Monooxygenase system of bacillus megaterium ALA2: Studies on linoleic acid epoxidation products

Article abstract of DOI:10.1007/s11746-006-5023-0

Bacillus megaterium ALA2 produces many oxygenated FA from linoleic acid: 12, 13-dihydroxy- 9(Z)-octadecenoic acid; 12,13,17-trihydroxy-9(Z)-octadecenoic acid; 12,13,16-trihydroxy-9(Z)-octadecenoic acid; 12-hydroxy-13,16-epoxy-9(Z)- octadecenoic acid; and 12,17;13,17-diepoxy-16-hydroxy-9(Z)-octadecenoic acid. Recently, we studied the monooxygenase system of B. megaterium ALA2 by comparing its palmitic acid oxidation products with those of the well-studied catalytically self-sufficient P450 monooxygenase of B. megaterium ATCC 14581 (NRRL B-3712) and of B. subtilis strain 168 (NRRL B-4219). We found that their oxidation products are identical, indicating that their monooxygenase systems (hydroxylation) are similar. Now, we report that strain ALA2 epoxidizes linoleic acid to 12,13-epoxy-9(Z)-octadecenoic acid and 9,10-epoxy-12(Z)-octadecenoic acid, the initial products in the linoleic acid oxidation. The epoxidation enzyme did not oxidize specific double bond of the linoleic acid. The epoxidation activity of strain ALA2 was compared with the above-mentioned two Bacillus strains. These two Bacillus strain also produced 12,13-epoxy-9(Z)- octadecenoic acid and 9,10-epoxy-12(2)-octadecenoic acid, indicating that their epoxidation enzyme systems might be similar. The ratios of epoxy FA production by, these three strains (ALA2, NRRL B-3712, and NRRL B-4219) were, respectively, 5.56:0.66:0.18 for 12,13-epoxy-9(Z)-octadecenoic acid and 2.43:0.41:0.57 for 9,10-epoxy-12(Z)-octadecenoic acid per 50 mL medium per 48 h. Copyright

Full text of DOI:10.1007/s11746-006-5023-0

Monooxygenase System of Bacillus megaterium ALA2:
Studies on Linoleic Acid Epoxidation Products
Ching T. Hou*
Microbial Genomics and Bioprocessing Research Unit, NCAUR, ARS, USDA, Peoria, Illinois 61604
ABSTRACT: Bacillus megaterium ALA2 produces many oxy- fungals (3,4). The main product, 12,13,17-THOA, was found to
genated FA from linoleic acid: 12,13-dihydroxy- 9(Z)-octade-
cenoic acid; 12,13,17-trihydroxy-9(Z)-octadecenoic acid;
12,13,16-trihydroxy-9(Z)-octadecenoic acid; 12-hydroxy-13,16-
epoxy-9(Z)-octadecenoic acid; and 12,17;13,17-diepoxy-16-hy-
droxy-9(Z)-octadecenoic acid. Recently, we studied the
monooxygenase system of B. megaterium ALA2 by comparing its
palmitic acid oxidation products with those of the well-studied
catalytically self-sufficient P450 monooxygenase of B. mega-
terium ATCC 14581 (NRRL B-3712) and of B. subtilis strain 168
(NRRL B-4219). We found that their oxidation products are iden-
tical, indicating that their monooxygenase systems (hydroxyla-
inhibit the growth of many plant pathogenic fungi (3). The struc-
ture of tetrahydrofuranyl FA (THFA) resemble those of known
anticancer agents (5). The diepoxy bicyclic FA are new chemi-
cal entities with many functional groups in their molecules. Their
applications in biomedical and specialty chemical industries are
expected, and the biosynthetic pathways for these oxygenated
FA are known (6–9).
Monooxygenases are known to hydroxylate alkanes and/or
epoxidate alkenes including saturated and unsaturated FA, e.g.,
the Pseudomonas oleovolans monooxygenase system (10–12);
tion) are similar. Now, we report that strain ALA2 epoxidizes the monooxygenase systems of methylotrophs (13); and Bacil-
linoleic acid to 12,13-epoxy-9(Z)-octadecenoic acid and 9,10-
epoxy-12(Z)-octadecenoic acid, the initial products in the linoleic
acid oxidation. The epoxidation enzyme did not oxidize specific
double bond of the linoleic acid. The epoxidation activity of
strain ALA2 was compared with the above-mentioned two Bacil-
lus strains. These two Bacillus strain also produced 12,13-epoxy-
9(Z)-octadecenoic acid and 9,10-epoxy-12(Z)-octadecenoic acid,
indicating that their epoxidation enzyme systems might be simi-
lar. The ratios of epoxy FA production by these three strains
(ALA2, NRRL B-3712, and NRRL B-4219) were, respectively,
5.56:0.66:0.18 for 12,13-epoxy-9(Z)-octadecenoic acid and
lus megaterium ATCC 14581 (14). The oxygenase system of
ALA2 was not studied. Strain ALA2 was recently reclassified
as Bacillus megaterium based on DNA analysis (15). This
opened a link to the well-studied Bacillus monooxygenase sys-
tem. Catalytically self-sufficient cytochrome P450 monooxy-
genase from B. megaterium ATCC 14581 (CYP102A1) has
been well-studied (16). Two genes from B. subtilis, CYP102A2
and CYP102A3, code for single-peptide monooxygenases
comprising both a heme and a FAD/FMN-containing reductase
domain and demonstrate a notable sequence similarity to
2.43:0.41:0.57 for 9,10-epoxy-12(Z)-octadecenoic acid per 50 CYP102A1 (17). CYP102A3 is involved in the hydroxylation
mL medium per 48 h.
of unsaturated, saturated, and branched-chain FA (18). Re-
cently, we compared the monoxygenase (hydroxylation) sys-
tems of strain ALA2 with the two just-mentioned well-studied
Bacillus strains and found that their palmitic acid oxidation
products were identical, indicating that their monooxygenase
(hydroxylation) systems are similar (19).
Paper no. J11351 in JAOCS 83, 677–681 (August 2006).
KEY WORDS: Bacillus megaterium ALA2, bioconversion,
linoleic acid, 12,13-epoxy-9(Z)-octadecenoic acid, monooxyge-
nase system.
The microbial enzyme (hydratase) attacks the double bond
of unsaturated FA at a specific position (20). Microbial hy-
dratases convert oleic, linoleic, and linolenic acids into their
10-hydroxy FA, indicating that the enzyme is carbon 10-posi-
tional specific. It would be interesting to know whether the
epoxidation enzyme of Bacillus strains including strain ALA2
has positional specificity.
In our previous studies of the strain ALA2 and linoleic acid
system, we focused on FA products having retention times
greater than 10 min in GC analysis (GCRT). In this paper, we
studied two product peaks having GCRT of 7.10 and 7.32 min
and identified them as 12,13-epoxy-9(Z)-octadecenoic acid and
9,10-epoxy-12(Z)-octadecenoic acid, the initial epoxidation
products of linoleic acid. We also compared the epoxidation
activity of strain ALA2 and the two previously mentioned well-
studied Bacillus strains: NRRL B-3712 and NRRL B-4219.
Microbial systems convert unsaturated FA to monohydroxy-, di-
hydroxy-, and trihydroxy-FA (1). Strain ALA2 is a unique mi-
crobe that produces a variety of hydroxy FA from linoleic acid
(2): 12,13-dihydroxy-9(Z)-octadecenoic acid; 12,13,17-trihy-
droxy-9(Z)-octadecenoic acid (12,13,17-THOA); 12,13,16-tri-
hydroxy-9(Z)-octadecenoic acid (12,13,16-THOA); 12-hy-
droxy-13,16-epoxy-9(Z)-octadecenoic acid (THFA); 7,12-dihy-
droxy-13,16-epoxy-9(Z)-octadecenoic acid (HO-THFA); 12,17;
13,17-diepoxy-16-hydroxy-9(Z)-octadecenoic acid (DEOA);
and 7-hydroxy-12,17;13,17-diepoxy-16-hydroxy-9(Z)-octade-
cenoic acid (HO-DEOA). Oxygenated FA can be used not only
as specialty chemicals but also as bioactive agents such as anti-
*Address correspondence at NCAUR, ARS, USDA, 1815 N. University St.,
Peoria, IL 61604. E-mail: houct@ncaur.usda.gov
Copyright © 2006 by AOCS Press
677
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678
C.T. HOU
MATERIALS AND METHODS
RESULTS AND DISCUSSION
Chemicals. Oleic acid (purity 99%) and 12,13-epoxy-9(Z)-oc- Strain ALA2 produced many oxygenated FA products from
tadecenoic acid methyl ester were purchased from Sigma (St. linoleic acid such as trihydroxy unsaturated FA and diepoxy
Louis, MO). Diazomethane was prepared from Diazald bicyclic unsaturated FA as well as tetrahydofuranyl unsaturated
(Sigma). All other chemicals were reagent grade and used with- FA (2,6–8,23). These novel oxygenated unsaturated FA prod-
out further purification.
ucts are our primary interest because of their potential indus-
Microorganisms. Strain ALA2 (NRRL B-21660) was iso- trial applications. However, to improve their yields through
lated from a dry soil sample collected from McCalla, Alabama molecular engineering, we need more information on the ini-
(21). Bacillus megaterium ATCC 14581 (NRRL B-3712) and tial step of FA oxidation: hydroxylation and epoxidation. The
B. subtilis 168 (NRRL B-4219) were obtained from the ARS epoxidation reaction should produce a maximum of two prod-
Culture Collection (Peoria, IL). Strains B-3712 and B-4219 ucts from linoleic acid since there are only two double bonds
were cultivated on TGY medium as reported (16). Strain ALA2 on the substrate molecule. In this study, we found two products
was cultivated aerobically in a 125-mL Erlenmeyer flask con- at GCRT 7.10 and 7.32 min that might be the products of the
taining 50 mL of medium with shaking at 200 rpm and 30°C. initial epoxidation reaction. We also compared these products
The medium (per liter) contained (22): dextrose 5 g; K2HPO4 with those produced by two known Bacillus strains: B-3712
5 g; yeast extract 15 g; tryptone 10 g; MgSO4⋅7H2O, 2.0 mM; and B-4219. Control experiments with the same condition but
FeSO4⋅7H2O, 0.5 mM; ZnSO4, 0.1 mM; NiCl2⋅6H2O, 0.01 without a microorganism or without linoleic acid all showed
mM; CoCl2⋅6H2O, 0.05 mM; and nicotinic acid 10 mg. The no production of oxygenated FA products, indicating all prod-
medium was adjusted to pH 6.8 with dilute phosphoric acid.
ucts are produced by microbial action. Both strains B-3712 and
Production of oxygenated FA. Strain ALA2 was aerobically B-4219 produced no product peaks having GCRT greater than
grown in 50 mL of medium for 1 d at 30°C and 200 rpm. Two 10 min.
hundred microliters (190 mg) of linoleic acid was added into a
Identification of GCRT 7.10 min product from linoleic acid
1-d-old culture, and the culture was then incubated for an addi- and strain ALA2. The preparative TLC-purified fractions were
tional 2 d. At the end of incubation, the culture was acidified to subjected to GC–MS analyses. GC–MS analysis of the methy-
pH 2 with 6 N HCl, and the lipid fraction was extracted twice lated TLC fraction 13 products with Rf of 0.6–0.75 from
with 100 mL of ethyl acetate followed by 100 mL of ethyl linoleic acid and strain ALA2 showed two peaks at GCRT
ether. The solvent was evaporated from the combined extracts 15.94 and 16.12 min, corresponding to the two products, GCRT
with a rotary evaporator.
7.10 and 7.32 min, in isothermal GC analysis. MS analysis of
(i) Purification of products. To prepare samples for GC–MS the GCRT 15.94 min product showed a molecular ion of 310.
analysis, the crude extract was further purified by preparative It also had the following m/z: 279, 207 [i.e., (C14H23O3) −
TLC. The solvent was diethyl ether/acetone (4:1 vol/vol). The (OCH3)], and 164 [i.e., (C14H23O3) − (C2O) − (OCH3)]. Both
silica gel on the plate was scraped in 1-cm fractions, collected, m/z 207 and 164 are characteristic for 12,13-epoxy-9(Z)-oc-
and extracted with a mixture of methylene chloride/methanol tadecenoic acid (Fig. 1). Other m/z values matched well with
(3:1 vol/vol). Each fraction was then methylated and subjected those of the authentic 12,13-epoxy-9(Z)-octadecenoic acid
to GC–MS analyses.
(24). Therefore, this product from linoleic acid and strain
(ii) Product analyses. The crude lipid extracts were methy- ALA2 is 12,13-epoxy-9(Z)-octadecenoic acid, the initial epox-
lated with diazomethane for GC analysis. Methyl ester deriva- idation product of linoleic acid. Based on this finding, we can
tives were injected into an Agilent Technologies 6890N Net- add 12,13-epoxy-9(Z)-octadecenoic acid to our previously pub-
work GC System equipped with an FID, a Supelco (Bellefonte, lished linoleic acid bioconversion pathway (2) as shown in Fig-
PA) SPB-1 capillary column (15 m by 0.32 mm inner diame- ure 2.
ter; 0.25 mm thickness), a 7683 series auto sample injector, and
Identification of GCRT 7.32 min product from strain ALA2.
a Chem Station A.10.02 [1757]. The column temperature was The mass spectrum of the GCRT 16.12 min product (corre-
kept at 200°C isothermally. The injection and detector temper- sponding to GCRT 7.32 min product in isothermal GC analy-
atures were 240 and 250°C, respectively. The relative yield of sis) obtained from methylated TLC fraction with Rf = 0.6–0.75
products was calculated as the ratio of product peak areas. was analyzed. It had a molecular ion of 310. Characteristic m/z
GC–MS analyses were performed with a Hewlett-Packard were: 279, 168 [i.e., (C11H19O3) − (OCH3)], 153 (C14H23O),
Model 5890 gas chromatograph interfaced with a Model 5971 and 111 (C8H15) (Fig. 3). Other m/z values matched well with
mass selective detector operating at 70 eV. The capillary col- those of the authentic 9,10-epoxy-12(Z)-octadecenoic acid
umn used was a Hewlett-Packard HP-5-MS cross-linked 5% (24). Therefore, this product from linoleic acid and strain
phenyl methyl silicone, 30 m by 0.25 mm inner diameter, film ALA2 was 9,10-epoxy-12(Z)-octadecenoic acid. This result in-
thickness 0.25 µm. The carrier gas was helium and its flow rate dicated that the epoxidation enzyme system of strain ALA2 is
was 0.65 mL/min. The GC column was programmed from not position-specific, as it acts on both double bonds at posi-
190°C for 3 min and then to 220°C at a rate of 2°C /min and tions 9 and 12. However, our data could not rule out the possi-
then to 240°C at 5°C per min and then kept at 240°C for 4 min. bility of two enzymes catalyzing the epoxidation reaction at
JAOCS, Vol. 83, no. 8 (2006)

LINOLEIC ACID EXPOXIDATION PRODUCTS BY BACILLUS MEGATERIUM ALA2
679
FIG. 1. Mass spectral analysis of methylated GCRT 7.10 min product from linoleic acid by Bacillus megaterium
ALA2. GCRT, gas chromatographic retention time.
each double bond position. Since there are no further biocon-
Linoleic acid epoxidation activities of strains NRRL B-3712
version products derived from 9,10-epoxy FA detectable on and NRRL B-4219. Strains NRRL B-3712 and NRRL B-4219
GC, it is not clear why strain ALA2 produced 9,10-epoxy- also produced GCRT 7.10 and 7.32 min products from linoleic
12(Z)-octadecenoic acid. It might be a dead-end product of acid. Their crude extracts were purified by preparative TLC as
linoleic acid bioconversion.
described in the Materials and Methods section. The methy-
FIG. 2. Biosynthetic pathway of conversion of linoleic acid to its oxygenated FA by Bacillus megaterium ALA2.
JAOCS, Vol. 83, no. 8 (2006)

680
C.T. HOU
FIG. 3. Mass spectral analysis of methylated GCRT 7.32 min product from linoleic acid by Bacillus megaterium
ALA2. For abbreviation see Figure 1.
lated TLC fraction products having Rf = 0.6–7.5 were then sub-
Previously, we reported that the hydroxylation enzyme sys-
jected to GC–MS analyses. GC–MS of the GCRT 7.10 min tems of strains ALA2 and NRRL B-3712 are quite similar; both
product from both strains NRRL B-3712 and NRRL B-4219 produced ω-1, ω-2, and ω-3 hydroxy FA from palmitic acid
showed typical m/z of 310, 207, and 164 for 12,13-epoxy-9(Z)- (19). The hydroxylase of strain ALA2 was more active than
octadecenoic acid. Other m/z values also matched well with that of B-3712 and produced equal amounts of ω-1, ω-2 hy-
those of the authentic 12,13-epoxy-9(Z)-octadecenoic acid. droxy FA products whereas the strain B-4219 hydroxylase sys-
Therefore, both strains NRRL B-3712 and NRRL B-4219 also tem was less active and produced only ω-1 hydroxy FA prod-
produced 12,13-epoxy-9(Z)-octadecenoic acid as their first uct. In this paper, we again showed that the epoxidation en-
epoxidation product from linoleic acid. The GC–MS spectra of zyme systems of these three well-studied Bacillus strains were
the GCRT 7.32 min product obtained from the methylated TLC similar, but strain ALA2 was the most active one.
fraction having Rf = 0.6–0.75 from both strains NRRL B-3712
Both strains B-3712 and B-4219 could not produce oxy-
and NRRL B-4219 were analyzed. They had a molecular ion genated FA products having GCRT greater than 10 min. This
of 310. Characteristic m/z values were: 279, 168, 153, and 111, indicated that, unlike strain ALA2, both of these strains lack
which were similar to those obtained by strain ALA2. Other the enzyme systems for further conversion of the initial epoxi-
m/z values matched well with those of the authentic 9,10- dation product.
epoxy-12(Z)-octadecenoic acid (24). Therefore, the GCRT 7.32
min product from both strains NRRL B-3712 and NRRL B- ACKNOWLEDGMENTS
4219 and from linoleic acid is 9,10-epoxy-12(Z)-octadecenoic
The excellent technical assistance of Amy Swartz of NCAUR is
acid. All three Bacillus strains produced two epoxy unsaturated highly appreciated. The mention of firm names or trade products
does not imply that they are endorsed or recommended by the
FA products from linoleic acid, indicating that their epoxida-
U.S.D.A. over other firms or similar products not mentioned.
tion enzyme systems may be similar.
Comparison of linoleic acid epoxidation products among
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