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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.