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 22.214.171.124) 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.
In addition, two new substrates are described that can be adopted for use in an auto-analyser format. 4,6-O-benzylidene-2-chloro-4-nitrophenyl-β-63-α-D-maltotriosyl-maltotrioside (BzCNPG3G3, Hexachrom) is not susceptible to transglycosylation and serves amiably as a routine quantitative assay tool with the potential to run kinetic assays due to the low pKa (∼5.5) of the chromogenic moiety while 4,6-O-benzylidene-4-methylumbelliferyl-β-63-α-D-maltotriosyl-maltotrioside (BzMUG3G3, Hexafluor) was found to be susceptible to transglycosylation with LD. It is anticipated that Hexafluor may find extensive use in applications where high sensitivity is required such as high throughput screening studies.
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).
Detection of a limit dextrinase inhibitor in barley.
Macri, L. J., MacGregor, A. W., Schroeder, S. W. & Bazin, S. L. (1993). Journal of Cereal Science, 18(2), 103-106.
Two proteins have been isolated from barley ( Hordeum vulgare cv. Harrington) that inhibited limit dextrinase from germinated barley. These inhibitors were purified by carboxymethyl cellulose ion exchange chromatography and chromatofocusing. They were shown to have approximate Mrs of 15k and isoelectric points of approximately 6•7 and 7•2 (measured by isoelectric focusing).
Purification and characterisation of limit dextrinase inhibitors from barley.
MacGregor, A. W., Macri, L. J., Schroeder, S. W. & Bazin, S. L. (1994). Journal of Cereal Science, 20(1), 33-41.
Two inhibitors of malt limit dextrinase were purified from a crude barley extract (cv. Harrington) by CM cellulose ion exchange chromatography and chromatofocusing. The inhibitors were heat-stable proteins of Mr approximately 15k and isoelectric points of 6•7 (low pI inhibitor) and 7•2 (high pI inhibitor). Both inhibitors were active over a wide pH range, and were most effective at pH 5•5 to 6•5, the pH optimum of the limit dextrinase enzyme. Inactivation of the limit dextrinase enzyme by either inhibitor could be reversed by warning the complex at 40°C in the presence of reducing agents.
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 126.96.36.199) 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.
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.
The survival of limit dextrinase during fermentation in the production of Scotch whisky.
Walker, J. W., Bringhurst, T. A., Broadhead, A. L., Brosnan, J. M. & Pearson, S. Y. (2001). Journal of the Institute of Brewing, 107(2), 99-106.
Limit dextrinase, is an important enzyme in the hydrolysis of starch from cereals to fermentable sugars. Work is described which demonstrates the importance of this enzyme in the production of Scotch whisky. The study considers the occurrence, survival and action of limit dextrinase (total and free) during fermentation in malt and grain distilleries. The results of both laboratory and distillery studies revealed that limit dextrinase can survive the conditions encountered during mashing and is not only present in the fermenter but its activity can increase during fermentation. This observation has important implications for the production of Scotch whisky, since the fermentation substrate (wash) is not boiled, the enzyme is therefore available to degrade dextrins into fermentable sugars, and can potentially increase the yield of alcohol.
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.
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.
The advantages of using natural substrate‐based methods in assessing the roles and synergistic and competitive interactions of barley malt starch‐degrading enzymes.
Osman, A. M. (2002). Journal of the Institute of Brewing, 108(2), 204-214.
Existing methods of assay of malt starch-degrading enzymes were critically appraised. New methods based on natural substrates, namely starch and its natural intermediate-derivative, were developed for all the enzymes, except limit dextrinase for which pullulan was used. Thermostability, optimal temperatures and pHs were established. α-Amylase and limit dextrinase were the most thermostable and β-amylase, α-glucosidase and maltase were the least stable while diastase occupied an intermediate position. The optimal temperatures were congruent with thermostability, β-amylase having the lowest (50°C) and α-amylase the highest (65°C) with the remaining enzymes, including diastase, falling in between. In contrast, α-amylase has the lowest optimal pH (pH 4.5) and β amylase the highest (pH 5.5) while the others have pHs in between the two values. The roles of the enzymes were evaluated taking into account the level of activity, thermostability, optimum pH, the nature of the product(s), and the relevance to brewing. β-Amylase production of maltose was synergistically enhanced, mostly by α-amylase but also limit dextrinase. α-Glucosidase and maltase are unimportant for brewing, because of their low activity and the negative impact on β-amylase activity and the negative effect of glucose on maltose uptake by yeast. The starch-degrading enzymes (diastase) in a gram of malt were able to degrade more than 8 g boiled starch into reducing sugars in 10 min at 65°C. The latter, suggests that it will be possible to gelatinise most of the malt starch at a higher temperature and ensure its hydrolysis to fermentable sugars by mixing with smaller portions of malt and mashing at lower temperatures e.g. 50–60°C.