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(Received for publication, February 6,
1995; and in revised form, July 6, 1995) From the
The amino acid sequences of
The enzyme The study of
these enzymes has been facilitated by the use of recombinant DNA
technology(1, 5, 6) . Although a number of
cellulase genes including several Cellvibrio
gilvus, a cellulose-metabolizing bacterium, has the unique
property of producing cellobiose in high yields from acid-swollen
cellulose(12) . The isolation and characterization of the
cellulase, xylanase, and
C. gilvus, A.
tumefaciens, and chimeric For determination of kinetic parameters, C.
gilvus
The amino acid sequences of
Figure 1:
Homology in amino acid sequences of
Figure 2:
Schematic representation of parental and
chimeric
Figure 3:
Restriction analysis of chimeric plasmids
on agarose gel electrophoresis. Lane 1, HindIII
digest of
The pH optima for C. gilvus and A. tumefaciens enzymes are 6.2-6.4 and 7.2-7.4,
respectively. These enzymes also show marked differences in their
temperature optima. The pH activity profiles of
chimeric
Figure 4:
pH
activity (A) and pH stability (B) profiles of
chimeric and parental
The chimeric
Figure 5:
Temperature optima (A) and heat
stability (B) profiles of chimeric
Heat
stability may be influenced by only a few amino acid substitutions (17, 25) . In general, protein stability increases
with the insertion into an Thus,
the pH activity and heat stability were changed distinctly by
substituting different segments of C. gilvus
While the K The relative rates
of hydrolysis of cello-oligosaccharides by parental and chimeric
enzymes are shown in Table 2.
4-Methylumbelliferyl-
Genetic construction of chimeric enzymes from two functionally
related proteins, sharing extensive sequence similarity, is expected
not only to provide valuable information on the structure-function
relationship of the parent proteins, but also to prepare enzymes with
improved properties. Enzymatic activities are one of the sensitive
criteria for judging the correct folding of engineered proteins. Our
results demonstrate that different combinations of homologous
C-terminal regions of
Volume 270,
Number 37,
Issue of September 15, pp. 21928-21933, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
-Glucosidases with Improved Enzymatic Properties (*)
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-glucosidases from Cellvibrio gilvus and Agrobacterium tumefaciens show
about 40% similarity. The pH/temperature optima and stabilities and
substrate specificities of the two enzymes are quite different. C.
gilvus -glucosidase exhibits an optimum pH of 6.2-6.4
and temperature of 35 °C, whereas the corresponding values for A. tumefaciens are 7.2- 7.4 and 60 °C, respectively. The
substrate specificity of A. tumefaciens enzyme toward
different aryl glycosides is broader than C. gilvus enzyme. To
analyze these properties further, three chimeric -glucosidases
were constructed by substituting segments from the C-terminal
homologous region of C. gilvus -glucosidase gene with
that of A. tumefaciens. The chimeric enzymes were
characterized with respect to pH/temperature activity and stability and
substrate specificity. Chimeric enzymes exhibited chromatographic
behavior similar to that of C. gilvus enzyme. However,
enzymatic properties of chimeras were admixtures of those of the two
parents. The chimeric enzymes were optimally active at 45-50
°C and pH 6.6-7.0. K values of
chimeric enzymes for the various saccharides were admixtures of both
parental enzymes. These results suggest that the two domains of C.
gilvus and A. tumefaciens enzymes probably can fold
independently. The homologous C-terminal region in
-glucosidase
appears to play an important role in determining enzyme
characteristics. Changes in the properties on substitution of segments
in this region might be related to the enzyme specificity, and
-glucosidases with improved properties can be prepared by
manipulating this region.
-glucosidase (EC 3.2.1.21) catalyzes the
hydrolysis of alkyl- and aryl--D-glucosides
(methyl--D-glucoside and p-nitrophenyl--D-glucoside) as well as
glycosides containing only carbohydrate residues (Cellobiose). On the
basis of substrate specificity, -glucosidases can be classified as
aryl--glucosidases, cellobiases, and those hydrolyzing both
aryl--glucosides and oligosaccharides. The last group is often
found in cellulolytic microorganisms(1, 2) . On the
basis of sequence homology, -glucosidases have been divided into
two subfamilies(2) : BGA (-glucosidases and
phospho--glucosidases from bacteria to mammals) and BGB
(-glucosidases from yeasts, molds, and rumen bacteria). It is one
of the components of the cellulase enzyme complex required for the
hydrolysis of cellulose to glucose by catalyzing the final step which
converts cellobiose to glucose(3, 4) .-glucosidases have been cloned
and expressed in both Escherichia coli and Saccharomyces
cerevisiae(7, 8, 9, 10) , their
enzymological properties, especially structure-function relationships,
have not been well understood, partially because most of the cellulases
show little sequence homology. Analysis of structure-function
relationships may be facilitated by the formation of chimeric
genes/enzymes produced by gene fusion(11) .-glucosidase systems of this organism (13, 14, 15) as well as the cloning,
analysis, and manipulation of the genes coding these enzymes (16, 17) have been investigated in our laboratory. The
-glucosidases from C. gilvus share conserved regions in
-glucosidases from different organisms. The nucleotide sequence of
the -glucosidase gene revealed that this enzyme belongs to the BGB
group of -glucosidases(15) . The amino acid sequences of
the C. gilvus -glucosidase gene show significant
similarity (about 40%) with those of a -glucosidase gene from Agrobacterium tumefaciens(18) . Despite this
similarity, their enzymatic properties, especially pH activity, thermal
stability, and substrate specificity, are quite different. To analyze
these properties further, chimeric -glucosidases were constructed
between them by substituting different segments from one enzyme in the
C-terminal homologous region of the other and comparing the enzyme
characteristics of parental and chimeric enzymes. The C-terminal region
seems to be important for -glucosidase activity, since deletion of
more than a 70-base pair fragment from the C-terminal part of C.
gilvus -glucosidase gene resulted in the loss of enzyme
activity. (
)Although, the deletion of about 100 amino acid
residues near the C-terminal region of the 
-amylase gene did not
affect enzyme activity(19) , cyclomaltodextrin
glucanotransferases lacking 30 amino acids (20) and an
endoglucanase lacking 75 amino acids (21) from the C-terminal
end showed no enzyme activity. Keeping in mind the importance of the
C-terminal region and the estimated location of the catalytic center of
Asp-291 in the N-terminal region of C. gilvus(15) ,
the C-terminal region was selected for the construction of chimeric
enzymes.
Bacterial Strains and Plasmids
E. coli NM522 hsd 
5 (lac-pro)
(F`prolacIqZ
M15) and E.
coli DH5 F-80d lacZ
M15 rec A1 end A1 gyr A96 thi-1 hsd R17
(r
m
) sup E44 rel A1 deoR
(lac ZYA-arg F)U169 were hosts for -glucosidase genes
from C. gilvus and A. tumefaciens, respectively. pCG5
and pcbg1 were the recombinant plasmids carrying C. gilvus(15, 16) and A. tumefaciens(18) -glucosidase genes, respectively.DNA Manipulation
Recombinant DNA techniques and
methods for agarose gel electrophoresis were followed as described by
Sambrook et al.(22) . Plasmid DNA was prepared by
alkaline lysis method. The digestion by restriction enzymes was carried
out in the appropriate buffer at concentrations of 1-10 units per
µg of DNA for 1-3 h at appropriate temperatures. The
completion of the reaction was confirmed by agarose gel
electrophoresis. Sephaglas BandPrep kit (Pharmacia LKB Biotechnology
Inc.) was used for the extraction and purification of DNA from agarose
gels.Construction of Chimeras
Plasmid pCG5 was used as
a vector. Three restriction nuclease sites, the BsmI, AgeI, and BsaBI sites in the C-terminal homologous
region of pCG5 were used to construct three chimeric -glucosidase
genes. Plasmid pCG5 was digested with BsmI followed by
blunting and dephosphorylation before ligation with insert DNA. A
1657-base pair fragment was obtained by digestion of pcbg1 with NdeI and HinfI. This fragment was recovered from
agarose gel, purified, and blunted. Plasmid pCHBSM1 was constructed by
ligation of this fragment with the BsmI-digested fragment of
pCG5. Plasmid pCHAGE1 was constructed by ligation of an AgeI-digested fragment of pCG5 and a 1670-base pair AvaII fragment of pcbg1. Plasmid pCHBSAB1 was constructed by
ligation of a BsaBI-digested fragment of pCG5 and a 1227-base
pair Sfi-HinfI fragment of pcbg1. The clones
producing -glucosidase were detected by fluorescence from
4-methylumbelliferone on a UV transilluminator after growing on LB-agar
plates containing 1 mM 4-methylumbelliferyl--glucoside.Production and Purification of
Cells were grown overnight in 5 ml of
Luria-Bertani (LB) medium at 30 °C. One milliliter of culture was
used to inoculate 1 liter of LB medium supplemented with 50 µg/ml
ampicillin. Cultures were incubated aerobically, and cells were
harvested in the late log phase of the growth by centrifugation. The
cells were suspended in 25 mM MOPS (-Glucosidases
)buffer (pH
6.5) and sonicated using a Branson Sonifier Model 250/450. Cell debris
was separated by centrifugation, and the supernatant was obtained as a
crude enzyme preparation.-glucosidase preparations were
partially purified by ion exchange chromatography. C. gilvus and the chimeric enzyme preparations were applied to a FPLC system
(Pharmacia LKB Biotechnology Inc.) using a column of SP Sepharose Fast
Flow HiLoad(TM) 26/10 (bed volume 53-58 ml) equilibrated with
25 mM acetate buffer (pH 5.0). The proteins were eluted with a
linear gradient of 0-1 M NaCl in the same buffer. In the
case of -glucosidases of A. tumefaciens, the enzyme
preparation was applied to a column of Q Sepharose Fast Flow
HiLoad 26/10 (bed volume 53-58 ml) equilibrated with
20 mM bis-tris propane (pH 6.5). The proteins were eluted with
a linear gradient of 0-1 M NaCl in the same buffer. The
partially purified fractions were used for the determination of enzyme
characteristics.
-glucosidase and chimeric CHBSM -glucosidase were
further purified on a large scales. Ten liters of cultures were
centrifuged at 5000 
g for 10 min, and cells were
suspended in 100 ml of 25 mM MOPS buffer (pH 6.5). The enzyme
solution was obtained after sonification of the cells and removal of
the cell debris by centrifugation. The enzyme solution was applied to a
column of SP Sepharose Fast Flow. The enzymes were eluted with a linear
gradient of 0-1 M NaCl in 20 mM acetate buffer,
pH 5.0. Active fractions were pooled, dialyzed, and applied to a column
of Mono Q (Pharmacia). The enzymes were eluted with a linear gradient
of 0-1 M NaCl. The active fractions were pooled,
concentrated, and finally applied to a gel filtration column of
Superose 6 (Pharmacia). The enzymes were eluted with 25 mM acetate buffer (pH 5.0) containing 0.15 M NaCl.
Homogeneity of the purified enzyme preparations was monitored by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis on a PhastSystem
(Pharmacia).
In the standard assay, an
aliquot of suitably diluted enzyme solution was incubated in the
reaction mixture containing 25 mM MOPS buffer (pH 6.5) and 2
mMp-nitrophenyl--Glucosidase Assay-glucoside (pNPG) for 30 min at
30 °C. The reaction was stopped by the addition of 0.2 M glycine-NaOH (pH 10.5), and the amount of p-nitrophenol
released was measured by the absorbance at 405 nm. One unit of
-glucosidase activity was defined as the amount of enzyme
releasing 1 µmol of p-nitrophenol per min under the above
conditions.Other Methods
Kinetic parameters were determined
by the double reciprocal plot method of Lineweaver and Burk (23) using p-nitrophenyl derivatives of saccharides
dissolved at various concentrations in 25 mM MOPS buffer, pH
6.5, as substrate. The released amount of p-nitrophenol was
measured by monitoring absorbance at 405 nm at 20-s intervals for 20
min by using a spectrophotometer (DU-7400, Beckman). Enzymatic action
on cello-oligosaccharides was measured in a reaction mixture containing
1 mM oligosaccharide, 25 mM MOPS buffer (pH 6.5), and
suitably diluted enzyme solution. The reaction mixture was incubated at
30 °C for 10 min and then boiled for 5 min to terminate the enzyme
reaction. Released glucose was measured by a glucose oxidase-peroxidase
kit, Glucose CII Test, Wako Chemical Industries. Products of enzymatic
action on cello-oligosaccharides were analyzed by the HPLC system
(DIONEX) equipped with a DIONEX pulsed amperometric detector. The
prepacked column was a DIONEX Carbopac[tm] PA1 (4 250
mm). Solvents A (100 mM NaOH) and B (100 mM NaOH,
NaOAc) were used as eluents (flow rate 1 ml/min) at 92% and 8%
concentrations, respectively. Sugars were identified and quantitated by
comparison with retention times of authentic saccharide standards.Enzymes and Chemicals
All the restriction enzymes
(TaKaRa and BioLabs), DNA ligation kit (TaKaRa), and Sephaglas BandPrep
DNA extraction and purification kit (Pharmacia) used in this study were
purchased from commercial suppliers and used according to the
manufacturers' recommendations.
4-Methylumbelliferyl--D-glucoside and aryl-glycosides
were obtained from Sigma and cello-oligosaccharides from Seikagaku
Kogyo. All other chemicals used were reagent grade.
Construction of Chimeric
-Glucosidases-Glucosidase genes of C. gilvus(15, 16) and A. tumefaciens(18) have been cloned previously, and their nucleotide
sequences have been reported. Despite the significant similarity of the
deduced amino acid sequences, most of the enzymatic features of the two
-glucosidases were quite distinct. -Glucosidase from A.
tumefaciens shows higher pH and temperature optimas as well as
broader substrate specificity as compared to the one from C.
gilvus. To analyze these properties further as well as to study
the effect of the shuffling of homologous segments in the C-terminal
region, three chimeric -glucosidases derived from two genes were
constructed.-glucosidases from C. gilvus and A. tumefaciens show significant
similarity on most of the parts. In particular, the region from Ala-541
to Pro-811 of -glucosidase from A. tumefaciens is quite
similar to the region from Ala-472 to Pro-741 of -glucosidase from C. gilvus (Fig. 1). Considering the translation frame
and similar regions of both genes, three regions in the C. gilvus -glucosidase gene (plasmid pCG5) were selected for
substitution with the A. tumefaciens -glucosidase gene.
Schematic representation of the structure of the chimeric
-glucosidase gene is shown in Fig. 2. The region between
the NdeI and HinfI sites starting from Cys-517 and
over stop codon in pcbg1 were substituted at the BsmI site of
pCG5 to obtain the chimeric enzyme CHBSM. Similarly, two other chimeric
enzymes were obtained by substituting the region between two AvaII sites starting from Ile-594 in pcbg1 at the AgeI site of pCG5 (CHAGE) and the region between SfiI
and HinfI sites starting from Asp-660 in pcbg1 at the BsaBI site of pCG5 (CHBSA). The clones expressing chimeric
enzymes were purified, and their plasmids were characterized by
restriction analysis. Plasmid pCHBSM encoding chimeric enzyme CHBSM has
two SfiI sites in the inserted fragment of pcbg1, whereas
pCHAGE1 and pCHBSAB1 encoding chimeras CHAGE and CHBSA, respectively,
have two EcoRV sites in the inserted fragments of pcbg1 (Fig. 3).
-glucosidases from C. gilvus and A. tumefaciens. AT and CG represent A. tumefaciens and C. gilvus, respectively. A, schematic representation
of sequence homology in N-terminal (solid) and C-terminal (hatched) regions of -glucosidase genes. B,
amino acid sequences of A. tumefaciens and C. gilvus -glucosidases in the C-terminal region. Identical and similar
amino acid residues are designated by and
, respectively.
Chimeric enzymes were constructed by shuffling the regions marked by arrowheads.
-glucosidase genes. Light and dark bars represent regions derived from C. gilvus and A.
tumefaciens, respectively. Restriction enzymes used for the
construction of chimeric enzymes are shown with open
arrowheads, whereas the restriction enzymes used for the
confirmation of chimeric plasmids are shown with filled
arrowheads.
DNA marker; lane 2, BioMarker; lane 3, SfiI-digested pCHBSM1; lane 4, BamHI/EcoRV-digested pCHAGE1; lane 5, BamHI-digested pCHBSAB1; lane 6, SfiI-digested pCG5; and lane 7, BamHI-digested pCG5. The 1.2-kilobase band in pCHBSM1 and
0.7-kilobase band in pCHAGE1 and pCHBSAB1 were created by the SfiI and EcoRV sites, respectively, in the inserted
gene.
Characteristics of Chimeric
Parental and chimeric -Glucosidases-glucosidases were
partially purified by the FPLC system using either SP Sepharose (cation
exchange) or Q Sepharose (anion exchange) columns in order to determine
enzymatic properties. Marked differences in the behavior of
-glucosidases from C. gilvus and A. tumefaciens have been observed during the course of purification using ion
exchange chromatography. The cation exchange column was used at pH 5.0
to elute -glucosidases from C. gilvus, whereas the anion
exchange column successfully eluted -glucosidases from A.
tumefaciens at pH 6.5. Chimeric -glucosidases showing
behavior similar to that of -glucosidase from C. gilvus in ion exchange chromatography were purified about 40-fold using
an SP Sepharose column.-Glucosidase from C. gilvus is
optimally active at 35 °C, whereas that of A. tumefaciens exhibits maximum activity at 60 °C. With regard to heat
stability, -glucosidase from C. gilvus shows complete
activity up to 30 °C, retains about 80% of its maximum activity at
35 °C, and inactivates completely at 55 °C. On the other hand, A. tumefaciens enzyme is stable up to 55 °C, and, even at
65 °C, it retains 60% of its maximum activity. A. tumefaciens enzyme specificity toward aryl-glycoside substrates is broader
than of C. gilvus enzyme.-glucosidases are shown in Fig. 4. Chimeric
enzymes showed intermediate profiles of their parents. CHBSM enzyme
exhibited the maximum activity at pH 6.6-7.0, about 40% at pH
8.0, and no activity at pH 10.0. The optimum pH of CHAGE enzyme was
6.8-7.0 with about 35% activity at pH 8.0. CHBSA enzyme was
optimally active at pH 6.6 and inactivated at pH 9.0. All the chimeras
were stable between pH 4 and 9, whereas the -glucosidases from C. gilvus and A. tumefaciens were stable at pH
4-8 and pH 5-10, respectively. Substitution of segments in
the homologous C-terminal region seems to have a marked influence on pH
activity and stability. In Bacillus cyclomaltodextrin
glucanotransferase (20) and cellulase(24) , pH activity
profiles were found to be influenced by the N- and the C-terminal
parts.
-glucosidases. The pH was adjusted with
buffers: citrate (pH 3.0-4.0), MES (pH 5.0-6.8), MOPS (pH
7.0-8.0), and CHES (pH 9.0-10.0). For pH stability
experiments, enzyme was incubated at different pH values for 1 h at 25
°C. The residual activities were measured under standard assay
conditions. 
--, CHBSM;
--
, CHAGE;
--, CHBSA;
--, C. gilvus;
--, A.
tumefaciens.
-glucosidases also exhibited a significant
variation in temperature optimum from their parent enzymes (Fig. 5). The chimeric enzymes were optimally active at
45-50 °C, showing an intermediate temperature optimum between C. gilvus and A. tumefaciens enzymes. CHBSM exhibited
maximum activity at 50 °C, and 61% of its maximum activity at 60
°C. On the other hand, CHAGE was optimally active at 50 °C and
exhibited 52% of its maximum activity at 60 °C. CHBSA showed the
temperature optima of 45 °C with no activity at 70 °C. Heat
stability experiments revealed that CHBSM enzyme was completely active
up to 45 °C, retained about 65% of its maximum activity at 55
°C, and was completely inactivated at 55 °C. CHAGE enzyme was
stable up to 40 °C, and, even at 55 °C, 50% of its maximum
activity was retained. CHBSA was least stable among the three chimeras.
It was stable up to 40 °C, and retained only 20% of its maximum
activity at 55 °C. The temperatures at which 50% loss of the enzyme
activities occurred were 41, 67, 57, 55, and 50 °C for C.
gilvus, A. tumefaciens, CHBSM, CHAGE, and CHBSA enzymes,
respectively. Thus heat stability of chimeric enzymes was increased by
9-16 °C as compared to C. gilvus enzyme.
-glucosidases. For
heat stability experiments, each enzyme at its optimum pH was treated
at different temperatures for 1 h. The residual activities were
measured under standard assay conditions.
--, CHBSM; --
,
CHAGE;
--, CHBSA; --, C. gilvus; --, A.
tumefaciens.
-helix of helix-forming amino acids
(alanine, glutamic acid etc.) and decreases with the insertion of
helix-breaking amino acids (proline, glycine etc.). The secondary
structures of the parental and chimeric enzymes were predicted by
Robson's method(26) . There were similar numbers of
helix-breaking but more helix-forming amino acid residues in the
-helix regions of chimeric enzymes than C. gilvus enzyme,
suggesting that it could be one of the factors influencing the heat
stability of chimeras. Hydrophobic interaction inside the protein
molecule is another important factor in stabilizing protein structure.
Hydrophobic cluster analysis (27, 28) of native and
chimeric enzymes revealed that the amino acid substitution from C.
gilvus to A. tumefaciens significantly increased the
hydrophobic properties of the chimeric enzymes. These substitutions
might be important for heat stability of -glucosidase.-glucosidase
gene with that of A. tumefaciens. It is interesting to note
that these changes were more pronounced with the increased size of the
insertion fragment. For example, CHBSM containing the largest insertion
fragment from A. tumefaciens -glucosidase exhibited
broader pH optima than the other two chimeras. Thermal stability was
also found to be in the order of CHBSM > CHAGE > CHBSA. In
chimeric isopropylmalate dehydrogenase from an extreme thermophile, Thermus thermophile, and a mesophile, Bacillus
subtilis, the stability of each chimeric enzyme was approximately
proportional to the content of the amino acid sequence from the T.
thermophile enzyme(29) .Substrate Specificity
The substrate specificity of
the chimeric and parental enzymes was studied using various
aryl-glycosides as shown in Table 1. -Glucosidase from C. gilvus has rather strict specificity toward glucose
residues in aryl-glycosides. This enzyme hydrolyzed p-nitrophenyl--D-xyloside at 0.81% of the level
of pNPG and p-nitrophenyl--D-galactoside at
0.15%. It had no activity (less than 0.002% of the level of pNPG) on
various other substrates tested. On the other hand, A. tumefaciens enzyme exhibited 14%, 63%, 20%, and 0.47% activity on p-nitrophenyl--D-xyloside, p-nitrophenyl--D-galactoside, p-nitrophenyl--D-fucoside, and p-nitrophenyl-N-acetyl--D-glucosaminide,
respectively. No distinct differences in the measured kinetic
parameters of K and V
for each substrate were found among the three chimeric enzymes of
CHBSM, CHAGE, and CHBSA. The three chimeric enzymes were found to be
closer to A. tumefaciens enzyme since they showed K and V
values
similar to that of A. tumefaciens enzyme for the substrate of p-nitrophenyl--D-xyloside, p-nitrophenyl--D-galactoside, and p-nitrophenyl--D-fucoside. However, when pNPG is
used as substrate, K values of the three
chimeric enzymes (0.270-0.293 mM) were just between the
two parental enzymes of C. gilvus (1.81 mM) and A. tumefaciens (0.032 mM). All enzymes including
three chimeric enzymes strictly recognize the hydroxy residue at the
C-2 position of
-D-glucose since p-nitrophenyl--D-mannoside was not hydrolyzed.
These results suggest that in chimeric enzymes, the two domains of C. gilvus and A. tumefaciens -glucosidases
probably can be folded into active form correctly. The catalytic
efficiency (K) of one of the chimeric enzymes of
CHBSM was measured to be 3.2
10
s, and it is slightly lower than that of the C. gilvus enzyme, 8.9
10 s. It might indicate that the stereostructure
of the chimeric enzyme is to a certain extent perturbed in limited
regions.
value of the
chimeric citrate synthases similarly have been found to be lower than
those of the parental enzymes(30) , substrate affinity
decreased by about 2-fold in active human-yeast chimeric
phosphoglycerate kinase engineered by domain interchanges(31) .
However, no significant differences were found between the K
values of parental and chimeric
isopropylmalate dehydrogenases(23) . Replacement of the
catalytic base Glu-400 by glutamine in Aspergillus niger glucoamylase was found to affect both substrate ground-state
binding and transition state stabilization(32) . K
values for maltose and maltoheptaose
were 12- and 3- fold higher for the Glu-400
Gln mutant, with K
values 35- and 60-fold lower, respectively, as
compared with those of the wild type enzyme. Similarly, in Aspergillus awamori glucoamylase mutants, Ser-119
Tyr,
Gly-183
Lys, and Ser-184
His, slightly higher activity
for maltose hydrolysis and lower activity for isomaltose as compared
with the wild type enzyme was observed by Sierks and Svensson (33) . The observed increase in selectivity was attributed to
the stabilization of the maltose transition-state complex for each
enzyme. Modulation of binding energy by mutation could be attributed to
modification in hydrogen
bonding(32, 34, 35) .
-glucoside was found to be the best substrate
for all of the enzymes. Both C. gilvus and A. tumefaciens -glucosidases releases 4-methylumbelliferone and glucose in
parallel from 4-methylumbelliferyl--glucoside (data not shown). C. gilvus enzyme hydrolyzes cellobiose only 11.9% as fast as
4-methylumbelliferyl--glucoside, whereas hydrolysis rates of
cellobiose by A. tumefaciens enzyme was 20.2%. Hydrolysis
rates of cellotetraose, cellopentaose, and cellohexaose by C.
gilvus enzymes were 3-5 times higher than that of the A.
tumefaciens enzyme. The hydrolysis rates of these oligosaccharides
by chimeric enzymes were similar to that of the C. gilvus enzyme and higher than that of the A. tumefaciens enzyme.
The products released by enzymatic action on different
cello-oligosaccharides were analyzed by HPLC. The C. gilvus enzyme hydrolyzed each oligosaccharide into smaller
oligosaccharides. However, cellobiose was hardly hydrolyzed. On the
other hand, the A. tumefaciens enzyme hydrolyzed each
oligosaccharide into glucose and the oligosaccharides smaller than the
original ones by one glucose unit. The chimeric enzymes more or less
exhibited patterns similar to that of C. gilvus enzyme.
-glucosidases from C. gilvus and A. tumefaciens resulted in the formation of enzymatically
active chimeric species. The C-terminal region in the -glucosidase
gene plays an important role in determining enzyme characteristics, and
the changes in enzymatic properties on substitution of the C-terminal
segments might be related to enzyme specificity. Chimeric
-glucosidases with improved enzymatic properties can be prepared
in a convenient and effective way by manipulating this region.
))-D-glucoside; FPLC, fast protein
liquid chromatography; HPLC, high performance liquid chromatography;
bis-tris,
2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol;
CHES, 2-(N-cyclohexylamino)ethanesulfonic acid.
We are thankful to Drs. L. A. Castle, University of
California, and R. O. Morris, University of Missouri, for useful
discussions and for providing the recombinant plasmid-carrying
-glucosidase gene from A. tumefaciens. Thanks are also
due to Drs. Y. Kashiwagi, H. Taniguchi, and S. Sasaki for critical
discussion. Chika Aoyagi provided excellent technical assistance.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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  S. Shipkowski and J. E. Brenchley Characterization of an Unusual Cold-Active {beta}-Glucosidase Belonging to Family 3 of the Glycoside Hydrolases from the Psychrophilic Isolate Paenibacillus sp. Strain C7 Appl. Envir. Microbiol., August 1, 2005; 71(8): 4225 - 4232. [Abstract] [Full Text] [PDF]
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 L. Verdoucq, M. Czjzek, J. Moriniere, D. R. Bevan, and A. Esen Mutational and Structural Analysis of Aglycone Specificity in Maize and Sorghum {beta}-Glucosidases J. Biol. Chem., June 27, 2003; 278(27): 25055 - 25062. [Abstract] [Full Text] [PDF]
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M. Cicek, D. Blanchard, D. R. Bevan, and A. Esen The Aglycone Specificity-determining Sites Are Different in 2,4-Dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA)-glucosidase (Maize beta -Glucosidase) and Dhurrinase (Sorghum beta -Glucosidase) J. Biol. Chem., June 23, 2000; 275(26): 20002 - 20011. [Abstract] [Full Text] [PDF]
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