Amylase measurement with 2-chloro-4-nitrophenyl maltotrioside as substrate

Foo, A Y; Bais, Renze

doi:10.1016/s0009-8981(98)00009-6

Cited by 53 publications

(36 citation statements)

References 10 publications

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“…Samples for the enzymatic activity analysis were homogenized in buffers according to the procedures described for: lipase (Winkler and Stuckman, 1979), trypsin (Erlanger et al, 1961), α-amylase (Foo and Bais, 1998), alkaline phosphatase (ALP) (Wenger et al, 1984), γ-glutamyltransferase (γ-GT) (Gendler, 1984) and leucine aminopeptidase (LAP) (Nagel et al, 1964) and centrifuged (4ºC, 15 min, 15000 G). The activity of all enzymes was measured at 25ºC (3 replicates each) and calculated for 1 mg of protein from the enzymatic extract (μmol of product 1 min -1 ).…”

Section: Methodsmentioning

confidence: 99%

Untitled

Kamaszewski¹,

Ostaszewska²,

Prusińska³

et al. 2014

Turk. J. Fish. Aquat. Sci.

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The aim of this study was to compare the physiology and morphology of the digestive tract of Russian sturgeon (Acipenser gueldenstaedtii) larvae fed Artemia sp. nauplii enriched or non-enriched with essential fatty acids (EFA). Physiology was evaluated by digestive enzyme activity analysis, while morphology was assessed with histological methods. The larvae were divided into two groups, in which fish were fed either pure Artemia sp. nauplii or Artemia enriched in EFA. Both groups had similar survival rates, but fish fed EFA-enriched Artemia displayed higher, body weight and length. At the end of the experiment (22 dph): 1) the activities of lipase and leucine aminopeptidase were similar in both groups; 2) the activities of trypsin, alkaline phosphatase and γ-glutamyltransferase were insignificantly higher in fish fed pure Artemia; 3) the activity of α-amylase was significantly higher in the EFA-enriched feeding group; 4) lower hepatocyte lipid vacuole diameters and hepatocyte proliferation were measured in the EFA-enriched group. Lipid accumulation was observed in the anterior intestine of fish fed pure Artemia on the 15 th and 22 nd dph. The epithelial turnover was significantly lower in the EFAenriched group, in the posterior intestine on the 15 th dph, but no differences occurred between the groups on the 22 nd dph, in either anterior or posterior intestine. In conclusion, the study revealed a positive effect of the EFA-enriched Artemia-based diet on the physiology and morphology of the digestive system of Russian sturgeon larvae.

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Section: Methodsmentioning

confidence: 99%

Untitled

Kamaszewski¹,

Ostaszewska²,

Prusińska³

et al. 2014

Turk. J. Fish. Aquat. Sci.

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“…The CNPG3 compound, with a 2-chloro-4-nitrophenol (CNP) group coupled to maltotriose (G3), is a suitable substrate for determining ␣-amylase activity (21). The assay is based on the detection of the released CNP group from the CNPG3 substrate by ␣-amylase activity.…”

Section: Methodsmentioning

confidence: 99%

Degradation of Granular Starch by the Bacterium Microbacterium aurum Strain B8.A Involves a Modular α-Amylase Enzyme System with FNIII and CBM25 Domains

Valk

Eeuwema

Sarian

et al. 2015

Appl Environ Microbiol

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The bacterium Microbacterium aurum strain B8.A, originally isolated from a potato plant wastewater facility, is able to degrade different types of starch granules. Here we report the characterization of an unusually large, multidomain M. aurum B8.A ␣-amylase enzyme (MaAmyA). MaAmyA is a 1,417-amino-acid (aa) protein with a predicted molecular mass of 148 kDa. Sequence analysis of MaAmyA showed that its catalytic core is a family GH13_32 ␣-amylase with the typical ABC domain structure, followed by a fibronectin (FNIII) domain, two carbohydrate binding modules (CBM25), and another three FNIII domains. Recombinant expression and purification yielded an enzyme with the ability to degrade wheat and potato starch granules by introducing pores. Characterization of various truncated mutants of MaAmyA revealed a direct relationship between the presence of CBM25 domains and the ability of MaAmyA to form pores in starch granules, while the FNIII domains most likely function as stable linkers. At the C terminus, MaAmyA carries a 300-aa domain which is uniquely associated with large multidomain amylases; its function remains to be elucidated. We concluded that M. aurum B8.A employs a multidomain enzyme system to initiate degradation of starch granules via pore formation. Starch is an excellent carbon and energy source for many microorganisms, which employ a dedicated set of proteins for extracellular hydrolysis of this polysaccharide, uptake of shorter oligosaccharides into the cell, and further degradation into glucose. Most studies on degradation of starch by microbial enzymes have focused on soluble starch. This has resulted in the identification and characterization of a large variety of enzymes cleaving either ␣(1¡4) or ␣(1¡6) linkages in amylose and amylopectin. Most of these enzymes belong to the glycoside hydrolase 13 (GH13) family (1). Sequence diversity is such that, at the moment, the GH13 family contains a total of 40 subfamilies (1). Most of the new members in subfamilies are identified in DNA sequencing projects, and biochemical information about the activity and specificity of these potentially new enzymes is highly lagging.Many plants produce starch in a granular form for the storage of carbohydrates. The crystallinity of such granules varies with the plant source. Potato starch granules have a relatively high degree of crystallinity, making them notoriously resistant to bacterial and fungal degradation (2-4). Nevertheless, some microorganisms have been reported to employ enzymes that are able to digest granular starch (5, 6).Amylases found to be involved in granular starch degradation are often multidomain enzymes that include one or more carbohydrate binding modules (CBMs), which aid in the binding of the enzyme to the granular substrate (7-10).In previous work, various bacteria able to grow on potato starch granules as a carbon source were isolated, and their enzymatic degradation mechanism was evaluated. Initially, this resulted in the identification of an enzyme mechanism involving peeling off layer afte...

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“…Serum α-amylase activity was determined according to the method described by YingFoo et al [15] using a kit supplied by Human, Germany.…”

Section: Serum α-Amylase Activitymentioning

confidence: 99%