This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Guillén, D.
Right arrow Articles by Rodríguez-Sanoja, R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Guillén, D.
Right arrow Articles by Rodríguez-Sanoja, R.
Agricola
Right arrow Articles by Guillén, D.
Right arrow Articles by Rodríguez-Sanoja, R.

 Previous Article  |  Next Article 

Applied and Environmental Microbiology, June 2007, p. 3833-3837, Vol. 73, No. 12
0099-2240/07/$08.00+0     doi:10.1128/AEM.02628-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Alpha-Amylase Starch Binding Domains: Cooperative Effects of Binding to Starch Granules of Multiple Tandemly Arranged Domains{triangledown}

D. Guillén, M. Santiago,{dagger} L. Linares, R. Pérez, J. Morlon,{ddagger} B. Ruiz, S. Sánchez, and R. Rodríguez-Sanoja*

Departamento de Biología Molecular y Biotecnología, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, A.P. 70228, Ciudad Universitaria, Coyoacán 04510, México D.F., México

Received 10 November 2006/ Accepted 22 April 2007


arrow
ABSTRACT
 
The Lactobacillus amylovorus alpha-amylase starch binding domain (SBD) is a functional domain responsible for binding to insoluble starch. Structurally, this domain is dissimilar from other reported SBDs because it is composed of five identical tandem modules of 91 amino acids each. To understand adsorption phenomena specific to this SBD, the importance of their modular arrangement in relationship to binding ability was investigated. Peptides corresponding to one, two, three, four, or five modules were expressed as His-tagged proteins. Protein binding assays showed an increased capacity of adsorption as a function of the number of modules, suggesting that each unit of the SBD may act in an additive or synergic way to optimize binding to raw starch.


arrow
INTRODUCTION
 
Adsorption of amylases is frequently considered a prerequisite for the hydrolysis of insoluble starch (16, 22, 24). For this adsorption, some amylases contain a carbohydrate-binding module (CBM). A CBM is defined as an ancillary module of 40 to 200 amino acids with a discrete fold that possesses carbohydrate-binding activity and is usually contiguous to a carbohydrate-active enzyme. The CBM does not have catalytic activity; it seems to allow the interaction between the insoluble substrate and the solubilized enzyme, bringing the substrate to the active site in the catalytic domain and consequently improving hydrolysis (23). CBMs have been classified into 49 families based on their sequence similarities, according to the CAZy database (http://www.cazy.org; last update on 2 April 2007). Starch binding domains (SBDs) belong to families CBM20, CBM21, CBM25, CBM26, CBM34, CBM41, and CBM45.

Approximately 10% of all amylolytic enzymes possess a distinct domain that facilitates binding to raw starch (13). This independent domain has been identified in such microorganisms as filamentous fungi, gram-positive bacteria, proteobacteria, actinomycetes, and archaea (12). The starch binding function has been demonstrated for only some glycoside hydrolases, {alpha}-amylases, cyclodextrin glucanotransferases, and acarviose transferases from glycoside hydrolase family 13 (GH13), ß-amylases from GH14, and glucoamylases from GH15, while in most it remains unverified (18).

The presence of SBDs has been demonstrated in three {alpha}-amylases from lactobacilli (10, 20, 22). The genes encoding these {alpha}-amylases (amyA) have been sequenced (11, 20). Amino acid sequence analysis of these enzymes reveals an identity of more than 96% and a structure that comprises at least two discrete functional domains, the N-terminal or catalytic domain (GH13) and the C-terminal domain or SBD (CBM-26) formed by direct tandem repeat units; four modules have been found in Lactobacillus plantarum and Lactobacillus manihotivorans {alpha}-amylases and five modules in the Lactobacillus amylovorus enzyme. In a previous report, we have shown that the protein encoded by the L. amylovorus amyA gene, which lacks tandem repeats, is unable to adsorb and hydrolyze raw starch despite the fact that it can hydrolyze soluble starch almost as well as complete {alpha}-amylase (22). After establishing the role of tandem repeats as SBDs, we identified in each module some of the residues reported as part of the carbohydrate binding sites in others SBDs; therefore, one single module is supposed to be able to bind raw starch; this capability was confirmed experimentally (26).

The observed modular arrangement, notably the presence of four or five CBM26 tandem units, represents an unusual architecture of the SBD related to amylases from family GH13. Nevertheless, two SBD modules (one from CBM25 and the other from CBM26) have recently been described for the maltohexaose-forming amylase from Bacillus halodurans (5). Two SBD modules, both from CBM25, were also found in the {alpha}-amylases from Bacillus sp. no. 195 (27) and Clostridium acetobutylicum ATCC 824 (21).

In this paper, we present an analysis of the functionality of the C-terminal direct tandem repeats of L. amylovorus {alpha}-amylase, using His-tagged derivatives of the entire SBD or one, two, three, or four modules. Their binding capacity and its possible cooperation were investigated with the aim of proposing a possible biological role.


arrow
MATERIALS AND METHODS
 
Microbial strains and media.
Escherichia coli strain DH5{alpha} [supE44 {Delta}lacU169 ({phi}80lacZ{Delta}M15) hsdR17 recA1 endA1 gyr96 thi-1 relA1] was used as the cloning host, and E. coli strain M15 (QIAGEN) (cells contain the pREP4 plasmid encoding the lac repressor in trans) was used for protein production. All strains were grown in LB medium (1% tryptone, 0.5% yeast extract, and 1% NaCl) supplemented with ampicillin (100 mg/liter) and kanamycin (25 mg/liter).

Lactobacillus amylovorus NRRL B-4540 was kindly provided by the ARS Culture Collection, U.S. Department of Agriculture, Peoria, IL. The strain was grown in MRS medium-starch (2%) at 30°C (9).

Construction of plasmids.
All DNA manipulations were performed using standard protocols (25). To produce different peptides equivalent to one, two, three, four, or five repeats, five different fragments were amplified from the region contained between amino acids 444 and 953 of the L. amylovorus amyA gene (GenBank accession number U62096) with the following primers: primer A, 5'-TGAAAACAAGGCTGGTTCAG-3'; primer B, 5'-GATGAACTGCTGGTATCGGC-3'; and primer C, 5'-TACTACCCCAACTTGAAGGC-3'. The conditions used in the PCRs were previously described (11). The amplified fragments were purified and ligated to pGEM-T Easy vector (Promega), following the manufacturer's instructions, and transformed into E. coli DH5{alpha} by electroporation (25).

Gene expression and peptide purification.
Obtained pGEM-T-easy constructs were digested with EcoRI, and released fragments were treated with mung bean nuclease and cut with BamHI. Gene derivatives were introduced into pQE31 (QIAGEN) treated with SmaI and BamHI. pQE31 carries six codons encoding His6. Five different gene fusions were constructed and verified by sequencing (Laragen, Inc). The first encoded the protein 1-Mod, containing one of the repeated units and the His6 tag at the N terminus. The second encoded 2-Mod, containing two units and the His6 tag at the N terminus. 3-Mod, 4-Mod, and 5-Mod contained three, four and five units, respectively. Constructs were electrotransformed into E. coli M15 for protein expression.

The strains were grown in LB medium supplemented with the required antibiotics for 10 h at 37°C. Expression of proteins was induced by the addition of isopropyl-ß-D-thiogalactopyranoside to a final concentration of 0.4 mM, and incubation was continued at 30°C for 8 h. Cells were harvested (8,000 x g for 10 min at 4°C) and washed in 20 mM Na2HPO4 at pH 7.4 before lysis. The obtained proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (15) and identified by Western blotting with a primary anti-His6 antibody (Roche) and a secondary anti-mouse immunoglobulin G antibody labeled with alkaline phosphatase (Perkin-Elmer Life Sciences), according to the manufacturer's instructions.

For protein purification, the E. coli pellet was resuspended to about 1/100 of the original culture volume in 20 mM Na2HPO4, 500 mM NaCl, and 50 mM imidazole at pH 7.4, with protease cocktail inhibitor (SIGMA). Cells were disrupted by sonication three times for 20 s at 60 Hz (Vibra-Cell; Sonics & Materials, Inc.). Obtained polypeptides were purified from clarified extract by immobilized metal affinity chromatography according to the manufacturer's protocols (Pharmacia Biotech).

Lactobacillus amylovorus {alpha}-amylase purification.
Following an 18-h batch culture (250 ml), the fermentation broth was collected and centrifuged at 9,000 x g for 15 min at 4°C. The L. amylovorus NRRL B-4540 {alpha}-amylase was purified from the supernatant by affinity chromatography as previously described (22), using a ß-cyclodextrin-epoxy-activated Sepharose 6B column (16 by 35 mm). After being washed with 0.1 M citrate-phosphate buffer, pH 5.5, the bound amylase was eluted with 8 mM ß-cyclodextrin in the same buffer at a flow rate of 0.5 ml min–1 for 400 min.

Electrophoresis analyses.
SDS-PAGE (7.5 to 12%) was performed according to the method of Laemmli (15). Proteins were visualized by Coomassie blue staining as described by Blakesley and Boezi (2).

Binding assay.
Before the binding assays, proteins were dialyzed extensively against 0.1 M citrate-phosphate buffer, pH 5, and purity was assessed by SDS-PAGE (15). Various amounts of peptides were added to a prewashed raw-cornstarch suspension (final concentration, 1%) in 0.1 M citrate-phosphate buffer, pH 5, to a final volume of 60 µl. The mixture was incubated at 4°C for 30 min with gentle shaking (20 rpm) and centrifuged at 13,000 x g for 5 min. The protein concentration in the supernatant was subtracted from the total protein to calculate the total amount of protein bound (1).

The protein concentration was determined by measuring the absorbance at 280 nm with the theoretical molar extinction coefficient of 20,416, 46,870, 62,800, 80,080 or 99,700 liter mol–1 cm–1 for 1-Mod, 2-Mod, 3-Mod, 4-Mod, or 5-Mod, respectively, or 207,680 for the complete {alpha}-amylase (8).


arrow
RESULTS AND DISCUSSION
 
Construction and purification of SBD modules.
The L. amylovorus {alpha}-amylase gene encodes a 953-amino-acid protein with a predicted molecular mass of 105 kDa. The mature form of the protein consists of a GH13 N-terminal catalytic domain and a C-terminal SBD composed of five identical modules (CBM26) in tandem. In order to study the role of these repeats, various deletion derivatives were constructed, as summarized in Fig. 1.


Figure 1
View larger version (11K):
[in this window]
[in a new window]

  		FIG. 1. PCR primers and molecular architecture of L. amylovorus {alpha}-amylase. Priming of oligonucleotides designed for PCR amplification of DNA encoding tandem CBM26 modules. Sequences of the primers are included in the text. {alpha}-Amylase CD, {alpha}-amylase catalytic domain; AmyC, {alpha}-amylase domain C.

Plasmids carrying the five tandem modules (the entire SBD) and the separated modules were introduced into E. coli M15 for production. After culture, the proteins were released by cell sonication and purified by metal affinity chromatography.

Adsorption to granular starch.
Fig. 2 shows the obtained adsorption isotherms. The amount of protein bound depends not only on the initial protein concentration but also on the number of modules constituting the peptide. One module is capable of adsorbing to starch granules. This ability increases as the number of modules increases, until reaching the five modules forming the L. amylovorus {alpha}-amylase SBD, which strongly adsorbs cornstarch, even more than the whole amylase. The inability of the {alpha}-amylase catalytic domain to bind to and hydrolyze raw starch in the absence of the SBD has previously been demonstrated (22).


Figure 2
View larger version (26K):
[in this window]
[in a new window]

  		FIG. 2. Adsorption isotherms (tandem units) for CBM26 SBD. (A) Binding curve, considering free protein after equilibrium; (B) initial adsorption isotherms; slopes describe partitioning of proteins between solid and liquid phases at low surface coverage. {circ}, 1-Mod; {square}, 2-Mod; •, 3-Mod; {blacksquare}, 4-Mod; {blacktriangleup}, intact SBD; {triangleup}, L. amylovorus {alpha}-amylase. Adsorption to cornstarch at 4°C in 0.1 M citrate-phosphate buffer, pH 5, was determined as described in Materials and Methods.

The affinity of these modules for starch was analyzed by binding isotherm measurements in accordance with the following Michaelis/Langmuir relationship:

Formula
where qad is the extent of protein adsorbed (µmol of protein/mg of starch), q is the free protein in solution (µM), Kp is a constant, and qmax is the maximum amount of protein adsorbed by the substrate (14).

The Langmuir equation presumes that the adsorption surface is homogeneous and that no interactions exist between adsorbing molecules. Although structural heterogeneity and interactions between adsorbed molecules are probably important factors in starch adsorption, the Langmuir relationship is the most commonly employed equation for describing the phenomena of adsorption of glycoside hydrolases to their substrates (1, 3, 14, 19, 27). The binding parameters for three, four, and five modules and of whole amylase are summarized in Table 1. The association constant increased with the number of modules present in the peptide, but in the case of the whole amylase, simultaneous interaction of the five CBMs seemed to be unfeasible.


View this table:
[in this window]
[in a new window]

  		TABLE 1. Affinities of CBM26 tandem modules for cornstarch as determined by depletion isotherms

The maximum amount of protein adsorbed, qmax, seems to be linked to the size of the protein (Fig. 3), given that the bound protein was directly proportional to the available surface on the starch granule, a condition previously reported for cellulose (7). The decrease in the qmax value with an increase in molecular weight can be described by the equation included in Fig. 3. In addition, when the Langmuir model was used for the 1-Mod and 2-Mod adsorption data, fitted curves did not correlate with experimental results and correlation coefficients were rather low. In both cases, saturation was not achieved even at a peptide concentration of 20 µM. The relationship appears to be linear, probably due to interactions between the proteins. For this reason, it is not possible to calculate the contribution of each module to adsorption, and the apparent free-energy change could only be estimated from the equation {Delta}Gapp = –RT lnKp (where {Delta}Gapp is the apparent {Delta}G, R is the ideal gas constant, and T is temperature) for three, four, and five modules and whole amylase (Fig. 2, inset). The {Delta}Gapp values calculated show a decrease in free energy (increasing affinity) with an increasing number of modules, with the exception of the whole amylase, the {Delta}Gapp value of which indicated a minor affinity compared to that observed in the peptide with three modules.


Figure 3
View larger version (12K):
[in this window]
[in a new window]

  		FIG. 3. qmax values of CBM26 tandem modules as a function of their molecular weights when binding to cornstarch.

Therefore, to compare the adsorption between all peptides, we decided to analyze the initial slopes of the isotherms shown in Fig. 2B, where lateral interactions between the adsorbed peptides do not influence binding. The data were fitted to the following linear equation:

Formula
where [Eb] is the concentration of SBD modules bound to the substrate (µmol/mg substrate), K is the linear coefficient (dimensionless), and [Et] is the total concentration of SBD modules in the reaction system (µM).

The linear coefficients are presented in Table 1. Binding levels of individual CBM peptides were compared by calculating the ratio of KSBD to the K value of every peptide constructed (Table 1). These ratios demonstrated that each peptide did not contribute equally to starch granule adsorption and confirmed that whole amylase did not bind to the substrate with all five modules present in its structure. In other words, a degree of synergy based on the initial binding of components (K) may be established as follows:

Formula
where SB is synergistic binding, KX mod is the K value for two, three, four, or five modules, and Ksingle mod is the K value for one module; an SB value greater than 1 indicates an enhancement in the adsorption of the subsequent module when a previous one was bound. Table 1 summarizes the SB value for each peptide; the values are greater than 1 for three, four, and five modules, showing that when combined in the same polypeptide chain, the modules display a significant increase in affinity for insoluble substrate.

Affinity improvement has also been reported in the SBD of the maltohexaose-forming amylase from Bacillus halodurans. The enhanced affinity is attributed to the simultaneous interaction of the two tandem CBMs present in the enzyme (one from family CBM25 and the other from family CBM26) through an avidity effect (5), a term originally used to describe the strength of binding of a molecule with multiple binding sites by a larger one, particularly the binding of a complex antigen by an antibody. Similar performances were also reported for some cellulases and xylanases (4, 6, 17). Nevertheless, this is the first report of a five-tandem-module SBD analyzed not only as separated modules but also as an element of the whole amylase.

Concerning the catalytic domain, a steric hindrance may explain why whole amylase adsorbs to starch with only two or three modules. Occasionally, amylases have the catalytic domain linked to the binding domain by a linker region of considerable internal mobility (28), but the L. amylovorus {alpha}-amylase does not present any linker structure between the catalytic domain and the SBD, nor among the modules. As a result, some of the modules may act as linkers. In conclusion, we propose that any of the CBMs may bind to starch with equal affinity by both intrachain and interchain interactions. Consequently, subsequent binding events are facilitated by previously established interactions. As a result, the number and flexibility of the neighbor CBMs in the molecule should determine the affinity of the SBD.

The origin of multiple binding modules remains unclear. It has been suggested that duplication of CBMs could be an evolutionary mechanism of thermophilic microorganisms to compensate for a loss of binding affinity in their glycoside hydrolases at elevated temperatures (6). An analysis was carried out with all the starch binding modules listed in the CAZy database (http://www.cazy.org), corresponding to families CBM20, -21, -25, -26, -34, -41, and -45. From the 504 entries, 2 or more modules were found only in 40 sequences, most of them from streptococci and lactobacilli. Given that lactic acid bacteria are mesophile microorganisms, module multiplication cannot be justified by growth temperature. The presence of tandem modules is likely an evolutionary adaptation designed to allow degradation of raw starch by improving lactobacillus {alpha}-amylase binding to the starch granules contained by vegetal material.


arrow
ACKNOWLEDGMENTS
 
This work was supported by CONACyT (National Council for Science and Technology) Mexico, grant 41222.

We thank Rafael Cervantes and Laura Escalante for technical assistance.


arrow
FOOTNOTES
 
* Corresponding author. Mailing address: Departamento de Biología Molecular y Biotecnología, Instituto de Investigaciones Biomédicas, UNAM, A.P. 70228, 04510 México D.F., México. Phone: 52 55 56 22 91 91. Fax: 52 55 56 22 92 12. E-mail: romina{at}correo.biomedicas.unam.mx Back

{triangledown} Published ahead of print on 27 April 2007. Back

{dagger} Present address: Facultad de Ciencias Marinas, Universidad Autónoma de Baja California, Km. 103, Carretera Tijuana-Ensenada, 22800 Baja California, México. Back

{ddagger} Present address: Unité Mixte de Recherche 5539 CNRS, Case 107, Département Biologie-Sante, Université Montpellier II, 34095 Montpellier, France. Back


arrow
REFERENCES
 
    1
  1. Belshaw, N. J., and G. Williamson. 1990. Production and purification of a granular-starch-binding domain of glucoamylase 1 from Aspergillus niger. FEBS Lett. 269:350-353.[CrossRef][Medline]
    2. 2
  2. Blakesley, R. W., and J. A. Boezi. 1977. A new staining technique for protein in polyacrylamide gels using Coomassie Brilliant Blue G-250. Anal. Biochem. 82:580-582.[CrossRef][Medline]
    3. 3
  3. Bolam, D. N., A. Ciruela, S. McQueen-Mason, P. Simpson, M. P. Williamson, J. E. Rixon, A. Boraston, G. P. Hazlewood, and H. J. Gilbert. 1998. Pseudomonas cellulose-binding domains mediate their effects by increasing enzyme substrate proximity. Biochem. J. 331:775-781.[Medline]
    4. 4
  4. Bolam, D. N., H. Xie, P. White, P. J. Simpson, S. M. Hancock, M. P. Williamson, and H. J. Gilbert. 2001. Evidence for synergy between family 2b carbohydrate binding modules in Cellulomonas fimi xylanase 11A. Biochemistry 40:2468-2477.[CrossRef][Medline]
    5. 5
  5. Boraston, A. B., M. Healey, J. Klassen, E. Ficko-Blean, A. Lammerts van Bueren, and V. Law. 2006. A structural and functional analysis of {alpha}-glucan recognition by family 25 and 26 carbohydrate-binding modules reveals a conserved mode of starch recognition. J. Biol. Chem. 281:587-598.[Abstract/Free Full Text]
    6. 6
  6. Boraston, A. B., B. W. McLean, G. Chen, A. Li, R. A. Warren, and D. G. Kilburn. 2002. Co-operative binding of triplicate carbohydrate binding modules from a thermophilic xylanase. Mol. Microbiol. 43:187-194.[CrossRef][Medline]
    7. 7
  7. Bothwell, M. K., G. Daughhetee, G. Y. Chaua, D. B. Wilson, and L. P. Walker. 1997. Binding capacities for Thermomonospora fusca E3, E4 and E5, the E3 binding domain, and Trichoderma reesei CBHI on avicel and bacterial microcrystalline cellulose. Biores. Technol. 60:169-178.[CrossRef]
    8. 8
  8. Combet, C., C. Blanchet, C. Geourjon, and G. Deléage. 2000. NPS@: network protein sequence analysis. Trends Biochem. Sci. 25:147-150.[CrossRef][Medline]
    9. 9
  9. De Man, J. C., M. Rogosa, and M. E. Sharpe. 1960. A medium for the cultivation of lactobacilli. J. Appl. Bacteriol. 23:130-135.[CrossRef]
    10. 10
  10. Florencio, J., D. Eiras-Stofella, C. Soccol, M. Raimbault, J. P. Guyot, and J. Fontana. 2000. Lactobacillus plantarum amylase acting on crude starch granules. Appl. Biochem. Biotechnol. 84:721-730.[CrossRef][Medline]
    11. 11
  11. Giraud, E., and G. Cuny. 1997. Molecular characterization of the {alpha}-amylase genes of Lactobacillus plantarum A6 and Lactobacillus amylovorus reveals an unusual 3' end structure with direct tandem repeats and suggests a common evolutionary origin. Gene 198:149-157.[CrossRef][Medline]
    12. 12
  12. Janecek, S., B. Svensson, and E. A. MacGregor. 2003. Relation between domain evolution, specificity, and taxonomy of the {alpha}-amylase family members containing a C-terminal starch binding-domain. Eur. J. Biochem. 270:635-645.[Medline]
    13. 13
  13. Janecek, S., and J. Sevcik. 1999. The evolution of starch-binding domain. FEBS Lett. 456:119-125.[CrossRef][Medline]
    14. 14
  14. Kyriacou, A., R. Neufeld, and C. R. MacKenzie. 1988. Effect of physical parameters on the adsorption characteristics of fractionated Trichoderma reesei cellulase components. Enzyme Microb. Technol. 10:675-681.[CrossRef]
    15. 15
  15. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685.[CrossRef][Medline]
    16. 16
  16. Leloup, V. M., P. Colonna, and S. G. Ring. 1991. {alpha}-Amylase adsorption on starch crystallites. Biotechnol. Bioeng. 38:127-134.[Medline]
    17. 17
  17. Linder, M., I. Salovuori, L. Ruohonen, and T. Teeri. 1996. Characterization of a double cellulose binding domain. J. Biol. Chem. 271:21268-21272.[Abstract/Free Full Text]
    18. 18
  18. Machovic, M., B. Svensson, E. A. MacGregor, and S. Janecek. 2005. A new clan of CBM families based on bioinformatics of starch-binding domains from families CBM20 and CBM21. FEBS J. 272:5497-5513.[CrossRef][Medline]
    19. 19
  19. Mihlbachler, K., M. A. De Jesus, K. Kaczmarski, M. J. Sepaniak, A. Seidel-Morgenstern, and G. Guiochon. 2006. Adsorption behavior of the (+/–)-Troger's base enantiomers in the phase system of a silica-based packing coated with amylose tri(3,5-dimethyl carbamate) and 2-propanol and molecular modeling interpretation. J. Chromatogr. A 1113:148-161.[CrossRef][Medline]
    20. 20
  20. Morlon-Guyot, J., F. Mucciolo-Roux, R. Rodríguez-Sanoja, and J. P. Guyot. 2001. Characterization of the L. manihotivorans {alpha}-amylase gene. DNA Seq. 12:27-37.[Medline]
    21. 21
  21. Nölling, J., G. Breton, M. V. Omelchenko, K. S. Makarova, Q. Zeng, R. Gibson, H. M. Lee, J. A. Dubois, D. Qiu, and J. Hitti. 2001. Genome sequence and comparative analysis of the solvent-producing bacterium Clostridium acetobutylicum. J. Bacteriol. 183:4823-4838.[Abstract/Free Full Text]
    22. 22
  22. Rodríguez-Sanoja, R., J. Morlon, J. Jore, J. Pintado, N. Juge, and J. P. Guyot. 2000. Comparative characterization of complete and truncated forms of Lactobacillus amylovorus {alpha}-amylase and role of the C-terminal direct repeats in raw starch binding. Appl. Environ. Microbiol. 66:3350-3356.[Abstract/Free Full Text]
    23. 23
  23. Rodríguez-Sanoja, R., N. Oviedo, and S. Sánchez. 2005. Microbial starch-binding domain. Curr. Opin. Microbiol. 8:260-267.[CrossRef][Medline]
    24. 24
  24. Rodríguez-Sanoja, R., B. Ruiz, J. P. Guyot, and S. Sánchez. 2005. Starch binding domain affects catalysis in two Lactobacillus {alpha}-amylases. Appl. Environ. Microbiol. 71:297-302.[Abstract/Free Full Text]
    25. 25
  25. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold. Spring Harbor Laboratory, Cold Spring Harbor, NY.
    26. 26
  26. Santiago, M., L. Linares, S. Sanchez, and R. Rodríguez-Sanoja. 2005. Functional characteristics of the starch binding domain of Lactobacillus amylovorus {alpha}-amylase. Biologia (Bratisl.) 60:111-114.
    27. 27
  27. Sumitani, J., T. Tottori, T. Kawaguchi, and M. Arai. 2000. New type of starch-binding domain: the direct repeat motif in the C-terminal region of Bacillus sp. no. 195 {alpha}-amylase contributes to starch binding and raw starch degrading. Biochem. J. 350:477-484.[CrossRef][Medline]
    28. 28
  28. Williamson, G., N. J. Belshaw, and M. P. Williamson. 1992. O-Glycosylation in Aspergillus glucoamylase. Conformation and role in binding. Biochem. J. 282:423-428.[Medline]

Applied and Environmental Microbiology, June 2007, p. 3833-3837, Vol. 73, No. 12
0099-2240/07/$08.00+0     doi:10.1128/AEM.02628-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Kiyohara, M., Sakaguchi, K., Yamaguchi, K., Araki, T., Ito, M. (2009). Characterization and Application of Carbohydrate-binding Modules of {beta}-1,3-xylanase XYL4. J Biochem 146: 633-641 [Abstract] [Full Text]  
  • Connaris, H., Crocker, P. R., Taylor, G. L. (2009). Enhancing the Receptor Affinity of the Sialic Acid-binding Domain of Vibrio cholerae Sialidase through Multivalency. J. Biol. Chem. 284: 7339-7351 [Abstract] [Full Text]  
  • Okano, K., Zhang, Q., Kimura, S., Narita, J., Tanaka, T., Fukuda, H., Kondo, A. (2008). System Using Tandem Repeats of the cA Peptidoglycan-Binding Domain from Lactococcus lactis for Display of both N- and C-Terminal Fusions on Cell Surfaces of Lactic Acid Bacteria. Appl. Environ. Microbiol. 74: 1117-1123 [Abstract] [Full Text]  
  • Abbott, D. W., Eirin-Lopez, J. M., Boraston, A. B. (2008). Insight into Ligand Diversity and Novel Biological Roles for Family 32 Carbohydrate-Binding Modules. Mol Biol Evol 25: 155-167 [Abstract] [Full Text]  

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Guillén, D.
Right arrow Articles by Rodríguez-Sanoja, R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Guillén, D.
Right arrow Articles by Rodríguez-Sanoja, R.
Agricola
Right arrow Articles by Guillén, D.
Right arrow Articles by Rodríguez-Sanoja, R.

Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»