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Studies on Coenzyme Q₁₀ Biosynthesis of Tobacco by Cell Culture Method

Author: LiYingHua
Tutor: FanHaiYan
School: Shenyang Agricultural University
Course: Botany
Keywords: Coenzyme Q10 Tobacco cell cultures Precursor substance Dynamics Elicitor
CLC: S572
Type: Master's thesis
Year: 2009
Downloads: 135
Quote: 1
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Abstract

The coenzyme Q ₁₀ is a fat-soluble natural vitamin-like substance from the in vivo delivery of the role of hydrogen donor cell energy generated elements. The the coenzyme Q ₁₀ as the activator of cell metabolism and natural antioxidants play an important role in the treatment of cardiovascular diseases and anti-tumor, can improve the body's immune system, widely used in the treatment of diseases of the human immune system. Therefore, more and more people pay attention as important biochemical drugs have medical value, good material of health food and cosmetics raw materials coenzyme Q ₁₀, the market prospect is broad. The the test tobacco varieties NC89 as test materials, using cell culture methods, to carry out the bio-coenzyme Q ₁₀ laboratory studies, to large-scale production of coenzyme Q for the future application of tobacco cell suspension culture technology ₁₀ provide experimental and theoretical basis. (1) leaves the three types of explants callus, suspension cultures of coenzyme Q ₁₀ high-yielding cell lines through state regulation good fit. Determine callus subculture medium: MS 1.0mg · L ^{-1 NAA 0.8mg · L ^{-1 6-BA 30g · L ^{-1 < / sup> sucrose 8g L ^{-1 Agar 0.5g · L ^{-1 hydrolyzed milk protein 0.5g · L ^{-1 Vitamin C 0.4g · L ^{-1 thiamine hydrochloride. Cell suspension cultures, the preferred training program: White medium, sucrose 20 g · L ^{-1 medium volume 20mL (100mL flask), inoculum size 75g · L ^{- 1 , an initial pH of 6.2, shaking speed 110r · min ^{-1 . The particle size of 0-2mm cell mass suspension culture system accounted for 36.7% ,3-4mm particle size of cell clusters accounted for 36.1%. The intracellular coenzyme Q ₁₀ content changes with the changes in particle size of the cell mass. (2) The system studied tobacco suspension cell growth and synthesis of coenzyme Q ₁₀ kinetics. Tobacco NC89 cell culture cycle for 17d, is divided into three stages of the lag phase, exponential growth phase and stationary phase. The opposite trend of the specific growth rate of the cells in the 4-17d and coenzyme Q ₁₀ ratio of the synthesis rate, the change in the concentration of total nitrogen with the cell dry weight of the cell growth is synchronized. Having a good linear relationship between the substrate concentration and biomass, the phosphate concentration changes with coenzyme Q ₁₀ Synthesis of change has a certain correlation. ② phosphate product was obtained by linear regression: the ① substrate cell biomass yield coefficient Y _{X / S} = 0.58g · g ^{-1 ; , the rate coefficient Y _{P / S = 33.4 mg · mg ^{-1 . Tobacco cell culture production of coenzyme Q ₁₀ dynamics model are as follows: ① cell growth kinetic equation: (?) ② substrate consumption dynamics equation: (?) ③ The product formation kinetic equation: (?) is determined by a nonlinear fitting parameters in the equation, the curve fitting a good description of the kinetic behavior of the tobacco cells during culture. (3) The effects of six kinds of precursor substances of tobacco NC89 cells growth and coenzyme Q ₁₀ synthesis regulation, results show that: the concentration of 1.0mg · L ^{-1 Coenzyme Q ₀ to promote cell dry weight and coenzyme Q ₁₀ production increased 48.6% and 81.6%, respectively, compared with the control; hydroxybenzoic acid concentration 5.0mg · L ^{( -10 cell dry weight and coenzyme Q ₁₀ production increased 22.6% and 186.8%, respectively, compared with the control L-tyrosine concentration of 1.0mg · L ^{-1 , cell dry weight ratio was increased by 57.8%, the coenzyme Q ₁₀ production corresponding increased 115%; L-phenylalanine concentration increased to 1.0mg L ^{-1 , cell dry weight and coenzyme Q ₁₀ production reaches a maximum 53.3%, respectively, compared with the control, 124.6%; methionine concentration of 5.0mg · L , ^{-1 10}}}}}}} yields are the highest, were increased 48.4% and 108.44% of the cell dry weight and coenzyme Q _{; solanesol promote intracellular coenzyme Q Synthesis ₁₀, but the role of cell growth is not obvious. determine the interaction of the tobacco cells coenzyme Q ₁₀ synthesis affect significantly: p-hydroxybenzoic acid and L-phenylalanine leucine, on the interaction of hydroxy benzoic acid and methionine, L-phenylalanine, alanine and methionine were significantly influence the intracellular content of the coenzyme Q ₁₀, parahydroxybenzoate 10} Yield 0:05 levels significantly. Reference precursor effects can try different stages of the culture with the interaction of L-phenylalanine on coenzyme Q _{The selection of appropriate coenzyme Q _{10 α-alanine be able to promote intracellular precursor substances and the concentration be added. (4) tobacco cell growth and coenzyme Q ₁₀ synthesis certain to add organic matter.} synthesis, but inhibition of cell growth; proline have to promote the role of the two; inositol weak influence cell growth, 10} synthesis of coenzyme Q _{significant role in promoting inositol concentration of 500mg · L ^{-1 the intracellular coenzyme Q ₁₀ production was increased by 117.5%. the leucine concentration 20mgmg · L ^{- 1 , the dry weight of the cells and coenzyme Q ₁₀ production increased by 120.6% and 61.8%, respectively, when the concentration of 30mg · L ^{-1 , cell The within Coenzyme Q ₁₀ content was increased by 36.2% (5) Add elicitor tobacco cell growth and coenzyme Q ₁₀ synthesis. concentration of 5.0mg · L ^{-1 salicylic acid allows intracellular coenzyme Q ₁₀ content increased by 25.8% compared with the control, the concentration of 2U · L ^{-1 fruit gelatinase promote the intracellular coenzyme Q ₁₀ content increased by 55.2% compared with the control, the concentration of 25mL · L ^{-1 of fulva elicitors also promote intracellular the the coenzyme Q ₁₀ content increased by 66.1% compared with the control, but all these three different degrees of inhibition of the growth of tobacco cells: mannitol on the growth of tobacco cells have a certain role, but the intracellular coenzyme Q ₁₀ synthesis inhibition; The cellulase and Verticillium dahliae elicitor NC89 cell dry weight and coenzyme Q increase in production have a certain role in promoting. ( 6) the tobacco coenzyme Q ₁₀ extraction methods of optimization and comparison results show that: the optimal conditions for the extraction of ethanol extraction: ethanol concentration of 90%, solid-liquid ratio of 1:10 -1:20, reflux time 120-150min. acetone grinding the best extraction liquid ratio of 1:3; determine the optimum extraction of alkali saponification conditions: acid digestion pH 3, the solid-liquid ratio of 1 : 10,80 ℃ reflux 90min, alkali saponification treatment pH 11, the solid-liquid ratio 1:15, reflow temperature of 100 ° C reflow time 30min. acetone grinding hexane extraction process is relatively simple, high extraction efficiency, suitable for coenzyme Q ₁₀ content of the preliminary determination. (7) studied the in vitro antioxidant activity in vitro cultured tobacco cells coenzyme Q ₁₀: the tobacco source coenzyme Q _{10 < / sub> concentration of 0.004-0.01mg · mL of ^{-1 the O pyrogallol autoxidation system that generated ₂ ^{- has · OH scavenging effect of vitamin C is roughly equal; scavenging effect and dose-effect relationship; When the concentration of 0.2-1.0mg · mL ^{-1 , when the concentration of 0.04-0.1mg · mL ^{-1 , coenzyme Q ₁₀ inhibition of mouse liver homogenate autoxidation stronger than vitamin C; concentration of 0.1 mg · mL ^{- 1 , coenzyme Q ₁₀ the mouse erythrocytes hemolysis inhibition rate 27.8% higher than the vitamin C.}}}}}}}}}}}}}}}}}}}}}}}}

Studies on Coenzyme Q10 Biosynthesis of Tobacco by Cell Culture Method

Abstract

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Studies on Coenzyme Q₁₀ Biosynthesis of Tobacco by Cell Culture Method