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

Xylan is one of the major components of hemicellulose
found in the plant cell walls. This compound has currently
regarded as a usable biomass product, which can be
converted into biofuels and chemicals (Woodward, 1984).
Biodegradation of xylan involves the action of three major
xylanolytic enzymes, namely endo β-1,4-xylanases (β -1,4-
D xylan xylanohydrolase, EC 3.2.1.8); β-1,4-xylosidases
(β-1,4 D xyloside xylohydrolase EC 3.2.1.37) and exoglu-
canases (β-1,4-D xylan xylohydrolase) (Coughland and
Hazlewood, 1993). The ability in degrading cellulose in
plant cell walls is related to the production of hemicellulase
enzymes including xylanase, acetylxylans esterase and
arabinofuranase (Forsberg et al., 1981).
Xylanases and xylosidases, the major enzymes that
involved in degradation of the xylose have been studied
from various microorganisms. The major cellulolytic and
xylanolytic bacteria include Ruminococcus flavofacians, R.
albus (Malburg et al., 1993), Bacillus coagulans 26
(Esteban et al., 1983); B. subtilis (Uchino and Nakane,
1981); B. circulans (Yang et al., 1989); and B. polymoxa
(Hespel, 1996). Many strains of F. succinogenes were
reported to possess a complement of fibrolytic enzymes
that extensively degrade plant cell walls polymers (Hu et
al., 1991; Forsberg and Cheng, 1992).
Fournier et al. (1985) reported that xylanolytic
microorganisms produce multiple forms of xylanase with
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
85 and S 135 (Forsberg et al., 1981; Hu et al., 1991;
Paradis et al., 1992). Previously, four unique xylanase
genes have been cloned from F. succinogenes S 85
(Malburg et al., 1993). Two of the cloned were reported
producing xylo-oligosaccharides as the major hydrolytic
product. The third cloned showed endonuclease and
xylanase activities, whereas the fourth cloned (xynC)
produce a large amount of xylose and xylobiose (Forsberg
and Cheng, 1992). Xylanase enzymes have molecular
weights ranging from 15 kDa to 63 kDa (Dekker and
Richards, 1976; Paradis et al., 1993). Matte and Forsberg
(1992) had studied two xylanase activities from F.
succinogenes; a 54-kDa arabinose debranching xylanase
and a 66-kDa xylanase with endonuclease activity. Paradis
et al. (1993) discovered xylanase carrying cloned xynC that
expressed in E. coli, had a molecular mass of 63 kDa. Sipat
et al. (1987) had cloned a xylanase gene (9.4 kb) from F.
succinogenes S 85 into E. coli HB 101 (pBX1) and studied
its hydrolytic properties. Deletion fragment of 3 kb
containing the xylanase gene from F. succinogenes S 85
was further cloned into pUC19 vector. The recombinant
plasmid namely pBX6 expressed xylanase activity when
transformed into E. coli HB 101, however, the properties of
pBX6 has not been well characterized. Studies on the
characterization of highly active xylanolytic enzymes
produce by recombinant plasmid cloned from bacteria may
be useful in development of technique to improve
degradation of plant hemicellulose.
This work was undertaken to study biochemical
characteristics of the recombinant plasmid pBX6 carrying a
xylanase gene from F. succinogenes S 85.
MATERIALS AND METHODS
General recombinant culture
Escherichia coli HB 101 containing a recombinant
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
capable of producing multiple xylanase (Sipat et al., 1987).
The bacterial culture containing recombinant plasmid
pBX6 was maintained on 2 Yeast Tryptone (YT) solid
mediums supplemented with antibiotic ampicylin (50
mg/ml). Plating serial dilution on 2 YT agar medium and
spectrophotometry using absorbance 550 nm checked
growth of colonies from preserved stock. An assay for
xylanase production was determined on remazol brilliant
blue (RBB)-xylan agar plate medium supplemented with
10 µg/ml ampicylin. For this purposes, six fold dilution
was spread out on 2 YT medium containing 0.2% RBB-
xylan (oat spelt xylan) as substrate (Biely et al., 1985).
Xylanase plaques showing clearing zone colony was
observed after incubation at 37oC for overnight. The
hydrolysis of xylan substrate, which showed by the clear
colony surrounding agar media, revealed the production of
xylanase. Growth of recombinant culture on various
environmental conditions was carried out on different
media, pH and temperature. Population growth was
measured as colony forming unit (CFU) at optical density
(OD) 550 nm.
Xylanolytic activity
Xylanase enzyme activity was assayed by incubating
the crude preparation from culture suspension with xylan as
a substrate at a given environmental condition (different pH
and temperature). Crude filtrate for xylanase enzyme
preparation of F. succinogenes S85 was prepared as
described by Matte and Forsberg (1992). E. coli HB 101
containing pBX6 was grown in 2 YT broth supplemented
with ampicylin (50 µg/ml). Triplicate flasks (250 ml)
containing 50 ml of medium were incubated at 37°C for
over night with vigorous shaking (250 rpm). Culture fluid
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
in the same volume of 25% sucrose in the presence of 0.1
mM EDTA. The cell free-culture supernatant was concen-
trated by ultrafiltration through an Amicon membrane (10
kDa cut-off). Approximately 25 µl of crude enzyme was
preincubated with 225 µl of Mc-Ilvaine buffer pH 7.2 at
various temperatures ranging from 20oC to 80oC for 30
min. The same procedure was followed using the pH
condition of 2 to 10. Reduction viscosity of the solution
(sugar released) was measured by Somogyi-Nelson's
method (Whitehead, 1993). Protein concentration was
measured by the method of Lowry (Lowry, 1951) using
bovine serum albumin (BSA) (Sigma, USA) as the stan-
dard, following the procedure stated in the manufacturer’s
instruction manual. The enzyme activity was determined by
pre incubating enzyme (0.1 ml of crude solution) with 2 g
oat spelt xylan in 100 ml of Mc-Ilvaine buffer (0.1 M citric
acid and 0.2 M Na2HPO4, pH 7.2), measured at 37oC. In
control assay, the enzyme had been inactivated by heating
in boiling water bath for 30 min. Specific activity was
defined as units per mg (U/mg) of protein (Whitehead,
1993). One unit of enzyme was defined as the amount
xylanase activity corresponding to 1 µmol of reducing
sugar released per minute using D-xylose as a standard.
RESULTS AND DISCUSSION
Growth kinetics of pBX6
The colony growing on 2 YT + RBB xylan agar
medium displayed xylanase activity by producing a
clearing zone (halo) surrounding the colony due to the
hydrolysis of xylan by enzyme activity. Biely et al. (1985)
stated that the formation of several halos was resulted
from diffusion and digestion of xylanase on RBB-
xylan. The result showed that plasmid pBX6 was found
to be capable of producing xylanase when grown in E. coli
HB 101. To monitor growth characteristics of recombinant
plasmid, E. coli HB 101 (pBX6) was cultured on 2 YT
broths in the presence or absence of ampicylin over a 72 h
incubation period at 37oC. Cultures forming colonies in the
subcultures (diameter 1 to 2 mm) were considered to be
capable of growth on xylan. Recombinant plasmid grown
on 2 YT broth medium containing ampicylin reached
optimum growth near lag phase after 24 h incubation. The
recombinant plasmid was stable grown on 2 YT broth
medium containing ampicylin which showed optimum
growth at 24 to 48-h incubation period. It is shown that
bacteria normally reproduce by binary fission, which
results in doubling of the number of viable bacterial cells
under optimal condition. During the stationary phase of
growth, the bacterial viability (CFU) increased and
declined rapidly thereafter. The cell viability, as indicated
by colony forming unit (CFU) was much higher in the
broth containing ampicylin compared with that of without
ampicylin (Table 1). The decline of the cell population was
slower in the presence of ampicylin. This result proofs the
presence of ampicylin maintained cell viability better, due
to the expression of the ampicylin resistance gene in
recombinant plasmid pBX6.
Table 1. Effect of ampicylin on growth of pBX6.
Incubation time (h) With ampicylin Without ampicylin
(log cfu/ml) (log cfu/ml)
0 0 0
12 9.53 9.46
24 9.57 9.54
36 9.49 9,42
48 9.24 9.20
60 9.23 9.20
72 9.20 9.10
The recombinant plasmid culture could also grow better
on others media such as on Luria broth (LB), sucrose broth
(SOB) and terrific broth. Abundance growth of the viscous
bacterial culture (E. coli HB 101) containing recombinant
plasmid pBX6 was observed on 2 YT broth than that of
Luria broth (LB), and sucrose peptone broth (SP) media
which contained different composition of minerals and
carbon source. Yeast extract enhanced the level of growth
to about 3 to 5 fold that of cultures grown without this
substance as mineral source. It was suggested that the
culture utilize its mineral better on 2YT than other media.
Culture on 2 YT broth media showed highest absorbance
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
4 BioSMART Vol. 5, No. 1, April 2003, hal. 1-4
70
60
specific activity (U/mg)
50
40
30
20
10
0 Esteban, R., A. Villanuera and T.G Villa. 1983. D-xylanases of B.
2 3 4 5 6 7
-10
pH
Figure 3. pH-Activity Profile of Crude Xylanase. Crude enzyme
of E. coli (pBX6) in Mc-Ilvaine buffer pH 7.2 was assayed under
various pH and the activity was measured by sugar reduction test
using oat-spelt xylan as substrate.
The mechanism of xylan degradation has been reported
by Dekker and Richards (1976). The instability of the
enzyme in degrading substrate may be attributed to the
structure of enzyme; hence, elucidation of the factors,
which limit the complete metabolism of xylan degrading
enzyme produced by F. succinogenes S 85, is necessary for
its efficient use.
On the basis of amino acid level (deduced from
nucleotide sequence), the predicted protein secondary
structure analyzed by GOR method of the Swiss Institute
for bioinformatics, demonstrated that pBX6 consists the
abundance of α helix in its structure that play an important
role in determining stability of the enzyme (Suryadi, 2001,
data not show), however; to facilitate the location of the
enzymatically functional domain, a further deletion by sub
cloning and expression study might be done.
CONCLUSION
Xylanolytic plasmid in E. coli HB 101 (pBX6) was
studied in 2YT medium in the presence and absence of
ampicylin (50 µg/ml). The cell growth revealed by cell
viability (CFU) reached maximum after 24 to 48-h
incubation period. The presence of ampicylin in culture
media slightly increases cell viability. The optimum pH
and temperature profile of crude xylanase activity was 7.4
and 37oC, respectively.
ACKNOWLEDGEMENT
IRPA Project No 135901-51115 from the Ministry of
Science and Technology Malaysia to Prof. Abdullah Sipat
funded this research. The first author was grateful to ARM
II Project-AARD, Indonesia for supporting the study.
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