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New Biotechnology �Volume 29, Number 3 � February 2012 RESEARCH PAPER
Strategy for purification of aggregationprone b-glucosidases from the cell wall ofyeast: a preparative scale approach
Mohammad Asif Shah1,2, Tapan Kumar Chaudhuri1 and Saroj Mishra2,
1 Structural Biology, Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology Delhi, Hauz Khas, New Delhi 110016, India2Biochemical Research Laboratory, Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology Delhi, Hauz Khas, New Delhi
110016, India
Purification of biotechnologically important proteins is of vital interest to the biotech industry.
b-Glucosidases, belonging to Family 1 and Family 3 of the glycosylhydrolases, have varied applications
as carbohydrate hydrolyzing and synthesizing enzymes. Obtaining high quantities of these enzymes is
important for exploring their biosynthetic potential, structural information and catalytic activities.
Classical methods for their preparation fail to deliver high yields because of adoption of several/
hydroxyapatite chromatography steps. We report here a preparative method for purification of large
quantities of two closely related cell bound b-glucosidases (BGL I and BGL II) from Pichia etchellsii that
belong to Family 3 glycosylhydrolases. A combination of ion-exchange and gel filtration
chromatography was used to process milligram quantities of protein with recoveries of up to 53%. A
simple affinity based separation resulted in resolution of BGL I and BGL II with high recovery and high
specific activities of 74 IU/mg and 32 IU/mg protein respectively. Peptide sequences of BGL II indicated
it to be a novel member of Family 3. Methods reported here present a successful strategy for obtaining
large quantities of these enzymes.
Introductionb-Glucosidases constitute a major group among glycosylhydro-
lases. They occur in all domains of living organisms (eubacteria,
archaea, and eukarya). These enzymes specifically cleave glyco-
sidic bonds (either O-linked b-glycosidic bonds, b-D-glucoside
glucohydrolase, EC 3.2.1.21, or S-linked b-glycosidic bonds, myr-
osinase, or b-D-thioglucoside glucohydrolase, EC 3.2.3.1) in oli-
gosaccharides and their aryl- and alkyl conjugates [1,2]. The
functional diversity exhibited by these enzymes has been utilized
in a multitude of biotechnological applications that include
cellulose to glucose conversion [3,4], cellulose to ethanol con-
version [5], release of flavored aromatic compounds [6–8] from
their flavorless glucosides. Under defined conditions, the
enzymes can synthesize glucosyl bonds by reverse hydrolysis
and transglycosylation approaches [9–11] leading to the synthesis
of carbohydrates, considered as potential therapeutics [12].
Corresponding author: Mishra, S. ([email protected])
1871-6784/$ - see front matter � 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.nbt.2011.06.010
Microarrays of synthetic carbohydrates have already been devised
for probing carbohydrate–protein interactions [13,14] demand-
ing the availability of sugars on large scale.
While several Family 1 enzymes have been used in synthesis, very
few studies have been conducted on Family 3 b-glucosidases. The
structure of only a single Family 3 glycosylhydrolase, Hordaeum
vulgare, has been solved by X-ray crystallography [15]. These pro-
teins belong to a category of large proteins (approximately 850 aa)
and are organized in multi-domain structures consisting of an (a/b)8
fold TIM barrel and an (a/b)6 fold b sandwich domain. They occur as
closely related isozymes which are difficult to resolve. Many of these
enzymes are cell surface bound with large hydrophobic patches
which make them prone to aggregation. Pichia etchellsii, a thermo-
tolerant yeast produces multiple b-glucosidases out of which two are
cell wall bound. Both the enzymes show good synthetic potential.
The synthesis of alkyl glucosides (and aryl glucosides or saccharides
with degree of polymerization from 3 or more) can occur by (i)
reverse hydrolysis or by (ii) transglycosylation. In the first method,
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RESEARCH PAPER New Biotechnology �Volume 29, Number 3 � February 2012
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enzyme-glycosyl intermediate is intercepted by another glucose (or
monosaccharide or an alcohol) resulting in the formation of sugars
of increasing chain length. This reaction is under thermodynamic
control and the equilibrium is shifted towards synthesis by reducing
the water activity and/or by increasing the substrate concentration.
In the second approach, an activated donor (e.g. lactose, cellobiose
or an aryl glucoside) is intercepted by an alcohol to generate a
glycosidic bond. This reaction is kinetically controlled. Both these
approaches have been used with the P. etchellsii b-glucosidases for
the synthesis of alkyl glucosides like hexyl-b-D-glucoside, heptyl b-D-
glucoside, octyl-b-D-glucoside, decyl-b-D-glucoside [16], oligosac-
charides like glucobiose, glucotriose and glucopentose [17]. Bio-
synthesis of monoterpenyl glucosides namely neryl, geranyl and
citronellyl-glucosides has also been reported [18]. Recently, the
whole cell suspensions of P. etchellsii, bearing the wall bound b-
glucosidases, have been reported to synthesize octyl-glucoside by
transglucosylation with conversions up to 60% [19]. Given the
importance of these enzymes, we report a preparative scale
approach for purification of these aggregation prone b-glucosidases.
The strategy presented here should be applicable for the purification
of other large molecular weight proteins of this category.
Materials and methodsStrain, maintenance medium and culture conditionsThe organism used in the study was an oenological yeast strain P.
etchellsii (DBEB-Y1015) JFG-2201 (Deutsche Sammlung von Mik-
roorganismen und Zellkulturen, DSMZ: Germany). The yeast was
maintained on YEPD slants or stabs containing (per liter); yeast
extract: 5 g, peptone: 2.5 g, glucose: 10 g, and agar: 20 g. Yeast
extract, peptone and agar were from HiMedia Laboratories, India
while glucose was from Qualigens Fine Chemicals, India. The pH
was adjusted to 5.5. The growth was carried out for 48 h on this
medium at 408C. The strain was maintained on YEPD and stored at
48C.
For enzyme production, a single colony was inoculated in 50 ml
of phosphate succinate (PS) minimal medium in 250 ml Erlen-
meyer flask, which contained (per liter); yeast extract: 2.5 g, pep-
tone: 5 g, succinic acid: 6 g, CaCl2: 0.3 g, K2HPO4: 8.7 g,
(NH4)2SO4: 4 g, MgSO4: 0.5 g, pH 4.7. All the components were
the highest grade available from Qualigens. The cells were allowed
to grow at 408C and 220 rpm in an orbital shaker (Scigenics, India)
for 8–10 h. This initial inoculum (2%, v/v) was transferred to
200 ml PS medium contained in 1000 ml Erlenmeyer flasks (12
in number) and allowed to grow for 14 h. Induction of b-gluco-
sidase was carried out by adding cellobiose (Sisco Research Labora-
tories, India) to a final concentration of 10 mM. The cells were
allowed to grow for another 6–8 h. Ten liters of cells was grown for
enzyme isolation.
Cell disruptionCells were harvested by centrifugation at 5000 � g for 10 min at
48C in a Sorvall RC 5B centrifuge. The pellet was washed with
50 mM sodium phosphate buffer, pH 7 (hereafter referred to as
buffer) three times by re-suspension and re-centrifugation. The
washed pellet was resuspended in phosphate buffer to make a cell
suspension of 15–25% (wcw/v) as described below. This suspen-
sion was used for the extraction of b-glucosidase enzymes using
French press or Dyno-Mill treatment. In the case of French press,
312 www.elsevier.com/locate/nbt
the cell pellet was resuspended in buffer to a final concentration of
15%, 20% or 25% (wcw/v). The cells were broken by passing the
suspension through the pressure cell at 1300 psi. A total of 5 passes
were carried out. The cell suspension was centrifuged at 10,000 � g
for 30 min at 48C and enzyme activity and protein was estimated
in the supernatant after every pass.
In the case of Dyno-Mill, three different modes of operation
(batch, continuous, and discontinuous batch) were evaluated for
maximum release of b-glucosidase activity from cell wall.
Batch operationFor batch mode, a 20% cell suspension of P. etchellsii was made in
buffer. Eighty milliliters of ice chilled glass beads was added to
20 ml of cell suspension. The cell suspension-glass bead mixture
was taken in a KD Dyno-Mill (Willy A Bachofen, Switzerland) and
agitated at 1500 rpm for various time periods ranging from 1 to
5 min. After each minute, samples were withdrawn and assayed for
enzyme and protein release. The operation was carried out at 48Cwhich was maintained by a circulating water bath (Pharmacia,
Sweden). The residual enzyme was recovered from glass beads by
washing the mill with same buffer. After disruption, the cell debris
was removed by centrifugation at 10,000 � g for 30 min at 48C in a
Sigma 3K30 centrifuge. The supernatant was used as a crude source
of enzyme. Protein concentration and enzyme activity were deter-
mined in the supernatant.
Continuous operationThe cell suspension was made as described above and the suspen-
sion was pumped continuously through the ice-chilled mill at
125–520 ml/min till 80–90% of cells were broken. The disruption
was monitored by examining the cells microscopically after dif-
ferent time intervals. After disruption, the mill was washed with
350–400 ml of buffer to remove the residual activity from the
beads. The cell debris was removed by centrifugation at
10,000 � g for 30 min at 48C. The cell-free extract was used as a
source of crude enzyme and enzyme activity and protein was
estimated in the cell-free extract.
Discontinuous batch operationThe cell suspension was prepared as described above. It was then
applied to Dyno-Mill at 125 ml/min for the first two passes. The
subsequent passes were performed at 525 ml/min. Samples were
collected after every pass and assayed for the release of protein and
b-glucosidase activity. A total of seven passes were carried out. The
mill was washed with l L of phosphate buffer to release the residual
enzyme from the glass beads. All the operations were carried out at
48C. The broken cell suspension was centrifuged at 10,000 � g for
30 min at 48C to remove the cell debris.
Column chromatographyThe cell-free extract was subjected to anion-exchange chromato-
graphy on Q-Sepharose Fast flow in XK16/30 or XK26/70 column
(Pharmacia, Sweden) (Amersham Biosciences, USA). The resin was
packed into the columns according to manufacturer’s instructions
(Pharmacia, Sweden) in Akta Explorer chromatographic system
(Amersham Biosciences, USA). The column was equilibrated with
buffer and cell-free extract applied at a flow rate of 0.5–1 ml/min
either with a peristaltic pump (Pharmacia, Sweden) or through
New Biotechnology �Volume 29, Number 3 � February 2012 RESEARCH PAPER
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P960 of Akta Explorer. The sample volume ranged from 0.5 to 3.0 L
containing a total protein of 0.25–3.3 g. The unbound proteins
were extensively washed with the same buffer. The washing was
monitored by observing the baseline stability. Elution was per-
formed with the same buffer containing KCl (130 or 200 mM).
Samples of 2–7 ml were collected and analyzed for the presence of
total protein and b-glucosidase activity.
Gel filtration was carried out by using Sephadex G 200 matrix or
Superdex 75 pre-packed column. In the case of Sephadex G 200
two columns were packed (XK 16/100 and XK 26/100, Pharmacia).
Relevant samples from anion exchange column were concentrated
by ammonium sulphate precipitation (80% saturation) and the
concentrate (in 2% of the bed volumes of the column) was applied
to Sephadex G 200 or Superdex 75 gel filtration columns. Fractions
of 2–5 ml size were collected at 0.2 ml/min flow rate, assayed for b-
glucosidase activity and checked on 10% SDS-PAGE. Those show-
ing BGL I and BGL II were pooled followed by the addition of solid
ammonium sulfate to a final concentration of 1 M. This was
applied to phenyl sepharose column (XK16/20, Pharmacia), pre-
equilibrated with buffer containing 1 M ammonium sulfate. The
unbound proteins were washed with the same buffer and elution
was carried out with a linear decreasing salt gradient (1–0 M
ammonium sulphate). Fractions of 1.5 ml size were collected at
0.25 ml/min. Those showing b-glucosidase activity were analyzed
on 10% SDS-PAGE. All steps were carried out at 208C.
Analytical methodsb-Glucosidase activity was measured on 4 mM pNPG (Sisco, India
and Aldrich) prepared in 50 mM phosphate citrate buffer, pH 6
according to the modified method [20]. A suitably diluted enzyme
(150 ml) was taken and added to 1.35 ml of pNPG solution pre-
incubated at 508C for 10 min. Reaction was allowed to occur for
10 min and stopped by adding 750 ml of 1 M Na2CO3. The absor-
bance of the resulting solution was measured at 420 nm against a
reagent blank. A p-nitrophenol (pNP, Sisco) standard curve (0.01–
0.1 mmol/ml) was made from a stock of 1 mM pNP prepared in
50 mM PC buffer, pH 6.0. Absorbance was taken at 420 nm. One
unit of b-glucosidase activity corresponded to the release of 1 mM
of pNP per min per ml of the reaction mixture. The specific activity
was expressed as IU/mg of protein.
Protein estimation was carried out by Bradford (Sigma–Aldrich)
dye binding method [21]. A suitably diluted 0.1 ml of protein
sample was taken and to this, 3.1 ml of Bradford reagent was
added. The amount of protein was estimated from a standard
curve of BSA (0.1–1.4 mg/ml) prepared in 50 mM sodium phos-
phate buffer, pH 7. Proteins from each purification step were
analyzed by SDS-PAGE on 10% gel containing 6% stacking gel,
and stained with Coomassie Brilliant Blue R-250.
Mass spectrometry and internal peptide sequencing of BGL IIThe purified proteins from phenyl sepharose chromatography
were dialyzed against Milli-Q water. The samples were concen-
trated by lyophilization. For analysis, the protein samples were
diluted in 1:1 ratio of acetonitrile and water containing 0.1%
acetic acid to a concentration of 10 ppm. The molecular weights
were determined by direct injection of 20 ml of the sample into
spectrometer (Applied Biosystems QSTAR XL fitted with Q/QTOF).
The analysis was done in the positive ion mode. Other MS settings
were as follows – flow rate: 10 ml/min; ion spray voltage: 5500 V;
nebulizer gas: 15 lb/in2; curtain gas: 20 lb/in2; declustering poten-
tial: 280 V; focusing potential: 265 V. The multiply charged pro-
tein peaks obtained in the mass spectrum were convoluted using
Bayesian Protein Reconstruct Software.
The internal peptide sequences of BGL II were determined by in-
gel digestion of the protein and sequencing of different peptides by
mass spectrometry. The product ion spectra were analyzed and
sequence was determined. The analysis was performed in the
Protein Chemistry Core Lab at Baylor College of Medicine, Hous-
ton, TX.
ResultsEnzyme isolationThe two enzymes BGL I and BGL II are cell wall bound proteins
[16]. The extraction of proteins from cell wall in an active form and
with high yield is a prerequisite for generating milligram quan-
tities of proteins. In general, for extraction of intracellular cell
components, both solid shear (high pressure homogenizers) and
liquid shear (bead mills) based methods have proven successful on
large scale [22,23]. Both these methods of cell disruption were
evaluated for the isolation of wall bound BGL I and BGL II and
enzyme released is shown in IU/l and IU/mg protein.
In the case of French press, the maximum release of enzyme was
observed with 20% cell suspension and highest specific activity of
1.10 IU/mg of protein was obtained. Because French press is sui-
table for volumes in the range of 5–50 ml, Dyno-Mill was used for
processing as this is generally a preferred method at large scale.
Different modes of operation were evaluated for enzyme release. In
the case of batch operation, a 20% (wcw/v) cell suspension was
made and maximum enzyme was released after agitating the cells
in the mill for 2 min. The extent of breakage was monitored
microscopically and estimated at 60–70%. In continuous opera-
tion, the cells were completely broken while in the case of dis-
continuous batch operation, the cells were found to be broken
partially. A comparison of these methods in terms of enzyme
release and specific activities is presented in Fig. 1. Highest recov-
ery was 840 IU/l and the specific activity was 6.4 IU/mg in the
discontinuous process, which was used in all subsequent opera-
tions. No colloidal glass from the beads was observed and thus the
material was suitable for the next stage.
Column chromatographiesThe cell-free extract obtained after centrifugation was directly
applied to Q-Sepharose FF anion-exchange column for both con-
centration (of protein of interest) and purification. This method is
also least expensive (compared to hydroxyapatite or affinity). A
pilot experiment was carried out on a small analytical (XK16/30,
bed height 30 cm) scale to determine optimum conditions for
elution. For this, a total protein of 250 mg (1180 units) was loaded
onto the column and elution was carried out at 200 mM and
130 mM KCl step gradients. The elution profile is shown in
Fig. 2a and b respectively. At 200 mM KCl, the eluted fractions
showed several contaminant proteins in addition to BGL I and
BGL II. Higher specific activity (27.47 IU/mg) was obtained with
130 mM KCl step gradient than with 200 mM KCl (15.02 IU/mg) at
a nearly equal (92%) recovery. The SDS-PAGE analysis from the
130 mM eluted fractions showed that a lot of contaminating
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RESEARCH PAPER New Biotechnology �Volume 29, Number 3 � February 2012
0
200
400
600
800
DiscontinousContinousBatch
Enz
yme
activ
ity (I
U/l)
0
2
4
6
8
Spec
ific
activ
ity (I
U/m
g)
FIGURE 1
Total b-glucosidase yield (IU/l) and specific activity (-&-) in different celldisruption processes. For all processes, a 20% wet cell weight (wcw) was
taken for reporting activities.
FIGURE 2
Elution profile and 10% SDS-PAGE analysis of fractions from ion-exchange column X
right side is shown 10% SDS-PAGE analysis of fractions. Lane 1: Marker lane, Lanes 2–
carried out with a step gradient of 130 mM KCl. On the right side is shown 10% Sappearing after the application of 130 mM KCl step gradient.
314 www.elsevier.com/locate/nbt
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proteins were indeed removed (Fig. 2b). On the basis of this, a large
column (XK26/70, bed height 50 cm) was used and the effect of
protein loadings (0.5–3.0 g) was investigated on enzyme recovery.
A quick scaled down quantitative b-glucosidase assay was done in
an ELISA plate. The specific activity and enzyme recovery for each
case are shown in Fig. 3. As observed, with an initial increase in
enzyme loading, there was an increase in specific activity up to a
loading of 1.76 g. However, further increase led to a decrease in the
same. While enzyme recovery was higher at 2.2 g, more contam-
inating proteins led to lower specific activities. Analysis of relevant
fractions by SDS-PAGE (Fig. 4a–c) confirmed that the preparation
was cleaner at a loading of 1.76 g (Fig. 4b). It was also observed that
enzyme recovery was initially as low as 64% at this protein loading
and could be enhanced to nearly 95% by the addition of 10 mM
cellobiose in the feed.
The concentrated ion-exchange fractions (5 mg total protein),
showing b-glucosidase activity, when applied to Sephadex G 200
column (XK16/100) gave a superior purity (70 IU/mg) compared to
that obtained on Superdex 75 column (15 IU/mg) while maintain-
K16/30. (a) Elution was carried out with a step gradient of 200 mM KCl. On the
6: fractions 2–6 appearing after the application of 200 mM KCl. (b) Elution was
DS-PAGE analysis of fractions. Lane 1: Marker lane, Lanes 2–6: fractions 7–12
New Biotechnology �Volume 29, Number 3 � February 2012 RESEARCH PAPER
10
20
30
40
50
60
70
80
3.532.521.510.50Enzyme lo ading (g)
Spec
ific
activ
ity (I
U/m
g)
30
45
60
75
90
105
Enz
yme
reco
very
(%)
FIGURE 3
Specific activity (-&-) and enzyme recovery (-~-) at different protein
loadings in XK 26/70 preparative column. Elution was carried out with a step
gradient of 130 mM KCl.
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ing the same level of yield (50%). On the basis of these results, 30–
40 mg protein sample was resolved on a preparative XK 26/100
column and the results are shown in Fig. 5a and b. As seen in the
SDS-PAGE, the two b-glucosidases were the major proteins. The
final separation of BGL I and BGL II was achieved on phenyl
Sepharose column. Two peaks were obtained, one was during
the flow-through (peak I) while the other peak (peak II) was eluted
at 100% concentration of elution buffer (Fig. 6a). Both showed b-
glucosidase activity and when checked on SDS-PAGE, peak 1
corresponded to BGL II while peak 2 to BGL I (Fig. 6b). The final
specific activity of BGL I was 74 IU/mg and that of BGL II 32 IU/
mg. A complete summary of purification is given in Table 1.
Purity of eluted b-glucosidases and internal peptide sequencesof BGL IIThe purity and the exact molecular weight of the proteins were
determined from the Electrospray spectrum shown in Fig. 7a (BGL
I) and b (BGL II). Typically, a protein will carry one charge per
1000 Da, or less if there are very few basic residues. As seen in Fig. 7,
multiply charged ions were formed for both the proteins and the
relative intensities were higher for BGL I than for BGL II. This was
advantageous as it improves the sensitivity at the detector and
allows a good analysis of high molecular weight molecules.
The presence of successive peaks differing from each by a single
charge confirmed the purity of the proteins. The data from both
the figures were convoluted by transformation of multiply charged
peaks in the spectrum using the algorithm in the mass spectro-
meter and molecular weight was determined as 97,000 for BGL I
and 94,000 for BGL II. The internal peptide sequences were deter-
mined for BGL II and were LSLSWPFK. . ., . . .NSDNPAYLNFHTER. . ..,
. . .EIYLEPFR. . ., . . .NDI/LDVTR. . .., . . .DQAVYF/MQVTPTR. . .. Two
of these (shown in bold) matched with the internal peptide
sequences of many Family 3 b-glucosidases.
DiscussionThis is the first report on the preparative scale approach for
resolution and purification of two closely related b-glucosidases.
The proteins are the cell wall bound enzymes of P. etchellsii and
belong to Family 3 of the glycosylhydrolase families. Relatively
little is known about this family and only one crystal structure is
available which is of a 657 aa enzyme [15]. Because the Family 3 b-
glucosidases occur as closely related isozymes, particularly in yeast,
their purification with concomitant high yield still remains a
challenge. In this work, we have introduced new strategies based
on the prior knowledge of substrate specificities of these enzymes
to resolve and purify them with high yield.
Breakage of yeast cells has been tried with solid shear and liquid
shear. Continuous shear, through the use of Dyno-Mill, is com-
mon for the release of enzymes from yeast cells [24] particularly for
large scale work. This is a larger device in which beads are agitated
by rapidly rotating discs. Because heat generation is a problem
with this device, adequate care has to be taken to maintain the
temperature at 48C. In our study, discontinuous liquid shear was
most successful with high recovery and very high specific activity
of b-glucosidases. Through this method, it is anticipated that
during the first two passes, as the flow rate is small, the cells
experience high shear to release the proteins. High flow rates kept
in the subsequent passes do not impart very high shear leading to
cells only being partially broken, which was observed under these
conditions. An increase in the release of total proteins is likely to
be due to stripping of proteins from the cell surface which also
explains increase in specific activity of b-glucosidases through this
method. In contrast to our previous work [16] where ammonium
sulphate precipitation was used as the first step in purification,
larger volumes of cell-free extract from Dyno-Mill (2–3 L), pre-
sented the problem of adding kilograms of ammonium sulphate.
This was circumvented by directly loading cell-free extract onto
anion-exchange column. It served dual purpose of eliminating the
use of DNase I (which is used for reducing the viscosity of the
solution) and preventing aggregation of the active protein by
maintaining low protein concentration in the cell-free extract
(0.5–0.7 mg/ml).
Because this is a high capacity technique, it was successfully
used to concentrate and purify proteins during the early stage.
Very large volumes could be loaded on to the column. Loading of
proteins at low concentrations minimized localized pH and ionic
strength fluctuations through rapid counter-ion release. This is
likely to have contributed to high recovery (above 60%) at this
stage. Recovery of the b-glucosidases from the column was further
increased to >90% by the addition of cellobiose to the cell-free
extract before loading. It served the purpose of ‘locking’ the active
site and thus prevented activity loss that may occur on account of
unfolding. We also used the strategy of absorbing the protein on
ion-exchange first followed by desorption as it allows a greater
degree of protein fractionation during the elution step. The fact
that it is also less expensive (when compared to hydrophobic
interaction, hydroxyapatite, metal chelate, covalent binding chro-
matography) justified its use at the early stage of purification.
Elution of b-glucosidases from ion-exchange matrix was
achieved by step gradient. This was first evaluated on a small
analytical column wherein b-glucosidases eluted around 200
and 130 mM KCl [Fig. 2a and b]. Higher specific activity
(27.4 IU/mg) with a yield of 88% was obtained at 130 mM KCl
and analysis of gel data indicated a purer preparation. It is also
important to note that nearly 60% of the column capacity was
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RESEARCH PAPER New Biotechnology �Volume 29, Number 3 � February 2012
FIGURE 4
Elution profile and 10% SDS-PAGE analysis from ion-exchange preparative column (XK26/70) at a protein loading of (a) 1.1 g, (b) 1.76 g and (c) 3.3 g. For all
protein loadings shown, elution was carried out with a step gradient of 130 mM KCl. Fractions loaded in the gel are numbered as they appear from the column(see the figure on the left in each case).
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taken up by the cell-free extract leaving about 40% for effective
separation. This worked well as step elution was being employed
which requires less volume. Two problems that normally occur
due to step elution viz. co-elution of other proteins resulting in the
loss of selective desorption and increase in partition coefficient,
316 www.elsevier.com/locate/nbt
which occurs as a protein is eluted so that a fraction of the protein
of interest is still bound, were largely avoided. Only a slight tailing
was observed. These results are better than our previous study [16]
wherein much lower yields were reported. In the scale-up opera-
tion on a preparative XK 26/70 column, the results could be
New Biotechnology �Volume 29, Number 3 � February 2012 RESEARCH PAPER
FIGURE 5
Elution profile and 10% SDS-PAGE analysis from Sephadex G 200 (XK26/100) column. On the left side are the data for OD280 (-*-) and b-glucosidase activity (-~-).Fractions testing positive for b-glucosidase activity (24–31) were analyzed on 10% SDS-PAGE (shown on the right side). M: Marker lane, IEX: pooled sample from
the previous ion-exchange chromatography step.
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reproduced. The loss in recovery of b-glucosidase (about 40% loss)
in this scale-up could be circumvented by the addition of 10 mM
cellobiose, a natural substrate of b-glucosidase. This inclusion led
to nearly 95% recovery of the enzyme. Thus efficient trapping of b-
glucosidases by the Q-Sepharose matrix was achieved. This might
be due to higher percentage of acidic residues on the surface of
these proteins [16]. The effect of protein loading on the recovery of
b-glucosidases was also studied. The results indicated that higher
amounts of b-glucosidases adsorbed to the matrix with an increase
in protein loading but nonspecific elution at higher loadings lead
to decrease in specific activity. The removal of the lower molecular
weight contaminants was achieved with Sephadex G 200 gel
filtration which gave better purification than Superdex 75.
Although the lower molecular weight proteins were successfully
removed in this step, there was about 50% loss in the recovery of
BGL I and BGL II. It is possible that the loss in b-glucosidase
activity was due to the removal of some contaminant proteins
FIGURE 6
(a) Elution profile from phenyl Sepharose column. (b) 10% SDS-PAGE analysis of
purified BGL II, appeared in fraction # 7. Peak II, corresponded to purified BGL I,
which might be providing non-specific force for maintaining the
native structure of the proteins. This was supported by the fact that
purified BGL I was stabilized against denaturation by the addition
of bovine serum albumin which might be providing the crowding
effect for stability. Multi-domain proteins are known to be stabi-
lized by molecular crowding effect [25]. Glycerol was found to be
more effective than BSA (data not shown). However, BGL II
appeared to be quite stable even in the absence of any crowding
agent. This may be explained in terms of its ability to tetramerize
which might be providing the ‘self crowding’ effect to the poly-
peptide chains. It is proposed that crowding provides a stabilizing
effect to the folded protein because of indirect compaction of the
denatured states [26–30].
The final resolution of BGL I and BGL II was attempted by
hydroxyapatite or phenyl sepharose chromatography. In the for-
mer method, low recovery (around 15–20%) was observed. The
major disadvantage with the use of this matrix is that it has a
fractions (7, 35–41) showing b-glucosidase activity. Peak I, corresponding towhich appeared in fractions # 35–41.
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RESEARCH PAPER New Biotechnology �Volume 29, Number 3 � February 2012
FIGURE 7
Electrospray spectrum of purified BGL I (a) and BGL II (b). The m/z (in Th) and the number of charges are indicated on each peak. The molecular mass was
determined as 97,000 Da for BGL I and 94,000 Da for BGL II.
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tendency to absorb carbon dioxide which can make a hard crust as
a layer on the column top. This leads to an increase in the back
pressure reducing the flow rate. Hydrophobic interaction chroma-
tography using phenyl sepharose was thus tried. It served both as
an affinity matrix for adsorption of the two enzymes, as BGL I and
318 www.elsevier.com/locate/nbt
BGL II are bound to the cell wall and do have hydrophobic patches,
as well as selective separation based on their preferences for the
aryl groups. BGL I shows preferential activity on aryl substrates
[16] and thus should bind to this matrix strongly and under
conditions of elution, BGL II should elute first. This strategy
New Biotechnology �Volume 29, Number 3 � February 2012 RESEARCH PAPER
TABLE 1
Summary of purification of BGL I and BGL II from Pichia etchellsii
Fraction Volume(ml)
Activity(IU/ml)
Proteinconcentration(mg/ml)
Specificactivity(IU/mg)
Total units(IU/ml)
Total protein(mg)
Yield(%)
Cell free extract 2000 4.21 1.10 3.81 8400 2200 100
Ion exchange 150 58.85 2.10 28 7980 315 95
Gel filtration 132 32.03 0.57 22 4229 75 53
301* 10*
Phenyl Sepharose
BGL I 12 10.62 0.143 74 164 1.70 54
BGL II 3 12.24 0.380 32 1.15
* Sample applied to phenyl sepharose column.
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worked and led to good and efficient separation of the two
enzymes. BGL I was eluted with much higher specific activity of
74 IU/mg as opposed to 35 IU/mg obtained with hydroxyapatite
[16]. Similarly, BGL II was also recovered to a specific activity of
32 IU/mg as opposed to 13.6 IU/mg reported earlier. It is also
possible that the hydrophobic matrix favored retention of enzyme
conformation during the process of elution. Although affinity
based matrices have been used for the purification of b-glucosi-
dases, these are limited to heterologous expression in Escherichia
coli employing His-Tag [31,32]. We also propose that phenyl
sepharose can be used as an affinity matrix for the separation of
isozymes based on their differential specificity towards aryl sub-
strates.
The high purity of the enzymes was confirmed by electronspray
ionization spectrum wherein ‘clean’ successive multiply charged
ions were obtained for the two enzymes. In this study, we also
identified a novel Family 3 glucosidase, namely BGL II; several
peptide sequences of which were novel. Availability of these
enzymes on large scale is likely to lead to a greater understanding
of Family 3 enzymes.
AcknowledgementsDepartment of Science and Technology (Govt. of India) is thanked
for providing financial support to one of the authors (SM) for
carrying out this work. The doctoral fellowship to MAS by Indian
Institute of Technology Delhi is gratefully acknowledged.
References
1 Bhatia, Y. et al. (2002) Microbial b-glucosidases: cloning, properties and
applications. Crit. Rev. Biotech. 22, 375–407
2 Morant, A.V. et al. (2008) b-Glucosidases as detonators of plant chemical defense.
Phytochemistry 69, 1795–1813
3 Barnett, C.C. et al. (1991) Cloning and amplification of the gene encoding an
extracellular b-glucosidase from Trichoderma reesei: evidence for improved rates of
saccharification of cellulosic substrates. Bio/Technology 9, 562–567
4 Bothast, R.J. and Saha, B.C. (1997) Ethanol production from agricultural biomass
substrates. Adv. Appl. Microbiol. 44, 261–286
5 Choa, K.M. et al. (1999) Integration of endo/exoglucanase and b-glucosidase genes
into the yeast chromosomes for direct conversion of cellulose to ethanol. Enzyme
Microb. Technol. 25, 23–30
6 Mamma, D. et al. (2004) Biochemical and catalytic properties of two intracellular
b-glucosidases from the fungus Penicillium decumbens. J. Mol. Catal. B 27, 183–190
7 Vasserot, Y. et al. (1995) Monoterpenyl glucosides in plants and their
biotechnological transformation. Acta Biotechnol. 15, 77–95
8 Winterhalter, P. and Skouroumounis, G.K. (1997) Glycoconjugated aroma
compounds: occurrence, role and biotechnological transformation. Adv. Biochem.
Eng. Biotechnol. 55, 73–105
9 Crout, D.H. and Vic, G. (1998) Glycosidases and glycosyl transferases in glycoside
and oligosaccharide synthesis. Curr. Opin. Chem. Biol. 2, 98–111
10 Hancock, S.M. et al. (2006) Engineering of glycosidases and glycosyltransferases.
Curr. Opin. Chem. Biol. 10, 509–519
11 Wang, L.-X. and Huang, W. (2010) Enzymatic transglycosylation for
glycoconjugate synthesis. Curr. Opin. Chem. Biol. 13, 592–600
12 Sharon, N. (2006) Carbohydrates as future anti-adhesion drugs for infectious
diseases. Biochim. Biophys. Acta 1760, 527–537
13 Huang, C. et al. (2006) Carbohydrate microarrays for profiling the antibodies
interacting with Globo H tumor antigen. Proc. Nat. Acad. Sci. U. S. A. 103,
15–20
14 Ratner, D.M. et al. (2004) Probing protein carbohydrate interactions with
microarrays of synthetic oligosaccharides. Chem. Biol. Chem. 5, 379–383
15 Varghese, J.N. et al. (1997) Three dimensional structure of barley b-D-glucan
exohydrolase, a family 3 glycosylhydrolase. Structure Fold. Des. 7, 179–190
16 Wallecha, A. and Mishra, S. (2003) Purification and characterization of two b-
glucosidases from a thermo-tolerant yeast Pichia etchellsii. Biochim. Biophys. Acta
1649, 74–84
17 Bhatia, Y. et al. (2002) Biosynthetic activity of Escherichia coli expressed Pichia
etchellsi b-glucosidase II. Appl. Biochem. Biotech. 102–103, 367–380
18 Bachhawat, P. et al. (2004) Enzymatic synthesis of oligosaccharides, alkyl and
terpenyl glucosides by recombinant Escherichia coli expressed Pichia etchellsii b-
glucosidase. Appl. Biochem. Biotech. 118, 269–282
19 Rather, M.Y. et al. (2010) b-Glucosidase catalysed synthesis of octyl-b-D-
glucopyranoside using whole cells of Pichia etchellsii in microaqueous media. J.
Biotechnol. 150, 490–496
20 Pandey, M. and Mishra, S. (1995) Cloning and expression of b-glucosidase gene
from the yeast Pichia etchellsii. J. Ferment. Bioeng. 80, 446–453
21 Bradford, M.M. (1976) Rapid and sensitive method for quantification of
microgram quantities of proteins utilizing the principle of protein dye binding.
Anal. Biochem. 72, 248–254
22 Chisti, Y. and Young, M. (1986) Disruption of microbial cells for intracellular
products. Enzyme Microb. Technol. 8, 194–204
23 Pingoud, A. et al. (2002) Biochemical Methods: A Concise Guide for Students and
Researchers. Wiley-VCH
24 Melendres, A.V. et al. (1991) A kinetic analysis of cell disruption by bead mill.
Bioseparation 2, 231–236
25 Ellis, R.J. (2001) Macromolecular crowding: obvious but underappreciated. Trends
Biochem. Sci. 26, 597–604
26 Berg, B. et al. (1999) Effects of macromolecular crowding on protein folding and
aggregation. EMBO J. 18, 6927–6933
www.elsevier.com/locate/nbt 319
RESEARCH PAPER New Biotechnology �Volume 29, Number 3 � February 2012
Research
Pap
er
27 Rivas, G. et al. (2004) Life in a crowded world: workshop on the biological
implications of macromolecular crowding. EMBO J. 5, 23–27
28 Zimmerman, S.B. and Minton, A.P. (1993) Macromolecular crowding:
biochemical, biophysical and physiological consequences. Ann. Rev. Biophys.
Biomol. Struct. 22, 27–75
29 Minton, A.P. (2005) Influence of macromolecular crowding upon the stability and
state of association of proteins: predictions and observations. J. Pharm. Sci. 94,
1668–1675
320 www.elsevier.com/locate/nbt
30 Stagg, L. et al. (2007) Molecular crowding enhances native structure and
stability of a/b protein flavodoxin. Proc. Natl. Acad. Sci. U. S. A. 104,
18976–18981
31 Opassiri, R. et al. (2004) b-Glucosidase, exo-b-glucanase and pyridoxine
transglucosylase activities of rice B Glu1. Biochem. J. 379, 125–131
32 Seidle, H.F. et al. (2006) Trp-49 of the family 3 b-glucosidase from Aspergillus niger is
important for its transglucosidic activity: creation of novel b-glucosidases with low
transglucosidic efficiencies. Arch. Biochem. Biophys. 445, 110–118