Characterization of recombinant Bacillus halodurans CM1 xylanase produced by Pichia pastoris KM71 and its potential application in bleaching process of bagasse pulp

Thermoalkalophilic xylanases promise potential application in pulp biobleaching to reduce the use of toxic chlorinated chemical agents, which are harmful to the environment. In this study, a thermoalkalophilic endoxylanase gene (bhxyn3) originating from Indonesian indigenous Bacillus halodurans CM1 was cloned into yeast expression vector pPICZα A and expressed in Pichia pastoris KM71 under the control of AOX1 promoter. Recombinant P. pastoris expressed the highest final level of xylanase (146 U/mL) on BMGY medium after five days of cultivation. Optimization of xylanase production on a small scale was carried out by varying the methanol concentrations and the optimal xylanase production by the recombinant P. pastoris was observed in the culture with 2% (v/v) methanol after four days of the induction phase. The recombinant xylanase (BHxyn3E) was thermotolerant and alkalophilic, with an optimal temperature at around 55‐65 °C and under pH 8.0. The enzyme activity was slightly induced by K+, Fe2+, and MoO4. Enzymatic bleaching of bagasse pulp with no prior pH adjustment (pH 9) using BHxyn3E at 200 U/g oven dried pulp increased the lightness index (L*) and changed substantially the color a index (a*); however, the treatments did not change the whiteness index in a significant way. Therefore, further optimization and assessment such as adjustment of incubation temperature and pH in biobleaching were needed to reduce the use of harmful chemical agents in industrial applications.


Introduction
Remarkable interest has been focused on integrating hy drolytic enzymes, such as xylanases, that degrade xylan components in plant cell walls into noncomplex sugars in industries, particularly in the pulp and paper industry. Xylanases (EC 3.2.1.8) are produced by microorganisms and considered as the key enzymes that hydrolyze β1,4 xylans to lead to the degradation of complex polysaccha rides (Jeya et al. 2009). On the other hand, the enzymes have widespread potential applications to be used in such a wide range of industrial fields (textile, feed, beverage, and biofuels) since its ability to facilitate the removal of xy lan through partial hydrolysis of lignin carbohydrate com plexes, thereby enabling lignin removal from raw materi als allowing it to be processed in the next stages of man ufacturing. In the pulp and paper process, this fragmenta tion of xylan polymer allows residual lignin attached to the remaining xylan complex, to be exposed by the efficient used bleaching agents. Thus, pretreatment of pulp using xylanases can reduce the load of chlorine and chlorine based compounds in the subsequent chemical bleaching stages. As a side effect, the utilization of cellulasefree xy lanase enable to enhance several physical pulp properties, such as brightness, tensile strength, and tear factor with out affecting the cellulose fiber strength of paper products (Zhao et al. 2015).
Currently, xylanases that are intended to be used in the industrial bleaching process must be robust to operate in extreme conditions since most operations were conducted under high temperature and strong alkaline pH for at least anhour long. Hence it must be adaptable in such an ex treme environment. Xylanases are produced by members of bacteria and fungi, commonly in group of bacterial xy lanases display an active at mesophilic temperatures (be low 50°C) in acidic or neutral pH (Walia et al. 2017).
Although several xylanases with thermo and alkali stabil ity were isolated, there is still high demand for thermo alkalophilic xylanases for application in paper processing (Bhardwaj et al. 2019).
In the previous study, thermoalkalophilic Bacillus halodurans CM1 from Cimanggu Hot Spring located in West Java area in Indonesia has been isolated. Xylanase produced by this bacterial strain was active in alkaline con dition with the optimum activity at 70°C and at pH 9 (Ulfah et al. 2011). Cloning of an endoxylanase encod ing gene (1191 bp) from this strain using pET21d vector in Escherichia coli BL21 star have been previously con ducted, and the obtained recombinant protein of BHxyn3E (46.7 kDa) showed a potential to promote xylan reduction in deinking process of waste paper (Helianti et al. 2018). However, the level of the secreted enzyme from recom binant E. coli was expressed very low. To increase en zyme productivity, changing the recombinant expression system is necessary. In this study, the gene encoded tar get xylanase was thus expressed into Pichia pastoris ex pression system. The yeast P. pastoris can neither utilize nor degrade xylan, but it is able to boost a higher expres sion level of heterologous gene extracellularly (Mohd Dali et al. 2011) with a high purity level of target protein after culturing in less expensive culture media without the pres ence of antibiotics for upscaled mass production (Shang et al. 2017). The cloned gene is also directly integrated into its chromosomal DNA through common genetic ma nipulation, thereby reducing the cost for plasmid mainte nance (Chantasingh et al. 2006). Hence, all mentioned features have made P. pastoris very useful for a protein expression system.
Considering these facts, our present research studies aim to clone the thermoalkalophilic xylanase gene orig inating from B. halodurans CM1 in an expression vec tor with a strong promoter, pPICZα A, and express it in P. pastoris KM71 to evaluate its performance in the pulp bleaching process. The enzyme production and biochemi cal characterization of the obtained xylanase were investi gated. Thus, crude xylanase was employed to evaluate its effectiveness in the biobleaching of bagasse pulp, which is one of the most available papermaking lignocellulosic fiber resources in many developing countries, including Thailand and Indonesia (Sudaryanto et al. 2008; Hidayat andYasuyuki 2012).

Culture preparation
E. coli DH5α (Invitrogen, Waltham, Massachusetts, USA) was used for cloning host and plasmid propagation. Fol lowing the transformation, E. coli DH5α transformants were selected at 37°C on the lowsalt Luria Bertani (LB) agar medium supplemented with 25 μg/mL zeocin. P. pas toris KM71 strain (Invitrogen, Waltham, Massachusetts, USA) was used as the host for xylanase expression us ing YPD containing 10 g/L yeast extract, 20 g/L peptone, and 20 g/L glucose to grow. The same media but with an additional 100 μg/mL zeocin was used for P. pastoris transformants selection. Recombinant plasmid pET21d harboring the target xylanase gene originating from B. halodurans CM1 (bhxyn3) with the accession number of KU759320 (Helianti et al. 2018) has been used for the insert, while pPICZαA for expression vector (Invitrogen, Waltham, Massachusetts, USA). All primers used in the study were synthesized and supplied by Integrated DNA Technologies, Inc. (IDT, Singapore).

Construction of the recombinant plasmid (pPICZαA-bhxyn3) and gene integration for secreted xylanase expression
An E. coli/ P. pastoris shuttle vector, pPICZα A, was used to express the target xylanase (BHxyn3E) extracellularly. The bhxyn3 gene is a 1191 bp DNA fragment encoding the mature BHxyn3E peptide (397 aa) (Helianti et al. 2018). The bhxyn3 gene was amplified using the following gene specific primers that included the forward primer (XynF; 5'GCATGAATTCGCTCAAGGGGACCAAAATCC 3') and the reverse primer (XynR; 5' CATATCTAGAGCATCGATAATTCTCGAGTAAGCA GGTTTC3') with its respective EcoRI and an XbaI restriction sites (underlined). The PCR program was started by initial denaturation at 98°C for 30 s, followed by 35 cycles of 98°C for 10 s, annealing at 55°C for 30 s and 72°C for 90 s and ended by the final extension at 72°C for 10 min. Afterward, the bhxyn3 gene obtained from PCR amplification was digested with EcoRI and XbaI (Thermoscientific, Waltham, Massachusetts, USA) and cloned into pPICZα A vector located right after AOX promoter through restriction cloning. The selected recombinant plasmid, pPICZαAbhxyn3 was confirmed by restriction analysis and sequencing.
After the sequence of bhxyn3 ORF was verified, the obtained recombinant plasmid pPICZαAbhxyn3 was lin earized with PmeI for P. pastoris integration. Linearized DNA was then transformed into P. pastoris KM71 by elec troporation, thus allowed the integration of cloned gene into its genomic DNA. The homolog sequence of AOX1 promoter in the plasmid and the genomic DNA of P. pas toris initiates the integration of the whole gene cassette through homologous recombination. Positive recombi nants were selected and monitored by colony PCR using 5'AOX1 and 3'AOX1 primers.

Expression of recombinant xylanase in P. pastoris
An overnight culture was prepared by cultivating a single colony of transformant into 5 mL of YPD at 30°C with shaking at 250 rpm. Seed culture was then added into 5 mL of fresh buffered minimal glycerol complex medium (BMGY; 1% yeast extract, 2% peptone, 0.1 M potassium phosphate buffer pH 6.0, 1.34% yeast nitrogen base, 0.4 µg/mL biotin, and 1% glycerol) containing zeocin (100 μg/mL) at 30°C with constant shaking (250 rpm) until cell density of 56 OD 600 was reached (Chantasingh et al. 2006). After that, the cell pellet was collected by centrifu gation at 5000 × g for 10 min and resuspended in about 1/5 volume of the initial culture of buffered minimal methanol medium (BMMY; 1% yeast extract, 2% peptone, 0.1 M potassium phosphate buffer pH 6.0, 1.34% yeast nitrogen base, 0.4 µg/mL biotin, and 2% methanol). The target gene was regulated under AOX1 promoter, which required methanol as an inducer. Since methanol is very easy to evaporate, in order to maintain its concentration during the induction phase, 100% methanol was regularly added into the culture every 24 h to a final concentration of 3% (v/v) (Chantasingh et al. 2006). The crude extract from culture supernatant from day 1 to 6 was subjected for activity as say and SDSPAGE analysis (Chantasingh et al. 2006).

Effect of zeocin on expression level of recombi nant xylanase
To investigate the effect of zeocin on xylanase expres sion, positive recombinant (P. pastoris KM71/pPICZαA bhxyn3) was grown in YPD medium overnight before about 0.5% (v/v) seed cultures were inoculated into 100 mL BMGY medium incorporated with zeocin (100 μg/mL) in a 1L flask and grown at 30°C overnight with shaking condition (250 rpm) or until the cell density reached 0.50.8 OD 600 culture. Afterward, the media was changed by the resuspension of harvested cells with 20 mL of BMMY medium to start the induction phase with the daily supplementation of methanol to the same final con centration of 2% (v/v) for four days. The culture broth was collected on days 3 and 4 to determine the xylanase activity. An additional experimental sample was also con ducted using BMGY medium devoid of zeocin.

Effect of methanol concentrations on the ex pression level of recombinant xylanase
The culture of the P. pastoris in BMMY medium was per formed in the same manner as described above, except the final concentrations of methanol were varied from 1 to 4% (v/v) throughout the induction phase.
Xylanase assay was performed by determining the re ducing sugar liberated from beechwood xylan (Sigma Aldrich, Munich, Germany) according to the method de scribed by Bailey et al. (1992) with triplicates. The re action mixture was composed of 20 µL of recombinant xylanase and 320 µL of 50 mM TrisHCl buffer (pH 8.0) containing 1.0% (w/v) beechwood xylan. After 5 min in cubation at 60°C, the reaction was cooled on ice to stop the reaction. The amount of reducing sugars liberated in the mixture was determined by detecting 3, 5dinitrosalicylic acid (DNS) reagent as xylose equivalents at 540 nm. One unit (U) of xylanase activity was defined as the amount of enzyme that releases reducing sugar at the rate of 1 µmol per minute under the assay conditions.

Effect of pH and temperature on enzyme activ ity and stability
The effect of pH and temperature on xylanase activity was investigated as described by Nimchua et al. (2012). Briefly, the optimal pH was determined by executing the enzyme activity assay in the pH range of 5.0 to 10.0. The buffers used were 50 mM sodium citrate (pH 5.0), 50 mM sodium phosphate (pH 6.0 to 8.0), 50 mM TrisHCl (pH 8.0 to 9.0) and 50 mM GlycineNaOH (pH 9.0 to 10.0), respectively. Assays at different temperatures were per formed at the optimal pH over a temperature range of 30 to 80°C. The pH stability of xylanase was measured by pre incubating the enzyme in various pHs ranged from 6 to 10 for one h, followed by activity determinations. Thermal stability was monitored by heating the enzyme for differ ent times (15, 30, 45 and 60 min) at various temperatures (50, 60 and 70°C). In the stability assays, the enzyme sam ples were determined for remained activity instantly after incubation.

Effect of ions and additives on xylanase activity
To study the effect of ions and additives on xy lanase activity, various ions, detergents, polymers and other additives were supplemented into the enzyme mixtures with final concentrations of 1 mM ( , 0.02% (w/v) for polymers (PEG4000 and PEG8000), 15% (w/v) for trehalose and 50% (v/v) for glycerol in 50 mM TrisHCl buffer (pH 8.0). The residual xylanase activities in reaction mixtures con taining ions and other additives were determined at 60°C for 60 and 180 min. The enzyme reaction without additive was used as the control.

Optimization of enzyme dosage and additive formulation for biobleaching
In this study, optimization of related reaction parameters for biobleaching was investigated as followed and using unbleached bagasse pulp from the Thai local pulp and paper company. Enzyme dose was optimized for pulp bleaching by treating the unbleached bagasse pulp with various enzyme concentrations, ranging from 0 to 200 U/g oven dried (OD) pulp. All the experiments were carried out using 10 g OD pulp in a plastic bag at 10% pulp con sistency under the temperature of 60˚C for one h with no pH adjustment (the pH of pulp solutions was 9.0 af ter preparing the bleaching reactions). Pulp consistency is measured by the percentage by weight of dry substances (in this study bonedry (%)). According to TAPPI T240, the value of pulp consistency used is essential as it defines control of the percentage of dry fibers in 100 g of pulp water mixture. Effect of nonionic surfactants and wetting agent to wards enzyme efficiency in pulp bleaching was studied by carrying out the enzymatic treatment of the unbleached pulp (0.075 g OD pulp) in 50mL test tube at 7.5% pulp consistency without pH adjustment. The treated enzyme solutions at optimal dosage were mixed with different sur factants (Tween 20, Tween 80, Triton X100, or Lutensol) and wetting agent (Sanmorin, Sanyo Kasei, Thailand) at the concentration of 0.25% (w/v) and immediately used the pulp at 60°C for 1 h under shaking (200 rpm). The ef fect of additive concentrations towards xylanase activity in bleaching of bagasse pulp was investigated using a se lected surfactant obtained from the screening experiment. The enzymatic treatment of the pulp was performed in the same manner as mentioned above, except doses of the se lected additive ranging from 0 to 2% (w/v).
The bleaching solutions were collected after enzyme treatments by filtering through Supor ® PES Membrane (Pall Corporation, Bangkok, Thailand) to remove the pulp debris and any particulates. The amount of re ducing sugar appeared in the filtrates was quantified us ing DNS assay by Miller (1959), with xylose used as a standard. In addition, all filtrated samples and reagent solutions were refiltered through 0.2 μm sterile mem brane and then degassed prior to HPLC characteriza tion of the specific solubilized sugars such as xylose and glucose. Analysis of monomeric sugars was performed using HLPCDIONEX ® UltiMate 3000 RS (Thermoscien tific, Waltham, Massachusetts, USA) and the conditions were: Column: Aminex®HPX87H column (300 x 7.8 mm; BioRad), mobile phase: 5 mM sulphuric acid, flow rate: 0.5 mL/h, column temperature: 65±1°C, Detector: Refractive Index (RI), injection volume: 20 μL and run time: 30 min. Control was prepared identically to the en zymatic treatment with enzyme replaced by distilled wa ter.

Pulp bleaching with enzyme treatment
Bleaching of the bagasse pulp was executed in 50 g batches in transparent plastic bags using 3step sequence (X)HEP: xylanase pretreatment (Xstage), followed by sodium hypochlorite bleaching (Hstage), and alkaline ex traction with sodium hydroxide in the presence of hydro gen peroxide (EPstage), respectively. The conditions for each bleaching step are indicated as followed.
In the Xstage condition, 10% pulp consistency at 60°C and pH 8.0 for one h; the pulp was pretreated with xy lanase (100 U/g OD pulp) and a mixture of xylanase (100 U/g OD pulp and Triton X100 (0.25% (w/v)), while con trol pulp was bleached identically without adding the en zyme. In the Hstage, the pulp was treated with 8% (w/w) sodium hypochlorite at 50°C and pH 3.04.0 for 2 h. This stage was followed by the final EPstage where treatment with 0.25% (w/w) NaOH and 5% (w/w) H 2 O 2 was per formed at 70°C and pH 1011 for one h.
In each step, the plastic bags containing pulp were im mersed in a water bath at designated temperatures and then washed several times with tap water prior to use in the next stage. Controls included unbleached pulp, pulp treated with distilled water and pulp treated with xylanase at X stage were prepared in the same manner. After bleaching sequence, the resulted pulp was homogenized for mak ing hand sheets according to TAPPI T218. The paper color and lightness of each hand sheet were determined with a spectrocolorimeter (Data Color Spectrum 650 TM, Lawrenceville, New Jersey, USA) using CIE L*a*b* no tation referring to the method of TAPPI T560.

Construction of expression vector and expression of B. halodurans CM1 xylanase gene
In this study we obtained our target gene by executing PCR on pET21d vector containing the bhxyn3 gene as a DNA template using designed XynF and XynR primers. A DNA fragment of 1191 bp was obtained and cloned in frame with αfactor secretion signal in the yeast expression vector, pPICZα A, under the control of AOX1 promoter. The obtained recombinant DNA was well confirmed with 100% sequence similarity to the B. halodurans CM1 xy lanase, and then the DNA was transformed into P. pastoris KM71. The transformants were screened by colony PCR technique using 5' AOX1 and 3' AOX1 primers to validate whether gene recombination has successfully occurred in the host genome. The AOX1 promoters region in the plas mid are homologous with AOX1 promoters in the genome of P. pastoris, thereby promoting the integration of our target gene and its expression became tightly regulated by this promoter. Among four recombinant clones tested, high level of xylanase induction was observed from an integrant, so called pPICZαAbhxyn3 after 36 d of induction (Figure  1a), furthermore, a fourday induction was chosen for the next experiment in order to reduce the process time and production cost. The transformant showed major bands with apparent molecular weights of approximately 46.7 kDa (Figure 1b), which was the size corresponded to the target xylanase from B. halodurans CM1. Qualitatively, the intensity of the bands also visualized the increment of protein expression from day 0 to 3.

Production of the recombinant xylanase by P. pastoris in shake flask
In this study, P. pastoris expressed optimum level of xy lanase in BMGY medium deprived of zeocin (135 U/mL) after four days ( Figure 1a). Comparatively, the enzyme productivity was slightly lower in yeast cell cultured in BMGY medium with zeocin (129 U/mL) after four days of induction (Figure 2a). The growth of P. pastoris in both treatments were in the same range throughout the process, yet enzyme production was consistently observed higher in the cultures with no zeocin from day 1 to 4, although not significant (Figure 2a). The zeocin has no role in plas mid maintenance and only function to minimalize contam ination, which was not the main issue in upscale produc tion. It is a good agreement with the report of . One of our targets was to estab lish protein production in the minimum cost. Thus, further experiments on cultivation of P. pastoris were performed using the medium lacking zeocin since no supplementa tion of it could reduce the cultivation cost.
To optimize the production of the recombinant xy lanase, the effect of various methanol concentrations to wards enzyme expression was investigated in a volume of 100 mL BMGY culture. Four different methanol con centrations (1, 2, 3 and 4% v/v) were added to the culture medium at the induction phase. After four days of induc tion, the secreted xylanase from culture medium induced with both 2% and 3% (v/v) methanol was higher in activi ties, of around 135 U/mL, than other concentrations (Fig  ure 2b). However, the greatest yield of specific activity of 358 U/mg was obtained from the culture supernatant in duced by 2% (v/v) methanol, thus with costefficiency for upscale consideration, methanol at 2% (v/v) was chosen for further experiment.

Effect of pH and temperature on xylanase ac tivity and stability
The enzyme activity of recombinant B. halodurans CM1 xylanase was determined in the pH range of 510 in the buffers with similar ionic concentrations. As the results in Figure 3, the maximum activity was found at pH 8 and its activity was stable over a board pH range of 610. Further more, this enzyme retained more than 80% of its optimal activity at higher pH (910) with the maximum activity at pH 8 (Figure 3a). The pH stability was investigated by preincubating the enzyme in various buffers with the pH range of 610 at 60°C for 1 h and the remaining activity was monitored at pH 8.0. The activity of enzyme was sta ble between pH 710, remaining over 75% of its maximal activity (Figure 3b). The effect of temperature on xylanase activity was accessed in the range of 3080°C in 50 mM TrisHCl buffer (pH 9.0) and the recombinant enzyme dis played the highest activity at 60°C. Over 50% of its max imal activity was found at 4565°C (Figure 3c). To in vestigate thermostability of the recombinant xylanase, the enzyme was incubated in the range of 5070°C for vari ous periods of time in the same buffer as described in the materials and methods section. As shown in Figure 3d, the enzyme was stable at only 50°C with more than 50% of its maximal activity was retained. At higher temperature (60 and 70°C), the enzyme rapidly become inactive after incubating for 15 min (Figure 3d).

Effect of metal ions and salts on xylanase activ ity
The effect of various metal ions on the enzyme activity was investigated. As indicated in

Application of recombinant xylanase from B. halodurans CM1 in bagasse pulp bleaching
The potential of using the recombinant xylanase as a pulp bleach booster was preliminarily monitored by analyz ing the hydrolysis efficacy of the enzyme on industrial bagasse pulp. The potential of pulp hydrolysis enzyme was preevaluated using various enzyme dosages with 10 g moisturefree pulp at 10% (w/v) consistency. Hence, the amount of reducing sugar liberated from the pulp was an alyzed. As shown in Table 2, increase of reducing sugars was observed with higher enzyme dosages. A maximum reducing sugar of 23.84 mg/g was obtained at 200 U/g be fore stationary condition (Table 2). Furthermore, xylose and glucose quantification of the solutions obtained from the enzymetreated pulp reactions was carried out using HPLC with Aminex® column. As shown in Table 2, the xylose content revealed a similar trend with the previous reducing sugar analysis, depicting the xylose amount of 1.73 mg/g at 200 U/g (Table 2) was the most reasonable amount, whereas no detectable amounts of glucose were observed from all samples tested in HPLC analysis (data not shown). Hence, the recombinant xylanase at 200 U/g was then chosen for further studies. This result suggested that the recombinant xylanase is cellulasefree xylanase.
To enhance the potential characterization of enzyme, various nonionic and anionic surfactants were supple mented in the enzymepulp reactions at the concentration of 0.25% (w/v) and the reducing sugar and xylose con tents released from the pulp were quantified. As indicated in Table 3, all nonionic surfactants evaluated (Tween 20, Tween 80, Triton X100 and Lutensol) had an activated ef fect on the recombinant enzyme and maximum release of reducing sugars (19.75 mg/g; 1.88 folds higher than con trol). The xylose content (2.71 mg/g; 4.65 folds higher than control) was observed in the enzymetreated solution supplemented with Triton X100. The pulp with Triton Effects of pH and temperature on the activity and stability, and Lineweaver-Burk plotting graph of recombinant BHxyn3E produced by P. pastoris. (a) Optimal pH: The reactions consisted of 1% (w/v) of beechwood xylan in 50 mM sodium citrate (pH 5.0), sodium phosphate (pH 6.0 to 8.0),Tris-HCl (pH 8.0 to 9.0), or Glycine-NaOH (pH 9.0 to 10.0) buffer were prepared and incubated at 60˚C for 5 min; (b) pH stability: The enzyme was pre-incubated in 50 mM buffers at 60°C for 1 hour and residual activity was measured under the standard assay conditions. The buffers used were sodium phosphate (pH 6.0 to 8.0), Tris-HCl (pH 8.0 to 9.0), and glycine-NaOH (pH 9.0 to 10.0); (c) Optimal temperature: the reactions were assayed over a temperature range of 30 to 80°C at optimal pH for 5 min; (d) Thermostability: The xylanase was pre-incubated over various temperatures (50, 60 and 70°C) at optimal pH for different times (15, 30, 45 and 60 min) prior to residual activity analysis under the assayed conditions. X100 only was used as a control to confirm the Triton X100 alone did not affect the xylan degradation. It was found that anionic surfactant (Sanmorin) acted as an in hibitor in reducing the amounts of reducing sugars (4.59 mg/g; 2.88 folds lower than control) and xylose (0.113 mg/g; 5.16 folds lower than control) ( Table 2). This result suggested that the nonionic surfactants, particularly Tri ton X100 plays essential role in enhancing the xylanase activity, leading to higher degradation enzymatic activity in pulp biobleaching. Among the pulp hydrolysis experiments carried out at various contents of Triton X100, the highest liberation of the reducing sugars was observed from the solution of the enzymatic treated pulp added with 0.25% (w/v) of Tri ton X100 (data not shown). Therefore, biobleaching of bagasse pulp was then carried out using the xylanase and Triton X100 at the concentrations of 200 U/g and 0.25% (w/v), respectively. Surfactants alone have no role in pulp hydrolysis, as we have observed a control with only Triton X100 in the reaction produced no reducing sugars (data not shown).
The bleaching efficiency of the enzyme on the bagasse pulp was evaluated based on its effects on pulp whiteness index (WI) using the 3step bleaching sequence [(X)HEp]. Without including the standard deviation, pretreatment of the bagasse pulps with the recombinant xylanase exhib ited an enhancement of pulp whiteness. The highest WI (83.85% CIE) although not significant was achieved from the pulp treated with Triton X100mixed enzyme, fol lowed by pulp treated with xylanase alone (83.09% CIE) ( Table 3). In addition, the component of the whiteness in dex, namely Lightness index (L*) and Color a index (a*) changed by the Triton X100mixed enzyme treatment over the untreated control (Table 3).

Discussion
This study described the characterization of recombinant xylanase from B. halodurans CM1 produced by P. pas toris KM71 and the potential application of the enzyme in biobleaching of bagasse pulp. The cloning and expres sion of xylanase gene originating from Japanese B. halo durans C125 have been reported, however, the xylanase produced was applied in the wheat straw pulp (Lin et al. 2013). This study is the first report of recombinant xy lanase bagasse pulp application from Indonesian B. halo durans expressed in P. pastoris. The cloning of an endo xylanase encoding gene from B. halodurans CM1 strain using pET21d vector in E. coli BL21 star has been previ ously conducted. Based on these results, the obtained re combinant enzymes showed a promising in deinking pro cess of waste paper (Helianti et al. 2018). However, the level of secreted enzyme from recombinant E. coli was very low, around 10 U/mL (19.73 U/mg) and only after pu rification with NiNTA it reached 543 U/mg. Therefore, in this study, we tried to increase the enzyme productivity by substitution of expression system in P. pastoris as a host cell.
The volumetric activity of extracellular xylanase by P. pastoris was more than 10folds higher compared to re combinant xylanase expressed in E. coli (Helianti et al. 2018) without downstream purification, thus showing that this P. pastoris is promising to be recombinant host in up scale production since it gave a better yield of enzyme productivity. In terms of purified enzyme, the recombi nant xylanase produced by P. pastoris also offers a more straightforward purification system compared to E. coli expression system. Therefore, the step of purification pro cess was shorter and no need to do a further succesive pu rification system, which can reduce the cost for the down stream process.
The recombinant xylanase produced by P. pastoris had optimum activity at pH 8 and temperature 60°C. This re sult was slightly different with the optimal pH and temper ature of recombinant xylanase expressed in E. coli (pH 9 and 65°C) or native enzyme in wildtype bacterium (pH 9 and 70°C). (Ulfah et al. 2011; Helianti et al. 2018. The difference in biochemical properties also found in the other enzymes, such as lipase and cellulase (Zhu et al. 2014; de Amorim Araújo et al. 2015; Tišma et al. 2019), that ex pressed in different host cell system. This result might be occurred due to the posttranslational protein modification in P. pastoris (GuerreroOlazarán et al. 2010; Gomes et al. 2016; Chae et al. 2017; Vieira Gomes et al. 2018. The other properties such as the effect of metal ions on this xylanase have not been studied previously, and this xylanase unexpectedly inactivated by Ca 2+ , which is sim ilar to Bacillus subtilis xylanase was reported by Sanghi et al. (2010) and different to Bacillus amyloliquefaciens xylanase reported by Kumar et al. (2017). Thus, different xylanase has their unique preference for metal ions. This information is useful in the application aspect, so that we have to omit the decreasing effect of metal ion both in fer mentation or in the application.
In order to enhance the efficient use of the recombi nant xylanase, various surfactants have been examined for their ability in promoting the increment of xylanase ac tivity on bagasse pulp hydrolysis. In this study, the ad dition of nonionic surfactant such as Triton X100 to the hydrolysis reaction allowed an increase in reducing sugars and xylose content of 1.88 and 4.65 folds higher than con trol, respectively. Corresponding to other reports, Triton X100, Tween 80, and Tween 20 were widely used and proven to increase hydrolysis of sugarcane bagasse and other types of raw straws by acting along in promoting various kind of lignocellulosedegrading enzymes such as xylanase, cellulase, and endoglucanase (Wang et al. 2011; Cheng et al. 2014. The bagasse pulp pretreated with crude recombinant xylanase and Triton X100 exhibited 1.40% CIE increase in the whiteness compared to the untreated control pulp, while only 0.40% CIE increase in whiteness was observed in the pulp treated with enzyme alone. How ever, Xylanase+Triton X100 treatment increased the light ness index (L*) and changed substantially the color a index (a*).
The increment of the whiteness index (WI) of the xylanaseprebleached pulp hand sheets in this study was somehow not significant. This could be due to the enzyme was not stable as the retaining activity was dropped below 20% in the first 15 min at bleaching temperature (60°C), although it could maintain its stability at more than 50% after an hour incubation at 50°C. Another factor to be put into consideration was the bleaching process occurred in no pH adjustment using pulp with pH 9.0, since the best pH stability of this recombinant xylanase was pH 8.0. There fore, the operation condition was not optimal for the en zyme to work steadily throughout the process. Although in many studies prebleaching experiments with enzymes were still primarily done at its optimum temperature (Nair et al. 2010; Weerachavangkul et al. 2012; Lin et al. 2013, the stability of the enzyme after a period of incubation at optimal temperature should also be observed. Based on Vena et al. (2013), the sugarcane bagasse usually contained 25.9% ±2.1 xylan. However, how much xylan present in pulp are diversely varied based on how the pulping was carried out, and from this paper as an exam ple, the mild alkaline pulp could have 6.2% xylan of the pulp. If we assumed this may be closed to the pulp we used in our experiment, it means, if we used 10 g of dried pulp, theoretically, there is 620 mg xylan in it. Therefore, if we use 200 U/g enzyme dosage, based on Table 2, the oretically 238.4 mg of xylan could be degraded and the effect could be seen, if it is in optimal and enzyme stable condition.
Optimal enzyme charge of 200 OD/g in this study was considered as high compared to another study of bleaching with B. halodurans xylanase with wheat straw pulp (Lin et al. 2013), however, the optimal dosage of enzyme var ied considerably depending on the type of pulp and micro bial xylanase characteristics used in the experiment. Apart from reporting optimal enzyme charge and Triton X100 positive effect in bagasse pulp bleaching, our results from this study has given the rise of concerns regarding the im portance of the optimization of bleaching conditions that is suitable for enzyme to operate for the next assessment.

Conclusions
In conclusion, characterization of alkaliphilic and ther mophilic xylanase originated from a new Indonesian strain (CM1) of B. halodurans produced by P. pastoris and its potential application in bleaching bagasse pulp has been reported in this work. The full characterization of the recombinant xylanase and its application in bleaching bagasse pulp is the new information. The ability of the enzyme for biobleaching of bagasse pulp has been con firmed in the first screening for further application, where it slightly increased the pulp whiteness with no neces sary for pH adjustment of an incoming pulp. However, further optimization and assessment in biobleaching were needed for industrial application, the usage of this en zyme could be an alternative solution for environmentally friendly pulp bleaching process in the industry.