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Limit-Dextrizyme Tablets

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Analysis of enzymes activity using carbohydrase tablet testing

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Chapter 1: Theory of endo-1, 4-Beta-D-Xylanase Assay Procedure
Chapter 2: Buffers & Reagents
Chapter 3: Assay Procedure
Product code: T-LDZ-200T



200 Tablets

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Content: 200 Tablets or 1,000 Tablets
Shipping Temperature: Ambient
Storage Temperature: Ambient
Physical Form: Solid
Stability: > 2 years under recommended storage conditions
Substrate For (Enzyme): Isoamylase, Isopullulanase, Neopullulanase, Pullulanase/Limit-Dextrinase
Assay Format: Spectrophotometer
Detection Method: Absorbance
Wavelength (nm): 590
Reproducibility (%): ~ 5%
Method recognition: RACI Standard Method

The T-LDZ-1000T pack size has been discontinued (read more).

High purity dyed and crosslinked Limit-Dextrizyme tablets for the measurement of enzyme activity, for research, biochemical enzyme assays and in vitro diagnostic analysis. 

New updated assay protocol employing amyloglucosidase into sample preparation. For the assay of limit-dextrinase and pullulanase. Containing AZCL-Pullulan.

Please note the video above shows the protocol for assay of endo-xylanase using xylazyme tablets. The procedure for the assays of pullulanase/limit-dextrinase, isopullulanase, neopullulanase and isoamylase using Limit-Dextrizyme Tablets is equivalent to this.

Browse our complete list of enzyme tablet tests.

Validation of Methods

Certificate of Analysis
Safety Data Sheet
FAQs Assay Protocol
Megazyme publication
Colourimetric and fluorimetric substrates for the assay of limit dextrinase.

Mangan, D., McCleary, B. V., Cornaggia, C., Ivory, R., Rooney, E. & McKie, V. (2015). Journal of Cereal Science, 62, 50-57.

The measurement of limit-dextrinase (LD) (EC in grain samples such as barley, wheat or rice can be problematic for a number of reasons. The intrinsic LD activity in these samples is extremely low and they often contain a limit-dextrinase inhibitor and/or high levels of reducing sugars. LD also exhibits transglycosylation activity that can complicate the measurement of its hydrolytic activity. A minor modification to the industrial standard Limit-Dextrizyme tablet test is suggested here to overcome this transglycosylation issue.

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Megazyme publication
Measurement of the content of limit-dextrinase in cereal flours.

McCleary, B. V. (1992). Carbohydrate Research, 227, 257-268.

Procedures for the quantitative extraction, activation, and assay of limit-dextrinase in cereal flours have been developed. Extraction and activation require incubation in buffer containing 20mm cysteine for at least 16 h or with 25mM dithiothreitol for 5 h. Activity is assayed with a soluble, dyed substrate (Red-Pullulan) or an insoluble, dyed, and cross-linked substrate (Azurine-CL-Pullulan) which is dispensed in tablet form (Limit-DextriZyme tablets).

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Variation in maltose in sweet wort from barley malt and rice adjuncts with differences in amylose structure.

Fox, G., Yu, W., Nischwitz, R. & Harasymow, S. (2019). Journal of the Institute of Brewing, 125(1), 18-27.

Starch from malt and solid adjuncts provides the majority of fermentable sugars for fermentation. However, there is no current data on the variation in starch structure (particularly long chained amylose) and its impact on the final wort composition of fermentable sugars, specifically maltose. This is the first study to report variation in amylose structure from barley malt and rice used as an adjunct and how this impacts the production of maltose. We compared four commercial malts with two rice adjuncts mashes, in solid and liquid additions, with an all‐malt mash used as a control. All combinations of malt and rice adjuncts were tested under two grist‐to‐liquor (G:L) ratios (1:3 and 1:4) in a 65°C ramped mash. After mashing, the wort original gravity and maltose concentration were measured. The commercial malts had different malt quality but very similar gelatinisation temperatures (~ 65°C). The malts varied in starch and amylose contents but had only minor variations in average amylose chain lengths. The two rice adjuncts also had similar average amylose chains lengths, but quite different amylose contents, and hence different gelatinisation temperatures. The results showed that liquid adjunct mashes had higher original gravity and maltose concentration for both G:L ratios. However, there was no consistent result in original gravity or maltose between G:L ratio or adjunct type, suggesting interactions between each malt and rice adjunct. Knowing amylose chain length could improve understanding of the potential maltose levels of the sweet wort prior to fermentation.

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Optimization of the production of an extracellular and thermostable amylolytic enzyme by Thermus thermophilus HB8 and basic characterization.

Akassou, M. & Groleau, D. (2017). Extremophiles, 1-14.

The objective of this study was to determine the potential of Thermus thermophilus HB8 for accumulating a high level of extracellular, thermostable amylolytic enzyme. Initial production tests indicated clearly that only very low levels of amylolytic activity could be detected, solely from cells after extraction using the mild, non-ionic detergent Triton X-100. A sequential optimization strategy, based on statistical designs, was used to enhance greatly the production of extracellular amylolytic activity to achieve industrially attractive enzyme titers. Focus was placed on the optimal level of initial biomass concentration, culture medium composition and temperature for maximizing extracellular amylolytic enzyme accumulation. Empirical models were then developed describing the effects of the experimental parameters and their interactions on extracellular amylolytic enzyme production. Following such efforts, extracellular amylolytic enzyme accumulation was increased more than 70-fold, with enzyme titers in the 76 U/mL range. The crude extracellular enzyme was thereafter partially characterized. The optimal temperature and pH values were found to be 80°C and 9.0, respectively. 100% of the initial enzyme activity could be recovered after incubation for 24 h at 80°C, therefore, proving the very high thermostability of the enzyme preparation.

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A chromogenic assay suitable for high-throughput determination of limit dextrinase activity in barley malt extracts.

Bøjstrup, M., Marri, L., Lok, F. & Hindsgaul, O. (2015). Journal of agricultural and Food Chemistry, 63(50), 10873-10878.

Twenty-four malt samples were assayed for limit dextrinase activity using a chromogenic assay developed recently in our group. The assay utilizes a small soluble chromogenic substrate which is hydrolyzed selectively by limit dextrinase in a coupled assay to release the chromophore 2-chloro-4-nitrophenol. The release of the chromophore, corresponding to the activity of limit dextrinase, can be followed by measuring the UV absorption at 405 nm. The 24 malt samples represented a wide variation of limit dextrinase activities, and these activities could be clearly differentiated by the assay. The results obtained were comparable with the results obtained from a commercially available assay, Limit-Dextrizyme from Megazyme International Ireland. Furthermore, the improved assay uses a soluble substrate. That makes it well suited for high-throughput screening as it can be handled in a 96-well plate format.

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Oligosaccharide and substrate binding in the starch debranching enzyme barley limit dextrinase.

Møller, M. S., Windahl, M. S., Sim, L., Bøjstrup, M., Hachem, M. A., Hindsgaul, O., Palcic, M., Svensson, B. & Henriksen, A. (2015). Journal of Molecular Biology, 427(6), 1263-1277.

Complete hydrolytic degradation of starch requires hydrolysis of both the α-1,4- and α-1,6-glucosidic bonds in amylopectin. Limit dextrinase (LD) is the only endogenous barley enzyme capable of hydrolyzing the α-1,6-glucosidic bond during seed germination, and impaired LD activity inevitably reduces the maltose and glucose yields from starch degradation. Crystal structures of barley LD and active-site mutants with natural substrates, products and substrate analogues were sought to better understand the facets of LD–substrate interactions that confine high activity of LD to branched maltooligosaccharides. For the first time, an intact α-1,6-glucosidically linked substrate spanning the active site of a LD or pullulanase has been trapped and characterized by crystallography. The crystal structure reveals both the branch and main-chain binding sites and is used to suggest a mechanism for nucleophilicity enhancement in the active site. The substrate, product and analogue complexes were further used to outline substrate binding subsites and substrate binding restraints and to suggest a mechanism for avoidance of dual α-1,6- and α-1,4-hydrolytic activity likely to be a biological necessity during starch synthesis.

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Efficient secretory expression of functional barley limit dextrinase inhibitor by high cell-density fermentation of Pichia pastoris.

Jensen, J. M., Vester-Christensen, M. B., Møller, M. S., Bønsager, B. C., Christensen, H. E. M., Hachem, M. A. & Svensson, B. (2011). Protein Expression and Purification, 79(2), 217-222.

The limit dextrinase inhibitor (LDI) from barley seeds acts specifically on limit dextrinase (LD), an endogenous starch debranching enzyme. LDI is a 14 kDa hydrophobic protein containing four disulfide bonds and one unpaired thiol group previously found to be either glutathionylated or cysteinylated. It is a member of the so-called CM-protein family that includes α-amylase and serine protease inhibitors, which have been extremely challenging to produce recombinantly in functional form and in good yields. Here, LDI is produced in very high yields by secretory expression by Pichia pastoris applying high cell-density fermentation in a 5 L fed-batch bioreactor. Thus about 200 mg of LDI, which showed twofold higher inhibitory activity towards LD than LDI from barley seeds, was purified from 1 L of culture supernatant by His-tag affinity chromatography and gel filtration. Electrospray ionization mass spectrometry verified the identity of the produced glutathionylated LDI-His6. At a 1:1 M ratio the recombinant LDI completely inhibited hydrolysis of pullulan catalyzed by 5–10 nM LD. LDI retained stability in the pH 2–12 range and at pH 6.5 displayed a half-life of 53 and 33 min at 90 and 93°C, respectively. The efficient heterologous production of LDI suggests secretory expression by P. pastoris to be a promising strategy to obtain other recombinant CM-proteins.

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Secretory expression of functional barley limit dextrinase by Pichia pastoris using high cell-density fermentation.

Vester-Christensen, M. B., Hachem, M. A., Naested, H. & Svensson, B. (2010). Protein Expression and Purification, 69(1), 112-119.

Heterologous production of large multidomain proteins from higher plants is often cumbersome. Barley limit dextrinase (LD), a 98 kDa multidomain starch and α-limit dextrin debranching enzyme, plays a major role in starch mobilization during seed germination and is possibly involved in starch biosynthesis by trimming of intermediate branched α-glucan structures. Highly active barley LD is obtained by secretory expression during high cell-density fermentation of Pichia pastoris. The LD encoding gene fragment without signal peptide was subcloned in-frame with the Saccharomyces cerevisiae α-factor secretion signal of the P. pastoris vector pPIC9K under control of the alcohol oxidase 1 promoter. Optimization of a fed-batch fermentation procedure enabled efficient production of LD in a 5-L bioreactor, which combined with affinity chromatography on β-cyclodextrin–Sepharose followed by Hiload Superdex 200 gel filtration yielded 34 mg homogenous LD (84% recovery). The identity of the recombinant LD was verified by N-terminal sequencing and by mass spectrometric peptide mapping. A molecular mass of 98 kDa was estimated by SDS–PAGE in excellent agreement with the theoretical value of 97419 Da. Kinetic constants of LD catalyzed pullulan hydrolysis were found to Km,app = 0.16 ± 0.02 mg/mL and Kcat,app = 79 ± 10 s-1 by fitting the uncompetitive substrate inhibition Michaelis–Menten equation, which reflects significant substrate inhibition and/or transglycosylation. The resulting catalytic coefficient, Kcat,app/Km,app = 488 ± 23 mL/(mg s) is 3.5-fold higher than for barley malt LD. Surface plasmon resonance analysis showed α-, β-, and γ-cyclodextrin binding to LD with Kd of 27.2, 0.70, and 34.7 µM, respectively.

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Thioredoxin in barley: could it have a role in releasing limit dextrinase in brewery mashes?

Heisner, C. B. & Bamforth, C. W. (2008). Journal of the Institute of Brewing, 114(2), 122-126.

There is not a strong correlation between the amount of thioredoxin in barley and the total protein content of the grain. The level of detectable thioredoxin decreases during germination, whereas the amount of limit dextrinase increases, for the most part in a bound form. Under no circumstances could a release of limit dextrinase in an active form be demonstrated through the agency of the thioredoxin system. By contrast the thiol agent dithiothreitol, especially in the presence of calcium, did promote the activation of limit dextrinase. Of most pronounced effect, however, was the lowering of pH. Three-fold higher limit dextrinase activity was achieved when the pH was lowered to 4.0.

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Limit dextrinase from germinating barley has endotransglycosylase activity, which explains its activation by maltodextrins.

McDougall, G. J., Ross, H. A., Swanston, J. S. & Davies, H. V. (2004). Planta, 218(4), 542-551.

Limit dextrinase (EC from germinating barley (Hordeum vulgare L) can be activated by millimolar concentrations of linear maltodextrins with a degree of polymerisation ≥ 2. The activation was assay-dependent; it was detected using assays based on the solubilisation of cross-linked dyed pullulan but not in assays that directly measured cleavage events such as the formation of new reducing termini. This strongly suggested that maltodextrins did not increase the catalytic rate of limit dextrinase i.e. this is not a true activation. On the other hand, considerable activation was noted in assays that measured pullulan degradation by reduction in viscosity. Taken together, this suggested that maltodextrins altered the mode of action of limit dextrinase, causing more rapid decreases in viscosity or greater solubilisation of dye-linked pullulan fragments per cleavage event. The proposed mechanism of activation by alteration in action pattern was reminiscent of initial work in the discovery of xyloglucan endotransglycosylase. Therefore, the ability of limit dextrinase to catalyse transglycosylation reactions into pullulan was tested and confirmed by an assay based on the incorporation of a fluorescently labelled maltotriose derivative into higher-molecular-weight products. The transglycosylation reaction was dependent on limit dextrinase activity and was enhanced in more highly purified preparations of limit dextrinase. Transglycosylation was inhibited by unlabelled maltotriose. How transglycosylation accounts for the apparent activation of limit dextrinase by maltodextrins and the physiological relevance of this novel reaction are discussed.

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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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