Agardh. The purified enzyme was sub

Agardh. The purified enzyme was subsequently used for evaluating
its abilities in improvement of agar quality based on 3–6 anhydrogalactose content, desulfation, gelling and melting temperature, gel
strength and viscosity and these values were compared with that
of untreated agar as control. The implications of our findings are
discussed in the context of desulfation and the improvement of gel
quality and yield for the commercial utilization of these sulfohydrolases in agar producing industries. We suggest that sulfohydrolase
ofG. duraallow agars to attain the conformation of agarose and
thus could be used as an alternate to alkali treatment.
2. Experimental
2.1. Materials
G. durawas collected from Veraval coast (20◦
54

N, 70◦
22

E) of
Gujarat, India. The healthy thalli were carried in a cool pack to
the laboratory under cool conditions. In order to make unialgal
material, the rhizoidal portions of the alga were removed to eliminate contaminants and were then cleaned manually with brush
in filtered autoclaved seawater to remove the epiphytic foreign
matters, freeze dried in liquid nitrogen and kept at−40
◦
C for
further use. Agar sample was purchased from Hi-Media (Product
Number RM666, Hi-Media Laboratories Pvt. Ltd, Mumbai, India),
para-nitrophenol and para-nitrophenylsulfate (p-NPS) which are
used as artificial substrates for sulfohydrolase were purchased from
Sigma–Aldrich (St. Louis, Missouri, USA). Ultra pure water used
for experiment was prepared in laboratory from a Milli-Q system
(Millipore, USA).
2.2. Enzyme extraction and purification
Seaweed extract for determination of sulfohydrolase activity was prepared under ice-cold conditions in the extraction
buffer [100 mM Tris–HCl (pH 9.5), 500 mM KCl and 10 mM-mercaptoethanol] at a proportion of 1:3 (w/v) by stirring the
suspension overnight at 4
◦
C. Extract was centrifuged at 12,000×g
for 30 min at 4
◦
C and proteins in the supernatant were precipitated between 30–70% ammonium sulfate saturation. Precipitate
was collected after centrifugation at 15,500×g for 60 min at
4
◦C and dissolved in the buffer containing 100 mM Tris–HCl (pH
7.1) and 10 mM-mercaptoethanol and dialysed against same
buffer for 24 h with the change of buffer at an interval of 4 h.
The dialysed enzyme solution was loaded on Sephadex G-50 gel
filtration chromatographic column equilibrated and eluted with
the buffer containing 100 mM Tris–HCl (pH 7.1) and 10 mM -mercaptoethanol. The active enzyme fractions were pooled and
again dialysed. The desalted enzyme solution was further purified
with DEAE-sepharose column previously equilibrated with aforementioned buffer and washed with the same buffer at a flow rate of
1mlmin−1
untill effluentA280was negligible. The enzyme solution
was eluted with increasing gradient of NaCl in aforesaid buffer and
the active fractions were pooled between 300 mM to 500 mM NaCl
gradient and used for further experiment. At each step of purification, the active fractions were analyzed by SDS–PAGE (Laemmli,
1970) using 10% polyacrylamide gel. Molecular weight markers
of 29–205 kDa (Genei, Bangalore) were also run simultaneously
at l20 V and proteins were quantified by Bradford method using
bovine serum albumin as a standard (Bradford, 1976).
2.3. Sulfohydrolase activity assay and determination of kinetic
parameters
Sulfohydrolase activity was determined by measuring the
amount of p-nitrophenol released from p-NPS. The assay mixture
containing diluted enzyme, 100 mM Tris–HCl (pH 8.0) and 25 mM
p-NPS was incubated for 30 min at 35
◦
C in water bath. The reaction was terminated by the addition of 0.2 ml of 0.2 N NaOH and the
product of the reaction p-nitrophenol was quantified spectrophotometrically by recording atA410nm. One unit of the sulfohydrolase
activity is defined as the amount of enzyme causing transformation
of 1mol of substrate per minute at optimal condition of temperature and pH. For the optimization of enzyme concentration
with agar as a substrate sulfohydrolase activity was determined
according toKim et al. (2004).
To obtain kinetics parameters sulfohydrolase activity was measured at various concentrations ranging from 2 to 20 mM of p-NPS.
The enzyme reaction was initiated by mixing aliquot of sulfohydrolase solution with the assay mixture containing 100 mM Tris–HCl
(pH 8.0) and indicated amount of substrate. ApparentKmandVmax
were determined using a Lineweaver–Burk plot.
2.4. Effect of temperature, pH and additives on enzyme activity
Optimal concentration of substrate was observed as 16 mM and
thus fixed the same for the determination of reaction rate under
different temperatures or pH conditions. Buffer solutions for the
determination of pH dependence of enzyme activity were prepared
as follows: 0.1 M acetic acid/0.1 M sodium acetate (pH 3–6.0), 0.1 M
Tris–HCl (pH 6.0–9.0), and 0.1 M glycine–NaOH (pH 9.0–12.0). To
eliminate any possibility of an influence of buffer species, enzyme
activity was measured in different buffers with overlapping pH
points. The optimum temperature for the purified sulfohydrolase
activity were measured at pH 8.0 over a temperature range of
25–65
◦
C and finally the enzyme activity was measured at pH 8.0
and temperature 35
◦
C at which activity was observed maximum.
Different metal ion and organic solvents were studied to examine
their effect on the enzymatic activity.
2.5. Desulfation of agar
Agar suspended in 100 mM Tris–HCl (pH 8.0) buffer at a proportion of 1:20 (w/v) was equilibrated for 30 min at 45
◦
C by stirring.
Sulfohydrolase enzyme (∼50 U) was added in the agar mixture
and kept at 35
◦
C for 12 h. The agar precipitate after centrifugation
was washed with 50% ethanol solution and dehydrated with acetone. The dehydrated agar sample was hydrolyzed (Wolnik, 1988)
and the amount of sulfate content in sample was determined by
inductively coupled plasma atomic emission spectroscopy (ICPAES, PerkinElmer, Optima 2000, USA). The 3,6-anhydrogalactose
content were determined by the method ofYaphe and Arsenault
(1965).
2.6. Determination of gel strength, gelling and melting
temperature and viscosity
The desulfated agar was rinsed with five volume of 50% ethanol
and dehydrated with two volume of acetone. The dehydrated agar
was dried at 80◦
C for 2 days and stored at room temperature for further analysis. For the determination of gel strength 1.5% solution of
agar was prepared in milliQ water and kept at 10
◦
C for 12 h and gel
strength (g/cm
2
at 20
◦
C) was measured using Nikkaksui-type gel
tester (Kiya Seisakusho, Ltd, Tokyo, Japan). The measurement was
performed on 1.5% (w/v) gel sample aged overnight at 4
◦
C, using a
cylindrical plunger of 1 cm in diameter. Gel strength was measured
as the required weight to break the gel. The gelling temperature was
measured by cooling a 1.5% (w/v) hot agar solution (25 ml) placed
in a glass test tube. Subsequently, temperature was allowed to
gradually drop and this drop in temperature was monitored every
1 min time interval. The temperature at which a clear depression
was formed after removing the digital thermometer was recorded
as gelling temperature. The melting temperature was determined

Hasil (Bahasa Indonesia) 1:

Agardh. Enzim dimurnikan kemudian digunakan untuk mengevaluasikemampuan dalam peningkatan kualitas agar yang didasarkan pada 3-6 anhydrogalactose konten, desulfation, gelling dan mencair suhu, gelkekuatan dan viskositas dan nilai-nilai ini dibandingkan dengan yangdari agar tidak diobati sebagai kontrol. Implikasi dari temuan kamidibahas dalam konteks desulfation dan peningkatan gelkualitas dan hasil untuk penggunaan komersial ini sulfohydrolases di agar memproduksi industri. Kami menyarankan bahwa sulfohydrolaseofG. duraallow agars untuk mencapai konformasi agarose dansehingga dapat digunakan sebagai alternatif untuk pengobatan alkali.2. eksperimental2.1. bahanG. durawas, dikumpulkan dari pantai Veraval (20◦54N, 70◦22E) dariGujarat, India. Thalli sehat dibawa dalam paket keren untukLaboratorium kondisi dingin. Untuk membuat unialgalbahan, Bagian rhizoidal alga telah disingkirkan untuk menghilangkan kontaminan dan kemudian dibersihkan secara manual dengan sikatdalam disaring air laut dengan autoclaved untuk menghapus epifit Asinghal-hal, beku kering di dalam nitrogen cair dan terus at−40◦C untukdigunakan lebih lanjut. Agar contoh dibeli dari Hi-Media (produkNomor RM666, Hi-Media laboratorium Pvt. Ltd, Mumbai, India),Para-nitrofenol dan para-nitrophenylsulfate (p-NPS) yangdigunakan sebagai buatan substrat untuk sulfohydrolase dibeli dariSigma-Aldrich (St. Louis, Missouri, USA). Ultra air murni yang digunakanuntuk percobaan ini dipersiapkan dalam laboratorium dari sistem Mili-Q(Millipore, USA).2.2. enzim ekstraksi dan pemurnianEkstrak rumput laut untuk penentuan kegiatan sulfohydrolase disiapkan dalam kondisi dingin di ekstraksipenyangga [100 mM Tris-HCl (pH 9,5), 500 mM KCl dan 10 mM - mercaptoethanol] di proporsi 1:3 (w/v) dengan pengadukansuspensi semalam di 4◦C. ekstrak disentrifugasi di 12, 000 × gselama 30 menit di 4◦C dan protein dalam supernatant yang diendapkan antara 30-70% amonium sulfat saturasi. Memicuitu dikumpulkan setelah sentrifugasi pada 15, g 500 × selama 60 menit di4◦C dan dilarutkan dalam buffer yang mengandung 100 mM Tris-HCl (pH7.1) dan 10 mM - mercaptoethanol dan dialysed terhadap samapenyangga untuk 24 h dengan perubahan penyangga pada interval 4 h.Solusi dialysed enzim yang dimuat pada gel Sephadex G-50penyaringan Kromatografi kolom equilibrated dan eluted denganbuffer yang mengandung 100 mM Tris-HCl (pH 7.1) dan 10 mM - mercaptoethanol. Pecahan enzim aktif yang menggenang danlagi dialysed. Solusi desalted enzim adalah lebih lanjut dimurnikandengan DEAE-sepharose kolom sebelumnya equilibrated dengan pengakuan buffer tersebut dan dicuci dengan buffer sama dengan laju aliran1mlmin−1sampai effluentA280was dapat diabaikan. Solusi enzimeluted dengan meningkatnya gradien NaCl dalam buffer tersebut di atas danpecahan aktif yang menggenang antara 300 mM sampai 500 mM NaClgradient and used for further experiment. At each step of purification, the active fractions were analyzed by SDS–PAGE (Laemmli,1970) using 10% polyacrylamide gel. Molecular weight markersof 29–205 kDa (Genei, Bangalore) were also run simultaneouslyat l20 V and proteins were quantified by Bradford method usingbovine serum albumin as a standard (Bradford, 1976).2.3. Sulfohydrolase activity assay and determination of kineticparametersSulfohydrolase activity was determined by measuring theamount of p-nitrophenol released from p-NPS. The assay mixturecontaining diluted enzyme, 100 mM Tris–HCl (pH 8.0) and 25 mMp-NPS was incubated for 30 min at 35◦C in water bath. The reaction was terminated by the addition of 0.2 ml of 0.2 N NaOH and theproduct of the reaction p-nitrophenol was quantified spectrophotometrically by recording atA410nm. One unit of the sulfohydrolaseactivity is defined as the amount of enzyme causing transformationof 1mol of substrate per minute at optimal condition of temperature and pH. For the optimization of enzyme concentrationwith agar as a substrate sulfohydrolase activity was determinedaccording toKim et al. (2004).To obtain kinetics parameters sulfohydrolase activity was measured at various concentrations ranging from 2 to 20 mM of p-NPS.The enzyme reaction was initiated by mixing aliquot of sulfohydrolase solution with the assay mixture containing 100 mM Tris–HCl(pH 8.0) and indicated amount of substrate. ApparentKmandVmax
were determined using a Lineweaver–Burk plot.
2.4. Effect of temperature, pH and additives on enzyme activity
Optimal concentration of substrate was observed as 16 mM and
thus fixed the same for the determination of reaction rate under
different temperatures or pH conditions. Buffer solutions for the
determination of pH dependence of enzyme activity were prepared
as follows: 0.1 M acetic acid/0.1 M sodium acetate (pH 3–6.0), 0.1 M
Tris–HCl (pH 6.0–9.0), and 0.1 M glycine–NaOH (pH 9.0–12.0). To
eliminate any possibility of an influence of buffer species, enzyme
activity was measured in different buffers with overlapping pH
points. The optimum temperature for the purified sulfohydrolase
activity were measured at pH 8.0 over a temperature range of
25–65
◦
C and finally the enzyme activity was measured at pH 8.0
and temperature 35
◦
C at which activity was observed maximum.
Different metal ion and organic solvents were studied to examine
their effect on the enzymatic activity.
2.5. Desulfation of agar
Agar suspended in 100 mM Tris–HCl (pH 8.0) buffer at a proportion of 1:20 (w/v) was equilibrated for 30 min at 45
◦
C by stirring.
Sulfohydrolase enzyme (∼50 U) was added in the agar mixture
and kept at 35
◦
C for 12 h. The agar precipitate after centrifugation
was washed with 50% ethanol solution and dehydrated with acetone. The dehydrated agar sample was hydrolyzed (Wolnik, 1988)
and the amount of sulfate content in sample was determined by
inductively coupled plasma atomic emission spectroscopy (ICPAES, PerkinElmer, Optima 2000, USA). The 3,6-anhydrogalactose
content were determined by the method ofYaphe and Arsenault
(1965).
2.6. Determination of gel strength, gelling and melting
temperature and viscosity
The desulfated agar was rinsed with five volume of 50% ethanol
and dehydrated with two volume of acetone. The dehydrated agar
was dried at 80◦
C for 2 days and stored at room temperature for further analysis. For the determination of gel strength 1.5% solution of
agar was prepared in milliQ water and kept at 10
◦
C for 12 h and gel
strength (g/cm
2
at 20
◦
C) was measured using Nikkaksui-type gel
tester (Kiya Seisakusho, Ltd, Tokyo, Japan). The measurement was
performed on 1.5% (w/v) gel sample aged overnight at 4
◦
C, using a
cylindrical plunger of 1 cm in diameter. Gel strength was measured
as the required weight to break the gel. The gelling temperature was
measured by cooling a 1.5% (w/v) hot agar solution (25 ml) placed
in a glass test tube. Subsequently, temperature was allowed to
gradually drop and this drop in temperature was monitored every
1 min time interval. The temperature at which a clear depression
was formed after removing the digital thermometer was recorded
as gelling temperature. The melting temperature was determined