High-density fermentation method of engineering bacteria containing nitrilase

A technology of genetically engineered bacteria and high-density fermentation, applied in microorganism-based methods, hydrolytic enzymes, biochemical equipment and methods, etc., can solve problems such as low economy, limited industrial applicability, harsh reaction conditions, etc., to avoid viscosity. The effect of increasing, preventing the accumulation of acid production, and improving the fermentation level

Inactive Publication Date: 2014-12-17
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This chemical method has many defects such as poor selectivity, low yield, harsh reaction conditions, low economy, and serious environmental pollution.
These drawbacks greatly limit the industrial applicability of this route

Method used

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  • High-density fermentation method of engineering bacteria containing nitrilase
  • High-density fermentation method of engineering bacteria containing nitrilase
  • High-density fermentation method of engineering bacteria containing nitrilase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Embodiment 1: construction contains the engineered bacterium of nitrilase gene

[0040] The present invention uses a rapid nucleic acid extractor and a microbial genome extraction kit (MP Company, USA) to extract the genome DNA of Acidovorax facilis ZJB09122. The strain is preserved in the China Center for Type Culture Collection with the preservation number CCTCC No.M 209044, which has been disclosed in the previously applied patent CN101629192B.

[0041] According to the Acidovorax facilis nitrilase gene and homology analysis reported on NCBI, a pair of cloning primers were designed to amplify the AcN gene:

[0042] Upstream primer (primer 1):

[0043] AcN(F)5'-AAT GGATCC ATGGTTTCGTATAACAGCAAG-3'

[0044] Downstream primer (primer 2):

[0045] AcN(R)5'-AGG GTC GAC CTACTTTGCTGGGACCGG-3'

[0046] In order to facilitate the construction of prokaryotic expression vectors, Nco I and Xho I restriction enzyme sites (underlined parts) were respectively introduced into ...

Embodiment 2

[0055] Example 2: Construction of strain E.coli BL21(DE3) / pET28b(+)-AfNIT

[0056] The present invention uses a rapid nucleic acid extractor and a microbial genome extraction kit (MP Company, USA) to extract the genomic DNA of Alcaligenes faecalis ZJUTB10. The strain is preserved in the China Center for Type Culture Collection, the preservation number is CCTCC No.M 208168, and the preservation date is October 22, 2008, which has been disclosed in the previously applied patent CN101392276B.

[0057] According to the nitrilase gene and homology analysis reported on NCBI, a pair of cloning primers were designed to amplify the AfN gene:

[0058] Upstream primer (primer 5):

[0059] AfN(F)5'-AAT GGATCC ATGCAGACAAGAAAAATCGTC-3'

[0060] Downstream primer (primer 6):

[0061] AfN(R)5'-AGG GTC GAC TCAGGACGGTTCTTGCAC-3'

[0062] In order to facilitate the construction of prokaryotic expression vectors, Nco I and Xho I restriction enzyme sites (underlined parts) were respectively...

Embodiment 3

[0115] Embodiment 3: DO-STAT feeding coupling step-by-step induction, and the application of the high-density fermentation technology of gradually increasing rotating speed in the high-density fermentation of nitrilase genetically engineered bacteria (1)

[0116] 1 Experimental process:

[0117] (1) Seed solution preparation: Take the E.coli BL21(DE3) / pET28b(+)-AcNIT slant strain containing the nitrilase gene constructed in Example 1, inoculate a loop in a 100mL container containing a final concentration of 5g / In a 500 mL shake flask of LB liquid culture medium of L kanamycin, cultivate overnight at 37° C. and 150 rpm to obtain a seed solution.

[0118] (2) Fermentor batch culture: the seed liquid that step (1) obtains is with the inoculum size of 3% (v / v), inoculates in the 5L that contains 3L final concentration 50g / L kanamycin fermentation medium that is housed In the fermentor, at 37 DEG C, 500rpm, under the condition of aeration ratio 1.3vvm, ferment and cultivate, feed...

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Abstract

The invention discloses a high-density fermentation method of engineering bacteria containing nitrilase. The method adopts a novel recombinant Escherichia coli fermentation and supplementary material culture medium formula, and uses a method of DO-STAT coupling step-by-step induction and gradual increase of the speed stage by stage. The method not only meets the need for mixing power in the culture process with biomass increase, effectively controls the growth rate of thalli, and prevents disadvantages of produced acid accumulation caused by fast growth of thalli, feedback inhibition and culture medium waste. In addition, the induced mode of batch-by-batch lactose supplement addition avoids the high viscosity of the fermentation broth caused by high concentration of lactose, resulting in resistance of oxygen transfer and mass transfer, but also improves the induction strength. The high-density fermentation technology of the invention increase the biomass of recombinant Escherichia coli from 10.35g DCW / L to 70g DCW / L, volume enzyme activity from 18205U / L to 103530U / L, and the fermentation level by 4.7 times.

Description

(1) Technical field [0001] The invention relates to a method for high-density cultivation of recombinant Escherichia coli producing nitrilase and high-efficiency expression of target genes. (2) Background technology [0002] Nitrilase can realize one-step hydrolysis of organic nitrile compounds to synthesize corresponding organic carboxylic acids. The nitrilase is used to realize the cyanohydrolysis reaction with mild conditions, high reaction efficiency, and relatively high regioselectivity and stereoselectivity. In recent years, the use of nitrilase to hydrolyze cyano groups to synthesize organic carboxylic acids has become a research hotspot, and a large number of documents and patents have reported the application of nitrilase in the synthesis of organic carboxylic acids. More importantly, the discovery of many nitrilases with regioselective catalytic activity for dinitriles, for the key chiral intermediate (R)-4-cyano-3-hydroxybutyric acid (Organic Process Research&De...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/78C12N15/70C12R1/19
CPCC12N9/78C12Y305/05001
Inventor 薛亚平郑裕国柳志强刘兆巍王应鹏徐喆
Owner ZHEJIANG UNIV OF TECH
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