Measurement of β-amylase in cereal flours and commercial enzyme preparations.
McCleary, B. V. & Codd, R. (1989), Journal of Cereal Science, 9(1), 17-33.
A procedure previously developed for the assay of cereal-flour β-amylase has been improved and standardised. The improved procedure uses the substrate p-nitrophenyl maltopentaose (PNPG5) in the presence of near saturating levels of α-glucosidase. PNPG5 is rapidly hydrolysed by β-amylase but less readily by cereal α-amylases. The substrate is hydrolysed by β-amylase to maltose and p-nitrophenyl maltotriose (PNPG3). With the levels of α-glucosidase used in the substrate mixture, PNPG3 is rapidly cleaved to glucose and p-nitrophenol, whereas PNPG5 is resistant to hydrolysis by the α-glucosidase. The assay procedure has been standardised for several β-amylases and the exact degree of interference by cereal α-amylases determined. The procedure can be readily applied to the selective measurement of β-amylase activity in cereal and malted cereal-flours.
Characterisation of the substituent distribution in hydroxypropylated potato amylopectin starch.
Richardson, S., Nilsson, G. S., Bergquist, K. E., Gorton, L. & Mischnick, P. (2000). Carbohydrate Research, 328(3), 365-373.
The distribution of substituents in hydroxypropylated potato amylopectin starch (amylose deficient) modified in a slurry of granular starch (HPPAPg) or in a polymer ‘solution’ of dissolved starch (HPPAPs), was investigated. The molar substitution (MS) was determined by three different methods: proton nuclear magnetic resonance (1H NMR) spectroscopy, gas-liquid chromatography (GLC) with mass spectrometry, and a colourimetric method. The MS values obtained by 1H NMR spectroscopy were higher than those obtained by GLC–mass spectrometry analysis and colourimetry. The relative ratio of 2-, 3-, and 6-substitution, as well as un-, mono-, and disubstitution in the anhydroglucose unit (AGU) were determined by GLC–mass spectrometry analysis. Results obtained showed no significant difference in molar distribution of hydroxypropyl groups in the AGU between the two derivatives. For analysis of the distribution pattern along the polymer chain, the starch derivatives were hydrolysed by enzymes with different selectivities. Debranching of the polymers indicated that more substituents were located in close vicinity to branching points in HPPAPg than in HPPAPs. Simultaneous α-amylase and amyloglucosidase hydrolysis of HPPAPg liberated more unsubstituted glucose units than the hydrolysis of HPPAPs, indicating a more heterogeneous distribution of substituents in HPPAPg.
High-performance anion-exchange chromatography–electrospray mass spectrometry for investigation of the substituent distribution in hydroxypropylated potato amylopectin starch.
Richardson, S., Cohen, A. & Gorton, L. (2001). Journal of Chromatography A, 917(1), 111-121.
The use of high-performance anion-exchange chromatography (HPAEC) with pulsed amperometric detection (PAD) coupled on-line with electrospray mass spectrometry (ESI-MS) for analysis of the substitution pattern in chemically modified starch, has been investigated. In order to characterise the distribution of substitution groups along the polymer chain, hydroxypropylated potato amylopectin starch (HPPAP) was subjected to enzymic hydrolysis, followed by analysis of the degradation products by HPAEC-PAD-MS. When using conventional chromatographic techniques for characterisation of enzymic hydrolysates, standard compounds are required for identification of the hydrolysis products. However, the on-line coupling with ESI-MS allowed identification of all products obtained, substituted as well as unsubstituted, and also of those compounds that co-eluted, without the need for standards. Further, HPAEC-PAD-MS was shown to be useful for analysis of the substitution pattern in modified starch; from results obtained it was suggested that the hydroxypropyl groups were homogeneously distributed in the amylopectin molecule. It was also shown that the starch hydrolysing enzymes were hindered by the hydroxypropyl groups and preferentially cleaved glucosidic linkages between unsubstituted glucose units.
Enzyme-aided investigation of the substituent distribution in cationic potato amylopectin starch.
Richardson, S., Nilsson, G., Cohen, A., Momcilovic, D., Brinkmalm, G. & Gorton, L. (2003). Analytical Chemistry, 75(23), 6499-6508.
The distribution of substituents along the polymer chain in cationic potato amylopectin starch, modified in solution, granular slurry, or dry state, was investigated. The starch derivatives were successively hydrolyzed by different enzymes, followed by characterization of the hydrolysis products obtained by means of electrospray mass spectrometry (ESI-MS) and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). ESI-MS and MALDI-MS were proved to be appropriate techniques for identification of the substituted hydrolysis products, for which there are no standard compounds available. No highly substituted oligomers were found in the hydrolysates, which was taken as an indication of a more or less homogeneous distribution of cationic groups in the amylopectin molecules. Furthermore, from the results obtained it was suggested that the enzymes cleave glucosidic linkages only between unsubstituted glucose units and, preferentially, linkages in sequences containing more than two adjacent unsubstituted units. The determination of the amount of unsubstituted glucose produced from every successive hydrolysis step revealed slight differences between the different starch samples with respect to the homogeneity of the substitution pattern. Among the three samples under investigation, starch cationized in solution was found to have the most and dry-cationized starch the least homogeneous distribution of substituents.
Rapid determination of enzyme purity by a microdialysis-based assay.
Richardson, S., Nilsson, G. S., Torto, N., Gorton, L. & Laurell, T. (1999). Analytical Communications, 36(5), 189-193.
Microdialysis was shown to be useful as a fast on-line sampling method for determining the purity of starch hydrolysing enzymes. The enzymes were characterised using their hydrolytic properties. β-Amylases and pullulanases from different sources and/or manufacturers were investigated, with maltose, maltoheptaose, pullulan, and potato amylopectin starch (PAP) as substrates. The hydrolysis products were sampled via an on-line microdialysis probe and determined in a high-performance anion-exchange chromatographic (HPAEC) system. Comparison between the expected (theoretical) hydrolysis products with those obtained in the experiments made it possible to determine impurities in the enzymes. Two of the β-amylases and one pullulanase released unwanted hydrolysis products, indicating trace impurities in the enzyme preparation. Microdialysis sampling allows on-line sampling and eliminates separate sample preparation and clean-up steps. On-line microdialysis coupled to a HPAEC system is therefore a fast and simple technique for analysing enzyme hydrolysates.
Microdialysis clean-up and sampling in enzyme-based methods for the characterisation of starch.
Nilsson, G. S., Richardson, S., Huber, A., Torto, N., Laurell, T. & Gorton, L. (2001). Carbohydrate Polymers, 46(1), 59-68.
Microdialysis was used for sampling enzyme hydrolysis products of starch hydrolysed with β-amylase, pullulanase, and/or isoamylase, to obtain information about the molecular structure of starch. Starches from waxy, normal, and high amylose maize, and from normal and genetically modified potato (amylose deficient) were used, and also commercial potato amyloses. The hydrolysis products were analysed using high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Simultaneous sampling and sample clean-up were achieved with microdialysis, thus enabling on-line injection into the liquid chromatographic system. The molecular weight cut-off of the membrane allowed for diffusion of small molecules such as oligosaccharides through the membrane, but hindered large molecules, e.g. enzymes and large polysaccharides, from entering the chromatographic system. With microdialysis sampling, it was possible to investigate the short chain fractions of debranched starch in the presence of amylose without pre-fractionation. The microdialysis–HPAEC-PAD system was also used for determination of the A:B chain ratio and the β-limit value. After β-amylolysis, only liberated maltose diffused through the dialysis membrane, which resulted in on-line sample clean-up from branched β-limit dextrin as well as from the enzyme. The proposed method is fast and easy to handle since clean-up of the hydrolysate is achieved on-line with the chromatographic system.
Maltose and glucose utilization during fermentation of barley and sorghum lager beers as affected by β-amylase or amyloglucosidase addition.
Espinosa-Ramírez, J., Pérez-Carrillo, E. & Serna-Saldívar, S. O. (2014). Journal of Cereal Science, 60(3), 602-609.
The effect of supplementation of amyloglucosidase or β-amylase during mashing of sorghum and barley beers was studied. The research focused in the increments in maltose and glucose concentrations, and their consumption during 144 h fermentation of lager beers. Barley, red sorghum and white sorghum malt beers, produced with regular or waxy sorghum adjuncts, and supplemented with these exogenous enzymes, were produced. The addition of exogenous enzymes increased up to 20% the total sugar content. Regression lines to describe sugar consumption were obtained (R2 >0.951). Amyloglucosidase treatments had higher glucose contents and up to 4 times higher glucose consumption rates compared with unsupplemented or with β-amylase treatments. The latter treatments had maltose as predominant sugar. Amyloglucosidase treatments had higher ethanol production rates, however the maximum production rate was delayed 24 h, compared with maltose-rich treatments. These worts observed incomplete fermentations and contained higher residual sugars. β-amylase treatments were not significantly different compared with their regular counterparts in terms of sugar and ethanol production. The selection of adequate sorghum cultivars for malt and adjuncts as well as the use of an osmotolerant yeast should be considered when exogenous enzymes are supplemented in brewing.