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<<ACC deaminase is an inducible enzyme whose synthesis is induced in the presence of its
substrate ACC. The minimum level of the substrate for induction was measured as 100 nM
in Pseudomonas sp. strain ACP and P. putida GR12-2. The induction of ACCD is a complex and slow
process. It exhibits activity within the first few hours of induction with the substrate, but the
activity decreases gradually after initial induction (Walsh et al., 1981; Jacobson et al., 1994). The
basal level of enzyme activity is observed in minimal medium supplemented with ammonium
sulfate as a nitrogen source. Honma (1983) demonstrated the induced activity after switching the
bacteria from nutrient rich medium to minimal medium supplemented with ACC as sole nitrogen
source. It illustrates that the induction of enzyme activity is directly correlated with substrate ACC.
Apart from ACC, other amino acids such as L-alanine, DL-alanine, D-serine also induce enzyme
activity and induce expression of ACCD to some extent. Moreover, the induced level of enzyme
activity by both ACC and aminoisobutyric acid was observed to be same in Pseudomonas sp. strain
ACP (Honma, 1983). Glick et al. (1998) proposed a model for functioning of bacterial ACC
deaminase which states that a significant portion of ACC is exuded from plant roots or seeds,
taken up by the soil microbes and hydrolyzed to ammonia and α-ketobutyrate. The uptake and
hydrolysis of ACC decrease the amount of ACC outside the plant roots. Furthermore, the
equilibrium between the internal and external ACC level is maintained through exudation of more
ACC into the rhizosphere. Thus, decrease in the level of ACC affects biosynthesis of the stress
hormone ethylene in host plants and stimulate plant growth (Honma et al., 1993; Glick et
al., 1998).>>
ORIGINAL ARTICLE
A colorimetric assay of 1-aminocyclopropane-1-carboxylate (ACC) based on
ninhydrin reaction for rapid screening of bacteria containing ACC deaminase
Aislamiento de cepas que utilizan ACC como única
fuente de nitrógeno (microbiológico)
1. Nitrogen-deficient DF medium
2. DF-ACC medium. ACC 3mM
*Diferencias entre medio DF deficiente en nitrógeno y medio DF-ACC
In the case of the specific reaction, a potential mechanism begins with ACC nucleophilically attacking the
central carbonyl of ninhydrin, the strained cyclopropane collapses and undergoes a decarboxylation 2,3. A
hydrolysis of the resulting ninhydrin adduct produces a leaving group composed of elements of the
cyclopropane from ACC. The ninhydrin derivative, possessing an amine β to the phenyl, nucleophilically
attacks a second ninhydrin resulting in the desired Ruhemann’s purple chromophore
was achieved by forming a ninhydrin reagent composed of 2.8 mmol ninhydrin, 0.085 mmol ascorbic acid in
1.076 moles ethylene glycol as solvent.
https://data.epo.org/publication-server/rest/v1.0/publication-
dates/20170308/patents/EP2735877NWB1/document.pdf
In addition, hydrindantin is insoluble in totally aqueous media. However, hydrindantin is soluble in
a number of organic solvents. Accordingly, organic solvents are generally added in relatively high
proportions to the reagent to reduce the possibility of precipitation of hydrindantin during storage
and use. Indeed, organic solvent contents up to as high as 75% by volume are typically used. The
solvent may also comprise a combination of two or more different types of solvent, to ensure
adequate dissolution of hydrindantin. Organic solvents used for this purpose typically include
dimethylsulfoxide (DMSO), methylcellosolve, ethylene glycol and sulfolane. Yet still, if insufficient
organic solvent is used or the hydrindantin concentration is too high, precipitation may occur on
standing or build up in the chromatographic apparatus, blocking the tubing.
Perhaps one of the most notable disadvantages to using ascorbic acid as a reducing agent is that it
is known to form brown coloured decomposition matter upon heating alone. US 3,778,230
suggests that the reduction products of reacting ascorbic acid with ninhydrin are not brown
coloured. However, there is a high risk that use of ascorbic acid as reducing agent results in the
formation of coloured by-products as a result of thermal decomposition of the ascorbic acid,
thereby adversely affecting the accuracy of the results recorded by the chromatogram.
Despite considerable research having been conducted with regard to finding alternative reducing
agents, manufacturers have generally reverted to providing separate solutions of hydrindantin and
ninhydrin. Typically, manufacturers provide two bottles, the first comprising a solution of
hydrindantin in an organic solvent and the second comprising a solution of ninhydrin, an aqueous
buffer and additional organic solvent; both bottles being tightly sealed under an inert gas
atmosphere. The bottles are then mixed to form the ninhydrin reagent prior to use in an amino
acid analyser. [0021] Most manufacturers claim a maximum shelf life of about 12 months before
mixing and 2 to 3 months after mixing. However, the life time of the mixed reagent is even lower
once attached to the analysis instrument and can be significantly below 1 month. [0022] Hitachi
appears to be the only
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