Method for efficiently introducing L-arabinose isomerase

A technology of arabinose and isomerase, which is applied in the field of efficient induction of L-arabinose isomerase, can solve the problems of unfavorable expression of L-arabinose isomerase activity, fast protein expression rate, and difficulty in folding inclusion bodies, etc., to achieve Reduce fermentation production costs, reduce production costs, and promote the effect of folding

Inactive Publication Date: 2013-01-23
J JIANGSU LIANGFENG FOODSTUFF GROUP
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  • Description
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  • Application Information

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Problems solved by technology

However, in actual operation, the protein expression rate is too fast, it is difficult to fold into active protein and form inclusion bodies, which is not conducive to the active expression of L-arabinose isomerase, thus affecting its fermentation enzyme activity

Method used

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  • Method for efficiently introducing L-arabinose isomerase
  • Method for efficiently introducing L-arabinose isomerase
  • Method for efficiently introducing L-arabinose isomerase

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Embodiment 1

[0018] Example 1 Adding D-galactose to promote the high expression of L-arabinose isomerase of A. cellulolyticus

[0019] The construction method of Escherichia coli BL21(DE3) containing A. cellulolyticus L-arabinose isomerase is as reported in the literature (Appl Microbiol Biotechnol, 2010, 86(4): 1089-1097), and the host cell used is large intestine Bacillus BL21(DE3), the source of the exogenous L-arabinose isomerase gene is Acidothermus cellulolytics ATCC 43068, and the expression vector plasmid is pET-22b(+) of the lactose operon.

[0020] When the recombinant bacteria were cultured in LB medium at 37°C until the light absorption value at 600nm was 0.6, 1mmol / L conventional inducer IPTG was added, and enzyme substrate D-galactose of different concentrations was added at the same time, induced at 30°C for 4h, and the bacteria were collected. The body was tested for fermentation enzyme activity, and the experimental results are shown in Table 1.

[0021] Table 1 Adding D-...

Embodiment 2

[0025] Example 2 Adding D-galactose to promote the high expression of L-arabinose isomerase of Bacillus stearothermophilus

[0026] The construction method of Escherichia coli BL21(DE3) containing Bacillus stearothermophilus L-arabinose isomerase is as reported in the literature (J Sci Food Agric 2010, 90(8): 1327-1333), and the host cell used is large intestine Bacillus BL21(DE3), the source of the exogenous L-arabinose isomerase gene is Bacillus stearothermophilus IAM 11001, and the expression vector plasmid is pET-22b(+) of the lactose operon.

[0027] When the recombinant bacteria were cultured in LB medium at 37°C until the light absorption value at 600nm was 0.6, 1mmol / L conventional inducer IPTG was added, and enzyme substrate D-galactose of different concentrations was added at the same time, induced at 30°C for 4h, and the bacteria were collected. The body was tested for fermentation enzyme activity, and the experimental results are shown in Table 2.

[0028] Table 2...

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Abstract

The invention discloses a method for efficiently introducing L-arabinose isomerase. According to the method, in the inducible expression process that an L-arabinose isomerase recombinant bacterium constructed by lactose operon plasmid pET-22b(+) expresses a targeted protein, enzyme substrate D-galactose is added, so that the effective folding of the L-arabinose isomerase is promoted, the activity expression of the L-arabinose isomerase is efficiently introduced, and the expression of active enzyme is effectively improved. By the method, the expression and the fermentation enzyme activity of the active L-arabinose isomerase by the L-arabinose isomerase recombinant bacterium constructed by the lactose operon plasmid pET-22b(+) are improved, so that the fermentation production cost of the g L-arabinose isomerase can be greatly reduced; and the obtained product L-arabinose isomerase is novel carbohydrase which can catalyze the D-galactose to be synthesized into high value-added functional rare sugar-D-tagatose, and has wide market prospect.

Description

technical field [0001] The invention relates to a method for efficiently inducing L-arabinose isomerase. Background technique [0002] With the increasing incidence of diseases such as diabetes and obesity, people pay more and more attention to dietary health, and try to avoid the intake of high-fat and high-energy foods. Sucrose is a traditional sweetener, but it has high energy. The development and research of new sucrose substitutes is an urgent task with important economic value and practical significance in the field of functional sweeteners. D-tagatose is a low-calorie functional monosaccharide discovered in recent years. D-tagatose is a rare ketohexose that occurs naturally. It and D-galactose are alkulose isomers. The sweetness characteristics of D-tagatose are very similar to sucrose, the sweetness is 92% of sucrose, and there is no bad smell or aftertaste, it can be used as an ideal flavor enhancer and sweetener; in addition, tagatose There are also many special...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/90C12R1/19
Inventor 季万兰江波沐万孟李晓卉丁美琴刘炯刘知远韩晓明
Owner J JIANGSU LIANGFENG FOODSTUFF GROUP
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