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Construction method and application of recombinant bacterium for producing chitosanase

A technology for producing chitosanase and chitosanase, applied in the biological field, can solve the problems of increased energy consumption and cost, and achieve the effects of high-efficiency expression, excellent hydrolysis characteristics, and good application prospects

Active Publication Date: 2019-04-19
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the substrate concentration reported by Chen et al. is 10%, it requires more than 24 hours of hydrolysis, which greatly increases energy consumption and cost (Chen et al. Biotechnology Letters, 2012, 34:689-694)

Method used

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  • Construction method and application of recombinant bacterium for producing chitosanase
  • Construction method and application of recombinant bacterium for producing chitosanase
  • Construction method and application of recombinant bacterium for producing chitosanase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Embodiment 1, the preparation of recombinant bacteria GS115 / BsCsn46 / VHb

[0067] 1. Using the Bacillus subtilis WY-34 genome as a template, PCR amplification was performed using primers BsCsn46-F and BsCsn46-R to obtain the Bacillus subtilis chitosanase gene BsCsn46. The primer sequences are as follows:

[0068] BsCsn46-F: AACCG GAATTC ATGGGACTGAATAAAGATCAAAAGC (the underline is the EcoRI restriction site);

[0069] BsCsn46-R:AAGAAT GCGGCCGC TTATTTGATTACAAAATTACCGTAC (the underline is the NotⅠ restriction site).

[0070] The Bacillus subtilis chitosanase gene BsCsn46 was codon-optimized, and the optimized BsCsn46 gene sequence was shown in Sequence 1. Using the codon-optimized sequence as a template, primers BsCsn46-F and BsCsn46-R were used for PCR amplification. increase to obtain PCR products.

[0071] 2. The PCR product obtained in step 1 and pPICZA were digested with EcoRI and NotI respectively, and ligated with T4 DNA ligase at 16°C to construct the recombi...

Embodiment 2

[0081] Embodiment 2, preparation and purification of recombinant chitosanase (BsCsn46)

[0082] 1. Preparation of recombinant chitosanase (BsCsn46) by high-density fermentation of recombinant bacteria

[0083] The high-density fermentation of the recombinant bacteria and the medium refer to the method in the Pichia Fermentation Process Guideline (Invitrogen), and the specific steps are as follows:

[0084] 1. Inoculate the recombinant bacterium that produces chitosanase activity higher (415U / mL) obtained by screening in Example 1 to 150mLYPD medium (tryptone 20g / L, yeast extract 10g / L, glucose 20g / L) , 30°C, 200rpm to OD 600 About 10, as a seed solution.

[0085] 2. Select a 5L fermenter (with a working volume of 1.5L) as a high-density fermentation vessel, add 150 mL of the seed liquid obtained in step 1 into a fermenter containing 1350 mL of fermentation medium for batch fermentation. During the fermentation process, 28% ammonia water was used to adjust the pH to 4.0, the...

Embodiment 3

[0102] Embodiment 3, the enzymatic property determination of recombinant chitosanase BsCsn46

[0103] 1. Determination of optimum pH and pH stability

[0104] Determination of optimum pH: use the purified chitosanase solution obtained in Example 2 as the enzyme solution to be tested, and determine the optimum pH within the pH range of 3.5-7.5. Enzyme activity was determined according to the standard method in Example 2. Taking the highest value of enzyme activity as 100%, the relative enzyme activities under different pH conditions were calculated respectively.

[0105] Determination of pH stability: Dilute the enzyme solution to be tested with different buffer solutions within the pH range of 3.0-11.0, incubate at 45°C for 30 minutes, immediately cool in an ice-water bath for 30 minutes, and then determine the residual enzyme under the optimal conditions according to the standard method vitality. With the enzyme solution without the above treatment (the above treatment ref...

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Abstract

The invention discloses a construction method and application of a recombinant bacterium for producing chitosanase. According to the method, a bacillus subtilis chitosanase gene and a vitreoscilla hemoglobin gene are co-expressed in pichia pastoris, and a recombinant strain is used for efficiently preparing the chitosanase through a high-density fermentation strategy. The enzyme activity of the chitosanase prepared through the method is as high as 50000 U / mL or above, and the protein content is up to 16.0 mg / mL. The specific enzyme activity of recombinant enzyme is 4065.7 U / mg, the optimum pHvalue is 6.0, and the optimum temperature is 55 DEG C. The enzyme is used for hydrolyzing high-concentration (larger than or equal to 10%) chitosan to prepare chitosan oligosaccharide, the yield of chitosan oligosaccharide is larger than 75%, and main products are disaccharide, trisaccharide and tetraose. The enzyme production level of the chitosanase preparation method is much higher than that reported in existing literature and patents, and the produced enzyme has excellent hydrolysis characteristics and has very good application value in industrial production of chitosanase and preparationof chitosan oligosaccharide.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a construction method and application of a chitosanase-producing recombinant bacterium. Background technique [0002] Chitosan is a product of partial deacetylation of chitin, a straight-chain natural polysaccharide composed of D-glucosamine and N-acetyl-D-glucosamine randomly linked by β-1,4-glucosidic bonds. Chitosanase (EC3.2.1.132) is an enzyme that specifically hydrolyzes chitosan. It randomly cuts β-1,4-glucosidic bonds from chitosan chains, and the products are chitosan and glucosamine. Based on their sequence homology, chitosanases are classified into glycoside hydrolases (GH) 5, 7, 8, 46, 75 and 80 families (Thadathil et al. Food Chemistry, 2014, 150: 392–399 ). [0003] In recent years, there have been many literatures and patent reports on the production of chitosanase by microorganisms, but most of them have low enzyme production levels and are difficult to be used in i...

Claims

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Application Information

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IPC IPC(8): C12N15/81C12N15/56C12N9/42C12N15/31C12P19/14C12P19/26
CPCC07K14/195C12N9/2434C12N15/815C12P19/14C12P19/26
Inventor 江正强马帅杨绍青刘翊昊闫巧娟
Owner CHINA AGRI UNIV
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