Organizing co mmittee

V1
0
30
60
90
120
0
2
4
6
8
10
mS
\cm
V, NaOH
V1
V2
0
100
0
1
2
3
4
5
6
mS
\cm
V, NaOH
DDА = 
203,2∗100
42+1000∗𝑚
𝐶𝑁𝑎𝑂𝐻 ∗ 𝑉𝑁𝑎𝑂𝐻
(2)

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Poster presentation 
218 
FERMENTATION OF THE METHYLOTROPHIC YEAST Pichia 
pastoris FOR RECOMBINANT PROTEIN EXPRESSION
 
O.N. Ashirov, T.X. Sadullayev, A.M. Yarilkaganova, J.M. Abdurakhmanov,
Sh. Sh. Khasanov, X.N. Niyozov, S.A. Sasmakov, Sh.S. Azimova 
 
S.Yu. Yunusov Institute of the Chemistry of Plant Substances Academy of sciences of the 
Republic of Uzbekistan st. Mirzo-Ulugbek, 77, 100170 Tashkent
e-mail: [e-mail]  
Methylotrophic yeast 
Pichia pastoris
is considered one of the widely used 
microorganisms for the production of heterologous proteins.
Pichia pastoris
cells growing in a Petri dish with MD medium were inoculated into 
BMGY medium in two 250 mL flasks. The seeded cells were grown for 24 h at 30 °C 
until the optical density (OD
600
) was 2-6. For the reproduction of 
Pichia pastoris
, 
mainly substrates such as glucose, glycerol and oleic acid, but also methanol can be 
used. Generally, a 3-step fermentation process is used for protein expression in the 
P. 
pastoris
system. At the first stage, 4% glycerol is used to increase the biomass. The 
second phase is the phase of glycerol saturation. Phase III is the initiation or induction 
phase of recombinant protein expression with methanol, which differs according to each 
expression phenotype. In this phase, the target protein is expressed. The goal of the first 
two stages is to produce enough cell biomass 
First, to start the fermentation process, 9.0 L BSM saline medium containing 4% 
glycerol was placed into the fermenter and sterilized at 121 °C for 20 min. After the 
temperature in the fermenter was cooled to 30 °C, the pH was raised to 5.0 using 25% 
ammonia. In addition, 4.35 ml of PTM1 saline solution per liter of nutrient medium was 
added to the medium. After that, 500 ml of cell culture grown for 24 hours was put into 
the fermenter and the total fermentation volume was 10 l. The rotation speed of the 
fermenter was 600 rpm, the amount of dissolved oxygen (DO) in the nutrient medium 
was 30%, and the temperature was adjusted to 30°C. Thus, the first stage (phase) of the 
fermentation process began. At the end of the above process, the glycerin contained in 
the nutrient medium was completely absorbed. This indicator was determined by the 
increase in the amount of dissolved oxygen in the environment by 100%. After that, 
50% glycerol containing 12 ml of PTM1/l was taken in the pre-sterilized container and 
18.15 ml per liter of nutrient medium was added, i.e. 181.5 ml per 10 L volume. In this 
case, the amount of soluble oxygen in the fermenter should not fall below 20%. Phase 
III is the methanol fed-batch culture or induction phase, which differs according to each 
expression host phenotype. In our case, the phenotype is Mut
+
, so we added 2% 
methanol in relation to the total volume of the fermenter since the literature says 1%.