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Enzyme and Microbial Technology 43 (2008) 130–136

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

David Nguyen a, Xiao Zhang a,∗, Zhi-Hua Jiang a, Andre 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 3J9b 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

bstract

The potential of a commercial lipase for bleaching kraft pulp was investigated. Enzymatic treatment of hardwood and softwood kraft pulps beforend after oxygen delignification resulted in a significant reduction of kappa number and hexenuronic acid content. Pretreatment of unbleachedardwood kraft pulp followed by a DE bleaching sequence gave the same kappa number as a control sequence but with considerably less chlorineioxide. Two enzyme fractions containing high accessory enzyme activities were purified by size exclusion chromatography. These fractions

emonstrated a high bleaching efficiency as well as a high selectivity, with a significant reduction in sugar released in the filtrate compared toommercial xylanase bleaching. This work provides the first evidence that the accessory enzymes from a commercial enzyme preparation canegrade hexenuronic acids and bleach kraft pulp.

2007 Elsevier Inc. All rights reserved.

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eywords: Biobleaching; Accessory enzymes; Hexenuronic acids; Kraft pulp

. Introduction

Biobleaching of kraft pulp using xylanase has providedulp mills with advantages such as improving environmentalerformance, reducing bleaching cost, increasing productivitynd enhancing pulp properties. This technology has been welldopted worldwide. In North America, approximately 2.5 mil-ion tonnes of kraft pulp is produced annually with xylanases a pre-bleaching step. However, several negative effects fromylanase bleaching recognized in the last few years have hin-ered the further application of xylanase bleaching. In industrialractice, xylanase prebleaching can typically cause a pulp yieldoss of up to 1% based on dry pulp [1]. This loss is mainlyue to the excessive hydrolysis of pulp hemicellulose by thenzymes. The solubilized hemicelluloses also lead to a signifi-

ant increase in the amount of COD and BOD delivered to theleaching effluent system. Mills with limited effluent treatmentapacity have been forced to stop using xylanase bleaching tech-

∗ Corresponding author.E-mail address: xxzhang@paprican.ca (X. Zhang).

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ology. Mechanistic studies have shown that xylanase treatmentnhances kraft pulp bleaching by either removing re-depositedylans on pulp fiber surfaces or cleaving the linkages betweenylan and residual lignin [2].

Conventional kraft pulping produces unbleached pulp with–5% residual lignin content. Due to its close interaction witharbohydrates in the pulp, this residual lignin is difficult toemove without significantly degrading cellulose and decreas-ng pulp strength. A recent study [3] has shown that the presencef hexenuronic acids in kraft pulp is another major factor con-ributing to high bleaching chemical consumption, decreasedrightness and increased brightness reversion. It is postulatedhat the presence of hexenuronic acids promotes the formationf lignin–carbohydrate complexes, as hexenuronic acid moietiesre suggested to be a site for lignin–carbohydrate linkages [4,5].t is conceivable that removing hemicelluloses that interact withesidual lignin and/or hexenuronic acids will help enhance pulpleaching selectivity.

Several enzymes, loosely described as accessory enzymes,ave attracted increasing attention in recent years due to theirpecific activity at the interfaces between lignin and carbohy-rates. For instance, feruloyl esterases can selective remove

D. Nguyen et al. / Enzyme and Microbia

Table 1Characteristics of the four kraft pulps

Type of kraft pulp Kappa number Hexenuronic acids(mmol/kg)

Unbleached hardwood KP (UBHW) 14.5 34.9Unbleached softwood KP (UBSW) 31.8 21.8HS

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ardwood-O2 delignification (O2HW) 7.6 33.5oftwood-O2 delignification (O2SW) 14.5 18.5

erulic acid, an analog of the lignin monomers, from arabinoxy-an present in plant cell walls [6]. Other accessory enzymes,uch as arabinofuranosidase and �-glucuronidase help debranchide chain groups from the hemicellulose backbone, for exam-le releasing glucuronic acids, a precursor of hexenuronic acids,rom cell wall xylan [7]. This group of enzymes has potentialor biobleaching applications. Accessory enzyme activities haveeen found in several commercial enzyme preparations [8]. Aommercial lipase from the fungus Aspergillus niger, lipase A,roduced by Amano Enzyme Inc. (Nagoya, Japan) was showno exhibit a high level of feruloyl esterase activity [8]. In theurrent study, we examined the potential of this commercialnzyme and its fractions for biobleaching of both hardwood andoftwood kraft pulps.

. Materials and methods

.1. Enzyme

Lipase A “Amano” 12 produced from Aspergillus niger was purchased frommano Enzyme Inc. (Nagoya, Japan). A crude lipase A enzyme solution wasrepared by dissolving an appropriate amount of the lyophilized powder in00 mL of a 50 mM phosphate buffer (pH 6.5). This crude enzyme solutionas then filtered through a 250 mL Nalgene CN filter unit with a 0.2 �m pore

ize and 50 mm diameter membrane (catalogue number: 126-0020) for steril-ty and to remove particulates. This sample was then desalted and concentratedn an Amicon ultrafiltration device through a regenerated cellulose YM mem-rane (molecular mass cut-off 1 kDa) (Millipore, Bedford, Massachusetts, USA)gainst a 50 mM phosphate buffer at pH 6.5. A commercial xylanase (producedrom Trichoderma reesei) which is currently employed in Canadian pulp millsor pulp bleaching was used in this study.

.2. Pulps

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

.3. Enzymatic pulp treatment

All pulp treatments were initially performed in a stainless steel Hobart mixer,here the pulp at a 10% consistency was mixed at 150 rpm with the required

nzyme dosage and pre-heated (50 ◦C) in 50 mM phosphate buffer at pH 6.5.his pulp mixture was then transferred to a small plastic bag and incubated

or 2 h in a water bath at 50 ◦C. The pulp mixture was then filtered through auchner funnel, washed with 2 L of deionized water, and made into handsheets

or kappa number and hexenuronic acids determination. The control samplesere treated in the same manner except there was no enzyme addition.

.4. Handsheet preparation

Handsheets were prepared according to PAPTAC Standard Testing Methods9].

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l Technology 43 (2008) 130–136 131

.5. Kappa number measurement and hexenuronic acidseasurement

Pulp kappa number was measured using an automated kappa number mea-urement device developed at Paprican [10]. The hexenuronic acid content forhe pulp samples was determined by an ion-chromatographic method describedreviously [11].

.6. Bleaching of kraft pulps following enzyme treatment (XD0E)

After the enzyme treatment (X) stage, the pulps were used in the subsequenthlorine dioxide (D0) stage after washing. In the D0 stage, pulps at 4% con-istency were placed into small plastic bags and incubated at 45 ◦C for 50 min.hree different dosages of chlorine dioxide, 0.26, 0.21, and 0.16 active chlorineultiple (ACM), were applied. The chlorine dioxide (ClO2) was pre-heated to

5 ◦C before it was added to the pulp to which enough sulfuric acid had beendded to assure a final pH between 2.2 and 2.6. After bleaching, the pulps wereltered through a Buchner funnel, and washed with deionized water. For thenal alkaline extraction (E) stage, D0 bleached pulps were placed in plasticags at 10% consistency and maintained at 75 ◦C for 60 min after mixing withpre-heated sodium hydroxide charge based on the initial kappa number of thearious pulps. For the hardwood and softwood kraft pulp, the sodium hydroxideharges were, respectively, 1.25%, and 2.0%, while for the oxygen delignifiedardwood and softwood kraft pulps the charges were 1.0%, and 1.25%. After0 min, the pulps were washed with deionized water, and made into handsheetsor kappa and hexenuronic acids determination.

.7. Protein purification of lipase A “Amano” 12

Desalting of the crude lipase A “Amano” 12 mixture was carried out usingio-Rad disposable desalting columns, Econo-PacTM 10DG (732–2010), as per

he manufacturer’s instruction manual at pH 6.0. The desalted enzyme was thenurified through a Superdex 200 HR 10/30 column (17-1088-01) using an eluentontaining 50 mM phosphate buffer with 0.15 M sodium chloride. The resultingrotein fractions were then individually concentrated in an Amicon ultrafiltrationevice through a regenerated cellulose YM membrane (molecular mass cut-offkDa) (Millipore, Bedford, Massachusetts, USA) against a 50 mM phosphateuffer at pH 6.0.

.8. Polyacrylamide gel electrophoresis

A Mini-PROTEAN® 3 electrophoresis cell was used with a precast Readyel Tris–HCl with linear gradient 4–15% to separate the proteins. The moleculararkers were purchased pre-stain SDS-PAGE standards in the low range cover-

ng molecular weights from 20,700 to 103,000. A SDS reducing buffer was usedo dilute the various protein fractions to same protein content and was heated at5 ◦C for four minutes. The gel was run at 200 V for approximately 35 min, theye band was stopped within 2–3 mm of the bottom of the gel. Coomassie Blueas then used to stain the gel to visualize the protein bands.

.9. Enzyme assays

Lipase activity was measured by a method previously described using p-itrophenol palmitate (PNPP) as a substrate [12] where one unit equals onemol of p-nitrophenol liberated from PNPP in one minute. Feruloyl esterasectivities were determined by the method of Mastihuba et al. [8] where one unitf enzyme activity is defined as the amount of enzyme releasing 1 �mol of 4-itrophenol from 4-nitrophenyl ferulate in one minute. �-l-Arabinofuranosidasectivity was determined by the method in Biely et al. [13] where one unit of �-l-

rabinofuranosidase activity is defined as the amount as the amount of enzymeiberating 1 �mol 4-nitrophenol in one minute. Xylanase activities were basedn the method of Miller [14] where one unit is defined as the amount of enzymehat produces 1 �mol of xylose per minute from birchwood xylan. The proteinontent of each enzyme fraction was determined as described by Bradford [15].

1 crobial Technology 43 (2008) 130–136

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Table 2Effects of lipase A treatment of four kraft pulps on kappa number and hex-enuronic acid content

Type ofkraft pulp

Treatment Kappa number(percent decrease)

Hexenuronic acids (mmol/kg)(percent decrease)

UBHW Control 14.4 32.7Lipase 12.7 (11.8 %) 27.5 (15.9 %)

UBSW Control 31.7 19.3Lipase 31.1 (1.9 %) 16.8 (13 %)

O2HW Control 7.5 32.3Lipase 6.6 (12 %) 26.9 (16.7 %)

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32 D. Nguyen et al. / Enzyme and Mi

. Results

Samples of unbleached hardwood kraft pulp (UBHW), oxy-en delignified hardwood kraft pulp (O2HW), unbleachedoftwood kraft pulp (UBSW), and oxygen delignified softwoodraft pulp (O2SW) were used in the study. These samples hadifferent kappa numbers as well as hexenuronic acid contentTable 1). The kappa number of the four pulp samples variedrom 7.6 to 31.8, while the hexenuronic acid content rangedrom 18.5 to 34.9 mmol/kg of o.d. pulp (mmol/kg).

To find an optimum enzyme dosage for pulp treatment, weested a series of enzyme dosages from 0 to 21 lipase units perram (LU/g) of oven dried pulp (UBHW). It was assumed thathe optimal dosage was the same for each pulp. The residualappa number and hexenuronic acid content were determinedfter 2 h treatment. As shown in Fig. 1, a 2-h lipase treatment ofBHW resulted in a significant reduction of both kappa num-er and hexenuronic acid content at all enzyme dosages tested.

high enzyme loading of 21 LU/g reduced the kappa num-er by about 2.5 points and the hexenuronic acid content bylmost 9 mmol/kg, while a low enzyme addition (0.42 LU/g)educed kappa number by 1.3 and the hexenuronic acid contenty 2.3 mmol/kg. Most of the achievable reduction occurred atnzyme dosages of 2.1 LU/g and higher. We selected a dosagef 2.1 LU/g for the subsequent experiments. The ability of lipaseto reduce kappa number and hexenuronic acid content of the

our pulp samples was then determined (Table 2). It is evidenthat the lipase treatment had a bleaching effect on the pulpslthough the effect with UBSW was small. The percent reduc-ion in kappa number and hexenuronic acid content is shownn brackets. Comparing hardwood and softwood kraft pulps,ipase A was more effective in reducing the kappa number ofardwood kraft pulp. The kappa removal after one-step lipase

treatment for hardwood and softwood kraft pulps is between

1.8–12% and 2–10%, respectively. The enzyme exhibited aigh reactivity towards hexenuronic acids in all four pulps withhe decrease ranging from 13% to 24.4%. It is also apparent that

ig. 1. The decrease of kappa number and hexenuronic acid content after lipasetreatment of unbleached hardwood kraft pulp (UBHW) at different enzyme

osages.

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2SW Control 14.3 19.7Lipase 12.8 (10.5 %) 14.9 (24.4 %)

he enzyme is more reactive towards oxygen delignified pulps.ipase A treatment degraded 16.7% and 24.4% of hexenuroniccids present in O2HW and O2SW respectively, comparing to aeduction between 13% and 16% in brownstock pulps (UBSWnd UBHW).

We next compared the lipase A with a commercial xylanasehich is currently used by Canadian pulp mills for pulp bleach-

ng. The optimum enzyme loading for pulp bleaching wasreviously determined at 1.3 U of xylanase units per gram ofven dried pulp (XU/g). As shown in Table 3, a 2-h treatmentf UBHW with commercial xylanase alone without subsequenthemical bleaching did not bring any significant changes toither pulp kappa number or hexenuronic acid content. Theylanase treatment was also carried out on the other three pulpamples, O2HW, UBSW and O2SW; no significant decreases inappa number or hexenuronic acid content were observed (dataot shown).

To determine the effects of lipase A and xylanase treatmentn pulp bleachability, a DE bleaching sequence was carriedut following enzyme treatments of UBHW. Three levels ofctive chlorine multiples, 0.16, 0.21 and 0.26, were used in the

stage. The ACMs were calculated on the basis of the origi-al kappa number of the pulps and not the kappa number afterhe enzyme treatment. As shown in Figs. 2 and 3, both lipase

and xylanase pretreatments resulted in bleached pulps withower kappa number and hexenuronic acid content compared tohose obtained from control experiments. Lipase A at the enzymeosage tested was more effective than the xylanase for improv-ng the bleaching of kraft pulp at all ACM levels. Bleaching of

ipase A pretreated UBHW using 0.16 ACM resulted in similarappa number and hexenuronic acid content to those obtainedrom control bleaching using 0.26 ACM.

able 3omparison of lipase A and commercial xylanase for their effects on bleachingf unbleached hardwood kraft pulp (UBHW)

ample Enzyme activity (pergram of pulp)

Kappa number Hexenuronic acids(mmol/kg)

ontrol – 14.4 32.7ipase A 2.1 LU 12.7 27.5ylanase 1.3 XU 13.8 31.1

D. Nguyen et al. / Enzyme and Microbial Technology 43 (2008) 130–136 133

Table 4The accessory enzymes and xylanase activities (pH 6.5) present in lipase A fractions and xylanase

Sample Lipase activity (U/mg) Feruloyl esterase activity (U/mg) Arabino-furanosidase activity (mU/mg) Xylanase activity (U/mg)

Lip Fraction 1 5.8 NDa ND NDLip Fraction 2 0.04 7.5 8.7 21Lip Fraction 3 0.2 1.6Xylanase ND 0.06

a 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 chemicalbleaching sequence.

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fractions with the commercial xylanase, we treated the UBHWwith these four enzymes based on the same protein content.Table 5 clearly demonstrates a significant bleaching effect for

ig. 3. The residual hexenuronic acid content of unbleached hardwood kraftulp (UBHW) after lipase A and commercial xylanase pretreatments followedy a DE chemical bleaching sequence.

Lipase A improved bleaching to a greater degree than com-ercial xylanase with lower kappa number and hexenuronic acid

ontent obtained after DE bleaching at all three ACM levels.owever, we recognized that this commercial enzyme con-

ains several enzyme activities. To further determine the activenzyme components that contribute to this improved bleach-

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ng, we fractionated the crude lipase mixture by size exclusionhromatography and obtained three major protein fractions. Ael electrophoregram of the fractions is shown in Fig. 4. Theajority of the protein (about 90% based on protein content)as collected as fraction 1, which contains predominantly lipase

ctivity with minor xylanase activity. Two other fractions, frac-ion 2 and 3, were collected later in the column separation.he enzyme activities present in the three fractions were deter-ined and compared with the commercial xylanase (Table 4).oth fraction 2 and 3 demonstrated xylanase activity whenirch xylan was used as substrate. The specific xylanase activ-ties in both fractions 2 and 3 were considerably lower thanhat of commercial xylanase. There was a significant level oferuloyl esterase activity detected in fraction 2 (7.5 U per mil-igram of protein (U/mg)), while the same enzyme activity inraction 3 was 1.6 U/mg. feruloyl esterase activity is negligi-le in the commercial xylanase. Fraction 2 also exhibited aigher arabinofuranosidase activity that either lipase A fractionor commercial xylanase. There was little accessory activity

etected in lipase A fraction 1.To compare the bleaching efficiency of the three lipase A

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

134 D. Nguyen et al. / Enzyme and Microbia

Table 5The 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 numberand hexenuronic acid content reduction

Sample Kappa number Hexenuronic acids (mmol/kg)

Control 14.4 32.7Xylanase 13.8 31.1Fraction 1 14.4 32.1Fraction 2 12.3 26.5Fraction 3 12.7 27.8

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

Sample Sugars released (mg/L)

Arabinose Galactose Glucose Xylose Mannose

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ipase Fraction 2 ND 27 50 1080 50ipase Fraction 3 ND ND ND 270 NDylanase ND ND 140 1200 ND

ipase fraction 2 and 3 treatments. At the same enzyme proteinontent, fraction 2 displayed the highest capability to removeexenuronic acids and reduce pulp kappa number. Fraction 3as also effective for removing hexenuronic acids and reduc-

ng kappa number. Neither fraction 1 nor commercial xylanaselone had a significant bleaching effect in the absence of furtherhemical bleaching. The selectivity of these enzymes or enzymeractions for biobleaching was also tested by determining themount of sugars released after enzyme treatment of UBHW.s seen in Table 6, a 2-h xylanase treatment released close to.2 g/L of total sugars into the filtrate. There was also a con-iderable amount of sugars hydrolyzed after lipase A fraction 2reatment (∼1.1 g/L), while only a small amount, 0.27 g/L waseleased from pulp after fraction 3 treatments. The results indi-ate that more selective enzymes for pulp bleaching are likelyresent in lipase A fractions 2 and 3.

. Discussion

The recalcitrant nature of the residual lignin is due in parto the presence of xylan in hardwood kraft pulp. The precip-tated xylan can form a barrier on fiber surfaces that hindershe diffusion of residual lignin from the fiber wall. Xylan basedemicelluloses also form covalent bonds with residual lignin.he effectiveness of xylanase pretreatment for bleaching boost-

ng has been demonstrated in industrial applications. Althoughhe exact mechanism involved in xylanase prebleaching is still inebate, it is conceivable that degrading pulp xylans by xylanasereatment will improve the porosity of the cell wall and helpolubilize lignin and chromophores that are otherwise boundo xylans. It is apparent that the use of xylanase will inevitablyause excessive hydrolysis of xylans that are not associated with

esidual lignin. The ability of accessory enzymes to debranchemicellulose side groups that may interact with residual ligninolds promise for more selective delignification [6,16,17]. Aecent study assessed the potential of a feruloyl esterase from

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l Technology 43 (2008) 130–136

spergillus niger to bleach wheat straw soda AQ pulp [17].hile this study concluded the feruloyl esterase is a promis-

ng enzyme for pulp bleaching, the results presented were notufficient to demonstrate that this enzyme has a direct bleachingapability.

In our initial work, we screened a number of accessorynzymes and several commercial enzymes for their bleachabil-ty. A commercial enzyme, lipase A from Amano, was showno be able to bleach kraft pulp. This enzyme has been previ-usly reported to contain a significant feruloyl esterase activitynd some other accessory enzyme activity [8]. The reductionf kappa number and hexenuronic acid content after lipase Areatment of different kraft pulps suggests that feruloyl esterasend/or other accessory enzymes may be associated with thisirect bleaching effect. The presence of di-ferulic acid bridgesetween polysaccharides and lignin in the cell walls has beenemonstrated in non-wood plants. The presence of linkagesetween lignin and glucuronic acid attached to the xylan back-one has also been identified in wood [18].

The kappa number was originally proposed to representhe amount of residual lignin in pulp. Recent studies havehown that hexenuronic acids also contribute to kappa num-er. Hexenuronic acids are formed from 4-O-methylglucuroniccid residues present on xylan during kraft pulping [19]. Thiscid and lignin have similar reactivities towards electrophilicleaching chemicals [3]. Previous work has proposed that theexenuronic acid moieties are likely sites in kraft pulps whereignin–carbohydrate linkages are formed [4,5]. Using a pre-iously proposed empirical equation [20], where 11.6 mmolexenuronic acids corresponds to 1 kappa number unit, we calcu-ated that the hexenuronic acids represent between 5% and 37%f the kappa measured in the four kraft pulp samples used in thistudy; the contribution of hexenuronic acids to kappa numbers much less significant in softwood pulp than in hardwood. Itas also found that oxygen delignification did not degrade hex-

nuronic acids but rather led to an increased percent contributionf hexenuronic acids to the pulp kappa number.

As mentioned, lipase A treatments led to a reduction of hex-nuronic acids of between 13% and 24.4% in the four kraft pulps.

hen the reduction in hexenuronic acids is calculated based onts percentage of kappa number, it was found that the reductionn hexenuronic acids represents 26%, 36%, 51% and 27% of theappa number removal in UBHW, UBSW, O2HW and O2SW,espectively, the ratio of molar consumption of permanganateetween one hexenuronic acid and phenylpropane unit is about–1.5 [21]. The presence of a complex of hexenuronic acid andignin has been proposed previously [5], and such a structure isossibly a major reason for the recalcitrance of residual lignin.he results imply that the lipase A specifically degraded hex-nuronic acids and in consequence removed the lignin attachedo these acids. This is the first evidence that an enzyme canpecifically remove hexenuronic acids from wood pulp. Ouresults also substantiate the theory that hexenuronic acids are a

ite for the formation of lignin–carbohydrate complexes (LCCs).

While a one-step enzyme treatment can significantly decreaseexenuronic acids and kappa number, a pulp treated with lipasealso demonstrated a better bleachability than one treated with

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ommercial xylanase. Bleaching lipase A pretreated UBHW in a0E sequence resulted in a chlorine dioxide saving of up to 38%

ompared to the control. This saving is much more significanthan that obtained with xylanase prebleaching. The reduction ofulp kappa numbers after lipase A treatment will lower the ACMequirement for the subsequent chlorine dioxide bleaching.

Fractionation of lipase A enabled us to further identify thective enzyme constituents present in the crude enzyme solu-ion. Three main fractions were obtained after size exclusionhromatography separation. The first fraction (FRC1) containshe majority of the protein from the enzyme and had a higheripase activity with trace xylanase activity (Table 4). Two sub-equent fractions (FRC2 and FRC3) showed negligible lipasectivity, while containing several accessory activities. There islso an appreciable amount of xylanase activity detected in bothractions, although the specific activity is significantly lowerhan that shown by commercial xylanase.

The feruloyl esterase activity present in Amano lipaseenzyme was previously determined by using either 4-

itrophenyl ferulate (4NPF) or ethyl ferulate as substrates [8].he specific activity of lipase A on 4NPF was about 1.1 U/mg,hile its activity on ethyl ferulate was 0.411 U/mg. Fractions 2

nd 3 from lipase A exhibited significant feruloyl esterase activ-ties of 7.5 and 1.7 U/mg, respectively, using 4NPF as substrate.he bleaching effect obtained from fraction 2 and 3 treatment ofBHW was similar. This result indicates that feruloyl esteraser its related enzymes enhances wood pulp bleachability. Theack of feruloyl esterase activity in commercial xylanase is the

ost probable reason for its lower bleaching efficiency. A notice-ble amount of arabinofuranosidase activity was detected in bothractions 2 and 3, and in commercial xylanase. The arabinofura-osidase has been shown previously to act synergistically withylanase in bleaching of wood pulp [7,22]. However, its reactionith hexenuronic acids requires further study.Bleaching selectivity is considered as one of the most impor-

ant criteria for a superior bleaching enzyme. To determine theelectivity of each enzyme and enzyme fraction, we measuredhe amount of sugar released in the filtrates after enzymatic treat-

ents of UBHW. As shown in Table 6, commercial xylanasereatment released a considerable amount of xylose with somelucose. It is not clear whether the glucose is hydrolyzed fromellulose or glucomannan. It is known that this xylanase con-ains some endoglucanase activity measured by CMC assay.reatment with lipase fraction 2 also solubilized an appre-iable amount of xylose with some galactose, glucose andannose. It should be mentioned that these enzyme fractions

re not pure after one step column separation; several proteinands were detected in all three fractions by polyacrylamide gellectrophoresis (Fig. 4). The release of galactose, glucose andannose suggests that fraction 2 also acted on glucomannan

resent in kraft pulp. The lipase fraction 3 gave the most promis-ng results in terms of sugar degradation. Although, this fractionppears to have a higher xylanase activity based on the DNS

ssay, it only released a small amount of xylose. The DNS assaysing soluble birch xylan as a substrate is a well accepted methodor measuring xylanase activity, however, it does not necessarilyredict the true hydrolytic potential of the enzyme on isolat-

l Technology 43 (2008) 130–136 135

ble substrate [23–25]. The ability of lipase fraction 3 to bleachraft pulp with minimum hemicellulose degradation reaffirmshe presence of enzymes in lipase A highly specific for removingyloses associated with either residual lignin and/or hexenuroniccids. Examining the effects of these fractions on pulp bleachingill be included in our future work. Further study is required to

dentify the key enzyme components present in Lipase A mix-ure and elucidate the mechanism of these enzyme reactions onCC and hexenuronic acid-xylan model compounds.

. Conclusions

We have demonstrated that a commercial lipase bleaches kraftulp and improves bleachability to a greater degree than com-ercial xylanase. The enzyme demonstrated a specific activity

oward degrading hexenuronic acids and subsequent release ofignin that is attached to these acid groups. The presence ofccessory enzyme activities including feruloyl esterase and ara-inofuranosidase are likely the major factors contributing tohe superior performance. The results from this study providehe first evidence that accessory enzymes from a commercialnzyme preparation can have a direct bleaching effect on kraftulp, removing hexenuronic acids and reducing kappa number.he application of accessory enzymes promises a more selectiveiobleaching strategy for the pulp and paper industry.

cknowledgments

The authors are thankful to the comments and suggestionsrom Drs. Jean Bouchard and Richard Berry. We would alsoike to thank Prof. Bernard Prior from University of Stellen-osch in South Africa for providing purified accessory enzymes.he financial support from Paprican Member Companies andenome Canada/Genome Quebec is greatly appreciated.

eferences

[1] Paice M, Renaud S, Bourbonnais R, Labonte S, Berry R. The effect ofxylanase on kraft pulp bleaching yield. J Pulp Paper Sci 2004;30:241–6.

[2] Kantelinen A, Hortling B, Sundquist J, Linko M, Viikari L. Proposedmechanism of the enzymatic bleaching of kraft pulp with xylanases. Holz-forschung 1993;47:318–24.

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