{alpha}-Amylase from Bacillus licheniformis mutants near to the catalytic site: effects on hydrolytic and transglycosylation activity

Manuel Heriberto Rivera, Agustín López-Munguía, Xavier Soberón and Gloria Saab-Rincón1

Instituto de Biotecnología, UNAM, Apartado Postal 510-3, Cuernavaca, Morelos 62271, México

1 To whom correspondence should be addressed. e-mail: gsaab{at}ibt.unam.mx


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The {alpha}-amylase from Bacillus licheniformis is the most widely used enzyme in the starch industry owing to its hyperthermostability, converting starch to medium-sized oligosaccharides. Based on sequence alignment of homologous amylases, we found a semi-conserved sequence pattern near the active site between transglycosidic and hydrolytic amylases, which suggested that hydrophobicity may play a role in modifying the transglycosylation/hydrolysis ratio. Based on this analysis, we replaced residue Val286 by Phe and Tyr in Bacillus licheniformis {alpha}-amylase. Surprisingly, the two resultant mutant enzymes, Val286Phe and Val286Tyr, showed two different behaviors. Val286Tyr mutant was 5-fold more active for hydrolysis of starch than the wild-type enzyme. In contrast, the Val286Phe mutant, differing only by one hydroxyl group, was 3-fold less hydrolytic than the wild-type enzyme and apparently had a higher transglycosylation/hydrolysis ratio. These results are discussed in terms of affinity of subsites, hydrophobicity and electrostatic environment in the active site. The engineered enzyme reported here may represent an attractive alternative for the starch transformation industries as it affords direct and substantial material savings and requires no process modifications.

Keywords: amylases/catalysis/site-directed mutagenesis/transglycosylation reaction


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The global market for starch processing enzymes is around US $156 million and the cost of the enzymes used in the liquefaction process represents 24% of the total process cost (Crabb and Mitchinson, 1997Go). Therefore, any improvement in either the enzyme production yield, thermostability or activity will have a direct impact in the process performance, economics and feasibility.

The industrial starch process requires several enzymes: the first step is carried out with Bacillus licheniformis {alpha}-amylase, which is used to depolymerize starch to maltodextrins and corn syrup solids by controlled hydrolysis, a process known as liquefaction. From this point, other enzymes are introduced, depending on the desired final products. For the production of glucose, the use of glucoamylase is necessary to complete hydrolysis. It would be highly desirable that a single enzyme would hydrolyze starch to glucose or, alternatively, that all the enzymes required for the process functioned under the same reaction conditions (Guzman-Maldonado and Paredes-Lopez, 1995Go).

{alpha}-Amylases ({alpha}-1,4-glucan 4-glucanohydrolase, EC 3.2.1.1) catalyze the hydrolysis of the {alpha}-1,4 glycosidic linkages of starch, found in amylose and amylopectin. They are classified into family 13 according to the classification of glycoside hydrolases (Henrissat et al., 1995Go; Brzozowski and Davies, 1997Go; Henrissat and Davies, 1997Go; Davies et al., 1998Go). The main structural feature of family 13 enzymes is the presence of the characteristic (ß/{alpha})8 barrel catalytic domain with a varying number of extra domains, depending on the type of amylase. In particular, {alpha}-amylases are characterized by the presence of three different domains: a central (ß/{alpha})8 barrel domain (domain A), which is interrupted by an irregular ß-domain (domain B) inserted between the third ß-strand and the third {alpha}-helix of the TIM barrel, and a third domain (domain C), which is a Greek key motif located on the opposite side of the barrel. The active site is situated in a cleft at the interface between domains A and B, where the C-termini of the ß-strands and loops joining the ß-strands to the {alpha}-helix in the TIM barrel are found (Nagano et al., 2001Go).

Protein engineering can greatly benefit from the analysis of sequences related to that of the target protein. Natural evolution provides relevant information through the differential conservation of residues in families of proteins with slightly different functions. One should keep in mind, however, that selective pressure operates on circumstances that usually deviate significantly from those required in bioprocesses.

In the case of {alpha}-amylases, most of the protein engineering work has been devoted to increasing its stability to temperature and its operative pH range (Declerck et al., 1995Go, 1997, 2000; Igarashi et al., 1998Go; Nielsen et al., 1999Go; Shaw et al., 1999Go), and also to dealing with successful changes in catalytic activity or in product specificity (Kuriki et al., 1996Go; Inohara-Ochiai et al., 1997Go; Matsui and Svensson, 1997Go; Wind et al., 1998aGo,b; Saab-Rincon et al., 1999Go; Beier et al., 2000Go; Brzozowski et al., 2000Go; Gottschalk et al., 2001Go). We have previously demonstrated in Bacillus stearothermophilus {alpha}-amylase that the introduction of tranglycosylation activity, present in saccharifying amylases, through the mutation of Ala289 to Tyr, changed the product specificity to higher yields of smaller oligosaccharides (Saab-Rincon et al., 1999Go). However, owing to its industrial importance and high thermostability, the {alpha}-amylase from B.licheniformis is the best option for protein engineering in order to extend its range of applications. Although it has been reported that {alpha}-amylase from B.licheniformis is mainly hydrolytic, higher transglycosylation activity could be obtained (Thompson et al., 1997Go; Marchal et al., 1999Go) by incorporating the same mutation previously designed for the B.stearothermophilus {alpha}-amylase (both enzymes share 63% identity). In this paper, we describe the enzymatic characterization of B.licheniformis {alpha}-amylase mutants Val286Tyr and Val286Phe (equivalent to Ala289 in the B.stearothermophilus enzyme). We found that the two mutants show different kinetic behaviors. From the wild-type (WT) crystal structure, no direct contact is observed between the position Val286 and any of the catalytic residues. We hypothesized that the effect of the mutation was indirect through an interaction with residue Trp263. To demonstrate this hypothesis, we constructed the double mutant Val286Tyr/Trp263Leu, which recovered the WT enzyme activity. The results reported here suggest that the introduction of a bulky aromatic residue at position 286 affects the position of residue Trp263, which is in the vicinity of the catalytic acid residue Glu261 (Trp263-C{epsilon} to Glu261-O{epsilon}2 3.35 Å). The different behavior of the Val286Phe highlights the importance of electrostatic interactions in this mutation.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Sequence alignment

A multiple sequence alignment was done with 23 starch-degrading enzymes using the programs Clustal X and pdb-viewer (Guex and Peitsch, 1997Go; Thompson et al., 1997Go). Some members of the glycoside hydrolase family are defined in Figure 1. The first three enzymes shown are liquefying amylases, while the remaining nine are saccharifying enzymes. In the lower part are shown the enzymes that are natural transferases.



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Fig. 1. Multiple structural alignment of the catalytic loops (4, 5, 6 and 7) between several members of the glycoside hydrolase family 13. On the left column are the enzymes ID {alpha}-amylases as follows: B. lichen, B.licheniformis {alpha}-amylase, PDB code 1VJS (henceforth given in parentheses) (Hwang et al., 1997Go); B. amylo, Bacillus amyloliquefaciens chimera {alpha}-amylase with decacarbose bound (1E43) (Brzozowski et al., 2000Go); B.stearo, B.stearothermophilus {alpha}-amylase (1HVX) (Suvd et al., 2001Go); B. sub, Bacillus subtilis 2633 {alpha}-amylase (1BAG) (Fujimoto et al., 1998Go); Taka-amy, Aspergillus orizae {alpha}-amylase (2TAA) (Matsuura et al., 1984Go); A. niger, Aspergillus niger {alpha}-amylase (2AAA) (Brady et al., 1991Go); H. sapiens sal, Homo sapiens salivary {alpha}-amylase (1JXK) (to be published); H. sapiens pan, Homo sapiens pancreatic {alpha}-amylase (1HNY) (Rydberg et al., 1999Go); Pig panc, Sus scrofa pancreatic {alpha}-amylase (pig) (1HX0) (Qian et al., 2001Go); Tenebrio, Tenebrio molitor {alpha}-amylase (1JAE) (Strobl et al., 1998Go); Alteromonas, Pseudoalteromonas haloplanktis {alpha}-amylase (1G94) (Aghajari et al., 1998Go, 2002); Barley, Hordeum vulgare {alpha}-amylase (1AMY) (Kadziola et al., 1994Go); B. circ1, Bacillus circulans 251 cyclodextrin glycosyltransferase (CGTase) (1CDG) (Lawson et al., 1994Go); B. circ2, Bacillus circulans 8 CGTase (1CGT) (Klein and Schulz, 1991Go); B. species, Bacillus sp. 1011 CGTase (1D7F) (Ishii et al., 2000Go); B. stearo, B.stearothermophilus CGTase (1CYG); Thermo sulfu, Thermoanaerobacter thermosulfurogenes CGTase (1A47) (Wind et al., 1998Go); Novamyl, B.stearothermophilus maltogenic {alpha}-amylase (1QHO) (Dauter et al., 1999Go); Thermus sp, Thermus sp. maltogenic amylase (1SMA) (Kim et al., 1999Go); Ther NPL, Thermoactinomyces vulgaris neopullulanase (1BVZ) (Kamitori et al., 1999Go); Ps Mtose, Pseudomonas stutzeri maltotetraose-forming amylase (2AMG) (Morishita et al., 1997Go); Olig gluc, Bacillus cereus oligo-glucosidase (1UOK) (Watanabe et al., 1997Go); and Nss suc, Neisseria polysaccharea amylosucrase (1G5A) (Mirza et al., 2001Go). The conserved residues are marked with an asterisk. The catalytic glutamic acid looks like a semi-conserved residue due to the Glu209Gln mutant-B. sub. The positions of the target (homologous aromatic residue) are indicated with a double asterisk.

 
Site-directed mutagenesis

The B.licheniformis {alpha}-amylase gene (ATCC-27811) was isolated by polymerase chain reaction (PCR) amplification from genomic DNA with the following primers: 5'-ATATGCTCTAGAAGGAGATATACATATGAAACAA CAAAAACGGC-3' and 5'-TGCCCCAAGCTTCTATCTTT GAACATATGAAAC-3'. The PCR product was cloned into the plasmid pET3a and sequenced. The amylase gene was overexpressed in the Escherichia coli strain BL21. The mega primer method for site-directed mutagenesis (Sarker and Sommer, 1990Go) was used to construct a DNA fragment carrying the mutations at positions 263 and 286 using the oligonucleotides 5'-GTCATTCTGCAGATATTCAGCT-3' and 5'-AATGAAGCGGATAGTCAAACAC-3', respectively, in combination with the oligonucleotide 5'-ATATGCTCTA GAAGGAGATATACATATGAAACAACAAAAACGGC-3' that anneals at the 5'-termini of the gene.

Enzyme purification

Enzyme production. For {alpha}-amylase production, transformants were cultured in 1 l of minimal medium M9 supplemented with appropriate antibiotics (ampicillin, 200 µg/ml and chloramphenicol, 20 µg/ml) and induced with dioxane free isopropyl-ß-D-thiogalactopyranoside (IPTG) 0.5 mM. Cell extracts containing wild-type and mutant enzymes were heated at 70°C for 1 h. Enzymes were purified using gel-filtration Superose 12 H/R (Pharmacia) in an AKTA FPLC system. Protein concentration was quantified using the Bradford method (Bio-Rad).

Activity assay

Starch. Depolymerization of starch (soluble starch from Sigma-Aldrich, St Louis, MO) was followed by measuring the formation of reducing sugars by the 3,5-dinitrosalicylic acid (DNS) method (Miller, 1959). The amount of enzyme was adjusted to give a linear response from 0 to 9 min, in order to measure the rate of formation of reducing sugars. Soluble starch was prepared by heating a suspension of starch at concentrations ranging from 0 to 10 mg/ml in the reaction buffer (20 mM Tris, 10 mM CaCl2, 1 mM NaCl at pH 7.5) until a homogeneous, viscous solution was obtained. Once the temperature had equilibrated at 80°C, the reaction was started by the addition of {alpha}-amylase and 200 µl samples were taken every 3 min and placed in DNS solution to stop the reaction and perform the analysis. After incubation for 5 min in boiling water, samples were cooled and the OD was read at 540 nm. The amount of reducing sugars was expressed as dextrose equivalents using a calibration curve. Lineweaver–Burk-type plots were built to obtain Michaelis–Menten parameters. A unit of enzyme activity is defined as 1 µmol of dextrose equivalent released per minute.

pNPG7. The hydrolytic activity of the {alpha}-amylase over benzylidene-blocked p-nitrophenyl maltoheptaoside (pNPG7) was determined for the WT enzyme and for the Val286Tyr variant using the Randox (UK) assay amylase kit at 37°C and 1.32 and 0.78 nM of enzyme, respectively. Parameters kcat and KM were calculated from initial rates in the 0.027–2.13 µM substrate concentration range. The activity assay also involved two indicator enzymes, glucoamylase to cleave the amylase reaction product and {alpha}-glucosidase to release the p-nitrophenol (the terminal glucose of the substrate is chemically blocked at both ends, preventing cleavage by the indicator enzymes). The p-nitrophenol released is determined spectroscopically at 405 nm, using a molar extinction coefficient of {epsilon} = 12 005 l/mol·cm (Lorentz, 2000Go) and is equivalent to the substrate hydrolyzed by the {alpha}-amylase (Kaufman et al., 1980; David, 1982; Rauscher et al, 1985).

Evidence of transglycosylation reactions using malto-oligosaccharides

In order to highlight the transglycosylation reactions, small oligosaccharides were used as substrates. Although they are bad substrates for hydrolysis, they allow us to distinguish transglycosylation products through the appearance of higher molecular weight oligosaccharides. Oligosaccharides from G3 to G7 were used to determine the product profile of each enzyme; 100 mM of each substrate were incubated with the same hydrolytic activity units (determined with starch) of each enzyme at 80°C. The reaction buffer was 20 mM Tris, 10 mM CaCl2, 1 mM NaCl at pH 7.5. Aliquots were taken at 10, 30 and 60 min and 49 h (when equilibrium was reached), diluted 1:10 and analyzed by HPLC in a Waters Millipore system with a refractive index detector, using a reversed-phase C18 column (250x4.6 mm i.d.). Elution was performed with pure water at a flow rate of 0.7 ml/min. The peak areas were measured and compared against those of a standard solution containing known amounts of oligosaccharides from G1 to G7.

Evidence of transglycosylation reactions using radioactive maltose (G2r) as acceptor

Radioactive maltose (Amershan Pharmacia Biotech) and maltotetraose (Sigma) were incubated at 80°C in 20 mM Tris buffer at pH 7.5 containing 10 mM CaCl2 and 1 mM NaCl. Reactions were initiated by addition of the same activity units of the different mutant enzymes to digest 100 mg/ml of substrate pre-incubated for 10 min at the reaction temperature and analyzed after 48 h of reaction. The reaction was stopped by incubation on ice and immediately loaded on a TLC plate. The plate was developed with {alpha}-naphthol and also visualized on a Molecular Dynamics PhosphorImager.

Test of thermostability

For the determination of the inactivation rate constants, the kinetics of irreversible loss of enzyme activity were measured as follows. Pure samples were dialyzed against 20 mM Tris, 1 mM CaCl2, 1 mM NaCl buffer at pH 7.5 and then the total content of protein was measured by the Bradford method. Samples were placed in Eppendorf tubes and incubated at 80°C in a water bath. The remaining activity of the incubated samples was measured at 80°C using DNS with 10 mg/ml of starch for different times (0, 10, 20, 30, 40 and 50 min ). The values were processed as reported previously (Declerck et al., 2000Go).


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Identifying potential residues important for specificity

We carried out a sequence analysis and structural comparison of amylases that have the ability to perform transglycosylation reactions with those which are mainly hydrolytic (Figure 1). From this analysis, conserved regions in the C-terminal end of the ß-strands (ß4, ß5, ß6 and ß7) of the TIM barrel domain were found, including the three classical regions previously identified in the {alpha}-amylase family (loops 4, 5 and 7), which contain the catalytic residues Asp231, Glu261 and Asp328, respectively (B.licheniformis {alpha}-amylase numbering) (Vihinen et al., 1990Go; Takata et al., 1992Go; Henrissat et al., 1995Go; Kuriki et al., 1996Go; Henrissat and Davies, 1997Go; Janecek, 2000Go). However, we also found one more conserved region corresponding to loop 6 (see Figure 1). Within this region, we identified a residue at position 286 (B.licheniformis {alpha}-amylase numbering) showing an altered pattern between liquefying and saccharifying enzymes. In the former, small residues are observed, whereas for the latter, aromatic residues are predominant. Based on these results, we constructed the Val286Tyr and Val286Phe mutants from B.licheniformis {alpha}-amylase, in order to evaluate how this position affects transferase activity and other enzyme properties including product specificity.

Activity

A comparison of the catalytic parameters of the Val286Tyr and Val286Phe variants with those of the WT enzyme was carried out using soluble starch as a substrate, measuring the release of reducing sugars. In the case of the Val286Tyr variant and the WT enzymes, benzylidene-blocked pNPG7 was also used, measuring the release of p-nitrophenol. All enzymes exhibited a Michaelis–Menten kinetic behavior. Table I shows the kinetic parameters obtained on starch, where it may be observed that the Val286Tyr mutant has a catalytic efficiency five times higher than the WT enzyme. This result is mainly due to an increase in the turnover number rather than to a change in KM. In contrast, the Val286Phe mutant, in which a Val residue is also replaced by an aromatic one, had a 3-fold decrement in catalytic efficiency as a result of a higher KM, leaving kcat practically unchanged. The Val286Tyr mutant showed the same behavior with benzylidene-blocked pNPG7 as with starch, yielding a 10-fold increment in turnover number relative to the WT enzyme (kcatWT = 8.4x10–1; kcatV286Y = 8.02 s–1, KMWT = 0.21 and KMV286Y = 0.43 µM). Owing to their similar structure, we think that the different behaviors observed for the two mutants can be attributed to the polarity and hydrogen-bonding capability of the hydroxyl group.


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Table I. Kinetic parameters obtained for starch by different WT and mutant {alpha}-amylases at different temperatures
 
The previous work on {alpha}-amylase from B.stearothermophilus suggested that the mutation of the equivalent position to an aromatic residue affected the orientation of a Trp residue that interacts with the catalytic Glu through a water molecule. In order to demonstrate if such an interaction could exist in the context of the B.licheniformis {alpha}-amylase, we constructed the double mutant Val286Tyr/Trp263Leu. The catalytic parameters of this mutant, also shown in Table I, are similar to those of the WT enzyme, suggesting that indeed there is an interaction between a bulkier amino acid such as Tyr with Trp at position 263 that changes the catalytic site giving rise to the modified activity.

Role of residue at positions 286 and 263 in the active site

In order to explain the differences found in catalytic properties between the mutantsVal286Tyr, Val286Phe, Trp263Leu/Val286Tyr and the WT enzyme in terms of the structure, we built models using a public Website, Swiss-Model (http://www.expasy.ch/swissmod/SWISS-MODEL.html), using the coordinates of the WT from B.licheniformis {alpha}-amylase (1VJS pdb entry) as template. Swiss-Model works with a Relax module that uses a library of allowed side-chain rotamers, sorted by increasing frequency of occurrence in known 3D structures. First the distorted but complete side chains are corrected. A van der Waals exclusion test and dihedral angle constraints are then used to select among allowed rotamers. The geometries of the side chains of the models were evaluated using the WHAT_IF (Vriend, 1990Go) and WHAT_CHECK programs (Hooft et al, 1996Go). We generated a structural superposition of the B.licheniformis {alpha}-amylase and chimera {alpha}-amylase (taking residues 1–300 from the Bacillus amyloliquefaciens {alpha}-amylase and residues 301–483 from the B.licheniformis {alpha}-amylase) and the model generated for the mutants Val286Tyr and Val286Phe (see Figure 2a). The models for the Phe and Tyr mutants are practically identical, except for the hydroxyl group of the Tyr which seems to be very close to the main-chain carbonyl of the catalytic Glu residue (OH-C{alpha} 2.7 Å and OH-C{gamma} 2.4 Å) (Figure 2b). The superposition of the decacarbose on the generated models, based on the structure of the chimeric {alpha}-amylase, B. amylo, suggests that close contact could occur between the Tyr and the sugar ring above it, involving subsites +2 and +3 (see Figure 2a).




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Fig. 2. View of the aligned active site of B.licheniformis {alpha}-amylase WT and modeled mutants (Val286Tyr, Val286Phe and Val286Tyr/Trp263Leu). The corresponding residues in WT enzyme are in green and the mutants in blue, red and yellow, respectively. (a) The units of glucose from acarbose superimposed show the subsites +2 and +3 and are located over the residue 263 as reference. (b) The distances between the hydroxyl group of Tyr at position 286 and the main-chain atoms of residue 261 are indicated.

 
The structural alignment between the modeled mutants and WT enzyme do not show positional differences for the side chain of Trp263 (see Figure 2a). Usually in {alpha}-amylases, those enzymes which carry an aromatic residue at the position equivalent to Val 286 do not contain an aromatic residue at the position equivalent to Trp263 (see Figure 1), with the only exception of barley {alpha}-amylase in which the equivalent position to 286 is occupied by a Phe residue and a Trp at the position equivalent to 263 (see Figure 1). It is noteworthy that in the crystal structure of this enzyme (Kadziola et al., 1998Go), the equivalent Phe residue orients the Trp equivalent to 263 by stacking interactions. This Trp residue is bridged to the main-chain carbonyl oxygen of the active site Glu residue through a water molecule. In our generated models, the orientation of the Trp residue is only slightly rotated relative to the WT structure and continues to be perpendicular to the Tyr286 residue. We cannot discard that the orientation of the Trp residue found in the models is biased by the WT structure used as template. We have to recognize that water molecules that play an important role in hydrogen-bond networks along the structure, and also the possible stabilization by stacking interactions among aromatic residues, are not taken into account in the modeling process. Our experimental results with the double mutant Val286Tyr/Trp263Leu clearly point to an interaction between the two positions when occupied by aromatic residues. The replacement of Trp263 by Leu removes the positive effect in the catalytic efficiency obtained upon Val286Tyr mutation. If the contributions to the activity of the two aromatic residues were independent of one another, their effect would be additive and then the {Delta}({Delta}Gr) values between the Val286Tyr relative to WT and Val286Tyr/Trp263Leu relative to the single mutant Trp263Leu would be the same. We constructed the single mutant Trp263Leu to corroborate this hypothesis. As can be observed in Table I, the deleterious effect of this mutation is much more dramatic in the context of the WT enzyme than in the context of the Val286Tyr mutant, demonstrating that there is a synergistic effect when these positions are both occupied by aromatic residues.

As for the difference in activity between the Val286Phe and Val286Tyr mutant enzymes, in the generated model of the Val286Tyr mutant the hydroxyl group points towards the main-chain of Glu261 (Figure 2a). Although the model does not predict any hydrogen bonding between these residues, an interaction through a water molecule could exist. Glu261 is the general acid catalyst that protonates the leaving group of natural substrates during the first step of the double displacement reaction and that activates the acceptor group (either water or another glycosyl residue) during the second step (Uitdehaag et al., 1999aGo,b; Rydberg et al., 2002Go). Thus, Tyr286 could be acting as a reservoir of water, favoring the hydrolytic reactions.

Specificity and product profile

Hydrolysis product profile using oligosaccharides (G1–G7) as substrate. In order to establish if the reaction rate observed in the mutants is a result of a change in the product specificity, we compared the hydrolysis digestion patterns among the constructed variants and the WT enzyme using starch and various oligosaccharides as substrates (G3, G4, G5, G6 and G7). Although, in general terms, the main starch digestion products reported previously for the commercial WT enzyme at pH 6.5–6.9 and 90°C (Marchal et al., 1999Go) are the same as those obtained with the recombinant enzyme used in the present study (G5, G3 and G2), a significant production of G1 was observed (data not shown). This pattern was the same for WT and all the mutants with the exception of the double mutant Val286Tyr/Trp263Leu, which showed a lower proportion of G1 (data not shown). As far as the use of small oligosaccharides as substrates is concerned, only the WT and Val286Phe enzymes showed activity with G3. For the other mutants, G4 was the smallest oligosaccharide they were able to hydrolyze at a very slow rate. During short incubation times (up to 1 h), only the WT enzyme shows high transglycosylation and hydrolysis activities with G4 as a substrate (data not shown). After periods of incubation as long as 49 h, when equilibrium is reached, the product profiles of WT and Val286Phe mutant are very similar, leaving only 6.09 and 6.25% of G4, respectively, and producing mainly G1 and G2 (see Table II and Figure 3) in addition to products of transglycosylation, such as G5, G6, G7 and larger oligosaccharides. In contrast, the mutants containing Tyr at position 286 were less efficient in the use of G4 (59.3 and 49.4% unreacted substrate, respectively), accumulating some G5 as result of transglycosylation and producing different amounts of G3, G2 and G1 (see Table II and Figure 3). When G5 was used as substrate, the Val286Tyr and Val286Tyr/Trp263Leu mutants showed some hydrolysis products early in the reaction. However, the increase of these products was very slow, reaching only ~9.6 and 14.45% of G1, ~37.89 and 35.9% of G2, 15.51 and 16.74% of G3 and 2.85 and 7.13% of G4, respectively, after 49 h of reaction. In contrast, the WT enzyme clearly showed the presence of transglycosylation products, in addition to the hydrolysis products, yielding a more efficient degradation of G5 at equilibrium, although also the accumulation of G6 and G7 as a result of transglycosylation reactions (see Figure 3). The Val286Phe mutant showed a slower but similar behavior to the WT enzyme, generating at the end a similar product profile (Table II). When G6 and G7 were used as substrates, activity was evident for all the mutants at short incubation times (10 min, data not shown). It is clear from the high molecular weight products that the mutants containing Tyr at position 286 are able to transgycosylate. However, at long incubation times, all these products are hydrolyzed, yielding a product pattern in which G2, G3 and G5 are the predominant species. In contrast, the WT and Val286Phe mutant enzymes yield a more spread product profile, with G1 and G2 being the main products, but showing the presence of larger oligosaccharides, products of transglycosylation reactions (Figure 3). Since these two enzymes are capable of using G6 and G7 as substrates, the accumulation of these and larger oligosaccharides at long incubation periods suggests a higher transglycosylation/hydrolysis ratio for them. The production of G5, G6 and G7 suggests transglycosylation activity for all the variants when G4 was used as substrate. This activity was also reported by Marchal et al. (Marchal et al., 1999Go), who observed an increased contribution of transglycosylation reactions to hydrolysis of small oligosaccharides (G3–G7) with commercial {alpha}-amylase from B.licheniformis (Maxamyl®, Genencor) at higher temperatures, reducing the end product specificity.


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Table II. Hydrolytic activities of the WT and mutant enzymes determined by HPLC after 49 h of reaction at 80°C
 


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Fig. 3. Comparative products profile using small oligosaccharides as substrates determined by HPLC after 49 h of reaction at 80°C. At the top of each series is indicated the oligosaccharide used as a substrate and on the left the variant enzyme that produced the digestion profile shown.

 
Transglycosylation profile using radioactive maltose (G2r) as acceptor with G4 as substrate detected by {alpha}-naphthol and visualized on a Molecular Dynamics PhosphorImager

The different product profile obtained with the variant enzymes suggests either a change in the reaction mechanism or a change in the specificity of the subsites. The results obtained are consistent with the existence of at least eight subsites for both WT and variants of {alpha}-amylase from B.licheniformis. When all of them are occupied (using substrates longer than G7), no difference in the product profile is observed. However, when the enzymes reach the limiting size substrates, differences start to show up, outlining the different affinity in at least one of the subsites among the different variants. In order to investigate this hypothesis, we decided to study the hydrolysis of G4 (see Figure 4) in the presence of traces of radioactively labeled G2. If transglycosylation reactions take place, then radioactively labeled maltose (that could act as an acceptor in transglycosylation reactions) would distribute among the products according to the enzyme specificity. It is important to take into account that transglycosylation products would be hydrolyzed relatively fast and this will be reflected in the product profile. Figure 4a and b show the incorporation of the labeled G2 after 48 h of reaction. WT and Val286Phe mutant enzymes showed a very similar product profile with the production of radiolabeled G1. In contrast, Val286Tyr and the double mutant Val286Tyr/Trp263Leu incorporated much less labeled G2, produced less G1 (observed both by radioactivity and by development with {alpha}-naphthol) and accumulated more G4. It is interesting to observe the difference in concentration of G4 for the Val286Tyr and the double mutant Val286Tyr/Trp 263Leu when observed by radioactivity (Figure 4b) compared to revealing with {alpha}-naphthol (Figure 4a). This indicates that, indeed, the higher accumulation of G4 for these two variants is due to a lower activity towards this substrate rather than the feedback as a result of transglycosylation, suggesting a decrement in affinity of a subsite on the reducing end positions (after the cleavage site). A recent report of the subsite mapping of B.licheniformis {alpha}-amylase demonstrates that subsite –5 is indeed the highest affinity subsite, followed by subsite –2, then subsite +3 and finally subsites +2 and –3, for which the authors report equal apparent binding energies (Kandra et al., 2002Go). Since the residue that we are mutating is between subsites +2 and +3, we do not expect the affinity of subsite –5 to be affected, and therefore the higher accumulation of G5 in the Tyr mutants must be attributed to a decrease in the affinity of subsites +2 and/or +3. In agreement with this hypothesis, a 2-fold increment in KM for the Val286Tyr mutant was observed when benzylidene-blocked pNPG7 was used as substrate relative to the WT enzyme. In the case of the Val286Phe mutant we do not have affinity constants for small oligosaccharides; however, its loss of catalytic efficiency is mainly attributed to an increase in the KM for starch, so that as it is as bad as the Val286Tyr mutant in binding small oligosaccharides in a productive way, the only explanation for its higher efficiency in the process of both G5 and G4 is its higher transglycosylation capability, so that the few productive events give rise to larger products that are more efficiently processed. These results further demonstrate the lower transglycosylation activity of the Tyr mutation at position 286. The presence of the hydroxyl group of tyrosine must favor the entrance of water over other acceptor groups, shifting the reaction towards hydrolysis, which is limited by the low affinity of the enzyme for such small substrates. Val286Phe and the WT enzyme, having more hydrophobic side chains, favor the entrance of less polar acceptors than water, as previously proposed by Matsui et al. (Matsui et al., 1994Go).



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Fig. 4. Hydrolytic activity on maltotetraose (G4) and radioactive maltose (G2r) of WT and mutants (Val286Tyr, Val286Phe, Trp263Leu and Trp263Leu/Val286Tyr) detected by TLC at 48 h. We used maltodextrins (G1–G7) as standard (Std), G2r without enzyme, G2r with WT enzyme, G4 without enzyme and G2r + G4 without enzyme as controls. (a) Hydrolysis of G4 detected by {alpha}-naphthol and (b) transglycosylation of labeled maltose visualized on a Molecular Dynamics PhosphorImager.

 
Thermostability

For industrial applications it is important to demonstrate the thermostability of the more active Val286Tyr mutant. When this variant and the WT enzyme were incubated at 80°C in buffer containing 10 mM Ca2+, no activity loss was observed after incubating over a period of 6 h for either of the two enzymes. This time is three times longer than that required for industrial liquefaction of starch. In order to stress any possible difference in stability, the irreversible thermal inactivation of the mutants Val286Tyr and Val286Phe, the double mutant Val286Tyr/Trp263Leu and WT were investigated by incubating the purified enzymes at 80°C for different intervals of time, reducing the concentration of Ca2+ to 1 mM to destabilize them. After different intervals of incubation, samples were taken and placed on ice for 15 min and their residual activity towards starch measured at 80°C. Figure 5 shows the residual activities against incubation time at 80°C for the WT and variants. All the curves were described by an exponential first-order decay model, yielding a half-life of 7 min for the Val286Tyr mutant, 8 min for the double mutant Val286Tyr/Trp263Leu and 40 min for the Val286Phe mutant compared with 10 min for the WT enzyme. In conclusion, under a high-stress assay, a decrease in stability was observed for the proteins carrying the Val286Tyr mutation. Interestingly, the Val286Phe was the most thermostable.



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Fig. 5. Irreversible thermal inactivation at 80°C, pH 7.5, 1 mM CaCl2, of WT (filled circles) and mutants Val286Tyr (filled triangles and dotted line), Val286Phe (filled squares) and Trp263Leu/Val286Tyr (filled inverted triangles and solid line). The linear regression of ln(activity) versus incubation time was used to determine the t1/2 of enzymes graphically. The t1/2 for the WT was ~10 min and that for the mutant Val286Tyr was ~7 min. The t1/2 forVal286Phe was ~40 min and that for the double mutant Trp263Leu/Val286Tyr was ~8 min.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
To our surprise, mutant Val286Tyr had an increased activity when compared with the WT enzyme. The same mutation in the context of the {alpha}-amylase from B.stearothermophilus resulted in a reduction of the hydrolytic activity and a change in product profile, explained by the introduction of transglycosylation activity (Saab-Rincon et al., 1999Go). This different behavior for the same mutation in both enzymes is unexpected considering the degree of identity between them (63%). However, these enzymes show some functional differences from the beginning. Whereas WT {alpha}-amylase from B.stearothermophilus was not able to transglycosylate at all, the WT {alpha}-amylase from B.licheniformis had some transglycosylation activity, particularly at high temperatures and with excess of substrate (Marchal et al., 1999Go). Thus, in the case of the {alpha}-amylase from B.stearothemophilus, it was easy to identify the new activity over a null background, whereas in the B.licheniformis enzyme it is difficult to observe clear differences in the transglycosylation or in the product profile when starch is used as substrate. However, we found some differences among the variant generated enzymes on reaction with small oligosaccharides. With the smallest oligosaccharide that all the enzymes were able to hydrolyze, G4, the WT and V286F variant enzymes yielded G1 and G2 in high proportion, whereas the V286Y mutant produced only a small amount of G1 and processed less G4. Since this mutant has been demonstrated to be more active than WT and V286F variant, this lower activity towards G4 can only be explained by a lower affinity for this substrate and/or to its lower capability of carrying out transglycosylation reactions. Since both transglycosylation and hydrolysis reactions are occurring simultaneously, it is complicated to quantify their individual contributions to the final result. However, in the case of the mutants containing Tyr at position 286, a loss of affinity for substrates smaller than G5 is clear, but also a lower incorporation of G2, suggesting a lower transglycosylation activity. In the case of the Val286Phe mutant enzyme, any possible loss of affinity is clearly compensated by its higher transglycosylation activity. Explaining these differences in structural terms is difficult, since an equivalent change in orientation of Trp263 is expected whether Tyr or Phe is at position 286. Both mutations seem to have a negative effect on KM and therefore on the catalytic efficiency. In the case of Tyr, the hydroxyl group points out towards the catalytic site, probably contributing to a more polar environment, which not only compensates for the loss in affinity, but also increases kcat. From the analysis of the behavior of the different mutants generated, two effects can be isolated: (i) the increase in activity seems to be directly related to the presence of the hydroxyl group in a certain geometry relative to the active site; a change in the position of this hydroxyl group, as possibly occurred in the Val286Tyr/Trp263Leu double mutant, cancelled the increment in activity observed upon the first mutation; and (ii) the decrease observed in the transglycosylation reaction is directly related to the polarity of the residue at position 286. This last statement is supported by the results with the double mutant, whose activity is closer to the WT; however, it is less transglycosidic, yielding a product profile more similar to the obtained with the Val286Tyr mutant.

The irreversible thermal inactivation of the B.licheniformis {alpha}-amylase was studied by Declerck et al. in detail (Declerck et al., 2000Go). These authors identified, by site-directed mutagenesis, the structural determinants for the stability, such as residues involved in salt bridges, calcium binding or potential deamidation processes. They demonstrated that the structural determinants that contribute to the thermal stability are concentrated in domain B and its interface with the catalytic domain (domain A). It was interesting to observe a 30% decrement in the half-life of the Val286Tyr mutant, whereas the Val286Phe mutant showed a 4-fold increase in half-life under the same conditions. Again, one would expect that any steric effect from the introduction of an aromatic ring would disturb the structure in a similar way; however, the presence of the hydroxyl group seems to be producing major rearrangements, at least when the concentration of Ca2+ is held below 1 mM. The change of the bulky Trp263 by a smaller Leu residue counteracted the disturbance produced by the introduction of the Tyr at position 286, showing a half-life similar to the WT enzyme. In practical terms, the conditions used for the determination of stability are more stressing than those used in the industrial process of starch, conditions under which we did not observe any change in thermal inactivation of the Val286Tyr mutant for several hours.

Conclusions

By modifying residues near the active site, we obtained an enzyme that is five times more active than the WT without altering in practical terms its thermostability or its product specificity towards starch. The different behaviors of the Phe and Tyr mutants allowed the identification of the importance of the polarity and/or hydrogen bonding for the conformation of the (general acid) active residue Glu261, and also the importance of the hydrophobicity at position 286 to increase transglycosylation. The results obtained here support the idea stated previously (Saab-Rincon et al., 1999Go) that an increase in transglycosylation is at the expense of the hydrolysis rate and vice versa. On the other hand, the introduction of transglycosylation shifts the product profile towards shorter oligosaccharides owing to the efficient recycling of medium-sized oligosaccharides to generate longer ones that can be more efficiently digested by the enzyme. The results shown here can be extended to identify other residues important to dictating the specificity of the reaction products and thus engineering proteins to modify their specificity and improve their catalysis, with important implications for the starch industrial process.


    Acknowledgements
 
We thank Fernando González Muñoz for technical assistance in the HPLC analysis of samples, Eugenio López and Paul Gaytán for the synthesis of oligonucleotides and René Hernández and Maricela Olvera for sequencing of clones. This work was supported by CONACyT Grant NC230 to X.S. and PAPIIT Grant IN222999 to A.L.-M.


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Received April 28, 2003; revised May 29, 2003; accepted June 2, 2003.





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