Recombinant glucose-oxidase-expressing Yarrowia lipolytica and application thereof

A technology of glucose oxidase and Yarrowia lipolytica, applied in the directions of oxidoreductase, recombinant DNA technology, enzymes, etc.

Inactive Publication Date: 2015-02-18
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the GOD gene has been expressed in Pichia pastoris GS115, Escherichia coli, Trichoderma reesei, etc., but it has not been studied in t

Method used

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  • Recombinant glucose-oxidase-expressing Yarrowia lipolytica and application thereof
  • Recombinant glucose-oxidase-expressing Yarrowia lipolytica and application thereof
  • Recombinant glucose-oxidase-expressing Yarrowia lipolytica and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 Construction of plasmids pINA1297-GOD and pINA1296-GOD.

[0027] Respectively, pINA1297 and pINA1296 were used as expression vectors for synthesizing exogenous genes of glucose oxidase.

[0028] The glucose oxidase gene fragment mutated at the Sfi1 site in the gene was obtained from the genome of Aspergillus niger with primers A1 and A2 by PCR, and transformed into Escherichia coli JM109. The glucose oxidase gene fragment with SfiⅠ and BamHI restriction sites was cloned from Escherichia coli JM109 genome by PCR using primers B1 and B2. The PCR product and the expression vector were digested with corresponding restriction endonucleases respectively, ligated and transformed, and the recombinant plasmid pINA1297-GOD was obtained by screening. The glucose oxidase gene fragment with HindIII and KpnI restriction sites was cloned from Escherichia coli JM109 genome by PCR using primers C1 and C2. The PCR product and the expression vector were digested with correspon...

Embodiment 2

[0029] Example 2 Construction of glucose oxidase-producing bacteria.

[0030] The NotⅠ linearized recombinant plasmids pINA1297-GOD and pINA1296-GOD were introduced into Yarrowia lipolytica by lithium acetate transformation method.

[0031] Due to the different selectable markers and integration mechanisms of plasmid pINA1296 (a single-copy integration plasmid based on pBR322) and plasmid pINA1297 (a multi-copy integration plasmid containing a zeta site), the recombinant expression vectors pINA1297-GOD and pINA1296-GOD was transformed into Y.Lipolytica Po1h (Ura-) strain and Y.Lipolytica Po1g (Leu-, containing pBR322 integration platform) respectively, and the positive transformant was screened by defective YNB plate, and it was verified by colony PCR. Positive single colonies were screened out to obtain recombinant bacteria Y.lipolytica (pINA1297-GOD) and Y.lipolytica (pINA1296-GOD). After linearization with NotI, the Kan resistance gene in the bacterial portion of pINA1297-...

Embodiment 3

[0032] Example 3 Production of glucose oxidase by shake flask fermentation of the recombinant strain.

[0033] The recombinant bacteria were activated and cultivated on the YPD plate for a certain stage, inoculated into the seed liquid medium YPD, and transferred to the fermentation medium PPB with 10% inoculum after 24 hours, cultivated in a shaker at 28°C and 200r / min, and determined Glucose oxidase activity of fermentation supernatant. Comparing the enzyme activities in the fermentation broth of different transformants, the glucose oxidase activity in the supernatant of the fermentation broth of Y.lipolytica (pINA1297-GOD) after 168 hours of cultivation reached 13.9U / ml, which was 9 times that of Y.lipolytica (pINA1296-GOD). Compared with the original Aspergillus niger enzyme production capacity of 4.6U / ml, it has increased twice.

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Abstract

The invention discloses a recombinant glucose-oxidase-expressing Yarrowia lipolytica and application thereof, belonging to the technical field of biology. By constructing a recombinant plasmid pINA1297-GOD, a lithium acetate conversion method is utilized to convert Yarrowia lipolytica po1h, and screening is performed to obtain the recombinant strain with favorable enzyme producing capacity. After the strain is fermented for 168 hours by using sucrose as the unique carbon source, the enzyme activity of the glucose oxidase reaches 13.9 U/ml, and is enhanced by two times as compared with the original Aspergillus niger.

Description

technical field [0001] The invention relates to a Yarrowia lipolytica recombinantly expressing glucose oxidase and an application thereof, belonging to the field of biotechnology. Background technique [0002] Glucose Oxidase (Glucose Oxidase, referred to as GOD, E.C.1.1.3.4) is a flavin glycoprotein that can specifically catalyze β-D-glucose to generate gluconic acid and hydrogen peroxide under aerobic conditions. GOD is widely distributed in animals, plants and microorganisms. Due to the rapid growth and reproduction of microorganisms and wide sources, they are the main source of GOD production. The main production strains are Aspergillus niger and Penicillium. At present, the application fields of GOD are continuously expanding, and the demand in domestic and foreign markets is increasing sharply. At present, the application fields of GOD are continuously expanding, and the demand in domestic and foreign markets is increasing sharply. At present, the GOD gene has been ...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/81C12N9/04C12R1/645
CPCC12N9/0006C12Y101/03004C12N1/145C12R2001/645
Inventor 陈坚刘晓筱张娟堵国成顾磊
Owner JIANGNAN UNIV
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