Biotransformation of 12-hydroxyoctadecanoic acid to 12-hydroxyoctadecanamide by Bacillus cereus 50

Article abstract of DOI:10.1007/s11746-997-0188-8

A new microbial isolate (Bacillus cereus 50) transformed 12-hydroxyoctadecanoic acid to 12-hydroxyoctadecanamide when grown aerobically in 1% yeast extract medium at 30°C, shaken at 250 rpm for 2 to 5 d. The compound was purified by thin-layer chromatography and characterized by infrared, gas chromatography, mass spectrometry, and nuclear magnetic resonance. The yields of 12-hydroxyoctadecanamide were 9.1 and 21.5% after 2 and 5 d, respectively.

Full text of DOI:10.1007/s11746-997-0188-8

Biotransformation of 12-Hydroxyoctadecanoic Acid
to 12-Hydroxyoctadecanamide by Bacillus cereus 50
a
b
a
a
J.-K. Huang , K.C. Keudell , W.E. Klopfenstein , L. Wen ,
c,
d
d
e
M.O. Bagby *, R.F. Vesonder , R.A. Norton , and D. Weisleder
Departments of Chemistry and Biological Science, Western Illinois University, Macomb, Illinois 61455, and Oil Chemical
a
b
c
d
e
Research, Mycotoxin Research and Analytical Chemistry Support, USDA, ARS, NCAUR, Peoria, Illinois 61604
ABSTRACT: A new microbial isolate (Bacillus cereus 50) and hexadecanoic acid (palmitic acid), were obtained from
transformed 12-hydroxyoctadecanoic acid to 12-hydroxyoc- Sigma (St. Louis, MO) with purities greater than 98% by GC
tadecanamide when grown aerobically in 1% yeast extract
medium at 30°C, shaken at 250 rpm for 2 to 5 d. The compound
was purified by thin-layer chromatography and characterized
by infrared, gas chromatography, mass spectrometry, and nu-
clear magnetic resonance. The yields of 12-hydroxyoctade-
canamide were 9.1 and 21.5% after 2 and 5 d, respectively.
JAOCS 74, 601–603 (1997).
analysis. The product, 12-hydroxyoctadecanamide, was puri-
fied to better than 97% purity, as determined by GC analysis.
YE was from Difco Laboratories (Detroit, MI).
Microorganisms and microbial conversion study. Strain 50
was isolated from the intestinal tract of a fish (large mouth
bass, Micropterus salmoides) (8). A single colony was cul-
tured in 5 mL of tryptone medium (containing 0.5% tryptone,
0
.2% KH PO , 0.4% Na HPO , 0.2% (NH ) SO , 0.01%
KEY WORDS: 12-Hydroxyoctadecanamide, 12-hydroxyoc-
tadecanoic acid, Bacillus cereus, biotransformation.
2 4 2 4 4 2 4
CaCl ·H O and 0.001% FeSO ·7H O, pH 7.2) at 30°C, 200
2
2
4
2
rpm for 18 h. The following day, 2-mL cultures were added
to 100 mL of 1% YE medium (containing 1% yeast extract,
Fatty amides have diverse applications as chemical additives, lu- 0.2% KH PO , 0.4% Na HPO , 0.2% (NH ) SO , 0.01%
2
4
2
4
4 2
4
bricants, slip agents, and nonsticky and protective coatings (1). CaCl ·H O and 0.001% FeSO ·7H O, pH 7.2). Ten mL of the
2
2
4
2
Fatty amides, such as erucamide, are chemically synthesized 1-to-50 diluted culture was transferred to each of the 125-mL
from 13(Z)-docosenoic acid (erucic acid) (2,3). Microbial trans- capacity Erlenmeyer flasks, and 12-HOA was added to a final
formation of 9(Z)-octadecenoic acid to 9(Z)-octadecenamide concentration of 0.2% to carry out the bioconversion reaction
(
(
6% yield on total lipids extracted) by Bacillus cereus B-14812 for up to 5 d. Pure dilution culture served as control. Tripli-
4), and to 9(Z)-octadecenamide, hexadecenamide, tetradece- cate samples were used.
namide, and tetradecanamide by B. megaterium B-3437 (5–7%
yield on total lipids extracted) (5) has been reported.
Three flasks (10 mL/flask) were removed on day 2 and on
day 5. The entire contents of triplicate flasks (10 mL) were
During our continuing efforts to isolate new microorgan- extracted. Palmitic acid (2 mg) was added as internal stan-
isms for biotransformation of 12-hydroxyoctadecanoic acid dard, and the acidified (with 3 N HCL) sample was extracted
(HOA) (6,7), we discovered another active strain of B. cereus twice with an equal volume of diethyl ether. The extracts were
5
1
0 (8). Bacillus cereus 50 produced significant amounts of washed once with an equal volume of water. The washed
2-hydroxyoctadecanamide (12-HOAM) when grown aero- ether was evaporated with N to a white residue.
2
bically in 1% yeast extract (1% YE) that contained 0.2%
For a larger-scale cultivation, a 1-to-50 dilution culture
(
wt/vol) of 12-HOA at 30°C when shaken at 250 rpm. In this (from overnight culture) was grown aerobically for 5 d at
paper, we report the conversion of 12-HOAM from 12-HOA 30°C in a one-liter flask with 250 mL of 1% YE medium
after 2 and 5 d, and its isolation and identification by infrared (total 500 mL from two flasks), and 12-HOA was added (final
(IR), gas chromatography–mass spectrometry (GC–MS), pos- concentration 0.2%, wt/vol). Samples (500 mL) were ex-
itive chemical ionization–mass spectrometry (PCI–MS), and tracted with diethyl ether and worked up in the usual manner.
nuclear magnetic resonance (NMR).
Thin-layer chromatography (TLC). The crude extracts were
subjected to partial purification before applying to TLC plates.
Crude extracts were dissolved in minimal amounts of methanol
in a flask, and diethyl ether was added to a final concentration
EXPERIMENTAL PROCEDURES
Materials. All chemicals and solvents were of ACS grade ob- of 98% (vol/vol). The flask was kept at −20°C for 1 h. The pre-
tained from commercial sources. The acids, R-(+)-12-HOA cipitate was collected and dried under N . Dried samples were
2
redissolved in methanol and reprecipitated once as above. The
compound (in methanol) was applied unto TLC plates (20 × 20
cm Silica gel 60 plate, 0.25 mm thickness; EM Science, Cherry
*
To whom correspondence should be addressed at Research Leader, USDA,
ARS, NCAUR. 1815 North University St., Peoria, IL 61604.
Copyright © 1997 by AOCS Press
601
JAOCS, Vol. 74, no. 5 (1997)

602
SHORT COMMUNICATION
Hill, NJ) as a strip 2.5 cm from the bottom of each plate. The nitrate reduction, degradation of gelatin and starch, and does
plates were developed with a CH Cl /MeOH (98:2, vol/vol) not produce gas from glucose (9).
2
2
system. Developed plates were wrapped with Saran Wrap
Identification of conversion products. Bacillus cereus 50
(Dow Brands, Indianapolis, IN) except for 1.5-cm margins on transformed 12-HOA to several compounds. One of these
the left and right of the plate. Sulfuric acid (30%) was sprayed compounds was purified by TLC. In our initial studies,
on the uncovered area, and color was developed by heating the methylation of the purified compound had no effect on GC
sprayed area at 150°C for 3–5 min with a heat gun. Color, de- retention time (6.8 min for the SPB-1 capillary column at
veloped on both edges, served as marker to locate 12-hydroxy- isothermal temperature and 17.6 min for the DB-5MS capil-
octadecanamide. 12-Hydroxyoctadecanamide was removed lary column with temperature programming). Therefore, the
from the plate, extracted with methanol, dried with N , and purified compound is not a free fatty acid. The IR spectrum
2
−
1
subjected to GC and GC–MS analyses.
indicated the presence of a hydroxy (3426 cm ) group, and
−1
Fourier transform infrared (FTIR) spectroscopy. IR spec- absorptions at 3320 and 3214 cm were indicative of the an-
tra of 12-hydroxyoctadecanamide or diazomethane-treated tisymmetric and symmetric stretch, respectively, of an amide
−
1
1
2-hydroxyoctadecanamide (thin film, KBr plate) were ob- group (C=O)NH . An absorption at 1659 cm was also con-
2
tained with a Mattson Galaxy 6020 IR spectrometer (Mattson sistent with the C=O stretch of a primary amide. Therefore,
Instruments, Inc., Madison, WI).
the compound is a hydroxy fatty amide.
GC and GC–MS. Crude extracts, partially purified samples
Low-resolution MS by PCI (reagent gas, isobutane) was
from crude extract, and a highly purified compound from the carried out on the purified compound. Results indicated the
+
TLC plate were analyzed by GC and GC–MS after being treated presence of a parent ion (m/z 299+H ) and an ion mass of m/z
with diazomethane or diazomethane and trimethylsilane (TMS) 282 (300 − 18). High-resolution PCI–MS of the parent peak
reagent. Treated samples were analyzed by chromatography indicated the presence of a compound with a chemical for-
with a Hewlett-Packard 5890A series II gas chromatograph mula of C H NO (m/z 300.2899, observed; calculated
1
8
37
2
(
Palo Alto, CA), equipped with a flame-ionization detector. 300.290255). Because 12-HOA was used as substrate for the
Samples were separated on an SPB-1 column (15 m × 0.32 mm conversion reaction, it is reasonable to believe that the puri-
i.d. and 0.25 mm thickness), either at isothermal (200°C) or fied compound is 12-hydroxyoctadecanamide. The location
temperature-programmed conditions as previously reported (6). of the hydroxyl group was determined by the presence of
Peak areas were determined with a Hewlett-Packard 3396A prominent ions from α-cleavage of the sigma bonds on the
electronic integrator. Methyl palmitate (retention time 1.6 min) left and right of the TMS groups (10). Electron-impact mass
was used as an internal standard, and 12-hydroxyoctade- spectra of 12-hydroxyoctadecanamide and its TMS deriva-
canamide (retention time 6.8 min) was used for quantitative tive indicated that the purified compound contains one hy-
analyses. Electron-impact mass spectra of the compounds men- droxyl group. For GC–MS of 12-hydroxyoctadecanamide,
tioned above were obtained on a Hewlett-Packard 5970 gas the prominent mass ions were m/z 214(37%) and 59(100%).
chromatograph, equipped with a DB-5ms capillary column (15 The m/z 214 ion results from α-cleavage of the sigma bond
m × 0.25 mm i.d.; J&W Scientific Co., Folsom, CA) coupled to between C –C ; the m/z 59 ion is the base peak and corre-
1
2
13
a Hewlett-Packard mass selective detector.
sponds to the McLafferty rearrangement product of the
In the later course of these studies, a Hewlett-Packard straight-chain monoamide (11). For 12-trimethyl-silyloxyoc-
5890A gas chromatograph equipped with an SP-2100, methyl tadecanamide, the prominent fragment ions m/z 187(40%),
fluid column (2.4 m × 2 mm i.d.; Supelco, Bellefonte, PA), 286(3%), and 358(100%). The mass ions of 187 and 286 re-
and a Hewlett-Packard 3396A electronic integrator was used. sulted from α-cleavage of the sigma bond between C –C
12
1
1
Esters were separated isothermally at 280°C (retention time and C –C , respectively. The mass ion of 358(359 − 1) re-
1
2
13
for 12-hydroxyoctadecanamide was 14.95 min).
sulted from the mass ion fragment of 286 with an additional
Low- and high-resolution MS by PCI was carried out on TMS attached to a carbonyl group (10). Therefore, the hy-
2-hydroxyoctadecanamide by The Nebraska Center for droxyl group is on carbon 12.
1
Mass Spectrometry, Department of Chemistry, University of
The proton NMR spectrum of the compound in CDCl so-
3
Nebraska-Lincoln (Lincoln, NE). Reagent gas was isobutane. lution indicated NH protons of the primary amide group
2
Exact mass measurements were made by using narrow-volt- (5.43 ppm, d, br.; 2H), a hydroxyl proton (3.57 ppm, m, 1H),
age scanning and ions of perfluorokerosene as standard mass. protons α and β to an acyl group, CH -C=O (2.18 ppm, m;
2
1
13
NMR spectra. H and C NMR spectra of 12-hydroxyoc- 2H) and CH -CH -C=O (1.65 ppm, m; 2H), methylene pro-
2
2
tadecanamide in CDCl or CD OD were obtained with a tons, -CH - (1.27 ppm, m; 27 H), and a CH (0.85 ppm, t,
3
3
2
3
1
3
Bruker ARX 400 Spectrometer (Burlington, Ontario, Canada). 3H). C NMR data indicated that the purified compound con-
tained 18 carbons and confirmed the presence of -C=O at C1
(
176 ppm); one -C-OH at 72.4 (C ); one CH -C-O- at 33.1
12 2
RESULTS AND DISCUSSION
Identification of microorganism. Strain 50 was identified as (C3–11,13–17); and -CH at 14.0 ppm (C ). It is a white solid
ppm and fourteen -CH -, ranging from 23 ppm to 39 ppm
2
3
18
B. cereus based on the following (8,9): It is a gram-positive that melts at 82–85°C.
rod, positive for oxidase and catalase activities, positive for
Production of hydroxy fatty amide. Diluted culture (10 mL
JAOCS, Vol. 74, no. 5 (1997)

SHORT COMMUNICATION
603
of a 1-to-50 dilution from an overnight culture (1% YE
medium) in a 125-mL Erlenmeyer flask that contained 0.2%
mobiles and Automobile Body Parts, Chem. Abstr. 109:212450r
1988).
. Kaneshiro, T., L.K. Nakamura, and M.O. Bagby, Oleic Acid
Transformations by Selective Strain of Sphingobacterium
thalpophilum and Bacillus cereus from Composted Manure,
Current Microbial. 31:62–67 (1995).
5. Kaneshiro, T., R.F. Vesonder, R.E. Peterson, D. Weisleder, and
M.O. Bagby, 9(Z)-Octadecenamide and Fatty Amides by Bacil-
lus megaterium (B-3437) Conversion of Oleic Acid, J. Am. Oil
Chem. Soc. 71:491–494 (1994).
. Huang, J.-K., K.C. Keudell, J. Zhao, W.E. Klopfenstein, L.
Wen, M.O. Bagby, A.C. Lanser, R.D. Plattner, R.E Peterson,
T.P. Abbott, and D. Weisleder, Microbial Transformation of 12-
Hydroxyoctadecanoic Acid to 5-n-Hexyl-Tetrahydrofuran-2-
Acetic Acid, Ibid. 72:323–326 (1995).
(
4
1
2-HOA was grown aerobically at 30°C, 250 rpm for 5 d.
Triplicate samples were prepared. Three flasks were removed
on day 2 and on day 5 for sample extractions and GC analy-
ses. The entire contents of the triplicate flasks were extracted.
The yields of 12-hydroxyoctadecanamide on day 2 and day 5
were 9.1% (from two experimental data: 10 and 8.2%) and
2
1.5% (from two experimental data: 23 and 20%), respec-
6
tively. The yield of 12-hydroxyoctadecanamide from a larger
cultivation (500 mL), after incubation for 5 d, was 24%.
Although the yield was low, enzymatic conversion of the
carboxylic group to an amide group provides a milder alter-
native way to alter the functionality of fatty acids.
7
. Huang, J.-K., K.C. Keudell, Su-Jin. Soeng, W.E. Klopfenstein,
L. Wen, M.O. Bagby, R.F. Vesendor, and R.A. Norton, Conver-
sion of 12-Hydroxyoctadecanoic Acid to 12,15-, 12,16-, and
1
2,17-Dihydroxyoctadecanoic Acids by Bacillus sp. U88,
ACKNOWLEDGMENTS
Biotechnol. Lett. 18:193–198 (1996).
8
9
. Kyaw, H., in Numerical Taxonomy of Bacterial Isolates from
Fish and Freshwater, edited by H. Kyaw, M.S. Thesis, Western
Illinois University, Macomb, 1991.
. Gibson, T., and R.E. Gordon, Part 15. Endospore-Forming Rods
and Cocci, Genus I. Bacillus, in Bergey's Manual of Determi-
native Bacteriology, 8th edn., edited by R.E. Buchanan and N.E.
Gibbons, Williams and Wilkins Co., Baltimore, 1974, pp.
This research was supported in part by the Department of Chemistry,
Western Illinois University (WIU) and Specific Cooperative Agree-
ment 58-5114-0-1024 between WIU and Agricultural Research Ser-
vice, U.S. Department of Agriculture. We thank The Nebraska Cen-
ter for Mass Spectrometry, Department of Chemistry, University of
Nebraska-Lincoln (Lincoln, NE), for doing low- and high-resolution
PCI-MS analysis on 12-HOAM.
5
29–550.
1
0. Kleiman, R., and G.F. Spencer, Gas Chromatography–Mass
Spectrometry of Methyl Esters of Unsaturated Oxygenated Fatty
Acids, J. Am. Oil Chem. Soc. 50:31–38 (1973).
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2
[Received March 12, 1996; accepted February 18, 1997]
JAOCS, Vol. 74, no. 5 (1997)