- Teks
- Sejarah
(Zinoun, Diouris, Potin, Floc’h, &
(Zinoun, Diouris, Potin, Floc’h, & Deslandes, 1997) have been
reported. Later, Genicot-Joncour et al. (2010)demonstrated the
potential conversion of-carrageen into-carrageen with the purified sulfurylase I and II fromC. crispus. The present study for the first
time reports the purification of sulfohydrolase from agarophyteG.
duraand its potential application in improvement of commercial
agar quality substantially. The purified enzyme had the ability to
catalyze the desulfation and consequent formation of 3,6-AG. Our
study clearly demonstrates that this desulfation event is reliant
on the enzymatic reaction that is carried out by a single protein. Thus, compatible with the nucleophilic substitution reactions
proposed for the formation of anhydro ring in alkaline solutions
(Cianca, Matulewicz, & Cerezo, 1997; Viana, Noseda, Duarte, &
Cerezo, 2004).
In the present study we achieved about 60% sulfate removal
from the agar subjected to enzymatic treatment together with
1.66-fold increase in the content of 3,6-AG which was primarily
responsible for the increased gel strength. Also, the improved gel
strength and other physical properties such as gelling and melting temperature with values 31
◦
C and 82
◦
C respectively, signify
the potentiality of the enzyme extracted fromG. dura. The enzymatic treatment of agar meets the acceptable ranges of gelling and
melting temperature for commercially available agaroses and thus
could be an alternative for alkali treatment which has been reported
to time dependent inGracilariopsis lemaneiformisfor obtaining the
gelling and melting temperature with 81
◦
C and 32
◦
C respectively
Fig. 4.FT-IR spectra of control agar and sulfohydrolase treated agar.
(Gonzalez-Leija et al., 2009). The findings observed in the present
study are quite comparable with the results obtained for alkali
treatment that results in 70% sulfate removal and 1.15-fold increase
in 3, 6-AG content. In contrast, no effect of alkali treatment on desulfation of agar and consequently on the concentration of 3, 6-AG has
been reported inG.cervicornis(Freile-Pelegrin & Murano, 2005). In
another study, approximately 90% sulfate removal with a considerable increase in 3,6-AG content (1.5–2.8-fold) and gel strength
(5–9-fold) have also been reported using either alkali treatment
alone or in combination with temperature (Gonzalez-Leija et al.,
2009; Meena, Prasad, Ganeshan, & Siddhanta, 2008). The alkali
based methods have been questioned for the environmental concerns as it generates the toxic effluent if emancipate untreated.
Also, such chemical treatments results in diminished polysaccharide yield. A practical alternative to these alkali or other chemical
based methods have been proposed recently with the use of dark
treatment (Villanueva, Hilliou, & Sousa-Pinto, 2009) with an advantage of higher polysaccharide yield. Further, the effect of irradiation
with decreased sulfate content from 1.93 to 1.68% combined with
increased agarose content from 12.13 to 19.72% on dry weight basis
has been well documented (Hong, Hien, & Son, 2007).
The sulfohydrolase purified in the present study showed the
higher activity at pH range 6.0–9.0 with an optimum being at pH 8
and 35
◦
C. Our results are in agreement with the earlier report (Rees,
1961a) from thePorphyra umbilicaliswhere the optimum activity
of enzyme being at pH 7.6–7.8. Similarly, the two enzymes namely
sulfurylase I (MW, 65 kDa) and II (MW, 32 kDa) fromC. crispuswith
optimum pH 8.0–9.0 for the former and 7–8 for the latter has also
been purified (Genicot-Joncour et al., 2010). On the contrary, the
enzyme fromC. crispuswith ability to convert-carrageenan into
the-carrageenan has exhibited the maximum activity at pH 6.5
and 40
◦
C(Wong & Craigie, 1978). Recently, sulfatase purified from
Sphingomonas(Kim et al., 2004) has been employed for improving
the agar quality substantially.
The effect of additives particularly EDTA, PMSF, Ca
2+
and Mg
2+
were of great interest as both EDTA and PMSF inhibit the enzyme
activity while divalent cations such as Ca
2+
and Mg
2+
enhanced
the activity of the enzyme. Inhibitory action of the PMSF suggests
the existence of serine residue at the active site. In addition, EDTA
induced enzyme inhibition indicates that divalent cations may play
a crucial role during catalytic process. The divalent ions (Ca
2+
and
Mg2+
) presumably stabilize the negative charges developed in the
sulfate during the nucleophilic attack by the hydroxyl group of
serine.
Gelation is preceded by disordered (or less ordered)–ordered
transition of the macromolecular conformation and many experimental data support the dimeric structure of agarose in the ordered
reported. Later, Genicot-Joncour et al. (2010)demonstrated the
potential conversion of-carrageen into-carrageen with the purified sulfurylase I and II fromC. crispus. The present study for the first
time reports the purification of sulfohydrolase from agarophyteG.
duraand its potential application in improvement of commercial
agar quality substantially. The purified enzyme had the ability to
catalyze the desulfation and consequent formation of 3,6-AG. Our
study clearly demonstrates that this desulfation event is reliant
on the enzymatic reaction that is carried out by a single protein. Thus, compatible with the nucleophilic substitution reactions
proposed for the formation of anhydro ring in alkaline solutions
(Cianca, Matulewicz, & Cerezo, 1997; Viana, Noseda, Duarte, &
Cerezo, 2004).
In the present study we achieved about 60% sulfate removal
from the agar subjected to enzymatic treatment together with
1.66-fold increase in the content of 3,6-AG which was primarily
responsible for the increased gel strength. Also, the improved gel
strength and other physical properties such as gelling and melting temperature with values 31
◦
C and 82
◦
C respectively, signify
the potentiality of the enzyme extracted fromG. dura. The enzymatic treatment of agar meets the acceptable ranges of gelling and
melting temperature for commercially available agaroses and thus
could be an alternative for alkali treatment which has been reported
to time dependent inGracilariopsis lemaneiformisfor obtaining the
gelling and melting temperature with 81
◦
C and 32
◦
C respectively
Fig. 4.FT-IR spectra of control agar and sulfohydrolase treated agar.
(Gonzalez-Leija et al., 2009). The findings observed in the present
study are quite comparable with the results obtained for alkali
treatment that results in 70% sulfate removal and 1.15-fold increase
in 3, 6-AG content. In contrast, no effect of alkali treatment on desulfation of agar and consequently on the concentration of 3, 6-AG has
been reported inG.cervicornis(Freile-Pelegrin & Murano, 2005). In
another study, approximately 90% sulfate removal with a considerable increase in 3,6-AG content (1.5–2.8-fold) and gel strength
(5–9-fold) have also been reported using either alkali treatment
alone or in combination with temperature (Gonzalez-Leija et al.,
2009; Meena, Prasad, Ganeshan, & Siddhanta, 2008). The alkali
based methods have been questioned for the environmental concerns as it generates the toxic effluent if emancipate untreated.
Also, such chemical treatments results in diminished polysaccharide yield. A practical alternative to these alkali or other chemical
based methods have been proposed recently with the use of dark
treatment (Villanueva, Hilliou, & Sousa-Pinto, 2009) with an advantage of higher polysaccharide yield. Further, the effect of irradiation
with decreased sulfate content from 1.93 to 1.68% combined with
increased agarose content from 12.13 to 19.72% on dry weight basis
has been well documented (Hong, Hien, & Son, 2007).
The sulfohydrolase purified in the present study showed the
higher activity at pH range 6.0–9.0 with an optimum being at pH 8
and 35
◦
C. Our results are in agreement with the earlier report (Rees,
1961a) from thePorphyra umbilicaliswhere the optimum activity
of enzyme being at pH 7.6–7.8. Similarly, the two enzymes namely
sulfurylase I (MW, 65 kDa) and II (MW, 32 kDa) fromC. crispuswith
optimum pH 8.0–9.0 for the former and 7–8 for the latter has also
been purified (Genicot-Joncour et al., 2010). On the contrary, the
enzyme fromC. crispuswith ability to convert-carrageenan into
the-carrageenan has exhibited the maximum activity at pH 6.5
and 40
◦
C(Wong & Craigie, 1978). Recently, sulfatase purified from
Sphingomonas(Kim et al., 2004) has been employed for improving
the agar quality substantially.
The effect of additives particularly EDTA, PMSF, Ca
2+
and Mg
2+
were of great interest as both EDTA and PMSF inhibit the enzyme
activity while divalent cations such as Ca
2+
and Mg
2+
enhanced
the activity of the enzyme. Inhibitory action of the PMSF suggests
the existence of serine residue at the active site. In addition, EDTA
induced enzyme inhibition indicates that divalent cations may play
a crucial role during catalytic process. The divalent ions (Ca
2+
and
Mg2+
) presumably stabilize the negative charges developed in the
sulfate during the nucleophilic attack by the hydroxyl group of
serine.
Gelation is preceded by disordered (or less ordered)–ordered
transition of the macromolecular conformation and many experimental data support the dimeric structure of agarose in the ordered
0/5000
(Zinoun, Diouris, Potin, Floc’h, & Deslandes, 1997) have beenreported. Later, Genicot-Joncour et al. (2010)demonstrated thepotential conversion of-carrageen into-carrageen with the purified sulfurylase I and II fromC. crispus. The present study for the firsttime reports the purification of sulfohydrolase from agarophyteG.duraand its potential application in improvement of commercialagar quality substantially. The purified enzyme had the ability tocatalyze the desulfation and consequent formation of 3,6-AG. Ourstudy clearly demonstrates that this desulfation event is relianton the enzymatic reaction that is carried out by a single protein. Thus, compatible with the nucleophilic substitution reactionsproposed for the formation of anhydro ring in alkaline solutions(Cianca, Matulewicz, & Cerezo, 1997; Viana, Noseda, Duarte, &Cerezo, 2004).In the present study we achieved about 60% sulfate removalfrom the agar subjected to enzymatic treatment together with1.66-fold increase in the content of 3,6-AG which was primarilyresponsible for the increased gel strength. Also, the improved gelstrength and other physical properties such as gelling and melting temperature with values 31◦C and 82◦C respectively, signifythe potentiality of the enzyme extracted fromG. dura. The enzymatic treatment of agar meets the acceptable ranges of gelling andmelting temperature for commercially available agaroses and thusbisa menjadi alternatif untuk pengobatan alkali yang telah dilaporkanuntuk waktu tergantung inGracilariopsis lemaneiformisfor memperolehgelling dan mencair suhu dengan 81◦C dan 32◦C masing-masingGambar 4.FT-IR spektrum kontrol agar-agar dan sulfohydrolase diperlakukan agar-agar.(Gonzalez-Leija et al., 2009). Temuan-temuan yang diamati di masa kiniStudi cukup sebanding dengan hasil yang diperoleh untuk alkalipengobatan yang mengakibatkan penghapusan sulfat 70% dan meningkatkan 1.15-fold3, 6-AG konten. Di sebaliknya, tidak ada efek pengobatan alkali pada desulfation agar dan akibatnya pada konsentrasi 3, memiliki 6-AGtelah dilaporkan inG.cervicornis (Freile-Pelegrin & Murano, 2005). DalamStudi lain, sekitar 90% sulfat penghapusan dengan peningkatan yang cukup isi 3,6-AG (1.5-2,8-lipat) dan kekuatan gel(5-9-lipat) juga telah dilaporkan menggunakan pengobatan alkali baiksendiri atau dalam kombinasi dengan suhu (Gonzalez-Leija et al.,2009; Meena, Prasad, Demus, & Siddhanta, 2008). AlkaliBerdasarkan metode telah dipertanyakan untuk masalah lingkungan hidup seperti itu menghasilkan limbah beracun jika membebaskan tidak diobati.Juga, seperti perawatan kimia hasil dalam hasil polisakarida berkurang. Alternatif yang praktis untuk alkali ini atau kimia lainnyaBerdasarkan metode telah diusulkan baru-baru ini dengan menggunakan gelappengobatan (Villanueva, Hilliou, & Sousa-Pinto, 2009) dengan keuntungan hasil polisakarida yang lebih tinggi. Selanjutnya, pengaruh iradiasidengan penurunan sulfat konten dari 1. 93% 1,68 dikombinasikan denganmeningkatkan agarose konten dari 12,13% 19.72 pada dasar berat keringtelah didokumentasikan dengan baik (Hong, Hien, & Son, 2007).Sulfohydrolase yang disucikan dalam penelitian ini menunjukkanaktivitas yang lebih tinggi pada pH berkisar 6,0-9,0 dengan menjadi optimal pada pH 8dan 35◦C. kami hasil yang sesuai dengan laporan sebelumnya (Rees,1961a) dari thePorphyra umbilicaliswhere aktivitas optimalenzim berada pada pH 7,6 – 7.8. Demikian pula, enzim dua yaitusulfurylase saya (MW, 65 kDa) dan fromC II (MW, 32 kDa). crispuswithoptimal pH 9.0-8,0 untuk mantan dan 7-8 untuk yang terakhir juga telahtelah dimurnikan (Genicot-Joncour et al., 2010). Sebaliknya,fromC enzim. crispuswith kemampuan untuk mengubah - carrageenan ke-carrageenan telah menunjukkan aktivitas maksimum pada pH 6.5dan 40◦C (Wong & Craigie, 1978). Baru-baru ini, sulfatase dimurnikan dariSphingomonas (Kim et al., 2004) telah bekerja untuk meningkatkankualitas agar-agar secara substansial.Efek dari aditif khususnya EDTA, PMSF, Ca2 +dan Mg2 +itu sangat menarik seperti EDTA dan PMSF menghambat enzimkegiatan sementara divalent kation seperti Ca2 +dan Mg2 +ditingkatkanaktivitas enzim. Penghambatan tindakan PMSF menunjukkanadanya Serin residu di situs aktif. Selain itu, EDTAinhibisi enzim diinduksi menunjukkan bahwa divalent kation mungkin memainkanperan penting selama proses katalitik. Ion divalent (Ca2 +danMG2 +) mungkin menstabilkan biaya negatif yang dikembangkan disulfat selama serangan nukleofilik oleh grup hidroksilSerin.Gelation didahului oleh teratur (atau kurang memerintahkan) – memerintahkantransisi konformasi makromolekul dan banyak data eksperimen mendukung struktur dimeric agarose memerintahkan
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