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BioSMART ISSN: 1411-321X

Volume 5, Nomor 1 April 2003


Halaman: 1-4

Xylanolytic Activity of the Recombinant Plasmid (pBX6) Cloned from


Fibrobacter succinogenes S85 Expressed in Escherichia coli HB 101

Y. SURYADI1, A. SIPAT2,3, K. M. YUSOFF2,3, H.M. YUSOFF2,3


1
Department of Protein Engineering and Immunology, RIFCB Bogor 16111
2
Department of Biochemistry and Microbiology, Faculty of Science and Environmental Sciences, Universiti Pertanian Malaysia
Serdang, Selangor DE, 43400 Malaysia
3
Fermentation Technology Center, Universiti Pertanian Malaysia, Serdang, Selangor DE, 43400 Malaysia

Received: 7 November 2002. Accepted: 25 December 2002

ABSTRACT

pBX6 is a recombinant plasmid containing a 3 kb fragment from Fibrobacter succinogenes S 85 genomic DNA encodes xylanase
activity which express in E. coli HB 101. Growth characteristics of the plasmid showed that xylanase activity was observed after 24 to
48 h incubation period. The growth kinetic of the recombinant plasmid pBX6 on 2 Yeast Tryphtone (YT) medium was higher in the
presence of ampicylin. pBX6 produced higher xylanase activity at pH 7.4 and 37oC.

Key words: pBX6, xylanase, Fibrobacter succinogenes S 85.

INTRODUCTION have properties characteristics of typical endoxylanase


producing xylo-oligosaccharides as the major hydrolytic
Xylan is one of the major components of hemicellulose product. The third cloned showed endonuclease and
found in the plant cell walls. This compound has currently xylanase activities, whereas the fourth cloned (xynC)
regarded as a usable biomass product, which can be produce a large amount of xylose and xylobiose (Forsberg
converted into biofuels and chemicals (Woodward, 1984). and Cheng, 1992). Xylanase enzymes have molecular
Biodegradation of xylan involves the action of three major weights ranging from 15 kDa to 63 kDa (Dekker and
xylanolytic enzymes, namely endo β-1,4-xylanases (β -1,4- Richards, 1976; Paradis et al., 1993). Matte and Forsberg
D xylan xylanohydrolase, EC 3.2.1.8); β-1,4-xylosidases (1992) had studied two xylanase activities from F.
(β-1,4 D xyloside xylohydrolase EC 3.2.1.37) and exoglu- succinogenes; a 54-kDa arabinose debranching xylanase
canases (β-1,4-D xylan xylohydrolase) (Coughland and and a 66-kDa xylanase with endonuclease activity. Paradis
Hazlewood, 1993). The ability in degrading cellulose in et al. (1993) discovered xylanase carrying cloned xynC that
plant cell walls is related to the production of hemicellulase expressed in E. coli, had a molecular mass of 63 kDa. Sipat
enzymes including xylanase, acetylxylans esterase and et al. (1987) had cloned a xylanase gene (9.4 kb) from F.
arabinofuranase (Forsberg et al., 1981). succinogenes S 85 into E. coli HB 101 (pBX1) and studied
Xylanases and xylosidases, the major enzymes that its hydrolytic properties. Deletion fragment of 3 kb
involved in degradation of the xylose have been studied containing the xylanase gene from F. succinogenes S 85
from various microorganisms. The major cellulolytic and was further cloned into pUC19 vector. The recombinant
xylanolytic bacteria include Ruminococcus flavofacians, R. plasmid namely pBX6 expressed xylanase activity when
albus (Malburg et al., 1993), Bacillus coagulans 26 transformed into E. coli HB 101, however, the properties of
(Esteban et al., 1983); B. subtilis (Uchino and Nakane, pBX6 has not been well characterized. Studies on the
1981); B. circulans (Yang et al., 1989); and B. polymoxa characterization of highly active xylanolytic enzymes
(Hespel, 1996). Many strains of F. succinogenes were produce by recombinant plasmid cloned from bacteria may
reported to possess a complement of fibrolytic enzymes be useful in development of technique to improve
that extensively degrade plant cell walls polymers (Hu et degradation of plant hemicellulose.
al., 1991; Forsberg and Cheng, 1992). This work was undertaken to study biochemical
Fournier et al. (1985) reported that xylanolytic characteristics of the recombinant plasmid pBX6 carrying a
microorganisms produce multiple forms of xylanase with xylanase gene from F. succinogenes S 85.
different physical and kinetics properties. Several studies
have been carried out to clone the xylanase gene from
various F. succinogenes strains, such as F. succinogenes S MATERIALS AND METHODS
85 and S 135 (Forsberg et al., 1981; Hu et al., 1991;
Paradis et al., 1992). Previously, four unique xylanase General recombinant culture
genes have been cloned from F. succinogenes S 85 Escherichia coli HB 101 containing a recombinant
(Malburg et al., 1993). Two of the cloned were reported xylanolytic activity was previously cloned from

© 2003 Jurusan Biologi FMIPA UNS Surakarta


2 BioSMART Vol. 5, No. 1, April 2003, hal. 1-4

Fibrobacter succinogenes S 85 and was found to be RESULTS AND DISCUSSION


capable of producing multiple xylanase (Sipat et al., 1987).
The bacterial culture containing recombinant plasmid Growth kinetics of pBX6
pBX6 was maintained on 2 Yeast Tryptone (YT) solid The colony growing on 2 YT + RBB xylan agar
mediums supplemented with antibiotic ampicylin (50 medium displayed xylanase activity by producing a
mg/ml). Plating serial dilution on 2 YT agar medium and clearing zone (halo) surrounding the colony due to the
spectrophotometry using absorbance 550 nm checked hydrolysis of xylan by enzyme activity. Biely et al. (1985)
growth of colonies from preserved stock. An assay for stated that the formation of several halos was resulted
xylanase production was determined on remazol brilliant from diffusion and digestion of xylanase on RBB-
blue (RBB)-xylan agar plate medium supplemented with xylan. The result showed that plasmid pBX6 was found
10 µg/ml ampicylin. For this purposes, six fold dilution to be capable of producing xylanase when grown in E. coli
was spread out on 2 YT medium containing 0.2% RBB- HB 101. To monitor growth characteristics of recombinant
xylan (oat spelt xylan) as substrate (Biely et al., 1985). plasmid, E. coli HB 101 (pBX6) was cultured on 2 YT
Xylanase plaques showing clearing zone colony was broths in the presence or absence of ampicylin over a 72 h
observed after incubation at 37oC for overnight. The incubation period at 37oC. Cultures forming colonies in the
hydrolysis of xylan substrate, which showed by the clear subcultures (diameter 1 to 2 mm) were considered to be
colony surrounding agar media, revealed the production of capable of growth on xylan. Recombinant plasmid grown
xylanase. Growth of recombinant culture on various on 2 YT broth medium containing ampicylin reached
environmental conditions was carried out on different optimum growth near lag phase after 24 h incubation. The
media, pH and temperature. Population growth was recombinant plasmid was stable grown on 2 YT broth
measured as colony forming unit (CFU) at optical density medium containing ampicylin which showed optimum
(OD) 550 nm. growth at 24 to 48-h incubation period. It is shown that
bacteria normally reproduce by binary fission, which
Xylanolytic activity results in doubling of the number of viable bacterial cells
Xylanase enzyme activity was assayed by incubating under optimal condition. During the stationary phase of
the crude preparation from culture suspension with xylan as growth, the bacterial viability (CFU) increased and
a substrate at a given environmental condition (different pH declined rapidly thereafter. The cell viability, as indicated
and temperature). Crude filtrate for xylanase enzyme by colony forming unit (CFU) was much higher in the
preparation of F. succinogenes S85 was prepared as broth containing ampicylin compared with that of without
described by Matte and Forsberg (1992). E. coli HB 101 ampicylin (Table 1). The decline of the cell population was
containing pBX6 was grown in 2 YT broth supplemented slower in the presence of ampicylin. This result proofs the
with ampicylin (50 µg/ml). Triplicate flasks (250 ml) presence of ampicylin maintained cell viability better, due
containing 50 ml of medium were incubated at 37°C for to the expression of the ampicylin resistance gene in
over night with vigorous shaking (250 rpm). Culture fluid recombinant plasmid pBX6.
cells were harvested by centrifugation at 16,000 g for 5 min
at room temperature, then they washed twice in 100 ml of
0.05 M sodium phosphate buffer (pH 7.0) and resuspended Table 1. Effect of ampicylin on growth of pBX6.
in the same volume of 25% sucrose in the presence of 0.1
Incubation time (h) With ampicylin Without ampicylin
mM EDTA. The cell free-culture supernatant was concen-
(log cfu/ml) (log cfu/ml)
trated by ultrafiltration through an Amicon membrane (10 0 0 0
kDa cut-off). Approximately 25 µl of crude enzyme was 12 9.53 9.46
preincubated with 225 µl of Mc-Ilvaine buffer pH 7.2 at 24 9.57 9.54
various temperatures ranging from 20oC to 80oC for 30 36 9.49 9,42
min. The same procedure was followed using the pH 48 9.24 9.20
condition of 2 to 10. Reduction viscosity of the solution 60 9.23 9.20
(sugar released) was measured by Somogyi-Nelson's 72 9.20 9.10
method (Whitehead, 1993). Protein concentration was
measured by the method of Lowry (Lowry, 1951) using The recombinant plasmid culture could also grow better
bovine serum albumin (BSA) (Sigma, USA) as the stan- on others media such as on Luria broth (LB), sucrose broth
dard, following the procedure stated in the manufacturer’s (SOB) and terrific broth. Abundance growth of the viscous
instruction manual. The enzyme activity was determined by bacterial culture (E. coli HB 101) containing recombinant
pre incubating enzyme (0.1 ml of crude solution) with 2 g plasmid pBX6 was observed on 2 YT broth than that of
oat spelt xylan in 100 ml of Mc-Ilvaine buffer (0.1 M citric Luria broth (LB), and sucrose peptone broth (SP) media
acid and 0.2 M Na2HPO4, pH 7.2), measured at 37oC. In which contained different composition of minerals and
control assay, the enzyme had been inactivated by heating carbon source. Yeast extract enhanced the level of growth
in boiling water bath for 30 min. Specific activity was to about 3 to 5 fold that of cultures grown without this
defined as units per mg (U/mg) of protein (Whitehead, substance as mineral source. It was suggested that the
1993). One unit of enzyme was defined as the amount culture utilize its mineral better on 2YT than other media.
xylanase activity corresponding to 1 µmol of reducing Culture on 2 YT broth media showed highest absorbance
sugar released per minute using D-xylose as a standard. reading on spectrophotometer OD550= 0.79. pBX6 grew
SURYADI et al. – Xylanolytic activity of F. succinogenes in E. coli 3

well in the media containing of glucose compare without enzyme activity at temperature of 37oC (specific activity
supplementation of this substance; however, the pBX6 119 U/mg), and at pH 7.4 (specific activity 61.99 U/mg),
failed to grow on minimal media (Figure 1). respectively.
The action of crude specific activity (U/mg) was
characterized by rapid decrease in the viscosity of the xylan
Effect of media on growth of pBX6
solution and relatively slow increase in the amount of
0.9 reducing sugar released. It was previously shown that total
0.8 endoxylanase activity of F. succinogenes S 85 culture was
optical density (OD) 550nm

0.7 expressed constitutively (Malburg et al., 1993). The


0.6 characteristics of the xylanase investigated by zymogram
0.5 analysis suggested that multiplicity of xylanases in E. coli
0.4 HB 101 were probably due to post translational
0.3 modification of a single gene product (Soong, 1996). The
0.2
recombinant plasmid pBX6, which was derived from
0.1
plasmid pBX1 grown in E. coli HB 101, produced an active
0
xylanase in the extracellular culture filtrate (Sipat et al.,
media
2 YT LB YE Min SOB Terr M17
1987). Malburg et al. (1993) stated that xylanase enzyme
activity associated with the cells obtained from F.
succinogenes S 85 was found in the periplasm.
Figures 2 and 3 shows temperature and pH-activity
Figure 1 . Effect of media on growth of pBX6. profiles of crude xylanase. Temperature optimum was
observed at 37oC. In comparison with other experiments,
Other environmental factors such as temperature and the endoxylanases from F. succinogenes S 85 showed a
pH in affecting the growth of the culture. The bacterial temperature optimum of 40oC (Matte and Forsberg, 1992).
population growth in 2 YT medium, as revealed by colony However, Soong (1996) observed temperature optimum for
forming unit (cfu/ml) at different temperatures and pH, xynA and xynB1 ranging from 40˚C to 50˚C. The optimum
showed mean average of 8.01 + 0.9 log cfu/ml and 8.28 + pH of the xylanase observed in this study was pH 7.0,
1.05 log cfu/ml, respectively. The temperature and pH whereas the optimum pH of the endoxylanase produced by
optimum for colony production was 37oC and pH 7, F. succinogenes S 85 as reported by Matte and Forsberg
respectively (Table 2). (1992) and Soong (1996) was pH 6.2 and 7.0, respectively.
Approximately 58% of the enzyme activity was retained at
Table 2. Effect of temperature and pH on growth of pBX6.
the pH range of 7 to 8. At low or high temperature the
Population Absorbance (OD660) enzyme loss specific activity approximately ranging from
Treatment 26% to 36%, while on acidic or alkaline pH condition the
(log cfu/ml) + sd
Temperature (oC) specific activity of the enzyme was reduced by 67% to
25 9.10 0.13 + 0.01 72%. The decrease in xylanolytic activity at pH value
28 9.30 0.84 + 0.20 above or below pH 7 is probably due to the effect of pH on
31 9.45 1.11 + 0.01 the regulation of enzyme synthesis or it could be attributed
34 9.48 1.92 + 0.19 to the inherent instability of the enzyme molecule.
37 9.50 2.02 +0.02
40 9.40 1.93 + 0.21
43 8.90 0.79 + 0.005
specific activity (U/mg)

46 8.68 0.43 + 0.002 140


120
pH
100
4 7.30 0.15 + 0.05
5 7.80 1.31 + 0.01 80
6 8.39 2.27 + 0.003 60
7 9.05 2.50 + 0.008
8 8.69 2.28 + 0.017 40
9 8.27 2.00 + 0.02 20
10 6.70 0.03 + 0.006 0
4 20 25 30 37 40 45 50 55 60 65 70 75 80
pH/temperature-profiles of pBX6
The xylanase gene of F. succinogenes S 85 released the temperature(oC)
enzyme into the extracellular culture fluid under optimal
environmental condition (Forsberg et al., 1981). The crude Figure 2. Temperature-Activity Profile of Crude Xylanase.
enzyme of pBX6 culture showed optimum sugar reduction Effect of Temperature on xylanase enzyme activity was measured
activity at temperature 37oC and pH 7.4. However, the in batch. Culture using Mc-Ilvaine buffer pH 7.2. Xylanase
activity was determined by measuring the reducing sugar liberated
specific activity was slightly slow at higher temperature
from the digestion of oat -spelt xylan using D-xylose as a
and pH condition. The crude xylanase enzyme of standard.
recombinant E. coli HB 101 (pBX6) showed optimum
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70
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