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Novel method for improving expression amount of alpha-galactosidase displayed on surfaces of saccharomyces cerevisiae cells prepared by fermentation tank

A technology of Saccharomyces cerevisiae cells and galactosidase, which is applied in the field of enzyme engineering to achieve the effects of increasing expression and promoting bacterial growth.

Active Publication Date: 2019-03-26
SHENYANG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] Aiming at the problems of large-scale cultivation and expression of enzyme proteins displayed on the surface of Saccharomyces cerevisiae cells in the prior art, the present invention discloses a new method for increasing the expression level of α-galactosidase displayed on the surface of Saccharomyces cerevisiae cells cultured in large quantities in fermenters

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  • Novel method for improving expression amount of alpha-galactosidase displayed on surfaces of saccharomyces cerevisiae cells prepared by fermentation tank
  • Novel method for improving expression amount of alpha-galactosidase displayed on surfaces of saccharomyces cerevisiae cells prepared by fermentation tank

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Experimental program
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Effect test

Embodiment 1

[0033] In a 10L fermentor, the seed solution of α-galactosidase displayed on the cell surface of S. %acid hydrolyzed casein, 2% galactose, 0.5% ammonium sulfate), the stirring speed is 100rpm, the air is fed at a speed of 0.5L / min, at 20°C, the induced expression is 30h, and the induced expression of α-galactosidase is centrifuged Collect the bacteria, resuspend the bacteria with a McIlvaine buffer solution of pH=5 at a concentration of 0.1 g / mL, and measure the unit enzyme activity. The results are as follows: figure 1 (corresponding to figure 1 The processing in 1) is shown. The unit enzyme activity obtained by adding 0.5% ammonium sulfate was 187.5U / g, and the recovered total enzyme activity was 4095.8U, which were 1.1 times and 1.7 times that of the control group, respectively.

Embodiment 2

[0039] In a 10L fermentor, the seed solution of α-galactosidase displayed on the surface of Saccharomyces cerevisiae cells was inoculated into the YNB-CAA medium (0.67% YNB, 0.5 % acid hydrolyzed casein, 2% galactose, 0.5% ammonium sulfate), the stirring speed is 100rpm, the air is fed in at a speed of 0.5L / min, and the expression is induced for 30h at 20°C. During the induction process, 20% of Galactose, ensure that the concentration of galactose in the medium is 1-3%, collect the bacteria by centrifugation, and resuspend the bacteria with McIlvaine buffer with pH=5 at a concentration of 0.1g / mL. The results are as follows: figure 2 (corresponding to figure 2 As shown in the treatment 3) in , the measured unit enzyme activity was 630.2U / g, and the total enzyme activity recovered was 19632.0U, which were 3.7 times and 7.9 times that of the control group, respectively.

[0040] Comparative Example 1: In Example 2, galactose was not added during the induction process of induc...

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Abstract

The invention discloses a novel method for improving the expression amount of alpha-galactosidase displayed on surfaces of saccharomyces cerevisiae cells prepared by a fermentation tank. The novel method comprises the following steps: adding 0.05 to 0.5 percent of ammonium sulfate with the limited concentration into an induced culture medium of the alpha-galactosidase displayed on the surfaces ofthe saccharomyces cerevisiae cells in a 10L fermentation tank system, and innovatively supplementing an inducing agent in a continuous flowing adding manner to keep the concentration of 1 to 3 percent, so as to greatly improve the thallus growth amount of saccharomyces cerevisiae and the expression amount of the alpha-galactosidase displayed on the surfaces of the saccharomyces cerevisiae cells. By adopting the method provided by the invention, the unit enzyme activity and recycling total enzyme activity of the alpha-galactosidase displayed on the surfaces of the saccharomyces cerevisiae cellsare 3.7 times and 7.9 times of those of common culture. The invention provides a novel way for industrially preparing and producing the alpha-galactosidase displayed on the surfaces of the saccharomyces cerevisiae cells.

Description

technical field [0001] The invention relates to the technical field of enzyme engineering, in particular to a method for increasing the expression level of α-galactosidase displayed on the surface of Saccharomyces cerevisiae cells prepared in a fermenter. Background technique [0002] α-galactosidase (α-galactosidase, EC3.2.1.22) is an exoglycosidase, which widely exists in plants, animals and microorganisms, and can specifically catalyze α-1 in galactooligosaccharides and polysaccharides. , Hydrolysis of 6-galactosidic bonds. It is widely used in food industry, feed industry, medical pharmaceutical industry, light industry and other fields. In the current market, naturally extracted or heterologously expressed α-galactosidase has the problems of poor stability, many separation and purification steps, high cost, and cannot be reused. [0003] Displaying α-galactosidase on the cell surface of Saccharomyces cerevisiae anchors α-galactosidase on the surface of Saccharomyces c...

Claims

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Application Information

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IPC IPC(8): C12N9/40C12N15/81C12N1/19C12R1/865
CPCC12N9/2465C12N15/81C12Y302/01022
Inventor 李苏红董墨思公云李拖平
Owner SHENYANG AGRI UNIV
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