Enzyme production in shake flasks
Shake flask experiments were carried out in 250-ml Erlenmeyer flasks. Seed cultures were prepared by inoculating 2 ml spore suspension (108 spores/ml) to 100 ml inoculum medium and they were incubated at 30°C and 200 rpm for 48 h. Elevenmilliliters aliquots of the inoculums were then used to initiate the growth in 250-ml Erlenmeyer flasks containing 100 ml enzyme production medium and the cultures were incubated at 30°C and 200 rpm for 7 days. Samples were with drawn daily and were centrifuged at 14,972×g, at 4°C for 5 min. Supernatants were then assayed for enzyme activities and the soluble protein content.