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Method for performing one-step purification and immobilization on alpha-amino acid fatty acyl transferase

An amino acid fatty acyl and ester acyl transferase technology, applied in transferase, biochemical equipment and methods, immobilized on or in biological cells, etc., can solve the problem of low expression of lipase, reduced production efficiency, inapplicability, etc. problems, to achieve the effect of improving the yield of enzyme activity, reducing purification steps, and saving operation steps

Active Publication Date: 2018-11-02
HEBEI FITNESS BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Two sets of inducible expression systems are used for the two genes. During the expression process, the expression of the FGE gene is firstly induced with arabinose, and then lipase is induced after a period of time. This step-by-step induction method is likely to cause the expression of lipase Relatively low, reducing production efficiency
In addition, the solid-phase carriers used in these studies are complex in production process and high in cost, and are not easy to be popularized in the field of immobilized enzyme production, and are not suitable for industrial immobilized enzyme production.
There is no report on the one-step purification and immobilization of α-amino acid ester acyltransferase through genetic modification

Method used

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  • Method for performing one-step purification and immobilization on alpha-amino acid fatty acyl transferase
  • Method for performing one-step purification and immobilization on alpha-amino acid fatty acyl transferase
  • Method for performing one-step purification and immobilization on alpha-amino acid fatty acyl transferase

Examples

Experimental program
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Effect test

Embodiment 1

[0026] Example 1 Gene acquisition and vector construction

[0027] The NCBI website obtained the gene sequence of Sphingobacterium α-amino acid fatty acyltransferase (SEAT gene, Accession No.AB610978.1) and the gene sequence of Streptococcus pneumoniae formylglycine synthetase (FGE gene, Accession No.NC_000962.3), Send to a third-party service agency to synthesize the full-length gene sequence, in which the nucleotide sequence encoding LCTPSR 5'CGAATTTTTCTGTCCTCAAAGAT3' (SEQ ID NO: 2) is added before the stop codon at the 3' end of the SEAT gene, followed by BamHI and HindIII on both sides of the gene Restriction site, add CACC before the start codon of FGE gene. The synthesized SEAT gene was ligated into the pET30a vector by restriction endonuclease ligation to obtain the SEAT-30a vector.

[0028] Using the Gateway system, the FGE gene was first connected to the Topo vector, and then recombined into the PDEST17 vector to obtain the FGE-pdest17 vector. Sequencing confirmed t...

Embodiment 2

[0030] Example 2 Obtaining of expression strains

[0031] Take 100ng each of SEAT-30a and FGE-pdest17 plasmids confirmed by sequencing, mix with 100μl BL21(DE3) competent cells, let stand on ice for 30min, heat shock at 42°C for 30sec, let stand at room temperature for 5min, then add liquid LB medium Shake culture at 37°C for 30min, centrifuge at 5000rpm for 1min, discard the supernatant, resuspend the pellet with 200μl sterile water, and coat a solid LB plate containing 50μg / mL ampicillin and 30μg / mL kanamycin. Incubate overnight at 37°C. The next day, pick positive single clones, culture overnight in liquid LB medium containing 50 μg / mL ampicillin and 30 μg / mL kanamycin, add IPTG to make the final concentration 1 mM / L, and induce protein expression for 4 hours at 25 °C . Take 1mL culture solution and centrifuge at 12000g for 1min, resuspend in 100μl distilled water, add 100μl 2×SDS protein loading buffer, mix well, heat at 95°C for 10min, centrifuge at 12000g for 5min, tak...

Embodiment 3

[0032] Example 3 Protein induced fermentation

[0033] Take the SQ05 strain and culture it overnight at 37°C in liquid LB medium containing 50 μg / ml ampicillin and 30 μg / mL kanamycin. The next day, take 20ml of culture medium and further expand the culture to 1L. When the cell concentration reaches OD 600 =0.6-0.8, add IPTG to make the final concentration 0.5mM. Induced at 25°C for 6h.

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Abstract

The invention relates to the technical field of enzyme immobilization and particularly discloses a method for performing one-step purification and immobilization on alpha-amino acid fatty acyl transferase. The method comprises the following steps: performing aldehyde labeling on the alpha-amino acid fatty acyl transferase, performing synchronous induction on the alpha-amino acid fatty acyl transferas and formylglycine synthetase in host cells to obtain a bacterial strain with high expression quantity, crushing the thallus, taking supernatant, and mixing the supernatant and an amino carrier torealize one-step immobilization of the alpha-amino acid fatty acyl transferase, wherein an additional purification step is not needed. By the method, the operation steps are saved, expression of the target protein is guaranteed, immobilization can be realized by the common amino carrier, and the method is suitable for industrialized production.

Description

technical field [0001] The invention relates to the technical field of enzyme immobilization, in particular to a method for one-step purification of immobilized α-amino acid fatty acyltransferase. Background technique [0002] Immobilization of enzymes (Immobilization of enzymes) is a kind of technology that binds or confines enzymes in a certain area with solid materials, and can still carry out its unique catalytic reactions, and can be recycled and reused. While maintaining its high-efficiency, specificity and mild enzyme-catalyzed reaction characteristics, the immobilized enzyme overcomes the shortcomings of free enzymes, showing high storage stability, easy separation and recovery, repeated use, and continuous and controllable operation. A series of advantages such as simple process. Moreover, it can save resources and energy, reduce or prevent pollution, and meet the strategic requirements of sustainable development. [0003] Immobilized enzyme technology can be divi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N15/70C12N11/16
CPCC12N9/1025C12N11/16C12N15/70
Inventor 李晨辉刘超王红权李燕飞康丽娟马福民姜召红梁云科李晓微
Owner HEBEI FITNESS BIOTECH CO LTD
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