Amylase recombinant plasmid

Production of a-amylase by Bacillus stearothermophilus (pAT9) and Gene Manipulation to Improve the Stability of the Recombinant Plasmid... 

PRODUCTION OF a-AMYLASE BY BACILLUS STEAROTHERMOPHILUS (pAT9) AND GENE MANIPULATION TO IMPROVE THE STABILITY OF THE RECOMBINANT PLASMID... 

Broth sampled from the culture was streaked on L agar plates, and they were Incubated at 48 C for 24 to 48 h. One hundred colonies were transferred from single colonies on the plate onto LS and LSK agar plates by replica plating. These plates were Incubated at 48 C for about 24 h to count the number of colonies having the Km or Km phenotype. The same plates were overlaid with an aqueous solution of I-l2 (0.01 M) to Identify colonies exhibiting a-amylase overproduction (Amy ) or absence of overproduction (Amy ). The fraction of colonies with phenotype Km Amy In the original one hundred colonies was defined as phenotypic stability of the recombinant plasmid. 

Although vector plasmid pTB90 isolated from the natural environment [13] has been used to clone various genes (penicillinase [8], a-amylase [1] and neutral protease [9]) in B. stearothermophilus, it was apparent from cultivation of the transformant that the recombinant plasmid of penicillinase, for instance, became unstable, having lost either the cloned gene or the entire plasmid when the cultivation temperature exceeded about 50°C [2]. Whenever the recombinant plasmid becomes unstable, the transformant cultivation per se turns out to be insignificant for evaluation of cloned... 

In the cultivation of the transformant B. stearothermophilus (pAT9) to produce a-amylase, the phenotypic stability and culture temperature of the recombinant plasmid, pAT9 could be improved and elevated, respectively by the use of newly constructed recombinant plasmids, pATHP9 and pZAM26, in place of pAT9. 

Khosravi, M., Webster, D. Stark, B. (1990). Presence of the bacterial hemoglobin gene improves alpha-amylase production of a recombinant Escherichia coli strain. Plasmid, 24, 190-4. 

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