Effect of oxygen supply on milk-clotting activity expressed by Paenibacillus spp. strain BD3526

Paenibacillus spp. BD3526, a novel bacteria species isolated from raw yak (Bos grunniens) milk collected in Tibet, is able to produce metalloproteinase with milk-clotting activity (MCA) in wheat bran broth. To determine the critical factor favoring enzyme productivity, response surface methodology and the BoxBehnken design were adopted to preliminarily optimize BD3526 cultivation conditions for maximum MCA in shaking flasks. The highest degree of MCA (7072 +/- 519 SU/mL) was obtained at a liquid volume of 30 mL and 300 r/min, which implies that oxygen supply is crucial for enzyme synthesis. MCA levels were enhanced as agitation speeds and aeration rates were increased in a 7-L scale fermentor. MCA in the supernatant was further boosted by adding hydrogen peroxide (H2O2) to the broth during cultivation. Maximum MCA (14778 +/- 1100 SU/mL) and MCA/proteolytic activity (PA) (33000 +/- 918) were achieved when cultivation was carried out at an agitation speed of 400 r/min, aeration rate of 5.5 L/min and H2O2 flow of 500 mmol/L at a feeding rate of 2.5 mL/h after 24 h of cultivation. (C) 2017 Elsevier Ltd. All rights reserved.