|Formulation:||In 3.2 M ammonium sulphate|
|Stability:||> 4 years at 4oC|
|Synonyms:||pullulanase; pullulan 6-alpha-glucanohydrolase|
|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.|
|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.
View all Carbohydrate Active EnZyme products available.
A simplified method of determining the internal structure of amylopectin from barley starch without amylopectin isolation.
Zhao, X., Andersson, M. & Andersson, R. (2020). Carbohydrate Polymers, 117503.
To determine the internal structure of barley starch without amylopectin isolation, whole starch was hydrolyzed using β-amylase to remove the linear amylose and obtain β-limit dextrins (β-LDs). The β-LDs were treated with extensive α-amylase to prepare α-limit dextrins (α-LDs), and the α-LDs were further hydrolyzed with β-amylase into building blocks. The chain-length distribution of β-LD and building block composition were analyzed by size-exclusion chromatography and anion-exchange chromatography. The internal structure of the barley whole starches had similar pattern to barley amylopectins analyzed by conventional methods. The starch of barley amo1-mutated varieties contained more short internal B-chains and less long internal B-chains than that of other varieties. The starch from amo1-mutated varieties had more large building blocks than that from waxy varieties. The simplified method presented in this study can effectively characterize starch internal structure that relates to physicochemical properties of starch, although some details of amylopectin structure are not assessable.Hide Abstract
Digestion kinetics of low, intermediate and highly branched maltodextrins produced from gelatinized starches with various microbial glycogen branching enzymes.
Zhang, X., Leemhuis, H. & van der Maarel, M. J. (2020). Carbohydrate Polymers, 247, 116729.
Twenty-four branched maltodextrins were synthesized from eight starches using three thermostable microbial glycogen branching enzymes. The maltodextrins have a degree of branching (DB) ranging from 5 % to 13 %. This range of products allows us to explore the effect of DB on the digestibility, which was quantified under conditions that mimic the digestion process in the small intestine. The rate and extent of digestibility were analyzed using the logarithm of the slope method, revealing that the branched maltodextrins consist of a rapidly and slowly digestible fraction. The amount of slowly digestible maltodextrin increases with an increasing DB. Surprisingly, above 10 % branching the fraction of slowly digestible maltodextrin remains constant. Nevertheless, the rate of digestion of the slowly digestible fraction was found to decline with increasing DB and shorter average internal chain length. These observations increase the understanding of the structural factors important for the digestion rate of branched maltodextrins.Hide Abstract
Removal of starch granule-associated proteins affects amyloglucosidase hydrolysis of rice starch granules.
Ma, M., Xu, Z., Li, P., Sui, Z. & Corke, H. (2020). Carbohydrate Polymers, 247, 116674.
Starch granule-associated proteins (SGAPs) include granule-surface proteins (SGSPs) and granule-channel proteins (SGCPs). To investigate impacts of SGAPs on amyloglucosidase (AMG) hydrolysis, waxy and non-waxy rice starches had their SGCPs or SGAPs removed. Removal of SGAPs or SGCPs did not affect morphology and amylopectin chain distribution but decreased relative crystallinity. Removal of SGAPs increased the digestion rate, AMG binding ability and pore diameter of hydrolyzed granules, and accelerated changes in relative crystallinity and destruction of crystalline region on hydrolysis. However, after removing SGCPs, AMG only bound to surface and attacked of the fingerprint of protein bodies on granules, with decreased hydrolysis rate. The degree of change in hydrolysis rate was not determined by SGCPs content of rice starch. These results implied that SGCPs had a more dominant role in AMG hydrolysis of rice starch than did SGSPs. This study provides novel information about the role of SGAPs in AMG hydrolysis mechanisms.Hide Abstract
On the role of the internal chain length distribution of amylopectins during retrogradation: double helix lateral aggregation and slow digestibility.
Roman, L., Yee, J., Hayes, A. M., Hamaker, B., Bertoft, E. & Martinez, M. M. (2020). Carbohydrate Polymers, 246, 116633.
A structure-digestion model is proposed to explain the formation of α-amylase-slowly digestible structures during amylopectin retrogradation. Maize and potato (normal and waxy) and banana starch (normal and purified amylopectin through alcohol precipitation), were analyzed for amylose ratio and size (HPSEC) and amylopectin unit- and internal-chain length distribution (HPAEC). Banana amylopectin (BA), like waxy potato (WP), exhibited a larger number of B3-chains, fewer BS- and Bfp-chains and lower S:L and BS:BL ratios than maize, categorizing BA structurally as type-4. WP exhibited a significantly greater tendency to form double helices (DSC and 13C-NMR) than BA, which was attributed to its higher internal chain length (ICL) and fewer DP6−12-chains. However, retrograded BA was remarkably more resistant to digestion than WP. Lower number of phosphorylated B-chains, more S- and Bfp-chains and shorter ICL, were suggested to result in α-amylase-slowly digestible structures through further lateral packing of double helices (suggested by thermo-rheology) in type-4 amylopectins.Hide Abstract
Cassava Metabolomics and Starch Quality.
Rosado‐Souza, L., David, L. C., Drapal, M., Fraser, P. D., Hofmann, J., Klemens, P. A., et al. (2019). Current protocols in Plant Biology, 4(4), e20102.
Cassava plays an important role as a staple food for more than 800 million people in the world due to its ability to maintain relatively high productivity even in nutrient‐depleted soils. Even though cassava has been the focus of several breeding programs and has become a strong focus of research in the last few years, relatively little is currently known about its metabolism and metabolic composition in different tissues. In this article, the absolute content of sugars, organic acids, amino acids, phosphorylated intermediates, minerals, starch, carotenoids, chlorophylls, tocopherols, and total protein as well as starch quality is described based on multiple analytical techniques, with protocols specifically adjusted for material from different cassava tissues. Moreover, quantification of secondary metabolites relative to internal standards is presented using both non‐targeted and targeted metabolomics approaches. The protocols have also been adjusted to apply to freeze‐dried material in order to allow processing of field harvest samples that typically will require long‐distance transport.Hide Abstract
Structural characterization of mixed-linkage α-glucans produced by mutants of Lactobacillus reuteri TMW 1.106 dextransucrase.
Münkel, F., Fischer, A., & Wefers, D. (2020). Carbohydrate Polymers, 231, 115697.
Dextrans and other bacterial α-glucans are versatile and structurally diverse polysaccharides which can be enzymatically synthesized by using glucansucrases. By substituting certain amino acids in the active site of these enzymes, the structure of the synthesized polysaccharides can be modified. In this study, such amino acid substitutions were applied (single and combined) to the dextransucrase from Lactobacillus reuteri TMW 1.106 and the structures of the synthesized polysaccharides were subsequently characterized in detail. Besides methylation analysis, α-glucans were hydrolyzed by several glycoside hydrolases and the liberated oligosaccharides were identified by comparison to standard compounds or by isolation and NMR spectroscopic characterization. Furthermore, two-dimensional NMR spectroscopy was used to analyze the untreated polysaccharides. The results demonstrated that structurally different α-glucans were formed, for example different highly O4-branched dextrans or several reuteran-like polymers with varying fine structures. Consequently, mutant Lactobacillus reuteri TMW 1.106 dextransucrases can be used to form structurally unique polysaccharides.Hide Abstract
A novel starch: Characterizations of starches separated from tea (Camellia sinensis (L.) O. Ktze) seed.
Huang, J., Kong, X., Wu, D., Zheng, Z. & Shu, X. (2019). International Journal of Biological Macromolecules, 139, 1085-1091.
The physicochemical, thermal and crystal properties of starches isolated from 3 different tea (Camellia sinensis (L.) O. Ktze) seeds were analyzed in this study. The shape of tea starch granules were flat spherical or oval shape, showed unimodal or bimodal distribution with average size of around 9 μm. Tea starch was typical A-type starch. Apparent amylose contents of three tea seed starches ranged from 27.06% to 33.17%. The chains having degree of polymerization (DP) 13-24 were over 50% of the total detectable chains for tea amylopectin. Peak gelatinization temperature of tea starch ranged from 65 to 77°C and the water solubility reached up to 9.70%. The peak viscosity of tea starches were as high as 5300 cP and final viscosity ranged from 4000 to 6700 cP. The results indicated that tea seed starch had potential as gel reagents and provide some guides for comprehensive utilization of tea starch in food and non-food applications.Hide Abstract
Physiochemical properties of rice with contrasting resistant starch content.
Ding, Y., Huang, J., Zhang, N., Rasmussen, S. K., Wu, D. & Shu, X. (2019). Journal of Cereal Science, 89, 102815.
RS2 and RS3 are two of the main resistant starch types in food, and the primary types found in raw and cooked rice respectively. To elucidate the physiochemical determinants for RS2 and RS3 content in rice, this study investigated the fine amylopectin structure, starch granules morphologies and starch pasting properties of nine different rice accessions. The results revealed that not all the accessions showed a decrease in resistant starch after cooking and cooling. Granular size, paste properties, fb1 and fb3 showed a significant negative correlation with RS3, whereas fa showed a significant positive correlation with RS3. RS2 had no significant correlation with granular size, but did have a significant positive correlation with HPV, CPV and fb2. Different rice types could be distinguished by the fine amylopectin structure of DP6-12 and DP13-24 chains. Taken together fa, fb2 and HPV and CPV values may have the potential to be used as indices for distinguishing or characterising rice with different RS2 and RS3 contents.Hide Abstract
Characterization of the GH13 and GH57 glycogen branching enzymes from Petrotoga mobilis SJ95 and potential role in glycogen biosynthesis.
Zhang, X., Leemhuis, H. & van der Maarel, M. J. (2019). PloS One, 14(7), e0219844.
Glycogen is a highly branched α-glucan polymer widely used as energy and carbon reserve by many microorganisms. The branches are introduced by glycogen branching enzymes (EC 184.108.40.206), that are classified into glycoside hydrolase families 13 (GH13) and 57 (GH57). Most microorganisms have typically only a single glycogen branching enzyme (gbe) gene. Only a few microorganisms carry both GH13 and GH57 gbe genes, such as Petrotoga mobilis and Mycobacterium tuberculosis. Here we report the basic characteristics of the GH13 and GH57 GBE of P. mobilis, both heterologously expressed in E. coli. The GH13 GBE has a considerably higher branching activity towards the linear α-glucan amylose, and produces a highly branched α-glucan with a high molecular weight which is very similar to glycogen. The GH57 GBE, on the contrary, makes a much smaller branched α-glucan. While the GH13 GBE acts as a classical glycogen branching enzyme involved in glycogen synthesis, the role of GH57 GBE remains unclear.Hide Abstract
Comparison of molecular structure of oca (Oxalis tuberosa), potato, and maize starches.
Zhu, F. & Cui, R. (2019). Food Chemistry, 296, 116-122.
Oca (Oxalis tuberosa) is an underutilized species and represents a novel starch source. Composition and structure of starches from tubers of two commercial oca varieties grown in New Zealand were compared to those of normal maize and potato starches. The phosphorus content of oca starch was ∼60% of that of potato starch. The amylose content of oca starch (∼21%) was lower than that of maize and potato starches (concanavalin A precipitation method). The fine structure of oca amylopectin was much more similar to that of potato amylopectin than to that of maize amylopectin. Oca amylopectin had a shorter internal chain length and less fingerprint B-chains than potato amylopectin. The two oca starches were structurally and compositionally similar. Oca starch granules had a volume moment mean size of 34.5 μm and B-type polymorph. Comparative analysis suggested that oca starch has the potential to be developed as a novel starch source.Hide Abstract
Synthesis of highly branched α-glucans with different structures using GH13 and GH57 glycogen branching enzymes.
Zhang, X., Leemhuis, H. & van der Maarel, M. J. (2019). Carbohydrate Polymers, 216, 231-237.
Glycogen branching enzymes (GBEs) convert starch into branched α-glucan polymers. To explore if the amylose content of substrates effects the structure of the branched α-glucans, mixtures of amylose and amylopectin were converted by four thermophilic GBEs. The degree of branching and molecular weight of the products increased with an increasing percentage of amylose with the GH57 GBEs of Thermus thermophilus and Thermococcus kodakarensis, and the GH13 GBEs of Rhodothermus marinus and Petrotoga mobilis. The only exception is that the degree of branching of the Petrotoga mobilis GBE products is not influenced by the amylose content. A second difference is the relatively high hydrolytic activity of two GH57 GBEs, while the two GH13 GBEs have almost no hydrolytic activity. Moreover, the two GH13 GBEs synthesize branched α-glucans with a narrow molecular weight distribution, while the two GH57 GBEs products consist of two or three molecular weight fractions.Hide Abstract
Organelle DNA contents and starch accumulation in potato tubers.
Niu, S., Zhang, G., Li, X., Haroon, M., Si, H., Fan, G. & Li, X. Q. (2019). Theoretical and Applied Genetics, 132(1), 205-216.
Starch is the main dry matter component of various staple food crops, including potato. Starch synthesis and accumulation is in plastids, uses sugar, consumes cellular energy, and requires active expression of starch synthesis genes. We hypothesized that the plastid/nuclear DNA ratios and mitochondrial/nuclear DNA ratios are involved in this accumulation. We analyzed the dry mater, starch, plastid DNA, mitochondrial DNA, and nuclear DNA in tuber stem ends and tuber bud ends in two potato cultivars and verified the results using whole tubers in nine potato cultivars. Dry matter contents (DMC) and organelle/nuclear DNA ratios increased rapidly during tuber bulking. DMC and starch contents were greater at the tuber stem ends than at the tuber bud ends. Both the comparisons between tuber ends and among whole tubers indicated that DMC and starch contents were positively correlated with both plastid/nuclear DNA ratios and mitochondrial/nuclear DNA ratios. The results suggest that pt/nuc and mt/nuc DNA ratios are important and may serve as a biomarker in selection, genetic engineering, and cytoplasm manipulation, for dry matter and starch accumulation in potato.Hide Abstract
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.Hide Abstract
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.Hide Abstract
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.Hide Abstract
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.Hide Abstract
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.Hide Abstract
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.).Hide Abstract
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.Hide Abstract
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.Hide Abstract