• Microbiology and Biotechnology Letters
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• v.27 no.3
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• pp.184-190
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• 1999

To acquire an industrially useful biocatalyst for the enzymatic synthesis and production of various D-amino acid aminotransferase (D-AAT) activity. The enzyme activity was found from 110 strains of isolated thermophiles revealing its wide occurrence in thermophiles. Enzyme activity and thermal stability of the D-AAT producers were compared. Finally we have selected four thermophiles as producers of potent biocatalysts for the D-amino acid production; two thermophiles, Bacillus sp. Lk-1 and LK-2, having higher specific activity and two thermophiles, B. stearothermophilus KL-01 and Bacillus sp. KLS-01, having higher thermal stability than the D-AAT producers. Taxonomic and physiological characteristics of the four isolated thermophiles were described herein.

• Journal of Microbiology
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• v.40 no.1
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• pp.33-37
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• 2002

An enzymatic reaction for the production of D-alanine from D-aspartic acid and pyruvate as substrates by a thermostable D-amino acid aminotransferase (D-AAT) was investigated at various conditions In the temperature range of 40-70$\^{C}$ and pH range of 6.0-9.5. The D-AAT was produced with recombinant E. coli BL21, which hosted the chimeric plasmid pTLK2 harboring the D-AAT from the novel thermophilic Bacillus sp. LK-2. The enzyme reaction was shown to follow the Ping Pong Bi Bi mechanism. The K$\_$m/ values for D-aspartic acid and pyruvate were 4.38 mar and 0.72 mM, respectively. It was observed that competitive inhibition by D-alanine, the product of this reaction, was evident with the inhibition constant K$\_$i/ value of 0.1 mM. A unique feature of this reaction scheme is that the decorboxylation of oxaloacetic acid, one of the products, spontaneously produces pyruvate. Therefore, only a catalytic amount of pyruvate is necessary for the enzyme conversion reaction to proceed. A typical time-course kinetic study skewed that D-alanine up to 88 mM could be produced from 100 mM of D-aspartic acid with a molar yield of 1.0.

• Journal of Food Hygiene and Safety
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• v.15 no.1
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• pp.51-54
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• 2000

In the 5'-flanking region of the D-AAT, AspAT and AlaDH gene, I found three or two pairs of sequences(designated as Pl, P2, P3) which show significant similarity to the E.coli consensus sequences of -35 and -10 for promoters. The spacing between -35 and -10 is 16 to 18bp in all the three putative promoters Pl, P2 and P3 which is in good agreement with the preferred spacer length in E.coli and in B.subtilis. Therefore, the putative promoters may also function to increase the efficiency of transcriptional initiation. The most stable, double-helical“Shine-Dalgarno”pairing is formed with a free energy change(ΔG) of -13.0 kcal/mol, -9.6 kcal/mol, -15.8 kcal/mol.

• Microbiology and Biotechnology Letters
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• v.35 no.1
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• pp.11-16
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• 2007

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.