Hasil Pencarian  ::  Simpan CSV :: Kembali

Abstrak :
Penelitian ini mencakup rangkaian oksidasi kolesterol berupa studi oksidasi kolesterol, oksidasi dengan menggunakan substrat hewani, oksidasi dengan enzim terimobilisasi material magnetit silikon dioksida (M-SiO2)/magnetit kitosan (M-Chit) dan oksidasi dengan protein rekombinan Rhodococcus erythropolis BL21(DE3) (RhoChoA). Enzim kolesterol oksidase yang diproduksi dengan metode submerged fermentation dari Streptomyces sp memiliki nilai aktivitas sebesar 5,12 U/mL dan aktivitas RhoChoA sebesar 17,9 U/mL. Studi kinetika dilakukan dengan menggunakan orde satu dengan reaksi irreversible. Optimasi produksi enzim dilakukan dengan memperhatikan faktor suhu dan jenis substrat. Untuk meningkatkan karakteristik enzim, imobilisasi dilakukan pada enzim kolesterol oksidase Streptomyces sp. Material magnetit disintesis dengan metode sol-gel dengan modifikasi menggunakan magnetit silikon dioksida dan magnetit kitosan yang diberi kode M-SiO2 dan M-Chit secara berurutan. Enzim hasil produksi diimobilisasi dengan menggunakan teknik cross-linking. 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. Uji oksidasi dilakukan dengan beberapa 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 untuk menganalisis konsentrasi substrat dan konsentrasi enzim yang optimum dalam oksidasi yang dijadikan sebagai referensi dalam penentuan uji biosensor kolesterol. Oksidasi kolesterol dengan menggunakan substrat hewani dilakukan dengan ekstraksi dengan pelarut lemak. Kuantifikasi kadar kolesterol dalam sampel menunjukkan susbtrat dari lemak hewani memiliki konsentrasi kolesterol tertinggi dari kuning telur dengan konsentrasi 1,94 mg/mL, hati ayam (0,93 mg/mL), daging sapi (0,25 mg/mL) dan daging ayam (0,23 mg/mL). Enzim kolesterol oksidase dengan konsentrasi 2 mg/mL dapat mengoksidasi ekstrak kasar kolesterol dari kuning telur, hati ayam dan daging ayam hingga teroksidasi 20%, sedangkan ekstrak kasar kolesterol dari daging sapi teroksidasi sebesar 10%. Hasil uji oksidasi dengan menggunakan HPLC diperoleh konsentrasi substrat secara optimal dioksidasi oleh enzim terimobilisasi M-SiO2 dengan konsentrasi 20 mg/mL serta konsentrasi kolesterol 1,94 mM sebesar 90%, sedangkan enzim kolesterol oksidase bebas mengoksidasi kolesterol sebesar 80%. Uji oksidasi kolesterol menggunakan enzim kolesterol oksidase terimobilisasi magnetit kitosan (M-Chit) ditemukan konsentrasi substrat yang optimum adalah 2,5 mg/mL dan konsentrasi enzim yang paling efektif adalah 2 mg/mL. Reaksi oksidasi kolesterol dengan kondisi optimum dan menggunakan enzim terimobilisasi M-Chit dapat mengoksidasi kolesterol sampai 10%. Uji penggunaan kembali material M-Chit dalam proses imobilisasi dapat digunakan sebanyak 2 kali. ......This research includes a series of cholesterol oxidation in the form of cholesterol oxidation studies, oxidation using animal substrates, oxidation with immobilized enzymes of magnetite silicon dioxide (M-SiO2) / magnetite chitosan (M-Chit) and oxidation with recombinant protein Rhodococcus erythropolis BL21 (DE3) ( RhoChoA). Cholesterol oxidase enzyme produced by the submerged fermentation method from Streptomyces sp has an activity value of 5.12 U / mL and a RhoChoA activity of 17.9 U / mL. The kinetic study was carried out using first order with an irreversible reaction. Optimization of enzyme production is carried out by controlling the temperatur and type of substrate. To improve the characteristics of the enzyme, immobilization was carried out on the cholesterol oxidase enzyme Streptomyces sp. The magnetite material was synthesized by the sol-gel method with modification using magnetite silicon dioxide and magnetite chitosan which were coded M-SiO2 and M-Chit, respectively. The immobilized enzymes are produced using a cross-linking technique. The FTIR characterization results of the magnetite material showed the M-O functional group at wave number 559.88; 598.91 and 680.1 cm-1, the Si-O group at wave numbers 1615.78 and 1761.65 cm-1. The oxidation test was carried out with several independent variables, namely 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 minutes), as well as the form of the enzyme (crude extract of the cholesterol oxidase enzyme and the immobilized cholesterol oxidase enzyme). The results of the oxidation test were quantified using HPLC to analyze the optimum substrate concentration and enzyme concentration in oxidation which were used as references in determining the cholesterol biosensor test. Cholesterol oxidation using animal substrates was carried out by extraction with fat solvents. The quantification of cholesterol levels in the sample showed that the animal fat substrate had the highest cholesterol concentration from egg yolks with a concentration of 1.94 mg / mL, chicken liver (0.93 mg / mL), beef (0.25 mg / mL) and chicken meat. (0.23 mg / mL). Cholesterol oxidase enzyme with a concentration of 2 mg / mL can oxidize the crude extract of cholesterol from egg yolk, chicken liver and chicken meat up to 20%, while the crude extract of cholesterol from beef is oxidized only 10%. The results of the oxidation test using HPLC showed that the optimal substrate concentration was oxidized by the immobilized enzyme M-SiO2 with a concentration of 20 mg / mL and a cholesterol concentration of 1.94 mM of 90%, while the free cholesterol oxidase enzyme was 80% oxidized. Cholesterol oxidation test using immobilized cholesterol oxidase enzyme magnetite chitosan (M-Chit) found that the optimum substrate concentration was 2.5 mg / mL and the most effective enzyme concentration was 2 mg / mL. Cholesterol oxidation reaction under optimum conditions and using the immobilized enzyme M-Chit can oxidize cholesterol up to 10%. The M-Chit reuse test in the immobilization process can be used 2 times

Abstrak :
Peningkatan kadar kolesterol dalam darah merupakan salah satu faktor utama dalam berbagai penyakit kardiovaskuler. Oleh karena itu, dibutuhkan metode praktis yang dapat mendeteksi kadar kolesterol secara berkala. Pengembangan terbaru alat pendeteksi kadar kolesterol saat ini cenderung mengarah pada biosensor menggunakan enzim kolesterol oksidase (CHOx). Untuk mengoptimalkan kinerja enzim dan memperoleh biokompatibilitas dan selektivitasnya yang tinggi, diperlukan teknik imobilisasi dengan material support. Selulosa merupakan salah satu biopolimer di alam dengan ketersediaan paling berlimpah dan serbaguna, serta memiliki karakteristik yang sensitif, akurat, dan biayanya yang murah. Aplikasi selulosa untuk imobilisasi enzim dikombinasikan dengan magnetit, yaitu logam oksida yang memiliki sifat paramagnetik tinggi. Tujuan dari penelitian ini adalah untuk mempelajari aplikasi dari penggabungan material magnetit dan selulosa sebagai support imobilisasi enzim CHOx. Penelitian ini menggunakan enzim CHOx Streptomyces sp. komersil. Parameter yang diuji dalam penelitian diantaranya ialah konsentrasi enzim optimum yang digunakan saat proses imobilisasi, serta aktivitas relatif dan residual dari enzim ChOx–baik setelah pengulangan penggunaan maupun setelah penyimpanan dalam waktu tertentu. Enzim yang telah direaksikan diuji dengan alat spektrofotometer UV-Vis. Sampel enzim terimobilisasi akan dicuci kembali menggunakan buffer untuk uji penggunaan kembali. Hasil yang diperoleh dari penelitian ini diantaranya; konsentrasi enzim CHOx (Ce) yang paling efektif digunakan untuk imobilisasi pada material magnetit-selulosa ialah 2 mg/10 mL, retensi aktivitas enzim terimobilisasi material dapat bertahan ~10% setelah digunakan ulang sebanyak empat kali, dan aktivitas relatif enzim terimobilisasi dapat bertahan hingga ~5% pada hari ke-10 penyimpanan–dengan tingkat stabilitas waktu simpan yang lebih baik daripada enzim bebas. ......Increasing levels of cholesterol in blood is one of the main causes in many cardiovascular diseases. Therefore, a practical method of detecting cholesterol levels on a regular basis is needed. The latest development of cholesterol level detection device currently leads to biosensors using cholesterol oxidase enzyme (CHOx). To optimize enzyme performance and obtain high biocompatibility and selectivity, an immobilization technique with material support is required. Cellulose is one of the most widely available and versatile biopolymers in nature, and has characteristics that are sensitive, accurate, and low-cost. The application of cellulose for enzyme immobilization is combined with magnetite, a metal oxide that has high paramagnetic properties. The purpose of this research was to study the application of the incorporation between magnetite and cellulose materials as a support for CHOx enzyme immobilization. This study uses commercial CHOx derived from Streptomyces sp. The parameters tested in this study include the optimal enzyme concentration used during the immobilization process, as well as the relative and residual activity of the ChOx enzyme–after repeated uses and after certain period of storage. The reacted enzymes were tested with a UV-Vis spectrophotometer. The immobilized enzyme sample was washed each time after use with buffer saline for reusability assays. The results obtained from this study include; the most effective concentration of CHOx enzyme (Ce) used for immobilization on magnetite-cellulose material was 2 mg/10 mL, retention of enzyme activity of immobilized material could last ~10% after being used four times, and the relative activity of immobilized enzyme could last up to ~5% on the 10th day of storage–with a better shelf-life than the free enzyme.

Abstrak :
Enzim kolesterol oksidase adalah enzim oksidoreduktase yang mampu mendegradasi kolesterol. Pada percobaan ini, dilakukan produksi enzim kolesterol oksidase oleh Streptomyces sp. melalui fermentasi submerged lalu dilakukan investigasi terhadap aktivitas dan kemampuan katalisis enzim kolesterol oksidase. Pada percobaan, substrat dan enzim divariasikan pada berbagai konsentrasi, yaitu 0,15, 0,075, dan 0,0375 U/mL dan 1,25, 2,5, dan 5 mg/mL. Perbandingan konstanta laju reaksi antara ekstrak kasar enzim dan enzim komersial diperoleh dari hasil penelitian. Perbandingan konstanta laju reaksi enzimatis oleh faktor yang mempengaruhi, diantaranya imobilisasi enzim dan suhu inkubasi enzim, dengan data yang diperoleh dari literatur. Enzim dan substrat mengalami reaksi oksidasi pada waktu inkubasi 5, 30, 65, 120, dan 240, lalu konsentrasi kolesterol residu pada sampel dilakukan plotting dengan waktu inkubasi, dan konstanta laju reaksi diperoleh melalui permodelan reaksi orde 1 dengan pendekatan integrasi numerik Euler. Aktivitas ekstrak kasar enzim yaitu 1,69 U/mL dengan konstanta laju reaksi yaitu 0,01/menit untuk ekstrak kasar enzim dan 0,014/menit untuk enzim komersial. Selanjutnya, diperoleh faktor yang mempengaruhi konstanta laju reaksi oksidasi kolesterol secara enzimatik oleh enzim kolesterol oksidase, yaitu konsentrasi enzim, jenis enzim, imobilisasi, dan suhu inkubasi. Reaksi oksidasi kolesterol oleh enzim kolesterol oksidase dari Streptomyces sp. mengikuti reaksi orde 1. ...... Cholesterol oxidase well known as oxidoreductase enzyme which able to degrade the cholesterol. Here, we produced cholesterol oxidase from Streptomyces sp. by submerged fermentation and investigate the activity and cholesterol oxidation kinetics of cholesterol oxidase. The enzyme and substrate are diluted in various concentration, 0.15, 0.075, 0.0375 U mL and 1.25, 2.5, 5 mg mL, respectively. Further step was comparing crude enzyme and commercial enzyme from Streptomyces sp. by oxidation constant rate of reaction. The enzyme and substrate were through the oxidation reaction, and the amount of cholesterol residue in the sample are determined by HPLC. In this work, we also compared the oxidation constant rate of reaction of previous experiment from literature with affecting factors, such as immobilization and incubating temperature. The cholesterol residue in the sample are plotted by time reaction and the rate constant is obtained by first order rate reaction using Euler integration method. The crude enzyme activity is 1.69 U mL and the reaction constant are 0.01 U mL and 0.014 for crude extract and commercial enzyme, respectively. Furthermore, several factors affecting constant rate of enzymatic oxidation of cholesterol were enzyme concentration, enzyme type, immobilization, and incubating temperature. Cholesterol oxidation by Streptomyces sp. cholesterol oxidase was follow the first order reaction.