Pullulanase from rice endosperm

Pullulanase (EC 3.2.1.41) in non-germinating seeds was compared with that in germinating seeds. Moreover, pullulanase from the endosperm of rice (Oryza sativa L., cv. Hinohikari) seeds was isolated and its properties investigated. The pI value of pullulanase from seeds after 8 days of germination was almost equal to that from non-germinating seeds, which shows that these two enzymes are the same protein. Therefore, the same pullulanase may play roles in both starch synthesis during ripening and starch degradation during germination in rice seeds. The enzyme was isolated by a procedure that included ammonium sulfate fractionation, DEAE-cellulofine column chromatography, preparative isoelectric focusing, and preparative disc gel electrophoresis. The enzyme was homogeneous by SDS/PAGE. The molecular weight of the enzyme was estimated to be 100 000 based on its mobility on SDS/PAGE and 105 000 based on gel filtration with TSKgel super SW 3000, which showed that it was composed of a single unit. The isoelectric point of the enzyme was 4.7. The enzyme was strongly inhibited by β-cyclodextrin. The enzyme was not activated by thiol reagents such as dithiothreitol, 2-mercaptoethanol or glutathione. The enzyme most preferably hydrolyzed pullulan and liberated only maltotriose. The pullulan hydrolysis was strongly inhibited by the substrate at a concentration higher than 0.1%. The degree of inhibition increased with an increase in the concentration of pullulan. However, the enzyme hydrolyzed amylopectin, soluble starch and β-limit dextrin more rapidly as their concentrations increased. The enzyme exhibited α-glucosyltransfer activity and produced an α-1,6-linked compound of two maltotriose molecules from pullulan.

On the other hand, remarkable pullulanase activity is detected in the endosperm of rice seeds.This enzyme may play a role in starch synthesis during ripening of starch and remain in the seed.However, pullulanase activity increased during germination of the rice seeds, while the starch content of seeds was markedly decreased.
We attempted to examine the difference between the pullulanase in non-germinating seeds and that in germinating seeds.

2008
Y. Yamasaki and others Moreover, we isolated pullulanase from the endosperm of rice seeds and investigated its properties.

EXPERIMENTAL PROCEDURE
Enzyme assay.Pullulanase activity was determined as follows.The reaction mixture containing 1 mg of pullulan and enzyme solution in 0.5 ml of 50 mM NaOAc buffer, pH 6.0, was incubated at 37 o C for 30 min.After incubation, the reaction was stopped by boiling the mixture for 5 min.The amount of maltotriose formed was measured according to the method of Somogyi (1952).
Determination of protein.Protein was determined by the method of Warburg and Christian (1942).The protein profiles from column chromatography were determined by measuring the absorbance of the eluates at 280 nm.
Electrophoresis.SDS/PAGE was performed on 7.5% gels according to the method of Laemmli (1970).After the run, the gels were stained for protein with Phast Blue R (R-350).
Estimation of molecular weight.The M r of the purified enzyme was estimated by SDS/PAGE (Laemmli, 1970) and gel filtration using HPLC.Gel filtration was performed on a column (0.46 × 60 cm) of TSKgel Super SW 3000: mobile phase, 50 mM phosphate buffer, pH 7.0, flow rate, 0.35 ml/min, temperature, 25 o C, detection, UV (280 nm).
Estimation of the isoelectric point.The isoelectric point of the enzyme was determined by electrofocusing (Yamasaki et al., 2007).

Pullulanase activity of endosperm rice seeds
Rice seeds showed remarkable pullulan-and soluble starch-hydrolyzing activities.Both of these activities in seeds were also found in the polished rice that had been threshed by a threshing machine (not shown).After 8 days of germination, the activity of pullulanase increased to three times that in nongerminating seeds, which shows that the enzyme will play a role in starch digestion (Fig. 1).The pI value of pullulanase in seeds after 8 days of germination was almost equal to that in non-germinating seeds (Fig. 1).This shows that the two enzymes are the same protein.Therefore, pullulanase will play a role in starch synthesis during the ripening of starch and remain in the seed.The same enzyme will play a role in starch degradation during germination.

Isolation of pullulanase
Rice (Oryza sativa L., cv.Hinohikari) seeds were homogenized in a homogenizer (Nissei Excel Auto-Homogenizer; Nihonseiki Co., Tokyo, Japan) with 25 mM NaOAc buffer, pH 5.3, containing 0.5 M NaCl and 5 mM mercaptoethanol.The supernatant was brought to 0.9 satn.with (NH 4 ) 2 SO 4 .After dissolving the precipitate in 25 mM NaOAc buffer, pH 5.3, the enzyme solution was dialysed against 25 mM Tris/HCl buffer, pH 8.0, and the dialysate was applied to a DEAE-cellulofine column (2 × 18 cm).After the column was washed with 25 mM Tris/HCl buffer, pH 8.0, a linear gradient of 0-1 M NaCl in the buffer was applied.The fraction that exhibited pullulanase activity was subjected to preparative Crude extracts of non-germinating seeds and germinating seeds after 8 days were subjected to isoelectric focusing (pH 3.5-10).After 24 h at 80 V, the two gels were cut into 3-mm sections and each section was crushed in 2 ml of deionized water.The fractions were measured to determine pullulan-hydrolyzing activities and pH.•, Pullulan-hydrolyzing activity, which maltotriose liberated from pullulan was measured by the method of Somogyi (1952); ---, pH.
Pullulanase from rice endosperm isoelectric focusing (IEF) (pH range, 3.5-10) using an HSI GT Tube Gel Electrophoresis Unit (Hoefer Scientific Instruments, CA, USA).Electrophoresis was performed at 100 V for 20 h.The enzyme solution was further purified by preparative disc gel electrophoresis, pH 9.0, using an HSI GT Tube Gel Electrophoresis Unit.Electrophoresis was performed at a constant current of 25 mA for 4 h.The enzyme solution was applied to a Vivapure Maxi D/Q spin column and the column was eluted with 25 mM Tris/HCl buffer, pH 8.0, containing 1 M NaCl.These procedures led to 37-fold purification with 10.1% recovery.The purified enzyme was homogenous by SDS/PAGE (Fig. 2).

General properties
The molecular weight of the enzyme was estimated to be 100 000 based on its mobility on SDS/PAGE and 105 000 based on gel filtration with TSKgel super SW 3000, which showed that it was composed of a single unit.Pullulanases from rice (Iwaki & Fuwa, 1981), sugar-beet (Li et al., 1992), spinach (Ludwig et al., 1984;Schindler et al., 2001) and millet (Yamasaki et al., 2007) are also single peptides.The isoelectric point of the enzyme was determined to be 4.7 by analytical IEF.
The optimum pH of the enzyme was determined to be 6.0.After 20 h of preincubation at 30 o C with 50 mM McIlvaine's buffer, the enzyme was stable in a pH range of 5.5-8.0.The optimum temperature for the enzyme activity was 55 o C after 15 min of incubation.After 15 min of preincubation with 50 mM NaOAc buffer, pH 6.0, at various temperatures, the enzyme was found to be stable at temperatures up to 50 o C.
β-Cyclodextrin strongly inhibited the enzyme activity in a non-competitive fashion.This inhibition of the enzyme is similar to those of rice (Iwaki & Fuwa, 1981), spinach (Ludwig et al., 1984) and sugar-beet (Li et al., 1992).The enzyme was not activated by thiol reagents such as dithiothreitol, 2-mer-Table 1.Substrate specificity of the purified pullulanase.
Substrates, 1 mg in 0.5 ml of 50 mM NaOAc buffer, pH 6.0, were incubated with the purified enzymes at 37 o C for 30 min.Malto-oligosaccharide liberated was determined by the method of Somogyi (1952) as described in Experimental Procedure.

Substrate specificity
Various substrates were incubated with the enzyme under standard conditions (Table 1).The enzyme most preferably hydrolyzed pullulan and liberated only maltotriose.However, pullulan hydrolysis was inhibited at a concentration higher than 0.1% (Fig. 3).The degree of inhibition increased with an increase in the concentration of pullulan.On the other hand, the enzyme also readily hydrolyzed βlimit dextrin, soluble starch and amylopectin, and hydrolyzed glycogen weakly.When the enzyme was incubated with amylopectin, the enzyme hydrolyzed amylopectin more rapidly as the concentration increased (Fig. 3).The same results were also obtained with soluble starch and β-limit dextrin (not shown).

α-Glucosyl transfer action
When the enzyme reacted with pullulan, unknown substances were produced.The reaction mixture was lyophilized and dissolved in a small amount of water.The solution was applied to a column (1.6 × 93 cm) of Bio-Gel P2 and eluted with 20 mM EDTA.The main substance (X) was eluted between maltoheptaose and maltopentaose.X was eluted between maltohexaose and maltoheptaose by HPLC and completely hydrolyzed by purified pullulanase to liberate only maltotriose.Therefore, we concluded that X should be Glcp α14 Glcp α14 Glcp α16 Glcp α14 Glcp α14 Glcp, which is an α-1,6-linked compound of two maltotriose molecules.

Figure 1 .
Figure 1.Isoelectric focusing of crude extracts of nongerminating seeds (upper figure) and germinating seeds (lower figure).Crude extracts of non-germinating seeds and germinating seeds after 8 days were subjected to isoelectric focusing (pH 3.5-10).After 24 h at 80 V, the two gels were cut into 3-mm sections and each section was crushed in 2 ml of deionized water.The fractions were measured to determine pullulan-hydrolyzing activities and pH.•, Pullulan-hydrolyzing activity, which maltotriose liberated from pullulan was measured by the method ofSomogyi (1952); ---, pH.

Figure 2 .
Figure 2. SDS/PAGE of purified rice pullulanase.SDS/PAGE was carried out as described in the Experimental Procedure.Left, pullulanase; right, Precision Plus Protein Standards (Dual Color, Bio-Rad).