Properties of a metagenome-derived β-glucosidase from the contents of rabbit cecum

Article abstract of DOI:10.1271/bbb.80664

In this study, a previously cloned β-glucosidase gene, umbgl3B, was heterologously expressed in Escherichia coli, and the biochemical properties of the purified enzyme were characterized. The recombinant enzyme was stable over a wide range of pH values (5.0-9.0) and below 30 °C. It displayed optimum enzymatic activity at pH 6.5 at 40 °C, under condition similar to that in the rabbit cecum, suggesting an active role of the native enzyme in vivo. The recombinant β-glucosidase Umbgl3B showed high activity to aryl β-D-glucosides and low activity to cellooligosaccharides, with a polymerization degree of less than 5. The enzyme had no activity toward long cellooligosaccharides or polysaccharides. The aspartic acid residue, D772, of the wildtype Umbgl3B was predicted as a nucleophile. Mutant D772A was constructed. It showed less than 1/10, 000 activity of the wild-type enzyme, but had the same properties, suggesting that residue D772 plays a key role in the enzyme's activity.

Full text of DOI:10.1271/bbb.80664

Biosci. Biotechnol. Biochem., 73 (7), 1470–1473, 2009
Properties of a Metagenome-Derived ꢀ-Glucosidase from the Contents
of Rabbit Cecum
y
1;
Yi FENG,1;2 Cheng-Jie DUAN,1 Li LIU,1 Ji-Liang TANG,1 and Jia-Xun FENG
1Guangxi Key Laboratory of Subtropical Bioresource Conservation and Utilization,
The Key Laboratory of the Ministry of Education for Microbial and Plant Genetic Engineering and College
of Life Science and Technology, Guangxi University, Nanning, Guangxi 530005, People’s Republic of China
2Department of Biochemistry, Guangxi Traditional Chinese Medical University,
179 Mingxiu Road, Nanning, Guangxi 530001, People’s Republic of China
Received September 18, 2008; Accepted April 7, 2009; Online Publication, July 7, 2009
[doi:10.1271/bbb.80664]
In this study, a previously cloned ꢀ-glucosidase gene,
umbgl3B, was heterologously expressed in Escherichia
coli, and the biochemical properties of the purified
enzyme were characterized. The recombinant enzyme
was stable over a wide range of pH values (5.0–9.0) and
below 30 ꢀC. It displayed optimum enzymatic activity
at pH 6.5 at 40 ꢀC, under condition similar to that in
the rabbit cecum, suggesting an active role of the
native enzyme in vivo. The recombinant ꢀ-glucosidase
Umbgl3B showed high activity to aryl ꢀ-D-glucosides
and low activity to cellooligosaccharides, with a poly-
merization degree of less than 5. The enzyme had no
activity toward long cellooligosaccharides or polysac-
charides. The aspartic acid residue, D772, of the wild-
type Umbgl3B was predicted as a nucleophile. Mutant
D772A was constructed. It showed less than 1/10,000
activity of the wild-type enzyme, but had the same
properties, suggesting that residue D772 plays a key role
in the enzyme’s activity.
ꢀ-Glucosidases are commonly present in both eu-
karyotic and prokaryotic organisms. The digestive tracts
of herbivores harbor symbiotic microorganisms to help
the host with the digestion of cellulosic feeds by
producing complete sets of cellulases. Many ꢀ-glucosi-
dases were isolated and characterized from cultured
bacteria4) and fungi5) in the rumens. Two ꢀ-glucosidase
genes have been identified from uncultured samples in
the cow rumen6) and the large-bowel of mice.7) How-
ever, the biochemical characterization of these metage-
nome-derived ꢀ-glucosidases has not been reported. We
recently constructed a cosmid library in Escherichia coli
with metagenomic DNA isolated directly from the
contents of the rabbit cecum. Seven ꢀ-glucosidase genes
and four ꢀ-1,4-endoglucanase genes were identified
from the metagenomic library, which contained about
32,500 clones and had a capacity of about 1:14 ꢀ
109 bp.8) Among these ꢀ-glucosidase genes, umbgl3B
(accession no. DQ182493) encoded a glycosyl hydrolase
family 3 (GHF 3) ꢀ-glucosidase that displayed the
highest activities when assayed on substrate-embedded
agar plates and by direct enzymatic activity assay with
cytoplasmatic extracts. To understand better the bio-
chemical properties of enzyme Umbgl3B, in this study,
we selected this gene for heterologous expression in
a bacterial system and further characterization of its
product.
Key words: ꢀ-glucosidase; expression; characterization;
mutagenesis; rabbit cecum
ꢀ-Glucosidase (EC 3.2.1.21) hydrolyzes the ꢀ-gluco-
sidic linkages of glucosides, such as cellobiose, celloo-
ligosaccharide, plant-derived ꢀ-glucosides and other
chemically related ꢀ-glucosides.1) It is one of the three
enzymes in the complete cellulase system composed
of ꢀ-1,4-endoglucanase (EC 3.2.1.4), exoglucanase
(EC 3.2.1.91), and ꢀ-glucosidase. While endoglucanase
and exoglucanase act together to solubilize crystalline
cellulose by digesting it into soluble sugars, ꢀ-glucosi-
dase hydrolyzes cellobiose and cellooligosaccharides
into glucose. Additionally, ꢀ-glucosidase stimulates the
rate and extent of cellulose hydrolysis by eliminating
cellobiose inhibition on endo- and exo-glucanase activ-
ities.2) Thus ꢀ-glucosidase plays an important role in the
utilization of biomass. It has also attracted considerable
attention in the food industry for its biotechnological
applications, such as enhancing the aromas of fruit juice
and tea, removing bitter components of citrus products,
and detoxification of cassava.3)
Materials and Methods
Expression of umbgl3B and purification of recombinant Umbgl3B.
The complete open reading frame of umbgl3B was amplified by
polymerase chain reaction (PCR) from the plasmid DNA, which
contained the original umbgl3B,8) with forward primer 50-GAC-
GGATCCGAGAAGTATTTTGCATCCACCTCTGATG-30 (umbgl3B-
F), and reverse primer 50-GCTGGATCCCAGGCTTCCGTGTCGCA-
CAAAACC-30 (umbgl3B-R), each containing a BamHI site at the 50
end (underlined). PCR was carried out for 30 cycles of 30 s at 94 ꢁC,
30 s at 43 ꢁC, and 3 min at 72 ꢁC, with a final 10 min extension at 72 ꢁC.
The PCR product was cloned into expression vector pET-30a(+)
(Novagen, Madison, WI), and the construct was transformed into
expression host E. coli BL21 (DE3) pLysS (Novagen, Madison, WI).
The bacterial transformants were screened first by activity assay on
Luria-Bertani (LB) agar plates embedded with 0.1% esculin hydrate,
y
To whom correspondence should be addressed. Tel: +86-771-323-9401; Fax: +86-771-323-9413; E-mail: jiaxunfeng@sohu.com
Abbreviations: CMC, carboxymethyl cellulose; GHF, glycosyl hydrolase family; IPTG, isopropyl D-thiogalactoside; LB, Luria-Bertani;
p-NP, p-nitrophenol; p-NPC, p-nitrophenyl ꢀ-D-cellobioside; p-NPG, p-nitrophenyl ꢀ-D-glucopyranoside; p-NPX, p-nitrophenyl ꢀ-D-xylopyranoside;
PCR, polymerase chain reaction; SDS–PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TEMED, N,N,N00,N00-tetramethylethy-
lenediamine

Characterization of a Novel ꢀ-Glucosidase
1471
0.2% ferric ammonium citrate, and 0.5 mM isopropyl D-thiogalacto-
pyranoside (IPTG), with a supplement of 25 mg/ml of kanamycin and
34 mg/ml of chloramphenicol, then by PCR to select the positive clones
that harbored the target gene.
Site-directed mutagenesis of umbgl3B. The previously predicted
nucleophile of Umbgl3B, the aspartic acid residue D772,8) was
replaced by an alanine through site-directed mutagenesis. The gene
encoding the mutant enzyme (D772A) was obtained by recombinant
PCR with primers designed to change the D772 codon (GAC) into an
Ala codon (GCC). The primers used to amplify the upstream fragment
were umbgl3B-F and 50-ACGTGAACCAGGCGGTCATGAT-30, and
those for the downstream fragment were 50-ATCATGACCGCCTGG-
TTCACGT-30 and umbgl3B-R. The mutant gene was sequenced and
compared with wild-type umbgl3B to verify the correct mutation.
Mutant enzyme D772A was expressed and purified in the same way
as recombinant Umbgl3B. Purified recombinant Umbgl3B and D772A
were subjected to SDS–PAGE. Before staining with Coomasie
Brilliant Blue G250, the gel was soaked and washed 3 times with a
refolding buffer (10 mM potassium phosphate buffer at pH 7.0) to
dilute SDS and allow renaturation of the denatured proteins, as
described by Ali et al.,16) then stained with 0.1 M citrate-phosphate
buffer (pH 6.0) containing 0.1% esculin hydrate and 0.2% ferric
ammonium citrate to visualize the activities on the gel. The activity of
D772A toward p-NPG was measured under routine conditions at a
protein concentration of 2.0 mg/ml. pH and temperature profiles of
D772A activity were also studied as for recombinant Umbgl3B.
The positive bacterial clone was cultivated in 150 ml of LB medium
containing 25 mg/ml of kanamycin and 34 mg/ml of chloramphenicol
at 37 ꢁC, and protein expression was induced with 0.5 mM IPTG. Cells
were harvested by centrifugation, resuspended in 2.5 ml of lysis buffer
(50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, with pH adjusted
to 8.0 with NaOH), and sonicated. After centrifugation, the supernatant
was collected as a crude extract of total proteins. Because the
recombinant protein was tagged at both the N and C terminals with
6 ꢀ His residues, the crude extract was purified first by affinity
chromatography using nickel-nitrilotriacetic acid agarose resin
(Qiagen, Valencia, CA) according to the manufacturer’s manual.
Secondary anion-exchange chromatography was performed on an
AKTA Explorer 100 system (Amersham Biosciences, Uppsala,
Sweden) with column Mono-Q 5/50 GL. The column was equilibrated
with 50 mM citrate-phosphate buffer at pH 6.0 and eluted with the
same buffer containing a linear gradient of 0–1.0 M NaCl at a flow rate
of 0.8 ml/min.
Protein and enzyme assays. Protein was quantified by the Bradford9)
method with bovine serum albumin as standard. Sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) was per-
formed by the method of Laemmli.10)
Results and Discussion
Expression of umbgl3B and purification of recombi-
nant Umbgl3B
Enzyme activity on p-nitrophenyl ꢀ-D-glucopyranoside (p-NPG,
Sigma, St. Louis, MO) was determined according to Odoux et al.,11)
with a minor modification. The purified enzyme was diluted 20–50
times in 50 mM citrate-phosphate buffer (pH 6.0) to a concentration of
14 mg/ml for the routine enzyme assay, in which 10 ml of the enzyme
was added to 130 ml of 0.1 M citrate-phosphate buffer (pH 6.0)
containing 2.5 mM p-NPG. The reaction was carried out at 28 ꢁC for
15 min and was stopped with 70 ml of 0.4 M Na2CO3. The release of
p-nitrophenol (p-NP) was measured at 420 nm. Enzyme assays were
performed under this condition, unless otherwise indicated. One unit
(U) of enzyme activity was defined as the amount of enzyme releasing
1 mmol of p-NP from p-NPG per min. Specific activity was expressed
in units of active enzyme in milligram of protein.
The bacterial transformants were screened by assays
on substrate-embedded LB agar plates and PCR to
confirm the presence of the umbgl3B gene (data not
shown). SDS–PAGE analysis detected a specific protein
band from crude extracts of the expression cells that
were induced by IPTG (Fig. 1, lane 3), but not in cells
without IPTG treatment (Fig. 1, lane 2). The expressed
recombinant Umbgl3B was estimated to have a mo-
lecular weight of about 100 kDa on the denatured gel,
Hydrolysis products from other substrates, such as p-NP from
p-nitrophenyl ꢀ-D-cellobioside (p-NPC) and p-nitrophenyl ꢀ-D-xylopy-
ranoside (p-NPX), glucose from cellooligosaccharides, and glucose-
equivalent reducing sugars from carboxymethyl cellulose (CMC) or
xylan, were measured. The enzyme activity on these substrates was
presented in velocity (mmol of product/min/mmol of protein).
Characterization of recombinant Umbgl3B. The effect of pH on
enzyme activity was assayed at 28 ꢁC, and the effect of temperature
on was then assayed at pH 6.0 (0.1 M citrate-phosphate buffer).
pH Stability was studied as described by Eckert and Schneider,12)
and thermal stability was evaluated according to the method of
Inoue et al.13)
p-NPC, p-NPX, CMC, oat spelt xylan, and cellooligosaccharides
(including cellobiose, cellotriose, cellotetraose, cellopentaose, and
cellohexaose) were applied to test the substrate specificity of the
recombinant Umbgl3B. All tested substrates were purchased from
Sigma. p-NPC (in 2.5 mM) and p-NPX (in 2.5 mM) were assayed
routinely as described above with a prolonged reaction time of 2 h. For
CMC and xylan, 20 ml of the diluted enzyme was added to 180 ml of
0.1 M citrate-phosphate buffer (pH 6.0) containing 1% (w/v) CMC or
xylan. Reactions were carried out for 2 h at 28 ꢁC and ended with
400 ml of dinitrosalicylic acid reagent. The released reducing sugars
were measured as glucose equivalents at 530 nm, as described by
Miller.14) The cellooligosaccharides were hydrolyzed under routine
assay conditions with replacement of p-NPG by 2.5 mM of each
cellooligosaccharide, an enzyme concentration of 140 mg/ml, and an
incubation time of 2 h. The reactions were terminated by boiling, and
the glucose generated was detected by the glucose oxidase method.15)
To investigate the effects of various metal ions and chelating agents
on the enzyme activity, 50 mM N,N,N00,N00-tetramethylethylenedia-
mine-HCl (TEMED-HCl) buffer (pH 6.0) containing 5.0 mM metal
ions or 5.0 mM EDTA was used in the routine enzyme assay, as
described above.
Fig. 1. SDS–PAGE Analysis of the Expression and Purification of
Recombinant Enzyme Umbgl3B and Mutant Enzyme D772A.
Lanes 1 to 6, SDS–PAGE (8.0%) stained with Coomasie Brilliant
Blue; lane 1, protein molecular weight marker (no. SM0431,
Fermentas, Vilnius, Lithuania); lane 2, crude extracts of uninduced
expression cells harboring recombinant umbgl3B; lane 3, crude
extracts of Umbgl3B from expression cells induced by IPTG; lane 4,
crude extracts of D772A; lane 5, recombinant Umbgl3B purified by
affinity chromatography and anion-exchange chromatography;
lane 6, D772A purified by affinity chromatography and anion-
exchange chromatography. Lanes 7 and 8, renaturated SDS–PAGE
gel treated with esculin solution. Purified Umbgl3B showed ꢀ-
glucosidase activity with a dark band on the gel (lane 7), while
purified D772A did not (lane 8).

1472
Y. FENG et al.
a
b
c
d
Fig. 2. Effects of pH and Temperature on Enzyme Activity and Stability.
a, Effects of pH on the activity of recombinant enzyme Umbgl3B (closed squares) and mutant enzyme D772A (open squares). Enzyme assays
were performed at the indicated pH values at 28 ꢁC for 15 min, with p-NPG as a substrate. The buffers were: 0.1 M citrate-phosphate buffer for
pH 3.0–7.0 and 0.1 M sodium phosphate buffer for pH 7.5–8.0. The activity of recombinant Umbgl3B on p-NPG at pH 6.0 was taken to be
100%, and it corresponded to a specific activity of 44.9 U/mg. b, pH stability of recombinant Umbgl3B. The purified enzyme was diluted to a
concentration of 14 mg/ml in the buffers (0.1 M citrate-phosphate buffer, pH 3.0–7.0; 0.1 M Tris–HCl buffer, pH 8.0–9.0; 0.1 M glycine–NaOH
buffer, pH 10.0) and incubated at 4 ꢁC for 24 h. Ten ml of pre-incubated enzyme (concentration, 14 mg/ml) was added to 130 ml 0.1 M citrate-
phosphate buffer (pH 6.0) containing the substrate for routine enzyme assay. The activity of the enzyme without pre-incubation was defined as
100%. c, Effects of temperature on the activity of recombinant enzyme Umbgl3B (closed squares) and mutant enzyme D772A (open squares).
Enzyme assays were performed at pH 6.0 (0.1 M citrate-phosphate buffer) and the indicated temperatures for 15 min. The activity of recombinant
Umbgl3B at 40 ꢁC was taken to be 100% (specific activity 72.6 U/mg). d, Thermal stability of recombinant Umbgl3B. Activity was measured
under routine conditions (0.1 M citrate-phosphate buffer at pH 6.0, 28 ꢁC, 15 min) after incubating the enzyme at the indicated temperatures for
30 min. The activity of the enzyme without pre-incubation was defined as 100% (specific activity 45.3 U/mg).
consistent with the calculated molecular size of 101 kDa
based on its amino acid sequence. These results
indicated expression of the recombinant umbgl3B in
E. coli BL21 (DE3) pLysS.
Two-step purification by affinity chromatography and
anion-exchange chromatography purified the recombi-
nant Umbgl3B to apparent homogeneity (Fig. 1, lane 5),
and increased the specific activity of the enzyme from
2.91 U/mg of the crude extract to 46.3 U/mg after
purification. Thus a 16-fold enrichment of the enzyme
was achieved.
that the native Umbgl3B protein might play an active
role of ꢀ-glucosidase under in vivo conditions in the
rabbit cecum.
ꢀ-Glucosidases mainly catalyze scission of the ꢀ-1,4-
glucosidic linkage in cellobiose, short-chain cellooligo-
saccharides (degree of polymerization, 3–8), and aryl
ꢀ-D-glucosides (such as p-NPG).3) The substrate speci-
ficity of recombinant Umbgl3B is shown in Table 1. The
enzyme had low activity toward p-NPC, p-NPX,
cellobiose, cellotriose, and cellotetraose. The hydrolytic
ability of recombinant Umbgl3B on p-NPG was 100
times greater than that on cellobiose, suggesting that
recombinant Umbgl3B is specific for aryl ꢀ-D-gluco-
sides. The hydrolytic activity of recombinant Umbgl3B
on cellooligosaccharides became weaker as the degree
of polymerization rose, and Umbgl3B did not hydrolyze
long-chain cellooligosaccharides such as cellopentaose
and cellohexaose. No hydrolysis of polysaccharides such
as CMC and xylan by the recombinant Umbgl3B was
detected (Table 1).
The presence of CoCl2 and CaCl2 significantly
increased enzyme activities, to 129% and 124% of the
baseline level respectively. MnCl2, FeCl2, and MgCl2
also increased enzyme activities to some degree, while
CuCl2 and CrCl2 inhibited enzyme activity to 86%
and 42% respectively. Chelating agent EDTA was an
effective inhibitor, dramatically reducing enzyme activ-
ity, to 32% of the baseline level (Table 2).
Characterization of recombinant Umbgl3B
Purified recombinant Umbgl3B hydrolyzed p-NPG
efficiently between pH 5.5 and 6.5, and had optimal
activity at pH 6.0 when assayed at 28 ꢁC (Fig. 2a). It
was stable over a broad range of pHs from 5.0 to 9.0,
retaining more than 90% of its activity (Fig. 2b). The
enzyme exhibited optimum activity at 40 ꢁC when
assayed at pH 6.0 for 15 min (Fig. 2c). However, the
thermal stability data showed that the enzyme was stable
at temperatures below 30 ꢁC with over 90% of activity
remaining, and lost its activity significantly at temper-
atures above 30 ꢁC (Fig. 2d). The pH-activity profile
was also investigated at 40 ꢁC. The enzyme reached its
highest activity (1.67-fold higher than at 28 ꢁC) at
pH 6.5, which is the physiological condition of the
rabbit cecum (pH 6.5 and 39 ꢁC).8) This result indicates

Characterization of a Novel ꢀ-Glucosidase
Table 1. Substrate Specificity of Recombinant Umbgl3B
1473
lane 4), and was also purified to apparent homogeneity
(Fig. 1, lane 6). The cells expressing the mutant D772A
showed no activity on plate assay. On the renaturated
gel, purified recombinant Umbgl3B showed activity
upon refolding (Fig. 1, lane 7), whereas no activity of
D772A was observed (Fig. 1, lane 8).
Substrate
Velocity (mmol/min/mmol)
p-NPG
p-NPC
p-NPX
0:324
1:09 ꢀ 10ꢂ3
7:92 ꢀ 10ꢂ4
Cellobiose
Cellotriose
Cellotetraose
Cellopentaose
Cellohexaose
CMC
2:57 ꢀ 10ꢂ3
9:41 ꢀ 10ꢂ4
However, hydrolysis of p-NPG by D772A was still
detectable in enzyme assay, with the concentration of the
protein increased to as high as 2.0 mg/ml, even though
the specific activity was negligible (3:15 ꢀ 10ꢂ3 U/mg).
D772A presented pH and temperature profiles on
enzyme activity similar to those of recombinant
Umbgl3B, except for low activity (Fig. 2a and c). The
mutant enzyme D772A also showed optimal pH and
temperature at 6.5 and 40 ꢁC. In the study of Li et al.,17)
mutation of D247 led to 30,000- to 200,000-fold lower
activity of the wild-type ꢀ-glucosidase from Flavobac-
terium meningosepticum. Mutant D772A retained less
than 1/10,000 activity of wild-type Umbgl3B, indicating
that D772 is a key residue for the enzyme’s activity.
9:90 ꢀ 10ꢂ5
0
0
0
0
Xylan
The protein concentration of the enzyme was quantified by the Bradford9)
method, with bovine serum albumin as standard. Enzyme assays were
performed at 28 ꢁC in 0.1 M citrate-phosphate buffer at pH 6.0 for 120 min.
The release of p-NP from p-NPG, p-NPC, and p-NPX, glucose from the
cellooligosaccharides, and glucose-equivalent reducing sugars was meas-
ured. The velocities presented in the table are the averages of triplicate
measurements.
Table 2. Effects of Metal Ions and Chelating Agents on Enzyme
Activities of Recombinant Umbgl3B
Acknowledgments
Reagent
Concentration
Relative activity (%)
This work was supported by Program for New Century
Excellent Talents in the Universities of China (NCET-
05-0752), and by the Hi-tech Research and Development
Program of China (863 Program 2007AA021307).
Control
CoCl2
CaCl2
MnCl2
FeCl2
MgCl2
ZnCl2
LiCl
—
100 ꢃ 3:8
129 ꢃ 4:5
124 ꢃ 4:0
108 ꢃ 3:4
107 ꢃ 3:6
106 ꢃ 3:8
103 ꢃ 3:5
101 ꢃ 3:6
99 ꢃ 3:8
97 ꢃ 2:8
86 ꢃ 2:7
42 ꢃ 1:8
32 ꢃ 1:3
5 mM
5 mM
5 mM
5 mM
5 mM
5 mM
5 mM
5 mM
5 mM
5 mM
5 mM
5 mM
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