Role of the linker region in the expression of Rhizopus oryzae glucoamylase

Background Rhizopus oryzae glucoamylase (RoGA) consists of three domains: an amino (N)-terminal raw starch-binding domain (SBD), a glycosylated linker domain, and a carboxy (C)-terminal catalytic domain. The 36-amino-acid linker region (residues 132–167) connects the two functional domains, but its structural and functional roles are unclear. Results To characterize the linker sequences of RoGA and its involvement in protein expression, a number of RoGA variants containing deletions and mutations were constructed and expressed in Saccharomyces cerevisiae. Deletion analyses demonstrate that the linker region, especially within residues 161 to 167, is required for protein expression. In addition, site-directed mutagenesis and deglycosylation studies reveal that the linker region of RoGA contains both N- and O-linked carbohydrate moieties, and the N-linked oligosaccharides play a major role in the formation of active enzyme. Although the linker segment itself appears to have no ordered secondary structural conformation, the flexible region indeed contributes to the stabilization of functional N- and C-terminal domains. Conclusion Our data provide direct evidence that the length, composition, and glycosylation of the interdomain linker play a central role in the structure and function of RoGA.


Background
Glucoamylase (1,4-α-D-glucan glucohydrolase, EC 3.2.1.3; GA) is an exohydrolase that catalyzes the release of β-D-glucose by hydrolyzing α-1,4and α-1,6-glucosidic linkages at the non-reducing ends of raw or soluble starches and related oligosaccharides [1][2][3]. GA has been used in industrial processes such as the production of glucose syrup and other food-processing applications [2,4]. Although many fungal species are capable of producing GA under different growth conditions [5], the industrial development of GA has focused only on GA from Aspergillus niger (AnGA; identical to Aspergillus awamori GA) and Rhizopus oryzae (RoGA) because of their stability and high activity [3,6,7]. The overall domain structure of AnGA consists of an N-terminal catalytic region and a C-terminal starch-binding domain (SBD). In contrast, the organization of that of the RoGA consists of an N-terminal SBD and a C-terminal catalytic region. The biochemical prop-erties of AnGA have been well characterized [2,[8][9][10][11][12][13][14][15][16], whereas less is known about RoGA [17,18]. This work focuses on functional analysis of RoGA.
RoGA is synthesized as a precursor containing a typical hydrophobic secretory signal sequence of 25 amino acids. The mature form of RoGA is a single-chain protein composed of three domains: an SBD (residues 26-131), a Thr/ Ser-rich linker region (residues 132-167), and a catalytic domain (residues 168-604) [19]. The schematic representation of RoGA is shown in Figure 1. TheN-terminal SBD belongs to the carbohydrate-binding module (CBM) family 21 and shows a relatively low level of similarity to SBDs in other starch-degrading enzymes [20][21][22][23]. The Cterminal catalytic domain of RoGA plays an active role in hydrolyzing starch and has a high degree of sequence similarity to those of other fungal GAs [6]. The linker region between these two functional domains is rich in hydroxylamino acid residues, but information about its function is quite limited [16,24,25]. In the CAZy classification based on amino acid sequences of the catalytic domain, GAs are classified into glycoside hydrolase (GH) family 15 [26], and on two recently published articles [27,28], the N-terminally positioned SBD (CBM21) and the C-terminally positioned SBD (CBM20) are classified to be grouped into a common CBM clan. However, the sequences of the linker regions are highly variable. Comparison of the primary structures of different fungal GAs reveals that the linker sequences vary greatly in length and composition (Table 1).
In some fungal hydrolases, the substrate-binding and catalytic domains are separated by a linker segment rich in proline and hydroxy amino acid residues, some of which have been shown to be involved in various functions including enzymatic activity, stability, protein secretion, and ligand binding [29,30]. In AnGA, the heavily O-glycosylated linker domain is essential for secretion and is responsible for enzyme stability as well as activity toward raw starch [11,16,24]. Aside from the knowledge that the linker region in RoGA acts as an interdomain spacer, very little is known about this specific sequence stretch. The linker region of RoGA contains high percentages of Thr (44%) and Ser (16%) residues. Along with numerous putative O-glycosylation sites, the RoGA linker also contains one potential N-glycosylation site adjacent to the catalytic domain (Asn 167 -Ser-Thr, Figure 1).
Here the baker's yeast, Saccharomyces cerevisiae, was used as a host system to study the structure and function of RoGA, with special attention to the interdomain linker region. The effects of the linker segment with specific length, composition, and glycosylation on the properties of the protein expression, ligand or substrate binding, enzyme activity and stability have been thoroughly investigated.

Expression of the full-length and deletion mutant GAs in S. cerevisiae
To investigate the function of the linker region of RoGA, several constructs containing full-length, truncated or partially deleted GA fragments were generated ( Figure 1). All constructs used in this study contained a leader signal sequence of the N-terminal 25 residues of RoGA. When the full-length GA was expressed in S. cerevisiae, secreted enzyme activity was detected in the culture medium following incubation at 30°C ( To further examine whether the linker region was essential for the formation of active starch-binding or catalytic domains, four deletion mutants devoid of either the substrate binding or catalytic domain in the presence or absence of the linker region were engineered to generate plasmids pS1-GA∆168-604 encoding the SBD with the Cterminal linker region; pS1-GA∆132-604 encoding the SBD alone; pS1-GA∆26-131 encoding the linker region preceding the catalytic domain; and pS1-GA∆26-167 encoding the catalytic domain only. It was found that two SBD-containing clones, one, GA∆168-604, containing the linker sequence and the other, GA∆132-604, without the linker sequence were both successfully expressed and secreted ( Figure 2B, upper panel, lanes 3 and 4). Since no effect was observed in the absence of the linker in both cases, the linker region appeared to be not essential for the formation and secretion of the SBD. As for the catalytic domain-containing variants, GA∆26-131 was successfully expressed and secreted in yeast, but GA∆26-167 lacking of the linker sequence was not expressed at all ( Figure 2B, lower panel, lanes 4 and 7), indicating that the linker region was specifically required for formation and secretion of the catalytic domain. To further define which region in the linker was crucial for its role in facilitating formation and secretion of the catalytic domain, several constructs encoding partial deletions of the linker region fused to the catalytic domain were subsequently expressed and characterized. Interestingly, both GA∆26-145 and GA∆26-160, unlike GA∆26-167, were successfully expressed in secretory forms ( Figure 2B, lower panel, lanes 5 and 6), indicating that the linker region from residues 161-167 was essential for in vivo folding and secretion of the active catalytic domain. It is of interest that a recently published article [7] on Rhizopus oryzar found a second GA gene, amyB, lacks the N-terminal SBD. The amyB gene contains the conserved residues in the catalytic domain important for starch hydrolysis, and the gene also contains a stretch of linker sequence (33 bp) preceding the catalytic domain. This finding also indicates that the small region of linker plays an important role in function of RoGA.

Characterization of recombinant GAs
In order to characterize RoGA and to study the function of the linker region, the recombinant proteins including fulllength GA, GA∆168-604, GA∆132-604, GA∆26-131, GA∆26-145, and GA∆26-160 were individually expressed in the budding yeast S. cerevisiae and were grown in SD media at 30°C. Extracellular (secreted) proteins were concentrated from culture supernatants and purified by cation-exchange chromatography. As shown in Figure 3A, purified full-length GA, GA∆168-604, GA∆132-604, GA∆26-131, GA∆26-145, and GA∆26-160 exhibited single protein bands with purity higher than 90%. SDS-PAGE and/or mass spectrometric determination revealed that the respective molecular mass of fulllength GA, GA∆168-604, GA∆132-604, GA∆26-131, GA∆26-145, and GA∆26-160 was approximately 78, 21, 12, 69, 60, and 56 kDa ( Table 2). The differences between the calculated and observed molecular weights ranged from 3% to 28%, indicating that post-translational modifications occurred in some of the recombinant proteins, presumably due to differential degree of glycosylation. There are two main types of protein glycosylation: N-glycosylation and O-glycosylation. The former refers to the attachment of oligosaccharides to a protein through the amide of asparagine residues, whereas the latter involves attachment of sugars to the hydroxyamino acids serine and threonine via their hydroxyl groups [31]. Potential Nglycosylation sites in RoGA analyzed using the on-line prediction server NetNGlyc version 1.0 [32] revealed that RoGA contained three putative N-linked glycosylation consensus sites, one (Asn 167 -Ser-Thr) located in the linker region and two (Asn 230 -Thr-Thr and Asn 236 -Lys-Thr) in the catalytic domain ( Figure 1). Although no specific consensus sequence for O-linked glycosylation has yet been reported [33,34], O-glycosylation may occur on any of the Ser or Thr residues within a short peptide region [35]. Interestingly, the program NetOglyc version 3.1 [36] predicted that potential O-glycosylation sites in RoGA were clustered only in the linker region. Of the residues in the 36-amino-acid linker region, 61% were Ser and Thr, among which some might serve as potential sites for Olinked glycosylation. MALDI fingerprint mass spectrum from the tryptic digestion fragments of RoGA (data not shown) also showed that no peptide fragment was mapped to the linker region, presumably due to the presence of heterogeneous glycosylation within this region.
To determine how much of the molecular weight discrepancy for each recombinant protein was derived from the addition of glycosyl groups, each protein was enzymatically deglycosylated by PNGase F or Jack bean α-mannosidase followed by SDS-PAGE analysis. PNGase F is an amidase that cleaves between the innermost GlcNAc and the asparagine residue of complex N-linked oligosaccharides from glycoproteins [37]. The fully glycosylated RoGA possessed a molecular mass of 78 kDa. After removal of N-linked carbohydrates with PNGase F, its molecular mass was reduced to 65 kDa ( Figure 3B), indicating that RoGA produced by S. cerevisiae was highly glycosylated and the N-linked carbohydrates contributed approximately 13 kDa to the molecular weight. Enzymatic deglycosylation of asparagine-linked glycans in the catalytic domain variants GA∆26-131, GA∆26-145, and GA∆26-160 also showed that these proteins were efficiently glycosylated to various degrees ( Figure 3B).
The linker sequences code for a 36 amino acid extension at the C-terminal end of GA∆168-604, which correspond to a molecular mass of approximately 3.5 kDa. The difference in molecular mass between GA∆168-604 and GA∆132-604 was 8.7 kDa, implying that the linker region contributed to the hyperglycosylation in GA∆168-604. However, treatment of GA∆132-604 and GA∆168-604 with the N-glycan-specific PNGase F did not reduce its apparent molecular weight; hence there seemed to be little or no N-glycosylation on either protein (data not shown). At present, no enzyme comparable to PNGase F is available for removing intact O-linked sugars [38]. In general, O-glycans are short linear oligosaccharides consisting of one to five mannose residues in yeast [24,34,39]. Therefore, α-mannosidase, an exoglycosidase capable of removing terminal mannose, was used to treat the recombinant proteins. If O-linked carbohydrates were present, the overall mass of the protein would decrease after treatment with α-mannosidase. The SBD variant GA∆132-604 did not respond to the treatment with αmannosidase, such that no change was detected in the electrophoretic mobility ( Figure 3C), suggesting that no major O-glycosylation occurred in this case. On the other hand, a clear shift in the mobility of GA∆168-604 was observed after treatment with α-mannosidase, indicating that presence of the linker region led to a considerable degree of modification by high-mannose-type O-glycans ( Figure 3C). Taken together, our data indicate that the linker region of RoGA is modified by both N-and Olinked glycosylation, but the exact positions and functions are not clearly understood and require further investigation.

Effects of mutations in the linker region of RoGA
The minimal active linker region Ala 161 to Asn 167 was previously demonstrated to be extremely important for GA function. In addition, N-terminal sequencing analysis of the GA∆26-160 yielded the following sequence: Ala 161 -Thr 162 -Phe 163 -Pro 164 -Xaa 165-Gly 166 -Xaa 167 , where Xaa represented unidentified residues. Generally speaking, biochemically modified amino acid residues were not identifiable by regular Edman degradation; hence residues Thr 165 and Asn 167 were possibly modified after translation. To abolish the glycosylation and evaluate the consequence, Thr 165 and Asn 167 in the linker region were individually mutated to generate T165A and N167D, respectively, and their effects on protein secretion and enzymatic activity were examined at 30°C. T165A mutation showed no effect on the phenotype as compared with wild-type full-length GA ( Figure 4A  Moreover, elimination of the N-glycosylation site at position Asn 167 (N167D) resulted in a faster relative electrophoretic mobility and lower apparent molecular weight ( Figure 4D), providing additional line of evidence that the only putative N-glycosylation site in the linker region, Asn 167 -Ser-Thr, was indeed modified in S. cerevisiae. The biological function of carbohydrates in GA is not, however, completely understood, although it directs protein folding, facilitates secretion, and enhances stability of Aspergillus GAs [24]. In this study, we have demonstrated that N-glycosylation at Asn 167 in the linker is required for functional expression of RoGA, whereas mutation abolishing N-glycosylation in the linker, N167D, decreased the efficiency of protein expression. Furthermore, similar result was obtained when Asn 167 was converted to Gln (data not shown). N-linked carbohydrates play important roles for a variety of structural and functional activities of glycoproteins [40]. Here, we have characterized the N-glycans attached to the linker region of RoGA to be important for folding process, especially for its catalytic domain.

The starch-binding domain
The SBD, the non-catalytic module of GA that binds raw starch [21], is found at the N terminus in RoGA and shares Purification and deglycosylation of recombinant RoGAs only 13.5% identity with that of AnGA [22]. Characterization of the key functional groups of this domain has been accomplished using sequence-based structure alignment and NMR spectroscopy [22,23]. The effects of the linker region on SBD adsorption on to insoluble starch were thus investigated. The binding assay was conducted at pH 4.5 using insoluble corn-starch as the affinity matrix. GA∆168-604 were measured as 35.12 µmol/g and 17.24 µmol/g, respectively. It was apparent that the 106-residue SBD, GA∆132-604, possessed stronger ligand affinity and higher capacity than those of the 142-residue SBD, GA∆168-604, indicating that the presence of the linker region in the SBD construct did not increase but instead slightly interfered with the raw starch-binding affinity. The difference in starch binding affinity between GA∆132-604 and GA∆168-604 might be due to higher sugar content in the linker region or steric hindrance caused by the linker tail. Binding of the starch-degrading enzyme to its substrate is a critical step in starch hydrolysis because it involves the phase transfer of a soluble enzyme to the insoluble substrate [15]. In our case the linker sequence influences the binding of the SBD to starch, possibly by affecting the ligand transfer process.
The catalytic domain GA activity was assayed by measuring the reducing sugar released from the reaction on starch. The standard assay was performed as previously described with minor modification [17]. The specific activities of the wild-type RoGA and the truncated mutants are listed in Table 3. Fulllength GA and GA∆26-131 exhibited similar activities, 4.58 × 10 3 U/µmol and 4.88 × 10 3 U/µmol, respectively, indicating that the absence of the N-terminal SBD had little or no effect on the ability to digest soluble starch. Moreover, comparison of the activities of the three catalytic domain variants GA∆26-131, GA∆26-145 and GA∆26-160 revealed that longer size of the linker region was correlated with enhanced catalytic activity. Figure 6 showed the effects of temperature and pH on enzyme activity. Regarding the thermal stability, the fulllength RoGA remained stable at 40°C for 30 min at pH 4.5 with almost 100% of its activity remained, whereas at the same pH only approximately 35% and 5% residual activity was respectively detected at 50°C and 60°C ( Figure 6, solid curve, open square). The three catalytic domain derivatives possessed inactivation profiles similar to that of the full-length GA ( Figure 6, solid curves), indicating that the linker region did not contribute as much as expected to the thermal stability. In addition, the pH stability of the full-length RoGA at 25°C was found to be quite high over the pH range between 4.0 and 6.0, and more than 70% and 50% of the activity remained after 2 h incubation (Tm, 25°C) at pH 3.0 and pH 8.0, respectively ( Figure 6, dotted curves). The truncated enzymes showed similar trends in stability over the pH range tested, indicating that the linker region was not crucial for the pH stability of GA either. Our data imply that the catalytic domain can function independently in terms of digesting soluble starch and the addition of linker sequences does not affect the thermal and pH stabilities of the enzyme.

Structure analysis
The three-dimensional structure of RoGA is currently unavailable; circular dichroism spectroscopy has been widely used to study the secondary structure, with the goal of understanding the role of the linker in protein structure. In this study, the far-UV spectrum of full-length RoGA displayed two broad minima at 208 and 222 nm, characteristic of the presence of a mainly α-helical structure ( Figure  7A, open square). The circular dichroism spectra of catalytic domain variants with different linker lengths were also indicative of a high content of helical conformation. In addition, the spectra of GA∆26-131, GA∆26-145, and GA∆26-160 were very similar to each other, mainly differing in absorbance intensity ( Figure 7A, closed symbols). GA∆26-131 possessed a higher fraction of ordered secondary structure than did the other mutants, suggesting that the linker sequence might increase the stability of the secondary structural motifs. It was thus concluded that the linker sequences or their high degree of glycosylation facilitated stabilization of conformation of the catalytic domain of RoGA.
As for the SBD, Figure 7A also showed the calculated circular dichroism spectra of GA∆132-604 and GA∆168-604 (open triangle and circle, respectively). The negative ellipticity peak centered at 215 nm indicated that the secondary structure of the protein was predominantly βsheet, but the latter showed a higher β-sheet conformation content. Similar to the case for the catalytic domain variants, the presence of the linker peptide in the recombinant SBD induced a substantial conformational change to a more ordered state. In addition, the thermal denaturation of GA∆132-604 and GA∆168-604 was monitored by circular dichroism at 215 nm. Figure 7B   the denatured state of GA∆168-604 possessed a greater negative ellipticity at 215 nm than did GA∆132-604, suggesting that the presence of linker sequence might induce the SBD to form a more compact structure at high temperature. Taken together, although the 36-amino-acid linker alone of RoGA is predicted to be disordered, it strongly influences the structural ordering of the independent functional domains of RoGA.

Conclusion
Many raw starch-degrading enzymes have a modular structure in which the functional domains are spatially separated by linker sequences. In AnGA, the highly O-glycosylated linker segment has been shown to be needed for the efficient digestion of raw starch [24,41]. In contrast, the raw starch-digesting α-amylase from the yeast Cryptococcus sp. strain S-2, lacking of a linker segment demonstrates that the linker region is not always essential for the discrete domains in raw starch-degrading enzymes to retain their proper function [42]. Our present study focuses on functional characterization of the linker region of RoGA. The linker between the SBD and the catalytic domain consists of 36 amino acids with several oligosaccharides attached, and the region facilitates the formation of functional RoGA in S. cerevisiae. The GA∆132-167 deletion mutant failed to produce detectable protein at 30°C, presumably because the distance between the two domains was too short for them to be folded and secreted correctly. Interestingly, prolonged incubation at low temperature was allowed to synthesize the mutant, which showed detectable activity as visualized on starch plate ( Figure  Prior to our present work, no three dimensional structure of intact RoGA has ever been reported. The catalytic domain of RoGA shows 36% identity and 52% similarity to that of AnGA, a functional domain with a characteristic α-helical structure. As for the three dimensional structure of SBD, it has been initially molecular modeled by structural bioinformatics methodology [22] and subsequently determined by NMR spectroscopy [23]. Interestingly, while the SBDs of RoGA and AnGA share extremely low similarity in their primary structures, their secondary and tertiary structures appear to be quite similar, strongly suggesting that SBDs from CBM20 and CBM21 can be grouped into a new clan with similar functional structures [27,28]. It has been clearly demonstrated that the presence of the linker region leads to much more stable con- formation of the two discrete functional domains. Taken together, our data provide several lines of direct evidence that the linker region of RoGA plays a crucial role in terms of holding structural integrity, enhancing structure stability, and facilitating functional protein expression.

Construction of the full-length RoGA construct
The full-length cDNA encoding RoGA was kindly provided by Simpson Biotech, and the set of primers F-RoGA and R-RoGA was used to clone the RoGA gene. Both primers included an additional EcoRI site at their 5'-ends. The sequences of all the primers used are listed in Table 4. PCR amplification was carried out with KlenTaq-1 DNA polymerase (Clontech). The 1.8-kb PCR product was gelpurified and subcloned into yT&A TA cloning vector (Yeastern Biotech) to generate a pT-RoGA plasmid, which was further digested with EcoRI and then subcloned into pS1 at the same enzyme site to generate a pS1-RoGA plasmid.

Construction of deletion mutants of RoGA
A series of deletion mutants was generated by a PCR-based technique using the pS1-RoGA plasmid as the template.
For the C-terminal truncation clone GA∆168-604, which lacks the catalytic domain, the template was amplified using the primer pair F-RoGA and R-GA∆168-604. For GA∆132-604, which lacks both the linker region and the catalytic domain, the template was amplified using primers F-RoGA and R-GA∆132-604. The resulting PCR products were separated on a 1% agarose gel and subcloned into pS1 as described above. The internal deletion clone GA∆132-167, which lacks the entire linker sequence, was generated by fusing an N-terminal fragment to a C-terminal fragment with a two-step PCR approach. In the first step, F-PS1 paired with R-GA∆132-167 and F-GA∆132-167 paired with R-PS1 were used to generate PCR products of 516 bp and 1517 bp, respectively. The PCR products thus obtained shared overlapping sequences that could anneal in a secondary PCR. In the second step, the two first-stage PCR products were purified, combined and reamplified together with the external primers F-PS1 and R-PS1. The generated PCR product was subcloned into pS1 and sequenced for confirmation. Similar technologies were employed to obtain the other internal deletion clones with the following primer pairs: GA∆26-131, F-GA∆26-131 and R-GA∆26-131; GA∆26-145, F-GA∆26-145 and R-GA∆26-145; GA∆26-160, F-GA∆26-160 and R-GA∆26-160; GA∆26-167, F-GA∆26-167 and R-GA∆26-167. All primer sequences are listed in Table 4.
Halo assay for GA activity S. cerevisiae transformants were patched on YPD agar plate containing 0.5% (w/v) soluble starch. Secreted GA activity was detected by observing halo formation on agar plates containing iodine solution (0.01%).

Purification of secreted proteins from S. cerevisiae
Yeast strains were transformed using the one-step transformation method as described [46]. The transformed yeast was cultivated in SD medium for 3 days at 30°C, and the supernatant of the culture medium was concentrated and dialyzed against 10 mM NaOAc (pH 4.5) using an Amicon stirred-cell concentrator (Millipore) equipped with a PM-10 membrane (10-kDa cut-off). For purification, the concentrated sample was loaded onto a 5-mL HiTrap SP cation-exchange column (Amersham Pharmacia Biotech) that was pre-equilibrated with the same buffer used for dialysis. The column was washed with 5  column volumes of 10 mM NaOAc buffer and then eluted with 10 volumes of a linear gradient of NaCl from 0 to 1 M in the same buffer at a flow rate of 2 mL/min. Fractions in 1-mL volumes were collected, and protein peaks were monitored by UV absorption at 280 nm. The fractions containing the desired proteins were pooled, dialyzed, and concentrated. The protein concentration was determined using the bicinchoninic acid (BCA) protein assay reagent kit (Pierce), with BSA as the reference standard. probed with polyclonal anti-RoGA antibody, used at a 1:5000 dilution. The secondary antibody was the horseradish peroxidase-conjugated anti-rabbit IgG, diluted to 1:5000. The bound complexes were detected with ECL reagents (ECL kit, Pierce) and exposure to X-ray film.

Mass spectrometric analysis
Molecular mass determination of the recombinant proteins was performed by Liquid Chromatography/Mass Spectrometer (LC/MS). The intact proteins (100 pmol) were acidified with 0.1% formic acid in 50% (v/v) acetonitrile, and the data were acquired over the 800-1800 m/z range under normal scan resolution. The original electrospray mass spectrum with multiply charged ion series was deconvoluted to give a mass spectrum.

N-terminal peptide sequence analysis
For N-terminal amino acid sequencing, purified protein was separated by SDS-PAGE, and electrophoretically transferred to a PVDF membrane. The blotted proteins were visualized using Coomassie Brilliant Blue R-250. After destaining, the blots were rinsed in deionized water and air-dried. N-terminal sequencing was performed by automated Edman degradation using an Applied Biosystems model 494 Procise sequencer at the National Taiwan University.

Deglycosylation assays
Peptide N-glycosidase F (PNGase F, New England Biolabs) and Jack bean α-mannosidase (Sigma-Aldrich) were used in enzymatic deglycosylation of the recombinant proteins according to the manufacturers' instructions. After enzymatic treatment, the samples were separated by SDS-PAGE and stained with Coomassie Brilliant Blue R-250 to detect any differences in protein migration.

Site-directed mutagenesis of amino acid residues in the linker
Site-directed mutagenesis was performed with a PCRbased technique to change Thr 165 to Ala and Asn 167 to Asp. The primers used were as follows: universal primers, F-PS1 and R-PS1; mutagenic primers F-T165A and R-T165A for the Thr 165 →Ala substitution; and F-N167D and R-N167D for the Asn 167 →Asp substitution. All primer sequences are listed in Table 4.

Adsorption assay
The adsorption of the SBD to granular corn-starch (Sigma-Aldrich) was performed as follows. The purified SBD-containing protein at a concentration ranging from 1.0 to 7.5 µM was mixed with 1 mg of prewashed corn-starch in a final volume of 1 mL in 10 mM NaOAc, pH 4.5. After incubation at 25°C for 16 h, the starch was removed by centrifugation at 16,000 g for 3 min at 4°C, and the amount of unbound protein remaining in the supernatant was determined by the BCA assay. The amount of adsorbed protein was calculated from the difference between the initial and unbound protein concentrations. The maximal amount of bound protein (B max ) and the dissociation constant (K d ) were determined by fitting to the non-linear regression of the binding isotherms, and the following equation was used for saturation binding with one binding site: B = B max F/(K d +F), where B (µmol) represents the bound protein, B max (µmol) is the maximal amount of bound protein, F (µmol) is the free protein in the system, and K d (µmol) is the equilibrium dissociation constant. The units of calculated B max and K d were converted to micromoles per gram and micromolar, respectively.

GA activity toward soluble starch
Substrate was prepared by boiling 1% soluble corn-starch in 10 mM NaOAc, pH 4.5. The amount of glucose liberated from the starch by enzymatic activity was determined using the glucose oxidase/peroxidase kit (Sigma-Aldrich). For each reaction, 50 µL of substrate solution was equilibrated in a 37°C water bath for 5 min, and the assay was initiated by adding 50 µL of appropriately diluted enzyme solution. After incubation at 37°C for 5 min, 200 µL of kit solution was added to each sample. The reaction mixtures were incubated for 5 min at 37°C and stopped by the addition of 200 µL of 6 M H 2 SO 4 . The absorbance was recorded at 540 nm using a U-3310 spectrophotometer (Hitachi). One unit of enzyme activity was defined as the amount of enzyme that produces 1 µmol glucose/min under the assay conditions described above.

Effect of temperature and pH on the activity of GA
Thermal stability of the enzyme was determined by preincubating the purified protein for 30 min at various temperatures ranging from 20 to 65°C. At time intervals, the