Flexible and expeditious assay for quantitative monitoring of alpha-amylase and amyloglucosidase activities

Oliveira, Hugo M.; Pinheiro, Alice Q.; Fonseca, António J.M.; Cabrita, Ana R.J.; Maia, Margarida R.G.

doi:10.1016/j.mex.2019.01.007

Cited by 11 publications

(6 citation statements)

References 13 publications

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“…Figure 3A shows the enzyme kinetics when it reacts with the substrate. An ANOVA revealed a statistic interaction (p < 0,05) with a 95,0% level of confidence between the fermentable sugar concentrations present in the hydrolyzed sample (g/g) and the enzyme concentration, showing a considerable increase in the monomeric sugars, due to the action that the enzyme exerts along any point of the carbohydrate chain (1-4 glucosidic bonds), hydrolyzing them in dextrin from amylose (Oliveira, Pinheiro, Fonseca, Cabrita, and Maia, 2019).…”

Section: Pretreatmentsmentioning

confidence: 94%

Study of Carbohydrate Hydrolysis From Arracacha Roots (Arracacia Xanthorriza Bancroft) to Produce Fermentable Sugars

et al. 2021

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In Colombia, approximately 855 840 tons of arracacha are produced each year. The unsalable postharvest arracacha root (Arracacia xanthorriza Bancroft) is not commercialized, mainly due to mechanical damage or small and misshapen roots. In this work, dry samples were characterized and subjected to two treatments: one using thermal hydrolysis, applying saturated steam at pressures of 0,1034 MPa, 0,2068 MPa, and 0,4137 MPa; and another one using hydrolysis with sulfuric acid in concentrations between 0,252,00 M. Then, the cake resulting from the hydrolysis and filtration process was enzymatically hydrolyzed (Liquozyme SC DS, Novozymes) at 1,5, 5 and 10 KNU/g (pH 6, 80 _C, 2 h). Fermentation inhibitors (acetic acid and furfural) were evaluated in the best pretreatment. The results showed that the treatment with sulfuric acid at 1,00 M (2 h) has high yields in reducing sugars added to enzymatic hydrolysis. The maximum level of fermentable carbohydrates per gram of dry sample (1,04 g/g) was also reached. Regarding the fermentation inhibitors of the reducing sugar, a higher concentration of acetic acid was found with a lower furfural content. Therefore, arracacha discards are a promising raw material to increase the supply of bioethanol.

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Section: Pretreatmentsmentioning

confidence: 94%

Study of Carbohydrate Hydrolysis From Arracacha Roots (Arracacia Xanthorriza Bancroft) to Produce Fermentable Sugars

et al. 2021

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“…Enzyme Activity Measurement. The iodine method was applied for the amylase activity test, referring to [20] work with some modifications. A total of 100 µl of enzyme solution and 650 µl of 50 mM acetate buffer pH 5 were kept at 37 o C. Then 750 µl of 1% starch solution was added and incubated for 30 minutes.…”

Section: Methodsmentioning

confidence: 99%

Purification of an Extracellular Amylase Produced by <i>Aspergillus niger</i> ICP2 through Submerged Fermentation

Winarsa

Putrasetya

Azizah

et al. 2023

Science and Technology Applications

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The future applications of amylase have sparked the interest of several industries, resulting in the discovery of possible amylase-producing microbes. Aspergillus niger ICP2 was an indigenous fungus isolated from coffee pulp that shows amylolytic activity after growing on a soluble starch agar. In this study, we optimized the production and successfully purified the amylase from Aspergillus niger ICP2. Amylase optimization production was performed for a 7-day of incubation under submerged fermentation. The amylase activity was measured using the iodin method, and the total protein was quantified spectrophotometrically at 280 nm. Purifying amylase crude extract conducted such as ammonium sulfate precipitation, dialysis, and anion-exchange chromatography. Amylase was maximally precipitated at 90% saturation, followed by dialysis on a 10 kDa column, increasing specific activity up to 12.17 U/mg and purity multiples of 9.31 times. Two peaks of amylase activity were formed when the dialyzed amylase was loaded onto anion exchange chromatography with specific activities of 14.47 U/mg (fraction I) and 32.73 U/mg (fraction II). The increase in specific activity and purity fold indicated that the amylase purification process was successful.

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“…Although the determination of reducing sugars obtained from the starch hydrolysis reaction is typically carried out using dinitrosalicylic acid, it is a costly method that requires a significant amount of reagents and has a complex protocol [ 87 ]. According to the literature, the α-amylase activity in the samples was found to be two times higher when measured by the iodine solution test compared to the DNS method [ 88 ], probably due to the fact that there are other reaction products that can form from the hydrolysis of starch, not only maltose.…”

Section: Introductionmentioning

confidence: 99%

An Accessible Method to Improve the Stability and Reusability of Porcine Pancreatic α-Amylase via Immobilization in Gellan-Based Hydrogel Particles Obtained by Ionic Cross-Linking with Mg2+ Ions

et al. 2023

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Amylase is an enzyme used to hydrolyze starch in order to obtain different products that are mainly used in the food industry. The results reported in this article refer to the immobilization of α-amylase in gellan hydrogel particles ionically cross-linked with Mg2+ ions. The obtained hydrogel particles were characterized physicochemically and morphologically. Their enzymatic activity was tested using starch as a substrate in several hydrolytic cycles. The results showed that the properties of the particles are influenced by the degree of cross-linking and the amount of immobilized α-amylase enzyme. The temperature and pH at which the immobilized enzyme activity is maximum were T = 60 °C and pH = 5.6. The enzymatic activity and affinity of the enzyme to the substrate depend on the particle type, and this decreases for particles with a higher cross-linking degree owing to the slow diffusion of the enzyme molecules inside the polymer’s network. By immobilization, α-amylase is protected from environmental factors, and the obtained particles can be quickly recovered from the hydrolysis medium, thus being able to be reused in repeated hydrolytic cycles (at least 11 cycles) without a substantial decrease in enzymatic activity. Moreover, α-amylase immobilized in gellan particles can be reactivated via treatment with a more acidic medium.

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Flexible and expeditious assay for quantitative monitoring of alpha-amylase and amyloglucosidase activities

Abstract: Graphical abstract

Cited by 11 publications

References 13 publications

Study of Carbohydrate Hydrolysis From Arracacha Roots (Arracacia Xanthorriza Bancroft) to Produce Fermentable Sugars

Study of Carbohydrate Hydrolysis From Arracacha Roots (Arracacia Xanthorriza Bancroft) to Produce Fermentable Sugars

Purification of an Extracellular Amylase Produced by <i>Aspergillus niger</i> ICP2 through Submerged Fermentation

An Accessible Method to Improve the Stability and Reusability of Porcine Pancreatic α-Amylase via Immobilization in Gellan-Based Hydrogel Particles Obtained by Ionic Cross-Linking with Mg2+ Ions

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