The crosslinked imprinted cyclodextrin glycosyltransferases obtained by imprinting with CD8 showed pH and temperature optima similar to those of the native and immobilized cyclodextrin g
Trang 1from Paenibacillus sp A11 and Bacillus macerans with
c-cyclodextrin
Jarunee Kaulpiboon1,2, Piamsook Pongsawasdi3 and Wolfgang Zimmermann2
1 Department of Pre-Clinical Science (Biochemistry), Faculty of Medicine, Thammasat University, Pathumthanee, Thailand
2 Department of Microbiology and Bioprocess Technology, Institute of Biochemistry, University of Leipzig, Germany
3 Starch and Cyclodextrin Research Unit, Department of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok, Thailand
Cyclodextrin glycosyltransferase (EC 2.4.1.19;
CGTase) catalyzes four different reactions: cyclization,
disproportionation, coupling, and hydrolysis
Cyclo-dextrins (CDs, cyclic oligosaccharides of glucose resi-dues) are formed by the intramolecular circularization reaction, whereas a linear malto-oligosaccharide is
Keywords
cross-linked imprinted proteins; cyclodextrin
glycosyltransferase; cyclodextrins; molecular
imprinting; product specificity
Correspondence
W Zimmermann, Department of
Microbiology and Bioprocess Technology,
Institute of Biochemistry, University of
Leipzig, Leipzig 04103, Germany
Fax: +49 341 97 36798
Tel: +49 341 97 36781
E-mail: wolfgang.zimmermann@
uni-leipzig.de
Website: http://www.biochemie.
uni-leipzig.de/agz
(Received 3 November 2006, revised 11
December, accepted 13 December 2006)
doi:10.1111/j.1742-4658.2007.05649.x
Cyclodextrin glycosyltransferase catalyzes the formation of a mixture of cyclodextrins from starch by an intramolecular transglycosylation reaction
To manipulate the product specificity of the Paenibacillus sp A11 and Bacillus maceranscyclodextrin glycosyltransferases towards the preferential formation of c-cyclodextrin (CD8), crosslinked imprinted proteins of both cyclodextrin glycosyltransferases were prepared by applying enzyme imprinting and immobilization methodologies The crosslinked imprinted cyclodextrin glycosyltransferases obtained by imprinting with CD8 showed
pH and temperature optima similar to those of the native and immobilized cyclodextrin glycosyltransferases However, the pH and temperature stability of the immobilized and crosslinked imprinted cyclodextrin glyco-syltransferases were higher than those of the native cyclodextrin glycosyl-transferases When the catalytic activities of the native, immobilized and crosslinked imprinted cyclodextrin glycosyltransferases were compared, the efficiency of the crosslinked imprinted enzymes for CD8 synthesis was increased 10-fold, whereas that for cyclodextrin hydrolysis was decreased Comparison of the product ratios by high-performance anion exchange chromatography showed that the native cyclodextrin glycosyltransferases from Paenibacillus sp A11 and Bacillus macerans produced CD6: CD7:
CD8:‡ CD9 ratios of 15 : 65 : 20 : 0 and 43 : 36 : 21 : 0 after 24 h of reaction at 40C with starch substrates In contrast, the crosslinked imprinted cyclodextrin glycosyltransferases from Paenibacillus sp A11 and Bacillus macerans produced cyclodextrin in ratios of 15 : 20 : 50 : 15 and
17 : 14 : 49 : 20, respectively The size of the synthesis products formed by the crosslinked imprinted cyclodextrin glycosyltransferases was shifted towards CD8 and ‡ CD9, and the overall cyclodextrin yield was increased
by 12% compared to the native enzymes The crosslinked imprinted cyclo-dextrin glycosyltransferases also showed higher stability in organic sol-vents, retaining 85% of their initial activity after five cycles of synthesis reactions
Abbreviations
A11, Paenibacillus sp A11; BM, Bacillus macerans; CD, cyclodextrin; CGTase, cyclodextrin glycosyltransferase; CLIP, crosslinked imprinted proteins; HPAEC, high-performance anion exchange chromatography; TNBS, 2,4,6-trinitrobenzene sulfonic acid.
Trang 2transferred to an acceptor sugar molecule in the
dis-proportionation reaction CGTase also catalyzes the
opening of a CD and transfer of the linear
malto-oligosaccharide to an acceptor sugar molecule in the
coupling reaction Furthermore, CGTase can catalyze
the hydrolysis of glycosidic linkages in starch [1] The
end-products, especially CD6, CD7 and CD8 (a-CD,
b-CD and c-CD), are extensively used in the food,
cos-metic and pharmaceutical industries, owing to their
ability to form inclusion complexes with appropriate
guest compounds [1–5] However, a major
disadvan-tage of the synthesis of CD by CGTases is that all
native enzymes usually produce a mixture of CD6,
CD7, CD8 and large-ring CD (‡ CD9), making
proces-ses to separate each type of CD unavoidable These
are time-consuming, cost-intensive and potentially
unsafe for the consumer and the environment To
resolve these problems, attempts to construct CGTases
with higher product specificity have been made, using
information on the three-dimensional structures of the
enzymes and genetic engineering techniques [6–8]
However, until now, no CGTase with a product
specif-icity for a single CD has been reported Recently, a
technique of crosslinking imprinted proteins (CLIP)
has been described [9,10] With a combination of
imprinting and enzyme immobilization methods, this
technique can be used for the production of
recogni-tion sites with predetermined selectivity in the enzyme
In the first step, the enzyme is derivatized with itaconic
acid anhydride and then imprinted with ligands such
as substrate analogs or inhibitors in aqueous medium
[10] Subsequently, the manipulated enzyme
conforma-tion is fixed by polymerizing it in a water-free organic
solvent The ligand is removed in the final step, and
the CLIP enzyme can be used either in aqueous
med-ium or organic solvent The CLIP enzymes show
altered substrate or product specificity and enhanced
stability in high concentrations of organic solvents
[11] CLIP enzymes are also more enantioselective than
the native enzyme [12] Furthermore, they are insoluble
and can be separated and recycled many times,
increasing their productivity These beneficial
proper-ties are especially useful in the areas of synthetic
organic chemistry, biomedical applications, and
envi-ronmental catalysis
In this study, the modification of the product
specif-icity and stability of two CGTases at the level of the
mature protein is described The native enzymes from
Paenibacillus sp A11 (A11) and Bacillus macerans
(BM) form CD7 and CD6 as their major products,
respectively [13,14] The imprinting of the enzymes
with CD8, resulting in high levels of the desired
prod-uct being formed, is reported
Results and Discussion
To provide enough attachment points for crosslinking
of the enzymes, the CGTases were derivatized with ita-conic anhydride in an aqueous medium Free amino groups of lysine, hydroxyl groups of tyrosine or sul-fhydryl groups of cysteine were covalently coupled with itaconic anhydride [12] By varying the protein⁄ itaconic anhydride ratio, different degrees of derivati-zation of the CGTases were obtained The remaining activity of the enzyme depended on the degree of deri-vatization (Table 1) The optimum ratio (w⁄ w) of both CGTases to itaconic anhydride was 1 : 5 The resulting degrees of derivatization of the A11 and BM CGTases determined by the 2,4,6-trinitrobenzene sulfonic acid (TNBS) assay were 62% and 65%, respectively The remaining activities of the derivatized A11 and BM CGTases were 90% and 77%, respectively With a ratio of 1 : 3, the remaining activity of both enzymes was not significantly different from a ratio of 1 : 5, but the derivatization degree was significantly lower With the use of higher ratios, higher levels of derivatization were possible, but resulted in a further decrease in activity In previous reports on the CLIP technique, Kronenburg et al [12] succeeded in manipulating the enantioselectivity of epoxide hydrolase with a derivati-zation degree of 70%, whereas Peißker et al [10] reported a 60% derivatization degree as optimum, con-sidering the remaining activity of the resulting deriva-tized protease
The derivatized A11 and BM CGTases were imprinted with CD8and crosslinked to obtain the cor-responding CLIP CGTases The derivatized nonim-printed enzymes were also crosslinked to obtain immobilized enzyme preparations for comparison The effect of pH on the activity of the different CGTase preparations from A11 and BM was
Table 1 Degree of derivatization of the CGTases from A11 and
BM obtained with different protein ⁄ itaconic anhydride ratios and remaining cyclization activity.
Ratio a
(w ⁄ w)
Paenibacillus sp A11 Bacillus macerans Derivatization
degree (%)
Remaining activity (%)
Derivatization degree (%)
Remaining activity (%)
a Protein ⁄ itaconic anhydride.
Trang 3determined in the pH range 5–11, as shown in
Fig 1A,B The optimum pH for the cyclization
activ-ity of the native, immobilized and CD8-imprinted
CLIP CGTases from A11 and BM was found to be
6.0 The pH activity profiles of the CGTases were
sim-ilar, showing 60% activity at pH 5.0, and decreasing
activity at higher pH values in the range of 7–11
However, the CGTase from A11 showed a broader pH
optimum, extending from 6.0 to 8.0 When the
immo-bilized and CD8-imprinted CLIP CGTases were
com-pared with the native enzymes, a higher activity in the
pH range from 8 to 11 was observed This effect was
more pronounced with the CGTase from BM The
immobilized and the CLIP CGTase from A11 were
more stable than the native enzyme in the pH ranges
from 3 to 6 and 8 to 11, whereas the immobilized and
CLIP CGTases from BM showed higher stability in the ranges from 3 to 7 and 9 to 11 (data not shown) The activities of the native, immobilized and CD8 -imprinted CLIP CGTases from A11 and BM were also determined at different temperatures in the range 30–
80C The optimum temperature for the cyclization activities of the different enzyme preparations were 40–
50C for the A11 CGTase (Fig 2A) and 60 C for the
BM CGTase (Fig 2B) The similar temperature optima of the different forms indicate that there was
no loss of enzyme activity through imprinting, immo-bilizing and crosslinking of the native enzyme The temperature stability of the immobilized and CLIP CGTases from A11 and BM at 60C and 70 C was considerably higher than that of the native enzymes (Fig 3A,B) This could be explained by a stabilizing effect of the covalent crosslinking of the enzymes The immobilized and CD8-imprinted CLIP CGT-ases from A11 showed 30% higher stability in phosphate buffer containing up to 50% ethanol or
pH
0
4 5 6 7 8 9 10 11 12
4 5 6 7 8 9 10 11 12
20
40
60
80
100
A
B
0
20
40
60
80
100
Fig 1 Effect of pH on the native (dotted line), immobilized (solid
line) and CD8-imprinted CLIP CGTase (dashed line) activity at 40 C.
The CGTases were from A11 (A) and BM (B) The buffers used
were 0.2 M potassium phosphate (pH 5.0–7.0) (s), Tris ⁄ HCl
(pH 7.0–9.0) (x), and glycine ⁄ NaOH (pH 9.0–11.0) (D).
0 20 40 60 80 100
0
20 30 40 50 60 70 80 90
20 30 40 50 60 70 80 90
20 40 60 80
100
A
B
Temperature (°C)
Fig 2 Effect of temperature on native (dotted line), immobilized (solid line) and CD 8 -imprinted CLIP CGTase (dashed line) activity at
pH 6.0 The CGTases were from A11 (A) and BM (B).
Trang 4cyclohexane compared to the native enzyme, whereas
the immobilized and CD8-imprinted CLIP CGTases
from BM showed 15% higher stability As ethanol and
other cosolvents have been shown to increase the yield
of CD produced by CGTases, the high stability of the
CD8-imprinted CLIP CGTases in the presence of
eth-anol could be used to further increase the product
yields of the CLIP enzymes [15] The effect of polar
cosolvents has been explained by suppression of the
intermolecular transglycosylation reaction, which
cau-ses partial degradation of the CD products formed
[16,17] With nonpolar solvents, CDs could form an
insoluble complex, resulting in their continuous
removal from the reaction by precipitation and a shift
of the equilibrium in favor of CD formation [18]
The reuse stability of the immobilized and CD8
-imprinted CLIP CGTases, which is an important
factor in the utilization of immobilized enzymes in large-scale applications, was also determined [19,20] More than 80% of the initial immobilized and CLIP CGTase activities from A11 and BM were retained for
up to five cycles of synthesis reactions
Comparison of the products obtained from the native, immobilized and CD8-imprinted CLIP
CGTas-es revealed that the native CGTasCGTas-es from A11 and
BM produced CD6: CD7 : CD8:‡ CD9 in ratios of
15 : 65 : 20 : 0 and 43 : 36 : 21 : 0, respectively, after
24 h of reaction at 40C In contrast, the CLIP CGTases from A11 and BM imprinted with CD8 pro-duced CD in ratios of 15 : 20 : 50 : 15 and
17 : 14 : 49 : 20, respectively (Table 2) The CLIP CGTases showed an increase in product specificity towards preferential formation of CD8 In addition to
a higher yield of CD8, the CD8-imprinted CLIP CGTases also produced a higher overall yield of CD compared with the native CGTases (Table 2) The immobilized and CD8-imprinted CLIP CGTases also produced larger amounts of large-ring CDs (‡ CD9) after 24 h of reaction at 40C (Fig 4A,B) As shown
in Fig 4C,D, large-ring CDs were predominantly pro-duced during the first 30 min of the reaction After
24 h, the amount of large-ring CDs was reduced, owing to their conversion to smaller CDs However, the conversion of large-ring CDs obtained with immo-bilized and CD8-imprinted CLIP CGTases was slower than with the native CGTases, indicating that the immobilized and CD8-imprinted CLIP enzymes had decreased hydrolysis and coupling activity
When the cyclization activities of the CGTase prepa-rations were compared, a 10-fold increase in the cata-lytic efficiency (kcat⁄ Km) of the CLIP CGTases from A11 and BM was observed, resulting from an increase
in the turnover rate (kcat) and the binding affinity (Km) (Table 3) The Kmvalues of the CLIP CGTases in the coupling reaction indicated stronger binding of the
CD8 substrate, whereas the turnover rates (kcat) were
0
20
40
60
80
100
A
0
20
40
60
80
100
B
20 30 40 50 60 70 80
20 30 40 50 60 70 80
Temperature (°C)
Fig 3 Effect of temperature on native (dotted line), immobilized
(solid line) and CD 8 -imprinted CLIP CGTase (dashed line) activity.
The CGTases were from A11 (A) and BM (B) The incubation was
performed at pH 6.0 for 30 min.
Table 2 Yields and product ratios of the native, immobilized and
CD 8 -imprinted CLIP CGTases from A11 and BM.
CGTase preparation
Yield (%) Product ratio (%)
CD6 CD7 CD8 ‡ CD 9
A11 CGTase
BM CGTase
Trang 5slower than with the native CGTases, resulting in
higher yields of CD8 after long reaction times This
result should, however, be interpreted with caution, as
the catalytic efficiency of the enzymes in the coupling
reaction was determined using cellobiose, which is not
the natural acceptor in the starch transglycosylation
reaction
The accumulation of large-ring CDs after 24 h of
reaction of the immobilized and CD8-imprinted CLIP
CGTases with starch can be explained by their
chan-ged hydrolytic activities The decreased kcatand overall
catalytic efficiency of both CLIP enzymes in the
hydro-lysis reaction clearly indicated their lower CD
hydroly-sis activity
In summary, the CD8-imprinted CLIP CGTases had
significantly higher catalytic efficiency for CD8
cycliza-tion and lower efficiency for CD hydrolysis, whereas their efficiency in the CD8coupling reaction was slightly increased when compared with the native enzymes
These results correspond to the observed higher yield of
CD8 and large-ring CDs obtained with the CLIP CGTases Whereas the immobilization of the CGTases alone resulted in increased yields of large-ring CDs, through reduction of their hydrolysis activity, the observed shift in product ratios of the CD8-imprinted CLIP CGTases suggests that the molecular imprinting had a pronounced effect on the structure of the active site of the enzymes Imprinting of the CGTases with CD
of different sizes should have similar effects on their preferential formation, which could, however, be expec-ted to be limiexpec-ted by the increasing flexibility of the ring structures of the larger CDs
Native A11 CGTase
Immobilized A11 CGTase
CLIP A11 CGTase
Native BM CGTase
Immobilized
BM CGTase
CLIP BM CGTase
6
10
6
7 8
10
Native A11 CGTase
Retention time (min)
Immobilized A11 CGTase
CLIP A11 CGTase
Immobilized
BM CGTase
CLIP BM CGTase
Retention time (min)
Native BM CGTase
15 6
6 7
7
24 24
Fig 4 HPAEC analysis of CD synthesized by different CGTase preparations from A11 (A, C) and BM (B, D) at 40 C for 24 h (A, B) and
30 min (C, D) The numbers above the peaks indicate the degree of polymerization of the CD.
Trang 6When the yields of CD8 obtained after different
reaction times with the enzyme preparations were
com-pared, most of the CD8 was found to be formed
dur-ing the first 30 min of incubation (Fig 5) After 24 h,
the CD8-imprinted CLIP CGTases produced 32%
(A11) and 25% (BM) CD8 In comparison to the
native enzymes, the yield of CD8 increased four-fold
with the CLIP CGTase from A11, and three-fold with
the CLIP CGTase from BM, after 24 h of reaction
The amount of CD8 produced by the CLIP CGTase
from BM slowly increased during the 24 h reaction
time, whereas no further increase occurred after 6 h of
reaction with the CLIP CGTase from A11 The differ-ences in the time course of CD8 formation detected depended on the type of CGTase used, and are in accordance with previously reported results Terada
et al [21] observed that the amount of CD8 increased when a CGTase from Bacillus sp A2-5a was incubated with starch for a long period of time, as a result of the conversion of large-ring CDs to smaller CDs In con-trast, longer reaction times with CGTase from the bac-terial isolate BT3 resulted in a 10% decrease in CD8 [22]
The highest yield (26% conversion to CD8) with the CLIP CGTase from A11 was found when synthetic lin-ear amylose (molecular mass 280 kDa) was used as substrate (Fig 6) Lower yields of CD8 were obtained with starches from potato, pea, and rice Corn starch (73% amylopectin) and corn amylopectin gave the lowest yields, owing to their branched structure Low yields were also obtained with dextrins, glucose oligo-saccharides of short chain length (degree of polymer-ization 23), indicating the preference of the CGTases for long chains of unbranched glucose polymers
In conclusion, the CGTases from Paenibacillus sp A11 and B macerans could be imprinted with CD8, which is not the major CD produced by the native enzymes CD8 was produced by the CD8-imprinted and crosslinked CLIP CGTases at much higher levels than by the native enzymes Moreover, the CLIP CGTases showed higher stability and yielded larger amounts of total CD in the synthesis reactions
Experimental procedures
Materials and enzymes
corn starch (molecular mass 340 kDa), rice starch, corn
Table 3 Comparison of the cyclization, coupling and hydrolysis activities catalyzed by native, immobilized and CD 8 -imprinted CLIP CGTases from A11 and BM.
CGTase preparation
CD 8 -cyclization activity CD 8 -coupling activity CD 6 )24-hydrolysis activity
Km,starch (mgÆmL)1)
kcat (s)1)
kcat⁄ K m
(mgÆmL)1Æs)1)
Km, CD8 (m M )
kcat (s)1)
kcat⁄ K m
( M )1Æs)1)
Km, CD6)24 (mgÆmL)1)
kcat (s)1)
kcat⁄ K m
(mgÆmL)1Æs)1) A11 CGTase
Native 0.83 ± 0.03 1.6 · 10 2 1.9 · 10 2 0.90 ± 0.20 1.2 · 10 2 1.3 · 10 5 1.25 ± 0.03 3.2 · 10 1 2.9 · 10 1
Immobilized 0.50 ± 0.02 2.5 · 10 2 5.0 · 10 2 0.55 ± 0.02 1.1 · 10 2 2.0 · 10 5 1.08 ± 0.01 8.0 · 10 0 6.4 · 10 0
CLIP imprinted
with CD 8
0.21 ± 0.01 4.9 · 10 2 2.3 · 10 3 0.26 ± 0.01 1.0 · 10 2 3.8 · 10 5 0.48 ± 0.04 5.5 · 10 0 1.1 · 10 1
BM CGTase
Native 0.55 ± 0.02 6.7 · 10 1 1.2 · 10 2 1.60 ± 0.20 4.2 · 10 2 2.6 · 10 5 1.30 ± 0.02 1.6 · 10 1 1.4 · 10 1
Immobilized 0.50 ± 0.01 9.0 · 10 1 1.8 · 10 2 0.63 ± 0.04 2.1 · 10 2 3.3 · 10 5 1.11 ± 0.05 6.3 · 10 0 4.8 · 10 0
CLIP imprinted
with CD8
0.20 ± 0.01 2.1 · 10 2
1.1 · 10 3
0.25 ± 0.04 9.4 · 10 1
3.8 · 10 5
0.59 ± 0.02 5.3 · 10 0
9.0 · 10 0
A
B
Incubation time (h)
0
0 5 10 15 20 25
0 5 10 15 20 25
10
20
30
40
0
10
20
30
40
Fig 5 Time course of CD8formation by native (m), immobilized (j)
and CD 8 -imprinted CLIP CGTases (s) from A11 (A) and BM (B).
Trang 7amylopectin, dextrin (degree of polymerization 23),
cellobi-ose, BSA, phenolphthalein, itaconic anhydride, TNBS,
2,2¢-azobis(2-methylpropionitrile), ethylene glycol
dimetha-crylate, water-free cyclohexane and n-propanol were
Germany) Pea starch (degree of polymerization 4000) was
kindly provided by Emsland-Sta¨rke GmbH (Emlichheim,
Germany) Synthetic amylose with an average molecular
mass of 280.9 kDa was prepared by the method of
were kindly provided by T Endo, Hoshi University, Tokyo,
Japan Rhizopus sp glucoamylase was obtained from
Toy-obo Co., Ltd (Osaka, Japan) BM CGTase was obtained
from Amano Enzyme Inc (Aichi, Japan) and had a specific
CGTase was purified using starch adsorption and ion
exchange chromatography (DEAE-Toyopearl 650M
col-umn; Tosoh Corporation, Tokyo, Japan) The enzyme had
dextrinizing activity [13]
CGTase assays and protein determination
Cyclization activity was determined as CD-forming activity
by the phenolphthalein method [24] CGTase (2.5 lg) was
0.2 m potassium phosphate buffer (pH 6.0) The reaction
was stopped by boiling for 10 min An aliquot (0.5 mL) was incubated with 2.0 mL of a solution containing 1.0 mL
of 4 mm phenolphthalein in ethanol, 4 mL of ethanol and
formed was calculated using a calibration curve One unit
of activity was defined as the amount of enzyme that
was determined by HPAEC
as donor with 50 mm cellobiose as glucosyl acceptor at
started by adding enzyme (2.5 lg) After 10 min, the reac-tion was stopped by boiling for 10 min Subsequently,
The released reducing sugars were determined with the di-nitrosalicylic acid method [25] One unit of activity was defined as the amount of enzyme that produced 1 lmol
The hydrolysis activity of the CGTase was determined by
kindly provided by M N Mokhtar, Leipzig University) at
The amount of glucose formed was determined by HPAEC One unit of activity was defined as the amount of enzyme
potassium phosphate buffer (pH 6.0) Lineweaver–Burk diagrams of the initial velocity against substrate concentra-tion were plotted, and kinetic parameters were determined using enzfitter software (Biosoft, Cambridge, UK) A reaction time of 30 min was used in the Lineweaver–Burk experiments By varying the reaction time with fixed sub-strate concentration, it was confirmed that the reaction velocity was linear at this time point
The protein concentrations were determined according to Bradford [26], using BSA as standard
Analysis of cyclodextrins HPAEC with pulsed amperometric detection was performed using a DX-600 system (Dionex Corp., Sunnyvale, CA, USA) to analyze and quantify the CD products A
was used A sample (25 lL) was injected and eluted with a linear gradient of sodium nitrate (0–10 min, increasing from 0% to 4%; 10–12 min, 4%; 12–32 min, increasing from 4%
to 8%; 32–48 min, increasing from 8% to 9%; 48–59 min,
A
0
5
10
15
20
25
30
0
5
10
15
20
25
30
h
cr
at
s
el
b
ul
os
ot
at
o
h cr at
s
ae
P
h cr at
s nr o C
h cr at
s i R
es ol y m
a cit e ht n
ni tc e p ol y m
a u
p o C
)3 2 D ( ni rt x e D
B
Substrates
Fig 6 Yield of CD 8 synthesized by the native (black bars),
immobi-lized (gray bars) and CD 8 -imprinted CLIP CGTases (white bars) with
different substrates at pH 6.0 for 30 min The CGTases were from
A11 (A) and BM (B).
Trang 8increasing from 9% to 18%; 59–79 min, increasing from
18% to 28%) in 150 mm NaOH containing 2% acetonitrile
Derivatization of the CGTases by acylation with
itaconic anhydride
Six milligrams of A11 and BM CGTase in 10 mL of 50 mm
potassium phosphate buffer (pH 6.0) was acylated by using
various amounts of itaconic anhydride The solution
mix-tures with different ratios of itaconic anhydride per mg of
monit-ored and maintained at 6.0 with 3 m NaOH Nonreacted
itaconic anhydride and other low molecular mass
com-pounds were removed by gel filtration (HiTrap desalting
column; Amersham Biosciences, Uppsala, Sweden) with
dis-tilled water as the eluent The fractions containing CGTase
activity were combined and lyophilized
Determination of free amino groups
of the CGTases
The relative amounts of amino groups of the native and
covalently derivatized CGTases were determined according
to Habeeb [27] and Hall et al [28] with TNBS The extent
of derivatization was calculated according to Shetty &
Kin-sella [29]:
with derivatized and native protein solutions, respectively
To a sample (0.3 mL) of native or derivatized protein
0.3 mL of TNBS (0.1%) were added The samples were
60 min, 0.47 mL of 1 m HCl was added, and the absorption
was measured at 335 nm against a blank treated as above
but containing 0.3 mL of deionized water instead of the
protein solution
Imprinting of the derivatized CGTases
dis-solved in 1 mL of 10 mm potassium phosphate buffer
The precipitate was collected by centrifugation at 13 520 g
(Het-tich GmbH & Co KG, Tuttlingen, Germany) The pellet
Crosslinking of imprinted derivatized CGTases
Imprinted derivatized CGTases (10 mg) were suspended in
1 mL of dry cyclohexane by using an ultrasonication bath for 15 min Four milligrams of 2,2¢-azobis(2-methylpropio-nitrile) and 200 lL of ethylene glycol dimethacrylate were added to the suspension The radical polymerization was
12 h The white polymer was washed with 2 mL of cyclo-hexane and with 50 mm potassium phosphate buffer
and enzyme activities were monitored during the different steps
Effect of pH and temperature on native, immobilized and CD8-imprinted CLIP CGTase activity
Each enzyme preparation (2.5 lg of protein) was incubated
temperatures, and the cyclization activity of the enzymes was assayed by the phenolphthalein method Potassium
(0.2 m) were used as buffers for pH 5.0–7.0, 7.0–9.0 and 9.0–11.0, respectively For determining the effect of tem-perature on the enzyme activity, the reactions were
Effect of pH on native, immobilized and
CD8-imprinted CLIP CGTase stability Each enzyme preparation (2.5 lg of protein) was incubated
remaining cyclization activity was assayed by the phenol-phthalein method The results were expressed as a percent-age of the highest activity determined, which was defined as 100%
Effect of temperature on native, immobilized and CD8-imprinted CLIP CGTase stability The thermostability of the enzyme preparations was
(2.5 lg of protein) in 10 mm potassium phosphate buffer
assayed by the phenolphthalein method The results were expressed as a percentage of the highest activity determined, which was defined as 100%
Trang 9Stability of native, immobilized and
CD8-imprinted CLIP CGTases in organic
solvents
The organic solvent tolerance of the native, immobilized
and CLIP CGTases in ethanol and cyclohexane was
phosphate buffer (pH 6.0) containing 10–50% of the
cyclization activity was assayed by the phenolphthalein
method
Reuse stability of immobilized and
CD8-imprinted CLIP CGTases
The immobilized and CLIP CGTases were recovered after
a synthesis reaction, and analyzed for their remaining
cycli-zation activity during five cycles of synthesis reactions
After each cycle, the enzymes were filtered off and washed
(pH 6.0)
Synthesis of CDs with native, immobilized and
CD8-imprinted CLIP CGTases
The native, immobilized and CLIP CGTases (2 U of
soluble potato starch in 0.2 m potassium phosphate buffer
was added for 3 h to convert the linear oligosaccharides to
glucose Subsequently, the glucoamylase was inactivated by
boiling for 10 min, and the reaction mixtures were analyzed
by HPAEC
Substrate specificity of native, immobilized and
CD8-imprinted CLIP CGTases
Soluble potato starch, pea starch, corn starch, rice starch,
synthetic amylose, corn amylopectin and dextrin substrates
were incubated with each CGTase preparation (2.5 lg of
0.6 mL Each reaction mixture was then analyzed by
HPAEC
Acknowledgements
JK was supported by a research fellowship of the
Alexander von Humboldt Foundation, Germany We
thank M N Mokhtar, Leipzig University, for his
advice on HPAEC analysis
References
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