Conversion of indoleacetaldehyde to indoleacetic acid by microbial contamination

Vol.3, No.3

BIOCHEMICAL

Sept. 1960

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

CORVZRSION OP INDOL&K!ETALDXMX TC INDOLXACETIC ACID BY MICROBIALCOI~i?WMI.I~TIOi'~* Martha Kent and Willis A. Gortner Pineapple Research Institute Honolulu, Ralraii

Received august 5,

1960

Our studies on the biosynthesis of indoleacetic

acid (UA) by the pine-

apple plant have shown that erroneous information may derive from enzyme studies where growth of microorganisms is not effectively tigation

pointed to a non-enzymic reaction,

were highly active.

Whenextracts

column, the IAc conversion activity ninhydrin-

and antbrone-positive

since boiled aqueous ex-

were fractionated

from 20 mg. of the bisulfite

The ether extract

several times with water, and an aliquot containing

the ether.

into a

50

50

to

addi-

of IAc was washed 60

pg. of the IXc

ml. flask which ms heated briefly

A reaction mixture of

phosphate buffer

containing both

constituents.

tion product according to Gray (1959).

in ether was pipetted

by a Sephadex-25

was highest in fractions

The assay method used IAcM liberated

pH 4.8

Invcs-

of indoleacetaldehyde (IAc) conversion to IAA by pineapple stem or

leaf extracts tracts

controlled.

to evaporate

ml. was added, which included 0.OU.l

5.0

and the boiled,

aqueous pineapple tissue extract,

which was active at a level representing 1.25 mg. fresh weight of tissue. The mixture was incubated overnight at &OOC. on a Dubnoff shaker. extractions IAC.

with peroxide-free

TVo

ether Were madeat pH 10 to remove the residual

The solution was then adjusted to pH 4.0-4.5

and extracted three times

*Published rrith the approval of the Director as Technical P3per Noof the Pineapple Research Institute of Hs.waii. *xSupplicd through the courtesy of R. 17. Lecper. 319

266

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BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

with 89 equal volume of ether. vith

I&$04,

!Phemoisture was removed from the ether extract

and the ether evaporated.

wing abnodified Salkowskl

Sept. 1960

IAA was determined quantitatively

(Gordon and Weber, 1951).

reagent

Following a

17-knar incubation period, about 33 pg. of IAA were consistently from ths 50~.

of IAc substrate.

The identity

chromatography of,an ether aliquot,

obtained

of IAA was confirmed by paper

,followed by Salkowski spray.

It ms found that glutamine or glutamic acid solutions cpuld promote conversion at a concentiation variously

weak or inactive

of 40 p.p.m.

in conversion.

Mono- and disaccharides were A solution of pineapple fruit

sacchsride and washings from ordinary filter sion of UC, but rarely sulfate-precipitated initially

higher.

Activity

paper effected

Bacteria were not

suspected as the cause of conversion since all

was nqt cloudy after

kept in the freezer.

solutions except the

Furthermore, the reaction mixture

the long incubation period.

In attempting to clarify that under sterile

a 25-56 conver-

was also shownby boiled snmudwn

enzyme from pineapple extracts.

buffer were routinely

somediscrepancies in results,

conditions,

the pineapple tissue extract

no conversion at all occurred.

it was found Culture of

showeda predominance of motile bacteria,

stained 8s gram negative rods, 1 to 4~. long and 0.5 u wide. modified malt agar, the organism was inactive transfer

poly-

which

Grown on

in IAc conversion.

Subsequent

to a solution or agar containing 0.5s glutamine or glutamic acid

(adjusted to pH 5.0) and including 0.01 M pH 4.8 phosphate buffer produced bacteria active

in the conversion.

was not required.

Without buffer,

&owth was reduced; sucrose

By increasing the inoculum in the standard conversion assay,

the time necessary for a 6555conversion of the substrate to IAA was reduced from 17 to 6 hours.

Buffer was not necessary for optimum conversion if 0.01%

glutsmic acid was present. A microbial contaminant of gluta.mic acid was readily in morphology and in IAc conversion activity extracts.

In addition,

isolated,

similar

to the one from pineapple tissue

a biack pigment-forming yeast was isolated from a

solution of the pineapple fruit

polysaccharide, 320

active

in converting about

Vol.3,No.3

25%

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Sept. 1960

of the IAc substrate to IAA. Observations madeearlier

more intelligible

in the course of the investigation

with the finding

became

that conversion was due to the presence

of microorganisms. In retrospect,'the

various treatments had either inhibited

growth of the organism, reduced the inoculum in other ways, or removed the amino-acid constituent were:

inhibition

necessary for growth.

by KCN, inhibition

Effects which had been noted

by 10-w Versene, removal of activity

by cation exchange resin, persistance of activity hydrolysis

of an extract,

substrate limitation free of bisulfite

reduction of activity

when the ether extract

after

alkaline or acid

on dialysis,

and marked

of IAc was not washed completely

from the addition product.

Millipore

filtration

only

reduced conversion because of leakage around the edges of the filter fact that not all solutions were filtered

and the

at the sametime.

It now appears that the active microorganism was introduced primarily with the tissue extract.

It

is probable that the contaminant grew optimally

in the amino acid mixture found naturally of the extract. or air,

in the extract

or Sephadex fractions

It is possible that the inoculum also camefrom the buffer

since fresh solutions of glutamic acid were active in conversion.

The glut‘amic acid becameincreasingly

active

in conversion a,s growth of the

contaminant increased in the stock solution. Since Pseudomonasfluorescens was reported by Jakoby and Narrod (1959) to oxidize aldehydes, a stock culture of the organism was obtained. on malt agar, there was no conversion activity. inoculated solution of glutamic acid and buffer

Grown

However, incubation of an solution on a shaker for one

or two days, produced growth which converted. the usual 655 of the IAc substrate to l&L

Viability

remained indefinitely

when the solution was stored

in the freezer,

but when grown on glutamic acid agar plates,

this and the

bacterial

contaminants remained viable for a few days only.

oxidation

of IAc to IAA was not included in the aldehydes tested by Jakoby

and Narrod, the oxidation reported.

Although the

of indole aldehyde to indolecarbowlic

The pineapple extract

acid was

contaminant also madethis conversion; 321

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BIOCHEMICAL

the identity

AND BIOPHYSICAL RESEARCH COMM&JNlCATlONS

of indolecarboxylic

Sept.1960

acid was confirmed by paper chromatography

and Salkowski color. A stock cultme of Proteus vule;aris was equally active in conversion once growth was adapted to glutamic acid. did not appear identical at identification

Microbial

artifact

and precipitated

enzymes, particularly

perioda of incubation are involved.

Gordon, S. A. and Weber, R. P., Plant Physiol. 26, 192 (1951). R. A., Arch. Biochem. Biophys., 81, 480 (1959).

Jakoby, W. B. and Narrod, S. A., J. Bacterial.,

322

acid meta-

in the production of I&A

RJZFESENCES

Gray,

attempts

of workers the possi-

in enzyme studies on indoleacetic

contamination may be a factor

from tryptophan by plant extracts where overnight

and no further

is intended to bring to the attention

of a biological

bolism.

to either known organism

were ma&.

This report bility

The morphology of the contaminants'

7'7, 410 (1959).