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Abstrak :
Pembentukan produk oksidasi kolesterol pada proses pangan maupun kesehatan melibatkan reaksi kimia dan biokimia, reaksi oksidasi kolesterol ini dapat dikontrol melalui model kinetika pada tren oksidasi. Penggunaan model kinetika menjadi salah satu aspek penting dalam memprediksi kadar kolesterol hasil oksidasi. Reaksi oksidasi kolesterol melibatkan enzim sebagai katalisator, yaitu enzim kolesterol oksidase. Bakteri yang digunakan sebagai penghasil enzim kolesterol oksidase yaitu Streptomyces sp. Enzim kolesterol oksidase diproduksi dengan menggunakan metode fermentasi submerged. Untuk meningkatkan efektifitas enzim kolesterol oksidase dalam oksidasi kolesterol, maka dibutuhkan matriks pendukung. Enzim kolesterol oksidase diimobilisasi dengan material magnetit silikon dioksida. Material magnetit silikon dioksida disintesis melalui metode hidrotermal dengan proses pelapisan silika pada partikel magnetit. Reaksi oksidasi dilakukan dengan metode oksidasi secara langsung terhadap substrat. Penelitian ini bertujuan untuk memperoleh enzim kolesterol oksidase dari Streptomyces sp., mengimobilisasi enzim terhadap material magnetit silikon dioksida, estimasi konstanta laju reaksi oksidasi kolesterol, serta membandingkannya dengan enzim bebas dan enzim terimobilisasi. Enzim yang telah diproduksi memiliki aktivitas sebesar 5,12 U/mL. Enzim yang telah diproduksi digunakan untuk imobilisasi dan reaksi oksidasi. Variabel bebas yang digunakan dalam penelitian ini yaitu konsentrasi enzim (5;10;20 mg/mL), konsentrasi substrat (1,64;3,23;6,46 mM) dan bentuk enzim (ekstrak kasar enzim kolesterol oksidase dan enzim kolesterol oksidase terimobilisasi). Hasil karakterisasi FTIR dari material magnetit menunjukkan gugus fungsi M-O di bilangan gelombang 559,88; 598,91 dan 680,1 cm-1 gugus Si-O di bilangan gelombang 1615,78 dan 1761,65 cm-1. Hasil uji oksidasi dengan menggunakan HPLC diperoleh konsentrasi substrat secara optimal dioksidasi oleh enzim terimobilisasi dengan konsentrasi 20 mg/mL serta konsentrasi kolesterol awal 1,94 mM dengan hasil akhir sebesar 0,49 mM, sedangkan enzim kolesterol oksidase bebas mengoksidasi kolesterol dengan hasil akhir sebesar 1,459 mM. ......The formation of products related to reactions and chemical reactions, these reactions can be carried out through the kinetics model on the oxidation trend. The use of kinetics is an important aspect in achieving oxidation cholesterol levels. A catalyst of cholesterol oxidation reaction is a cholesterol oxidase enzyme. The bacteria that used as a producer of cholesterol oxidase enzyme is Streptomyces sp. Cholesterol oxidase enzymes are produced using submerged fermentation methods. To increase the effectiveness of cholesterol enzymes in cholesterol oxidation, a support matrix is needed. The cholesterol oxidase enzyme is immobilized with magnetite silicon dioxide. Magnetite silicon dioxide material is synthesized by using hydrothermal method with silica coating process. The oxidation reaction is carried out by the oxidation method directly against the substrate. The aims of this study are to produce the enzymes from Streptomyces sp., immobilize enzymes to magnetite silicon dioxide, obtaining the rate of oxidation reactions, and comparing data between free enzyme and immobilized enzyme. The produced enzyme activity was 5,12 U/mL, this enzyme is used for immobilization and oxidation reaction. In this study, the independent variables are enzyme concentration (5;10;20 mg/mL), substrate concentration (1,94; 3,23;6,46) and enzyme forms (crude extract cholesterol oxidase enzyme and immobilized enzyme). The FTIR characterization of materials have shown that metal oxide functional groups appeared at wavenumber of 559,88; 598,91 dan 680,1 cm-1- and Si-O groups at wavenumber of 1615,78 dan 1761,65 cm-1. The oxidation test by HPLC showed that the substrate concentration optimizely oxidized by immobilized enzyme with the initial concentration 20 mg/mL and substrate concentration 1,94 mM with final oxidation concentration was 1,94 mM, meanwhile the free enyme with same concentration showed 1,459 mM at final concentration.

Abstrak :
Enzim kolesterol oksidase merupakan enzim yang dapat membantu mempercepat reaksi oksidasi kolesterol. Salah satu pemanfaatan enzim kolesterol oksidase adalah biosensor kolesterol. Metode enzimatis lebih unggul karena memiliki spesifitas tinggi dan tidak berbahaya. Namun metode enzimatis memiliki kelemahan yaitu enzim yang mudah terdegradasi sehingga perlu dilakukan imobilisasi. Penambahan material imobilisasi dapat menambah biaya karena material imobilisasi yang terbilang cukup mahal, sehingga studi aktivitas material imobilisasi perlu dilakukan. Imobilisasi enzim kolesterol oksidase dilakukan pada material kitosan-magnetit. Penelitian ini dimulai dengan memproduksi enzim kolesterol oksidase dari Streptomyces sp., yang hasilnya kemudian akan diimobilisasi dan digunakan dalam reaksi oksidasi kolesterol. Penelitian ini dilakukan dengan variabel bebas yaitu konsentrasi enzim (0,5;1;2 mg/mL), konsentrasi substrat (0,75;1,25;2,5 mg/mL), waktu oksidasi (5,30,60,120,180 menit), serta bentuk enzim (ekstrak kasar enzim kolesterol oksidase dan enzim kolesterol oksidase terimobilisasi). Hasil uji oksidasi dikuantifikasi dengan menggunakan HPLC. Material imobilisasi akan dicuci dengan menggunakan buffer agar dapat digunakan kembali dalam proses imobilisasi. Dari penelitian dihasilkan konsentrasi substrat yang optimum adalah 2,5 mg/mL, konsentrasi enzim yang paling efektif adalah 2 mg/mL. Reaksi oksidasi kolesterol dengan kondisi optimum dapat mereduksi kolesterol sampai 10%. Sedangkan uji penggunaan kembali material kitosan-magnetit dalam proses imobilisasi menghasilkan bahwa material dapat digunakan kembali saat digunakan sebanyak 2 kali penggunaan. ......Cholesterol oxidase enzyme is an enzyme which can be the catalyst for cholesterol oxidation reaction. One of the cholesterol oxidase utilizations is cholesterol biosensor. Enzymatic method has more advantages which highly substrate specific, and non-toxic. However, the enzymatic method has some weakness which are easily-degraded and loss its activity which makes enzyme immobilization needs to be done. Immobilization can add additional cost because the material is expensive in average, so the material repeatability study is important. In this research, cholesterol oxidase enzyme will be immobilized in Chitosan-Magnetite. Novelty of this research is the usage of Chitosan-Magnetite material for cholesterol oxidase immobilization, and material repeatability test. This research started with enzyme production from Streptomyces sp., The enzyme will be immobilized and used for the cholesterol oxidation reaction. The independent variables of this research are enzyme concentration (0.5; 1; 2 mg/mL), substrate concentration (0.75; 1.25; 2.5 mg/mL), oxidation time (5; 30; 60; 120; 180 mg/mL), and enzyme forms (crude cholesterol oxidase and immobilized cholesterol oxidase). The immobilization material was washed with buffer, so it can be used in repetition. The research resulted the optimum substrate concentration in cholesterol enzymatic oxidation was 2.5 mg/mL, and enzyme optimum concentration was 2 mg/mL. With the optimum condition of cholesterol oxidation reaction, immobilized cholesterol oxidase can reduce substrate up to 10%. The repeatability study of the chitosan-magnetite material with 2 times repeatability test resulted the material can be used to immobilize cholesterol oxidase enzyme without losing its ability for immobilization.