ABSTRAK

Kitin dan kitosan oligosakarida atau lebih dikcnal dongan kitooligosakarida merupakan hasil konversi hidrolisis kitin dan kitosan. Kitooligosakarida dilaporkan memiliki berinacammaeam fungsi fisiologis yang bormanfaat bagi manusia. Oligosakarida lurunan kitin dan kitosan tersebut dapat dibuat dengan rnemanfaatkan limbah industri pengolahan udang dan kepiting. Kitooligosakarida biasanya diproduksi secara kimia dengan hidrolisis asam. Namun cara tersebut mempunyai kelemahan utama yaitu sulil dikontrol dan mencemari lingkungan.

Pcnclitian ini bertujuan unluk mendapatkan cnzim kitinase yang berasal dari mikrobia yang hidup di lingkungan okstrim. Knzim kitinase dari mikrobia lingkungan ekstrim dihaiapkan memiliki sifal"sifal unggul sehingga dapat dimanfaalkan sebagai katalis dalam hidrolisis secara en/.imatis untuk menggantikan proses hidrolisis asam yang selama ini dipergunakan dalam produksi kitooligosakarida. Proses hidrolisis secara enzimatis memiliki beberapa keunggulan yaitu- bersifat spesifik, mudah dikontrol dan ramah terhadap lingkungan.

Lingkungan ekstrim yang dijadikan sumber mikroba adalab sumber air panas Cangar dan sumber lumpur panas Lapindo Branlas. lsolasi dari lumpur panas Lapindo Brantas dipei-oleh 1 buah isolat yaitu LB7OL LB7C2a, LB7C2b dan LB7C3. Kmpal isolat hasil isolasi masing-masing memiliki polensi menghasilkan en/.im kitinase yang ditunjukkan dengan kemampuan mendegradasi kitin. baik pada media eair maupun media padat. Isolat LB7C2b memiliki aktivilas yang paling linggi sehingga dipilih untuk produksi en/.im kitinase. Produksi cnzim kitinase dilakukan pada kultur isolat. LB7C2b pada media koloidal kitin broth yang berumur 60 jam. Purifikasi en/im kitinase LB7C2b dengan afinitas kolidal kitin dapat menghasilkan enzim yang kemurniannya 19 kali lebih linggi dari eskstrak kasar. Karakter en/.im kitinase yang diperoleh yaitu memiliki berat molekul 69,1 kl)a, optimum pada suhu 55°C dan pll 7. stabil pada suhu 30 sampai 60°C dan pi I 5,5-7,5, memiliki nilai Km sebesar 0,676 umol/ml sedangkan nilai Vmaks sebesar 0,052 umol/menit. Aktivitas en/.im kitinase isolat LB7O2b meningkat sebesar 9,4% dengan penambahan 1 mM Ca2+. dan menurun sebesar 11,7% dan 7.9% dengan penambahan 1 mM Ag+ dan Hg2+. Bnisim kitinase isolat LB7C2b memiliki spesifisitas substrat yang luas, yaitu pada substrat 0,3% koloidal kitin, koloidal kitosan. elilen glikol kitin, serbuk kitin dan serbuk kitosan. Knzim tersebut memiliki aktivitas lerlinggi pada koloidal kitin, dan diklasifikasikan sebagai eksokitinase karena menghasilkan GlcNAc dan diduga juga menghasilkan OlcNAc2 dari hidrolisis koloidal kitin.

Isolasi bakteri kitinohtik dari sumber air panas Cangar, Kab. Malang-Jawa Tirnur, didapatkan 5 isolat bakteri yang dapat menghasilkan cnzim kitinase, yaitu isolat 1A, 2A, 2B, 3A, 3B. Isolat 215 mempunyai aktivitas hidrolisis paling linggi dari isolat lainnya. Isolat ini yang kemudian digunakan untuk memproduksi en/.im kitinase. Sebuah en/.im dengan pita lunggal berhasil dipurifikasi dengan menggunakan melode koloidal kitin afinitas dengan kemurnian sebesar 1.79 kali. l(]n/im kitinase hasil pemurman mempunyai aktivitas optimum pada pll 7 dan suhu 55°C. Nilai Vmaks sebesar 0,01-1 j.imol menit1 dan Km sebesar 0,061 |.tmol ml. Hasil SOS-PAGIO menunjukkan satu pita dengan berat molekul en/.im kitinase sebesar 74,99 kl)a. Knzim kitinase hasil isolasi dan pemurnian mempunyai aktivitas tertinggi pada substi'at koloidal kitin dibanding substrat etilen ghkol kitin, koloidal kit.isan, serbuk ktin dan serbuk kitosan. Ion-ion logam Mg-% Ou-'. (^a2', Mn-\ Ag-', dan Hg-' konsentrasi lmMyang ditambahkan pada reaksi antara en/.im ktinase dengan koloidal kitin berperan sebagai inhibitor. Kir/.im kitinase isolat Cangar 215 memiliki spesifisitas substral yang luas, yaitu pada substrat 0,3% koloidal kitin, koloidal kitosan. elilen glikol kitin, serbuk kitin dan serbuk kitosan. Knzim tersebut memiliki aktivitas tertinggi pada koloidal kitin. Kata kunci: afinitas koloidal kitin, bakteri kitinolitik. kitinase, kitooligosakarida, purifikasi-, termofilik

SUMMARY Chilma.se have a wide range of applications such as chilin waste: management, and chitin do

rival production, such as Nacelyl glucosamine. The enzyme can be isolated from bacteria and the thermostable ones have advantages in industry and biotechnology. The objectives of this research were to obtain chitinolytic thermophilic bacteria isolates from Lapindo mud and Cangar Hot spring, to observe the potency of bacteria isolates in producing chitinase, to evaluate activities and characteristics of purified chitinase and its potency in producing chiloohgosacharides from chitin hydrolysis.

This research was conducted in several steps, include isolation and characterization of chitinolytic thermophilic: bacteria from Lapindo mud and Cangar hot spring, production chitinase from the best chitinolytic isolate, purification of chitinase by collidal chitin affinity and dialysis, characterization of enzyme property, include determination of molecular weigh, determination of optimum condition and stability to temperature and pll, determination of Km and Vnmkti, effect of metal ion to chitinase activity, substrate specificity and detection of colloidal chitin hydrolysis.

Lapindo hot mud and Cangar hot spring were used as sources of migroorganis. Isolation from Lapindo mud resulted 1 bacteria isolates, then namely LB7C1, LB7C2a, LB7C2b and LB7C3. Kach isolate has potency in producing chitinase indicated by ability in degrading chitin, both in agar and broth medium. Among these. LB7C2b has the highest activity, then selected for chitinase production. Chitinase were isolated from 60 hours LB7C2b culture on colloidal chitin broth medium. The purification by colloidal chilin affinity was able to purify the enzyme 19 fold higher than thai of crude extract. The chitinase have molecular weight 69,1 kDa,. optimum at 55°C and pil 7, stable at temperature ranges 3O60°C and pll 5,5-7,5. The Km and Vmax value were 0.(576 pmol/ml and 0,052 umol/min. Addition of 1 mM Ca2' increased chitinase activity up to 9,1%. whereas addition of 1 mM Ag' dan l.lg2' inhibited chitinase activity up to .11,7% and 7,9% respectively. Chitinase from LB7C2b showed wide range of substrate specificity, toward 0.3% colloidal chilin. colloidal chitosan, ethylene glycol chitin, chitin powder and chitosan powder. The enzyme was most active toward colloidal chitin and was classified as exoch.iti.nase because of its ability in producing GlcNAc and presumable GlcNAc^ from colloidal chitin hydrolyzis.

Rive chitinolitic thermophilic bacteria were isolated from Cangar hot spring, i.e. LA, 2A, 2B. 3A, 3B. Isolate 2B has the highest chitinolitic activity among them. Colloidal chitin affinity could purify the enzyme 4.79 fold. The enzyme showed that it work optimumly at pll 7 and temperature of ~>~>°C. The Vm»ks and Km value are 0,014 j.nnol min ' and 0,061 |imol ml ' respectively. SI)S-IV\(iK analysis showed that the molecular mass of the enzyme is about 4,99 kDa. Th purified chitinase was most active in colloidal chilin substrate than in ethylene glycol chitin. colloidal chitosan. chitin powder and chitosan powder. Mg-\ Cu2\ Ca-', Mn-\ Ag-', dan llg2' at concentration of 1 mM inhibit the enzyme. The hydrolysis of colloidal chitin produced monomer, dinner and longer kitoligosacharides. The chitinase of isolate 2B can be used in functional chitooligosacharides production. Key words: : colloidal chitin affinity, chitinolytic bacteria, chitooligosacharide, chitinase,

purification, thermophilic

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