Alternative method for quantification of alfa-amylase activity

Abstract

A modification of the sensitive agar diffusion method was developed for macro-scale determination of alfa-amylase. The proposed modifications lower costs with the utilisation of starch as substrate and agar as supporting medium. Thus, a standard curve was built using alfa-amylase solution from Aspergillus oryzae, with concentrations ranging from 2.4 to 7,500 U.mL -1. Clear radial diffusion zones were measured after 4 hours of incubation at 20 °C. A linear relationship between the logarithm of enzyme activities and the area of clear zones was obtained. The method was validated by testing α-amylase from barley at the concentrations of 2.4; 60; 300 and 1,500 U.mL -1 . The proposed method turned out to be simpler, faster, less expensive and able to determine on a macro-scale α-amylase over a wide range (2.4 to 7,500 U.mL -1 ) in scientific investigation as well as in teaching laboratory activities.Keywords: alfa-amylase, alternative method, quantification. Alternativa para quantificação de alfa-amilase ResumoModificações foram propostas ao método sensível de difusão em ágar para a macrodeterminação de alfa-amilase. As modificações propostas diminuem os custos, com a utilização de amido como substrato e ágar como meio solidificante. Assim, foi construída uma curva padrão utilizando uma solução de alfa-amilase de Aspergillus oryzae com concentrações variando de 2,4 a 7.500 U.mL -1. Em seguida, as zonas claras de difusão radial foram mensuradas depois de 4 horas de incubação a 20 °C. Foi obtida uma relação linear entre o logaritmo da atividade enzimática e os diâmetros das zonas claras. O método foi validado utilizando-se soluções de alfa-amilase de cevada nas concentrações de 2,4; 60; 300 e 1.500 U.mL -1. O método tornou-se mais simples, rápido, com baixo custo e passível de ser utilizado para macrodeterminação de alfa-amilase em ampla faixa (2,4 a 7.500 U.mL -1 ) na investigação científica e para fins didáticos em aulas práticas.Palavras-chave: alfa-amilase, método alternativo, quantificação.

Assessment of α-Amylase Activity in a Microanalysis System: Experimental Optimization and Evaluation of Type of Inhibition

“…Several authors have published pedagogical articles describing experiments on α-amylase activity. ,,− Most of them are starch–-iodine methods, which underestimate the α-amylase inhibitory potential of some compounds and extracts. , Moreover, the published studies evaluate α-amylase from saliva, which physiologically accounts for 10–30% of starch cleavage to shorter oligomers . On the other hand, α-amylase from the pancreas plays an important role in postprandial hyperglycemia. , As mentioned above, we used porcine pancreatic α-amylase in the present study, which has a high level of similarity when compared to the human pancreatic enzyme.…”

Proteomic Analysis Reveals the Importance of Exudates on Sclerotial Development in Sclerotinia sclerotiorum

“…Detection and Activity of Enzymatic Proteins. Cell wall degrading enzyme activities in mycelia and sclerotial exudates were visually screened by published protocols, including GLU, 32 MAN, 33 AMY, 34 and PG. 35 Briefly, a mycelial plug or 20 μL of sclerotial exudates was incubated on detection media (i.e., water agar medium with the corresponding substrates) and the plates were investigated at 24 h after inoculation (hai).…”

Critical review on conventional spectroscopic α‐amylase activity detection methods: merits, demerits, and future prospects

“…The enzyme glucose oxidase (GOD) is used to determine blood glucose level, total starch content in cereal products, and serum α ‐amylase activity because it is cheap, stable, and is highly specific for glucose. Since GOD is highly specific for glucose, the product of α ‐amylase (maltose and other dextrins; Eqn (5)) cannot be used directly with GOD to determine α ‐amylase activity and, therefore, is concomitantly hydrolysed by an amyloglucosidase to yield glucose (Eqn (6)): …”