INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion References

Raw-starch-degrading amylases are commercially important enzymes in the beverage, food, and textile industries (13). Starch is commonly used as a renewable raw material for the industrial production of lactic acid (6). The amylolytic lactic acid bacteria are attractive alternatives for this type of process, since they can directly produce lactic acid from starch (37, 38). Various Lactobacillus strains exhibit amylase activity: Lactobacillus cellobiosus (32), Lactobacillus amylovorus (26), Lactobacillus amylophilus (27), Lactobacillus plantarum (10, 30), Lactobacillus manihotivorans (24), and Lactobacillus amylolyticus (2). The -amylase genes of L. plantarum A6 (12), L. amylovorus (12), and L. manihotivorans (J. Morlon-Guyot, I. Jacobé, de Haut, J. Boniface, and J. P. Guyot, Abstr. Aff. VI4, 9ème Colloque Club Bact. Lact., 1998) have been sequenced. These lactobacillus amyA genes have more than 98% nucleotide sequence identity and a similar level of identity for the deduced amino acid sequence (Morlon-Guyot et al., Abstr. Aff. VI4, 9ème Colloque Club Bact. Lact., 1998). With such high homologies, it is assumed that the three enzymes possess similar structure-function relationships. It has been demonstrated that the first 410 amino acids of L. amylovorus are sufficient to transfer an amylolytic activity to a nonamylolytic strain of L. plantarum (17), suggesting that the N-terminal parts of these enzymes contains the active site. The 3'-terminal halves of the three genes exhibit a special tandem repeated-unit structure (12; Morlon-Guyot et al., Abstr. Aff., VI4, 9ème Colloque Club Bact. Lact., 1998) whose role is still unknown. Giraud and Cuny (12) suggested that the region of repeated sequences might be responsible for raw-starch binding. A recent study showed that some proteolytic fragments of the L. plantarum A6 amylase lose the raw-starch-digesting ability (9), but their positions in the primary structure were not determined. In the present work, the functional role of the carboxyl-terminal repeat sequences of the L. amylovorus amylase (AmyA) is investigated using clones encoding either the entire AmyA or the truncated amylase AmyA, which has the repeated sequences deleted (17). Comparison of the enzymatic properties of the two amylases reveals that the C-terminal part of the enzyme containing the repeated sequences is involved in the ability of this enzyme to bind to raw starch.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Material. Soluble potato starch, calcium carbonate, dinitrosalicylic acid (DNS), and glucose were from Prolabo, Paris, France. Raw corn starch, pullulan maltose, amylose (type III from potato), amylopectin (from corn), glycogen (from mussels), and -, -, and -cyclodextrins were from Sigma Chemical Co. (St. Louis, Mo.). Sepharose was from Pharmacia Biotech (Uppsala, Sweden).

Bacterial strains, plasmids, and culture conditions. L. plantarum LMG9211 was from the Laboratorium voor Microbiologie Universiteit Gent (Ghent, Belgium) culture collection. Plasmids pLPCR2-3 carrying the 5.5-kb amyA gene from L. amylovorus and pLPCR2-3B/X carrying the 4.0-kb truncated amyA gene were constructed as described previously (17). MRS medium (7) was used to grow the lactobacillus strains at 30°C. This medium was first made without glucose, and, depending on the experiments, the carbon source added was either 1% (wt/vol) glucose, maltose, or soluble or raw starch, with or without 4% calcium carbonate. For enzyme preparation, cultures were grown in a 2-liter bioreactor (Biolafitte, Poissy, France) at 30°C and agitated at 300 rpm. The pH was maintained at 6.0 by automatic addition of 5 N NaOH.

Transformation of L. plantarum and selection of transformants. Plasmids pLPCR2-3 and pLPCR2-3B/X (Fig. 1) were introduced into L. plantarum LMG9211 by electroporation, as described previously (32). The transformants were selected by adding erythromycin (2.5 µg/ml) to the solid culture medium. The production of -amylase by the recombinant clones was detected by visualization of halos due to starch degradation on 1% starch-containing plates after staining them with iodine vapors.

View larger version (10K): [in this window] [in a new window]   FIG. 1.   Restriction map of pLPCR2-3 and pLPCR2-3B/X plasmids. (A) pLPCR2-3 plasmid, carrying the 2.8-kb amyA gene of L. amylovorus, ligated at the BglII site of the pLPCR2 plasmid (dashed line). (B) pLPCR2-3B/X plasmid derived from pLPCR2-3 after deletion of the 2-kb BamHI-XhoI fragment.

Enzyme purification. Following an 18-h batch culture, the fermentation broth was collected and centrifuged at 9,000 × g for 15 min at 4°C.

1

Electrophoresis analyses. Sodium dodecyl sulfate-7.5% polyacrylamide gel electrophoresis (SDS-7.5% PAGE) was performed according to the method of Laemmli (19). Proteins were visualized by Coomassie blue staining as described by Blakesly and Boezi (1). Activity staining was performed in the gel after renaturation of the enzymes, using the method described by Lacks and Springhorn (18).

Enzyme activity assay. Routine -amylase activity assay was used to determine the activity of -amylase in the fractions collected throughout the purification. The activity of -amylase towards soluble potato starch was determined by measurement of its iodine-complexing ability, using the protocol described by Giraud et al. (11) with minor modifications: enzyme incubation was performed at pH 5.0 and 63°C. One enzyme unit is defined as the amount of enzyme that permits the hydrolysis of 10 mg of soluble starch over 30 min.

Effects of pH and temperature. The amylase activity was determined at various pH values (ranging from 3 to 7) and at various temperatures (from 45 to 75°C) in 0.1 M citrate-phosphate buffer. A second-order factorial design was used (3, 25) in order to study the combined effect of pH and temperature on amylase activity.

Adsorption of -amylase on raw starch. Various amounts (from 0 to 140 µg/ml) of recombinant enzyme (AmyA or AmyA) were added to a raw-corn-starch suspension (10 mg/ml) 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 under gentle shaking (6 rpm) and centrifuged at 13,000 × g for 5 min. The protein concentration in the supernatant was assayed, and the amount of adsorbed enzyme was calculated by subtraction (35). Adsorption constants (K_ad [in milliliters per milligram of starch]) were calculated from the slopes obtained from the linear adsorption using 10 initial concentrations of purified enzyme (5).

SEM. Samples were prepared from L. plantarum(pLPCR2-3) and L. plantarum(pLPCR2-3B/X) cultures incubated for various times (up to 48 h) with 1% raw starch. The samples were then centrifuged, and the resulting pellets were lyophilized and submitted to homogenous gold metallization. SEM examination was performed using a JEOL JSM-6300F microscope (Montpellier).

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Amylase production. -Amylase constructs (Fig. 1) were transferred by electroporation into L. plantarum LMG9211, and the resulting transformants, L. plantarum(pLPCR2-3) and L. plantarum(pLPCR2-3B/X), were tested for the production of -amylase in the medium. Recombinant clones from both transformants formed halos on starch-containing plates, which indicate secretion of active -amylase (data not shown). In order to select transformants with the best amylase secretion efficiencies, L. plantarum transformants were grown for 24 h on various carbon sources, using L. plantarum LMG9211 as a negative control. The recombinant transformants displayed comparable growth rates in all media tested. The cultures were harvested in the late logarithmic growth phase (optical density at 600 nm, 2), and amylolytic activities on soluble starch were measured in the supernatant. Amylase produced by the L. plantarum(pLPCR2-3B/X) recombinant strain was found to be secreted at a much higher yield (from 2- to 15-fold, depending on the carbon source) than that produced by L. plantarum(pLPCR2-3) (Table 1). The carbon source had an effect on the amylase production of L. plantarum(pLPCR2-3): the highest amylolytic activity was obtained using soluble starch as a carbon source, while glucose decreased amylase production (from 1.5- to 8.5-fold, depending on the carbon source to which it is compared). Such an effect was not observed with the L. plantarum(pLPCR2-3B/X) transformant, which exhibited the same level of -amylase activity independent of the carbon source. For both recombinant strains, the presence of calcium carbonate in the growth medium slightly increased amylase production, except for the transformants grown on soluble starch, where the effect was negligible.

View this table: [in this window] [in a new window]   TABLE 1.   Levels of -amylase activity in the culture supernatants of L. plantarum strains in response to medium compositiona

Purification of the recombinant AmyA and AmyA amylases. The amylase (AmyA) produced by the L. plantarum(pLPCR2-3) transformant (grown on soluble starch) was purified from the culture supernatant by affinity chromatography on -cyclodextrin-Sepharose. The elution pattern showed a unique peak of protein which was superimposable on the amylase activity of the enzyme. The recombinant enzyme, purified to homogeneity, migrated as a single band on SDS-PAGE which was active on the zymogram (data not shown). Thus, using a single chromatography step, about 75-fold purification was obtained with a yield of 69% (Table 2).

View this table: [in this window] [in a new window]   TABLE 2.   Purification steps of recombinant -amylases secreted by L. plantarum strainsa

1

1

Effects of temperature and pH. Response surfaces of AmyA and AmyA amylase activities as a function of pH and temperature are shown in Fig. 2. AmyA showed optimal activity at pH 5.0 and 62°C, whereas AmyA had optimal activity at pH 4.8 and 64°C. A given percentage of AmyA activity was maintained over a broader range of temperatures than was AmyA activity; as an example, a minimum of 80% of optimum activity was obtained between 47 and 75°C for AmyA, whereas for AmyA, the same percentage was observed between 59 and 69°C.

View larger version (33K): [in this window] [in a new window]   FIG. 2.   Effect of pH and temperature on the amylase activities of AmyA (A) and AmyA (B).

View larger version (18K): [in this window] [in a new window]   FIG. 3.   Thermostabilities of AmyA (A) and AmyA (B) at 75 (), 65 (), 55 (), and 45°C ().

Substrate specificity. AmyA and AmyA relative activities towards various polysaccharide substrates were tested (Table 3). For both enzymes, the best hydrolyzing substrates were found to be amylopectin, soluble starch, and -cyclodextrin, while amylose was hydrolyzed at a lower rate. Both enzymes were unable to hydrolyze either pullulan or - and -cyclodextrins. With glycogen, a relatively high activity was observed with AmyA, whereas it was very low with AmyA. AmyA was active towards raw starch, while AmyA was unable to hydrolyze it.

1

1

1

1

1

1

1

1

Ability to bind to raw starch. Adsorption of AmyA and AmyA to raw-starch granules was assayed at various protein concentrations (5). As shown in Fig. 4, AmyA was adsorbed to raw starch whereas AmyA was unable to bind to it. For AmyA, the K_ad (see Material and Methods) was 0.26 ml of protein suspension per mg of raw starch, and the curve reached a plateau when 30 µg of protein was bound to 1 mg of raw starch, indicating limited availability or accessibility of raw starch for the enzyme. This maximum is reached when the protein forms a monolayer on the starch surface (35).

View larger version (13K): [in this window] [in a new window]   FIG. 4.   Adsorption of AmyA () and AmyA () on raw starch. The linear adsorption isotherms indicate the apparent equilibrium distribution of enzymes between the solid phase (bound protein) and the liquid phase (unbound protein) at various protein concentrations.

SEM observation of raw-starch granules after fermentation with the recombinant L. plantarum strains. Enzymatic attack on raw-starch granules was observed by SEM during fermentation. In each fermentation sample, the aspects of starch granules were relatively homogenous (Fig. 5A, C, and D). When the fermentation was carried out with L. plantarum(pLPCR2-3B/X), the initially smooth granules (Fig. 5A and B) did not show any measurable degradation after 24 (Fig. 5C and E) or 48 (Fig. 5G) h. In contrast, the starch granules became rougher and perforated after 24 h when fermentation was carried out with L. plantarum(pLPCR2-3) (Fig. 5D and F). After 48 h of fermentation with this transformant, many starch granules displayed large cavities, and their lamellar organization could be observed (Fig. 5H).

View larger version (137K): [in this window] [in a new window]   FIG. 5.   Scanning electron micrographs showing the effects of -amylases produced by recombinant L. plantarum strains on raw corn starch granules after fermentation. Shown are native raw starch (A and B) and raw-starch granules after 24 (C and E) or 48 (G) h of fermentation with L. plantarum(pLPCR2-3B/X) and after 24 (D and F) or 48 (H) h of fermentation with L. plantarum(pLPCR2-3).

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

Expression and secretion. Since L. plantarum is easily electroporable (31) and had already been used for efficient L. amylovorus -amylase gene expression and secretion (8, 17), it was chosen as the host for transformation of the two -amylase constructs, pLPCR2-3 and pLPCR2-3B/X. Despite identical promoter and signal sequences, AmyA was expressed at a higher level of amylase than AmyA. This variation in amylase production could be due to a difference in plasmid copy number among the transformants or to the folding of the recombinant protein.

Enzymatic activities. AmyA specific activity was very similar to that of AmyA on all substrates tested. These data are in agreement with those obtained previously with AmyA and AmyA on soluble starch (17), suggesting that the C-terminal part of the amylase is not involved in the activity of the enzyme. These data also suggest that the truncated amylase folds correctly, as already shown for Bacillus stearothermophilus and Bacillus subtilis C-terminally truncated amylases (28, 33). However, AmyA showed lower activity for soluble starch, with one-third-fold increase in K_M and one-third decrease in k_cat compared to AmyA, indicating altered substrate binding in AmyA. A similar effect of C-terminal truncation was observed with the B. stearothermophilus amylase (33).

Binding to raw starch. Only weak amylolytic activity was obtained in the culture supernatant of L. plantarum(pLPCR2-3) grown in the presence of raw starch compared to that obtained with L. plantarum(pLPCR2-3B/X) on the same medium. These data can be explained by the fact that the intact amylase (AmyA) was adsorbed on raw starch whereas AmyA was not retained on raw starch and was thus totally recovered in the supernatant.