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/lp/springer-journals/improving-the-thermostability-of-gh49-dextranase-aodex-by-site-QUB0LgqEGE
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- Springer Journals
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- Copyright © The Author(s) 2023
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- 2191-0855
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- 10.1186/s13568-023-01513-2
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Abstract
As an indispensable enzyme for the hydrolysis of dextran, dextranase has been widely used in the fields of food and medicine. It should be noted that the weak thermostability of dextranase has become a restricted factor for industrial applications. This study aims to improve the thermostability of dextranase AoDex in glycoside hydrolase (GH) family 49 that derived from Arthrobacter oxydans KQ11. Some mutants were predicted and constructed based on B-factor analysis, PoPMuSiC and HotMuSiC algorithms, and four mutants exhibited higher heat resistance. Compared with the wild-type, mutant S357P showed the best improved thermostability with a 5.4-fold increase of half-life at 60 °C, and a 2.1-fold increase of half-life at 65 °C. Furthermore, S357V displayed the most obvious increase in enzymatic activ- ity and thermostability simultaneously. Structural modeling analysis indicated that the improved thermostability of mutants might be attributed to the introduction of proline and hydrophobic effects, which generated the rigid opti- mization of the structural conformation. These results illustrated that it was effective to improve the thermostability of dextranase AoDex by rational design and site-directed mutagenesis. The thermostable mutant of dextranase AoDex has potential application value, and it can also provide references for engineering other thermostable dextranases of the GH49 family. Keywords Dextranase, Thermostability, Arthrobacter oxydans, Site-directed mutagenesis, GH49 dextrans are determined by their corresponding molec- Introduction ular weights (Naessens et al. 2005; Xue et al. 2022). For Dextran is a kind of high molecular α-glucan (Bhavani example, medium molecular weight dextrans could be and Nisha 2010). Its main chain is made up of D-glucose served as additives in the food industry, and low molecu- molecules linked by α-(1 → 6) glycosidic bonds, and lar weight dextrans could be used as plasma substitutes there are also possible α-(1 → 2), α-(1 → 3), or α-(1 → 4) in the medical field (Falconer et al. 2011; Kothari and bonds in a few branches (Díaz-Montes 2021). Dextran Goyal 2016). However, the low or medium molecular has broad application prospects in the food and phar- weight dextrans are obtained by hydrolysis of dextranase, maceutical industry, and the application areas of diverse which can specifically hydrolyze α-(1 → 6) glycosidic bonds of high molecular weight dextran (Khalikova et al. *Correspondence: 2005). It was found that different dextranases belonged to Zhen Wei multiple glycosidic hydrolase (GH) families by searching 2019000036@jou.edu.cn Shujun Wang the classification in the Carbohydrate-Active Enzymes shjwang@hhit.edu.cn (CAZy) database (http:// www. cazy. org/). According to Jiangsu Key Laboratory of Marine Bioresources and Environment, the searching results of Protein Data Bank (https:// www. Co-Innovation Center of Jiangsu Marine Bio-Industry Technology, Jiangsu Ocean University, Lianyungang 222005, China rcsb. org/), only several molecular structures of dextra- School of Food Science and Engineering, Jiangsu Ocean University, nases were identified, and they were distributed in the Lianyungang 222005, China GH15, GH27, GH49 and GH66 families (Larsson et al. Jiangsu Institute of Marine Resources Development, Jiangsu Ocean University, Lianyungang 222005, China © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Wei et al. AMB Express (2023) 13:7 Page 2 of 10 2003; Mizuno et al. 2004; Okazawa et al. 2015; Suzuki the thermostability of AoDex was further enhanced by et al. 2016). the site-directed mutagenesis strategy, which was based In addition to preparing small molecular weight dex- on a series of predicted software. Our results indicated tran, dextranase has two main application directions. that the AoDex mutants with improved thermostability On the one hand, dextranase can hydrolyze dextran in a could be contributed to industrial applications. sucrose solution in quantity so as to reduce the consump- tion of glucose and the energy losses of the heat transfer Materials and methods process in the sugar industry (Purushe et al. 2012). On Plasmids construction of mutant dextranases the other hand, dextranase is widely used to treat den- The plasmid pCold III-KQ that contains complete gene tal plaque. Some bacteria, such as Streptococcus mutans, for dextranase from A. oxidans KQ11 (GenBank Acces- can ferment sucrose and produce dextran in the mouth, sion No. MK118723.1) was constructed in our previous where adherent bacteria reproduce and generate inflam - study (Ren et al. 2019). Based on the prediction of an mation (Lai et al. 2019; Otsuka et al. 2015). Therefore, online tool SignalP 5.0 (Almagro Armenteros et al. 2019), dextranase was added to toothpaste, mouthwash, and the N-terminal of the full-length dextranase AoDex has other oral hygiene products for the further prevention a signal peptide of 32 amino acid residues. In order to and treatment of dental caries (Jiao et al. 2014; Junta- realize the intracellular expression of AoDex, the signal rachot et al. 2020). However, the industrial applications sequence was cleaved. Subsequently, the gene segments mentioned above depend mostly on the thermostabil- that remove the N-terminal signal peptide sequences of ity of dextranase. Both operating temperatures in sugar dextranase were cloned and ligated into the pCold III manufacturing processes and the long-term storage of vector, and then the new plasmid pCold III-KQ-WT was toothpaste at ambient temperatures put higher require- formed. Recombinant plasmids containing genes of dex- ments on the heat resistance of dextranase. tranase mutants were constructed by the QuikChange Some microorganisms have been reported to synthe- method using appropriate primers and templates (Addi- size dextranase, such as Thermotoga lettingae, Flavobac- tional file 1: Table S1). terium johnsoniae, Chaetomium globosum and so forth (Gozu et al. 2016; Kim and Kim 2010; Yang et al. 2018). However, the optimal temperature for most dextranases Protein expression and purification was known to be 30–60 °C, and they could remain active All recombinant plasmids that contain wild-type and for several hours. The optimal temperature of several mutant dextranase genes were transformed into Escheri- dextranases was also reported to reach 65–70 °C, but chia coli BL21(DE3). The strains were incubated in a they were originated from Thermoanaerobacter species, constant temperature shaker at 37 °C, and when the whose bacterial culture and enzyme purification were absorbance of the bacterial cells reached approximately relatively complicated (Hoster et al. 2001; Park et al. 0.6, isopropyl β-D-thiogalactopyranoside was added with 2012; Wynter et al. 1996). The dextranases available in a final concentration of 0.2 mM. The cells were then cul - commerce have limited thermostability and half-life. tured on an incubator shaker at 16 °C for 24 h to induce Consequently, improving the thermostability of dextra- the expression of dextranase gene. nase derived from microorganisms has been a crucial 2+ Mutant dextranases were primarily purified by Ni project in recent years. affinity chromatography. Cell pellets were centrifuged at Previously, a dextranase AoDex secreted by Arthro- 6000 g for 10 min and resuspended in a binding buffer of bacter oxydans KQ11 was expressed and purified in 20 mM Na HPO -NaH PO , 500 mM NaCl, 20 mM imi- 2 4 2 4 our laboratory. It belongs to the GH49 family and has a dazole at pH 7.0. The cells were then broken by ultrasoni - molecular weight of 66 kDa, and exhibits excellent activ- cation, and the supernatant was loaded onto a Histrap ity at 50–55 °C and pH 7.0–9.0 (Liu et al. 2019; Wang HP column (GE Healthcare, USA) after centrifugating at et al. 2014a). Furthermore, the thermostability of AoDex 13,700 g for 30 min. The target proteins were eluted with was tried to improve by using diversified methods. An buffer containing 20 mM Na HPO -NaH PO , 500 mM 2 4 2 4 atmospheric and room-temperature plasma (ARTP) NaCl, 500 mM imidazole at pH 7.0. Subsequently, the method was used to mutate the wild-type strain, and impure proteins with smaller molecular weights were the optimum temperature of a mutant strain was 5 °C removed using a centrifugal filter device (Millipore Ultra- higher than the wide strain (Wang et al. 2014b). The 15 30 K device, Germany). The buffer for the final dex - crystal structure of AoDex was also determined and the tranases was replaced with 20 mM Na HPO -NaH PO , 2 4 2 4 mutagenesis was preliminarily performed on AoDex, and 50 mM NaCl, 20% (v/v) glycerol at pH 7.0 for further sta- the mutant S357F showed increased thermostability com- ble preservation. All purified proteins were detected by pared with the wild-type (Ren et al. 2019). In this study, SDS-PAGE. Protein concentration was measured using W ei et al. AMB Express (2023) 13:7 Page 3 of 10 the Bradford protein assay kit (Beyotime Biotechnology, improved thermostability were obtained in our previ- China). ous work (Ren et al. 2019). In this study, the systematic rational design of AoDex was further performed based Activity assay of mutant dextranases on B-FITTER software, PoPMuSiC and HotMuSiC Web Dextranase activity was evaluated by the ratio of the servers. The B-FITTER is used to analyze the B-factor increased concentration of reducing sugar based on the value for each residue, which could reflect the flexible reaction between 3,5-dinitrosalicylic and the reducing regions from the X-ray structure of a protein (Sun et al. sugar using dextran-20 (Sangon Biotech, China) as a sub- 2019). The PoPMuSiC algorithm evaluates thermody - strate (Miller 1959). One unit of dextranase activity was namic stability with changes in the folding free energy defined as the amount of enzyme that released 1 µmol of (ΔΔG) between the wild-type and mutants (Dehouck glucose from dextran per min. Each experiment was car- et al. 2011). The HotMuSiC algorithm predicts the ther - ried out in three replicates. mostability of mutated proteins by calculating the melt- ing temperature (ΔT ) of each residue (Pucci et al. 2020). Analysis of kinetic parameters Consequently, the higher B-values, the more negative The assays were carried out at 55 °C and pH 7.0 with values of ΔΔG and the more positive values of ΔT indi- three repeat experiments. The concentration of dextran cated more stable mutants. was ranged from 0.05 to 1.50 mM. Kinetic parameters Based on the results of the B-FITTER software, the top including K , v , k and k /K were calculated by five B-values came from residues S354 to N358. These m max cat cat m fitting the data to the Michaelis–Menten equation using selected residues formed the random coil, which indi- the Linewaver-Burk plot. cated that this region might be beneficial to improve the thermostability (Fig. 1a). Further estimation for this Determination of the thermostability region from the PoPMuSiC and HotMuSiC algorithm The thermostability of dextranase was evaluated by half- calculated three sites (S354, A356 and S357) for muta- life (T ). Wild-type and mutant dextranases were incu- tion with both ΔΔG < 0 and ΔT > 0 (Fig. 1b, Additional 1/2 m bated at 60 °C or 65 °C for 10–40 min at pH 7.0, and file 1: Table S2). Synthesizing the above results, S357 and then placed on ice for 10 min. The remaining enzyme its mutants were predicted to have the biggest B-value activity was measured in each case. For data process- and ΔT , respectively, and thus we identified site-satura - ing, the untreated dextranase activity was defined as tion mutagenesis at the site of residue S357. In addition, 100%, and the remaining activity was calculated as its S354 and A356 were also selected to perform some site- relative enzyme activity. The inactivation kinetics of the directed mutagenesis as putative sites to improve ther- enzymes followed the first-order reaction rate and T mostability (Additional file 1: Table S2). 1/2 was calculated based on the first-order rate constant (k) (Ó’Fágáin 2003). Each experiment was carried out in Activity screen of mutant dextranases three replicates. On the basis of the residues selected above, the mutant dextranases were successfully expressed and purified. Molecular dynamics simulation The mutants were predicted with a molecular weight of The crystal structure of AoDex was obtained from PDB 69 kDa, which was consistent with the results of SDS- 6NZS, and the designed mutants of AoDex were con- PAGE (Additional file 1: Fig. S1). The active mutants were structed by SWISS-MODEL (https:// swiss model. expasy. then preliminarily screened at 55 °C. As shown in Fig. 2, org/). The molecular dynamics (MD) simulation was per - ten mutants of S357 showed enzymatic activities. The formed using Gromacs version 2020.3 program with the nine remaining mutants at this site tended to be inactive Amber ff99sb force field. The wild-type and mutants of or precipitated (data not shown). Among the activated AoDex were executed at 328 K for 20 ns. All structures mutants, S357K, S357D and S357R retained less than were filled with water, and counterions were added to 70% activities compared to the wild-type, hence the three balance the charge. The structures were then optimized mutants were not continued to be studied. Although through the steepest descent methods. After the simu- mutants of S354Q, S354H, A356C and A356V could also lation was complete, the root mean square deviation be expressed, their activities were not detected at any (RMSD) values were calculated for further analysis. temperature. The optimal temperature of the active mutants was fur - Results ther determined within the temperature range of 35 °C Identification of mutation sites to 65 °C at pH 7.0. Results showed that the optimal tem- The crystal structure of the dextranase AoDex had been perature for these mutants was still 55 °C, which was the determined (PDB: 6NZS), and several mutants with same as the wild-type (Fig. 3a). However, some mutants Wei et al. AMB Express (2023) 13:7 Page 4 of 10 ab ΔΔG ΔT -5 Fig. 1 Data statistics of thermostability of dextranase AoDex with predicted software. a The top 10 highest B-factor values of residues calculated by the B-FITTER software. b Aggregate data of ΔΔG (kcal/mol) and ΔT (°C) that calculated by the PoPMuSiC and HotMuSiC Web servers, respectively. The undetected values are not shown in the figure (S357V, S357L, S357P, and S357F) exhibited higher activ- ity than wild-type in the temperature range of 55–60 °C. Compared to other mutants, S357P retained more than 80% activity at 60 °C, indicating that the thermostability of S357P appeared to be higher than other mutants. In addition, the optimal pH of the active mutants was also measured within the pH range from 5.0 to 9.0 at the opti- mal temperature. Changes in the amino acid residue at S357 also did not affect the optimal pH of AoDex, and individual mutants such as S357V showed greater activity in the pH range of 6.0–8.0 (Fig. 3b). Eec ff t of mutated residues on thermostability The thermostability of the active mutant dextranases was measured at 60 °C (Fig. 4). Results showed that the half- Fig. 2 Preliminary screening of the activities of dextranase AoDex lives (T ) of S357P, S357V, S357I and S357L were longer and its mutants 1/2 a b 100 100 80 80 60 60 40 40 35 40 45 50 55 60 65 70 5.06.0 7.08.0 9.0 pH Fig. 3 Enzymatic properties of dextranase AoDex and its mutants. Relative activity is defined as the percentage of maximum enzymatic activity under the corresponding experimental conditions. a The optimal temperature of AoDex and mutants. The activities were determined at pH 7.0. b The optimal pH of AoDex and mutants. The activities were determined at 55 °C WT WT S357P S357P S357F S357F S357A S357A S357V S357V S357L S357L S357I S357I S357E S357E S357 G355 A356 S354 N358 N349 A52 S361 E258 A580 S357 G355 A356 S354 N358 WT S357P S357F S357A S357V S357L S357K S357D S357I S357R S357E Relative activity (%) Relative activity (%) B-value ΔΔG Relative activity (%) (kcal/mol) W ei et al. AMB Express (2023) 13:7 Page 5 of 10 increased thermostability, S357P and S357V showed higher T values, which were about 5.4 and 2.9 times of 1/2 the wild-type, respectively. Previous studies had shown that the mutant S357F was also more stable than the wild-type, so the thermostability of S357F was reassessed. As shown in Fig. 4 and Table 1, the T value of S357F 1/2 was higher than the wild-type, which was consistent 20 with our previous results. However, S357F had a lower T value than S357P and S357V, indicating that S357P 1/2 and S357V had significantly improved in the aspect of heat resistance. Furthermore, we also measured the half- lives of S357P, S357F and S357V at 65 °C, and the three mutants showed higher T values than the wild-type 1/2 (Table 1). S357P exhibited the maximum value of T of 1/2 14.0 min, which was 2.1 times of the wild-type. Mean- Fig. 4 Parameters of thermostability of AoDex and the active mutants. The T values were detected at 60 °C and pH 7.0 while, the time course of the activities for these mutants 1/2 during the incubation at 60 °C and 65 °C were shown in Fig. 5. After incubating at 60 °C for 55 min, S357P per- formed best heat-resistance, and retained more than 55% Table 1 Specific activities and half-lives of wild-type and mutant of the initial enzymatic activity compared with other dextranase AoDex mutants (Fig. 5a). S357V showed higher residual activity Dextranase Specific T at 60 °C (min) T at 65 °C (min) than S357F in 45 min (Fig. 5a). When the incubation tem- 1/2 1/2 activity (U/ perature reached 65 °C, the activities of all the enzymes mg) exhibited dramatic declines, but the residual activity of Wild-type 859 ± 9.2 10.2 ± 0.1 6.8 ± 0.1 S357P was still higher than other mutants within 25 min S357P 780 ± 6.1 55.4 ± 2.2 14.0 ± 0.2 (Fig. 5b). To further assess the thermostability of S357P and S357V, MD simulations were performed at 328 K for S357F 1104 ± 6.1 17.4 ± 0.3 9.0 ± 0.1 20 ns. Results showed that the structures including wild- S357V 1069 ± 10.8 29.6 ± 0.1 9.9 ± 0.4 type, S357P and S357V tended to reach the stable states with the RMSDs of 0.05–0.15 nm (Additional file 1: Fig. S2). After 5 ns simulation, the overall structural fluctua - than those of the wild-type, indicating the enhanced tion of the mutants was less than that of the wild-type, thermostability of these mutants. S357A and S357E dis- suggesting that these mutants had better thermostabil- played similar or lower T values compared to the wild- 1/2 ity than wild-type. The mutant S357V showed an aver - type, illustrating that the thermostability did not improve age RMSD value of 0.110, while S357P displayed a lower although they were active. Among the four mutants with ab 100 100 WT WT S357P S357P S357F S357F S357V S357V 10 20 30 40 50 60 10 20 30 40 50 60 Time (min) Time (min) Fig. 5 Thermostability of the wild-type and mutant dextranase AoDex during the incubation at 60 °C a and 65 °C b. Relative activity is defined as the percentage of maximum enzymatic activity under the corresponding experimental conditions. The activities were determined at pH 7.0 WT S357P S357F S357A S357V S357L S357I S357E T (min) 1/2 Relative activity (%) Relative activity (%) Wei et al. AMB Express (2023) 13:7 Page 6 of 10 RMSD value of 0.106, indicating that S357P was more thermostability (Radestock and Gohlke 2011). Therefore, stable than S357V, and the results basically matched their in this study, reasonable predictions of flexible sites for half-life experiments. Therefore, S357P exerted the prop - dextranase AoDex were made using relevant software erty with the highest thermostability of these mutants and Web servers to investigate strategies to improve the despite the slightly lower activity. thermostability of dextranase. The above results suggested that S357P was the most Enzymatic characterizations of the thermostable mutants stable dextranase among heat-resistance mutants, indi- The kinetic parameters of the mutants with improved cating that the introduction of proline significantly thermostability were determined at the optimal tempera- improved thermostability. Proline contains a pyrro- ture of 55 °C. As shown in Table 2, the K values of the lidine ring on its side chain, resulting in its special mutants increased to different degrees, suggesting that rigid conformation (Allen et al. 2004). Based on the all of these mutant dextranases reduced affinity for the structural and statistical analysis, the thermostabil- substrate of dextran-20. The k values of S357V and ity of a protein could be improved through rigidify- cat S357I improved 1.6 and 1.2 times, respectively, and ing the flexible regions by introducing prolines to the showed enhanced catalytic rate constants. However, the structure (Arnold and Raines 2016; Xie et al. 2020; Yu k values of S357P and S357L were similar or slightly et al. 2015). Besides, the positions where residues were cat decreased to the wild-type. S357V exhibited a maximal replaced could also affect the thermostability of the k /K value, which means that it had a higher catalytic protein. Studies showed that it was more conducive cat m efficiency. We also compared the kinetics of S357F with to improve the thermostability when proline replaced other mutants, and it was found that although the cata- other amino acids in the second positions of β-turns or lytic efficiency of S357F was increased, the affinity for N1 positions of α-helices (Trevino et al. 2007; Xu et al. the substrate decreased significantly. From the above 2020). Furthermore, prolines in loop regions played results, it was concluded that the thermostable mutant a significant role in maintaining the thermostabil - of S357P showed a decreased affinity for substrates and ity (Farhat-Khemakhem et al. 2013; Yu et al. 2015). In a lower catalytic efficiency, but remained higher ther - this study, the substituted position of proline for S357 mostability. Although the thermostability of S357V was in AoDex is located in an exposed long loop between lower than that of S357P, its catalytic activity increased two β-sheets of the catalytic domain, as well as at the significantly. Therefore, mutant S357V could enhance entrance of the substrate binding channel. This unique the thermostability and catalytic efficiency of dextranase location of proline may lead to a sharp bend in the pep- synchronously. tide chain; hence, it may help rigidify flexible regions of the dextranase AoDex, or form the hydrophobic inter- Discussion action between its own side chain and other hydro- Improving the thermostability of enzymes has become a phobic residues, thus increasing the rigidity of the hot and difficult issue of enzymology. Enzymes with high peptide chains and making the structure more com- heat resistance could be more conducive to their stable pact (Fig. 6b). The replacement of proline in flexible preservation and promote their application in related regions provided new possibilities for the thermostable fields. Generally, the factors affecting the thermostabil - modification of dextranases. The mutant S357F could ity of proteins mainly include the non-covalent interac- enhance thermostability, which was also verified in tions of residues such as ionic bonds, hydrogen bonds our previous study. The substitution of phenylalanine and hydrophobic interactions, and some covalent bind- was analyzed to form an aromatic interaction with sur- ing such as disulfide bonds (Xu et al. 2020). Additionally, rounding aromatic amino acids such as W507 (Fig. 6f ) rigid regions in proteins may be crucial for maintaining (Ren et al. 2019). As is known, the side chains of valine, Table 2 The kinetic parameters of wild-type and mutant dextranase AoDex −1 −1 −1 −1 Dextranase v (mmol·L ·min ) K (μmol·L ) k (s ) k /K max m cat cat m −1 −1 3 (μmol ·L·s ) × 10 Wild-type 2.83 ± 0.05 50.9 ± 1.5 12.6 ± 0.2 247.8 ± 8.3 S357P 4.83 ± 0.04 62.0 ± 1.3 11.1 ± 0.1 179.1 ± 4.1 S357F 4.71 ± 0.15 115.6 ± 6.6 25.0 ± 0.8 217.0 ± 14.2 S357V 4.26 ± 0.05 58.0 ± 1.5 20.3 ± 0.2 350.2 ± 9.7 S357I 5.84 ± 0.02 121.5 ± 1.4 14.7 ± 0.1 121.0 ± 1.6 S357L 4.89 ± 0.11 128.4 ± 5.2 12.8 ± 0.3 99.9 ± 4.6 W ei et al. AMB Express (2023) 13:7 Page 7 of 10 Fig. 6 Changes in intramolecular interactions of dextranase AoDex that are caused by mutants at residue S357. The structural models of the mutants were determined by SWISS-MODEL. The ribbon representation of dextranase is shown in gray. The mutant residues are labeled as navy-blue sticks. The predicted catalytic residues are labeled as orange sticks. a The relative positions of key residues of the wild-type. b–f Relative positions of key residues of AoDex mutants. g The overview of relative positions of substrate binding channels, catalytic residues, and mutant residues (take mutant V357 for example) leucine, isoleucine, and phenylalanine are all hydropho- formed new hydrophobic interactions with adjacent bic. In this study, the thermostability of S357V, S357I L353, which would probably increase the thermostabil- and S357L was also improved, although these mutants ity of dextranase (Fig. 6c, d, e). Thus, the improved heat were less heat resistant than S357P and S357F. Accord- resistance of mutant S357F could also be attributed to ing to the structural model of single point mutation increase hydrophobic forces. Furthermore, the thermo- for S357, the hydrophobic residues replaced serine and stability of S357A and S357E was similar to that of the Wei et al. AMB Express (2023) 13:7 Page 8 of 10 wild-type, suggesting that the interactions generated by there have been few studies on the thermostability of the replacement of the two residues have little effect on dextranase, and this is probably due to the fact that there the overall structural rigidity. are not many dextranases whose structures have been In addition, an increase in the activity of mutant dextra- resolved. Except for dextranase AoDex, the other GH49 nases was also found in this study. Based on our previous dextranase with a known structure was Dex49A. It was studies, the catalytic residues of the dextranase AoDex derived from Penicillium minioluteum, and its three- were predicted to be D420 and D439, and the substrate dimensional structure resembled that of AoDex (Lars- channel tended to form in the void outside the catalytic son et al. 2003; Ren et al. 2019). Compared with Dex49A, domain (Ren et al. 2019). The structure of AoDex showed AoDex was found to have several extended loops on the that the mutant site of S357 was just close to the entrance surface of the structure. Moreover, the residues of AoDex of the substrate channel. When the mutants of S357 gen- from S354 to N358 that were predicted to be beneficial erated hydrophobic interactions with adjacent L353, the to improve thermostability were also located in these size and shape of the substrate channel could be changed. exposed loop regions, and this feature was absent in the And it probably promoted the binding of substrates and structure of Dex49A. It has been reported that some catalytic residues, which also might explain the reason deletions in the exposed loop regions of a thermophilic for the increased activity of mutants S357V, S357I, S357L protein are more likely to help to lower its unfolding and S357F (Fig. 6g). S357F was slightly more active than entropy and increase the thermostability (Suzuki et al. S357V, indicating that the conformation of phenylalanine 2016; Thompson and Eisenberg 1999). The mutations might be more favorable for substrate binding than other of S357 from the loop regions might be speculated to hydrophobic residues. Valine had a shorter side chain result in the broken of the conformational entropy of the compared to leucine and isoleucine, and, in the mean- original structure, and thus the thermostability of rel- time, S357V had higher enzymatic activity compared to evant mutants was improved. Based on the structure of S357I and S357L. Therefore, it appeared that the length Dex49A, a GH49 dextranase that originated from Lipo- of the side chain of an amino acid could also change the myces starkeyi was modeled, and enhanced its optimal shape of the substrate binding channel and then affect temperature by introducing disulfide bonds (Chen et al. the catalytic activity. In addition, the activities of S357A 2009). For the dextranase of P.minioluteum, studies had and S357E were similar to the wild-type, demonstrat- found that the recombinant expression of dextranase in ing that these substitutions of residues did not appear to Pichia pastoris could also significantly improve thermo - affect the flexibility of the loop. However, the activity of stability (Beldarrain et al. 2003). The above results were S357P with excellent thermostability decreased slightly. specific to the dextranase Dex49A, which was derived It was previously reported that the increase in the ther- from fungi. Dextranase AoDex also belonged to GH49, mostability of enzymes was usually accompanied by the and a favorable mutant S357F had been mined in our decrease in the activities, which might explain the reason previous research. In this study, we further attempted for the above results (Xie et al. 2014). S357K, S357D and to improve the thermostability of dextranase AoDex by S357R with decreased activities illustrated that the struc- rational design and obtained several mutants with better tural conformation of the three mutations significantly heat resistance, including S357P and S357V. Enzymes of affected catalytic activities. the same glucoside hydrolase have the similar substrate Currently, there are several strategies to obtain the heat binding pocket and catalytic mechanism; hence, the find - resistant dextranases. One way is to screen the thermo- ings of AoDex could also provide some references for the philic dextranases of thermophiles (Hoster et al. 2001; thermostability of other dextranase in GH49. Park et al. 2012). Nonetheless, both the rigorous culture conditions of thermophilic microorganisms and the lim- Abbreviations ited stability of natural enzymes put higher requirements CAZy Carbohydrate-active enzymes on the studies. Another option is to achieve the thermo-GH Glycoside hydrolase WT Wild type stability of dextranases by directed evolution or rational SDS-PAGE S odium dodecyl-sulfate polyacrylamide gel electrophoresis design, and these techniques have been widely used in RMSD Root mean square error other multiple enzymes such as xylanases and proteinases (Rigoldi et al. 2018). Several variants of a dextranase that Supplementary Information originated from Paenibacillus sp. had been reported to The online version contains supplementary material available at https:// doi. increase the half-lives by 2.3–6.9 times through random org/ 10. 1186/ s13568- 023- 01513-2. mutagenesis (Hild et al. 2007). A GH97 dextranase from Additional file 1. Table S1 Primer sequences used for plasmids Pseudoalteromonas sp. K8 increased thermostability at mutagenesis in this study. Table S2 Data statistics of selected mutants of 33 °C by rational design (Zhang et al. 2020). Presently, W ei et al. AMB Express (2023) 13:7 Page 9 of 10 Díaz-Montes E (2021) Dextran: sources, structures, and properties. Polysaccha- dextranase AoDex from B-FITTER, PoPMuSiC and HotMuSiC. Figure. S1 rides 2(3):554–565. https:// doi. org/ 10. 3390/ polys accha rides 20300 33 SDS-PAGE of the wild-type ( WT ) and mutants of dextranase AoDex. Fig- Falconer DJ, Mukerjea R, Robyt JF (2011) Biosynthesis of dextrans with different ure. S2 The RMSD values of the wild-type and mutants of AoDex at 328 K. molecular weights by selecting the concentration of Leuconostoc mes- enteroides B-512FMC dextransucrase, the sucrose concentration, and the temperature. Carbohydr Res 346(2):280–284. https:// doi. org/ 10. 1016/j. Acknowledgements carres. 2010. 10. 024 Not applicable. Farhat-Khemakhem A, Ali MB, Boukhris I, Khemakhem B, Maguin E, Bejar S, Chouayekh H (2013) Crucial role of Pro 257 in the thermostability of Bacil- Author contributions lus phytases: biochemical and structural investigation. Int J Biol Macromol ZW and JC conceived and designed the experiments. ZW and JC performed 54:9–15. https:// doi. org/ 10. 1016/j. ijbio mac. 2012. 11. 020 the experiments. ZW, LX, NL and JY analyzed data. ZW wrote the manuscript. Gozu Y, Ishizaki Y, Hosoyama Y, Miyazaki T, Nishikawa A, Tonozuka T (2016) A SW reviewed the manuscript. All authors approved the final manuscript. glycoside hydrolase family 31 dextranase with high transglucosylation activity from Flavobacterium johnsoniae. Biosci Biotechnol Biochem Funding 80(8):1562–1567. https:// doi. org/ 10. 1080/ 09168 451. 2016. 11828 52 This work was funded by Natural Science Foundation of Jiangsu Province (No. Hild E, Brumbley SM, O’Shea MG, Nevalainen H, Bergquist PL (2007) A Paeni- BK20201028), Open-end Funds of Jiangsu Institute of Marine Resources Devel- bacillus sp dextranase mutant pool with improved thermostability and opment (No. JSIMR202113), Natural Science Foundation of Jiangsu Higher activity. Appl Microbiol Biotechnol 75(5):1071–8. https:// doi. org/ 10. 1007/ Education Institutions of China (No. 22KJB180015), Jiangsu Natural Resources s00253- 007- 0936-6 Development Special Project (Marine Science and Technology Innovation) Hoster F, Daniel R, Gottschalk G (2001) Isolation of a new Thermoanaerobac- (No. JSZRHYKJ202008). This work was supported by the Research Start-up terium thermosaccharolyticum strain (FH1) producing a thermostable Fund of Jiangsu Ocean University and Priority Academic Program Develop- dextranase. J Gen Appl Microbiol 47(4):187–192. https:// doi. org/ 10. 2323/ ment of Jiangsu Higher Education Institutions (PAPD). jgam. 47. 187 Jiao Y-L, Wang S-J, Lv M-S, Jiao B-H, Li W-J, Fang Y-W, Liu S (2014) Characteriza- Availability of data and materials tion of a marine-derived dextranase and its application to the prevention The datasets generated during and/or analyzed during the current study are of dental caries. 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Journal
AMB Express – Springer Journals
Published: Jan 19, 2023
Keywords: Dextranase; Thermostability; Arthrobacter oxydans; Site-directed mutagenesis; GH49