Novo Nordisk A/S, Novo Allé, DK-2800 Bagsvaerd, Denmark
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Abstract |
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Keywords: amylase/CGTase/product specificity/site-directed mutagenesis/structural homology
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Introduction |
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The -amylases (EC 3.2.1.1) are a family of endo-amylases that catalyze the hydrolysis of
-1,4-glycosidic linkages in polymers of
-D-glucose and are thus needed for most organisms. The
-amylases are widely distributed in micro-organisms, plants and higher organisms and show varying cleavage patterns (Svensson, 1994
). They are used in a wide variety of industrial applications such as in liquefaction, brewing and detergents.
-Amylases from different organisms exhibit similar three-dimensional structures, despite great differences in primary structure. As most starch-hydrolyzing and related enzymes, the
-amylase family has a (ß/
)8- or TIM-barrel catalytic domain. Thus, the
-amylases belong to family 13 in the glycoside hydrolase classification as all other starch-degrading enzymes containing a TIM-barrel. The catalytic domain consists of eight inner parallel ß-strands surrounded by eight
-helices. This central domain is referred to as domain A. The active site in domain A is formed by the loops connecting the C-terminus end of the ß-strands to the N-terminus of the
-helices. Domain B is a small folding module, inserted between ß-strand 3 and
-helix 3 in the TIM-barrel. After the (ß/
)8-barrel and at the opposite side of the A domain with respect to the B domain the
-amylases contain a third folding module called domain C, which displays a Greek key motif. A function has been assigned to each of the three domains: the catalytic A domain, the B domain is involved in functional diversity and stability, while the terminal C domain besides conformational stability has not been assigned a particular function yet, even though it has been suggested that it is a starch granule-binding domain (SBD) (Svensson, 1994
). Also common in
-amylases is the requirement for calcium which maintains the structural integrity.
The CGTases (EC 2.4.1.19), also belonging to family 13, convert starch into CDs composed of primarily six, seven or eight (-, ß- and
-CDs, respectively) glucose units bound by
-1,4-bonds. In addition, various amounts of linear oligosaccharides and CDs with higher DP are produced. CGTases contain a catalytic core composed of three domains (A, B and C) sharing structural homology with the
-amylase, as well as two C-terminal domains (D and E), of which E is thought to be involved in substrate binding. The CGTases are classified as
-, ß- and
-CGTases according to their most predominant CD product. The ability of the CDs to form inclusion complexes with small hydrophobic molecules has led to applications in the pharmaceutical, food, cosmetic and agrochemical industries. By the molecular inclusion the chemical and physical properties of the included compounds are altered. Examples of the effects of inclusion are protection against heat and light, enhanced solubility and reduction of undesirable compounds (Wind et al., 1998
). Besides the ability of CGTases to degrade starch into CDs through an intramolecular reaction called cyclization, CGTases are also able to perform intermolecular transglycosylation reactions such as coupling and disproportionation.
Novamyl (EC 3.2.1.133) is a 686-residue thermostable maltogenic -amylase originally cloned from a strain of Bacillus during a systematic screening programme for
-amylase producing micro-organisms. Novamyl received its description as a maltogenic
-amylase because prolonged hydrolysis of starch with Novamyl results primarily in the production of maltose (Outtrup and Norman, 1984
; Diderichsen and Christiansen, 1988
). Novamyl was subsequently shown also to possess endo-amylase activity (Christophersen et al., 1998
) and is used in the baking industry as an antistaling agent owing to its ability to reduce retrogradation of amylopectin. Novamyl shows the highest sequence homology with the CGTases and also belongs to family 13 in the glycoside hydrolase classification (Henrissat, 1991
); see Table I
for comparisons with other glycoside hydrolases. Recently, the three-dimensional structure of Novamyl was obtained by X-ray crystallography and found to be very similar to the three-dimensional structure of the CGTases (Dauter et al., 1999
). This distinguishes Novamyl from the other
-amylases within glycoside hydrolase family 13 that normally possess the conventional three-domain
-amylase structure. Novamyl's catalytic mechanism is also expected to be similar to the catalytic mechanism known for CGTases and
-amylases, since the active site conformation in Novamyl is very much like the active site conformations in
-amylases and CGTases. Hence Novamyl is likely to catalyze hydrolysis using a double-displacement mechanism leading to overall retention of anomeric configuration.
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Owing to the structural similarities between Novamyl and CGTases, it was of interest to see if Novamyl could be converted into a CGTase-like enzyme capable of producing CDs. Previously it has been shown that an aromatic residue at the 195 position (Bacillus circulans strain 251, CGTase numbering) is critical to an efficient cyclization reaction; however, CGTase variants with a leucine occupying the 195 position are known (Penninga et al., 1995; Wind et al., 1998
). In contrast,
-amylases typically have a small residue at the structurally homologous position.
Here we report the conversion of Novamyl from a maltose-producing enzyme into a CGTase-like enzyme capable of producing CDs, by placing the aromatic residue at the position essential to an efficient cyclization reaction and deletion of a loop consisting of five residues likely to be a steric hindrance to cyclization. Both deletion of the five amino acid loop and correct insertion of an aromatic residue in the active site were required to alter the product specificity of this enzyme.
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Materials and methods |
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Site-directed mutagenesis was carried out using sequence overlap extension PCR (Higuchi et al., 1988) with Pwo polymerase as recommended by the manufacturer (Boehringer Mannheim) for amplification. Fragments were cloned into a suitable vector by digestion with HindIII and SalI as recommended by the manufacturer (Promega) and ligated with T4 DNA Ligase also as recommended by the manufacturer (Promega). Ligations were transformed into an amylase-depleted Bacillus subtilis strain and positive transformants were sequenced in order to confirm the desired alterations. Sequencing was performed using DyeDeoxy terminators and an Applied Biosystems 377 DNA Sequencer.
Sequences of mutagenic primers were as follows (incorporated restriction site underlined):
NM001:
Primer 1: 5'-CTTGTACGATCTTGCGTCGCAGGAAAATGG-3'
Primer 2: 3'-CTCCGCGTTACCTTTTTGAACATGCTAGAACGCAG-5'
NM002:
Primer 1: 5'-CTTCACTGCCGATTTGTCGCAGGAAAATGGC-3'
Primer 2: 3'-GCTCCGCGTTACCTTTTTGAAGTGACGGCTAAACAGC-5'
Expression and purification of wt enzyme and variants
Cells were fermented for 5 days at 30°C in 500 ml shake flasks (270 r.p.m.) containing 100 ml of a complex medium mainly consisting of sucrose soy meal.
The culture supernatant was flocculated using a mixture of cationic (C521, American Cyanmide Company) and anionic (A130, American Cyanmide Company) flocculants. The culture supernatant was diluted (2:1, v/v) with Milli-Q water, adjusted to pH 7.5 and CaCl2 added to a final concentration of 0.5% (w/v). Sodium aluminate was added to a final concentration of 0.25% (w/v), while maintaining the pH around 7.5 by titration with 20% (v/v) formic acid. The cationic flocculant C521 was subsequently added to a final concentration of 0.25% (v/v) followed by careful addition of the anionic flocculant to a final concentration of around 0.006% (w/v). The flocculated culture supernatant was centrifuged in a Sorvall RC-3B centrifuge, equipped with a GSA rotor head (4500 r.p.m. for 35 min at 4°C). Novamyl variants were purified on individual columns of -cyclodextrin coupled to activated agarose. The centrifuged culture supernatant was applied to an
-cyclodextrinagarose column (1.6x5 cm) in 50 mM sodium acetate, pH 5.0, 1 mM CaCl2, 0.5 M NaCl at a flow rate of 300 ml/h. The column was washed using 50 mM sodium acetate, pH 5.0, 1 mM CaCl2, 0.5 M NaCl ~10 column volumes) and Novamyl variants were subsequently eluted in the same buffer containing 2% (w/v)
-cyclodextrin. The variants were homogeneous as estimated using SDSPAGE and stained using Coomassie Brilliant Blue. Protein concentrations were determined spectrophotometrically at 280 nm using
= 132 710 M1 cm1 and a molecular weight of 75 kDa.
Enzyme assays
Amylase activity was determined using the Phadebas Amylase Test (Pharmacia Diagnostics, Uppsala, Sweden). The assay was performed by preincubating 4 ml of buffer (50 mM sodium acetate, pH 5.0, 1 mM CaCl2) at 60°C. A 100 µl volume of enzyme solution (for wild-type typically 0.00235 mg/ml) was added, followed by one tablet of Phadebas insoluble blue starch. The activity was quenched after 90 min of incubation at 60°C by addition of 1 ml of 1 M NaOH. After centrifugation or filtration, the absorbance was measured at 650 nm. Activities are given as PSU, which is defined as A650/min at 60°C at pH 5.0.
Activity towards maltotriose (1%, w/v) was measured at 60°C in 50 mM sodium acetate, pH 5.0, 1 mM CaCl2. 200 µl substrate in Eppendorf tubes was preincubated at 60°C. A 20 µl aliquot (t = 0) was transferred into a microtiter plate well containing 50 µl of 0.1 M NaOH. Novamyl-catalyzed hydrolysis was initiated by addition of 10 µl of enzyme solution (for wild-type around 0.00235 mg/ml) and at different time points aliquots of 20 µl were removed, typically after 2, 4, 6, 8 and 10 min, and transferred into microtiter plate wells containing 50 µl 0.1 M NaOH. A 200 µl volume of GOD-Perid was added and the absorbance was measured at 650 nm after 30 min of incubation at room temperature. MANU is defined as the amount of enzyme that cleaves 1 µmol maltotriose per minute at 60°C and pH 5.0.
Thermostability was determined by incubating Novamyl and variants at various temperatures (5095°C) in 50 mM sodium acetate, pH 5.0, 1 mM CaCl2 for 5 min. The residual activity was determined as described above. Tm is defined as the temperature at which 50% of the starting activity is retained.
The activity of ß-CD formation was determined using 5% Paselli SA2 starch (AVEBE, Foxhol, The Netherlands) with an average DP of 50 as substrate in 10 mM citrate buffer at pH 6.0 at 50°C. ß-CD formed was determined on the basis of its ability to form a stable, colorless inclusion complex with phenolphthalein (Vikmon, 1982).
Determination of the distribution of the CDs produced and the overall conversion from starch was performed by incubating amylopectin (waxy maize Cerestar we5676) with NM001 and samples were analyzed on an HPLC system to determine the distribution of -, ß- and
-CD expressed in percentages. The determination was carried out using 5% dry starch at 50°C and pH 5.5.
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Results and discussion |
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Interestingly, both mutants were found to have decreased activity towards maltotriose when the 191195 loop was deleted. NM001 and NM002 had only 13 and 4% activity, respectively, compared with Novamyl (see Table II). This might suggest that the loop facilitates small oligosaccharides to be in the optimum orientation for hydrolysis. However, further studies are needed to verify this.
These results demonstrate that it is possible to convert a five-domain -amylase showing up to 67% sequence similarity to CGTases into a CGTase-like enzyme capable of producing CD only by introducing two changes in the active site structure: placing the essential aromatic residue at the optimum position and deleting a loop that sterically would interfere with the cyclization reaction.
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Notes |
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Acknowledgments |
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References |
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Received February 18, 2000; accepted March 29, 2000.