by heating the 1.5% (w/v) agar gel

by heating the 1.5% (w/v) agar gel with a temperature increase of
about 0.5
◦
Cmin−1
recording the temperature at which a glass bead
(15.78 mm diameter, 4.93 g) placed on top of the gel sank (Murano
et al., 1992). Apparent viscosity was measured (in 1.5% agar solution) on a Brookfield DV-II + Pro viscometer with spindle number
SC-18 (Synchrolectric Viscometer, Stoughton, MASS 02072), using
Spindle No. 1 at the speed of 60 rpm at 80
◦
C.
2.7. Preparation of xerogels of agar and desulfated agar
Phase inversion of the respective agar hydro gel samples was
done by immersing the respective samples in acetone for 48 h followed by de-solvation under vacuum for 24 h. The xerogels thus
obtained were subjected for the SEM analyses. For recording SEM
image, vacuum dried samples were mounted on a sample holder,
coated with gold and the micrographs were recorded on a LEO scanning electron microscope-LEO 1430VP (Carl-Zeiss, Germany) at an
accelerating voltage of 20 kV and 202×magnification.
2.8. FT-IR spectroscopy
The native agar and enzyme treated agar was characterized by
FT-IR analysis using a PerkinElmer FT-IR (PerkinElmer Spectrum GX
FT-IR System, USA) by using 10.0 mg of sample in 600 mg of KBr.
All spectra were the average of two counts with 10 scans each.
3. Results
3.1. Purification of sulfohyrolase
The purification steps and their results are summarized in
Table 1. The sulfohydrolase from G. durawas purified following a
combination of ammonium sulfate precipitation, ion exchange and
gel filtration chromatography. The purification steps resulted in
obtaining an enzyme with a purification fold of 86 times and a yield
of 4.1%. The specific activity of purified enzyme was 209.19 U/mg
protein at pH 8.0 and 35
◦
C. The purified sulfohydrolase appeared
as a single band in SDS–PAGE (Fig. 1) indicating that the enzyme
was purified to apparent homogeneity. The molecular weight of the
Fig. 1.SDS–poly acrylamide gel electrophoresis (SDS–PAGE) of purified sulfohyrolase. Lane 1: Protein marker with molecular weight ranging from 29 to 205 kDa
(29 kDa, carbonic anhydrase; 43 kDa, ovalbumin; 66 kDa, bovin serum albumin;
97.4 kDa phosphorylase b and 205 kDa, myosin from rabit muscle). Lane 2: Sulfohydrolase after DEAE-sepharose chromatography.
purified sulfohydrolase was estimated to be∼50 kDa, based on its
mobility calculated with the help of standard protein marker. The
results of SDS–PAGE revealed a monomeric protein to be the active
conformation of the enzyme.
The pH and temperature dependence of the purified enzyme
is shown in Fig. 2. The enzyme activity for hydrolysis of sulfate
ester bonds in p-NPS (16 mM) was determined by analyzing the
content of sulfate release in the assay mixture. For hydrolysis of pNPS the optimum pH was 8.0, though the enzyme showed higher
activities from pH 6.0–8.0. The pH dependence varied significantly
from acidic (pH 3.0–6.0) to alkaline (pH 9.0–12.0) ranges and were
relatively higher in the acidic range. The average relative activity at
acidic pH (9.0) was 34.37%
but the maximum being at pH 7.0–9.0 with 89.23%

Hasil (Bahasa Indonesia) 1:

dengan memanaskan agar-agar 1,5% (w/v) gel dengan kenaikan suhusekitar 0.5◦Cmin−1merekam suhu di mana manik-manik kaca(diameter 15.78 mm, 4.93 g) ditempatkan di atas gel tenggelam (Muranoet al., 1992). Jelas viskositas diukur (dalam larutan agar 1,5%) pada Brookfield DV-II + Pro viscometer dengan nomor spindleSC-18 (Synchrolectric Viscometer, Stoughton, massa 02072), menggunakanGelendong No. 1 pada kecepatan 60 RPM di 80◦C.2.7. persiapan xerogels agar-agar dan desulfated agarPembalikan fase sampel hydro gel agar masing-masing adalahdilakukan oleh membenamkan sampel masing-masing di aseton untuk 48 jam, diikuti oleh de solvation di bawah vakum untuk 24 h. Xerogels dengan demikianmemperoleh menjadi sasaran untuk analisis SEM. Untuk merekam SEMGambar, vakum kering sampel yang dipasang pada dudukan sampel,berlapis emas dan dipreparasi tercatat pada 1430VP mikroskop elektron scanning-LEO LEO (Carl-Zeiss, Jerman) padamempercepat tegangan sebesar 20 kV dan 202 × pembesaran.2.8. FT-IR spektroskopiAsli agar-agar dan enzim agar-agar diperlakukan ditandai denganFT-IR analisis menggunakan PerkinElmer FT-IR (PerkinElmer spektrum GXSistem FT-IR, USA) dengan menggunakan 10.0 mg sampel di 600 mg KBR.Semua spektrum yang rata-rata dua hal dengan 10 scan.3. hasil3.1. pemurnian sulfohyrolaseLangkah-langkah pemurnian dan hasilnya dirangkum dalamTabel 1. Sulfohydrolase dari durawas G. dimurnikan berikutkombinasi presipitasi amonium sulfat, pertukaran ion dangel kromatografi filtrasi. Langkah-langkah pemurnian mengakibatkanmemperoleh enzim dengan lipat pemurnian 86 kali dan hasil4.1%. Aktivitas tertentu dimurnikan enzim adalah 209.19 U/mgprotein pada pH 8,0 dan 35◦Sulfohydrolase c. dimurnikan munculsebagai satu band di SDS-halaman (Fig. 1) menunjukkan bahwa enzimdimurnikan untuk keseragaman jelas. Berat molekulGbr 1.SDS–poly Akrilamida Elektroforesis (SDS-halaman) dari murni sulfohyrolase. Lane 1: Protein penanda dengan berat molekul yang berkisar 29-205 kDa(29 kDa, karbonat anhydrase 43 kDa, ovalbumin; kDa 66, bovin serum albumin;97.4 kDa phosphorylase b dan 205 kDa, myosin dari rabit otot). Lane 2: Sulfohydrolase setelah kromatografi DEAE-sepharose.sulfohydrolase murni diperkirakan kDa be∼50, berdasarkan yangmobilitas dihitung dengan bantuan protein standar penanda. Thehasil SDS-halaman mengungkapkan protein monomeric menjadi aktifkonformasi enzim.Ketergantungan pH dan suhu enzim dimurnikanditampilkan dalam Fig. 2. Aktivitas enzim untuk hidrolisis sulfatEster Obligasi di p-NPS (16 mM) ditentukan oleh menganalisiskandungan sulfat rilis dalam campuran assay. Hidrolisis pNPS pH optimal adalah 8.0, walaupun enzim menunjukkan lebih tinggikegiatan dari pH 6,0-8,0. Ketergantungan pH bervariasidari asam (pH 3.0 – 6.0) untuk rentang alkali (pH 9.0-12,0) danrelatif lebih tinggi di kisaran asam. Aktivitas relatif rata-rata diasam pH (< 6.0) adalah 63.27% sementara pada pH basa (> 9.0) adalah 34.37%tetapi maksimum pada pH 7,0-9,0% 89.23