ABSTRAK EKSPLORASI POTENSI DAN PENGEMBANGAN … · ektoin dari mikroba potensial. Pengembangan...
Transcript of ABSTRAK EKSPLORASI POTENSI DAN PENGEMBANGAN … · ektoin dari mikroba potensial. Pengembangan...
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ABSTRAK
EKSPLORASI POTENSI DAN PENGEMBANGAN PRODUKSI
EKTOIN DARI BAKTERI HALOFILIK ISOLAT KAWAH
LUMPUR ”BLEDUG KUWU” PURWODADI JAWA TENGAH
Oleh
I Putu Parwata
NIM: 30514010
(Program Studi Doktor Kimia)
Ektoin (asam 1,4,5,6-tetrahidro-2-metil-4-pirimidin karboksilat) adalah molekul
organik kompatibel yang banyak dimanfaatkan dalam berbagai bidang seperti
farmasi, kosmetika, serta aplikasi bioteknologi. Molekul ini dapat melindungi sel
serta biomolekul seperti protein dan membran sel dari berbagai stres lingkungan
seperti tekanan osmosis, pemanasan, pembekuan, kekeringan, sinar UV, atau
kontak dengan bahan toksik. Seiring meningkatnya permintaan pasar terhadap
molekul aktif ini, berbagai upaya telah dilakukan untuk meningkatkan produksi
ektoin dari mikroba potensial. Pengembangan produksi ektoin dilakukan baik dari
mikroba yang secara alami mampu menghasilkan ektoin (wild type) maupun dari
mikroba yang tidak mampu memproduksi molekul ini dengan menerapkan teknik
rekayasa genetika. Selain itu, eksplorasi mikroba yang memiliki potensi unggul
dalam produksi ektoin juga menjadi kecenderungan penelitian dewasa ini. Salah
satu mikroba potensial penghasil ektoin adalah bakteri halofilik, kelompok
ekstrimofilik yang dapat hidup di dalam lingkungan dengan kadar garam tinggi.
Produksi ektoin oleh bakteri ini adalah sebagai salah satu strategi perlindungan sel
terhadap tekanan osmosis yang disebabkan oleh kadar garam tinggi di dalam
habitatnya.
Beberapa bakteri halofilik telah diisolasi dari sampel air garam yang diperoleh
dari Kawah Lumpur ―Bledug Kuwu‖ yang berlokasi di Desa Kuwu, Kecamatan
Kradenan, Kabupaten Grobogan, Provinsi Jawa Tengah. Hasil penelitian
pendahuluan menunjukkan bakteri-bakteri halofilik yang diperoleh memiliki
toleransi yang baik terhadap kadar garam (0,5-30% [b/v]). Karakteristik ini sangat
mendukung untuk produksi ektoin. Untuk itu, penelitian ini dirancang untuk
mengeksplorasi lebih lanjut potensi bakteri halofilik yang telah diperoleh dalam
produksi ektoin. Penelitian dilakukan dalam empat tahap, pertama dilakukan
seleksi bakteri penghasil ektoin berdasarkan toleransinya terhadap kadar garam
tinggi, profil pertumbuhan, dan produksi ektoin per sel kering. Tahap kedua
dilakukan optimalisasi produksi ektoin dari bakteri halofilik terbaik yang diperoleh dari tahap pertama. Pada tahap ketiga dilakukan produksi ektoin dari E.
coli rekombinan yang membawa kelompok gen biosintesis ektoin (ectABC) yang
diisolasi dari bakteri halofilik terbaik. Produksi ektoin oleh sel rekombinan
kemudian dioptimalisasi. Pada tahap akhir dilakukan uji aplikasi ektoin sebagai
penstabil lipase terhadap pemanasan, metanol, dan garam.
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Hasil uji potensi menunjukkan lima bakteri halofilik, yaitu: isolat BK-AB12, BK-
AG13, BK-AB18, BK-AG18, dan BK-AG25 mampu memproduksi ektoin dengan
produktivitas masing-masing sebesar 33,65; 8,21; 10,31; 9,71; dan 61,73 mg
ektoin/g sel kering. Berdasarkan hasil tersebut, isolat BK-AG25 adalah bakteri
penghasil ektoin terbaik dan dipilih untuk tahap selanjutnya. Hasil identifikasi
menggunakan urutan gen 16s rRNA menunjukkan isolat BK-AG25 memiliki
kedekatan homologi dengan Halomonas elongata. Untuk itu, isolat ini selanjutnya
diberi nama Halomonas elongata BK-AG25.
Optimalisasi produksi ektoin dari H.elongata BK-AG25 dilakukan melalui
kultivasi dua tahap, pertama untuk meningkatkan kadar biomassa (densitas sel)
dan kedua untuk meningkatkan produksi ektoin. Optimalisasi produksi biomassa
pada kultivasi pertama dilakukan dengan response surface methodology (RSM)
terhadap faktor-faktor: kadar glukosa, (NH4)2SO4, MgSO4, NaCl dan temperatur
inkubasi. Hasil percobaan menghasilkan model regresi yang menunjukkan bahwa
produksi biomassa optimal diperoleh jika bakteri diinkubasi pada temperatur 37,4
°C di dalam media MM63 dengan kadar NaCl, glukosa, (NH4)2SO4 dan MgSO4
masing-masing sebesar 8,9% [b/v]; 1,1% [b/v]; 0,37% [b/v]; dan 0,04% [b/v].
Kultivasi bakteri pada kondisi tersebut menghasilkan biomassa dengan kadar
sebesar 4,92 + 0,028 mg/mL
Pada kultivasi kedua, H. elongata BK-AG25 diinokulasi pada media MM63 yang
telah dioptimalisasi tapi mengandung kadar NaCl yang lebih tinggi untuk
menstimulasi biosintesis ektoin. Optimalisasi produksi ektoin pada kultivasi
kedua dilakukan dengan RSM terhadap dua parameter, yaitu kadar NaCl dan
temperatur inkubasi. Berdasarkan model regresi yang diperoleh dari data
percobaan, bakteri diprediksi menghasilkan ektoin dengan kadar dan produktivitas
optimum pada kadar NaCl 18% [b/v] dan temperatur 33 °C. Hasil percobaan pada
kondisi tersebut mampu menghasilkan ektoin dengan kadar 1,17 + 0,015 g/L dan
produktivitas sebesar 179,9 + 8,52 mg ektoin/g sel kering. Selanjutnya,
optimalisasi kadar glukosa dan waktu inkubasi berhasil meningkatkan produksi
ektoin dari bakteri menjadi 1,57 g/L dengan produktivitas sebesar 269 mg
ektoin/g sel kering pada kadar glukosa optimum sebesar 0,8% [b/v] dan waktu
inkubasi 35 jam.
Setelah optimalisasi kondisi untuk biosintesis ektoin dari H. elongata BK-AG25,
langkah selanjutnya adalah optimalisasi ekstraksi ektoin menggunakan teknik
osmotic shock dan ―bacterial milking‖. Bakteri ditumbuhkan dengan teknik
kultivasi dua tahap yang telah dioptimalisasi sebelumnya. Sel bakteri kemudian
dipindahkan secara aseptik ke dalam air steril yang mengandung garam dengan
kadar rendah (osmotic downshock) untuk menstimulasi ekskresi ektoin. Dengan
teknik ini, sekitar 80% ektoin diekskresikan ke luar sel. Ketahanan sel bakteri
setelah proses osmotic downshock menggunakan air dengan kadar NaCl 1,5% dan
3% cukup tinggi (di atas 70%), namun pada kadar NaCl 0% bakteri hanya mampu
mempertahankan sekitar 9% selnya. Setelah proses osmotic downshock, sel
bakteri diinokulasi kembali di dalam media MM63 yang mengandung kadar
garam tinggi (osmotic upshock) untuk sintesis ektoin. Proses osmotic upshock dan
osmotic downshock diulang beberapa kali untuk menghasilkan lebih banyak
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ektoin, dikenal dengan nama ―bacterial milking‖. Data penelitian menunjukkan
empat siklus ―bacterial milking‖ dari bakteri mampu menghasilkan total ektoin
sekitar 2,26 g/L.
Selain dari kultur alami H. elongata BK-AG25, produksi ektoin juga
dikembangkan menggunakan bakteri non halofilik rekombinan yang membawa
kelompok gen yang mengkode enzim-enzim yang terlibat dalam biosintesis
ektoin. Kelompok gen biosintesis ektoin (ectABC) dari H. elongata BK-AG25
telah berhasil diamplifikasi dengan panjang total 2.438 pasang basa (pb), terdiri
dari gen ectA (579 pb), ectB (1.266 pb) dan ectC (414 pb) yang tersusun dalam
satu operon. Operon disisipkan ke dalam vektor ekspresi pET30a(+) dan
ditransformasi ke dalam sel E. coli BL21 (DE3). Sel rekombinan ditumbuhkan di
dalam media LB dan ekspresi operon diinduksi dengan IPTG. Ketiga gen dalam
operon ectABC berhasil diekspresikan oleh sel rekombinan, dua gen (ectA dan
ectB) diekspresikan secara kuat, sementara ectC terekspresi secara lemah.
Produksi ektoin dari sel rekombinan kemudian diuji menggunakan media MM63.
Hasil uji menunjukkan E. coli rekombinan mampu memproduksi ektoin dan
sebagian besar (> 70%) diekskresikan ke luar sel. Inkubasi sel rekombinan selama
14 jam setelah induksi mampu menghasilkan ektoin ekstraseluler sebanyak 0,23
g/L dengan produktivitas sebesar 69 mg ektoin/g sel kering. Ini merupakan
laporan pertama tentang ekspresi kelompok gen biosintesis ektoin dari Halomonas
elongata di bawah kontrol promotor T7 pada sel E. coli BL21.
Produksi ektoin dari E. coli rekombinan selanjutnya dioptimalisasi. Optimalisasi
kadar glukosa dan NaCl di dalam media MM63 serta temperatur inkubasi
menghasilkan model regresi untuk kadar ektoin intraseluler dan ekstraseluler yang
dihasilkan oleh sel rekombinan. Model regresi menunjukkan kadar ektoin
ekstraseluler optimal diperoleh pada kadar glukosa 0,92% [b/v], kadar NaCl
0,28% [b/v], dan temperatur inkubasi 34 °C. Hasil percobaan pada kondisi
tersebut menghasilkan ektoin ekstraseluler dengan kadar sebesar 0,37 + 0,027 g/L.
Sementara itu, ektoin intraseluler dengan kadar sebesar 0,05 + 0,005 g/L
dihasilkan oleh sel rekombinan pada kadar NaCl 1,78% [b/v] dan temperatur 32
°C. Optimalisasi produksi ektoin dari E. coli rekombinan juga dilakukan pada
nilai OD kultur sebelum induksi dan konsentrasi akhir senyawa penginduksi
(IPTG), menghasilkan model regresi untuk kadar ektoin ekstraseluler dan
produktivitas bakteri. Hasil prediksi model regresi menunjukkan sel rekombinan
menghasilkan ektoin ekstraseluler optimal pada OD awal kultur 0,74 dan
konsentrasi akhir IPTG 0,62 mM. Selain itu, produktivitas sel rekombinan terbaik
diperoleh pada OD awal kultur sebesar 0,3 dan konsentrasi akhir IPTG 1,5 mM.
Pada kondisi optimum tersebut, sel rekombinan mampu menghasilkan ektoin
ekstraseluler dengan kadar 0,71 + 0,03 g/L dan produktivitas bakteri sebesar 376
+ 2,3 mg ektoin/g sel kering. Selanjutnya, optimalisasi waktu inkubasi berhasil
meningkatkan kadar ektoin ekstraseluler yang dihasilkan oleh sel rekombinan
menjadi 0,75 g/L dengan produktivitas sebesar 418 mg ektoin/g sel kering setelah
diinkubasi selama 12 jam.
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Ektoin yang dihasilkan oleh H. elongata BK-AG25 diaplikasikan sebagai
penstabil lipase. Hasil percobaan menunjukkan ektoin mampu mempertahankan
bahkan meningkatkan aktivitas katalitik lipase setelah dipanaskan atau diinkubasi
di dalam pelarut metanol. Penambahan 60-150 mM ektoin mampu meningkatkan
aktivitas lipase hingga 20% setelah dipanaskan selama 1 jam pada temperatur di
bawah 80 °C. Sementara itu, aktivitas lipase yang ditambahkan 40-125 mM ektoin
juga berhasil ditingkatkan hingga 50% setelah diinkubasi selama 1 jam di dalam
metanol dengan kadar hingga 78% [v/v].
Kata-kata kunci: ektoin, bakteri halofilik, Halomonas elongata, Kawah Lumpur
―Bledug Kuwu‖, response surface methodology, penstabil lipase
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ABSTRACT
EXPLORATION AND DEVELOPMENT FOR ECTOINE
PRODUCTION BY HALOPHILIC BACTERIA ISOLATED
FROM THE MUD CRATER OF ”BLEDUG KUWU”
PURWODADI, CENTRAL JAVA
By
I Putu Parwata
NIM: 30514010
(Program Studi Doktor Kimia)
Ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidine carboxylic acid) is compatible
organic molecules widely used in many fields, such as pharmaceuticals,
cosmetics, and biotechnological applications. This molecule can protect cell’s
biomolecules, such as proteins and cell membrane, from various environmental
stresses like osmotic pressure, heating, freezing, dryness, UV rays, or contact with
toxic materials. An increase of commercial demand of ectoine as an active
biocompound has led a number of effort to improve the production of this
molecule from microorganisms. The development of ectoine production is carried
out both from microbes that are naturally capable of producing ectoine as well as
from those that are unable to produce ectoine by applying genetic engineering
techniques. In addition, exploration of microbes that have superior ability to
produce ectoine become one of the research trends today. One of the potential
ectoine-producing bacteria is halophile, which is the class of extremophile that
favor to live in hypersaline environment. The aim of ectoine production by this
type of bacteria is as one of the protection strategies against high osmotic pressure
exerted by hypersaline environment in order to prevent difusion of liquid out of
the cell.
Several local halophilic bacteria have been isolated from brine samples obtained
from the mud crater of "Bledug Kuwu" located at Kuwu Village, Kradenan
District, Grobogan Regency, Central Java. The results of the preliminary study
showed that the halophilic bacteria exhibited relatively high tolerance to salt
levels (0.5-30% [w/v]), which is the important characteristic for ectoine producing
bacteria. The present study is aimed to further explore the potency of those local
halophilic bacteria in producing ectoine. This study was conducted in four steps:
the first one is screening the potential ectoine-producing bacteria in terms of their
tolerance against high level of salt, the growth and ectoine yield per dry cell
weight. The second step was the optimization of ectoine production yield from the
best halophilic bacterium obtained from the first step. The third step was
producing ectoine from recombinant E. coli, which has been transformed to carry
the ectoine gene cluster (ectABC) isolated from the best halophilic bacterium. The
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ectoine production by the recombinant cell was subjected to the optimization. The
last step was application of ectoine as a stabilizer for protein.
The results of the ectoine production test of five halophilic bacteria BK-AB12,
BK-AG13, BK-AB18, BK-AG18, and BK-AG25 gave the respective yield of 33,
65; 8,21; 10.31; 9.71; and 61.73 mg/g cdw. The highest ectoine yield was thus
produced by BK-AG25 isolate and hence it was selected for further study. The
identification of the bacterial isolate by ribotyping method was phylogenetically
closest to Halomonas elongata. Afterwards, it was labeled as Halomonas
elongata BK-AG25.
The optimization of ectoine production by H. elongata BK-AG25 was conducted
by two-stage cultivation method. The first cultivation stage was intended to
increase the yield of bacterial biomass (cell density), while the second one was
aimed to enhance ectoine production yield. There were four parameters, i.e. the
levels of glucose, (NH4)2SO4, MgSO4 and NaCl as well as the incubation
temperatures, which were optimized by response surface methodology (RSM) in
the first cultivation stage. The result of optimization gave the regression model
suggesting that the highest yield of biomass can be achieved when H. elongata
BK-AG25 is cultivated at 37.4 °C in MM63 medium containing 8.9% [w/v] NaCl,
1.1% [w/v] glucose, 0.37% [w/v] (NH4)2SO4 and 0.04% [w/v] MgSO4. The
bacterial cultivation at such conditions gave biomass about 4.92 0.028 mg/mL.
In the second stage of cultivation, H. elongata BK-AG25 was inoculated in the
optimized MM63 medium but containing higher NaCl concentration to stimulate
ectoine biosynthesis. The production yield of ectoine was optimized by RSM,
which targeted NaCl concentration and temperature of cultivation as optimization
parameters. The obtained regression model suggest the highest yield of ectoine
production by H. elongata BK-AG25 can be achieved when it is cultivaled in
MM63 medium containing 18% [w/v] NaCl at 33 °C. At these conditions, the
yield of ectoine production was about 1.17 0.015 g/L and the bacterial
productivity was about 179.9 8.52 mg ectoine/g cdw (cell dry weight). The
production yield was further optimized by targeting glucose concentration and
incubation time. The optimization successfully improved the yield of ectoine
production up to 1.57 g/L with a bacterial productivity of about 269 mg ectoine/g
cdw when the cultivation was conducated at glucose concentration of 0.8% [w/v]
and an incubation time about 35 hours.
After the condition of ectoine biosynthesis by H. elongata BK-AG25 was
optimized, the next stage is to optimize ectoine extraction using the osmotic shock
process and the "bacterial milking". The bacterial inoculum was grown by two-
stage cultivation optimized above. The bacterial culture was then transferred
aseptically into sterile water containing a lower level of salt to stimulate osmotic
downshock in order to drive ectoine excretion. By this way about 80% of ectoine
was excreted out of the cell. The survival rate of bacteria was relatively high,
which was about 70%. One trial of osmotic downshock exerted by pure distilled
(0% NaCl) siginificantly lowered the survival rate of the bacterial cell to only
about 9%. After osmotic downshock, the cells were reinoculated in fress MM63
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medium containing high level of salt (osmotic upshock) to stimulate ectoine
biosynthesis. The osmotic upshock and downshock were repeated several times to
produce high level of ectoine. Such proccess is known as ―bacterial milking‖. Our
experimental data showed that four cycles of "bacterial milking" by H. elongata
BK-AG25 were successfully produced about 2.26 g/L of ectoine.
Besides producing ectoine by the wild type H. elongata BK-AG25, in this study,
ectoine was also produced by recombinant cell of nonhalophile bacteria carrying
genes cluster encoding enzymes involved in ectoine biosynthesis. The ectoine
gene cluster of H. elongata BK-AG25 (ectABC) was successfully amplified with a
total length of 2,438 base pairs (bp), consisting of ectA (579 bp), ectB (1,266 bp)
and ectC (414 bp), which were constructed as one operon. The operon was
inserted into the expression vector pET30a(+) and was transfered into E. coli
BL21 (DE3). The recombinant cell was then grown in LB medium and the
expression of the operon was induced by IPTG. The expression results showed
that all genes in ectABC operon were successfully expressed by the recombinant
cells but in different levels, in which ectA and ectB genes were strongly expressed,
while ectC was weakly expressed. The production of ectoine by the recombinant
E. coli was than tested using MM63 medium. The results showed that the
recombinant E. coli was able to produce ectoine and most of them (> 70%) were
excreted in to the medium. Incubation of the recombinant cell for 14 hours after
induction using IPTG enabled the bacteria produced around 0.23 g/L extracellular
ectoine with the productivity of about 69 mg ectoine/g cdw. Up to present, this is
the first report on the expression of the ectoine gene cluster of Halomonas
elongata under the control of T7 promoter in E. coli BL21.
Ectoine production by the recombinant E. coli was then optimized. Optimization
of the levels of glucose and NaCl in MM63 medium as well as the incubation
temperature produced a regression model for the concentration of intracellular and
extracellular ectoine produced by the recombinant. The regression model
suggested an optimal extracellular ectoine production by the recombinant E. coli
at glucose level of 0.92% [w/v] and NaCl level of 0.28% [w/v] at 34 °C. The
experimental results at these conditions yield about 0.37 + 0.027 g/L extracellular
ectoine. Meanwhile, about 0.05 + 0.005 g/L of intracellular ectoine was produced
by the recombinant cell at NaCl level of 1.78% [w/v] and 32 °C. Further
optimization was targeting optical density and IPTG concentration, resulted
regression model for the concentration of extracellular ectoine and the bacterial
produtivity. The regression model predicted that the optimum concentration of
extracellular ectoine produced by the recombinant cells at the initial OD value of
0.74 and the final concentration of IPTG of 0.62 mM. In addition, the optimum
bacterial productivity was predicted at the initial OD of 0.3 and the final
concentration of IPTG of 1.5 mM. The experimental results at these optimum
conditions were enable the bacteria to produce about 0.71 0.03 g/L of
extracellular ectoine with the bacterial productivity of 376 2.3 mg ectoine/g cdw. Furthermore, the production of ectoine from the recombinant E. coli against
the incubation time showed that the optimum ectoine concentration of 0.75 g/L
with the bacterial productivity of 418 mg ectoine/g cdw were produced by the
bacteria after 12 hours of incubation.
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Ectoine produced by H. elongata BK-AG25 was applied as a stabilizer of lipase.
The experimental results showed that ectoine was able to maintain and increase
the catalytic activity of lipase against deleterious effect of high temperature and
methanol. Addition of 60-150 mM ectoine could increase lipase activity up to
20% after heating for 1 hour at temperature below 80 °C. Meanwhile, the activity
of lipase containing 40-125 mM ectoine was succesfully increased up to 50% after
incubated for 1 hour in methanol with the level up to 78% [v/v].
Keywords: ectoine, halophilic bacteria, Halomonas elongata, the mud crater of
―Bledug Kuwu‖, response surface methodology, lipase stabilizer.