|Formulation:||In 3.2 M ammonium sulphate|
|Stability:||> 1 year under recommended storage conditions|
|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.
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Characterization of the Glucan-Branching Enzyme GlgB Gene from Swine Intestinal Bacteria.
Shao, Y., Wang, W., Hu, Y. & Gänzle, M. G. (2023). Molecules, 28(4), 1881.
Starch hydrolysis by gut microbiota involves a diverse range of different enzymatic activities. Glucan-branching enzyme GlgB was identified as the most abundant glycosidase in Firmicutes in the swine intestine. GlgB converts α-(1→4)-linked amylose to form α-(1→4,6) branching points. This study aimed to characterize GlgB cloned from a swine intestinal metagenome and to investigate its potential role in formation of α-(1→4,6)-branched α-glucans from starch. The branching activity of purified GlgB was determined with six different starches and pure amylose by quantification of amylose after treatment. GlgB reduced the amylose content of all 6 starches and amylose by more than 85% and displayed a higher preference towards amylose. The observed activity on raw starch indicated a potential role in the primary starch degradation in the large intestine as an enzyme that solubilizes amylose. The oligosaccharide profile showed an increased concentration of oligosaccharide introduced by GlgB that is not hydrolyzed by intestinal enzymes. This corresponded to a reduced in vitro starch digestibility when compared to untreated starch. The study improves our understanding of colonic starch fermentation and may allow starch conversion to produce food products with reduced digestibility and improved quality.Hide Abstract
The location of octenyl succinate anhydride groups in high-amylose maize starch granules and its effect on stability of pickering emulsion stability.
Li, J., Wang, Q., Blennow, A., Herburger, K., Zhu, C., Nurzikhan, S., wei, J., Zhong, Y. & Guo, D. (2022). LWT, 169, 113892.
Octenyl succinate anhydride (OSA) modified high amylose maize starch (HAMS) showed limited emulsion stability due to the OSA groups were located on the surface of starch granules. However, amylose is enriched in the internal region of HAMS and provides the reaction site for OSA groups. In this study, five types of starches with different amylose content were prepared using optimized reaction conditions. Our data showed that amylose content (AC) was the most important parameter controlling the degree of substitution (DS), in comparison with temperature, pH, starch concentration and time. DS reached a maximum (2.1%) at AC of 58%. Importantly, OSA groups were mainly located in the internal regions of HAMS under the optimized reaction conditions. This was supported by (1) the decrease of the crystallinity, (2) the weaker fluorescence intensity of the starch fluorophore probe 8-amino-1,3,6-pyrenetrisulfonic acid (APTS) in the internal regions of HAMS, and (3) unchanged signals from Fourier transform infrared spectroscopy which characterized the granular surface structure ordering. This new OSA reaction pattern permits HAMS to be used in stabilizing Pickering emulsions This is the first report that OSA groups located in the internal regions of HAMS granules.Hide Abstract
The influence of amylose content on the modification of starches by glycogen branching enzymes.
Gaenssle, A. L., van der Maarel, M. J. & Jurak, E. (2022). Food Chemistry, 133294.
Glycogen branching enzymes (GBEs) have been used to generate new branches in starches for producing slowly digestible starches. The aim of this study was to expand the knowledge about the mode of action of these enzymes by identifying structural aspects of starchy substrates affecting the products generated by different GBEs. The structures obtained from incubating five GBEs (three from glycoside hydrolase family (GH) 13 and two from GH57) on five different substrates exhibited minor but statistically significant correlations between the amount of longer chains (degree of polymerization (DP) 9-24) of the product and both the amylose content and the degree of branching of the substrate (Pearson correlation coefficient of ≤−0.773 and ≥0.786, respectively). GH57 GBEs mainly generated large products with long branches (100-700 kDa and DP 11-16) whereas GH13 GBEs produced smaller products with shorter branches (6-150 kDa and DP 3-10).Hide Abstract
Slowly digestible property of highly branched α-limit dextrins produced by 4, 6-α-glucanotransferase from Streptococcus thermophilus evaluated in vitro and in vivo.
Ryu, J. J., Li, X., Lee, E. S., Li, D. & Lee, B. H. (2021). Carbohydrate Polymers, 275, 118685.
Starch molecules are first degraded to slowly digestible α-limit dextrins (α-LDx) and rapidly hydrolyzable linear malto-oligosaccharides (LMOs) by salivary and pancreatic α-amylases. In this study, we designed a slowly digestible highly branched α-LDx with maximized α-1,6 linkages using 4,6-α-glucanotransferase (4,6-αGT), which creates a short length of α-1,4 side chains with increasing branching points. The results showed that a short length of external chains mainly composed of 1–8 glucosyl units was newly synthesized in different amylose contents of corn starches, and the α-1,6 linkage ratio of branched α-LDx after the chromatographical purification was significantly increased from 4.6% to 22.1%. Both in vitro and in vivo studies confirmed that enzymatically modified α-LDx had improved slowly digestible properties and extended glycemic responses. Therefore, 4,6-αGT treatment enhanced the slowly digestible properties of highly branched α-LDx and promises usefulness as a functional ingredient to attenuate postprandial glucose homeostasis.Hide Abstract
Fine structure impacts highly concentrated starch liquefaction process and product performance.
Kong, H., Yu, L., Gu, Z., Li, Z., Ban, X., Cheng, L., Hong, Y. & Li, C. (2021). Industrial Crops and Products, 164, 113347.
Designing a highly concentrated (45 %, w/w) starch liquefaction process is a green method to enhance the productivity of starch syrup and related fermentation products. Previous studies mainly focused on handling highly concentrated normal corn starch slurries, but the production efficiency and product performance cannot perfectly match the conventional liquefaction process (30 %, w/w). In the present research, four starches from various botanical sources were selected with an objective to accelerate highly concentrated starch liquefaction process. The results demonstrated that with potato starch or tapioca starch as a substrate, liquefaction process was more feasible as observed from the obvious reduction in paste viscosity and acceleration in amylolysis. To clarify the mechanism of these differences, changes in the fine structure during liquefaction were further characterized. The long external chains (16.2 glucose units on average) in potato starch and long internal chains (5.1 glucose units on average) in tapioca starch, which indicated high proportion of consecutive α-1,4 linkages, seemed more susceptible to enzymatic attack under highly concentrated substrate condition. This caused rapid degradation of starch molecules. The liquefied products were suitable for glucose syrup production. By comparison, normal corn starch and waxy corn starch, which contain relatively shorter linear fragments, were less accessible to α-amylase. This suppressed liquefaction process led to the survival of large molecules, thereby being unsuitable for subsequent saccharification process. The results suggest that selecting an appropriate substrate is an effective strategy to accelerate highly concentrated starch liquefaction and improve product performance.Hide Abstract
Two 1, 4-α-glucan branching enzymes successively rearrange glycosidic bonds: A novel synergistic approach for reducing starch digestibility.
Yu, L., Kong, H., Gu, Z., Li, C., Ban, X., Cheng, L., Hong, Y. & Li, Z. (2021). Carbohydrate Polymers, 262, 117968.
Enzymatically rearranging α-1,4 and α-1,6 glycosidic bonds in starch is a green approach to regulating its digestibility. A two-step modification process successively catalyzed by 1,4-α-glucan branching enzymes (GBEs) from Rhodothermus obamensi STB05 (Ro-GBE) and Geobacillus thermoglucosidans STB02 (Gt-GBE) was investigated as a strategy to reduce the digestibility of corn starch. This dual GBE modification process caused a reduction of 25.8 % in rapidly digestible starch fraction in corn starch, which were more effective than single GBE-catalyzed modification with the same duration. Structural analysis indicated that the dual GBE modified product contained higher branching density, more abundant short branches, and shorter external chains than those in single GBE-modified product. These results demonstrated that a moderate Ro-GBE treatment prior to starch gelatinization caused several suitable alterations in starch molecules, which promoted the transglycosylation efficiency of the following Gt-GBE treatment. This dual GBE-catalyzed modification process offered an efficient strategy for regulating starch digestibility.Hide Abstract
Influence of microwave treatment on the structure and functionality of pure amylose and amylopectin systems.
Zhong, Y., Tian, Y., Liu, X., Ding, L., Kirkensgaard, J. J. K., Hebelstrup, K., Putaux, J. L. & Blennow, A. (2021). Food Hydrocolloids, 119, 106856.
Pure granular amylose (AM) and pure granular amylopectin (waxy) starch (AP) granules have the high nutritional value in food industry. Effects of microwave treatment (400 W/g DW, 1-8 min) on the structure and properties of transgenic AM granules and AP granules were investigated in direct comparison. Microwave treatment, especially during the first 3 min, decreased the molecular weight of molecules in both the AM and the AP samples. The crystallinity of AM starch initially increased from 15.6% to 20.6%, which was associated with the formation of new Vh-type crystals. After that, crystallinity decreased alongside to 11.3% with the complete disruption of B-type crystals. In contrast, the crystallinity of AP starch initially decreased from 18.9% to 10.8% followed by an increase to 20.0%. Upon prolonged treatment of AM granules, the resistant starch and water solubility was significantly increased. Our data demonstrate notable different microwave-dependent reorganization patterns for pure granular AM and AP molecules as native granular systems, which is helpful to the improvement of functionality of these two starches.Hide Abstract
Structural elements determining the transglycosylating activity of glycoside hydrolase family 57 glycogen branching enzymes.
Xiang, G., Leemhuis, H. & van der Maarel, M. (2021). Authorea Preprints, In Press.
Glycoside hydrolase family 57 glycogen branching enzymes (GH57GBE) catalyze the formation of an α-1,6 glycosidic bond between α-1,4 linked glucooliogosaccharides. As an atypical family, a limited number of GH57GBEs have been biochemically characterized so far. This study aimed at acquiring a better understanding of the GH57GBE family by a systematic sequence-based bioinformatics analysis of almost 2500 gene sequences and determining the branching activity of several native and mutant GH57GBEs. A correlation was found in a very low or even no branching activity with the absence of a flexible loop, a tyrosine at the loop tip, and two β-strands.Hide Abstract
New insights into the alleviating role of starch derivatives on dough quality deterioration caused by freeze.
Li, Y., Li, C., Ban, X., Cheng, L., Hong, Y., Gu, Z. & Li, Z. (2021). Food Chemistry, 130240.
The alleviating role of starch derivatives on the quality deterioration of frozen steamed bread dough was investigated in terms of derivative structure, the bread characteristics and dough properties including freezable water contents, yeast activity as well as dough viscoelasticity. The addition of starch derivatives including short-clustered maltodextrin (SCMD), DE2 maltodextrin (MD) and pregelatinized starch (PGS) significantly increased the specific volume and decreased the hardness of steamed bread compared with Control bread after 8-week frozen storage. Lower freezable water content was found in PGS dough than SCMD dough, which was consistent with the results of water absorption index of starch derivatives. The analysis of dough gassing rate and yeast survival ratio demonstrated SCMD could provide more cryoprotection for yeast cells. Meanwhile, a higher elastic module and a more continuous gluten-network structure of SCMD dough were found after 8-week frozen storage. These results indicated starch derivatives especially SCMD were promising to be used as the alternative improvers in frozen dough production.Hide Abstract
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