Process for extracellularly producing recombinant alpha-cyclodextrin glucosyltransferase

Abstract

The invention discloses a process for producing recombinant alpha-cyclodextrin glucosyltransferase in a fermenting way by culturing escherichia coli in high density through a temperature two-stage control strategy and a constant oxygen-dissolved and feed-supplemented batch technology, belonging to the technical field of fermentation engineering. The method comprises the following steps of: inoculating recombinant escherichia coli BL21(DE3) as a production strain at the inoculation quantity of 5-10 percent and fermenting in batch; starting adding a supplementing liquid in a flowing mode when dissolved oxygen rises to about 80-100 percent, ensuring that a strain body exponentially grows, controlling the temperature of the growth phrase of the strain body to be 33-37DEG C and maintaining the dissolved oxygen to 20-30 percent; adding 0.75-1.5 percent (mass volume percent) of glycine when the strain body OD600 reaches about 15-30; reducing the temperature to be 23-27DEG C and continuously supplementing 0.2-0.4g.l<-1>.h<-1> when the strain body OD600 reaches 45-60 and supplementing the supplementing liquid by adopting a gradient diminishing mode at the same time; and enabling the enzymeactivity of the extracellular alpha-cyclodextrin glucosyltransferase to reach 200-280U / ml by fermenting culture for 30-35 hours. By adopting the strategy to ferment, the invention realizes the high-efficiency extracellular expression of the alpha-cyclodextrin glucosyltransferase and greatly improves the production intensity. The invention has the advantages of wide source of raw materials, simple and feasible process and suitability for large-scale production; with effective extracellular expression, the pollution on host bacteria hybrid proteins can be greatly reduced and the purification efficiency of the protein can be improved. The invention lays a foundation for large-scale production of the alpha-cyclodextrin glucosyltransferase.

Description

technical field

[0001] The invention relates to a production process for extracellular production of recombinant α-cyclodextrin glucosyltransferase, which belongs to the technical field of fermentation engineering. Background technique

[0002] Cyclodextrin glucosyltransferase (CGTase for short, EC 2.4.1.19) is an extracellular enzyme capable of converting starch and related substrates to synthesize cyclodextrins (CDs) through intramolecular transglycosylation reactions, which can be divided into α- , β- and γ-three types. Cyclodextrin has a unique hydrophobic ring-shaped barrel-shaped molecular structure, which can perform inclusion complexation and molecular recognition on various organic compounds, thereby changing and protecting the physical and chemical properties of the guest compound. Therefore, cyclodextrin has a wide range of applications in food, medicine, cosmetics and other fields. However, the current production of cyclodextrins is too expensive due to low con...

Examples

Embodiment 1

[0030] Construction of E.coli BL21(DE3)(pET-20b(+) / cgt)

[0031]The α-CGTase gene (Chen Jian, Wu Jing, Li Zhaofeng, Li Bin, Cheng Cheng. A kind of α -Cloning and expression of cyclodextrin glucosyltransferase gene [P]. Chinese patent, application number 200810024162.3, 2008.) inserted into the downstream of the signal peptide sequence of the commercialized plasmid pET-20b (+), and constructed the expression vector pET- 20b(+) / cgt, and transform it into commercial host strain E.coli BL21(DE3).

Embodiment 2

[0033] 1. Bacterial strain: The laboratory constructed Escherichia coli E.coli BL21(DE3)(pET-20b(+) / cgt) by itself.

[0034] 2. Seed culture: Inoculate the strains stored in glycerol tubes at -80°C into the seed medium (peptone 10g / L, yeast powder 5g / L, NaCL 10g / L, ampicillin 100mg / L, pH 7.10), and use a rotary constant temperature The culture was carried out on a speed-regulating shaker, the speed of which was controlled at 200 rpm / min, the culture temperature was 35° C., the initial pH value was 7.10, and the culture time was 10 hours.

[0035] 3. Enzyme production by fermentation:

[0036] Batch fermentation stage: the seed liquid (peptone 10g / L, yeast powder 5g / L, NaCL 10g / L, pH 7.10) was inserted into the fermentation medium (glycerol 8g / L, peptone 1g / L, yeast Powder 2g / L, KH 2 PO 4 13.5g / L, (NH 4 ) 2 HPO 4 4g / L, citric acid 1.7g / L, MgSO 4 0.68g / L, trace element solution 10mL / L, ampicillin 100mg / L), maintain dissolved oxygen at 20% by adjusting the rotation speed...

Embodiment 3

[0042] 1. Bacterial strain: The laboratory constructed Escherichia coli E.coli BL21(DE3)(pET-20b(+) / cgt) by itself.

[0043] 2. Seed culture: Inoculate the strains stored in glycerol tubes at -80°C into the seed medium (peptone 10g / L, yeast powder 5g / L, NaCL 10g / L, ampicillin 100mg / L, pH 7.10), and use a rotary constant temperature The culture was carried out on a speed-regulating shaker, the speed of which was controlled at 200 rpm / min, the culture temperature was 35° C., the initial pH value was 7.10, and the culture time was 10 hours.

[0044] 3. Enzyme production by fermentation:

[0045] Batch fermentation stage: the seed liquid (peptone 10g / L, yeast powder 5g / L, NaCL 10g / L, pH 7.10) was inserted into the fermentation medium (glycerol 8g / L, peptone 1g / L, yeast Powder 2g / L, KH 2 PO 4 13.5g / L, (NH 4 ) 2 HPO 4 4g / L, citric acid 1.7g / L, MgSO 4 0.68g / L, trace element solution 10mL / L, ampicillin 100mg / L), by adjusting the rotating speed or introducing oxygen-enriched a...

technical field

[0001] The invention relates to a production process for extracellular production of recombinant α-cyclodextrin glucosyltransferase, which belongs to the technical field of fermentation engineering. Background technique

[0002] Cyclodextrin glucosyltransferase (CGTase for short, EC 2.4.1.19) is an extracellular enzyme capable of converting starch and related substrates to synthesize cyclodextrins (CDs) through intramolecular transglycosylation reactions, which can be divided into α- , β- and γ-three types. Cyclodextrin has a unique hydrophobic ring-shaped barrel-shaped molecular structure, which can perform inclusion complexation and molecular recognition on various organic compounds, thereby changing and protecting the physical and chemical properties of the guest compound. Therefore, cyclodextrin has a wide range of applications in food, medicine, cosmetics and other fields. However, the current production of cyclodextrins is too expensive due to low con...

Examples

Embodiment 1

[0030] Construction of E.coli BL21(DE3)(pET-20b(+) / cgt)

[0031]The α-CGTase gene (Chen Jian, Wu Jing, Li Zhaofeng, Li Bin, Cheng Cheng. A kind of α -Cloning and expression of cyclodextrin glucosyltransferase gene [P]. Chinese patent, application number 200810024162.3, 2008.) inserted into the downstream of the signal peptide sequence of the commercialized plasmid pET-20b (+), and constructed the expression vector pET- 20b(+) / cgt, and transform it into commercial host strain E.coli BL21(DE3).

Embodiment 2

[0033] 1. Bacterial strain: The laboratory constructed Escherichia coli E.coli BL21(DE3)(pET-20b(+) / cgt) by itself.

[0034] 2. Seed culture: Inoculate the strains stored in glycerol tubes at -80°C into the seed medium (peptone 10g / L, yeast powder 5g / L, NaCL 10g / L, ampicillin 100mg / L, pH 7.10), and use a rotary constant temperature The culture was carried out on a speed-regulating shaker, the speed of which was controlled at 200 rpm / min, the culture temperature was 35° C., the initial pH value was 7.10, and the culture time was 10 hours.

[0035] 3. Enzyme production by fermentation:

[0036] Batch fermentation stage: the seed liquid (peptone 10g / L, yeast powder 5g / L, NaCL 10g / L, pH 7.10) was inserted into the fermentation medium (glycerol 8g / L, peptone 1g / L, yeast Powder 2g / L, KH 2 PO 4 13.5g / L, (NH 4 ) 2 HPO 4 4g / L, citric acid 1.7g / L, MgSO 4 0.68g / L, trace element solution 10mL / L, ampicillin 100mg / L), maintain dissolved oxygen at 20% by adjusting the rotation speed...

Embodiment 3

[0042] 1. Bacterial strain: The laboratory constructed Escherichia coli E.coli BL21(DE3)(pET-20b(+) / cgt) by itself.

[0043] 2. Seed culture: Inoculate the strains stored in glycerol tubes at -80°C into the seed medium (peptone 10g / L, yeast powder 5g / L, NaCL 10g / L, ampicillin 100mg / L, pH 7.10), and use a rotary constant temperature The culture was carried out on a speed-regulating shaker, the speed of which was controlled at 200 rpm / min, the culture temperature was 35° C., the initial pH value was 7.10, and the culture time was 10 hours.

[0044] 3. Enzyme production by fermentation:

[0045] Batch fermentation stage: the seed liquid (peptone 10g / L, yeast powder 5g / L, NaCL 10g / L, pH 7.10) was inserted into the fermentation medium (glycerol 8g / L, peptone 1g / L, yeast Powder 2g / L, KH 2 PO 4 13.5g / L, (NH 4 ) 2 HPO 4 4g / L, citric acid 1.7g / L, MgSO 4 0.68g / L, trace element solution 10mL / L, ampicillin 100mg / L), by adjusting the rotating speed or introducing oxygen-enriched a...