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 188.8.131.52) 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 pK (~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.
The Multi Domain Caldicellulosiruptor bescii CelA Cellulase Excels at the Hydrolysis of Crystalline Cellulose.
Brunecky, R., Donohoe, B. S., Yarbrough, J. M., Mittal, A., Scott, B. R., Ding, H., Taylor II, L., E., Russell, J. F., Chung, D., Westpheling, J., Teter, S. A., Himmel, M. E. & Bomble, Y. J. (2017). Scientific Reports, 7, 9622.
The crystalline nature of cellulose microfibrils is one of the key factors influencing biomass recalcitrance which is a key technical and economic barrier to overcome to make cellulosic biofuels a commercial reality. To date, all known fungal enzymes tested have great difficulty degrading highly crystalline cellulosic substrates. We have demonstrated that the CelA cellulase from Caldicellulosiruptor bescii degrades highly crystalline cellulose as well as low crystallinity substrates making it the only known cellulase to function well on highly crystalline cellulose. Unlike the secretomes of cellulolytic fungi, which typically comprise multiple, single catalytic domain enzymes for biomass degradation, some bacterial systems employ an alternative strategy that utilizes multi-catalytic domain cellulases. Additionally, CelA is extremely thermostable and highly active at elevated temperatures, unlike commercial fungal cellulases. Furthermore we have determined that the factors negatively affecting digestion of lignocellulosic materials by C. bescii enzyme cocktails containing CelA appear to be significantly different from the performance barriers affecting fungal cellulases. Here, we explore the activity and degradation mechanism of CelA on a variety of pretreated substrates to better understand how the different bulk components of biomass, such as xylan and lignin, impact its performance.