ISBN : 978-602-17761-0-0
205
© 2013 Published by Center for Pulp and Paper through REPTech2012
Xylanase, An Environmentally Friendly Bleaching Agent for Pulp and Paper: Characterization and Stability Study of Bacillus halodurans CM1 Crude Xylanase
Nuur Faridatun Hasanah, Dian Fajar Vitia Ningrum, Budiasih Wahyuntari
1
Center for Bioindustrial Technology, Laboratoriesfor Technology Development of Agro-Biomedical Industries, LAPTIAB- BPPT, Puspiptek-Serpong 611, Tangerang Selatan 15314, Indonesia
1
budiasih.wahyuntaribppt.go.id,
1
solichin.budiasihgmail.com
ABSTRACT
Thermoalkalophilic xylanases have many important applications especially in the pulp and paper industry. Bacillus halodurans CM1 a thermoalkalophilic bacterium recently has been isolated from sediment of Cimangguhot
spring, West Java. The isolate produced extracellular xylanase. The experiment observed the effect of pH and fermentation time on xylanase production and partial characterization of the enzyme that related to the enzyme
application as bleaching agent in pulp and paper processing. The highest production of xylanase was observed at pH 10 and temperature 55
o
C after 21 hours of fermentation. The optimal xylanase activity was at pH 7.0 and 70
o
C. The molecular weight of the enzyme was approximately 27.67 kDa. The enzyme hydrolyzed xylan into xylobiose,
xylotriose and other longer xylooligosaccharides. Stability study was conducted at pH 7.0 – 9.0 and temperature 60- 80ºC. The stability study showed that the enzyme only stable at pH 7 and 60
o
C, which retained 60 of its activity after 105 minutes, therefore the enzyme might be used for biobleaching of kraft pulp and deinking of used
paper. Keywords: Bacillus halodurans, xylanase enzyme, thermoalkalophilic bacterium
1. Introduction
Xylan is a major component of plant cell wall hemicellulose. The major constituents of
hemicelluloses are the hetero-1,4-β-D-Xylan and hetero-1,4-β-D-Mannans. Xylanis present
predominantly in hard wood and graminaceous plant. For complete hydrolysis of xylan, many
xylanolytic microorganisms often synthesize the multiple groups of xylanolytic enzymes [13].
Interest in the application of xylanases in the pulp and paper industry has increased during
recent years. Xylanases may be used in pulp- prebleaching process to remove the hemicelluloses
which bind to the pulp. The hydrolysis of pulp- bound hemicelluloses releases the lignin in the
pulp, reducing the amount of chlorine required for conventional chemical bleaching and minimizing
the toxic [13].The removal of lignin is essentially required during paper manufacturing. In some
study, mix enzyme preparation having both xylanase and laccase activity can be used for
biobleaching at 55
o
C, pH 9 [3, 7]. However, most of the xylanases known to date are optimally active
at temperatures below 50ºC and are active in acidic or neutral pH. Conversely, only a few xylanases
are reported to be active and stable at alkaline pH and high temperature [20, 22, 26].In this study, we
reported characterization of the enzyme produced by a newly isolated bacterium, including effect of
pH and temperature on xylanase activity, stability, and molecular weight of the enzyme.
2. Materials and Methods 2.1. Microorganism and Effect of pH and
Fermentation Time on Enzyme Production Bacillus halodurans CM1 culture collection of
Bioindustry Laboratory, BPPT was refreshed at 55
o
C in modiied Luria Broth medium containing 0.5
wv bacto peptone, 0.25 wv yeast extract, 0.25 wv sodium chloride and 1 wv xylan as a
substrate. The culture of Bacillus halodurans CM1 was fermented at 55
o
C for 24 h in the modiied Nakamura et al 1994 medium using 1 wv xylan as a substrate
at different pHs pH 8; 9; 10 11. The composition of modiied Nakamura et al 1994 was as follows: 0.25
wv bactopeptone, 0.25 wv yeast extract, 0.05 wv K
2
HPO
4
.3H
2
O, 0.01 wv MgSO
4
.7H
2
O and 1 wv beechwoodxylan Sigma Aldrich Co,
St. Louis, MO [14]. After 20 hours of fermentation, the sample was taken for measuring the activity and
protein content of the cells free supernatant. The cells were removed from fermented broth by centrifugation
at 5600 x g using High-Speed Refrigerated Centrifuge Himac CR 21G and R10A3 rotor for 15 min at 4
o
C and the supernatant from extracellular secretion was used
for characterization of the enzyme.
2.2. Effect of Temperature and pH on Enzyme Activity
The inluence of pHs on xylanase activities were measured at 50°C fermentation temperature at pHs
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© 2013 Published by Center for Pulp and Paper through REPTech2012
ranging from 7.0 to 11.0 buffer used at pH 7 8 was sodium phosphate, pH 9 was Tris HCL, pH 10 11
was glycine-NaOH. The inluence of temperature on enzyme activity
was determined by incubating the enzyme at optimum pH resulted from previous study at temperatures
ranging from 60-100
o
C.
2.3. Enzyme Activity and Protein Concentration
Xylanase activity was assayed using Dinitrosalicylic Acid DNS method, according to Bailey et al.
1992 [2]. Beechwoodxylan was suspended in 50 mM sodium phosphate buffer pH 7.0. The reaction
mixture contained 50 µL of crude enzyme and 450 µL of 1 wv xylan was incubated for 5 min in
the thermomixer at the optimum temperature for enzyme activity. The reaction was terminated by the
addition of 750 µL of DNS reagent. The mixture was centrifuged at 14.000 rpm using Centrifuges MiniSpin
plus from Eppendorf for 2 min and the supernatant was heated at 100
o
C for 5 min. The mixture was cooled in running water, and then added 250 µL of distilled
water. Reducing sugar released during incubation was measured as xylose equivalents at l540 nm. One unit
xylanase activity is deined as the amount of enzyme required to liberate 1 µmol of xylose per minute at the
assay condition [23, 24]. Protein concentration was determined by Bio-Rad
Protein Assay, based on the method of Bradford 1976 with Bovine Serum Albumin BSA as a standard
protein[7].
2.4. Stability of Enzyme
The pH of crude enzyme was adjusted at pH7.0; 8.0 9.0, then the reaction mixture was incubated
in different temperature 60, 70 80
o
C for 120 min. Residual activity was observed every 15 minutes.
2.5. Electrophoresis
The molecular weight of the crude xylanase was estimated by sodium dodecyl sulphate-polyacrylamide
gel electrophoresis in 12 vv acrylamide gel using low molecular weight markers Amersham
Low Molecular Weight Calibration Kit for SDS Electrophoresis. A suspension of beechwoodxylan
at a inal concentration 0.1 wv was incorporated into the separating gel before addition of ammonium
persulphate. Proteins were visualized by silver staining with silver nitrate solution.
2.6. Zymogram Analysis
