Isomaltulose Is Actively Metabolized in Plant Cells

Abstract

Isomaltulose is a structural isomer of sucrose (Suc). It has been widely used as a nonmetabolized sugar in physiological studies aimed at better understanding the regulatory roles and transport of sugars in plants. It is increasingly used as a nutritional human food, with some beneficial properties including low glycemic index and acariogenicity. Cloning of genes for Suc isomerases opened the way for direct commercial production in plants. The understanding that plants lack catabolic capabilities for isomaltulose indicated a possibility of enhanced yields relative to Suc. However, this understanding was based primarily on the treatment of intact cells with exogenous isomaltulose. Here, we show that sugarcane (Saccharum interspecific hybrids), like other tested plants, does not readily import or catabolize extracellular isomaltulose. However, among intracellular enzymes, cytosolic Suc synthase (in the breakage direction) and vacuolar soluble acid invertase (SAI) substantially catabolize isomaltulose. From kinetic studies, the specificity constant of SAI for isomaltulose is about 10% of that for Suc. Activity varied against other Suc isomers, with V_max for leucrose about 6-fold that for Suc. SAI activities from other plant species varied substantially in substrate specificity against Suc and its isomers. Therefore, in physiological studies, the blanket notion of Suc isomers including isomaltulose as nonmetabolized sugars must be discarded. For example, lysis of a few cells may result in the substantial hydrolysis of exogenous isomaltulose, with profound downstream signal effects. In plant biotechnology, different V_max and V_max/K_m ratios for Suc isomers may yet be exploited, in combination with appropriate developmental expression and compartmentation, for enhanced sugar yields.

Isomaltulose (palatinose; α-d-glucopyranosyl-1,6-d-fructofuranose) is a naturally occurring structural isomer of Suc (α-d-glucopyranosyl-1,2-d-fructofuranose) with some advantages for food uses, including acariogenicity and low glycemic index (Lina et al., 2002). It is produced on an industrial scale from Suc by an enzymatic rearrangement using immobilized bacterial cells (Schiweck et al., 1991). With the characterization of Suc isomerase (SI) enzymes and the cloning of corresponding genes (Mattes et al., 1995; Börnke et al., 2001; Wu and Birch, 2005), direct production in plants became feasible, with potential cost savings over fermentative conversion of Suc harvested from plants (Börnke et al., 2002b; Kunz et al., 2002; Wu and Birch, 2007; Basnayake et al., 2011; Hamerli and Birch, 2011).

It has proven difficult by metabolic engineering to increase the yield of primary metabolites above those in crops highly selected through conventional breeding (Capell and Christou, 2004), possibly because of the high redundancy and plasticity in plant metabolic pathways and control systems for endogenous biomaterials (Morandini, 2009). Based mainly on short-term studies of cells supplied with exogenous sugars, isomaltulose is widely considered as nonmetabolizable in plants (Loreti et al., 2000; Fernie et al., 2001; Börnke et al., 2002b; Sinha et al., 2002; Atanassova et al., 2003). This property is commonly exploited in physiological studies aimed at better understanding of the regulatory roles and transport of sugars. Extracellular isomaltulose appears to be differentially sensed by plants (Loreti et al., 2000; Roitsch et al., 2000; Sinha et al., 2002; Atanassova et al., 2003), but known Suc transporters distinguish between isomers and do not detectably bind or transport isomaltulose (Li et al., 1994; Sivitz et al., 2007; Sun et al., 2010).

The apparent inability of plants to metabolize isomaltulose also raised the possibility of enhanced sugar yields through the conversion of Suc into a nonmetabolized isomer. Substantial isomer accumulation (approximately 440 μmol g⁻¹ fresh weight) has been achieved in sugarcane (Saccharum interspecific hybrids) expressing transgenes for SI enzymes targeted to the Suc storage vacuoles (Wu and Birch, 2007). However, isomer levels in field-grown sugarcane have not exceeded the usual Suc concentrations (more than 500 μmol g⁻¹ fresh weight) in elite sugarcane cultivars (Basnayake et al., 2011; Hamerli and Birch, 2011). Much lower isomaltulose concentrations (approximately 15 μmol g⁻¹ fresh weight) have been achieved in potato (Solanum tuberosum) tubers expressing an apoplast-targeted SI (Börnke et al., 2002b). There was no evidence for enzymes able to metabolize isomaltulose in potato tuber extracts (Fernie et al., 2001), yet the concentration of this isomer decreased substantially during storage of the transgenic tubers (Hajirezaei et al., 2003).

We have used sugarcane suspension cell lines to investigate sugar metabolism in plant cells engineered for SI activity (Wu and Birch, 2010). Fluctuations in isomer concentration during the culture cycle led us to reconsider the proposition that isomaltulose is nonmetabolizable in plants. To address this question directly, we prepared enzyme extracts from sugarcane cells and compared activities on Suc and isomaltulose under routine physiological assay conditions. The results show that isomaltulose is a substrate for some sugarcane enzymes, including vacuolar acid invertase. Because this substantially changes the assumptions about “nonmetabolized sugars” for physiological studies and has profound biotechnological implications, we extended the analysis to include several other Suc isomers and plant soluble acid invertases (SAIs). The effects varied between isomers and plant species, with important implications for both physiological analyses using nonmetabolized sugars and for plant metabolic engineering aimed at enhanced sugar accumulation.

RESULTS

Isomaltulose Was Rapidly Removed from the Culture Medium Late in the Culture Cycle

Previous studies of isomaltulose metabolism by plants were generally conducted using exogenous sugar applied to intact cells and analyzed for 2 to 48 h. Consistent with these earlier studies, we found that during most of the sugarcane cell culture cycle, exogenous isomaltulose concentration was unaltered, whereas exogenous Suc and hexoses were depleted (Fig. 1, A and B, up to 10 d). However, after sugarcane cell cultures entered the decline phase (Fig. 1C), we observed a rapid depletion of exogenous isomaltulose (Fig. 1, A and B, after 10 d).

Figure 1.

Concentrations of Suc (squares), isomaltulose (IM; inverted triangles), and hexose (in disaccharide equivalents; circles) in the culture medium originally supplied with 55 mm Suc (A) or 55 mm Suc plus 55 mm isomaltulose (B), relative to growth (C), of a sugarcane Q117 cell line during a 14-d batch culture. Values are means with se from four replicate cultures.

Growth typically ceased at 8 to 10 d followed by a decline in cell mass under these culture conditions, so it is likely that some cells lysed before 9 d. Therefore, the late decline in isomaltulose concentration might reflect a release of intracellular enzymes for disaccharide metabolism into the medium as the cultures enter the decline phase. There are well-established assays for the major enzymes active in the cleavage of Suc in plants: apoplastic cell wall invertase (CWI), vacuolar SAI, cytosolic Suc synthase (SuSy; in the breakage direction), and cytosolic neutral invertase (NI). Therefore, we compared these activities from actively growing sugarcane suspension cells against Suc versus isomaltulose as substrates.

Isomaltulose Was Not Hydrolyzed by CWI and Had No Allosteric Effect on CWI

Considering first the apoplastic enzyme, the wall fraction from actively growing sugarcane cells showed strong hydrolysis of Suc under routine assay conditions for CWI, but there was no detectable hydrolysis of isomaltulose. Furthermore, isomaltulose at tested concentrations up to 100 mm had no effect on the hydrolysis of Suc by sugarcane CWI (Fig. 2).

Figure 2.

Hydrolysis by sugarcane CWI of Suc in the presence or absence of isomaltulose (IM). The enzyme source was a washed cell wall preparation from actively growing sugarcane Q117 suspension cells. Isomaltulose tested as a sole substrate underwent no measurable hydrolysis during the 3-h assay. Values are means with se from four replicate CWI preparations, normalized against total soluble protein from the same cell samples. The presence of isomaltulose had no significant effect on activity against Suc at any tested sugar concentration (P > 0.05 by ANOVA with Bonferroni posttests).

Isomaltulose Was Not Hydrolyzed by NI and Slightly Inhibited NI Activity against Suc

Turning next to cytosolic enzymes, there was strong sugarcane NI activity against Suc but no detectable activity against isomaltulose under routine assay conditions (10 min) and barely detectable activity in an extended assay (3 h; Fig. 3A). There was slight inhibition of NI activity against Suc when isomaltulose concentration was higher than 10 mm (Fig. 3B).

Figure 3.

Effects of sugarcane cell extracts on Suc (squares) and isomaltulose (IM; inverted triangles) under NI assay conditions (A), and hydrolysis of Suc by sugarcane NI in the presence or absence of isomaltulose (B). The enzyme source was desalted soluble fraction from actively growing sugarcane Q117 suspension cells. Values are means with se from four replicate cell extracts. In the presence of 10 or 100 mm isomaltulose, there was a significant reduction in NI activity against Suc (P < 0.001 by ANOVA with Bonferroni posttests).

Isomaltulose Was Digested by SuSy

The breakage reaction of SuSy converts the substrates Suc plus UDP into the products Fru plus UDP-Glc. Under SuSy breakage reaction conditions, there was substantial production of Fru from isomaltulose as substrate. The relationship between Fru production rate and substrate concentration up to 100 mm appeared linear for isomaltulose and hyperbolic for Suc (Fig. 4). Our measured K_m for Suc was 41 mm, close to that determined previously for sugarcane SuSy (Schäfer et al., 2004).

Figure 4.

Cleavage of Suc (squares) and isomaltulose (inverted triangles) by sugarcane cell extracts under SuSy breakage assay conditions. The enzyme source was desalted soluble fraction from actively growing sugarcane Q117 suspension cells. Values are means with se from three replicate cell extracts.

In the Michaelis-Menten model, reaction rate is directly proportional to substrate concentration when the substrate concentration is well below K_m, which could apply here if SuSy has low affinity for isomaltulose but efficient cleavage of isomaltulose once bound. Although this SuSy breakage activity could have physiological significance if isomaltulose were produced in the cytosol, it is unlikely that sufficient UDP is released into the cell culture medium to account for the removal of isomaltulose shown in Figure 1.

Isomaltulose Was Hydrolyzed by Sugarcane SAI

Most of the Suc stored in sugarcane culms is located in the large vacuolar compartment within the storage parenchyma cells, from which it can be mobilized through the developmentally controlled activity of SAI (Vorster and Botha, 1999). In marked contrast to the CWI results, the SAI in sugarcane suspension cells was active against both Suc and isomaltulose. The hydrolysis reaction showed classic Michaelis-Menten kinetics on each substrate (Fig. 5), but isomaltulose relative to Suc showed a lower V_max (221 versus 318 nmol mg⁻¹ protein min⁻¹) and a higher K_m (5.11 versus 0.77 mm; Table I).

Figure 5.

Hydrolysis of Suc (squares), isomaltulose (inverted triangles), trehalulose (triangles), and leucrose (diamonds) by sugarcane cell extracts under SAI assay conditions. The enzyme source was desalted soluble fraction from actively growing sugarcane Q117 suspension cells. Hyperbolic (Michaelis-Menten) curves are fitted, and Hanes-Woolf plots are shown as an inset. Values are means with se from four replicate cell extracts.

Table I. Kinetic characteristics of Suc isomer cleavage under SAI assay conditions.

The enzyme source was desalted soluble fraction from actively growing sugarcane Q117 suspension cells.

Sugar	Vmax	Km	Specificity Constant (Vmax/Km)
	nmol mg−1 protein min−1	mm
Suc	318	0.77	412.9
Isomaltulose	211	5.11	41.3
Trehalulose	135	2.34	57.7
Leucrose	1,811	4.26	425.1

SAI requires no cofactor other than water for cleavage of its substrates, and it is highly active in the pH range (5.0–5.5) of the culture medium during the decline phase. This indicates that SAI released into the cell culture medium during the decline phase could account for the removal of isomaltulose shown in Figure 1. Assays for SAI activity in the culture supernatant at intervals during the culture cycle confirmed this interpretation (Fig. 6). It is not surprising that the hydrolysis products (Glc and Fru) do not substantially accumulate in the culture medium during this phase, as they are known to be readily taken up and metabolized by sugarcane cells (Wu and Birch, 2010). The effect in decline-phase cultures is that lysis of some cells releases SAI, which hydrolyzes extracellular isomaltulose into hexoses that are rapidly removed from the culture medium by the remaining living cells. The decline in cultured cell weight is slowed relative to cultures without added isomaltulose (Fig. 1C), as is the onset of necrotic browning in the cell suspension.

Figure 6.

Activity of the cell-free culture supernatant at various times after subculture, measured in SAI assays conducted on isomaltulose or Suc substrates. Values are means with se from four replicate cultures.

Trehalulose and Leucrose Were Also Hydrolyzed by Sugarcane SAI

The surprising discovery of sugarcane SAI activity against isomaltulose led us to test activity against other Suc isomers. Trehalulose (α1-1) and leucrose (α1-5) were also hydrolyzed with Michaelis-Menten kinetics under routine assay conditions for sugarcane SAI (Fig. 5). The K_m values for the isomers were in the range 2 to 6 mm compared with 0.77 mm for Suc. While these differences in K_m may be physiologically relevant in actively growing tissues with low sugar concentrations, it is evident that the reaction rate on any of these substrates would be close to V_max at sugar levels (greater than 100 mm) typical of sugarcane storage tissues. The spread of V_max values was much wider, and from a metabolic engineering perspective, it is interesting that trehalulose is expected to undergo the slowest hydrolysis by SAI whereas leucrose would be hydrolyzed even faster than the native substrate, Suc (Table I). The V_max/K_m (specificity constant) values indicate that in an equimolar mixture, isomaltulose or trehalulose would be hydrolyzed at only 10% to 15% of the rate for Suc.

SAI from Different Plant Sources Varied in Activity on Different Suc Isomers

Consistent with the results from Fernie et al. (2001), we found that the SAI activity from potato tubers did not detectably hydrolyze isomaltulose (Fig. 7A). Although SAI activity in potato tubers is low relative to some other plant tissues such as grape (Vitis vinifera) fruits (Fig. 7D), the potato extracts hydrolyzed both Suc and leucrose to at least 60 times the detection limit under routine SAI assay conditions (Fig. 7A). In routine SAI assays using sugarcane suspension cell extracts, the rate of Glc production from isomaltulose was 61% to 66% of that from Suc (Table I; Fig. 7B). SAI activity in sugarcane young stem tissue was lower than in potato tubers, but it digested isomaltulose at 30% to 40% of the rate on Suc (Fig. 7C). Thus, the substrate specificity of SAI activity in crude extracts varies between plant species.

Figure 7.

Hydrolysis of isomaltulose, leucrose, Suc, trehalulose, and turanose by plant tissue extracts under SAI assay conditions with 5 mm (white bars) and 100 mm (black bars) substrate. The enzyme source was desalted extract prepared from cv Desiree potato tuber (A), sugarcane Q117 suspension cells (B), internode 3 tissues from glasshouse-grown sugarcane Q117 (C), and ripe fruits of cv Globe grape (D) or white-fleshed nectarine (E). Values are means with se from three (C) to four (A, B, D, and E) replicate tissue extracts.

We extended the analysis to two additional plant species. In grape fruit extracts, SAI activity against tested Suc isomers was detectable but below 1% of the very high activity against Suc (Fig. 7D). In nectarine (Prunus persica) fruit extracts, hydrolysis rates of Suc and its tested isomers were similar (Fig. 7E).

DISCUSSION

Our results necessitate a correction to the widespread view that isomaltulose is a nonmetabolized sugar in plants, with important implications for both basic physiological studies and applied plant metabolic engineering.

A sugar can only be metabolized in a compartment that contains relevant metabolic enzymes and cofactors. Several previous studies using incubation periods of 15 min to 48 h have indicated no transport of isomaltulose across the plant plasmalemma (Schmitt et al., 1984; M’Batchi et al., 1985; M’Batchi and Delrot, 1988; Roitsch et al., 2000; Fernie et al., 2001; Sinha et al., 2002). In contrast, one study of plants engineered for apoplastic isomaltulose production interpreted nonaqueous fractionation results to indicate substantial redistribution of isomaltulose from the leaf apoplast to cytosolic and vacuolar compartments (Börnke et al., 2002a). Our results indicate no depletion of isomaltulose from the culture medium during a full culture cycle through logarithmic and stationary phases, in contrast with the rapid depletion of extracellular Suc and hexoses (Fig. 1; Wu and Birch, 2010). We conclude that intact sugarcane suspension cells lack the ability to import isomaltulose. Our results pose no difficulties for existing conclusions based on the interpretation that isomaltulose is nonmetabolized in the apoplast of intact plant cells. However, caution is required in systems that may include lysed or damaged cells, because the release of vacuolar SAI can allow rapid hydrolysis of extracellular isomaltulose (Figs. 1, 5, and 6), yielding Glc and Fru, which have very different physiological consequences.

Only one previous study has examined Suc isomer metabolism by intracellular enzymes, and the results indicated that isomaltulose was not metabolized by any of the sucrolytic activities in potato tuber extracts (Fernie et al., 2001). In contrast, we found that isomaltulose is efficiently cleaved by two of the four tested sucrolytic enzymes from sugarcane: cytosolic SuSy breakage reaction (Fig. 4) and vacuolar SAI (Fig. 5). Apoplastic CWI neither detectably cleaved isomaltulose nor was inhibited by it under routine assay conditions (Fig. 2). Cytosolic NI could not detectably hydrolyze isomaltulose but was slightly inhibited by it (Fig. 3).

The affinities of SuSy and SAI for isomaltulose were lower than for Suc. Strong constitutive expression of cytosolic SI is damaging in plants, an effect previously attributed to starvation through the sequestration of Suc into a nonmetabolizable isomer (Börnke et al., 2002a; Wu and Birch, 2007). Although the isomaltulose concentrations reached in this situation are unknown, it remains plausible that the relatively low affinity of SuSy (and the inactivity of NI) substantially limit isomaltulose cleavage at cytosolic disaccharide concentrations in growing cells, resulting in the observed plant growth retardation.

Sugars are important signal molecules in plants, but knowledge of the compartments and receptors involved is incomplete (Hanson and Smeekens, 2009). Some Suc transporters can distinguish between isomers, for example, transporting turanose but not isomaltulose (Sivitz et al., 2007; Sun et al., 2010). The structural basis for this recognition is unknown (Kühn and Grof, 2010; Geiger, 2011). Known Suc isomerases produce various ratios of isomaltulose and trehalulose from Suc in transgenic plants (Wu and Birch, 2007; Hamerli and Birch, 2011). The discovery of isomaltulose and trehalulose cleavage activities in plant cells, reported here, increases the complexity of mechanistic models needed to understand enhanced total sugar accumulation observed under some conditions in certain SI-expressing sugarcane lines (Wu and Birch, 2007, 2010).

Current strategies for plant metabolic engineering aimed at the commercial production of isomaltulose avoid SI activity in the cytosol and direct it toward the Suc storage compartment, the vacuole in plants like sugarcane. In this situation, isomer levels in mature stem vacuoles greatly exceed the K_m of SAI for isomaltulose, and the rate of hydrolysis is expected to approach V_max. Prolonged selection of sugarcane by humans for high sweetness has resulted in cultivars with very low SAI activity in mature internodes (Zhu et al., 2000). Physiologically, this phenotype likely involves a combination of transcriptional regulation and SAI inhibitors (Huang et al., 2007). Nevertheless, there is substantial “futile” cycling of stored Suc in mature sugarcane culms (Vorster and Botha, 1999), and there has been an interest to further decrease SAI activity in order to enhance sugar yield (Ma et al., 2000; Botha et al., 2001).

V_max/K_m ratios provide a measure of enzyme selectivity among substrates (Parkin, 2003), and the calculated values of 414 for Suc versus 43 for isomaltulose (Table I) imply that sugarcane SAI would hydrolyze isomaltulose at about one-tenth the rate for Suc from an equimolar sugar mixture at any concentration. In practice, it is desirable to achieve high isomer purity in the storage compartment of sugarcane engineered for isomaltulose production, and the ratio of isomaltulose to Suc is likely to change with internode maturity. This will change the relative rates of substrate cleavage by SAI. For example, the expected SAI digestion rate on isomaltulose would be half that on Suc when isomaltulose constituted 83% of disaccharide in the vacuole, and the rates of hydrolysis of the two sugars would be equal when isomaltulose constituted 91% of disaccharide in the vacuole.

While the notion of accumulation as a nonmetabolized sugar must be discarded, the lower V_max and V_max/K_m ratio (specificity constant) of sugarcane SAI for isomaltulose relative to Suc may yet prove an advantage, when other limitations, including the developmental expression pattern of the SI transgene and the stability of the SI enzyme in the hostile sugarcane vacuole, are further refined for practical use. Extending this reasoning, leucrose appears to have less potential for enhanced accumulation, whereas trehalulose is promising based on the kinetics of hydrolysis by SAI (Table I). Indeed, there is evidence from field trials for high-level production of trehalulose in engineered sugarcane (Hamerli and Birch, 2011).

The relative activities on particular isomers vary between SAI activities in extracts from different plant tissues, but the capacity to hydrolyze Suc isomers is widespread (Fig. 7). Overall, it is clearly unsafe to assume that Suc isomers are nonmetabolized sugars in plants. Our results indicate that the capacity for cleavage varies over a wide range, from undetectable to faster than Suc, depending on the tested isomer, concentration, cellular compartmentation, plant species, and possibly tissue type.

Invertases appear particularly relevant in the context of both physiological studies and metabolic engineering, because of their high activity in some potential sugar-storage compartments and the lack of cofactors likely to be lost when compartmentation is breached. The structural and regulatory complexity of plant invertases is still being elucidated. Further studies are required to clarify the mechanistic basis for the diverse substrate selectivities of different invertases and to elucidate potential interactions between Suc isomers as novel substrates in plants engineered for SI activity.

CONCLUSION

Isomaltulose is catabolized by cytosolic SuSy and by vacuolar SAI in sugarcane cells at rates that are substantial, although lower than Suc. Specificity constants of sugarcane SAI vary between isomers, with leucrose similar to Suc due to a high V_max. Isomaltulose and trehalulose are expected to be digested at about 10% to 15% of the rate for Suc in an equimolar mixture. SAIs from other tested plants vary in specificity across Suc and its isomers, and all except potato showed substantial activity against isomaltulose. Therefore, isomaltulose should not be used as a nonmetabolized sugar in plant physiology research without caution about likely hydrolysis on SAI release from lysed or damaged cells. The expectation that isomers should accumulate as nonmetabolized sugars in engineered plants must be refined. However, with appropriate developmental expression and compartmentation of Suc isomerase activity, the differences in V_max and V_max/K_m ratios for key enzymes acting on different Suc isomers, and in different plant species, may yet be exploited for enhanced sugar yields.

MATERIALS AND METHODS

Plant Tissue Samples

Internodal tissues were from sugarcane (Saccharum hybrid ‘Q117’) grown for 7 months under glasshouse conditions described previously (Wu and Birch, 2007). Tubers of potato (Solanum tuberosum ‘Desiree’) and ripe fruits of grape (Vitis vinifera ‘Globe’) and white-fleshed nectarine (Prunus persica) were from a grocer.

Sugarcane Cell Cultures

Homogenous suspension cultures generated from sugarcane genotype Q117 were maintained as described (Wu and Birch, 2010). To determine sugars in the culture medium, cells were removed from culture aliquots by gentle centrifugation (46g) onto a support grid (QIAshredder; Qiagen) and the filtrate was analyzed by high-performance anion-exchange chromatography (HPAEC; Wu and Birch, 2007). For experiments to measure enzyme activities, a 5-d-old culture was subcultured at a cell density of 0.025 g fresh weight mL⁻¹ into MSC3 medium. Fifty-milliliter cultures were grown in 250-mL Cellstar culture flasks (Greiner Bio-One) with gentle rocking at 28°C in the dark. After 2 d, the suspension cells were harvested by gentle centrifugation (46g), washed in 1 culture volume equivalent of MSC3 medium without Suc and coconut water, filtered using a gentle vacuum to remove the medium, and frozen at −80°C.

Crude Enzyme Extraction

Enzymes were extracted by grinding 3 g of frozen cells in a chilled mortar using 3 volumes of extraction buffer that contained 0.1 m HEPES-KOH buffer (pH 7.5), 10 mm MgCl₂, 2 mm EDTA, 2 mm EGTA, 10% (v/v) glycerol, 5 mm dithiothreitol, 2% (w/v) polyvinylpyrrolidone, and 1× complete protease inhibitor (Roche). The homogenate was immediately centrifuged at 10,000g for 15 min at 4°C. The pellet was used for the CWI assay as described below. The supernatant was immediately desalted using PD-10 columns (GE Healthcare) that were preequilibrated and eluted using the extraction buffer without glycerol. This desalted extract was used for all intracellular enzyme assays. Protein concentration was assayed by the Bradford reaction using a Bio-Rad kit with bovine serum albumin standards.

Enzyme Assays

Activities of CWI, NI, and SAI were calculated based on Glc production. SuSy breakage activity was calculated based on Fru production and confirmed based on UDP-Glc levels. Hexose products were measured by HPAEC (Wu and Birch, 2007). The Glc levels determined by HPAEC were confirmed by Glc oxidase assay as described below. UDP-Glc was quantified using the dehydrogenase assay described below. All assays included a negative control in which the enzyme component was pretreated at 95°C for 5 min. This defined any background level of the assayed products from sources other than the enzyme reaction. Any such background was subtracted before the calculation of enzyme activity.

The CWI enzyme assay was conducted as described (Albertson et al., 2001) with some modifications. The cell pellet mentioned above was suspended into 10 mL of extraction buffer to wash off the soluble invertase and then centrifuged at 2,500g for 10 min at 4°C, and the supernatant was discarded. The washing step was repeated once, then the pellet was suspended into 20 mL of extraction buffer. To start the assay, 200 μL of this cell wall suspension was added to 400 μL of 2× citrate/phosphate buffer (pH 3.2) containing the designated concentration of Suc or isomaltulose, and three glass balls of 1 to 2 mm diameter, in a 2-mL round-bottom microfuge tube. The closed reaction tubes were placed horizontally on a shaker at 100 rpm at 37°C for 3 h. To stop the reaction, the tubes were placed on ice, 10.5 μL of 10 n ice-cold KOH was added to neutralize the pH, and the tubes were heated at 95°C for 5 min.

For determination of SAI, 25 μL of the desalted enzyme extract mentioned above was incubated with 25 μL of 2× citrate/phosphate buffer (pH 4.2) containing the designated Suc or isomer concentration. For assays of activity in the culture medium, the enzyme source was 25 μL of culture supernatant after centrifugation to pellet the cells. The reaction was incubated at 30°C for 10 min. Pretests indicated that the reaction rate was linear for at least 15 min. To stop the reaction, the tubes were placed on ice, the required amount of 1 n ice-cold KOH was added to neutralize the pH, and the tubes were heated at 95°C for 5 min.

NI activity was started by mixing 25 μL of crude enzyme extract with 25 μL of 2× citrate/phosphate buffer, pH 7.3, containing the designated concentration of Suc or isomaltulose. The reaction was incubated at 30°C for 10 min and then stopped by placing the tube at 95°C for 5 min.

SuSy (breakage) activity was assayed in a reaction mixture comprising 100 mm Tris-HCl buffer (pH 7.0), 2 mm MgCl₂, 2 mm UDP, and the designated concentration of Suc or isomaltulose. Blank reactions without UDP were included as an additional negative control. After 10 min at 30°C, the assay was terminated by boiling for 10 min.

For Glc determination using the Glc oxidase assay, a 5-μL reaction sample was mixed with 45 μL of Glc oxidase reagent (Sigma-Aldrich; containing 500 units of Glc oxidase, 100 units of peroxidase, and 4 mg of o-dianisidine 40 mL⁻¹ Na acetate buffer, pH 5.5) and incubated at 37°C for 30 min. Then, 50 μL of 12 n H₂SO₄ was added to stop the reaction and develop the color. A₅₄₀ was corrected for blank readings and compared with a standard curve from a range of Glc concentrations (0–320 ng mL⁻¹).

For UDP-Glc determination, 160 μL of SuSy reaction sample was mixed with 10 μL of 20 mm NAD⁺, 28 μL of water, and 2 μL (6.7 units) of UDP-Glc dehydrogenase; then, A₃₄₀ was recorded until no further increase could be noted, and readings were interpolated on a standard curve from known UDP-Glc concentrations.

Statistical Analyses

Statistical tests specified with the results were performed using Prism 5.0 software (GraphPad).

Biochemicals

Unless specified below, chemicals, enzymes, and cofactors were purchased from Sigma-Aldrich. All solvents and biochemicals were of analytical grade. Isomaltulose was purchased from the Tokyo Kasei Cogco Co.