of no more than 190 rpm this mixture must continue mixing for no less than 5 minutes step 11 the enzyme solution from step 6 is added to the mixture from step 10 and the now complete reagent is mixed at a rate of no less than 190 rpm for a period of no less than 30 minutes step 12 the reagent can now be filtered as needed by the manufacturing equipment by passing it through a 100 micron sieve bag or through a 100 micron filter integral to a pumping system the apoenzyme of quinoprotein glucose dehygrogenase specified above is obtained from boehringer mannheim gmbh in germany boehringer marinheim gmbh identification number 1464221 alternatively this apoenzyme may be obtained from acinetobacter calcoaceticus by the following protocol recited in duine et al febs letters vol 108 no 2 pps 443 46 acinetobacter calcoaceticus are grown on a mineral salt medium supplemented with 0 02 molar m sodium succinate or 0 10 m ethanol at 22 u00b0 c with good aeration the cells are harvested at the end of the logarithmic phase and a wet cell yield of u02dc 4 g l can be obtained frozen cells 10 g are thawed and mixed with 15 milliliters ml of 36 millimolar mm tris 39 mm glycine buffer after adding 6 milligrams mg lysozyme the suspension is stirred at room temperature for 15 min and centrifuged for 10 min at 48 000 u00d7 g the supernatant is discarded and the pellet extracted twice with 36 mm tris 39 mm glycine buffer containing 1 triton x 100 surfactant the supernatants of the centrifugation steps are.