Enhanced Trypsin Thermostability in Pichia Pastoris Through Truncating

Liu et al. Microb Cell Fact (2018) 17:165 https://doi.org/10.1186/s12934-018-1012-x Microbial Cell Factories

RESEARCH Open Access Enhanced trypsin thermostability in Pichia pastoris through truncating the fexible region Lin Liu1,2,3, Haoran Yu1,2,3,4, Kun Du1,2,3, Zhiyan Wang1,2,3, Yiru Gan1,2,3 and He Huang1,2,3*

Abstract Background: High thermostability is required for trypsin to have wider industrial applications. Target mutagenesis at fexible regions has been proved to be an efcient protein engineering method to enhance the protein thermostability. Results: The fexible regions in porcine trypsin were predicted using the methods including molecular dynamic simulation, FlexPred, and FoldUnfold. The amino acids 78–90 was predicted to be the highly fexible region simultaneously by the three methods and hence selected to be the mutation target. We constructed fve variants (D3, D5, D7, D9, and D11) by truncating the region. And the variant D9 showed higher thermostability, with a 5 °C increase in Topt, T 10 5.8 °C rise in 50 , and a 4.5 °C rise in Tm, compared to the wild-type. Moreover, the half-life value of the variant D9 was also found to be dramatically improved by 46 min. Circular dichroism and intrinsic fuorescence indicated that the structures had no signifcant change between the variant D9 and the wild-type. The surface hydrophobicity of D9 was measured to be lower than that of wild-type, indicating the increased hydrophobic interaction, which could have contributed to the improved thermostability of D9. Conclusions: These results showed that the thermostability of variant D9 was increased. The variant D9 could be expected to be a promising tool enzyme for its wider industrial applications. The method of truncating the fexible region used in our study has the potential to be used for enhancing the thermostability of other proteins. Keywords: Thermostability, Trypsin, Flexible region, Truncation, Ω-loop

Background hydrolyzing peptides under high temperature conditions. Trypsin (EC 3.4.21.4) is a serine protease, which hydro- Terefore, it is necessary to improve the thermostability lyzes peptide bonds on the carboxylic sides of lysine of trypsins for satisfying the industrial applications. and arginine [1]. It exists widely in nature, and has been In recent years, a number of efective approaches, discovered in bacteria, fungus and mammals [2]. As an such as enzyme immobilization, protein engineering, essential tool enzyme, trypsin can be applied in variety and use of cosolvents, have been successfully utilized to of felds including pharmaceutical, leather processing, improve the thermostability of trypsin [5–7]. For exam- and food processing, especially in proteomic applica- ple, Pazhang et al. reported that polyols (sorbitol and tions about the use of mass spectrometry (MS) to ana- glycerol) stabilized trypsin by decreasing the polypep- lyze the peptides obtained [3, 4]. Te peptides must be tide chain fuctuations. Among various methods from denatured to achieve efective digestion, since the native improving protein stability, the protein engineering peptide could not be digested by trypsin. However, the attracted more attention due to requiring changes to the peptides denatured by chemicals would complicate the primary structure of the enzyme to confer the desired further analytical steps. Te problem could be solved by features [8]. Up to date, protein engineering comprises two main approaches including directed evolution and rational design. Te process of directed evolution is *Correspondence: [email protected] 3 Collaborative Innovation Center of Chemical Science and Engineering, heavily experimental and time-consuming. And high- Tianjin 300350, China throughput screening is usually required in directed evo- Full list of author information is available at the end of the article lution. However, not all enzyme stabilities are amenable

© The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Liu et al. Microb Cell Fact (2018) 17:165 Page 2 of 10

to developing a high-throughput screening method. (amino acids 78–90) was overlapped with the Ω-loop Recently, rational design has been widely applied for region (amino acids 78–91) (Figs. 1, 2a). Five variants engineering the protein thermostability. Compared to were constructed by truncating the fexible Ω-loop. Te directed evolution, rational design is faster and universal efects of loop truncation on the kinetic properties, ther- because it focuses more on several specifc target sites. mostability and structural properties were determined. With fexible sites as target sites, the strategy of RFS Te best variant (D9) showed signifcantly improved (rigidifying fexible sites) has been a powerful method to thermal stability without compromising enzymatic engineer the protein thermostability [9]. Identifying the activity. fexible regions was the critical procedure for design- ing thermostable proteins using this strategy. Molecular dynamics (MD) simulation was commonly applied to Results identify the highly fexible regions of proteins. However, Design and purifcation of the trypsin variants disadvantages, such as computational expense and inac- In this study, we attempted to enhance the thermosta- curacy, limit the use of MD simulation [10]. Two other bility of trypsin by the approach of rational design. Te methods (FlexPred and FoldUnfold) could also be used to fexible regions were predicted by three methods (MD precisely predict the fexible regions, which are faster and simulation, FoldUnfold, and FlexPred) and listed (Addi- cheaper than the MD simulation [11]. When the three tional fle 1: Fig. S1 and Table S1). We selected four fex- methods are used in combination, it has the potential to ible regions from each method and compared with each be more efective to fnd the highly fexible region. other. Te amino acids 78–90 was predicted to be the Rigidifying the fexible regions was another step in highly fexible region of the trypsin, which is overlapped the RFS strategy for engineering the protein thermosta- with the Ω-loop region (amino acids 78–91) (Figs. 1, 2a). bility [9]. Among various approaches applied to rigidify Te Ω-loop (amino acids 78–91) was placed between the the fexible regions, the method of truncating fexible two short β-strands based on the analysis of three-dimen- regions has received attention in recent years [12–14]. sional porcine trypsin structure, which seemingly inter- Ban reported that truncation of a fexible portion of rupts the formation of one long strand. Moreover, based the C-terminal end in GBEGt (1,4-α-glucan branching on previous reports, loops were generally considered as enzyme from G. thermoglucosidans STB02) enhanced potential targets for engineering proteins with improved the thermostability of the enzyme without signifcantly properties [13, 22–24]. Ten fve variants (D3, D5, D7, compromising its enzyme activity [15]. Truncating the D9, and D11) were designed by truncating the fexible fexible N-terminal contributed to increasing thermal loop by three residues (D3), fve residues (D5), seven resi- stability of mannanase Man 1312 without activity loss dues (D7), nine residues (D9) and eleven residues (D11) [16]. Tus, it is possible to enhance the thermal stability (Fig. 2b, Additional fle 1: Fig. S2). A SP-Sepharose col- of proteins through truncating the fexible regions. umn was used for purifying the variants. Te results of Ω-loop, a nonregular secondary structure, commonly SDS-PAGE indicated that all purifed enzymes showed a connects α-helices and β-strands [17]. As a special loop, single band of ~ 24 kDa (Fig. 3). Ω-loop usually consists of 6–16 amino acids and locates on the surface of proteins, which plays essential roles in protein folding, function and stability [18]. Structural analysis of psychrophilic, mesophilic and thermophilic enzymes revealed that shorter and fewer surface loops are usually existed in thermophilic enzymes due to the higher structural rigidity [19]. Tompson et al. found that the thermostability of proteins could be improved by deleting the dynamic surface loop [20]. Similarly, the thermostability of HCA II could be improved through truncating the surface loop [21]. Terefore, truncation could be an efective way to rigidify the fexible loop and improve the thermostability of trypsin. Fig. 1 Prediction of the fexible regions of porcine trypsin. MD simulations were carried out in Gromacs package. The RMSF values In this study, we aimed to enhance the thermostability of WT were collected at 300 K for a minimum of 10 ns. FlexPred of the trypsin expressed in Pichia pastoris GS115 using predicted residue fexibility in trypsin using SVM approach. The the strategy of RFS. MD simulation, FlexPred, and Fol- approach of FoldUnfold used the expected average number of dUnfold were frstly used to predict the highly fexible contacts per residue calculated from the amino acid sequence as an regions. It was found that the predicted fexible region indicator of whether the given region is folded or unfolded Liu et al. Microb Cell Fact (2018) 17:165 Page 3 of 10

Fig. 2 Design of the truncation of the Ω-loop region. a Crystal structure of porcine trypsin visualized by the PyMOL software from the Protein Data Bank (PDB ID: 4AN7). The Ω-loop is colored blue and the active site is colored red. b The design of the truncation of the Ω-loop region

and Table 1. Te kd values of variants were lower than that of WT except D11, indicating that WT had deacti- vated more slowly than D11 and more rapidly than other variants. After incubation at 50 °C for 60 min, WT, D7 and D9 had 70% remaining activities, while the residual activity of D11 was nearly 60% (Additional fle 1: Fig. S3). Meanwhile, both variants D3 and D5 retained more than 85% of the original activities. Te half-life value of WT was 155.76 min, while the half-life times of D3, D5. D7, D9, and D11 were 209.41 min, 199.18 min, 171.57 min, Fig. 3 SDS-PAGE analysis for purifed WT and variants (D3, D5, D7, D9, 202.08 min, and 124.44 min, respectively (Table 1). Except and D11) (10 μL; protein concentration 3 μg). M, molecular weight D11, all other variants showed longer half-life time than marker; 1, WT; 2–6, D3, D5, D7, D9, and D11 WT, indicating that the kinetic stability of trypsin was improved by truncating the Ω-loop region. Te inactivation curves of WT and variants were obtained by incubating the enzymes at temperatures rang- Furthermore, we measured the protein yield based ing from 25 to 85 °C for 10 min. After heat treatment, on the purifed enzymes. Te protein yield of WT was enzymes were cooled in the icebox for 5 min. As shown in 43.32 mg/L, while the yields of D3, D5, D7, D9, and D11 Fig. 5a, WT almost lost their enzyme activities at 85 °C, but were 25.82 mg/L, 29.92 mg/L, 53.44 mg/L, 40.58 mg/L, other variants remained nearly 15% of its original activity. and 33.49 mg/L, respectively. Apparently, the protein Moreover, the residual activity of all variants at 60 °C was yields of other variants were lower than that of WT except above 50%, while WT only retained 20% of initial activity. T 10 for D7. Te results suggested that the protein yield might Te 50 values of D3, D5, D7, D9, and D11 were deter- be infuenced after truncating the fexible region of trypsin. mined to be 65.8 °C, 62.0 °C, 61.2 °C, 60.3 °C, and 60.5 °C, respectively, while that of WT was 54.5 °C (Table 1). Analysis of thermostability properties Te term “protein stability” commonly includes kinetic Melting temperature measurement stability and thermodynamic stability [25]. To investigate To investigate the efect of truncation on the thermody- kinetic stability of WT and variants, purifed enzymes namic stability, the thermofour assay using the SYPRO were incubated at 50 °C for diferent time (30 min, 60 min, Orange as a dye was determined. SYPRO Orange is 90 min, 120 min, and 180 min, respectively) and then the weakly fuorescent in the hydrophilic environment but residual activity was measured at 25 °C. Te efect of the has a high fuorescence intensity in hydrophobic regions heat treatment on enzyme activities was shown in Fig. 4 of proteins [26]. Along with the protein unfolding, the Liu et al. Microb Cell Fact (2018) 17:165 Page 4 of 10

dye binds to the exposed hydrophobic regions of the protein which could lead to a signifcant increase in the fuorescence intension. As shown in Table 1 and Additional fle 1: Fig. S4, Tm value for WT was 51.0 °C, whereas Tm values of variants were 50.5 °C, 62.3 °C, 54.4 °C, 55.5 °C, and 53.3 °C for D3, D5, D7, D9, and D11, respectively. Te thermodynamic stability of variants of D5, D7, D9, and D11 was higher than that of WT, while the thermodynamic stability of D3 was decreased.

Optimum temperature for WT and variants Te infuence of temperature on the enzymatic activity of WT and variants was investigated. Te enzyme activity profle was measured between 25 and 85 °C, with 5 °C intervals. As shown in Fig. 5b and Table 1, the optimum Fig. 4 Natural-logarithmic plot of residual activity of WT and variants temperatures of D3 and D9 were 65 °C, 5 °C higher than (D3, D5, D7, D9, and D11). After incubation at 50 °C for diferent that of WT, while the optimum temperatures of D7 and minutes, the residual activity of each trypsin was assayed. The D11 were decreased by 5 °C and 10 °C, respectively. Te original activity was defned as 100%. Each value is the mean of three optimum temperature of D5 was 60 °C, which was con- independent experiments sistent with that of WT. At 85 °C, the variant D9 retained nearly 30% of its initial activity, while the residual activity of other variants and WT was about 15%. Table 1 Thermostability constants of WT and variants

10 1 Characterization of kinetic properties Enzymes Topt (°C) Tm (°C) T50 (°C) t1/2 (50 °C) (min) kd (min − ) Kinetic parameters of WT and variants were shown 3 WT 60.0 51.0 54.5 155.76 4.45 10− in Table 2. Te Km and kcat values of WT were × 3 D3 65.0 50.5 65.8 209.41 3.31 10− 28.80 2.94 μM and 0.634 0.030 s−1, respectively. Te × 3 ± ± D5 60.0 62.3 62.0 199.18 3.48 10− Km values of D7 and D11 were drastically decreased, and × 3 D7 55.0 54.4 61.2 171.57 4.04 10− that of D3 was increased by more than 50%. Further- × 3 D9 65.0 55.5 60.3 202.08 3.43 10− more, the Km values of D5 and D9 were 33.40 4.11 μM × 3 ± D11 50.0 53.3 60.5 124.44 5.57 10− × and 27.04 ± 3.35 μM, respectively. Although kcat values Each value was calculated from triplicate experiments displayed no signifcant diference between WT and

Fig. 5 Thermostability properties of WT and variants (D3, D5, D7, D9, and D11). a Comparison of thermal tolerance for WT and variants. The residual activities of the enzymes were measured after incubating at diferent temperatures (25–85 °C) for 10 min. b Efect of temperature on enzyme activity. The residual activities were determined under diferent temperatures (25–85 °C). Values are presented as the average of three experiments Liu et al. Microb Cell Fact (2018) 17:165 Page 5 of 10

Table 2 Kinetic parameters of WT and variants

1 1 1 Enzymes Km (μM) kcat (s − ) kcat/Km (mM − s− ) Specifc activity (U/mg)

WT 28.80 2.94 0.634 0.030 22.014 1.45 105,614.2 4154.28 ± ± ± ± D3 58.60 3.30 0.676 0.007 11.536 0.87 73,850.3 1385.34 ± ± ± ± D5 33.40 4.11 0.652 0.007 19.521 1.05 102,234.8 5238.41 ± ± ± ± D7 11.01 0.94 0.604 0.024 54.910 3.56 196,961.5 8182.93 ± ± ± ± D9 27.04 3.35 0.624 0.099 23.111 2.19 108,760.9 5205.32 ± ± ± ± D11 7.50 0.29 0.580 0.019 77.330 4.98 340,425.6 9314.77 ± ± ± ± Each value was calculated from triplicate experiments. Means standard deviations ± The enzyme activities were measured at 25 °C

variants, the kcat/Km values of D7 and D11 were around 150% and 250% higher than that of WT, respectively. In addition, the kcat/Km value of D3 was decreased by nearly 50%, while other variants showed no considerable change compared with WT. Te specifc activity of WT was 105,614.2 ± 4154.28 U/mg, while the specifc activity of D3, D5, D7, D9, and D11 was 73,850.3 ± 1385.34 U/mg, 102,234.8 ± 5238.41 U/mg, 196,961.5 ± 8182.93 U/mg, 108,760.9 ± 5205.32 U/mg, and 340,425.6 ± 9314.77 U/ mg, respectively (Table 2). Te specifc activity of trypsin displayed the same tendency of changes with the catalytic efciency after truncating the Ω-loop region.

CD spectroscopy analysis CD spectra and fuorescence spectra were performed to further explore the impact of the truncation on structures. As shown in Fig. 6, CD spectra of WT and all variants Fig. 6 CD spectra of WT and variants (D3, D5, D7, D9, and D11) were exhibited a similar shape. Te spectrum of WT and its measured at room temperature (25 °C). The concentration of protein used for the far-UV CD spectrum (190–260 nm) was 0.2 mg/mL. Each variants all showed a single negative band near to 205 nm, enzyme equilibrated in Tris–HCl bufer (10 mM, pH 8.0) indicating that the percentage of the secondary structure had no considerable alteration (Additional fle 1: Table S2). a signifcant decrease in the surface hydrophobicity com- Fluorescence spectroscopy analysis pared with WT. Intrinsic fuorescence experiment was carried out to esti- mate the infuence of mutations on the tertiary structure Discussion of trypsin. As shown in Fig. 7a, except for D11, the result Trypsin is the most widely used protease for the studies of fuorescence spectra displayed that WT and other about proteins. In many instances, a high thermostabil- variants had a maximum emission at around 334 nm. It ity of trypsin is usually required in some industrial and indicated that there was no signifcant change in tertiary biotechnological applications. Some methods, such as structures after the truncation the Ω-loop region. How- immobilization, the addition of polyol and enzymatic gly- ever, a redshift of 3 nm was found between WT and D11, cation, have been used for improving the thermostability which indicated partial tryptophan residues of D11 more of trypsin [6, 27, 28]. In the present study, we improved exposed to the hydrophilic environment. In addition, the the thermostability of trypsin by truncating the fexible emission intensity of D3, D5, D7, and D9 were notably region. Te highly fexible region (amino acids 78–90) was higher than that of WT, suggesting that the tryptophan identifed, which is overlapped with the Ω-loop region residues were buried away from the aqueous solvent. (amino acids 78–91). We designed fve variants (D3, D5, Te surface hydrophobicity of trypsin was determined D7, D9, and D11) in this fexible loop. Among these vari- by fuorescence spectra. As shown in Fig. 7b, the fuores- ants, D9 variant showed signifcantly improved thermo- cence intensity of WT was higher than variants (D5, D7, stability with no loss of enzyme activity. In addition, the and D9). Te results indicated that the three variants had variant D9 showed no signifcant change in structures Liu et al. Microb Cell Fact (2018) 17:165 Page 6 of 10

Fig. 7 Fluorescence spectra of WT and variants (D3, D5, D7, D9, and D11). a Intrinsic fuorescence spectra of WT and variants. The fuorescence emission spectra were monitored at 300–400 nm. b Surface hydrophobicity of WT and variants. The fuorescence emission spectra were recorded between 400 and 600 nm

when comparing with WT. Te properties of D9 suggest of D9 was lower than that of WT, indicating the increased that truncating the fexible regions could be an efective hydrophobic interaction. strategy for improving the thermostability of proteins. Enhancing stability without sacrifcing enzyme activity is In this work, MD simulation was used to determine the critical for protein engineering [34]. Te improvement of overall and local fexibility of WT and D9 variant. Te thermostability generally leads to the decrease of enzyme overall fexibility of proteins was evaluated using RMSD activity due to the so-called stability-activity trade-of values. And the local fexibility of proteins was investi- [35]. As shown in Table 2, we observed that the change in gated by analyzing the RMSF values. As shown in Addi- enzyme activity was mainly caused by an alteration of the tional fle 1: Figs. S5, S6, the variant D9 showed lower Km value but not the kcat value. Te thermostability of D3 RMSD and RMSF values compared with WT. From the was signifcantly increased, while the catalytic efciency of results, we found that the overall and local fexibility of D9 D3 was decreased by nearly 50% compared with WT. Te variant were all decreased, which might be a reason that Km of D3 was increased by 1.03-fold, indicating that the the thermostability of D9 variant was increased. Previous substrate binding afnity was afected after deleting the studies have showed that the proteins with better ther- three residues at the Ω-loop region. In contrast, the variant mostability which usually displayed higher rigidity [9, 29]. D11 has the lowest melting temperature and shortest half- Furthermore, we also predicted the residue fuctuation life among these variants. Te Km of D11 was drastically profle by submitting the protein structures to CABS-fex decreased and its kcat value was decreased only slightly, sever. As shown in Additional fle 1: Fig. S7, the Ω-loop resulting in the highest catalytic efciency (Table 2). Te located at a region with high mobility, suggesting that reason for this might be the change of surface charge these residues in this region underwent large disturbance. between WT and D11 (Additional fle 1: Fig. S8). Vidya And the fuctuations in this region were considerably alle- et al. found that the alteration of surface charge afected viated when the Ω-loop was truncated. Tis result might the thermal properties of l-asparaginase II [36]. And Niu be related to the enhancement of the hydrophobic inter- et al. reported that the variant BglT with high activities action in variant D9. Hydrophobic interaction plays an displayed the biased distribution of surface charge com- essential role in the stability of protein structure [30]. Due pared with the wild-type BglT [29]. In this research, we to the energetic cost of solvating nonpolar side chains, the investigated the electrostatic surfaces of WT and D11 exposure of hydrophobic residues at the protein surface using PyMOL. Te potential distribution of WT exhib- may lead to a decrease in protein thermostability [31]. ited neutral in the Ω-loop region, while the variant D11 Te increase in the hydrophobicity of protein’s internal displayed almost all negative in this region. Furthermore, residues or the decrease in the hydrophobicity of protein’s we inferred that the decrease of thermostability of D11 surface residues improves the hydrophobic interaction of might be related to the loss of aromatic π–π interactions. proteins [32, 33]. In this study, the surface hydrophobicity Te aromatic π–π interaction plays an important role in Liu et al. Microb Cell Fact (2018) 17:165 Page 7 of 10

protein stability and the loss of aromatic π–π interaction above prediction, we modifed this region by the method could decrease the protein stability. For instance, Deng of truncation and acquired fve variants. Compared with et al. found that aromatic π–π interaction was more pow- WT, the D9 variant showed a 5 °C, 5.8 °C, and 4.5 °C T 10 erful than hydrogen bonds and salt bridges in stabilizing increasement in Topt, 50 , and Tm, respectively. Te half- α-amylase [37]. Tus, we analyzed aromatic π–π interac- life value of D9 was 202.08 min, 46 min longer than that tions of D11 by PIC server, and it revealed that the variant of WT. Tese results demonstrated that truncating the D11 lacked three aromatic π–π interactions when com- fexible regions could beneft the improvement of ther- pared with WT. Nevertheless, the activity-stability trade- mostability. In addition, this study provides an efcient of could be overcome through creating chemically and/ method that could be applied to engineer other proteins or genetically modifed nonnatural enzymes [19]. In this for better thermostability. study, the thermostability and catalytic efciency of D9 are all increased compared with WT. Based on the crys- Methods tal structure, we found the active site of Asp89 was located Analysis of fexible regions at the Ω-loop region (Fig. 2a). Te active site was usu- MD simulation was measured using the Gromacs package, ally covered by the adjacent loops, which might infuence version 5.0 with the Gromos force feld (53A6). Te initial the enzyme activity [38]. Moreover, Zhang et al. reported structure was solvated in a cubic simulation box with a that rigidifying the fexible residues in the active center layer of water at least 1.1 nm from the protein surface. Te improved the thermostability of Candida rugosa lipase1, whole system was minimized using the steepest descent without a decrease in catalytic activity [39]. Tus, truncat- method (1000 steps) in addition to the conjugate gradient ing the Ω-loop region might result in the improvement of method (5000 steps). Ten two 100 ps position-restricted the thermostability and enzyme activity at the same time. simulations were measured under NVT and NPT ensem- CD spectra and fuorescence spectra were measured to bles, respectively. Finally, a 10 ns MD simulation was per- investigate the structural changes between the WT and formed at 300 K. Te values of RMSD (root mean square variants. Compared with WT, the CD spectra of all vari- deviation) and RMSF (root mean square fuctuation) were ants showed no obvious change. It indicated the trunca- calculated using g_rmsd and g_rmsf, respectively. tion of the Ω-loop region have almost no infuence on Similarly, the FoldUnfold and FlexPred were also used the secondary structure of trypsin. Moreover, according to predict the fexible regions. Te FlexPred web server to the results of fuorescence experiments, the intrinsic analyzes the fexibility of proteins using support vector fuorescence intensity of variants (D3, D5, D7, and D9) machine (SVM). Moreover, the three-dimensional struc- showed a superior increase compared with WT, suggest- ture was used as an input for the two methods to pre- ing the structure of enzyme became tighter after truncat- dict fexible regions of proteins. Te crystal structure of ing the Ω-loop region. Tis result was coincident with the porcine trypsin (PDB ID: 4AN7) [40] was acquired from work of Kheirollahi and Khajeh [34]. Tey found that the the Protein Data Bank and the structures of variants higher intrinsic fuorescence intensity might be the result were constructed by homology modeling in the SWISS- of the increase of structure compactness in the variant MODEL web server (http://www.swissmodel.expasy.org) Glu138Pro. Moreover, Boone et al. suggested that the [41]. Te residue fuctuation profle of proteins was ana- thermal stability of HCA II can be increased by improv- lyzed by CABS-fex server [42]. Visualization and analysis ing the surface compactness via loop truncation [21]. of structures were performed by PyMOL 2.7.6. Accurate prediction of fexible regions is critical for engineering the thermostability of proteins through the Strains, plasmids, and chemicals method of RFS. Based on the analysis of MD simula- Escherichia coli Top 10 (TransGen, Beijing, China) was tion, the fexible region (amino acids 78–90) is not one of used as the host strain for cloning. Pichia pastoris GS115 the most fexible regions (Additional fle 1: Fig. S1). Tis and the pPIC9K vector were from our laboratory collec- means that the fexible region could be eliminated if only tion. Pfu DNA polymerase and restriction endonucleases predicted by MD simulation. Terefore, it is important to were purchased from Termo Fisher Scientifc (Waltham, use several predictive methods simultaneously for engi- USA). Bradford Protein Assay Kit (TIANGEN, Beijing, neering the protein thermostability. China) was used to determine the concentration of proteins. All other chemicals and reagents were analytical Conclusions grade. In this study, the fexible regions of trypsin were predicted by three methods (MD simulation, FlexPred, and Recombinant plasmid construction FoldUnfold) and the highly fexible region were identifed Te gene sequence of the porcine trypsinogen (GenBank through the combination of three methods. Based on the Accession No. CS583166.1) was synthesized (GENEWIZ Liu et al. Microb Cell Fact (2018) 17:165 Page 8 of 10

Inc., Tianjin, China) and inserted into the vector pPIC9K Measurement of thermostability to construct the plasmid pPIC9K-Try. Five variants Digestion with trypsin is usually carried out at 20–40 °C (residues86–88/D3, residues84–88/D5, residues82–88/D7, [27]. It provides the possibility of carrying out digestions residues80–88/D9, and residues 78–88/D11) were generated at higher temperatures if the porcine trypsin was found by deleting three amino acids, fve amino acids, seven to be remarkably stable at 50 °C. Tus, for the thermal amino acids, nine amino acids and eleven amino acids, stability assay, the purifed trypsin (200 μg/mL protein respectively, from the N-terminal of Asp89 at the Ω-loop in 67 mM NaH2PO4 bufer, pH 7.6) were heat-treated at region. Te gene sequences of variants used in this study 50 °C for 30, 60, 90,120, and 180 min, and then cooled were achieved by overlapping extension PCR. Te ampli- on ice for 30 s. Te enzyme activities were measured fed PCR products and the pPIC9 K vector were digested at 25 °C, and the residual activity was recorded as the with EcoRI and NotI, ligated, and then transformed into percentage of its initial activity. Te frst-order rate con- E. coli Top 10. Te recombinant plasmids were confrmed stant (kd) was obtained by linear regression of ln (resid- by DNA sequencing (GENEWIZ Inc., Tianjin, China), ual activity) versus the duration time. Te half-time of and then transformed in P. pastoris GS115. All the prim- WT and variants at 50 °C were calculated by the equa- ers used to acquire the variants in this study were shown tion. All experiments data were duplicated three times. T 10 in Additional fle 1: Table S3. Te protein half-inactivation temperature ( 50 ) was determined by measuring residual activity at diferent Expression and purifcation of trypsin variants temperatures. Te enzyme solutions were incubated at Te positive clones of the wild-type (WT) and variants the temperature range of 25–85 °C for 10 min and then were inoculated into shake fask containing 25 mL BMGY cooled on ice for 5 min. Finally, the residual activity was T 10 (bufered glycerol-complex) media and cultured at 30 °C determined at 25 °C as described above. Te value of 50 with 250 rpm shaking speed until the OD reached 2–6. was defned as the temperature when the enzyme lost half of its activity after the above treatment. Te experi- Ten centrifuged at 3000×g for 5 min to collect the cells and transferred into a 500 mL conical fask contained ments were repeated three times. 100 mL of BMMY (bufered methanol-complex) medium to induce protein expression for 96 h. As an inducer, methanol was added to a fnal concentration of 1% (v:v) Thermofour assay every 24 h. Te crude enzyme solutions were purifed by Melting temperatures of WT and variants were deter- a SP-Sepharose column (Bona INC., Jinan, China). More- mined with the Termofour method [26, 45]. Sam- over, the protein was eluted using a 0–500 mM sodium ples containing 2.0 μM protein and 5 × SYPRO Orange chloride gradient. Te purifcation of WT and variants (diluted from a 5000 × stock supplied in DMSO) were was performed by AKTA system (GE Healthcare, Swe- prepared in sample bufer (50 mM HEPES, 0.5 mM den). Te fnal concentration of enzymes was measured TCEP and 67 mM NaH2PO4 bufer, pH 7.6). Denatura- through Bradford method, and 15% SDS-PAGE was used tion curves were determined in a LightCyder 480 Real- to certify the eluted protein [43]. time PCR machine (Roche, Mannheim, Germany). Te temperature was ranged from 25 to 95 °C at intervals of Enzyme activity assays 0.5 °C. Te wavelengths for excitation and emission were Te activity of trypsin was determined using Nabenzoyl 465 and 580 nm, respectively. All determinations were l-arginine ethyl ester hydrochloride (BAEE) as a sub- carried out in triplicate. Te data were analyzed using strate, as described previously [44]. Initially, the assay a plug-in named LightCycler Termal Shift Analysis was the trypsinogen activated by enterokinase. Further- (Roche, Mannheim, Germany) and melting temperature more, BAEE was hydrolyzed by trypsin to Nabenzoyl (Tm) was acquired by taking a negative frst derivative l-arginine (BA). Trypsin reaction was conducted in a plot. 3 mL of assay bufer (67 mM NaH PO bufer, contain- 2 4 Measurement of optimum temperature ing 0.25 mM BAEE, pH 7.6). Te change in absorbance at 253 nm was measured in a UV-765 PC spectropho- Te optimal temperature was assayed at various tempera- tometer (Youke Co., Ltd, Shanghai, China). One unit tures ranging from 25 to 85 °C at intervals of 5 °C. For (U) was defned as enzyme required for producing an WT and variants, the optimal activity assayed was considered as 100%. ∆OD253 of 0.001 per minute at 25 °C. Liu et al. Microb Cell Fact (2018) 17:165 Page 9 of 10

Determination of kinetic parameters Abbreviations RFS: rigidifying fexible sites; MD: molecular dynamic; RMSD: root mean square Kinetic parameters (Km and kcat) were measured at deviation; RMSF: root mean square fuctuation; SVM: support vector machine; various concentrations of substrate BAEE in 67 mM BMGY: bufered glycerol-complex; BMMY: bufered methanol-complex; BAEE: NaH PO bufer (pH 7.6). Te kinetic parameters were Nabenzoyl l-arginine ethyl ester hydrochloride; TCEP: Tris(2-carboxyethyl) 2 4 phosphine; ANS: 8-Anilino Naphthalene Sulfonic acid. calculated by linear regression analysis of Lineweaver– Burk double-reciprocal plot. Te measurements were Authors’ contributions performed by three times. All of measurements were per- LL, YRG and HH designed the experiments. LL and KD performed the experiments. LL, HRY and ZYW wrote the manuscript. All authors read and approved formed by three times. the fnal manuscript.

Circular dichroism analysis Author details 1 Department of Biochemical Engineering, School of Chemical Engineering Circular dichroism (CD) experiment was conducted on and Technology, Tianjin University, Tianjin 300350, China. 2 Key Laboratory a J-810 spectrometer (Jasco, Japan). Samples (0.2 mg/ of System Bioengineering, Ministry of Education, Tianjin University, Tian- 3 mL) in 10 mM Tris–HCl bufer (pH 8.0) were added to jin 300350, China. Collaborative Innovation Center of Chemical Science and Engineering, Tianjin 300350, China. 4 Present Address: Department a 1 mm path length quartz cuvette for CD measurement. of Biochemical Engineering, University College London, Gordon Street, Lon- Spectra were collected with a scan speed of 200 nm/min don WC1H 0AH, UK. and a response time of 1 s. Te spectra data were moni- Acknowledgements tored from 190 to 260 nm and were averages of three Not applicable. scans. Te secondary structures of WT and variants were analyzed by the CDPro software (http://sites.bmb.colos Competing interests The authors declare that they have no competing interests. tate.edu/sceeram/CDPro/). Availability of data and materials Fluorescence spectroscopy analysis All data generated or analyzed during this study are included in this published article. Te intrinsic fuorescence spectra of trypsin were detected on a Hitachi F-2500 Fluorescence Spectropho- Consent for publication tometer (Hitachi, Japan) at a concentration of 10 μg/mL. Not applicable. Samples were excited at 280 nm, and the emission was Ethics approval and consent to participate recorded ranging from 300 to 400 nm. Te slit widths Not applicable. were 10 nm. All experiments were carried out at room Funding temperature (25 °C), while all required background cor- This study was supported by the National Science and Technology Major rections were made. Projects of China (Grant No. 2014ZX09508006-002-002).

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