Effect of polyols on thermostability of xylanase from a tropical isolate of Aureobasidium pullulans and its application in prebleaching of rice straw pulp
© Bankeeree et al.; licensee Springer. 2014
Received: 1 November 2013
Accepted: 14 January 2014
Published: 18 January 2014
In an attempt to find a thermostable xylanase enzyme for potential application in the pretreatment prior to H2O2 bleaching of paper pulp for industry, an extracellular xylanase from Aureobasidium pullulans CBS 135684 was purified 17.3-fold to apparent homogeneity with a recovery yield of 13.7%. Its molecular mass was approximately 72 kDa as determined by SDS-PAGE. The optimal pH and temperature for activity of the purified enzyme were pH 6.0 and 70°C, respectively. The enzyme was relatively stable at 50°C, retaining more than half of its original activity after 3-h incubation. The thermostability of the enzyme was improved by the addition of 0.75 mM sorbitol prolonging the enzyme’s activity up to 10-fold at 70°C. When the potential of using the enzyme in pretreatment of rice straw pulp prior to bleaching was evaluated, the greatest efficiency was obtained in a mixture containing xylanase and sorbitol. Treatment of the rice straw pulp with xylanase prior to treatment with 10% (v/v) H2O2 and production of hand sheets increased the ISO sheet brightness by 13.5% and increased the tensile and tear strengths of the pulp by up to 1.16 and 1.71-fold, respectively, compared with pulps treated with H2O2 alone. The results suggested the potential application of the enzyme before the bleaching process of paper pulp when the maintenance of high temperature and enzyme stability are desirable.
Xylanolytic enzymes form a group involved in the hydrolysis of xylans and arabinoxylans, the most abundant hemicellulosic polymers in plant biomass. Within this group, endo-1,4-β-xylanase is of special interest for use in various industrial applications, especially biopulping and bleaching. Xylanases are used in the pretreatment of pulp prior to bleaching to increase the liberation of lignin through the hydrolysis of hemicellulose (Suurnäkki et al. 2004). They have been employed to reduce the subsequent use of toxic chemicals, such as chlorine and hydrogen peroxide (H2O2) (Beg et al. 2001). Although xylanases potentially offer a number of advantages over conventional chemical reagents, their application at an industrial scale remains limited. In the case of biobleaching, the incoming pulp for the enzyme-catalyzed process usually employs a high temperature (70–100°C) (Beg et al. 2001), at which commercial xylanases, such as Cartazyme®, Ecopulp X200®, and Resinase®, are not sufficiently stable (Viikari et al. 2002). Consequently, there is on-going search for more potent strains of xylanase producers, especially those that can produce thermostable enzymes with greater yields (Viikari et al. 2007).
Xylanolytic enzymes from Aureobasidium pullulans, generally known as black yeast, can be efficiently produced (Leathers 1986; Li et al. 1993; Ohta et al. 2010). The cellulase-free xylanases from A. pullulans produced efficiently (Leathers 1986; Ohta et al. 2010) are advantageous to the pulp and paper industry in that the hydrolysis of cellulose fibers is avoided resulting in greater yields of recovered pulp. From a number of xylanase-producing A. pullulans isolated from a range of different Thai habitats (Manitchotpisit et al. 2009), one strain (A. pullulans CBS 135684), a color variant, produced cellulase-free xylanase that was relatively thermostable. The stabilization of enzymes remains an important concern especially during thermal processing. The loss of enzyme activity throughout the elevated temperature ranges is related to changes of enzyme conformation (Cui et al. 2008; Fu et al. 2010). In order to prevent the conformational changes of the enzyme, addition of chemicals such as polyols to can promote numerous hydrogen bond or salt-bridge formation between amino acid residues which make the enzyme molecule more rigid, and therefore more resistant to the thermal unfolding (George et al. 2001; Costa et al. 2002). However, the selection of the appropriate additive depends on the nature of the enzyme. The objectives of this study were to (i) characterize the biochemical properties of purified thermally stable xylanase from an A. pullulans yeast strain, (ii) determine the effect of polyols on the thermostability of the enzyme, and (iii) investigate the potential application of the xylanase in the pulp bleaching of non-woody material. Rice straw was utilized as it is an alternative raw material that can be used for pulping with advantages of its porous fiber structure, greater concentration of holocellulose and less lignin levels (Rodrígueza et al. 2008). In addition, fibers from rice straw are shorter than softwood fibers which results in superior paper that can replace hardwood chemically treated pulps for printing and writing paper.
Materials and methods
Organism and culture conditions
Aureobasidium pullulans previously isolated, was maintained at the fungal culture collection of the Plant Biomass Utilization Research Unit, Department of Botany, Faculty of Science, Chulalongkorn University, Bangkok, Thailand (Manitchotpisit et al. 2009). It was deposited at the Centraalbureau voor Schimmelcultures, The Netherlands (CBS number 135684). The yeast was grown in yeast malt (YM) agar medium (Atlas 1993) at room temperature for 2 days and short-term stock cultures were stored at 4°C. For long-term storage, the strain was stored at -20°C in YM broth containing 20% (v/v) glycerol.
Seed culture was prepared by growing A. pullulans in basal medium (Leathers 1986) containing 1% (w/v) glucose at 30°C with 150-rpm agitation for 72 h. The inoculum was adjusted to 2.5 × 106 cells. mL-1 and 100 μL was transferred into a 250-mL Erlenmeyer flask containing 100 mL of basal medium supplemented with 1% (w/v) agricultural wastes including wheat germ, wheat bran or corncob as the sole carbon source. The cultures were incubated at 30°C with 150-rpm agitation for 3 days. The cells were separated from the culture broth by centrifugation (18,000 × g, 20 min) at 4°C. The supernatant was used as the crude enzyme solution for further studies.
Xylanase and cellulase activities were assayed (Bailey et al. 1992) at 70°C using 1% (w/v) beech wood xylan (Fluka, USA) and 0.5% (w/v) carboxymethyl cellulose with the degree of substitution of 0.65 (Sigma, USA) as the substrates in 50 mM acetate buffer (pH 6.0). The amount of released reducing sugars was determined by the 3,5-dinitrosalicylic acid (DNS) method (Miller 1959). One unit (U) of xylanase/cellulase was defined as the amount of enzyme required to release 1 μmol xylose/glucose equivalent per min under the optimal conditions. Results are reported as the mean value of three replicates.
The extracellular xylanase was purified at 4°C. Culture supernatant (500 mL) from late log-phase (72 h) was concentrated ten-fold by ultrafiltration (10 kDa MW membrane cut-off, Amicon, Beverly, MA, USA) prior to precipitation using ammonium sulfate (50–80% saturation). After recovery (centrifugation 10,000 × g for 20 min), the pellet was dissolved in 20 mL 20 mM Tris–HCl buffer (pH 8.0) and dialyzed for 24 h against the same buffer. The enzyme solution (~28 mg total protein.mL-1) was applied (5 mL each run) to a pre-equilibrated anion exchange DEAE Sepharose FF (Sigma-Aldrich Co., USA) column. After washing the column with five-column-volumes of 20 mM Tris–HCl buffer (pH 8.0), elution was performed with a linear NaCl gradient (0 to 1.0 M over 50 mL) in the same buffer at a flow rate of 1.0 mL.min-1. Five-mL fractions were collected and analyzed for xylanase activity and protein content. The protein content was estimated by the Lowry method (Lowry et al. 1951), using bovine serum albumin (BSA) as the standard. Fractions with xylanase activity were pooled, dialyzed against 50 mM acetate buffer (pH 6), and then concentrated by ultrafiltration. Five mL of the concentrated enzyme solution (~4 mg total protein/mL) was applied to a 30 × 5 cm gel filtration column (Sephacryl S-100 HR, Sigma, USA) pre-equilibrated with 50 mM acetate buffer (pH 6.0). Elution was carried out at a flow rate of 0.5 mL.min-1, collecting 3 mL fractions that were assayed for xylanase activity and protein content as described above. The protein profiles of the collected fractions were determined by resolution through SDS-PAGE (12.5% (w/v) acrylamide resolving gel) as described (Laemmli 1970).
Optimal conditions for xylanase activity
The optimal temperature for the purified xylanase activity was determined by incubating the reaction mixture (4.5 U.mL-1 enzyme and 1% w.v-1 xylan, total volume 5 ml) at various temperatures, ranging from 40–90°C and at different pHs. To determine the optimal pH for enzyme activity, 50 mM sodium acetate buffers of pH 3.0–6.0 and 50 mM phosphate buffers of pH 6.0–10.0 were used. The relative activity was calculated as the percentage of enzyme activity in comparison to the maximum activity.
Effects of ions
To investigate the effect of ions on the enzyme activity, CaCl2, CuCl2, MgCl2, FeSO4.7H2O, CoCl2, ZnCl2 and EDTA were separately added to the reaction mixture at two different final concentrations of 1 and 10 mM, respectively, prior to performing the enzyme assay under the optimum conditions. The relative activity was calculated as a percentage of enzyme activity without the addition of ion.
The substrate specificity of the purified xylanase was tested with each of the followings; viz. beech wood xylan, oat spelt xylan, rice straw xylan, α-cellulose and carboxymethyl cellulose (CMC). Oat spelt and rice straw xylans were prepared according to the method of Höijea et al. (2005). The xylanase activity was assayed under the optimum conditions.
Effect of temperature on enzyme stability and thermodynamic analysis
where R is the gas constant (8.314 J.mol-1.K-1).
where h is the Planck constant (11.04 × 10-36 J.min) and kB is the Boltzman constant (1.38 × 10-23 J.K-1).
Effect of polyols on xylanase thermostability
In order to improve the thermal stability of the xylanase, polyols including ethylene glycol (2C), glycerol (3C), xylitol (5C), sorbitol (6C) and mannitol (6C) were added to separate enzyme solutions at 0.5 M final concentration prior to incubation at 70°C. Aliquots were withdrawn every 30 min, ice-cooled and then the residual xylanase enzyme activity was assayed under the optimal conditions. The stability of the enzyme was expressed as a percentage of residual activity (% RA) compared with activity of the initial enzyme activity (before incubation and no polyols). The polyol that most improved the thermostability was selected for further study over a range of concentration on the optimal (0.25–1.00 M) at 70°C.
Pulp treatment and property determination
The rice straw was collected from a local rice field in Suphanburi province, Thailand. The pulping of rice straw was carried out with the soda process (Chaiarrekij et al. 2011) before being extensively washed with tap water to remove the alkali. The xylanase pretreatment (18.6 U crude xylanase.g-1 dry pulp) with or without sorbitol at a final concentration of 0.75 M, was performed in transparent plastic bags with 10% (w/v) rice straw pulp suspended in 50 mM sodium acetate buffer (pH 6.0). The reaction was performed at 70°C for 2 h (Viikari et al. 2007). Reducing sugars in the hydrolysates were determined by the DNS method. For H2O2 treatment, the enzyme-treated pulp (or the enzyme-untreated pulp as the control) was transferred to the H2O2 solution (10% (v/v) final concentration) and incubated for 1 h at the same temperature. The resultant bleached pulp was made into 60 g.m-2 hand sheets on a Rapid-Köthen sheet former (RK-2A KWT, PTI, Austria) according to the ISO Standard Method 5269–2. The brightness and opacity of the hand sheets were measured using an optical tester (Color Touch PC, Technidyne, U.S.A.), based on the ISO Standard Methods 2470 and 2471, respectively. The tensile and tear indexes were determined after tensile strength and tear resistance were measured using a tensile strength tester (Strograph E-S, Toyo Seiki, Japan) and a tear strength tester (Protear, Thwing-Albert, U.S.A.) according to TAPPI Standard Method T 494 om-01 and T414 om-04, respectively. Fiber morphology was also analyzed using a fiber quality analyzer (FQA LDA02, OpTest Equipment, Canada) according to the TAPPI Standard Method T271 om-12. Untreated pulp was used as the control.
Statistical differences among the means of data were calculated using one-way analysis of variance (ANOVA) and Duncan’s Multiple Range Test (DMRT) or Student’s t-test (2 tailed) with the SPSS 17.0 software package (SPSS Inc., Chicago, U.S.A.). Differences at P < 0.05 were considered significant.
Results and discussion
Xylanase activity produced by A. pullulans CBS 135684 cultivated in basal medium containing 1% (w/v) agricultural waste as the sole carbon source at room temperature (28 ± 3°C) for 3 days
Xylanase activity (U.mL-1)*
3.25 ± 0.13b
2.59 ± 0.08a
4.10 ± 0.10c
Purification of the native extracellular xylanase from A. pullulans CBS 135684
Purification steps of xylanase isolated from A. pullulans CBS 135684 cultivated in basal medium containing 1% (w/v) corncob
Total protein (mg)*
Total activity (U)a,*
Specific activity (U.mg-1)*
2,030.00 ± 32.24
4,850.00 ± 36.42
2.39 ± 0.04
1,376.00 ± 26.18
4,431.00 ± 27.31
3.22 ± 0.06
(NH4)2SO4 precipitation (50–80% saturation)
560.00 ± 1.24
2,440.00 ± 14.24
4.36 ± 0.02
21.00 ± 1.04
834.00 ± 9.84
39.70 ± 1.76
16.00 ± 1.24
662.00 ± 10.46
41.40 ± 2.85
Optimal conditions for xylanase activity
The optimal pH (pH 6.0) and temperature (70°C) of the purified xylanase were similar to those of the crude enzyme (data not shown). Xylanases that are active in an alkaline environment and at high temperature are uncommon in yeasts (Techapun et al. 2002). Even among A. pullulans strains, previous reports on xylanase from A. pullulans (NRRL Y-2311-1 and ATCC 42023) showed that the enzymes were active at acidic pH (4.5-4.8) (Leathers 1989; Li et al. 1993; Vadi et al. 1996). Alkaline tolerant and thermotolerant xylanases have been reported in a number of bacteria, including Bacillus sp. (Zheng et al. 2000) and Clostridium absonum CFR-702 (Rani and Nand 2000). With its broad optimum pH and thermophilic properties, the xylanase from A. pullulans CBS 135684 may be used in several industrial applications not only for pulp bleaching but also for the bioconversion of lignocellulosic materials that is more optimally performed at a high pH and temperature (Parachin et al. 2009).
Effects of metal ions
Effects of different cations with the purified xylanase from A. pullulans CBS 135684 cultivated in basal medium containing 1% (w/v) corncob
Relative enzyme activity (%)* at an additive concentration of:
100.0 ± 2.0abc, NS
100.0 ± 1.0C, NS
99.9 ± 4.5abc, 2
74.1 ± 1.2B, 1
105.9 ± 3.0c, 1
111.7 ± 5.1E, 2
112.4 ± 1.5d, NS
112.4 ± 2.8E, NS
95.5 ± 2.2a, 2
29.9 ± 2.7A, 1
103.4 ± 4.1bc, 1
106.1 ± 3.5D, 2
99.2 ± 2.5ab, NS
98.4 ± 2.4C, NS
100.9 ± 5.3abc, NS
99.6 ± 1.2C, NS
The activity of the purified xylanase on various substrates was determined. The enzyme showed high specificity towards different xylans. Among them, the highest activity was observed with 1% (w/v) beech wood xylan (4.10 ± 0.1 U.mL-1), followed by oat spelt xylan (3.87 ± 0.23 U.mL-1) and rice straw xylan (3.48 ± 0.12 U.mL-1), respectively. It might be due to the fact that the substitution of side chains in the cereal xylans was higher than that of the hardwood xylan (Voragen et al. 1992). In contrast, no activity was observed when using α-cellulose or CMC as substrate which indicated that the xylanase enzyme from A. pullulans CBS 135684 was cellulase-free (data not shown).
Thermostability of the purified xylanase
Thermodynamic parameters for the irreversible thermal inactivation of the purified xylanase from A. pullulans CBS 135684 cultivated in basal medium containing 1% (w/v) corncob
Effect of polyols on the thermostability of the purified xylanase
Prebleaching of rice straw pulp
The effect of xylanase pretreatment on the physical and optical properties of rice straw pulp
39.5 ± 0.10a
39.8 ± 5.90
7.68 ± 0.50a
0.93 ± 0.05
49.3 ± 0.34a
0.07 ± 0.002a
1.48 ± 0.04a
55.5 ± 1.00b
35.8 ± 7.00
6.56 ± 0.61a
1.01 ± 0.05
45.2 ± 0.14b
0.07 ± 0.003a
1.42 ± 0.03b
Xylanase + H2O2
62.8 ± 0.40c
42.4 ± 3.90
12.2 ± 0.75b
0.93 ± 0.05
40.6 ± 0.90c
0.09 ± 0.002b
1.59 ± 0.02c
Xylanase + Sorbitol + H2O2
63.0 ± 0.58c
46.3 ± 4.90
13.1 ± 1.30b
1.00 ± 0.16
39.7 ± 0.30c
0.10 ± 0.008c
1.65 ± 0.02d
From the observation of fiber morphology, no significant changes were noticed in the fiber length while the fines content was decreased by the xylanase-pretreatment of the pulp. The lower fines content led to the higher numeric average of fiber length and thus the paper strength was enhanced (Liu et al. 2012). In general, the strength of paper is influenced by the fiber morphology which can be adversely affected by the kink and curl of fibers (Page 1969). However, in this study, the paper strength, in terms of the tear and tensile indexes, increased along with the curl and kink indexes of the fibers after xylanase pretreatment. This may be due to the digested fibers becoming more flexible, thus the moderate curl of the fibers could enhance the interweaving between them which led to the strengthened bonding between the pulp fibers (Liu et al. 2012) These results clearly indicated that the crude xylanase from A. pullulans CBS 135684 had potential for treatment of rice straw before bleaching for paper manufacture since it significantly improved the paper brightness without compromising fiber quality.
The extracellular xylanase produced by a Thai strain of A. pullulans CBS 135684 grown on corncob based media was cellulase-free and showed an enhanced activity at a high temperature (70°C). The purified enzyme was active over a broad pH range (> 70% activity at pH 4.0-10.0, and optimal at pH 6.0) which was uncommon among yeasts. The negative entropy change at all temperatures suggested that xylanase proceeds towards compaction during denaturation. The thermostability of the enzyme at high temperatures was improved by 0.75 mM sorbitol supplementation in the enzyme preparation. In the bleaching of rice straw fibers, enzyme pretreatment prior to H2O2 mediated bleaching significantly increased the fiber brightness and boosted the bleaching effect of H2O2. This new thermophilic xylanase from A. pullulans CBS 135684 has potential for use in the bleaching of rice straw fibers.
This work was financially supported by the Ratchadaphiseksomphot Endowment Fund of Chulalongkorn University (RES560530024-EN) and Eveleigh-Fenton Fund (Rutgers University).
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