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EXTRACTO:

<<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

Ensayo de ninhidrina (colorimétrico)


1. 500 mg ninhidrina y15 mg ácido ascórbico se disolvieron en 60 ml de etilenglicol
Una solución de etilenglicol al
- 0.8333% o 833mg% de ninhidrina
- 0.025% o 25mg%de ácido ascórbico
Existen varias pruebas colorimétricas para determinar la existencia de aminoácidos,
resumidas en la siguiente tabla:

La Ninhidrina, (tricetohidrindeno) es un agente oxidante fuerte. Causa una deshidratación


oxidativa de los alfa-aminoácidos produciendo dióxido de carbono, amonio y aldehído y una
forma reducida de ninhidrina, la hidrindantina. La hidrindantina nuevamente reacciona con
amonio y un mol de ninhidrina para producir el complejo de Ruhemann, o púrpura de
Ruhemann, el cual absorbe luz a una longitud de onda de 570nm.
La reacción con ninhidrina involucra a los grupos carboxílicos y amino de los aminoácidos.
Debido que Prolina carece de un grupo alfa-amino, da un color amarillo con esta prueba.
De la misma manera, asparagina, al tener un grupo amida libre, da un producto de color
marrón (Dubey 2014: 468).
Todos los aminoácidos cuando son calentados con ninhidrina pueden formar complejos
rosados, púrpuras o azules. El complejo de color se llama púrpura de Ruhemann.
Prolina e hidroxiprolina dan color amarillo. Aminoácidos con grupos amida (glutamina,
asparagina) producen color marrón (Vasudenvan et al. 2016: 31).
2. Almacenado a -20° y mezclado con 60 ml buffer citrato (pH 6) 1M antes de usar
*Por qué buffer citrato
Ajustar pH?
Volumen de solución final: 120ml
3. CURVA DE CALIBRACIÓN por una repetición.
ACC en mM en Solución de Reactivo
el medio DF trabajo ninhidrina
0 (medio DF)
1ml 2ml
BLANCO
0.005 1ml 2ml
0.01 1ml 2ml
0.015 1ml 2ml
0.02 1ml 2ml
0.03 1ml 2ml
0.04 1ml 2ml
0.05 1ml 2ml
0.1 1ml 2ml
0.15 1ml 2ml
0.2 1ml 2ml
0.25 1ml 2ml
0.3 1ml 2ml
0.4 1ml 2ml
0.5 1ml 2ml
Volumen total 15 ml 30ml
Differentiation of 1-aminocyclopropane-1-carboxylate (ACC)
deaminase from its homologs is the key for identifying bacteria
containing ACC deaminase

Actividad ACC desaminasa (enzimático)


the colorimetric 2,4-dinitrophenylhydrazine assay of α-ketobutyrate, as described by Penrose and
Glick (2003) for non-rhizobia and Duan et al. (2009) for rhizobia.

suspended in 0.1 M MgSO4 × 7H2O


bacterial density of each suspension was determined using a calibration curve assessed
by turbidity (λ = 600 nm) an adjusted to 6 × 108 colony forming units per mL (cfu mL−1).
seeded in point form onto surface of solid (16 g L−1 agar) DF mineral medium alone
(negative control)
or supplemented either with ammonium sulfate (2 g L−1) or ACC (3 mM) (Sigma–Aldrich,
USA) as the sole source of nitrogen
ACC was also confirmed by ninhydrin-ACC reaction according to the procedure described
by Li et al. (2011).

The method differs from the α-ketobutyrate assay in that it


measures the ACC remaining in the media versus measuring a
metabolite by-product.
The reaction of ninhydrin with α-amino acids (such as ACC) is
well established in the literature and produces 2,
4 diketohydrindylidene-2diketohydrindamine, colloquially referred
to as Ruhmenann’s Purple

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
CONDICIONES DE CULTIVO

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