To establish a rapid quantification method for heparinase I during its production in recombinant Es-cherichia coli, a translational fusion vector was constructed by fusing the N terminus of heparinase I to the C ter-minus of a green fluorescent protein mutant (GFPmut1). As a result, not only was the functional recombinant ex-pression of heparinase I in E. coli accomplished, but also a linear correlation was obtained between the GFP fluores-cence intensity and heparinase I activity, allowing enzyme activity to be quantified rapidly during the fermentation.
Aiming at development of transformation methods for alkalophilic Bacillus pseudofirmus JCM 9141 (growth at pH>10), screening conditions of transformants by using antibiotics were first examined to overcome the antibiotic inactivation at high pH. Then, different transformation methods including competent cells, protoplast and electroporation were studied, respectively. The transformation efficiencies of competent cells and protoplast were low, while a modified electroporation method achieved higher transformation efficiency under 3000 V·cm -1. This method was also applicable to other alkalophilic Bacillus sps.
