RECOMBINANT PLASMID, BACILLUS SUBTILIS TRANSFORMED THEREWITH AND PRODUCTION OF PULLULANASE-ANALOG ENZYME USING SAID BACILLUS

摘要

PURPOSE:To easily obtain a pullulanase-analog enzyme useful in the field of starch saccharification industry, by culturing Bacillus subtilis transformed with a specific plasmid in a medium. CONSTITUTION:A DNA fragment (A) capable of producing a pullulanase-analog enzyme having a molecular weight of about 2.2 Md is produced by treating a chromosome DNA of Bacillus stearothermophilus (TRS40) with a restriction enzyme HindIII. The fragment A is linked with a vector fragment (B) produced by treating vector plasmid pTB522, etc., with HindIII to obtain plasmid pPP10 (C) which is a recombinant DNA. Bacillus subtilis (D) transformed with the plasmid C is cultured in a medium containing 1-10wt.% of starch, etc., and meat extract, etc., at pH 6.5-7.3 and about 37 deg.C for 24-72hr under aeration. The obtained culture product is separated and purified to form a pullulanase- analog enzyme having the following properties. Molecular weight, 62,000 Dalton; working pH, 5.0-7.5; optimum working temperature, 60-65 deg.C; action and substrate specificity, acting to pullulan to produce panose, glucose and mannose; etc.