Purifikasi Dan Karakterisasi Enzim Linamarase Kacang Arbila (Phaseolus Lunatus L)

Pramitha, Asti Rizkiana and Dr. Siti Narsito Wulan,, S.TP, M.P, M.Sc and Tri Dewanti W, Prof. Dr. Ir. (2021) Purifikasi Dan Karakterisasi Enzim Linamarase Kacang Arbila (Phaseolus Lunatus L). Magister thesis, Universitas Brawijaya.

Abstract

Kacang arbila merupakan salah satu jenis kacang lokal yang ditemukan di daerah Nusa Tenggara Timur, Indonesia. Kacang arbila memiliki kandungan nutrisi yang dapat bermanfaat sebagai sumber makanan. Namun, kacang arbila mengandung senyawa beracun berupa glikosida sianogenik yaitu linamarin. Senyawa tersebut berpotensi menghasilkan sianida karena melepaskan racun berupa hidrogen sianida (HCN) yang terbentuk dari hidrolisis linamarin oleh enzim linamarase (β-glukosidase). Senyawa glikosida sianogenik apabila dikonsumsi secara lansung dapat mengaktifkan enzim linamarase didalam pencernaan sehingga berpotensi untuk menghasilkan senyawa HCN yang menyebabkan keracunan. Beberapa upaya telah dilakukan bertujuan untuk meminimalkan efek senyawa beracun salah satunya dengan proses pengolahan tetapi ditemukan adanya sisa senyawa residu sianida yang masih beracun setelah proses pengolahan. Hal ini terjadi karena kurangnya mengoptimalkan kondisi optimum enzim linamarase dalam menghidrolisis linamarin selama proses pengolahan. Aktivitas enzim linamarase merupakan kunci awal dari pembentukan senyawa HCN bebas dan aseton sianohidrin yang menyebabkan kacang menjadi beracun. Penelitian ini bertujuan untuk memperoleh dan mengevaluasi karakteristik enzim linamarase hasil purifikasi dari kacang arbila, menguji pengaruh pH dan suhu optimum aktivitas enzim linamarase dapat diaplikasikan dalam proses detoksifikasi sianida, serta menguji engaruh penambahan enzim linamarase eksogen dalam memaksimalkan kontak antara enzim dan substrat sehingga terjadi pembebasan sianida pada kacang arbila.Tahapan penelitian meliputi penelitian pendahuluan dan penelitian utama yang terdiri dari penelitian tahap satu dan penelitian tahap dua. Tahap penelitian pendahuluan bertujuan untuk menganalisa bahan baku kacang arbila meliputi : analisa kadar sianida, air, abu, lemak, protein. Selanjutnya penelitian tahap satu meliputi: ekstraksi linamarin, ekstraksi enzim linamarase dengan buffer fosfat, pengkuruan kadar protein enzim linamarase, pengukuran aktivitas enzim linamarase, penentuan konsentrasiii optimum ammonium sulfat, presipitasi enzim linamarase, dialisis, karakterisasi enzim linamarase terhadap pH dan suhu dengan beberapa tingkatan serta dilakukan pengujian stabilitas enzim terhadap pH dan suhu. Kemudian penelitian Tahap dua yaitu aplikasi enzim linamarase hasil purifikasi untuk menguji pengaruh penambahan enzim dalam memaksimalkan kontak antara enzim linamarase dan substrat. Diharapkan dengan adanya aplikasi tersebut dapat menunjukan aktivitas linamarase dalam menghidrolisis linamarin secara maksimal sebagai upaya untuk membantu penurunan kadar sianida. Hasil penelitian menunjukkan bahwa enzim linamarase mengalami peningkatan aktivitas spesifik setelah proses purifikasi dialisis yaitu 9,42 U/mg, dibandingkan dengan sebelum crude enzimnya yaitu 2,80 U/mg. Enzim mengalami peningkatan kemurnian 3 kali lipat dari crude enzimnya. Pengaruh aktivitas enzim linamarase terhadap variasi pH dan suhu yaitu diperoleh pH optimum 5,5 dan stabil pada kisaran pH 5 -6, sementar itu untuk suhu optimum diperoleh 50oC dan stabil pada kisaran , sementara itu pengaruh variasi pH dan suhu terhadap stabilitas enzim diperoleh 45 – 55oC. Penambahan enzim linamarase sebanyak 200 µl mampu membebaskan total sianida sebesar 1933,76 ppm dibanding dengan tanpa penambahan enzim, total sianida yang terukur hanya sebesar 422 ppm

English Abstract

Arbila beans are the local beans found in East Nusa Tenggara, Indonesia. Arbila beans contain nutrients that can be useful as a food source. However, arbila beans contain a toxic compound in the form of a cyanogenic glycoside, namely linamarin. This compound has the potential to produce cyanide because it releases toxins in the form of hydrogen cyanide (HCN) which is formed from the hydrolysis of linamarin by the enzyme linamarase (β-glucosidase). Cyanogenic glycoside compounds when consumed directly can activate the linamarase enzyme in the digestive tract so that it has the potential to produce HCN compounds that cause poisoning. Several attempts have been made to minimize the effects of toxic compounds, one of which is by processing, but it was found that there were residual cyanide compounds that were still toxic after processing. This is due to the lack of optimizing the optimum conditions for the linamarase enzyme to hydrolyze linamarin during the processing. The activity of the linamarase enzyme is the initial key to the formation of free HCN compounds and acetone cyanohydrin which causes bean to become toxic The aimed of study to obtain and evaluate the characteristics of the purified linamarase enzyme from arbila beans, to determine the effect of pH and optimum temperature on the activity of the linamarase enzyme that can be applied in the cyanide detoxification process, and to examine the effect of adding exogenous linamarase enzyme in maximizing the contact between the enzyme and the substrate so that cyanide liberation occurs. on arbila beans. The research stages include preliminary research and main research consisting of stage one research and stage two research. The preliminary research phase aims to analyze the raw materials of arbila beans include : analysis of cyanide levels, water, ash, fat, protein. Furthermore, the first stage of research includes: linamarin extraction, linamarase enzyme extraction with phosphate buffer, measurement of linamarase enzyme protein levels, measurement of linamarase enzyme activity, determination of the optimum concentration of ammonium sulfate, linamarase enzymeiv precipitation, dialysis, characterization of linamarase enzyme to pH and temperature with several levels and Enzyme stability testing was carried out to pH and temperature. Then the second stage of research is the application of the purified linamarase enzyme to test the effect of adding the enzyme in maximizing the contact between the linamarase enzyme and the substrate. It is hoped that this application can show the activity of linamarase in hydrolyzing linamarin maximally as an effort to help decrease cyanide levels. The results showed that the linamarase enzyme had increased specific activity after the dialysis purification process, which was 9.42 U/mg, compared to before the crude enzyme was 2.80 U/mg. Enzymes have increased purity 3 times from crude enzymes. The effect of linamarase enzyme activity on variations in pH and temperature is that the optimum pH is 5.5 and stable in the pH range of 5-6, meanwhile the optimum temperature is obtained at 50oC and is stable in the range of 45-45, while the effect of variations in pH and temperature on enzyme stability is obtained at 45- 55oC. The addition of the enzyme linamarase as much as 200 l was able to liberate the total cyanide of 1933.76 ppm compared to without the addition of the enzyme, the total cyanide measured was only 422 ppm

Item Type:	Thesis (Magister)
Identification Number:	0420100015
Subjects:	300 Social sciences > 338 Production > 338.1 Agriculture > 338.16 Production efficiency
Divisions:	S2/S3 > Magister Teknologi Hasil Pertanian, Fakultas Teknologi Pertanian
Depositing User:	soegeng sugeng
Date Deposited:	03 Jan 2022 04:32
Last Modified:	09 Oct 2024 03:58
URI:	http://repository.ub.ac.id/id/eprint/187747

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