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

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Abstract

Limit dextrinase (EC 3.2.1.41) 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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Abbreviations

APTS :

8-Amino-1,3,6-pyrene trisulphonic acid

DP :

Degree of polymerisation

G :

Glucose

G2 :

Maltose

G3–G7 :

Linear maltodextrins of DP 3–7, i.e. maltotriose to maltoheptaose

G3–APTS :

Maltotriose APTS derivative

LD :

Limit dextrinase

PAHBAH :

p-Hydroxybenzoic acid hydrazide

UPW :

Ultra-pure water

References

  • Blake, MS, Johnston KH, Russell-Jones GJ, EC Gotsclich (1984) A rapid, sensitive method for detection of alkaline phosphatase-conjugated anti-antibody on Western blots. Anal Biochem 136:175–179

    CAS  PubMed  Google Scholar 

  • Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilising the principle of protein–dye binding. Anal Biochem 72:248–254

    Article  CAS  PubMed  Google Scholar 

  • Bringhurst TA, Broadhead AL, Brosnan, JM, Pearson SY, Walker JW (2001) The identification and behaviour of branched dextrins in the production of Scotch whisky. J Inst Brew London 107:137–149

    CAS  Google Scholar 

  • Burton RA, Zhang X-Q, Hrmova M, Fincher GB (1999) A single limit dextrinase gene is expressed both in the developing endosperm and in germinated grains of barley. Plant Physiol 199:859–871

    Article  Google Scholar 

  • Burton RA, Jenner H, Carrangis L, Fahy B, Fincher GB, Hylton C, Laurie DA, Parker M, Waite D, van Wegan S, Verhoeven T, K Denyer (2002) Starch granule initiation and growth are altered in barley mutants that lack isoamylase activity. Plant J 31:97–112

    Article  CAS  PubMed  Google Scholar 

  • Cho M-J, Wong JH, Marx C, Jiang W, Lemaux PG, Buchanan BB (1999) Over-expression of thioredoxin h leads to enhanced activity of starch debranching enzyme (pullulanase) in barley grain. Proc Natl Acad Sci USA 96:14641–14646

    CAS  PubMed  Google Scholar 

  • Coutinho PM, Henrissat B (1999) Carbohydrate-active enzymes: an integrated database approach. In: Gilbert HJ, Davies G, Henrissat B, Svensson, B (eds) Recent advances in carbohydrate bioengineering. The Royal Society of Chemistry, Cambridge, pp 3–12

  • Dubois M, Gilles KA, Hamilton JK, Rebers PA, Smith FA (1956) Colorimetric method for determination of sugars and related substances. Anal Chem 28:350–356

    CAS  Google Scholar 

  • Dunn G, Manners DJ (1975) The limit dextrinases from ungerminated oats (Avena sativa L.) and ungerminated rice (Orzya sativa L.). Carbohydr Res 39:283–293

    Article  CAS  PubMed  Google Scholar 

  • Enevoldsen BS, Bathgate GN (1969) Structural analysis of wort dextrins by means of beta-amylase and the debranching enzyme, pullulanase. J Inst Brew London 75:433–443

    CAS  Google Scholar 

  • Fry SC (1997) Novel dot blot assays for glycosyltransferases and glycosylhydrolases: optimisation for xyloglucan endotransglycosylase (XET) activity. Plant J 11:1141–1150

    Article  CAS  Google Scholar 

  • Fry SC, Smith RC, Renwick KF, Martin DJ, Hodge SK, Matthews KJ (1992) Xyloglucan endotransglycosylase, a new wall-loosening enzyme activity from plants. Biochem J 282:821–828

    CAS  Google Scholar 

  • Hamilton LM, Kelly CT, Fogarty WM (2000) Review: cyclodextrins and their interaction with amylolytic enzymes. Enzyme Microbial Technol 26:561–567

    Article  CAS  Google Scholar 

  • Hardie DG, Manners DJ, Yellowlees D (1976) The limit dextrinase from malted sorghum (Sorghum vulgare) Carbohydr Res 50:75–85

    Google Scholar 

  • Jespersen HM, MacGregor EA, Henrissat B, Sierks MR, Svensson B (1993) Starch- and glycogen-debranching and branching enzymes: prediction of structural features of the catalytic (β/α)8-domain and evolutionary relationship to other amylolytic enzymes. J Protein Chem 12:791–805

    CAS  PubMed  Google Scholar 

  • Kitahata S, Tanimoto T, Ikuta A, Tanaka K, Fujita K, Hashimoto H, Murakami H, Nakano H, Koizumi K (2000) Synthesis of novel heterobranched beta-cyclodextrins from 4(2)–O-beta-d-galactosyl-maltose and beta-cyclodextrin by the reverse action of pullulanase, and isolation and characterisation of the products. Biosci Biotechnol Biochem 64:1223–1229

    CAS  PubMed  Google Scholar 

  • Kristensen M, Planchot V, Abe J-I, Svensson B (1998) Large-scale purification and characterisation of barley limit dextrinase, a member of the alpha-amylase structural family. Cereal Chem 75:473–479

    CAS  Google Scholar 

  • Kristensen M, Lok F, Planchot V, Svendson I, Leah R, Svensson B (1999) Isolation and characterisation of the gene encoding the starch debranching enzyme limit dextrinase from germinating barley. BBA 1431:538–546

    Article  CAS  PubMed  Google Scholar 

  • Kubo A, Fujita N, Harada K, Matsuda T, Satoh H, Nakamura Y (1999) The starch debranching enzymes isoamylase and pullulanase are both involved in amylopectin biosynthesis in rice endosperm. Plant Physiol 121:399–409

    Article  CAS  PubMed  Google Scholar 

  • Lee EY, Marshall JJ, Whelan WJ (1971) The substrate specificity of amylopectin-debranching enzymes from sweet corn. Arch Biochem Biophys 143:365–374

    CAS  PubMed  Google Scholar 

  • Lee WJ, Pyler RE (1984) Barley malt limit dextrinase: varietal, environmental and malting effect. J Am Soc Brew Chemists 42:11–17

    CAS  Google Scholar 

  • Lever M (1972) A new reaction for colorimetric determination of carbohydrates. Anal Biochem 47:273–279

    CAS  PubMed  Google Scholar 

  • Longstaff MA, Bryce JH (1993) Development of limit dextrinase in germinated barley (Hordeum vulgare L). Evidence of proteolytic activation. Plant Physiol 101:881–889

    CAS  PubMed  Google Scholar 

  • MacGregor AW (1996) Malting and brewing science: challenges and opportunities. J Inst Brew 102:97–102

    Google Scholar 

  • MacGregor AW, Fincher GB (1993) Carbohydrates of the barley grain. In: MacGregor AW, Bhatty RS (eds) Barley: chemistry and technology. American Association of Cereal Chemists, St. Paul, MN, pp 73–130

  • MacGregor AW, Macri LJ, Schroeder SW, Bazin SL (1994) Purification and characterisation of limit dextrinase inhibitors from barley. J Cereal Sci 20:33–41

    Article  CAS  Google Scholar 

  • MacGregor AW, Bazin SL, Macri LJ, Babb JC (1999) Modelling the contribution of alpha-amylase, beta-amylase and limit dextrinase to starch degradation during mashing. J Cereal Sci 29:161–169

    Article  CAS  Google Scholar 

  • MacGregor AW, Bazin SL, Schroeder SW (2002) Effect of starch hydrolysis products on the determination of limit dextrinase and limit dextrinase inhibitors in barley and malt. J Cereal Sci 35:17–28

    Article  CAS  Google Scholar 

  • Macri LJ, MacGregor AW, Shroeder SW, Bazin SL (1993) Detection of a limit dextrinase inhibitor in barley. J Cereal Sci 18:103–106

    Article  CAS  Google Scholar 

  • McCleary BV (1992) Measurement of the content of limit dextrinase in cereal flours. Carbohydr Res 227:257–268

    Article  CAS  Google Scholar 

  • McDougall GJ, Fry SC (1990) Xyloglucan oligosaccharides promote growth and activate cellulase: evidence for a role for cellulase in cell expansion. Plant Physiol 93:1042–1048

    CAS  Google Scholar 

  • Merji M, Mathlouthi M (2001) Effect of small carbohydrates on the catalytic activity of a protease and two glycohydrolases. Carbohydr Polym 45:161–167

    Article  Google Scholar 

  • Mouille G, Maddelein ML, Libessart N, Talaga P, Decq A, Del-rue B, Ball S (1996) Preamylopectin processing: a mandatory step for starch biosynthesis in plants. Plant Cell 8:1353–1366

    CAS  Google Scholar 

  • Morell MK, Samuel MS, O’Shea MG (1998) Analysis of starch structure using fluorophore-assisted carbohydrate electrophoresis. Electrophoresis 19:2603–2611

    CAS  PubMed  Google Scholar 

  • Nakamura Y, Kubo A, Shimamune T, Matsuda T, Harada K, Satoh H (1997) Correlation between activities of starch de-branching enzyme and alpha-polyglucan structure in endosperms of sugary-1 mutants of rice. Plant J 12:143–153

    CAS  Google Scholar 

  • Pan D, Nelson OE (1984) A debranching enzyme deficiency in endosperm of the sugary-1 mutants of maize (Zea mays). Plant Physiol 74:324–328

    CAS  Google Scholar 

  • Panchal CJ, Stewart GG (1979) Utilisation of wort carbohydrates. Brew Dig 54:36–43

    CAS  Google Scholar 

  • Sissons MJ, Lance RCM, Sparrow DHB (1993) Studies on limit dextrinase in barley. 3. Limit dextrinase in developing kernels. J Cereal Sci 17:19–24

    Article  CAS  Google Scholar 

  • Stenholm K (1997) Malt limit dextrinase and its importance in brewing. VTT publication no 323, VTT, Technical Research Centre of Finland, Espoo, Finland

  • Svensson B (1994) Protein engineering in the α-amylase family: catalytic mechanism, substrate specificity, and stability. Plant Mol Biol 25:141–157

    CAS  PubMed  Google Scholar 

  • Swanston JS, Taylor K (1990) The effects of different steeping regimes on water uptake, germination rate, milling energy and hot water extract. J Inst Brew 96:3–6

    Google Scholar 

  • Takaha T, Yanase M, Okada S, Smith SM (1993) Disproportionating enzyme (4-α-glucanotransferase; EC 2.4.1.25) of potato. Purification, molecular cloning, and potential role in starch metabolism. J Biol Chem 268:1391–1396

    CAS  PubMed  Google Scholar 

  • Takata H, Kuriki T, Okada S, Takesada Y, Iizuka M, Minamiura N, Imanaka T (1992) Action of neopullulanase. Neopullulanase catalyzes both hydrolysis and transglycosylation at the α-(1-4)- and α-(1-6)-glucosidic linkages. J Biol Chem 267:18447–18452

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

The Scottish Crop Research Institute acknowledges grant-in-aid from the Scottish Executive Environment and Rural Affairs Department. We are indebted to Prof. Birte Svensson for her gift of polyclonal antibodies against LD and her helpful advice. We thank Dr. Barry McCleary of Megazyme International Ireland Ltd for his enthusiasm, advice, time and kind gifts of materials; Dr. Steve Smith (University of Edinburgh) for his gift of polyclonal antibodies against D-enzyme, and for his advice; and Dr. Sandy MacGregor of the Canadian Grain Commission for helpful discussions. We thank Dr. Derek Stewart for his help; Dr. Nigel Deighton for LC–MS/MS analyses; Julie Sungurtas and Laurence Ducreux for their technical help; Tom Geoghegan for his photographic expertise; and Ian Pitkeithly for help with graphics.

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  1. Quality Health and Nutrition Programme, Genes to Products Theme, Scottish Crop Research Institute, Invergowrie, Dundee, DD2 5DA, UK

    Gordon J. McDougall, Heather A. Ross, J. Stuart Swanston & Howard V. Davies

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  1. Gordon J. McDougall
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  2. Heather A. Ross
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  3. J. Stuart Swanston
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Correspondence to Gordon J. McDougall.

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McDougall, G.J., Ross, H.A., Swanston, J.S. et al. Limit dextrinase from germinating barley has endotransglycosylase activity, which explains its activation by maltodextrins. Planta 218, 542–551 (2004). https://doi.org/10.1007/s00425-003-1141-1

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