed twice in dialysate A (50 mM sodium phosphate buffer, 10 mM ethylenediaminetetraacetic acid (EDTA), pH 7.0) at 4 °C, to remove metal ions. Further dialysis was done twice in dialysate B (50 mM sodium phosphate buffer, pH 7.0) at 4 °C, to remove EDTA.

2.3 Determination of molecular mass and protein concentration The molecular mass and purity of the recombinant enzyme were estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using SurePAGEᵀᴹ, Bis-Tris, 8%, 10 wells (GenScript, Nanjing, China). For protein staining, coomassie brilliant blue R250 was used.

Protein concentration was determined according to the bicinchoninic acid (BCA) assay using BCA Protein Assay Kit from TIANGEN BIOTECH (Beijing, China).

2.4 Enzyme assay Pullulanase activity was determined to measure the release of reducing sugars from pullulan. The reactions were carried out in sodium acetate buffer (0.2 M, pH 5.0) with 0.5% (w/v) pullulan at 80 °C. 20 μL of 1% (w/v) pullulan in sodium acetate buffer (0.2 M, pH 5.0) was preheated at 80 °C for 5 min, and then 20 μL of appropriately diluted enzyme solution was quickly added. After 10 min, 60 μL of 3,5-dinitrosalicylic acid solution was immediately added to terminate the reaction. After the mixture coloration at 99.9 °C for 10 min, it was then diluted with deionized water to 200 μL. Finally, 100 μL of the diluted mixture was added to 96-well plates, and the absorbance was measured at 540nm using the Spark Multimode Microplate Reader (Tecan Trading AG, Switzerland). One unit of pullulanase activity was defined as the amount of enzyme required to release 1 μmol of reducing sugars (with glucose as the standard) from pullulan per min under the assay conditions specified.

2.5 pH and temperature conditions on FN-pullulanase activity The effect of pH on activity of pullulanase was examined using 1% (w/v) pullulan in different buffers at 80 °C. Two buffer systems (sodium acetate(0.2M, pH 3.5-6.0) and sodium phosphate (0.2M, pH 6.0-7.5)) were used. Similarly, the effect of temperature (at a range of 4 °C to 90 °C) on the activity of pullulanase was investigated using 1% (w/v) pullulan in sodium acetate buffer (0.2 M, pH 5.0). The activities at each pH condition and temperature were expressed as relative value to the highest activity (100%).

2.6 pH and temperature conditions on FN-pullulanase stability To study its pH stability, pullulanase was pre-incubated on ice for 30 min or 24 h in different buffers(0.2 M) with pH values ranging from 3.5 to 9.0, and its residual activity was measured under standard conditions. The thermal stability of the enzyme was investigated by incubating it at temperatures of 65 °C, 70 °C, 75 °C and 80 °C, at intervals of 12 h, 2 h, 10 min and 2 min

respectively in sodium acetate buffer (0.2 M, pH 5.0). The residual activity of enzyme was immediately determined at each time interval under standard conditions. The initial activity of the enzyme in the absence of incubation conditions was defined as 100%.

2.7 Metal ions and chemical agents conditions The chlorides of magnesium ion (Mg2+), calcium ion (Ca2+), manganese ion (Mn2+), ferrous ions (Fe2+), copper ion (Cu2+), cobalt ion (Co2+), nickel ion (Ni2+), cadmium ion (Cd2+), barium ion (Ba2+), zinc ion (Zn2+), ferric ion (Fe3+) and chromium ion (Cr3+) with final concentrations of 1 mM, 5 mM and 10 mM were simultaneously used to determine the effects of metal ions on the enzyme activity. The final concentrations of DTT were 1mM, 5mM and 10mM, and urea were 1mM, 10mM, 100mM and 1M. In order to ensure consistency of the ions and chemicals in the enzyme reaction system, twice their final concentration was added to the enzyme solution and was properly diluted with deionized water. The mixture was incubated on ice for 1 h, and the relative enzyme activity as a function of each ion or chemical agent, was determined under standard conditions.

2.8 Kinetic parameters Various concentrations of pullulan (0.5-10 mg/mL) in sodium acetate buffer (0.2 M, pH 5.0) were used as substrates to investigate the kinetic parameters of FN-pullulanase. Its enzymatic activity was measured at standard conditions, and the kinetic parameters were determined using the Lineweaver-Burk equation.

2.9 Products analysis by Thin-layer chromatography (TLC) The product formations of pullulan, soluble starch and amylopectin were analyzed using silica gel thin-layer chromatography. The moderate pullulanase was separately incubated with 0.5% (w/v) pullulan, 0.5% (w/v) soluble starch and 0.5% (w/v) amylopectin at 75 °C. After 6 h, the mixture with 0.5% pullulan was boiled for 10 min to stop the reaction; and α-glucosidase from yeast was added. The mixture was incubated at 37 °C for another 6 h. All other incubated mixtures were boiled for 10 min. The products were loaded onto silica gel and put in developing system

(n-buty alcohol/ethanol/water 5:3:2) to separate the samples. After 3 h, the gel plate was taken out, air dried, and then sprayed with 10% (V/V) sulphuric acid. The gel plate was briefly placed in hot oven with 105 °C for 5-10 min and viewed under ChemiScope 6000 Series (Clinx Science Instruments, China). Glucose (G1), maltose (G2), maltotriose (G3), maltotetraose (G4), maltopentaose (G5) and maltoheptaose (G7) were purchased from Solarbio (Beijing Solarbio Science & Technology Co., Ltd.) and used as standard markers.

3. Results and discussion 3.1 Gene cloning, purification and molecular mass of FN-pullulanase In this study, a signal peptide (MVNFSKKALVFLFLLAVVFSYA) was found in putative pullulanase. The gene fragment without a signal peptide-encoding sequence was cloned and expressed in E.coli BL21(DE3). The recombination pullulanase was expressed in the form of a fusion protein with 6× His-tag and had a theoretical molecular mass of 94.1 kDa (https://web.expasy.org/compute_pi/). After purification by nickel affinity chromatography, the expressed protein showed a single band on the gel and its molecular mass was estimated as 95 kDa which was consistent with the theoretical molecular weight (Fig. 1).

3.2 Sequence analysis of FN-pullulanase Nineteen reported sequences of type I pullulanases were collected, which included the pullulanase from Thermus thermophiles HB8 [32], Geobacillus thermoleovorans US105 [25], Bacillus stearothermophilus TRS128 [36,37], Anoxybacillus sp. LM18-11 [38], Anoxybacillus sp. LM14-2 [39], Anoxybacillus sp. Sk3-4 [40], Bacillus subtilis 168 [41], Paenibacillus barengoltzii [42], Bacillus sp. CICIM 263 [33], Exiguobacterium acetylicum YH5 [43], Bacillus acidopullulyticus [44], Anaerobranca gottschalkii DSM 13577 [29], Thermotoga neapolitana KCCM 41025 [34], Thermotoga maritima MSB8 [30,31], Fervidobacterium pennivorans Ven5 [24], Caldicellulosiruptor saccharolyticus [28], Shewanella arctica 40-3 [45], Klebsiella pneumoniae [46], and Bacillus flavocaldarius KP 1228 [35]. The phylogenetic tree of FN-pullulanase in relation with the nineteen reported pullulanases was shown in Fig. 2A. It showed that FN-pullulanase had a closer relationship with the enzymes

from B. acidopullulyticus, A. gottschalkii DSM 13577, T. neapolitana KCCM 41025, T. maritima MSB8 and F. pennivorans Ven5, marked with a yellow background. The comparison of the amino acid sequences of identified pullulanases was shown in Fig. 2B. It was observed that FN-pullulanase had the highest amino acid identity (86%) with F. pennivorans pullulanase. The increase was closely followed by the sequence from T. maritima MSB8, which had a high amino acid homology with FN-pullulanase at 60%. Among the pullulanases with crystal structure, the sequence from B. acidopullulyticus had the highest amino acid homology with FN-pullulanase at 41%. For a better understanding of the sequence characteristics of FN-pullulanase, five pullulanases which had a much closer relationship with it, were selected for sequence alignment (Fig. 1S). The result of multiple sequence alignment showed that there were four typically amylolytic conserved regions [47] in the FN-pullulanase sequence (summarized in Table 1). Furthermore, YNWGYDP (a typical motif of type I pullulanase) [48], was also found in FN-pullulanase and indicated its classification as type I pullulanase. All type I pullulanases belong to the glycoside hydrolase family 13 (GH13), which adopts a (α/β)8-barrel fold as the catalytic domain. Typically, in catalytic A domain, β4-aspartate, β5-glutamate and β7-aspartate are considered as the catalytic sites [49,50]. Therefore, Asp-515, Glu-544, Asp-631 were inferred as the catalytic sites in FN-pullulanase.

3.3 Effect of pH and temperature on enzyme activity As shown in Fig. 3A, the relative enzyme activity increased with an increase in pH value. A gradual decrease was observed in the enzyme activity at an increased pH (>5.0). The highest activity of the enzyme FN-pullulanase in 0.2 M sodium acetate buffer was therefore recorded at pH 5.0. The result (Fig. 3B) showed that between temperature ranges of 4 °C to 60 °C, the enzyme activity increased slowly with the increasing temperature. The activity increased consistently as the temperature kept rising, and its optimal activity was reached at 80 °C. A further increase above 80 °C led to a sharp decline in the activity of the enzyme. Thus, the specific activity of the purified pullulanase in 0.2 M sodium acetate, was 25.93 U/mg at 80 °C. The properties of type I pullulanase (optimal pH, optimal temperature and specific activity)

from various sources were summarized in Fig. 4. It could be seen from the figure that the optimal pH for varying pullulanases ranged from 5.0 to 8.0, and mostly about 6.0. The optimal temperatures of reported pullulanases were between 35-90 °C. Pullulanase from T. maritima [30,31,51] had the optimal temperature at 90 °C and was closely followed by those from T. neapolitana [34], F. pennivorans [24,52] and F. nodosum (this study) with optimal temperature slightly lower than 90 °C. Usually, saccharification occurs at pH 4.5-5.5 and 55-65 °C (or at higher temperatures). Therefore, when compared with other pullulanases, FN-pullulanase not only had a greater optimal temperature, but also had a suitable optimal pH. However, our result showed that specific activities greater than that of FN-pullulanase was observed in pullulanases from Klebsiella pneumoniae [53], Anoxybacillus sp. LM18-11 [38] and Bacillus acidopullulyticus [54–56], whose specific activities were 7000 U/mg, 750 U/mg and 219 U/mg, respectively.

3.4 Effect of pH and temperature on the enzyme stability The effect of pH (Fig. 5A) and temperature (Fig. 5B) on stability of FN-pullulanase were studied. After incubation on ice for 30 min or 24 h at pH 3.5-9.0, more than 80% of initial activity was retained, which inferred that FN-pullulanase had high pH stability. Although the residual activity at 24 h incubation was lower than that of 30 min, it could be observed from the figure that the enzyme activity decreased very slowly with an increase in incubation time. Similarly, it was observed that different pH conditions applied in this study had little effect on the residual activity. The thermostability was investigated at 65 °C, 70 °C and 75 °C for varying time intervals. FN-pullulanase exhibited a good heat resistance at 65 °C and about 74% of its initial activity was retained after incubation for 47 h. As shown in Fig. 4B, the decay constants (kd) at 65 °C, 70 °C and 75 °C were 0.006 h-1, 0.0644 h-1 and 1.2884 h-1, respectively. Using the equation t1/2=ln(2)/kd, the half-life (t1/2) at 65 °C, 70 °C and 75°C, was calculated as 115.5245 h, 10.7632 h and 0.6140 h, respectively. The enzymatic properties (pH stability, thermostability and metal ions effect) of FN-pullulanase as well as other type I pullulanases were outlined in Table 2. Relatively, almost all enzymes applied in this study had good stability in a wide range of pH, and FN-pullulanase is not exempted. Also, our result illustrated that the most heat-resistant pullulanase was obtained from T.

maritima [30,31,51], whose t1/2 was 3.5 h at 90 °C. Besides, the enzymes from B. flavocaldarius [35,57], F. pennivorans [24,52] and T. neapolitana [34] also displayed good thermostability. Although FN-pullulanase was not the most heat-resistant (compared with other pullulanases), it had a t1/2 of 115.5 h at 65 °C, which implied that the t1/2 at 60 °C would be much longer than 115.5 h. Usually, the saccharification process lasts for more than 48-60 h at 60 °C, thus FN-pullulanase could fully meet the requirements of industrial application in terms of heat resistance. It is interesting to note that FN-pullulanase was completely inactivated after incubation at 80 °C for 2 min even though it had the highest activity at 80 °C (data not shown). We speculated that the thermostability of FN-pullulanase was enhanced by the presence of pullulan during the determination of enzyme activity. Yuzuru Suzuki et al. [57] found that when the substrate concentration increased from 1% to 5%, the optimal temperature of the pullulanase increased by 0-5°C, which provided a basis for our speculation.

3.5 Effect of metal ions and chemical agents The relative activity of the FN-pullulanase in the presence of various metal ions and chemical agents were listed in Table 3. The results showed that Co2+, Ba2+, Ca2+, Mg2+, Ni2+ could activate the activity of FN-pullulanase at all concentrations tested. On the contrary, Cu2+, Fe3+, Zn2+ and urea had an obvious inhibitory effect on FN-pullulanase. Among them, Cu2+ and Fe3+ completely inactivated the enzyme at all tested concentrations, while 1 mM Zn2+ completely inhibited the enzyme activity. In addition, at some concentrations, Fe2+, Mn2+, Cr3+ and Cd2+ exhibited inhibitory effect on FN-pullulanase, while they activated it in other concentrations. DTT, used as a reducing agent, significantly activated FN-pullulanase at concentrations of 5 mM and 10 mM. Also, in the presence of 1 M urea, the residual activity of FN-pullulanase was retained at about 50%, which implied that the enzyme was relatively tolerant to denaturants. Particularly, among FN-pullulanase activators, Co2+ displayed the strongest activation, which increased the relative activity to about 150% or more (≤ 226%). As shown in Table 2, with the exception of pullulanase from E. acetylicum [43], Cu2+ and Zn2+ inhibited almost all pullulanases. The pullulanases from B. flavocaldarius [35,57], F. pennivorans [24,52] and A. gottschalkii [29] did not require any metal ion as activator. Ca2+ and Mg2+ activated

most of the pullulanase except those from B. flavocaldarius [35,57] and E. acetylicum [43]. Co2+ acted as an activator of pullulanase from E. acetylicum [43], P. barengoltzii [42], B. stearothermophilus [36,37] and S. arctica [45], but inhibited pullulanase from T. maritima [30,31,51], B. flavocaldarius [35,57], T. neapolitana [34], A. gottschalkii [29] and Anoxybacillus sp. SK3-4 [40].

3.6 Products analysis and kinetic parameters The products of pullulan, soluble starch and amylopectin, under the action of FN-pullulanase, were analyzed using silica gel thin-layer chromatography (Fig. 6). Our results showed that in Lane 1, pullulan was completely converted to trisaccharide after catalysis of FN-pullulanase for 6 h. In order to determine the type of trisaccharide in Lane 1, the hydrolysate of pullulan and α-glucosidase were incubated together for another 6 h. Only glucose was detected in Lane 2, thus confirmed that the product of pullulan was maltotriose, and not panose or isopanose. Lane 3 and Lane 4 showed that both soluble starch and amylopectin produced G2, G3, G4, G5 and G6 after catalysis of FN-pullulanase for 12 h. By comparison, when starch was used as substrate, G2 and G3 were produced by the pullulanases obtained from F. pennivorans [24] and B. cereus [58], and G2, G3, G4 were produced by the pullulanase which was from Bacillus sp. CICIM 263 [33]. Furthermore, G2, G3, GO (oligosaccharides ≥ G8) were produced by the pullulanases obtained from E. acetylicum [43] and T. neapolitana [34]. When amylopectin was used as substrate, G1-G7 and GO were produced by the pullulanases obtained from A. gottschalkii [29]; while G2, G3 were produced by pullulanases obtained from Bacillus sp. CICIM 263 [33] and F. pennivorans [24]. Finally, G2, G3, GO were produced by pullulanases obtained from E. acetylicum [43] and T. neapolitana [34]. Although the products of pullulanases from various sources were slightly different, G2 and G3 were their common products. Compared to isoamylase, pullulanase could hydrolyze the smaller substrates (with side chains of 2- or 3-glucose units) [60]. Therefore, the formation of G2 and G3 also reflected the difference between pullulanase and isoamylase. In addition to product analysis, the kinetic parameters of FN-pullulanase were also determined. The Km and Vmax were computed to be 6.17±1.38 mg/mL and 37.95±9.23 μmol·min-1·mg-1, respectively.

4. Conclusion In summary, this study identified a novel type I pullulanase from Fervidobacterium nodosum. The sequence characteristics, enzymatic properties and product characteristics of FN-pullulanase were studied in detail. FN-pullulanase showed a more suitable optimal pH with a relatively high thermostability, thus exhibited the potential to act as a starch-converting enzyme especially in industrial processes. However, in the aspect of specific activity, FN-pullulanase was still inadequate when compared with pullulanases from K. pneumoniae, Anoxybacillus sp. LM18-11 and B. acidopullulyticus. Therefore, further studies are required to improve the specific activity of FN-pullulanase.

Acknowledgements This work was supported by the National Natural Science Foundation of China [grant number 31871833]; and National Key R&D Program of China [grant number 2017YFC1600903]. Ethical Statement This article does not contain any studies with human participants performed by any of the authors.

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Figure legents Figure 1. SDS-PAGE analysis. Lane M, protein marker; Lane 1, purified recombinant FN-pullulanase.

Figure 2. (A) Phylogenetic tree analysis of identified type I pullulanases from various microorganisms. The tree was built by the neighbor-joining method with MEGA7, and was showed as the formation of bootstrap consensus tree. (B) Comparison of the amino acid sequences of identified type I pullulanases from various microorganisms. The capital letters in the figure are abbreviations for microorganisms. FENO (F. nodosum), FEPE (F. pennivorans), THMA (T. maritima), THNE (T. neapolitana), ANGO (A. gottschalkii), BAAC (B. acidopullulyticus), PABA (P. barengoltzii), BAST (B. stearothermophilus), BASP (Bacillus sp. CICIM 263), EXAC (E. acetylicum), CASA (C. saccharolyticus), GETH (G. thermoleovorans), THTH (T. thermophiles), ANSK (Anoxybacillus sp. Sk3-4), ANLM18 (Anoxybacillus sp. LM18-11), ANLM14 (Anoxybacillus sp. LM14-2), BASU (B. subtilis), KLPN (K. pneumoniae), SHAR (S. arctica)

Figure 3. (A) Effect of pH on FN-pullulanase activity. (B) Effect of temperature on FN-pullulanase activity.

Figure 4. A summary of the optimum pH, optimum temperature and specific activity of type I pullulanases from various microorganisms. FN-pullulanase (red solid circle), pullulanases with known specific activity (green solid circles) and pullulanases with unknown specific activity (hollow circles) were shown. The size of each circle area represented the specific activity of each pullulanase. a. This study; b. Bacillus acidopullulyticus [54–56]; c. Paenibacillus barengoltzii [42]; d. Geobacillus thermoleovorans [25]; e. Thermus thermophiles [32]; f. Thermotoga maritima [30,31,51]; g. Fervidobacterium pennivorans [24,52]; h. Bacillus stearothermophilus

[36,37]; i.

Anoxybacillus sp. LM18-11 [38]; j. Klebsiella pneumoniae [53]; k. Bacillus subtilis 168 [41]; l.

Bacillus flavocaldarius [35,57]; m. Thermotoga neapolitana [34]; n. Bacillus sp. CICIM 263 [33]; o. Shewanella arctica [45]; p. Anaerobranca gottschalkii [29].

Figure 5. (A) Effect of pH on the enzyme stability, 30 min (square), 24 h (triangle). (B) Effect of temperature on the enzyme stability, 65°C (blue square), 70°C (red triangle) and 75°C (green circle).

Figure 6. Products analysis on TLC. Lane M, glucose (G1), maltose (G2), maltotriose (G3), maltotetraose (G4), maltopentaose (G5) and maltoheptaose (G7) were mixed as marker; Lane 1, hydrolysis products from pullulan by FN-pullulanase; Lane 2, the hydrolysis products from hydrolysate of pullulan by α-glucosidase; Lane 3, hydrolysis products from soluble starch in the presence of FN-pullulanase; Lane 4, hydrolysis products from amylopectin in the presence of FN-pullulanase.

Table 1. Regions conserved among type I pullulanases Ⅰ

YNWGYDP

Microbial sourcec

position

sequence

position

Ⅱ

sequence

Ⅲ

Ⅳ

position

sequence

position

sequencer

position

sequence

542

YGEPWGG

620

PEETINYVACHDNHTLWDK

F. nodosum

396

YNWGYDP

437

GIRVILDMVFPHT

510

DGFRFDQMGLMDa

B. acidopullulyticus

505

YNWGYDP

545

RIGVNMDVVYNHT

617

DGFRFDLMALLG

649

YGEPWTG

725

PSETINYVTSHDNMTLWDK

F. pennivorans

424

YNWGYDP

465

GIRVILDMVFPHT

538

DGFRFDQMGLMD

570

YGEPWGG

648

PQETINYVEVHDNHTLWDK

562

YGEPWGG

641

PEETINYVACHDNHTLWDK

T. neapolitana

416

YNWGYDP

457

DIGVIMDMVFPHT

530

DGFRFGQMGLIDb

T. maritima

416

YNWGYDP

457

GIGVIMDMVFPHT

530

DGFRFDQMGLID

562

YGEPWGG

641

PEETINYAACHDNHTLWDK

A. gottschalkii

446

YNWGYDP

487

GIRVIKDVVYNHT

559

DGFRFDLMALHD

591

YGEPWQA

666

PTESIVYVSCHDNLTLWDK

a

Underlined amino acid residues are activity sites of type I pullulanase. acid in bold fonts might be a database entry error. cAbbreviations and accession numbers of the sequences: F. nodosum (this study), Fervidobacterium nodosum (accession number WP_011994577.1); B. acidopullulyticus, Bacillus acidopullulyticus (PDB ID 2WAN), F. pennivorans, Fervidobacterium pennivorans (accession number AAD30387); T. neapolitana, Thermotoga neapolitana (accession number ACN58254); T. maritima, Thermotoga maritima (accession number CAA04522); A. gottschalkii, Anaerobranca gottschalkii (accession number AAS47565). Table 2. Comparison of pH stability, thermostability and metal ions effect between various type I pullulanases. bAmino

Half-life (min)c Enzymea

pH stabilityb 40 °C

45 °C

50 °C

55 °C

60 °C

65 °C

70 °C

75 °C

80 °C

85 °C

87 °C

90 °C

107 °C

Activators

Inhibitors

Reference

Co2+ Al3+ Zn2+ Mn2+ Fe3+ Cu2+ Fe2+

[30,31,51]

THMA

-

-

-

-

-

-

-

-

(360)

-

-

-

210

-

Li+ Na+ K+ Ca2+ Mg2+

BAFL

3-9 (15h, 25°C)

-

-

-

-

-

-

-

-

-

-

-

(10)

10

None

Cd2+ Pb2+ Zn2+ Cu2+ Fe3+ [35,57] Ni2+ FEPE THNE FENO ANGO

3.5-9 (24h, on ice) 4-10 (days, 4°C)

-

-

-

-

-

6931.5 -

645.8 1320

(240) 36.8 <10

120 88 -

44 -

2 -

-

-

Co2+ Ba2+ Ca2+ Mg2+ Ni2+

None Mg2+ Co2+

Ba2+

Ca2+

Ca2+ None

Co2+ Hg2+ Sn2+ Ba2+ Ca2+

Mg2+

Zn2+

Ni2+

Ni2+ Cu2+

Co2+

Mn2+

Cu2+

Cu2+

Fe3+

Zn2+

Co2+

Zn2+

Hg2+

Hg2+

[24,52] [34] This study

Cd2+

[29]

ANLM14

-

-

-

-

-

-

3360

-

-

-

-

-

-

-

-

-

[39]

BASP

4.5-9 (1h, 60°C)

-

-

-

-

-

-

(60)

-

-

-

-

-

-

Mg2+ Mn2+ Ca2+

Fe3+

[33]

ANLM18

-

-

-

-

4200

2880

90

-

-

-

-

-

-

-

-

2+

2+

[38] 4+

2+

Sr Ba NH Ni Mn ANSK

-

-

-

-

-

540

-

-

-

-

-

-

-

2+

K+ Na+ Mg2+

-

[40] Zn2+ Co2+ Cu2+ Fe2+ Fe3+

BAAC EXAC

4-8.5 (24h, 4°C) 4-10 (30min, 50°C)

-

-

(30)

-

34.9 -

-

-

-

-

-

-

-

-

-

-

2+

-

5.5-10.5 (30min, 50°C)

501

253

33

-

-

-

-

-

-

-

-

-

2+

2+

Co Mn Fe Zn Co2+

PABA

2+

Fe3+

Ni2+

Mg2+

[54–56]

2+

2+

Ca2+

Zn2+

Mg Cu Hg+

[43] Na+

-

[42] Mn2+ Cr3+ Fe2+ K+

Ba2+ Sn2+ Cu2+ Ag+

KLPN

5.5-12 (60min, 40°C)

(30)

-

-

-

-

-

-

-

-

-

-

-

-

-

-

[53]

BAST

6-8.5 (60min, 60°C)

-

-

-

-

-

(60)

-

-

-

-

-

-

-

Ca2+ Mn2+ Ba2+ Mg2+ Co2+ Fe2+

Zn2+ Cu2+

[36,37];

SHAR

5-11 (24h, on ice)

<15

-

-

-

-

-

-

-

-

-

-

-

-

Ca2+ Co2+

Fe2+ Ni2+

[45]

a

Abbreviations means type I pullulanase was from: THMA (Thermotoga maritima), BAFL (Bacillus flavocaldarius), FEPE (Fervidobacterium pennivorans), THNE (Thermotoga neapolitana), FENO (Fervidobacterium nodosum), ANGO (Anaerobranca gottschalkii), ANLM14 (Anoxybacillus sp. LM14-2), BASP (Bacillus sp. CICIM 263), ANLM18 (Anoxybacillus sp. LM18-11), ANSK (Anoxybacillus sp. SK3-4), BAAC (Bacillus acidopullulyticus), EXAC (Exiguobacterium acetylicum), PABA (Paenibacillus barengoltzii), KLPN (Klebsiella pneumoniae), BAST (Bacillus stearothermophilus), SHAR (Shewanella arctica). bpH stability was expressed as “stable pH range (incubation time, incubation temperature)”. cThermostability was reflected by half-life at different temperatures. The bracketed numbers in the table indicate that the enzyme was stable during the incubation period. For example, (60) means that the enzyme was stable after incubation at the corresponding temperature for 60 minutes. “-”, not reported. Table 3. Effect of metal ions and chemical agents on FN-pullulanase activity Relativity activity (%) Reagent

1 mM

5 mM

10 mM

100 mM

1000 mM

Co2+

226.14±0.68

172.76±5.85

152.24±5.06

-

-

Ba2+

163.05±3.64

146.86±2.31

140.15±6.26

-

-

Ca2+

146.58±7.12

148.86±0.55

140.68±0.43

-

-

Mg2+

129.56±0.89

111.95±5.84

121.13±1.37

-

-

Ni2+

124.17±5.04

109.55±1.92

101.98±1.94

-

-

Mn2+

110.7±5.28

87.94±6.52

129.89±0.87

-

-

Cd2+

152.2±3.96

116.21±2.19

93.18±1.54

-

-

Cr3+

128.84±3.06

91.58±2.64

65.1±1.72

-

-

Fe2+

59.01±5.34

93.32±1.32

160.58±2.85

-

-

Zn2+

28.34±1.09

24.43±1.54

ND

-

-

a

ND-not detectable.

Fe3+

NDa

ND

ND

-

-

Cu2+

ND

ND

ND

-

-

DTT

93.68±4.35

113.78±5.63

148.91±4.8

-

-

Urea

-

-

86.81±2.46

82.17±2.41

51.37±4.86

Figure S1. Sequence alignment between FN-pullulanase and reported type I pullulanases