Bleaching of kraft pulp by a commercial lipase: Accessory enzymes degrade hexenuronic acids

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Enzyme and Microbial Technology 43 (2008) 130–136

Bleaching of kraft pulp by a commercial lipase: Accessory enzymes degrade hexenuronic acids David Nguyen a , Xiao Zhang a,∗ , Zhi-Hua Jiang a , Andr´e Audet a , Michael G. Paice a , Sylvie Renaud a , Adrian Tsang b a

FPInnovations - Paprican Division (PAPRICAN), 570 boul. St-Jean, Pointe-Claire, Quebec, Canada H9R 3J9 b Centre for Structural and Functional Genomics, Concordia University, 7141 Sherbrooke Street West, Montreal, Quebec, Canada H4B 1R6 Received 10 July 2007; received in revised form 16 November 2007; accepted 20 November 2007

Abstract The potential of a commercial lipase for bleaching kraft pulp was investigated. Enzymatic treatment of hardwood and softwood kraft pulps before and after oxygen delignification resulted in a significant reduction of kappa number and hexenuronic acid content. Pretreatment of unbleached hardwood kraft pulp followed by a DE bleaching sequence gave the same kappa number as a control sequence but with considerably less chlorine dioxide. Two enzyme fractions containing high accessory enzyme activities were purified by size exclusion chromatography. These fractions demonstrated a high bleaching efficiency as well as a high selectivity, with a significant reduction in sugar released in the filtrate compared to commercial xylanase bleaching. This work provides the first evidence that the accessory enzymes from a commercial enzyme preparation can degrade hexenuronic acids and bleach kraft pulp. © 2007 Elsevier Inc. All rights reserved. Keywords: Biobleaching; Accessory enzymes; Hexenuronic acids; Kraft pulp

1. Introduction Biobleaching of kraft pulp using xylanase has provided pulp mills with advantages such as improving environmental performance, reducing bleaching cost, increasing productivity and enhancing pulp properties. This technology has been well adopted worldwide. In North America, approximately 2.5 million tonnes of kraft pulp is produced annually with xylanase as a pre-bleaching step. However, several negative effects from xylanase bleaching recognized in the last few years have hindered the further application of xylanase bleaching. In industrial practice, xylanase prebleaching can typically cause a pulp yield loss of up to 1% based on dry pulp [1]. This loss is mainly due to the excessive hydrolysis of pulp hemicellulose by the enzymes. The solubilized hemicelluloses also lead to a significant increase in the amount of COD and BOD delivered to the bleaching effluent system. Mills with limited effluent treatment capacity have been forced to stop using xylanase bleaching tech-

∗

Corresponding author. E-mail address: [email protected] (X. Zhang).

0141-0229/$ – see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2007.11.012

nology. Mechanistic studies have shown that xylanase treatment enhances kraft pulp bleaching by either removing re-deposited xylans on pulp fiber surfaces or cleaving the linkages between xylan and residual lignin [2]. Conventional kraft pulping produces unbleached pulp with 3–5% residual lignin content. Due to its close interaction with carbohydrates in the pulp, this residual lignin is difficult to remove without significantly degrading cellulose and decreasing pulp strength. A recent study [3] has shown that the presence of hexenuronic acids in kraft pulp is another major factor contributing to high bleaching chemical consumption, decreased brightness and increased brightness reversion. It is postulated that the presence of hexenuronic acids promotes the formation of lignin–carbohydrate complexes, as hexenuronic acid moieties are suggested to be a site for lignin–carbohydrate linkages [4,5]. It is conceivable that removing hemicelluloses that interact with residual lignin and/or hexenuronic acids will help enhance pulp bleaching selectivity. Several enzymes, loosely described as accessory enzymes, have attracted increasing attention in recent years due to their specific activity at the interfaces between lignin and carbohydrates. For instance, feruloyl esterases can selective remove

D. Nguyen et al. / Enzyme and Microbial Technology 43 (2008) 130–136 Table 1 Characteristics of the four kraft pulps Type of kraft pulp Unbleached hardwood KP (UBHW) Unbleached softwood KP (UBSW) Hardwood-O2 delignification (O2 HW) Softwood-O2 delignification (O2 SW)

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2.5. Kappa number measurement and hexenuronic acids measurement Kappa number 14.5 31.8 7.6 14.5

Hexenuronic acids (mmol/kg) 34.9 21.8 33.5 18.5

Pulp kappa number was measured using an automated kappa number measurement device developed at Paprican [10]. The hexenuronic acid content for the pulp samples was determined by an ion-chromatographic method described previously [11].

2.6. Bleaching of kraft pulps following enzyme treatment (XD0 E)

ferulic acid, an analog of the lignin monomers, from arabinoxylan present in plant cell walls [6]. Other accessory enzymes, such as arabinofuranosidase and ␣-glucuronidase help debranch side chain groups from the hemicellulose backbone, for example releasing glucuronic acids, a precursor of hexenuronic acids, from cell wall xylan [7]. This group of enzymes has potential for biobleaching applications. Accessory enzyme activities have been found in several commercial enzyme preparations [8]. A commercial lipase from the fungus Aspergillus niger, lipase A, produced by Amano Enzyme Inc. (Nagoya, Japan) was shown to exhibit a high level of feruloyl esterase activity [8]. In the current study, we examined the potential of this commercial enzyme and its fractions for biobleaching of both hardwood and softwood kraft pulps.

After the enzyme treatment (X) stage, the pulps were used in the subsequent chlorine dioxide (D0 ) stage after washing. In the D0 stage, pulps at 4% consistency were placed into small plastic bags and incubated at 45 ◦ C for 50 min. Three different dosages of chlorine dioxide, 0.26, 0.21, and 0.16 active chlorine multiple (ACM), were applied. The chlorine dioxide (ClO2 ) was pre-heated to 45 ◦ C before it was added to the pulp to which enough sulfuric acid had been added to assure a final pH between 2.2 and 2.6. After bleaching, the pulps were filtered through a Buchner funnel, and washed with deionized water. For the final alkaline extraction (E) stage, D0 bleached pulps were placed in plastic bags at 10% consistency and maintained at 75 ◦ C for 60 min after mixing with a pre-heated sodium hydroxide charge based on the initial kappa number of the various pulps. For the hardwood and softwood kraft pulp, the sodium hydroxide charges were, respectively, 1.25%, and 2.0%, while for the oxygen delignified hardwood and softwood kraft pulps the charges were 1.0%, and 1.25%. After 60 min, the pulps were washed with deionized water, and made into handsheets for kappa and hexenuronic acids determination.

2. Materials and methods

2.7. Protein purification of lipase A “Amano” 12

2.1. Enzyme Lipase A “Amano” 12 produced from Aspergillus niger was purchased from Amano Enzyme Inc. (Nagoya, Japan). A crude lipase A enzyme solution was prepared by dissolving an appropriate amount of the lyophilized powder in 100 mL of a 50 mM phosphate buffer (pH 6.5). This crude enzyme solution was then filtered through a 250 mL Nalgene CN filter unit with a 0.2 ␮m pore size and 50 mm diameter membrane (catalogue number: 126-0020) for sterility and to remove particulates. This sample was then desalted and concentrated in an Amicon ultrafiltration device through a regenerated cellulose YM membrane (molecular mass cut-off 1 kDa) (Millipore, Bedford, Massachusetts, USA) against a 50 mM phosphate buffer at pH 6.5. A commercial xylanase (produced from Trichoderma reesei) which is currently employed in Canadian pulp mills for pulp bleaching was used in this study.

2.2. Pulps All pulp samples used in the study were collected from Paprican Member Companies. The characteristics of these pulp samples are presented in Table 1.

Desalting of the crude lipase A “Amano” 12 mixture was carried out using Bio-Rad disposable desalting columns, Econo-PacTM 10DG (732–2010), as per the manufacturer’s instruction manual at pH 6.0. The desalted enzyme was then purified through a Superdex 200 HR 10/30 column (17-1088-01) using an eluent containing 50 mM phosphate buffer with 0.15 M sodium chloride. The resulting protein fractions were then individually concentrated in an Amicon ultrafiltration device through a regenerated cellulose YM membrane (molecular mass cut-off 1 kDa) (Millipore, Bedford, Massachusetts, USA) against a 50 mM phosphate buffer at pH 6.0.

2.8. Polyacrylamide gel electrophoresis A Mini-PROTEAN® 3 electrophoresis cell was used with a precast Ready Gel Tris–HCl with linear gradient 4–15% to separate the proteins. The molecular markers were purchased pre-stain SDS-PAGE standards in the low range covering molecular weights from 20,700 to 103,000. A SDS reducing buffer was used to dilute the various protein fractions to same protein content and was heated at 95 ◦ C for four minutes. The gel was run at 200 V for approximately 35 min, the dye band was stopped within 2–3 mm of the bottom of the gel. Coomassie Blue was then used to stain the gel to visualize the protein bands.

2.3. Enzymatic pulp treatment All pulp treatments were initially performed in a stainless steel Hobart mixer, where the pulp at a 10% consistency was mixed at 150 rpm with the required enzyme dosage and pre-heated (50 ◦ C) in 50 mM phosphate buffer at pH 6.5. This pulp mixture was then transferred to a small plastic bag and incubated for 2 h in a water bath at 50 ◦ C. The pulp mixture was then filtered through a Buchner funnel, washed with 2 L of deionized water, and made into handsheets for kappa number and hexenuronic acids determination. The control samples were treated in the same manner except there was no enzyme addition.

2.4. Handsheet preparation Handsheets were prepared according to PAPTAC Standard Testing Methods [9].

2.9. Enzyme assays Lipase activity was measured by a method previously described using pnitrophenol palmitate (PNPP) as a substrate [12] where one unit equals one ␮mol of p-nitrophenol liberated from PNPP in one minute. Feruloyl esterase activities were determined by the method of Mastihuba et al. [8] where one unit of enzyme activity is defined as the amount of enzyme releasing 1 ␮mol of 4nitrophenol from 4-nitrophenyl ferulate in one minute. ␣-l-Arabinofuranosidase activity was determined by the method in Biely et al. [13] where one unit of ␣-larabinofuranosidase activity is defined as the amount as the amount of enzyme liberating 1 ␮mol 4-nitrophenol in one minute. Xylanase activities were based on the method of Miller [14] where one unit is defined as the amount of enzyme that produces 1 ␮mol of xylose per minute from birchwood xylan. The protein content of each enzyme fraction was determined as described by Bradford [15].

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3. Results Samples of unbleached hardwood kraft pulp (UBHW), oxygen delignified hardwood kraft pulp (O2 HW), unbleached softwood kraft pulp (UBSW), and oxygen delignified softwood kraft pulp (O2 SW) were used in the study. These samples had different kappa numbers as well as hexenuronic acid content (Table 1). The kappa number of the four pulp samples varied from 7.6 to 31.8, while the hexenuronic acid content ranged from 18.5 to 34.9 mmol/kg of o.d. pulp (mmol/kg). To find an optimum enzyme dosage for pulp treatment, we tested a series of enzyme dosages from 0 to 21 lipase units per gram (LU/g) of oven dried pulp (UBHW). It was assumed that the optimal dosage was the same for each pulp. The residual kappa number and hexenuronic acid content were determined after 2 h treatment. As shown in Fig. 1, a 2-h lipase treatment of UBHW resulted in a significant reduction of both kappa number and hexenuronic acid content at all enzyme dosages tested. A high enzyme loading of 21 LU/g reduced the kappa number by about 2.5 points and the hexenuronic acid content by almost 9 mmol/kg, while a low enzyme addition (0.42 LU/g) reduced kappa number by 1.3 and the hexenuronic acid content by 2.3 mmol/kg. Most of the achievable reduction occurred at enzyme dosages of 2.1 LU/g and higher. We selected a dosage of 2.1 LU/g for the subsequent experiments. The ability of lipase A to reduce kappa number and hexenuronic acid content of the four pulp samples was then determined (Table 2). It is evident that the lipase treatment had a bleaching effect on the pulps although the effect with UBSW was small. The percent reduction in kappa number and hexenuronic acid content is shown in brackets. Comparing hardwood and softwood kraft pulps, lipase A was more effective in reducing the kappa number of hardwood kraft pulp. The kappa removal after one-step lipase A treatment for hardwood and softwood kraft pulps is between 11.8–12% and 2–10%, respectively. The enzyme exhibited a high reactivity towards hexenuronic acids in all four pulps with the decrease ranging from 13% to 24.4%. It is also apparent that

Table 2 Effects of lipase A treatment of four kraft pulps on kappa number and hexenuronic acid content Type of kraft pulp

Treatment Kappa number (percent decrease)

Hexenuronic acids (mmol/kg) (percent decrease)

UBHW

Control Lipase

14.4 12.7 (11.8 %)

32.7 27.5 (15.9 %)

UBSW

Control Lipase

31.7 31.1 (1.9 %)

19.3 16.8 (13 %)

O2 HW

Control Lipase

7.5 6.6 (12 %)

32.3 26.9 (16.7 %)

O2 SW

Control Lipase

14.3 12.8 (10.5 %)

19.7 14.9 (24.4 %)

the enzyme is more reactive towards oxygen delignified pulps. Lipase A treatment degraded 16.7% and 24.4% of hexenuronic acids present in O2 HW and O2 SW respectively, comparing to a reduction between 13% and 16% in brownstock pulps (UBSW and UBHW). We next compared the lipase A with a commercial xylanase which is currently used by Canadian pulp mills for pulp bleaching. The optimum enzyme loading for pulp bleaching was previously determined at 1.3 U of xylanase units per gram of oven dried pulp (XU/g). As shown in Table 3, a 2-h treatment of UBHW with commercial xylanase alone without subsequent chemical bleaching did not bring any significant changes to either pulp kappa number or hexenuronic acid content. The xylanase treatment was also carried out on the other three pulp samples, O2 HW, UBSW and O2 SW; no significant decreases in kappa number or hexenuronic acid content were observed (data not shown). To determine the effects of lipase A and xylanase treatment on pulp bleachability, a DE bleaching sequence was carried out following enzyme treatments of UBHW. Three levels of active chlorine multiples, 0.16, 0.21 and 0.26, were used in the D stage. The ACMs were calculated on the basis of the original kappa number of the pulps and not the kappa number after the enzyme treatment. As shown in Figs. 2 and 3, both lipase A and xylanase pretreatments resulted in bleached pulps with lower kappa number and hexenuronic acid content compared to those obtained from control experiments. Lipase A at the enzyme dosage tested was more effective than the xylanase for improving the bleaching of kraft pulp at all ACM levels. Bleaching of lipase A pretreated UBHW using 0.16 ACM resulted in similar kappa number and hexenuronic acid content to those obtained from control bleaching using 0.26 ACM. Table 3 Comparison of lipase A and commercial xylanase for their effects on bleaching of unbleached hardwood kraft pulp (UBHW)

Fig. 1. The decrease of kappa number and hexenuronic acid content after lipase A treatment of unbleached hardwood kraft pulp (UBHW) at different enzyme dosages.

Sample

Enzyme activity (per gram of pulp)

Kappa number

Hexenuronic acids (mmol/kg)

Control Lipase A Xylanase

– 2.1 LU 1.3 XU

14.4 12.7 13.8

32.7 27.5 31.1

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Table 4 The accessory enzymes and xylanase activities (pH 6.5) present in lipase A fractions and xylanase Sample Lip Fraction 1 Lip Fraction 2 Lip Fraction 3 Xylanase a

Lipase activity (U/mg)

Feruloyl esterase activity (U/mg)

Arabino-furanosidase activity (mU/mg)

Xylanase activity (U/mg)

5.8 0.04 0.2 ND

NDa

ND 8.7 2.9 4.0

ND 21 72 670

7.5 1.6 0.06

ND: not detected.

Fig. 2. The residual kappa number of unbleached hardwood kraft pulp (UBHW) after lipase A and commercial xylanase pretreatments followed by a DE chemical bleaching sequence.

ing, we fractionated the crude lipase mixture by size exclusion chromatography and obtained three major protein fractions. A gel electrophoregram of the fractions is shown in Fig. 4. The majority of the protein (about 90% based on protein content) was collected as fraction 1, which contains predominantly lipase activity with minor xylanase activity. Two other fractions, fraction 2 and 3, were collected later in the column separation. The enzyme activities present in the three fractions were determined and compared with the commercial xylanase (Table 4). Both fraction 2 and 3 demonstrated xylanase activity when birch xylan was used as substrate. The specific xylanase activities in both fractions 2 and 3 were considerably lower than that of commercial xylanase. There was a significant level of feruloyl esterase activity detected in fraction 2 (7.5 U per milligram of protein (U/mg)), while the same enzyme activity in fraction 3 was 1.6 U/mg. feruloyl esterase activity is negligible in the commercial xylanase. Fraction 2 also exhibited a higher arabinofuranosidase activity that either lipase A fraction 3 or commercial xylanase. There was little accessory activity detected in lipase A fraction 1. To compare the bleaching efficiency of the three lipase A fractions with the commercial xylanase, we treated the UBHW with these four enzymes based on the same protein content. Table 5 clearly demonstrates a significant bleaching effect for

Fig. 3. The residual hexenuronic acid content of unbleached hardwood kraft pulp (UBHW) after lipase A and commercial xylanase pretreatments followed by a DE chemical bleaching sequence.

Lipase A improved bleaching to a greater degree than commercial xylanase with lower kappa number and hexenuronic acid content obtained after DE bleaching at all three ACM levels. However, we recognized that this commercial enzyme contains several enzyme activities. To further determine the active enzyme components that contribute to this improved bleach-

Fig. 4. Polyacrylamide gel electrophoresis of protein molecular weight markers and three lipase fractions (lane 1: protein markers; lane 2: fraction 3; lane 3: fraction 2; lane 4: fraction 1).

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Table 5 The effects of treatment (pH 6.5, 50 ◦ C, 2 h) of unbleached hardwood kraft pulp (UBHW) by commercial xylanase and three lipase A fractions on kappa number and hexenuronic acid content reduction Sample

Kappa number

Hexenuronic acids (mmol/kg)

Control Xylanase Fraction 1 Fraction 2 Fraction 3

14.4 13.8 14.4 12.3 12.7

32.7 31.1 32.1 26.5 27.8

Table 6 Sugars released in a one-step enzyme treatment of unbleached hardwood kraft pulp (UBHW) using lipase A fractions and commercial xylanase Sample

Lipase Fraction 2 Lipase Fraction 3 Xylanase

Sugars released (mg/L) Arabinose

Galactose

Glucose

Xylose

Mannose

ND ND ND

27 ND ND

50 ND 140

1080 270 1200

50 ND ND

lipase fraction 2 and 3 treatments. At the same enzyme protein content, fraction 2 displayed the highest capability to remove hexenuronic acids and reduce pulp kappa number. Fraction 3 was also effective for removing hexenuronic acids and reducing kappa number. Neither fraction 1 nor commercial xylanase alone had a significant bleaching effect in the absence of further chemical bleaching. The selectivity of these enzymes or enzyme fractions for biobleaching was also tested by determining the amount of sugars released after enzyme treatment of UBHW. As seen in Table 6, a 2-h xylanase treatment released close to 1.2 g/L of total sugars into the filtrate. There was also a considerable amount of sugars hydrolyzed after lipase A fraction 2 treatment (∼1.1 g/L), while only a small amount, 0.27 g/L was released from pulp after fraction 3 treatments. The results indicate that more selective enzymes for pulp bleaching are likely present in lipase A fractions 2 and 3. 4. Discussion The recalcitrant nature of the residual lignin is due in part to the presence of xylan in hardwood kraft pulp. The precipitated xylan can form a barrier on fiber surfaces that hinders the diffusion of residual lignin from the fiber wall. Xylan based hemicelluloses also form covalent bonds with residual lignin. The effectiveness of xylanase pretreatment for bleaching boosting has been demonstrated in industrial applications. Although the exact mechanism involved in xylanase prebleaching is still in debate, it is conceivable that degrading pulp xylans by xylanase treatment will improve the porosity of the cell wall and help solubilize lignin and chromophores that are otherwise bound to xylans. It is apparent that the use of xylanase will inevitably cause excessive hydrolysis of xylans that are not associated with residual lignin. The ability of accessory enzymes to debranch hemicellulose side groups that may interact with residual lignin holds promise for more selective delignification [6,16,17]. A recent study assessed the potential of a feruloyl esterase from

Aspergillus niger to bleach wheat straw soda AQ pulp [17]. While this study concluded the feruloyl esterase is a promising enzyme for pulp bleaching, the results presented were not sufficient to demonstrate that this enzyme has a direct bleaching capability. In our initial work, we screened a number of accessory enzymes and several commercial enzymes for their bleachability. A commercial enzyme, lipase A from Amano, was shown to be able to bleach kraft pulp. This enzyme has been previously reported to contain a significant feruloyl esterase activity and some other accessory enzyme activity [8]. The reduction of kappa number and hexenuronic acid content after lipase A treatment of different kraft pulps suggests that feruloyl esterase and/or other accessory enzymes may be associated with this direct bleaching effect. The presence of di-ferulic acid bridges between polysaccharides and lignin in the cell walls has been demonstrated in non-wood plants. The presence of linkages between lignin and glucuronic acid attached to the xylan backbone has also been identified in wood [18]. The kappa number was originally proposed to represent the amount of residual lignin in pulp. Recent studies have shown that hexenuronic acids also contribute to kappa number. Hexenuronic acids are formed from 4-O-methylglucuronic acid residues present on xylan during kraft pulping [19]. This acid and lignin have similar reactivities towards electrophilic bleaching chemicals [3]. Previous work has proposed that the hexenuronic acid moieties are likely sites in kraft pulps where lignin–carbohydrate linkages are formed [4,5]. Using a previously proposed empirical equation [20], where 11.6 mmol hexenuronic acids corresponds to 1 kappa number unit, we calculated that the hexenuronic acids represent between 5% and 37% of the kappa measured in the four kraft pulp samples used in this study; the contribution of hexenuronic acids to kappa number is much less significant in softwood pulp than in hardwood. It was also found that oxygen delignification did not degrade hexenuronic acids but rather led to an increased percent contribution of hexenuronic acids to the pulp kappa number. As mentioned, lipase A treatments led to a reduction of hexenuronic acids of between 13% and 24.4% in the four kraft pulps. When the reduction in hexenuronic acids is calculated based on its percentage of kappa number, it was found that the reduction in hexenuronic acids represents 26%, 36%, 51% and 27% of the kappa number removal in UBHW, UBSW, O2 HW and O2 SW, respectively, the ratio of molar consumption of permanganate between one hexenuronic acid and phenylpropane unit is about 1–1.5 [21]. The presence of a complex of hexenuronic acid and lignin has been proposed previously [5], and such a structure is possibly a major reason for the recalcitrance of residual lignin. The results imply that the lipase A specifically degraded hexenuronic acids and in consequence removed the lignin attached to these acids. This is the first evidence that an enzyme can specifically remove hexenuronic acids from wood pulp. Our results also substantiate the theory that hexenuronic acids are a site for the formation of lignin–carbohydrate complexes (LCCs). While a one-step enzyme treatment can significantly decrease hexenuronic acids and kappa number, a pulp treated with lipase A also demonstrated a better bleachability than one treated with

D. Nguyen et al. / Enzyme and Microbial Technology 43 (2008) 130–136

commercial xylanase. Bleaching lipase A pretreated UBHW in a D0 E sequence resulted in a chlorine dioxide saving of up to 38% compared to the control. This saving is much more significant than that obtained with xylanase prebleaching. The reduction of pulp kappa numbers after lipase A treatment will lower the ACM requirement for the subsequent chlorine dioxide bleaching. Fractionation of lipase A enabled us to further identify the active enzyme constituents present in the crude enzyme solution. Three main fractions were obtained after size exclusion chromatography separation. The first fraction (FRC1) contains the majority of the protein from the enzyme and had a higher lipase activity with trace xylanase activity (Table 4). Two subsequent fractions (FRC2 and FRC3) showed negligible lipase activity, while containing several accessory activities. There is also an appreciable amount of xylanase activity detected in both fractions, although the specific activity is significantly lower than that shown by commercial xylanase. The feruloyl esterase activity present in Amano lipase A enzyme was previously determined by using either 4nitrophenyl ferulate (4NPF) or ethyl ferulate as substrates [8]. The specific activity of lipase A on 4NPF was about 1.1 U/mg, while its activity on ethyl ferulate was 0.411 U/mg. Fractions 2 and 3 from lipase A exhibited significant feruloyl esterase activities of 7.5 and 1.7 U/mg, respectively, using 4NPF as substrate. The bleaching effect obtained from fraction 2 and 3 treatment of UBHW was similar. This result indicates that feruloyl esterase or its related enzymes enhances wood pulp bleachability. The lack of feruloyl esterase activity in commercial xylanase is the most probable reason for its lower bleaching efficiency. A noticeable amount of arabinofuranosidase activity was detected in both fractions 2 and 3, and in commercial xylanase. The arabinofuranosidase has been shown previously to act synergistically with xylanase in bleaching of wood pulp [7,22]. However, its reaction with hexenuronic acids requires further study. Bleaching selectivity is considered as one of the most important criteria for a superior bleaching enzyme. To determine the selectivity of each enzyme and enzyme fraction, we measured the amount of sugar released in the filtrates after enzymatic treatments of UBHW. As shown in Table 6, commercial xylanase treatment released a considerable amount of xylose with some glucose. It is not clear whether the glucose is hydrolyzed from cellulose or glucomannan. It is known that this xylanase contains some endoglucanase activity measured by CMC assay. Treatment with lipase fraction 2 also solubilized an appreciable amount of xylose with some galactose, glucose and mannose. It should be mentioned that these enzyme fractions are not pure after one step column separation; several protein bands were detected in all three fractions by polyacrylamide gel electrophoresis (Fig. 4). The release of galactose, glucose and mannose suggests that fraction 2 also acted on glucomannan present in kraft pulp. The lipase fraction 3 gave the most promising results in terms of sugar degradation. Although, this fraction appears to have a higher xylanase activity based on the DNS assay, it only released a small amount of xylose. The DNS assay using soluble birch xylan as a substrate is a well accepted method for measuring xylanase activity, however, it does not necessarily predict the true hydrolytic potential of the enzyme on isolat-

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able substrate [23–25]. The ability of lipase fraction 3 to bleach kraft pulp with minimum hemicellulose degradation reaffirms the presence of enzymes in lipase A highly specific for removing xyloses associated with either residual lignin and/or hexenuronic acids. Examining the effects of these fractions on pulp bleaching will be included in our future work. Further study is required to identify the key enzyme components present in Lipase A mixture and elucidate the mechanism of these enzyme reactions on LCC and hexenuronic acid-xylan model compounds. 5. Conclusions We have demonstrated that a commercial lipase bleaches kraft pulp and improves bleachability to a greater degree than commercial xylanase. The enzyme demonstrated a specific activity toward degrading hexenuronic acids and subsequent release of lignin that is attached to these acid groups. The presence of accessory enzyme activities including feruloyl esterase and arabinofuranosidase are likely the major factors contributing to the superior performance. The results from this study provide the first evidence that accessory enzymes from a commercial enzyme preparation can have a direct bleaching effect on kraft pulp, removing hexenuronic acids and reducing kappa number. The application of accessory enzymes promises a more selective biobleaching strategy for the pulp and paper industry. Acknowledgments The authors are thankful to the comments and suggestions from Drs. Jean Bouchard and Richard Berry. We would also like to thank Prof. Bernard Prior from University of Stellenbosch in South Africa for providing purified accessory enzymes. The financial support from Paprican Member Companies and Genome Canada/Genome Quebec is greatly appreciated. References [1] Paice M, Renaud S, Bourbonnais R, Labonte S, Berry R. The effect of xylanase on kraft pulp bleaching yield. J Pulp Paper Sci 2004;30:241–6. [2] Kantelinen A, Hortling B, Sundquist J, Linko M, Viikari L. Proposed mechanism of the enzymatic bleaching of kraft pulp with xylanases. Holzforschung 1993;47:318–24. [3] Jiang ZH, van Lierop B, Berry R. Hexenuronic acid groups in pulping and bleaching chemistry. TAPPI J 2000;83:167–75. [4] Jiang ZH, van Lierop B, Nolin A, Berry R. A new insight into the bleachability of kraft pulps. J Pulp Paper Sci 2003;29(2):167–75. [5] Jiang ZH, Bouchard J, Berry R. Evidence for the formation of lignin-hexenuronic acid-xylan complexes during modified kraft pulping processes. Holzforschung 2006;60(2):137–42. [6] de Vries RP, Kester HCM, Poulsen CH, Benen JAE, Visser J. Synergy between enzymes from Aspergillus involved in the degradation of plant cell wall polysaccharides. Carbohydr Res 2000;327:401–10. [7] Saha BC. Alpha-l-arabinofuranosidases: biochemistry, molecular biology and application in biotechnology. Biotechnol Adv 2000;18:403–23. [8] Mastihuba V, Kremnicky L, Mastihubova M, Willett JL, Cote GL. A spectrophotometric assay for feruloyl esterases. Anal Biochem 2002;309:96–101. [9] PAPTAC. Forming Handsheets for Physical Tests of Pulp, in Pulp and Paper Technical Association of Canada Standard Testing Methods. PAPTAC 1997;C:4. Montreal, Quebec, Canada.

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