2.2 Cell cultivation and hLYZ induction methods
The fed-batch cultivation for hLYZ production by MutSP. pastoris was implemented in a 5 L fermentor (BLBIO-5GJ-3-H, Bailun Bio Co., China). The working volumes were about 3.0 L. Inoculation and aeration rate were at 20% (v v-1) and 4 vvm. Temperature and pH were maintained at and 6.0. In the glycerol batch phase, DO was maintained above 10% by manually increasing agitation rate. When glycerol was exhausted and DO suddenly rose up, the glycerol fed-batch phase was initiated. With the help of glycerol feeding control strategies A, B or C, to be described below, the glycerol feeding rate was automatically/manually regulated by the programmable peristaltic pump (BT00-100 M, Langer Co., China) and the duration of this phase were about 23 h. The methanol induction phase was initiated by feeding methanol after glycerol was used out. During the methanol induction phase, methanol concentration was controlled around 5-10 g L-1 using the same procedures described in our previous reports.1,10 Air was used for aeration throughout the entire methanol induction phase, the aeration rate, temperature and pH remained unchanged.
Strategy A (high DO-low glycerol concentration): with the help of DO-stat control mode, DO was controlled at 20-80% and glycerol concentration naturally stayed at 0-1 g L-1. When the base-line of DO could not be control over 20%, pure oxygen was used to aerate.
Strategy B (low DO-high glycerol concentration): glycerol concentration was controlled at 2-5 g L-1 by manually regulating glycerol feeding rate based off-line measured data and DO naturally stayed at almost zero level under the ordinary air-aeration conditions.
Strategy C (periodic DO control): with the aid of strategy A and B, the strategy periodically switched the cultivation environment from “high DO-low glycerol concentration” (DO 20-80%, glycerol concentration 0-1 g L-1; T1≈5 h) to “low DO-high glycerol concentration” (DO ~0%, glycerol concentration 2-5 g L-1; T2≈1 h) for 4 cycles.