Method for fermentation production of hydroxyproline by adopting escherichia coli JL-HYP

Abstract

The invention relates to a method for fermentation production of hydroxyproline by adopting escherichia coli JL-HYP. The method comprises the following steps that an escherichia coli JL-HYP strain is subjected to slant activation, the activated escherichia coli JL-HYP strain is subjected to gradient dilution, the diluent coats an LB culture medium, after culture, single colonies are selected and respectively inoculated to an LB slant culture medium, the LB slant culture medium is placed in a biochemical incubator, and culture is carried out for 12h at 30-36 DEG C; single colonies are selected from the LB slant culture medium and inoculated into a seed bottle filled with a seed culture medium to be cultured; the seed liquid is inoculated into a fermentation tank filled with a fermentation culture medium to be cultured for 30-45 h, and thus fermentation liquor containing hydroxyproline is obtained; and hydroxyproline in the fermentation liquor containing hydroxyproline is extracted. For the process, the fermentation period is short, the density of thallus is high, and the output and yield of hydroxyproline are high. The method has the advantages that the fermentation period is short, the density of thallus is high, and the output and yield of hydroxyproline are high.

Description

technical field

[0001] The invention relates to a method for producing hydroxyproline, in particular to a method for producing hydroxyproline by fermentation of Escherichia coli JL-HYP. Background technique

[0002] Hydroxyproline (Hyp) is an imino acid, its full name is cis-4-hydroxyl-L-proline; it is the product of hydroxylation of L-proline, and its molecular formula is C 5 h 9 NO 3 . Hydroxyproline is white flaky crystal or crystalline powder, slightly sweet, with a melting point of 274°C, easily soluble in water and slightly soluble in ethanol.

[0003] At present, the methods for producing hydroxyproline reported at home and abroad mainly include proteolytic extraction, chemical synthesis, microbial fermentation and enzymatic conversion. Among them, proteolytic extraction is currently the most commonly used industrial production method. However, the protein hydrolysis extraction method needs to be treated with strong acid and strong alkali, the purification steps a...

Examples

Embodiment 1

[0023] Escherichia coli JL-HYP and common hydroxyproline-producing Escherichia coli were inoculated on the slant, cultured in a biochemical incubator at 37°C for 20 hours, then diluted by gradient, spread on the complete medium (LB), and picked Take a single colony and inoculate it on the slant, and incubate at 30-36°C for about 12 hours in a biochemical incubator. Inoculate into a 500ml seed bottle, culture conditions: temperature 30-36°C, shaker speed 200rmp, culture time 6-8 h; when the OD value grows to about 1.0-2.0, inoculate into a 500ml fermentation bottle, inoculum size: 7 %, culture conditions: temperature 30-36°C, shaker speed 200rmp, fermentation cycle 30-45h. After the fermentation, the hydroxyproline content was detected to be 23.65g / L and 18.37g / L, and the yield increased by 28.74%.

Embodiment 2

[0025] Pick one ring of Escherichia coli JL-HYP and ordinary hydroxyproline-producing Escherichia coli from fresh slant and inoculate them into 500mL seed bottle, culture conditions: temperature 30-36℃, shaker speed 200rmp, culture The time is 6-8h; when the OD value grows to about 1.0-4.0, put the shake flask seeds into a 5L automatic fermenter with 3L fermentation medium at an inoculation amount of 10% for secondary seed cultivation. The culture temperature is 30-36°C, the tank pressure is 0.01-0.05MPa, the air flow rate is 1-8L / min and the stirring speed is 200-800r / min to maintain the dissolved oxygen at 10-50%, and the pH value is controlled between 7.0-7.2. The time is 5-8h. The secondary seeds were inserted into a 30L fully automatic fermenter with 18L fermentation medium at a 10% inoculum size for acidogenic fermentation. The fermentation temperature is 30-36°C, the tank pressure is 0.01-0.15MPa, the air flow rate is 5-50L / min and the stirring speed is 200-800r / min to...

Embodiment 3

[0027] Pick one ring of Escherichia coli JL-HYP and ordinary hydroxyproline-producing Escherichia coli from fresh slant and inoculate them into 500mL seed bottle, culture conditions: temperature 30-36℃, shaker speed 200rmp, culture The time is 6-8h; when the OD value grows to about 1.0-4.0, insert the shake flask seeds into a 0.6m 3 2m of fermentation medium 3Secondary seed cultures were carried out in fermenters. Cultivation temperature 30-36℃, tank pressure 0.01-0.05MPa, air flow 0.1-0.6m 3 / min and stirring speed 100-300r / min to maintain the dissolved oxygen at 10-50%, the pH value is controlled between 7.0-7.2, and the culture time is 5-8h. Insert the secondary seeds with 10% inoculum into the 6m 3 10m of fermentation medium 3 Acidogenic fermentation in fermenter. Fermentation temperature 30-36℃, tank pressure 0.01-0.15MPa, air flow 0.5-7 m 3 / min and stirring speed 200-800r / min to maintain the dissolved oxygen at 10-40%, the fermentation pH value is controlled betwe...

technical field

[0001] The invention relates to a method for producing hydroxyproline, in particular to a method for producing hydroxyproline by fermentation of Escherichia coli JL-HYP. Background technique

[0002] Hydroxyproline (Hyp) is an imino acid, its full name is cis-4-hydroxyl-L-proline; it is the product of hydroxylation of L-proline, and its molecular formula is C 5 h 9 NO 3 . Hydroxyproline is white flaky crystal or crystalline powder, slightly sweet, with a melting point of 274°C, easily soluble in water and slightly soluble in ethanol.

[0003] At present, the methods for producing hydroxyproline reported at home and abroad mainly include proteolytic extraction, chemical synthesis, microbial fermentation and enzymatic conversion. Among them, proteolytic extraction is currently the most commonly used industrial production method. However, the protein hydrolysis extraction method needs to be treated with strong acid and strong alkali, the purification steps a...

Examples

Embodiment 1

[0023] Escherichia coli JL-HYP and common hydroxyproline-producing Escherichia coli were inoculated on the slant, cultured in a biochemical incubator at 37°C for 20 hours, then diluted by gradient, spread on the complete medium (LB), and picked Take a single colony and inoculate it on the slant, and incubate at 30-36°C for about 12 hours in a biochemical incubator. Inoculate into a 500ml seed bottle, culture conditions: temperature 30-36°C, shaker speed 200rmp, culture time 6-8 h; when the OD value grows to about 1.0-2.0, inoculate into a 500ml fermentation bottle, inoculum size: 7 %, culture conditions: temperature 30-36°C, shaker speed 200rmp, fermentation cycle 30-45h. After the fermentation, the hydroxyproline content was detected to be 23.65g / L and 18.37g / L, and the yield increased by 28.74%.

Embodiment 2

[0025] Pick one ring of Escherichia coli JL-HYP and ordinary hydroxyproline-producing Escherichia coli from fresh slant and inoculate them into 500mL seed bottle, culture conditions: temperature 30-36℃, shaker speed 200rmp, culture The time is 6-8h; when the OD value grows to about 1.0-4.0, put the shake flask seeds into a 5L automatic fermenter with 3L fermentation medium at an inoculation amount of 10% for secondary seed cultivation. The culture temperature is 30-36°C, the tank pressure is 0.01-0.05MPa, the air flow rate is 1-8L / min and the stirring speed is 200-800r / min to maintain the dissolved oxygen at 10-50%, and the pH value is controlled between 7.0-7.2. The time is 5-8h. The secondary seeds were inserted into a 30L fully automatic fermenter with 18L fermentation medium at a 10% inoculum size for acidogenic fermentation. The fermentation temperature is 30-36°C, the tank pressure is 0.01-0.15MPa, the air flow rate is 5-50L / min and the stirring speed is 200-800r / min to...

Embodiment 3

[0027] Pick one ring of Escherichia coli JL-HYP and ordinary hydroxyproline-producing Escherichia coli from fresh slant and inoculate them into 500mL seed bottle, culture conditions: temperature 30-36℃, shaker speed 200rmp, culture The time is 6-8h; when the OD value grows to about 1.0-4.0, insert the shake flask seeds into a 0.6m 3 2m of fermentation medium 3Secondary seed cultures were carried out in fermenters. Cultivation temperature 30-36℃, tank pressure 0.01-0.05MPa, air flow 0.1-0.6m 3 / min and stirring speed 100-300r / min to maintain the dissolved oxygen at 10-50%, the pH value is controlled between 7.0-7.2, and the culture time is 5-8h. Insert the secondary seeds with 10% inoculum into the 6m 3 10m of fermentation medium 3 Acidogenic fermentation in fermenter. Fermentation temperature 30-36℃, tank pressure 0.01-0.15MPa, air flow 0.5-7 m 3 / min and stirring speed 200-800r / min to maintain the dissolved oxygen at 10-40%, the fermentation pH value is controlled betwe...

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