Recombinant Bacillus subtilis for producing chondroitinase and application thereof

A technology of Bacillus subtilis and chondroitin sulfate, which is applied in the field of bioengineering, can solve the problems of large loss of enzyme activity, cumbersome crushing process, and not easy to be broken completely, and achieves the effect of improving enzyme activity and wide application value.

Active Publication Date: 2016-11-23
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] There are two main problems in the fermentation production of ChSase ABC in China: 1. Most of the strains used for the fermentation and production of ChSase ABC are intracellular production methods, and the obtained enzyme activity is low, and the crushing process is relatively cu

Method used

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  • Recombinant Bacillus subtilis for producing chondroitinase and application thereof
  • Recombinant Bacillus subtilis for producing chondroitinase and application thereof
  • Recombinant Bacillus subtilis for producing chondroitinase and application thereof

Examples

Experimental program
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Example Embodiment

[0031] Example 1 Construction of recombinant plasmids P43-H6cslA1, Pppnk-H6cslA1, PcsfB-H6cslA1

[0032] Primers AmyX-F and AmyX-R were designed, and a standard PCR amplification system and program were used to amplify the obtained sequence of signal peptide amyX as shown in SEQID NO.5.

[0033] Primers CSL-F2 and CSL-R2 were designed, using the genomic DNA of P. vulgaris ATCC6896 as a template, using standard PCR amplification systems and procedures to amplify the chondroitin sulfate lyase gene cslABC ​​(shown in SEQ ID NO.1) .

[0034] promoter P ppnk and P csfB From Bacillus subtilis 168, the B. subtilis 168 strain was inoculated into 5ml of LB liquid medium, and cultured at 37°C and 200rpm for 16h. Bacteria were collected, and the genomic DNA of B. subtilis 168 strain was extracted using a bacterial genome extraction kit. According to the published genome information sequence, primers Pppnk-F / Pppnk-R and PcsfB-F / PcsfB-R were designed respectively, and the extracted B. ...

Example Embodiment

[0050] Example 2 Shake flask culture and enzyme activity assay of 6 strains of recombinant Bacillus subtilis

[0051] The 6 recombinant Bacillus subtilis strains constructed above and the control bacteria B.subtilis 168 and B.subtilisWB600 were selected respectively, single clones were inoculated into 5 mL LB medium, and cultured overnight at 37°C at 200 rpm. After 16 hours, it was transferred to a 250 mL conical flask at 10% of the inoculum. The culture medium was a fermentation medium with a liquid filling volume of 25 mL, and cultured at 200 rpm at 37° C. for 48 hours.

[0052] Eight samples of the 48-h fermentation broth in each shake flask were centrifuged at 5000 × g for 5 min at 4°C, the supernatant was retained, and the crude enzyme activity in the supernatant was determined. from the attached figure 2 It can be seen that when using strong constitutive promoter, strong RBS and adding histidine tag, 6 strains of recombinant B. subtilis B.subtilis 168 / P43-H6cslA1; B.su...

Example Embodiment

[0053] Example 3 3L tank cultivation of recombinant Bacillus subtilis B.subtilisWB600 / P43-H6cslA1

[0054] The recombinant Bacillus subtilis strain B.subtilis WB600 / P43-H6cslA1 was picked, single clone was inoculated into 15mL LB medium, and cultured at 200rpm at 37°C overnight. After 16 hours, it was transferred to a 250ml conical flask according to 10% of the inoculum volume, and the liquid filling volume was 150ml, and continued to be cultured at 200rpm at 37°C overnight as a seed solution. After 16 hours, it was transferred to a 3L fermenter according to 10% of the inoculum volume, the liquid filling volume was 1.5L, the culture was maintained at 37°C at a constant temperature, the ventilation volume was 2vvm, and the pH was maintained at 7.0 by adjusting the 5M NaOH solution. The stirring speed was 400 rpm for the first 4 h, and then changed to 600 rpm. At the 7th hour, the initial sugar was exhausted, and the feed was started. The feed was 10g / L / h from the 7th to the 9t...

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Abstract

The invention discloses recombinant Bacillus subtilis for producing chondroitinase and application thereof and belongs to the technical field of bioengineering. Chondroitinase of common Proteus origin is heterologously expressed in Bacillus subtilis, signal peptide amyX is selected, 3 strong constitutive promoters and strong RBS (ribosome binding site) of pP43NMK vector itself are utilized, histidine tags are added to facilitate subsequent purification, and expressive enzyme activity of chondroitinase in Bacillus subtilis is improved. Certain basis is laid for efficiently producing chondroitinase from food-grade microbes, and the recombinant Bacillus subtilis is suitable for industrial application.

Description

technical field [0001] The invention relates to a recombinant bacillus subtilis producing chondroitin sulfate lyase and an application thereof, belonging to the technical field of bioengineering. Background technique [0002] Chondroitin sulfate (Chondroitin sulfate, CS) is a kind of disaccharide that combines D-glucuronic acid and 2-acetylamino-2-deoxysulfate-D-galactose through β-1,3 glycosidic bonds as the basic unit. A class of macromolecular polysaccharides formed by polymerization, the molecular weight is between 5-50kDa. According to the structure and molecular weight of CS, CS can be divided into categories such as A, B, C, D, E, F, H, K, L and M. The common ones are CS A, B and C. CS A is also called 4-sulfate chondroitin, the sulfate group is on the fourth carbon of galactosamine, and CS C is also called 6-sulfate chondroitin, and the sulfate group is on the amino semi At the 6th carbon of lactose. CS B, also known as dermatan sulfate, is isomer with CS A. Curr...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70C12N9/88C12R1/125
Inventor 康振陈坚堵国成张琳培周正雄
Owner JIANGNAN UNIV
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