Direct recovery of Bacillus subtilis xylanase from

Journal of Bioscience and Bioengineering VOL. xx No. xx, 1e5, 2017 www.elsevier.com/locate/jbiosc

Direct recovery of Bacillus subtilis xylanase from fermentation broth with an alcohol/salt aqueous biphasic system Hui Suan Ng,1 Cindy Xin Yi Chai,1 Yin Hui Chow,2 Wai Leng Carmen Loh,1 Hip Seng Yim,1 Joo Shun Tan,3 and John Chi-Wei Lan4, * Faculty of Applied Sciences, UCSI University, UCSI Heights, 56000 Cheras, Kuala Lumpur, Malaysia,1 School of Engineering, Taylor’s University Lakeside Campus, 47500 Subang Jaya, Selangor, Malaysia,2 Bioprocess Technology, School of Industrial Technology, Universiti Sains Malaysia, 11800 Gelugor, Pulau Pinang, Malaysia,3 and Biorefinery and Bioprocess Engineering Laboratory, Department of Chemical Engineering and Materials Science, Yuan Ze University, Chungli, Taoyuan, 320, Taiwan4 Received 16 October 2017; accepted 13 December 2017 Available online xxx

Xylanase enzyme degrades linear polysaccharide b-1,4 xylan and the hemicellulose of the plant cell wall. There is a growing demand in finding a cost-effective alternative for industrial scale production of xylanase with high purity for pharmaceutical applications. In this study, an alcohol/salt aqueous biphasic system (ABS) was adopted to recover xylanase from the Bacillus subtilis fermentation broth. The effects of several ABS parameters such as types and concentrations of alcohols and salts (i.e., sulphate, phosphate, and citrate), amount of crude loading and pH of the system on the recovery of xylanase were investigated. Partition coefficient of xylanase (KE), selectivity (S) and yield (YT) of xylanase in top phase of the ABS were measured. Highest KE (6.58 ± 0.05) and selectivity (4.84 ± 0.33) were recorded in an ABS of pH 8 composed of 26% (w/w) 1-propanol, 18% (w/w) ammonium sulphate. High YT of 71.88% ± 0.15 and a purification fold (PFT) of 5.74 ± 0.33 were recorded with this optimum recovery of xylanase using alcohol/salt ABS. The purity of xylanase recovered was then qualitatively verified with sodium dodecyl sulphate (SDS) gel electrophoresis. The SDS profile revealed the purified xylanase was successfully obtained in the top phase of the one-step 1-propanol/sulphate ABS with a distinct single band. Ó 2017, The Society for Biotechnology, Japan. All rights reserved. [Key words: Aqueous biphasic system; Recovery; Xylanase; Bacillus subtilis; Fermentation]

Enzyme xylanase degrades the linear polysaccharide b-1,4 xylan to xylose, a monosaccharide and thus is able to breakdown the hemicellulose of plant cell wall. Xylanase can be applied in a wide array of industries such as bleaching of craft pulp in the pulp and paper industry; food additives and clarifying agents of juices in food industry; and antimicrobial agents for the production of pharmacologically active polysaccharides in pharmaceutical industry (1). In recent study, xylanase was applied to improve the rumen fermentation and reduced the production of greenhouse gases (2). In view of the great potential of xylanase, considerable research interest has drawn towards the production of xylanase over the past two decades. The organisms that are responsible for the production of xylanase can range from prokaryotes to eukaryotes (e.g., bacteria, algae, fungi, snail, insect, protozoa, anthropods, gastropods). However, bacterial fermentation of xylanase is generally more feasible for large-scale production. Bacillus subtilis which is naturally found in the soil and vegetation is among the most industrially important xylanase producer because of the inherent higher enzyme activity. Thus, the xylanase was fermented using the B. subtilis strains in this study for the optimum production of xylanase. * Corresponding author at: Biorefinery and Bioprocess Engineering Laboratory, Department of Chemical Engineering and Materials Science, Yuan Ze University, No. 135, Yuan Tung Road, Chungli, Taoyuan, Taiwan. Tel.: þ886 3 4638800x3550; fax: þ886 3 4559373. E-mail address: [email protected] (J.C.-W. Lan).

The economic feasibility of the large scale enzyme production relies on the advances applied in both the upstream and the downstream processing. Aqueous biphasic system (ABS) is a simple liquideliquid separation method for purification of various biological molecules and particles (3). ABS consists of two immiscible aqueous phases which can be formed by dissolving two mutually incompatible liquid components above a critical concentration (4). ABS has successfully purified various enzymes, bioactive compounds, proteins, and nucleic acids in previous literature (5,6). As compared to conventional enzyme separation and purification methods, ABS offers several advantages such as biocompatibility, low energy consumption, easy to scale-up and feasibility of process integration (3). Several polymer/salt ABSs have been applied for the recovery of bacterial xylanase from fermentation broth of different strains in previous studies (7). Xylanase sourced from Bacillus pumilus fermentation can be purified up to 33-fold with a yield of 98% with ABS composed of 22% (w/w) polyethylene glycol (PEG) 6000 and 10% (w/w) phosphate with an addition of 12% (w/w) sodium chloride (NaCl) (7). A yield of more than 92% of recombinant Bacillus halodurans xylanase was recovered with ABS composed of 18.3% (w/w) PEG 1000 and 14.4% (w/w) phosphate at pH 8.5 (8). Polymer/salt ABS exhibits high viscosity, slow phase separation, and complication in the recycling of the phase-forming components despite the fact that it is simple to conduct. In view of this, alcohol/salt ABS is applied in this study for recovery of B. subtilis

1389-1723/$ e see front matter Ó 2017, The Society for Biotechnology, Japan. All rights reserved. https://doi.org/10.1016/j.jbiosc.2017.12.010

Please cite this article in press as: Ng, H. S., et al., Direct recovery of Bacillus subtilis xylanase from fermentation broth with an alcohol/salt aqueous biphasic system, J. Biosci. Bioeng., (2017), https://doi.org/10.1016/j.jbiosc.2017.12.010

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J. BIOSCI. BIOENG.,

xylanase from crude feedstock to overcome the above-mentioned limitations of polymer/salt ABS. Alcohol/salt ABS on the contrary is of lower cost and viscosity as compared to the conventional polymer/salt ABS (9). Moreover, the enzyme extracted to the alcohol-rich phase can be easily recovered by evaporation, and thus enabling the recycling and reuse of the phase-forming component (10). In this study, the effects of types and concentrations of alcohol and salt, crude load, and pH of the system were examined for optimum recovery of B. subtilis xylanase.

S ¼

KE KP

(3)

Purification fold (PFT) was determined as the ratio of the xylanase specific enzyme activity in the top phase (SAT) to the xylanase specific enzyme activity of the crude feedstock (SAF) as shown in Eq. 4: PFT ¼

SAT SAF

(4)

The percentage yield of xylanase in the top phase of the ABS (YT, %) was calculated according to Eq. 5: YT ð%Þ ¼

1þ

100 1 VR KE

(5)

MATERIALS AND METHODS

where VR is the ratio of volume of the top phase to the volume of bottom phase of the ABS.

Materials Bicinchoninic acid (BCA) protein assay kit was purchased from Bio Basic (Markham, Canada). Ethanol, 1-propanol, 2-propanol, ammonium sulphate, potassium phosphate and sodium citrate were purchased from Merck (Darmstadt, Germany). Sodium dodecyl sulphate (SDS) and 30% acrylamide/bris solution were obtained from Bio-Rad (Hercules, CA, USA). Glacial acetic acid, sodium chloride (NaCl), 3,5-dinitrosalicylic acid (DNS), and sodium potassium tartrate were obtained from Friendemann Schimidt (Australia). All the chemicals and reagents used in this study were of analytical grade.

Sodium dodecyl sulphate polyacrylamide gel electrophoresis Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was performed with Bio-Rad Mini-PROTEAN electrophoresis system. The 12% resolving gel and the 4% stacking gel solutions were prepared as described (12). The casting frame was set on the casting stands. An appropriate amount of resolving gel solution was added into the gap between the glass plates. The gel was left for 20e30 min for the gelation process. The stacking gel solution was then added into the gap between the glass plates after the resolving gel gelated. The resultant ABS purified xylanase sample was then subjected to the SDS-PAGE analysis. Electrophoresis was conducted with a voltage of 120 V for 1.5 h.

Bacterial cultivation of B. subtilis for xylanase production B. subtilis was obtained from Microbiological Laboratory, UCSI University, Malaysia. Inoculation was performed by transferring a loopful of B. subtilis culture into 10 mL of nutrient broth and incubated in 37 C for 24 h with an agitation speed of 150 rpm (11). Fermentation media was prepared by mixing 2 g of sucrose; 0.05 g of K2HPO4; 0.02 g of NaCl; 0.016 g of MgSO4$7H2O; 0.05 g of yeast extract; and 2 g of sugarcane bagasse (11). After the 10% (v/v) inoculation, the fermentation broth was incubated in 37 C for 48 h with the agitation speed of 150 rpm. Crude xylanase (supernatant) was collected after centrifuged with a centrifugal force of 8000 g for 10 min. Aqueous biphasic system Alcohol/salt ABS was prepared as described in previous literature (10). ABS of 5 g was prepared with 15 mL centrifuge tube. The aqueous system consists of appropriate amount of alcohol/salt solutions and crude xylanase was then centrifuged at 8000 g for 5 min for complete phase formation. The volumes of the top and bottom phases were noted. The two separated phase samples were collected for enzyme activity assay and BCA protein assay analysis. The effects of the concentrations of alcohol and salt, crude load and pH of the system on the ABS recovery of xylanase were investigated. Determination of xylanase enzyme activity The xylanase enzyme activities exhibited by different phase samples were determined using the xylanase activity assay (11). Substrate solution of 1% (w/w) birchwood xylan solution was prepared in citrate buffer. A total of 0.5 mL of diluted enzyme sample was added to 0.5 mL of 1% (w/w) birchwood xylan solution. The resulted mixture was incubated for 15 min at 50 C. Next, the mixture was added with 7.5 mL of 3,5-dinitrosalicylic acid and left in the boiling water bath for 10 min to stop the enzyme reaction. The mixture was then left to cool to room temperature before the absorbance was measured at 550 nm. Triplicate measurements were taken and the results were expressed as mean standard error. One unit of xylanase activity was defined as the amount of enzyme needed to release 1 mmol of reducing sugar equivalent to xylose per min under the assay condition as above-mentioned. Determination of total protein concentration The protein concentration in the phase samples was determined using the Bio Basic Bicinchoninic Acid Assay (BCA) protein assay kit (Lot: SK30210-K1045R0J). A total of 20 mL of the phase samples were pipetted into the 96 wells microtiter plate, followed by the addition of 200 mL of BCA working reagent. The resulted mixture were mixed gently and incubated at 37 C for 30 min. After the incubation, the absorbance of the mixture was measured at 562 nm. Bovine serum albumin (BSA) standard curve was constructed to determine the protein concentration in the phase samples. All the measurements were quantified in triplicates and expressed as mean standard error. Selectivity (S), purification factor (PFT) and yield (YT) The partition coefficient of xylanase (KE) is defined as the ratio of xylanase activity in top phase (AT) to the xylanase activity in bottom phase (AB) of the alcohol/salt ABS (Eq. 1) and the partition coefficient of total protein (KP) is determined as the ratio of protein concentration in the top phase (PT) to the protein concentration in the bottom phase (PB) of the alcohol/salt ABS (Eq. 2): KE ¼

AT AB

(1)

KP ¼

PT PB

(2)

whereas, selectivity (S) was calculated as the ratio of KE to KP as indicated in Eq. 3:

RESULTS AND DISCUSSION Effect of different types of alcohol/salt ABS on recovery of xylanase ABS with high water content is extremely suitable for recovery of enzyme due to the high biocompatibility (6). Whereas, organic solvents such as alcohols can maintain the enzyme’s open conformation and enhance the activity of an enzyme (10,13). Thus, ethanol, 1-propanol and 2-propanol are suitable to be applied as the phase-forming components for the ABS to recover target enzyme from crude feedstock. To identify the type of alcohol/salt ABS for better recovery efficiency of B. subtilis xylanase, crude xylanase was added to ABS with different types and concentrations of alcohol and salt as referred (10). The amount of crude xylanase and the pH of the ABS were fixed at 20% (w/w) crude xylanase and pH 7, respectively in this ABS partition experiment. The effect of different types of alcohol/salt ABS on the KE and YT of B. subtilis xylanase was evaluated (Table 1). Results showed that the partition of xylanase to the alcohol-rich top phase of the alcohol/salt ABS was considerably enhanced by the sulphate salt, but to a lesser extent, by phosphate salt and citrate salt. Sulphate ions which exhibit higher salting-out effect as compared to phosphate ions will repel more xylanase molecules to the alcohol-rich top phase and therefore further strengthen the interaction between the xylanase and the alcohol molecules (14). This resulted in high KE values (KE > 2.77 0.04) for all alcohol/sulphate ABSs investigated (Table 1), and thus ammonium sulphate is the most suitable phase-forming salt for the ABS recovery of B. subtilis

TABLE 1. Partition coefficient and yields of B. subtilis xylanase with different types of alcohol/salt ABS. Types of ABS Ethanol/sulphate Ethanol/phosphate Ethanol/citrate 2-Propanol/sulphate 2-Propanol/phosphate 2-Propanol/citrate 1-Propanol/sulphate 1-Propanol/phosphate 1-Propanol/citrate

Phase composition, % (w/w)

KE

YT (%)

24/26 20/20 24/26 18/20 16/16 18/20 18/20 16/16 18/20

2.77 0.04 1.31 0.08 1.22 0.06 3.39 0.14 1.32 0.35 2.32 0.01 4.83 0.26 1.32 0.16 2.51 0.25

40.91 0.35 37.07 1.26 34.36 1.17 12.37 0.46 35.74 0.30 8.80 0.02 39.68 0.27 24.80 0.29 21.80 1.72


MICROBIAL XYLANASE RECOVERY BY AQUEOUS BIPHASIC SYSTEM

Effect of different phase composition of 1-propanol/ sulphate on ABS recovery efficiency of xylanase The effect of different phase composition of 1-propanol/sulphate ABS on the recovery efficiency of xylanase from crude feedstock was investigated (Table 2). The recovery efficiency of alcohol/salt ABS for xylanase purification is dependent on the salting-out mechanism (18). Xylanase will be salted out by the sulphate salt component to the alcohol-rich top phase of the ABS when there

TABLE 2. Effects of different phase composition of 1-propanol/sulphate ABS efficiency on recovery of B. subtilis xylanase. Phase composition, % (w/w) 1-propanol/sulphate 16/20 16/22 16/24 18/18 18/20 18/22 18/24 20/18 20/20 20/22 20/24 22/18 22/20 22/22 22/24 24/18 24/20 24/22 24/24 26/18 26/20 26/22 26/24 28/18 28/20 28/22 28/24

KE

YT (%)

4.26 0.14 4.02 0.09 4.73 0.01 3.53 0.11 3.53 0.07 2.79 0.04 3.37 0.13 3.44 0.11 4.66 0.12 2.70 0.07 2.75 0.26 2.82 0.07 4.15 0.10 3.23 0.01 5.04 0.10 5.31 0.18 4.99 0.30 4.86 0.13 4.80 0.05 5.66 0.03 4.40 0.10 5.62 0.06 4.48 0.28 2.19 0.11 1.59 0.06 2.12 0.020 1.87 0.080

32.13 0.74 35.42 0.49 43.53 0.05 32.47 0.66 32.47 0.41 27.53 0.31 35.43 0.87 35.88 0.74 38.89 0.60 23.07 0.43 40.62 2.25 31.46 0.52 40.33 0.60 44.65 0.07 48.96 0.49 57.21 0.82 55.49 1.47 54.84 0.66 60.25 0.26 64.12 0.14 60.70 0.56 58.44 0.26 63.50 0.44 35.40 1.10 33.48 0.85 47.60 0.23 44.48 1.05

3

are insufficient free water molecules to accommodate the xylanase in the salt-rich bottom phase. The optimum recovery of xylanase from crude feedstock was achieved with ABS composed of 26% (w/w) 1-propanol and 18% (w/ w) ammonium sulphate with a KE of 5.66 0.03 and YT of 64.12% 0.14. Overall, xylanase exhibited a top phase preference as the values of KE were greater than one for all the 27 systems investigated, indicating the B. subtilis xylanase preferred a relatively more hydrophobic alcohol-rich phase as compared to the hydrophilic salt-rich phase. In contrast to the significant enhancement in the KE of the xylanase with the increased concentration of 1propanol from 22% (w/w) to 24% (w/w), the effect of increased concentration of ammonium sulphate on the recovery efficiency of xylanase was insignificant. When the concentrations of 1-propanol and ammonium sulphate were increased, the YT of xylanase was increased because of the intensified salting-out effect. However, further increase in the concentration of alcohol beyond 26% (w/w) has led to a drastic decrease in KE and YT. The reduction of the free volume whereby the salted-out xylanase could not dissolve in the alcohol-rich top phase resulted in a lower KE and YT (10). The mass transfer process of the xylanase to the alcohol-rich top phase was in great balance in ABS composed of 26% (w/w) 1-propanol and 18% (w/w) ammonium sulphate. Effect of crude load on 1-propanol/sulphate ABS recovery efficiency of xylanase The amount of the crude feedstock loaded into an ABS will alter the volume ratio of the ABS and thus affects the xylanase recovery efficiency of the alcohol/salt ABS (16). To identify the crude loading amount of xylanase for the best recovery efficiency of xylanase in the 1-propanol/sulphate ABS, the amount of crude load ranging from 10% (w/w) to 40% (w/w) was loaded into the system and the resultant KE and YT were measured (Fig. 1). A maximum KE (3.52 0.03) and YT (60.10% 0.20) were achieved with 20% (w/w) crude xylanase feedstock loaded into the 1-propanol/sulphate ABS, indicating the maximum capacity for the 1-propanol/sulphate ABS was 20% (w/w) of crude feedstock loading of xylanase. When the crude load increased from 20% (w/w) to 30% (w/w), the YT of the xylanase decreased significantly from 60.10% 0.20 to 54.14% 0.38. Inter-phase precipitation was observed when crude xylanase of 30% (w/w) was loaded into the 1propanol/sulphate ABS due to the phase saturation whereby there is no available free volume to dissolve the xylanase, and thus resulted in loss of xylanase. Moreover, protein aggregation due to the enhancement of the intra-molecular bonding between the xylanase molecules would also resulted in inter-phase precipitation (19,20).

Yield (%)

Partition coefficient 70

5

60

4

50 40

3 30

Yield, YT (%)

xylanase. However, alcohol/citrate ABS displayed lower KE values irrespective of the highest salting-out ability of citrate ions because sodium ions are among the weakest chaotropic ions compared to ammonium ions and potassium ions (15). For the sulphate-based ABS composed using different alcohols, it was found that the KE of xylanase decreases in an order of 1propanol > 2-propanol > ethanol. In general, the solubility of enzymes in the alcohol-rich top phase increases with the alkyl chain length of the alcohol which is corresponds to the hydrophobicity of alcohol (10). Both isomers of propanol contain longer chain length as compared to the ethanol and 1-propanol has a higher hydrophobicity as compared to the 2-propanol (16). The interactions between the alcohol molecules and xylanase were most optimum in 1-propanol/salt ABS, results in a high KE and YT when compared to 2-propanol/salt ABS (17), whereby a maximum KE of 4.83 0.26 was achieved in the ABS composed of 18% (w/w) of 1-propanol and 20% (w/w) of ammonium sulphate. Although a maximum YT (40.91% 0.35) was attained in ABS composed of 24% (w/w) ethanol and 26% (w/w) ammonium sulphate, it is not feasible for the recovery of xylanase due to the occurrence of protein precipitation at high concentrations of ABS phase composition (10). Moreover, 1-propanol/sulphate ABS showed an overall better KE of 4.83 0.26 as compared to the other types of alcohol/salt ABS, indicating a more efficient partition of xylanase to the top phase of the ABS. Therefore, 1-propanol/sulphate ABS was chosen as the type of alcohol/salt ABS for subsequent partition experiments to obtain the optimum recovery of B. subtilis xylanase.

Partition Coefficient, KE

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20

2

10 1

0 10

20 30 [Crude load], % (w/w)

40

FIG. 1. Yield and KE of xylanase with 1-propanol/sulphate ABS of different crude loading amount.


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J. BIOSCI. BIOENG.,

Yield (%)

Partition coefficient 74

6

72

5

70

4

68

3

66

2

64

1

62

Partition Coefficient, KE

7

0

Yield, YT (%)

4

60 5

6

7 pH

8

9

FIG. 2. Yield and KE of xylanase in 1-propanol/sulphate ABS of different pH with 20% (w/w) crude feedstock.

When the amount of crude load increased from 20% (w/w) to 40% (w/w), the YT of xylanase decreased despite a relatively consistent trend of KE was observed (Fig. 1). The higher amount of crude feedstock loaded into the ABS introduced a considerably higher content of contaminant proteins into the ABS and thus altered the overall mass transfer process in the system (21). Therefore, 20% (w/w) of crude load was selected as the desirable crude load for the maximum recovery of xylanase from B. subtilis fermentation broth. Effect of pH on 1-propanol/sulphate ABS recovery efficiency of xylanase Different proteins exhibit different surface charges dependent on their amino acids composition and the surrounding pH. The partition behaviour of a target protein is highly dependent on the isoelectric point (pI) and resultant surface charges of the protein which is prompted to change based on the pH of the ABS. Therefore, the system pH of an ABS is one of the main determinants in the protein purification process of xylanase using alcohol/salt ABS. The ABS recovery efficiency of xylanase was investigated with 1-propanol/sulphate ABS of different system pH ranging from pH 5 to pH 9 as shown in Fig. 2. The pHs of the sulphate salt solutions were adjusted using sulphuric acid. B. subtilis xylanase has a pI value of 9.63 (22) and appears to be a positively charged protein at pH < pI, and thus xylanase was preferentially partition to the salt-rich bottom phase of the 1propanol/sulphate ABS in the system with pH lower than 9.63. However, the partition of the xylanase in the ABS was also affected by the hydrophobic interactions between the xylanase and the phase-forming components despite the surface charges of the protein. Fig. 2 shows there was an increasing trend for KE when the system pH increased from pH 5 to pH 8 (6.57 0.05) where xylanase showed top phase preference in all developed ABS with different pHs. The relatively hydrophobic alcohol molecules driven the xylanase to partition to the top phase and the increase of the pH further enhanced the partition of the xylanase to the alcohol-rich top phase of the 1-propanol/sulphate ABS. Moreover, Highest YT (71.89% 0.15) of xylanase was recorded with 1-propanol/salt ABS of pH 8. There was a drastic decrease in the KE (4.45 0.09) and YT (65.60% 0.05) of xylanase when pH of the system was further increased to pH 9, which was probably due to the migration of more of the unwanted proteins to the top phase of the ABS that altered the mass transfer efficiency of the ABS upon approaching the xylanase’s pI value (23). Nevertheless, a maximum selectivity of xylanase (4.42 0.88) and a high PFT (4.19 0.70) were also obtained with 1-propanol/sulphate of pH 8. These results indicated that a fine balance between the electrostatic effect and hydrophobic interaction of xylanase with the phase-forming components was attained and thereby promoted the exclusive partition of

FIG. 3. SDS-PAGE profile on purity of B. subtilis xylanase recovered from 1-propanol/ sulphate ABS.

xylanase to the alcohol-rich top phase. In addition, xylanase sourced from bacterial strains often exhibit high level of catalytic activity at alkaline pH (22). As a result, recovery of xylanase in the top phase of the alcohol/salt ABS was performed with 1-propanol/ sulphate ABS of pH 8. SDS-PAGE profile of purified B. subtilis xylanase from 1-propanol/sulphate ABS The highest recovery of xylanase from crude feedstock was achieved with 1-propanol/phosphate ABS of 26% (w/w) 1-propanol, 18% (w/w) of ammonium sulphate at pH 8. The purity of the partitioned xylanase was assessed using the 12% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDSPAGE) (Fig. 3). Lane 1 is the standard protein marker with molecular weight ranged from 11 kDa to 190 kDa. Lane 2 is the crude feedstock, whereas, lanes 3 and 4 were top phase sample and bottom phase sample, respectively. Multiple bands were observed in lane 2 (crude xylanase), indicated the crude xylanase contained high content of impurities. A single distinct band was observed in the phase sample recovered from the top phase of the 1-propanol/sulphate ABS with a molecular weight of 37 kDa. A high purity of xylanase was attained using the one-step ABS where highest xylanase activity was detected in this top phase sample. Lane 4 showed multiple bands of unwanted proteins indicated other unwanted proteins were driven to the bottom phase of the ABS. In summary, B. subtilis xylanase was successfully purified with a one-step 1-propanol/ammonium sulphate ABS. Overall, ABS parameters such as the types of and concentrations of phase-forming components, crude loading and pH determined the ABS recovery efficiency of xylanase. An optimum ABS recovery condition for purification of xylanase was achieved in an ABS with a composition of 26% (w/w) 1-propanol, 18% (w/w) ammonium sulphate of pH 8 and loaded with 20% (w/w) crude xylanase. The 1-propanol/sulphate ABS is a potential system in replacement to the conventional polymer/salt ABS in view of its high recovery efficiency and low investment cost on the system setup in large-scale production of xylanase. Supplementary data related to this article can be found at https://doi.org/10.1016/j.jbiosc.2017.12.010.

ACKNOWLEDGMENTS This study was supported by the Ministry of Education Malaysia Fundamental Research Grant (FRGS/2/2014/SG05/UCSI/03/1), UCSI University Pioneer Scientist Incentive Funds (PSIF) PROJ-In-FAS-


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MICROBIAL XYLANASE RECOVERY BY AQUEOUS BIPHASIC SYSTEM

027 and the Taylor’s University Research Grant Scheme (TRGS)Emerging Researchers Funding Scheme (TRGS/ERFS/2/2016/SOE/ 002) from the Taylor’s University, Malaysia and Ministry of Science and Technology Taiwan (MOST104-2632-E-155-001).

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