Seleksi dan Karakterisasi Enzim Kitinase dari Bakteri Pengendali Fusarium proliferatum
Date
2023Author
Khairah, Miftahul
Mubarik, Nisa Rachmania
Manaf, Lisdar A
Metadata
Show full item recordAbstract
Tanaman bawang merah merupakan tanaman yang memiliki nilai nutrisi dan ekonomi yang penting bagi masyarakat. Produksi bawang merah terkendala oleh serangan cendawan patogen salah satunya Fusarium proliferatum. Cendawan Fusarium proliferatum menyebabkan penyakit layu dan busuk pada bawang merah. Upaya pengendalian terhadap cendawan ini umumnya menggunakan pestisida, namun penggunaan psetisida yang berlebihan akan mengakibatkan kerusakan lingkungan, resistensi patogen dan berdampak negatif terhadap kesehatan manusia. Pengendalian hayati yang dilakukan dengan memanfaatkan agen bakteri endofit. Bakteri endofit dapat menghasilkan enzim hidrolitik seperti kitinase yang bermanfaat sebagai biokontrol cendawan patogen, termasuk F. proliferatum. Penelitian bertujuan menyeleksi, mencirikan karakter enzim, dan melakukan purifikasi parsial enzim kitinase dari bakteri endofit asal bawang merah sebagai pengendali cendawan patogen F. proliferatum.
Penelitian diawali dengan menapiskan empat isolat bakteri yaitu ABS 4.1.2, ABP 5.2.2, ABS 5.1 dan BBP 5.21 yang diperoleh dari penelitian sebelumnya. Penapisan dilakukan dengan menguji kemampuan bakteri tersebut dalam menghambat pertumbuhan F. proliferatum secara in vitro dengan metode dual culture dan penentuan aktivitas enzim kitinase. Isolat yang menunjukkan penghambatan terbesar dan aktivitas kitinase yang tinggi ditetapkan sebagai isolat terpilih dan diidentifikasi berdasarkan gen penyandi 16S rRNA. Isolat bakteri terpilih lalu dikultur pada media produksi dan enzim yang dihasilkan diendapkan menggunakan ammonium sulfat dan diukur aktivitas optimum kitinase berdasarkan pH dan suhu. Bobot molekul kitinase dianalisis dengan eletroforesis SDS-PAGE. Kultur sel, enzim ekstrak kasar dan enzim hasil pemekatan diuji dengan metode agar well diffusion dalam menghambat pertumbuhan F. proliferatum. Pengamatan mikroskopis dilakukan untuk mengevaluasi kerusakan hifa F. proliferatum yang disebabkan oleh kitinase.
Isolat bakteri ABS 4.1.2 mampu menghasilkan enzim kitinase dan menghambat pertumbuhan F. proliferatum secara in vitro. Enzim kitinase yang dihasilkan dapat diendapkan dengan ammonium sulfat pada konsentrasi 60 %. Aktivitas optimum enzim ekstrak kasar dan enzim hasil pemekatan pada pH 7 dengan aktivitas optimum enzim ekstrak kasar pada suhu 35 oC dan enzim hasil pemekatan pada 45 oC. Bobot molekul enzim ekstrak kasar sebesar 65 kDa dan 32 kDA dan enzim hasil pemekatan sebesar 32 kDa. Isolat ABS 4.1.2 diidentifikasi sebagai Pseudomonas aeruginosa. Kitinase yang dihasilkan ABS 4.1.2 menunjukkan penghambatan pertumbuhan terhadap F. proliferatum. Perlakukan enzim ektrak kasar dan enzim hasil pemekatan menunjukkan perubahan morfologi meliputi lisis, pembengkakan dan pembentukan vakulola pada hifa F. proliferatum. Hasil tersebut menunjukkan bahwa isolat ABS 4.1.2 dan enzim kitinase yang dihasilkan dapat dijadikan kandidat biokontrol F. proliferatum. The onion (Allium cepa L.) is a plant with essential nutritional and economic
value for the community. The phytopathogenic fungal attacks, such as Fusarium
proliferatum, constrain onion production. Fusarium proliferatum causes wilt and
rot diseases in onions. Pesticides are generally used to control this fungus, but
excessive use of pesticides causes environmental damage, pathogen resistance, and
negative impacts on human health. Biological control is carried out by utilizing
endophytic bacterial agents. Endophytic bacteria can produce hydrolytic enzymes
such as chitinase, which are useful for the biocontrol of pathogenic fungi, including
F. proliferatum. This study aims to select, characterize, and obtain partial
purification of the chitinase enzyme from onion endophytic bacteria as a control for
the phytopathogenic fungus F. proliferatum.
The study was started by screening four bacterial isolates, ABS 4.1.2, ABP
5.2.2, ABS 5.1, and BBP 5.21, obtained from previous studies. The screening was
carried out by testing the ability of these bacteria to inhibit the growth of F.
proliferatum in vitro using the dual culture method and determining the activity of
the chitinase enzyme. The isolate showing the most significant inhibition against F.
proliferatum and high chitinase activity was designated as the selected isolate and .
were identified based on the 16S rRNA gene. The selected bacterial isolates were
then cultured on production media. The chitinase enzymes were precipitated using
ammonium sulfate, and the optimum chitinase activity was measured based on pH
and temperature. SDS-PAGE analyzed the molecular weight of chitinase. Cell
culture, crude extract enzymes, and precipitated enzymes were tested using the
agar-well diffusion method to inhibit the growth of F. proliferatum. Microscopic
observation was carried out to assess the damage to F. proliferatum hyphae caused
by chitinase.
ABS 4.1.2 bacterial isolates were able to produce chitinase enzymes and
inhibit the growth of F. proliferatum in vitro. The chitinase enzyme produced could
be precipitated with ammonium sulfate at a concentration of 60%. Optimum
activity of crude extract and precipitated enzymes at pH 7 and optimum temperature
of crude extract at 35 oC and 45 oC, respectively SDS-PAGE showed that there were
two bands in the crude enzyme, and one band precipitated chitinase with an
estimated molecular weight of 65 kDa and 32. ABS isolate 4.1.2 was identified
as Pseudomonas aeruginosa. Chitinase produced by ABS 4.1.2 showed growth
inhibition and antagonistic activity against F. proliferatum. Enzyme treatment of
crude extract and precipitated enzymes showed morphological changes in lysis,
swelling, and vacuolization formation in F. proliferatum hyphae. Therefore, these
results indicate that the ABS 4.1.2 isolate and its chitinase enzyme can be biocontrol
candidates for F. proliferatum.