Application of cyanobacteria as biocatalysts for the reduction of

science
Application of cyanobacteria as biocatalysts for
the reduction of diethyl 2-oxopropylphosphonate
Monika GÓRAK*, Ewa ŻYMAŃCZYK-DUDA – Department of Bioorganic Chemistry, Faculty
of Chemistry, Wrocław University of Technology, Wrocław, Poland
Please cite as: CHEMIK 2014, 68, 2, 123–128
Introduction
The asymmetric reduction of ketones is one of the most
important and practical reactions for producing non-racemic chiral
alcohols, which can be transformed into industrially important
chemicals such as pharmaceuticals and agrochemicals. The
reduction of ketones can be carried out by chemical and biological
methodologies. The strategy based on biological methods focuses
on non-photosynthetic and heterotrophic microorganisms or their
isolated enzymes [1].
Cyanobacteria are a diverse group of prokaryotes, widespread
and able to adapt to grow in various types of water bodies [2, 3].
These microorganisms are the oldest oxygenic photosynthetic
organisms, producing various organic compounds from inorganic
carbon source – CO2, using solar light energy.
Cyanobacteria have gained a lot of attention because of their
potential applications in biotechnology. They have been identified as
a rich source of bioactive compounds and have been considered as
one of the most promising groups of organisms to produce them
[4, 5]. These cyanobacterial metabolites include antibacterial [6],
antifungal [7], antiviral [8], anticancer [9] and immunosuppressive
agents [10] as well as algicide [11].
In the past few years, many research groups examined
the possibility of applying of autotrophic microorganisms as
a biocatalyst in the reduction of aldehydes and ketones [12].
For example, the cyanobacterium Synechococcus sp. was found
to reduce limonene [13], enones [14], acetophenone and its
halogenated derivatives [15] to the corresponding alcohols with
high stereoselectivity.
The aim of the study was to examine which strains of
cyanobacteria are capable to reduce diethyl β- oxoalkylphosphonates.
This paper reports for the first time, effective use of phototrophic
microorganisms as a potential biocatalyst for the production of
chiral 2-hydroxyalkylphosphonate esters. The applying of bluegreen algae for reduction is relatively rare due to the difficulty in
their cultivation and the lack of knowledge on the stereochemical
control of reactions.
Experimental part
Material and methods
Substrate – diethyl 2-oxopropylphosphonate – was synthesized
according to standard procedure described in literature [16].
Microorganisms
Arthrospira maxima (CCALA 27), Geitlerinema sp. (CCALA 138),
Leptolyngbya foveolarum (CCALA 76), Nodularia sphaerocarpa (CCALA
114), Nostoc cf-muscorum (CCALA 129), Synechococcus bigranulatus
(CCALA 187) were purchased from the Culture Collection of
Autotrophic Organisms (CCALA) of the Institute of Botany of the
Academy of Sciences of the Czech Republic.
Corresponding author:
Monika GÓRAK – M.Sc., e-mail: [email protected]
126 •
Culture conditions
All species were cultivated in 250 ml Erlenmeyer flasks containing
100 ml of a suitable medium. Geitlerinema sp., Leptolyngbya foveolarum,
Nodularia sphaerocarpa and Synechococcus bigranulatus were grown
in BG-11 medium [17], Nostoc cf-muscorum was grown in BG-11
medium without NaNO3 and Arthrospira maxima – in Spirulina medium
[18]. All species of cyanobacteria were cultivated under continuous
illumination provided by fluorescent lamp (Power-Glo, 8W, Hagen) at
25°C under stationary conditions. The experiments were conducted
under sterile conditions.
Biotransformation – general procedure
After pre-cultivation for about 3 weeks, 1mM of diethyl
2-oxopropylphosphonate (20µL) was added to the axenic culture
of cyanobacteria. The bioconversion process was carried out for
7 days, under continuous illumination at 25°C under stationary
conditions. Experiments were completed by the biomass removing
by centrifugation (4500 rpm, 20 min) and supernatant was extracted
twice with ethyl acetate. Subsequently, organic layer was dried over
anhydrous MgSO4 and evaporated under reduced pressure (Rotary
evaporators IKA® RV10digital).
The control experiment was carried out in culture medium
without cells of cyanobacteria. The experiment was conducted and
completed, as in the case of biotransformations described above. In
this case, the lack of the reaction was observed.
The mixture of substrate and product – diethyl 2-hydroxypropylphosphonate was analyzed by nuclear magnetic resonance spectroscopy: diethyl 2- oxopropylphosphonate (31P NMR) δ 20.34 ppm;
diethyl 2- hydroxypropylphosphonate (31P NMR) δ 30.60 ppm.
NMR spectra were recorded on Bruker Avance DRX300 instrument operating at 300 MHz, measurements were made in CDCl3
(99.5 at % D) at temperature of 298K.
Optical purity of the products was determined by means of
31
P NMR spectroscopy with the addition of quinine as a chiral
discriminator [19].
Results
This paper presents, effective use of phototrophic
microorganisms as a potential biocatalyst for the production of
β- hydroxyalkylphosphonate esters. The research was preceded
by the screening, which allowed identifying the strains of blue-green
algae having the potential to reduce oxophosphonates.
Fig. 1. Microbial bioconversion of diethyl 2- oxopropylphosphonate
to diethyl 2-hydroxypropylphosphonate
The applying of blue-green algae for reduction is relatively
rare due to the difficulty in their cultivation and maintaining axenic
cultures of cyanobacteria. The presence of unwanted organisms can
lead to incorrect or conflicting results and conclusion. Therefore, it
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Fig. 2. 31P NMR spectra (with quinine; δ 20.34 ppm diethyl 2- oxopropylphosphonate and δ 30.60 ppm diethyl 2- hydroxypropylphosphonate) for biotransformation catalyzed by Nodularia sphaerocarpa
Among the tested blue- green algae, only the applying
of Arthrospira maxima and Nodularia sphaerocarpa cultures in
bioconversion of diethyl 2-oxopropylphosphonate allowed
obtaining the corresponding diethyl 2- hydroxypropylphosphonate,
within 7 days. The degree of conversion of the substrate (calculated
based on 31 P NMR spectra) was respectively 26.4% and 12.9%, and
the optical purity of the product in both cases was over 99%.
In the case of the other strains the desired product was not
observed. It could arise from the low photosynthetic activity of
strains under experimental conditions. Leptolyngbya foveolarum and
Geitlerinema sp. are mat- forming cyanobacteria – the biomass
of cells fall to the bottom. That may affect the limited availability
of substrate for the cells of these strains of cyanobacteria under
stationary conditions.
Summary and Conclusion
Screening of cyanobacteria shown that among the tested,
morphologically different strains, only filamentous strains of
Arthrospira maxima and Nodularia sphaerocarpa are efficient
biocatalysts in reduction of chosen substrate. Appropriate culture
and bioconversion conditions allowed obtaining the desired
product. The degree of conversion of the substrate was 26.4%
when Arthrospira maxima were applied, for biotransformation
process carried out for 7 days. The lower value of the degree of
conversion of the substrate (12.9%) was obtained in the case of
nr 2/2014 • tom 68
applying of Nodularia sphaerocarpa strain. Both cyanobacteria of
the genus Nodularia and Arthrospira are microorganisms inhabiting
different environments. Similar to other strains of cyanobacteria
occurred worldwide, they have developed a mechanism for survival
under conditions of limited nutrient availability.
Application of mat- forming strains – Leptolyngbya foveolarum and
Geitlerinema sp., did not allowed obtaining the desired product.
Screening of microorganisms allowed identifying the potential
cyanobacterial strains capable to reduce β-oxoalkilphosphonates.
Furthermore, the using of photosynthetic organisms in the
biotransformation process has expanded the knowledge of the
application of this group of microorganisms in the process of receiving
β-hydroxyalkilphosphonates.
Acknowledgement
This work was financed form Project „Biotransformation for pharmaceutical
and cosmetics industry” no POIG.01.03.01–00–158/09–07 part – financed
by the European Union within the European Regional Development Fund for
the Innovative Economy.
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• 127
science
is important to work with axenic cultures of cyanobacteria. In this
study were used axenic cultures of cyanobacterial strains, what was
confirmed on agar- solidified BG-11 medium.
Axenic, morphologically different strains of cyanobacteria: unicellular photoautotrophic Synechococcus bigranulatus, heterocystous photoheterotrophic cyanobacterium Nostoc cf-muscorum and
filamentous strains of Nodularia sphaerocarpa, Arthrospira maxima,
Leptolyngbya foveolarum, Geitlerinema sp. have been used in the
screening. Cyanobacteria of the genus Synechococcus and Nostoc
were also tested, since literature data indicate that these strains are
capable to reduce ketones [15, 20].
An important factor influencing the reaction is light intensity,
therefore during cultivation and bioconversion continuous
illumination was used. The reduction of exogenously added
ketones in cyanobacterial cells is dependent upon photosynthetic
activity [21]. Moreover, literature data indicate that the enzymes
involved in the cyanobacterial reduction reaction are NADPH –
dependent. The reduced form of nicotinamide adenine dinucleotide
phosphate – NADPH, generated during photosynthesis can be
used for the reduction of exogenous ketones to produce the
corresponding alcohols [21].
science
16. Ryglowski A., Kafarski P.: Preparation of 1-aminoalkylphosphonic acids
and 2-aminoalkylphosphonic acids by Reductive amination of oxoalkylphosphonates Tetrahedron 1996, 52, 10685.
17. Rippka R., Deruelles J., Waterbury J.B., Herdman M., Stainer R.Y.: Generic assignments, strain histories and properties of pure cultures of
cyanolobacteria. Journal of General Microbiology 1979, 111, 1.
18. Aiba S., Ogawa T.: Assessment of growth yield of a blue-green alga
Spirulina platensis in axenic and continuous culture. Journal of General
Microbiology 1977, 1, 179.
19. Żymańczyk-Duda E.; Skwarczyński M.; Lejczak B.; Kafarski P.: Accurate
assay of enantiopurity of 1-hydroxy- and 2-hydroxyalkylphosphonate
esters. Tetrahedron: Asymmetry 1996, 7, 1277.
20. Havel J. Weuster-Botz D.: Cofactor regeneration in phototrophic cyanobacteria applied for asymmetric reduction of ketones. Applied Microbiology and Biotechnology 2007, 75, 1031.
21. Yamanaka R., Nakamura K., Murakami A.: Reduction of exogenous ketones depends upon NADPH generated photosynthetically in
cells of the cyanobacterium Synechococcus PCC 7942. AMB Express
2011, 1, 24.
Ewa ŻYMAŃCZYK-DUDA – Sc.D., (Eng), graduated from the Faculty
of Chemistry, Wrocław University of Technology. She currently works for
Department of Bioorganic Chemistry. Research interests: the application
of biocatalysts – whole microbial cells as well as pure enzymes for
bioconversion of xenobiotics into the chiral products of define absolute
configuration; biocatalyzed synthesis of organophosphorus compounds
with one or two stereogenic center.
* Monika GÓRAK – M.Sc., graduated from the Faculty of Chemistry,
Wrocław University of Technology (2010). She is a PhD student at the
Department of Bioorganic Chemistry of the Faculty of Chemistry. Research
interests: the application of cyanobacteria as biocatalysts for the reduction
of oxoalkilphosphonates.
e-mail: [email protected], phone: +48 71 320 46 14
Translation into English by the Author
Z prasy światowej – innowacje: odkrycia, produkty
i technologie
From the world press – innovation: discoveries, products and technologies
dokończenie ze strony 125
Oznaczanie kortykosteroidów w kosmetykach
Kortykosteroidy znane są jako bardzo skuteczne leki szeroko stosowane w leczeniu chorób zapalnych. W dermatologii stosowane są
do łagodzenia objawów zapalnych chorób skórnych, takich jak łuszczyca i egzemy, są również szeroko stosowane w leczeniu bólu stawów i stanów zapalnych. Preparaty zawierające steroidy (walerianian
betametazonu, dipropionian betametazonu, aceton idu triamcinolonu)
są dostępne w postaci kremów, żeli i maści, inne steroidy (dipropionian betametazonu, budezonid) są podawane jako roztwory do inhalacji. Poprzez podawanie doustne, steroidy (prednizon, prednizolon)
można stosować do leczenia zapalenia wątroby, tocznia rumieniowatego i choroby zapalnej jelit. Z drugiej strony, stosowanie kortykosteroidów bez kontroli lekarskiej może mieć poważne skutki uboczne,
takie jak atrofia skóry i łuszczyca. Dlatego też kortykosteroidy są
zakazane w kosmetykach. Obecność na rynku podrabianych produktów, zawierających kortykosteroidy, stanowi zagrożenie dla zdrowia
konsumentów. Ze względu na obawy zaistnienia skutków ubocznych
stosowania kortykosteroidów, chemia analityczna ma decydujące znaczenie w kwestii bezpieczeństwa kosmetyków poprzez kontrolę jakości surowców i gotowych preparatów farmaceutycznych. Naukowcy
z Uniwersytetu Bolońskiego na łamach „Journal of Pharmaceutical and
Biomedical Analysis” opisują innowacyjną metodę do równoczesnego
oznaczania sześciu kortykosteroidów (walerianianu betametazonu,
beklometazonu, dipropionian beklometazonu, metyloprednizolonu,
budezonidu, flunizolidu), opartą na spektrometrii mas i chromatografii
cieczowej. Opisana metoda pozwala na badanie próbek w postaci kremów, proszków, roztworów i tabletek oraz charakteryzuje się dobrą
precyzją i dokładnością. (kk)
(Jessica Fiori, Vincenza Andrisano: LC–MS method for the simultaneous determination of sixglucocorticoids in pharmaceutical formulations and counterfeitcosmetic
products, Journal of Pharmaceutical and Biomedical Analysis 91 (2014) 185– 192)
Syntetyczne pochodne ADEP dużo silniejsze w działaniu
Naukowcy opracowali syntetyczne analogi naturalnie występujących, nowych antybiotyków – acyldepsypeptydów ADEP. Otrzymali
tym samym potencjalne leki o bardzo dużej sile bakteriobójczej, nawet
na oporne szczepy bakteryjne. Ich mechanizm działania jest zupełnie
inny niż obecnie dostępnych na rynku leków przeciwbakteryjnych.
Acydepsypeptydy ADEP zabijają bakterie poprzez wiązanie z białkami
w komórce bakteryjnej, które działają jako „komórkowy śmietnik”.
Te struktury w kształcie „beczki” zwane ClpP pełnią rolę niszczarek
nieprawidłowo uformowanych, uszkodzonych lub zniszczonych białek. ADEP po związaniu z ClpP upośledzają selektywność tych struktur
w usuwaniu nieprawidłowych białek. W efekcie z komórki usuwane są
zarówno „śmieci”, jak i zdrowe komórki. Wówczas dla bakterii ścieżka
usuwania ClpP staje się zabójcza.
Zespół profesora Jasona Sello z Brown University and the
Massachusetts Institute of Technology zsyntetyzował kilka nowych
cząsteczek ADEP. Zamienili oni niektóre aminokwasy w naturalnie występującej cząsteczce ADEP na takie, które mogą zwiększyć
sztywność cząsteczki. Naukowcy następnie sprawdzili zdolność wiązania się nowych cząsteczek z ClpP. Eksperymenty in vitro pokazały,
że siła wiązania syntetycznych analogów jest siedem razy większa
niż naturalnych ADEP. Dodatkowo efekt ten jest osiągany już przy
niewielkich stężeniach. Co więcej, test toksyczności na komórki
bakteryjne wykazał, że nowe ADEP mają dużo większy potencjał.
Niemal 32 razy silniej działają na Staphylococcus aureus, 600 razy
silniej na Enterococcus faecalis i 1200 razy silniej na Streptococcus
pneumonie. Zespół naukowców zachęcony wynikami pracuje dalej,
aby rozwinąć nową generację leków przeciwbakteryjnych ADEP.
Kolejny krok, to eksperymenty, które pokażą jak badane związki
działają in vivo na myszach. (kk)
(http://biotechnologia.pl, 28.01.2014)
dokończenie na stronie 159
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