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Recombinant Streptococcus zooepidemicus for fermentation of micromolecule hyaluronic acid

A technology of Streptococcus zooepidemicus and hyaluronic acid, applied in the field of bioengineering, can solve the problems of complex reaction conditions, low level of recombinant expression of hyaluronidase, difficulty in obtaining molecular weight, etc., and achieve the effect of wide application value

Active Publication Date: 2017-10-03
JIANGNAN UNIV
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Problems solved by technology

Physical methods include heating, ultrasonic waves, and ray radiation to promote random breakage of hyaluronic acid. Although the process is simple, the efficiency is low and the product stability is poor.
The chemical methods are mainly hydrolysis and oxidative degradation. They are easy to introduce pollution by chemical reagents, and the reaction conditions are complicated. They not only affect the properties of hyaluronic acid, make purification difficult, but also generate a large amount of industrial wastewater.
In addition, it has also been reported to use de novo synthesis of hyaluronic acid oligosaccharides, but this method has many problems such as expensive substrates, cumbersome steps, and low synthesis efficiency, making it difficult to achieve large-scale preparation of oligosaccharides
[0004] Compared with the above methods, enzymatic catalytic degradation is the most promising method. In recent years, hyaluronan lyase from microorganisms has been expressed by heterologous recombination and used in vitro enzymatic hydrolysis to prepare small molecule hyaluronic acid. However, the analysis of its hydrolysis mechanism and products found that the continuous exo-cleavage mode was adopted in the process of bacterial lyase catalyzed hydrolysis, and unsaturated disaccharide units were released one by one from the reducing end to the non-reducing end until one chain was completely degraded. This hydrolysis mechanism leads to a wide range of molecular weight distribution of hydrolyzed products, and it is difficult to effectively obtain oligosaccharide products with concentrated molecular weight, especially the structure of hyaluronic acid has also changed.
In addition, analysis of hyaluronic acid degradation process in vitro by commercialized bovine testicular hyaluronidase (BTH) found that the distribution of end products was wide (from 4-52 disaccharide units), and BTH had transglycosidic activity to As for the difficulty in obtaining a final product with a relatively single molecular weight, in fact, BTH has low enzyme activity and is expensive, while the recombinant expression levels of hyaluronidase from other sources are all low, which are not suitable for large-scale fermentation, resulting in hyaluronic acid. There are always technical shortcomings in the preparation method of oligosaccharides

Method used

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  • Recombinant Streptococcus zooepidemicus for fermentation of micromolecule hyaluronic acid
  • Recombinant Streptococcus zooepidemicus for fermentation of micromolecule hyaluronic acid

Examples

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Embodiment 1

[0034] Example 1 Construction of recombinant plasmids secreting and expressing hyaluronidase

[0035] Primers HyaL-F and HyaL-R were designed, and a standard PCR amplification system and program was used to amplify the hyaluronidase gene Hyal whose sequence was obtained as shown in SEQ ID NO.1.

[0036]Design primers AmyX-F / AmyX-R, Bpr-F / Bpr-R, NprE-F / NprE-R, OppA-F / OppA-R, Vpr-F / Vpr-R, WapA-F / WapA-R, YvgO-F / YvgO-R, YweA-F / YweA-R, YxaL-F / YxaL-R, respectively with plasmid pMA0911-amyX, pMA0911-bpr, pMA0911-nprE, pMA0911-oppA, pMA0911-vpr, pMA0911-wapA , pMA0911-yvgO, pMA0911-yweA, pMA0911-yxaL as templates, using standard PCR system and procedures, amplified to obtain signal peptides AmyX, Bpr, NprE, OppA, Vpr, WapA, YvgO, YweA, YxaL (SEQ ID NO. 2-10), wherein the above-mentioned signal peptide upstream primers are all introduced into the streptococcal RBS sequence AAGGAGGATGT, and the downstream primers are all introduced into the N-terminal 20bp sequence of hyaluronidase Hya...

Embodiment 2

[0060] Embodiment 2 Shake flask culture and enzyme activity assay of 9 strains of recombinant Streptococcus zooepidemicus

[0061] Pick the 9 recombinant Streptococcus zooepidemicus strains constructed above and the control bacteria (transformed pEU308 empty plasmid), inoculate the monoclonal in 5mL seed medium, culture at 200rpm37°C overnight, then transfer to 250mL with 10% inoculum Erlenmeyer shake flask, the culture medium is the fermentation medium, the filling volume is 25mL, it is cultured at 200rpm at 37°C for 20h, and 0.1mMIPTG is added for induction.

[0062] The 20h fermented liquid samples in shake flasks of 9 strains were taken respectively and centrifuged at 5000r / min at 4°C for 5min, the supernatant was retained, and the crude enzyme activity in the supernatant was determined. From attached figure 2 It can be seen that in the inducible promoter P spac Under the mediation effect, nine recombinant strains S.zooepidemicus HyalA, HyalC, S.zooepidemicus HyalE, S.z...

Embodiment 3

[0063] Example 3 Determination of Hyaluronic Acid Production and Molecular Weight of Recombinant Streptococcus zooepidemicus

[0064] Take the fermentation broth samples of the recombinant bacteria fused with the signal peptide and the control bacteria to the 20th hour, centrifuge at 5000r / min, 4°C for 5min, save the supernatant, add 3 times the volume of 100% ethanol for precipitation, mix well and place After 30 minutes, centrifuge at 5000r / min for 10 minutes, add an equal volume of distilled water, and redissolve the precipitate at 4°C. This purification step is repeated three times. The content of hyaluronic acid was determined by the Bitter-Muir carbazole colorimetric method. Add 1 mL of borax sulfuric acid reagent and 200 μL of hyaluronic acid sample diluted by a certain multiple into the colorimetric tube, mix well and cook in boiling water for 15 min, and cool to At room temperature, add 50 μL carbazole reagent, mix well and cook in boiling water for 15 minutes, measur...

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Abstract

The invention discloses recombinant Streptococcus zooepidemicus for fermentation of micromolecule hyaluronic acid and belongs to the technical field of bioengineering. Hyaluronidase derived from leeches is heterologously expressed in Streptococcus zooepidemicus, nine kinds of signal peptides are selected and are fused with the terminal N of the hyaluronidase, and an appropriate signal peptide is selected through comparison of enzyme activity through a Pspac-induced promoter and Streptococcus RBS so that efficient secretion and expression of hyaluronidase in Streptococcus zooepidemicus is realized. Through degradation of macromolecule hyaluronic acid produced by the Streptococcus zooepidemicus, active micromolecule hyaluronic acid is obtained and a yield is improved. The recombinant Streptococcus zooepidemicus lays a foundation for the efficient fermentation preparation of micromolecule hyaluronic acid based on a microbial system and is suitable for industrial production and application.

Description

technical field [0001] The invention relates to a recombinant Streptococcus zooepidemicus fermenting small molecule hyaluronic acid, which belongs to the technical field of bioengineering. Background technique [0002] Hyaluronic acid is a kind of high-molecular viscous polysaccharide. It is currently mainly produced by animal tissue extraction or microbial fermentation. Its biological function is closely related to its molecular weight. Macromolecular hyaluronic acid has been widely used in the medical field and cosmetics industry. , small molecular hyaluronic acid oligosaccharides have unique biological functions and have important application prospects in the fields of health care and medicine. [0003] The current methods for preparing small molecule hyaluronic acid mainly focus on physical and chemical methods. Physical methods include heating, ultrasonic waves, and ray radiation to promote random fracture of hyaluronic acid. Although the process is simple, the efficie...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12P19/26C12R1/46
CPCC12N9/2474C12P19/26C12Y302/01035
Inventor 康振陈坚堵国成张琳培韦朝宝
Owner JIANGNAN UNIV
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