3. Results and discussion3.1. Immob

3. Results and discussion
3.1. Immobilization of PLA1
3.1.1. Carrier screening
Immobilization of an enzyme yields the enzyme reusable, min- imizes product contamination by the enzyme, and simplifies separation of product and enzyme. The type of carrier is a crucial factor
in such an enzyme immobilization process. Table 1 shows the effect of the selected carriers on the fixation level (%) and specific activity (lmol/g protein/min) of the immobilized enzyme. Among
the eight carriers examined, Lewatit VP OC 1600, Accurel MP 1000, Amberlite XAD 4 and Octyl silica were hydrophobic. Since hydrophobic carriers are not easily suspended in the enzyme suspension, pre-wetting hydrophobic carriers with ethanol prior to immobilization
was suggested to solve this problem in the previous studies (Vikbjerg et al., 2007; Zhang, Hellgren, & Xu, 2007). In this study, Pre-wetting indeed made it possible to suspend these carriers in the enzyme suspension, resulting also in increased fixation level of PLA1. Lewatit VP OC 1600 (fixation level, 79.5%) exhibited the highest fixation, followed by Amberlite XAD 7HP (78.2%) and Duolite A568 (73.2%). The catalytic activities of the various immobilized PLA1 were
compared by measuring the specific activities based on its hydrolytic activity, according to Vikbjerg et al. (2007). Lewatit VP OC 1600 (specific activity, 6.7  10 3 lmol/g protein/min), Amberlite XAD7HP (6.6  103 lmol/g protein/min) and Duolite A568 (6.3  103 lmol/g protein/min) as carriers resulted not only in significantly higher fixation levels but also higher specific activities, compared with the other carriers tested. However, low fixation did not necessarily lead to low specific activity of the immobilized enzyme. For instance, in case of Accurel MP 1000, even though fixation level was low, specific activity was significantly higher, compared with Amberlite XAD 4, which showed very similar fixation level to Accurel MP 1000. Among the carriers,
Lewatit VP OC 1600 had the highest protein fixation as well as specific activity. Thus, Lewatit VP OC 1600 was judged a suitable carrier for further experimentation.
3.1.2. Protein concentration of initial enzyme suspension
The initial enzyme concentration is also an important factor to be considered for the efficiency of the immobilization. The initial PLA1 suspension, whose protein concentration ranged from 2.9
to 29.6 mg/mL, was tested for the immobilization of PLA1. Increased protein concentration in the PLA1 suspension increased the amount of protein immobilized (mg/g, Fig. 1a). Meanwhile,
the fixation level remained constant up to a protein concentration of 14.9 mg/g, but deceased significantly with further increased protein concentration. This indicated that, after the carrier was saturated with loaded protein, binding ability of the carrier decreased with the increasing protein concentration of the initial PLA1 suspension. The apparent activity increased steadily when protein amount in the carrier was increased from 16.9 to 147.9 mg/g, which corresponded to a protein concentration of 2.9–20.6 mg/mL, (Fig. 1b). However, there was no significant difference in apparent activity as the protein amount in the carrier increased further. Meanwhile,the specific activity of immobilized enzyme increased when the protein amount of the immobilized enzyme was increased from 16.9 to 56.9 mg/g and the specific activity remained constant when the protein amount of the immobilized enzyme was increased up to 147.9 mg/g. However, the specific activity decreased with further increase of protein amount in immobilized enzyme. The decline in specific activity was related to the increased steric restrictions and the mass-transfer limitations of the enzyme. Due to the lack of surface area, the enzyme tends to be packed into more than a monolayer on the surface, causing decrease in activity (Madoery, Gattone, & Fidelio, 1995). Therefore, an initial enzyme concentration of 20.6 mg/g was selected for the immobilization of PLA1 and the resulting immobilized enzyme preparation was
used for subsequent acidolysis reactions.

Hasil (Bahasa Indonesia) 2:

3. Hasil dan diskusi
3.1. Imobilisasi PLA1
3.1.1. Pembawa skrining
Imobilisasi enzim menghasilkan enzim dapat digunakan kembali, min imizes kontaminasi produk oleh enzim, dan pemisahan fi penyederhanaan es produk dan enzim. Jenis pembawa merupakan faktor penting
dalam seperti proses imobilisasi enzim. Tabel 1 menunjukkan efek dari operator yang dipilih pada tingkat fi xation (%) dan aktivitas c spesifik (lmol / g protein / min) dari enzim amobil. Di antara
delapan operator diperiksa, Lewatit VP OC 1600, Accurel MP 1000, Amberlite XAD 4 dan Oktil silika yang hidrofobik. Karena operator hidrofobik tidak mudah tersuspensi dalam suspensi enzim, pra-membasahi operator hidrofobik dengan etanol sebelum imobilisasi
disarankan untuk memecahkan masalah ini dalam penelitian sebelumnya (Vikbjerg et al, 2007;. Zhang, Hellgren, & Xu, 2007). Dalam penelitian ini, Pre-pembasahan memang memungkinkan untuk menangguhkan ini operator di suspensi enzim, dihasilkan juga meningkat tingkat fi xation dari PLA1. Lewatit VP OC 1600 (tingkat xation fi, 79,5%) dipamerkan tertinggi fi xation, diikuti oleh Amberlite XAD 7HP (78,2%) dan Duolite A568 (73,2%). Kegiatan katalitik dari berbagai PLA1 amobil yang
dibandingkan dengan mengukur spesifik kegiatan c fi berdasarkan aktivitas hidrolitik, menurut Vikbjerg et al. (2007). Lewatit VP OC 1600 (spesifik aktivitas c fi, 6.7? 10? 3 lmol / g protein / min), Amberlite XAD7HP (6,6? 10? 3 lmol / g protein / min) dan Duolite A568 (6,3? 10? 3 lmol / g protein / min) sebagai pembawa mengakibatkan tidak hanya di signi fi tingkat cantly fi yang lebih tinggi xation tetapi juga lebih tinggi spesifik kegiatan fi c, dibandingkan dengan operator lain yang diuji. Namun, rendah fi xation tidak selalu menyebabkan rendah spesifik aktivitas fi k dari enzim amobil. Misalnya, dalam kasus Accurel MP 1000, meskipun tingkat fi xation rendah, spesifik aktivitas fi c secara signifikan lebih tinggi, dibandingkan dengan Amberlite XAD 4, yang menunjukkan tingkat fi xation sangat mirip dengan Accurel MP 1000. Di antara operator,
Lewatit VP OC 1600 memiliki tertinggi protein fi xation serta spesifik aktivitas fi c. Dengan demikian, Lewatit VP OC 1600 dihukum pembawa cocok untuk eksperimen lebih lanjut.
3.1.2. Konsentrasi protein suspensi enzim awal
Konsentrasi enzim awal juga merupakan faktor penting yang harus dipertimbangkan untuk e fi siensi imobilisasi. Suspensi PLA1 awal, yang protein konsentrasi berkisar antara 2,9
sampai 29,6 mg / mL, diuji untuk imobilisasi PLA1. Peningkatan konsentrasi protein dalam suspensi PLA1 meningkatkan jumlah protein amobil (mg / g, Gambar. 1a). Sementara itu,
tingkat fi xation tetap konstan hingga konsentrasi protein sebesar 14,9 mg / g, tapi meninggal secara signifikan dengan konsentrasi protein lebih meningkat. Hal ini menunjukkan bahwa, setelah pembawa jenuh dengan protein dimuat, kemampuan mengikat pembawa menurun dengan konsentrasi protein meningkatnya suspensi PLA1 awal. Kegiatan jelas terus meningkat ketika jumlah protein dalam carrier meningkat 16,9-147,9 mg / g, yang berhubungan dengan konsentrasi protein dari 2,9-20,6 mg / mL, (Gambar. 1b). Namun, tidak ada perbedaan signifikan dalam kegiatan nyata sebagai jumlah protein dalam carrier meningkat lebih lanjut. Sementara itu, spesifik aktivitas fi c enzim amobil meningkat ketika jumlah protein enzim amobil meningkat 16,9-56,9 mg / g dan spesifik aktivitas fi c tetap konstan ketika jumlah protein enzim amobil meningkat hingga 147,9 mg / g. Namun, aktivitas spesifik fi c menurun dengan peningkatan lebih lanjut dari jumlah protein enzim amobil. Penurunan aktivitas spesifik fi c terkait dengan pembatasan sterik yang meningkat dan keterbatasan transfer massa dari enzim. Karena kurangnya luas permukaan, enzim cenderung dikemas dalam monolayer lebih dari pada permukaan, menyebabkan penurunan aktivitas (Madoery, Gattone, & Fidelio, 1995). Oleh karena itu, konsentrasi enzim awal 20,6 mg / g dipilih untuk imobilisasi PLA1 dan persiapan enzim amobil yang dihasilkan
digunakan untuk reaksi acidolysis berikutnya.