Mutagenesis

The DCase gene of Agrobacterium sp. KNK 712 was separated from recombinant plasmid pAD108 and then inserted into M13mp18. Random mutation was performed using two mutagens, hydroxylamine hydrochloride and NaNO2 (14,15). For the mutation with hydroxylamine, recombinant phage particles containing the DCase gene were treated with 0.25 M mutagen (pH 6.0) for 1-8 h at 37 °C. For the mutation with NaNO2, single-strand recombinant phage DNA was treated with 0.9 M mutagen (pH 4.3) for 30 min at 25 °C. After preparation of double-strand phage DNA, the 1.8-kb EcoRI-HindIII fragment corresponding to the DCase gene was prepared, inserted into pUC19, and then transformed into E. coli JM109.

The recombinant clones were screened by means of a newly developed colorimetric enzyme assay to select ther-mostabilized enzyme-producing colonies. The method is as follows. For first screening for colony assaying, recombinant E. coli colonies on the plate medium (the plates being called the master plates) were transferred to sterilized filter paper and then lysed for 30 min at 37 °C by adding a lysis solution containing lysozyme and Triton X-100. After inactivation of the heat-sensitive enzymes (65 °C, 5 min), a color reagent (1 mL), a mixture of two solutions (Sol. A, 30 mM potassium phosphate buffer, pH 7.4, 3 mg/mL A-carbamoyl-D-phenylglycine, 10 mM phenol, and 0.8 u/mL D-amino acid oxidase; Sol. B, 70 u/mL horseradish peroxidase and 50 mM 4-aminoantipyrine: Sol. A:Sol. B = 100:1, mixed just before use) was added to the filter paper, followed by incubation at 37 °C. The candidate colonies, which were colored red, were separated from the master plates. These clones were then subjected to the following second screening for cell-free assaying to confirm the mutant clones.

Recombinant E. coli cells were disrupted by sonication and the heat-sensitive enzymes were then inactivated (65 °C, 10 min). The crude enzyme sample was dispensed in 150-mL aliquots onto a 96-well microtiter plate; these ali-quots were serially diluted twofold and 50 iL of the modified color reagent (Sol. A:Sol. B = 19:1) was added. The plate was incubated at 37 °C, and then the enzyme activity was measured as absorbance at 505 nm.

The candidate clones that exhibited high absorbance were separated and the enzyme genes were analyzed.

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