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.