CHEMICAL ENGINEERING TRANSACTIONS VOL. 79, 2020 A publication of The Italian Association of Chemical Engineering Online at www.cetjournal.it Guest Editors: Enrico Bardone, Antonio Marzocchella, Marco Bravi Copyright © 2020, AIDIC Servizi S.r.l. ISBN 978-88-95608-77-8; ISSN 2283-9216 Rapid Screening of Additive Formulations for Enhancing Xylanase Stability in Pulp Bleaching and Storage Conditions Nongluck Jaitoa, Lily Eurwilaichitrb, Thidarat Nimchuaa,* a Enzyme Technology Research Team, Biorefinery and Bioproduct Technology Research Group, National Center for Genetic Engineering and Biotechnology (BIOTEC) 113 Thailand Science Park, Pahonyothin Rd., Khlong Luang, Patumthani 12120, Thailand b Thailand Bioresource Research Center (TBRC), National Center for Genetic Engineering and Biotechnology (BIOTEC) 143 Thailand Science Park, Pahonyothin Rd., Khlong Luang, Patumthani 12120, Thailand thidarat.nim@biotec.or.th Enzyme deactivation is a significant parameter to restrain the industrial commercialization of enzymes. In this study, a high-throughput screening technique using differential scanning fluorimetry (DSF) incorporated with enzyme activity analysis was thus developed in order to identify a suitable additive formulation for promoting enzyme stability in terms of conformational stability and functional activity. To demonstrate the concept feasibility, endo-xylanase obtained from metagenome of termite gut-inhabiting bacteria (XYN12) was used as a study model and several group of additives including salts, polymers, polyols and sugars at various concentrations and different combination patterns (single, double and triple pairing) were applied for investigating their effects on enzyme stability. As a result, a total of 80 reaction mixtures were retrieved including a native control (enzyme without any additives). DSF assay was performed using real-time PCR instrument to detect unfolding of protein in the presence of a florescence dye. Accurate melting temperature (Tm) values of each enzyme formulation were calculated using Boltzmann equation and compared to the Tm of the native control in order to find ∆Tm. After that, the residual activity of xylanase in all reaction mixtures were measured using pHBAH method in automated liquid handling system. For single additives, glycerol had a stabilizing effect to the target enzyme, as observed by a significant increase in the ∆Tm, while (NH4)2SO4, CaCl2, NaCl destabilized it. In addition, cocktail additives included glycerol, PEG4000 and trehalose dramatically enhanced the enzyme stability by positive shifting of Tm from 54 to 56 οC and increasing of relative activity by more than 4 folds compared to single additive. Storage stability of the enzyme was improved when mixed with this cocktail additive as indicated by remaining of the enzyme activity (86 %) after kept at 30 οC, pH 7.4 for 262 days, whereas native control lost almost of its activity within 40 days when stored at the same condition. 1. Introduction Enzymes have been used for a long time in several applications and recognized as an alternative greener technology for reducing or replacing the use of harsh chemicals in the industrial processes (Masutti et al., 2012). Although enzymes are remarkable catalysts in terms of high catalytic and specific activity with ability to function under mild conditions, however they are generally unstable and can be inactivated rapidly by heat, chemical agents, autolysis (proteases) or ionic strength, resulting in enzyme denaturation during manufacturing, storage and application in industry. Generally, increase of the enzyme stability could be achieved by the addition of the protective and stabilizing additives such as salts, polyols, polyelectrolytes, bovine serum albumine (BSA),organic osmolytes, sugars and others. However, the selection of the suitable additive formulation relies on the nature of the enzyme and requires automated- and integrated- screening platform to develop the reliable and appropriate formulation at a record speed under a variety of stress conditions. In this study, the stability of β-1,4-endoxylanase enzyme obtained from metagenome of termite gut inhabiting microbes (XYN12) was of interest in order to use as a bleach boosting agent in pulp bleaching process (Nimchua et al., 2012). In enzymatic bleaching process, xylanases, particularly β-1,4-endoxylanase DOI: 10.3303/CET2079052 Paper Received: 19 September 2019; Revised: 26 November 2019; Accepted: 6 March 2020 Please cite this article as: Jaito N., Eurwilaichitr L., Nimchua T., 2020, Rapid Screening of Additive Formulations for Enhancing Xylanase Stability in Pulp Bleaching and Storage Conditions, Chemical Engineering Transactions, 79, 307-312 DOI:10.3303/CET2079052 307 are the core enzymes responsible for partial hydrolysis of xylan in pulp structure through the random cleavage of β-(1,4) glycosidic linkages of heteroxylan backbone (Ashikin et al., 2017), which leads to the removal of xylan in lignin-carbohydrate complexes (LCC) produced during pulping process. By eliminating of the LCC, the enzyme-pretreated pulp is thus more permeable to chemical bleaching, leading to a significant reduction in the dosage of chemicals required for bleaching with improved pulp qualities included brightness and strength. However, an aggressive operating conditions of pulp bleaching process at alkaline pH and relatively high temperature range are considered to affect the xylanase stability. Although the functional properties of this enzyme is most effective in the temperature range of 40-60 oC and pH 8-9 (Nimchua et al., 2012), but almost half of its activity lost within 20 min in these conditions. Therefore, the aim of this work was to improve the stability of the XYN12 during pulp-bleaching simulated conditions and long-term storage by using the high- throughput approach for rapid screening of appropriate additive stabilizers. 2. Experimental 2.1 Substrate and additives Beechwood xylan purchased from sigma (Karlsruhe, Germany) was used as a substrate in all experiments. The salts, sugars and glycerol used in these experiments were obtained from Carlo erba (Val de Reuil Cedex, France). Poly (ethylene glycol) (PEG) with molecular mass of 3350 and 4000 was obtained from sigma-aldrich (Darmstadt, Germany). 2.2 Enzyme preparation The enzyme was produced from E. coli RosettaTM strain transformed with pET28a(+) (Invitrogen, USA) harboring gene encoded endo-xylanase obtained from metagenome of termite gut-inhabiting bacteria (XYN12). To achieve high productivity of XYN12, high cell density fermentation of the E. coli expressing XYN12 was carried out in 5 L bioreactor by maintaining dissolved O2 concentration at 30 % of saturation. The total gas flow rate was restricted between 1 and 2 VVM and the pH was controlled at 7.0 ± 0.2 by adding 25 % (v/v) NH4OH and 10 % (v/v) H3PO4. All experiments were conducted at 37 ± 0.5 °C (growth and induction phases) by controlling the temperature of the water re-circulating in bioreactor jacket. After culturing, the cells were harvested from 1 L of culture broth by centrifugation at 7,000 rpm at 4 °C for 20 min, then re-suspended in 200 mL of 50 mM sodium phosphate buffer (pH 7.4), and disrupted using high pressure homogenizer (Microfluidizer/M-110P, PCL Holding, USA). Cell-free extract was prepared by centrifugation in order to separate cell debris at 19,000 rpm at 4 °C for 30 min. The target enzyme was further purified from the crude extract using a HisTrap column (GE Healthcare, USA) following the manufacturer’s protocol. After that, the purified enzyme was concentrated on an Amicon Ultrafiltration Unit (Millipore, Billerica, USA) and determined the protein concentration by Bradford assay (Bio-Rad, USA) using bovine serum albumin (BSA) as a standard. 2.3 Enzyme activity assay Xylanase activity was determined based on the amount of released reducing sugars by the Para- hydroxylbenzoic acid hydrazide (pHBAH) method modified from Mellitzer et al. (2012). Briefly, the assay solutions contained 20 µl of proper dilutions of the enzyme and 30 µl of 1 % (w/v) of beechwood xylan in 100 mM Tris-HCl buffer, pH 8.5 as the substrate were transferred to a 96-well plate. The plates were then incubated at 55 ºC for 10 min. The quantity of reducing sugars was determined by adding of 150 µl of 1 % (w/v) pHBAH solution (prepared freshly by diluting a stock solution of 5 % (w/v) pHBAH (Sigma, USA) in 0.5 % (v/v) HCl with 0.5 M NaOH at a ratio of 1:4 v/v) into the reaction plates. After that, 120 µl of the reaction mixtures were transferred to a new 96-well PCR plate and incubated at 95 ºC for 5 min prior to cooling at 4 ºC in PCR thermal cycler (Applied Biosystems, USA). The absorption in the reaction samples was investigated at 410 nm in a spectra MAX M5 (Molecular Device, USA). All 96-well pipetting steps were performed by robotic liquid handler (Multiprobe II PLUS EX, PerkinElmer, USA). One unit of enzyme activity was defined as the amount of enzyme which releases 1 µmol of reducing sugar per minute under the assay conditions and was calculated by use of the formula: Where ∆OD410 is the absorbance at 410 nm of the sample subtracted from blank, D is dilution factor, V1 is total reaction volume (μL), V2 is enzyme volume, m is the slope of the standard curve (μmol/ml) and t is the incubation time (min). =Activity (Ս/ml) ∆OD D t × V1 V 2 410 × m × 308 2.4 Thermal shift determination To investigate the influence of additives towards enzyme thermal stability, 20 µl reactions of 50X SYPRO (2 µl) Orange dye (Sigma, USA), test additives (Table 1; 5-15 µl), 1 mg/ml protein (2 µl) and distilled water were mixed in a 96-well PCR plate. DSF assay was performed according to the protocol of Seabrook and Newman (2013) by heating the plates in RT-PCR (Bio-RAD, USA) under FRET (Fӧrster resonance energy transfer) scanning mode at raising rate of 0.5 ºC/min starting from 25 to 95 ºC. The controls were prepared as the same manner using distilled water instead of test compound. The wavelengths at 470 and 570 nm were used for excitation and emission, respectively. The changes of fluorescence intensity in the reactions were detected simultaneously by a fluorescence reporter molecule at a particular wavelength in every cycle and was plotted as a function of temperature, generating a sigmoidal curve of two-state transition of enzyme. The temperature midpoint for enzyme unfolding transition (Tm) was calculated from Boltzman equation as follows: where I0 and I1 are the values of minimum and maximum intensities respectively, whereas a is the slope of the curve within Tm. The Tm value measured from enzyme-additive mixture was compared with the Tm value of the control, and the difference in unfolding temperature (∆Tm) was calculated. After DSF assay, all reaction mixtures were determined for the remaining xylanase activity using pHBAH method as described above. All samples were carried out in triplicate. Table 1: List of additives used in the thermal shift assay Additive pairing Additive group Additive compound Concentration single salts (NH4)2SO4, CaCl2, or NaCl 50, 100, 150, 200 and 250 mM sugars Fructose (Fru), Maltose (Mal), or Trehalose (Tre) 10, 20, 30, 40 and 50 mM polyol Glycerol (Gly) 5, 10, 15, 20 and 25 % (v/v) polymers PEG3350 (P3350) or PEG4000 (P4000) 0.1, 0.2, 0.3, 0.4 and 0.5 % (w/v) double glycerol and sugars Fructose, Maltose or Trehalose glycerol:sugar = 5 or 25 % (v/v): 10 or 50 mM glycerol and polymers PEG3350 and PEG4000 glycerol:polymer = 5 or 25 % (v/v): 0.1 or 0.5 % (w/v) triple glycerol, polymers and sugars PEG3350 or PEG4000 and Fructose, Maltose or Trehalose glycerol:polymer:sugar = 5 or 25 % (v/v):0.1 or 0.5 % (w/v):10 or 50 mM 2.5 Effect of additives on enzyme stability The cocktail additive formulation (0.5 % w/v PEG4000, 25 % v/v glycerol and 50 mM trehalose) showed the best stabilizing effect on enzyme by means of positive shift of Tm and remaining activity was added to 1 mg/ml xylanase, pH 8.5 (50 mM Tris-HCl) for determination its efficiency in the pulp bleaching simulated conditions (pH 8.5, 45 ºC for 1 h). Control was prepared in parallel at the same enzyme concentration without addition of additives. The residual activity was measured after incubation at 0, 10, 20, 30, 40, 50 and 60 min. 2.6 Storage stability of native and xylanase incorporated with additives Enzyme samples containing 10 mg/ml xylanase was mixed with the selected additive solution (0.5 % w/v PEG4000, 25 % v/v glycerol and 50 mM trehalose). The reaction mixture was incubated in 50 mM sodium phosphate buffer, pH 7.4 at 30oC, for 262 days Samples were drawn from the reaction mixtures at proper time intervals for enzyme activity assay. Control was prepared in the same manner without any additives. All samples were conducted in triplicate and the results were reported as mean values. 3. Results 3.1 Enzyme production After high cell density fermentation, the E. coli expressed a high level of the alkalo-tolerant xylanase (activity of 10,179 u/ml and specific activity of 481 u/mg) on synthetic medium (José da Silva et al., 2013) incorporated with 80 g/L lactose as an inducer after 16 h of cultivation. The induced protein with His6 tag was further = γ I 0 + I 1 − I 0 + T m − T x a 1 e 309 purified using His-trap column and the target protein fraction was eluted with 200 mM imidazole. The active fractions were pooled and concentrated by an Amicon Ultrafiltration Unit, resulted in a target enzyme yield of 8.63 % and purification fold of 8.13. The purified protein appeared as a single band of approximately 35 kDa against the protein marker on 10 % SDS-PAGE (Figure 1). Figure 1: Analysis of the purified enzyme using SDS–PAGE (10 % polyacrylamide gel). Lane M protein marker, Lane 1 purified recombinant xylanase. 3.2 Additive screening In this study, four additive groups at different concentrations and combination patterns (Table 1) which generated a total of 80 condition treatments have been investigated for the stabilizing effect towards the protein using DSF and pHBAH assays. Among the single additives tested, glycerol was the only compound that gave the greatest ∆Tm value (≥ 0.5 ºC; Figure 2A), while other additives gave a positive shift of ∆Tm value less than 0.5. All tested salts including (NH4)2SO4, CaCl2, NaCl induced a negative shift of ∆Tm value (data not shown). A positive ∆Tm can be linked to an increase of conformational compactness, while negative ∆Tm displays a sign of misfolding. As the result in enhancing of protein stabilization by single additives is rather limited, thus a new approach that use of cocktail additives by combining more than one compound to significantly increase the protein stability has been demonstrated. In cocktail approach, glycerol, the best stabilizer in single additives was combined with the other 2 groups of additives (polymers and sugars) in double and triple pairing, which allowed approximately 34 treatment conditions to be investigated. After screening, only 13 of 80 additive formulations were found to stabilize the enzyme by increasing of ∆Tm (≥ 0.5 ºC; Figure 2A) and the relative activity of enzyme (≥ 100 %; Figure 2B). Of all combined formulations tested, almost of the triple formulations (no. 9-13; Figure 2A) showed the maximal ∆Tm of 2 ºC, while double additive mixtures (no. 2-7; Figure 2A) showed relatively less ∆Tm values, indicated that triple combinations appear to be more favourable for protein stabilization. Among the triple mixtures, the formulation containing glycerol, PEG4000 and trehalose gave the highest relative activity (442.2±6.9 %; Figure 2B) compared to the control, thus it was selected for study it efficiency in stabilization of the enzyme in pulp-bleaching simulated conditions and storage. 3.3 Effect of the selected additive mixture on xylanase stability The stabilizing efficiency of the selected cocktail additive (0.5% w/v PEG4000, 25% v/v glycerol and 50 mM trehalose) on the activity of enzyme at pulp bleaching simulated condition was investigated with respect to the temperature of 45 ºC and pH 8.5. The enzyme activity of native and additive-combined enzyme was measured by pHBAH assay after incubation at 10 min-intervals for 1 h. After adding the cocktail additive, the activity of enzyme revealed that the use of additives led to an increase of xylanase activity by approximately 30 % compared to the activity of control sample before starting the incubation (Figure 3). In addition, almost of the enzyme activity was rapidly loss (more than 90%) after incubation at 45 ºC for 40 min in the enzyme without additives, whereas no sign of inactivation of xylanase activity was observed in additive incorporated enzyme (Figure 3). The obtained results showed consistent trend as reported in the study of Bӧrjesson et al. (2006) that revealed the synergistic interactions of enzyme and its additive formulations by increasing the enzymatic hydrolysis of lignocellulose. In fact, addition of polyols, sugars and polymers to liquid solutions of enzymes has been demonstrated in enhancing strength of the hydrophobic interactions among non-polar amino acid residues, leading to protein stiffening and promoting the enzyme stability against operating environments. In this study, the result suggested that the presence of the synthesis additive mixture has a positive stabilizing effect on enzyme, making it more resistant to unfolding environments, particularly industrial pulp-bleaching process. 310 3.4 Storage stability of xylanase The storage stability of native and enzyme incorporated with the additives (0.5% w/v PEG4000, 25% v/v glycerol and 50 mM trehalose in 50 mM Sodium phosphate buffer, pH 7.4) was investigated at 30 ºC for 262 days. The activity profile of xylanase showed that negligible activity was detected from native enzyme after 40 days of storage, while additive mixture treated xylanase remained a relative activity of more than 86 % throughout the preserved time. The remarkable increase in storage time by the designed additive mixture might be explained by a preferential exclusion theory, proposes that the addition of additives such as PEG4000, glycerol and trehalose to the bulk water solution secludes water molecules away from the enzyme surface, leading to reduced hydration radius and increased compactness of the enzyme molecules, consequently stabilizing effect. Figure 2: (A) Difference of the midpoint temperature (∆Tm) and (B) relative activity of XYN12 in the selected additive formulations that showed ∆Tm ≥ 0.5 ºC and relative activity ≥ 100%. For abbreviation, see Table 1. Figure 3: Activity profile of xylanase in presence and absence of additive mixture at pulp bleaching simulated conditions (pH 8.5 and 45 ºC for 1 h) 0 20 40 60 80 100 120 140 160 180 0 10 20 30 40 50 60 R e la tiv e a ct iv ity ( % ) Time (min) Native enzyme Enzyme additive mixture 311 Figure 4: Activity profile of xylanase in presence and absence of additive mixture at storage conditions (50 mM sodium phosphate buffer pH 7.4 and 30 ºC for 262 days) 4. Conclusions This research assesses the stabilization efficiency of several additives in single and cocktail combinations on conformation and biological function of the alkalo-tolerant xylanase obtained from termite gut using DSF incorporated with activity analysis. The DSF assay recognized as a rapid and simple method, was successfully established to carry out a high-throughput screening technique for potential additives that helps to stabilize and enhance the enzyme activity. The data obtained from DSF assay can be used in complement with the data gathered from pHBAH method, where the same set of additives was applied to determine the residual activity. By co-considering of the DSF and pHBAH datasets, additive formulations showing positive result in both assays become the potential candidates for further study in stabilizing the enzyme at operating and storage conditions. In this study, compared to single additive approach, the DFS and pHBAH results revealed that the triple pairing of glycerol with polymers and sugars had a dramatic stabilizing effect, increasing the ∆Tm by up to 2 ºC and the activity of at least 300 %. The best stabilizing performance in pulp- bleaching simulated conditions was observed from the additive mixture comprising glycerol, PEG4000 and trehalose. Correspondingly, it appeared to be appropriate for long-time storage stabilization of the target enzyme at 30 ºC. Therefore, this result clearly establishes the proof of principle that the “cocktail approach” in which more than one additive are used, is a potential method to efficiency enhance the thermal stability and prevent the activity loss of protein in both pulp-bleaching simulated and storage conditions. Acknowledgments This study was supported by a post-doctoral fellowship program from the National Center for Genetic Engineering and Biotechnology (BIOTEC, Patumthani, Thailand) and a research grant (P-13-50429) from the National Science and Technology Development Agency (NSTDA, Patumthani, Thailand). References Ashikin N., Wahab N., Illias R., Fuzu S., 2017, Comparison of Thermostable Xylanase Production by Escherichia coli Immobilised onto Different Nanoparticles, Chem. Eng. Transact., 56, 1825-1830. Bӧrjesson J., Peterson R., Tjerneld F., 2006, Enhance enzymatic conversion of softwood lignocellulose by poly (ethylene glycol) addition, Enzyme Microb. Technol., 40, 754-762. Nimchua T., Thongaram T., Uengwetwanit T., Pongpattanakitshote S. Eurwilaichitr L., 2011, Metagenomic analysis of novellignocellulose-degrading enzymes from higher termite guts inhabiting microbes, J. Microbiol. Biotechnol., 22, 462–469. Masutti D.C., Borgognone A., Setti L., 2012, Production of Enzymes from Rice Husks and Wheat Straw in Solid State Fermentation, Chem. Eng. Transact., 27, 133-138. Mellitzer A., Glieder A., Weis R., Reisnger C., Flicker K., 2012, Sensitive high-throughput screening for detection of reducing sugars, Biotechnol. J., 7, 155-162. Seabrook S.A., Newman J., 2013, High-throughput thermal scanning for protein stability: making a good technique more robust, J. Am. Chem. Soc., 15, 387-392. 0 40 80 120 160 200 0 30 60 90 120 150 180 210 240 270 R e la tiv e a ct iv ity ( % ) Time (day) Native enzyme Enzyme additive mixture 312