Conversion of the maltogenic {alpha}-amylase Novamyl into a CGTase

Lars Beier, Allan Svendsen1, Carsten Andersen, Torben P. Frandsen, Torben V. Borchert and Joel R. Cherry

Novo Nordisk A/S, Novo Allé, DK-2800 Bagsvaerd, Denmark


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Novamyl is a thermostable five-domain maltogenic {alpha}-amylase that shows sequence and structural homology with the cyclodextrin glycosyltransferases (CGTases). Comparing X-ray crystal structures of Novamyl and CGTases, two major differences in the active site cleft were observed: Novamyl contains a loop insertion consisting of five residues (residues 191–195) and the location of an aromatic residue known to be essential to obtain an efficient cyclization reaction. To convert Novamyl into a cyclodextrin (CD)-producing enzyme, the loop was deleted and two substitutions, F188L and T189Y, were introduced. Unlike the parent Novamyl, the obtained variant is able to produce ß-CD and showed an overall conversion of starch to CD of 9%, compared with CGTases which are able to convert up to 40%. The lower conversion compared with the CGTase is probably due to additional differences in the active site cleft and in the starch-binding E domain. A variant with only the five-residue loop deleted was not able to form ß-CD.

Keywords: amylase/CGTase/product specificity/site-directed mutagenesis/structural homology


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Several enzymes are capable of catalyzing the hydrolysis of {alpha}-1,4 linkages in starch polymers, as starch and similar polymers are almost universally used as energy sources. However, the product specificity of the almost 80 families in the glycoside hydrolase classification (Henrissat, 1991Go; Henrissat and Bairoch, 1993Go; Coutinho and Henrissat, 1999Go) is different, e.g. {alpha}-amylases produce oligosaccharides with different degree of polymerization (DP) while cyclodextrin glycosyltransferases (CGTases) produce both cyclodextrins (CDs) and oligosaccharides.

The {alpha}-amylases (EC 3.2.1.1) are a family of endo-amylases that catalyze the hydrolysis of {alpha}-1,4-glycosidic linkages in polymers of {alpha}-D-glucose and are thus needed for most organisms. The {alpha}-amylases are widely distributed in micro-organisms, plants and higher organisms and show varying cleavage patterns (Svensson, 1994Go). They are used in a wide variety of industrial applications such as in liquefaction, brewing and detergents.

{alpha}-Amylases from different organisms exhibit similar three-dimensional structures, despite great differences in primary structure. As most starch-hydrolyzing and related enzymes, the {alpha}-amylase family has a (ß/{alpha})8- or TIM-barrel catalytic domain. Thus, the {alpha}-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 {alpha}-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 {alpha}-helices. Domain B is a small folding module, inserted between ß-strand 3 and {alpha}-helix 3 in the TIM-barrel. After the (ß/{alpha})8-barrel and at the opposite side of the A domain with respect to the B domain the {alpha}-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, 1994Go). Also common in {alpha}-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 ({alpha}-, ß- and {gamma}-CDs, respectively) glucose units bound by {alpha}-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 {alpha}-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 {alpha}-, ß- and {gamma}-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., 1998Go). 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 {alpha}-amylase originally cloned from a strain of Bacillus during a systematic screening programme for {alpha}-amylase producing micro-organisms. Novamyl received its description as a maltogenic {alpha}-amylase because prolonged hydrolysis of starch with Novamyl results primarily in the production of maltose (Outtrup and Norman, 1984Go; Diderichsen and Christiansen, 1988Go). Novamyl was subsequently shown also to possess endo-amylase activity (Christophersen et al., 1998Go) 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, 1991Go); see Table IGo 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., 1999Go). This distinguishes Novamyl from the other {alpha}-amylases within glycoside hydrolase family 13 that normally possess the conventional three-domain {alpha}-amylase structure. Novamyl's catalytic mechanism is also expected to be similar to the catalytic mechanism known for CGTases and {alpha}-amylases, since the active site conformation in Novamyl is very much like the active site conformations in {alpha}-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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Table I. Similarities of Novamyl to other amylolytic enzymes: SWISSPROT sequence name, PDB file name, EC number and name, family in the glycosyl hydrolase classification (Henrissat, 1991Go) and percentage similarity to Novamyl
 
In the literature there are only a few examples of changing the catalytic mechanism and/or product specificity. Substitution of the aromatic residue essential to cyclization in CGTases increases the saccharifying activity (Penninga et al., 1995Go), conversion of a galactosidase to a fucosidase done by random mutagenesis (Zhang et al., 1997Go), product change of lysozyme from {alpha}- to ß-anomer (Kuroki et al., 1995Go) and conversion of a neopullulanase-{alpha}-amylase to an amylopullulanase-type enzyme (Ibuka et al., 1998Go) are some examples of similar conversions/alterations.

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., 1995Go; Wind et al., 1998Go). In contrast, {alpha}-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.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Site-directed mutagenesis

Site-directed mutagenesis was carried out using sequence overlap extension PCR (Higuchi et al., 1988Go) 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 {alpha}-cyclodextrin coupled to activated agarose. The centrifuged culture supernatant was applied to an {alpha}-cyclodextrin–agarose 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) {alpha}-cyclodextrin. The variants were homogeneous as estimated using SDS–PAGE and stained using Coomassie Brilliant Blue. Protein concentrations were determined spectrophotometrically at 280 nm using {varepsilon} = 132 710 M–1 cm–1 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 {Delta}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 (50–95°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, 1982Go).

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 {alpha}-, ß- and {gamma}-CD expressed in percentages. The determination was carried out using 5% dry starch at 50°C and pH 5.5.


    Results and discussion
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
In order to convert Novamyl into a CGTase-like enzyme, sequence alignments of Novamyl with the structurally homologous CGTases were performed followed by superimposing the X-ray structures of Novamyl (Dauter et al., 1999Go) and B.circulans strain 251 CGTase (PDB entry 1CDG) (Lawson et al., 1994Go) to identify differences in the active site. Figure 1Go shows the alignment of Novamyl and CGTases around position 195. Figure 2Go shows the superimposition of the selected Novamyl and CGTase structures with only the most relevant residues displayed. Comparing these structures and the alignment regarding cyclization reaction, it was obvious that the aromatic residue had to be placed at the 189 position in Novamyl. In addition, the loop consisting of residues 191–195 close to the position of the essential aromatic residue needed to be deleted to facilitate the cyclization reaction, as the loop is likely to hinder sterically the CD product formation. Hence the F188L and T189Y substitutions were made to introduce the aromatic residue at the position known to be essential to an efficient cyclization reaction and the five amino acid loop consisting of residues 191–195 located in the active site next to the location of the aromatic residue deleted. In Table IIGo the mutant enzymes are listed; NM001 contains the alterations described above, whereas NM002 only had the loop (residues 191–195) deleted. The temperature of denaturation (Table IIGo) shows that although the two variants are destabilized compared with Novamyl, they retain stability at the relevant assay temperature (60°C).



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Fig. 1. Alignment of Novamyl and CGTase sequences in the region around the aromatic residue essential to cyclization. B. circ 251, B.circulans strain 251 – cdgu_bacci (SWISSPROT sequence name); T. ther, T.thermosulfuigenes EM1 – amy_thetu; B. lich, B.licheniformis – cdgt_bacst; B. spec 1011, Bacillus sp. 1011 – cdgt_bacs0; B. spec 38–2, Bacillus sp. 38–2 – cdgt_bacs3; B. ohb, B.ohbensis – cdgt_bacoh.

 


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Fig. 2. Superimposed structures of Novamyl and B.circulans strain 251 CGTase structures. Only the three catalytic residues and the neighborhood of the aromatic 195 position are shown in order of clarity. The three catalytic residues of Novamyl D228, E258 and D329 are colored yellow, the Novamyl residues 188–196 magenta and B.circulans strain 251 CGTase residues 194–197 cyan.

 

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Table II. List of Novamyl and variants thereof, melting points, amylase activities and activities towards maltotriose (MANU)
 
Novamyl and the two variants were tested for their ability to produce ß-CD. As shown in Figure 3Go, it is clear that only NM001 possesses the ability to produce ß-CD. Thus, the total CD distribution and the total conversion of starch into CDs were measured for the CD-producing variant NM001 to determine how the constructed CGTase performed. Figure 4Go shows the distribution of the CDs produced, with ß-CD as the most dominant one and, hence, NM001 can be classified as a ß-CGTase. The most efficient industrial CGTases are able to convert up to 40% of the starch into CDs, while the converted {alpha}-amylase NM001 converted 9% of the starch into CDs. The lower conversion efficiency of NM001 compared with the best CGTases is likely to be due to additional differences in loops in other parts of the active site cleft and in the E domain. Dauter et al. (1999) reported a number of significant differences between Novamyl and B.circulans strain 251 CGTase. In the active site cleft, in addition to those already mentioned, there is an insertion of two residues in the loop consisting of residues 262–267 in Novamyl that is not present in CGTase, together with slight changes in the adjacent loop orientation; this provides a different environment at the reducing end of the active site cleft that makes it more amenable to extended linear polymers. This is in contrast to the CGTases in which the cleft is closed by the loop from residues 260 to 264 (B.circulans numbering). Furthermore, a loop consisting of residues 615–622 in the E-domain of Novamyl provides extensive additional binding between the E- and A-domains and between the E- and D-domains. Finally, in the X-ray crystal structure Novamyl binds one maltose molecule in the E-domain, while the B.circulans CGTase X-ray crystal structure displays two molecules of maltodextrins in this domain. Hence it should be possible to optimize the CD production of NM001 even further, as a number of differences still exist between NM001 and CGTases, although those most important to the cyclization reaction have been minimized.



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Fig. 3. Accumulated ß-CD formation of wild-type Novamyl, NM001 and NM002.

 


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Fig. 4. Distribution of CD production of NM001.

 
The {alpha}-amylase activities of the three enzymes were also measured. NM001 had only 34% amylase activity compared with Novamyl when measured on long-chain starch molecules (see Table IIGo). This is as expected, as CGTases possess lower saccharifying activity than amylases, hence making Novamyl more CGTase-like would be likely to decrease the {alpha}-amylase hydrolyzing activity. NM002 displayed an almost 4-fold increase in amylase activity compared with Novamyl (see Table IIGo). This is not surprising, either, as the active site environment in NM002 is more similar to that in other {alpha}-amylases, since no three-domain {alpha}-amylases contain a loop like the 191–195 loop in Novamyl. The decreased long chain {alpha}-amylase activity of NM001 relative to both Novamyl and NM002 must be attributed to the F188L and T189Y mutations reducing the efficiency of hydrolysis.

Interestingly, both mutants were found to have decreased activity towards maltotriose when the 191–195 loop was deleted. NM001 and NM002 had only 13 and 4% activity, respectively, compared with Novamyl (see Table IIGo). 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 {alpha}-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.


    Notes
 
1 To whom correspondence should be addressed Back


    Acknowledgments
 
The authors thank Barrie E.Norman for carrying out the experiment showing the overall cyclodextrin distribution of NM001.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Christophersen,C., Otzen,D.E., Norman,B.E., Christensen,S. and Schäfer,T. (1998) Starch/Stärke, 50, 39–45.

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Received February 18, 2000; accepted March 29, 2000.