Chapter 1: Principle of the Assay Procedure
Chapter 2: Substrate & Kit Description
Chapter 3: Dissolution of Azo-CM-Cellulose
Chapter 4: Precipitant Solution
Chapter 5: Preparation of Buffer Solution
Chapter 6: Assay Procedure
Chapter 7: Calculation
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|Stability:||> 5 years under recommended storage conditions|
|Substrate For (Enzyme):||α-amylase|
|Assay Format:||Spectrophotometer, Petri-dish (Qualitative)|
|Reproducibility (%):||~ 7%|
High purity dyed, soluble Red Starch for the measurement of enzyme activity, for research, biochemical enzyme assays and in vitro diagnostic analysis.
An excellent soluble substrate for the assay of α-amylase activity.
Please note the video above shows the protocol for assay of endo-cellulase using Azo-CM cellulose. The procedure for the assay of α-amylase using Red Starch is equivalent to this.
Browse our full range of soluble chromogenic enzyme substrates.
Biochemical features of the novel Tail Tubular Protein A of Yersinia phage phiYeO3-12.
Anna, P., Natalia, U., Karolina, F., Katherine, T. & Ewa, B. (2020). Scientific Reports (Nature Publisher Group), 10(1).
Tail Tubular Protein A (TTPA) was long thought to be strictly a structural protein of environmental bacteriophages. However, our recent work has suggested that some TTPAs have additional functional features and thus are dual-function proteins. This study introduces a new TTPA family member, TTPAgp11, which belongs to Yersinia phage phiYeO3-12. We cloned the gene, expressed it and then purified the phage protein. The protein, including its hydrolytic activity, was characterized. Our enzymatic activity tests showed that TTPAgp11 displayed hydrolytic activity towards Red-starch, suggesting that this enzyme could be classified as part as the α-1, 4-glucosidase family. Protein folding and aggregation tests indicated that TTPAgp11 is a single-domain protein whose aggregation can be induced by maltose or N-acetylglucosamine. The spatial structure of TTPAgp11 seemed to resemble that of the first reported dual-function TTPA, TTPAgp31, which was isolated from Klebsiella pneumoniae phage 32.Hide Abstract
Whole-Genome Sequence of Lachnospiraceae Bacterium NBRC 112778, Isolated from a Mesophilic Methanogenic Bioreactor.
Morimura, K. & Ueda, K. (2020). Microbiology Resource Announcements, 9(8).
Here, we report the draft genome sequence of Lachinospiraceae bacterium NBRC 112778. This anaerobic bacterium, isolated from a sweet potato-digesting mesophilic methanogenic bioreactor, represents a new genus related to the genus Anaerosporobacter. The 4.52-Mb circular genome harbored many genes involved in carbohydrate utilization, suggesting its adaptation to a saccharolytic environment.Hide Abstract
Nabubuya, A., Namutebi, A., Byaruhanga, Y., Narvhus, J. & Wicklund, T. (2017). Food Science & Nutrition, 5(6), 1088-1097.
Changes in total starch and reducing sugar content in five sweetpotato varieties were investigated weekly during root development and following subjection of the roots to different postharvest handling and storage conditions. Freshly harvested (noncured) roots and cured roots (spread under the sun for 4 days at 29-31°C and 63-65% relative humidity [RH]) were separately stored at ambient conditions (23°C-26°C and 70-80% RH) and in a semiunderground pit (19-21°C and 90-95% RH). Changes in pasting properties of flour from sweetpotato roots during storage were analyzed at 14-day intervals. Significant varietal differences (p < .05) in total starch, sucrose, glucose, maltose, and fructose concentrations were registered. The total starch and sucrose content of the roots did not change significantly (p < .05) during root development (72.4 and 7.4%, respectively), whereas the average concentrations of glucose, maltose, and fructose decreased markedly (0.46-0.18%, 0.55-0.28%, and 0.43-0.21%), respectively. Storage led to decrease in total starch content (73-47.7%) and increase in sucrose and glucose concentrations (8.1-11.2% and 0.22-1.57%, respectively). Storage also resulted in reduction in sweetpotato flour pasting viscosities. Curing resulted in increased sucrose and glucose concentrations (9.1-11.2% and 0.45-0.85%, respectively) and marked reduction (p < .05) in total starch content (72.9-47.6%). This resulted in low pasting viscosities compared to flour from storage of uncured roots. These findings show that significant changes occur in the carbohydrate components of sweetpotato roots during storage compared to development and present an opportunity for diverse utilization of flours from sweetpotato roots in the food industry.Hide Abstract
Brzozowska, E., Pyra, A., Pawlik, K., Janik, M., Górska, S., Urbańska, N., Drulis-Kawa, Z. & Gamian, A. (2017). Scientific Reports, 7(1), 18048.
In this paper, the enzymatic activity, substrate specificity and antibiofilm feature of bacteriophage dual-function tail proteins are presented. So far, tail tubular proteins A–TTPAgp31 and TTPAgp44-have been considered as structural proteins of Klebsiella pneumoniae bacteriophages KP32 and KP34, respectively. Our results show that TTPAgp31 is able to hydrolyze maltose as well as Red-starch. The activity of 1 µM of the protein was calculated as 47.6 milli-Units/assay relating to the α-amylase activity. It degrades capsular polysaccharides (cPS), slime polysaccharides (sPS) and lipopolysaccharide (LPS) of K. pneumoniae PCM 2713 and shows antibiofilm reactivity towards S. aureus PCM 519 and E. faecalis PCM 2673. TTPAgp44 hydrolyses trehalose and cPS of E. faecium PCM 1859. TTPAgp44′s activity was also observed in the antibiofilm test against P. aeruginosa PCM 2710 and B. subtilis PCM 2021. TTPAgp31 has been identified as α-1,4-glucosidase whereas, TTPAgp44 exhibits trehalase-like activity. Both proteins contain aspartate and glutamate residues in the β-stranded region which are essential for catalytic activity of glycoside hydrolases. The significant novelty of our results is that for the first time the bacteriophage tubular proteins are described as the unique enzymes displaying no similarity to any known phage hydrolases. They can be used as antibacterial agents directed against bacterial strains producing exopolysaccharides and forming a biofilm.Hide Abstract
Vennemann, M., Scott, G., Curran, L., Bittner, F. & Tobe, S. S. (2014). Forensic Science, Medicine, and Pathology, 10(1), 69-75.
Purpose: Despite their wide use, the limits of presumptive tests can be poorly understood. The aim of this study was to investigate the specificity and sensitivity of conventional, as well as innovative, presumptive tests for blood, semen and saliva. Methods: We investigated Kastle–Meyer (KM) and leucomalachite green (LMG) tests for blood with regard to their sensitivity and specificity in the presence of oxidizing (hypochlorite) and anti-oxidizing (ascorbic acid) agents. The suitability and specificity of the red starch paper (RSP) test for saliva was assessed. Finally, the inhibitory effect of detergent on the acid phosphatase (AP) test for semen was investigated along with possible cross reactions to tea stains. Results: Our results confirm previous findings of higher sensitivity and specificity of the KM test compared to LMG test for blood. Contrary to previous studies, no statistically significant difference was observed in the sensitivity of the tests between dry and wet stains. The novel RSP test was found to successfully detect saliva. We demonstrated that acid phosphatase (AP) testing for semen is possible on used RSP. A common multipurpose detergent had an inhibitory effect on AP tests. False positive results were obtained from tea stains. Testing different sorts of tea (black, green and herbal teas) revealed that only Camellia varieties produce positive result with the AP test, due to AP being present in the plants. Conclusions: From our results we conclude that specific knowledge of each test, including substances that may affect the test outcome, is imperative to ensure correct interpretation of presumptive test results.Hide Abstract
Forester, S. C., Gu, Y. & Lambert, J. D. (2012). Molecular Nutrition & Food Research, 56(11), 1647-1654.
Scope: Green tea has been shown to ameliorate symptoms of metabolic syndrome in vivo. The effects could be due, in part, to modulation of postprandial blood glucose levels. Methods and results: We examined the effect of coadministration of (−)-epigallocatechin-3-gallate (EGCG, 100 mg/kg, i.g.) on blood glucose levels following oral administration of common corn starch (CCS), maltose, sucrose, or glucose to fasted CF-1 mice. We found that cotreatment with EGCG significantly reduced postprandial blood glucose levels after administration of CCS compared to control mice (50 and 20% reduction in peak blood glucose levels and blood glucose area under the curve, respectively). EGCG had no effect on postprandial blood glucose following administration of maltose or glucose, suggesting that EGCG may modulate amylase-mediated starch digestion. In vitro, EGCG noncompetitively inhibited pancreatic amylase activity by 34% at 20 μM. No significant change was induced in the expression of two small intestinal glucose transporters (GLUT2 and SGLT1). Conclusions: Our results suggest that EGCG acutely reduces postprandial blood glucose levels in mice when coadministered with CCS and this may be due in part to inhibition of α-amylase. The relatively low effective dose of EGCG makes a compelling case for studies in human subjects.Hide Abstract
Gu, Y., Hurst, W. J., Stuart, D. A. & Lambert, J. D. (2011). Journal of Agricultural and Food Chemistry, 59(10), 5305-5311.
We determined the in vitro inhibitory effects of cocoa extracts and procyanidins against pancreatic α-amylase (PA), pancreatic lipase (PL), and secreted phospholipase A2 (PLA2) and characterized the kinetics of such inhibition. Lavado, regular, and Dutch-processed cocoa extracts as well as cocoa procyanidins (degree of polymerization (DP) = 2–10) were examined. Cocoa extracts and procyanidins dose-dependently inhibited PA, PL, and PLA2. Lavado cocoa extract was the most potent inhibitor (IC50 = 8.5–47 μg/mL). An inverse correlation between log IC50 and DP (R2 > 0.93) was observed. Kinetic analysis suggested that regular cocoa extract, the pentamer, and decamer inhibited PL activity in a mixed mode. The pentamer and decamer noncompetitively inhibited PLA2 activity, whereas regular cocoa extract inhibited PLA2 competitively. This study demonstrates that cocoa polyphenols can inhibit digestive enzymes in vitro and may, in conjunction with a low-calorie diet, play a role in body weight management.Hide Abstract
ApuA, a multifunctional α-glucan-degrading enzyme of Streptococcus suis, mediates adhesion to porcine epithelium and mucus.
Ferrando, M. L., Fuentes, S., de Greeff, A., Smith, H. & Wells, J. M. (2010). Microbiology, 156(9), 2818-2828.
We have identified apuA in Streptococcus suis, which encodes a bifunctional amylopullulanase with conserved α-(amylase and pullulanase substrate-binding domains and catalytic motifs. ApuA exhibited properties typical of a Gram-positive surface protein, with a putative signal sequence and LPKTGE cell-wall-anchoring motif. A recombinant protein containing the predicted N-terminal α-(amylase domain of ApuA was shown to have α-(1,4) glycosidic activity. Additionally, an apuA mutant of S. suis lacked the pullulanase α-(1,6) glycosidic activity detected in a cell-surface protein extract of wild-type S. suis. ApuA was required for normal growth in complex medium containing pullulan as the major carbon source, suggesting that this enzyme plays a role in nutrient acquisition in vivo via the degradation of glycogen and food-derived starch in the nasopharyngeal and oral cavities. ApuA was shown to promote adhesion to porcine epithelium and mucus in vitro, highlighting a link between carbohydrate utilization and the ability of S. suis to colonize and infect the host.Hide Abstract
Martin, N. C., Clayson, N. J. & Scrimger, D. G. (2006). Science & Justice, 46(2), 97-105.
The detection of saliva in forensic casework is extremely important in many types of case. This study describes a relatively sensitive method, based on a red dye bound to starch, for the detection of amylase. The sensitivity and specificity of the method has been examined by testing over 50 household products, various body fluids and five laboratory chemicals. This study demonstrated for the first time that positive results can be obtained from certain washing powders as well as other household products. As well as detecting amylase in saliva, positive Red-Starch results were obtained from faeces and urine. The method was found to be suitable for the detection of mixtures of saliva and semen in conjunction with the Brentamine test for the detection of acid phosphatase.Hide Abstract
Mota, M., Teixeira, J. A., Yelshin, A., Dias, R. & Cortez, S. (2005). 10th World Filtration Congress, 392-396.
Columns packed with commercial glass beads of 5 and 19 microns average diameter and binary mixtures with finest fraction of 5 micron (30 % volume fraction of the mixture) were used to analyse starch by hydrodynamic chromatography (HDC). Experiments were carried out at 3 and 15°C. The observed resolution increased with the application of binary packing as compared with single-size packing. The best results were obtained at starch’s amylopectin and amylose separation with a glass beads mixture (5 + 19 micron) at 3°C. In what concerns amylopectin and amylose separation, a lower pressure drop were obtained for the mixed binary packing when compared with the packing containing uniform 5 micron glass beads. For the Hylon VII starch RRT were 0.777 and 0.964 for amylopectin (AP) and amylose (AM), respectively, while for the Tapioca starch the obtained RRTs were 0.799 and 0.923. Application of unbound glass beads as column packing might reduce equipment and running costs in preparative scale separations.Hide Abstract