反应详情

Transposon mutagenesis was carried out (Manning R. F. et al., U.S. Pat. No. 5,082,785) to construct L-sorbose reductase deficient strain from 2-keto-L-gulonic acid-producing L42-9 strain. Any strains which produce L-sorbose from D-sorbitol and assimilate the resulting L-sorbose by L-sorbose reductase of the present invention can be obtained from G. melanogenus IFO 3293 by means of multi-step mutations with chemical mutagens including NTG and ICR170, ultraviolet irradiation etc. E. coli W3110 carrying P1::Tn5 maintained on LB agar plate containing 30 μg/ml of kanamycin was inoculated into 5 ml of P1 medium (LB supplemented with 0.01 M MgSO47H2O and 10 μg/ml thymine) and cultivated at 30° C. overnight. One ml of this culture was transferred to 100 ml of the same medium in a 500 ml-Erlenmyer flask and grown to OD550 of approximately 0.09 at 30° C. for 95 min. The resulting culture was cooled on ice for 10 min and centrifuged at 3500 rpm at 4° C. for 20 min. The cells were suspended in 25 ml of P1 medium and the cell suspension was transferred to a 300 ml-flask and incubated at 42° C. for 20 min without shaking. When lysis of the cell was observed by standing at 37° C. for 90 min, the cells were lysed by adding 0.5 ml of chloroform. The mixture was then vortexed thoroughly, stood at room temperature for 10 min, and centrifuged at 10,000 rpm at 4° C. for 15 min. After the supernatant was transferred into a sterile screw-top glass bottle, 0.5 ml of chloroform was added again and the lysate was stored at 4° C.

1. customproducing L42-9 strain