Fermentation process for producing hyaluronidase

Abstract

The invention discloses a fermentation process for producing hyaluronidase, and relates to the technical field of bioengineering, the fermentation process comprises the following steps: inoculating a strain from a strain preservation glycerol tube to a YPD plate through a plate streak method, activating the strain, and culturing at 30 DEG C for 2-3 days; picking a single colony, inoculating the single colony to a shake flask culture medium YPD as a first-stage seed solution, and culturing at 30 DEG C for 16-20 hours to obtain the first-stage seed solution; inoculating the primary seed solution into a fermentation culture medium in a first fermentation tank in a fermentation tank group according to a certain inoculum size, and obtaining hyaluronidase fermentation liquor by a culture method of combining intermittent fermentation and continuous fermentation by correlating dissolved oxygen and stirring speed and regulating and controlling material supplementation and thallus growth. Through combined regulation and control of intermittent fermentation and continuous fermentation culture modes in the hyaluronidase fermentation process, the fermentation time is shortened, the equipment utilization rate is improved, meanwhile, the stable growth rate of strains is kept, and stability of the performance of hyaluronidase produced through fermentation is facilitated.

Description

Technical Field

[0001] The invention relates to the technical field of bioengineering, and in particular to a fermentation process for producing hyaluronidase. Background Art

[0002] Hyaluronan (HA) is a type of acidic glycosaminoglycan, which is composed of glucuronic acid (GlcA) and N-acetylglucosamine (GlcNAc) connected alternately by β-1,3 and β-1,4 glycosidic bonds. Due to its unique structure, HA has biocompatibility and biological activity, and is widely used in cosmetics, medicine and food. High molecular weight HA is generally used in lubricants, moisturizers and injectable fillers; low molecular weight HA can penetrate the skin barrier and tissue repair, and is mostly used in anti-aging skin care products and drug carriers.

[0003] The production methods of low molecular weight HA mainly include chemical method, physical method and enzymatic method. The chemical method mainly breaks the chemical bonds by adding chemical reagents, but the position of hydrolysis is random and it is easy to change the molecular structure of HA, making it difficult to purify the product. The physical method mainly uses high temperature heating, acid / base solution and ultrasonic crushing for treatment. Similar to the chemical method, the position of hydrolysis is random, difficult to control, and the molecular weight is uneven. The enzymatic method mainly acts on high molecular weight HA through hyaluronidase. The reaction conditions are mild, and the molecular weight can be controlled by controlling the reaction time and the amount of enzyme added. In addition, the position of HA hydrolysis by enzymatic hydrolysis is specific and has strong specificity. Therefore, the preparation of low molecular weight HA by enzymatic hydrolysis has attracted widespread attention. At present, commercialized hyaluronase relies on tissue extraction, has low yield and potential immune response. Leech hyaluronidase has been expressed in microorganisms, however, its low production efficiency and high cost make it difficult to produce on a large scale. Therefore, the development of a fermentation process for producing hyaluronidase is of great significance for the production of low molecular weight hyaluronic acid. Summary of the invention

[0004] The purpose of the present invention is to address the defects and shortcomings of the prior art and provide a fermentation process for producing hyaluronidase. By combining the intermittent fermentation and continuous fermentation culture methods in the hyaluronidase fermentation process, the fermentation time is shortened, the equipment utilization rate is improved, and the stable growth rate of the strain is maintained, which is also conducive to the stability of the performance of the hyaluronidase produced by fermentation.

[0005] To achieve the above object, the present invention adopts the following technical scheme: its operation steps are as follows:

[0006] Step 1: Inoculate the strain from the glycerol tube for strain preservation onto a YPD plate (yeast extract peptone dextrose agar medium) by streaking to activate the strain and culture at 30°C for 2-3 days;

[0007] Step 2: Pick a single colony formed in step 1 and inoculate it into a shake flask medium YPD (yeast extract peptone dextrose medium) as a primary seed solution, and culture it at 30° C. for 16-20 hours to obtain a primary seed solution;

[0008] Step 3: Inoculate the primary seed liquid into the fermentation medium in the first fermenter in the fermenter group at a certain inoculation amount, and obtain the hyaluronidase fermentation liquid by linking the dissolved oxygen and stirring speed, regulating the feed and bacterial growth, and using a culture method combining intermittent fermentation and continuous fermentation.

[0009] Preferably, the certain inoculation amount in step three refers to the seed liquid being inoculated into the fermentation medium of the first fermenter at an inoculation amount of 1%-10%, the volume of the fermentation medium being 1 / 5-1 / 2 of the fermenter, the pH being always controlled at 5.0, the ventilation volume being 1-2.0 vvm, and the fermentation temperature being 30° C. for 0-30 h and 25° C. for 30-60 h.

[0010] Preferably, the fermentation medium is BSM (g / L) containing: glycerol 40, K 2 SO 4 18. MgSO4·7H 2 O14.9, KOH4.13, 85% H 3 PO 4 26.7mL / L, CaSO 4 ·2H 2 O0.93, PTM1 trace elements 4.35mL / L.

[0011] Preferably, the PTM1 trace elements (g / L) include: CuSO 4 ·5H 2 O6, KI0.09, MnSO 4 ·H 2 O3、H 3 BO 3 0.02, MoNa 2 O 4 ·2H 2 O0.2、CoCl 2 6H 2 O0.92、ZnCl 2 20. FeSO 4 7H 2 O65, biotin 0.2, H 2 SO 4 5.0mL.

[0012] Preferably, the intermittent fermentation culture method in step three refers to no feeding during 0-16 hours, supplementing the carbon source at 10-15 g / L / h during 16-30 hours, and continuously supplementing the carbon source (containing 8-12 m / LPTM1) at 7-15 g / L / h after 48 hours, and the carbon source includes but is not limited to glycerol and glucose.

[0013] Preferably, the fermentation tank group is composed of a group of several fermentation tanks, including but not limited to 3-6 fermentation tanks.

[0014] Preferably, the continuous fermentation culture method in step three means that when the fermentation is 60 hours, the first fermenter releases 1 / 2 volume of the fermentation liquid to the second fermenter every 0.5-1 hour, and the first fermenter and the second fermenter are supplemented with sterilized fermentation medium at the same flow rate, and then the second fermenter repeats the operation of the first tank for 0-60 hours, and the second fermenter releases 1 / 2 volume of the fermentation liquid to the third fermenter, and so on, until all tanks are fermented and released.

[0015] Preferably, the association of dissolved oxygen and stirring speed in step three refers to the association of dissolved oxygen below 30% and stirring speed of 200-800rpm, specifically: when the dissolved oxygen is greater than 30%, the stirring speed is as low as 200rpm; when the dissolved oxygen is lower than 30%, the stirring speed is slowly increased from 200rpm until the dissolved oxygen reaches 30%, and the maximum stirring speed is 800rpm, which is applied to the whole fermentation process.

[0016] Preferably, the regulation of feed supplement and bacterial growth in step three refers to adding carbon source, nitrogen source and other nutrients at different time periods during the fermentation process to design the growth of OD600.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: the present invention provides a fermentation process for producing hyaluronidase, which shortens the fermentation time and improves the equipment utilization rate by combining the intermittent fermentation and continuous fermentation culture methods in the hyaluronidase fermentation process, while maintaining a stable growth rate of the strain, which is also beneficial to the stability of the performance of the hyaluronidase produced by fermentation. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is the relationship between cell growth (OD600) and enzyme activity (U / mL) over time during the fermentation process of the present invention. DETAILED DESCRIPTION

[0019] The technical solution of the present invention will be clearly and completely described below in conjunction with the accompanying drawings. The preferred embodiments in the description are only used as examples, and all other embodiments obtained by those skilled in the art without making any creative work are within the scope of protection of the present invention.

[0020] This specific implementation adopts the following technical solutions:

[0021] Step 1. Prepare yeast extract peptone glucose medium (g / L): 10% yeast powder, 20% peptone, 20% glucose; add 2% agar powder to yeast extract peptone glucose agar medium.

[0022] Step 2. Activation culture of hyaluronidase-producing bacteria: Pick a ring of recombinant Pichia pastoris from the glycerol tube, streak it on YPD solid culture medium (YPD plate), and culture it in a 30°C incubator for 2-3 days until colonies grow on the plate.

[0023] Step 3, preparation of seed solution: pick a single colony of the recombinant Pichia yeast formed in step 2, inoculate it into a 500mL conical flask containing 50mL of YPD liquid medium (shake flask medium YPD), culture it at 30°C and 220rpm for 20h, measure OD600 to be 32, and obtain the first-level seed solution.

[0024] Step 4, fermentation tank expansion culture of hyaluronidase: The primary seed liquid formed in step 3 is inoculated into 3 liters of fermentation medium (liquid) in the first fermentation tank in the fermentation tank group at a 10% inoculation amount, the volume of the fermentation medium is 2 / 3 of the volume of the fermentation tank, pH 5.0, and the ventilation volume is 2.0vvm. No feeding is performed during 0-16h, and a carbon source is supplemented at 15g / L / h during 16-30h. After 48h, a carbon source (containing 12m / L PTM1) is continuously supplemented at 7g / L / h, and the source includes but is not limited to glycerol and glucose. The whole process is associated with dissolved oxygen and stirring speed, that is, the dissolved oxygen below 30% is associated with the stirring speed of 200-800rpm; the fermentation tank group consists of 3-6 fermentation tanks, when the fermentation is 60h, the first fermentation tank releases its 1 / 2 volume of fermentation liquid to the second fermentation at 0.5-1h, and at the same time, the first fermentation tank and the second fermentation tank are supplemented with sterilized fermentation medium at the same flow rate, and then the second fermentation tank repeats the operation of the first fermentation tank for 0-60h, and the second fermentation tank releases its 1 / 2 volume of fermentation liquid to the third tank, and so on, until all the fermentation tanks are fermented for 60h to obtain hyaluronidase fermentation liquid.

[0025] Step 5. Hyaluronidase activity assay method: Add 800μL HA substrate (50mM citric acid buffer pH 5.5) and an appropriate amount of fermentation broth supernatant to a total volume of 1mL of reaction solution, and make up to 1mL with buffer. The control group is 800μL HA substrate and inactivated enzyme solution. The reaction system was placed in a 38°C water bath for 15min. After the reaction, it was immediately placed in a boiling water bath for 2min, and 2mL DNS solution was added and boiled in a boiling water bath for 10min. After cooling, 7mL of deionized water was added, mixed, and the absorbance was detected at 540nm. Enzyme activity definition unit: The amount of enzyme required to release 1μg of glucose reducing sugar from HA per hour at pH 5.5 and 38°C. Figure 1 As shown, the enzyme activity of hyaluronic acid reached 2.47 million U / mL.

[0026] Compared with the prior art, the beneficial effects of the present invention are as follows: the present invention provides a fermentation process for producing hyaluronidase, which shortens the fermentation time and improves the equipment utilization rate by combining the intermittent fermentation and continuous fermentation culture methods in the hyaluronidase fermentation process, while maintaining a stable growth rate of the strain, which is also beneficial to the stability of the performance of the hyaluronidase produced by fermentation.

[0027] For those skilled in the art, they can modify the technical solutions described in the aforementioned embodiments and make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A fermentation process for producing hyaluronidase, characterized in that: Its operation steps are as follows: Step (i) inoculate the strain from the glycerol tube for strain preservation onto a YPD plate by streaking, activate the strain, and culture at 30°C for 2-3 days; Step (ii), picking up a single colony formed by culture in step (i) and inoculating it into a shake flask medium YPD as a primary seed solution, culturing at 30° C. for 16-20 hours to obtain a primary seed solution; Step (iii), inoculating the primary seed liquid into the fermentation medium in the first fermenter in the fermenter group at a certain inoculation amount, and obtaining the hyaluronidase fermentation liquid by associating dissolved oxygen with stirring speed, regulating feed and bacterial growth, and using a culture method combining intermittent fermentation and continuous fermentation.

2. A fermentation process for producing hyaluronidase according to claim 1, characterized in that: The certain inoculation amount in step (iii) refers to that the seed liquid is inoculated into the fermentation medium of the first fermenter at an inoculation amount of 1%-10%, the volume of the fermentation medium is 1 / 5-1 / 2 of the fermenter, the pH is always controlled at 5.0, the ventilation volume is 1-2.0vvm, and the fermentation temperature is 30°C for 0-30h and 25°C for 30-60h.

3. A fermentation process for producing hyaluronidase according to claim 1, characterized in that: The fermentation medium was BSMg / L, containing: glycerol 40, K2SO4 18, MgSO4·7H2O 14.9, KOH 4.13, 85% H3PO4 26.7 mL / L, CaSO4·2H2O 0.93, and PTM1 trace elements 4.35 mL / L.

4. A fermentation process for producing hyaluronidase according to claim 3, characterized in that: The PTM1 trace elements g / L include: CuSO4·5H2O 6, KI 0.09, MnSO4·H2O 3, H3BO30.02, MoNa2O4·2H2O 0.2, CoCl2·6H2O 0.92, ZnCl220, FeSO4·7H2O 65, biotin 0.2, and H2SO45.0 mL.

5. A fermentation process for producing hyaluronidase according to claim 1, characterized in that: The intermittent fermentation culture method in step (iii) means that no feed is added during 0-16 hours, carbon source is supplemented at 10-15 g / L / h during 16-30 hours, and carbon source is continuously supplemented at 7-15 g / L / h after 48 hours. The carbon source includes but is not limited to glycerol and glucose.

6. A fermentation process for producing hyaluronidase according to claim 1, characterized in that: The fermentation tank group is a group of several fermentation tanks, including but not limited to 3-6 fermentation tanks.

7. A fermentation process for producing hyaluronidase according to claim 1, characterized in that: The continuous fermentation culture method in step (iii) means that when the fermentation is 60h, the first fermentation tank releases 1 / 2 volume of fermentation liquid to the second fermentation tank in 0.5-1h, and at the same time, the first fermentation tank and the second fermentation tank are supplemented with sterilized fermentation medium at the same flow rate, and then the second fermentation tank repeats the operation of the first tank for 0-60h, and the second fermentation tank releases 1 / 2 volume of fermentation liquid to the third fermentation tank, and so on, until all tanks are released after fermentation.

8. A fermentation process for producing hyaluronidase according to claim 1, characterized in that: The associated dissolved oxygen and stirring speed in step (iii) refers to the association between dissolved oxygen below 30% and stirring speed of 200-800rpm. Specifically, when the dissolved oxygen is greater than 30%, the stirring speed is as low as 200rpm; when the dissolved oxygen is lower than 30%, the stirring speed is slowly increased from 200rpm until the dissolved oxygen reaches 30%, and the maximum stirring speed is 800rpm, which is applied to the whole fermentation process.

9. A fermentation process for producing hyaluronidase according to claim 1, characterized in that: The regulation of feed supplement and bacterial growth in step (iii) refers to the addition of carbon source, nitrogen source and other nutrients at different time periods during the fermentation process to design the growth of OD600.

Images