Construction of a thermostable pullulanase producing bacillus strain and its fermentation studies

A codon optimized thermostable pullunase gene(Bn pul B) was synthesized and cloned behind an artificial tandom promter Pga2. The synthetic cassette was used to generate a suicide plasmid p GE-BPB;transformation of the plasmid into Bacillus subtilis 168 led to a pullulanase producing recombinant strain CH-1with native npr E deleted. Initially,30.3 U/m L of extracellular pullulanase was detected by CH-1 on regular medium supplemented with glucose,up to 268 U/m L of enzyme was detected by fermentation at 32 ℃ in medium with initial p H at 6.2,using 45 g/L of sucrose as carbon source and 60 g/L of wheat bran plus bean pulp as nitrogen source.