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Microbiology and Biotechnology Letters

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Microbiol. Biotechnol. Lett. 2007; 35(1): 11-16

E. coli에서 Pseudoalteromonas carageenovora 유래 Arylsulfatase의 구성적 발현과 Agarose 제조에의 응용

Constitutive Expression of Arylsulfatase from Pseudoalteromonas carageenovora in E. coli and Its Application to Preparation of Agarose

Mi-Jin Kim , Yhon-Hwa Jang , Moon-Hee Sung , Yeon-Hee Kim and Soo-Wan Nam

Department of Biotechnology and Bioengineering, Department of Biomaterial Control, Dong-Eui University, 1Department of Biotechnology and Bioengineering, Department of Biomaterial Control, Dong-Eui University, 2BioLeaders Corp, 3Department of Biotechnology, Osaka University, 4Department of Biotechnology and Bioengineering, Department of Biomaterial Control, Dong-Eui University

Abstract

The arylsulfatase gene (astA, 984 bp ORF) from Pseudoalteromonas carrageenovora genome was amplified by PCR and subcloned into the pHCE-IA vector, in which the hyper consitutive expression (HCE) promoter from the D-amino acid aminotransferase (D-AAT) gene of Geobacillus toevii was employed. The transformant cell, Escherichia coli BL21 (DE3)/pHCE-AST, on LB agar plate containig 4-methylumbelliferyl sulfate, showed an intense fluorescence at 360 nm, indicating that 4-methylumbelliferone was liberated by desulfatate activity. When BL21 (DE3)/pHCE-AST was grown on LB media containing 0.4% glucose or 0.4% glycerol, the arylsulfatase activity was higher at glycerol rather than at glucose. On 2% glycerol medium, the arylsulfatase activity reached 15.0 unit/ml, which was 2.6-fold higher expression level than that with 1% glycerol. The DNA ladder in agarose prepared from agar by this recombinant enzyme revealed similar resolution and migration patterns with a commercial agarose. This results suggests that arylsulfatase overexpressed in E. coli could be applicable to the economic production of electrophoretic-grade agarose.

Keywords: Arylsulfatase, constitutive HCE promoter, Pseudoalteromonas carrageenovora, agarose, E. coli

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