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Andrea Ballabio - One of the best experts on this subject based on the ideXlab platform.
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molecular and biochemical characterisation of a novel sulphatase gene Arylsulfatase g arsg
European Journal of Human Genetics, 2002Co-Authors: Paola Ferrante, Germana Meroni, Silvia Messali, Andrea BallabioAbstract:Molecular analysis has provided important insights into the biochemistry and genetics of the sulphatase family of enzymes. Through bioinformatic searches of the EST database, we have identified a novel gene consisting of 11 exons and encoding a 525 aa protein that shares a high degree of sequence similarity with all sulphatases and in particular with arylsulphatases, hence the tentative name Arylsulfatase G (ARSG). The highest homology is shared with Arylsulfatase A, a lysosomal sulphatase which is mutated in metachromatic leukodistrophy, particularly in the amino-terminal region. The 10 amino acids that form the catalytic site are strongly conserved. The murine homologue of Arylsulfatase G gene product shows 87% identity with the human protein. To test the function of this novel gene we transfected the full-length cDNA in Cos7 cells, and detected an Arylsulfatase G precursor protein of 62 kDa. After glycosylation the precursor is maturated in a 70 kDa form, which localises to the endoplasmic reticulum. Northern blot analysis of Arylsulfatase G revealed a ubiquitous expression pattern. We tested the sulphatase activity towards two different artificial substrates 4-methylumbelliferyl (4-MU) sulphate and p-nitrocatechol sulphate, but no arylsulphatase activity was detectable. Further studies are needed to characterise the function of Arylsulfatase G, possibly revealing a novel metabolic pathway.
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molecular and biochemical characterisation of a novel sulphatase gene Arylsulfatase g arsg
European Journal of Human Genetics, 2002Co-Authors: Paola Ferrante, Germana Meroni, Silvia Messali, Andrea BallabioAbstract:Molecular and biochemical characterisation of a novel sulphatase gene: Arylsulfatase G (ARSG)
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biochemical characterization of Arylsulfatase e and functional analysis of mutations found in patients with x linked chondrodysplasia punctata
American Journal of Human Genetics, 1998Co-Authors: Aurora Daniele, Giancarlo Parenti, Andrea Ballabio, Maddalena Daddio, Generoso Andria, Germana MeroniAbstract:X-linked chondrodysplasia punctata (CDPX) is a congenital disorder characterized by abnormalities in cartilage and bone development. Mutations leading to amino acid substitutions were identified recently in CDPX patients, in the coding region of the Arylsulfatase E (ARSE) gene, a novel member of the sulfatase gene family. Transfection of the ARSE full-length cDNA, in Cos7 cells, allowed us to establish that its protein product is a 60-kD precursor, which is subject to N-glycosylation, to give a mature 68-kD form that, unique among sulfatases, is localized to the Golgi apparatus. Five missense mutations found in CDPX patients were introduced into wild-type ARSE cDNA by site-directed mutagenesis. These mutants were transfected into Cos7 cells, and the Arylsulfatase activity and biochemical properties were determined, to study the effect of these substitutions on the ARSE protein. One of the mutants behaves as the wild-type protein. All four of the other mutations resulted in a complete lack of Arylsulfatase activity, although the substitutions do not appear to affect the stability and subcellular localization of the protein. The loss of activity due to these mutations confirms their involvement in the clinical phenotype and points to the importance of these residues in the correct folding of a catalytically active ARSE enzyme.
Xueyan Wang - One of the best experts on this subject based on the ideXlab platform.
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enzymatic desulfation of the red seaweeds agar by marinomonas Arylsulfatase
International Journal of Biological Macromolecules, 2016Co-Authors: Xueyan Wang, Delin Duan, Xiaoting FuAbstract:Agar and sulfated galactans were isolated from the red seaweeds Gracilariopsis lemaneiformis and Gelidium amansii. A previously purified Arylsulfatase from Marinomonas sp. FW-1 was used to remove sulfate groups in agar and sulfated galactans. After enzymatic desulfation, the sulfate content decreased to about 0.16% and gel strength increased about two folds. Moreover, there was no difference between the DNA electrophoresis spectrum on the gel of the Arylsulfatase-treated agar and that of the commercial agarose. In order to reveal the desulfation ratio and site, chemical and structural identification of sulfated galactan were carried out. G. amansii sulfated galactan with 7.4% sulfated content was composed of galactose and 3,6-anhydro-L-galactose. Meanwhile, G. lemaneiformis sulfated galactan with 8.5% sulfated content was composed of galactose, 3,6-anhydro-L-galactose, 2-O-methyl-3,6-anhydro-L-galactose and xylose. Data from C-13 NMR, FT-IR, GC-MS provided evidence of sulfate groups at C-4 and C-6 of d-galactose and C-6 of I-galactose both in GRAP and GEAP. Data from GC-MS revealed that desulfation was carried out by the Arylsulfatase at the sulfate bonds at C-4 and C-6 of d-galactose and C-6 of l-galactose, with a desulfation ratio of 83.4% and 86.0% against GEAP and GRAP, respectively. (C) 2016 Elsevier B.V. All rights reserved.
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enzymatic desulfation of the red seaweeds agar by marinomonas Arylsulfatase
International Journal of Biological Macromolecules, 2016Co-Authors: Xueyan Wang, Delin DuanAbstract:Agar and sulfated galactans were isolated from the red seaweeds Gracilariopsis lemaneiformis and Gelidium amansii. A previously purified Arylsulfatase from Marinomonas sp. FW-1 was used to remove sulfate groups in agar and sulfated galactans. After enzymatic desulfation, the sulfate content decreased to about 0.16% and gel strength increased about two folds. Moreover, there was no difference between the DNA electrophoresis spectrum on the gel of the Arylsulfatase-treated agar and that of the commercial agarose. In order to reveal the desulfation ratio and site, chemical and structural identification of sulfated galactan were carried out. G. amansii sulfated galactan with 7.4% sulfated content was composed of galactose and 3,6-anhydro-l-galactose. Meanwhile, G. lemaneiformis sulfated galactan with 8.5% sulfated content was composed of galactose, 3,6-anhydro-l-galactose, 2-O-methyl-3,6-anhydro-l-galactose and xylose. Data from 13C NMR, FT-IR, GC-MS provided evidence of sulfate groups at C-4 and C-6 of d-galactose and C-6 of l-galactose both in GRAP and GEAP. Data from GC-MS revealed that desulfation was carried out by the Arylsulfatase at the sulfate bonds at C-4 and C-6 of d-galactose and C-6 of l-galactose, with a desulfation ratio of 83.4% and 86.0% against GEAP and GRAP, respectively.
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characterization of a novel alkaline Arylsulfatase from marinomonas sp fw 1 and its application in the desulfation of red seaweed agar
Journal of Industrial Microbiology & Biotechnology, 2015Co-Authors: Xueyan Wang, Jiachao Xu, Delin Duan, Xin Gao, Xiaoting FuAbstract:A bacterial strain capable of hydrolyzing sulfate ester bonds of p-nitrophenyl sulfate (pNPS) and agar was isolated from the coast area of Qingdao, China. It was identified as Marinomonas based on its 16S rRNA gene sequence and named as Marinomonas sp. FW-1. An Arylsulfatase with a recovery of 13 % and a fold of 12 was purified to a homogeneity using ion exchange and gel filtration chromatographies. The enzyme was composed of a single polypeptide chain with the molecular mass of 33 kDa estimated using SDS-PAGE. The optimal pH and temperature of Arylsulfatase were pH 9.0 and 45, respectively. Arylsulfatase was stable over pH 8–11 and at temperature below 55 °C. The K m and V max of this enzyme for the hydrolysis of pNPS were determined to be 13.73 and 270.27 μM/min, respectively. The desulfation ratio against agar from red seaweed Gelidium amansii and Gracilaria lemaneiformis were 86.11 and 89.61 %, respectively. There was no difference between the DNA electrophoresis spectrum on the gel of the Arylsulfatase-treated G. amansii agar and that of the commercial agarose. Therefore, this novel alkaline Arylsulfatase might have a great potential for application in enzymatic conversion of agar to agarose.
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characterization of a novel alkaline Arylsulfatase from marinomonas sp fw 1 and its application in the desulfation of red seaweed agar
Journal of Industrial Microbiology & Biotechnology, 2015Co-Authors: Xueyan Wang, Delin Duan, Xin GaoAbstract:A bacterial strain capable of hydrolyzing sulfate ester bonds of p-nitrophenyl sulfate (pNPS) and agar was isolated from the coast area of Qingdao, China. It was identified as Marinomonas based on its 16S rRNA gene sequence and named as Marinomonas sp. FW-1. An Arylsulfatase with a recovery of 13 % and a fold of 12 was purified to a homogeneity using ion exchange and gel filtration chromatographies. The enzyme was composed of a single polypeptide chain with the molecular mass of 33 kDa estimated using SDS-PAGE. The optimal pH and temperature of Arylsulfatase were pH 9.0 and 45, respectively. Arylsulfatase was stable over pH 8-11 and at temperature below 55 A degrees C. The K (m) and V (max) of this enzyme for the hydrolysis of pNPS were determined to be 13.73 and 270.27 mu M/min, respectively. The desulfation ratio against agar from red seaweed Gelidium amansii and Gracilaria lemaneiformis were 86.11 and 89.61 %, respectively. There was no difference between the DNA electrophoresis spectrum on the gel of the Arylsulfatase-treated G. amansii agar and that of the commercial agarose. Therefore, this novel alkaline Arylsulfatase might have a great potential for application in enzymatic conversion of agar to agarose.
Lutz Fischer - One of the best experts on this subject based on the ideXlab platform.
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A natural variant of Arylsulfatase from Kluyveromyces lactis shows no formylglycine modification and has no enzyme activity
Applied Microbiology and Biotechnology, 2018Co-Authors: Timo Stressler, Katrin Reichenberger, Paul Swietalski, Ines Seitl, Claudia Gluck, Sebastian Leptihn, Andreas Kuhn, Jens Pfannstiel, Lutz FischerAbstract:Kluyveromyces lactis is a common fungal microorganism used for the production of enzyme preparations such as β-galactosidases (native) or chymosin (recombinant). It is generally important that enzyme preparations have no unwanted side activities. In the case of β-galactosidase preparations produced from K. lactis, an unwanted side activity could be the presence of Arylsulfatase (EC 3.1.6.1). Due to the action of Arylsulfatase, an unpleasant “cowshed-like” off-flavor would occur in the final product. The best choice to avoid this is to use a yeast strain without this activity. Interestingly, we found that certain natural K. lactis strains express Arylsulfatases, which only differ in one amino acid at position 139. The result of this difference is that K. lactis DSM 70799 (expressing R139 variant) shows no Arylsulfatase activity, unlike K. lactis GG799 (expressing S139 variant). After recombinant production of both variants in Escherichia coli, the R139 variant remains inactive, whereas the S139 variant showed full activity. Mass spectrometric analyses showed that the important posttranslational modification of C56 to formylglycine was not found in the R139 variant. By contrast, the C56 residue of the S139 variant was modified. We further investigated the packing and secondary structure of the Arylsulfatase variants using optical spectroscopy, including fluorescence and circular dichroism. We found out that the inactive R139 variant exhibits a different structure regarding folding and packing compared to the active S139 variant. The importance of the amino acid residue 139 was documented further by the construction of 18 more variants, whereof only ten showed activity but always reduced compared to the native S139 variant.
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Detection, production, and application of microbial Arylsulfatases
Applied Microbiology and Biotechnology, 2016Co-Authors: Timo Stressler, Ines Seitl, Andreas Kuhn, Lutz FischerAbstract:Arylsulfatases are enzymes which catalyze the hydrolysis of arylsulfate ester bonds to release a free sulfonate. They are widespread in nature and are found in microorganisms, most animal and human tissues, and plant seeds. However, this review focuses on Arylsulfatases from microbial origin and gives an overview of different assays and substrates used to determine the Arylsulfatase activity. Furthermore, the production of microbial Arylsulfatases using wild-type organisms as well as the recombinant production using Escherichia coli and Kluyveromyces lactis as expression hosts is discussed. Finally, various potential applications of these enzymes are reviewed.
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homologous expression and biochemical characterization of the Arylsulfatase from kluyveromyces lactis and its relevance in milk processing
Applied Microbiology and Biotechnology, 2016Co-Authors: Timo Stressler, Desiree Leisibach, Sabine Lutzwahl, Andreas Kuhn, Lutz FischerAbstract:The industrial manufacturing process of lactose-free milk products depends on the application of commercial β-galactosidase (lactase) preparations. These preparations are often obtained from Kluyveromyces lactis. There is a gene present in the genome of K. lactis which should encode for an enzyme called Arylsulfatase (EC 3.1.6.1). Therefore, this enzyme could also be present in β-galactosidase preparations. The Arylsulfatase is suspected of being responsible for an unpleasant “cowshed-like” off-flavor resulting from the release of p-cresol from milk endogenous alkylphenol sulfuric esters. So far, no gene/functionality relationship is described. In addition, no study is available which has shown that Arylsulfatase from K. lactis is truly responsible for the flavor generation. In this study, we cloned the putative Arylsulfatase gene from K. lactis GG799 into the commercially available vector pKLAC2. The cloning strategy chosen resulted in a homologous, secretory expression of the Arylsulfatase. We showed that the heretofore putative Arylsulfatase has the desired activity with the synthetic substrate p-nitrophenyl sulfate and with the natural substrate p-cresol sulfate. The enzyme was biochemically characterized and showed an optimum temperature of 45–50 °C and an optimum pH of 9–10. Additionally, the Arylsulfatase was activated by Ca2+ ions and was inactivated by Zn2+ ions. Moreover, the Arylsulfatase was inhibited by p-cresol and sulfate ions. Finally, the enzyme was added to ultra-heat treated (UHT) milk and a sensory triangle test verified that the Arylsulfatase from K. lactis can cause an unpleasant “cowshed-like” off-flavor.
Christoph Peters - One of the best experts on this subject based on the ideXlab platform.
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Juvenile form of mucopolysaccharidosis VI (Maroteaux-Lamy syndrome). A C-terminal extension causes instability but increases catalytic efficiency of Arylsulfatase B.
The Journal of biological chemistry, 1994Co-Authors: G Arlt, Doug A. Brooks, John J. Hopwood, Julie Bielicki, Dirk Isbrandt, K Von Figura, Tessa M. Bradford, C A Bindloss-petherbridge, Christoph PetersAbstract:Abstract A deficiency of the enzyme Arylsulfatase B results in the lysosomal storage disorder Maroteaux-Lamy syndrome or mucopolysaccharidosis type VI. Severe, intermediate and mild forms of this autosomal recessively inherited disease can be clinically differentiated. To determine the molecular defect in a patient with the intermediate form of the disorder, DNA fragments generated from the patient's mRNA by reverse transcription and subsequent amplification by the polymerase chain reaction were subcloned and sequenced. The mRNA transcribed from one allele contains a 244-base pair deletion causing a frameshift and a truncation of the open reading frame. The C-terminal third of the encoded mutant polypeptide has a nonsense sequence. This mutation is due to a deletion of exon 5 in this allele. A silent A to G transition at nucleotide 1191 was present in the same allele, and the second allele was characterized by a T to C transition at nucleotide 1600 causing a mutation of the translational stop codon to a glutamine codon (*534Q) and extending the encoded polypeptide by 50 amino acids. Stable expression of the *534Q allele in LTK- cells resulted in a mutant precursor 4 kDa larger than the wild-type precursor. The majority of the mutant precursor appears to be degraded before reaching the trans Golgi. This is consistent with an altered polypeptide structure, where a number of missing or masked epitopes were observed in an enzyme immunobinding assay using a panel of monoclonal antibodies. Immunoquantification analysis showed that epitopes were most likely masked, as missing epitopes could be reformed by binding the mutant protein to a polyclonal antibody of Arylsulfatase B. It is suggested that the additional amino acids at the C terminus of the Arylsulfatase B polypeptide induce a protein conformational change. *534Q mutant polypeptide escaping degradation is sorted to dense lysosomes. The mutant polypeptide has an approximately 9-fold higher catalytic efficiency than wild-type Arylsulfatase B.
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Mucopolysaccharidosis VI (Maroteaux-Lamy syndrome). An intermediate clinical phenotype caused by substitution of valine for glycine at position 137 of Arylsulfatase B.
The Journal of biological chemistry, 1991Co-Authors: G Wicker, Doug A. Brooks, John J. Hopwood, Gary Gibson, K Von Figura, V Prill, Christoph PetersAbstract:The Maroteaux-Lamy syndrome (mucopolysaccharidosis type VI) is a lysosomal storage disease with autosomal recessive inheritance caused by deficiency of the enzyme Arylsulfatase B. Severe, intermediate, and mild forms of the disease have been described. The molecular correlate of the clinical heterogeneity is not known at present. To identify the molecular defect in a patient with the intermediate form of the disease, Arylsulfatase B mRNA from his fibroblasts was reverse-transcribed, amplified by the polymerase chain reaction, and subcloned. Three point mutations were detected by DNA sequence analysis, two of which, a silent A to G transition at nucleotide 1191 and a G to A transition at nucleotide 1126 resulting in a methionine for valine 376 substitution, were polymorphisms. A G to T transversion at nucleotide 410 causing a valine for glycine 137 substitution (G137V) was identified as the mutation underlying the Maroteaux-Lamy phenotype of the patient, who was homozygous for the allele. The kinetic parameters of the mutant Arylsulfatase B enzyme toward a radiolabeled trisaccharide substrate were normal excluding an alteration of the active site. The G137V mutation did not affect the synthesis but severely reduced the stability of the Arylsulfatase B precursor. While the wild type precursor is converted by limited proteolysis in late endosomes or lysosomes to a mature form, the majority of the mutant precursor was degraded presumably in a compartment proximal to the trans Golgi network and only a small amount escaped to the lysosomes accounting for the low residual enzyme activity in fibroblasts of a patient with the juvenile form of the disease.
Delin Duan - One of the best experts on this subject based on the ideXlab platform.
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enzymatic desulfation of the red seaweeds agar by marinomonas Arylsulfatase
International Journal of Biological Macromolecules, 2016Co-Authors: Xueyan Wang, Delin Duan, Xiaoting FuAbstract:Agar and sulfated galactans were isolated from the red seaweeds Gracilariopsis lemaneiformis and Gelidium amansii. A previously purified Arylsulfatase from Marinomonas sp. FW-1 was used to remove sulfate groups in agar and sulfated galactans. After enzymatic desulfation, the sulfate content decreased to about 0.16% and gel strength increased about two folds. Moreover, there was no difference between the DNA electrophoresis spectrum on the gel of the Arylsulfatase-treated agar and that of the commercial agarose. In order to reveal the desulfation ratio and site, chemical and structural identification of sulfated galactan were carried out. G. amansii sulfated galactan with 7.4% sulfated content was composed of galactose and 3,6-anhydro-L-galactose. Meanwhile, G. lemaneiformis sulfated galactan with 8.5% sulfated content was composed of galactose, 3,6-anhydro-L-galactose, 2-O-methyl-3,6-anhydro-L-galactose and xylose. Data from C-13 NMR, FT-IR, GC-MS provided evidence of sulfate groups at C-4 and C-6 of d-galactose and C-6 of I-galactose both in GRAP and GEAP. Data from GC-MS revealed that desulfation was carried out by the Arylsulfatase at the sulfate bonds at C-4 and C-6 of d-galactose and C-6 of l-galactose, with a desulfation ratio of 83.4% and 86.0% against GEAP and GRAP, respectively. (C) 2016 Elsevier B.V. All rights reserved.
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enzymatic desulfation of the red seaweeds agar by marinomonas Arylsulfatase
International Journal of Biological Macromolecules, 2016Co-Authors: Xueyan Wang, Delin DuanAbstract:Agar and sulfated galactans were isolated from the red seaweeds Gracilariopsis lemaneiformis and Gelidium amansii. A previously purified Arylsulfatase from Marinomonas sp. FW-1 was used to remove sulfate groups in agar and sulfated galactans. After enzymatic desulfation, the sulfate content decreased to about 0.16% and gel strength increased about two folds. Moreover, there was no difference between the DNA electrophoresis spectrum on the gel of the Arylsulfatase-treated agar and that of the commercial agarose. In order to reveal the desulfation ratio and site, chemical and structural identification of sulfated galactan were carried out. G. amansii sulfated galactan with 7.4% sulfated content was composed of galactose and 3,6-anhydro-l-galactose. Meanwhile, G. lemaneiformis sulfated galactan with 8.5% sulfated content was composed of galactose, 3,6-anhydro-l-galactose, 2-O-methyl-3,6-anhydro-l-galactose and xylose. Data from 13C NMR, FT-IR, GC-MS provided evidence of sulfate groups at C-4 and C-6 of d-galactose and C-6 of l-galactose both in GRAP and GEAP. Data from GC-MS revealed that desulfation was carried out by the Arylsulfatase at the sulfate bonds at C-4 and C-6 of d-galactose and C-6 of l-galactose, with a desulfation ratio of 83.4% and 86.0% against GEAP and GRAP, respectively.
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characterization of a novel alkaline Arylsulfatase from marinomonas sp fw 1 and its application in the desulfation of red seaweed agar
Journal of Industrial Microbiology & Biotechnology, 2015Co-Authors: Xueyan Wang, Jiachao Xu, Delin Duan, Xin Gao, Xiaoting FuAbstract:A bacterial strain capable of hydrolyzing sulfate ester bonds of p-nitrophenyl sulfate (pNPS) and agar was isolated from the coast area of Qingdao, China. It was identified as Marinomonas based on its 16S rRNA gene sequence and named as Marinomonas sp. FW-1. An Arylsulfatase with a recovery of 13 % and a fold of 12 was purified to a homogeneity using ion exchange and gel filtration chromatographies. The enzyme was composed of a single polypeptide chain with the molecular mass of 33 kDa estimated using SDS-PAGE. The optimal pH and temperature of Arylsulfatase were pH 9.0 and 45, respectively. Arylsulfatase was stable over pH 8–11 and at temperature below 55 °C. The K m and V max of this enzyme for the hydrolysis of pNPS were determined to be 13.73 and 270.27 μM/min, respectively. The desulfation ratio against agar from red seaweed Gelidium amansii and Gracilaria lemaneiformis were 86.11 and 89.61 %, respectively. There was no difference between the DNA electrophoresis spectrum on the gel of the Arylsulfatase-treated G. amansii agar and that of the commercial agarose. Therefore, this novel alkaline Arylsulfatase might have a great potential for application in enzymatic conversion of agar to agarose.
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characterization of a novel alkaline Arylsulfatase from marinomonas sp fw 1 and its application in the desulfation of red seaweed agar
Journal of Industrial Microbiology & Biotechnology, 2015Co-Authors: Xueyan Wang, Delin Duan, Xin GaoAbstract:A bacterial strain capable of hydrolyzing sulfate ester bonds of p-nitrophenyl sulfate (pNPS) and agar was isolated from the coast area of Qingdao, China. It was identified as Marinomonas based on its 16S rRNA gene sequence and named as Marinomonas sp. FW-1. An Arylsulfatase with a recovery of 13 % and a fold of 12 was purified to a homogeneity using ion exchange and gel filtration chromatographies. The enzyme was composed of a single polypeptide chain with the molecular mass of 33 kDa estimated using SDS-PAGE. The optimal pH and temperature of Arylsulfatase were pH 9.0 and 45, respectively. Arylsulfatase was stable over pH 8-11 and at temperature below 55 A degrees C. The K (m) and V (max) of this enzyme for the hydrolysis of pNPS were determined to be 13.73 and 270.27 mu M/min, respectively. The desulfation ratio against agar from red seaweed Gelidium amansii and Gracilaria lemaneiformis were 86.11 and 89.61 %, respectively. There was no difference between the DNA electrophoresis spectrum on the gel of the Arylsulfatase-treated G. amansii agar and that of the commercial agarose. Therefore, this novel alkaline Arylsulfatase might have a great potential for application in enzymatic conversion of agar to agarose.
