1National Laboratory for Functional Food Carbohydrate, Center for Agricultural Bio-Materials and School of Agricultural Biotechnology and 5Department of Biological Resources and Materials Engineering, College of Agriculture and Life Sciences, Seoul National University, Seoul 151-742, 3Department of Food Science and Technology, Chungbuk National University, Cheongju 361-763, 4Department of Biology, University of Incheon, Incheon 402-749, Korea and 6Novo Nordisk A/S, Krogshojvej 36, 2880 Bagsvaerd, Denmark 2Present address: Department of Chemistry, University of British Columbia, Vancouver, BC V6T 1Z1, Canada
7 To whom correspondence should be addressed. e-mail: parkkh{at}plaza.snu.ac.kr
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Abstract |
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Keywords: antistaling enzyme/CGTase/error-prone PCR/random mutagenesis/retrogradation
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Introduction |
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-Amylases have an antistaling effect on bread because they retard retrogradation of the loaf (De Stefanis and Turner, 1981
; Sahlström and Bråthen, 1997
). Effective antistaling enzymes, such as amylases, hydrolyze amylose and amylopectin chains into smaller molecules, leading to less crystallization and limited crystal size (Boyle and Hebeda, 1990
). Low molecular weight dextrins produced by antistaling enzymes decrease the retrogradation rate of bread by inhibiting the interaction between starch and the continuous protein matrix (Martin and Hoseney, 1991
). In addition, shorter amylopectin chains form more complexes of amylopectin and lipid, consequently retarding retrogradation of bread (Kweon et al., 1994
). Leon et al. (2002
) investigated the effect of amylaselipase mixtures on retrogradation of bread. They reported that maltose and water-soluble dextrin (DP 46) were most effective in preserving crumb softness. However, the use of
-amylase has become limited despite its antistaling effect because too much of the enzyme can cause stickiness in bread. De Stefanis and Turner (1981
) explained that it is the production of branched maltooligosaccharides of DP 20100 by
-amylase that leads to gumminess in bread. To solve the problem, Carroll et al. (1987
) used a mixture of
-amylase and pullulanase in bread baking. Pullulanase hydrolyzes the branched maltooligosaccharides of DP 20100 produced by the
-amylase into smaller maltooligosaccharides, which improves the quality of baked goods (Kim et al., 2000
). Min et al. (1998
) employed an amylase that produces mainly maltooligosaccharides as an antistaling agent for bread baking. The maltotriose and maltotetraose produced by this enzyme seem to retard retrogradation of bread. Cyclodextrin glucanotransferases (CGTases; EC 2.4.1.19) produce various cyclodextrins (CDs) via intramolecular transglycosylation of maltooligosaccharides. CDs produced by the action of CGTase in bread could be a problem in countries where CDs are not allowed as food additives. Owing to this restriction, the development of mutant CGTases that produce large amounts of maltooligosaccharides but no CD has been attempted. The analysis of tertiary structure and site-directed mutagenesis of CGTase have been carried out in order to understand the structurefunction relationship (Fujiwara et al., 1992a
; Lawson et al., 1994
; Penninga et al., 1996
; Beier et al., 2000
; Shin et al., 2000
). The results revealed that substitution of the conserved hydrophobic aromatic residues involved in cyclization of CGTase leads to an increase in the saccharifying activity of the enzyme (Fujiwara et al., 1992b
; Leemhuis et al., 2002
). Recently, we reported the potential adaptation of CGTase from Bacillus stearothermophilus ET1 as an antistaling agent for bread by eliminating the cyclization activity of the enzyme via site-directed mutagenesis at F191 and F255 (Lee et al., 2002
). However, to improve the properties of enzymes, the site-directed mutagenesis limit needs to be overcome.
CGTase I-5 was isolated from an alkalophilic Bacillus sp. and had an optimal temperature and pH of 60°C and 6.0, respectively. It produced mainly ß-CD from starch. The purpose of this study was to engineer CGTase I-5 by random mutagenesis using error-prone polymerase chain reaction (PCR) such that the enzyme produced no CD or less CD than is produced by other antistaling enzymes. CGTase I-5 was used for this purpose, because substantial amounts of this enzyme can be purified easily via DNA manipulation of the structural gene. The enzymatic properties of the engineered enzymes were analyzed and the enzymes were also evaluated as antistaling agents for bread.
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Materials and methods |
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Escherichia coli MC1061 [F, araD139, recA13, (araABC-leu)7696, galU, galK,
lacX74, rpsL, thi, hsdR2, mcrB] was used as a host for DNA manipulation and transformation. Escherichia coli MC1061 was grown in LB medium [1% (w/v) Bacto-tryptone, 0.5% (w/v) yeast extract, 0.5% (w/v) NaCl]. Escherichia coli transformants were grown in LB medium supplemented with ampicillin (100 µg/ml). An alkalophilic Bacillus sp. strain I-5 was isolated from Korean soil in our laboratory and used as the source of the CGTase gene (cgtI5; GenBank accession No. AY478421) in this study.
Construction of pR2CGT
The cgtI5 gene was cloned in pBR322 and the resulting construction was designated as pCGTJ322. The 2.5 kb SalIHindIII fragment containing cgtI5 was subcloned into pR2BS, an expression vector, by placing the gene under the control of the amyR2 promoter on the vector to enhance the expression level of the gene product (Figure 1). The resulting recombinant DNA, pR2CGT, was transformed into E.coli MC1061 as described by Sambrook et al. (1989) and used for DNA manipulation and enzyme production.
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Random mutagenesis of cgtI5 was carried out by error-prone PCR (Song and Rhee, 2000). Two synthetic oligonucleotides, CGTI-5N (5'-GACTGGGTCGACCGATGAGGAGGTATA GTATG-3') and CGTI-5C (5'-CCTTAAGCTTGTGCGTG TGGAGGCAAGACCC-3'), were designed to have a SalI and a HindIII restriction site (underlined), respectively. PCR conditions for random mutagenesis were optimized such that mutation at a desirable level (three or four amino acid substitutions) and without bias in base substitution could be achieved. The PCR reaction mixture (100 µl) consisted of 10 mM TrisHCl (pH 8.3), 50 mM KCl, 7 mM MgCl2, 0.1 mM MnCl2, 0.2 mM dATP, 0.2 mM dGTP, 1 mM dCTP, 1 mM dTTP, 25 pmol of each oligonucleotide primer, 5 ng of template DNA (pR2CGT) and 5 U of Taq polymerase (Takara Shuzo, Japan). PCR was performed in an automatic thermal cycler (GeneAmp 9600, Perkin-Elmer, USA) for 30 cycles, each of which consisted of denaturation at 94°C for 1 min, annealing at 50°C for 1 min and extension at 72°C for 1 min. The resulting mutant PCR products were digested with SalI and HindIII restriction enzymes and then ligated into the corresponding restriction sites on pR2BS. The ligation mixture was transformed into E.coli MC1061 and spread on LB agar plates containing ampicillin (100 µg/ml). Three rounds of PCR were carried out using a mutant selected from the previous step as a template. The mutants were screened as described below.
Site-directed mutagenesis
To check the effects of the mutation at each of the three sites (M234T, F259I and V591A), the CGTase I-5 gene was mutated using a QuikChange site-directed mutagenesis kit (Stratagene, USA). The primers M234T-N (5'-GCGGTCAAGCATACG CCATTCGGCTGGCAG-3') and M234T-C (5'-CTGCCA GCCGAATGGCGTATGCTTGACCGC-3') were used to construct the M234T mutant. For the F259I mutation, the primers F259I-N (5'-AAGCCGGTCTTCACCATTGGCGAATGG TTCCTT-3') and F259I-C (5'-AAGGAACCATTCGCC AATGGTGAAGACC GGCTT-3') were used. Another set of primers, V591A-N (5'-GTCACCGTTCGGTTCGCAATC AACAATGCC-3') and V591A-C (5'-GGCATTGTTGAT TGCGAACCGAACGGTGAC-3'), was used to replace a valine at 591 with an alanine. The underlined nucleotides correspond to the desired substitution of amino acid residues. All mutations were confirmed by dideoxy chain-termination sequencing using an ABI377 PRISM DNA sequencer (Perkin-Elmer).
Screening of the mutants
Escherichia coli MC1061 transformants carrying mutant CGTases with enhanced hydrolyzing activity but no cyclization activity were screened by the iodine and phenolphthalein tests using LBS agar plates [LB broth containing 1% (w/v) soluble starch, 1.5% (w/v) agar and 100 µg/ml ampicillin], in terms of the size of clear zone and color change around colonies, respectively. After 10 h of incubation on LBS plates at 37°C, 10 ml of iodine solution (0.203 g I2, 5.2 g KI, distilled H2O to 100 ml) or 5 ml of phenolphthalein top agar [2% (w/v) Na2CO3, 0.6% (w/v) agar, 0.1% (w/v) K2HPO4, 0.02% (w/v) phenolphthalein, 0.02% (w/v) methyl orange] were overlaid on the plates (Park et al., 1989). About 1000 colonies from each round were selected primarily based on the size of clear zone formed around the colony in the iodine test. Among them, 100 colonies with a smaller orange-colored zone than that of wild-type in the phenolphthalein test were selected for further analyses. The activities of 100 positive colonies from each round were assayed using their crude cell extracts; 20 mutants from each round with appropriate activity were selected, purified and analyzed further. DNA sequencing of the genes for selected mutants was carried out as described above to confirm the introduced mutations.
Purification of CGTases
Escherichia coli MC1061 carrying the wild-type or mutant CGTase clone was cultured in 100 ml of LB broth at 37°C for 12 h in a 500 ml baffled flask with shaking at 200 r.p.m. The cells were then transferred to a 5 l jar fermenter (KFC, Korea) containing 3 l of LB broth and were cultured at 37°C for 24 h with sufficient aeration and agitation at 200 r.p.m. The cells were harvested by centrifugation at 4°C, resuspended in 300 ml of 50 mM sodium acetate buffer (pH 6.0) and sonicated in an ice-bath using a sonicator (VC-600, Sonics and Materials, USA) (output 45 minx3 times, 60% duty). The enzyme in the crude cell extract was purified using a ß-CD affinity column as described by Chung et al. (1998). Active fractions were collected, concentrated by ultrafiltration (Amicon, USA) and dialyzed against 50 mM sodium acetate buffer (pH 6.0). Protein concentration was determined by the Bradford method (Bradford, 1976
) with bovine serum albumin as the standard. The yield of the recombinant enzymes was about 5 mg/l of culture. The purity and molecular weight of the purified proteins were analyzed by 10% SDSPAGE.
Enzyme assay
The hydrolytic activity of CGTase was measured by the DNS method (Miller, 1959). A mixture of 0.25 ml of 1% (w/v) soluble starch (average molecular weight 1x105 g/mol) dissolved in 50 mM sodium acetate buffer (pH 6.0) and 0.2 ml of the same buffer was pre-warmed at 60°C for 5 min. CGTase (1 µg for mutant, 10 µg for wild-type) was added to the solution and the reaction was carried out at 60°C for 10 min. The reaction was stopped by the addition of 0.5 ml of DNS solution (10.6 g of 3,5-dinitrosalicylic acid, 19.8 g of NaOH, 306 g of sodium potassium tartrate, 1416 ml of distilled water, 7.6 ml of phenol and 8.3 g of sodium metabisulfite). The solution was boiled for 5 min and cooled immediately under running tap water. The absorbance of the solution was measured at 575 nm (Ultrospec III, Pharmacia, Sweden). The blank was run in the same way but without the enzyme. One unit (U) of the hydrolyzing activity was defined as the amount of enzyme that split 1 µmol equivalent of glycosidic bond in soluble starch per minute. The cyclization activity of CGTase was assayed by the phenolphthalein method (Kaneko et al., 1987
). The enzyme (2 µg for mutant, 0.2 µg for wild-type) was added to 1 ml of gelatinized soluble starch solution [4% (w/v) in 50 mM sodium acetate (pH 6.0)] and incubated at 60°C for 10 min. The reaction was terminated by the addition of 3.5 ml of 30 mM NaOH. Then 0.5 ml of phenolphthalein solution [0.02% (w/v) phenolphthalein in 5 mM Na2CO3] was added to the reaction mixture and it was left to stand at room temperature for 15 min. The concentration of ß-CD was determined by extrapolating the change in absorbance at 550 nm using a standard curve. One unit of cyclization activity was defined as the amount of enzyme producing 1 mg of ß-CD per minute.
Kinetic studies of wild-type and mutant CGTases
The kinetics of the wild-type and CGTase[318] for hydrolyzing soluble starch were measured by mixing appropriate concentrations of the enzyme (0.12 µg). The reducing sugar formed during the reaction was determined by the copperbicinchoninate method (Fox and Robyt, 1991). The kinetics of cyclization activity were measured by determining the ß-CD forming activity of the enzyme (0.33 µg) using the phenolphthalein method. The soluble starch concentrations used in the determination of kinetic parameters were 0.55 and 0.510 times the Km values for the hydrolyzing and the cyclization activity, respectively. The kinetic parameters were determined by fitting a LineweaverBurk plot with the SigmaPlot program (version 5.0; SPSS, Chicago, IL).
Structure modeling
The three-dimensional structure of CGTase[318] was modeled using the SWISS-MODEL version 3.51 program at the ExPASy server. The structures of various CGTases reported previously were used as modeling templates. The RCSB Protein Data Bank entries for these proteins are 1A47, 2DIJ and 1D7F. The modeled structure was visualized and analyzed using the Swiss-PdbViewer version 3.51. The figures were created with MOLSCRIPT version 2.1.
Bread baking
All four groups of bread loaves were baked with White Pan Bread Mix II (Cheil Jedang, Korea) as described previously (Lee et al., 2002). Bread loaves were baked in the following four set-ups: control using the bread mix alone; bread mix supplemented with 0.02% (w/w) Novamyl (
2000 U); bread mix supplemented with 0.012% (w/w) wild-type CGTase (120 U); and bread mix supplemented with 0.001% (w/w) CGTase[318] (120 U). Bread loaves were stored at 4°C in polyethylene bags. Novamyl 1500MG, an antistaling amylase, was kindly donated by Novozyme (Korea). The volume of each bread loaf was measured by the rice grain displacement procedure.
Analysis of CD in bread
Bread crumbs (10 g) were extracted with 100 ml of distilled water by stirring vigorously for 1 h at room temperature. The mixture was centrifuged at 7000 g for 10 min; the supernatant was diluted with distilled water (1:99, v/v) and filtered through a 0.45 µm filter (Gelman Sciences, USA). A 25 ml volume of the supernatant was dried using a freeze-dryer (Ilshin Engineering, Korea) and then dissolved in 5 ml of water. The concentration of CD in the bread was analyzed using the phenolphthalein method as described above. HPLC was also carried out to measure the CD present in the loaves. The HPLC system used in this study (SLC-100, Samsung Electronics, Korea) was equipped with a LiChrosorb NH2 column (10 µm, 4x250 mm) (Merck, Germany) and a differential refractometer (400R, Waters, USA). The samples were eluted at a flow rate of 1.0 ml/min using a mixture of acetonitrile and water (65:35, v/v).
Differential scanning calorimetry (DSC)
The retrogradation rate of each loaf was determined through DSC using a DSC 120 instrument (Seiko, Japan) at days 3 and 7 during the storage of bread at 4°C (Kweon et al., 1994). DSC was calibrated with indium (156.6°C, 28.591 J/g) and tin (232.2°C, 60.62 J/g). Distilled water was used as a reference. Bread samples (10 mg) were weighed and hermetically sealed in aluminum pans. The pans were then heated from 20 to 130°C at a rate of 5°C/min. The degree of retrogradation was expressed as the enthalpy calculated from the area of the endothermic peak between 40 and 80°C.
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Results and discussion |
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The 2.5 kb SalIHindIII fragment of pR2CGT carrying the cgtI5 gene from alkalophilic Bacillus sp. strain I-5 was isolated and used as a template for the construction of mutants by error-prone PCR. Six mutants with the most appropriate phenotypes were finally selected and their genetic and enzymatic characteristics were analyzed as summarized in Table I. CGTase[17] was used as the template for the second round of PCR and CGTase[25], [228], [232] and [240] for the third round. Nucleotide sequencing analysis of the mutations introduced into the enzyme indicated that mild random mutation had accumulated in the mutant obtained from the final round during the sequential error-prone PCR processes. Among the mutants obtained from the final round, CGTase[318] showed the highest hydrolyzing and the lowest cyclization activities as compared with those of wild-type CGTase I-5.
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The wild-type and mutant CGTase I-5 enzymes were purified using a ß-CD column and the purity of the enzymes was verified by SDSPAGE (data not shown). The optimal temperatures for the hydrolyzing activity of wild-type CGTase and CGTase[318] were 60 and 50°C, respectively (Table II). The optimal pH for the hydrolyzing activity of wild-type and mutant CGTase I-5 was 6.0, even though the wild-type was more stable than the mutant enzyme at acidic (pH 45) and alkaline (pH 810) pHs (data not shown). The mutant enzyme was as stable as the wild-type at the optimal pH.
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The production of CDs in a bread loaf is undesirable when CGTase is added to the bread mix. HPLC analysis of the bread loaves revealed that -, ß- and
-CD and G3 were produced in the bread loaf treated with wild-type CGTase I-5 (Figure 4B), whereas no CD was detected in the bread loaf treated with CGTase[318] (Figure 4C). CGTase I-5 produced 2.13 mg of ß-CD per 1 g of bread crumbs on its addition to bread mix. The fact that CD was not detected in the bread loaf treated with CGTase[318] despite its residual cyclization activity (Table II) might have been due to the reaction conditions and structural differences between soluble starch and the starch in the bread mix. Maltose was produced in a larger amount and G3 in a smaller amount in the bread loaf treated with CGTase[318] (Figure 4C), as compared with levels in the loaf treated with wild-type CGTase I-5 (Figure 4B). Maltooligosaccharides larger than maltotriose were not detected in the bread loaf treated with CGTase[318], indicating very limited disproportionation activity by the mutant enzyme. The bread loaf treated with CGTase ET1 and CGTase ET1-F255I (corresponding to F259 in CGTase I-5) contained significant amounts of maltooligosaccharides with DP of G4G5 (Lee et al., 2002
).
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Generally, the first endothermic peak of DSC at 4080°C is considered to be the staling endotherm that results from the melting of crystalline amylopectin when starch solution or starchy food is heated (Russell, 1983
; Kweon et al., 1994
). The degree of bread staling during storage can be monitored using DSC as the increase of the first endothermic peak area, because amylopectin is the main component responsible for retrogradation of starch (Kweon et al., 1994
).
The addition of an antistaling enzyme, Novamyl, CGTase I-5 or CGTase[318], significantly retarded retrogradation of the bread loaf (Figure 6). Throughout the storage period of 7 days, the retrogradation rates of the experimental loaves were much lower than that of the control loaf to which no enzyme was added. Little difference was found among the retrogradation rates of the bread loaves supplemented with the three enzymes, although the lowest retrogradation rates were seen in the loaf treated with the mutant enzyme CGTase[318]. Therefore, CGTase[318] is probably as effective an antistaling agent as Novamyl. The production of maltooligosaccharides and the significant reduction in the molecular weights of amylose and amylopectin are likely to be the main causes of the retarded retrogradation in the bread loaf treated with CGTase. However, no significant increase in maltooligosaccharides was observed in the bread loaves treated with CGTase I-5 or CGTase[318], indicating that factors other than the molecular weight of amylopectin might be related to the retarded retrogradation of the bread loaf.
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Acknowledgements |
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Received November 19, 2003; revised April 1, 2004; accepted April 5, 2004 Edited by Stephen Withers
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