1Department of Food Science and Human Nutrition and 2Department of Chemical Engineering, 2114 Sweeney Hall, Iowa State University, Ames, IA 50011-2230, USA
3 To whom correspondence should be addressed. e-mail: reilly{at}iastate.edu
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Abstract |
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Keywords: Aspergillus niger/cyclomaltodextrin glucanotransferase/glucoamylase/Paenibacillus macerans/shuffling/starch-binding domain
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Introduction |
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All GAs have fairly homologous catalytic domains (Coutinho and Reilly, 1997). Their tertiary structures are similar, consisting of an (
,
)6 barrel (Aleshin et al., 1992
) with a peripheral 13th
-helix present in fungal forms. The active site is located in a well (Aleshin et al., 1992
). GAs from filamentous fungi have linkers of varying primary sequences, decreasing in length from the
40 residues found in GAs from most Aspergillus species to
20 residues in Corticium rolfsii GA (Coutinho and Reilly, 1997
).
SBDs are found in carbohydrate-binding module Families 20, 21, 25 and 26 (Coutinho and Henrissat, 1999a,b). Family 20 members include all SBDs located at the C-termini of GAs, along with SBDs from cyclomaltodextrin glucanotransferases (EC 2.4.1.19, CGTases, Domain E),
-amylases (EC 3.2.1.1), ß-amylases (EC 3.2.1.2), maltotetraose-forming exo-amylases (EC 3.2.1.60), maltogenic
-amylases (EC 3.2.1.133) and other hydrolases. GA and CGTase SBDs are quite homologous (Figure 1), with those from
-amylases intermediate to them, suggesting that perhaps they could be interchanged.
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The functional domain boundaries of A.niger GA are somewhat different than those suggested by its tertiary structure. For instance, the catalytic domain requires a portion of its glycosylated region to achieve full secretion and thermostability. In a C-terminal truncation study of GAI, yeast expressing A.niger GA residues 1460 had a little activity on a starch-clearing plate but no measurable secreted GA activity. Forms with residues 1482 and 1496 were fully active but were slightly lower in high-temperature thermostability than wild-type GAI and GAII (Evans et al., 1990). Furthermore, when residues 466512, 485512 and 466483 were deleted, the GA resulting from the first deletion was almost undetectable, while the GAs resulting from the second and third were expressed extracellularly to
60 and
20% of the activities of wild-type GAI and GAII, which were essentially equal (Libby et al., 1994
). Activities of these latter two forms on soluble starch were about the same as those of GAI and GAII, while those on insoluble starch were similar to GAI. However, their thermostabilities were somewhat lower.
The optimal functional binding domains of fusions of the A.niger GA SBD to ß-galactosidase expressed in Escherichia coli also contain some of the glycosylated linker region. The SBD possessed the greatest binding capacity when the last 11 amino acid residues of the glycosylated region were included. A further addition of 14 residues or the removal of 16 residues causes decreased binding capacity (Chen et al., 1991). This suggests that a 119-residue SBD is a better functional domain than the 108-residue SBD defined by its tertiary structure. Therefore, the boundaries of the functional domains used in this research are residues 1482 for the catalytic domain and residues 497616 for the SBD. The role of the remaining 14-residue glycosylated region (residues 483496) is unclear, except to separate the catalytic and SBDs.
Given that different GAs have SBDs at either their C- and N-termini, the possibility exists that A.niger GA may function with its SBD at its N-terminus. In addition, considering that many GAs and CGTases have similar C-terminal SBDs, it is possible that substitution of a CGTase SBD for the A.niger GA SBD may yield a functional enzyme. The goals of this project therefore were to determine whether A.niger GA with its SBD at its N-terminus rather than its C-terminus and whether A.niger GA with a C-terminal substitution of the Paenibacillus (formerly Bacillus) macerans CGTase SBD can still hydrolyze soluble starch and bind and hydrolyze insoluble starch. Although there is no known tertiary structure for the latter, its primary sequence is so similar to the SBD of Bacillus circulans CGTase (Figure 1) that its tertiary structure should be almost identical to that of the latter shown in Figure 2.
To achieve these goals, genetic engineering was used to reverse the domain order of GAI and GAII to give RGAI and RGAII, respectively. In addition, a reduced-linker version of RGAI (RGAIL) was made. Finally, a GAI with a CGTase SBD (GAE) was constructed. All four rearranged GAs and the wild-type forms, GAI and GAII, were expressed in Saccharomyces cerevisiae and purified. The abilities of the purified enzymes to bind insoluble starch and to hydrolyze soluble and insoluble starch were then compared.
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Materials and methods |
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Construction and sequencing of GA variants were carried out in plasmid pBS+ from Stratagene. Plasmid pDS4, expressing a truncated GA designated as GACDO (Suominen et al., 1993), and the yeast expression vector YEpPM18 (Cole et al., 1988
), containing the GAI cDNA from A.awamori, a gift from Cetus, were the sources of the GA coding and expression vector sequences. The plasmid pRE513 (Evans et al., 1990
) was used to express GAII. The plasmid pLCGT1 (Lee and Tao, 1994
), containing the CGTase cDNA from P.macerans, was obtained from Dr Zivko Nikolov.
All cloning was done in E.coli TG1 [supE, hsd5, thi
(lac-proAB)F'(traD36, proAB+, lacIq, lacZ,
M15)]. GAs were expressed by S.cerevisiae strain C468 (
, leu2-3, leu2-112, his3-11, his3-15, mal) (Innis et al., 1985
), also from Cetus.
Escherichia coli strains were grown in LB + Amp medium (10 g/l Difco Bacto tryptone, 5 g/l Difco Bacto yeast extract, 10 g/l NaCl, pH adjusted to 7.5 with NaOH, with or without 1.5% Difco agar and 60 mg/l ampicillin). Yeast strains were grown in SD + His medium (1.7 g/l Difco yeast nitrogen base without amino acids, 5 g/l ammonium sulfate, 2% glucose, 100 mg/l L-histidine, with or without 1.5% Difco agar).
Reagents, enzymes and oligonucleotides
Reagents were from Sigma or Fisher. T4 DNA ligase, restriction enzymes and buffers were purchased from Boehringer Mannheim Biochemicals, Promega Biotech, Stratagene or New England Biolabs. HKTM phosphatase from Epicentre Technologies was used for dephosphorylation. Acarbose was donated by Miles Laboratories. Maltose, glucose oxidase and peroxidase were from Sigma. The Iowa State Nucleic Acid Facility supplied the oligonucleotides used for adaptors and sequencing primers.
Construction of plasmids
A cloning scheme for the three domain-reversed GAs consisting of 11 plasmids was necessary to achieve the desired constructs. It was designed to take advantage of existing restriction sites in the GA gene. Adaptors were synthesized to code for residues 483496 and 606616. They were also used to add restriction sites for subsequent plasmid construction. Unique restriction sites to be added were chosen for compatibility so that one type of sticky end could be re-annealed to another. The first eight plasmids were constructed in the pBS+ cloning vector. The last three were constructed in the expression vector portion of YEpPM18 while maintaining the pre-pro leader sequence cleavage sites and the terminator.
Constructs were produced in four stages. The first of these was to join adaptors to GA gene regions to obtain plasmids pCG1 and pCG2. The next stage involved combining coding sequences from pCG1 and pCG2 with another adaptor, resulting in pCG3, the progenitor to pRGAI. Deletions were then made to produce pCG4 and pCG5, the progenitors of pRGAIL and pRGAII, respectively. In the final stage, the coding sequences were completed by adding the C-terminal half of the catalytic domain to pCG3, pCG4 and pCG5 and placing the coding sequences in the expression vector. The specific details follow and are also shown in Figure 3.
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pCG2A. This plasmid was the end-product of a spontaneous deletion in pCG2. Inverted repeat sequences of BamHI, XbaI and BamHI at the vector/adaptor joint were most likely responsible for a homologous recombination event.
pCG2B. This was constructed to replace the deleted section. pCG2A was restricted with EcoRI and SpeI and Adaptor 3 (Figure 4) was inserted. pCG2B then contained a synthetic glycosylated linker region surrounded by engineered XbaI and SpeI sites.
pCG2C. A 5.5 kb EcoRI fragment derived from YEpPM18 was cloned into EcoRI-restricted and dephosphorylated pCG2B. This step was necessary to achieve complete restriction of the plasmid with EcoRI and XbaI for pCG3 construction.
pCG3. The EcoRI/SalI SBD coding region of pCG1 and a SalI/XbaI-synthesized adaptor (Adaptor 4) (Figure 4) were annealed to EcoRI- and XbaI-restricted pCG2C. This clone yielded a completed SBD upstream of the N-terminal half of the catalytic domain with the linker region between them. This is the parent of pRGAI.
pCG4.
The progenitor of pRGAIL was constructed by restricting pCG3 with XbaI and SpeI to remove the linker region. The sticky ends were re-annealed, destroying both restriction sites.
pCG5. The progenitor of pRGAII was constructed by restricting pCG3 with NheI and XbaI to remove the SBD. The NheI and XbaI sites were destroyed upon ligation of their cohesive ends.
pRGAI, pRGAIL and pRGAII.
The final expression vectors for the domain-reordered GAs were all constructed by three-part directional cloning. Sequences upstream of the catalytic domain PstI site were joined to the C-terminal half of the catalytic domain and placed in the expression vector. This was accomplished by annealing the BssHII/PstI fragments from pCG3, pCG4 and pCG5 to the PstI/HindIII fragment of pDS4 and the BssHII/HindIII vector portion of YEpPM18.
GAE. The fusion gene GAE was constructed by using a fragment containing the GAI cDNA, cleaving it to obtain the GA gene holding residues 1514 and ligating it to that part of the CGTase gene containing SBD residues 579687. Specifically, the fusion gene of GAE was constructed by using modified pGEM-7Z(+) (the BstXI site had been destroyed) as a cloning vector. The small XhoI/EcoRI fragment, which contained the GAI cDNA, of YEpPM18 was inserted into the modified pGEM-7Z(+) to construct pGEM7m-GA. The big BstXI/EcoRI fragment containing the GA gene from amino acid residues 1514 of pGEM7m-GA, the small HindIII/EcoRI fragment (containing the CGT gene from amino acid residues 579687) of pLCGT1 and a single-strand adaptor (5'-AGCTGGCG-3') were ligated to construct pGEM7m-GAE. Then, the small XhoI/HindIII fragment of pGEMm-GAE, containing the fusion gene of GAE, was ligated to the big XhoI/HindIII fragment of YEpPM18 to reconstruct a yeast expression vector YEpPM-GAE.
Qiagen columns from Diagen were used for purification of plasmid DNA for fragment preparation. Electrophoresed DNA fragments were extracted from agarose gels with the GeneClean product from Bio 101, Inc. Other cloning work was done using standard molecular biology protocols (Sambrook et al., 1989).
Sequencing
Adaptors were verified by sequencing through the regions containing them. All complete gene constructs were verified by restriction analysis and DNA sequencing, the latter being done by the Iowa State Nucleic Acid Facility. The secreted sequences of the four constructed GAs are as follows: RGAI, Ala(497616)SerArg(483496)ThrSerIleGluGlyArg(1484)MetAlaTyr; RGAIL, Ala(497616)SerSerIleGluGlyArg(1484)MetAlaTyr; RGAII, AlaAlaArg(483496)ThrSerIleGluGlyArg(1484)MetAlaTyr; GAE, (1514)(CGTase 579687).
Therefore, using domains defined by function, RGAI consists of the SBD followed by the linker and the catalytic domain, while RGAIL is the SBD followed by the catalytic domain, RGAII is the linker followed by the catalytic domain and GAE is GAII followed by the CGTase SBD. In terms of tertiary structure, RGAI consists of the last part of the linker followed by the SBD, then two short adaptors around the middle of the linker and, finally, the catalytic domain with the first part of the linker. RGAI
L is the same less one of the adaptors and the middle part of the linker. RGAII is the middle of the linker, followed by an adaptor, the catalytic domain and the first part of the linker. GAE is GAII followed by domain E of CGTase.
Transformation of yeast
Saccharomyces cerevisiae strain C468 was transformed with the constructed expression plasmids pRGAI, pRGAIL and pRGAII, as well as with YEpPM18 (wild-type GAI), pRE513 (wild-type GAII) and pAC1 (negative control) by the lithium acetate method (Ito et al., 1983
). It was transformed with YEpPM-GAE by electroporation. Cells containing expression plasmids were then selected by leucine prototrophy, which was conferred by the expression vector.
Starch-clearing plate assay
A starch-clearing plate assay verified GA activity in the resulting yeast strains. Aliquots of 5 ml of selective yeast medium (SD + His minimal medium) were inoculated with the appropriate yeast strain and grown at 30°C with shaking. At the end of the exponential phase, when OD600 was 0.5, equivalent numbers of cells were plated on SD + His medium containing 1% (w/v) soluble starch. Plates were incubated at 30°C for 5 days and then at 50°C for 5 h. Plates were stained for 1 min with iodine vapors. Clear halos develop around colonies producing active GA.
GA production and purification
GAs were secreted from the yeast strains in shake-flask fermentations. Six liters of selective yeast medium containing 2% glucose were inoculated with the appropriate yeast strain and shaken at 30°C and 2.1 g for 5 days. Cells were removed from the culture by centrifugation. Medium containing GA was concentrated 20-fold in an Amicon ultrafiltration system. Concentrate was then diafiltered with three times its volume of wash/diafiltration buffer (0.1 M NaOAc, pH 4.3/1.5 M NaCl) and then reconcentrated.
GA in the diafiltered concentrate (120 ml) was purified by acarbose affinity chromatography (Chen et al., 1994
). The loaded column was washed with wash/diafiltration buffer and eluted with 1.7 M TrisHCl, pH 7.6. Column loading, washing and elution were monitored with a UV detector. Eluted GA was dialyzed extensively with water and the purified GA was lyophilized for storage. The entire harvesting, purification and lyophilization process was completed in under 32 h, largely at 4°C.
SDSPAGE of GA
GAs were electrophoresed under standard SDSPAGE conditions on a Bio-Rad 415% TrisHCl/2.6% cross-linker linear gradient gel to determine purity and apparent molecular mass. Each GA-containing lane was loaded with 7 µg of protein sample, electrophoresed under the suggested conditions and stained with Coomassie Blue.
GA concentration
Concentrations of rehydrated GAs were determined by one of two methods. GAs used for SDSPAGE and soluble and insoluble starch hydrolysis assays were quantified with the Pierce bicinchoninic acid kit. GA concentrations in solutions used in the insoluble starch-binding assays were determined with the Bio-Rad Bradford assay kit. Bovine serum albumin was the reference standard in both methods.
Soluble starch hydrolysis
Soluble starch substrate was prepared daily by boiling Fisher soluble starch (1.8%, w/v) in 50 mM NaOAc, pH 4.4, the optimal for GA activity. For each reaction, 1.0 ml of starch substrate was equilibrated in a 35°C water bath for 5 min, a sufficiently low temperature that no enzyme activity was lost during the assay. Assays were initiated with 0.22 µg of enzyme in a total volume of 200 µl. At times ranging from 5 to 90 min, 100 µl samples were removed and quenched with 40 µl of 4 M TrisHCl, pH 7.0.
Glucose concentrations were determined by the Sigma glucose oxidase/peroxidase/o-dianisidine assay kit. Activities were determined from slopes of glucose concentration versus time plots. Specific activities were then calculated on a IU/mg and kcat basis, where 1 IU is defined as the enzyme necessary to produce 1 µmol/min of glucose at 35°C in a 1.5% soluble starch reaction mixture. Protein molecular masses were calculated from the number of amino acid residues.
Insoluble starch hydrolysis
Insoluble starch substrate was prepared by suspending 1.8% (w/v) Sigma corn starch in 50 mM NaOAc, pH 4.4, at 35°C. Reactions were initiated with 4 µg of GA solution plus reaction buffer in 200 µl. Reaction mixture samples (150 µl) were taken at 10 min intervals for 1 h and quenched with 60 µl of 4 M TrisHCl, pH 7.0, before being microfuged to pellet the unreacted starch. A 140 µl portion of each microfuged sample was removed and analyzed for glucose content. The activity of GA on insoluble starch was determined from the plot of glucose released over time and recorded on a IU/mg and kcat basis as described above.
Insoluble starch binding
Insoluble starch substrate was prepared by washing Sigma corn starch twice with water. The water was drawn off by suction and the starch was air-dried for several days, with occasional repowdering with a mortar and pestle. A 0.2 g/ml stock mixture of the washed starch in 50 mM NaOAc, pH 4.4, was made fresh for each set of assays and chilled on ice. Chilled GA and buffer were added to aliquots of the stock mixture, resulting in 030 µg of GA in a 0.1 g/ml starch mixture. Reaction tubes were shaken at 5°C for 30 min. The starch was pelleted by centrifugation and the supernatant was assayed for mass of unbound GA. Equilibrium binding constants (Kad) were calculated from the linear slopes of plots of nanomoles of bound GA per gram of starch versus nanomoles of unbound GA per liter of solution.
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Results and discussion |
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Soluble starch hydrolysis was initially characterized by starch-clearing plate assays (Figure 5). The negative control did not show GA activity. GAI, RGAI and RGAIL produced similarly sized cleared zones. The cleared zones from GAII and RGAII were larger than those from the other GAs, perhaps enhanced by differences in diffusion due to enzyme size. GAE gave a smaller cleared zone.
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Purified GAs were subjected to SDSPAGE (Figure 6). The apparent molecular mass for GAI, RGAI and RGAIL is 110 kDa. GAII has an apparent molecular mass of
90 kDa, with that of RGAII being a little lower. GAE had two bands, one slightly greater than 110 kDa and the other
90 kDa, suggesting that in some molecules the SBD was cleaved during processing. These molecular masses are much higher than the 81.7 and 69.2 kDa found by MALDI-TOF for glycosylated GAI and GAII expressed by S.cerevisiae C468 containing plasmid YEpPM18 (Khan et al., 2000
). GAI and GAII produced from pGAC9, a vector with one-fifth the expression ability of the YEpPM18-based vector, gave apparent molecular masses of 97 and 87 kDa, respectively, measured by SDSPAGE (Innis et al., 1985
; Evans et al., 1990
). This suggests that SDSPAGE yields erroneously high molecular masses for GA forms. YEpPM18 produces GAI of higher molecular mass than pGAC9 (H.-M.Chen, personal communication), probably caused by more glycosylation (Innis et al., 1985
).
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GA-specific activities on soluble starch substrate are compared in Table I. GAII and RGAII have similar activities based on protein mass, somewhat higher than that of GAI. RGAI, RGAIL and GAE have similar activities, slightly less than that of GAI. These results generally agree with the results from the starch-clearing assay. When specific activities are converted to kcat values, differences between enzyme forms become smaller, these values ranging from 13.6 to 22.7 s1.
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Insoluble starch hydrolysis
GAI, RGAI and RGAIL have high specific activities on insoluble starch (Table I). GAII, RGAII and GAE specific activities are much lower. GAs lacking SBDs have 15% or less the activity on insoluble starch as those containing SBDs, as shown earlier (Svensson et al., 1982
). GAE activity on insoluble starch is much closer to the activities of GAII and RGAII, which lack SBDs, than it is to those GAs with SBDs. The already mentioned loss of an SBD in some GAE molecules (Figure 4) may explain some but not all of its low activity. The very low kcat values of three GA forms indicate that activity on any soluble starch associated with the insoluble starch substrate was close to negligible.
It is apparent that the GA domains can cooperate to hydrolyze insoluble starch whether the SBD is N- or C-terminal to the catalytic domain; however, the former is less effective than the latter. The positioning of the SBD relative to the catalytic domain affects insoluble starch hydrolysis much more than it affects soluble starch hydrolysis. GAI has 45 and 80% greater activity than RGAIL and RGAI, respectively, on insoluble starch, whereas the differences for soluble starch hydrolysis are 20 and 25%. The presence of the whole linker in RGAI significantly inhibits insoluble starch hydrolysis, demonstrated by RGAI
L possessing 25% more activity than RGAI.
Insoluble starch binding
GAI has the greatest ability to bind insoluble starch, while RGAI and RGAIL bind insoluble starch less strongly. GAE, GAII and RGAII have very little binding ability (Figure 7 and Table I).
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The very low ability of GAE to bind insoluble starch, 3% that of GAI, appears to be the main reason for its low activity on this substrate. The CGTase SBD (domain E) is not separate from the rest of the enzyme in its native state, and it is quite possible that its structure is so severely modified when detached from the other CGTase domains and attached to GA through a linker that its binding ability is diminished. In addition, as mentioned earlier, not all GA molecules retain their fused CGTase SBDs after processing. These factors appear to overcome the fact that GA and CGTase SBDs in their native states have very similar primary and tertiary structures (Figures 1 and 2).
Two other studies of CGTase SBD fused to other proteins also shed light on this difference. Dalmia et al. (Dalmia et al., 1995) fused P.macerans CGTase SBD or A.niger GA SBD to the C-terminus of ß-galactosidase, finding that both constructs followed Langmuir adsorption isotherms, the former binding half as much insoluble corn starch but about the same amount of cross-linked amylose as the latter at high enzyme concentrations. Furthermore, the GA SBDß-galactosidase fusion protein has the same Kad as the CGTase SBDß-galactosidase protein on insoluble corn starch, but double its value on cross-linked amylose. They explained these results as being caused by the CGTase SBD being potentially more susceptible to unfolding and proteolysis, countered in the case of amylose by the greater evolutionary advances of the CGTase SBD toward small-molecule binding.
In a second study, Ohdan et al. (Ohdan et al., 2000) fused a Bacillus sp. CGTase SBD, with and without the immediately preceding domain D, to the C-terminus of a Bacillus subtilis
-amylase not previously possessing an SBD. The fused protein lacking domain D hydrolyzed soluble corn starch at the same rate as the parent
-amylase, while that with domain D was only one-eighth as active, apparently because of structural distortion caused by the extra domain. Both fusion proteins had several times the activity of
-amylase on insoluble corn starch, the form with domain D being more active, perhaps because of its role as a linker. No comparison with other SBDs was made.
In all three cases when CGTase SBDs were fused to other proteins, to GA in this study and to ß-galactosidase and -amylase earlier, diminished binding and/or activity on either soluble or insoluble starch was noted. These effects appear to be caused by enhanced susceptibility to proteolysis and to changes in tertiary structure.
Comparison of insoluble starch hydrolysis and binding data
Consideration of soluble and insoluble starch hydrolysis as well as insoluble starch binding provides insight into the nature of the cooperative interaction between binding and hydrolysis. When the three data sets for GAI, RGAI and RGAIL are considered simultaneously, it appears that the relative positioning of the binding domain to the catalytic domain is an important factor influencing insoluble starch hydrolysis. The lower ability of RGAI and RGAI
L than of GAI to hydrolyze insoluble starch is not mainly the result of one catalytic domain having greater activity than the other, since RGAI and RGAI
L both hydrolyze soluble substrate at 80% the rate of GAI. Nor does their difference in insoluble starch hydrolysis rate appear to be completely correlated with starch-binding ability; in fact, RGAI binds starch over twice as well as RGAI
L, yet it hydrolyzes insoluble starch less effectively than RGAI
L, perhaps because the orientation of its SBD places the insoluble substrate in a less optimal position for hydrolysis than does the orientation of the RGAI
L SBD, where no linker is present. Three-dimensional enzyme/substrate studies would be needed to substantiate this hypothesis.
Furthermore, it may be noted that the rates by which GAII, RGAII and GAE hydrolyze insoluble starch are not well correlated with Kad beyond the fact that they all bind and hydrolyze insoluble starch poorly compared with GAI, RGAI and RGAIL.
Conclusions
This study has produced significant information regarding GA structure and function. It is now clear that the A.niger GA catalytic domain can hydrolyze soluble starch with an SBD attached to its N-terminus and that the SBD can bind insoluble starch with a catalytic domain attached to its C-terminus. The results also demonstrate that the catalytic and SBDs can cooperate to hydrolyze insoluble starch when their order is reversed from that of wild-type A.niger GA. In addition, insoluble starch binding does not solely correlate with insoluble starch hydrolysis, as evidenced by the results from GAI, RGAI and RGAIL on the one hand and from GAII, RGAII and GAE on the other.
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Acknowledgements |
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Received June 20, 2002; revised May 23, 2003; accepted June 6, 2003.