Colourimetric and fluorimetric substrates for the assay of limit dextrinase

The measurement of limit-dextrinase (LD) (EC 3.2.1.142) 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-beta-6(3)-alpha-D-maltotriosyl-maltotrioside (BzCNPG(3)G(3), 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 pK(a) (similar to 5.5) of the chromogenic moiety while 4,6-O-benzylidene-4-methylumbelliferyl-beta-6(3)-alpha-D-maltotriosyl-maltotrioside (BzMUG(3)G(3), 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. (C) 2015 Elsevier Ltd. All rights reserved.