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Pullulanase M1 (Klebsiella planticola)

Pullulanase M1 Klebsiella planticola E-PULKP
Product code: E-PULKP

700 Units

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Content: 700 Units
Shipping Temperature: Ambient
Storage Temperature: 2-8oC
Formulation: In 3.2 M ammonium sulphate
Physical Form: Suspension
Stability: > 4 years at 4oC
Enzyme Activity: Pullulanase/Limit-dextrinase
EC Number:
CAZy Family: GH13
CAS Number: 9075-68-7
Synonyms: pullulanase; pullulan 6-alpha-glucanohydrolase
Source: Klebsiella planticola
Molecular Weight: 109,000
Concentration: Supplied at ~ 650 U/mL
Expression: Purified from Klebsiella planticola
Specificity: Hydrolysis of (1,6)-α-D-glucosidic linkages in pullulan, amylopectin and glycogen, and in the α- and β-limit dextrins of amylopectin and glycogen.
Specific Activity: ~ 30 U/mg (40oC, pH 5.0 on pullulan)
Unit Definition: One Unit of pullulanase activity is defined as the amount of enzyme required to release one µmole of glucose reducing-sugar-equivalents per minute from pullulan (5 mg/mL) in sodium acetate buffer (100 mM), pH 5.0 at 40oC.
Temperature Optima: 40oC
pH Optima: 5
Application examples: Applications in the cereals, food and feeds industries particularly in starch saccharification and production of high glucose or maltose syrups.

High purity Pullulanase M1 (Klebsiella planticola) for use in research, biochemical enzyme assays and in vitro diagnostic analysis.

Recommended pullulanase for research on starch structure.

Certificate of Analysis
Safety Data Sheet
FAQs Booklet
Molecular structure of Maori potato starch.

Zhu, F. & Hao, C. (2018). Food Hydrocolloids, 80, 206-211.

New Zealand Maori potatoes (Taewa) represent unique genetic resources for potato quality, though they are much underutilized. In this report, the composition and molecular structure of starches from 5 Maori potato varieties were studied. In particular, the internal unit chain composition of the amylopectins in the form of β-limit dextrins were highlighted. Starches from a commercial modern potato variety and a maize variety with normal amylose contents were employed for comparison. Genetic diversity in the amylose (e.g., 22.6% in Moemoe to 28.6% in Turaekuri) and phosphorus (5.4 mg/100 g in Turaekuri to 7.0 mg/100 g in Kowiniwini) contents as well as the molecule structure of the starches (e.g., external chain length of amylopectin ranged from 13.0 glucosyl residues in Turaekuri to 15.8 glucosyl residues in Karuparera) has been revealed. Maori potato amylopectins have the highest amount of long unit and internal chains and the lowest amount of these chains among amylopectins from different sources. Overall, Maori potato starch appeared to be structurally and compositionally similar to modern potato starch.

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Effect of hydroxypropylation and beta‐amylase treatment on complexation of debranched starch with naringenin.

Gonzalez, A., Wang, Y. J., Staroszczyk, H., Brownmiller, C. & Lee, S. O. (2018). Starch‐Stärke, In Press.

Naringenin exhibits many health benefits but it has limited water solubility and consequently low bioavailability. The objective of this study was to investigate the effect of hydroxypropylation and enzymatic treatments on starch complexation with naringenin. Potato starch and Hylon VII were hydroxypropylated to two substitution degrees and then debranched or debranched/β-amylase treated prior to complexing with naringenin. Both soluble and insoluble complexes were recovered and characterized. An increase in hydroxypropylation level improved recovery of soluble complexes, while total recovery remained unchanged; the β-amylase treatment further increased soluble complex recovery. For the same treatment, the naringenin content was greater in Hylon VII complexes (6.72-15.15mg/g) than in potato starch complexes (2.45-11.18 mg/g). Insoluble complexes comprised greater naringenin contents (3.91-15.15 mg/g) compared to soluble counterparts (2.45-9.43 mg/g). All complexes exhibited a mixture of B+V X-ray diffraction pattern. This work is the first one to demonstrate that hydroxypropylated starch formed complexes with naringenin, and an appropriate level of beta-amylase hydrolysis further improved their complexation.

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Biochemical characterization of two GH70 family 4, 6-α-glucanotransferases with distinct product specificity from Lactobacillus aviarius subsp. aviarius DSM 20655.

Meng, X., Gangoiti, J., de Kok, N., van Leeuwen, S. S., Pijning, T. & Dijkhuizen, L. (2018). Food Chemistry, In Press.

Nine GtfB-like 4,6-α-glucanotransferases (4,6-α-GTs) (represented by GtfX of L. aviaries subsp. Aviaries DSM 20655) were identified to show distinct characteristics in conserved motifs I-IV. In particular, the “fingerprint” Tyr in motif III of these nine GtfB-type 4,6-α-GTs was found to be replaced by a Trp. In L. aviarius subsp. aviarius DSM20655, a second GtfB-like protein (GtfY), containing the canonical GtfB Tyr residue in motif III, was located directly upstream of GtfX. Biochemical characterization revealed that both GtfX and GtfY showed GtfB-like 4,6-α-GT activity, cleaving (α1→4) linkages and catalyzing the synthesis of (α1→6) linkages. Nonetheless, they differ in product specificity; GtfY only synthesizes consecutive (α1→6) linkages, yielding linear α-glucan products, but GtfX catalyzes the synthesis of (α1→6) linkages predominantly at branch points (22%) rather than in linear segments (10%). The highly branched α-glucan produced by GtfX from amylose V is resistant to digestion by α-amylase, offering great potential as dietary fibers.

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Control of secondary cell wall patterning involves xylan deacetylation by a GDSL esterase.

Zhang, B., Zhang, L., Li, F., Zhang, D., Liu, X., Wang, H., Xu, Z., Chu, C. & Zhou, Y. (2017). Nature Plants, 3, 17017.

O-acetylation, a ubiquitous modification of cell wall polymers, has striking impacts on plant growth and biomass utilization and needs to be tightly controlled. However, the mechanisms that underpin the control of cell wall acetylation remain elusive. Here, we show a rice brittle leaf sheath1 (bs1) mutant, which contains a lesion in a Golgi-localized GDSL esterase that deacetylates the prominent hemicellulose xylan. Cell wall composition, detailed xylan structure characterization and enzyme kinetics and activity assays on acetylated sugars and xylooligosaccharides demonstrate that BS1 is an esterase that cleaves acetyl moieties from the xylan backbone at O-2 and O--3 positions of xylopyranosyl residues. BS1 thus plays an important role in the maintenance of proper acetylation level on the xylan backbone, which is crucial for secondary wall formation and patterning. Our findings outline a mechanism for how plants modulate wall acetylation and endow a plethora of uncharacterized GDSL esterases with surmisable activities.

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Responses of Synechocystis sp. PCC 6803 to heterologous biosynthetic pathways.

Vavitsas, K., Rue, E. Ø., Stefánsdóttir, L. K., Gnanasekaran, T., Blennow, A., Crocoll, C., Gudmundsson, S. & Jensen, P. E. (2017). Microbial cell factories, 16(1), 140.

Background: There are an increasing number of studies regarding genetic manipulation of cyanobacteria to produce commercially interesting compounds. The majority of these works study the expression and optimization of a selected heterologous pathway, largely ignoring the wholeness and complexity of cellular metabolism. Regulation and response mechanisms are largely unknown, and even the metabolic pathways themselves are not fully elucidated. This poses a clear limitation in exploiting the rich biosynthetic potential of cyanobacteria. Results: In this work, we focused on the production of two different compounds, the cyanogenic glucoside dhurrin and the diterpenoid 13R-manoyl oxide in Synechocystis PCC 6803. We used genome-scale metabolic modelling to study fluxes in individual reactions and pathways, and we determined the concentrations of key metabolites, such as amino acids, carotenoids, and chlorophylls. This allowed us to identify metabolic crosstalk between the native and the introduced metabolic pathways. Most results and simulations highlight the metabolic robustness of cyanobacteria, suggesting that the host organism tends to keep metabolic fluxes and metabolite concentrations steady, counteracting the effects of the heterologous pathway. However, the amino acid concentrations of the dhurrin-producing strain show an unexpected profile, where the perturbation levels were high in seemingly unrelated metabolites. Conclusions: There is a wealth of information that can be derived by combining targeted metabolite identification and computer modelling as a frame of understanding. Here we present an example of how strain engineering approaches can be coupled to ‘traditional’ metabolic engineering with systems biology, resulting in novel and more efficient manipulation strategies.

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Understanding the fine structure of intermediate materials of maize starches.

Han, W., Zhang, B., Li, J., Zhao, S., Niu, M., Jia, C. & Xiong, S. (2017). Food Chemistry, 233, 450-456.

Here we concern the molecular fine structure of intermediate material (IM) fraction in regular maize starch (RMS) and Starpro 40 maize starch (S40). IM had a branching degree and a molar mass (M w ) somewhere between amylopectin (AP) and amylose (AM). Compared with AP, IM had more extra-long (Fr I) and long (Fr II) chains and fb3-chains (degree of polymerization (DP) > 36), with a higher average chain length (CL). Also, IM contained less A-chains but more B-chains (both BS-chains with DP 3-25 and BL-chains with DP ≥ 26), accompanied by longer B- and BL-chains, total internal chains (TICL) and average internal chains (ICL), and a similar average external chain length (ECL). Furthermore, relative to RMS-IM, the IM of S40 (with higher apparent amylose content than RMS) showed increases in relatively-long chains, e.g., Fr II, fb3-chains and BL-chains, but reductions in Mw, relatively-short chains (those with DP 6-12, etc.).

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Effect of microwave irradiation on internal molecular structure and physical properties of waxy maize starch.

Yang, Q., Qi, L., Luo, Z., Kong, X., Xiao, Z., Wang, P. & Peng, X. (2017). Food Hydrocolloids, 69, 473-482.

Native waxy maize starch was treated at a moisture content of 30% by microwave irradiation for 5 min, 10 min and 20 min, respectively. The molecular structure and physical properties of waxy maize starch were characterized. Compared with native maize starch, lower population of short chains of amylopectin (A chain), higher proportion of short B1 and long B2 and B2 were observed in irradiated starches. 1 H NMR data showed that α-(1,6) glycosidic linkages were destroyed more easily than α-(1,4) glycosidic linkages during microwave treatment. A increase in gelatinization temperatures and a decrease in the molecular weight, the relative crystallinity, ΔH, viscosities and syneresis were observed after microwave treatment. Gelatinization temperatures were positively correlated with long chains B3 with DP > 36, while ΔH and syneresis were negatively correlated with them. The extent of the changes induced by microwave treatment for different times revealed that the major degradation occurred in internal chain (amorphous region) at the first stage (microwave treatment for 5 min), the external chain (crystalline region) mostly destroyed at the second stage (microwave treatment for above 10 min). The foregoing data indicated that the molecular structure of amylopectin is a critical factor determining physical properties.

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Small differences in amylopectin fine structure may explain large functional differences of starch.

Bertoft, E., Annor, G. A., Shen, X., Rumpagaporn, P., Seetharaman, K. & Hamaker, B. R. (2016). Carbohydrate Polymers, 140, 113-121.

Four amylose-free waxy rice starches were found to give rise to gels with clearly different morphology after storage for seven days at 4°C. The thermal and rheological properties of these gels were also different. This was remarkable in light of the subtle differences in the molecular structure of the amylopectin in the samples. Addition of iodine to the amylopectin samples suggested that not only external chains, but also the internal chains of amylopectin, could form helical inclusion complexes. It is suggested that these internal helical segments participate in the retrogradation of amylopectin, thereby stabilising the gels through double helical structures with external chains of adjacent molecules. Albeit few in number, such interactions appear to have important influences on starch functional properties.

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Molecular and thermal characterization of starches isolated from African rice (Oryza glaberrima).

Gayin, J., Bertoft, E., Manful, J., Yada, R. Y. & Abdel‐Aal, E. S. M. (2016). Starch‐Stärke, 68(1-2), 9-19.

Starch is the principal component of rice that affects its cooking and nutritional quality. This study investigated molecular and thermal properties of starches isolated from seven Africa rice accessions (ARAs) in comparison with two commonly produced Asian rice varieties (ARVs) and a developed cross (sativa × glaberrima) variety (NERICA 4). All starch granules were polyhedral and tightly packed with size distribution ranging from 2-22 µm and displayed type-A X-ray diffraction pattern. ARAs starch granules had higher ratio of absorbance to scattering when exposed to iodine vapor exhibiting greater flexibility and availability of glucan chains to form complexes with iodine as compared to ARVs. The enthalpies of starch gelatinization (15.1-15.8 J/g) and retrograded gel melting (9.2-10.8 J/g) were higher in ARAs than in NERICA 4 (14.5 and 9.2 J/g, respectively) and ARVs, (13.3-14.3 and 6.4-7.3 J/g, respectively) possibly due to their higher amylose content and longer chains. Significant (p < 0.05) differences in peak, trough, final, breakdown, and setback viscosities were also observed among the starches with Koshihikari Asian rice having the highest peak viscosity (310 RVU). These differences in molecular structure and thermal properties between the ARAs and ARVs are likely to influence the cooking and eating quality of the ARAs.

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Technological, nutritional and functional properties of wheat bread enriched with lentils or carob flours.

Turfani, V., Narducci, V., Durazzo, A., Galli, V. & Carcea, M. (2016). LWT-Food Science and Technology, 78, 361-366

Blending wheat flour with carob seed or green lentil flour at 5-6%, 10-12% or 24% level modified the dough's technological properties and bread characteristics. Carob seed flours increased dough tenacity while reducing extensibility, whereas lentil flour reduced dough tenacity, extensibility and strength. Carob seed flours increased water absorption up to about 40%. Flours from all legumes increased dough development time (carob flours more than lentils flour). Lentil flour strongly decreased the stability due to weakening of the gluten network. In a baking test, however, all blends gave acceptable loaves. Blending the wheat flour with 5-6% legume flour generally did not alter the loaf volume. However, increasing the legume flour to 10-12% or 24% reduced the loaf volume, except when supplemented with refined carob seed flour at 10%, which increased it. Carob flour and especially lentil flour enriched bread with lysine-rich proteins, dietary fibre, phenolic compounds and lignans and in general increased its antioxidant power. The nutritional value of lentils and the technological properties of carob are useful in increasing nutritional and functional value of wheat bread.

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Alanine aminotransferase 1 (OsAlaAT1) plays an essential role in the regulation of starch storage in rice endosperm.

Yang, J., Kim, S. R., Lee, S. K., Choi, H., Jeon, J. S. & An, G. (2015). Plant Science, 240, 79-89.

Alteration of storage substances, in particular the major storage form starch, leads to floury endosperm. Because floury mutants have physical attributes for milling processes, identification and characterization of those mutants are valuable. In this study we identified a floury endosperm mutant caused by a T-DNA insertion in Oryza sativa alanine-aminotransferase1 (OsAlaAT1). OsAlaAT1 is localized in the cytosol and has aminotransferase enzyme activity. The osalaat1 mutant has less amylose and its amylopectin is structurally altered. OsAlaAT1 is predominantly expressed in developing seeds during active starch synthesis. AlaAT catalyzes the interconversion of pyruvate to alanine, and this pathway is activated under low-oxygen conditions. Consistently, OsAlaAT1 is induced by such conditions. Expression of the starch synthesis genes AGPasesOsSSIOsSSIIa, and OsPPDKB is decreased in the mutant. Thus, our observations suggest that OsAlaAT1 plays an essential role in starch synthesis in developing seeds that are exposed to low concentrations of oxygen.

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Mechanistic insights into glucan phosphatase activity against polyglucan substrates.

Meekins, D. A., Raththagala, M., Auger, K. D., Turner, B. D., Santelia, D., Kötting, O., Gentry, M. S. & Vander Kooi, C. W. (2015). Journal of Biological Chemistry, 290(38), 23361-23370.

Glucan phosphatases are central to the regulation of starch and glycogen metabolism. Plants contain two known glucan phosphatases, Starch EXcess4 (SEX4) and Like Sex Four2 (LSF2), which dephosphorylate starch. Starch is water-insoluble and reversible phosphorylation solubilizes its outer surface allowing processive degradation. Vertebrates contain a single known glucan phosphatase, laforin, that dephosphorylates glycogen. In the absence of laforin, water-soluble glycogen becomes insoluble, leading to the neurodegenerative disorder Lafora Disease. Because of their essential role in starch and glycogen metabolism glucan phosphatases are of significant interest, yet a comparative analysis of their activities against diverse glucan substrates has not been established. We identify active site residues required for specific glucan dephosphorylation, defining a glucan phosphatase signature motif (CζAGΨGR) in the active site loop. We further explore the basis for phosphate position-specific activity of these enzymes and determine that their diverse phosphate position-specific activity is governed by the phosphatase domain. In addition, we find key differences in glucan phosphatase activity toward soluble and insoluble polyglucan substrates, resulting from the participation of ancillary glucan-binding domains. Together, these data provide fundamental insights into the specific activity of glucan phosphatases against diverse polyglucan substrates.

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Effects of Chemical and Enzymatic Modifications on Starch–Oleic Acid Complex Formation.

Arijaje, E. O. & Wang, Y. J. (2015). Journal of Agricultural and Food Chemistry, 63(16), 4202-4210.

The solubility of starch-inclusion complexes affects the digestibility and bioavailability of the included molecules. Acetylation with two degrees of substitution, 0.041 (low) and 0.091 (high), combined without or with a β-amylase treatment was employed to improve the yield and solubility of the inclusion complex between debranched potato starch and oleic acid. Both soluble and insoluble complexes were recovered and analyzed for their degree of acetylation, complexation yields, molecular size distributions, X-ray diffraction patterns, and thermal properties. Acetylation significantly increased the amount of recovered soluble complexes as well as the complexed oleic acid in both soluble and insoluble complexes. High-acetylated debranched-only starch complexed the highest amount of oleic acid (38.0 mg/g) in the soluble complexes; low-acetylated starch with or without the β-amylase treatment resulted in the highest complexed oleic acid in the insoluble complexes (37.6–42.9 mg/g). All acetylated starches displayed the V-type X-ray pattern, and the melting temperature generally decreased with acetylation. The results indicate that starch acetylation with or without the β-amylase treatment can improve the formation and solubility of the starch–oleic acid complex.

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Differences in structures of starch hydrolysates using saliva from different individuals.

Nantanga, K. K. M., Chan, E., Suleman, S., Bertoft, E. & Seetharaman, K. (2013). Starch‐Stärke, 65(7‐8), 709-713.

High salivary amylase activity is associated with improved glycemic homeostasis in humans. Therefore, high salivary amylase activity is associated with greater digestion of starch. However, it is unclear if the structures of the hydrolysates from different individuals with different salivary amylase activity are the same. To test this, cooked starch (1:2 starch/water ratio) was treated with saliva from six participants at equal activity and conditions mimicking oral digestion. Salivary amylase activities ranged from 470 × 103 to 118 × 103 U/mL among the participants. The composition of the hydrolysates was characterised by gel-permeation chromatography. All samples gave rise to different and complex mixtures of hydrolysates with different breakdown structures. While saliva from participant 2 (high amylase activity) greatly reduced the high MW fraction, the saliva from participant 6 (low amylase activity) more extensively hydrolysed the starch to small MW fractions of oligosaccharides. These results show that different starch hydrolysates are produced during oral digestion by saliva from different individuals. Further research is therefore needed to understand if hydrolysate structure, rather than level of amylase activity, impacts glucose homeostasis.

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Structures of human salivary amylase hydrolysates from starch processed at two water concentrations.

Nantanga, K. K. M., Bertoft, E. & Seetharaman, K. (2013). Starch‐Stärke, 65(7‐8), 637-644.

Digestion of starch in humans starts in the mouth and progresses to the small intestine. A thorough understanding of the progression of digestion, of consequence to glycemic and possibly insulinemic responses, requires a better characterization of the digestion products along the gut – products that are the substrates in the subsequent hydrolysis by sucrase-isomaltase and maltase-glucoamylase. This submission focuses on the first step of digestion, i.e., impact of human salivary amylase on the structure of hydrolysis products obtained from cooked starch. Starch was cooked at 1:0.7 (T0.7) or 1:2 (T2) starch:water ratios. To remove the effect of granular structure, starch was also dispersed using DMSO (TD) prior to amylase treatment. Cooked and dispersed starches were subjected to salivary amylase at conditions mimicking oral digestion. All samples gave rise to different and complex mixtures of hydrolysates with broad size-distributions as measured by gel-permeation chromatography (GPC). Following hydrolysis, the smallest dextrins (DP <30) constituted 35% in TD and only ∼20% in both T0.7 and T2. Cooking appeared to protect amylose molecules from hydrolysis with less hydrolysis in T0.7. These results show that the amount of water present during processing of starch affects structures of salivary amylase hydrolysates, which potentially impact on glucose homeostasis.

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Deficiency of maize starch-branching enzyme i results in altered starch fine structure, decreased digestibility and reduced coleoptile growth during germination.

Xia, H., Yandeau-Nelson, M., Thompson, D. B. & Guiltinan, M. J. (2011). BMC Plant Biology, 11(1), 95-107.

Background: Two distinct starch branching enzyme (SBE) isoforms predate the divergence of monocots and dicots and have been conserved in plants since then. This strongly suggests that both SBEI and SBEII provide unique selective advantages to plants. However, no phenotype for the SBEI mutation, sbe1a, had been previously observed. To explore this incongruity the objective of the present work was to characterize functional and molecular phenotypes of both sbe1a and wild-type (Wt) in the W64A maize inbred line. Results: Endosperm starch granules from the sbe1a mutant were more resistant to digestion by pancreatic α-amylase, and the sbe1a mutant starch had an altered branching pattern for amylopectin and amylose. When kernels were germinated, the sbe1a mutant was associated with shorter coleoptile length and higher residual starch content, suggesting that less efficient starch utilization may have impaired growth during germination. Conclusions: The present report documents for the first time a molecular phenotype due to the absence of SBEI, and suggests strongly that it is associated with altered physiological function of the starch in vivo. We believe that these results provide a plausible rationale for the conservation of SBEI in plants in both monocots and dicots, as greater seedling vigor would provide an important survival advantage when resources are limited.

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Residual amylopectin structures of amylase-treated wheat starch slurries reflect amylase mode of action.

Leman, P., Goesaert, H. & Delcour, J. A. (2009). Food Hydrocolloids, 23(1), 153-164.

Some amylases can delay bread staling and/or starch (amylopectin) retrogradation, but the molecular basis of this effect remains little understood. In order to increase our insight in these aspects of amylase functionality, several amylases were added in a pure wheat-starch-containing model system and subjected to a heating step corresponding to that in the baking phase in bread making. Next, the effects of the limited amylolytic degradation on the rapid visco analyser (RVA) rheological properties of starch were studied and the accompanying changes in the amylopectin molecular properties (such as chain length distribution) investigated. The different amylases clearly affected the molecular structure of amylopectin to a different extent, which could be related to their mode of action and the enzyme activity levels added. Bacillus subtilis and Aspergillus oryzae α-amylases had only a limited impact on the side chain distribution of the amylopectin molecules, presumably due to their preferential hydrolysis of internal chain segments and the low enzyme activity added in the RVA. In contrast, porcine pancreatic α-amylase and Bacillus stearothermophilus maltogenic α-amylase, both with higher degree of multiple attack and used at higher enzyme activity levels, had a marked influence on the amylopectin molecular structure. More in particular, under the test conditions, the maltogenic α-amylase reduced the average chain length of the outer chains by 50%. Presumably, this will affect amylopectin retrogradation to a large extent. The results contribute to a better understanding of amylase functionality in starchy foods.

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Debranching of β-dextrins to explore branching patterns of amylopectins from three maize genotypes.

Xia, H. & Thompson, D. B. (2006). Cereal Chemistry, 83(6), 668-676.

The amylopectin (AP) branching pattern is a fundamental feature of AP fine structure but a little-studied one. In this work, we followed enzyme digestion over time for AP from three maize genotypes (wx, du wx, and AP of ae VII). The objective was to describe differences in the progress of β-amylolysis and in subsequent debranching of β-limit dextrins (β-LD). During the progress of β-amylolysis, changes in the distribution of short residual chains show that the enzyme favors hydrolysis farthest from branch points. On treating β-LD with isoamylase (IA) alone, debranching was incomplete. Using IA and pullulanase (PUL) sequentially, a similar increase in the DP 5–7 region and the peak at DP 6 were observed for all samples, indicating a common element in the branching pattern. This similarity suggests that, despite differences in the proportion of short to long B chains, the most closely associated branch points may be arranged in a similar way for these AP. We suggest that the increase in DP 6 after PUL digestion would result from debranching of linear DP 6 residual B chains that originally had two branch points, consistent with interior segment length (ISL) of 1 or 2.

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Enzyme-aided investigation of the substituent distribution in cationic potato amylopectin starch.

Richardson, S., Nilsson, G., Cohen, A., Momcilovic, D., Brinkmalm, G. & Gorton, L. (2003). Analytical Chemistry, 75(23), 6499-6508.

The distribution of substituents along the polymer chain in cationic potato amylopectin starch, modified in solution, granular slurry, or dry state, was investigated. The starch derivatives were successively hydrolyzed by different enzymes, followed by characterization of the hydrolysis products obtained by means of electrospray mass spectrometry (ESI-MS) and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). ESI-MS and MALDI-MS were proved to be appropriate techniques for identification of the substituted hydrolysis products, for which there are no standard compounds available. No highly substituted oligomers were found in the hydrolysates, which was taken as an indication of a more or less homogeneous distribution of cationic groups in the amylopectin molecules. Furthermore, from the results obtained it was suggested that the enzymes cleave glucosidic linkages only between unsubstituted glucose units and, preferentially, linkages in sequences containing more than two adjacent unsubstituted units. The determination of the amount of unsubstituted glucose produced from every successive hydrolysis step revealed slight differences between the different starch samples with respect to the homogeneity of the substitution pattern. Among the three samples under investigation, starch cationized in solution was found to have the most and dry-cationized starch the least homogeneous distribution of substituents.

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Rapid determination of enzyme purity by a microdialysis-based assay.

Richardson, S., Nilsson, G. S., Torto, N., Gorton, L. & Laurell, T. (1999). Analytical Communications, 36(5), 189-193.

Microdialysis was shown to be useful as a fast on-line sampling method for determining the purity of starch hydrolysing enzymes. The enzymes were characterised using their hydrolytic properties. β-Amylases and pullulanases from different sources and/or manufacturers were investigated, with maltose, maltoheptaose, pullulan, and potato amylopectin starch (PAP) as substrates. The hydrolysis products were sampled via an on-line microdialysis probe and determined in a high-performance anion-exchange chromatographic (HPAEC) system. Comparison between the expected (theoretical) hydrolysis products with those obtained in the experiments made it possible to determine impurities in the enzymes. Two of the β-amylases and one pullulanase released unwanted hydrolysis products, indicating trace impurities in the enzyme preparation. Microdialysis sampling allows on-line sampling and eliminates separate sample preparation and clean-up steps. On-line microdialysis coupled to a HPAEC system is therefore a fast and simple technique for analysing enzyme hydrolysates.

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Safety Information
Symbol : GHS08
Signal Word : Danger
Hazard Statements : H334
Precautionary Statements : P261, P284, P304+P340, P342+P311, P501
Safety Data Sheet
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