Organizing co mmittee

 
 

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Poster presentation 
196 
EXPRESSION OF RECOMBINANT EXOINULINASE Helianthus 
tuberosus IN Pichia pastoris GS115
Kh.Kh. Dolimov
1
, B.U. Zokirov
2
, D.S. Ulashev
2
, F.S. Umurqulova
2
,
Ch.F. Rakhmonova
2
, G.S. Sobirjonova
2 
 
1) 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] 
2) Shahrisabz branch of Tashkent Institute of Chemical Technology 
st. Shahrisabz Street, 20, 181306 Shahrisabz
e-mail: [e-mail] 
Today, there are more than 470 million people worldwide with diabetes and 2.1 
million people die every year.
Diabetes is one of the leading causes of blindness, kidney 
failure, heart attacks, stroke and lower limb amputations. However, more than 70% of 
people with diabetes do not have access to prevention and healthy nutrition. Currently, 
the world uses fructose monosaccharide obtained from ground pear (
Helianthus 
tuberosus
) for healthy nutrition and prevention of this disease. 
At the present time, inulin, which is a valuable ingredient of Jerusalem artichoke 
(
Helianthus tuberosus
), is hydrolyzed to fructose only by yeast enzymes. However, 
recent studies have shown that native 
Helianthus tuberosus
inulinase is 1.5 times more 
active than the yeast inulinase enzyme. Based on this, to use of the native artichoke 
inulinase on a technological scale remains an urgent task. In the course of our research, 
we studied the change in the expression level of 
Helianthus tuberosus 
recombinant 
exoinulinase in 
Pichia pastoris
yeast depending on time, the amount of methanol in the 
nutrient medium and the secretion level of the target protein (Table 1).
Table 1. Changes in the expression level of 
Helianthus tuberosus
recombinant 
exoinulinase depending on the methanol amount and time 
Methanol 
concentration (%) 
Incubation time 
Dilution rate 
ELISA results 
(OD-450 nm) 
Amount of target 
protein (mg/ml) 
0.5 
48 
1:30000 
1.504 
0.811 
72 
1:30000 
1.996 
1.11 
96 
1:30000 
1.78 
0.96 
1 
48 
1:30000 
1.517 
0.823 
72 
1:30000 
2.05 
1.115 
96 
1:30000 
1.79 
0.967 
1.5 
48 
1:30000 
1.501 
0.803 
72 
1:30000 
1.802 
1.017 
96 
1:30000 
1.74 
0.937 
Standard sample (commercial inulinase, 
“Evergreen Chemical Factory Co., Ltd”, China) 
1:10000 
≥ 3000 (out) 
≥1.5 
1:30000 
2.915 
1.5 
Negative control (
Pichia pastoris
yeast lysate) 
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0.087 
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The table shows that the highest level of enzyme expression occurs at the 72 hours of 
cultivation when 1.0% methanol is added to the nutrient medium every 24 hours. 
Nevertheless, we can observe a very close result at 72 hours of cultivation, when every 24 
hours 0.5% methanol is added to the nutrient medium. The difference between these two 
results does not justify the cost of methanol. In this regard, we determined that the 
methanol amount - 0.5% and the duration of cultivation - 72 hours are the optimal 
conditions for the expression of recombinant 
Helianthus tuberosus
exoinulinase in 
Pichia 
pastoris
GS115. 

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Poster presentation 
197